Assembly, activation, and substrate specificity of cyclin D1/Cdk2 complexes.

Previous studies have shown conflicting data regarding cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes, and considering the widespread overexpression of cyclin D1 in cancer, it is important to fully understand their relevance. While many have shown that cyclin D1 and Cdk2 form active complexes, others have failed to show activity or association. Here, using a novel p21-PCNA fusion protein as well as p21 mutant proteins, we show that p21 is a required scaffolding protein, with cyclin D1 and Cdk2 failing to complex in its absence. These p21/cyclin D1/Cdk2 complexes are active and also bind the trimeric PCNA complex, with each trimer capable of independently binding distinct cyclin/Cdk complexes. We also show that increased p21 levels due to treatment with chemotherapeutic agents result in increased formation and kinase activity of cyclin D1/Cdk2 complexes, and that cyclin D1/Cdk2 complexes are able to phosphorylate a number of substrates in addition to Rb. Nucleophosmin and Cdh1, two proteins important for centrosome replication and implicated in the chromosomal instability of cancer, are shown to be phosphorylated by cyclin D1/Cdk2 complexes. Additionally, polypyrimidine tract binding protein-associated splicing factor (PSF) is identified as a novel Cdk2 substrate, being phosphorylated by Cdk2 complexed with either cyclin E or cyclin D1, and given the many functions of PSF, it could have important implications on cellular activity.

Discovery of novel DNA gyrase inhibitors by high-throughput virtual screening.

The bacterial type II topoisomerases DNA gyrase and topoisomerase IV are validated targets for clinically useful quinolone antimicrobial drugs. A significant limitation to widely utilized quinolone inhibitors is the emergence of drug-resistant bacteria due to an altered DNA gyrase. To address this problem, we have used structure-based molecular docking to identify novel drug-like small molecules that target sites distinct from those targeted by quinolone inhibitors. A chemical ligand database containing approximately 140,000 small molecules (molecular weight, <500) was molecularly docked onto two sites of Escherichia coli DNA gyrase targeting (i) a previously unexplored structural pocket formed at the dimer interface of subunit A and (ii) a small region of the ATP binding pocket on subunit B overlapping the site targeted by coumarin and cyclothialidine drugs. This approach identified several small-molecule compounds that inhibited the DNA supercoiling activity of purified E. coli DNA gyrase. These compounds are structurally unrelated to previously identified gyrase inhibitors and represent potential scaffolds for the optimization of novel antibacterial agents that act on fluoroquinolone-resistant strains.

Induction of petite mutants in yeast Saccharomyces cerevisiae by the anticancer drug dequalinium.

Dequalinium (DEQ), a drug with both antimicrobial and anticancer activity, induced the formation of petite (respiration-deficient) mutants in the yeast Saccharomyces cerevisiae. DEQ was found to be approximately 50-fold more potent than ethidium bromide (EB) at inducing petites. Analysis of the DEQ-induced petite mutants indicated a complete loss of mitochondrial DNA (<1 copy/cell). Prior to the loss of mtDNA, DEQ caused cleavage of the mtDNA into a population of fragments 30-40kbp in size suggesting that this drug causes petites by inducing a breakdown of mtDNA. The selective effect of DEQ on yeast mtDNA may underlie the antifungal activity of this chemotherapeutic agent.

Expression and characterization of the ATP-binding domain of a malarial Plasmodium vivax gene homologous to the B-subunit of the bacterial topoisomerase DNA gyrase.

We have previously reported the presence of a DNA gyrase-like topoisomerase activity associated with the 35kb apicoplast DNA in the malarial parasite Plasmodium falciparum [Weissig V, Vetro-Widenhouse TS, Rowe TC. Topoisomerase II inhibitors induce cleavage of nuclear and 35kb plastid DNAs in the malarial parasite Plasmodium falciparum. DNA Cell Biol 1997;16:1483]. Sequences encoding polypeptides homologous to both the A and B subunits of bacterial DNA gyrase have been identified in the genome sequence of P. falciparum among data produced by the Malaria Genome Consortium and the University of Florida Malaria Gene Sequence Tag Project. Based on these findings, we have cloned and expressed a region of the Plasmodium vivax GyrB gene encoding a 43kDa polypeptide homologous to the ATP-binding domain of Escherichia coli DNA gyrase. The 43kDa PvGyrB polypeptide was found to have intrinsic ATPase activity with a K(m) of 0.27mM and a k(cat) of 0.051s(-1). The PvGyrB ATPase was also sensitive to the bacterial DNA gyrase inhibitor coumermycin. The implications of these findings are discussed.