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Inhibitors of Leishmania mexicana CRK3 Cyclin-Dependent Kinase: Chemical Library Screen and Antileishmanial Activity. Karen M. Grant, 2004.The CRK3 cyclin-dependent kinase of Leishmania has been shown by genetic manipulation of the parasite to be essential for proliferation . We present data which demonstrate that chemical inhibition of CRK3 impairs the parasite's viability within macrophages, thus further validating CRK3 as a potential drug target . A microtiter plate-based histone H1 kinase assay was developed to screen CRK3 against a chemical library enriched for protein kinase inhibitors . Twenty-seven potent CRK3 inhibitors were discovered and screened against Leishmania donovani amastigotes in vitro . Sixteen of the CRK3 inhibitors displayed antileishmanial activity, with a 50% effective dose (ED50) of less than 10 µM . These compounds fell into four chemical classes: the 2,6,9-trisubstituted purines, including the C-2-alkynylated purines; the indirubins; the paullones; and derivatives of the nonspecific kinase inhibitor staurosporine . The paullones and staurosporine derivatives were toxic to macrophages . The 2,6,9-trisubstituted purines inhibited CRK3 in vitro, with 50% inhibitory concentrations ranging from high nanomolar to low micromolar concentrations . The most potent inhibitors of CRK3 (compounds 98/516 and 97/344) belonged to the indirubin class; the 50% inhibitory concentrations for these inhibitors were 16 and 47 nM, respectively, and the ED50s for these inhibitors were 5.8 and 7.6 µM, respectively . In culture, the indirubins caused growth arrest, a change in DNA content, and aberrant cell types, all consistent with the intracellular inhibition of a cyclin-dependent kinase and disruption of cell cycle control . Thus, use of chemical inhibitors supports genetic studies to confirm CRK3 as a validated drug target in Leishmania and provides pharmacophores for further drug development . Disruption of the Subtilase Gene, albin1, in Ophiostoma piliferum. Brad Hoffman, 2004.Wood sapstaining fungi produce multiple proteases that break down wood protein . Three groups of subtilases have been identified in sapstaining fungi; however, it is not known if these groups have distinct physiological roles (B . Hoffman and C . Breuil, Curr . Genet . 41:168-175, 2002) . In this work we examined the role of the subtilase Albin1 from Ophiostoma piliferum . Reamplification of cDNA ends PCR was used to obtain the albin1 gene sequence . The encoded subtilase is probably extracellular and involved in nutrient acquisition . This gene was disrupted with an Agrobacterium tumefaciens-mediated transformation system . Two of the disruptants obtained had significantly lower levels of proteolytic activity, slower growth in bovine serum albumin, and significantly reduced growth on wood . Thus, albin1 plays an important role in O . piliferum's ability to acquire nitrogen from wood proteins .
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