ABSTRACT
Hypoxic pulmonary vasoconstriction (HPV) occurs with ascent to high altitude and can contribute to development of high altitude pulmonary edema (HAPE). Vascular smooth muscle contains carbonic anhydrase (CA), and acetazolamide (AZ), a CA inhibitor, blunts HPV and might be useful in the prevention of HAPE. The mechanism by which AZ impairs HPV is uncertain. Originally developed as a diuretic, AZ also has direct effects on systemic vascular smooth muscle, including modulation of pH and membrane potential; however, the effect of AZ on pulmonary arterial smooth muscle cells (PASMCs) is unknown. Since HPV requires Ca2+ influx into PASMCs and can be modulated by pH, we hypothesized that AZ alters hypoxia-induced changes in PASMC intracellular pH (pH(i)) or Ca2+ concentration ([Ca2+](i)). Using fluorescent microscopy, we tested the effect of AZ as well as two other potent CA inhibitors, benzolamide and ethoxzolamide, which exhibit low and high membrane permeability, respectively, on hypoxia-induced responses in PASMCs. Hypoxia caused a significant increase in [Ca2+](i) but no change in pH(i). All three CA inhibitors slightly decreased basal pH(i), but only AZ caused a concentration-dependent decrease in the [Ca2+](i) response to hypoxia. AZ had no effect on the KCl-induced increase in [Ca2+](i) or membrane potential. N-methyl-AZ, a synthesized compound lacking the unsubstituted sulfonamide group required for CA inhibition, had no effect on pH(i) but inhibited hypoxia-induced Ca2+ responses. These results suggest that AZ attenuates HPV by selectively inhibiting hypoxia-induced Ca2+ responses via a mechanism independent of CA inhibition, changes in pH(i), or membrane potential.
Subject(s)
Acetazolamide/pharmacology , Calcium/physiology , Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/metabolism , Hypoxia/physiopathology , Muscle, Smooth, Vascular/physiology , Pulmonary Artery/physiology , Benzolamide/pharmacology , Calcium Signaling/drug effects , Ethoxzolamide/pharmacology , Hydrogen-Ion Concentration , Methazolamide/pharmacologyABSTRACT
Ab initio calculations at the MP2/6-31G* level have shown that variously substituted di- and trifluorobenzenes form non-covalent complexes with benzene that adopt either aromatic-aromatic or H--F binding, the choice being determined by the pattern of fluorination. The binding energies of these structures are from 3.4 to 4.5 kcal mol(-1). This range is large enough to account for observed variations in the binding affinity of a library of fluoroaromatic inhibitors of carbonic anhydrase. This enzyme has an aromatic amino acid at a central position in the active site. The diverse modes of binding of the dimers also suggest that aggregates of fluorobenzenes might adopt specified 3-dimensional shapes in the solid state.
Subject(s)
Carbonic Anhydrase II/chemistry , Fluorobenzenes/chemistry , Benzene Derivatives , Binding Sites , Carbonic Anhydrase II/metabolism , Carbonic Anhydrase Inhibitors/chemistry , Computer Simulation , Hydrocarbons, Aromatic , Models, Chemical , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Binding , SoftwareABSTRACT
QSAR has been used to elucidate the origin of the hydrophobicity and binding affinity of a small library of fluoroaromatic inhibitors of F131V carbonic anhydrase II. Our analysis predicted the presence of a twisted amide conformation for several bound inhibitors, which we confirmed crystallographically. We also determined that the hydrophobicity of the inhibitors as a whole results from the fragment hydrophobicities of their fluorobenzyl rings, corrected for field effects and the presence of an intramolecular F.H contact in solution. The loss of this interaction on binding to the enzyme makes the affinity sensitive to the same terms, but with the opposite dependence on the F.H contact. In the case of the four inhibitors bound as twisted amides, this F.H contact must be retained to some extent in the bound state in order for their affinities to be consistent with our QSAR analysis of the entire set of 17 molecules.
Subject(s)
Amides/chemistry , Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrase Inhibitors/chemical synthesis , Carbonic Anhydrase Inhibitors/metabolism , Chemical Phenomena , Chemistry, Physical , Combinatorial Chemistry Techniques , Drug Design , Magnetic Resonance Spectroscopy , Molecular Structure , Quantitative Structure-Activity RelationshipABSTRACT
We have developed a versatile tool for the delivery of inhibitors of carbonic anhydrase II, which allows modification of a hydrophobic drug with either a water-solubilizing, photolabile cage or a hydrophobic, photolabile cage. The former mask is useful for direct delivery of hydrophobic molecules in an aqueous prodrug form. The latter may find application if delivery from a surface is desirable. In our system, where the target enzyme is found in the eye, both approaches may be useful for the delivery of hydrophobic drugs having subnanomolar dissociation constants from the enzyme.
