Biology subjects

Singhal, J.

Publications and source records attributed to Singhal, J..

11 recordsLinked to original sources

bioRxiv2026

Mosquito-directed PROTACs to block malaria transmission

The mosquito stage of the Plasmodium falciparum life cycle is an attractive target for intervention since it is crucial for the sexual reproduction and transmission of parasites to human host. Mosquito determinants crucial for parasite infection and growth pose as lucrative targets for transmission blockers. Owing to the fact that p38 MAPK has role in immune response and vector competence, we have evaluated the potential of PROTAC molecule (NR-7h) to degrade Anopheles stephensi p38 MAPK (Asp38 MAPK), a conserved serine/threonine kinase involved in stress reactions, midgut homeostasis, and parasite survival. PROTAC-mediated degradation of Asp38 MAPK led to the disrupted development of the parasite, suggesting its crucial function in vector competence. Furthermore, NR-7h-treated mosquitoes showed higher expression of immune genes such Rel-2, TEP1, APL1, and NOS, suggesting that p38 MAPK regulates host immunity in a way that promotes parasite persistence. PROTAC-mediated degradation of target proteins, provides a more persistent and resistance-proof therapeutic effect than traditional kinase inhibitors. Our findings establish PROTACs as a novel vector-targeted strategy for the development of endectocides to limit malaria transmission.

molecular biology↗

bioRxiv2025

Immunometabolic determinants of long-term response in leukemia patients receiving CD19 CAR T cell therapy

Although most patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) receiving CD19-targeted chimeric antigen receptor (CAR) T cell therapy achieve remission, loss of CAR T cell functionality and subsequent relapse remains an unmet therapeutic need. We applied an integrative approach to study the immunometabolism of pre- and post-infusion CD19-CAR T cells of patients with relapsed/refractory B-ALL. Pre-infusion CAR T cells of long-term responders (LTR) had increased oxidative phosphorylation, fatty acid oxidation, and pentose phosphate pathway activities, higher mitochondrial mass, tighter cristae, and lower mTOR expression compared to products of short-term responders. Post-infusion CAR T cells in bone marrow (BM) of LTR had high immunometabolic plasticity and mTOR-pS6 expression supported by the BM microenvironment. Transient inhibition of mTOR during manufacture induced metabolic reprogramming and enhanced anti-tumor activity of CAR T cells. Our findings provide insight into immunometabolic determinants of long-term response and suggest a therapeutic strategy to improve long-term remission.

immunology↗

bioRxiv2025

CRISPR/Cas9-mediated identification of human macrophage SphK1 as druggable target for development of anti-leishmanial chemotherapeutics

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates apoptosis, autophagy, inflammation, and intracellular pathogen survival. While S1P signaling has been implicated in Leishmania donovani infection, the specific roles of its biosynthetic enzymes Sphingosine kinases SphK1 and SphK2 in host macrophage remains poorly defined. Here, we delineate the role of SphK1 in modulating host-pathogen interactions using CRISPR/Cas9-mediated knockout and pharmacological inhibition in THP-1 macrophages. We generated and validated CRISPR constructs targeting SphK1 (promoter, SBS, exon 6) and SphK2 (exon 3). SphK1 Knockout was confirmed at transcript and protein levels, accompanied by a marked reduction in SphK1 enzymatic activity and S1P levels. Functionally, SphK1 knockout macrophages exhibited decreased intracellular L. donovani burden, elevated TNF-, and reduced IL-10, and increased autophagic and apoptotic markers, suggesting a pro-inflammatory, cell-death-prone state. Pharmacological inhibition using the selective SphK1 inhibitor, PF-543 recapitulated these findings, showing reduced phosphorylated SphK1, enhanced p38MAPK activation, and augmented autophagy and apoptosis. Conversely, the SphK2 inhibitor ABC294640 had minimal effect, reinforcing the predominant role of SphK1. Together, our study identifies SphK1 as a critical host factor that facilitates L. donovani survival by modulating lysosomal stress, immune evasion, and cell fate pathways. Targeting SphK1-S1P signalling may offer a novel therapeutic approach for visceral leishmaniasis.

