Pharmacological Interventions to Circadian Clocks and Their Molecular Bases

Journal Pre-proof Pharmacological interventions to circadian clocks and their molecular bases Simon Miller, Tsuyoshi Hirota PII:

S0022-2836(20)30019-X

DOI:

https://doi.org/10.1016/j.jmb.2020.01.003

Reference:

YJMBI 66384

To appear in:

Journal of Molecular Biology

Received Date: 29 November 2019 Revised Date:

30 December 2019

Accepted Date: 2 January 2020

Please cite this article as: S. Miller, T. Hirota, Pharmacological interventions to circadian clocks and their molecular bases, Journal of Molecular Biology, https://doi.org/10.1016/j.jmb.2020.01.003. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 The Author(s). Published by Elsevier Ltd.

Graphical abstract

Pharmacological interventions to circadian clocks and their molecular bases

Simon Miller and Tsuyoshi Hirota

Institute of Transformative Bio-Molecules, Nagoya University, Nagoya 464-8601, Japan. Correspondence to Tsuyoshi Hirota: Email [email protected]; Tel. +81-52-7476356

RESEARCH HIGHLIGHTS •

Clock proteins are regulated post-translationally by protein kinases.

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Small-molecule compounds targeting clock proteins have been identified through targetbased and phenotypic screening approaches.

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Clock-modulating compounds regulate activities of protein kinases and core clock proteins.

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X-ray crystallographic studies facilitate the understanding of mechanisms of action and derivatization of clock-modulating compounds.

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Clock-modulating compounds have therapeutic potential in the treatment of a host of diseases.

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ABSTRACT Daily physiological rhythms are regulated by the body’s internal timekeeper known as the circadian clock. Expression, post-translational modification, and degradation of clock proteins constituting the circadian clock are precisely controlled in a rhythmic manner. Perturbation of these processes by nature and nurture results in physiological dysfunction and diseases. Small-molecule modulators of clock or clock-related proteins can adjust clock functions, and thus represent a promising method of therapeutic treatment for a range of clock-related diseases. In this review, we will introduce the identification and development of small-molecule compounds that target clock proteins, as well as X-ray crystal structures of protein-compound complexes that facilitate the understanding of clock protein regulation and drug derivatization. Furthermore, we describe the effects of these compounds in a diseased setting and discuss the therapeutic potential of clock modulators.

Keywords Circadian clock; small-molecule compounds; protein kinases; clock proteins; clock-related diseases

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INTRODUCTION The circadian clock is a molecular timekeeping mechanism that regulates daily rhythms of sleep-wake behavior, hormone secretion, body temperature, and metabolism via transcription-translation negative feedback loops of clock genes at a cellular level [1,2]. External cues such as light and diet entrain the circadian clock to synchronize with environmental 24-hour periodicity. The suprachiasmatic nucleus (SCN) in the hypothalamus is the timekeeper of behavioral rhythms and therefore called the central clock. The SCN is located above the optic chiasm and receives signals from retinal ganglion cells, inducing neuronal and hormonal signalling to synchronize molecular clocks in almost all tissues and cells throughout the body, called peripheral clocks that control local rhythms in each tissue. Circadian rhythms in tissues and cells can be perturbed by changes to diurnal/nocturnal routines, such as irregular sleep patterns, night-shift work, intercontinental travel, and changes to the timing of dietary intake. Disruptions to the circadian rhythm have been implicated in many diseases including sleep disorders, cardiovascular disease, obesity, diabetes, and cancer by accelerating the onset of predisposed diseases and also exacerbating the severity of pre-existing diseases [3,4]. In order to understand how the circadian clock regulates cellular homeostasis, and how its dysfunction can lead to disease, it is important to modulate circadian clock functions by targeting clock-related proteins. Since many important genes are duplicated in mammals, pan-specific regulation of homologs or different isoforms can have a greater effect on clock functions, compared to single gene knockouts. Therefore, the development of pharmacological modulators of clock and clockrelated proteins is critical for dissecting the molecular mechanisms and enabling regulation of the clock in a clinical setting. Furthermore, the efficacy of many drugs can be influenced by the timing of their delivery [5], thus selective regulation of clock proteins combined with optimized dosing time could improve therapeutic outcomes. This review will focus on smallmolecule modulators of clock proteins, and provide insights into their mechanisms of regulation and therapeutic potential.

