International Journal of Molecular Sciences

Review Cdc-Like (CLKs): Biology, Chemical Probes, and Therapeutic Potential

1, 1,2, 1,2, Paula Martín Moyano †,Václav Nˇemec † and Kamil Paruch * 1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic; [email protected] (P.M.M.); [email protected] (V.N.) 2 International Clinical Research Center, Center for Biomolecular and Cellular Engineering, St. Anne’s University Hospital in Brno, 602 00 Brno, Czech Republic * Correspondence: [email protected] These authors contributed equally to this work. †  Received: 31 August 2020; Accepted: 9 October 2020; Published: 13 October 2020 

Abstract: kinases represent a very pharmacologically attractive class of targets; however, some members of the family still remain rather unexplored. The biology and therapeutic potential of cdc-like kinases (CLKs) have been explored mainly over the last decade and the first CLK inhibitor, compound SM08502, entered clinical trials only recently. This review summarizes the biological roles and therapeutic potential of CLKs and their heretofore published small-molecule inhibitors, with a focus on the compounds’ potential to be utilized as quality chemical biology probes.

Keywords: CLK; cdc-like ; inhibitor; chemical biology; tool compound; chemical probe; T3; SGC-CLK-1; MU1210; SM08502

1. Introduction Protein kinases play pivotal roles in nearly every aspect of cellular function [1]. Chemically, they transfer the γ-phosphate group from nucleoside triphosphate (typically ATP or GTP) to the hydroxyl group of serine, threonine, or tyrosine residues of the physiological substrate. Once the substrate is phosphorylated, it is released from the kinase and the remaining nucleoside diphosphate is recycled. The transfer of electrostatically charged phosphate group to an side chain of the target protein is often accompanied with conformational changes of the protein; thus, can act as a molecular “on-off” switch that strongly increases or decreases the enzymatic activity and/or the stability of the phosphorylated protein [2,3]. It has been estimated that about up to 30% of all human can be phosphorylated by kinases [4,5]. Phosphorylation of proteins can lead to activation or deactivation of signal-transduction pathways that are important in various biological processes such as metabolism, transcription, cell cycle progression, cell movement, , and differentiation. Over the past three decades, the family has emerged as one of the most important classes of drug targets because mutations and dysregulations of protein kinases are frequently involved in the initiation and progression of cancers and other diseases [4,6–9]. Some kinases (e.g., BCR-ABL, BTK, AKT, VEGFR, etc.) have been established as attractive targets for pharmacological inhibition for a relatively long time [10,11] while others, with the increasing knowledge of the cellular mechanisms, have come to the forefront only recently [11–14]. The kinase-driven cellular processes include also alternative and constitutive splicing, which are pivotal for expression [15,16]. Alternative splicing gives rise to higher complexity and diversity of transcriptome via the production of alternatively spliced mRNAs, which is consequently mirrored by the higher diversity of the proteome [16,17]. Aberrant splicing is responsible for the inaccurate

Int. J. Mol. Sci. 2020, 21, 7549; doi:10.3390/ijms21207549 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 7549 2 of 31 translation of genetic information into the structure of proteins, which has often a negative impact on variousInt. J. Mol. cellular Sci. 2020, 21 processes, x FOR PEER [18 REVIEW–21]. The splicing process is, to a large extent, regulated 2by of protein 31 kinasesby the that higher phosphorylate diversity of splicingthe proteome factors—central [16,17]. Aberrant components splicing is ofresponsible the spliceosome, for the inaccurate for example, serinetranslation/arginine-rich of genetic proteins information (SR proteins)—and into the structure less of directly proteins, also which by otherhas often kinases a negative involved impact in on cellular signalingvarious pathways cellular processes [19,22]. [18–21]. The splicing process is, to a large extent, regulated by protein kinasesSR proteins that phosphorylate are splicing factors splicing that factors—centra interact simultaneouslyl components withof the RNA spliceosome, (via the RNAfor example, recognition motifserine/arginine-rich RRM) and other proteins proteins (SR (via proteins)—and the RS domain less directly containing also arginineby other /serinekinases residueinvolved repeats). in Theycellular are employed signaling inpathways cellular [19,22]. processes such us constitutive and alternative pre-mRNA splicing, mRNA nuclearSR proteins export, are splicing nonsense-mediated factors that interact decay, simultaneously and mRNA with translation RNA (via [ 23the– 26RNA]. As recognition summarized motif RRM) and other proteins (via the RS domain containing arginine/serine residue repeats). They in recent reviews [16,19,22,27], SR-protein phosphorylation can be executed by SR protein kinases are employed in cellular processes such us constitutive and alternative pre-mRNA splicing, mRNA (SRPKs),nuclear topoisomerase export, nonsense-mediated 1 (TOP1), proteindecay, and kinase mRNA B translation (PKB/AKT), [23–26]. NIMA-related As summarized kinase2 in recent (NEK2), PRP4reviews kinase (PRP4K),[16,19,22,27], dual-specificity SR-protein phosphorylation tyrosine phosphorylation-regulated can be executed by SR kinaseprotein 1Akinases (DYRK1A) (SRPKs), [28 ,29], cAMP-dependenttopoisomerase protein1 (TOP1), kinase protein (PKA) kinase [30 B,31 (PKB/AKT], and by), the NIMA-related family of cdc-like kinase2 kinases (NEK2), (CLKs), PRP4 kinase which are the subject(PRP4K), of dual-specificity this review. tyrosine phosphorylation-regulated kinase 1A (DYRK1A) [28,29], cAMP- dependentOther kinases protein involved kinase (PKA) in [30,31], the regulation and by the family of splicing of cdc-like include kinases MAPKs (CLKs), which (acting are the via the RAS-RAF-MEK-ERKsubject of this review. signaling cascade), tyrosine kinases (BRK, FYN, SRC, c-ABL), Fas-activated serine/threonineOther kinases kinase involved (FAST) in [32 the], andregulation possibly of splicing also AURKA include [MAPKs33]. Those (acting kinases via the can RAS-RAF- influence the splicingMEK-ERK via phosphorylation signaling cascade), of splicing tyrosine factors kinases such as SAM68(BRK, FYN, (e.g., RAS-RAF-MEK-ERKSRC, c-ABL), Fas-activated cascade) [34] serine/threonine kinase (FAST) [32], and possibly also AURKA [33]. Those kinases can influence the or more indirectly via phosphorylation of their upstream effectors. splicing via phosphorylation of splicing factors such as SAM68 (e.g., RAS-RAF-MEK-ERK cascade) [34]Despite or more significant indirectly scientific via phosphor progressylation in of the their area upstream over the effectors. past decade, the biology of CLKs has not been completelyDespite significant elucidated scientific and, progress particularly, in the thearearoles over ofthe individual past decade, CLK the biology isoforms of remainCLKs has largely unexplored.not been completely These questions elucidated could and, be, particularly, at least partially, the roles addressedof individual by CLK the isoforms utilization remain of thelargely recently discoveredunexplored. highly These selective questions tool could compounds be, at least (quality partially, probes) addressed that areby the summarized utilization of in the this recently review. discovered highly selective tool compounds (quality probes) that are summarized in this review. 2. Classification and Structure of CLKs 2. Classification and Structure of CLKs Protein kinases form a large family of more than 500 proteins with common characteristics that can be clusteredProtein kinases into phylogenetic form a large groupsfamily of (Figure more than1)[35 500]. proteins with common characteristics that can be clustered into phylogenetic groups (Figure 1) [35].

Figure 1. Kinome tree with CLKs marked by circles: CLK1 (blue), CLK2 (red), CLK3 (green), CLK4 Figure 1. Kinome tree with CLKs marked by circles: CLK1 (blue), CLK2 (red), CLK3 (green), CLK4 (yellow). Source: KinMap software. Illustration reproduced courtesy of Technology, (yellow). Source: KinMap software. Illustration reproduced courtesy of Cell Signaling Technology, Inc. Inc. (www.cellsignal.com) (www.cellsignal.com).

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The group CMGC (named after initials of some key members) includes several subgroups: Cyclic Dependent Kinases (CDKs), -Activated Protein Kinase Kinasess (MAPKs), Glycogen Synthase Kinases (GSKs), and CLKs, firstfirst reported in 1991 [[36,37].36,37]. Overall, CMGCCMGC isis a a large large and and well-conserved well-conserved group group of kinasesof kinases that that have have pivotal pivotal roles roles in cell in cycle cell cycleregulation regulation and intracellular and intracellular signal . transduction. The CLK family consists of CLK1, CLK2, CLK3,CLK3, and CLK4, which are brieflybriefly summarized in Table1 1,, withwith highlightedhighlighted didifferencesfferences inin thethe ATP-bindingATP-binding region.region. The full amino acid sequences are given in Figure2 2..

Table 1.1. Human CLKs: amino acid residues count, mass in kDa, and am aminoino acids present in the ATP phosphates’ binding region (the residues specificspecific forfor individualindividual isoformisoform areare inin bold)bold) [[38].38].

CLKCLK Form Form AminoAmino Acid Acid Amino Acids in the ATP Phosphates’ kDakDa Amino Acids in the ATP Phosphates’ Binding Region (Uniprot(UniprotRef.) Ref.) ResiduesResidues Binding Region CLK1CLK1 167–175167–175 484 484 57 57 (P49759)(P49759) (LGEG(LGEGAAFGFGKV)KV) CLK2CLK2 169–177169–177 499 499 60 60 (P49760)(P49760) (LGEG(LGEGTFGTFGRV)RV) CLK3 310–318 CLK3 638 73 310–318 (P49761) 638 73 (LGEGTFGKV) (P49761) (LGEGTFGKV) CLK4 165–173 CLK4 481 57 165–173 (Q9HAZ1) 481 57 (LGEGAFGKV) (Q9HAZ1) (LGEGAFGKV)

CLKs amino acid residues 10 20 30 40 50 60 70 80 CLK1 MRHSKRTYCP DWDDKDWDYG KWRSSSSHKR RKRSHSSAQE NKRCKYNHSK MCDSHYLESR SINEKDYHSR RYIDEYRNDY CLK2 MPHPRRYHSS ERGSRGSYRE HYRSRKHKRR RSRSWSSSSD RTRRRRREDS YHVRSRSSYD DRSSDRRVYD RRYCGSYRRN CLK3 MPVLSARRRE LADHAGSGRR SGPSPTARSG PHLSALRAQP ARAAHLSGRG TYVRRDTAGG GPGQARPLGP PGTSLLGRGA CLK4 MRHSKRTHCP DWDSRESWGH ESYRGSHKRK RRSHSSTQEN RHCKPHHQFK ESDCHYLEAR SLNERDYRDR RYVDEYRNDY 90 100 110 120 130 140 150 160 CLK1 TQGCEPGHRQ RDHESRYQNH SSKSSGRSGR SSYKSKHRIH HSTSHRRSHG KSHRRKRTRS VEDDEEGHLI CQSGDVLSAR CLK2 DYSRDRGDAY YDTDYRHSYE YQRENSSYRS QRSSRRKHRR RRRRSRTFSR SSSQHSSRRA KSVEDDAEGH LIYHVGDWLQ CLK3 RRSGEGWCPG AFESGARAAR PPSRVEPRLA TAASREGAGL PRAEVAAGSG RGARSGEWGL AAAGAWETMH HCKRYRSPEP CLK4 CEGYVPRHYH RDIESGYRIH CSKSSVRSRR SSPKRKRNRH CSSHQSRSKS HRRKRSRSIE DDEEGHLICQ SGDVLRARYE 170 180 190 200 210 220 230 240 CLK1 YEIVDTLGEG AFGKVVECID HKAGGRHVAV KIVKNVDRYC EAARSEIQVL EHLNTTDPNS TFRCVQMLEW FEHHGHICIV CLK2 ERYEIVSTLG EGTFGRVVQC VDHRRGGARV ALKIIKNVEK YKEAARLEIN VLEKINEKDP DNKNLCVQMF DWFDYHGHMC CLK3 DPYLSYRWKR RRSYSREHEG RLRYPSRREP PPRRSRSRSH DRLPYQRRYR ERRDSDTYRC EERSPSFGED YYGPSRSRHR CLK4 IVDTLGEGAF GKVVECIDHG MDGMHVAVKI VKNVGRYREA ARSEIQVLEH LNSTDPNSVF RCVQMLEWFD HHGHVCIVFE 250 260 270 280 290 300 310 320 CLK1 FELLGLSTYD FIKENGFLPF RLDHIRKMAY QICKSVNFLH SNKLTHTDLK PENILFVQSD YTEAYNPKIK RDERTLINPD CLK2 ISFELLGLST FDFLKDNNYL PYPIHQVRHM AFQLCQAVKF LHDNKLTHTD LKPENILFVN SDYELTYNLE KKRDERSVKS CLK3 RRSRERGPYR TRKHAHHCHK RRTRSCSSAS SRSQQSSKRS SRSVEDDKEG HLVCRIGDWL QERYEIVGNL GEGTFGKVVE CLK4 LLGLSTYDFI KENSFLPFQI DHIRQMAYQI CQSINFLHHN KLTHTDLKPE NILFVKSDYV VKYNSKMKRD ERTLKNTDIK 330 340 350 360 370 380 390 400 CLK1 IKVVDFGSAT YDDEHHSTLV STRHYRAPEV ILALGWSQPC DVWSIGCILI EYYLGFTVFP THDSKEHLAM MERILGPLPK CLK2 TAVRVVDFGS ATFDHEHHST IVSTRHYRAP EVILELGWSQ PCDVWSIGCI IFEYYVGFTL FQTHDNREHL AMMERILGPI CLK3 CLDHARGKSQ VALKIIRNVG KYREAARLEI NVLKKIKEKD KENKFLCVLM SDWFNFHGHM CIAFELLGKN TFEFLKENNF CLK4 VVDFGSATYD DEHHSTLVST RHYRAPEVIL ALGWSQPCDV WSIGCILIEY YLGFTVFQTH DSKEHLAMME RILGPIPQHM 410 420 430 440 450 460 470 480 CLK1 HMIQKTRKRK YFHHDRLDWD EHSSAGRYVS RRCKPLKEFM LSQDVEHERL FDLIQKMLEY DPAKRITLRE ALKHPFFDLL CLK2 PSRMIRKTRK QKYFYRGRLD WDENTSAGRY VRENCKPLRR YLTSEAEEHH QLFDLIESML EYEPAKRLTL GEALQHPFF CLK3 QPYPLPHVRH MAYQLCHALR FLHENQLTHT DLKPENILFV NSEFETLYNE HKSCEEKSVK NTSIRVADFG SATFDHEHHT CLK4 IQKTRKRKYF HHNQLDWDEH SSAGRYVRRR CKPLKEFMLC HDEEHEKLFD LVRRMLEYDP TQRITLDEAL QHPFFDLLKK 490 500 510 520 530 540 550 560 CLK1 KKSI CLK2 RLRAEPPNKL WDSSRDISR CLK3 TIVATRHYRP PEVILELGWA QPCDVWSIGC ILFEYYRGFT LFQTHENREH LVMMEKILGP IPSHMIHRTR KQKYFYKGGL CLK4 K 570 580 590 600 610 620 630 640 CLK1 CLK2 CLK3 VWDENSSDGR YVKENCKPLK SYMLQDSLEH VQLFDLMRRM LEFDPAQRIT LAEALLHPFF AGLTPEERSF HTSRNPSR CLK4

Figure 2. Amino acid sequences of CLK1-4 with color-coded insertions: MAPK-like insertion (red), FigureLAMMER 2. Amino motif (yellow),acid sequencesβ-hairpin: of CLK1-4β-9 (green), with andcolor-codedβ-10 (blue) insertions: [38]. MAPK-like insertion (red), LAMMER motif (yellow), β-hairpin: β-9 (green), and β-10 (blue) [38].

