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Boxazomycins A-C are potent broad-spectrum antibiotics isolated from Actinomycetes strain G495-1 in 1987. We now report that boxazomycin A inhibits bacterial growth by selectively inhibiting protein synthesis, its effect is bacteriostatic, and it is equally active against drug resistant bacterial strains. No cross-resistance to protein synthesis inhibitors was observed suggesting that its inhibition is distinct from clinical protein synthesis inhibitors. We also report in vivo efficacy in a Staphylococcus aureus murine infection model supported by corresponding pharmacokinetic studies., (© 2024. Merck & Co., Inc., Rahway, NJ, USA and its affiliates, under exclusive licence to the Japan Antibiotics Research Association.)

Background and Objectives: The 9-valent human papillomavirus (9vHPV) vaccine Phase III immunogenicity study in 9- to 15-year-old boys and girls was extended to assess immunogenicity and effectiveness through 10 years after the last vaccine dose (NCT00943722)., Methods: Boys (n = 301) and girls (n = 971) who received three 9vHPV vaccine doses in the base study (day 1, months 2 and 6) enrolled in the extension. Serum was collected through month 126 for antibody assessments by competitive Luminex immunoassay and immunoglobulin G-Luminex immunoassay. For effectiveness analysis starting at age 16 years, genital swabs were collected (to assess HPV DNA by polymerase chain reaction) and external genital examinations conducted every 6 months. Primary analyses were conducted in per-protocol populations., Results: Geometric mean antibody titers peaked around month 7, decreased sharply between months 7 and 12, then gradually through month 126. Seropositivity rates remained ≥81% by competitive Luminex immunoassay and ≥95% by immunoglobin G-Luminex immunoassay at month 126 for each 9vHPV vaccine type. After up to 11.0 (median 10.0) years of follow-up postdose 3, there were no cases of HPV6/11/16/18/31/33/45/52/58-related high-grade intraepithelial neoplasia or condyloma in males or females. Incidence rates of HPV6/11/16/18/31/33/45/52/58-related 6-month persistent infection in males and females were low (54.6 and 52.4 per 10000 person-years, respectively) and within ranges expected in vaccinated cohorts, based on previous human papillomavirus vaccine efficacy trials., Conclusions: The 9vHPV vaccine demonstrated sustained immunogenicity and effectiveness through ∼10 years post 3 doses of 9vHPV vaccination of boys and girls aged 9 to 15 years.

Aim: Critical virus reagents in regulated bioanalytical assays require stability monitoring. Although stability at ultra-low frozen temperatures is generally assumed, published data are limited and real-time studies are time consuming. Materials & methods: The authors reviewed literature data, typical mechanisms of molecular degradation, glass transition temperatures of commonly used buffers and available real-time storage data to model frozen virus reagent stability. Results: Storage at ultra-low temperatures below the glass transition temperature was critical for virus stability. Modeling of real-time data suggested that virus potency remained within 0.5 log 10 of its starting potency at a probability of >99, 90 and 73% after 10, 20 and 30 years, respectively. Conclusion: The study supports the practice of virus storage at -70°C or below for 20-30 years.

Emerging evidence demonstrates a connection between microbiome composition and suboptimal response to vaccines (vaccine hyporesponse). Harnessing the interaction between microbes and the immune system could provide novel therapeutic strategies for improving vaccine response. Currently we do not fully understand the mechanisms and dynamics by which the microbiome influences vaccine response. Using both mouse and non-human primate models, we report that short-term oral treatment with a single antibiotic (vancomycin) results in the disruption of the gut microbiome and this correlates with a decrease in systemic levels of antigen-specific IgG upon subsequent parenteral vaccination. We further show that recovery of microbial diversity before vaccination prevents antibiotic-induced vaccine hyporesponse, and that the antigen specific IgG response correlates with the recovery of microbiome diversity. RNA sequencing analysis of small intestine, spleen, whole blood, and secondary lymphoid organs from antibiotic treated mice revealed a dramatic impact on the immune system, and a muted inflammatory signature is correlated with loss of bacteria from Lachnospiraceae , Ruminococcaceae , and Clostridiaceae . These results suggest that microbially modulated immune pathways may be leveraged to promote vaccine response and will inform future vaccine design and development strategies.

