ve

dnocvale

es

typically bind small molecules (300500 A ) (Fuller et al.,

dynamically adjust to bind a drug-like molecule. By 2005, about

inhibitors have been larger and more hydrophobic than

et al., 2013; Bernal et al., 2010; Boersma et al., 2012; Changbeing developed that might be well suited to PPIs. For instance,

fragment-based lead discovery (FBLD) has had a particularly

strong impact. FBLD used biophysical methods, including crys-

Although PPIs come in many shapes and sizes, most of the

clinical-stage inhibitors target PPIs in which the hot-spot resi-

dues are concentrated in small binding pockets (250900 A2)tallography, surface plasmon resonance, and nuclear magnetic

resonance (NMR), or disulfide trapping (tethering) to identify

low-molecular-weight, low-complexity molecules that bound

(Basse et al., 2013; Smith and Gestwicki, 2012) and partner

proteins are characterized by short primary sequences at the

interface (London et al., 2013; Perkins et al., 2010). At onea half-dozen small molecules had been reported to bind with

the affinities one would expect for drug leads, at binding sites

defined by high-resolution structures (Wells and McClendon,

2007). In parallel, computation and chemical technologies were

et al., 2013; DeLano et al., 2000; Gavenonis et al., 2014). While

these approaches are outside the scope of the current review,

they represent a parallel strategy that can also inform small-

molecule design.2009). Unlike enzymes or G protein-coupled receptors (GPCRs),

nature did not offer simple small molecules that can start a

chemical discovery process, and high-throughput screening

(HTS) had not provided validated hits.

Between 1995 and 2005, hopeful signs were emerging. A

clinically approved integrin antagonist (tirofiban) and natural

products like taxanes, rapamycin, and cyclosporine inspired

confidence that PPIs could be modulated by small molecules.

Mutational analysis of protein interfaces showed that not all res-

idues at the PPI interface were critical, but rather that small hot

spots conferred most of the binding energy (Arkin and Wells,

2004; Clackson and Wells, 1995). Hot spots tended to cluster

at the center of the interface, to cover an area comparable to

the size of a small molecule, to be hydrophobic, and to show

conformational adaptivity. These features suggested that at least

some PPIs might have small-molecule-sized patches that could

typical orally available drugs (Wells and McClendon, 2007).

Two commonly used metrics to assess the drug-like quality of

a compound (or to compare a series of compounds) are ligand

efficiency (LE; DG/HA) and lipophilic ligand efficiency (LLE;

pIC50 logD or logP) (Hopkins et al., 2014). The LE for small-

molecule inhibitors of PPIs have hovered around 0.24, whereas

a LE of0.3 or higher is desired. Values of LLE >5 are consideredfavorable for in vivo activity. Encouragingly, recent PPI inhibitors

are approaching these drug-like values for several targets (see

below). Even inhibitors with properties outside average ranges

for oral drugs have been made orally bioavailable. Clinically suc-

cessful PPI inhibitorsmay therefore expand our understanding of

the types of molecules that can be made into drugs.

Also during the past fifteen years, there has been very prom-

ising progress with designing peptides that target PPIs and

show promising cell-based (and even in vivo) activities (AzzaritoSmall-Molecule Inhibitors oInteractions: Progressing to

Michelle R. Arkin,1,* Yinyan Tang,1 and James A. Wells11Department of Pharmaceutical Chemistry, School of Pharmacy, Uni*Correspondence: michelle.arkin@ucsf.eduhttp://dx.doi.org/10.1016/j.chembiol.2014.09.001

The past 20 years have seen many advances in our unhow to target themwith small-molecule therapeutics. Ipotent inhibitors have been developed for diverse prtrials for six targets. Surprisingly, many of these PPIlead-like or drug-like molecules and are orally amultiple disciplines andmake use of all themodern tooscreening, and biomarkers. PPIs become progressivcomplex, i.e., as binding epitopes are displayed on primthe last 10 years of progress, focusing on the propertiand prospects for the future of PPI drug discovery.

Protein-protein interactions (PPIs) represent a vast class of ther-

apeutic targets both inside and outside the cell. PPIs are central

to all biological processes and are often dysregulated in disease.

Despite the importance of PPIs in biology, this target class has

been extremely challenging to convert to therapeutics. Twenty

years ago, PPIs were deemed intractable. High-resolution

structures in the 1980s to 1990s showed that PPI interfaces

are generally flat and large (roughly 1,0002,000 A2 per side)

(Hwang et al., 2010), in stark contrast to the deep cavities that21102 Chemistry & Biology 21, September 18, 2014 2014 Elsevier LtChemistry & Biology

Review

f Protein-Proteinward the Reality

rsity of California, San Francisco, San Francisco, CA 94158, USA

erstanding of protein-protein interactions (PPIs) and2004, we reviewed some early successes; since then,tein complexes, and compounds are now in clinicallinical candidates have efficiency metrics typical ofilable. Successful discovery efforts have integrateds of target-based discoverystructure, computation,ly more challenging as the interfaces become moreary, secondary, or tertiary structures. Here, we reviewof PPI inhibitors that have advanced to clinical trials

weakly to subsites on the protein surface (Erlanson et al., 2004;

Hajduk and Greer, 2007; Winter et al., 2012).

The last decade has seen amazing progress in tackling chal-

lenging PPI targets with synthetic molecules. More than 40

PPIs have now been targeted (Basse et al., 2013; Higueruelo

et al., 2009; Labbe et al., 2013), and several inhibitors have

reached clinical trials. With this advance, it is important to recon-

sider the distinction between ligandability (druggability) and

our ability to convert PPI inhibitors into drugs. Historically, PPId All rights reserved

extreme, the peptide epitope is recapitulated by a linear peptide

epitope 14 amino acids long; there are four clinical-stage exam-

ples of small-molecule mimetics of these epitopes. PPIs that

contain a single unit of secondary structure, such as an alpha he-

lix, binding to a hydrophobic groove have also proven tractable

Figure 1. The Complexity of the PPI Interface Affects DruggabilityPPIs can be classified bywhether one side of the interface consists of a primary(linear) protein sequence (green), a single region of secondary structure (suchas an alpha helix, yellow), or multiple sequences requiring tertiary structure(red). There are fewer examples of small-molecule inhibitors of PPIs as theinterface becomes more complex (from primary, to secondary, to tertiary epi-topes). Structures shown are BRDt/histone (green; Protein Data Bank [PDB]:2WP1), MDM2/p53 (yellow; PDB: 1YCR), and IL-2/IL-2Ra (red; PDB: 1Z92).

