Chemical Biology Discussion Group

Abstracts

Keynote Presentation

Expanding the Druggable Proteome: Finding Small Molecule Inhibitors of Protein-Protein Interactions

Adrian Whitty, Boston University

Developing small molecule inhibitors of protein-protein interaction (PPI) interfaces remains among the most difficult challenges facing contemporary drug discovery. In this talk I will discuss some of the factors that determine the "druggability" of PPI targets, what we have learned to date about the strengths and weaknesses of fragment-based approaches for addressing such targets, and what to look for in target sites and in fragment hits to determine which are most likely to be advanceable to pharmaceutically-relevant lead compounds. The talk will be illustrated using unpublished data obtained during a multi-target collaboration between Biogen Idec and Sunesis Pharmaceuticals, co-led by the author, in which Sunesis's proprietary Tethering® technology was used to search for leads against TNFa and other highly challenging protein-protein interaction targets.

Short Presentations

An Asymmetric Synthesis of a Multiscaffold Library for Discovery Screening: A Tethered Cycloaddition and Cycloisomerization Approach

Renato Bauer
Memorial Sloan-Kettering Cancer Center (Derek Tan Laboratory)

Diversity-oriented synthesis (DOS) is a major research area through which the potentials of organic synthesis are currently being tapped to impact biology and medicine. In practice, collections of compounds derived from DOS are screened in a high-throughput manner to find novel small molecules that interact with target proteins in biochemical assays or that modulate cellular pathways in phenotypic assays. Here, we present a DOS strategy that exploits optically active t-butylsulfinamides as lynchpins for the transition metal-mediated cyclizations of enynes or diynes. The required enynes and diynes were synthesized enantioselectively in three steps and, upon treatment with transition metal-based reagents, produced functionalized mono- and bicycles as end products. The present work addresses reactivity patterns of important cycloaddition and cycloisomerization reactions in terms of yield, regioselectivity, and diastereoselectivity, and also demonstrates how a strategically designed synthetic route can rapidly yield novel architectures for biological evaluation. Our particular strategy gives rise to eight different scaffolds based on those found in polycyclic terpenoid and alkaloid natural products.

Synthesis and Molecular Modeling of a New Nitrogen Mustards Interstrand Crosslink

Angelo Guainazzi
Stony Brook University (Orlando D. Schärer Laboratory)

Nitrogen mustards (NM) are a group of bifunctional alkylating agents that react with the N(7) atom of guanine residues forming interstrand crosslinks (ICLs). ICLs are very cytotoxic since they inhibit vital cellular processes such as transcription and replication by covalently linking two opposite DNA strands. Despite the importance of ICL-forming agents in cancer chemotherapy, the mechanism by which these lesions are repaired remains poorly understood. A major impediment in studying ICLs repair has been the limited availability of well-defined substrates. We have developed a new strategy that enables the synthesis of defined site-specific NM-like ICLs in high yields and purity. Our strategy relies on the incorporation of ICL precursors bearing reactive aldehyde functionality on complementary strands of DNA, followed by ICL formation via double reductive amination. The synthetic substrates, which bear chemical modification with respect to therapeutic NM ICLs, were validated through molecular dynamic studies, confirming that the mimic had identical structural features to its natural counterpart. Our synthetic approach furthermore allows for the synthesis of major groove ICLs with different amount of distortion, providing unique and valuable tools for biochemical and cell biological studies of ICL repair.

A Bipedal DNA Brownian Motor with Coordinated Legs

Tosan Omabegho
New York University (Nadrian C. Seeman Laboratory)

Biological bipedal motors, such as kinesin, myosin, and dynein are all examples of coordinated activity between two motor domains that lead to processive linear movement along directionally polar tracks. How such directed motion emerges from domain coordination is a major issue in the effort to create synthetic molecular motors that can cyclically bias Brownian motion using chemical energy as input (1). Synthetic DNA walking devices (2 - 5) are useful systems to explore these questions, due to DNA's programmability and structural robustness. A benchmark goal is the design and construction of controlled autonomous translocators, for example to use in synthetic molecular assembly procedures that emulate nucleic acid polymerases or the ribosome.