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CLOCK REGULATORS Circadian oscillation is driven by clock proteins. The transcription factors circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1) dimerize and bind to E-box elements to activate transcription of Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes [2]. PER1/2 and CRY1/2 proteins are regulated post-translationally: Phosphorylation of PER proteins by casein kinase I (CKI), CK2, and glycogen synthase kinase 3 (GSK-3) regulates both their degradation and subcellular localization [6]. CKIdependent phosphorylation of PER2 at residue Ser478 facilitates the recruitment of the Skp1-Cullin1-F-box (beta-transducin repeats-containing protein) E3 ubiquitin ligase complex SCFβ-TrCP, initiating proteasomal degradation [7,8]. Other F-box proteins FBXL3 and FBXL21 have distinct subcellular localization (the nucleus and cytosol), and bind to CRYs inducing proteasomal degradation and stabilization, respectively [9–13]. PER and CRY proteins associate with CKIδ and other proteins in the cytoplasm to form large complexes ~1 MDa. The complexes contain GAPVD1, a cytoplasmic trafficking factor, which is required for nuclear localization [14]. After translocation to the nucleus, PER/CRY/CKIδ complexes interact with CLOCK-BMAL1 and inhibit transcription of Per and Cry genes closing the negative feedback loop. PER/CRY/CKIδ complexes can also sequester CLOCK-BMAL1 away from thousands of other CLOCK-BMAL1 DNA-binding sites [15]. Furthermore, Bmal1 is regulated by the inhibitory nuclear hormone receptors REV-ERBα/β (Nr1d1 and Nr1d2), and the activatory retinoic acid receptor-related orphan receptors RORα/β/γ (Rora, Rorb, and Rorc) to form the secondary loop. REV-ERBs and RORs compete for binding to RORE consensus sequences, thereby downregulating and upregulating Bmal1 transcription, respectively [16,17]. Mutations in core clock genes have been associated with disruptions to circadian rhythms. Familial advanced sleep phase disorder (FASP) induces early sleep/wake times due to shortening of the circadian period [18]. Three mutations responsible for FASP have

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been identified: 1) A PER2 Ser662Gly missense mutation in the Ser/Thr cluster that is phosphorylated by CKI (called the FASP site) [19], which accelerates PER2 degradation [20]; 2) A missense mutation in the kinase domain of CKIδ (Thr44Ala amino acid substitution) [21], which favors the phosphorylation of Ser478 in PER2 over the FASP site, thus recruiting β-TrCP and enhancing PER2 degradation [22,23]; or 3) A CRY2 Ala260Thr missense mutation in the phosphate binding loop, which increases the binding affinity of FBXL3 to CRY2 with subsequent ubiquitination and proteasomal degradation [24]. In contrast, familial delayed sleep phase disorder (DSPD) results in a late phase of sleep/wake cycles due to lengthening of the circadian period, and is a common cause of insomnia [25]. A CRY1 splice site mutation, which lacks exon 11 (24 amino acid residues), was identified in DSPD families. The mutant CRY1 protein exhibits increased affinity for CLOCK-BMAL1, resulting in inhibition of CLOCK-BMAL1 target genes and a delay in the speed of the clock [25]. CKI is a Ser/Thr protein kinase with 7 identified isoforms, among which CKIδ/ε have been well-characterized in circadian clock regulation [6]. CKI is one of the few protein kinases that requires a phosphorylated site as a recognition module pS/pT-x-x-S/T [26]. Therefore, the first Ser residue of the human PER2 Ser/Thr cluster that is mutated in FASP (Ser662Gly) has been considered to be phosphorylated by an unknown priming kinase for the following phosphorylation by CKI. However, recent studies showed that CKIε and a splice variant of CKIδ called CKIδ2 initiate a priming phosphorylation reaction of Ser659 in mouse PER2 (corresponding to Ser662 in human PER2). Subsequent phosphorylation of Ser662, Ser665, Ser668, and Ser671 in mouse PER2 is catalyzed by the CKIδ1 splice variant [27]. The methylation state of CkIδ mRNA drives the alternative splicing of CKIδ1 (415 amino acid residues) and CKIδ2 (409 amino acids) [28]. The C-terminal region of CKIδ2 resembles CKIε, whereas the final 16 residues in CKIδ1 differ from those in CKIδ2, implicating the C-terminal region of CKI in the regulation of circadian timing. The human PER2 Ser662Gly FASP mutation inhibits the Ser/Thr cluster hyperphosphorylation and

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favors Ser480 (Ser478 in mouse PER2) phosphorylation by CKI, which recruits β-TrCP to initiate PER degradation with resulting period shortening [8]. CK2, formerly known as casein kinase 2, is a holoenzyme composed of two catalytic subunits, CK2α and/or CK2α’, and two regulatory subunits CK2β that form a tetramer [29]. The role of CK2 in mammalian clock regulation was identified by RNAi screening, where its downregulation lengthened the circadian period [30,31]. CK2 is known to phosphorylate hundreds of substrates, including the clock proteins BMAL1 and PER2. CK2-mediated phosphorylation of Ser90 in BMAL1 is required for nuclear localization [32]. CK2 also phosphorylates N-terminal residues in PER2, including Ser53, to fine-tune PER2 stability [30,33]. A more recent phosphoproteomic analysis identified other potential phosphorylation sites in BMAL1, PER2, CRY1, and CLOCK [34]. The Ser/Thr protein kinase GSK-3 has two isoforms GSK-3α and GSK-3β, and has been associated with the phosphorylation of more than 100 substrates, including the clock proteins PER2, CRY2, CLOCK, BMAL1, and REV-ERBα [35–39]. GSK-3 is inhibited by phosphorylation resulting from the activation of pathways such as insulin and Wnt signalling [40]. GSK-3β recognises substrates that have been phosphorylated by priming kinases, with the consensus sequence S/T-x-x-x-pS/pT [41,42]. For example, GSK-3β-mediated phosphorylation of mouse CRY2 at Ser553 requires a priming phosphorylation at Ser557 by dual-specificity tyrosine-phosphorylated and regulated kinase 1A (DYRK1A) [43]. Sequential Ser557/Ser553 phosphorylation enhances proteasomal degradation of CRY2 [36]. Together, these clock proteins and clock-regulating kinases provide potential targets for pharmacological regulation of circadian clock function.

