Biomedical subjects
Publications and source records attributed to A A Patchett.
A series of C-terminus capped dipeptides and tripeptides was synthesized as growth hormone (GH) secretagogues. Among them, tripeptide Aib-D-Trp-D-homoPhe-OEt showed low nanomolar activity in the rat pituitary assay. Thus, we have demonstrated that the GH secretagogue activity of the hexa-hepta-GH releasing peptides can be mimicked at the tripeptide level.
Systematic SAR studies of the different regioisomers and homologues of the spiro(indane-1,4-piperidine) moiety in the growth hormone secretagogue L-162,752 are presented. Among them, spiro(3H-1-benzopyran-2,3-piperidine) was found to afford secretagogues with low nanomolar in vitro activity.
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Antibodies raised against an intracellular and extracellular domain of the GH secretagogue receptor (GHS-R) confirmed that its topological orientation in the lipid bilayer is as predicted for G protein-coupled receptors with seven transmembrane domains. A strategy for mapping the agonist-binding site of the human GHS-R was conceived based on our understanding of ligand binding in biogenic amine and peptide hormone G protein-coupled receptors. Using site-directed mutagenesis and molecular modeling, we classified GHS peptide and nonpeptide agonist binding in the context of its receptor environment. All peptide and nonpeptide ligand classes shared a common binding domain in transmembrane (TM) region 3 of the GHS-R. This finding was based on TM-3 mutation E124Q, which eliminated the counter-ion to the shared basic N+ group of all GHSs and resulted in a nonfunctional receptor. Restoration of function for the E124Q mutant was achieved by a complementary change in the MK-0677 ligand through modification of its amine side-chain to the corresponding alcohol. Contacts in other TM domains [TM-2 (D99N), TM-5 (M213K, S117A), TM-6 (H280F), and extracellular loop 1 (C116A)] of the receptor revealed specificity for the different peptide, benzolactam, and spiroindolane GHSs. GHS-R agonism, therefore, does not require identical disposition of all agonist classes at the ligand-binding site. Our results support the hypothesis that the ligand-binding pocket in the GHS-R is spatially disposed similarly to the well characterized catechol-binding site in the beta2-adrenergic receptor.
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We have reported that MK-0677 is a novel, orally active GH secretagogue that stimulates an immediate and long-lasting increase in serum GH levels in dogs. Significant elevations in IGF-I levels were associated with the increased GH secretion. Cortisol secretion was also increased following MK-0677 administration. In the current study, we determined the effect of repeat oral administration of MK-0677 on GH, IGF-I and cortisol levels; we also investigated if the GH and cortisol responses to MK-0677 are influenced by circulating IGF-I concentrations. Following the initial oral administration of MK-0677, GH secretion (area under the time-response curve (AUC) ng/ml per h) was increased 7.9- to 9.8-fold (1.0 mg/kg), 5.6-fold (0.5 mg/kg) or 3.9-fold (0.25 mg/kg). With repeat MK-0677 administration, the GH response was decreased by 41-77%; GH concentrations remained significantly above control in the 0.5 mg/kg and 1.0 mg/kg groups. Individual beagle GH profiles indicated that the increased GH concentration was associated with an amplified GH pulsatile profile. Serum IGF-I levels were significantly increased over control levels at all dosage levels by 480 min on the first day of MK-0677 administration. With repeated administration, IGF-I levels were increased up to 126% and remained elevated through 14 days, the longest treatment period evaluated. While daily MK-0677 administration appeared to increase IGF-I levels over 24 h, as evidenced by significant increases in the pretreatment IGF-I levels on days 4-14, no such increase was noted with alternate day MK-0677 administration; thus the dosage regimen modulated circulating IGF-I levels. MK-0677 stimulated increases in cortisol secretion (AUC microgram/dl per h) on the first day of treatment. A decreased cortisol response was observed following repeated daily treatment with MK-0677; in contrast, with alternate day treatment, no decrease in cortisol response to MK-0677 occurred. A marked increase in circulating IGF-I concentrations following administration of exogenous GH resulted in a significant decrease in both the GH and cortisol response to MK-0677 compared with control animals. Our findings suggested, therefore, that circulating IGF-I concentrations regulate GH and cortisol response to MK-0677. In summary, chronic oral administration of MK-0677 was associated with significant increases in GH and IGF-I levels that were maintained for the duration of the treatment. The GH profile following MK-0677 administration consisted of episodic increases above control. Compared with day 1, repeated daily treatment with MK-0677 resulted in an attenuated GH response that was associated with an increase in circulating IGF-I levels. The cortisol response was similarly reduced during chronic MK-0677 treatment, suggesting that IGF-I mediated negative feedback on both the GH and cortisol axes. The fact that similar attenuation of the GH and cortisol responses to MK-0677 on day 1 was observed if IGF-I levels were increased by treating animals with exogenous GH suggested that the attenuated response to MK-0677 that occurred during chronic treatment was mediated by increases in IGF-I rather than desensitization to MK-0677. Thus, a regulatory feedback loop apparently prevents hyperstimulation of the GH axis by MK-0677. We conclude that MK-0677 offers the potential of an orally active GH secretagogue that can maintain elevated IGF-I levels when administered chronically.
