Analysis of combinatorial chemistry samples by micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method has been developed that can evaluate the purity of samples generated in combinatorial chemistry libraries. This method uses an open tube capillary (27 cm x 50 microm) along with a run buffer composed of sodium dodecyl sulfate (SDS), hydroxypropyl-beta-cyclodextrin, and sodium tetraborate coupled with UV detection. Neutral compounds and compounds that were insoluble in aqueous buffers could be analyzed under these conditions in approximately 3 min. The concentration of SDS and the concentration of hydroxypropyl-beta-cyclodextrin effected the separation. The affect on selectivity resulting from the addition of an organic modifier to the run buffer was examined. The low background absorbency of the run buffer made for easy detection of compounds that absorbed at low UV wavelengths. The quick analysis time made this suitable for analysis of combinatorial chemistry samples.

Novel nonsecosteroidal vitamin D mimics exert VDR-modulating activities with less calcium mobilization than 1,25-dihydroxyvitamin D3.

BACKGROUND: The secosteroid 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) acts through the vitamin D receptor (VDR) to elicit many activities that make it a promising drug candidate for the treatment of a number of diseases, including cancer and psoriasis. Clinical use of 1,25(OH)2D3 has been limited by hypercalcemia elicited by pharmacologically effective doses. We hypothesized that structurally distinct, nonsecosteroidal mimics of 1,25(OH)2D3 might have different activity profiles from vitamin D analogs, and set out to discover such compounds by screening small-molecule libraries. RESULTS: A bis-phenyl derivative was found to activate VDR in a transactivation screening assay. Additional related compounds were synthesized that mimicked various activities of 1,25(OH)2D3, including growth inhibition of cancer cells and keratinocytes, as well as induction of leukemic cell differentiation. In contrast to 1, 25(OH)2D3, these synthetic compounds did not demonstrate appreciable binding to serum vitamin D binding protein, a property that is correlated with fewer calcium effects in vivo. Two mimics tested in mice showed greater induction of a VDR target gene with less elevation of serum calcium than 1,25(OH)2D3. CONCLUSIONS: These novel VDR modulators may have potential as therapeutics for cancer, leukemia and psoriasis with less calcium mobilization side effects than are associated with secosteroidal 1,25(OH)2D3 analogs.

Synthesis of retinoid X receptor-specific ligands that are potent inducers of adipogenesis in 3T3-L1 cells.

A novel series of oxime ligands has been synthesized that displays potent, specific activation of the retinoid X receptors (RXRs). The oximes of 3-substituted (tetramethyltetrahydronaphthyl)carbonylbenzoic acids are readily available by condensation with hydroxyl- or methoxylamine; alkylation of the hydroxyl oxime provides a variety of analogues. Oximes and variously substituted oxime derivatives demonstrate high binding affinity for the RXRs and specific RXR activation and, hence, are called rexinoids. These oxime rexinoids are activators of the RXR:PPARgamma heterodimer and are potent inducers of differentiation of 3T3-L1 preadipocytes to adipocytes. We have recently reported that ligands which activate the RXR:PPARgamma heterodimer in this manner are effective in the treatment of type II diabetes (non-insulin-dependent diabetes mellitus, NIDDM). Thus, these new oxime rexinoids are potential therapeutic agents for the treatment of metabolic disorders, such as obesity and diabetes.

Matrix metalloproteinase inhibitors: a structure-activity study.

Modifications around the dipeptide-mimetic core of a hydroxamic acid based matrix metalloproteinase inhibitor were studied. These variations incorporated a variety of natural, unnatural, and synthetic amino acids in addition to modifications of the P1' and P3' substituents. The results of this study indicate the following structural requirements: (1) Two key hydrogen bonds must be present between the enzyme and potent substrates. (2) Potent inhibitors must possess strong zinc-binding functionalities. (3) The potential importance of the hydrophobic group at position R3 as illustrated by its ability to impart greater relative potency against stromelysin when larger hydrophobic groups are used. (4) Requirements surrounding the nature of the amino acid appear to be more restrictive for stromelysin than for neutrophil collagenase, 72 kDa gelatinase, and 92 kDa gelatinase. These requirements may involve planar fused-ring aryl systems and possibly hydrogen-bonding capabilities.

Sensitization of diabetic and obese mice to insulin by retinoid X receptor agonists.

