CHIMERA_NA: A Customizable Mutagenesis Tool for Structural Manipulations in Nucleic Acids and Their Complexes.

Studying the structure and dynamics of nucleic acids and their complexes is crucial for understanding fundamental biological processes and developing therapeutic interventions. However, the limited availability of experimentally characterized nucleic acid structures poses a challenge for exploring their properties comprehensively. To address this, we developed a customizable mutagenesis tool, CHIMERA_NA, to manipulate nucleic acid structures and their complexes. Utilizing the user-friendly CHIMERA_NA, researchers can perform mutations in nucleic acid structures, enabling the exploration of diverse structural configurations and dynamic behaviors. The tool offers the flexibility to generate all possible combinations of mutations or specific user-defined mutations based on research requirements. CHIMERA_NA leverages the capabilities of UCSF Chimera software, a widely used platform for molecular structure analysis, to facilitate the generation of mutations in nucleic acids. Our tool modifies the reference structure of nucleic acids or their complexes to generate initial coordinates of mutated structures/complexes within seconds for further computational exploration. This capability allows users to extend their investigations beyond structural repositories, enabling the study of DNA/RNA drug recognition, nucleic acid-protein interactions, and the intrinsic structural and dynamic properties of nucleic acids. By providing a user-friendly and customizable approach to nucleic acid mutagenesis, CHIMERA_NA contributes to advancing our understanding of nucleic acid biology and facilitating drug discovery efforts targeting nucleic acid-based mechanisms. CHIMERA_NA is freely available in the Supporting Information of this article.

Design and Characterization of Neutral Linker-Based Bis-Intercalator via Computer Simulations: Balancing DNA Binding and Cellular Uptake.

Bis-intercalators refer to a class of chemical compounds known for their unique ability to simultaneously intercalate, or insert, into DNA at two distinct sites. These molecules typically feature two intercalating moieties connected by a linker, allowing them to engage with DNA base pairs at multiple locations. The bis-intercalation phenomenon plays a significant role in altering the DNA structure, affecting its stability, and potentially influencing various cellular processes. These compounds have gained considerable attention in medicinal chemistry and biochemistry due to their potential applications in cancer therapy, where they may interfere with DNA replication and transcription, leading to anticancer effects. Traditionally, these molecules often possess a high positive charge to enhance their affinity for the negatively charged DNA. However, due to a high positive charge, their cellular uptake is compromised, along with their enhanced potential off-target effects. In this study, we utilized bis-intercalator TOTO and replaced the charged linker segment (propane-1,3-diammonium) with a neutral peroxodisulphuric acid linker. Using molecular modeling and computer simulations (500 ns, 3 replicas), we investigated the potential of the designed molecule as a bis-intercalator and compared the properties with the control bis-intercalator bound to DNA. We observed that the designed bis-intercalator exhibited improved DNA binding (as assessed through MM-PBSA and Delphi methods) and membrane translocation permeability. With an overall reduced charge, significantly less off-target binding of the designed molecule is also anticipated. Consequently, bis-intercalators based on peroxodisulphuric linkers can potentially target DNA effectively, and their role in the future design of bis-intercalators is foreseen.

Flexible RNA aptamers as inhibitors of Bacillus anthracis ribosomal protein S8: Insights from molecular dynamics simulations.

Bacillus anthracis, the causative agent of anthrax, poses a substantial threat to public health and national security, and is recognized as a potential bioweapon due to its capacity to form resilient spores with enduring viability. Inhalation or ingestion of even minute quantities of aerosolized spores can lead to widespread illness and fatalities, underscoring the formidable lethality of the bacterium. With an untreated mortality rate of 100%, Bacillus anthracis is a disconcerting candidate for bioterrorism. In response to this critical scenario, we employed state-of-the-art computational tools to conceive and characterize flexible RNA aptamer therapeutics tailored for anthrax. The foundational structure of the flexible RNA aptamers was designed by removing the C2'-C3' in each nucleotide unit. Leveraging the crystal structure of Bacillus anthracis ribosomal protein S8 complexed with an RNA aptamer, we explored the structural, dynamic, and energetic aspects of the modified RNA aptamer - S8 protein complexes through extensive all-atom explicit-solvent molecular dynamics simulations (400 ns, 3 replicas each), followed by drawing comparisons to the control system. Our findings demonstrate the enhanced binding competencies of the flexible RNA aptamers to the S8 protein via better shape complementarity and improved H-bond network compared to the control RNA aptamer. This research offers valuable insights into the development of RNA aptamer therapeutics targeting Bacillus anthracis, paving the way for innovative strategies to mitigate the impact of this formidable pathogen.

