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 [K ؉ ] versus noncompetitive inhibition in respect to external [Cl ؊ ].fluorescence ͉ high-througput screening ͉ Na-K-2Cl cotransporter ͉ thallium C ation-chloride cotransporters have received much attention in the past decade for the role they play in the nervous system. In particular KCC2, a neuronal-specific K-Cl cotransporter, has been shown to modulate inhibitory neurotransmission both in the brain and in the spinal cord. By reducing the intracellular Cl Ϫ concentration below its thermodynamic equilibrium potential in central neurons, KCC2 strengthens synaptic inhibition. Several studies have shown that loss of KCC2 function in central neurons results in the development of CNS hyperexcitability (1-3). Moreover, a paper from Coull and coworkers also showed that disinhibition in the dorsal horn of the spinal cord triggered by peripheral nerve injury was mediated by a significant decrease in KCC2 expression (4). Local blockade or knockdown of spinal KCC2 in intact rats markedly reduced the nocicepetive threshold. By linking change in KCC2 function to disruption of Cl Ϫ homeostasis in lamina I neurons, the study clearly linked the loss of KCC2 function to a loss of inhibition in this region of the spinal cord and to an increase in chronic pain. The relationship between KCC2 and nerve or spinal cord injury has been confirmed in subsequent studies that have shown that inflammatory response due to intraplantar injection of formalin (5, 6) or hind paw injection of complete Freund's adjuvant (7), or to loose ligation of the sciatic nerve (8), or contusive spinal cord injury at T9 (9), all led to a down-regulation of cotransporter expression. Altogether, these studies also point to a role of KCC2 in neuropathic pain.The pharmacology of the cation-chloride cotransporters is dominated by 2 classes of drugs: the thiazide a...

The melanocortin MC4 receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC4 receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC4 receptor, we created HEK293 cell lines coexpressing the human melanocortin MC4 receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z’=0.50). A pilot screen run on the Microsource Spectrum compound library (n= 2,000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β2AR agonists –the β2AR being endogenously expressed in HEK293 cells-, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of an HTS for allosteric modulators for a Gs protein coupled receptor.

Injectable, degradation-resistant peptide agonists for the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R), such as exenatide and liraglutide, activate the GLP-1R via a complex orthosteric-binding site and are effective therapeutics for glycemic control in type 2 diabetes. Orally bioavailable orthosteric small-molecule agonists are unlikely to be developed, whereas positive allosteric modulators (PAMs) may offer an improved therapeutic profile. We hypothesize that allosteric modulators of the GLP-1R would increase the potency and efficacy of native GLP-1 in a spatial and temporally preserved manner and/or may improve efficacy or side effects of injectable analogs. We report the design, optimization, and initial results of a duplexed high-throughput screen in which cell lines overexpressing either the GLP-1R or the glucagon receptor were coplated, loaded with a calcium-sensitive dye, and probed in a three-phase assay to identify agonists, antagonists, and potentiators of GLP-1, and potentiators of glucagon. 175,000 compounds were initially screened, and progression through secondary assays yielded 98 compounds with a variety of activities at the GLP-1R. Here, we describe five compounds possessing different patterns of modulation of the GLP-1R. These data uncover PAMs that may offer a drug-development pathway to enhancing in vivo efficacy of both endogenous GLP-1 and peptide analogs.

Escherichia coli diacylglycerol kinase (DAGK) is a homotrimeric helical integral membrane protein in which a number of single-site mutations to cysteine are known to promote misfolding. Here, effects of other amino acid replacements have been explored using a folding assay based on the dilution of acidic urea/DAGK stock solutions into detergent/lipid mixed micelles. DAGK with an I110P or I110R mutation in the third transmembrane helix could not be purified because its expression was toxic to the E. coli host, most likely because of severe folding defects. Other mutations at Ile110 enhanced irreversible misfolding to varying degrees that generally correlated both with the polarity of the inserted amino acid and with the degree of protein destabilization. However, the I110W mutant was an exception in that it was highly misfolding prone while at the same time being more stable than the wild-type protein. This contrasts with I110Y, which also exhibited enhanced stability but folded with an efficiency similar to that of the wild type. For most mutants, the critical step leading to irreversible misfolding occurred for monomeric DAGK prior to trimerization and independent of association with mixed micelles. Misfolding of DAGK evidently involves the formation of incorrect monomer tertiary structure. Mutations appear to enhance misfolding by disfavoring the formation of correct structure rather than by directly stabilizing the misfolded state. Finally, when urea-solubilized DAGK was diluted into detergent/lipid-free buffer, it retained a significant degree of folding competency over a period of minutes. This property may be relevant to membrane protein folding in cells under conditions where the usual machinery associated with membrane integration is saturated, dysregulated, or dysfunctional.

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