Retinoic acid receptors (RAR), thyroid hormone receptors (TR), peroxisome proliferator activated receptors (PPARs) and the orphan receptor, LXR, bind preferentially to DNA as heterodimers with a common partner, retinoid X receptor (RXR), to regulate transcription. We investigated whether RXR-selective agonists replicate the activity of ligands for several of these receptors? We demonstrate here that RXR-selective ligands (referred to as rexinoids) function as RXR heterodimer-selective agonists, activating RXR: PPARgamma and RXR:LXR dimers but not RXR:RAR or RXR:TR heterodimers. Because PPARgamma is a target for antidiabetic agents, we investigated whether RXR ligands could alter insulin and glucose signalling. In mouse models of noninsulin-dependent diabetes mellitus (NIDDM) and obesity, RXR agonists function as insulin sensitizers and can decrease hyperglycaemia, hypertriglyceridaemia and hyperinsulinaemia. This antidiabetic activity can be further enhanced by combination treatment with PPARgamma agonists, such as thiazolidinediones. These data suggest that the RXR:PPARgamma heterodimer is a single-function complex serving as a molecular target for treatment of insulin resistance. Activation of the RXR:PPARgamma dimer with rexinoids may provide a new and effective treatment for NIDDM.

A novel retinoic acid receptor-selective retinoid, ALRT1550, has potent antitumor activity against human oral squamous carcinoma xenografts in nude mice.

We have identified a novel retinoid, ALRT1550, that potently and selectively activates retinoic acid receptors (RARs). ALRT1550 binds RARs with Kd values of approximately equal to 1-4 nM, and retinoid X receptors with low affinities (Kd approximately equal to 270-556 nM). We studied the effects of ALRT1550 on cellular proliferation in squamous carcinoma cells. ALRT1550 inhibited in vitro proliferation of UMSCC-22B cells in a concentration-dependent manner with an IC50 value of 0.22 +/- 0.1 (SE) nM. 9-cis-Retinoic acid (ALRT1057), a pan agonist retinoid that activates RARs and retinoid X receptors, inhibited proliferation with an IC50 value of 81 +/- 29 nM. In vivo, as tumor xenografts in nude mice, UMSCC-22B formed well-differentiated squamous carcinomas, and oral administration (daily, 5 days/week) of ALRT1550, begun 3 days after implanting tumor cells, inhibited tumor growth by up to 89% in a dose-dependent manner over the range of 3-75 micrograms/kg. ALRT1550 (30 micrograms/kg) also inhibited growth of established tumors by 72 +/- 3% when tumors were allowed to grow to approximately equal to 100 mm3 before dosing began. In comparison, 9-cis retinoic acid at 30 mg/kg inhibited growth of established tumors by 73 +/- 5%. Interestingly, retinoids did not appear to alter tumor morphologies in UMSCC-22B tumors. Notably, ALRT1550 produced a therapeutic index of approximately equal to 17 in this model, indicating a separation between doses that inhibited tumor growth and that induced symptoms of hypervitaminosis A. In summary, ALRT1550 potently inhibits cellular proliferation in vitro and in vivo in this squamous cell carcinoma tumor model. These data support additional study of ALRT1550 for its potential for improving anticancer therapy in human clinical trials.

Activation of specific RXR heterodimers by an antagonist of RXR homodimers.

Retinoid X receptor (RXR) plays a central role in the regulation of many intracellular receptor signalling pathways and can mediate ligand-dependent transcription, acting as a homodimer or as a heterodimer. Here we identify an antagonist towards RXR homodimers which also functions as an agonist when RXR is paired as a heterodimer to specific partners, including peroxisome proliferator-activated receptor and retinoic acid receptor. This dimer-selective ligand confers differential interactions on the transcription machinery: the antagonist promotes association with TAF110 (TATA-binding protein (TBP)-associated factor 110) and the co-repressor SMRT, but not with TBP, and these properties are distinct from pure RXR agonists. This unique class of RXR ligands will provide a means to control distinct target genes at the level of transcription and allow the development of retinoids with a new pharmacological action.

Discovery of novel retinoic acid receptor agonists having potent antiproliferative activity in cervical cancer cells.

