A community effort in SARS-CoV-2 drug discovery.

The COVID-19 pandemic continues to pose a substantial threat to human lives and is likely to do so for years to come. Despite the availability of vaccines, searching for efficient small-molecule drugs that are widely available, including in low- and middle-income countries, is an ongoing challenge. In this work, we report the results of an open science community effort, the "Billion molecules against COVID-19 challenge", to identify small-molecule inhibitors against SARS-CoV-2 or relevant human receptors. Participating teams used a wide variety of computational methods to screen a minimum of 1 billion virtual molecules against 6 protein targets. Overall, 31 teams participated, and they suggested a total of 639,024 molecules, which were subsequently ranked to find 'consensus compounds'. The organizing team coordinated with various contract research organizations (CROs) and collaborating institutions to synthesize and test 878 compounds for biological activity against proteases (Nsp5, Nsp3, TMPRSS2), nucleocapsid N, RdRP (only the Nsp12 domain), and (alpha) spike protein S. Overall, 27 compounds with weak inhibition/binding were experimentally identified by binding-, cleavage-, and/or viral suppression assays and are presented here. Open science approaches such as the one presented here contribute to the knowledge base of future drug discovery efforts in finding better SARS-CoV-2 treatments.

A molecular signature defining exercise adaptation with ageing and in vivo partial reprogramming in skeletal muscle.

Exercise promotes functional improvements in aged tissues, but the extent to which it simulates partial molecular reprogramming is unknown. Using transcriptome profiling from (1) a skeletal muscle-specific in vivo Oct3/4, Klf4, Sox2 and Myc (OKSM) reprogramming-factor expression murine model; (2) an in vivo inducible muscle-specific Myc induction murine model; (3) a translatable high-volume hypertrophic exercise training approach in aged mice; and (4) human exercise muscle biopsies, we collectively defined exercise-induced genes that are common to partial reprogramming. Late-life exercise training lowered murine DNA methylation age according to several contemporary muscle-specific clocks. A comparison of the murine soleus transcriptome after late-life exercise training to the soleus transcriptome after OKSM induction revealed an overlapping signature that included higher JunB and Sun1. Also, within this signature, downregulation of specific mitochondrial and muscle-enriched genes was conserved in skeletal muscle of long-term exercise-trained humans; among these was muscle-specific Abra/Stars. Myc is the OKSM factor most induced by exercise in muscle and was elevated following exercise training in aged mice. A pulse of MYC rewired the global soleus muscle methylome, and the transcriptome after a MYC pulse partially recapitulated OKSM induction. A common signature also emerged in the murine MYC-controlled and exercise adaptation transcriptomes, including lower muscle-specific Melusin and reactive oxygen species-associated Romo1. With Myc, OKSM and exercise training in mice, as well habitual exercise in humans, the complex I accessory subunit Ndufb11 was lower; low Ndufb11 is linked to longevity in rodents. Collectively, exercise shares similarities with genetic in vivo partial reprogramming. KEY POINTS: Advances in the last decade related to cellular epigenetic reprogramming (e.g. DNA methylome remodelling) toward a pluripotent state via the Yamanaka transcription factors Oct3/4, Klf4, Sox2 and Myc (OKSM) provide a window into potential mechanisms for combatting the deleterious effects of cellular ageing. Using global gene expression analysis, we compared the effects of in vivo OKSM-mediated partial reprogramming in skeletal muscle fibres of mice to the effects of late-life murine exercise training in muscle. Myc is the Yamanaka factor most induced by exercise in skeletal muscle, and so we compared the MYC-controlled transcriptome in muscle to Yamanaka factor-mediated and exercise adaptation mRNA landscapes in mice and humans. A single pulse of MYC is sufficient to remodel the muscle methylome. We identify partial reprogramming-associated genes that are innately altered by exercise training and conserved in humans, and propose that MYC contributes to some of these responses.

Muscle-Specific Cellular and Molecular Adaptations to Late-Life Voluntary Concurrent Exercise.

