Discovery and characterization of small molecule SIRT3-specific inhibitors as revealed by mass spectrometry.

Sirtuins play a prominent role in several cellular processes and are implicated in various diseases. The understanding of biological roles of sirtuins is limited because of the non-availability of small molecule inhibitors, particularly the specific inhibitors directed against a particular SIRT. We performed a high-throughput screening of pharmacologically active compounds to discover novel, specific, and selective sirtuin inhibitor. Several unique in vitro sirtuin inhibitor pharmacophores were discovered. Here, we present the discovery of novel chemical scaffolds specific for SIRT3. We have demonstrated the in vitro activity of these compounds using label-free mass spectroscopy. We have further validated our results using biochemical, biophysical, and computational studies. Determination of kinetic parameters shows that the SIRT3 specific inhibitors have a moderately longer residence time, possibly implying high in vivo efficacy. The molecular docking results revealed the differential selectivity pattern of these inhibitors against sirtuins. The discovery of specific inhibitors will improve the understanding of ligand selectivity in sirtuins, and the binding mechanism as revealed by docking studies can be further exploited for discovering selective and potent ligands targeting sirtuins.

Discovery of Mycobacterium tuberculosis α-1,4-glucan branching enzyme (GlgB) inhibitors by structure- and ligand-based virtual screening.

GlgB (α-1,4-glucan branching enzyme) is the key enzyme involved in the biosynthesis of α-glucan, which plays a significant role in the virulence and pathogenesis of Mycobacterium tuberculosis. Because α-glucans are implicated in the survival of both replicating and non-replicating bacteria, there exists an exigent need for the identification and development of novel inhibitors for targeting enzymes, such as GlgB, involved in this pathway. We have used the existing structural information of M. tuberculosis GlgB for high throughput virtual screening and molecular docking. A diverse database of 330,000 molecules was used for identifying novel and efficacious therapeutic agents for targeting GlgB. We also used three-dimensional shape as well as two-dimensional similarity matrix methods to identify diverse molecular scaffolds that inhibit M. tuberculosis GlgB activity. Virtual hits were generated after structure and ligand-based screening followed by filters based on interaction with human GlgB and in silico pharmacokinetic parameters. These hits were experimentally evaluated and resulted in the discovery of a number of structurally diverse chemical scaffolds that target M. tuberculosis GlgB. Although a number of inhibitors demonstrated in vitro enzyme inhibition, two compounds in particular showed excellent inhibition of in vivo M. tuberculosis survival and its ability to get phagocytosed. This work shows that in silico docking and three-dimensional chemical similarity could be an important therapeutic approach for developing inhibitors to specifically target the M. tuberculosis GlgB enzyme.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic ß Cells.

Pancreatic ß cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic ß cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals. We show here that NAADP-mediated Ca(2+) release from endolysosomal Ca(2+) stores activates inward membrane currents and depolarizes the ß cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca(2+) release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca(2+) signals, and insulin secretion. Our findings implicate NAADP-evoked Ca(2+) release from acidic Ca(2+) storage organelles in stimulus-secretion coupling in ß cells.

Crystal structure of the N-terminal domain of human SIRT7 reveals a three-helical domain architecture.

Human SIRT7 is an NAD(+) dependent deacetylase, which belongs to sirtuin family of proteins. SIRT7, like other sirtuins has conserved catalytic domain and is flanked by N- and C-terminal domains reported to play vital functional roles. Here, we report the crystal structure of the N-terminal domain of human SIRT7 (SIRT7(NTD) ) at 2.3 Å resolution as MBP-SIRT7(NTD) fusion protein. SIRT7(NTD) adopts three-helical domain architecture and comparative structural analyses suggest similarities to some DNA binding motifs and transcription regulators. We also report here the importance of N- and C-terminal domains in soluble expression of SIRT7. Proteins 2016; 84:1558-1563. © 2016 Wiley Periodicals, Inc.

Mycobacterium tuberculosis keto-mycolic acid and macrophage nuclear receptor TR4 modulate foamy biogenesis in granulomas: a case of a heterologous and noncanonical ligand-receptor pair.

The cell wall of Mycobacterium tuberculosis is configured of bioactive lipid classes that are essential for virulence and potentially involved in the formation of foamy macrophages (FMs) and granulomas. Our recent work established crosstalk between M. tuberculosis cell wall lipids and the host lipid-sensing nuclear receptor TR4. In this study, we have characterized, identified, and adopted a heterologous ligand keto-mycolic acid from among M. tuberculosis lipid repertoire for the host orphan NR TR4. Crosstalk between cell wall lipids and TR4 was analyzed by transactivation and promoter reporter assays. Mycolic acid (MA) was found to transactivate TR4 significantly compared with other cell wall lipids. Among the MA, the oxygenated form, keto-MA, was responsible for transactivation, and the identity was validated by TR4 binding assays followed by TLC and nuclear magnetic resonance. Isothermal titration calorimetry revealed that keto-MA binding to TR4 is energetically favorable. This keto-MA-TR4 axis seems to be essential to this oxygenated MA induction of FMs and granuloma formation as evaluated by in vitro and in vivo model of granuloma formation. TR4 binding with keto-MA features a unique association of host nuclear receptor with a bacterial lipid and adds to the presently known ligand repertoire beyond dietary lipids. Pharmacologic modulation of this heterologous axis may hold promise as an adjunct therapy to frontline tuberculosis drugs.

SMMRNA: a database of small molecule modulators of RNA.

We have developed SMMRNA, an interactive database, available at http://www.smmrna.org, with special focus on small molecule ligands targeting RNA. Currently, SMMRNA consists of ∼770 unique ligands along with structural images of RNA molecules. Each ligand in the SMMRNA contains information such as Kd, Ki, IC50, ΔTm, molecular weight (MW), hydrogen donor and acceptor count, XlogP, number of rotatable bonds, number of aromatic rings and 2D and 3D structures. These parameters can be explored using text search, advanced search, substructure and similarity-based analysis tools that are embedded in SMMRNA. A structure editor is provided for 3D visualization of ligands. Advance analysis can be performed using substructure and OpenBabel-based chemical similarity fingerprints. Upload facility for both RNA and ligands is also provided. The physicochemical properties of the ligands were further examined using OpenBabel descriptors, hierarchical clustering, binning partition and multidimensional scaling. We have also generated a 3D conformation database of ligands to support the structure and ligand-based screening. SMMRNA provides comprehensive resource for further design, development and refinement of small molecule modulators for selective targeting of RNA molecules.

Mechanistic insight into the enzymatic reduction of truncated hemoglobin N of Mycobacterium tuberculosis: role of the CD loop and pre-A motif in electron cycling.

Many pathogenic microorganisms have evolved hemoglobin-mediated nitric oxide (NO) detoxification mechanisms, where a globin domain in conjunction with a partner reductase catalyzes the conversion of toxic NO to innocuous nitrate. The truncated hemoglobin HbN of Mycobacterium tuberculosis displays a potent NO dioxygenase activity despite lacking a reductase domain. The mechanism by which HbN recycles itself during NO dioxygenation and the reductase that participates in this process are currently unknown. This study demonstrates that the NADH-ferredoxin/flavodoxin system is a fairly efficient partner for electron transfer to HbN with an observed reduction rate of 6.2 µM/min(-1), which is nearly 3- and 5-fold faster than reported for Vitreoscilla hemoglobin and myoglobin, respectively. Structural docking of the HbN with Escherichia coli NADH-flavodoxin reductase (FdR) together with site-directed mutagenesis revealed that the CD loop of the HbN forms contacts with the reductase, and that Gly(48) may have a vital role. The donor to acceptor electron coupling parameters calculated using the semiempirical pathway method amounts to an average of about 6.4 10(-5) eV, which is lower than the value obtained for E. coli flavoHb (8.0 10(-4) eV), but still supports the feasibility of an efficient electron transfer. The deletion of Pre-A abrogated the heme iron reduction by FdR in the HbN, thus signifying its involvement during intermolecular interactions of the HbN and FdR. The present study, thus, unravels a novel role of the CD loop and Pre-A motif in assisting the interactions of the HbN with the reductase and the electron cycling, which may be vital for its NO-scavenging function.

Microwave-Assisted Synthesis of Functionalized Carbon Nanospheres Using Banana Peels: pH-Dependent Synthesis, Characterization, and Selective Sensing Applications.

