Chemical Biology Discussion Group: Special Year-End Meeting
Monday, June 2, 2008
Presented by Chemical Biology Discussion Group
Organizer: Paramjit Arora, New York University
Organizer: Paramjit Arora, New York University
Recent years have seen an increasing level of dialogue between chemists and biologists, the lines of communication consolidated by the availability of recombinant biotechnology tools for manipulating the chemical structure of genes and the proteins they encode. This has led to an explosion of interdisciplinary activity at the chemistry-biology interface, now coined chemical biology. The goal of the Chemical Biology Discussion Group is to bring together chemists and biologists working in the New York area who are interested in hearing about the latest ideas in this rapidly growing field. This group will provide a forum for lively discussion and for establishing connections, and perhaps collaborations, between chemists armed with novel technologies and biologists receptive to using these approaches to solve their chosen biological problem.
Presentations and Abstracts
Exploring Chemical Space with Aptamers
Michael Famulok, Universität Bonn
Small molecule inhibitors of proteins are invaluable tools in Chemical Biology. Their identification can be tedious, because most screening methods have to be tailored to the corresponding drug target. We have developed modular assays based on aptamer displacement or protein-dependent reporter ribozymes for the screening of small-molecule inhibitors. As aptamers can be generated for virtually any protein, the assay potentially identifies inhibitors for targets or individual protein domains for which no functional screen is available. Thereby, chemical space is explored in a rapid, focused, and modular manner, by indirectly taking advantage of the highest molecular diversity currently amenable to screening, namely that of 1016 different nucleic acid sequences. I will discuss the application of these approaches to find new inhibitors for several target proteins. Examples showing that these modulators can be used as tools for gaining novel biological insight are provided.
Interlocking Molecules by Coupling Across a DNA Duplex Helical Turn
Yu Liu, New York University (Canary Group)
Two uridine monomers containing 2'-thio-alkyl linkers functionalized with ω-amine or ω-carboxylate groups were synthesized and incorporated into oligonucleotides (ODNs). Amide coupling across one full helical turn of DNA with these modified ODNs was examined under the templation of a complementary DNA. The coupling efficiency was evaluated by denaturing gel electrophoresis and MALDI MS, and the formation of coupled product was also characterized by complete nuclease digestion. The construction of a hybrid catenane based on this strategy proved the interlocked structure. The free 3' and 5' ends of the coupled oligonucleotides also provided useful handles for post-synthetic labeling or modification.
Aminoacyl-tRNA Substrate Specificity of the Translation Machinery
Phillip Effraim, Columbia University (Cornish Group)
The translation machinery is able to catalyze the mRNA-template directed synthesis of polypeptides with an error rate of only one in every 103 to 104 peptide bonds synthesized. Significant advances in the last decade make it possible for the first time to examine how the translation machinery is able to work with the chemically diverse 20 natural amino acids, but at the same time have such a low error rate. Several lines of evidence suggest that the current kinetic model of the elongation cycle does not fully account for substrate selection, and that there are other factors in addition to the codon anticodon interaction that are important for substrate selection. We have developed assays that, combined with our purified in vitro translation system, allow us to test the hypothesis that the ribosome is able to read-out the amino acid-tRNA combinations during translation.
Mechanism of Signal Transduction by a Staphylococcal Quorum Sensing Receptor
Elizabeth A. George Cisar, Eddie Geisinger, Richard P. Novick, Tom W. Muir, The Rockefeller University (Muir Group)
Virulence factor production in Staphylococcus aureus is largely under the control of the accessory gene regulator (agr) quorum sensing system. In order to elucidate the mechanism of signal transduction by binding of the peptide ligand to its receptor histidine kinase, AgrC, we designed complementary AgrC phosphorylation site and ATP binding mutants. The mutants are completely inactive individually but recover ligand-dependent activity when co-expressed, showing that AgrC is activated as a dimer via transautophosphorylation. Co-expression of complementary AgrC mutants with different ligand specificities indicated that while there are two potential binding sites per AgrC dimer, only one cognate binding interaction is required for activation. Furthermore, pairs of complementary mutants in which one AgrC construct contains a second mutation that confers constitutive activity maintained ligand-independent activity. Taken together, our results suggest that AgrC is activated by a symmetric signaling mechanism, in which ligand-binding of either protomer triggers a conformational change that can be transferred to the sister protomer, resulting in transautophosphorylation.
