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Article
Discovery of BAY-985 a highly selective TBK1/Ikk# inhibitor
Julien Lefranc, Volker K. Schulze, Roman Christian Hillig, Hans Briem, Florian Prinz, Anne Mengel, Tobias Heinrich, József Bálint, Srinivasan Rengachari, Horst
Irlbacher, Detlef Stöckigt, Ulf Bömer, Benjamin Bader, Stefan Nikolaus Gradl, Carl F. Nising, Franz von Nussbaum, Dominik Mumberg, Daniel Panne, and Antje Wengner
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01460 • Publication Date (Web): 20 Dec 2019
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Discovery of BAY-985 a highly selective TBK1/IKKε inhibitor
Julien Lefranc,*,# Volker Klaus Schulze,# Roman Christian Hillig,# Hans Briem,# Florian Prinz,# Anne Mengel,# Tobias Heinrich,# Jozsef Balint,║ Srinivasan Rengachari,§,† Horst Irlbacher,# Detlef Stöckigt,# Ulf Bömer,# Benjamin Bader,# Stefan Nikolaus Gradl,# Carl Friedrich Nising,# Franz von Nussbaum,# Dominik Mumberg,# Daniel Panne,§,‡ Antje Margret Wengner#
#Bayer AG, Pharmaceuticals, Research and Development, 13353 Berlin, Germany
║ASCA GmbH (Angewandte Synthesechemie Adlershof), 12489 Berlin, Germany
§Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Lancaster Road, Leicester, LE1 7RH, U. K.
‡European Molecular Biology Laboratory, 38042 Grenoble, France
ABSTRACT. The serine/threonine kinase TBK1 (TANK-binding kinase 1) and its homologue IKKε are non-canonical members of the inhibitor of nuclear factor κB (IκB) kinase family. These kinases play important roles in multiple cellular pathways and, in particular, in inflammation. Herein, we describe our investigations on a family of benzimidazoles and the identification of the potent and highly selective TBK1/IKKε inhibitor BAY-985. BAY-985 inhibits the cellular
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phosphorylation of interferon regulatory factor 3 (IRF3) and displays antiproliferative efficacy in the melanoma cell line SK-MEL-2, but showed only weak antitumor activity in the SK-MEL-2 human melanoma xenograft model.
INTRODUCTION
The serine/threonine kinase TBK1 (TANK-binding kinase 1) and its homologue IKKε are non- canonical members of the inhibitor of nuclear factor κB (IκB) kinase family. These kinases play important roles in multiple cellular pathways and, in particular, in inflammation.1,2 IKKs are activated following engagement of pattern recognition receptors such as toll-like receptors (TLRs), cytosolic DNA receptors, and retinoic acid inducible gene I (RIG-I)-like receptors. Activated non- canonical IKKs phosphorylate interferon regulatory factors (IRFs) 3, 5, and 7, which then dimerize and enter the nucleus to induce expression of pro-inflammatory and antiviral genes.1
TBK1 also plays an important role in autophagy,3,4 a quality control process in which the cell recycles proteins or disposes of excessive or damaged organelles and which contributes to the clearance of intracellular pathogens.5-9 Furthermore, TBK1 maintains T cell homeostasis and negatively regulates both autoimmunity and antitumor immunity by promoting immune tolerance in dendritic cells, likely by regulating a subset of type I interferon receptor induced genes.10
In view of its critical role in inflammatory signaling and oncology, TBK1 has attracted considerable interest as a potential target in cancer therapy. TBK1 is overexpressed and activated in bladder, lung, breast, and colon cancers.11–13 Knockdown experiments have shown that TBK1 is crucial for cellular survival and synthetically lethal with mutated KRAS in lung cancer.14 The RalB/Sec5 effector complex, downstream of KRAS signaling, directly recruits and activates TBK1, thus preventing induction of apoptosis.15 TBK1 also regulates an autocrine circuit of
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CCL5/RANTES and interleukin 6 to promote KRAS-driven tumorigenesis.16 TBK1 is further thought to promote vascularization,13 to directly drive oncogenesis by phosphorylating and activating Akt1,11,17 and to regulate prostate cancer dormancy by inhibition of mTOR.18
Although a number of studies suggest that TBK1 is a potentially interesting target in cancer therapy, recent data have challenged this view.19,20 Considering that most studies were performed using relatively unselective TBK1 inhibitors or by protein depletion, it remains unclear if selective kinase inhibition of TBK1 is a viable therapeutic strategy. Clinical studies targeting TBK1 in KRAS-mutant cancers using momelotinib, a compound that inhibits both Janus kinases and TBK1, have been terminated early without release of further information.21 Over the last decade, several attempts have been made to identify TBK1/IKKε inhibitors; however, it has become apparent that the identification of such highly selective inhibitors is very challenging.22,23
TBK1 is an 84 kD, 729 amino acid protein comprising an N-terminal kinase domain, an ubiquitin-like domain, an α-helical scaffold dimerization domain, and a C-terminal adaptor binding domain (CTD).24 Depending on the context, cell type, and stimulus, the CTD binds to a number of different adaptors including TANK, NFκB activating kinase (NAK)-associated protein
1(NAP1), similar to NAP1 TBK1 adaptor (SINTBAD), and Optineurin.25–27 Thus, in addition to its enzymatic activity, TBK1 also has an important scaffolding function, and an unravelling of the relative contributions to cellular signaling is highly desirable. To that end, we have developed BAY-985, a highly potent and selective TBK1 inhibitor. BAY-985 has been donated as a probe molecule to the Structural Genomics Consortium (SGC) to enable access to the scientific community for future studies of TBK1 and its function. Similarly, selective inhibitors of other potential drug targets have proven valuable tool compounds for the scientific community,28 and we anticipate that BAY-985 will broaden the understanding of the biology of TBK1.
