•Piperidine and acetonitrile react to amidine in the presence of zinc(II).•Zinc(II) oxide, zinc(II) chloride or zinc(II) acetate were used.•Additionally, picolinate or pyrazinoate was used as an ...N,O-chelating agent.•Products were various amidinium salts.
The influence of the zinc(II) starting material in the nucleophilic addition of piperidine (abbreviated as pipe) to acetonitrile to form amidine, piperidinoacetamidine (pipeam), was assessed. Thus, zinc(II) oxide, zinc(II) chloride or zinc(II) acetate were reacted with picolinic acid or pyrazinoic acid, piperidine and acetonitrile. The acid was used as a source of an N,O-donor ligand. The reactions with picolinic acid (picH) gave pipeamHZn(pic)3·0.5H2O (1), an amidinium salt of a homoleptic complex Zn(pic)3–. The same product was obtained when Zn(pic)(picH)Cl, an already known picolinate complex, was reacted under similar conditions. The chloride-containing mixtures produced another amidine compound, (pipeamH)Cl. The reactions with pyrazinoic acid (pyrazH) gave pipeamHZn(pyraz)3 (3), the amidinium salt of a homoleptic complex Zn(pyraz)3–. The acetate-containing mixture yielded acetamidinium acetate. The latter product indicates the presence of ammonia in the reaction. Notably, no amidine species was isolated when the coordinatively saturated pyrazinoate complex, Zn(pyraz)2n (2), was reacted with piperidine and acetonitrile. A detailed structural characterization of the new compounds and a discussion of their formation are presented.
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Pyrazinoic acid (POA) is a metabolite of the anti-tuberculosis drug pyrazinamide (PZA), and its detection can be used to assess the resistance of Mycobacterium tuberculosis in cultures, as only ...sensitive strains of the bacteria can metabolize PZA into POA. Prussian blue is a well-known metal-organic framework compound widely used in various sensing platforms such as electrochemical, photochemical, and magnetic sensors. In this study, we present a novel sensing platform based on Prussian blue-modified gold nanoparticles (AuNPs) designed to enhance the affinity of POA towards the sensing surface and to capture POA molecules from aqueous solutions. This SERS-based method allows for the selective enrichment of POA, which can be detected in both pure aqueous solution and in the presence of its pro-drug PZA. The limit of detection (LOD) for POA was estimated to be 1.08 μM in pure aqueous solution and 0.18 mM in the presence of PZA. Furthermore, the precision of the SERS method was verified by the relative standard deviation (RSD) of 3.34–12.02% for three parallel samples using different matrices, i.e. aqueous solution, spiked river water and spiked simulated saliva. The recoveries of the samples ranged from 92.65 to 118.51%. These all demonstrate the potential application of the proposed detection scheme in medical research.
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•SERS-based platform employing Prussian Blue (PB) for capturing the target analyte from aqueous solution.•High affinity of pyrazinoic acid (POA) towards the PB surface to enrich the analyte in close vicinity to the SERS-active surface.•Detection scheme in presence of the pro-drug pyrazinamide (PZA) shows selective capturing of POA.•High potential to be used in assessing resistance of Mycobacterium tuberculosis in culture medium.
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Combining the advantage of higher efficacy due to local pulmonary administration of pyrazinoic acid (POA) and potent effect of pyrazinoic acid ester (PAE) delivered as an aerosol ...would aid in tuberculosis therapy. A combination spray dried dry powder, composed of POA, PAE (n-propyl POA), maltodextrin and leucine, was prepared for aerosol delivery to animals. Solid-state characteristics of morphology (scanning electron microscopy) crystallinity (X-ray powder diffraction), thermal properties (thermogravimetric analysis and differential scanning calorimetry) and moisture content (Karl Fisher) were evaluated. Particle size distributions, by volume (laser diffraction) for the dispersed powder and by mass (inertial impaction) were determined. Efficient delivery of the powder to a nose only animal exposure chamber employed a novel rotating brush/micro-fan apparatus. Spherical, crystalline particles were prepared. The volume median diameter, ∼1.5μm, was smaller than the mass median aerodynamic diameter, ∼3.0μm, indicating modest aggregation. Drug content variations were observed across the particle size distribution and may be explained by PAE evaporative losses. Delivery to the nose-only exposure chamber indicated that boluses could be administered at approximately 3min intervals to avoid aerosol accumulation and effect uniform dose delivery with successive doses suitable for future pharmacokinetic and pharmacodynamic studies.
