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The ever-evolving and versatile VLP technology is becoming an increasingly popular area of science. This study presents surface decorated reporter-tagged VLPs of CHIKV, an enveloped RNA virus of the genus alphavirus and its applications. Western blot, IFA and live-cell imaging confirm the expression of reporter-tagged CHIK-VLPs from transfected HEK293Ts. CryoEM micrographs reveal particle diameter as ∼67nm and 56-70 nm, respectively, for NLuc CHIK-VLPs and mCherry CHIK-VLPs. Our study demonstrates that by exploiting NLuc CHIK-VLPs as a detector probe, robust ratiometric luminescence signal in CHIKV-positive sera compared to healthy controls can be achieved swiftly. Moreover, the potential activity of the Suramin drug as a CHIKV entry inhibitor has been validated through the reporter-tagged CHIK-VLPs. The results reported in this study open new avenues in the eVLPs domain and offer potential for large-scale screening of clinical samples and antiviral agents targeting entry of CHIKV and other alphaviruses.

The nucleotide-binding pockets (NBPs) in virus-specific proteins have proven to be the most successful antiviral targets for several viral diseases. Functionally important NBPs are found in various structural and non-structural proteins of SARS-CoV-2. In this study, the first successful multi-targeting attempt to identify effective antivirals has been made against NBPs in nsp12, nsp13, nsp14, nsp15, nsp16, and nucleocapsid (N) proteins of SARS-CoV-2. A structure-based drug repurposing in silico screening approach with ADME analysis identified small molecules targeting NBPs in SARS-CoV-2 proteins. Further, isothermal titration calorimetry (ITC) experiments validated the binding of top hit molecules to the purified N-protein. Importantly, cell-based antiviral assays revealed antiviral potency for INCB28060, darglitazone, and columbianadin with EC

In present work, the recombinant cytoplasmic 2-Cys peroxiredoxin from Citrus sinensis (CsPrx) was purified and characterized. The peroxidase activity was examined with different substrates using DTT, a non-physiological electron donor. The conformational studies, in oxidized and reduced states, were performed using circular dichroism (CD) and fluorescence measurement. The CD analysis showed higher α-helical content for reduced state of the protein. The thermal stability studies of CsPrx by Differential Scanning Calorimetry (DSC) showed that oxidized state is more stable as compared to the reduced state of CsPrx. In vitro studies showed that the CsPrx provides a protective shield against ROS and free radicals that participate in the degradation of plasmid DNA. The pre-treatment of 10 μM CsPrx provide almost 100% protection against peroxide-mediated cell killing in the Vero cells. CsPrx showed significant cell proliferation and wound healing properties. The superior morphology of viable cells and wound closure was found at 20 μM CsPrx treated for 12 h. The results demonstrated that CsPrx is a multifaceted protein with a significant role in cell proliferation, wound healing and protection against hydrogen peroxide-induced cellular damage. This could be the first report of a plant peroxiredoxin being characterized for biomedical applications.

Alphaviruses are continuously re-emerging and pose a global threat to human health and currently no antiviral drug is commercially available for alphaviral infections. Alphavirus non-structural protein nsP4, which possesses RNA-dependent RNA polymerase (RdRp) activity, is a potential antiviral target. To date, no antiviral drug is commercially available against alphaviruses. Since RdRp is the key virus-specific enzyme involved in viral genome replication, this study identifies and validates the antiviral efficacy of small molecules targeting alphavirus RdRp. Purified nsP4 was characterized using the surface plasmon resonance (SPR) assay, and the binding affinities of divalent metal ions, ribonucleotides, and in vitro transcribed viral RNA oligonucleotides were obtained in the micromolar (μm) range. Further, four potential inhibitors, piperine (PIP), 2-thiouridine (2TU), pyrazinamide (PZA), and chlorogenic acid (CGA), were identified against nsP4 RdRp using a molecular docking approach. The SPR assay validated the binding of PIP, 2TU, PZA, and CGA to purified nsP4 RdRp with K

The reemergence of SARS-CoV named, as SARS-CoV-2 has been highly infectious and able to infect a large population around the globe. The World Health Organization (WHO) has declared this SARS-CoV-2 associated Coronavirus Disease 2019 (COVID-19) as pandemic. SARS-CoV-2 genome is translated into polyproteins and has been processed by its protease enzymes. 3CLprotease is named as main protease (Mpro) enzyme which cleaves nsp4-nsp16. This crucial role of Mpro makes this enzyme a prime and promising antiviral target. The drug repurposing is a fast alternative method than the discovery of novel antiviral molecules. We have used high-throughput virtual screening approach to examine FDA approved LOPAC1280 library against Mpro. Primary screening have identified few potential drug molecule for the target among which 10 molecules were studied further. Molecular docking of selected molecules was done to detailed study about their binding energy and binding modes. Positively, Etoposide, BMS_195614, KT185, Idarubicin and WIN_62577 were found interacting with substrate binding pocket of Mpro with higher binding energy. These molecules are being advanced by our group for in vitro and in vivo testing to study the efficacy of identified drugs. As per our understanding, these molecules have the potential to efficiently interrupt the viral life cycle and may reduce or eliminate the expeditious outspreading of SARS-CoV-2.

