ABSTRACT
The rise of pyrazinamide (PZA)-resistant strains of Mycobacterium tuberculosis (MTB) poses a major challenge to conventional tuberculosis (TB) treatments. PZA, a cornerstone of TB therapy, must be activated by the mycobacterial enzyme pyrazinamidase (PZase) to convert its active form, pyrazinoic acid, which targets the ribosomal protein S1. Resistance, often associated with mutations in the RpsA protein, complicates treatment and highlights a critical gap in the understanding of structural dynamics and mechanisms of resistance, particularly in the context of the G97D mutation. This study utilizes a novel integration of computational techniques, including multiscale biomolecular and molecular dynamics simulations, physicochemical and medicinal chemistry predictions, quantum computations and virtual screening from the ZINC and Chembridge databases, to elucidate the resistance mechanism and identify lead compounds that have the potential to improve treatment outcomes for PZA-resistant MTB, namely ZINC15913786, ZINC20735155, Chem10269711, Chem10279789 and Chem10295790. These computational methods offer a cost-effective, rapid alternative to traditional drug trials by bypassing the need for organic subjects while providing highly accurate insight into the binding sites and efficacy of new drug candidates. The need for rapid and appropriate drug development emphasizes the need for robust computational analysis to justify further validation through in vitro and in vivo experiments.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Multidrug-Resistant , Tuberculosis , Humans , Pyrazinamide/chemistry , Pyrazinamide/metabolism , Pyrazinamide/pharmacology , Mycobacterium tuberculosis/genetics , Antitubercular Agents/chemistry , Antitubercular Agents/metabolism , Antitubercular Agents/pharmacology , Tuberculosis/microbiology , Mutation , Microbial Sensitivity TestsABSTRACT
Both latent and multidrug-resistant tuberculosis (TB) have been causing significant concern worldwide. A novel drug, pretomanid (PA-824), has shown a potent bactericidal effect against both active and latent forms of Mycobacterium tuberculosis (MTb) and a synergistic effect when combined with pyrazinamide and moxifloxacin. This study aimed to develop triple combination spray dried inhalable formulations composed of antitubercular drugs, pretomanid, moxifloxacin, and pyrazinamide (1:2:8 w/w/w), alone (PaMP) and in combination with an aerosolization enhancer, L-leucine (20 % w/w, PaMPL). The formulation PaMPL consisted of hollow, spherical, dimpled particles (<5 µm) and showed good aerosolization behaviour with a fine particle fraction of 70 %. Solid-state characterization of formulations with and without L-leucine confirmed the amorphous nature of moxifloxacin and pretomanid and the crystalline nature of pyrazinamide with polymorphic transformation after the spray drying process. Further, the X-ray photoelectron spectroscopic analysis revealed the predominant surface composition of L-leucine on PaMPL dry powder particles. The dose-response cytotoxicity results showed pyrazinamide and moxifloxacin were non-toxic in both A549 and Calu-3 cell lines up to 150 µg/mL. However, the cell viability gradually decreased to 50 % when the pretomanid concentration increased to 150 µg/mL. The in vitro efficacy studies demonstrated that the triple combination formulation had more prominent antibacterial activity with a minimum inhibitory concentration (MIC) of 1 µg/mL against the MTb H37Rv strain as compared to individual drugs. In conclusion, the triple combination of pretomanid, moxifloxacin, and pyrazinamide as an inhalable dry powder formulation will potentially improve treatment efficacy with fewer systemic side effects in patients suffering from latent and multidrug-resistant TB.
