A novel class of antimicrobial drugs selectively targets a Mycobacterium tuberculosis PE-PGRS protein
Hoonhee Seo, Sukyung Kim, Hafij Al Mahmud, MdImtiazul Islam, Youjin Yoon, Hyun-Deuk Cho, Kung-Woo Nam, Jiwon Choi, Young Sig Gil, Byung-Eui Lee, Ho-Yeon Song
Abstract
The continued spread of drug-resistant tuberculosis is one of the most pressing and complex challenges facing tuberculosis management worldwide. Therefore, developing a new class of drugs is necessary and urgently needed to cope with the increasing threat of drug-resistant tuberculosis. This study aims to discover a potential new class of tuberculosis drug candidates different from existing tuberculosis drugs. By screening a library of compounds, methyl (S)-1-((3-alkoxy-6,7-dimethoxyphenanthren-9-yl)methyl)-5-oxopyrrolidine-2-carboxylate (PP) derivatives with antitubercular activity were discovered. MIC ranges for PP1S, PP2S, and PP3S against clinically isolated drug-resistant Mycobacterium tuberculosis strains were 0.78 to 3.13, 0.19 to 1.56, and 0.78 to 6.25 μg/ml, respectively. PPs demonstrated antitubercular activities in macrophage and tuberculosis mouse models, showing no detectable toxicity in all assays tested. PPs specifically inhibited M. tuberculosis without significantly changing the intestinal microbiome in mice. Mutants selected in vitro suggest that the drug targets the PE-PGRS57, which has been found only in the genomes of the M. tuberculosis complex, highlighting the specificity and safety potency of this compound. As PPs show an excellent safety profile and highly selective toxicity specific to M. tuberculosis, PPs are considered a promising new candidate for the treatment of drug-resistant tuberculosis while maintaining microbiome homeostasis.
Introduction
Although it has been nearly 70 years since the first clinical trial of isoniazid (INH) for human tuberculosis [1], this disease remains a significant health concern worldwide. It is one of the leading causes of death from infectious diseases, ranking even above AIDS [2]. One-third of the world’s population (i.e., approximately 2 to 3 billion people) are infected with Mycobacterium tuberculosis, and approximately 7 million people are treated for active tuberculosis, with 1.5 million deaths annually [3]. The rise in people with AIDS worldwide has further complicated this issue [4]. To make matters worse, the emergence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and now totally drug-resistant (TDR) strains of M. tuberculosis necessitates the development of new drugs to control this epidemic more urgently than ever [5].
Methods
Synthesis of PPs derivatives and analogues
According to a published method [23], 3, 6, 7-trimethoxy-9-phenanthrenemethanol was synthesized using homoveratric acid and 4-methoxybenzaldehyde as starting materials (S1 Scheme). (S)-N-[(2, 3, 6-trimethoxy-10-phenanthryl)methyl]-pyroglutamic acid methyl ester (PP1S) was prepared with a reported method [24] as shown in S1 Scheme from compound 3, 6, 7-trimethoxy-9-phenanthrenemethanol in situ. Compound PP2S was prepared using the same procedures and materials as PP1S except that homoveratric acid and 4-(n-butyloxy)benzaldehyde were used as starting materials. Compound PP3S and PP3R were synthesized as previously described [25]. PP analogues were synthesized (S2 Scheme). All new compounds were characterized by 1H-NMR and mass spectroscopy. Their purities were measured by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis. Enantiomers were identified with a circular dichroism detector (Chirascan plus, Applied Photophysics, United Kingdom) (S1 Fig). Full details of chemical synthesis for compounds described in this paper are shown in Supporting information (S1 Supporting Chemistry Schemes).
Results
In vitro antitubercular activities of PPs
As a result of the resazurinmicrotiter assay for 11 types of DPG analogs, DPGA1 (PP1S) had the lowest MIC and showed no cytotoxicity within the tested concentration range (S1 Table). Next, derivatives of PP1S were synthesized, and further in vitro evaluation was performed on them.
Structures of PPs and their in vitro antitubercular effects are shown in Fig 1. PPs are composed of PP1S, PP2S, and PP3S, in which the alkoxy R group has methyl, butyl, and benzyl group, respectively (Fig 1A). PPs have a chiral center. With an S-form, PP1S, PP2S, and PP3S had in vitro antitubercular activities, whereas PP1R, PP2R, and PP3R with an R-form did not (S2 Table). Testing antitubercular effects of PPs against M. tuberculosis H37Rv with the resazurinmicrotiter assay showed that MICs of PP1S and PP3S were the same at 1.6 μg/ml, while the MIC of PP2S was lower at 0.4 μg/ml (Fig 1B). Effects of PPs on XDR M. tuberculosis were confirmed through colony-forming unit (CFU) enumeration assay (Fig 1C). PPs at both 0.8 μg/ml and 1.6 μg/ml showed significant effects. At 0.8 μg/ml, PP1S, PP2S, and PP3S showed log reductions of 0.8, 1.8, and 0.5, respectively. At 1.6 μg/ml, they showed log reductions of 1.3, 1.9, and 0.6, respectively. However, INH and rifampicin (RIF) showed log reductions of 1.0 and 0.6, respectively, at 12.5 μg/ml, a much higher concentration than PPs.
