1

Discovery of Inhibitors of Protein-Protein Interactions from

Combinatorial Libraries

María J. Vicent1, Enrique Pérez-Payá1,2 and Mar Orzáez1,*

1 Dept. Química Médica. Centro de Investigación Príncipe Felipe. E-46013 Valencia,

Spain.

2 Instituto de Biomedicina de Valencia (CSIC). E-46010 Valencia, Spain.

• Corresponding author: Dr. Mar Orzáez. Centro de Investigación Príncipe Felipe,

Avda. Autopista del Saler, 16, E-46013 Valencia, Spain. Phone: +34-963289680;

Fax: +34-963289701; E-mail: morzaez@cipf.es

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Abstract: Protein-protein interactions play a central role within numerous processes in the cell.

The relevance of the processes in which this type of interactions are implicated make

them responsible of many pathological situations. In the last decade protein-protein

interfaces have shown their potential as new drug targets, and combinatorial chemistry

has been defined as an useful tool in this line. This review gives a global vision of the

actual situation of combinatorial chemistry, highlighting its applicability to high-

throughput drug discovery and giving some crucial examples of its contribution to find

modulators of protein-protein interactions.

Abbreviations:

Acquired immunodeficiency syndrome (AIDS), conformationally restricted libraries

(CRL), hightroughput methods of screening (HTS), human immunodeficiency virus

type 1 (HIV-1), nuclear magnetic resonance (NMR), rabies virus Nucleoprotein (N),

rabies virus Phosphoprotein (P), structure activity relationship (SAR).

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PROTEIN-PROTEIN INTERACTIONS AS DRUG TARGETS.

The most generalised approach used to develop new therapeutic agents has been

mostly based on the choice of a single gene product of clinical relevance as target.

However, as it was early postulated and more recently experimentally confirmed, a

large number of proteins inside the cell rarely stays alone. Molecular signalling and

signal transduction pathways at cellular level require massive protein usage in order to

transmit signals through protein-protein interactions producing cell-answerings in

response to different environmental changes or modifications. Not only proteins in

cellular pathways are busy seeking for interactions but structural proteins are also

forming part of macromolecular structures and enzymes have regulatory and

catalytically active subunits. In this crowded scenario, in which protein-protein

interactions are the main actors, it is reasonable to speculate that the modulation of

protein-protein interactions could open new alternatives to the development of

molecules that could modulate cellular pathways that in turn could have relevant future

therapeutic applications. Special attention is devoted to the drawing of the protein

interaction maps. In fact, there are now available cellular-protein interaction maps of

model organisms[1], human[2,3], and some cellular pathways and, in addition, many

genomes have been completely sequenced (see http://www.ensembl.org). However,

these maps represent only a very partial picture of the complete set of real interactions.

Because detecting and analysing all protein-protein interactions by classical

experimental methods is not as fast as genome sequencing, there is an unbalance

between our knowledge on the basic pieces of the puzzle (the proteins) and the higher

order relationships between them. Thus, it would be useful for the identification of new

potential targets the use of predictive bioinformatics methods based on different types

of evidences such as sequence features [4] or different indirect experimental evidences

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such as co-expression [5], domain-domain co-occurrence [6] and others methods.

These approaches are becoming more and more accurate and now it is reasonable to

take them as a starting point in absence of previous experimental evidence. In addition,

systematic whole-cell mass spectrometry approaches are currently used to identify

protein complexes [7,8], which includes strong, transient and weak interactions between

proteins that will increase our knowledge of interacting networks [9,10]. Nevertheless,

data derived from all these methodologies clearly point to the need of taking into

consideration protein-protein interactions as targets for the development of new drugs.

In fact a study of the pairwise interactions among 8100 human open reading frames

(that represents only about 10% of the total protein-coding genes), showed that at least

in 424 of them one of the partners is implicated in human diseases [3] .

Figure 1

The development of modulators for protein-protein interactions is not exempt of

difficulties. Among others, one of the first identified problems is related to the nature of

the surfaces that define these interactions. While the ‘classical’ protein sites targeted by

drug discovery programs are ligand binding sites, defined by small-concave

cartographic spots, the protein-protein interaction surfaces are flat and large. An

extensive network of weak molecular interactions maintains the structure of the

complex, then at a first glance, they were classified as extremely difficult target for

small-molecules, that in turn were the favourite choice for drugs. Years of investigation

are now providing information on the detailed nature of these interactions that for sure

will provide the hints to afford disruption of this type of complexes. For example, now

we know that the packing between molecules is not as tight as the core packing of

