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AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes. [ABSTRACT FROM AUTHOR]

A duplexed, functional multiaddition high throughput screen and subsequent optimization effort identified the first orally bioavailable and CNS penetrant glucagon-like peptide-1 receptor (GLP-1R) noncompetitive antagonist. Antagonist 5d not only blocked exendin-4-stimulated insulin release in islets but also lowered insulin levels while increasing blood glucose in vivo. [ABSTRACT FROM AUTHOR]

KCC2, a neuronal-specific K-Cl cotransporter, plays a major role in maintaining intracellular Cl concentration in neurons below its electrochemical equilibrium potential, thus favoring robust GABA hyperpolarizing or inhibitory responses. The pharmacology of the K-Cl cotransporter is dominated by loop diuretics such as furosemide and bumetanide, molecules used in clinical medicine because they inhibit the loop of Henle Na-K-2Cl cotransporter with much higher affinity. To identify molecules that affect KCC2 activity, we developed a fluorescence-based assay suitable for highthroughput screening (HTS) and used the assay to screen a library of 234,000 small molecules. We identified a large number of molecules that either decrease or increase the activity of the cotransporter. Here, we report the characterization of a small number of inhibitors, some of which inhibit KCC2 activity in the submicomolar range without substantially affecting NKCC1 activity. Using medicinal chemistry, we synthesized a number of variants, tested their effect on KCC2 function, and provide an analysis of structure/activity relationships. We also used one of the compounds to demonstrate competitive inhibition in regard to external tK] versus noncompetitive inhibition in respect to external [Cl]. [ABSTRACT FROM AUTHOR]

Abstract: We have examined the impact of the inflammatory cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNFα) on assembly of nAChRs from subunit mixtures of nAChRα4, β2 and β4 transiently transfected into 293 cells. In control transfections approximately 55% of α4 associated preferentially with β4, but less than 15% complexed with β2 and the remainder was associated with both β subunits. These relative ratios were modified by pro-inflammatory cytokines. IL-1β strongly enhanced α4/β2 association and decreased α4/β4, whereas TNFα promoted mixed α4/β2/β4 interactions. These results show that the emerging rules governing assembly of nAChRs are subject to modification by the pro-inflammatory cytokine environment. [Copyright &y& Elsevier]

[Display omitted] Kir6.2/SUR1 is an ATP-regulated potassium channel that acts as an intracellular metabolic sensor, controlling insulin and appetite-stimulatory neuropeptides secretion. In this Letter, we present the SAR around a novel Kir6.2/SUR1 channel opener scaffold derived from an HTS screening campaign. New series of compounds with tractable SAR trends and favorable potencies are reported. [ABSTRACT FROM AUTHOR]

Vector-borne diseases such as dengue fever and malaria, which are transmitted by infected female mosquitoes, affect nearly half of the world's population. The emergence of insecticide-resistant mosquito populations is reducing the effectiveness of conventional insecticides and threatening current vector control strategies, which has created an urgent need to identify new molecular targets against which novel classes of insecticides can be developed. We previously demonstrated that small molecule inhibitors of mammalian Kir channels represent promising chemicals for new mosquitocide development. In this study, high-throughput screening of approximately 30,000 chemically diverse small-molecules was employed to discover potent and selective inhibitors of Aedes aegypti Kir1 (AeKir1) channels heterologously expressed in HEK293 cells. Of 283 confirmed screening ‘hits’, the small-molecule inhibitor VU625 was selected for lead optimization and in vivo studies based on its potency and selectivity toward AeKir1, and tractability for medicinal chemistry. In patch clamp electrophysiology experiments of HEK293 cells, VU625 inhibits AeKir1 with an IC50 value of 96.8 nM, making VU625 the most potent inhibitor of AeKir1 described to date. Furthermore, electrophysiology experiments in Xenopus oocytes revealed that VU625 is a weak inhibitor of AeKir2B. Surprisingly, injection of VU625 failed to elicit significant effects on mosquito behavior, urine excretion, or survival. However, when co-injected with probenecid, VU625 inhibited the excretory capacity of mosquitoes and was toxic, suggesting that the compound is a substrate of organic anion and/or ATP-binding cassette (ABC) transporters. The dose-toxicity relationship of VU625 (when co-injected with probenecid) is biphasic, which is consistent with the molecule inhibiting both AeKir1 and AeKir2B with different potencies. This study demonstrates proof-of-concept that potent and highly selective inhibitors of mosquito Kir channels can be developed using conventional drug discovery approaches. Furthermore, it reinforces the notion that the physical and chemical properties that determine a compound's bioavailability in vivo will be critical in determining the efficacy of Kir channel inhibitors as insecticides. [ABSTRACT FROM AUTHOR]

