Editorial

A Donny StrosbergChief Executive Officer Hybrigenics, 3–5 Impasse Reille, 75014 Paris, France Tel.: +33 15 810 3810 Fax: +33 15 810 3840 adstrosberg@hybrigenics.fr

© Future Drugs Ltd. All rights reserved. ISSN 1478-9450 141

Breaking the spell: drug discovery based on modulating protein–protein interactions‘...protein chemistry is providing the basic and appliedknowledge for future advances in drug discovery.’Expert Rev. Proteomics 1(2), 141–143 (2004)

Target-based drug discovery has generallybeen accepted as the preferred method to dis-cover new chemical entities and develop corre-sponding therapeutic compounds. Mem-brane-bound receptors, intracellular kinasesand proteases are widely explored as exqui-sitely specific tools for screening and selectionof small molecule drugs. Just a few years ago,these so-called druggable tools represented nomore than 500 distinct targets. The availabil-ity of the sequences of the circa 30,000 genescontained in the humangenome has considerablyincreased the number ofpredicted available pro-teins to be explored byexpression, purificationand systematic screen-ing. The G-protein-cou-pled membrane recep-tors and the kinase family of proteins eachrepresent over 500 distinct members, and pro-teases of all types are likely to be considerablymore abundant.

Nevertheless, many of the proteins encodedby the human genome have no obvious bind-ing or enzymatic activity, and even when theyare predicted to have a catalytic site on thebasis of their structure, substrates oftenremain unknown.

Thus, to further expand the realm of targetsavailable for drug development, scientists havelong considered the possibility of identifyingsmall compounds that disturb or stabilize pro-tein–protein interactions. However, for this tobe possible, one must first ensure reliability ofthe available data.

A number of recent studies suggest that pro-tein–protein interactions can finally be analyzedon a larger and more reliable scale than wasbelieved to be possible only a few years ago.Even though comparison between comprehen-sive reports indicate that not all problems ofreproducibility have yet been solved [1], itappears that results obtained in high-through-put analyses performed using well-standardized,quality-controlled procedures yield data thatcan indeed be validated with various alternate

methods (FIGURE 1) [2,3].Most promising are the

technologies that notonly identify pairs ofinteracting proteins, butalso allow the precisedelineation of the actualprotein domains that areinvolved in the interac-

tions [4]. The ability to precisely evaluate proce-dures for modulating interactions that havebeen shown to play a role in the physiology ofcells has emerged with the availability ofnumerous pairs of protein partners.

Modulation of protein–protein interactionshad been predicted to require bulky, long pep-tides or synthetic molecules, supposedly sinceinteractions between proteins would involvemultiple contact of amino acid residues, overextended, mostly flat areas of the molecules.Failure to actually identify such modulatorsappeared to confirm the difficulty of developingsuch compounds.

However, binding sites of antibodies for avariety of protein antigens have been well char-acterized by x-ray crystallography and nuclear

‘Scientists have long considered the possibility of

identifying small compounds that disturb or

stabilize protein–protein interactions for drug

development.’

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Strosberg

142 Expert Rev. Proteomics 1(2), (2004)

magnetic resonance studies, and have generally not supportedthe view that protein–protein interactions actually involve largesurfaces. In fact, inhibition of antigen–antibody interactions bypeptides bearing single epitopes has been the mainstay ofimmunochemists for many years. Other examples exist wheresmall molecule inhibitors can indeed prevent binding of largeproteins to their interacting partner. For example, cell-boundintegrins can be blocked from their binding partner on othercells by tripeptides, for example, LFA-1 binding to ICAM-1 [5].

However, it has not generally been easyto discover such small modulators of pro-tein interactions. Now, new methods allowone to find, in a systematic manner, proof-of-principle peptides that achieve such amodulation, for almost any pair of inter-acting proteins. Colland and coworkersdemonstrated that an oligonucleotideencoding a peptide derived from a flagellaprotein, when transfected into Helicobacterpylori, could prevent synthesis of such anorganelle, and resulted in bacteria thatwere unable to move around [6].

In fact, the protein interaction map-ping method used by these authors actu-ally generates the precise domains ofinteractions that can be used as peptidicmodulators of the protein–protein inter-actions (FIGURE 2) [4]. Early examples ofsmall molecule modulation of protein–protein interaction acting on, for exam-ple, a calcium channel [7] or on the BH3domain interacting with Bcl-xL proteins[8] have been reported sporadically (for

recent reviews see [9,10]). However, severalrecent reports suggest that the spellfinally appears to be broken. Potent smallmolecule antagonists of the MDM2 pro-tein prevent the ability of this ligase E3ubiquitin to bind to the tumor suppres-sor protein p53 and to promote its degra-dation [11]. Such molecules, by activatingthe p53 pathway in cancer cells, wereshown to promote cell cycle arrest, apop-tosis and growth inhibition of humantumor xenografts in nude mice.

Similarly, other small molecules wereshown to block binding between T-cellfactor (Tcf) and β-catenin, a key proteinoften mutated in colon cancer. By disrupt-ing the formation of Tcf/β-catenin com-plexes, these compounds potently antago-nized cellular effects of β-catenin-mediatedactivities, including cell proliferation [12].

