Rajasree Golla

A Homogeneous Enzyme Fragment Complementation Cyclic AMP Screen for GPCR Agonists

In the new high-throughput screening (HTS) campaign, receptor functional assays, 3’,5’-cyclic adenosine mono-phosphate (cAMP), intracellular [Ca2+]i, phosphatidylinositol turnover, and reporter-based assays are being used as primary screens as they are now developed as homogeneous and automation-friendly assays. FlashPlate assay and scintillation proximity assay using radiolabeled cAMP have been used for measuring cAMP. A nonradioactive homogeneous HTS assay using HitHunter™ enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Gαs-coupled receptor. In the EFC-cAMP assay, the β-galactosidase (β-gal) donor fragment-cAMP (ED-cAMP) conjugate complements with the β-gal enzyme acceptor (EA) fragment to form an active β-gal enzyme. Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form an active enzyme. Cyclic AMP in the samples compete with ED-cAMP to bind to the anti-cAMP antibody, thus increasing the free ED-cAMP that can complement with the EA fragment to form an active enzyme that is assayed with a luminescent substrate. Thus, this assay results in a positive signal unlike other technologies, wherein the signal is completed by cAMP in the sample. Glucagon-like peptide (GLP)-1 binds to GLP-1 receptor (with a Kd of 0.2 nM) signals through Gαs to activate adenylate cyclase, which results in an increase of intracellular cAMP (EC50 of 0.3 nM). GLP-1 stimulation of cAMP levels measured by the EFC method was similar in both adherent and suspension cell formats (EC50 ~0.3 nM) at different cell numbers. The assay was further validated with forskolin, exendin, and several active GLP-1 peptide analogues. The stimulation of cAMP by GLP-1 and forskolin was effectively inhibited by the adenylate cyclase inhibitors MDL-12330A and SQ-22536, confirming that the increased cAMP is through the AC pathway. The assay tolerates dimethyl sulfoxide (DMSO) up to 10%, and tartrazine does not interfere with the assay with the adherent cells up to 1 mM and affects minimally up to 10 μM in suspension cells. The assay is very robust, with a Z value of 0.7 to 0.8. The assay was validated with several plates of low molecular weight nonpeptide compounds and peptide agonists with different potencies. The suspension cell protocol is a robust homogeneous assay that involves fewer steps than the adherent cell protocol and is suitable for HTS. The cAMP assay using EFC technology is advantageous in that it has a greater dynamic range of detection; is nonradioactive, very sensitive, robust; has minimal interference from DMSO and colored compounds; and is amenable for automation. An added advantage of this assay is that the cAMP is measured as a positive signal, thereby reducing the incidence of false positives.

Eleven Amino Acid Glucagon-like Peptide-1 Receptor Agonists with Antidiabetic Activity

Glucagon-like peptide 1 (GLP-1) is a 30 or 31 amino acid peptide hormone that contributes to the physiological regulation of glucose homeostasis and food intake. Herein, we report the discovery of a novel class of 11 amino acid GLP-1 receptor agonists. These peptides consist of a structurally optimized 9-mer, which is closely related to the N-terminal 9 amino acids of GLP-1, linked to a substituted C-terminal biphenylalanine (BIP) dipeptide. SAR studies resulted in 11-mer GLP-1R agonists with similar in vitro potency to the native 30-mer. Peptides 21 and 22 acutely reduced plasma glucose excursions and increased plasma insulin concentrations in a mouse model of diabetes. These peptides also showed sustained exposures over several hours in mouse and dog models. The described 11-mer GLP-1 receptor agonists represent a new tool in further understanding GLP-1 receptor pharmacology that may lead to novel antidiabetic agents.

Pyridine amides as potent and selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1

Several series of pyridine amides were identified as selective and potent 11beta-HSD1 inhibitors. The most potent inhibitors feature 2,6- or 3,5-disubstitution on the pyridine core. Various linkers (CH(2)SO(2), CH(2)S, CH(2)O, S, O, N, bond) between the distal aryl and central pyridyl groups are tolerated, and lipophilic amide groups are generally favored. On the distal aryl group, a number of substitutions are well tolerated. A crystal structure was obtained for a complex between 11beta-HSD1 and the most potent inhibitor in this series.

Generation of 3,8-substituted 1,2,4-triazolopyridines as potent inhibitors of human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1).

A series of pyridyl amide/sulfonamide inhibitors of 11β-HSD-1 were modified to incorporate a novel 1,2,4-triazolopyridine scaffold. Optimization of substituents at the 3 and 8 position of the TZP core, with a special focus on enhancing metabolic stability, resulted in the identification of compound 38 as a potent and metabolically stable inhibitor of the enzyme.

Characterization of a new class of selective nonsteroidal progesterone receptor agonists.

The identification of a new series of selective nonsteroidal progesterone receptor (PR) agonists is reported. Using a high-throughput screening assay based on the measurement of transactivation of a mouse mammary tumor virus promoter-driven luciferase reporter (MMTV-Luc) in human breast cancer T47D cells, a benzimidazole-2-thione analog was identified. Compound 1 showed an apparent EC50 of 53 nM and efficacy of 93% with respect to progesterone. It binds to PR with high affinity (Ki nM), but had no or very low affinity for other steroid hormone receptors. Structure-activity relationship studies of a series of benzimidazole-2-thione analogs revealed critical positions for high PR binding affinity and transactivation potency as well as receptor selectivity, as exemplified by 25. Compound 25 binds to human PR with high affinity (Ki nM) and had at least > 1000-fold selectivity for PR versus other steroid receptors. Molecular modeling studies suggested that these agonists overlap favorably with progesterone in the ligand-binding domain of PR. In T47D cells, compound 25 acted as a full agonist in the MMTV-Luc reporter assay, as well as in the induction of endogenous alkaline phosphatase activity with apparent EC50 values of 4 and 9 nM, respectively. In the immature rat model, compound 25 provided a significant suppression of estrogen-induced endometrium hypertrophy as measured by luminal epithelial height. In contrast, compound 25 was inactive in the luteinizing hormone release assay in young ovariectomized rats. These benzimidazole-2-thione analogs constitute a new series of nonsteroidal PR agonists with an excellent steroid receptor selectivity profile. The differential activities observed in the in vivo progestogenic assays in rat models suggest that these analogs can act as selective PR modulators.

N-Aryl-oxazolidin-2-imine Muscle Selective Androgen Receptor Modulators Enhance Potency through Pharmacophore Reorientation

A novel selective androgen receptor modulator (SARM) scaffold was discovered as a byproduct obtained during synthesis of our earlier series of imidazolidin-2-ones. The resulting oxazolidin-2-imines are among the most potent SARMs known, with many analogues exhibiting sub-nM in vitro potency in binding and functional assays. Despite the potential for hydrolytic instability at gut pH, compounds of the present class showed good oral bioavailability and were highly active in a standard rodent pharmacological model.

A Sensitive, Robust High-Throughput Electrochemiluminescence Assay for Rat Insulin

In diabetes research, mouse and rat models are used for in vivo experiments, and quantification of insulin in serum samples under different pathophysiological conditions and after treatment with compounds is essential. There are few commercial radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) assay kits to determine the rat/mouse plasma levels of insulin. However, reliability in insulin measurements using the available biological assays is a great concern. The authors report a robust, extremely sensitive electrochemiluminescence (ECL) insulin assay using the Origen technology platform. The assay performance, as judged by the Z′ value of 0.82±0.03 and the signal-to-background (S/B) ratio of 133, suggests that this is a robust and reliable assay. The intra-assay and interassay variation is less than 5%. The dynamic range of detection for insulin is 5 pg to 5 ng in the ECL assays. Recovery of insulin was about 100% when different volumes of serum were spiked with exogenous insulin. These results suggest that the ECL insulin assay is an extremely sensitive, robust, nonradioactive homogeneous assay and can be used successfully to determine the insulin levels in rodent serum samples. (Journal of Biomolecular Screening 2004:62-70)

Potent biphenyl- and 3-phenyl pyridine-based inhibitors of acetyl-CoA carboxylase.

We report the synthesis and enzymatic evaluation of potent inhibitors of acetyl-CoA carboxylases (ACCs) containing biphenyl or 3-phenyl pyridine cores. These compounds inhibit both ACC1 and ACC2, or are moderately selective for either enzyme, depending on side chain substitution. Typical activities of the most potent compounds in this class are in the low double-digit to single-digit nanomolar range in in vitro assays using human ACC1 and ACC2 enzymes.

Optimization of 1,2,4-Triazolopyridines as Inhibitors of Human 11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD-1).

Small alkyl groups and spirocyclic-aromatic rings directly attached to the left side and right side of the 1,2,4-triazolopyridines (TZP), respectively, were found to be potent and selective inhibitors of human 11β-hydroxysteroid dehydrogenase-type 1 (11β-HSD-1) enzyme. 3-(1-(4-Chlorophenyl)cyclopropyl)-8-cyclopropyl-[1,2,4]triazolo[4,3-a]pyridine (9f) was identified as a potent inhibitor of the 11β-HSD-1 enzyme with reduced Pregnane-X receptor (PXR) transactivation activity. The binding orientation of this TZP series was revealed by X-ray crystallography structure studies.

Discovery of 3-hydroxy-4-cyano-isoquinolines as novel, potent, and selective inhibitors of human 11β-hydroxydehydrogenase 1 (11β-HSD1).

Derived from the HTS hit 1, a series of hydroxyisoquinolines was discovered as potent and selective 11β-HSD1 inhibitors with good cross species activity. Optimization of substituents at the 1 and 4 positions of the isoquinoline group in addition to the core modifications, with a special focus on enhancing metabolic stability and aqueous solubility, resulted in the identification of several compounds as potent advanced leads.