Startingfrom a singleton chromanone high throughput screening(HTS) hit, we describe a focused medicinal chemistry optimizationeffort leading to the identification of a novel series of phenoxymethyl-dihydrothiazolopyridonederivatives as selective positive allosteric modulators (PAMs) ofthe metabotropic glutamate 5 (mGlu5) receptor. These dihydrothiazolopyridonespotentiate receptor responses in recombinant systems. In vitro andin vivo drug metabolism and pharmacokinetic (DMPK) evaluation allowedus to select compound 16afor its assessment in a preclinicalanimal screen of possible antipsychotic activity. 16awas able to reverse amphetamine-induced hyperlocomotion in ratsin a dose-dependent manner without showing any significant motor impairmentor overt neurological side effects at comparable doses. Evolutionof our medicinal chemistry program, structure activity, and propertiesrelationships (SAR and SPR) analysis as well as a detailed profilefor optimized mGlu5receptor PAM 16aare described. [ABSTRACT FROM AUTHOR]

The human Ether-à-go-go-related gene (hERG)-encoded K+ current, IKr is essential for cardiac repolarization but is also a source of cardiotoxicity because unintendedhERGinhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes IKr block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC70 of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC50 of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: We report the optimization of a series of non-MPEP site metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) based on a simple acyclic ether series. Modifications led to a gain of MPEP site interaction through incorporation of a chiral amide in conjunction with a nicotinamide core. A highly potent PAM, 8v (VU0404251), was shown to be efficacious in a rodent model of psychosis. These studies suggest that potent PAMs within topologically similar chemotypes can be developed to preferentially interact or not interact with the MPEP allosteric binding site. [Copyright &y& Elsevier]

Abstract: This Letter describes a chemical lead optimization campaign directed at VU0108370, a weak M1 PAM hit with a novel chemical scaffold from a functional HTS screen within the MLPCN. An iterative parallel synthesis approach rapidly established SAR for this series and afforded VU0405652 (ML169), a potent, selective and brain penetrant M1 PAM with an in vitro profile comparable to the prototypical M1 PAM, BQCA, but with an improved brain to plasma ratio. [Copyright &y& Elsevier]

Perturbations in Ca2+ homeostasis contribute to β-cell failure in diabetes, limiting insulin secretion and exacerbating hyperglycemia. TALK-1 is the most islet-restricted and most abundantly expressed K+ channel of the β-cell. ER TALK-1 channels provide K+ countercurrent for [Ca2+]ER release, thereby tuning [Ca2+]ER storage and release. During β-cell Ca2+ influx, TALK-1-enhanced [Ca2+]ER release leads to activation of the hyperpolarizing Ca2+ activated K+ channels, thus increasing the silent phase duration between Ca2+ oscillations. A gain-of-function mutation in TALK-1(L114P) causes MODY, by disrupting cytoplasmic and ER Ca2+ handling. However, the ability to probe ER TALK-1 channels as a therapeutic target to normalize β-cell Ca2+ handling and reduce ER stress in diabetes has not been determined. Here, using a high throughput fluorescence-based thallium (TI+) flux assay (z' =0.64) in a TALK-1 overexpression cell line, we identified 80 TALK-1 selective inhibitors from a small molecule library of 50,000 compounds. 4 potent inhibitors that also inhibited the ER membrane TALK-1 channels were identified by monitoring cyclopiazonic acid-induced [Ca2+]ER release (e.g. K2P16-i7(IC50=5.3μM); 63.36% [Ca2+]ER recovery, p-value=0.02). We also tested these inhibitors for their efficacy in controlling mouse islet [Ca2+]ER storage as well as cytosolic Ca2+ oscillations. TALK-1 inhibitors significantly increased islet [Ca2+]ER storage and Ca2+ oscillation frequency (e.g.K2P16-i7; [Ca2+]ER AUC=60.9a.u., peaks/min=0.45 vs. DMSO; [Ca2+]ER AUC=49.7a.u., peaks/min= 0.17, p-value<0.05). Importantly, the inhibitors did not alter [Ca2+]ER and glucose-stimulated Ca2+ oscillation frequency in TALK-1 KO islets. In summary, we have identified the first ER TALK-1 selective inhibitors that can not only be utilized to assess the role of TALK-1 in human β-cell Ca2+ handling and GSIS, but also potentially reduce ER dysfunction during diabetes pathogenesis. Disclosure: A. Y. Nakhe: None. P. Dadi: None. D. Westover: None. E. Days: None. J. Denton: None. D. Jacobson: None. [ABSTRACT FROM AUTHOR]

