Identification and Further Development of Potent ... - ACS Publications

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Identification and Further Development of Potent TBK1 Inhibitors André Richters,† Debjit Basu,† Julian Engel,† Meryem S. Ercanoglu,‡ Hyatt Balke-Want,‡ Roberta Tesch,†,§ Roman K. Thomas,‡ and Daniel Rauh*,† †

Department of Chemistry and Chemical Biology, Technical University of Dortmund, Otto-Hahn-Straße 6, 44227 Dortmund, Germany ‡ Department of Translational Genomics, University of Cologne, Weyertal 115b, 50931 Cologne, Germany § Programa de Pós-Graduaçaõ em Farmacologia e Química Medicinal, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-901 Brazil S Supporting Information *

ABSTRACT: The cytosolic Ser/Thr kinase TBK1 was discovered to be an essential element in the mediation of signals that lead to tumor migration and progression. These findings meet the need for the identification of novel tool compounds and potential therapeutics to gain deeper insights into TBK1 related signaling and its relevance in tumor progression. Herein, we undertake the activity-based screening for unique inhibitors of TBK1 and their subsequent optimization. Initial screening approaches identified a selection of TBK1 inhibitors that were optimized using methods of medicinal chemistry. Variations of the structural characteristics of a representative 2,4,6-substituted pyrimidine scaffold resulted in improved potency. Prospective use as tool compounds or basic contributions to drug design approaches are anticipated for our improved small molecules.

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chemokine 5 (CCL5) and intereukin 6 (IL-6) further substantiate a potential role in cancer therapy.8 However, TBK1 was also demonstrated to participate in tumor metastasis and epithelial−mesenchymal transition (EMT), and therefore, modulating TBK1 activity may curtail tumor growth and propagation. Prevailing findings support this assumption, since small molecule inhibitors have been discovered that block TBK1 transferase activity and thereby hinder the ability of tumor cells to further metastasize, proliferate, or undergo EMT processes.9 Recently, TBK1 has been shown to activate estrogen receptor α (ERα) by phosphorylation, thereby stabilizing ERα and inducing transactivational activity and modulating breast cancer growth. Ectopic expression of TBK1 in this context renders breast cancer cells resistant to tamoxifen while suppression of TBK1 activity by treatment with BX795 (Figure 1),10,11 a known 3-phosphoinositide-dependent protein kinase 1 (PDK1) and TBK1 inhibitor, in combination with tamoxifen leads to synergistic antiproliferative effects in breast cancer cells. Therefore, TBK1 may serve as a predictive marker for tamoxifen resistance and as a potential therapeutic target in breast cancer.12 Additionally, TBK1 is involved in mouse double minute 2 (MDM2) mediated repression of estrogen

ANK-binding kinase 1 (TBK1) is a critical regulator of the antiviral response and initiates type I interferon (IFN) transcription through an endosomal toll-like receptor 3 (TLR3) pathway. Subsequently, the interferon regulatory factors 3 and 7 (IRF3/7) are activated and then translocated to the nucleus to initiate IFN gene transcription.1−4 Alternatively, cytosolic activation occurs by the recognition of infiltrated viral RNA through sensor proteins such as melanoma differentiationassociated protein 5 (MDA5) or the retinoic acid-inducible gene 1 protein (RIG-I). Consecutive TBK1 activation is induced by adapter protein mediated phosphorylation of TBK1, thereby initiating the same downstream cascades of IFN production as in the endosomal pathway. TBK1 is involved in the formation of a higher ordered multi enzyme complex and interacts with several adapter proteins such as nuclear factor κB (NFκB)-activating kinase-associated protein 1 (NAP1), TBK1binding protein 1 (SINTBAD), and TRAF family memberassociated NFκB activator (TANK) in an orchestrated trafficking network.1,5 Besides its implication in the mediation of antiviral responses, TBK1 has been proposed as a target for cancer therapy since TBK1 knockout studies led to antiproliferative effects in KRAS-mutated human alveolar adenocarcinoma cells (A549).6 Based on this analysis, TBK1 has become a focus of medicinal chemistry approaches to decipher the complex regulation and activation mechanism and its associated signaling pathway.1,7 Constitutive explorations investigating the role of TBK1 in promoting KRAS-driven tumorigenicity and survival by activating autocrine C−C motif © 2014 American Chemical Society

Special Issue: New Frontiers in Kinases Received: November 7, 2014 Accepted: December 26, 2014 Published: December 26, 2014 289

