Article (2002) Inhibition of Protein-protein

7/27/2019 Article (2002) Inhibition of Protein-protein

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Perspective

Inhibition of Protein-Protein Association by Small Molecules: Approaches and

Progress

Peter L. Toogood*

Department of M edi cinal Chemi stry, Pfizer Global Research an d Development, 2800 Plymouth Road,A n n A r b o r , M i ch ig a n 4 8 1 05

Received October 10, 2001

Scientific discovery consists in the interpretation for our own convenience of a system of existence,which has been made with no eye to our convenience at all.

sNorbert Wiener

Introduction

The a bility of proteins to a ssociat e rests a t the coreof biology. Cellular ar chitecture, informat ion tra nsfer,

and chemical specificity rely upon highly precise rec-ognit ion events; frequently these events involve theassembly of t wo or more proteins. The binding of t woproteins ma y occur with low or high a ffinity, but clearlyprotein association in cells does not occur in a ra ndom,disorganized way. Rather, protein associat ion is care-fu lly s cr ipt e d t o ac h ie ve s pe ci f ic g oa ls , be t h e y t h eassembly of part icular subcellular ar chitectures or t herelay of informat ion (signal t ra nsduction). P rotein-p ro t e in as s o c ia t io n e v e n t s may be re c o g n ize d in a l l

aspects of cell biochemistry. The mammalian immuneresponse relies in large part upon the recognit ion ofproteins and peptides by antibodies. Cell-cell recogni-

t i on a n d a t t a ch m en t t o t h e e xt r a c el lu la r m a t r i x i smediated by cell surface receptors (cadherins and inte-grins) that are ligated by protein partners such as actinan d f ibron e ct ion . S ig n al t ran s du c t ion f rom t h e ce llsurfa ce to the nucleus is frequently m ediat ed by one ormore protein-protein associations, e.g., Grb-2 bindingto p185erbB2 to recruit i ts downstream t arget S OS to themembra ne. Then, tra nscription itself is orchestra ted bya p le t h ora of t ran s c r ip t ion fac t ors , ac t iva t o rs , a n dsuppressors, whose assembly is poorly understood butclearly important. Given the ubiquitous nature of thesere la t io n s h ip s , an d t h e k n o wle dg e t h at in ap p ro p ria t eprotein-protein binding can lead t o disease, i t shouldnot be surprising that protein-protein interactions have

attracted the attention of scientists in the pharmaceuti-ca l i n du s t ry a n d e ls ew h e r e w h o a r e i n t er es t ed i nproducing inhibitors for use a s biochemical tools ortherapeutic agents. In deed, there a re a mple examplesin t h e l i t e ra t u re o f t h e u s e o f an t ibo die s , do min a n tnegative proteins, or medium-sized peptides to inhibitparticular protein-protein assemblies. In contrast, thediscovery of small drug-like molecules that can per-form a similar function has proven difficult.

A n u mbe r of s p ecia l ch al len g es are p res en t e d byta rget ing protein-protein binding in a drug discovery

Abbreviations: AcpYEEK, N-acetyl-O-phospho-tyrosyl-glutamyl-glutamyl-lysine; Apaf-1, apoptotic protease activating factor-1; bcl-2,B-cell leukemia/lymphoma 2; bFG F, ba sic fibroblast growth factor;BH 3, Bcl-2 homology doma in 3; CHO, chinese hamster ovar y; DH FR,

dihydrofolate reductase ; EG Fr, epidermal growth factor receptor;ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticu-lum; FKBP, FK506-binding protein; FRET, fluorescence resonanceenergy transfer; GA, geldanamycin; Grb2, Growth factor receptorbound protein 2; GST, gluta thione S-tra nsferase; HIV, huma n immu-nodeficiency virus; GH, growth hormone; HRP, horseradish peroxidase;IL, interleukin; LPS, lipopolysaccharide; MMP, matrix metallopro-teinase; MW, molecular weight ; NGF, nerve growth factor; NMR,nuclear magn etic resonan ce; NOx, nitric oxides; iNOS, inducible nitricoxide synthase; PARP, polyadenosylribose polymerase; PBMC, periph-eral blood mononuclear cells; SAR, structure-activity relationship;SH2, src homology domain-2; P I, phosphatidyl-inositol; S OS, son-of-sevenless; TNF, tumor necrosis factor; zVADfmk, N-benzyloxycarbonyl-valyl-ala nyl-aspa rtyl-fluoromethyl ketone..

* E-mail: P eter.t [email protected]. Tel: (734) 622 1335. F a x:(734) 622 1407.

Copyri ght 2002 by t he American Chemical Society

Volume 45, Number 8 April 11, 2002

10.1021/jm010468s CCC : $22.00 2002 American C hemical SocietyP ubl ish ed on Web 03/15/2002


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program, some of which may apply generally, and othersof wh ich a lmost certa inly do not. P rotein-protein bind-ing typically occurs over a relatively large surface area(a pproxima tely 800 A2 per protein on a vera ge); however,it is a common misconception that binding must com-prise many low aff inity interact ions rat her than a smallnumber of essential high a ffinity intera ctions.1,2 In fact ,in the association of huma n growth h ormone (hG H) withits receptor, only 8 of 31 buried hGH residues at the

binding int erface account for a pproxima tely 85%of thebinding energy.3 These eight residues a re ma tched bya complementary set of nine essential residues of 33buried residues on the receptor side.

The overall binding aff inity between two proteinsdepends on the funct ion of the protein complex. Forexample, obligate h omodimers usua lly associate st rongly(Kd ) 10-9-10-12 M), and their interfaces consist oflarge numbers of hydrophobic residues which disfavorthe monomeric sta te.2 An exception is th e DNA helicaseU v r D w i t h Kd ) 1.4 M. 4 Both interface surface areaand binding aff inity generally increase w ith increasingmolecular weight . In contrast , proteins that associateand dissociate in response to changes in their environ-

ment, which are likely to include the majority of signaltra nsduction mediat ors, tend to bind more weakly a ndexhibit correspondingly more hydr ophilic residues at t hebinding interfa ce. The different char acter of th e bindinginterfa ce is dictat ed by the req uirement for exposure oft h is p at c h t o s o lv e n t in t h e mo n o me ric s t a t e o f t h eprotein.

The relative affinity of protein-protein binding t endsto be overemphasized when discussing the potential ofs mal l molecu les t o in h ibi t s u ch in t erac t ion s . H ig haffinity binding itself has not presented a n insurmount-able obsta cle to the discovery of enzyme inhibit ors, andenzyme-l igand aff init ies span a similar range to pro-tein-protein binding a ffinities. This is tr ue even when

the ligan d itself is a nother proteins

a protease substratefor example. Furth ermore, even proteins tha t bind w itha re la t iv e ly h ig h a f f in i t y wi l l be ou t -comp et e d by acompar at ively wea k small molecule ligand, if tha t sma llmolecule is present at high enough concentra t ion.5

Another considerat ion is that , at least in some cases,the mere disruption of a binding equilibrium will besufficient to produce a significant biochemical effectwit hout th e need to completely inhibit protein-proteinbinding.

A more pragm at ic concern is th at the binding surfa cesbetween tw o proteins tend t o be relat ively f lat , lackingcrevices a nd pockets t ha t might provide snug binding

sites for small molecules. Although a structural char-ac t e r iza t ion of p rot e in h omodimers in dica t e s t h a t asignifican t degree of protrusion is observed a t someprotein interfaces, this situa tion is less likely to be trueat the int erface betw een nonobligatory heterodimers.2

Nonetheless, i t is clear that protein-protein bindinginterfaces var y w idely in na ture a nd some are likely topresent bet ter targets than others for drug discovery.

I n t h is re v ie w, I h av e a t t e mp t ed t o p res en t re ce n texamples of small molecule protein-protein bindinginhibitors (PPBIs) that demonstrate the progress thathas been made in the last t wo years towa rd the goal ofspecifica lly inhibiting protein-protein binding for thera-peutic adva nta ge using a sma ll molecule. I ha ve avoided

discussing peptide inhibitors 6 except in the context oft h e ir u s e as t e mplat e s fo r t h e de sig n o f re la t e d n o n -peptide molecules or where a part icular example servesto illustrat e importa nt approaches to inhibitor discoveryor d es ig n . F u r t h er m or e, I h a v e e nd ea v or ed n ot t odu plica t e t h e w ork o f C och ran t o w h os e re v ie w t h ereader is referred for a summary of work published priorto 1999.5 Finally, I have chosen to omit any discussionof integrin inhibitors; recent reviews of the extensiveliterature in this a rea ar e provided by Holzeman 7 a n dby Saman e n .8 In orga nizing this review, I have chosent o div ide i t in t o t h re e bro ad s e c t io n s , wh ic h I h a v eloosely entitled Inhibitors by Screening, Inhibitors byDesign, and Future Directions. This arbitrary division

h e lp e d me t o o rg an ize my t h o u g h t s a l t h o u g h i t h a sre su l t ed in t h e s e para t ion of s o me ac cou n t s t h a t a reclosely related, such as the many excellent approachesto the identification of SH2 inhibitors. Finally, I concedet h a t t h i s r e vi ew i s u n li ke ly t o b e com pr eh en s iv e,although I hope it is representa t ive. Where pert inentwork has been omit ted I welcome the opportunity toe xp an d my own k n owle dge of t h is e xci t in g f ield ofdiscovery.

Inhibitors by Screening

W h ile man y ap p ro ac h e s t o t h e dis c o v e ry o f s mal lmolecule ligan ds involve some element of screening, t hissect ion will describe recent work that has relied pre-dominantly on screening to identify inhibitors of theta rgeted protein-protein binding interact ion. Thesea pproa ches w ill be furth er sub-divided by the na tur e ofthe assay employed in the screening process.

Competitive Binding. The most direct meth od tha th as bee n e mploy e d for t h e iden t i f ica t ion of s mal lmolecule inhibitors of a protein-protein association isa competitive binding a ssa y in w hich one of the proteinpartn ers is labeled, typically with a ra dioisotope. Ones u ch e xamp le is t h e dis cov ery by O w alabi e t a l . o f asma ll, non-peptide inhibitor of the peptide nerve growt hfactor (NGF) binding to its r eceptor p75 and the kina seTrk A.9 NG F is essentia l for the survival of sympa thetic

and immat ure sensory neurons and increases the painresponse. Antagonists of NGF act ion are expected tobehave as nonantiinflammatory analgesics.

To identify NGF an ta gonists, mouse NG F w as la beledwit h iodin e-125 t o p rodu ce a re ag e nt t h a t cou ld bedetected using sta ndar d ra diometric methods. In bind-ing assays that employed either purified Trk A, or p75and Trk A expressing PC12 cells, low volume screeningof commercia lly a vaila ble compound librar ies identifiedN-{5-nitro-1H-benz[d e]isoquinoline-1,3(2H)-dione}-2-aminoethanol (ALE-0540; 1, Figure 1) as a competitivein h ibi t o r o f NG F bin din g wi t h an I C 50 ) 5.88 ( 1.87M for binding t o Trk A and an IC 50 ) 3.72 ( 1.3 Mfor binding t o p75 an d Trk A bearing P C12 cells. ALE -

Figure 1. Compound 1, ALE-0540; 2, TAK-779.

