Anthony C. Bishop

A chemical switch for inhibitor-sensitive alleles of any protein kinase

Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.

Structural basis for selective inhibition of Src family kinases by PP1.

BACKGROUND Small-molecule inhibitors that can target individual kinases are powerful tools for use in signal transduction research. It is difficult to find such compounds because of the enormous number of protein kinases and the highly conserved nature of their catalytic domains. Recently, a novel, potent, Src family selective tyrosine kinase inhibitor was reported (PP1). Here, we study the structural basis for this inhibitors specificity for Src family kinases. RESULTS A single residue corresponding to Ile338 (v-Src numbering; Thr338 in c-Src) in Src family tyrosine kinases largely controls PP1s ability to inhibit protein kinases. Mutation of Ile338 to a larger residue such as methionine or phenylalanine in v-Src makes this inhibitor less potent. Conversely, mutation of Ile338 to alanine or glycine increases PP1s potency. PP1 can inhibit Ser/Thr kinases if the residue corresponding to Ile338 in v-Src is mutated to glycine. We have accurately predicted several non-Src family kinases that are moderately (IC(50) approximately 1 microM) inhibited by PP1, including c-Abl and the MAP kinase p38. CONCLUSIONS Our mutagenesis studies of the ATP-binding site in both tyrosine kinases and Ser/Thr kinases explain why PP1 is a specific inhibitor of Src family tyrosine kinases. Determination of the structural basis of inhibitor specificity will aid in the design of more potent and more selective protein kinase inhibitors. The ability to desensitize a particular kinase to PP1 inhibition of residue 338 or conversely to sensitize a kinase to PP1 inhibition by mutation should provide a useful basis for chemical genetic studies of kinase signal transduction.

Magic bullets for protein kinases

A chemical-genetic method for the generation of target-specific protein kinase inhibitors has been developed recently. This strategy utilizes a functionally silent active-site mutation to sensitize a target kinase to inhibition by a small molecule that does not inhibit wild-type kinases. Tyrosine and serine/threonine kinases are equally amenable to the drug-sensitization approach, which has been used to generate selective inhibitors of mutant Src-family kinases, Abl-family kinases, cyclin-dependent kinases, mitogen-activated kinases, p21-activated kinases and Ca(2+)/calmodulin-dependent kinases. The designed inhibitors are specific for the sensitized kinase in a cellular background where the wild-type kinase has been inactivated. By these means, kinase-sensitization has been used systematically to generate and analyze conditional alleles of several yeast protein kinases in vivo.

Generation of Monospecific Nanomolar Tyrosine Kinase Inhibitors via a Chemical Genetic Approach

Selective protein kinase inhibitors are highly sought after as tools for studying cellular signal transduction cascades, yet few have been discovered due to the highly conserved fold of kinase catalytic domains. Through a combination of small molecule synthesis and protein mutagenesis, a highly potent (IC50 = 1.5 nM) and uniquely specific inhibitor (4-amino-1-tert-butyl-3-(1‘-naphthyl)pyrazolo[3,4-d]pyrimidine) of a rationally engineered v-Src tyrosine kinase (Ile338Gly v-Src) has been identified. Both the potency and specificity of this compound surpass those of any known Src family tyrosine kinase inhibitors. The molecule strongly inhibits the engineered v-Src in whole cells but does not inhibit tyrosine phosphorylation in cells that express only wild-type tyrosine kinases. In addition, the inhibitor selectively disrupts transformation in cells that express the target v-Src. The structural degeneracy of kinase active sites should allow the same complementary inhibitor/protein design strategy to be widel...

Chemical genetic analysis of the budding-yeast p21-activated kinase Cla4p.

The p21-activated kinases (PAKs) are effectors for the Rho-family GTPase Cdc42p. Here we define the in vivo function of the kinase activity of the budding yeast PAK Cla4p, using cla4 alleles that are specifically inhibited by a cell-permeable compound that does not inhibit the wild-type kinase. CLA4 kinase inhibition in cells lacking the partially redundant PAK Ste20p causes reversible SWE1-dependent cell-cycle arrest and gives rise to narrow, highly elongated buds in which both actin and septin are tightly polarized to bud tips. Inhibition of Cla4p does not prevent polarization of F-actin, and cytokinesis is blocked only in cells that have not formed a bud before inhibitor treatment; cell polarization and bud emergence are not affected by Cla4p inhibition. Although localization of septin to bud necks is restored in swe1Δ cells, cytokinesis remains defective. Inhibition of Cla4p activity in swe1Δ cells causes a delay of bud emergence after cell polarization, indicating that this checkpoint may mediate an adaptive response that is capable of promoting budding when Cla4p function is reduced. Our data indicate that CLA4 PAK activity is required at an early stage of budding, after actin polarization and coincident with formation of the septin ring, for early bud morphogenesis and assembly of a cytokinesis site.