Probing the Nucleic Acid Flexibility to Disarm the Viral Counter-Defense Machinery: Design and Characterization of Potent p19 Inhibitors.

Plant viruses are highly destructive and significant contributors to several global pandemics and epidemics in plants. A viral disease outbreak in plants can cause a scarcity of food supply and is a severe concern to humanity. The siRNA (small interfering RNA)-mediated RNA-induced silencing complex (RISC) formation is a primary defense mechanism in plants against viruses, where the RISC binds and degrades viral mRNAs. As a counter-defense, many viruses encode RNA-silencing suppressor proteins (e.g., the p19 protein from the Tombusviridae family) for viral proliferation in plants. The functional form of p19 (homodimer) binds to plant siRNA with high affinities, thereby interrupting the RISC formation and thus preventing the viral mRNA silencing in plants. By altering the RISC formation, the p19 protein helps the virus invasion in the plant and ultimately stunts host growth. In this study, we designed several modified siRNA-based molecules for p19 inhibition. The viral p19 protein is known to interact predominantly through H-bonds with 2'-OH and phosphates of the plant siRNA. We utilized this information and in silico-designed flexible substituents of siRNA, where we removed the C2'-C3' bond in each nucleotide unit. We performed all-atom explicit-solvent molecular dynamics simulations (400 ns, 3 replicates each) for control/modified siRNA─p19 complexes (8 in total) followed by energetic estimations. Strikingly, in a few modified complexes, the siRNA not only retained the double-helical structural integrity but also displayed remarkably enhanced p19 binding compared to the control siRNA; hence, we consider it important to perform biological and chemical in vitro and in vivo studies on proposed flexible nucleic acids as p19 inhibitors for crop protection.

Identification of potential anti-mucor agents by targeting endothelial cell receptor glucose-regulated protein-78 using in silico approach.

Mucormycosis is a fungal infection of the sinuses, brain and lungs that is the cause of approximately 50% mortality rate despite the available first-line therapy. Glucose-Regulated Protein 78 (GRP78) is already reported to be a novel host receptor that mediates invasion and damage of human endothelial cells by Rhizopus oryzae and Rhizopus delemar, the most common etiologic species of Mucorales. The expression of GRP78 is also regulated by the levels of iron and glucose in the blood. There are several antifungal drugs in the market but they pose a serious side effect to the vital organs of the body. Therefore, there is an immediate need to discover effective drug molecules having increased efficacy with no side effects. With the help of various computational tools, the current study was attempted to determine potential antimucor agents against GRP78. The receptor molecule GRP78 was screened against 8820 known drugs deposited in DrugBank library using high-throughput virtual screening method. Total top 10 compounds were selected based on the binding energies greater than the reference co-crystal molecule. Furthermore, molecular dynamic (MD) simulations using AMBER were performed to calculate the stability of the top-ranked compounds in the active site of GRP78. After extensive computational studies, we propose that two compounds (CID439153 and CID5289104) have inhibitory potency against mucormycosis and can serve as potential drugs that can form the basis of treating mucormycosis disease.Communicated by Ramaswamy H. Sarma.

The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties.

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

Ceftriaxone induces glial EAAT-2 promotor region via NF-kB conformational changes: An interaction analysis using HADDOCK.

Excitotoxicity, depletion of energy metabolites, and ionic imbalance are the major factors involved in neurodegeneration mediated through excitatory amino acid transporter-2 (EAAT-2) dysfunction in ischemic insult. Recent studies have revealed that ceftriaxone expresses EAAT-2 via nuclear transcription factor kappa-B (NF-kB) signaling pathway, stimulation of EAAT-2 expression in the ischemic, and excitotoxic conditions that could provide potential benefits to control neurodegeneration. In this study, we have predicted the in silico model for interaction between NF-kB and EAAT-2 promoter region to rule out the conformational changes for the expression of EAAT-2 protein. Using homology-built model of NF-kB, we identified ceftriaxone-induced conformational changes in gene locus -272 of DNA where NF-kB binding with EAAT-2 promoter region through protein-DNA docking calculation. The interaction profile and conformational dynamics occurred between ceftriaxone predocked and postdocked conformations of NF-kB with DNA employing HADDOCK 2.2 web server followed by 250 ns long all atom explicit solvent molecular dynamics simulations. Both the protein and DNA exhibited modest conformational changes with respect to HADDOCK score, energy terms (desolvation energy [Edesolv ]), van der waal energy (Evdw ), electrostatic energy (Eelec ), restraints energy (Eair ), buried surface area, root mean square deviation, RMSF, radius of gyration, total hydrogen bonds when ceftriaxone pre- and postdocked NF-kB conformations were bound to DNA. Hence, the conformational changes in the C-terminal domain could be the reason for EAAT-2 expression through ceftriaxone specific binding pocket of -272 of DNA.

Bicyclo-DNA mimics with enhanced protein binding affinities: insights from molecular dynamics simulations.

DNA-protein interactions occur at all levels of DNA expression and replication and are crucial determinants for the survival of a cell. Several modified nucleotides have been utilized to manipulate these interactions and have implications in drug discovery. In the present article, we evaluated the binding of bicyclo-nucleotides (generated by forming a methylene bridge between C1' and C5' in sugar, leading to a bicyclo system with C2' axis of symmetry at the nucleotide level) to proteins. We utilized four ssDNA-protein complexes with experimentally known binding free energies and investigated the binding of modified nucleotides to proteins via all-atom explicit solvent molecular dynamics (MD) simulations (200 ns), and compared the binding with control ssDNA-protein systems. The modified ssDNA displayed enhanced binding to proteins as compared to the control ssDNA, as seen by means of MD simulations followed by MM-PBSA calculations. Further, the Delphi-based electrostatic estimation revealed that the high binding of modified ssDNA to protein might be related to the enhanced electrostatic complementarity displayed by the modified ssDNA molecules in all the four systems considered for the study. The improved binding achieved with modified nucleotides can be utilized to design and develop anticancer/antisense molecules capable of targeting proteins or ssRNAs.Communicated by Ramaswamy H. Sarma.

Bases immediate upstream of the TATAAT box of the sigma 70 promoter of Escherichia coli significantly influence the activity of a model promoter by altering the bending angle of DNA.

Different workers have found different bases of the spacer of the sigma 70 promoter of Escherichia coli to be important, depending on the base sequence of the two hexameric boxes of the naturally occurring promoter they were working on. Besides, there was no clue as to why particular bases worked better than others in particular positions. This necessitated a fresh look at the spacer region of a model promoter comprising all the consensus promoter elements. Randomisation of the three bases of the spacer in positions -15 to -13 with respect to the transcription initiation site, has elicited more than 50-fold variation in activity of the promoter, the highest and the lowest activities being 14,391(the three bases being GCA) and 264 Miller units (the three bases being AAA) respectively. Pairs of promoters of very similar activities were observed, even when the bases in these three positions were very different. The promoters with similar activities had similar three dimensional structures of the promoter DNA, as determined by molecular dynamics simulations. Randomisation of the three bases in positions -18 to -16 of the promoter that contained the triplet GCA in positions -15 to -13, resulted in promoters with highest activity of 15,759 (the triplet upstream of GCA being TAT) and lowest activity of 1,882 (the triplet upstream of GCA being AAA). Good correlation between the bending angles of the promoter DNAs and promoter activities could be observed, the R2 value being 0.8724. Retardation of electrophoretic mobility of the promoter DNAs correlated well with activity.

Harmonizing Interstrand Electrostatic Repulsion by Conformational Rigidity in Counterion-Deprived Z-DNA: A Molecular Dynamics Study.

