Valerian root extracts are widely used as mild sedatives to promote sleep, with clinical studies confirming their efficacy. Their sleep-promoting effects are associated with the adenosine A1 receptor (A1AR), a key regulator of sleep through neural activity inhibition. Adenosine, a neuromodulator that accumulates during wakefulness, activates A1ARs to facilitate sleep transitions. Using advanced analytics, we detected adenosine at 0.05% in the valerian extract Ze 911, supporting direct A1AR activation in vitro. Additionally, we explored A1ARs’ allosteric sites for modulatory activity. Valerenic acid and pinoresinol, key constituents of Ze 911, were identified as positive allosteric modulators (PAMs) of A1ARs. Valerenic acid exhibited strong PAM activity, with high cooperativity (αβ = 4.79 for adenosine and αβ = 23.38 for CPA) and intrinsic efficacy (τB = 5.98 for adenosine and τB = 3.14 for CPA). Pinoresinol displayed weaker PAM activity, with moderate cooperativity (αβ = 3.42 for adenosine and αβ = 0.79 for CPA) and limited efficacy (τB = 0.93 for adenosine and τB = 1.66 for CPA). The allosteric modulation observed in valerian extract Ze 911 suggests a mechanism of action in which valerenic acid and pinoresinol enhance receptor activation through allosteric interactions, potentially amplifying the effects of endogenous adenosine. By targeting A1ARs’ allosteric sites, valerian extract Ze 911 offers increased therapeutic selectivity and reduced off-target effects, emphasizing its potential for managing sleep disorders.

Predicting the pathogenic effect of rare genetic variants is significantly hampered by the limited cohort of diagnosed patients and the difficulty in functionally evaluating the effect of existing and novel variants. To address this issue, we leveraged a protein Multiplexed Assay of Variants Effect (MAVE) to functionally assess the effect of ~ 2200 missense variants of the TP63 gene, some of which cause autosomal dominant developmental disorders. The activity of each variant was measured using an optimized fibroblast-to-keratinocyte conversion protocol, and a subset of mutants was validated through multiple orthogonal biochemical and genomics functional assays to assess consistency and robustness. To systematically expand MAVEs beyond predefined assay, we here introduce SCRAMseq (Single Cell RNAseq Associated with MAVEs by sequencing). This generalizable platform leverages full-length scRNAseq for high-throughput, single-cell genotype-to-phenotype mapping. We validate SCRAMseq across different experimental and genetic contexts to demonstrate its reproducibility and accuracy. SCRAMseq provides a robust and versatile workflow poised to accelerate the discovery and dissection of gene variant effects on any potential disease-driving pathway, thereby significantly broadening MAVEs applicability to virtually any possible disease-driving gene.