1544 J ou r n al of M ed i ci n al Ch em i st r y, 2002, V ol . 45, N o. 8 Per spect i ve


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0540 did not bind to NGF and wa s further shown t o beinactive at a selection of other receptors, e.g., 5HT-2A,endothelin, and opioid receptors. ALE-0540 was effectivein cell-based assays as indicated by the inhibition of TrkA (auto)phosphorylation and inhibition of neurite out-growth. In addition, ALE-0540 displayed antiallodynice ff ect s i n v iv o i n r a t m od el s of i nf la m m a t o r y a n dneuropathic pain.

A similar a p proac h wa s t a k en by B a ba e t a l . in t h e

discovery of antagonists of HIV-1 cell attachment.10 Thisgroup prepared CHO cells expressing the -chemokinereceptor CCR5, which is a target for HIV-1 and mediatesme mbran e fu sion a n d ce ll e n t ry by t h is re t rov iru s .H I V-1 bin din g t o CCR 5 is in h ibi t e d by t h e n at u ra lr ecep t or l ig a n d s w h i ch a r e l a r g e p ep t id es s u ch a sR ANTE S. B y e mploy in g a s cre en for 125I-RANTESbinding t o CHO/CC R5 cells, Ba ba et al. ident ified froma Takeda compound library a small molecule inhibitordubbed TAK-779 (2, F igure 1). This non-peptide inh ibi-tor completely prevented th e ass ociat ion of 125I-RANTESwit h CH O/CC R5 cells at a concentra tion of 100 nM w ithI C 50 ) 1.4 nM. The inh ibition w a s specific, a nd n o effectwas o bs e rv e d o n l ig an ds bin din g t o CCR 1, CCR 3, o r

CCR 4, a l t h o ug h TAK779 did in h ibi t t h e bin din g ofchemotact ic protein to CCR2b with an IC 50 ) 27 nM.TAK779 inhibit ed RANTES -indu ced Ca 2+ mobilizationin CCR5 expressing CHO cells but not CCR1 expressingC H O ce ll s. Th e g oa l of t h i s s t u d y w a s t o i de nt i fyinhibitors of HIV replication; as desired, TAK779 po-t e n t ly in h ibit e d t h e re pl ica t io n of H I V-1 in CCR 5expressing MAGI cells wit h a n IC 50 ) 1.2 nM. N o effectwa s observed on the replica tion of CC R4 specific HIV-1w i t h u p t o 2 0 M TAK779. HIV-2 replicat ion wa ssimilarly unaffected. The effect of TAK779 extended toth e inhibit ion of CC R5 specific clinical isola tes of HI V-1i n P B M C s.

Although the exa ct binding site for TAK779 on CC Rha s not been defined, it w as found to inhibit the bindingof only one of tw o monoclona l a ntibodies specific to thesecond extracellular loop, suggesting tha t t his loop ma yassociate directly with the inhibitor. Chemokine recep-tor antagonists, similar to other small molecule ligandsfor G -protein coupled receptors, a re genera lly believedto funct ion allosterically by binding to the receptorprotein tr an smembra ne region. A growing set of chemok-in e re c e p t o r an t ag o n is t s h a s be e n ide n t i f ie d , an d a l-though a sma ll number of representat ive examples areincluded in this review, they a re generally not regar dedas a uthent ic inhibitors of protein-protein binding.11-13

The groups of Boger and Cheresh recently collabo-

rated to discover compounds that disrupt the bindingof mat rix meta lloproteinase 2 (MMP2) to th e integrinRv3 as potentia l a ntia ngiogenics.14 Combinatorial mix-t u re s of comp ou n ds de sig n ed t o fa v or in h ibi t ion ofprotein-protein binding w ere screened using an E LIS Atype a ssay . To perform the screen, purified int egrin wa sad sorbed onto microtiter plat e wells a nd the MMP 2 waslabeled with biot in. Integrin-MMP2 associat ion couldthen be detected using a horseradish peroxidase (HRP )-linked ant i-biotin mAb and a peroxidase subst ra te. Thein it ia l in h ibi t ors iden t i f ie d u s in g t h is s cre en (e .g . ,A6B10C4; 3, Figur e 2) possessed a very high molecula rweight and were unsuitable for biochemical studies;however, further refinement of the structure with a view

to approa ching more drug-like molecules resulted in t hedesign of TSR1265 (4, Figu re 2). Although t his m oleculei s s t il l l a r ge b y ph a r m a ceu t ica l s t a n da r d s, i t w a s

suitable for demonstrating the principle that inhibitionof MMP2-Rv3 binding in cells can disrupt angiogen-esis.15

TSR1265 la beled w ith carbon-14 wa s demonstra tedto bind select ively to Rv3 bu t n o t R51 o r M M P2.Binding w as independent of l igand binding to the highaffinity RG D binding site, indicat ing tha t the tw o sitesare different and not allosterically linked. In a cell-basedas s ay , h ams t e r CS-1 me lan o ma c e l ls t h a t h ad be e nt ran s fe ct e d wi t h t h e h u man 3-integrin subunit wereprevented by TSR 1265 from degra ding collagen IV. Thiseffect was at tributed to the inability of these cells torecruit MMP2 when TSR1265 was present . This hy-pothesis was supported by data indicating that TSR1265

inhibits the binding of MMP2 to 3-integrin tra nsfectedcells. Significantly, TSR1265 did not prevent the deg-ra da tion of collagen I V by MMP2 a lone. In vivo studieswere performed using 10-day old chick chorioallantoicme mbran e (CAM ). I n t h is mode l s y s t em, TSR 1265almost completely abolished bFGF-st imulated angio-genesis despite levels of MMP2 activation and expres-sion equivalent to controls. Consistent with this result,the growth of Rv3-negative CS-1 tumors in chick CAMwas reduced following a single IV injection of TSR1265,and treated tumors displayed less surface vasculaturean d overa ll blood vessel density tha n untr eat ed tumors.The use of Rv3-negat ive tumors in this experimentsupports t he conclusion tha t the ant iangiogenic effect

arises through a specific effect on the vasculature rathertha n by direct inhibit ion of tumor growth .Enzyme Assay. I n i ns t a n ces w h er e on e of t h e

partners in a protein-protein recognition event is anenzyme, the act ivity of this enzyme may be altered byassociat ion with its protein partner. Examples of thissignaling mechanism a re not uncommon, alt hough theyare frequently triggered by an init ial catalyzed eventsuch as phosphorylation. When the result of protein-protein associat ion is a cha nge in enzyme activity, th enthe level of this a ct ivity can be used to provide a read-out of the extent to which protein-protein associationhas occurred. F or example, t he phenothia zine a nt ipsy-chotic t rifluoroperazin e (5, Figure 3) wa s found to inhibit

Figure 2. Compound 3, A6B10C4; 4, TSR1265.

Per spect i ve J ou r n al of M ed i ci n al Ch em i st r y, 2002, V ol . 45, N o. 8 1545


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(Ca2+

-Mg2+

)-ATP a se in a calcium /calm odulin-dependentm a n n er con s is t en t w i t h a n i n di r ect a c t ion on t h eenzyme through binding to calmodulin.16,17 The inhibi-tory effect of trifluoroperaz ine on the ATP a se (a nd otherdownstream proteins) could be overcome by excesscalmodulin.

For proteins that are catalytically active only in theirhomodimeric state, an inhibitor of dimerization may bede t e c t able as an in h ibi t o r o f t h e c a t a ly t ic ac t iv i t y . 18

P ar ticular care is required in t his case to employ controlexperiments to establish that inhibit ion is not of themore classical competit ive or noncompetit ive form.Scientists a t P ha rma copeia a nd B erlex ha ve successfullyused this approach to discover small molecule inhibitors

of inducible nitric oxide synth a se (iNOS),19

a n e n zy metha t is implicat ed in the pa thogenesis of inflamma toryand autoimmune diseases. The assay consisted of A-172cells tha t w ere induced to produce iNOS by the a dditionof interferon-, I L -, and TNF-R; NO production wa smeasured by standard methods 22 h following induc-t ion. Using this assay, workers screened an encodedsolid-phase library of pyrimidine imidazoles, a struc-tural class that was chosen based on prior knowledgeo f h e me l ig an ds t h at d is p lay s e le c t iv i t y fo r iNO S v sendothelial NOS. None of these compounds showeds ig n i f ic an t ac t iv i t y ag a in s t p ar t ia l ly p u ri f ie d h u maniNOS; however, in the cell-based assay 53 compoundswere identif ied tha t d isplayed >60%inhibition of NO

production. The most potent library inhibitor (6, Figure4) an d a related, less chemically react ive inhibitor (7,F ig ure 4) we re me as u red t o h a v e I C 50 ) 1.1 n M an d0.6 nM, respectively. Control experiments indicated thatthese compounds did not alter iNOS transcript ion ortranslation and that the potency of inhibition increasedwit h t ime fo llowin g in duct ion of iNOS (t1/2 8 h).Subsequent electrophoresis a nd size-exclusion chroma-tography experiments demonstra ted tha t t hese inhibi-t o rs p re v en t d imeriza t ion of t h e iNOS mo n ome rs .Compound 7 bound to iNOS monomers with Ki ) 2.2nM but was inact ive against dimeric iNOS in enzymeactivity assays. This compound also was effect ive invivo, suppressing LP S-st imulated plasma NOx produc-

t ion in a dose-dependent manner in rats with ED 50 )1.2 mg/kg. Further chemical modificat ion led t o im-proved selectivity for iNOS vs neurona l an d endothelialNOS.

Furth er structura l insight into this inhibition of iNOSwas derived from a crystal structure of an inhibitor-iNOS complex at 2.25 r esolut ion. This structurerevealed that the inhibitor bound via its imidazole ringto th e sixth coordin a tion position on the heme iron. This

coordination appears to allosterically inhibit protein-protein intera ct ions between monomers by disruptinghelix 7a leading to partial disorder in helix 8, which ispart of the dimer interface.

Fluorometry. The a bility of certa in molecules tofluoresce wh en irra diated with light ma y be exploitedin a v ar ie t y o f way s in t h e de v e lo p me n t o f b in din gas s ay s .20,21 F or e xa m p le , t h e a c t of s eq u es t er i ng afluorophore from solvent upon binding t o a protein ca nresult in an increase in the emitted light. This techniquecan be used to observe the binding of small moleculesbut is genera lly ra ther insensitive. The proximity of tw oproteins can be observed by exploiting a phenomenonknown as f luorescence resonance energy transfer or

FRE T. A fluorescence output from t he d etector fluoro-phore is only observed when it is sufficiently close tothe donor fluorophore to absorb the light that it emits.Another elegant use of f luorescence employs probemolecules to observe and measure binding events byt a k i ng a d v a n t a g e of t h e s low e r t u m bl in g of l a r g ermolecules or molecular complexes. When a fluorescentcompound is irradiated with polarized light, the extentof polar izat ion of the emit ted light is a funct ion of thetumbling rate of the molecule. Rapid tumbling leads toinhomogeneous fluorescence emission, causing a low ers i g n a l t o b e m e a s u r e d a t a d e t e c t o r , w h i c h b e a r s ap olar izer corre s po ndin g t o t h e i rradia t e d l ig h t . I ncontr ast , a m olecule tha t tu mbles more slowly will emita higher proport ion of i ts signal in the same plane ast h e i rradia t e d l ig h t , leadin g t o a h ig he r s ig na l a t t h edetector. This very sensit ive technique can be w idelya p pl ie d t o t h e ob se rv a t i on of b in d in g t h r ou g h t h eemployment of a probe f luorescent l igand that can bedisp lac ed in t o s olu t ion by e xp erimen t a l l ig an ds orpartn er proteins.

