Novel chemical scaffold as potential drug against Leishmania donovani: Integrated computational and experimental approaches.

In this study, we have screened a large number of Food and Drug Administration-approved compounds for novel anti-leishmanial molecules targeting the citrate synthase enzyme of the parasite. Based on their docking and molecular dynamic simulation statistics, five compounds were selected. These compounds followed Lipinski's rule of five. Additionally, in vitro, antileishmanial and cytotoxicity studies were performed. The three compounds, Abemaciclib, Bazedoxifene, and Vorapaxar, had shown effective anti-leishmanial activities with IC50 values of 0.92 ± 0.02, 0.65 ± 0.09, and 6.1 ± 0.91 against Leishmania donovani promastigote and with EC50 values of 1.52 ± 0.37, 2.11 ± 0.38, 10.4 ± 1.27 against intramacrophagic amastigote without significantly harming macrophage cells. Among them, from in silico and antileishmanial activities studies, Abemaciclib had been selected based on their less binding energy, good antileishmanial activities, and also a significant difference in their binding energy with human citrate synthase for cell death mechanistic studies using flow cytometry and a DNA fragmentation assay. The action of this compound resulted in an increased reactive oxygen species production, depolarization of mitochondrial membrane potential, DNA damage, and an increase in the sub-G1 cell population. These properties are the hallmarks of apoptosis which were further confirmed by apoptotic assay. Based on the above result, this anticancer compound Abemaciclib could be employed as a potential treatment option for leishmaniasis after further confirmation.

Repurposing the in-house generated Alzheimer's disease targeting molecules through computational and preliminary in-vitro studies for the management of SARS-coronavirus-2.

Covid-19 was declared a world pandemic. Recent studies demonstrated that Covid-19 impairs CNS activity by crossing the blood-brain barrier and ensuing cognitive impairment. In this study, we have utilized Covid-19 main protease (Mpro) as a biological target to repurpose our previously reported multifunctional compounds targeting Alzheimer's disease. Molecular docking, spatial orientation, molecular dynamics simulation, MM-GBSA energy calculation, and DFT studies were carried out with these molecules. Among all the compounds, F27, F44, and F56 exhibited higher binding energy (- 8.03, - 8.65, and - 8.68 kcal/mol, respectively) over the co-crystal ligand O6K (- 7.00 kcal/mol). In MD simulation, compounds F27, F44, and F56 could make a stable complex with Mpro target throughout the simulation. The compounds were synthesized following reported methods and subjected for cytotoxicity, and assessment of their capability to cross the blood-brain barrier in PAMPA assay, and antioxidant property evaluation through DPPH assay. The compounds F27, F44, and F56 exhibited cytocompatibility with the SiHA cell line and also displayed significant antioxidant properties with IC50 = 45.80 ± 0.27 µM, 44.42 ± 0.30 µM, and 42.74 ± 0.23 µM respectively. In the PAMPA assays, the permeability coefficient (Pe) value of F27, F44, and F56 lies in the acceptable range (Pe > 4). The results of the computational and preliminary in-vitro studies strongly corroborate the potential of F27, F44, and F56 as a lead for further optimization in treating the CNS complications associated with Covid-19.

Ligand conjugated lipid-based nanocarriers for cancer theranostics.

Cancer is one of the major health-related issues affecting the population worldwide and subsequently accounts for the second-largest death. Genetic and epigenetic modifications in oncogenes or tumor suppressor genes affect the regulatory systems that lead to the initiation and progression of cancer. Conventional methods, including chemotherapy/radiotherapy/appropriate combinational therapy and surgery, are being widely used for theranostics of cancer patients. Surgery is useful in treating localized tumors, but it is ineffective in treating metastatic tumors, which spread to other organs and result in a high recurrence rate and death. Also, the therapeutic application of free drugs is related to substantial issues such as poor absorption, solubility, bioavailability, high degradation rate, short shelf-life, and low therapeutic index. Therefore, these issues can be sorted out using nano lipid-based carriers (NLBCs) as promising drug delivery carriers. Still, at most, they fail to achieve site-targeted drug delivery and detection. This can be achieved by selecting a specific ligand/antibody for its cognate receptor molecule expressed on the surface of the cancer cells. In this review, we have mainly discussed the various types of ligands used to decorate NLBCs. A list of the ligands used to design nanocarriers to target malignant cells has been extensively undertaken. The approved ligand-decorated lipid-based nanomedicines with their clinical status have been explained in tabulated form to provide a wider scope to the readers regarding ligand-coupled NLBCs.

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.

