Synthesis and characterization of zinc derivatized 3, 5-dihydroxy 4', 7-dimethoxyflavone and its anti leishmaniasis activity against Leishmania donovani.

This study reports the synthesis and characterization of zinc derivatized 3,5-dihydroxy 4', 7- dimethoxyflavone (DHDM-Zn) compound for the development of new antileishmanial agents. The interaction studies of DHDM with zinc were carried out by UV spectra and fluorescence spectra analysis. Characterization of the complex was further accomplished by multi-spectroscopic techniques such as FTIR, Raman, HRMS, NMR, FESEM-EDX. The morphological and topographical studies of synthesized DHDM-Zn were carried out using FESEM with EDX. Further, it was demonstrated that DHDM-Zn exhibited an excellent in vitro antagonistic effect against the promastigote form of L. donovani. In addition, the possible mechanisms of promastigote L. donovani cell death, by involvement of derivatized compound in arrest of the cell cycle in the G1 phase and residual cell count reduction were investigated. Promastigote growth kinetics performed in the presence of the derivatized compound revealed a slow growth rate. The combination of growth kinetics and cell cycle analysis, made it possible to interpret and classify the cause of leishmanial cell death accurately. These results support that zinc derivatized complex (DHDM-Zn) might work as a lead compound for designing and developing a new antileishmanial drug.

Identification of an anti-SARS-CoV-2 receptor-binding domain-directed human monoclonal antibody from a naïve semisynthetic library.

There is a desperate need for safe and effective vaccines, therapies, and diagnostics for SARS- coronavirus 2 (CoV-2), the development of which will be aided by the discovery of potent and selective antibodies against relevant viral epitopes. Human phage display technology has revolutionized the process of identifying and optimizing antibodies, providing facile entry points for further applications. Herein, we use this technology to search for antibodies targeting the receptor-binding domain (RBD) of CoV-2. Specifically, we screened a naïve human semisynthetic phage library against RBD, leading to the identification of a high-affinity single-chain fragment variable region (scFv). The scFv was further engineered into two other antibody formats (scFv-Fc and IgG1). All three antibody formats showed high binding specificity to CoV-2 RBD and the spike antigens in different assay systems. Flow cytometry analysis demonstrated specific binding of the IgG1 format to cells expressing membrane-bound CoV-2 spike protein. Docking studies revealed that the scFv recognizes an epitope that partially overlaps with angiotensin-converting enzyme 2 (ACE2)-interacting sites on the CoV-2 RBD. Given its high specificity and affinity, we anticipate that these anti-CoV-2 antibodies will be useful as valuable reagents for accessing the antigenicity of vaccine candidates, as well as developing antibody-based therapeutics and diagnostics for CoV-2.

Synthesis and Evaluation of Methyl 4-(7-Hydroxy-4,4,8-Trimethyl-3-Oxabicyclo[3.3.1]Nonan-2-yl)Benzoate as an Antileishmanial Agent and Its Synergistic Effect with Miltefosine.

In our interest in oxabicyclic compounds as potent antileishmanial agents, the present work deals with the chemical synthesis of a new oxabicyclic derivative, methyl 4-(7-hydroxy-4,4,8-trimethyl-3-oxabicyclo[3.3.1]nonan-2-yl)benzoate (PS-207). This oxabicyclic derivative showed a good antileishmanial effect on the parasite, on both the promastigote and the amastigote. The mode of parasitic death from PS-207 seemed to be apoptosis-like. Interestingly, the combination of PS-207 with a low dose of miltefosine showed a synergistic effect against the parasite.

Kennedy Epitope (KE)-dependent Retrograde Transport of Efficiently Cleaved HIV-1 Envelopes (Envs) and its Effect on Env Cell Surface Expression and Viral Particle Formation.