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proteins, probably due to the less hydrophobic interaction surfaces and the presence of

interfacial water molecules [11]. This flexibility allows multiple forms of protein-

protein complexes that can be initially classified as permanent, with no individual

components found in the cell during protein lifetime and, transient complexes that are

formed and disrupted in response to different cell situations. The total extension of the

interactive surface, considered a key parameter of the discovery strategy, would then

probably vary when facing a small (close to 1000 Å2) or a middle to large (> 4000 Å2)

interacting interface [11]. In addition, it is well established now that when comparing

the folding of an individual protein versus the structural arrangements needed for a

protein-protein interaction there are differences on the role of the structural elements. A

correct ‘hydrophobic-driven’ single protein packing tends to collect hydrophobic

elements of secondary structure inside the protein core regardless of their secondary

structure. However, it has been observed that in protein-protein interaction surfaces

there is a ranking of preferences on packing, from high to low, between β-sheet/β-sheet,

loop/loop, α-helix/α-helix while α-helix/β-sheet interactions are less favoured [11].

Furthermore, statistical data also suggests that some defined amino acids have tendency

to be more present than others at the protein-protein interfaces. Methionine, tryptophan,

cysteine, phenylalanine, tyrosine, arginine and histidine are overrepresented at transient

molecular interfaces opposed to hydrophobic residues that are in general

underrepresented in this type of surfaces [12]. Some amino acid pairs are frequently

found in association like tryptophan and proline or phenylalanine and isoleucine. It

should be also noted that exposed hydrophobic interfaces tend to be small and although

hydrophobic residues contribute to the binding affinity, these types of interactions are

less specific than those composed of polar or charged residues.

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Taking all this information together it seems that there is an infinite diversity in the

universe of protein-protein interactions difficult to cope with from the point of view of

the design of new therapeutics. Nevertheless proteins are often constructed of modular

domains that are used repeatedly in distinct molecules to mediate their interactions [13].

For example, proline rich motifs are recognized by conserved interaction modules such

as SH3, WW and EVH1 domains or phosphotyrosine motifs are recognized by SH2 or

PTB domains. These common features facilitate the development of general strategies

for the inhibition of different molecular targets, which share an interaction motif

although their cellular function could be completely different.

In any case the remaining question is whether or not relatively small molecules, a

desirable characteristic to consider a molecule as a potential drug candidate, could or

not be capable of disrupting protein-protein interaction surfaces. Recent progress in the

field has provided a large number of examples [14,15] in which this strategy of

modulation has prospered opening a new field for drug discovery. Relevant and

difficult to target diseases such as acquired immunodeficiency syndrome (AIDS),

Alzheimer or cancer are being favoured with recent discoveries from methodologies

using this strategy.

A second key question is related to the methodological strategy that could be used to

ensure the identification of active compounds able to inhibit these protein-protein

interactions. Is it necessary to use traditional searching or it is maybe better to use a

combinatorial approach?. The limiting factor for rational design is the requirement of

an exhaustive knowledge of the targets in order to identify ‘hot spots’ along the

interaction surface of proteins where small drugs could disrupt the complexes.

Unfortunately, our current knowledge about protein-protein surfaces, even with enough

structural data available, is extremely limited to permit the use of this rational strategy

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as a general approach to modulate interactions. Moreover in this new technological era

where the information about protein interactions emerge as global interactome maps [3]

in which thousands of proteins are inter-connected, it seems necessary to find a global

strategy to perform the search of modulators for all these interactions. It is in these

situations where combinatorial approaches could be extremely beneficial and profitable,

offering a great diversity of compounds to test protein-protein disruption activity and

therefore overcoming the problem of lack of information. From the technological point

of view the use of combinatorial chemistry requires the development of hightroughput

methods of screening (HTS) for protein-protein interactions in order to increase the

probability of finding a lead compound. There are many techniques available to

identify protein partners such as yeast two hybrid system, mass spectrometry, protein

chips (reviewed in [16]), POSSYCCAT for the study of interactions between

transmembrane fragments [17], etc. Once an active product has been identified, deeper

studies could permit the location of the interaction site with its target and give the

necessary information to improve the drug-protein interaction surface. This site could

probably be considered as a ‘hot spot’ on the interaction surface between the proteins.

Furthermore, the discovery of a new inhibitor could facilitate the identification of the

cellular processes in which this interaction is implicated. In the cases where enough

information about the target is available to initiate an adequate drug rational design,

combinatorial chemistry could also contribute offering the possibility of improving its

inhibition capabilities through structural modifications of the defined lead compound

[18,19].

