Oats Lower Age-Related Systemic Chronic Inflammation (iAge) in Adults at Risk for Cardiovascular Disease.

Despite being largely preventable, cardiovascular disease (CVD) is still the leading cause of death globally. Recent studies suggest that the immune system, particularly a form of systemic chronic inflammation (SCI), is involved in the mechanisms leading to CVD; thus, targeting SCI may help prevent or delay the onset of CVD. In a recent placebo-controlled randomized clinical trial, an oat product providing 3 g of ß-Glucan improved cholesterol low-density lipoprotein (LDL) levels and lowered cardiovascular risk in adults with borderline high cholesterol. Here, we conducted a secondary measurement of the serum samples to test whether the oat product has the potential to reduce SCI and improve other clinical outcomes related to healthy aging. We investigated the effects of the oat product on a novel metric for SCI called Inflammatory Age® (iAge®), derived from the Stanford 1000 Immunomes Project. The iAge® predicts multimorbidity, frailty, immune decline, premature cardiovascular aging, and all-cause mortality on a personalized level. A beneficial effect of the oat product was observed in subjects with elevated levels of iAge® at baseline (>49.6 iAge® years) as early as two weeks post-treatment. The rice control group did not show any significant change in iAge®. Interestingly, the effects of the oat product on iAge® were largely driven by a decrease in the Eotaxin-1 protein, an aging-related chemokine, independent of a person's gender, body mass index, or chronological age. Thus, we describe a novel anti-SCI role for oats that could have a major impact on functional, preventative, and personalized medicine.

In silico design of a novel nucleotide antiviral agent by free energy perturbation.

Nucleoside analogs are the backbone of antiviral therapies. Drugs from this class undergo processing by host or viral kinases to form the active nucleoside triphosphate species that selectively inhibits the viral polymerase. It is the central hypothesis that the nucleoside triphosphate analog must be a favorable substrate for the viral polymerase and the nucleoside precursor must be a satisfactory substrate for the host kinases to inhibit viral replication. Herein, free energy perturbation (FEP) was used to predict substrate affinity for both host and viral enzymes. Several uridine 5'-monophosphate prodrug analogs known to inhibit hepatitis C virus (HCV) were utilized in this study to validate the use of FEP. Binding free energies to the host monophosphate kinase and viral RNA-dependent RNA polymerase (RdRp) were calculated for methyl-substituted uridine analogs. The 2'-C-methyl-uridine and 4'-C-methyl-uridine scaffolds delivered favorable substrate binding to the host kinase and HCV RdRp that were consistent with results from cellular antiviral activity in support of our new approach. In a prospective evaluation, FEP results suggest that 2'-C-dimethyl-uridine scaffold delivered favorable monophosphate and triphosphate substrates for both host kinase and HCV RdRp, respectively. Novel 2'-C-dimethyl-uridine monophosphate prodrug was synthesized and exhibited sub-micromolar inhibition of HCV replication. Using this novel approach, we demonstrated for the first time that nucleoside analogs can be rationally designed that meet the multi-target requirements for antiviral activity.

The Mechanism of Action of Hepatitis B Virus Capsid Assembly Modulators Can Be Predicted from Binding to Early Assembly Intermediates.

Interfering with the self-assembly of virus nucleocapsids is a promising approach for the development of novel antiviral agents. Applied to hepatitis B virus (HBV), this approach has led to several classes of capsid assembly modulators (CAMs) that target the virus by either accelerating nucleocapsid assembly or misdirecting it into noncapsid-like particles, thereby inhibiting the HBV replication cycle. Here, we have assessed the structures of early nucleocapsid assembly intermediates, bound with and without CAMs, using molecular dynamics simulations. We find that distinct conformations of the intermediates are induced depending on whether the bound CAM accelerates or misdirects assembly. Specifically, the assembly intermediates with bound misdirecting CAMs appear to be flattened relative to those with bound accelerators. Finally, the potency of CAMs within the same class was studied. We find that an increased number of contacts with the capsid protein and favorable binding energies inferred from free energy perturbation calculations are indicative of increased potency.