The NO-sGC-cGMP signaling pathway plays an important role in the cardiovascular system. Loss of nitric oxide tone or impaired signaling has been associated with cardiovascular diseases, such as hypertension, pulmonary hypertension and heart failure. Direct activation of sGC enzyme independent of NO represents a novel approach for modulating NO signaling with tremendous therapeutic potential. Herein, we describe the design of a structurally novel class of heme-dependent sGC stimulators containing the 3,3-dimethylpyrrolidin-2-one moiety which resulted in the identification of the potent, selective stimulator 30 (MK-2947) for the treatment of hypertension., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: In this study we evaluated the utility of in-vitro screening tools for predicting the in-vivo behavior of six cyclic peptides with different solubility and permeability properties (BCS class II and III), intended for oral delivery in presence of permeation enhancer Labrasol., Methods: An in vitro flux assay was used to assess peptide permeation across a biomimetic, lipid-based membrane and in vivo studies in rats were used to determine oral peptide bioavailability in the presence of Labrasol., Results: The in vitro flux was significantly increased for BCS class III peptides, while it significantly decreased or remained unchanged for BCS class II peptides with increasing Labrasol concentrations. The different flux responses were attributed to the combination of reduced effective free peptide concentration and increased membrane permeability in the presence of Labrasol. In vivo studies in male Wistar-Hans rats indicated improved oral bioavailability at different extents for all peptides in presence of Labrasol. On comparing the in vitro and in vivo data, a potential direct correlation for BCS class III peptides was seen but not for BCS class II peptides, due to lower free concentrations of peptides in this class., Conclusion: This study assessed the utility of in vitro screening tools for selecting peptides and permeation excipients early in drug product development. Graphical Abstract Graphical Abstract and Figure 1 contains small text.Graphical Abstract text is made larger. The Figure 1 text cannot be made larger.

A series of structurally diverse azaspirodecanone and spirooxazolidinone analogues were designed and synthesized as potent and selective somatostatin receptor subtype 5 (SSTR5) antagonists. Four optimized compounds each representing a subseries showed improvement in their metabolic stability and pharmacokinetic profiles compared to those of the original lead compound 1 while maintaining pharmacodynamic efficacy. The optimized cyclopropyl analogue 13 demonstrated efficacy in a mouse oral glucose tolerance test and an improved metabolic profile and pharmacokinetic properties in rhesus monkey studies. In this Communication, we discuss the relationship among structure, in vitro and in vivo activity, metabolic stability, and ultimately the potential of these compounds as therapeutic agents for the treatment of type 2 diabetes. Furthermore, we show how the use of focused libraries significantly expanded the structural class and provided new directions for structure-activity relationship optimization., Competing Interests: The authors declare no competing financial interest.

We report new SSTR5 antagonists with enhanced potency, subtype selectivity, and minimal off-target activities as compared to previously reported compounds. Starting from the reported SSTR5 antagonist 1 , we systematically surveyed changes in the central core and head piece while maintaining the diphenyl tail group constant. From this study the azaspirodecanone 10 emerged as a new highly potent and selective SSTR5 antagonist. Compound 10 lowered glucose excursion by 94% in an oral glucose tolerance test (OGTT) in mice following a 3 mg/kg oral dose. The compound increased both total and active circulating incretin hormone GLP-1 levels in mice at a dose of 10 mg/kg. A synergistic effect was also demonstrated when compound 10 was coadministered with a DPP-4 inhibitor, substantially increasing circulating active GLP-1[7-36] amide and insulin in response to a glucose challenge., Competing Interests: The authors declare no competing financial interest.

