Cinnamic Acid Derivatives as Inhibitors of Oncogenic ...

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Cinnamic Acid Derivatives as Inhibitors of Oncogenic Protein Kinases – Structure, Mechanisms and Biomedical Effects# Marcin Mielecki* and Bogdan Lesyng Bioinformatics Laboratory, Mossakowski Medical Research Center, Polish Academy of Sciences, Warsaw, Poland Please provid e Abstract: Cinnamic acid belongs to phenolic-acid class of polyphenols, one of the most corresp on din g author(s) p hotograp h abundant plant secondary metabolites. These substances are widely studied because of plethora of their biological activities. In particular, their inhibition of protein kinases contributes to the pleiotropic effects in the cell. Protein kinases are essential in controlling cell signaling networks. Selective targeting of oncogenic protein kinases increases clinical anticancer efficacy. Cinnamic acid and related compounds have inspired researchers in the design of numerous synthetic and semisynthetic inhibitors of oncogenic protein kinases for the past three decades. Interest in cinnamoyl-scaffold-containing compounds revived in recent years, which was stimulated by modern drug design and discovery methodologies such as in vitro and in silico HTS. This review presents cinnamic acid derivatives and analogs for which direct inhibition of protein kinases was identified. We also summarize significance of the above protein kinase families – validated or promising targets for anticancer therapies. The inhibition mode may vary from ATP-competitive, through bisubstrate-competitive and mixedcompetitive, to non-competitive one. Kinase selectivity is often correlated with subtle chemical modifications, and may also be steered by an additional non-cinnamoyl fragment of the inhibitor. Specific cinnamic acid congeners may synergize their effects in the cell by a wider range of activities, like suppression of additional enzymes, e.g. deubiquitinases, influencing the same signaling pathways (e.g. JAK2/STAT). Cinnamic acid, due to its biological and physicochemical properties, provides nature-inspired ideas leading to novel inhibitors of oncogenic protein kinases and related enzymes, capable to target a variety of cancer cells.

Keywords: Anticancer, BCR-ABL, cinnamic acid, CK2, curcumin, EGFR, HER2, JAK2, kinase, mTOR, PIM1, PIM2, RIO1, RSK2, STAT, tyrphostin. 1. INTRODUCTION Plant products like fruits, vegetables, herbs, spices, cereals, essential oils and propolis, have been considered to be an important source of bioactive compounds for centuries. There is growing statistical evidence that fruits and vegetables present in the diet may prolong the average life expectancy [1] and are strongly implicated in cancer chemoprevention [2]. One of the most abundant group of plant secondary metabolites are polyphenols, in particular: phenolic acids (hydroxybenzoates and hydroxycinnamates), flavonoids, stilbenes, curcuminoids, coumarins, lignans and quinones. They have drawn significant scientific attention due to their *Address correspondence to this author at the Bioinformatics Laboratory, Mossakowski Medical Research Center, Polish Academy of Sciences, Pawińskiego 5, 02-106 Warsaw, Poland, E-mail: [email protected] # Dedicated to the memory of Professor Krystyna Grzelak 0929-8673/16 $58.00+.00

numerous and widely studied biological activities. Their antioxidant, immunomodulatory and anticancer properties are the most frequently studied [2-12]. Many polyphenols exert cytotoxic potential with some level of selectivity – inducing apoptosis of neoplastic cells. Thus, dietary polyphenols are linked to the maintenance of health [13]. Disproportionately less experiments were carried out to establish molecular interactions of polyphenols with macromolecular components. In perspective, this is crucial to understand the synergy of the bioactive constituents consumed in plant foods [14]. Derivatives of cinnamic acid (Fig. 1), are ubiquitous in plant products and have low toxicity as significant dietary components. A number of biological activities of natural or synthetic cinnamic acid derivatives were described [15]. Anticancer (antineoplastic) potential of cinnamic acids, esters, amides, hydrazides and other related synthetic compounds was also comprehensively © 2016 Bentham Science Publishers

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Mielecki and Lesyng

reviewed [16] and is a subject of several patent applications, e.g. [17, 18]. Physicochemical properties of these nature-derived compounds make them attractive scaffolds for a design of new drug candidates. Cinnamic acid (Fig. 1) exposes several possible points of derivatization: a phenyl ring, a carboxyl (or amide) group, and a (trans)-α,β-unsaturated carbonyl, which is a Michael acceptor [16]. Interestingly, cellular thiols (e.g.: glutathione, cysteine) may alkylate the β-position, leading to thiol derivatives and modulating properties of cinnamates in the cell. This is evident that cinnamic erbstatin OH

acid derivatives exert their broad chemopreventive biological effects, in particular: anticarcinogenic, cardioprotective, and antineurodegenerative, through their strong antioxidant potential and scavenging free radicals in the cell [19]. Additionally, these compounds may more or less specifically, directly or indirectly interact with macromolecular components of the cell, especially with receptor or intracellular proteins. Antineoplastic (antiproliferative and antimetastatic) agents may, for example, intervene in the signaling networks controlling the cell fate. genistein

cinnamic acid

OH

O

H N

OH

O

O OH HO

O

OH from 2007

from 1989

modern derivatives

tyrphostin-related derivatives

O

O HO

OH

HO

N

NH2

HO

N

A1-A4

S

HO

A

O

O

HO

O

A1-A4

N H

A

nanomolar inhibitors of protein kinases NH2

HO

ERBB1 ABL CK2 PIM1 PIM2 mTOR RSK2

N

O X1-X4

L

L

N H

L

Y X1-X4

N

micromolar inhibitors of protein kinases

A = any substituent L = C or N (pyridine ring)

ERBB1 ERBB2 JAK2 ABL RIO1

X = any, offen hydroxyl, halogen, metoxyl Y = short straight or branched alkyl chain

Fig. (1). Two routes of biochemical evolution of the cinnamic acid-inspired inhibitors of protein kinases: historical (starting from 1989) and modern one (starting from 2007). The first one was based on the rational inhibitor design and lead to development of the tyrphostin-related derivatives, the second one – based mainly on the cinnamoyl-derivatization of existing inhibitor fragments and high throughput screening (virtual in silico and biochemical in vitro) methodologies. Year above the arrow indicates invention of the first inhibitors.

Cinnamic Acid-Inspired Inhibitors of Protein Kinases

The main classes of proteins reported to be affected by cinnamic acid derivatives are: transcription factors, matrix metalloproteinases, oxidative stress-and inflammation-related enzymes, DNA-modifying enzymes and kinases [11, 12, 16, 19, 20]. Cinnamic acid derivatives may affect both tyrosine-specific and serine/threonine-specific protein kinases in the cell: ERBB1 (EGFR), ERBB2 (HER2), BCR-ABL, JAK2, TPK, PKC, ERK, JNK, p38 MAPK, IKK, PAK1, MEKK3, FAK, AKT, FYN, FLT3, CK2 [11, 12, 16, 19, 20]. It should be stressed, however, that these effects on cellular proteins may be highly heterogenic (for example on the level of gene expression, protein folding, enzymatic activity and/or upstream regulators) and may not result from direct protein – smallmolecule interactions. There is an increasing awareness that often, despite of the in-detail described cellular effects of these derivatives, primary cellular targets have yet to be defined. This review focuses on interactions of the synthetic and semisynthetic derivatives of cinnamic acid with protein kinases (ERBB1, ERBB2, JAK2, ABL, RIO1, CK2, PIM1, PIM2, mTOR, RSK2) as the crucial components of the signaling networks and one of the most important and widely exploited targets of anticancer drugs. 2. PROTEIN KINASES AS TARGETS IN ANTICANCER THERAPY Protein kinases comprise one of the largest protein superfamilies. They are classified as phosphotransferases catalyzing transfer of a phosphate moiety from purine nucleoside triphosphates to specific substrates in proteins. Protein kinases are classified according to homology of their catalytic domains consisting of 250300 amino-acid residues. This, in particular, refers to the characteristic bilobal structure – a β-stranded Nlobe and an α-helical C-lobe connected by a flexible hinge region [21]. Protein kinases are encoded by ca. 1.7 % of human genes (518 genes, [22]). They play critical roles in such fundamental cellular processes as: metabolism, cell cycle, cell division, differentiation and apoptosis. About a half of all known human kinase genes are involved in oncogenesis and other major diseases [23, 24]. Protein kinases (as well as G-protein coupled receptors) are one of the most important groups of oncogenic molecular targets [25]. 145 tyrosine kinases are under evaluation in drug discovery [26] and more than 34 are validated anticancer drug targets [27]. Examples of the most important are: BCR-ABL, ERBBs, CDKs and VEGFR [28]. The tyrosine kinase domain has become

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a proved and successful target for drug design. A lot of laboratories all over the world search for small molecule compounds that could serve as effective inhibitors of protein kinases and further on as anticancer drugs. More than 250 compounds are being evaluated in various phases of clinical trials [29, 30] and the vast majority is ATP-competitive. Several kinase inhibitors received US Food and Drug Administration approval for cancer treatment [31, 32]. The breakthrough discovery made in 2001 by Novartis scientists showed that imatinib (STI571, Signal Transduction Inhibitor 571 or Gleevec) is a clinically effective inhibitor of the oncogenic fusion kinases BCR-ABL and TEL-ABL [33-35]. The inhibitor was called a new paradigm for anticancer targeted therapies as a successful treatment for chronic myelogenous leukemia (CML). Unfortunately, the problem of relapse quickly appeared – de novo mutations resulted in resistance against Gleevec in the new cancer cell clones [36]. Compounds that circumvent the acquired resistance to the first generation tyrosine kinase inhibitors were tested in patients with the refractory disease, e.g. nilotinib – Tasigna [37]. Agents directed against new molecular targets are as well being explored. There is growing opinion among specialists that molecules interfering with multiple kinases (multipletarget inhibitors) might be more effective than singletarget agents [38, 39]. For example, imatinib, sorafenib and sunitinib effectively block the tyrosine kinases from different families [40]. Advantages of such inhibitors may include synergized cellular effects and potentially reduced sensitivity to resistant de novo kinase mutants. Gleevec was already employed for the treatment of not only FIP1L1-PDGFRα-positive but also FIP1L1-PDGFRα-negative eosynophilic syndromes (e.g. chronic eosynophilic leukemia and idiopathic hypereosynophilic syndrome) [41, 42]. However, possible toxicity of the compounds with reduced selectivity may be unfavorable or disapproving. Definitely more scaffolds should be scrutinized. Natural polyphenol-derived inhibitors of protein kinases – derivatives of cinnamic acid might fulfill the above-mentioned requirements and compete with the existing drugs. 3. EPIDERMAL GROWTH FACTOR RECEPTORS (ERBB) ERBB1 (EGFR or HER1) is a member of an ERBB family of receptor tyrosine kinases. The protein molecule was discovered by Stanley Cohen in the late 1970s as a phosphorylated protein band which appeared upon EGF-stimulation [43]. Further on EGFR was reported

