作者: Whang, Jake ; Park, Shinhyun ; Seok Lee, Jong ; Lee, Aram ; Cui, Lianji ; Soon Son, Eun ; Seong Yang, Jeong ; Kim, Kyungjong ; Mi Kim, Young ; Park, Yumi ; Eun Ahn, Ji ; Bang, Seorin ; Choi, Inhee ; Kang, Minji ; Ji Jeong, Mi

We present the development of a phenyl oxazole methyl (POM) core structure with spirocyclic derivatives as part of our efforts to discover innovative anti-tuberculosis agents. Derivatives of spirocyclic POM were synthesized and evaluated for their inhibitory effects on M.tuberculosis (M. tb) H37Rv. Notably, compound 5c displayed potent anti-tubercular activity with MIC value of 0.206 μM in culture broth medium. Furthermore MIC values of compound 5c against DS/MDR/pre-XDR clinical isolates ranged from 0.34 to 0.68 μg/mL, 0.17 to 0.68 μg/mL, and 0.17 to 0.34 μg/mL, respectively. Also, compound 5c with favorable ADME and PK properties was not cytotoxic to THP-1 human cells. Based on the spontaneous mutant generation, we have identified the target of compound 5c to be MmpL3. The computational docking study suggested its plausible binding mode against MmpL3. There is no approved drug targeting this target yet, and the outcomes of the presented research will contribute to the future discovery of novel anti-tuberculosis drugs.

2024-11-01·International Journal of Biological Macromolecules

Integrating computational and experimental approaches in discovery and validation of MmpL3 pore domain inhibitors for specific labelling of Mycobacterium tuberculosis

Article

作者: Wang, Qiantao ; Ni, Xincheng ; Zhang, Xiaorui ; Yang, Yang ; Zhou, Yuanzheng ; Li, Jin ; He, Zhiqun ; Yang, Tao ; Zhang, Jiangnan ; Zeng, Jumei ; Luo, Youfu ; Qiu, Zhiqiang ; Dong, Baoyu

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to pose a significant global health threat. Identifying new druggable targets is crucial for the advancement of drug development. Equally critical is the development of precise methods for monitoring Mtb to effectively combat this disease. Addressing these needs, our study pinpointed the pore domain (PD) of MtbMmpL3 as a new binding site for virtual screening, which led to the discovery of the small molecule ZY27. To confirm the binding site and action mode of ZY27, we employed cosolvent molecular dynamics (CMD), steered molecular dynamics (SMD), and long timescale molecular dynamics (MD) simulations of 5 μs. These in silico studies verified that ZY27 binds to the PD of MtbMmpL3. In antimicrobial activity tests, ZY27 exhibited potent anti-Mtb activity and high selectivity among mycobacterial species. Whole-genome sequencing of spontaneous ZY27-resistant Mtb variants, complemented by acid-fast staining experiments, confirmed that ZY27 specifically targets MtbMmpL3. Utilizing the ligand-protein binding data, we designed and synthesized two solvatochromic fluorescent probes, 27FP1 and 27FP2, based on ZY27. Further investigations through flow cytometry and confocal microscopy confirmed that these probes specifically label Mtb cells via the MtbMmpL3 binding mechanism.

2024-10-16·mBio

Allosteric coupling of substrate binding and proton translocation in MmpL3 transporter from Mycobacterium tuberculosis

Article

作者: Jackson, Mary ; Yang, Lixinhao ; Gumbart, James C. ; Grzegorzewicz, Anna E. ; Li, Wei ; Zgurskaya, Helen I. ; Babii, Svitlana

ABSTRACT Infections caused by Mycobacterium spp. are very challenging to treat, and multidrug-resistant strains rapidly spread in human populations. Major contributing factors include the unique physiological features of these bacteria, drug efflux, and the low permeability barrier of their outer membrane. Here, we focus on MmpL3 from Mycobacterium tuberculosis , an essential inner membrane transporter of the resistance–nodulation–division superfamily required for the translocation of mycolic acids in the form of trehalose monomycolates (TMM) from the cytoplasm or plasma membrane to the periplasm or outer membrane. The MmpL3-dependent transport of TMM is essential for the growth of M. tuberculosis in vitro , inside macrophages, and in M. tuberculosis- infected mice. MmpL3 is also a validated target for several recently identified anti-mycobacterial agents. In this study, we reconstituted the lipid transport activity of the purified MmpL3 using a two-lipid vesicle system and established the ability of MmpL3 to actively extract phospholipids from the outer leaflet of a lipid bilayer. In contrast, we found that MmpL3 lacks the ability to translocate the same phospholipid substrate across the plasma membrane indicating that it is not an energy-dependent flippase. The lipid extraction activity was modulated by substitutions in critical charged and polar residues of the periplasmic substrate-binding pocket of MmpL3, coupled to the proton transfer activity of MmpL3 and inhibited by a small molecule inhibitor SQ109. Based on the results, we propose a mechanism of allosteric coupling wherein substrate translocation by MmpL3 is coupled to the energy provided by the downhill transfer of protons. The reconstituted activities will facilitate understanding the mechanism of MmpL3-dependent transport of lipids and the discovery of new therapeutic options for Mycobacterium spp. infections. IMPORTANCE MmpL3 from Mycobacterium tuberculosis is an essential transporter involved in the assembly of the mycobacterial outer membrane. It is also an important target in undergoing efforts to discover new anti-tuberculosis drugs effective against multidrug-resistant strains spreading in human populations. The recent breakthrough structural studies uncovered features of MmpL3 that suggested a possible lipid transport mechanism. In this study, we reconstituted and characterized the lipid transport activity of MmpL3 and demonstrated that this activity is blocked by MmpL3 inhibitors and substrate mimics. We further uncovered the mechanism of how the binding of a substrate in the periplasmic domain is communicated to the transmembrane proton relay of MmpL3. The uncovered mechanism and the developed assays provide new opportunities for mechanistic analyses of MmpL3 function and its inhibition.

100 项与 MmpL3 Inhibitors (AbbVie) 相关的药物交易

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