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International Journal of Environmental... Jan 2022Tuberculosis (TB), the most frequent bacterium-mediated infectious disease caused by , has been known to infect humans since ancient times. Although TB is common...
Tuberculosis (TB), the most frequent bacterium-mediated infectious disease caused by , has been known to infect humans since ancient times. Although TB is common worldwide, the most recent report by the WHO (World Health Organization) listed the three countries of India, China, and Russia with 27%, 14%, and 8% of the global burden of TB, respectively. It has been reported that resistance to TB drugs, particularly by the gene to the pyrazinamide drug due to mutations, significantly affects the effective treatment of TB. Understanding the mechanism of drug resistance using computational methods is of great interest to design effective TB treatment, exploring the structural features with these tools. Thus, keeping in view the importance of these methods, we employed state-of-the-art computational methods to study the mechanism of resistance caused by the W68L, L85P, and T87A mutations recently reported in 2021. We employed a molecular docking approach to predict the binding conformation and studied the dynamic properties of each complex using molecular dynamics simulation approaches. Our analysis revealed that compared to the wildtype, these three mutations altered the binding pattern and reduced the binding affinity. Moreover, the structural dynamic features also showed that these mutations significantly reduced the structural stability and packing, particularly by the W68L and L85P mutations. Moreover, principal component analysis, free energy landscape, and the binding free energy results revealed variation in the protein's motion and the binding energy. The total binding free energy was for the wildtype -9.61 kcal/mol, W68L -7.57 kcal/mol, L85P -6.99 kcal/mol, and T87A -7.77 kcal/mol. Our findings can help to design a structure-based drug against the MDR (multiple drug-resistant) TB.
Topics: Amidohydrolases; Antitubercular Agents; Drug Resistance, Bacterial; Microbial Sensitivity Tests; Molecular Docking Simulation; Mutation; Mycobacterium tuberculosis; Pyrazinamide
PubMed: 35162636
DOI: 10.3390/ijerph19031615 -
Medicina (Kaunas, Lithuania) Jan 2023: There is a lack of information regarding the effective duration of treatment necessary to prevent the development of acquired resistance when fluoroquinolones (FQ),...
: There is a lack of information regarding the effective duration of treatment necessary to prevent the development of acquired resistance when fluoroquinolones (FQ), and/or pyrazinamide (Z) resistance has occurred in patients with polydrug-resistant tuberculosis and isoniazid resistance. The management of these kinds of patients should be carried out in experienced centers according to drug susceptibility test results, clinical status of the patient and the extensity of the disease. : We evaluated treatment regimens, treatment outcomes, and drug adverse effects in seven patients with polydrug-resistant tuberculosis, including those with Z and/or FQ resistance in a retrospective analysis : Regarding the patients with polydrug-resistant tuberculosis in addition to isoniazid (H) resistance, three had Z, two had FQ, and the remaining two had both Z and FQ resistance. In the intensive phase of the treatment, the patients were given at least four drugs according to drug susceptibility tests, and at least three drugs in the continuation phase. The duration of treatment was 9-12 months. Two of the patients were foreign nationals, and could not be followed up with due to returning to their home countries. Regarding the remaining five patients, three of them were terminated as they completed treatment, and two as cured. No recurrence was observed in the first year of the treatment. The most common, and serious drug side effect was seen for amikacin. : In patients with polydrug-resistant TB, if Z and/or FQ resistance is detected in addition to H resistance, the treatment of these patients should be conducted on a case-by-case basis, taking into account the patient's resistance pattern, clinical condition, and disease prognosis. Close monitoring of the side effects will increase the success rate of the treatment.
Topics: Humans; Antitubercular Agents; Isoniazid; Retrospective Studies; Tuberculosis; Pyrazinamide; Fluoroquinolones; Tuberculosis, Multidrug-Resistant; Mycobacterium tuberculosis
PubMed: 36837448
DOI: 10.3390/medicina59020246 -
Bioorganic & Medicinal Chemistry Letters Jan 2013A series of 19 new compounds related to pyrazinamide were synthesized, characterized with analytical data and screened for in vitro whole cell antimycobacterial activity...
A series of 19 new compounds related to pyrazinamide were synthesized, characterized with analytical data and screened for in vitro whole cell antimycobacterial activity against Mycobacterium tuberculosis H37Rv, Mycobacterium kansasii and two types of Mycobacterium avium. The series consisted of 3-(benzylamino)-5-cyanopyrazine-2-carboxamides and 3-(benzylamino)pyrazine-2,5-dicarbonitriles with various substituents on the phenyl ring. RP-HPLC method was used to determine the lipophilicity of the prepared compounds. Nine compounds exerted similar or better activity against Mycobacterium tuberculosis compared to pyrazinamide (MIC=6.25-12.5 μg/mL). 3-(Benzylamino)pyrazine-2,5-dicarbonitrile inhibited all of the tested mycobacterial strains with MIC within the range 12.5-25 μg/mL. Although not the most active, 4-NH(2) substituted compounds possessed the lowest in vitro cytotoxicity (hepatotoxicity), leading to selectivity index SI=5.5 and SI >21.
Topics: Antitubercular Agents; Benzylamines; Cells, Cultured; Humans; Inhibitory Concentration 50; Microbial Sensitivity Tests; Molecular Structure; Mycobacterium; Pyrazinamide
PubMed: 23237840
DOI: 10.1016/j.bmcl.2012.11.052 -
Rapid Communications in Mass... Jan 2023Tuberculosis (TB) remains a challenging global infectious disease, mainly affecting the lungs. First-line anti-TB drugs play a crucial role in slowing down the rapid...
RATIONALE
Tuberculosis (TB) remains a challenging global infectious disease, mainly affecting the lungs. First-line anti-TB drugs play a crucial role in slowing down the rapid spread of TB. In addition, the patient might benefit from therapeutic drug monitoring since it has become an accepted clinical tool for optimizing TB treatment.
METHODS
A simple and sensitive liquid chromatography/tandem mass spectrometry method was developed to monitor the plasma level of isoniazid, ethambutol and pyrazinamide in plasma samples. A one-step extraction procedure using an Ostro™ plate was applied, and extracts were analyzed by gradient elution followed by detection on a mass spectrometer by multiple reaction monitoring mode.
RESULTS
The analytes were separated within 4.2 min and over the concentration range of 0.2-10 μg/ml for isoniazid and ethambutol and 1-65 μg/ml for pyrazinamide. The method was successfully validated according to the European Medicine Agency guideline for the selectivity, linearity and lower limit of detection, precision and accuracy, matrix effect, extraction recovery, carryover, dilution integrity and stability, and applied for quantification of analytes in clinical samples from TB patients.
CONCLUSIONS
The presented method allows sensitive and reproducible determination of selected anti-TB drugs with advantages such as low sample volume requirement, short run time of analysis, one-step sample preparation procedure with capabilities for phospholipids removal, and a low quantification limit as well as a high degree of selectivity.
Topics: Humans; Ethambutol; Pyrazinamide; Isoniazid; Chromatography, Liquid; Tandem Mass Spectrometry; Antitubercular Agents; Tuberculosis; Chromatography, High Pressure Liquid
PubMed: 36329637
DOI: 10.1002/rcm.9425 -
ACS Infectious Diseases Nov 2020Pyrazinamide, a first-line antibiotic used against , has been shown to act in a pH-dependent manner . Why pyrazinamide, an antitubercle prodrug discovered more than 65...
Pyrazinamide, a first-line antibiotic used against , has been shown to act in a pH-dependent manner . Why pyrazinamide, an antitubercle prodrug discovered more than 65 years ago, exhibits this pH-dependent activity was unclear. Upon entering mycobacterial cells, pyrazinamide is deamidated to pyrazinoate by an enzymatic process and exists in an acid-base equilibrium with pyrazinoic acid. Thus, the effects of total pyrazinoic acid (pyrazinoic acid + pyrazinoate) on growth, pH homeostasis, and proton motive force over a range of pH values found in host tissues were investigated. Although was able to maintain pH homeostasis over an external pH range of 7.0 to 5.5, total pyrazinoic acid induced growth inhibition increased as culture medium pH was decreased from 7.3 to 6.4. Consistent with growth inhibition, total pyrazinoic acid increased both acidification of the bacterial cytoplasm and dissipation of membrane potential as the environmental pH decreased when added to the bacterial suspensions. The results suggest pyrazinoic acid is the active form of the drug, which acts as an uncoupler of proton motive force, likely a protonophore, providing a mechanistic explanation for the pH dependence of the drug activity.
Topics: Acid-Base Equilibrium; Antitubercular Agents; Mycobacterium tuberculosis; Pyrazinamide
PubMed: 33078607
DOI: 10.1021/acsinfecdis.0c00507 -
The Journal of Antimicrobial... Feb 2004Pyrazinamide is a paradoxical frontline tuberculosis drug characterized by high in vivo sterilizing activity but poor in vitro activity. This separation in pyrazinamide...
