Biomedical subjects

Keith T Demarest

Publications and source records attributed to Keith T Demarest.

31 records · Page 2Linked to original sources

PubMed2004

Synthesis and biological evaluation of novel macrocyclic bis-7-azaindolylmaleimides as potent and highly selective glycogen synthase kinase-3 beta (GSK-3 beta) inhibitors.

Palladium catalyzed cross-coupling reactions were used to synthesize two key intermediates 3 and 5 that resulted in the synthesis of novel series of macrocyclic bis-7-azaindolylmaleimides. Among the three series of macrocycles, the oxygen atom and thiophene containing linkers yielded molecules with higher inhibitory potency at GSK-3 beta (K(i)=0.011-0.079 microM) while the nitrogen atom containing linkers yielded molecules with lower potency (K(i)=0.150->1 microM). Compound 33 and 36 displayed 1-2 orders of magnitude selectivity at GSK-3 beta against CDK2, PKC beta II, Rsk3 and little or no inhibitions to the other 62 protein kinases. Compound 46 was at least 100-fold more selective towards GSK-3 beta than PKC beta II, and it had little or no activity against a panel of 65 protein kinases, almost behaved as a GSK-3 beta 'specific inhibitor'. All three compounds showed good potency in GS assay. Molecular docking studies were conducted in an attempt to rationalize the GSK-3 beta selectivity of azaindolylmaleimides. The high selectivity, inhibitory potency and cellular activities of these non-crown-ether typed molecules may provide them as a valuable pharmacological tools in elucidating the complex roles of GSK-3 beta in cell signaling pathways and the potential usage for the treatment of elevated level of GSK-3 beta involved diseases.

Amino Acid Sequence↗

PubMed2004

Benzoxazinones as PPARgamma agonists. 2. SAR of the amide substituent and in vivo results in a type 2 diabetes model.

A series of benzoxazinones has been synthesized and tested for PPARgamma agonist activity. Synthetic approaches were developed to provide either racemic or chiral compounds. In vitro functional potency could be measured through induction of the aP2 gene, a target of PPARgamma. These studies revealed that compounds with large aliphatic chains at the nitrogen of the benzoxazinone were the most potent. Substitution of the chain was tolerated and in many cases enhanced the in vitro potency of the compound. Select compounds were further tested for metabolic stability, oral bioavailability in rats, and efficacy in db/db mice after 11 days of dosing. In vivo analysis with 13 and 57 demonstrated that the series has potential for the treatment of type 2 diabetes.

Amides↗

PubMed2004

Development of a sensitive and HTS-compatible reporter gene assay for functional analysis of human adenosine A2a receptors in CHO-K1 cells.

Adenosine A2a receptor, a member of the G protein-coupled receptor superfamily, has been demonstrated to be an important pharmacological target. It couples to stimulatory G protein and activates adenylate cyclase upon agonist stimulation. Here we attempted to stably transfect Chinese hamster ovary (CHO-K1) cells, which lack any known subtypes of adenosine receptors, with recombinant human adenosine A2a receptors (hA2aR). Rapid down-regulation of hA2aR in a clonal cell line, CHOA2a-2, was observed over a short period of time in culture. This is consistent with other groups' findings of low expression and poor G protein coupling of this receptor in several cell systems. To facilitate pharmacological profiling for hA2aR ligand, we introduced a cyclic AMP response element (CRE)-linked beta-galactosidase reporter gene into CHOA2a-2 cells to generate a stable cell line, CHOA2a-2CREbetagal#26. Robust cyclic AMP signal amplification was obtained using a colorimetric assay measuring beta-galactosidase activity. The EC(50) of 5'-N-ethylcarboxamidoadenosine (NECA), a potent A2a agonist, for inducing beta-galactosidase activity was 23.3 +/- 3.5 nM, similar to 22.7 +/- 3.9 nM, which was the NECA EC(50) in the direct measurement of cyclic AMP of CHOA2a-2 cells in early culture. Subsequently we validated this assay for high throughput screening for hA2aR agonists. The Z' factor for robotic assay performance was 0.79 +/- 0.03, the ratio of signal/noise was 157 +/- 36, and the ratio of signal/background was 10.6 +/- 1.2, demonstrating that this assay is well suitable for quality high throughput screening. High throughput screening of Johnson & Johnson libraries uncovered a couple of distinct series of nonadenosine small molecules, in addition to adenosine analogues, as potential hA2aR agonists with EC(50) values of 2-6 microM. Preliminary characterization of those compounds was presented.

Adenosine A2 Receptor Agonists↗

PubMed2004

Reduction in glucagon receptor expression by an antisense oligonucleotide ameliorates diabetic syndrome in db/db mice.

