1521-0081/65/2/710–778$25.00https://www.doczj.com/doc/d85015178.html,/10.1124/pr.112.006833 P HARMACOLOGICAL R EVIEWS Pharmacol Rev65:710–778,April2013 Copyright?2013by The American Society for Pharmacology and Experimental Therapeutics
ASSOCIATE EDITOR:DIANNE M.PEREZ
Nuclear Receptors and Their Selective
Pharmacologic Modulators
Thomas P.Burris,Laura A.Solt,Yongjun Wang,Christine Crumbley,Subhashis Banerjee,Kristine Griffett,
Thomas Lundasen,Travis Hughes,and Douglas J.Kojetin
The Scripps Research Institute,Jupiter,Florida
Abstract (711)
I.Introduction (711)
A.Discovery of Nuclear Receptors (712)
B.Ligand Regulation of Nuclear Receptor Function (713)
C.Nuclear Receptor Modulators and Selective Modulators (716)
II.Selective Estrogen Receptor Modulators (718)
A.Estrogen Receptor Structure (718)
B.Estrogen Receptor Function (719)
C.Selective Estrogen Receptor Modulators (720)
III.Vitamin D Receptor Modulators (725)
A.Vitamin D Receptor Structure (725)
B.Vitamin D Receptor Function (725)
C.Vitamin D Receptor Modulators (728)
IV.Thyroid Hormone Receptor Modulators (735)
A.Thyroid Hormone Receptor Structure (735)
B.Thyroid Hormone Receptor Action (735)
C.Selective Thyroid Hormone Receptor Modulators (737)
V.Selective Androgen Receptor Modulators (739)
A.Androgen Receptor Structure (739)
B.Androgen Receptor Function (740)
C.Selective Androgen Receptor Modulators (740)
VI.Selective Glucocorticoid Receptor Modulators (742)
A.Glucocorticoid Receptor Structure (742)
B.Glucocorticoid Receptor Action (743)
C.Selective Glucocorticoid Receptor Modulators (744)
VII.Selective Peroxisome Proliferator-Activated Receptor g Modulators (748)
A.Peroxisome Proliferator-Activated Receptor g Structure (748)
B.Peroxisome Proliferator-Activated Receptor g Function (749)
C.Selective Peroxisome Proliferator-Activated Receptor g Modulator (750)
VIII.Liver X Receptor Modulators (754)
A.Liver X Receptor Structure (754)
B.Liver X Receptor Function (755)
C.Selective Liver X Receptor Modulators (755)
IX.Selective Progesterone Receptor Modulators (758)
A.Progesterone Receptor Structure (758)
B.Progesterone Receptor Function (759)
C.Selective Progesterone Receptor Modulators (759)
References (763)
Address correspondence to:Thomas P.Burris,The Scripps Research Institute,130Scripps Way2A1,Jupiter,FL33458.E-mail: tburris@https://www.doczj.com/doc/d85015178.html,
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Abstract——Nuclear receptors are ligand-activated transcription factors and include the receptors for steroid hormones,lipophilic vitamins,sterols,and bile acids.These receptors serve as targets for development of myriad drugs that target a range of disorders. Classically defined ligands that bind to the ligand-binding domain of nuclear receptors,whether they are endogenous or synthetic,either activate receptor activ-ity(agonists)or block activation(antagonists)and due to the ability to alter activity of the receptors are often termed receptor“modulators.”The complex pharmacol-ogy of nuclear receptors has provided a class of ligands distinct from these simple modulators where ligands display agonist/partial agonist/antagonist function in a tissue or gene selective manner.This class of ligands is defined as selective modulators.Here,we review the development and pharmacology of a range of selective nuclear receptor modulators.
I.Introduction
The nuclear receptor(NR)superfamily constitutes a group of48transcription factors in humans,which includes the receptors for steroid hormones,thyroid hormone,lipophilic vitamins,and cholesterol metab-olites(Evans,1988;Mangelsdorf et al.,1995)(Table1).Approximately half of NRs are classified as orphan receptors because they do not have well characterized ligands(O’Malley and Conneely,1992;Mangelsdorf and Evans,1995;Giguere,1999;Kliewer et al.,1999).The NRs regulate a wide range of physiologic and deve-lopmental processes,and virtually all the NRs that have
ABBREVIATIONS:A276575,2,5-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5-(1-methylcyclohexen-3-y1)-1H-[1]benzopyrano[3,4-f]quinoline;A-348441,4-[(3S,7R,12S)-7,12-dihydroxy-3-[2-[4-[(8S,11R,13S,14S,17S)-17-hydroxy-13-methyl-3-oxo-17-prop-1-ynyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-11-yl]-N-methylanilino]ethoxy]-2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17-hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid;AC-262536,4-(3-hydroxy-8-aza-bicyclo[3.2.1]octyl)-naphthalene-1-carbonitrile;A/B,amino-terminal domain;AF,activation function; AL-438,10-methoxy-5-(2-propenyl)-2,5-dihydro-2,2,4-trimethyl-1H-(1)benzopyrano(3,4-f)quinoline;AP,activator protein;AR,androgen receptor;BMD, bone mineral density;BMS-564929,(7R,7aS)-2-chloro-4-(7-hydroxy-1,3-dioxotetrahydropyrrolo[1,2-c]imidazol-2-yl)-3-methylbenzonitrile;BXL-2198,1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor vitamin D3;BXL-01-0029,1,3-di-O-acetyl-1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor-cholecalciferol;BXL-62,1a,25(OH)2-16-ene-20-cyclopropyl-vitamin D3;CBP,CREB–binding protein;CDB-2914,ulipristal acetate;CDB-4124, telapristone acetate;CE,conjugated estrogens;CETP,cholesteryl ester transfer protein;CGH509A,2-(4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenyl)-2-(4-(3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)acetic acid;CGS23425,N-[3,5-dimethyl-4-(49-hydroxy-39-isopropylphenoxy)-phenyl]-oxamic acid;CKD,chronic kidney disease;CLX-0921(THR0921),(E)-methyl3-(3,5-dimethoxyphenyl)-2-(4-(4-((2,4-dioxothiazolidin-5-yl)methyl)phenoxy)phenyl)acrylate;CNS,central nervous system;CpdA,Compound A;CREB, cAMP response element-binding protein;Cyp24,25-hydroxyvitamin D3-24hydroxylase;DBD,DNA-binding domain;DBP,vitamin D-binding protein;DHT,dihydrotestosterone;DIO1,type I iodothyronine59-deiodinase;DIOII,type II iodothyronine59-deiodinase;DRIP,vitamin D interacting protein;DT56a,femarelle;EAE,experimental autoimmune encephalomyelitis;ED-71,1a,25-dihydroxy-2b-(3-hydroxypropoxy) vitamin D3;ER,estrogen receptor;ERBA a,v-erbA,c-erbA;FDA,Food and Drug Administration;FK614,3-(2,4-dichlorobenzyl)-2-methyl-N-(pentylsulfonyl)-3-H-benzimidazole-5-carboxamide;F-L-Leu,Fmoc-L-leucine;GC-1,3,5-dimethyl-4(49-hydroxy-39-isopropylbenzyl)-phenoxy)acetic acid;GR,glucocorticoid receptor;GRE,glucocorticoid-responsive element;GSK-9772,4-[[N-butyl-4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl) anilino]methyl]-2,6-dichlorophenol;GW0072,4-[4-[(2S,5S)-5-[2-(dibenzylamino)-2-oxoethyl]-2-heptyl-4-oxo-1,3-thiazolidin-3-yl]butyl]benzoic acid; GW3965,2-[3-[3-[[2-chloro-3-(trifluoromethyl)phenyl]methyl-(2,2-diphenylethyl)amino]propoxy]phenyl]acetic acid;GW9662,2-chloro-5-nitro-N-phenylbenzamide;HDL,high-density lipoprotein;HDX,hydrogen/deuterium exchange;HDXMS,hydrogen deuterium exchange mass spectroscopy; Hsp,heat shock protein;IBD,irritable bowel disease;ICI182,780,fulvestrant;IL,interleukin;INF-g,interferon g;INT-131,N-(3,5-dichloro-4-quinolin-3-yloxyphenyl)-2,4-dihydroxybenzenesulfonamide;KAT-681,sodium;3-[4-[3-[(4-fluorophenyl)-hydroxymethyl]-4-hydroxyphenoxy]-3,5-dimethylanilino]-3-oxopropanoate;KB141,3,5-dichloro-4-[4-hydroxy-3-(propan-2-yl)phenoxy]phenyl acetic acid;KB2115,3-[[3,5-dibromo-4-[4-hydroxy-3-(1-methylethyl)-phenoxy]-phenyl]-amino]-3-oxopropanoic acid;KR-62980,1-(trans-methylimino-N-oxy)-6-(2-morpholinoethoxy)-3-phe-nyl-1H-indene-2-carboxylic acid ethyl ester;L-94901,3,5-dibromo-3-pyridazinone-L-thyronine;LBD,ligand-binding domain;LBP,ligand-binding pocket;LDL,low-density lipoprotein;LG100268,6-[1-(3,5,5,8,8-pentamethyl-6,7-dihydronaphthalen-2-yl)cyclopropyl]pyridine-3-carboxylic acid; LGD-2226,6-(bis-(2,2,2-trifluoroethyl)amino)-4-trifluoromethyl-1H-quinolin-2-one;LGD-3303,9-chloro-2-ethyl-1-methyl-3-(2,2,2-trifluoroethyl)-3H-pyrrolo-[3,2-f]quinolin-7(6H)-one;LGD-5552,(5Z)-5-[(2-fluoro-3-methylphenyl)methylidene]-10-methoxy-2,2,4-trimethyl-1H-chromeno[3,4-f]quino-lin-9-ol;LH,luteinizing hormone;LPS,lipopolysaccharide;LSN,(R)-(+)-7,9-difluoro-5-[4-(2-piperidin-1-ylethoxy)phenyl]-5H-6-oxachrysen-2-ol; LXR,liver X receptor;LXR-623,2-[(2-chloro-4-fluorophenyl)methyl]-3-(4-fluorophenyl)-7-(trifluoromethyl)indazole;LXRM,liver X receptor modulator;LY353381,arzoxifene;MB07811,(2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(49-hydroxy-39-isopropylbenzyl) phenoxy)methyl]-2-oxido-[1,3,2]-dioxaphosphonane;2MD,2-methylene-19-nor-(20S)-1a,25(OH)2D3;MK0533,(2R)-2-(3-3-[(4-methoxy-phenyl)carbonyl]-2-methyl-6-(trifluoromethoxy)-1H-indol-1-ylphenoxy)butanoic acid;MK-2866,(2S)-3-(4-cyanophenoxy)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylpropanamide;MMTV,mouse mammary tumor virus;MR,mineralocorticoid receptor; MRL-24,3-(3-((1-(4-methoxybenzoyl)-2-methyl-5-(trifluoromethoxy)-1H-indol-3-yl)methyl)phenyl)-2-methylpropanoic acid;NCoR,nu-clear receptor corepressor;NFAT1,nuclear factor of activated T cells1;NF-k B,nuclear factor k B;NR,nuclear receptor;1a(OH)D3,1-a-hydroxyvitamin D3;1,25-(OH)2D3,1a,25-dihydroxyvitamin D3;OPG,osteoprotegerin;OVX,ovariectomized;P4,pregn-4-ene-3,20-dione;PA-082,1-(3,4-dimethoxy-benzyl)-6,7-dimethoxy-4-[4-(2-methoxy-phenyl)-piperidin-1-ylmethyl]-isoquinoline;PBMC,peripheral blood mononuclear cell;PPAR g,peroxisome proliferator-activated receptor g;PPRE,peroxisome proliferator-activated receptor response element;PR,progesterone receptor;PRE,progesterone receptor DNA-response element;PRKO,progesterone receptor knockout/ null mutation;PTH,parathyroid hormone;PXR,pregnane X receptor;QRX-431,3,5-dimethyl-4(49-hydroxy-39-isopropylbenzyl)-phenoxy)acetic acid;RANKL,receptor activator of nuclear factor k B ligand;RBC,red blood cell;RIP,receptor-interacting protein;Ro-26-9228,1a-fluoro-16-ene-20-epi-23-ene-26,27-bishomo-25-hydroxyvitamin D3;RAR,retinoid acid receptor;ROR,retinoic acid receptor–related orphan receptor;RTH,resistance to thyroid hormone syndrome;RU24858,3-((9R,10S,11S,13S,16R)-9-fluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-3-oxopropanenitrile;RU24782,(9R,10S,11S,13S,16R)-9-fluoro-11-hydroxy-10,13,16-trimethyl-17-(2-(methylthio)acetyl)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one
Nuclear Receptors and Their Selective Modulators711
identified ligands are well-characterized targets for the development of drugs to treat myriad diseases in-cluding cancer,diabetes,atherosclerosis,inflammation, and endocrine/reproductive disorders.
A.Discovery of Nuclear Receptors
Biochemical evidence for the existence of cellular receptors for steroid hormones was first demonstrated by the use of radiolabeled estrogens and examination of specific binding within estrogen-responsive tissues (Glascock and Hoekstra,1959;Jensen and Jacobson, 1960,1962).Autoradiographic analysis suggested that these estrogen receptors(ERs)were nuclear in localization rather than associated with the plasma membrane as were the other receptors that were be-ing characterized at the time(Jensen et al.,1967). Localization of ER within the nucleus and experiments that suggested that the receptor was associated with chromatin(King et al.,1966;Maurer and Chalkley, 1967;Teng and Hamilton,1968;Shyamala and Gorski, 1969;Mainwaring,1971;Mainwaring and Peterken, 1971;Spelsberg et al.,1971;Steggles et al.,1971)were consistent with the early suggestions that estrogen action might be associated with regulation of RNA synthesis(Mueller et al.,1958).Studies in the early to mid-1960s suggested that ER was a protein,and in 1966,Toft and Gorski were the first to biochemically isolate a steroid hormone receptor(Toft and Gorski, 1966).Characterization of purified ER as well as other steroid receptors over the next two decades led to the following key observations:1)steroid receptors stimu-late mRNA synthesis of specific genes by stimulating RNA polymerase activity(Gorski,1964;Greenman et al.,1965;Means and Hamilton,1966;O’Malley et al.,1968a,b,1972a,b;O’Malley and McGuire,1968; Comstock et al.,1972;Glasser et al.,1972;Means et al.,1972;Mohla et al.,1972;Rosenfeld et al.,1972; Chan et al.,1973;Parks et al.,1974;Ringold et al., 1975,1977;Scolnick et al.,1976;Young et al.,1977);2) steroid receptors bind to double-stranded DNA via specific base sequences(hormone response elements)(King and Gordon,1972;Musliner and Chader,1972; Toft,1972;Andre and Rochefort,1973;Beato et al., 1973;Gehring and Tomkins,1974;Yamamoto and Alberts,1974,1975;Yamamoto et al.,1974);and3) hormone response elements are located in the pro-moters of steroid-responsive target genes. Characterization of purified steroid receptor pro-teins by limited proteolysis clearly indicated that the receptors were composed of several domains that retained specific functions(e.g.,DNA binding or steroid binding)when examined in isolation(Wrange and Gustafsson,1978;Carlstedt-Duke et al.,1982).Molec-ular cloning of the glucocorticoid receptor(GR)in1985 provided the first glimpse into the genetic organiza-tion of a nuclear receptor(Hollenberg et al.,1985; Weinberger et al.,1985a).On the basis of the predicted amino acid sequence and domain structure of the receptor,the location of the DNA-binding domain (DBD)and steroid-binding domain(ligand-binding do-main;LBD)was correctly predicted(Weinberger et al., 1985b).The cDNAs for all of the steroid receptors were rapidly identified,including the estrogen receptor (ER)(Walter et al.,1985;Green et al.,1986;Greene et al.,1986;Krust et al.,1986;Kumar et al.,1986),the progesterone receptor(PR)(Conneely et al.,1986,1987a; Jeltsch et al.,1986;Loosfelt et al.,1986;Gronemeyer et al.,1987;Misrahi et al.,1987),the mineralocorticoid receptor(MR)(Arriza et al.,1987;Patel et al.,1989), and the androgen receptor(AR)(Chang et al.,1988a,b; Lubahn et al.,1988;Trapman et al.,1988;Tilley et al., 1989).Comparison of the sequences of all of these re-ceptors demonstrated a highly conserved structure that was defined as six subregions(regions A through F; Fig.1)based on degree of homology(Krust et al.,1986). The amino-terminal region,considered the A/B region, was the most divergent among the receptors and was shown to contain a hormone-independent transactiva-tion function in many receptors(activation function1; AF-1).Region C,the most conserved domain,is rich in cysteines and basic amino acids,with the position of the cysteines absolutely conserved among the receptors
RU40066,(10S,13S,17S)-17-hydroxy-10,13-dimethyl-17-(prop-1-yn-1-yl)-6,7,8,10,12,13,14,15,16,17-decahydro-3H-cyclopenta[a]phenanthren-3-one;RU486,mifepristone;RXR,retinoic acid X receptor;S-4,(2S)-3-(4-acetamido-phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoro-methyl-phenyl)-propionamide;S-23,(S)-N-(4-cyano-3-trifluoromethyl-phenyl)-3-(3-fluoro,4-chlorophenoxy)-2-hydroxy-2-methyl-propanamide; S-40503,2-[4-(dimethylamino)-6-nitro-1,2,3,4-tetrahydroquinolin-2-yl]-2-methylpropan-1-ol;SARM,selective androgen receptor modulator; SeGRA,selective glucocorticoid receptor agonist;SERM,selective estrogen receptor modulator;SGRM,selective glucocorticoid receptor modulator;SHPT,secondary hyperparathyroidism;SLXRM,selective liver X receptor modulator;SMRT,silencing mediator for retinoid and thyroid hormone receptor;SPPAR g M,selective peroxisome proliferator-activated receptor g modulator;SPRM,selective progesterone receptor modulator;SRC,steroid receptor coactivator;SREBP1c,sterol regulatory element-binding protein1c;T-0681,3-[4-[3-[(4-fluorophenyl)-hydroxymethyl]-4-hydroxyphenoxy]-3,5-dimethylanilino]-3-oxopropanoate;SR1664,(S)-49-((5-((1-(4-nitrophenyl)ethyl)carbamoyl)-1H-indol-1-yl) methyl)-[1,19-biphenyl]-2-carboxylic acid;T0901317,N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzene-sulfonamide;T3,triiodothyronine;T4,thyroxine;TAZ,transcriptional coactivator with PDZ-binding motif;TDZ,thiazolidinedione drug;TGF b, transforming growth factor b;TH,thyroid hormone;T H,T helper;TIF,transcriptional mediators/intermediary factor;TNF a,tumor necrosis factor a;TR,thyroid receptor;TSH,thyroid-stimulating hormone;VDR,vitamin D receptor;VDRE,vitamin D3response element;VDRM,vitamin D receptor modulator;WAY-140424,bazedoxifene;WAY-252623,2-[(2-chloro-4-fluorophenyl)methyl]-3-(4-fluorophenyl)-7-(trifluoromethyl)indazole; YT-32,(22E)-ergost-22-ene-1a,2b diol;ZK156979,22-ene-25-oxa-vitamin D;ZK216348,3-dihydro-1-benzofuran-7-yl)-2-hydroxy-4-methyl-N-(4-methyl-1-oxo-2,3-benzoxazin-6-yl)-2-(trifluoromethyl)pentanamide;ZK-245186,(2R)-1,1,1-trifluoro-4-(5-fluoro-2,3-dihydro-1-benzofuran-7-yl)-4-methyl-2-[[(2-methylquinolin-5-yl)amino]methyl]pentan-2-ol.
