Optimizing Pyrazolopyrimidine Inhibitors of Calcium Dependent Protein Kinase 1 for Treatment of Acute and Chronic Toxoplasmosis.

Calcium dependent protein kinase 1 (CDPK1) is an essential Ser/Thr kinase that controls invasion and egress by the protozoan parasite Toxoplasma gondii. The Gly gatekeeper of CDPK1 makes it exquisitely sensitive to inhibition by small molecule 1H-pyrazolo[3,4-d]pyrimidine-4-amine (PP) compounds that are bulky ATP mimetics. Here we rationally designed, synthesized, and tested a series of novel PP analogs that were evaluated for inhibition of CDPK1 enzyme activity in vitro and parasite growth in cell culture. Optimal substitution on the PP scaffold included 2-pyridyl ethers directed into the hydrophobic pocket and small carbocyclic rings accessing the ribose-binding pocket. Further optimization of the series led to identification of the lead compound 3a that displayed excellent potency, selectivity, safety profile, and efficacy in vivo. The results of these studies provide a foundation for further work to optimize CDPK1 inhibitors for the treatment of acute and chronic toxoplasmosis.

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

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.

Full-length hepatitis B virus core protein packages viral and heterologous RNA with similarly high levels of cooperativity.

A critical feature of a viral life cycle is the ability to selectively package the viral genome. In vivo, phosphorylated hepatitis B virus (HBV) core protein specifically encapsidates a complex of pregenomic RNA (pgRNA) and viral polymerase; it has been suggested that packaging is specific for the complex. Here, we test the hypothesis that core protein has intrinsic specificity for pgRNA, independent of the polymerase. For these studies, we also evaluated the effect of core protein phosphorylation on assembly and RNA binding, using phosphorylated core protein and a phosphorylation mimic in which S155, S162, and S170 were mutated to glutamic acid. We have developed an in vitro system where capsids are disassembled and assembly-active core protein dimer is purified. With this protein, we have reassembled empty capsids and RNA-filled capsids. We found that core protein dimer bound and encapsidated both the HBV pregenomic RNA and heterologous RNA with high levels of cooperativity, irrespective of phosphorylation. In direct competition assays, no specificity for pregenomic RNA was observed. This suggests that another factor, such as the viral polymerase, is required for specific packaging. These results also beg the question of what prevents HBV core protein from assembling on nonviral RNA, preserving the protein for virus production.