Tw o examples of t he use of fluorescence polariza tionto examin e inhibition of protein-protein binding comefrom studies of the apoptosis regulat ing Bcl-2 familyproteins.22 Apoptosis is an energy requiring process ofcellular suicide.23,24 W h ile o u r u n de rs t an din g o f t h eevents involved in apoptosis is still growing, it is clear

tha t the B cl-2 family of proteins play a crit ical role inre lay in g s ign als t o t h e mit och on drion wh e re t h e s esignals a re a ggregated. P ro-apoptotic signa ls lead t o lossof the mitochondrial membrane potential and releaseof cytochrome c w h i ch r es u lt s i n a c t iv a t i on of t h ecaspa se-3/caspa se-9 cas cad e of protea ses. The preciseme ch an is m of ac t ion of B c l-2 p rot e in s is n ot fu llyunderstood; however, i t a ppears t hat certa in memberspromote cell death whereas others inhibit cell death,a nd t he bala nce of these tendencies is controlled by thedistribution of heterodimers a nd h omodimers of var iousfamily members. NMR studies and site-directed mu-ta genesis ha ve implicat ed conserved protein doma insknown as B H3 domains as m ediators of protein-protein

Figure 3. Trifluoroperazine.

Figure 4. Inhibitors of iNOS.



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as s ocia t ion in t h e B c l-2 fa mily , a n d s h ort p ep t ide sencompassing the BH3 domains of pro-apoptotic Bcl-2family members can trigger apoptosis in cells.

H y p ot h e s iz in g t h at s mal l mo le cu le mimics of B H 3

domains migh t simila rly induce a poptosis by inhibitingthe association of anti-apoptotic Bcl-2 proteins with pro-apoptotic Bcl-2 proteins, Degterev and co-workers setout to identify such m olecules by screening a libra ry of16 320 compound s.25 A fluorescence polarization assaywa s e mploy e d in wh ich a f lu ore sce n t ly labe le d B H 3domain from B ak (a Bcl-2 family protein) wa s used a sthe reference ligand for binding to a recombinant GST-Bcl-xL fusion protein. Three compounds with micromolaraffinit y for B cl-xL (BH3I-1, BH3I-1, and B H3I-2; 8-10,Figure 5) were selected from the screen for furtheranalysis. To control for binding to the fluorescent tag,all thr ee inhibitors were eva luat ed using unlabeledB H 3peptide to measure their association with immobilized

Bcl-xL on a surfa ce-enha nced la ser desorption/ionizat ion(SELDI) chip. Association of the BH3 peptide and Bcl-xL was o bs e rv e d by mas s s p e c t ro me t ry . B in din g wasinhibited by all three BH3 inhibitor (BH3I) molecules.These compounds a lso inhibited th e B H3 peptide bind-ing to B cl-2 (a nother B cl-2 family protein) a nd preventedtruncated Bid from associat ing with (His-tagged) Bcl-xL immobilized on Ni2+ bea ds . F in a l ly, Ki values forB H 3I s bin din g t o B c l-xL we re de t e rmin e d by NM Rtitra t ion. Thus four different t echniques w ere used todemonstrate binding of these small molecules to variousBcl-2 family members. These same techniques wereused to demonstrat e the select ivity of these BH 3Is forB cl-2 family proteins versus a va riety of other protein-

protein interactions including splicing factors U2AF35

/U2AF 65, Apaf-1/CARD , a nd CI DE -B /DF F40. This la stexperiment un derscores a part icular obsta cle to t hediscovery of specific inhibit ors of protein-protein bind-ing; it is genera lly difficult t o define a ppropria te contr olsfor nonspecific effects. More st ructura l da ta on proteininterfa ces an d physiologica lly relevan t protein part ner-ing w ill be required before sequence dat a, or better st ill,three-dimensiona l structura l da ta , w ill be sear chable forpotential recognition sites that might compete with theintended target .

The described BH3I molecules induced apoptosis inJ K cells as indicated by TUNEL staining, the appear-an c e o f an n e x in V, D NA frag me n t a t ion , an d f low cy -

tometry. C ytochrome cwa s released from the mitochon-drion, a lthough the mitochondrial membra ne potentialdid n ot ap p are n t ly de cre as e. Cas p as e ac t iva t io n oc-cu r r ed w i t h ca s p a s e-9 a c t iv it y i n cr e a s in g p r ior t ocaspase-8 activity, consistent with apoptosis triggeredthrough the mitochondrial path wa y (as distinct from theFAS pathway). The cytotoxicity of BH3Is followed thesame order a s th eir binding aff inity for Bcl-xL in vitro.

To examine whether BH3Is disrupt the heterodimer-

izat ion of Bcl-2 family members in cells , the authorsused an intr acellular FRE T assa y. HE K-293 cells werecotra nsfected w ith yellow-fluorescent protein (YFP )-fused Bax and cyan fluorescent protein (CFP)-fused Bcl-xL . The extent of FRET betw een t he tw o proteins w asdetermined a s th e ra tio between the fluorescence at 527nm a nd t he fluorescence a t 475 nm followin g excita tionat 433 nm. Cotransfect ion of the two tagged proteinsincreased t his ra t io to 1.7 compar ed to 1.0 for the t woproteins expressed separa tely, indicat ing th at the pro-teins a re in close proximity in the cell (probably a ssoci-ated). The BH3Is all reduced this ratio, with the extentof reduction correla ting to the potency of the compoundsin cytotoxicity and binding assays. A time course showed

that loss of FRET preceded cell death.C om pa r i s on of t h e B H 3 Is a n d B a k B H 3 p ep t id e

binding to Bcl-xL was performed using NMR. A 2-D 15N/1H heteronuclear single-qua ntum correlat ion (HSQC)spectru m of 15N-labeled Bcl-xL in th e presence of B H3peptide showed that peptide binding primarily affectedresidues at the BH 1-B H3 h ydrophobic cleft , consistentwit h the known str ucture of this complex. Similar ly, allthree BH 3Is induced significan t changes a t t he surfaceof Bcl-xL in th e hydrophobic cleft formed by the B H1,B H 2 , a n d B H 3 d om a i n s, a l t h ou g h d i f fer en t B H 3 I saffected slight ly different residue sets on the Bcl-x Lprotein. Nuclear Overhauser effects between BH3I-1and residues Tyr-65 a nd P he-107 of B cl-xL furtherpinpointed the binding locus for this inhibitor. Interest-ingly, Phe-107 is buried in the structure of free Bcl-x Land is inaccessible to solvent ; however, this residuebecomes surfa ce-exposed upon B H3 peptide binding,mak in g c o n t ac t s wi t h a le u c in e re s idu e in t h e B H 3peptide. The NMR dat a suggest tha t B cl-xL undergoesa similar conformational change upon binding of thesmall molecule inhibitor BH3I-1.

O v eral l , t h e e viden ce p rov ide d by D e g t ere v e t a l .s u pp ort s t h e h y p ot h e s is t h a t t h e ir B H 3I s a c t a s B H 3mimics, inhibiting heterodimerization between Bcl-x Lor B cl-2 a nd pro-apoptot ic fam ily members, th erebyreleasing these pro-apoptot ic members of t he B cl-2

protein family t o promote cell death.26

In related w ork,Tzu n g an d co-wo rke rs de mon s t ra t e d t h at a s imila rmechanism of action may also hold for the inhibitor ofelectron tra nsfer complex III known a s a ntimycin A (11,Figure 5).27 Wh ile i t i s d i ff icu l t t o k n ow wh e t h e rprecisely this mechanism operates in cells, these resultsdemonstrate the potential of small organic moleculesto inhibit importa nt protein-protein interactions in acellular environment . In addit ion, they highlight theimporta nce of B cl-2 fam ily protein-protein associationevents in th e regulat ion of cell homeosta sis. The bala ncethat exists between a pro-apoptot ic state and an ant i-apoptot ic state appears to be controlled by the relat ivelevels of multiple heterodimeric complexes, a nd it ma y

Figure 5. Compound 8, BH3l-1; 9, BH3l-1; 10, B H3l-2; 11,Antimycin A.



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be t h at complete(100%) inhibit ion of a ny one complexis unnecessary to shift t he equilibrium significan t ly inone direction or the other.

Virtual Screening. Ra pid developments in comput-

ing power and the increasing incorporation of compu-t a t ion al me t h ods in t o ch e mis t ry a n d biolog y made i tinevita ble tha t sma ll molecule screening w ould becomea virtual experiment . An illustrat ive example of thisparsimonious approach to l igand identif icat ion is pro-vided in the w ork of Wa ng a nd co-w orkers who a lso wereconcerned w ith inhibitors of Bcl-2 function.28 Wa ng usedan homology model for Bcl-2, derived from the solutionstru cture of B cl-xL . A t otal of 193 383 compounds w erescreened in silico using the progra m D OCK 3.5 to scores h ap e co mpleme n t ar i t y for e ach v ir t u al c omp ou n dbound to the B cl-2 model in a var iety of conforma tions.The 1000 best results were optimized using SYBYL6.2 t o as s ig n an a p p ro p ria t e g e o me t ry a n d t h e n t h e

binding energy for each was calculated. The 53 bestcompounds then were selected manually using bindinge n e rg y , s h ap e c o mp le me n t a r i t y , a n d h y dro g e n bo n dforming potentia l as criteria for t he selection. A tota l of28 of these compounds w ere ava ilable commercia lly, an dthese were obtained for test ing. Binding to Bcl-2 wasmeasured using a f luorescence polarizat ion assay inwhich the reference ligand was a 5-carboxyfluoresceinlabeled peptide derived from Bak with KD 0.20 Mfor binding to B cl-2. A compound dubbed HA14-1 (12,Figur e 6) proved to be th e best ligan d for Bcl-2 with IC 50 9 M. At 50 M, H A14-1 caused cell dea th in >90%of cultured HL60 human leukemia cells. Cell death w a saccompanied by DNA fragmentat ion, consistent with

apoptosis, a nd t his fra gmentat ion w as completely pre-vented by the caspase inhibitor zVADfmk. Addit iona levidence for apoptotic cell death included cleavage ofcaspase-9 and caspase-3, PARP cleavage, and decreasein t h e mit och on dria l me mbra n e p ot e n t ia l . H o we ve r ,HA14-1-induced a poptosis wa s not inhibited by Cr mA,indict ing t hat HA14-1 signaled via the B cl-2 pathw ayand not the FAS pathway. Bcl-2 inhibits Apaf-1 activa-tion of procaspase-9 cleavage and cyctochrome creleasefrom the mitochondrion, whereas FAS-mediated apop-tosis does not req uire Apa f-1. It w a s found th a t H A14-1ha d litt le effect on Apaf-1 deficient cells but w a s a potentinducer of a poptosis in Apaf-1 posit ive cells . In ag-gregate these results support the h ypothesis th at HA14-1

induces apoptosis by inh ibiting t he function of Bcl-2 aswould be expected for a compound tha t inh ibited B cl-2heterodimeriza tion w ith pro-a poptotic protein par tners.

