A promising drug lead that inhibits HCV infectivity in a genotype-independent manner

E2 glycoprotein plays a significant role in the HCV life cycle, but only crystal structures of short peptides, or epitopes were present until Kong et al. resolved the new HCV E2 core (E2c) crystal structure. We have created a new HCV E2 homology model based on the new E2c crystal structure, selected 3 potential binding sites located near residues critical for HCV entry, and used computer docking to identify a set of ligands that should bind to the sites. We tested the set for E2 binding using surface plasmon resonance and performed inhibition assays of HCV infection. One of these compounds, E2216, inhibited HCV infectivity. The homology model was created using AS2TS and Smith-Waterman, FASTA, BLAST and PSI-BLAST sequence alignments. Three potential ligand-binding sites were selected and 3 corresponding grid parameter files were created using Autodock 1.5.6 to guide the virtual screening of ~4,000 ligands (NCI_DSII library). Recombinant HCV E2 protein was used to identify 40 virtual screening hits by using Biacore t100. Pseudotyped retroviral particles harboring HCV envelope proteins (HCVpp) of genotype 2a and cell culture-produced HCV particles (HCVcc) based on the JFH1 strain were used in testing the ligands for HCV infectivity inhibition. E2216 was observed to selectively block the HCV infectivity of both HCVcc and HCVpp with an IC50 of 6.2uM and 3.3uM respectively in a genotype-independent manner. It was also found to block the cell to cell transmission. E2216 appears to be a promising drug lead that targets HCV E2 and can be further optimized to help in blocking HCV entry into hepatocytes and prevent the progression of the infection

Identification of drug leads against HCV and malaria using different target proteins

Hepatitis C Virus (HCV) infects 170 million individuals worldwide. Although several newly FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating all HCV genotypes and subtypes to be used in an interferon-free regimen. On the other hand, malaria is another public health burden that causes 219 million clinical episodes, and 660,000 deaths per year. In addition, 3.3 billion people live in areas at risk of malaria transmission in 106 countries. It is alarming that 86% of deaths caused by malaria globally were in children. Several challenges are faced when treating malaria, such as resistance against drugs that are used in treatment. This necessitates the development of new classes of drugs to overcome resistance. CD81 is a target protein that plays an essential role in the internalization of HCV into hepatocytes. Thus it was also targeted to identify sets of small molecule ligands predicted to bind to several sites that were identified to be involved in HCV infection. Thirty-six ligands predicted by AutoDock to bind to these sites were tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 23 out of 36 of the ligands bound in vitro to the recombinant CD81-LEL protein. In an effort to create new drugs that block hepatitis C virus entry into hepatocytes, we have designed and synthesized a small molecule that targets the HCV E2 glycoprotein binding site on CD81. A selective high affinity ligand (SHAL) (11) was created by linking together two small molecules that were predicted by docking and were shown by experimental methods to bind to the same site on CD81 where E2 binds. SH7153 was found to bind to recombinant CD81-LEL with a Kd of 21 µM but wasn’t found to inhibit HCV infection when tested using Raji cells (antibody neutralizing assays) and HCV infection inhibition assays. This led to the conclusion that the linkers’ lengths should be optimized so as to have a SHAL that fits properly in the desired binding sites. The HCV glycoprotein E2 has also been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. Recently, 2 research groups were able to resolve the core structure of HCV E2 which will largely help providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. By targeting conserved E2 residues among different genotypes and subtypes in the CD81 binding site on HCV E2, one might also be able to develop drugs that block HCV infection in a genotype-independent manner. Using the E2c structure as a template, we have used homology modeling methods to develop a structural model of the E2 protein core (residues 421-645) that includes the three amino acid segments that are not present in the E2c structure. Blind docking to this model was then performed using a library of ~4000 small molecules and a set of 40 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction were selected for experimental testing. Surface Plasmon resonance was used to screen the ligands for binding to recombinant E2 protein and the best binders were subsequently tested to identify compounds that inhibit the infection of hepatocytes by HCV. One compound, 281816, inhibited infection by HCV genotypes 1a, 1b, 2a, 2b, 4a and 6a with IC50’s ranging from 2.2 uM to 4.6 uM. Such inhibitors may represent a new paradigm for HCV treatment. In an attempt to make 281816 more promising, a SHAL prototype was designed using an analogue of 281816 (SH2216). It would be tempting to test the SHAL inhibitory effect and compare it to the 281816’s inhibitory effect. To date, human CD81 (hCD81) is the only human surface protein known to play a role in the process by which sporozoites of several Plasmodium species infect human hepatocytes. Blocking a human receptor that is exploited for the entry process of pathogens has been proven to be a good strategy for fighting drug-resistant mutants. Hence, we targeted the 21 amino acid stretch on CD81 large extracellular loop that was found to be involved in Plasmosium yoleii invasion via virtual screening runs, preliminary binding assays and sporozoite invasion assays. This led to the identification of 4 drug leads that range between moderate and strong inhibitors of infection by Plasmodium yoleii and Plamodium falciparum. Additionally one ligand was found to potentiate the invasion of Plasmodium yoleii

Identification of ligands that target the HCV-E2 binding site on CD81

Hepatitis C is a global health problem. While many drug companies have active R&D efforts to develop new drugs for treating Hepatitis C virus (HCV), most target the viral enzymes. The HCV glycoprotein E2 has been shown to play an essential role in hepatocyte invasion by binding to CD81 and other cell surface receptors. This paper describes the use of AutoDock to identify ligand binding sites on the large extracellular loop of the open conformation of CD81 and to perform virtual screening runs to identify sets of small molecule ligands predicted to bind to two of these sites. The best sites selected by AutoLigand were located in regions identified by mutational studies to be the site of E2 binding. Thirty-six ligands predicted by AutoDock to bind to these sites were subsequently tested experimentally to determine if they bound to CD81-LEL. Binding assays conducted using surface Plasmon resonance revealed that 26 out of 36 (72 %) of the ligands bound in vitro to the recombinant CD81-LEL protein. Competition experiments performed using dual polarization interferometry showed that one of the ligands predicted to bind to the large cleft between the C and D helices was also effective in blocking E2 binding to CD81-LEL

A Randomized Controlled Trial of Sertraline in Young Children With Autism Spectrum Disorder.