Retinoic acid receptor (RAR) active retinoids have proven therapeutically useful for treating certain cancers and dermatological diseases. Herein, we describe the discovery of two new RAR active trienoic acid retinoids, (2E,4E,6E)-7-(3,5-di-tert-butylphenyl)-3-methylocta-2, 4,6-trienoic acid (10a, ALRT1550) and (2E,4E,6Z)-7-(3,5-di-tert-butylphenyl)-3-methylocta-2, 4,6-trienoic acid (10b, LG100567). ALRT1550 is a RAR selective retinoid which exhibits exceptional potency in both competitive binding and cotransfection assays. Moreover, it is the most potent antiproliferative retinoid described to date and thus has implications for the treatment of certain cancers. LG100567 is a potent panagonist which activates both RARs and retinoid X receptors.

Characterization of the novel CCK analogs JMV-180, JMV-320, and JMV-332 in H345 cells.

The interaction of the novel CCK analogs JMV-180, JMV-320, and JMV-332 with CCK-B/gastrin receptors on small cell lung cancer (SCLC) cells was investigated. JMV-180, JMV-320, and JMV-332 potently inhibited specific binding of 125I-CCK-8 to CCK-B/gastrin receptors expressed on the SCLC cell line NCI-H345 (H345) with IC50 values of 4.9, 1.8, and 7.0 nM, respectively. JMV-320 and JMV-332 stimulated intracellular calcium ([Ca2+]i) release in a dose-dependent manner in cells preloaded with indo-1. JMV-180 did not stimulate [Ca2+]i but inhibited the [Ca2+]i release elicited by 10 nM CCK-8 in a dose-dependent manner. These data indicate that JMV-320 and JMV-332 function as CCK-B/gastrin receptor agonists while JMV-180 functions as a CCK-B/gastrin receptor antagonist in H345 cells.

Intraventricular CCK-8 reduces single meal size in the baboon by interaction with type-A CCK receptors.

Intraventricular cholecystokinin COOH-terminal octapeptide (CCK-8) decreases meal size in the meal-trained baboon. In the present study, we tested whether this action is mediated by CCK-A receptors, CCK-B receptors, or both. Intraventricular administration of the selective CCK-A receptor agonist A71623 at 1 and 10 nmol/kg suppressed 30-min meal size 69 +/- 22% and 75 +/- 7%, respectively. Additionally, intraventricular A71623 was equipotent to CCK-8 at 1 nmol/kg (% suppression of meal by CCK = 59 +/- 17). However, intraventricular administration of the CCK-B receptor agonist A63387 at 10 nmol/kg had no effect on 30-min meal size (% suppression = 18 +/- 29). Intravenous administration of 10 nmol/kg A71623 did not result in an alteration of meal size, but prandial plasma insulin and glucose responses were delayed and blunted. Basal plasma insulin levels doubled after intravenous administration of A71623. Both behavioral and metabolic responses to A71623 in the baboon are virtually identical to those we have previously observed after CCK-8 treatment. Thus we conclude that the predominant receptor population with which intraventricular CCK-8 interacts are type-A CCK receptors that are accessible to the ventricular system of the baboon.

Evaluation of a stable CCK agonist (A68552) in conditioned avoidance responding in mice, rats, and primates: comparison with typical and atypical antipsychotics.

In a variety of in vivo and in vitro tests, cholecystokinin (CCK) has been shown to produce effects that would suggest a functional antagonism of dopamine. On that basis, it has been hypothesized that CCK could have antipsychotic effects. We compared the CCK agonist, A68552, to the antipsychotics haloperidol (HAL), clozapine (CLOZ) and sulpiride (SULP) in various forms of conditioned avoidance using rats, mice, and cynomolgus monkeys. In rats, HAL disrupted both acquisition of a conditioned shelf-jump avoidance response and performance of the response by previously trained animals. CLOZ and SULP were ineffective in suppressing performance by previously trained rats but blocked acquisition of the response. CLOZ disrupted avoidance responding on the first 3 of 4 consecutive days of acquisition. SULP significantly suppressed avoidance responding on the last 3 days and significantly increased escape failures on day 2. A68552 administered during acquisition failed to significantly suppress avoidance responding. In mice, both HAL and CLOZ blocked performance of two-way shuttle conditioned avoidance at doses (0.1 and 3.0 mg/kg, IP, respectively) that had no effect on escape responding. A68552 at doses up to 1.07 mg/kg IP had no effect on performance. Mice treated with A68552 during acquisition showed a mild but statistically significant suppression of avoidance and an equivalent suppression of escape responding. Cynomolgus monkeys trained in a conditioned avoidance procedure were sensitive to the disruptive effects of HAL at a dose of 0.03 mg/kg IM while A68552 was without significant effect at doses up to those producing emesis (0.214 mg/kg, IM). A68552 does not resemble either HAL or the "atypical" antipsychotics, CLOZ or SULP, in conditioned avoidance tests.

