Insights into the Spectrum of Activity and Mechanism of Action of MGB-BP-3

Abstract

MGB-BP-3 is a potential first-in-class antibiotic, a Strathclyde Minor Groove Binder (S-MGB), that has successfully completed Phase IIa clinical trials for the treatment of Clostridioides difficile associated disease. Its precise mechanism of action and the origin of limited activity against Gram-negative pathogens are relatively unknown. Herein, treatment with MGB-BP-3 alone significantly inhibited the bacterial growth of the Gram-positive, but not Gram-negative, bacteria as expected. Synergy assays revealed that inefficient intracellular accumulation, through both permeation and efflux, is the likely reason for lack of Gram-negative activity. MGB-BP-3 has strong interactions with its intracellular target, DNA, in both Gram-negative and Gram-positive bacteria, revealed through ultraviolet−visible (UV−vis) thermal melting and fluorescence intercalator displacement assays. MGB-BP-3 was confirmed to bind to dsDNA as a dimer using nano-electrospray ionization mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Type II bacterial topoisomerase inhibition assays revealed that MGB-BP-3 was able to interfere with the supercoiling action of gyrase and the relaxation and decatenation actions of topoisomerase IV of both Staphylococcus aureus and Escherichia coli. However, no evidence of stabilization of the cleavage complexes was observed, such as for fluoroquinolones, confirmed by a lack of induction of DSBs and the SOS response in E. coli reporter strains. These results highlight additional mechanisms of action of MGB-BP-3, including interference of the action of type II bacterial topoisomerases. While MGB-BP-3′s lack of Gram-negative activity was confirmed, and an understanding of this presented, the recognition that MGB-BP-3 can target DNA of Gram-negative organisms will enable further iterations of design to achieve a Gram-negative active S-MGB.

Antibiotics in the clinical pipeline as of December 2022

“…MGB-BP-3 ( 16 ) was discovered at the University of Strathclyde (Glasgow, UK) and was inspired by the actinomycetes-derived minor groove binders, distamycin, netropsin and thiazotropsin [ 109 , 110 ]. In addition to activity against C. difficile , 16 has activity against a range of G+ve bacteria including S. aureus and Enterococcus faecalis but is not active against G-ve bacteria due to a lack of intracellular accumulation [ 111 ]. It was shown that two molecules of 16 bound to the minor groove of dsDNA, which then interfered with transcription, the supercoiling action of gyrase, and the relaxation and decatenation by topoisomerase IV enzymes in vitro [ 111 ].…”

“…In addition to activity against C. difficile , 16 has activity against a range of G+ve bacteria including S. aureus and Enterococcus faecalis but is not active against G-ve bacteria due to a lack of intracellular accumulation [ 111 ]. It was shown that two molecules of 16 bound to the minor groove of dsDNA, which then interfered with transcription, the supercoiling action of gyrase, and the relaxation and decatenation by topoisomerase IV enzymes in vitro [ 111 ]. This is mechanistically distinct from fluoroquinolones that cause an increase in double strand breaks, as well as induce recA and lexA SOS responses.…”

The allure of targets for novel drugs

“…In an efficacy model, S-MGB-363 was as active as the standard of care drug, posaconazole, in combating infection in a mouse model of invasive aspergillosis. 58 Through its fluorescent properties, it was possible to observe that S-MGB-363 associates with the fungal cell wall before transporting to the nucleus. Association at the cell membrane was not observed in a lung epithelial cell line (A549), nor was uptake, suggesting that entry into the target cell is a primary reason for the selectivity of S-MGB-363.…”

“…Experiments have shown that this is because MGB-BP-3 fails to accumulate in cells of Gram-negative bacteria because in most cases, it is removed by the action of efflux pumps. 58 However, if efflux is blocked by an inhibitor such as PAβN or a mutant lacking an efflux pump is tested, MGB-BP-3 becomes effective again. It is a challenge to incorporate such features into new S-MGBs molecules themselves.…”

Tracking the mechanism of covalent molecular glue stabilization using native mass spectrometry

“…Signicant advances in native mass spectrometry (MS) have enabled the high-resolution detection of protein complexes in their native state, enabling protein function elucidation. 18,19 Further, native MS is a powerful tool to investigate ligandprotein complexes, [20][21][22] ligand-DNA complexes, 23 and molecular glue protein complex stabilization. 24,25 In contrast to protein denaturing MS techniques, native MS preserves protein complexes from solution into the gas phase enabling the analyses of protein complexes in their native state.…”