DOI: 10.1021/cb500908d ACS Chem. Biol. 2015, 10, 289−298

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potential target in cancer therapy.8,14 Since TBK1 may serve as a potential therapeutic target, numerous studies have been conducted to identify and develop new TBK1 inhibitors such as (2- and 6-aryl-)azabenzimidazoles, 6-aminopyrazolopyrimidines, and pyrimidines, respectively (Figure 1).15−18 The recently reported crystal structure of the full-length TBK1 kinase in complex with the type I inhibitor BX795 has provided the structural basis for medicinal chemistry approaches.19 TBK1 consists of three superordinate domains: a catalytic domain facilitating Ser/Thr kinase activity, a ubiquitin-like domain (ULD) for substrate specificity, and a scaffolding and dimerization domain (SDD) for structural integrity and mediating adapter protein interactions to form a multi-enzyme complex.19 Moreover, the presence of SDD and ULD provided evidence for TBK1 existing as a homodimer.1,20,21 Although TBK1 was proposed as a target in various cancer types and particularly in facilitating EMT processes, its associated tumor biology to large extent remains unclear.9,22 Therefore, tool compounds for deciphering TBK1 biology as well as specific inhibitors are needed to more deeply investigate its function with respect to the orchestrated network of cellular signaling of the antiviral response, as well as its role in cancer progression and metastasis. Herein, we report the identification of a new class of pyrimidine-based inhibitors of TBK1 by activity-based screening approaches. Subsequent ligand optimization by using

Figure 1. Representative scaffolds (6-aminopyrazolopyrimidine (1), 2aryl-azabenzimidazole (2), and pyrimidines (BX795, CYT387)) of currently known TBK1 inhibitors revealing broad target specificity.

induced protein kinase B (Akt) activation by facilitating phosphorylation induced degradation of hematopoietic PBXinteraction protein (HPIP).13 Broader studies indicate potential therapeutic use of TBK1 inhibitors, since they attenuate radiation-induced EMT of human lung cancer cells (A549) via activation of glycogen synthase kinase-3 β (GSK3β) and simultaneous repression of zinc finger E-box-binding homeobox 1 (ZEB1).9 Similarly, studies on the impact of TBK1 as a therapeutic target in HER2 positive breast cancer and KRASdriven tumorigenicity further emphasized that TBK1 is a

Figure 2. (A) Activity-based screening results as cluster analysis according to inhibitory effect and structural similarity. Colors encode for the remaining TBK1 activity elicited at 13.2 μM compound concentrations. Ring sizes represent the discontinuity of binding: Marginal differences in the compound’s structural composition result in dramatic differences of the inhibitory effect. The clustering was performed using SARANEA.36 (B) Simplified screening workflow for Collections I and II as well as illustration of redundant scaffolds revealing divergent substitution patterns for subsequent SAR studies. (C) Validated hit compounds derived from Collection I (% remaining activity in gray). 290



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Table 1. Collection of Tozasertib Derivatives and Screening Hits and Related IC50 Valuesa

a

compd

R1

R2

R3

X

IC50 [μM]

13 14 15a 15b 15c 15d 15e 15f 15g 15h 15i 15j 15k 22 23 24 tozasertib axitinib bosutinib dovitinib erlotinib K252a sunitinib tofacitinib

H H NO2 t Bu i Pr Me OMe F Cl CF3 Cl Cl NH2 H H NHCOCH2CH3 NHCO(cyclopropane)

H NO2 H H H H H H H H F CF3 H H NHCOCH2CH3 H H

NO2 H H H H H H H H H H H H NHCOCH2CH3 H H H

O O O O O O O O O O O O O O O O S

2.35 ± 1.41 ± 0.06 ± 2.66 ± 0.34 ± 0.38 ± 0.65 ± 0.84 ± 1.19 ± 0.29 ± 0.72 ± 3.00 ± 0.73 ± nib >10 2.50 ± 6.22 ± 0.47 ± 0.67 ± 0.06 ± 2.20 ± 95%). Notably, the ten prime targets of 15a within this panel are represented by tyrosine kinases, and GSK3β (91% inhibition) is the most effectively inhibited Ser/Thr kinase followed by Aurora A (88%). TBK1 was likewise demonstrated to be efficiently inhibited by 15a (79%), but the vast diversity of addressed targets illustrates that selective inhibition of TBK1 is challenging. Remarkably, PDK1 (33% inhibition) as well as IKKε (13% inhibition), which represent prime targets of the known TBK1 inhibitor BX795, were not significantly impaired. In addition to profiling studies, we further investigated the binding mode of 15a within the catalytic domain of TBK1 by performing docking studies using the free available receptor− ligand docking server DockThor (Supporting Information Figure S5).34 These analyses support our initially proposed binding mode based on the superposition of tozasertib in complex with Aurora A and TBK1. According to our assumption that the pyrazole moiety accounts for the formation of hydrogen interactions to the backbone amides within the hinge region a similar binding mode was observed. However, the docking studies solely revealed the formation of two instead of three hydrogen bond interactions to the peptide backbone of Glu87 and Phe88. In addition, we identified a vice versa orientation of the piperazine and para-nitrophenyl moieties most likely due to the formation of favorable ionic interactions of the piperazine with Asp157 and due to the lack of an aromatic amino acid within the glycine-rich loop that allows for flexible ligand rearrangement (see Supporting Information Figure S5 for detailed information). Conclusions. In the present study, we report the successful identification of TBK1 inhibitors by activity-based screening endeavors. Compilations of industry provided compounds (GSK and ROCHE) were scrutinized for inhibitory activity with respect to TBK1. Since this collection was comprised of molecules that were already subject to drug development endeavors, we derived a plethora of compounds with remarkable inhibitory effects and were able to retrace SARs among the identified scaffolds. Among these, oxindole 77 (IC50 = 0.003 μM) from ROCHE and imidazopyrimidine 28 (IC50 = 0.14 μM) from GSK were shown to constitute the most potent representatives of each library. In addition, two independently compiled small molecule inhouse libraries afforded 2,4,6-substituted pyrimidine scaffolds

Figure 5. qRT-PCR-based examination of IFN-pathway interference in RAW macrophages rendered by compounds BX795, 15a, 15c, and 15f at 1 μM compound concentration (ctrl: unstimulated; DMSO: LPS-induced).

Notably, for 15a, we observed a 6-fold increased mRNA level of IFN-β at 1 μM compound concentration. The respective pathway was apparently still induced and therefore 15a was expulsed from representing a potent cellular modulator of TBK1 and its associated signaling cascade. In contrast, 15c and 15f, previously highlighted as moderate TBK1 inhibitors in activity-based analyses, were capable of blocking the pathway activation more dramatically. In detail, 15c merely led to a 2fold increase in mRNA levels as compared to unstimulated cells though 15f likewise impaired the pathway activation ending up into 4-fold IFN-β expression above control level. Hereby, we confirmed that our inhibitors significantly impact the TBK1 associated signaling cascade on a cellular level. We can only speculate on the reasons for the converse result of 15a representing the most potent inhibitor in activity-based assays, 295



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the fluorophore XL665 were added. FRET between europium cryptate and XL665 was measured to quantify the phosphorylation of the substrate peptide. ATP concentrations were adjusted to 30 μM while a substrate concentration of 1 μM was used. Kinase and inhibitor were preincubated for 30 min before the reaction was started by addition of ATP and substrate peptide. A Tecan infinite M1000 plate reader was used to measure the fluorescence of the samples at 620 nm (Eulabeled antibody) and 665 nm (XL665 labeled streptavidin) 60 μs after excitation at 317 nm. The quotient of both intensities for reactions made with eight different inhibitor concentrations was fit to a Hill four-parameter equation to determine IC50 values. Each reaction was performed in duplicate, and at least three independent determinations of each IC50 were made. For screening purposes, we utilized the Cisbio system HTRF KinEASE-STK as stated above and TBK1 from Millipore (#14-628, Lot# D9SN044N-A). Robotics assisted compound transfers (2 × 6.6 nL for 13.2 μM screening concentration) were processed using a PinTool system from Tecan, or compounds were manually transferred where appropriate (10 μM screening concentration). At least 8 data points for positive and negative controls were included for each assay plate using BX795 (13.2 μM or 10 μM) as 0% and DMSO as 100% TBK1 kinase activity control. The assay readout was performed using a Tecan infinite M1000 plate reader, and a Z′-factor of 0.81 was determined for the entire screen. qRT-PCR in RAW Macrophages. RAW 264.7 macrophages were preincubated for 1 h with DMSO or TBK1 inhibitors as indicated and then stimulated with 100 ng/mL LPS (Invivogen) for 2 h. Hereafter, RNA was extracted using Quiageńs RNeasy Mini Kit. cDNA was prepared from 500 ng RNA using the Superscript III reverse transcriptase (Invitrogen #18064). qRT-PCR was performed using a 7300 Real-Time PCR System (Applied Biosystems) and Power SYBR Green PCR Master Mix (Applied Biosystems) with primers for mouse GAPDH and Interferon-β (both at 60 °C) as described by Orzalli et al.33 ΔCt-values were determined using the 7300 System Software (Applied Biosystems) using GAPDH as reference control and gene expression was calculated by the ΔΔCt-method.35

