Figure 1.
Expression and purification of the Leishmania CRK3:CYC6.
(A) CRK3his and CYC6his were expressed in E. coli individually (lanes 1 and 2 respectively). Coomassie-stained SDS-PAGE. (B) Co-expressed CRK3his and CYC6his purified by Nickel-chelate and gel filtration chromatography. Coomassie-stained SDS-PAGE. (C) Co-expression of CRK3 and CYC6his and purification of the CRK3:CYC6his by Nickel-chelate and ion-exchange chromatography. Coomassie-stained SDS-PAGE. (D) Activation of CRK3his histone H1 kinase in the presence of increasing concentrations of CYC6his. All lanes contain 1.25 µg of CRK3 and 0.025 µg, 0.05 µg, 0.075 µg, 0.1 µg of CYC6 (lanes 2–5).
Table 1.
Analysis of the peptide substrates identified from the IMAP substrate finder assay.
Table 2.
Lexicon azapurine HTS hits.
Figure 2.
Predicted binding of azapurine pharmacophore and model of CRK3 active site.
a) Sequence alignment of LmajCRK3, Human CDK2 and Human CDK4 showing the percentage identity in shades of blue. The active site regions are boxed in green with key differences boxed in red. b) A model of Lm CRK3 with an azapurine derivative (compound 11) docked in to the ATP site to show the predicted binding mode. c) Schematic overview of the predicted binding mode of azapurine derivatives with Lm CRK3 detailing the A-D-A motif not possible in CDK2 due to the Tyr101 - Phe82 difference. d) CDK2 binding mode with NU6102 [39] showing the D-A-D motif.
Figure 3.
Route of synthesis of the Azapurine derivatives.
(i) NaN3, acetonitrile, 100°C, 90 mins; or H2SO4, NaNO2, H2O, 5°C, 10 mins, then NaN3, hexane 5°C – RT, 2 hrs. (ii) NaOEt, ethanol, cyanoacetamide, ethyl cyclohexylacetate (or ethyl phenylacetate), 110°C, overnight. (iii) POCl3, microwave, 130°C, 10 mins. (iv) HNR2R3, Et3N, dichloromethane, microwave, 110°C, 10 mins.
Table 3.
Azapurine derivatives.
Table 4.
BioFocus SFK48 HTS hits.