Erika A. Currier

Discovery of the first nonpeptide agonist of the GPR14/urotensin-II receptor: 3-(4-chlorophenyl)-3-(2- (dimethylamino)ethyl)isochroman-1-one (AC-7954).

A functional cell-based screen identified 3-(4-chlorophenyl)-3-(2-(dimethylamino)ethyl)isochroman-1-one hydrochloride (AC-7954, 1) as a nonpeptidic agonist of the urotensin-II receptor. Racemic 1 had an EC50 of 300 nM at the human UII receptor and was highly selective. Testing of the enantiopure (+)- and (-)- 1 revealed that the UII receptor activity of racemic 1 resides primarily in (+)-1. Being a selective nonpeptidic druglike UII receptor agonist, (+)-1 will be useful as a pharmacological research tool and a potential drug lead.

Identification of the First Synthetic Steroidogenic Factor 1 Inverse Agonists: Pharmacological Modulation of Steroidogenic Enzymes

Steroidogenic factor SF-1, a constitutively active nuclear hormone receptor, is essential to the development of adrenal and gonadal glands and acts as a shaping factor of sexual determination and differentiation. Its effects are exerted primarily through the control of the synthesis of steroid hormones. The functional cell-based assay Receptor Selection and Amplification Technology (R-SAT) was used to identify potent and selective SF-1 inverse agonists through the screening of a chemical library of drug-like small-molecule entities. Among them, 4-(heptyloxy)phenol (AC-45594), a prototype inverse agonist lead, was used to show that SF-1 constitutive activity can be pharmacologically modulated by a synthetic ligand. In a physiological system of endocrine function, the expression of several reported SF-1 target genes, including SF-1 itself, was inhibited by treatment with AC-45594 and analogs. Thus, pharmacological modulation of SF-1 is critical to its function as an endocrine master regulator and has potentially important consequences to diseases in which SF-1 activity is critical.

Integrative Functional Assays, Chemical Genomics and High Throughput Screening: Harnessing Signal Transduction Pathways to a Common HTS Readout

Chemical genomics is a drug discovery strategy that relies heavily on high-throughput screening (HTS) and therefore benefits from functional assay platforms that allow HTS against all relevant genomic targets. Receptor Selection and Amplification Technology (R-SAT) is a cell-based, high-throughput functional assay where the receptor stimulus is translated into a measurable cellular response through an extensive signaling cascade occurring over several days. The large biological and chronological separation of stimulus from response provides numerous opportunities for enabling assays and increasing assay sensitivity. Here we review strategies for building homogeneous assay platforms across large gene families by redirecting and/or amplifying signal transduction pathways.

The ras-related GTPase rac1 regulates a proliferative pathway selectively utilized by G-protein coupled receptors

Ras and rac are each members of the superfamily of monomeric GTPases and both function as molecular switches to link cell-surface signals to intracellular responses. Using a novel assay of cellular proliferation called R-SATTM (Receptor Selection and Amplification Technology), we examined the roles of ras and rac in mediating the proliferative responses to a variety of cell-surface receptors. Activated, wild-type and dominant-negative mutants of rac and ras were tested for their effects on cellular proliferation either alone or in combination with receptors. Activated rac (rac Q61L, henceforth rac*) and ras (ras G12V, henceforth ras*) each induced strong proliferative responses. Dominant-negative rac (rac T17N, henceforth rac(−)) dramatically suppressed proliferative responses to G-protein coupled receptors (GPCRs) including the m5 muscarinic receptor and the α1B adrenergic receptor. In contrast, rac(−) had little or no effect upon responses to the tyrosine kinase receptor TrkC, and only partially suppressed responses to the Janus kinase (JAK/STAT) linked granulocyte macrophage colony stimulating factor (GM-CSF) receptor. Dominant-negative ras (ras T17N, henceforth ras(−)) blocked the proliferative responses to all of the tested receptors. Compared to rac(−) and ras(−), wild-type rac and ras had only modest effects on the tested receptors. Overall these results demonstrate that rac mediates the proliferative effects of G-protein coupled receptors through a pathway that is distinct from the proliferative signaling pathway utilized by tyrosine kinase linked and JAK-linked receptors.

Synthesis and Evaluation of Dibenzothiazepines: A Novel Class of Selective Cannabinoid-1 Receptor Inverse Agonists

A novel class of CB1 inverse agonists was discovered. To efficiently establish structure-activity relationships (SARs), new synthetic methodologies amenable for parallel synthesis were developed. The compounds were evaluated in a mammalian cell-based functional assay and in radioligand binding assays expressing recombinant human cannabinoid receptors (CB1 and CB2). In general, all of the compounds exhibited high binding selectivity at CB1 vs CB2 and the general SAR revealed a lead compound 11-(4-chlorophenyl)dibenzo[b,f][1,4]thiazepine-8-carboxylic acid butylamide (12e) which showed excellent in vivo activity in pharmacodynamic models related to CB1 receptor activity. The low solubility that hampered the development of 12e was solved leading to a potential preclinical candidate 11-(3-chloro-4-fluorophenyl)dibenzo[b,f][1,4]thiazepine-8-carboxylic acid butylamide (12h).

Discovery of non-peptidergic MrgX1 and MrgX2 receptor agonists and exploration of an initial SAR using solid-phase synthesis

A class of small molecules displaying comparable activities with peptide ligands BAM22 and corticostatin-14 at both the human and rhesus monkey MrgX1 and MrgX2 receptors, respectively, was discovered. A comparative study to compare solid-phase and solution-phase chemistries for the efficient synthesis of the active class, tetracyclic benzimidazoles, was undertaken. The solid-phase chemistry was found to be superior both for the synthesis of analogs and for the synthesis of gram quantities.

Discovery of Potent and Selective Small-Molecule PAR-2 Agonists

Proteinase activated receptor-2 plays a crucial role in a wide variety of conditions with a strong inflammatory component. We present the discovery and characterization of two structurally different, potent, selective, and metabolically stable small-molecule PAR-2 agonists. These ligands may be useful as pharmacological tools for elucidating the complex physiological role of the PAR-2 receptors as well as for the development of PAR-2 antagonists.

Optimization of isochromanone based urotensin II receptor agonists

A series of novel isochromanone based urotensin II receptor agonists have been synthesized and evaluated for their activity using a functional cell based assay (R-SAT). Several potent and efficacious derivatives were identified, with 3-(3,4-dichlorophenyl)-6,7-dimethyl-3-(2-dimethylaminoethyl)isochroman-1-one being the most potent compound showing an EC₅₀-value of 51 nM, thereby being the most potent compound so far within the isochromanone series. In addition, two other heterocyclic systems (isochromanes and tetrahydroisoquinolinones) were investigated and these derivatives were found to be both potent and efficacious. The activity of the isochromane derivatives implies that the carbonyl group of the isochromanone is not necessary for activity. Furthermore it was found that the geometry of the heterocycles was more important for receptor interaction than the composition of the heteroatoms present.

Identification of the Atypical L-Type Ca2+ Channel Blocker Diltiazem and Its Metabolites As Ghrelin Receptor Agonists

Using a high-throughput functional screen, the atypical L-type Ca2+ channel blocker diltiazem was discovered to be an agonist at the human ghrelin (GHSR1a) receptor. In cellular proliferation, Ca2+ mobilization, and bioluminescence resonance energy transfer (BRET-2) assays, diltiazem was a partial agonist at GHSR1a receptors, with 50 to 80% relative efficacy compared with the GHSR1a peptide agonist GHRP-6, and high nanomolar to low micromolar potency, depending upon the assay. Seven of the known primary metabolites of diltiazem were synthesized, and three of them (MA, M1, and M2) were more efficacious and/or more potent than diltiazem at GHSR1a receptors, with a rank order of agonist activity of M2 > M1 > MA > diltiazem, whereas M4 and M6 metabolites displayed weak agonist activity, and the M8 and M9 metabolites were inactive. Binding affinities of diltiazem and these metabolites to GHSR1a receptors followed a similar rank order. In vivo tests showed that diltiazem and M2 each stimulated growth hormone release in male Sprague-Dawley neonatal rats, although to a lesser degree than GHRP-6. Thus, diltiazem and chemical analogs of diltiazem represent a new class of GHSR1a receptor agonists. The possible contributions of GHSR1a receptor activation to the clinical actions of diltiazem are discussed in the context of the known beneficial cardiovascular effects of ghrelin.

Discovery of a class of calcium sensing receptor positive allosteric modulators; 1-(benzothiazol-2-yl)-1-phenylethanols.

1-(Benzothiazol-2-yl)-1-(4-chlorophenyl)ethanol (1) was identified as a positive allosteric modulator (PAM) of the CaSR in a functional cell-based assay. This compound belongs to a class of compounds that is structurally distinct from other known positive allosteric modulators, for example, the phenylalkylamines cinacalcet, a modified analog (13) potently suppressed parathyroid hormone (PTH) release in rats, consistent with its profile as a PAM of CaSRs.