The structures of CLKs are quite typical and consist of the N- and C-lobes connected by the “hinge” region of the protein backbone including the ATP , which is conserved as in other

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The structures of CLKs are quite typical and consist of the N- and C-lobes connected by the Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 31 “hinge” region of the protein backbone including the ATP binding site, which is conserved as in other kinases [39–41]. [39–41]. As As for for other other kinases, kinases, the the binding binding of of th thee purine purine part part of of ATP ATP is very likely likely provided by hydrogen bonding to the NH (donor) and CO (acceptor) motifs in the protein backbone (Figure3). hydrogenInt. J. Mol. bonding Sci. 2020 ,to 21 , thex FOR NH PEER (donor) REVIEW and CO (acceptor) motifs in the protein backbone (Figure 4 of 31 3). Additional CO is available as an acceptor site [[42].42]. kinases [39–41]. As for other kinases, the binding of the purine part of ATP is very likely provided by hydrogen bonding to the NH (donor) and CO (acceptor) motifs in the protein backbone (Figure 3). Additional CO is available as an acceptor site [42].

Figure 3. OverlapOverlap of of the the crystal crystal structures structures of CLK2 (p (pink,ink, PDB: 3NR9) [43] [43] and and PKA PKA with with bound bound ATP ATP (blue,Figure PDB: 1ATP)3. Overlap [[44].44 ].of Keythe crystal amino structures acid residues of CLK2 in the(pink, ATP PDB: binding 3NR9) region[43]region and areare PKA highlightedhighlighted with bound byby ATP boxes.boxes. (blue, PDB: 1ATP) [44]. Key amino acid residues in the ATP binding region are highlighted by boxes. The Glu Glu residue residue in in the the back back region region of the of theATP-bi ATP-bindingnding site forms site forms an ion an pair ion with pair Lys, with which Lys, whichfacilitates facilitates Thethe Glutransfer the residue transfer of thein the γ of-phosphate back the γ region-phosphate from of the ATP fromATP-bi [40]. ATPnding [40 site]. forms an ion pair with Lys, which Thefacilitates structuresstructures the transfer of of CLKs CLKs of containthe contain γ-phosphate catalytic catalytic from domains ATPdomains [40]. with βwith-strands β-strands and α-helixes and α-helixes distributed distributed between The structures of CLKs contain catalytic domains with β-strands and α-helixes distributed betweenthe N- and the C-regions. N- and TheC-regions. N-lobe The contains N-lobe three containsβ-strands three followed β-strands by α followed-helix and by additional α-helix twoand between the N- and C-regions. The N-lobe contains three β-strands followed by α-helix and additionalβ-strands (Figuretwo β-strands3)[39,45 (Figure]. 3) [39,45]. additional two β-strands (Figure 3) [39,45]. The tertiary structures of all CLKs are quite similar (Figure 4), nevertheless the structure of CLK3 The tertiaryThe tertiary structures structures of allof all CLKs CLKs are are quite quite similarsimilar (Figure (Figure 4),4), nevertheless nevertheless the the structure structure of CLK3 of CLK3 containscontains some some additional additional motifs motifs (Figure (Figure2 2),), 2), asas as describeddescribed described below belowbelow [39,45,46]. [ 39[39,45,46].,45,46].

Figure 4. Overlay of X-ray crystal structures: CLK1 (yellow, ligand:12h, PDB: 6I5H) [47], CLK2 (green, Figure 4. Overlay of X-ray crystal structures: CLK1 (yellow, ligand:12h, PDB: 6I5H) [47], CLK2 (green, PDB:PDB: 3NR9) 3NR9) [43], [43], CLK3 CLK3 (blue, (blue, PDB: PDB: 6YTW 6YTW [48 [48],], CLK4 CLK4 (pink,(pink, ligand: ligand: CX CX4945,4945, PDB: PDB: 6FYV) 6FYV) [45,49]. [45, 49]. Figure 4. Overlay of X-ray crystal structures: CLK1 (yellow, ligand:12h, PDB: 6I5H) [47], CLK2 (green, PDB: CLK33NR9) contains [43], CLK3 a small (blue, insert PDB: between 6YTW [48], stands CLK4 β6 (pink, and β ligand:9 in the CX kinase4945, core PDB: that 6FYV) interacts [45,49]. with a hydrophobic pocket near the hinge region that connects the N- and C-lobes [50]. The C-lobe of CLK3

CLK3 contains a small insert between stands β6 and β9 in the kinase core that interacts with a hydrophobic pocket near the hinge region that connects the N- and C-lobes [50]. The C-lobe of CLK3

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Int. J. Mol. Sci.CLK3 2020 contains, 21, x FOR a smallPEER REVIEW insert between stands β6 and β9 in the kinase core that interacts with a5 of 31 hydrophobic pocket near the hinge region that connects the N- and C-lobes [50]. The C-lobe of CLK3 is is formedformed by by three three conserved regions: regions: the the LAMMER LAMMER motif, motif, mitogen-activated mitogen-activated insertion, insertion, the MAPK-like the MAPK- like insertion containing containing an anα8 α (residues8 (residues 419–427), 419–427), and βand-hairpin β-hairpin insertion insertion (Figures (Figures2 and5)[ 245 and,50]. 5) [45,50].

FigureFigure 5. CLK3 5. CLK3 crystal crystal structure. structure. The The grey grey circle circle (A) representsrepresents the the most most common common binding binding pocket pocket for for small-moleculesmall-molecule inhibitors. inhibitors. PDB: PDB: 2EU9 2EU9 [51]. [51].

There are two “extra” elements that are not present in CLK1: the first one consists of inserted There are two “extra” elements that are not present in CLK1: the first one consists of inserted residues in the hairpin (residues 136–145) folding above the N-terminal domain, the second one is a residues in the hairpin (residues 136–145) folding above the N-terminal domain, the second one is a supplementary helical segment at the C terminus (residues 476–481) [39]. supplementaryAlso, CLK3 helical possesses segment one at Lys248the C terminus residue in (residues the entrance 476–481) of the [39]. pocket (Figure5), whichAlso, modifiesCLK3 possesses the electrostatic one Lys248 surface chargeresidue distribution in the entrance therein—this of the might active be site unfavorable pocket for(Figure the 5), whichentry modifies of some the CLK electrostatic inhibitors [45 surface,46]. charge distribution therein—this might be unfavorable for the entryGiven of some the CLK structural inhibitors features [45,46]. of CLK3 described above, it is perhaps not surprising that various small-moleculeGiven the structural inhibitors features frequently of CLK3 exhibit described different above, affinities it towardis perhaps CLK3 not compared surprising to the that other various small-moleculeCLKs (see the inhibitors chapter “Small-molecule frequently exhibit CLK inhibitors”different affinities below). toward CLK3 compared to the other CLKs (see the chapter “Small-molecule CLK inhibitors” below). 3. Biology of CLKs

3. BiologyCLKs of CLKs are evolutionary conserved dual-specificity kinases that are able to phosphorylate serine, threonine, and tyrosine residues [36,52]. CLKs catalyze the phosphorylation of SR proteins, serine,CLKs andare evolutionary arginine-rich splicing conserved factors dual-specificity 1-12 (SRSF1-12), kinases which regulatethat are theable spliceosome to phosphorylate molecular serine, threonine,machinery and [ 53tyrosine–55]. SR residues proteins seem[36,52]. to haveCLKs very cata distinctlyze the functions phosphorylation based on their of phosphorylationSR proteins, serine, and statearginine-rich [56], as reported, splicing for factors example, 1-12 for (SRSF1-1 SRSF10 (SRp38)2), which [57 ,58regulate]. Upon the hyper-phosphorylation, spliceosome molecular machinerySR proteins [53–55]. bind SR to pre-mRNAproteins seem and helpto have to stabilize very distinct interactions functi betweenons based spliceosome on their phosphorylation components, statewhich [56], resultsas reported, in spliceosome for example, assembly for [ 27SRSF10,59–61]. (SRp38) Conversely, [57,58]. de-phosphorylation Upon hyper-phosphorylation, of SR proteins by SR proteinsphosphatases bind to ispre-mR requiredNA in theand second help stepto stabilize for the pre-mRNA interactio splicingns between [59–61 spliceosome] and subsequently components, for whichthe results export in of splicedspliceosome mRNA assembly to the cytoplasm [27,59–61]. [26,62 Co]. Vianversely, this mechanism, de-phosphorylati CLKs regulateon of pre-mRNASR proteins by splicing and consequently also the translation of genetic information into protein structure [63,64]. phosphatases is required in the second step for the pre-mRNA splicing [59–61] and subsequently for All CLK isoforms have been found in most tissues and cell types (e.g., prostate, leukocytes, the export of spliced mRNA to the cytoplasm [26,62]. Via this mechanism, CLKs regulate pre-mRNA testes, muscle, brain, liver, lung, kidney, thyroid), but their expression levels differ in some tissues. splicing and consequently also the translation of genetic information into protein structure [63,64]. All CLK isoforms have been found in most tissues and cell types (e.g., prostate, leukocytes, testes, muscle, brain, liver, lung, kidney, thyroid), but their expression levels differ in some tissues. For instance, CLK1 expression level in the testes was significantly lower than those of CLK2-4 [53,65– 67]. All CLK isoforms are mostly localized in the nucleus; however, in the testes, CLK3 was found to be localized mostly in stress granules in the cytoplasm [16,52,68]. In contrast, SRPKs were found mostly in the cytoplasm and only a minor fraction was observed in the nucleus [69,70].

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For instance, CLK1 expression level in the testes was significantly lower than those of CLK2-4 [53,65–67]. All CLK isoforms are mostly localized in the nucleus; however, in the testes, CLK3 was found to be localized mostly in stress granules in the cytoplasm [16,52,68]. In contrast, SRPKs were found mostly in the cytoplasm and only a minor fraction was observed in the nucleus [69,70]. Interestingly, CLK1/4 activity is highly responsive to physiological temperature changes, which is caused by reversible temperature-dependent rearrangements in the kinase activation segment, reflected in temperature-dependent SR protein phosphorylation, splicing, and [71]. Specifically, it has been shown that lower body temperature causes activation of CLK1/4, which leads to increased SR protein phosphorylation. The mRNAs of all CLK isoforms are alternatively spliced. The spliced variants form heterodimers with full-length CLKs, showing that alternative splicing mediated by CLKs is auto-regulated [69]. CLKs are mostly localized in the nucleus, but their localization has been described also in the cytoplasm (e.g., in stress granules) [16,52,68], although less abundantly than SRPKs [69,70]. CLKs are capable of auto-phosphorylation on Tyr residues, while their substrates are phosphorylated on Ser/Thr residues [53]. In contrast to SRPKs, which phosphorylate only Arg-Ser dipeptides, CLKs can phosphorylate both Arg-Ser and Ser-Pro dipeptides—common motifs in all SR proteins [50]. It has been reported that CLK1/2/4 isoforms phosphorylate Aurora B–S331 in the midbody during late cytokinesis, which leads to Aurora B activation and consequently to the regulation of abscission checkpoint [72]. Via regulation of splicing, CLKs (indirectly) affect a variety of biological processes [73–75]. For instance, it has been described that CLKs modulate alternative splicing of key Wnt-related and consequently the Wnt signaling pathway [76]. CLKs regulate also the splicing of the TAU protein which plays an essential role in the stabilization of microtubules and thereby impacts processes such as cell division and neuronal activity [77–79]. The CLK family consists of four isoforms (CLK1-4); however, the knowledge about their individual biological roles is still rather limited.

3.1. CLK1 CLK1 (alternatively referred to as STY) represents one of the first kinases with dual specificity described in the literature and also the most explored CLK isoform up today [36]. CLK1 has been found to regulate alternative splicing of its own gene clk1 via exon 4 skipping and intron 4 retention [80]. Thus, CLK1 forms an auto-regulatory loop where the catalytically active CLK1 triggers the expression of truncated isoforms CLK1T1 and CLKT2 [80]. In contrast, extrinsic stress factors or CLK1 inhibition promote the expression of full-length CLK1 [80]. CLK1 possesses a diffuse nuclear localization sequence (NLS) in the N terminus that is responsible for the formation of oligomeric CLK1, which is likely unable to pass through nuclear pores [81]. On the other hand, NLS strongly interacts with its substrate SRSF1 (SR protein), which is very likely important for the nuclear import of CLK1 via a “piggyback” mechanism, where CLK1 is transported inside the nucleus together with the TRN-SR2/SRSF1 complex (Figure6). This mechanism has been supported by various experiments, for example, disruption of SRSF1 protein transport by TRN-SR2 knockdown or mutation of the SRSF1 NLS impaired CLK1 nuclear localization [68]. Int. J. Mol. Sci. 2020, 21, 7549 7 of 31 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 7 of 31

