Perspective Dual-Specificity Tyrosine Phosphorylation-Regulated

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Perspective

Dual Specificity Tyrosine-Phosphorylation-Regulated Kinase 1a (DYRK1a) Kinase Inhibitors as Potential Therapeutics Dnyandev B Jarhad, Karishma K Mashelkar, Hong-Rae Kim, Minsoo Noh, and Lak Shin Jeong J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00185 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 9, 2018

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Journal of Medicinal Chemistry

Perspective Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A) Inhibitors as Potential Therapeutics Dnyandev B. Jarhad,† Karishma K. Mashelkar,† Hong-Rae Kim, Minsoo Noh, Lak Shin Jeong*

Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 08826, Korea ABSTRACT: Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a member of an evolutionarily conserved family of protein kinases that belongs to the CMGC group of kinases. DYRK1A, encoded by a gene located in the human chromosome 21q22.2 region, has attracted attention due to its association with both neuropathological phenotypes and cancer susceptibility in patients with Down syndrome (DS). Inhibition of DYRK1A attenuates cognitive dysfunctions in animal models for both DS and Alzheimer's disease (AD). Furthermore, DYRK1A has been studied as a potential cancer therapeutic target because of its role in the regulation of cell cycle progression by affecting both tumor suppressors and oncogenes. Consequently, selective synthetic inhibitors have been developed to determine the role of DYRK1A in various human diseases. Our perspective includes a comprehensive review of potent and selective DYRK1A inhibitors and their forthcoming therapeutic applications.

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■ INTRODUCTION Protein kinases constitute one of the largest superfamily of proteins in eukaryotes and have evolved as key enzymes for the regulation of basic cellular functions and processes involving the chemical addition of phosphate groups to the proteins, a process known as phosphorylation.1

Dual-specificity

tyrosine-regulated

kinases

(DYRKs)

constitute

an

evolutionarily conserved family of protein kinases and belong to the CMGC (named after the initials of some of its members) group of kinases, which also includes other kinase families such as cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), glycogen synthase kinase-3 (GSK3) and CDK-like kinases (CLKs).2 DYRKs have been identified in organisms such as baker’s yeast (YAK1), fission yeast (POM1), and Drosophila (minibrain, mnb).3 Two major classes of its mammalian homolog DYRK have been recognized: class I DYRKs, which include DYRK1A and DYRK1B, and class II DYRKs, which include DYRK2, DYRK3 and DYRK4.4 DYRK-related kinases have distinct structural and enzymatic features5 and play diverse roles in cell cycle control and cell differentiation.4 Among the mammalian DYRKs, DYRK1A has attracted therapeutic attention because of its genetic locus in human chromosome 21q22.2, covering the Down syndrome critical region. DYRK possesses dual specificity because of its capability to autophosphorylate tyrosine residue Y321 in its own activation loop as well as to phosphorylate its substrates at serine or threonine residues.6,7 In addition to the role of DYRK1A in apoptosis, cell cycle regulation, splicing and signaling pathways, it has also been shown to be involved in brain growth8, neuronal development9 and synaptic transmission10. Hyperactivity of DYRK1A has been correlated with abnormal brain development, neurodegeneration, cancer, cognitive disabilities, and early onset Alzheimer’s disease (AD) in individuals with Down syndrome (DS).11-14 Studies have shown that 2 ACS Paragon Plus Environment

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the loss-of-function mutations of DYRK1A are responsible for the syndromic form of intellectual disabilities with microcephaly, epilepsy and autistic troubles, which is generally referred as autosomal dominant mental retardation 7 syndrome (MRD7).15,16 As estimated by the Centers for Disease Control and Prevention, approximately 5 million people suffer from Alzheimer's disease in the United States alone, the country with the highest aging population, and approximately 83,000 people die from this condition each year. Thus, the need for DYRK1A inhibitors has become a major concern and has motivated scientists to develop potential therapeutic inhibitors for applications in DS15, AD17,18, cancer19-21 and diabetes22-24. Herein, we present a comprehensive review of the literature on the currently available potent and selective DYRK1A inhibitors and their forthcoming therapeutic applications.

■ ROLE OF DYRK1A ACTIVITY IN VARIOUS DISEASES Neurodegenerative diseases Alzheimer’s disease: Alzheimer’s disease is a neurodegenerative disease known to be caused by β-amyloid protein deposits, called amyloid plaques, and neurofibrillary tangles (NFTs).25 It is one of the leading causes of deaths worldwide as the aging population increases. NFTs are formed as a result of hyperphosphorylation of the microtubule-binding protein Tau and its intracellular aggregation in the brain.26 Researchers have postulated that DYRK1A phosphorylates Tau protein at Thr212 in neuroblastoma cells, which leads to the formation of NFTs.27 It has also been found that DYRK1A phosphorylates amyloid precursor protein (APP) at the Thr668 residue in vitro.28 Thus, the overexpression of DYRK1A enhances the levels of phospho-APP, and hence β-amyloid, in the brain. A greater than theoretically expected (1.5-fold) 3 ACS Paragon Plus Environment


overexpression of the APP gene in DS has been observed, thereby causing an increase in βamyloid production. This could explain the appearance of early onset AD-like pathologies in DS patients.29 As yet, no cure or effective treatment has been found for Alzheimer’s disease despite intense research since its discovery in 1906. A potent inhibitor of DYRK1A, harmine, inhibits the DYRK1A-mediated phosphorylation of Tau, with an IC50 value of 0.7 µM.30 Down syndrome: Down syndrome (DS) is a common genetic disorder that causes retardation in physical growth and neuronal abnormalities in DS individuals. Down syndrome is likely to be related with the trisomy of human chromosome 21, which arises from chromosomal nondisjunction during meiotic cell division.31 DYRK1A maps to human chromosomal region 21q22.2 and is therefore associated with this disease.32 The overexpression of DYRK1A has been observed in patients with DS, causing splicing aberrations and leading to changes in mRNA expression levels.33 However, the mechanism by which trisomy 21 is actually responsible for DS is not yet understood in detail. Similar to AD, an effective treatment for DS has not yet been reported; therefore, DYRK1A is an attractive therapeutic target for this disease. Cancer As mentioned earlier, DYRK1A plays an important role in cell cycle control. Hence, the overexpression of DYRK1A may cause deregulation of the cell cycle. Examination of tumor cells showed overexpression of kinases such as DYRK and CLK. Sprouty2 (Spry2) protein negatively modulates epidermal growth factor receptor (EGFR) signaling, whose mutation might promote oncogenesis. However, DYRK1A phosphorylates T75 on spry2 and thus prevents EGFR degradation.34 In addition, DYRK1A increases the expression of the antiapoptotic enzyme Bcl-XL, thereby contributing to cancer.35 DYRK1A phosphorylates CDK inhibitor p27Kip1 (CDKN1B) and Cyclin D1 (CCND1), which has been investigated in cancer cell cycle 4 ACS Paragon Plus Environment

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progression, and thus, regulates cell proliferation.36 Caspase 9, which is associated with apoptosis processing, is also known to be phosphorylated by DYRK1A at Thr 125 which interrupts apoptosis.37 Most solid tumors are resistant to pro-apoptotic stimuli and are therefore resistant to chemotherapy, as in the case of leukemias, melanomas and glioblastomas. Diabetes Approximately 5% of the world’s population suffers from diabetes. The diabetic condition (type I) arises from a deficiency of insulin-secreting β-cells. NFATc (nuclear factor of activated Tcells, cytoplasmic) plays a crucial role in the activation of human β-cell proliferation. DYRK1A phosphorylates NFAT, which is further phosphorylated by GSK3β and CK1 (casein kinase 1); its removal from the nucleus leads to its inactivation. Inhibition of DYRK1A prompts translocation of NFAT into the nucleus, which promotes β-cell proliferation.22,23 Harmine, a DYRK1A inhibitor, has been known to induce β-cell proliferation.38 Because higher than normal levels of DYRK1A have adverse effects on human health, its consequences cannot be neglected. Thus, it is important to design a potent and specific DYRK1A inhibitor

for

the

benefit

of

appropriate

patients.