712Burris et al.
consistent with their function in coordination of Zn2+ ions within the two zinc-finger structures located within this DNA binding domain.The D region,also termed the“hinge domain”due to its localization be-tween the DBD and the LBD,is a relatively short region with a low degree of conservation.The hinge domain plays a role in modulation of DNA binding for some receptors.The E region encompasses the LBD and contains the ligand-dependent transactivation domain(activation function2;AF-2).A region further toward the carboxy-terminal to the E region,referred to as the F domain,was noted in only some receptors; to date,it still has an unclear role in receptor function. Significant homology between the steroid receptors and the v-erbA oncogene led to the prediction that the c-erbA gene may encode a receptor for a steroid or steroid-like molecule(Weinberger et al.,1985a;Green et al.,1986).Functional analysis of the protein en-coded by the c-erbA gene verified this prediction when it was shown to encode a receptor for thyroid hormone(TH)(Sap et al.,1986;Weinberger et al.,1986b). Receptors for a range of lipophilic vitamins were soon identified as belonging to the same class of mol-ecules,including vitamin A(retinoic acid)(Giguere et al.,1987;Petkovich et al.,1987)and vitamin D (McDonnell et al.,1987;Baker et al.,1988).A number of additional“receptors”also were identified based on sequence homology to the steroid receptors but with no known ligands.These include v-erbA-related receptor and estrogen-related receptor,which were identified based on low-stringency cross-hybridization strategies (Giguere et al.,1988;Miyajima et al.,1988).More orphan receptors would be identified with this method; however,in addition,a number would be identified with other methods including biochemical methods (e.g.,chicken ovalbumin upstream promoter transcrip-tion factor)(Wang et al.,1989),positional cloning(e.g., DAX-1)(Zanaria et al.,1994;Burris et al.,1996),or interaction cloning[e.g.,receptor-interacting protein (RIP)14,RIP15and SHP](Seol et al.,1995,1996). Since their initial characterization as orphan nuclear receptors,endogenous ligands have been identified for many of these including bile acids for farnesoid X receptor(Makishima et al.,1999;Parks et al.,1999), oxysterols for liver X receptors(LXRs)(Janowski et al., 1996;Lehmann et al.,1997),9-cis-retinoic acid for retinoic acid X receptor(RXR)(Heyman et al.,1992; Levin et al.,1992;Mangelsdorf et al.,1992),heme for REV-ERB(Raghuram et al.,2007;Yin et al.,2007), xenobiotics for pregnane X receptor(PXR)and consti-tutive active/androstane receptor(Willson and Kliewer, 2002),and fatty acids for peroxisome proliferator-activated receptors(PPARs)(Keller et al.,1993;Yu et al.,1995).
B.Ligand Regulation of Nuclear Receptor Function
As indicated earlier,NRs typically function as ligand-dependent transcription factors.A number of or-phan NRs still have no characterized endogenous or synthetic ligands;however,this review focuses on the action of ligands on NRs,so only those receptors that have characterized ligands will be discussed.NRs rec-ognize specific DNA-response elements in the promoters/ enhancers of their cognate target genes where they can respond to ligands by altering their ability to recruit a range of other transcriptional proteins that alter the
TABLE1
Human Nuclear Receptor Superfamily
Common Symbol Endogenous Ligand
Subfamily1
NR1A1TR a Thyroxine
NR1A2TR b Thyroxine
NR1B1RAR a Retinoic acid
NR1B2RAR b Retinoic acid
NR1B3RAR g Retinoic acid
NR1C1PPAR a Fatty acids
NR1C2PPAR d Fatty acids
NR1C3PPAR g Fatty acids
NR1D1REV-ERB a Heme
NR1D2REV-ERB b Heme
NR1F1ROR a Oxysterols
NR1F2ROR b Oxysterols
NR1F3ROR g Oxysterols
NR1H2LXR b Oxysterols
NR1H3LXR a Oxysterols
NR1H4FXR Bile acids
NR1I1VDR Vitamin D
NR1I2PXR Xenobiotics
NR1I3CAR Xenobiotics
Subfamily2
NR2A1HNF4a
NR2A2HNF4g
NR2B1RXR a9-Cis-retinoic acid
NR2B2RXR b9-Cis-retinoic acid
NR2B3RXR g
NR2C1TR2
NR2C2TR4
NR2E1TLX
NR2E3COUPTF1
NR2F1COUPTF2
NR2F6EAR2
Subfamily3
NR3A1ER a Estradiol
NR3A2ER b Estradiol
NR3B1ERR a
NR3B2ERR b
NR3B3ERR g
NR3C1GR Glucocorticoids
NR3C2MR Mineralocorticoids
NR3C3PR Progestins
NR3C4AR Androgens
Subfamily4
NR4A1NGFIB
NR4A2NURR1
NR4A3NOR1
Subfamily5
NR5A1SF-1
NR5A2LRH-1
Subfamily6
NR6A1GCNF
Subfamily0
NR0B1DAX1
NR0B2SHP
REV-ERB,reverse strand ERBA oncogene;FXR,farnesoid X receptor;CAR,
constitutive androstane receptor;HNF,hepatocyte nuclear factor;TLX,Tailless gene
homolog;COUPTF,chicken ovalbumin upstream promoter transcription factor;EAR,
erbA-related gene;ERR,estrogen receptor-related orphan receptor;NGFIB,nerve
growth factor-induced gene B;NURR,nuclear receptor related1;NOR,neuron-
derived orphan receptor;SF1,steroidogenic factor1;GCNF,germ cell nuclear factor;
DAX1,dosage-sensitive sex reversal,adrenal hypoplasia critical region,on
chromosome X,gene1;SHP,short heterodimer partner.
Nuclear Receptors and Their Selective Modulators713
rate of gene expression(Fig.1).Of course,the various NRs have distinct preferences for both ligands and DNA sequences that they recognize as response elements within the genome,providing for the distinct functions of the48members of this class of transcrip-tion factors.Most of the NRs function as dimers,either homodimers(as is the case for the steroid receptors) or heterodimers with a common NR,the RXR.A subset of NRs also functions as monomers and include many of the orphan members of the superfamily.
Some NRs,including several steroid hormone recep-tors,undergo a translocation from the cytoplasm to the nucleus that is ligand dependent,as discussed in the previous section.In this case,they are unable to exert an effect on the transcription of target genes in the absence of ligands.Most NRs appear to bind their response elements both in the absence and presence of ligand,and in many cases may take an active role in the regulation of target gene regulation even in the absence of ligand.One clear example of this is the
Fig.1.Nuclear receptor domain structure and mechanism of action.(A)Nuclear receptors display a conserved modulator domain architecture with an N-terminal AF-1region(A/B region),followed by zinc-finger DBD(C region),a hinge domain(D region),an LBD containing the AF-2region(E region), and some receptors have a C-terminal F domain.(B)Mechanistically,nuclear receptors are regulated by small molecule ligands,which generally stabilize the receptor into a conformation suitable to bind coregulator proteins(coactivators or corepressors).Ligands can also modulate posttranslational modification of the receptor.Ultimately,these events have an impact on the expression of receptor-specific target genes by modulating coregulator recruitment at specific DNA-response element sites in the target gene promoter.(C)Schematic illustrating the principle of selective receptor modulation.
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thyroid hormone receptor(TR),which can actively silence target gene transcription in the absence of TH but transforms into a transcriptional activator in the presence of TH(Cheng et al.,2010).These types of functional transformations are due to induction of conformational changes that alter the array of protein cofactors to which the NR binds,as will be discussed in detail later.
Based on the observation of transcriptional interfer-ence or“squelching”of transactivation activity of PR by overexpression of ER,it was proposed that limiting the amount of accessory or coactivator proteins was re-quired for NRs to regulate transcription(Meyer et al., 1989).It became clear that NRs compete for coactivator proteins within the cell,which mediates activation of transcription,and it also appeared that distinct trans-activation domains may use distinct coactivator proteins (Tasset et al.,1990).Interestingly,transcriptional si-lencing mediated by TR or the retinoic acid receptor (RAR)could also be squelched,suggesting the exis-tence of proteins that mediated suppression of target gene transcription(corepressors)(Baniahmad et al., 1995).By use of biochemical methods,putative in-termediary proteins that interacted with the ER’s LBD were identified that were potential coactivators as they only interacted in the presence of an agonist,antago-nists blocked the agonist-dependent interaction,and the proteins could not interact with transcriptionally defective ERs(Halachmi et al.,1994).Similar putative coactivators were identified as GR-interacting proteins that display ligand-dependent interaction(Eggert et al.,1995).The first coactivator,steroid receptor coactivator1(SRC1),was cloned using a two-hybrid system using the LBD of PR as bait and shown to function as a coactivator for a range of NRs(Onate et al.,1995).SRC1displayed all of the expected char-acteristics of an authentic coactivator,including agonist-dependent interaction with the LBD,which could be blocked by an antagonist,and the ability to rescue squelched NRs(Onate et al.,1995).It rapidly became clear that SRC1was only the first of a family of coactivators,which includes SRC2and SRC3(Hong et al.,1996,1997;Voegel et al.,1996;Anzick et al., 1997;Chen et al.,1997;Li et al.,1997a;Takeshita et al.,1997;Torchia et al.,1997).Additionally,a range of other classes of coactivators were soon characterized, and the list of various proteins with transcriptional coactivator activity is now in the hundreds(Horwitz et al.,1996;Xu et al.,1999;McKenna and O’Malley, 2002,2010;Lonard and O’Malley,2006,2007;Lonard et al.,2007).Corepressor proteins were also identified, such as nuclear receptor corepressor1(NCoR1)and si-lencing mediator for retinoid and thyroid hormone receptor(SMRT)(Chen and Evans,1995;Horlein et al.,1995),but this class of proteins appears to be far fewer in number than coactivators.These coactivator/ corepressor proteins display an array of activities associated with regulation of transcription,including
histone acetyltransferase activity,histone deacetylase
activity,arginine methyltransferase activity,ubiquitin
ligase activity,and ATP-dependent chromatin-remodeling
activity.
Structural studies of the LBD of NRs have provided
significant information about how ligands modulate the
structure of the receptor leading to recruitment of these
cofactors.The LBDs of NRs display a very conserved
tertiary structure,which is a globular domain composed
almost exclusively of a-helices arranged in a three-layer “sandwich”(Wurtz et al.,1996).NR ligands bind to a ligand-binding pocket(LBP)within the interior of this
globular domain,which is consistent with the typical
hydrophobic character of NR ligands.There are11
a-helices within the globular structure that vary in size
between various NRs.Helix12(H12)forms a mobile “lid”over the entrance to the LBP and contains critical residues for the function of AF-2.Ligand-dependent positioning of H12proves to be critical for formation of the coactivator-binding surface of the LBD that allows for ligand-dependent recruitment of coactivator proteins. In the absence of ligand,H12is believed to be quite mobile,but ligand binding stabilizes the position of H12against the globular domain so as to complete the formation of a hydrophobic groove on the surface of the LBD that recognizes a signature motif found on many coactivator proteins known as an NR box or LXXLL motif(L=leucine,X=any amino acid)(Feng et al.,1998; Heery et al.,1997;Savkur and Burris,2004).The coactivator-binding surface of the LBD is composed of helices3,5,and12and serves as a docking site for the a-helical NR box(Fig.2).The hydrophobic leucine side chains of the NR box become buried within the hydrophobic LBD groove while absolutely conserved glutamic acid(H12)and lysine(H3)residues form a charge clamp by forming hydrogen bonds with the peptide backbones of the leucines flanking the NR box (Nolte et al.,1998).Unexpectedly,corepressors
were
Fig.2.Coactivator binding surface in the LBD composed of a surface formed at the intersection of several structural elements in the LBD, including helices3,4,and12.This surface is called the AF-2site. Coactivator regions containing a canonical LXXLL motif bind to this surface,docking the three hydrophobic leucine side chains(show in yellow)into a hydrophobic groove.Two key residues in the LBD form a“charge clamp”that helps stabilize this interaction,including a Lysine residue on helix3and a Glutamic acid residue on helix12,which make interactions with the backbone of the LXXLL motif peptide.
Nuclear Receptors and Their Selective Modulators715
found to use a similar signature sequence for recognition of the LBD,a longer amphipathic a -helical sequence known as a CoRNR box (Fig.3)(Hu and Lazar,1999;Hu et al.,2001).As one would anticipate,H12cannot be in the agonist position,as we indicated earlier,for effective CoRNR box binding to the LBD;in fact,agonist binding places H12into a position that physically precludes binding of the CoRNR box motif.This was predicted before a corepressor was cloned or the structure of the LBD was determined,as deletion of the AF-2region (later determined to be H12)of TR created a constitutive silencing receptor,which indicated that AF-2(H12)was required for displacement of a putative corepressor protein (Baniahmad et al.,1995).C.Nuclear Receptor Modulators and Selective Modulators
Because of the role that many of the NRs play in disease,these receptors have been a rich source for the development of small molecule synthetic ligands that either mimic the action of typical endogenous ligands (agonists)or block the action of endogenous ligands (antagonists).For the vast majority of receptors,agonist binding to the LBD results in a conformational change that leads to recruitment of coactivator pro-teins,resulting in increased transcription of target genes.An antagonist (in the purist sense,or as some would call it,a “neutral ”antagonist)simply binds to the LBD and prevents the conformational change that an agonist would cause,thus preventing coactivator recruitment and subsequent stimulation of transcrip-tion.This is the simplest case,but the pharmacology of the NRs turns out to be much more complex.Partial agonists bind to the LBD,and the resulting conforma-tional change provides only a partial activation of transcription,which is likely due to less proficient recruitment of coactivators.Partial agonists will also
compete with full agonists so as to reduce the level of transactivation intrinsic to that of the full agonist.Inverse agonists are also commonly found,and they are particularly important when a receptor has some degree of basal activity (and basal level of recruitment of coactivator);binding of this class of ligand results in a conformational change that reduces the basal level of activity (reduces basal coactivator binding).In many cases,it has become clear that the conformation induced by inverse agonists may also result in re-cruitment of corepressor proteins,resulting in active silencing of target gene transcription.In this case,a particular NR may not need to display any basal coactivator binding for the inverse agonist to cause a decrease in target gene transcription.
The resulting positioning of H12in response to ligand binding is the critical determinate of the func-tion of the ligand (agonist versus antagonist),and this has been clearly shown in several cocrystal structures of NR LBDs with various ligands.An example is shown in Fig.4for ER.When ER is bound to an agonist (estradiol),H12is positioned in a manner to complete the formation of the coactivator-binding groove,which allows for recruitment of the NR box of coactivator proteins.In the case of antagonist binding,such as raloxifene or ICI 182,780(fulvestrant),the LBD as-sumes a conformation that does not allow coactivator NR box recognition.In the case of raloxifene binding,H12is placed in such a position so as to physically block NR box binding (Brzozowski et al.,1997;Shiau et al.,1998);in the case of ICI 182,780binding,the long aliphatic extension of this compound itself exits the LBP entryway and folds along the coactivator binding groove,thus preventing NR box recognition (Pike et al.,1999,2001).
Significantly more complex is the situation of NR ligands classified as selective modulators.Selective NR modulators display tissue and/or target gene specificity in terms of their agonist,antagonist,or inverse agonist activity.The first examples of selective modulators were identified targeting ER,where compounds such as tamoxifen function as an antagonist in breast tissue but as an agonist in bone and uterus.It is clear that the tissue specificity can be altered,given that compounds such as the selective ER modulator (SERM)raloxifene were identified that had the clinically superior tissue specificity profile of antagonist in the breast and uterus but agonist in bone.These types of NR ligands are the focus of this review;in the subsequent sections,the pharmacologic profile of major modulators for several NRs are described in detail.The mechanism of action that underlies the tissue/target gene specificity is still not clear,but several sound theories have been proposed.It is highly likely that various selective modulators function via distinct mechanisms and that one mechanism will not be able to explain how they all display their unique pharmacologic profiles.
Before
Fig.3.Corepressor binding surface in the LBD.This surface is similar to the coactivator-binding surface but uses a conserved LXXIIXXXI motif to interact with the LBD.The conserved hydrophobic residues (shown in yellow)bind in a hydrophobic groove formed the intersection of helices 3and 4,but this motif does not engage helix 12via the “charge clamp.”
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turning to specific selective modulators,the potential mechanisms that may be responsible for modulator pharmacology will be discussed.