This work presents a microwave-based green synthesis method for producing carbon nanospheres (CNSs) and investigates the impact of presynthesis pH on their size and assembly. The resulting CNSs are monodispersed, averaging 35 nm in size, and exhibit notable characteristics including high water solubility, photostability, and a narrow size distribution, achieved within a synthesis time of 15 min. The synthesized CNS features functional groups such as -OH, -COOH, -NH, -C-O-C, =C-H, and -CH. This diversity empowers the CNS for various applications including sensing. The CNS exhibits a distinct UV peak at 282 nm and emits intense fluorescence at 430 nm upon excitation at 350 nm. These functionalized CNSs enable selective and specific sensing of Cu2+ ions and the amino acid tryptophan (Trp) in aqueous solutions. In the presence of Cu2+ ions, static-based quenching of CNS fluorescence was observed due to the chelation-enhanced quenching (CHEQ) effect. Notably, Cu2+ ions induce a substantial change in UV spectra alongside a red-shift in the peak position. The limits of detection and quantification for Cu2+ ions with CNS are determined as 0.73 and 2.45 µg/mL, respectively. Additionally, on interaction with tryptophan, the UV spectra of CNS display a marked increase in the peak at 282 nm, accompanied by a red-shift phenomenon. The limits of detection and quantification for l-tryptophan are 4.510 × 10-3 and 1.50 × 10-2 µg/mL, respectively, indicating its significant potential for biological applications. Furthermore, the practical applicability of CNSs is demonstrated by their successful implementation in analyzing real water samples and filter paper-based examination, showcasing their effectiveness for on-site sensing.

Corrigendum to "Exploiting cheminformatic and machine learning to navigate the available chemical space of potential small molecule inhibitors of SARS-CoV-2″ [Computational and Structural Biotechnology Journal 19 (2021) 424-438].

[This corrects the article DOI: 10.1016/j.csbj.2020.12.028.].

Quinoxalinone substituted pyrrolizine (4h)-induced dual inhibition of AKT and ERK instigates apoptosis in breast and colorectal cancer by modulating mitochondrial membrane potential.

AKT and ERK 1/2 play a pivotal role in cancer cell survival, proliferation, migration, and angiogenesis. Therefore, AKT and ERK 1/2 are considered crucial targets for cancer intervention. In this study, we envisaged the role of AKT and ERK signaling in apoptosis regulation in presence of compound 4h, a novel synthetic derivative of quinoxalinone substituted spiropyrrolizines exhibiting substantial antiproliferative activity in various cancer cell lines. Structurally 4h is a spiropyrrolizine derivative. Molecular docking analysis revealed that compound 4h shows strong binding affinity with AKT-1 (-9.5 kcal/mol) and ERK2 (-9.0 kcal/mol) via binding at allosteric sites of AKT and active site of ERK2. The implications of 4h binding with these two survival kinases resulted in the obstruction for ATP binding, hence, hampering their phosphorylation dependent activation. We demonstrate that 4h mediated apoptotic induction via disruption in the mitochondrial membrane potential of MCF-7 and HCT-116 cells and 4h-mediated inhibition of survival pathways occurred in a wild type PTEN background and is diminished in PTEN-/- cells. In 4T1 mammary carcinoma model, 4h exhibited pronounced reduction in the tumor size and tumor volume at significantly low doses. Besides, 4h reached the highest plasma concentration of 5.8 µM within a period of 1 h in mice model intraperitoneally. Furthermore, 4h showed acceptable clearance with an adequate elimination half-life and satisfactory pharmacokinetic behaviour, thus proclaiming as a potential lead molecule against breast and colorectal cancer by specifically inhibiting simultaneously AKT and ERK1/2 kinases.

Design of a bioactive small molecule that targets the myotonic dystrophy type 1 RNA via an RNA motif-ligand database and chemical similarity searching.

Myotonic dystrophy type 1 (DM1) is a triplet repeating disorder caused by expanded CTG repeats in the 3'-untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The transcribed repeats fold into an RNA hairpin with multiple copies of a 5'CUG/3'GUC motif that binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1). Sequestration of MBNL1 by expanded r(CUG) repeats causes splicing defects in a subset of pre-mRNAs including the insulin receptor, the muscle-specific chloride ion channel, sarco(endo)plasmic reticulum Ca(2+) ATPase 1, and cardiac troponin T. Based on these observations, the development of small-molecule ligands that target specifically expanded DM1 repeats could be of use as therapeutics. In the present study, chemical similarity searching was employed to improve the efficacy of pentamidine and Hoechst 33258 ligands that have been shown previously to target the DM1 triplet repeat. A series of in vitro inhibitors of the RNA-protein complex were identified with low micromolar IC(50)'s, which are >20-fold more potent than the query compounds. Importantly, a bis-benzimidazole identified from the Hoechst query improves DM1-associated pre-mRNA splicing defects in cell and mouse models of DM1 (when dosed with 1 mM and 100 mg/kg, respectively). Since Hoechst 33258 was identified as a DM1 binder through analysis of an RNA motif-ligand database, these studies suggest that lead ligands targeting RNA with improved biological activity can be identified by using a synergistic approach that combines analysis of known RNA-ligand interactions with chemical similarity searching.