This review will try to illustrate in a first stage the principles and types of

combinatorial chemistry and how with this combinatorial approach it is possible to

identify inhibitors for key protein-protein interactions.

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COMBINATORIAL CHEMISTRY: A VALID APPROACH FOR TARGET

VALIDATION AND DRUG DISCOVERY

Combinatorial vs. traditional chemistry. Principles of combinatorial chemistry

Traditionally, natural product extracts or industrial collections of randomly

synthesised organic molecules (synthesised one at a time) were screened to discover

“hits”, which were then optimised in an iterative process by the synthesis of

derivatives. Many important drugs were identified following this approach however

the ratio of novel to previously discovered compounds started to diminish with time.

This fact together with the cost constraints found on pharmaceutical research forced

the investigation of methods in the pharmaceutical industry that could offer higher

productivity at lower expenses. By that time, genomics and proteomics had

experienced an exponential growth and more potent and efficient technological devices

for biological screening had also been achieved. Altogether, triggered the appearance

of a new discipline in the early 1980s: Combinatorial Chemistry [20].

Combinatorial chemistry could be defined as “the generation of large collections,

or ‘libraries’, of compounds by synthesising all possible combinations of a set of

smaller chemical structures, or ‘building blocks’, in a time and submitted for

pharmacological assay” [19-28]. In this way, the chemist can synthesise up to

thousands of compounds at once instead of preparing only a few by simple

methodology. The traditional approach of organic synthesis to sequentially synthesise

a logically designed set of analogues based on a lead compound has been surpassed by

the ability to screen whole compound libraries accumulated over the years by large

pharmaceutical companies. Therefore, with the combinatorial strategy the productivity

has been amplified beyond the levels obtained as routine in the last century [29,30].

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Solid-phase and solution-phase combinatorial chemistry

Solid phase peptide synthesis methodology, introduced by Merrifield in 1963,

paved the way for solid-phase combinatorial approaches (extensively reviewed in

[27,28,31-35]). Geysen [36,37] was the pioneer in this field synthesising peptides on

pin-shaped solid supports followed by Houghten with his “teabag” approach to epitope

mapping [38]. Solid-phase combinatorial chemistry has been widely implemented for

hit discovery as well as lead optimisation due to its advantages and simplicity of use,

advantages such as: (i) the compounds can be isolated once they are attached to solid

supports, (ii) the isolation of the immobilised product by simple filtration permits the

use of large reagent excesses to get high-yield conversions for each of the steps, (iii)

pseudo-dilution effects are present and (iv) the use of split-and-mix synthesis

simplifies the preparation of large libraries [15].

The split-and-mix solid–phase synthesis on beads was introduced by Furka et

al.[39,40]. This technique has been enthusiastically exploited by many others since its

first disclosure. For example, Houghten has used split-and-mix on a macro scale in a

"teabag" approach for the generation of large libraries of peptides [41]. Lam et al. [42-

44] was able to enhance the production and rapid evaluation of random libraries of

millions of peptides in a way that acceptor-binding ligands of high affinity could be

rapidly identified and sequenced, on the basis of a 'one-bead, one-peptide' approach.

Consequently to the large acceptance achieved by the split-and-mix strategy, several

identification techniques have been also developed, such as nucleotide- [45,46],

peptide- [47,48], chemical- [49], radiofrecuency- [50], color- [51], and shape- [52-54]

encoded or iterative [55-57] and recursive [58,59] deconvolution.

Unfortunately, there is not an ideal general technique, therefore, among the

limitations of solid-phase combinatorial synthesis should be mentioned the restriction

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of being only suitable in linear synthetic strategies and the requirements for linkage or

cleavage steps that may restrict the possible chemical reactions. A hydroxyl, amine,

carboxyl, or other polar group must be present on a molecule to be able to attach it to a

support. This is a potentially undesirable constraint on the structure of compounds

synthesised on solid phase, as products retain the polar group even after they are

cleaved from the support. Several groups have been devoted to diminish these

drawbacks in order to continue improving the implementation of solid-phase synthesis.

The use of ‘Traceless linkers’ [12,60-62] have been one of the most remarkable

implementations. For instance, Mori and coworkers [60,63] have developed an aniline

linker for solid-phase synthesis of azomethines or Ellman et al. [64] have reported an

acylsulfonamide linker able to add diversity to a library when displaced by various

nucleophiles at the same time that is completely removed from products during the

cleavage process. Furthermore, other innovative techniques for solid-phase synthesis

have been developed, including: react-and-release type chemistry [65], in which

reagents used to cleave products from the solid support are incorporated into the

product; use of scavenger resins [66]; the introduction of safety-catch linkers [67-69]

or the improvement of resin properties and loading capacities [70-72].