It is interesting to note that the ant imitotic an titumordrug paclitaxel (Taxol; 13, F i g u r e 6 ) a l s o h a s b e e ndemonstra ted t o bind to Bcl-2.29 The sign ifican ce of th isobservation is not clear, although paclitaxel is a potentinducer of apoptosis. This effect ma y be at lea st pa rtia llymediated by the inhibit ion of Bcl-2 binding to other

Bcl-2 family members.Phenotypic Assays. Wh e n a p ar t icu la r ce llu la r

behavior or phenotype is anticipated to result from theinhibit ion of a specific protein-protein int eraction, thenthis phenotype can be used t o provide the rea d-out in ascreen for inh ibitors. While a risk of fa lse positives dueto alternative mechanisms is inherent to this approach,these fa lse positives can r eadily be removed by use of amore specific secondary assay. Meanwhile, the pheno-type-based screen possesses the benefit of identifyingas h i t s comp ou n ds t h at (a) are ce ll-p erme a ble, (b)produce the desired effect on living cells, and (c) if theywork via the intended mechanism are sufficiently potentand specific to work in the cellular milieu. Haggarty et

a l. used a cell-ba sed screen t o identify compounds t ha tinhibit mitosis.30 Reasoning that the majority of com-p o u n d s t h a t a r e k n o w n t o i n h i b i t m i t o s i s d o s o b yinterfering with the polymerization of R a n d tubulin,t h e ir s e c o n dary as s ay was fo r in h ibi t io n o f in v i t rotubulin polymerizat ion. An an tibody (TG -3) ag a inst t hephosphorylat ed form of nucleolin t ha t is expressedspecifically by cells in mitosis was used to determinethe extent to w hich A549 lung epithelial cells w ereblocked in mitosis by 16 320 diverse compounds from acommercially available library (Chembridge, Diverse Eset). This screen identified 139 compounds tha t in-creased the a mount of phosphorylated n ucleolin by atleast 2.5-fold. Of these 139 compounds, 52 destabilizedmicrotubules but had no effect on the polymerizat iono f ac t in ( e .g . , Sy n s t ab, 14, F ig u re 6) , s imila r t o t h eknown a nt imitotics colchicine an d nocoda zole. Alth oughno detailed mechanist ic studies were reported, thesecompounds w ere proposed to inhibit microtubule forma -t ion by preventing t he a ssociat ion of R a n d tubulin.

A fo rm o f p h e n o t y p ic as s ay was u s e d re c e n t ly bySh arma e t a l .31 The phenotype in th is example w a s cellgrowth. Yeast cells overexpressing the tyrosine kinasev-src under control of the Gal-I promoter grow poorlyin culture on glucose medium. Allevia tion of this grow tharrest by a compound provided in the form of a soakedfilter-paper disk was visible in the form of a halo of

g r ow i n g y ea s t a r o un d t h e d i sk . A t ot a l of 20 000compounds were screened using this technique whichidentified several known src-kinase inhibitors and a newcompound (15, Figure 6), which was named UCS15A.This new compound decreased the levels of phosphory-lat ed v-src substra tes in tr an sformed NIH cells, includ-ing cortact in and Sam68 even though the expressionlevels of these proteins w ere una ltered. However, t hekinase a ct ivity of v-src aga inst a Cdc2 peptide in vitrowas unaffected by UCS15A. In addit ion, UCS15A wasde mon s t ra t e d n ot t o in h ibit t h e ac t iva t io n of v -s rcthr ough phosphoryla tion a t ty rosine-416 or to desta blizethe v-src protein. It w as hypothesized that UC S15A ma yprevent the association of v-src with it s substra tes, an d

Figure 6. Compound 12, HA14-1; 13, Taxol; 14, S ynst a b A;15, U CS15A.



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this hypothesis was supported by immunoprecipitationstudies in wh ich U CS 15A prevented t he coprecipitat ionof Sam68 and an unknown 64 KDa protein (pp64) in adose-dependent manner. UCS15A inhibited bone re-sorption by osteoclast-like multinucleated cells and bythe mouse calvaria organ in culture without significant

cytotoxicity. The molecula r m echa nism of U CS 15A hasy e t t o be de lin ea t e d; h owe v er , p rel imin a ry s t u die simplicate the SH3 module as a potential target. Inspec-tion of the molecular structure of UC S15A suggests tha tit could form a covalent a tta chment t o its ta rget thr oughany of several potential alkylat ing groups, part icularlythe epoxide ring.

Another form of phenotypic assay is called the yeasttw o-hybrid assa y (Figure 7a). This t echnique, originallydescribed by Fields an d S ong,32 detects protein-proteinbinding through the expression of a reporter gene whosetranscription is activated by a heterodimeric transcrip-t ion fac t or . Two fu sion p rot e in s a re re q u ire d, e ac hcomprising one of the transcript ion factor monomers

fused to one of two par tner proteins. When th e part nerp rot e in s bin d t o e a ch ot h e r , t h e ac t ive t ra n s cr ipt ionfactor heterodimer is formed. If th e tw o partner proteinsdo not norma lly associate w ith each other, they may beinduced to do so by a heterodimeric ligand comprisingone ligan d for each protein tethered to each other via al in k er (F ig ure 7b). Th is la t t e r t e ch n iq u e h as bee nreferred to as a three-hybrid system a nd is a n exampleof chemica l-induced dimeriza tion (CID , see below ).33,34

Alternat ively, the partner proteins may be inhibitedfro m as s o c ia t in g by a PPB I ( F ig u re 7c ) . I f a g ro wt hcr i t ica l g en e is p la c ed u n der t h e re gu la t ion of t h et ran s c r ip t ion fa c t or , t h e n t h e P P B I wi l l c a u s e g rowt hinhibit ion. For example, in one report , a yeast strain

was used that requires hist idine to be supplied in theg rowt h me diu m u n les s t h e re port e r g en e H I S 3 isexpressed. 35 Thus, protein-protein binding was indi-cated by the a bility of the yeast t o grow in the a bsenceo f e x o g e n o u s h is t id in e . I n t h is c as e , a n in h ibi t o r o fprotein association resulted in the phenotype, inabilityto grow on histidine-deficient medium.

Huang and Schreiber turned this concept around anddeveloped an assay for PPBIs in which the phenotype

indicating successful inhibition was survival not death.36

The DNA-binding protein lexA and activation domainB42 were separately fused to either FKBP12 or to theR 1 s u bu n it of t ran s formin g g ro wt h fac t or- t y p e Ireceptor. In the absence of a PPBI, LexA bound to itsD N A b i nd in g s it e , a n d B 4 2 w a s b r ou g ht i n t o cl os eproximity as a result of the a ssociat ion between FKB P 12and R1 result ing in transcript ion of a U R A 3 reportergene. When th e ura 3 protein w a s expressed in cells, theywere rendered sensitive t o th e pro-toxin 5-fluorooroticacid (5-FOA). The FKBP12 ligand, FK506, inhibitedFKBP binding to R1 and thus blocked the delivery ofB42 t o the LexA DNA complex. The protein ura 3 w asnot expressed, an d t he cells w ere insensitive to 5-FOA.

In principle, with some at tent ion to the molecularbiology, this reverse two-hybrid sy stem ma y be appliedto any protein-protein binding event to discover newPPBIs. Indeed, by random generation and coexpressionof fusion proteins in distinct yeast colonies, it may bepossible to simultaneously select for protein-proteinbinding pairs a nd identify sma ll molecules th at inhibitthem. As yet, the full potential of the reverse two-hybridsystem does not a ppear to ha ve been realized.

Inhibitors by Design

Peptide-Derived Ligands. A common approach tothe design of PPBIs is to dissect the binding locus of

one of th e proteins a nd to prepar e trun cated peptidest h at con t a in t h e e s se n t ia l re sidu es f rom t h e bindin gepitope. F or a skilled m olecular biologist or peptidechemist this approach may well provide the fastest routet o a c o mp o u n d t h at c an be u s e d t o t e s t t h e re s u l t o fin t er fer in g wi t h a s pe ci f ic p rot e in-protein bindingevent. Fr om a phar ma ceutical sta ndpoint, however, thisapproach is generally unsa t isfactory because t he pep-tides themselves ar e unlikely to make useful drugs dueto poor bioavailability , and the path from peptides tosmall molecule peptide mimetics is fraught with dif-f ic u l t ie s . D e s p i t e t h e s e c av e at s , t h e re a re ma n y e x -a mples of effective peptide inhibit ors of protein-proteinbinding.5,6 For the purposes of this review, we will focus

only on examples where the initial peptide inhibitor leadhas been further adapted toward a non-peptide drug-like molecule.

Sr c-homology 2 (SH 2) domain s offer importa nt ta rgetsfor P PB I s du e t o t h e ir import an c e in re lay in g s ig n alsbetween cell surface receptors and cytosolic proteins.The SH 2 doma in media tes th e association of src-fam ilykinases with downstream signaling proteins by bindingto phosphorylat ed tyrosine residues (pTyr) in thesedownstream proteins. Many peptides and peptide mi-metic inhibitors of SH2 are known, and these may beused as a basis for the design of non-peptide ligands. 37

The protein p56lck is a SH2 domain-containing tyrosinekinase, which is involved in T-cell activation. Inhibitors

Figure 7. (a) Two-hybrid system: detects a ssociat ion betw eenproteins A and B. (b) Three-hybrid system. The tethered dimerx-y b ri dg es A a n d B , b ri ng in g t h e t w o s u bu n it s of t h etranscription factor into association. I f x is a know n l igandfor A, the three-hybrid system can be used to identify proteint ar get s for l igand y. (c) Reverse tw o-hybrid syst em: detectsinhibitors of A-B association.



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of p56lck have the potential to block the intera ct ion oft h is p ro t e in wi t h t h e T -c e l l re c e p t o r an d de ra i l t h esigna ling ca scade tha t leads t o cytokine production andcell proliferation. Starting with the peptide inhibitor Ac-pYEE I (Kd ) 0.1 M), wh ich bear s five negative chargesat p h y siolog ica l p H , B o eh rin g er I n g elh eim wo rke rsdesigned a singly charged peptide mimetic which is aweaker inhibitor of the p56lck SH 2 doma in (Kd ) 1 M)but is cell permea ble (16, Figure 8).38 The steps involvedin this t ran sforma tion included replacement of the t woC-terminal a mino acids with a p-methoxybenzyl group,effectively removing two charges at essentially no costin term s of potency. The phosphat e group wa s replacedby a carboxylic acid, removing one more charge, but alsodecreasing the binding affinity by 200- to 500-fold. Theintroduction of lipophilic substituents recovered some

of this lost potency. Fina lly, removal of the last glutam at eresidue and introduction of a conformation-constrainingbridge between tw o adjacent r esidues ga ve compound16 which displayed some permeability across a Caco-2membrane in vitro (21%permeated in 3 h) and boundto p56lck w i t h Kd ) 1 ( 0.2 M. Compound 16 inhibitedcalcium release in J urkat cells following exposure toT-cell receptor cross-linking antibodies with EC 50 ) 10M.

