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WEDNESDAY, APRIL 30

8:00 am Breakout Group Discussions: Meet the Experts
Brainstorming discussion groups moderated by experts in the area. Attendees are invited to choose the topic according to their main interest; however, they may switch between groups. We emphasize that this discussion is for an interactive informal exchange among scientists and is not meant to be, in any way, a corporate or product discussion.

METHODS FOR IDENTIFYING PPI BINDING SITES & LEAD COMPOUNDS

9:00 Chairperson’s Remarks
David C. Fry, Ph.D., F. Hoffmann-La Roche Inc.

9:05 High-Throughput Screening for Protein-Protein Interaction Modulators
Haian Fu, Ph.D., Professor of Pharmacology and Director of Emory Chemical Biology Discovery Center, Emory University

This presentation will highlight a major effort by an academic high-throughput screening center to target protein-protein interactions for molecular probe identification and drug discovery. Case studies will be presented to illustrate the design of robust protein-protein interaction assays for uHTS and the utility of various multiplexed screenings for effective hit identification with build-in target selectivity.

9:35 Protein-Protein Docking Simulations for Hot-Spot Identification and Drug Design
Juan Fernandez-Recio, Ph.D., Research Group Leader, Life Sciences, Barcelona Supercomputing Center
We have developed computational tools for ab initio structural prediction of protein-protein complexes (docking). More specifically, our efforts have focused on devising a scoring function, based on electrostatics and desolvation, which has been optimized for evaluation of rigid-body docking poses. This scoring function has shown one of the best structural predictive rates of protein-protein interactions in the blind test CAPRI (http://capri.ebi.ac.uk).

We have also developed tools for accurate and fast binding-site identification, based on desolvation of protein surfaces. Recently, on the basis of unrestricted rigid-body docking and using our binding energy as scoring function, we were able to identify protein-protein interaction hot spots with positive predictive value of 80%. In summary, I will describe how computational docking tools are providing structural models for the protein-protein interaction and are helping to identify key residues of the protein surface that could be potential candidates for initial drug targeting.

10:05 The Rapid Creation and Screening of Conformation-Restricted Synthetic Libraries against Protein-Protein Interaction Targets
Stephen Hale, Ph.D., Senior Director, Discovery, Ensemble Discovery
Conformation-restricted macrocyclic structures can present functionally diverse chemical groups over a relatively large and distributed surface. This class of molecules is well suited to bind to the extended binding surfaces typical of protein-protein interactions that define key therapeutically relevant pathways. DNA-programmed-chemistry is applied to build libraries that are rapidly screened for families of macrocyclic compounds that selectively block the anti-apoptotic BCL-XL:BAK protein complex formation.

10:35 Networking Coffee Break, Poster and Exhibit Viewing

11:15 Transient Pockets on Protein Surfaces Involved in Protein-Protein Interaction
Volkhard Helms, Ph.D., Chair of Computational Biology, Center for Bioinformatics, University of Saarland
Protein surfaces involved in formation of protein-protein contacts are typically quite flat and unstructured both in the unbound and in the complex forms. Nonetheless, X-ray crystallographic studies showed that small molecules are sometimes able to induce opening of suitable pockets at this interface and bind competitively. Mimicking this behavior by computational means would open up interesting possibilities for structure-based drug design. Interestingly, we could show that in molecular dynamics simulations of the solvated unbound proteins at room temperature transient pockets opened frequently nearly everywhere on the protein surface. These pockets opened and disappeared on timescales much shorter than nanoseconds, and we suggest that this is driven by the tight coupling of protein and solvent dynamics. Moreover, we could show for a number of prominent protein interaction partners that such transient pockets are suitable conformations for docking small-molecule inhibitors.

11:45 Monitoring the Antagonist-Protein and Protein-Protein Interactions with NMR Spectroscopy
Tad A. Holak, Ph.D., Department of Structural Cell Biology, Max Planck Institute for Biochemistry
We describe an NMR method that directly monitors the influence of ligands on protein-protein interactions. The method shows whether an antagonist releases proteins in their wild-type folded states or whether it induces their denaturation, partial unfolding or precipitation. We illustrate the method by studying lead compounds that have recently been reported to block the MDM2-p53 interaction. Activation of p53 in tumor cells by inhibiting its interaction with MDM2 offers a new strategy for cancer therapy. We also studied the interaction of the retinoblastoma tumor suppressor protein (pRb) with the pRb binding proteins. pRb is a fundamental negative regulator of cell proliferation that is frequently targeted in human cancer.