Murine exercise models can provide information on factors that influence muscle adaptability with aging, but few translatable solutions exist. Progressive weighted wheel running (PoWeR) is a simple, voluntary, low-cost, high-volume endurance/resistance exercise approach for training young mice. In the current investigation, aged mice (22-mo-old) underwent a modified version of PoWeR for 8 wk. Muscle functional, cellular, biochemical, transcriptional, and myonuclear DNA methylation analyses provide an encompassing picture of how muscle from aged mice responds to high-volume combined training. Mice run 6-8 km/d, and relative to sedentary mice, PoWeR increases plantarflexor muscle strength. The oxidative soleus of aged mice responds to PoWeR similarly to young mice in every parameter measured in previous work; this includes muscle mass, glycolytic-to-oxidative fiber type transitioning, fiber size, satellite cell frequency, and myonuclear number. The oxidative/glycolytic plantaris adapts according to fiber type, but with modest overall changes in muscle mass. Capillarity increases markedly with PoWeR in both muscles, which may be permissive for adaptability in advanced age. Comparison to published PoWeR RNA-sequencing data in young mice identified conserved regulators of adaptability across age and muscles; this includes Aldh1l1 which associates with muscle vasculature. Agrn and Samd1 gene expression is upregulated after PoWeR simultaneous with a hypomethylated promoter CpG in myonuclear DNA, which could have implications for innervation and capillarization. A promoter CpG in Rbm10 is hypomethylated by late-life exercise in myonuclei, consistent with findings in muscle tissue. PoWeR and the data herein are a resource for uncovering cellular and molecular regulators of muscle adaptation with aging.

One-Year Postfracture Mortality Rate in Older Adults With Hip Fractures Relative to Other Lower Extremity Fractures: Retrospective Cohort Study.

BACKGROUND: Hip fracture in older adults is tied to increased mortality risk. Deconvolution of the mortality risk specific to hip fracture from that of various other fracture types has not been performed in recent hip fracture studies but is critical to determining current unmet needs for therapeutic intervention. OBJECTIVE: This study examined whether hip fracture increases the 1-year postfracture mortality rate relative to several other fracture types and determined whether dementia or type 2 diabetes (T2D) exacerbates postfracture mortality risk. METHODS: TriNetX Diamond Network data were used to identify patients with a single event of fracture of the hip, the upper humerus, or several regions near and distal to the hip occurring from 60 to 89 years of age from 2010 to 2019. Propensity score matching, Kaplan-Meier, and hazard ratio analyses were performed for all fracture groupings relative to hip fracture. One-year postfracture mortality rates in elderly populations with dementia or T2D were established. RESULTS: One-year mortality rates following hip fracture consistently exceeded all other lower extremity fracture groupings as well as the upper humerus. Survival probabilities were significantly lower in the hip fracture groups, even after propensity score matching was performed on cohorts for a variety of broad categories of characteristics. Dementia in younger elderly cohorts acted synergistically with hip fracture to exacerbate the 1-year mortality risk. T2D did not exacerbate the 1-year mortality risk beyond mere additive effects. CONCLUSIONS: Elderly patients with hip fracture have a significantly decreased survival probability. Greatly increased 1-year mortality rates following hip fracture may arise from differences in bone quality, bone density, trauma, concomitant fractures, postfracture treatments or diagnoses, restoration of prefracture mobility, or a combination thereof. The synergistic effect of dementia may suggest detrimental mechanistic or behavioral combinations for these 2 comorbidities. Renewed efforts should focus on modulating the mechanisms behind this heightened mortality risk, with particular attention to mobility and comorbid dementia.

Late-life exercise mitigates skeletal muscle epigenetic aging.

