Identification of novel molecular scaffolds for the design of MMP-13 inhibitors through Virtual Screening Methods

Osteoarthritis (OA) is the leading cause of joint pain and disability in middle-aged and elderly patients. It is characterized by progressive loss of articular cartilage that eventually leads to denudation of the joint surface. The cartilage loss observed in OA is the result of a complex process involving degradation of various components of the cartilage matrix. Particularly, degradation of cartilage-specific type II collagen by mammalian collagenases (MMPs) is a key step in the loss of structural and functional integrity of cartilage.1 Among all known MMPs, MMP-13 is considered the principal target in OA. Indeed, today there are overwhelming data on the role of MMP-13 in the pathogenesis of OA,2 and inhibition of its activity has proven to be efficacious in a variety of models of experimentally induced as well as spontaneously occurring OA.3 Unfortunately, none of the known MMP inhibitors (MMPIs) have been successfully utilized as therapeutic agents so far. This was due to the lack of selectivity for a specific isozyme, leading to heavy dose- and durationdependent musculoskeletal side effects.4 Therefore, current drug development strategies for treatment of OA are focused on selective inhibition of MMP-13, although recent evidences suggest that other MMPs, such as MMP-1, may also contribute to the collagen degradation process.5 However, the design of a selective MMPI is not a trivial task, as Pag. 7 MMPs share an high similarity in the overall three-dimensional fold and many conserved amino acids exist in the inhibitor binding site, besides the conserved catalytic zinc ion. The major structural difference observed between the MMP enzymes resides in the relative size and shape of the S1’ subsite, which is located in proximity of the catalytic metal. From a structural point of view, almost all MMPIs known so far are based on a zinc-binding group (ZBG) and a hydrophobic portion protruding into the hydrophobic S1’ subsite. These compounds behave as competitive inhibitors since the ZBG can mimic one of the transition states occurring during the substrate hydrolysis. Currently, two successful strategies to confer selectivity of action to an MMP inhibitor are known: the first resides in the proper modification of the P1’ substituent on MMPI to take advantage of the differences between the diverse MMPs; the second is the finding of an allosteric inhibitor,6 which binds tightly to the S1’ and S1’* subsite without chelating the metal that is thought to contribute to the promiscuous inhibition of multiple MMPs.Errore. Il segnalibro non è definito.c Recently, as a result of the first strategy, it has been designed a Nisopropoxy- arylsulfonamide-based hydroxamate inhibitor, which showed low nanomolar activity for MMP-13 and high selectivity over some other tested MMPs.7 In parallel to further studies aiming to assess the activity of this promising compound using in vivo models of OA, it has been decided to seek for novel scaffolds as allosteric inhibitors on one hand, and as zincchelating non-hydroxamate inhibitors on the other. In fact, a debate is still open on the advisability of using hydroxamates as ZBG due to toxicity and metabolic stability issues.8,9 In this respect, we have taken advantage of the availability of several MMP-13 crystal structures and have used two different in silico methods to screen the Life Chemicals and the Maybridge databases, respectively. Experimental tests of a limited selection of candidate compounds (60) verified nine novel leads, structurally unrelated to the known MMPIs

Selective Arylsulfonamide Inhibitors of ADAM-17: Hit Optimization and Activity in Ovarian Cancer Cell Models

Activated Leukocyte Cell Adhesion Mol. (ALCAM) is expressed at the surface of epithelial ovarian cancer (EOC) cells and is released in a sol. form (sALCAM) by ADAM-17-mediated shedding. This process is relevant to EOC cell motility and invasiveness, which is reduced by inhibitors of ADAM-17. In addn., ADAM-17 plays a key role in EGFR signalling and thus may represent a useful target in anticancer therapy. Herein we report our hit optimization effort to identify potent and selective ADAM-17 inhibitors, starting with previous mol. 1. A new series of secondary sulfonamido-based hydroxamates was designed and synthesized. The biol. activity of the newly synthesized compds. was tested in vitro on isolated enzymes and human EOC cell lines. The optimization process led to compd. 21, which showed an IC50 of 1.9 nM on ADAM-17 with greatly increased selectivity. This compd. maintained good inhibitory properties on sALCAM shedding in several in vitro assays

Identification of a Novel p53 Modulator Endowed with Antitumoural and Antibacterial Activity through a Scaffold Repurposing Approach

Intracellular pathogens, such as Chlamydia trachomatis, have been recently shown to induce degradation of p53 during infection, thus impairing the protective response of the host cells. Therefore, p53 reactivation by disruption of the p53-MDM2 complex could reduce infection and restore pro-apoptotic effect of p53. Here, we report the identification of a novel MDM2 inhibitor with potential antitumoural and antibacterial activity able to reactivate p53. A virtual screening was performed on an in-house chemical library, previously synthesised for other targets, and led to the identification of a hit compound with a benzo[a]dihydrocarbazole structure, RM37. This compound induced p53 up-regulation in U343MG glioblastoma cells by blocking MDM2-p53 interaction and reduced tumour cell growth. NMR studies confirmed its ability to dissociate the MDM2-p53 complex. Notably, RM37 reduced Chlamydia infection in HeLa cells in a concentration-dependent manner and ameliorated the inflammatory status associated with infection

