1:35 Protein-Protein Interactions as Small Molecule Drug Targets: What Has Been Learned?
David C. Fry, Ph.D., Senior Research Leader, Roche Research Center, F. Hoffmann-La Roche, Inc.
Protein-protein interactions regulate a wide variety of important cellular pathways, and therefore represent a highly populated class of targets for drug discovery. However, such systems have proven to be very challenging. This overview presents an analysis of individual protein-protein interaction systems which have recently yielded success in the discovery of drug-like inhibitors. The structural characteristics of the protein binding sites and the attributes of the small molecule ligands are focused upon, in an attempt to derive commonly shared principles that may be of general usefulness in future drug discovery efforts within this difficult target class.

2:05 Co-receptor Blockade as an Anti-HIV Strategy- The CXCR4 Example
Rebecca S.Y. Wong, Ph.D, Senior Scientist/Medical Writer, AnorMED Corp of Genzyme Corporation, Langley, BC, Canada
The development of drug resistance and toxicities in the current anti-HIV drug classes has prompted the investigation into novel therapeutic agents with a unique target of action. Coreceptor blockade, via either the CCR5 or CXCR4 HIV co-receptor, has been validated as one of the potential novel therapeutic strategies for HIV. AMD11070 is a selective and reversible inhibitor of CXCR4 discovered at AnorMED (now part of Genzyme Corp). AMD11070 is a first-in-class, orally bioavailable CXCR4 inhibitor that entered clinical development in the treatment of HIV infection. This presentation will describe the screening platform used in our CXCR4 antagonist research program. It will also include our research efforts to understand the interactions between CXCR4 and its small molecule inhibitors. Results from a phase Ib/IIa clinical study, which indicated the effectiveness of AMD11070 in reducing the X4-using viral load in HIV-infected patients, will also be presented.

2:35 Interactome Project: Overview, Current Status, and Future Direction
David E. Hill, Ph.D., Associate Director and Senior Research Scientist, Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute
It is becoming increasingly clear that genes and their products interact in complex biological networks with local and global properties and perturbations of these networks contribute to the disease state. We propose that a further understanding of the mechanisms involved in many different diseases, and the development of new therapeutic strategies, can be gained by i) studying genes and their products in the context of the molecular networks in which they function, and ii) investigating how such networks are altered in disease cells compared to their unaffected counterparts. To generate the information necessary to eventually address how complex cellular networks relate to biology, we initiated, at the scale of the whole proteome, an integrated approach for modeling protein-protein interaction or "interactome" networks. In addition, we are developing tools and strategies for determining the dynamics of interactions involving proteins having multiple interacting partners as well as identifying interaction surfaces for each partner. By generating libraries of missense mutations in a candidate disease gene, we are able to identify single amino acid changes that target one interaction while not altering all other interactions as well as pleiotropic mutations that alter multiple interactions of a target protein. This knowledge of "interaction surfaces" and the amino acids required for specific interactions should help drive the development of techniques for disrupting protein-protein interactions.

3:05 Networking Refreshment Break, Poster and Exhibit Viewing

4:15 Inhibitors of Apoptosis Proteins: Application of Structure-Based Drug Design to the Taming of a Protein-Protein Interaction Target
Christopher S. Straub, Ph.D., Senior Scientist, Oncology Chemistry, Novartis Institutes for BioMedical Research, Inc.
A lead is ideally a small, low molecular weight compound that can be built upon to traverse the hurdles towards a clinical candidate. In the pursuit of inhibitors to protein-protein interaction targets, it is often the case that leads are difficult to find. Our efforts to seek antagonists of the inhibitors of apoptosis proteins (IAP’s), a target with high potential in cancer treatment, began with no identified small molecule leads. As such, we commenced by minimizing the native ligand for this protein, Smac, and then utilized structure-based drug design to optimize towards a drug candidate. The result: several peptidomimetic scaffolds that exhibit good in vitro activity in multiple tumor cell lines. Consistent with the hypothesized mechanism of action, these compounds also demonstrate strong synergy when combined with cytotoxics against a broader panel of cell lines. This activity has translated well to in vivo models of human cancer. This presentation will detail the design efforts in this program and will highlight the evolution of a drug like small molecule from a natural ligand in this protein-protein interaction target.