Subject(s)
Nitroimidazoles , Pyrazinamide , Tuberculosis, Multidrug-Resistant , Humans , Pyrazinamide/pharmacology , Pyrazinamide/chemistry , Moxifloxacin/pharmacology , Moxifloxacin/chemistry , Powders/chemistry , Leucine/chemistry , Aerosols/chemistry , Antitubercular Agents/pharmacology , Antitubercular Agents/chemistry , Tuberculosis, Multidrug-Resistant/drug therapy , Administration, Inhalation , Dry Powder Inhalers/methods , Particle SizeABSTRACT
As one of the oldest infectious diseases in the world, tuberculosis (TB) is the second most deadly infectious disease after COVID-19. Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), which can attack various organs of the human body. Up to now, drug-resistant TB continues to be a public health threat. Pyrazinamide (PZA) is regarded as a sterilizing drug in the treatment of TB due to its distinct ability to target Mtb persisters. Previously we demonstrated that a D67N mutation in Mycobacterium tuberculosis polynucleotide phosphorylase (MtbPNPase, Rv2783c) confers resistance to PZA and Rv2783c is a potential target for PZA, but the mechanism leading to PZA resistance remains unclear. To gain further insight into the MtbPNPase, we determined the cryo-EM structures of apo Rv2783c, its mutant form and its complex with RNA. Our studies revealed the Rv2783c structure at atomic resolution and identified its enzymatic functional groups essential for its phosphorylase activities. We also investigated the molecular mechanisms underlying the resistance to PZA conferred by the mutation. Our research findings provide structural and functional insights enabling the development of new anti-tuberculosis drugs.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Humans , Mycobacterium tuberculosis/genetics , Polyribonucleotide Nucleotidyltransferase/genetics , Cryoelectron Microscopy , Amidohydrolases , Microbial Sensitivity Tests , Antitubercular Agents/pharmacology , Pyrazinamide/chemistry , Pyrazinamide/therapeutic use , Tuberculosis/drug therapy , Tuberculosis/microbiology , Mutation , RNAABSTRACT
First-line tuberculostatic agents, Rifampicin (RIF), Isoniazid (ISH), Ethambutol (ETB), and Pyrazinamide (PZA) are generally administered as a fixed-dose combination (FDC) for improving patient adherence. The major quality challenge of these FDC products is their variable bioavailability, where RIF and its solid state are key factors. In this work, the analysis of the impact of the polymorphism in the performance of RIF in RIF-ISH and PZA-RIF-ISH combined products was carried out by an overall approach that included the development and validation of two methodologies combining near-infrared (NIR) spectroscopy and partial least squares (PLS) to the further evaluation of commercial products. For NIR-PLS methods, training and validation sets were prepared with mixtures of Form I/Form II of RIF, and the appropriate amount of ISH (for double associations) or ISH-PZA (for triple associations). The corresponding matrix of the excipients was added to the mixture of APIs to simulate the environment of each FDC product. Four PLS factors, reduced spectral range, and the combination of standard normal variate and Savitzky-Golay 1st derivative (SNV-D') were selected as optimum data pre-treatment for both methods, yielding satisfactory recoveries during the analysis of validation sets (98.5±2.0%, and 98.7±1.8% for double- and triple-FDC products, respectively). The NIR-PLS model for RIF-ISH successfully estimated the polymorphic purity of Form II in double-FDC capsules (1.02 ± 0.02w/w). On the other hand, the NIR-PLS model for RIF-ISH-PZA detected a low purity of Form II in triple FDC tablets (0.800 ± 0.021w/w), these results were confirmed by X-ray powder diffraction. Nevertheless, the triple-FDC tablets showed good performance in the dissolution test (Q=99-102%), implying a Form II purity about of 80% is not low enough to affect the safety and efficacy of the product.
Subject(s)
Antitubercular Agents , Rifampin , Humans , Rifampin/chemistry , Antitubercular Agents/chemistry , Isoniazid/chemistry , Pyrazinamide/chemistry , Ethambutol/chemistry , Tablets/chemistryABSTRACT
Tuberculosis (TB) is an airborne infection caused by the bacteria Mycobacterium Tuberculosis (MTB). It mainly affects the lungs and causes severe coughing, fever, and chest pains. With the rising prevalence of drug-resistant and inactive Tuberculosis (TB), there is an essential need to discover more effective molecules capable of combating this heinous illness. Pyrazinamide is a first-line tuberculosis therapy that shortens prophylactic duration from twelve to six months. The majority of presently used tuberculosis medications were found by a mix of serendipity and innovative chemical alterations of an existing lead drug. Given that the majority of these discoveries occurred years ago, there is a definite need to use fresh methodologies and technology for discovery to meet the grave danger posed by tuberculosis and the rise of treatment resistance strains. Although current research has provided significant insight into TB transmission, diagnosis, and treatment in the last four years, much more progress is needed to successfully reduce tuberculosis prevalence and eventually eradicate it. The disease continues to be a public health concern, second only to HIV/AIDS in high fatality rates. This review focuses on current efforts to translate the anti-tubercular activity of all known pyrazinamide analogues and proposes a novel approach for developing new anti-tubercular drugs based on the fusion of pyrazinamide with various heterocyclic rings that shorten treatment for drug-sensitive and multidrug-resistant tuberculosis.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis, Multidrug-Resistant , Tuberculosis , Humans , Pyrazinamide/pharmacology , Pyrazinamide/therapeutic use , Pyrazinamide/chemistry , Antitubercular Agents/pharmacology , Antitubercular Agents/therapeutic use , Antitubercular Agents/chemistry , Tuberculosis/drug therapy , Tuberculosis/microbiology , Tuberculosis, Multidrug-Resistant/drug therapy , Structure-Activity RelationshipABSTRACT
Drug products used for treating tuberculosis are one of the most widely reported medicines to be classified as falsified or substandard in low- and middle-income countries, representing a major hazard to health. The aim of this study was, firstly, to develop an ultra-performance liquid chromatography (UPLC) method which is able to analyze fixed combination tablets with up to four active pharmaceutical ingredients, including isoniazid, pyrazinamide, rifampicin, and ethambutol. Secondly, we aimed to optimize it through the design of experiments and multi-linear regression based on a central composite design and to validate it according to the guidelines of the International Conference on Harmonization. The application of this tools enabled the identification of the influential factors (flow rate, formic acid, and temperature) and their effects on the studied responses (retention factor and percentage for each drug) as part of the quality by design approach. The method proved to be to be linear in the range from 5.0 to 15 µg/mL for isoniazid, pyrazinamide, and rifampicin, being precise (<1%) and accurate (97−101%). In addition, the method validated for ethambutol proved to be linear from 1.4 to 4.2 µg/mL, as well as precise (0.54%) and accurate (97.3%). The method was stability indicated for all the active pharmaceutical ingredients studied and was able to detect two substandard formulations sampled on the African market.
Subject(s)
Substandard Drugs , Tuberculosis , Humans , Ethambutol/chemistry , Pyrazinamide/therapeutic use , Pyrazinamide/chemistry , Isoniazid/therapeutic use , Isoniazid/chemistry , Rifampin/therapeutic use , Rifampin/chemistry , Antitubercular Agents/therapeutic use , Antitubercular Agents/chemistry , Tuberculosis/drug therapy , Chromatography, Liquid , TabletsABSTRACT
Tuberculosis (TB), the most frequent bacterium-mediated infectious disease caused by Mycobacterium tuberculosis, 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 pncA 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.
