New Noncovalent Inhibitors of Penicillin-Binding Proteins from Penicillin-Resistant Bacteria

BACKGROUND: Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs beta-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for beta-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs. METHODOLOGY/PRINCIPAL FINDINGS: Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains. CONCLUSIONS: We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.Eur-Intafa

Assay platform for clinically relevant metallo-beta-lactamases

Metallo-β-lactamases (MBLs) are a growing threat to the use of almost all clinically used β-lactam antibiotics. The identification of broad-spectrum MBL inhibitors is hampered by the lack of a suitable screening platform, consisting of appropriate substrates and a set of clinically relevant MBLs. We report procedures for the preparation of a set of clinically relevant metallo-β-lactamases (i.e., NDM-1 (New Delhi MBL), IMP-1 (Imipenemase), SPM-1 (São Paulo MBL), and VIM-2 (Verona integron-encoded MBL)) and the identification of suitable fluorogenic substrates (umbelliferone-derived cephalosporins). The fluorogenic substrates were compared to chromogenic substrates (CENTA, nitrocefin, and imipenem), showing improved sensitivity and kinetic parameters. The efficiency of the fluorogenic substrates was exemplified by inhibitor screening, identifying 4-chloroisoquinolinols as potential pan MBL inhibitors

A novel three-enzyme reaction cycle for the synthesis of N-acetyllactosamine with in situ regeneration of uridine 5'-diphosphate glucose and uridine 5'-diphosphate galactose

A new three-enzyme reaction cycle consisting of sucrose synthase, UDP glucose 4‘-epimerase, and human β-1,4-galactosyltransferase was established for the synthesis of N-acetyllactosamine (LacNAc) with in situ regeneration of UDP galactose. We found that UDP glucose 4‘-epimerase is reductively inactivated in the presence of UMP and acceptor substrates of β-1,4-galactosyltransferase. Reactivation of UDP glucose 4‘-epimerase by the transition state analogues dUDP or dTDP 6-deoxy-d-xylo-4-hexulose in combination with the repetitive batch technique enabled us to use the native enzymes for 11 days in this cycle. With 10 U of sucrose synthase, 5 U of UDP glucose 4‘-epimerase, and 1.25 U of β-1,4-galactosyltransferase, 594 mg of LacNAc could be synthesized. N-Acetyllactosamine was also subsequently converted to Neu5Acα2,6Galβl,4GlcNAc with α-2,6-sialyltransferase and CMP-Neu5Ac