AS-silencing of Mtb targets under replicating in-vitro growth conditions.

<p>The survival kinetics of target AS-recombinants of Mtb enumerated up to 63-days = almost ~70 generations; are shown here as log₁₀ cfu/ml <i>vs</i>. the vector control. Under replicating growth condition, <i>ilvB</i> demonstrated the maximum AS-repression among different magnitude of cidality in comparison to <i>ppk</i>. The order of cidality (log₁₀ cfu/ml) was <i>ilvB</i> (5.4)<i>> ppk</i>(4.8)<i>> rpoC</i>(3.5)<i>> rpoB</i>(2.5)<i>> aroK</i>(2.2).</p

Unravelling the Secrets of Mycobacterial Cidality through the Lens of Antisense

<div><p>One of the major impediments in anti-tubercular drug discovery is the lack of a robust grammar that governs the in-vitro to the in-vivo translation of efficacy. <i>Mycobacterium tuberculosis</i> (Mtb) is capable of growing both extracellular as well as intracellular; encountering various hostile conditions like acidic milieu, free radicals, starvation, oxygen deprivation, and immune effector mechanisms. Unique survival strategies of Mtb have prompted researchers to develop in-vitro equivalents to simulate in-vivo physiologies and exploited to find efficacious inhibitors against various phenotypes. Conventionally, the inhibitors are screened on Mtb under the conditions that are unrelated to the in-vivo disease environments. The present study was aimed to (1). Investigate cidality of Mtb targets using a non-chemical inhibitor antisense-RNA (AS-RNA) under in-vivo simulated in-vitro conditions.(2). Confirm the cidality of the targets under in-vivo in experimental tuberculosis. (3). Correlate in-vitro <i>vs</i>. in-vivo cidality data to identify the in-vitro condition that best predicts in-vivo cidality potential of the targets. Using cidality as a metric for efficacy, and AS-RNA as a target-specific inhibitor, we delineated the cidality potential of five target genes under six different physiological conditions (replicating, hypoxia, low pH, nutrient starvation, nitrogen depletion, and nitric oxide).In-vitro cidality confirmed in experimental tuberculosis in BALB/c mice using the AS-RNA allowed us to identify cidal targets in the rank order of <i>rpoB>aroK>ppk>rpoC>ilvB</i>. <i>RpoB</i> was used as the cidality control. In-vitro and in-vivo studies feature <i>aroK</i> (encoding shikimate kinase) as an in-vivo mycobactericidal target suitable for anti-TB drug discovery. In-vitro to in-vivo cidality correlations suggested the low pH (R = 0.9856) in-vitro model as best predictor of in-vivo cidality; however, similar correlation studies in pathologically relevant (Kramnik) mice are warranted. In the acute infection phase for the high fidelity translation, the compound efficacy may also be evaluated in the low pH, in addition to the standard replication condition.</p></div

Cidality of Mtb targets by AS-silencing in lung infection in mice.

<p>(A). Overall survival kinetics of Mtb AS-recombinants and the WT and vector controls in the lungs of mice (n = 3) on the days-3, 7, 14, 28, 42, 56. The control strains showed expected course of infection in the lungs; there was no difference in both these strains (WT and vector, ns, P = 0.3105, two-way ANOVA). The treatment control (Rifampicin treatment of WT Mtb) showed expected cidality pattern of ~3.8 log₁₀ cfu reduction, represented by the turquoise and brown chequered bars on day-3 and day-28. Target <i>ilvB</i> (however, significantly different from control, *P = 0.0402, two-way ANOVA) was non-cidal in-vivo. The graph is a plot of log₁₀ cfu/lung of mice <i>vs</i>. number of days. The cidality emerged in the rank order of <i>rpoB>aroK>ppk>rpoC</i>. Data was statistically significant (**P = 0.0086 to *P = 0.0402, two-way ANOVA) from 14 day onwards. (B). Lung pictures on the day 28 (4^thweek), visually demonstrate in-vivo cidality by a clearing of infection in the lungs with the healing of granuloma due to the killing of the respective AS-recombinant of Mtb in the order of <i>rpoB>aroK>rpoC>ppk</i>; correlating with the cfu data outcome (panel <b>C</b>). The maximum healing was visible in the rifampicin treated lungs, correlating with the cidality shown in the cfu histogram. The control strains (WT and V) demonstrated the fully formed visible granulomas in the lungs. (C). Final histograms of AS-based cidality on Day-56 (8^th week) graphs. A statistically significant robust data with error bars (SEM) from triplicates (n = 3), shows the cidality pattern in the order of <i>rpoB</i>(3.9)> <i>aroK</i>(2.4)<i>> ppk</i>(1.6)<i>> rpoC</i>(1.59)><i>ilvB</i>(0.36).</p

TB or No TB: A delicate balance between host-pathogen interactions.

