Association of Anxiety-Related Traits with a Polymorphism in the Serotonin ‘Transporter Gene Regulatory Region Klaus-Peter Lesch; Dietmar Bengel; Armin Heils; Sue Z. Sabol; Benjamin D. Greenberg; Susanne Petri; Jonathan Benjamin; Clemens R. Muller; Dean H. Hamer; Dennis L. Murphy Science, New Series, Volume 274, Issue 5292 (Nov. 29, 1996), 1527-1531. Stable URL: hhupflinks,jstor-org/siisici=0036-8075% 28 1996 1129%293%3A274%3A5292%3C1527%3A AOATWA%3E2.0,CO%3B2-C Your use of the ISTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at hutp:/wwww,jstor orglabout/terms.html. ISTOR’s Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. Each copy of any part of @ JSTOR transmission must contain the same copyright notice that appears on the sereen or printed page of such transmission, Science is published by American Association for the Advancement of Science, Please contact the publisher for further permissions regarding the use of this work. Publisher contact information may be obtained at bup:/wwwwjstor.org/journals/aaas hil. Science (©1996 American Association for the Advancement of S JSTOR and the JSTOR logo are trademarks of JSTOR, and are Registered in the U.S. Patent and Trademark Office. For more information on JSTOR contact jstor-info@umich edu, ©2003 JSTOR hupslwww jstor.org/ Fei Ape 25 14:45:37 2003Poe, cramosoms 88,81 (198, igang [kad pretis wore obtain talng oF eDNA fragments compreing the ara (OF andro 0 2089 for ye respec 4 Modolol, 1. Bondar, M. Mesalson, Proc. Nat ‘oss So USA. 80, 16781985) BC Lease an. G. Zier, The Gono of Drosophila mesnogster cadre Yor i952, FLL aaron 5. Pade ¥.G.Co 6, 1129 (196 Genet of ein es sy: widtyperetes to cur toga Organ Rod PAA sn genomic DNA wore std tom Pasa ec Yeh BY ar Tp Fh este Ionorin was ao used Ans bdeson te owice. Soman scanning nes were olan fromthe Goomieton sack contr 25, 6.8 Moana, AB Arbach A Conlon, AL. ‘oyna Roser, Gunes Da: 6, 681 (19) 2 Kaka, COB, 707 (899. eka eta 6p. 721 ML. Foro a, p73 Moga Rano. Parr. Corse Ma Gat a! 6, 5851065, ‘iota NP. Brown Dave, JC Een, S. Ego. Hig C.D Laren AP. Mahon Moyer 6; Poona JW Taman ©. Tuma Pols, Mt Vstanaon ana he Stomngton oc ‘Caner fr reagents, Gotsching, Sen Neu ond Pobori r ete adie othe ‘manure ® Gandunforhap th mogosnsya “DTorgerson or esetanco th romance ae ‘nema of the Pruret,Eerman, end Et Sorters or advice a suppor Furdos by Ne tora! tits of Peat Nona Carer neta (NH "gants POIGAST96. to. ANE). and AOVGITBE2 fo SMP), a Portia Felon ‘SiofomtnoFonas Natend Suissa toP.G)arcan NG ipanete Fordation for Canear esearch {raning Progam f tbe US-lpan Cooperstie Caner Comentoe (2% 28, 22, 23 September 1986; soup 1 Nowe 1996 Association of Anxiety-Related Traits with a Polymorphism in the Serotonin Transporter Gene Regulatory Re: Klaus-Peter Lesch,* Dietmar Bengel, Armin Heils, Sue Z. Sabol, Benjamin D. Greenberg, Susanne Petri, Jonathan Benjamin, Clemens R. Miller, Dean H. Hamer, Dennis L. Murphy Transporter-facilitated uptake of serotonin (S-hydroxytryptamine or 5-HT) has been Implicated in anxiety in humans and animal models and isthe site of action of widely used Uuptake-inhibiting antidepressant and antianxiety drugs. Human 5-HT transporter (5- HTT) gene transcription is modulated by a common polymorphism