7-Deaza-7-fluoro-2'-C-methyladenosine inhibits Zika virus infection and viral-induced neuroinflammation.

Zika virus (ZIKV) has gained a lot of attention in the past few years due to its rapid spread worldwide and its close association to severe neurological outcomes, such as microcephaly and Guillain-Barre syndrome. In this study, the in vitro and in vivo anti-ZIKV activity of 7-deaza-7-fluoro-2'-C-methyl-adenosine (DFMA) was evaluated. In vitro, using primary mouse neuronal cells and human neural stem cells infected by ZIKV, treatment with DFMA resulted in impaired viral replication and protection against virus-induced cell death. In vivo, when administrated prior to infection, DFMA prevented lethality and markedly reduced viral loads and neuroinflammation, including microgliosis and overall brain damage. Additionally, as an early therapeutic treatment, DFMA increased survival rates in mice. Collectively, these findings demonstrate that the nucleoside analog DFMA inhibits ZIKV infection and viral-induced neuroinflammation in vitro and in vivo without apparent untoward effects, suggesting it may be useful in individuals infected with ZIKV.

Nucleoside Analogs with Selective Antiviral Activity against Dengue Fever and Japanese Encephalitis Viruses.

Dengue virus (DENV) and Japanese encephalitis virus (JEV) are important arthropod-borne viruses from the Flaviviridae family. DENV is a global public health problem with significant social and economic impacts, especially in tropical and subtropical areas. JEV is a neurotropic arbovirus endemic to east and southeast Asia. There are no U.S. FDA-approved antiviral drugs available to treat or to prevent DENV and JEV infections, leaving nearly one-third of the world's population at risk for infection. Therefore, it is crucial to discover potent antiviral agents against these viruses. Nucleoside analogs, as a class, are widely used for the treatment of viral infections. In this study, we discovered nucleoside analogs that possess potent and selective anti-JEV and anti-DENV activities across all serotypes in cell-based assay systems. Both viruses were susceptible to sugar-substituted 2'-C-methyl analogs with either cytosine or 7-deaza-7-fluoro-adenine nucleobases. Mouse studies confirmed the anti-DENV activity of these nucleoside analogs. Molecular models were assembled for DENV serotype 2 (DENV-2) and JEV RNA-dependent RNA polymerase replication complexes bound to nucleotide inhibitors. These models show similarities between JEV and DENV-2, which recognize the same nucleotide inhibitors. Collectively, our findings provide promising compounds and a structural rationale for the development of direct-acting antiviral agents with dual activity against JEV and DENV infections.

Tetrahydroisoquinoline CXCR4 Antagonists Adopt a Hybrid Binding Mode within the Peptide Subpocket of the CXCR4 Receptor.

The rationale for the structural and mechanistic basis of a tetrahydroisoquinoline (THIQ) based series of CXCR4 antagonists is presented. Using the previously reported crystal structures which reveal two distinct binding sites of CXCR4 defined as the small molecule (IT1t or minor) binding pocket and peptide (CVX15 or major) binding pocket, we hypothesized our THIQ small molecule series could bind like either molecule in these respective receptor configurations (IT1t versus CVX15 based poses). To this end, a thorough investigation was performed through a combination of receptor mutation studies, medicinal chemistry, biological testing, conformational analysis, and flexible docking. Our findings showed that the CVX15 peptide-based CXCR4 receptor complexes (red pose) were consistently favored over the small molecule IT1t based CXCR4 receptor configurations (blue pose) to correctly explain the computational and mutational studies as well as key structural components of activity for these small molecules.

The Bioactive Protein-Ligand Conformation of GluN2C-Selective Positive Allosteric Modulators Bound to the NMDA Receptor.