12:15 pm Luncheon Technology Workshop 
(Sponsorship Available) or Lunch on Your Own

1:15 Break

CASE STUDIES: SMALL-MOLECULE PPI MODULATORS

1:40 Chairperson’s Remarks
Sudhir Sahasrabudhe, Ph.D., Chief Scientific Officer, Prolexys Pharmaceuticals

1:45 Inducing Programmed Cell Death in Cancer: A New Therapeutic Approach
Kurt Deshayes, Ph.D., Senior Scientist, Department of Protein Engineering, Genentech, Inc.
Inhibitor of apoptosis (IAP) proteins are antiapoptotic regulators that block cell death in response to diverse stimuli. They are expressed at elevated levels in human malignancies and are targets for small-molecule cancer therapeutics under current clinical development. We have recently demonstrated that small-molecule IAP antagonists bind to select baculovirus IAP repeat (BIR) domains, resulting in dramatic induction of autoubiquitination activity and rapid proteasomal degradation of c-IAPs. The IAP antagonists also induce cell death that is dependent on TNF signaling and de novo protein biosynthesis.

2:15 Discovery of the Oral Thrombopoietin Receptor Agonist Eltrombopag: A Small-Molecule PPI Modulator
Juan I. Luengo, Ph.D., Director, Chemistry, Oncology CEDD, GlaxoSmithKline
Thrombopoietin (TPO) is a 332-amino acid cytokine that regulates the production of platelets through interaction with its own receptor (TPO-R). To investigate the possibility of discovering small-molecule TPO receptor agonists, a high-throughput screen was carried out, using a cell-based reporter gene assay. From the several hits obtained, one of them was selected for medicinal chemistry optimization, eventually resulting in the identification of Eltrombopag (SB-497115). Eltrombopag is a potent TPO-R agonist in cell proliferation and human bone marrow megakaryocyte differentiation studies (EC₅₀’s of 30–100 nM, with full maximal TPO efficacy). TPO agonist activity was confirmed in advanced clinical studies, by its dose-dependent elevation of platelet counts in patients suffering from immune thrombocytopenic purpura (ITP). The identification of Eltrombopag as a TPO-R agonist provides the first example of a small non-peptidyl molecule that can induce the selective activation of a cytokine receptor, and the potential mechanism for such TPO-R modulating activity will be discussed.

3:00 Networking Refreshment Break, Poster and Exhibit Viewing

3:30 Allosteric Stabilization with a Small Molecule as a PPI Inhibition Strategy: Application to Alpha1-Antitrypsin
Ruben Abagyan, Ph.D., Professor, Department of Molecular Biology, The Scripps Research Institute, La Jolla
Two strategies for inhibiting protein-protein interactions are described. The orthosteric strategy uses “druggable” pockets directly at the protein-protein interface. The allosteric strategy aims at stabilizing the unproductive conformation of one of the interacting proteins. The allosteric site does not need to reside at the interface and, therefore, has a better chance of having favorable pocket characteristics. In collaboration with the David Lomas laboratory at the Cambridge University, we used the allosteric-stabilization strategy to identify the first small-molecule inhibitors of the pathological polymerization of the Z mutant of alpha1-antitrypsin, which provides a novel therapy for the treatment of liver disease. Atypical kinase modulators can follow a similar approach. We also review the induced fit at the protein interfaces and new methods to generate and identify the small-molecule druggability of a putative target pocket for both strategies.

4:00 Structure-Based Investigation of Wnt Signaling Inhibitors
Jie Zheng, Ph.D., Associate Member, Department of Structural Biology, St. Jude Children’s Research Hospital
Through structure-based virtual ligand screening and NMR spectroscopy, several drug-like compounds have been identified and those compounds have been shown to be able to disrupt the protein-protein interaction events at different points of the Wnt signaling pathway. Wnt signaling plays a critical role in embryonic development and in the regulation of cell growth. Inappropriate activation of Wnt signaling has been implicated in cancers and other human diseases. Therefore, the small-molecule inhibitors we obtained will be helpful in formulating rational approaches to the development of novel pharmaceutical agents that can interfere with specific Wnt signal events that contribute to human diseases. In the presentation, we will focus on two targets in the Wnt pathways: the PDZ domain of Dishevelled (Dvl) and LRP5/6.

4:30 In Vitro Characterization of Potent Bcl-2 Selective Antagonists
David Mann, Ph.D., Lead Senior Scientist, Chemistry Department, Infinity Pharmaceuticals, Inc.
At Infinity we have used Diversity Oriented Synthesis (DOS) to generate a complex series of library compounds. From these libraries we were able to identify multiple novel Bcl2 ligands having sub-micromolar affinities and, using the initial SAR obtained, evolved the potency in the lead series into the picomolar affinity range. In parallel we have developed a series of Biological assays to thoroughly explore the mechanism of action of these selective Bcl2 antagonists. The assays developed include a suite of microplate assays performed on mitochondria isolated from Bcl2-dependent cell lines to assess in situ Bcl2 affinity, Bax conformation change and cross-linking, and ultimately Cytochrome C release. Additionally, in line apoptosis marker time course assays (Caspase activation, Annexin V staining, and subsequent Propidium Iodide uptake) performed in cellulo have been demonstrated to differentiate between compounds acting though Bcl2 as a target versus cytotoxic agents that act via other mechanisms to induce cell death.

5:00 Close of Conference