9:15 Identification of Inhibitors of Protein-Protein Interactions by Fragment Screening
William Metzler, Ph.D., Associate Director, Macromolecular NMR, Bristol-Myers Squibb Pharmaceutical Research Institute
Protein-protein interactions are often given low priority when potential new targets for therapeutic intervention are considered. This reluctance is primarily due to the difficulties usually encountered in identifying tractable small-molecule leads against these targets. By leveraging capabilities that we have utilized for fragment screening of enzyme targets, compounds have been identified that are capable of inhibiting several diverse types of protein-protein interactions, including those involved in cell adhesion, P53 modulation, nuclear localization and T-cell recognition. This presentation will describe our efforts in two programs targeting protein-protein interactions. The first involves inhibiting the active transport of proteins into the nucleus, which is facilitated by the interaction of the nuclear localization sequence of the imported protein with karyopherin. A hypothesis-driven fragment screening approach was used to identify sub-micromolar inhibitors of the binding of karyopherin to the transcription factor NF-kB. In the second program, we initiated studies to identify small molecules that could block the engagement of the T-cell receptor CD28 with its B-cell counter-receptors CD80 and CD86, thereby preventing T-cell proliferation and inducing antigen-specific unresponsiveness. A fragment screen of CD28 identified numerous small molecules that bound CD28 with mM affinity. To improve affinity, we built a homology model of CD28 and performed a virtual screen biased by the fragment hits. After subsequent deck mining, several small molecules have been identified that have improved, albeit still limited, ability to block CD28 binding to CD80 and CD86.

9:45 Targeting Protein-Protein Interactions With Small Molecules
Ruben Abagyan, Ph.D., Professor, Department of Molecular Biology, The Scripps Research Institute

Targeting protein interactions with small molecules is a challenging task.

• What interface to target? Can it be targeted with a small molecule?

• What are the possible conformational changes upon protein-protein or protein-small molecule interaction?

• Can we implement a robust docking and virtual ligand screening procedure for interfaces with a potential for induced conformational changes?

Presented will be the results of a collaboration with the team of Professor David Lomas from Cambridge University, UK to identify the first series of lead molecules for the treatment of conditions associated with Z alpha-1-antitrypsin deficiency. The compounds identified block polymerization for Z alpha1 antitrypsin and reduce intracellular accumulation of Z alpha1 antitrypsin by 70% in cell models of disease.

11:15 FAST-NMR: Functional Annotation Screening Technology 
Kelly A. Mercier, Researcher, The Powers Research Group, Chemistry, University
of Nebraska Lincoln
More than 50% of proteins identified from structural genomics projects have no known function due to a lack of sequence and structural homology to proteins of known function. Our Functional Annotation Screening Technology using Nuclear Magnetic Resonance (FAST-NMR) was developed to aid in the functional annotation of hypothetical or novel proteins. FAST-NMR utilizes NMR-based small molecule screening combined with robotics, molecular modeling, structural biology, and bioinformatics that includes our Comparison of Protein Active-Site Structures (CPASS) database and software. FAST-NMR maps the structural details of the protein's active site and the identity of ligands with known biological activity that bind the protein. A putative biological function can be attributed to the novel protein by comparison of this information against proteins with an identified function using CPASS. Furthermore, the FAST-NMR results provide a starting point for a structure-based drug design program, where CPASS may aid in inhibitor selectivity. FAST-NMR would precede a drug discovery effort by identifying novel drug targets and the results of FAST-NMR provide a starting point for fragment-based drug design. Also, FAST-NMR uses similarly technology that could be similarly applied to a fragment-based drug design approach. Our differential approach to NMR-based metabolomics is complimentary to FAST-NMR by providing in vivo information on drug efficacy and selectivity. This presentation will focus on the various components of FAST-NMR and their application in determining the function of SAV1430, a hypothetical protein from Staphylococcus aureus.

1:30 Design of Beta-Secretase (BACE-1) Inhibitors Through In Silico Property-Based Fragment Scanning
Bradley P. Feuston, Ph.D., Senior Investigator, Molecular Systems, Merck Research Laboratories
Beta-Secretase is a transmembrane aspartyl protease intimately involved in the neurodegenerative disorder, Alzheimer’s disease. With a significant amount of in-house structural knowledge of BACE-1, chemotype-specific scoring functions for rank-ordering virtual compounds has proven useful for explaining SAR. Combining these scoring functions with Merck’s unique virtual compound library tools provided an opportunity for focused library designs to directly impact lead finding/optimization in a timely manner. To facilitate the design and optimization of virtual BACE-1 compound libraries, Merck’s Virtual Library ToolKit (VLTK) has been enhanced to include 3D library construction. The modular tool kit, VLTK, comprises several components which allow users to select reagents, analyze synthons, enumerate libraries, calculate properties, optimize libraries and finally track the synthesized compounds through biological assays. The virtual 3D library scanning component which preserves initial scaffold positions also includes conformational sampling of part or the entire molecule for subsequent docking and/or scoring evaluations. This presentation will concentrate on the various aspects of focused library designs and specifically, the application to BACE-1 inhibitors.

2:00 Substrate Activity Screening (SAS): A Fragment-Based Method for the Identification of Nonpeptidic Lead Scaffolds and Optimization to Potent Protease Inhibitors 
Andrew W. Patterson, Researcher, Chemistry, University of California, Berkeley
SAS is a fragment-based method for the rapid development of novel nonpeptidic enzyme inhibitors. The method consists of three steps: (1) a diverse library of low molecular weight substrates is screened against the enzyme target to identify lead fragments, (2) the identified fragments are rapidly optimized by subsequent rounds of analogue synthesis and evaluation, and (3) the optimized substrates are converted to inhibitors by direct incorporation of mechanism-based inhibitor pharmacophores. Because the assay requires productive enzyme/substrate binding and turnover, false positives due to aggregation or micelle formation that can be seen in high-throughput inhibitor screens are eliminated. Additionally, catalytic substrate turnover results in signal amplification allowing for the identification of very weakly active lead fragments. Initial studies have focused on the cysteine protease cathepsin S, producing two distinct classes of novel, potent and selective nonpeptidic inhibitors.

2:30 To Be Announced

3:00 Application of Fragment Screening, Computational Modeling and Structure-Based Design to Identify and Progress Millimolar Affinity Fragments to Nanomolar b-Secretase Inhibitors
James R. Arnold, Ph.D., Senior Scientist, Computational Chemistry and Informatics, AstraZeneca Pharmaceuticals
Alzheimer's Disease (AD) is characterized by the progressive formation in the brain of amyloid plaques and vascular deposits composed of the b-amyloid peptide (Ab). Ab is generated in vivo through the proteolytic cleavage of the membrane-anchored b-amyloid precursor protein (APP) by b and g-secretases. Inhibition of b-secretase (BACE) has emerged as a therapeutic target for the treatment and prevention of AD.

Molecular scaffolds targeting BACE were identified using multiple fragment screening techniques and large-scale crystallography. Structure guided evolution of millimolar affinity fragments afforded compounds with single digit micromolar affinity and good ligand efficiency. We describe how the team than combined techniques from Medicinal Chemistry, Computational Chemistry and Structural Biology to produce nanomolar, non-peptidic, BACE inhibitors.