molecular biology↗

bioRxiv2025

Targeting Leishmania donovani Sphingosine Kinase 1 using PF-543 enhances immune response and limits parasite load

BackgroundSphingosine-1-phosphate (S1P) is a bioactive lipid mediator regulating apoptosis, proliferation, and immune responses. While S1Ps presence in Leishmania donovani phagolysosomes has been reported, the role of sphingosine kinases, especially SphK1, in parasite survival and host immune modulation remains underexplored. This study investigates the molecular and functional role of L. donovani SphK1 (LdSphK1) and evaluates the antileishmanial potential of PF-543, a specific SphK1 inhibitor. MethodsLdSphK1 and human SphK1 (rhSphK1) were cloned, expressed in E. coli, purified, and analyzed by SDS-PAGE. Enzymatic activity and inhibition by PF543 were assessed using NBD-S1P-based fluorometric assays. Protein-ligand interactions were analyzed using Microscale Thermophoresis (MST). Leishmania promastigotes overexpressing LdSphK1 were studied via confocal microscopy, and their viability and infectivity were assessed in vitro. THP-1 macrophages infected with L. donovani were treated with PF543 alone or with Amphotericin B and analyzed by MTT assay, RT-PCR, Giemsa staining, ELISA and immunoblotting. In vivo efficacy was tested in L. donovani-infected Swiss mice. ResultsrLdSphK1 ([~]102 kDa) and rhSphK1 ([~]50 kDa) were enzymatically active and significantly inhibited by PF-543. MST confirmed high-affinity binding of PF-543 (KD [~]29 microMolar). In L. donovani SphK1 overexpressor (LdSphKa) promastigotes, PF543 inhibited SphK1 activity and reduced parasite infectivity, more than in wildtype L. donovani promastigotes. Notably, PF543 treatment reduced parasite infectivity in vitro, lowered amastigote load by [~]40%, and promoted a pro-inflammatory cytokine shift ({uparrow}IL-12, {uparrow}TNF-, {downarrow}IL-10). Inhibition of ceramide synthesis and S1P supplementation revealed that S1P rescues ceramide-induced parasite death, implicating SphK1 in parasite survival. PF543 and Amphotericin B demonstrated synergistic anti-parasitic effects both in vitro and in vivo, with >90% reduction in parasite burden in mice. ConclusionPF543 is a potent inhibitor of SphK1, impairing parasite survival and modulating host immune responses. When combined with Amphotericin B, it offers a synergistic therapeutic strategy against visceral leishmaniasis, warranting further clinical exploration. Author SummaryLeishmaniasis, a neglected tropical disease, has limited available treatments and is becoming more resistant to medications. In this study, we explored the therapeutic potential of PF-543, a potent sphingosine kinase 1 (SphK1) inhibitor (demonstrated anticancer effects in various preclinical models) against Leishmania donovani. We successfully cloned and purified both Leishmania and human SphK1 proteins and confirmed PF-543 binding through biochemical and biophysical assays. Overexpression of LdSphK1 in parasites enhanced their survival and infectivity. In vitro, PF-543 treatment of infected macrophages decreased amastigote burden, shifted cytokine profiles towards a pro-inflammatory state, and enhanced host cell apoptosis. Notably, PF-543 acted synergistically with Amphotericin B, the current clinical drug, both in vitro and in vivo in Swiss mice, drastically lowering parasite burden. This study highlights the possibility of combination therapy and finds PF-543 to be a promising irresistible host-targeted antileishmanial drug. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/652326v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1926575org.highwire.dtl.DTLVardef@4305f4org.highwire.dtl.DTLVardef@3c10c7org.highwire.dtl.DTLVardef@171801e_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2024