N-methyl-d-aspartate (NMDA) receptors are ligand-gated, cation-selective channels that mediate a slow component of excitatory synaptic transmission. Subunit-selective positive allosteric modulators of NMDA receptor function have therapeutically relevant effects on multiple processes in the brain. A series of pyrrolidinones, such as PYD-106, that selectively potentiate NMDA receptors that contain the GluN2C subunit have structural determinants of activity that reside between the GluN2C amino terminal domain and the GluN2C agonist binding domain, suggesting a unique site of action. Here we use molecular biology and homology modeling to identify residues that line a candidate binding pocket for GluN2C-selective pyrrolidinones. We also show that occupancy of only one site in diheteromeric receptors is required for potentiation. Both GluN2A and GluN2B can dominate the sensitivity of triheteromeric receptors to eliminate the actions of pyrrolidinones, thus rendering this series uniquely sensitive to subunit stoichiometry. We experimentally identified NMR-derived conformers in solution, which combined with molecular modeling allows the prediction of the bioactive binding pose for this series of GluN2C-selective positive allosteric modulators of NMDA receptors. These data advance our understanding of the site and nature of the ligand-protein interaction for GluN2C-selective positive allosteric modulators for NMDA receptors.

Synthesis and anti-HCV activity of a series of ß-d-2'-deoxy-2'-dibromo nucleosides and their corresponding phosphoramidate prodrugs.

Several ß-d-2'-deoxy-2'-substituted nucleoside analogs have displayed potent and selective anti-HCV activities and some of them have reached human clinical trials. In that regard, we report herein the synthesis of a series of 2'-deoxy,2'-dibromo substituted U, C, G and A nucleosides 10a-d and their corresponding phosphoramidate prodrugs 13a-d. The synthesized nucleosides 10a-d and prodrugs 13a-d were evaluated for their inhibitory activity against HCV as well as cellular toxicity. The results showed that the most potent compound was prodrug 13a, which exhibited micromolar inhibitory activity (EC50 = 1.5 ±â€¯0.8 µM) with no observed toxicity. In addition, molecular modeling and free energy perturbation calculations for the 5'-triphosphate formed from 13a and related 2'-modified nucleotides are discussed.

Jak Inhibitors Modulate Production of Replication-Competent Zika Virus in Human Hofbauer, Trophoblasts, and Neuroblastoma cells.

Zika Virus (ZIKV) is a flavivirus that has been implicated in causing brain deformations, birth defects, and microcephaly in fetuses, and associated with Guillain-Barre syndrome. Mechanisms responsible for transmission of ZIKV across the placenta to the fetus are incompletely understood. Herein, we define key events modulating infection in clinically relevant cells, including primary placental macrophages (human Hofbauer cells; HC), trophoblasts, and neuroblastoma cells. Consistent with previous findings, HC and trophoblasts are permissive to ZIKV infection. Decrease of interferon signaling by Jak ½ inhibition (using ruxolitinib) significantly increased ZIKV replication in HC, trophoblasts, and neuroblasts. Enhanced ZIKV production in ruxolitinib-treated HC was associated with increased expression of HLA-DR and DC-SIGN. Nucleoside analogs blocked ruxolitinib-mediated production of extracellular virus. Although low-level ZIKV infection occurred in untreated HC and trophoblasts, replicating virions were incapable of infecting naive Vero cells. These deficient virions from untreated HC have "thin-coats" suggesting an immature structure. Blocking Jak ½ signaling (with ruxolitinib) restored replication competence as virions produced under these conditions confer cytopathic effects to naive Vero cells. These data demonstrate that Jak-STAT signaling directly impacts the ability of primary placental cells to produce replication-competent virus and is a key determinant in the production of mature virions in clinically relevant cells, including HC and trophoblasts. Design of targeted agents to prevent ZIKV replication in the placenta should consider Jak ½ signaling, the impact of its block on ZIKV infection, and subsequent transmission to the fetus.

Synthesis of sulfamoylbenzamide derivatives as HBV capsid assembly effector.