Chemistry & Biology

Reviewto small-molecule inhibition. Globular interfaces, requiring ter-

tiary structure on both sides of the PPI, have fewer published

successes. In developing guidelines for PPI inhibitor discovery,

it is instructive to consider each clinical success story in the

context of the type of interface (Figure 1).

Primary Peptide Epitopes: Short, Continuous, LinearPeptidesOne of the first examples of a clinically successful PPI inhibitor is

tirofiban,whichbinds to the integrin IIbIIIa. Tirofibanwasdesigned

to mimic the linear tripeptide Arg-Gly-Asp (RGD), the epitope of

fibrinogen that binds to the I-like domain of IIbIIIa (Hartman

et al., 1992). While this small, linear peptide epitope seems like a

special case of PPI, this motif is observed in turns, at protein

termini, and in unstructured regions (Perkins et al., 2010). Unsur-

prisingly, these types of interfaces have been particularly

amenable to inhibition by small molecules, and four such targets

have recently entered clinical trials, which we discuss below.

LFA1

Leukocyte function-associated antigen-1 (LFA-1) has been the

target of drug discovery efforts aimed at reducing inflammatory

immune responses. LFA-1 is a beta2 integrin involved in T cell

activation and adhesion (Ford and Larsen, 2009) through binding

to its ligand ICAM1. LFA-1 is a heterodimer consisting of an alpha

chain (CD11a/aL) and a beta chain (CD18/b2). ICAM1 binds to an

inserted domain (I domain) located on CD11a. CD18 also has an

I-like domain that is located near the I domain. Three types of

drugs have been in the clinic for LFA-1. The anti-LFA-1 antibody

efalizumab was approved for psoriasis in 2003, but it was with-

drawn in 2009 due to rare but fatal viral infections associated

Chemistry & Biology 2with immunosuppression (Schwab et al., 2012). Small molecules

that bound to an allosteric site on the I domain were in clinical

trials for psoriasis, but no development has been reported since

2010 (Guckian and Scott, 2013). Lifitegrast (SAR1118), however,

recently completed its registrational phase 3 trial, and a new

drug application will be filed in early 2015 (Shire, 2014).

Lifitegrast was discovered at Sunesis, building from a series

originally published by Genentech (Gadek et al., 2002), and

was developed clinically by SARcode/Shire (Zhong et al.,

2012). There are no crystal structures of lifitegrast or its analogs

bound to LFA-1. The mechanism of action of the compound is

still under debate; it either binds directly to the ICAM site on

the I domain (Keating et al., 2006) or to the related site on the

I-like domain (Shimaoka et al., 2003) (Figure 2), analogous to

the binding of tirofiban. SARcode developed lifitegrast for the in-

flammatory disorder dry eye syndrome, the most common eye

disease in humans (Schaumberg et al., 2003). SAR1118 has an

optimal pharmacokinetic profile for ocular formulation, with low

systemic exposure and high aqueous solubility. Since this indi-

cation does not require systemic exposure, lifitegrast could

achieve the efficacy that eluded I domain antagonists (Guckian

and Scott, 2013) and avoid the toxicity that led to efalizumabs

withdrawal (Schwab et al., 2012).

IAPs

Inhibitor of apoptosis proteins (IAPs), including cIAP1, cIAP2,

and XIAP, are important regulators of cell fate, including

apoptotic cell death and immunity (Dubrez et al., 2013; Fulda

and Vucic, 2012). The IAPs have PPI interaction domains called

BIR domains and a RING domain E3 ubiquitin-ligase domain.

The BIRs bind directly to an N-terminal tetrapeptide sequence

exposed during proteolytic activation of caspase-3, caspase-7,

and caspase-9, thus inhibiting the activity of these proapoptotic

proteases. IAPs ubiquitin-ligase activity also leads to the ubiq-

uitylation and degradation of caspases (Dueber et al., 2011)

and other proteins involved in apoptosis and NF-kB signaling

(reviewed in Fulda and Vucic, 2012). Current thinking holds

that XIAP inhibition directly affects caspase activity, whereas

cIAP inhibition leads to stronger ubiquitin-mediated effects.

Development of homolog-selective inhibitors of the IAPs will

allow evaluation of these pathways in cancer therapy and toxicity

(Condon et al., 2014; Ndubaku et al., 2009; Sun et al., 2014).

An endogenous inhibitor of IAPs, called Smac (second mito-

chondrial activator of caspases), competeswith caspase binding

to BIR domains and thereby stimulates apoptosis (Liu et al.,

2000). Small-molecule IAP inhibitors mimic the tetrapeptide-

binding motif on Smac and are therefore called Smac mimetics

(SMs). The Smac peptide motif, Ala-Val-Pro-Ile (AVPI), binds to

two adjacent subpockets on BIR domains (Liu et al., 2000).

Hot-spot residues on the XIAP BIR3 domain focus on the pocket

that binds the N-terminal amine (E314) and alanine residue (L307

and W310) and W323, which interacts with proline. Smac pep-

tides are impressively tight binders; AVPI-amide binds cIAP1

with a Kd value of 2 nM (Condon et al., 2014). Since the first pep-tidomimetic inhibitor of the IAP/Smac (and IAP/caspase) interac-

tion was disclosed (Oost et al., 2004), several groups have devel-

oped inhibitors with improved potency, PK, and in vivo activity.

These compounds mimic the key interactions between AVPI

and BIR domains (Figure 2). Seven SMs have reached clinical tri-

als, and five compounds remain active in clinical development.

1, September 18, 2014 2014 Elsevier Ltd All rights reserved 1103

Clinical IAP inhibitors fall into two structural camps. The

monovalent antagonists, LCL161 (Novartis) (Dubrez et al.,

2013), GDC-0917/CUDC-427 (Genentech/Curis) (Flygare et al.,

2012; Wong et al., 2013) and SM-406/AT-406 (Wang lab/

Ascenta) (Cai et al., 2011) were designed from the AVPI peptide

and have IC50 values in the 260 nM range and a LE of0.3 (LLEsare in the drug-like range of 67.5). The bivalent antagonists,

birinapant (TetraLogic) (Benetatos et al., 2014; Condon et al.,

2014) and SM-1387 (Bai et al., 2014), are dimerized SMs, de-

signed based on the idea that the Smac homodimer simulta-

neously binds to XIAPs BIR2 and BIR3 domains (Huang et al.,

2003). These dimers demonstrate better cellular potencies

than their monovalent analogs. Unlike the orally active, monova-

lent compounds, the bivalent drug candidates are dosed intrave-

nously. SM-406 and birinapant bind 50-fold more tightly tocIAP1 than XIAP and may allow clinical evaluation of whether

XIAP inhibition is required for efficacy.

