The therapeutic potential of small molecule signaling inhibitors is often limited by off-target effects. Recently, in a screen for compounds that perturb zebrafish embryonic dorsoventral axis, we identified dorsomorphin, the first selective inhibitor of bone morphogenetic protein (BMP) signaling. Here we show that dorsomorphin has significant “off-target” effects against the VEGF (vascular endothelial growth factor) type-2 receptor (Flk1/KDR) and disrupts zebrafish angiogenesis. Since both BMP and VEGF signals are known to be involved in vascular development, we sought to determine whether dorsomorphin’s anti-angiogenic effects are due to its impact on the BMP or VEGF signals through the development of analogs that target BMP but not VEGF signaling, and vise versa. In a structure activity relationship (SAR) study of dorsomorphin analogs based primarily on their effects on live zebrafish embryos, we identified highly selective and potent BMP inhibitors as well as selective VEGF inhibitors. One of the BMP inhibitors, DMH1, which exclusively targets the BMP, but not VEGF, pathway, dorsalized the embryonic axis without disrupting angiogenic process, demonstrating that BMP signaling was not involved in angiogenic process. This is one of the first full-scale SAR study performed in vertebrates, and demonstrates the potential of zebrafish as an attractive complementary platform for drug development that incorporates an assessment of in vivo bioactivity and selectivity in the context of a living organism.

The presence of druggable, topographically distinct allosteric sites on a wide range of receptor families has offered new paradigms for small molecules to modulate receptor function. Moreover, ligands that target allosteric sites offer significant advantages over the corresponding orthosteric ligands in terms of selectivity, including subtype selectivity within receptor families, and can also impart improved physicochemical properties. However, allosteric ligands are not a panacea. Many chemical issues (e.g., flat structure-activity relationships) and pharmacological issues (e.g., ligand-biased signaling) that are allosteric centric have emerged. Notably, the fact that allosteric sites are less evolutionarily conserved leads to improved selectivity; however, this can also lead to species differences that can hinder safety assessment. Many allosteric ligands possess molecular switches, wherein a small structural change (chemical or metabolic) can modulate the mode of pharmacology or receptor subtype selectivity. As the field has matured, as described here, key principles and strategies have emerged for the design of ligands/drugs for allosteric sites.

Previous clinical and animal studies suggest that selective activators of M 1 and/or M 4 muscarinic acetylcholine receptors (mAChRs) have potential as novel therapeutic agents for treatment of schizophrenia and Alzheimer's disease. However, highly selective centrally penetrant activators of either M 1 or M 4 have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors. We previously identified VU10010 [3-amino-N-(4-chlorobenzyl)-4, 6-dimethylthieno[2,3-b]pyridine-2-carboxamide] as a potent and selective allosteric potentiator of M 4 mAChRs. However, unfavorable physiochemical properties prevented use of this compound for in vivo studies. We now report that chemical optimization of VU10010 has afforded two centrally penetrant analogs, VU0152099 [3-amino-N-(benzo[d], that are potent and selective positive allosteric modulators of M 4 . VU0152099 and VU0152100 had no agonist activity but potentiated responses of M 4 to acetylcholine. Both compounds were devoid of activity at other mAChR subtypes or at a panel of other GPCRs. The improved physiochemical properties of VU0152099 and VU0152100 allowed in vivo dosing and evaluation of behavioral effects in rats. Interestingly, these selective allosteric potentiators of M 4 reverse amphetamine-induced hyperlocomotion in rats, a model that is sensitive to known antipsychotic agents and to nonselective mAChR agonists. This is consistent with the hypothesis that M 4 plays an important role in regulating midbrain dopaminergic activity and raises the possibility that positive allosteric modulation of M 4 may mimic some of the antipsychotic-like effects of less selective mAChR agonists.To date, five muscarinic acetylcholine receptor (mAChR) subtypes have been identified (M 1 -M 5 ) and play important roles in mediating the actions of ACh in the peripheral and central nervous systems (Wess, 1996). Of these, M 1 and M 4 are the most heavily expressed in the CNS and represent attractive therapeutic targets for cognition, Alzheimer's disease, and schizophrenia (Bymaster et al., 2002;Messer, 2002;Raedler et al., 2007). In contrast, the adverse effects of cholinergic agents are thought to be primarily due to activation of peripheral M 2 and M 3 mAChRs (Bymaster et al., 2003a,b).

This report describes the discovery and initial characterization of the first positive allosteric modulator of muscarinic acetylcholine receptor subtype 5 (mAChR5 or M5). Functional HTS, identified VU0119498, which displayed micromolar potencies for potentiation of acetylcholine at M1, M3, and M5 receptors in cell-based Ca2+ mobilization assays. Subsequent optimization led to the discovery of VU0238429, which possessed an EC50 of approximately 1.16 µM at M5 with >30-fold selectivity versus M1 and M3, with no M2 or M4 potentiator activity.

A functional high throughput screen and subsequent multi-dimensional, iterative parallel synthesis effort identified the first muscarinic acetylcholine receptor (mAChR) negative allosteric modulator (NAM) selective for the M5 subtype. ML375 is a highly selective M5 NAM with sub-micromolar potency (human M5 IC50 = 300 nM, rat M5 IC50 = 790 nM, M1–4 IC50 >30 μM), excellent multi-species PK, high CNS penetration, and enantiospecific inhibition.