Excess glucagon levels contribute to the hyperglycemia associated with type 2 diabetes. Reducing glucagon receptor expression may thus ameliorate the consequences of hyperglucagonemia and improve blood glucose control in diabetic patients. This study describes the antidiabetic effects of a specific glucagon receptor antisense oligonucleotide (GR-ASO) in db/db mice. The ability of GR-ASOs to inhibit glucagon receptor mRNA expression was demonstrated in primary mouse hepatocytes by quantitative real-time RT-PCR. Intraperitoneal administration of GR-ASO at a dosage of 25 mg/kg twice a week in db/db mice for 3 weeks resulted in 1) decreased glucagon receptor mRNA expression in liver; 2) decreased glucagon-stimulated cAMP production in hepatocytes isolated from GR-ASO-treated db/db mice; 3) significantly reduced blood levels of glucose, triglyceride, and free fatty acids; 4) improved glucose tolerance; and 5) a diminished hyperglycemic response to glucagon challenge. Neither lean nor db/db mice treated with GR-ASO exhibited hypoglycemia. Suppression of GR expression was also associated with increased ( approximately 10-fold) levels of plasma glucagon. No changes were observed in pancreatic islet cytoarchitecture, islet size, or alpha-cell number. However, alpha-cell glucagon levels were increased significantly. Our studies support the concept that antagonism of glucagon receptors could be an effective approach for controlling blood glucose in diabetes.

Animals↗

PubMed2003

2,5-disubstituted 3,4-dihydro-2H-benzo[b][1,4]thiazepines as potent and selective V2 arginine vasopressin receptor antagonists.

A number of 2,5-disubstituted benzothiazepines were synthesized and screened for their ability to inhibit arginine vasopressin binding to the human V(2) and V(1a) receptor subtypes. The more active compounds were subsequently analyzed for their antagonist activity in in vitro functional assays. The SAR showed a preference for an acidic unit appended from the benzothiazepine scaffold. This substitution pattern afforded the most potent and selective analogues in the series. The carboxymethyl analogue 4, showed a 140-fold greater selectivity for the V(2) over the V(1a) receptor in the binding assay. In the cell-based functional assays this analogue was a potent and selective antagonist of the V(2) receptor. The in vitro SAR of the series and a description of the in vivo studies around compound 4 is described.

Animals↗

PubMed2003

Human cardiomyocytes express high level of Na+/glucose cotransporter 1 (SGLT1).

We have quantitatively measured gene expression for the sodium-dependent glucose cotransporters 1 and 2 (SGLT1 and SGLT2) in 23 human tissues using the method of real time PCR. As predicted, our results revealed that the expression of SGLT1 was very high in the small intestine (1.2E + 6 molecules/microg total RNA) relative to that in the kidney (3E + 4 molecules/microg total RNA). Surprisingly, we observed that the expression of SGLT1 in human heart was unexpectedly high (3.4E + 5 molecules/microg total RNA), approximately 10-fold higher than that observed in kidney tissue. DNA sequencing confirmed that the PCR amplified fragment was indeed the human SGLT1 gene. Moreover, in situ hybridization studies using a digoxigenin (DIG)-labeled antisense cRNA probe corresponding to human SGLT1 cDNA confirm that human cardiomyocytes express SGLT1 mRNA. In contrast, the expression of SGLT2 in human tissues appears to be ubiquitous, with levels ranging from 6.7E + 4 molecules/microg total RNA (in skeletal muscle) to 3.2E + 6 molecules/microg total RNA (in kidney), levels 10-100-fold higher than the expression of SGLT1 in the same tissues. Our finding that human cardiomyocytes express high levels of SGLT1 RNA suggests that SGLT1 may have a functional role in cardiac glucose transport. Since several SGLT inhibitors are currently in development as potential anti-diabetic agents, it may be important to assess the functional consequences of inhibition of SGLT1 in the heart.

Humans↗

PubMed2003

Macrocyclic bisindolylmaleimides as inhibitors of protein kinase C and glycogen synthase kinase-3.

Efficient methods were developed to synthesize a novel series of macrocyclic bisindolylmaleimides containing linkers with multiple heteroatoms. Potent inhibitors (single digit nanomolar IC(50)) for PKC-beta and GSK-3beta were identified, and compounds showed good selectivity over PKC-alpha, -gamma, -delta, -epsilon, and -zeta. Representative compound 5a also had high selectivity in a screening panel of 10 other protein kinases. In cell-based functional assays, several compounds effectively blocked interleukin-8 release induced by PKC-betaII and increased glycogen synthase activity by inhibiting GSK-3beta.

Antineoplastic Agents↗

PubMed2003

Synthesis and discovery of macrocyclic polyoxygenated bis-7-azaindolylmaleimides as a novel series of potent and highly selective glycogen synthase kinase-3beta inhibitors.