Pyrazinamide is an essential first‐line antitubercular drug which plays pivotal role in tuberculosis treatment. It is a prodrug that requires amide hydrolysis by mycobacterial pyrazinamidase enzyme ...for conversion into pyrazinoic acid (POA). POA is known to target ribosomal protein S1 (RpsA), aspartate decarboxylase (PanD), and some other mycobacterial proteins. Spontaneous chromosomal mutations in RpsA have been reported for phenotypic resistance against pyrazinamide. We have constructed and validated 3D models of the native and Δ438A mutant form of RpsA protein. RpsA protein variants were then docked to POA and long range molecular dynamics simulations were carried out. Per residue binding free‐energy calculations, free‐energy landscape analysis, and essential dynamics analysis were performed to outline the mechanism underlying the high‐level PZA resistance conferred by the most frequently occurring deletion mutant of RpsA. Our study revealed the conformational modulation of POA binding site due to the disruptive collective modes of motions and increased conformational flexibility in the mutant than the native form. Residue wise MMPBSA decomposition and protein‐drug interaction pattern revealed the difference of energetically favorable binding site in the wild‐type (WT) protein in comparison with the mutant. Analysis of size and shape of minimal energy landscape area delineated higher stability of the WT complex than the mutant form. Our study provides mechanistic insights into pyrazinamide resistance in Δ438A RpsA mutant, and the results arising out of this study will pave way for design of novel and effective inhibitors targeting the resistant strains of Mycobacterium tuberculosis.
Mutations in mycobacterial ribosomal protein S1 (RpsA) have been reported to cause resistance against pyrazinamide. Our study revealed the conformational modulation of drug‐binding site due to the disruptive collective modes of motions and increased conformational flexibility in the RpsA mutant. Analysis of size and shape of minimal energy landscape area delineated higher stability of the wild‐type complex than the mutant form. We have provided the mechanistic insights into pyrazinamide resistance in Δ438A RpsA mutant, and the results arising out of this study will pave way for design of novel and effective inhibitors targeting the resistant strains of Mycobacterium tuberculosis.
Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug-susceptible and multidrug-resistant tuberculosis (MDR-TB). PZA is a prodrug that is ...converted to its active form, pyrazinoic acid (POA), by a nicotinamidase/pyrazinamidase encoded by the pncA gene, the mutation of which is the major cause of PZA resistance. Although RpsA (ribosomal protein S1, involved in trans-translation) has recently been shown to be a target of POA/PZA, whole-genome sequencing has identified mutations in the panD gene encoding aspartate decarboxylase in PZA-resistant strains lacking pncA and rpsA mutations. To gain more insight into a possible new target of PZA, we isolated 30 POA-resistant mutants lacking mutations in pncA and rpsA from M. tuberculosis in vitro, and whole-genome sequencing of 3 mutants identified various mutations in the panD gene. Additionally, sequencing analysis revealed that the remaining 27 POA-resistant mutants all harbored panD mutations affecting the C-terminus of the PanD protein, with PanD M117I being the most frequent mutation (24/30, 80%). Conditional overexpression of panD from M. tuberculosis, M. smegmatis or E. coli, or of M. tuberculosis mutant PanD M117I, all conferred resistance to POA and PZA in M. tuberculosis. β-alanine and pantothenate, which are downstream products of PanD, were found to antagonize the antituberculosis activity of POA. In addition, the activity of the M. tuberculosis PanD enzyme was inhibited by POA at therapeutically relevant concentrations in a concentration-dependent manner but was not inhibited by the prodrug PZA or the control compound nicotinamide. These findings suggest that PanD represents a new target of PZA/POA. These results have implications for a better understanding of this peculiar persister drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of death with 