DEVELOPMENT OF SMALL-MOLECULE CLOCK MODULATORS Approaches to develop clock modulators There are two major approaches to identify small molecules targeting clock proteins: targetbased and phenotypic screening approaches. A target-based approach first narrows down

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proteins of interest, followed by the development of an in vitro assay to measure activity of recombinant proteins. Candidate compounds can be identified by high-throughput screening and derivatization of “hit” compounds. This approach facilitates drug development for known clock proteins with measurable in vitro activities. Enzymes such as protein kinases can be easily assessed, but the development of in vitro assays for non-enzymatic proteins is more challenging. In contrast, a phenotypic screening approach uses reporter cells. For example, clock gene reporters Bmal1-dLuc and Per2-dLuc have been used to measure cellular circadian rhythms. This assay evaluates period, phase, and amplitude of the rhythms, but not overall reporter intensity, enabling a significant reduction of false-positives that is a general problem for high-throughput screening [44]. Modulators of clock function can be identified by the screening of well-characterized compound libraries [45–47], as well as uncharacterized compounds [34,48–51]. Repositioning of existing drugs provides an efficient way to identify in vivo effective clock modulators [52]. When uncharacterized compounds are used, target proteins of hit compounds need to be identified. For example, an affinity probe will be developed through derivatization of hit compounds, and interacting proteins will be affinity-purified and then identified by mass spectrometry analysis. This approach is important for the identification of novel modulators for known clock proteins and also has the potential to discover new clock proteins.

Protein Kinases CKI A high-throughput phenotypic screen of clock modulators with a following structural-activity relationship (SAR) study of a hit compound containing a purine scaffold identified the periodlengthening compound longdaysin (Fig. 1). A subsequent affinity-based target deconvolution approach revealed that longdaysin inhibits both CKIα and CKIδ. The effect of longdaysin towards CKIα led to the elucidation of its role in regulating PER1 stability and circadian period. The period-lengthening effect of longdaysin was observed in a variety of mammalian

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cells and tissues, and also in live zebrafish [48]. Docking studies predicted that the purine moiety and the amine of longdaysin together form two hydrogen bonds to the backbone of the hinge residue Leu93 in CKIα and Leu85 in CK1δ [53,54]. An SAR study of longsdaysin derivatives identified a more potent inhibitor NCC007 (Fig. 1) that has enhanced effects against CK1α and CK1δ with more favorable binding modes in docking studies compared to longdaysin. NCC007 infusion into mouse brain induces period lengthening of behavioral rhythms [53]. A recent study utilizing phenotypic proteomic profiling [55] supported the selectivity of longdaysin against CKI, compared to other period-lengthening kinase inhibitors such as SP600125 [46]. Photopharmacology approaches provide a way to tightly control the timing of drug binding to specific targets via the photoactivation of compounds [56]. Longdaysin was modified to attach photoremovable protecting groups (PPGs): 2-nitrobenzyl (DK325) and 6nitroveratryloxycarbonyl (DK359) (Fig. 1) to the secondary amine, which inhibited hydrogen bonding to the hinge region of CKIα and CKIδ in docking studies [54]. The caged compounds are inactive against CKIα and CKIδ, and upon UV (365 nm) or violet (400 nm) light irradiation, they produce the active longdaysin for period lengthening in cells, tissues, and zebrafish in vivo, enabling temporal control of the clock period. CKIδ and CKIε are closely related homologs. CKIδ has a much larger effect on circadian period than CKIε [57], whereas CKIε reduces the capacity of phase shifts in response to environmental stimuli such as light [58]. Thus, CKIδ and CKIε appear to regulate the clock in an isoform-specific manner, highlighting the importance of isotype-selective inhibition. CKIδ and CKIε have very high sequence conservation, making selective compound design difficult. High-throughput screening of small-molecules in enzymatic inhibition assays of CKIε followed by modification of a hit compound identified PF670462 (Fig. 1), a potent dual inhibitor of CKIδ (IC50 = 13 nM) and CKIε (IC50 = 7.7 nM), displaying >30-fold selectivity compared to 42 other kinases. PF670462 causes phase delays of behavioral rhythms when administered to rats [59]. Directed chemistry efforts identified a