BACKGROUND: Emergence of new pathogenic viruses along with adaptive potential of RNA viruses has become a major public health concern. Hence it becomes even more important to explore and evaluate the antiviral properties of nanocomposites which is an ever-evolving field of medical biology. METHODS: In this study, series of metal/metal oxide (Ag/NiO : NiO, AN-5%, AN-10% and AN-15%) and ternary metal oxide nanocomposites (Ag2O/NiO/ZnO : N/Z, A/N/Z-1, A/N/Z-2 and A/N/Z-3) have been synthesized and characterized. Cellular uptake of nanocomposites was confirmed by ICP-MS. RESULTS: Intriguingly, molecular docking of metal oxides in the active site of nsP3 validated the binding of nanocomposites to chikungunya virus replication protein nsP3. In-vitro antiviral potential of nanocomposites were tested by performing plaque reduction assay, cytopathic effect (CPE) analysis and qRT-PCR. The nanocomposites showed significant reduction in virus titre. Half-maximal inhibitory concentration (IC50) for A/N/Z-3 and AN-5% were determined to be 2.828 and 3.277 g/mL, respectively. CPE observation and qRT-PCR results were consistent with the data obtained from plaque reduction assay for A/N/Z-3 and AN-5%. CONCLUSION: These results, have opened new avenues for development of nanocomposites based antiviral therapies.

The SARS-CoV-2 pandemic has reinforced efforts for developing effective vaccination strategy for existing and emerging viruses. Currently there are various vaccine technology available for treating viral diseases, however it is imperative to develop and investigate second-generation vaccines such as chimeric virus-like particles (chi-VLPs) vaccine for increased immunogenicity, ease of production and scalability to supplement the worldwide vaccine supply. Intriguingly, chi-VLPs expresses more than one antigenic epitope on its surface, hence it is expected to be a more effective vaccine candidate. Hereby, this study reports, a novel bivalent vaccine design of chimeric alphavirus coronavirus virus-like particles (ChAC-VLPs), displaying fusion glycoproteins of CHIKV and receptor binding domain (RBD) of SARS-CoV-2 on its surface. Uniqueness and versatility of ChAC-VLPs has been demonstrated via a various techniques including Western blot, Immunofluorescence, cryoEM, and dynamic light scattering (DLS). The multimeric epitope display of immunogenic antigens, i.e CHIKV envelop glycoprotein and SARS-CoV-2 RBD was validated by cell-based assays. ChAC-VLP immunized mice has shown substantial neutralization titres for CHIKV (PRNT50 of 1:25) from the serum collected after 2ndbooster doses. Similarly, serum antibodies were detected for SARS-CoV2 RBD as observed by antigen specific ELISA and validated using surface plasmon resonance (SPR). SPR binding response was detected to be >200 RU for anti-RBD antibody in post-immunized mice sera. In conclusion, present study proposes ChAC-VLPs as a potential hybrid vaccine candidate for CHIKV and SARS-CoV-2 infection and contributes valuable insights in chi-VLPs domain.

Emerging variants of SARS-CoV-2 still threaten the effectiveness of currently deployed vaccines, and antivirals can prove to be an effective therapeutic option for attenuating it. The papain-like protease (PLpro) is an attractive target due to its sequence conservation and critical role in the replication and pathogenesis of SARS-CoV-2. PLpro also plays very important role in modulation of host immune responses by deubiquitinating (DUBs) or deISGylating host proteins. Thus, targeting PLpro serves as a two-pronged approach to abate SARS-CoV-2. Due to its structural and functional similarities with the host DUB enzymes, anin-houselibrary of DUB inhibitors was constituted in this study. Five promising compounds exhibiting high binding affinities with the substrate binding site of PLpro were identified from a library of 81 compounds within silicoscreening, docking, and simulation studies. Interestingly, lithocholic acid, linagliptin, teneligliptin, and flupenthixol significantly inhibited the proteolytic activity of PLpro. Each of these compounds abrogatedin vitroreplication of SARS-CoV-2 with EC50values in the range of 5-21 μM. In addition, crystal structure of SARS-CoV-2 PLpro and its complex with inhibitors have been determined that revealed their inhibitory mechanism. The findings of this study provide the proof-of-principle that the DUB inhibitors hold high potential as a new class of therapeutics against SARS-CoV-2. Additionally, this is the first study that has opened a new avenue towards not only targeting PLpro active site but also simultaneously directing towards restoration of antiviral immune response of the host for deterring SARS-CoV-2.Graphical abstract

Molecular interactions of Chikungunya virus (CHIKV) non-structural protein 3 (nsP3) with GTPase Activating Protein SH3 Domain Binding Protein 1 (G3BP1) host protein is important for efficient replication of CHIKV. CHIKV nsP3 protein binds to the nuclear transport factor 2 (NTF2) -like domain of G3BP1 via two tandem FGDF motifs and disrupts the stress granules (SGs) formation. Interestingly, this makes the G3BP1 host protein an additional drug target for antiviral research. In this study, seven potential small molecules-targeting FGDF motif binding pocket of G3BP1 have been identified using structure-based virtual screening approach. Binding energies and binding modes of the molecules were further analyzed in detail through molecular docking, dynamics and simulations. Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) experiments confirmed the binding of identified molecules to purified G3BP1 with binding affinities in micromolar (μM) range. The antiviral efficacy of molecules targeting G3BP1 was evaluated byin vitrocell culture-based antiviral studies. All seven molecules L-7, WIN, SB2, NAL, DHD, GSK and FLU effectively diminished the CHIKV replication with EC50values of 1.996, 0.403, 5.387, 1.528, 7.394, 3.664, and 0.618 µM, respectively. Moreover, the CHIKV infected cells treated with these molecules seemed to have fewer virus-induced SGs than virus infected control cells. Interestingly, the inhibitors showed no adverse effect on SG formation in oxidative stress condition. It further substantiated that these inhibitors effectively bind to the NTF2-like domain of G3BP1, obstruct the nsP3-G3BP1 interactions, and halts virus replication. This is the first report of small molecules targeting G3BP1, the host protein playing a crucial role in CHIKV viral replication and in the formation of SGs for host antiviral response.

Capsid protein (CP) of Chikungunya virus (CHIKV) is a multifunctional protein with a conserved hydrophobic pocket that plays a crucial role in the capsid assembly and virus budding process. This study demonstrates antiviral activity of thymoquinone (TQ), a natural compound targeting the hydrophobic pocket of CP. The binding of TQ to the hydrophobic pocket of CHIKV CP was analysed by structure-based molecular docking, isothermal titration calorimetry and fluorescence spectroscopy. The binding constant KD obtained for TQ was 27 μM. Additionally, cell-based antiviral studies showed that TQ diminished CHIKV replication with an EC50 value 4.478 μM. Reduction in viral RNA copy number and viral replication as assessed by the qRT-PCR and immunofluorescence assay, confirmed the antiviral potential of TQ. Our study reveals that TQ is an effective antiviral targeting the hydrophobic pocket of CHIKV CP and may serve as the basis for development of a broad-spectrum therapy against alphaviral diseases.

Arthropod-borne viruses of the alphavirus and flavivirus genera are human pathogens of significant concern, and currently, no specific antiviral treatment is available for these viruses. This study has elucidated the antiviral mechanisms of natural small molecules against the dengue (DENV) and chikungunya virus (CHIKV). Herbacetin (HC) and Caffeic acid phenethyl ester (CAPE) depleted polyamine levels in Vero cells, which has been demonstrated by thin-layer chromatography (TLC). As polyamines play an essential role in the replication and transcription of RNA viruses, the depletion of polyamines by HC and CAPE was anticipated to inhibit the virus replication. To test this hypothesis, HC and CAPE were evaluated for antiviral activities using a cell-based virus yield assay by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), plaque reduction assay, and immunofluorescence assay (IFA). HC and CAPE displayed potent inhibition with EC50of 463 nM and 0.417 nM for CHIKV and 8.5 µM and 1.15 µM for DENV, respectively. However, the addition of exogenous polyamines did not ultimately rescue the virus titer in both CHIKV and DENV-infected cells. This finding suggested additional antiviral mechanisms for HC and CAPE. Further,in silicoanalysis revealed that HC and CAPE may directly target the viral methyltransferases (MTase) of CHIKV and DENV. The inhibition of virus-specific MTases by HC and CAPE was confirmed using purified viral MTase of CHIKV and DENV. Altogether, the dual targeting of the host pathway and the viral MTase using potent natural antiviral molecules is expected to facilitate the development of effective biological therapies.IMPORTANCEVector-borne diseases caused by DENV and CHIKV has a catastrophic impact on human health worldwide. There are no effective vaccines and antiviral drugs present in the market till date against these viruses. In the present study, natural small molecules have been identified as antivirals against DENV and CHIKV. These molecules directly inhibit the virus and impede the synthesis of essential host molecules required for efficient virus replication. Hence, these broad-spectrum antivirals have high therapeutic potential.

Alphaviruses cause animal or human diseases that are characterized by febrile illness, debilitating arthralgia, or encephalitis. Selective estrogen receptor modulators (SERMs), a class of FDA-approved drugs, have been shown to possess antiviral activities against multiple viruses, including Hepatitis C virus, Ebola virus, dengue virus, and vesicular stomatitis virus. Here, we evaluated three SERM compounds, namely 4-hydroxytamoxifen, tamoxifen, and clomifene, for plausible antiviral properties against two medically important alphaviruses, chikungunya virus (CHIKV) and Sindbis virus (SINV). In cell culture settings, these SERMs displayed potent activity against CHIKV and SINV at non-toxic concentrations with EC50 values ranging between 400 nM and 3.9 μM. Further studies indicated that these compounds inhibit a post-entry step of the alphavirus life cycle, while enzymatic assays involving purified recombinant proteins confirmed that these SERMs target the enzymatic activity of non-structural protein 1 (nsP1), the capping enzyme of alphaviruses. Finally, tamoxifen treatment restrained CHIKV growth in the infected mice and diminished musculoskeletal pathologies. Combining biochemical, cell culture-based studies, and in vivo analyses, we strongly argue that SERM compounds, or their derivatives, may provide for attractive therapeutic options against alphaviruses.