It is quite clear that various modulators with distinct tissue/target gene specificity profiles induce unique conformations within the LBD of the NRs.In many cases ER is used as a model because the pharmacology of selective modulators is most well developed with this particular https://www.doczj.com/doc/d85015178.html,ing techniques such as phage display or hydrogen deuterium exchange mass spec-troscopy (HDXMS),it has been demonstrated that binding of distinct classes of SERMs results in distinct conformations on the surface of the LBD (Chang et al.,1999;Norris et al.,1999;Dai et al.,2008,2009).In fact,we were able to predict the tissue specificity profile of various SERMS using LBD conformation information gleaned from HDXMS data (Dai et al.,2009).The unique conformations induced by the various selective modulators are believed to be associated with distinct patterns of recruitment of coactivators and corepres-sors that lead to the tissue/target gene specificity profile.We and others were also able to demonstrate this by showing that ER LBD bound to various selective modulators displayed distinct affinities for different coactivators and/or NR boxes (Gee et al.,1999;Bramlett et al.,2001).Various cell types express different levels of the coactivators and corepressors that may lead to the tissue-specific activity.Variation of the ratio of coactivator to corepressor in a particular cell type modulates the agonist/antagonist activity of the SERM tamoxifen (Smith et al.,1997).SRC1-dominant expression over SMRT led to tamoxifen displaying significant agonist activity,whereas in the reverse conditions,tamoxifen functioned as an antagonist (Smith et al.,1997).Shang and Brown (2002)took this one step farther by comparing two SERMs,raloxifene and tamoxifen,that display distinct tissue specificity profiles.In mammary cells,both these SERMS are effective in recruitment of corepressor to target promoters (hence both are antagonists),but in uterine cells SRC1is at a much higher level of expression than in mammary cells and tamoxifen is much more sensitive to SRC1than raloxifene in terms of “transformation ”into an agonist.This results in tamoxifen effectively recruiting SRC1to various ER target genes in uterine cells (agonist)but not mam-mary cells,whereas raloxifene is not effective in recruitment of SRC1in either cell type (antagonist).In cases such as ER,where receptor subtypes (ER a and ER b )mediate the action of the modulators,distinct pharmacologic action at the various subtypes may also play a role in tissue selective action.Receptor subtypes are often expressed differentially;as a single modulator may function differently on the various subtypes,dis-tinct tissue specificity profiles may emerge (Barkhem et al.,1998).
There are examples of differential,target-gene spe-cific actions of a single modulator within a single cell type (Bramlett and Burris,2003).The distinct action of the selective modulator cannot be attributed to dif-ferential expression of cofactor proteins or receptor subtypes in this case.One potential mechanism that may explain these actions is the target gene itself may convey specific information to the LBD so that it may respond differentially to various selective modulator ligands.For example,ER has been demonstrated to alter its affinity for various coactivators depending on the DNA-response element to which it is bound
(Hall
Fig.4.Agonist and antagonist LBD conformations observed in ER crystal structures.Crystal structures of the ER LBD have suggested structural features contributing to ligand-induced agonism and antagonism.(A)The natural agonist,17b -estradiol,docks in the LBP and positions helix 12into a conformation referred to as the agonist or active conformation.This conformation forms the coactivator-binding surface as described in Fig.2.(B)In contrast,when raloxifene is bound in the LBP,the position of helix 12is rotated with respect to the agonist conformation such that it binds in the AF-2coactivator-binding surface and thus blocks binding of coactivators via the LXXLL motif.This is referred to as the antagonist,repressive,or inactive conformation.(C)Other ligands such as ICI 182,780can physically block the AF-2coactivator-binding surface and do not stabilize helix 12;thus,these ligands are termed pure antagonists.In these panels,helix 12is blue,and ligands are black.
Nuclear Receptors and Their Selective Modulators
717
et al.,2002).In addition,the specific DNA-response element to which GR is bound has been shown to play an important role in its selection of coactivators (Meijsing et al.,2009).Furthermore,the relatively re-cent X-ray structure of an intact NR heterodimer (PPAR g /RXR a )complexed with DNA (Fig.5)demon-strated that the DBD of RXR a made critical contacts with the LBD of PPAR g that were required for function of the complex,suggesting that critical communication between DNA,DBD,and LBD occurs.HDXMS data with intact vitamin D receptor (VDR)/RXR a on DNA (Fig.6)demonstrated that binding of the dimer to DNA resulted in major conformational alterations in H12,clearly demonstrating the potential for DNA to relay critical information to regions of the LBD that are important for coactivator recognition (Zhang et al.,2011).
The molecular events underlying selective NR mod-ulator pharmacology are myriad,and this discussion has only touched on a few highlights of various mech-anisms that have been implicated in this unique profile of ligands that target the NRs.Posttranslational mod-ifications of receptors and regulation of the modifi-cations has been shown to lead to target-gene specific regulation;additionally,the nongenomic actions (re-ferring to actions of NRs independent of their classic
action as transcription factors,i.e.,genomic action)of various receptors are likely to also play a role in the action of at least some of the selective NR modulators that have been identified to date.This review will summarize some of the key breakthroughs that have been made in the development of selective modulators for a range of NRs and will discuss how many of them have been effective in targeting human diseases.II.Selective Estrogen Receptor Modulators A.Estrogen Receptor Structure
The estrogen receptors (ERs)have a deep history in the realm of selective modulators,whose ligands are referred to as selective estrogen receptor modulators (SERMs).ERs were discovered in the laboratory of Elwood Jensen in the 1950s and 1960s when experi-ments suggested that estrogenic effects observed in the uterus were mediated by a specific receptor.Since this discovery,the field of ER pharmacology has exploded,resulting in large numbers of synthetic ligands to explore the biology of ERs.In 1996,a second ER isoform,ER b (ESR2;NR3A2),was discovered in the laboratory of Jan-Ake Gustafsson;the ER discovered earlier is now known as ER a (ESR1;NR3A1)(Kuiper et al.,
1996).
Fig.5.Intact structure of PPAR g /RXR a complex on DNA.At the time of this review,only one crystal structure has been reported of an intact nuclear receptor complex:PPAR g /RXR a bound to DNA,ligands,and coactivator peptides.In this structure,the receptors form an asymmetric complex where the LBD of PPAR g contacts not only the LBD of RXR a through the canonical helix 11heterodimerization surface but also contacts the RXR a DBD,suggesting a feature that could allow molecular communication between the LBD (the site of ligand binding)and DBD (the site of DNA binding).The N-terminal AF-1region of the receptors was not captured in this complex due to its highly dynamic properties.In this figure,PPAR g is orange,RXR a is blue,ligands are green,and DNA is
white.
Fig. 6.HDX study of intact VDR/RXR a complex on DNA.HDX is a powerful technique to monitor the effect of ligand and DNA binding on the dynamics of intact receptor complexes.HDX studies on the in-tact VDR/RXR a complex were the first to show that binding of the heterodimer to DNA can allosterically permeate changes in receptor dynamics,not only in the DBD but extending to the AF-2regions in the LBD of the receptors.In this figure,regions colored blue show protection from HDX upon binding DNA,whereas regions colored yellow show increased HDX upon binding DNA.Ligand binding to the LBD was also observed to affect the dynamics of the DBD (not shown).
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ERs display the canonical domain organization found
in other members of the nuclear receptor superfamily.
However,the ERs have longer N-terminal(A/B domain;
contains the AF-1region)and C-terminal(F domain)
domains.The F domain in particular appears to have an
effect on the function of ER ligands,though the exact
molecular mechanisms are not well understood(Kojetin
et al.,2008;Skafar and Zhao,2008).In addition,ER
splice variants have been shown to influence the phar-
macology of synthetic ER compounds,and site-specific
phosphorylation of ER has been shown to modulate
the gene-specific transcriptional response(Taylor et al.,
2010;Duplessis et al.,2011).
The natural ligand for the ERs is the steroid
hormone17b-estradiol,which binds to ERs with high
affinity(K d;0.2nM)and activates the receptors,re-sulting in increased transcription of genes containing
estrogen response element(ERE)promoter sequences
(59-GGTCANNNTGACCT-39)(Maurer and Notides,1987).
ERs can function as homodimers(ER a-ER a;ER b-ER b)
or heterodimers(ER a-ER b)(Cowley et al.,1997).The
ER LBD is probably the most studied NR domain in
terms of NR structure,and,as discussed earlier and il-
lustrated in Fig.4,these structural studies have served
as a model for understanding the molecular basis of NR
agonism and antagonism.
B.Estrogen Receptor Function
Both ERs are widely expressed,although ER a is more
abundant in breast,endometrial,ovarian,and hypo-
thalamus tissues,whereas ER b is more abundant in
brain,bone,endothelial,heart,intestine,kidney,lung,
and prostate tissues.Furthermore,ER a and ER b have
both overlapping and nonoverlapping functions,which
combine with the tissue-specific differences in the
relative abundance of the receptors.This suggests that
the specific targeting of ER isoforms could prove useful
in the therapeutic treatment of diseases with perhaps
limited cross-talk to other tissues or ER isoform-specific
genes—essentially the primary basis of selective ligand
modulation.The actions of ER b in many cases oppose
the actions of ER a,suggesting a role for a single ligand
to have different functional outcomes depending on the
specific ER target(Matthews and Gustafsson,2003).
ERs work in concert with a variety of coregulator
proteins,many of which are differentially expressed in
various tissues,providing what is considered the mole-
cular basis of SERM activity:ligand-dependent and
tissue-specific coregulator recruitment.
A variety of ER splice variants and mutations have
been identified,some of which from were obtained from
clinical samples from patients with cancer and other
diseases(Herynk and Fuqua,2004).The mutations
are primarily found in AF-1and LBD/AF-2.Many of
the natural ER mutations found in clinical samples
were in fact derived from tissues deemed to be ER
negative in terms of their therapeutic classification using a ligand-binding assay.This suggests that natural mutations in ERs can affect the ligand-binding proper-ties of ERs.Thus,while some cancers might be classified as ER negative,this could mean either that ER is not expressed or that a mutation has rendered ER incapable of binding ligand or has reduced its binding affinity and thus other modes of treatment are warranted.
Because of their broad tissue expression patterns and important roles in development and physiology, ERs are drug targets for a variety of diseases(Riggs and Hartmann,2003).Breast tissue development and physiology is substantially influenced by ERs,and treatment of ER-positive breast cancer is currently one of the primary clinical uses of SERMs.In these treatments,SERMs are given as an adjuvant therapy after a primary therapy such as a mastectomy or lumpectomy and radiation therapy.ER-based adjuvant cancer therapies focus on blocking estrogen action in these tissues.As will be discussed more later,because many breast cancers occur in postmenopausal women, who may also suffer from diseases such as osteoporosis and hot flashes,SERMs that display antagonist block-ing action in the breast and agonist action in the bone serve in a sense a dual role in these patients.
As discussed previously,estrogens also have a strong influence in bone,which expresses both ER isoforms (Bord et al.,2001;Riggs et al.,2002).ER a is highly expressed in developing cortical bone,and ER b levels are higher in developing cancellous bone.One of the primary diseases affecting bone is osteoporosis,which is in part caused by estrogen deficiencies in the body. Cancellous bone(ER b rich)is less rigid compared with cortical bone(ER a rich)and is affected more in osteoporosis than cortical bone,supporting the selec-tive targeting of ER b for osteoporosis treatment.
In the cardiovascular system,estrogens and SERMs provide a generally favorable serum lipid profile. Estrogen increases high-density lipoprotein(HDL) and triglycerides and lowers low-density lipoprotein (LDL)levels.SERMs can display both similar and ligand-specific effects on lipids.SERMs generally de-crease LDL and triglyceride levels,but some increase HDL(toremifene)while others increase HDL levels (Saarto et al.,1996).Estrogen and SERMs thus gen-erally provide a benefit in cardiovascular disease in postmenopausal women,among whom it is estimated that about one-third die from coronary artery disease (Wenger,1997).
In the central nervous system(CNS),where ER b expression levels are generally high,ER b function and targeting has been linked to several brain processes, including cognition and memory(McEwen and Alves, 1999).In addition,further,studies have implicated ER agonism in affording relief from hot flashes associated with the postmenopause period in women(Marttunen et al.,1998;Johnston et al.,2000).
Nuclear Receptors and Their Selective Modulators719
C.Selective Estrogen Receptor Modulators
SERMs have been useful for clinical treatment of a variety of disorders,including breast cancer,osteoporo-sis,and menopausal health symptoms via hormone re-placement therapy.However,the need for better SERM therapeutics is illustrated by the complex pharmacology of tamoxifen(Fig.7),a commonly prescribed SERM for breast cancer treatment.Tamoxifen displays mixed phar-macology in different tissues,as it acts as an antagonist in breast tissue but as an agonist in bone and the uterus. Thus,patients who take tamoxifen as a breast cancer therapy receive benefits in the breast where it acts as an ER antagonist and in the bone where it acts as an ER agonist(increased bone mineral density);however,pa-tients also experience negative side effects,such as in the uterus where tamoxifen acts as an agonist that increases cell proliferation.
The notion that the same drug can have such op-posing actions in different tissues,not all of which are favorable for the patient,supports the need for SERMs with better tissue-selective pharmacologic properties. An ideal SERM may be one that antagonizes in breast and endometrial tissues,but agonizes in bone and CNS tissues(O’Regan and Jordan,2001).On a molecular level, an ideal SERM may be one that recruits a particular coregulator protein over another,perhaps in a gene/ promoter-specific manner.
Tamoxifen,perhaps the most well known SERM,is used in the treatment of ER-positive breast cancer. Tamoxifen was discovered by ICI Pharmaceuticals (now AstraZeneca,London,UK)under the internal name ICI46474(Harper and Walpole,1967a,b;Jordan, 2006).It is a chemical derivative of the first non-steroidal antiestrogen MER25,which was discovered in the1950s and identified as an inhibitor of estrogen actions(Lerner et al.,1958).Both these compounds are chemical derivatives of the highly potent synthetic nonsteroidal estrogen diethylstilbestrol(Furr and Jordan,1984).Early studies revealed that tamoxifen could compete with estrogens,in terms of preventing
Fig.7.ER modulators. 720Burris et al.
the accumulation of[3H]estradiol in ER target tissues (e.g.,uterine,vaginal,and mammary)of mice,rats, and humans(Emmens,1971;Lunan and Green,1974; Jordan,1975;Jordan and Dowse,1976).These studies also provided insight into the pharmacokinetic/ pharmacodynamic properties of tamoxifen,including its conversion into the metabolite4-hydroxytamoxifen (Fig.7),which is the primary form that binds to ERs in vivo(Rochefort et al.,1979;Rochefort and Borgna, 1981).Tamoxifen shows maximal levels in tissues approximately6-hours after dosing,with elevated plasma levels detected for the entire duration of the experiment(120hours)(Major et al.,1976).Additional biochemical studies in the form of[3H]estradiol com-petition assays validated that tamoxifen binds directly to ER and competes https://www.doczj.com/doc/d85015178.html,ter,struc-tural validation via X-ray crystallography confirmed this interaction and provided molecular details concern-ing the interactions between ER a with17b-estradiol and tamoxifen(Brzozowski et al.,1997;Shiau et al., 1998).Namely,helix12in the LBD acts as a molec-ular switch,adopting an“active”conformation in the presence of an agonist such as diethylstilbestrol or17b-estradiol,allowing the interaction of coregulator pro-teins with the AF-2coregulator-binding surface.In the presence of4-hydroxytamoxifen,helix12rotates and occupies the AF-2coregulator-binding surface,resulting in transcriptional antagonism.
As already described,tamoxifen displays a mixed agonist/antagonist pharmacologic profile in different tissues.Generally,this is a good property for SERMs, in the sense that it is possible to develop compounds with differential actions that could potentially benefit one tissue while not affecting others.However,for tamoxifen these differential effects provide significant side effects.Notable evidence suggests that the source of this mixed pharmacology depends on the tissue-selective expression of coregulator proteins,which interact with ERs at the promotor and enhance or repress transcription of ER-dependent genes.A pri-mary example is the tamoxifen-dependent recruitment steroid receptor coactivator1(SRC1/NCOA1)(Shang and Brown,2002).SRC1is expressed at higher levels in the uterus and lower levels in breast tissue.Tamoxifen induces SRC1recruitment to ER a-dependent gene pro-moters in uterine tissue,which is not the case for other SERMs including raloxifene.However,like many other SERMs,tamoxifen induces corepressor recruitment to ER gene promotors in breast tissue.
Raloxifene(Fig.7)was discovered and is marketed by Eli Lilly and Company(Indianapolis,IN)as a SERM used in the prevention of osteoporosis in postmenopausal women.Raloxifene also shows efficacy in reducing the risk of invasive breast cancer without affecting the risk of primary coronary events(Barrett-Connor et al.,2006). Similar to tamoxifen,when raloxifene binds to the ER LBD,it induces a conformation that repositions helix12to the AF-2surface and blocks coactivator binding (Brzozowski et al.,1997).Additionally,raloxifene pre-serves or increases bone density and inhibits the growth of breast cancer(Clemens et al.,1983;Gottardis and Jordan,1987;Jordan et al.,1987;Black et al.,1994; Sato et al.,1994,1995;Turner et al.,1994;Anzano et al.,1996).Raloxifene does not agonize endometrial growth to as large an extent as tamoxifen(Gottardis et al.,1990).The reduced level of agonism in uterine tissue is likely a consequence of its inability to stimulate SRC1recruitment to ER gene promotors in endometrial cells as is the case for tamoxifen(Shang and Brown, 2002).However,both tamoxifen and raloxifene induce recruitment of corepressors to ER gene promotors in breast cancer cells(Shang and Brown,2002). Lasofoxifene(Fig.7),discovered in a collaboration between Pfizer(New York,NY)and Ligand Pharma-ceuticals(La Jolla,CA),is currently under develop-ment for the prevention and treatment of osteoporosis and the treatment of vaginal https://www.doczj.com/doc/d85015178.html,sofoxifene is a potent SERM that does not agonize uterine cell growth but decreases total cholesterol,fat body mass, and bone loss in female rats,and displays similar properties in male rats(Ke et al.,1998,2000;Rosati et al.,1998).In addition,lasofoxifene increases vaginal mucus formation without inducing cell proliferation. This suggests that it would be useful in the treatment of vaginal and vulvar atrophy in postmenopausal women(Wang et al.,2006).It is thought that these effects are likely due to the ability of lasofoxifene to increase the expression levels of ER b and AR in vaginal tissues,which other SERMs do not(Wang et al.,2006).Clinical data support the use of lasofox-ifene in the prevention and treatment of osteoporosis and treatment of vaginal atrophy in postmenopausal women without an increased risk of endometrial cancer but with an increased risk of venous thromboembolic events(Bachmann et al.,2005;McClung et al.,2006b; Taylor,2009;Cummings et al.,2010;Gennari et al., 2010).From a structural perspective,much like ta-moxifen and raloxifene,lasofoxifene displaces helix12 in the LBD from the agonist position to block the AF-2 coregulator-binding surface(Vajdos et al.,2007). Toremifene(Fig.7)is a tamoxifen analog licensed under the brand name Fareston(marketed by GTx)as a treatment approved by the U.S.Food and Drug Administration(FDA)for advanced metastatic breast cancer.It is also currently under development for prevention of prostate cancer.Clinical studies have shown that toremifene is equally as efficacious as tamoxifen in the treatment of metastatic breast cancer and may have fewer negative side effects(Holli,2002; Lewis et al.,2010).Biochemical studies have shown that toremifene downregulates the expression of breast cancer resistance protein(BCRP),which is a multidrug resistance transporter for a variety of antitumor agents (Zhang et al.,2010).
