Malaria and Other Parasitic Diseases

Area Co-Ordinators

  • Dr. Saman Habib

    Malaria

  • Dr. Sanjay Batra

    Malaria


Vision and Goal:

▶ 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.

Malaria

YiHA GTPases in biogenesis of organellar ribosomes of the malaria parasite

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

  • Scheme Domain Organisation of pf-RBR E3 Ligase

  • IB: Anti-FLAG
    IB: Anti-HA

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.



Core Competencies and Activities:

▶ 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




1.2.4 Functional Characterization of TCP1γ of L. donovani

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.

1.2.5 Characterization of leishmanial dipeptidylcarboxypeptidase as a potential vaccine molecule against visceral leishmaniasis

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

1.2.6 Characterization of RNA Editing Ligase 1 (REL1) of Leishmania donovani as drug target

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.

1.2.7 Development of Leishmania vaccine

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.

1.3 Filariasis

1.3.1 Immunobiology of Lymphatic Filariasis: Immunogenicity and protective efficacy of recombinase-A protein of Wolbachia, an endosymbiont of filarial nematode Brugia malayi.

Filarial parasites cause global morbidity. Wolbachia, an endo-symbiotic intracellular bacterium of the filarial nematode helps in their growth and development, regulates fecundity in female worms and contributes to the immunopathogenesis of the disease. It is believed that several genes and proteins of Wolbachia are intricately involved in either suppression, diversion, or polarization of the host immune response. However, genes and proteins of Wolbachia that may act as putative vaccine candidates are not known. We cloned recombinase-A protein of Wolbachia from Brugia malayi (wBmRecA) and carried out its detailed biochemical and immunological characterization. Bioinformatics analysis, circular dichroism and fluorescence spectral studies showed significant sequence and structural similarities between wBmRecA and RecA of other alpha-proteo- bacterial species (Fig.).




↖ Fig. Multiple sequence alignment of wBmRecA amino acid sequence with other RecA sequences was performed using CLUSTALW software. Identical and similar residues are denoted by red color background and boxed, respectively. The secondary structure is displayed onto the alignment. The amino acid residues involved in ATP binding (phosphate binding P-loop), single and double strand DNA binding are represented as bold underline. (B) Evolutionary relationship of wBmRecA among different bacterial species.

Notably, wBmRecA was ubiquitously expressed in all the three major life stages of B. malayi, including excretory-secretory products of the adult worm (Fig. 8 A-C).

Fig. 8. Expression of wBmRecA in different life stages of B. malayi (A) RT-PCR analysis using cDNA from different stages of B. malayi. (B) Protein lysate from different stages of B. malayi probed with anti-wBmRecA antibody. (C) Cross reactivity of anti- wBmRecA antibody with excretory-secretory (ES) product from female worms (lane 1). No reactivity was observed in case of control sera (lane 2).


In silico studies suggested immunogenic potential of wBmRecA, and mice immunized with wBmRecA exhibited elevated levels of immunoglobulins IgG1, IgG2a, IgG2b and IgG3 in their serum (Fig. 9) along with increased percentages of CD4+, CD8+ T cells and CD19+ B cells in their spleens.

Interestingly, splenocytes from immunized mice showed increased m-RNA expression of T-bet, elevated proinflammatory cytokines IFN-γ and IL-12, while peritoneal MΦs exhibited increased levels of iNOS, downregulated Arg-1 and secreted copious amounts of nitric oxide which contributed to severely impaired development of the third stage infective larvae (Bm-L3).

Fig. Host protective nature of wBmRecA. Peritoneal exudate cells from naive mice were separately cultured with (A) Bm-L3 and (C) Mf in the presence or absence of sera from r-wBmRecA immunized animals and (B) Bm-L3 and (D) Mf killing was observed microscopically. (E) Recovery of Bm-L3 in mice from different treatment groups. (F) Sero-reactivity of wBmRecA with human bancroftian serum samples. Lane 1- Protein molecular weight marker; lane 2- serum from endemic normal (EN); lane 3- serum from asymptomatic microfilariaemic carriers (MF+); lane 3- serum from symptomatic microfilariaemic (SYMPT) and lane 4- serum from non-endemic normal (NEN) controls.

Interestingly, sera from immunized mice promoted significant cellular adherence and cytotoxicity against microfilariae and Bm-L3 (Fig. 10 A-E) and wBmRecA demonstrated strong immuno-reactivity with bancroftian sera from endemic normal individuals (Fig. 10F) which suggest that wBmRecA is highly immunogenic, and should be explored further as a putative vaccine candidate against lymphatic filariasis (Gangwar et al., Vaccine, 2019).