in its upstream Fegulatory region. The short variant of the polymorphism reduces the transcriptional efficiency of the 5-HTT gene promoter, resulting in decreased 5-HTT expression and 5-HT uptake in lymphoblasts, Association studies in twa independant samples totaling 508 individuals revealed that the 5-H T polymorphism accounts for 3 to 4 percent of total, variation and 7 to 9 percent of inherited variance in anxiety-related personality traits in individuals as well as sibships. Avnxiety-related traits are fundamental, en- luring, and continuously distributed dimen- sions of normal human personality (J-3). Although vin studies have indicated that inlividual variation in measuces of anxiery- related personality traits is 40 t0 60% her- itable (4), none of the relevant genes has yet been identified. Variance in personality traits, including those related to anxiety, is thought to he generated by a complex in- teraction of environmental and experiential FP Lasch A Hol ©: Pai Deparment ol Pann, Universty of Wozburg, Fuchaonstacse 15, 97080 Wirzbry, Geman, .Bongal 8D. Grantor, J Banain 0. L Murphy, Laneratery of Cina Sacre, Nora nt fen ‘a Heath, Notional eee of Hea, Berheea, MD eee, USA £51 Sil and. H Hamer, Laboratory tlhe, tna’ Cancer tut, Nona! het of Heath, Batada, Mb 20602 USA C.F Mil ett cf Humen Genes, Unies of ‘rsp, Ameena FOP Wrst, Germany To wham corespandance ous be adressed Ena ipbscieamacnrinergde factors with a number of gene products involving distinct brain systems such as midbrain raphe serotonin (5-HT) system, (4). Neurotransmission mediated by 5-HT contributes to many physiologic functions such as motor activity, food intake, sleep, and reproductive activity, a8 well as to co nition snd emotional stares including mood and anxiety (5). By regulating the magai- tude and duration of serotonergic responses, the S-HT transporter (5-HTT) is central t0 the fine-tuning of brain serotonergic neuto: transmission and of the peripheral actions of S-HT. In the brain, 5-HTT expression is particularly abundant in cortical and limbic ‘reas involved in emotional aspects of be- havior (5). The human 5-HTT is encoded bya single gene (SLC6A4) on chromosome 17ql2 (6-8). Although 3-HTT has long been suspected to play'a role in behavioral, and psychiatric disorders, previous stxles id. noe reveal any common, replicated SHIT gene sequence variation in either SCIENCE * VOL.274 + 29 NOVEMBER 1996 neuropsychiatric patients or healthy indi- viduals (9). Recently, we reported a polymorphism in the transcripcional conteol region upstream of the 5-HTT coding sequence (10). Initial ‘experiments demonstrated that the long and short variants of this S-HITT gene-linked polymorphic region (5-HTTLPR) had differ ‘ent transcriptional efficiencies when fused 10 a reporter gene and transfected into human, placental choriocarcinoma (JAR) cells (10). ‘The 5-HTTLPR is locates ~1 kb upstream, of the 5-HTT gene transcription initiation site and is composed of 16 repeat elements ‘The polymorphisin consists of a 44-base pair (bp) insertion oF deletion involving repent elements 6 to 8 (Fig. 1A). In the present study, polymerase chain reaction. (PCR)- based genotype analysis of 505 subjects re- vealed allele frequencies of 57% for the long (and 43% forthe shore (3) allele (11). The S-HTTLPR genotypes were distributed ac- cconling. to Hardy-Weinberg. equilibrium 532% If, 49% Us, and 19% sf Because appropriate cell models for human. serotonergic neurons do not exist and JAR cells are monozypotic forthe §- HTTLPR, we stalied S-HTT gene expression in hurnan Iymphoblastoid cell lines. Like 5-HT neurons and JAR cells, lymphoblasts constitutively ex- press functional 5-HTT and exhibit adeno- sine 3'5'-monophosphate (cAMP)- 0.0, NEO T soe (mean = SO} oo RE a Pessimism (P = 0.011), Fear of Uncertainty {P = 0.043), and Fatigabiliry (P = 0.009), but not Shyness. ‘These results with three different per sonality assessment scales show that the S-HTTLPR influences a constellation of traits related to anxiety. Across the three personality measures, the 5-HTT polymor- phism contributes a modest but replicable 3 to 4% of che total variance and 7 t0 9% ‘of the genetic variance. These percentages are based on estimates from twin. studies, using these and related measures, that have consistently demonstrated that ge- netic factors contribute 40 to 60% of the variance in Neuroticism, Harm Avoid- ance, and other anxiety-related personali- ty traits in large population samples (4). Population associations between a ge- netic marker and a phenotypic ait can arise either ftom population stratification ot from genetic transmission. Because sibling pairs ate by definition eehnically an racial ly homogeneous, any difference in trait scores between genetically disconlant sib- lings mast reflect true genetic transinission, ‘Accordingly, our study was designed to al low family-based as well as population- based measurements of gene-tait associa tions. The combined seudy population in- cluded 459 siblings from 210 independent families, of which 78 sib-pairs from 61 in- Table 2. Familal association between the 5: HITLPR and anwey-rated tats (27). Results for tha NEO factor of Nauroticism and tho estat: 60 TPO factor of Harm Avoidance are nT score Units (as in Table 1); those forthe 1GPF factor of Tension (4) are in Sten scoro uns tatich havea mean of 5.6 and SD of nthe norralve popula tion) For association soross podiraes. S Lis Genotype ‘agree Conscien- te Maximum tkethood estate of (score for S 1 Newroticim Extaversion Openness {BTS Core en sore for Linda across all fames|~2 nt. = ~2Pogtkeinood of data with Es n= 221) (OUtS-HTTLPR effect) = logikelnood of data wih (group L) 72 S342 120 S252 105 S72*129 4542119 435+ 11.6 SHTTLPA ofectl: ond P was calated by tok- ts 108 5782192 S122 122 581=123 4262118 405+ 195 ing—ZhL lobedstbsiedasay’ satstcatone sis 43 §062112 8232104 §592141 4092117 4212124 degree of freedom, For association vithin pedk- list sis (qoupS) 49 5742126 G29217 G612128 4212117 4102192 gees, S— Lis the mean of [score for S sb) ~ Ff ‘a of od 58. 