Deoxyribonucleic acid (DNA) is a vital biomacromolecule. Although the right-handed B-DNA type helical structure is the most abundant and extensively studied form of DNA, several noncanonical forms, such as triplex, quadruplex, Z-DNA, A-DNA, and ss-DNA, have been probed from time to time to gain insights into the DNA's function. Z-DNA was recently found to be involved in cancer and several autoimmune diseases. In the present Article, we evaluated the conformational stability of locked-sugar-based Z-DNA via all-atom explicit-solvent molecular dynamics simulations and found that the modified DNA maintained the left-handed conformation even in the absence of counterions, wherein the structural rigidity dominates over the electrostatic repulsion between the complementary strands. The control Z-DNA without counterions, as expected, instantaneously resulted in unfolded states. The remarkable stability of the conformationally locked model system was thoroughly investigated via structural and energetic perspectives and was probably the result of the backbone widening in tandem with enhanced electrostatics between complementary strands. We believe that the design of the proposed modified Z-DNA construct could help understand the otherwise delicate Z-DNA conformation even in salt-deprived conditions. The design could also motivate the medicinal use of short segments of such modified nucleotides and could be utilized in more advanced modeling techniques, such as DNA origami which has gained popularity in recent years.

Discovery of the Lead Molecules Targeting the First Step of the Histidine Biosynthesis Pathway of Acinetobacter baumannii.

Acinetobacter baumannii is a multidrug-resistant, opportunistic, nosocomial pathogen for which a new line of treatments is desperately needed. We have targeted the enzyme of the first step of the histidine biosynthesis pathway, viz., ATP-phosphoribosyltransferase (ATP-PRT). The three-dimensional structure of ATP-PRT was predicted on the template of the known three-dimensional structure of ATP-PRT from Psychrobacter arcticus (PaATPPRT) using a homology modeling approach. High-throughput virtual screening (HTVS) of the antibacterial library of Life Chemicals Inc., Ontario, Canada was carried out followed by molecular dynamics simulations of the top hit compounds. In silico results were then biochemically validated using surface plasmon resonance spectroscopy. We found that two compounds, namely, F0843-0019 and F0608-0626, were binding with micromolar affinities to the ATP-phosphoribosyltransferase from Acinetobacter baumannii (AbATPPRT). Both of these compounds were binding in the same way as AMP in PaATPPRT, and the important residues of the active site, viz., Val4, Ser72, Thr76, Tyr77, Glu95, Lys134, Val136, and Tyr156, were also interacting via hydrogen bonds. The calculated binding energies of these compounds were -10.5 kcal/mol and -11.1 kcal/mol, respectively. These two compounds can be used as the potential lead molecules for designing antibacterial compounds in the future, and this information will help in drug discovery programs against Acinetobacter worldwide.

Assessing the DNA structural integrity via selective annihilation of Watson-Crick hydrogen bonds: Insights from molecular dynamics simulations.

Understanding the role of base pairing and stacking displayed by polynucleotide chains interwind together resulting in a double-helical B-DNA type structure is crucial to gaining access to the sophisticated structural arrangement of DNA. Several computational and experimental studies hinted towards the dominance of base pairing over stacking for duplex stability. To find out how significant the individual Watson-Crick hydrogen bonds are in maintaining the double-helical integrity of the DNA, in the present article, we selectively switched off the hydrogen bonds (one specific bond or their combinations in all the base pairs at a time) via manipulating the force fields for A-T and G-C base pairs. We studied 12 systems in total via all-atom explicit-solvent molecular dynamics simulations (200 ns each). The MD output structures were compared with the control system by means of structural, dynamic, and energetic properties to monitor the overall consequences of removing H-bond(s) on the B-DNA characteristics of the model systems. Our findings suggest that all the individual hydrogen bonds involved in base pairing are vital for maintaining the DNA structural integrity as any possible alteration in Watson-Crick hydrogen bond(s) leads to the disintegration/collapse of DNA strands resulting in unfolded states.

Structure prediction and discovery of inhibitors against phosphopantothenoyl cysteine synthetase of Acinetobacter baumannii.

