Peptidomimetics as potent dual SARS-CoV-2 cathepsin-L and main protease inhibitors: In silico design, synthesis and pharmacological characterization.

In this paper we present the design, synthesis, and biological evaluation of a new series of peptidomimetics acting as potent anti-SARS-CoV-2 agents. Starting from our previously described Main Protease (MPro) and Papain Like Protease (PLPro) dual inhibitor, CV11, here we disclose its high inhibitory activity against cathepsin L (CTSL) (IC50 = 19.80 ± 4.44 nM), an emerging target in SARS-CoV-2 infection machinery. An in silico design, inspired by the structure of CV11, led to the development of a library of peptidomimetics showing interesting activities against CTSL and Mpro, allowing us to trace the chemical requirements for the binding to both enzymes. The screening in Vero cells infected with 5 different SARS-CoV-2 variants of concerns, highlighted sub-micromolar activities for most of the synthesized compounds (13, 15, 16, 17 and 31) in agreement with the enzymatic inhibition assays results. The compounds showed lack of activity against several different RNA viruses except for the 229E and OC43 human coronavirus strains, also characterized by a cathepsin-L dependent release into the host cells. The most promising derivatives were also evaluated for their chemical and metabolic in-vitro stability, with derivatives 15 and 17 showing a suitable profile for further preclinical characterization.

Exploiting the Features of Short Peptides to Recognize Specific Cell Surface Markers.

Antibodies are the macromolecules of choice to ensure specific recognition of biomarkers in biological assays. However, they present a range of shortfalls including a relatively high production cost and limited tissue penetration. Peptides are relatively small molecules able to reproduce sequences of highly specific paratopes and, although they have less biospecificity than antibodies, they offer advantages like ease of synthesis, modifications of their amino acid sequences and tagging with fluorophores and other molecules required for detection. This work presents a strategy to design peptide sequences able to recognize the CD44 hyaluronic acid receptor present in the plasmalemma of a range of cells including human bone marrow stromal mesenchymal cells. The protocol of identification of the optimal amino acid sequence was based on the combination of rational design and in silico methodologies. This protocol led to the identification of two peptide sequences which were synthesized and tested on human bone marrow mesenchymal stromal cells (hBM-MSCs) for their ability to ensure specific binding to the CD44 receptor. Of the two peptides, one binds CD44 with sensitivity and selectivity, thus proving its potential to be used as a suitable alternative to this antibody in conventional immunostaining. In the context of regenerative medicine, the availability of this peptide could be harnessed to functionalize tissue engineering scaffolds to anchor stem cells as well as to be integrated into systems such as cell sorters to efficiently isolate MSCs from biological samples including various cell subpopulations. The data here reported can represent a model for developing peptide sequences able to recognize hBM-MSCs and other types of cells and for their integration in a range of biomedical applications.

Anti-Angiogenic Effects of Natural Compounds in Diet-Associated Hepatic Inflammation.

Alcoholic liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD) are the most common causes of chronic liver disease and are increasingly emerging as a global health problem. Such disorders can lead to liver damage, resulting in the release of pro-inflammatory cytokines and the activation of infiltrating immune cells. These are some of the common features of ALD progression in ASH (alcoholic steatohepatitis) and NAFLD to NASH (non-alcoholic steatohepatitis). Hepatic steatosis, followed by fibrosis, lead to a continuous progression accompanied by angiogenesis. This process creates hypoxia, which activates vascular factors, initiating pathological angiogenesis and further fibrosis. This forms a vicious cycle of ongoing damage and progression. This condition further exacerbates liver injury and may contribute to the development of comorbidities, such as metabolic syndrome as well as hepatocellular carcinoma. Increasing evidence suggests that anti-angiogenic therapy may have beneficial effects on these hepatic disorders and their exacerbation. Therefore, there is a great interest to deepen the knowledge of the molecular mechanisms of natural anti-angiogenic products that could both prevent and control liver diseases. In this review, we focus on the role of major natural anti-angiogenic compounds against steatohepatitis and determine their potential therapeutic benefits in the treatment of liver inflammation caused by an imbalanced diet.

Metabolomics-assisted discovery of a new anticancer GLS-1 inhibitor chemotype from a nortopsentin-inspired library: From phenotype screening to target identification.