Synthesis and use of cell-permeant cyclic ADP-ribose.

Cyclic ADP-ribose (cADPR) is a second messenger that acts on ryanodine receptors to mobilize Ca(2+). cADPR has a net negative charge at physiological pH making it not passively membrane permeant thereby requiring it to be injected, electroporated or loaded via liposomes. Such membrane impermeance of other charged intracellular messengers (including cyclic AMP, inositol 1,4,5-trisphosphate and nicotinic acid adenine dinucleotide phosphate) and fluorescent dyes (including fura-2 and fluorescein) has been overcome by synthesizing masked analogs (prodrugs), which are passively permeant and hydrolyzed to the parent compound inside cells. We now report the synthesis and biological activity of acetoxymethyl (AM) and butoxymethyl (BM) analogs of cADPR. Extracellular addition of cADPR-AM or cADPR-BM to neuronal cells in primary culture or PC12 neuroblastoma cells induced increases in cytosolic Ca(2+). Pre-incubation of PC12 cells with thapsigargin, ryanodine or caffeine eliminated the response to cADPR-AM, whereas the response still occurred in the absence of extracellular Ca(2+). Combined, these data demonstrate that masked cADPR analogs are cell-permeant and biologically active. We hope these cell-permeant tools will facilitate cADPR research and reveal its diverse physiological functions.

Fluorescence "Turn-Off" and Colorimetric Sensor for Fe2+, Fe3+, and Cu2+ Ions Based on a 2,5,7-Triarylimidazopyridine Scaffold.

Two cyanoimidazopyridine-based sensors (SS1 and SS2) were explored for the colorimetric and fluorometric detection of Fe2+, Fe3+, and Cu2+ ions in the semi-aqueous medium. The "turn-off" fluorescence response of both sensors to these ions was due to the restriction in internal charge transfer. Job's plot and semi-empirical calculations revealed that SS1 and SS2 complexed with Cu2+ ions in a 1:1 ratio and Fe2+/3+ ions in a 2:1 ratio, respectively. The sensors were found to have high binding constant (K a) values and low detection limit values. FMO analysis using the semi-empirical quantum mechanics method revealed the decrease in energy gap after complexation with metal ions. Sensor-coated filter paper strips were prepared and analyzed, where the color changes in the strips could be utilized for the real-time detection of Fe2+, Fe3+, and Cu2+ ions.

NMR spectroscopy and molecular dynamics simulation of r(CCGCUGCGG)2 reveal a dynamic UU internal loop found in myotonic dystrophy type 1.

The NMR structure of an RNA with a copy of the 5'CUG/3'GUC motif found in the triplet repeating disorder myotonic dystrophy type 1 (DM1) is disclosed. The lowest energy conformation of the UU pair is a single-hydrogen bond structure; however, the UU protons undergo exchange indicating structural dynamics. Molecular dynamics simulations show that the single hydrogen bond structure is the most populated one but the UU pair interconverts among zero, one, and two hydrogen bond pairs. These studies have implications for the recognition of the DM1 RNA by small molecules and proteins.

Myotonic dystrophy type 1 RNA crystal structures reveal heterogeneous 1 × 1 nucleotide UU internal loop conformations.

RNA internal loops often display a variety of conformations in solution. Herein, we visualize conformational heterogeneity in the context of the 5'CUG/3'GUC repeat motif present in the RNA that causes myotonic dystrophy type 1 (DM1). Specifically, two crystal structures of a model DM1 triplet repeating construct, 5'r[UUGGGC(CUG)(3)GUCC](2), refined to 2.20 and 1.52 Å resolution are disclosed. Here, differences in the orientation of the 5' dangling UU end between the two structures induce changes in the backbone groove width, which reveals that noncanonical 1 × 1 nucleotide UU internal loops can display an ensemble of pairing conformations. In the 2.20 Å structure, CUGa, the 5' UU forms a one hydrogen-bonded pair with a 5' UU of a neighboring helix in the unit cell to form a pseudoinfinite helix. The central 1 × 1 nucleotide UU internal loop has no hydrogen bonds, while the terminal 1 × 1 nucleotide UU internal loops each form a one-hydrogen bond pair. In the 1.52 Å structure, CUGb, the 5' UU dangling end is tucked into the major groove of the duplex. While the canonically paired bases show no change in base pairing, in CUGb the terminal 1 × 1 nucleotide UU internal loops now form two hydrogen-bonded pairs. Thus, the shift in the major groove induced by the 5' UU dangling end alters noncanonical base patterns. Collectively, these structures indicate that 1 × 1 nucleotide UU internal loops in DM1 may sample multiple conformations in vivo. This observation has implications for the recognition of this RNA, and other repeating transcripts, by protein and small molecule ligands.