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compound called PF4800567 (Fig. 1) that exhibits >22-fold selectivity for CK1ε over CK1δ [60]. Treatment with PF670462 and PF4800567 showed that CKIδ inhibition causes period lengthening in the SCN and peripheral tissues as well as mouse behavior, whereas CKIε inhibition has little effect [61]. In contrast, these compounds similarly inhibit ovarian cancer cell growth, indicating that CKIε is the main driver of disease progression, as shown by CKIε knockdown [62]. Crystal structure determination of CKI isoforms with compounds could be important in the pursuit of isotype-selective inhibition, by potentially revealing induced structural changes such as side chain conformations, nearby loop interactions, or changes in bindingpocket volume. Structure-guided functional group exchange can be important not only for interactions with variant residues, but also invariant residues that might have reduced conformational freedom by restraints imposed by differences in adjacent secondary structure elements or loops. The X-ray crystal structure of CKIδ-PF670462 complex (PDB ID: 3UYT and 3UZP) revealed that the selectivity of PF670462 for CKIδ/ε over many other kinases is due to a small residue (Pro66), which is generally larger in other kinases. The reduced steric bulk of Pro66 enables Met82 to avoid a steric clash with the compound via a conformational change that picks up an interaction with Pro66, which is inhibited by bulkier amino acids at this position in other kinases [63]. The crystal structures of PF4800567 in complex with CK1δ (PDB ID: 4HNF) and CK1ε (PDB ID: 4HNI), as well as the apo form of CK1ε (PDB ID: 4HOK), revealed that a variant residue in the αC helix (Ile55 in CK1δ and Phe55 in CK1ε) enabled Phe55 to insert into a hydrophobic pocket normally occupied by Phe150 (in the DFG motif), facilitating the rotation of Phe150, “DFG” out conformation, which is then free to interact with the compound [64]. Recent studies reported selective CKIδ inhibitors. Among a series of pteridine derivatives, meta-substituents at the C6 phenyl ring were found to have profound effects on inhibitory activity and selectivity. Epiblastin A (Fig. 1), a meta-chloro substituent is a selective CKIδ inhibitor (IC50 = 500 nM) 10-fold and 18-fold selective over CK1ε and CK1α,

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respectively. In contrast, Epiblastin C (Fig. 1), a meta-1-naphthyl substituent is a selective CK1ε inhibitor (IC50 = 1 µM) ~36-fold selective over both CK1α and CKIδ [65]. The cinnamic acid side chain of the previously reported nanomolar dual inhibitor of p38α and CKIδ, diarylisoxazole 8 [66], was modified by attaching a chiral pyrrolidine scaffold. Pictet-Spengler cyclized compounds 31a and 31b derived by chance have ~5-fold higher binding affinity to CK1δ than CK1ε [67]. These compounds together represent attractive targets for utilization in circadian studies.

CK2 Administration of a CK2 inhibitor DMAT to lung explants of Per2::Luc knock-in mice lengthens the circadian period [30]. Although DMAT (Fig. 1) is more selective than tetrabromobenzotriole (TBB) (Fig. 1) [68], re-evaluation of CK2 inhibitors with a larger panel of 78 kinases revealed that DMAT also strongly inhibits PIM1/2/3, PKD1, HIPK2, and DYRK1a [69]. CX-4945 (Silmitasertib) (Fig. 1) is more potent and specific, and the first small-molecule inhibitor of CK2 to enter human clinical trials for cancer therapy [70]. CX4945 has high oral bioavailability, a long half-life, low toxicity and high potency with an IC50 value of 1 nM [71]. CX-4945 lengthens circadian period in Bmal1-dLuc U2OS cells [34]. The crystal structure of CK2α/CX-4945 (PDB IDs: 3NGA and 3PE1) revealed a canonical hydrogen bond to the hinge region, and its tight affinity was apparent from multiple hydrogen bonds, together with extensive hydrophobic and stacking interactions (Fig. 2A) [72,73]. However, CX-4945 has still been found to inhibit DYRK, HIPK, and PIM kinases [73] as well as CLKs [74]. High-throughput phenotypic screening of clock modulators and target deconvolution identified a new CK2-selective inhibitor GO289 (Fig. 1) that strongly lengthens circadian period [34]. GO289 inhibits CK2 with high potency (IC50 = 7 nM) and >1000-fold selectivity over DYRK, HIPK, PIM, and CLK. The crystal structure of the CK2α-GO289 complex (PDB ID: 6A1C) provided molecular insights into the selectivity of the binding mode. Most protein

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kinase inhibitors make canonical interactions with the hinge region in an ATP-competitive manner; however, GO289 does not interact directly with the conserved hinge region of CK2α, and instead forms a hydrogen bond via a bromine atom in its bromoguaiacol group to a water molecule that is coordinated by the hinge residues Glu114 and Val116 (Fig. 2B). Two hydrogen bonds to Lys68, mediated by hydroxyl and methoxy groups on the bromoguaiacol moiety, and a cation-π interaction, between His160 and a phenyl group, revealed a planar conformation and an efficient binding mode in the ATP-binding site. Hydrophobic interactions with Val66 and Met163 are also formed. The GO289-interacting residues Val66, His160, and Met163 are not conserved in other kinases (Ala, Glu, and Leu, respectively), underlying the selectivity of the compound [34]. Dysfunctional CK2 is often observed in cancer [29]. GO289 was further shown to strongly inhibit growth of human renal cell carcinoma cell lines and mouse MLL-AF9 acute myeloid leukaemia cells in a cell typedependent manner [34]. Therefore, selective CK2 inhibitors such as GO289 provide a promising candidate for drug development for clock-mediated cancer therapy.

GSK-3 Lithium is a well-known inhibitor of GSK-3, which enhances the inhibitory phosphorylation of GSK-3 by other kinases such as protein kinase B (PKB), and has long been a treatment for mood disorders such as bipolar disease and depression [42]. In contrast to period lengthening by lithium, phenotypic screening of 1,280 well-characterized compounds, LOPAC, revealed that synthetic GSK-3/CDK dual inhibitors cause period shortening in U2OS cells harboring a Bmal1-dluc reporter. More selective GSK-3 inhibitors (such as CHIR99021 and 1-azakenpaullone) also shorten the period, suggesting off-target effects of lithium for circadian regulation [45]. GSK-3 downregulates glycogen synthesis by phosphorylating and inhibiting the activity of glycogen synthase, thus GSK-3 inhibition represents a target for the treatment of glucose intolerance and type 2 diabetes [75]. Since the IC50 of lithium against GSK-3 is high

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(~2 mM) and lithium also inhibits key kinases involved in insulin signalling, including phosphoinositide 3-kinases (PI3Ks), phosphoinositide-dependent kinase 1 (PDK1), and PKB [76], more selective inhibitors were required for potential clinical applications. Screening of combinatorial libraries of substituted dihydropyrimidines that inhibit GSK-3 activity, followed by derivatization identified CHIR99021 (Fig. 1) as a potent inhibitor of GSK-3α/β (IC50 = 5-10 nM) [77]. CHIR99021 displays >10,000-fold selectivity to GSK-3 than PI3K, PDK1, and PKB [77,78]. Oral administration of CHIR99021 to insulin-resistant rodents increases glycogen synthase activity, enhances insulin sensitivity, and reduces hyperglycaemia [79]. Crystal structures of GSK-3β/CHIR99021 (PDB ID: 5HLN and 6B8J) showed ATP-competitive binding to the hinge residue Val135, interactions with hydrophobic regions I and II, and interactions with the ribose region and DFG motif in the P-loop [80]. GSK-3β has a rare variant Pro136 residue that induces an inward conformation of the Val135 carbonyl (towards the ATP-binding site), enabling hydrogen bonding of the compound to Val135, and could account for the moderately high selectivity of CHIR99021 for GSK-3β [79]. However, CHIR99021 inhibits 18 other kinases from a broad panel assay. A GSK-3β kinase assay screen followed by an SAR analysis and structure-guided modification identified the highly selective compounds BRD1652 (Fig. 1) and BRD0209, which have shown good pharmacokinetic properties and have therapeutic potential in mood disorders [80]. Considering the higher selectivity of BRD1652 and its analogs, compared to CHIR99021, it would be interesting to observe their efficacy on circadian functions.

Clock Proteins CRY A new period lengthening compound KL001 (Fig. 1) was identified from high-throughput circadian phenotypic screens, and affinity-based target deconvolution identified CRY proteins as the target. KL001, the first-in-class CRY modulator, competes with FAD for binding to the FAD pocket of CRY, and KL001 treatment resulted in the inhibition of

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ubiquitin-dependent degradation of CRY1 and CRY2 [51]. Consistent with the inhibitory role of CRYs in fasting hormone-induced gluconeogenesis through interaction with the Gsα subunit of heterotrimeric G protein [81] and the glucocorticoid receptor [82], KL001 inhibits glucagon-induced gluconeogenesis in primary hepatocytes [51]. Furthermore, the compound reduces proliferation of patient-derived glioblastoma stem cells (GSCs) [83]. Orally available derivatives of KL001 (compound 41 and compound 50 (Fig. 1)) reduce blood glucose levels in diabetic model mice [84,85], and extend the survival of GSC transplanted mice [83]. These results show that CRY proteins provide an attractive target to regulate the circadian function as potential therapeutics against clock-related diseases. KL001 is based on a carbazole scaffold with a methanesulfonamide and a furan. The crystal structure of CRY2 in complex with KL001 (PDB ID: 4MLP) revealed how KL001 and FAD bind to the same region of the FAD pocket: the carbazole moiety of KL001 superposed onto the isoalloxazine moiety of FAD (Fig. 3). The methanesulfonamide of KL001 overlaps with the FBXL3 C-terminal region at the FAD pocket, which is required for recognition and degradation of CRY [86,87]. Together, KL001 inhibits CRY degradation through competition with FBXL3 for CRY-binding. SAR studies showed that the carbazole group is essential for the activity of KL001 [88,89]. Furthermore, the substitution of the methanesulfonamide and furan moiety (KL001) for an acetamide and chloro-cyano-phenyl group (KL044) (Fig. 1) increased potency tenfold [88]. Furan substituents produced by C−H activation reactions identified a new class of CRY modulators that display period-shortening properties (GO044, GO200 (Fig. 1) and GO211) [89]. Screening of 1,000 compounds using E box-luciferase reporter cells identified a derivative of 2-ethoxypropanoic acid designated as compound 15 (KS15) (Fig. 1) that increases reporter activity. Pulldown assays using biotinylated compound 15 against overexpressed clock proteins revealed its interactions with CRY1 and CRY2. The compound blocks CRY-dependent inhibition of CLOCK-BMAL1 and reduces the amplitude of Per2dLuc reporter rhythms in cultured fibroblasts [90,91]. KS15 also has anti-proliferative and

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pro-apoptotic properties. KS15 treatment of breast cancer MCF-7 cells increases the expression of apoptotic genes and cell-cycle regulators such as p53 and Bax, which correlates to reduced proliferation and cell viability [92]. In contrast, KS15 treatment does not reduce the viability of non-cancerous MCF-10A cells. A subsequent SAR analysis of KS15 identified critical sites required for CRY inhibition, but the molecular mechanisms of CRY interaction remain elusive [91]. Because KL001 and KS15 target both CRY1 and CRY2, identification of isoform-selective compounds will be important for the elucidation of redundant and distinct functions of CRY isoforms.