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to share sequence homology with the v-erb-b avian erythroblastic leukemia viral oncogene and hence the name ERBB. Other three members of the ERBB family are: ERBB2 (HER2), ERBB3 (HER3) and ERBB4 (HER4) [30]. Their molecular masses range between 175 and 190 kDa and are higher than the sequencepredicted (134-148 kDa) ones due to glycosylation. These receptor kinases consist of three domains: a ligand-binding (glycosylated) ectodomain, a transmembrane domain and a cytoplasmic tyrosine-kinase domain with the extensively phosphorylated C-terminal extension. They are specifically activated by growth factor ligands and may form homodimers and heterodimers [44, 45]. EGF, TGFα, amphiregulin, and epigen exclusively bind ERBB1. Betacellulin, HBEGF, and epiregulin may stimulate the ERBB1 and ERBB4 receptors. Neuregulins 1-4 may bind ERBB4, and neuregulins 1-2 stimulate also ERBB3. The latter may interact in a unique way with neuroglycan C. The differential stimulation pattern and formation of heterodimers enhance the ERBB signaling complexity [44, 45]. The process of ligand-binding and EGFR dimerization enables autophosphorylation of the cytoplasmic region of the receptor and creates efficient docking phosphotyrosine sites for effector proteins. This leads to activation of downstream proliferation and prosurvival signaling pathways: PI3K/AKT/ mTOR, MAPK (RAS/RAF/MEK/ERK), SRC and JAK/STAT [44-46]. 3.1. ERBB1 and ERBB2 Kinases as Oncogenes Clinical significance of ERBB kinases was recently comprehensively and expertly discussed by Artaega end Engelman [47]. Overexpression, gene amplifications and/or constitutively active mutants of ERBB1 and ERBB2 are altered in many epithelial tumors (Table 1), and clinical studies indicate that they have important roles in tumor etiology and progression. The most significant EGFR mutations occur in the ectodomain and in the kinase domain, and enable constitutive dimerization and ligand-independent activation or increased ligand-dependent kinase activity. The deletion of exons 2-7 (amino acids 6-273, EGFRvIII) of the ectodomain is observed in 40% of high grade glioblastoma multiforme, and the deletion of amino acids 521602 (EGFRc958) – in 20% of glioblastoma multiforme [44, 47]. Activating mutations in the kinase domain (exons 18-21) of EGFR are often characteristic of nonsmall cell lung (adeno)carcinomas [44]. Gene amplification of HER2 and resulting overexpression occurs in 20-30% of breast cancer [44]. Significantly, targeting the wild-type EGFR receptor with antibodies in some

Mielecki and Lesyng

colorectal cancers and head and neck cancers efficiently blocks tumor growth. Upregulation of ERBBs is often associated with a poor clinical outcome. EGFR and HER2 are highly validated drug targets and treatment paradigms against several types of neoplasms and metastatic carcinomas. Two classes of ERBB-targeted drugs used in the clinic are monoclonal antibodies (e.g.: trastuzumab – Herceptin, pertuzumab – Omnitarg, cetuximab – Erbitux, panitumumab) and small-molecule kinase inhibitors [59, 60]. These drugs rapidly suppress the ERBB-dependent downstream signaling. Clinically approved EGFR-targeting ATPcompetitive inhibitors are quinazoline derivatives: gefitinib-Iressa (AstraZeneca), erlotinib – Tarceva (Genentech/OSI Pharmaceuticals) against non-small cell lung cancer and pancreatic cancer, lapatinib – Tykerb (GlaxoSmithKline) against breast cancer, and afatinib – Gilotrif (Boehringer Ingelheim Pharmaceuticals) against non-small cell lung cancer. Lapatinib and afatinib also target HER2. Gefitinib was developed from tyrphostin AG1478 – a potent anilinoquinazoline (with no cinnamoyl scaffold) inhibitor of the EGFR kinase [61]. Recently developed and clinically tested EGFR- and/or HER2-targeting inhibitors are: daconitinib, icotinib, neratinib, poziotinib, vandetanib, BMS599626, TAK-285, with such irreversible ATPcompetitive inhibitors of EGFRT790M as: AZD-9291, CO-1686 – rociletinib, and WZ4002 [30]. Advantages of pan-HER inhibitors (afatinib and daconitinib) with undergoing clinical trials, and their potential to improve patient outcomes in non-small cell lung cancers and head and neck cancers were recently discussed by Wang and coworkers [62]. Inhibitors of the ERBB receptor tyrosine kinases may be used in combination with inhibitors of their corresponding downstream signaling to synergize the cancer-cell suppressive effect. 3.2. Cinnamic Acid Derivatives as Inhibitors of ERBB1 and ERBB2 Erbstatin and its stable analogs – hydroxycinnamates (Fig. 1) were commonly exploited as micromolar non-ATP-competitive inhibitors of tyrosine phosphorylation in various cellular processes [63-66]. Methyl 2,5dihydroxycinnamate, for example, inhibited the EGFinduced phenotypic changes in the EGFRoverexpressing NIH3T3 cells [67]. This analog was also reported to inhibit the G2/M transition of the immortalized human lymphoid cells at concentrations below IC50 for the EGFR inhibition and also below that reported for the induction of protein cross-linking [68]. However, the kinase selectivity of these hydroxycin-

Cinnamic Acid-Inspired Inhibitors of Protein Kinases

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Table 1. Selected examples of EGFR and HER2 genetic aberrations detected in solid tumors. Nb

EGFR or HER2 Aberration

Cancer Type

Reference

1

EGFR amplification and overexpression Deletions: EGFRc958, EGFRvI (N-terminal), EGFRvII (exons 14-15), EGFRvIII (exons 27), EGFRvIV (exons 25-27), EGFRvV (C-terminal)

glioblastoma

[44, 47]

2

EGFR amplification and overexpression Insertions in exon 20 of EGFR Deletion: EGFR 746-A750 (ELREA, exon 19) Substitutions: EGFRG719S/C/A (exon 18), EGFRT790M (exon 20), EGFRL858R (exon 21), EGFRL861Q/R (exon 21)

lung

[44, 48]

3

HER2 amplification and overexpression EGFR overexpression Deletions: EGFRvIII, HER2 L755-T759 (exon 19) Substitutions: HER2L755S (exon 19), HER2S760A (exon 19), HER2R896C (exon 22)

breast

[49, 50]

4

EGFR amplification and overexpression HER2 amplification and overexpression Substitutions: EGFRY801C (exon 20), EGFRL858R (exon 21), EGFRG863D (exon 21), HER2K724N (exon 18), HER2T733I (exon 18), HER2L755S (exon 19), HER2D769H (exon 19), HER2V777L (exon 20), HER2Q799P (exon 20), HER2L869Q (exon 21)

gastric

[49, 51]

5

EGFR amplification and overexpression HER2 amplification and overexpression Substitutions: HER2V777L/M (exon 20), HER2V842I (exon 21)

colorectal

[49, 52]

6

EGFR amplification and overexpression HER2 amplification and overexpression Deletion: EGFRΔE746-A750 (exon 19) Substitution: EGFRL858R (exon 21)

salivary gland

[53]

7

EGFR overexpression Deletion: EGFRvIII

head and neck

[54]

8

EGFR and HER2 overexpression

bladder

[55]

9

HER2 amplification and overexpression

endometrial

[56]

10

EGFR amplification and overexpression HER2 amplification and overexpression

ovarian

[57]

11

EGFR amplification and overexpression HER2 amplification and overexpression

esophageal

[58]

ΔΕ

Δ

namate analogs of erbstatin has not been extensively studied. Tyrphostins – tyrosine phosphorylation inhibitors – were the first inhibitors of EGFR and signal transduction. They were designed and synthetized in group of Levitzki and Gazit in Hebrew University of Jerusalem in the late 1980s based on a scaffold of such natural polyphenols as: erbstatin, quercetin, genistein, lavendustin A, herbimycin A, aeroplysinin, and also a tyrosyl moiety of substrate proteins [69-72]. Thus, tyr-

phostins were invented to bind to the substrate subsite of the tyrosine kinase domain and to block binding of peptide substrates in the active site. Subsequent studies found that tyrphostins also exerted ATP-competitive, bisubstrate-competitive, and mixed-competitive mechanisms of inhibition [61]. The identified scaffold responsible for the potency of the EGFR inhibitors was polyhydroxycyanocinnamate (Fig. 1). These compounds were competitive with the poly(GAT) substrate and also mixed-competitive, with low micromolar IC50

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values, e.g.: 25 µM for compound 36 (3,4-dihydroxyα-cyanocinnamate), 10 µM for compound 46 (3,4dihydroxycinnamide), 6 µM for compound 24 (3,4,5trihydroxybenzylidenemalononitrile) and 3 µM for compound 25 (3,4-dihydroxy-5-methoxybenzylidenemalononitrile) (Fig. 2). Extending the side chain of the cinnamonitrile core with the substitution of -COOH with -C(NH2)=C(CN)2 increased the inhibitor potency with IC50 down to 0.125 µM (compound 48) (Fig. 2) and shifted the mechanism of inhibition towards the mixed- competitive. The above work inspired other specialists to design small molecule inhibitors of the ERBB kinases for anticancer targeted therapies. compound 36, IC50= 25 µM O HO

OH

HO

N

compound 46, IC50= 10 µM O HO

NH2

HO

N

compound 24, IC50= 6 µM N

HO

Mielecki and Lesyng

From the series of 3,4-dihydroxy-α-cyano-cinnamic acid derivatives compound 42 – AG490 (2-cyano-3-(3,4-dihydroxyphenyl)-N-benzyl-2-propenamide) (Fig. 3) appeared to be the most potent in inhibiting the EGFR autophosphorylation and significantly less potent in inhibiting the HER2 autophosphorylation (IC50 = 0.1/13.5 µM) in membrane extracts of the EGFR-dependent HER14 cell line. The AG490 IC50 for inhibiting the EGFR-catalyzed poly(GAT) phosphorylation was 2.0 µM and GI50 for the EGFR-dependent proliferation was 3.5 µM. Methyl derivative of AG490 (compound 44(-), IC50 = 0.4 µM) was also among the compounds with the highest ratio in inhibiting the EGFR/HER2 autophosphorylation (2.5/37 µM). The best antiproliferative activity was in the range of 2.53.0 µM and was 4-20-times higher than the IC50 values observed in the kinase experiments. Selectivity of the AG490-related compounds as inhibitors of the EGFR autophosphorylation was confirmed in further studies [73]. The example EGFR/HER2 IC50 potencies were: 1.1/45 µM for AG494, 0.5/12.1 µM for AG490, and 1.0/9.4 µM for AG698 (Fig. 3). The authors also reported the HER2-autophosphorylation inhibitors: AG658 (IC50 = 0.13 µM), AG800 (IC50 = 0.2 µM), AG825 (IC50 = 0.35 µM) (Fig. 4) and postulated ATPcompetitive mechanism of inhibition of the studied derivatives of α-cyanocinnamides (shown for AG494 and AG825). AG 494, IC50= 1.1/45 µM

HO O

N

compound 25, IC50= 3 µM

µ AG 490, IC50= 0.5/12.1 M

N

HO

O HO

HO OH

HO

compound 48, IC50= 0.125 µM NH2

HO

N H

N

N

N

µ AG 698, IC50= 1.0/9.4 M

N

N

Fig. (2). Structures of the first inhibitors of the EGFR kinase – tyrphostin-related cinnamic acid derivatives (with IC50 referring to inhibition of the EGFR-catalyzed peptide substrate phosphorylation).