There are functional benefits to exercise in muscle, even when performed late in life, but the contributions of epigenetic factors to late-life exercise adaptation are poorly defined. Using reduced representation bisulfite sequencing (RRBS), ribosomal DNA (rDNA) and mitochondrial-specific examination of methylation, targeted high-resolution methylation analysis, and DNAge™ epigenetic aging clock analysis with a translatable model of voluntary murine endurance/resistance exercise training (progressive weighted wheel running, PoWeR), we provide evidence that exercise may mitigate epigenetic aging in skeletal muscle. Late-life PoWeR from 22-24 months of age modestly but significantly attenuates an age-associated shift toward promoter hypermethylation. The epigenetic age of muscle from old mice that PoWeR-trained for eight weeks was approximately eight weeks younger than 24-month-old sedentary counterparts, which represents ~8% of the expected murine lifespan. These data provide a molecular basis for exercise as a therapy to attenuate skeletal muscle aging.

Development & validation of LC-MS/MS assay for 5-amino-1-methyl quinolinium in rat plasma: Application to pharmacokinetic and oral bioavailability studies.

5-Amino-1-methyl quinolinium (5-AMQ) is a potent Nicotinamide N-methyl transferase (NNMT) inhibitor. NNMT is an enzyme that catalyzes the N-methylation of the endogenous substrate nicotinamide, as well as exogenous xenobiotics. NNMT is fundamental to cellular metabolism; NNMT is overexpressed in select tissues (e.g., adipose tissue, skeletal muscle, etc.) in pathophysiological conditions, making it a clinically relevant target for drug development in several chronic diseases including obesity and diabetes. The objective of this study was to develop and validate a simple, sensitive, and reproducible liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 5-AMQ in rat plasma and urine samples. 5-AMQ was extracted from plasma and urine by protein precipitation. Chromatographic separation was achieved using an ACE® Excel™ C18 column (2 µm, 50 × 2.1 mm) with a binary gradient solvent system comprising of water (A) and acetonitrile (B) containing 0.1 % formic acid as the mobile phase. Analysis was performed using an API 4000 QTRAP hybrid triple quadruple mass spectrometer and multiple reaction monitoring (MRM) in positive mode at m/z transitions of 159.100 → 90.00 and 162.200 → 117.200 for 5-AMQ and the internal standard, respectively. The standard curves of 5-AMQ in rat urine and plasma samples were linear in the concentration range of 10-2500 ng/mL. The intra-day and inter-day precisions and accuracies for 5-AMQ at four concentration levels in rat plasma and urine samples were found to be within the 15 % FDA acceptance range. Similarly, the accuracy and precision of 5-AMQ quantification in samples diluted up to 20-fold using blank plasma were within the 15 % acceptable range. Furthermore, the extraction recoveries and matrix effects at three concentration levels of rat plasma samples ranged from 99.5 %-110.6 % and -6.1 %-14.1 %, respectively. 5-AMQ was stable in rat plasma samples subjected to standard storage, preparation, and handling conditions, with less than 15 % variation noted at two concentration levels. The validated, sensitive, and reproducible LC-MS/MS method for 5-AMQ in rat plasma and urine samples was effectively applied to a pharmacokinetic study in rats with IV and oral administration of 5-AMQ. 5-AMQ displayed substantial plasma exposures via IV and oral route, with a mean maximum plasma concentration of 2252 ng/mL after oral administration, mean area under the curve (AUC0-∞) of 3708 h.ng/mL and 14431 h.ng/mL for the IV and oral groups, respectively, mean terminal elimination half-life of 3.80 ± 1.10 h and 6.90 ± 1.20 h respectively after intravenous and oral dose, and a good oral bioavailability (F % = 38.4).

Combined nicotinamide N-methyltransferase inhibition and reduced-calorie diet normalizes body composition and enhances metabolic benefits in obese mice.

Obesity is a large and growing global health problem with few effective therapies. The present study investigated metabolic and physiological benefits of nicotinamide N-methyltransferase inhibitor (NNMTi) treatment combined with a lean diet substitution in diet-induced obese mice. NNMTi treatment combined with lean diet substitution accelerated and improved body weight and fat loss, increased whole-body lean mass to body weight ratio, reduced liver and epididymal white adipose tissue weights, decreased liver adiposity, and improved hepatic steatosis, relative to a lean diet substitution alone. Importantly, combined lean diet and NNMTi treatment normalized body composition and liver adiposity parameters to levels observed in age-matched lean diet control mice. NNMTi treatment produced a unique metabolomic signature in adipose tissue, with predominant increases in ketogenic amino acid abundance and alterations to metabolites linked to energy metabolic pathways. Taken together, NNMTi treatment's modulation of body weight, adiposity, liver physiology, and the adipose tissue metabolome strongly support it as a promising therapeutic for obesity and obesity-driven comorbidities.