REV-ERB REV-ERB is a nuclear hormone receptor whose ligand is heme, and the first hemecompetitive agonist, GSK4112 (Fig. 1), was identified by the measurement of REV-ERB interactions with one of its corepressors called nuclear receptor corepressor-1 (NCoR) in a biochemical assay. GSK4112 treatment of rat-1 fibroblasts expressing a Per2-Luc reporter as well as lung fibroblasts from PER2::Luc knock-in mice causes phase-dependent phase shifts [93]. It inhibits expression of Bmal1 and gluconeogenic genes in the HepG2 liver cell line and reduces glucose output in mouse primary hepatocytes [94]. GSK4112-derived REVERB agonists SR9009 (Fig. 1) and SR9011 regulate metabolic pathways such as lipogenesis, cholesterol/bile synthesis, glucose production, and lipid oxidation, then lower plasma levels of triglycerides, total cholesterol, non-esterified fatty acids, and glucose in mice [95]. SR9009 and SR9011 can also reduce anxiety and induce wakefulness, with implications in the treatment of sleep disorders [96]. SR9009 and SR9011 further exhibit cytotoxic properties to a range of cancers, including brain, leukaemia, breast, colon, and melanoma, and in vivo glioblastoma growth was inhibited after treatment with these compounds in mice [97]. SR9009/SR9011 potentially have off-target effects, as shown by a mouse model with a conditional deletion of REV-ERBα and REV-ERBβ [98]. The development of an SR9009/SR9011 derivative with extensive modifications to the tertiary amine scaffold produced a more potent agonist called SR10067 (Fig. 1) that displayed sleep

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and mood regulation in mice, with improved pharmacokinetic properties [96]. Another GSK4112 derivative, GSK2945 (Fig. 1) displays a long half-life and good oral bioavailability (23%), whereas compounds 10, 16, and 23 have short half-lives, amenable to acute time-ofday injection treatment. A major advantage of these compounds is the increased selectivity (>1,000-fold) for REV-ERB over liver X receptor alpha (LXRα). They inhibit Bmal1 and interleukin 6 (IL-6) expression in human THP-1 cells in response to lipopolysaccharide stimulation [99], with implications in nuclear factor kappa B (NF-κB)-mediated regulation of circadian rhythm and clock genes, and an association with inflammatory diseases [100,101]. A derivative of GSK4112, called SR8278 (Fig. 1), was found to be a REV-ERB antagonist that enhances luciferase reporter expression of the REV-ERB target genes Bmal1, G6Pase, and phosphoenolpyruvate carboxykinase (PEPCK) [102]. SR8278 has poor pharmacokinetic properties, but its use as an investigative tool has shown that REV-ERB antagonism inhibits glucose-stimulated insulin release in MIN-6 cells [103], and administration to mice affects mood and behavior [104]. Fluorine nuclear magnetic resonance-based screening for recombinant REV-ERBβ identified a new class of REV-ERB antagonist called ARN5187 (Fig. 1). This compound increases Bmal1 and PEPCK gene expression and is cytotoxic to a range of cancerous cells via dual inhibition of REV-ERB and autophagy [105].

ROR Despite similarities in the DNA-binding domains (DBDs) of REV-ERB and ROR, their ligandbinding domains (LBDs) interact with different molecules: ROR binds cholesterol, cholesterol sulphate, and oxysterols, whereas REV-ERB binds heme. Furthermore, ROR contains a specific helix 12 that recruits co-activators [106]. By using cell-based nuclear receptor profiling, T0901317 (Fig. 1) a benzenesulfonamide agonist of liver X receptors (LXRs) [107] was found to act also as an inverse agonist of RORα/γ [108]. SR1001 (Fig. 1), a derivative of T0901317, is an inverse agonist selective for RORα/γ. SR1001 inhibits the binding of RORγ