Identifying patients who will respond to IAP inhibitors as single

agents has been challenging. SMs induce apoptosis in 15% ofcancer cell lines (Oost et al., 2004), suggesting that single-agent

activity of clinical compounds could be unpredictable. Transcrip-

tion profiling and genetic data suggest that certain tumor types

might be more sensitive, particularly MALT lymphomas with

constitutively active IAP protein fusions (Fulda and Vucic, 2012).

Importantly, preclinical and clinical studies have identified serum

cytokines and cIAP levels as potential biomarkers of response

(Dubrez et al., 2013), which could be used to quickly assess

whether a patient is resistant to SM treatment. Furthermore, pre-

clinical data provides a strong rationale for several combinations

with radiation and chemotherapeutics, including the apoptotic

agonist TRAIL (Fulda and Vucic, 2012). The preclinical data also

scription, and epigene

histones. Transcriptio

contain multiple enzym

ing proteins (Lessard

represent multiple opp

tion using small-molec

Bromodomains are

ylated lysines (Kacs)

complexes to turn o

2014). Bromodomains

formed by a left-hand

of various lengths and

ordered water molecu

100 mM affinity (Vollbeen exploited by syn

higher affinity than the

examples, (+)-JQ1, sti

containing protein 4 (B

ability of bromodomain

Vidler et al., 2012). (+

49 nM. Crystal struct

cupies the acetyl-lysin

interactions not seen

(Figure 3). In the pas

been described and h

bromodomains in canc

kopoulos and Knapp,

Five compounds h

I-BET762 (Glaxo Smit

(Constellation) (Gehlin

2012), and OTX15 (On

1104 Chemistry & Biology 21, September 18, 2014 2014 Elsevier Ltd All rights reservedrequires the complex integration of signal

transduction, the action of DNA-binding

proteins that repress or stimulate tran-

tic signals on the DNA and DNA-bound

nal complexes are highly dynamic and

es, scaffolding proteins, and DNA-bind-

and Crabtree, 2010). These complexes

ortunities to activate or repress transcrip-

ule modulators of PPIs.

epigenetic readers that recognize acet-

on histone tails and direct transcription

n genes (Filippakopoulos and Knapp,

share a conserved hydrophobic pocket

ed four-helical bundle and loop regions

charges. An asparagine residue and fiveFigure 2. Peptide-like Inhibitors of PPIswith Primary Epitopes(A) SAR1118 (lifitegrast) inhibits binding of LFA1(CD11a/CD18) to its ligand ICAM. The mechanismeither involves binding directly to the ICAM site inthe I domain of CD11a (Keating et al., 2006) orallosteric inhibition through binding to the I-likedomain in CD18 (Shimaoka et al., 2003).(B) X-ray structure of a cIAP1/XIAP chimera (whitesurface) bound to GDC-0152 (green sticks; PDB:3UW4) and overlaid with SMAC peptide (magentacartoon; PDB: 1G73). Chemical structures ofSMAC mimetics in clinical trials are shown.

point to the potential for on-target toxic-

ities (Erickson et al., 2013; Yang and

Novack, 2013). To date, clinical trials are

too early to assess anticancer activity,

but the clinical compounds appear to

behave well and are tolerated at doses

that show strong pharmacodynamic ef-

fects (Infante et al., 2014; A.W. Tolcher

et al., 2013, J. Clin. Oncol., abstract).

Bromodomains

The cellular decision to transcribe a gene

Chemistry & Biology

Reviewles recognize Kac-histone peptides with

muth et al., 2009). This deep pocket has

thetic Kac mimetics that bind with much

native peptide. One of the first published

mulated strong interest in bromodomain-

RD4) as a cancer target and in the drugg-

s as a class (Filippakopoulos et al., 2010;

)-JQ1 binds to BRD4 with an affinity of

ures show that (+)-JQ1 completely oc-

e binding groove, including hydrophobic

in the histone H3 peptide/BRD4 complex

t 5 years, several potent inhibitors have

ave been used to explore the biology of

er and inflammation (reviewed in Filippa-

2014).

ave already advanced to clinical trials.

h-Klein) (Mirguet et al., 2013), CPI-0610

g et al., 2013), Ten-010 (Tensha) (Rohn,

cEthix) (P. Herait et al., 2014, Am. Assoc.

Chemistry & Biology

ReviewCancer Res., conference) are based on the JQ1 scaffold

(Figure 3) and are in clinical trials for cancer, including NUT

midline carcinoma, a rare cancer caused by BRD4-NUT fusions

(Filippakopoulos et al., 2010). RVX-208 is a quinazolinone that

binds deeply into the Kac pocket, making a hydrogen bond

to the Kac-recognizing asparagine residue; unlike the JQ1-like

compounds, it binds preferentially to the second bromodomain

of BRD3 (McLure et al., 2013). RVX-208 has been in phase 2

trials for atherosclerosis, with promising results. The clinical

compounds were identified through a range of methods. For

instance, I-BET762 and RVX-208 were found in cell-based HTS

focused on ApoA1 upregulation (for atherosclerosis) and were

later found to bind BET bromodomains (McLure et al., 2013; Mir-

guet et al., 2013); compound 3, a published analog of CPI-0610,

was evolved from an isoxazole fragment merged with the core

structure of JQ1 (Gehling et al., 2013). BET proteins may repre-

sent a best-case scenario for PPI inhibition, since the primary

recognition element is a single amino acid. However, the ability

to develop potent, ligand-efficient (average LE = 0.34; Table 1)

tecting IN from prot

catalytic activity (Enge

of LEDGF binding dom

shows a short turn o

dimer interface, with

(Cherepanov et al., 20

peptides provided a p

IN PPI, and stimulat

based on virtual scree

viewed inDe Luca et al

tert-Butoxy-(4-phenyl-

tives (Figure 3), inclu

(Boehringer-Ingelheim

viral potency, LE = 0.3

2014; Fenwick et al., 2

signed to identify inh

DNA; structural studie

LEDGF binding pocke

core positions the tw

Chemistry & Biology 21, September 18, 2014 mimics the binding of a peptide derived fromHIF1a (PDB: 4AJY; not shown).

inhibitors, even though histone peptides

bind with low affinity, speaks to the po-

tential to gain binding energy that is not

used by the native PPI.

