Evaluation of vanillic acid as inhibitor of carbonic anhydrase isozyme III by using a modified Hummel–Dreyer method: approach for drug discovery

Abstract

α-3 carbonic anhydrase isozyme (CAIII) is the most abundant protein in adipocytes and considered insensitive to sulfonamide inhibitors. It was reported recently that the knock-down of CAIII is attributed with controlling lipogenesis. Thus inhibition of this target may lead to the discovery of new therapies against obesity and insulin resistance. Vanillic acid as a small molecule with coordinating groups and has a potential to bind zinc atoms in CA binding sites. Inhibition of CAIII by vanillic acid was evaluated by Hummel-Dreyer chromatography because it provides free interaction between ligand and macromolecule and introduces solution for faulty results obtained by current colorimetric assays. HPLC system of vanillic acid produces vacancy (negative) peak representing the amount of attached vanillic acid with CAIII. It was found that vanillic acid is able to bind with CAIII through two equilibria, one at equimolar ratio and another at 2:1 (vanillic acid-CAIII) ratio. The affinity constant of equimolar binding between CAIII and vanillic acid was found to be 14,400 m(-1) . It was found that vanillic acid binding with CAIII is much stronger than phenol and acetazolamide (positive controls).

Synthesis and evaluation of 2,4,5-trisubstitutedthiazoles as carbonic anhydrase-III inhibitors

“…The column temperature was gradually increased from 25 to 50 C (which is the maximum tolerated temperature by the column). Selected temperatures were: 25,30,35,36,37,38,40,45, and 50 C. There was a sharp increase in the interaction by increasing temperature, however, increasing temperature could affect the column longevity, thereby 37 C was chosen as the optimum temperature.…”

“…CA-III inhibitory activities of all the 17 target compounds (12-16b) as shown in Table 1 were carried out by using size exclusion HDM chromatography [35][36][37] . The HDM chromatography was preferred over colorimetric assay of CA inhibitors, due to the susceptibility of the colorimetric methods to the intrinsic acidic or alkaline properties of analytes (inhibitors), including the lower catalytic activity of CA-III in CO 2 hydration.…”

“…The HDM chromatography was preferred over colorimetric assay of CA inhibitors, due to the susceptibility of the colorimetric methods to the intrinsic acidic or alkaline properties of analytes (inhibitors), including the lower catalytic activity of CA-III in CO 2 hydration. Moreover, in HDM chromatographic method, the dilution effect of the mobile phase not only provides comparatively larger space for interaction between the macromolecule (CA-III) and analytes, but also lowers the probability of selfaggregation of macromolecules [35][36][37]41 . The HDM was further optimised prior to use, where mobile phase was buffered to maintain the pH ¼ 6.5 to mimic the physiological condition and to minimise the intrinsic properties of analytes.…”

“…The chromatographic analysis by HDM is basically based on the measurement of the intensity of the formation of vacancy (negative) peak due to the subtraction of CA-III bound portion of analytes from the mobile phase. This method is also independent of the enzyme catalytic activity, thereby considered as more sensitive method for the estimation of inhibitory activities of analytes as compared to the colorimetric assay [35][36][37]41 . Thus, weak inhibitors created weak vacancy peaks independent of the enzyme concentrations.…”

Safety Assessment of Vanillic Acid: Subacute Oral Toxicity Studies in Wistar Rats

“…In addition, ameliorates glomerulonephritis, 18 had the cognitive effect in diabetic mice 19 and its presence was reported in cerebospinalfluid 20 . It was reported to inhibit carbonic anhydrase isozyme III 21 and snake venom 5'-nucleotidas 22 . It controls transgene expression in mammalian cells and mice 23 .…”