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RESULTS AND DISCUSSION
HTS. We first performed a TBK1 high-throughput screening (HTS) campaign using a biochemical kinase assay as an entry point. Due to the limited set of compounds identified with this approach, we performed a second HTS, this time with a TBK1-dependent cellular reporter gene assay. This second approach was expected to allow identification of compounds that inhibit TBK1 in its cellular environment, thus potentially revealing novel modes of action. The reporter cell line expressed luciferase under the control of multiple interferon-stimulated response elements (ISREs). Co-incubation with poly dA:dT stimulates TBK/Ikke driven ISRE activation and hence luciferase expression. In the HTS we screened 3.05 million compounds for their ability to inhibit the luciferase signal by more than 40%. Screening hits were tested for toxicity and unspecific luciferase or transcriptional/translational inhibition (see also supporting information). They were further characterized using two different biochemical assays to measure TBK1 inhibition in vitro (referred to as ‘low ATP assay’ and ‘high ATP assay’). These two assays varied in the ATP concentration (10 µM, low ATP assay; 1 mM, high ATP assay), where a high potency in the high ATP assay is desirable as this increased ATP concentration reflects more realistically the situation in the cell and in vivo. The cellular HTS campaign allowed us to identify compound 1 (Figure 1A) which was ATP competitive in the biochemical assays.
X-ray Crystallography and Initial Optimization. Starting from the HTS hit 1, we explored the 6-position of the benzimidazole moiety with regard to potency and selectivity toward potential off-target kinases, most importantly the CDK family of kinases. TBK1 contains a methionine (Met86, see Figure 1B) at the so-called gatekeeper position, the residue immediately preceeding the hinge which connects the N- and C-terminal lobe of the kinase domain. This feature can be exploited to develop potentially selective inhibitors as CDKs contain a phenylalanine at this
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position. Guided by a co-complex crystal structure published for a similar 6-cyanobenzimidazole inhibitor binding to the ATP pocket of the kinase CK1, which also contains a methionine gatekeeper,29 we predicted that the cyano group of compound 1 would not interact with and modulate the side-chain conformation of the Met gatekeeper of TBK1. Potentially, this could explain the low selectivity of compound 1 toward kinases which feature a bulky Phe gatekeeper, such as CDKs (data not shown). As has been shown for JNK3 kinase inhibitors, selectivity can be improved by exploiting the high flexibility of a Met gatekeeper side chain.30–32 An appropriate substituent on the inhibitor is expected to push away the Met side chain and to open up the so- called ‘hydrophobic back pocket’, whereas a more rigid Phe side chain at the gatekeeper position, such as in CDKs, would restrict access to this back pocket. Based on this rationale, we synthesized compound 2 where the 6-cyano side chain of 1 was replaced by a bulkier N-cyclopropylmethyl amide group. While this substitution only modestly improved potency (see Table 1, TBK1 low ATP assay), it resulted in a suprisingly drastic improvement in selectivity toward the off-target kinase CDK9 (IC50 = 5 nM for 1 vs >20000 nM for 2).