Small molecule nicotinamide N-methyltransferase inhibitor activates senescent muscle stem cells and improves regenerative capacity of aged skeletal muscle.

Aging is accompanied by progressive declines in skeletal muscle mass and strength and impaired regenerative capacity, predisposing older adults to debilitating age-related muscle deteriorations and severe morbidity. Muscle stem cells (muSCs) that proliferate, differentiate to fusion-competent myoblasts, and facilitate muscle regeneration are increasingly dysfunctional upon aging, impairing muscle recovery after injury. While regulators of muSC activity can offer novel therapeutics to improve recovery and reduce morbidity among aged adults, there are no known muSC regenerative small molecule therapeutics. We recently developed small molecule inhibitors of nicotinamide N-methyltransferase (NNMT), an enzyme overexpressed with aging in skeletal muscles and linked to impairment of the NAD+ salvage pathway, dysregulated sirtuin 1 activity, and increased muSC senescence. We hypothesized that NNMT inhibitor (NNMTi) treatment will rescue age-related deficits in muSC activity to promote superior regeneration post-injury in aging muscle. 24-month old mice were treated with saline (control), and low and high dose NNMTi (5 and 10 mg/kg) for 1-week post-injury, or control and high dose NNMTi for 3-weeks post-injury. All mice underwent an acute muscle injury (barium chloride injection) locally to the tibialis anterior (TA) muscle, and received 5-ethynyl-2'-deoxyuridine systemically to analyze muSC activity. In vivo contractile function measurements were conducted on the injured TA muscle and tissues collected for ex-vivo analyses, including myofiber cross-sectional area (CSA) measurements to assess muscle recovery. Results revealed that muscle stem cell proliferation and subsequent fusion were elevated in NNMTi-treated mice, supporting nearly 2-fold greater CSA and shifts in fiber size distribution to greater proportions of larger sized myofibers and fewer smaller sized fibers in NNMTi-treated mice compared to controls. Prolonged NNMTi treatment post-injury further augmented myofiber regeneration evinced by increasingly larger fiber CSA. Importantly, improved muSC activity translated not only to larger myofibers after injury but also to greater contractile function, with the peak torque of the TA increased by ∼70% in NNMTi-treated mice compared to controls. Similar results were recapitulated in vitro with C2C12 myoblasts, where NNMTi treatment promoted and enhanced myoblast differentiation with supporting changes in the cellular NAD+/NADH redox states. Taken together, these results provide the first clear evidence that NNMT inhibitors constitute a viable pharmacological approach to enhance aged muscle regeneration by rescuing muSC function, supporting the development of NNMTi as novel mechanism-of-action therapeutic to improve skeletal muscle regenerative capacity and functional recovery after musculoskeletal injury in older adults.

Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice.