Efficiently cleaved HIV-1 Envs are the closest mimics of functional Envs as they specifically expose only bNAb (broadly neutralizing antibody) epitopes and not non-neutralizing ones, making them suitable for developing vaccine immunogens. We have previously identified several efficiently cleaved Envs from clades A, B, C and B/C. We also described that truncation of the CT (C-terminal tail) of a subset of these Envs, but not others, impairs their ectodomain conformation/antigenicity on the cell surface in a CT conserved hydrophilic domain (CHD) or Kennedy epitope (KE)-dependent manner. Here, we report that those Envs (4 - 2.J41 and JRCSF), whose native-like ectodomain conformation/antigenicity on the cell surface is disrupted upon CT truncation, but not other Envs like JRFL, whose CT truncation does not have an effect on ectodomain integrity on the cell surface, are also defective in retrograde transport from early to late endosomes. Restoration of the CHD/KE in the CT of these Envs restores wild-type levels of distribution between early and late endosomes. In the presence of retrograde transport inhibitor Retro 2, cell surface expression of 4 - 2.J41 and JRCSF Envs increases [as does in the presence of Rab7a DN and Rab7b DN (DN: dominant negative)] but particle formation decreases for 4 - 2.J41 and JRCSF Env pseudotyped viruses. Our results show for the first time a correlation between CT-dependent, CHD/KE regulated retrograde transport and cell surface expression/viral particle formation of these efficiently cleaved Envs. Based on our results we hypothesize that a subset of these efficiently cleaved Envs use a CT-dependent, CHD/KE-mediated mechanism for assembly and release from late endosomes.

Biophysical and Biochemical Characterization of the Receptor Binding Domain of SARS-CoV-2 Variants.

The newly emerging SARS-CoV-2 variants are potential threat and posing new challenges for medical intervention due to high transmissibility and escaping neutralizing antibody (NAb) responses. Many of these variants have mutations in the receptor binding domain (RBD) of SARS-CoV-2 spike protein that interacts with the host cell receptor. Rapid mutation in the RBD through natural selection to improve affinity for host receptor and antibody pressure from vaccinated or infected individual will greatly impact the presently adopted strategies for developing interventions. Understanding the nature of mutations and how they impact the biophysical, biochemical and immunological properties of the RBD will help immensely to improve the intervention strategies. To understand the impact of mutation on the protease sensitivity, thermal stability, affinity for the receptor and immune response, we prepared several mutants of soluble RBD that belong to the variants of concern (VoCs) and interest (VoIs) and characterize them. Our results show that the mutations do not impact the overall structure of the RBD. However, the mutants showed increase in the thermal melting point, few mutants were more sensitive to protease degradation, most of them have enhanced affinity for ACE2 and some of them induced better immune response compared to the parental RBD.

Generation of soluble, cleaved, well-ordered, native-like dimers of dengue virus 4 envelope protein ectodomain (sE) suitable for vaccine immunogen design.

Dengue virus is transmitted by Aedes mosquitoes and dengue is endemic in many regions of the world. Severe dengue results in complications that may lead to death. Although some vaccine candidates are in clinical trials and one vaccine Dengvaxia, with restricted efficacy, is available, there are currently no specific therapies to completely prevent or treat dengue. The dengue virus structural protein E (envelope) exists as a head-to-tail dimer on mature virus, is targeted by broadly neutralizing antibodies and is suitable for developing vaccine immunogens. Here, we have used a redesigned dengue prME expression construct and immunoaffinity chromatography with conformational/quaternary antibody A11 to purify soluble DENV4 sE(A259C) (E ectodomain) dimers from mammalian expression system to ~99 % purity. These dimers retain glycosylation reported for native DENV E, display the three major broadly neutralizing antibody epitopes, and form well-ordered structure. This strategy can be used for developing subunit vaccine candidates against dengue and other flaviviruses.

Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein Based Novel Epitopes Induce Potent Immune Responses in vivo and Inhibit Viral Replication in vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) initiates infection by attachment of the surface-exposed spike glycoprotein to the host cell receptors. The spike glycoprotein (S) is a promising target for inducing immune responses and providing protection; thus the ongoing efforts for the SARS-CoV-2 vaccine and therapeutic developments are mostly spiraling around S glycoprotein. The matured functional spike glycoprotein is presented on the virion surface as trimers, which contain two subunits, such as S1 (virus attachment) and S2 (virus fusion). The S1 subunit harbors the N-terminal domain (NTD) and the receptor-binding domain (RBD). The RBD is responsible for binding to host-cellular receptor angiotensin-converting enzyme 2 (ACE2). The NTD and RBD of S1, and the S2 of S glycoprotein are the major structural moieties to design and develop spike-based vaccine candidates and therapeutics. Here, we have identified three novel epitopes (20-amino acid peptides) in the regions NTD, RBD, and S2 domains, respectively, by structural and immunoinformatic analysis. We have shown as a proof of principle in the murine model, the potential role of these novel epitopes in-inducing humoral and cellular immune responses. Further analysis has shown that RBD and S2 directed epitopes were able to efficiently inhibit the replication of SARS-CoV-2 wild-type virus in vitro suggesting their role as virus entry inhibitors. Structural analysis revealed that S2-epitope is a part of the heptad repeat 2 (HR2) domain which might have plausible inhibitory effects on virus fusion. Taken together, this study discovered novel epitopes that might have important implications in the development of potential SARS-CoV-2 spike-based vaccine and therapeutics.