A-71623, a selective CCK-A receptor agonist, suppresses food intake in the mouse, dog, and monkey.

The anorectic actions of cholecystokinin (CCK)-8 and of a selective CCK-A agonist, A-71623, were examined in CD1 mice, beagle dogs, and cynomolgus monkeys. A-71623 suppressed intakes in all species tested, and the effects were blocked by a selective CCK-A antagonist, A-70104. In the dog only, CCK-8 was more potent on a molar basis compared to A-71623, although the effects of both CCK agonists were more short-lived in the dog compared to the other species tested. Our results support other evidence suggesting that the anorectic actions of exogenous application of CCK-8 in these species are mediated via stimulation of the CCK-A receptor subtype.

Behavioral effects of A71623, a highly selective CCK-A agonist tetrapeptide.

We studied the behavioral effects of a novel cholecystokinin tetrapeptide (CCK-4) analogue, A71623, with full agonist activity and high affinity and selectivity for the CCK-A receptor subtype relative to the CCK-B receptor. In tests for anorectic activity, A71623 was found to suppress 60-min intakes of a liquid diet in both deprived and sated rats, and the effects were blocked by a selective CCK-A antagonist, A70104. Compared with CCK-8, A71623 was found to have improved potency and duration of action; the most potent route of administration was intraperitoneal. A71623 also suppressed the intake of a liquid diet and a 0.2 M sucrose solution in lean and obese Zucker rats. In daily injection studies, the anorectic activity of CCK-8 diminished rapidly, whereas the suppressant effects of A71623 on food intakes and body weight gains persisted throughout the 11-day treatment period. Finally, A71623 reduced the spontaneous locomotor activity of rats at doses above those required to suppress intakes. These studies are the first to describe the behavioral effects of a potent and highly selective CCK-A receptor agonist.

Development of potent and selective CCK-A receptor agonists from Boc-CCK-4: tetrapeptides containing Lys(N epsilon)-amide residues.

A series of Boc-CCK-4 derivatives represented by the general structure Boc-Trp-Lys(N epsilon-COR)-Asp-Phe-NH2, where R is an aromatic, heterocyclic, or aliphatic group, are potent and selective CCK-A receptor agonists. These amide-bearing compounds complement the previously described urea-based tetrapeptides (Shiosaki et al. J. Med. Chem. 1991, 34, 2837-2842); structure-activity studies revealed parallel as well as divergent trends between these two series. A significant correlation was observed between pancreatic binding affinity and the resonance constant R of the phenyl substituent in one particular series of derivatives. Sulfation of phenolic amides appended onto the epsilon-amino group of the lysine did not affect affinity for the CCK-A receptor in contrast to the 500-fold increase in binding potency observed upon sulfation of CCK-8, suggesting that the lysine appendage and the sulfated tyrosine in CCK-8, both key structural elements that impart high affinity for the CCK-A receptor, are interacting differently with the receptor. The amide-bearing tetrapeptides are full agonists relative to CCK-8 in stimulating pancreatic amylase release while being partial agonists in eliciting phosphoinositide (PI) hydrolysis. Both effects were blocked by selective CCK-A receptor antagonists.

Effects of selective CCK receptor agonists on food intake after central or peripheral administration in rats.

In this paper report the effects of peripheral (intraperitoneal, i.p.) and central (intracerebroventricular, i.c.v.) injection of selective cholecystokinin (CCK) receptor agonists on food intake in the rat. Stimulation of peripheral and central CCK-A receptors by the selective CCK-A receptor agonist A-71623 suppressed intakes of a liquid diet in both deprived and sated rats. In contrast, i.c.v., but not i.p., injections of the selective CCK-B receptor agonist A-63387, reduced food intakes, although on a molar basis the effect was much less than that seen with A-71623. Although these results stress the relative importance of the CCK-A receptor in the effects of exogenous CCK-8 administration on feeding, stimulation of the CCK-B receptor may still be involved in the control of feeding following the endogenous release of CCK.

Cholecystokinin antagonists: (R)-tryptophan-based hybrid antagonists of high affinity and selectivity for CCK-A receptors.

