In vitro potency of 2-(((2-hydroxyphenyl)amino)methylene)-5,5-dimethylcyclohexane-1,3-dione against drug-resistant and non-replicating persisters of Mycobacterium tuberculosis.

OBJECTIVES: New antituberculosis agents active against drug-resistant and non-replicating tubercle bacilli are required. We evaluated a previously identified hit, 2-(((2-hydroxyphenyl)amino)methylene)-5,5-dimethylcyclohexane-1,3-dione (PAMCHD), against several clinical Mycobacterium tuberculosis isolates, including multidrug-resistant (MDR) strains and non-replicating drug-tolerant persisters of M. tuberculosis H37Rv. METHODS: PAMCHD's potential against drug-resistant M. tuberculosis was investigated by broth microdilution. CFU enumeration was performed to determine PAMCHD's activity against five types of dormant bacilli. RESULTS: No significant differences in MICs of PAMCHD were observed against M. tuberculosis H37Rv (2.5-5 µg/mL) and eight drug-susceptible strains (1.25-5 µg/mL) as well as drug-resistant strains including six isoniazid (INH)-resistant (2.5-10 µg/mL), one INH + ethambutol (EMB)-resistant (5 µg/mL), one rifampicin (RIF) + EMB-resistant (5 µg/mL) and three MDR (2.5-10 µg/mL) strains. Thus, PAMCHD maintains activity against all kinds of clinical strains, especially MDR. Regarding drug-tolerant persisters, INH and RIF killed, respectively, 0.5 and 5.0 log10 CFU of non-replicating persisters developed by hypoxia and 1.5 and 2.5 log10 CFU developed by nutrient starvation at 64 × of their respective MIC against actively dividing cultures. In contrast, PAMCHD sterilised persister cultures developed by hypoxia (killed 6.5 log10 CFU) or starvation (killed 7.5 log10 CFU). PAMCHD sterilised RIF-tolerant (tolerance level up to 100 µg/mL of RIF) 100-day-old static persisters at 64 × MIC, while moxifloxacin killed only 1.0 log10 CFU of these persisters at 64 × MIC. CONCLUSION: PAMCHD offers significant potential against MDR-TB and exhibits notable potency against non-replicating drug-tolerant M. tuberculosis persisters. These findings warrant further studies of PAMCHD for further anti-TB drug development.

In vitro bactericidal activity of 3-cinnamoyl-4-hydroxy-6-methyl-2-pyrone (CHP) against drug-susceptible, drug-resistant and drug-tolerant isolates of Mycobacterium tuberculosis.

OBJECTIVES: Tuberculosis (TB) poses a serious global threat to humans. New bactericidal agents that can shorten treatment duration and target drug resistance still remain a top priority in the discovery of anti-TB drugs. The objective of this study was to investigate the bactericidal potential of 3-cinnamoyl-4-hydroxy-6-methyl-2-pyrone (CHP) against drug-susceptible, drug-resistant clinical isolates and drug-tolerant Mycobacterium tuberculosis. METHODS: The minimum bactericidal concentration (MBC) was determined by colony-forming unit (CFU) enumeration. The kill curve analysis was done at different concentrations spanning over 16 days. Drug combination studies with antituberculosis drugs were done to investigate possible synergy. The potential against drug- resistant isolates of M. tuberculosis was done by broth dilution assay. CFU enumeration was done to determine its activity against nutrient-starved drug tolerants, and its feasibility for oral administration was tested by serum inhibitory titre. RESULTS: CHP displayed bactericidal activity with an MBC of 4 µg/mL against M. tuberculosis H37Rv. The kill curve analysis exhibited a biphasic pattern of killing. CHP showed synergy with rifampicin, isoniazid and amikacin but was indifferent towards ethambutol and levofloxacin. CHP retained its full activity against drug-susceptible, monoresistant and multidrug-resistant (MDR) clinical isolates. CHP showed very strong bactericidal activity against nondividing, drug-tolerant M. tuberculosis that on comparison was highly superior to rifampicin. Furthermore, CHP significantly improved the bactericidal activity of rifampicin and isoniazid in a combination study. The serum inhibitory titre in mice indicated its high oral bioavailability. CONCLUSION: Our results show strong bactericidal potential of CHP against M. tuberculosis that warrant its immediate mechanistic, pharmacokinetic and pharmacodynamic studies.

In vitro evaluation of dinactin, a potent microbial metabolite against Mycobacterium tuberculosis.