The synthesis of novel series of sulfamoylbenzamides as HBV capsid assembly effector is reported. The structure was divided into five parts which were independently modified as part of our lead optimization. All synthesized compounds were evaluated for their anti-HBV activity and toxicity in human hepatocytes, lymphocytes and other cells. Additionally, we assessed their effect on HBV cccDNA formation in an HBeAg reporter cell-based assay. Among the 27 compounds reported, several analogs exhibited submicromolar activities and significant reduction of HBeAg secretion. Selected compounds were studied under negative-stain electron microscopy for their ability to disrupt the HBV capsid formation. Structures were modeled into a binding site recently identified in the HBV capsid protein for similar molecules to rationalize the structure-activity relationships for this family of compounds.

Structural analysis of CXCR4 - Antagonist interactions using saturation-transfer double-difference NMR.

CXCR4 is a GPCR involved in leukocyte trafficking. Small molecule antagonists of the receptor may treat inflammatory disease, cancer and HIV. Here we probe the binding of a tetrahydroisoquinoline-based antagonist (TIQ-10) to CXCR4 using saturation transfer double-difference (STDD) NMR. STDD spectra were acquired using extracts from Chinese Hamster Ovary cells expressing membrane-embedded CXCR4. The experiments demonstrate competitive binding between TIQ-10 and established antagonists and provide the TIQ-10 - CXCR4 binding epitope. Molecular modeling of TIQ-10 into the binding pocket provides a pose consistent with STDD-derived interactions. This study paves the way for future investigations of GPCR-ligand interactions in a biological milieu for use in chemical biology, biochemistry, structural biology, and rational drug design.

Pyrazolo-Piperidines Exhibit Dual Inhibition of CCR5/CXCR4 HIV Entry and Reverse Transcriptase.

We report novel anti-HIV-1 agents with combined dual host-pathogen pharmacology. Lead compound 3, composed of a pyrazole-piperidine core, exhibits three concurrent mechanisms of action: (1) non-nucleoside reverse transcriptase inhibition, (2) CCR5-mediated M-tropic viral entry inhibition, and (3) CXCR4-based T-tropic viral entry inhibition that maintains native chemokine ligand binding. This discovery identifies important tool compounds for studying viral infectivity and prototype agents that block HIV-1 entry through dual chemokine receptor ligation.

Predicting Zika virus structural biology: Challenges and opportunities for intervention.

BACKGROUND: Zika virus is an emerging crisis as infection is implicated in severe neurological disorders-Guillain-Barré syndrome and fetal microcephaly. There are currently no treatment options available for Zika virus infection. This virus is part of the flavivirus genus and closely related to Dengue Fever Virus, West Nile Virus, and Japanese Encephalitis Virus. Like other flaviviruses, the Zika virus genome encodes three structural proteins (capsid, precursor membrane, and envelope) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Currently, no structural information exists on these viral proteins to facilitate vaccine design and rational drug discovery. METHODS: Structures for all Zika virus viral proteins were predicted using experimental templates available from closely related viruses using the online SwissModel server. These homology models were compared to drug targets from other viruses using Visual Molecular Dynamics Multiseq software. Sequential alignment of all Zika virus polyproteins was performed using Clustal Omega to identify mutations in specific viral proteins implicated in pathogenesis. RESULTS: The precursor membrane, envelope, and NS1 proteins are unique to Zika virus highlighting possible challenges in vaccine design. Sequential differences between Zika virus strains occur at critical positions on precursor membrane, envelope, NS2A, NS3, NS4B, and NS5 as potential loci for differential pathogenesis. Druggable pockets in Dengue Fever Virus and West Nile Virus NS3 and NS5 are retained in predicted Zika virus structures. CONCLUSIONS: Lead candidates for Zika virus can likely be established using NS3 and NS5 inhibitors from other flaviviruses, and the structures presented can provide opportunities for Zika virus intervention strategies.

Anti-HIV small-molecule binding in the peptide subpocket of the CXCR4:CVX15 crystal structure.

