RESUMO
The protozoan protein kinase calcium-dependent protein kinase 1 (CDPK1) has emerged as a potential therapeutic target for the treatment of cryptosporidiosis. A focused screen of known kinase inhibitors identified a pyridopyrimidinone as a new chemotype of Cryptosporidium parvum (Cp) CDPK1 inhibitors. Structural comparison of CpCDPK1 to two representative human kinases, RIPK2 and Src, revealed differences in the positioning of the αC-helix that was used in the design of a potent pyridopyrimidinone-based CpCDPK1 inhibitor 7 (a.k.a. UH15-16, IC50 = 10â¯nM), which blocked the growth of three C. parvum strains (EC50 = 12-40â¯nM) as well as C. hominis (EC50 = 85â¯nM) in HCT-8 host cells. Pharmacokinetic and tissue distribution analyses indicated that 7 had low systemic exposure after oral administration, but high gastrointestinal concentration, as well as good Caco-2 cell permeability. Finally, 7 demonstrated partial efficacy in an IL-12 knock-out mouse model of acute cryptosporidiosis.
RESUMO
Toxoplasma gondii and Neospora caninum are major worldwide morbidity-causing pathogens. Bumped kinase inhibitors (BKIs) are a compound class that has been optimized to target the apicomplexan calcium-dependent protein kinase 1 (CDPK1) - and several members of this class have proven to be safe and highly active in vitro and in vivo. BKI-1708 is based on a 5-aminopyrazole-4-carboxamide scaffold, and exhibited in vitro IC50 values of 120 nM for T. gondii and 480 nM for N. caninum ß-galactosidase expressing strains, and did not affect human foreskin fibroblast (HFF) viability at concentrations up to 25 µM. Electron microscopy established that exposure of tachyzoite-infected fibroblasts to 2.5 µM BKI-1708 in vitro induced the formation of multinucleated schizont-like complexes (MNCs), characterized by continued nuclear division and harboring newly formed intracellular zoites that lack the outer plasma membrane. These zoites were unable to finalize cytokinesis to form infective tachyzoites. BKI-1708 did not affect zebrafish (Danio rerio) embryo development during the first 96 h following egg hatching at concentrations up to 2 µM. Treatments of mice with BKI-1708 at 20 mg/kg/day during five consecutive days resulted in drug plasma levels ranging from 0.14 to 4.95 µM. In vivo efficacy of BKI-1708 was evaluated by oral application of 20 mg/kg/day from day 9-13 of pregnancy in mice experimentally infected with N. caninum (NcSpain-7) tachyzoites or T. gondii (TgShSp1) oocysts. This resulted in significantly decreased cerebral parasite loads and reduced vertical transmission in both models without drug-induced pregnancy interference.
Assuntos
Coccidiose , Fibroblastos , Neospora , Pirazóis , Toxoplasma , Animais , Neospora/efeitos dos fármacos , Toxoplasma/efeitos dos fármacos , Camundongos , Coccidiose/tratamento farmacológico , Coccidiose/parasitologia , Pirazóis/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/parasitologia , Humanos , Antiprotozoários/farmacologia , Concentração Inibidora 50 , Toxoplasmose/tratamento farmacológico , Toxoplasmose/parasitologia , Modelos Animais de Doenças , Inibidores de Proteínas Quinases/farmacologia , Proteínas QuinasesRESUMO
Metastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Nonspecific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small-molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. We tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell-cycle, metabolic, and enzymatic assays were used to demonstrate their mechanism of action. A human patient-derived xenograft model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. We demonstrate a new class of small-molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. This work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer.