研究者詳細 - 廣田　毅

Structural differences in the FAD-binding pockets and lid loops of mammalian CRY1 and CRY2 for isoform-selective regulation. 査読有り国際誌

Miller S, Srivastava A, Nagai Y, Aikawa Y, Tama F, Hirota T

Proceedings of the National Academy of Sciences of the United States of America 118 巻 ( 26 ) 2021年6月

The circadian clock is a biological timekeeper that operates through transcription–translation feedback loops in mammals. Cryptochrome 1 (CRY1) and Cryptochrome 2 (CRY2) are highly conserved core clock components having redundant and distinct functions. We recently identified the CRY1- and CRY2-selective compounds KL101 and TH301, respectively, which provide useful tools for the exploration of isoform-selective CRY regulation. However, intrinsic differences in the compound-binding FAD (flavin adenine dinucleotide) pockets between CRY1 and CRY2 are not well understood, partly because of nonoptimal properties of previously reported apo form structures in this particular region constituted by almost identical sequences. Here, we show unliganded CRY1 and CRY2 crystal structures with well-defined electron densities that are largely free of crystal contacts at the FAD pocket and nearby lid loop. We revealed conformational isomerism in key residues. In particular, CRY1 W399 and corresponding CRY2 W417 in the FAD pocket had distinct conformations (“out” for CRY1 and “in” for CRY2) by interacting with the lid loop residues CRY1 Q407 and CRY2 F424, respectively, resulting in different overall lid loop structures. Molecular dynamics simulations supported that these conformations were energetically favorable to each isoform. Isoform-selective compounds KL101 and TH301 preferred intrinsic “out” and “in” conformations of the tryptophan residue in CRY1 and CRY2, respectively, while the nonselective compound KL001 fit to both conformations. Mutations of lid loop residues designed to perturb their isoform-specific interaction with the tryptophan resulted in reversed responses of CRY1 and CRY2 to KL101 and TH301. We propose that these intrinsic structural differences of CRY1 and CRY2 can be targeted for isoform-selective regulation.

DOI： 10.1073/pnas.2026191118

Reversible modulation of circadian time with chronophotopharmacology. 査読有り国際共著国際誌

Kolarski D, Miró-Vinyals C, Sugiyama A, Srivastava A, Ono D, Nagai Y, Iida M, Itami K, Tama F, Szymanski W, Hirota T, Feringa BL

Nature communications 12 巻 ( 1 ) 頁： 3164 - 3164 2021年5月

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Nature Communications

The circadian clock controls daily rhythms of physiological processes. The presence of the clock mechanism throughout the body is hampering its local regulation by small molecules. A photoresponsive clock modulator would enable precise and reversible regulation of circadian rhythms using light as a bio-orthogonal external stimulus. Here we show, through judicious molecular design and state-of-the-art photopharmacological tools, the development of a visible light-responsive inhibitor of casein kinase I (CKI) that controls the period and phase of cellular and tissue circadian rhythms in a reversible manner. The dark isomer of photoswitchable inhibitor 9 exhibits almost identical affinity towards the CKIα and CKIδ isoforms, while upon irradiation it becomes more selective towards CKIδ, revealing the higher importance of CKIδ in the period regulation. Our studies enable long-term regulation of CKI activity in cells for multiple days and show the reversible modulation of circadian rhythms with a several hour period and phase change through chronophotopharmacology.

DOI： 10.1038/s41467-021-23301-x

Photopharmacological Manipulation of Mammalian CRY1 for Regulation of the Circadian Clock. 査読有り国際共著国際誌

Kolarski D, Miller S, Oshima T, Nagai Y, Aoki Y, Kobauri P, Srivastava A, Sugiyama A, Amaike K, Sato A, Tama F, Szymanski W, Feringa BL, Itami K, Hirota T

Journal of the American Chemical Society 143 巻 ( 4 ) 頁： 2078 - 2087 2021年1月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of the American Chemical Society

