Mycobacterium tuberculosis (Mtb) aspartate decarboxylase PanD is required for biosynthesis of the essential cofactor coenzyme A and targeted by the first line drug pyrazinamide (PZA). PZA is a ...prodrug that is converted by a bacterial amidase into its bioactive form pyrazinoic acid (POA). Employing structure–function analyses we previously identified POA-based inhibitors of Mtb PanD showing much improved inhibitory activity against the enzyme. Here, we performed the first structure–function studies on PanD encoded by the nontuberculous mycobacterial lung pathogen Mycobacterium abscessus (Mab), shedding light on the differences and similarities of Mab and Mtb PanD. Solution X-ray scattering data provided the solution structure of the entire tetrameric Mab PanD, which in comparison to the structure of the derived C-terminal truncated Mab PanD1–114 mutant revealed the orientation of the four flexible C-termini relative to the catalytic core. Enzymatic studies of Mab PanD1–114 explored the essentiality of the C-terminus for catalysis. A library of recombinant Mab PanD mutants based on structural information and PZA/POA resistant PanD mutations in Mtb illuminated critical residues involved in the substrate tunnel and enzymatic activity. Using our library of POA analogues, we identified (3-(1-naphthamido)pyrazine-2-carboxylic acid) (analogue 2) as the first potent inhibitor of Mab PanD. The inhibitor shows mainly electrostatic- and hydrogen bonding interaction with the target enzyme as explored by isothermal titration calorimetry and confirmed by docking studies. The observed unfavorable entropy indicates that significant conformational changes are involved in the binding process of analogue 2 to Mab PanD. In contrast to PZA and POA, which are whole-cell inactive, analogue 2 exerts appreciable antibacterial activity against the three subspecies of Mab.
Pyrazinoic acid (PA) is structurally similar to nicotinic acid which acts on G-protein-coupled receptor (GPR109A). GPR109A expresses in colonic and intestinal epithelial sites, and involves in DNA ...methylation and cellular apoptosis. Therefore, it may be assumed that PA has similar action like nicotinic acid and may be effective against colorectal carcinoma (CRC). CRC was produced via subcutaneous injection of dimethylhydrazine (DMH) at 40 mg/kg body weight once in a week for 4 weeks. After that, PA was administered orally at 2 doses of 10 and 25 mg/kg daily for 15 d to observe the antiproliferative effect. Various physiologic, oxidative stress, molecular parameters, histopathology, RT-PCR and NMR based metabolomics were performed to evaluate the antiproliferative potential of PA. Our results collectively suggested that PA reduced body weight, tumor volume and incidence no. to normal. It restored various oxidative stress parameters and normalized IL-2, IL-6, and COX-2 as compared with carcinogen control. In molecular level, overexpressed IL-6 and COX-2 genes became normal after PA administration. Again, normal tissue architecture was prominent after PA administration. Score plots of PLS-DA models exhibited that PA treated groups were significantly different from CRC group. We found that CRC rat sera have increased levels of acetate, glutamine, o-acetyl-glycoprotein, succinate, citrulline, choline, o-acetyl choline, tryptophan, glycerol, creatinine, lactate, citrate and decreased levels of 3-hydroxy butyrate, dimethyl amine, glucose, maltose, myoinositol. Further the PA therapy has ameliorated the CRC-induced metabolic alterations, signifying its antiproliferative properties. In conclusion, our study provided the evidence that PA demonstrated good antiproliferative effect on DMH induced CRC and thus demonstrated the potential of PA as a useful drug for future anticancer therapy.
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 IC
50
of 6.11, 10.64, 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 able to reactive oxygen species (ROS) induce DNA cleavage in the presents of H
2
O
2
(1.0 mM) in high doses. 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.