Subject(s)
Antitubercular Agents , Drug Resistance, Bacterial/genetics , Mycobacterium tuberculosis , Pyrazinamide , Amidohydrolases/chemistry , Amidohydrolases/genetics , Antitubercular Agents/pharmacology , Microbial Sensitivity Tests , Molecular Docking Simulation , Mutation , Mycobacterium tuberculosis/genetics , Pyrazinamide/chemistry , Pyrazinamide/pharmacologyABSTRACT
The emergence of drug resistance continues to afflict TB control where drug resistant strains have become a global health concern. Contrary to drug-sensitive TB, the treatment of MDR/XDR-TB is more complicated requiring the administration of second-line drugs that are inefficient than the first line drugs and are associated with greater side effects. The emergence of drug resistant Mtb strains had coincided with an innovation void in the field of drug discovery of anti-mycobacterials. However, the approval of bedaquiline and delamanid recently for use in MDR/XDR-TB has given an impetus to the TB drug discovery. The review discusses the drug discovery efforts in the field of tuberculosis with a focus on the strategies adopted and challenges confronted by TB research community. Here, we discuss the diverse clinical candidates in the current TB drug discovery pipeline. There is an urgent need to combat the current TB menace through multidisciplinary approaches and strategies making use of the recent advances in understanding the molecular biology and pathogenesis of Mtb. The review highlights the recent advances in drug discovery, with the host directed therapeutics and nanoparticles-drug delivery coming up as important tools to fight tuberculosis in the future.
Subject(s)
Antitubercular Agents/chemistry , Extensively Drug-Resistant Tuberculosis/drug therapy , Mycobacterium tuberculosis/drug effects , Antitubercular Agents/administration & dosage , Antitubercular Agents/adverse effects , Antitubercular Agents/pharmacokinetics , Diarylquinolines/pharmacology , Diarylquinolines/standards , Drug Therapy, Combination , Ethambutol/chemistry , Ethambutol/pharmacology , Humans , Isoniazid/chemistry , Isoniazid/pharmacology , Nitroimidazoles/pharmacology , Nitroimidazoles/standards , Oxazoles/pharmacology , Oxazoles/standards , Pyrazinamide/chemistry , Pyrazinamide/pharmacology , Rifampin/chemistry , Rifampin/pharmacologyABSTRACT
Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNAPro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC50 values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.
Subject(s)
Adenosine Triphosphate/metabolism , Amino Acyl-tRNA Synthetases/antagonists & inhibitors , Biological Assay/methods , Computer Simulation , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Pyrazinamide/chemistry , Binding Sites , Crystallography, X-Ray , Enzyme Inhibitors/isolation & purification , Humans , Ligands , Models, Molecular , Protein ConformationABSTRACT
A common strategy employed in antibacterial drug discovery is the targeting of biosynthetic processes that are essential and specific for the pathogen. Specificity in particular avoids undesirable interactions with potential enzymatic counterparts in the human host, and it ensures on-target toxicity. Synthesis of pantothenate (Vitamine B5), which is a precursor of the acyl carrier coenzyme A, is an example of such a pathway. In Mycobacterium tuberculosis (Mtb), which is the causative agent of tuberculosis (TB), pantothenate is formed by pantothenate synthase, utilizing D-pantoate and ß-Ala as substrates. ß-Ala is mainly formed by the decarboxylation of l-aspartate, generated by the decarboxylase PanD, which is a homo-oliogomer in solution. Pyrazinoic acid (POA), which is 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 analogues with modifications at position 3 of the pyrazine ring. Analogue 2, which incorporates a bulky naphthamido group at this position, displayed a 1000-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.
Subject(s)
Antitubercular Agents/pharmacology , Carboxy-Lyases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Mycobacterium tuberculosis/enzymology , Pyrazinamide/analogs & derivatives , Antitubercular Agents/chemistry , Carboxy-Lyases/metabolism , Enzyme Inhibitors/chemistry , Humans , Models, Molecular , Mycobacterium tuberculosis/drug effects , Pyrazinamide/chemistry , Pyrazinamide/pharmacology , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
The urge for the development of a more efficient antibiotic crystalline forms led us to the disclosure of new antibiotic coordination frameworks of pyrazinamide, a well-known drug used for the treatment of tuberculosis, with some of the novel compounds unravelling improved antimycobacterial activity. Mechanochemistry was the preferred synthetic technique to yield novel compounds, allowing the reproduction of a 1D zinc framework, the synthesis of a novel hydrogen bonding manganese framework, and three new compounds with silver. The structural characterization of the novel forms is presented along with stability studies. The increased antimicrobial activity of the new silver-based frameworks against Escherichia coli, Staphylococcus aureus, and Mycobacterium smegmatis is particularly relevant.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Coordination Complexes/chemical synthesis , Manganese/chemistry , Organometallic Compounds/chemical synthesis , Pyrazinamide/chemistry , Silver/chemistry , Zinc/chemistry , Anti-Bacterial Agents/pharmacology , Coordination Complexes/pharmacology , Drug Stability , Escherichia coli/drug effects , Escherichia coli/growth & development , Hydrogen Bonding , Microbial Sensitivity Tests , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/growth & development , Organometallic Compounds/pharmacology , Pyrazinamide/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/growth & development , Structure-Activity RelationshipABSTRACT
A new algorithm called Fast and Flexible CrystAl Structure Predictor (FFCASP) was developed to predict the structure of covalent and molecular crystals. FFCASP is massively parallel and able to handle more than 200 atoms in the unit cell (in other terms, it allows global optimization around 100 individual parameters). It uses a global optimizer specialized for Crystal Structure Prediction (CSP) which combines particle swarm and simulated annealing optimizers. Three different molecular crystals, including diverse intermolecular interactions, namely, cytosine, coumarin, and pyrazinamide, have been selected to evaluate the performance of FFCASP. While cytosine polymorphs have been searched by employing two different force fields (a DFT-SAPT based intermolecular potential and generalized amber force field (GAFF)) up to Z = 16, only GAFF has been used both in coumarin and pyrazinamide polymorph searches up to Z = 4. For these three molecular crystals, FFCASP generated more than 20â¯000 crystal structures, and the unique ones have been further treated by DFT-D3. A combination of data mining and a machine learning approach was introduced to determine the unique structures and their distribution into different clusters, which ultimately gives an opportunity to retrieve the common features and relations between the resulting structures. There are two known experimental crystal structures of cytosine, and both were successfully located with FFCASP. Two of the reported crystal structures of coumarin have been reproduced. Similarly, in pyrazinamide, three known experimental structures have been rediscovered. In addition to finding the experimentally known structures, FFCASP also located other low-energy structures for each considered molecular crystals. These successes of FFCASP offer the possibility to discover the polymorphic nature of other important molecular crystals (e.g., drugs) as well.
Subject(s)
Algorithms , Coumarins/chemistry , Cytosine/chemistry , Density Functional Theory , Pyrazinamide/chemistry , Crystallography, X-Ray , Models, Molecular , Molecular StructureABSTRACT
Favipiravir is an established antiviral that is currently being assessed as an investigational drug for the treatment of COVID-19. Favipiravir is strikingly similar to two molecules that the World Health Organization (WHO) lists as essential medicines, which also consist of a six-membered aromatic N-heterocycle bearing a carboxamide function: the anti-tuberculosis agent, pyrazinamide, and nicotinamide, also known as vitamin B3 . We demonstrate the utility of 1 H nuclear magnetic resonance (NMR) profiling, an emerging pharmacopoeial tool, for the highly specific identification, selective differentiation of congeners, and subsequent detection of drug falsification or adulteration of these medicines. The straightforward comparison of basic 1-D 1 H NMR spectra, obtained with benchtop or advanced NMR instruments alike, offers a rapid identity assay and works independently of physical reference materials. This approach accelerates and advances pharmaceutical quality control measures under situations of increased drug demand and altered economy, such as during a pandemic.
Subject(s)
Amides/analysis , Antiviral Agents/analysis , Drug Contamination/prevention & control , Niacinamide/analysis , Pyrazinamide/analysis , Pyrazines/analysis , Quality Control , Amides/chemistry , Antiviral Agents/chemistry , Niacinamide/chemistry , Proton Magnetic Resonance Spectroscopy , Pyrazinamide/chemistry , Pyrazines/chemistry , World Health OrganizationABSTRACT
BACKGROUND: Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the main killers of people all over the world. The major hurdles with existing therapy are the lengthy regimen and appearance of multi drug resistant (MDR) and extensively drug resistant (XDR) strains of M.tuberculosis. AIMS: The present work was aimed to synthesize and determine antitubercular and antimicrobial potential of some novel 3-chloro-4-aryl-1-[4-(5-pyrazin-2-yl[1,3,4]oxadiazole-2-ylmethoxy)-phenyl]-azetidin-2-one derivatives 7: (A: -H: ) from pyrazinoic acid as precursor, which is a well-established antitubercular agent. Here we report the synthesis of a new class of heterocyclic molecules in which pyrazine, 1, 3, 4-oxadiazole and azetidinone moieties were present in one frame work. METHODS: Pyrazinoic acid (1: ) was esterified first (2: ) followed by amination to produce hydrazide (3: ) which was refluxed with POCl3 to obtain 2-chloromethyl-5pyrazino-1, 3, 4-oxadiazole (4: ). This was then further reacted with 4-amino phenol to obtain 4-[5-pyrazino-1, 3, 4-oxadiazol-2-yl-methoxy]-phenyl amine (5: ) which on condensation with various aromatic aldehydes afforded a series Schiff's bases 6(A-H): . Dehydrative annulations of 6(A-H): in the presence of chloroacetyl chloride and triethylamine yielded 3-chloro-4-aryl-1-[4-(5-pyrazin-2-yl-[1, 3, 4]oxadiazole-2-ylmethoxy)-phenyl]-azetidin-2-one derivatives 7(A-H): . Antibacterial, antifungal and antitubercular potential of all the synthesized compounds were assessed. Docking study was performed using the software VLife Engine tools of Vlifemds 4.6 on the protein lumazine synthase of M. tuberculosis (PDB entry code 2C92). RESULTS: The present studies demonstrated that synthesized oxadiazole derivatives have good antimicrobial activity against the various microorganisms. Among the synthesized derivative, 7B: and 7G: were found to be prominent compounds which have potential antibacterial, antifungal and antitubercular activity (with MIC 3.12 µg/ml and high dock score ranging from -59.0 to -54.0) against Mycobacterium tuberculosis. CONCLUSIONS: Derivatives 7B: and 7G: would be effective lead candidates for tuberculosis therapy.
Subject(s)
Antifungal Agents/pharmacology , Antitubercular Agents/pharmacology , Pyrazinamide/analogs & derivatives , Antifungal Agents/therapeutic use , Antitubercular Agents/chemistry , Antitubercular Agents/therapeutic use , Aspergillus niger/drug effects , Azetidines/chemistry , Azetidines/pharmacology , Azetidines/therapeutic use , Candida albicans/drug effects , Drug Evaluation, Preclinical , Humans , Microbial Sensitivity Tests , Molecular Docking Simulation , Mycobacterium tuberculosis/drug effects , Mycoses/drug therapy , Mycoses/microbiology , Oxadiazoles/chemistry , Oxadiazoles/pharmacology , Oxadiazoles/therapeutic use , Pyrazinamide/chemistry , Pyrazinamide/pharmacology , Pyrazinamide/therapeutic use , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
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.
Subject(s)
Antitubercular Agents/toxicity , Drug Resistance, Bacterial , Mycobacterium tuberculosis/drug effects , Pyrazinamide/analogs & derivatives , Pyrazinamide/toxicity , Animals , Antibodies/chemistry , Antibodies/immunology , Enzyme-Linked Immunosorbent Assay/methods , Immunoconjugates/chemistry , Immunoconjugates/immunology , Inhibitory Concentration 50 , Pyrazinamide/chemistry , Pyrazinamide/immunology , Rabbits , Serum Albumin, Bovine/chemistry , Toxicity Tests/methodsABSTRACT
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. METHOD: 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.
Subject(s)
Pyrazinamide/analogs & derivatives , 1-Octanol/chemistry , Animals , Cell Line , Drug Stability , Humans , Hydrogen-Ion Concentration , Microsomes, Liver/metabolism , Permeability , Pyrazinamide/chemistry , Pyrazinamide/pharmacokinetics , Rats , Water/chemistryABSTRACT
The severity of tuberculosis (TB) in children is considered a global crisis compounded by the scarcity of pharmaceutical formulations suitable for pediatric use. The purpose of this study was to optimally develop and evaluate a pyrazinamide containing edible orodispersible film formulation potentially suitable for use in pediatrics actively infected with TB. The formulation was prepared employing aqueous-particulate blending and solvent casting methods facilitated by a high performance Box Behnken experimental design template. The optimized orodispersible formulation was mechanically robust, flexible, easy to handle, exhibited rapid disintegration with initial matrix collapse occurring under 60 s (0.58 ± 0.05 min ≡ 34.98 ± 3.00 s) and pyrazinamide release was controlled by anomalous diffusion coupled with matrix disintegration and erosion mechanisms. It was microporous in nature, light weight (57.5 ± 0.5 mg) with an average diameter of 10.5 mm and uniformly distributed pyrazinamide load of 101.13 ± 2.03 %w/w. The formulation was physicochemically stable with no evidence of destructive drug-excipient interactions founded on outcomes of characterization and environmental stability investigations. Preliminary inquiries revealed that the orodispersible formulation was cytobiocompatible, palatable and remained intact under specific storage conditions. Summarily, an edible pyrazinamide containing orodispersible film formulation was optimally designed to potentially improve TB pharmacotherapy in children, particularly the under 5 year olds.
Subject(s)
Antitubercular Agents/chemistry , Drug Compounding/methods , Drug Delivery Systems/methods , Drug Design , Mycobacterium tuberculosis , Pyrazinamide/chemistry , Tuberculosis, Pulmonary/drug therapy , Antitubercular Agents/adverse effects , Cell Survival/drug effects , Child , Child, Preschool , Drug Liberation , Drug Stability , Excipients/chemistry , Hep G2 Cells , Humans , Pyrazinamide/adverse effects , Solubility , Solvents/chemistry , Tuberculosis, Pulmonary/microbiologyABSTRACT
A pharmacophore design approach, based on the coordination chemistry of an intimate molecular hybrid of active metabolites of pro-drugs, known to release active species upon enzymatic oxidative activation, is devised. This is exemplified by combining two anti-mycobacterial drugs: pyrazinamide (first line) and delamanid (third line) whose active metabolites are pyrazinoic acid (PyzCOOH) and likely nitroxyl (HNO (or NO.)), respectively. Aiming to generate those active species, a hybrid compound was envisaged by coordination of pyrazine-2-hydroxamic acid (PyzCONHOH) with a Na3[FeII(CN)5] moiety. The corresponding pentacyanoferrate(II) complex Na4[FeII(CN)5(PyzCONHO-)] was synthesized and characterized by several spectroscopic techniques, cyclic voltammetry, and DFT calculations. Chemical oxidation of this complex with H2O2 was shown to induce the release of the metabolite PyzCOOH, without the need of the Mycobacterium tuberculosis (Mtb) pyrazinamidase enzyme (PncA). Control experiments show that both H2O2- and N-coordinated pyrazine FeII species are required, ruling out a direct hydrolysis of the hydroxamic acid or an alternative oxidative route through chelation of a metal center by a hydroxamic group. The release of HNO was observed using EPR spectroscopy in the presence of a spin trapping agent. The devised iron metal complex of pyrazine-2-hydroxamic acid was found inactive against an actively growing/non-resistant Mtb strain; however, it showed a strong dose-dependent and reversible vasodilatory activity with mostly lesser toxic effects than the reference drug sodium nitroprussiate, unveiling thus a potential indication for acute or chronic cardiovascular pathology. This is a priori a further indirect evidence of HNO release from this metal complex, standing as a possible pharmacophore model for an alternative vasodilator drug.
Subject(s)
Antitubercular Agents/chemical synthesis , Coordination Complexes/chemical synthesis , Ferrous Compounds/chemical synthesis , Hydroxamic Acids/chemistry , Iron/chemistry , Mycobacterium tuberculosis/drug effects , Nitrogen Oxides/chemistry , Amidohydrolases/metabolism , Antitubercular Agents/pharmacology , Coordination Complexes/pharmacology , Drug Discovery , Electron Spin Resonance Spectroscopy , Hydrogen Peroxide/chemistry , Ligands , Nitrogen Oxides/metabolism , Oxidation-Reduction , Pyrazinamide/analogs & derivatives , Pyrazinamide/chemistry , VasodilationABSTRACT
Nanoparticles-based drug delivery is at the forefront in the field of pharmaceutical and medicinal research to eradicate or alleviate the associated impediments, such as prolonged treatment time, high doses, toxicity and resistance problem of anti-tuberculosis drugs for the treatment of age-old tuberculosis disease. Herein, the first-line anti-tuberculosis drugs were loaded into the biodegradable starch nanocrystals and native starch to improve the therapeutic profile addressing the existing issues related to conventional drugs. The loading performance of anti-tuberculosis drugs with starch nanocrystals and native starch was found in the range of 65-95%. According to the release study, the native starch was not appropriate, however, the starch nanocrystals demonstrated sustained release drug delivery for isoniazid and pyrazinamide ranging from 50 to 93% for 24 h; the burst release for streptomycin was reported at pH 2 in 6.5 h while only 14% rifampicin was released at pH 8 buffer. An anti-mycobacterium analysis of strain H37Rv showed that minimum inhibition concentration of starch nanocrystals loaded with isoniazid and pyrazinamide (0.033 µg/mL and 1.25 µg/mL, respectively) were more effective than the parent isoniazid (0.2 µg/mL) and pyrazinamide (25.0 µg/mL) at pH 5.5.
Subject(s)
Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Nanoparticles/chemistry , Starch/pharmacology , Antitubercular Agents/chemistry , Microbial Sensitivity Tests , Molecular Conformation , Pyrazinamide/chemistry , Pyrazinamide/pharmacology , Rifampin/chemistry , Rifampin/pharmacology , Starch/chemistry , Streptomycin/chemistry , Streptomycin/pharmacologyABSTRACT
Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb), each year causing millions of deaths. In this article, we present the synthesis and biological evaluations of new potential antimycobacterial compounds containing a fragment of the first-line antitubercular drug pyrazinamide (PZA), coupled with methyl or ethyl esters of selected amino acids. The antimicrobial activity was evaluated on a variety of (myco)bacterial strains, including Mtb H37Ra, M. smegmatis, M. aurum, Staphylococcus aureus, Pseudomonas aeruginosa, and fungal strains, including Candida albicans and Aspergillus flavus. Emphasis was placed on the comparison of enantiomer activities. None of the synthesized compounds showed any significant activity against fungal strains, and their antibacterial activities were also low, the best minimum inhibitory concentration (MIC) value was 31.25 µM. However, several compounds presented high activity against Mtb. Overall, higher activity was seen in derivatives containing Ê-amino acids. Similarly, the activity seems tied to the more lipophilic compounds. The most active derivative contained phenylglycine moiety (PC-á´ /Ê-Pgl-Me, MIC < 1.95 µg/mL). All active compounds possessed low cytotoxicity and good selectivity towards Mtb. To the best of our knowledge, this is the first study comparing the activities of the á´ - and Ê-amino acid derivatives of pyrazinamide as potential antimycobacterial compounds.