|Anthony C. Bishop
Assistant Professor of Chemistry, Amherst College
Ph.D.: Princeton University
Bio-Organic Chemistry and Chemical Biology
The research interests of my laboratory are predominantly focused at the interface between organic chemistry and molecular biology. We use a combination of chemical and biochemical a pproaches to study important biological processes, particularly cellular signal transduction and protein synthesis. The two major ongoing projects of the lab are described below.
I. Generation of highly selective protein phosphatase inhibitors through convergent engineering of a protein/small molecule interface
Phosphorylation of tyrosine is a central control element in eukaryotic signal transduction. This chemical switch is regulated by two large families of enzymes, the protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). PTKs catalyze the transfer of the gamma phosphate of ATP to tyrosine in a protein substrate. PTPs catalyze the hydrolytic dephosphorylation of phosphotyrosine. This interplay between kinase and phosphatase activity allows the cell precise control of many distinct protein phosphorylation states. Aberrant regulation of phosphorylation-dependent signaling pathways has been widely implicated in the pathogenesis of cancer, diabetes, and a variety of immune deficiencies.
Small molecule inhibitors that are specific for each cellular PTP would be invaluable tools in dissecting phosphorylation networks. However, it is not currently possible to design target-specific ligands based on analysis of a gene sequence. Very few selective PTP inhibitors are known and no general methods for specifically targeting a given PTP have been described. Because the human genome encodes more than 100 PTPs, the identification of inhibitors that are specific for each PTP through the methods of traditional medicinal chemistry is not a practical prospect. Therefore, we are attempting to generate highly selective PTP inhibitors through the convergent engineering of PTP/inhibitor interfaces (Fig. 1). This strategy uses a combined chemical/genetic approach to inhibitor design and employs the following steps. The strategy involves: i.) introduction of a functionally silent point mutation to create a unique pocket in the active site of the target PTP of interest, ii.) rational derivatization of a known chemical inhibitor with a bulky group designed to fit the target's novel active site pocket (and which is sterically incompatible with similar wild-type PTPs), iii.) expression of the mutated target in a cell type of interest to monitor the effects of target-specific inhibition. Successful generation of PTP-specific inhibitors will allow for the functional characterization of individual PTPs and for the validation of PTPs as suitable therapeutic targets.
II. Biochemical characterization of a newly discovered tRNA-modification enzyme family
Transfer RNA acts as an essential link between the cellular pool of amino acids and the messenger RNA (mRNA) decoding machinery of the ribosome. These RNA molecules of ~76 nucleotides contain many modified bases in addition to the four canonical bases A, C, G, and U. Although the functions of some modified tRNA bases have been well characterized, the biological role of most of these chemically interesting nucleotides remains unclear.
5,6-Dihydrouridine is the most abundant modified base in prokaryotic and eukaryotic tRNAs. This non-aromatic base is found almost exclusively at conserved positions in the D-loop and is thought to be formed post-transcriptionally by the reduction of uridines in tRNA transcripts (Fig. 2). Despite the widespread occurrence of dihydrouridine, little is known about its functional roles.
Recently, it was shown that a previously uncharacterized protein family is responsible for dihydrouridine formation. Thus, this protein family has been re-named as the dihydrouridine synthase (DUS) family of enzymes. Our goal is to gain a molecular level understanding of how DUSs act to reduce D-loop uridines to dihydrouridine. The identification of a previously unknown enzyme family allows many lines of questioning to emerge. The work in my laboratory is focused upon the macromolecular protein/RNA interactions that must occur before and during dihydrouridine formation. The two major thrusts of the proposed research are described below.
i) How do DUSs recognize and bind tRNA? DUSs contain no known RNA binding domains, and are not predicted to interact with RNA in annotated genomic databases. Thus, it is very likely that DUSs utilize a yet undiscovered RNA-binding motif. One of the primary objectives of our work is to develop an understanding of how DUSs recognize and bind tRNA. The identification of a novel DUS RNA-binding motif may have broad implications outside the DUS family, as similar RNA-binding moieties are often found in otherwise unrelated protein families.
ii) What is the tRNA-substrate specificity of the DUSs and how it is achieved? The genome of the bacterium E. coli encodes three members of the DUS family. From the initial published study on E. coli DUSs, it is clear that these enzymes have non-redundant tRNA-substrate specificities. However, the determination of the substrate specificities for the three E. coli DUSs has not been carried out. We are attempting to map the precise tRNA positions that are modified by each of the E. coli DUSs. In addition, the portions of the enzymes that give rise to the specificity will be delineated.
