Date of Award

1-2020

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

John Siekierka

Committee Member

Nina Goodey

Committee Member

David Rotella

Abstract

Determining the binding affinity and potency in vitro is one of the significant steps that can give a clue for a new candidate drug during the drug discovery process. Thermofluor is a method used in measuring binding affinity (Kd) of protein-ligands interaction through determining the change in thermal denaturation temperature of protein using real time PCR (RT-PCR). Kinetic analysis assay is used to screen a library of compounds to calculate their potencies (IC50) and inhibition constants (Ki) and it can be performed by spectrophotometer technique. In this study, we used bovine carbonic anhydrase II (BCA II) enzyme, and four of its inhibitors as a model to compare drug affinities, which were determined either by Fluorescence Thermal Shift Assay (FTSA) using Sypro Orange dye or kinetic assay using 4-Nitrophenyl acetate as a substrate to measure the nonphysiologically esterase activity of CA. The inhibitors studied were Methazolamide, Brinzolamide, Dorzolamide HCl and Mafenide HCl. The Kd values were determined to be 5.4±0.085 μM, 1.2±0.44 μM, 2.08±0.63 μM, and IC50 values were 0.148±0.024 μM 0.129±0.015 μM 0.092±0.01 μM 1.715±0.16 μM whereas the Ki values were 4±0.55 nM, 3.5±0.5 nM, 2.5±0.5 nM and 46.5±6.5 nM for Methazolamide, Brinzolamide, Dorzolamide HCl and Mafenide HCl, respectively. The potencies (IC50) of the inhibitors were10-50 fold lower than that of the Kd values. In addition, Kd values were higher compared to Ki values. Therefore, kinetic analysis is a more sensitive technique and requires a lower amount of the enzyme to measure drug affinity than FTSA.

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