HIV Integrase

Viruses hijack host proteins to facilitate

their replication and survival; these host/

pathogen complexes represent impor-

tant clinical targets. The homotetrameric

protein HIV integrase (IN) integrates the

viral genome into human DNA by cata-

lyzing 30 processing and strand transfer.The HIV drugs raltegravir and elvitegravir

bind to the active site of IN and inhibit its

enzymatic activity. Recently, a second

class of IN inhibitors targeting a host/

pathogen PPI have reached the clinic

(Engelman et al., 2013).Figure 3. Small-Molecule Inhibitors of PPIswith Primary Epitopes(A) Crystal structure of BRD4 bromodomain (whitesurface) bound to (+)-JQ1 (green sticks; PDB:3MXF) overlaid with acetylated histone peptide(magenta cartoon; PDB: 2WP1). Clinical com-pounds or their closest published analogs areshown below.(B) Crystal structure of the dimerization interface ofHIV integrase (white and cyan surface) bound tocompound 16 (green sticks; PDB: 4NYF) withoverlaid epitope from LEDGF (magenta; PDB:2B4J). Compound 16 is a precursor to the clinicalcompound BI 224436.(C) Von-Hippel Lindau (VHL) protein (white sur-face) with bound inhibitor compound 51 (greensticks; PDB: 4B9K). The central hydroxyprolineThe human protein lens endothelial

growth factor (LEDGF) acts as an IN

cofactor, facilitating integration of the viral

genome into the host chromosome, pro-

eolytic degradation, and stimulating its

lman et al., 2013). The cocrystal structure

ain with a dimer of IN catalytic domain

f helix from LEDGF pointing into the IN

400 A2 buried surface area on IN

05; Basse et al., 2013). LEDGF-mimicking

roof of concept for inhibiting the LEDGF/

ed small-molecule discovery programs

ning, structure-aided design, and HTS (re-

., 2011). Themost potent PPI inhibitors are

quinolin-3-yl)-acetic acid (tBPQA) deriva-

ding the clinical compound BI-224436

, Gilead), which has low nanomolar anti-

3, and LLE = 11.5 (Table 1) (Fader et al.,

014). tBPQAs were found in an HTS de-

ibitors of IN-catalyzed 30 processing ofs later revealed the compound bound to

t at the IN dimer interface. The quinolone

o side-chain mimetics; as with BRD4

2014 Elsevier Ltd All rights reserved 1105

to

)Table 1. Properties of Selected Protein-Protein Interaction Inhibi

PPI Target/Partnera Compound Name MW (Da

Primary Epitope

LFA1/ICAM1 SAR1118 (lifitegrast) 615

cIAP/SMAC GDC-0152 499

GDC-0917/CUDC-427 565

AT-406/SM-406 562

TL-32711 (birinapant) 809

Bromodomain/histone I-BET762 (GSK 525762) 424

compound 3e 400

RVX-208 370

Integrase/LEDGF BI 224436 443inhibitors, BI-224436 binds to pockets that the LEDGF turn does

not (Figure 3).

tBPQAs are dual inhibitors. In addition to inhibiting the LEDGF/

IN complex, they also allosterically inhibit IN catalytic activity

by preventing the formation of functional IN tetramer. Recent

studies suggest that the mechanism of these inhibitors is com-

plex and altering tetramerization may be more therapeutically

important than inhibiting LEDGF per se (Balakrishnan et al.,

2013; Sharma et al., 2014). BI-224436 could provide a new

mechanism for HIV treatment that will most likely have a different

resistance profile than active site IN inhibitors. Furthermore, its

story highlights the fact that enzyme/protein complexes have

multiple effects on the enzyme; small-molecule inhibitors might

alter one or more of these functions.

Prospects for Primary Sequence Epitopes

Inhibitors of PPIs that consist of primary sequence epitopes have

been discovered using multiple approaches. Fragment-based,

biochemical, and cell-based screening have all yielded hits for

this PPI class (Fader et al., 2014; Gehling et al., 2013; Mirguet

VHL/HIF1a compound 51 450

Secondary Epitope

BCL family/BH3 ABT-263 (navitoclax) 975

ABT-199 868

compound 14 437

MDM2/p53 RG7112 (Ro5045337) 728

RG7388 (Ro5503781) 616

MI-888f 548

PDK1/PIF-tide PS210 380

Menin/MLL MIV-6R 418

MIV-2-2 416

p300 CH1 domain/HIF1a OHM 1 495

Tertiary Epitope

IL-2/IL-2Ra SP4206 663

HPV11 E1/E2 BILH 434 608

MW, molecular weight; nd, not determined.aItalicized compounds are in active clinical development. For PPI families (e.

IC50 is reported. When available, Kd values are reported.bLE = DG / number of heavy atoms.cLLE = pIC50 clogD.dActivity is

Chemistry & Biology

ReviewSecondary Structure Epitopes: a Helix, b Sheet, orExtended PeptidesIt has been estimated that up to 40% of PPIs consist of a single

peptide from one protein that binds into a groove on the other

(Petsalaki and Russell, 2008). Sixty percent of PPI in the protein

databank utilize an a helix, and 60%of these bind a single face of

the helix (Raj et al., 2013). In general, hot spots on the target pro-

tein are centered on two to three subpockets that bind hydro-

phobic residues on the partner peptide. Such interfaces have

been described as hot sequences (London et al., 2010) or

secondary-structural epitopes, reflecting the fact that the

key residues on the peptide are not next to each other in the pri-

mary sequence, but that an isolated peptide can often mimic the

binding affinity and pose of the PPI.

BCL Family

BCL family proteins are central effectors of apoptosis. Proapo-

ptotic family members, including BAD and BAK, are kept in an

inhibited state through binding to the antiapoptotic family mem-

bers, including BCL-2, BCL-xL, and MCL1. The antiapoptotic

members are helical proteins with an open groove that binds

to a single helix (the BH3 domain) on the proapoptotic partner.

