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Identification of the first selective Activin receptorLike Kinase 1 inhibitor, a reversible version of L-783277 Hanna Cho, Sandip Sengupta, Sean SH Jeon, Wooyoung Hur, Hwan Geun Choi, Hong Seog Seo, Byung Joo Lee, Jeong Hun Kim, Minhwan Chung, Noo Li Jeon, Nam Doo Kim, and Taebo Sim J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01679 • Publication Date (Web): 19 Jan 2017 Downloaded from http://pubs.acs.org on January 20, 2017

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Journal of Medicinal Chemistry

Identification of the first selective Activin receptor-Like Kinase 1 inhibitor, a reversible version of L783277 Hanna Cho,‡ 1 Sandip Sengupta,† 1 Sean SH Jeon,‡ Wooyoung Hur,† Hwan Geun Choi,† Hong-Seog Seo, ‡￡ Byung Joo Lee,⊥∇ Jeong Hun Kim,⊥∇# Minhwan Chung,￥ Noo Li Jeon,￥ Nam Doo Kim, § Taebo Sim‡ † * ‡

KU-KIST Graduate School of Converging Science and Technology, Korea University, 145 Anam-

ro, Seongbuk-gu, Seoul 02841, Republic of Korea †

Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), 5

Hwarangro 14-gil, Seongbuk-gu, Seoul 02792, Republic of Korea ￡

Cardiovascular Center, Korea University Guro Hospital, 80 Guro-dong, Guro-gu, Seoul 152-703,

Republic of Korea ⊥

Fight Against Angiogenesis-related Blindness Laboratory, Clinical Research Institute, Seoul

National University Hospital, 101, Daehak-ro, Jongno-gu, Seoul 03080, Republic of Korea ∇

Department of Biomedical Sciences, College of Medicine, Seoul National University, 103,

Daehakro, Jongro-gu, Seoul 03080, Republic of Korea #

Department of Ophthalmology, College of Medicine, Seoul National University, 101, Daehak-ro,

Jongno-gu, Seoul 03080, Republic of Korea ￥Mechanical

engineering, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826,

Republic of Korea §

Daegu-Gyeongbuk Medical Innovation Foundation, 2387 dalgubeol-daero, Suseong-gu, Daegu

42019, Republic of Korea

1

These authors contributed equally to this work.

1

ACS Paragon Plus Environment


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ABSTRACT

We synthesized 1 (San78-130), a reversible version of L-783277, as a selective and potent ALK1 inhibitor. Our study showed that 1 possesses great kinase selectivity against a panel of 342 kinases and more potent activity against ALK1 than L-783277. Among the six ALK isotypes (ALK1-6), ALK1 is most significantly inhibited by compound 1. Compound 1 suppresses the BMP9-induced Smad1/5 pathway by mainly inhibiting ALK1 in C2C12 cells. Our molecular dynamics simulations suggest that H-bonding interaction between the C-4’ hydroxyl group of 1 and Arg334 of ALK1 substantially contributes to the ALK1 inhibition. To the best of our knowledge, 1 is the first selective ALK1 inhibitor. Furthermore, compound 1 promoted angiogenesis in both endothelial tube formation and microfluidic chip based 3D angiogenesis assays, suggesting that 1 could be a lead compound for therapeutic angiogenesis agents. Our study may provide an insight to designing selective and potent inhibitors against ALK1.