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to SRC2, a coactivator that binds to helix 12 in the LBD of RORγ [109]. RORα/γ have been shown to enhance T helper 17 (TH17) cell differentiation and IL-17 expression in vitro and in vivo [110]. Consistently, SR1001 treatment of murine splenocytes inhibits TH17 differentiation and IL-17 production, and administration to differentiated murine and human TH17 cells inhibits cytokine expression. In a multiple sclerosis mouse model, this compound reduces cytokine expression, including IL-17, and delays disease onset and progression [109]. Two SR1001-derived compounds, SR2211 and SR1555 (Fig. 1), are RORγ-selective inverse agonists [111,112]. In collagen-induced arthritis model mice, SR2211 administration reduces inflammatory T-cell function and inflammatory cytokine production, resulting in reduced joint inflammation [113]. Like T0901317 and SR2211, SR1555 suppresses TH17 differentiation and IL-17 production, but unlike other RORγ-targeting inverse agonists, SR1555 also upregulates T regulatory (TReg) cell production, which could be beneficial in retaining immune function while inhibiting excessive pro-inflammatory effects of TH17 signalling [112]. It will be interesting to observe the effects of additional chemical modifications in the development of compounds that can differentially regulate T cell and cytokine homeostasis. Substitution of the benzenesulfonamide (T0901317) for a thiophenesulfonamide (SR3335) (Fig. 1) led to the identification of the RORα-specific inverse agonist. SR3335 suppresses transcription of the ROR target genes G6Pase and PEPCK in HepG2 cells. In correlation with these observations, administration of SR3335 to diet-induced obese mice suppresses gluconeogenesis and improves glucose homeostasis with significantly lower blood glucose levels [114]. To the best of our knowledge, the effects of ROR inverse agonists on circadian clock functions have not been reported. A derivative of T0901317, called SR1078 (Fig. 1), was found to be an agonist of ROR that activates target gene expression, such as G6Pase and fibroblast growth factor 21, in HepG2 cells and in mouse liver [115]. SR1078 also inhibits HepG2 and Hep3B hepatoma cell growth both in vitro and in vivo [116]. Reduced RORα expression has been observed in many individuals with autism, and the administration of SR1078 to BTBR autism mouse

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models resulted in increased RORα activity and reduced repetitive behavior [117]. Nobiletin (Fig. 1) is a naturally occurring polymethoxylated flavonoid found predominantly in tangerines and oranges. A high-throughput chemical screen using Clock∆19/+ Per2::Luc mouse fibroblasts, which display circadian rhythms with weak amplitude, identified nobiletin as an amplitude enhancer. RORα/γ were found to be the direct targets of nobiletin in a competitive radio-ligand binding assay for RORs. Administration of nobiletin to diabetic or diet-induced obese mice results in improved glucose and lipid homeostasis, increased energy expenditure, and a reduction in white adipose tissue in WT mice, but not Clock∆19/∆19 mice, indicative of Clock-dependent regulation [47]. Furthermore, nobiletin promotes healthy aging against metabolic challenge by activating mitochondrial respiratory chain genes to improve mitochondrial respiration [118]. Nobiletin and its metabolites have become drug candidates due to their natural occurrence, non-toxicity and their implication in protective effects against a host of diseases including cancer, cardiovascular disease, diabetes, neurodegeneration, and inflammatory diseases, as reviewed in [119,120]. It is interesting that nobiletin (a RORα/γ agonist) and SR3335 (a RORα inverse agonist) both improve glucose homeostasis despite their stimulation and inhibition of ROR-mediated gene expression, respectively. Another naturally occurring compound, neoruscogenin (Fig. 1), was identified as a RORα agonist via high-throughput cell-free screening of plant extract fractions followed by extract deconvolution. Neoruscogenin increases the interaction of RORα with TIF2 (also called NCOA2, a coactivator important for hepatic glucose regulation). It also activates RORα-mediated gene expression of Bmal1 and the metabolic genes Cyp7b1, G6Pase, Lipin2, and AngptI4, with potential implications in atherosclerosis, lipid and glucose homeostasis [121]. For REV-ERB and ROR, crystal structures have been reported only for the natural ligands heme (PDB ID: 3CQV) and cholesterol (PDB ID: 1N83), respectively [122,123]. Elucidation of structures in complex with synthetic compounds will provide insights into the development of more potent and selective compounds.

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CONCLUDING REMARKS Pharmacological regulation of the clock has the potential to treat a host of diseases by controlling the clock to restore periodicity and homeostatic balance in diseased cells or tissues. The discovery of small molecule modulators for CRY, REV-ERB, and ROR have made the circadian clock druggable. However, those for other clock proteins BMAL1, CLOCK, and PER are still missing. Some of these proteins are also known to interact with cofactors, such as NAD and/or heme. Therefore, future studies will enable the discovery of new compounds to regulate all clock proteins. Structure-guided drug design can provide valuable insights into how to develop new modulators with improved specificity, enabling more detailed dissection of intracellular regulatory mechanisms. In mammals, there are two or three homologs for each clock protein. Considering that existing CRY modulators, REVERB modulators, and ROR agonists exhibit no isoform selectivity, the development of compounds with greater isoform selectivity will facilitate the elucidation of redundant and distinct functions of each isoform. In addition to compound selectivity, potency is also important for the prevention of deleterious side effects, by potentially reducing toxicity in a therapeutic

setting.

The

development

of

new

small-molecules

with

improved

pharmacokinetic properties represents a promising opportunity to both understand and treat clock-related diseases.

Acknowledgements We thank Daniel Bader for assistance in the preparation of Fig. 1. T.H. is receiving supports from JSPS Grants 18H02402 and 18K19171; Takeda Science Foundation; Uehara Memorial Foundation; and Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care.

FIGURE LEGENDS

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Fig. 1. Representative small-molecule compounds targeting clock and clock-related proteins. PDB accession codes for reported crystal structures of protein-compound complexes are also shown.

