Boulos, J. | Search bioRxiv

bioRxiv2025

Antagonistic properties of 4-(hexyloxy)benzoate derivatives and their N-methyl ammonium salts at muscarinic acetylcholine receptors

Muscarinic acetylcholine receptors (mAChRs) are key regulators of diverse physiological processes and longstanding therapeutic targets. Building on the long-acting antagonist KH-5, we synthesised and evaluated a series of 4-(hexyloxy)benzoate derivatives and their quaternary N-methylated analogues to explore how structural modifications influence receptor affinity and the duration of functional antagonism. Our structure-activity analysis revealed that introducing a rigid azabicyclo[2.2.2]octan-1-ium group boosted binding affinity (up to 250-fold compared to parental compounds) yet reduced the half-life of functional antagonism. In contrast, analogues with moderate flexibility maintained high potency while preserving longer receptor residence time. Computational docking and molecular dynamics (MD) simulations demonstrated that stable hydrogen bonding with residue N6.52 and salt-bridge formation with D3.32 were critical for sustained ligand binding to the receptor, with MD-derived metrics outperforming docking energies in predicting biological activity. Crucially, a positively charged nitrogen and a 4-hexyloxy substituent are essential features for high-affinity binding and prolonged antagonism. Shortening the alkyl chain resulted in a marked loss of affinity and abolished sustained activity. These findings underscore the need to balance molecular rigidity with conformational flexibility and charge distribution in the design of long-residence mAChR antagonists, offering a framework for further development of mAChR-targeted long-acting antagonists. HighlightsO_LINew analogues show up to 250x higher affinity at muscarinic receptors C_LIO_LIN6.52 H-bonding during MD predicts compound binding better than docking energies C_LIO_LICharged nitrogen and 4-hexyloxy are key to high affinity and sustained action C_LIO_LIRigid azabicyclo groups boost potency but shorten antagonism duration C_LIO_LIFlexible analogues balance potency with longer receptor residence time better C_LI

pharmacology and toxicology↗

bioRxiv2022

Recruitment of transcriptional effectors by Cas9 creates cis regulatory elements and demonstrates distance-dependent transcriptional regulation

It is essential to regulate the expression of genes, such as those encoding the proteins of the cardiac sarcomere. This regulation is often mediated by cis regulatory elements termed enhancers and repressors that recruit transcription factors to gene-distal sites. However, the relationship between transcription factors recruitment to gene-distant sites and the regulation of gene expression is not fully understood. Specifically, it is unclear if such recruitment to any genomic site is sufficient to form an enhancer or repressor at the site, and what is the relationship between the cis regulatory elements position and its ability to control the transcription of distant genes. Using dead Cas9 to recruit either viral or endogenous transcription factor activation domains, we demonstrate that targeting naive genomic sites lacking open chromatin or active enhancer marks is sufficient to alter the chromatin signature of the target site, the distant gene promoter, and significantly induce the distant gene expression, even across chromatin insulating loci. The magnitude of induction is affected by the distance between the activation site and the cognate gene in a non-linear manner. Dead Cas9 mediated recruitment of repression domains behave similarly to activation in that targeting of non-regulatory regions could repress gene expression with a nonlinear distance dependence and across chromatin insulating loci. These findings expand the models of enhancer generation and function by showing that an arbitrary genomic site can become a regulatory element and interact epigenetically and transcriptionally with a distant promoter. They also provide new fundamental insights into the rules governing gene expression.

genomics↗