Upregulation of the antiapoptotic BCL family members is an

important mechanism through which cancer cells avoid cell

death in the face of pathway dysregulation, radiation, and

chemotherapy. The structure of BCL-xL bound to BAK peptide

(16-mer, Kd = 340 nM) suggested that this PPI might be particu-

larly druggable (Sattler et al., 1997). The binding site onBCL-xL is

comprised of two large (200 A2) nearby pockets and three hy-drophobic residues from the BH3 peptides define the PPI hot

spot (Oltersdorf et al., 2005). Indeed, between 2000 and 2004,

several laboratories published small-molecule inhibitors with

(Oltersdorf et al., 200

available analog ABT-2

1 and 2 clinical trials

leukemia (CLL) (Rober

these early clinical tria

an important dose-lim

Current clinical BCL

BCL-xL. ABT-199 was

avoid thrombocytopen

tivity into ABT-263, a s

with BCL-2. A trypto

molecule sat very nea

that position led to AB

tightly than ABT-263 a

compared to BCL-xL

the BAK-binding pock

bone structure or side

currently in phase 1 tr

very promising, with re

2014). BCL-xL-selectiv

have also been develo

while avoiding the lym

2014). The L-shaped B

ingmode fromABT-19

the peptide-binding gr

and compound 14 are

than ABT-737 and AB

activity for BCL family

weaker thanbindingaf

teinbinding,or thecellu

for several members o

Chemistry & Biology 21, September 18, 2014 0.21. ABT-737 demonstrated impressive

single-agent activity in cancer xenograft

models, but was not orally bioavailable

5). In 2008, Abbott disclosed the orally

63 (Tse et al., 2008), which entered phase

for solid tumors and chronic lymphocytic

ts et al., 2012; Rudin et al., 2012). During

ls, thrombocytopenia (platelet loss) was

iting toxicity linked to BCL-xL inhibition.

family inhibitors are selective for BCL-2 or

designed to be selective toward BCL-2 to

ia (Souers et al., 2013). To engineer selec-

tripped-down scaffold was cocrystalized

phan residue from a neighboring BCL-2

rby to the inhibitor; linking of an indole atFigure 4. BCL-2/BCL-xL and MDM2Interfaces Contain Secondary Epitopes(A) Structure of BCL-2 (white surface) bound toABT-199 (green sticks; PDB: 4MAN) with overlaidBAX BH3 peptide (magenta cartoon; PBD: 2XA0).Chemical structures of selected BCL-2 and BCL-xL inhibitors are shown.(B) Structure of MDM2 (white surface) boundto RG7112 (green sticks; PDB: 4IPF) with over-laid p53 peptide (magenta cartoon; PDB: 1YCR).Chemical structures of compounds in clinicaltesting or their closest published analogs areshown.

potencies in the 0.110 mM range that

bound in the BAK-binding site (reviewed

in Arkin and Wells, 2004).

The major preclinical breakthrough

was the 2005 disclosure of ABT-737,

which was discovered through NMR-

based fragment discovery, structure-

based design, and medicinal chemistry

(Oltersdorf et al., 2005). ABT-737 binds

to BCL-2, BCL-xL, and BCL-W with sub-

nanomolar affinity, but it is also a large

compound (MW = 813 Da), with LE =T-199, which binds BCL-2 10-fold more

nd shows 5,000-fold selectivity for BCL-2

. Like its precursors, ABT-199 accesses

ets, but does not mimic the peptide back-

-chain orientation (Figure 4). ABT-199 is

ials for CLL, and preliminary results look

sponse rates of >80% (Cancer Discovery,

e inhibitors (e.g., compound 14; Figure 4)

ped, with the aim of treating solid tumors

phocytic effects of BCL-2 (Koehler et al.,

CL-xL-selective series has a distinct bind-

9 and appears to be buriedmore deeply in

oove. It is noteworthy that both ABT-199

more ligand efficient and have better LLE

T-263 (Table 1). Interestingly, cell-based

inhibitors is generally 100- to 1,000-fold

finity. Thismaybedue topermeability, pro-

lar context ofBCL-2-xLandseems tohold

f this target class. Thus, very-high-affinity

2014 Elsevier Ltd All rights reserved 1107

compounds have been required (Souers et al., 2013). While it is

still early days in the clinical life of BCL family antagonists, the

impressive story of their discovery highlights the realities of drug

discovery for a novel and challenging target; often, several itera-

tions of clinical candidates are needed to strike the ideal balance

of efficacy, safety, and dosing regimen.

MDM2

The transcription factor p53 is a master tumor suppressor that

regulates cell-fate decisions such as senescence, cell-cycle

arrest, and apoptosis. p53 or its regulators are mutated or

altered in most cancers, resulting in reduction or loss of p53

function. The ubiquitin E3 ligase responsible for p53 degradation

is MDM2 (murine double minute 2) and the MDM2/MDMx heter-

odimer (Khoo et al., 2014). This system is overexpressed or

amplified in several cancers with wild-type p53, and inhibition

of p53 ubiquitylation by MDM2 should lead to increased p53

levels and activity. The MDM2/p53 complex can be minimized

to an a helix from the N-terminal transactivation domain of p53

that binds into a pocket on the surface of the N-terminal domain

of MDM2 (Kussie et al., 1996). The MDM2/p53 interface is more

compact than that found for BCL-xL/BH3 peptide: the helix is

only two turns, and the three critical hot-spot residues (Phe19,

Trp23, and Leu26) point toward a deep pocket in the center of

the peptide-binding groove. The isolated peptide binds with

500 nM affinity to the N domain of MDM2.The breakthrough for inhibiting p53/MDM2 came from the dis-

covery of the Nutlins, identified in a large high-throughput screen

(Vassilev et al., 2004). The optimized,100 nM inhibitor containsa central imidazole ring with four substitutions. Three hydropho-

bic substitutionsmimic the binding of the three key residues from

p53, and the fourth substituent binds on the protein surface.

Soon after the discovery of the Nutlins, several labs described

compounds with different cores but similar molecular-recogni-

tion features (Fry, 2012; Khoo et al., 2014). A general feature is

a tripod shape that pushes at least two hydrophobic elements

deep into the binding site; these are fairly rigid structures that

use a central ring to enforce the side-chain geometries. Some

compounds also bind further along the p53-binding groove.

Compounds also differ in their ability to bindMDMX, whichmight

be important for efficacy and overcoming resistance in some

contexts (Khoo et al., 2014). The key observation from these

diverse MDM2 ligands is that many different scaffolds (including

peptides) can direct the hydrophobic groups into the p53-bind-

ing pocket. (Fry, 2012). Thus, it is not critical to mimic precisely

the peptide backbone or the peptides side-chain orientation.

One of the puzzling aspects ofMDM2 inhibitor design has been

the role of the fourth substituent. Modifications to this group alter

the compounds physicochemical characteristics and have a

modest effect on binding to the N-terminal domain of MDM2.

However, changes often have a disproportionate effect on cell-

based potency (Ding et al., 2013; Zhao et al., 2013). Recent

data suggest that parts ofMDM2outside thep53-bindingdomain

may play a role in the binding interaction. For instance, MDM2mutations that render cells resistant to Nutlins often occur

outside the p53-binding domain (Wei et al., 2013). The direct

demonstration of a structural interaction has yet to be made,

but thegeneral point rings true: inminimizing thePPI for biochem-

ical and structural studies, we are likely to miss long-range inter-

actions that the compounds will face when they enter the cell.