2.0 (score fork. sb wth each nuclear lary: the S-L 20 a fa 4 ns 8601, conservativly corected forthe noninde. Pp Goze he fe re fs Bendre of sb-par om a sno tary: and TEE ‘was calculated bya two-sided est. grup Or S282111 6562105 6102101 s0S=97 478+ 100 ts ta] 5552110 S35=104 598+ 101 48> 102 426+ 108 Factor sis 52 S612113 S21=111 599*89 498=112 454*108 staigic lis+ sis (oupS) 183 5872110 8312108 597208 4912108 477 = 107 Newel —Fangon Hath F 42 36 14 1 0 ies avodance S-L 29 7s i rs ns P dos rs 8 ns ns ‘Across pedigrees test Total (n = 505) (f= 468 amly members, 7 unvlted nicl) An (group 163 5312115 5425106 594*116 4822107 4or109 SL St 06 26 tis Dar 852120 SAS=N2 SB2Z112 422113 412125 ~2NL BF 113 63 sis % 5692112 S222107 578x116 4582122 aaox116 P Sons 0.0008 ont is-+sis (group S) 942 S642 118 5302111 SA12113 4612116 448» 123 _ Wihinpodirees test m = 78 -pais) F ‘93 1a 13 40 10” S-L AB. o8 54 sou 34 ne re 22 rst 22 24 30 P 0.002 oy rs ‘04s re ces 0.022 0.004 SCIENCE + VOL.274 © 29 NOVEMBER 1996 1529dependent families had discordant (that is, Uf versus iso ss) 5-HTTLPR genotypes. It was fist necessary to analyze the association between 5-HTTLPR genotype and anxiet)~ related measures after correcting for the statistical nonindependence of family mem- bers resulting from factors unrelated 10 SHIT. Elston and colleagues have de- scribed a maximum likelihood method! for estimating quantitative trait associations that rakes into account polygenic inheri- tance (27), An aetoss-peigrees analysis of the major 5-HTTLPR-associated traits of Neuroticism (NEO-PL-R), Tension (16PF), and Harm Avoidance (TPQ) (Table 2) re” vealed that there was a significant associa sion for each trait with 5-HTTLPR geno- type, and that the effect sizes and signif cance levels were comparable to those ob- tained by population association analysis. In the 78 sib-pairsthae were discordant for the S-HTTTLPR, the average difference in Net- roticism scores between the Land S siblings was 4.6 T-score units (Table 2), which vas inlistinguishable from the 3.4 T-score dif- ference seen in all L andl S indivicuals. Despite the reduction in sample size, the clifference hetween the L and $ siblings as statistically significant, even after conserva tively correcting for the nonindependence of sib-pairs from the same family (17, 20, 26). Similar results were obeained for Ten” sion and Harm Avoidance; the scores of group S probands were significantly higher than those oftheir group L siblings, and the effect sizes wore similar to those obtained by population-based or across-pedigres analy ses (Table 2). These within-pedigrees 1e- sults demonstrate that the observed associ ations between 5-HTTLPR genotype and personality are the result of genetic trans: mission rather than population stratitica- tion, Overall, however, the associations fe- ported here represent only a small portion Of the genetic contribution to anxiety-relat- ced rats observed in this nonrandom pop- ulation sample. Considerable evidence indicates that in- creased serotonergic neurotransmission (which would be an evident consequence of the reduced 5-HT uptake capacity found in individuals with the short allele of the 5.HTT polymorphism) is anxiogenie in an- imal models as well as in humans (2, 3, 22, 23, 28). Atthe clinical level, reduced 5-HT. uptake or reduced inhibitor binding to 5-HTT has been one of the most consistent biological findings in individuals with de- presion and several ansiety disonlers (29) ‘Our findings chat individuals with che short S-HTTLPR allele and reduced 5-HTT function have greater anxiety-related per- sonaligy characteristics would at first seem to conflict with the fact that