There is a critical need for new mechanism-of-action drugs that reduce the burden of obesity and associated chronic metabolic comorbidities. A potentially novel target to treat obesity and type 2 diabetes is nicotinamide-N-methyltransferase (NNMT), a cytosolic enzyme with newly identified roles in cellular metabolism and energy homeostasis. To validate NNMT as an anti-obesity drug target, we investigated the permeability, selectivity, mechanistic, and physiological properties of a series of small molecule NNMT inhibitors. Membrane permeability of NNMT inhibitors was characterized using parallel artificial membrane permeability and Caco-2 cell assays. Selectivity was tested against structurally-related methyltransferases and nicotinamide adenine dinucleotide (NAD+) salvage pathway enzymes. Effects of NNMT inhibitors on lipogenesis and intracellular levels of metabolites, including NNMT reaction product 1-methylnicotianamide (1-MNA) were evaluated in cultured adipocytes. Effects of a potent NNMT inhibitor on obesity measures and plasma lipid were assessed in diet-induced obese mice fed a high-fat diet. Methylquinolinium scaffolds with primary amine substitutions displayed high permeability from passive and active transport across membranes. Importantly, methylquinolinium analogues displayed high selectivity, not inhibiting related SAM-dependent methyltransferases or enzymes in the NAD+ salvage pathway. NNMT inhibitors reduced intracellular 1-MNA, increased intracellular NAD+ and S-(5'-adenosyl)-l-methionine (SAM), and suppressed lipogenesis in adipocytes. Treatment of diet-induced obese mice systemically with a potent NNMT inhibitor significantly reduced body weight and white adipose mass, decreased adipocyte size, and lowered plasma total cholesterol levels. Notably, administration of NNMT inhibitors did not impact total food intake nor produce any observable adverse effects. These results support development of small molecule NNMT inhibitors as therapeutics to reverse diet-induced obesity and validate NNMT as a viable target to treat obesity and related metabolic conditions. Increased flux of key cellular energy regulators, including NAD+ and SAM, may potentially define the therapeutic mechanism-of-action of NNMT inhibitors.

Host oxidative folding pathways offer novel anti-chikungunya virus drug targets with broad spectrum potential.

Alphaviruses require conserved cysteine residues for proper folding and assembly of the E1 and E2 envelope glycoproteins, and likely depend on host protein disulfide isomerase-family enzymes (PDI) to aid in facilitating disulfide bond formation and isomerization in these proteins. Here, we show that in human HEK293 cells, commercially available inhibitors of PDI or modulators thereof (thioredoxin reductase, TRX-R; endoplasmic reticulum oxidoreductin-1, ERO-1) inhibit the replication of CHIKV chikungunya virus (CHIKV) in vitro in a dose-dependent manner. Further, the TRX-R inhibitor auranofin inhibited Venezuelan equine encephalitis virus and the flavivirus Zika virus replication in vitro, while PDI inhibitor 16F16 reduced replication but demonstrated notable toxicity. 16F16 significantly altered the viral genome: plaque-forming unit (PFU) ratio of CHIKV in vitro without affecting relative intracellular viral RNA quantities and inhibited CHIKV E1-induced cell-cell fusion, suggesting that PDI inhibitors alter progeny virion infectivity through altered envelope function. Auranofin also increased the extracellular genome:PFU ratio but decreased the amount of intracellular CHIKV RNA, suggesting an alternative mechanism of action. Finally, auranofin reduced footpad swelling and viremia in the C57BL/6 murine model of CHIKV infection. Our results suggest that targeting oxidative folding pathways represents a potential new anti-alphavirus therapeutic strategy.

Structure-Activity Relationship for Small Molecule Inhibitors of Nicotinamide N-Methyltransferase.

Nicotinamide N-methyltransferase (NNMT) is a fundamental cytosolic biotransforming enzyme that catalyzes the N-methylation of endogenous and exogenous xenobiotics. We have identified small molecule inhibitors of NNMT with >1000-fold range of activity and developed comprehensive structure-activity relationships (SARs) for NNMT inhibitors. Screening of N-methylated quinolinium, isoquinolinium, pyrididium, and benzimidazolium/benzothiazolium analogues resulted in the identification of quinoliniums as a promising scaffold with very low micromolar (IC50 ∼ 1 µM) NNMT inhibition. Computer-based docking of inhibitors to the NNMT substrate (nicotinamide)-binding site produced a robust correlation between ligand-enzyme interaction docking scores and experimentally calculated IC50 values. Predicted binding orientation of the quinolinium analogues revealed selective binding to the NNMT substrate-binding site residues and essential chemical features driving protein-ligand intermolecular interactions and NNMT inhibition. The development of this new series of small molecule NNMT inhibitors direct the future design of lead drug-like inhibitors to treat several metabolic and chronic disease conditions characterized by abnormal NNMT activity.

Noncoupled Fluorescent Assay for Direct Real-Time Monitoring of Nicotinamide N-Methyltransferase Activity.