Lipid A constitutes the outer monolayer of the outer membrane of Gram-negative bacteria and is essential for bacterial growth. Synthetic antibacterials were identified that inhibit the second enzyme (a unique deacetylase) of lipid A biosynthesis. The inhibitors are chiral hydroxamic acids bearing certain hydrophobic aromatic moieties. They may bind to a metal in the active site of the deacetylase. The most potent analog (with an inhibition constant of about 50 nM) displayed a minimal inhibitory concentration of about 1 microgram per milliliter against Escherichia coli, caused three logs of bacterial killing in 4 hours, and cured mice infected with a lethal intraperitoneal dose of E. coli.
Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.
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The potential application of small molecules in GH therapy has recently become a topic of increasing interest. The spiroindoline MK-0677, the benzolactam L-692,429, and the peptides, GHRP-6 and hexarelin, have been shown to possess potent and selective GH-secretory activity in several species including human. Moreover, these synthetic GH secretagogues act on a signal transduction pathway distinct from that of GHRH. A specific high affinity binding site in porcine and rat anterior pituitary membranes that mediates the activity of these secretagogues has now been identified. The binding affinity of these structurally diverse secretagogues is tightly correlated with GH-secretory activity. The binding is Mg(2+)-dependent, is inhibited by GTP-gamma-S, and is not displaced by GHRH and somatostatin. The receptor is distinct from that for GHRH and has the properties of a new G-protein-coupled receptor. It is speculated that these GH secretagogues mimic an unidentified natural hormone that regulates GH secretion in concert with GHRH and somatostatin.
A potent, orally active growth hormone (GH) secretagogue L-163,191 belonging to a recently synthesized structural class has been characterized. L-163,191 releases GH from rat pituitary cells in culture with EC50 = 1.3 +/- 0.09 nM and is mechanistically indistinguishable from the GH-releasing peptide GHRP-6 and the prototypical nonpeptide GH secretagogue L-692,429 but clearly distinguishable from the natural GH secretagogue, GH-releasing hormone. L-163,191 elevates GH in dogs after oral doses as low as 0.125 mg/kg and was shown to be specific in its release of GH without significant effect on plasma levels of aldosterone, luteinizing hormone, thyroxine, and prolactin after oral administration of 1 mg/kg. Only modest increases in cortisol were observed. Based on these properties, L-163,191 has been selected for clinical studies.
MK-996, N-(4'-(5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridin-3-yl- methyl)1,1'-biphenyl-2-yl)-sulfonylbenzamide, is a potent, orally active, highly selective, nonpeptide angiotensin II (AII) receptor antagonist. MK-996 prevents the pressor response to intravenous AII in the conscious rat, dog, and rhesus monkey (ED50, mg/kg; oral/intravenous = 0.067/0.014, 0.035/0.017, and 0.1/0.036, respectively). In the anesthetized chimpanzee, MK-996 (1 mg/kg, iv) produces 100% (peak) inhibition of the AII pressor response and is still active (52%) at 24 h. To our knowledge this pharmacologic profile in the rat, dog, rhesus monkey, and chimpanzee presents the least species variability of any AII receptor antagonist yet described. Responses to methoxamine and arginine vasopressin are not affected by MK-996. In aortic coarcted (high renin) rats, MK-996 (3 mg/kg, by mouth) reduces blood pressure to normotensive (< 120 mm Hg) levels without reflex tachycardia. This dose of MK-996 reduces blood pressure to approximately the same level as both losartan (3 mg/kg, by mouth) and enalapril (3 mg/kg, by mouth) in this model. The duration of antihypertensive activity of MK-996 is similar to enalapril and shorter than losartan at the doses tested. Additionally, in the rat MK-996 does not potentiate the vasodepressor response to bradykinin and completely prevents the ability of AII to stimulate an increase in plasma levels of aldosterone. Therefore, MK-996 is a potent, orally active, nonpeptide AII receptor antagonist with a long duration of action, little species variability, and anti-hypertensive activity.