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INTRODUCTION L-783277 is a naturally occurring 14-membered resorcylic acid lactone (RAL) bearing a cis-enone moiety.1 L-783277 and its epoxide analogue, hypothemycin, possesses inhibitory activities against multiple oncogenic kinases. It is known that the electrophilic cisenone functionality group in these natural products form Michael adducts with conserved cysteines located just prior to the DFG motif of the activation loop of the kinases (e.g. Cys166 of ERK2), which enhances their binding affinities to target kinases.2 A number of derivatives of L-783277 have been synthesized to improve inhibitory activity and selectivity.1-4 The reversible binding of RALs is also an important factor in determining the kinaseinhibitory activities of these substances. For example, although hypothemycin does not inhibit GSK3β, which possesses a conserved cysteine, it is able to inhibit Src, TrkA and TrkB, which have no conserved cysteines.2 Even though various derivatives of L-783277 have been probed, 5-7 reversible non-covalent analogues of L-783277 have not been fully investigated in terms of kinase selectivity. We postulated that a reversibly version of L-783277 might be superior to L-783277 in terms of overall kinase selectivity and that it might possess higher inhibitory potencies against certain kinases if it has larger conformational flexibility than does L-783277. To test this hypothesis, we synthesized 1 (San78-130), a reversibly version of L-783277, in which the alkene moiety of the cis-enone of L-783277 was saturated. Results derived from kinome-wide selectivity profiling and biochemical kinase assays show that compared with L-783277, 1 is a more potent inhibitor of Activin A like kinase 1 (ALK1) and that it displays a better kinase selectivity against a panel of 342 kinases.

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ALK1 is a serine-threonine kinase expressed predominantly in endothelial cells where it functions as a type I receptor for TGF-β and BMP9 (bone morphogenetic protein 9).8-9 Upon ligand binding, ALK1 phosphorylates Smad1/5/8 which activates the common mediator Smad4 to upregulate transcription of a range of target genes involved in vasculogenic and angiogenic processes.10-11 In addition, ALK1 activates non-Smad pathways including MAPK (ERK, p38, and JNK) and PI3K/AKT, which regulate survival and proliferation.8 Because of its regulatory role in tumor angiogenesis, ALK1 has been a therapeutic target for development of anti-cancer agents.8 Dorsomorphin and 26 (LDN193189) (Figure 1) display broad inhibitory activities against BMP type I receptor kinase and attenuate both Smad and non-Smad signaling induced by BMPs.11 However, these inhibitors also block other signaling pathways, causing undesired off-target effects.9 Several inhibitors of BMP signaling pathway have been reported.12-14 Biologic drugs such as PF-34469629 (human monoclonal antibody) and ALK1-Fc fusion protein15 (soluble extracellular domain trap) effectively block the proliferation and tube formation of VEGF-induced endothelial cells (ECs) in vitro. Although the BMP9-ALK1 pathway has been studied thoroughly, its precise role in angiogenesis is still unclear.9 Thus, identification of a selective inhibitor capable of blocking BMP9 signaling could be useful to investigate the role of ALK1 in angiogenesis. Herein, we report that compound 1 is a potent and selective ALK1 inhibitor that selectively blocks BMP9-induced ALK1 signaling in C2C12 cells.

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RESULT Chemistry We devised and developed routes for the synthesis of the macrocyclic lactone 1 (Schemes 1 and 2), which contains a saturated ketone group in place of the cis-enone moiety that is present in L-783277. We anticipated that 1 would be a reversibly binding version of L783277. In addition, we synthesized the two isomeric trihydroxy analogues of L-783277, 13 and 14, analogue 15 containing an external olefin group, and the lactam derivative 25 in order to carry out SAR analysis. The convergent route designed to synthesize these substances, outlined in a retrosynthetic fashion in Scheme 1, proceeds through the common intermediate 2. The routes to 1, 13, 14 and 15 involve three key steps involving a Suzuki cross-coupling reaction between an aromatic triflate and olefin, addition of an acetylene to an aldehyde, and Mitsunobu macrolactonization. In the pathway, the required triflate 4 and olefin 5, prepared from

commercially

available

2,4,6-trihydroxybenzoic

acid

and

1,2,5,6-bis-O-(1-

methylethylidene)-D-mannitol, respectively,4 would be joined by use of a Suzuki crosscoupling reaction to produce the key intermediate 2. The preparation of 1 began with synthesis of alkyne 3 by reaction between commercially available (R)-propylene oxide and TMS-acetylene (Scheme 1).4 In a parallel route, Suzuki cross-coupling between triflate 4 and olefin 56 generated the key intermediate 2 (Scheme 2). This process was carried out by treating alkene 5 with 9-BBN and then reacting the formed B-alkyl-9-BBN with aromatic triflate 4 in presence of K3PO4 and Pd(0) to afford the desired 2 in 92% yield. The propargylic alcohol 6 (dr = 2:1, 1H NMR analysis of crude 5