Nicotinamide N-methyltransferase (NNMT) is an important biotransforming enzyme that catalyzes the transfer of a labile methyl group from the ubiquitous cofactor S-5'-adenosyl-l-methionine (SAM) to endogenous and exogenous small molecules to form methylated end products. NNMT has been implicated in a number of chronic disease conditions, including metabolic disorders, cardiovascular disease, cancer, osteoarthritis, kidney disease, and Parkinson's disease. We have developed a novel noncoupled fluorescence-based methyltransferase assay that allows direct ultrasensitive real-time detection of the NNMT reaction product 1-methylquinolinium. This is the first assay reported to date to utilize fluorescence spectroscopy to directly monitor NNMT product formation and activity in real time. This assay provided accurate kinetic data that allowed detailed comparative analysis of the NNMT reaction mechanism and kinetic parameters. A reaction model based on a random bireactant mechanism produced global curve fits that were most consistent with steady-state initial velocity data collected across an array of substrate concentrations. On the basis of the reaction mechanism, each substrate could independently bind to the NNMT apoenzyme; however, both substrates bound to the complementary binary complexes with an affinity ∼20-fold stronger compared to their binding to the apoenzyme. This reaction mechanism implies either substrate-induced conformational changes or bireactant intermolecular interactions may stabilize the binding of the substrate to the binary complex and formation of the ternary complex. Importantly, this assay could rapidly generate concentration response curves for known NNMT inhibitors, suggesting its applicability for high-throughput screening of chemical libraries to identify novel NNMT inhibitors. Furthermore, our novel assay potentially offers a robust detection technology for use in SAM substrate competition assays for the discovery and development of SAM-dependent methyltransferase inhibitors.

Drug search for leishmaniasis: a virtual screening approach by grid computing.

The trypanosomatid protozoa Leishmania is endemic in ~100 countries, with infections causing ~2 million new cases of leishmaniasis annually. Disease symptoms can include severe skin and mucosal ulcers, fever, anemia, splenomegaly, and death. Unfortunately, therapeutics approved to treat leishmaniasis are associated with potentially severe side effects, including death. Furthermore, drug-resistant Leishmania parasites have developed in most endemic countries. To address an urgent need for new, safe and inexpensive anti-leishmanial drugs, we utilized the IBM World Community Grid to complete computer-based drug discovery screens (Drug Search for Leishmaniasis) using unique leishmanial proteins and a database of 600,000 drug-like small molecules. Protein structures from different Leishmania species were selected for molecular dynamics (MD) simulations, and a series of conformational "snapshots" were chosen from each MD trajectory to simulate the protein's flexibility. A Relaxed Complex Scheme methodology was used to screen ~2000 MD conformations against the small molecule database, producing >1 billion protein-ligand structures. For each protein target, a binding spectrum was calculated to identify compounds predicted to bind with highest average affinity to all protein conformations. Significantly, four different Leishmania protein targets were predicted to strongly bind small molecules, with the strongest binding interactions predicted to occur for dihydroorotate dehydrogenase (LmDHODH; PDB:3MJY). A number of predicted tight-binding LmDHODH inhibitors were tested in vitro and potent selective inhibitors of Leishmania panamensis were identified. These promising small molecules are suitable for further development using iterative structure-based optimization and in vitro/in vivo validation assays.

T(H)17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26.

Interleukin 17-producing helper T cells (T(H)17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. We found that interleukin 26 (IL-26), a human T(H)17 cell-derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, T(H)17 cell-derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26-DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of T(H)17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death.

Identification of a novel inhibitor of dengue virus protease through use of a virtual screening drug discovery Web portal.

