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Protein–protein interactions as targets for small-molecule therapeutics in cancer

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Protein–protein interactions as targets for small-molecule therapeutics in cancer
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  Protein–protein interactions as targetsfor small-molecule therapeuticsin cancer  Alex W. White*, Andrew D. Westwell and Ghali Brahemi Small-moleculeinhibitionofthedirectprotein–proteininteractionsthatmediatemany important biological processes is an emerging and challenging area indrug design. Conventional drug design has mainly focused on the inhibition of asingle protein, usually an enzyme or receptor, since these proteins often containa clearly defined ligand-binding site with which a small-molecule drug can bedesigned to interact. Designing a small molecule to bind to a protein–proteininterface and subsequently inhibit the interaction poses several challenges,including the initial identification of suitable protein–protein interactions, thesurface area of the interface (it is often large), and the location of ‘hot spots’(small regions suitable for drug binding). This article reviews the general approachto designing inhibitors of protein–protein interactions, and then focuses on recentadvances in the use of small molecules targeted against a variety of protein–protein interactionsthat have therapeutic potentialforcancer. Fundamental processes in living cells, suchas intracellular signal transduction andmaintenance of cytoskeletal architecture, arelargely controlled by proteins, often acting inconcert with other protein partners throughprotein–protein interactions (PPIs).Inappropriate protein–protein recognition cancontribute fundamentally to many diseases,including cancer. Selective, small-moleculemodulation of PPIs is therefore an area of growing interest to pharmaceutical science(Refs 1, 2, 3, 4, 5, 6, 7, 8, 9).Despite the pivotal role of PPIs in manyprocesses relevant to cancer development,targeting PPIs as a therapeutic strategy incancer is still in its infancy, and examples of modulators of protein complexes as potentialtherapeutic agents are few relative to the morewidely studied area of small-molecule enzyme(e.g. kinase) inhibitors (Ref. 10). The reasons forthe dominance of kinase (and other enzyme)inhibitors as targets for modern cancertherapeutics are centred on the enzymesubstrate-binding site. In the case of enzymeinhibition, the design methodology for thesecompounds is well established, and active sitesare usually contained within deep clefts thatclearly define the ligand-binding site andcontain the critical amino acid residues requiredfor ligand interaction. Furthermore, allWelsh School of Pharmacy, King Edward VII Avenue, Cardiff University, Cardiff, CF10 3NB, UK.*Corresponding author: Alex W. White, Welsh School of Pharmacy, King Edward VII Avenue,Cardiff University, Cardiff, CF10 3NB, UK. Tel: +44 (0)29 2087 6308; Fax: +44 (0)29 2087 4149;E-mail: whiteaw@cf.ac.uk expert  reviews http://www.expertreviews.org/ in molecular medicine 1  Accession information:  doi:10.1017/S1462399408000641; Vol. 10; e8; March 2008 & 2008 Cambridge University Press      P    r    o    t    e     i    n  –    p    r    o    t    e     i    n     i    n    t    e    r    a    c    t     i    o    n    s    a    s    t    a    r    g    e    t    s     f    o    r    s    m    a     l     l  -    m    o     l    e    c    u     l    e    t     h    e    r    a    p    e    u    t     i    c    s     i    n    c    a    n    c    e    r  molecules in a biological system are bathed in anaqueous environment, and enzyme active-siteclefts tend to shield binding sites from watermolecules that can otherwise hinder ligandinteractions.Small-molecule drug discovery againstprotein–protein targets is more challengingthan for conventional small-molecule proteintargets but represents an innovative directionfor 21st century medicine. Although the fieldhas already been the subject of several reviews(for a selection see Refs 1, 2, 3, 4, 5, 6, 7, 8, 9), inthis article we aim to give a broad overview of emerging small-molecule PPI inhibitors in theanticancer field. The concept of protein–protein interactionand ‘hot spots’ Among the reasons for the lack of modulators of protein complexesastherapeutic agents hasbeenthe notion that protein interfaces representdifficult or intractable targets because of thegenerally large and noncontiguous nature of PPI surfaces. This perceived problem