Cryptochrome 1 (CRY1) and CRY2 are core regulators of the circadian clock, and the development of isoform-selective modulators is important for the elucidation of their redundant and distinct functions. Here, we report the identification and functional characterization of a small-molecule modulator of the mammalian circadian clock that selectively controls CRY1. Cell-based circadian chemical screening identified a thienopyrimidine derivative KL201 that lengthened the period of circadian rhythms in cells and tissues. Functional assays revealed stabilization of CRY1 but not CRY2 by KL201. A structure-activity relationship study of KL201 derivatives in combination with X-ray crystallography of the CRY1-KL201 complex uncovered critical sites and interactions required for CRY1 regulation. KL201 bound to CRY1 in overlap with FBXL3, a subunit of ubiquitin ligase complex, and the effect of KL201 was blunted by knockdown of FBXL3. KL201 will facilitate isoform-selective regulation of CRY1 to accelerate chronobiology research and therapeutics against clock-related diseases.

隐铬1（CRY1）和CRY2是昼夜节律的核心调节器，异构形式选择性调节剂的开发对于阐明其冗余和独特功能很重要。在这里，我们报告选择性地控制CRY1的哺乳动物生物钟的小分子调节剂的鉴定和功能表征。基于细胞的昼夜节律化学筛选确定了噻吩并嘧啶衍生物KL201，可延长细胞和组织中的昼夜节律周期。功能分析显示KL201使CRY1稳定，但没有使CRY2稳定。KL201衍生物的结构-活性关系研究与CRY1-KL201复合物的X射线晶体学相结合，揭示了CRY1调控所需的关键位点和相互作用。绑定到CRY1的KL201与FBXL3重叠，FBXL3。KL201将促进CRY1的同工型选择性调节，以加速针对时钟相关疾病的年代生物学研究和疗法。