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

Abstract

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 (ED 50 ) of less than 10 M. 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 ED 50 s for these inhibitors were 5.8 and 7.6 M, 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.

Pharmacological Assessment Defines Leishmania donovani Casein Kinase 1 as a Drug Target and Reveals Important Functions in Parasite Viability and Intracellular Infection

“…Twenty micrograms of total protein extracts was analyzed by Western blotting with SY3535 antibody. We detected one band corresponding to endogenous CK1.2 present in all the samples and a higher band between 40 and 55 kDa, corresponding to CK1.2-V5-His 6 , that was absent in the mock-transfected controls (Fig. 4B, top).…”

“…Expression and purification of recombinant LmaCK1.2-His 6 . The open reading frame of the Leishmania major CK1.2 homolog LmjF.…”

“…Phosphorylation is indeed essential for almost all cellular processes, and as a result, inhibition of protein kinases should lead to interference with the regulation of key processes in metabolism, proliferation, or differentiation. In Leishmania, different protein kinases have already been identified as potential drug targets, such as mitogen-activated protein kinases (MAPKs) or cdc2-related kinase 3 (CRK3) (5)(6)(7). Members of another family of kinases have emerged as potential drug targets, casein kinase 1 (CK1), a family of multifunctional Ser/Thr protein kinases that are characterized by a highly conserved kinase domain and a specific C-terminal domain implicated in its regulation and localization (8).…”

Development of new antileishmanial drugs – current knowledge and future prospects

“…Included among the potential inhibitors of CRK3 cyclin-dependent kinase of Leishmania, indirubin derivatives exhibited growth arrest and change in the DNA content [104]. Evidence that PKC plays a critical role in the invasion process is highlighted by a study demonstrating that pre-treatment of intact parasites by imidazolidinone compounds inhibited PKC activity and the parasitehost cell invasion process [105].…”

Kinetoplastida: new therapeutic strategies

“…Other opportunities lie in understanding of metabolic pathways and enzyme targets, with studies on isoprenoid biosynthesis leading to studies on bisphosphonates (Yardley et al, 2002), and on kinases to paullones (Grant et al, 2004).…”