Targeting Artemisinin-Resistant Malaria by Repurposing the Anti-Hepatitis C Virus Drug Alisporivir.

The emergence of Plasmodium falciparum resistance raises an urgent need to find new antimalarial drugs. Here, we report the rational repurposing of the anti-hepatitis C virus drug, alisporivir, a nonimmunosuppressive analog of cyclosporin A, against artemisinin-resistant strains of P. falciparum. In silico docking studies and molecular dynamic simulation predicted strong interaction of alisporivir with PfCyclophilin 19B, confirmed through biophysical assays with a Kd value of 354.3 nM. Alisporivir showed potent antimalarial activity against chloroquine-resistant (PfRKL-9 with resistance index [Ri] 2.14 ± 0.23) and artemisinin-resistant (PfKelch13R539T with Ri 1.15 ± 0.04) parasites. The Ri is defined as the ratio between the IC50 values of the resistant line to that of the sensitive line. To further investigate the mechanism involved, we analyzed the expression level of PfCyclophilin 19B in artemisinin-resistant P. falciparum (PfKelch13R539T). Semiquantitative real-time transcript, Western blot, and immunofluorescence analyses confirmed the overexpression of PfCyclophilin 19B in PfKelch13R539T. A 50% inhibitory concentration in the nanomolar range, together with the targeting of PfCyclophilin 19B, suggests that alisporivir can be used in combination with artemisinin. Since artemisinin resistance slows the clearance of ring-stage parasites, we performed a ring survival assay on artemisinin-resistant strain PfKelch13R539T and found significant decrease in parasite survival with alisporivir. Alisporivir was found to act synergistically with dihydroartemisinin and increase its efficacy. Furthermore, alisporivir exhibited antimalarial activity in vivo. Altogether, with the rational target-based Repurposing of alisporivir against malaria, our results support the hypothesis that targeting resistance mechanisms is a viable approach toward dealing with drug-resistant parasite.

Targeting PfProhibitin 2-Hu-Hsp70A1A complex as a unique approach towards malaria vaccine development.

Malaria parasite invasion to host erythrocytes is mediated by multiple interactions between merozoite ligands and erythrocyte receptors that contribute toward the development of disease pathology. Here, we report a novel antigen Plasmodium prohibitin "PfPHB2" and identify its cognate partner "Hsp70A1A" in host erythrocyte that plays a crucial role in mediating host-parasite interaction during merozoite invasion. Using small interfering RNA (siRNA)- and glucosamine-6-phosphate riboswitch (glmS) ribozyme-mediated approach, we show that loss of Hsp70A1A in red blood cells (RBCs) or PfPHB2 in infected red blood cells (iRBCs), respectively, inhibit PfPHB2-Hsp70A1A interaction leading to invasion inhibition. Antibodies targeting PfPHB2 and monoclonal antibody therapeutics against Hsp70A1A efficiently block parasite invasion. Recombinant PfPHB2 binds to RBCs which is inhibited by anti-PfPHB2 antibody and monoclonal antibody against Hsp70A1A. The validation of PfPHB2 to serve as antigen is further supported by detection of anti-PfPHB2 antibody in patient sera. Overall, this study proposes PfPHB2 as vaccine candidate and highlights the use of monoclonal antibody therapeutics for future malaria treatment.

Characterization of Plasmodium falciparum prohibitins as novel targets to block infection in humans by impairing the growth and transmission of the parasite.

