Identification of potent inhibitors of the sortilin-progranulin interaction.

High-throughput screening methods have been used to identify two novel series of inhibitors that disrupt progranulin binding to sortilin. Exploration of structure-activity relationships (SAR) resulted in compounds with sufficient potency and physicochemical properties to enable co-crystallization with sortilin. These co-crystal structures supported observed SAR trends and provided guidance for additional avenues for designing compounds with additional interactions within the binding site.

Structural characterization of nonactive site, TrkA-selective kinase inhibitors.

Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research of novel targets against pain. Although originally identified as an oncogene, Tropomyosin-related kinase A (TrkA) is linked to pain and elevated levels of NGF (the ligand for TrkA) are associated with chronic pain. Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief. Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases. The X-ray structures reveal a binding site outside the kinase active site that uses residues from the kinase domain and the juxtamembrane region. Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes. The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes. The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition. This finding highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information because of substantial differences in the interacting residues. These structures illustrate the flexibility of binding to sequences outside of-but adjacent to-the kinase domain of TrkA. This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins.

Discovery of highly potent and selective orexin 1 receptor antagonists (1-SORAs) suitable for in vivo interrogation of orexin 1 receptor pharmacology.

While a correlation between blockade of the orexin 2 receptor (OX2R) with either a dual orexin receptor antagonist (DORA) or a selective orexin 2 receptor antagonist (2-SORA) and a decrease of wakefulness is well established, less is known about selective blockade of the orexin 1 receptor (OX1R). Therefore, a highly selective orexin 1 antagonist (1-SORA) with suitable properties to allow in vivo interrogation of OX1R specific pharmacology in preclinical species remains an attractive target. Herein, we describe the discovery of an optimized 1-SORA series in the piperidine ether class. Notably, a 4,4-difluoropiperidine core coupled with a 2-quinoline ether linkage provides OX1R selective compounds. The combination with an azabenzimidazole or imidazopyridine amide substituent leads to analogs 47 and 51 with >625-fold functional selectivity for OX1R over OX2R in rat. Compounds 47 and 51 possess clean off-target profiles and the required pharmacokinetic and physical properties to be useful as 1-SORA tool compounds.

Site specific modification of the human plasma proteome by methylglyoxal.

Increasing evidence identifies dicarbonyl stress from reactive glucose metabolites, such as methylglyoxal (MG), as a major pathogenic link between hyperglycemia and complications of diabetes. MG covalently modifies arginine residues, yet the site specificity of this modification has not been thoroughly investigated. Sites of MG adduction in the plasma proteome were identified using LC-MS/MS analysis in vitro following incubation of plasma proteins with MG. Treatment of plasma proteins with MG yielded 14 putative MG hotspots from five plasma proteins (albumin [nine hotspots], serotransferrin, haptoglobin [2 hotspots], hemopexin, and Ig lambda-2 chain C regions). The search results revealed two versions of MG-arginine modification, dihydroxyimidazolidine (R+72) and hydroimidazolone (R+54) adducts. One of the sites identified was R257 in human serum albumin, which is a critical residue located in drug binding site I. This site was validated as a target for MG modification by a fluorescent probe displacement assay, which revealed significant drug dissociation at 300 µM MG from a prodan-HSA complex (75 µM). Moreover, twelve human plasma samples (six male, six female, with two type 2 diabetic subjects from both genders) were analyzed using multiple reaction monitoring (MRM) tandem mass spectrometry and revealed the presence of the MG-modified albumin R257 peptide. These data provide insights into the nature of the site-specificity of MG modification of arginine, which may be useful for therapeutic treatments that aim to prevent MG-mediated adverse responses in patients.

Discovery of piperidine ethers as selective orexin receptor antagonists (SORAs) inspired by filorexant.

Highly selective orexin receptor antagonists (SORAs) of the orexin 2 receptor (OX2R) have become attractive targets both as potential therapeutics for insomnia as well as biological tools to help further elucidate the underlying pharmacology of the orexin signaling pathway. Herein, we describe the discovery of a novel piperidine ether 2-SORA class identified by systematic lead optimization beginning with filorexant, a dual orexin receptor antagonist (DORA) that recently completed Phase 2 clinical trials. Changes to the ether linkage and pendant heterocycle of filorexant were found to impart significant selectivity for OX2R, culminating in lead compound PE-6. PE-6 displays sub-nanomolar binding affinity and functional potency on OX2R while maintaining >1600-fold binding selectivity and >200-fold functional selectivity versus the orexin 1 receptor (OX1R). PE-6 bears a clean off-target profile, a good overall preclinical pharmacokinetic (PK) profile, and reduces wakefulness with increased NREM and REM sleep when evaluated in vivo in a rat sleep study. Importantly, subtle structural changes to the piperidine ether class impart dramatic changes in receptor selectivity. To this end, our laboratories have identified multiple piperidine ether 2-SORAs, 1-SORAs, and DORAs, providing access to a number of important biological tool compounds from a single structural class.

Novel oxazolidinone calcitonin gene-related peptide (CGRP) receptor antagonists for the acute treatment of migraine.

In our efforts to develop CGRP receptor antagonists as backups to MK-3207, 2, we employed a scaffold hopping approach to identify a series of novel oxazolidinone-based compounds. The development of a structurally diverse, potent (20, cAMP+HS IC50=0.67 nM), and selective compound (hERG IC50=19 µM) with favorable rodent pharmacokinetics (F=100%, t1/2=7h) is described. Key to this development was identification of a 3-substituted spirotetrahydropyran ring that afforded a substantial gain in potency (10 to 35-fold).

The EPIC Kids Study: a randomized family-focused YMCA-based intervention to prevent type 2 diabetes in at-risk youth.

BACKGROUND: It is well established that behavioral lifestyle interventions resulting in modest weight reduction in adults can prevent or delay type 2 diabetes mellitus; however in children, successful weight management interventions are rarely found outside of controlled clinical settings. The lack of effective community-based programs is a barrier to reducing obesity prevalence and diabetes risk in children. The objective of our study is to develop and test a group-randomized family-centered community-based type 2 diabetes prevention intervention targeting at-risk children, 9- to 12-years-old. METHODS/DESIGN: Using participatory methods, the adult-focused YMCA Diabetes Prevention Program was adapted for families, creating a novel lifestyle behavior change program focused on healthy eating, physical activity, and a supportive home environment. The program will be tested in sixty 9- to 12-year-old children at risk of diabetes and sixty parents over 12 consecutive weeks with two intervention formats randomized by location: a face-to-face instructor-led program, or a hybrid program with alternating face-to-face and mobile technology-delivered content. Anthropometric, behavioral, psychosocial and physiological outcomes will be assessed at baseline, post-intervention (12 weeks), and follow-up (24 weeks). Secondary outcomes are participant acceptability, feasibility, and adherence. The RE-AIM framework (reach, efficacy, adoption, implementation, and maintenance) will guide intervention implementation and evaluation. Changes at 12 weeks will be assessed using a paired t-test combining both delivery formats. Exploratory models using linear regression analysis will estimate the magnitude of the difference between the face-to-face and hybrid format. The sample size of 60 children, informed by a previous YMCA intervention in which -4.3 % change in overweight (SE = 1.1) was observed over 6 months, will give us 80 % power to detect an effect size of this magnitude, assuming a one-sided test at alpha = 0.05. DISCUSSION: The proposed study capitalizes on a partnership with the YMCA, a popular and widespread community organization, and uses mobile technologies to extend program reach while potentially reducing burden associated with weekly attendance. The long-term goal is to create a scalable, replicable, and sustainable pediatric "diabesity" prevention program that overcomes existing barriers to the translation of efficacious interventions into effective community programs. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02421198 on April 15, 2015.

(E)-Alkenes as replacements of amide bonds: development of novel and potent acyclic CGRP receptor antagonists.

A new class of CGRP receptor antagonists was identified by replacing the central amide of a previously identified anilide lead structure with ethylene, ethane, or ethyne linkers. (E)-Alkenes as well as alkynes were found to preserve the proper bioactive conformation of the amides, necessary for efficient receptor binding. Further exploration resulted in several potent compounds against CGRP-R with low susceptibility to P-gp mediated efflux.

MK-8825: a potent and selective CGRP receptor antagonist with good oral activity in rats.

Rational modification of the clinically tested CGRP receptor antagonist MK-3207 (3) afforded an analogue with increased unbound fraction in rat plasma and enhanced aqueous solubility, 2-[(8R)-8-(3,5-difluorophenyl)-8-methyl-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(6S)-2'-oxo-1',2',5,7-tetrahydrospiro[cyclopenta[b]pyridine-6,3'-pyrrolo[2,3-b]pyridin]-3-yl]acetamide (MK-8825) (6). Compound 6 maintained similar affinity to 3 at the human and rat CGRP receptors but possessed significantly improved in vivo potency in a rat pharmacodynamic model. The overall profile of 6 indicates it should find utility as a rat tool to investigate effects of CGRP receptor blockade in vivo.

Identification of potent, highly constrained CGRP receptor antagonists.

A novel series of potent CGRP receptor antagonists containing a central quinoline ring constraint was identified. The combination of the quinoline constraint with a tricyclic benzimidazolinone left hand fragment produced an analog with picomolar potency (14, CGRP K(i)=23 pM). Further optimization of the tricycle produced a CGRP receptor antagonist that exhibited subnanomolar potency (19, CGRP K(i)=0.52 nM) and displayed a good pharmacokinetic profile in three preclinical species.

The discovery of highly potent CGRP receptor antagonists.

Rational modification of a previously identified spirohydantoin lead structure has identified a series of potent spiroazaoxindole CGRP receptor antagonists. The azaoxindole was found to be a general replacement for the hydantoin that consistently improved in vitro potency. The combination of the indanylspiroazaoxindole and optimized benzimidazolinones led to highly potent antagonists (e.g., 25, CGRP K(i)=40pM). The closely related compound 27 demonstrated good oral bioavailability in dog and rhesus.

Novel CGRP receptor antagonists through a design strategy of target simplification with addition of molecular flexibility.

A novel class of CGRP receptor antagonists was rationally designed by modifying a highly potent, but structurally complex, CGRP receptor antagonist. Initial modifications focused on simplified structures, with increased flexibility. Subsequent to the preparation of a less-potent but more flexible lead, classic medicinal chemistry methods were applied to restore high affinity (compound 22, CGRP Ki=0.035 nM) while maintaining structural diversity relative to the lead. Good selectivity against the closely related adrenomedullin-2 receptor was also achieved.

Feasibility of a family-focused YMCA-based diabetes prevention program in youth: The E.P.I.C. Kids (Encourage, Practice, and Inspire Change) Study.

Efficacious lifestyle modification programs for children at risk of type 2 diabetes (T2D) have not been well established outside of clinical settings. In this study, the feasibility of a family-focused, YMCA-based prevention program for children at risk of T2D was evaluated between September 2015 and July 2016 in Tucson, Arizona. A 12-week YMCA-led lifestyle intervention was adapted for 9-12-year-old children and their families to encourage healthy eating, physical activity, and supportive home environments. Two YMCA locations were randomized to offer either a face-to-face lifestyle coach-led intervention or an alternating face-to-face and digitally-delivered intervention. Program feasibility and preliminary effects on child anthropometric and behavioral outcomes were assessed at baseline and post-intervention. Changes were assessed using linear regression combining delivery formats, with adjustment for clustering of participants within site/format. Forty-eight children (10.9 ±â€¯1.2 years old; 45% female; 40% Hispanic; 43% White; 87% obese) and their parents enrolled, and 36 (75%) completed 12-week measures. Weekly program attendance averaged 61%. Participants and coaches highly rated program content and engagement strategies. Statistically significant changes in child BMI-z score (-0.05, p = 0.03) and family food and physical activity environment (+5.5% family nutrition and physical activity score, p = 0.01) were observed. A YMCA-led family-focused T2D intervention was feasible for the YMCA and participants and effects on child weight, behavior, and the home environment warranted further investigation.

Rosuvastatin, a 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitor, decreases cardiac oxidative stress and remodeling in Ren2 transgenic rats.

Angiotensin-II (Ang-II)-stimulated increases in nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity and oxidative stress are known to play a key role in cardiac remodeling. Inhibition of isoprenylation and activation of small G proteins, such as Rac1, a component of NADPH oxidase, may mediate the antioxidant actions of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins). In this study, we investigated the effects of rosuvastatin on cardiac oxidative stress and remodeling in transgenic rats (Ren2) overexpressing the mouse renin gene with elevated cardiac levels of Ang-II. We treated 6- to 7-wk-old Ren2 rats and age-matched Sprague-Dawley (SD) rats with rosuvastatin (10 mg/kg.d) or vehicle for 3 wk. At the end of the treatment period, left ventricular mass, wall thickness, ejection fraction (by echocardiography), and cardiac remodeling (by light microscopy and immunohistochemistry) were assessed. In addition, myocardial content of nitrotyrosine, malondialdehyde, NADPH-oxidase subunits (gp91(phox), p40(phox), and p22(phox)), and Rac1 were analyzed by immunochemistry. Systolic blood pressure was significantly higher in Ren2 rats, compared with SD rats (P < 0.05); rosuvastatin had no significant effect on systolic blood pressure in either group. In Ren2, but not SD rats, rosuvastatin significantly improved the ventricular ejection fraction, cardiac hypertrophy, and perivascular fibrosis (P < 0.05). In addition, rosuvastatin administration significantly decreased the accentuated myocardial gp91(phox), p40(phox), p22(phox), and Rac1 expression. These changes were accompanied by a parallel reduction in myocardial lipid peroxidation (nitrotyrosine and malondialdehyde content) (P < 0.05). These results suggest that in vivo statin treatment through its direct actions on the heart reduces oxidative stress and remodeling including ventricular mass regression in the Ang-II-dependent Ren2 model.