The CXC chemokine receptor 4 (CXCR4) is involved in chemotaxis and serves as a coreceptor for T-tropic HIV-1 viral entry, thus making this receptor an attractive drug target. Recently, crystal structures of CXCR4 were reported as complexes with the small molecule IT1t and the CVX15 peptide. Follow-up efforts to model different antagonists into the small molecule CXCR4:IT1t crystal structure did not generate poses consistent with either the X-ray crystal structure or site-directed mutagenesis (SDM). Here, we compare the binding pockets of the two CXCR4 crystal structures, revealing differences in helices IV, V, VI, and VII, with major differences for the His203 residue buried in the binding pocket. The small molecule antagonist AMD11070 was docked into both CXCR4 crystal structures. An AMD11070 pose identified from the CXCR4:CVX15 model presented interactions with Asp171, Glu288, Trp94, and Asp97, consistent with published SDM data, thus suggesting it is the bioactive pose. A CXCR4 receptor model was optimized around this pose of AMD11070, and the resulting model correlated HIV-1 inhibition with MM-GBSA docking scores for a congeneric AMD11070-like series. Subsequent NAMFIS NMR results successfully linked the proposed binding pose to an independent experimental structure. These results strongly suggest that not all small molecules will bind to CXCR4 in a similar manner as IT1t. Instead, the CXCR4:CVX15 crystal structure may provide a binding locus for small organic molecules that is more suitable than the secondary IT1t site. This work is expected to provide modeling insights useful for future CXCR4 antagonist and X4-tropic HIV-1 based drug design efforts.

Defining the communication between agonist and coactivator binding in the retinoid X receptor α ligand binding domain.

Retinoid X receptors (RXRs) are obligate partners for several other nuclear receptors, and they play a key role in several signaling processes. Despite being a promiscuous heterodimer partner, this nuclear receptor is a target of therapeutic intervention through activation using selective RXR agonists (rexinoids). Agonist binding to RXR initiates a large conformational change in the receptor that allows for coactivator recruitment to its surface and enhanced transcription. Here we reveal the structural and dynamical changes produced when a coactivator peptide binds to the human RXRα ligand binding domain containing two clinically relevant rexinoids, Targretin and 9-cis-UAB30. Our results show that the structural changes are very similar for each rexinoid and similar to those for the pan-agonist 9-cis-retinoic acid. The four structural changes involve key residues on helix 3, helix 4, and helix 11 that move from a solvent-exposed environment to one that interacts extensively with helix 12. Hydrogen-deuterium exchange mass spectrometry reveals that the dynamics of helices 3, 11, and 12 are significantly decreased when the two rexinoids are bound to the receptor. When the pan-agonist 9-cis-retinoic acid is bound to the receptor, only the dynamics of helices 3 and 11 are reduced. The four structural changes are conserved in all x-ray structures of the RXR ligand-binding domain in the presence of agonist and coactivator peptide. They serve as hallmarks for how RXR changes conformation and dynamics in the presence of agonist and coactivator to initiate signaling.

Conformational Analysis of Retinoic Acids: Effects of Steric Interactions on Nonplanar Conjugated Polyenes.

Retinoic acids and other vitamin A analogs contain a trimethylcyclohexenyl ring in conjugation with a polyene chain joined at carbon-6 (C6) and carbon-7 (C7). A MP2-SCS/cc-pVDZ// B3LYP/6-31G(d) 2-D potential energy surface was computed for all-trans retinoic acid, which had 6 minima (3 enantiomeric pairs). The global minima were distorted s-gauche enantiomers (6-7 = 53°) with half-chair conformations of the ring. Distorted s-gauche enantiomers (6-7 = 55°) with inverted half-chair ring conformations were 1.7 kJ/mol above the global minima. The s-trans enantiomers (6-7 = 164°) were 11.3 kJ/mol above the global minima. Steric energies were computed by the method of Guo and Karplus to identify key structural elements in retinoic acids which determines their conformation. Small molecule crystal structures in the CCDC database with trimethylcyclohexenyl ring and exocyclic double bonds have ring-chain geometries near to one of the 6 energy minima of retinoic acids, except for retinaldehyde iminium cations.

A CXCL8 receptor antagonist based on the structure of N-acetyl-proline-glycine-proline.