IFN-γ+ CD4+T cell-driven prophylactic potential of recombinant LDBPK_252400 hypothetical protein of Leishmania donovani against visceral leishmaniasis.

Visceral leishmaniasis (VL) is a potentially fatal parasitic disease causing high morbidity and mortality in developing countries. Vaccination is considered the most effective and powerful tool for blocking transmission and control of diseases. However, no vaccine is available so far in the market for humans. In the present study, we characterized the hypothetical protein LDBPK_252400 of Leishmania donovani (LdHyP) and explored its prophylactic behavior as a potential vaccine candidate against VL. We found reduced hepato-splenomegaly along with more than 50% parasite reduction in spleen and liver after vaccination in mice. Protection in vaccinated mice after the antigen challenge correlated with the stimulation of antigen specific IFN-γ expressing CD4+T cell (~4.6 fold) and CD8+T cells (~2.1 fold) in vaccinated mice in compared to infected mice, even after 2-3 months of immunization. Importantly, antigen-mediated humoral immunity correlated with high antigen specific IgG2/IgG1 responses in vaccinated mice. In vitro re-stimulation of splenocytes with LdHyP enhances the expression of TNF-α, IFN-γ, IL-12 and IL-10 cytokines along with lower IL-4 cytokine and IL-10/IFN-γ ratio in vaccinated mice. Importantly, we observed ~3.5 fold high NO production through activated macrophages validates antigen mediated cellular immunity induction, which is critical in controlling infection progression. These findings suggest that immunization with LdHyP mount a very robust immunity (from IL-10 towards TFN-γ mediated responses) against L. donovani infection and could be explored further as a putative vaccine candidate against VL.

Molecular docking, binding mode analysis, molecular dynamics, and prediction of ADMET/toxicity properties of selective potential antiviral agents against SARS-CoV-2 main protease: an effort toward drug repurposing to combat COVID-19.

The importance of the main protease (Mpro) enzyme of SARS-CoV-2 in the digestion of viral polyproteins introduces Mpro as an attractive drug target for antiviral drug design. This study aims to carry out the molecular docking, molecular dynamics studies, and prediction of ADMET properties of selected potential antiviral molecules. The study provides an insight into biomolecular interactions to understand the inhibitory mechanism and the spatial orientation of the tested ligands and further, identification of key amino acid residues within the substrate-binding pocket that can be applied for structure-based drug design. In this regard, we carried out molecular docking studies of chloroquine (CQ), hydroxychloroquine (HCQ), remdesivir (RDV), GS441524, arbidol (ARB), and natural product glycyrrhizin (GA) using AutoDock 4.2 tool. To study the drug-receptor complex's stability, selected docking possesses were further subjected to molecular dynamics studies with Schrodinger software. The prediction of ADMET/toxicity properties was carried out on ADMET Prediction™. The docking studies suggested a potential role played by CYS145, HIS163, and GLU166 in the interaction of molecules within the active site of COVID-19 Mpro. In the docking studies, RDV and GA exhibited superiority in binding with the crystal structure of Mpro over the other selected molecules in this study. Spatial orientations of the molecules at the active site of Mpro exposed the significance of S1-S4 subsites and surrounding amino acid residues. Among GA and RDV, RDV showed better and stable interactions with the protein, which is the reason for the lesser RMSD values for RDV. Overall, the present in silico study indicated the direction to combat COVID-19 using FDA-approved drugs as promising agents, which do not need much toxicity studies and could also serve as starting points for lead optimization in drug discovery.

Identification of high affinity and low molecular alternatives of boceprevir against SARS-CoV-2 main protease: A virtual screening approach.

SARS-CoV-2 has posed global challenge for healthcare due to COVID-19. The main protease (Mpro) of this virus is considered as a major target for drug development efforts. In this work, we have used virtual screening approach with molecular dynamics simulations to identify high affinity and low molecular weight alternatives of boceprevir, a repurposed drug currently being evaluated against Mpro. Out of 180 compounds screened, two boceprevir analogs (PubChem ID: 57841991 and 58606278) were reported as potential alternatives with comparable predicted protease inhibitor potential and pharmacological properties. Further experimental validation of the reported compounds may contribute to the ongoing investigation of boceprevir.

Potential alternatives to current cholinesterase inhibitors: an in silico drug repurposing approach.