FigureFigure 6. 6.Schematic Schematic representation representation of the phosphorylation of the phosphorylation mechanism involving mechanism CLK1 [39involving,49,55,68 ,82CLK1–85]. [39,49,55,68,82–85]. Not only nuclear import but also nuclear function has been described most thoroughly for the CLK1Not isoform, only nuclear namely itsimport regulation but also mechanism nuclear functi of theon SR has protein been SRSF1described [55, 68most,85, 86thoroughly]. In this process, for the CLK1CLK1 andisoform, SRPK1 namely work its co-operatively regulation mechanism as a complex of the [ 55SR]. protein The complex SRSF1 [55,68,85,86]. containing CLK1 In this in process, active formCLK1 first and recruits SRPK1 hypo-phosphorylatedwork co-operatively as SRSF1 a complex from nuclear[55]. The speckles complex (also containing termed interchromatinCLK1 in active granuleform first clusters), recruits whichhypo-phosphorylated act as a pool of SRSF1 SR proteins from nuclear in the nucleus,speckles whereby(also termed a ternary interchromatin complex CLK1-SRPK1-SRSF1granule clusters), which is formed act as a (Figurepool of 6SR). protei The ternaryns in the complex nucleus, executeswhereby fulla ternary phosphorylation complex CLK1- of SRSF1SRPK1-SRSF1 and subsequently is formed (Figure releases 6). itThe (Figure ternary6)[ comp55,84lex,85 executes]. Alternatively, full phosphorylation CLK1 itself of can SRSF1 form and a complexsubsequently with releases SRSF1 and it (Figure catalyze 6) full[55,84,85]. phosphorylation Alternatively, first. CLK1 Subsequently, itself can SRPK1form a cancomplex engage with to createSRSF1 the and ternary catalyze complex full phosphorylation CLK1-SRPK1-SRSF1 first. Subs [85].equently, The interaction SRPK1 can between engage CLK1 to create N-terminus the ternary and SRPK1complex kinase CLK1-SRPK1-SRSF1 domain holds the [85]. complex The interaction CLK1-SRPK1 between together CLK1 but N-terminus also facilitates and SRPK1 the release kinase of hyper-phosphorylateddomain holds the complex SRSF1 fromCLK1-SRPK1 the ternary together complex, but which also is facilitates a prerequisite the forrelease the assembly of hyper- of spliceosomephosphorylated [55]. SRSF1 It has been from also the suggested ternary thatcomplex, the interaction which is between a prerequisite CLK1 and for SRPK1 the assembly anchors the of SRPK1spliceosome in the [55]. nucleus, It has thereby been also increasing suggested SRPK1 that concentrationthe interaction in between the nucleoplasm CLK1 and [55 SRPK1]. In addition, anchors CLK1the SRPK1 phosphorylates in the nucleus, the splicing thereby factor increasing SPF45 (non-SR SRPK1 protein)concentration on eight in serinethe nucleoplasm residues, regulating [55]. In celladdition, migration CLK1 and phosphorylat invasion (SPF45es the overexpression splicing factor promotesSPF45 (non-SR both processes) protein) on [87 eight]. serine residues, regulatingCLK1 cell can migration be phosphorylated and invasion by(SPF45 AKT2, overexpression which promotes promotes CLK1-mediated both processes) SR[87]. protein phosphorylationCLK1 can [be88 ].phosphorylated In addition, CLK1by AKT2, also activates which KKT2promotes via phosphorylationCLK1-mediated atSR the protein S508 residue,phosphorylation which is crucial[88]. In foraddition kinetochore, CLK1assembly also activates [89], KKT2 documenting via phosphorylation that the CLK1 at is the a component S508 residue, of awhich wider is signaling crucial for network. kinetochore assembly [89], documenting that the CLK1 is a component of a wider signalingIn addition, network. CLK1 can regulate autophagy—CLK1 inhibition or knockout induces autophagy via activationIn addition, of the mTOR CLK1/ PI3Kcan regulate pathway autophagy—CLK1 [90,91]. inhibition or knockout induces autophagy via activationThese recent of the findings mTOR/PI3K suggest pathway it may [90,91]. be desirable to evaluate the effect of CLK inhibitors on splicingThese in therecent context findings of the suggest SRPK1-CLK1 it may complex, be desirabl rathere to thanevaluate using the the effect isolated of CLK CLK1 inhibitors kinase [85 on]. splicing in the context of the SRPK1-CLK1 complex, rather than using the isolated CLK1 kinase [85]. 3.2. CLK2 3.2. CLK2 Specifically for CLK2, it has been found that it can be stabilized by AKT in response to feeding and actsSpecifically as a suppressor for CLK2, of the it has peroxisome been found proliferator-activated that it can be stabilized receptor byγ AKTcoactivator in response (PGC-1 to αfeeding), thus modulatingand acts as a fatty suppressor acid oxidation of the peroxi and ketogenesissome proliferator-activated [92]. In addition, receptor it has been γ coactivator reported that (PGC-1 CLK2α), phosphorylatesthus modulating the fatty protein acid oxidatio phosphatasen and ketogenesis 2A (PP2A), [92]. which In leadsaddition, to assembly it has been of reported the heterotrimeric that CLK2 PP2Aphosphorylates holoenzyme, the protein subsequent phosphatase AKT dephosphorylation, 2A (PP2A), which andleads attenuation to assembly of of AKTthe heterotrimeric activity [93]. PP2A holoenzyme, subsequent AKT dephosphorylation, and attenuation of AKT activity [93]. CLK2 phosphorylation by activated AKT is an important regulatory mechanism promoting cell survival following ionizing radiation [94].

Int. J. Mol. Sci. 2020, 21, 7549 8 of 31

CLK2 phosphorylation by activated AKT is an important regulatory mechanism promoting cell survival following ionizing radiation [94]. Besides the role of CLK2 in SR protein phosphorylation and splicing regulation, it has been documented that CLK2 and CLK1 also phosphorylate the protein-tyrosine phosphatase PTP-1B, whereby increasing its activity [95]. Recent findings show that CLK2 hyper-phosphorylates Prostate-Associated Gene 4 (PAGE) at multiple Ser- and Tyr-residues. Interestingly, it has been documented that HIPK1-phosphorylated PAGE4 potentiates c-JUN whereas CLK2-phosphorylated PAGE4 diminishes c-JUN activity [96].

3.3. CLK3 High levels of CLK3 were detected in mature spermatozoa in mouse testes. Interestingly, CLK3 in these cells is mainly located in the cytoplasm [97].

3.4. CLK4 CLK4 and CLK1 are almost identical in amino acid sequence. CLK1/4 proteins supplied through stress-induced splicing of the intron-retaining pre-mRNAs have been found to be essential for the rapid recovery of the phosphorylation state of SR proteins and contribute to the restart of splicing reactions after stress removal. They may thus serve as a guardian maintaining the phosphorylation state of SR proteins to improve the survival of cells exposed to stress [98].

4. Effects of Altered CLK Expression On the molecular level, an increased level of CLKs has been found to lead to the following. Overexpression of active CLKs causes the redistribution of SR proteins within the nucleus and dissolution of speckles [54,56,99], dispersion of eIF4E nuclear speckles [100], complete redistribution of interchromatin granule clusters [101], and specific targeting of SRp55 for degradation by proteasome [102]. The overexpression of CLK1 also affects splicing site selection of pre-mRNA of both its own transcript and adenovirus E1A transcripts in vivo [103]. CLK1 regulates expression of the alternative splicing factor 45 (SPF45), which is frequently overexpressed in cancer. Expression of CLK1 enhances SPF45-induced exon 6 exclusion from Fas mRNA, whereas CLK1 inhibition reduces it [87]. In human stem cells, overexpression of CLK2 leads to the dispersal of speckles and a significant drop in the number of genes in shared neighborhoods [104]. On the morphological and phenotypical levels, altered expression of CLK has been found to elicit different changes, summarized below. Expression of CLKs in PC12 cells mimic nerve growth factor (NGF)-dependent events, including morphological differentiation and elaboration of neurites up-regulated during HMBA-induced erythroleukemia cell differentiation [105]. Rather conversely, a reduction in transcript levels for CLK1 (and other genes encoding proteins involved in neurite outgrowth, namely NGF, M6a, and GNAQ) was observed in chronically stressed mice [106]. In Xenopus embryos, overexpression of CLK2 induces expression of both anterior and posterior neural marker genes, thereby promoting early neural development [107]. Overexpression of CLK2 significantly increases the growth of HeLa cells and inhibits radiation-induced cell death [94]. CLK2 also acts as an oncogene in —it is amplified and overexpressed in a significant fraction of breast tumors and its downregulation inhibits breast cancer growth in cell culture as well as in xenograft models [108]. Recently, CLK3 was found to be markedly upregulated in hepatocellular carcinoma tissues and its expression levels were closely associated with TNM stages and prognosis [109]. Overexpression of CLK2 in the mediobasal thalamus in mice on a high-fat diet can partially reverse the obese phenotype [110]. CLK2 regulates CREB via the attenuation of CREB inhibitory dephosphorylation by PP2Am, which affects diet-induced thermogenesis in brown adipose tissue via increased CREB-dependent expression of UCP1 [93]. There are indications that BAT CLK2 increases Int. J. Mol. Sci. 2020, 21, 7549 9 of 31 diet-mediated energy expenditure and protects against obesity [93]. Moreover, it has been shown that CLK2 is an insulin-regulated suppressor of hepatic gluconeogenesis [111].

5. CLKs as Therapeutic Targets Compromised accuracy of alternative splicing can have a profound impact on human pathogenesis, in particular on tumor development and progression [112]. Recent reports described recurrent change-of-function mutations in splicing factors in a variety of cancers and suggested that abnormal splicing can trigger tumor growth [113–115]. As briefly illustrated in the previous chapter and described in greater detail here, there has been increasing experimental evidence that abnormal function and levels of CLKs are present in different cancers. Cancer cells have general as well as cancer type-specific and subtype-specific alterations in the splicing process that can have prognostic value and contribute to cancer progression. These splicing alterations are often linked to the occurrence of cancer driver mutations in genes encoding either core components or regulators of the splicing machinery, which can make the cells particularly vulnerable to the pharmacological inhibition of splicing [116,117]. More specifically, there has been emerging evidence that splicing kinases are dysregulated in cancer and play important roles in both tumorigenesis as well as therapeutic response to radiation and [16]. A recent report demonstrated that MYC activation, which altered pre-mRNA splicing without the transcriptional regulation of CLKs, can render cancer cells vulnerable to CLK inhibitors with resulting synergistic cell death [118]. The potential therapeutic benefit of CLK inhibition was also demonstrated in vivo in an allograft model of spontaneous, MYC-driven breast cancer [118]. Numerous investigations of the cancer-related biology of CLKs have been supported by the use of small-molecule inhibitors (described in the following chapter) with varying degrees of selectivity. Of those, the inhibitor SM08502 recently entered clinical trials for the treatment of advanced solid tumors [76], and the compound CX-4945/silmitasertib (which inhibits CLK2 as well as CK2) [119] for the treatment of , medulloblastoma, basal cell carcinoma, and multiple myeloma [120]. CX-4945 could be also applicable in the treatment of glioblastoma where CLK2 modulates phosphorylation of Forkhead box O3a (FOXO3a)/p27 and, consequently, cell cycle and survival of tumor cells [121]. As exemplified below, CLKs could be attractive targets in other therapeutic areas beyond oncology. Despite their high , overexpression of CLK1 vs. CLK2 was found to have opposite effects on the replication of the HIV-1 virus, while changing the levels of CLK3 and CLK4 had no significant effect [122]. As described previously, the CLK-mediated phosphorylation process is involved in RNA splicing; however, aberrant splicing involving the microtubule-associated protein tau is associated with neurodegenerative diseases such as frontotemporal dementia and Parkinson’s disease and other tauopathies [78,123,124]. Recent reports indicate that CLKs (and other kinases) can be involved in the molecular mechanism of Alzheimer’s disease and [123,125,126] the CLK/DYRK inhibitor Leucettine 41 can activate the autophagic mTOR/PI3K pathway, which may represent a therapeutic option [91]. Pharmacological inhibition of CLK1 has been investigated pre-clinically (in mice) for the treatment of Duchenne muscular dystrophy—it resulted in enhanced mutated exon 31 skipping, which led to the production of functional exon 31 skipped dystrophin in cells derived from Duchenne muscular dystrophy patients [127]. CLK1 inhibition and CLK1 knockout effect also splicing of the viral RNA transcript. Specifically, it has been shown that CLK1 knockdown or inhibition reduced influenza A/WSN/33 virus replication in A549 cells [128]. Moreover, CLK1 / mice infected with influenza A/England/195/2009 (H1N1pdm09) − − virus had lower levels of virus replication in comparison to wild-type mice, and CLK1 inhibition had a similar effect [128]. CLK2 is also considered as an insulin-regulated suppressor of hepatic gluconeogenesis, contributing to hyperglycemia in diabetes. The mechanism is principally based on direct Int. J. Mol. Sci. 2020, 21, 7549 10 of 31 phosphorylation of the SR domain of the transcriptional coactivator (PGC-1α) by CLK2. This phosphorylation process causes repression of gene expression in the gluconeogenesis, resulting in hypoglycemia [111]. Along this line, it has been suggested that the levels of insulin could be regulated by CLK2 inhibition [88,111]. In neurology, inhibition of CLK2 was reported to restore normal sociability of Shank3-deficient mice (likely via activation of AKT activation) and thus could be therapeutically relevant for the treatment of the autistic spectra disorder Phelan-McDermid syndrome (PMDS) [129]. Finally, it has been reported that inhibition of CLK3 in Plasmodium falciparum (Pf CLK3) represents a promising approach for the treatment of malaria via preventing the splicing of essential parasite genes. It has been demonstrated that CLK3 inhibition can kill multiple species of malaria parasites at the blood stage and at the liver-stage and block transmission of the parasite to mosquitoes [130,131]. Another indication where CLK inhibition could be therapeutically relevant is Legionellosis, where CLK inhibition (by TG003) was demonstrated to reduce Legionella growth within mouse macrophages [132].

6. Small-Molecule CLK Inhibitors Over the last two decades, structurally diverse CLK inhibitors have been described in the literature, with the majority of them being published in recent years. This chapter provides a chronological summary of individual compounds and/or selected representatives of individual structural series, with the focus on substances that have been most thoroughly profiled and could thus serve as chemical probes for chemical biology studies (Table2). For better comparison, Table2 also contains data for structurally similar DYRKs and HIPKs, since achieving selectivity against those two sub-families is not trivial and represents a significant challenge in the development of selective CLK inhibitors. Additional typical off-targets of many CLK inhibitors are PIM and/or CK1 kinases. Of note, most of the reported CLK inhibitors interact relatively weakly with CLK3—only 4 out of 24 compounds exhibit IC50/Kd values towards CLK3 < 100 nM. Table2 contains the published values obtained in primary biochemical assays; however, for their direct comparison it is important to keep in mind that these data were often obtained from various sources/assays or from the same (similar) assay but under unequal conditions (e.g., using different concentrations of ATP). On the other hand, some studies report a direct comparison of several (usually two or three) inhibitors from different classes/publications, profiling them in the same assay [76,133–136]. An additional brief summary is given for each of the inhibitors, with the main focus on their selectivity and (therefore) potential to be used as quality CLK chemical probes in chemical biology. It is likely that in the near future, highly selective tool compounds will help further unravel the complexity of CLK biology and validate the therapeutic potential of CLK inhibition. Of the listed CLK inhibitors, we highlight those that (in our opinion) fulfill the criteria for a quality probe. However, it is highly recommended to confirm the context of the obtained biological data in original reports prior to use and (whenever possible) use at least two chemically different quality chemical biology probes to minimize the risk of compound-specific off-target effects. Some of the compounds exhibiting sufficient activity in the cell and in vivo have been pre-clinically profiled as anti-tumor agents [76,135,137]. Of note, the therapeutic potential of CLK inhibitors might be enhanced by simultaneous inhibition of additional targets [136,137]. Int. J. Mol. Sci. 2020, 21, 7549 11 of 31

Table 2. In vitro activities of known CLK inhibitors, including their typical off-targets. The IC50 values (in nM) are given as numbers, Kd values (in nM) are specified. In cases where IC50/Kd is not available, the compound’s potency is expressed as a percentage of the ’s residual activity at the concentration of the inhibitor specified in brackets. CLK probes recommended by SGC are highlighted in green. ND: no data found.