Although the solid-phase combinatorial chemistry is still the most widely used,

several groups have been applying an alternative solution-phase approach to

complement solid-phase techniques. Solution-phase synthesis offers the possibility of

expanding the repertoire of chemical reactions and allows the application of

convergent synthetic strategies, the synthesis of mixture libraries or the use of dynamic

libraries [73,74].

Dynamic combinatorial chemistry [29,73,74] is based on continuous

interconversion between the library constituents, it uses reversible connections

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between the initial building blocks producing flexible and adaptive libraries. This

approach is driven by the interactions of the library constituents with the target site.

This strategy allows a target-driven generation or amplification of active constituents

by performing a self-screening process where the active species are preferentially

expressed and retrieved from the library [73]. A similar concept is found in Phage

displayed libraries (biologically displayed libraries) [75-77] first published on 1985 by

Smith et al.[78]. These offer a strategy to isolate peptide ligands to target proteins and

to define interaction sites between proteins [79]. In phage displayed libraries, instead

of using inter-convertible building blocks as it is done in dynamic libraries, possible

active peptides or oligonucleotides (phage population) are screened by incubation with

the target molecule adsorbed to a solid support. Active phages bind the target, then

target-bound phages are isolated and propagated by infection of E. coli and subjected

to an additional round of adsorption to the immobilised target. The use of biological

displayed libraries for the isolation of peptide ligands is an interesting alternative to

chemical libraries.

Both, solid-phase and solution-phase strategies can be easily automated, however,

the major limitation to solution-phase is the isolation and purification of the library

compounds [80,81]. To date, most of the isolation/purification steps are based on acid-

base chemistry and sequestration-enabling reagents [82-84]. The pioneering acid-base

extraction approach of Boger et al. [85], clearly showed the possibility of generate

large libraries of pure molecules. His work has been mainly focused on the main

concept raised in this review, the discovery of small compounds (mainly

peptidomimetics) inhibitors of protein-protein interactions [15]. Mixture synthesis

provides for solution-phase synthesis what split-and-mix synthesis provides for solid-

phase approaches (reviewed in [86,87]).

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Analogous to encoding approaches for split-and-mix synthesis of large libraries

(also known as iterative approach), there are two key and complementary

deconvolution strategies that permit the immediate identification of active lead

compounds from large mixture libraries: positional scanning[88,89] and deletion

synthesis [90,91] techniques. The positional scanning approach was first described by

Houghten et al. [88,89] and was developed as an alternative to the iterative strategy

where the lead identification was achieved through successive steps of mixture

selection with the active compound, synthesis of sublibrary and subsequent evaluation,

that is, only one residue can be identified at a time by a synthesis-assay cycle. When

using a positional scanning approach [92,93] several sublibraries can be synthesised at

a time, in each sublibrary an individual component is placed in one position but

mixtures are used in all others. The most active sublibrary in each set indicates the

optimal substituent for that position so, an increase in activity is measured that allows

the identification of active lead structures. On the other hand, in deletion synthesis

deconvolution libraries [90], an individual component is missing in each sublibrary but

full mixtures are used in all other positions. The least active sublibrary in each set

indicates the optimal substituent for that position as the missing component that in

turn, will be the most active. This strategy provides less global information that

positional-scanning but is better at identifying a uniquely potent library member. It is

important to note that, with either of both techniques, the lead identification comes out

from a single round of screening.

SUITABILITY OF COMBINATORIAL APPROACH TO TARGET PROTEIN-

PROTEIN INTERACTIONS

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Despite the already described inherent difficulties in the process to identify small

compounds modulators of protein-protein interactions, there are several important

modulators of this type of complexes that have been defined using combinatorial

approaches. Some of the more representative examples are described in more detail

below (Table 1).

Apoptosis inhibitors

Programmed cell death or apoptosis is a highly regulated process of cell deletion

that plays crucial roles in development and maintenance of tissue homeostasis in

multicellular organisms. Due to the relevance of this cellular mechanism its

deregulation is a key issue in the pathogenesis of several human diseases such as cancer

or neurodegenerative disorders [94]. This type of cellular death can be triggered by a

variety of extrinsic and intrinsic signals [95].

Inhibitors of apoptosome formation.

