Chirality is an important consideration in drug development: it can influence recognition of the intended target, pharmacokinetics, and off-target effects. Here, we investigate how chirality affects the activity and mechanism of action of RJW100, a racemic agonist of the nuclear receptors liver receptor homolog-1 (LRH-1) and steroidogenic factor-1 (SF-1). LRH-1 and SF-1 modulators are highly sought as treatments for metabolic and neoplastic diseases, and RJW100 has one of the few scaffolds shown to activate them. However, enantiomer-specific effects on receptor activation are poorly understood. We show that the enantiomers have similar binding affinities, but RR-RJW100 stabilizes both receptors and is 46% more active than SS-RJW100 in LRH-1 luciferase reporter assays. We present an LRH-1 crystal structure that illuminates striking mechanistic differences: SS-RJW100 adopts multiple configurations in the pocket and fails to make an interaction critical for activation by RR-RJW100. In molecular dynamics simulations, SS-RJW100 attenuates intramolecular signalling important for coregulator recruitment, consistent with previous observations that it weakly recruits coregulators in vitro. These studies provide a rationale for pursuing enantiomerically pure RJW100 derivatives: they establish RR-RJW100 as the stronger LRH-1 agonist and identify a potential for optimizing the SS-RJW100 scaffold for antagonist design.

手性是药物开发中的一个重要考虑因素：它可以影响对预期靶标的识别、药代动力学和脱靶效应。在这里，我们研究手性如何影响 RJW100 的活性和作用机制，RJW100 是核受体肝受体同源物 1 (LRH-1) 和类固醇生成因子 1 (SF-1) 的外消旋激动剂。 LRH-1 和 SF-1 调节剂作为代谢和肿瘤疾病的治疗方法而备受追捧，而 RJW100 是少数能激活它们的支架之一。然而，对映体特异性对受体激活的影响尚不清楚。我们表明，对映体具有相似的结合亲和力，但 RR-RJW100 可以稳定两种受体，并且在 LRH-1 荧光素酶报告基因测定中比 SS-RJW100 活性高 46%。我们提出了一种 LRH-1 晶体结构，它阐明了显着的机械差异：SS-RJW100 在口袋中采用了多种配置，并且未能形成对 RR-RJW100 激活至关重要的相互作用。在分子动力学模拟中，SS-RJW100 减弱了对辅调节因子招募很重要的分子内信号传导，这与之前的观察结果一致，即它在体外弱招募辅调节因子。这些研究为追求对映体纯的 RJW100 衍生物提供了理论依据：它们将 RR-RJW100 确立为更强的 LRH-1 激动剂，并确定了优化 SS-RJW100 支架用于拮抗剂设计的潜力。