Evaluation of Immunopharmacological Efficacy of Live Leishmania donovani Overexpressing Ld_ζ1domain for Protection Against Experimental Human Visceral Leishmaniasis

ObjectiveTo evaluate the efficacy and immunogenicity of the zeta domain over-expressing Leishmania donovani (Ld_{zeta}1domain) as a vaccination candidate against visceral leishmaniasis (VL). MethodsIn this study, Leishmania overexpressor Ld_{zeta}1domain (OE) were transformed by electroporation using a GFP-tagged Ld_{zeta}1domain recombinant plasmid. The resulting overexpressing cells were analysed in vitro to assess their growth dynamics and infectivity. We also investigated the immune-protective potential of these overexpressor in a mouse model challenged with Leishmania donovani. The immune response, including Th1 and Th2 pathways, was thoroughly characterized using RT-PCR and ELISA assays. In addition, the study conducted a thorough evaluation of the mouses spleen and liver parasites, as well as quantitative evaluation of tissue pathological changes. ResultsLd_{zeta}1 domain (OE) parasites exhibited significantly lower viability and replication rates than WT parasites, and in vivo studies showed that mice immunized with the Ld_{zeta}1(OE) domain had lower parasite numbers than mice infected with LdWT. Spleen and liver showed significant histological changes suggestive of protection. Parasite burden in the spleen and liver of vaccinated mice were significantly reduced. The immune response showed increased IFN-{gamma} levels and lower IL-10 production, resulting in a greater IFN-{gamma}/IL-10 ratio, indicating parasite elimination. The vaccination also caused a significant IgG humoral response and increased nitric oxide production in immunized mice. ConclusionOur findings demonstrated that overexpressing the zeta toxin resulted in controlled parasite attenuation, lowering pathogenicity while retaining immunogenic features. Our work established the zeta over-expressors protective efficacy, immunogenicity, and proliferation in response to a Leishmania challenge in vitro and in vivo. This preliminary prototype study suggested that Ld_{zeta}1domain (OE) parasites may be suitable for developing an attenuated vaccine against leishmaniasis. Graphical representation O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/630021v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@32dfc0org.highwire.dtl.DTLVardef@1ffe89dorg.highwire.dtl.DTLVardef@d9feorg.highwire.dtl.DTLVardef@1e7bae7_HPS_FORMAT_FIGEXP M_FIG C_FIG A schematic representation on the protective effectiveness, immunogenicity, and proliferation of the zeta over-expressor in response to Leishmania challenge in vitro and in vivo model.

pathology↗

bioRxiv2024

Chemical degradation identified Human p38-MAPK as a host target to combat parasitic infections

The interplay between host and parasite determines parasite burden and disease outcome. Parasite exploits host signaling pathways like p38-MAPK for its survival and pathogenesis. NR-7h, a proteolysis-targeting chimera (PROTAC) targeting human p38-MAPK was used to assess p38-MAPKs role in Leishmania donovani and Plasmodium falciparum infection in their respective hosts. NR-7h degraded host p38-MAPK in a time- and dose-dependent manner. Degradation of host p38-MAPK by NR-7h reduced parasite load in host cells dose-dependently, implicating the role of p38-MAPK in parasite survival. The modulation of cytokine profiling and oxidative burst upon NR-7h mediated degradation of host p38-MAPK was further correlated with parasite death. The synergistic effect of host p38-MAPK degradation by NR-7h with Amphotericin B enhances the efficacy of parasite-directed therapy. This study underscores the importance of host p38-MAPK for L. donovani and P. falciparum progression and highlights NR-7hs potential in antiparasitic therapy by targeting this pathway.

microbiology↗

bioRxiv2024

A first-in-Plasmodium study on tRNA intron splicing endonuclease PfTSEN1 and its substrate expression in clinical stage malaria

Mature tRNAs play critical role in several cellular processes including protein translation, post-translational-modifications and programmed-cell-death. Maturation of pre-tRNAs require removal of 5-leaders, 3-trailers, splicing of introns and addition of conserved 3-terminal CCA sequence. The tRNA splicing mechanism, an essential step in tRNA maturation govern by a tRNA splicing endonuclease. While the existence of functional tRNA splicing endonuclease(s) in Plasmodium falciparum has not been identified, its significance in other eukaryotes suggests a potential role in tRNA splicing event. Our study identified total tRNAs in Plasmodium and characterize a PftRNA splicing endonuclease (annotated as PfTSEN1) recognised recently as a component of ribonucleoprotein (RNP) complex, and synthesized a naphthoquinone derivative as a novel anti-malarial compound ( TSENi) targeting the functional activity of this protein. Enzyme activity assays elucidated that PfTSEN1 catalyses splicing of in vitro transcribed pre-tRNAleu, the expression of which was confirmed during the clinical stages of malaria parasite by RT-PCR. Interestingly, TSENi binds to and inhibits enzymatic activity of PfTSEN1, and showed potent anti-malarial activity against chloroquine-sensitive 3D7 and resistant strains Dd2 of P. falciparum. Overall, our study deliver key knowledge towards the functional role of PftRNA splicing endonuclease, and its inhibitor TSENi as potent anti-malarial.

molecular biology↗

bioRxiv2023

Host inducible-HSP70A1A is an irresistible drug target to combat SARS-CoV-2 infection and pathogenesis

One of the fundamental mechanisms developed by the host to contain the highly infectious and rapidly proliferating SARS coronavirus is elevation of body temperature, a natural fallout of which is Heat Shock Protein (HSP) over-expression. Here, for the first time, we demonstrate that the SARS-CoV-2 virus exploits the host Hsp70 chaperone for its entry and propagation and blocking it can combat the infection. SARS-CoV-2 infection as well as febrile temperature enhanced Hsp70 overexpression in host Vero E6 cells. In turn, Hsp70 overexpression elevated the host cell autophagic response that is a prerequisite for viral propagation. Suppressive and prophylactic treatment of Vero E6 cells with HSP70 inhibitor PES-Cl, a small molecule derivative of Pifithrin , abrogated viral infection more potently than the currently used drug Remdesivir by suppressing host HSP70 and autophagic response. In conclusion, our study not only provides a fundamental insight into the role of host Hsp70 in SARS-CoV-2 pathogenesis, it paves the way for the development of potent and irresistible anti-viral therapeutics.

microbiology↗

bioRxiv2023

Identification of merozoite secreted repertoire and immuno-pharmacological inhibition of a novel host-parasite interaction to block malarial infection

BackgroundDuring the intra-erythrocytic proliferation of Plasmodium falciparum, the host erythrocyte invasion is regarded as a complex and tightly regulated process comprising multiple receptor-ligand interactions, and numerous secretory molecules. Proteins secreted sequentially from apical organelles of merozoites serve as adhesins that play a crucial role in RBC invasion and can serve as vaccine and therapeutic targets. MethodsPurified merozoites were triggered to discharge apical organelle contents by exposure to ionic conditions mimicking that of blood plasma. The secreted proteins were subjected to tandem mass spectrometry, and a well-characterized invasion ligand, RhopH3, was identified. A novel RhopH3 receptor, 14-3-3{square} was unearthed using a Bacterial two-hybrid approach. This interaction was confirmed using multiple biophysical and biochemical approaches. We were successful in disrupting this interaction using a de novo peptide binder of 14-3-3{square}, and we subsequently assessed its effect on merozoite invasion. ResultsA total of 66 proteins were identified in the secretory fraction with apical organellar or merozoite membrane localization. The well-known adhesin, RhopH3 was also identified and its interaction with the host phosphopeptide-binding protein, 14-3-3{square} was established. We also discovered a de novo peptide with the potency to disrupt this crucial interaction, thereby blocking merozoite invasion. ConclusionWe, for the first time, report the secretory repertoire of plasmodium merozoite. Our study shows the importance of the erythrocyte protein, 14-3-3{square} during the invasion process and paves the way for developing anti-malarial peptides or small molecules that inhibit the host-pathogen interaction, hence abrogating the invasion process.