Nuclear Receptors and Their Selective Modulators721
Fulvestrant(ICI182,780)(Fig.7)is a selective estrogen receptor downregulator(SERD)initially dis-covered by ICI Pharmaceuticals and marketed under the brand name Faslodex by AstraZeneca.Recently, fulvestrant has been used for the treatment of metasta-tic breast cancer in postmenopausal women(Wakeling et al.,1991).Fulvestrant is considered a pure ER antagonist,as it has no ER agonist effects.Interest-ingly,fulvestrant has a greater binding affinity than estradiol for ER,and is more effective than tamoxifen at inhibiting MCF-7breast cancer cell proliferation and tumor progression in patients.Fulvestrant also has a much longer half-life when compared with ta-moxifen(Wakeling et al.,1991;Bundred et al.,2002; Robertson et al.,2003;Robertson and Harrison,2004). Fulvestrant has been shown to be effective in tamox-ifen sensitive and insensitive cell lines(Coopman et al., 1994;Osborne et al.,1994).The mechanism of action for fulvestrant involves the direct blocking of the ER AF-2coregulator-binding surface,impairing receptor dimerization and increasing receptor degradation and turnover,perhaps via fulvestrant-dependent inter-action with cytokeratins8and18(CK8,CK18)and colocalization to proteasomes(Robertson,2001;Long and Nephew,2006;Long et al.,2010).It has been suggested that resistance to fulvestrant is mediated by the tyrosine kinase c-ABL(Zhao et al.,2011a).Clinical trials revealed that fulvestrant is well tolerated in breast cancer patients without the negative side effects associated with most ER SERMS such as partial agonist activity in the uterus(Addo et al.,2002; Vergote and Robertson,2004).
Clomifene(clomiphene)(Fig.7)is marketed under the brand names Clomid and Serophene.Since its introduction in the1960s,clomifene has been used in the management of infertility via induction of ovula-tion(Goldstein et al.,2000).Clomifene is a mixture E and double-bond Z isomers,one of which is an ER agonist/antagonist and the other a strict antagonist (Glasier,1990).In terms of isoform specificity,clomi-fene has been shown to agonize and antagonize ER a but only antagonize ER b(Kurosawa et al.,2010).The mechanism of action ascribed to clomifene in infertility treatment involves antagonism in the hypothalamus, which increases levels of gonadotropin-releasing hor-mone(GnRH)and subsequently increases follicle-stimulating hormone(FSH)and luteinizing hormone (LH)secretion(Dickey and Holtkamp,1996;Tarlatzis and Grimbizis,1998).It has also been suggested that clomifene may inhibit the estradiol-dependent pro-liferation of endometrial epithelial cells by inhibiting the recruitment of SRC1to ER a and thus estradiol-dependent ER transactivation(Amita et al.,2010).
A summary of several clinical trials revealed that clomifene is as effective as tamoxifen in inducing ovulation for infertility management,although re-sults from another study suggest that the actions on gonadotropin-releasing hormone levels may be non-genomic in nature(Garas et al.,2004;Steiner et al., 2005).
Ormeloxifene(centchroman)(Fig.7)is a SERM that has been licensed under the brand names Saheli, Novex-DS,Centron,and Sevista by Torrent Pharma-ceuticals(Ahmedabad,India)and later HLL Lifecare (Thiruvananthapuram,Kerala,India)for birth control and dysfunctional uterine bleeding.Ormeloxifene dis-plays higher affinity and selectivity for ER a(K i=250 nM)versus ER b(K i=750nM)(Blesson et al.,2006). Early studies reported on the anti-inflammatory properties of ormeloxifene in acute and chronic models of inflammation,and both estrogenic and antiestro-genic effects in the uterus at low and high doses, respectively(Dhawan and Srimal,1973;Kamboj et al., 1973).Ormeloxifene shows estrogenic activity in the uterus and fallopian tubes,which was suggested to contribute to its antifertility efficacy(Imam et al., 1975).Structure-activity relationship analysis revealed regions of ormeloxifene important for ER binding and function(Salman et al.,1983).Pharmacokinetic data have revealed the half-life of a single dose of ormelox-ifene(approximately170hours)as well as the tissue distribution of ormeloxifene and its metabolite7-desmethyl ormeloxifene(Paliwal et al.,1989;Lal et al.,1995;Paliwal and Gupta,1996).Ormeloxifene induces caspase-dependent apoptosis in both ER-positive (MCF-7)and ER-negative(MDA-MB-231)breast cancer cell lines(Nigam et al.,2008).However,the ER de-pendency of these cell lines is in reference to ER a.Two ER b variants are expressed in these cells,suggesting an ER b-mediated effect.Ormeloxifene appears to function by inhibiting the interaction of the coactivator SRC1with ER a while enhancing the interaction with the coactivator RIP140and corepressor NCoR as well as interaction of NCoR with ER b in the rat uterus (Daverey et al.,2009).Antimutagenic effects from treatment with ormeloxifene have also been described, as it reduces sister chromatid exchange and chromo-some aberrations in female Swiss albino mice exposed to genotoxic compounds(Giri et al.,1999).A phase2 trial revealed that ormeloxifene has efficacy in the treatment of advanced breast cancer(Misra et al., 1989).
Femarelle(DT56a)is a SERM used for the treatment of menopause and bone health(Somjen et al.,2007). Femarelle is an extract of tofu and flaxseed oil con-taining phytoestrogens,and is thus sometimes referred to as the“Tofu pill”or a“natural”SERM.The exact identity of the pharmacologically active phytoestrogens in the extract are unclear,but femarelle competes with17b-estradiol and stimulates or agonizes creatine kinase activity,which is an estrogenic marker,in rat skeletal tissue in a manner similar to17b-estradiol (Malnick et al.,1983;Somjen and Yoles,2003; Somjen et al.,2007).However,unlike17b-estradiol,
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femarelle does not agonize estrogenic activity in the uterus or other reproductive tissues(Somjen and Yoles, 2003;Oropeza et al.,2005).In addition,femarelle does not effect cell proliferation in the human MCF-7breast cancer cell line(Yoles and Lilling,2007).Femarelle shows efficacy in preserving bone mineral density (BMD)in both female rats and clinically in post-menopausal women without risk of thrombogenicity (Yoles et al.,2003,2004;Somjen et al.,2005,2006; Nachtigall et al.,2011).Femarelle and other ER agonists have been shown to abolish fat cell content in rat bone marrow.It has also been suggested that femarelle can relieve menopausal vasomotor symptoms not by affecting hormone levels or the endometrium directly,but rather in brain responsiveness via estrogenic action on the brain involved in regulating mood,cognition,and homeostasis(Pluchino et al., 2009).
Bazedoxifene(WAY-140424)(Fig.7),discovered as a result of collaboration between Wyeth(Madison,NJ) and Ligand Pharmaceuticals,has been approved by the FDA under the brand name Conbriza for the pre-vention and treatment of osteoporosis in postmeno-pausal women.Bazedoxifene was discovered using a stringent preclinical selection process,as it was understood that the discovery of a new ligand would be compared with the already established SERM ralox-ifene(Komm and Lyttle,2001;Anonymous2008). Bazedoxifene displays general properties of a dual ER a/ER b SERM,with K i values of0.1and0.3nM, respectively,with no cross reactivity with other nu-clear receptors.In its early discovery,it displayed the prototypical SERM gene-selective activation pheno-type,where it antagonized expression on a2?-ERE (estrogen response element)promoter,but agonized expression driven by a hepatic lipase promoter in the same cell line(Komm and Lyttle,2001).Bazedoxifene antagonizes estrogen-stimulated proliferation of MCF-7 breast cancer cells with little to no effects in uterine and CNS tissue,and also maintains bone density, reduces cholesterol in rats,and causes regression of endometriosis in mice(Komm and Lyttle,2001; Komm et al.,2005;Ronkin et al.,2005;Kulak et al., 2011).Bazedoxifene inhibits the proliferation of estrogen-dependent(MCF-7and T47D)and estrogen-independent(MCF-7:5C and MCF-7:2A)cell lines (Lewis-Wambi et al.,2011).Its ability to antagonize growth of MCF-7:5C cells in particular is unique among SERMs and occurs as a result of downregula-tion of ER a(via protein degradation)and suppress-ing cyclin D1expression.Bazedoxifene has also shown some efficacy in inflammation,where it reduces lipopolysaccharide(LPS)-induced expression of interleukin-6(IL-6)and interferon-g-inducible protein-10(IP-10)through ER-dependent inhibition of nuclear factor k B(NF-k B)p65transactivation(Cerciat et al., 2010).
During early phase1and2clinical trials,it was shown that bazedoxifene did not increase hot flashes, which is a common side effect of SERMs.Bazedoxifene also preserved bone density and lowered cholesterol levels(Komm and Lyttle,2001).It appears that this SERM does not stimulate but rather antagonizes endometrial growth in postmenopausal women(Ron-kin et al.,2005).Other clinical trials revealed that bazedoxifene treatment prevents bone loss and reduces bone turnover with similar efficacy to raloxifene in postmenopausal women who have normal-to-low BMD without increasing mammographic breast density,the incidence of hot flashes(Miller et al.,2008;Silverman et al.,2008,2012;Archer et al.,2009;Harvey et al., 2009;Kanis et al.,2009;Pinkerton et al.,2009a; Christiansen et al.,2010;Bachmann et al.,2011;de Villiers et al.,2011;Xu et al.,2011).Bazedoxifene also displays efficacy for treating vasomotor symp-toms and preventing endometrial hyperplasia in post-menopausal women(Pickar et al.,2009;Pinkerton et al.,2009b).
Preclinical data have suggested that the combination treatment of bazedoxifene and conjugated estrogens (CE)might lead to a more favorable benefit-risk profile in the treatment of menopause symptoms, including favorable vasomotor,lipid,and skeletal response with minimal stimulation in the uterus (Kharode et al.,2008;Peano et al.,2009).This com-bination therapy also shows efficacy in the treat-ment of osteoporosis in a manner that prevents uterine growth,with decreased uterine wet weight and lower cholesterol levels(Komm et al.,2011).In particular,gene expression analysis via microarray experiments have revealed that a subset of CE-inducible genes were antagonized by bazedoxifene alone or in combination with CE(Chang et al., 2010b).The combination of bazedoxifene and CE [BZA-CE;or tissue-selective estrogen complex(TSEC)] displays efficacy and was determined to be safe in treating menopausal symptoms,decreasing bone turnover and bone loss in postmenopausal women at risk for osteoporosis,and treating vulvar/vaginal atrophy(Lindsay et al.,2009;Lobo et al.,2009;Utian et al.,2009;Bachmann et al.,2010;Kagan et al., 2010).
Arzoxifene(LY353381)(Fig.7),developed by Eli Lilly and Company,is structurally related to ralox-ifene;however,early studies revealed improved in vivo potency and efficacy(Palkowitz et al.,1997;Sato et al., 1998a).In addition,whereas raloxifene shows more specificity for ER a versus ER b(21versus560nM), arzoxifene displays properties of a dual ER a/ER b SERM(22versus66nM)(Overk et al.,2007). Arzoxifene binds directly to the ER and prevents increased body weight and cholesterol levels in ovariectomized rats with similar efficacy as estrogen and raloxifene but with more potency than raloxifene
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in reducing these parameters(Sato et al.,1998a;Suh et al.,2001).In addition,arzoxifene prevented bone loss in ovariectomized rats with an efficacy similar to parathyroid hormone(PTH),including during long-term dosing(Sato et al.,1998b;Ma et al.,2002).It has been reported that arzoxifene displays negligible effects in uterine cells and increased efficacy in antag-onizing uterine hypertrophy stimulated by estrogen when compared with tamoxifen(Sato et al.,1998a; Suh et al.,2001).Other studies have revealed that arzoxifene shows some efficacy in activating insulin-like growth factor I in uterine signaling.This causes an increase in proliferating cell nuclear antigen expres-sion and the number of mitotic cells in the uterus,as well as simulation of EnCa101endometrial tumors that were previously stimulated with estrogen or ta-moxifen(Klotz et al.,2000;Dardes et al.,2001). Arzoxifene was also shown to act as a chemoprotective agent in a rat model for mammary carcinogenesis(Suh et al.,2001).It was shown that arzoxifene was30–100 times more potent than raloxifene in regulating these in vivo parameters.Arzoxifene also displayed several advantages for use as a SERM over raloxifene,in-cluding preventing increased body weight,cholesterol, and bone loss,while also acting as an antagonist in the uterus(Sato et al.,1998a).Arzoxifene antagonizes estrogen-stimulated growth in ER-positive MCF-7 human breast cancer cells in a manner similar to ta-moxifen,including the propensity for cross-resistance, although resistance to either modulator is independent of the other(Schafer et al.,2001;Detre et al.,2003; Freddie et al.,2004b).This suggests that if a patient becomes resistant to one SERM,another may be beneficial for future treatments(Freddie et al.,2004a). Resistance to arzoxifene has been linked to over-expression of cyclin D1,which occurs in approximately 40%of all breast cancer patients,in a manner that converts arzoxifene from an antagonist to an agonist as a result of a ligand-dependent increase in the stabili-zation of SRC1complexed with ER a(Zwart et al., 2009).Arzoxifene has also been shown to synergize with the retinoid LG100268(6-[1-(3,5,5,8,8-pentamethyl-6,7-dihydronaphthalen-2-yl)cyclopropyl]pyridine-3-carboxylic acid),which binds to RXRs,in prevention and treat-ment rat models for breast cancer at concentrations with treatment with one ligand alone displayed negligible effects(Suh et al.,2002;Liby et al.,2006). The mechanism by which the combined treatment of arzoxifene and LG100268influences apoptosis in breast cancer involves the induction of transforming growth factor b(TGF b)by arzoxifene and inhibition of NF-k B and phosphatidylinositol39kinase signaling by LG100268(Rendi et al.,2004).One study suggests that arzoxifene is an agonist of ER b within the serotonin neuron,increasing the expression of tryptophyan hy-droxylase and the serotonin reuptake transporter(SERT) (Bethea et al.,2002).
A phase1clinical trial of arzoxifene determined that daily oral dosing was safe and well tolerated;and combined with all previous studies,this suggests that arzoxifene may be useful in the treatment of meta-static breast cancer(Munster et al.,2001;Fabian et al.,2004).A phase2clinical trial determined that arzoxifene is effective in treating tamoxifen-sensitive and tamoxifen-refractory patients with advanced or metastatic breast cancer(Chan,2002;Baselga et al., 2003;Buzdar et al.,2003).Other phase2trials have revealed that arzoxifene is effective in the treatment of recurrent or advanced endometrial cancer,as well as suppressing bone turnover in the treatment of osteo-porosis(Burke and Walker,2003;McMeekin et al., 2003).However,phase3clinical trials revealed that arzoxifene did not significantly differ in terms of clinical output compared with the standard mode of treatment with tamoxifen for advanced and metas-tatic breast cancer(Deshmane et al.,2007).In fact, the results revealed that tamoxifen produced longer survival and time-to-treatment failure rates.Despite this,a clinical study revealed that arzoxifene might still be useful for treatment of bone loss in post-menopausal women at doses that do not have a signif-icant effect in the uterus and endometrium(Jackson et al.,2008).Another phase3trial revealed that although arzoxifene displayed significantly greater effects on BMD and turnover compared with ralox-ifene,it did not translate into greater nonvertebral fracture efficacy or a more favorable adverse event profile(Kendler et al.,2012).
Afimoxifene(Fig.7)is the4-hydroxy derivative of tamoxifen,the active metabolite of tamoxifen.Afimox-ifene is typically formulated as a gel containing4-hydroxytamoxifen,and shows clinical efficacy in the treatment of cyclical mastalgia(breast pain/discomfort) in premenopausal women(Mansel et al.,2007).
SERM drug discovery has been categorized under three broad categories.Tamoxifen is generally consid-ered to be a first-generation SERM as it was the one of the first discovered and is perhaps the most used clinically.Because tamoxifen has less than optimal tissue specificity,such as agonist activity in the uterus, discovery efforts concentrated on second-generation SERMs.Raloxifene is considered a second-generation SERM,and it was selected for its selective tissue specificity,such as its antagonist activity in the uterus, as compared with tamoxifen.The third-generation of SERMs include the compounds discovered after raloxifene that display further improvements in tissue specificity.An example is LSN2120310,(R)-(+)-7,9-difluoro-5-[4-(2-piperidin-1-ylethoxy)phenyl]-5H-6-oxachrysen-2-ol,which is based on the raloxifene chemical scaffold;it displays agonist activity in the bone and antagonist activity in the breast and uterus, but unlike tamoxifen and raloxifene it may actually pre-vent hot flashes(Wallace et al.,2006).Going forward,
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the next generation of SERMs will build upon our un-derstanding of ligand-specific regulation of individual genes within a single tissue,not only displaying tissue-specific agonist or antagonist activity but also differen-tial upregulation or downregulation of specific genes within a single cell type(Bramlett and Burris,2003).