Further work on tyrphostins succeeded in developing novel inhibitors and confirmed that substituted cinnamide family can be EGFR- or HER2-selective [70]. From the series of 3,4-dihydroxy-α-cyano-

Fig. (3). Structures of the inhibitors of the EGFR kinase – tyrphostin-related cinnamic acid derivatives, also tested on the HER2 kinase (with IC50 referring to inhibition of the EGFR/HER2 autophosphorylation).

According to our best knowledge, there have been no further reports on the structure of a complex of an ERBB tyrosine kinase with a compound built on the αcyanocinnamic core. Nevertheless, this would un-

Cinnamic Acid-Inspired Inhibitors of Protein Kinases

doubtedly help to understand molecular interactions of these cinnamic acid derivatives with the receptor tyrosine kinases, and their mechanism of action. This would also facilitate designing novel more potent and selective EGFR/HER2-ligands based on the αcyanocinnamic core. AG 658, IC50= 130 nM

S

AG 800, IC50= 200 nM

O O

NH2 S

HO

N

AG 825, IC50= 350 nM

S S

N

Fig. (4). Structures of the inhibitors of the HER2 kinase – tyrphostin-related cinnamic acid derivatives (with IC50 referring to inhibition of the HER2 autophosphorylation).

Despite the lack of any structural data for these ERBB inhibitors, quite natural conclusion is that molecular interactions responsible for binding and stabilization of the inhibitor in the ATP-pocket of the EGFR and/or HER2 kinases differ enough for some degree of selectivity. Appropriate modifications of the same αcyanocinnamic core may guide a mode of binding and a mechanism of inhibition as well as selectivity of an inhibitor. The similar antiproliferative potency of the EGFR-directed and HER2-directed inhibitors was explained by plausible inhibition of other ERBBdownstream signaling kinases. It was also observed that the designed compounds were approximately 10times less effective as blockers of EGFR autophosphorylation than blockers of EGFR-catalyzed poly(GAT) substrate phosphorylation [69]. The same was reported for cinnamic acid benzyl amide derivatives as more potent inhibitors of JAK2-catalyzed STAT5-peptide substrate phosphorylation than inhibitors of JAK2 autophosphorylation [74].

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The ability of tyrphostins to specifically inhibit EGF-dependent cell proliferation was reported soon after their discovery [69]. α-cyanocinnamic derivatives were effective antiproliferative and proapoptotic agents of the EGF-dependent HER14 cell line (AG490 GI50 = 3.5 µM, Fig. 3), the EGF-dependent keratinocytes (AG490 IC50 = 0.2 µM for inhibition of the EGFR autophosphorylation compared with GI50 = 7.0 µM) and the HPV16-transformed keratinocytes HF-1 with EGFR overexpression [70, 75-77]. The antiproliferative effect on the HF-1 cells, however, did not necessarily correlate with the inhibition of EGFR autophosphorylation as shown for AG494 (GI50 = 4.7 µM, Fig. 3) and AG555 (GI50 = 6.4 µM, Fig. 6) [78]. The authors demonstrated the potential of tyrosine kinase inhibitors as selective antiproliferative agents for proliferative diseases caused by overactivation of a protein tyrosine kinase (e.g. psoriasis). Compounds including the derivatives of α- cyanocinnamates, α-cyanocinnamides, and α-cyanocinnathioamides (and also benzylidenemalononitriles) can be used to treat proliferative disorders characterized by overactivation of the receptor tyrosine kinases, in particular EGFR and HER2 [79]. A series of derivatives of metronidazole cinnamate led to a relatively potent inhibitor of the EGFR (IC50 = 0.62 µM) and HER2 (IC50 = 2.15 µM) autophosphorylation, which was metronidazole paraphenylcinnamate (compound 3h, Fig. 5) [80]. The compound suppressed proliferation of the EGFR-overexpressing MCF-7 cell line with GI50 = 0.36 µM. The authors showed via molecular docking that this inhibitor was a potential blocker of the ATP pocket of EGFR. Atoms of the cinnamoyl moiety contributed to the binding affinity of the inhibitor which might be stabilized in the active site by three possible hydrogen bonds with amino acids of the ATP-pocket. Virtual screening and extensive molecular modeling allowed identification of four ligands of EGFR from the Traditional Chinese Medicine Database also targeting the ATP-pocket of EGFR [81]. These ligands had docking score and predicted biological activity (pIC50) higher that gefitinib (Iressa). Two of these compounds were 2-O-caffeoyl tartaric acid (3,4-dihydroxycinnamoyl tartaric acid) and 2-Oferuloyl tartaric acid (3- methoxy-4-hydroxycinnamoyl tartaric acid) (Fig. 5). The predicted binding affinities of caffeoyl/feruloyl tartaric acid in the ATP-pocket of EGFR were in the low-nanomolar range with pIC50 = 8.386/8.359 for Multiple Linear Regression method (approximated IC50 = 4 nM) and 7.041/7.242 for Support Vector Machine method (approximated IC50 = 90/57 nM). Importantly, the modeled complexes were

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Mielecki and Lesyng AG 82, IC50= 7 µM

stable during 20-ns molecular dynamics simulations. The authors [81] concluded that the identified compounds were highly plausible bioactive EGFR inhibitors.

N

HO

HO

AG 490, IC50= 48 µM

N

O

N

OH

compound 3h, IC50= 0.62/2.15 µM

N

O

NO2

AG 555, IC50= 88 µM O

2-O-caffeoyl tartaric acid, IC50= 90 nM OH HO O HO

HO

HO

O

N H

AG 556, IC50= 16 µM

N

O

O OH

HO

2-O-feruloyl tartaric acid, IC50= 57 nM

LS-104, IC50= 2.52 µM O

OH O

HO

O

HO N H

O O

O OH

HO

N

Fig. (5). Structures of the inhibitors of the EGFR and HER kinases – modern cinnamic acid derivatives (with IC50 referring to inhibition of the EGFR/HER2 autophosphorylation by compound 3h and EGFR-IC50 estimated computationally for cinnamoyl tartaric acids).

Fig. (6). Structures of the inhibitors of the JAK2 kinase – AG490-related cinnamic acid derivatives (with IC50 referring to inhibition of the JAK2 autophosphorylation for AG490, AG82, AG555, AG556, and referring to inhibition of the JAK2-catalyzed peptide substrate phosphorylation for LS104).

4. JANUS KINASES (JAK)

4.1. JAK2 Kinase as Oncogene

Human kinases JAK1 and JAK2 were discovered in 1989 by Wilks [82], who identified their coding sequences based on nested PCR. The JAK family of nonreceptor tyrosine kinases includes also JAK3 and TYK2. They are multidomain proteins of 120-140 kDa. The catalytic domain in the C-terminus is a functional tyrosine kinase. The neighboring pseudokinase domain is responsible for regulation of the first domain by suppressing its activity. These two almost identical domains inspired another name of the kinase family – Janus Kinases [83]. The third and fourth domains, FERM and SH2, were identified in the N-terminus. Their main role is association with the cytosolic portion of receptors for cytokines, growth factors and hormones. JAK kinases are a part of JAK/STAT extracellular signal transduction pathways, which influence many cellular processes: proliferation, differentiation, migration and apoptosis, particularly in blood and marrow cells. JAK/STAT pathways are composed of a number of effector proteins with regulatory roles, e.g.: PIAS, PTP, SH2/Lnk/APS, SOCS, STAM, and StIP [84-88].

A number of myeloproliferative neoplasms and autoimmune diseases are characterized by constitutive and extracellular signal-independent activation of the JAK/STAT pathways, frequently caused by oncogene alleles of the JAK2 tyrosine kinase [89-91]. Overactivation of JAK2 contributes to development of a number of leukemias and a smaller number of solid tumors, whereas JAK3 has an important role in the activation of T-lymphocytes in autoimmune disorders. Possible fusion alleles of JAK3 have not yet been identified [92]. Somatic mutations of the jak2 gene can be divided into two groups: substitutions and deletions of several amino acids in quite narrow area of the pseudokinase domain, and chromosomal translocations producing fusion tyrosine kinases (FTK, [92], Table 2). Mutations in the pseudokinase domain abrogate its suppressing capability towards the catalytic domain of the oncogene kinase, causing its overactivation. In chromosomal translocations a fusion partner of the variable-length C-terminal part of JAK2 is able to form dimers or oligomers, and this permits autophosphorylation of the JAK2 kinase. Oncogenes of JAK1, JAK3

HO

Cinnamic Acid-Inspired Inhibitors of Protein Kinases

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Table 2. Oncogenic JAK2 kinases in hematologic neoplasms. Nb

JAK2 Oncogene

1

JAK2V617F

2

L611S

K607N D620E

ΔIREED T875N

3 4 5

JAK2 JAK2

JAK2 JAK2

6

JAK2

7

TEL-JAK2

8

9

PCM1-JAK2

BCR-JAK2

Hematologic Neoplasm

Reference

polycythemia vera (erythremia)

[93]

essential thrombocytosis and primary myelofibrosis

[94]

acute lymphoblastic leukemia

[95]

acute myelogenous leukemia AML

[96]

non-classified myeloproliferative syndrome

[97]

precursor B-cell acute lymphoblastic leukemia B

[98]

acute megakaryoblastic leukemia

[99]

acute lymphoblastic leukemia

[100]

atypical chronic myeloid leukemia

[101]

T-cell lymphoma

[102, 103]

atypical chronic myeloid leukemia

[104]

acute erythroid leukemia

[105]

atypical acute lymphoblastic leukemia

[106]

typical chronic myeloid leukemia

[106]

acute myeloid leukemia

[107]

10

RPN1-JAK2

chronic idiopathic myelofibrosis (bone marrow fibrosis)

[108]

11

SSBP2-JAK2

precursor B-cell acute lymphoblastic leukemia B

[109]

and TYK2 occur less frequently compared to oncogenes of JAK2. A vast number of synthetic JAK2 kinase inhibitors were developed – at least 45 potent JAK2-inhibitory scaffolds were described so far [110]. Chemically they can be classified as azaheterocycles: pyrimidines, pyridines, benzimidazoles, nicotinonitriles, azaindoles and azaspirans. Although IC50 values of these inhibitors are in the low-nanomolar range, their selectivity is rather restricted because of the conservation of the ATPbinding pocket. Nonetheless, 11 JAK2 inhibitors entered clinical trials [111]. They are promising candidate drugs against myeloproliferative neoplasms and malignant neoplasms (leukemias and lymphomas) with JAK2 oncogenes. Unfortunately, side effects are still a serious problem and often dose-limiting (dose-limiting toxicity). Two JAK2 kinase inhibitors were improved by the FDA in 2011 and 2012: ruxolitinib against myelofibrosis (Jakafi, Incyte) and tofacitinib against rheumatoid arthritis (a.k.a. tasocitinib or CP-690550, Xeljanz, Pfizer) [111]. Xeljanz was not accepted by the EMA [112] because of the serious concerns of its side effects. They included: myelosuppressions (anemia, neutropenia, limphopenia), opportunistic tuberculosis and bacterial, fungal, and viral infections, malignancies

(lymphomas and solid tumors), Epstein-Barr virusassociated post-transplant lymphoproliferative disorder in renal transplant patients treated with concomitant immunosuppressive medications, gastrointestinal perforations or increased cholesterol and liver enzymes concentrations. The main cause is probably low selectivity of tofacitinib which inhibits all members of the JAK family with the similar potency. 4.2. Cinnamic Acid Derivatives as Inhibitors of JAK2 The first inhibitor of JAK2, commercially available from many companies, was tyrphostin AG490 (2cyano-3-(3,4-dihydroxyphenyl)-N-benzyl-2-propenamide) (Fig. 6). Nonetheless, it is neither potent nor JAK2-selective inhibitor with relatively high IC50 (50100 µM) required to achieve suppression of the JAK2/STAT pathway in the cancer cell [92, 113, 114]. The reported IC50 values for the JAK2 inhibition are: 36.4 µM, 145.3 µM [115], 48 µM [113] and 30.4 µM [74]. AG490 has higher inhibitory activity for the receptor tyrosine kinases HER1 (IC50 = 0.5 µM) and HER2 (IC50 = 12.1 µM, [73]) and may also noncompetitively inhibit the atypical serine/threonine protein kinase RIO1 with the similar potency (IC50 = 20-60

10 Current Medicinal Chemistry, 2016, Vol. 23, No. ??

µM, [116]). The AG490-analogous compounds inhibit JAK2 with IC50 also in the low-micromolar range (7-88 µM, [113]), e.g.: AG82 – 7 µM, AG556 – 16 µM, and AG555 – 88 µM (Fig. 6). The authors observed that minor structural modifications to the structure of AG490 did not significantly affect the ability of such compounds to inhibit JAK2 autophosphorylation [113]. The above data suggest that AG490 and structurally related compounds may behave like non-specific protein kinase inhibitors. Likewise, screening of smallmolecule libraries also suggests that the selected polyphenols may nonspecifically suppress protein kinase activity [117]. Relatively high JAK2-inhibitory values for tyrphostins correspond to rather weak effects in the cancer cell [118]. LS104, an AG490 analog (Fig. 6), inhibited the JAK2-catalyzed peptide substrate phosphorylation with low-micromolar IC50, the JAK2 autophosphorylation in the JAK2V617F-expressing Ba/F3-EpoR-VF cells and induced their apoptosis (GI50 = 1.5 µM) [119]. These data suggested that the inhibitor interacted with the enzyme in area of the substrate binding site. LS104 also targeted the FLT3 kinase, induced apoptosis and chemosensitized the FLT3-ITD-expressing acute myeloid leukemia 32D (GI50 = 4 µM) and MV4;11 (GI50 = 3 µM) cell lines [120]. LS104 was reported to enter phase I clinical trials for refractory hematological malignances and JAK2V617F-positive myeloproliferative disorders, however, there have been no further reports on the compound. Tyrphostin AG490 served as a scaffold for the design and synthesis of compounds structurally closely related to cinnamic acid benzyl amide (CABA) with improved pharmacological properties [114, 121-123]. They showed antiproliferative and proapoptotic activity in numerous cancer cell lines and this correlated with the suppression of JAK2/STAT pathways in the cancer cell. For example, WP1066 ((E)-2-cyano-N-((S)1-phenylethyl)-3-(6-bromopyridin-2-yl)acrylamide) (Fig. 7) exhibited stronger antiproliferative activity than AG490 in the cells with the JAK2 oncogene [124]. It induced apoptosis in the acute myeloid leukemia cells (GI50 < 3.0 µM, [125]), human erythroleukemia cells (GI50 = 3.0 µM, [124]), and suppressed proliferation of the cells derived from patients suffering from polycythemia vera (GI50 = 2.3 µM, [124]). Disproportionately fewer studies were devoted to establish thermodynamics and mechanism of interactions of CABA derivatives with elements of the JAK2/STAT pathway, particularly with the JAK2 kinase. Recent studies [74] performed with the aid of

Mielecki and Lesyng

molecular modeling and the analysis of the recombinant JAK2 enzymatic activity demonstrated mechanistic diversity of CABA derivatives. The authors concluded that the tested CABA analogs behaved like nonclassical inhibitors of the activated (phosphorylated) JAK2-catalyzed peptide substrate phosphorylation, although markedly weaker than clinically tested ATPcompetitive JAK2 inhibitors. Relatively small structural changes in the studied compounds affected interactions with JAK2, and their mode of action ranged from allosteric-noncompetitive to bisubstrate-competitive. These studies demonstrated that direct inhibition of JAK2 by WP1065 ((E)-2-cyano-N-((S)-1phenylethyl)-3-(pyridin-2- yl)acrylamide, IC50 = 14.8 µM), WP1130 ((E)-2-cyano-N-((S)-1-phenylbutyl)-3(3-bromopyridin-2-yl)acrylamide, IC50 = 3.8 µM), and WP1702 ((E)-2-cyano-N-((S)-1,4-diphenylbutyl)-3-(3bromopyridin-2-yl)acrylamide, IC50 = 2.9 µM) (Fig. 7) potentially contributes, albeit minimally, to suppression of the JAK2/STAT signaling pathway in the cancer cell. Additional specific structural modifications may amplify the JAK2-inhibitory effects and increase chances to overcome de novo and acquired resistance of cancer cells to the existing inhibitors [74]. WP1066 and an analogous compound WP1130 (degrasyn) are potent suppressors of the JAK2/STAT pathway but their direct inhibition of JAK2 has only a moderate contribution to these effects. The recent data suggest that the suppression of the JAK2/STAT pathway may result mainly from the inhibitor interactions with deubiquitinases. WP1066 increased ubiquitination, promoted JAK2 protein degradation and induced caspase- dependent apoptosis in the acute myelogenous leukemia cells [126] and erythroid human cells carrying the JAK2V617F mutation [124]. WP1130 inhibited deubiquitinases (e.g.: USP5, USP9x, USP14, UCHL5) of the JAK2 protein thereby blocking the kinase signaling [127, 128]. The similar mechanism of action was also reported for WP1130-downregulation of BCRABL, including the T315I mutant kinase [129, 130]. Degrasyn-like compounds are now being developed as very promising ubiquitinase-targeting anticancer agents and candidate drugs against multiple myeloma [131, 132]. 5. ABELSON PROTEIN KINASES (ABL) Abl is a family of non-receptor tyrosine kinases comprising ABL and ARG. The ABL protein was discovered as a fusion gene gag-abl expressed by the leukemogenic Abelson murine leukemia retrovirus (P120 or v-ABL) in the late 70’s independently by Witte and coworkers, and Reynolds and coworkers [133-135]. It

Cinnamic Acid-Inspired Inhibitors of Protein Kinases

was then showed for the first time for v-ABL that this protein has tyrosine-specific autocatalytic phosphotransferase activity. Subsequently, a homologous coding sequence was identified in the mouse genome (c-ABL). ABL (c-ABL) is a multidomain ubiquitously expressed 123-kDa protein. The N-terminal kinase core is composed of SH3, SH2, and a kinase domain [136]. SH2 is preceded by a variable N-terminal cap region. C- terminal to the kinase domain are functional motifs responsible for ABL subcellular localization: nuclear localization signals (three NLSs), DNA-binding HMGlike boxes (three HLBs), a nuclear export signal (NES), and actin binding domains (ABD). The main role of the ABL kinases is centered on regulating processes involving cytoskeleton reorganization in response to extracellular and intracellular stimuli and is directly connected to cell proliferation and survival: cell morphogenesis, migration, adhesion, endocytosis, autophagy, immune and neuronal synapse formation. Dozens of substrate and adaptor proteins linking the ABL kinases to the ABL-regulated processes were identified [136-138]. ABL can be activated by growth factor receptors (like: PDGFR, EGFR, IGF1R, EphB4R, MuSK), SRC kinases, adhesion receptors (integrins, cadherins), and immunoreceptors (BCR, TCR). Numerous proliferation and prosurvival pathways can be activated by v-ABL and BCR-ABL in hematopoietic cells, promoting cytokine-independent growth: RAS/E2F, PI3K/AKT, JNK, MAPK, and JAK/STAT as well as antiapoptotic BCL-2 family members [139, 140]. Regulation of the ABL catalytic activity is complex and controlled by intramolecular as well as intermolecular interactions with adaptor and substrate proteins [136, 140, 141]. The interactions correlated with the inactive conformation involve: SH3 – kinase domain/SH2 linker, SH2 – C-lobe, and myristoylated Nterminal cap – C-lobe pocket. This conformation may additionally be stabilized by the cell proteins (e.g.: Factin, retinoblastoma, peroxiredoxin 1, FUS1, PSTPIP1). Autocatalytic or SRC-catalyzed phosphorylation on tyrosine residues of the SH2/kinase linker (Y245) and activation loop (Y412) promotes disruption of the ABL inactive assembly and full activation of the kinase. In the active state the SH2 domain interacts with the kinase N-lobe and allosterically activates the kinase. Direct interaction of the SH2 and SH3 domains of ABL with the RAS effector protein RIN1 stabilizes the active conformation independently of the phosphorylation status of ABL [138].

Current Medicinal Chemistry, 2016, Vol. 23, No. ??

11

5.1. ABL Kinase as Oncogene ABL is largely known for its fusion oncoprotein BCR-ABL and as such is a paradigm of anticancer targeted therapies. This chromosomal translocation (between chromosomes 9 and 22, Philadelphia chromosome) is a driving aberration of about 95% of chronic myeloid leukemia and also some cases of B-cell acute lymphoblastic leukemia (20% – 30%), acute myeloid leukemia, and chronic neutrophilic leukemia [140, 142]. After the prototypic leukemiogenic fusion genes, gag-abl1 (v-abl1, causative for murine pre-B leukemia) and bcr-abl1, other abl1-involving chromosomal translocations were identified. They include: etv6 (tel)–abl1, eml1– abl1, nup214–abl1, zmiz1–abl1, rcsd1–abl1, sfpq–abl1, foxp1–abl1, and snx2–abl1 [140, 142]. Protein products of the above genes usually retain the SH2 domain (part or entire) and often also the SH3 domain of ABL but are deprived of the N-terminal cap. This artificial topography destabilizes the inactive conformation of the ABL tyrosine kinase domain. Additional domains/motifs of the partner proteins might also promote oligomerization of the tyrosine fusion kinase and contribute to the ABL activation / autophosphorylation. Transforming potential of these new translocations in development and progression of such human hematologic neoplasm as: chronic myelogenous leukemia, T-cell and B-cell acute lymphoblastic leukemias, and acute myelomonocytic leukemia remains to be fully established. Enhanced ABL expression and activation may also contribute to progression and invasiveness of selected solid tumors [140]. Imatinib mesylate – an ATP-competitive ABL inhibitor targets and locks the inactive conformation of the kinase domain (IC50 = 100 nM, [143]). It is an effective drug for treatment of chronic myeloid leukemia in its chronic phase. Unfortunately, resistance to the drug is often developed in an accelerated and a blast phases. A number of new low-nanomolar inhibitors of ABL have been developed so far and are clinically tested or already approved antileukemic drugs [37, 140, 143]. They include imatinib-related molecules and similarly blocking the ATP-pocket of the kinase domain (e.g.: nilotinib (Tasigna, Novartis), ponatinib (Iclusig, Ariad Pharmaceuticals), dasatinib (Sprycel, Bristol-Myers Squibb), bosutinib (Bosulif, Pfizer)), and also allosteric non-competitive inhibitors targeting the myristoyl pocket located in the kinase C-lobe of ABL and stabilizing the inactive conformation (e.g.: DPH, GNF-2, GNF-5).

