The Problems of Applying Classical Pharmacology Analysis to Modern In Vitro Drug Discovery Assays: Slow Binding Kinetics and High Target Concentration.

The analysis framework used to quantify drug potency in vitro (e.g., Kd or Ki) was initially developed for classical pharmacology bioassays, for example, organ bath experiments testing moderate-affinity natural products. Modern drug discovery can infringe the assumptions of the classical pharmacology analysis equations, owing to the reduction of assay volume in miniaturization, target overexpression, and the increase of compound-target affinity in medicinal chemistry. These assumptions are that (1) the compound concentration greatly exceeds the target concentration (i.e., minimal ligand depletion), and (2) the compound is at equilibrium with the receptor (i.e., rapid ligand binding kinetics). Unappreciated infringement of these assumptions can lead to substantial underestimation of compound affinity, which negatively impacts the drug discovery process, from early-stage lead optimization to prediction of human dosing. This study evaluates the real-world impact of these factors on the target interaction assays used in drug discovery using literature examples, database searches, and simulations. The ranges of compound affinity and the assay types that are prone to depletion and equilibration artifacts are identified. Importantly, the highest-affinity compounds, usually the highest value chemical matter in drug discovery, are the most affected. Methods and simulation tools are provided to enable investigators to evaluate, manage, and minimize depletion or equilibration artifacts. This study enables the correct application of pharmacological data analysis to accurately quantify affinity using modern drug discovery assay technology.

Quantifying the Kinetics of Signaling and Arrestin Recruitment by Nervous System G-Protein Coupled Receptors.

Neurons integrate inputs over different time and space scales. Fast excitatory synapses at boutons (ms and µm), and slow modulation over entire dendritic arbors (seconds and mm) are all ultimately combined to produce behavior. Understanding the timing of signaling events mediated by G-protein-coupled receptors is necessary to elucidate the mechanism of action of therapeutics targeting the nervous system. Measuring signaling kinetics in live cells has been transformed by the adoption of fluorescent biosensors and dyes that convert biological signals into optical signals that are conveniently recorded by microscopic imaging or by fluorescence plate readers. Quantifying the timing of signaling has now become routine with the application of equations in familiar curve fitting software to estimate the rates of signaling from the waveform. Here we describe examples of the application of these methods, including (1) Kinetic analysis of opioid signaling dynamics and partial agonism measured using cAMP and arrestin biosensors; (2) Quantifying the signaling activity of illicit synthetic cannabinoid receptor agonists measured using a fluorescent membrane potential dye; (3) Demonstration of multiplicity of arrestin functions from analysis of biosensor waveforms and quantification of the rates of these processes. These examples show how temporal analysis provides additional dimensions to enhance the understanding of GPCR signaling and therapeutic mechanisms in the nervous system.

Pharmacological characterisation of novel adenosine A3 receptor antagonists.

The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics-Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 µM) competitive antagonist. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.

Analyzing kinetic signaling data for G-protein-coupled receptors.

In classical pharmacology, bioassay data are fit to general equations (e.g. the dose response equation) to determine empirical drug parameters (e.g. EC50 and Emax), which are then used to calculate chemical parameters such as affinity and efficacy. Here we used a similar approach for kinetic, time course signaling data, to allow empirical and chemical definition of signaling by G-protein-coupled receptors in kinetic terms. Experimental data are analyzed using general time course equations (model-free approach) and mechanistic model equations (mechanistic approach) in the commonly-used curve-fitting program, GraphPad Prism. A literature survey indicated signaling time course data usually conform to one of four curve shapes: the straight line, association exponential curve, rise-and-fall to zero curve, and rise-and-fall to steady-state curve. In the model-free approach, the initial rate of signaling is quantified and this is done by curve-fitting to the whole time course, avoiding the need to select the linear part of the curve. It is shown that the four shapes are consistent with a mechanistic model of signaling, based on enzyme kinetics, with the shape defined by the regulation of signaling mechanisms (e.g. receptor desensitization, signal degradation). Signaling efficacy is the initial rate of signaling by agonist-occupied receptor (kτ), simply the rate of signal generation before it becomes affected by regulation mechanisms, measurable using the model-free analysis. Regulation of signaling parameters such as the receptor desensitization rate constant can be estimated if the mechanism is known. This study extends the empirical and mechanistic approach used in classical pharmacology to kinetic signaling data, facilitating optimization of new therapeutics in kinetic terms.

A kinetic method for measuring agonist efficacy and ligand bias using high resolution biosensors and a kinetic data analysis framework.

