Protein–protein interactions as targets for small molecule drug discovery

Protein–Protein Interactionsas Targets for Small MoleculeDrug Discovery

David C. FryRoche Research Center,Hoffmann-La Roche Inc.,

Nutley, NJ 07110

Received 31 July 2006;revised 14 September 2006;accepted 16 September 2006

Published online 28 September 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20608

Abstract: Protein–protein interactions represent a highly populated class of targets for drug dis-covery. However, such systems present a number of unique challenges. This review presents ananalysis of individual protein–protein interaction systems which have recently yielded success indiscovering drug-like inhibitors. The structural characteristics of the protein binding sites and theattributes of the small molecule ligands are focused upon, in an attempt to derive commonly sharedprinciples that may be of general usefulness in future drug discovery efforts within this targetclass. # 2006 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 84: 535–552, 2006

This article was originally published online as an accepted preprint. The \Published Online" datecorresponds to the preprint version. You can request a copy of the preprint by emailing theBiopolymers editorial office at [email protected]

Keywords: protein–protein interactions; PPI; drug discovery; peptidomimetics

INTRODUCTION

Large scale efforts are underway to survey and catalog

all of the protein–protein interactions that occur in

cells, and it is becoming clear that the number of such

interactions is quite high. It appears that almost every

important pathway is partly composed of, or is crit-

ically influenced by, protein–protein interactions.

Therefore, such interactions are most likely to be

closely linked to disease states across a wide variety of

therapeutic areas, and represent a highly populated

class of targets for drug discovery. However, owing to

the relatively large surfaces that are typically involved

in protein–protein pairing, a perception has arisen that

the likelihood of successfully modulating protein–

protein interactions with small molecules is low. This per-

ception has been supported by a track record, wherein

protein–protein interactions have proven exceptionally

complex and resistant as pharmaceutical targets. These

systems, therefore, have been branded as high-risk and

have been avoided. However, this type of blanket char-

acterization is not appropriate. Substantial progress has

been made in identifying drug-like small molecule

inhibitors for several protein–protein systems. It has

become apparent that, while there are unique hurdles

involved in developing a protein–protein inhibitor as a

drug, these hurdles can be identified and assessed. Fur-

ther, it is clear that cases will be found where these hur-

dles can be overcome, and where the risk in pursuing a

drug discovery effort will be judged to bewithin accept-

able limits. As successes in discovering protein–protein

inhibitors accumulate, principles should begin emerg-

ing that will make successive attempts more effective.

This review will describe the unique difficulties

faced when trying to modulate a protein–protein

interaction with a small molecule, and the key factors

Correspondence to: David C. Fry; e-mail: [email protected].

Biopolymers (Peptide Science), Vol. 84, 535–552 (2006)

# 2006 Wiley Periodicals, Inc.

535

involved in judging whether or not a particular system

is amenable. It will then provide detailed examples of

programs where a drug-like inhibitor was developed

against a protein–protein interaction, focusing on the

molecular-level aspects of the system. Accordingly, it

will be limited to those cases for which sufficient

structural information is available. An attempt will be

made to identify common attributes shared among

these successful examples, in terms of characteristics

of the binding sites and properties of the small mole-

cule inhibitors, which may be useful for guiding

future drug discovery efforts within this target class.

HURDLES ASSOCIATED WITHDISCOVERING PROTEIN–PROTEININHIBITORS

Among the properties a molecule must possess in order

to function as a drug, two are directly related to the pri-

mary binding site: affinity and selectivity. Achieving

these with regard to modulating a protein–protein inter-

action can present substantial difficulties. Many pro-

tein–protein interaction sites are large and relatively

flat, and affinity is achieved through summing up a

large number of weak interactions. A small molecule

cannot effectively duplicate these interactions, because

they are too numerous and widely spaced. However,

pioneering work by Clackson and Wells1 demonstrated

that not all protein–protein interaction surfaces are reli-

ant upon a wide distribution of weak interactions. This

was exemplified by the growth hormone system, where

they showed that a limited number of amino acid resi-

dues mediate nearly all of the key interactions that con-

tribute to binding affinity. This subregion was termed

the \hot spot," and its dimensions were comparable to

the size of a small organic molecule.

Because a small molecule is only able to partici-

pate in a limited number of interactions, it must maxi-

mize the energetic contribution from each of them. A

key factor toward this goal is the ability to shield

atomic interactions from competition with solvent

molecules. To allow opportunities for such protec-

tion, the surface of the protein target must not be too

shallow, and must offer reasonably deep subpockets.

Judgments concerning the shallowness of a protein

binding surface have typically been made by visual

inspection. A more quantitative and consistent

approach is to use a computer program that accu-

rately measures and reports the dimensions of the

binding site.2 The output of the program can be pre-

calibrated by examining a set of protein binding sites

that are known to support high affinity binding by

drug-like molecules.3 Once calibrated in this way, the

program can be used to objectively assess the poten-

tial of a protein site for drug discovery.

A flat interaction surface can also present a chal-

lenge with regard to selectivity. On such a surface,

there is not enough contact to interrogate the ligand’s

contours, and thereby achieve specificity through

shape recognition. Rather, selectivity is encoded by a

particular spatial arrangement of functional groups

through which interactions are mediated. A small

molecule may be capable of matching a subset of

these, and thereby achieve binding, but if this same

subset appears on the surface of another protein, the

small molecule will not be able to discriminate. Fur-

thermore, proteins can achieve selectivity by employ-

ing charged groups or protrusions far from the pri-

mary site that can create repulsions or steric clashes

with unwanted partners. These features can be

thought of as \anti-binding" elements. A drug-like

molecule is too compact to be able to position such

elements sufficiently far from its binding epitope.

There are three other issues associated with protein–

protein interactions that may present problems with

regard to drug discovery. First, unlike an enzyme–

ligand or receptor–ligand interaction, where one partici-

pant possesses a pocket and the other simply occupies

that pocket, protein–protein interaction surfaces are

more likely to be topologically complex. That is, both

participants may contribute protrusions and subpockets.