SRIs such as fluoxetine, which competitively inhibit 1530 5-H uptake, are therapeutic agents in anx- iety and depressive disorders (21-23). How- ever, the therapeutie effects of the SRIs have primarily been demonstrated in neue ropsychiatric patients who may have some Trimary S-HT or other ncurotransmitce Uystunetion that is ameliorated by the SRs, sshereas our findings are ina sample ofthe general population. The SRIs also have oth- et pharmacological properties that. may contre co ther therapeutic effets 30), The lifelong duration of the genetically chriven diferences in 5-HT uptake, incl ing possible influences during extly brain development (31), may also lead to differ ent efleets from those produced by SRI ‘ulministeation later in Ie “The associations reported here represent conly a small portion of the genetic contti- bution to anxiety-related personality tits. other genes were hyposhesize to contsb- ue similar gene dosige effects to anxiety, approximately 10 co 15 genes might be predicted tobe involved. Small, aitive, or interactive contributions of this sie have been four in studies of other quantitative traits in plants and vertebrates, including hhumans (17, 32). As other anxiety-related genes ate. identified, including perhaps Some with effects thae are lager than or fiweract with this polymorphism, ie might become posible to use this information to enhance individualized pharmacologic treatment of neuropsychintrie disorder, just as for other medical dsonders (17, 32). Whether this particular polymorphism con. tributes to the general tendency for individ uals who score higher on neuroticism oF ssety factors in diferent personality tests to be at high risk for analy or personal ity disorders ap well av depresion will re quire frther study (33) Ie likewise remains tobe seen whether therapeutic responses to serotonergic agents are influenced hy this polymorphism REFERENCES AND NOTES 1. A. Gay, Ta Neuronsychiogy of Anny: An ‘uty io the Functor of he Sept apace! ‘Sytem (Ono Sone, Hem Yr 180) CF Clrgar, Paya Dav 4 167 (1985) Bp fence in Hanno of ancrmat HS Fjeonel Ea Fran, London 157) pp. 181-158 2. CLR Genego Ach Gan, PSyChRIY 44, 573 987. 3, peyona Dv. 68508. 4G Lost, m,Pajce 44, 1285 (19895 A. Heat 6. F Goninger NG Narn, J Pars. Soe jena! 66, 7621904 F, Pome, Ms. Ov, VoSufin Somnes 264773 90d; CS Boe Iman ta Faye Aging 9 229 (188: NL Pe. tromneta Per Soe Pajcha! 88,0808 Si Uanoor ana J. Scherk Sconce 268, 2087 {i098 15, HT. Chan, M4, lar ©. Gara, Aharmaca! Tontot Metods 27, 209(7092 JG. Heke Syrao 17, 11968 P. Woo Sara a 5 Pertti, Aon. HY Aa. Se 600, # (1990 HG. Wistar, BL. Murphy, Oa Boo, Ea SCIENCE + VOL27# © 29 NOVEMBER 1996 ‘Advances in the Neweosaegy of Amity Deore: fey. New Yr 1906, KP Lesh ofa. Tana. 91, 67 (1968, P Leseh at 8 86 197 (954) 4 Geer Pak, KH, Hm. Gana. 6,077 1065, 5 Farameorty ctl, oc Nat Acad. Sth USA 0, 2542(1003, ICP tasch eal, Bet. Pychiany 37,215 (1005; ‘Aru et aA Moa. Ganot Nauropsyenee: Goro 67, 409106) D.D Balsa al, Pera Gare, 100 /1996)4. Opi at al Lancet ‘47. 