Exploiting Ligand Conformation for the Selective Inhibition of Non-Ribosomal Peptide Synthetase Adenylation
Justin S. Cisar,1,2 Julian A. Ferreras,3 Rajesh K. Soni,3 Luis E. N. Quadri,3 Derek S. Tan1,2 1. Tri-Institutional Training Program in Chemical Biology, 2. Tri-Institutional Research Program and Molecular Pharmacology & Chemistry Program, Memorial Sloan–Kettering Cancer Center (Tan Group) 3. Department of Microbiology & Immunology and Molecular Biology Program, Weill Medical College of Cornell University
Bacteria synthesize and utilize nonribosomal peptides (NRPs) for a variety of functions including iron acquisition, biofilm formation, and virulence. Small-molecule probes that inhibit NRP biosynthesis would be valuable tools to study these processes and may have therapeutical applications. The initial steps of NRP biosynthesis are facilitated by NRP synthetase amino acid adenylation domains, which generate a tightly bound aminoacyl-AMP intermediate that adopts a distinct cisoid conformation. To inhibit these enzymes, we designed macrocyclic AMP-analogs that are nonhydrolyzable and conformationally locked into the cisoid conformation. The macrocyclic constraint prevents a transoid conformation that is observed in ligand binding to the mechanistically related aminoacyl-tRNA synthetases, which are required for host and bacterial protein translation. We have demonstrated these compounds are potent inhibitors of the cysteine adenylation domain of yersiniabactin synthetase HMWP2 and, unlike the corresponding linear aminoacyl-AMP analogs, do not inhibit protein translation in vitro. Selective small molecule inhibitors of non-ribosomal peptide synthesis should provide a powerful means to study the biological functions of non-ribosomal peptide natural products and a potential avenue to develop novel antibiotics.
Beta-Peptide Ligands for hDM2: Cellular Entry and p53 Activation.
Liz Harker, Yale University (Schepartz Group)
We have recently designed and characterized two 3 14 –helical β –peptides in which nonnatural side chains have been substituted into the hDM2¯recognition epitope to improve affinity. Western blots indicate that these peptides effectively inhibit the p53•hDM2 interaction in cell culture where the degree of p53 activation corresponds to the affinity of each peptide for hDM2. Additionally, we have found that incorporation of arginine side chains into the β –peptide scaffold promotes cellular entry. These results demonstrate the utility of the 3 14 –helical β –peptide scaffold for targeting a protein recognition surface both in vitro and in cells.
Probing the Mechanisms Behind Inhibition of Fertilization and Activation of Development by Fertilinβ Derived Oligopeptide Polymers
Keith A. Baessler,1 Younjoo Lee,2 and Nicole S. Sampson2.* 1Biochemistry and Structural Biology Graduate Program, 2Department of Chemistry, Stony Brook University (Sampson Group)
Polymer probes containing the three-amino acid binding sequence of sperm protein fertilin β have been developed to investigate the mechanisms by which mammalian sperm bind and fuse to the egg plasma membrane. The β1 integrin has been implicated as a receptor for sperm fertilin β. However, β1 integrin knockout mice are fertile. The polymer probes were tested as inhibitors of in vitro fertilization using wild-type and β1 integrin knockout eggs. Inhibition by polymer probes required β1 integrin on the egg. Furthermore, inhibition is due to competition with sperm binding, not activation of the egg block to polyspermy.