iscompounded by the lack of high-throughputscreening technologies and paucity of smallmolecules in compound libraries with the sizeand functionality to modulate protein-complextargets selectively. In recent years, however,several reports have appeared that challengethe dogma that PPIs represent difficult targetsto modulate using small molecules. Inparticular, the concept of PPI ‘hot spots’ hasaided the development of small molecules thatinteract with a small area of an entire protein–protein interface to exert high-affinity andselective binding (Ref. 11).The initial challenge in developing PPIinhibitors is the discovery of specific PPIs andin turn identification of those that are‘druggable’. Two types of PPI can be defined:transient and tight. Tight interactions can beidentified more easily, through biochemicalco-purification; in contrast, transientinteractions have been characterised by site-directed mutagenesis and molecular modellingas it is often difficult to co-crystallise transientpartners (Ref. 12). Computational techniqueshave also been used to identify PPIs and theirinhibitors, and have been recently reviewed(Ref. 13). Following the identification of asuitable PPI, the next step involves study of the binding interface and ligand design. This is alsochallenging compared with designing a ligandto interact with an active-site cleft. Interactionsites are typically relatively flat with a largesurface area, although shallow grooves aresometimes seen. Ligand-binding sites may beexposed only after conformational changesconcomitant with binding of protein partners(Ref. 14).To understand better the nature of protein–protein interfaces, a study of 75 PPIsestablished a ‘standard size’ of 1600  400 A˚ 2 for an interaction surface. The smallestinterfaces were approximately 1150 A˚ 2 and thelargest interfaces identified were up to 4660 A˚ 2 (Ref. 15). Given the large sizes involved, itwould be difficult to target a small molecule tothe whole interface. Similarly, large peptide- based inhibitors can be less effective since asingle continuous peptide sequence may poorlymatch the binding surface (Ref. 12).Fortuitously, within a binding interface only asmall number of highly conserved amino acidresidues – ‘hot spots’ – are crucial for theinteraction, and thus these are often the targetfor drug design. Hot spot residues within aprotein–protein interface are less obvious toidentify than, for example, important residueswithin an enzyme active site. Although alaborious process, site-directed mutagenesis has been commonly employed to locate hot spotresidues, leading to a definition of a hot spot as‘a residue that, when mutated to Alanine, givesa distinct drop in the binding constant, typicallyten-fold or higher’ (Ref. 16). Phage display hasalso been used to detect hot spots (Ref. 17).Basedontheanalysisofcurrentlyidentifiedhotspots, the amino acids tryptophan (Trp), tyrosine(Tyr) and arginine (Arg) occur frequently, oftensurrounded by hydrophobic regions. Theseamino acids are thought to exclude solventfrom the important interacting residues and arefavoured as they can form multiple interactions.For example, despite structural similarity,phenylalanine is three times less likely to occur ina hot spot than Tyr, presumably due to the abilityof Tyr to form an additional hydrogen bond.Hot spots contain leucine, methionine, serine,threonine and valine residues less frequently(Ref. 11). However, a review of thethermodynamic aspects of PPIs concludesthat hydrophobic interactions are also a keydriving force for PPIs, and that the complexthermodynamics of PPIs are ultimately a function expert  reviews http://www.expertreviews.org/ in molecular medicine 2  Accession information:  doi:10.1017/S1462399408000641; Vol. 10; e8; March 2008 & 2008 Cambridge University Press      P    r    o    t    e     i    n  –    p    r    o    t    e     i    n     i    n    t    e    r    a    c    t     i    o    n    s    a    s    t    a    r    g    e    t    s     f    o    r    s    m    a     l     l  -    m    o     l    e    c    u     l    e    t     h    e    r    a    p    e    u    t     i    c    s     i    n    c    a    n    c    e    r  of how protein–protein binding changes theinternal structure of the molecule(s) (Ref. 18). Scope of the review Overthepastdecade,theprincipalexploitationof PPIs has been through therapeutic antibodies,such as Herceptin, which are showing greatpromise as a new class of drugs in cancertreatment (Ref. 19). Therapeutic antibodies arelarge proteins that interact directly, for example,with membrane-bound receptor proteins, suchas growth factor receptors, to elicit a biologicalresponse. Although an important class of cancertherapeutic agents exploiting protein–proteinrecognition, therapeutic antibodies (or other‘biologics’) are not included in this review;instead, we discuss the emerging class of small-molecule inhibitors of PPIs, focusing on theirapplication to anticancer therapy. We considerestablished clinical agents that fall into the PPImodulator class (such as tubulin inhibitors), butconcentrate on agents in preclinicaldevelopment (such as the p53–MDM2inhibitors). The mechanism of action of small-molecule PPI inhibitors differs from that of theantibodies in that a small, nonphysiologicalcompound designed to bind preferentially to aPPI site blocks the binding of a further protein,preventing the formation of a protein dimer/multimer that would otherwise be essential forregulating a biological process. Protein–protein interactions astherapeutic targets in cancer Microtubules One of the most successful therapeutic targetsin cancer treatment is tubulin. There areseveral natural, semisynthetic and syntheticcompounds that have significant clinicalactivity by affecting tubulin PPIs. They have been extensively reviewed elsewhere (Refs 20,21, 22, 23), but we summarise the topic herefor completeness.Microtubules are large cylindrical proteins –25 nm in diameter and up to several  m m inlength (Ref. 21). A microtubule is composed of copolymers of two monomers –  a - and b -tubulin – binding in an alternating manner.Microtubules undergo constant dynamicpolymerisation and depolymerisation at bothends of the filament (referred to as plus andminus ends), although the process is faster atthe plus end (Refs 20, 21, 24). Microtubuledynamicity is required for several biologicalprocesses including maintenance of cell shape,cellular signalling and movement; however,their best-studied role is in cell division(Ref. 21). Several drugs exert their antitumoureffect by interfering with tubulin PPIs and areclassified according to the site to which they bind on microtubules.A crystal structure of the taxane analoguetaxol (a natural product from the Pacific yewtree  Taxus brevifolia ) bound to tubulin indicatesthat it binds within a hydrophobic pocket. It issuggested that taxol stabilises microtubules by strengthening PPIs, thus inhibiting celldivision (Ref. 20). Several synthetic taxanederivatives are under clinical investigation. Inaddition, nontaxane compounds are known to bind at the taxane site and several are underpreclinical investigation, including theepothilones (Ref. 25), eleutherobins (Ref. 26),discodermolides (Ref. 27), sarcodicyins andlaulimalides (Ref. 21).By contrast to taxol, colchicine (a naturalproduct from the plant genus  Colchicum ) and itsanalogues destabilise microtubules. Althoughthe crystal structure of the tubulin–colchicinecomplex is not available, studies suggest thatcolchicine interacts with  b -tubulin at theinterface between two tubulin monomers(Refs 28, 29, 30).Vincristine and vinblastine (natural productsfrom the periwinkle plant  Catbaranthus roseus ),and new-generation analogues vinflunine andvinorelbine, also destabilise microtubules(Ref. 31). Vinblastine has been shown to interactwith  b -tubulin (Refs 20, 23, 32). Other naturalproducts, including rhyzoxin, cryptophycinsand dolastatin 10, have similar effects to thevincas (Ref. 33). p53 The tumour suppressor protein p53, knowninformally as ‘the guardian of the genome’, has been extensively studied in the field of cancer.It protects biological tissues from malignanttransformation and forms a central part of theDNA-damage response. It is estimated thataround 50% of all human tumours havemutations in the p53 gene ( TP53 ), implying thatcancer cells sustain viability by reducing the biological activity of p53 (Refs 34, 35, 36).Humanp53contains393aminoacidsarrangedin four functional domains. The N-terminus expert  reviews http://www.expertreviews.org/ in molecular medicine 3  Accession information:  doi:10.1017/S1462399408000641; Vol. 10; e8; March 2008 & 2008 Cambridge University Press      P    r    o    t    e     i    n  –    p    r    o    t    e     i    n     i    n    t    e    r    a    c    t     i    o    n    s    a    s    t    a    r    g    e    t    s     f    o    r    s    m    a     l     l  -    m    o     l    e    c    u     l    e    t     h    e    r    a    p    e    u    t     i    c    s     i    n    c    a    n    c    e    r  interacts positively with transcription