1108 Chemistry & Biology 21, September 18, 2014 2014 Elsevier LtThree scaffolds have been developed into experimental thera-

peutics. Roche initially took RG7112 (Figure 4) into phase 1 trials

(Tovar et al., 2013) and is currently testing the more potent and

ligand-efficient analog RG7388 (Ding et al., 2013). The spirooxin-

dole class of compounds, exemplified by MI-888, was discov-

eredby theWang lab atUniversity ofMichigan and is beingdevel-

oped by Sanofi (MI-773, structure not disclosed) (Zhao et al.,

2013). Roche recently disclosed Ro8994, a 5 nM (95% bioavail-

able) analog based on a spiroindolinone scaffold that combines

features of RG7388 andMI-888 (Zhang et al., 2014). Finally, Daii-

chi Sankyo is developing an undisclosed MDM2 inhibitor based

on a dihydroimidazothiazole scaffold (Miyazaki et al., 2013).

These clinical-stage inhibitors show impressive tumor regres-

sions in xenograft models and selective killing of cells containing

wild-type p53 versus those with mutant p53 (Tovar et al., 2013).

Their drug-like properties are also promising, with LE in the range

of 0.270.35, LLE in the range of 4.36.7, and very high oral bio-

availabilities despite molecular weights in the range of 550730.

MDM2 inhibitors are being developed for single-agent use and

in combination with existing drugs. Early trials for RG7112 have

shown pharmacodynamic activity, including 3- to 5-fold in-

creases in levels of p53 and p53 target genes (Ray-Coquard

et al., 2012), and potential therapeutic activity in acute leukemia

(M. Andreeff et al., 2012, Am. Soc. Hematol., conference). The

biological challenges facing clinical positioning of these com-

pounds has been recently discussed (Khoo et al., 2014). Despite

decades of work on p53, its precise roles in survival and death

pathways remain to be elucidated; these clinical MDM2 antago-

nists are poised to address some of these critical issues.

Selected Emerging Examples of Secondary Structure

Epitope Inhibitors

Diverse methods have been used to develop secondary struc-

tural mimetics and prospects for developing inhibitors of this

class of PPI are good. One instructive example is the N lobe of

the kinase PDK1. This site binds to a 10-residue peptide called

a PDK1-interacting fragment (PIF-tide) from substrate proteins.

Binding serves to colocalize the proteins and to allosterically acti-

vate PDK1 kinase activity (Gold et al., 2006). Fragments that

mimic thePIF-tide havebeen found throughvirtual screening (En-

gel et al., 2006), NMR (Stockman et al., 2009), and tethering

(Sadowsky et al., 2011). For tethering, six cysteine residues

were introduced around the PIF-tide binding site and were

screened for binding to a library of disulfide-containing frag-

ments. Remarkably, fragments tethered at the same cysteine

residues could be either activators or inhibitors of PDK-1 kinase

activity. Cocrystal structures showed that activators pushed

the C helix toward the active site and better organized the cata-

lytic apparatus, whereas inhibitors shifted the C helix outward

into a less competent state (Figure 5). Thus, small changes in

the small-molecule ligand can have positive or negative effects

onenzymeallostery. Virtual screening, followedbychemical opti-

mization, provided cell active inhibitors of the PDK1/PIF-tide

interaction. PS210 (LE=0.35)mimics two hot-spot phenylalanine

Chemistry & Biology

Reviewresidues on the PIF-tide (Busschots et al., 2012). In biochemical

assays, this compound activates the kinase toward peptide sub-

strates, asdoes thePIF-tide. Paradoxically, in cells, a cell-perme-

able version of PS210 inhibits phosphorylation of the subset of

PDK1 substrates that require binding at the PIF-tide site. We

recently identified cell active compounds from an HTS that

d All rights reserved

Chemistry & Biology

Reviewmonitored displacement of an optimized PIF-tide (unpublished

data). These compounds serveas additional examples of peptide

mimicry by small molecules and provide ligand efficient scaffolds

that may be further optimized.

Multiple approaches have also led to inhibitors of the transcrip-

tion factor mixed-lineage leukemia (MLL) protein at two different

domains. MLL binds to the scaffolding protein menin, and this

interfacecanbeminimized toan11-merpeptide from theN-termi-

nal domain ofMLL.HTS followedby structural biology andmedic-

inal chemistry led to MIV2-2 and MIV-6, potent, ligand-efficient,

and cell-active inhibitors that closely mimic the MLL peptide hot

spots (Figure 5 and Table 1) (He et al., 2014; Shi et al., 2012).

MLLalsobinds to theKIXdomainof theacetyltransferaseCBPus-

ing an a helix in the C-terminal domain of MLL. Mapp and

colleagues have designed synthetic molecules that bind to the

KIX domain at the MLL site (Buhrlage et al., 2009). They have

also used tethering to identify disulfide-bound compounds that

bind in the MLL site and allowed the first X-ray structure of the

KIX domain (Wang et al., 2013b). Their ultimate goal is to develop

bifunctional molecules that activate transcription by bringing

together DNA-binding proteins and transcriptional coactivators.

Prospects for Secondary Structure Epitope Design

Design approaches have focused on both the binding pocket

and the partner peptide. When focusing on the binding pocket,

(Figure 5 and Table 1),

and inhibits tumor grow

Computational appro

residues have also se

2014). This type of epi

peptide approaches (

Boersma et al., 2012;

of PPIs with secondar

low LE; however, the

and the PDK1, menin

more favorable ranges

Tertiary Structural EBinding SitesInterleukin-17

The larger, shallower i

discovery remain chall

disclosed the develop

hibits binding of interle

et al., 2012), which

hormone/receptor int

undisclosed structure

a LE of 0.24. We eand preclinical data su

Chemistry & Biology 21, September 18, 2014 interfaces often show small motions that

create deeper binding pockets (Johnson

and Karanicolas, 2013; Wells and

McClendon, 2007). Pocket-based design

is justified by the observation that inhibi-

tors such as ABT-199 and RG7112 mimic

the function of hot-spot residues but

rarely mimic trajectories seen in the pep-

tide structures. Others have focused on

strategies to mimic the peptide partner

directly. For example, Lao et al. designed

an a-helical mimetic of the HIF1a transac-

tivation domain that binds to the cysteine-

histidine rich 1 (CH1) domain on CBP/

p300. The optimized compound OHM 1

is based on a bis-oxopiperazine scaffold;

OHM 1 binds CH1 with a Kd of 500 nM

downregulates hypoxia-inducible genes,

th in a xenograft model (Lao et al., 2014).