Objective: Selective serotonin reuptake inhibitors like sertraline have been shown in observational studies and anecdotal reports to improve language development in young children with fragile X syndrome (FXS). A previous controlled trial of sertraline in young children with FXS found significant improvement in expressive language development as measured by the Mullen Scales of Early Learning (MSEL) among those with comorbid autism spectrum disorder (ASD) in post hoc analysis, prompting the authors to probe whether sertraline is also indicated in nonsyndromic ASD. Methods: The authors evaluated the efficacy of 6 months of treatment with low-dose sertraline in a randomized, double-blind, placebo-controlled trial in 58 children with ASD aged 24 to 72 months. Results: 179 subjects were screened for eligibility, and 58 were randomized to sertraline (32) or placebo (26). Eight subjects from the sertraline arm and five from the placebo arm discontinued. Intent-to-treat analysis showed no significant difference from placebo on the primary outcomes (MSEL expressive language raw score and age equivalent combined score) or secondary outcomes. Sertraline was well tolerated, with no difference in side effects between sertraline and placebo groups. No serious adverse events possibly related to study treatment occurred. Conclusion: This randomized controlled trial of sertraline treatment showed no benefit with respect to primary or secondary outcome measures. For the 6-month period, treatment in young children with ASD appears safe, although the long-term side effects of low-dose sertraline in early childhood are unknown. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT02385799

Identification of a Novel Drug Lead That Inhibits HCV Infection and Cell-to-Cell Transmission by Targeting the HCV E2 Glycoprotein

Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421–645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50’s ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment

Assessment of Molecular Measures in Non-FXTAS Male Premutation Carriers

Approximately 30–40% of male and 8–16% of female carriers of the Fragile X premutation will develop a neurodegenerative movement disorder characterized by intentional tremor, gait ataxia, autonomic dysfunction, cognitive decline, and Parkinsonism during their lifetime. At the molecular level, premutation carriers have increased expression levels of the FMR1 and the antisense FMR1 (ASFMR1) mRNAs. Both genes undergo alternative splicing giving rise to a number of different transcripts. Alteration in the alternative splicing process might be associated with FXTAS. In this study, we have investigated the correlation between objective measures of movement (balance and tremor using the CATSYS battery) and the expression of both the FMR1 and the ASFMR1 genes. In addition, we investigated whether their expression level and that of the ASFMR1 131 bp splice isoform could distinguish between premutation carriers with FXTAS and non-FXTAS premutation carriers. Confirming previous findings, the expression levels of transcripts at the FMR1 locus positively correlated with the CGG repeat number and significantly differentiated the premutation carriers from the control groups. Furthermore, premutation carriers with and without FXTAS, showed a significant difference in the expression level of the ASFMR1 131 bp splice isoform when compared to age and gender matched controls. However, there was no significant difference in the ASFMR1 131 bp splice isoform expression level when comparing premutation carriers with and without FXTAS. Finally, our results indicate significant group differences in CATSYS dominant hand reaction time and postural sway with eyes closed in premutation carriers without FXTAS compared to controls. In addition, a significant inverse association between the tremor intensity and the expression level of ASFMR1 131 bp splice isoform in premutation carriers compared to controls, was observed, suggesting a potential role in the pathogenesis of FXTAS

Assessment of Molecular Measures in Non-FXTAS Male Premutation Carriers.

Approximately 30-40% of male and 8-16% of female carriers of the Fragile X premutation will develop a neurodegenerative movement disorder characterized by intentional tremor, gait ataxia, autonomic dysfunction, cognitive decline, and Parkinsonism during their lifetime. At the molecular level, premutation carriers have increased expression levels of the FMR1 and the antisense FMR1 (ASFMR1) mRNAs. Both genes undergo alternative splicing giving rise to a number of different transcripts. Alteration in the alternative splicing process might be associated with FXTAS. In this study, we have investigated the correlation between objective measures of movement (balance and tremor using the CATSYS battery) and the expression of both the FMR1 and the ASFMR1 genes. In addition, we investigated whether their expression level and that of the ASFMR1 131 bp splice isoform could distinguish between premutation carriers with FXTAS and non-FXTAS premutation carriers. Confirming previous findings, the expression levels of transcripts at the FMR1 locus positively correlated with the CGG repeat number and significantly differentiated the premutation carriers from the control groups. Furthermore, premutation carriers with and without FXTAS, showed a significant difference in the expression level of the ASFMR1 131 bp splice isoform when compared to age and gender matched controls. However, there was no significant difference in the ASFMR1 131 bp splice isoform expression level when comparing premutation carriers with and without FXTAS. Finally, our results indicate significant group differences in CATSYS dominant hand reaction time and postural sway with eyes closed in premutation carriers without FXTAS compared to controls. In addition, a significant inverse association between the tremor intensity and the expression level of ASFMR1 131 bp splice isoform in premutation carriers compared to controls, was observed, suggesting a potential role in the pathogenesis of FXTAS