Non-neutralizing SARS CoV-2 directed polyclonal antibodies demonstrate cross-reactivity with the HA glycans of influenza virus.

The spike protein of the SARS-CoV-2 virus is the foremost target for the designing of vaccines and therapeutic antibodies and also acts as a crucial antigen in the assessment of COVID-19 immune responses. The enveloped viruses; such as SARS-CoV-2, Human Immunodeficiency Virus-1 (HIV-1) and influenza, often hijack host-cell glycosylation pathways and influence pathobiology and immune selection. These glycan motifs can lead to either immune evasion or viral neutralization by the production of cross-reactive antibodies that can lead to antibody-dependent enhancement (ADE) of infection. Potential cross-protection from influenza vaccine has also been reported in COVID-19 infected individuals in several epidemiological studies recently; however, the scientific basis for these observations remains elusive. Herein, we show that the anti-SARS-CoV2 antibodies cross-reacts with the Hemagglutinin (HA) protein. This phenomenon is common to both the sera from convalescent SARS-CoV-2 donors and spike immunized mice, although these antibodies were unable to cross-neutralize, suggesting the presence of a non-neutralizing antibody response. Epitope mapping suggests that the cross-reactive antibodies are targeted towards glycan epitopes of the SARS-CoV-2 spike and HA. Overall, our findings address the cross-reactive responses, although non-neutralizing, elicited against RNA viruses and warrant further studies to investigate whether such non-neutralizing antibody responses can contribute to effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or ADE.

Mutational studies on Leishmania donovani dihydrolipoamide dehydrogenase (LdBPK291950.1) indicates that the enzyme may not be classical class-I pyridine nucleotide-disulfide oxidoreductase.

We report biochemical studies on two Cys residues mutation (Cys15Thr, Cys38Gly) nearest to the active site and three other amino acid substitution mutations expected to be the part of active site of LdDLDH_Variant1. Our biochemical studies show that the replacement of Cys15 increases the Km for dihydrolipoamide (DLD) substrate by five folds and NAD+ by three fold indicating that this mutation affects the binding of DLD and NAD+ significantly. Cys38 was also mutated to 'Gly' which resulted in nine fold greater Km for NAD+ without affecting Km for DLD. However, even after these mutations (Cys15Thr and Cys38Gly), reduced enzyme activity suggests that both the 'Cys' residues are not involved in disulfide bond formation but affect the binding of substrates. The data hints towards the possibility of a different catalytic mechanism from the classical class I - pyridine nucleotide-disulfide oxidoreductase. Remaining other mutated residues Ala48Ile, Asp49Gly, and Ala54Ile showed an increase in two to three-folds Km value for NAD+, which means these residues are important for the binding of NAD+ to the enzyme. However, Ala48Ile and Asp49Gly mutations showed a decrease of Km for DLD. Apart from the mutational studies, localization of LdDLDH_Variant2 of LdDLDH was also analyzed.

BLIMP-1 Mediated Downregulation of TAK1 and p53 Molecules Is Crucial in the Pathogenesis of Kala-Azar.

Precise regulation of inflammasome is critical during any pathogenic encounter. The whole innate immune system comprising of pattern recognition receptors (PRRs) relies on its ability to sense microbes. The fate of cellular death in infected cells depends mostly on the activation of these inflammasome, the dysregulation of which, due to functional manipulation by various pathogens, leads to be the cause of many human diseases. Here, an interesting finding has been observed which is related to how Leishmania donovani parasites exploit various host mediator molecules to cause immunosuppression. Here we report for the first time that the parasites check pyroptosis in the infected cells in-vitro by BLIMP-1 mediated suppression of TAK1 and p53 proteins. This might be one of the reasons how parasites evade the pro-inflammatory response of the host cells. Further understandings and validations are required to come up with better therapeutic approaches against kala-azar.