Current long duration treatment options and the emergence of drug resistance in tuberculosis (TB) have led to renewed interest in discovery of novel anti-tubercular agents or the scaffolds exhibiting enhanced efficacy with current anti-TB drugs. Herein, dinactin, a potent bioactive macrotetrolide isolated from Streptomyces puniceus AS13, was evaluated against Mycobacterium tuberculosis H37Rv and other susceptible and drug-resistant clinical isolates of M. tuberculosis. In vitro pharmacological assays showed that dinactin is bactericidal against laboratory standard strain M. tuberculosis H37Rv (minimum inhibitory concentration [MIC] 1 µg/mL and minimum bactericidal concentration [MBC] 4 µg/mL). Dinactin also retained its activity against various clinical isolates, including multidrug-resistant strains of M. tuberculosis. Whole cell interaction assays with standard first- and second-line anti-TB drugs showed the synergistic interaction of dinactin with rifampicin or amikacin, reflecting its suitability for use in combination regimens. The killing kinetics studies of dinactin against M. tuberculosis H37Rv revealed that it has strong concentration-dependent anti-TB activity that is also dependent on time. The kill curve also showed dynamic killing capacity of dinactin as it exhibited bactericidal potential at all concentrations tested. Kill curve data demonstrated that dinactin, like isoniazid, exerts its strong tuberculocidal activity within the first two days of exposure. This evidence strongly supports further evaluation of dinactin as a new option in the treatment of TB.

Synthesis and in vitro evaluation of substituted 3-cinnamoyl-4-hydroxy-pyran-2-one (CHP) in pursuit of new potential antituberculosis agents.

Tuberculosis is an ever-evolving infectious disease that urgently needs new drugs. In the search for new antituberculosis agents, a library of 3-cinnamoyl-4-hydroxy-6-methyl-2H-pyran-2-ones (CHPs) (2a-2y) was synthesized and evaluated against a standard virulent laboratory strain of Mycobacterium tuberculosis H37Rv. Out of 25 compounds, 11, 5, 7 and 2 (2a and 2u) showed least, moderate, good and appreciable activities, respectively, based on minimum inhibitory concentrations (MICs). Both 2a and 2u exhibited an MIC value of 4 µg ml-1, which was close to those of standard antituberculosis drugs ethambutol, streptomycin and levofloxacin. Neither 2a nor 2u showed any activity against Gram-positive or Gram-negative bacteria and even against non-tuberculous mycobacterium, i.e. Mycobacterium smegmatis. Thus, like the antituberculosis drugs rifampicin, isoniazid and pretomanid, they are highly TB specific. All the pyrone-based chalcones showed no recognizable level of cytotoxicity against normal human kidney cell line (HEK-293) up to 80 µM concentration and 11 exhibited an IC50 ≤ 100 µM (highest tested concentration). On further investigation, both 2a and 2u proved to be nontoxic against four human cell lines but 2a proved to be a better choice as it did not reach IC50 even at 100 µM (highest tested concentration) while the IC50 of 2u was around 80 µM. In conclusion, our results demonstrate that 2a is specific against M. tuberculosis with no appreciable toxicity; its activity matches that of some clinically approved antituberculosis drugs and it therefore merits further evaluation.

Drug targets exploited in Mycobacterium tuberculosis: Pitfalls and promises on the horizon.

Tuberculosis is an ever evolving infectious disease that still claims about 1.8 million human lives each year around the globe. Although modern chemotherapy has played a pivotal role in combating TB, the increasing emergence of drug-resistant TB aligned with HIV pandemic threaten its control. This highlights both the need to understand how our current drugs work and the need to develop new and more effective drugs. TB drug discovery is revisiting the clinically validated drug targets in Mycobacterium tuberculosis using whole-cell phenotypic assays in search of better therapeutic scaffolds. Herein, we review the promises of current TB drug regimens, major pitfalls faced, key drug targets exploited so far in M. tuberculosis along with the status of newly discovered drugs against drug resistant forms of TB. New antituberculosis regimens that use lesser number of drugs, require shorter duration of treatment, are equally effective against susceptible and resistant forms of disease, have acceptable toxicity profiles and behave friendly with anti-HIV regimens remains top most priority in TB drug discovery.

In vitro antimycobacterial activity of 2-(((2-hydroxyphenyl)amino)methylene)-5,5-dimethylcyclohexane-1,3-dione: a new chemical entity against Mycobacterium tuberculosis.

