Restoring susceptibility to ß-lactam antibiotics in methicillin-resistant Staphylococcus aureus.

Infections by Staphylococcus aureus have been treated historically with ß-lactam antibiotics. However, these antibiotics have become obsolete in methicillin-resistant S. aureus by acquisition of the bla and mec operons. The presence of the ß-lactam antibiotic is detected by the sensor domains of BlaR and/or MecR, and the information is transmitted to the cytoplasm, resulting in derepression of the antibiotic-resistance genes. We hypothesized that inhibition of the sensor domain would shut down this response system, and ß-lactam susceptibility would be restored. An in silico search of 11 million compounds led to a benzimidazole-based hit and, ultimately, to the boronate 4. The X-ray structure of 4 is covalently engaged with the active-site serine of BlaR. Compound 4 potentiates by 16- to 4,096-fold the activities of oxacillin and of meropenem against methicillin-resistant S. aureus strains. The combination of 4 with oxacillin or meropenem shows efficacy in infected mice, validating the strategy.

Lytic transglycosylase Slt of Pseudomonas aeruginosa as a periplasmic hub protein.

Peptidoglycan is a major constituent of the bacterial cell wall. Its integrity as a polymeric edifice is critical for bacterial survival and, as such, it is a preeminent target for antibiotics. The peptidoglycan is a dynamic crosslinked polymer that undergoes constant biosynthesis and turnover. The soluble lytic transglycosylase (Slt) of Pseudomonas aeruginosa is a periplasmic enzyme involved in this dynamic turnover. Using amber-codon-suppression methodology in live bacteria, we incorporated a fluorescent chromophore into the structure of Slt. Fluorescent microscopy shows that Slt populates the length of the periplasmic space and concentrates at the sites of septation in daughter cells. This concentration persists after separation of the cells. Amber-codon-suppression methodology was also used to incorporate a photoaffinity amino acid for the capture of partner proteins. Mass-spectrometry-based proteomics identified 12 partners for Slt in vivo. These proteomics experiments were complemented with in vitro pulldown analyses. Twenty additional partners were identified. We cloned the genes and purified to homogeneity 22 identified partners. Biophysical characterization confirmed all as bona fide Slt binders. The identities of the protein partners of Slt span disparate periplasmic protein families, inclusive of several proteins known to be present in the divisome. Notable periplasmic partners (KD < 0.5 µM) include PBPs (PBP1a, KD = 0.07 µM; PBP5 = 0.4 µM); other lytic transglycosylases (SltB2, KD = 0.09 µM; RlpA, KD = 0.4 µM); a type VI secretion system effector (Tse5, KD = 0.3 µM); and a regulatory protease for alginate biosynthesis (AlgO, KD < 0.4 µM). In light of the functional breadth of its interactome, Slt is conceptualized as a hub protein within the periplasm.

Structural characterization of lytic transglycosylase MltD of Pseudomonas aeruginosa, a target for the natural product bulgecin A.

Natural product bulgecin A potentiates the activity of ß-lactam antibiotics by inhibition of three lytic transglycosylases in Pseudomonas aeruginosa, of which MltD is one. MltD exhibits both endolytic and exolytic reactions in the turnover of the cell-wall peptidoglycan and tolerates the presence or absence of stem peptides in its substrates. The present study reveals structural features of the multimodular MltD, presenting a catalytic module and four cell-wall-binding LysM modules that account for these attributes. Three X-ray structures are reported herein for MltD that disclose one unpredicted LysM module tightly attached to the catalytic domain, whereas the other LysM modules are mobile, and connected to the catalytic domain through long flexible linkers. The formation of crystals depended on the presence of bulgecin A. The expansive active-site cleft is highlighted by the insertion of a helical region, a hallmark of the family 1D of lytic transglycosylases, which was mapped out in a ternary complex of MltD:bulgecinA:chitotetraose, revealing at the minimum the presence of eight subsites (from -4 to +4, with the seat of reaction at subsites -1 and + 1) for binding of sugars of the substrate for the endolytic reaction. The mechanism of the exolytic reaction is revealed in one of the structures, showing how the substrate's terminal anhydro-NAM moiety could be sequestered at subsite +2. Our results provide the structural insight for both the endolytic and exolytic activities of MltD during cell-wall-turnover events.