SARS-CoV-2 nucleocapsid protein (N-protein) is a virus specific multitasking protein, responsible for recognition and encapsidation of the viral genome. The N-terminal domain (NTD) of N-protein has a major role of packaging viral RNA genome into a long helical nucleocapsid structure. In this study, using structure-based drug repurposing strategy, small molecules from a FDA approved, natural product, and LOPAC1280 libraries have been virtually screened against the RNA binding pocket of SARS-CoV-2 NTD and twelve candidate molecules with high binding affinity were identified. Highly sensitive isothermal titration calorimetry (ITC) method was utilized to confirm binding of these molecules to purified NTD protein. In vitro cell-based SARS-CoV-2 antiviral assays demonstrate that nine of these identified molecules are highly efficacious in inhibiting virus replication with half maximal effective concentration (EC50) ranging from 0.98 μM-10 μM. FDA approved drugs: Telmisartan, an angiotensin II type 1 (AT1) receptor antagonist used in the management of hypertension and Bictegravir, an HIV-1 integrase inhibitor showed significant inhibitory activity against SARS-CoV-2 with a EC50 values of 1.02 μM and 8.11 μM respectively. Additionally, Bisdemethoxycurcumin, a natural analogue of curcumin and MCC-555, an antidiabetic drug exerted antiviral activity with EC50 values of 1.64 μM and 4.26 μM, respectively. Taken together, this is the first report of drug molecules targeting the NTD of SARS-CoV-2 N-protein and the data presented in this study exhibit high potential for development of COVID-19 therapy based on drug repurposing.

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDOKF1 from Comamonas testosteroni KF1 and found that it had an apparent kcat/Km for phthalate of 0.58 ± 0.09 μM−1s−1, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 A resolution revealed that it is a hexamer comprising two stacked α3 trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDOKF1 larger than that of other characterized ROs. Complexes of PDOKF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.

The native pumpkin 2S albumin, a multifunctional protein, possess a variety of potential biotechnologically exploitable properties. The present study reports the characterization of recombinant pumpkin 2S albumin (rP2SA) and unraveling of its potential DNA/RNA binding site. The purification and characterization of the rP2SA established that it retains the characteristic α-helical structure and exhibited comparable DNase, RNase, antifungal and anti-proliferative activities as native protein. In vitro studies revealed that rP2SA exhibits potent antiviral activity against chikungunya virus (CHIKV) at a non-toxic concentration with an IC50 of 114.5 μg/mL. In silico studies and site-directed mutagenesis were employed to unravel the potential DNA/RNA binding site. A strong positive charge distribution due to presence of many arginine residues in proximity of helix 5 was identified as a potential site. The two of the arginine residues, conserved in some 2S albumins, were selected for the mutation studies. The mutated forms of recombinant protein (R84A and R91A) showed a drastic reduction in DNase and RNase activities suggesting their presence at binding site and involvement in the nuclease activity. A metal binding site was also identified adjacent to DNA/RNA binding site. The present study demonstrated the structural and functional integrity of the rP2SA and reports potential antiviral activity against CHIKV. Further, potential DNA/RNA binding site was unraveled through mutation studies and bioinformatics analysis.

Graphical abstract, The ongoing COVID-19 pandemic, periodic recurrence of viral infections, and the emergence of challenging variants has created an urgent need of alternative therapeutic approaches to combat the spread of viral infections, failing to which may pose a greater risk to mankind in future. Resilience against antiviral drugs or fast evolutionary rate of viruses is stressing the scientific community to identify new therapeutic approaches for timely control of disease. Host metabolic pathways are exquisite reservoir of energy to viruses and contribute a diverse array of functions for successful replication and pathogenesis of virus. Targeting the host factors rather than viral enzymes to cease viral infection, has emerged as an alternative antiviral strategy. This approach offers advantage in terms of increased threshold to viral resistance and can provide broad-spectrum antiviral action against different viruses. The article here provides substantial review of literature illuminating the host factors and molecular mechanisms involved in innate/adaptive responses to viral infection, hijacking of signalling pathways by viruses and the intracellular metabolic pathways required for viral replication. Host-targeted drugs acting on the pathways usurped by viruses are also addressed in this study. Host-directed antiviral therapeutics might prove to be a rewarding approach in controlling the unprecedented spread of viral infection, however the probability of cellular side effects or cytotoxicity on host cell should not be ignored at the time of clinical investigations.

Alphaviruses, including Chikungunya (CHIKV) and Venezuelan equine encephalitis virus (VEEV), are among the leading causes of recurrent epidemics all over the world. Alphaviral nonstructural protein 1 (nsP1) orchestrates the capping of nascent viral RNA via its S-adenosyl methionine-dependent N-7-methyltransferase (MTase) and guanylyltransferase activities. Here, we developed and validated a novel capillary electrophoresis (CE)-based assay for measuring the MTase activity of purified VEEV and CHIKV nsP1. We employed the assay to assess the MTase inhibition efficiency of a few adenosine analogs and identified 5-iodotubercidin (5-IT) as an inhibitor of nsP1. The antiviral potency of 5-IT was evaluated in vitro using a combination of cell-based assays, which suggest that 5-IT is efficacious against CHIKV in cell culture (EC50 : 0.409 µm).