73 (1908 0. Cater al, Noworere 7 "er 1906, ‘Hoe, Mawar 102, 247 (108) A Fete, haoctom 68,262 1098. ‘Bod to GNA ton and stale wos canes from heathy hanen sola Oigerucosie pment SHFTLPA and conespendeg {2's natboute postions 1416 0-139" (ays, S-GSCGTTGCOGCTOTGAATE) and -910 10 “659 (G01, 5 GAGSGACTOAGCTEGACANCC: ‘Glories HTT gore 5 tarkng risen ope ‘here wea to ganoale 204. o S2B.p hagas oR arpltesion as cardeutinatealveumoct ‘0 contig a 0 ng gore CRA 2.5 mt oaytonuscosces (SSTP7 dawa2dGTP ~ Die a't ng of soroe and entennas pans: 10 mht s-Help63) somM KCL | Send MgCl oat Vor Taq GN potmorso.Anreang was cae butet et Sterdoe,ensonat Fak mn, ae ‘nett o 9 tor 30 a or 2 oye than ot ay Cat nme 15,7 [19855 8. A Fara 2 Oka Joka mc ‘phar! 16,53 (934 Eosoh ar vive ransomed robs wth Iho gonotyas p=, fon ~ Shanes = 3) vse gown MFM 1640 speed wth 10% ‘Beatom caf soum at 37°C n 9 humid o> “pho 5% CO, Fox nducton ol HTT expos on, hrpheblsts weet wt 510200 gl foro 08 to 2 nM PRA sec own fr an ‘sor 24 nee {Tho human SHY gore 8° rogusery serene (brome sonnet dre HGS MTTBHG EMBL-Gordark accession ‘uber 27675} tat ws oats ton aan Senor Ieearyin ZAP eerese(Statagoe| ac des, {helong arent ofthe HTT ugaetorysoqsnce SHITP, bas pre 1440 #22 wit remot tho varcereteon maton te) was ged mo He rometafess lutrase We) experaon vecor [BGLS base Promega The shor vot oe SHrT gare prema HTTP, basopars ~ 1096 19122) wa gored by aang Pe short, Nr comtaning 404 bp PCR procuct Pe and losing t ma he Pat ote of ho SHTTP het anal ser bo Wagers fankod bythe Pst Sos etnostotcapostore 1388 and 1102 een moved eens and hen cor bound ‘ae veo by saquence naj Lary a short fuman ST uot conatust and cons we ‘ana exes in renobists wih arnt onotyos (3), and wer gone orrastn Woe flue ane oho pL Snae and pOLS cond ‘ecors Tancecbn afl wes aseasosby co ‘Tonlacton wit oS Bal orogt Fr arent ‘Serssen, ymhodats B70" cal) were ex Bowes for 34 urs 5 ya of conebuct DNA cs foe wih nf Tansee ioclcin reagent [Promaga! Simic APM Tea. Cate wor rom {eran atonal 2s hare tor hanvec 91 0 Nojeraee ye blr Brats wore say to ‘hears acy by ackton e 10 yo cl hese ‘Scat 15 iw to 100 rl of eso ge Chemimmnescanee wos courted ion 10 Borst time 903 ar reagent mated fenton spectrometer Tol RNA seated fom het 19) by (unc toeynate coun pieion (agen “285/185 bans of aboctral ANA wero ara edi dentomanytacorote vations nA Sorestaton, ana able stared CON (7°06 fig was syria wh rancor primer. ATT IRENA wes measured by seiquatsve compel18 a te emige rracrpten POR wth a SHIT cONA- ‘afted else contanrg a 72-8 dacen ase pats 16 fo 008) a8 lena andar. Tne POR Lmpltcaten 20s a 8°C, 503 818°C, # mr 72°C 85 oe of o6- 1827 bp raga as ara out mah the amines 303 -ATCON GMAGCOATAGCCAAGATG. base pare 1457 1 1459 wien spect tothe varereion ton at and ‘Ste (6 AGATOAGGTTCCTATOCAGTANG, Baio ple 2147 192167) SHIT mn concer tens of rchobst oo ies wth th genes iro re tetod agaraticraanalconceratons ‘fcompative ample rengrg rom O.0t 10 Ong, So cancaratn ef the compalive terete a {arottanelte cain was thn usod oom fare AWA coneentatone semcustishly Fmenobast cal ree ‘nth cient panty 13) ore nd aeringucom of ST gana ns. fon To conta! or difrences nthe etoency rovaca vareraon of MENA, we paerrod COMA She and subeoquort compte FOR Wc uplests. Tho feacon products wae do-wcrho. Tosod tyeugh 2% apres, vausaod by uve! Irenaten into presence of atm bem, snd quand by derstorene andj ter binding oe SHIT poten nts ss Dy eeveotng monbranes