Resistance to First-Line Anti-TB Drugs Is Associated with Reduced Nitric Oxide Susceptibility in Mycobacterium tuberculosis

Background and objective: The relative contribution of nitric oxide (NO) to the killing of Mycobacterium tuberculosis in human tuberculosis (TB) is controversial, although this has been firmly established in rodents. Studies have demonstrated that clinical strains of M. tuberculosis differ in susceptibility to NO, but how this correlates to drug resistance and clinical outcome is not known. Methods: In this study, 50 sputum smear- and culture-positive patients with pulmonary TB in Gondar, Ethiopia were included. Clinical parameters were recorded and drug susceptibility profile and spoligotyping patterns were investigated. NO susceptibility was studied by exposing the strains to the NO donor DETA/NO. Results: Clinical isolates of M. tuberculosis showed a dose- and time-dependent response when exposed to NO. The most frequent spoligotypes found were CAS1-Delhi and T3_ETH in a total of nine known spoligotypes and four orphan patterns. There was a significant association between reduced susceptibility to NO (>10% survival after exposure to 1mM DETA/NO) and resistance against first-line anti-TB drugs, in particular isoniazid (INH). Patients infected with strains of M. tuberculosis with reduced susceptibility to NO showed no difference in cure rate or other clinical parameters, but a tendency towards lower rate of weight gain after two months of treatment. Conclusion: There is a correlation between resistance to first-line anti-TB drugs and reduced NO susceptibility in clinical strains of M. tuberculosis. Further studies including the mechanisms of reduced NO susceptibility are warranted and could identify targets for new therapeutic interventions

DHFR INHIBITOR TRIAZA-COUMARIN IS A F420 REDOX-ACTIVATED DUAL MECHANISM ANTITUBERCUR

Ph.DDOCTOR OF PHILOSOPHY (SOM

Characterization of In Vitro Resistance to Linezolid in Mycobacterium abscessus

ABSTRACT Single-step selection of Mycobacterium abscessus mutants resistant to linezolid yielded high-level resistance at a low frequency that was associated with mutations in 23S rRNA or the ribosomal protein L3. Surprisingly, linezolid-resistant rRNA mutations conferred cross-resistance to several unrelated antibiotics. Low-level linezolid-resistant mutants were isolated at a higher frequency and were due to loss-of-function mutations in the transcriptional regulator MAB_4384, the repressor of the drug efflux pump MmpL5-MmpS5. IMPORTANCE The protein synthesis inhibitor linezolid is used for the treatment of lung disease caused by Mycobacterium abscessus. However, many strains of the bacterium show poor susceptibility to the antibiotic. For most clinical isolates, resistance is not due to mutations in the target of the drug, the ribosome. The mechanism responsible for non-target-related, indirect linezolid resistance is unknown. Here, we analyzed the development of linezolid resistance in the M. abscessus reference strain in vitro. We found, as expected, resistance mutations in the ribosome. In addition, we identified mutations in a system that involves a drug pump, suggesting drug efflux as a mechanism of resistance to linezolid. This finding may inform the analysis of clinical resistance to linezolid. Surprisingly, a subset of linezolid-resistant ribosome mutations conferred cross-resistance to several structurally and mechanistically unrelated drugs, uncovering a novel multidrug resistance mechanism

1,3,5-triazaspiro[5.5]undeca-2,4-dienes as selective Mycobacterium tuberculosis dihydrofolate reductase inhibitors with potent whole cell activity

The emergence of multi- and extensively-drug resistant tubercular (MDR- and XDR-TB) strains of mycobacteria has limited the use of existing therapies, therefore new drugs are needed. Dihydrofolate reductase (DHFR) has recently attracted much attention as a target for the development of anti-TB agents. This study aimed to develop selective M. tuberculosis DHFR inhibitors using rationale scaffolding design and synthesis, phenotype-oriented screening, enzymatic inhibitory study, whole cell on-target validation, molecular modeling, and in vitro DMPK determination to derive new anti-TB agents. 2,4-diamino-1-phenyl-1,3,5-triazaspiro[5.5]undeca-2,4-dienes 20b and 20c were identified as selective M. tuberculosis DHFR inhibitors, showing promising antimycobacterial activities (MIC50: 0.01 μM and MIC90: 0.025 μM on M. tuberculosis H37Rv). This study provided compelling evidence that compound 20b and 20c exerted whole cell antimycobacterial activity through DHFR inhibition. In addition, these two compounds exhibited low cytotoxicity and low hemolytic activity. The in vitro DMPK and physiochemical properties suggested their potential in vivo efficacy.NMRC (Natl Medical Research Council, S’pore

Mycobacterium tuberculosis PanD StructureFunction Analysis and Identification of a Potent Pyrazinoic Acid-Derived Enzyme Inhibitor

