SARS-CoV-2 is a type of coronavirus responsible for the international outbreak of respiratory illness termed COVID-19 that forced the World Health Organization to declare a pandemic infectious disease situation of international concern at the beginning of 2020. The need for a swift response against COVID-19 prompted to consider different sources to identify bioactive compounds that can be used as therapeutic agents, including available drugs and natural products. Accordingly, this work reports the results of a virtual screening process aimed at identifying antiviral natural product inhibitors of the SARS-CoV-2 Mpro viral protease. For this purpose, ca. 2000 compounds of the Selleck database of Natural Compounds were the subject of an ensemble docking process targeting the Mpro protease. Molecules that showed binding to most of the protein conformations were retained for a further step that involved the computation of the binding free energy of the ligand-Mpro complex along a molecular dynamics trajectory. The compounds that showed a smooth binding free energy behavior were selected for in vitro testing. From the resulting set of compounds, five compounds exhibited an antiviral profile, and they are disclosed in the present work.
Previous studies support the hypothesis that the envelope GB virus C (GBV-C) E1 protein interferes the HIV-1 entry and that a peptide, derived from the region 139-156 of this protein, has been defined as a novel HIV-1 entry inhibitor. In this work, we firstly focus on the characterization of the structural features of this peptide, which are determinant for its anti-HIV-1 activity and secondly, on the study of its interaction with the proposed viral target (i.e., the HIV-1 fusion peptide). We report the structure of the peptide determined by NMR spectroscopy in dodecylphosphocholine (DPC) micelles solved by using restrained molecular dynamics calculations. The acquisition of different NMR experiments in DPC micelles (i.e., peptide-peptide titration, diffusion NMR spectroscopy, and addition of paramagnetic relaxation agents) allows a proposal of an inhibition mechanism. We conclude that a 18-mer peptide from the non-pathogenic E1 GBV-C protein, with a helix-turn-helix structure inhibits HIV-1 by binding to the HIV-1 fusion peptide at the membrane level, thereby interfering with those domains in the HIV-1, which are critical for stabilizing the six-helix bundle formation in a membranous environment.
In the present study, we report the discovery of a novel allosteric inhibitory site for p38α, a subclass of the mitogen-activated protein kinases (MAPK) family. The putative site was discovered after inspection of the crystallographic structure of the p38α-MK2 complex. MK2 (MAPK-activated protein kinase 2) is an interesting protein playing a dual role as modulator and substrate of p38α. This intriguing behavior is due to the ability of the two proteins to form distinctive heterodimers when p38α is phosphorylated or not. We hypothesized that the regulatory action of MK2 is due to its capability to keep p38α in an inactive conformation and consequently, we investigated the atomic structure of the p38α-MK2 complex to understand such regulatory behavior at the molecular level. After inspection of the complex structure, two peptides designed from the MK2 regulatory loop in contact with p38α with sequences Tyr1-Ser2-Asn3-His4-Gly5-Leu6 (peptide-1) and [Phe0]-peptide-1 (peptide-2) in their zwitterionic form were investigated for their phosphorylation inhibitory capability in vitro. Since both peptides exhibited inhibitory capability of the p38α kinase mediated phosphorylation of MEF2A, in a subsequent step we pursued the discovery of small molecule peptidomimetics. For this purpose we characterized in detail the peptide-p38α interaction using molecular dynamics simulations, leading to the definition of a pharmacophore for the peptide-protein interaction. This hypothesis was used as query for a in silico screening, leading to the discovery of a fused ring compound with micromolar inhibitory activity. Site-directed mutagenesis studies support that the compound binds to the putative novel allosteric site in p38α.
Astrocytes actively participate in neuro-inflammatory processes associated to Alzheimer's disease (AD), and other brain pathologies. We recently showed that an astrocyte-specific intracellular signaling pathway involving an interaction of the phosphatase calcineurin with the transcription factor FOXO3 is a major driver in AD-associated pathological inflammation, suggesting a potential new druggable target for this devastating disease. We have now developed decoy molecules to interfere with calcineurin/FOXO3 interactions, and tested them in astrocytes and neuronal co-cultures exposed to amyloid-β (Aβ) toxicity. We observed that interference of calcineurin/FOXO3 interactions exerts a protective action against Aβ-induced neuronal death and favors the production of a set of growth factors that we hypothesize form part of a cytoprotective pathway to resolve inflammation. Furthermore, interference of the Aβ-induced interaction of calcineurin with FOXO3 by decoy compounds significantly decreased amyloid-β protein precursor (AβPP) synthesis, reduced the AβPP amyloidogenic pathway, resulting in lower Aβ levels, and blocked the expression of pro-inflammatory cytokines TNFα and IL-6 in astrocytes. Collectively, these data indicate that interrupting pro-inflammatory calcineurin/FOXO3 interactions in astrocytes triggered by Aβ accumulation in brain may constitute an effective new therapeutic approach in AD. Future studies with intranasal delivery, or brain barrier permeable decoy compounds, are warranted.
Abstract:Bradykinin (BK) is a member of the kinin family, released in response to inflammation, trauma, burns, shock, allergy and some cardiovascular diseases, provoking vasodilatation and increased vascular permeability among other effects. Their actions are mediated through at least two G-protein coupled receptors, B1 a receptor upregulated during inflammation episodes or tissue trauma and B2 that is constitutively expressed in a variety of cell types. The goal of the present work is to carry out a structure-activity study of BK B2 antagonism, taking into account the stereochemical features of diverse non-peptide antagonists and the way these features translate into ligand anchoring points to complementary regions of the receptor, through the analysis of the respective ligand-receptor complex. For this purpose an atomistic model of the BK B2 receptor was built by homology modeling and subsequently refined embedded in a lipid bilayer by means of a 600 ns molecular dynamics trajectory. The average structure from the last hundred nanoseconds of the molecular dynamics trajectory was energy minimized and used as model of the receptor for docking studies. For this purpose, a set of compounds with antagonistic profile, covering maximal diversity were selected from the literature. Specifically, the set of compounds include Fasitibant, FR173657, Anatibant, WIN64338, Bradyzide, CHEMBL442294, and JSM10292. Molecules were docked into the BK B2 receptor model and the corresponding complexes analyzed to understand ligand-receptor interactions. The outcome of this study is summarized in a 3D pharmacophore that explains the observed structureactivity results and provides insight into the design of novel molecules with antagonistic profile. To prove the validity of the pharmacophore hypothesized a virtual screening process was also carried out. The pharmacophore was used as query to identify new hits using diverse databases of molecules. The results of this study revealed a set of new hits with structures not connected to the molecules used for pharmacophore development. A few of these structures were purchased and tested. The results of the binding studies show about a 33% success rate with a correlation between the number of pharmacophore points fulfilled and their antagonistic potency. Some of these Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporationstructures are disclosed in the present work.Response to Reviewers: We sincerely appreciate the involvement of reviewer #1 and are deeply obliged. The reviewer was right in pointing the distortion of the ring and the amide in Figure 8 that has now been fixed in the revised version of Figure 8. We have also modified the viewpoint of Figure 9 to clearly show the protonation status of the terminal amine that was unfortunately hidden in the previous version of Figure 9 this study is summarized in a 3D pharmacophore that explains the observed structure-activity results and provides insight into the design of novel molecules with antagonistic profile. ...
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