Mycobacterium tuberculosis infection remains one of the most dangerous infectious diseases globally, prompting the development of various therapeutic agents. Rifampicin, one of such drugs, induces the drug-metabolizing enzyme cytochrome P450 (CYP), which accelerates the degradation of co-administered drugs and diminishes their efficacy. Consequently, there is a critical need to develop compounds that inhibit excessive CYP-mediated metabolism. Previous studies have identified the heme group as the active site of CYP and demonstrated that the coordination bond between the central heme iron and inhibitors is essential for CYP inhibition. Conventional molecular simulations have struggled to accurately reproduce this coordination bond. In response, this study constructed a novel molecular force field that incorporates coordination bonding and charge transfer around the heme iron, successfully reproducing the experimentally observed structure of a CYP-inhibitor complex [Publication Information 1]. Additionally, the fragment molecular orbital (FMO) method was employed to analyze the electronic-structure binding characteristics between CYP and the inhibitor, thereby identifying the amino acid residues critical for inhibitor binding to CYP, as illustrated in the accompanying figure.

In this study [Publication Information 2], in collaboration with researchers in Thailand, various substituents were introduced at the positions indicated by the red circle in the figure to systematically identify candidate compounds with enhanced binding affinity for CYP and improved inhibitory potential. Eleven novel compounds exhibiting favorable drug properties and low toxicity were selected as candidates. Utilizing an advanced molecular simulation method and supercomputing resources, the binding characteristics of CYP with these novel compounds were analyzed. Results demonstrated that two of the novel compounds exhibit stronger binding to CYP than existing inhibitors and are expected to be effective CYP inhibitors.

Mr. Nagura and Miss Chimura, both master's students and lead authors of the two published papers, reflect on their experience: "The CYP protein targeted in this research contains heme iron in its active site; thus, molecular simulation capable of accurately describing the binding state around the iron was essential. However, conventional classical calculation methods could not reproduce the experimental structure, and we encountered significant challenges in developing a method that accurately accounted for the coordination bonds and charge distribution around iron. Furthermore, when analyzing the binding properties between various compounds and CYP using the new method, we benefited greatly from the advice of the professor and students at our joint research laboratory in Thailand, which enabled us to search for new compounds more efficiently. I believe that spending two months at the research laboratory in Thailand and engaging in collaborative research through direct discussions contributed substantially to the success of this project."

The novel molecular simulation method proposed in this paper will be applied to additional enzyme proteins to identify new compounds that effectively inhibit their function. These compounds will be synthesized in a laboratory in Thailand, and their inhibitory effects will be evaluated through cell-based experiments. To expedite this collaborative research, Associate Professor Kurita and the laboratory's graduate students will visit the Thai laboratory to develop a detailed research implementation plan. Collaborative research and student exchanges with the Thai laboratory have been ongoing for over 10 years. These exchanges are expected to continue, contributing to the development of new tuberculosis therapies.

This research was conducted through the International Internship Program of the Japan Student Services Organization (JASSO) and the Student Exchange and Research Exchange Program between Toyohashi University of Technology and Ubon Ratchathani University in Thailand. We thank Associate Professor Pornpan Pungpo of the Department of Chemistry, Faculty of Science, Ubon Ratchathani University, and the graduate students in her laboratory for their valuable contributions to this collaborative research. Additionally, part of this research was carried out as an activity of the FMO Drug Discovery Consortium, and the computational results were obtained using the supercomputer Fugaku of RIKEN in Japan (Project Number: hp250154).