12 Current Medicinal Chemistry, 2016, Vol. 23, No. ??

5.2. Cinnamic Acid Derivatives as Inhibitors of BCR-ABL Emergence of drug resistance during the imatinibbased targeted therapies of chronic myeloid leukemia poses a challenge for the treatment of hematologic malignancies. Although combination of allosteric and ATP-competitive inhibitors of ABL presents a great promise for the imatinib-resistant ABL mutants, including the most problematic T315I gatekeeper mutation [144], natural compounds are still underrepresented in the arsenal of inhibitors of the ABL kinases. WP 1065, IC50= 14.8 µM

polyG1u6Ala3Tyrl substrate phosphorylation with lower IC50 (AG82 – 3.0 µM, AG114 – 2.5 µM, AG213 – 2.4 µM, AG538 – 0.37 µM, AG776 – 1.7 µM). Tyrphostins with no cinnamoyl scaffold were more potent inhibitors of the human ABL and BCR-ABL fusions with IC50 as low as 1.1 µM for inhibition of ABL by AG514. This inhibitor had significantly higher IC50 for the EGFR inhibition (94 µM) and was ABL-selective in the presented study. The authors also postulated the selective interactions of some tyrphostins with the wild type and chimeric fusions of ABL. AG 82, IC50= 3.6 µM N

HO

O N

Mielecki and Lesyng

N H N

WP 1066

HO OH

AG 99, IC50= 21.6 µM O HO

NH2

HO

WP 1130, IC50 = 3.8 µM

O Br

N

N

AG 213, IC50= 5.8 µM S

N

N H

HO

NH2

HO

N

N

WP 1702, IC50= 2.9 µM

AG 776, IC50= 2.7 µM S HO

NH2

HO OH

Fig. (7). Structures of the inhibitors of the JAK2 kinase – tyrphostin-related derivatives of cinnamic acid benzyl amide (with IC50 referring to inhibition of the JAK2-catalyzed peptide substrate phosphorylation).

Kawada et al. [145] reported inhibition of BCRABL by ethyl 2,5-dihydroxycinnamate and concluded that this contributed to suppression of the oncogenic functions of ABL in the K562 human chronic myelogenous leukemia cells. Methyl 2,5-dihydroxycinnamate also inhibited activation of v-ABL and morphological transformation of the v-ablts-NIH3T3 cells. Cinnamide-related tyrphostins were also demonstrated to inhibit the human ABL and BCR-ABL chimeric proteins, for example α-cyano-3,4-dihydroxycinnamide (AG99 IC50 = 21.6 µM) [146]. Similar tyrphostins had lowmicromolar IC50 values, e.g.: AG82 – 3.6 µM, AG114 – 13.0 µM, AG213 – 5.8 µM, AG538 – 4.0 µM, and AG776 – 2.7 µM (Fig. 8). These compounds, however, were not ABL-selective and inhibited EGFR-catalyzed

N

AG 114, IC50= 13.0 µM NH2

HO

N

N

N

AG 538, IC50= 4.0 µM O HO

HO

OH

N

OH

Fig. (8). Structures of the inhibitors of the ABL kinase – tyrphostin-related cinnamic acid derivatives (with IC50 referring to inhibition of the ABL-catalyzed peptide substrate phosphorylation).

Novel 2-phenylaminopyrimidine cinnamides, structurally related to imatinib, were 2-3 times more potent in inhibiting the human K562 chronic myelogenous leukemia cells than the parent compound (GI50 = 0.23 µM): 2-chlorocinnamide (compound 12c, GI50 = 0.1

Cinnamic Acid-Inspired Inhibitors of Protein Kinases

µM) and 2-bromocinnamide (compound 12d, GI50 = 0.07 µM) derivatives (Fig. 9) [147]. The authors performed molecular docking of 2-bromocinnamide derivative to the ABL kinase domain and proposed the imatinib-analogous binding mode. Both inhibitors shared the same hydrogen bonds (with Met318, Thr315, Glu286, Phe382) and pi-pi interactions with the ABL amino-acid residues. Notably, the authors [147] also demonstrated the importance of the cinnamoyl double bond for antiproliferative potency of the new inhibitor. Although these novel semisynthetic compounds would probably not inhibit the ABLT315I mutant, they constitute an attractive alternative to the existing drugs due to their plausibly novel pharmacokinetic properties. compound 12c, GI50= 100 nM

O

Cl

NH N N H

compound 12d, GI50= 70 nM

N

Br N

Fig. (9). Structures of the modern inhibitors of the ABL kinase-imatinib mesylate-related cinnamic acid derivatives (with GI50 referring to inhibition of the human K562 chronic myelogenous leukemia cells).

6. ATYPICAL PROTEIN KINASES RIO Comprehensive analysis of the human genome performed by Manning and coworkers [22] allowed discovery of 13 atypical protein kinase families (aPKs). RIO kinases are classified as atypical because the sequence motifs of their kinase domains differ significantly from the motifs of the canonical kinase domain, despite the overall structural similarity [148, 149]. Four RIO subfamilies were distinguished: RIO1, RIO2, RIO3, and RIOB. They control crucial cellular processes: ribosome biogenesis, cell cycle progression, and chromosome maintenance [150-153]. It was shown that RIO1 and RIO2 kinases autophosphorylate on their serine residues [154, 155]. A possible role of this autophosphorylation has not yet been established. Autophosphorylation has no impact on the kinase activity, so, it may have a distinct function than it has in typical kinases - where the autophosphorylation regulates (usually increases or "switches on") kinase activity. RIO1 can in vitro phosphorylate MBP, casein and histone H1 but little is known about possible natural substrates phosphorylated by the RIO kinases. RIO1 from Haloferax volcanii phosphorylates α1 protein - 20S proteasome core particle subunit [156].

Current Medicinal Chemistry, 2016, Vol. 23, No. ??

13

Crystallographic structures of RIO1 and RIO2 from Archaeoglobus fulgidus were solved by LaRonde- LeBlanc and coworkers [154, 155, 157]. The RIO domain is truncated by deletion of the loops important for substrate binding in canonical protein kinases. On the other hand, it includes two additional parts: a helix Nterminal to the canonical N-lobe and a loop inserted between the third β-strand of the N-lobe and the αhelix C (flexible loop). The P-loop motifs (STGKEA in RIO1, GXGKES in RIO2, STGKES in RIO3, and SGKEA in RIOB) differ significantly from the ones seen in canonical protein kinases (GXGXXG) [158]. The hinge region of RIO2 is similar to the one of the canonical kinase domain where it usually consists of 56 amino acids and forms just an extended chain. In RIO1, however, there is a β-hairpin in this region, not seen in any other protein kinases. The mode of binding of purine nucleosides also differs from other kinases. Metal cation is coordinated between the α- and βphosphates of ATP in RIO1 compared with the α- and γ-phosphates in canonical kinases. The proteins have subfamily-specific ATP-binding loops with the sequences significantly different than their counterparts in typical eukaryotic kinases. 6.1. RIO Kinases as Oncogenes and Potential Targets of Anticancer Therapies A possible role of RIO kinases in pathogenesis, particularly in cancerogenesis, is not as clear as that of many other kinases (like: EGFR, HER2, BRC-ABL, and JAK2). Nevertheless, a direct linkage between deregulation of protein biosynthesis and cancerogenesis, tumor growth, and metastasis implicates that targeting ribosome biogenesis may affect rapidly growing cancer cells. Distinguishing characteristic of the cancer cell are enlarged nucleoli – the center of rRNA synthesis and processing [159]. Several tumor suppressors (e.g. p53 and retinoblastoma) have been found to affect formation of ribosomes. On the other hand, some protooncogenes have been shown to regulate the activity of translation factors [160]. Dysfunction of proteins engaged in ribosome biogenesis – ribosomal proteins (e.g. S19), nucleophosmin (NPM), dyskeratosis congenita 1 protein (dyskerin1, DKC1) – was strictly associated with many types of cancer [160]. The authors claim that components of the translation machinery that are overexpressed or deregulated in the cancer cell represent targets for anticancer therapy. The p53 tumor suppressor senses not only DNA damage and inappropriate cell proliferation but also the state of protein synthesis [161]. The latter ability of p53 is crucial for suppression of the tumor growth.

14 Current Medicinal Chemistry, 2016, Vol. 23, No. ??

The RIO kinases were found to be upregulated in some malignances (Table 3). Very interesting data, presented by Kimmelman and coworkers [166], link the rio3 gene aberration to pathogenesis and progression of the extremely invasive pancreatic ductal adenocarcinoma. The authors showed that knockdown of the rio3 gene resulted in a significant decrease in the ability to form colonies by the 8898T cells (harboring the rio3 gene amplification) and in reduced tumor growth and progression. The RIO3 kinase was important for small GTPase Rac activation, a protein crucial for cytoskeletal reorganization and cell motility. The kinase was found in complex with PAK1, which is a key downstream effector of Rac. According to the same authors, the rio3 and pak4 proinvasion genes indicated that the Rho-signaling pathway might also be a target for anticancer drugs. Furthermore, pharmacological inhibition of Rac was able to reverse the morphological changes to the ones characteristic of control cells and to decrease pancreatic ductal cell migration. Read and coworkers [163] suggested important role for RIO1 and RIO2 in proliferation and survival of glioblastoma. Overexpression of RIO2, which formed a complex with RIO1 and mTOR, facilitated AKT-signaling via AKT and induced cancerogenic transformation of the mouse astrocytes. Down-regulation of RIO1 or RIO2 expression resulted in deregulation of the AKT signaling and induced apoptosis. The unique features make the RIO proteins novel and attractive targets for inhibitor-based anticancer therapies. 6.2. Cinnamic Acid Derivatives as Inhibitors of RIO1 The only so far known inhibitors of the RIO kinases are natural and nature-derived compounds. Kiburu and LaRonde-LeBlanc [167] showed that a natural suppressor of ribosome biogenesis and mRNA translation – toyocamycin was a nanomolar pyridine inhibitor of the atypical kinase RIO1. This compound mimicked binding of the ATP molecule in the kinase structure and