In the apoptosis pathway in mammals the formation of a multiprotein complex

called apoptosome between cytochrome c, Apaf-1 (apoptotic protease-activating factor)

and procaspase-9 links mitochondria dysfunction with the activation of the effector

caspases starting the cascade of cell death [8,96-98]. Our laboratory focused on the

identification of compounds that inhibit the apoptosome-mediated activation of

procaspase-9 from the screening of a positional scanning diversity-oriented

combinatorial library of trimers of N-alkylglycines [99]. These type of molecules also

known as peptoids present a series of characteristics that make them good candidates to

cope with the disruption of protein-protein interactions. Its main chain is quite similar

to a polypeptide but with the lateral side chain on the nitrogen atom instead of the

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carbon thus giving the advantage of the similarity at the same time that conferring

flexibility to the scaffold which could favour the interaction with the target surface

[100]. Moreover, due to this chemical difference with proteins, cellular proteases are

not able to degrade this type of substrates increasing the lifetime of the drug inside the

organism. The library consisted in 52 controlled mixtures and a total of 5120

compounds. Mixtures 1 to 20 (O1XX) contain as defined position one of 20 selected

commercially available primary amines, while at the ‘X’ positions (mixture positions) a

set of 16 primary amines was present. Mixtures 21 to 36 (XO2X) and 37 to 52 (XXO3)

contain at the defined position only a set of 16 amines. The mixtures making up each

sublibrary were screened for their ability to prevent the apoptosome-dependent

activation of procaspase-9. For this goal the apoptosome was assembled in vitro by

incubating rApaf-1, cytochrome c, dATP and [35S]-Met procaspase-9.

After the identification of a lead compound (peptoid 1) rescued from the library a

rational strategy was used to improve its solubility as well as its cellular internalisation

capability, obtaining finally a drug with antiapoptotic activity in different cell lines, see

Fig (2).

Figure 2

Inhibitors of interaction between pro- and anti-apoptotic proteins.

The response of mitochondria to apoptotic signals is a key point of regulation of

apoptosis that is controlled by the Bcl-2 family of proteins. In particular by the balance

between pro- and anti-apoptotic proteins in the mitochondrial membrane. Anti-

apoptotic Bcl-2 proteins (for example, Bcl-2 and Bcl-XL) are the target of pro-apoptotic

proteins (like Bad, Bax, Bak) that through their BH3 domain bind to a hydrophobic cleft

on the surface of Bcl-2 or Bcl-XL. Several lines of evidence indicate that BH3-mediated

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binding has a key role in regulating apoptotic functions. In fact, short peptides derived

from BH3 domains of various pro-apoptotic Bcl-2 family members are sufficient to

induce apoptosis in cells [101]. To identify small-molecule inhibitors of Bcl-XL-BH3

domain interaction, Degterev et al. [102] screened a commercially available library of

16320 chemicals and found hit compounds that after chemical optimisation rendered

biologically active compounds that inhibited the interaction between Bcl-XL and BH3

domains and induced apoptosis in Jurkat cells. The same protein-protein complex was

selected as target by Oltersdorf et al. [103]. However in this study they applied the

methodology of SAR by NMR. In this methodology a library of small chemical

fragments is screened against the protein target to identify low-affinity binding

compounds. Then the chemical linkage of proximal fragments would render a new

molecule with high binding affinity to the target. Using this technique a small

molecule, named ABT-737, with high affinity (Ki< 1 nM) to Bcl-XL, was identified, see

Fig (3). ABT-737 displayed synergism with chemotherapeutics and radiation.

Furthermore, the compound showed in vivo anti-tumor activity causing complete

regression of established tumor xenografts.

Figure 3

Viral inhibitors

Combinatorial libraries have also contributed to the inhibition of the life cycle of

certain viruses such as the human immunodeficiency virus or the rabies virus.

HIV protease dimerisation inhibitors

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus with a very complex

life cycle in which at least 15 protein are implicated [104]. Several drugs targeting

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different steps of this cycle have been developed. One of the main pharmacological

targets is the virus protease (PR), a pivotal enzyme in viral maturation responsible of

processing the polyproteins encoded by the virus rendering active proteins needed for

the assembly and the infection steps of the virus particle [105]. PR is an homodimeric

aspartil protease with the active site located at the interface of the dimmer [106]. This

dimerisation interface is a highly conserved region which makes it a relevant target for

drug discovery. The main dimerisation region is folded as an interdigitating N-and C-

terminal four stranded β-sheet. The group of Chmielewski using rational design

developed inhibitors of the PR dimerisation derived from crosslinked interfacial

peptides corresponding to this conserved β-sheet region and identified the minimal

structure necessary for the activity of these peptides [107]. Moreover in an attempt to

improve its activity a focused library on the basis of this minimized molecule was

designed (compound 6) in which certain side chains were substituted by different

natural and non natural amino acid side chains, see Fig (4). The analysis of the library

not only gave information about the groups that confer better characteristics in each

position but also rendered a molecule (compound 36, Fig (3)) with improved activity

respect to the initial scaffold.

