Perfluoroalkyl Substances of Significant Environmental Concern Can Strongly Inhibit Human Carbonic Anhydrase Isozymes

Abstract

Perfluoroalkyl substances (PFASs) persist and are ubiquitous in the environment. The origins of PFAS toxicity and how they specifically affect the functions of proteins remain unclear. Herein, we report that PFASs can strongly inhibit the activity of human carbonic anhydrases (hCAs), which are ubiquitous enzymes that catalyze the hydration of CO2, are abundant in the blood and organs of mammals, and involved in pH regulation, ion homeostasis, and biosynthesis. The interactions between PFASs and hCAs were investigated using stopped-flow kinetic enzyme-inhibition measurements, native mass spectrometry (MS), and ligand-docking simulations. Narrow-bore emitters in native MS with inner diameters of ∼300 nm were used to directly and simultaneously measure the dissociation constants of 11 PFASs to an enzyme, which was not possible using conventional emitters. The data from native MS and stopped-flow measurements were in excellent agreement. Of 15 PFASs investigated, eight can inhibit at least one of four hCA isozymes (I, II, IX, and XII) with submicromolar inhibition constants, including perfluorooctanoic acid, perfluorooctanesulfonamide, and perfluorooctanesulfonic acid. Some PFASs, including those with both short and long perfluoromethylene chains, can effectively inhibit at least one hCA isozyme with low nanomolar inhibition constants.

Native mass spectrometry of human carbonic anhydrase I and its inhibitor complexes

“…In this case, the kinetic energy applied to the system results too high to keep intact the inhibitors bound to the protein. Since the main signal in all the spectra at 300 V belongs to the holo-CA (see SI), this is suggestive for a different binding energy between Zn-protein and inhibitor-Zn adduct, resulting this latter one less stable with higher acceleration energy [74][75][76]. This is perfectly consistent with the great stabilization that the Zn ion receives from the coordination to the three histidine residues, while the inhibitor establishes only one bond with the metal ion [76].…”

Carbonic Anhydrase Inhibitors Targeting Metabolism and Tumor Microenvironment

“…For these figures, the uncatalyzed reaction was not subtracted in the curves plotted in the supplementary figures, but those values were automatically subtracted when the IC 50 was calculated due to the use of the algorithm in the computer software that is used for the stopped-flow instrument. Despite these minor errors (now corrected through submission of errata), the inhibitory constants ( K i s) determined by the University of Florence stopped-flow kinetic measurements of CA enzyme inhibition have been validated by native mass spectrometry (MS) measurements performed in another laboratory on a number of sulfonamides, which gave results in excellent agreement with the kinetic inhibition data [ 130 , 131 ]. Dissociation constants ( K d s) were obtained from native mass spectrometry measurements of buffered aqueous solutions containing hCA I and hCA II with acetazolamide, ethoxzolamide, brinzolamide, furosemide, dichlorophenamide, and indapamide, which are well known CAIs [ 20 ].…”

“…Moreover, K d values obtained by native MS for the micromolar inhibitors were within 50% of the K i values. More recently, native MS was used to measure the K d values of 15 perfluoroalkyl substances with either carboxylate, sulfate, or sulfonamide zinc-binding groups to hCA I and hCA II [ 131 ]. The 30 measured K d values obtained from native MS measurements were also in excellent agreement with the corresponding K i measurements (data not shown here) [ 131 ].…”

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

“…Compounds 7-11 were synthesised following well known Drazen's reaction procedure 40 by treating various aldehydes (1-5) with methyldichloroacetate (6) in dry ether under basic condition using sodium methoxide. Compounds 7-11 on further reaction with thiourea under reflux condition in methanol resulted in the formation of methyl 2-amino-5-substituted-4-carboxylate derivatives (12)(13)(14)(15)(16). Hydrolysis of compounds 12-16 with aqueous sodium hydroxide solution resulted in the formation of 2-amino-5-substituted-4-carboxylic acid derivatives (12a-16a).…”

“…A large number of potential small-molecule inhibitors have been developed and some are under current clinical trial investigations targeting hCAs as potential therapeutic agents for the treatment of various diseases including diuretics, glaucoma, edema, obesity, osteoporosis, epilepsy, pain, malaria, and cancer [8][9][10][11][12][13][14][15] . It is worth mentioning that carboxylic acid group-containing environmental organic pollutants like perfluorinated alkyl substances (PFASs) do exhibit toxicity by inhibiting hCAs, which in turn lead to the disturbance of normal physiological functions 16 . However, hCA inhibitors (hCAIs) are found to be associated with various side effects mainly due to their lack of isoform specificity and off-target activity.…”