■ ACTIVATION AND INHIBITION OF DYRK1A DYRK1A kinases must be activated before they can phosphorylate their substrates. During translation, DYRK1A activates itself by intramolecular (Cis) autophosphorylation at the conserved tyrosine (Y321) residue39 in its activation loop, as demonstrated by the mutation of Mg2+ ATP-binding lysine in the catalytic domain.6 This binding leads to the formation of a transient protein intermediate,6 which produces an active conformation of DYRK1A. This active 5 ACS Paragon Plus Environment


form is believed to be stabilized by Y321, which is the primary mediator of a network of interactions.40 Interestingly, in vitro studies have shown that Y321 cannot be rephosphorylated after phosphatase activity.6,41 DYRK1A kinase activity is no longer influenced by tyrosine phosphorylation once the protein is completely translated, as dephosphorylating Y321 in the active kinase does not significantly decrease its kinase activity in vitro.41 Matured and active DYRK1A kinase phosphorylates its substrates only at serine (S) and threonine (T) residues that have a proline residue situated carboxyl-terminal to the target site42, thereby expressing its activity in diverse biological functions. Substitution of the second Y321 in the activation domain by phenylalanine, histidine or glutamine resulted in a substantial decrease of DYRK1A activity by >80%.6,43 Although intense efforts have been made to develop DYRK1A inhibitors, highly potent and specific inhibitors are still lacking. Many inhibitors of DYRK1A from natural and synthetic sources have been reviewed.18,21,44-46 However, it is difficult to design a rationale for the chemical synthesis of an inhibitor, since its exact mechanism of action is still unclear. Small cellpermeant molecules are important from a medicinal chemistry point of view in determining the molecular interactions of molecules with the target enzyme. To interpret its mechanism of action, different docking and co-crystallization experiments were performed with competitive ATP inhibitors that gave insight into the necessary interactions between the amino acid residues of DYRK1A and the inhibitors.46 Currently, it appears that most of the inhibitors suppress DYRK1A kinase activity by interacting with residues in the ATP-binding site, resulting in the prevention of protein autophosphorylation or inhibition of mature protein activity. The activity of inhibitors against DYRK1A and their mode of action can now be analyzed using a simple and rapid ELISA DYRK1A non-radioactive kinase assay.47 6 ACS Paragon Plus Environment

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Figure 1. Structure of DYRK1A inhibitor 1 that has been co-crystalized with DYRK1A kinase.40 The crystal structure analysis of DYRK1A has been reported by co-crystallization with various inhibitors such as harmine46 and INDY48 ((Z)-1-(3-ethyl-5-hydroxy-2(3H)-benzothiazolylidene)2-proanone)). Recently, Elkins and colleagues reported the crystal structure of DYRK1A complexed with ATP-mimetic inhibitor 1 (Figure 1).40 Compound 1 binds to the ATP binding site and interacts with the hinge backbone through three hydrogen bonds. The primary amine of 1 forms two additional hydrogen bonds with the side chains of Asn292 and the DFG motif aspartate Asp307.40 Compound 1 was also found to interact with Asp307 through electrostatic interaction between the amide nitrogen of the inhibitor and the nitrogen of Asp307 via an ion (modeled as chloride). Moreover, hydrogen bonding occurs with the backbone carbonyl of Glu291 via a water molecule. The reader is referred to the original reference describing the detailed binding interactions of inhibitor 1 with the DYRK1A kinase domain for further clarification.40 Surprisingly, the potent natural polyphenol epigallocatechin gallate (EGCG, 2) is noncompetitive with ATP49, and its mechanism of action is still uncertain.

■ CLASSIFICATION OF DYRK1A INHIBITORS



Several DYRK1A inhibitors were initially designed or postulated to specifically target other protein kinases. Nevertheless, over the years, several classes of DYRK1A inhibitors have been reported, and few are used as pharmacological tools. Kinase inhibitors in general are categorized as type I, type II or type III inhibitors. Type I inhibitors, known as ATP competitors, bind to the ATP binding site of the kinase domain when the activation loop is phosphorylated. However, type II and III are non-ATP competitors. Type II inhibitors bind to the ATP binding site but partially bind to an allosteric pocket, whereas type III inhibitors particularly bind to an allosteric pocket. Type II and III inhibitors induce conformational changes in the target enzyme that result in the loss of kinase activity. The alkaloid compound, harmine (an ATP competitor) has been identified as the conventional DYRK1A inhibitor.46 Herein, we have classified the DYRK1A inhibitors into two classes: i) natural DYRK1A inhibitors and their derivatives; and ii) synthetic DYRK1A inhibitors. In addition, the synthetic inhibitors are further divided into subclasses based on their heterocyclic skeleton.

NATURALLY OCCURRING DYRK1A INHIBITORS AND THEIR DERIVATIVES There are various DYRK1A inhibitors obtained from natural sources, and these inhibitors mainly contain either nitrogen heterocycles or polyphenols. Selective profiling studies against a panel of kinases have identified two natural compounds (2 and 3) as DYRK1A inhibitors. The potent and selective inhibitors of DYRK1A are described below. Epigallocatechin gallate The antioxidant, anti-inflammatory and antitumor properties of polyphenols make them a unique class of phytochemicals. Compound 2, a catechin derivative, was extracted as a major 8 ACS Paragon Plus Environment

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component from green tea and has been identified as the first DYRK inhibitor in animals and humans (Figure 2). Bain and colleagues identified that 2 specifically inhibited DYRK1A (IC50 = 330 nM) from among 29 tested kinases that were structurally and functionally related to DYRK1A.50 However, it does not selectively inhibit DYRK1A. It also inhibits p38regulated/activated kinase (PRAK) (IC50 = 1 µM) activity by binding to PRAK, resulting in inhibition of PRAK activation.51 In 2006, Adayev et al., using a combination of biochemical and genetic studies, postulated that 2 inhibits DYRK1A by binding to a site in/around the kinase domain and identified it as a non-ATP-competitor.49 DYRK1A-overexpressing transgenic mice, fed on a green tea diet from incubation to adulthood, displayed brain structure development (brain weight and thalamus-hypothalamus volume) and improved cognitive deficits in long- and short-term memory.52

Figure 2. Structure of EGCG (2) as a DYRK1A inhibitor that contains bis-catechol PAINS motifs (shown in red). Furthermore, compound 2 also exhibits anticancer activity, and several mechanisms of action have been suggested to describe its anticancer effect.53 However, it has disadvantages for use in 9 ACS Paragon Plus Environment


cell and animal experiments due to poor bioavailability, complex pharmacokinetic properties and heterogeneous effects on signaling pathways.54 Although several preclinical studies and clinical trials have revealed that 2 has improved some DS-associated phenotypes by inhibiting DYRK1A, the beneficial effects of 2 have been questioned due to inconsistent results and a lack of necessary controls.55 For example, Dyrk1a expression differs over the lifespan in DS mouse models, but little attention has been given to these developmental changes in preclinical therapeutic treatments targeting Dyrk1a.55,56 There was significant heterogeneity observed in behavioral outcomes when various compositions and doses of 2 or 2-containing supplements were administered to transgenic Dyrk1a or Ts65Dn mice, as well as when different mouse models, ages and routes of administration were employed.55 Therefore, the necessity of controlling or monitoring the administered dose or composition of 2 in all studies is of great importance. Additionally, the identification of the effective dose and the level of EGCG accumulation in subjects over time remain important questions. However, there is no definitive proof that any of the observed improvement of some DS-associated phenotypes was a result of DYRK1A inhibition.55 Several studies have used 2-containing supplements as the source of 2 for administration. However, these supplements also contain other components, such as catechins (epigallocatechin, epicatechin gallate, epigallate, and epicatechin), sucrose, and/or caffeine, that could have acted separately or synergistically with 2.57 For instance, different supplements containing 2 showed differences in degradation, polyphenol content, and effects on trisomic bone.57 Thus, it is essential to precisely evaluate the dose of 2 and the composition of the EGCGcontaining supplements used in different experiments. Importantly, compound 2 has been classified as a promiscuous pan-assay interference compound (PAINS) due to its interference in biological assays leading to false positive results.58 It 10 ACS Paragon Plus Environment

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undergoes oxidative degradation in cell culture methods, forming oxidized degradation products that are more toxic than the parent EGCG molecule, which can be the reason for the cytotoxicity of 2.59 It is known that the PAINS motif catechol readily undergoes oxidation to form the highly reactive orthoquinone, which binds covalently to proteins, and it has even been shown to chelate metals.60 Compound 2 contains bis-catechol (pyrogallol), which is an additional PAINS motif, and thus may interfere in assays. Furthermore, the moiety is known to cause nonspecific membrane perturbation rather than specific protein binding, thereby interfering in ion channel assays.61 As a consequence of this detrimental effect, it can activate several different pathways in cells and exhibits diverse biological activities. Unfortunately, it is being reported in the literature extensively as a ‘promising DYRK1A inhibitor’ in spite of these well-known promiscuous effects in unrelated cell-based assays and target-based assays.61 Harmine and its derivatives Compound 3 belongs to a β-carboline alkaloid with a pyrido[3,4-b]indole ring and was first isolated from the South American vine Banisteriopsis caapi (Figure 3). It was initially used in 1928 in the treatment of Parkinson’s disease patients for improving the mental status of the patients.53 Compound 3 was found to be a potent inhibitor of DYRK1A (IC50 = 80 nM) in vitro and specifically inhibited DYRK1A from among 69 protein kinases. However, it also strongly inhibited other DYRKs (IC50 = 0.9 µM for DYRK2, 0.8 µM for DYRK3).62 Studies based on molecular docking and the co-crystal structure of 3 with the DYRK1A kinase domain have shown that it binds to the ATP binding site of DYRK1A and inhibits the serine/threonine phosphorylation activity by competing directly with ATP.46,63 A study has shown that 3 inhibits the tyrosine autophosphorylation of DYRK1A in vitro, though a higher concentration was required to inhibit the autophosphorylation in vivo.64 11 ACS Paragon Plus Environment


Figure 3. General structures of harmine (3) and its analogues (4-8) as potent DYRK1A inhibitors.