Chemical inhibition of the Pho85 cyclin-dependent kinase reveals a role in the environmental stress response

In addition to its well-established role in responding to phosphate starvation, the cyclin-dependent kinase Pho85 has been implicated in a number of other physiological responses of the budding yeast Saccharomyces cerevisiae, including synthesis of glycogen. To comprehensively characterize the range of Pho85-dependent gene expression, we used a chemical genetic approach that enabled us to control Pho85 kinase activity with a cell-permeable inhibitor and whole genome transcript profiling. We found significant phenotypic differences between the rapid loss of activity caused by inhibition and the deletion of the genomic copy of PHO85. We demonstrate that Pho85 controls the expression of not only previously identified glycogen synthetic genes, but also a significant regulon of genes involved in the cellular response to environmental stress. In addition, we show that the effects of this inhibitor are both rapid and reversible, making it well suited to the study of the behavior of dynamic signaling pathways.

Acquisition of inhibitor-sensitive protein kinases through protein design.

Protein phosphorylation is the major post-translational modification used by eukaryotic cells to control cellular signaling. Protein kinases have emerged as attractive drug targets because heightened protein kinase activity has been associated with several proliferative diseases, most notably cancer and restenosis. Until now, it has been very difficult to confirm the utility of protein kinases as inhibitor targets because very few small molecules that selectively inhibit one particular kinase are known. Discovery of highly specific kinase inhibitors has been slow because the protein family contains approximately 2000 members, all of which share a conserved active site fold. Recent work in several laboratories has sought to circumvent the problem of kinase structural degeneracy by engineering drug sensitivity into Src family tyrosine kinases and mitogen-activated protein kinases through site-directed mutagenesis. By introducing a unique non-naturally occurring amino acid into a conserved region of the enzymes binding site, a target protein kinase can be rapidly sensitized to a small molecule. Introduction of the engineered kinase into a cell line or animal model should greatly expedite the investigation of protein kinase inhibition as a viable drug treatment. The purpose of this review is to summarize these recent advances in protein kinase drug sensitization.

Discovery of Carbohydrate Sulfotransferase Inhibitors from a Kinase-Directed Library

, compound 1 was not found to form a gel even at concentrations as high as 0.2m. As discussed in the text, we rationalize this in terms of 1, but not these other compounds, being present in a syn conformation. The observation that the less sterically hindered compound, (2’,3’,5’-tri-O-isobutyrylribofuranosidyl)-2-aminopurin-6one, which lacks an aryl substituent and which should be present mostly in anti conformation, forms a gel at concentrations as low as 0.02m is considered consistent with this theory.

Blocking site-to-site translocation of a misactivated amino acid by mutation of a class I tRNA synthetase.

The genetic code is established by the aminoacylation reactions of tRNA synthetases. Its accuracy depends on editing reactions that prevent amino acids from being assigned to incorrect codons. A group of class I synthetases share a common insertion that encodes a distinct site for editing that is about 30 Å from the active site. Both misactivated aminoacyl adenylates and mischarged amino acids attached to tRNA are translocated to this site, which, in turn, is divided into subsites—one for the adenylate and one for the aminoacyl moiety attached to tRNA. Here we report that a specific mutation in isoleucyl-tRNA synthetase prevents editing by blocking translocation. The mutation alters a widely conserved residue that is believed to tether the amino group of mischarged tRNA to its subsite for editing. These and other data support a model where editing is initiated by translocation of the misacylated amino acid attached to tRNA to create an “editing complex” that facilitates subsequent rounds of editing by translocation of the misactivated adenylate.

Modified AutoDock for accurate docking of protein kinase inhibitors

Protein kinases are an important class of enzymes controlling virtually all cellular signaling pathways. Consequently, selective inhibitors of protein kinases have attracted significant interest as potential new drugs for many diseases. Computational methods, including molecular docking, have increasingly been used in the inhibitor design process [1]. We have considered several docking packages in order to strengthen our kinase inhibitor work with computational capabilities. In our experience, AutoDock offered a reasonable combination of accuracy and speed, as opposed to methods that specialize either in fast database searches or detailed and computationally intensive calculations.However, AutoDock did not perform well in cases where extensive hydrophobic contacts were involved, such as docking of SB203580 to its target protein kinase p38. Another shortcoming was a hydrogen bonding energy function, which underestimated the attraction component and, thus, did not allow for sufficiently accurate modeling of the key hydrogen bonds in the kinase-inhibitor complexes.We have modified the parameter set used to model hydrogen bonds, which increased the accuracy of AutoDock and appeared to be generally applicable to many kinase-inhibitor pairs without customization. Binding to largely hydrophobic sites, such as the active site of p38, was significantly improved by introducing a correction factor selectively affecting only carbon and hydrogen energy grids, thus, providing an effective, although approximate, treatment of solvation.