aches to systematically replace peptideFigure 5. Preclinical Examples of Inhibitorsof PPIs with Secondary Epitopes(A) Crystal structures of PDK-1, showing the PIF-tide binding site bound with Tethered activator(JS30, green sticks; PDB: 3OTU) or inhibitor (1F8,magenta sticks; PDB: 3ORX). JS30 pushes the Chelix into a catalytically competent conformation.The chemical structure of noncovalent PIF-tideinhibitor PS210 is shown.(B) Crystal structure of menin (white surface)shown bound to MIV-6R (green sticks; PDB:4OG8) with MLL peptide overlaid (magentacartoon; PDB: 3U85). Structures of MLL inhibitorsMIV-6R and MIV 2-2 are shown.(C) Structure of OHM 1, which binds to CBP at theHIF1a peptide-binding site.

virtual methods should allow for side-

chain flexibility, since hot spots at theseen some promising success (Guo et al.,

tope is also highly amenable to stabilized

Azzarito et al., 2013; Bernal et al., 2010;

Chang et al., 2013). Historically, inhibitors

y-structural epitopes were large and had

second-generation clinical compounds

, and HIF1a lead compounds all fall into

for LE and LLE.

pitopes: Discontinuous

nterfaces that originally discouraged drug

enging. Recently, Ensemble Therapeutics

ment of a macrocyclic compound that in-

ukin-17 (IL-17) to its receptor (Livingston

represents a breakthrough in targeting

erfaces with synthetic macrocyles. The

has 3 nM affinity, a MW of 750 Da, andagerly await publication of the structural

rrounding this program.

2014 Elsevier Ltd All rights reserved 1109

IL-2 and HPV-11

Two examples of PPI inhibitors highlight the dynamic nature

of globular PPIs. A series of compounds, exemplified by

SP4206, binds to IL-2 at the receptor-alpha interface (Figure 6)

(reviewed in Wilson and Arkin, 2011). These compounds were

identified serendipitously and optimized through fragment-

based methods, including tethering. IL-2 presents a relatively

flat surface in the apo- and IL-2Ra bound states but shows

pockets upon binding SP4206. SP4206 binds at the receptor-binding hot spot and mimics the receptors hot-spot residues

and charge distribution, even though the residues on are sepa-

rated over multiple regions of the receptors structure. The

conformational changes seen across the SP4206-binding sur-

face include side-chain rotations and loop rearrangements,

larger changes than those observed for the peptide epitopes

described above. Molecular dynamics simulations suggest that

these structural adaptations are better thought of as part of the

protein conformational ensemble rather than induced fits.

BILH 434 (Figure 6) is another example of an inhibitor of a ter-

tiary structural epitope (Wang et al., 2004). Human papilloma

virus-11 (HPV11) requires the replication initiation factor E1 heli-

case to bind to the E2 transcription factor at specific DNA sites.

BILH 434 was identified in a screen for inhibitors of the E1/E2/

DNA complex and binds to the transactivation domain (TAD) of

E2 protein with a Kd of 40 nM (LE = 0.25). The X-ray structure

of the compound/E2 complex shows the compound nestled

into the elbow of the E2 TAD. Several side chains in this protein

interface change conformations to create a deep pocket for BILH

434 to bind. Again, changes to the structure were unanticipated

and were crucial for creating a binding site at an otherwise flat

surface. Importantly, both the IL-2 and E2 TAD discoveries relied

heavily on biophysical validation of the binding mode that was

confirmed by X-ray crystallography.

Prospects for Tertiary Epitope Binders

This class is probably the most challenging, and there are only

a handful of successful programs that have broken into the nano-

molar level of potency with modest LEs (typically 0.24 kcal/HA).

Allosteric MechanisFunctional screening

tously led to allosteric

BIO8898, an inhibitor

conformation of the trim

vianet al., 2011). Simila

and disrupt trimer form

component complexe

gray box screening,

in vitro. This approach

HSP70 chaperone netw

and how these HSP7

effects on folding and

et al., 2013; Wang et a

dipitous) allosteric mo

forward for the most d

How Has Our Under10 Years?The discovery of small

tinues to be challengin

strate that it is possible

being sought, but it is l

a target class analogo

extremely diverse in t

the binding affinities an

are (conservatively) 10

1110 Chemistry & Biology 21, September 18, 2014 2014 Elsevier Ltd All rights reservedms of Inhibitionfor PPI inhibitors has also serendipi-

mechanisms of inhibition. For instance,

of the trimeric ligand CD40L, alters the

er, thus inhibiting its binding toCD40 (Sil-

rly, compoundshavebeen found that bind

ation for TNF (He et al., 2005). For multi-

s, one way to harness this serendipity is

where the protein network is assembled

has yielded interesting modulators of the

ork. Depending on the biological context

0 modulators bind, they have opposite

thus utility in different disease states (LiFigure 6. Small-Molecule Inhibitors of PPIswith Tertiary Epitopes(A) Structure of IL-2 (white surface) bound toSP4206 (green sticks; PDB: 1PY2).(B) Structure of transactivation domain of E2 pro-tein fromHPV11 (white surface) bound to BILH 434(green sticks; PDB: 1R6N).

These interfaces tend to be much more

dynamic than the primary and secondary

class epitopes and thus less amenable

to virtual screening. Small molecules

can, however, find deeper pockets in

these interfaces and thus bind with higher

LE than their protein partner counterpart.

The small molecules also bind hot spots

predicted from alanine scanning. This

class is probably themost prevalent class

for extracellular PPIs. Strong validation

of the biology will certainly make this

class temptingif high-hangingfruit.

For example, four groups have recently reported allosteric and

orthosteric PPI inhibitors of Ras, a very challenging cancer target

with no apparent deep pockets in the interface. Interestingly,

three groups took fragment-based approaches, using NMR

(Maurer et al., 2012; Sun et al., 2012) and tethering (Ostrem

et al., 2013), and one group identified inhibitors through virtual

screening (Shima et al., 2013). Finally, PPI-binding peptides

discovered from phage display could provide another avenue

toward peptide-based therapies (DeLano et al., 2000).

Chemistry & Biology

Reviewl., 2013a). Directed (as opposed to seren-

dulation of PPIs provides potential way

ifficult targets (Nussinov and Tsai, 2014).

standing of PPIs Evolved in the Past

, drug-like molecules that inhibit PPIs con-

g, but the diverse success stories demon-

for some systems. General strategies are

ikely that PPI is not properly thought of as

us to class I GPCRs or kinases. PPIs are

he size and shapes of the interfaces and

d dynamics of assembly. Given that there

0,000 PPIs (Venkatesan et al., 2009), our

current database of PPI inhibitors is small and biased. Neverthe-

less, here is what we know so far.