This will be problematic if the target protein’s role is

predominantly a contributor of protrusions, for it is

much more likely that one could develop a small mole-

cule that tightly occupies a pocket than one that

sheathes a protrusion. A second issue is related to the

observation that numerous protein–protein interactions

are regulated via phosphorylation. There are systems in

which one partner binds only if a key side chain in its

binding epitope is phosphorylated. A protein site that

has evolved to discriminate toward such a partner is not

likely to be an attractive site for pharmacological inter-

vention, since any small molecule mimicking the

charged species would be too polar, and would not

achieve sufficient bioavailability to serve as a drug. A

third issue concerns the functional status of the target.

Conceptually, it is much easier to develop an inhibitor

than an agent that enhances or restores activity. This is

because stimulation requires not only binding, but also

the precise mimicking of an interaction that triggers a

response, whereas inhibition can be accomplished in a

less exact way via any strategy that effectively prevents

binding of one of the partner proteins. Disease states are

often caused when the function of a key protein is dis-

rupted by mutation, and such cases, where functional

restoration would be the desired therapeutic strategy, do

not represent attractive situations for drug discovery.

536 Fry

Biopolymers (Peptide Science) DOI 10.1002/bip

In the following sections, an analysis is presented of

individual protein–protein interaction systems for

which drug-like inhibitors have been discovered. This

examination focuses on the structural characteristics of

the binding sites and the attributes of the small molecule

ligands, in an attempt to derive commonly shared prin-

ciples that may be of general usefulness in future drug

discovery efforts within this challenging target class.

INTEGRINS

The first example of drug-like protein–protein inhibi-

tors came from the field of integrins. Integrins are

cell surface receptors that mediate a variety of func-

tions involving cell–cell interaction and communica-

tion, and they have been the subject of widespread

drug discovery efforts toward alleviating such condi-

tions as asthma, arthritis, heart disease, and cancer.

Each member of the integrin family is composed of

one � and one � chain, which associate noncova-

lently. There are at least 18 � chains and 8 � chains

known, and these combine to produce at least 24 dif-

ferent family members.

One of the first integrins to be targeted for drug

discovery was �IIb�3. This receptor binds a variety of

protein ligands, such as fibronectin and vitronectin,

all of which possess an Arg-Gly-Asp motif. A key de-

velopment that set the stage for drug design efforts

was the identification of highly potent cyclic peptides

containing the Arg-Gly-Asp motif. Cyclization effec-

tively restricted the backbones of these peptides, so

that their conformations could be accurately deter-

mined by NMR. Since an X-ray structure of an integ-

rin complexed with a ligand was not forthcoming,

design of small molecules was based on the assump-

FIGURE 1 Inhibitors of the �IIb�3 integrin receptors from a variety of chemical classes: (A) a

benzodiazepine4; (B) a pyrrolo-benzodiazepine-dione5; (C) an oxoisoquinoline6; (D) an isoindoli-

none7; (E) a thienothiophene—known as L-7397588; (F) a piperidynylbutoxy-phenylpropionic

acid—known as tirofiban10; and (G) an inhibitor of the �4�1 integrin receptor—a benzylpyroglu-

tamyl-phenylalanine.13

Protein–Protein Interactions for Small Molecule Drug Discovery 537


tion that the constrained peptide backbone configura-

tion was maintained upon binding. Multiple research

groups pursued this replication strategy, by which

attempts were made to mimic, with moieties

appended to an organic scaffold, the critical Arg gua-

nidine group and Asp carboxylate group in their

proper spatial arrangement. This approach proved to

be successful, as several compound classes were

developed that exhibited nanomolar levels of inhibi-

tory activity. Derivatized benzodiazepines,4 pyrrolo-

benzodiazepine-diones,5 oxoisoquinolines,6 isoindoli-

nones,7 and thienothiophenes8 (Figure 1) were found

to be effective scaffolds for properly positioning

the critical basic and acidic functional groups. Ulti-

mately, from widespread efforts targeting the �IIb�3receptor, two molecules achieved clinical approval,

for use in preventing platelet aggregation—epifiba-

tide (which retains substantial peptide character)9 and

tirofiban (which is nonpeptidic)10 (Figure 1).

A slightly different ligand motif, namely Leu-Asp-

Val, is sought by the integrin family member desig-

nated �4�1. A cyclic peptide with potent binding ac-

tivity toward this receptor was developed—(cyclo)-

Arg-Cys-Asp-thiaPro-Cys11—and the structure of

this peptide was determined by NMR. Remarkably,

not only was the backbone rigidly constrained, but

the thioproline residue was fixed completely in the

cis conformation. At Roche, a series of peptide ana-

logs was designed in which the Asp-thiaPro portion

was replaced with 1-(2-aminoethyl)cyclopentyl-car-

boxylic acid with no loss of potency.12 By using

NMR-derived structural information from these ana-

logs, nonpeptidic small molecule inhibitors were

developed, based on a N-benzylpyroglutamyl-phenyl-

alanine core.13 Ultimately, small molecule inhibitors

of this compound class were produced that exhibited

subnanomolar activity14 (Figure 1).

Recently, X-ray structures have started to become

available for integrins complexed with ligands. A struc-

ture of �V�3 has been solved with the cyclic pep-

tide (cyclo)Arg-Gly-Asp-(D)Phe-N-methyl-Val bound15

(Figure 2). It reveals that the peptide contacts both the

FIGURE 2 Structures of ligands complexed with integrin receptors: (A) a cyclic peptide repre-

senting a natural Arg-Gly-Asp-containing partner bound to �V�315; and the small molecule inhibi-

tors (B) tirofiban16 and (C) L-73975816 bound to �IIb�3. Also shown are superpositions of the

bound position of the cyclic peptide with those of (D) tirofiban and (E) L-739758. The receptor

backbone is depicted as a violet ribbon; and key receptor side chains and small molecule inhibitors

are colored by atom type in stick format. The position of the magnesium ion in the MIDAS domain

is represented by a teal sphere. The peptide is colored brown, except for the nitrogens of the guani-

dino moiety which are blue. The superpositions were accomplished by aligning the sequences of

the two receptors, then maximizing the superposition of the backbone atoms from the key binding

regions of the receptors; and then turning off the display of all receptor atoms.

