Burger's Medicinal Chemistry and Drug Discovery || Design of Peptidomimetics

Burger’s Medicinal Chemistry, Drug Discovery, and Development, Seventh Edition,edited by Donald J. Abraham and David P. RotellaCopyright � 2010 John Wiley & Sons, Inc.

DESIGN OF PEPTIDOMIMETICS

JUAN J. PEREZ1, FRANCESC J. CORCHO

1

JAIME RUBIO-MARTINEZ2

1Universitat Politecnica de Catalunya,Departament d’Enginyeria Quimica,Barcelona, Spain

2Universitat deBarcelona and the Institut deRecerca en Qu�ımica Teòrica iComputacional (IQTCUB), Departament deQuimica Fisica, Barcelona, Spain

1. INTRODUCTION

Peptides are important mediators in the reg-ulation of most of the physiological processeseither through the endocrine signaling path-way or sometimes through the local paracrineand autocrine signaling pathways. Peptideselicit a plethora of functions being identifiedto serve as hormones, neurotransmitters,enzyme substrates or inhibitors, neuromodu-lating agents, and immunomodulators [1].Representative examples include angiotensinII, bombesin, bradykinin, cholecystokinin,endothelins, enkephalins, oxytocin, secretin,somatostatin, or tachykinins. The wide spec-trum of signaling activities exerted makesthem very appealing molecules for pharma-ceutical intervention, aimed at playing rolesas agonists, antagonists, or inhibitors. In ad-dition, the repertoire of possible roles for ther-apeutical intervention rendered by peptidescan be further expanded if we take into con-sideration that fragments of proteinsmay alsobe used to mediate or inhibit biological ac-tions, playing the same role as those proteinepitopes where they are originally found [2].This is currently an area of an enormousactivity, mostly focused on the inhibition ofprotein–protein interactions.

Although peptides are important signalmediators, they exhibit a low profile as ther-apeutic agents: poor oral bioavailability, lowabsorption, are easily metabolized by pepti-dases, and are immunogenic. About 15 yearsago, it was thought that biotechnology couldcope with some of the pharmacokinetic pro-blems of peptides. However, in spite of theadvances in delivery techniques in the past10 years [3–5], peptides cannot compete with

small-molecule compounds as therapeuticagents. At present, there are only a few pep-tides approved for therapeutic intervention,including oxytocin, the segment 17–36 of theglucagon-like peptide 1 receptor agonist, theparathyroid hormone, or calcitonin. All thesepeptides are about 30 residues long and canonly be delivered intracerebroventricularly orthrough nasal administration. Interestinglyafter the human genome sequencing, it hasbeen estimated that among the 5000–10,000potential drug targets available, two thirdscould be amenable to traditional small-mole-cule drugs whereas the nearly one-third left tobiopharmaceuticals [6]. Thus, there may stillbe specific treatments that could only beamenable using peptides. Accordingly, intoday’s view, peptides should be consideredas valuable molecules for therapeutic inter-vention where important research efforts inachieving more efficient delivery proceduresshould be spent and, on the other hand, asvaluable source of inspiration for the design ofsmall-molecule compounds.

The term peptidomimetic concerns the de-velopment of small-molecule mimics of pep-tides. However, it has been used in differentways in the past 30 years. Historically, theterm was coined and used in the medicinalchemistry arena, aimed at finding new leadsfor therapeutical intervention, although withtime the term has been widened and used todescribe molecules designed to emulate thestructural features of peptides. Throughoutthis review, we will specifically use the termpeptidomimetic to describe small moleculesaimed at therapeutical intervention, whileleaving the term peptide surrogate or pseudo-peptide for those molecules inspired by thestructural features (either primary or second-ary) of a peptide that still keep a peptidestructural appearance. The former view is thefocus of this review in line with the scope ofthis book.

The concept of peptidomimetic can be con-sidered to be coined with the discovery of theenkephalins, the endogenous peptide analge-sics discovered in 1975. Isolated in 1804 fromthe Papaver somniferum and synthesized in1952, morphine had long been known toelicit its analgesic action through the opiatereceptor with important adverse effects.

1

The endogenous ligand had been sought for along time, in order to get a better understand-ing of its analgesic action and with the hopethat an endogenous opiate would not exhibitthe same adverse effects. Interestinglyenough, the enkephalin sequence exhibitsmoieties that are already present in mor-phine [8]. Moreover, a more elaborated ana-lysis could be argued that enkephalins adopt atype I b-turn conformation when bound to thereceptor, mimicking well the 3D structure ofmorphine [9]. The perfect match between en-kephalins and morphine marked the startingpoint and the paradigm of peptidomimeticdesign. However, it is known nowadays fromsite-directed mutagenesis studies that pep-tides and small molecules do not necessarilyinteract in the samemannerwith the receptor,but share some receptor points at recognition,withpeptides usually exhibiting a larger num-ber of anchoring points [10].

In simple terms, a peptidomimetic may beconsidered as a small molecule that mimicsthe pharmacological action of a peptide. How-ever, most of the natural peptides mediatetheir actions in the signaling process as ago-nists, and a systematic replacement of theresidues of a peptide sequence may lead todiscover analogs with an antagonistic profile,inhibitors of enzymes, or inhibitors of otherproteins. A definition of a peptidomimeticshould be broadened to include these consid-erations. Accordingly, we will consider a pep-tidomimetic as a small molecule designed toexhibit the same pharmacodynamical profileof a set of peptideanalogsand toelicit a similarpharmacological response mediated throughthe same receptor. Alternatively, a peptido-mimetic can be considered at the molecularlevel as a small molecule that mimics some ofthe molecular recognition interactions of apeptide with its receptor, mediating the samepharmaceutical action on the therapeutictarget.

Excellent reviewsonpeptidomimeticswerewritten in the early 1990s pointing their viewson medicinal chemistry aspects. In these re-views, several definitions of peptidomimeticwere provided. Thus, Giannis and Kolter [11]defined a peptidomimetic as “a compoundthat, as the ligand of a receptor, can imitateor block the biological effect of a peptide at the

receptor level.” Wiley and Rich [12] definedpeptidomimetics as “chemical structuresdesigned to convert the information containedin peptides into small nonpeptide structures.”A year later, Gante [13] defined a peptidomi-metic as “a substance having a secondarystructure as well as other structural featuresanalogous to that of the original peptide,which allows to displace the original peptidefrom receptors or enzymes.” More recently,there is still a strong interest in designingpeptidomimetics as orally active compoundsinspired by the structures of endogenous pep-tides and peptide epitopes [14], although witha strong tendency to offer a broadened defini-tion of peptidomimetics that includes thedesign of new architectures inspired by thestructure of peptides, oriented to the design ofnanomaterials [15,16].

2. PEPTIDE FEATURES

Peptides as well as proteins are polymers ofamino acids linked through peptide bonds.Traditionally, peptides have been consideredpolymers with lengths of up to 30 residues,leaving the term protein for larger polymers.However, recently the term evolved to specifi-cally consider proteins as those amino acidpolymers that exhibit a defined tertiary struc-ture, whereas the term peptides was left tothose amino acid polymers that are unstruc-tured insolution. Indeed, thisdefinitioncoverswell the traditional one, but is more specificsince short peptide segments with precise 3Dstructures have already been reported [17].

Unstructured in solution implies that pep-tides are flexible molecules in a constantinterchange among different conformations(states). This is a consequence of the roughtopology of the potential energy surface, withmany low-energy conformations accessible atroom temperature. Since the amino acid se-quence contains all the information necessaryfor a polypeptide to adopt its three-dimen-sional structure, it can be inferred that thetopology of the potential energy surface thatdefines its conformational profile is a conse-quence of its own sequence. In the first ap-proximation, it can be considered that theconformational profile of the peptide is the

2 DESIGN OF PEPTIDOMIMETICS

result of a combination of the conformationalfeatures of each of the residues constitutingthe peptide chain. This approach, known asthe rotational isomeric model, assumes thatresidues are independent beads of a chain andthat the final conformations are the super-position of the different contributions of eachof them. Although very simplistic, this modelprovides a rough estimate of the number ofconformations attainable by the peptide. Spe-cifically, considering that if a standard residuecan attain typically three different low-energyconformations, a peptide of N residues willadopt around 3N�10N/2 different conforma-tions. More interestingly, the model also pro-vides a qualitative description of the density ofstates [18]. However, residues are not inter-dependent to each other and peptides mayexhibit conformations that result from coop-erative interactions among them. On the onehand, intramolecular interactions may pro-vide new conformations not featured in therotational isomeric model, and on the other,they may also discard other structures asunattainable due to steric hindrance. Thebalance between new and unattainable con-formations may be the explanation of thebimodal distribution of states observed in realcharacterizations of the conformationalspace [19].