Acidic NAADP-sensitive calcium stores in the endothelium: agonist-specific recruitment and role in regulating blood pressure.

Accumulating evidence implicates nicotinic acid adenine dinucleotide phosphate (NAADP) in the control of Ca(2+)-dependent functions. Little, however, is known concerning its role in the vascular endothelium, a major regulator of blood pressure. Here, we show that NAADP acetoxymethyl ester (NAADP-AM), a cell-permeant NAADP analog, increases cytosolic Ca(2+) concentration in aortic endothelial cells. We demonstrate that these signals and those evoked by acetylcholine are blocked by disrupting acidic organelles with bafilomycin A1. In contrast, Ca(2+) signals in response to thrombin are only partially inhibited by bafilomycin A1 treatment, and those to ATP were insensitive, suggesting that recruitment of acidic stores is agonist-specific. We further show that NAADP-evoked Ca(2+) signals hyperpolarize endothelial cells and generate NO. Additionally, we demonstrate that NAADP dilates aortic rings in an endothelium- and NO-dependent manner. Finally, we show that intravenous administration of NAADP-AM into anesthetized rats decreases mean arterial pressure. Our data extend the actions of NAADP to the endothelium both in vitro and in vivo, pointing to a previously unrecognized role for this messenger in controlling blood pressure.

Identification of a chemical probe for NAADP by virtual screening.

Research into the biological role of the Ca(2+)-releasing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) has been hampered by a lack of chemical probes. To find new chemical probes for exploring NAADP signaling, we turned to virtual screening, which can evaluate millions of molecules rapidly and inexpensively. We used NAADP as the query ligand to screen the chemical library ZINC for compounds with similar three-dimensional shape and electrostatic properties. We tested the top-ranking hits in a sea urchin egg bioassay and found that one hit, Ned-19, blocks NAADP signaling at nanomolar concentrations. In intact cells, Ned-19 blocked NAADP signaling and fluorescently labeled NAADP receptors. Moreover, we show the utility of Ned-19 as a chemical probe by using it to demonstrate that NAADP is a key causal link between glucose sensing and Ca(2+) increases in mouse pancreatic beta cells.

Chemical Space Exploration of DprE1 Inhibitors Using Chemoinformatics and Artificial Intelligence.

Tuberculosis (TB), entrained by Mycobacterium tuberculosis, continues to be an enfeebling disease, killing nearly 1.5 million people in 2019, with 2 billion people worldwide affected by latent TB. The multidrug-resistant and totally drug-resistant emerging strains further exacerbate the TB infection. The cell wall of bacteria provides critical virulence components such as cell surface proteins, regulators, signal transduction proteins, and toxins. The cell wall biosynthesis pathway of Mycobacterium tuberculosis is exhaustively studied to discover novel drug targets. Decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1) is an important enzyme involved in the arabinogalactan biosynthetic pathway of Mycobacterium tuberculosis cell wall and is essential for both latent and persistent bacterial infection. We analyzed all known ∼1300 DprE1 inhibitors to gain deep insights into the chemogenomic space of DprE1-ligand complexes. Physicochemical descriptors of the DprE1 inhibitors showed a marked lipophilic character forming a cluster distinct from the existing TB drugs, as revealed by the principal component analysis. Similarity analysis using Murcko scaffolds and rubber band scaling revealed scarce representation of the chemical space. Further, Murcko scaffold analysis uncovered favorable and unfavorable scaffolds, where benzo and pyridine-based core scaffolds exhibit the highest biological activity, as evidenced by their MIC and IC50 values. Automatic SAR and R-group decomposition analysis resulted in the identification of substructures responsible for the inhibitory activity of the DprE1 enzyme. Further, with activity cliff analysis, we observed prominent discontinuity in the SAR of DprE1 inhibitors, where even simple structural modification in the chemical scaffold resulted in significant potency difference, presumably due to the binding orientation and interaction in the active site. Thiophene, 6-membered aromatic rings, and unsubstituted benzene ring-based toxicophores were identified in the DprE1 chemical space using an artificial intelligence approach based on inductive logic programming. This paper, hence, ushers in new insights for the design and development of potent covalent and non-covalent DprE1 inhibitors and guides hit and lead optimization for the development of non-hazardous small molecule therapeutics for Mycobacterium tuberculosis.