Compound 3 significantly inhibits endogenous DYRK1A in HeLa cells with an IC50 of 48 nM.64 In addition, 3 was found to be cytostatic and/or cytotoxic against cancer cells65,66 and to possess antidiabetic activity67. Although 3 has been considered to be a potent and orally bioavailable inhibitor of DYRK1A, it also exhibited an even stronger inhibition of monoamine oxidase (MAO)-A, causing side effects that led to a significant limitation of its potential therapeutic application.68 However, despite this limitation in terms of DYRK1A inhibition, it may find other therapeutic uses. For example, N,N-dimethyltryptamine (DMT), a hallucinogen, is rapidly degraded by MAO-A enzymes via the deamination metabolic pathway in the liver and gut when ingested in the absence of 3.69 Remarkably, when DMT is co-administered with 3, the degradation of DMT is effectively prevented, thus enabling its transfer into the bloodstream to induce the desired psychoactive effects. Therefore, the synergistic interaction between DMT and 3 is necessary for the enhancement of the psychoactive effects from DMT oral consumption.69 12 ACS Paragon Plus Environment

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To overcome the hallucinogenic and toxic effect observed with 3, several harmine and βcarboline analogues, 4-8, have been synthesized as potent DYRK1A inhibitors. Demethylation of a methoxy group resulted in harmol (4), which showed low inhibition of MAO-A; however, it also showed only a slight effect on DYRK1A inhibition.70 Compound 4 inhibited DYRK1Amediated phosphorylation of Tau (IC50 = 90 nM) with a greater potency than 3 (IC50 = 700 nM). A similar inhibition was shown by 9-ethylharmine (5), with an IC50 value of 400 nM.30 In 2014, Drung et al. synthesized various β-carboline derivatives, evaluated them for DYRK1A activity and concluded that compound 6 was more effective than 3.71 Compound 6 (IC50 = 130 nM) displayed a 97% inhibition of DYRK1A at 10 µM and inhibited MAO-A to a lesser extent (34% inhibition at 10 µM), whereas 3 showed complete inhibition of MAO-A (103% inhibition at 1 µM). The higher inhibition of MAO-A in the case of 3 revealed that the amine of the central ring is essential for MAO-A binding but not for DYRK1A binding.70 Interestingly, compound 6 inhibited DYRK1A (85% inhibition at 1 µM) specifically among other DYRKs and cyclin-like kinases.71 To enhance the activity against DYRK1A while reducing the activity of MAO-A, harmine analogues 7a and 7b were synthesized, and their activities were compared with 3 using a kinaseGLO assay.72 The results showed a greater than 60% inhibition of DYRK1A by inhibitor 3 at 1 µM, whereas 7a (IC50 = 81 nM) and 7b (IC50 = 181 nM) showed improved inhibition (8% and 10% uninhibited control, respectively). Compound 7a inhibited MAO-A to a far lesser extent (IC50 = 3.24 µM), whereas compound 7b showed a significant drop in MAO-A inhibition (IC50 >10 µM). Similar behavior was shown by compound 8. Replacing the methyl group at the 1position for a trifluoromethyl or chloro group resulted in the complete loss of the activity against 13 ACS Paragon Plus Environment


MAO-A, while retaining the activity against DYRK1A, demonstrating that the 1-position of the β-carboline plays an important role in discriminating DYRK1A and MAO-A inhibition.70 The effect of 7b on the tyrosine autophosphorylation during translation of DYRK1A and the threonine (Thr434) phosphorylation in substrate protein splicing factor 3b1 (SF3B1) was evaluated in a coupled in vitro-transcription-translation system.72 Compound 7b inhibited the tyrosine autophosphorylation of DYRK1A at concentrations >1 µM, whereas higher concentrations of 7b were required to inhibit threonine phosphorylation in SF3B1.72 Strikingly, in the peptide assay, preferential inhibition of threonine phosphorylation over tyrosine autophosphorylation of DYRK1A was observed, owing to the fact that autophosphorylation assays in the in vitro translation mix cannot be performed under conditions of substrate saturation.73 Acrifoline

Figure 4. Natural alkaloid 9 as a potent DYRK1A inhibitor.

In 2014, an alkaloid termed acrifoline (compound 9) (Figure 4) was isolated from the stem bark of Glycosmis chlorosperma along with other acridone alkaloids such as chlorospermines A, B and atalaphyllidine.74 Compound 9 showed potent inhibition (IC50 = 75 nM) against DYRK1A, with an inhibitory activity comparable to that of 3, but provided a better molecular scaffold 14 ACS Paragon Plus Environment

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against DYRK1A. The evaluation of the selectivity profile against fourteen protein kinases on the basis of IC50 values revealed that compound 9 had selectivity towards DYRK1A. Molecular docking studies revealed that the strong interactions of 9, with both conserved Lys188 and backbone atoms in the hinge region (Glu239 and/or Leu241), were key interactions for enhanced biological activity. Leucettines

Figure 5. Structures of leucettamine B (10) and leucettine L41 (11) as potent DYRK1A inhibitors, containing a Michael acceptor that is recognized as a PAINS motif.

Leucettines appear to be promising drug candidates for the inhibition of DYRK1A and its closely related kinases, CLKs. The natural product leucettamine B (10) was isolated from the marine calcareous sponge Leucetta microraphis75, and leucettine L41 (11) was derived from alkaloid 10 (Figure 5). Compound 10 inhibited protein kinases such as DYRK1A (IC50 = 420 nM), DYRK2 (IC50 = 0.49 µM), CLK1 (IC50 = 0.1 µM) and CLK2 (IC50 = 0.91 µM) in the micromolar/submicromolar range76, which are associated with Alzheimer’s disease at different stages of disease progression.77 However, compound 11 specifically inhibited DYRKs (preferentially DYRK1A, IC50 = 32 nM)78a and CLKs, (IC50 = 60-70 nM), but unlike 10, it also inhibited GSK-3 (IC50 = 0.41 µM), PIM1 (proto-oncogene serine/threonine-protein kinase, IC50 15 ACS Paragon Plus Environment


= 4.1 µM) and CK2 (IC50 = 0.32 µM).78b Interestingly, compound 11 targets only DYRK1A in the mouse brain, as analyzed by affinity chromatography.79 Additionally, it showed better affinity and potency than 10, likely for two reasons. First, the phenyl ring being less flexible probably increases the stabilizing hydrophobic interactions with residues of the Gly loop. Second, the secondary amine adjacent to the phenyl group is involved in hydrogen bonding. Thus, leucettines (2-aminoimidazolones) appear to serve as important moieties for designing potent DYRK1A inhibitors.78 Although initial results showed promising activity, leucettines should be carefully examined when developed as DYRK1A inhibitors. Their structure contains an unsaturated enone, which is typically regarded as a PAINS chemotype; therefore, it may be prone to react with biological nucleophiles, such as cysteines, via Michael addition.80 In particular, the exocyclic double bond is considered to be a potent Michael acceptor that binds covalently with cellular structures and modifies the protein. In addition, leucettines may interfere with biochemical assays due to the aggregate-based inhibition of enzymes.80 Meridianins Meridianins are naturally occurring compounds that have 3-(pyrimidin-4-yl)-1H-indole as a core structure. They were isolated from a marine source, the south Atlantic tunicate Aplidium meridianum.81 Meridianins were found to be active against various protein kinases82a and to possess antitumor activity82b.


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H2 N N 5

N

H2 N

N

OMe

N

Br 6 7

N H

Meridianin (12a-14a) 12a, a5-Br (IC50 = 0.33 M) 13a, a6-Br (IC50 = 0.13 M) 14a, 7-Br (IC50 = 0.068 M) a - isolated from natural source

N

I

5

Br

NH2

N

6 7

N H

(12b-14b)

N

N N H2 N

Variolin B (15, IC50 = 80 nM)

12b, 5-Br (IC50 = 0.33 M) 13b, 6-Br (IC50 = 0.34 M) 14b, 7-Br (IC50 = 0.039 M)

Figure 6. Meridianins 12a-14b and their structural analogue variolin B (15).

The meridianin series (12a-14a) and its synthetic derivatives, the meridianin-like series (12b14b), were tested against DYRK1A (Figure 6) and other kinases such as CDK5/p25, CK1δ/ε, GSK-3R/β and CLK1.83 Compounds 14a, 13b and 14b were remarkably active against DYRK1A (IC50 = 0.068, 0.34, 0.039 µM, respectively) and CLK1 (IC50 = 0.065, 0.032, and 0.042 µM, respectively). Variolin B (15), a marine natural product extracted from the Antarctic sponge Kirkpatrickia variolosa, structurally resembles meridianins.84 It was found to be cytotoxic to various human cancer cell lines.85a Surprisingly, it exerted a profound inhibitory activity against CK1 (IC50 = 5 nM) and DYRK1A (IC50 = 80 nM).85b However, it has limited use in clinical trials due to its low solubility in water and complex pharmacokinetic properties.86



Figure 7. Structurally modified meridianins 16 and 17 and their aza-analogue meriolin (18).

Compound 16, prepared from 14b by Suzuki coupling, displayed submicromolar activity against DYRK1A but was less active than 14b (Figure 7).83 The N-Acylated meridianin 17 showed an improved inhibition of DYRK1A (IC50 = 0.5 µM) over DYRK2 (IC50 = 1.4 µM) and DYRK3 (IC50 = 2.2 µM) and exhibited no cytotoxicity up to 100 µM in human neuroblastoma SH-SY5Y cells.87 It displayed a 56.7% inhibition of DYRK1A, similar to 12a. The authors proposed that the meridianin pyrimidine N-1 and the 2-amino group of the pyrimidine ring interacted with the hinge region of DYRK1A and facilitated the binding of meridianins to the ATP binding site, while the indole ring was involved in π–π interactions with the phenylalanine gatekeeper.83 Compound 17 also showed similar interactions as the parent meridianins. In addition to a carbonyl group interaction with the ATP binding pocket, a bulky bromine interacted with Asp307 and Lys188 side chains through hydrophobic and ionic interactions.87 Meriolin (18) showed DYRK1A inhibition with an IC50 value of 0.029 µM; however, it also showed potent inhibition of CDK9 (IC50 = 0.006 µM).85b Staurosporine


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Figure 8. Structures of the DYRK1A inhibitors 19 and 20 with an indolocarbazole ring.

