John L. Wood

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.

Identifying the proteins to which small-molecule probes and drugs bind in cells

Most small-molecule probes and drugs alter cell circuitry by interacting with 1 or more proteins. A complete understanding of the interacting proteins and their associated protein complexes, whether the compounds are discovered by cell-based phenotypic or target-based screens, is extremely rare. Such a capability is expected to be highly illuminating—providing strong clues to the mechanisms used by small-molecules to achieve their recognized actions and suggesting potential unrecognized actions. We describe a powerful method combining quantitative proteomics (SILAC) with affinity enrichment to provide unbiased, robust and comprehensive identification of the proteins that bind to small-molecule probes and drugs. The method is scalable and general, requiring little optimization across different compound classes, and has already had a transformative effect on our studies of small-molecule probes. Here, we describe in full detail the application of the method to identify targets of kinase inhibitors and immunophilin binders.

Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase

In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in‐frame UAG codons in the mRNAs of some methyltransferases. Pyl is directly acylated onto a suppressor tRNAPyl by pyrrolysyl‐tRNA synthetase (PylRS). Due to the lack of a readily available Pyl source, we looked for structural analogues that could be aminoacylated by PylRS onto tRNAPyl. We report here the in vitro aminoacylation of tRNAPyl by PylRS with two Pyl analogues: N‐ε‐d‐prolyl‐l‐lysine (d‐prolyl‐lysine) and N‐ε‐cyclopentyloxycarbonyl‐l‐lysine (Cyc). Escherichia coli, transformed with the tRNAPyl and PylRS genes, suppressed a lacZ amber mutant dependent on the presence of d‐prolyl‐lysine or Cyc in the medium, implying that the E. coli translation machinery is able to use Cyc‐tRNAPyl and d‐prolyl‐lysine‐tRNAPyl as substrates during protein synthesis. Furthermore, the formation of active β‐galactosidase shows that a specialized mRNA motif is not essential for stop‐codon recoding, unlike for selenocysteine incorporation.

Evolution of a Synthetic Strategy : Total Synthesis of (±)-Welwitindolinone A Isonitrile

An efficient and highly stereoselective total synthesis of the natural product (+/-)-welwitindolinone A isonitrile (1) is described. The bicyclo[4.2.0]octane core of 1 was established by a regio- and diastereoselective [2+2] ketene cycloaddition. The C12 quaternary center and vicinal stereogenic chlorine were installed in a single operation with excellent stereocontrol via a chloronium ion mediated semipinacol rearrangement. Described strategies for construction of the spiro-oxinole include a SmI2-LiCl mediated reductive cyclization and a novel anionic cyclization that simultaneously constructs the spiro-oxindole and vinyl isonitrile moieties.

The K252a derivatives, inhibitors for the PAK/MLK kinase family selectively block the growth of RAS transformants.

BACKGROUNDOncogenic RAS mutants such as v-Ha-RAS activate members of Rac/CDC42-dependent kinases (PAKs) and appear to contribute to the development of more than 30% of all human cancers. PAK1 activation is essential for oncogenic RAS transformation, and several chemical compounds that inhibit Tyr kinases essential for the RAS-induced activation of PAK1 strongly suppress RAS transformation either in cell culture or in vivo (nude mice). Although we have developed a cell-permeable PAK-specific pep-tide inhibitor called WR-PA18, so far no chemical (metabolically stable) compound has been developed that directly inhibits PAK1 in a highly selective manner. Thus, we have explored such a PAK1 inhibitors) among synthetic derivatives of an adenosine triphosphate antagonist. RESULTSFrom the naturally occurring adenosine triphosphate antagonist K252a, we have developed two bulky derivatives, called CEP-1347 and KT D606 (a K252a dimer), which selectively inhibit PAKs or mixed-lineage kinases both in vitro and in cell culture and convert v-Ha-RAS-transformed NIH 3T3 cells to flat fibroblasts similar to the parental normal cells. Furthermore, these two K252a analogues suppress the proliferation of v-Ha-RAS transformants, but not the normal cells. CONCLUSIONThese bulky adenosine triphosphate antagonists derived from K252a or related indolocarbazole compounds such as staurosporine would be potentially useful for the treatment of RAS/PAK1-induced cancers, once their anti-PAK1 activity is significantly potentiated by a few additional chemical modifications at the sugar ring suggested in this paper.

PAK is essential for RAS-induced upregulation of cyclin D1 during the G1 to S transition.

Oncogenic RAS mutants such as v-Ha-RAS induce cell cycling, in particular the G1 to S transition, by up-regulating cyclin D1 and down-regulating p27, an inhibitor for cyclin-dependent kinases (CDKs). PI-3 kinase appears to be involved in the regulation of both cyclin D1 and p27. In this report, using two distinct inhibitors specific for PAK1-3 (CEP-1347 and WR-PAK18), we present the first evidence indicating that the PIX/Rac/CDC42-dependent Ser/Thr kinases PAK1-3, acting downstream of PI-3 kinase and up-stream of the Raf/MEK/ERKs kinase cascade, is essential for RAS-induced up-regulation of cyclin D1, but not down-regulation of p27. Since these PAK-inhibitors block selectively the malignant growth of RAS transformants, in which PAK1 is constitutively activated, but not normal cell growth, it is suggested that RAS transformants are addicted to the high levels of PAK1 for their malignant entry to S phase.

An Enantioselective Total Synthesis and Stereochemical Revision of (+)-Citrinadin B

This manuscript describes an enantioselective synthesis of the naturally occurring alkaloid citrinadin B. The synthetic effort revealed an anomaly in the original structural assignment that has led to the proposal of a stereochemical revision. This revision is consistent with the structures previously reported for a closely related family of alkaloids, PF1270A-C. The synthesis is convergent and employs a stereoselective intermolecular nitrone cyloaddition reaction as a key step.

Progress Toward the Total Synthesis of (±)-Actinophyllic Acid

This paper describes ongoing progress toward the synthesis of the novel indole alkaloid actinophyllic acid via a synthetic strategy that allows for the installation of all C-atoms (highlighted in red) requisite for completion of a total synthesis.

CP-263,114 synthetic studies. Construction of an isotwistane ring system via rhodium carbenoid C - H insertion

Abstract Described is a rhodium carbenoid C–H insertion strategy for the construction of an isotwistane ring system needed for a late-stage fragmentation en route to the total synthesis of CP-263,114.

Collaborative Total Synthesis: Routes to (±)-Hippolachnin A Enabled by Quadricyclane Cycloaddition and Late-Stage C–H Oxidation

Described herein are synthetic efforts toward the synthesis of hippolachnin A. Two independently devised routes from the Brown and Wood groups allowed for the synthesis of hippolachnin A from the unusual starting material, quadricyclane, by harnessing the power of late-stage C-H oxidation. Collaborative union of the best features of the two routes allowed for preparation of the molecule with improved efficiency.