BackgroundAt a molecular level, insects utilize members of several highly divergent and unrelated families of cell-surface chemosensory receptors for detection of volatile odorants. Most odors are detected via a family of odorant receptors (ORs), which form heteromeric complexes consisting of a well-conserved OR co-receptor (Orco) ion channel and a non-conserved tuning OR that provides coding specificity to each complex. Orco functions as a non-selective cation channel and is expressed in the majority of olfactory receptor neurons (ORNs). As the destructive behaviors of many insects are principally driven by olfaction, Orco represents a novel target for behavior-based control strategies. While many natural and synthetic odorants have been shown to agonize Orco/Or complexes, only a single direct Orco modulator, VUAA1, has been described. In an effort to identify additional Orco modulators, we have investigated the structure/activity relationships around VUAA1.ResultsA search of our compound library identified several VUAA1 analogs that were selected for evaluation against HEK cells expressing Orco from the malaria vector Anopheles gambiae (AgOrco). While the majority of compounds displayed no activity, many of these analogs possess no intrinsic efficacy, but instead, act as competitive VUAA1 antagonists. Using calcium mobilization assays, patch clamp electrophysiology, and single sensillum in vivo recording, we demonstrate that one such candidate, VU0183254, is a specific allosteric modulator of OR signaling, capable of broadly inhibiting odor-mediated OR complex activation.ConclusionsWe have described and characterized the first Orco antagonist, that is capable of non-competitively inhibiting odorant-evoked activation of OR complexes, thereby providing additional insight into the structure/function of this unique family of ligand-gated ion channels. While Orco antagonists are likely to have limited utility in insect control programs, they represent important pharmacological tools that will facilitate the investigation of the molecular mechanisms underlying insect olfactory signal transduction.

Staphylococcus aureus is a significant infectious threat to global public health. Acquisition or synthesis of heme is required for S. aureus to capture energy through respiration, but an excess of this critical cofactor is toxic to bacteria. S. aureus employs the heme sensor system (HssRS) to overcome heme toxicity; however, the mechanism of heme sensing is not defined. Here, we describe the identification of a small molecule activator of HssRS that induces endogenous heme biosynthesis by perturbing central metabolism. This molecule is toxic to fermenting S. aureus , including clinically relevant small colony variants. The utility of targeting fermenting bacteria is exemplified by the fact that this compound prevents the emergence of antibiotic resistance, enhances phagocyte killing, and reduces S. aureus pathogenesis. Not only is this small molecule a powerful tool for studying bacterial heme biosynthesis and central metabolism; it also establishes targeting of fermentation as a viable antibacterial strategy.

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

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This Letter describes a chemical lead optimization campaign directed at VU0108370, a weak M 1 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 M 1 PAM with an in vitro profile comparable to the prototypical M 1 PAM, BQCA, but with an improved brain to plasma ratio.The muscarinic acetylcholine receptors (mAChRs) are members of the family A G-proteincoupled receptors (GPCRs) and include five subtypes denoted M 1 -M 5 . All five of the mAChRs are known to play critical roles in multiple basic physiological processes and represent attractive therapeutic targets for a number of peripheral and CNS pathologies.1 -3 Within the mAChRs, a major challenge has been a lack of subtype selective ligands to study the specific contribution of discrete mAChRs in various disease states.3 , 4 To address this limitation, we have focused on targeting allosteric sites on mAChRs as a means to develop subtype selective small molecules, both allosteric agonists and positive allosteric modulators (PAMs).5 -9 Moreover, the emerging phenomenon of ligand-biased signaling requires the development of diverse chemical scaffolds of M 1 ligands to successfully dissect of the roles of M 1 activation through multiple, discrete ligand-biased signaling pathways.10 , 11As members of the Molecular Libraries Production Center Network (MLPCN),12 we performed a real-time cell-based calcium-mobilization assay employing a rat M 1 /CHO cell Correspondence to: Michael R. Wood. † these authors contributed equally to this work Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Perusal of the HTS data, which also yielded the non-selective hit 5, identified a second weak M 1 PAM hit 7, VU0108370, with an EC 50 of ∼13 μM. Confirmation of 7 from fresh powder and counter-screening against M 2 -M 5 increased our enthusiasm for this highly M 1 mAChR selective hit (Fig. 2); however, the CRC did not plateau, suggesting the M 1 EC 50 was actually >13 μM. Despite the weak potency, the confirmation of a novel M 1 PAM scaffold with high M 1 selectivity initiated a lead optimization campaign to improve M 1 potency while maintaining the high M 2 -M 5 selectivity. NIH Public AccessOur initial optimization strategy is outlined in Figure 3, and as SAR with allosteric ligands is often shallow, we employed an iterative parallel synthesis approach, along with targeted syntheses for structures encompassing more speculative modifications. Attempted mod...