CLKs Structurally Similar Kinases Other Significant Targets Kinases Screened Compound CLK1 CLK2 CLK3 CLK4 DYRK1A DYRK1B DYRK2 HIPK1 HIPK2 HIPK3 CK1δ = 150 CK1ε = 300 CK1γ2 = 270 19 95 30 12 130 CK1γ3 = 290 TG003 [64,134,138] inactive ND ND ND ND ~402 kinases (Kd) (Kd) (Kd) (Kd) (Kd) PIM1 = 130 PIM3 = 280 YSK4 = 290 (Kd) 37 680 470 50 27 430 ML106 [134,138] ND ND ND ND EGFR = 230 (Kd) 402 kinases (Kd) (Kd) (Kd) (Kd) (Kd) (Kd) ML167 [138] 1522 1648 inactive 136 inactive 4420 ND ND ND ND Not published 442 kinases CK2α = 1.5 PIM1 = 216 CX-4945 5% 11% 15% 7% TBK1 3.3 2.9 67 23 14 ND ~235 kinases [133,139,140] (0.5 µM) (0.5 µM) (0.5 µM) (0.5 µM) DAPK2 ZIPK FLT1 106 kinases PIM1/3 KH-CB19 [141] 20 ND 530 ND 55 ND ND ND ND ND (thermal shift SGK085 assay) GSK3α = 210 Leucettine L 1% 11% 4% PIM1 41 71 720 inactive 64 60 44 73 402 kinases [142] (10 µM) (10 µM) (10 µM) IRAK1 TAOK1 PRKCE ML315 [143] 68 231 inactive 68 282 1156 ND ND ND ND MAP3K1 442 kinases CK1ε 22% 69% 1% 91% Thiophene 48 [144] 110 100 70 40 ND ND - 102 kinases (5 µM) (5 µM) (5 µM) (5 µM) 61% 3A5 [145] 51 68 346 ND 260 ND inactive inactive inactive CK1γ2 ~140 kinases (1 µM) SRPK1 = 200 Cpd-2 [146] 1.1 2.4 ND ND ND ND ND ND ND ND SRPK2 = 310 28 kinases SRPK3 = 230 Int. J. Mol. Sci. 2020, 21, 7549 12 of 31

Table 2. Cont.

CLKs Structurally Similar Kinases Other Significant Targets Kinases Screened Compound CLK1 CLK2 CLK3 CLK4 DYRK1A DYRK1B DYRK2 HIPK1 HIPK2 HIPK3 95% SRI-29329 [147] 78 16 >1000 86 ND ND ND ND ND - 29 kinases (1 µM) T3 [135,148] 0.67 15 110 ND 260 230 ND inactive inactive ND - 71 kinases Compound 25 [90] 2 31 inactive 8 138 690 inactive inactive inactive inactive - 368 kinases 85% 88% 16% 23% TG693 [127] 113 ND ND inactive ND inactive HASPIN ~313 kinases (1 µM) (1 µM) (1 µM) (1 µM) Indazole1 [133] 12 10 2250 12 73 ND ND ND ND ND - 34 kinases

KuWal151 [149] 88 510 inactive 28 inactive inactive inactive ND ND ND - IC50 for 14 kinases HIPK4 YSK4 4.8 0.096 6.5 0.61 0.074 1.5 32 55 96 5% T-025 [118] IRAK4 468 kinases (K ) (K ) (K ) (K ) (K ) (K ) (K ) (K ) (K ) (0.3 µM) d d d d d d d d d FLT ERK8 TTK = 5 60% 97% 96% 92% DYRK3 = 99 CC-671 [137] 300 6.3 ND 104 157 ND 225 kinases (3 µM) (3 µM) (3 µM) (3 µM) PhKγ1 = 136 TSSK1 = 452 Pyrido CDK5 39% [3,4-g]quinazoline 18 ND ND ND ND ND ND ND ND CK1 - (1 µM) 9m [150] GSK3 ERK8 NEK7 SGC-CLK-1 [151] 13 4 363 46 ND ND ND 50 42 ND 403 kinases PIP5K2B STK16 additional 14 kinases with SM08502 [76] 8 1 22 1 1 1 3 ND 23 21 466 kinases IC50 < 50 nM GSK3α 210 MU1210 [47,152] 8 20 Inactive 12 213 956 1309 187 29 159 PIM1/1 kinases HASPIN BTK = 2 AB1 [89] ND ND ND ND ND ND ND ND ND ND ND hERGFR = 2 MNK1 MAP4K3 CAMKKb TCMDC-135051 17% 71% 99% 83% 56% 93% ND 40 ND ND CDK9 140 kinases [130,131] (1 µM) (1 µM) (1 µM) (1 µM) (1 µM) (1 µM) IRAK1 TGFBR1 PHK Int. J. Mol. Sci. 2020, 21, 7549 13 of 31 Int.Int. J.J. Mol.Mol. Sci.Sci. 20202020,, 2121,, xx FORFOR PEERPEER REVIEWREVIEW 14 14 of of 31 31

TG003 (Figure(Figure7 )7) represents represents one one of the of firstthe CLKfirst inhibitorsCLK inhibitors (published (published in 2004) in [ 64 2004)] and [64] its activities and its activitiesin biochemical in biochemical assays have assays been have determined been determ severalinedined times severalseveral with slightlytimestimes withwith diff erentslightlyslightly results differentdifferent[64,134 resultsresults,153]. [64,134,153].TG003[64,134,153]. is active TG003TG003 also isis in activeactive cell-based alsoalso inin assays cell-basedcell-based and in assaysassays vivo. and Inand the inin cell, vivo.vivo. the InIn inhibitor thethe cell,cell, thethe can inhibitorinhibitor elicit suppression cancan elicitelicit suppressionofsuppression the exon ofof skipping thethe exonexon and skippingskipping serine /andandarginine-rich serine/argininserine/arginin proteine-rich phosphorylation, protein phosphoryl dissociationation, dissociation of nuclear of nuclearspeckles, speckles, and Clk1 and/Sty-dependent Clk1/Sty-dependent alternative alternat splicingiveive splicingsplicing [64]. A broader [64].[64]. AA broaderbroader screening screeningscreening against 402againstagainst kinases 402402 kinasesrevealed revealed significant significant off-target off-target activities, activities, indicating indicating that the that use ofthe TG003 use of as TG003 a chemical as a chemical probe is probe likely isproblematicis likelylikely problematicproblematic [134]. Despite [134].[134]. DespiteDespite this fact, thisthis the fact,fact, compound thethe compoundcompound is still isis being stillstill beingbeing widely widelywidely used usedused in life inin sciences lifelife sciencessciences as a asCLK a CLK inhibitor. inhibitor.

Figure 7. Structure of the compound TG003. Figure 7. Structure of the compound TG003. The quinazoline-based inhibitor ML106 (Figure8) was published in 2009 [ 134]. ML106 (analog The quinazoline-based inhibitor ML106 (Figure 8) waswas publishedpublished inin 20092009 [134].[134]. ML106ML106 (analog(analog of ML167) inhibits CLK1/2/3/4 with Kd values of 37 nM, 680 nM, 470 nM, and 50 nM, respectively. of ML167) inhibits CLK1/2/3/4 with K d valuesvalues ofof 3737 nM,nM, 680680 nM,nM, 4470 nM, and 50 nM, respectively. ML106 was profiled in a panel of 402 kinases; detailed results of the profiling are not available, ML106 was profiled in a panel of 402 kinases; detailediled resultsresults ofof thethe profilingprofiling areare notnot available,available, butbut but significant off-targets are DYRK1A, DYRK1B, and EGFR with Kd d values 27 nM, 430 nM, and 230 nM, significantsignificant off-targetsoff-targets areare DYRK1A,DYRK1A, DYRK1B,DYRK1B, andand EGFREGFR withwith KKd valuesvalues 2727 nM,nM, 430430 nM,nM, andand 230230 nM,nM, respectively [134]. Of note, the published IC50 50 values (CLK150 IC50 = 59 nM, CLK250 IC50 = 1902 nM, respectivelyrespectively [134].[134]. OfOf note,note, thethe publishedpublished ICIC50 valuesvalues (CLK1(CLK1 ICIC50 == 5959 nM,nM, CLK2CLK2 ICIC50 == 19021902 nM,nM, CLK3CLK3 CLK350 IC50 = 6936 nM, CLK450 IC50 = 39 nM) do not correlate well withd the Kd values above [153]. ICIC50 == 69366936 nM,nM, CLK4CLK4 ICIC50 == 3939 nM)nM) dodo notnot correlatecorrelate wellwell withwith thethe KKd valuesvalues aboveabove [153].[153]. OtherOther Other quinazoline-based analogs were also profiled in panels of 402 or 442 kinases, which revealed quinazoline-based analogs were also profiled in panels of 402 or 442 kinases, which revealed their their very good selectivity [153]. Some of them were further tested in the Caco-2 permeability assay or very good selectivity [153]. Some of them were furtherrther testedtested inin thethe Caco-2Caco-2 permeabilitypermeability assayassay oror forfor for cellular efflux, suggesting that selected analogs might be used as tool compounds for cell-based cellular efflux, suggesting that selected analogss mightmight bebe usedused asas tooltool compoundscompounds forfor cell-basedcell-based experiments; however, no cellular activities have been published thus far [153]. experiments; however, no cellular activities have been published thus far [153].

Figure 8. StructureStructure of the compound ML106.

To ourour bestbest knowledge,knowledge, thethe compoundcompound ML167 (F (Figureigureigure 9 9)9)) represents representsrepresents the thethe most mostmost selective selectiveselective CLK4 CLK4CLK4 inhibitor (IC50 = 136 nM), according to the data obtained in biochemical screening (Table2)[ 153]. inhibitorinhibitor (IC(IC50 == 136136 nM),nM), accordingaccording toto thethe datadata obtainedobtained inin biochemicalbiochemical screeningscreening (Table(Table 2)2) [153].[153]. Although ML167 is not the most potent CLK inhibito inhibitor,r,r, it it is is selective selective with with respect respect to to other other family family members CLK1/2/3 CLK1/2/3 andand (importantly) DYRK1ADYRK1A/B/B (Table(Table2 2).). The The selectivity selectivity was was determineddetermined by by testingtesting inin a a panelpanel of of 442442 kinaseskinases [153]; [[153];153]; unfortunately,unfortunately, neitherneither thethe resultsresults ofof thisthis kinome-widekinome-wide screening screening nor nor informationinformation about about additional additional off-targets ooff-targetsff-targets areareare available.available.available. Likewise, Likewise, there there are are no no data data on on the the compound’scompound’s activity in the cell.cell. Therefore, Therefore, any any utilization utilization of of ML167 ML167 as as a a chemical probe probe for CLK4 should be considered withwith caution.caution. Many Many ML167 ML167 analogs analogs with with di ff differentdifferenterent CLK CLKCLK and andand DYRK DYRKDYRK selectivity selectivityselectivity profiles profilesprofiles have havebeen synthesized;been synthesized; more more detailed detailed information informat canionion be cancan found bebe foundfound in the inin original thethe originaloriginal report reportreport [153]. [153].[153]. CX-4945 (Figure 10) has been primarily reported, marketed, and used as a highly selective and potent inhibitor of CK2 since 2011 [140]. However, CX-4945 strongly inhibits also CLKs and DYRK1A [133]. In addition, it interacts significantly with other kinases such as HIPKs or PIM kinases [133,139].

Figure 9. Structure of the compound ML167.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 14 of 31

TG003 (Figure 7) represents one of the first CLK inhibitors (published in 2004) [64] and its activities in biochemical assays have been determined several times with slightly different results [64,134,153]. TG003 is active also in cell-based assays and in vivo. In the cell, the inhibitor can elicit suppression of the exon skipping and serine/arginine-rich protein phosphorylation, dissociation of nuclear speckles, and Clk1/Sty-dependent alternative splicing [64]. A broader screening against 402 kinases revealed significant off-target activities, indicating that the use of TG003 as a chemical probe is likely problematic [134]. Despite this fact, the compound is still being widely used in life sciences as a CLK inhibitor.

Figure 7. Structure of the compound TG003.

The quinazoline-based inhibitor ML106 (Figure 8) was published in 2009 [134]. ML106 (analog of ML167) inhibits CLK1/2/3/4 with Kd values of 37 nM, 680 nM, 470 nM, and 50 nM, respectively. ML106 was profiled in a panel of 402 kinases; detailed results of the profiling are not available, but significant off-targets are DYRK1A, DYRK1B, and EGFR with Kd values 27 nM, 430 nM, and 230 nM, respectively [134]. Of note, the published IC50 values (CLK1 IC50 = 59 nM, CLK2 IC50 = 1902 nM, CLK3 IC50 = 6936 nM, CLK4 IC50 = 39 nM) do not correlate well with the Kd values above [153]. Other quinazoline-based analogs were also profiled in panels of 402 or 442 kinases, which revealed their very good selectivity [153]. Some of them were further tested in the Caco-2 permeability assay or for cellular efflux, suggesting that selected analogs might be used as tool compounds for cell-based experiments; however, no cellular activities have been published thus far [153].

Figure 8. Structure of the compound ML106.

To our best knowledge, the compound ML167 (Figure 9) represents the most selective CLK4 inhibitor (IC50 = 136 nM), according to the data obtained in biochemical screening (Table 2) [153]. Although ML167 is not the most potent CLK inhibitor, it is selective with respect to other family members CLK1/2/3 and (importantly) DYRK1A/B (Table 2). The selectivity was determined by testing in a panel of 442 kinases [153]; unfortunately, neither the results of this kinome-wide screening nor information about additional off-targets are available. Likewise, there are no data on the compound’s activity in the cell. Therefore, any utilization of ML167 as a chemical probe for CLK4 should be Int.considered J. Mol. Sci. with2020, 21 caution., 7549 Many ML167 analogs with different CLK and DYRK selectivity profiles14 of 31 have been synthesized; more detailed information can be found in the original report [153].

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 31 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 31 CX-4945 (Figure 10) has been primarily reported, marketed, and used as a highly selective and potentCX-4945 inhibitor (Figure of CK2 10) since has 2011been [140].primarily However, reported, CX-4945 marketed, strongly and inhibits used as also a highly CLKs andselective DYRK1A and potent[133]. In inhibitor addition, of itCK2 interacts since 2011significantly [140]. However, with other CX-4945 kinases strongly such as inhibits HIPKs oralso PIM CLKs kinases and DYRK1A[133,139]. [133]. In addition, it interacts significantly with other kinases such as HIPKs or PIM kinases [133,139]. Figure 9. Structure of the compound ML167.

Cl NH Cl NH N N N N HO HO O CX-4945 O CX-4945 Figure 10. Structure of the compound CX-4945. Figure 10. Structure of the compound CX-4945. KH-CB19 (Figure 11), published in 2011, was among the first CLK inhibitors found to be superior to TG003KH-CB19 [141]. (Figure Based 1111), on), published IC50 values in 2011,obtained was amongin biochemical the firstfirst CLK assays, in inhibitorshibitors the compound found to be is superiora superior potent toinhibitor TG003TG003 [ of141[141]. CLK1]. Based Based and on DYRK1A.on IC 50ICvalues50 values The obtained selectivity obtained in biochemical profin biochemicalile, evaluated assays, assays, by the thermal compound the compound shift is assay a potent isagainst a inhibitor potent 106 ofinhibitorkinases, CLK1 andindicatesof CLK1 DYRK1A. andalso DYRK1A. strong The selectivity inhibition The selectivity profile, of CLK4 evaluated prof andile, someevaluated by thermal off-target by shiftthermal activity assay shift against assay 106PIM1/3against kinases, and106 indicateskinases,SGK085 [141].indicates also strong KH-CB19 also inhibition strong also exhibits inhibition of CLK4 CLK and of inhibitory someCLK4 o andff-target activity some activity inoff-target the againstcell [141].activity PIM1 Utilization /against3 and SGK085 PIM1/3of KH-CB19 [ 141and]. KH-CB19SGK085as a chemical [141]. also probe KH-CB19 exhibits should CLK also be inhibitory exhibits done with CLK activity caution inhibitory in and the cellideallyactivity [141 after in]. Utilizationthe further cell [141]. profiling of KH-CB19Utilization across as of the a KH-CB19 chemical kinome. probeasThe a chemicalutilization should beprobe donein vivo should with might caution be done be andproblematic with ideally caution after sinc and furthere ideally the molecule profiling after further acrosscontains profiling the kinome.potentially across The thelabile utilization kinome. ester inThefunctionality. vivo utilization might be in problematic vivo might since be problematic the molecule sinc containse the potentiallymolecule contains labile ester potentially functionality. labile ester functionality.