4:45 Privileged Scaffolds Targeting Reverse-Turn and Helix Recognition Garland R. Marshall, Ph.D., Professor, Biochemistry & Molecular Biophysics, Washington University School of Medicine Preorganization of an inhibitor into its protein-bound conformation reduces the entropy of binding and enhances the inhibitor's affinity. Novel privileged scaffolds with limited flexibility have been designed to bind to reverse-turn and helical recognition sites. An example targeting two-component regulatory systems in Yersinia pestis will be discussed in detail.

5:15 End of Day

 

Wednesday, May 16 7:30 am Interactive Round Table Discussions Topic: Structure-Based Drugability Assessment of Protein-Protein Interfaces Moderator: S. Don Emerson, Ph.D., Research Fellow, Protein NMR and Biophysics Group, Pfizer Inc. Software for analysis and scoring of potential drug binding sites Discussion of property ranges for surface area, concavity, polarity, hydrophobicity, H-bond acceptors/donors, etc. Topic: "Specificity" in Protein: Protein Interactions Beatrice D. Darimont, Ph.D., Assistant Professor of Chemistry, University of Oregon Specificity and Affinity Relations Exploitation of Structural Details Binding Kinetics and Dynamics of Conformational Changes 8:55 Chairperson's Remarks Richard Eglen, Ph.D., Vice President & General Manager, Life & Analytical Sciences, PerkinElmer Inc.     9:00 Computational Design and Analysis of Promiscuous and Highly Specific Protein Interfaces Tanja Kortemme, Ph.D., Assistant Professor, Biopharmaceutical Sciences, California Institute for Quantitative Biomedical Research, University of California-San Francisco Our lab develops and applies a protein modeling program, Rosetta, to predict and design protein-protein interactions. We have previously described a Rosetta protocol to identify binding energy hot spots in protein interfaces from structure ("computational alanine scanning"). I will present recent advances in the development and application of computational prediction and design methods in 2 areas: (1) We have used RosettaInterface to successfully redesign several protein-protein interfaces to create new protein pairs. (2) We have developed a new "multi-constraint" computational design method that allows us to simultaneously optimize a protein interface sequence for multiple functional and structural requirements. By applying this technology to analyze multi-specific protein binding sites, we identify two classes of molecular mechanisms for promiscuity, which use either shared or distributed "multi-faceted" binding hot spots. These findings suggests routes to target each type of interface. In particular, multi-constraint computational methods may allow the redesign of multi-faceted promiscuous signaling interfaces to yield new predetermined interaction patterns. Results from designer proteins as control elements of cellular signaling pathways involving Rho-type GTPases will be presented. 9:45 Targeting Protein-Protein Interactions in the Beta-Catenin and HSP20 Pathways Sudhir R. Sahasrabudhe, Ph.D., Chief Scientific Officer, Prolexys Pharmaceuticals Inc. With a focus on discovering small molecule agents that inhibit protein interactions in pathways relevant to disease biology, we have identified novel and druggable therapeutic targets for cancer and asthma. Using yeast-based and biochemical high-throughput screens, we have identified small molecules which have been tested in cell and animal models of disease. This presentation will highlight the current results of lead programs: a) small molecules that inhibit the protein interactions in the beta-catenin pathway, and b) protein interaction modulators in the HSP20 pathway. 10:15 Networking Coffee Break, Poster and Exhibit Viewing 10:45 CDK2/CYCLIN A Recruitment Site Inhibitors: A Peptide to Small Molecule Approach Daniel Sutherlin, Ph.D., Scientist, Medicinal Chemistry, Genentech Inc. The CDK2/CyclinA complex plays and important role in the cell cycle and is also an important cancer target. Where most small molecule drug discovery programs have focused on the active site of the Cyclin Dependent Kinase, we have pursued inhibitors that bind to a substrate recognition groove on the surface of CyclinA 50 angstroms away from the kinase active site. Peptides that mimic endogenous substrates and inhibitors of the kinase complex have been shown to bind to this site in the 10 to 100 nM range. Furthermore, the inhibition of cyclinA with cell penetrating peptides has been shown to selectively kill cancer cells in vitro and in vivo. In order to validate this site as a small molecule target, nanomolar octa-peptide leads were examined through amino acid substitution and truncation of the sequence in order to identify a smaller, albeit significantly less potent, tetrapeptide lead. A 1000 fold loss in affinity was recovered by finding new contacts with the protein surface and by rigidification of the peptide backbone using a combination of solid-phase parallel synthesis and structure based design. Finally, two guanidine functionalities were eliminated from the inhibitors to result in neutral small molecules that are equal to the activity of the starting peptides and represent a significant advance in the discovery of small molecule drugs targeted specifically to cyclins. 