Probing the Surface of Human Carbonic Anhydrase for Clues towards the Design of Isoform Specific Inhibitors

Abstract

The alpha carbonic anhydrases (α-CAs) are a group of structurally related zinc metalloenzymes that catalyze the reversible hydration of CO2 to HCO3 −. Humans have 15 different α-CAs with numerous physiological roles and expression patterns. Of these, 12 are catalytically active, and abnormal expression and activities are linked with various diseases, including glaucoma and cancer. Hence there is a need for CA isoform specific inhibitors to avoid off-target CA inhibition, but due to the high amino acid conservation of the active site and surrounding regions between each enzyme, this has proven difficult. However, residues towards the exit of the active site are variable and can be exploited to design isoform selective inhibitors. Here we discuss and characterize this region of “selective drug targetability” and how these observations can be utilized to develop isoform selective CA inhibitors.

Mapping Selective Inhibition of the Cancer-Related Carbonic Anhydrase IX Using Structure–Activity Relationships of Glucosyl-Based Sulfamates

“…hCA IX inhibitor development is complicated by the fact that the 15 CA isoforms share a conserved active site architecture with limited sequence variation between residues. 19,20 However, isoform selectivity among hCA inhibitors has been shown to be feasible by utilizing structural information on the active sites, and a method termed the "tail approach," whereby inhibitors are designed utilizing a chemical group that coordinates to the active site zinc (zinc-binding group, ZBG) conjugated to a variable "tail" region designed to interact with residues toward the surface of the active site (discussed in detail by Pinard et al 19 ). We have previously shown that a series of glucosyl sulfamates, where the sulfamate acts as a ZBG and the glucose or acetylglucose group acts as the "tail" (Figure 1), are excellent hCA IX inhibitors that show capabilities to inhibit the enzyme better than off-target CA isoforms (hCA I and II) and also show limited membrane permeability allowing for location specific targeting of the extracellular hCA IX over other cytosolic hCAs.…”

Targeting Aggressive Cancers with an Artificial Sweetener: Could Saccharin be a Lead Compound in Anticancer Therapy?

“…This comes from data that have shown saccharin's ability to inhibit the carbonic anhydrase (CA) family of enzymes, and especially the cancer-related isoforms IX and XII [8][9][10][11]. CAs are zinc metalloproteins that catalyze the interconversion of CO 2 to bicarbonate and a proton, a reaction that is important for many physiological processes including respiration, fluid secretion and pH regulation [12]. Interestingly, CA IX and XII, two of the 15 CA isoforms found in humans, have been shown to have an elevated expression profile in aggressive cancers [13][14][15].…”

“…These results, in combination with the enzymes differential expression patterns in several cancers, have established both CA IX and XII as targets for aggressive cancer. Despite the positive effect observed when targeting CA IX and XII with inhibitors for treating a wide array of cancers, drug development has been challenging due to the structural homology between them and other off-target CA isoforms [12,14]. Specifically, the active sites of CA IX and XII are highly conserved compared with those that are ubiquitously expressed, such as CA I and II [12].…”

“…Despite the positive effect observed when targeting CA IX and XII with inhibitors for treating a wide array of cancers, drug development has been challenging due to the structural homology between them and other off-target CA isoforms [12,14]. Specifically, the active sites of CA IX and XII are highly conserved compared with those that are ubiquitously expressed, such as CA I and II [12]. This also negates the possibility of using clinically approved CA inhibitors in the treatment of cancer since these compounds do not exhibit isoform selectivity [12,21].…”

“…Specifically, the active sites of CA IX and XII are highly conserved compared with those that are ubiquitously expressed, such as CA I and II [12]. This also negates the possibility of using clinically approved CA inhibitors in the treatment of cancer since these compounds do not exhibit isoform selectivity [12,21]. As such, this has prompted directed research into the design and discovery of isoform selective inhibitors to target both CA IX and XII [14,22].…”