Inhibition of Calcium Dependent Protein Kinase 1 (CDPK1) by Pyrazolopyrimidine Analogs Decreases Establishment and Reoccurrence of Central Nervous System Disease by Toxoplasma gondii

Abstract

Calcium dependent protein kinase 1 (CDPK1) is an essential enzyme in the opportunistic pathogen Toxoplasma gondii. CDPK1 controls multiple processes that are critical to the intracellular replicative cycle of T. gondii including secretion of adhesins, motility, invasion, and egress. Remarkably, CDPK1 contains a small glycine gatekeeper residue in the ATP binding pocket making it sensitive to ATP-competitive inhibitors with bulky substituents that complement this expanded binding pocket. Here we explored structure-activity relationships of a series of pyrazolopyrimidine inhibitors of CDPK1 with the goal of increasing selectivity over host enzymes, improving antiparasite potency, and improving metabolic stability. The resulting lead compound 24 exhibited excellent enzyme inhibition and selectivity for CDPK1 and potently inhibited parasite growth in vitro. Compound 24 was also effective at treating acute toxoplasmosis in the mouse, reducing dissemination to the central nervous system, and decreasing reactivation of chronic infection in severely immunocompromised mice. These findings provide proof of concept for the development of small molecule inhibitors of CDPK1 for treatment of CNS toxoplasmosis.

Toxoplasma Calcium-Dependent Protein Kinase 1 Inhibitors: Probing Activity and Resistance Using Cellular Thermal Shift Assays

“…1A. Several optimized inhibitors have now been used successfully in animal infection models (9,(13)(14)(15). We also demonstrated that CDPK1 with a methionine substitution for the gatekeeper residue (G128M) has higher 50% inhibitory concentrations (IC 50 s) for BKIs, and when parasites overexpress CDPK1(G128M), 50% effective concentrations (EC 50 s) are higher than those for parasites overexpressing the wild-type (WT) enzyme (5,12,16).…”

Treatment of Toxoplasmosis: Historical Perspective, Animal Models, and Current Clinical Practice

“…Recently, inhibitors of the calcium-dependent protein kinase 1 were found to penetrate the CNS and significantly reduced the number of brain cysts in acute and latent mouse models of T. gondii infection (237). Most importantly, treatment of gamma interferon (IFN-␥) receptor 1-deficient mice, which are normally extremely susceptible to infection, not only prolonged survival after reactivation but also cured some of the animals (238). It was hypothesized that interfering with tachyzoite invasion and egress contributed to the results, but a blockage of bradyzoite invasion in vitro suggested a direct effect on cyst formation and turnover (238).…”

Unpacking the Pathogen Box—An Open Source Tool for Fighting Neglected Tropical Disease

“…However, compound 185 was shown to have improved metabolic characteristics in addition to encouraging in vivo activity for the prevention and treatment of infection . A comprehensive follow‐up study identified compound 186 as an orally bioavailable agent which was able to decrease acute infections in immunocompetent and compromised animals in vivo, resulting in lower cyst counts in chronically infected animals . Related compounds MMV688364 ( 187 ) and MMV688469 ( 188 ) are currently unreported in the literature.…”