The conformational profile of a peptide isalso modulated by the immediate environ-ment. Depending on its nature, the solventmay enhance or damp certain conformationsin solution. Thus, in water peptides tend toexhibit a more flexible behavior than in othersolvents. On the other hand, there are struc-turing solvents such as trifluoroethanol thatare capable of enhancing the structure of apeptide in solution. This is also found incrystal structures where depending on themother liquor the peptide may exhibit differ-ent conformations [20]. There are two solventfeatures that may affect the attainability tothe accessible states of peptides in solution:on the one hand, the solvent dielectric con-stant, affecting long-range interactions, andon the other, the capability of the solvent toelicit direct solute–solvent hydrogen bondinginteractions. This is particularly evidentin shorter peptides that do not have a suffi-cient number of intramolecular interactions.

Accordingly, it can be said that peptides areexpected to be flexible molecules due to theirrough conformational landscape that givesrise to consider in general their observablestructure as an average image of differentstructures attainable according to theirsequence and subject to the immediateenvironment.

3. PEPTIDOMIMETIC DESIGN PROCESS

In order to design a small molecule that ex-hibits a similar pharmacodynamic profile ofthe original peptide with improved pharma-cokinetics, it is necessary to have a reason-able hypothesis of the chemical moieties ofthe peptide responsible for the interactionwith the therapeutic target as well as knowl-edge of their spatial distribution. This is re-ferred as construction of a pharmacophorehypothesis.

The very first step in the design process isto establish structure–activity relationshipfrom a set of analogs derived from the originalpeptide. The goal of this first step is twofold: toestablish the shortest peptide segment thatretains activity and to identify those residuesthat are important for function. The formerinformation is established through themeasurement of the activity and affinity ofsystematically shorter segments of the origi-nal peptide, whereas the latter information isnormally obtained through the synthesis ofanalogs obtained by replacement of the resi-dues derived from the original sequence. Inthis process, it is very useful to perform analanine scan, where different analogs aresynthesized from a systematic replacementwith alanine at each position of the sequence.Another strategy involves conservativesubstitutions of the residues through the re-placement of residues with similar chemicalproperties, or less conservative substitutionsthat will provide information about the neces-sity of specific interactions in certain posi-tions. Also, the substitution of L- for D-aminoacids may provide information about thebioactive conformation. Itmaybe the case thatin the process of residue substitutions an ana-log with antagonistic profile may be produced.For this reason, it is necessary to characterize

PEPTIDOMIMETIC DESIGN PROCESS 3

both the binding and the biological activity inregard to the reference peptide along theprocess.

In the second step, additional structure–activity relationships need to be worked out tounderstand the conformational features ofthe bioactive conformation. Accordingly, allefforts need to be devoted to develop a phar-macophoric hypothesis of the interaction ofthe peptide with the receptor. Obviously, thebest scenario is to have an experimental struc-ture of the peptide bound to its receptor (bioac-tive conformation). In this way, we know thebound conformation and the spatial distribu-tion of the side chain moieties or backboneelements involved in the recognition with thereceptor. This is possible nowadays for thedesign of enzyme inhibitors, with great suc-cess aswill be shown later. This procedure canalso be successfully used in the design of pro-tein–protein inhibitors. In contrast, this is notthe case for peptides that mediate their func-tion through the interaction with membraneproteins such as G-protein-coupled receptor(GPCR) for which only the structures of rho-dopsin and a- and b-adrenergic receptors areavailable.

In theabsence of anexperimental structureof the complex, a pharmacophoric hypothesisof the peptide-receptor recognition needs to bedeveloped. This represents a combined effortinvolving peptide synthesis, biophysicalmethods, computationalmethods, pharmacol-ogy, and molecular biology [20]. In the case ofGPCRs, in contrast to previous models, aimedat providing an explanation to the basal activ-ity shown by certain GPCRs and the existenceof reverse agonists, the accepted model for theactivation is the so-called two-state model. Inthis model, antagonists are supposed to bindto the inactive conformation whereas agonistsare suited to bind to the active conformation.Although most of the GPCR-mediated pepti-domimetics designed are antagonists, agon-ism will represent to find a different boundconformation and consequently the samemethodology might be applied.

Figure 1 shows pictorially a generalscheme regarding the process of peptidomi-metic design. At the top of the left-hand side, anew sequence has been identified as impor-tant biological mediator. A process of analog

synthesis is followed in order to understandthe key residues involved in the interactionand the minimum length required. The nextstep involves the synthesis of conformation-ally constrained analogs including cyclic ana-logs and peptidomimetics that still incorpo-rate much of the peptide chain. This results inthe establishment of the chemical require-ments needed for recognition as well as theirspatial arrangement. On the other hand, dif-ferent information is accumulated on theright-hand side of Fig. 1. Screening of differentsources can identify new hits providing infor-mation about the chemical structures of bin-ders, augmenting our knowledge about thediversity of scaffolds possible. On the otherhand, the use of molecular modeling techni-ques and biophysical methods can provide awealth of information regarding the hypoth-esis of ligand–target interaction throughpharmacophore development or the charac-teristics of the binding through structuralanalysis. The information provided by thesemethods together with the information pro-vided from the first-generation peptidomi-metics and the new hits can be put togetherto design second-generation peptidomimeticsready for optimization.

4. LOCAL CONSTRAINTS ON PEPTIDECONFORMATION

Design of analogs in this category involvesmodifications of the peptide backbone, suchas methylation of the amide nitrogen or the a-carbon, modifications of the peptide bond,modifications of the side chains, or the synth-esis of cyclic analogs. These modifications areaimed at modifying the conformational fea-tures of the amino acids, basically restrictingtheir conformational freedom or impeding theformation of secondary structures by the elim-ination of typical hydrogen bonding sites [14].

Modifications of the backbone includemethylation of the hydrogen amide giving riseto N-methylated residues (structure 1 inFig. 2) [21] or substitution of the alpha hydro-gen by an alkyl chain giving rise to the Ca,a

disubstituted amino acids (structure 2 inFig. 2) [22]. Modifications of the amino acidicnature of the backbone are referred to as


isosteric or isoelectronic exchange of the con-stituting units. These modifications changethe capacity of the backbone to elicit hydrogenbond interactions and may also change itsconformational degrees of freedom. For exam-ple, the amino nitrogen can be substituted byoxygen or sulfur atoms giving rise to the cor-responding depsi derivatives (structure 3 inFig. 2) found in nature [23]; substitution of theCa group by a nitrogen atom gives rise to theso-called aza derivatives (structure 4 inFig. 2) [24]; reduction of the peptide bondrepresents another interesting backbonemod-ification (structure 5 in Fig. 2) [25]. Largermodifications of the backbone include theretro-inverso peptides (structure 6 in Fig. 2),where the configuration of the Ca is changed

and the direction of peptide bond is re-versed [26], or peptoids (structure 7 in Fig. 2),polymers constituted by amino acids wherethe side chain is linked to the backbone amidenitrogen [27,28]. Alternatively, the number ofcarbon atoms of the residue can be enlarged byattaching the amino group in an atom differ-ent from that in a- in regard to the carboxylicmoiety, giving rise tov-amino acids (structure8 in Fig. 2). In particular, b- [29] and g-resi-dues [30] have been used extensively. Thesemodifications provide a plethora of possibili-ties that can be used to generate new analogsto help understand the role of backbone atomsin the recognition process.

Modifications of the side chains can be donewith the aim to complement the repertoire of

Residue identificationminimum length

Screeningcomputer modelingbiophysical methods

Hitsstructural information

Contrained residuesdipeptide analogs

First generation peptidomimetics Second generation peptidomimetics

Figure 1. Scheme of the process followed to design peptidomimetics.