Episomal expression of human glutathione reductase (HuGR) in Leishmania sheds light on evolutionary pressure for unique redox metabolism pathway: Impaired stress tolerance ability of Leishmania donovani.

Trypanothione based redox metabolism is unique to the Trypanosomatida family. Despite extensive studies on redox metabolism of Leishmania parasites, a prominent question of why Leishmania adopt this unique redox pathway remains elusive. We have episomally expressed human glutathione reductase (HuGR) in Leishmania donovani (LdGR+) and investigated its effect. LdGR+ strain has slower growth compared to the wild type (Ld) indicating decreased survival ability of the strain. Further, LdGR+ strain showed enhanced accumulation of intracellular reactive oxygen species (ROS) and more sensitivity to the anti-leishmanial drug, Miltefosine, inferring increased stress level. In contrast, the expression analyses of genes specific to redox metabolism were increased significantly in LdGR+ strain compared to wild type. Lower infectivity index of the LdGR+ strain substantiated the above findings and indicated that the expression of HuGR reduces the stress tolerance ability of the parasite. From molecular docking studies with HuGR, it was observed that oxidized trypanothione (TS2) binds much better than oxidized glutathione (GS2). These results also give us hints that the parasite is losing infectivity potential due to an overall increase in intracellular stress caused with the expression of HuGR, showcasing a possible role of evolutionary pressure on the Leishmania parasites posed by HuGR.

Dihydrolipoamide dehydrogenase from Leishmania donovani: New insights through biochemical characterization.

Dihydrolipoamide dehydrogenase (DLDH) regulates many crucial metabolic pathways as a multi-enzyme complex. Leishmania donovani dihydrolipoamide dehydrogenase (LdDLDH) has two variants present on two different chromosomes with very less sequence similarities. In the current study, we cloned both the variants in pET28a (+) vector and expressed in Rosetta-gami (DE3) E. coli strain. Expressed proteins were finally purified from pellets using Ni-NTA affinity chromatography. Purified enzymes were biochemically characterized and different kinetic parameters were studied. Both the variants showed maximum activity in pH range of 7.0-8.0 and temperature 50±5°C in the physiological direction. The estimated Km for dihydrolipoamide (DLA) and NAD+ were 2.7±0.48mM and 171.23±11.59µM respectively for variant 1 (LdBPK291950.1). In the case of variant 2 (LdBPK323510.1), Km values for DLA and NAD+ were found to be 829.85±37µM and 226±1.56µM respectively. The variant 2 was more efficient in terms of activity. While both the forms of the enzymes showed diaphorase activity, variant 1 was found to be better. Sequence dissimilarities of both forms were analyzed for biological insights.

Design of commercially comparable nanotherapeutic agent against human disease-causing parasite, Leishmania.

Nanotherapeutic agents (NTA) play a crucial role in clinical medicine, if their unique properties are well understood and well exploited. In this direction, we report synthesis and characterization of highly potent phytofabricated silver nanoparticles (AgNPs) using Sechium edule, which served the purpose of both reducing and capping agent. The designed AgNPs were characterized using UV-Vis spectroscopy, XRD, FTIR, HR-TEM, and TGA techniques. The formation of AgNPs was also confirmed using electrochemistry, which to the best of our knowledge has never been reported before for biosynthesized nanoparticles. The antileishmanial potential of AgNPs was examined on the clinical isolates of Leishmania donovani promastigote cells in an in vitro experimental setting. A dose dependent killing activity of the AgNP was observed with an IC50 value of 51.88 ± 3.51 µg/ml. These results were also compared using commercially available drug, miltefosine. Furthermore, the clinical applicability of AgNP, as antileishmanial agent was proven by testing them against normal mammalian monocyte cell line (U937). The results were statistically analyzed and no significant toxicity of AgNPs on the normal mammalian cells was observed.

Evaluation of CAAX prenyl protease II of Leishmania donovani as potential drug target: Infectivity and growth of the parasite is significantly lowered after the gene knockout.

Prenylation pathway is responsible for post translational modification of various signal proteins, including proteins of Ras superfamily. CAAX prenyl proteases are known to be key players in prenylation pathway. In the current study, we have evaluated CAAX prenyl protease II as a possible drug target against Leishmania donovani parasite, the causative agent of visceral leishmaniasis. Gene knockout strategy was employed to target CAAX prenyl protease II and subsequent effects were studied. CAAX prenyl protease II knockout resulted in significant decrease in growth and infectivity.