FigureFigure 11.11. Structure ofof thethe compoundcompound KH-CB19.KH-CB19. Figure 11. Structure of the compound KH-CB19. LeucettineLeucettine 41 (Figure 12)12) was was developed developed by by modifi modificationcation of of the the natural natural product product Leucettamine Leucettamine B B[[142].142Leucettine ].The The compound compound 41 (Figure strongly strongly 12) was in inhibitshibits developed CLK1/4 CLK1 by/4 and andmodifi DYRK1A/B DYRK1Acation /ofB[ [142] the142 natural].. However, However, product the data Leucettamine for DYRK2DYRK2 B are[142].are ratherrather The inconsistentcompoundinconsistent strongly (IC(IC5050 = 73 in hibitsnM; Kd CLK1/4 = 450450 andnM), DYRK1A/B since for a [142]competitive. However, inhibitor, the data KKdd forisis typicallytypically DYRK2 smalleraresmaller rather thanthan inconsistent ICIC5050 [142].[142]. (IC Leucettine Leucettine50 = 73 nM; 41 41 Kd is is only = 450 weakly nM), since active for towards a competitive CLK2CLK2/3 /inhibitor,3 (Table3 3). ).K dTestingTesting is typicallyin in a a µ panelsmallerpanel ofof than 402402 kinaseskinasesIC50 [142]. atat 1010 Leucettine µMM concentrationconcentration 41 is only revealedrevealed weakly aaactive goodgood towards selectivity CLK2/3 profile;profile; (Table additional 3). Testing ooff-targetsff-targets in a beingpanelbeing HIPK1ofHIPK1/3 402 kinases/3 andand GSK3A GSK3Aat 10 µM [[142].142 concentration]. Interestingly, revealed GSK3AGSK3 aA good inhibitioninhibition selectivity inin thethe profile; cellcell waswas additional notnot observedobserved off-targets whilewhile inbeingin cellcell HIPK1/3 inhibitioninhibition and ofof GSK3A DYRK1ADYRK1A [142]. waswas Interestingly, confirmedconfirmed [[142].142 GSK3]. LeucettineA inhibition 41 in could the cell be awas usefuluseful not chemicalchemicalobserved probe,while butinbut cell additionaladditional inhibition profilingprofiling of DYRK1A wouldwould was bebe desirable desirableconfirmed (such(such [142]. asas determinationLeucettinedetermination 41 could ofof thethe be CLKCLK a useful engagementengagement chemical ininthe theprobe, cellcell orbutor inin additional cellcell activityactivity profiling againstagainst would HIPKs).HIPKs). be desirable (such as determination of the CLK engagement in the cell or in cell activity against HIPKs).

Figure 12. Structure of Leucettine 41. Figure 12. Structure of Leucettine 41.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 15 of 31

CX-4945 (Figure 10) has been primarily reported, marketed, and used as a highly selective and potent inhibitor of CK2 since 2011 [140]. However, CX-4945 strongly inhibits also CLKs and DYRK1A [133]. In addition, it interacts significantly with other kinases such as HIPKs or PIM kinases [133,139].

Cl NH

N N

HO

O CX-4945 Figure 10. Structure of the compound CX-4945.

KH-CB19 (Figure 11), published in 2011, was among the first CLK inhibitors found to be superior to TG003 [141]. Based on IC50 values obtained in biochemical assays, the compound is a potent inhibitor of CLK1 and DYRK1A. The selectivity profile, evaluated by thermal shift assay against 106 kinases, indicates also strong inhibition of CLK4 and some off-target activity against PIM1/3 and SGK085 [141]. KH-CB19 also exhibits CLK inhibitory activity in the cell [141]. Utilization of KH-CB19 as a chemical probe should be done with caution and ideally after further profiling across the kinome. The utilization in vivo might be problematic since the molecule contains potentially labile ester functionality.

Figure 11. Structure of the compound KH-CB19.

Leucettine 41 (Figure 12) was developed by modification of the natural product Leucettamine B [142]. The compound strongly inhibits CLK1/4 and DYRK1A/B [142]. However, the data for DYRK2 are rather inconsistent (IC50 = 73 nM; Kd = 450 nM), since for a competitive inhibitor, Kd is typically smaller than IC50 [142]. Leucettine 41 is only weakly active towards CLK2/3 (Table 3). Testing in a panel of 402 kinases at 10 µM concentration revealed a good selectivity profile; additional off-targets being HIPK1/3 and GSK3A [142]. Interestingly, GSK3A inhibition in the cell was not observed while in cell inhibition of DYRK1A was confirmed [142]. Leucettine 41 could be a useful chemical probe, Int.but J. additional Mol. Sci. 2020 profiling, 21, 7549 would be desirable (such as determination of the CLK engagement in the15 ofcell 31 or in cell activity against HIPKs).

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 16 of 31

Table 3. Kd values for kinases significanFigure 12.12.tly Structure inhibited of by Leucettine Leucettine 41. 41 at 10 µM concentration [142].

Table 3. Kd values for kinases significantlyKinases inhibited Kd by (nM) Leucettine 41 at 10 µM concentration [142]. CLK4 70.0 Kinases Kd (nM) DYRK1A 7.8 CLK4CLK1 75.0 70.0 DYRK1A 7.8 DYRK2 450.0 CLK1 75.0 DYRK2HIPK1 320.0 450.0 HIPK1CLK3 1100.0 320.0 CLK3IRAK1 930.0 1100.0 IRAK1HIPK3 230.0 930.0 HIPK3 230.0 DYRK1BDYRK1B 140.0 140.0 CLK2CLK2 360.0 360.0 TAOK1TAOK1 1700.0 1700.0 TYK2TYK2 n.t n.t GSK3A 550.0 GSK3A 550.0

ML315ML315 (Figure (Figure 13)13 )was was published published in in2013 2013 as a as selective a selective CLK/DYRK1A CLK/DYRK1A inhibitor inhibitor [143]. [143The]. compoundThe compound inhibits inhibits both CLK1/4 both CLK1 with/4 IC with50 of IC68 50nMof and, 68 nM somewhat and, somewhat more weakly, more CLK2 weakly, and CLK2 DYRK1A and (ICDYRK1A50 = 231 (IC nM50 =and231 282 nM nM, and 282respectively) nM, respectively) [143]. A [143 very]. A good very selectivity good selectivity profile profile was wasconfirmed confirmed by screeningby screening in a inpanel a panel of 442 of kinases 442 kinases whereby whereby PRKCE PRKCE was revealed was revealed as the as only the off-target only off-target showing showing <10%

FigureFigure 13. 13. StructureStructure of of the the compound compound ML315. ML315.

BasedBased on on the natural product Harmine, which which is is moderately active active against against CLK1 CLK1 and and DYRK DYRK kinases,kinases, Thiophene Thiophene 48 48 (Figure (Figure 14)14) has has been been iden identifiedtified as as a a CLK1/DYRK1/DYRK2 CLK1/DYRK1/DYRK2 inhibitor inhibitor [144]. [144]. ProfilingProfiling in in a a panel of 102 kinases at at 5 µMµM concentration revealed decent decent selectivity, selectivity, with strong inhibitioninhibition of of CLK4 CLK4 [144]. [144]. In In cell cell activity activity has has been been demonstrated demonstrated as as well well [144]. [144]. Further Further profiling profiling would would bebe necessary necessary to to determine determine whether whether Thiophene Thiophene 48 48 fulfills fulfills the the criteria criteria for for a a quality quality chemical chemical probe probe (for (for CLK1/4CLK1/4 and DYRKs).

Figure 14. Structure of the compound Thiophene 48.

The compound 3A5 (Figure 15) was reported in 2015 as a CLK inhibitor with a novel benzobisthiazole scaffold [145]. Furthermore, 3A5 has a strong affinity towards CLK1/2 and only moderate activity against CLK3 and DYRK1A. Importantly, it is inactive against structurally similar HIPKs and only weakly active against DYRK2. Unfortunately, the selectivity was determined in a

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 16 of 31

Table 3. Kd values for kinases significantly inhibited by Leucettine 41 at 10 µM concentration [142].

Kinases Kd (nM) CLK4 70.0 DYRK1A 7.8 CLK1 75.0 DYRK2 450.0 HIPK1 320.0 CLK3 1100.0 IRAK1 930.0 HIPK3 230.0 DYRK1B 140.0 CLK2 360.0 TAOK1 1700.0 TYK2 n.t GSK3A 550.0

ML315 (Figure 13) was published in 2013 as a selective CLK/DYRK1A inhibitor [143]. The compound inhibits both CLK1/4 with IC50 of 68 nM and, somewhat more weakly, CLK2 and DYRK1A (IC50 = 231 nM and 282 nM, respectively) [143]. A very good selectivity profile was confirmed by screening in a panel of 442 kinases whereby PRKCE was revealed as the only off-target showing <10% residual activity at 10 µM concentration [143]. Although the microsomal stability of ML315 is low, it has appropriate physicochemical properties for in cell studies and represents a useful chemical probe for CLK/DYRK biology [143].

Figure 13. Structure of the compound ML315.

Based on the natural product Harmine, which is moderately active against CLK1 and DYRK kinases, Thiophene 48 (Figure 14) has been identified as a CLK1/DYRK1/DYRK2 inhibitor [144]. Profiling in a panel of 102 kinases at 5 µM concentration revealed decent selectivity, with strong inhibition of CLK4 [144]. In cell activity has been demonstrated as well [144]. Further profiling would beInt. necessary J. Mol. Sci. 2020 to determine, 21, 7549 whether Thiophene 48 fulfills the criteria for a quality chemical probe16 of(for 31 CLK1/4 and DYRKs).

Figure 14. StructureStructure of the compound Thiophene 48.

The compound 3A5 (Figure 15)15) was reported in in 2015 as as a CLK inhibitor with a novel benzobisthiazole scaffold scaffold [145]. [145]. Furthermore, Furthermore, 3A5 3A5 has has a a strong strong affinity affinity towards towards CLK1/2 CLK1/2 and only moderateInt.Int. J.J. Mol.Mol. Sci.Sci. activity 20202020,, 2121 ,against, xx FORFOR PEERPEER CLK3 CLK3 REVIEWREVIEW and and DYRK1A. Importan Importantly,tly, it is inactive against structurally similar 17 17 of of 31 31 HIPKs and only weakly active against DYRK2. Unfo Unfortunately,rtunately, the selectivity was determined in a panelpanel of of only only 142 142 kinases kinases at at 1 1 µMµMµM concentration concentration and and the the CLK4 CLK4 and and DYRK1B DYRK1B inhibitory inhibitory activities activities have have notnot been been determined determined atat all.all. Although Although 3A5 3A5 shows shows so somesomeme selectivity selectivity for for CLKs CLKs vs.vs. DYRKs, DYRKs, its its utilization utilization asas a a probe probe cannot cannot bebe recommendedrecommended asas itsits inin cellcell activitiesacactivitiestivities havehavehave notnotnot beenbeenbeen demonstrateddemonstrateddemonstrated thusthusthus far. far.far.

FigureFigure 15. 15. StructureStructure of of the the compound compound 3A5. 3A5.

TheThe CLK1/2 CLK1CLK1/2/2 inhibitor inhibitor cpd-2 cpd-2 (Figure (Figure 16)1616)) was was published published in in 2015, 2015, together together with with two two analogs analogs cpd-1 andand cpd-3 cpd-3 [146]. [[146].146]. Cpd-2 possesses high selectivity for for CLK1/2 CLK1CLK1/2/2 vs. vs. SRPK1/2/3. SRPK1SRPK1/2/3./2/3. However,However, the the inhibitors inhibitors havehave been been screened screened against against only only 2828 kinases.kinases. Th Therefore,Therefore,erefore, the the compound compound cannot cannot be be recommended recommended as as a a chemicalchemical probe probe atat thisthis point.point.

FigureFigure 16. 16. StructureStructure of of the the compound compound cpd-2. cpd-2.

TheThe CLK1CLK1/2/4CLK1/2/4/2/4 inhibitor inhibitorinhibitor SRI-29329 SRI-29329SRI-29329 (Figure (Figure(Figure 17 )17)17) was waswas published publishedpublished in 2016, inin 2016,2016, together togethertogether with withwith its analogs itsits analogsanalogs [147]. [147].Some[147]. SomeSome members membersmembers of the ofof series thethe seriesseries show showshow dual dualdual inhibitory inhibitoryinhibitory activity activityactivity towards towardstowards CLK1 CLK1/24CLK1/24/24 and andand CDK1 CDK1/4CDK1/4/4 (such (such(such as asCGP-74514A,as CGP-74514A,CGP-74514A, which whichwhich is is incorrectlyis incorrectlyincorrectly being beingbeing sold soldsold and andand used usedused as asas a aa selective selectiveselective CDK1 CDK1CDK1 inhibitor) inhibitor)inhibitor) [[147].[147].147]. Of note,note, SRI-29329SRI-29329 isisis inactive inactiveinactive against againstagainst DYRK1A DYRK1ADYRK1A [147 [147].[147].]. Since SiSincence SRI-29329 SRI-29329SRI-29329 has beenhashas profiledbeenbeen profiledprofiled against againstagainst only 29 onlyonly kinases, 2929 kinases,itkinases, is not recommended itit isis notnot recommendedrecommended to use it astoto auseuse chemical itit asas aa chemicalchemical probe [147 probeprobe]. [147].[147].

Figure 17. Structure of the compound SRI-29329. FigureFigure 17.17. StructureStructure ofof thethe compoundcompound SRI-29329.SRI-29329.

