Biology subjects

Bhanot, P.

Publications and source records attributed to Bhanot, P..

5 recordsLinked to original sources

bioRxiv2026

AP2-L-Dependent Processes Promote PVM Maintenance and Plasmodium Liver-stage Development

Successful malaria infection requires transformation of Plasmodium sporozoites into replicative exo-erythrocytic forms (EEFs) within hepatocytes, a process requiring a functional parasitophorous vacuole membrane (PVM) and interactions with host cellular pathways. ApiAP2 transcription factors regulate developmental progression throughout the Plasmodium life cycle. AP2-L consists of an extended amino terminal region and two AP2 domains and is highly expressed in asexual stage trophozoites, sporozoites and early EEFs, but its role in these stages is poorly understood. We characterized the functional consequences of deleting the entire P. berghei ap2-l coding sequence on the erythrocytic cycle in vivo, sporozoite gene expression, liver-stage development, and hepatocyte-parasite interactions. AP2L-KO parasites maintained erythrocytic replication but sporozoite inoculation failed to establish blood-stage infection in mice. AP2-L loss decreased transcripts encoding proteins involved in sporozoite gliding motility, host-cell entry, and rhoptry secretion. Motif discovery upstream of a subset of these genes suggests possible direct targets of AP2-L. Infected HepG2 cells showed reduced EEF numbers and sizes, impaired parasite DNA replication, and abnormal parasite positioning relative to the host nucleus. PVMs of AP2L-KO parasites exhibited aberrant localization of proteins, absence of tubular vacuolar network extensions, and sensitivity to detergent. These abnormalities were accompanied by increased accumulation of the host's autophagy-associated protein around parasites, suggesting efficient host-mediated elimination. Additionally, AP2L-KO parasites displayed reduced association with the host microtubule-organizing center, Golgi compartments, and COPB2-positive vesicular structures, suggesting an impairment in uptake of nutrients. Together, these findings demonstrate that AP2-L directs both vacuolar biogenesis and host cytoskeletal/vesicular capture in liver-stage parasites.

microbiology↗

bioRxiv2021

Discovery of imidazole-based inhibitors of P. falciparum cGMP-dependent protein kinase

The discovery of new targets for treatment of malaria and in particular those aimed at the pre-erythrocytic stage in the life cycle, advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, this manuscript presents initial structure-activity relationships in an imidazole scaffold at four positions, representative in vitro ADME, hERG characterization and cell-based anti-parasitic activity. This series of PfPKG inhibitors has good in vitro PfPKG potency, low hERG activity and cell-based anti-parasitic activity against multiple Plasmodium species that appears to correlate with in vitro potency.

pharmacology and toxicology↗

bioRxiv2021

Discovery of novel potent, cell-permeable inhibitors of P. falciparum cGMP-dependent protein kinase.

The discovery of new targets for treatment of malaria advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, we recently reported the discovery and optimization of novel, potent isoxazole-based PfPKG inhibitors that lacked any obvious safety warnings. This manuscript presents representative in vitro ADME, hERG characterization and cell-based antiparasitic activity of these PfPKG inhibitors. We also report the discovery and structure-activity relationships of a new series with good potency, low hERG activity and cell-based anti-parasitic activity comparable to a literature standard.

pharmacology and toxicology↗

bioRxiv2021

Characterization of novel competitive inhibitors of P. falciparum cGMP-dependent protein kinase

P. falciparum cGMP-dependent protein kinase (PfPKG) is an enticing anti-malarial drug target. Structurally novel isoxazole-based compounds were shown to be ATP competitive inhibitors of PfPKG. Isoxazoles 3 and 5 had Ki values of 0.7 {+/-} 0.2 and 2.3 {+/-} 0.9 nM, respectively, that are comparable to a known standard, 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine (1.4 {+/-} 0.5 nM). They also exhibited excellent selectivity for PfPKG over the human ortholog and the gatekeeper mutant T618Q PfPKG, which mimics the less accessible binding site of the human ortholog. The human orthologs larger binding site volume was predicted to explain the selectivity of the inhibitors for the P. falciparum enzyme. Analogs 4 and 6 were at least 20-fold less potent compared to 3 and 5, suggesting that removing the carbonyl group in 3 or altering the diethylamino moiety in 5 reduced affinity.

biochemistry↗

bioRxiv2019

Overlapping and distinct roles of CDPK family members in the pre-erythrocytic cycle of the rodent malaria parasite, Plasmodium berghei

Invasion of, development within, and exit from hepatocytes by Plasmodium is essential for the parasite to establish the malaria-causing erythrocytic cycle. Identification of signaling pathways that operate during the pre-erythrocytic cycle provides insights into a critical stage of infection and potential targets for chemoprevention of disease. Calcium Dependent Protein Kinases (CDPK) represent a kinase family that is present in Plasmodium but absent in mammals. We demonstrate that P. berghei homologs of CDPK1, CDPK4 and CDPK5 play overlapping but distinct roles in sporozoite invasion and parasite egress from hepatocytes. All three kinases are expressed in sporozoites. All three are required for optimal motility of sporozoites and consequently their invasion of hepatocytes. Increased cGMP compensates for the functional loss of CDPK1 and CDPK5 during sporozoite invasion but cannot overcome CDPK4s loss. CDPK1 and CDPK5 expression is downregulated after sporozoite invasion. CDPK5 reappears in a subset of late stage liver stages and is present in all merosomes. Chemical inhibition of CDPK4 and depletion of CDPK5 in liver stages suggests that these kinases play a role in the formation and/or release of merosomes from mature liver stages. Furthermore, depletion of CDPK5 in merosomes significantly delays a merosome-initiated erythrocytic cycle without affecting the infectivity of hepatic merozoites. These data suggest that CDPK5 is required for the release of hepatic merozoites from merosomes. Our work provides the first evidence that sporozoite invasion requires CDPK1 and CDPK5 and that the release of hepatic merozoites is a regulated process.\n\nSignificanceThe malaria-parasite Plasmodium begins its mammalian cycle by infecting hepatocytes in the liver. A single parasite differentiates into tens of thousands of hepatic merozoites which exit the host cell in vesicles called merosomes. Hepatic merozoites initiate the first round of erythrocytic infection that leads to disease symptoms. We show that optimal invasion of liver cells by Plasmodium requires the action of three closely-related parasite kinases, CDPK1, 4 and 5. Loss of any of the three enzymes in the parasite significantly reduces infection of liver cells. CDPK5 is also required for the release of hepatic merozoites from merosomes and therefore for initiating the erythrocytic cycle. A better understanding of how these kinases function could lead to drugs that prevent malaria.

microbiology↗