machineryand with MDM2, an E3 ubiquitin ligase. Thesecond domain constitutes the DNA-bindingregion, and harbours 90% of the reported p53mutations. The third domain is responsible forthe formation of the p53 tetramer. The currentmodel of p53 function suggests that DNAinteractions occur with a tetramer form of p53(actually a dimer of a dimer) (Refs 34, 36, 37).Severaleventsareknownto induce theactivityof p53, including DNA damage, hypoxia,oncogene overexpression and viral infections(Refs 36, 38, 39). Numerous biological functionshave been attributed to p53 such as apoptosis(Refs 40, 41) and senescence (Ref. 42), G1 arrest,cell cycle control, DNA repair (Ref. 43) andangiogenesis inhibition. Among the mostimportant of these functions is apoptosis(programmed cell death), a conserved processthat mediates the elimination of a cell uponexposure to genomic stress. The completemechanism through which p53 mediatesapoptosis is still under study; it is mediatedprimarily but not exclusively by p53. Followinga stressful event, p53 activates through itsN-terminus many pro-apoptotic genes such as POXA  and  PUMA . Another suggestedmechanism is linked to the mitochondria,whereby p53 binds to the mitochondrialmembrane resulting in the formation of poresand the release of cytochrome  c ; this in turnactivates caspases, which degrade the nucleusresulting in cell death (Refs 44, 45).Under normal conditions, cellular levels of p53 are barely detectable: it has a short half-lifeof 10–20 min as a result of a PPI with MDM2that results in the negative regulation of p53(Fig. 1) (Refs 34, 35). MDM2 catalyses the bindingof ubiquitin to p53, thereby directing it todegradation by the proteasome (Ref. 46). In atypical feedback loop, p53 increases thetranscription of MDM2 (Ref. 47). Under stressfulconditions, p53 concentrations are increased dueto a reduction in its MDM2-mediated degradationand a concurrent increase in p53 transcription.As increased p53 activity has an antitumoureffect, there have been many attempts to elevatethe biological activity of p53 by increasing itsaccumulation in the cell. The main strategiesemployed have been stabilisation of p53 orprotection from ubiquitination and proteasomaldegradation, primarily by blocking the p53–MDM2 interaction (Refs 48, 49, 50). Closeexamination of the p53–MDM2 interfacereveals that it is the side chains of only threekey p53 amino acids that make most of theinteractions within a hydrophobic groove inthe surface of MDM2 (Figs 1 and 2). In anX-ray-crystallography-derived model of thep53–MDM2 interaction shown in Figure 2a, ap53-derived peptide (green) is showncomplexed with an MDM2 fragment (surfacerender) containing the hydrophobic bindingcleft. The p53 fragment forms an  a -helix fromwhich the side chains of the three amino acidsLeu26, Trp23 and Phe19 (shown left to right)project into the binding cleft, promoting thep53–MDM2 interaction (Refs 51, 52). Furtheranalysis of this hot spot revealed that the areaof the interaction site is about 300 A˚ 2 (Ref. 50)The hot spot is a good target for drug design –the strategy being to design a drug that mimicsthe conformation and interaction of the threep53 residues. Small-molecule inhibitors of p53 PPIs Although a p53–MDM2 PPI inhibitor has yet toreach clinical trials, several small-moleculeinhibitors with significant potency have beenreported in the literature. Most of thesemolecules are useful experimental tools forresearch. Chalcones (compounds derived from1,3-diphenylprop-2-en-1-one) were early-reported molecules with such activity. Theyinterfere with p53–MDM2 interactions by binding to the p53 transactivation domain withrelatively low potency: IC 50 s were in the range50–250  m M  (IC 50  is the concentration of inhibitorrequired to achieve 50% inhibition) (Ref. 53).The natural product chlorofusin is a fungalmetabolite containing a peptide segment thatwas discovered by screening over 53 000microbial extracts. It inhibits the p53–MDM2interaction with an IC 50  of 4.6  m M ; however, it isa large (molecular weight of 1363.7),structurally complex molecule that is lesspractical for drug development or clinical usethan a smaller molecule (Ref. 54). A more drug-like molecule called RITA (‘reactivation of p53and induction of tumour cell apoptosis’)(Fig. 3a) was similarly discovered by screeninga molecular library against cancer cell lines. Ittoo has micromolar potency and was reportedto interfere with p53–MDM2 interactions by binding to p53 (Ref. 55), although more-recentnuclear magnetic resonance (NMR) studies expert  reviews http://www.expertreviews.org/ in molecular medicine 4  Accession information:  doi:10.1017/S1462399408000641; Vol. 10; e8; March 2008 & 2008 Cambridge University Press      P    r    o    t    e     i    n  –    p    r    o    t    e     i    n     i    n    t    e    r    a    c    t     i    o    n    s    a    s    t    a    r    g    e    t    s     f    o    r    s    m    a     l     l  -    m    o     l    e    c    u     l    e    t     h    e    r    a    p    e    u    t     i    c    s     i    n    c    a    n    c    e    r  arguethatRITAdoesnotinfactblockp53–MDM2 binding in vitro (Ref. 56).The nutlins are a series of highly promisingmolecules for p53–MDM2 inhibition and werediscovered by high-throughput screeningfollowed by structure-based optimisation (foran example structure, see Fig. 3b). The nutlinsinterfere with p53–MDM2 interactions bymimicking the interaction of the three p53residues within the MDM2 hot spot (Fig. 1),and are highly potent (activities range between100 and 300n M ). The interaction of nutlininhibitors with MDM2 has been studied usingX-ray crystallography (Fig. 2b): the imidazolinesubstituents of the nutlin inhibitor occupysimilar regions within the binding cleft as thep53 side chains (Refs 50, 57). The BCL2 family The B-cell lymphoma 2 (BCL2) family of proteinsis composed of more than 20 members (Refs 58,59, 60). Some members are anti-apoptotic[e.g. BCL2, BCL2L1 (Bcl-xL), BCL2L2 andMCL1], whereas others are pro-apoptotic [e.g.BAX, BAK1, BID and BCL2L11 (BIM)]. Thefamily members cooperate through PPIs tomediate the intrinsic apoptotic pathway(Refs 61, 62). Anti-apoptotic BCL2 familymembers protect cells from apoptosis byinhibiting the action of pro-apoptotic members(Ref. 63). Three-dimensional NMR studies of anti-apoptotic BCL2 proteins revealed thepresence of a hydrophobic groove that acts as a binding site for the BH3 peptide domain of pro-apoptotic BCL2 members (Ref. 64). Agentsdesigned to target the binding grooves of anti-apoptotic BCL2 proteins are predicted to induceapoptosis in cancer cells (which overexpressthese anti-apoptotic proteins) by antagonisingtheir protective effect.Over the past ten years, several nonpeptidicsmall-molecule inhibitors to anti-apoptoticBCL2 proteins have been introduced andsome of them are already in clinical trials as  A conceptual diagram of a protein–protein interaction (PPI) inhibitor targeting a PPI (p53–MDM2) hot spot Expert Reviews in Molecular Medicine © 2008 Cambridge University Press LeuMDM2p53 degradationNutlinTrpPhe H 3 CCH 3 MDM2p53p53 NH Antitumour effect       N     u      t      l      i     n Figure 1. A conceptual diagram of a protein–protein interaction (PPI) inhibitor targeting a PPI(p53–MDM2) hot spot.  In this diagram, the inhibition of the p53–MDM2 interaction by a nutlin inhibitor isused as an example to illustrate the generic concept of the action of all PPI inhibitors discussed in thisarticle, since it is one of the best-characterised examples of PPI inhibition. MDM2 normally negativelyregulates the antitumour protein p53 by inducing its proteasomal degradation; however, in response tocellular stress, particularly DNA damage, p53 levels rise as a result of increased p53 transcription andreduced MDM2-mediated degradation (not shown). The nutlin inhibitors are similarly thought to increase p53levels, by blocking MDM2-mediated degradation. The PPI of p53 and MDM2 is mediated by the side chainsof three p53 amino acids (Leu, Trp and Phe) that are recognised by MDM2. The nutlin inhibitors mimic thebinding of these amino acid side chains and preferentially bind to MDM2. This physically blocks theinteraction of the two proteins, thus releasing p53 that is free to exert a beneficial antitumour effect. Thisschematic illustration is derived from the X-ray data presented in Fig. 2a, and comparison of Figs 1 and 2ashould be instructive. Abbreviation: MDM2, ‘mouse double minute 2’ homologue. expert  reviews http://www.expertreviews.org/ in molecular medicine 5  Accession information:  doi:10.1017/S1462399408000641; Vol. 10; e8; March 2008 & 2008 Cambridge University Press      P    r    o    t    e     i    n  –    p    r    o    t    e     i    n     i    n    t    e    r    a    c    t     i    o    n    s    a    s    t    a    r    g    e    t    s     f    o    r    s    m    a     l     l  -    m    o     l    e    c    u     l    e    t     h    e    r    a    p    e    u    t     i    c    s     i    n    c    a    n    c    e    r
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