We report the discovery of a novel small-molecule inhibitor of the dengue virus (DENV) protease (NS2B-NS3pro) using a newly constructed Web-based portal (DrugDiscovery@TACC) for structure-based virtual screening. Our drug discovery portal, an extension of virtual screening studies performed using IBM's World Community Grid, facilitated access to supercomputer resources managed by the Texas Advanced Computing Center (TACC) and enabled druglike commercially available small-molecule libraries to be rapidly screened against several high-resolution DENV NS2B-NS3pro crystallographic structures. Detailed analysis of virtual screening docking scores and hydrogen-bonding interactions between each docked ligand and the NS2B-NS3pro Ser135 side chain were used to select molecules for experimental validation. Compounds were ordered from established chemical companies, and compounds with established aqueous solubility were tested for their ability to inhibit DENV NS2B-NS3pro cleavage of a model substrate in kinetic studies. As a proof-of-concept, we validated a small-molecule dihydronaphthalenone hit as a single-digit-micromolar mixed noncompetitive inhibitor of the DENV protease. Since the dihydronaphthalenone was predicted to interact with NS2B-NS3pro residues that are largely conserved between DENV and the related West Nile virus (WNV), we tested this inhibitor against WNV NS2B-NS3pro and observed a similar mixed noncompetitive inhibition mechanism. However, the inhibition constants were ∼10-fold larger against the WNV protease relative to the DENV protease. This novel validated lead had no chemical features or pharmacophores associated with adverse toxicity, carcinogenicity, or mutagenicity risks and thus is attractive for additional characterization and optimization.

Intrinsic flexibility of West Nile virus protease in solution characterized using small-angle X-ray scattering.

West Nile virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection can cause severe neurological disease and fatality in humans. Efforts are ongoing to develop antiviral drugs that inhibit the WNV protease, a viral enzyme required for polyprotein processing. Unfortunately, little is known about the solution structure of recombinant WNV protease (NS2B-NS3pro) used for antiviral drug discovery and development, although X-ray crystal structures and nuclear magnetic resonance (NMR) studies have provided valuable insights into the interactions between NS2B-NS3pro and peptide-based inhibitors. We completed small-angle X-ray scattering and Fourier transform infrared spectroscopy experiments to determine the solution structure and dynamics of WNV NS2B-NS3pro in the absence of a bound substrate or inhibitor. Importantly, these solution studies suggested that all or most of the NS2B cofactor was highly flexible and formed an ensemble of structures, in contrast to the NS2B tertiary structures observed in crystallographic and NMR studies. The secondary structure of NS2B-NS3pro in solution had high ß-content, similar to the secondary structure observed in crystallographic studies. This work provided evidence of the intrinsic flexibility and conformational heterogeneity of the NS2B chain of the WNV protease in the absence of substratelike ligands, which should be considered during antiviral drug discovery and development efforts.

An assembly model of rift valley Fever virus.

Rift Valley fever virus (RVFV) is a bunyavirus endemic to Africa and the Arabian Peninsula that infects humans and livestock. The virus encodes two glycoproteins, Gn and Gc, which represent the major structural antigens and are responsible for host cell receptor binding and fusion. Both glycoproteins are organized on the virus surface as cylindrical hollow spikes that cluster into distinct capsomers with the overall assembly exhibiting an icosahedral symmetry. Currently, no experimental three-dimensional structure for any entire bunyavirus glycoprotein is available. Using fold recognition, we generated molecular models for both RVFV glycoproteins and found significant structural matches between the RVFV Gn protein and the influenza virus hemagglutinin protein and a separate match between RVFV Gc protein and Sindbis virus envelope protein E1. Using these models, the potential interaction and arrangement of both glycoproteins in the RVFV particle was analyzed, by modeling their placement within the cryo-electron microscopy density map of RVFV. We identified four possible arrangements of the glycoproteins in the virion envelope. Each assembly model proposes that the ectodomain of Gn forms the majority of the protruding capsomer and that Gc is involved in formation of the capsomer base. Furthermore, Gc is suggested to facilitate intercapsomer connections. The proposed arrangement of the two glycoproteins on the RVFV surface is similar to that described for the alphavirus E1-E2 proteins. Our models will provide guidance to better understand the assembly process of phleboviruses and such structural studies can also contribute to the design of targeted antivirals.

Use of parallel validation high-throughput screens to reduce false positives and identify novel dengue NS2B-NS3 protease inhibitors.