We report the discovery and hit-to-lead optimization of a structurally novel indazole series of CYP11B2 inhibitors. Benchmark compound 34 from this series displays potent inhibition of CYP11B2, high selectivity versus related steroidal and hepatic CYP targets, and lead-like physical and pharmacokinetic properties. On the basis of these and other data, the indazole series was progressed to lead optimization for further refinement., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Although the physiologic role of muscarinic receptors in bladder function and the therapeutic efficacy of muscarinic antagonists for the treatment of overactive bladder are well established, the role of β 3 -adrenergic receptors (β 3 ARs) and their potential as therapeutics is just emerging. In this manuscript, we characterized the pharmacology of a novel β 3 AR agonist vibegron (MK-4618, KRP-114V) and explored mechanistic interactions of β 3 AR agonism and muscarinic antagonism in urinary bladder function. Vibegron is a potent, selective full β 3 AR agonist across species, and it dose dependently increased bladder capacity, decreased micturition pressure, and increased bladder compliance in rhesus monkeys. The relaxation effect of vibegron was enhanced when combined with muscarinic antagonists, but differentially influenced by muscarinic receptor subtype selectivity. The effect was greater when vibegron was co-administered with tolterodine, a nonselective antagonist, compared with coadministration with darifenacin, a selective M3 antagonist. Furthermore, a synergistic effect for bladder strip relaxation was observed with the combination of a β 3 AR agonist and tolterodine in contrast to simple additivity with darifenacin. To determine expression in rhesus bladder, we employed a novel β 3 AR agonist probe, [ 3 H]MRL-037, that selectively labels β 3 receptors in both urothelium and detrusor smooth muscle. Vibegron administration caused a dose-dependent increase in circulating glycerol and fatty acid levels in rhesus and rat in vivo, suggesting these circulating lipids can be surrogate biomarkers. The translation of our observation to the clinic has yet to be determined, but the combination of β 3 AR agonists with M2/M3 antimuscarinics has the potential to redefine the standard of care for the pharmacological treatment of overactive bladder., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Herein we report the discovery and hit-to-lead optimization of a series of spirocyclic piperidine aldosterone synthase (CYP11B2) inhibitors. Compounds from this series display potent CYP11B2 inhibition, good selectivity versus related CYP enzymes, and lead-like physical and pharmacokinetic properties.

GPR142 has been identified as a potential glucose-stimulated insulin secretion (GSIS) target for the treatment of type 2 diabetes mellitus (T2DM). A class of triazole GPR142 agonists was discovered through a high throughput screen. The lead compound 4 suffered from poor metabolic stability and poor solubility. Lead optimization strategies to improve potency, efficacy, metabolic stability, and solubility are described. This optimization led to compound 20e , which showed significant reduction of glucose excursion in wild-type but not in GPR142 deficient mice in an oral glucose tolerance test (oGTT) study. These studies provide strong evidence that reduction of glucose excursion through treatment with 20e is GPR142-mediated, and GPR142 agonists could be used as a potential treatment for type 2 diabetes.

Aim: A traditional oral fatty acid challenge assesses absorption of triacylglycerol (TG) into the periphery through the intestines, but cannot distinguish the composition or source of fatty acid in the TG. Stable isotope-labeled tracers combined with LC-MRM can be used to identify and distinguish TG synthesized with dietary and stored fatty acids., Results: Concentrations of three abundant TGs (52:2, 54:3 and 54:4) were monitored for incorporation of one or two (2)H11-oleate molecules per TG. This method was subjected to routine assay validation and meets typical requirements for an assay to be used to support clinical studies., Conclusion: Calculations for the fractional appearance rate of TG in plasma are presented along with the intracellular enterocyte precursor pool for 12 study participants.

DGAT2 plays a critical role in hepatic triglyceride production, and data suggests that inhibition of DGAT2 could prove to be beneficial in treating a number of disease states. This article documents the discovery and optimization of a selective small molecule inhibitor of DGAT2 as well as pharmacological proof of biology in a mouse model of triglyceride production.

A novel, potent series of glucagon receptor antagonists (GRAs) was discovered. These indazole- and indole-based compounds were designed on an earlier pyrazole-based GRA lead MK-0893. Structure-activity relationship (SAR) studies were focused on the C3 and C6 positions of the indazole core, as well as the benzylic position on the N-1 of indazole. Multiple potent GRAs were identified with excellent in vitro profiles and good pharmacokinetics in rat. Among them, GRA 16d was found to be orally active in blunting glucagon induced glucose excursion in an acute glucagon challenge model in glucagon receptor humanized (hGCGR) mice at 1, 3 and 10mg/kg (mpk), and significantly lowered acute glucose levels in hGCGR ob/ob mice at 3 mpk dose., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Hit-to-lead efforts resulted in the discovery of compound 19, a potent CYP11B2 inhibitor that displays high selectivity vs related CYPs, good pharmacokinetic properties in rat and rhesus, and lead-like physical properties. In a rhesus pharmacodynamic model, compound 19 displays robust, dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Inhibition of hepatic transporters such as organic anion transporting polypeptides (OATPs) 1B can cause drug-drug interactions (DDIs). Determining the impact of perpetrator drugs on the plasma exposure of endogenous substrates for OATP1B could be valuable to assess the risk for DDIs early in drug development. As OATP1B orthologs are well conserved between human and monkey, we assessed in cynomolgus monkeys the endogenous OATP1B substrates that are potentially suitable to assess DDI risk in humans. The effect of rifampin (RIF), a potent inhibitor for OATP1B, on plasma exposure of endogenous substrates of hepatic transporters was measured. From the 18 biomarkers tested, RIF (18 mg/kg, oral) caused significant elevation of plasma unconjugated and conjugated bilirubin, which may be attributed to inhibition of cOATP1B1 and cOATP1B3 based on in vitro to in vivo extrapolation analysis. To further evaluate whether cynomolgus monkeys are a suitable translational model to study OATP1B-mediated DDIs, we determined the inhibitory effect of RIF on in vitro transport and pharmacokinetics of rosuvastatin (RSV) and atorvastatin (ATV). RIF strongly inhibited the uptake of RSV and ATV by cOATP1B1 and cOATP1B3 in vitro. In agreement with clinical observations, RIF (18 mg/kg, oral) significantly decreased plasma clearance and increased the area under the plasma concentration curve (AUC) of intravenously administered RSV by 2.8- and 2.7-fold, and increased the AUC and maximum plasma concentration of orally administered RSV by 6- and 10.3-fold, respectively. In contrast to clinical findings, RIF did not significantly increase plasma exposure of either intravenous or orally administered ATV, indicating species differences in the rate-limiting elimination pathways., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

We report the discovery of a benzimidazole series of CYP11B2 inhibitors. Hit-to-lead and lead optimization studies identified compounds such as 32, which displays potent CYP11B2 inhibition, high selectivity versus related CYP targets, and good pharmacokinetic properties in rat and rhesus. In a rhesus pharmacodynamic model, 32 produces dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Modification of the previously disclosed (S)-N-(2-(aminomethyl)-5-chlorobenzyl)-1-((R)-2-hydroxy-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamide 2 by optimization of the P3 group afforded novel, low molecular weight thrombin inhibitors. Heterocycle replacement of the hydroxyl functional group helped maintain thrombin in vitro potency while improving the chemical stability and pharmacokinetic profile. These modifications led to the identification of compound 10, which showed excellent selectivity over related serine proteases as well as in vivo efficacy in the rat arteriovenous shunt. Compound 10 exhibited significantly improved chemical stability and pharmacokinetic properties over 2 and may be utilized as a structurally differentiated preclinical tool comparator to dabigatran etexilate (Pro-1) to interrogate the on- and off-target effects of oral direct thrombin inhibitors.