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material) was prepared from 2 using the same sequence of reactions described in our report of the synthesis of L-783277.4 The diastereomeric mixture was subjected to Pd/C-catalyzed hydrogenation to give the saturated alcohol 7 in 88% yield. Removal of the TBS group in 7 using TBAF formed the dihydroxy ester 8 (84% yield), which was then saponified using NaOH to produce the dihydroxy acid 9. Without purification, 9 was subjected to Mitsunobu macrolactonization16 in toluene at 0 °C to generate the desired 14-membered macrocyclic lactone 10 (65% yield for 2 steps). The route was completed by Dess-Martin oxidation of the hydroxyl group in 10 giving ketone 11 (85%), which underwent simultaneous acetonide and MOM deprotection using TFA to produce the target lactone 1 (94% for 2 steps).

Scheme 1. Retrosynthetic analysis for the synthesis of 1, 13, 14 and, 15

The advanced intermediates 10 and 11 in the route for synthesis of 1 were used in pathways for preparation of the isomeric trihydroxy analogues of L-783277, 13 and 14, and 6


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external olefin containing analogue 15 (Scheme 2). The diastereomeric alcohols 10A (C-6´R anti/anti) and 10B (C-6´S anti/syn) (2:1)16 were readily separated by using silica gel column chromatography. The stereochemistry of these substances was assigned by using a modification of Mosher’s empirical method.17-18 Specifically, treatment of the major isomer 10A independently with (-)-(S)- and (+)-(R)-α-methoxy-α-(trifluromethyl)-phenylacetic acid (MTPA), DCC and a catalytic amount of DMAP led to formation of the corresponding diastereomeric (S)-10A and (R)-10A MTPA esters, which were subjected to 1H NMR analysis. The chemical shifts for the C-6´-H and C-4´-H protons of (S)-10A MTPA are at lower fields than those of the corresponding hydrogens in (R)-10A MTPA (Scheme 2, ∆δ = 0.058 and 0.031 respectively). In addition, the resonance for the C-5´-H proton of (S)-10A MTPA is at a higher field than that in (R)-10A MTPA ester (Scheme 2, ∆δ = ˗0.078). The ∆δ obtained from these measurements are consistent with assignment of the “R” configuration at C-6´-H in the major isomer 10A. Resonances for the C-6´-H and C-4´-H protons in the 1H NMR spectrum of (S)-10A MPTA are shifted to lower field due to the influence of the methoxy group, whereas that of the C-5´-H proton in the (S)-10A MTPA appears at higher field due to the influence of the phenyl group. In contrast, resonances for the C-6´-H and C-4´-H protons in the (R)-10A MPTA are shifted to higher field and the resonance for the C-5´-H proton appears at lower field. The combined observations demonstrate that the major isomer 10A has C-6´R anti/anti stereochemistry and that the minor isomer 10B has C-6´S anti/syn stereochemistry. Both the 10A and 10B were subjected to global deprotection using TFA to generate the respective targets 13 and 14 in over 90% yields. The olefin derivative 15 was generated from the intermediate 11 through Wittig olefination to form 12 (79%). Global deprotection of 7



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12 using TFA then produced 15 (95%). Scheme 2. Synthesis of compound 1, 13, 14 and 15

(a) 9-BBN, K3PO4(3 M), Pd(PPh3)4, DME, THF, 80 °C, 2 h, 92%; (b) Et3N, Pd-C, MeOH, rt, 4 h, 88%; (c) TBAF, THF, 0 °C to rt, 4 h, 84%; (d) NaOH, EtOH:H2O, 85 °C, 12 h; (e) TPP, DIAD, THF, 0 °C to rt, 1 h, 65% for 2 steps; (f) DMP,CH2Cl2, NaHCO3, 0 °C to rt, 1 h, 85%; (g) THF:TFA:H2O, rt, 1-2 h; (h) C-1 salt, nBuLi, THF, -78 °C to rt, 2 h, 79%; ∆δ values [∆δ (in ppm) = δS-MTPA - δR-MTPA] obtained for the (S)- and (R)-MTPA esters of 10A. Finally, the amide derivative of L-783277, 25, was synthesized by utilizing the pathway displayed in Scheme 3. The sequence began with preparation of hydroxyl-ester 16 from the key intermediate 2.4 Oxidation of 2 using DMP generated the corresponding aldehyde 17, which was subjected to Pinnick oxidation19 to form acid 18 (68% for two steps). 8