<p>There are various hostile or stringent conditions encountered by Mtb in-vivo in the host. Mtb enters the host via inhalation. The first encounter in-vivo is with the immune cells: the macrophages which phagocytose and attack Mtb with their ammunition like low pH, hydrolases, free-radicals, etc. This first encounter is unanimously responded by all the hosts irrespective of their immune status. Beyond this point, the countering of the pathogen is host population specific. The outcome as TB or no-TB is a delicate balance and is outcome result of the battles between the pathogen and the immuno-competence of the host to outsmart. Further, it progresses into a larger immune structure: the granuloma. During the process, the other stresses in the granuloma, especially in the caseating/ necrotizing lesions are the gradually decreasing levels of O₂, N, C, etc. representing hypoxia, the arrest of various biosynthesis processes and poor nutrition nearly close to starvation. If the host wins, the pathogen is contained in the solid granuloma, which may gradually heal with time. But in case the pathogen Mtb overpowers the immune pressure, the granuloma progresses as a caseating and necrotising granuloma. In this case Mtb bacilli multiply to finally break open from granuloma, and disseminate to other organs of the body or come out into the bronchi and get coughed out to infect other hosts. We attempted to simulate some of these stress conditions in the form of various in-vitro screens to identify the ideal in-vitro screen/ condition.</p

Maximum fold target repression during the course of infection.

<p>The data validates IPTG-inducible in-vivo AS-repression of Mtb targets. The net target transcript levels, as evaluated by RTPCR of Mtb from lung homogenates; showed a variable -fold down regulation (13- to 103-fold), during the entire course of in-vivo studies. The maximum fold repression of targets equated that the in-vivo transcript translation into cidality is target-vulnerability-dependent. Target <i>rpoB</i> translated into maximum cidality of 3.9 log₁₀ cfu reduction with mere 13-fold transcript level repression; whereas, in the case of <i>ppk</i>, only 1.3 log₁₀ cfu reduction could be achieved in-vivo despite a maximum of 103-fold transcript repression (as in Table C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154513#pone.0154513.s002" target="_blank">S1 File</a>).</p

Survival kinetics of AS-silenced Mtb under different in-vitro physiologies.

<p>Survival kinetics of AS-recombinants: (A) <i>rpoB</i>, (B) <i>rpoC</i>, (C) <i>ppk</i>, (D) <i>aroK</i>, and (E) <i>ilvB</i>; under granuloma simulated (F), replicating and non-replicating (dormant) in-vitro growth conditions, showed target vulnerability specific cidality. Various conditions tested: Replicating (REP) growth condition as solid circles, Nutrient starvation (NSM) as solid square, Hypoxia (Hpx) as solid vertical triangle, Nitric oxide (NO) model as solid inverted triangle, Low pH (LpH) as bottom solid square, Nitrogen depletion (Msx) condition as the left solid square. The graph represents plots of log₁₀ cfu/ml <i>vs</i>. no. of days, studied up to 35 days. V and WT are plotted with all the genes as controls for a comparison. All the symbols have been kept uniform for the respective assay conditions throughout. WT is RED; Vector is BLUE, and the gene-specific AS-recombinants of Mtb are in GREEN colour. The error bars (SEM) from independent triplicates represent the robustness of data. The data for WT and V are common in all the graphs, for an easy comparison with the green ones- the gene specific. Although Mtb can withstand and emerge successfully from various physiological pressures encountered in-host; a target is superior if, it is bactericidal under all or most of those physiological constraints upon specific inhibition. The targets were significantly (P<0.001) silenced with different cidality magnitudes: <i>ppk</i> (7.5)><i>ilvB</i> (7.3)><i>rpoB</i> (7.1)><i>rpoC</i> (5.2)><i>aroK</i> (3.2).</p

Cidality SCORE of Mtb targets by in-vitro AS-silencing.