11:15 Cellular Consequences of Inhibiting Bcl2 / BH3 Protein-Protein Interactions: Tools to Evaluate Candidate Bcl-2 Antagonists Christian Fritz, Ph.D., Senior Director, Cancer Biology, Infinity Pharmaceuticals, Inc. The interaction between Bcl-2 and its pro-apoptotic protein family members is an attractive target for intervention in cancer. Bcl-2 is over-expressed in several cancers and is thought to play a key role in tumor survival and resistance to chemotherapy. Programs in both academia and industry have produced a host of Bcl-2 binding compounds, some of which are also cytotoxic to cancer cells. To follow up on these molecules, a key question is whether this cytotoxicity is caused by Bcl-2 antagonism or simply by an off-target effect of those molecules. To answer some of these questions, we have started to define the consequences of inhibiting Bcl-2 in cells that depend on Bcl-2 for their survival. These studies have uncovered a unique cell death profile for Bcl-2 antagonists that is different from that induced by standard chemotherapeutics. This profile now allows us to test putative Bcl-2 binders for on-target cellular activity. Surprisingly, very few of the published molecules seem to elicit cell death by neutralizing Bcl-2 function. 11:45 Technology Watch Yeast as a Screening platform to identify, characterize and modulate therapeutic protein-protein interactions Daniel Auerbach, Ph.D., Chief Scientific Officer, Dualsystems Biotech AG   Sponsored By Interactions between proteins play pivotal roles in most cellular processes and aberrant protein interactions are the underlying cause for many diseases. Many protein classes, in particular integral membrane proteins are difficult to analyze. Screening of cDNA libraries identifies novel interactors of a protein of interest. Dualsystems has developed several yeast based screening assays which cover all major protein classes. Using these systems, cDNA libraries can be exhaustively screened in a high-throughput setting and novel protein interactions of therapeutic interest can be identified and characterized. 12:00 Informal Q&A 12:15 pm Luncheon Technology Workshop Sponsored by Novel Cell-Based Technologies to Accelerate High-Throughput Screening at GPCRs Richard Eglen, Ph.D., Vice President & General Manager, Life & Analytical Sciences, PerkinElmer,Inc.  1:15 Session Break 1:40 Chairperson’s Remarks 1:45 Fragment-Based Approaches to Disrupt Protein-Protein Interactions by NMR Chaohong Sun, Ph.D., Associate Research Investigator, Protein NMR Research, Abbott Laboratories  Protein-protein interactions play critical roles in a variety of biological processes and represent novel targets for therapeutic intervention. However, designing small molecule inhibitors to disrupt protein-protein interactions has been challenging due to the unique features of the binding site (relatively large and less well defined) and the difficulty in identifying drug-like chemical leads. An NMR-driven fragment-based approach has been proven to be successful for tackling this class of protein targets. Here, examples of using NMR to target protein-protein interactions for survivin and c-Jun N-terminal kinase (JNK) are presented. 2:15 Foldamer-Based Strategies For Inhibiting Protein-Protein Interactions Samuel H. Gellman, Ph.D., Professor, Chemistry, University of Wisconsin - Madison Foldamers are unnatural oligomers with well-defined conformational propensities. Results reported by several groups over the past decade have established folding rules for oligomers of beta-amino acids ("beta-peptides") and, more recently, oligomers containing both alpha- and beta-amino acid residues ("alpha/beta-peptides"). Such oligomers adopt predictable secondary structures, and they can be designed to display very high conformational stability. In addition, these foldamers are moderately to very highly resistant to proteolytic degradation. Efforts to disrupt protein-protein interactions involved in human cytomegalovirus infection and in aberrant apoptotic signaling (involving Bcl-family proteins) will be discussed.  