microbiology↗

bioRxiv2021

Targeting the artemisinin resistant malaria by repositioning of the anti-Hepatitis C Virus drug Alisporivir

The rapid emergence of P. falciparum-resistant strains raises an urgent need to find new antimalarial drug candidates. This study reports the rational repositioning of the anti-Hepatitis C Virus drug, Alisporivir, a non-immunosuppressive analog of cyclosporin A (CsA) against multiple, drug-resistant strains of P. falciparum. Alisporivir being non-hemolytic has been proven to be a better drug than CsA. Indeed, our study also demonstrated the same. Alisporivir inhibited chloroquine-sensitive parasite growth with an IC50 of 196.6nM. Alisporivir also inhibited the growth of chloroquine-resistant parasites with an IC50 of 422.1nM. Alisporivir exhibited, anti-malarial activity in in vivo. Further, we exploited the Cyclophilins targeting potential of Alisporivir against artemisinin-resistant malaria parasite owing to the fact that PfCyP-19B is one of the genes that is overexpressed in artemisinin-resistant parasite revealed by a population transcriptomic study. Our semiquantitative real-time transcript and immunofluorescence analysis confirmed the overexpression of PfCyP-19B in Artemisinin-resistant P. falciparum (PfKelch13R539T). Artemisinin resistance is attributed to slow clearance of ring stage parasites. Ring survival assay (RSA) is designed to access the potency of compounds on these dormant slow clearing parasites leading to drug resistance. Thus, the potency of Alisporivir against PfKelch13R539T was evaluated by RSA. A 2.5-fold decrease in parasite survival was detected with Alisporivir. Further, combination of Alisporivir with DHA found to potentiate the efficacy of DHA by 4.55-fold. These results support the hypothesis that targeting of resistance mechanism is a potential approach to deal with resistant parasite. Overall, this study demonstrates the rational reposition of Alisporivir against resistant malaria resistance.

microbiology↗

bioRxiv2020

Discovery of tREP-18, a novel class of tRNA encoded peptide with potent leishmanicidal activity

In the post genomic era, tRNA-derived fragments have emerged as a new class of non-coding gene regulators, those play crucial roles both at the transcriptional and translational levels, in different cellular biogenesis. However, none of the studies has ever asked whether tRNAs can also be translated into peptides with any biological significance. Thus, we present a novel hypothesis which suggested that; design and synthesis of tRNA-derived peptides from prokaryotic genome can be exploited for developing unique chemotherapeutics against neglected tropical diseases, like Visceral leishmaniasis (VL) and its aggressive form known as post kalazar dermal leishmaniasis (PKDL). To achieve this aim, we have used a novel system biology-based strategy, which involved; i) mining of unique tRNAs from E. coli genome and their translation into peptide in silico, ii) designing of theoretical 3D models to evaluate their stability, iii) prediction of their biological activity by screening against anti-parasitic database to filter the lead peptide. Based on this strategy, a unique tRNA-derived peptide (tREP-18) was selected, chemically synthesized, and used in vitro for elucidating its therapeutic significance against L. donovani, a causative agent of VL and PKDL. Our findings demonstrated that, tREP-18 can impose high level toxicity to L. donovani promastigotes, by disrupting the ultrastructural cellular architect, destabilizing the mitochondrial membrane potential ({Delta}{Psi}m), thus leading to drastic reduction in cell viability and proliferation. It also imparted high level of toxicity to BS12 a clinical isolate of PKDL. Conceivably, we for the first time reports a novel tRNA-derived peptide "tREP18" with excellent anti-leishmanial property, which can be further utilized for developing it as antileishmanial drug. One Sentence SummaryHere we report a novel first-in-class tRNA encoded peptide and its super anti-leishmanial characteristics.

synthetic biology↗