Discussion
As a pathogenic organism, M. tuberculosis continues to evolve resistance to frontline drugs, leading to the emergence of MDR, XDR, and ultimately TDR M. tuberculosis [13]. Accordingly, the development of a new class of antituberculosis agents is urgently needed. Here, we report antituberculosis effects of PPs, a new class of drugs utterly different from existing tuberculosis medications structurally. PPs are derivatives of DPGA1, one of the DPG analogues (DPGA) discovered during the study to improve the antitubercular activity and toxicity issue of DPG (S1 Supporting Chemistry Schemes). In terms of structure–activity relationship (SAR), when the hexahydro-indolizine structure of DPG was substituted with methyl-5-oxopyrrolidine-2-carboxylate, antituberculous activity was increased but the toxicity was lowered. On the other hand, substitution with a methoxycarbonyl group or a hydroxymethyl group did not increase its antitubercular activity. It has been confirmed that the cleaved form of phenanthrene compounds have antitubercular activities, and it is worth conducting further studies. Among PPs having 3 alkoxy groups, PP2S, in which the alkoxy substituent is a butyl group, showed a better in vitro antitubercular effect. From this observation, it was clear that the substitution of the alkoxy group on carbon 3 of the phenanthromethyl group was related to the antitubercular effect. Unlike S-form, R-form had no antitubercular effect, confirming that the chirality of PPs was also an essential factor in the antitubercular effect. In particular, PP2S has not been reported in the existing literature. Further studies on its stability in blood and activation process/activation form are needed.
Acknowledgments
We wish to thank all participants of this study.
Citation: Seo H, Kim S, Mahmud HA, Islam MI, Yoon Y, Cho H-D, et al. (2022) A novel class of antimicrobial drugs selectively targets a Mycobacterium tuberculosis PE-PGRS protein. PLoSBiol 20(5): e3001648. https://doi.org/10.1371/journal.pbio.3001648
Academic Editor: Matthew K. Waldor, Brigham and Women’s Hospital, UNITED STATES
Received: December 1, 2021; Accepted: April 26, 2022; Published: May 31, 2022
Copyright: © 2022 Seo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All metadata files are available from the National Center for Biotechnology Information (accession number(s) PRJNA787746). https://www.ncbi.nlm.nih.gov/sra/?term=Composition%20of%20gut%20microbiota%20changed%20with%20drug%20treatment.
Funding: This research was supported by a grant (HI13C0828) from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare, Republic of Korea. It was also supported by Soonchunhyang University Research Fund. H-Y S received all the funding described. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: BCG, bacillus Calmette–Guérin; BLAST, Basic Local Alignment Search Tool; CLSI, Clinical and Laboratory Standards Institute; DPG, deoxypergularinine; EMB, ethambutol; GFP, green fluorescent protein; HE, hematoxylin–eosin; HPLC, high-performance liquid chromatography; INH, isoniazid; MDR, multidrug-resistant; MOI, multiplicity of infection; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible Staphylococcus aureus; NCBI, National Center for Biotechnology Information; NTM, nontuberculosis mycobacteria; PERMANOVA, permutational multivariate analysis of variance; PP, methyl (S)-1-((3-alkoxy-6,7-dimethoxyphenanthren-9-yl)methyl)-5-oxopyrrolidine-2-carboxylate; PP1S, (S)-N-[(2,3,6-Trimethoxy-10-phenanthryl)methyl]-pyroglutamic acid methyl ester; PP2S, Methyl (S)-1-((3-butoxy-6,7-dimethoxyphenanthren-9-yl)methyl)-5-oxopyrrolidine-2-carboxylate; PP3S, Methyl (S)-1-{[3-(Benzyloxy)-6,7-dimethoxy-9-phenanthryl]methyl}-5-oxopyrrolidine-2-carboxylate; PP3R, Methyl (R)-1-{[3-(Benzyloxy)-6,7-dimethoxy-9-phenanthryl]methyl}-5-oxopyrrolidine-2-carboxylate; PZA, pyrazinamide; RIF, rifampicin; SAR, structure–activity relationship; SD, Sprague Dawley; STR, streptomycin; TDR, totally drug-resistant; TLC, thin-layer chromatography; VAN, vancomycin; XDR, extensively drug-resistant
https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001648#abstract0