Staurosporine (19), with an indolocarbazole ring, was isolated from the bacterium Streptomyces staurosporeus (Figure 8). It showed potent activity against DYRK1A (IC50 = 19 nM) but induced toxicity, as it also inhibited many other kinases such as AurA, AurB, Chk1, Ftl3, FGFR1, HGK, Ikkb, Jak2, KDR, and SYK.88 Nevertheless, it has been used as an ideal control molecule for the screening of DYRK1A inhibitors. Compound 20 also showed potent inhibition of DYRK1A but was non-selective, which limited its development as a therapeutic agent.88

SYNTHETIC INHIBITORS Despite the substantial efforts accomplished so far by derivatizing naturally occurring DYRK1A inhibitors, the search for highly potent and selective DYRK1A inhibitors remains a priority. To overcome the disadvantages of natural DYRK1A inhibitors, several synthetic inhibitors have been developed that mainly have a higher potency against serine and threonine phosphorylation than against autophosphorylation. We have categorized these compounds into several classes



according to their heteroaromatic scaffolds: benzothiazoles, indole, carbazoles, azaindole, purines, β-carboline derivatives, naphthyridines, quinazolines, and polyphenols. Benzothiazoles There are many synthetic DYRK1A inhibitors with benzothiazole as their core structure, such as 21 (Figure 9). In 2010, Ogawa et al. reported that 21 is three times more potent (IC50 = 240 nM) than TG003 (22), (Z)-1-(3-ethyl-5-methoxy-2,3-dihydrobenzothiazol-2-ylidene)propan-2-one) (IC50 = 930 nM).48 Compared to 3, compound 21 exhibited equipotent activity and similar substrate selectivity, suggesting a similar structural mechanism of action. Remarkably, it did not have affinity for monoamine oxidase. However, 21 also inhibited other DYRK family members, such as DYRK1B, with comparable potency. The co-crystal structure of DYRK1A/21 in complex revealed considerable hydrophobic interactions of 21 with the active site of DYRK1A, where the phenolic hydroxyl group and the carbonyl oxygen of 21 form hydrogen bonds with the hinge backbone amide NH of Leu241 and with the side chain NH2 group of Lys188, respectively.48 Further, the same group described the efficacy of an acetylated prodrug 23, which was hydrolyzed in vivo to generate 21, which restored the abnormal development of Xenopus laevis tadpoles.


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Figure 9. Representative benzothiazole analogues as potent DYRK1A inhibitors, with recognized PAINS shown in red. Rothweiler et al. reported benzothiazole derivatives 24-26, with an acetamido group at the C2 position of the thiazole moiety, as novel DYRK1A inhibitors for neurodegenerative therapies.89 The structure-activity relationship (SAR) of the benzene ring of the benzothiazoles indicated the 5-hydroxy derivative 24 (IC50 = 800 nM), 5-methoxy derivative 25 (IC50 = 400 nM), and 6-nitro derivative 26 (IC50 = 700 nM) to be the most potent inhibitors. Furthermore, a combination of Xray crystallography, dynamic NMR and computational simulation techniques were performed to interpret the binding interactions between fragment-sized variants of the benzothiazole scaffold and the DYRK1A kinase.89 Benzofuro-fused INDY (27) ((Z)-1-(1-ethylbenzo[2,3]benzofuro[4,5d]thiazol-2(1H)-ylidene)propan-2-one, referred to as BINDY) was reported as a novel DYRK1A inhibitor (IC50 = 25.1 nM).90 Compound 27 was designed and synthesized based on the crystal structure of the DYRK1A/21 complex by replacing the phenol group of 21 with dibenzofuran. However, the presence of a Michael acceptor moiety in 21-23 and in 27 raises concern over false positive results in the assays, as discussed above.80



Figure 10. Structures of selective DYRK1A inhibitors that contain recognizable rhodanine PAINS motifs (red). In 2016, by examination of a synthetic chemical library, Kii et al. identified a folding intermediate-selective inhibitor of DYRK1A, compound 28 (Figure 10). is referred to as FINDY ((Z)-5-(4-methoxy-3-((trimethylsilyl)ethynyl) benzylidene)-2-thioxothiazolidin-4-one) and was found to be a highly selective DYRK1A inhibitor compared to other DYRK family members, such as DYRK1B and DYRK2.91 It precisely suppressed DYKR1A intramolecular Ser97autophosphorylation (IC50 = 110 nM), resulting in its degradation by proteasomes, but did not affect tyrosine phosphorylation. In addition, the structure of 28 is analogous to the competitive ATP inhibitor RD0392 ((Z)-5-(3-ethoxy-4-hydroxybenzylidene)-2-thioxothiazolidin-4-one). Sonamoto et al. developed a bioluminescent reporter assay for evaluating DYRK1A inhibitors and

identified

29

(Z)-5-[(2,3-dihydrobenzofuran-5-yl)methylene]-2-iminothiazolidin-4-one

(referred to as CaNDY: CDC37 association inhibitor for DYRK1A) as a potent and selective inhibitor of the DYRK kinase family.92 Compound 29 is a competitive ATP inhibitor that antagonizes the kinase-specific co-chaperone CDC37 interaction with DYRK1A and inhibits the substrate phosphorylation of recombinant DYRK1A (IC50 = 7.9 nM). It also displayed activity against DYRK1B (IC50 = 24.1 nM), CLK1, CLK2, and haspin from among a panel of 275 recombinant kinases. Replacement of the oxygen atom attached to the benzene ring of 29 by a NH group led to a decrease in inhibitory activity. This demonstrates that the oxygen atoms attached to the aromatic and iminothiazolidinone of 29 are key pharmacophores for the inhibitory activity against DYRK1A. Interestingly, 29 selectively destabilizes newly synthesized DYRK1A molecules in cells but does not decrease the level of pre-accumulated DYRK1A. However, these compounds (28 and 29) may have associated PAINS, as their scaffolds contain 22 ACS Paragon Plus Environment

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the recognizable rhodanine PAINS motif or molecules containing rhodanine-related motifs.80 Rhodanines can interact nonspecifically with target proteins to form aggregates. It also display multiple behaviors that could give false positive assay readouts, such as Michael acceptor ability, metal chelation, and exertion of light-induced reactivity. The ALARM NMR (a La assay to detect reactive molecules by NMR) technique has been used to identify the reactivity of thiols towards rhodanine moiety-containing compounds.93 Furthermore, the observed potency of rhodanine- and thiazolidine-2,4-dione-based analogs against a variety of targets reflects the role of exocyclic, thioxo groups present in rhodanines. In comparison with thiazolidine-2,4-dione, rhodanines have a capability to participate in several interactions because of the unique electronic properties of the exocyclic thioxo group.80 Indoles and carbazoles

Figure 11. Structures of representative indole and carbazole derivatives as DYRK1A inhibitors, with PAINS shown in red.



Dichloroindolyl enaminonitrile (30) (Figure 11) was identified as a potent and highly specific non-ATP-competitive inhibitor of CLK kinases.94 It also displayed effective inhibition of DYRK1A, with an IC50 of 55 nM in enzyme kinetic assays. Guillou et al. evaluated the inhibitory activity of paprotrain (31) on a panel of CNS kinases that were apparently involved in Alzheimer's disease.95 Compound 31 showed a moderate activity against DYRK1A kinase (IC50 = 5.5 µM). In a further study inspired by the 6,5,6-fused tricyclic skeleton of 3, they reported the synthesis of a series of cyclized pyridocarbazoles, such as 32 from 31, which were screened against a panel of five protein kinases, including DYRK1A.96 Among an array of final 11Hpyrido[4,3-a]carbazole derivatives, compound 32 exhibited high inhibitory activity against DYRK1A (IC50 = 18 nM). However, it also showed equipotency against DYRK1B (IC50 = 23 nM) and submicromolar values for CDK5 and GSK3. The SAR of 32 concluded that the presence of the nitrile group at the 6-position was essential for the strong inhibition of DYRK1A and CLK1. It was also suggested that the rigidity of the scaffold should be taken into consideration when designing CNS kinases inhibitors, particularly with DYRK1A and CLK1. Indirubin has been identified as a promising protein kinase inhibitor scaffold (represented by 33).97 The modified bis-indole derivative 33 displayed relatively higher inhibitory potencies against DYRK1A and DYRK2 versus other kinases, such as CDK5, GSK3, and CK1. The simultaneous introduction of the carboxylate moiety at position 5′ and a bromine group at position 7 of the indirubin scaffold resulted in changing the nonselective bis-indole indirubin into a potent and selective DYRK inhibitor. The co-crystal structure of the 33/DYRK2 complex revealed that the carboxylic acid group interacted with Lys178, whereas the bulky bromine created a steric clash between 33 and the kinase hinge region.97 However, the indirubin moietycontaining compounds should be treated with caution, as indirubin itself has been reported to 24 ACS Paragon Plus Environment

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behave as a promiscuous compound by forming aggregates, at least at micromolar concentrations.98 In 2012, Routier and colleagues demonstrated the utility of chromeno[3,4-b]indoles, represented by 34, as DYRK1A inhibitors for the development of potent treatments for neurological or oncological disorders.99 Chromenoindoles have been developed as Lamellarin isosters, which are potent anticancer agents and nonselective inhibitors of several kinases. The SAR analysis indicated that the presence of phenolic OH was essential for the kinase activity. Compound 34 was the most active DYRK1A inhibitor (IC50 = 0.067 µM) in the library of chromeno[3,4b]indoles. Furthermore, the same group described the biological evaluation of a V-shaped molecule

library

comprising

3-(6-hydroxyindol-2-yl)-5-(phenyl)-pyrazine

and

pyridine

derivatives against DYRK1A, CDK5 and GSK3β.100 It was observed that compound 35 showed significant inhibitory activity against DYRK1A (IC50 = 60 nM). Indazole

Figure 12. Indazole derivatives 36 and 37 as DYRK1A inhibitors.