(tozasertib and Scaffold I) that were considered to serve as basis for further rational design approaches to improve these core structures with respect to their inhibitory activity toward TBK1. Consecutive validation studies and optimization of tozasertib resulted in the linear synthesis of a small focused library of derivatives (13−15a−k and 22−24). We paid particular attention to the derivatization of the para-phenyl position, which was found to be crucial for modulating TBK1 activity in vitro. Candidates in our focused library were then tested for enhanced inhibitory effects resulting in distinct SARs. Notably, the nature of the para-connected functional group with respect to its spatial arrangement combined with electron withdrawing and, in turn, electron donating properties was pivotal for the evoked inhibitory effect. We illustrated the ability of terminal tert-butyl moieties to exhibit weak inhibition (15b, 2.7 μM), which was significantly improved by decreasing the spatial arrangement with isopropyl and methyl derivatives for improved potency (15c and 15d; 0.34 and 0.38 μM, respectively). The most significant perturbation of TBK1 revealed an inhibitory effect within the midnanomolar range (IC50 = 0.06 μM) and was elicited by nitro substituted compound 15a providing evidence for the advantageous effect of electron withdrawing functional groups. Ultimately, we conducted cellular studies using RAW macrophages and methods of qPCR to investigate the compounds impact on the expression of interferons as a marker for the TBK1-pathway modulation. Significant downregulation of the IFN-β expression was confirmed for compounds 15c and 15f as the copy number of IFN-β was decreased, at 1 μM compound concentrations, to approximately 2- and 4-fold, respectively, as compared to the relative expression in nonstimulated macrophages. Consecutive profiling studies revealed a plethora of kinases that is significantly inhibited by the most potent compound 15a and underline the need for further design approaches to tune the specificity of our 2,4,6-substituted pyrimidine-based inhibitors. Docking studies further illuminated the potential binding mode of 15a and provide a substantial basis for broader design endeavors. In conclusion, our activity-based screening procedure allowed us to identify compounds that inhibited TBK1. These compounds were then optimized by using methods of medicinal chemistry. Potential use of these compounds to decipher TBK1 related signaling or to provide starting materials for subsequent drug design approaches is anticipated for our optimized derivatives.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental procedures for compound synthesis, experimental screening workflows, cluster analysis of screening hits from Collection II, superposition of Aurora A and TBK1, qPCR validation studies, docking studies, profiling studies as well as IC50 data for identified screening hits derived from GSKand ROCHE-libraries. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 (0)231−755 7080. Fax: +49 (0)231−755 7082. E-mail: [email protected].

METHODS

Notes

The authors declare no competing financial interest.

Reagents and Materials. Unless otherwise noted, all reagents and solvents were purchased from Acros, Fluka, Sigma-Aldrich, or Merck and used without further purification. Small volume (20 μL fill volume) white round-bottom 384-well plates were obtained from Greiner Bio-One GmbH (Solingen, Germany). All supplies for the TBK1 HTRF assay kit were purchased from CisBio (Bagnols-surCèze, France). Axitinib, bosutinib, dovitinib, erlotinib, K252a, sunitinib, tofacitinib, and tozasertib were purchased from LC Laboratories (Woburn, MA). Activity-Based Assay for IC50 Determination and Screening. IC 50 determinations for TBK1 (Millipore, #14-628M, Lot# D9SN044N-B) were performed with the KinEASE-STK assay from Cisbio according to the manufacturer’s instructions. A biotinylated substrate peptide (STK3 from Cisbio) was phosphorylated by TBK1. After completion of the reaction, an antiphosphoserine/-threonine antibody labeled with europium cryptate and streptavidin labeled with

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ACKNOWLEDGMENTS This work was cofunded by the German federal state North Rhine Westphalia (NRW) and the European Union (European Regional Development Fund: Investing in Your Future), the German Federal Ministry for Education and Research (NGFNPlus and e:Med) (Grant No. BMBF 01GS08104, 01ZX1303C). R.T. is funded and supported by CAPES and FAPERJ.

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ABBREVIATIONS Akt, protein kinase B; CCL5, C−C motif chemokine 5; FGFR, fibroblast growth factor receptor; GSK3β, glycogen synthase 296



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kinase-3 β; HER2, receptor tyrosine-protein kinase erbB-2; HPIP, hematopoietic PBX-interaction protein; IL-6, intereukin 6; MDA5, melanoma differentiation-associated protein 5; NAP1, NFκB-activating kinase-associated protein 1; NFκB, nuclear factor NFκB p105 subunit; PBX, pre-B-cell leukemia transcription factor; PKC, protein kinase C; RIG-I, retinoic acid-inducible gene 1 protein; RTK, receptor tyrosine kinase; SAR, structure−activity relationship; SINTBAD, TBK1-binding protein 1; TANK, TRAF family member-associated NFκB activator; TBK1, TANK binding kinase 1; TNF, tumor necrosis factor; TRAF, TNF receptor associated factor; qRT-PCR, quantitative real-time polymerase chain reaction; VEGFR, vascular endothelial growth factor receptor; ZEB1, zinc finger E-box-binding homeobox 1

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