Acetazolamida

Communication pubs.acs.org/JACS

Neutron Diffraction of Acetazolamide-Bound Human Carbonic Anhydrase II Reveals Atomic Details of Drug Binding
S. Zoë Fisher,† Mayank Aggarwal,‡ Andrey Y. Kovalevsky,† David N. Silverman,§ and Robert McKenna*,‡
† ‡

Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, United States Department of Biochemistry and Molecular Biology,University of Florida, P.O. Box 100245, Gainesville, Florida 32610, United States § Department of Pharmacology and Therapeutics, University of Florida, P.O. Box 100247, Gainesville, Florida 32610, United States
S * Supporting Information

ABSTRACT: Carbonic anhydrases (CAs) catalyze the hydration of CO2 forming HCO3− and a proton, an important reaction for many physiological processesincluding respiration, fluid secretion, and pH regulation. As such, CA isoforms are prominent clinical targets for treating various diseases. The clinically used acetazolamide (AZM) is a sulfonamide that binds with high affinity to human CA isoform II (HCA II). There are several X-ray structures available of AZM bound to various CA isoforms, but these complexes do not show the charged state of AZM or thehydrogen atom positions of the protein and solvent. Neutron diffraction is a useful technique for directly observing H atoms and the mapping of H-bonding networks that can greatly contribute to rational drug design. To this end, the neutron structure of H/D exchanged HCA II crystals in complex with AZM was determined. The structure reveals the molecular details of AZM binding and the charged state ofthe bound drug. This represents the first determined neutron structure of a clinically used drug bound to its target.

arbonic anhydrase (CA) is a ubiquitous metalloenzyme found in all kingdoms of life, from plants to humans, catalyzing CO2 hydration (and bicarbonate dehydration). Humans have 15 isoforms that are expressed in diverse tissues. These isoforms can be cytosolic, transmembrane, ormembrane-bound.1 Human CA II (HCA II) is a monomeric ∼29 kDa soluble cytosolic isoform that contains zinc in the active site. HCA II is found predominantly in red blood cells and has the fastest catalytic turnover (106 s−1) among all the CAs characterized to date. Most CAs use a zinc-hydroxide (Zn− OH−) mechanism to reversibly convert CO2 to HCO3− and a proton, as illustrated in reactions 1 and 2:2EZnOH− + CO2 ↔ EZnHCO3− ↔ EZnH 2O + HCO3−
(1)

C

EZnH 2O + B ↔ EZnOH + BH

−

+

(2)

CA inhibitors (CAIs) have been used for decades as diuretics and antiglaucoma drugs. CAIs can be broadly classified into two groups based on their binding mechanism: (a) binding to the tetrahedral configuration of the Zn metal center, thus displacing the Zn-bound solvent (e.g., sulfonamides), or (b)by forming a
© 2012 American Chemical Society

trigonal-bipyramidal species through expansion of the metal coordination geometry without displacing the Zn-bound solvent (e.g., cyanates). The use of CAIs has now been expanded to include treatments for convulsions, obesity, and cancer, as well as being developed for use as diagnostic tools.3,4 In cancer, CA IX is believed to be responsible for theacidification of the extracellular matrix (ECM) when tumors become hypoxic.5 Due to the sequence conservation among CA isoforms in humans, clinically used CAIs target many isoforms. It should be noted that HCA II inhibition dominates as its expression is widespread, and HCA II has one of the highest catalytic rates. Most structure-based CAI designs have focused on HCA II Xray crystallographicstudies. There are numerous crystal structures in the Protein Data Bank (www.rcsb.pdb.org) of CAs bound to a variety of CAIs: the sulfonamides, their bioisosteres, small molecule anions, phenols, coumarins, and polyamines.6,7 Sulfonamides are still of significant interest with more than 30 derivatives being used clinically.8 The primary sequence conservation among CA isoforms causes cross-reactivity...