Acinetobacter baumannii is an extremely dangerous multidrug-resistant (MDR) gram-negative pathogen which poses a serious life-threatening risk in immunocompromised patients. Phosphopantothenoyl cysteine synthetase (PPCS) catalyzes the formation of an amide bond between L-cysteine and phosphopantothenic acid (PPA) to form 4'- Phosphopantothenoylcysteine during Coenzyme A (CoA) biosynthesis. CoA is a crucial cofactor for cellular survival and inhibiting its synthesis will result in cell death. Bacterial PPCS differs from eukaryotic PPCS in a number of ways like it exists as a C-terminal domain of a PPCDC/PPCS fusion protein whereas eukaryotic PPCS exists as an independent protein. This difference makes it an attractive drug target. For which a conventional iterative approach of SBDD (structure-based drug design) was used, which began with three-dimensional structure prediction of AbPPCS using PHYRE 2.0. A database of FDA-approved compounds (Drug Bank) was then screened against the target of interest by means of docking score and glide energy, leading to the identification of 6 prominent drug candidates. The shortlisted 6 molecules were further subjected to all-atom MD simulation studies in explicit-solvent conditions (using AMBER force field). The MD simulation studies revealed that the ligands DB65103, DB449108 and DB443210, maintained several H-bonds with intense van der Waals contacts at the active site of the protein with high binding free energies: -11.42 kcal/mol, -10.49 kcal/mol and -10.98 kcal/mol, respectively, calculated via MM-PBSA method. Overall, binding of these compounds at the active site was found to be the most stable and robust highlighting the potential of these compounds to serve as antibacterials.Communicated by Ramaswamy H. Sarma.

Virtual screening of quinoline derived library for SARS-COV-2 targeting viral entry and replication.

The COVID-19 pandemic infection has claimed many lives and added to the social, economic, and psychological distress. The contagious disease has quickly spread to almost 218 countries and territories following the regional outbreak in China. As the number of infected populations increases exponentially, there is a pressing demand for anti-COVID drugs and vaccines. Virtual screening provides possible leads while extensively cutting down the time and resources required for ab-initio drug design. We report structure-based virtual screening of a hundred plus library of quinoline drugs with established antiviral, antimalarial, antibiotic or kinase inhibitor activity. In this study, targets having a role in viral entry, viral assembly, and viral replication have been selected. The targets include: 1) RBD of receptor-binding domain spike protein S 2) Mpro Chymotrypsin main protease 3) Ppro Papain protease 4) RNA binding domain of Nucleocapsid Protein, and 5) RNA Dependent RNA polymerase from SARS-COV-2. An in-depth analysis of the interactions and G-score compared to the controls like hydroxyquinoline and remdesivir has been presented. The salient results are (1) higher scoring of antivirals as potential drugs (2) potential of afatinib by scoring as better inhibitor, and (3) biological explanation of the potency of afatinib. Further MD simulations and MM-PBSA calculations showed that afatinib works best to interfere with the the activity of RNA dependent RNA polymerase of SARS-COV-2, thereby inhibiting replication process of single stranded RNA virus. Communicated by Ramaswamy H. Sarma.

Lactosmart: A Novel Therapeutic Molecule for Antimicrobial Defense.

The problem of antibiotic resistance has prompted researchers around the globe to search for new antimicrobial agents. Antimicrobial proteins and peptides are naturally secreted by almost all the living organisms to fight infections and can be safer alternatives to chemical antibiotics. Lactoferrin (LF) is a known antimicrobial protein present in all body secretions. In this study, LF was digested by trypsin, and the resulting hydrolysates were studied with respect to their antimicrobial properties. Among the hydrolysates, a 21-kDa basic fragment of LF (termed lactosmart) showed promise as a new potent antimicrobial agent. The antimicrobial studies were performed on various microorganisms including Shigella flexneri, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli as well as fungal pathogens such as Candida albicans, Candida tropicalis, and Candida glabrata. In addition, the lipopolysaccharide (LPS)-binding properties of lactosmart were studied using surface plasmon resonance technique in vitro, along with docking of LPS and molecular dynamics (MD) simulation studies. The results showed that lactosmart had better inhibitory effects against pathogenic microorganisms compared to LF. The results of docking and MD simulation studies further validated the tighter binding of LPS to lactosmart compared to LF. The two LPS-binding sites have been characterized structurally in detail. Through these studies, it has been demonstrated that in native LF, only one LPS-binding site remains exposed due to its location being on the surface of the molecule. However, due to the generation of the lactosmart molecule, the second LPS-binding site gets exposed too. Since LPS is an essential and conserved part of the bacterial cell wall, the pro-inflammatory response in the human body caused by LPS can be targeted using the newly identified lactosmart. These findings highlight the immense potential of lactosmart in comparison to native LF in antimicrobial defense. We propose that lactosmart can be further developed as an antibacterial, antifungal, and antibiofilm agent.

Cdc4 phospho-degrons allow differential regulation of Ame1CENP-U protein stability across the cell cycle.

Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. It is still poorly understood how efficient, centromere-dependent kinetochore assembly is accomplished from hundreds of individual protein building blocks in a cell cycle-dependent manner. Here, by combining comprehensive phosphorylation analysis of native Ctf19CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U, which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-phase and targets the protein for degradation. Binding of the Mtw1Mis12 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic in SCF mutants, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits facilitates efficient centromere-dependent kinetochore assembly. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.

Symmetric Nucleosides as Potent Purine Nucleoside Phosphorylase Inhibitors.

Nucleic acids are one of the most enigmatic biomolecules crucial to several biological processes. Nucleic acid-protein interactions are vital for the coordinated and controlled functioning of a cell, leading to the design of several nucleoside/nucleotide analogues capable of mimicking these interactions and hold paramount importance in the field of drug discovery. Purine nucleoside phosphorylase is a well-established drug target due to its association with numerous immunodeficiency diseases. Here, we study the binding of human purine nucleoside phosphorylase (PNP) to some bidirectional symmetric nucleosides, a class of nucleoside analogues that are more flexible due to the absence of sugar pucker restraints. We compared the binding energies of PNP-symmetric nucleosides to the binding energies of PNP-inosine/Imm-H (a transition-state analogue), by means of 200 ns long all-atom explicit-solvent Gaussian accelerated molecular dynamics simulations followed by energetics estimation using the MM-PBSA methodology. Quite interestingly, we observed that a few symmetric nucleosides, namely, ν3 and ν4, showed strong binding with PNP (-14.1 and -12.6 kcal/mol, respectively), higher than inosine (-6.3 kcal/mol) and Imm-H (-9.6 kcal/mol). This is rationalized by an enhanced hydrogen-bond network for symmetric nucleosides compared to inosine and Imm-H while maintaining similar van der Waals contacts. We note that the chemical structures of both ν3 and ν4, due to an additional unsaturation in them, resemble enzymatic transition states and fall in the category of transition-state analogues (TSAs), which are quite popular.

Identification of potential drug candidates to combat COVID-19: a structural study using the main protease (mpro) of SARS-CoV-2.

The recent outbreak of the SARS-CoV-2 virus leading to the disease COVID 19 has become a global pandemic that is spreading rapidly and has caused a global health emergency. Hence, there is an urgent need of the hour to discover effective drugs to control the pandemic caused by this virus. Under such conditions, it would be imperative to repurpose already known drugs which could be a quick and effective alternative to discovering new drugs. The main protease (Mpro) of SARS-COV-2 is an attractive drug target because of its essential role in the processing of the majority of the non-structural proteins which are translated from viral RNA. Herein, we report the high-throughput virtual screening and molecular docking studies to search for the best potential inhibitors against Mpro from FDA approved drugs available in the ZINC database as well as the natural compounds from the Specs database. Our studies have identified six potential inhibitors of Mpro enzyme, out of which four are commercially available FDA approved drugs (Cobicistat, Iopromide, Cangrelor, and Fortovase) and two are from Specs database of natural compounds (Hopeaphenol and Cyclosieversiodide-A). While Cobicistat and Fortovase are known as HIV drugs, Iopromide is a contrast agent and Cangrelor is an anti-platelet drug. Furthermore, molecular dynamic (MD) simulations using GROMACS were performed to calculate the stability of the top-ranked compounds in the active site of Mpro. After extensive computational studies, we propose that Cobicistat and Hopeaphenol show potential to be excellent drugs that can form the basis of treating COVID-19 disease.Communicated by Ramaswamy H. Sarma.