The preparation and in vitro prolyl endopeptidase (PEP) inhibitory activity of a series of alpha-keto heterocyclic compounds is described. The design is based on the introduction of alpha-keto heterocycles at the C-terminal end of substrate-like peptides. Many of the compounds including those substituted with thiazole, benzothiazole, benzoxazole, imidazole, and pyridine groups exhibit IC50 potencies of PEP inhibition at nanomolar levels. Structure-activity studies of the C-terminal heterocyclic groups indicate the importance of an sp2 nitrogen atom at a beta-position from the adjoining ketone carbonyl group. This heterocyclic nitrogen atom would provide a critical hydrogen-bond interaction with the histidine residue of the catalytic triad in PEP. Our inhibitors would extend the generality of the alpha-keto heterocycle design to another serine protease.
Several series of 2,4-dihydro-2,4,5-trisubstituted-3H-1,2,4-triazol-3-ones with acidic sulfonamide replacements of tetrazole at the 2'-position of the biphenyl-4-ylmethyl side chain at N4 were prepared and tested as angiotensin II (AII) antagonists. Preferred substituents on the triazolinone ring were n-butyl at C5 and 2-(trifluoromethyl)phenyl at N2. Subnanomolar IC50 values at the AT1 receptor subtype were observed for a variety of acylsulfonamides, including aroyl, heteroaroyl, and cycloalkylcarbonyl derivatives. Certain other acidic sulfonamides, such as sulfonylcarbamates and disulfimides also displayed high affinity for the AT1 receptor. In addition, AT2 binding for some of these compounds was increased by as much as 1000-fold over the corresponding tetrazole (e.g., AT2 IC50 17 nM for the tert-butyl sulfonylcarbamate 92). When evaluated for inhibition of the AII pressor response, the benchmark benzoylsulfonamide 9 (L-159,913) was efficacious in several species and was superior to losartan (1a) in conscious rhesus monkeys. Several subsequent analogues, including the 2-chlorobenzoyl (18), (3-chlorothiophene-2-yl)carbonyl (51), ((S)-2,2-dimethylcyclopropyl)carbonyl (80), and tert-butoxycarbonyl (92) derivatives, were highly effective in rats, surpassing 9 and losartan in duration of action and/or potency. Compound 18 (L-162,223) displayed very prolonged AII antagonism in the rat model (> 24 h at 1 mg/kg iv). At 1 mg/kg po in rats, 18 and 92 (L-162,234) produced 85-87% peak inhibition of the AII pressor response with duration exceeding 6 h. The identification of triazolinone-based sulfonamide derivatives combining high AT1 affinity, considerably enhanced AT2 potency, and favorable in vivo properties provides insights relevant to the design of dual AT1/AT2 receptor antagonists.
A systematic search has been used to derive a hypothesis for the receptor-bound conformation of A-II antagonists at the AT1 receptor. The validity of the pharmacophore hypothesis has been tested using CoMFA, which included 50 diverse A-II antagonists, spanning four orders of magnitude in activity. The resulting cross-validated R2 of 0.64 (conventional R2 of 0.76) is indicative of a good predictive model of activity, and has been used to estimate potency for a variety of non-peptidyl antagonists. The structural model for the non-peptide has been compared with respect to the natural substrate, A-II, by generating peptide to non-peptide overlays.
BACKGROUND: The family of G protein coupled receptors is the largest and perhaps most functionally diverse class of cell-surface receptors. Due to the difficulty of obtaining structural data on membrane proteins there is little information on which to base an understanding of ligand structure-activity relationships, the effects of receptor mutations and the mechanism(s) of signal transduction in this family. We therefore set out to develop a structural model for one such receptor, the human angiotensin II receptor. RESULTS: An alignment between the human angiotensin II (type 1; hAT1), human beta 2 adrenergic, human neurokinin-1, and human bradykinin receptors, all of which are G protein coupled receptors, was used to generate a three-dimensional model of the hAT1 receptor based on bacteriorhodopsin. We observed a region within the model that was congruent with the biogenic amine binding site of beta 2, and were thus able to dock a model of the hAT1 antagonist L-158,282 (MK-996) into the transmembrane region of the receptor model. The antagonist was oriented within the helical domain by recognising that the essential acid functionality of this antagonist interacts with Lys199. The structural model is consistent with much of the information on structure-activity relationships for both non-peptide and peptide ligands. CONCLUSIONS: Our model provides an explanation for the conversion of the antagonist L-158,282 (MK-996) to an agonist by the addition of an isobutyl group. It also suggests a model for domain motion during signal transduction. The approach of independently deriving three-dimensional receptor models and pharmacophore models of the ligands, then combining them, is a powerful technique which helps validate both models.