Dengue virus (DENV), a mosquito-borne member of the family Flaviviridae, is a significant global pathogen affecting primarily tropical and subtropical regions of the world and placing tremendous burden on the limited medical infrastructure that exists in many of the developing countries located within these regions. Recent outbreaks in developed countries, including Australia (Hanna et al., 2009), France (La Ruche et al., 2010), Taiwan (Kuan et al., 2010), and the USA (CDC, 2010), lead many researchers to believe that continued emergence into more temperate latitudes is likely. A primary concern is that there are no approved vaccines or antiviral therapies to treat DENV infections. Since the viral NS2B-NS3 protease (DENV NS2B-NS3pro) is required for virus replication, it provides a strategic target for the development of antiviral drugs. In this study, proof-of-concept high-throughput screenings (HTSs) were performed to unambiguously identify dengue 2 virus (DEN2V) NS2B-NS3pro inhibitors from a library of 2000 compounds. Validation screens were performed in parallel to concurrently eliminate insoluble, auto-fluorescing, and/or nonspecific inhibitors. Kinetic analyses of the hits revealed that parallel substrate fluorophore (AMC) interference controls and trypsin inhibition controls were able to reduce false positive rates due to solubility and fluorophore interference while the trypsin inhibition control additionally eliminated non-specific inhibitors. We identified five DEN2V NS2B-NS3pro inhibitors that also inhibited the related West Nile virus (WNV) protease (NS2B-NS3pro), but did not inhibit the trypsin protease. Biochemical analyses revealed various mechanisms of inhibition including competitive and mixed noncompetitive inhibition, with the lowest K(i) values being 12±1.5 µM for DEN2V NS2B-NS3pro and 2±0.2 µM for WNV NS2B-NS3pro.

Using free energy of binding calculations to improve the accuracy of virtual screening predictions.

Virtual screening of small molecule databases against macromolecular targets was used to identify binding ligands and predict their lowest energy bound conformation (i.e., pose). AutoDock4-generated poses were rescored using mean-field pathway decoupling free energy of binding calculations and evaluated if these calculations improved virtual screening discrimination between bound and nonbound ligands. Two small molecule databases were used to evaluate the effectiveness of the rescoring algorithm in correctly identifying binders of L99A T4 lysozyme. Self-dock calculations of a database containing compounds with known binding free energies and cocrystal structures largely reproduced experimental measurements, although the mean difference between calculated and experimental binding free energies increased as the predicted bound poses diverged from the experimental poses. In addition, free energy rescoring was more accurate than AutoDock4 scores in discriminating between known binders and nonbinders, suggesting free energy rescoring could be a useful approach to reduce false positive predictions in virtual screening experiments.

Anthracene-based inhibitors of dengue virus NS2B-NS3 protease.

Dengue virus (DENV) is a mosquito-borne flavivirus that has strained global healthcare systems throughout tropical and subtropical regions of the world. In addition to plaguing developing nations, it has re-emerged in several developed countries with recent outbreaks in the USA (CDC, 2010), Australia (Hanna et al., 2009), Taiwan (Kuan et al., 2010) and France (La Ruche et al., 2010). DENV infection can cause significant disease, including dengue fever, dengue hemorrhagic fever, dengue shock syndrome, and death. There are no approved vaccines or antiviral therapies to prevent or treat dengue-related illnesses. However, the viral NS2B-NS3 protease complex provides a strategic target for antiviral drug development since NS3 protease activity is required for virus replication. Recently, we reported two compounds with inhibitory activity against the DENV protease in vitro and antiviral activity against dengue 2 (DEN2V) in cell culture (Tomlinson et al., 2009a). Analogs of one of the lead compounds were purchased, tested in protease inhibition assays, and the data evaluated with detailed kinetic analyses. A structure activity relationship (SAR) identified key atomic determinants (i.e. functional groups) important for inhibitory activity. Four "second series" analogs were selected and tested to validate our SAR and structural models. Here, we report improvements to inhibitory activity ranging between ∼2- and 60-fold, resulting in selective low micromolar dengue protease inhibitors.