These examples confirm that given theright collection of compounds, and the

appropriate pair of interacting proteins, one will indeed be ableto optimize small modulators that will alter phenotypic proper-ties of the living cell and, ultimately, result in development ofpotent therapeutic compounds.

Such modulators have long been predicted to constitute atreasure trove of novel drugs. Indeed, they considerably extendthe range of targets beyond the usual enzymes or receptors nowwell explored by many groups. Basically, any protein interactsat one time or another in its life cycle with other proteins, and

At least fivefold coverage of the library: at least 50 million interactions tested

Library of ten millionBait X

All positive clones sequenced and clustered:identification of the proteins interacting with the bait

Novel proteins of interest

Selected prey = new bait

quantitative scoring and classification of interacting preys

Figure 1. High-throughtput PIM yeast two-hybrid technology. In the Rain and coworkers format of the yeast two-hybrid technology, extensive and complex libraries of complementary DNA or genome DNA fragments are repeatedly screened against a single bait protein or domain of interest. A huge database of interactions (PIM Builder) has been constituted and regroups data for over 20,000 individual human proteins. Selected subsets (PIM Riders) are generated for specific pathway analysis: several have been made available [101].PIM: Protein interaction mapping.

SID®

Prey fragment library

Mating and selection Random drawing

Observed fragment pattern Theoretical background

Bait

Local PBS® E-value

SID®

Figure 2. Evaluation of protein interactions. Specific bioinformatics tools integrated in PIM technology described by Rain and coworkers [4] provides confidence scores (PBS) that are calculated for each interaction: the PBS corresponds to the probability for an interaction to be nonspecific and allows a prioritization of putative target proteins. Moreover, the extensive screening of complex libraries allows the identification of actual domains of interactions between two individual proteins. These SIDs constitute useful tools for experimental biology: SIDs can be used as dominant-negative mutants [6], as positive controls for high-throughput screening of small-molecule libraries or as proof-of-principle concept ligand for rational drug design.PIM: Protein interaction mapping; SID: Selected interacting domain.

Target-based drug discovery

www.future-drugs.com 143

can therefore become, through this interaction, a target forintervention. It is likely that novel sensitive procedures to ana-lyze the modulation of these interactions, combined with novelsources of natural or synthetic small compounds, will rapidly

provide drug developers with increasing numbers of new chem-ical entities, so rarely discovered in recent years. Now morethan ever, protein chemistry is providing the basic and appliedknowledge for future advances in drug discovery.

References

1 Titz B, Schlesner M, Uetz P. What do we learn from high-throughput protein interaction data? Expert Rev. Proteomics 1(1), 111–121 (2004).

2 Tewari M, Hu PJ, Ahn JS et al. Systematic interactome mapping and genetic perturbation analysis of a C. elegans TGF-β signaling network. Mol. Cell. 13(4), 469–482 (2004).

3 Colland F, Jacq X, Trouplin V et al. Functional proteomic mapping of a human signaling pathway. Genome Res. 14, 1324–1332 (2004).

4 Rain JC, Selig L, De Reuse H et al. The protein–protein interaction map of Helicobacter pylori. Nature 409, 211–215 (2001).

5 Liu G. Small molecule antagonists of the LFA-1/ICAM-1 interaction as potential therapeutic agents. Expert Opin. Ther. Pat. 11, 383–393 (2001).

6 Colland F, Rain JC, Gounon P et al. Identification of the Helicobacter pylori anti-σ28 factor. Mol. Microbiol. 41, 477–487 (2001).

7 Young K, Lin S, Sun L et al. Identification of a calcium channel modulator using a high-throughput yeast two-hybrid screen. Nature Biotechnol. 16, 946–950 (1998).

8 Degterev A, Lugovskoy A, Cardone M et al. Identification of small-molecule inhibitors of interaction between the BH3 domain and Bcl-xL. Nature Cell. Biol. 3(2), 173–182 (2001).

9 Gadek TR, Nicholas JB. Small molecule antagonists of proteins. Biochem. Pharmcol. 65, 1–8 (2003).

10 Arkin MR, Wells JA. Small-molecule inhibitors of protein–protein interactions: progressing towards the dream. Nature Rev. Drug Discov. 3, 301–317 (2004).

11 Vassilev LT, Vu BT, Graves B et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Sciencexpress January 2, 1–10 (2004).

12 Lepourcelet M, Chen YNP, France DS et al. Small-molecule antagonists of the oncogenic Tcf/β-catenin protein complex. Cancer Cell 5, 91–102 (2004).

Website

101 Hybrigenics – PIMRider®

http://pim.hybrigenics.com/pimriderext/common(Viewed July 2004)

Affiliation• A Donny Strosberg, PhD

Chief Executive Officer, Hybrigenics, 3–5 Impasse Reille 75014 Paris, FranceTel.: +33 15 810 3810Fax: +33 15 810 3840adstrosberg@hybrigenics.fr