Mielecki and Lesyng

blocked the ATP pocket of RIO1. Importantly, this natural antibiotic was a selective and potent antiproliferative agent of the stem cell-like melanoma cells with effective concentration as low as 0.1 µM [168]. Mielecki and coworkers [116] demonstrated that certain derivatives of α-cyanocinnamic acid benzyl amide were low-micromolar inhibitors of the autophosphorylation activity of RIO1 with the following IC50: 3.8 µM for WP1609 ((E)-2-cyano-N-(4-fluorobenzyl)-3-(pyridin-4-yl)acrylamide), 4.5 µM for WP1086 ((E)-Nbenzyl-2-cyano-3-(pyridin-4-yl)acrylamide) and 8.6 µM for WP1683 ((E)-2-cyano-N-(4-methoxybenzyl)-3(pyridin-4-yl)acrylamide) (Fig. 10). These compounds, similarly to toyocamycin, were ATP-competitive and bound in the ATP pocket of RIO1 as it was shown for WP1086 (Fig. 13, Fig. 14). Inhibition of the RIO atypical protein kinases by the above compounds may retard the translation machinery and ribosome biogenesis and may contribute to their antiproliferative activities in the cancer cell. 7. SERINE/THREONINE PROTEIN KINASES AS TARGETS FOR CINNAMIC ACID DERIVATIVES 7.1. CK2 Kinase CK2 is a pleiotropic protein kinase with the serine / threonine specificity. Its function is important for many fundamental cellular processes like: cell cycle progression, proliferation and survival. Its overexpression was implicated in many types of solid tumors, what makes it a therapeutically important target. A new class of CK2 inhibitors, simple derivatives of cinnamic acid, were mimetics of TBB (4,5,6,7-tetrabromo-benzotriazole) – a widely employed ATP-competitive inhibitor of CK2 (IC50 = 560 nM) [169, 170]. The most potent and very efficient CK2-binder was 4,5,6,7tetrabromocinnamic acid (TBCA, Fig. 11) with KI = 77 nM and IC50 = 110 nM. This compound competed for the ATP-binding pocket of CK2. The authors showed

Table 3. Aberrations of human RIO kinases in carcinomas. Nb

RIO Kinase

Aberration

Cancer Type

Reference

1

RIO1

overexpression

colorectal

[162]

2

RIO1/RIO2

overexpression

glioblastoma

[163]

3

RIO3

overexpression

melanoma

[164]

4

RIO3

overexpression

metastatic head and neck squamous cell carcinoma

[165]

5

RIO3

gene amplification and overexpression

pancreatic

[166]

Cinnamic Acid-Inspired Inhibitors of Protein Kinases

Current Medicinal Chemistry, 2016, Vol. 23, No. ??

through molecular docking simulations that TBCA occupied the same pocket as TBB. TBCA was more potent and also more selective than TBB, without having any comparable effect on DYRK1 as judged on a panel of 28 kinases [169]. TBCA decreased cell viability and induced apoptosis of the Jurkat cells (GI50 = 7.7 µM, [169]). Negative charge and orientation of the carboxyl functionality of TBCA played crucial role in the inhibitor binding as demonstrated by the structure-activity relationship studies. Mutations in the hydrophobic pocket, interacting with TBB and its derivatives, also compromised binding of TBCA [169]. Pagano and coworkers [169] concluded that TBCA was a promising therapeutic CK2-targeting agent. WP 1086, IC50= 4.5 µM

15

µM and targeted the ATP-binding site of PIM1. Two of the most potent compounds were: 3-(6-(4-aminocyclohexylamino)-pyrazin-2-yl)cinnamic acid (compound 1; PIM1 KD = 28 nM and IC50 = 17 nM, [172]; PIM2 IC50 = 150 nM, [173]) and 3- (6-(4methyl[1,4]diazepan-1-yl)pyrazin-2-yl)cinnamic acid (compound 2; PIM1 IC50 = 57 nM and PIM2 IC50 = 40 nM, [171]; PIM1 IC50 = 200 nM, [172]) (Fig. 11). Extensive structure-activity relationship experiments and crystallographic structures of the above compounds with the PIM1 kinase revealed that the cinnamoyl moiety played a crucial role in binding and positioning at the hinge region (Fig. 13, Fig. 14). This inhibitor fragment also significantly contributed to the inhibitor potency with the carboxyl moiety forming a salt bridge or a network of hydrogen bonding. Noteworthy, some cross reactivity with CK2 was also identified [171]. TBCA, IC50= 110 nM Br

O

Br

WP 1609, IC50= 3.8 µM

O

Br

fragment 3, IC50= 130 µM

N H

N N

OH

Br

O

F

OH N

WP 1683, IC50= 8.6 µM

compound 1, IC50= 17/150 nM

O

Fig. (10). Structures of the inhibitors of the RIO1 kinase – tyrphostin-related derivatives of cinnamic acid benzyl amide (with IC50 referring to inhibition of the RIO1 autophosphorylation).

NH2

N

O

N

N H

OH

compound 2, IC50= 57/40 nM

N N

N N

7.2. PIM Kinases Selectivity profiling on a 72-protein-kinase panel and with 1 µM TBCA revealed that TBCA also significantly inhibited PIM family of serine/threonine kinases: PIM1, PIM2, and PIM3 [170]. Overexpression of PIM1 in such cancers as prostate cancer and lymphomas as well as the unique hinge region (LERPXPX) make it a potential and attractive target for anticancer therapy. Two additional kinases were also effectively suppressed by TBCA: ERK8 and MNK1. Another studies, based on a crystallographic fragment screen and high-throughput screening, identified a series of cinnamic acid derivatives as inhibitors of the PIM kinases [171-173]. Pyridine congener of cinnamic acid (fragment 3, Fig. 11) inhibited PIM1 with IC50 = 130

C-KbBA, KD= 2.1 µM H

O

H H

H

O O H HO

H O

Fig. (11). Structures of the modern cinnamic acid derivatives as inhibitors of the serine/threonine protein kinases with indicated kinase affinities – TBCA with CK2-IC50, fragment 3 with PIM1-IC50, compound 1 with PIM1/2-IC50, compound 2 with PIM1/2-IC50, and C-KβBA with computationally estimated mTOR- K D.

16 Current Medicinal Chemistry, 2016, Vol. 23, No. ??

Mielecki and Lesyng

compound 14, IC50= 13 nM O

compound 15, IC50= 5 nM

O H2N N

N N

N H

N

compound 16, IC50= 7 nM

HO

O

indazole 1, IC50= 360 nM O NH2

N N H

N

azaindole 8, IC50= 120 nM O NH2 N O

N HN

indazole 11, IC50= 15 nM

O

O

O O

NH2

indazole 12, IC50= 12 nM

N N H

N

N H

OH

Fig. (12). Structures of the modern cinnamic acid-inspired covalent inhibitors of the serine/threonine protein kinase RSK2 with indicated IC50.

Dual CK2 and PIM inhibitors could be of special interest for targeted therapies of cancers with overexpression of both kinases, like prostate adenocarcinoma. Noteworthy, highly-selective dual inhibitors of CK2 and PIM kinases, 2,8-difuranodicarboxylic acid derivatives were also identified through automated screening and structure-activity relationship studies [174]: 2,8dicarboxynaphtho[2,1-b:7,6-b’]difuran (CPA), 2,8dicarboxyanthra[2,1-b:7,6-b’]difuran (CPB), and 2,9dicarboxynaphtho[2,1-b:7,8-b’]difuran (AMR). These potent low-nanomolar inhibitors targeted the ATPbinding pocket of the CK2/PIM kinases. Structural

studies (crystallography and molecular docking) of the complexes of the CK2 and PIM1 kinases with the inhibitors allowed defining the binding mode and structural determinants of the inhibitor potency [174]. Interestingly, they revealed no contacts with the hinge regions. While the aromatic core of the inhibitors made mostly van der Waals and hydrophobic contacts, one of the carboxyl functionalities stabilized an inhibitor via direct (e.g. with Lys68 of CK2) and water mediated hydrogen bonds. These results [174] support the essential role of the carboxyl group of cinnamic acid derivatives in their positioning in ATP-pockets of the CK2 and PIM kinases through ionic (salt bridges) and/or hydrogen bond interactions with basic amino acid side chains (like Lys68) or basic subpockets. 7.3. mTOR Kinase The serine/threonine protein kinase mTOR (also called FRAP1) belongs to phosphatidylinositol 3kinase-related kinases and is a multidomain protein with a molecular mass of 288.9 kDa [175]. Starting from the N-terminus mTOR consists of the following domains: HEAT (Huntingtin-EF2-A subunit of PP2ATOR1), FAT (FRAP-ATM-TRAP), FRB (FKBP12 rapamycin binding), kinase domain, NR (negative regulatory), and FATC (FRAP-ATM-TRAP C-terminal). Access of substrates to the active site of mTOR is sterically controlled by the FRB and NR domains, and also by the mLST8 protein. FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with substrates to permit entrance to the restricted active site [175]. Rapamycin–FKBP12 inhibits the kinase by directly blocking substrate recruitment and by further restricting the active-site access. mTOR functions in two distinct complexes: TORC1 and TORC2, which integrate several key cellular pathways controlling growth, proliferation and metabolism in response to extracellular and intracellular stimuli, like: hormones, growth factors, nutrients (amino acids), energetic status (AMP/ATP), and also oxidative and genotoxic stress [176]. TORC1 regulates protein and lipid biosynthesis, ATP synthesis, angiogenesis, autophagy, and TORC2 – cell proliferation and cytoskeleton arrangement. Deregulation and overactivation of mTOR are very often associated with tumorigenesis and human malignances [177]. Rapamycin, a natural macrocyclic lactone, is an allosteric inhibitor of TORC1. Several rapalogs (analogs of rapamycin) were approved by the Food and Drug Agency as immunosuppressants and anticancer drugs, e.g.: rapamycin as Rapamune (Pfizer) or Sirolimus (Zydus Pharmaceuticals), temsirolimus as

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17

Fig. (13). Crystallographic structures of the complexes reveal noncovalent interactions and covalent bonds of the cinnamic acid derivatives with amino acid residues of the ATP pocket in serine/threonine protein kinases. Cinnamic acid moiety of the inhibitors binds in the deep subpocket of the ATP pocket and derivatization has a little impact on its positioning. Dashed lines represent for hydrogen bonding. Molecular surfaces of the kinases were colored according to their Poisson-Boltzmann electrostatic field.

18 Current Medicinal Chemistry, 2016, Vol. 23, No. ??

Mielecki and Lesyng

(Legend Fig. 13) contd….

A – RIO1 and WP1086 ((E)-N-benzyl-2-cyano-3-(pyridin-4-yl)acrylamide), PDB: 4JIN. B – PIM1 and compound 1 (3-(6-(4-aminocyclohexylamino)-pyrazin-2-yl)cinnamic acid), PDB: 2XJ1. C – PIM1 and compound 2 (3-(6-(4-methyl[1,4]diazepan-1-yl)pyrazin-2-yl)cinnamic acid), PDB: 2XJ2. D – RSK2 and azaindole 8 (3-(3-(1H-pyrrolo[2,3-b]pyridin-3-ylcarbonyl)-2-cyanocinnamide), PDB: 4JG7. E – RSK2 and indazole 12 (N-(1-hydroxy-2-methylpropan-2-yl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-indazol-5-yl)-2cyanoacrylamide), PDB: 4JG8.

