Screening of coformers for quercetin cocrystals through mechanochemical methods

Quercetin (QUE) is a nutraceutical compound that exhibits pharmacological properties such as antioxidant, cardioprotective, anti-ulcer, and anti-inflammatory effects. Although QUE is well-known for its benefits, its efficacy is limited due to low solubility. Thus, cocrystallization acts as an interesting approach to improve the solubility�among other properties�of this compound. In this work, cocrystallization screening was applied through neat grinding (NG) and liquid-assisted grinding (LAG), in which QUE and four cocrystal formers (benzamide,�picolinamide, isonicotinamide, and pyrazinoic acid) were tested. The precursors and QUE-coformer systems were characterized using thermoanalytical techniques (TG-DTA), X-ray powder diffraction (XRPD), and Fourier transform infrared (FTIR) spectroscopy. The results showed the formation of QUE cocrystals with picolinamide and isonicotinamide coformers in a 1:1 stoichiometric ratio. Furthermore, although coformers are isomers, spectroscopic and thermal data suggest that the supramolecular synthons involved in cocrystallization are different.

Synthesis, and Biological Evaluations of Novel Pyrazinoic Acid Derivatives as Anticancer Agents

Pyrazinoic acid or pyrazine-2-carboxylic acid (PA), due to its nitrogenous heteroaromatic ring, can be explored as an anticancer agent. Here, a series of twenty novels PA derivatives have been synthesized and characterized using IR, NMR, and mass spectrums. Their cytotoxic activity was evaluated against three different cancer cell lines, including lung (A549), breast (MCF-7), and colon (HT-29). P16, the most potent compound, showed moderate cytotoxicity with IC50 of 6.11 μM, 10.64 μM, and 14.92 μM, against the A549, MCF-7, and HT-29 cell lines, respectively. Furthermore, the effect of this compound against MRC5 as a non-tumoral lung cell line, exhibited a selectivity index of 9.02. The apoptotic induction activity of P16 was also performed on the A549 cell line. The results showed that as the concentration of the compound increases (from 3 to 6 μM), the percentage of induction of apoptosis increases from 8.54% to 72.4%. Electrophoretic gel mobility shift assays showed that P16 was also able to ROS induce DNA cleavage in the presents of H2O2 (1.0 mM) in dose-dependent manner. Molecular docking was also applied to anticipate the binding locations and the binding of the synthesized compound with Bcl-2 apoptosis regulator and DNA as their proposed targets.

Factors affecting the pharmacokinetics of pyrazinamide and its metabolites in patients co-infected with HIV - implications for individualized dosing

Pyrazinamide is a first-line drug used in the treatment of tuberculosis. High exposure of pyrazinamide and its metabolites may result in hepatotoxicity whereas low exposure of pyrazinamide has been correlated to treatment failure by first-line antitubercular therapy. The aim of this study was to describe the pharmacokinetics and metabolism of pyrazinamide in patients co-infected with tuberculosis and HIV. We further aimed to identify demographic and clinical factors which affect the pharmacokinetics of pyrazinamide and its metabolites in order to suggest individualized dosing regimens. Plasma concentrations of pyrazinamide, pyrazinoic acid and 5-hydroxy pyrazinamide from 63 Rwandan patients co-infected with tuberculosis and HIV were determined by liquid chromatography tandem mass spectrometry followed by non-linear mixed effects modelling. Females had a close to 50% higher pyrazinamide bioavailability than males. The distribution volumes of pyrazinamide and both metabolites were lower in patients on concomitant efavirenz-based HIV therapy. Furthermore, there was a linear relationship between serum creatinine and oral clearance of pyrazinoic acid. Simulations indicated that increasing doses from 25 mg/kg to 35 mg/kg and 50 mg/kg in females and males, respectively would result in adequate exposure with regard to suggested thresholds and increase probability of target attainment to >0.9 for a minimal inhibitory concentration of 25 mg/L. Further, lowering the dose by 40% in patients with high serum creatinine would prevent accumulation of toxic metabolites. Individualized dosing is proposed to decrease variability in exposure to pyrazinamide and its metabolites. Reducing the variability in exposure may lower the risk of treatment failure and resistance development.

Urine metabolome of tuberculosis patients receiving intensive phase of treatment show diurnal variations.

Diurnal variation in biofluid metabolome as observed in a healthy human may alter in perturbed conditions. Biofluids like urine are rich in molecular constituents including metabolites, and infectious disease conditions like tuberculosis (TB) may influence diurnal differences for which limited reports are available in the literature. In this study, we present an optimized gas chromatography coupled to a quadrupole mass spectrometry (GC-MS) method to analyze processed and trimethylsilyl (TMS) derivatized urine metabolites. Urine samples were collected at four time points (0, 6, 12 and 24 hours) of study subjects [n=15; mean age 37 (24-70) in years] including controls [n=7; mean age 29.3 (24-35) years] and culture-confirmed active TB patients [ATB; n=8; mean age 43.7 (25-70) years] receiving treatment in the intensive phase. Global urine metabolite profiling was carried out using the optimized GC-MS method. Higher urine analyte diversity was observed in ATB patients (74) than in controls (36) during the day. Diurnal variations of the parent anti-TB drugs and their breakdown products (pyrazinamide, pyrazinoic acid, 5-hydroxy pyrazinoic acid, isonicotinic acid and alpha amino butyric acid) were observed with maximum abundance at 6 h. Interestingly, urine of ATB subjects at 6 h showed the highest metabolic diversity, whereas it was at 12 h in controls. Many analytes including glycine and alanine amino-acids showed diurnal variation in ATB and controls. These changes could be attributed to the altered host metabolic activities due to disease, treatment-associated decrease in total body bacterial burden and gut microbiota dysbiosis. And the optimized spiked-in internal standard, urine sample volume and GC-MS method could be used for global urine metabolome analysis in healthy and different perturbed conditions.

Atypical Genetic Basis of Pyrazinamide Resistance in Mono-resistant Mycobacterium tuberculosis

Pyrazinamide (PZA) is a widely used antitubercular chemotherapeutic. Typically, PZA resistance (PZA-R) emerges in M. tuberculosis strains with existing resistance to isoniazid and rifampicin (MDR) and is conferred by loss-of-function pncA mutations that inhibit conversion to its active form, Pyrazinoic acid (POA). PZA-R departing from this canonical scenario is poorly understood. Here, we genotype pncA and purported alternative PZA-R genes (panD, rpsA, and clpC1) with long-read sequencing of nineteen phenotypically PZA mono-resistant isolates collected in Sweden and compare their phylogenetic and genomic characteristics to a large set of MDR PZA-R (MDRPZA-R) isolates. We report the first association of ClpC1 mutations with PZA-R in clinical isolates, in the ClpC1 promoter (clpC1p-138) and N-terminal (ClpC1Val63Ala). Mutations have emerged in both these regions under POA selection in vitro and ClpC1N-terminal has been implicated further, through its POA-dependent efficacy in PanD proteolysis. ClpC1Val63Ala mutants spanned 4 Indo-oceanic sublineages. Indo-oceanic isolates invariably harbored ClpC1Val63Ala and were starkly overrepresented (OR=22.2, p <0.00001) among PZA mono-resistant isolates (11/19) compared to MDRPZA-R isolates (5/80). The genetic basis of Indo-oceanic isolates’ overrepresentation in PZA mono-resistant TB remains undetermined, but substantial circumstantial evidence suggests ClpC1Val63Ala confers low-level PZA resistance. Our findings highlight ClpC1 as potentially clinically relevant for PZA-R and reinforce the importance of genetic background in the trajectory of resistance development.

Computational Insight into the Binding Mechanism of Pyrazinoic Acid to RpsA Protein