This study reports on the in vitro antituberculosis potential of 2-(((2-hydroxyphenyl) amino)methylene)-5,5-dimethylcyclohexane-1,3-dione (PAMCHD) against Mycobacterium tuberculosis H37Rv. PAMCHD has been proven to be a tuberculostatic as well as a tuberculocidal agent by agar and broth dilution methods with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values equivalent to some standard antituberculosis drugs (ATDs). The dynamics of M. tuberculosis killing revealed the time- as well as concentration-dependent antituberculosis activity of PAMCHD and it sterilized M. tuberculosis culture at or above 10.0 µg/mL. PAMCHD acts either synergistically or additively with ATDs. Isoniazid (INH) and PAMCHD post-antibiotic effects increased with concentration from 16.18 ± 13.30 and 31.64 ± 13.30 to 127.9 ± 27.60 and 138.71 ± 16.42 h, respectively, from 1 × MIC to 8 × MIC; no significant difference was observed between INH and PAMCHD post-antibiotic effects. M. tuberculosis mutation frequency against PAMCHD is lower than that of INH. Mutant prevention concentration (MPC) of INH, rifampin (RIF) and PAMCHD were observed to be 40, 160 and 160 µg/mL, respectively, and their MPC/MIC values were 128, 2051 and 64, respectively; this lowest MPC/MIC highlights the advantage of PAMCHD over RIF and INH.

Synthesis and in vitro evaluation of 2-(((2-ether)amino)methylene)-dimedone derivatives as potential antimicrobial agents.

The study was designed with an aim to synthesize a series of 2-(((2-ether)amino)methylene)-dimedone derivatives and evaluate the synthesized compounds for antimicrobial activity. Compound library was synthesized by reaction with alkyl, alkenyl, alkynyl and alicyclic bromo-compounds. Characterization of the synthesized compounds was performed by 1H NMR, 13C NMR and mass spectral techniques. The compounds were evaluated for their antibacterial activity against Gram-positive (Staphylococcus aureus, Bacillus subtilis, Clostridium sporogenes) and Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli). The activity of these compounds was also evaluated against fungi (Aspergillus fumigatus, Penicillium chrysogenum, Fusarium oxysporum, Candida albicans) and molds (A. niger and A. oryzae). Broth microdilution method and CLSI guidelines with minor modification were used for the determination of anti-bacterial and antifungal activity, respectively. Although four compounds (4i, 4j, 4k and 4l) showed good antibacterial activity but compound 4k was found to be most active chemotype in the series. Compound 4k was found to be active against S. aureus, B. cereus and B. subtilis bacterial strains at one dilution lower compared to the control ciprofloxacin. Antibacterial activity of compound 4k was comparable to ciprofloxacin against S. pyogenes and M. luteus. The compound 4d, 4e and 4s showed good antifungal and antimold activity compared to other chemotypes. However, in comparison to fluconazole both the compounds showed lower activity. The results merit the antimicrobial promise of the 2-(((2-ether)amino)methylene)-dimedone analogs.

α-pyrones and their hydroxylated analogs as promising scaffolds against Mycobacterium tuberculosis.

Tuberculosis ranks as the leading cause of global human mortality from a single infectious agent. To address the uprising issues of drug resistance, intense research efforts have been directed towards drug discovery. However, it is a long and economically challenging process that is often associated with high failure rates. Therefore, it seems prudent to take forward the core scaffolds that have already acclaimed clinical relevance. In this direction, hydroxylated α-pyrone scaffold has received US FDA approval for human use against HIV. Interestingly, literature review reveals the potential applicability of α-pyrones in TB drug discovery. On one hand, α-pyrones play a vital role in the cell wall of Mycobacterium tuberculosis and on the other hand natural α-pyrones display appreciable anti-TB activity. This review aims to rekindle the interest of researchers toward α-pyrone as a new anti-TB drug that may possibly tackle drug resistance and open a dual frontier in TB and HIV drug discovery.

Design and synthesis of indolopyridone hybrids as new antituberculosis agents.

Tuberculosis continues to be the most dangerous infectious disease globally and need for development of new therapies is of utmost importance. In this study we describe the rationale design for synthesis using molecular hybridization and subsequent in-vitro antimycobacterial activity of various indolo-pyridone hybrid molecules against Mycobacterium tuberculosis H37Rv. A total of 16 indolo-pyridone hybrid molecules were synthesized with 85-90% yields and characterized by various spectroscopic techniques. Four compounds were ineffective with MIC >256 µg/ml (highest concentration tested), six exhibited poor activity with MIC > 100 µg/ml, four showed moderate activity with MIC > 50 µg/ml and two had notable anti-TB activity with MIC values 32 µg/ml. Interestingly the last two compounds were observed equally effective against drug susceptible and various drug resistant strains including multidrug-resistant (MDR) strains, thereby clearly demonstrating their potential against MDR-TB. Our results showed that un-substituted aryl rings posses better antituberculosis activity than those having any kind of substitution and derivatives with small sized electron withdrawing groups in aryl ring exhibited activity while bigger groups lead to considerable loss in activity. The results of this study open up a new door for further SAR guided synthesis on one hand and on the other hand provide a promising opportunity that may lead to the discovery of a new class of antituberculosis agents.