BACKGROUND
Pyrazinamide is a paradoxical frontline tuberculosis drug characterized by high in vivo sterilizing activity but poor in vitro activity. This separation in pyrazinamide activity reflects differences between the in vivo tissue environment and in vitro culture conditions. The well-known acid pH requirement for pyrazinamide activity was discovered previously based on such reasoning but does not completely explain the discrepancy between in vivo and in vitro activity of pyrazinamide. This study examined the effect of iron, which could potentially be elevated in local inflammatory lesions, on pyrazinamide activity in vitro.
MATERIALS AND METHODS
The effect of iron on the activity of pyrazinamide or its active derivative pyrazinoic acid against Mycobacterium tuberculosis was assessed in liquid medium in a drug exposure assay or in solid medium with pyrazinamide plus iron or pyrazinamide alone. The effect of iron on pyrazinamide or pyrazinoic acid was expressed as percentage of growth inhibition.
RESULTS
We have shown that iron enhances the activity of pyrazinamide and pyrazinoic acid against M. tuberculosis in both liquid and solid media at acid pH 5.6. Iron enhanced the activity of pyrazinoic acid but not pyrazinamide against the naturally pyrazinamide-resistant Mycobacterium bovis BCG. Other metal ions such as magnesium, calcium and zinc did not enhance the activity of pyrazinamide or pyrazinoic acid.
CONCLUSIONS
Iron increased the activity of pyrazinamide or pyrazinoic acid against M. tuberculosis in vitro. These findings may have implications for the study of mechanism of action of pyrazinamide and possible iron supplement for improving the activity of pyrazinamide.
Topics: Antitubercular Agents; Calcium; Culture Media; Drug Synergism; Hydrogen-Ion Concentration; Iron; Magnesium; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Pyrazinamide; Zinc
PubMed: 14729751
DOI: 10.1093/jac/dkh042 -
PloS One 2020Pyrazinamide (PZA) susceptibility testing in Mycobacterium tuberculosis (Mtb) is a current area of development and PZA-resistant strains are increasingly prevalent....
Pyrazinamide (PZA) susceptibility testing in Mycobacterium tuberculosis (Mtb) is a current area of development and PZA-resistant strains are increasingly prevalent. Previous studies have demonstrated that the detection of pyrazinoic acid (POA), the metabolite produced by the deamidation of PZA, is a good predictor for PZA resistance since a resistant strain would not convert PZA into POA at a critical required rate, whereas a susceptible strain will do, expelling POA to the extracellular environment at a certain rate, and allowing for quantification of this accumulated analyte. In order to quantify POA, an indirect competitive ELISA (icELISA) test using hyperimmune polyclonal rabbit serum against POA was developed: for this purpose, pure POA was first covalently linked to the highly immunogenic Keyhole Limpet Hemocyanine, and inoculated in rabbits. A construct made of bovine serum albumin (BSA) linked to pure POA and fixed at the bottom of wells was used as a competitor against spiked samples and liquid Mtb culture supernatants. When spiked samples (commercial POA alone) were analyzed, the half maximal inhibitory concentration (IC50) was 1.16 mg/mL, the limit of detection 200 μg/mL and the assay was specific (it did not detect PZA, IC50 > 20 mg/mL). However, culture supernatants (7H9-OADC-PANTA medium) disrupted the competition and a proper icELISA curve was not obtainable. We consider that, although we have shown that it is feasible to induce antibodies against POA, matrix effects could damage its analytical usefulness; multiple, upcoming ways to solve this obstacle are suggested.
Topics: Animals; Antibodies; Antitubercular Agents; Drug Resistance, Bacterial; Enzyme-Linked Immunosorbent Assay; Immunoconjugates; Inhibitory Concentration 50; Mycobacterium tuberculosis; Pyrazinamide; Rabbits; Serum Albumin, Bovine; Toxicity Tests
PubMed: 33151985
DOI: 10.1371/journal.pone.0241600 -
Journal of Chemical Information and... Jan 2019In an effort to discover the mechanism of resistance offered by Mycobacterium tuberculosis (Mtb) toward the pyrazinamide (PZA) drug, an extensive molecular dynamics...
In an effort to discover the mechanism of resistance offered by Mycobacterium tuberculosis (Mtb) toward the pyrazinamide (PZA) drug, an extensive molecular dynamics strategy was employed. PZA is a first-line prodrug that effectively cuts therapy time by 33% (from 9 to 6 months). Pyrazinamidase enzyme (PZase), encoded by the pncA gene, is responsible for the activation of prodrug PZA into pyrazinoic acid (POA). POA is toxic and potently inhibits the growth of latent Mtb even at low pH values. PZA resistance is caused by three genes pncA, rpsA, and panD. Among them, the pncA gene contributes 72-99% to the resistance. Hence, the present study focused on the novel mutations N11K, P69T, and D126N in the pncA gene. In the present study, the possible mechanism of these three mutations was studied through molecular dynamics simulation and docking techniques. Our in-depth analysis and results are in strong agreement with our experimental observation. The binding pocket analysis showed that mutations decrease the volume of the active site and hinder the correct orientation of PZA drug in the active site. Moreover, the Patchdock score was found to be low as compared to WT showing the disturbance of shape complementarity between PZase and PZA drug. These mutations were found to disturb the position of the Fe ion. Among the mutations, D126N allosterically disturbed the position of the Fe ion. MMGBSA analyses showed that these mutations decrease the binding affinity toward the PZA drug. In conclusion, mutations N11K, P69T, and D126N result in weak binding affinity with PZA and also cause significant structural deformations that lead to PZA resistance. This study provides useful information that mutations in other than active parts may also cause protein folding and ligand displacement effects, altering the biological functions.
