Background and objectives: The presence of penicillin-binding protein 2a (PBP2a) is the cause of Methicillin-resistant Staphylococcus aureus (MRSA), which is important nosocomial pathogens worldwide and now are also of growing importance in community-acquired infection. The PBP2a resistance depends upon a supplementary peptidoglycan transpeptidase, which continues to function when normal PBPs have been inactivated by beta-lactam antibiotics. Analysis and quantitative study of the molecular interactions of PBP2a against β-lactam antibiotics are required, as they support explaining and enhance understanding of the structure-activity relationship of antibiotic resistance. Methods: Bioinformatics and computational methods have been highly effective tools for β-lactams targeting PBPs to tackle the urgent threat of antimicrobial resistance. Regarding β-lactam antibiotics targeting PBP2a and PBPs, we applied different docking programs to illustrate inhibition mode, MM/GBSA to estimate the binding free energies, and molecular dynamic simulation to validate and analyze the molecular interactions. Results: Based on β-lactam antibiotics targeting PBPs as covalent inhibitors, covalent docking was employed to provide explicit models of PBP2a against susceptible β-lactam antibiotics. The simultaneous use of non-covalent docking enhances our comprehensive comprehension of the resistance of PBP 2a, which resulted from the lack of covalent linked to β-lactam antibiotics. The selected antibiotics strongly interact with PBP2a, revealing the essential amino acid residues and binding affinity for inhibition. MD simulations were performed for the ligand-bound state of PBP-2a to explain their interaction and conformational changes. These findings are also strongly supported by root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF) and Hydrogen bond analysis of the protein-ligand complex. Conclusions: Our research offers extensive knowledge of the PBP2a-lactam interactions for the ability of known antibiotics to combat MRSA. The simulation results indicating stability and accuracy provide valuable insights for the advancement of pharmaceutical interventions against infectious diseases Abstract Figure: Figure The interaction of Methicillin with PBP2a of MRSA.: The protein is shown as a cartoon model, and the covalent binding of the ligand and serine active site is shown as a stick model.

Yeast surface display represents a commonly used platform suitable for the generation and screening of antibodies as well as the selection of high-producer clones. The methods of yeast display rely on the genetic fusion of recombinant antibodies to an abundant cell wall protein of yeast. Here, the study of proof of concept showed that the conventional strategy of expression of fusion antibodies was replaced by non-covalent binding to antibodies for yeast surface display. The use of cell surface display of an epitope tag will endow the cells with new arms for immobilizing, absorbing or targeting the proteins. Staphylococcal protein A, which is characterized by its ability to bind selectively to the Fc region of IgG, was examined to be expressed on the surface of Saccharomyces cerevisiae using a secretion signal of Rhizopus oryzae glucoamylase and C-terminal half of α−agglutinin including glycosylphosphatidylinositol (GPI) anchor attachment signal under the control of the glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) promoter. On the other hand, an Fc-fused enzyme was created to construct a molecular fusion of Rhizopus oryzae Lipase with the spacer and the Fc region of IgG heavy chain. The secretion of fusion protein was carried out using pre-α-factor leader region as secretion signal under the control of the 5 ^’ -upstream region of the Candida tropicalis isocitrate lyase gene ( UPR-ICL ) in S. cerevisiae . The Fc-fused lipase was captured by Staphylococcal protein A as an adaptor protein displayed on the surface of yeast cells. The method of this switchable yeast display takes advantage of the “secretion-and-capture” strategy and can be applied to improve the efficiency of yeast display of full-length IgG. graphical Staphylococcal protein A, which has the ability to binding to the Fc region of IgG and leaving the antigen combing site free, has been assembled on the surface of yeast cells to target antibodies or enzymes with Fc fusion.

Background: Phage display technology has been established as a powerful screening approach to select ligands or peptides for binding to proteins. Despite rapid growth in the field, there has been a relative dearth of quantitative criteria to measure the effectiveness of the process of phage display screening. Since human serum albumin (HSA) has been extensively studied as a drug carrier to extend the plasma half-life of protein therapeutics, the use of phage display technology is required for identifying albumin-binding peptides as the very promising strategy of albumin-binding against albumin fusion. The construction of albumin-binding drug requires the assessment of a large quantity of HSA-binding peptide (HSA binder) candidates for conjugation with therapeutic proteins. The use of the linear epitope mapping method has allowed researchers to discover many HSA-binding peptides. However, it may be inefficient to select these peptides based on sequence identity via randomly sequencing individual phage clones from enrichment pools. Method: Here, a simple assessment method to facilitate phage display selection of HSA-binding peptides was recommended. With experimentally determined phage titer, one can calculate the specificity ratios, the recovery yields and the relative dissociation constants, which are defined as quantitative criteria for panning and characterization of the binding phage fused peptides. Results: Consequently, this approach may not only enable more rapid and low-cost phage display screening, but also efficiently reduce pseudo-positive phages selected as HSA binders for conjugation with therapeutic proteins.

The spread and evolution of antimicrobial resistance (AMR) pose significant threats to public health, food safety, and sustainable development. Metallo-β-lactamases (MBLs), a class of carbapenemases that hydrolyze carbapenems—often considered last-resort antibiotics—are of particular concern and underscore the urgent need to develop effective inhibitors. Recent advancements in bicyclic boronate inhibitors represent significant progress toward this goal. Although the mechanistic basis of NDM-1 inhibition by bicyclic boronate inhibitors remains incompletely elucidated, this study systematically characterizes the binding profiles and intermolecular interaction landscapes between NDM-1 and four cyclic boronic acid-based inhibitors—vaborbactam (RPX7009), taniborbactam (VNRX 5133), xeruborbactam (QPX7728), and ledaborbactam (VNRX-5236). We established the NDM-1-mediated hydrolysis of two representative carbapenem antibiotics, meropenem and imipenem, as the reference system for comparative analysis. While conventional docking workflows often struggle to model metalloenzyme active sites accurately, the AMDock (Assisted Molecular Docking) platform, integrated with AutoDock’s optimized zinc force field, enables robust and reliable docking simulations for this enzyme family. Our AMDock-based molecular docking not only identified the critical NDM-1 binding-site residues governing hydrogen-bond networks with ligands, but also revealed that bicyclic boronate inhibitors show substantially greater binding stability than both carbapenems and their monocyclic boronate counterparts. A follow-up 100 ns all-atom molecular dynamics simulation in GROMACS provided atomistic-level evidence for this enhanced performance: RMSD, RMSF, radius of gyration, continuous hydrogen-bonding occupancy, and 2D free-energy landscape analyses all consistently supported the higher binding affinity of bicyclic boronates. Ultimately, this study characterizes two promising drug candidates, QPX7728 and VNRX-5133, as competitive inhibitors that can effectively rescue carbapenem activity against NDM-1 MBL-mediated antimicrobial resistance. This visualization elucidates interactions between NDM-1 and its two ligand classes—carbapenem antibiotics and cyclic boronate inhibitors. It reveals the stability and interaction strength underlying the distinct inhibition pattern of bicyclic boronate inhibitors in the enzyme’s active site, offering a promising strategy to combat antibiotic resistance.