Graphical abstract
Pyrazinamide (PYZ)—an essential component of primary drug regimen used for the treatment and management of multidrug resistant or latent tuberculosis—is well known for its hepatoxicity. However, the ...mechanism of PYZ-induced hepatotoxicity is still unknown to researchers. Studies have shown that the drug is metabolized in the liver to pyrazinoic acid (PA) and 5-hydroxy pyrazinoic acid (5-OHPA) which individually may cause different degrees of hepatotoxicity. To evaluate this hypothesis, PYZ, PA, and 5-OHPA were administered to albino Wistar rats orally (respectively, at 250, 125, and 125 mg kg−1 for 28 days). Compared to normal rats, PYZ and its metabolic products decreased the weights of dosed rats and induced liver injury and a status of oxidative stress as assessed by combined histopathological and biochemical analysis. Compared to normal controls, the biochemical and morphological changes were more aberrant in PA- and 5-OHPA-dosed rats with respect to those dosed with PYZ. Finally, the serum metabolic profiles of rats dosed with PYZ, PA, and 5-OHPA were measured and compared with those of normal control rats. With respect to normal control rats, the rats dosed with PYZ and 5-OHPA showed most aberrant metabolic perturbations in their sera as compared to those dosed with PA. Altogether, the study suggests that PYZ-induced hepatotoxicity might be associated with its metabolized products, where 5-OHPA contributes to a higher degree in its overall toxicity than PA.
Lipophilic pyrazinoic acid amide and ester prodrugs Simões, Marta Filipa; Valente, Emília; Gómez, M. José Rodríguez ...
European journal of pharmaceutical sciences,
6/2009, Volume:
37, Issue:
3
Journal Article
Peer reviewed
Pyrazinamide (PZA) is active against
M. tuberculosis and is a first line agent for the treatment of human tuberculosis. PZA is itself a prodrug that requires activation by a pyrazinamidase to form ...its active metabolite pyrazinoic acid (POA). Since the specificity of cleavage is dependent on a single bacterial enzyme, resistance to PZA is often found in tuberculosis patients. Esters of POA have been proposed in the past as alternatives to PZA however the most promising compounds were rapidly degraded in the presence of serum. In order to obtain compounds that could survive during the transport phase, we synthesized lipophilic ester and amide POA derivatives, studied their activity against
M. tuberculosis, their stability in plasma and rat liver homogenate and also their activation by a mycobacterial homogenate. The new lipophilic ester prodrugs were found to be active in concentrations 10-fold lower than those needed for PZA to kill sensitive
M. tuberculosis and also have a suitable stability in the presence of plasma. Amides of POA although more stable in plasma have lower activity. The reason can probably be found in the rate of activation of both types of prodrugs; while esters are easily activated by mycobacterial esterases, amides are resistant to activation and are not transformed into POA at a suitable rate.
The title 1:1 salt, C
7
H
8
NO
2
+
·C
5
H
3
N
2
O
2
−
(systematic name: 4-carboxyanilinium pyrazine-2-carboxylate), was synthesized successfully by slow evaporation of a saturated solution from ...water–ethanol (1:1
v
/
v
) mixture and characterized by X-ray diffraction (SCXRD, PXRD) and calorimetry (DSC). The crystal structure of the salt was solved and refined at 150 and 293 K. The salt crystallizes with one molecule of 4-aminobenzoic acid (PABA) and one molecule of pyrazinoic acid (POA) in the asymmetric unit. In the crystal, the PABA and POA molecules are associated via COOH...N
arom
heterosynthons, which are connected by N—H...O hydrogen bonds, creating zigzag chains. The chains are further linked by N—H...O hydrogen bonds and π–π stacking interactions along the
b
axis centroid-to-centroid distances = 3.7377 (13) and 3.8034 (13) Å at 150 and 293 K, respectively to form a layered three-dimensional structure.
The propensity of monocytes to migrate into sites of mycobacterium tuberculosis (TB) infection and then become infected themselves makes them potential targets for delivery of drugs intracellularly ...to the tubercle bacilli reservoir. Conventional TB drugs are less effective because of poor intracellular delivery to this bacterial sanctuary. This study highlights the potential of using semicrystalline delta inulin particles that are readily internalised by monocytes for a monocyte-based drug delivery system. Pyrazinoic acid was successfully attached covalently to the delta inulin particles via a labile linker. The formation of new conjugate and amide bond was confirmed using zeta potential, Proton Nuclear Magnetic Resonance (
HNMR) and Fourier transform infrared spectroscopy (FTIR). Scanning electron microscopy (SEM) confirmed that no significant change in size after conjugation which is an important parameter for monocyte targeting. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were used to establish the change in thermal properties. The analysis of in-vitro release demonstrated pH-triggered drug cleavage off the delta inulin particles that followed a first-order kinetic process. The efficient targeting ability of the conjugate for RAW 264.7 monocytic cells was supported by cellular uptake studies. Overall, our finding confirmed that semicrystalline delta inulin particles (MPI) can be modified covalently with drugs and such conjugates allow intracellular drug delivery and uptake into monocytes, making this system potentially useful for the treatment of TB.
The pro-drug pyrazinamide is hydrolyzed to pyrazinoic acid (POA) in its use for the treatment of tuberculosis. As a molecule with bactericidal activity, POA binds to the C-terminal S1 domain of ...ribosomal protein S1 from
Mycobacterium tuberculosis
(
Mt
RpsA
CTD
_S1) to inhibit trans-translation. Trans-translation is a critical component of protein synthesis quality control, and is mediated by transfer-messenger RNA. Here, we have determined the solution structure of
Mt
RpsA
CTD
_S1(280–368), and analyzed its structural dynamics by NMR spectroscopy. The solution structure of
Mt
RpsA
CTD
_S1(280–368) mainly consists of five anti-parallel β strands, two α helices, and two 3
10
helices. Backbone dynamics reveals that the overall structure of
Mt
RpsA
CTD
_S1(280–368) is rigid, but segment L326–V333 undergoes large amplitude fluctuations on picosecond to nanosecond time scales. In addition, residues V321, H322, V331 and D335 with large
R
ex
values exhibit significant chemical or conformational exchange on microsecond to millisecond time scale. Titration of the truncated
Mt
RpsA
CTD
_S1(280–368) with POA shows similar characteristics to titration of
Mt
RpsA
CTD
_S1(280–438) with POA. In addition, diverse length fragments of
Mt
RpsA
CTD
_S1 show various HN resonance signals, and we find that the interaction of
Mt
RpsA(369–481) with
Mt
RpsA
CTD
_S1(280–368)
K
d = (4.25 ± 0.15) mM is responsible for the structural difference between
Mt
RpsA
CTD
_S1(280–368) and
Mt
RpsA
CTD
_S1. This work may shed light on the underlying molecular mechanism of
Mt
RpsA
CTD
recognizing and binding POA or mRNA, as well as the detailed mechanism of interactions between
Mt
RpsA
CTD
_S1(280–368) and the additional C-terminal
Mt
RpsA(369–481).
Pyrazinamide, the first-line antitubercular drug, has been regarded the basic component of tuberculosis treatment for over sixty years. Researchers have investigated its effect on
for this long time, ...and as a result, new potential targets of pyrazinamide or its active form, pyrazinoic acid, have been found. We have designed and prepared 3-(phenyl-carbamoyl)pyrazine-2-carboxylic acids as more lipophilic derivatives of pyrazinoic acid. We also prepared methyl and propyl derivatives as prodrugs with further increased lipophilicity. Antimycobacterial, antibacterial and antifungal growth inhibiting activity was investigated in all prepared compounds. 3-(4-Nitrophenyl)carbamoylpyrazine-2-carboxylic acid (
) exerted high antimycobacterial activity against
H37Rv with MIC = 1.56 μg·mL
(5 μM). Propyl 3-{4-(trifluoromethyl)phenylcarbamoyl}pyrazine-2-carboxylate (
) showed also high antimycobacterial activity against
H37Rv with MIC = 3.13 μg·mL
. In vitro cytotoxicity of the active compounds was investigated and no significant cytotoxic effect was observed. Based to structural similarity to known inhibitors of decaprenylphosphoryl-β-d-ribose oxidase, DprE1, we performed molecular docking of the prepared acids to DprE1. These in silico experiments indicate that modification of the linker connecting aromatic parts of molecule does not have any negative influence on the binding.
Tuberculosis (TB) caused by
(
) has become a frequently deadly infection due to increasing antimicrobial resistance. This serious issue has driven efforts worldwide to discover new drugs effective ...against
. One research area is the synthesis and evaluation of pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the synthesis and biological evaluations of a series of ureidopyrazines. Compounds were synthesized by reacting alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate. Reactions were performed in pressurized vials using a CEM Discover microwave reactor with a focused field. Purity and chemical structures of products were assessed, and the final compounds were tested in vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5-(3-phenylureido)pyrazine-2-carboxylate (compound
, MIC
= 1.56 μg/mL, 5.19 μM) and propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound
, MIC
= 6.25 μg/mL, 18.91 μM) had high antimycobacterial activity against
H37Rv with no in vitro cytotoxicity on HepG2 cell line. Therefore
and
are suitable for further structural modifications that might improve their biological activity and physicochemical properties. Based on the structural similarity to 1-(2-chloropyridin-4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds were evaluated for similar activity as abiotic elicitors.
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