Inhibition of the assembly of Plasmodium Hsp70-1 and Hsp40 complex blocks DNA replication by destabilizing ribonucleotide reductase subunit-2.

Plasmodium falciparum undergoes endoreduplication, in which its nuclear, mitochondrial, and apicoplast genomes are replicated multiple times without cytokinesis,; enabling replication as a promising target for arresting parasite growth and pathogenicity. Previously, it was reported that the inhibition of expression of small subunit of ribonucleotide reductase (PfR2) leads to the inhibition of DNA synthesis and growth of the parasites. Here, we report the molecular determinant that is necessary for aiding early folding intermediates of PfR2. We find that PfR2 interacts with the cytosolic Type-I Hsp40-cochaperone PfYdj1 (Pf3D7_1437900), which subsequently interacts with PfHsp70-1 through its Histidine-Proline-Aspartate (HPD) motif. Such association of PfR2 is specific toward PfYdj1 and not with other cytosolic Type II Hsp40-cochaperone PfSis1 (PF3D7_0213100). We show that perturbation of association between PfYdj1 and PfHsp70-1 by small molecule 116-9e results in the disruption of PfR2 homeostasis within the parasite. As a result, there is a significant reduction in dNTP production leading to a replication arrest. We demonstrate that the combination of 116-9e and benzo-hydroxamate, a catalytic inhibitor of PfR2, works in a synergistic fashion. Together, our work suggests that targeting PfYdj1-PfHsp70-1 complex assembly in the context of ribonucleotide reductase function can be used as an attractive strategy to curb malaria.IMPORTANCERibonucleotide reductase is an important enzyme which catalyzes the reduction of ribonucleotides to deoxyribonucleotides, and inhibition of the synthesis of its catalytic subunit (PfR2) leads to significant growth inhibition in malaria parasites. Our study deciphers the molecular determinants that are essential for the maturation of PfR2. We show that Plasmodium Hsp40 cochaperone, PfYdj1 (Pf3D7_1437900), is the molecular cochaperone that facilitates the binding of PfR2 with PfHsp70-1, which is crucial for PfR2 folding and stability. We show that perturbation of the assembly between PfYdj1-PfHsp70-1 by a small molecule 116-9e destabilizes PfR2 and subsequently inhibits dNTP formation, resulting in replication arrest in the parasite. We demonstrate that 116-9e and the catalytic inhibitor of PfR2, benzo hydroxamate, potentiate each other's action. Also, the combination of both the inhibitors displays profound synergism in 3D7 parasites. We propose that the combination of 116-9e and benzo hydroxamate can be employed as an attractive anti-malaria strategy.