LOCAL CONSTRAINTS ON PEPTIDE CONFORMATION 5

chemical moieties available from the 20 nat-ural amino acids. For example, the a-amino-butyric acid (Abu) belongs to this category andcan be considered as a threonine lacking thehydroxyl group (structure 9 in Fig. 3); or dif-ferent aromatic moieties, for example,nitrophenylalanine (Phe(NO2)) (structure10 in Fig. 3) or naphthalenealanine (Nap)(structure 11 in Fig. 3).

More interesting are those amino acidswith side chains that limit the conformationalfreedom of the amino acid [22,31]. There is animportant group of residues generated by re-placement of the alpha hydrogen by an alkylchain giving rise to the so-called Ca,a disub-stituted amino acids. The substitution can becarried out with another linear chain or boththe Ca and the Cb can be part of a cyclicstructure. The simplest representative of theformer group is the a-aminoisobutyric acid(Aib) (structure 12 in Fig. 3), which can beconsidered as an alanine with a methyl group

in place of the alpha hydrogen. In the lattergroup, the Ca is the vertex of a cycle, the 1-aminocyclopropanecarboxylic acid (c3c) (struc-ture 13 in Fig. 3) being its simplest represen-tative. Another example is the 2-aminoin-dane-2-carboxylic acid (Aic), shown as struc-ture14 inFig. 3. Also in this category dehydro-amino acids exhibiting a double bond betweenthe Ca and the Cb can be included. As exampleof this kind of residues, structure 15 in Fig. 3depicts pictorially dehydrophenylalanine(DPhe) [32]. Another class of residues exhibitscyclic structures involving two atoms of thebackbone like in proline, for example, thepipecolic acid (structure 16 in Fig. 3) or morecomplex residues such as the tetrahydroiso-quinoline-3-carboxylic acid (Tic) shown asstructure 17 in Fig. 3. All these residues, inaddition to offering a great deal of side chaindiversity, also constrain the conformationalfreedom of the residue, resulting in useful

Figure2. Diverse isostere replacements and substitutions Sof thepeptide backbone: (1) a regular amino acidthat is represented for comparisonpurposes; (2)Ca substituted residue; (3) depsi derivative; (4) azaderivative;(5) peptide bond reduction; (6) retro-inverso peptide; (7) peptoid; v-amino acids.


tools to get insight into the features of thebound conformation of the peptide.

An alternative procedure to the synthesis ofconstrained analogs using unnatural aminoacids consists of designing dipeptide analogsby bridging atoms of the peptide backbone,backbone-side chains, or even side chains gen-erating heterocycles of different lengths[11,13,33,34]. Molecules produced in thisway are half peptide and half small molecule.

These compounds in addition to being lessvulnerable to peptidase degradation, nor-mally exhibit a bent structure that may helpto produce analogs closer to the bioactiveconformation.

This has been published in the literature indifferentways. For example, bridging throughbackboneatomstypically yieldspiperazinones(structure18 inFig. 4), or if thebridgingoccursbetween the side chains of two consecutive

Figure 3. Side chain modifications: (9) a-aminobutyric acid (Abu); (10) nitrophenylalanine; (11) naphthy-lalanine; (12) a-aminoisobutyric acid (Aib); (13) 1-aminocyclopropanecarboxylic acid (c3c); (14) the 2-ami-noindane-2-carboxylic acid (Aic); (15) dehydrophenylalanine; (16) tetrahydroisoquinoline-3-carboxylic acid(Tic)

LOCAL CONSTRAINTS ON PEPTIDE CONFORMATION 7

residues, it yields mono- and bicyclic lactams(structure 19 in Fig. 4). Whereas cyclizationperformed between the backbone amide nitro-genandsideatomsyieldsafive-memberedringlactam (structure 20 in Fig. 4), bridging theCa

atom of one residue with the nitrogen amideyields either five- or six-membered ringlactams (structure 21 in Fig. 4). Similarly,bridging between the side chain of cysteine,threonine, or serine and the amide nitrogenyields oxazolones, oxazolines, and oxazoli-dines (structures 22–24 in Fig. 4) or the corre-sponding thio-derivatives [35].

An alterative strategy to produce local geo-metrical restrictions is to use an organic scaf-fold where different peptide chains are at-tached in a suitable orientation. This ap-proach has originated a series of interestingcontributions in which peptidomimetics weredesigned in this way. Different scaffolds havebeen used including cyclohexane or steroidsalthough monosaccharides have been exten-sively used for this purpose [13,36]. However,these analogs have the disadvantage to bedesigned without using a clear model of thesecondary structure. There is a long list ofdipeptidemimetics thatworkas scaffoldswiththe purpose of providing a specific secondary

structure to the peptide chain that will bediscussed later. Scaffold peptidomimetic de-sign offers the great advantage that can beused in combinatorial chemistry to producepeptidomimetic libraries [37].

5. GLOBAL CONSTRAINTS ON PEPTIDECONFORMATION

Whereas substitution of different residues of apeptide chain by constrained amino acids pro-vides localbents to thestructureof thepeptide,cyclization represents a procedure to globallyrestrain its conformational space [38]. Cyclicpeptideshaveshownto(1)increaseagonistandantagonist potency; (2) withstand proteolyticdegradation; (3) increase receptor selectivity;(4) enhance bioavailability; and (5) provideconformational insight for receptor bind-ing [39]. Cyclization can be done through con-densation of theN- and C-termini, by connect-ing functionalized side chains, through a link-agebetweentheN-terminusandasidechainortwo backbone atoms. There are different ex-amples of cyclic peptides in nature [40]: forexample, oxytocin and vasopressin are con-nected through a disulfur bridge, whereas in

Figure 4. Structures of dipeptide isostere analogs to constraint the peptide backbone conformation. See textfor details.


cyclosporinthecyclization isperformedbycon-densation.However, it should be stressed thatfor designing purposes cyclic peptidesmay notnecessarily provide good results, since cycliza-tion may force moieties important for ligandrecognition to adopt a conformation facing theinterior of the structure [41].

6. ASSESSMENT OF THE BIOACTIVECONFORMATION

The synthesis and evaluation of a series ofpeptide analogs may provide enough informa-tion to generate a hypothesis about the im-portant moieties involved in recognition andabout the three-dimensional arrangement.Depending on howwell defined is this hypoth-esis, it could be used to find hits using an insilico approach [42–44]. These results willimprove the knowledge of the necessary re-quirements for binding and eventually activa-tion of the receptor.

Theoretical procedures concern the assess-ment of the bound conformation of the peptideusing indirectmethods through the analysis ofthe conformational profile of a set of analogsincluding binders and nonbinders. Once theirconformational profile is established, thebound conformation may be assessed usingthe paradigm that active compounds share incommon a conformation (the bound conforma-tion) that is not found in the conformationalprofile of the nonbinders. A critical step is theselection of the analogs to perform the analy-sis, since the reason for an analog to be anonbinder can be that it does not exhibit thenecessary chemicalmoieties or because it can-not adopt the right conformation to have themproperly arranged. There are a number ofissues to be considered for the computationalapproach including the representation of thepeptide, the inclusion of the solvent, and eventhe strategy to search the conformationalspace. The reader is referred to previousreviews in this regard [44].

7. FIRST-GENERATIONPEPTIDOMIMETICS

If there is not enough information to builda pharmacophoric hypothesis, medicinal

chemistry can help to provide a step furtherin the design of a first generation of peptido-mimetics. One strategy is to suppose thesequence exhibits either a b- or a g-turn andsubstitute the dipeptide responsible with arigid organic scaffold [45–48]. g- or b-turnschange the direction of the peptide chain andare consequently assumed to be on the sur-face, and therefore they may be importantepitopes in the ligand-receptor recognition.Figure 5 depicts pictorially the most relevantturns found in proteins and peptides. Fordesigning purposes, it is useful to learn thata b-turn forms 10-membered cycles, whereasa g-turn renders a 7-membered cycle.