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Acid 18 and commercially available (S)-4-aminobutan-2-ol were then coupled using EDCI/HOBt to produce amide 19 (87%). To accomplish macrolactonization of 19,16 hydrolysis of the methyl ester in 19 was attempted using NaOH, but the process resulted in concomitant amide hydrolysis and formation of the undesired diacid. Owing to this problem, the route to 25 was altered. The TBDPS group in 2 was removed using TBAF to generate primary alcohol 20 (85%), which upon Dess-Martin periodinane oxidation followed by Pinnick oxidation19 generated acid 21 (69% for two steps). Coupling of acid 21 with (S)-4-aminobutan-2-ol using EDCI/HOBt produced the required amide 22 (88%) that underwent smooth hydrolysis using NaOH at room temperature to form acid 23 (71%). Macrolactonization16 was then accomplished using TPP/DIAD to produce the 14-membered macrolactone 24, which upon removal of the acetonide group using TFA formed the desired final compound 25 (44% for two steps). Scheme 3. Synthesis of amide derivative 25

9



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(a) TBAF, THF, 0 °C, 4 h, 85%; (b) DMP, CH2Cl2, 0 °C to rt, 1 h; (c) NaClO2, NaH2PO4, 2methyl 2 butene, tBuOH:H2O, 0 °C to rt, 69% for 2 steps; (d) EDCI, HOBt, CH3CN, DIPEA, rt, 12 h, 88%; (e) NaOH, EtOH:H2O (1:1), 0 °C to rt, 6 h, 71%; (f) TPP, DIAD, Toluene, 0 °C to rt, 1 h; (g) TFA, CH2Cl2, rt, 2 h, 44% for 2 steps

Kinome-wide profiling of compound 1 and L-783277 To evaluate the kinome-wide selectivity, we examined kinase-inhibitory activities of L-783277 and 1 against a panel of 342 kinases (Figure 2A and Supplementary Table 1 and 2). We found 18 kinases inhibited more than 80% by 1 µM of L-783277. Among the 18 kinases, we identified 11 kinases as novel target kinases of L-783277, including TGFβR2, ZAK, PDGFRβ, MKK6, PKCmu, PKCnu, PKD2, MNK1, MNK2, TAK1 and MKK4. In contrast, 1 10


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at 1 µM inhibited (>80%) only 6 kinases including ALK1, FLT3, FLT4, KDR, MEK2 and MKK6 (Figure 2A). This finding indicates that 1 has a greater kinase selectivity than does L783277. The IC50 values of the two compounds against ALK1, FLT3/4, KDR, MEK and MKK6 were then determined (Figure 2B). L-783277 was found to inhibit strongly FLT3, FLT4, KDR and MEK with IC50 values in the range of 1 to 8 nM, whereas 1 serves as only a moderate inhibitor of the same kinases (IC50 = 114-188 nM) (Figure 2B). Compound 1 turned out to be inactive against MKK6 from a titration experiment (Figure 2B), which is inconsistent with the single-point inhibition data (89% inhibition at 1 µM) from the kinomewide profiling analysis (Supplementary Table 2). We believe that it was an experimental error on MKK6 inhibition data from kinome-wide profiling analysis of compound 1. To our surprise, compound 1 turned out to be more potent against ALK1 (IC50 = 62 nM) than L783277 (IC50 = 125.5 nM). These results indicate that 1 possesses a higher activity against ALK1 and MKK6 with greater selectivity compared with L-783277. Sequence alignment20 (Figure 2C) revealed that FLT3, FLT4, MEK2, MKK6, and KDR contain the conserved cysteine. Compound 1 exhibited 20 to 110-fold less activity against these kinases than L-783277. Unlike these 5 kinases, ALK1 does not possess a cysteine analogous to Cys166 of ERK2. Interestingly, ALK1 has DLG motif in the activation loop instead of the conventional DFG motif. ALK1 lacking the conserved cysteine does not form a covalent bond with L-783277, which is a potential reason why L-783277 has a much lower potency against ALK1 than FLT3, FLT4, and KDR. Although both ALK1 and FLT4 are inhibited by 1 to similar degrees, ALK1 was selected as the primary target kinase for further studies because it is more strongly inhibited by 1 than by L-783277. We tested whether compound 1 retains the in vitro potency against MEK2 (IC50 = 186 nM) in cellular level. First, we tested whether 1 has in vitro inhibitory 11