In our effort to discover DPP-4 inhibitors with added benefits over currently commercially available DPP-4 inhibitors, MK-3102 (omarigliptin), was identified as a potent and selective dipeptidyl peptidase 4 (DPP-4) inhibitor with an excellent pharmacokinetic profile amenable for once-weekly human dosing and selected as a clinical development candidate. This manuscript summarizes the mechanism of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and human efficacy data for omarigliptin, which is currently in phase 3 clinical development.

We report herein the identification of MK-4409, a potent and selective fatty acid amide hydrolase (FAAH) inhibitor. Starting from a high throughput screening (HTS) hit, medicinal chemistry efforts focused on optimizing of FAAH inhibition in vitro potency, improving the pharmacokinetic (PK) profile, and increasing in vivo efficacy in rodent inflammatory and neuropathic pain assays.

A series of novel tri-2,3,5-substituted tetrahydropyran analogs were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-4) for the treatment of type 2 diabetes. Optimization of the series provided inhibitors with good DPP-4 potency and selectivity over other peptidases (QPP, DPP8, and FAP). Compound 23, which is very potent, selective, efficacious in the diabetes PD model, and has an excellent pharmacokinetic profile, is selected as a clinical candidate., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [(11)C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain.

In the course of the development of an aminobenzimidazole class of human glucagon receptor (hGCGR) antagonists, a novel class of cyclic guanidine hGCGR antagonists was discovered. Rapid N-dealkylation resulted in poor pharmacokinetic profiles for the benchmark compound in this series. A strategy aimed at blocking oxidative dealkylation led to a series of compounds with improved rodent pharmacokinetic profiles. One compound was orally efficacious in a murine glucagon challenge pharmacodynamic model and also significantly lowered glucose levels in a murine diabetes model., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A novel class of N-aryl-2-acylindole human glucagon receptor (hGCGR) antagonists is reported. These compounds demonstrate good pharmacokinetic profiles in multiple preclinical species. One compound from this series, indole 33, is orally active in a transgenic murine pharmacodynamic model. Furthermore, a 1mg/kg oral dose of indole 33 lowers ambient glucose levels in an ob/ob/hGCGR transgenic murine diabetes model. This compound was deemed suitable for preclinical safety studies and was found to be well tolerated in an 8-day experimental rodent tolerability study. The combination of preclinical efficacy and safety observed with compound 33 highlights the potential of this class as a treatment for type 2 diabetes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The design, synthesis, and structure-activity relationship (SAR) for a series of β-substituted 3-(4-aryloxyaryl)propanoic acid GPR40 agonists is described. Systematic replacement of the pendant aryloxy group led to identification of potent GPR40 agonists. In order to identify candidates suitable for in vivo validation of the target, serum shifted potency and pharmacokinetic properties were determined for several compounds. Finally, further profiling of compound 7 is presented, including demonstration of enhanced glucose tolerance in an in vivo mouse model., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Platensimycin (PTM) is a recently discovered broad-spectrum antibiotic produced by Streptomyces platensis. It acts by selectively inhibiting the elongation-condensing enzyme FabF of the fatty acid biosynthesis pathway in bacteria. We report here that PTM is also a potent and highly selective inhibitor of mammalian fatty acid synthase. In contrast to two agents, C75 and cerulenin, that are widely used as inhibitors of mammalian fatty acid synthase, platensimycin specifically inhibits fatty acid synthesis but not sterol synthesis in rat primary hepatocytes. PTM preferentially concentrates in liver when administered orally to mice and potently inhibits hepatic de novo lipogenesis, reduces fatty acid oxidation, and increases glucose oxidation. Chronic administration of platensimycin led to a net reduction in liver triglyceride levels and improved insulin sensitivity in db/+ mice fed a high-fructose diet. PTM also reduced ambient glucose levels in db/db mice. These results provide pharmacological proof of concept of inhibiting fatty acid synthase for the treatment of diabetes and related metabolic disorders in animal models.

A series of amide derived beta(3)-adrenergic receptor (AR) agonists is described. The discovery and optimization of several series of compounds derived from 1, is used to lay the SAR foundation for second generation beta(3)-AR agonists for the treatment of overactive bladder., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Systematic structure-activity relationship (SAR) studies of a screening lead led to the discovery of a series of thiazolidinediones (TZDs) as potent GPR40 agonists. Among them, compound C demonstrated an acute mechanism-based glucose-lowering in an intraperitoneal glucose tolerance test (IPGTT) in lean mice, while no effects were observed in GPR40 knock-out mice., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Objective: Acute activation of G protein-coupled receptor 40 (GPR40) by free fatty acids (FFAs) or synthetic GPR40 agonists enhances insulin secretion. However, it is still a matter of debate whether activation of GPR40 would be beneficial for the treatment of type 2 diabetes, since chronic exposure to FFAs impairs islet function. We sought to evaluate the specific role of GPR40 in islets and its potential as a therapeutic target using compounds that specifically activate GPR40., Research Design and Methods: We developed a series of GPR40-selective small-molecule agonists and studied their acute and chronic effects on glucose-dependent insulin secretion (GDIS) in isolated islets, as well as effects on blood glucose levels during intraperitoneal glucose tolerance tests in wild-type and GPR40 knockout mice (GPR40(-/-))., Results: Small-molecule GPR40 agonists significantly enhanced GDIS in isolated islets and improved glucose tolerance in wild-type mice but not in GPR40(-/-) mice. While a 72-h exposure to FFAs in tissue culture significantly impaired GDIS in islets from both wild-type and GPR40(-/-) mice, similar exposure to the GPR40 agonist did not impair GDIS in islets from wild-type mice. Furthermore, the GPR40 agonist enhanced insulin secretion in perfused pancreata from neonatal streptozotocin-induced diabetic rats and improved glucose levels in mice with high-fat diet-induced obesity acutely and chronically., Conclusions: GPR40 does not mediate the chronic toxic effects of FFAs on islet function. Pharmacological activation of GPR40 may potentiate GDIS in humans and be beneficial for overall glucose control in patients with type 2 diabetes.

Background: Insulin induces vascular smooth muscle cell (VSMC) proliferation, which is an important step in the atherosclerotic process. Recently, a nonpeptidyl fungal metabolite originally referred to as L-783,281, but also known as demethylasterriquinone B-1 (DMAQB-1), was found to have hypoglycemic activity in diabetic mice through interaction with the intracellular beta subunit of the insulin receptor. This study was designed to determine whether DMAQB-1 has an insulin-like proliferative effect on human infragenicular VSMCs., Methods: Human infragenicular VSMCs were isolated from diabetic patients undergoing amputations. DMAQB-1 cell culture dose response was measured in both serum-free media and media with 1% fetal bovine serum (FBS). A working concentration of DMAQB-1 that ranged from 0.5 to 500 nmol/L was studied in the presence of varying concentrations of glucose and insulin. The ability of DMAQB-1 to stimulate glucose transport at less than or equal to 100 nmol/L was determined by [(14)C]-2-deoxyglucose uptake. DNA synthesis was used as the marker for proliferative stimulus and detected by [(3)H]-thymidine uptake measured at 24 hours. Analysis of variance was used to compare the results among the groups; a P value less than.05 was considered significant. Polynomial regression was used to calculate the median lethal dose., Results: In normal glucose media (100 mg/dL), various concentrations of DMAQB-1 demonstrated a small but statistically significant decrease in DNA synthesis at 0.5 nmol/L in serum-free media and at 5 nmol/L in media supplemented with 1% FBS. The corresponding median lethal dose was 107 nmol/L in serum-free media and 650 nmol/L in media supplemented with 1% FBS. A DMAQB-1 concentration of 5 nmol/L induced glucose transport that was equivalent to an insulin concentration of 100 microU/mL. In serum-free, high glucose media (200 mg/dL), DMAQB-1 concentrations up to 500 nmol/L did not cause a statistically significant change in DNA synthesis. When serum-free, high glucose media was combined with mild (100 microU/mL) or moderate (250 microU/mL) concentrations of insulin, DMAQB-1 caused no statistically significant increase in DNA synthesis., Conclusion: Nontoxic doses of DMAQB-1 can induce glucose transport equivalent to insulin in the physiologic range. However, DMAQB-1 does not have an insulin-like proliferative effect on human VSMCs in normal-glucose, high-glucose, or high-insulin environments.