Cell wall: A versatile fountain of drug targets in Mycobacterium tuberculosis.

Tuberculosis is the leading infectious disease responsible for an estimated one and a half million human deaths each year around the globe. HIV-TB coinfection and rapid increase in the emergence of drug resistant forms of TB is a dangerous scenario. This underlines the urgent need for new drugs with novel mechanism of action. A plethora of literature exist that highlight the importance of enzymes involved in the biosynthesis of mycobacterial cell wall responsible for its survival, growth, permeability, virulence and resistance to antibiotics. Therefore, assembly of cell wall components is an attractive target for the development of chemotherapeutics against Mycobacterium tuberculosis. The aim of this review is to highlight novel sets of enzyme inhibitors that disrupt its cell wall biosynthetic pathway. These include the currently approved first and second line drugs, candidates in clinical trials and current structure activity guided endeavors of scientific community to identify new potent inhibitors with least cytotoxicity and better efficacy against emergence of drug resistance till date.

α-pyrones: Small molecules with versatile structural diversity reflected in multiple pharmacological activities-an update.

The investigations in the chemistry and biology of α-pyrone (2-pyrone) are of vital importance as they constitute an essential pharmacophore in many naturally occurring and biologically active synthetic agents. They are a promising class of biorenewable platform chemicals that provide access to an array of chemical products and intermediates. Literature survey reveals that a simple change in the substitution pattern on the 2-pyrone ring system often leads to diverse biological activities. In this review, we present a brief overview of 2-pyrone pharmacophore followed by highlighting their pharmacological properties and potential applicability till date. Particular attention is focused on the distinctive chemotherapeutic activities of 2-pyrones as anti-HIV, anti-TB and anti-cancer agents followed by their potential role against neurodegeneration, hypercholesterolemia, microbial infections, chronic obstructive lung disease, inflammation, antinociception and immunomodulation. Since 2005, when 2-pyrones came in limelight, their detailed pharmacological activities have been well documented. This review has mainly been prepared on the basis of original reports published in recent two decades with an aim to attract the attention of researchers towards this versatile scaffold for future endeavors that may lead to the development of potential drug candidates against above diseases.

The synthesis, biological evaluation and structure-activity relationship of 2-phenylaminomethylene-cyclohexane-1,3-diones as specific anti-tuberculosis agents.

The present study utilised whole cell based phenotypic screening of thousands of diverse small molecules against Mycobacterium tuberculosis H37Rv (M. tuberculosis) and identified the cyclohexane-1,3-dione-based structures 5 and 6 as hits. The selected hit molecules were used for further synthesis and a library of 37 compounds under four families was synthesized for lead generation. Evaluation of the library against M. tuberculosis lead to the identification of three lead antituberculosis agents (37, 39 and 41). The most potential compound, 2-(((2-hydroxyphenyl)amino)methylene)-5,5-dimethylcyclohexane-1,3-dione (39) showed an MIC of 2.5 µg mL-1, which falls in the range of MICs values found for the known antituberculosis drugs ethambutol, streptomycin and levofloxacin. Additionally, this compound proved to be non-toxic (<20% inhibition at 50 µM concentration) against four human cell lines. Like first line antituberculosis drugs (isoniazid, rifampicin and pyrazinamide) this compound lacks activity against general Gram positive and Gram negative bacteria and even against M. smegmatis; thereby reflecting its highly specific antituberculosis activity.

Comparative analysis of bioactive N-alkylamides produced by tissue culture raised versus field plantlets of Spilanthes ciliata using LC-Q-TOF (HRMS).

Spilanthes ciliata (S. ciliata) is a perennial herb of global importance owing to its luscious source of bioactive fatty acid derived amides known as N-alkylamides. It finds application in skin creams, mouth gels and toothpastes. Despite multifaceted applications, a major limitation associated for its commercial application is the scarcity of contamination free plant source, fluctuations in active metabolites due to variation in extraction procedures, and lack of rapid qualitative method for alkylamide profiling. In the current work, attempts were made to 1) optimize conditions for mass propagation of contamination free plants of S. ciliata by tissue culture using leaf discs as explants, 2) establish an optimum extraction ratio of plant/solvent (w/v) for maximum elution of alkylamides and 3) develop a rapid method for qualitative estimation of alkylamide from in vitro raised plants in comparison with that of the field grown counterpart by using LC-Q-TOF (HRMS). To the best of our knowledge, this is the first qualitative report on alkylamide profile of micropropagated whole plant of Spilanthes. The correlation pattern reported in this study may form the basis for using tissue culture raised plantlets of S. ciliata as potential source of bioactive alkylamides on industrial scale.