Dipeptidemimicking is carried out throughthe substitution of the turn cycle by a hetero-cycle framework that restricts the geometry ofthe peptide backbone in a similar fashion.Specifically, in the case of b-turns the hetero-cyclemimics the features of residues i þ 1 andi þ 2. Several scaffolds have been suggestedin the literature for this purpose [49], includ-ing a nine-membered ring lactam [50] (struc-ture 25 in Fig. 6); other cycles include the 4-(2-aminoethyl)-6-dibenzofuranpropionic acid(structure 26 in Fig. 6) or the 6,60-bis(acyla-mino)-2,20-bipyridine-based amino acid(structure 27 in Fig. 5) [13,51]. Other scaffoldsare the N-substituted benzimidazoles (struc-ture 28 in Fig. 5 [52]; structure 29in Fig. 6 [53]). Benzodiazepines have also beenwidely used to mimic b-turns with success(structure 30 in Fig. 6) [54]. The 1-azaoxobi-cycloalkane skeleton has demonstrated to ex-hibit a high degree of versatility that gives riseto a range of different bicycles including var-ious ring sizes and heteroatoms embedded.Figure 6 depicts pictorially different struc-tures available (structures 31–33) [55,56].

A great deal of interest has also beendevoted to design cyclic structure mimeticsof g-turns. According to the features of thissecondary structure motif (see Fig. 5), seven-membered rings have been widely chosen astemplate scaffolds [13] including azepinones(structure 34 in Fig. 7) [57,58], diazepinones(structure 35 in Fig. 7) [59], and even six-membered ring lactams have also been usedsatisfactorily [60] (structure 36 in Fig. 7).

FIRST-GENERATION PEPTIDOMIMETICS 9

Figure 5. Schematic representation of the interaction involved in a b-turn (types I and II) and a g-turn

Figure 6. Structures of different dipeptide isosteres designed to provide a b-turn. See text for details.


8. SECOND-GENERATIONPEPTIDOMIMETICS

First-generation peptidomimeticsmay be con-sidered molecules halfway between peptidesand small molecules. Although they may notyet exhibit a good pharmacokinetic profile,they can provide useful information to under-stand the spatial arrangement of the neces-sary moieties involved in the ligand–peptideinteraction and can be considered as a logicalstep to refine the pharmacophore of the inter-action. A note of caution should be raisedregarding definition of a peptide–receptorpharmacophore since the interaction surfacebetween a peptide and its receptor is normallylarge and a competitive inhibitor or anantago-nist may be blocking only part of this largerarea. In this sense, it should be also taken intoaccount that different families of compounds,although competing with the same peptideligand, may not actually bind at the same spotand, consequently, may not obey the samepharmacophores.

Second-generationpeptidomimeticsshouldbeconsideredmoredrug-likemolecules,whosedesign isbasedonall the informationgatheredfrom the previous structure–activity studies.These molecules in a general sense can bethought as scaffolds either based or not onconformational restrictions and on the struc-ture of possible hits obtained from screeningeither through high-throughput screening or

insilico,with thepropermoietiesarrangedinasuitable manner.

9. PEPTIDOMIMETIC DESIGN CASESTUDIES

There is an abundant literature describinginteresting stories about the improvementand discovery of new peptidomimetics in dif-ferent areas and using different approaches.Instead of providing an exhaustive list of ex-amples, we have decided for the present re-view to describe a few example case studiesselected on different kinds of peptide targets,suggesting the reader to browse on the differ-ent reviews on peptidomimetics quoted in thereference list for further information. Specifi-cally,webrieflydescribe a fewexamples cover-ing three broad groups of peptidomimetics:enzyme inhibitors, antagonists of GPCRs, andprotein–protein inhibitors.

10. ENZYME INHIBITORS

Enzymes are involved in the regulation of keybiological processes. Precise regulation oftheir activity is critical for organism survival,as deviations from their activity can be harm-ful. Accordingly, enzyme inhibition can beconsidered in some cases as a good strategyfor therapeutic intervention. Inhibitors havespecifically been designed in viral infections,as antimalarial agents, as regulators of bloodclot formation, as anti-inflammatory agents,or as anticancer agents, among others.

Fromastructuralpointofview,thecapacityof enzymes to catalyzebiochemical reactions isdue to their ability to stabilize transition-statespecies [61]. The use of this hypothesis to de-sign inhibitors was successfully used for thefirst time in the early 1970s [62], and thisparadigm still represents a valid approach todesign first-generation enzyme inhibitors.Transition-state analog inhibitors are peptideanalogs that exhibit a structure similar to thesubstrate, but with the scissile amide bond ofthe substrate sequence replaced by an aminoacid containing groups suchashydroxymethy-lene, hydroxyethylene, or hydroxymethyla-mine or with a chelating moiety in the case ofmetalloproteases that mimic the transition

Figure7. Structures of different isosteresdesignedto provide a g-turn. See text for details.

ENZYME INHIBITORS 11

state [63]. From the information gathered,subsequent structural studies involving theenzyme–inhibitor interaction of these first-generation peptidomimetics provide the op-portunity to develop hypothesis of the impor-tant features involved in the enzyme–ligandinteraction,whichinturnallowsthedesignofanew generation of inhibitors with improvedcharacteristics [64].

10.1. Inhibitors of the HIV-1Protease [65,66]

Among the different groups of enzymes de-scribed in the literature, proteases representone of the largest. These enzymes are respon-sible for the cleavage of a peptide bond of apeptide sequence with high specificity andefficacy. Proteases can be classified into sixdifferent groups depending on the key resi-dues involved in the active site, includingserine, threonine, cysteine, asparticacid, glu-tamic acid, and metalloproteases. The me-chanism of action of the former three involvesa nucleophilic attack of a serine, a threonine,or a cysteine to the peptide bond, respectively,whereas the latter three require the nucleo-philic attack of a water molecule through atetrahedral intermediate state. In order forthe different proteases to exhibit substrateselectivity, the active site of a protease typi-cally consists on a cleft capable of accommo-dating three to nine residues [67].

The HIV-1 aspartic protease is responsiblefor processing the viral polypeptide chain ne-cessary for the organization of structural pro-teins and the release of viral enzymes duringinfection. Thus, inhibition of the protease re-sults in immature and noninfectious virions.Stories of the design of potent inhibitors havebeen recently reviewed [65,66,68]. HIV-1 pro-tease was shown to cleave Tyr-Pro or Phe-Pro sequences in the viral polypeptide. Thisinformation leads to the design of an initialcollection of inhibitors of HIV-1 designed astransition-state analogs based on previousexperience of other proteases [69]. After sometime, the first two inhibitor–protease struc-tures with bound MVT-101 (structure 37 inFig. 8) and JG-365 (structure 38 in Fig. 8),respectively, gave structural clues that wereused to design more potent compounds.

The structure of the HIV protease is adimer consisting of two identical 99 aminoacid chains, each exhibiting a four-strandedb-sheet formed by N- and C-terminal b-strands. The enzyme active site is located atthe interface of the two subunits and containsthe catalytic triad (Asp25-Thr26-Gly27) respon-sible for the protease activity. Interestingly,the 3D structure reveals that each monomerexhibits a flap whose conformation changessignificantly when an inhibitor is bound. Thebinding cleft contains hydrophobic residuesand is long enough to accommodate peptidesof approximately six to eight amino acids.Inhibitor residues are numbered toward theC-terminus of the polypeptide chain in regardto the scissile bond asP0

1,P02, andP0

3, whereasthey are called P1, P2, and P3 toward the N-terminus. Similarly, the corresponding sub-sites of the protease binding cleft accommo-dating the different inhibitor side chains aretermed S1–S3 and S01--S03, respectively.

The crystallographic structures of the com-plexeswithMVT-101 and JG-365were used todesign improved inhibitors. These new mole-cules were developed simultaneously in dif-ferent laboratories following a similar strat-egy: the design of different in silico analogsand the assessment of their binding affinitiesusing different methods. Interestingly, thepredicted binding affinities correlated well inall the cases with the IC50 obtained experi-mentally [70–72]. These studies led to thefirst-generation inhibitors of the protease pre-sently in use, including the first one approvedby the FDA: saquinavir (39), ritonavir (40),indinavir (41), or nelfinavir (42) in Fig. 9.