orm ele! Inver ‘at colle wth (IFTSS(OGSIo ANN ‘our st 3-0 a dens LD, Paamoarty et aL dot Chom 270, 17168 095], Nonspete ‘Bh wras dtomed nthe presan o 5 yht srr, ATS Beeentereitenn 28 Sao Ba poloty eis 5 HT yp sod ons {oBHTT wantin sent i) 1. W- Bop etal eyanine Rocptors and Traepores, 18 Neri Deer, Now York, 194), pp. BYT-844) Wo amnes SIs uptake by nosbalng = 10" fended enpetists wih 0 1 at PHT er Some at 25° te anenoe prance oft nM ipramne. {I Bonne at Nt Gane. 12,81 (906) {wo nacpandet groups ct preset} ab lege, eer ary mabe and raked naka. ‘wre sta.) The NAA sel (17a {utad tor be NH adel cologo causes by ‘ating forpare of eters and pata of atte fora soy of personaly tats enc cross “ho sempe conarted of 221 siecle har 98 were mam and 7 war fame, The srrsge ages 23.3 6 jer fang 1810 yeu, Pe ‘erage ecucatoral lvl wo 158 = 21 yours trang 20 ear) andre erage Kiroay Soe ‘wea0.2 = 07 parged to 56 hore Ol excuvey Feta an 8s excharvy Peoson. The Sine compen a 9.15 ute non Hepa, {0.0% sarc lender, & 9 Hip Ese Acan AmoscanBlac snd 27% ator Tae fam) suck beta sarpo wae 208 abn tom 108 omit and 3 wevstog ed ‘The NO srg (0H. Hamer al, Sonn 261 321 foaoy S. Hse a, hte Gant 11, 26 195] wat coli wom NI cis a ae tena henophie ogancatrs a acy a \Glarensten HW pest, and paycnoogea ata. Te sanpb conaeied of 784 subeos of ‘hon S2% were nei and 8% wee anal The ‘rage age was 37.897 yous fenge Taio 72 oar the evra odustend etwas 173 =28 eos yange 1216 20 yar) aretha wera Kae SEerewan 42 = 20 ange tad). The eric com ‘oston was 88.5% wrt caspni.9 2 PanciLatna, 07% Asean knercanck, 049% ate Arca Abskan, na. ober. The on Istucive offre Nc sample wae 95" als tom {stance Sparen and 26 urvasodindis BUR MeGrge and PT. 1. Cos, Porson ‘Aabihod ute, Now Yok, 100) Pia costa and RP. MoS, n Handbook of Parsonally vero, J. Cred an Me Devaruo, Ea enum, Now Yor res} A Hering, Poctophannsaogy The Foun Garoaten af Press, FE. Beem ard Da opt es Faye, Now York. 1005) p471:0.L sry eta m Seren, Pram Ca op) ‘Pranraciogy and Texaputcs Kuna Acad {Doro Netherlands, 1950, ep. 229-230 Barwa, H. Weal A Seago. ne Cin Psero pharmaco! po), 3 (1989 "Geb Aramco Ter 66, 3191995, SSL Nn 06, 03 (255) 2 campbte tbe a the SL derances and F “lie frat the NEO Nein fet, 122F Secondo and pinay factors, estnstad TFO ‘ects and TPO Ham Avorance eubeede 8 {rae om te authors by equ. PLB Cate, Adnan. Soe Pj, 38, 476 (1949) 18. Cat Tho Desapten ana Mnswe™ Iman of Persanaty (Word Book, Yerkars, NY, {doy J. S.Wopee and AL Pa, An Payoh 48, 473 (100) Popuaton asocatlon wee assostod by one-way nays ef vrs ANOVA wang SPSS Stata Softwar, VT Gearge and RC. tn. Gane. cea 4 180 (187 crss-peigiees assocton was =: Second ung the ASSOG prog ofa SAGE Pacsaga | C Seton, ¥.T George, A M, Sra IISAGE User Gus Aaesoe? 2 Depart lomeny and Gonatg, Leis State Urey adel Cantar, New Orang 104) 1. Nek et ft Gone, ac 2, 145 (909), thn-po (gave sesoeton wae deerinac oy 9p es (Bing genecaty dcordatsb-pare and wes con. Serta coreced fortwo renndependence oh Stings fan te sana tary (17 {'x\Bon Boa af are Cin. Paychaphamacat 9 (sree 0, 7 (908) Mt'Fana and C Nemarc Cin. Cham. 40, 288 (1988) Lye al. al Poy 96,281 {994} 6. Fa, Toes, Tortus, Payot fy News 1, 1001068) ‘Crue oa, Newraprarmacotgy 22,36 (1883 8 Discovering High-Affi Ru a (MBs. N. Masia JH. Potun, Pato ‘phanacoog/ 1071982) PB snd aot fy, ends Paanrace Se. 15.220 (5, E- (Cock seta. Newecharnsea! Newetouca! 