1.4 Medicinal Chemistry

1.4.1 Malaria

Aminopropanols

In continued effort to assess the antiplasmodial efficacy of the aminopropanols, 30 new analogues were synthesized and submitted for activity. The synthesis involved the incorporation of heterocyclic scaffolds in the aminopropanol unit. These compounds displayed significant activity against 3D7 as well as K1 strains of P. falciparum. Encouraged by these results, synthesis of a novel class of diaminopropanol compounds by incorporating an additional amino group in the molecule and their derivatives was accomplished. All 10 new compounds of this class, displayed enhanced bioactivity in the range of IC50 0.04-0.250 µM for 3D7 CQS and 0.07-0.91 µM for K1 CQR strains of P. falciparum. Two compounds S-019-190 & S-019-0192 were resynthesized for carrying out the in vivo studies. However, the 10 new compounds bearing nitropyridine and 3-aminoquinoline scaffolds were found to eleicit poor biological response.

1-(Arylsulfonyl)pyrrolidine-2-carboxamides

During this period around 20 new 1-(substituted phenylsulfonyl)pyrrolidine-2-carboxamides were prepared and subjected for evaluation as antiplasmodial agent. However the compounds were found to be inactive against the 3D7 CQS strain of P. falciparum.

Heterocycles

Compounds belonging to various heterocycles including oxaboroles, triazoles, indolinone, spiroindolinones, pyrazoles, isoxazoles, pyrrolo[1,2-a]isoquinolines, 4-aminoquinolines, phosphorylated naphthalenes were prepared and submitted for evaluation as antiplasmodial agents. Of these a few analogues displayed antiplasmodial efficacy ranging between 2-4 M against 3D7 CQS strain of P. falciparum.


1.5 Anti-parasitic screening for drug discovery

1.5.1 Anti-malarial screening

633 compounds were screened against the human malaria parasite, P. falciparum (CQ-sensitive PF3D7 and CQ-resistant K1 strain). These compounds belong to diverse chemical classes such as quinoline triazols, oxazenes, oxazole salt, di-aminopropanol, pyrimidine based hetrocyclic derivatives, HDAC inhibitors, spiroindoles, peptidyl hydroxamic and dihydronaphthalene.

Quinoline triazol (20) derivatives (S019-0546, 0547, 0548, 0549, 0550, 0551, 0552, 0553, 0554, 0555, 0556, 0557, 0558, 0559, 0560, 0561, 0562, 0563, 0564 and 0565) had IC50 in the range of 0.03 to 0.35 µM against CQ-sensitive (PF3D7) and 0.03 to 1.91 µM against CQ-resistant strain (K1). Oxazene class (10) compounds (S019-0472, 0473, 0474, 0475, 0476, 0477, 0478, 0479, 0510 and 0511) exhibited activity with IC50 in the range of 0.11 to 0.97 µM against PF3D7 and 0.07 to 0.48 µM against K1. Oxazole salt derivatives (23) (S019-0129, 0131, 0132, 0133, 0134, 0135, 0136, 0137, 0139, 0140, 0141, 0142, 0156, 0157, 0158, 0159, 0160, 0161, 0163, 0164, 0165, 0168, 0169) showed activity with IC50 values between 0.24 to 0.97 µM against 3D7 and 0.23 to 2.34 µM against K1 strain. Di-aminopropanols (10) compounds (S019-0117, 0118, 0119, 0120, 0182, 0184, 0187, 0190, 0192 and 0212) exhibited antimalarial effect with IC50 values of 0.04 to 0.25 µM against PF3D7 and 0.24 to 1.96 µM against K1. Aminopropanol derivatives (S018-0507, 0251 and 0509) exhibited IC50 ranging from 0.12 -0.78 µM against Pf3D7 and <0.07 - 2.94 µM against K1 strain. Compounds (S019-0464, 0065 and 0067) belonging to pyrimidine class exhibited activity in the range of 0.17 and 0.46 µM (IC50 values) against Pf3D7and 0.24 to 0.61 µM against K1. Compound designed based on HDAC inhibitor (S019-0077) had IC50 values of 0.22 µM against 3D7 and 0.66 µM against K1. Spiroindole derivatives (S018-0309 and S018-0312) showed IC50 of 0.88 and 0.48 µM against 3D7 and 1.47 and 3.11 µM against K1 strain. Peptidyl hydroxamic acid (2) derivatives (S018-0396 and 0411) had IC50 values of 0.94 and 0.59 µM, respectively, PF3D7 strain and 0.81 and 0.54 µM, respectively against K1 strain. Indole based scaffold, S019-0010 had IC50 values of 1.04 µM against 3D7 and 0.62 µM against K1. Dihydronaphthalene derivatives (S018-0489, 0490 and S017-0670) exhibited IC50 values 0.97, 0.03 and 0.72µM respectively, against PF3D7 and 0.63, 0.02 and 3.99 µM respectively, against K1. Furan based compound (S018-0485) exhibited IC50 at 0.86 µM against 3D7 and 0.91 µM against K1 strain. Imidazopyridine class compound (S019-0019) showed IC50 at 0.60 µM against PF3D7 and 0.15 µM against K1 strain. S019-0124, S019-0200, S019-077 and S019-0608 showed antimalarial activity in the range of 0.2-0.5 µM in CQ-sensitive and resistant strains. These compounds were evaluated for cytotoxic profile against Vero cell line and were found to have minimal or no cytotoxicity.

