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We performed a systematic comparison of three label-free methods for quantitative assessment of binding strengths of proteins interacting with small molecule ligands. The performance of (1) nanoelectrospray ionization mass spectrometry (nESI-MS), (2) surface plasmon resonance (SPR), and (3) isothermal titration calorimetry (ITC) was compared for the determination of dissociation constants (K(D)). The model system studied for this purpose was the human carbonic anhydrase I (hCAI) with eight known and well characterized sulfonamide inhibitors (Krishnamurthy et al., Chem. Rev. 2008, 108: 946-1051). The binding affinities of the inhibitors chosen vary by more than four orders of magnitude e.g., the K(D) value determined for ethoxzolamide by nESI-MS was 5 +/- 1 nM and the K(D) value for sulfanilamide was 145.7 +/- 10.0 microM. The agreement of the determined K(D) values by the three methods investigated was excellent for ethoxzolamide and benzenesulfonamide (variation with experimental error), good for acetazolamide and 4-carboxybenzenesulfonamide (variation by approximately one order of magnitude), but poor for others e.g., sulpiride. The accuracies of the K(D) values are determined, and advantages and drawbacks of the individual methods are discussed. Moreover, we critically evaluate the three examined methods in terms of ease of the measurement, sample consumption, time requirement, and discuss their limitations., ((c) 2009 John Wiley & Sons, Ltd.)

The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90alphaN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 masculineC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90alphaN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding.

Chloroplast carbonic anhydrase is dependent on a reducing environment to retain its catalytic activity. To investigate the properties of the three accessible cysteine residues of pea carbonic anhydrase, mutants were made in which Ala or Ser substituted for C165, C269, and C272. The mutants at position 165 were found to be spectroscopically similarly to the wild-type. They have a high catalytic activity, and are also sensitive to oxidation. In contrast, both C269 and C272 were found to be critical both for the structure and for the catalytic activity. All mutants with substitutions at either of these two positions had to be co-overexpressed with GroES/EL chaperones to give soluble enzyme in Escherichia coli. The k(cat) values were decreased by 2 and 3 orders of magnitude for the C272A and C269A mutants, respectively, and the Km values were increased approximately 7 times. However, the binding of the inhibitor ethoxyzolamide was only slightly weakened. The near-UV CD spectra were found to be changed in both sign and intensity compared to that of the wild-type, and the far-UV spectra indicate some loss of alpha-helix structure. Moreover, the quaternary structure was changed from the wild-type octameric to tetrameric in these mutants. The results indicate that mutation of either of these cysteines causes minor structural changes around at least one of the two tryptophans of the subunit. Furthermore, the data demonstrate that C269 and C272 are involved in the interaction between subunits and are necessary for a proper structure at the tetramer-tetramer interface.

We have previously determined that 6-amino-2-benzothiazolesulfonamide (aminozolamide) significantly lowers IOP in rabbits and, more importantly, in ocular hypertensive human subjects. Results from in vitro experiments established that both the inhibitory activity of aminozolamide against carbonic anhydrase B as well as the penetration rate across excised rabbit corneas were equivalent to ethoxzolamide. Consequently, we have investigated the ocular disposition of aminozolamide to explain its activity when instilled topically to the eye. A constant concentration of 67.4 micrograms/ml of drug was applied for 90 min to the left eye of anesthetized rabbits. Drug and metabolite were measured in both aqueous humor and iris/ciliary body over time. The metabolite was collected and purified. Identification using mass spectroscopy, high pressure liquid chromatography (HPLC) and fluorescence scans indicated that the metabolite was 6-acetamido-2-benzothiazolesulfonamide. Relatively high levels of metabolite were identified in the cornea and iris/ciliary body but were much lower in aqueous humor. Tissue concentrations over time for the metabolite in iris/ciliary body were approximately 2-fold higher than levels of metabolite measured in aqueous humor. When compared to drug levels measured in either aqueous humor or iris/ciliary body, metabolite levels in these respective tissues were much higher. It is hypothesized that topical activity is a consequence of both metabolite retention in the iris/ciliary body as well as inhibition of 99+% of carbonic anhydrase. Both of these events must occur over a sufficient time period to effect a significant lowering of IOP.

An analogue series representing modification to the benzene ring of ethoxzolamide has been evaluated for solubility, pKa, partitioning, and permeability across excised rabbit corneas. These physical parameters were correlated to Hammett sigma (para) and/or Hansch pi parameter values for each compound. From these correlations, a mathematical model was developed relating corneal permeability to molecular modifications of ethoxzolamide. A three-dimensional plot of maximum attainable penetration rate versus sigma (para) and pi yielded an optimal range of pi and sigma values from which an optimally penetrating analogue could be designed.

Ethoxzolamide (1) and two analogues, representing a hydroxyethoxy and a hydrogen substitution on the 6-position of the benzene ring (2 and 3), were applied to rabbit eyes using a topical infusion method designed to provide a constant rate into aqueous humor. Statistical-moment theory was applied to the topical infusion data to describe disposition of each compound within the rabbit eye. For each analogue, it was possible to compare the corneal absorption rate constant (ka), aqueous humor elimination rate constant (k10), disposition mean residence time (MRTd), apparent steady-state volume of distribution (Vdss), and total ocular clearance (Qe). In vivo ocular ka values were related to maximum in vitro corneal penetration rates determined across excised rabbit corneas. In particular, 2 had a much longer residence time and a slower clearance than 1 and 3, which may be responsible for its ability to lower intraocular pressure when dosed topically to the rabbit eye, whereas 1 and 3 show no activity.

In the past five years we have studied the penetration of locally applied sulfonamides into the eye, with a view toward developing new topical carbonic anhydrase inhibitors for the treatment of glaucoma. The drugs varied by 400 fold in their permeability to the anterior chamber and 20,000 fold in permeability to the posterior chamber. We report now on two particular findings related to drug structure: 1) Transcorneal permeability of the ionic or dissociated form of the drug is relatively high -- some 1/4 that of the undissociated form. 2) Depending on the structure, certain compounds are sequestered in the cornea (presumably the stroma) and form a release system into the anterior aqueous.

A specific high-performance liquid chromatographic method is described for ethoxzolamide following the extraction of the material from iris/ciliary body eye tissue in rabbits. The steps consist of base extraction and protein and enzyme deactivation, followed by acid treatment, extraction into ethyl acetate, evaporation, and solubilization with a 50% aqueous methanol solution. The samples were chromatographed on a reverse-phase phenyl column with a mobile phase consisting of 50% methanol in 1% acetic acid. The recovery was 74.3% over a 10-fold range of tissue concentrations. The sensitivity was 0.03 microgram/mL, and the response was linear over the concentration range (0.03-0.5 microgram/mL) used in the study. Intravenous (2.0- and 6.0-mg/kg) and topical (1% suspension) doses of ethoxzolamide were administered to rabbits. Iris/ciliary body tissues were excised 45 min after drug administration. The tissue levels after a dose of 6 mg/kg were statistically greater than the levels obtained after a dose of 2 mg/kg. The smaller intravenous dose represented the lowest dose for which a reduction in intraocular pressure could be measured. An initial transitory drop in intraocular pressure was detected for the topical dose. Iris/ciliary body levels in the treated eye could be detected for the 2-mg/kg iv and topical doses.