1.6 million deaths worldwide reported in 2021. Oral pyrazinamide (PZA) is an integral part of ...anti-TB regimens, but its prolonged use has the potential to drive the development of PZA-resistant Mtb. PZA is converted to the active moiety pyrazinoic acid (POA) by the Mtb pyrazinamidase encoded by pncA, and mutations in pncA are associated with the majority of PZA resistance. Conventional oral and parenteral therapies may result in subtherapeutic exposure in the lung; hence, direct pulmonary administration of POA may provide an approach to rescue PZA efficacy for treating pncA-mutant PZA-resistant Mtb. The objectives of the current study were to (i) develop novel dry powder POA formulations, (ii) assess their feasibility for pulmonary delivery using physicochemical characterization, (iii) evaluate their pharmacokinetics (PK) in the guinea pig model, and (iv) develop a mechanism-based pharmacokinetic model (MBM) using in vivo PK data to select a formulation providing adequate exposure in epithelial lining fluid (ELF) and lung tissue. We developed three POA formulations for pulmonary delivery and characterized their PK in plasma, ELF, and lung tissue following passive inhalation in guinea pigs. Additionally, the PK of POA following oral, intravenous, and intratracheal administration was characterized in guinea pigs. The MBM was used to simultaneously model PK data following administration of POA and its formulations via the different routes. The MBM described POA PK well in plasma, ELF, and lung tissue. Physicochemical analyses and MBM predictions suggested that POA maltodextrin was the best among the three formulations and an excellent candidate for further development as it has: (i) the highest ELF-to-plasma exposure ratio (203) and lung tissue-to-plasma exposure ratio (30.4) compared with POA maltodextrin and leucine (75.7/16.2) and POA leucine salt (64.2/19.3) and (ii) the highest concentration in ELF (C maxELF: 171 nM) within 15.5 min, correlating with a fast transfer into ELF after pulmonary administration (K PM: 22.6 1/h). The data from the guinea pig allowed scaling, using the MBM to a human dose of POA maltodextrin powder demonstrating the potential feasibility of an inhaled product.
Pyrazinamidase (PZase) is a member of Fe-dependent amidohydrolases that activates pyrazinamide (PZA) into active pyrazinoic acid (POA). PZA, a nicotinamide analogue, is an essential first-line drug ...used in Mycobacterium tuberculosis (Mtb) treatment. The active form of PZA, POA, is toxic and potently inhibits the growth of latent Mtb, which makes it possible to shorten the conventional 9-month tuberculosis treatment to 6 months. In this study, an extensive molecular dynamics simulation was carried out to the study the resistance mechanism offered by the three mutations Q10P and D12A and G97D. Our results showed that two regions Gln10-His43, Phe50-Gly75 are profoundly affected by these mutations. Among the three mutations, Q10P and D12A mutations strongly disturb the communication among the catalytic triad (Asp8, Lys98 and Cys138). The oxyanion hole is formed between the backbone nitrogen atoms of A134 and C138 which stabilizes the hydroxyl anion of nicotinamide. The D12A mutation greatly disturbs the oxyanion hole formation followed by the Q10P and G97D. Our results also showed that these mutations destabilize the interaction between Fe
2+
ion and Asp49, His51, His57 and His71. The binding pocket analysis showed that these mutations increase the cavity volume, which results in loose binding of PZA. MMGBSA analyzes have shown that these mutations reduce the binding affinity to the PZA drug. Our results may provide useful information for the design of new and effective PZase inhibitors based on structural information of WT and mutant PZases.
Communicated by Ramaswamy H. Sarma
•First LC–MS/MS method for the simultaneous determination of pyrazinamide and its metabolites.•Most sensitive and rapid method in human plasma compared to all existing methods.•Chromatographic ...separation of the analytes within 4min.•Consistent and quantitative recovery with minimal matrix interference for all three analytes.•Successful clinical study with healthy subjects and incurred sample reanalysis.