TheThe pan-CLKpan-CLK inhibitorinhibitor T3T3 (Figur(Figuree 18),18), basedbased onon cpd-1/2/3,cpd-1/2/3, waswas publishedpublished inin 20172017 [135].[135]. TheThe compoundcompound isis aa potentpotent inhibitorinhibitor ofof CLK1,CLK1, CLK2,CLK2, andand CLK3—theCLK3—the ICIC5050 valuesvalues determineddetermined inin biochemicalbiochemical assaysassays areare 0.670.67 nM,nM, 1515 nM,nM, andand 110110 nM,nM, respectively.respectively. T3T3 exhibitsexhibits goodgood selectivityselectivity againstagainst DYRK1ADYRK1A andand DYRK1BDYRK1B (IC(IC5050 == 260260 nMnM andand 230230 nM,nM, respectively)respectively) [135].[135]. OtherOther significantsignificant off-targetsoff-targets havehave notnot beenbeen foundfound thusthus far.far. However,However, thethe compoundcompound hashas beenbeen screenedscreened againstagainst onlyonly 7171 kinaseskinases atat 0.10.1 µMµM andand 11 µMµM concentrationsconcentrations [135].[135]. CellularCellular actiactivityvity ofof T3T3 hashas beenbeen confirmedconfirmed inin severalseveral assays;assays; itsits cellularcellular potencypotency basedbased onon NanoBRETNanoBRET assayassay isis 44 nM,nM, 1717 nM,nM, andand 22 nMnM towardstowards CLK1/2/4,CLK1/2/4, andand 3232 nMnM andand 6767 nMnM towardstowards DYRK1A/B,DYRK1A/B, respectivelyrespectively [148].[148]. ItIt hashas beenbeen alsoalso shownshown thatthat T3T3 inducesinduces aa decreasedecrease inin thethe phosphorylationphosphorylation statestate ofof SRSFSRSF proteinprotein inin aa time-time- andand dose-dependentdose-dependent mannermanner atat 10–10– 10001000 nMnM concentrationconcentration inin HeLaHeLa cellscells [148].[148]. Importantly,Importantly, thethe testingtesting waswas performedperformed inin parallelparallel withwith aa negativenegative controlcontrol compoundcompound [148].[148]. AsAs aa result,result, T3T3 cancan bebe recommendedrecommended asas aa qualityquality chemicalchemical probeprobe forfor CLK1/2/4,CLK1/2/4, ideallyideally inin combinationcombination withwith anotheranother selectiveselective inhibitor.inhibitor.

Int. J. Mol. Sci. 2020, 21, 7549 17 of 31

The pan-CLK inhibitor T3 (Figure 18), based on cpd-1/2/3, was published in 2017 [135]. The compound is a potent inhibitor of CLK1, CLK2, and CLK3—the IC50 values determined in biochemical assays are 0.67 nM, 15 nM, and 110 nM, respectively. T3 exhibits good selectivity against DYRK1A and DYRK1B (IC50 = 260 nM and 230 nM, respectively) [135]. Other significant off-targets have not been found thus far. However, the compound has been screened against only 71 kinases at 0.1 µM and 1 µM concentrations [135]. Cellular activity of T3 has been confirmed in several assays; its cellular potency based on NanoBRET assay is 4 nM, 17 nM, and 2 nM towards CLK1/2/4, and 32 nM and 67 nM towards DYRK1A/B, respectively [148]. It has been also shown that T3 induces a decrease in the phosphorylation state of SRSF protein in a time- and dose-dependent manner at 10–1000 nM concentration in HeLa cells [148]. Importantly, the testing was performed in parallel with a negative control compound [148]. As a result, T3 can be recommended as a quality chemical probe for CLK1/2/4, Int.ideallyInt. J. J. Mol. Mol. in Sci. Sci. combination 2020 2020, ,21 21, ,x x FOR FOR with PEER PEER another REVIEW REVIEW selective inhibitor. 18 18 of of 31 31

FigureFigureFigure 18. 18.18. Structure Structure of ofof the thethe compound compoundcompound T3. T3.T3.

InInIn 2017, 2017,2017, Compound CompoundCompound 25 2525 (Figure (Figure(Figure 19) 1919)) was waswas published publishedpublished as asas a aa CLK1 CLK1CLK1 inhibitor inhibitorinhibitor [90]. [[90].90]. While WhileWhile the thethe compound compoundcompound exhibitsexhibitsexhibits the the highest highest potency potency potency towards towards towards CLK1 CLK1 CLK1 (IC (IC (IC5050 50= = 2 2= nM), nM),2 nM), it it is isit also also isalso highly highly highly active active active towards towards towards CLK2 CLK2 CLK2 and and CLK4andCLK4 CLK4 (IC(IC5050 (IC == 503131 = nMnM31 nM andandand 88 nM,nM, 8 nM, respectively),respectively), respectively), butbut but practicallypractically practically inactiveinactive inactive againstagainst against CLK3. CLK3. The The most mostmost significantsignificantsignificant off-target ooff-targetff-target outside outside of of the the CLK CLK subfamilysubfamily subfamily isis is DYRK1ADYRK1A DYRK1A (IC(IC (IC505050 = = 138 138138 nM). nM).nM). Compound CompoundCompound 25 25′0s′ss high highhigh ininin vitro vitrovitro selectivity selectivityselectivity was waswas confirmed confirmedconfirmed in inin the thethe panel panelpanel of ofof 387 387387 human human protein protein kinases kinases at at 10 10 µM µµMM concentration. concentration. TheTheThe cellular cellularcellular activity activityactivity was was investigated investigated via viavia the the effect eeffectffect onon SRSR proteinproteinprotein phosphorylation.phosphorylation.phosphorylation. In In addition, addition,addition, CompoundCompoundCompound 2525 induced inducedinduced autophagy autophagyautophagy in inin BNL BNLBNL CL.2 CL.2CL.2 and andand SKOV-3 SKOV-3SKOV-3 (human (human(human ovarian ovarianovarian cancer cancercancer cell line) cellcell line) cellline) lines. cellcell lines.Thelines. pharmacokinetic TheThe pharmacokineticpharmacokinetic profile (inprofileprofile mice) (in(in is also mice)mice) available isis alsoalso in availableavailable the original inin publicationthethe originaloriginal [90 publicationpublication]. Unfortunately, [90].[90]. Unfortunately,anUnfortunately, inactive control anan compoundinactiveinactive controlcontrol as well compound ascompound the determination asas wellwell as ofas cellularthethe determinationdetermination target engagement ofof cellularcellular are missing. targettarget engagementCompoundengagement 25are are can missing. missing. be considered Compound Compound a useful 25 25 can can tool be be cons compoundconsideredidered afor a useful useful CLK1 tool tool/2/4, compound compound but it would for for beCLK1/2/4, CLK1/2/4, desirable but but to itsupplementit would would be be desirable desirable it with an to to inactive supplement supplement control it it with compound.with an an inactive inactive control control compound. compound.

FigureFigureFigure 19. 19.19. Structure Structure of ofof Compound CompoundCompound 25. 25.25.

TheTheThe orally orallyorally available availableavailable CLK CLKCLK inhibitor inhibitorinhibitor TG693 TG693TG693 (Figure (Figure(Figure 20) 2020)) was waswas reported reportedreported inin 2017 20172017 [127]. [[127].127]. The TheThe compound compoundcompound showsshowsshows goodgoodgood selectivity, selectivity,selectivity, based basedbased on on screeningon screeningscreening against againsagains a panelt t aa panelpanel of 313 ofof kinases 313313 kinaseskinases at 1 µM at concentration;at 11 µMµM concentration;concentration; however, however,itshowever,in vitro itsitsactivity in in vitrovitro towards activityactivity CLK1 towardstowards (IC50 CLK1=CLK1113 (ICnM,(IC5050 biochemical= = 113 113 nM,nM, biochemicalbiochemical assay) is somewhat assay) assay) isis lower somewhatsomewhat compared lowerlower to comparedothercompared known to to other CLKother known inhibitors.known CLK CLK TG693 inhibitors. inhibitors. exhibits TG693 TG693 only exhibi exhibi a weaktsts only aonlyffinity a a weak weak for CLK2affinity affinity/3; for andfor CLK2/3; CLK2/3; to our knowledge, and and to to our our knowledge,theknowledge, activity the againstthe activity activity CLK4 against against has notCLK4 CLK4 been has has determined. not not been been determined. determined. The most The significantThe most most significant significant off-targets off-targets off-targets are HASPIN, are are HASPIN,DYRK1a,HASPIN, andDYRK1a,DYRK1a, DYRK2, andand with DYRK2,DYRK2, residual withwith activities residualresidual of activities7%,activities 16%, and ofof 7%,7%, 23%, 16%,16%, respectively, andand 23%,23%, upon respectively,respectively, treatment uponupon with treatmenttreatment with with 1 1 µM µM TG693. TG693. Although Although TG693 TG693 does does not not fulfill fulfill the the criteria criteria for for a a quality quality probe, probe, it it still still mightmight be be one one of of the the best best tools tools for for selective selective inhibition inhibition of of CLK1. CLK1. TG693 TG693 exhibits exhibits activity activity in in the the cell cell and and inin vivo vivo and and its its pharmacokinetic pharmacokinetic profile profile in in mice mice is is superior superior to to that that of of TG003 TG003 [127]. [127].

FigureFigure 20. 20. Structure Structure of of the the compound compound TG693. TG693.

Indazole1Indazole1 (Figure (Figure 21) 21) was was published published recently recently as as an an inhibitor inhibitor of of CLKs, CLKs, active active in in vivo vivo [133]. [133]. The The compoundcompound potently potently inhibits inhibits CLK1/2/4 CLK1/2/4 and and DYRK1A DYRK1A in in vitro. vitro. However, However, the the data data on on the the selectivity selectivity of of

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 18 of 31

Figure 18. Structure of the compound T3.

In 2017, Compound 25 (Figure 19) was published as a CLK1 inhibitor [90]. While the compound exhibits the highest potency towards CLK1 (IC50 = 2 nM), it is also highly active towards CLK2 and CLK4 (IC50 = 31 nM and 8 nM, respectively), but practically inactive against CLK3. The most significant off-target outside of the CLK subfamily is DYRK1A (IC50 = 138 nM). Compound 25′s high in vitro selectivity was confirmed in the panel of 387 human protein kinases at 10 µM concentration. The cellular activity was investigated via the effect on SR protein phosphorylation. In addition, Compound 25 induced autophagy in BNL CL.2 and SKOV-3 (human ovarian cancer cell line) cell lines. The pharmacokinetic profile (in mice) is also available in the original publication [90]. Unfortunately, an inactive control compound as well as the determination of cellular target engagement are missing. Compound 25 can be considered a useful tool compound for CLK1/2/4, but it would be desirable to supplement it with an inactive control compound.

Figure 19. Structure of Compound 25.

The orally available CLK inhibitor TG693 (Figure 20) was reported in 2017 [127]. The compound shows good selectivity, based on screening against a panel of 313 kinases at 1 µM concentration; however, its in vitro activity towards CLK1 (IC50 = 113 nM, biochemical assay) is somewhat lower

Int.compared J. Mol. Sci. to2020 other, 21, known 7549 CLK inhibitors. TG693 exhibits only a weak affinity for CLK2/3; and 18to ofour 31 knowledge, the activity against CLK4 has not been determined. The most significant off-targets are HASPIN, DYRK1a, and DYRK2, with residual activities of 7%, 16%, and 23%, respectively, upon 1treatmentµM TG693. with Although 1 µM TG693. TG693 Although does not TG693 fulfill thedoes criteria not fulfill for a the quality criteria probe, for a it quality still might probe, be oneit still of themight best be tools one forof the selective best tools inhibition for selective of CLK1. inhibition TG693 of exhibits CLK1. TG693 activity exhibits in the cell activity and inin vivothe celland and its pharmacokineticin vivo and its pharmacokinetic profile in mice isprofile superior in mice to that is superior of TG003 to [127 that]. of TG003 [127].

FigureFigure 20.20. Structure of the compound TG693.TG693.

Int.Int. J.J. Mol.Mol.Indazole1Indazole1 Sci.Sci. 20202020 ,,(Figure (Figure 2121,, xx FORFOR 21) 21 PEERPEER) was was REVIEWREVIEW published published recently recently as asan aninhibitor inhibitor of CLKs, of CLKs, active active in vivoin vivo [133]. 19 19 [ of133 ofThe 31 31]. Thecompound compound potently potently inhibits inhibits CLK1/2/4 CLK1/ 2and/4 and DYRK1A DYRK1A in vitro.in vitro However,. However, the the data data on onthe the selectivity selectivity of Indazole1ofIndazole1 Indazole1 areare are notnot not sufficient,sufficient, sufficient, sincesince since thethe the compoundcompound compound haha hasss beenbeen been profiledprofiled profiled againstagainst against onlyonly only 3434 34 kinaseskinases kinases [133].[133]. [133]. Therefore,Therefore, itsits useuse asas aa chemicalchemical probeprobe shouldshould bebe avoidedavoided atat thisthis point.point.point.

FigureFigure 21. 21. StructureStructure of of the the compound compound Indazole1. Indazole1.

TheThe potent potent CLK CLK inhibitor inhibitor KuWal151 KuWal151 (Figure(Figure 2222)22)) waswas publishedpublished inin 20182018 [[149].[149].149]. The The determined determined IC values for CLK1/2/4 are 88 nM, 510 nM and 28 nM, respectively [149]. Interestingly, KuWal151 is ICIC5050 valuesvalues forfor CLK1/2/4CLK1/2/4 areare 8888 nM,nM, 510510 nMnM andand 2828 nMnM,, respectivelyrespectively [149].[149]. Interestingly,Interestingly, KuWal151KuWal151 isinactiveis inactiveinactive towards towardstowards DYRK1A DYRK1A/BDYRK1A/B/B and andand DYRK2 DYRK2DYRK2 [ 149 [149].[149].]. However, However,However,the thethe compound’s compound’scompound’s selectivityselectivityselectivity profileprofileprofile across across the kinome has not been published thus far and only IC values for 14 kinases have been reported. thethe kinomekinome hashas notnot beenbeen publpublishedished thusthus farfar andand onlyonly ICIC5050 valuesvalues forfor 1414 kinaseskinases havehave beenbeen reported.reported. TheThe antiproliferativeantiproliferative activity activityactivity of ofof KuWal151 KuWal151KuWal151 was waswas demonstrated demodemonstratednstrated in ainin panel aa panelpanel of 57 ofof cancer 5757 cancercancer cell lines—in cellcell lines—inlines—in most mostcases,most cases,cases, the compound thethe compoundcompound was active waswas activeactive at sub-micromolar atat sub-micromolarsub-micromolar concentrations concentrationsconcentrations [149]. [149].[149].

FigureFigure 22. 22. StructureStructure ofof thethe compound compound KuWal151. KuWal151.

T-025T-025 (Figure (Figure 23)2323)) was was reported reported in in 2018 2018 as as a a pote potentpotentnt pan-CLK pan-CLK inhibitor, which which (unlike (unlike most CLK inhibitors) exhibits also high potency against CLK3 (K = 3.5 nM) [118]. The analysis of the crystal inhibitors)inhibitors) exhibitsexhibits alsoalso hihighgh potencypotency againstagainst CLK3CLK3 (K(Kdd == 3.53.5 nM)nM) [118].[118]. TheThe analysisanalysis ofof thethe crystalcrystal structurestructure revealed revealed that that the the inhibitor inhibitor interacts interacts with with Glu244 Glu244 and and Leu246Leu246 inin thethe CLK2CLK2 hinge hinge regionregion region [118].[118]. [118]. TheThe compound’s compound’s good good selectivity selectivity profile profileprofile was was reveal revealedrevealeded via via screening screening against against 468 468 kinases kinases at at 0.3 0.3 µMµµMM concentrationconcentration [118]. [[118].118]. InIn In comparisoncomparison comparison toto to thethe the inhibitor inhibitor T3,T3, T-025T-025 exhibits exhibits lower selectivity selectivity towards towards DYRK1A/B and it inhibits also DYRK2, HIPKs, IRAK4, and YSK4 with K < 100 nM [118]. In addition, DYRK1A/BDYRK1A/B andand itit inhibitsinhibits alsoalso DYRK2,DYRK2, HIPKs,HIPKs, IRAK4,IRAK4, andand YSK4YSK4 withwith KKddd << 100100 nMnM [118].[118]. InIn addition,addition, T-025T-025 exhibitsexhibits cellularcellular andand inin vivo activityactivity and and accord accordingaccordinging to to the the authors,authors, itit representsrepresents the the firstfirst first reportedreported CLKCLK inhibitor inhibitor with with anti-tumor anti-tumor efficacy eefficacyfficacy [118]. [[118].118]. UnfortUnfort Unfortunately,unately,unately, the the cellular potency and selectivity have notnot beenbeen determined.determined.determined. Although AlthoughAlthough T-025 T-025T-025 does doesdoes not notnot fulfill fulffulf theillill thethe criteria criteriacriteria for aforfor quality aa qualityquality chemical chemicalchemical probe probeprobe for CLKs, forfor CLKs,stillCLKs, it mightstillstill itit might bemight a useful bebe aa usefuluseful tool compound tooltool compoundcompound in some inin somesome cases, cases,cases, considering consideringconsidering its potency itsits potencypotency against againstagainst CLK3 CLK3CLK3 and andinand vivo inin vivovivo activity. activity.activity.