To understand the binding mode of this compound class, we co-crystallized three compounds with close to full-length TBK1S172E24 (see Supporting Information, Table S4. TBK1 only crystallized when ATP site inhibitors were added prior to crystal setup. The vast majority of crystals diffracted to a minimum Bragg spacing of 4–5 Å which required testing of 20–50 crystals/compound to identify a specimen that diffracted to better than 3.5 Å resolution. We collected a 3.15 Å data set for compound 2 and determined the structure by molecular replacement. Despite the modest resolution, omit maps of the ATP site of the kinase domain showed clear difference density, which allowed modeling of the ligand (Figure 1B). The inhibitor inserts into the flat and mainly hydrophobic adenine pocket of the ATP-binding site at the hinge region where
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the central conjugated bi-aromatic ring system is sandwiched between Leu15, Val23, and Ala33 at the roof of the ATP site, and Gly92 and Met142 at its floor. The inhibitor forms two hydrogen- bond interactions to Cys89 of the hinge region of TBK1, one via the central amino group (3.1 Å) and one via a nitrogen atom of the benzimidazole ring (3.5 Å). The piperazine moiety points toward the solvent, where the amide oxygen forms a 3.5 Å hydrogen bond to the side chain of Arg25. The cyclopropyl moiety inserts into the back pocket behind the ATP site and forms a hydrophobic contact with the gatekeeper residue Met86. The nitrogen atom of the adjacent amide group forms a hydrogen bond to Thr156 (2.7 Å). However, compound 2 does not interact with the conserved Lys38 at the back wall of the ATP site nor with Asp157 (of the DFG motif at the start of the activation loop), two polar and conserved kinase residues which line this back pocket behind the ATP site (Figure 1B).
Figure 1. (A) Compound 1 identified during our cellular HTS and compound 2 as an early variant. (B) Crystal structure of TBK1 in complex with 2 (PDB accession code 6RSR). Left, overall view of one TBK1 monomer with the kinase domain in cyan (N-terminal lobe) and blue (C-terminal lobe), the ubiquitin-like domain (ULD) in yellow, and the scaffold dimerization domain (SDD) in green. Right, 2 bound to the hinge region within the ATP site of the kinase domain. The 3.1 Å resolution mFo-DFc electron density omit map contoured at 2 is shown in blue.
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A comparison of the binding mode of 2 with the less selective TBK1 inhibitor BX-795 (PDB accession code 4EUU)33 revealed that, indeed, the gatekeeper Met86 of TBK1 can accommodate the cyclopropyl moiety of inhibitor 2 by adopting an alternative side-chain conformation (Figure 2) relative to the one observed with BX-795 which does not occupy the back pocket.
Figure 2. Conformational flexibility of the TBK1 gatekeeper Met86. Superimposition of the kinase domains of TBK1 in complex with 2 (PDB accession code 6RSR, green carbon atoms) and BX-795 (PDB accession code 4EUU, orange carbon atoms). View into the hinge region of the ATP-binding site. Hydrogen-bond interactions between the inhibitors and TBK1 shown as yellow dotted lines. The gatekeeper residue Met86 can accommodate back-pocket binders such as 2 by adopting a new side-chain conformation (red arrow).
Compounds 1 and 2 showed low permeability and strong efflux in Caco-2 assays: Papp A–B = 32 nm/s, Papp B–A = 268 nm/s, ER = 8.3 and Papp A–B = 3.9 nm/s, Papp B–A = 155 nm/s, ER = 40,
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respectively. We further explored the 6-position of the benzimidazole moiety with the aim of improving selectivity and potency. We focused our attention on small heterocycles that could potentially be bioisosteric to an amide side chain. In addition to the desired shift of the Met86 gatekeeper side chain, computational design focused on the addition of a possible hydrogen bond with the catalytic Lys38, which is in close proximity to the gatekeeper (Figure 1B). This led to the design of compound 3 where the amide was replaced by its 1,2,4-oxadiazole bioisostere. This compound inhibited TBK1 and IKKε with IC50 values of 129 nM (low ATP assay) and 168 nM, respectively (see Table 1). Compound 3 also showed improved permeability and efflux ratio in the Caco-2 assay (Papp A–B = 42 nm/s, Papp B–A = 142 nm/s, ER = 3.4) compared to compound 1.
In parallel, we tested several of our more selective TBK1 inhibitors for antiproliferative activity in more than 100 cell lines. Contrary to results we had obtained in RNAi experiments (data not shown), most of the cell lines tested did not respond to these TBK1 inhibitors. Among the cell lines with higher sensitivity, we selected two cell lines, ACHN (CDKN2A mutated) and SK-MEL-
2(NRAS and TP53 mutated),34 as models to further determine the antiproliferative activity. Compound 3 showed micromolar antiproliferative activity in both cell lines (Table 1).
