Identification of IspD as a novel target for tuberculosis treatment using compound M6

Lijun Dong; Hui Qi; Yue Zhu; Yuma Yang; Yue Zhao; Sihan Zhang; Yongqiang Su; Taiyun Yue; Xiancai Du; Hetian Lei; Yanhui Yang

doi:10.3389/fmicb.2024.1461227

Identification of IspD as a novel target for tuberculosis treatment using compound M6

Front Microbiol. 2024 Nov 13:15:1461227. doi: 10.3389/fmicb.2024.1461227. eCollection 2024.

Authors

Lijun Dong^#^{1

2}, Hui Qi^#², Yue Zhu¹, Yuma Yang¹, Yue Zhao¹, Sihan Zhang¹, Yongqiang Su¹, Taiyun Yue¹, Xiancai Du³, Hetian Lei⁴, Yanhui Yang¹

Affiliations

¹ Ningxia Key Laboratory of Clinical and Pathogenic Microbiology, The School of Basic Medical Sciences, Ningxia Medical University, Yinchuan, China.
² Shenzhen Eye Hospital, Jinan University, Shenzhen Eye Institute, Shenzhen, China.
³ School of Medicine, Southern University of Science and Technology, Shenzhen, China.
⁴ Department of Ophthalmology, The First People's Hospital of Chenzhou, Chenzhou, China.

^# Contributed equally.

Abstract

Introduction: Tuberculosis (TB) is a serious infectious disease that endangers human health, and TB becomes more difficult in eradiation due to its multidrug resistance (MDR). The objective of this research was to identify novel targets for treating TB.

Methods: A 2-fold serial dilution method was used to determine minimal inhibitory concentrations (MIC) of compound M6 against Mycobacterium smegmatis (M. smegmatis). Compound M6 was subjected to reverse molecular docking with seven Mycobacterium tuberculosis proteins, and the best binding protein with the highest LibDock score was evaluated. The target protein with the highest score was purified through prokaryotic expression. Isolated target proteins were investigated for the enzyme activities and for the kinetic effect of compound M6 by absorbance detection. Subsequently, the CRISPR/Cas9 technology was employed to inhibit target gene expression for detecting MIC changes. Finally, potential targets were evaluated for the effect of the compound M6 in bacteria.

Results: The MIC values of compound M6 against M. smegmatis were 32 μg/mL. The results from reverse molecular docking show that IspD has the highest LibDock score of 142.50, followed by Rv0674, IspF, and Dxr, with docking scores of 110.762, 71.6955, and 57.7446, respectively. IspD is a key enzyme in the 2-C-methyl-D-erythritol 4-phosphate pathway of MTB. The aKi and Ki values of M6 for the substrate MEP are 609.58 μM and 81.33 μM. For CTP, the aKi and Ki values are 657.89 μM and 40.07 μM. With tetracycline inducing CRISPR/Cas9 to suppress the expression of IspD, the MIC value of M6 against IspD went down significantly from 32 μg/mL to 4 μg/mL.

Conclusion: IspD is a novel target of the compound M6 for treating TB.

Keywords: CRISPR/Cas9; IspD; Mycobacterium tuberculosis; N-(1, 3, 4-oxazole-2-)-benzolamide; molecular docking.

Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Research Projects for Social Development of Ningxia Hui Autonomous Region under Grant (No. 2021BEG03072) and Scientific Research Project of Ningxia Education Department (No. NYG2024122) and Ningxia Natural Science Foundation (No. 2024AAC03209) to YY, National Natural Science Foundation of China (No. 82070989) to HL. No funding bodies had any role in study design, data collection and analysis, decision to publisher preparation of the manuscript.