Antiviral therapy is crucial for the circumvention of viral epidemics. The unavailability of a specific antiviral drug against the chikungunya virus (CHIKV) disease has created an alarming situation to identify or develop potent chemical molecules for remedial management of CHIKV. In the present investigation, in silico studies of dihydrorugosaflavonoid derivatives (5a-f) with non-structural protein-3 (nsP3) were carried out. nsP3 replication protein has recently been considered as a possible antiviral target in which crucial inhibitors fit into the adenosine-binding pocket of the macrodomain. The 4'-halogenated dihydrorugosaflavonoids displayed intrinsic binding with the nsp3 macrodomain (PDB ID: 3GPO) of CHIKV. Compounds 5c and 5d showed docking scores of -7.54 and -6.86 kcal mol-1, respectively. Various in vitro assays were performed to confirm their (5a-f) antiviral potential against CHIKV. The non-cytotoxic dose was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and was found to be

With increasing environment pollution and bacterial transmitted viral diseases globally, the development of new, effective, and low-cost materials/strategies is the current major challenge. To combat with this alarming problem, three new multi-functional and thermally stable SnO2NPs@ZIF-8 composites (NC1, NC2, and NC3) were synthesized by a facile and sustainable approach involving in situ encapsulation of SnO2NPs (150, 300, and 500 μL suspension in methanol) within zeolitic imidazole framework at room temperature. The morphology and crystallinity of ZIF-8 remained unchanged upon the proper encapsulation of SnO2NPs in its matrix. Herein, for the first time, the antiviral potential of ZIF-8 and SnO2NPs@ZIF-8 against chikungunya virus is reported by investigating their cytotoxicity against Vero cell line (employing MTT ((3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)) assay). The maximum non-toxic doses were 0.04 mg mL−1 for ZIF-8 and SnO2NPs@ZIF-8 and 0.1 mg mL−1 for SnO2NPs. Further, NC1 exhibited (based on plaque assay) reduction in viral load/titers up to > 80% during post-treatment and > 50% during pre-treatment, greater than that of ZIF-8 and SnO2NPs due to synergistic effect. Further, NC1 (10 mg) exhibited enhanced photocatalytic efficiency (≥ 96%) for degradation of methylene blue (0.5 × 10−5 M) at pH ˃ 7.0. The probable mechanism for their antiviral activity and photocatalytic activity has been discussed. The multi-functional composites can effectively be used to reduce water pollution and as remedy for mosquito/bacterial transmitted viral diseases.

Klebsiella pneumonia is known to cause several nosocomial infections in immunocompromised patients. It has developed resistance against a broad range of presently available antibiotics, resulting in high mortality rates in patients and declared an urgent threat. Therefore, exploration of possible novel drug targets against this opportunistic bacteria needs to be undertaken. In the present study, we performed an extensive in-silico analysis for functional and structural annotation and characterized HP CP995_08280 from K. pneumonia as a drug target and aimed to identify potent drug candidates. The functional and structural studies using several bioinformatics tools and databases predicted that HP CP995_08280 is a cytosolic protein that belongs to the β-lactamase family and shares structural similarity with FmtA protein from Staphylococcus aureus (PDB ID: 5ZH8). The structure of HP CP995_08280 was successfully modeled followed by structure-based virtual screening, docking, molecular dynamics, and Molecular mechanic/Poisson-Boltzmann surface area (MMPBSA) were performed to identify the potential compounds. We have found five potent antibacterial molecules, namely BDD 24083171, BDD 24085737, BDE 25098678, BDE 33638819, and BDE 33672484, which exhibited high binding affinity (-7.5 kcal/mol) and were stabilized by hydrogen bonding and hydrophobic interactions with active site residues (Ser42, Lys45, Tyr126, and Asp128) of protein. Molecular dynamics and MMPBSA revealed that HP CP995_08280 - ligand(s) complexes were less dynamic and more stable than native HP CP995_08280. Hence, the present study may serve as a potential lead for developing inhibitors against drug-resistant Klebsiella pneumonia.

The ongoing COVID-19 pandemic demands a novel approach to combat and identify potential therapeutic targets. The SARS-CoV2 infection causes a hyperimmune response followed by a spectrum of diseases. Limonoids are a class of triterpenoids known to prevent the release of IL-6, IL-15, IL-1α, IL-1β via TNF and are also known to modulate PI3K/Akt/GSK-3β, JNK1/2, MAPKp38, ERK1/2, and PI3K/Akt/mTOR signaling pathways and could help to avoid viral infection, persistence, and pathogenesis. The present study employs a computational approach of virtual screening and molecular dynamic (MD) simulations of such compounds against RNA-dependent RNA polymerase (RdRp), Main protease (Mpro), and Papain-like protease (PLpro) of SARS-CoV2. MD simulation, Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA), and Essential dynamics revealed that the macromolecule-ligand complexes are stable with very low free energy of binding. Such compounds that could modulate both host responses and inhibit viral machinery could be beneficial in effectively controlling the global pandemic., Graphical abstract Image 1

