Phosphonomycin: structure and synthesis.

Synthesis and resolution of the antibiotic phosphonomycin are described. The structure is (-)(IR, 2S)-1,2-epoxypropylphosphonic acid.

A potent nonpeptide cholecystokinin antagonist selective for peripheral tissues isolated from Aspergillus alliaceus.

A new, competitive, nonpeptide cholecystokinin (CCK) antagonist, asperlicin, was isolated from the fungus Aspergillus alliaceus. The compound has 300 to 400 times the affinity for pancreatic, ileal, and gallbladder CCK receptors than proglumide, a standard agent of this class. Moreover, asperlicin is highly selective for peripheral CCK receptors relative to brain CCK and gastrin receptors. Since asperlicin also exhibits long-lasting CCK antagonist activity in vivo, it should provide a valuable tool for investigating the physiological and pharmacological actions of CCK.

Antibacterial agents that inhibit lipid A biosynthesis.

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.

Receptor for motilin identified in the human gastrointestinal system.

Motilin is a 22-amino acid peptide hormone expressed throughout the gastrointestinal (GI) tract of humans and other species. It affects gastric motility by stimulating interdigestive antrum and duodenal contractions. A heterotrimeric guanosine triphosphate-binding protein (G protein)-coupled receptor for motilin was isolated from human stomach, and its amino acid sequence was found to be 52 percent identical to the human receptor for growth hormone secretagogues. The macrolide antibiotic erythromycin also interacted with the cloned motilin receptor, providing a molecular basis for its effects on the human GI tract. The motilin receptor is expressed in enteric neurons of the human duodenum and colon. Development of motilin receptor agonists and antagonists may be useful in the treatment of multiple disorders of GI motility.

Rapid identification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry.

Nonpeptide agonists of each of the five somatostatin receptors were identified in combinatorial libraries constructed on the basis of molecular modeling of known peptide agonists. In vitro experiments using these selective compounds demonstrated the role of the somatostatin subtype-2 receptor in inhibition of glucagon release from mouse pancreatic alpha cells and the somatostatin subtype-5 receptor as a mediator of insulin secretion from pancreatic beta cells. Both receptors regulated growth hormone release from the rat anterior pituitary gland. The availability of high-affinity, subtype-selective agonists for each of the somatostatin receptors provides a direct approach to defining their physiological functions.

A receptor in pituitary and hypothalamus that functions in growth hormone release.

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.

Orphan G-protein-coupled receptors and natural ligand discovery.

The superfamily of seven-transmembrane-domain G-protein-coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Each of the approximately 1000 family members found in vertebrates responds to stimuli as diverse as hormones, neurotransmitters, odorants and light, which selectively activate intracellular signaling events mediated by heterotrimeric G proteins. Because GPCRs are centrally positioned in the plasma membrane to initiate a cascade of cellular responses by diverse extracellular mediators, it is not surprising that modulation of GPCR function has been successful in the development of many marketed therapeutic agents. It has become clear that GPCRs for which a natural activating ligand has not yet been identified (orphan GPCRs) might provide a path to discovering new cellular substances that are important in human physiology. The process of 'de-orphanizing' these novel proteins has accelerated significantly and opened up new avenues for research in human physiology and pharmacology.

Structural model of antagonist and agonist binding to the angiotensin II, AT1 subtype, G protein coupled receptor.

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.

Identification of a new G-protein-linked receptor for growth hormone secretagogues.

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.

Ligand activation domain of human orphan growth hormone (GH) secretagogue receptor (GHS-R) conserved from Pufferfish to humans.

Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.

Structural requirements for the activation of the human growth hormone secretagogue receptor by peptide and nonpeptide secretagogues.

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.

Substituted pyrazolo corticoids as topical antiinflammatory agents.

The synthesis of a series of substituted pyrazolo corticoids is described. Of these 11beta,17alpha,21-trihydroxy-6,16alpha-dimethyl-4,6-pregnadieno[3,2-c]-2'-(4-pyridly)pyrazole (21) shows an excellent separation of systemic to local activity in the model animal test. Compound 21 exhibits high vasoconstriction activity in human volunteers and is clinically effective in the treatment of psoriasis.

Synthesis of an analogue of tabtoxinine as a potential inhibitor of D-alanine:D-alanine ligase (ADP forming).

The design and synthesis of a potential inhibitor of D-alanine:D-alanine ligase (ADP forming) (EC 6.3.2.4) are described. This enzyme, which catalyzes the second step in the biosynthesis of bacterial peptidoglycan, is believed to generate D-alanyl phosphate as an enzyme-bound intermediate. With tabtoxinine, a potent inhibitor of glutamine synthetase, as a model, beta-lactams 9R and 9S were synthesized as potential precursors of a D-alanyl phosphate mimic.