Rational design of second-generation non-peptide inhibitors began with the discovery ofthe conserved water molecule mediating con-tacts between the carbonyl oxygen atoms ofthe transition-state analog inhibitors and theamide group of Ile50 of the enzyme pocket.This tetrahedrally coordinated water is re-ported in practically all the HIV proteasestructures, and its displacement had beenpostulated as a possible way of obtaininghighly specific protease inhibitors with in-creased affinity. Chenera et al. [73] identifiedcore structures containing trans-1,4-cyclohex-anediol or hydroquinone by using molecularmodeling and showed that six-membered


ringswith twooxygenatoms inparamimic thewater function. Adequate substitutions in thescaffold led to the discovery of inhibitors withaffinities as low as 7mM. However, the bestcandidate obtained in this series lackedhydro-phobic moieties to fill the S2 subsite. A moresuccessful approachwas carried out atDupontMerck [74] that led to the design of very potentinhibitors. The process started with the gen-eration of pharmacophores derived from 3Dmodels of a previously designed diol transi-tion-state analog bound to the active site ofHIV protease. The search of this pharmaco-phore in different chemical databases pro-vided 43 (Fig. 10) that, in addition to fit theinitial search criteria, also exhibited a meth-oxy phenyl ring mimicking the structuralwatermolecule.Due to the fact that thephenylring does not adequately orient all substitu-ents of the inhibitor, a cyclohexanone ringcontaining the water mimic was proposedinstead. Subsequent enlargement of this ring

to a seven-membered ring allowed the incor-poration of a diol moiety and was shown tobind the receptor with high affinity. Furthermodeling studies were performed to deter-mine the optimal stereochemistry and confor-mation required for an optimal interactionwith the active site. Conformational analysisof designed cyclic ureas established the pre-ferred conformations of the ringwhen theureanitrogen is substituted. The preferred confor-mation subsequently confirmed by small-molecule crystallography studies was the4R,5S,6S,7R stereoisomer. These new scaf-folds were considered as good potential HIVprotease inhibitors because they were able toproperly place their substituents into the dif-ferent subsites of the binding cleft, to displacethe structural water 301, to interact with bothAsp25, and finally, they were syntheticallyfeasible. Substitutions in the ring predictedto fill S1 and S2 sites led to subnanomolarinhibitors suchasDMP-450 (44) andDMP-323

Figure 8. Structures of different first-generation HIV protease inhibitors. See text for details.


Figure 9. Structures of diverse second-generation HIV protease inhibitors. See text for details.


(45) (see Fig. 10). The predicted interactions ofthese inhibitors were later confirmed by X-raystudies. The development of DMP-450 is nowhalted after early clinical trials.

10.2. Inhibitors of Ras FarnesylTransferase [75,76]

In the previous case study, the inhibition of aprotease was shown to be a useful antiviralstrategy. Enzyme inhibition can also be usedto avoid a harmful protein function. Specifi-cally, there are many proteins that are notactive without certain posttranslational mod-ifications that are catalyzed by enzymes. Rasproteins are involved in the signal transduc-tion process and require a farnesyl group forbeing fully active. Inhibition of this posttran-slational modification was proposed as aninteresting mechanism for the treatment ofcertain kinds of cancer [77]. Unfortunately,recent results have demonstrated lower effi-cacy for these drugs than expected [78]. Atpresent, tipifarnib is a Ras inhibitor in clinicaltrials for the treatment of certain types of

hematological andsolid tumors. Interestingly,some of the inhibitors developed have beendemonstrated effective in parasites and arepresently being studied for the treatment ofinfectious diseases such as malaria [79].

Ras proteins are plasma membrane-boundGTP binding proteins involved in mitotic sig-nal transduction [80]. Mutations that consti-tutively activate Ras result in uncontrolledcell growth and play a critical role in malig-nant transformations [81]. As mentioned be-fore, Ras requires farnesylation and subse-quent plasma membrane association for itstransforming activity. This posttranslationalmodification is mediated by farnesyltransfer-ase (FT), which transfers a farnesyl moietyfrom farnesylpyrophosphate to the cysteine ofthe CA1A2X motif (C¼ cysteine; A1 and A2¼isoleucine, leucine, or valine; X¼methionineor serine)C-terminal tetrapeptide ofRas. Pep-tide analogs with the sequence CAAX, such asCys-Val-Phe-Met (CVFM), show inhibition ofRas farnesylation.

Different conformationally constrained in-hibitors of FT have been described in the past.They are modified tetrapeptides with the se-quence CAAX where the middle two residueshave been replaced by conformationally con-strainedaminoacids, byanorganic scaffold, orby N-alkylated glycine derivatives.

Compound BZA-5B (46, Fig. 11) was thebest inhibitor of a series of compounds basedon the replacement of the aliphatic portionof the CAAX tetrapeptide by a 3-amino-1-carboxymethyl-5-phenyl-benzodiazepin-2-one (BZA) [82,83]. A different approach usedthe hydrophobic spacer 4-aminobenzoic acid(4ABA) to link Cys and Met [84,85], Cys-4ABA-Met (47, Fig. 11) being the most potentanalog. The rationale used in this work was tosuppose that the N-terminal cysteine of theCAAX peptide binds to the zinc ion through athiol with the subsequent looping around thezinc ion in order to allow additional coordina-tion to the metal through the terminal carbox-ylate. Following the same hypothesis, a che-mical series of compounds using an imidazolegroup prompted a series of quinolones thatlead to tipifarnib (48, Fig. 11) (R115777) pre-sently in clinical trials for the treatment ofsolid cancers [86].

Figure10. Structuresof diverse second-generationHIV protease inhibitors. See text for details.


A different group of inhibitors was discov-ered using alternative procedures. Thus, froma random screening of an extensive chemicalcollection of antihistamines, the 8-clorobenzo-cycloheptapyridine derivative SCH44342 (49,Fig. 11) was found to be a FT inhibitor, andconstituted an early lead compound (IC50¼250nM) [87]. From this compound, a series ofderivatives was developed with the aim ofoptimizing its affinity and selectivity. Thesestudies resulted in molecules with FT inhibi-tion constants in the lownanomolar range andabout10,000-foldmoreselectiveforFTthanforthe structurally related geranylgeranyltrans-ferase (GGT). All these compounds are char-

acterized by a tricycle core composed of twosix-membered aromatic rings, fused to a cen-tral seven-memberedring.Oneexampleof thisseries is the tricyclic compound Lona-farnib(SCH66336) (50, Fig. 11), which is presentlyundergoing clinical trials as anticanceragent [88,89]. Attempts made to define thebound conformation of this class of inhibitorsby using molecular modeling techniques wereunsatisfactory.ThiscanbeduetothesizeoftheFT active site cavity that binds farnesylpyro-phosphate and the carboxyl terminal residuesof the Ras protein that is much larger than asingle tricycle inhibitor. Another family of dis-closed FT inhibitors is the 2,3,4,5-tetrahydro-

Figure 11. Structures of different Ras farnesyltransferase inhibitors. See text for details.


1-(imidazol-4-ylalkyl)-1,4-benzodiazepines(BMS-214662) (51, Fig. 11) [90]. Low nanomo-lar inhibitors were identified that are able torevertthetransformedphenotypeofRastrans-formed cells at submicromolar concentrationsand that can prevent the anchorage-indepen-dent growth in soft agar of Ras transformedcells at concentrations as low as 160nM.

11. GPCR LIGANDS

The problem of designing peptidomimeticsassociated to GPCR ligands has been ham-pered in the past by the lack of an experimen-tal structure of the ligand–receptor complex.At present, there are only three GPCR struc-tures available and there is an interestingdiscussion about the analysis of their simila-rities [91,92]. Accordingly, all the informationgathered needs to be retrieved from the diffe-rent structure–activity studies available.There are many stories regarding this kind ofligands, mostly related to the design ofantagonists.

11.1. Design of Bradykinin B2 Antagonists

Bradykinin (BK) is a linear nonapeptide hor-mone of sequence Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9, produced from its pre-cursor kininogen by the action of a group ofproteases called kallikreins. The peptide hor-mone is released in response to inflammation,trauma, burns, shock, allergy, and some car-diovascular diseases, influencing vasculartone and permeability and decreasing bloodpressure [93]. BK actions are mediatedthrough two G-protein-coupled receptors: theB1 andB2 kinin receptors. B1 has a low level ofexpression in normal tissues, being expressedduring trauma or inflammation [94]. On theother hand, the B2 kinin receptor is constitu-tively expressed by different cell types [93]. B2

receptor agonists have been sought in the pastfor the treatment and prevention of variouscardiovascular diseases, such as hyperten-sion, ischemic heart disease, congestive heartfailure, as well as diabetic disorders. B2 an-tagonists are also sought to reduce the effectsmediated by BK, such as pain and inflamma-tion in several diseases (asthma, rhinitis, andseptic shock) [95].