Identification of promising drug candidates against NSP16 of SARS-CoV-2 through computational drug repurposing study.

The recent outbreak of the SARS-CoV-2 virus leading to the disease COVID 19, a global pandemic has resulted in an unprecedented loss of life and economy worldwide. Hence, there is an urgent need to discover effective drugs to control this pandemic. NSP16 is a methyltransferase that methylates the ribose 2'-O position of the viral nucleotide. Taking advantage of the recently solved structure of NSP16 with its inhibitor, S-Adenosylmethionine, we have virtually screened FDA approved drugs, drug candidates and natural compounds. The compounds with the best docking scores were subjected to molecular dynamics simulations followed by binding free energy calculations using the MM-PBSA method. The known drugs which were identified as potential inhibitors of NSP16 from SARS-CoV-2 included DB02498, DB03909, DB03186, Galuteolin, ZINC000029416466, ZINC000026985532, and ZINC000085537017. DB02498 (Carba-nicotinamide-adenine-dinucleotide) is an approved drug which has been used since the late 1960s in intravenous form to significantly lessen withdrawal symptoms from a variety of drugs and alcohol addicts and it has the best MM-PBSA binding free energy of-12.83 ± 0.52 kcal/mol. The second best inhibitor, Galuteolin is a natural compound that inhibits tyrosinase enzyme with MM-PBSA binding free energy value of -11.21 ± 0.47 kcal/mol. Detailed ligand and protein interactions were analyzed and common residues across SARS-CoV, SARS-CoV-2, and MERS-CoV were identified. We propose Carba-nicotinamide-adenine-dinucleotide and Galuteolin as the potential inhibitors of NSP16. The results in this study can be used for the treatment of COVID-19 and can also form the basis of rational drug design against NSP16 of SARS-CoV-2.

5-HT1A targeting PARCEST agent DO3AM-MPP with potential for receptor imaging: Synthesis, physico-chemical and MR studies.

Contrast enhancement in MRI using magnetization or saturation transfer techniques promises better sensitivity, and faster acquisition compared to T1 or T2 contrast. This work reports the synthesis and evaluation of 5-HT1A targeted PARACEST MRI contrast agent using 1,4,7,10-tetraazacycloDOdecane-4,7,10-triacetAMide (DO3AM) as the bifunctional chelator, and 5-HT1A-antagonist methoxyphenyl piperazine (MPP) as a targeting unit. The multi-step synthesis led to the MPP conjugated DO3AM with 60% yield. CEST-related physicochemical parameters were evaluated after loading DO3AM-MPP with paramagnetic MRI active lanthanides: Gadolinium (Gd-DO3AM-MPP) and Europium (Eu-DO3AM-MPP). Luminescence lifetime measurements with Eu-DO3AM-MPP and computational DFT studies using Gd-DO3AM-MPP revealed the coordination of one water molecule (q = 1.43) with metal-water distance (rM-H2O) of 2.7 Å and water residence time (τm) of 0.23 ms. The dissociation constant of Kd 62 ± 0.02 pM as evaluated from fluorescence quenching of 5-HT1A (protein) and docking score of -4.81 in theoretical evaluation reflect the binding potential of the complex Gd-DO3AM-MPP with the receptor 5-HT1A. Insights of the docked pose reflect the importance of NH2 (amide) and aromatic ring in Gd-DO3AM-MPP while interacting with Ser 374 and Phe 370 in the antagonist binding pocket of 5-HT1A. Gd-DO3AM-MPP shows longitudinal relaxivity 5.85 mM-1s-1 with a water residence lifetime of 0.93 ms in hippocampal homogenate containing 5-HT1A. The potentiometric titration of DO3AM-MPP showed strong selectivity for Gd3+ over physiological metal ions such as Zn2+ and Cu2+. The in vitro and in vivo studies confirmed the minimal cytotoxicity and presential binding of Gd-DO3AM-MPP with 5-HT1A receptor in the hippocampus region of the mice. Summarizing, the complex Gd-DO3AM-MPP can have a potential for CEST imaging of 5-HT1A receptors.