538 Fry


� and � subunits of the heterodimer. The backbone

conformation of the cyclic peptide in the bound state is

essentially identical to that found in the free state, as

was hypothesized in earlier drug design efforts. There

is a magnesium ion in the � subunit, in a region called

the \I-like" domain, which is coordinated by a highly

conserved integrin motif called \MIDAS." This mag-

nesium interacts with the critical Asp carboxylic acid

group of the peptide. The Arg of the peptide forms a

salt bridge with an aspartate residue that resides in a �-propeller domain of the � subunit. Other structures

have been determined, in which �IIb�3 is complexed

with the small molecule inhibitors tirofiban and L-

739758. These structures reveal a consistent binding

strategy, in which the basic moiety of the inhibitor is

mimicking the Arg of the peptide and the acidic moiety

is mimicking the Asp16 (Figure 2). This replication is

exactly what was hoped for during the design of these

Arg-Gly-Asp mimetics. The backbone of the peptide is

not mimicked at all by either of these nonpeptidic

inhibitors. This indicates that as long as a small mole-

cule inhibitor effectively duplicates the key binding

interactions, it can achieve high affinity binding—it

does not have to exactly replicate the natural protein

partner. As for the Leu-Asp-Val mimetics from the

�4�1 system, there is no X-ray structure yet available

for this complex, but it is predicted that they will simi-

larly bridge the heterodimer, and that their carboxylic

acid moieties will interact with the MIDAS motif.

There is a second class of integrins that contain an

additional domain, termed the \I-domain," located

within the � subunit. The most extensively studied

member of this class is �L�2, which is expressed

exclusively on leukocytes and plays a crucial role in

inflammation. Its ligand is a protein called ICAM-1.

An X-ray structure of the complex between this

ligand and �L�2 has been obtained,17 and reveals that

a Glu residue from ICAM-1 directly ligates a magne-

sium ion associated with the MIDAS motif in the I-

domain of the receptor. This critical role of an acidic

group in the ligand is similar to the binding strategy

utilized by the other integrins. However, the small

molecule inhibitors that have been discovered for

�L�2 do not bind by replicating this strategy, but

instead are located at an allosteric site, which is

remote from the MIDAS region. Lovastatin (Figure

3) was the first such inhibitor to be reported, and it

was identified via high-throughput screening.18 NMR

and X-ray studies have established that it binds to an

allosteric site on �L�2 (Figure 4), which is now desig-

nated the \L-site," and thereby induces a conforma-

tional change that prohibits binding of ICAM-1. Ela-

borated versions of lovastatin have since been devel-

oped, which exhibit higher potency.19 Inhibitors from

other chemical classes have also been reported, such

as p-arylthio-cinnamides20 and 1,4-diazepane-2,5-

diones21 (Figure 3). X-ray structures have been solved

for these types of inhibitors bound to �L�218,21,22

(Figure 4), and they confirm that all of these small

molecule inhibitors bind to the L-site and function

similarly via an allosteric mechanism.

The integrin field demonstrates that potent drug-

like small molecules can be developed that inhibit a

protein–protein interaction, and that despite their size

they can effectively mimic the key binding interac-

tions normally mediated by a protein. It also indicates

that an alternative strategy is possible—that is, the

small molecule can bind to a secondary site and trig-

ger a transmitted conformational change that disrupts

the normal binding ability of the target protein.

FIGURE 3 Inhibitors of the �L�2 integrin receptor: (A) lovastatin18; (B) a p-arylthio-cinna-mide20; and (C) a 1,4-diazepane-2,5-dione.21



These first successes within the target class com-

prising protein–protein interactions were viewed with

admiration but not with general optimism. Integrins

were judged to be an uniquely amenable system,

because they provided an accessible metal site to

assist binding of competitive inhibitors, and an oppor-

tunistic secondary site for the binding of allosteric-

type inhibitors.

INTERLEUKIN-2

Interleukin-2 (IL-2) is a cytokine consisting of 133

residues, which plays a key role in the growth, activa-

tion, and differentiation of T cells. IL-2 acts by bind-

ing to a heterotrimeric receptor on the T-cell surface,

composed of subunits designated IL-2R�, IL-2R�,and IL-2R�. Monoclonal antibodies have been devel-

oped that recognize IL-2Ra and block the binding of

IL-2, and these antibodies have shown clinical effec-

tiveness as agents that suppress the immune response

associated with organ transplant rejection.23 There-

fore, inhibition of IL-2 signaling via a small molecule

that blocks the interaction between IL-2 and IL-2R�has been an actively pursued strategy toward the dis-

covery of new immunosuppressive drugs.

The three-dimensional structure of IL-2 has been

characterized by both X-ray crystallography24,25 and

NMR spectroscopy.26,27 Its fold is similar to other

cytokines, such as IL-428 and GM-CSF,29 which form

four-helix bundles with an up-up–down-down ar-

rangement of the four main �-helices (referred to as

helices A, B, C, and D). The residues of IL-2 that are

critical for binding to IL-2R� were identified by mu-

tagenesis30,31 and by NMR spectroscopy.32 These res-

idues were found to be clustered in three-dimensional

space, although located within various segments of

secondary structure, namely the AB loop, the N-

terminal end of the B helix, the BC loop, the CD

loop, and the N-terminal end of the D helix.

At Roche, in an attempt to find a small, nonpepti-

dic inhibitor that would bind to IL-2R� and block its

interaction with IL-2, a series of acylphenylalanine

derivatives was prepared. These compounds were

FIGURE 4 Structures of ligands complexed with the �L�2 integrin receptor I-domain, showing

the binding site for the natural protein partner and the remote allosteric site utilized by small mole-

cule inhibitors: (A) the natural protein partner ICAM17; and the small molecule allosteric inhibitors:

(B) lovastatin18; (C) a representative p-arylthio-cinnamide22; and (D) a representative 1,4-diaze-

pane-2,5-dione.21 The backbones of the receptor and ICAM are depicted in ribbon format. The

position of the magnesium ion in the MIDAS domain is represented by a lavender sphere. In (A),

key interacting side chains of ICAM are colored by atom type in stick format. In (B–D), the small

molecule inhibitors are colored brown, and a portion of the surface of �L�2, representing the allo-

steric binding site, is shown in grey-white as a Connelly depiction.