Acetylcholinesterase/Butyrylcholinesterase inhibitors are considered an effective method for treating Alzheimer's disease (AD). In this current work, we have computationally analyzed 11 new small molecule drugs used in various neurological diseases and Donepezil, a known inhibitor of acetylcholinesterase, as a positive control. We investigated these drugs for possible fundamental interactions with acetylcholinesterase and butyrylcholinesterase as both are critical in the pathophysiology of Alzheimer's disease. We have selected FDA approved compounds for repurposing as possible inhibitors of these enzymes and novel therapeutic option for Alzheimer's disease. We selected the top two molecules for each protein for their binding energies, interactions, and Donepezil, the most commonly used drug for AD treatment. Molecular simulation and dynamics studies of the top 2 drugs in each case and free energy analysis helped us reach further conclusions about the best possible drugs for repurposing. Brexipirazole and Deutetrabenazine produce encouraging results as butyrylcholinesterase and acetylcholinesterase inhibitors, respectively.

Structure-guided approach to identify a novel class of anti-leishmaniasis diaryl sulfide compounds targeting the trypanothione metabolism.

Leishmania protozoans are the causative agent of leishmaniasis, a neglected tropical disease consisting of three major clinical forms: visceral leishmaniasis (VL), cutaneous leishmaniasis, and mucocutaneous leishmaniasis. VL is caused by Leishmania donovani in East Africa and the Indian subcontinent and by Leishmania infantum in Europe, North Africa, and Latin America, and causes an estimated 60,000 deaths per year. Trypanothione reductase (TR) is considered to be one of the best targets to find new drugs against leishmaniasis. This enzyme is fundamental for parasite survival in the human host since it reduces trypanothione, a molecule used by the tryparedoxin/tryparedoxin peroxidase system of Leishmania to neutralize the hydrogen peroxide produced by host macrophages during infection. Recently, we solved the X-ray structure of TR in complex with the diaryl sulfide compound RDS 777 (6-(sec-butoxy)-2-((3-chlorophenyl)thio)pyrimidin-4-amine), which impairs the parasite defense against the reactive oxygen species by inhibiting TR with high efficiency. The compound binds to the catalytic site and engages in hydrogen bonds the residues more involved in the catalysis, namely Glu466', Cys57 and Cys52, thereby inhibiting the trypanothione binding. On the basis of the RDS 777-TR complex, we synthesized structurally related diaryl sulfide analogs as TR inhibitors able to compete for trypanothione binding to the enzyme and to kill the promastigote in the micromolar range. One of the most active among these compounds (RDS 562) was able to reduce the trypanothione concentration in cell of about 33% via TR inhibition. RDS 562 inhibits selectively Leishmania TR, while it does not inhibit the human homolog glutathione reductase.

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.

Geobacillus yumthangensis sp. nov., a thermophilic bacterium isolated from a north-east Indian hot spring.

A thermophilic, spore-forming, rod-shaped bacterium isolated from the Yumthang hot spring in North Sikkim, India was subjected to taxonomic studies. The thermophilic bacterial isolate was designated as strain AYN2T. Cells were Gram-stain-positive, aerobic, motile, rod-shaped, catalase-positive and methyl red-negative. Strain AYN2T was able to grow in the pH range from 6 to 10 (optimum, pH 7.5-8.0), at 40-70 °C (60 °C) and in NaCl concentrations of 0-4 % (1 %). The major cellular fatty acids were iso-C15 : 0 (12.8 %), iso-C16 : 0 (13.9 %) and iso-C17 : 0 (13.8 %). No matches were found in the rtsba6 Sherlock libraries. The G+C content of the genomic DNA was 42.11 mol%. Based on phylogenetic analysis of the 16S rRNA gene sequences, strain AYNT showed highest sequence similarity to the type strain of Geobacillus toebii (96 %). However, the phenotypic properties of strain AYN2T were clearly distinct from those of G. toebii and related species. On the basis of polyphasic analysis, strain AYN2T represents a novel species in the genus Geobacillus, for which the name Geobacillus yumthangensis sp. nov. is proposed. The type strain is AYN2T(MTCC=12749=KCTC=33950= JCM 32596).

Understanding the importance of conservative hypothetical protein LdBPK_070020 in Leishmania donovani and its role in subsistence of the parasite.

The genome of Leishmania donovani, the causative agent of visceral leishmaniasis, codes for approximately 65% of both conserved and non-conserved hypothetical proteins. Studies on 'conserved hypothetical' proteins are expected to reveal not only new and crucial aspects of Leishmania biochemistry, but it could also lead to discovery of novel drug candidates. Conserved hypothetical protein, LdBPK_070020, is a 31.14 kDa protein, encoded by an 810 bp gene. BLAST analysis of LdBPK_070020, performed against NCBI non-redundant database, showed 80-99% similarity with conserved hypothetical proteins of Leishmania belonging to other species. Using homologues recombination method, we have performed gene knockout of LdBPK_070020 and effects of the same were investigated on the parasite. The gene knocked out strain shows significant retardation in growth with respect to wild type. Detailed biochemical studies indicated towards important role of LdBPK_070020 in the parasite survival and growth.

