20 research outputs found
Simple and Rapid Method To Determine Antimycobacterial Potency of Compounds by Using Autoluminescent Mycobacterium tuberculosis
A major obstacle in the process of discovery of drugs against Mycobacterium tuberculosis is its extremely slow growth rate and long generation time (similar to 20 to 24 h). Consequently, determination of MICs and minimum bactericidal concentrations (MBCs) of potential drug candidates using current methods requires 7 days (resazurin-based MIC assay [REMA]) and 1 month (CFU enumeration), respectively. We employed a synthetic luciferase operon optimized for expression in high-GC-content bacteria and adapted it for use in mycobacteria. Using luminescence-based readouts, we were able to determine the MICs and bactericidal activities of approved tuberculosis (TB) drugs, which correlated well with currently used methods. Although luminescence-based readouts have been used previously to determine the MICs and bactericidal activities of approved TB drugs, in this study we adapted this assay to carry out a pilot screen using a library of 1,114 compounds belonging to diverse chemical scaffolds. We found that MICs derived from a 3-day luminescence assay matched well with REMA-based MIC values. To determine the bactericidal potencies of compounds, a 1:10 dilution of the cultures from the MIC plate was carried out on day 7, and the bactericidal concentrations determined based on time to positivity in 2 weeks were found to be comparable with MBC values determined by the conventional CFU approach. Thus, the luminescent mycobacterium-based approach not only is very simple and inexpensive but also allowed us to generate the information in half the time required by conventional methods
Aminopyrazinamides: Novel and Specific GyrB Inhibitors that Kill Replicating and Nonreplicating <i>Mycobacterium tuberculosis</i>
Aminopyrazinamides originated from a high throughput
screen targeting
the <i>Mycobacterium smegmatis</i> (Msm) GyrB ATPase. This
series displays chemical tractability, robust structure–activity
relationship, and potent antitubercular activity. The crystal structure
of Msm GyrB in complex with one of the aminopyrazinamides revealed
promising attributes of specificity against other broad spectrum pathogens
and selectivity against eukaryotic kinases due to novel interactions
at hydrophobic pocket, unlike other known GyrB inhibitors. The aminopyrazinamides
display excellent mycobacterial kill under <i>in vitro</i>, intracellular, and hypoxic conditions
Discovery of benzothiazoles as antimycobacterial agents: Synthesis, structure-activity relationships and binding studies with Mycobacterium tuberculosis decaprenylphosphoryl-beta-D-ribose 2 '-oxidase
We report the discovery of benzothiazoles, a novel anti-mycobacterial series, identified from a whole cell based screening campaign. Benzothiazoles exert their bactericidal activity against Mycobacterium tuberculosis (Mtb) through potent inhibition of decaprenylphosphoryl-beta-D-ribose 2'-oxidase (DprE1), the key enzyme involved in arabinogalactan synthesis. Specific target linkage and mode of binding were established using co-crystallization and protein mass spectrometry studies. Most importantly, the current study provides insights on the utilization of systematic medicinal chemistry approaches to mitigate safety liabilities while improving potency during progression from an initial genotoxic hit, the benzothiazole N-oxides (BTOs) to the lead-like AMES negative, crowded benzothiazoles (cBTs). These findings offer opportunities for development of safe clinical candidates against tuberculosis. The design strategy adopted could find potential application in discovery of safe drugs in other therapy areas too. (c) 2015 Elsevier Ltd. All rights reserved
Simple and Rapid Method To Determine Antimycobacterial Potency of Compounds by Using Autoluminescent Mycobacterium tuberculosis
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
Aminopyrazinamides: Novel and Specific GyrB Inhibitors that Kill Replicating and Nonreplicating Mycobacterium tuberculosis
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
4-Aminoquinolone Piperidine Amides: Noncovalent Inhibitors of DprE1 with Long Residence Time and Potent Antimycobacterial Activity
4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-beta-D-ribose 2'-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of similar to 100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure-activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb