Previous studies suggest that selective antagonists of specific subtypes of muscarinic acetylcholine receptors (mAChRs) may provide a novel approach for the treatment of certain central nervous system (CNS) disorders, including epileptic disorders, Parkinson's disease, and dystonia. Unfortunately, previously reported antagonists are not highly selective for specific mAChR subtypes, making it difficult to definitively establish the functional roles and therapeutic potential for individual subtypes of this receptor subfamily. The M 1 mAChR is of particular interest as a potential target for treatment of CNS disorders. We now report the discovery of a novel selective antagonist of M 1 mAChRs, termed VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl) VU0255035 has excellent brain penetration in vivo and is efficacious in reducing pilocarpine-induced seizures in mice. We were surprised to find that doses of VU0255035 that reduce pilocarpine-induced seizures do not induce deficits in contextual freezing, a measure of hippocampus-dependent learning that is disrupted by nonselective mAChR antagonists. Taken together, these data suggest that selective antagonists of M 1 mAChRs do not induce the severe cognitive deficits seen with nonselective mAChR antagonists and could provide a novel approach for the treatment certain of CNS disorders.Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors (GPCRs) that are widely expressed in the central nervous system (CNS) and are critical for the modulation of activity in multiple brain circuits (Langmead et al., 2008). Previous studies suggest that mAChRs play important roles in a broad range of CNS functions, including

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 renal outer medullary potassium channel (ROMK) is expressed in the kidney tubule and critically regulates sodium and potassium balance. The physiological functions of other inward rectifying K ϩ (Kir) channels expressed in the nephron, such as Kir7

Cholinergic transmission in the forebrain is mediated primarily by five subtypes of muscarinic acetylcholine receptors (mAChRs), termed M1-M5. Of the mAChR subtypes, M1 is among the most heavily expressed in regions that are critical for learning and memory, and has been viewed as the most critical mAChR subtype for memory and attention mechanisms. Unfortunately, it has been difficult to develop selective activators of M1 and other individual mAChR subtypes, which has prevented detailed studies of the functional roles of selective activation of M1. Using a functional HTS screen and subsequent diversity-oriented synthesis approach we have discovered a novel series of highly selective M1 allosteric agonists. These compounds activate M1 with EC50 values in the 150 nM to 500 nM range and have unprecedented, clean ancillary pharmacology (no substantial activity at 10μM across a large panel of targets). Targeted mutagenesis revealed a potentially novel allosteric binding site in the third extracellular loop of the M1 receptor for these allosteric agonists. Optimized compounds, such as VU0357017, provide excellent brain exposure after systemic dosing and have robust in vivo efficacy in reversing scopolamine-induced deficits in a rodent model of contextual fear conditioning. This series of selective M1 allosteric agonists provides critical research tools to allow dissection of M1-mediated effects in the CNS and potential leads for novel treatments for Alzheimer’s disease and schizophrenia.

The three-dimensional structure of the N-terminal domain of an archaeal TFIIB, which has high sequence homology with eucaryal analogues, is strikingly similar to that of the C-terminal zinc ribbon of the eucaryal transcription elongation factor TFIIB.