Insulin levels are inadequate to maintain euglycemia in patients with type-2 diabetes (T2D), which is due in part to perturbations in pancreatic ꞵ-cell Ca2+ homeostasis. TALK-1 K+ channels are key regulators of ꞵ-cell electrical excitability, Ca2+ handling and glucose-stimulated insulin secretion (GSIS). KCNK16, which encodes TALK-1 is the most abundant ꞵ-cell K+ channel transcript and also the most islet-restricted ion channel transcript. Furthermore, a nonsynonymous coding sequence polymorphism in KCNK16 causes a predisposition for developing T2D. However, the ability to utilize TALK-1 as a therapeutic target to normalize ꞵ-cell Ca2+ homeostasis in T2D has not been determined. Here, we optimized a high throughput fluorescence-based thallium (TI+) flux assay (z' =0.64) to screen for TALK-1 inhibitors, which resulted in identification of 286 TALK-1 inhibitors with potent drug-like properties (from a 50,000 compound screen). Of these hits, 78 exhibit TALK-1 selectivity and do not influence the activity of other K+ channels including TALK-2, TREK-2, TASK-1, TASK-3, KATP or hERG channels. We then performed dose response curve analyses and identified 9 potent inhibitors with IC50s < 2.5μM that inhibit TALK-1 activity (>65%). To test the efficacy of the inhibitors for their ability to augment islet function, we assessed their impact on mouse islet Ca2+ handling. TALK-1 inhibitors significantly increased islet Ca2+ oscillation frequency (e.g., VU0233408, 2.47 peaks/min vs. DMSO, 1.82 peaks/min, p-value= 0.011), similar to what was previously observed in TALK-1 KO islets. Moreover, the inhibitors did not increase glucose-stimulated Ca2+ oscillation frequency in TALK-1 KO islets. Thus, pharmacological inhibition of TALK-1 is predicted to increase GSIS. In conclusion, we have identified the first potent and selective TALK-1 inhibitors that can be utilized to assess the role of TALK-1 during human ꞵ-cell GSIS under physiological and diabetic conditions. Disclosure: A.Y. Nakhe: None. P. Dadi: None. D. Westover: None. E. Days: None. J. Denton: None. D. Jacobson: None. Funding: National Institute of Diabetes and Digestive and Kidney Diseases (R01DK115620VDC); Vanderbilt University Diabetes and Research Training Center (P60DK20593) [ABSTRACT FROM AUTHOR]

Insecticide resistance is a growing threat to mosquito control programs around the world, thus creating the need to discover novel target sites and target-specific compounds for insecticide development. Emerging evidence suggests that mosquito inward rectifier potassium (Kir) channels represent viable molecular targets for developing insecticides with new mechanisms of action. Here we describe the discovery and characterization of VU041, a submicromolar-affinity inhibitor of Anopheles (An.) gambiae and Aedes (Ae.) aegypti Kir1 channels that incapacitates adult female mosquitoes from representative insecticide-susceptible and -resistant strains of An. gambiae (G3 and Akron, respectively) and Ae. aegypti (Liverpool and Puerto Rico, respectively) following topical application. VU041 is selective for mosquito Kir channels over several mammalian orthologs, with the exception of Kir2.1, and is not lethal to honey bees. Medicinal chemistry was used to develop an analog, termed VU730, which retains activity toward mosquito Kir1 but is not active against Kir2.1 or other mammalian Kir channels. Thus, VU041 and VU730 are promising chemical scaffolds for developing new classes of insecticides to combat insecticide-resistant mosquitoes and the transmission of mosquito-borne diseases, such as Zika virus, without harmful effects on humans and beneficial insects. [ABSTRACT FROM AUTHOR]