540 Fry


intended to mimic key residues of the IL-2 binding

epitope—in particular, Arg38 and Phe42. One mem-

ber of this initial series was found to be an inhibitor of

the IL-2/IL-2R� interaction in a scintillation proxim-

ity assay, with an IC50 of *45 �M. Synthesis of

derivatives of the lead compound ultimately resulted

in an inhibitor designated compound 24 (Figure 5),

which exhibited an IC50 value of 3 �M.33 Although

these acylphenylalanine derivatives were designed to

bind to IL-2R�, NMR studies revealed that compound

24 bound to IL-2.33 This represented the first example

of a small molecule inhibitor of a protein–protein

interaction, in which the inhibitor bound to the partner

classified as the ligand, rather than to the receptor.

X-ray structures subsequently became available

for IL-2 in complex with compound 24,36 and for

IL-2R� in complex with IL-2,37 allowing a retro-

spective analysis of the drug discovery effort. First,

the structure of IL-2 bound to its receptor showed

that the strategy to mimic IL-2 residues Arg38 and

Phe42 with a small molecule was reasonable, as

these two side chains are oriented toward IL-2R�

and are involved in critical interactions (Figure 6).

However, the binding pocket on IL-2R� that accom-

modates these two residues is quite shallow, which

may explain why this compound was unable to

achieve satisfactory binding there. Second, a com-

parison of the two structures reveals that compound

24, despite its intended design, is an effective mimic

of IL-2R� (Figure 6). Specifically, its piperyl-guani-

dino group replicates Arg 36 of IL-2R�, by making

a salt bridge with Glu62 of IL-2. Additionally, one

of its carbonyl groups occupies a similar position as

the carboxylate of Glu29 from IL-2R�. Further, itsbiaryl alkene group fills a hydrophobic groove on

IL-2, which is normally accessed by IL-2R� resi-

dues Leu2, Met25, Asn27, Tyr43, and His120. It

extends exceptionally deeply into this groove, and in

order for this to occur, the side chain of IL-2 residue

Phe 42 is partially displaced, and there is a slightly

enhanced separation between the B helix and the AB

loop.

An optimization effort starting with compound

24 was able to achieve compounds with affinities to

FIGURE 5 Inhibitors of the interaction between IL-2 and the � subunit of its receptor (IL-2R�).(A) An acylphenylalanine known as compound 2433; and (B–C) more potent derivatives

thereof.34,35



the 60 nM level,34 and further drug development

efforts have yielded second-generation inhibitors

with completely nonpeptidic scaffolds that exhibit

potencies to 600 nM35 (Figure 5). These experiences

with IL-2 reinforced the theme that in amenable

cases a small molecule can effectively mimic a pro-

tein by simply replicating key side-chain interac-

tions, without a requirement for duplicating the

backbone. This system also introduced the concepts

that either partner in protein–protein interaction may

be the preferred binding target, and that a suffi-

ciently rigid small molecule can bring about confor-

mational changes at a protein site that facilitate and

enhance its binding.

Z-INTERACTING PROTEIN A

Z-interacting protein A (\ZipA") is a bacterial pro-

tein that plays an essential role in the formation of

new cell walls during cell division. It is recruited to

an organelle called the septal ring at the beginning of

the cell division cycle, via interaction with FtsZ, a

protein that is a component of the ring. It has been

demonstrated that cells depleted of functional ZipA

can no longer divide, and it is believed that inhibitors

of the ZipA-FtsZ interaction may show usefulness as

novel antibiotics.

The structure of ZipA in complex with a 17-resi-

due peptide from FtsZ has been solved by X-ray crys-

tallography.38 It shows that the peptide forms a partial

� helix when bound, and that several of its side

chains, in particular Tyr371, Leu372, Ile374, Phe377,

Leu378, and Gln381, are seated in a shallow hydro-

phobic groove on ZipA (Figure 7).

Small molecule inhibitors of the ZipA-FtsZ inter-

action have been developed, although all of them

show relatively weak binding in terms of what would

be expected for a viable drug candidate. High-

throughput screening identified a pyridyl-pyrimidine

derivative as a 12 �M inhibitor39 (Figure 8), and this

binding affinity is comparable to that displayed by

the FtsZ peptide. An X-ray structure has been deter-

mined for this inhibitor bound to ZipA39 (Figure 7),

and a comparison of this structure with that of the

ZipA-peptide complex reveals by what strategies the

small molecule is able to mimic the peptide. Nota-

bly, the peptide backbone is not specifically repli-

cated by the small molecule. Rather, the small mole-

cule primarily mimics key side chains from the pep-

tide. The hydrophobic bulk of Phe377 is duplicated

by part of the 3-chloro-4-methyl-phenyl group of the

inhibitor, which also partially emulates the role of

Leu372. Ile374 is mimicked by the pyrimidine ring,

while the pyridine ring mimics Leu378. Other side

chains from the peptide that make significant contact

with ZipA are too distant to be replicated by this

small molecule.

Inhibitors of the ZipA-FtsZ interaction from

another chemical class, featuring an indolo-quinolizi-

FIGURE 6 (A) Structure of IL-2 bound to IL-2R�.37 The protein backbones are depicted in rib-

bon format. IL-2R� is on the top, colored green. IL-2 is on the bottom, colored blue. Key interact-

ing side chains are colored by atom type, depicted in stick format. For IL-2R� (top), these residues

are (left to right) as follows: Arg36, Glu29, Asn27, Tyr43, Met25, His120, and Leu2. For IL-2

(bottom), these residues are (left to right) as follows: Glu62, Phe42, and Arg38. (B) Superposition

of IL-2 complexed with IL-2R�37 and IL-2 complexed with compound 24.36 IL-2R� is on the top,

colored green. IL-2 is on the bottom, colored blue. Compound 24 is colored brown, except for the

nitrogens which are blue and the oxygens which are red. The superposition was accomplished by

maximizing alignment of the backbone atoms of IL-2 (both IL-2 backbones are shown).