ABSTRACT:The M 1 muscarinic acetylcholine receptor is thought to play an important role in memory and cognition, making it a potential target for the treatment of Alzheimer's disease (AD) and schizophrenia. Moreover, M 1 interacts with BACE1 and regulates its proteosomal degradation, suggesting selective M 1 activation could afford both palliative cognitive benefit as well as disease modification in AD. A key challenge in targeting the muscarinic acetylcholine receptors is achieving mAChR subtype selectivity. Our lab has previously reported the M 1 selective positive allosteric modulator ML169. Herein we describe our efforts to further optimize this lead compound by preparing analogue libraries and probing novel scaffolds. We were able to identify several analogues that possessed submicromolar potency, with our best example displaying an EC 50 of 310 nM. The new compounds maintained complete selectivity for the M 1 receptor over the other subtypes (M 2 −M 5 ), displayed improved DMPK profiles, and potentiated the carbachol (CCh)-induced excitation in striatal MSNs. Selected analogues were able to potentiate CCh-mediated nonamyloidogenic APPsα release, further strengthening the concept that M 1 PAMs may afford a disease-modifying role in the treatment of AD. KEYWORDS: Muscarinic, acetylcholine, positive allosteric modulator (PAM), ML169, Alzheimer's disease (AD), medium spiny neurons (MSNs), MLPCN T he muscarinic acetylcholine receptors (mAChRs) are members of the family A G-protein-coupled receptors (GPCRs) and comprise five subtypes (M 1 −M 5 ) that mediate a broad range of actions of the neurotransmitter acetylcholine (ACh, 1) in the central nervous system and other tissues (Figure 1) and play key roles in memory and attention mechanisms, motor control, nociception, regulation of sleepwake cycles, cardiovascular function, renal function, and GI function. 1 mAChR subtypes M 1 , M 3 , and M 5 are coupled to G q and stimulate phospholipase C and intracellular Ca 2+ release, while M 2 and M 4 are coupled to G i and block adenylyl cyclase. 2 Data from brain expression, cellular localization, and knockout mice have implicated that M 1 plays a vital role in both memory and cognition, 3 and numerous studies have shown efficacy in preclinical cognition models with selective M 1 activators. 4−7 Recently, studies have conclusively shown that M 1 interacts with BACE1 to regulate its protesomal degradation and that M 1 activation activates α-secretase, which leads to an increase in sAPPα thereby preventing the formation of Aβ via MAPK-and PKC-dependent pathways. 8 In addition, M 1 activation decreases τ phosphorylation; therefore, M 1 activation affects the major pathological hallmarks of AD. 8,9 Beyond the cholinergic and NMDA hypofunction hypotheses of schizophrenia where M 1 could play a critical therapeutic role, 10 BACE1 has also been linked to schizophrenia, via neuregulin 1; thus, the ability of M 1 activation to regulate BACE1 suggests that pharmacological agents that can selectivity activate M 1 represent a nove...

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Small molecules active in the pathogenic bacterium Staphylococcus aureus are valuable tools for the study of its basic biology and pathogenesis, and many molecules may provide leads for novel therapeutics. We have previously reported a small molecule, 1, which activates endogenous heme biosynthesis in S. aureus, leading to an accumulation of intracellular heme. In addition to this novel activity, 1 also exhibits toxicity towards S. aureus growing under fermentative conditions. To determine if these activities are linked and establish what features of the molecule are required for activity, we synthesized a library of analogs around the structure of 1 and screened them for activation of heme biosynthesis and anaerobic toxicity to investigate structure–activity relationships. The results of this analysis suggest that these activities are not linked. Furthermore, we have identified the structural features that promote each activity and have established two classes of molecules: activators of heme biosynthesis and inhibitors of anaerobic growth. These molecules will serve as useful probes for their respective activities without concern for the off target effects of the parent compound.