Using a Molecular Modeling and Native State Mass Spectrometry Quick Screening Approach to Understand the Inhibition of IMP-1 Variants

Introduction: Resistance to B-lactam antibiotics has become increasingly prevalent since the introduction of antibiotics. Each year in the United States approximately two million people acquire bacterial infections that are resistant to antibiotics. A common mechanism for B-lactam resistance is the production of B-lactamases that hydrolyze the B-lactam ring, thus rendering the drugs inactive. Today there are more than 2000 B-lactamases, but this study will focus on the B1 subclass known as metallo-B-lactamases (MBLs). MBLs are capable of inactivating all B-lactam antibiotics, except monobactams, and do not have any known clinical inhibitors. Thus, the development of MBL inhibitors is crucial. Most studies focus on the three most clinically relevant MBLs which include: Imipenemase MBL (IMP-1); Verona integrin-encoded MBL (VIM-2); and New Delhi MBL (NDM-1). Over time each of these MBLs have evolved and now have many variants whose inhibition is poorly studied. The variant IMP-78 will be the focus of this study due to its two mutations (S262G/V67F) near the active site. The primary purpose of this study is to investigate whether an inhibitor of IMP-1 is capable of having the same mechanism of inhibition with the variant IMP-78. Results: Six previously published novel MBL inhibitors were chosen for this study. The native state electrospray ionization mass spectrometry (ESI-MS) showed that 3 out of 6 inhibitors were capable of binding to both IMP-1 and IMP-78. While the remaining 3 inhibitors were capable of binding only to IMP-1 and not to IMP-78. The preliminary molecular modeling results suggest a difference in the size of the binding pockets of IMP-1 and IMP-78. Future work: As this study is currently ongoing, there is future work to be completed. To investigate whether one or both amino acid mutations in IMP-78 prevent inhibitor binding, IMP-6 (S262G) and IMP-10 (V67F) will be analyzed using native state ESI-MS. The molecular modeling and molecular dynamic (MD) simulations will be completed on each inhibitor with IMP-1 and IMP-78. Molecular modeling and MD simulations will be performed for the 3 inhibitors that bind IMP-1 but not IMP-78 using IMP-6 and IMP-10. The binding affinity of each inhibitor for IMP-1 and IMP-78 will be determined using ITC. Lastly, microbiological studies (MIC’s) will be conducted.