Catalytic process of anhydro-N-acetylmuramic acid kinase from Pseudomonas aeruginosa.

The bacterial cell envelope is the structure with which the bacterium engages with, and is protected from, its environment. Within this envelop is a conserved peptidoglycan polymer which confers shape and strength to the cell envelop. The enzymatic processes that build, remodel, and recycle the chemical components of this cross-linked polymer are preeminent targets of antibiotics and exploratory targets for emerging antibiotic structures. We report a comprehensive kinetic and structural analysis for one such enzyme, the Pseudomonas aeruginosa anhydro-N-acetylmuramic acid (anhNAM) kinase (AnmK). AnmK is an enzyme in the peptidoglycan-recycling pathway of this pathogen. It catalyzes the pairing of hydrolytic ring opening of anhNAM with concomitant ATP-dependent phosphoryl transfer. AnmK follows a random-sequential kinetic mechanism with respect to its anhNAM and ATP substrates. Crystallographic analyses of four distinct structures (apo AnmK, AnmK:AMPPNP, AnmK:AMPPNP:anhNAM, and AnmK:ATP:anhNAM) demonstrate that both substrates enter the active site independently in an ungated conformation of the substrate subsites, with protein loops acting as gates for anhNAM binding. Catalysis occurs within a closed conformational state for the enzyme. We observe this state crystallographically using ATP-mimetic molecules. A remarkable X-ray structure for dimeric AnmK sheds light on the precatalytic and postcatalytic ternary complexes. Computational simulations in conjunction with the high-resolution X-ray structures reveal the full catalytic cycle. We further report that a P. aeruginosa strain with disrupted anmK gene is more susceptible to the ß-lactam imipenem compared to the WT strain. These observations position AnmK for understanding the nexus among peptidoglycan recycling, susceptibility to antibiotics, and bacterial virulence.

Application of 2D-ITC to the Elucidation of the Enzymatic Mechanism of N-Acetylmuramic Acid/N-Acetylglucosamine Kinase (AmgK) from Pseudomonas aeruginosa.

Characterization of the turnover mechanism of bisubstrate enzymes is a tedious task. Molecular tools for studying the enzymatic mechanism are not readily available for all enzymes (e.g., radioactive substrates, substrate-competitive inhibitors, etc.). Wang and Mittermaier recently introduced two-dimensional isothermal titration calorimetry (2D-ITC) for determining the bisubstrate mechanism at high resolution while simultaneously quantifying the kinetic parameters for substrate turnover in a single reporter-free experiment. We demonstrate the utility of 2D-ITC in studying N-acetylmuramic acid/N-acetylglucosamine kinase (AmgK) from Pseudomonas aeruginosa. This enzyme is involved in cytoplasmic cell-wall-recycling events as a step in the peptidoglycan salvage pathway. Furthermore, AmgK phosphorylates N-acetylglucosamine and N-acetylmuramic acid, linking the recycling events to de novo cell-wall synthesis. We document in a 2D-ITC experiment that AmgK follows an ordered-sequential mechanism, where ATP binds first and ADP is released last. We also show that classical enzyme kinetic methods support the results of 2D-ITC and that 2D-ITC could overcome the shortcomings of these classical methodologies. We provide evidence for inhibition of AmgK by the catalytic product ADP, but not by the phosphorylated sugar product. These results provide a full kinetic characterization of the bacterial kinase AmgK. This work highlights 2D-ITC as a versatile tool for the mechanistic evaluation of bisubstrate enzymes, as an alternative for classical methods.