Figure 4

Rabies virus infection inhibition

Infection by rabies virus is the cause of thousands of deaths by encephalomyelitis

per year in the world. The clear need to have a post-exposure treatment led the group of

Yves Jacob to work on the development of a new virucidal drug [108]. Rabies virus is a

member of the lyssaviruses family. Among other viral constituents the Phosphoprotein

(P) is crucial for the formation of the transcription-replication complex, in which

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interacts with the RNA-polymerase and the nucleoprotein (N). Moreover P also

interacts in the cell with Dynein LC8 a protein implicated in retrograde transport. These

characteristics made P a good target to develop a new virucidal drug, although there was

no structural information about the complexes in which it participates. Ybes Jacob and

coworkers designed two genetically encoded combinatorial peptide libraries, called

coactamer libraries. In these libraries the peptide scaffold is structurally constrained

due to the presence of either prolines or cysteines. This structure mimics conotoxins

and insect antimicrobial peptides, which easily reach the cellular interior, in an attempt

to increase drug bioavailability. A two hybrid assay was used to identify peptides

interacting with phosphoproteins from two different lyssaviruses. Peptides selected

from the library were tested for its viral transcription-replication complex interference

activity. To be sure that their activity were related with an alteration of the N-P

interaction a ProteinChip with an anti-Flag M2 monoclonal antibody cross-linked was

used to immunoprecipitate P and its associated proteins from cell extracts in the

presence or absence of the co-transfected inhibitor peptides. Peaks corresponding to

the interaction between P and N were analysed by SELDI-TOF, and four peptides

modulators of the ratio P/N (indicator of an altered interaction) were identified.

Table 1

Inhibitors of cell migration

Integrin αvβ3 / MMP-2

Integrin αvβ3 is an heterodimeric cell surface receptor that promotes cell attachment

to the extracellular matrix. MMP-2 (gelatinase A) a protein secreted by vascular

endothelial cells is crucial for the degradation of collagen matrix which permits new

vessels to proliferate. Moreover, the interaction between integrin αvβ3 and MMP-2

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seems to be responsible for the activation of MMP-2 in invasive endothelial cells [109].

Disruption of this protein-protein interaction is one of therapeutic strategies that are

being exploited for tumor growth inhibition. Screening of a library composed of ten

mixtures of 60 small organic compounds designed to mimic potential protein–protein

interactive moieties have led to the identification of a lead compound with in vitro

activity. Posterior refinement experiments permitted to obtain a more water-soluble

molecule with antitumor and antiangiogenic in vivo activity [110]. One of the active

compounds, termed TSRI265, was shown to prevent collagen IV degradation in hamster

CS-1 melanoma cells transfected with the human 3-integrin, see Fig (3). Furthermore,

TSRI265 almost completely inhibited FGF-stimulated angiogenesis in 10-day-old

chick chorioallantoic membrane. The data obtained from this study set the basis to

consider inhibition of MMP-2/ v 3 binding as a crucial molecular target to block the

angiogenic process [111].

Integrin α4β1 / Paxillin

The interaction between the signal adaptor protein Paxillin and the cytoplasmic tail

of integrin α4β1, a cell surface receptor, has been implicated in several pathogenic

situations including enhanced rates of cell migration, reduced rates of cell spreading,

focal adhesion, and stress fibber formation [112]. All these processes contribute to

leukocyte migration into tissues and to the expression of genes involved in chronic

inflammation. From the screening of a combinatorial library in an ELISA assay using

immobilised His-tagged α4 and examining the binding of Paxillin, Boger and coworkers

obtained a lead molecule that inhibits this interaction [92]. The library consisted of a

scaffold composed of three different variable subunits (X,Y and Z) linked by amide

bonds, and a basic side chain 4-(dimethylamino) butyric acid linked to the X subunit,

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see Fig (5). For each variable position ten aromatic amino acids were used. The library

was prepared in solution using two different formats, as 100 mixtures of 10 compounds

and using a positional scanning approach in which 30 sublibraries were generated.

Results using the two types of library formats were quite similar. The lead molecule

(11 X7-Y7-Z7 ), see Fig (3), obtained from the screening not only disrupted Paxillin/

Integrin α4β interaction but also demonstrated a potent inhibition activity of human

Jurkat T cell migration.