III.Vitamin D Receptor Modulators
A.Vitamin D Receptor Structure
Similar to other NRs,the427-amino acid vitamin D receptor(VDR)protein can be functionally divided into three regions with well-characterized functions.The short amino terminus,also referred to as the A/B domain,contains the ligand-independent transactiva-tion function AF-1.However,unlike many other NRs, the A/B domain is small,consisting of only20amino acids.The AF-1region with the A/B domain is not well developed in VDR,and it remains to be determined whether this region plays a significant role in VDR-mediated transactivation(Sone et al.,1991).The central region of VDR contains the DBD consisting of two zinc-finger motifs,which target the receptor to vitamin D3response elements(VDREs),followed by a flexible“hinge”region(D domain).This section contains the nuclear localization sequence,which allows the entry of the RXR/VDR heterodimer into the nucleus.The carboxy terminus of VDR contains a multifunctional domain harboring the LBD,the RXR heterodimerization motif,and a ligand-dependent transactivation function(AF-2).When ligand binds to VDR,a conformational change ensues,resulting in the enhancement of RXR/VDR heterodimer formation (Cheskis and Freedman,1994;Prufer et al.,2000; Pinette et al.,2003;Sutton and MacDonald,2003). There is only one VDR isoform encoded by a single gene in both humans and other organisms.
The amino acid residues165–215in the VDR LBD is an“insertion”domain that is poorly conserved between different species and does not appear to have any biologic significance.Additionally,this region has made resolving the crystal structure extremely diffi-cult.In2000,the Moras laboratory resolved the crystal structure of a mutant VDR LBD,lacking residues 165–215,bound to1,25-(OH)2D3,thus proving that VDR bound this ligand and was capable of trans-activation(Rochel et al.,2000).The crystal structure VDR’s LBD resembles that of other NRs,displaying a three-stranded b-and12-a helices arranged to form three layers that completely encompass the ligand, resting in a hydrophobic core.The C-terminal helix12 contributes to transactivation by forming the bottom portion of a surface that has a high affinity for coactivator molecules(Renaud and Moras,2000;Xu and O’Malley,2002).Several laboratories published follow-up studies describing the VDR LBD bound to four superagonist analogs of1,25-(OH)2D3or other known agonists,thereby confirming the original report (Tocchini-Valentini et al.,2001,2004;Shaffer and Gewirth,2002;Ciesielski et al.,2004;Vanhooke et al., 2004).All the human VDR X-ray crystal structures subsequently cited are of the mutant VDR LBD protein,as residues165–215form an“undefined”loop in the hinge region of domain D.This region has been removed to facilitate crystal growth,and studies have found that deletion of this region does not influence VDR LBD structure or genomic function(Rochel et al., 2001).
The crystal structure of the receptor-ligand complex has revealed many aspects of VDR biology.The b-sheet residues contact the ligand and Trp-286,which is specific to VDR,plays an essential role in positioning of the ligand(Rochel et al.,2000).The ligand-binding pocket is composed of mainly hydrophobic res-idues.The crystal structure has revealed that bound 1,25-(OH)2D3curves around helix H3,with its A ring interacting with the C terminus of helix H5,and the 25-hydroxyl end is close to helices H7and H11(Rochel et al.,2000).Furthermore,Rochel et al.(2000)de-termined that the positioning of helix H12is critical for coactivator binding and transcriptional activation of VDR,and the position of helix H12is stabilized by a number of hydrophobic contacts and polar interactions. Hydrogen/deuterium exchange(HDX)coupled with mass spectrometry is a rapid and sensitive approach for characterization of protein folding,protein-protein interactions,and protein-ligand interactions.This technique has provided complementary information to that gained by X-ray crystallography(Hamuro et al., 2006;Chandra et al.,2008;Hsu et al.,2009;Iacob et al.,2009).HDX was used to probe the conforma-tional dynamics of the LBD of VDR in complex with three ligands,one being1,25-(OH)2D3.While HDX anal-ysis did not provide direct information about the position of H12,it did indicate that ligand binding1,25-(OH)2D3 did stabilize this domain,thereby aiding the ability of the receptor to stimulate the classic AF-2-dependent VDR transactivation(Zhang et al.,2011).
B.Vitamin D Receptor Function
VDR has traditionally been associated with calcemic activities(modulation of calcium and phosphate ho-meostasis involved in the development and mainte-nance of bone)(Mizwicki and Norman,2009).VDR is a ligand-dependent transcription factor with the hormonally active form of vitamin D3,the secosteroid 1a-25-dihydroxyvitamin D3[1,25-(OH)2D3],or calci-triol as its natural ligand(Mizwicki and Norman, 2009).However,the scope of VDR biology has ex-panded due to the observations that VDR is present in cells other than those of the intestine,bone,kid-ney,and parathyroid gland leading to the conclusion that there an noncalcemic actions of VDR ligands that regulate a wide range of physiologic cellular
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responses including cell proliferation,differentiation, and immunomodulation.
Thirty-seven tissues are known to possess a VDR, the expression of which,coupled with the increased evidence involving VDR in processes other than min-eral homeostasis,has prompted the generation and testing of therapeutic VDR ligands in inflammation, dermatological conditions,osteoporosis,cancers,sec-ondary hyperparathyroidism,and autoimmune dis-eases(Corbett et al.,2006;Norman,2008).These efforts have led to the development of VDR ligands for the treatment of psoriasis,secondary hyperparathy-roidism,and osteoporosis(Nagpal et al.,2005).While the therapeutic potential for targeting VDR is im-mense,the limiting factor for the clinical application of VDR ligands has been the increased incidence of hypercalcemia/hypercalciuria(Cheskis and Freedman, 1994;Crofts et al.,1998;Prufer et al.,2000;Thompson et al.,2002;Dong et al.,2003).Therefore,there is an unmet clinical need for the identification of VDR ligands that exhibit an improved therapeutic index while limiting the untoward side effects.The goal of this section is to review the biologic actions of vitamin D and its analogs with future perspectives for the next generation of noncalcemic vitamin D3analogs.
In1919,Sir Edward Mellanby’s observation that rickets was caused by a nutritional deficiency led to the isolation of a fat-soluble antirachitic substance in fish oil and other foods that was further identified as vi-tamin D2(Mellanby,1919).Concurrently,Huldschinsky (1919)and Hess and Unger(1921)discovered that exposing children to ultraviolet(UV)light could cure them of rickets and that antirachitic activity could be induced in various foods by ultraviolet radiation.Sub-sequent studies led to the structural identification of vitamin D2(ergocalciferol)and vitamin D3(cholecal-ciferol)as secosterols,which were derived from the photolytic cleavage of the B rings of ergosterol and 7-dehydrocholesterol.These two sterols were consid-ered the biologically active forms of vitamin D until the mid-1960s when25-hydroxyvitamin D3[25-(OH)D3] was found to be the major circulating metabolite of vitamin D3,produced primarily in the liver.Sub-sequently,1,25-dihydroxyvitamin D3[1,25-(OH)2D3], a metabolite more polar than25-(OH)D3,was identi-fied and is now known to be the most active metabolite of vitamin D.
In1968,the discovery of a high-affinity receptor for 1,25-(OH)2D3in the intestine of vitamin D-deficient chicks further advanced vitamin D research(Haussler and Norman,1969).This50–70kDa protein,found to be associated with nuclear chromatin,displayed sat-urable binding of1,25-(OH)2D3and had specificity for other vitamin D metabolites.Cloning of VDR revealed considerable similarity to other members of the NR superfamily,as it possessed the characteristic two zinc-finger motifs in the DBD.This sequence homology suggested that VDR was also a ligand-activated tran-scription factor.Although VDR was initially detected in classic vitamin D target organs,including intestine, bone,kidney,and parathyroid glands,all involved in mineral homeostasis,VDR has since been demon-strated to be present in many other tissues and cell types as well.
Adequate availability of vitamin D3depends on the photochemical production of vitamin D3in the skin plus the dietary intake of vitamin D3.Few food sources naturally contain significant amounts of vitamin D2 and D3,but many foods are now fortified with the vitamin,so minimum daily requirements are easily met.Vitamin D3does not have significant biologic activity and must be metabolized to its active form 1,25-(OH)2D3,which occurs in a two-step process.The first step toward the activation of vitamin D3occurs primarily in the liver,where vitamin D3is hydroxyl-ated at carbon25by25-hydroxylase,yielding25-hydroxycholecalciferol[25(OH)D3],or calcidiol.The resulting25(OH)D3is the more stable metabolite and is transported by the vitamin D-binding protein(DBP) to the kidneys for the final step in vitamin D bio-activation,where25(OH)D3serves as a substrate for the25(OH)2D3-1a-hydroxylase enzyme.The conversion of25(OH)D3results in the steroid hormone1,25-(OH)2D3, or calcitriol,the active circulating metabolite,which is released into the general circulatory system(Henry and Norman,1974;Bikle et al.,1975;Goltzman et al., 2004;Chlon et al.,2008).DBP also delivers25(OH)D3 to at least10other organs and tissues that have low levels of the25(OH)2D3-1a-hydroxylase enzyme,which re-sults in the paracrine or local production of1,25-(OH)2D3 (Norman,2008).This restricted production produces limited quantities of1,25-(OH)2D3in the local envi-ronment to regulate a given biologic effect.The potency of1,25-(OH)2D3requires the circulating levels to be tightly regulated(Dreier et al.,2008;Nemere and Hintze,2008).Control of serum usually involves joint reciprocal changes in the rate of synthesis and deg-radation.Collectively,the VDR-containing tissues de-fine locations where1,25-(OH)2D3can initiate biologic processes via receptor-ligand complexes to produce bio-logic responses through genomic mechanisms.
The natural ligand for VDR is the conformationally flexible secosteroid1,25-(OH)2D3,which binds VDR with an affinity K D in the range of0.1nM to5nM (Wecksler and Norman,1980ab;Wecksler et al., 1980ab).The parent vitamin D3binds to VDR with an affinity of.100m M.VDR functions as a hetero-dimer with another NR,the RXR.RXR,a nuclear receptor for9-cis-retinoic acid,is an obligate partner of VDR in mediating1,25-(OH)2D3action(Yu et al.,1991; Kliewer et al.,1992;Pinette et al.,2003;Sutton and MacDonald,2003).In the absence of ligand,the majority of VDR is present in the cytoplasm(Barsony et al.,1990).Upon ligand binding,VDR undergoes
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a conformational change that promotes RXR-VDR heterodimerization and complex nuclear translocation (Cheskis and Freedman,1994;Prufer et al.,2000). Once in the nucleus,the RXR-VDR complex binds to VDREs present in the promoter regions of responsive genes.Canonical VDREs are a direct repeat of59-AGG/ TTCA-39motifs or a minor variation of this motif separated by three nucleotides and commonly referred to as direct repeat-3motifs.Upon binding to VDREs on VDR target genes,the ligand-bound heterodimer recruits or dissociates the coactivator/corepressor pro-teins that ultimately modulate the transcriptional activity of the complex.The molecular details sur-rounding much of RXR/VDR transactivation have been described,including the chromatin environment,inter-acting protein partners,and temporal kinetics,all of which are highly diverse,depending on the genomic target and cellular context in which the regulation is occurring(Meyer et al.,2007).
Ligand binding increases the RXR/VDR heterodimer interaction with coactivators,transcriptional proteins that mediate induction of gene transcription.Ligand binding induces a conformational change in the re-ceptor,creating a hydrophobic cleft that renders NRs receptive to coactivator binding through their NR boxes, distinct amino acid sequences(LXXLL motifs).Unable to bind DNA itself,coactivator proteins enhance transcriptional activity through a range of enzymatic activities and protein-protein interactions.Several co-activators have been identified that interact with VDR, including those of the steroid receptor coactivator family(SRC1,SRC2,and SRC3)as well as CBP[cAMP response element binding protein(CREB)binding protein]/p300,pCAF(p300/CBP-associated factor),and thyroid receptor interacting protein1/Sug1,to name a few(Lee et al.,1995;Hermanson et al.,2002).VDR also directly interacts with certain components of the basal transcriptional machinery,including TF-IIB,TF-IIA, and TAF(Lavigne et al.,1999;Mengus et al.,1997, 2000;Barry et al.,2003).
A multiprotein complex that functions as a transcrip-tional coactivator for VDR is the vitamin D interacting protein(DRIP)complex(Rachez et al.,1998).The RXR/ VDR heterodimer recruits the DRIP complex by ligand-mediated recruitment of DRIP205,a component of the complex.Ligand-dependent targeted recruitment of the VDR-DRIP and VDR-SRC complex occurs in a sequential manner.The VDR-SRC complex is recruited to the VDR responsive promoter to promote the destabilization of the nucleosomal core,allowing the VDR-DRIP complex to bind to the unwound DNA and interact with basal transcriptional machinery(Rachez et al.,2000).
In the absence of ligand,corepressor proteins bind VDR,which results in chromatin compaction and gene silencing.Three corepressors,NCoR-1,NCoR-2,and Hairless,have been found to interact with VDR (Hermanson et al.,2002).Motifs related to NR boxes are present in the corepressors called CoRNR boxes (I/LXXI/VI motifs).These motifs are shown to be essen-tial for the interaction of corepressors with unliganded NRs(Hu and Lazar,1999;Webb et al.,2000;Hu et al., 2001).The number of cofactors and corepressors identified to date gives insight into the complexity of VDR activity and have increased our understanding of tissue-and gene-selective transcription mediated by VDR and its natural and synthetic ligands.
By its very definition,a transcription factor mod-ulates gene expression,and VDR is no exception to that rule.However,in addition to its ability to positively regulate the expression of genes,VDR can also negatively regulate the expression of other genes and antagonize the action of other transcription factors. Genes that are positively regulated by VDR ligands include those that regulate extracellular bone matrix formation,bone remodeling,adhesion molecules,dif-ferentiation,antiproliferation,and metabolism.Inter-estingly,genes known to be downregulated in response to VDR ligands are involved in hyperproliferation and anti-inflammatory functions,and may indicate why VDR ligands have therapeutic effects in inflammatory diseases.VDR ligands have been demonstrated to inhibit the expression of cytokines and chemokines, including IL-12,IL-2,tumor necrosis factor a(TNF a), and interferon g(INF-g),to name a few(Nagpal et al., 2005).Genes involved in mineral homeostasis,in-cluding PTH and PTH-related peptide(PTHrP),are also downregulated by VDR ligands(Kremer et al., 1991;Falzon,1996).Negative regulation of PTH, PTHrP,and Rel B appears to occur through a negative DNA motif called a negative VDRE(nVDRE)(Demay et al.,1992;Nishishita et al.,1998;Dong et al.,2003). Finally,VDR can compete with nuclear factor of activated T cells-1(NFAT1)for binding to the NFAT1–activator protein(AP-1)enhancer motif where it then interacts with c-Jun.The displacement of AP-1leads to the inhibition of gene expression.Both RXR/VDR heterodimers and VDR monomers have been demon-strated to be involved in the inhibition(Alroy et al., 1995;Harant et al.,1997;Takeuchi et al.,2002). Vitamin D metabolism is strictly regulated to per-form its principle function:govern calcium and phos-phate homeostasis.This regulation occurs in the“classic”mineral-regulating organs:the intestine,bone,para-thyroid glands,and kidneys.Elegant genetic studies in VDR-deficient mice have provided insight into the physiologic function of VDR as well as confirmed its crucial role in the regulation of bone development. Although they are phenotypically normal at birth, VDR2/2mice develop hypocalcemia,hyperparathyroid-ism,osteomalacia(rickets),and alopecia after weaning (Li et al.,1997b;Yoshizawa et al.,1997;Kato et al., 1999;Van Cromphaut et al.,2001;Zeitz et al.,2003). These mice die between4and6months of age.However,
Nuclear Receptors and Their Selective Modulators727
all symptoms are abolished in these animals when they are fed a diet rich in calcium phosphate and lactose, with the exception of the hair abnormalities.These findings suggest that intestinal calcium absorption is critical for1,25-(OH)2D3action on bone and calcium homeostasis.
We will summarize the role of vitamin D and VDR activity in the classic mineral-regulating organs.The intestine absorbs dietary calcium and phosphate.This occurs along the length of the entire intestine,but calcium transport occurs primarily in the duodenum and phosphate absorption in the jejunum and ileum.The absorption of calcium is facilitated by the presence of bile salts,which increase the absorption efficiency to50%. The most crucial function of the active form of vitamin D in mineral homeostasis is to enhance the small in-testine’s efficiency in absorbing dietary calcium and phosphate.Both hypocalcemia and hypophosphatemia increase the production of1,25-(OH)2D3,thereby in-creasing the intestine’s efficiency in absorbing calcium and phosphate.Vitamin D deficiency causes calcium malabsorption and a negative calcium and phosphate balance.In addition,1,25-(OH)2D3directly stimulates intestinal calcium and phosphate transport by acting on duodenal enterocytes to induce the calcium transport protein1,which channels calcium from the intestinal lumen into the cell(Bronner,2003).
In skeletal development,once the epiphyses and metaphyses fuse,longitudinal growth ceases.From this point forward,bone mineralization and turnover occur to maintain skeletal strength and integrity. Vitamin D is essential for the development and main-tenance of a mineralized skeleton.Bone is one of the major target organs of vitamin D,and VDR ligands regulate bone formation and resorption. Vitamin D deficiency results in rickets in the young and osteomalacia in adults.Also,1,25-(OH)2D3stim-ulates the mobilization of calcium stores from bone by inducing the dissolution of bone mineral and matrix(Holick,1996).Therefore1,25-(OH)2D3can enhance bone formation and growth.The primary target cells of1,25-(OH)2D3are osteoblasts and os-teoblast precursors.The effects of1,25-(OH)2D3are mediated by VDR and lead to the expression of se-veral genes associated with osteoblast proliferation and differentiation.