CRY1 and CRY2 proteins are highly conserved components of the circadian clock that controls daily physiological rhythms. Disruption of CRY functions are related to many diseases, including circadian sleep phase disorder. Development of isoform-selective and spatiotemporally controllable tools will facilitate the understanding of shared and distinct functions of CRY1 and CRY2. Here, we developed CRY1-selective compounds that enable light-dependent manipulation of the circadian clock. From phenotypic chemical screening in human cells, we identified benzophenone derivatives that lengthened the circadian period. These compounds selectively interacted with the CRY1 photolyase homology region, resulting in activation of CRY1 but not CRY2. The benzophenone moiety rearranged a CRY1 region called the "lid loop"located outside of the compound-binding pocket and formed a unique interaction with Phe409 in the lid loop. Manipulation of this key interaction was achieved by rationally designed replacement of the benzophenone with a switchable azobenzene moiety whose cis-trans isomerization can be controlled by light. The metastable cis form exhibited sufficiently high half-life in aqueous solutions and structurally mimicked the benzophenone unit, enabling reversible period regulation over days by cellular irradiation with visible light. This study revealed an unprecedented role of the lid loop in CRY-compound interaction and paves the way for spatiotemporal regulation of CRY1 activity by photopharmacology for molecular understanding of CRY1-dependent functions in health and disease.

DOI： 10.1021/jacs.0c12280

TARGETING THE CIRCADIAN CLOCK WITH SMALL MOLECULE COMPOUNDS RESULTS IN ANTI-TUMOR EFFECTS SPECIFICALLY IN GLIOBLASTOMA STEM CELLS 国際共著

Chan, P; Wu, L; Hovsepyan, A; Mkhitaryan, S; Karapetyan, G; Shah, K; Wakimoto, H; Kamenecka, T; Solt, L; Cope, J; Moats, R; Hirota, T; Rich, J; Kay, S

NEURO-ONCOLOGY 25 巻 2023年11月

概日時計タンパク質CRY1とCRY2をアイソフォーム選択的に制御する化合物招待有り査読有り

廣田毅, 羽鳥恵

生化学 95 巻 ( 5 ) 頁： 604 - 608 2023年10月

Development of a novel PET ligand, [(11)C]GO289 targeting CK2 expressed in the brain. 査読有り国際誌

Ogata A, Yamada T, Hattori S, Ikenuma H, Abe J, Tada M, Ichise M, Suzuki M, Ito K, Kato T, Amaike K, Hirota T, Kakita A, Itami K, Kimura Y

Bioorganic & medicinal chemistry letters 90 巻頁： 129327 - 129327 2023年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Bioorganic and Medicinal Chemistry Letters

Positron emission tomography (PET) is a powerful imaging tool that enables early in vivo detection of Alzheimer's disease (AD). For this purpose, various PET ligands have been developed to image β-amyloid and tau protein aggregates characteristically found in the brain of AD patients. In this study, we initiated to develop another type of PET ligand that targets protein kinase CK2 (formerly termed as casein kinase II), because its expression level is known to be altered in postmortem AD brains. CK2 is a serine/threonine protein kinase, an important component of cellular signaling pathways that control cellular degeneration. In AD, the CK2 level in the brain is thought to be elevated by its involvement in both phosphorylation of proteins such as tau and neuroinflammation. Decreased CK2 activity and expression levels lead to β-amyloid accumulation. In addition, since CK2 also contributes to the phosphorylation of tau protein, the expression level and activity of CK2 is expected to undergo significant changes during the progression of AD pathology. Furthermore, CK2 could act as a potential target for modulating the inflammatory response in AD. Therefore, PET imaging targeting CK2 expressed in the brain could be a useful another imaging biomarker for AD. We synthesized and radiolabeled a CK2 inhibitor, [11C]GO289, in high yields from its precursor and [11C]methyl iodide under basic conditions. On autoradiography, [11C]GO289 specifically bound to CK2 in both rat and human brain sections. On baseline PET imaging, this ligand entered and rapidly washed out of the rat brain with its peak activity rather being small (SUV < 1.0). However, on blocking, there was no detectable CK2 specific binding signal. Thus, [11C]GO289 may be useful in vitro but not so in vivo in its current formulation. The lack of detectable specific binding signal in the latter may be due to a relatively high component of nonspecific binding signal in the overall rather weak PET signal, or it may also be related to the known fact that ATP can competitively binds to subunits of CK2, reducing its availability for this ligand. In the future, it will be necessary for PET imaging of CK2 to try out different non-ATP competitive formulations of CK2 inhibitor that can also provide significantly higher in vivo brain penetration.

