An allosteric potentiator of M 4 mAChR modulates hippocampal synaptic transmission Jana K Shirey 1,5 , Zixiu Xiang 1,5 , Darren Orton 2,3 , Ashley E Brady 1 , Kari A Johnson 1 , Richard Williams 2,3 , Jennifer E Ayala 1 , Alice L Rodriguez 1,2 , Ju ¨rgen Wess 4 , David Weaver 1,2 , Colleen M Niswender 1,2 & P Jeffrey Conn 1,2 Muscarinic acetylcholine receptors (mAChRs) provide viable targets for the treatment of multiple central nervous system disorders. We have used cheminformatics and medicinal chemistry to develop new, highly selective M 4 allosteric potentiators. VU10010, the lead compound, potentiates the M 4 response to acetylcholine 47-fold while having no activity at other mAChR subtypes. This compound binds to an allosteric site on the receptor and increases affinity for acetylcholine and coupling to G proteins. Whole-cell patch clamp recordings revealed that selective potentiation of M 4 with VU10010 increases carbachol- induced depression of transmission at excitatory but not inhibitory synapses in the hippocampus. The effect was not mimicked by an inactive analog of VU10010 and was absent in M 4 knockout mice. Selective regulation of excitatory transmission by M 4 suggests that targeting of individual mAChR subtypes could be used to differentially regulate specific aspects of mAChR modulation of function in this important forebrain structure. One of the most important neuromodulatory systems responsible for regulation of multiple aspects of central nervous system (CNS) function is a widespread network of neurons that use acetylcholine (ACh; 1) as the primary neurotransmitter. Cholinergic projections from the basal forebrain provide major modulatory inputs to the cortex and hippocampus 1 and have a critical role in memory and attention mechanisms 2–4 . Furthermore, abundant evidence suggests that the clinical syndrome associated with Alzheimer’s disease results at least in part from failed neurotransmission at cholinergic synapses in the hippocampus and neocortex. There are also a number of other critical cholinergic systems in the CNS, including cholinergic interneurons in the striatum and other midbrain regions, as well as cholinergic projection neurons in various brain stem regions 1,5 . Together, these cholinergic pathways have been implicated in a wide variety of CNS functions including nociception, regulation of sleep/wake cycles, motor control and arousal. Agents that regulate cholinergic transmission have been proposed to have potential efficacy in a wide variety of CNS and neurodegenerative disorders, including chronic and neuropathic pain, sleep disorders, epilepsy, schizophrenia, Alzheimer’s disease and Parkinson’s disease 6–9 . Based on this broad influence of cholinergic systems in the CNS, it is surprising that there have not been greater advances in development of therapeutic agents that target cholinergic signaling. Efforts to develop agents that enhance cholinergic transmission for ameliorating the loss of cognitive function in people with Alzheimer’s disease and other memory disorders have been partially successful, and clinical trials with tacrine (2) and other acetylcholinesterase (AChE) inhibitors have established dose-related improvements in measures of cognitive performance and quality of life 10–13 . More recently, cholinergic agents have been shown to reduce behavioral disturbances and psychotic symptoms in people suffering from Alzheimer’s disease as well as a variety of other neurodegenerative disorders including Lewy body dementia, Parkinson’s disease dementia, vascular dementia and schizophrenia 14–18 . However, though this clinical validation of the efficacy of cholinergic agents is encouraging, all cholinergic agents developed so far have dose-limiting adverse effects that prevent widespread use in the clinic. These adverse effects are primarily due to activation of mAChRs in the periphery. To take advantage of the therapeutic potential of manipulating cholinergic systems, it will be critical to develop new approaches for selectively regulating choliner- gic signaling in central circuits involved in CNS disorders while avoiding the peripheral adverse effects associated with currently available treatments. Evidence suggests that cholinergic transmission in many of the most critical CNS circuits is mediated primarily by mAChRs 1 . Of the five mAChR subtypes that have been identified (termed M 1 –M 5 ), M 1 and M 4 are most heavily expressed in the CNS and are the most likely candidates for mediating the effects on cognition, attention mechan- isms and sensory processing 19–21 . In contrast, the most prominent adverse effects of cholinergic agents (bradycardia, gastrointestinal Received 23 March; accepted 16 October; published online 2 December 2007; doi:10.1038/nchembio.2007.55 1 Department of Pharmacology, Vanderbilt Program in Drug Discovery, 23rd Avenue South at Pierce, Nashville, Tennessee 37232-6600, USA. 2 Vanderbilt Institute for Chemical Biology, 802 Robinson Research Building, Nashville, Tennessee 37232, USA. 3 Department of Chemistry, Vanderbilt University, 7330 Stevenson Center, Station B 351822, Nashville, Tennessee 37235, USA. 4 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 8A, Room B1A-05, 8 Center Drive MSC 0810, Bethesda, Maryland 20892-0810, USA. 5 These authors contributed equally to this work. Correspondence should be addressed to P.J.C. (jeff.conn@vanderbilt.edu). 42 VOLUME 4 NUMBER 1 JANUARY 2008 NATURE CHEMICAL BIOLOGY ARTICLES