5, 1745 938 Maen Many Nt Tash 8,43 (18 SSW. Sidr 1M La Toby AHL Patron ota, Gates 127,181 (9916. ‘Sear tl 192,823 (1002, Fo ‘Solace 26, 152 (185) RP Eben a a Me fe Gant 1.791805, Otro SR "Tamora, Metabaen 48 218996). Come et 1, Nate 358,64 (1082), EA ata a7, 51999; F Cain, cn Gara 46, Seysad-A Gardeneen etalon, 2795 (1938) RT. Muar, Joyce, C8. lange, Came. ena 36,225 (194) DM. Salsa, Ach Gan Pay 60,005 (19896, fecronset a ‘acts Deo 18,23 (100) eS. Kener ‘rch Gen. Pye 0, 8631908 A Wace tha PM. Mic. arcbook of Dawosson and ‘recy J Dan Boer ae tt Atco, Es (Ose fr Ne York, 1094) pp. T#-119: KS Kane et aL, eh Gan Payeniay 4,451 (987). Ctl tal Mol Pochany, eras. Wis thank MO Senad . Oeapa, and Jats for Teenne ssitnoo, IW, Cavs and Drake oo Foralaselstaoa snd Atami Mary, and A «late frogsteal suppor. Supoarea by te Deut ce Foctursecerenscrat he Buncosineo. ‘utr eungura Fees. fho European Com Iman. and th rarural Renan Programe of Inara NOL CPL eupporedy sa Horn {nd iy Shhg Foun, a 18 Juno 1006; soap 22 October 1006 ty Ligands for Proteins: SAR by NMR Suzanne B. Shuker, Philip J. Hajduk, Robert P. Meadows, ‘Stephen W. Fesik* ‘Anuclear magnetic resonance (NMR) based method is described in which small organic ‘molecules that bind to proximal subsites of a protein are identified, optimized, and linked together to produce high-affinity ligands. The approach is called "SAR by NMR” because structure-activty relationships (SAR) are obtained from NMR. With this technique, com- pounds with nanomolar affinities for the FK506 binding protein were rapialy discovered by tethering two ligands with micromolar affinities. The method reduces the amount of ‘chemical synthesis and time required for the discovery of high-affinity ligands and ‘appears particularly useful in target-directed drug research. ‘Digs are typically discovered by identify- ing active compounds from screening chemical libraries or natural products and ‘optitnizing their properties through the syn- thesis of structurally related analogs. This is a costly and time-consuming process. Suit- able compounds with the requisite potency, compound availability, or desited chemical and physical properties cannot always be found, Furthermore, even when such com- pounds are found, optimization often re- quires the synthesis of many analogs. ‘Prarracaical Diovan Oven, ASO Labor, ‘Aboot Par 60064, USA Towwhom corsepondonon ae be ERE, SCIENCE * VOL.274 + 29 NOVEMBER 1996 We now describe a method for ident ing high-affinity ligands that can aid in the dug. discovery process. The, technique, which iscalled “SAR by NMR." isa inked fragment approach wherein ligands ae con- seructed fom building blocks that have been optimized for binding 0 invividal protein suites (Fig 1)- Inthe ist step of this, proces, a library” of low molecular ‘weight compounds (1) is screene to iden- tify: molecules that bind to the prove. Binding s determined by the observation of "SN. of 'Hlamide chenseal shit changes in two-dimensional N-heteronaclear sngle- gquancum cortelation (U°N-HSQC) spectra (2) (Fig. 2) upon the adtion ofa liga ro 1531