<p>The normal or stringent physiological conditions used are: Msx = Nitrogen depletion, LpH = low pH, NO2 = Nitric oxide, NSM = Nutrient Starvation Model, Hpx = Hypoxia, REP = logarithmically replicating condition. The graph depicts the net compounded effect, the Cidality SCORE, of respective genes under various physiological conditions (a total of) as the inhibition on the upper scale, and growth on the lower scale. It represents the behaviour of the respective target under a diseased situation. WT = Wild-type Mtb strain, V = WT strain of Mtb containing the blank vector. The rest are all the gene-specific recombinants of Mtb. The blue colour boxes show the cidality SCORE representing the overall cidality potential of a target based on the AS-RNA gene silencing magnitudes as <i>ppk</i>(7.5)><i>ilvB</i>(7.3)><i>rpoB</i>(7.1)><i>rpoC</i>(5.2)><i>pyrH</i>(3.6)><i>aroK</i>(3.2). Statistically significant (***), the error bars (SEM) represent the robustness of data from the triplicates.</p

Biarylmethoxy Nicotinamides As Novel and Specific Inhibitors of <i>Mycobacterium tuberculosis</i>

A whole cell based screening effort on a focused library from corporate collection resulted in the identification of biarylmethoxy nicotinamides as novel inhibitors of <i>M. tuberculosis</i> (Mtu) H37Rv. The series exhibited tangible structure–activity relationships, and during hit to lead exploration, a cellular potency of 100 nM was achieved, which is an improvement of >200-fold from the starting point. The series is very specific to Mtu and noncytotoxic up to 250 μM as measured in the mammalian cell line THP-1 based cytotoxicity assay. This compound class retains its potency on several drug sensitive and single drug resistant clinical isolates, which indicate that the compounds could be acting through a novel mode of action

2‑Phenylindole and Arylsulphonamide: Novel Scaffolds Bactericidal against <i>Mycobacterium tuberculosis</i>

A cellular activity-based screen on <i>Mycobacterium tuberculosis</i> (Mtb) H37Rv using a focused library from the AstraZeneca corporate collection led to the identification of 2-phenylindoles and arylsulphonamides, novel antimycobacterial scaffolds. Both the series were bactericidal <i>in vitro</i> and in an intracellular macrophage infection model, active against drug sensitive and drug resistant Mtb clinical isolates, and specific to mycobacteria. The scaffolds showed promising structure–activity relationships; compounds with submicromolar cellular potency were identified during the hit to lead exploration. Furthermore, compounds from both scaffolds were tested for inhibition of known target enzymes or pathways of antimycobacterial drugs including InhA, RNA polymerase, DprE1, topoisomerases, protein synthesis, and oxidative-phosphorylation. Compounds did not inhibit any of the targets suggesting the potential of a possible novel mode of action(s). Hence, both scaffolds provide the opportunity to be developed further as leads and tool compounds to uncover novel mechanisms for tuberculosis drug discovery

Pyrazolopyrimidines Establish MurC as a Vulnerable Target in <i>Pseudomonas aeruginosa</i> and <i>Escherichia coli</i>

The bacterial peptidoglycan biosynthesis pathway provides multiple targets for antibacterials, as proven by the clinical success of β-lactam and glycopeptide classes of antibiotics. The Mur ligases play an essential role in the biosynthesis of the peptidoglycan building block, <i>N</i>-acetyl-muramic acid-pentapeptide. MurC, the first of four Mur ligases, ligates l-alanine to UDP-<i>N</i>-acetylmuramic acid, initiating the synthesis of pentapeptide precursor. Therefore, inhibiting the MurC enzyme should result in bacterial cell death. Herein, we report a novel class of pyrazolopyrimidines with subnanomolar potency against both <i>Escherichia coli</i> and <i>Pseudomonas aeruginosa</i> MurC enzymes, which demonstrates a concomitant bactericidal activity against efflux-deficient strains. Radio-labeled precursor incorporation showed these compounds selectively inhibited peptidoglycan biosynthesis, and genetic studies confirmed the target of pyrazolopyrimidines to be MurC. In the presence of permeability enhancers such as colistin, pyrazolopyrimidines exhibited low micromolar MIC against the wild-type bacteria, thereby, indicating permeability and efflux as major challenges for this chemical series. Our studies provide biochemical and genetic evidence to support the essentiality of MurC and serve to validate the attractiveness of target for antibacterial discovery