Constantine Kreatsoulas

Selective activation of the M1 muscarinic acetylcholine receptor achieved by allosteric potentiation

The forebrain cholinergic system promotes higher brain function in part by signaling through the M1 muscarinic acetylcholine receptor (mAChR). During Alzheimers disease (AD), these cholinergic neurons degenerate, therefore selectively activating M1 receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the M1 mAChR. BQCA reduces the concentration of ACh required to activate M1 up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 μM. Furthermore studies in M1−/− mice demonstrates that BQCA requires M1 to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and site-directed mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to M1 allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces β-arrestin recruitment to M1, suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the M1 receptor and represents a promising therapeutic strategy for cognitive disorders.

The M1 Muscarinic Receptor Allosteric Agonists AC-42 and 1-[1′-(2-Methylbenzyl)-1,4′-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one Bind to a Unique Site Distinct from the Acetylcholine Orthosteric Site

Activation of M1 muscarinic receptors occurs through orthosteric and allosteric binding sites. To identify critical residues, site-directed mutagenesis and chimeric receptors were evaluated in functional calcium mobilization assays to compare orthosteric agonists, acetylcholine and xanomeline, M1 allosteric agonists AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride), TBPB (1-[1′-(2-methylbenzyl)-1,4′-bipiperidin-4-yl]-1,3-dihydro-2H-benzimidazol-2-one), and the clozapine metabolite N-desmethylclozapine. A minimal epitope has been defined for AC-42 that comprises the first 45 amino acids, the third extracellular loop, and seventh transmembrane domain (Mol Pharmacol 61:1297–1302, 2002). Using chimeric M1 and M3 receptor constructs, the AC-42 minimal epitope has been extended to also include transmembrane II. Phe77 was identified as a critical residue for maintenance of AC-42 and TBPB agonist activity. In contrast, the functional activity of N-desmethylclozapine did not require Phe77. To further map the binding site of AC-42, TBPB, and N-desmethylclozapine, point mutations previously reported to affect activities of M1 orthosteric agonists and antagonists were studied. Docking into an M1 receptor homology model revealed that AC-42 and TBPB share a similar binding pocket adjacent to the orthosteric binding site at the opposite face of Trp101. In contrast, the activity of N-desmethylclozapine was generally unaffected by the point mutations studied, and the docking indicated that N-desmethylclozapine bound to a site distinct from AC-42 and TBPB overlapping with the orthosteric site. These results suggest that structurally diverse allosteric agonists AC-42, TBPB, and N-desmethylclozapine may interact with different subsets of residues, supporting the hypothesis that M1 receptor activation can occur through at least three different binding domains.

Pyridyl aminothiazoles as potent inhibitors of Chk1 with slow dissociation rates.

Pyridyl aminothiazoles comprise a novel class of ATP-competitive Chk1 inhibitors with excellent inhibitory potential. Modification of the core with ethylenediamine amides provides compounds with low picomolar potency and very high residence times. Investigation of binding parameters of such compounds using X-ray crystallography and molecular dynamics simulations revealed multiple hydrogen bonds to the enzyme backbone as well as stabilization of the conserved water molecules network in the hydrophobic binding region.

Scoring of KDR Kinase Inhibitors: Using Interaction Energy as a Guide for Ranking

SummaryWithin a congeneric series of ATP-competitive KDR kinase inhibitors, we determined that the IC50 values, which span four orders of magnitude, correlated best with the calculated ligand-protein interaction energy using the Merck Molecular Force Field (MMFFs(94)). Using the ligand-protein interaction energy as a guide, we outline a workflow to rank order virtual KDR kinase inhibitors prior to synthesis. When structural information of the target is available, the ability to score molecules a priori can be used to rationally select reagents. Our implementation allows one to select thousands of readily available reagents, enumerate compounds in multiple poses and score molecules in the active site of a protein within a few hours. In our experience, virtual library enumeration is best used when a correlation between computed descriptors/properties and IC50 or Ki values has been established.

P4-322: Exploring the pharmacology of BQCA, a highly selective allosteric M1 potentiator

Background: The cholinergic system is a major neuromodulatory neurotransmitter system, affecting learning and memory functions. Loss of cholinergic function is an invariant pathological feature of Alzheimer’s disease (AD), contributing to cognitive decline. Muscarinic acetylcholine receptors (mAChRs) play key roles in facilitating cognitive processes, and non-selective muscarinic receptor agonists have shown efficacy in the treatment of AD. However these compounds were not tolerated, suggesting greater selectivity is required. Of the five muscarinic subtypes (M1-M5), regional localization and transgenic mouse studies indicate M1 has greatest potential for the treatment of cognitive impairment, but due to high conservation of the acetylcholine binding site, truly selective ligands have not been identified. Methods: The FLIPR fluorometric imaging plate reader was used for Ca flux measurements in CHO cells stably expressing mAChRs. Molecular modeling was used to define the binding epitopes for allosteric potentiators for M1. Results: We now describe a drug-like molecule, Benzyl Quinolone Carboxylic Acid (BQCA), which is a highly selective positive allosteric modulator of the M1 receptor. In CHO cells expressing human M1, BQCA potentiated threshold responses to acetylcholine more than 15-fold with an EC50 of approximately 853 nM. At 100 M, BQCA sensitized the M1 receptor approximately 100-fold. No other agonist, antagonist, or potentiator activity was observed on other muscarinic receptors up to 100 M. IP1 assays confirmed the potentiation activity of BQCA in rat cortical neurons and selectivity in human neuroblastoma SH-SY5Y cells. BQCA had no effect on [H]N-methylscopolamine binding to M1 but reduced the concentration of agonist required to overcome antagonism and recruit GTP S, indicating that it promotes the active form of the receptor without altering equilibrium binding of orthosteric ligands. Chimeric receptor studies suggested that extracellular region II is important for BQCA selectivity. Modeling and site directed mutagenesis were used to clarify the role of specific residues in the binding sites, and indicate that Y179 and W400 in the second and third extracellular loops are critical interaction sites. Conclusions: In summary, BQCA is a highly selective pharmacological reagent for the M1 muscarinic acetylcholine receptor that should enable studies addressing the potential of M1 as a therapeutic target for AD.