To address this problem, we have contructed an autonomous bipedal walker made of DNA that walks along a directionally polar DNA track that is consumed during the walking cycle. This device displays true motor behavior by coordinating the stepping cycle of its two legs as it walks along its track; it does this by having its leading leg catalyze the release of its trailing leg. The release signal, sent from the leading leg to the trailing leg, is mediated by metastable DNA fuel strand complexes (4 - 7), and aided by the structural asymmetry of the track. The basis of our demonstration entails crosslinking aliquots of the walker covalently to its track in successive walking states, showing that the walker can complete a full walking cycle on a stiff linear track whose length could be extended for longer walks.

Bacterial Isoprenoid Biosynthesis as an Antibiotic Target

Scott Lefurgy
Albert Einstein College of Medicine Yeshiva University (Tom Leyh Laboratory)

Streptococcus pneumoniae is a leading cause of death among children worldwide. The increasing prevalence of multi-drug resistant S. pneumoniae continually requires new approaches to combat this threat. Our laboratory has discovered an antibiotic target in this organism—mevalonate kinase (MK), which catalyzes the first step in the conversion of mevalonate to the isoprenoid building block, isopentenyl diphosphate. Mevalonate kinase is potently, allosterically inhibited by diphosphomevalonate (DPM), whereas human MK is not inhibited by DPM. To assess the spectrum of DPM inhibition, MK homologs from pathogenic bacteria were assessed for their sensitivity to DPM. Surprisingly, these homologs are inhibited via a completely different mechanism that appears to hinge on the oligomeric state of the enzyme. This result suggests that DPM may be an exquisitely narrow-spectrum antibiotic capable of killing numerous subspecies of S. pneumoniae without affecting even their closest bacterial relatives. To extend DPM inhibition to a downstream target in the mevalonate pathway, DPM analogs were designed to inactivate DPM decarboxylase by producing a highly reactive carbocation immediately prior to decarboxylation. The absence of covalent adduct formation suggests that, counter to existing dogma, the decarboxylation transition state is concerted. Determination of the transition state structure is underway.

Intrinsically Disordered RTX Motifs as Scaffolds for Engineering Allosterically Controlled Biomolecular Recognition

Mark Blenner
Columbia University (Scott Banta Laboratory)

Directed evolution techniques have matured over recent years and high affinity binders are readily discoverable using numerous protein scaffolds, such as peptides, antibodies and repeat proteins just to name a few. It would be advantageous to be able to control the binding event with an orthogonal effector. Intrinsically disordered proteins are able to form ordered secondary and tertiary structures upon binding a ligand. We describe a Repeat in Toxin (RTX) motif from the adenylate cyclase toxin of Bordetella pertussis. This motif is comprised of 8 glycine and aspartic acid rich nonamers. Calcium binding causes this unstructured protein to form a parallel beta-helix where the calcium binding causes the first six residues to form a turn and last three form a beta-strand. These assemble into a beta-helix, where the strands form parallel beta sheets that present two highly variable residues. This work explores the calcium-induced RTX transition from a disordered to ordered state. CD, FRET and fluorescent spectroscopic methods are used to study this RTX motif and assess potential application as a useful scaffold for designing allosterically controlled biomolecular recognition.

Chemical Reporters for the Visualization and Identification of Fatty-acylated Proteins in Mammalian Cells during Salmonella Infection

Guillaume Charron
The Rockefeller University (Howard C. Hang Laboratory)

Salmonella enterica serovars are a group of Gram-negative facultative intracellular bacteria that infect a wide variety of animals. Salmonella infections are common in humans, causing typhoid fever and gastrointestinal diseases, and are an important public health concern worldwide. Once inside macrophages, Salmonella reside in a niche for their proliferation, Salmonella-containing vacuoles (SCVs), maintained by secreted bacterial protein effectors that modulate the composition of SCVs. Protein lipidation is believed to be an important process in maintaining SCVs since lipidated protein in host cells are differentially recruited or excluded from the SCVs. The lipidation of proteins has traditionally been studied with radioactive lipids, which are cumbersome to use and present low specificity, limiting the detection of less abundant lipidated proteins. Here, we present new chemical tools designed for the detection and identification of fatty-acylated proteins during Salmonella infection. Proteomic analysis of these changes should reveal insights into the specific role of secreted bacterial proteins effectors in reorganizing SCVs.