FigureFigure 23.23. StructureStructure ofof thethe compoundcompound T-025.T-025.

InIn 2018,2018, CC-671CC-671 (Figure(Figure 24)24) waswas reportedreported asas CLCLK/TTKK/TTK inhibitorinhibitor [137].[137]. Interestingly,Interestingly, thisthis compoundcompound exhibitsexhibits somesome selectivityselectivity forfor CLK2CLK2 (IC(IC5050 == 66 nM)nM) vs.vs. CLK1CLK1 (IC(IC5050 == 300300 nM)nM) andand especiallyespecially CLK3CLK3 (60%(60% residualresidual activityactivity atat 33 µMµM concentration),concentration), basedbased onon biochemicalbiochemical assayassay [137].[137]. Unfortunately,Unfortunately,

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 19 of 31

Indazole1 are not sufficient, since the compound has been profiled against only 34 kinases [133]. Therefore, its use as a chemical probe should be avoided at this point.

Figure 21. Structure of the compound Indazole1.

The potent CLK inhibitor KuWal151 (Figure 22) was published in 2018 [149]. The determined IC50 values for CLK1/2/4 are 88 nM, 510 nM and 28 nM, respectively [149]. Interestingly, KuWal151 is inactive towards DYRK1A/B and DYRK2 [149]. However, the compound’s selectivity profile across the kinome has not been published thus far and only IC50 values for 14 kinases have been reported. The antiproliferative activity of KuWal151 was demonstrated in a panel of 57 cancer cell lines—in most cases, the compound was active at sub-micromolar concentrations [149].

Figure 22. Structure of the compound KuWal151.

T-025 (Figure 23) was reported in 2018 as a potent pan-CLK inhibitor, which (unlike most CLK inhibitors) exhibits also high potency against CLK3 (Kd = 3.5 nM) [118]. The analysis of the crystal structure revealed that the inhibitor interacts with Glu244 and Leu246 in the CLK2 hinge region [118]. The compound’s good selectivity profile was revealed via screening against 468 kinases at 0.3 µM concentration [118]. In comparison to the inhibitor T3, T-025 exhibits lower selectivity towards DYRK1A/B and it inhibits also DYRK2, HIPKs, IRAK4, and YSK4 with Kd < 100 nM [118]. In addition, T-025 exhibits cellular and in vivo activity and according to the authors, it represents the first reported CLK inhibitor with anti-tumor efficacy [118]. Unfortunately, the cellular potency and selectivity have not been determined. Although T-025 does not fulfill the criteria for a quality chemical probe for

Int.CLKs, J. Mol. still Sci. it2020 might, 21, 7549be a useful tool compound in some cases, considering its potency against 19CLK3 of 31 and in vivo activity.

Figure 23. Structure of the compound T-025.

In 2018,2018, CC-671 CC-671 (Figure (Figure 24) was24) reportedwas reported as CLK as/TTK CL inhibitorK/TTK inhibitor [137]. Interestingly, [137]. Interestingly, this compound this exhibits some selectivity for CLK2 (IC = 6 nM) vs. CLK1 (IC = 300 nM) and especially CLK3 Int.compound J. Mol. Sci. 2020exhibits, 21, x someFOR PEER selectivity REVIEW for50 CLK2 (IC50 = 6 nM) vs. CLK150 (IC50 = 300 nM) and especially 20 of 31 Int. J. Mol. Sci. 2020, 21, x FOR PEERµ REVIEW 20 of 31 (60%CLK3 residual (60% residual activity activity at 3 Mat 3 concentration), µM concentration), based based on biochemical on biochemical assay assay [137 ].[137]. Unfortunately, Unfortunately, the thecompound’s compound’s ability ability to inhibit to inhibit CLK4 CLK4 is unknown is unknown as well as as well its selectivity as its selectivity profile in profile the cell. in Although the cell. AlthoughCC-671the compound’s does CC-671 not fulfillabilitydoes thenot to criteriafulfillinhibit the forCLK4 criteria a quality is unknown for probe,a quality as further wellprobe, profilingas furtherits selectivity (and profiling potentially profile (and inpotentially structural the cell. structuralmodifications)Although modifications)CC-671 may does potentially not may fulfill potentially lead the to criteria an lead interesting forto an a qualityinteresting tool compound probe, tool further compound for specific profiling for inhibition specific (and potentially inhibition of CLK2. ofInstructural CLK2. addition, In modifications) addition, CC-671 has CC-671 been may has usedpotentially been in various used lead in cell-basedtovarious an interesting cell-based and in tool vivo and compoundassays in vivo where assays for specific it where elicited inhibitionit growthelicited growthinhibitionof CLK2. inhibition In and addition, induction and CC-671 induction of apoptosis has beenof apoptosis inused various in various in types various cell-based of cancer types cellsandof cancer [in137 vivo]. Thece assaysllsin [137]. vivo where Theeffi itcacy inelicited vivo was efficacyshowngrowth inwasinhibition two shown cell line-derived andin two induction cell line-derived and of oneapoptosis patient and on in tumor-derivede vapatientrious tumor-derived types xenograft of cancer xeno models cellsgraft [137]. of models triple-negative The of in triple- vivo negativebreastefficacy cancer. was breast shown This cancer. therapeuticin two This cell therapeutic line-derived effect was effect credited and was one tocredited patient dual inhibition tumor-derived to dual inhibition of CLK2 xeno/ TTKofgraft CLK2/TTK [137 models]. [137].of triple- negative breast cancer. This therapeutic effect was credited to dual inhibition of CLK2/TTK [137].

Figure 24. Structure of the compound CC-671. Figure 24. Structure of the compound CC-671. The family of pyrido[3,4-g]quinazoline-based compounds inhibiting CLK1, DYRK1A, CDK5, The family of pyrido[3,4-g]quinazoline-based compounds inhibiting CLK1, DYRK1A, CDK5, CK1, CK1, Theand familyGSK3 wasof pyrido[3,4- published gin]quinazoline-based 2019 [150]. More th compoundsan 20 different inhibiting analogs CLK1, with varying DYRK1A, selectivity CDK5, and GSK3 was published in 2019 [150]. More than 20 different analogs with varying selectivity were wereCK1, anddescribed GSK3 wasin thepublished report. in The2019 most[150]. activeMore thcompoundan 20 different towards analogs CLK1 with isvarying the pyrido[3,4- selectivity describedwere described in the report.in the Thereport. most activeThe most compound active towardscompound CLK1 towards is the pyrido[3,4- CLK1 isg ]quinazolinethe pyrido[3,4- 9m g]quinazoline 9m (Figure 25), exhibiting IC50 of 18 nM [150]. The inhibitory activities against (Figure 25), exhibiting IC50 of 18 nM [150]. The inhibitory activities against DYRK1A, CDK5, CK1, DYRK1A,g]quinazoline CDK5, 9m CK1, (Figure and 25),GSK3 exhibiting were determined IC50 of 18 at nMfixed [150]. concentrations The inhibitory (10 µM activities and 1 µM); against the and GSK3 were determined at fixed concentrations (10 µM and 1 µM); the residual kinase activities residualDYRK1A, kinase CDK5, activities CK1, and at 1µMGSK3 concentration were determined are: DYRK1Aat fixed concentrations = 39%, CDK5 =(10 73%, µM CK1 and =1 98%,µM); and the at 1 µM concentration are: DYRK1A = 39%, CDK5 = 73%, CK1 = 98%, and GSK3 = 70%. However, GSK3residual = 70%. kinase However, activities more at 1µM detailed concentration information are: about DYRK1A the kinome-wide = 39%, CDK5 selectivity = 73%, CK1 is not = 98%,available and more detailed information about the kinome-wide selectivity is not available [150]. [150].GSK3 = 70%. However, more detailed information about the kinome-wide selectivity is not available [150].

Figure 25. StructureStructure of of the the compound compound pyrido[3,4- g]quinazoline]quinazoline 9m. Figure 25. Structure of the compound pyrido[3,4-g]quinazoline 9m. The SGC developed CLK1/2/4 inhibitor SGC-CLK-1 (Figure 26) in collaboration with Luceome BiotechnologiesThe SGC developed [151]. The CLK1/2/4 compou nd’sinhibitor activity SGC-CLK-1 towards (FigureCLK1/2/4 26) was in collaboration determined inwith biochemical Luceome Biotechnologies [151]. The compound’s activity towards CLK1/2/4 was determined in biochemical assays and confirmed in the cell by NanoBRET assay (in cell IC50 = 165/100/70 nM for CLK1/2/4, respectively)assays and confirmed [151]. SGC-CLK-1 in the cell was by testedNanoBRET in a panelassay of(in 403 cell kinases IC50 = 165/100/70and the potential nM for off-targetsCLK1/2/4, (HIPK1/2,respectively) ERK8, [151]. NEK7, SGC-CLK-1 PIP5K2B, was STK16) tested were in thena panel evaluated of 403 inkinases NanoBRET and the assay potential [151]. As off-targets a result, the(HIPK1/2, only off-target ERK8, NEK7, found PIP5K2B, to be partially STK16) inhibitedwere then in evaluated the cell wasin NanoBRET STK16 [151]. assay A [151].negative As acontrol result, compoundthe only off-target was tested found in toparallel be partially with inhibitedSGC-CLK-1. in theThis cell inhibitor was STK16 represents [151]. A an negative excellent control tool compound forwas cell-based tested in experiments parallel with and SGC-CLK-1.it can be recommended This inhibitor as a qualityrepresents probe an for excellent CLK1/2/4. tool compound for cell-based experiments and it can be recommended as a quality probe for CLK1/2/4.

Figure 26. Structure of the compound SGC-CLK-1. Figure 26. Structure of the compound SGC-CLK-1.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 20 of 31 the compound’s ability to inhibit CLK4 is unknown as well as its selectivity profile in the cell. Although CC-671 does not fulfill the criteria for a quality probe, further profiling (and potentially structural modifications) may potentially lead to an interesting tool compound for specific inhibition of CLK2. In addition, CC-671 has been used in various cell-based and in vivo assays where it elicited growth inhibition and induction of apoptosis in various types of cancer cells [137]. The in vivo efficacy was shown in two cell line-derived and one patient tumor-derived xenograft models of triple- negative breast cancer. This therapeutic effect was credited to dual inhibition of CLK2/TTK [137].

Figure 24. Structure of the compound CC-671.

The family of pyrido[3,4-g]quinazoline-based compounds inhibiting CLK1, DYRK1A, CDK5, CK1, and GSK3 was published in 2019 [150]. More than 20 different analogs with varying selectivity were described in the report. The most active compound towards CLK1 is the pyrido[3,4- g]quinazoline 9m (Figure 25), exhibiting IC50 of 18 nM [150]. The inhibitory activities against DYRK1A, CDK5, CK1, and GSK3 were determined at fixed concentrations (10 µM and 1 µM); the residual kinase activities at 1µM concentration are: DYRK1A = 39%, CDK5 = 73%, CK1 = 98%, and GSK3 = 70%. However, more detailed information about the kinome-wide selectivity is not available [150].

Int. J. Mol. Sci. 2020, 21, 7549 20 of 31 Figure 25. Structure of the compound pyrido[3,4-g]quinazoline 9m. The SGC developed CLK1/2/4 inhibitor SGC-CLK-1 (Figure 26) in collaboration with Luceome BiotechnologiesThe SGC developed [151]. The CLK1/2/4 compound’s inhibitor activity SGC-CLK-1 towards (Figure CLK1/2 /26)4 was in collaboration determined inwith biochemical Luceome Biotechnologies [151]. The compound’s activity towards CLK1/2/4 was determined in biochemical assays and confirmed in the cell by NanoBRET assay (in cell IC50 = 165/100/70 nM for CLK1/2/4, respectively)assays and confirmed [151]. SGC-CLK-1 in the cell was by NanoBRET tested in a panelassay of(in 403cell kinases IC50 = 165/100/70 and the potential nM for oCLK1/2/4,ff-targets (HIPK1respectively)/2, ERK8, [151]. NEK7, SGC-CLK-1 PIP5K2B, was STK16) tested were in thena panel evaluated of 403 inkinases NanoBRET and the assay potential [151]. Asoff-targets a result, the(HIPK1/2, only o ffERK8,-target NEK7, found PIP5K2B, to be partially STK16) inhibited were then in evaluated the cell was in NanoBRET STK16 [151 assay]. A [151]. negative As a control result, compoundthe only off-target was tested found in parallel to be withpartially SGC-CLK-1. inhibited This in inhibitorthe cell was represents STK16 an [151]. excellent A negative tool compound control forcompound cell-based was experiments tested in andparallel it can with be recommended SGC-CLK-1. asThis a quality inhibitor probe represents for CLK1 /an2/4. excellent tool compound for cell-based experiments and it can be recommended as a quality probe for CLK1/2/4.

Int.Int. J.J. Mol.Mol. Sci.Sci. 2020,, 21,, xx FORFOR PEERPEERFigureFigure REVIEWREVIEW 26.26. Structure of the compound SGC-CLK-1.SGC-CLK-1. 21 21 of of 31 31

SM08502 (Figure 27)27) represents the first first CLKCLK inhibitorinhibitor thatthat enteredentered clinicalclinical trialstrials [[76].76]. The exact structure of this isoquinoline-basedisoquinoline-based compound ha hass not been disclosed so far.far. Since the compoundcompound inhibitsinhibits several several off-targets ooff-targetsff-targets (19(19 additionaladditional kinaseskinases withwithwith ICICIC505050 << 505050 nM),nM), nM), itit isis notnot suitable suitable for for utilization utilization as a chemicalchemical probe.probe. On On the other hand, its favorablefavorable pharmacological properties and anti-tumor efficacyefficacy enabledenabled thethe initiation initiation of of Phase Phase I of I of clinical clinical trials trials using using SM08502 SM08502 in patients in patients with with advanced advanced solid solidtumors tumors [76]. It[76]. has beenIt has suggested been suggested that the compound’sthat thethe compound’scompound’s pan-CLK inhibitorypan-CLKpan-CLK inhibitory activityinhibitory is substantialactivityactivity isis substantialfor sufficient for in sufficient cell and in in vivo cell anti-tumorand in vivo activity anti-tumor [76]. Particularly,activity [76]. it Particularly, seems to be importantit seems to that be importantSM08502important inhibits thatthat SM08502SM08502 also CLK3, inhibitsinhibits which alsoalso is CLK3,CLK3, often not whichwhich the isis case oftenoften for notnot other thethe CLK casecase inhibitors forfor otother CLK [76]. inhibitors [76].

