▶ Development of new drugs/drug combinations as therapeutic interventions for malaria, leishmaniasis and filariasis;
▶ Identification of unique targets and pathways for future interventions;
▶ Investigations on parasite biology and host-parasite interactions.

1.1.1 Organellar base excision repair endonuclease with restricted enzymatic functions Apicoplast and mitochondria of the malaria parasite are sites of active protein synthesis but exhibit differences in ribosome composition and translation factor requirement in comparison with organelles of other eukaryotes. Plasmodium mitochondria have highly fragmented rRNA and are predicted to carry a reduced ribosomal protein repertoire. We have recently identified two GTPases (EngA and Obg1) and a dimethyltransferase (KsgA1) with a role in biogenesis of parasite mitochondrion. This was extended to invetatigate another important GTPase identified by us-- PfYihA, that has three nuclear-encoded homologs in P. falciparum. Immunofluorescence localization assays using specific antibodies generated against the three proteins revealed that PfYihA1 targeted to the parasite apicoplast, PfYihA2 to the mitochondrion, and PfYihA3 was found in both the apicoplast and cytosol suggesting their roles in biogenesis of organellar and cytosolic ribosomes. The three recombinant PfYihA proteins were active GTPases and interacted with surrogate E. coli ribosomes in a nucleotide-independent manner. In vivo complexation of PfYihA with organellar and/or cytosolic LSU was confirmed by co-immunoprecipitation. Mitochondrial PfYihA2 carries a large C-ter extension with a strongly positively charged stretch. We hypothesise that this is important in compensating for the absence of helices 80-88 of the central protuberance in the fragmented rRNA of Plasmodium mitoribosomes and may provide additional contact sites to aid in complex assembly. Our results indicate that P. falciparum mitochondria assemble ribosomes with the aid of PfEngA, PfObg1 and PfYihA2 GTPases while apicoplast ribosomes use PfYihA1 and 3 with additional involvement of a putative apicoplast PfRbgA GTPase homolog (Parasitology 2018, PMID: 29642957).

1.1.2 Organellar base excision repair endonuclease with restricted enzymatic functions Organellar base excision repair endonuclease with restricted enzymatic functions

We explored DNA repair mechanism(s) operative within the apicoplast and mitochondrion of P. falciparum by mining its nuclear genome for sequences encoding proteins of major DNA repair pathways with predicted targeting to either organelle. Of the panel of enzymes identified for base excision repair (BER), we characterized the apurinic/apyrimidinic (AP) endonuclease PfApn1- an EndoIV whose homolog is not known in humans. PfApn1 targeted to the mitochondrion and functioned as an AP-endonuclease requiring both Zn2+ and Mn2+ ions for maximal activity. Mutation of the critical third metal-binding site residue H542 resulted in loss of Mn2+ (but not Zn2+) binding indicating that Mn2+ bound PfApn1 at this site; this was further supported by molecular dynamic simulation. CD spectra analysis further showed requirement of both metal ions for attainment of PfApn1 β-strand rich optimal conformation. PfApn1 also functioned as a 3′-phosphatase that would enable removal of 3′-blocks for DNA polymerase activity during BER. Interestingly, unlike E. coli and yeast EndoIV homologs, PfApn1 lacked 3′-5′ exonuclease activity and also did not cleave damaged bases by nucleotide incision repair (NIR). Uncoupling of endonuclease/phosphatase and exonuclease/NIR in PfApn1 suggested that amino acid residues distinct from those critical for endonuclease function are required for exonuclease activity and NIR. Characterization of a critical mitochondrion-targeted AP endonuclease provided evidence for a functional BER pathway in the parasite organelle (FEBS Journal 2019, PMID: 31386260).