Torisel (Pfizer), and ewerolimus as Zortress, Certican or Afinitor (Novartis) [111]. Anticancer efficacy of rapalogs as monotherapy agents is limited and this makes them cytostatic rather than cytotoxic [177]. They may induce a feedback loop of the TORC signaling networks leading for example to upregulation of prosurvival, antiapoptotic, and thereby oncogenic pathways PI3K/AKT and MEK/ERK. There is a requirement for developing inhibitors of mTOR with new mechanisms of action. For example, second-generation mTOR inhibitors, like OSI-027 and OXA-01, target the ATP pocket of the kinase [178]. A triterpenoid derivative C-KβBA, 3-cinnamoyl-11keto-β-boswellic acid (Fig. 11), is a semisynthetic potent, selective, and allosteric inhibitor of mTOR in vitro and in vivo [179]. It bound to the FRB domain of mTOR with a high affinity, competing with an endogenous allosteric mTOR activator, phosphatidic acid. The presence of 10 µM C-KβBA markedly reduced the experimental apparent KD for phosphatidic acid binding to the mTOR kinase (from 37.1 to 329.3 µM). The cinnamoyl moiety of C-KβBA largely contributed to its antiproliferative and proapoptotic activity. A congener of C-KβBA, 3-acetyl-11-keto-β-boswellic acid (AKβBA) was a less potent antiproliferative agent. Molecular docking (Molegro Virtual Docker 5) revealed significant affinity of C-KβBA to the FRB domain of mTOR (Fig. 14), with computationally estimated KD = 2.1 µM and interaction energy -189.6 kJ·M-1, but not to S6K1, PP2A, AKT1, ERK2, IKKa, neither IKKb. Predicted affinity for the AKβBA binding to mTOR (KD = 35.7 µM and interaction energy 130.7 kJ·M-1) was significantly higher than that for the cinnamic congener. This inhibitor might interact with the same amino acid residues in the FRB pocket as the phosphatidic acid. The above studies [179] suggested that C-KβBA (Fig. 11) might overcome the therapeutic disadvantages of rapalogs. This compound exhibited antiproliferative and proapoptotic activity in the prostate and breast cancer cell lines: PC-3 (GI50 = 3.6 µM), LNCaP, DU-145, and MDA-MB-231. In the PC-3 cells it suppressed protein translation, down-regulated eIF4E and cyclin D1

expression, as well as induced G1 cell-cycle arrest, and also down-regulated phosphorylation of S6K. Importantly, it did not induce any feedback-loop activation of AKT and ERK pathways and associated hyperphosphorylation of GSK-3b. Thus, C-KβBA had the improved pharmacodynamic characteristics and was the better proapoptotic agent than rapamycin. This makes it a very promising candidate to enter clinical trials. Importantly, the molecular modeling data obtained by Morad and coworkers [179] correlated very well with the results of biochemical and biological experiments performed by the same authors. 7.4. RSK2 Kinase Modern strategies of designing and synthetizing irreversible inhibitors, forming covalent bonds with cysteine and other nucleophilic residues of protein kinases, exploit various electrophiles, e.g.: acrylates, acrylamides, vinyl sulfonates, quinones, and alkynyl amides [180]. However, specific structural and physicochemical properties (like electron density distribution) of cinnamic acid inspired the design and synthesis of entirely novel covalent inhibitors of protein kinases. This natural molecule represents α,β-unsaturated carbonyl compounds which are considered to be soft electrophiles and may selectively react with nucleophilic thiolates of cysteine residues in proteins via Michael addition [181, 182]. It was demonstrated that doubly activated methyl α-cyanocinnamate reversibly reacted with a free-cysteine thiol (KD = 9.4 mM, [183]). A series of α-cyanocinnamide derivatives also reacted with this thiol with low-milimolar affinities. The preliminary results stimulated the design of RSK2 kinase blockers and provided a model for developing inhibitors with a unique mechanism of action. RSK protein kinases are involved in PI3K/AKT/mTOR signaling and, thus, participate in regulation of cell proliferation, survival, migration, invasion, and metabolism. They are activated, similarly to their structural homologs – MSK kinases, by receptor tyrosine kinases, SRC kinases, PDK1, and RAS/RAF/MEK/ERK pathways [184, 185]. They may support tumorigenesis and metastasis in some cancer types, although, their genes

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Fig. (14). Structural insight into the molecular mechanistic diversity of the protein kinase inhibition by cinnamic acid derivatives. Molecular surfaces of the kinases were colored according to their Poisson-Boltzmann electrostatic field. A – Binding of the following derivatives – WP1086 ((E)-N-benzyl-2-cyano-3-(pyridin-4-yl)acrylamide; PDB: 4JIN), compound 1 (3-(6-(4-aminocyclohexylamino)-pyrazin-2-yl)cinnamic acid; PDB: 2XJ1) and compound 2 (3-(6-(4methyl[1,4]diazepan-1-yl)pyrazin-2-yl)cinnamic acid; PDB: 2XJ2) – in the ATP pocket of RIO1 or PIM1. These crystallographic complexes were superposed onto the AMP-PNP-PIM1 complex (PDB: 1XR1). Aryl ring of the cinnamoyl moiety is perfectly positioned in the adenine subpockets of the kinases and cinnamic acid penetrates the subpocket not occupied by an ATP substrate. B – Binding of WP1065 ((E)-2-cyano-N-((S)-1-phenylethyl)-3-(pyridin-2-yl)acrylamide) in the allosteric helix C pocket, adjacent to the ATP pocket, of the JAK2 kinase domain. It represents the noncompetitive mode of inhibition of JAK2 (the structure modeled by Mielecki and coworkers [74]). C – Binding of C-KβBA (3-cinnamoyl-11-keto-β-boswellic acid) in the allosteric phosphatidic acid pocket of the FRB domain of the mTOR kinase. It represents the noncompetitive mode of inhibition of mTOR (the structure modeled by Morad and coworkers and provided as the supplementary data in [179]). D – Binding of reversible covalent analogs of cinnamic acid in the ATP pocket of the RSK2 kinase: pyrrolopyrimidine 16 (tertbutyl 3-(4-amino-7-(3-hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2- cyanoacrylate; PDB: 4D9U), indazole 1 (3-(1H-indazol-5-yl)-2-cyanoacrylamide; PDB: 4JG6), azaindole 8 (3- (3-(1H-pyrrolo[2,3-b]pyridin-3-ylcarbonyl)-2cyanocinnamide; PDB: 4JG7), and indazole 12 (N-(1-hydroxy-2- methylpropan-2-yl)-3-(3-(3,4,5-trimethoxyphenyl)-1H-indazol5-yl)-2-cyanoacrylamide; PDB: 4JG8).

are not frequently amplified or mutated and the data are so far incomplete, depending on the RSK and tumor type. However, chemical inhibition of the RSK kinases was effective in suppressing invasion and metastasis of several solid tumors in the preclinical models [185]. Thus, these kinases emerge as promising targets for antimetastatic anticancer therapies.

It was demonstrated that selectivity and reversibility of the Michael addition might be tuned by specific modifications of the inhibitor core [183]. Conjugation of an α-cyanoacryl functionality with a kinaserecognizing pyrrolopyrimidine scaffold resulted in formation of slowly-dissociating, highly-selective, reversible, and covalent inhibitors of RSK2 (Fig. 12): compound 14 (methyl 3-(4-amino-7-(3-hydroxy-pro-

20 Current Medicinal Chemistry, 2016, Vol. 23, No. ??

pyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)-2-cyanoacrylate, IC50 = 13 nM), compound 15 (3-(4-amino7-(3-hydroxypropyl)5-p-tolyl-7H-pyrrolo[2,3d]pyrimidin-6-yl)-2-cyano-N-isopropylacrylamide, IC50 = 5 nM), and compound 16 (tert-butyl 3-(4-amino-7-(3hydroxypropyl)-5-p-tolyl-7H-pyrrolo[2,3-d]pyrimidin6-yl)-2-cyanoacrylate, IC50 = 7 nM) [183]. Pyrrolopyrimidine 15 was also a potent inhibitor of other RSK kinases: RSK1 (IC50 = 0.54 nM) and RSK4 (IC50 = 1.2 nM). Crystal structure of the complex revealed that the inhibitor 16 targeted the non-catalytic cysteine residue (C436) in the ATP-pocket of RSK2. Hydrogen bonds with the RSK2 hinge-region and with T493, and other noncovalent interactions stabilized and positioned the reactive inhibitor moiety in the proximity of C436 (Fig. 13, Fig. 14). It was concluded that specific noncovalent interactions drove covalent bond formation. Importantly, this strategy may allow improvement of an inhibitor potency avoiding permanent covalent modifications of proteins and resulting off-target toxicity. Fragment-based development of reversible covalent inhibitors of related cysteine-containing kinases proved the above concepts. Novel derivatives and analogs of α-cyanocinnamic acid, heteroaryl-substituted α-cyanoacrylamides, were potent and highly-selective inhibitors of the RSK/MSK family of protein kinases [186]. Indazole 1 (3-(1H-indazol-5-yl)-2-cyanoacryl-amide, Fig. 12) was a submicromolar inhibitor of RSK2 (IC50 = 0.36 µM), as well as NEK2 (IC50 = 1.4 µM) and PLK1 (IC50 = 0.57 µM). The inhibitor was stabilized in the ATP-pocket of RSK2 by noncovalent interactions, like hydrogen bonds with L495 and M496 of the hinge region, and also by a thioether bond to C436. Ligands with other scaffolds, still containing the α-cyanoacryl functionality, were more selective RSK2 inhibitors (Fig. 12): azaindole 8 (3-(1H-pyrrolo[2,3-b]pyridin-3ylcarbonyl)-2-cyanocinnamide, IC50 = 120 nM), indazole 11 (3-(3-(3,4,5-trimethoxyphenyl)-1H-indazol-5yl)-2- cyanoacrylamide, IC50 = 15 nM) and indazole 12 (N-(1-hydroxy-2-methylpropan-2-yl)-3-(3-(3,4,5trimethoxyphenyl)-1H-indazol-5-yl)-2-cyanoacrylamide, IC50 = 12 nM). The same authors [186] also reported the NEK2- (purine 7, 4-(9H-purin-6-yl)-2cyanocinnamide, IC50 = 1.1 µM) and PLK1-selective (azaindole 9, 3- (1H-pyrrolo[2,3-b]pyridin-3-yl)-2cyanoacrylamide, IC50 < 0.62 µM) derivatives. The crystallographic structures of the RSK2 kinase domain with the derivatives 8 and 12 (Fig. 13, Fig. 14) confirmed that specific noncovalent interactions were required to stabilize the unstrained covalent thioether bond, which might be broken upon unfolding of the