Re-emergence and global expansion of Chikungunya virus (CHIKV) from Africa to Indian Subcontinent in 2013, has significantly resulted in chronic morbidities in infected individuals. The burden of CHIKV on human population is still uncertain, owing to lack of vaccine and underdiagnosis. Due to the absence of vaccine or antiviral therapeutics, timely diagnosis and detection of CHIKV is vital for minimizing virus transmission. Commercially available diagnostic and titration kits relies on the traditional methods such as real-time PCR (RT-PCR), serodiagnostic assays, and plaque assay, which are expensive, time-consuming and technically challenging. To overcome these limitations and to increase the diagnostic coverage of CHIKV infections, a rapid and economical antigen capture assay has been developed in this study for serological diagnosis of CHIKV, using tamarind chitinase (chi)-like lectin (TCLL). TCLL extracted and purified from tamarind seeds (Tamarindus indica), has been reported recently to bind to N-acetylglucosamine (GlcNAc) containing glycan on the envelope protein of virus. Evaluation of antigen capture assay for serological diagnosis of CHIKV signified that the developed assay is able to detect CHIKV in both laboratory and clinical samples efficiently. Furthermore, a standard graph using different concentrations of CHIKV has been established using samples with known virus titer, to assist in quantification of viral load in a given sample. The feasibility of antigen capture assay for broad-spectrum diagnosis of alphaviral infections was evaluated using Sindbis virus (SINV) belonging to the same alphavirus genus, and the results obtained were in agreement with those of CHIKV. In summary, the developed glycan-based virus capture assay can be potentially applied as point-of-care routine diagnostic and titration assay for CHIKV as well for other re-emerging alphaviral infections.

The global spread of SARS-CoV-2 has resulted in millions of fatalities worldwide, making it crucial to identify potent antiviral therapeutics to combat this virus. We employed structure-assisted virtual screening to identify phytochemicals that can target the two proteases which are essential for SARS-CoV-2 replication and transcription, the main protease and papain-like protease. Using virtual screening and molecular dynamics, we discovered new phytochemicals with inhibitory activity against the two proteases. Isoginkgetin, kaempferol-3-robinobioside, methyl amentoflavone, bianthraquinone, podocarpusflavone A, and albanin F were shown to have the best affinity and inhibitory potential among the compounds, and can be explored clinically for use as inhibitors of novel coronavirus SARS-CoV-2. Communicated by Ramaswamy H. Sarma

The Coronavirus Disease 2019 (COVID-19), caused by the SARS-CoV-2 virus has raised severe health problems in china and across the world as well. CoVs encode the nucleocapsid protein (N-protein), an essential RNA-binding protein that performs different roles throughout the virus replication cycle and forms the ribonucleoprotein complex with viral RNA using the N-terminal domain (NTD) of N-protein. Recent studies have shown that NTD-N-protein is a legitimate target for the development of antiviral drugs against human CoVs. Owing to the importance of NTD, the present study focuses on targeting the NTD-N-protein from SARS-CoV-2 to identify the potential compounds. The pharmacophore model has been developed based on the guanosine monophosphate (GMP), a RNA substrate and further pharmacophore-based virtual screening was performed against ZINC database. The screened compounds were filtered by analysing the in silico ADMET properties and drug-like properties. The pharmacokinetically screened compounds (ZINC000257324845, ZINC000005169973, and ZINC000009913056) were further scrutinized through computational approaches including molecular docking and molecular dynamics simulations and revealed that these compounds exhibited good binding affinity as compared to GMP and provide stability to their respective complex with the NTD. Our findings could disrupt the binding of viral RNA to NTD, which may inhibit the essential functions of NTD. These findings may further provide an impetus to develop the novel and potential inhibitor against SARS-CoV-2.

Chlorogenic acid (CGA) is a phenolic compound with well-known antibacterial properties against pathogens. In this study, structural and biochemical characterization was used to show the inhibitory role of CGA against the enzyme of the shikimate pathway, a well-characterized drug target in several pathogens. Here, we report the crystal structures of dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, from Providencia alcalifaciens (PaDHQS), in binary complex with NAD and ternary complex with NAD and CGA. Structural analyses reveal that CGA occupies the substrate position in the active site of PaDHQS, which disables domain movements, leaving the enzyme in an open and catalysis-incompetent state. The binding analyses by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) show that CGA binds to PaDHQS with KD (equilibrium dissociation constant) values of 6.3 μM and 0.5 μM, respectively. In vitro enzyme inhibition studies show that CGA inhibits PaDHQS with a Ki of 235 ± 21 μM, while it inhibits the growth of Providencia alcalifaciens, Moraxella catarrhalis, Staphylococcus aureus, and Escherichia coli with MIC values of 60 to 100 μM. In the presence of aromatic amino acids supplied externally, CGA does not show the toxic effect. These results, along with the observations of the inhibition of the 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) regulatory domain by CGA in our previous study, suggest that CGA binds to shikimate pathway enzymes with high affinity and inhibits their catalysis and can be further exploited for designing novel drug-like molecules. IMPORTANCE The shikimate pathway is an attractive target for the development of herbicides and antimicrobial agents, as it is essential in plants, bacteria, and apicomplexan parasites but absent in humans. The enzymes of shikimate pathway are conserved among bacteria. Thus, the inhibitors of the shikimate pathway act on wide range of pathogens. We have identified that chlorogenic acid targets the enzymes of the shikimate pathway. The crystal structure of dehydroquinate synthase, the second enzyme of the pathway, in complex with chlorogenic acid and enzymatic inhibition studies explains the mechanism of inhibition of chlorogenic acid. These results suggest that chlorogenic acid has a good chemical scaffold and have important implications for its further development as a potent inhibitor of shikimate pathway enzymes.