Bain and colleagues identified compound 36 (A-443654, (2S)-1-(1H-indol-3-yl)-3-[5-(3-methyl2H-indazol-5-yl)pyridin-3-yl]oxypropan-2-amine) as a potent protein kinase B (PKB) inhibitor



(Figure 12).62 This compound showed activity against DYRK1A, but with a lower potency and poor selectivity. 5-(pyridin-3-yl)-1H-indazole (37), named as ALGERNON, is a selective inhibitor of the DYRK1A and CLK families.101 Nakano-Kobayashi et al. demonstrated that 37 suppresses endogenous DYRK1A, as indicated by the suppression of phosphorylation of endogenous tau in primary hippocampal neurons. Importantly, in a DS mice model, it prevented cognitive deficits by balancing DYRK1A activity and restoring the aberrant brain development caused by DYRK1A expression. DYRK1A negatively regulates neural stem cell (NSC) proliferation.101 The concentration of compound 37 used in the study showed an enhancement of NSC proliferation, both in vitro and in vivo, through inhibition of DYRK1A. Notably, compound 37 may serve as a better template than 2 or 3, since it does not contain a promiscuous profile. Moreover, a higher concentration was required to inhibit MAO-A than the concentration required to inhibit DYRK1A. Benzimidazoles and imidazopyridines


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Br

Br Br

N

Br

N

Br

N

N

X Br Br

N H

Br

38a (X = N, IC50 = 4.36 M) 38b (X = CH, IC50 = 2.1 M) 38c (X = C(NMe2), IC50 = 0.4 M) [ATP] = 50 M

39

N N

N Ar

NH

N

N Ar

40 Imidazo[1,2-a]pyridine series less active

R 41 R = CN, SO2Me, ether, amine, acid, ester, amide Benzimidazole series more active

Figure 13. General structures of benzimidazoles (38-39, and 41) and imidazopyridine (40).

The tetrabromobenzimidazole derivatives such as 38a (4,5,6,7-tetrabromo-1H-benzotriazole, TBB), 38b (4,5,6,7-tetrabromo-1H-benzimidazole, TBI), and 38c (2-dimethylamino-4,5,6,7tetrabromo-1H-benzimidazole, DMAT) were first identified as casein kinase 2 (CK) inhibitors.102 (Figure 13). These compounds (38a-38c) showed inhibitory activity against DYRK1A (IC50 = 4.36, 2.1, and 0.41 µM, respectively). Additionally, 38c inhibited DYRK2 (IC50 = 0.35 µM) with almost equimolar potency and had the highest affinity to CK2 (IC50 = 0.13 µM). However, the promiscuous profile needs to be addressed to develop these compounds as tools for therapeutic use. Recently, a novel benzimidazole series was discovered to possess promising kinase inhibitory activities. Compound 39 displayed significant affinity for DYRK1A and Pim kinases.102b In 2017, a series of imidazo[1,2-a]pyridines (40) and benzimidazoles (41) were 27 ACS Paragon Plus Environment


reported as DYRK1A inhibitors.103 Compound 41 exhibited a more potent inhibition against DYRK1A (60-100% inhibition at 10 µM) than did 40. Recently, Branca et al. evaluated the effects of 41 against DYRK1A on the AD-like pathology developed by 3xTg-AD mice and established that DYRK1A inhibition decreased APP and insoluble Tau phosphorylation.17

Figure 14. Imidazo[4.5-b]pyridine analogue 42 as a dual DYRK1A/CLK1 kinase inhibitor.

In 2017, the novel imidazo[4.5-b]pyridine analogue 42 was reported to be a dual DYRK1A/CLK1 inhibitor (Figure 14).104 Compound 42, which is structurally similar to the Vshaped molecules, displayed potent inhibitory activity against DYRK1A (IC50 = 4 nM) and CLK1 (IC50 = 15 nM) in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay.

Azaindoles


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F

R OMe

OH MeO

HO

7

N

N H

7

43a (R = OH, IC50 = 3 nM) 43b (R = F, IC50 = 20.7 nM) [ATP] = 50 to 800 M

N

N H

44 (IC50 = 1.43 M)

OH R

N

2

R1 6 7

N

N

N H

45 (R1 = NH2, R2 = H, IC50 = 43 nM) 46 (R1 = NHCOMe, R2 = H, IC50 = 6.6 nM)

N H

47 (IC50 = 1 nM)

Figure 15. DYRK1A inhibitors based on the 6- and 7- azaindole scaffold and a prominent PAINS motif (catechol, shown in red).

Gourdain et al. demonstrated a series of 3,5-diaryl-1H-pyrrolo[2,3-b]pyridines for the inhibition of DYRK1A in vitro.105 Compound 43a, also called DANDY (Figure 15), was the most potent inhibitor (IC50 = 3 nM) of DYRK1A in this series. In addition to a strong affinity to CLK1 kinase, compounds with hydroxyl groups on the aryl moieties exhibited a high inhibitory potency (IC50 = 3-20 nM). In further structure-activity relationship studies, fluoro analogues of 43a have been recently synthesized and evaluated for their inhibitory activity against DYRK1A.106 Replacing one or two of the phenolic OH groups of the DANDY derivatives by fluoride atoms had a minimal effect on DYRK1A inhibitory activity (compound 43b, IC50 = 20.7 nM). However, the introduction of methoxy or benzyloxy groups to 43b in place of the free phenolic 29 ACS Paragon Plus Environment


hydroxyl groups resulted in a significant loss of inhibitory potency against DYRK1A (e.g., 44, 1.43 µM). These results suggest the possibility of pan-assay interference, since catechol is recognized as a PAINS motif, as described earliar.58,60,61 Based on the potent inhibitory activity of 43a with a 7-azaindole moiety, a library of novel 7azaindole derivatives served as a lead for the development of DYRK1A inhibitors with submicromolar IC50 values.107 This series was screened for anticancer efficacy in glioblastoma cell lines, among which 45 (IC50 = 43 nM) and 46 (IC50 = 6.6 nM) were found to be the most potent; however, submicromolar IC50 values were determined for DYRK1B, DYRK2 and CLK1 kinases. Both compounds significantly decreased the viability, clonogenic survival, migration, and invasion of glioblastoma cells. Shaw et al. reported a series of meridianin-derived 6azaindoles for the inhibition of DYRK1A and their effects on the transcription factor NFAT (nuclear factor of activated T cells) in the nucleus.108 Compound 47 was the most potent inhibitor (IC50 = 1 nM) of DYRK1A and exhibited potent activity (IC50 = 2.98 µM) in NFAT translocation. The SAR study indicated that introduction of nitrogen at the 6-position of the indole scaffold resulted in strong DYRK1A binding with the inhibitor and an increased NFAT residence time in the nucleus. The increased biological activity arises primarily from the additional interaction between the azaindole nitrogen and the Lys-188 in the back pocket of the ATP binding site of DYRK1A.108 Purine derivatives


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Figure 16. Purine derivatives 48-53 as DYRK1A inhibitors.

In the structure-based development and iterative biological evaluation of CDK1/2 inhibitors, Davies et al. showed that compound 48 (Figure 16) is a potent inhibitor of CDK1/2 (IC50 = 9 nM and 6 nM, respectively) and possesses low inhibitory activity against DYRK1A (IC50 = 0.9 µM) at higher (100 µM) concentrations of ATP.109 The 6-aminopurine analogues purvalanol A (49) and roscovitine (50) were originally developed as potent competitive ATP inhibitors of CDK1, CDK2, and CDK5, but they also showed moderate activity against DYRK1A, with IC50 values of 0.3 µM and 3.1 µM, respectively in cell-based assays.50 Moreover, the use of 49 has assisted in the identification of DYRK1A, not CDK5, as being the kinase responsible for the phosphorylation of specific sites (S1392) in MAP1B (microtubule-associated protein 1B), whereas 50 only slightly reduced S1392 phosphorylation.110 Compound 50 has entered into 31 ACS Paragon Plus Environment


clinical trials against various cancers such as leukemia and non-small cell lung cancer. To improve the antitumor activity of 50, Meijer et al. synthesized a bioisostere of roscovitine (51) and found that it was 2- to 3-fold more potent than 50. Similarly, 51 had a higher affinity for CDKs than for DYRK1A (IC50 = 1.3 µM).111 Based on CDK/50 co-crystallization structures, the same group designed and reported the more active N6-biaryl-substituted purine analogue 52, bearing a 2-pyridyl substituent on the para position of the phenyl ring.112 A kinase assay performed on a panel of 108 kinases demonstrated that 52 was a selective inhibitor of CDKs compared to DYRK1A (IC50 = 0.9 µM). Remarkably, 52 caused apoptotic cell death, with an approximately 50-fold greater potency than compound 50.112 Meijer et al. also reported the tri/tetra-substituted imidazo[4,5-b]pyridines exemplified by compound 53 as CDK and DYRK1A inhibitors.113 Among the tested derivatives, 53 exhibited the most potent activity (IC50 = 2.8 µM) against DYRK1A. Thiazolo[ 5,4-f]quinazoline derivatives


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Figure 17. Thiazolo[ 5,4-f]quinazoline derivatives as the most potent inhibitors of DYRK1A.

Besson et al. reported the design and synthesis of thiazolo[5,4-f]quinazoline compounds as novel inhibitors of the DYRK family kinases.114 They described the effectiveness of thiazolo[5,4f]quinazolines against DYRK1A and DYRK1B, and most of them displayed similar or better inhibitory activity than 3. In this series, compounds 54-58 exhibited strong inhibitory activities against DYRK1A (IC50 = 0.22−0.99 nM) and DYRK1B (IC50 = 0.28−2.83 nM) (Figure 17). Among these, 54 and 55 displayed the most potent and selective inhibition against DYRK1A and DYRK1B (IC50 of 0.22/0.28 nM and 0.36/0.59 nM, respectively) of 339 tested kinases. They belong to the most potent and selective DYRK1A/1B inhibitors reported so far. Recent studies conducted by Coutadeur et al. suggest that 54 inhibits Tau phosphorylation at multiple ADrelevant sites induced by DYRK1A.115 Hedou et al. evaluated a series of thiazolo[5,4f]quinazolin-9(8H)-one derivatives, among which 59 exhibited potent inhibitory activities against CDK5, GSK-3, CLK1, CK1, and DYRK1A.116 N8-aminoquinazolin-9-ones exhibited poor inhibitory activities compared to the C9-amino-substituted quinazolines. These findings suggested that the presence of 3-, 4- or 5-membered cycloalkyl groups linked to the front pocket is favorable for the development of further SAR studies. Compound 59 showed nanomolar activity against DYRK1A but also possessed strong affinity for CLK1. Debray et al. investigated the inhibitory activity of a library of heterocycle-fused quinazolin-4-ones against DYRK1A, CDK5 and GSK3 to highlight compound 60 as a selective inhibitor (IC50 = 0.61 µM) of DYRK1A.117 Naphthyridines



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Figure 18. Naphthyridine derivatives 61 and 62 as potent inhibitors of DYRK1A and DYRK1B.

A series of naphthyridine derivatives exhibited potent inhibitory activity against DYRK1A and DYRK1B.118

Among

these,

CX-4945

(61,

(5-[(3-

chlorophenyl)amino]benzo[c][2,6]naphthyridine-8-carboxylic acid)) (Figure 18) showed strong inhibitory activity against DYRK1A (IC50 = 6.8 nM) and DYRK1B (IC50 = 6.4 nM) and activity against casein kinase 2 (CK2) and Cdc2-like kinases (IC50 = ∼3-10 nM), which regulate alternative splicing.118 This compound is currently in phase 1b/2 clinical trials as an anticancer agent and was approved as an orphan drug in the USA for the treatment of advanced cholangiocarcinoma. It was also reported that 61 could be used to prevent and treat degenerative brain diseases associated with DYRK1A.119 It serves as a potent competitive ATP inhibitor and was approximately 20 times more potent in vitro (IC50 = 6.8 nM) than 3, 21 and 23. Interestingly, the binding modes of 61 in relation to 3 with DYRK1A were remarkably identical, which highlighted the importance of tricyclic scaffolds to enhance the inhibitory activity against DYRK1A. As stated previously, 3 displayed potent inhibition of MAO-A. Thus, it would be important to evaluate the effects of 61 against MAO-A. Furthermore, 61 effectively reversed the abnormal phosphorylation of Tau, amyloid precursor protein (APP) and presenilin 1 (PS1) in 34 ACS Paragon Plus Environment

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mammalian

cells.120

However,

its

oral

administration

apparently

blocked

Tau

hyperphosphorylation in the hippocampus of DYRK1A-overexpressing mice. Hoffman-La Roche, AG identified another 1,6-naphthrydine derivative (62) as a potent DYRK1A/1B inhibitor (IC50 = 4.6 and 13.8 nM, respectively), which exhibited antitumor activity.121 β-Carboline derivatives

Figure 19. Structures of β-carboline derivatives 63 and 64.

There has been considerable interest in the development of DYRK1A inhibitors based on the βcarboline scaffold. Falke et al. identified compound 63 as a potent DYRK1A inhibitor with considerable selectivity against DYRK1A (Figure 19).122 Compound 63 selectively inhibited DYRK1A within the CMGC family of protein kinases. Remarkably, higher selectivity for DYRK1A over DYRK1B (IC50 values of 6 nM and 600 nM, respectively) was observed, although the catalytic domains of these kinases have 85% amino acid sequence similarity. This study suggested that substituents at the C10 position of the parent scaffold induce a strong and selective inhibitory activity against DYRK1A: the 10-iodo atom forms a bond with Leu241 through a water molecule via hydrogen bonding in the hinge region, while the deprotonated 35 ACS Paragon Plus Environment


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carboxylic group forms a salt bridge to the conserved Lys188. As a result, the inhibitor is positioned in the ATP binding site of DYRK1A.123 Currently, compound 63 appears to be one of the most selective and potent DYRK1A inhibitors. However, as this molecule contains a lipophilic, flat, aromatic structure, solubility and cell permeability are major drawbacks. Modification of the pyridine ring of 3 yielded 2,3,4,7-tetrahydro-1H-indolo[2,3-c]quinolinone (64) that inhibited DYRK1A to control Tau phosphorylation.124 Compound 64 was tested for its effects on phosphorylated Tau, and the total Tau expression was compared with those of 3-5. Compounds 3-5 showed remarkable effects in reducing total Tau and phosphorylated Tau levels at doses of ≥ 1 µM, 30 whereas 64 significantly reduced Tau levels at 50 µM. Quinoline derivatives

Figure

20.

Examples

of

quinoline

derivatives

65-68


as

DYRK1A

inhibitors.

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Quinolines represent an important class of DYRK1A inhibitors. They have higher binding affinity towards the ATP binding site of proteins due to its structural similarities with the adenine moiety. A series of substituted 2-oxo-1,2-dihydro-quinoline analogues (65-67) as DYRK1A/1B inhibitors (Figure 20) and their cellular activity against colorectal adenocarcinoma cell lines were reported by HoffmannLa Roche AG.125 Compounds 65-67 inhibited DYRK1A selectively in the nanomolar range, while compound 65 showed better inhibition of a cancer cell line, with an EC50 of 0.306 µM. Compound 68 displayed inhibition of DYRK1A; however, it revealed good selectivity towards Pim-1 kinase, a protooncogene that encodes a serine/threonine kinase that is known to play a critical role in the control of cell growth and apoptosis.126 Quinazoline derivatives

Figure 21. Quinazoline derivatives 69-72 as DYRK1A inhibitors.

In a combination of in silico, in vitro, and cell-based screenings, 2,4-diamino-quinazoline derivative 69 inhibited DYRK1A substrate phosphorylation (8% at 10 µM) (Figure 21).127 It also exhibited activity against the autophosphorylation of DYRK1A, with an IC50 value of 5 µM, but did not show significant anti-DYRK1A activity at this concentration. Thomas et al. found that 70 showed selective inhibition against CLK1, CLK4, and DYRK1A (IC50 = 27 nM) in the screening of 402 kinases.128 The mechanism of action suggests that 70 inhibited the CLK isozymes by binding at the ATP binding domain. Subsequently, the same group also identified another quinazolin-4-amine derivative (71) as a selective 37 ACS Paragon Plus Environment


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inhibitor of DYRK1A, CLK1, and CLK4.129 Interestingly, SAR studies emphasized that the presence of a benzo[1,3]dioxole ring at the C6 position is essential for the inhibitory activity. Compounds 70 and 71 displayed strong affinity and selectivity for DYRK1A (IC50 = 14-27 nM), CLK1 and CLK4, compared to CLK2, CLK3, and DYRK1B. Recently Esvan et al. designed and synthesized a series of pyrido[3,4g]quinazoline derivatives (exemplified by 72) based on meridianin scaffolds by controlling the conformation between the aminopyrimidine and indole moieties and evaluated their activity against five members of the CMGC family kinases (CDK5/p25, CK1δ/ε, GSK-3α/β, CLK1 and DYRK1A).130 Compound 72 displayed nanomolar potency against DYRK1A and CLK1. The X-ray co-crystal structure of 72 with the ATP binding site of CLK1 showed that the aminopyrimidine moiety is positioned in the hinge region, while the amino group is located towards the ribose pocket.130 Pyrimidine derivatives

Figure

22.

Representative pyrido[3,2-d]pyrimidine (73), pyrrolo[2,3-d]pyrimidine (74)

and

pyrazolo[3,4-d]pyrimidine (75) as DYRK1A inhibitors.