Exploiting cheminformatic and machine learning to navigate the available chemical space of potential small molecule inhibitors of SARS-CoV-2.

The current life-threatening and tenacious pandemic eruption of coronavirus disease in 2019 (COVID-19) has posed a significant global hazard concerning high mortality rate, economic meltdown, and everyday life distress. The rapid spread of COVID-19 demands countermeasures to combat this deadly virus. Currently, there are no drugs approved by the FDA to treat COVID-19. Therefore, discovering small molecule therapeutics for treating COVID-19 infection is essential. So far, only a few small molecule inhibitors are reported for coronaviruses. There is a need to expand the small chemical space of coronaviruses inhibitors by adding potent and selective scaffolds with anti-COVID activity. In this context, the huge antiviral chemical space already available can be analysed using cheminformatic and machine learning to unearth new scaffolds. We created three specific datasets called "antiviral dataset" (N = 38,428) "drug-like antiviral dataset" (N = 20,963) and "anticorona dataset" (N = 433) for this purpose. We analyzed the 433 molecules of "anticorona dataset" for their scaffold diversity, physicochemical distributions, principal component analysis, activity cliffs, R-group decomposition, and scaffold mapping. The scaffold diversity of the "anticorona dataset" in terms of Murcko scaffold analysis demonstrates a thorough representation of diverse chemical scaffolds. However, physicochemical descriptor analysis and principal component analysis demonstrated negligible drug-like features for the "anticorona dataset" molecules. The "antiviral dataset" and "drug-like antiviral dataset" showed low scaffold diversity as measured by the Gini coefficient. The hierarchical clustering of the "antiviral dataset" against the "anticorona dataset" demonstrated little molecular similarity. We generated a library of frequent fragments and polypharmacological ligands targeting various essential viral proteins such as main protease, helicase, papain-like protease, and replicase polyprotein 1ab. Further structural and chemical features of the "anticorona dataset" were compared with SARS-CoV-2 repurposed drugs, FDA-approved drugs, natural products, and drugs currently in clinical trials. Using machine learning tool DCA (DMax Chemistry Assistant), we converted the "anticorona dataset" into an elegant hypothesis with significant functional biological relevance. Machine learning analysis uncovered that FDA approved drugs, Tizanidine HCl, Cefazolin, Raltegravir, Azilsartan, Acalabrutinib, Luliconazole, Sitagliptin, Meloxicam (Mobic), Succinyl sulfathiazole, Fluconazole, and Pranlukast could be repurposed as effective drugs for COVID-19. Fragment-based scaffold analysis and R-group decomposition uncovered pyrrolidine and the indole molecular scaffolds as the potent fragments for designing and synthesizing the novel drug-like molecules for targeting SARS-CoV-2. This comprehensive and systematic assessment of small-molecule viral therapeutics' entire chemical space realised critical insights to potentially privileged scaffolds that could aid in enrichment and rapid discovery of efficacious antiviral drugs for COVID-19.

Analogues of the nicotinic acid adenine dinucleotide phosphate (NAADP) antagonist Ned-19 indicate two binding sites on the NAADP receptor.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca(2+)-releasing messenger. Biological data suggest that its receptor has two binding sites: one high-affinity locking site and one low-affinity opening site. To directly address the presence and function of these putative binding sites, we synthesized and tested analogues of the NAADP antagonist Ned-19. Ned-19 itself inhibits both NAADP-mediated Ca(2+) release and NAADP binding. A fluorometry bioassay was used to assess NAADP-mediated Ca(2+) release, whereas a radioreceptor assay was used to assess binding to the NAADP receptor (only at the high-affinity site). In Ned-20, the fluorine is para rather than ortho as in Ned-19. Ned-20 does not inhibit NAADP-mediated Ca(2+) release but inhibits NAADP binding. Conversely, Ned-19.4 (a methyl ester of Ned-19) inhibits NAADP-mediated Ca(2+) release but cannot inhibit NAADP binding. Furthermore, Ned-20 prevents the self-desensitization response characteristic of NAADP in sea urchin eggs, confirming that this response is mediated by a high-affinity allosteric site to which NAADP binds in the radioreceptor assay. Collectively, these data provide the first direct evidence for two binding sites (one high- and one low-affinity) on the NAADP receptor.