The enzyme glutaminase-1 (GLS-1) has shown a clear and coherent implication in the progression and exacerbation of different aggressive tumors such as glioblastoma, hepatocarcinoma, pancreas, bone, and triple-negative breast cancer. Few chemotypes are currently available as selective GLS-1 inhibitors, and still, fewer of them are at the clinical stage. In the present paper, starting from a naturally-inspired antitumor compound library, metabolomics has been used to putatively identify the molecular mechanism underlying biological activity. GLS-1 was identified as a potential target. Biochemical analysis confirmed the hypothesis leading to the identification of a new hit compound acting as a GLS-1 selective inhibitor (IC50 = 3.96 ± 1.05 µM), compared to the GLS-2 isoform (IC50 = 12.90 ± 0.87 µM), with remarkable antitumor potency over different aggressive tumor cell lines. Molecular modelling studies revealed new insight into the drug-target interaction providing robust SAR clues for the rational hit-to-lead development. The approach undertaken underlines the wide potential of metabolomics applied to drug discovery, particularly in target identification and hit discovery following phenotype screening.

In Silico Identification and In Vitro Evaluation of New ABCG2 Transporter Inhibitors as Potential Anticancer Agents.

Different molecular mechanisms contribute to the development of multidrug resistance in cancer, including increased drug efflux, enhanced cellular repair mechanisms and alterations of drug metabolism or drug targets. ABCG2 is a member of the ATP-binding cassette superfamily transporters that promotes drug efflux, inducing chemotherapeutic resistance in malignant cells. In this context, the development of selective ABCG2 inhibitors might be a suitable strategy to improve chemotherapy efficacy. Thus, through a multidisciplinary approach, we identified a new ABCG2 selective inhibitor (8), highlighting its ability to increase mitoxantrone cytotoxicity in both hepatocellular carcinoma (EC50from 8.67 ± 2.65 to 1.25 ± 0.80 µM) and transfected breast cancer cell lines (EC50from 9.92 ± 2.32 to 2.45 ± 1.40 µM). Moreover, mitoxantrone co-administration in both transfected and non-transfected HEK293 revealed that compound 8 notably lowered the mitoxantrone EC50, demonstrating its efficacy along with the importance of the ABCG2 extrusion pump overexpression in MDR reversion. These results were corroborated by evaluating the effect of inhibitor 8 on mitoxantrone cell uptake in multicellular tumor spheroids and via proteomic experiments.

Identification of a dual acting SARS-CoV-2 proteases inhibitor through in silico design and step-by-step biological characterization.

COVID-19 pandemic, starting from the latest 2019, and caused by SARS-CoV-2 pathogen, led to the hardest health-socio-economic disaster in the last century. Despite the tremendous scientific efforts, mainly focused on the development of vaccines, identification of potent and efficient anti-SARS-CoV-2 therapeutics still represents an unmet need. Remdesivir, an anti-Ebola drug selected from a repurposing campaign, is the only drug approved, so far, for the treatment of the infection. Nevertheless, WHO in later 2020 has recommended against its use in COVID-19. In the present paper, we describe a step-by-step in silico design of a small library of compounds as main protease (Mpro) inhibitors. All the molecules were screened by an enzymatic assay on Mpro and, then, cellular activity was evaluated using Vero cells viral infection model. The cellular screening disclosed compounds 29 and 34 as in-vitro SARS-CoV-2 replication inhibitors at non-toxic concentrations (0.32 < EC50 < 5.98 µM). To rationalize these results, additional in-vitro assays were performed, focusing on papain like protease (PLpro) and spike protein (SP) as potential targets for the synthesized molecules. This pharmacological workflow allowed the identification of compound 29, as a dual acting SARS-CoV-2 proteases inhibitor featuring micromolar inhibitory potency versus Mpro (IC50 = 1.72 µM) and submicromolar potency versus PLpro (IC50 = 0.67 µM), and of compound 34 as a selective SP inhibitor (IC50 = 3.26 µM).

NMR-based metabolomic profile of hypercholesterolemic human sera: Relationship with in vitro gene expression?

Hypercholesterolaemia is considered an important cause of atherosclerotic cardiovascular disease. In a previous investigation, we demonstrated that cultured hepatoma cells treated with hypercholesterolaemic sera compared with cells treated with normocholesterolaemic sera show overexpression of mRNAs related to mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS2). In the present work, using an NMR metabolomic analysis, we demonstrate that the hypercholesterolaemic blood sera previously used to treat cultured hepatoma cells are characterized by a metabolomic profile that is significantly different from the normocholesterolaemic sera. Acetate, acetone, 2-hydroxybutyrate, cysteine, valine, and glutamine are the metabolites distinguishing the two groups. Abnormalities in the concentrations of these metabolites reflect alterations in energy-related pathways, such as pantothenate and CoA biosynthesis, pyruvate, glycolysis/gluconeogenesis, the citrate cycle, and ketone bodies. Regarding ketone bodies, the pathway is regulated by HMGCS2; therefore, serum samples previously found to be able to increase HMGCS2 mRNA levels in cultured cells also contain higher amounts of the metabolites of its encoded enzyme protein product.