Fig. 2. Crystal structures of CK2α in complex with small-molecule inhibitors bound to the ATP-binding pocket. CK2 structures are shown as white cartoons with the hinge region colored dark gray. Compound-interacting residues of CK2α are shown as sticks. Nitrogen, oxygen, sulphur, chlorine and bromine atoms are colored blue, red, gold, green, and brown, respectively. Hydrogen bonds, halogen bonds, and cation-π interactions are indicated by red, yellow, and blue dashed lines, respectively. Water molecules are shown as red spheres. (A) CK2α in complex with CX-4945 (light pink) (PDB: 3PE1). The pyridine nitrogen in CX-4945 makes a canonical hydrogen bond to the backbone of V116 in the hinge. The carboxylate group of the compound forms a hydrogen bond with K68, and is stabilized by the coordination of two water molecules (W1 and W2). (B) CK2α in complex with GO289 (cyan) (PDB: 6A1C). The bromoguaiacol group of GO289 binds indirectly via a water molecule (W2) to the hinge backbone carbonyl of E114, and V116 amide in a non-canonical interaction. Hydroxyl and methoxy groups form hydrogen bonds to K68, and a network of hydrophobic and stacking interactions with V66, H160, and M163 support the high selectivity of the compound for CK2.

Fig. 3. Crystal structures of CRY2 in complex with KL001 (PDB: 4MLP) and FAD (4I6G). Key residues interacting with the compounds are represented as sticks. For clarity, only CRY2 residues from 4MLP are displayed (white sticks). Nitrogen, oxygen, sulphur, and phosphorus atoms are colored blue, red, gold, and orange, respectively. Superposition of FAD (magenta) onto KL1001 (cyan) shows how the isoalloxazine moiety of FAD and the carbazole of KL001 occupy the same location in the pocket, with a slight positional shift.

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Figure 1 CKI

Longdaysin (CKIα/δ inhibitor) IC50: CKIα 5.6 µM, CKIδ 8.8 µM

NCC007 (CKIα/δ inhibitor) IC50: CKIα 1.8 µM, CKIδ 3.6 µM

DK359 (caged longdaysin)

Ref [48]

Ref [53]

Ref [54]

CKI

PF4800567 (CK1ε inhibitor) IC50: CKIδ 711 nM, CKIε 32 nM PDB: 4HNF; 4HNI Ref [60]

PF670462 (CKIδ/ε inhibitor) IC50: CKIδ 13 nM, CKIε 7.7 nM PDB: 3UYT; 3UZP Ref [59]

CK2

Epiblastin A (CKIδ inhibitor) IC50: CKIα 8.9 µM, CKIδ 500 nM, CKIε 4.7 µM Ref [65]

CK2

Epiblastin C (CKIε inhibitor) IC50: CKIα 36 µM, CKIδ 36 µM, CKIε 1.0 µM Ref [65]

DMAT (CK2 inhibitor) IC50: CK2 130 nM

TBB (CK2 inhibitor) IC50: CK2 150 nM

Ref [68]

Ref [68]

CX-4945 (CK2 inhibitor) IC50: CK2 1 nM PDB: 3NGA; 3PE1 Ref [70]

GSK-3

GO289 (CK2 inhibitor) IC50: CK2 7 nM PDB: 6A1C Ref [34]

CHIR99021 (GSK-3α/β inhibitor) IC50: GSK-3α 10 nM, GSK-3β 6.7 nM PDB: 5HLN; 6B8J Ref [77]

BRD1652 (GSK-3α inhibitor) IC50: GSK-3α 0.4 nM, GSK-3β 4 nM Ref [80]

Figure 1 CRY

KL001 (CRY activator) PDB: 4MLP Ref [51]

CRY

Compound 50 (CRY activator)

KL044 (CRY activator)

GO200 (CRY modulator)

Ref [85]

Ref [88]

Ref [89]

REV-ERB

KS15 (CRY inhibitor) Ref [90]

SR9009 (REV-ERB agonist) Ref [95]

GSK4112 (REV-ERB agonist) Ref [93]

SR10067 (REV-ERB agonist) Ref [96]

REV-ERB Cl

S Cl O

N S NO2

GSK2945 (REV-ERB agonist) Ref [99]

N

O O O

SR8278 (REV-ERB antagonist) Ref [102]

ARN5187 (REV-ERB antagonist) Ref [105]

Figure 1 ROR

T0901317 (LXR agonist, RORα/γ inverse agonist) Ref [107-108]

SR1001 (RORα/γ inverse agonist)

SR2211 (RORγ inverse agonist)

Ref [109]

Ref [111]

ROR

SR1555 (RORγ inverse agonist) Ref [112]

SR3335 (RORα inverse agonist) Ref [114]

ROR

Nobiletin (RORα/γ agonist) Ref [47]

Neuroruscogenin (RORα agonist) Ref [121]

SR1078 (ROR agonist) Ref [115]

FIGURE 2 A

B

CK2/CX-4945

V66

W1

CK2/GO289

K68 V66

W2 E114

E114 Hinge

K68 W1

W2

Hinge V116

V116 M163

H160

M163

H160

FIGURE 3 CRY2/KL001 CRY2/FAD W310 W417 Q307 F314 S414 H373

H377

A380

R376

F399 L403