Inhibition Profiling of Human Carbonic Anhydrase II by High-Throughput Screening of Structurally Diverse, Biologically Active Compounds

Abstract

Human carbonic anhydrase II (CA II), a zinc metalloenzyme, was screened against 960 structurally diverse, biologically active small molecules. The assay monitored CA II esterase activity against the substrate 4-nitrophenyl acetate in a format allowing high-throughput screening. The assay proved to be robust and reproducible with a hit rate of approximately 2%. Potential hits were further characterized by determining their IC(50) and K(d) values and tested for nonspecific, promiscuous inhibition. Three known sulfonamide CA inhibitors were identified: acetazolamide, methazolamide, and celecoxib. Other hits were also found, including diuretics and antibiotics not previously identified as CA inhibitors, for example, furosemide and halazone. These results confirm that many sulfonamide drugs have CA inhibitory properties but also that not all sulfonamides are CA inhibitors. Thus many, but not all, sulfonamide drugs appear to interact with CA II and may target other CA isozymes. The screen also yielded several novel classes of nonsulfonamide inhibitors, including merbromin, thioxolone, and tannic acid. Although these compounds may function by some nonspecific mechanism (merbromin and tannic acid), at least 1 (thioxolone) appears to represent a genuine CA inhibitor. Thus, this study yielded a number of potentially new classes of CA inhibitors and preliminary experiments to characterize their mechanism of action.

Inhibition of Carbonic Anhydrase II by Thioxolone: A Mechanistic and Structural Study

“…The CA II expression plasmid, pACA, was a generous gift from Dr. Carol Fierke, University of Michigan. Bacterial expression and purification of CA II on a SP Sepharose Fast Flow (Amersham Biosciences/GE Healthcare, Piscataway, NJ) cation exchange column were performed as described previously (1). SDS-PAGE analysis indicated that the CA II had greater than 95% purity.…”

“…The upper limit of substrate concentration (0.5 mM) was within the typical range of 0.4 to 1.0 mM 4-NPA concentration used in CA activity assays (1,52,65) and is far in excess of the enzyme concentration (1 µM). The substrate concentration is well below experimental estimates of K M for this enzyme-substrate combination, which, because of the relatively low solubility of 4-NPA in aqueous solution, vary between 4 and 22 mM (52,81).…”

“…Thioxolone (compound 1, (1). The mechanism of CA II inhibition was not immediately obvious from the thioxolone structure.…”

“…ABSTRACT: This paper examines the functional mechanism of thioxolone, a compound recently identified as a weak inhibitor of human carbonic anhydrase II by Iyer et al (2006) J. Biomol. Screening 11,[782][783][784][785][786][787][788][789][790][791].…”

“…We recently performed a screening study of a model library of 960 structurally diverse, biologically active compounds against human CA II, using a well-known esterase assay with 4-nitrophenyl acetate (4-NPA) (1,(49)(50)(51)(52). This screen yielded a number of CA inhibitors, including thioxolone (6-hydroxy-1,3-benzoxathiol-2-one), a biologically active compound not previously described as a CA inhibitor (compound 1, Table 1).…”