In the present work the plasma levels of PZA and its two active metabolites, pyrazinoic acid (PA) and 5-hydroxy pyrazinoic acid (5-OH PA) were determined by a sensitive and rapid LC–MS/MS method. The analytes and their labeled internal standards were extracted from 200μL plasma samples by liquid-liquid extraction with methyl tert-butyl ether: diethyl ether (90:10, v/v) under acidic conditions. Their separation was achieved on a Zorbax Eclipse XDB C18 (100×4.6mm, 3.5μm) column using methanol and 0.1% acetic acid (65:35, v/v) as the mobile phase within 4.0min. Detection and quantitation were done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions, m/z 124.1→81.1,m/z 125.0→80.9 and m/z 141.0→81.0 for PZA, PA and 5-OH PA respectively in the positive ionization mode. All the analytes were baseline resolved with a resolution factor of 3.3 and 6.4 between PZA and its metabolites, PA and 5-OH PA respectively. The calibration curves were linear from 0.100–30.0μg/mL, 0.03–9.00μg/mL and 0.002–0.600μg/mL for PZA, PA and 5-OH PA respectively with r2≥0.9980 for all the analytes. The intra-batch and inter-batch accuracy and precision (% CV) across quality controls varied from 93.5–106.7% and 1.10–4.57 respectively for all the analytes. The mean extraction recovery of PZA, PA and 5-OH PA was 83.7%, 89.2% and 80.8% respectively, which was consistent at higher as well as lower concentration levels. The% change in the stability of analytes under different storage conditions ranged −6.7 to 7.1 for all the analytes. The method was applied to assess the comparative bioavailability of a 500mg PZA test and reference formulation in healthy subjects. The assay reproducibility was also tested by reanalysis of 22 incurred subject samples.
One of the most effective approaches to discovering novel drugs for cancer treatment involves the exploration of new synthetic compounds. The pyrazinoic acid or pyrazine‐2‐carboxylic acid ...(PA)‐derivative compounds can be explored as a new anticancer agent due to their nitrogenous heteroaromatic ring. In this study, ten novels PA derivatives were synthesized by Ugi multicomponent reaction and characterized using IR, NMR, and mass spectroscopy. The cytotoxic activity was assayed in three different cancer cell lines, including colon (HT‐29), lung (A549), and breast (MCF‐7). The U10 was the most potent compound, exhibiting moderate cytotoxicity with IC50 of 8.26 μM, 8.23 μM, and 22.58 μM against HT‐29, A549, and MCF‐7 cell lines, respectively. In addition, the effect of U10 exposure in the MRC‐5 cell line as a non‐tumoral lung cell line showed a selectivity index of 3.76. The apoptotic activity and intracellular ROS level induction of U10 were assayed in MCF‐7 cells. The results demonstrated that apoptosis increases from 23.94 % at 10 μM to 36.8 % at 25 μM. Intracellular ROS level assay showed that U10 was able to significantly increase intracellular ROS by increasing the concentration. Molecular docking was utilized to predict the binding sites and interactions between the synthesized compound and DNA, as well as the Bcl‐2 apoptosis regulator.
Ten novels PA derivatives were synthesized by Ugi multicomponent reaction. The cytotoxic activity was explored against colon (HT‐29), lung (A549), and breast (MCF‐7) cell lines. The apoptotic activity and intracellular ROS level induction of them were assayed in MCF‐7 cells. Molecular docking was explored on DNA and Bcl‐2 apoptosis regulator.
Pyrazinamide (PZA) is a critical component of first- and second-line treatments of tuberculosis (TB), yet its mechanism of action largely remains an enigma. We carried out a genetic screen to isolate ...Mycobacterium bovis BCG mutants resistant to pyrazinoic acid (POA), the bioactive derivative of PZA, followed by whole genome sequencing of 26 POA resistant strains. Rather than finding mutations in the proposed candidate targets fatty acid synthase I and ribosomal protein S1, we found resistance conferring mutations in two pathways: missense mutations in aspartate decarboxylase panD, involved in the synthesis of the essential acyl carrier coenzyme A (CoA), and frameshift mutations in the vitro nonessential polyketide synthase genes mas and ppsA-E, involved in the synthesis of the virulence factor phthiocerol dimycocerosate (PDIM). Probing for cross resistance to two structural analogs of POA, nicotinic acid and benzoic acid, showed that the analogs share the PDIM- but not the CoA-related mechanism of action with POA. We demonstrated that POA depletes CoA in wild-type bacteria, which is prevented by mutations in panD. Sequencing 10 POA-resistant Mycobacterium tuberculosis H37Rv isolates confirmed the presence of at least 2 distinct mechanisms of resistance to the drug. The emergence of resistance through the loss of a virulence factor in vitro may explain the lack of clear molecular patterns in PZA-resistant clinical isolates, other than mutations in the prodrug-converting enzyme. The apparent interference of POA with virulence pathways may contribute to the drug’s excellent in vivo efficacy compared to its modest in vitro potency.
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