The discovery of the antagonistic profile ofthe [D-Phe7]bradykinin analog led, in the late1980s, to the characterization of the first-gen-eration B2 bradykinin antagonists, includingNPC349, NPC567, and NPC17731 [96]. Ana-lysis of the results obtained from biophysicalstudies [97] suggested that the peptide exhi-bits ab-turn involving the last four residues ofthe peptide. These studies led to the synthesisof HOE140 (icatibant) (52, Fig. 12), a peptideanalog of sequence D-Arg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-D-Tic7-Oic8-Arg9 (where Hypstands for hydroxyproline; Thi, b-(2-thienyl)-alanine; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Oic, (2S,3aS,7aS)-octahy-droindole-2-carboxylic acid) that contains sev-eral conformational constrained residues toenhance the secondary motif at the C-termi-nus [97]. The pharmacology of this compoundhas been extensively studied and presentlyhas been approved for the treatment of acutehereditary angioedema [98].

The first nonpeptide antagonist describedwas WIN64338 (53, Fig. 13) [99] based on alow-profile pharmacophore. Although thismo-lecule exhibits a reasonable binding affinityfor the B2 receptor in rat tissue models, thecompound exhibits a low affinity for the hu-man receptor.A fewyears later, the first orallybioavailable B2 antagonists were described,such as FR173657 (FK3657) (54, Fig. 13) [100]or LF-16-0335C (55, Fig. 13) [101], and morerecently the compound MEN16132 (56,Fig. 13) [102] about 30 times more effectiveB2 antagonist than HOE140. These com-pounds are presently in clinical trials.

11.2. Design of Endothelin Antagonists [103]

Endothelins (ET) are 21-residue peptides par-tially constrained by two disulfide bonds withpotent vasoconstrictor properties. There arethree isoforms described, known as ET-1, ET-2, and ET-3 [104,105], that mediate their ac-tion through at least two different G-protein-coupled receptors: ETA and ETB [106].The former receptor is selective for ET-1 andET-2, whereas the latter binds ET-1, ET-2,and ET-3with equal affinity [107]. Upon bind-ing ET initiate a complex signal transductioncascade including activation of variouskinase-mediated pathways involved in

GPCR LIGANDS 17

Figure 12. Structure of HOE140.

Figure 13. Structures of different B2 bradykinin antagonists.


mitogenic responses [108]. ET affects cell pro-liferation in various types of cells; they alsomodulate apoptosis induced by serum starva-tion and chemical treatment and act as survi-val factors for fibroblasts, endothelial, andsmooth muscle cells [109]. ET-1 may play apivotal role in the pathogenesis of cell growthdisorders such as cancer, restenosis, and be-nign prostatic hyperplasia. Great efforts havebeen devoted in the past to block the endothe-lin system, leading to the discovery of a largenumber of peptide and nonpeptide receptorantagonists. Most preclinical and clinicalpharmacological studies reported on endothe-lin receptor antagonism are focused on their

therapeutic potential in relation to pulmonaryarterial hypertension (PAH)andheart failure.

Early efforts to design ET antagonists pro-duced two results simultaneously. On the onehand, the identification of the cyclic peptideBE-18257B (57, Fig. 14) as anETA antagonist,found as a fermentation by-product of Strep-tomyces misakiensis [110], and on the other,the peptide analog [Dpr1,Asp15]ET-1 (whereDpr is diaminopropionic acid), designed byreplacement of the Cys1–Cys15 disulfide bondof ET-1 by an amide bond between the sidechains of Dpr1 and Asp15 [111]. Shortly after,chemical modification of BE-18257B yieldedBQ-123 (58, Fig. 14), an antagonist in thenanomolar range [112]. The same authors, in

Figure 14. Chemical structure of BE-18257B (57) and BQ-123 (58), two cyclic peptide antagonists ofendothelin.

GPCR LIGANDS 19

the course of a structure–activity relation-ships study of BQ-123, observed that somestructural alterations intensified ETB ratherthanETAaffinity of someof theanalogs. Theseresults led to the development of BQ-788(59, Fig. 15), a selective ETB antagonist [113].

In parallel, structure–activity relationshipstudies carried out on the peptide establishedthat the C-terminal hexapeptide His-Leu-Asp-Ile-Ile-Trp or (16–20)ET-1 was the mini-mum fragment preserving biological activityin some, but not all the tissues responding toET [114,115]. These results together with theinformation provided by different reportsaimed at understanding the conformationalfeatures of the peptides in solution [116–118]permitted scientists at Parke Davis to designthe pseudopeptide analogs PD 145065 (60,Fig. 16) and PD 156252 (a modified version

of PD 145065 where an N-methyl amino acidwas added at position 20 to stabilize fromproteolytic degradation) [119]. An alternativestrategy to design endothelin peptidomi-metics was also followed in the same labora-tories. The lead compound PD 012527 (61,Fig. 17) found through library screening wasused to generate a series of compounds. Usingthe Topliss Decision Tree approach for leadoptimization [120,121], different ETA-selec-tive antagonists were found, the best repre-sentatives being PD 155080 (62, Fig. 17) andPD 156707 (63, Fig. 17) [122,123].

Design of ET antagonists at SmithKlineBeecham began by identification of compoundSK&F 66861 (64, Fig. 18) by screening aG-protein-coupled receptor ligand data-base [124]. Subsequently, comparison of thelead with 1H NMR-derived conformational

Figure 15. Structure of BQ-788, a small-molecule selective antagonist of ETB.

Figure 16. Structure of the pseudopeptide PD 245065.


models of ET-1 led to the hypothesis that thephenyl groups in positions 1 and 3 in SK&F66861weremimicking a combination of two ofthe aromatic side chains of Tyr13, Phe14, andTrp21 in ET-1. Furthermore, the carboxylicacid of SK&F 66861 was proposed to mimiceither Asp18 side chain or C-terminal carboxylof ET-1. Subsequent medicinal chemistry

work substituting the indene scaffold by anindane to increase stability together with theincorporation of a second carboxylic acid moi-ety to mimic the C-terminal carboxyl of ET-1yielded compound SB 209670 (65, Fig. 18), anETA partially selective compound in the na-nomolar range [125]. Further studies aimed atimproving its poor bioavailability, intestinal

Figure 17. Structures of PD 012527 (61), PD 155080 (62), and PD 156707 (63).

GPCR LIGANDS 21

permeability screening yielded the compoundSB 217242 (Enrasentan) (66, Fig. 18), unfor-tunately with poor results for the treatmentof chronic heart failure as found in clinicalstudies [126]. Using SB209670 as a template

and following the Kohonen neural networkmethod, scientists at Merck identified thebenzothiadiazole moiety as a surrogate of themethylendioxyphenyl group of themodel com-pound [127]. These studies yielded the com-

Figure 18. Structures of SK&F 66861 (64), SB 209670 (65), Enrasentan (SB 217242) (66), and EMD 122801(67).


pound EMD 122801 with an IC50 for ETA of0.30 nM (67, Fig. 18) [128].

The first orally active endothelin ETA/ETB

antagonists came after optimization of Ro46-2005 (68, Fig. 19), found in a libraryscreening [129]. Optimization of the com-pound yielded Ro 47-0203 (Bosentan) (69,Fig. 19) [130] that was the first competitiveantagonist at ETA and ETB receptors thatpassed clinical phases and reached the mar-

ket in 2002 for the treatment of pulmonaryarterial hypertension and chronic heartfailure. Scientists at the same laboratory de-signed Ro 46-8443 (70, Fig. 19), the firstnonpeptide ETB-selective antagonist of theendothelin receptor [131]. This compound hasthe same structure as Bosentan, where thesecond pyrimidine and the hydroxyethoxyside chain were replaced by a p-methoxyphe-nyl moiety and a glycerol residue, respec-

Figure 19. Structures of Ro 46-2005 (68), Bosentan (Ro 47-0203) (69), and Ro 46-8443 (70).

GPCR LIGANDS 23

tively. Wu et al. [132] investigated further theuse of amide sulfonamides as scaffold to deignETA-selective endothelin receptor antago-nists aiming at improving their oral bioavail-ability and half-life. A series of compoundswere produced by replacement of the amidebond with various linker groups including,urethane, imide, ureido, and other extendedamide linkages. They found carbonyl group tobe the optimal spacer, leading to the discov-ery of TBC-11251 (71, Fig. 20), a potent ETA

antagonist with good bioavailability and half-life.