The reemergence of SARS-CoV named, as SARS-CoV-2 has been highly infectious and able to infect a large population around the globe. The World Health Organization (WHO) has declared this SARS-CoV-2 associated Coronavirus Disease 2019 (COVID-19) as pandemic. SARS-CoV-2 genome is translated into polyproteins and has been processed by its protease enzymes. 3CLprotease is named as main protease (Mpro) enzyme which cleaves nsp4-nsp16. This crucial role of Mpro makes this enzyme a prime and promising antiviral target. The drug repurposing is a fast alternative method than the discovery of novel antiviral molecules. We have used high-throughput virtual screening approach to examine FDA approved LOPAC1280 library against Mpro. Primary screening have identified few potential drug molecule for the target among which 10 molecules were studied further. Molecular docking of selected molecules was done to detailed study about their binding energy and binding modes. Positively, Etoposide, BMS_195614, KT185, Idarubicin and WIN_62577 were found interacting with substrate binding pocket of Mpro with higher binding energy. These molecules are being advanced by our group for in vitro and in vivo testing to study the efficacy of identified drugs. As per our understanding, these molecules have the potential to efficiently interrupt the viral life cycle and may reduce or eliminate the expeditious outspreading of SARS-CoV-2.

Chikungunya virus (CHIKV), a mosquito-borne pathogenic alphavirus is a growing public health threat. No vaccines or antiviral drug is currently available in the market for chikungunya treatment. nsP2pro, the viral cysteine protease, carries out an essential function of nonstructural polyprotein processing and forms four nonstructural proteins (nsPs) that makes the replication complex, hence constitute a promising drug target. In this study, crystal structure of nsP2pro has been determined at 2.59 A, which reveals that the protein consists of two subdomains: an N-terminal protease subdomain and a C-terminal methyltransferase subdomain. Structural comparison of CHIKV nsP2pro with structures of other alphavirus nsP2 advances that the substrate binding cleft is present at the interface of two subdomains. Additionally, structure insights revealed that access to the active site and substrate binding cleft is blocked by a flexible interdomain loop in CHIKV nsP2pro. This loop contains His548, the catalytic residue, and Trp549 and Asn547, the residues predicted to bind substrate. Interestingly, mutation of Asn547 leads to three-fold increase in Km confirming that Asn547 plays important role in substrate binding and recognition. This study presents the detailed molecular analysis and signifies the substrate specificity residues of CHIKV nsP2pro, which will be beneficial for structure-based drug design and optimization of CHIKV protease inhibitors.

Chikungunya virus has emerged as one of the most important global arboviral threats over the last decade. Inspite of large scale morbidity, with long lasting polyarthralgia, so far no licensed vaccine or antiviral is available. CHIKV nsP2 protease is crucial for processing of viral nonstructural polypeptide precursor to release enzymes required for viral replication, thus making it a promising drug target. In this study, high cell density cultivation (HCDC) of Escherichia coli in batch process was carried out to produce rCHIKV nsP2pro in a cost-effective manner. The purified nsP2pro and fluorogenic peptide substrate have been adapted for fluorescence resonance energy transfer (FRET) based high throughput screening (HTS) assay with Z’ value and CV of 0.67 ± 0.054 and

Catabolite repressor activator (Cra) is a member of the LacI family transcriptional regulator distributed across a wide range of bacteria and regulates the carbon metabolism and virulence gene expression. In numerous studies to crystallize the apo form of the LacI family transcription factor, the N-terminal domain (NTD), which functions as a DNA-binding domain, has been enigmatically missing from the final resolved structures. It was speculated that the NTD is disordered or unstable and gets cleaved during crystallization. Here, we have determined the crystal structure of Cra from Escherichia coli (EcCra). The structure revealed a well-defined electron density for the C-terminal domain (CTD). However, electron density was missing for the first 56 amino acids (NTD). Our data reveal for the first time that EcCra undergoes a spontaneous cleavage at the conserved Asn 50 (N50) site, which separates the N-terminal DNA binding domain from the C-terminal effector molecule binding domain. With the site-directed mutagenesis, we confirm the involvement of residue N50 in the spontaneous cleavage phenomenon. Furthermore, the Isothermal titration calorimetry (ITC) assay of the EcCra-NTD with DNA showed EcCra-NTD is in a functional conformation state and retains its DNA binding activity.

FmtA is a penicillin-recognizing protein (PRP) with novel hydrolytic activity toward the ester bond between d -Ala and the backbone of teichoic acids. Teichoic acids are polyol-phosphate polymers found in the Staphylococcus aureus cell wall, and they play important roles in antibiotic resistance and pathogenesis. Two of the PRPs conserved motifs, namely, SXXK and Y(S)XN, are involved in the hydrolysis by FmtA, but the catalytic mechanism remains elusive. Here we determined the crystal structure of FmtA. FmtA shares the core structure of PRPs: an all α-helical domain and α/β domain sandwiched together. However, it does not have the typical PRPs active-site cleft. Its active site is shallow, solvent-exposed, and enlarged. Furthermore, our mutagenesis and kinetic studies suggest that the SXXK and Y(S)XN motifs of FmtA offer all that is necessary for catalysis, and more: the active-site nucleophile (serine), the general base (lysine) required for the acylation step and the deacylation step, and an anchor (tyrosine) to hold the active-site serine, and possibly the substrate, in an optimum conformation for catalysis. Our study establishes that the FmtA esterase activity represents an expansion of the catalytic activity repertoire of the PRPs core structure, which typically displays peptidase activity. This finding points toward a novel mechanism of ester bond hydrolysis by a PRP. The structure of FmtA provides insights to the design of inhibitor molecules with the potential to serve as leads in the development of novel antibacterial chemotherapeutic agents.