Novel Agents against Miltefosine-Unresponsive Leishmania donovani.

Visceral leishmaniasis is a deadly endemic disease. Unresponsiveness to the only available oral drug miltefosine poses a big challenge for the chemotherapy of the disease. We report a novel molecule, PS-203 {4-(4,4,8-trimethyl-7-oxo-3-oxabicyclo[3.3.1]non-2-yl)-benzoic acid methyl ester}, as effective against a miltefosine-unresponsive strain of the parasite. Further, combinations of PS-203 with miltefosine were also evaluated and showed promising results against a miltefosine-unresponsive strain.

Probing the molecular mechanism of hypericin-induced parasite death provides insight into the role of spermidine beyond redox metabolism in Leishmania donovani.

Hypericin, a natural compound from Hypericum perforatum (St. John's wort), has been identified as a specific inhibitor of Leishmania donovani spermidine synthase (LdSS) using integrated computational and biochemical approaches. Hypericin showed in vitro inhibition of recombinant LdSS enzyme activity. The in vivo estimation of spermidine levels in Leishmania promastigotes after hypericin treatment showed significant decreases in the spermidine pools of the parasites, indicating target specificity of the inhibitor molecule. The inhibitor, hypericin, showed significant antileishmanial activity, and the mode of death showed necrosis-like features. Further, decreased trypanothione levels and increased glutathione levels with elevated reactive oxygen species (ROS) levels were observed after hypericin treatment. Supplementation with trypanothione in the medium with hypericin treatment restored in vivo trypanothione levels and ROS levels but could not prevent necrosis-like death of the parasites. However, supplementation with spermidine in the medium with hypericin treatment restored in vivo spermidine levels and parasite death was prevented to a large extent. The data overall suggest that the parasite death due to spermidine starvation as a result of LdSS inhibition is not related to elevated levels of reactive oxygen species. This suggests the involvement of spermidine in processes other than redox metabolism in Leishmania parasites. Moreover, the work provides a novel scaffold, i.e., hypericin, as a potent antileishmanial molecule.

An insight into plant lipase research - challenges encountered.

Lipases from bacterial, fungal, and animal sources have been purified to homogeneity with very few of them being contributed from plants. Plant lipases are mostly found in energy reserve tissues, for example, oilseeds. They act as biocatalysts which are attractive due to their high substrate specificity, low production cost and easy pharmacological acceptance due to their eukaryotic origin. Hence plant lipases represent better potential for commercial applications in organic synthesis, food, detergent and pharmacological industries. However, low expression, uneconomical fold purity and the plethora of difficulties related to their recombinant expression has been limiting their commercial applicability and posing challenges to many researchers. This article focuses on comprehensive approaches that have been reported to date to address these challenges.

Procerain B, a cysteine protease from Calotropis procera, requires N-terminus pro-region for activity: cDNA cloning and expression with pro-sequence.

We have previously reported isolation and characterization of a novel plant cysteine protease, Procerain B, from the latex of Calotropis procera. Our initial attempts for active recombinant Procerain B in Escherichiacoli expression system was not successful. The reason for inactive enzyme production was attributed to the absence of 5' pro-region in the Procerain B cDNA that may be involved in proper folding and production of mature active protein. The current manuscript reports the cloning of full length Procerain B for the production of the active protein. The complete cDNA sequence of Procerain B with pro-region sequence was obtained by using RNA ligase mediated rapid amplification of 5' cDNA ends (RLM-RACE). The N-terminus pro-sequence region consists of 127 amino acids and characterized as the member of inhibitory I29 family. Further the three dimensional structure of full length Procerain B was modelled by homology modelling using X-ray crystal structure of procaricain (PDB ID: 1PCI). N-terminus pro-sequence of full length Procerain B runs along the active site cleft. Full length Procerain B was expressed in prokaryotic system and activated in vitro at pH 4.0. This is the first study reporting the production of active recombinant cysteine protease from C.procera.

A leishmaniasis study: structure-based screening and molecular dynamics mechanistic analysis for discovering potent inhibitors of spermidine synthase.