Figure 27. GenericGeneric structure structure of the compound SM08502.

In vitro profilingprofiling of a a series series of of 3,5-disubstituted 3,5-disubstituted furo[3,2- furo[3,2-b]pyridines revealed highly potent and selective CLK1/2/4 CLK1/2/4 inhibitors [[47].47]. The The analog analog most most active active in in the the cell, cell, compound compound MU1210 MU1210 (Figure (Figure 28),28), was profiledprofiled in in greater greater detail detail using using radioenzymatic radioenzymatic and NanoBRETand NanoBRET assays, assays, in parallel in parallel with the with negative the negativecontrol MU140 control (Table MU1404). (Table 4).

Figure 28. StructureStructure of the compound MU1210.

Table 4. Biochemical and cellular activity of MU1210 and negative control MU140. The cellular Kii values were determined by NanoBRET assays in HEK293T cells.

Kinase MU140 Activity [nM] MU1210 Activity [nM] (Negative Control) Biochemical IC5050 CellularCellular KKii BiochemicalBiochemical ICIC5050 CellularCellular KKii CLK1 8 84 >3000 >10,,000 CLK2 20 91 >10,000 >10,000 CLK3 >3000 - >3000 - CLK4 12 23 >10,000 >10,000 DYRK1A 213 6580 >3000 >10,000 DYRK1B 956 >10,000 >3000 >10,000 DYRK2 1309 1700 >10,,000 >10,,000 HIPK1 187 - >10,000 - HIPK2 29 >10000 >10,000 - HIPK3 159 - >3000 - HIPK4 - 5410 - -

Int.Int. J.J. Mol.Mol. Sci. 2020, 21, 7549x FOR PEER REVIEW 2221 of 31 31

HIPK2 was revealed as the most significant off-target in vitro (biochemical assay); however, it was HIPK2not inhibited was revealed by MU1210 as the in most the significantcell (based o ffon-target the NanoBRETin vitro (biochemical assay). Similarly, assay); however,the NanoBRET it was notassay inhibited demonstrated by MU1210 only invery the weak cell (based activity on against the NanoBRET DYRKs (typical assay). Similarly,problematic the off-targets NanoBRET of assay CLK demonstratedinhibitors) and only GSK3 veryα, and weak overall activity excellent against selectivity DYRKs (typical profile problematic in the cell. oAdditionalff-targets of weak CLK inhibitors)off-targets anddetermined GSK3α, andin the overall primary excellent biochemical selectivity screen profile were in the GSK3 cell.α, Additional PIM1, PIM2, weak and off HASPIN-targets determined exhibiting inresidual the primary activity biochemical of 10%, 16%, screen 19%, were and GSK326%, respα, PIM1,ectively, PIM2, at 1 and µM HASPIN concentration exhibiting of MU1210. residual MU1210 activity of 10%, 16%, 19%, and 26%, respectively, at 1 µM concentration of MU1210. MU1210 exhibited exhibited anti-tumor activity in MCF7, MDA MB 231, and MCF10A breast cancer cell lines (with EC50 anti-tumorvalues of 1.1, activity 1.3, and in 1.5 MCF7, µM, MDArespectively) MB 231, as and well MCF10A as in HeLa, breast U2OS, cancer PANC1, cell lines HEK293T, (with ECAGP01,50 values and ofMEF 1.1, cancer 1.3, and cell 1.5 lines.µM, respectively)In addition, asMU1210 well as affected in HeLa, phosphorylatio U2OS, PANC1,n HEK293T,of SR protein AGP01, in HeLa and MEFcells cancer(while cellthe lines.negative In addition, control MU1210MU140 had affected no effect) phosphorylation and modulated of SR proteinalternative in HeLa mRNA cells splicing (while the of negativeMDM4 [47,152]. control MU140 had no effect) and modulated alternative mRNA splicing of MDM4 [47,152]. Overall,Overall, thethe compoundcompound MU1210MU1210 isis aa potentpotent CLK1CLK1/2/4/2/4 inhibitorinhibitor inin vitrovitro asas wellwell asas inin thethe cellcell thatthat exhibitsexhibits excellentexcellent selectivityselectivity profileprofile acrossacross thethe kinome,kinome, fulfillingfulfilling thethe criteriacriteria forfor aa qualityquality chemicalchemical probeprobe [[154–156].154–156]. TheThe compound compound has has been been recommended recommended by by the the Structural Structural Genomic Genomic Consortium Consortium as the as state-of-the-artthe state-of-the-art tool tool compound compound for CLK1for CLK1/2/4/2/4 [152 ].[152]. TheThe irreversibleirreversible inhibitor inhibitor of Trypanosomaof Trypanosoma brucei bruceiCLK1, CLK1, compound compound AB1 (Figure AB1 29(Figure), was published29), was verypublished recently very [89 recently]. The phenotypic[89]. The phenotypic screen of thescreenT. bruceiof thebloodstream T. brucei bloodstream forms with forms the Novartiswith the kinase-focusedNovartis kinase-focused inhibitor library inhibito (2.3r library million (2.3 compounds) million compounds) identified compounds identified withcompounds pan-kinetoplastid with pan- activity.kinetoplastid Further activity. testing Further against testingT. brucei againstmutants T. thatbrucei overexpress mutants that known overexpress essential known protein essential kinases revealedprotein kinases CLK1 as revealed the primary CLK1 target as the and primary subsequent target SAR and studies subsequent provided SAR the optimizedstudies provided compound the AB1.optimized The irreversible compound competitive AB1. The inhibitionirreversible of CLK1competitive is provided inhibition by the of Michael CLK1 acceptoris provided acryl by amide the motif,Michael which acceptor forms acryl a covalent amide motif, bond withwhich the forms C215 a residue covalent in bond the active with sitethe cavity.C215 residue That residue in the active is not presentsite cavity. in human That residue CLK1, is thereby not present providing in human the compound’sCLK1, thereby selectivity providing for theTrypanosoma compound’s brucei selectivityCLK1 overfor Trypanosoma the human variant. brucei CLK1 AB1 exhibits over the also human potent variant. inhibitory AB1 activity exhibits against also hERGFRpotent inhibitory and BTK kinasesactivity (IC values are 2 nM and 2 nM, respectively); additional information on the selectivity has not been against50 hERGFR and BTK kinases (IC50 values are 2 nM and 2 nM, respectively); additional publishedinformation yet on [89 the]. selectivity has not been published yet [89].

HN O N O NH N N

N

AB1 FigureFigure 29.29. Structure ofof thethe compoundcompound AB1.AB1.

TheThe compoundcompound Lead-30Lead-30 as well as its precursor,precursor, thethe screeningscreening hithit TCMDC-135051TCMDC-135051 (Figure 3030),), werewere publishedpublished veryvery recentlyrecently [130[130,131].,131]. Both TCMDC-135051 and Lead-30Lead-30 inhibitinhibit PfPfCLK3 inin PlasmodiumPlasmodium falciparum (IC(IC5050 = =4040 nM nM and and 19 19nM, nM, respectively). respectively). Screening Screening of TCMDC-135051 of TCMDC-135051 (at 1 µM (at 1concentration)µM concentration) against against 140 kinases 140 kinases revealed revealed 14 addi 14tional additional kinases kinases showing showing residual residual activity activity below below20%. TCMDC-135051 20%. TCMDC-135051 was found was found to be to also be also active active in invivo—its vivo—its dose-dependent dose-dependent efficacy efficacy waswas demonstrateddemonstrated inin P.P. bergheiberghei-infected-infected micemice [[130].130]. TheseThese resultsresults makemake TCMDC-135051TCMDC-135051 andand Lead-30Lead-30 goodgood candidatescandidates forfor furtherfurther development;development; however, thethe compoundscompounds havehave not been susufficientlyfficiently profiledprofiled forfor selectivityselectivity toto bebe utilizedutilized asas qualityquality chemicalchemical probes.probes.

Int. J. Mol. Sci. 2020, 21, 7549 22 of 31 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 23 of 31

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 23 of 31

FigureFigure 30. 30. StructuresStructures of of the the compounds compounds TCMDC-135051 TCMDC-135051 and Lead-30. Figure 30. Structures of the compounds TCMDC-135051 and Lead-30. The chemical structures of the CLK inhibitors reviewed above are collectively represented in The Thechemical chemical structures structures of ofthe the CLK CLK inhibitors inhibitors reviewed above above are are collectively collectively represented represented in in Figure 31. FigureFigure 31. 31.

FigureFigure 31. 31.Chemical Chemical structuresstructures of of th thee reviewed reviewed CLK CLK inhibitors. inhibitors.

Figure 31. Chemical structures of the reviewed CLK inhibitors.

Int.Int. J. Mol.J. Mol. Sci. Sci. 2020 2020, 21, 21, x, FORx FOR PEER PEER REVIEW REVIEW 21 21 of of 31 31

SM08502SM08502 (Figure (Figure 27) 27) represents represents the the first first CLK CLK inhibitor inhibitor that that entered entered clinical clinical trials trials [76]. [76]. The The exact exact structurestructure of of this this isoquinoline-based isoquinoline-based compound compound ha has snot not been been disclosed disclosed so so far. far. Since Since the the compound compound inhibitsinhibits several several off-targets off-targets (19 (19 additional additional kinases kinases with with IC IC50 50< <50 50 nM), nM), it itis isnot not suitable suitable for for utilization utilization asas a achemical chemical probe. probe. On On the the other other hand, hand, its its favo favorablerable pharmacological pharmacological properties properties and and anti-tumor anti-tumor efficacyefficacy enabled enabled the the initiation initiation of of Phase Phase I ofI of clinical clinical trials trials using using SM08502 SM08502 in in patients patients with with advanced advanced solidsolid tumors tumors [76]. [76]. It Ithas has been been suggested suggested that that the the compound’s compound’s pan-CLK pan-CLK inhibitory inhibitory activity activity is is substantialsubstantial for for sufficient sufficient in in cell cell and and in in vivo vivo anti-tumor anti-tumor activity activity [76]. [76]. Particularly, Particularly, it itseems seems to to be be importantimportant that that SM08502 SM08502 inhibits inhibits also also CLK3, CLK3, which which is isoften often not not the the case case for for ot otherher CLK CLK inhibitors inhibitors [76]. [76].

FigureFigure 27. 27. Generic Generic structure structure of of the the compound compound SM08502. SM08502.

InIn vitro vitro profiling profiling of of a seriesa series of of 3,5-disubstituted 3,5-disubstituted furo[3,2- furo[3,2-b]pyridinesb]pyridines revealed revealed highly highly potent potent and and selectiveselective CLK1/2/4 CLK1/2/4 inhibitors inhibitors [47]. [47]. The The analog analog most most active active in in the the cell, cell, compound compound MU1210 MU1210 (Figure (Figure 28), 28), waswas profiled profiled in in greater greater detail detail using using radioenzymatic radioenzymatic and and NanoBRET NanoBRET assays, assays, in in parallel parallel with with the the negativenegative control control MU140 MU140 (Table (Table 4). 4).

Int. J. Mol. Sci. 2020, 21, 7549 23 of 31 FigureFigure 28. 28. Structure Structure of of the the compound compound MU1210. MU1210.

TableTableTable 4. 4. 4.Biochemical BiochemicalBiochemical and andand cellular cellularcellular acti activityactivityvity of ofof MU1210 MU1210MU1210 and andand negative negativenegative control controlcontrol MU140. MU140.MU140. The TheThe cellular cellularcellular K KiK ii valuesvaluesvalues were werewere determined determineddetermined by byby Nano NanoBRETNanoBRETBRET assays assaysassays in inin HEK293T HEK293THEK293T cells. cells.cells.

Kinase KinaseKinase MU140MU140MU140 Activity Activity Activity [nM][nM] [nM] MU1210MU1210MU1210 Activity Activity [nM] [nM][nM] (Negative(Negative(Negative Control) Control)

BiochemicalBiochemicalBiochemical ICIC 50IC50 50 CellularCellular K Ki Ki i BiochemicalBiochemicalBiochemical IC IC50 IC50 50 CellularCellularCellular Ki K Ki i CLK1CLK1CLK1 8 8 84 84 84 > >30003000 >3000 >10,000>10>10,000,000 CLK2CLK2CLK2 20 20 91 91 91 > >10,00010,000 >10,000 >10,000>10,000>10,000 CLK3CLK3CLK3 >3000>3000>3000 -- - >>30003000>3000 - - - CLK4CLK4CLK4 12 12 23 23 23 > >10,00010,000 >10,000 >10,000>10,000>10,000 DYRK1A 213 6580 >3000 >10,000 DYRK1ADYRK1ADYRK1B 213 956213 >10,000 6580 6580 > >30003000 >3000 >10,000 >10,000 >10,000 DYRK1BDYRK1BDYRK2 1309956956 >10,000 1700 >10,000 >10,000 >3000 >3000 >10,000 >10,000 >10,000 DYRK2DYRK2HIPK1 1309 1871309 1700 - 1700 > >1010,000 >10,000,000 >10 ->10,000,000 HIPK1HIPK1HIPK2 187 29187 >10,000 - - >>10,00010,000>10,000 - - - HIPK2HIPK2HIPK3 1592929 >10000 >10000 - >>10,0003000>10,000 - - - HIPK4 - 5410 - - HIPK3HIPK3 159159 - - >3000 >3000 - - HIPK4HIPK4 - - 54105410 - - - - 7. Conclusions Over the last two decades, a growing number of studies not only elucidated various aspects of the relatively complex biology of CLKs but also pre-clinically investigated their therapeutic potential. Some of the recently discovered structurally diverse small-molecule CLK inhibitors (summarized in this review) demonstrate sufficient selective activity to fulfill the criteria for quality chemical biology probes. The use of those selective compounds will very likely help further investigate different aspects of the CLK biology, including the role of the individual isoforms and therapeutic potential of their specific inhibition, which may be important in some contexts. The first CLK inhibitor SM08502 that entered clinical trials quite recently (and other compounds that are likely to follow) will demonstrate whether and how inhibition of CLKs can be used for the development of novel .

Author Contributions: Writing—original draft preparation, P.M.M., V.N. and K.P.; writing—review and editing, P.M.M., V.N. and K.P.; funding acquisition, K.P. All authors have read and agreed to the published version of the manuscript. Funding: The research was funded by the following sources: European Structural and Investment Funds, Operational Programme Research, Development and Education, Preclinical Progression of New Organic Compounds with Targeted Biological Activity (Preclinprogress)-CZ.02.1.01/0.0/0.0/16_025/0007381, project no. LQ1605 from the National Program of Sustainability II (MEYS CR), the project CZ-OPENSCREEN: National Infrastructure for Chemical Biology (LM2015063), and Bader Philanthropies. Conflicts of Interest: The authors declare no conflict of interest.

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