Mineralocorticoid receptor blockade attenuates chronic overexpression of the renin-angiotensin-aldosterone system stimulation of reduced nicotinamide adenine dinucleotide phosphate oxidase and cardiac remodeling.

The renin-angiotensin-aldosterone system contributes to cardiac remodeling, hypertrophy, and left ventricular dysfunction. Angiotensin II and aldosterone (corticosterone in rodents) together generate reactive oxygen species (ROS) via reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which likely facilitate this hypertrophy and remodeling. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo mineralocorticoid receptor (MR) blockade in a rodent model of the chronically elevated tissue renin-angiotensin-aldosterone system, the transgenic TG (mRen2) 27 rat (Ren2). The Ren2 overexpresses the mouse renin transgene with resultant hypertension, insulin resistance, proteinuria, and cardiovascular damage. Young (6- to 7-wk-old) male Ren2 and age-matched Sprague-Dawley rats were treated with spironolactone or placebo for 3 wk. Heart tissue ROS, immunohistochemical analysis of 3-nitrotyrosine, and NADPH oxidase (NOX) subunits (gp91(phox) recently renamed NOX2, p22(phox), Rac1, NOX1, and NOX4) were measured. Structural changes were assessed with cine-magnetic resonance imaging, transmission electron microscopy, and light microscopy. Significant increases in Ren2 septal wall thickness (cine-magnetic resonance imaging) were accompanied by perivascular fibrosis, increased mitochondria, and other ultrastructural changes visible by light microscopy and transmission electron microscopy. Although there was no significant reduction in systolic blood pressure, significant improvements were seen with MR blockade on ROS formation and NOX subunits (each P < 0.05). Collectively, these data suggest that MR blockade, independent of systolic blood pressure reduction, improves cardiac oxidative stress-induced structural and functional changes, which are driven, in part, by angiotensin type 1 receptor-mediated increases in NOX.

Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia.

We investigated whether previously reported muscle mitochondrial dysfunction and altered gene transcript levels in type 2 diabetes might be secondary to abnormal blood glucose and insulin levels rather than an intrinsic defect of type 2 diabetes. A total of 13 type 2 diabetic and 17 nondiabetic subjects were studied on two separate occasions while maintaining similar insulin and glucose levels in both groups by 7-h infusions of somatostatin, low- or high-dose insulin (0.25 and 1.5 mU/kg of fat-free mass per min, respectively), and glucose. Muscle mitochondrial DNA abundance was not different between type 2 diabetic and nondiabetic subjects at both insulin levels, but the majority of transcripts in muscle that are involved mitochondrial functions were expressed at lower levels in type 2 diabetes at low levels of insulin. However, several gene transcripts that are specifically involved in the electron transport chain were expressed at higher levels in type 2 diabetic patients. After the low-dose insulin infusion, which achieved postabsorptive insulin levels, the muscle mitochondrial ATP production rate (MAPR) was not different between type 2 diabetic and nondiabetic subjects. However, increasing insulin to postprandial levels increased the MAPR in nondiabetic subjects but not in type 2 diabetic patients. The lack of MAPR increment in response to high-dose insulin in type 2 diabetic patients occurred in association with reduced glucose disposal and expression of peroxisome proliferator-activated receptor-gamma coactivator 1alpha, citrate synthase, and cytochrome c oxidase I. In conclusion, the current data supports that muscle mitochondrial dysfunction in type 2 diabetes is not an intrinsic defect, but instead a functional defect related to impaired response to insulin.

Albumin activation of NAD(P)H oxidase activity is mediated via Rac1 in proximal tubule cells.