A common strategy employed in antibacterial drug discovery is targeting of biosynthetic processes which are essential and specific for the pathogen. Specificity in particular avoids undesirable interactions with potential enzymatic counterparts in the human host, and ensures on-target toxicity. Synthesis of pantothenate (Vitamine B5), a precursor of the acyl carrier coenzyme A, is an example of such a pathway. In Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), pantothenate is formed by pantothenate synthase, utilizing D-pantoate and β-Ala as substrates. β-Ala is mainly formed by the decarboxylation of L-aspartate, generated by the decarboxylase PanD, a homo-oliogomer in solution. Pyrazinoic acid (POA), the bioactive form of the TB prodrug pyrazinamide, binds and inhibits PanD activity weakly. Here, we generated a library of recombinant Mtb PanD mutants based on structural information and PZA/POA resistance mutants. Alterations in oligomer formation, enzyme activity and/or POA binding were observed in respective mutants, providing insights into essential amino acids for Mtb PanD’s proper structural assembly, decarboxylation activity and drug interaction. This information provided the platform for the design of novel POA analogs with modifications at position 3 of the pyrazine ring. Analog 2, incorporating a bulky naphthamido group at this position, displayed a 1,000-fold increase in enzyme inhibition compared to POA, along with moderately improved antimycobacterial activity. The data demonstrate that an improved understanding of mechanistic and enzymatic features of key metabolic enzymes can stimulate design of more potent PanD inhibitors

Structural and mechanistic insights into Mycobacterium abscessus as-partate decarboxylase PanD and a pyrazinoic acid-derived inhibitor

Mycobacterium tuberculosis (Mtb) aspartate decarboxylase PanD is required for biosynthesis of the essential cofactor coenzyme A and targeted by the first line drug pyrazinamide (PZA). PZA is a prodrug that is converted by a bacterial amidase into its bioactive form pyrazinoic acid (POA). Employing structure-function analyses we previously identified POA-based inhibitors of Mtb PanD showing much improved inhibitory activity against the enzyme. Here, we performed the first structure-function studies on PanD encoded by the non-tuberculous mycobacterial lung pathogen Mycobacterium abscessus (Mab), shedding light on the differences and similarities of Mab and Mtb PanD. Solution X-ray scattering data provided the solution structure of the entire tetrameric Mab PanD, which in comparison to the structure of the derived C-terminal trun-cated Mab PanD1-114 mutant, revealed the orientation of the four flexible C-termini relative to the catalytic core. Enzymatic studies of Mab PanD1-114 explored the essentiality of the C-terminus for catalysis. A library of recombinant Mab PanD mutants based on structural information and PZA/POA resistant PanD mutations in Mtb, illuminated critical residues involved in the substrate tunnel and enzymatic activity. Using our library of POA analogs, we identified (3-(1-naphthamido)pyrazine-2-car-boxylic acid) (analog 2) as the first potent inhibitor of Mab PanD. The inhibitor shows mainly electrostatic- and hydrogen bonding interaction with the target enzyme as explored by isothermal titration calorimetry and confirmed by docking studies. The observed unfavorable entropy indicates that significant conformational changes are involved in the binding process of analog 2 to Mab PanD. In contrast to PZA and POA, which are whole-cell inactive, analog 2 exerts appreciable antibacterial activity against the three subspecies of Mab.Ministry of Education (MOE)National Research Foundation (NRF)Submitted/Accepted versionResearch reported in this publication is supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers 2R01AI106398-05 (T.D., (T.D., (T.D., C.A.,C.A., G.G.) G.G.) . The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The studies are also supported by the National Research Founda-tion (NRF) Singapore, NRF Competitive Research Programme (CRP), Grant Award Number NRF–CRP18–2017–01 (G.G., T.D.), and the Singapore Ministry of Education (MOE) Academic Research Fund Tier 1 (RG107/20) to G.G

Reduced NO susceptibility in spoligotype-based clusters of <i>M. tuberculosis</i>.

<p>Survival of clinical isolates 24 hours after exposure to the NO donor DETA/NO. Presence of resistance to first-line anti-TB drugs is indicated with circles; isoniazid (INH), streptomycin (SM) and rifampin (RIF). Each point represents a mean value of duplicates and the dashed line is the median survival of all 50 isolates.</p

Multiple logistic regression analysis comparing strains of <i>M. tuberculosis</i> susceptible to NO with strains of reduced susceptibility to NO.

<p>Multiple logistic regression analysis comparing strains of <i>M. tuberculosis</i> susceptible to NO with strains of reduced susceptibility to NO.</p

Characteristics for patients infected with strains of <i>M. tuberculosis</i> susceptible to NO or with reduced susceptibility to NO.

<p>Q1–Q3 (quartile 1 to quartile 3); NO (nitric oxide); BMI (body mass index); ESR (sedimentation rate); INH (isoniazid); SM (streptomycin); EMB (ethambutol); RIF (rifampin). NO-susceptible and reduced NO-susceptible strains defined as ≤10% and >10% survival respectively after exposure to 1 mM DETA/NO. All patients were smear positive at week 0. Continuous data were tested with Mann-Whitney <i>U</i>-test and discrete data with Fisher’s exact test or Pearson’s Chi-square test.</p

Dose- and time-dependent killing of <i>M. tuberculosis</i> exposed to NO.

<p>Survival of three clinical strains, H37Rv and BCG after exposure to 1 mM of the NO donor DETA/NO for 4 and 24 hours (A). Survival of the three clinical strains exposed to different doses of DETA/NO for 24 hours (B). Survival was determined through viable count (colony forming units, CFU) and each point represents a mean value of duplicates.</p