5,11-dimethyl-2,9-bis(phenylacetyl)-5,11-diazatetracyclo[6.2.2.0(2,7).0(4,9)]dodecane, a potent, novel analgesic.

5,11-Dimethyl-2,9-bis(phenylacetyl)-5,11-diazatetracyclo[6.2.2.0(2,7)90(4,9)]dodecane (2a) has been found to be a potent narcotic analgesic of unusual structure. All of the analgesic activity was attributable to the levorotatory isomer 2b which was approximately three times as potent as morphine in the rat. Removal of one N-methyl group from 2a reduced, but did not abolish, the analgesic activity. However N-allyl analogues were neither agonists nor antagonists. Replacement of one of the phenyls of 2a with a cyclohexyl group yielded an analogue with considerable activity. Structural similarities with derivatives of ethenooripavine are noted.

Synthesis and structure-activity relationships of peptidyl alpha-keto heterocycles as novel inhibitors of prolyl endopeptidase.

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.

Synthesis and biological activity of flavipucine analogues.

A series of analogues of flavipucine was prepared possessing side chain as well as nuclear variants. The analogue with an octyl side chain (5d) was found to exhibit enhanced activity against several bacteria and fungi as compared with the natural product itself. The separation and characterization of the individual diastereoisomeric pairs both spectroscopically and with respect to chromatographic mobility have been effected.

Cholecystokinin antagonists. Synthesis and biological evaluation of 3-substituted benzolactams.

A series of 1,3-substituted benzolactams are reported that are potent nonpeptidal antagonists of the peptide hormone cholecystokinin (CCK). Design considerations were based upon the natural product CCK antagonist asperlicin and the potent benzodiazepine antagonist series exemplified by L-364,718 (1). Compound 19, the most potent compound in the benzolactam series, had an IC50 = 3 nM for inhibition of binding of 125I-CCK-8 to CCK receptors in rat pancreatic tissue, and its racemic analogue 8 was found to be orally active in inhibiting CCK-induced gastric emptying in mice, with an ED50 = 2.6 mg/kg po. The effects of ring size, substitution at positions 1 and 3, and stereochemistry at position 3 are discussed. Conformational studies of compound 19 and L-364,718 have delineated similarities that these molecules share in their core conformations and substituent orientations.

Angiotensin-converting enzyme inhibitors: synthesis and biological activity of acyl tripeptide analogues of enalapril.

The synthesis and biological activity of a series of inhibitors of angiotensin-converting enzyme (EC 3.4.15.1) are described. Incorporation of the substituted N-carboxymethyl dipeptide design of enalapril (MK-421) into acyl tripeptides and larger peptides yielded potent inhibitors of the enzyme. These can be viewed as substrate analogues in which the carbonyl of the scissile peptide bond is replaced by a CHCO2H group. Several of the analogues described possess inhibitory potency equal to that of enalaprilat (MK-422), but none achieves an increase in potency which would demonstrate additional binding interactions contributed by the extended peptide chain. Application of the design described may be useful for inhibition of other metallopeptidases.

(3-Amino-2-oxoalkyl)phosphonic acids and their analogues as novel inhibitors of D-alanine:D-alanine ligase.

The dipeptide D-alanyl-D-alanine is an essential precursor of bacterial peptidoglycan; thus, blocking its formation is a possible target for the design of novel antibacterial agents. The synthesis of this dipeptide by bacterial D-alanine:D-alanine ligase requires ATP. In analogy with glutamine synthetase, we hypothesized a mechanism for this enzyme involving the intermediacy of D-alanyl phosphate. Several (3-amino-2-oxoalkyl)phosphonic acids and their analogues have been synthesized as possible inhibitory mimics of this proposed intermediate. The most active of them, (3(R)-amino-2-oxobutyl)phosphonic acid (8a) and the corresponding aza analogue (22), were effective ligase inhibitors although they had no significant antibacterial activity. The ligase inhibition of these compounds is consistent with an acyl phosphate displacement step in the mechanism of DAla-DAla ligase.

Design and synthesis of P2-P1'-linked macrocyclic human renin inhibitors.

Using a computer model of the active site of human renin developed at Merck, we designed a series of novel P2-P1'-linked, macrocyclic renin inhibitors 3-10. These unique inhibitors incorporate a transition-state isostere within a 13- or 14-membered ring. The three most active compounds in this family were 13-membered-ring glutamine-derived inhibitor 3, 14-membered-ring diaminopropionic acid derived inhibitor 6, and 13-membered-ring diol 9 (IC50 0.61, 0.59, 0.65 microM, respectively). Modification of inhibitor 3 at P4 led to 56 nM macrocyclic renin inhibitor 39. This study shows the viability of renin inhibitor designs which incorporate a scissile-bond replacement within a macrocycle.