Mielecki and Lesyng

protein structure. Upstream regulators of the RSK/MSK kinases – MEK1, ERK1, and p38 MAPK – also showed minor inhibition by indazole 12 [186]. These data clearly demonstrated the potential of αcyanocinnamic derivatives and analogs as reversible covalent inhibitors of protein kinases and other cysteine-exposing enzymes (e.g.: deubiquitinases). 8. SUMMARY AND PERSPECTIVES Cinnamic acid derivatives as inhibitors of protein kinases were fully inspired by the plant secondary metabolites. Structural and physicochemical properties of these compounds make them attractive leads, and present several advantages for potential kinase inhibitors. These unique properties include a (trans)-α,βunsaturated carbonyl, which is a reactive Michael electrophile, and is crucial for certain bioactivities of these compounds. Structure-activity relationship studies repeatedly reveal that α,β-saturation significantly reduces cytotoxic and/or enzyme-inhibitory potencies of cinnamic acid derivatives. One should mention here: α,β-saturated cinnamic acid (tested as inhibitors of 17hydroxysteroid dehydrogenase), methyl-2,5-dihydroxycinnamate (tested as a cytotoxic agent), cinnamoyl analogs of fumagillin (tested for antiproliferation of endothelial cells), and benzotriazolyl derivatives of cinnamic acid (tested for inhibition of a liver transglutaminase) [16]. Similar results presented for the cinnamoyl inhibitors of protein kinases: BCR-ABL [147], CK2 [169], PIM1-2 [172, 173], and RSK/MSK [183, 186] confirm the importance of the α,β-unsaturated carbonyl. This may be partially explained by an increase of the conformational entropy of saturated cinnamoyl analogs, unfavorable for their binding free energies. Proper positioning of the carboxyl anion to form hydrogen/ion bonding with amino acid residues in a kinase pocket increases the inhibitor binding affinity. On the other hand, specific reactivity of the α,βunsaturated carbonyl may be harnessed to improve the inhibitor residence-time. One should note that selectivity and a mode of binding / inhibition strongly depend on particular modifications. Nature-derived tyrosine kinase inhibitors, including cinnamic acid derivatives and analogs, have been opening the new era of anticancer targeted therapies. They already played a great role in inspiring the conceptual way leading, in particular, to imatinib, a selective and potent inhibitor of the BCR-ABL oncogene, and finally leading to an efficient drug against chronic myelogenous leukemia. The best studied synthetic analogs of cinnamic acid are α-cyanocinnamides (derived histori-

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cally from tyrphostins, Fig. 1) which were reported to inhibit the following oncogenic kinases: ERBB1, ERBB2, JAK2, and ABL. In particular, congeners of α-cyanocinnamic acid benzyl amide are covered by several patents. However, the synthetic α-cyanocinnamoyl-scaffold-based compounds did not gain significant success as inhibitors of tyrosine protein kinases. The only, although very effective compound, which clinical (safety/efficacy) trials are going to be initiated in 2016 by M.D. Anderson Cancer Center, is WP1066, 2-bromopyridine congener of 1-phenethyl αcyanocinnamide [111]. Current research focuses, among other things, on finding the highest tolerable dose that can be given to patients with melanoma of the central nervous system or with recurrent glioblastoma multiforme. Some studies, however, suggest that the protein kinase inhibitors comprising cinnamoyl moieties/ fragments (Fig. 1) emerge as very promising anticancer drug candidates with improved pharmacodynamics. They were identified using such techniques as highthroughput screening or crystallography molecularfragments screening. The reported inhibitors include mainly azaheteroaryl derivatives and analogs of cinnamic acid – pyrazine, pyrrolopyridine, pyrrolopirymidine – and also bromocinnamic and cinnamoyl-boswellic acids. They are potent (nanomolar or low-micromolar) and highly selective inhibitors of the serine/threonine protein kinases: CK2, mTOR, PIM1, PIM2, and RSK2 (Fig. 13, Fig. 14). The cinnamoyl moiety of these compounds is crucial for inhibitor

binding and potency, and may form strong interactions with amino acid side chains, like salt bridges, networks of hydrogen bonding, as well as covalent bonds. One model compound should be especially emphasized here. Curcumin – di(3-metoxy-4- hydroxycinnamoyl)methane (diferuloylmethane) – is now probably the most successful natural derivative of cinnamic acid considered to be an anticancer agent. This compound is currently tested in 114 clinical trials, either alone or in a combination therapy with tyrosine kinase inhibitors in not only neoplasms (EGFR-mutant advanced non-small cell lung carcinoma, breast cancer, colorectal adenoma, familial adenomatous polyposis, prostate cancer, head and neck cancer, multiple myeloma, advanced osteosarcoma, pancreatic adenocarcinoma, endometrial carcinoma, myelodysplastic syndrome, chronic lymphocytic leukemia, and small lymphocytic lymphoma) but also in other pathological states (psoriasis, chronic obstructive pulmonary disease, vascular aging, rheumatoid arthritis, diabetes, chronic periodontitis, cardiovascular disease, cystic fibrosis, atopic asthma, schizophrenia, Parkinson's disease, Alzheimer's disease, and bipolar disorder) [111]. Curcumin emerges as an antineoplastic, neuroprotective and immunomodulatory panacea. Direct molecular interactions of curcumin with a plethora of its cellular targets were comprehensively reviewed by Gupta and coworkers [187]. Protein kinases are one of the most critical targets for curcumin vinylogs and analogs (Table 4). The effective curcumin-kinase affinities are usually in the low-micromolar range. The inhibition

Table 4. Kinases for which direct intermolecular interactions with curcumin were confirmed. Nb

Kinase

Experimental Stability of Binding (IC50*, µM)

Reference

1

PhK

75 (KI, noncompetitive inhibition)

[188]

2

cAK kinase domain

4.8

[189]

3

CDPK

41

[189]

4

PKC

15; 4-11 (EC50)

[189, 190]

5

IKK

-

[191]

6

SRC

-

[192]

7

ERBB2 kinase domain

-

[193]

8

GSK-3β

0.066 nM

[194]

9

CAMKII

10

CAMK4 kinase domain

* otherwise indicated in the brackets

21

33 (CAMKII autophosphorylation) 2+

13 (Ca -dependent CAMKII-catalyzed phosphorylation ) 0.037 (KD)

[195] [196]

22 Current Medicinal Chemistry, 2016, Vol. 23, No. ??

mechanism may be of the ATP competitive, peptide substrate-competitive or bisubstrate-competitive type. The possible interactions of curcumin with key amino acids of PKC, ERBB2 kinase domain, GSK-3β and CAMK4 kinase domain were revealed using molecular docking and molecular dynamics simulations. This is now evident that blocking protein kinases by curcumin contributes to its broad biological activities, in particular to its antiproliferative and anticancer properties. Curcumin may also potentiate the cytotoxic effects of chemotherapeutic agents. Notably, novel derivatives of curcumin may be very promising protein kinase inhibitors. Pyrazole-curcumin has a higher inhibitory potency towards Ca2+-dependent activity of CAMKII (IC50 = 1.49 µM) and CAMKII autophosphorylation (IC50 = 6.5 µM) than its parent compound [195]. Despite the fact that plant secondary metabolites, including derivatives of cinnamic acid, have been consumed for centuries, we only recently began to dissect their molecular mechanisms of action responsible for their widely studied biological activities. The arsenal of entirely natural compounds was expanded to semisynthetic and synthetic nature-inspired inhibitors and this often helps to unveil bioactivities of the parent compounds. Although cinnamoyl-scaffold based compounds are not very strong inhibitors / binders of protein kinases, their pleiotropic non-canonical modes of inhibition still drew interest. These compounds still hold promise as inhibitors and anticancer agents, and their further studies should lead to valuable results.

Mielecki and Lesyng

DOK1

=

downstream of tyrosine kinases 1

DYRK1

=

dual-specificity tyrosinephosphorylated and regulated kinase

EGFR

=

epidermal growth factor receptor 1 (HER1)

eIF4E

=

eukaryotic translation initiation factor 4E

EML1

=

echinoderm microtubuleassociated protein-like 1

EphB4R

=

ephrin activated EphB4 receptor

ERBB2/neu

=

epidermal growth factor receptor 2 (HER2)

ERK

=

extracellular signal regulated kinase

FAK

=

focal adhesion kinase

FIP1L1-PDGFRαnegative =

factor interacting with PAP1 like 1-platelet derived growth factor receptor α

FLT3

=

Fms-related tyrosine kinase 3,

FOXP1

=

forkhead box P1,

FRAP1

=

FKBP12 (FK506-binding protein 12)-rapamycin complex associated protein

FUS1

=

cell fusion protein 1,

LIST OF ABBREVIATIONS

FYN

=

tyrosine-protein kinase Fyn

ABL

=

Abelson kinase

GSK-3β

=

glycogen synthase kinase 3β

AKT

=

v-akt murine thymoma viral oncogene homolog 1 (protein kinase B)

HIF1α

=

hypoxia induced factor 1 alpha

IGF-1R

=

insulin-like growth factor 1 receptor

IKK

=

IκBα kinase

JAK2

=

just another kinase 2 (janus kinase 2)

JNK

=

c-Jun N-terminal kinase

MBP

=

myelin basic protein

MEKK3

=

mitogen activated protein kinase kinase kinase 3

mLST8

=

mammalian lethal with SEC13 protein 8

MSK

=

mitogen- and stress-activated kinase

BCR

=

breakpoint cluster region

cAK

=

cyclic AMP dependent kinase

CAMKII

=

calcium/calmodulin dependent protein kinase II

CAMK4

=

calcium/calmodulin dependent protein kinase IV

CDK

=

cyclin dependent kinase

CDPK

=

plant Ca2+-dependent protein kinase

CK2

=

casein kinase 2

CRK

=

cellular homolog of oncogene from CT10 avian sarcoma virus

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Current Medicinal Chemistry, 2016, Vol. 23, No. ??

23

mTOR

=

mechanistic (mammalian) target of rapamycin

STAM

=

signal transduction adapter molecules

MuSK

=

agrin-stimulated muscle-specific receptor tyrosine kinase

STAT

=

signal transducer and activator of transcription

NUP214

=

nucleoporin 214 kDa

STIP

=

STAT-interacting proteins

p190RhoGAP =

190 kDa GTPase activating protein for Rho A

TEL

=

translocation E-26 transforming specific leukemia

p38

=

mitogen activated protein kinase

TPK

=

tyrosine protein kinase

PAK1

=

p21-activating serine/threonine kinase 1

VEGF

=

vascular endothelial growth factor

PCM1

=

pericentriolar material 1

VEGFR

=

PhK

=

phosphorylase kinase

vascular endothelial growth factor receptor

PIAS

=

protein inhibitor of activated STATs

ZMIZ1

=

zinc finger MIZ-typecontaining 1

PIM

=

provirus integration site for Moloney murine leukemia virus

CONFLICT OF INTEREST

PKC

=

protein kinase C

The authors confirm that this article content has no conflict of interest.

PSTPIP1

=

proline-serine-threonine phosphatase-interacting protein 1

ACKNOWLEDGEMENTS

PTP

=

protein tyrosine phosphatases

Rac

=

ras-related C3 botulinum toxin substrate

These studies were partially supported by funds of IMDiK PAS. REFERENCES

Ras

=

rat sarcoma

RCSD1

=

CapZ interacting protein (RCSD domain containing 1)

Rho

=

ras homology

[2]

RIN1

=

Ras and Rab interactor 1

[3]

RPN1

=

ribophorin 1

RSK2

=

p90 ribosomal S6 kinase

S6K

=

p70 ribosomal S6 kinase

SFPQ

=

splicing factor, proline/glutamine-rich

SH2/Lnk/APS =

Src homology 2 / linker of T-cell receptor pathways / prostatespecific antigen (kallikrein- related peptidase)

SH3

=

Src homology 3

SNX2

=

sorting nexin 2

SOCS

=

suppressors of cytokine signaling

SSBP2

=

single-stranded DNA-binding protein 2

[1]

[4]

[5]

[6] [7] [8] [9]

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[12]

[13]

[14] [15] [16] [17] [18]

[19] [20]

[21]

[22] [23] [24]

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Received: November 09, 2015

Revised: January 27, 2016

Accepted: March 10, 2016

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