BACKGROUND: Rac1 is a Rho-family small GTP-ase, when activated is pivotal in NAD(P)H oxidase (NOX) activation and generation of reactive oxygen species (ROS). Evidence links Rac1 activation to receptor-mediated albumin endocytosis in the proximal tubule cell (PTC). Thus in states of albumin overload, Rac1 activation could lead to NOX activation and ROS formation in the PTC. Furthermore, accumulating evidence supports that HMG-CoA reductase inhibition may reduce oxidative stress and albuminuria. METHODS: To investigate the role of HMG-CoA reductase inhibition of Rac1 and oxidative stress we used the opossum kidney PTC. ROS generation in the PTC was confirmed using oxidative fluorescent dihydroethidium staining. RESULTS: We observed time-dependent increases in NOX activity with bovine serum albumin (albumin) stimulation (500 microg/dl, maximum at 20 min, p < 0.05) that was inhibited in a concentration-dependent manner with the HMG-CoA reductase inhibitor rosuvastatin (1 microM, p < 0.05). Additionally, the Rac1 inhibitor NSC23766 (100 ng/ml) attenuated albumin activation of NOX. Western blot analysis confirmed Rac1 translocation to plasma membrane in the PTC following albumin stimulation and subsequent inhibition by rosuvastatin and NSC23766. CONCLUSIONS: These data demonstrate that albumin-mediated increases in NOX activity and ROS in PTC are reversed by inhibition of Rac1 signaling with the use of rosuvastatin.

Inactivity induces increases in abdominal fat.

Previously, inducing inactivity for 53 h after 21 days of voluntary running resulted in a 25 and 48% increase in epididymal and omental fat pad weights, respectively, while rats continued to eat more than a group that never had access to a running wheel (J Physiol 565: 911-925, 2005). We wanted to test the hypothesis that inactivity, independent of excessive caloric intake, could induce an increase in fat pad mass. Twenty-one-day-old rats were given access to voluntary running wheels for 42-43 days so that they were running approximately 9 km/day in the last week of running, after which wheels were locked for 5, 53, or 173 h (WL5, WL53, WL173) before the rats were killed. During the 53 and 173 h of inactivity, one group of animals was pair fed (PF) to match sedentary controls, whereas the other continued to eat ad libitum (AL). Epididymal and retroperitoneal fat masses were significantly increased in the WL173-PF vs. the WL5 group, whereas epididymal, perirenal, and retroperitoneal fat masses were all significantly increased in the WL173-AL group compared with the WL5 group. Additionally, hyperplasia, and not hypertrophy, of the epididymal fat mass was responsible for the increase at WL173-AL as demonstrated by a significant increase in cell number vs. WL5, with no change in cell diameter or volume. Thus two important findings have been elucidated: 1) increases in measured abdominal fat masses occur in both AL and PF groups at WL173, and 2) adipocyte expansion via hyperplasia occurred with an ad libitum diet following cessation of voluntary running.

Methods in the evaluation of cardiovascular renin angiotensin aldosterone activation and oxidative stress.

Renin angiotensin aldosterone system (RAAS) activation plays an essential role in the development of cardiovascular disease (CVD). Multiple pathophysiologic processes are able to activate RAAS, among which hypertension, obesity, diabetes mellitus 2, and chronic kidney disease deserve special attention, because they are the main contributors to CVD. Adding to the well-known effects of RAAS overactivity on the vasculature and water and electrolyte balance, current evidence links abnormal activation of the RAAS to increased production of reactive oxygen species (ROS) and oxidative stress. This association is mediated at least partially through interaction of angiotensin II (Ang II) with its receptor angiotensin receptor 1 (AT1R) in cardiovascular tissue, and subsequent activation of the nicotinamide adenine dinucleotide phosphate (NADPH) enzymatic complex, which finally leads to increased ROS production. This resulting state of enhanced oxidative stress contributes largely to generalized atherosclerosis and finally to CVD. The generation of animal models of increased RAAS and Ang II expression, in particular the Ren2 rodent model, provides important opportunities to better characterize the relationship between this system and the production of ROS. This chapter describes methods to evaluate, characterize, and quantify the activity of the RAAS and NADPH oxidase, as well as the production of ROS production in animal model of RAAS.

Effect of antihypertensive agents on the development of type 2 diabetes mellitus.

People with hypertension have a high prevalence of insulin resistance and are at relatively high risk of developing type 2 diabetes mellitus. It is becoming increasingly evident that antihypertensive agents have disparate metabolic effects. For example, recent clinical trials indicate that agents that interrupt the renin-angiotensin axis reduce the risk of developing diabetes compared with other classes of antihypertensive agents. Blockade of the effects of angiotensin II might improve blood flow to insulin-sensitive tissues. Furthermore, interruption of the renin-angiotensin system might provide metabolic benefit through such mechanisms as reduced oxidative stress and restored nitric oxide production, which could lead to improved insulin signaling. Alternatively, collective trials suggest that both diuretics and beta-blockers accelerate the appearance of new-onset type 2 diabetes mellitus in patients with hypertension. Therefore, the risk of new-onset diabetes-associated cardiovascular risks should be factored into future treatment recommendations for patients who require antihypertensive therapy. This will become even more important as the number of insulin-resistant patients with hypertension increases in parallel with the steady growth in the number of sedentary, obese, and aged persons in our population.