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.

The synthesis and SAR of analogues prepared from novel insulin receptor activator 1 are described. Changes to the dihydroxyquinone core were not tolerated while functionalization of the two indoles contained in 1 resulted in little effect upon activation of the insulin receptor.

Hypothemycin, a resorcylic acid lactone antibiotic, was identified as active in a screen for inhibitors of T cell activation. It was found to inhibit the proliferation of mouse and human T cells stimulated with anti-CD3 mAb + PMA and of human PBMC stimulated with anti-CD3 mAb alone. This inhibition was partially reversed by exogenous IL-2 indicating that it is not due to non-specific toxicity. Hypothemycin potently suppressed the production of IL-2 (IC50: 9 nM) but affected IL-2-induced proliferation to a lesser extent (IC50: 194 nM). Hypothemycin also inhibited IL-6, IL-10, IFN-gamma and TNF-alpha production. By contrast, it markedly enhanced the production of IL-4, IL-5 and IL-13. These effects were seen both at the mRNA and protein secretion levels. Analysis of the effect of hypothemycin on CD69 induction suggested that it disrupts calcineurin-independent rather than calcineurin-dependent signaling. Furthermore, hypothemycin was able to inhibit the phosphorylation of ERK1/2 induced by PMA treatment of T cells. Therefore, hypothemycin represents an inhibitor of T cell activation with a novel mode of action and unique modulatory activity on cytokine production.

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.

Quinoxapeptin A and B are novel chromodepsipeptides which were isolated from a nocardioform actinomycete with indeterminant morphology. Quinoxapeptins A and B are potent inhibitors of HIV-1 and HIV-2 reverse transcriptase and almost equally active against two single mutants forms as well as a double mutant form of HIV-1 reverse transcriptase. Quinoxapeptin A and B are specific inhibitors of HIV-1 and HIV-2 reverse transcriptase because they did not inhibit human DNA polymerase alpha, beta, gamma and delta. Quinoxapeptin A and B are structurally similar to luzopeptin A which was also active against HIV-1 and HIV-2 reverse transcriptase.

Measurement of 4-methylumbelliferyl p-guanidinobenzoate (MUGB)-hydrolyzing activity in the plasma of normal controls, cystic fibrosis (CF) heterozygotes, and CF homozygotes did not support previously reported (35) differences in MUGB-hydrolyzing activity. We identified human plasma albumin as the major source of MUGB-hydrolyzing activity by comparison of our plasma results to those obtained with physiologic concentrations of commercial albumin samples. Substantiating evidence was obtained from gel filtration experiments and correlation of albumin levels in CF plasma with MUGB titers. We found essentially no proteolytic activity towards dinitrophenylprotamine sulfate associated with commercial albumin samples. It appears that the reaction between human albumin and MUGB represents a weak esterase activity, perhaps involving the acylation of a specific site(s) on the protein. Hypoalbuminemia has been documented (8) in some CF patients. Low albumin concentrations, indicated by MUGB titers less than 190 nmole methylumbelliferone/ml plasma, were found in 42% of CF homozygotes, 6% of heterozygotes, and 4% of controls. Gel filtration studies of a normal amniotic fluid supernatant indicated that albumin was the major MUGB-hydrolyzing substance in this fluid. We conclude that MUGB abnormalities are not associated with the basic gene defect in CF and cannot be used as the basis of a test for intrauterine or heterozygote detection.