Protozoa Leishmania donovani (Ld) is the main cause of the endemic disease leishmaniasis. Spermidine synthase (SS), an important enzyme in the synthetic pathway of polyamines in Ld, is an essential element for the survival of this protozoan. Targeting SS may provide an important aid for the development of drugs against Ld. However, absence of tertiary structure of spermidine synthase of Leishmania donovani (LSS) limits the possibilities of structure based drug designing. Presence of the same enzyme in the host itself further challenges the drug development process. We modeled the tertiary structure of LSS using homology modeling approach making use of homologous X-ray crystallographic structure of spermidine synthase of Trypanosoma cruzi (TSS) (2.5Å resolution). The modeled structure was stabilized using Molecular Dynamics simulations. Based on active site structural differences between LSS and human spermidine synthase (HSS), we screened a large dataset of compounds against modeled protein using Glide virtual screen docking and selected two best inhibitors based on their docking scores (-10.04 and -13.11 respectively) with LSS and having least/no binding with the human enzyme. Finally Molecular Dynamics simulations were used to assess the dynamic stability of the ligand bound structures and to elaborate on the binding modes. This article is part of a Special Issue entitled: Computational Methods for Protein Interaction and Structural Prediction.

Exploring critical determinants of protein amyloidogenesis: a review.

The transformation of polypeptide chains from their globular native structure to fibrillar aggregates has been a matter of great concern because of the involvement of these aggregates in the onset of several degenerative diseases. These aggregates exhibit highly ordered cross ß sheet structures and are known as 'amyloids'. Formation of amyloids in the body is associated with cytotoxicity due to direct interaction of the aggregated species with the cell membrane leading to cellular permeability or due to loss of functionality of the proteins involved in the amyloid formation. The preference of polypeptide chains to remain in their native conformation or to aggregate into amyloids is guided by several factors such as its conformation at specific condition, concentration, physicochemical properties of the amino acid sequence and so on. In the current review, we have reviewed the different factors that guide the transition of proteins from their natively folded state to the amyloidogenic state. Understanding the critical determinants of amyloidogenesis is vital towards deciphering the molecular mechanism of amyloidogenesis and for the development of effective therapeutics against amyloidosis.

Evaluation of a diospyrin derivative as antileishmanial agent and potential modulator of ornithine decarboxylase of Leishmania donovani.

World health organization has called for academic research and development of new chemotherapeutic strategies to overcome the emerging resistance and side effects exhibited by the drugs currently used against leishmaniasis. Diospyrin, a bis-naphthoquinone isolated from Diospyros montana Roxb., and its semi-synthetic derivatives, were reported for inhibitory activity against protozoan parasites including Leishmania. Presently, we have investigated the antileishmanial effect of a di-epoxide derivative of diospyrin (D17), both in vitro and in vivo. Further, the safety profile of D17 was established by testing its toxicity against normal macrophage cells (IC50∼20.7 µM), and also against normal BALB/c mice in vivo. The compound showed enhanced activity (IC50∼7.2 µM) as compared to diospyrin (IC50∼12.6 µM) against Leishmania donovani promastigotes. Again, D17 was tested on L. donovani BHU1216 isolated from a sodium stibogluconate-unresponsive patient, and exhibited selective inhibition of the intracellular amastigotes (IC50∼0.18 µM). Also, treatment of infected BALB/c mice with D17 at 2mg/kg/day reduced the hepatic parasite load by about 38%. Subsequently, computational docking studies were undertaken on selected enzymes of trypanothione metabolism, viz. trypanothione reductase (TryR) and ornithine decarboxylase (ODC), followed by the enzyme kinetics, where D17 demonstrated non-competitive inhibition of the L. donovani ODC, but could not inhibit TryR.

Krebs cycle enzymes for targeted therapeutics and immunotherapy for anti-leishmanial drug development using: Pathways, potential targets, and future perspectives.

Leishmaniasis is a parasitic and neglected tropical disease which majorly impacts poor and developing nations. One of the significant factors that impacts the severity of the pathological condition includes the socioeconomic background of the affected region. The rise of drug-resistant Leishmania is a serious concern for the effectiveness of the present treatment. As a result, the drug options need to be relooked immediately. Leishmania employs Krebs cycle intermediates for its needs after infection for establishing various defense mechanisms to escape the host immune responses. Nevertheless, a variety of immunological reactions are also seen during infection, which clear the parasites. One of the more promising strategies in this regard would involve combining targeted therapy and immunotherapy. The targeted treatments work by obstructing vital pathways that are required for Leishmania to grow and survive. The mechanism of action of immunotherapy is the control of the host immune response, which entails the blockage of molecular pathways essential for the growth and maintenance of the parasite. The Krebs cycle intermediates have important biochemical roles. Additionally, in macrophages and dendritic cells, they play roles as signalling molecules for controlling inflammatory responses. The review brings together the available literature about the importance of Krebs cycle metabolites as potential treatment targets for leishmaniasis.