VDR is expressed at high levels in primary osteoblasts and various osteoblast cell lines.Thus,1,25-(OH)2D3 is classically considered to be a stimulator of bone resorption because it induces osteoclastogenesis by en-hancing the expression of receptor activator of NF-k B ligand(RANKL)in bone marrow and stromal cells (Suda et al.,1999).Additionally,the human and mouse RANKL promoter contains a functional VDRE that demonstrates RXR-VDR heterodimer-mediated ligand-dependent activation(Kitazawa and Kitazawa,2002; Kitazawa et al.,2003).
Given the profound role of VDR in bone formation;it is no surprise that vitamin D insufficiency,particularly in the elderly,results in osteoporosis and is thought to be a major factor in fracture risk(Chapuy et al.,1997). However,there is a large body of evidence suggesting that VDR plays a role in regulating the neuromuscular system,and that a combination VDR action in bone and muscle increases the risk of falls and fractures in the elderly(Fraix,2012).Support for VDR in muscle comes from analyses of myoblast cell lines generated from VDR2/2mice.These studies revealed that VDR2/2 mice had fiber sizes20%smaller than wild-type con-trols.VDR2/2mice also exhibited increased expression of the myogenic transcription factors myf5,E2A,and myogenin(Endo et al.,2003).These findings support a direct role for VDR transcriptional regulation of skeletal muscle.Because calcium is a critical modula-tor of skeletal muscle function,it is reasonable to hypothesize that VDR would have a significant impact on muscle function and fracture risk(Hamilton,2010). The parathyroid glands act as a calcium sensor in the body and hence play a central role in calcium homeostasis.The parathyroid glands sense the circu-lating levels of calcium in the body and secrete PTH, a calciotropic hormone that stimulates the release of calcium from bone.PTH can stimulate1,25-(OH)2D3 synthesis in the kidneys,whereas calcium is the dominant regulator of PTH synthesis and secretion. Therefore,PTH and calcium regulate the synthesis and secretion of each other and act as an important feedback loop in regulating calcium homeostasis.
In the kidney,vitamin D regulates calcium and phosphate resorption and controls its own synthesis and degradation.Perhaps the most important effect of 1,25-(OH)2D3in the kidney is the suppression of1a-hydroxylase activity and the stimulation of24-hydrox-ylase activity.This homeostatic feedback loop ensures that the proper amount of1,25-(OH)2D3will be released by the kidney.Under conditions of hypocalcemia or hypophosphatemia,renal VDR expression is decreased to prevent feedback until mineral levels are normalized.
C.Vitamin D Receptor Modulators
Osteoporosis is a common metabolic disease charac-terized by decreased bone mass due to deterioration of bone tissue and loss of spatial architecture,thus resulting in increase bone fragility and risk of deve-loping fractures.Osteoporosis involves the loss of both organic and mineral contents of the bone.Various con-ditions that can lead to osteoporosis include estrogen or androgen deficiency,glucocorticoid excess,hyper-thyroidism,hyperparathyroidism,and/or severe in-activity.A number of antiresorptive agents that treat these various conditions are currently on the market and prevent further bone loss,but they do not rebuild bone once it has been lost.Currently,there is only one FDA-approved anabolic bone-building agent for the
728Burris et al.
treatment of osteoporosis,recombinant human para-thyroid hormone(1-34)[rhPTH(1-34)],also known as teriparatide.This agent rebuilds bone mass and restores bone architecture while reducing the risk of vertebral and nonvertebral bone fractures in osteopo-rotic patients(Neer et al.,2001).However,teriparatide use is limited because daily subcutaneous injections are required,which may prove difficult for the elderly. Additionally,its high cost,the warnings regarding osteosarcoma,and the restriction of therapy to a max-imum2years adds to this drug’s limitations.There-fore,the need for bioactive bone-building agents with reduced side effects persists.
A second,new antiresorptive agent emerged from the discovery that RANKL is the principal regulator of osteoclastic bone resorption.From this discovery came the development of denosumab,a fully human mono-clonal antibody to RANKL(McClung et al.,2006a). This agent,which is administered at60mg sub-cutaneously every6months,has recently received regulatory approval for the treatment of women with postmenopausal osteoporosis who are at high risk for fracture(Lewiecki et al.,2010).Denosumab has been shown to inhibit the resorptive component of the bone-remodeling system,increase BMD,and reduce the risk of vertebral fractures,hip fractures,and nonvertebral fractures in women with postmenopausal osteoporosis (Hattner et al.,1965;Gallagher and Sai,2010).
The most commonly used osteoporosis treatments are antiresorptive agents,including bisphosphonates and selective estrogen modulators(Lewiecki et al.,2010). Less effective antiresorptive agents include calcitriol, 1-a-hydroxyvitamin D3[1a(OH)D3](Fig.8),and1a,25-dihydroxyvitamin D3[1,25-(OH)2D3].VDR ligands regulate both bone formation and resorption,and 1a,25-(OH)2D3increases the expression and/or protein levels of osteocalcin and osteopontin in osteoblasts,thus supporting a role in bone matrix formation.Therefore, designing drugs to target VDR for the treatment of osteoporosis and osteomalacia is a viable option. However,the use of VDR ligands is limited by its margin of safety,as there is a high risk of developing side effects such as hypercalcemia and hypercalciuria. These side effects result from increased calcium ab-sorption in the intestine,leading to increased plasma and urine levels of calcium that can ultimately result in the mineralization of soft tissue and kidney stone formation.Osteoporosis is a debilitating disease,with a growing prevalence among the elderly population. Therefore,there is an urgent need for bone-building agents that are orally bioavailable yet have reduced side effects(Nagpal et al.,2005).
Several VDR ligands,both natural and synthetic, have been used for the treatment of osteoporosis and osteomalacia,including calcitriol,alfacalcidol, ED-71[1a,25-dihydroxy-2b-(3-hydroxypropoxy)vitamin D3],Ro-26-9228[1a-fluoro-16-ene-20-epi-23-ene-26,27-bishomo-25-hydroxyvitamin D3],and2MD[2-methy-lene-19-nor-(20S)-1a,25(OH)2D3](Fig.8).These ligands enhance their beneficial bone anabolic effects by en-hancing intestinal calcium absorption and by inhibiting the synthesis of PTH.Several reports have demon-strated the prevention and decrease of vertebral frac-tures and an increase in total body and spine BMD in osteoporotic patients after calcitriol(1,25-(OH)2D3) treatment.However,2years of calcitriol treatment resulted in a57%increase in intestinal calcium ab-sorption,a100%increase in urinary calcium,and a32% decrease in PTH serum levels.Several of the patients treated with calcitriol also developed hypercalciuria. Clearly,the treatment of osteoporosis with calcitriol is limited by its margin of safety,which appears to be very narrow(Sairanen et al.,2000).
Prodrug and medicinal chemistry approaches have been explored in an effort to identify less calcemic vitamin D3analogs or VDRMs(selective VDR modu-lators)that are suitable for the treatment of https://www.doczj.com/doc/d85015178.html,ing the prodrug approach,alfacalcidol(1a-hydroxyvitamin D3),a precursor to1,25-(OH)2D3,was identified.This precursor gets enzymatically converted to the active hormone1,25-(OH)2D3in the liver by the action of25-hydroxylase.Alfacalcidol is superior to1,25-(OH)2D3because enterocytes,the intestinal absorptive cells found in the small intestine and colon,lack25-hydroxylase.Therefore,alfacalcidol’s actions are reduced compared with calcitriol because it does not induce intestinal calcium absorption in the first pass when it is absorbed from the intestine.It was determined that in vivo,this analog is converted rapidly to1,25-(OH)2D3in both the liver and bone.Thus,treatment with alfacalci-dol reduced the incidence of vertebral fractures and increased bone mass in several clinical trials(Lau and Baylink,1999).The success of this drug is believed to be the result of the ability to administer higher doses of alfacalcidol than calcitriol before hypercalcemia occurs (Lau and Baylink,1999;Shiraishi et al.,1999). Calcitriol and alfacalcidol(Fig.8)are currently used in Japan for treatment of osteoporosis.Clinical evi-dence of improvement of fracture rates after alfacalci-dol and1,25-(OH)2D3treatment has been published, although the results from these studies are still being debated(Orimo et al.,1987;Gallagher et al.,1989; Ott and Chesnut,1989;Gallagher and Goldgar,1990; Papadimitropoulos et al.,2002;O’Donnell et al.,2008). This mode of treatment is viable in Japan and other countries outside the United States because their diets have modest calcium intake,making the side effects more manageable and hypercalcemia and hypercalciu-ria less common.In a controlled environment,the use of1,25-(OH)2D3and alfacalcidol incurs few side effects and can effectively treat osteoporosis.