Figure 5

Cell cycle inhibitors

Cell growth in eukaryotic cells is under control of a series of concerted molecular

mechanisms defined as the cell cycle. In cell life cycle there are strict checkpoints

controlling cells prepared to enter mitosis. The p16-cyclinD-pRB-E2F pathway

controls the G1/S transition of the mammalian cell cycle. E2Fs are a family of

transcription factors (E2F1 to E2F7 in mammalian cells) that require heterodimerization

with proteins of the DP family to bind DNA and exert its regulatory function over genes

implicated in DNA replication [113,114]. Activation of E2F dependent transcription

promotes progression from G1 to S and conversely its inhibition arrests cells in G1

[115]. Sardet and coworkers [116] identified, from the screening of a combinatorial

library of thiorredoxin-20mer peptide (called aptamers), a molecule that interacts with

E2F1 dimerization domain. For this purpose, a two hybrid assay with E2F1

dimerization domain as a bait, was used. Among the molecules identified there was one

peptide (Apt5) that shared notable sequence similarity with a region of DP1. More

extensive studies demonstrated that this molecule interferes with the interaction between

E2F/DP. Moreover, deeper studies in mammalian cells demonstrated that Apt5 is

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capable of blocking fibroblasts in G1 reinforcing the hypothesis that this interaction

could be considered as a key target for the development of pharmaceutical

antiproliferative agents.

Inhibitors of G-proteins

Heterotrimeric G-proteins (Gαβγ) transform the signal produced by G-protein

coupled receptors in an intracellular signal. In humans the wide diversity of receptors

together with the great number of isoforms of G-proteins generate a pool of complexes

implicated in a wide variety of cellular processes ranging from neurotransmission or

embryonic development [117] to respiratory control [118]. Drug discovery efforts that

initially were directed to G-coupled receptors now have been focused in G-proteins and

its intracellular partners.

In an attempt of interfere with these interactions the group of Scott JK used different

phage displayed peptide libraries (Table 1) that were screened against immobilized βγ

subunits as target [119]. Selected peptides were grouped into different families and one

of the groups identified was shown to share notable homology with peptides derived

from Phospholipase C β2 (PLC β2). These peptides in fact prevented activation of PLC

β2 by Gβγ subunits but did no block Gβγ-mediated of voltage-gated calcium channels

which remarks the pathway specificity of the molecules. Moreover all peptides selected

were predicted to bind to the same site of the Gβγ-subunits. In this sense the screening

of the library not only rendered active molecules but also gave information about a ‘hot

spot’ site in Gβγ for binding interaction [120]. This information could be of great

interest for the posterior refinement of the peptide/protein interactions as well as for the

development of new pharmaceuticals using a traditional strategy.

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Conformationally restricted libraries (CRL).

The main characteristic of these libraries is that the random sequences (the

diversity) are grafted onto a rigid natural protein domain or into stable secondary

structural motifs usually named as the scaffold of the CRL [121,122]. The sequence of

the scaffold is kept in its major part and only a few positions are combinatorialised.

Hence, the library presents dual diversity, namely of sequences and structures. An

adequate selection of the combinatorialised positions in the scaffold allows the

generation of molecules that virtually populate the conformational space between the

random and the fully folded conformation which, in turn, depends on the selected

scaffold. The aims of the design of a monomeric α-helix CRL was based on its

potential use as source for the identification of molecules that would modulate protein-

protein interactions. In this context, the α-helical based CRL was screened in two

different biological assays. In particular, we were interested in the modulation of

protein-protein interactions that mediate membrane fusion. The mechanism of

virus-cell fusion of some enveloped viruses and in particular for rhabdoviruses

revealed that, to fuse with the cellular membranes, the G protein trimeric spikes find

and bind to their target cells. However, the molecular mechanisms involved in

rhabdovirus fusion are not well understood and it might involve new proteins or

principles yet to be discovered. The screening of the library allowed the identification

of peptides that enhanced the infectivity of rhabdoviruses [8,123]. These peptides are

currently being used in studies addressed to understand the molecular mechanism that

control the fusion of the rhabdovirus to the target cell. In a different biological assay

we addressed the identification of novel modulators of the SNARE complex as

inhibitors of regulated exocytosis. Calcium-dependent exocytosis in excitable cells is

mediated by the precise docking and fusion of neurotransmitter-loaded cargo vesicles

