Structure-function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a.

The recently discovered growth hormone secretagogue, ghrelin, is a potent agonist at the human growth hormone secretagogue receptor 1a (hGHSR1a). To elucidate structural features of this peptide necessary for efficient binding to and activation of the receptor, several analogues of ghrelin with various aliphatic or aromatic groups in the side chain of residue 3, and several short peptides derived from ghrelin, were prepared and tested in a binding assay and in an assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Bulky hydrophobic groups in the side chain of residue 3 turned out to be essential for maximum agonist activity. Also, short peptides encompassing the first 4 or 5 residues of ghrelin were found to functionally activate hGHSR1a about as efficiently as the full-length ghrelin. Thus the entire sequence of ghrelin is not necessary for activity: the Gly-Ser-Ser(n-octanoyl)-Phe segment appears to constitute the "active core" required for agonist potency at hGHSR1a.

Discovery of a potent, highly selective, and orally efficacious small-molecule activator of the insulin receptor.

A series of 3,6-diaryl-2,5-dihydroxybenzoquinones were synthesized and evaluated for their abilities to selectively activate human insulin receptor tyrosine kinase (IRTK). 2, 5-Dihydroxy-6-(1-methylindol-3-yl)-3-phenyl-1,4-benzoquinone (2h) was identified as a potent, highly selective, and orally active small-molecule insulin receptor activator. It activated IRTK with an EC(50) of 300 nM and did not induce the activation of closely related receptors (IGFIR, EGFR, and PDGFR) at concentrations up to 30 000 nM. Oral administration of the compound to hyperglycemic db/db mice (0.1-10 mg/kg/day) elicited substantial to nearly complete correction of hyperglycemia in a dose-dependent manner. In ob/ob mice, the compound (10 mg/kg) caused significant reduction in hyperinsulinemia. A structurally related compound 2c, inactive in IRTK assay, failed to affect blood glucose level in db/db mice at equivalent exposure levels. Results from additional studies with compound 2h, aimed at evaluating classical quinone-related phenomena, provided sufficient grounds for optimism to allow more extensive toxicologic evaluation.

Activation of insulin signal transduction pathway and anti-diabetic activity of small molecule insulin receptor activators.

We recently described the identification of a non-peptidyl fungal metabolite (l-783,281, compound 1), which induced activation of human insulin receptor (IR) tyrosine kinase and mediated insulin-like effects in cells, as well as decreased blood glucose levels in murine models of Type 2 diabetes (Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M. T. , Pelaez, F., Ruby, C., Kendall, R. L., Mao, X., Griffin, P., Calaycay, J., Zierath, J. R., Heck, J. V., Smith, R. G. & Moller, D. E. (1999) Science 284, 974-977). Here we report the characterization of an active analog (compound 2) with enhanced IR kinase activation potency and selectivity over related receptors (insulin-like growth factor I receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor). The IR activators stimulated tyrosine kinase activity of partially purified native IR and recombinant IR tyrosine kinase domain. Administration of the IR activators to mice was associated with increased IR tyrosine kinase activity in liver. In vivo oral treatment with compound 2 resulted in significant glucose lowering in several rodent models of diabetes. In db/db mice, oral administration of compound 2 elicited significant correction of hyperglycemia. In a streptozotocin-induced diabetic mouse model, compound 2 potentiated the glucose-lowering effect of insulin. In normal rats, compound 2 improved oral glucose tolerance with significant reduction in insulin release following glucose challenge. A structurally related inactive analog (compound 3) was not effective on insulin receptor activation or glucose lowering in db/db mice. Thus, small molecule IR activators exert insulin mimetic and sensitizing effects in cells and in animal models of diabetes. These results have implications for the future development of new therapies for diabetes mellitus.

Discovery of a small molecule insulin mimetic with antidiabetic activity in mice.

Insulin elicits a spectrum of biological responses by binding to its cell surface receptor. In a screen for small molecules that activate the human insulin receptor tyrosine kinase, a nonpeptidyl fungal metabolite (L-783,281) was identified that acted as an insulin mimetic in several biochemical and cellular assays. The compound was selective for insulin receptor versus insulin-like growth factor I (IGFI) receptor and other receptor tyrosine kinases. Oral administration of L-783,281 to two mouse models of diabetes resulted in significant lowering in blood glucose levels. These results demonstrate the feasibility of discovering novel insulin receptor activators that may lead to new therapies for diabetes.

Synthesis and activity of 2-(sulfonamido)methyl-carbapenems: discovery of a novel, anti-MRSA 1,8-naphthosultam pharmacophore.

A series of 1beta-methyl carbapenems substituted at the 2-position with lipophilic, acyclic and cyclic (sulfonamido)methyl groups was prepared and evaluated for activity against resistant gram-positive bacteria. From these studies, the 1,8-naphthosultamyl group emerged as a novel, PBP2a-binding, anti-MRSA pharmacophore worthy of further exploration.

Synthesis and properties of 2-(naphthosultamyl)methyl-carbapenems with potent anti-MRSA activity: discovery of L-786,392.

A series of 1beta-methyl-2-(naphthosultamyl)methyl-carbapenems bearing dicationic groups on the naphthosultamyl moiety was prepared and evaluated for activity against resistant gram-positive bacteria. Based on a combination of excellent in vitro antibacterial activity, acceptable mouse acute toxicity, and a desirable fragmentation pattern on beta-lactam ring opening, the analog 2g (L-786,392) was selected for extended evaluation.

Reduced immunotoxicity and preservation of antibacterial activity in a releasable side-chain carbapenem antibiotic.

A carbapenem antibiotic, L-786,392, was designed so that the side chain that provides high-affinity binding to the penicillin-binding proteins responsible for bacterial resistance was also the structural basis for ameliorating immunopathology. Expulsion of the side chain upon opening of the beta-lactam ring retained antibacterial activity while safely expelling the immunodominant epitope. L-786,392 was well tolerated in animal safety studies and had significant in vitro and in vivo activities against methicillin- and vancomycin-resistant Staphylococci and vancomycin-resistant Enterococci.

Preparation and structure-activity relationships of simplified analogues of the antifungal agent cilofungin: a total synthesis approach.

The echinocandins are a well-known class of lipopeptides characterized by their potent antifungal activity against Candida species. The mechanism of action of the echinocandins is generally thought to be the inhibition of beta-1,3-glucan synthesis, an important structural component in the cell wall of Candida species. Extensive structure-activity studies on the fatty acid side chain of echinocandin B (1) led to the preparation of the clinical candidate cilofungin (4). However, little is known about the cyclic peptide. We now report the preparation, by solid-phase synthesis, of a series of simplified analogs of cilofungin in which the unusual amino acids found in the echinocandins were replaced with more readily accessible natural amino acids. The solid-phase approach to the total synthesis of these analogs allowed us to conveniently explore structural modifications that could not be accomplished by chemical modification of the natural product. The simplest analog 5 showed no biological activity. Structural complexity was then returned to the system in a systematic fashion so as to reapproach the original cilofungin structure. Antifungal activity and the inhibition of beta-1,3-glucan synthesis were monitored at each step of the process, thereby revealing the basic structure-activity relationships of the amino acids and the minimal structural requirements for biological activity in the echinocandin ring system. The results suggests that the 3-hydroxy-4-methylproline residue enhances activity but the L-homotyrosine residue is crucial for both antifungal activity and the inhibition of beta-1,3-glucan synthesis.