Investigation of the effects of pyrazole-3,4-dicarboxamide derivatives on carbonic anhydrase isoenzymes in vitro conditions

Carbonic anhydrase (CA) (E.C. 4.2.1.1) is a metalloenzyme with Zn2+ in its active site that catalyses the reversible hydration of carbon dioxide and water. CA is expressed by eight gene families (α, β, γ, δ, η, θ, ι). α-CA is the CA family found in mammals. α-CA has 15 isoenzymes in humans and 16 isoenzymes in other mammals. hCA I and hCA II isoenzymes show a wider distribution compared to other hCA isoenzymes. Since CA isoenzymes are involved in a wide variety of physiological and pathological processes, it is thought that they can be used for the treatment of diseases such as obesity, neuropathic pain, arthritis, in addition to the use of diuretics, glaucoma, cancer in the clinic. In the study, hCA I and hCA II isoenzymes were purified from human erythrocytes using affinity chromatography. hCA I isoenzyme was purified with 17.23% yield and its specific activity was calculated as 1027.06 EU/mg protein. hCA II isoenzyme was purified with 24.71% yield and its specific activity was determined as 1818.97 EU/mg protein. Qualitative and quantitative protein determinations were performed on the purified enzymes and enzyme purity was checked by SDS-PAGE. The inhibition potential of 10 inhibitor compounds was determined by esterase activity method. IC50 and Ki values of the compounds with inhibition potential were calculated. Ki values for hCA I were between 0.024 - 0.496 µM, while Ki values for hCA II were between 0.006 - 5.441 µM. In silico inhibition effects of the compounds with high inhibition potential were also examined. Keywords: Carbonic Anhydrase, IC50 Values, Inhibition, Ki Values, Pyrazole Carboxamide