Apoptosis Therapy in Cancer: The First Single-molecule Co-activating p53 and the Translocator Protein in Glioblastoma

In the complex scenario of cancer, treatment with compounds targeting multiple cell pathways has been emerging. In Glioblastoma Multiforme (GBM), p53 and Translocator Protein (TSPO), both acting as apoptosis inducers, represent two attractive intracellular targets. On this basis, novel indolylglyoxylyldipeptides, rationally designed to activate TSPO and p53, were synthesized and biologically characterized. The new compounds were able to bind TSPO and to reactivate p53 functionality, through the dissociation from its physiological inhibitor, murine double minute 2 (MDM2). In GBM cells, the new molecules caused Δψm dissipation and inhibition of cell viability. These effects resulted significantly higher with respect to those elicited by the single target reference standards applied alone, and coherent with the synergism resulting from the simultaneous activation of TSPO and p53. Taken together, these results suggest that TSPO/MDM2 dual-target ligands could represent a new attractive multi-modal opportunity for anti-cancer strategy in GBM

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Human recombinant beta-secretase immobilized enzyme reactor for fast hits\u2019 selection and characterization from a virtual screening library

In the present work, a human recombinant BACE1 immobilized enzyme reactor (hrBACE1-IMER) has been applied for the sensitive fast screening of 38 compounds selected through a virtual screening approach. HrBACE1-IMER was inserted into a liquid chromatograph coupled with a fluorescent detector. A fluorogenic peptide substrate (M-2420), containing the \u3b2-secretase site of the Swedish mutation of APP, was injected and cleaved in the on-line HPLC-hrBACE1-IMER system, giving rise to the fluorescent product. The compounds of the library were tested for their ability to inhibit BACE1 in the immobilized format and to reduce the area related to the chromatographic peak of the fluorescent enzymatic product. The results were validated in solution by using two different FRET methods. Due to the efficient virtual screening methodology, more than fifty percent of the selected compounds showed a measurable inhibitory activity. One of the most active compound (a bis-indanone derivative) was characterized in terms of IC(50) and K(i) determination on the hrBACE1-IMER. Thus, the hrBACE1-IMER has been confirmed as a valid tool for the throughput screening of different chemical entities with potency lower than 30\u3bcM for the fast hits' selection and for mode of action determination

State-of-the-art methodologies for the discovery and characterization of DNA G-quadruplex binders.

Nowadays, the molecular basis of interaction between low molecular weight compounds and biological macromolecules is the subject of numerous investigations aimed at the rational design of molecules with specific therapeutic applications. In the last decades, it has been demonstrated that DNA quadruplexes play a critical role in several biological processes both at telomeric and gene promoting levels thus providing a great stride in the discovery of ligands able to interact with such a biologically relevant DNA conformation. So far, a number of experimental and computational approaches have been successfully employed in order to identify new ligands and to characterize their binding to the DNA. The main focus of this review is the description of these methodologies, placing a particular emphasis on computational methods, isothermal titration calorimetry (ITC), mass spectrometry (MS), nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence spectroscopies

From garcinol to barbituric acid derivatives: development of a novel cell-permeable, selective, and noncompetitive inhibitor of KAT3 histone acetyltransferases.

Lysine acetylation is among the prominent posttranslational modifications in eukaryotic cells. Protein acetylation level is a consequence of the balance between the opposite activities of protein acetyltransferases (KATs) and deacetylases (KDACs), and its deregulation has been linked to several diseases, including cancer, inflammation and neurodegenerative diseases. At present, only a small number of KATs modulators have been reported and just a few of them show selectivity between KATs isoforms. Among these, a polyisoprenylated benzophenone, garcinol, isolated from Garcinia Indica, is a potent inhibitor of histone acetyltransferases p300 and PCAF. Starting from the garcinol hardly optimizable and not very cell-permeable core structure, we applied a molecular pruning approach and prepared many analogues that were screened for their inhibitory effects using biochemical and biophysical (SPR) assays. Further optimization led to the discovery of the benzylidenebarbituric acid derivative 7h (EML425) as a potent and selective reversible inhibitor of CBP/p300, non-competitive versus both acetyl-CoA and a histone H3 peptide, and endowed with good cell permeability. Furthermore, in human leukemia U937 cells, it induced a marked and time-dependent reduction in the acetylation of lysine H4K5 and H3K9, a marked arrest in the G0/G1 phase and a significant increase in the hypodiploid nuclei percentage