2:45 Stapled Peptides: A New Class of Targeted Therapeutics that Modulate Intracellular Protein-Protein Interactions Huw M. Nash, Ph.D., Vice President , Corporate Development, AILERON Therapeutics, Inc. Many of the key protein-protein interactions that participate in disease-associated signaling pathways utilize alpha-helical interfaces. For these targets, the “naturally optimized” modulator of the protein-protein interaction is an isolated alpha-helical peptide derived from one of the proteins in the complex. Stabilization of an alpha-helical structure by installing an all-hydrocarbon bridge along the non-binding face provides resistance to proteolysis, improved affinity and most interestingly cell penetrability for alpha helical peptides; this technology has been termed “stapled peptides”. A hydrocarbon “stapled” version of the BH3-domain alpha-helix of BID, a pro-apoptotic BCL-2 family member, has recently been shown to possess potent in vitro and in vivo anti-cancer activities for leukemias. Further studies have shown that the stapled BID-BH3 peptide functionally mimics the parent BID protein by directly activating BAX/BAK, a property that differentiates it from all other small molecule antagonists of Bcl-2 and Bcl-XL. This hydrocarbon stapling strategy has been extended to generate potent and specific leads from the BH3 domains of other Bcl-2 family proteins and from other unrelated alpha-helical peptides, and in vivo pharmacological profiling of the BID-BH3 peptide has shown it to possess good drug-like properties. 3:15 Networking Refreshment Break, Poster and Exhibit Viewing 3:45 REPLACE: A Strategy for Iterative Design of Cyclin Dependant Kinase Inhibitors Campbell McInnes, Ph.D., Assistant Professor, Pharmaceutical Sciences, University of South Carolina A fragment-based design strategy termed REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) is described in which nonpeptidic surrogate fragments for specific determinants of known peptide ligands are identified in silico using a core peptide-bound protein structure as a design anchor. In the REPLACE application example the effective replacement of two critical binding motifs in a lead protein-protein interaction inhibitor pentapeptide with more drug-like phenyltriazole and diphenyl ether groups is presented. Peptide small molecule hybrid crystal structures confirmed the expected mode of binding.  4:15 Finding Specificity in Unlikely Places: Recognition and Binding of Client Proteins by the Heat-Shock Protein 90 Beatrice D. Darimont, Ph.D., Assistant Professor of Chemistry, University of Oregon Hsp90 is an unusual chaperone that assists the folding and function of a restricted yet diverse set of structurally distinct regulatory proteins involved in cell cycle regulation and other vital biological processes. It is essential for the viability of eukaryotic cells and has become a prime anticancer target. Little is known about how Hsp90 recognizes, interacts and regulates the activity of its "client proteins". Thus far most Hsp90 inhibitors target the ability of Hsp90 to bind and hydrolyze ATP, which is required for most Hsp90 activities. By studying the interaction of Hsp90 with the glucocorticoid receptor (GR), we identified that Hsp90 binds client proteins with the help of specific docking sites that are able to distinguish particular client protein conformations. Our results open new possibilities for the manipulation of specific signaling pathways via Hsp90 and provide evidence for an unexpected role of Hsp90 in diversifying the cellular response to steroid hormones. 4:45 ROSETTA, a Swiss Army Knife of Molecular Modeling: Cutting Edge Tools for Analyzing and Designing Interactions of Molecules Small and Large Xavier I. Ambroggio, Ph.D., Post-Doctoral Fellow, Department of Biochemistry & Biophysics, University of North Carolina; Managing Partner, Rosetta Design Group LLC An accurate model is a prerequisite for the rational design of drugs targeting macromolecular systems. The complexity inherent in modeling protein-protein interfaces requires improved methods for modeling small molecules. In this presentation, we will review the RosettaInterface, RosettaLigand, RosettaDesign, and the RosettaDock modes of the ROSETTA modeling package and show how they integrate to address difficult problems in the modeling of protein-protein and small molecule-protein interactions. In addition, the general framework of ROSETTA and how ROSETTA can be extended or modified to model specific problems of interest will be discussed. 5:15 Close of Protein-Protein Interactions as Drug Targets Conference

 

Scientific Advisory Board

• Don Emerson, Ph.D., Research Fellow, Protein NMR and Biophysics Group, Pfizer Inc.
• David C. Fry, Ph.D., Senior Research Leader, Roche Research Center, F. Hoffmann-La Roche, Inc.
• Marina Nelen, Ph.D., Senior Scientist, Lead Generation Biology, Johnson & Johnson PRD
• John Van Drie, Ph.D., Director, Computer-Aided Drug Discovery, Novartis Institutes for BioMedical Research