Machine Learning Models Identify Inhibitors of New Delhi Metallo-ß-lactamase.

The worldwide spread of the metallo-ß-lactamases (MBL), especially New Delhi metallo-ß-lactamase-1 (NDM-1), is threatening the efficacy of ß-lactams, which are the most potent and prescribed class of antibiotics in the clinic. Currently, FDA-approved MBL inhibitors are lacking in the clinic even though many strategies have been used in inhibitor development, including quantitative high-throughput screening (qHTS), fragment-based drug discovery (FBDD), and molecular docking. Herein, a machine learning-based prediction tool is described, which was generated using results from HTS of a large chemical library and previously published inhibition data. The prediction tool was then used for virtual screening of the NIH Genesis library, which was subsequently screened using qHTS. A novel MBL inhibitor was identified and shown to lower minimum inhibitory concentrations (MICs) of Meropenem for a panel of E. coli and K. pneumoniae clinical isolates expressing NDM-1. The mechanism of inhibition of this novel scaffold was probed utilizing equilibrium dialyses with metal analyses, native state electrospray ionization mass spectrometry, UV-vis spectrophotometry, and molecular docking. The uncovered inhibitor, compound 72922413, was shown to be 9-hydroxy-3-[(5-hydroxy-1-oxa-9-azaspiro[5.5]undec-9-yl)carbonyl]-4H-pyrido[1,2-a]pyrimidin-4-one.

The directed evolution of NDM-1.

ß-Lactam antibiotics are among the most frequently prescribed therapeutic agents. A common mechanism of resistance toward ß-lactam antibiotics is the production of ß-lactamases. These enzymes are capable of hydrolyzing the ß-lactam bond, rendering the drug inactive. Among the four described classes, the metallo- ß-lactamases (MBLs, class B) employ one or two zinc ions in the active site for catalysis. One of the three most clinically relevant MBLs is New Delhi Metallo- ß-Lactamase (NDM-1). The current study sought to investigate the in vitro protein evolution of NDM-1 ß-lactamase using error-prone polymerase chain reaction. Evaluation revealed that variants were not found to confer higher levels of resistance toward meropenem based on amino acid substitutions. Thus, we postulate that increases in transcription or changes in zinc transport may be clinically more relevant to meropenem resistance than amino acid substitutions.

Carbapenem Use Is Driving the Evolution of Imipenemase 1 Variants.

Metallo-ß-lactamases (MBLs) are a growing clinical threat because they inactivate nearly all ß-lactam-containing antibiotics, and there are no clinically available inhibitors. A significant number of variants have already emerged for each MBL subfamily. To understand the evolution of imipenemase (IMP) genes (blaIMP) and their clinical impact, 20 clinically derived IMP-1 like variants were obtained using site-directed mutagenesis and expressed in a uniform genetic background in Escherichia coli strain DH10B. Strains of IMP-1-like variants harboring S262G or V67F substitutions exhibited increased resistance toward carbapenems and decreased resistance toward ampicillin. Strains expressing IMP-78 (S262G/V67F) exhibited the largest changes in MIC values compared to IMP-1. In order to understand the molecular mechanisms of increased resistance, biochemical, biophysical, and molecular modeling studies were conducted to compare IMP-1, IMP-6 (S262G), IMP-10 (V67F), and IMP-78 (S262G/V67F). Finally, unlike most New Delhi metallo-ß-lactamase (NDM) and Verona integron-encoded metallo-ß-lactamase (VIM) variants, the IMP-1-like variants do not confer any additional survival advantage if zinc availability is limited. Therefore, the evolution of MBL subfamilies (i.e., IMP-6, -10, and -78) appears to be driven by different selective pressures.

MBLinhibitors.com, a Website Resource Offering Information and Expertise for the Continued Development of Metallo--Lactamase Inhibitors.

In an effort to facilitate the discovery of new, improved inhibitors of the metallo--lactamases (MBLs), a new, interactive website called MBLinhibitors.com was developed. Despite considerable efforts from the science community, there are no clinical inhibitors of the MBLs, which are now produced by human pathogens. The website, MBLinhibitors.com, contains a searchable database of known MBL inhibitors, and inhibitors can be searched by chemical name, chemical formula, chemical structure, Simplified Molecular-Input Line-Entry System (SMILES) format, and by the MBL on which studies were conducted. The site will also highlight a "MBL Inhibitor of the Month", and researchers are invited to submit compounds for this feature. Importantly, MBLinhibitors.com was designed to encourage collaboration, and researchers are invited to submit their new compounds, using the "Submit" function on the site, as well as their expertise using the "Collaboration" function. The intention is for this site to be interactive, and the site will be improved in the future as researchers use the site and suggest improvements. It is hoped that MBLinhibitors.com will serve as the one-stop site for any important information on MBL inhibitors and will aid in the discovery of a clinically useful MBL inhibitor.