The kinetics/dynamics of signaling are of increasing value for G-protein-coupled receptor therapeutic development, including spatiotemporal signaling and the kinetic context of biased agonism. Effective application of signaling kinetics to developing new therapeutics requires reliable kinetic assays and an analysis framework to extract kinetic pharmacological parameters. Here we describe a platform for measuring arrestin recruitment kinetics to GPCRs using a high quantum yield, genetically encoded fluorescent biosensor, and a data analysis framework to quantify the recruitment kinetics. The sensor enabled high temporal resolution measurement of arrestin recruitment to the angiotensin AT1 and vasopressin V2 receptors. The analysis quantified the initial rate of arrestin recruitment (kτ), a biologically-meaningful kinetic drug efficacy parameter, by fitting time course data using routine curve-fitting methods. Biased agonism was assessed by comparing kτ values for arrestin recruitment with those for Gq signaling via the AT1 receptor. The kτ ratio values were in good agreement with bias estimates from existing methods. This platform potentially improves and simplifies assessment of biased agonism because the same assay modality is used to compare pathways (potentially in the same cells), the analysis method is parsimonious and intuitive, and kinetic context is factored into the bias measurement.

The importance of target binding kinetics for measuring target binding affinity in drug discovery: a case study from a CRF1 receptor antagonist program.

In drug discovery, it is essential to accurately measure drug-target binding affinity. Here, we revisit the fact that target binding kinetics impact the measurement of affinity, using a case study: development of corticotropin-releasing factor antagonists. Slow dissociation of the drug-target complex results in affinity assays being far from equilibrium, which results in erroneous estimates of affinity. This scenario can impair prediction of human dosing, assessment of target selectivity, identification of high-affinity ligands and determination of SAR. We describe strategies to detect lack of equilibration in affinity assays and methods to correctly measure affinity of slowly dissociating compounds. These considerations will facilitate drug discovery by ensuring reliable measurement of drug-target binding affinity.

Receptor binding kinetics equations: Derivation using the Laplace transform method.

INTRODUCTION: Measuring unlabeled ligand receptor binding kinetics is valuable in optimizing and understanding drug action. Unfortunately, deriving equations for estimating kinetic parameters is challenging because it involves calculus; integration can be a frustrating barrier to the pharmacologist seeking to measure simple rate parameters. Here, a well-known tool for simplifying the derivation, the Laplace transform, is applied to models of receptor-ligand interaction. The method transforms differential equations to a form in which simple algebra can be applied to solve for the variable of interest, for example the concentration of ligand-bound receptor. METHODS: The goal is to provide instruction using familiar examples, to enable investigators familiar with handling equilibrium binding equations to derive kinetic equations for receptor-ligand interaction. First, the Laplace transform is used to derive the equations for association and dissociation of labeled ligand binding. Next, its use for unlabeled ligand kinetic equations is exemplified by a full derivation of the kinetics of competitive binding equation. Finally, new unlabeled ligand equations are derived using the Laplace transform. These equations incorporate a pre-incubation step with unlabeled or labeled ligand. RESULTS: Four equations for measuring unlabeled ligand kinetics were compared and the two new equations verified by comparison with numerical solution. Importantly, the equations have not been verified with experimental data because no such experiments are evident in the literature. Equations were formatted for use in the curve-fitting program GraphPad Prism 6.0 and fitted to simulated data. DISCUSSION: This description of the Laplace transform method will enable pharmacologists to derive kinetic equations for their model or experimental paradigm under study. Application of the transform will expand the set of equations available for the pharmacologist to measure unlabeled ligand binding kinetics, and for other time-dependent pharmacological activities.

Pharmacologic Characterization of Valbenazine (NBI-98854) and Its Metabolites.