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potency against MEK2 (IC50 = 186 nM) at the cellular level. The results show that, in contrast to L-783277 that completely suppresses MEK phosphorylation, 1 displays only moderate inhibition at the same concentration (1 µM) in the A375 cell line (Supplementary Figure 1).

N N

O

N N

N

N N

N

O NH2 N

O

N N

O HN

Dorsomorphin

OH

LDN193189

Figure 1 Known inhibitors of ALK family kinases. (A)

(B)

(C)

12


K02288

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IC50 (nM) Kinase

L-783277

1

ALK1/ACVR1

125.50

62

FLT3

1.59

188.00

KDR/VEGFR2

6.34

162.60

FLT4/VEGFR3

1.13

114.50

MEK2

8.16

186.70

MKK6

388.20

>10,000

Figure 2. (A) Kinases showing >80% inhibition are clustered on kinome tree. Eighteen out of 336 kinases are inhibited more than 80% by L-783277. Six out of 342 kinase are inhibited more than 80% by 1. The size of each circle indicates over 80 percent inhibition of each kinase.

Illustration

reproduced

courtesy

of

Cell

Signaling

Technology,

Inc.

(www.cellsignal.com) (B) IC50 values of 1 and L-783277 against selected kinases. (C) Amino acid sequences of selected kinases are aligned. ALK1 has no cysteine analogous to Cys168 in ERK2 (left arrow) and possesses a non-conventional DLG motif (right arrow).

SAR study of compound 1 analogues against ALK1 We performed the SAR study for the five new L-783277 analogues (Figure 3A) based on biochemical kinase-inhibitory activity against ALK1. The results show that both isomeric hydroxyl analogues, 13 and 14, have much lower inhibitory activities than does 1 (IC50 = 0.062 µM). The α-hydroxyl isomer 14 is a slightly better inhibitor of ALK1 (IC50 = 0.770 µM) than is the β-hydroxyl isomer 13 (IC50 = 1.55 µM) (Figure 3B). The external olefin 15 displayed a reasonable activity (IC50 = 1.2 µM), which suggests that ketone group at 13



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C-6’ position in L-783277 is not critical for inhibition of ALK1. Contrary to our expectations, 25 bearing an amide instead of ketone functionality turned out to be inactive against ALK1 (Figure 3B). In order to determine if the enzymatic SAR data have relevance to a cellular context, Western blot analysis of 1 and its analogues 13, 14, 15 and 25 were performed using the C2C12 cell line. The murine mesenchymal precursor cell line C2C12, which undergoes rapid osteogenic differentiation under BMP stimulation, is a common model for studying ALK1 signaling.11 C2C12 cells were treated with these L-783277 analogues (10 µM) for 2 h with BMP9 stimulation and their abilities to block Smad1/5 signaling were determined. Western blot analysis showed that compound 1 completely inhibited the phosphorylation of Smad1/5. The results show that 1 completely inhibits phosphorylation of Smad1/5, while 14 and 15 decreases Smad1/5 phosphorylation by 70-80%. In addition, 13 causes a moderate reduction in Smad1/5 phosphorylation and 25 has no effect on this process (Figure 3C). The level of Smad1/5 phosphorylation inhibition by these substances is consistent with biochemical kinase assay data (Figure 3B), which indicates that the enzyme SAR data translate into parallel effects in C2C12 cells. (A)

(B)

(C)

14


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Compound

IC50 (µM)

ALK1/ACVRL1

1

0.062 ± 0.014

13

1.785 ± 0.332

14

0.770 ± 0.028

15

1.256 ± 0.374

25

inactivea

L-783277

0.125

Figure 3. (A) Chemical structures of L-783277, 1 and its analogues (B) IC50 values of 1 and its analogues. aInactive means less than 40% inhibition at 10 µM of compound. (C) Western blot analysis of 1 and its analogues on C2C12 cell line. Compound 1 completely attenuated Smad1/5 phosphorylation at 10 µM concentration while the other analogues showed moderate or no inhibitory activity.