The SH2 domain bearing adapter protein Grb2 hasbeen a ta rget for severa l groups interested in disruptingmitogenic signaling.39 I n t h i s c a s e , t h e m i n i m a l s e -quence necessary for achieving micromolar bindingaffinity to th e Gr b2-SH 2 doma in is the relat ively short

tripeptide pTyr-Ile-Asn. G uided by st ructura l informa -t ion on the SH2 domain of Grb2, workers at Novart isin corp orat e d s ev era l modifica t io n s in t o t h is s impleligand and arrived at 3-benzyloxycarbonylamino-pTyr-1-aminocyclohexane carboxylic acid-Asn-NH 2 which isa potent ant agonist of the G rb2-SH 2 doma in with IC 50) 1 nM as mea sured in an E LISA.40 Further structuralmodifica tions w ere ma de, less to improve potency41 t h a nt o d ecr ea s e t h e p ep t id ic n a t u r e of t h is l ig a n d . F ore xamp le, re place men t of t h e a s p ara g in e re s idu e by(1S,2R)-2-amino-cyclohex-3-ene carboxylic acid provideda l ig an d (17, Figure 8) containing 3 nonproteinogenica m i n o a c id s a n d d is pl a y in g a n I C 50 ) 1.6 n M forinhibition of phospho-EGFr (intracellular domain) bind-

in g t o G rb2-SH 2.42 R em a r k a b ly , t h e i nh i bi t or w a sspecific and did not bind to the r elated sr c-family kinasep56 lck at concentra t ions up to 10 M.

B u ildin g o n t h e wo rk o f F u re t , 39-43 B u rk e a n d c o -wo rk ers h av e u s ed a s imilar t r ipe pt ide s c a f fold t oevalua te bioisosteres of the phosphotyrosine residue.44,45

Guided by molecular modeling, this group discoveredt h a t NR-oxalyl p-(2-malonyl)phenylalanine is able tomimic both the charge st at e and the size of a phospho-tyrosine residue. While the dicarboxylate mimics theparent phosphate group, the oxalat e a ppears to conferaddit ional potency for the Grb2-SH2 domain throughintera ctions w ith Arg-67. This phospha ta se-insensitivephosphotyrosine replacement possesses t he a dded ben-efit that it does not significantly inhibit compounds fromcrossing cell membranes. Thus, compound 18 (MW )

763, Figure 8) is a potent inhibitor of Grb2-SH2 bindingin vitro (IC 50 ) 50 nM) and blocks the associat ion ofc-Met (the hepatocyte growth factor receptor) with Grb2in Oka jima cells by 50%a t a concentra tion of 30 nM.46

Time course experiments indicated t ha t optima l inhibi-t ion was achieved after approximately 8 h suggest ingt h at t h e in h ibi t or e n t ers ce lls by p as s iv e di f fu sion .Replacement of the na phthy l group in compound 18 bya meth yl indole provided a compound th a t is 5-fold morepotent (19, F igure 8).45

Pe p t ide an t a g on is t s o f t h e C5a re ce pt o r w e re f i rstobt a in e d by s i t e-direct e d mu t at io n o f C5a , an d t h esynthesis of peptide fragments conta ining t he effectordomain. Finch and co-workers have taken this effort one

step further to produce cyclic peptides th at mimic thes t ru ct u re of ac t ive p ep t ide an t a g on is t s .47 The mostpotent of these inhibitors (20, Figure 9) bound to thereceptor with IC 50 ) 0.3 M as measured in a competi-t io n as s ay u s in g 125I-labeled C5a. This same peptidedisplayed a nta gonist potency in t he presence of 100 nMC5a with IC 50 ) 20 nM as measured by myeloperoxidasere le as e f ro m c y t och alas in B s t imu la t e d h u man p oly -morphonuclear cells. C5a is believed to be a pat hogenicfac t or in a ran g e of immu n o in f lamma t o ry dis eas e sincluding sepsis. C5a receptor ant agonists thereforema y be useful as a nt iinflamma tory agents. C yclic pep-t ide 20 was dosed intravenously in anesthesized ratsw h i ch t h en w e r e c h a ll en g ed w i t h e it h er C 5a or l i-

Figure 8. P eptide-derived SH2 ligands.



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popolysaccharide (which stimulates an increase in en-dogenous C5a).48 Compared to control animals, peptide20 significantly reduced neutropenia (decrease in cir-c u la t in g PM Ns ) an d blo c k e d t h e e le v at io n o f s e ru mTNF-R an d IL6, tw o pro-inflamma tory cytokines.

The w ork of Ga rca-Echeverra provides an excellenti l lu s t ra t io n o f t h e v ar ie t y o f t e c h n iq u e s t h a t may beemployed for the discovery of inhibitors of protein-protein binding.49 Although t he f inal inhibitor, with apeptidic backbone and a molecular weight of 1206, doesnot qualify as a small molecule, i ts derivat ion nicelyillustrat es the various tools tha t ma y be brought t o bearon the discovery of PPBIs. The binding region between

p53 and huma n double minute 2 (hdm2) wa s init iallyprobed using antibodies to identify the binding regionon each protein. Furth er ma pping of the binding r egionon hdm2 employed synt hetic peptides derived from th eN-termin us of p53. A hexapeptide comprising r esidues18-23 of p53 was found to bind to hdm2 with IC 50 )700 M . Wi t h a f f in i t y t h i s l ow , a h ex a pep t id e w a sconsidered to be the minim a l binding epitope for hdm2recognition. A longer peptide comprising 12 residuesfrom p53 displayed an IC 50 ) 8.7 M, and this peptidewa s used as t he star t ing point for further optimizat ion.Increased binding affinity was pursued through the useof phage displa y. This t echniq ue provides a convenientmethod for screening peptide sequences for aff inity

towa rd a ny given molecular ta rget . A 12-mer w ith 28-fold improv ed bindin g a f f in i t y wa s iden t i f ie d , a n dsynthetic peptides representing truncates of this se-quence were prepared to determine the minimum lengthrequired for micromolar aff inity toward hdm2. Thisminima l length tur ned out to be eight a mino acids. Theava ilability of structural da ta from both X-ra y crystal-lography 50 an d NMR spectroscopy 51 for various p53-derived peptides bound to hdm2 proved valuable ing u idin g e f for t s t o op t imize bin din g of s u ch a s h ortpeptide. For exam ple, crysta llography dat a revealedtha t a 15-mer p53-derived peptide bound in a deephydrophobic cleft on hdm2, adopting a helical conforma-tion. Both solid-phase and solution-based studies identi-

f ie d r es id u es t h a t f or m ed con t a c t s w i t h t h e h d m 2p ro t e in as we l l as re s idu e s t h at may be s t ru c t u ra l lyimporta nt but more appropriat e for modificat ion.

To bias their unbound p53-derived peptides towarda helica l conforma tion a nd so decrease th e entropic costof binding, G arc a-Echeverra elected to intr oduce R,R-disubst ituted amino acids at posit ions shown to formno, or weak, direct interactions with hdm2. In addition,based on crysta l structure data , a t yrosine residue wa s

replaced by phosphonomethylphenyla lan ine to introducean electrosta t ic interact ion with Lys-94 in hdm2; sub-s t i t u t io n o n t h e t ry p t o p h an re s idu e was in c lu de d t obetter complement a hydrophobic hole in the hdm2protein. These final replacements led to a peptide (21,Figure 9) w ith IC 50 ) 5 nM for inhibit ion of p53 bindin gto h dm2-G ST. This result represents a 1700-fold im-provement in overall binding affinity.

Structure-Based Design. A long-sta nding goal ofmedicinal chemists is to develop techniques for the denovo design of compounds based on knowledge of aproteins three-dimensional structure. Not surprisingly,t h is g e n e ra l ap p ro ac h h as be e n p u rs u e d a ls o in t h esear ch for inhibit ors of protein-protein association. One

s t ra t e g y t h at h as p ro v e n u s e fu l is t h e s e le c t io n o f ascaffolding templat e upon w hich may be a ppended sidechains specifically oriented t o occupy th e sam e relat iveregions of space as key side chains of a known ligand(where the ligand here is i tself a peptide or protein).T h e s e s ide c h ain s may be o p t imize d in an i t e ra t iv efashion either through mult iple rounds of synthesis,assa y, structure a nd design, or combinat orially. An earlyexam ple of such an approach wa s described by Hirsh-mann and co-workers who used a glucose template tobuild simple but effect ive ligands for the soma tostat inreceptor.52 So f t ware h as be e n wri t t e n t o h e lp s e le c tscaffolding molecules, e.g., from known ring systems,that will posit ion groups in a defined relat ive spat ial

a n d d ir ect i on a l m a n n er , n ot a b l y t h e p rog r a m C A-VEAT.53

A g ro u p f ro m H o ffman n -L a R och e led by O lsone mploy e d be n zen e a s a t e mplat e in t h e ir de sig n o finterleukin-1 receptor antagonists.54 Sit e-directed mu-ta genesis studies w ere used to ma p the binding epitopesof IL-1R a nd to a common region in their crystalo-graphically determined t hree-dimensiona l structures.Three IL-1 residues were identif ied as essential forreceptor binding bas ed on their rela tive contr ibution tobinding affinity, namely Arg-4, Phe-46, and Lys-93. A1,2,4-trisubst ituted benzene ring was selected as thescaffold because it a llowed a ppenda ge of the side cha ins

in a man n e r t h a t wo u ld a l low for t h e p ot e n t ia l in t er-digi t a t io n of I L -1 an d I L -1 re ce pt o r re sidu es u p onbinding. Since the location of the lysine -a mine differedbet we e n t h e t wo I L -1 c ry st a l s t ru ct u re s, a t e t h er wa sused that would permit access to either of the docu-mented orientat ions.

One of the a dvant ages of th e structure-based designapproach is tha t synthet ic considerat ions can be builtinto the liga nd design, fa cilita ting a ccess to compoundstha t can be easily a ssembled to test t he binding models.The corresponding disadvantage is that it is easy to letsynthetic ease subvert the original model such tha t t heligands produced are not optimal from a binding per-spective. Olsons group prepared putative IL-1 antago-

Figure9. Compound 19, AcF[OP-DCH a -WR]; 20, ant agonistof hdm2 binding t o p53.



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nists in eight steps from 2,4-dihydroxybenzene. Whilethe initially designed ligand (22, Figure 10) an ta gonizedIL1-R binding to the IL-1 receptor with IC 50 ) 770 Mas me as u re d by E L I SA, fu r t h e r o p t imiza t io n le d t ocompounds 23 a n d 24 (Figure 10), which inhibited bothIL-1R and IL-1 bin din g wi t h I C 50 values of


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a ppears to depend on a co-cha perone protein called p23;however, t he release of fully funct iona l t arget proteinhas been proposed to require the hydrolysis of ATP.Indeed, the N-terminus of Hsp90 bears a wea k a ff inity(Kd ) 400 M) ATP /ADP b indin g site. The discovery tha tt h e an s amy c in an t ibio t ic s radidic o l (32, Figure 13),herbimycin A (33), a nd geldana mycin (GA, 34) bind t o

Hsp90 and reverse the transformed phenotype of v-srctra nsformed cells identified Hsp90 as a potentia l ta rgetfor ant itumor therapy.60 Indeed, the GA analogue 17-allylamino-geldanamycin (17-AAG) (35) is current ly inphase I clinical trials for treating cancer. Although thesena tur a l products bind t o the ATP /ADP site on H sp90,their mechanism of act ion is proposed to involve thein h ibi t io n o f as s o c ia t io n be t we e n H s p 90 an d i t s c o -chaperone p23.61,62

Th e cry s t a l s t ru ct u re s of t h e H s p 90 N-t e rmin aldomain bound to GA and to radidicol have been pub-lished.63,64 By examinat ion of these structures, Chiosisan d co-workers ident ified key intera ctions betw een thel ig an d an d H s p 90 t h at t h e y be l ie v e d c o n t r ibu t e d t o

protein inh ibition, includi ng conta cts w ith Asp-93/Ser -52, hydr ogen bonds t o Lys-112/Lys -58, an d int era ctionwith a hydrophobic pocket comprising contributionsfrom six hydrophobic a mino acid side chain s.65 Selectingpurine as a sta rting point, partly t o fa vor good bioa vail-ability and cell permeability , these workers designedcompound P U 3 (36, Figure 13) to ta ke adva nta ge of thekey interactions noted. Computer docking of this mol-ecule into t he H sp90 ADP /ATP binding site sh owed t ha tit should satisfy all of the important binding interactionsexcept the one with Lys-58. B y using a n a ffinity columnof immobilized G A, t he competit ion between P U3 an dGA for binding to Hsp90 wa s examined to est imate therelat ive aff inity of PU3 for Hsp90. An EC 50 of 15-20

M for inhibition of Hsp90 binding to immobilized GAwa s determined, compar ed to EC 50 ) 1 M for 17-AAG(GA binds to H sp90 w ith Kd ) 1.2 M).

As expected for an inhibitor of Hsp90, PU3 destabi-lized the estrogen receptor and promoted its protea-some-dependent degradation. Similarly, Her2 levels inM CF 7 breas t can c er ce lls de cre as ed in re sp on s e t ot r ea t m e n t w i t h 1 0 M P U 3. P r ot ei ns t h a t a r e n o tsensit ive to GM were similar ly unaffected by P U3. P U3

showed antiprolifera tive activity a gainst MC F-7 and tw oother cell lines in vitr o at simila r concentra tions to thoserequired to compete for Hsp90 binding and to induceprotein degrada t ion. In a ddit ion, the tr an sformed phe-notype of MCF7 cells , such a s th eir r ound shape w ithindist inct cell margins and prominent nucleoli , werere ve rs ed by t re a t me n t wi t h P U 3.

Collect ively, t he da ta on Hsp90 inhibitors suggeststha t P U 3 functions as a n inhibitor of Hsp90 in cells andthat it most likely works by preventing the associationof Hsp90 with p23 or its target proteins. PU3 thereforerepresents one of the first exa mples of a P P B I designedde novo from a crystal st ructure of the t ar get protein.As its inventors point out in their art icle, PU3 has a

molecula r w eight of 371 a nd is rule-of-5 complia nt , i.e.,by current standards it would be defined as a drug-likemolecule.

A group at P ar ke-Da vis used molecular modeling a ndan X-ray crystal structure of a phosphopeptide-boundpp60c-sr c protein to design a benzylamine-based SH2ligan d for the c-src tyrosine kina se.66 Noticing that muchof the binding of an 11-mer peptide to the c-src SH2domain could be ascribed to two primary recognit ionelements, Lunney et a l . set out to design a less highlycharged, non-peptide ligand tha t incorpora ted th ese twoe ss en t ia l con t ac t s . F ro m a s earc h of t h e Ca mbridg eCrysta logra phic Da ta base, a benzoxazinone ring struc-t u re in i t a l ly w as s elect e d a s a p ot e n t ia l t e mp la t e forpositioning th e key recognition elements in the proteinbinding site. Sy nthetic considerat ions led to the m odi-fica tion of this templat e to a ring-opened am inometh-ylbenzene carboxam ide. The first genera tion of designedinhibitors displayed moderate affinity for c-src (IC 50 )6-10 M), supporting the design strategy. The crystalstructur e of one of these designed inhibitors (37, Figure14) confirmed tha t t he tw o tar geted contacts ha d beenachieved; however, it a lso revealed a bindin g mode thatdiffered from the original model as a consequence ofreorientation of the key phenyl phosphate group in theprotein binding site compared to the peptide ligandreferenced in the design process. This study serves to

underscore the flexibility of proteins in adapting to evensimilar recognit ion elements w hen presented in a dif-ferent context. It reinforces the necessity for structuredetermina tion, modeling, an d synt hesis to be performeditera tively to compensa te for the complexity of designingligands for f lexible proteins ba sed on sta t ic structurald a t a .

Other recent examples of structure-based design havebeen published by a group at ARIAD. This team wasin t ere st e d in in h ibit o rs o f t h e S H 2 do main a s a n t ire -sorption agents for possible use aga inst osteoporosis.The ARIAD group employed crystal structure data forSrc an d t h e re la t e d k in as e L c k in c o n ju n c t io n wi t hmolecular modeling to design two non-peptide ligands

Figure 13. Compound 32, radidicol; 33, h erbimycin A; 34,geldanamycin; 35, 17-AAG ; 36, P U 3 .



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of the SH 2 doma in pTyr binding site (38 a nd 39, Figure14).67,68 Design considerations included (1) the incorpo-ra tion of an edge to fa ce conta ct betw een the ligan d an dTyr-181; (2) the addition of two hydrogen bonds; (3)buria l of hyd rophobic surfa ce (a cyclohexane ring) in theIle (pY + 3) bindin g s i t e; an d (4) t h e in clu sion ofaddit ional hydrophobic conta cts by building off of t heligands benzene ring to complement the S H2 doma inhydrophobic architecture.

T wo s e p ara t e a p p ro a c h e s we re u s e d t o t a rg e t t h e

inibitor to Src vs related tyrosine kinases. The proteinSrc is unique a mong its class in possessing a cysteineresidue (C188) in t he SH 2 domain tha t is not presentin other S rc tyrosine kina se family m embers (e.g., Lck,Fyn, Yes, Yrk, Hck, Fgr, B lk, Lyn, Fr k/Ra k, an d Iy k/B sk). The environment of the cysteine thiol, surr oundedby p os i t iv ely ch arg e d re sidu es t h at a re p res e nt t ofac i l i t a t e p h o s p h a t e bin din g , lo we rs t h e pKa o f t h isresidue making it more a reactive nucleophile. 69 AP22161(38) wa s designed to exploit t his unique cysteine residueto form a covalent at tachment to the protein throughaddition to a benzaldehyde, which w as positioned to tra pthe cysteine thiol as a hemithioacetal. This compoundinhibited Src binding t o a fluorescein-ta gged SH 2 ligandpeptide with IC 50 ) 5.5 M.

The a bility of AP 22161 to penetra te cells wa s a ssessedusing a sophist icated mammalian two-hybrid assay. 67

Br iefly, expression of a secreted a lkaline phosphata sereporter protein w a s engineered to be dependent on t heappropriat e recognit ion of a SH 2 binding peptide by aSrc family k in a s e . I n t h e p re s e n c e o f a SH 2 do maininhibitor, no alkaline phosphatase is produced. Withap p ro p ria t e c o n t ro ls , t h is as s a y was u s e d t o de mo n -strate that AP22161 selectively inhibits Src SH2 vs ZAPSH2 in cells , albeit at relat ively high concentrat ions(IC 50 ) 60-80 M). No cell toxicity was observed att h e s e c on ce n t ra t ion s . Co n sis t en t wi t h t h e s e re su l t s ,

AP22161 was a relatively weak inhibitor of bone resorp-t ion w i t h I C 50 ) 42.9 M.

An observat ion that the protein Src cocrystallizedwit h a m olecule of citra te bound in the SH 2 doma in ledto a second approach for specifically target ing Src inbone.68 The phosphona te-conta ining a mino a cid Dpp (40,Figure 14) was introduced a s both a pTyr mimic and apotential bone-targeting residue. The incorporation ofDpp into the non-peptide SH2 ligan d led to the synt hesis

of AP 22408 (39) which inhibited Src with I C 50 ) 0.3 M,as measured using a f luorescence polarizat ion assay.The aff inity of AP22408 for bone wa s confirmed byhydroxyapaptite chromatography and the association oftritium-labeled AP22408 with dentine (a bone substi-tute).68 AP22408 inhibited the absorption of dentineslices by ra bbit osteoclasts in vitro with an IC 50 ) 1.6M, providing the osteoclast s w ere preincubat ed w iththe inhibitor. Moreover, in a n esta blished anima l modelfor the in vivo evaluation of antiresorptive compoundsag a in s t t h y ro id h o rmon e -in du ce d bon e re s orp t ion ,AP 22408 significa ntly decreased serum calcium (Ca 2+)compared to control consistent with a robust ant ire-sorptive effect. This in vivo demonst ra tion of a pheno-

typically observable outcome from the use of a smallmolecule SH 2 inhibitor provides one of th e few existingvalidations for the use of inhibitors of protein-proteinin t erac t ion s t o modu la t e s ign al in g in a biolog ica l lyrelevant set t ing. I t should provide significant impetusto the field of SH 2 inhibitor design a s well a s encoura gerelated efforts towa rd t he inhibit ion of other signalingp at h way s t h at de p e n d o n SH 2-me dia t ie d p ro t e in a s -sociated events.

Future Directions

Allosteric Regulation and Protein Stabilization.Although it may be conceptually simpler to imagine

inhibit ing protein-

protein associat ion by interferingdirect ly at the binding interface, nature has taught ustha t remotely binding inhibitors m ay be equally effec-tive.70 This is t he concept of allosteric control t ha t isused so effectively by enzymes. The binding of a smallmolecule allosteric effector can result in large confor-ma tional changes in a protein which relay informationto the act ive site with the result tha t either substrat esno longer bind or the catalyt ic residues are no longercorrectly aligned to perform chemistry. 71 In principle,this strategy may equally well apply to the inhibit ionof protein-p rot e in bindin g . I n de ed, we h av e a lre a dyseen one example in the form of an NOS inhibitor thatbound t o the cata lyt ic iron, inducing a conforma tional

change tha t prevented dimerizat ion of the NOS mono-mer.19 M an y me mbe rs o f t h e g ro win g c la s s o f s mal lmolecule antagonists of G-protein-coupled receptors maywo rk a l los t e r ica l ly by bin din g t o a t ran s me mbra n eregion of the receptor and thereby preventing bindingof the natural l igand to an extracellular site.9-13

The feasibility of discovering small molecules thata l t e r p ro t e in c o n fo rmat io n was i l lu s t ra t e d in re c e n twork from a group at P fizer w ho devised a method forsta bilizing t he protein p53. The p53 tumor suppressorprotein is frequently mut at ed in cancer cells, diminish-ing its a bility t o respond to DNA dama ge. Consequently,the processes of cell cycle arrest or apoptosis that wouldn o rmally be in i t ia t e d by p 53 fa i l t o o c c u r , a n d c e l ls

Figure 14. S H 2 l ig a n d s . C o mp ou n d 38, AP22161; 39,AP22408; 40, D P P .