DOI： 10.1016/j.bmcl.2023.129327

Analysis of the phosphoproteome of CK2α((-/-))/Δα' C2C12 myoblasts compared to the wild-type cells. 査読有り国際共著国際誌

Borgo C, Cesaro L, Hirota T, Kuwata K, D'Amore C, Ruppert T, Blatnik R, Salvi M, Pinna LA

Open biology 13 巻 ( 2 ) 頁： 220220 - 220220 2023年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Open Biology

CK2 is a Ser/Thr protein kinase composed of two catalytic (α/α′) subunits and a non-catalytic β-subunit dimer, whose activity is often abnormally high in cancer cells. The concept that CK2 may be dispensable for cell survival has been challenged by the finding that viable CK2α/α′ knock-out myoblast clones still express small amounts of an N-terminally deleted α′ subunit generated during the CRISPR/Cas9 procedure. Here we show that, although the overall CK2 activity of these CK2α (-/-) /Δα′ (KO) cells is less than 10% compared to wild-type (WT) cells, the number of phosphosites with the CK2 consensus is comparable to that of WT cells. A more in-depth analysis, however, reveals that the two phosphoproteomes are not superimposable according to a number of criteria, notably a functional analysis of the phosphoproteome found in the two types of cells, and variable sensitivity of the phosphosites to two structurally unrelated CK2 inhibitors. These data support the idea that a minimal CK2 activity, as in KO cells, is sufficient to perform basic housekeeping functions essential for cell survival, but not to accomplish several specialized tasks required upon cell differentiation and transformation. From this standpoint, a controlled downregulation of CK2 would represent a safe and valuable anti-cancer strategy.

DOI： 10.1098/rsob.220220

A methylbenzimidazole derivative regulates mammalian circadian rhythms by targeting Cryptochrome proteins. 招待有り査読有り国際誌

Yagi M, Miller S, Nagai Y, Inuki S, Sato A, Hirota T

F1000Research 11 巻頁： 1016 - 1016 2022年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：F1000Research

Background: Impairment of the circadian clock has been associated with numerous diseases, including sleep disorders and metabolic disease. Although small-molecules that modulate clock function may form the basis of drug discovery of clock-related diseases, only a few compounds that selectively target core clock proteins have been identified. Three scaffolds were previously discovered as small-molecule activators of the clock protein Cryptochrome (CRY), and they have been providing powerful tools to understand and control the circadian clock system. Identifying new scaffolds will expand the possibilities of drug discovery. Methods: A methylbenzimidazole derivative TH401 identified from cell-based circadian screens was characterized. Effects of TH401 on circadian rhythms were evaluated in cellular assays. Functional assays and X-ray crystallography were used to elucidate the effects of the compound on CRY1 and CRY2 isoforms. Results: TH401 lengthened the period of circadian rhythms and stabilized both CRY1 and CRY2. The compound repressed Per2 reporter activity, which was reduced by Cry1 or Cry2 knockout and abolished by Cry1/Cry2 double knockout, indicating the dependence on CRY isoforms. Thermal shift assays showed slightly higher interaction of TH401 with CRY2 over CRY1. The crystal structure of CRY1 in complex with TH401 revealed a conformational change of the gatekeeper W399, which is involved in isoform-selectivity determination. Conclusions: The present study identified a new small-molecule TH401 that targets both CRY isoforms. This compound has expanded the chemical diversity of CRY activators, and will ultimately aid in the development of therapeutics against circadian clock-related disorders.