The vesicular monoamine transporter 2 (VMAT2) is an integral presynaptic protein that regulates the packaging and subsequent release of dopamine and other monoamines from neuronal vesicles into the synapse. Valbenazine (NBI-98854), a novel compound that selectively inhibits VMAT2, is approved for the treatment of tardive dyskinesia. Valbenazine is converted to two significant circulating metabolites in vivo, namely, (+)-α-dihydrotetrabenazine (R,R,R-HTBZ) and a mono-oxy metabolite, NBI-136110. Radioligand-binding studies were conducted to assess and compare valbenazine, tetrabenazine, and their respective metabolites in their abilities to selectively and potently inhibit [3H]-HTBZ binding to VMAT2 in rat striatal, rat forebrain, and human platelet homogenates. A broad panel screen was conducted to evaluate possible off-target interactions of valbenazine, R,R,R-HTBZ, and NBI-136110 at >80 receptor, transporter, and ion channel sites. Radioligand binding showed R,R,R-HTBZ to be a potent VMAT2 inhibitor in homogenates of rat striatum (Ki = 1.0-2.8 nM), rat forebrain (Ki = 4.2 nM), and human platelets (Ki = 2.6-3.3 nM). Valbenazine (Ki = 110-190 nM) and NBI-136110 (Ki = 160-220 nM) also exhibited inhibitory effects on VMAT2, but with lower potency than R,R,R-HTBZ. Neither valbenazine, R,R,R-HTBZ, nor NBI-136110 had significant off-target interactions at serotonin (5-HT1A, 5-HT2A, 5-HT2B) or dopamine (D1 or D2) receptor sites. In vivo studies measuring ptosis and prolactin secretion in the rat confirmed the specific and dose-dependent interactions of tetrabenazine and R,R,R-HTBZ with VMAT2. Evaluations of potency and selectivity of tetrabenazine and its pharmacologically active metabolites were also performed. Overall, the pharmacologic characteristics of valbenazine appear consistent with the favorable efficacy and tolerability findings of recent clinical studies [KINECT 2 (NCT01733121), KINECT 3 (NCT02274558)].

Binding kinetics redefine the antagonist pharmacology of the corticotropin-releasing factor type 1 receptor.

Corticotropin-releasing factor (CRF) receptor antagonists are under preclinical and clinical investigation for stress-related disorders. In this study the impact of receptor-ligand binding kinetics on CRF1 receptor antagonist pharmacology was investigated by measuring the association rate constant (k1), dissociation rate constant (k₋1), and kinetically derived affinity at 37°C. Three aspects of antagonist pharmacology were reevaluated: comparative binding activity of advanced compounds, in vivo efficacy, and structure-activity relationships. Twelve lead compounds, with little previously noted difference of affinity, varied substantially in their kinetic binding activity with a 510-fold range of kinetically derived affinity (k₋1/k1), 170-fold range of k₋1, and 13-fold range of k1. The k₋1 values indicated previous affinity measurements were not close to equilibrium, resulting in compression of the measured affinity range. Dissociation was exceptionally slow for three ligands (k₋1 t(1/2) of 1.6-7.2 h at 37°C). Differences of binding behavior were consistent with in vivo pharmacodynamics (suppression of adrenocorticotropin in adrenalectomized rats). Ligand concentration-effect relationships correlated with their kinetically derived affinity. Two ligands that dissociated slowly (53 and 130 min) produced prolonged suppression, whereas only transient suppression was observed with a more rapidly dissociating ligand (16 min). Investigating the structure-activity relationship indicated exceptionally low values of k1, approaching 100,000-fold less than the diffusion-limited rate. Retrospective interpretation of medicinal chemistry indicates optimizing specific elements of chemical structure overcame kinetic barriers in the association pathway, for example, constraint of the pendant aromatic orthogonal to the ligand core. Collectively, these findings demonstrate receptor binding kinetics provide new dimensions for understanding and potentially advancing the pharmacology of CRF1 receptor antagonists.

Discovery of NBI-77860/GSK561679, a potent corticotropin-releasing factor (CRF1) receptor antagonist with improved pharmacokinetic properties.

Antagonists of the corticotropin-releasing factor (CRF) neuropeptide may prove effective in treating stress and anxiety related disorders. In an effort to identify antagonists with improved physico-chemical properties a new series of CRF(1) antagonists were designed to substitute the propyl groups at the C7 position of the pyrazolo[1,5-a]pyrimidine core of 1 with heterocycles. Compound (S)-8d was identified as a high affinity ligand with a pK(i) value of 8.2 and a functional CRF(1) antagonist with pIC(50) value of 7.0 in the in vitro CRF ACTH production assay.

Analytical method for simultaneously measuring ex vivo drug receptor occupancy and dissociation rate: application to (R)-dimethindene occupancy of central histamine H1 receptors.