Attempts to design the macrocyclic maleimides as selective protein kinase C gamma inhibitors led to the unexpected discovery of a novel series of potent and highly selective glycogen synthase kinase-3beta (GSK-3beta) inhibitors. Palladium-catalyzed cross-coupling reactions were used to synthesize the key intermediates 17 and 22 that resulted in the synthesis of novel macrocycles. All three macrocyclic series (bisindolyl-, mixed 7-azaindoleindolyl-, and bis-7-azaindolylmaleimides) were found to have submicromolar inhibitory potency at GSK-3beta with various degrees of selectivity toward other protein kinases. To gain the inhibitory potency at GSK-3beta, the ring sizes of these macrocycles may play a major role. To achieve the selectivity at GSK-3beta, the additional nitrogen atoms in the indole rings may contribute to a significant degree. Overall, the bis-7-azaindolylmaleimides 28 and 29 exhibited little or no inhibitions to a panel of 50 protein kinases. Compound 29 almost behaved as a GSK-3beta specific inhibitor. Both 28 and 29 displayed good potency in GS cell-based assay. Molecular docking studies were conducted in an attempt to rationalize the GSK-3beta selectivity of azaindolylmaleimides.

Adipocytes↗

PubMed2003

Benzoxazinones as PPARgamma agonists. part 1: SAR of three aromatic regions.

A series of benzoxazinones was synthesized as PPARgamma agonists. The compounds were obtained in seven steps, and SAR was developed by variations to the core shown below. The compounds were tested as functional agonists in the induction of the aP2 gene in preadipocytes, and the most potent compound in the series has an EC(50)=0.51 microM. The potency was further confirmed through a PPAR-Gal4 construct. Efficacy has been demonstrated in the db/db mouse model of hyperglycemia.

Adaptor Protein Complex 2↗

PubMed2003

Synthesis and biological evaluation of novel indoloazepine derivatives as non-peptide vasopressin V2 receptor antagonists.

A series of novel 3,4,5,6-tetrahydro-1H-azepino[4,3,2-cd]indoles was synthesized and tested for vasopressin receptor antagonist activity. We identified compounds with high affinity for the human V2 receptor and good selectivity over the human V1a receptor. Compound 6c bound to V2 receptors with an IC(50) value of 20 nM, had >100-fold selectivity over V1a receptors, and inhibited cAMP formation in a cellular V2 functional assay with an IC(50) value of 70 nM.

Antidiuretic Hormone Receptor Antagonists↗

PubMed2003

Validation of a cell-based screen for insulin receptor modulators by quantification of insulin receptor phosphorylation.

Stimulation of a cell with insulin initiates a signal transduction cascade that results in cellular activities that include phosphorylation of the receptor itself. Measurement of the degree of phosphorylation can serve as a marker for receptor activation. Receptor phosphorylation has been measured using Western blot analysis, which is very low throughput and not easily quantifiable. The goal of this project was to develop a cell-based assay to measure receptor phosphorylation in high throughput. This report describes a cell-based assay for insulin receptor phosphorylation that is robust and amenable to high-volume screening in a microwell format.

Animals↗

PubMed2002

Bridged bicyclic vasopressin receptor antagonists with V(2)-selective or dual V(1a)/V(2) activity.

The synthesis and biological testing of a novel series of nonpeptide vasopressin receptor antagonists, containing a bridged bicyclic nucleus, are reported. Variation of substituents (R(1)-R(3)) in general formula 3, and the configuration of the stereocenter, resulted in potent V(2)-selective (e.g., 5) and balanced dual V(1a)/V(2) (e.g., 10) compounds. Data from receptor binding, cell-based functional, and in vivo assays are presented [corrected]

Animals↗

PubMed2002

The use of biosensors to study GPCR function: applications for high-content screening.

Plasma membrane-associated G protein-coupled receptors (GPCRs) initiate the transmission of multiple intracellular signals leading to a myriad of physiological and pathophysiological effects. The downstream signaling events associated with occupation of the GPCR and activation of the G-protein include the generation of numerous second messenger molecules to provide the necessary signal amplification within the appropriate intracellular compartment to transmit a specific signal from the cell surface to the cell interior. The complex process of signal transmission also requires a series of highly orchestrated events which includes the translocation of cellular proteins to discreet intracellular destinations. A better understanding of these events has made it possible to design assays to examine multiple endpoints within whole cells. In this review we describe recent advances in assay biology and instrumentation useful for broadening our understanding of the molecular events associated with GPCR activation. This review will focus on novel cell-based approaches using fluorescent biosensors, such as fluorescent dyes and fluorescent protein tags, to generate information-rich data from multiple cellular targets--a process that has been referred to as high-content screening. High-content screening applications will be discussed as they pertain to specific signal transduction cascades initiated upon GPCR activation and examples of specific biosensors will be provided.

Animals↗