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

Novel Transthyretin Amyloid Fibril Formation Inhibitors: Synthesis, Biological Evaluation, and X-Ray Structural Analysis

Transthyretin (TTR) is one of thirty non-homologous proteins whose misfolding, dissociation, aggregation, and deposition is linked to human amyloid diseases. Previous studies have identified that TTR amyloidogenesis can be inhibited through stabilization of the native tetramer state by small molecule binding to the thyroid hormone sites of TTR. We have evaluated a new series of β-aminoxypropionic acids (compounds 5–21), with a single aromatic moiety (aryl or fluorenyl) linked through a flexible oxime tether to a carboxylic acid. These compounds are structurally distinct from the native ligand thyroxine and typical halogenated biaryl NSAID-like inhibitors to avoid off-target hormonal or anti-inflammatory activity. Based on an in vitro fibril formation assay, five of these compounds showed significant inhibition of TTR amyloidogenesis, with two fluorenyl compounds displaying inhibitor efficacy comparable to the well-known TTR inhibitor diflunisal. Fluorenyl 15 is the most potent compound in this series and importantly does not show off-target anti-inflammatory activity. Crystal structures of the TTR∶inhibitor complexes, in agreement with molecular docking studies, revealed that the aromatic moiety, linked to the sp(2)-hybridized oxime carbon, specifically directed the ligand in either a forward or reverse binding mode. Compared to the aryl family members, the bulkier fluorenyl analogs achieved more extensive interactions with the binding pockets of TTR and demonstrated better inhibitory activity in the fibril formation assay. Preliminary optimization efforts are described that focused on replacement of the C-terminal acid in both the aryl and fluorenyl series (compounds 22–32). The compounds presented here constitute a new class of TTR inhibitors that may hold promise in treating amyloid diseases associated with TTR misfolding

A common feature pharmacophore for FDA-approved drugs inhibiting the Ebola virus [v1; ref status: indexed, http://f1000r.es/4qh]

We are currently faced with a global infectious disease crisis which has been anticipated for decades. While many promising biotherapeutics are being tested, the search for a small molecule has yet to deliver an approved drug or therapeutic for the Ebola or similar filoviruses that cause haemorrhagic fever. Two recent high throughput screens published in 2013 did however identify several hits that progressed to animal studies that are FDA approved drugs used for other indications. The current computational analysis uses these molecules from two different structural classes to construct a common features pharmacophore. This ligand-based pharmacophore implicates a possible common target or mechanism that could be further explored. A recent structure based design project yielded nine co-crystal structures of pyrrolidinone inhibitors bound to the viral protein 35 (VP35). When receptor-ligand pharmacophores based on the analogs of these molecules and the protein structures were constructed, the molecular features partially overlapped with the common features of solely ligand-based pharmacophore models based on FDA approved drugs. These previously identified FDA approved drugs with activity against Ebola were therefore docked into this protein. The antimalarials chloroquine and amodiaquine docked favorably in VP35. We propose that these drugs identified to date as inhibitors of the Ebola virus may be targeting VP35. These computational models may provide preliminary insights into the molecular features that are responsible for their activity against Ebola virus in vitro and in vivo and we propose that this hypothesis could be readily tested

A common feature pharmacophore for FDA-approved drugs inhibiting the Ebola virus [v2; ref status: indexed, http://f1000r.es/4wt]

We are currently faced with a global infectious disease crisis which has been anticipated for decades. While many promising biotherapeutics are being tested, the search for a small molecule has yet to deliver an approved drug or therapeutic for the Ebola or similar filoviruses that cause haemorrhagic fever. Two recent high throughput screens published in 2013 did however identify several hits that progressed to animal studies that are FDA approved drugs used for other indications. The current computational analysis uses these molecules from two different structural classes to construct a common features pharmacophore. This ligand-based pharmacophore implicates a possible common target or mechanism that could be further explored. A recent structure based design project yielded nine co-crystal structures of pyrrolidinone inhibitors bound to the viral protein 35 (VP35). When receptor-ligand pharmacophores based on the analogs of these molecules and the protein structures were constructed, the molecular features partially overlapped with the common features of solely ligand-based pharmacophore models based on FDA approved drugs. These previously identified FDA approved drugs with activity against Ebola were therefore docked into this protein. The antimalarials chloroquine and amodiaquine docked favorably in VP35. We propose that these drugs identified to date as inhibitors of the Ebola virus may be targeting VP35. These computational models may provide preliminary insights into the molecular features that are responsible for their activity against Ebola virus in vitro and in vivo and we propose that this hypothesis could be readily tested

Liquid chromatography-mass spectrometry-based protocol to measure drug accumulation in Mycobacterium tuberculosis and its host cell

Summary: The extent to which a drug accumulates in Mycobacterium tuberculosis (Mtb) and its host cell can affect treatment efficacy. We describe protocols measuring drug accumulation in Mtb, macrophages, and Mtb-infected macrophages. The method leverages drug extraction from the cellular lysate and drug-level quantification by liquid chromatography-mass spectrometry. The general methodology has broad applicability and can quantify drug accumulation in other cell types, while being extended to quantification of drug metabolites formed within the cell under study.For complete details on the use and execution of this protocol, please refer to Lavin et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Kinase TB dual event dataset

<p>dataset used in PMID:24144044 and elsewhere</p