542 Fry


none core, have been reported, and their affinities are

also in the high micromolar range40 (Figure 8). This

series was also derived via optimization of a hit

obtained from high-throughput screening. A crystal

structure is available for one of these compounds

bound to ZipA40 (Figure 7). It reveals that the small

molecule replicates three of the key side chains from

the peptide, but not the backbone. Phe377 and Ile374

are mimicked by the indolo-quinolizinone ring sys-

tem, while Leu372 is mimicked by the propyl amide

appendage.

The small molecule inhibitors of the ZipA-FtsZ

interaction share a number of strategic concepts in

terms of how they mimic the natural protein ligand.

First, there is no replication of the protein backbone,

but rather a mimicry of a subset of critical side-chain

interactions. Second, these side chains are not

exactly duplicated by moieties of the small mole-

cule, in terms of shape, electronic character, or ori-

entation. For example, the role of an all-carbon aro-

matic ring is partly fulfilled by chlorine. Aliphatic

side chains are replicated by heterocyclic ring sys-

tems. The shapes of the side chains are not precisely

FIGURE 8 Small molecule inhibitors of the interaction

between ZipA and FtsZ. (A) a pyridyl-pyrimidine39; (B) an

indoloquinolizinone.40

FIGURE 7 (A) Structure of Z-interacting Protein (ZipA) complexed with a peptide fragment of its

natural binding partner FtsZ.38 ZipA is depicted in space-filling format and colored purple. The back-

bone of the FtsZ peptide is depicted in tube format and colored green. Key interacting side chains of

the FtsZ peptide are depicted in stick format and colored brown. (B–C) Superpositions of the ZipA-

bound position of the FtsZ peptide with those of small molecule inhibitors: (B) a pyridyl-pyrimi-

dine39; (C) an indoloquinolizinone.40 The backbone of the FtsZ peptide is depicted in tube format and

colored green. Key interacting side chains of the FtsZ peptide are depicted in stick format and colored

brown. These residues are (left to right) as follows: Leu372, Ile374, Phe377, and Gln381 (numbering

corresponds to that of the parent FtsZ protein). The small molecule inhibitors are colored by atom

type and depicted in stick format. The superposition was accomplished by maximizing alignment of

the backbone atoms of ZipA, and then turning off the display of all ZipA atoms.



duplicated, but rather the general hydrophobic vol-

ume is matched. The side chains enter the ZipA

binding groove on a downward trajectory from the

peptide backbone, while the small molecules lay

more flatly in the groove. These findings were

encouraging, because they suggested that, because

there is no requirement to precisely match the natu-

ral ligand, there will be a variety of opportunities for

finding scaffolds that function successfully against

a particular protein–protein system. However, the

low affinity of these ZipA inhibitors raised the con-

cern that such imprecision might routinely lead to

compounds lacking sufficient potency to qualify as

drugs.

NITRIC OXIDE SYNTHASE

Nitric oxide synthase (NOS) is a heme-containing di-

meric enzyme that is responsible for creating the sig-

nal transduction molecule nitric oxide, via oxidation

of L-arginine. An inducible isoform of the enzyme

(\iNOS") has been implicated in tissue damage asso-

ciated with various inflammatory and autoimmune

diseases. Therefore, an inhibitor that selectively tar-

gets iNOS has been actively pursued, since such a

compound could find therapeutic usefulness in these

disease areas.

Screening of a combinatorial synthetic chemistry

library was one approach that was tried, and it

resulted in the discovery of potent, selective inhibi-

tors of iNOS41 (Figure 9). These molecules were

found to act by a unique mechanism, namely disrup-

tion of dimer formation, leading to the inactive mono-

meric state. An X-ray structure has been reported for

one of these compounds bound to iNOS41 (Figure

10). It shows that the inhibitor binds to the active site,

in the same location as the natural substrate L-argi-

nine.42 Therefore, it occupies a functional pocket,

which is remote from the dimerization interface.

Dimerization is inhibited not via direct competition,

but through a conformational change propagated out-

ward from the binding site, which ultimately alters

the structure at the interface, such that binding is

abrogated. An analysis of the strategy by which the

inhibitor binds to the active site pocket indicates that

the imidazole moiety of the inhibitor mimics the gua-

nidino portion of L-arginine. In that regard, the imid-

azole coordinates directly to the heme group of the

enzyme, and it forms hydrogen bonds to the side

chain of Glu371. The benzodioxolane ring and the

pyrimidine ring attain a stacked juxtaposition, and

they are seated in a hydrophobic region normally

occupied by the aliphatic portion of the side chain of

L-arginine.

This class of inhibitor of iNOS avoids the prob-

lems associated with replicating a protein–protein

interaction surface by binding to an alternate region,

where a preexisting appropriately sized pocket is

available, and eliciting inhibition by an allosteric

mechanism. This strategy requires an opportunistic

structural link between this type of remote pocket and

the interaction region, and this situation may not be

in place for many protein–protein targets.

FIGURE 9 Small molecule inhibitor of inducible nitric

oxide synthase (iNOS).41

FIGURE 10 Superposition of iNOS complexed with its

natural substrate L-arginine42 and with a small molecule in-

hibitor.41 The backbone of iNOS is depicted in ribbon for-

mat and colored red and green. The heme group of iNOS is

depicted in stick format and colored orange. The bound

substrate L-arginine is depicted in stick format and colored

brown, except for the nitrogens of the guanidino group

which are blue. The small molecule inhibitor is colored by

atom type and depicted in stick format. The superposition

was accomplished by maximizing alignment of the back-

bone atoms from the active site of iNOS (the single iNOS

backbone shown is from the complex with the small mole-

cule inhibitor).

544 Fry


TUMOR NECROSIS FACTOR a

Tumor necrosis factor � (TNF-�) is a cytokine,

which serves as a key promoter of the inflammatory

response. Inhibitory antibodies directed against TNF-

� have been developed as drugs, and go by the trade

names Enbrel, Remicade, and Humira. These agents

have proven to be effective therapeutics, particularly

for treating rheumatoid arthritis. Small molecule inhib-

itors would be desirable, because of their expected

advantages in terms of cost and mode of delivery.