Molecular dynamics simulation study for 1 on ALK1 To gain insight into the factors governing kinase-inhibitory activities, molecular dynamics simulations were carried out to elucidate the modes of binding L-783277, 1 and 25 on ALK1 (Figure 4A-C). The results show that the phenolic hydroxyl group of the three substances makes a hinge contact with His280 of ALK1. Interestingly, the ketone functionality at C-6’ in these compounds contributes little to interactions with ALK1, which is in agreement with the observed activity of 15 that possesses an exo-methylene group at this position. The hydroxyl group at C-4’ in both L-783277 and 1 interacts significantly Arg334 of ALK1. As depicted in the interactions fraction bar graph displayed in Figure 4A and 4B, Hbonding of Arg334 with the hydroxyl group at C-4’ in L-783277 and 1 is the most substantial 15



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contributor to their interactions with ALK1. In contrast, hydroxyl group at C-4’ in 25 is not capable of making H-boding contact with Arg334, presumably because of its unfavorable location caused by the preferred conformation of the amide group. This lack of H-bonding interaction of hydroxyl group at C-4’ with Arg334 would make 25 inactive on ALK1. (A)

(B)

(C)

Figure 4. Molecular dynamics simulation study and interaction profile analysis of (A) L783277, (B) 1, and (C) 25 within the ATP-pocket of ALK1. Hydrogen bonds are represented 16


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as dashed lines.

Compound 1 attenuated Smad1/5/8 pathway via ALK1 inhibition Western blot analysis was carried out to determine the cellular activity of 1 in C2C12 cells. Luo et al reported that expression level of ALK1 is much higher than that of ALK2 in C2C12 cells.21 Also, David et al reported that ALK1 overexpression leads to high BMP9induced reporter gene activation, while ALK2 overexpression influences no effect, which indicates that BMP9 signaling is largely dependent on ALK1, and is not related with ALK2.22 Based on these previous reports, we treated BMP9 as ligand to activate mainly ALK1 signal in C2C12 cells and examined whether the compounds affect ALK1 downstream Smad1/5 phosphorylation. The results showed that administration of 1 (1 µM) leads to complete abolishment of phosphorylation of Smad1/5 and that p-Smad1/5 activity is strongly blocked by even 0.1 µM of 1. Compound 1 inhibited BMP9 induced Smad1/5 phosphorylation in C2C12 cells by primarily blocking ALK1 activity. It should be noted that 1 displays a more potent

activity in C2C12 cells than does L-783277 (Figure 5A). Non-Smad pathways were also investigated to examine whether 1 displays off-target effects. While L-783277 was found to suppress p38, ERK1/2, and MEK phosphorylation, compound 1 had almost no effect on these kinases even at a concentration of 10 µM. The results of both the in vitro kinase assay and cellular assay indicate that 1 possesses a greater selectivity and potency against ALK1 than does L-783277. The well-known pan-ALK family kinase inhibitor, 26, attenuated TGFβ1-induced Smad1/5 phosphorylation (Figure 5B). Compound 26 displayed a complete inhibition of pSmad1/5 induced by TGFβ1 at 0.1 µM concentration. In contrast, 1 at the high concentration 17



