High-throughput screening of compounds library to identify novel inhibitors against latent Mycobacterium tuberculosis using streptomycin-dependent Mycobacterium tuberculosis 18b strain as a model

Highlights

• Screening of 30,000 chemDiv library identified seven novel compounds having activity against SS18b.

• All compounds were non-toxic on HepG2 cell line. Finally compounds 8002–7516 and 3013–0377 selected for further evaluation.

• Compound 8002–7516 found to be the most promising that could be optimized to develop as a lead drug candidate.

Abstract

One of the significant challenges to treat tuberculosis is the phenotypic resistance adapted by the latent or dormant Mycobacterium tuberculosis (M. tuberculosis) cells against most of the available drugs. Different in-vitro assay such as oxygen depletion model and nutrient starvation models have contributed to unravelling the pathogen phenotypic resistance but are too cumbersome for application to high-throughput screening (HTS) assays. In this context, non-replicating streptomycin-starved 18b (SS18b) mutant strain of M. tuberculosis provided a simple and reproducible model. This model mimics latent tuberculosis and is best suited for screening medicinally appropriate libraries. Using SS18b strain in a resazurin reduction microplate assay (REMA), high-throughput screening of ChemDiv library constituting of 30,000 compounds resulted in the identification of 470 active compounds. Clustering and scaffolding based medicinal chemistry analysis characterized these hits into 15 scaffolds. Seven most potent compounds exhibiting an MIC ≤ 1 μg/ml against SS18b were non-toxic in HepG2 cell line (selective Index ≥ 160). Our screening revealed seven novel compounds exhibiting activity against the non-replicating form of M tuberculosis. 8002–7516 was the most promising compound showing intracellular killing and could be optimized to develop a lead drug candidate.

Introduction

Tuberculosis is one of the top 10 causes of death and the leading cause of a single infectious agent. According to the global tuberculosis report 2018 by World Health Organization (WHO) an estimated 1.3 million deaths were caused by TB alone, with an additional 300, 000 deaths in association with HIV in 2017. Globally, 10.0 million people developed TB disease in 2017, 558,000 people developed TB resistant to the most effective first-line anti-tuberculosis drug, rifampicin (RR-TB), where 82% of the population had multidrug-resistant TB (MDR-TB) in the year 2017. China accounts for almost half of the world's population cases of MDR/RR-TB (13%), India (24%), and the Russian Federation (10%) alone [1].

From the past four decades, the therapy to treat TB has remained unchanged. Currently used directly observed therapy short-course (DOTS) involving a combination of four drugs (rifampicin, isoniazid, pyrazinamide, and ethambutol) for treating TB is required for two months, followed by rifampin and isoniazid for a period of 4 months [2]. The crisis is further convoluted by multidrug-resistant MDR and extensively drug-resistant XDR-TB strains that have become resistant to common antibiotics due to poor adherence to such a lengthy treatment [3,4]. Several drug discovery programs have initiated worldwide, aiming to find therapeutic agents to overcome such phenotypic resistant pathogens [5] either by replacing or complementing already existing DOTS. Once the pathogen encounters the host, it has to cope with the ability of the pathogen to enter the latent phase [6,7]. An estimated 1.7 billion people that contribute 23% of the world population have latent TB infection are thus at the risk of developing active TB disease during their lifetime.

The pathogen achieves the dormant phase as a result of complex host-pathogen interactions [8,9]. The pathogen encounters various stresses inside the host; these stresses have been mimicked in vitro by different models, like, the oxygen depletion model and nutrient starvation model. Nitric oxide treatment and acidic medium represent two additional stresses that M. tuberculosis has to counter during infection. Such models either alone or in combination [[10], [11], [12], [13], [14], [15]] have contributed to unravelling the pathogen phenotypic resistance, or so there role cannot be neglected. Still, they are too tedious for application to HTS assays. Hence, a candidate that is simple, reproducible, and easy to mimic in-vitro, as well as in-vivo conditions, was required. In this context, SS18b was used to mimic dormant bacteria. This strain was isolated in Japan in 1955 from a patient diagnosed with streptomycin-resistant tuberculosis. The phenotype is recognized as streptomycin dependent, as the microorganism was unable to grow in absence of streptomycin. Additionally, when maintained in streptomycin media, it did not lose viability for several weeks and grew again upon addition of streptomycin [16]. Molecular characterization revealed novel mutation in rrs gene that encodes for 16S rRNA to be responsible for a streptomycin-dependent mutant of Mycobacterium tuberculosis. Insertion of an additional cytosine in the 530 loop region of 16S rRNA may have streptomycin dependence. The presence of streptomycin presumably stabilizes the interaction in a way that leads to normal functioning of the ribosome in such strain [17]. In the present study, ChemDiv library of 30,000 drugs like compounds procured from ChemDiv library was screened to find new lead compounds using HTS assay against non-replicating SS18b strain of M. tuberculosis. Compounds with promising MICs were further investigated.