Not only does the risk of osteoporosis increase with age,it is well recognized that muscle strength declines with age(Iannuzzi-Sucich et al.,2002).This inverse
Nuclear Receptors and Their Selective Modulators729
受体酪氨酸蛋白激酶的研究 曹 川,戴 霞(综述),李世荣(审校) (中国人民解放军第三军医大学附属西南医院整形外科,重庆400038) 中图分类号:Q78 文献标识码:A 文章编号:100622084(2008)1221780202 摘要:酪氨酸蛋白激酶是细胞信号转导的主要信号酶之一,对细胞的生长、发育与功能调控起着重要的作用。受体型酪氨酸蛋白激酶是细胞内段具有酪氨酸激酶活性跨膜结构的酶蛋白受体,其胞外区与生长因子配体结合,然后激活胞内段的酶活性区启动信号转导,参与细胞的生长、增殖、转化及胚胎发育和肿瘤形成。主要介绍受体型酪氨酸蛋白激酶的结构、分类及其信号转导途径。 关键词:受体酪氨酸蛋白激酶;信号转导;丝裂原活化蛋白激酶 Research Progress of Receptor Tyrosi n e Prote i n K i n a se CAO Chuan,DA I X ia,L I Shi2rong.(D epart2 m ent of P lastic Surgery,Southw estern Hospital,Third M ilitary M edical U niversity of PLA,Chongqing400038, China) Abstract:Tyr osine p r otein kinase is one of the key kinases in cell signal transducti on,which p lays an i m portant r ole in cell gr owth,devel opment and functi onal regulati on.Recep t or tyr osine p r otein kinase is a trans me mbrane kinase p r otein recep t orwith tyr osine p r otein kinase activity on intracellular part,it can actuate the signal transducti on by activating the active z one of its intracellular part resulting fr om the combinati on of its extr ocellular part with its gr owth fact or ligand and then takes part in the cell gr owth,cell p r oliferati on,cell transdifferentiati on,e mbryonic devel opment and tumor genesis.This article mainly revie ws recep t or tyr osine p r otein kinase about its structure,classificati on and its signal transducti on pathway. Key words:Recep t or tyr osine p r otein kinase;Signal transducti on;M it ogen activited p r otein kinase 细胞信号转导的基本方式是蛋白磷酸化,磷酸化过程在 胞内多种蛋白激酶的催化作用下进行,细胞内一类主要的蛋 白激酶是酪氨酸蛋白激酶(tyr osine p r otein kinases,TPK),其 磷酸化作用位点为蛋白质的酪氨酸残基。TPK是信号转导中 一类与受体及多种信号蛋白活化有关的信号酶,对细胞的增 殖、分化及功能具有重要的调节作用,为一个大的结构多样的 酶家族,分为受体依赖型TPK(recep t or tyr osine kinases,RTK) 和非受体依赖型TPK。RTK即具有酶活性的细胞膜受体(又 称催化性受体),是细胞内段具有酪氨酸激酶活性的跨膜结 构的酶蛋白受体,其胞外区与生长因子配体结合,然后激活胞 内段的酶活性区启动信号转导,大多数生长因子如表皮生长 因子(ep ider mal gr owth fact or,EGF)、血小板衍生生长因子 (p latelet2derived gr owth fact or,P DGF)、成纤维细胞生长因子 (fibr oblast gr owth fact or,FGF)、胰岛素样生长因子(insulin2like gr owth fact or,I GF)等的受体均有RTK活性。有一些受体本身 不具有酶活性,但在其胞内段有TPK特异结合的位点,配体 与受体结合后,须通过该位点结合胞内TPK再磷酸化胞内靶 蛋白的酪氨酸残基,启动信号转导过程,这类受体也称为细胞 因子受体超家族,包括干扰素、白细胞介素、集落刺激因子等。 具有TPK活性的受体是外界刺激信息传递至细胞核,转化成 细胞效应的信号通路的关键组成部分,参与细胞的生长、增 殖、转化及胚胎发育和肿瘤形成,具有重要的生理功能[1]。 1 RTK的共同结构 所有RTK都是膜结合的Ⅰ型糖蛋白,基本结构模式可分 成3个部分:细胞外结构区、跨膜区以及细胞内激酶区。其结 构的共同特点是胞内段均具有TPK活性区,再根据胞外区结 构的不同将其聚类[1,2]。 1.1 胞外区 胞外区为500～850个氨基酸组成的亲水性配 体结合区,糖基化位点大都在此区。每一种RTK的胞外区都 含有一个以上的可识别结构域,包括半胱氨酸富含区、甘氨酸富含区、亮氨酸富含区、纤维粘连蛋白Ⅲ样重复区、类I g区、类Ⅷ因子区、EGF样结构域、cadherin 样结构域、kringle样结构域、dis2 coidin样结构域等。 根据RTK胞外区的结构特点,可将其分为4个类型:Ⅰ型为胞外区具有富含半胱氨酸的2个重复序列,包括EGFR、HER2/ neu、HER3/c2erb23和Xm rk。Ⅱ型为异四聚体结构,由2个α及β亚单位以二硫键相连。α亚单位含一富含半胱氨酸的重复序 列,构成配体结合区,β亚单位穿膜后与酪氨酸激酶区相连,包括胰岛素受体、I GFR。Ⅰ、Ⅱ型RTK富含半胱氨酸的重复序列构成坚固的能抵抗蛋白酶消化的构型。Ⅲ型没有胞外区富含半胱氨酸的重复序列,由5个I g样结构组成,RTK激酶区有一77～107个亲水性氨基酸构成的插入序列将ATP结合位点与底物磷酸化位点分开,包括P DGFR和v2f m s/c2kit。Ⅳ型结构同Ⅲ型类似,胞外区仅由3个I g样结构组成,包括FGFR、flg和bek。4种RTK引起的最终细胞生物效应并不相同,但结构上的相似性说明它们可能源于同一受体结构不断进化的结果,从而提示它们在信号转导途径及调节机制方面有共同之处。 1.2 跨膜区 跨膜区由高度疏水、缺乏带电荷的氨基酸残基构成,长度保守(22～26个氨基酸),但氨基酸序列并不保守。RTK的单跨膜区域明显不同于其他跨膜受体(如β2肾上腺素能受体等),且在信号转导中没有直接的作用。 1.3 胞内区 胞内区由500～600个氨基酸组成,负责产生转导信号,其启动信号的分子机制包括胞内分子的一系列变构激活和与蛋白底物及调节因子(如蛋白激酶C)的相互作用。胞内区分为以下几个亚结构:①近膜区:由41～50个氨基酸组成,将酪氨酸激酶区与胞膜分开,4种RTK的近膜区氨基酸序列各不相同,但每一类型RTK近膜区的氨基酸序列高度保守,对信号转导有调节作用。②激酶区:氨基酸序列高度保守,包括ATP结合位点、底物结合区、自身磷酸化位点和调节位点;③羧基末端:由70～200个氨基酸组成,一级结构在不同种属间不同,甚至在同一类型的RTK中亦不相同。该区富含分子质量小的氨基酸,构成亲水性强的多肽链,赋予羧基末端极大的柔韧性。 2 RTK的分类 到目前为止,已发现50多种不同的RTK,根据它们胞外区的结构特点与功能,主要分为9个亚族,其中大部分为细胞
1、核素nuclide ：指质子数和中子数均相同，并且原子核处于相同能态的原子称为一种核素。 2、同位素isotope：具有相同质子数而中子数不同的核素互称同位素。同位素具有相同的化学性质和生物学特性，不同的核物理特性。 3、同质异能素isomer：质子数和中子数都相同，处于不同核能状态的原子称为同质异能素。 4、放射性活度radioactivity：简称活度：单位时间内原子核衰变的数量。 5、放射性核纯度:也称为放射性纯度,指所指定的放射性核素的放射性活度占药物中总放射性活度的百分比,放射性纯度只与其放射性杂志的量有关. 6、放射化学纯度(放化纯):指特定化学结构的放射性药物的放射性占总放射性的百分比. 7、放射性药物：指含有一个或多个放射原子(放射性核素)而用于医学诊断和治疗用的一类特殊药物。 8、正电子发射型计算机断层仪（PET）：利用发射正电子的放射性核素及其标记物为显像剂，对脏器或组织进行功能，代谢成像的仪器。 9、单光子发射型计算机断层仪（SPECT）：利用注入人体的单光子放射性药物发出的γ射线在计算机辅助下重建影响，构成断层影像的仪器。 10、“闪烁”现象 (flare phenomenon): 在肿瘤病人放疗或化疗后，临床表现有显著好转，骨影像表现为原有病灶的放射性聚集较治疗前更为明显，再经过一段时间后又会消失或改善，这种现象称为“闪烁”现象。 1、核医学的定义及核医学的分类. 答：核医学是一门研究核素和核射线在医学中的应用及其理论的学科.及应用放射性核素诊治疾病和进行生物医学研究.核医学包括实验核医学和临床核医学.实验核医学主要包括核衰变测量,标记,示踪.体外放射分析,活化分析和放射自显影.临床诊断学是利用开放型放射性核素诊断和治疗疾病的临床医学学科.由诊断
核医学讲义 一、核医学概述 概述 *核医学又称核子医学或原子医学，旧称"同位素"，在我国属于一门独立医学学科。 *核素显像是影像医学的一部分。 *核医学就是利用放射性核素及其标记的化合物进行疾病的诊断和治疗一门学科。 分类 *实验核医学 *临床核医学 *核药学 *核仪器和核电子学 核医学的发展史 *1934年Enrico Fermi发明核反应堆，生产第一个碘的放射性同位素。 *1936年John Lawrence 首先用32P治疗白血病，这是人工放射性同位素治疗疾病的开始。 *1937年Herz首先在兔进行碘[128I]半衰期（半衰期T1/2 25分）的甲状腺试验，以后被131I（8.4天）替代。 *1942年Joseph Hamilton首先应用131I测定甲状腺功能和治疗甲状腺功能亢进症 * 1943年至1946年用131I治疗甲状腺癌转移 *1946年7月14日，美国宣布放射性同位素可以进行临床应用，开创了核医学的新纪元 *1951年Benedict Cassen 发明线性扫描机 *1958年Hal O.Anger发明Anger照相机 *1959年Solomon A.Berson 和Rosalyn S. Yalow发明放射免疫分析等对影像核医学和体外测定的发展都起到了很大的推动作用 *50年代，钼[99Mo]-锝[99mTc] (99Mo-99mTc)发生器的出现 *70年代单光子断层仪的应用和80年代后期正电子断层仪进入临床应用，使影像核医学在临床医学中的地位有了显著提高 反应堆 最早的扫描机 最早的伽玛相机 最早的摄碘试验 钼[99Mo]-锝[99mTc] (99Mo-99mTc)发生器 放射性核素显像技术特点 *功能性显像 *定量显像 *化学或代谢显像 放射性核素的生产方式 *核反应堆生产 *加速器生产 *核素发生器生产 二、核物理基础 基本概念
1、核医学的定义及核医学的分类. 答：核医学是一门研究核素和核射线在医学中的应用及其理论的学科.及应用放射性核素诊治疾病和进行生物医学研究.核医学包括实验核医学和临床核医学.实验核医学主要包括核衰变测量,标记,示踪.体外放射分析,活化分析和放射自显影.临床诊断学是利用开放型放射性核素诊断和治疗疾病的临床医学学科.由诊断和治疗两部分组成.诊断和医学包括以脏器显像和功能测定为主要内容的体内诊断法和以体外放射分析为主要内容的体外诊断法.治疗核医学是利用放射性核素发射的核射线对病变进行高密度集中治疗. 2、分子核医学的主要研究内容。 答：分子医学的概念：是建立在分子细胞学、分子生物化学、分子药理学及计算机技术基础上的一门边缘学科，是在大分子、蛋白、核酸水平上研究疾病的发生、发展规律，最终达到对疾病进行特异性诊断和个性化治疗的一门学科。 研究内容：代谢显像、受体显像、反义与基因显像、放射免疫显像、凋亡显像。 3、原子的结构.元素、同位素、核素、同质异能素、放射性活度的概念,放射性衰变的类型。 答：原子是由处于原子中心的原子核和带负电荷核外电子组成,原子核由质子和中子组成,他们统称核子. 核素：指质子数和中子数均相同，并且原子核处于相同能态的原子称为一种核素。 同位素：具有相同质子数而中子数不同的核素互称同位素。同位素具有相同的化学性质和生物学特性，不同的核物理特性。 同质异能素：质子数和中子数都相同，处于不同核能状态的原子称为同质异能素。 放射性活度：简称活度：单位时间内原子核衰变的数量。 放射性衰变：α衰变(alpha decay)、β—衰变(beta decay)、正电子衰变、电子俘获（electron capture）、γ衰变(gamma decay)。 4、什么是放射性药物，按理化性质如何分类，放射性药物与普通药物有何不同，医用放射性药物由哪些途径产生，放射性核纯度和放化纯的概念？ 答：放射性药物指含有一个或多个放射原子(放射性核素)而用于医学诊断和治疗用的一类特殊药物；分类：离子型、胶体型、放射性标记化合物、放射性标记生物活性物质。与普通药物不同点：放射性，理化特性取决于被标记物固有特性，有特定物理半衰期和有效半衰期，脱标及辐射自分解，计量单位用活度为基本单位，治疗作用机理不同于普通药物。 产生：加速器生产，反应堆生产，从裂变产物中提取，放射性核素发生器淋洗。 放射性核纯度:也称为放射性纯度,指所指定的放射性核素的放射性活度占药物中总放射性活度的百分比,放射性纯度只与其放射性杂志的量有关. 放射化学纯度(放化纯):指特定化学结构的放射性药物的放射性占总放射性的百分比. 5、治疗常用的放射性核素。 答：常用的放射性核素多是发射纯β-射线（32P、89Sr、90Y等）或发射β-射线时伴有γ射线（131I、153Sm、188Re、117Sn m、117Lu等）的核素。131I（NaI）甲状腺疾病诊断、治疗；133Xe肺通气显像；99mTc-MIBI心肌灌注显像；99mTc-MDP骨显像；99mTc-ECD 脑灌注显像；99mTc-MAA肺灌注显像；99mTc-RBC肝血池显像；99mTc-寡核苷酸肿瘤基因反义显像。 6、目前常用的脏器显像仪有哪些，什么是PET，SPECT？ 答：γ照相机 ECT，单光子发射型计算机断层仪（SPECT），正电子发射型计算机断层仪（PET），脏器功能测定仪 CT。 正电子发射型计算机断层仪（PET）：利用发射正电子的放射性核素及其标记物为显像剂，对脏器或组织进行功能，代谢成像的仪器。PET主要由探测系统包括晶体、电子准直、符合线路和飞行时间技术，计算机数据处理系统图像显示和断层床等组成。原理：是用正电子衰变和工业苏标记的放射性药物，在人体内放出的正电子与组织相互作用，发生正电子湮灭，向相反方向发射光子，与γ光子检测仪互相作用，产生荧光子，并形成一个电子脉冲，经过显像系统及计算机处理形成PET图像，与SPECT比较具有空间分辨率高、探测效率高、能准确地显示受检脏器内显像剂浓度提供的代谢影像和各种定量生理参数等优点。 单光子发射型计算机断层仪（SPECT）：利用注入人体的单光子放射性药物发出的γ射线在计算机辅助下重建影响，构成断层影像的仪器。 7、肿瘤常用的显像剂 答：67Ga，201Tl,99mTc-MIBI，18F-FDG，99mTc-PMT，99mTc-DMSA， 99mTc-octreotide，111In-DTPA-D-phel-octreotide，特点：均为亲肿瘤显像剂。 8、幅射防护的原则及外照射防护的措施？ 答：辐射防护基本原则是：1实践的正当化，要求产生电离辐射的实践给个人和社会带来利益大于代价，抵偿其所造成危害。2防护最优化，指用最小代价获得最大净利益，避免一切不必要的照射，使一切必要照射保持在合理达到的最低水平。3个人剂量的限制，在实施上述两项原则时，要同时保证个人的当量剂量不超过规定的限值。外照射防护原则：1时间防护，尽量减少接触放射源的时间。2距离防护，尽量增大人体与放射源的距离。3屏蔽防护，在人体和放射源之间安装屏蔽物，借助于物质对射线的吸收减少人体受照剂量。 9、免疫分析基本原理，非放射性标记免疫分析包括那些方法，免疫放射分析技术的主要特点和分析质控指标。 答：（1）免疫分析是利用特异抗体与标记抗原和非标记抗原的竞争结合反应，用过测定放射性复合物量来计算出非标记抗原量的一种超微量分析技术。（2）非放射性的标记免疫分析包括时间分辨荧光分析法；酶标记的免疫分析法；化学发光免疫分析法。（3）免疫放射分析技术的特点：以标记抗体作为示踪剂，反应动力学，因标记抗体是过量的，且反应是非竞争性的，抗原抗体是全量反应，故反应速度比RIA快，灵敏度明显高于放射免疫分析，约为放射免疫分析的10~100倍，标准曲线工作范围宽，特异性高，稳定性好。（4）质控指标：稳定性、精密度、灵敏度、准确度、特异性。 10、脑灌注显像的原理、正常及异常图像特点、主要的适应症，常用的显像剂及显像特点。了解乙酰唑胺介入显像及PET脑显像的主要内容。 答：原理：根据血脑屏障的特殊功能，选择一些具有脂溶性的、电中性的小分子（<500）放射性示踪剂，它能自由通过完整无损的血脑屏障，并大部分被脑细胞所摄取，且在脑内的存留量与血流量成正
蛋白激酶 1、按底物蛋白被磷酸化的氨基酸残基种类（5类） ①.丝氨酸/苏氨酸（Ser/Thr）蛋白激酶 ②.酪氨酸（Tyr）蛋白激酶： 1.EGFR（EGFR、HER2/ErbB2、ErbB3、ErbB4）：表皮生长因子受体 2.PDGFR（PDGFRα、PDGFRβ）：血小板衍生生长因子受体 CSF1R: 集落刺激因子1受体 c-Kit：干细胞生长因子受体 Flk2：胎肝激酶2 3.InsR：胰岛素受体 IGF-1R：类胰岛素生长因子受体 IRR：胰岛素相关受体 4.NGFR：神经生长因子受体 5.FGFR（FGFR1、FGFR2、FGFR3、FGFR4）：成纤维细胞生长因子受体 6.VEGFR（VEGFR1、VEGFR2/FLK-1、VEGFR3/FLT4）：血管内皮生长因子受体 7.HGFR：肝细胞生长因子受体 8.c-Met Ron sea 9.Ltk Alk 10.c-RET 11.Ros 12.Eph、Eck、Eek、Erk、Elk 13.Tie、Tie-2 Src家族 Tec家族（Btk、Itk/Tsk/Emt、Tec、Txk和Bmx等） ZAP70家族 、TYK1）等 Abl Wee等 ③.组氨酸蛋白激酶（组氨酸、精氨酸或赖氨酸的碱性残基被磷酸化，见于双组分信号系统） ④.色氨酸蛋白激酶 ⑤.天冬氨酰基/谷氨酰基蛋白激酶 2、按序列相似性及功能（7类） ①.AGC组：核苷酸依赖家族（PKA、PKG、PKC家族） ②.CaMK组：Ca2+/钙调素调节的蛋白激酶家族、snfl/AMPK家族 ③.CMGC组：CDK、MAPK、GSK3、CLK家族
④.CKI：酪氨酸激酶家族I ⑤.TK：酪氨酸蛋白激酶 ⑥.TKL：类似酪氨酸激酶 ⑦.STE 癌症 1、癌症主要有四种： 1、癌瘤：影响皮肤、粘膜、腺体及其他器官； 2、血癌：即血液方面的癌； 3、肉瘤：影响肌肉、结缔组织及骨头； 4、淋巴瘤：影响淋巴系统。 常见的癌症有血癌（白血病）、骨癌、淋巴癌（包括淋巴细胞瘤）、肠癌、肝癌、胃癌、盆腔癌（包括子宫癌,宫颈癌）、肺癌（包括纵隔癌）、脑癌、神经癌、乳腺癌、食道癌、肾癌等。
疾病分类知识及问答 疾病分类是根据疾病的病因、解剖部位、临床表现和病理等特性，将疾病进行排列分组，使其成为一个有序的组合。 ICD-10：国际疾病分类 疾病分类是一个类目系统，根据疾病的某些特征，按照一定的规则把疾病分门别类，建立标准确立疾病条目。（疾病分类实际上也是一种分组，有时一个组别可以包含若干种相同或相似性质的疾病，有时仅单纯地包含某种疾病）。SNDO：疾病和手术标准命名法。 疾病和手术标准命名法是一个双重分类系统，每个疾病分为两部分，一部分表达疾病的发生部位，另一部分表示疾病的原因。手术也分为两部分，一部分表示手术操作的部位，另一部分表示手术操作方式。 疾病（诊断）和操作SNDOMED（医学系统命名法）的每个术语（词条）均有一个编码与之对应，而且还容纳了多个国际性编码系统和资料，如ICD-9，ICD-10，ICD-O,ICD-9CM等，交互检索功能强，该系统适用于信息的计算机存储和自动编码，已逐渐成为临床病理学界统一的语言。 疾病分类的目的是为了按照所设定的方案进行资料收集、整理、分析和利用。ICD-10分类系统的特点：科学性、准确性、完整性、适用性、可操作性。 ICD-10不是标准疾病命名，但是是标准疾病分类。 ICD-10由三卷组成，第一卷为类目表，第二卷为指导手册，第三卷为字母顺序索引。 强烈优先分类章：第十五章妊娠、分娩产褥期。 一般优先分类章：第一章某些传染病和寄生虫病；第二章肿瘤；第五章精神和行为障碍；第十六章起源于围生期的某些情况；第十七章先天畸形、变形和染色体异常；第十九章损伤、中毒和外因的某些其他后果。 最后分类章：第十八章症状、体征和临床与实验室异常所见。 类目：指三位数编码，包括一个字母和两位数字。 亚目：指四位数编码，包括一个字母、三位数字和一个小数点。 细目：指五位数编码，包括一个字母、四位数字和一个小数点。 残余类目（剩余类目）：指含有亚目标题“其他”和“未特指”字样的亚目。残余类目是分类那些不能归类到该类目其他特指亚目的疾病。在ICD-9中，这些疾病特定分类在.8和.9,因此也称.8和.9为残余类目。在ICD-10中，这些疾病绝大多数还是分类在.8和.9 ；但也有例外，如其他慢性胰腺炎。（.8特指，.9未特指） 双重分类（星剑号分类系统）：指星号和剑号编码，剑号表明疾病的原因，星号表明疾病的临床表现。 疾病分类编码的查找方法分为3个步骤。首先要确定主导词；其次是在第三卷索引中查找编码；最后是在第一卷中核对编码。对于肿瘤的编码操作，由于它具有两个编码，所以要两次操作。 主导词的选择：1、疾病的主导词主要是由疾病诊断中的临床表现担任，常常被置于诊断术语的尾部。。2、疾病的病因常常可以作为主导词，但细菌、病毒虽然是病因，却不能作为主导词，此时还要以临床表现为主导词。3、以人名