Small heterocyclic molecules such as piperazine are potential pharmacotherapeutic agents and binding of these molecules to the hydrophobic pocket of capsid protein (CP) offers a new perspective for therapeutic intervention. Here, we report the crystal structure of CP from Aura virus (AVCP) in complex with piperazine at 2.2 Å resolution. Piperazine binds to the conserved hydrophobic pocket of CP where dioxane based antivirals bind. Comparative structural studies of the piperazine-bound AVCP structure with the apo, active and dioxane-bound AVCP structures provide insights into the conformational variations in the pocket. Additionally, the molecular docking studies showed that piperazine binds into the hydrophobic pocket of Chikungunya virus CP (CVCP) with more affinity than with AVCP. Furthermore, the antiviral activity of piperazine against Chikungunya virus (CHIKV) was investigated by plaque reduction and immunofluorescence assays. The AVCP-piperazine complex may serve as a lead scaffold for structure-based design of piperazine derivatives as alphaviral inhibitors. The antiviral properties of piperazine provide its usefulness for further investigations towards the development of piperazine based anti-alphaviral drugs.

Chikungunya virus (CHIKV) is a small spherical shaped mosquito-borne alphavirus. CHIKV infection is a major public health concern in many tropical and subtropical regions of the biosphere. It has single-stranded positive-sense RNA genome (11,811 nucleotides) that organises as 5′UTR-nsP1-nsP2-nsP3-nsP4-J-C-E3-E2-6K-E1-polyA-3′UTR. The non-structural precursor protein is proteolytically cleaved by non-structural protein 2 (nsp2) and this makes it a potential antiviral drug target. In the viral replication, nsP2 display three key roles as helicase, triphosphatase and protease. The C-terminal half of the nsP2 protein consists of two structural domains: a protease domain and a methyltransferase-like domain. These domains function together and are crucial for protease activity. Till date, there is no commercially available drug against CHIKV and launching a new drug into the market is a complicated and time-consuming process. Therefore, drug repurposing is a new but familiar approach to reduce the time and cost of drug discovery. Recently, crystal structure of CHIKV nsP2 protease domain (PDB ID: 4ZTB) was determined by our group. The mechanism of nsP2 protease function is related to deprotonation of the thiol group of cysteine, the catalytic dyad residue at the active site (Cys478 and His548). In this study, nsP2 protease active site has been targeted by virtual screening of compound libraries and molecular docking of potential inhibitory molecules. Based on the binding energies, hit molecules on the top were selected from Pharmacologically Active Compounds (LOPAC1280) library. Inhibitory molecules are being further analyzed by MD simulations to find the stable binding poses. Based on the analysis of docking and MD simulation compounds hve been selected, the protease inhibitory activity and antiviral efficacy have been tested for two of these molecules against CHIKV using cell-based in vitro assays. Based on kinetic studies, top compounds inhibited CHIKV nsP2 protease with inhibition constant (Ki) values of 36.5 M and 86.5 M. Interestingly, these two compounds inhibited CHIKV replication in Vero cells at concentrations much lower than their cytotoxic concentrations as validated by in vitro antiviral plaque assay experiments. Thus, structure-based approach to identify inhibitors is a very effective approach to identify and develop drug molecules against viral pathogens.

The latest advancements in drug development are structure-assisted and computer-aided identification, design, and synthesis of target-specific antiviral. Structural virology of animal viruses has made valuable contribution to our understanding of viruses in general, replication, evolution, and interaction with the host. Structure–function relation studies are definitely the need of the hour for rational design of drugs and vaccines to effectively treat animal viral diseases. These studies are important for economical, veterinary, and human medical perspectives. X-ray crystallography, nuclear magnetic resonance and cryo-electron microscopy techniques elaborate the structure–function relation studies by utilizing various computational approaches for structure-assisted designing of drug molecules and antiviral against viruses. Availability of protein three-dimensional structures, high-performance computing, data management software, and internet are enhancing the modern day drug discovery process. Computational tools offer the advantage of delivering new drug candidates more quickly and at a lower cost. Various viral proteins that are necessary for the survival of the virus inside the host are discussed in this chapter such as viral polymerase, helicase, protease, etc. Various antiviral drug molecules identified and designed on the basis of the structure of viral proteins which are highlighted, and disclosed details of the available inhibitors and potential antiviral are discussed. Further improvement in identified inhibitors, the need of novel structure-based drugs and their clinical testing is emphasized. This chapter describes briefly the structural techniques and advances made in animal virology toward structure-based identification and development of antiviral for treatment of animal viruses. In the future, the studies discussed may serve as the basis for development of potential antiviral against animal viral diseases.