The intriguing structural similarities of glutamic acid based cholecystokinin (CCK) antagonists (A-64718 and A-65186) and the benzodiazepine CCK antagonist MK-329 (L-364,718) have been reported. Efforts to include the weak CCK antagonist benzotript into this construct utilizing a similar approach have resulted in a novel series of benzotript-based hybrid antagonists N alpha-(3'-quinolylcarbonyl)-(R)-tryptophan di-n-pentylamide (9, A-67396), N alpha-(4',8'-dihydroxy-2'-quinolylcarbonyl)-(R)-tryptophan di-n-pentylamide (23, A-70276), and N alpha-(3'-quinolylcarbonyl)-(R)-5'-hydroxytryptophan di-n-pentylamide (36, A-71134) which possess respectively binding affinities of 23, 21, and 11 nM for the pancreatic CCK-A receptor and which inhibit CCK8-induced amylase secretion. Compound 9 possesses a selectivity of greater than 500-fold for the pancreatic CCK-A receptor over the CCK-B receptor.

Boc-CCK-4 derivatives containing side-chain ureas as potent and selective CCK-a receptor agonists.

Novel Boc-CCK-4 derivatives were communicated recently as having high potency and selectivity for the CCK-A receptor (Shiosaki et al. J. Med. Chem. 1990, 33, 2950-2952). While Boc-CCK-4 binds selectively to the CCK-B receptor, replacement of the methionine with an N epsilon-substituted lysine dramatically reversed receptor selectivity, leading to the development of this novel series of tetrapeptides. A detailed structure-activity analysis of a series of urea-substituted tetrapeptides, represented by the general structure Boc-Trp-Lys(N epsilon-CO-NHR)-Asp-Phe-NH2, revealed that a number of substituted phenyl, naphthyl, and aliphatic urea residues in the lysine side chain yielded potent and selective CCK-A ligands. These tetrapeptides elicit full agonist responses in stimulating pancreatic amylase release that are effectively blocked by a selective CCK-A receptor antagonist. Conversion of the urea to a thiourea significantly reduced CCK-A binding potency as did replacement of the lysine with the homologous ornithine or homolysine. Tetrapeptides that were partial agonists (less than 80% efficacy) in phosphoinositide (PI) hydrolysis relative to CCK-8 did not exhibit high-dose inhibition of amylase secretion in guinea pig acini.

Characterization of two novel cholecystokinin tetrapeptide (30-33) analogues, A-71623 and A-70874, that exhibit high potency and selectivity for cholecystokinin-A receptors.

Based on their relative affinities for cholecystokinin octapeptide (26-33) (CCK-8), cholecystokinin tetrapeptide (30-33) (CCK-4), desulfated CCK-8, and gastrin, cholecystokinin (CCK) receptors have been classified as CCK-A (alimentary) and CCK-B (brain). Selective nonpeptide antagonists of CCK-A and CCK-B receptors, as well as highly selective CCK-A and CCK-B peptide agonists, have been described. We report here the characterization of two novel CCK-4-based peptides, A-71623 and A-70874. In radioligand binding assays, the IC50 values for A-71623 and A-70874 were 3.7 and 4.9 nM in guinea pig pancreas (CCK-A) and 4500 and 710 nM in cerebral cortex (CCK-B), respectively. Both were agonists in stimulating pancreatic amylase release, and their stimulatory effects were potently inhibited by the CCK-A antagonist L-364,718. A-71623 was a full agonist and A-70874 was a partial agonist (approximately 80%) in stimulating phosphoinositide breakdown in pancreas. Both peptides also were potent agonists in stimulating CCK-A receptors in the ileum. They were, however, weak and behaved as partial agonists in calcium studies in NCI-H345 cells, which possess CCK-B/gastrin receptors. In guinea pig gastric glands, the affinities of A-71623 and A-70874 for the CCK-B/gastrin receptor were 11 and 1.6 microM, respectively. These results demonstrate that A-71623 and A-70874 are potent and selective agonists at CCK-A receptors. The preferential interaction of these novel CCK-4 analogs with CCK-A receptors is in contrast to other CCK-4-based peptides, which are primarily selective for CCK-B receptors. In addition, A-71623 and A-70874 are the first two examples of potent CCK-A agonists that do not contain a tyrosine residue whose sulfation is required for potent CCK-A agonist activity of larger peptides.