Topics: Amidohydrolases; Binding Sites; Iron; Molecular Docking Simulation; Mutation; Mycobacterium tuberculosis; Protein Conformation; Pyrazinamide
PubMed: 30481017
DOI: 10.1021/acs.jcim.8b00525 -
Pharmacotherapy Jun 2002To determine population pharmacokinetic parameters of pyrazinamide after multiple oral doses given to children and adults with tuberculosis. (Clinical Trial)
Clinical Trial Comparative Study
STUDY OBJECTIVE
To determine population pharmacokinetic parameters of pyrazinamide after multiple oral doses given to children and adults with tuberculosis.
DESIGN
Prospective, multiple-dose population pharmacokinetic study.
SETTING
Five hospitals in the United States.
PATIENTS
Sixty-seven adults and 23 children with active tuberculosis.
INTERVENTION
The 90 patients received multiple oral doses of pyrazinamide as part of their treatment, based on the best clinical judgment of the attending physicians and in keeping with standard clinical practices at each institution. The patients also received other antituberculosis drugs empirically or based on in vitro susceptibility data.
MEASUREMENTS AND MAIN RESULTS
Serum samples were collected over 12 hours after dosing and were assayed with a validated gas chromatography assay with mass selective detection. Concentration-time data were analyzed by using population methods. Pyrazinamide concentrations increased linearly with increasing oral doses (185-3550 mg). Median maximum serum concentration values were 41.0 microg/ml with daily dosing and 66.1 microg/ml with larger, twice-weekly dosing. Incomplete (18%) or delayed (30%) absorption was more common in children than in adults (1% for each). Pharmacokinetic parameters of pyrazinamide were independent of human immunodeficiency virus status and patient demographics, except for body weight. Population elimination half-life values in pediatric and adult patients were 3.5 and 6.0 hours, respectively. Median volume of distribution (L/kg) was 32% larger in children, and median clearance (L/hr/kg) was 106% larger in children, with a resultant median half-life 43% shorter in children.
CONCLUSION
Pyrazinamide concentrations and most pharmacokinetic parameters were comparable to those previously published. Apparent half-life was somewhat shorter than that in previous reports. Compared with adults, absorption of pyrazinamide in children appeared more likely to be incomplete or delayed.
Topics: Adult; Aging; Antitubercular Agents; Area Under Curve; Child; Chromatography, Gas; Drug Monitoring; HIV Infections; Half-Life; Humans; Models, Biological; Pyrazinamide; Tuberculosis
PubMed: 12066959
DOI: 10.1592/phco.22.9.686.34067 -
Spectrochimica Acta. Part A, Molecular... Feb 2018The α and γ polymorphs of drug pyrazinamide have been detected with the help of temperature dependent Raman spectroscopic technique. Pyrazinamide is a very useful drug...
The α and γ polymorphs of drug pyrazinamide have been detected with the help of temperature dependent Raman spectroscopic technique. Pyrazinamide is a very useful drug used for the treatment of tuberculosis (TB) and plays a significant role in destroying the dormant tubercle bacilli which are not destroyed by other common TB drugs. Temperature dependent Raman spectra suggest polymorphic phase change from α→γ form of pyrazinamide between 145 and 146°C. In situ Raman spectra of pyrazinamide between 145 and 146°C show the conversion of α→γ form by the shift in CO stretching vibration accompanied by several other changes. The phase change is characterized by the breaking of two linear NH⋯O type hydrogen bonds associated with CO stretching vibration in α dimer and formation of one linear NH⋯N type hydrogen bond along with a weak intramolecular CH⋯O type hydrogen bond in the γ dimer.
Topics: Dimerization; Models, Molecular; Molecular Conformation; Pyrazinamide; Quantum Theory; Spectrum Analysis, Raman; Temperature; Vibration; X-Ray Diffraction
PubMed: 28922644
DOI: 10.1016/j.saa.2017.09.016