Routier et al. identified a group of 2,7-disubstituted pyrido[3,2-d]pyrimidine derivatives, represented by 73, which were able to inhibit DYRK1A, CDK5 and GSK3 kinases.131 The SAR study disclosed that pyrido[3,2-d]pyrimidine derivatives selectively inhibited DYRK1A and CDK5 and had a low inhibitory activity towards GSK3. The amide link at the 7-position on the pyrido[3,2-d]pyrimidine core appears to 38 ACS Paragon Plus Environment

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be important for selectivity towards DYRK1A. Compound 73 exhibited inhibitory activity against DYRK1A, CDK5 and GSK3, with IC50 values of 60 nM, 2.2 µM, and 4.6 µM, respectively. Compound 73 had the highest ligand and lipophilic efficacy among the screened pyrido[3,2-d]pyrimidine derivatives. The pyrrolo[2,3-d]pyrimidine derivatives illustrated by 74 also showed inhibitory effects on DYRK1A and CLK1.132 Compound 74 inhibited DYRK1A, DYRK1B and CDK9, with IC50 values of 2.1, 8 and 10 nM, respectively. Hoffman-La Roche AG evaluated a library of pyrazolo[3,4-d]pyrimidines derivatives, represented by 75, for their inhibitory activity against DYRK1A and DYRK1B.133 This study mainly focused on the addition of an aromatic ring or lipophilic alkyl chains to the C3 and C6 positions of the pyrazolo[3,4d]pyrimidine moiety. Compound 75 displayed a highly effective inhibition of DYRK1A and DYRK1B (IC50 = 5.9 and 18.3 nM, respectively) compared to other screened pyrazolo[3,4-d]pyrimidines derivatives.

Figure 23. Pyrido[2,3-d]pyrimidine derivative 76 as a potent inhibitor of DYRK1A and DYRK1B. The same group also synthesized the pyrido[2,3-d]pyrimidine analogues, represented by 76, and evaluated their activity against DYRK1A and DYRK1B (Figure 23), among which 18 compounds exhibited nanomolar inhibitory activity against DYRK1A and DYRK1B (IC50 = 4.6-15 nM).134 However, selectivity among the two subtypes, DYRK1A and DYRK1B, was not achieved in this series. For instance, the best activity was achieved with 76 towards DYRK1A (IC50 = 4.6 nM) versus DYRK1B (IC50 = 10.5 nM). 39 ACS Paragon Plus Environment


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Pyridazine derivatives

Figure 24. Pyridazine derivatives 77 and 78 as DYRK1A inhibitors.

Bendjeddou et al. described the inhibitory activity of various 3,6-disubstituted imidazo[1,2-b]pyridazine derivatives against CLKs, CDKs and DYRKs.135 In this series, the oxime derivative 77 (Figure 24) exhibited significant inhibitory activities against DYRK1A (IC50 = 33 nM), DYRK1B (IC50 = 170 nM) and CLK2 (IC50 = 59 nM), as well as moderate activity for CDK1 (1.1 µM). Compound 78, bearing a pyridine moiety at the 6-position on the pyridazin-3(2H)-one moiety showed moderate inhibitory activity against DYRK1A (IC50 = 0.61 µM).136 However, no activity was observed against CDK5, GSK3 and PI3Kα kinases.

Polyphenols


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Journal of Medicinal Chemistry CH3 4

HO

9

O

H3CO

OH

OCH3

O

R

CH3O

79 (R = NO2, DYRK1A, IC50 = 15.0 M; CK2, IC50 = 0.37 M) 80 (R = H, DYRK1A, IC50 = 0.6 M; CK2, IC50 = 32.34 M)

O 81

OMe OH HO

O

O

OH O 82 DYRK1A (IC50 = 0.62 M) DYRK1B (IC50 = 6.4 M)

OH HO 83 DYRK1A (IC50 = 2.9 M) DYRK1B (IC50 = 2.3 M) CLK1(IC50 = 1.9 M)

Figure 25. Polyphenols 79-83 as DYRK1A inhibitors, with recognized PAINS motifs highlighted in red.

Polyphenols have by no means been completely explored in the study of DYRK1A, and much attention needs to be focused on these moieties. However, few polyphenolic compounds (79-83) showed inhibitory activity towards DYRK1A, as is illustrated in Figure 25. Compound 79 was found to be a potent inhibitor of CK2 (IC50 = 0.37 µM), an enzyme that has been known to prevent programmed cell death.137 Nevertheless, it also showed inhibition of DYRK1A (IC50 = 15.0 µM), though to a lesser extent. Interestingly, compound 80, replacing the nitro group at the 9-position of 79 with hydrogen, showed a remarkable increase in its activity against DYRK1A (IC50 = 0.6 µM), while a loss of the binding affinity for CK2 was observed (IC50 > 30 µM). Docking studies regarding CK2 and DYRK1A proposed that compound 80 was deeply buried in the binding cleft of DYRK1A compared to 79 to 41 ACS Paragon Plus Environment


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interact with both the hinge region and the phosphate binding region. The methyl group at C4 was positioned between Ile165 and Val173, and thus protected from the solvent. The C4 methyl of 80 was placed in the opposite direction from how it was oriented in the 79-CK2 complex. This type of interaction suggests that the nitro group at C9 (79) is unfavorable for DYRK1A activity due to the restricted approach of the bulky group by the gatekeeper Phe238 in DYRK1A.137 Folmer et al. evaluated the effect of 81, known as flavokavain A, on a panel of 52 protein kinases and found that at 50 µM it reduced the activity of DYRK1A, PRAK and Aurora B by 90%, 91% and 84%, respectively, in a kinase inhibition assay.138 In this study, protein kinase activities were reported as a percentage relative to untreated K562 cells (= 100%). The flavonoid derivative 82 was found to show anticancer activity by targeting DYRK1A (IC50 = 0.62 µM).139 It also displayed less potency towards the inhibition of DYRK1B (IC50 = 6.4 µM). Compound 83 decreased the activity of DYRK1A/1B and CLK1,

with

IC50

values

of

2.9,

2.3

and

1.9

µM,

respectively.140

Moreover,

compounds 81 and 82 displayed features associated with frequent-hitters because they possessed a Michael acceptor and a flavonoid moiety in their respective structures. These groups can interfere in the assays, as they are covalent modifiers. These compounds seem to be highly susceptible to aggregation in biochemical assay conditions. Flavonoids are frequently identified as false positives in fluorescence- or absorbance-based assays due to their colored extracts or intrinsic fluorescence. Flavonoids have been reported to generate reactive oxygen species (ROS), causing cysteine oxidation.141

■ PERSPECTIVES Due to the apparent involvement of DYRK1A in cancer, DS, AD and other neurodegenerative diseases, DYRK1A inhibitors have been recommended for use as potential therapeutics, since these molecules are capable of diffusing across the blood brain barrier (BBB). The discovery of selective DYRK1A inhibitors has slowly evolved, and this process still largely relies on the development of competitive 42 ACS Paragon Plus Environment

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ATP inhibitors that act by binding within the DYRK1A kinase domain in its active conformation when the activation loop is phosphorylated. Most of the inhibitors developed to date possess chemical structures resembling an adenine moiety and include various nitrogen heterocyclic compounds such as imidazopyridines, purines, quinoline, and quinazolines. These heteroaromatic motifs, due to their shape and hydrophobic nature, usually involve multiple interactions with protein, resulting in highly efficient binding to the kinase domain. Moreover, the extent of target selectivity is derived from the rigidity of inhibitors and the hydrogen bonding potential from their heteroatoms. However, despite the advantages their physical properties bring, they also have a major limitation in their low aqueous solubility. From a medicinal chemistry perspective, it is rather complicated and difficult to draw conclusions from the extensive lists of natural and synthetic DYRK1A inhibitors reported so far to direct further design. Docking experiments and various three-dimensional structures of DYRK1A complexed with diverse competitive ATP inhibitors could suggest which DYRK1A amino acid residues are essential for binding interaction. Obviously, additional studies are required to clarify the exact mechanisms of DYRK1A kinase inhibition by various inhibitors. In short, a deeper understanding of multiple interactions between the DYRK1A substrate and different inhibitors will help develop the rationale for discovering and developing novel DYRK1A inhibitors. The majority of DYRK1A inhibitors discussed here were originally developed to target structurally related kinases, such as other kinases in the DYRK family and CDKs. It has been observed that many DYRK1A inhibitors have a higher affinity for serine/threonine phosphorylation than for tyrosine autophosphorylation. One of these examples is harmine, which inhibits the substrate phosphorylation of DYRK1A with higher potency than tyrosine autophosphorylation.64 Moreover, DYRK1A kinase activity is thoroughly controlled by two sequential conformational states, one of which has an immature transitory folding intermediate and the second has a mature conformation of the kinase. However, these processes are kinetically extremely different. The inhibitors that preferentially inhibit tyrosine 43 ACS Paragon Plus Environment


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autophosphorylation in contrast to serine and threonine phosphorylation of substrates induce conformational changes in DYRK1A, resulting in loss of function; these inhibitors might show a new direction for developing potent and specific inhibitors. For example, β-carboline DYRK1A inhibitor 7b preferentially inhibits tyrosine autophosphorylation of DYRK1A at concentrations >1 µM, whereas higher concentrations of 7b were required to inhibit threonine phosphorylation in SF3B1.72 In contrast, excessive inhibition of DYRK1A may be deleterious due to long-term function loss in other biological signaling pathways. On that account, in most of the cases, it would be more beneficial to design inhibitors that specifically inhibit substrate phosphorylation over tyrosine autophosphorylation. It is extremely challenging to achieve selectivity and specificity among the closely related kinases, such as the CMGC group of kinases, as these kinases share a high structural similarity in the ATP binding sites, with only slight differences. Notably, DYRK1B shares 85% identical amino acid sequence similarity in the N-terminal and catalytic domains of DYRK1A.142 Designing selective DYRK1A inhibitors would be very useful to investigate the role of DYRK1A in pathobiochemical and physiological processes. The DYRK1A inhibitors known thus far show poor selectivity among other closely related protein kinases of the CMGC superfamily, which includes CDKs, MAP kinases (such as extracellular signal–regulated kinases, known as ERKs), GSK-3, and CDC-like kinases. As discussed earlier, most of the known DYRK1A inhibitors act by competing with ATP in the binding pocket of the kinase. Therefore, further studies should be directed towards exploring the interactions of DYRK1A inhibitors with the DYRK1A kinase conformations or kinase subfamilies that can help distinguish the target kinase from other kinases.2 Additionally, the competitive ATP inhibitors must resolve the difficulties encountered from high intracellular ATP levels, which may cause inconsistencies between the IC50 values measured by biochemical versus cellular assays. To overcome these problems, the design of non-ATP-competitive inhibitors provides an opportunity for the further development of selective and specific inhibitors with novel chemical scaffolds, as these inhibitors do not compete with the 44 ACS Paragon Plus Environment

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intracellular concentrations of ATP and are more specific than ATP-competitive inhibitors. It also targets more diverse regions or premature kinases; furthermore, the non-ATP-mimetic inhibitors interfere with the functionality of the ATP binding site by inducing a conformational shift in the target enzyme such that the kinase is no longer able to function. Nevertheless, some novel interesting compounds have been in development as selective and more specific inhibitors of DYRK1A. For example, we discussed novel compound 63 as a potent and selective inhibitor of DYRK1A compared to other DYRKs and members of the CLK kinase family. Currently, major efforts are being made in developing strong and selective DYRK1A inhibitors for therapies of neurodegenerative disorders, myeloid leukemias and gliomas. Since the use of the most potent and orally bioavailable archetypical DYRK1A inhibitor (3), progressive changes have been made to this class of inhibitors over the past few years. Compound 3 exhibited potent activity against cell growth and tumorigenesis in myeloid leukemias and gliomas. Harmine analogues 7a and 7b could also be developed as chemical probes for DYRK1A in terms of selectivity and potency. Furthermore, considering the relevance of DYRK1A in cancer, it appears that newly reported DYRK1A inhibitors, which are mainly developed for their applicability in neurodegenerative diseases, could also be considered as anticancer agents for acute megakaryoblastic leukemia (AMKL) and glioblastoma multiforme (GBM). It is desirable to study the efficacy of DYRK1A inhibitors in other neural tumors that display overexpression of DYRK1A, such as oligodendrogliomas. Harmine-inspired heterocyclic compound 61, a potent DYRK1A inhibitor, has demonstrated its therapeutic potential for DYRK1A-associated diseases, and this molecule is currently in clinical trials (Phase II) for cancer treatment. Therefore, it is foreseeable that more potent and selective DYRK1A drug candidates with different and new mechanisms of action will considerably increase in clinical trials in the near future.



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To determine the target of action in the treatment of animal models and patients with DYRK1A drug candidates, it is necessary to develop practical, sensitive detection methods that enable a rapid and accurate investigation of the effects of the inhibitors in cells, animals, and patients. From a development perspective, the biggest challenge is to design DYRK1A inhibitors in such a way that it does not suppress the DYRK1A activity completely, but only to a certain extent where it does not affect the healthy individual. For example, excessive inhibition of DYRK1A in the case of MRD7 caused loss-of-function mutations of DYRK1A.

■ CONCLUSION Inhibition of DYRK1A serves as an emerging area of investigation, and a large number of DYRK1A inhibitors have been actively discovered, mainly by targeting the ATP competitive binding site. Some of the discovered DYRK1A inhibitors have already entered clinical trials. Few DYRK1A inhibitors are specific for DYRK1A from among the CMGC group of kinases; hence, the issues of selectivity and specificity need to be addressed to utilize DYRK1A inhibitors as tools for understanding the role of DYRK1A in physiological and pathological processes. These studies will lead to the validation of DYRK1A as a potential drug target, particularly in Down syndrome and Alzheimer’s disease. In the future, we anticipate the possibility of substantial changes in the diagnosis and treatment of various neurodegenerative disorders. ■ AUTHOR INFORMATION Corresponding Author *

Lak Shin Jeong: Phone, 82-2-880-7850; E-mail: [email protected] 46 ACS Paragon Plus Environment

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ORCID Karishma K. Mashelkar: 0000-0002-6125-5272 Minsoo Noh: 0000-0002-4020-5372 Lak Shin Jeong: 0000-0002-3441-707X Author Contributions †J.

D. B. and M. K. K. contributed equally to this work. The manuscript was written through

contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. Biographies Dnyandev B. Jarhad received his Ph.D. degree in 2016 from Indian Institutes of Technology Bombay (IIT Bombay), India, where he worked with Professor V. K. Singh on the total synthesis of natural products and their analogues. Since 2016, he is pursuing postdoctoral research at Seoul National University under the guidance of Professor Lak Shin Jeong. His current research interests include development of therapeutic nucleosides, particularly antiviral and antitumor nucleosides. Karishma K. Mashelkar obtained her M.S. degree in Organic Chemistry from Goa University (India) and then she joined Syngenta research and development, India as a Research Associate in their discovery group. She is currently pursuing her doctoral degree under the supervision of Prof. Lak Shin Jeong at Seoul National University. Her current research is related to the medicinal chemistry field and mainly focuses on the design and synthesis of nucleosides as kinase inhibitors. Hong-Rae Kim earned his bachelor’s degree in chemistry from the University of Washington, Seattle, USA in 2012. He is currently pursuing his doctoral degree at Seoul National University under the 47 ACS Paragon Plus Environment


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supervision of professor Lak Shin Jeong. His current research focuses on the synthesis of bioactive modified nucleosides. Professor Minsoo Noh received his bachelor’s and master’s degrees from Seoul National University and completed his Ph.D. at the Massachusetts Institute of Technology, USA. After working at Bioscience & Skin Research department of Amorepacific Corporation as a principal investigator for few years, he joined College of Pharmacy, Ajou University as a faculty member in 2010 and moved to College of Pharmacy, Seoul National University in 2013. He currently is an associate professor at Seoul National University where he is conducting active research. Professor Lak Shin Jeong received his BS (1984) and MS (1986) degrees from Seoul National University and a Ph.D. (1992) from University of Georgia working under the direction of Prof. Chung K. Chu. After carrying out postdoctoral research at the National Cancer Institute, USA, he joined the faculty of the College of Pharmacy at Ewha Womans University as Assistant Professor (1995) and was promoted to full professor in 2002. In 2013, he moved to the College of Pharmacy, Seoul National University. His research interests include development of novel antiviral nucleosides using chiral templates, development of methodologies for antiviral and antitumor nucleosides, computer-aided drug design, synthesis of antisense oligonucleotides, development of adenosine receptor ligands, and development of novel next-generation 4’-selenonucleosides.

■ ACKNOWLEDGMENTS This research was supported by the grants from Mid-career Research Program (370C-20160046) and the Ministry of Science, ICT & Future Planning (370C-20170057) of the National Research Foundation (NRF) of Korea ■ ABBREVIATIONS USED 48 ACS Paragon Plus Environment

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AD, Alzheimer’s disease; ALARM NMR, a La assay to detect reactive molecules by nuclear magnetic resonance; AMKL, Acute megakaryoblastic leukemia; APP, amyloid precursor protein; AurA, Aurora A kinase; AurB, Aurora B kinase; BINDY, Benzofuro-fused inhibitor of DYRK; CaNDY, CDC37 association inhibitor for DYRK1A; Cdc2, cell division control protein 2 homolog; CDKs, cyclindependent kinases; Chk1, checkpoint kinase 1; CK1/2, casein kinase 1/2; CK5, casein kinase 5; CLKs, cyclin-dependent-like kinases; CMGC, cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAP kinases), glycogen synthase kinases (GSK) and CDK-like kinases; DANDY, diarylazaindole inhibitors of DYRK1A; DMAT, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; DMT, N, N-dimethyltryptamine; DS, Down syndrome; DYRK1A, Dual-specificity tyrosine-regulated kinase 1A; EGCG,

epigallocatechin gallate; EGFR, epidermal growth factor receptor; ERKs,

extracellular signal–regulated kinases; FDA, Food and Drug Administration; FGFR1, Fibroblast growth factor receptor 1; Ftl3, fms like tyrosine kinase 3; GBM, glioblastoma multiforme; GSK3, glycogen synthase kinase-3; HGK, hepatocyte progenitor kinase-like/germinal center kinase-like kinase; Ikkb, inhibitor of nuclear factor kappa-B kinase subunit beta; INDY, inhibitor of DYRK; Jak2, Janus kinase 2; KDR, Kinase insert domain receptor; MAO-A, monoamine oxidase A; MAP, mitogen-activated protein kinase; MAP1B, microtubule-associated protein; MAPKs, mitogen-activated protein kinases; MRD7, Mental Retardation Disease 7; NFAT, nuclear factor of activated T-cells; NFATc, nuclear factor of activated T-cells, cytoplasmic; NFTs, neurofibrillary tangles; PAINS, pan assay interfering compounds; PI3K, phosphoinositide 3-kinase; PIM1, proto-oncogene serine/threonine-protein kinase; PKB ,Protein kinase B; PRAK, p38 regulated/activated kinase; PS1, presenilin 1; ROS, reactive oxygen species; SAR, structure–activity relationship; SF3B1, splicing factor 3b1; Spry2, Sprouty2; SYK, Spleen tyrosine kinase; TBB, 4,5,6,7-tetrabromo-1H-benzotriazole; TBI, 4,5,6,7-tetrabromo-1H-benzimidazole

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