Prohibitins (PHBs) are highly conserved pleiotropic proteins as they have been shown to mediate key cellular functions. Here, we characterize PHBs encoding putative genes ofPlasmodium falciparum by exploiting different orthologous models. We demonstrated that PfPHB1 (PF3D7_0829200) and PfPHB2 (PF3D7_1014700) are expressed in asexual and sexual blood stages of the parasite. Immunostaining indicated hese proteins as mitochondrial residents as they were found to be localized as branched structures. We further validated PfPHBs as organellar proteins residing in Plasmodium mitochondria, where they interact with each other. Functional characterization was done in Saccharomyces cerevisiae orthologous model by expressing PfPHB1 and PfPHB2 in cells harboring respective mutants. The PfPHBs functionally complemented the yeast PHB1 and PHB2 mutants, where the proteins were found to be involved in stabilizing the mitochondrial DNA, retaining mitochondrial integrity and rescuing yeast cell growth. Further, Rocaglamide (Roc-A), a known inhibitor of PHBs and anti-cancerous agent, was tested against PfPHBs and as an antimalarial. Roc-A treatment retarded the growth of PHB1, PHB2, and ethidium bromide petite yeast mutants. Moreover, Roc-A inhibited growth of yeast PHBs mutants that were functionally complemented with PfPHBs, validating P. falciparum PHBs as one of the molecular targets for Roc-A. Roc-A treatment led to growth inhibition of artemisinin-sensitive (3D7), artemisinin-resistant (R539T) and chloroquine-resistant (RKL-9) parasites in nanomolar ranges. The compound was able to retard gametocyte and oocyst growth with significant morphological aberrations. Based on our findings, we propose the presence of functional mitochondrial PfPHB1 and PfPHB2 in P. falciparum and their druggability to block parasite growth.

Host SUMOylation Pathway Negatively Regulates Protective Immune Responses and Promotes Leishmania donovani Survival.

SUMOylation is one of the post-translational modifications that have recently been described as a key regulator of various cellular, nuclear, metabolic, and immunological processes. The process of SUMOylation involves the modification of one or more lysine residues of target proteins by conjugation of a ubiquitin-like, small polypeptide known as SUMO for their degradation, stability, transcriptional regulation, cellular localization, and transport. Herein, for the first time, we report the involvement of the host SUMOylation pathway in the process of infection of Leishmania donovani, a causative agent of visceral leishmaniasis. Our data revealed that infection of L. donovani to the host macrophages leads to upregulation of SUMOylation pathway genes and downregulation of a deSUMOylating gene, SENP1. Further, to confirm the effect of the host SUMOylation on the growth of Leishmania, the genes associated with the SUMOylation pathway were silenced and parasite load was analyzed. The knockdown of the SUMOylation pathway led to a reduction in parasitic load, suggesting the role of the host SUMOylation pathway in the disease progression and parasite survival. Owing to the effect of the SUMOylation pathway in autophagy, we further investigated the status of host autophagy to gain mechanistic insights into how SUMOylation mediates the regulation of growth of L. donovani. Knockdown of genes of host SUMOylation pathway led to the reduction of the expression levels of host autophagy markers while promoting autophagosome-lysosome fusion, suggesting SUMOylation-mediated autophagy in terms of autophagy initiation and autophagy maturation during parasite survival. The levels of reactive oxygen species (ROS) generation, nitric oxide (NO) production, and pro-inflammatory cytokines were also elevated upon the knockdown of genes of the host SUMOylation pathway during L. donovani infection. This indicates the involvement of the SUMOylation pathway in the modulation of protective immune responses and thus favoring parasite survival. Taken together, the results of this study indicate the hijacking of the host SUMOylation pathway by L. donovani toward the suppression of host immune responses and facilitation of host autophagy to potentially facilitate its survival. Targeting of SUMOylation pathway can provide a starting point for the design and development of novel therapeutic interventions to combat leishmaniasis.

Pathogen induced subversion of NAD+ metabolism mediating host cell death: a target for development of chemotherapeutics.

Hijacking of host metabolic status by a pathogen for its regulated dissemination from the host is prerequisite for the propagation of infection. M. tuberculosis secretes an NAD+-glycohydrolase, TNT, to induce host necroptosis by hydrolyzing Nicotinamide adenine dinucleotide (NAD+). Herein, we expressed TNT in macrophages and erythrocytes; the host cells for M. tuberculosis and the malaria parasite respectively, and found that it reduced the NAD+ levels and thereby induced necroptosis and eryptosis resulting in premature dissemination of pathogen. Targeting TNT in M. tuberculosis or induced eryptosis in malaria parasite interferes with pathogen dissemination and reduction in the propagation of infection. Building upon our discovery that inhibition of pathogen-mediated host NAD+ modulation is a way forward for regulation of infection, we synthesized and screened some novel compounds that showed inhibition of NAD+-glycohydrolase activity and pathogen infection in the nanomolar range. Overall this study highlights the fundamental importance of pathogen-mediated modulation of host NAD+ homeostasis for its infection propagation and novel inhibitors as leads for host-targeted therapeutics.