At Bristol-Myers Squibb, Stein et al. [133]used a benzene sulfonamide scaffold to designendothelin antagonists leading to the potent,orally bioavailable, highly selective ETA re-ceptor antagonist BMS-182874 (72, Fig. 20).At Abbot Laboratories, Wu-Wong et al. [134]reported a series of compounds derived fromthe same scaffold that showed different selec-tivities for ETA and ETB. Thus, ABT-627(73, Fig. 21) and ABT-546 (74, Fig. 21)were ETA selective, A-182086 (75, Fig. 21) wasnonselective, and A-192621 was ETB selective(76, Fig. 21). Of these compounds, ABT-627,

also known as Atrasentan, is undergoing clin-ical trials for the treatment of different typesof cancers [135].

Lead compounds acting as ETA/ETB an-tagonists have been identified from thesearch in 3D chemical databases usingdifferent pharmacophores. Thus, Funket al. [136] used HypoGen and HipHopalgorithms implemented in the Catalyst mo-lecular modeling software, to create twopharmacophores of five and six features, re-spectively. These pharmacophores were vali-dated for their activity prediction capabilityand were used to screen the Maybridge 3Ddatabase of chemical compounds. As a result,two new potential lead compounds were iden-tified that displaced ET-1 from its receptorout of six compounds tested with IC50 valuesof 220nM (77, Fig. 22) and 6200nM (78,Fig. 22).

12. PROTEIN–PROTEIN INHIBITORS

Protein–protein interactions are involved innumerous signaling cascades. Disruption ofthis process can become a useful process for

Figure 20. Structures of TBC-11251 (71) and BMS-182874 (72).


therapeutic intervention [137]. Many caseshave been reported recently where shortstretches of peptides are capable of mimickingprotein epitopes and act as inhibitors [138]. Inthis section, we briefly describe two storiesrelated to the discovery of small-molecule in-hibitors for the treatment of cancer. On theone hand, small-molecule mimics of the BH3domain of the proapoptotic members of theBcl2 family of proteins and, on the other,

small-molecule inhibitors of a fragment of theSmac protein involved in inhibiting the inhi-bitor of apoptosis proteins (IAPs).

12.1. Design of Bcl-2 Inhibitors [139–141]

Apoptosis or programmed cell death is anessential process designed for cells to commitsuicide under certain conditions, providing amechanism for a normal development of anorganism. Deregulation of this process can

Figure 21. Structures of ABT-627 (73), ABT-546 (74), A-182086 (75), and A-192621 (76).

PROTEIN–PROTEIN INHIBITORS 25

result in an undesirable increase of cell num-bers as in cancer, or in cell loss as found inautoimmune or neurodegenerative dis-eases [142]. Apoptosis is executed by a familyof cysteine aspartyl proteases called cas-pases [143–145], which are activated by twomajor interconnected pathways [146,147]: theextrinsic pathway activated by cell surfacereceptors and the intrinsic pathway that isactivated by numerous signals in which cyto-chrome c is released from the mitochondria.

The intrinsic pathway, and in some cases theextrinsic one, is regulated by the B-cell lym-phoma-2 (Bcl-2) family of proteins [148–152].More than 20 members of this family havebeen identified so far; some of them exhibitproapoptotic activity whereas the rest exhibitan antiapoptotic profile. Sequence analysis ofthese proteins shows that all members shareat least one out of four homology domains(BH1–BH4), and there is a relation betweenthe function of the proteins and theirsequence. Specifically, the antiapoptoticmembers, such as Bcl-2, Bcl-xL, Bcl-w, orMcl-1 are characterized for exhibiting the fourhomology domains, whereas the proapoptoticmembers are characterized by exhibitingeither theBH3domain only, suchasBad,Bim,Bid, or Noxa, or domains from BH1 to BH3such as Bax, Bak, or Bok. Several studiessupport the hypothesis that overexpression ofBcl-2 and Bcl-xL proteins is connected to theinitiation and development of different typesof cancer, as well as to an elevated resistanceto conventional anticancer therapies.

In spite of the tremendous progress madein the past few years, the mechanism of apop-tosis regulation by the Bcl-2 family of proteinsis far from being understood. However, it isknown that the relative amount of pro- andantiapoptotic proteins in a cell, together withthe capability of these proteins to form hetero-dimers, determines cell susceptibility to un-dergo apoptosis [153–155]. Specifically, whenthe BH3 domain of proapoptotic proteins issuppressed, binding to antiapoptotic proteinsis prevented with the subsequent loss of theirapoptotic capability [156,157]. Accordingly, itcan be considered that proapoptotic proteinstrigger apoptosis through an inhibitory func-tion of the antiapoptotic ones. This hypothesiswas demonstrated when different peptidefragments of this domain were shown to bindat the nanomolar level and induce apoptosis incell-free systems and HeLa cells [158,159].Furthermore, cell-permeable BH3 peptidesproduce apoptosis in different cellularlines [160] demonstrating the pharmacologi-cal potential benefit of these peptides. Effortshave been devoted to improve the naturalsequences in order to achieve a consensussequence for the BH3–antiapoptotic proteininteraction [161,162]. Furthermore, the struc-

Figure 22. Structures of two hits obtained fromdatabase search. See text for details.


tural analysis of complexes of Bcl-xL withshort peptide fragments of the BH3 domainof Bak [163] and Bad [164] reveals that thesepeptides adopt an a-helix when bound to theantiapoptotic protein, with their hydrophobicside facing to ahydrophobic cleft formedby theBH1, BH2, and BH3 domains. Based on thisinformation, different strategies have beenworked out.

One strategy to augment the pharmacolo-gical profile of the BH3 peptides focused onimproving the stability of the helical struc-ture. One approach is to enhance the helicalcontent through a lactam cross-link (79,Fig. 23) [165], unfortunately although the he-lical contents increase, this kind of cross-linkdoes not solve some of peptide shortcomings toact as a drug. Amore successful approach is toadd a hydrocarbon staple (80) [166] as shownin Fig. 23. These results can be improved if inaddition some of the residues is replaced bya,a-disubstituted nonnatural amino acidsthat reinforce the tendency of the sequence toadopt a helical structure. This process hasdemonstrated to augment the helical content

of the peptide and improve their pharmacoki-netical profile by decreasing their resistanceto proteolytic cleavage, their binding affinity,and permeability.

Other approaches include the constructionof synthetic scaffolds that have attached thecorresponding side chains of the BH3 motifs.Terphenyl, terpyridine, or terephthalaminehave been used for this purpose [167,168].This strategy puts the right-hand side chainsexposed for interaction while preserving theamino acid skeleton from protease hydrolysis.Other strategies include the use of foldamers,chimerical peptides that juxtapose nativeBH3 domain sequencewith alternatinga- andb-peptide foldamer sequence yielding com-pounds with nanomolar affinities [169].

A different approach focused on findingsmall-molecule mimics of the BH3domains. Different groups have reportedsmall-molecule inhibitors of Bcl-2/Bcl-xLusing diverse strategies. Some inhibitorshave been found from high-throughputscreening of libraries. These compounds ex-hibit inhibitory constants in the micromolarrange. One of the early compounds describedis Antimycin A3 (81) [170], or BH3I-1 (82) andBH3I-2 (83) shown in Fig. 24 [171]. Naturalproducts such as chelerythrine (84) [172] orgossypol (85) [173] shown in Fig. 25 have alsobeen identified. Natural polyphenols isolatedfrom tea extracts, such as epigallocatechingallate (86) (from green tea) or theaflavi-nin (87) (from black tea), shown in Fig. 25,compete with the BH3 domain at the submi-cromolar range [174]. Obatoclax (GX15-070)(88, Fig. 26) is a derivative from a hitidentified by screening of a natural productlibrary that competitively binds to Bcl-xL,Bcl-w, and Mcl-1 in the high nanomolarrange [175].