1.5.2 In vitro antimalarial activity report of compounds received under DBT Twinning project during Jan 2019 – Dec 2019

During the reported period, 40 compounds were received under DBT Twining project and were screened against PF3D7 and K1 strains. Out of these, 10 compounds namely SKQ-1C, 1D, 1E, 2C, 2D, 2E, 3B, 3C, 3D and 3E have shown IC50 as 0.05, 0.04, 0.35, 0.14, 0.05, 0.04, 0.17, 0.14, 0.07 and 0.08 µM against Pf3D7 chloroquine sensitive and 0.99, 0.94, 0.31, 0.34, 0.79, 0.28, 0.38, 0.63, 0.22 and 0.29 µM against PfK1 chloroquine resistant strain, respectively. These molecules were also evaluated for cytotoxic profile in Vero cell line and found to be safe.


1.5.3 In vitro antimalarial activity report of plant extracts received across the country (Jan 2019 – Dec 2019)

During the reporting period, two plant extracts received from other research institutes were screened against the P. falciparum. Both plant extracts, namely RMRC-001 & RMRC-002 have shown antimalarial activity with their IC50 values of 27.45 and 19.65 µg/ml against CQ sensitive Pf3D7 strain and 5.10 & 36.85 µg/ml against CQ resistant PfK1 strain, respectively. These molecules are found to be safe in Vero cells.

1.5.4 In vivo antimalarial profile of CSIR-CDRI compounds against P. yoelii nigeriensis N67 (CQ resistant) (Jan 2019 – Dec 2019)

Four compounds, namely S018-0248 (aminopropanol class), S017-0383 (Nitropyridine class), S019-0065 and S019-0067 (pyrimidine based hetrocyclic class) were administered for 4 days, at 100 mg/kg in swiss mice infected with P. yoelii nigeriensis N67 infected. Out of these, only S018-0248 showed 38.34% parasite suppression on day 4 post treatment but was not curative on day 28. S018-0485 (furan derivatives), S018-0490 and S018-0489 (dihydronaphthalene derivatives) were dosed for 4 consecutive days, at 100 mg/kg which resulted in 18.87, 21.60 and 0%, respectively, parasite suppression on day 4. S018-0248 (aminopropanol class) was also tested at the same dose and showed 38.34% parasite suppression on day 4. S017-0383 (nitropyridine) was dosed for 4 consecutive days (100 mg/kg) and it did not show any parasite suppression on day 4.

1.5.5 Anti-leishmanial screening

Novel synthetic moieties representing several prototypes viz., quinoline-imidazole hybrids, pyrolo quinolinone, chalcones, stilbenes, phenyl allylindole, imidazo pyrolo quinolone derivatives, quinoline tetrahydropyrimidine etc. were synthesized and tested for their efficacy against experimental model of visceral leishmaniasis. Total sixty seven synthetic compounds were evaluated at 25 μM and 12.5 μM concentrations respectively against in vitro macrophage-amastigote model for lead identification. Compounds belonging to pyrimidine-based heterocyclic derivates (S-018-0699, S-018-0701) series showed significant anti-amastigote activity (>80% inhibition of amastigote multiplication and SI >5). Half-maximal inhibitory concentrations (IC50) of S-018-0699 and S-018-0701 were found to be 9.28 µM and 8.77 μM respectively against intracellular amastigotes. S-018-0699 and S-018-0701 will be further evaluated for their anti-leishmanial activity in L. donovani- golden hamster model.

153 Novel synthetic moieties representing several prototypes viz., Isoxazide, Triazol, Nitropyridine, Aminothiophenol, Aminopyridine, Naphthol Hetrocyclic, Isoxazole, Oxazoline, Benzofuran Nitropyridine, Oxazoline, Isoxazole, Isoxazide amide, Dihydroxy-hex-2-en-1-one, and 16 analogues of lead compound 96/261 were screened in vitro against promastigotes and amastigotes. Out of these 27 compounds exhibited IC50 < 10µM and SI index > 5.

Six compounds were evaluated for their in vivo efficacy in L. donovani / Hamster model at 50mg/Kg I.P or 100mg/Kg oral dose. Only one compound (analogue of 96/261) showed >70% anti-leishmanial efficacy in L. donovani golden hamster model at 100mg/Kg oral dose for 5 days.