Background: Resistance to the critical first line anti-tubercular drug, Pyrazinamide, is a significant obstacle to achieving the global end to tuberculosis targets. Approximately 50% of multidrug-resistant tuberculosis and over 90% of extensively drug-resistant tuberculosis strains are also Pyrazinamide resistant. Pyrazinamide is a pro-drug that reduce the duration of tuberculosis therapy time by 9-12 months, while used as an anti-biotic in the 1st- & 2nd-line tuberculosis treatment regimens. Pyrazinamidase is an enzyme, encoded by pncA gene, is responsible for the amide hydrolysis of Pyrazinamide into active Pyrazinoic acid. Pyrazinoic acid, could inhibit trans-translation by binding to Ribosomal protein S1 and competing with tmRNA, the natural cofactor of Ribosomal protein S1. Although pncA mutations have been commonly associated with Pyrazinamide resistance, a small number of resistance cases have been associated with mutations in Ribosomal protein S1. Ribosomal protein S1was recently identified as a possible target of Pyrazinamide based on its binding activity to Pyrazinoic acid and the capacity to inhibit trans-translation. Objective: Despite the critical role played by Pyrazinamide, its mechanisms of action are not yet fully understood. Therefore, an effort to explore the resistance mechanism toward Pyrazinamide drug in Mycobacterium (M.) tuberculosis. Methods: An extensive molecular dynamics simulation was performed using the AMBER software package. We mutated residues of the binding site (i.e., F307A, F310A, and R357A) in the RpsA S1 domain to address the drug-resistant mechanism of RpsA in complex that might be responsible for Pyrazinamide resistance. Moreover, it is challenging to collect the drug mutant combine complex of a protein by single-crystal X-ray diffraction. Thus, the total three structures were prepared by inducing mutations in the wild-type protein using PyMol. Results: The dynamics results revealed that mutation in binding pocket produced Pyrazinamide resistance due to the specificity of these residues in binding pockets which result in scarcity of hydrophobic and hydrogen bonding interaction with Pyrazinoic acid, which increases the CA-distance between the binding pocket residues as compared to wild type RpsA that lead to structural instability. Conclusion: The overall dynamic results will provide useful information behind the drug resistance mechanism to manage tuberculosis and also helps in better management for future drug resistance.

Assessment of the Physicochemical Properties and Stability for Pharmacokinetic Prediction of Pyrazinoic Acid Derivatives

Background: Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, which still has high prevalence worldwide. In addition, cases of drug resistance are frequently observed. In the search for new anti-TB drugs, compounds with antimycobacterial activity have been developed, such as derivatives of pyrazinoic acid, which is the main pyrazinamide metabolite. In a previous study, the compounds were evaluated and showed moderate antimycobacterial activity and no important cytotoxic profile; however, information about their pharmacokinetic profile is lacking. Objective: The aim of this work was to perform physicochemical, permeability, and metabolic properties of four pyrazinoic acid esters. Methods: The compounds were analyzed for their chemical stability, n-octanol:water partition coefficient (logP) and apparent permeability (Papp) in monolayer of Caco-2 cells. The stability of the compounds in rat and human microsomes and in rat plasma was also evaluated. Results: The compounds I, II and IV were found to be hydrophilic, while compound III was the most lipophilic (logP 1.59) compound. All compounds showed stability at the three evaluated pHs (1.2, 7.4 and 8.8). The apparent permeability measured suggests good intestinal absorption of the compounds. Additionally, the compounds showed metabolic stability under action of human and rat microsomal enzymes and stability in rat plasma for at least 6 hours. Conclusion: The results bring favorable perspectives for the future development of the evaluated compounds and other pyrazinoic acid derivatives.

In-silico study of antituberculous activity of Zn-pyrazinamide in pyrazinamidase

Tuberculosis is caused by Mycobacterium tuberculosis and is one of the leading causes of death. Treatment with pyrazinamide depends on the formation of the bioactive species, pyrazinoic acid (POA), catalyzed by the enzyme pyrazinamidase (PZAse). New mutant strains show resistance to PZA, therefore, it is necessary to search for new drugs. Metallodrugs can offer a synergistic effect on the biological activity of the metal and the drug. Recent studies by our group show anti-tuberculosis activity of pyrazinamide coordinated with Zn, however, the mechanism of action is unknown. In this work, an in-silico study was carried out in three stages: Quantum mechanical, molecular docking and molecular dynamics simulations. ZnPZA (Egap = 4.12 eV) presented greater chemical reactivity than PZA (Egap = 4.97 eV). Greater binding energy was found in ZnPZA-PZAse (-6.98 kcal/mol) than in PZA-PZAse (-6.48 kcal/mol). RMSD and RMSF show stability in PZA-PZAse and ZnPZA-PZAse dockings. Hydrogen bonds interaction of ZnPZA with the catalytic amino acids Asp8 and Lys96 occurs for 83 and 40 ns, respectively. It is concluded that ZnPZA could serve as a transporter of PZA to the active site of PZAse, to promote the production of POA and the antituberculous effect; however, further experimental studies are needed.