Repurposing of Some Natural Product Isolates as SARS-COV-2 Main Protease Inhibitors via In Vitro Cell Free and Cell-Based Antiviral Assessments and Molecular Modeling Approaches.

The emergence of the SARS-CoV-2 pandemic has prompted scientists to search for an efficient antiviral medicine to overcome the rapid spread and the marked increase in the number of patients worldwide. In this regard natural products could be a potential source of substances active against coronavirus infections. A systematic computer-aided virtual screening approach was carried out using commercially available natural products found on the Zinc Database in addition to an in-house compound library to identify potential natural product inhibitors of SARS-CoV-2 main protease (MPRO). The top eighteen hits from the screening were selected for in vitro evaluation on the viral protease (SARS-CoV-2 MPRO). Five compounds (naringenin, 2,3',4,5',6-pentahydroxybenzophenone, apigenin-7-O-glucoside, sennoside B, and acetoside) displayed high activity against the viral protein. Acteoside showed similar activity to the positive control GC376. The most potent compounds were tested in vitro on SARS-CoV-2 Egyptian strain where only naringenin showed moderate anti-SARS-CoV-2 activity at non-cytotoxic micromolar concentrations in vitro with a significant selectivity index (CC50/IC50 = 178.748/28.347 = 6.3). Moreover; a common feature pharmacophore model was generated to explain the requirements for enzyme inhibition by this diverse group of active ligands. These results pave a path for future repurposing and development of natural products to aid in the battle against COVID-19.

New quinoxalinone inhibitors targeting secreted phospholipase A2 and α-glucosidase.

Elevated blood glucose and increased activities of secreted phospholipase A2 (sPLA2) are strongly linked to coronary heart disease. In this report, our goal was to develop small heterocyclic compound that inhibit sPLA2. The title compounds were also tested against α-glucosidase and α-amylase. This array of enzymes was selected due to their implication in blood glucose regulation and diabetic cardiovascular complications. Therefore, two distinct series of quinoxalinone derivatives were synthesised; 3-[N'-(substituted-benzylidene)-hydrazino]-1H-quinoxalin-2-ones 3a-f and 1-(substituted-phenyl)-5H-[1,2,4]triazolo[4,3-a]quinoxalin-4-ones 4a-f. Four compounds showed promising enzyme inhibitory effect, compounds 3f and 4b-d potently inhibited the catalytic activities of all of the studied proinflammatory sPLA2. Compound 3e inhibited α-glucosidase (IC50 = 9.99 ± 0.18 µM); which is comparable to quercetin (IC50 = 9.93 ± 0.66 µM), a known inhibitor of this enzyme. Unfortunately, all compounds showed weak activity against α-amylase (IC50 > 200 µM). Structure-based molecular modelling tools were utilised to rationalise the SAR compared to co-crystal structures with sPLA2-GX as well as α-glucosidase. This report introduces novel compounds with dual activities on biochemically unrelated enzymes mutually involved in diabetes and its complications.

Epigenetic Pathways of Oncogenic Viruses: Therapeutic Promises.

Cancerous transformation comprises different events that are both genetic and epigenetic. The ultimate goal for such events is to maintain cell survival and proliferation. This transformation occurs as a consequence of different features such as environmental and genetic factors, as well as some types of infection. Many viral infections are considered to be causative agents of a number of different malignancies. To convert normal cells into cancerous cells, oncogenic viruses must function at the epigenetic level to communicate with their host cells. Oncogenic viruses encode certain epigenetic factors that lead to the immortality and proliferation of infected cells. The epigenetic effectors produced by oncogenic viruses constitute appealing targets to prevent and treat malignant diseases caused by these viruses. In this review, we highlight the importance of epigenetic reprogramming for virus-induced oncogenesis, with special emphasis on viral epigenetic oncoproteins as therapeutic targets. The discovery of molecular components that target epigenetic pathways, especially viral factors, is also discussed.