22

[124,125]. Mechanistically, neuronal exocytosis is an orchestrated cascade of protein-

protein interactions that involve several proteins. At the centre of the process are

found the so-called SNARE proteins that assemble into a highly stable, ternary

complex known as the SNARE core complex. [124-127]. Structurally, the SNARE

complex is a highly stable parallel four helix bundle formed by coiled coil

arrangements. The high stability of the SNARE complex has hampered the discovery

of small molecules that modulate the assembly of the proteins and only clostridial

neurotoxins and peptides patterned after protein domains of SNARE proteins have

been reported to have an effect on the assembly [128-130]. However, the discovery of

amino acid sequences unrelated to the SNARE proteins capable to inhibit the assembly

of the core complex remained elusive. From the screening of the α-helix CRL we

identified up to 8 peptides that inhibited in vitro the formation of the SDS resistant

SNARE complex. The most active 17-mer peptide abrogated the Ca2+-dependent

release of L-glutamate in intact hippocampal neurons [130].

Conclusion

The network of protein interactions that defines the full interactome of proteins of

cellular regulatory mechanisms is far broader than the subject matter reviewed here.

Consequently, the identification of molecules that inhibit protein-protein interactions

reviewed here should be viewed as a starting point upon which to build the concept

that such protein-protein interactions are pharmacologically accessible by small

molecules. The actual and future molecules that will modulate interactions between

proteins, not only would have the chance to be promote to ‘more promising hits to

drugs’ but also will define valuable tools for the study of cell biology. The use of

collections of molecules or compound libraries together with the availability of

23

structural and bioinformatics information for key protein-protein complexes have

significantly open new views to the understanding of how modulators of protein-

protein interaction could be developed. In turn, these findings open important

questions and perspectives that will be the driving force for future research. Questions

ahead will look upon the ‘strength’ of the interactions between proteins and how many

transient interactions (difficult to identify in the cellular context) that are still

undiscovered, will be classified as key points of regulation in cellular pathways.

However, combinatorial chemistry in its different formats will be always a valuable

and useful tool to face research programs for the identification of modulators of

protein-protein interactions, specially when structural information of the complex is

not available but a biological activity could be evaluated.

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Tables Table 1. Libraries and screening methods used in the different protein-protein

interaction assays.

PROTEIN-PROTEIN INTERFACE

METHOD OF SCREENING

TYPE OF LIBRARY REF.

Apaf-1/apoptosome [S35] Procaspase 9 processing

Trimers of N-alkylglycines [99]

Bcl-XL/BH3 Bak Fluorescence polarization assay

16320 organic compounds [102]

HIV-protease dimerization interface Enzymatic assay Focused library of

interfacial peptides [107]

Phosphoprotein/ nucleprotein from Rabies virus

Yeast two hybrid assay Coactamer libraries [108]

Integrin αvβ3/ MMP-2 Solid phase receptor-binding assays

Mixtures of small organic compounds [111]

Paxillin/ integrin α4β1 ELISA assay with immobilized His-tagged α4

4-(dimethylamino) butyric acid-A-B-C [131]

E2F/DP Yeast two hybrid assay Thioredoxin-20mer peptides (aptamers) [116]

Gβγ Phage displayed libraries against immobilized βγ subunits

Linear Disufide-bridged-loop Half –Cys α-conotoxin

[119]

Rhabdovirus Trimeric G protein Virus infectivity α-helical CRL [123]

Snare complex

SDS-PAGE α-helical CRL [129]

34

Legend to figures

Figure 1. Representative examples of solved strutures of known protein-protein

interfaces. (A) CARD domain of Apaf-1/prodomain of caspase (3YGS) [132] (B)

Dimeric HIV protease (1AID) [133] (C) Integrin alpha2 I domain/collagen complex

(1DZI) [134]

Figure 2. Apaf 1 inhibition by identified peptoids. (A) Structure of the lead compound

obtained from the screening, peptoid 1, and a more soluble derivative peptoid 1a. (B)

Apoptosome-dependent activation of procaspase-9 followed by incubating in vitro

transcribed-translated [35S]-Met procaspase-9 and rApaf-1 in the presence cytochrome

at different concentrations of peptoid 1a. (C) Structures peptoid 1 third generation

derivatives: penetratin-GG-peptoid 1, cyclo-peptoid 1a and PGA-GG-peptoid 1.

Figure 3. Structure of active compounds obtained from the screening of different

libraries in protein-protein interaction assays.

Figure 4. Structure of the focused library designed to inhibit HIV protease dimerisation.

The library consisted of 49 single modifications of the parent compound in the four

marked different positions.

Figure 5. General structure of the library designed to interfere with the Paxillin/α4

interaction. The positions marked as X, Y and Z were substituded by ten different

aromatic amino acids.