Fig. 1: Ectopic expression of Pf-RBR-E3 ligase (FLAG-tagged) alongwith HA-Ub constructs in mammalian cells and immunoprecipitation with FLAG antibody and immunoblotting with HA antibody. * denotes the non-specific signal from heavy chain of antibody

1.1.3 Understanding the role of RBR-E3 Ubiquitin ligase in Human malaria parasite

Human malaria parasite (P. falciparum) has evolved versatile protein quality control (PQC) system which includes the protein folding and degradation machinery. Interestingly, the components of PQC machinery have diverged significantly from their human orthologs and have acquired potential moonlighting roles which gives survival advantage to the parasite (Anas et al, 2019, Cell Stress and Chaperones). Our lab is investigating the functions of different components of protein folding and degradation machinery. One of the critical component which facilitates efficient removal of misfolded or aggregated species is E3 ligase family which selectively ubiquitinates protein substrates for various cellular activities. Amongst different E3 ligases, we found that parasite has only one Ring-between-Ring (RBR) E3 ligase which is distinct from human ortholog with additional interfaces which may potentially expand its repertoire of interaction. Our immunoprecipitation experiment revealed that parasite’s RBR-E3 ligase catalyzes both K63 and K48 mediated ubiquitination suggestive of its involvement in diverse cellular processes. Ongoing experiments include mutagenesis experiments to identify catalytic cysteines involved in ubiquitination and immunoprecipitation to identify the substrates or protein interactors of RBR-E3 ligase in parasite.

1.1.4 Protein kinases as potentially novel drug targets against malaria

In order to develop novel strategies against malaria, protein kinases may serve as promising drug targets. We created knockouts of two protein kinases in Plasmodium berghei, PKAc and STK2. We found that PKAc is indispensable in malaria blood stages. Due to the essentiality of PKAc in erythrocytic stages, conditional knockout was created by disrupting the PKAc locus in sporozoites using Flp/FRT conditional mutagenesis system. We found that PKAc cKO sporozoites were able to glide, invade hepatocytes, and mature into hepatic schizonts which developed successfully into merosomes (Fig.2A-C) However, these failed to initiate blood stage infection when injected in mice (Fig.2D) (Choudhary et al., 2019)

Fig. (A) Gliding motility of PKAc cKO sporozoites was similar to the gliding activity observed for TRAP/FlpL parasites. Scale bar, 25 μm. (B) Bar graph showing percent sporozoites inside versus outside. (C) PKAc cKO merosomes show normal loss of the PV membrane and normal segregation of merozoite membranes. Scale bar, 5 μm. (D) PKAc cKO merosomes have impaired infectivity in mice. Swiss mice were inoculated i.v. with the indicated number of merosomes with or without mechanical rupture (mr).

STK2 was dispensable in malaria blood stages and STK2 KO parasites completed normal mosquito stage development. STK2 KO sporozoites were able to successfully invade hepatocytes and developed into EEFs and remain indistinguishable from wild-type until 48 h.p.i. Late EEFs harvested at 62 h.p.i.) showed significant reduction in merozoite development as assessed by MSP1 staining and nuclei count (Fig. 3 A and B). Impairment in merozoites formation in STK KO parasites led to delay in appearance of parasites in blood stages (Fig. 3C) (Jillapalli et al., 2019). .

Fig. 3. (A) In vitro EEF development at 62 h.p.i. revealed significantly reduced MSP1 expression in STK KO parasites. Scale bars: 5 μm. (B) Quantification of hepatic merozoite nuclei. (C) Analysis of pre-patent period in STK KO parasites.
References:
Choudhary HH, Gupta R, Mishra S. PKAc is not required for the preerythrocytic stages of Plasmodium berghei. Life Science Alliance. 2019; 2: 1-11.

Jillapalli R, Narwal SK, Kolli SK, Mastan BS, Reddy SR, Srivastava PN, Mishra S, Kumar KA. A Plasmodium berghei serine-threonine kinase PBANKA_0311400 is required for late liver stage development and timely initiation of blood stage infection. Biology Open. 2019; 8: bio042028.

1.1.5 Deciphering the role of toll-interacting protein during malaria infection

Plasmodium parasite has evolved intricate mechanisms to avoid the development of TLR-mediated effector immune responses. Toll-interacting protein (Tollip), a negative regulator of TLR-signaling plays a prominent role in pathogenesis in visceral leishmaniasis; though, its role during Plasmodium infection has yet not been explored. We investigated the role of Tollip in Plasmodium yoelii MDR (1×106) infected Balb/c mice on days 3-7 post-infection (having 5-40 % blood parasitemia, respectively). In our results, we have witnessed that the level of Tollip increases at early time points and then goes down and again flares up at later stage when parasitemia was 40% (Fig. 4). This data suggests the dual role of Tollip during early and late malaria infection. During early infection, Tollip regulates excess TLR activation and might help in parasite multiplication. However, at later stage, when pro-inflammatory cytokines up-regulate, Tollip again flares up to control the high inflammation and might play host-protective role. Further, the effect of Tollip expression on cytokine response, parasitemia level and host survival is being validated using Tollip-knockdown mice.