Virtual screening provided several hits inthe micromolar range including HA14-1(89) [176], compound 6 (90) [177], YC137(91) [178], or the recently discovered NMNB(92) [179] shown in Fig. 26. Structure–activ-ity relationships using NMR (fragmentscreening) have also provided small-moleculeinhibitors of Bcl-2. This is the case of ABT-737 (93,Fig. 26) [180], a small-molecule in-hibitor of Bcl-2, Bcl-xL, and Bcl-w in thesubnanomolar range, with demonstrated

HNO

79

80

Figure 23. Structures of two peptidomimetics de-signed to stabilize the helical structure.


antitumor activity in vitro and in vivo. Othersmall-molecule leads using similar frag-ments have been described using thismethodology [181,182].

12.2. Design of XIAP Inhibitors [183]

The secondmitochondrial-derived activator ofcaspases (Smac) is a protein released frommitochondria into the cytosol in response to

an apoptotic stimulus. The protein is an en-dogenous inhibitor of different IAPs, its actionbeing to prevent the binding of IAPs to cas-pase-3, caspase-7, and caspase-9, critical ac-tors of the apoptotic process. IAPs are foundhighly expressed in tumor cells, playing animportant role in the resistance of tumor cellsto current chemotherapeutic agents [184]. IAPinhibition by Smac induces the cell to initiatethe apoptotic process. Accordingly, mimics of

Figure 24. Structures of Antimycin A3 (81), BH3I-1 (82), and BH3I-2 (83)


the protein could be used as therapeuticagents for the treatment of cancer.

The X-linked inhibitor of apoptosis (XIAP)is one of the most potent IAPs. Structuralanalysis of the complex XIAP/Smac reveals

that the two proteins interact via the N-term-inal peptide segment of Smac: Ala-Val-Pro-Ile [185,186] (94, Fig. 27), which binds to theso-called baculovirus IAP repeat (BIR3) do-main of XIAP. Moreover, the isolated peptideinhibits the protein with a Ki¼ 290nM [187].Accordingly, mimetics of the Smac N-termi-nus tetrapeptide could be transformed intouseful therapeutic agents.

Early structure–activity relationship stu-dies demonstrated the importance of Alain position 1, although the ethyl side chain(a-aminobutyric acid) improves affinity.Similarly, it was shown that the bindingaffinity diminishes when Pro is replaced inposition 3. In contrast, there is tolerance forpositions 2 and 4. Specifically, most of theamino acids are admitted in position 2, withexception of Gly, Asp, and Pro. On the otherhand, residues with bulky hydrophobic sidechains such as Trp or Phe fit well in position4. N-methylation of the amide moiety ofresidue 4 was tolerated in contrast to theN-methylation carried out on the other resi-dues [187]. These analogs exhibit single-digitnanomolar affinity and promote cell death indifferent human cancer cell lines [188]. In afurther step, the side chain of valine wassubstituted with several five-membered het-erocyclic moieties while maintaining the Alaamino acid achieving submicromolar activ-ity [189]. Li et al. using combinatorial synth-esis and trying to mimic the last residue ofthe tetrapeptide by a heterocycle described aseries of hybrid mimetics of the peptide [190],the most potent being the oxazoline contain-ing pseudopeptide (95, Fig. 27). Further workin this direction has been pursued by otherauthors [191].

Several studies have been publishedreporting the design of conformationally con-strained peptidomimetics of the tetrapeptide.One of the first attempts came from the ana-lysis of the crystal structure of the Smac/XIAPcomplex. Inspection of the structure revealsthat the isopropyl side chain of residue 2 issolvent exposed, whereas the proline ring sitson a hydrophobic pocket. These features sug-gested the design of a peptidomimetic fusingthe side chains of valine and proline through abicyclic scaffold. Although the first analogsynthesized (96) (Fig. 28) exhibits a slight

Figure 25. Structures of chelerythrine (84), gossy-pol (85), epigallocatechin gallate (86), and theafla-vinin (87)


lower affinity than the original peptide, aftersubsequent optimization, lownanomolar com-poundswere obtained [192].Compound (97) inFig. 28 can be used as a proof of principle.Unfortunately, these series of compoundsshowed low ex vitro activity due to a low cellpermeability, forcing to develop further a ser-

ies of new analogs with improved cell penetra-tion properties. As a result of an extensivestudy, several compounds were designed,compound 98 being an example of an analogthat exhibits good activity ex vivo(Fig. 28) [193].

Figure26. Structure of obatoclax (GX15-070) (88),HA14-1 (89), compound6 (90), YC137 (91),NMB(91), andABT-737 (93).


Figure 27. Structure of the peptide Ala-Val-Pro-Ile (94) and peptidomimetic 95.

Figure 28. Structure of different bicyclic com-pounds peptidomimetics of 94.

Other peptidomimetics based on bicyclicscaffolds were reported. Thus, Genetech de-scribed inhibitors using a bicyclic [7,5]-lactamscaffold (99, Fig. 29). These compounds ex-hibit both single-agent cell killing and addi-tivity with doxorubicin in cancer celllines [194]. More recently, the use of azabi-cyclooctane as scaffold has yielded orallybioavailable inhibitors (100, Fig. 29) [195].Interestingly, this compound decreased theviability of cancer cell lines without affectingnontumor cells. Novartis also reported thedesign of peptidomimetic inhibitors of XIAPbased on a pyrido[3,4b]pyrrole scaffold, theirrepresentative compound being LBW242(101, Fig. 29) [196]. Similarly, taking as start-ing point the compound SM-130 described bySun et al. [197], Bolognesi and colleaguessynthesized a library of 4-subtituted azabicy-clo[5.3.0]alkane compounds displaying highaffinities (102, Fig. 29). X-ray crystallographyand molecular modeling simulations wereused to properly analyze the structural fea-tures of XIAP domain–Smac mimeticcomplexes [198,199].


Other approaches have also been used todesign peptidomimetics of the tetrapeptide.An early discovery was the discovery of theinhibition profile of embelin (103, Fig. 30), anactive compound foundby screening in silico ofa traditional herbal medicine database. Thecompound was found to inhibit XIAP with an

affinity in the low micromolar concentra-tion [200]. The workwas pursued beyondwiththe synthesis of different analogs with im-proved affinities [201]. Very recently, a newset of lead compounds obtained by fragmentscreening combined with NMR spectroscopywas disclosed. Of these compounds, molecule

Figure 29. Structure of different bicyclic compounds peptidomimetics of 94 (cont).


Figure 30. Structure of embelin (103) and compound 104.

Figure 31. Structure of the inhibitor 105 and its monomer (106).


104 (Fig. 30) exhibits cellular activity in exvivo experiments [202].

In the early studies carried out by Harranet al. [189], the authors found that dimers ofthe analogs were more effective than themonomers. The reason for this was basedon the fact that XIAP contains three BIRdomains. Accordingly, it could be thought thatit binds to two different protein domains si-multaneously. Thus, compound 105 (Fig. 31)exhibits 1.5 times higher affinity than itsmonomer (106) (Fig. 31). In this direction,recently a cell permeable, bivalent Smac mi-metic (107, Fig. 32) [203] and a cell permeable,bivalent and cyclicmimetic (108, Fig. 32) [204]that concurrently target both BIR2 and BIR3domains of the XIAP protein with activity atthe nanomolar range have been reported.

13. CONCLUSIONS

In this review, we have intended to providean overall picture of the knowledge accumu-

lated during the past 30 years in peptidomi-metic design. The review stresses thenecessity of a multidisciplinary approach tocope with the design, where medicinal che-mists, structural biophysicists, and computa-tional chemists need to work hand in hand inorder to assemble the different pieces of thepuzzle. The original idea of mimicking thestereospecific arrangement of the key moi-eties of a peptide at its recognition site hasprovided a much deeper insight into the fea-tures of peptide–protein interactions. Wealso have outlined that the strategies to de-sign a small moleculemimicking the featuresof a peptide as well as its pharmacologicalprofile vary depending on the structural in-formation available. The area of peptidomi-metic design can be considered mature, witha vast literature on peptide analogs designedas modifications of the peptide backbone orthe side chains, as well as a wealth of infor-mation regarding organic scaffolds to mimicgeometrical features of a peptide backbone.

Figure 32. Structure of bivalent linear and cyclic Smac mimetics.


Moreover, the combined action of structuralmethods and the use of computational meth-ods have demonstrated to be very useful inthe medicinal chemistry of this kind of com-pounds. However, there is still an amplespace to explore. We expect to witness in thenear future newachievementsmostly relatedto the design of small-molecule ligands forGPCRs or protein–protein inhibitors.

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