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conta ining dam aged D NA are allowed to divide leadingto tumor formation. Evidence suggests t ha t t he struc-tura l basis of this funct ional failure is a d estabilizat ionof the p53 DNA binding doma in. Foster a nd co-workersdiscovered a small molecule, CP-31398 (41, Fig ure 15),from a collection of greater than 100 000 compounds,t h at s t abi l ize d mu t a n t p 53 in a wi ld-t y p e s t ru ct u recap able o f b in din g D NA a s in dica t e d in a g el-s h i fta s s a y .72 Mutan t p53, when t reated w ith C P -31398, wa sthermally more stable than untreated protein. More-ov er , H 1299 c el ls t ran s fe ct e d wi t h mu t an t p 53 e x-pressed a p53-regula ted reporter gene in r esponse to theaddit ion of CP -31398 in a concentra t ion-dependentmanner. Similar effects were evident in vivo in mice

with the growth of A375

S2 melanoma and DLD-1 coloncarcinoma , both inhibited with daily or tw ice daily dosesof CP-31398, indicating that the function of p53 couldbe restored even in an an imal m odel.

The concept of protein stabilization has been success-fully, albeit unwit t ingly, exploited through use of thecl a s s of a n t i t u mor a g e nt s k n ow n a s a n t i m it ot i cs .73

Microtubule sta bilizing drugs such as Taxol appear tofunction by st abilizing a form of -tubulin that wouldotherw ise be unsta ble once GTP hydr olysis occurred. 74

Thus, Ta xol allow s -tubulinG DP complexes to part ici-p at e in microt u bu le forma t io n wh e n t h e y n ormallywould not . In this exam ple, t he sma ll molecule facili-tates protein-protein binding rather than inhibiting it ;

however, it is easy to imagine how this outcome couldbe re v e rs e d. I n de e d, t h e o r ig in al an t imit o t ic dru g s ,including colchicine and nocodazole, do funct ion bydestabilizing microtubules, not by stabilizing them. Asseen a bove, the compounds identif ied by Ha ggart y a ndco-workers also are proposed to operate by this mech-an is m.30 An excellent discussion of how various smallmolecules, including colchicines and vinca alkaloids,may affect microtubule dynamics is provided by Down-ing.75

The potential of sma ll molecules to affect proteins t a b i l i t y w a s f u r t h e r i l l u s t r a t e d b y S c h u l t z a n d c o -wo rke rs u s in g a mode l s y s t em de riv ed f rom h u mangrowth hormone (hG H).76 A mutat ion wa s made in hGH

to delibera tely introduce a cavity tha t desta bilized theprotein so th at i t bound its receptor (hGHbp) 106-foldmore weakly (Kd(wt) ) 0.3 nM, Kd(mutant) > 1 mM).Se lect ion f rom a s mal l l ibra ry of in dole a n alog u esidentified 5-chloro-2-trichloromethylimidazole (E8; 42,Figure 15) as a compound capa ble of restoring receptorbinding a ffinity. In the presence of 100 M ligand, hG Hbound to hGH bp with Kd ) 260 nM. In cells, E 8 restoredt h e ag o n is t p o t en t ia l o f mu t a n t h G H , a s in dica t e d bycell proliferat ion, a nd fa cilita ted mitogenic signa ling asindicated by the phosphorylat ion of the downstreamprotein J ak2.

S e ve ra l g r ou ps h a v e b ee n s t u dy in g m et h od s f ordimerizing a nd r everse-dimerizing proteins t o control

ma cromolecular interact ions in vivo. The ability oftethered ligands to bring t heir respect ive part ner pro-teins together is now w ell esta blished.77 In one recentexample, Lin et a l. used a dexametha zone-methotrexatedimer to a ct ivat e tra nscript ion of a LacZ reporter genein yeast via a LexA-DHFR fusion protein and a B42-GR fusion protein.78 An extension of this idea proposesthe use of chemical-induced dimerizat ion to hijack ap rot e in t h a t i s n o t a n a t u r a l p a r t n er p rot e in i n t o

blocking a targeted protein-protein binding interaction.For example, a tethered dimer of FK506 and a peptidel ig a n d f or t h e S H 2 d om a i n of t h e s r c k i na s e F y npromotes the formation of a complex between FKBP,the ligand dimer, a nd Fyn, which might be expected toin t er fere wi t h F y n bin din g t o i t s n a t u ra l down s t re a mproteins.79 A similar a pproach w as used by C hiosis andco-w orkers to inhib it P I-3-kina se.80 Other workers havetethered geldanamycin (GA) to steroid molecules suchas t e s t eot e ron e an d e st radio l t o t arg e t s p eci fic c el ltypes.81,82 In a ddition, a four-carbon tethered G A dimerexhibits, as yet unexplained, selectivity for promotingthe degra dat ion of HE R-2 kinase over other kina ses.83

From studies on the prototypical dimerizer, FK506,

an d i t s t a rg e t prot e in F KB P , a p oin t mu t an t of F KB P(F M) was discovered tha t rendered t his normally m on-omeric protein a weakly stable dimer (Kd ) 30 M). 84

I n t h is c as e , t h e dime r c o u ld be dis s o c ia t e d by t h eaddi t io n o f an F KB P l ig an d s u c h as 43 or 44 (Figure16). This phenomenon was subsequently exploited invivo to regulate protein secretion thr ough the endoplas-mic ret iculum.85 Fusion proteins of F M (four copies)joined to either hGH or human proinsulin via a furincleavage sequence were shown to aggregate in the ERof HT1080 cells (a human fibrosarcoma line) in culture.Addit ion of a t ight binding ligand (Kd ) 1 n M ) t h at isselective for F M vs FKBP caused disaggregat ion of thecomp le xe s an d s u bse q u en t re le as e of t h e corre ct lyprocessed and folded hGH or insulin proteins whichwere secreted from the cells a nd could be detected int h e s u pe rn a t a n t . R eg u la t e d s ecr et i on of h G H a n dinsulin was also achieved in mice, demonstrat ing thatthese sma ll molecule inhibitors of F M aggregat ion werealso fully functional in vivo.

Challenges Ahead. Th e g rowin g n u mber of e x-am ples of small molecules tha t ha ve been demonstr at edto inhibit protein-protein binding should provide en-cou rag e men t t o t h os e w h o be l ie ve t h a t t h e re a re op -portu nities for exploiting t his a pproa ch in drug d iscov-ery. Clearly, high molecular weight is not a prerequisitefor a P P B I t o be effective, even in vivo. Moreover, there

does not appear to be a n int rinsic limit on t he level ofaffinity achievable (witness the 9 nM inhibitor of CaM-dependent PDE act ivat ion 30 an d t he 1.6 nM inhibitorof SH2, 17). I t wo u ld a p pe ar t h a t w e a re o n t h e v erg eof s e e in g t h e f i rs t e xamp les of de s ign e d P P B I s a p -proaching preclinical development. Yet, it is prema tureto claim that the difficulties accompanying the discoveryof effect ive P P BI s ha ve been overcome. F or sure, thisapproach will work only in well-chosen situations, andbetter m ethods for chara cterizing protein-protein in-terfaces will play an important role in helping to selectthe most pliable cases for drug discovery. Theoreticaland experimental approaches to this problem are al-ready being developed. For example, Ringe and Mattos

Figure 15. Compound 41, C P -31398; 42, E8.



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ha ve advoca ted the crysta llization of proteins in variousorganic solvents to identify surface regions t ha t mightprovide binding sites for sm a ll organic molecules by t helocation of bound solvent molecules.86 Weiss and co-workers have developed a combinatorial approach toalanine scanning using phage-display technology andh av e u s e d t h is ap p ro a c h t o ide n t i fy k e y s ide c h ain sinvolved in binding between hGH and its receptor. 87

Massova a nd Kollman ha ve described a computat iona l

v ers ion of a lan in e s ca n n in g t h at a ls o may be u s ed t oprobe the contr ibution of individua l residues to protein-protein binding energies.88

Cu rre n t ly , t h e re are n o g e n e ra l t e c h n iq u e s o r ap -proaches that will reliably i l luminate the path towardthe synt hesis of potent a nd effect ive drug-like P P B Is.However, these techniques are being developed and withincreased use and experience should provide someguidelines for inhibitor design. As in other areas of drugdiscovery, the judicious employment of combinatorialchemistry has the potential t o speed up t he discoveryof P P B I s .89 Also, NMR methods such as SAR by NMR 90

an d re la t e d t e ch n iq u es ma y be u s ed t o iden t i fy we akbinding ligands as lead structures for inhibitor optimi-

zat ion. Covalent l inkage of two or more weak bindingligands is a nt icipated, under optima l circumstances, toproduce a compound of substa ntia lly improved a ffinity.91

Alterna tively, specific noncova lent liga nds m a y be con-v e rt e d t o c o v ale n t l ig an ds by t h e in c o rp o rat io n o f areactive functional group.92

In conclusion, a combination of screening and struc-ture-based design will provide start ing points for thed is cov er y of n ov el P P B I s , a n d s t r uct u r a l d a t a f r omprotein-l igan d c omp le xe s s h ou ld be a ble t o g u idefurth er optimizat ion. Success will likely hinge on beingable to bury sufficient hydrophobic surface w ithoutinordinately increasing the size of the small molecule.I f t h is is n o t p o s s ible a t t h e p ro t e in in t e r fac e , t h e n

allosteric sites ma y be considered. It is to be hoped tha tover t ime some themes may emerge, highlighting forexam ple part icular generic structures tha t ma y form ab a s is f or P P B I s . C om pou n d l ib r a r ie s t h e n ca n b epopulated with compounds exemplifying these struc-tures, increas ing the cha nces of lead discovery th roughhigh throughput screening. Perhaps then the trepida-t ion that protein-protein binding currently imbues inma ny m edicinal chemists w ill be overcome, and t he richopportunit ies a vailable for drug discovery f inally w illbe recognized.

Acknowledgment. T h e au t h o r t h an k s D r . E l le nDobrusin for the inspiration and encouragement to wr ite

this review and Dr. Bruce Roth for issuing the challenge.Drs. J ack Li, Ellen Dobrusin, Beth Lunney, an d Ch uckOmer a re gra tefully a cknowledged for th eir insightfulcomments a nd correct ions t o early dr aft s .

Note Added in Proof

For a complementa ry review of this field, see: Ockey,D . A. ; G a d ek , T. R . I n h ib it or s of P r o t ei n-ProteinInteract ions. E x p er t O p i n . T h er . P a t . 2002, 12, 393-400.