A small molecule inhibitor of TNF-� has been

reported43 (Figure 11), and it acts via a unique mech-

anism. The functional form of TNF-� is a homo-

trimer, and the inhibitor has been shown to disrupt

the trimeric state. A series of elegant biophysical

studies established that the compound binds to the

trimer and actively promotes dissociation of one sub-

unit, leaving the inactive dimeric form. An X-ray

structure is available for the small molecule inhibitor

bound to the TNF-� dimer43 (Figure 12). It shows

that the inhibitor is located within a cleft between the

two protein subunits. Within the small molecule, the

phenyl-indole and chromone rings are in a stacked

conformation. The inhibitor acts as a wedge and indu-

ces local conformational changes, which ultimately

results in widening of the angle between two subu-

nits. This change ostensibly alters the interface uti-

lized for binding by the third subunit, to a point that it

can no longer achieve sufficient affinity to remain

part of the complex.

The TNF-� situation represents another case of al-

losteric inhibition, with the end result of preventing

formation of a functional oligomeric protein state. It

also relies on utilization of an opportunistic binding

pocket. It remains to be seen whether the ability to

employ this type of strategy is a rare occurrence, or a

situation that is commonly associated with the mobile

nature of protein structural elements, particularly

near exposed interfaces.

MDM2

MDM2 regulates the level and activity of a key pro-

tein involved in cell cycling and repair, namely the

tumor suppressor p53. It accomplishes this primarily

by two methods: by binding directly to p53 and steri-

cally blocking its transcription activation domain,

and by serving as a specific E3 ligase that targets p53

for ubiquitination and destruction by the proteosome.

MDM2 has been found to be overexpressed in many

human tumors, and restoring p53 function by inhibi-

ting its interaction with MDM2 is viewed as a viable

anticancer strategy. Therefore, the search for small

molecule inhibitors of this interaction has been an

ongoing focus of oncology programs in many phar-

maceutical research centers.

The region of MDM2 that interacts with p53 has

been known to be contained within a domain consist-

ing of the N-terminal 120 residues, and it was demon-

strated that the p53 binding epitope was even smaller,

and could be effectively reproduced as a 15-residue

peptide fragment.44 The structure of this peptide in

complex with MDM245 showed that the p53 peptide

adopts an � helical conformation when bound, and

achieves affinity by inserting three hydrophobic side

chains into subpockets within a binding cleft on

MDM2. At Roche, the dimensions of the MDM2

binding site were analyzed with a computer program

called cavSearch and, while it was somewhat small,

FIGURE 11 Allosteric small molecule inhibitor of tumor

necrosis factor-� (TNF-�).43

FIGURE 12 Structure of an allosteric small molecule in-

hibitor bound to the dimer form of TNF-�.43 The backbone

of TNF-� is depicted in ribbon format, with one subunit col-

ored brown and the other green. The small molecule inhibitor

is colored by atom type and depicted in stick format. A por-

tion of the surface of the dimer, representing the allosteric

binding site, is shown in grey-white as a Connelly depiction.



this site was found to fall within the range expected

for a site that can bind a small molecule effectively.46

Following a high-throughput screen using a diverse

library of synthetic chemicals, a number of com-

pounds were identified as active antagonists of the

p53-MDM2 interaction. NMR was used to identify

which of those compounds were authentic—i.e.,

which bound reversibly and nondestructively to the

primary binding pocket of MDM2.47 One such class

of compounds was a group of imidazolines, which

were subsequently optimized. Ultimately, compounds

were developed that exhibited in vitro binding poten-

cies as low as 90 nM, and showed encouraging in

vivo activity and safety in animal tumor models48

(Figure 13).

High-resolution structures of these compounds

bound to MDM248,49 revealed that the rigidity and ge-

ometry of the imidazoline scaffold drives effective

binding by directing substituents into the three sub-

pockets normally occupied by hydrophobic side

chains of p53. This was the first system involving a

small-molecule inhibitor of a protein–protein interac-

tion, where high resolution complex structures were

available for both the inhibitor and the natural ligand

it was meant to emulate. Such information makes it

possible to evaluate the strategy by which the drug-

FIGURE 13 Small molecule inhibitors of the interaction between MDM2 and p53. (A) An imid-

azoline; (B) a benzodiazepindione.

FIGURE 14 Superpositions of the MDM2-bound positions of a peptide fragment of the natural

protein partner p5345 and those of small molecule inhibitors: (A) an imidazoline48; (B) a benzodia-

zepindione.50 The backbone of the peptide is depicted in tube format and colored green. Key inter-

acting side chains of the peptide are depicted in stick format and colored brown. These residues are

(left to right) as follows: Phe19, Trp23, and Leu26. The small molecule inhibitors are colored by

atom type and depicted in stick format. The superpositions were accomplished by maximizing

alignment of MDM2 backbone atoms from residues at the active site, and then turning off the dis-

play of all MDM2 atoms.

546 Fry


like molecule replicates the natural protein ligand. An

overlay of the bound structure of the imidazoline with

that of the bound p53 peptide (Figure 14) shows that

the three peptide side chains known to be essential for

binding are successfully mimicked by the inhibitor.

Leu26 and Trp23 are each mimicked by a bromo-

phenyl group, and Phe19 is mimicked by an ethoxyl

group. This follows the theme that a proteomimetic

need not precisely duplicate the protein backbone.

The imidazoline core shows that, in this case, it is pos-

sible with a small molecule to economically bridge an

eight-residue segment of an � helix. Further, the exact

nature of the side chains need not be matched—in this

case, an aromatic side chain is replicated by an ali-

phatic group, and the converse is also observed.

Subsequent to this work, another class of small mol-

ecule was developed to yield potent inhibitors of the

MDM2-p53 interaction. These benzodiazepindiones

were optimized to levels of potency as high as 80 nM50

(Figure 13). An X-ray structure of one of these com-

pounds complexed with MDM2 was reported,50 and it

revealed that the binding strategy is based on side-chain

replication (Figure 14). Leu26 and Trp23 are mimicked

by chlorophenyl groups, and Phe19 is mimicked by an

iodophenyl group. This strategy is very similar to that

employed by the imidazoline inhibitors. However,

although the two central scaffolds present substituents

with equal effectiveness in terms of orientation and ri-

gidity, they are completely different chemically.