We introduce a novel experimental method to determine both the extent of ex vivo receptor occupancy of administered compound and its dissociation rate constant (k4). [Here, we reference k4 as the rate of offset of unlabeled ligand in convention with Motulsky and Mahan (1)]. We derived a kinetic rate equation based on the dissociation rate constant for an unlabeled compound competing for the same site as a labeled compound and describe a model to simulate fractional occupancy. To validate our model, we performed in vitro kinetics and ex vivo occupancy experiments in rat cortex with varying concentrations of (R)-dimethindene, a sedating antihistamine. Brain tissue was removed at various times post oral administration, and histamine H1 receptor ligand [3H]-doxepin binding to homogenates from drug-treated or vehicle-treated rats was measured at multiple time points at room temperature. Fractional occupancy and k4 for (R)-dimethindene binding to H1 receptors were calculated by using our proposed model. Rats dosed with 30 and 60 mg/kg (R)-dimethindene showed 42% and 67% occupancy of central H1 receptors, respectively. These results were comparable to occupancy data determined by equilibrium radioligand binding. In addition, drug k4 rate determined by using our ex vivo method was equivalent to k4 determined by in vitro competition kinetics (dissociation half-life t(1/2) approximately 30 min). The outlined method can be used to assess, by simulation and experiment, occupancy for compounds based on dissociation rate constants and contributes to current efforts in drug optimization to profile antagonist efficacy in terms of its kinetic drug-target binding parameters. Data described by the method may be analyzed with commercially available software. Suggested fitting procedures are given in the appendix.

Pharmacological and pharmacokinetic characterization of 2-piperazine-alpha-isopropyl benzylamine derivatives as melanocortin-4 receptor antagonists.

A series of 2-piperazine-alpha-isopropylbenzylamine derivatives were synthesized and characterized as melanocortin-4 receptor (MC4R) antagonists. Attaching an amino acid to benzylamines 7 significantly increased their binding affinity, and the resulting compounds 8-12 bound selectively to MC4R over other melanocortin receptor subtypes and behaved as functional antagonists. These compounds were also studied for their permeability using Caco-2 cell monolayers and metabolic stability in human liver microsomes. Most compounds exhibited low permeability and high efflux ratio possibly due to their high molecular weights. They also showed moderate metabolic stability which might be associated with their moderate to high lipophilicity. Pharmacokinetic properties of these MC4R antagonists, including brain penetration, were studied in mice after oral and intravenous administrations. Two compounds identified to possess high binding affinity and selectivity, 10d and 11d, were studied in a murine cachexia model. After intraperitoneal (ip) administration of 1mg/kg dose, mice treated with 10d had significantly more food intake and weight gain than the control animals, demonstrating efficacy by blocking the MC4 receptor. Similar in vivo effects were also observed when 11d was dosed orally at 20mg/kg. These results provide further evidence that a potent and selective MC4R antagonist has potential in the treatment of cancer cachexia.

Allosteric ligands for the corticotropin releasing factor type 1 receptor modulate conformational states involved in receptor activation.

Allosteric modulators of G-protein-coupled receptors can regulate conformational states involved in receptor activation ( Mol Pharmacol 58: 1412-1423, 2000 ). This hypothesis was investigated for the corticotropin-releasing factor type 1 (CRF(1)) receptor using a novel series of ligands with varying allosteric effect on CRF binding (inhibition to enhancement). For the G-protein-uncoupled receptor, allosteric modulation of CRF binding was correlated with nonpeptide ligand signaling activity; inverse agonists inhibited and agonists enhanced CRF binding. These data were quantitatively consistent with a two-state equilibrium underlying the modulation of CRF binding to the G-protein-uncoupled receptor. We next investigated the allosteric effect on CRF-stimulated G-protein coupling. Ligands inhibited CRF-stimulated cAMP accumulation regardless of their effect on the G-protein-uncoupled state. The modulators reduced CRF E(max) values, suggesting that they reduced the efficacy of a CRF-bound active state to couple to G-protein. Consistent with this hypothesis, the modulators inhibited binding to a guanine nucleotide-sensitive state. Together, the results are quantitatively consistent with a model in which 1) the receptor exists in three predominant states: an inactive state, a weakly active state, and a CRF-bound fully active state; 2) allosteric inverse agonists stabilize the inactive state, and allosteric agonists stabilize the weakly active state; and 3) antagonism of CRF signaling results from destabilization of the fully active state. These findings imply that nonpeptide ligands differentially modulate conformational states involved in CRF(1) receptor activation and suggest that different conformational states can be targeted in designing nonpeptide ligands to inhibit CRF signaling.

Identification and characterization of pyrrolidine diastereoisomers as potent functional agonists and antagonists of the human melanocortin-4 receptor.

A series of trans-4-phenylpyrrolidine-3-carboxamides were synthesized and characterized as potent ligands of the human melanocortin-4 receptor. Interestingly, a pair of diastereoisomers 13b displayed potent functional agonist and antagonist activity, respectively. Thus, the 3S,4R-pyrrolidine 13b-1 possessed a Ki of 1.0 nM and an EC50 of 3.8 nM, while its 3R,4S-isomer 13b-2 exhibited a Ki of 4.7 and an IC50 of 64 nM. Both compounds were highly selective over other melanocortin receptor subtypes. The MC4R agonist 13b-1 also demonstrated efficacy in a diet-induced obesity model in rats.

Design, synthesis, in vitro, and in vivo characterization of phenylpiperazines and pyridinylpiperazines as potent and selective antagonists of the melanocortin-4 receptor.

Benzylamine and pyridinemethylamine derivatives were synthesized and characterized as potent and selective antagonists of the melanocortin-4 receptor (MC4R). These compounds were also profiled in rodents for their pharmacokinetic properties. Two compounds with diversified profiles in chemical structure, pharmacological activities, and pharmacokinetics, 10 and 12b, showed efficacy in an established murine cachexia model. For example, 12b had a K(i) value of 3.4 nM at MC4R, was more than 200-fold selective over MC3R, and had a good pharmacokinetic profile in mice, including high brain penetration. Moreover, 12b was able to stimulate food intake in the tumor-bearing mice and reverse their lean body mass loss. Our results provided further evidence that a potent and selective MC4R antagonist with appropriate pharmacokinetic properties might potentially be useful for the treatment of cancer cachexia.

Synthesis and characterization of pyrrolidine derivatives as potent agonists of the human melanocortin-4 receptor.

A series of trans-4-phenylpyrrolidine-3-carboxamides were synthesized and characterized as potent ligands of the human melanocortin-4 receptor. Interestingly, a pair of diastereoisomers 20f-1 and 20f-2 displayed potent functional agonist and antagonist activity, respectively. Thus, the 3S,4R-compound 20f-1 possessed a K(i) of 11nM and an EC(50) of 24nM, while its 3R,4S-isomer 20f-2 exhibited a K(i) of 8.6 and an IC(50) of 65nM. Both compounds were highly selective over other melanocortin receptor subtypes. The MC4R agonist 20f-1 also demonstrated efficacy in diet-induced obese rats.

Discovery of 1-[2-[(1S)-(3-dimethylaminopropionyl)amino-2-methylpropyl]-4-methylphenyl]-4-[(2R)-methyl-3-(4-chlorophenyl)-propionyl]piperazine as an orally active antagonist of the melanocortin-4 receptor for the potential treatment of cachexia.

A potent and selective antagonist of the melanocortin-4 receptor, 1-[2-[(1S)-(3-dimethylaminopropionyl)amino-2-methylpropyl]-6-methylphenyl]-4-[(2R)-methyl-3-(4-chlorophenyl)propionyl]piperazine (10d), was identified from a series piperazinebenzylamine attached with a N,N-dimethyl-beta-alanine side chain. This compound possessed high water solubility and exhibited good metabolic profiles. In animals, 10d showed moderate to good oral bioavailability and promoted food intake in tumor-bearing mice after oral administration.

Pyrrolidinones as potent functional antagonists of the human melanocortin-4 receptor.

A series of pyrrolidinones derived from phenylalaninepiperazines were synthesized and characterized as potent and selective antagonists of the melanocortin-4 receptor. In addition to their high binding affinities, these compounds displayed high functional potencies. 12a had a K(i) of 0.94 nM in binding and IC(50) of 21 nM in functional activity. 12a also demonstrated efficacy in a mouse cachexia model.

Pyrrolidinones as orally bioavailable antagonists of the human melanocortin-4 receptor with anti-cachectic activity.

A series of pyrrolidinones derived from phenylalanines were synthesized as potent antagonists of the human melanocortin-4 receptor. These compounds showed high potencies and selectivities, and several of them had good oral bioavailabilities. In addition, 12e demonstrated in vivo efficacy in a murine cachexia model.

Allosteric modulators of class B G-protein-coupled receptors.

Class B GPCR's are activated by peptide ligands, typically 30-40 amino acid residues, that are involved in major physiological functions such as glucose homeostasis (glucagon and glucagon-like peptide 1), calcium homeostasis and bone turnover (parathyroid hormone and calcitonin), and control of the stress axis (corticotropin-releasing factor). Peptide therapeutics have been developed targeting these receptors but development of nonpeptide ligands, enabling oral administration, has proved challenging. Allosteric modulation of these receptors provides a potential route to developing nonpeptide ligands that inhibit, activate, or potentiate activation of these receptors. Here the known mechanisms of allosteric modulators targeting Class B GPCR's are reviewed, particularly nonpeptide antagonists of the corticotropin-releasing factor 1 receptor and allosteric enhancers of the glucagon-like peptide-1 receptor. Also discussed is the potential for antagonist ligands to operate by competitive inhibition of one of the peptide binding sites, analogous to the Charniere mechanism. These mechanisms are then used to discuss potential strategies and management of pharmacological complexity in the future development of allosteric modulators for Class B GPCR's.