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of 10 µM slightly attenuated p-Smad1/5 level induced by TGFβ. Since TFGβ1-induced Smad1/5 phosphorylation was mainly mediated by ALK5,23 this result suggests that 1 has only weak inhibitory activity against ALK5. To confirm kinase selectivity, IC50 values of 1 and 26 against ALK kinase family members were determined (Table 1). While 26 inhibited all ALK family members with IC50 values of single or two digit nM, 1 inhibited ALK1 with IC50 of 62 nM and displayed a moderate activity against ALK2 with (IC50 = 217 nM) and much less activities against ALK3-6. We also examined the IC50’s using 200 µM of ATP which is close to a physiological ATP concentration (ca. 1 mM). Compound 1 showed 1.21 µM and 3.35 µM IC50’s against ALK1 and ALK2 respectively, and showed almost no inhibition against the remaining ALKs. For both 10 µM and 200 µM ATP conditions, compound 1 showed about 3-fold higher activity against ALK1 than against ALK2, and exhibited much less activity against ALK3-6. These results confirmed the selectivity against ALK1/2 over ALK3-6, and showed that compound 1 moderately discriminated ALK1 over ALK2. Together, these findings demonstrate that 1 suppresses the BMP9-induced Smad1/5 pathway by selectively inhibiting ALK1 in C2C12 cells. In addition, 1 and its analogues do not suppress the proliferation of MDA-MB-231 breast cancer cells, suggesting that no serious off-target effects are induced by these compounds (Supplementary Figure 2). Table1. IC50 values of 1 and 26 against ALK isotypes IC50 (nM)a Compound 1

ALK1/ACVRL1

Compound 26

10 µM ATP

200 µM ATP

10 µM ATP

62

1213

11.13

18


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a

ALK2/ACVR1

217

3353

3.26

ALK3/BMPR1A

6267

inactiveb

2.15

ALK4/ACVR1B

2761

inactiveb

43.29

ALK5/TGFBR1

7213

inactiveb

27.15

ALK6/BMPR1B

727

inactiveb

0.94

Radiometric biochemical kinase assay.

b

Inactive means less than 40% inhibition at 10 µM of

compound

Id1 is a well-known target gene of the BMP9-Smad1/5/8 pathway.24 Id1 possesses a helix-loop-helix (HLH) domain and plays a role in differentiation and cell cycle progression by binding differentiation-promoting transcription factors.25-26 Both mRNA and protein levels of Id1 were increased upon stimulation with BMP9 and were significantly downregulated by 10 µM of 1 (Figure 5C). This indicates that 1 suppresses Id1 transcription via ALK1 inhibition. (A)

(B)

(C)

Figure 5. (A) BMP9-mediated Smad1/5/8 pathway is inhibited by 1 but Akt and MAPK pathways are not in C2C12 cells. (B) 1 marginally inhibits TGFβ1-induced phosphorylation of Smad1/5 and does not inhibit Smad2/3 phosphorylation, while L-783277 potently inhibits 19



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Smad1/5 phosphorylation and moderately inhibits Smad2/3 phosphorylation. Compound 26 completely inhibits Smad1/5 phosphorylation but does not inhibit Smad2/3 phosphorylation. (C) 1 downregulates the BMP9-induced transcription/translation of Id-1 gene in C2C12 cells.

The effect of compound 1 on angiogenesis. Anti-angiogenic function of ALK1 has been demonstrated in several studies.27-28 Thus, pharmacological inhibition of ALK1 should promote angiogenesis. To test the proangiogenic properties of the ALK1 inhibitor 1 in vitro, an endothelial tube formation assay was carried out. The results showed that 1 promoted vascular endothelial tube formation in a dose dependent manner starting from 2 µM (Figure 6A and 6B). A microfluidic chip based 3D angiogenesis assay, which recapitulates to functional 3D vessel,29 was performed to evaluate the angiogenic capability of 1 more comprehensively. We found that 1 robustly stimulated sprouting angiogenesis even at 2 µM. Both the vessel areas and lengths of sprouts were significantly increased by treatment with 1. Moreover, 1 also displayed a synergistic pro-angiogenic effect with recombinant rhVEGF165. Compared with the rhVEGF165 monotherapy group, the vessel area coverage was significantly increased in the group co-treated with rhVEGF165 and 1 (Figure 7A and 7B). The robust pro-angiogenic capability of 1 suggests that 1 could be a promising lead compound for the development of therapeutic angiogenesis agents.

(A)

(B)

20


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Figure 6. (A) Representative photograph of endothelial cell tube formation as a function of the concentration of 1. (B) Quantitative comparison of branch nodes number. Scale bar, 500 ㎛. *p

Identification of the First Selective Activin Receptor ... - ACS Publications

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