The MDM2 system, particularly because of the

success achieved in vivo and the absence of any

unique binding elements such as a metal site, gener-

ated renewed enthusiasm for finding drug-like inhibi-

tors of protein–protein targets. It confirmed that in-

hibiting a protein–protein interaction requires nothing

overly exotic—as long as a small molecule can prop-

erly mimic the key interactions made by the natural

protein ligand, it can be an effective and potent mod-

ulator. It also suggested that, for any given target, it

will be possible to find compounds from multiple

chemical classes that exhibit sufficient potency.

BCL-2/BCL-XL

The Bcl-2 family is a group of small proteins that are

critical mediators of apoptosis, and they function pri-

marily by forming protein–protein complexes with

other members of the group. Bcl-2 and Bcl-XL have

received special attention in oncology because they

are antiapoptotic, and are found to be overexpressed

in many cancer cells. Inhibiting their functional inter-

actions has emerged as a promising strategy for drug

development.

The molecular aspects of interactions involving

Bcl-2 family members have been revealed by the

structure of a prototypical complex in which Bcl-

XL is bound to a peptide fragment of its partner,

FIGURE 15 Small molecule inhibitors of Bcl-XL: (A) a chlorobiphenyl-piperazinyl-benzoyl-

sulfonamide known as ABT-737; and (B) a smaller analog thereof.



Bak.51 In this structure, the Bak peptide attains an

�-helical conformation, and sits in a cleft formed

between two helices of Bcl-XL. Bak inserts four

branched aliphatic side chains into subpockets

located along the binding cleft. The overall dimen-

sions of the cleft have been examined, and fall

within the range compatible with tight binding of a

drug-like molecule.3

Small molecule inhibitors of Bcl-2 and Bcl-XL

have been reported from numerous research groups,

and they exhibit varying levels of binding affinity.

The most potent Bcl-2 inhibitor described to date,

which has a reported Kd <1 nM, is a compound

which is composed of a linear array of five aromatic

rings and a piperazine, and features a sulfonamide as

one of the linkers52 (Figure 15). Its size and linearity

are more excessive than what is normally expected

for a drug molecule, but it does verify that tight bind-

ing by an organic molecule is attainable in the Bcl-2

pocket. An NMR structure of a similar molecule

from this class, with a potency of 36 nM, bound to

Bcl-XL has recently been reported,53 and it verifies

that the inhibitor sits in the primary binding groove

and fills subpockets that are normally occupied by

amino acid side chains from the natural binding part-

ner. A superposition of the structure of the bound in-

hibitor with that of the bound Bak peptide indicates

how the small molecule mimics one of the natural

protein partners (Figure 16). The two rings of the

phenylsulfanyl-ethyl-amino-benzylamide stack with

each other, positioning the S-phenyl to effectively

mimic the hydrophobic side chain of Ile85. This ori-

entation is reinforced by further stacking on either

side, to form an aromatic ladder, with residues Phe97

and Tyr195 of Bcl-XL. Ile81 and Leu78 are repli-

cated by the two rings of the fluorodiphenyl moiety.

Val78, the fourth residue of the peptide that appears

to make critical contacts with Bcl-XL, is not dupli-

cated at all by the inhibitor; however, this does not

seem to have a grave effect on the potency attained

by the inhibitor. This system represents another case

in which aliphatic side chains are successfully mim-

icked by aromatic moieties of the small molecule. As

was the case with the MDM2 system, a relatively

small molecule has proven capable of effectively

covering a span equivalent to an eight-residue stretch

of an �-helix.

X-LINKED INHIBITOR OFAPOPTOSIS

The protein known as the X-linked inhibitor of apo-

ptosis (XIAP) is another mediator of programmed

cell death, and it acts by participating in a series of

protein–protein interactions. XIAP binds directly to

caspases, and thereby maintains them in a catalyti-

cally inactive condition. Inhibiting this interaction

could help to restore apoptosis, and thereby offset the

cancerous state. For this reason, the XIAP system has

found popularity as a potential drug discovery target

in oncology.

The interaction of XIAP with caspases is mediated

by a region called the BIR domain. This interaction

can be abrogated via intervention by a naturally

occurring competitor protein called SMAC, which

FIGURE 16 Superposition of the Bcl-XL-bound posi-

tions of a peptide fragment of one of the natural protein

partners Bak51 and that of a small molecule inhibitor from

the ABT-737 family.53 The backbone of the peptide is

depicted in tube format and colored green. Key interacting

side chains of the peptide are depicted in stick format and

colored brown. These residues are (left to right) as follows:

Val74, Leu78, Ile81, and Ile85. The small molecule inhibi-

tor is colored by atom type and depicted in stick format.

The superposition was accomplished by maximizing align-

ment of Bcl-XL backbone atoms, and then turning off the

display of all Bcl-XL atoms.

FIGURE 17 Small molecule that binds to the BIR3 do-

main of the X-linked inhibitor of apoptosis (XIAP) and

inhibits its interaction with caspases.56

548 Fry


also binds to the BIR domain. A nine-residue peptide

fragment from SMAC was shown to be fully compe-

tent as an inhibitor, with an affinity of 430 nM.54 A

structure of this peptide bound to the BIR domain54

revealed that it sits in a surface groove, and showed

that only the first four residues make significant con-

tact. The corresponding tetrapeptide was tested and,

as expected from the structure, exhibited comparable

binding affinity of 480 nM.55 Analysis of the dimen-

sions of the XIAP binding site suggested that it may

be too small to support tight binding of a drug-like

small molecule.3 However, varying the composition

of the tetrapeptide ultimately resulted in a version

with 20 nM binding affinity.55

These efforts have been followed by design and

synthesis programs, which have succeeded in produc-

ing organic molecules that are comparable in size to

the tetrapeptides but with less peptidic character,

while maintaining affinity in the low nanomolar range

(Figure 17). A structure has been reported for one of

the most potent of these molecules bound to the BIR

domain,56 and it indicates that affinity is achieved

without significant expansion in the dimensions of

the binding pocket. Rather, the inhibitor fills the

pocket more effectively, and benefits additionally

from rigidification and better electronic interactions.

