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Antimicrobial Agents and Chemotherapy, August 2004, p. 3033-3042, Vol. 48, No. 8
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.8.3033-3042.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Inhibitors of Leishmania mexicana CRK3 Cyclin-Dependent Kinase: Chemical Library Screen and Antileishmanial Activity

Karen M. Grant,^1* Morag H. Dunion,¹ Vanessa Yardley,² Alexios-Leandros Skaltsounis,³ Doris Marko,⁴ Gerhard Eisenbrand,⁴ Simon L. Croft,² Laurent Meijer,⁵ and Jeremy C. Mottram¹

Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow,¹ Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom,² Laboratory of Pharmacognosy and Natural Product Chemistry, University of Athens, Athens, Greece,³ Department of Chemistry, University of Kaiserslautern, Kaiserslautern, Germany,⁴ CNRS Station Biologique, Roscoff, France⁵

Received 23 February 2004/ Accepted 30 April 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.

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* Corresponding author. Mailing address: Wellcome Centre for Molecular Parasitology, University of Glasgow, Anderson College, 56 Dumbarton Rd., Glasgow G11 6NU, United Kingdom. Phone: 44 141 330 6766. Fax: 44 141 330 5422. E-mail: k.grant{at}vet.gla.ac.uk.

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Antimicrobial Agents and Chemotherapy, August 2004, p. 3033-3042, Vol. 48, No. 8
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.8.3033-3042.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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