DOI： 10.12688/f1000research.124658.1

概日時計の分子機構をケミカルバイオロジーで紐解く招待有り査読有り

松田智宏, 廣田毅

化学と生物 60 巻 ( 5 ) 頁： 212 - 214 2022年5月

Phosphorylation of RNA Polymerase II by CDKC;2 Maintains the Arabidopsis Circadian Clock Period. 査読有り国際共著

Uehara TN, Nonoyama T, Taki K, Kuwata K, Sato A, Fujimoto KJ, Hirota T, Matsuo H, Maeda AE, Ono A, Takahara TT, Tsutsui H, Suzuki T, Yanai T, Kay SA, Itami K, Kinoshita T, Yamaguchi J, Nakamichi N

Plant & cell physiology 63 巻 ( 4 ) 頁： 450 - 462 2022年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Plant and Cell Physiology

The circadian clock is an internal timekeeping system that governs about 24 h biological rhythms of a broad range of developmental and metabolic activities. The clocks in eukaryotes are thought to rely on lineage-specific transcriptional-translational feedback loops. However, the mechanisms underlying the basic transcriptional regulation events for clock function have not yet been fully explored. Here, through a combination of chemical biology and genetic approaches, we demonstrate that phosphorylation of RNA polymerase II by CYCLIN DEPENDENT KINASE C; 2 (CDKC;2) is required for maintaining the circadian period in Arabidopsis. Chemical screening identified BML-259, the inhibitor of mammalian CDK2/CDK5, as a compound lengthening the circadian period of Arabidopsis. Short-term BML-259 treatment resulted in decreased expression of most clock-associated genes. Development of a chemical probe followed by affinity proteomics revealed that BML-259 binds to CDKC;2. Loss-of-function mutations of cdkc;2 caused a long period phenotype. In vitro experiments demonstrated that the CDKC;2 immunocomplex phosphorylates the C-terminal domain of RNA polymerase II, and BML-259 inhibits this phosphorylation. Collectively, this study suggests that transcriptional activity maintained by CDKC;2 is required for proper period length, which is an essential feature of the circadian clock in Arabidopsis.

DOI： 10.1093/pcp/pcac011

Structural and Chemical Biology Approaches Reveal Isoform-Selective Mechanisms of Ligand Interactions in Mammalian Cryptochromes. 招待有り査読有り国際誌

Miller S, Hirota T

Frontiers in physiology 13 巻頁： 837280 - 837280 2022年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers in Physiology

Cryptochromes (CRYs) are core components of the circadian feedback loop in mammals, which regulates circadian rhythmicity in a variety of physiological processes including sleep–wake cycles and metabolism. Dysfunction of CRY1 and CRY2 isoforms has been associated with a host of diseases, such as sleep phase disorder and metabolic diseases. Accumulating evidence for distinct roles of CRY1 and CRY2 has highlighted the need for CRY isoform-selective regulation; however, highly conserved sequences in CRY ligand-binding sites have hindered the design of isoform-selective compounds. Chemical biology approaches have been identifying small-molecule modulators of CRY proteins, which act in isoform-non-selective and also isoform-selective manners. In this review, we describe advances in our understanding of CRY isoform selectivity by comparing X-ray crystal structures of mammalian CRY isoforms in apo form and in complexes with compounds. We discuss how intrinsic conformational differences in identical residues of CRY1 and CRY2 contribute to unique interactions with different compound moieties for isoform selectivity.

DOI： 10.3389/fphys.2022.837280

Cell-Based Phenotypic Screens to Discover Circadian Clock-Modulating Compounds. 招待有り査読有り国際誌

Hatori M, Hirota T

Methods in molecular biology (Clifton, N.J.) 2482 巻頁： 95 - 104 2022年

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Methods in Molecular Biology

There is increasing demand to control circadian clock functions in a conditional manner for deeper understanding of the circadian system as well as for potential treatment of clock-related diseases. Small-molecule compounds provide powerful tools to reveal novel functions of target proteins in the circadian clock mechanism, and can be great therapeutic candidates. Here we describe the detailed methods of measuring cellular circadian rhythms in a high-throughput manner for chemical screening to identify compounds that affect circadian rhythms by targeting clock-related proteins.

DOI： 10.1007/978-1-0716-2249-0_6

Preface 招待有り国際共著

Hirota T., Hatori M., Panda S.

Neuromethods 186 巻 2022年

Modulation of circadian clock by crude drug extracts used in Japanese Kampo medicine. 査読有り国際誌

Zhang M, Kobayashi K, Atsumi H, Katada Y, Nakane Y, Chen J, Nagano R, Kadofusa N, Nishiwaki-Ohkawa T, Kon N, Hirota T, Sato A, Makino T, Yoshimura T

Scientific reports 11 巻 ( 1 ) 頁： 21038 - 21038 2021年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Circadian rhythm is an approximately 24 h endogenous biological rhythm. Chronic disruption of the circadian clock leads to an increased risk of diabetes, cardiovascular disease, and cancer. Hence, it is important to develop circadian clock modulators. Natural organisms are a good source of several medicines currently in use. Crude drugs used in Japanese traditional Kampo medicine or folk medicines are an excellent source for drug discovery. Furthermore, identifying new functions for existing drugs, known as the drug repositioning approach, is a popular and powerful tool. In this study, we screened 137 crude drug extracts to act as circadian clock modulators in human U2OS cells stably expressing the clock reporter Bmal1-dLuc, and approximately 12% of these modulated the circadian rhythm. We further examined the effects of several crude drugs in Rat-1 fibroblasts stably expressing Per2-luc, explant culture of lung from Per2::Luciferase knockin mice, and zebrafish larvae in vivo. Notably, more than half of the major ingredients of these crude drugs were reported to target AKT and its relevant signaling pathways. As expected, analysis of the major ingredients targeting AKT signaling confirmed the circadian clock-modulating effects. Furthermore, activator and inhibitor of AKT, and triple knockdown of AKT isoforms by siRNA also modulated the circadian rhythm. This study, by employing the drug repositioning approach, shows that Kampo medicines are a useful source for the identification of underlying mechanisms of circadian clock modulators and could potentially be used in the treatment of circadian clock disruption.

DOI： 10.1038/s41598-021-00499-w

Effects of cryptochrome-modulating compounds on circadian behavioural rhythms in zebrafish. 招待有り査読有り国際誌

Iida M, Nakane Y, Yoshimura T, Hirota T

Journal of biochemistry 171 巻 ( 5 ) 頁： 501 - 507 2021年9月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biochemistry

The circadian clock controls daily rhythms of various physiological processes, and impairment of its function causes many diseases including sleep disorders. Chemical compounds that regulate clock function are expected to be applied for treatment of circadian clock-related diseases. We previously identified small-molecule compounds KL001, KL101 and TH301 that lengthen the period of cellular circadian clock by directly targeting clock proteins cryptochromes (CRYs) in mammals. KL001 targets both CRY1 and CRY2 isoforms, while KL101 and TH301 are isoform-selective compounds and require CRY C-terminal region for their effects. For further application of these compounds, the effects on locomotor activity rhythms at the organismal level need to be investigated. Here we used zebrafish larvae as an in vivo model system and found that KL001 lengthened the period of locomotor activity rhythms in a dose-dependent manner. In contrast, KL101 and TH301 showed no effect on the period. The amino acid sequences of CRY C-terminal regions are diverged in zebrafish and mammals, supporting the importance of this region for the effects of KL101 and TH301. This study demonstrated efficacy of CRY modulation for controlling circadian behavioural rhythms in organisms and suggested species-dependent differences in the effects of isoform-selective CRY-modulating compounds.

DOI： 10.1093/jb/mvab096

体内時計のケミカルバイオロジー研究と創薬の可能性招待有り

廣田毅, 松田智宏

MEDCHEM NEWS 31 巻 ( 2 ) 頁： 62 - 67 2021年5月

担当区分：筆頭著者,　責任著者記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：公益社団法人日本薬学会

概日時計は地球上の生物に普遍的なシステムであり、生体内の1日周期のリズムを支配する。概日時計を構成する時計タンパク質の発現、翻訳後修飾、分解のサイクルにより、多様な生理機能がリズミカルな制御を受けている。現代社会において、シフトワークや社会的な時差ぼけなどによる概日リズムの乱れが深刻化しており、睡眠障害だけでなく、がんや代謝性疾患などのさまざまな疾患につながることが報告されている。概日時計に関連する疾患の分子メカニズム解明や治療に向け、時計タンパク質を標的とする低分子化合物は有用なツールとなる。本稿では、これまでに同定されたユニークな時計調節化合物と、創薬の可能性について解説する。