Fig. Expression status of Tollip at different parasitemia

1.1.6 Exploration of antimalarial potential of existing therapeutics

Various existing drugs (anticancer drugs, immunomodulator and antibiotics) were evaluated for their in vitro activity against human malaria parasite Plasmodium falciparum 3D7 (chloroquine-sensitive strain) and K1 (chloroquine-resistant strain). The IC50 values of selected drugs are reported in the fig. 5. Amongst all tested drugs, 3 drugs (5-Florouracil, Imiquimod and Moxifloxacin) from each group were selected for further screening in in vivo system on the basis of their promising in vitro activity. In Plasmodium yoelii infected swiss mice, these three drugs showed approximately 82-97% parasitemia suppression along with increased host mean survival time as compared to untreated group (fig. below).

1.2 Leishmaniasis

1.2.1 Synthesis, Biological Evaluation, Structure−Activity Relationship and Mechanism of Action Studies of Quinoline-metronidazole Derivatives against Experimental Visceral Leishmaniasis

To identify novel chemical scaffolds for the development of antileishmanial agents, a series of quinoline-metronidazole was synthesized and tested against murine model of visceral leishmaniasis. Among all synthesized derivatives, 15b and 15i showed promising antileishmanial efficacy against both extracellular promastigote (IC50 9.54 µM and 5.42 µM respectively) and intracellular amastigote (IC50 9.81 µM and 3.75 µM respectively) form of L. donovani with negligible cytotoxicity towards host (J774 macrophages, Vero cells). However compound 15i showed better in vivo efficacy and effectively eliminated spleen and liver parasite burden (>80 %) in BALB/c mice model of VL. Mechanistic studies revealed that 15i triggers oxidative stress, induces bioenergetic collapse and apoptosis of the parasite by depleting ATP production and loss in mitochondrial membrane potential. Structure−activity analyses and pharmacokinetic studies revealed 15i as a promising antileishmanial lead and suggests quinoline-metronidazole series as a suitable platform for future development of antileishmanial agents. (J Med Chem. (2019) 62(11):5655-5671)

1.2.2 Differential induction of SOCS isoforms by Leishmania donovani impairs macrophage- T cell crosstalk and host defense

Immune evasion strategies adopted by Leishmania donovani involve the exploitation of SOCS proteins that are well-known negative regulators of the JAK/STAT pathway. However, the cellular mechanism underpinning the induction of SOCS isoforms and their role in breaching the multilevel regulatory circuit connecting the innate and adaptive arms of immunity are still ambiguous during experimental visceral leishmaniasis. Using bone marrow macrophages (BMMфs) and CD 4+ T cells, we observed that L. donovani preferentially upregulates SOCS1 and SOCS3 expression in macrophages and T cells respectively, while SOCS1 level remains consistently high in BMMфs, SOCS3 expression is pronounced and long-lasting in T cells. Consequently, this inhibits STAT1 mediated IL-12 induction in macrophages & STAT4 mediated IFN-γ synthesis in T cells. Mechanistically, PI3K/Akt-mediated SRF activation promotes nuclear translocation and binding of Egr2 to SOCS1 promoter for its early induction in infected BMMфs. Additionally, L. donovani activates

IDO/kynurenine/AHR signaling in BMMфs in order to maintain prolonged SOCS1 expression. Later, prostaglandin E2, secreted from infected BMMфs induces cAMP-PKA pathway by binding to the EP2/EP4 receptor of CD 4+ T cells, leading to SP1, CREB and GATA1 activation and SOCS3 expression. SiRNA-mediated silencing of SOCS1 and SOCS3 in macrophage and T cells respectively restored IL-12 and IFN-γ cytokine levels and BMMфs-T cell interaction. Vivo morpholino-mediated silencing of SOCS1 and SOCS3 resulted in protective cytokine responses, thereby reducing organ parasite burden significantly in L. donovani-infected Balb/c mice. Collectively, our results imply that L. donovani orchestrates different SOCS isoforms to impair macrophage-T cell crosstalk and preserve its own niche. (J Immunol, 2019, PMID: 31882519)