A comparison of the structure of the bound inhibitor

to that of a bound peptide from SMAC shows how

the small molecule mimics one of the natural protein

binding partners (Figure 18). The terminal Ala1 is

mimicked by the N-methyl-alanine moiety of the in-

hibitor. Val2 is mimicked by the tert-butyl group.

The ring of Pro3 is closely replicated by the pyrroli-

dine of the inhibitor. The phenoxy substituent is

unique to the inhibitor, and accesses a subpocket that

the peptide does not exploit. Presence of the phenoxy

has been shown to enhance affinity of the small mole-

cule, and it appears to accomplish this by making a

favorable stacking interaction with Trp323 of XIAP.

Finally, the hydrophobic side chain of Ile4 of the pep-

tide is effectively replicated by the tetrahydronaph-

thalene ring of the inhibitor.

The XIAP system is a case where an extended

conformation of a peptide, rather than an �-helicalone, is being mimicked. In this case, characteristics

of the backbone are retained by the small molecule.

In particular, there are two carbonyls and one NH of

the peptide that participate in hydrogen bonds with

residues from XIAP, and these are all specifically

retained in the small molecule. Otherwise, the princi-

ple of duplicating side-chain interactions is still an

essential component of the binding strategy. This sys-

tem also presents a new tactic, by which the small

molecule takes advantage of a nearby interaction op-

portunity that is not utilized by the natural ligand.

CONCLUSIONS

The preceding examples clearly demonstrate that

potent drug-like inhibitors can be discovered for a va-

riety of protein–protein interaction systems. When

these inhibitors are viewed collectively, some com-

mon themes emerge in terms of their physical proper-

ties and the strategies by which they accomplish

effective binding and inhibition:

1. They are on the high end of the molecular weight

range normally considered feasible for serving as

an administerable drug. This appears to be neces-

sary, since the binding region for most of the

protein–protein interactions being targeted is rel-

atively large. Even though the inhibitor is dupli-

cating only a subset of the natural contacts, these

are still spaced so far apart, that reaching them

cannot be achieved with a molecule of low mo-

lecular weight. Nevertheless, for many of these

molecules, it has been possible to optimize to a

form for which acceptable pharmacokinetics are

obtained.

2. They have distinct three-dimensional confor-

mations and are rigid. In some cases, this is

FIGURE 18 Superposition of the XIAP-bound positions

of a tetrapeptide fragment (Ala-Val-Pro-Ile) from one of

the natural protein partners Smac54 and that of a small mol-

ecule inhibitor.56 The surface of XIAP is shown in grey-

white as a Connolly depiction. The peptide is colored

brown and depicted in stick format. The small molecule in-

hibitor is colored by atom type and depicted in stick format.

The superposition was accomplished by maximizing align-

ment of XIAP backbone atoms of key residues from the

binding region (the single XIAP surface shown is from the

complex with the small molecule inhibitor).



achieved by projecting substituents off of a

rigid cyclic core, or by having multiple rings in

succession causing a restricted set of relative

orientations. A few of the molecules which

appear linear and flexible actually exhibit in-

herent preferences for intramolecular stacking,

and this imparts rigidity. The main advantage

of these properties is that functional groups can

be presented in an optimal orientation for mak-

ing key interactions within the binding site on

the target protein. Preorganization also reduces

the energetic penalty associated with loss of en-

tropy upon binding. Additionally, a rigid inhib-

itor is more capable of displacing flexible por-

tions of the target protein, thus creating addi-

tional subpockets and increasing the extent of

the intermolecular interaction surface. Dis-

placement of protein elements may also be par-

ticularly important for allosteric inhibitors,

where this action can trigger a conformational

change that is transmitted to the primary inter-

action site.

3. They do not precisely mimic the natural protein

ligand. Early efforts at designing proteomimetics

attempted to carefully match the key protein ele-

ments—for example, an aromatic ring bound at

a certain orientation would need to be duplicated

by an aromatic ring with an identical entry tra-

jectory. In the examples presented, there are

many cases of nonexact matches that are fully

effective. Aliphatic groups substitute for aro-

matic groups and vice-versa. The protein back-

bone is often not duplicated at all. Entry angles

into a subpocket are often quite variable—all

that seems to matter are the interactions that are

ultimately made within the subpocket.

In summary, there are both encouraging signs, and

remaining apprehensions, concerning the prospect of

finding small molecule modulators of protein–protein

interactions. On the favorable side, the existing suc-

cess stories indicate that high potency can be

achieved at a given protein–protein interface via a va-

riety of binding strategies, and by a variety of chemi-

cal types. In cases where the primary site does not

appear amenable, hoping to discover an allosteric

modulator may still be a realistic option, as there is

ample precedent for this situation. On the negative

side, the most potent known protein–protein inhibi-

tors tend to be complex, particularly in terms of ring

types and number of stereocenters, and may be repre-

sented poorly in corporate screening libraries and dif-

ficult to synthesize. Also, the existing success stories

presumably comprise only a small percentage of the

protein–protein systems that have been pursued, sug-

gesting that a certain set of characteristics is required

of an amenable target protein. Fortunately, some of

these attributes can be preassessed if sufficient struc-

tural information is available. Additionally, the issue

of selectivity has not been specifically addressed in

the stories presented, and it is not clear how one

should deal with this concern. The existence of sites,

among the tens of thousands of proteins in the cell,

sufficiently similar to that of the targeted protein to

allow nonselective binding cannot be assessed, and

such analysis probably will not be possible in the

near future. Currently, all that can be done is to test a

drug candidate in an in vivo setting and hope for the

absence of toxicity caused by binding to a nontar-

geted protein. The rigidity and complex conformation

exhibited by a typical potent protein–protein inhibitor

should reduce the chances for this sort of nonspecific

binding.

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