Biochemical and Antiparasitic Properties of Inhibitors of the Plasmodium falciparum Calcium-Dependent Protein Kinase PfCDPK1

Abstract

PfCDPK1 is a Plasmodium falciparum calcium-dependent protein kinase, which has been identified as a potential target for novel antimalarial chemotherapeutics. In order to further investigate the role of PfCDPK1, we established a high-throughput in vitro biochemical assay and used it to screen a library of over 35,000 small molecules. Five chemical series of inhibitors were initially identified from the screen, from which series 1 and 2 were selected for chemical optimization. Indicative of their mechanism of action, enzyme inhibition by these compounds was found to be sensitive to both the ATP concentration and substitution of the amino acid residue present at the “gatekeeper” position at the ATP-binding site of the enzyme. Medicinal chemistry efforts led to a series of PfCDPK1 inhibitors with 50% inhibitory concentrations (IC50s) below 10 nM against PfCDPK1 in a biochemical assay and 50% effective concentrations (EC50s) less than 100 nM for inhibition of parasite growth in vitro. Potent inhibition was combined with acceptable absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties and equipotent inhibition of Plasmodium vivax CDPK1. However, we were unable to correlate biochemical inhibition with parasite growth inhibition for this series overall. Inhibition of Plasmodium berghei CDPK1 correlated well with PfCDPK1 inhibition, enabling progression of a set of compounds to in vivo evaluation in the P. berghei rodent model for malaria. These chemical series have potential for further development as inhibitors of CDPK1.

Applying chemical genetic tools to the study of phospho-signalling pathways in malaria parasites

“…Although a number of small molecule screens have identified pharmacological inhibitors against members of this kinase family [10,42,43], chemical genetics has played a prominent role in providing insights into the functional role of the CDPKs ( Table 1). Several of these chemical genetic studies have exploited the fact that the CDPK1 in T. gondii has a glycine gatekeeper residue.…”

Designing selective inhibitors for calcium-dependent protein kinases in apicomplexans

“…The subset of small gatekeeper-containing CDPKs includes PfCDPK1 (Thr), TgCDPK1 (Gly), CpCDPK1 (Gly) and PfCDPK4 (Ser) (Figure 1B). The first three have been the targets of a significant portion of medicinal chemistry efforts invested on parasite kinases 74-80 .…”

“…One of the most potent of these called purfalcamine blocked late stage schizogony 93 , although the specific target of this inhibitor in the parasite remains uncertain. Using similar in vitro enzyme assays for small molecule screens, a number of other chemical series have emerged as potential inhibitors of PfCDPK1, including pyrazolopyrimidines, azabenzimididazoles, and imidazopyridazines 74, 75, 77 . The small Thr gatekeeper of PfCDPK1 was important in its susceptibility to these inhibitors in vitro ; however, the potency of enzyme inhibition in vitro was not correlated with inhibition of parasite growth in red blood cells 74 .…”

“…Using similar in vitro enzyme assays for small molecule screens, a number of other chemical series have emerged as potential inhibitors of PfCDPK1, including pyrazolopyrimidines, azabenzimididazoles, and imidazopyridazines 74, 75, 77 . The small Thr gatekeeper of PfCDPK1 was important in its susceptibility to these inhibitors in vitro ; however, the potency of enzyme inhibition in vitro was not correlated with inhibition of parasite growth in red blood cells 74 . Furthermore, the imidazopyridazine series was independently identified in a whole cell screen using a kinase inhibitor library, with specific analogs found to be effective in vivo 94 .…”

“…Furthermore, the imidazopyridazine series was independently identified in a whole cell screen using a kinase inhibitor library, with specific analogs found to be effective in vivo 94 . Synthesis of additional imidazopyridazine analogs was used to improve their ADME properties, for example by reducing Log P (Log D), enhancing microsome stability, and improving selectivity over human kinases 74-77 . Despite these improvements, treatment of mice infected with P. berghei with some of the most potent derivatives only resulted in modest efficacy, despite achieving what should have been therapeutic serum levels 95 .…”