A role for the collagen-derived tripeptide, N-acetyl proline-glycine-proline (NAc-PGP), in neutrophil recruitment in chronic airway inflammatory diseases, including COPD and cystic fibrosis, has recently been delineated. Due to structural similarity to an important motif for interleukin-8 (CXCL8) binding to its receptor, NAc-PGP binds to CXCR1/2 receptors, leading to neutrophil activation and chemotaxis. In an effort to develop novel CXCL8 antagonists, we describe the synthesis of four chiral isomers of NAc-PGP (NAc-L-Pro-Gly-L-Pro (LL-NAc-PGP), NAc-L-Pro-Gly-D-Pro (LD-NAc-PGP), NAc-D-Pro-Gly-L-Pro (DL-NAc-PGP), and NAc-D-Pro-Gly-D-Pro (DD-NAc-PGP)), characterize them by circular dichroism and NMR spectroscopy, compare their structures to the equivalent region of CXCL8, and test them as potential antagonists of ll-NAc-PGP and CXCL8. We find that LL-NAc-PGP superimposes onto the CXCR1/2 contacting E(29)S(30)G(31)P(32) region of CXCL8 (0.59A rmsd for heavy atoms). In contrast, DD-NAc-PGP has an opposing orientation of key functional groups as compared to the G(31)P(32) region of CXCL8. As a consequence, DD-NAc-PGP binds CXCR1/2, as demonstrated by competition with radiolabeled CXCL8 binding in a radioreceptor assay, yet acts as a receptor antagonist as evidenced by inhibition of CXCL8 and LL-NAc-PGP mediated neutrophil chemotaxis. The ability of DD-NAc-PGP to prevent the activation of CXC receptors indicates that DD-NAc-PGP may serve as a lead compound for the development of CXCR1/2 inhibitors. In addition, this study further proves that using a different technical approach, namely preincubation of NAc-PGP instead of simultaneous addition of NAc-PGP with radiolabeled CXCL8, the direct binding of NAc-PGP to the CXCL8 receptor is evident.

Structure, energetics, and dynamics of binding coactivator peptide to the human retinoid X receptor α ligand binding domain complex with 9-cis-retinoic acid.

Retinoid X receptors (RXRs) are ligand-dependent nuclear receptors, which are activated by the potent agonist 9-cis-retinoic acid (9cRA). 9cRA binds to the ligand binding domain (LBD) of RXRs and recruits coactivator proteins for gene transcription. Using isothermal titration calorimetry, the binding of a 13-mer coactivator peptide, GRIP-1, to the hRXRα-LBD homodimer complex containing 9cRA (hRXRα-LBD:9cRA:GRIP-1) is reported between 20 and 37 °C. ΔG is temperature independent (-8.5 kcal/mol), and GRIP-1 binding is driven by ΔH (-9.2 kcal/mol) at 25 °C. ΔC(p) is large and negative (-401 cal mol(-1) K(-1)). The crystal structure of hRXRα-LBD:9cRA:GRIP-1 is reported at 2.05 Å. When the structures of hRXRα-LBD:9cRA:GRIP-1 and hRXRα-LBD:9cRA ( 1FBY ) homodimers are compared, E453 and E456 on helix 12 bury and form ionic interactions with GRIP-1. R302 on helix 4 realigns to form new salt bridges to both E453 and E456. F277 (helix 3), F437 (helix 11), and F450 (helix 12) move toward the hydrophobic interior. The changes in the near-UV spectrum at 260 nm of the hRXRα-LBD:9cRA:GRIP-1 support this structural change. Helix 11 tilts toward helix 12 by ≈1 Å, modifying the ring conformation of 9cRA. Hydrogen-deuterium exchange mass spectroscopy indicates GRIP-1 binding to hRXRα-LBD:9cRA significantly decreases the exchange rates for peptides containing helices 3 (F277), 4 (R302), 11 (F437), and 12 (E453, E456). The structural changes and loss of dynamics of the GRIP-1-bound structure are used to interpret the energetics of coactivator peptide binding to the agonist-bound hRXRα-LBD.