A series of N-acylated indoles (12-18), N-alkylated indoles (19-24), N-acylated dihydroindoles (26-30), and N-alkylated dihydroindoles (31-34) were synthesized and evaluated in the in vitro AT1 (rabbit aorta) and AT2 (rat midbrain) binding assay. The carboxylic acid 3-[[N-(2-carboxy-3,6-dichlorobenzoyl)-5-indolyl]methyl]-5,7-dimeth yl- 2-ethyl-3H-imidazo[4,5-b]pyridine (14b) was found to be the most potent AT1 (IC50 = 0.8 nM) antagonist in the N-acylated indole series and displayed a 25-fold higher potency than the parent unsubstituted derivative 14a (AT1 IC50 = 20 nM) and a 22-fold greater potency than the corresponding dihydroindole analog 27 (AT1 IC50 = 18 nM). Replacement of the terminal carboxyl (COOH) of 14a with the bioisostere tetrazole in 16 (AT1 IC50 = 5 nM, AT2 IC50 = 130 nM) not only improved the AT1 potency by 4-fold but also resulted in a 50-fold increase in AT2 activity. In the N-alkylated indole series, the tetrazole 3-[[N-(2-tetrazol-5-yl-6-chlorobenzyl)-5- indolyl]methyl]-5,7-dimethyl-2-ethyl-3H-imidazo[4,5-b]pyridine (24) exhibited the highest AT1 (IC50 = 1 nM) activity, revealing a 230-fold increase in AT1 activity as a result of the incorporation of the isosteric tetrazole for the carboxyl (COOH) of 20 and a nearly 9-fold increase over the corresponding deschloro analog 22 (AT1 IC50 = 8.7 nM). Tetrazole 34 was identified as the most potent (AT1 IC50 = 18 nM) AT1 receptor antagonist in a structurally distinct series of compounds derived from N-alkylation of dihydroindole 25. A new class of highly potent (14b, AT1 IC50 = 0.8 nM; 24, AT1 IC50 = 1 nM) AT1-selective non-peptide AII receptor antagonists derived from N-substituted indoles and dihydroindoles is disclosed. Tetrazole 24 of the N-alkylated indole series displayed good in vivo activity by blocking the AII-induced pressor response for 5.5 h after intravenous administration in conscious normotensive rats at a 1.0 mg/kg dose level.
The design, synthesis, and biological activity of a new class of highly potent non-peptide AII receptor antagonists derived from N-substituted (phenylamino)phenylacetic acids and acyl sulfonamides which exhibit a high selectivity for the AT1 receptor are described. A series of N-substituted (phenylamino)phenylacetic acids (9) and acyl sulfonamides (16) and a tetrazole derivative (19) were synthesized and evaluated in the in vitro AT1 (rabbit aorta) and AT2 (rat midbrain) binding assay. The (phenylamino)phenylacetic acids 9c (AT1 IC50 = 4 nM, AT2 IC50 = 0.74 microM), 9d (AT1 IC50 = 5.3 nM, AT2 IC50 = 0.49 microM), and 9e (AT1 IC50 = 5.3 nM, AT2 IC50 = 0.56 microM) were found to be the most potent AT1-selective AII antagonists in the acid series. Incorporation of various substituents in the central and bottom phenyl rings led to a decrease in the AT1 and AT2 binding affinity of the resulting compounds. Replacement of the carboxylic acid (CO2H) in 9c, 9d, and 9e with the bioisostere acyl sulfonamide (CONHSO2Ph) resulted in a (5-7)-fold increase in the AT1 potency of 16a (AT1 IC50 = 0.9 nM, AT2 IC50 = 0.2 microM), 16b (AT1 IC50 = 1 nM, AT2 IC50 = 2.9 microM), and 16c (AT1 IC50 = 0.8 nM, AT2 IC50 = 0.42 microM) and yielded acyl sulfonamides with subnanomolar AT1 activity. Incorporation of the acyl sulfonamide (CONHSO2Ph) for the CO2H of 9c not only enhanced the AT1 potency but also effected a marked increase in the AT2 potency of 16a (AT2 IC50 = 0.74 microM of 9c vs 0.2 microM of 16a) and made it the most potent AT2 antagonist in this study. Replacement of the CO2H of 9b with the bioisostere tetrazole resulted in 19 (AT1 IC50 = 15 nM) with a 2-fold loss in the AT1 and a complete loss in the AT2 binding affinity. (Phenylamino)phenylacetic acid 9c demonstrated good oral activity in AII-infused conscious normotensive rats at an oral dose of 1.0 mg/kg by inhibiting the pressor response for > 6 h. Acyl sulfonamides 16a-c displayed excellent in vivo activity by blocking the AII-induced pressor response for > 6 h after oral administration in conscious rats at a 3.0 mg/kg dose level. Both acyl sulfonamides 16a and 16c exhibited superior in vivo activity in rats compared to that of (phenylamino)phenylacetic acid 9c.