Supramolecular modification of isoniazid : an in vitro and in silico analysis of combination drug efficacy against mycobacteria species

Abstract

Tuberculosis was for the longest of time a leading infectious killer instigated by a single pathogen until the emergence of SARS-CoV-2. The disease continues to be a major global health problem causing high rates of morbidity and mortality. Unfortunately, both the estimated disease incidence and mortality related to tuberculosis infections have been reported to be on the rise as treatment of the disease has been neglected due to a shift in attention to address the COVID-19 global pandemic. The remnants thereof still pose a direct negative impact on tuberculosis diagnosis and treatment, resulting in the partial reversal of any progress that had been made in the quest to end tuberculosis prior to the global COVID-19 pandemic. It is presumed that the challenges around tuberculosis treatment and misdiagnosis are as a result of the continuous ability of the pathogen to evade the host’s immune system and development of resistance against tuberculosis drugs. However, diagnosis seems to be the weakest link contributing to delayed treatment thereof. If detected early, tuberculosis can be cured. However, if tuberculosis is not detected, it definitely cannot be treated; and, if it cannot be treated, it certainly cannot be eradicated. In addition, there is also an emergence of nontuberculous mycobacteria which are caused by mycobacteria species other than those belonging to the Mycobacterium tuberculosis complex. Some of these organisms cause pulmonary infections indistinguishable to those caused by M. tuberculosis infections, presenting with pulmonary and extrapulmonary tuberculosis-like diseases. The prevalence of these diseases has steadily gained traction in recent years, reported and isolated from clinical isolates around the world. Yet, these mycobacteria are not targeted when screening for tuberculosis in clinical isolates. This is a public health problem as nontuberculous mycobacteria are generally resistant to the tuberculosis treatment regimen used in screening assays and if not screened, they can easily be misdiagnosed as Mycobacterium tuberculosis. If there are no obvious ways in which these organisms can be distinguished during diagnosis, it would be beneficial if drugs that are designed could target both Mycobacterium tuberculosis and nontuberculous mycobacteria as nontuberculous mycobacteria are not susceptible to first- and second-line tuberculosis treatment regimens. The aim of this study was to design and synthesise isoniazid derivatives that are effective against clinically infectious Mycobacterium tuberculosis as well as nontuberculous mycobacteria isolates. To achieve this, methods such as the one-pot reflux method were employed for the synthesis of the isoniazid derivative. Crystallographic data were collected using the Bruker APEX II CCD area detector diffractometer. Data were reduced and corrected using the SAINT-Plus software and the SADABS software, respectively. Diagrams were generated using WinGX and PLATON software. To test for the efficacy of the derivatives against mycobacteria of choice (M. tuberculosis H37Ra, M. bovis BCG, M. smegmatis, M. avium and M. fortuitum) in comparison to the efficacy of isoniazid and that of rifampicin, a number of disciplines with varying techniques were employed and these include microbiology, where minimum inhibitory concentration experiments were conducted, and cell biology where cell viability was assessed using real-time cell analysis techniques and flow cytometry analysis conducted using the xCELLigence RTCA system and the BD FACSArai™ III cell sorter, respectively. The potential safety of these compounds was also assessed on RAW 264.7 murine macrophage cells where methods such as the MTT assay and real-time cell analysis assay as well as flow cytometry assay (Muse® Cell Analyzer) were employed to conduct cell viability assessment. Lastly, molecular docking and modelling experiments were conducted to determine the target protein and binding of the ligands thereof (protein-ligand binding). Unit cells were collected for all nineteen synthesised derivatives, where nine have been previously synthesised and ten were found to be novel. Of the ten, six were refined successfully and five of those published in peer-reviewed journals. All derivatives were screened against mycobacteria of choice and only a few were found to have inhibitory effects against some of the mycobacteria with some noteworthy concentrations varying between 0.25 μg/mL and 11.36 μg/mL as single compounds, and 0.09 μg/mL and 0.59μg/mL for combination drug treatments. Isoniazid was comparable against single and combination derivatives with concentrations ranging between 3.9 μg/mL and 22.73 μg/mL, and rifampicin gave competitive results at concentrations ranging between 0.16 μg/mL and 15.65 μg/mL against single and derivatives. In most cases, the derivatives were observed to cause direct apoptotic effects on mycobacterial cells at competitive percentages of between 59% and 90%, thus better than the apoptosis percentage induced by isoniazid and rifampicin of less than 10%. Nonetheless, the related apoptosis results were in accordance with RTCA and flow cytometry results that saw most derivatives posing less toxicity against murine macrophage cells and the ability of the said cells to recover as compared to when exposed to isoniazid and rifampicin. The molecular modelling results also saw a few molecules and combinations possessing exceptional binding abilities with the catalase-peroxidase enzyme with no degree of displacement and good drug residence time, thus suggestive of some derivatives being effective mycobacteria inhibitors targeting the catalase-peroxidase enzyme with docking scores ranging from -4.879 kcal/mol, -5.390 kcal/mol, to -6.065 kcal/mol.