DOI： 10.14894/medchem.31.2_62

CiNii Research

Comparing the efficacy and selectivity of Ck2 inhibitors. A phosphoproteomics approach. 査読有り国際共著国際誌

Borgo C, Cesaro L, Hirota T, Kuwata K, D'Amore C, Ruppert T, Blatnik R, Salvi M, Pinna LA

European journal of medicinal chemistry 214 巻頁： 113217 - 113217 2021年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：European Journal of Medicinal Chemistry

CK2 (an acronym derived from the misnomer “casein kinase 2”) denotes a ubiquitous, highly pleiotropic protein kinase which has been implicated in global human pathologies, with special reference to cancer. A large spectrum of fairly selective, cell permeable CK2 inhibitors are available, one of which, CX4945 is already in clinical trials for the treatment of neoplasia. Another recently developed CK2 inhibitor, GO289, displays in vitro potency and selectivity comparable to CX4945. Here the cellular efficiency of these two inhibitors has been evaluated by treating C2C12 myoblasts for 5 h with each of them at 4 μM concentration and running a quantitative phosphoproteomics analysis of phosphosites affected by the two compounds. A small but significant proportion of the quantified phosphosites is decreased by treatment with CX4945 and, even more with GO289. This figure substantially increases if a subset of quantified phosphosites conforming to the CK2 consensus (pS/pT-x-x-D/E/pS/pT) is considered. Also in this case GO289 is more effective than CX4945. By adopting stringent criteria two shortlists of 70 and 35 sites whose phosphorylation is decreased >50% by GO289 and CX4945, respectively, have been generated. All these phosphosites conform to the consensus of CK2 with just sporadic exceptions. Their WebLogos are indistinguishable from that of bona fide CK2 phosphosites and their Two-Sample Logos rule out any significant contribution of Pro-directed and basophilic protein kinases to their generation. To sum up, we can conclude that by treating C2C12 cells for 5 h with either CX4945 or GO289 off-target effects are negligible since almost all the phosphosites undergoing a substantial reduction are attributable to CK2, with a higher inhibitory efficacy displayed by GO289. CX4945 and GO289 provide highly selective tools to control the CK2-dependent phosphoproteome compared with previously developed CK2 inhibitors.

DOI： 10.1016/j.ejmech.2021.113217

Reductive stability evaluation of 6-azopurine photoswitches for the regulation of CKIα activity and circadian rhythms. 査読有り国際共著国際誌

Kolarski D, Sugiyama A, Rodat T, Schulte A, Peifer C, Itami K, Hirota T, Feringa BL, Szymanski W

Organic & biomolecular chemistry 19 巻 ( 10 ) 頁： 2312 - 2321 2021年

担当区分：責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Organic and Biomolecular Chemistry

Photopharmacology develops bioactive compounds whose pharmacological potency can be regulated by light. The concept relies on the introduction of molecular photoswitches, such as azobenzenes, into the structure of bioactive compounds, such as known enzyme inhibitors. Until now, the development of photocontrolled protein kinase inhibitors proved to be challenging for photopharmacology. Here, we describe a new class of heterocyclic azobenzenes based on the longdaysin scaffold, which were designed to photo-modulate the activity of casein kinase Iα (CKIα) in the context of photo-regulation of circadian rhythms. Evaluation of a set of photoswitchable longdaysin derivatives allowed for better insight into the relationship between substituents and thermal stability of thecis-isomer. Furthermore, our studies on the chemical stability of the azo group in this type of heterocyclic azobenzenes showed that they undergo a fast reduction to the corresponding hydrazines in the presence of different reducing agents. Finally, we attempted light-dependent modulation of CKIα activity together with the accompanying modulation of cellular circadian rhythms in which CKIα is directly involved. Detailed structure-activity relationship (SAR) analysis revealed a new potent reduced azopurine with a circadian period lengthening effect more pronounced than that of its parent molecule, longdaysin. Altogether, the results presented here highlight the challenges in the development of light-controlled kinase inhibitors for the photomodulation of circadian rhythms and reveal key stability issues for using the emerging class of heteroaryl azobenzenes in biological applications.

DOI： 10.1039/D1OB00014D