Analyses of PPIs in which crystal structures are available for

both the protein-protein and protein-small-molecule reveal

someprovocative themes (JohnsonandKaranicolas, 2013;Smith

and Gestwicki, 2012; Wells and McClendon, 2007). First, PPI

target proteins tend to have extended binding grooves that may

be divided into three or more (visible) subpockets (Fuller et al.,

2009). Hot-spot residues are centered in or around these pockets

and are complementary on both sides of the interface. Second,

small conformational changes in thebinding site present a deeper

pocket to small molecules than to the partner protein. These

pockets are also more readily formed at the PPI interfaces than

elsewhere on the protein surfaces (Johnson and Karanicolas,

2013). The consensus is that ligand-binding surfaces (including

PPIs) haveahigherpropensity forbinding (tocognate ligands, sol-

vent molecules, inhibitors) than the rest of the protein surface.

Third, PPIs with small-molecule inhibitors tend to have small,

high-affinity interfaces and include a hot segment that can reca-

pitulate the binding of the partner protein. The observation that

high-affinity complexes havemore small-molecule ligands (Smith

andGestwicki, 2012) could imply that they are inherently sticky.

Fortunately, however, there are many examples where peptides,

mutated proteins, and small moleculessuch as bromodomain

inhibitorsbind to hot spot sites with higher affinities than the

native PPI. The preponderance of inhibitors for primary- and sec-

ondary-structural epitopes is probably due to their inherent

druggability, the fact that there are many such interfaces (Petsa-

laki and Russell, 2008), and the availability of design approaches.

In general, success seems to follow the simplicity of the epitopes

to mimic: primary is easier than secondary is easier than tertiary.

PPI inhibitors bind at the hot spot and tend tomimic the types of

binding interactions (e.g., hydrophobic)madeby thepartner.How-

ever, they generally use different functional groups and approach

thebinding site fromdifferent orientations than in thePPI. To reach

each subpocket in an extended interface, molecules often have

three distinct pharmacophores arrayed on a scaffold that serves

to orient them. Hence, orthosteric inhibitors have tended to be

larger than 500Da and to bemore 3D than the average compound

inanHTS library (Basseetal., 2013; Labbe etal., 2013). Toaddress

the potential lack of PPI-inhibitor-like compounds in HTS libraries,

investigators have proposed design of libraries that mimic the

shapes of current PPI inhibitors or protein secondary structures.

To date, more traction has been gained through virtual screening

and the use of nontraditional libraries, including larger, more natu-

ral-product-like macrocycles on the one hand and fragment-

based lead discovery on the other. Fragment screening has

been a particularly successful strategy, perhaps because the

pocket sizes at PPIs are close to fragment size and the bias in

chemical composition is lower. Experimental and virtual fragment

screens have even be used to define the druggability of a PPI (Haj-

duk and Greer, 2007; Kozakov et al., 2011).

On the other hand, second-generation clinical candidates are

Chemistry & Biology

Reviewgenerally smaller andmore efficient than their precursors, and so

perhaps the inherency of undruglikeness of PPI inhibitors has

been overstated (Table 1), particularly for small interfaces. Con-

cerns for LE and LLE originate from the observation that larger

molecules are more difficult to tune for in vivo properties. More

important than molecular weight per se is finding the balance

Chemistry & Biology 2of hydrophobicity and electrostatics that provides tight, specific

binding to the protein target and acceptable pharmacokinetics

and pharmacodynamics. These issues are by no means specific

to PPI drug discovery.

Screening methodologies have also become more sophisti-

cated in the past 10 years. Foremost, there is an expanded

use of multiple drug discovery technologies, including an inte-

grated use of biochemistry, biophysics, structural biology, and

computational studies. Initial PPI inhibitors have been discov-

ered using a wide variety of methods; we conclude that the

screening format itself is less crucial than the compounds going

into the screen and the postscreening evaluation of hits. True

positives should be validated by demonstrating that the inhibitor

binds to a single site on the PPI target using biophysical and

structural methods. The importance of mechanism of action

over potency at the screening stage cannot be overstated.

What We Expect to See More of in the Near FutureTwenty years ago, PPIs were considered impossible fruit to

reach. Today, there are dozens of targets for which early stage

compounds have been discovered, and nearly a dozen com-

pounds have been in the clinic. Clearly, much progress has

been made, and this has generated the courage to move

forward, even with enthusiasm.

Ten years from now, how far will we have come? In terms of

methodology, new computational methods will allow better un-

derstanding of allostery and conformational ensembles that are

grist for improved virtual screening algorithms. There will be

a continued emphasis on fragment-based design, and also a

resurgence in natural products and natural-product-like mole-

cules, since biology has evolved these molecules for binding at

protein interfaces. Popular target classes will include epigenetic

and transcription factor complexes, signal transduction net-

works, and protein quality control systems, reflecting the need

for chemical tools to dissect these complex protein networks.

Building on the function of natural products, there will also be

greater emphasis on synthetic stabilizers of PPIs (Milroy et al.,

2014). Advances in cell biologywill allow faster discovery of com-

pounds mechanism of action and more translatable cell-based

assays. Importantly, the PPI inhibitors in clinical trials today will

be drugs benefitting patients. To us the writing is on the wall:

PPIs are a challenging but tractable class for small-molecule dis-

covery if the biology is compelling and the will is strong.

ACKNOWLEDGMENTS

We thank our colleagues in the PPI inhibitor field for their beautiful work andapologize to those whose work we could not describe due to space consider-ations. We also thank Jason Gestwicki for his thoughtful edits. This work wassupported by NIH grants AG046526 and CA136779.

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Small-Molecule Inhibitors of Protein-Protein Interactions: Progressing toward the RealityPrimary Peptide Epitopes: Short, Continuous, Linear PeptidesLFA1IAPsBromodomainsHIV IntegraseProspects for Primary Sequence Epitopes

Secondary Structure Epitopes: Helix, Sheet, or Extended PeptidesBCL FamilyMDM2Selected Emerging Examples of Secondary Structure Epitope InhibitorsProspects for Secondary Structure Epitope Design

Tertiary Structural Epitopes: Discontinuous Binding SitesInterleukin-17IL-2 and HPV-11Prospects for Tertiary Epitope Binders

Allosteric Mechanisms of InhibitionHow Has Our Understanding of PPIs Evolved in the Past 10 Years?What We Expect to See More of in the Near FutureAcknowledgmentsReferences