Pharmacologic antagonism of the oral aversive taste-directed response to capsaicin in a mouse brief access taste aversion assay.

Chemosensory signaling by the tongue is a primary determinant of ingestive behavior and is mediated by specific interactions between tastant molecules and G protein-coupled and ion channel receptors. The functional relationship between tastant and receptor should be amenable to pharmacologic methods and manipulation. We have performed a pharmacologic characterization of the taste-directed licking of mice presented with solutions of capsaicin and other transient receptor potential vanilloid-1 (TRPV1) agonists using a brief access taste aversion assay. Dose-response functions for lick-rate suppression were established for capsaicin (EC(50) = 0.5 microM), piperine (EC(50) = 2 muM), and resiniferatoxin (EC(50) = 0.02 microM). Little or no effect on lick rate was observed in response to the full TRPV1 agonist olvanil. Capsaicin lick rates of wild-type and transient receptor potential melastatin-5 (TRPM5) knockout mice were equivalent, indicating that TRPM5, a critical component of aversive signaling for many bitter tastants, did not contribute to the capsaicin taste response. The selective TRPV1 antagonists N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (10 microM) and (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide (AMG9810) (10 microM) effectively blocked capsaicin- and piperine-mediated lick suppression. However, (E)-3-(4-chlorophenyl)-N-(3-methoxyphenyl)-N-phenylprop-2-enamide (SB 366791) and capsazepine, also TRPV1 antagonists, were without effect at test concentrations of up to 30 and 100 microM, respectively. Our results demonstrate that TRPV1-mediated oral aversiveness presents a pharmacologic profile differing from what has been reported previously for TRPV1 pain signaling and, furthermore, that aversive tastes can be evaluated and controlled pharmacologically.

Applications of calorimetric methods to drug discovery and the study of protein interactions.

Recent studies report the application of isothermal titration calorimetry and differential scanning calorimetry to the study of protein-ligand interactions, allosteric cooperativity and aspects of protein folding. New methods of data analysis compare alternative methods for determining ligand binding enthalpy and analyze potential sources of error in the experimental measurement of other thermodynamic parameters. Several reports examine issues concerning drug design and the correlation of thermodynamic and X-ray structural data. New instruments allow volumetric effects in biochemical systems to be evaluated calorimetrically and to substantially expand the throughput of differential scanning calorimetry measurements in drug discovery and other high-throughput applications.

Combinatorial informatics in the post-genomics ERA.

The multitude of potential drug targets emerging from genome sequencing demands new approaches to drug discovery. A chemogenomics strategy, which involves the generation of small-molecule compounds that can be used both as tools to probe biological mechanisms and as leads for drug-property optimization, provides a highly parallel, industrialized solution. Key to the success of this strategy is an integrated suite of chemi-informatics applications that can allow the rapid and directed optimization of chemical compounds with drug-like properties using 'just-in-time' combinatorial chemical synthesis. An effective embodiment of this process requires new computational and data-mining tools that cover all aspects of library generation, compound selection and experimental design, and work effectively on a massive scale.

Chemical genomics as an emerging paradigm for postgenomic drug discovery.

Chemical genomics approaches are evolving to overcome key problems limiting the efficiency of drug discovery in the postgenomic era. Many of these stem from the low success rates in finding drugs for novel genomics targets whose biochemical properties and therapeutic relevance is poorly understood. The fundamental objective of chemical genomics is to find and optimize chemical compounds that can be used to directly test the therapeutic relevance of new targets revealed through genome sequencing. An integrated approach to chemical genomics encompasses a diverse set of tools including quantitative affinity-based screens, computer-directed combinatorial chemistry, and structure-based drug design. The approach is most effectively applied across targets classes whose members are structurally related, and where some members are known to have bona fide therapeutic relevance.

A high throughput in vivo assay for taste quality and palatability.

Taste quality and palatability are two of the most important properties measured in the evaluation of taste stimuli. Human panels can report both aspects, but are of limited experimental flexibility and throughput capacity. Relatively efficient animal models for taste evaluation have been developed, but each of them is designed to measure either taste quality or palatability as independent experimental endpoints. We present here a new apparatus and method for high throughput quantification of both taste quality and palatability using rats in an operant taste discrimination paradigm. Cohorts of four rats were trained in a modified operant chamber to sample taste stimuli by licking solutions from a 96-well plate that moved in a randomized pattern beneath the chamber floor. As a rat's tongue entered the well it disrupted a laser beam projecting across the top of the 96-well plate, consequently producing two retractable levers that operated a pellet dispenser. The taste of sucrose was associated with food reinforcement by presses on a sucrose-designated lever, whereas the taste of water and other basic tastes were associated with the alternative lever. Each disruption of the laser was counted as a lick. Using this procedure, rats were trained to discriminate 100 mM sucrose from water, quinine, citric acid, and NaCl with 90-100% accuracy. Palatability was determined by the number of licks per trial and, due to intermediate rates of licking for water, was quantifiable along the entire spectrum of appetitiveness to aversiveness. All 96 samples were evaluated within 90 minute test sessions with no evidence of desensitization or fatigue. The technology is capable of generating multiple concentration-response functions within a single session, is suitable for in vivo primary screening of tastant libraries, and potentially can be used to evaluate stimuli for any taste system.

Quantitative assessment of TRPM5-dependent oral aversiveness of pharmaceuticals using a mouse brief-access taste aversion assay.

Many orally administered pharmaceuticals are regarded by humans as aversive, most often described as 'bitter'. Taste aversiveness often leads to patient noncompliance and reduced treatment effectiveness. 'Bitter' taste is mediated by T2R G-protein coupled receptors through a peripheral signaling pathway critically dependent upon function of the TRPM5 ion channel. The brief-access taste aversion (BATA) assay operationally defines aversive taste as suppression of the rate at which a rodent licks from sipper tubes that deliver tastant solutions or suspensions. We have used a mouse BATA assay for rapid quantification of oral aversiveness from a set of 20 active pharmaceutical ingredients (APIs). Robust lick-rate dose-response functions were obtained from both C57BL/6J wild type (WT) and C57BL/6J/TRPM5-/- (TRPM5 knockout) mouse strains, generating reliable determinations of potency and relative maximal oral aversiveness for each API. A subset of APIs was also evaluated in a human bitterness assessment test; effective concentrations for half-maximum responses (EC50s) from both the human test and WT mouse BATA were equivalent. Relative to WT potencies, EC50s from TRPM5 knockout mice were right-shifted more than 10-fold for most APIs. However, APIs were identified for which EC50s were essentially identical in both mouse strains, indicating a TRPM5-independent alternative aversive pathway. Our results suggest the BATA assay will facilitate formulation strategies and taste assessment of late development-phase APIs.

Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor.

ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.

Decrypting the biochemical function of an essential gene from Streptococcus pneumoniae using ThermoFluor technology.

The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.