Hoffman HE, Blair ER, Johndrow JE & Bishop AC. Allele-specific inhibitors of protein tyrosine phosphatases. Journal of the American Chemical Society 127 , 2824-2825 (2005).
Kunkel EJ, Plavec I, Nguyen D, Melrose J, Rosler ES, Kao LT, Wang Y, Hytopoulos E, Bishop AC, Bateman R, Shokat KM, Butcher EC & Berg EL. Rapid structure-activity and selectivity analysis of kinase inhibitors by BioMAP analysis in complex human primary cell-based models. Assay and Drug Development Technologies 2 , 431-441 (2004).
Bishop AC. A hot spot for protein kinase inhibitor sensitivity. Chemistry & Biology 11 , 587-589 (2004).
Sreenivasan A, Bishop AC, Shokat KM & Kellogg DR. Specific inhibition of Elm1 kinase activity reveals functions required for early G1 events. Molecular and Cellular Biology 23 , 6327-6337 (2003).
Bishop AC, Beebe K & Schimmel PR. Interstice mutations that block site-to-site translocation of a misactivated amino acid bound to a class I tRNA synthetase. Proceedings of the National Academy of Sciences of the United States of America 100 , 490-494 (2003).
Buzko OV, Bishop AC & Shokat KM. Modified AutoDock for accurate docking of protein kinase inhibitors. J. Comput. Aided Mol. Des. 16 , 113-127 (2002).
Bishop AC, Xu J, Johnson RC, Schimmel P & de Crécy-Lagard V. Identification of the tRNA-Dihydrouridine Synthase Family. J. Biol. Chem . 277 , 25090-25095 (2002).
Bishop AC, Nomanbhoy TK & Schimmel P. Blocking site-to-site translocation of a misactivated amino acid by mutation of a class I tRNA synthetase. Proc. Natl. Acad. Sci. USA 99 , 585-590 (2002).
Carroll AS, Bishop AC, DeRisi JL, Shokat KM & O'Shea EK. Chemical inhibition of the Pho85 cyclin-dependent kinase reveals a role in the environmental stress response. Proc. Natl. Acad. Sci. USA 98 , 12578-12583 (2001).
Bishop AC, Buzko O & Shokat KM. Magic bullets for protein kinases. Trends Cell Biol. 11 , 167-172 (2001).
Liu Y, Witucki LA, Shah K, Bishop AC & Shokat KM. Src-Abl tyrosine kinase chimeras: replacement of the adenine binding pocket of c-Abl with v-Src to swap nucleotide and inhibitor specificities. Biochemistry 39 , 14400-14408 (2000).
Weiss EL, Bishop AC, Shokat KM & Drubin DG. Chemical genetic analysis of the budding-yeast p21-activated kinase Cla4p. Nature Cell Biol. 2 , 677-685 (2000).
Bishop AC, Ubersax JA, Petsch DT, Matheos DP, Gray NS, Blethrow J, Shimizu E, Tsien JZ, Schultz PG, Rose MD, Wood JL, Morgan DO & Shokat KM. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407 , 395-401 (2000).
Bishop AC, Buzko O, Heyeck-Dumas S, Jung I, Kraybill B, Liu Y, Shah K, Ulrich S, Witucki L, Yang F, Zhang C & Shokat KM. Unnatural ligands for engineered proteins: new tools for chemical genetics. Annu. Rev. Biophys. Biomol. Struct. 29 , 577-606 (2000).
Weintraub BC, Jun JE, Bishop AC, Shokat KM, Thomas ML & Goodnow CC. Entry of B cell receptor into signaling domains is inhibited in tolerant B cells. J. Exp. Med. 191 , 1443-1448 (2000).