疾病简介 肝血管瘤是一种较为常见的肝脏良性肿瘤，临床上以海绵状血管瘤最多见，自然人群尸检发现率为0.35-7.3%，占肝良性肿瘤的5-20%。近年来，随着人们健康体检的意识提高及各种影像诊断技术的进步，无症状的小血管瘤发现率明显升高。多数病例临床无症状或症状轻微，病程长、生长缓慢，预后良好。目前对该病的基础和临床研究不多，缺乏成熟而严格的诊治规范，治疗技术方案、适应征的界定存在诸多模糊甚至是错误的认识，传统外科手术治疗与射频消融术、肝动脉栓塞、放射治疗、术中微波固化术、冷冻和硬化剂注射治疗等多种治疗手段共存，治疗技术方案尚未形成较为统一的临床路径供医患抉择。[1][2] 发病原因 目前对肝血管瘤的确切确切发病原因尚不明确，主要有以下几种学说：（1）先天性发育异常学说：目前多数学者认为血管瘤的发生为是先天性肝脏末梢血管畸形引起，一般认为在胚胎发育过程中由于肝血管发育异常，引起血管内皮细胞异常增生而形成肝血管瘤； （2）激素刺激学说：有学者观察到在女性青春期、怀孕、口服避孕药等可使血管瘤的生长速度加快，认为女性激素可能也是血管瘤的致病机制之一； （3）其它：如毛细血管组织感染后变形，导致毛细血管扩张，肝组织局部坏死后血管扩张形成空泡状，其周围血管充血扩张；肝内区域性血循环停滞，致使血管形成海绵状扩张。[3][4] 疾病分类 肝血管瘤依据其纤维组织多少，病理上可分为4型： （1）海绵状血管瘤，是最为常见的类型； （2）硬化性血管瘤； （3）血管内皮细胞瘤；
（4）毛细血管瘤，此种少见。目前多采用按直径大小分类：<5cm（小血管瘤）；5-10cm（血管瘤）；10cm-15cm（巨大血管瘤）；>15cm（特大血管瘤），可能具有一定的指导肝血管瘤患者治疗技术方案的意义，为肝血管瘤的诊治提供有效参考。[5] 疾病危害 肝血管瘤在任何年龄段均可发病，以30-50岁多见，文献报道女性多于男性，男女比例约1：3-6。但53859例健康人群体检资料分析显示，肝血管瘤的发生率为3.11%，男性和女性的发病率相当（3.36% VS.2.88%，P>0.05），这一现象与文献报告的结果不同，可能与以往的文献报告分析的均是门诊或住院暴露病例而非大样本的人群普查，多数小血管瘤未被统计纳入有关。深入分析该普查病例的男性和女性血管瘤的大小构成比，可以看到在>5cm的肝血管瘤病例占所有病例的比例中，女性是男性的2.56倍（2.90% VS.1.26%, P>0.05），这一结果支持了先前的设想。 进一步分析年龄与发病率关系时可见随着年龄的增大，发病率有所增加，到40-60岁时达高峰，随后有所下降。这一现象可能的解释是随着年龄的增加，初始较难查出的隐匿血管瘤因逐渐生长而被检查到，使发病率增加；在40-60岁后，部分血管瘤停止生长甚至部分血管瘤有所消退致发病率有所下降。 这一现象在随访超过5年的131例病例的分析中得到验证，随着年龄的增加，血管瘤增大的病例比例明显下降，血管瘤最大径增大的程度也明显缩小。在性别、年龄与血管瘤大小的关系分析中发现女性血管瘤在各年龄段均较男性血管瘤为大，且血管瘤的大小随年龄的增大而明显增大，到40-60岁达高峰，此后略有缩小。从以上资料不难得出血管瘤发展可能受到激素水平的变化的影响，雌激素的影响可能更为明显，这可能也能解释为何在直径>5cm的肝血管瘤中，女性发病率远高于男性的原因。[6][7][8] 疾病症状 多数肝血管瘤无明显不适症状，多在健康体检常规行B超检查或行腹部手术时被发现，尚无证据说明它们有恶变可能，但偶可与肝脏的其他恶性肿瘤相混淆导致误诊。当血管瘤增大至5cm以上时，可能出现非特异性的腹部症状，包括： （1）腹部包块：包块有囊性感，无压痛，表面光滑或不光滑，在包块部听诊有时可听到传导的血管杂音； （2）胃肠道症状：可出现右上腹隐痛和不适，以及食欲不振、恶心、呕吐、嗳气、食后胀饱和消化不良等；
昆虫分类学基础知识(一) 昆虫分类学基础知识昆虫是自然界中最昌盛的动物类群，其种类及数量极多。据报道，全世界现有昆虫1000 万种，已描述的昆虫种类约110 万种，约占整个已知动物种类总数的60%以上，并且每年仍以7000 种的速度增加。我国地域辽阔，环境复杂多样，生物资源极为丰富，是世界上昆虫种类最多的国家之一。据报道，我国的昆虫种类约占世界昆虫种类的1/10 ，按这个比率，我国昆虫种类应超过100 万种，可是我国目前已记载鉴定的昆虫种类不超过8 万种，还有更多的昆虫尚未被发现和开发，而且，有不少种类在未被我们认识之前就已灭绝。因此，查清自然界昆虫资源及区系是当代科学上一项重要的内容和任务。在这方面，我国的任务尤为繁重。昆虫不仅种类繁多，数量庞大，而且分布范围之广也是惊人的，地球上的每个角落几乎都有它们的踪迹，其中有很多种类与人类有着极为密切的利害关系。人类在生产活动和科学实验中，不但有许多害虫和益虫要认识，而且有许多在生产上迫切需要解决的近似种类或易混淆的种类要区别。昆虫分类学( insect taxonomy )是昆虫学( entomology )的一个分支学科，是研究昆虫种的鉴定( identification )、分类（classification ）和系统发育（phylogeny ）的科学。在数以百万计的昆虫种类中，存在着血缘的远近和亲疏关系。亲缘关系越近，其形态特征和对环境的要求、生活习性以及发生发展规律也愈相近。而昆虫分类就是在这种亲缘关系的基础上，运用“分析、比较、综合、归纳”的科学方法，对地质年代中的化石昆虫与现存的昆虫种类之间，现存昆虫彼此之间以及近缘生物间进行对比研究，以了解种与种、类与类间的异同，反映
实验七昆虫分类（一）——目的分类 目的： 了解昆虫纲分目的特征； 掌握常见农业昆虫中重要目的主要形态鉴定特征； 学习使用和编制检索表； 原理：（自己写） 材料用具： 跳虫、蜚蠊、飞蝗、蠼螋、蜻蜓、蓟马、蝽象、蜡蝉、草蛉、地 老虎、粉蝶、金龟子、胡蜂、蜜蜂、食蚜蝇、大蚊标本等。户外采集 的昆虫。 体视显微镜、镊子、解剖针和大头针等工具；多媒体课件。 内容与方法：（参考以下这些） 一、检索表 检索表的编制，是用对比分析和归纳的方法，将不同种类昆虫的 特征，从中选定比较重大而稳定的特征，做成简短的文字条文排列而 成。因此检索表的运用和编制是昆虫分类工作重要的基础。 1、类型：双项式（两项式）、单项式、退格式或包孕式。 2、步骤：选取特征→列表→成文 例如： 目名口器翅其它特征 弹尾目咀嚼式无腹末有弹器 缨尾目咀嚼式无腹末有尾须1对和中尾丝1条直翅目咀嚼式前翅皮革质，后翅膜质后足适于跳跃，或前足适于开掘鞘翅目咀嚼式前翅为鞘翅，后翅膜质 半翅目刺吸式前翅为半鞘翅，后翅为膜质喙着生于头前端
同翅目刺吸式前后翅膜质，或前翅稍加厚喙着生于头腹面后端 两项式 1.无翅 (2) 有翅 (3) 2.腹末有弹器……………………………………………………………………………弹尾目 腹末有尾须1对和中尾丝1条...............................................................缨尾目3.口器刺吸式 (4) 口器咀嚼式 (5) 4.前翅为半鞘翅…………………………………………………………………………半翅目 前后翅膜质……………………………………………………………………………同翅目5.前翅皮质，后翅膜质…………………………………………………………………直翅目 前翅为鞘翅，后翅膜质………………………………………………………………鞘翅目3、编制检索表的注意事项 尽可能地选用明显而稳定的外部特征 绝对性状 对称性状 文字简洁 二、重要农业昆虫的分目 观察标本，掌握主要农业昆虫的分类鉴定特征。 目名口器翅其它特征直翅目 缨翅目 半翅目 同翅目 脉翅目 鞘翅目 鳞翅目 双翅目 膜翅目
学的定义及核医学的分类. 答：核医学是一门研究核素和核射线在医学中的应用及其理论的学科.及应用放射性核素诊治疾病和进行生物医学研究.核医学包括实验核医学和临床核医学.实验核医学主要包括核衰变测量,标记,示踪.体外放射分析,活化分析和放射自显影.临床诊断学是利用开放型放射性核素诊断和治疗疾病的临床医学学科.由诊断和治疗两部分组成.诊断和医学包括以脏器显像和功能测定为主要内容的体内诊断法和以体外放射分析为主要内容的体外诊断法.治疗核医学是利用放射性核素发射的核射线对病变进行高密度集中治疗. 2、分子核医学的主要研究内容。 答：分子医学的概念：是建立在分子细胞学、分子生物化学、分子药理学及计算机技术基础上的一门边缘学科，是在大分子、蛋白、核酸水平上研究疾病的发生、发展规律，最终达到对疾病进行特异性诊断和个性化治疗的一门学科。 研究内容：代谢显像、受体显像、反义与基因显像、放射免疫显像、凋亡显像。 3、原子的结构.元素、同位素、核素、同质异能素、放射性活度的概念,放射性衰变的 答：原子是由处于原子中心的原子核和带负电荷核外电子组成,原子核由质子和中子组成,他们统称核子. 核素：指质子数和中子数均相同，并且原子核处于相同能态的原子称为一种核素。 同位素：具有相同质子数而中子数不同的核素互称同位素。同位素具有相同的化学性质和生物学特性，不同的核物理特性。 同质异能素：质子数和中子数都相同，处于不同核能状态的原子称为同质异能素。 放射性活度：简称活度：单位时间内原子核衰变的数量。 放射性衰变：α衰变(alpha decay)、β—衰变(beta decay)、正电子衰变、电子俘获（electron capture）、γ衰变(gamma decay)。 4、什么是放射性药物，按理化性质如何分类，放射性药物与普通药物有何不同，医用放射性药物由哪些途径产生，放射性核纯度和放化纯的概念？ 答：放射性药物指含有一个或多个放射原子(放射性核素)而用于医学诊断和治疗用的一类特殊药物；分类：离子型、胶体型、放射性标记化合物、放射性标记生物活性物质。与普通药物不同点：放射性，理化特性取决于被标记物固有特性，有特定物理半衰期和有效半衰期，脱标及辐射自分解，计量单位用活度为基本单位，治疗作用机理不同于普通药物。 产生：加速器生产，反应堆生产，从裂变产物中提取，放射性核素发生器淋洗。 放射性核纯度:也称为放射性纯度,指所指定的放射性核素的放射性活度占药物中总放射性活度的百分比,放射性纯度只与其放射性杂志的量有关. 放射化学纯度(放化纯):指特定化学结构的放射性药物的放射性占总放射性的百分 5、治疗常用的放射性核素。 答：常用的放射性核素多是发射纯β-射线（32P、89Sr、90Y等）或发射β-射线时伴有γ射线（131I、153Sm、188Re、117Sn m、117Lu等）的核素。131I（NaI）甲状腺
G蛋白偶联受体激酶的研究进展 摘要：G蛋白偶联受体激酶(GRKs)是一簇与G蛋白偶联受体(GPCRs)失敏相关的激酶。GRKs的主要功能是介导GPCRs磷酸化，并与抑制蛋白arrestins协同作用促进GPCRs内化和与G蛋白失偶联，从而导致GPCRs的失敏。 关键词：G蛋白偶联受体激酶；G蛋白偶联受体；抑制蛋白；失敏；内化G蛋白偶联受体激酶(G protein-coupled receptorkinases，GRKs)是一簇与G蛋白偶联受体(GPCRs)快速失敏(desensitization)相关的激酶。许多GPCRs如阿片受体、血栓素受体、5一羟色胺受体、肾上腺素能受体等在激动剂持续刺激时易发生转导信号的快速衰减，发生机制主要与3类调节分子即GRKs，抑制蛋白arrestins和第二信使调节激酶，如PKA，PKC有关。其中以GRKs最为引人关注。 1 GRKs的结构和功能 GRKs家族由7个结构上有同源序列的家族成员组成[1]。每种GRKs都含有共同的功能结构，包括1个中心催化区、1个底物识别和含有G蛋白信号调节蛋(regulators of G protein signaling，RGS)样结构的氨基末端区，以及1个作用于胞膜的羧基末端区。根据序列和功能的相似性可分为3个亚家族。第一个亚家族包括GRKl和GRK7。GRKl是视紫质(rhodopsin)激酶，仅在视网膜光受体细胞表达，作用底物是视网膜的视蛋白。人GRK7基因位于3q21，长度约10 kb，由4个外显子组成。人GRK7仅在视网膜表达，包括所有的视网膜神经元和锥体外节段。重组的人GRK7在光照条件下可催化视紫质磷酸化。有实验证实，GRKl和GRK7在人视锥细胞均有表达；与此相反，GRK7在小鼠的许多组织包括视网膜均有表达，而视网膜的光受体则不表达GRK7。人锥体外节段表达GRK7而小鼠不表达，提示GRK7可能为GRKl基因缺失的小口氏病(一种夜盲症)患者提供正常的光觉[2]。 第二个亚家族的GRK2和GRK3又分别称为β-肾上腺素能受体激酶1(β-ARKl)和β-肾上腺素能受体激酶2(β-ARK2)。Oppermann等[3]将GRK2，GRK3的单抗加入家兔心肌细胞，结果异丙肾上腺素诱导的受体磷酸化77％受到抑制；而GRK4的单抗却无抑制作用。鸡神经元细胞可表达GRK3样蛋白，细胞内注入GRK2或GRK3的特异性单抗，结果消除了α2一AR介导的钙流抑制失敏。 第三个亚家族包括GRK4，GRK5和GRK60。 GRK4仅在睾丸高水平表达，提示该
1、核医学的定义及核医学的分类. 答：核医学是一门研究核素和核射线在医学中的应用及其理论的学科.及应用放射性核素诊治疾病和进行生物医学研究.核医学包括实验核医学和临床核医学.实验核医学主要包括核衰变测量,标记,示踪.体外放射分析,活化分析和放射自显影.临床诊断学是利用开放型放射性核素诊断和治疗疾病的临床医学学科.由诊断和治疗两部分组成.诊断和医学包括以脏器显像和功能测定为主要内容的体内诊断法和以体外放射分析为主要内容的体外诊断法.治疗核医学是利用放射性核素发射的核射线对病变进行高密度集中治疗. 2、分子核医学的主要研究内容。答：分子医学的概念：是建立在分子细胞学、分子生物化学、分子药理学及计算机技术基础上的一门边缘学科，是在大分子、蛋白、核酸水平上研究疾病的发生、发展规律，最终达到对疾病进行特异性诊断和个性化治疗的一门学科。研究内容：代谢显像、受体显像、反义与基因显像、放射免疫显像、凋亡显像。 3、原子的结构.元素、同位素、核素、同质异能素、放射性活度的概念,放射性衰变的类型。答：原子是由处于原子中心的原子核和带负电荷核外电子组成,原子核由质子和中子组成,他们统称核子. 核素：指质子数和中子数均相同，并且原子核处于相同能态的原子称为一种核素。同位素：具有相同质子数而中子数不同的核素互称同位素。同位素具有相同的化学性质和生物学特性，不同的核物理特性。同质异能素：质子数和中子数都相同，处于不同核能状态的原子称为同质异能素。放射性活度：简称活度：单位时间内原子核衰变的数量。放射性衰变：α衰变(alpha decay)、β—衰变(beta decay)、正电子衰变、电子俘获（electron capture）、γ衰变(gamma decay)。 4、什么是放射性药物，按理化性质如何分类，放射性药物与普通药物有何不同，医用放射性药物由哪些途径产生，放射性核纯度和放化纯的概念？答：放射性药物指含有一个或多个放射原子(放射性核素)而用于医学诊断和治疗用的一类特殊药物；分类：离子型、胶体型、放射性标记化合物、放射性标记生物活性物质。与普通药物不同点：放射性，理化特性取决于被标记物固有特性，有特定物理半衰期和有效半衰期，脱标及辐射自分解，计量单位用活度为基本单位，治疗作用机理不同于普通药物。产生：加速器生产，反应堆生产，从裂变产物中提取，放射性核素发生器淋洗。放射性核纯度:也称为放射性纯度,指所指定的放射性核素的放射性活度占药物中总放射性活度的百分比,放射性纯度只与其放射性杂志的量有关. 放射化学纯度(放化纯):指特定化学结构的放射性药物的放射性占总放射性的百分比. 5、治疗常用的放射性核素。答：常用的放射性核素多是发射纯β-射线（32P、89Sr、90Y等）或发射β-射线时伴有γ射线（131I、153Sm、188Re、117Sn m、117Lu等）的核素。131I（NaI）甲状腺疾病诊断、治疗；133Xe肺通气显像；99mTc-MIBI心肌灌注显像；99mTc-MDP骨显像；99mTc-ECD 脑灌注显像；99mTc-MAA肺灌注显像；99mTc-RBC肝血池显像；99mTc-寡核苷酸肿瘤基因反义显像。 6、目前常用的脏器显像仪有哪些，什么是PET，SPECT？答：γ照相机ECT，单光子发射型计算机断层仪（SPECT），正电子发射型计算机断层仪（PET），脏器功能测定仪 CT。正电子发射型计算机断层仪（PET）：利用发射正电子的放射性核素及其标记物为显像剂，对脏器或组织进行功能，代谢成像的仪器。PET主要由探测系统包括晶体、电子准直、符合线路和飞行时间技术，计算机数据处理系统图像显示和断层床等组成。原理：是用正电子衰变和工业苏标记的放射性药物，在人体内放出的正电子与组织相互作用，发生正电子湮灭，向相反方向发射光子，与γ光子检测仪互相作用，产生荧光子，并形成一个电子脉冲，经过显像系统及计算机处理形成PET图像，与SPECT比较具有空间分辨率高、探测效率高、能准确地显示受检脏器内显像剂浓度提供的代谢影像和各种定量生理参数等优点。单光子发射型计算机断层仪（SPECT）：利用注入人体的单光子放射性药物发出的γ射线在计算机辅助下重建影响，构成断层影像的仪器。 7、肿瘤常用的显像剂答：67Ga，201Tl,99mTc-MIBI，18F-FDG，99mTc-PMT，99mTc-DMSA，99mTc-octreotide，111In-DTPA-D-phel-octreotide，特点：均为亲肿瘤显像剂。 8、幅射防护的原则及外照射防护的措施？答：辐射防护基本原则是：1实践的正当化，要求产生电离辐射的实践给个人和社会带来利益大于代价，抵偿其所造成危害。2防护最优化，指用最小代价获得最大净利益，避免一切不必要的照射，使一切必要照射保持在合理达到的最低水平。3个人剂量的限制，在实施上述两项原则时，要同时保证个人的当量剂量不超过规定的限值。外照射防护原则：1时间防护，尽量减少接触放射源的时间。2距离防护，尽量增大人体与放射源的距离。3屏蔽防护，在人体和放射源之间安装屏蔽物，借助于物质对射线的吸收减少人体受照剂量。 9、免疫分析基本原理，非放射性标记免疫分析包括那些方法，免疫放射分析技术的主要特点和分析质控指标。答：（1）免疫分析是利用特异抗体与标记抗原和非标记抗原的竞争结合反应，用过测定放射性复合物量来计算出非标记抗原量的一种超微量分析技术。（2）非放射性的标记免疫分析包括时间分辨荧光分析法；酶标记的免疫分析法；化学发光免疫分析法。（3）免疫放射分析技术的特点：以标记抗体作为示踪剂，反应动力学，因标记抗体是过量的，且反应是非竞争性的，抗原抗体是全量反应，故反应速度比RIA快，灵敏度明显高于放射免疫分析，约
1、核素nuclide :指质子数和中子数均相同，并且原子核处于相同能态的原子称为一种核 2、同位素isotope :具有相同质子数而中子数不同的核素互称同位素。同位素具有相同的化学性质和生物学特性，不同的核物理特性。 3、同质异能素isomer:质子数和中子数都相同，处于不同核能状态的原子称为同质异能素。 4、放射性活度radioactivity :简称活度：单位时间内原子核衰变的数量。 5、放射性核纯度：也称为放射性纯度，指所指定的放射性核素的放射性活度占药物中总放射性活度的百分比，放射性纯度只与其放射性杂志的量有关. 6、放射化学纯度（放化纯）:指特定化学结构的放射性药物的放射性占总放射性的百分比. 7、放射性药物：指含有一个或多个放射原子（放射性核素）而用于医学诊断和治疗用的一类特殊药物。 &正电子发射型计算机断层仪（PET :利用发射正电子的放射性核素及其标记物为显像齐腹对脏器或组织进行功能，代谢成像的仪器。 9、单光子发射型计算机断层仪（SPECT :利用注入人体的单光子放射性药物发出的丫射线在计算机辅助下重建影响，构成断层影像的仪器。 10、“闪烁”现象（flare phe no me non）: 在肿瘤病人放疗或化疗后，临床表现有显着好转，骨影像表现为原有病灶的放射性聚集较治疗前更为明显，再经过一段时间后又会消失或改善，这种现象称为“闪烁”现象。 1、核医学的定义及核医学的分类. 答：核医学是一门研究核素和核射线在医学中的应用及其理论的学科.及应用放射性核素诊 治疾病和进行生物医学研究.核医学包括实验核医学和临床核医学.实验核医学主要包括核衰变测量,标记,示踪.体外放射分析，活化分析和放射自显影?临床诊断学是利用开放型放射性核