1.2.3 Effect of overexpression of of LdMAPK1 on Leishmania proteome:

Mitogen-activated protein kinases (MAPKs) are well-known mediators of signal transduction of eukaryotes, regulating important processes, like proliferation, differentiation, stress response, and apoptosis. In Leishmania, MAPK1 plays various roles in regulating the critical cellular activities like parasite survival, infectivity and drug resistance. Earlier, we have shown that LdMAPK1 modulates antimony susceptibility by downregulating P-glycoprotein (P-gp) efflux pump and plays a vital role in the post-translational modification and possibly the regulation of heat shock proteins. With an aim to identify LdMAPK1 modulated phosphoproteins by comparative phosphoproteomics analysis of wild type (Dd8+/+), LdMAPK1 over-expressing (Dd8++/++) and LdMAPK1 single deletion (Dd8+/-) mutant parasites, iTRAQ labeled quantitative analysis was carried out. Biological triplicates were run on orbitrap fusion and the protein search was performed against L. donovani database down loaded from Uniprot using Proteome Discoverer 2.2 software. Comparatively, iTRAQ labeling based quantitative analysis identified 420, 512, 320 phosphopeptides for 210, 255 and 142 phosphoproteins in biological triplicates of wild type (Dd8+/+), LdMAPK1 over-expressing (Dd8++/++) and single deletion (Dd8+/-) mutant parasites respectively. Out of these, only 8 phosphoproteins namely, acetyl-coenzyme A synthetase, heat shock protein 83-1, serine/threonine-protein phosphatase, elongation factor 1-alpha, nucleolar protein, and 3 uncharacterized protein exhibited ˃1.5 fold upregulation in LdMAPK1 overexpressing parasites while 7 proteins, (eukaryotic translation initiation factor 5a, eukaryotic release factor-3, glyceraldehydes-3-phosphate dehydrogenase, enolase, heat shock protein 70 and two ribonucleoside diphosphate reductase small chain proteins showed >1.6 fold down-regulation. However, except one (eukaryotic translation initiation factor 5a), none of the either up or down regulated proteins, in overexpressing parasites, exhibited differential expression in single deletion mutant parasites. The study suggests that LdMAPK1 over-expression modulates the expression levels of phosphoproteins related to diverse pathways mostly related to metabolism, signal transduction, translation and molecular chaperones.

▶ Design and synthesis of novel molecules as potential parasiticidal agents;
▶ Bioevaluation of synthetic molecules and natural products for antimalarial, antileishmanial and antifilarial activities against in vitro and in vivo models;
▶ Preclinical development of combination therapy regimens with novel compounds/known drugs;
▶ Mechanism of drug action / drug resistance;
▶ Characterization of drug targets using molecular approaches;
▶ Development of immunoprophylactic modalities;
▶ Development of improved screening models/drug delivery systems.
   ☑ Malaria
   ☑ Leishmaniasis
   ☑ Filariasis

As drug target: T-complex polypeptide-1 (TCP1), a group II chaperonin class of protein (HSP60 family) is involved in intracellular assembly and folding of various proteins in eukaryotes. In Leishmania, only the TCP1 subunit has been cloned and characterized from our lab. It forms homo-oligomeric complex and exhibited chaperonin activities. In the present study, we evaluated the essentiality of LdTCP1γ gene using both molecular and chemical validation strategies. Gene replacement studies indicate that LdTCP1γ is essential for parasite survival as efforts to generate null mutant failed and single-allele replacement mutants exhibited retarded growth and decreased infectivity in mouse macrophages compared to wild-type parasites. Modulation of LdTCP1γ expression in promastigotes also modulated cell cycle progression. Suramin, initially developed as a treatment for human African sleeping sickness, exhibited significant inhibition of LdTCP1γ refolding activity and multiplication of amastigotes with low toxicity to mammalian cells. The interaction of suramin with LdTCP1γ was observed both by isothermal titration calorimetry and computational molecular docking studies. The study suggests that LdTCP1γ is an essential gene, hence a potential drug target. It also provides a framework for the development of a new class of drugs.