Biography

Peter L. Toogood w a s bor n and ed ucat ed in England . Heobt ained his B .S c . and Ph.D. a t I m per ial College, Lond on,where he performed research on the synthesis and antifeedant

properties of aza dirachtin under th e supervision of P rofessorSteven V. Ley. Subsequently, he held a NATO postdoctoralf el low s h i p a t H a r v a r d U n i v er s it y , w h e r e h e w o r ke d w i t hProfessor J eremy R. Knowles to prepare and study bivalentinhibitors of influenza hemagglutinin. In 1992, D r. Toogoodmoved to the University of Michiga n as a n Assista nt P rofessor,where he init iated a progra m of research in organic synthesisand biochemistry that led to a formal synthesis of althiomycin,a tota l synth esis of motuporin, a nd a proposed revision of thestructure of keramamide F. His research on the mechanismof a c t ion of d i de mn i n B p r ov id ed n ew i n si g ht s i n t o t h einhibition of protein synth esis by this na tura l product a nd ledto a reinterpretation of the role of elongation factor-1Rduringpolypeptide elonga tion. In 1998, Dr. Toogood joined themedicinal chemistry depart ment of the Par ke-Da vis Pha rma -ceut ical Com pany t o w or k on cancer d r ug d iscover y . Hecurrently h olds the position of Assista nt D irector for Oncologyat Pfizer Global Research and Development, Ann Arbor.

References

(1) Stites, W. E. Protein-Protein Interactions: Interface Structure,Binding Thermodynamics, and Mutat ional Analysis. Chem. Rev.1997, 97, 1233-1250.

(2) J ones, S.; Thornton, J . M. P rinciples of P rotein-Protein Interac-tions. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13-20.

(3) Wells, J . A. Bin ding in th e Grow th H ormone Receptor Complex .Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 1-6.

(4) Ratcliffe, G. C.; Erie, D. A. A Novel Single-Molecule Study toDetermine Protein-Pr otein Association Consta nts. J. Am . Chem.Soc. 2001, 123, 5632-5635.

(5) Cochran, A. G. Antagonists of Protein-Protein Interact ions .Chem. Biol. 2000, 7, R85-R94.

(6) Zutshi , R. ; Brickner, M.; Chmielewski , J . Inhibit ion of t heAssembly of P rotein-Pr otein In terfaces. Curr . Opin. Chem. Bi ol.1998, 2, 62-66.

(7) Holzeman n, G. Recent Advances in Rv3 Integrin Inhibitors.I D r u g s 2001, 4, 72-81.

(8) Sa man en, J . G PI Ib/IIIa Antagonists. Annu. Rep. Med. Chem.1996, 31, 91-100.

(9) Owa labi, J . B.; Rikalla, G.; Tehim, A.; Ross, G . M.; Riopelle, R.J .; Kambolj, R.; Ossipov, M,; Bian, D.; Wegert, S.; Porreca, F.;Lee, D. K. H. Characterization of Antiallodynic Actions of ALE-0540, a Novel Nerve Growth Factor Receptor Anatagonist, int h e R at . J. Pharmacol. Exp. Therap. 1999, 289, 1271-1276.

(10) Ba ba, M.; Nishimura, O.; Ka nzaki , N.; Okamoto, M.; Sa wa da,

H.; Iizawa, Y.; Shiraishi, M.; Aramaki, Y.; Okonogi, K.; Ogawa,Y.; Megurp, K.; Fujino, M. A Small-Molecule Nonpeptide CCR5Antagonist w ith H ighly Potent an d S elective Anti-HIV-1 Activ-i ty . Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5698-5702.

(11) Ca scieri, M.; Spr inger, M. S . The Chem okine/Ch emokine-Recep-tor Family: Potential and Progress for Therapeutic Intervention.Cu r r . O p in . Ch em . Bi o l . 2000, 4, 420-427.

(12) Horuk, R.; Ng, H . P. Ch emokine Receptor Antagonists. M ed. R es.Rev. 2000, 20, 155-168.

(13) Trivedi , B . K . ; Low, J . E . ; Ca rson, K .; La Rosa, G. J . C hemok-ines: Tar gets for Novel Therapeutics. Ann. Rep. Med. Chem.2000, 35, 191-200.

(14) B oger, D . L.; G oldberg, J .; Silletti , S.; K essler, T.; Cheresh, D .A. Identification of a Novel Class of Small-Molecule Antiangio-genic Agents Through the Screening of Combinatorial LibrariesWhich Function by Inhibiting the Binding and Localization ofProteinase MMP2 to I ntegrin Rv3. J. Am . Ch e m . So c. 2001,123, 1280-1288.

(15) S illetti , S .; Kessler, T.; G oldberg, J .; Boger, D. L.; Cheresh, D .

A. Disruption of Mat rix Metalloproteinase 2 B inding to IntegrinRv3 by an Organic Molecule Inhibits Angiogenesis and TumorGrowth in vivo. Pr o c. N a t l . Acad . Sci . U. S. A. 2001, 98, 119-124.

(16) Bar-Sagi, D.; Prives, J . Trifluoroperazine, a Calmodulin Antago-nist, In hibits Muscle Cell Fusion. J. Cel l Bio l . 1983, 97, 1375-1380.

(17) Levin, R. M.; Weiss , B. Inhibit ion by Tri fluoroperazine ofCalmodulin-Induced Activation of ATPase Activity of Rat Eryth-rocyte. Neuropharmacology1980, 19, 169-174.

(18) Schultz, M. D.; B owma n, M. J .; Ham , Y.-W.; Zhao, X.; Tora, G .;Chmielewski, J . Small-Molecule Inhibitors of HIV-1 ProteaseDimerization from Cross-Linked Interfacial Peptides. Angew.Ch em . , I n t . Ed . 2000, 39, 2710-2713.

(19) McMillan, K.; Adler, M.; Auld, D. S.; B aldw in, J . J .; B lasko, E.;Browne, L. J . ; Chelsky, D. ; Da vey, D. ; Dol le , R. ; Ea gan, K . A.;Erickson, S.; Feldman, R. I.; Glaser, C. B.; Mallari, C.; Morrissey,M. M.; Ohlmeyer, M. H. J .; P an, G. ; Pa rkinson, J . F. ; P hi ll ips ,



15/16

G. B.; Polokoff, M. A.; Sigal, N. H.; Vergona, R.; Whitlow, M.;Young, T. A.; Devlin, J . J . Allosteric Inhibitors of Inducible NitricOxide S ynthase Dimerizat ion D iscovered via CombinatorialChemistry . Proc. Natl. A cad. Sci. U .S.A. 2000, 97, 1506-1511.

(20) Hemm ila , I.; Webb, S. Time-Resolved Fluoromet ry: An Overviewof the Labels and Core Technologies for Drug Screening Ap-plications. Dr ug D iscovery Today 1997, 2, 373-381.

(21) Hertzberg, R. P .; Pope, A. J . High-Throughput S creening: NewTechnology for the 21st Century. Curr. Opin. Chem. Biol. 2000,4, 445-451.

(22) Hua ng, Z. Bcl-2 Family P roteins as Tar gets for Anticancer DrugDesign. Oncogene2000, 19, 6627-6631.

(23) Reed, J . C. Mechanism s of Apoptosis. Am. J. Pathol. 2000, 157,1415-1430.(24) Strasser, A.; O Conner, L.; Dixit, V. M. Apoptosis Signaling.

Annu. Rev. Biochem. 2000, 69, 217-245.(25) Degterev, A.; Lugovsky, A.; Cardone, M.; Mulley, B.; Wagner,

G.; Mitchison, T.; Yuan, J . Identification of Sma ll-MoleculeInhibitors of Interaction Between the BH3 Domain and Bcl-xL.Na t u r e, Cel l Bio l . 2001, 3, 173-182.

(26) Following subm ission of this review, an ar ticle appeared describ-ing the photochemically induced covalent attachment to TNFreceptor 1 of compounds similar to BH3I-1 and BH3I-1 . Carter,P. H.; Scherle, P. A.; Muckelbauer, J . A.; Voss, M. E.; Liu, R.Q.; Thompson, L. A.; Tebben, A. J .; Soloman, K. A.; Lo, Y. C.;Li , Z . ; Strzemienski , P . ; Yang, G. ; Falahatpisheh, N.; Xu, M.;Wu, Z.; Farrow, N. A.; Ramnarayan, K.; Wang, J .; Rideout, D.;Yala moori, V.; Domaille, P.; Und erwood, D. J .; Trzaskos, J . M.;Friedma n, S. M.; Newt on, R. C.; Decicco, C. P . P hotochemicallyEnhanced Binding of Small Molecules to the Tumor NecrosisFactor Receptor-1 Inhibits the Binding of TNF-R. Pr o c. N a t l .

Acad. Sci. U.S.A. 2001, 98, 11879-11884.(27) Tzung, S .-P.; Kim, K . M.; B asa nez, G.; Giedt, C. D .; S imon, J .;

Simmerberg, J .; Zhang, K. Y. J .; Hockenbery, D. M. AntimycinA Mimics a Cell-Deat h-Inducing B cl-2 Homology Doma in 3.Na t u r e Cel l Bio l . 2000, 3, 183-191.

(28) Wan g, J .-L.; Liu, D.; Zhan g, Z.-J .; Sha n, S.; Ha n, X.; Sriniva sula ,S. M.; Croce, C. M.; Alnemri, E. S.; Huang, Z. Structure-BasedDiscovery of an Organic Compound that Binds Bcl-2 Protein andInd uces Apoptosis of Tumor Cells. Proc. N atl. A cad. Sci. U .S.A.2000, 97, 7124-7129.

(29) Rodi, D. J .; J anes, R. W.; Sanga nee, H. J .; Holton, R. A.; Walla ce,B. A.; Makowski, L. Screening of a Library of Phage-DisplayedPeptides Identifies Huma n B cl-2 as a Taxol-Binding P rotein. J .M o l. Bio l . 1999, 285, 197-203.

(30) Ha ggart y, S. J .; Mayer, T. U.; Miyam oto, D. T.; Fat hi, R.; King,R. W.; Mitchison, T. J .; S chreiber, S . L. Dissecting CellularPr ocesses Using S ma ll Molecules: Identification of Colchicine-Like, Taxol-Like a nd Other S mall Molecules t hat Perturb

Apoptosis. Chem. B iol. 2000, 7, 275-

286.(31 ) S h ar m a, S . V. ; O n ey am a, C . ; Tam as h i t a , Y .; N akan o, H . ;Sugaw ara , K.; H ama da, M.; Kosaka, N.; Tama oki, T. UC S15A,a Non-Kinase Inhibitor of Src Signal Transduction. Oncogene2001, 20, 2068-2079.

(32) Fields , A.; S ong, O. K . A Novel Genetic System to DetectProtein-Protein Interactions. N a t u r e 1989, 340, 245-246.

(33 ) B e l sh aw , P . ; H o , S . N . ; C r ab t r e e, G . R . ; S c h r ei b er , S . L .Controlling Protein Association and Subcellular Localizationwith a Synthetic Ligand that Induces Dimerization of Proteins.Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4604-4607.

(34) Licitra , E . J .; Liu, J . O. A Three-Hybrid System for DetectingSmall Ligand-Protein Receptor Interactions. Proc. Natl. Acad.Sci. U.S.A. 1996, 93, 12817-12821.

(35) Igara shi , K. ; S higeta, K. ; Kohno, S. ; Isohara , T.; Yama no, T.;Uno, I . A Novel Indication of the Activities of Small MoleculeInhibitors of Protein-Pr otein Interactions: Applicat ion of theYeast Two-Hybrid System. J. An t ib io t . 1998, 996-888.

(36) Huang, J .; Schreiber, S. L. A Yeast Genetic Sy

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