As immunogen: Treatment of VL is associated with the generation of Th1 type of cellular response and antigens that are involved in Th1 stimulation are considered as a suitable vaccine candidate. Interestingly, the recombinant protein, LdTCP1γ was found to be potent immunogenic in nature as it exhibited strong Th1 type response. It exhibited strong LTT response along with significant NO and ROS production in cure hamsters as compared to untreated infected controls. The study suggested that LdTCPγ is a promising molecule which has to be further evaluated for its prophylactic efficacy.

Peptidase from parasite origin are becoming important as vaccine candidate, among them cell surface metallopeptidase and lysosomal cysteine peptidase have shown immunoprophylactic activity. From our lab, dipeptidylcarboxypeptidase (LdDCP) a zinc metallopeptidase has been reported as a potent drug target. In the present study, LdDCP was evaluated for its immunogenicity in cured hamster by XTT, NO and RO production. The study suggested that LdDCP has potential of developing as vaccine candidate against VL infection

RNA editing is a unique posttranscriptional modification of mitochondrial mRNAs that is shared in all trypanosomatid pathogens. Modification of specific editing sites, dictated by complementary guide RNAs (gRNAs), constitutes essential steps to ensure the production of translatable mRNAs that encode essential components of the mitochondrial oxidative phosphorylation system which is indispensible for survival of Leishmania parasite inside host. One of the important components of RNA editing is RNA editing ligase 1 (REL1). We aim to characterize RNA editing pathway, particularly the enzyme, REL1 of L. donovani (LdREL1) in the context of parasite survival and infectivity.

Peptidase from parasite origin are becoming important as vaccine candidate, among them cell surface metallopeptidase and lysosomal cysteine peptidase have shown immunoprophylactic activity. From our lab, dipeptidylcarboxypeptidase (LdDCP) a zinc metallopeptidase has been reported as a potent drug target. In the present study, LdDCP was evaluated for its immunogenicity in cured hamster by XTT, NO and RO production. The study suggested that LdDCP has potential of developing as vaccine candidate against VL infection.

LdREL1 has been expressed and purified. We now started preparing over-expression and knock-out construct of LdREL1 to establish its function in Leishmania parasites. A 0.8 kb flanking sequence upstream (5′F) and 0.725 kB flanking sequence downstream (3′F) of LdREL1 were PCR-amplified from L. donovani g-DNA, cloned in pCR-2.1-TOPO TA vector and then sub-cloned in pX63NEO and pX63HYG vectors in between HindIII and SalI as well as SmaI and BglII sites to generate 5′F-pX63HYG-3′F and 5′F-pX63NEO-3′F constructs respectively. Both of these circular constructs were then digested with HindIII and BglII to generate linear constructs for transfection in Leishmania parasites. Transfection of knock-out construct will be started to generate LdRELl deleted parasites to see the effect of this knockout in parasite survival and infection

To prepare over-expression construct, LdREL1 ORF was PCR amplified from a pCR- 2.1-TOPOTA-LdREL1 construct using primers with restriction sites HindIII and BamHI and cloned into Leishmania expression vector pLp-NEO2 in between HindIII and BamHI sites. Clone pLp-NEO2-LdSir2 (ORF in the right orientation) has been transfected into Leishmania parasites by electroporation. Transfectants were selected and maintained in the presence of 40 mg/mL G418 and further selected in 80 mg/mL G418. Taking these LdREL1 knockout and over-expression parasites, the role of REL1 in RNA editing, oxidative phosphorylation, parasite survival and infectivity will be explored in future.

Previously identified 6 Th1 stimulatory proteins are being evaluated for their prophylactic as well as therapeutic efficacy against visceral leishmaniasis (VL). In continuation, we have now developed a chimeric protein by fusing eldolase and triosephosphate isomerase (TPI) and evaluated its therapeutic efficacy against experimental VL. When compared to the individual proteins, the fusion product has shown significantly increased therapeutic efficacy. The suppression of infection was more than 75% when used with BCG as an immune modulator. The immunological parameters such as DTH response, lympho-proliferation as well as cytokine responses for both Th1 type (IFN-γ, TNF-α and IL-12) and Th2 type (IL4 and TGF-β) in the splenic tissue samples as well as in the sera samples indicated inclination towards Th1 response. Development of chimeras of aldolase-enolase and aldolase-TPI is underway.