The selectivity of 3 was further assayed in an internal kinase panel and at 100 nM in the DiscoverX KINOMEscan (see Figure 3 and Supporting Information Table S1). This analysis revealed low kinase selectivity, with eight other kinases in the panel being more potently inhibited than TBK1.
SAR. Starting from compound 3, we investigated the amide substitution on the piperazine moiety (Table 1). Synthetic precursors of 3, namely compounds 4 and 5, with a Boc protecting group or presenting an unsubstituted piperazine ring, were tested and showed reduced potency in the TBK1 and IKKε assays. Alkylation of the nitrogen also led to reduced potency compared to
compound 3: N-propyl as well as N-hydroxyethyl substitution (compounds 6 and 7) resulted in only micromolar inhibition of TBK1 in both the low and high ATP assays. Compounds 8–10 with a fluorinated alkyl substituent were also assayed: 8 inhibited TBK1 at an IC50 value of 281 nM in the low ATP assay and at 5290 nM in the high ATP assay; however, although 8 showed no antiproliferative activity in SK-MEL-2 cells, it inhibited proliferation in ACHN cells with an IC50 of 350 nM. Compound 10 bearing a shorter trifluoromethylated side chain behaved similarly to 8 in the TBK1 assay, while difluoromethyl analogue 9 had slightly higher potency (IC50 = 98 nM, low ATP assay) and a moderate antiproliferative activity, with an IC50 of 1.6 µM in SK-MEL-2 cells. We further investigated amide substituents (compounds 11–14). Aromatic amides such as furan analogue 14 showed reduced potency toward TBK1; however, compounds 11–13, each bearing a small cycloalkyl substituent, proved to be as potent as compound 3 and didn’t show major differences with regards to Caco-2 permeability (Papp A–B = 68 nm/s, Papp B–A = 151 nm/s, ER = 2.2 for 11, Papp A–B = 52 nm/s, Papp B–A = 124 nm/s, ER = 2.4 for compound 12) . Despite substantial efforts, no substituent superior to the initial trifluoroethylcarbonyl group was found; therefore, this group was retained throughout the rest of our investigations.
Table 1. SAR at the piperazine ring (Targeting the Entrance of the ATP-Binding Site)a
a IC50 values are reported as arithmetic means of several measurements (except stated otherwise); n.d.: not determined. b single measurement. c IC50 reported at the median value of several measurements.
Next, we investigated the benzylic position. The introduction of a methyl group at that position had a significant impact on potency (Table 2). While racemic compound 15 had an IC50 value of 34 nM in the TBK1 low ATP assay, testing of the separated enantiomers revealed that one antipode is superior: (S)-configured 16 inhibited TBK1 with an IC50 value of 106 nM (low ATP assay), while (R)-configured 17 displayed a fivefold increase in potency (IC50 = 19 nM, low ATP assay; 372 nM, high ATP assay). Compound 17 was also highly potent toward IKKε (IC50 = 37 nM) and was active in our cellular mechanistic pIRF3 assay and SK-MEL-2 antiproliferation assay, with IC50 values of 1.2 μM and 3.5 μM, respectively. An additional benefit of the introduction of a substituent at the benzylic position was the increased kinase selectivity relative to the unsubstituted analogue (see Table 3, 17 vs 3). In contrast, replacement of the benzylic methylene by a carbonyl group (compound 18) significantly reduced potency toward both TBK1 and IKKε. Interestingly, compound 16 and 17 showed improved permeability compare to 3 in our Caco-2 assay with Papp A–B = 114 nm/s, Papp B–A = 79 nm/s, ER = 0.7 and Papp A–B = 108 nm/s, Papp B–A = 86 nm/s, ER = 0.8 respectively.
Table 2. SAR at the Benzylic Positiona
a IC50 values are reported as arithmetic means of several measurements; n.d.: not determined. b
IC50 reported at the median value of several measurements.
After some preliminary work on the benzimidazole core (data not shown), our further efforts were focused on the substituent at the 6-position, as summarized in Table 3. We had previously established that the introduction of a methyl group at the benzylic position (with (R) configuration) improved the kinase selectivity profile of our compounds (cf. 3 and 17); however, compound 17 still had micromolar off-target activity toward kinases such as CDK9 or RSK4, and also nanomolar activity toward FLT3 and DRAK1 (with IC50 values of 30 and 10 nM, respectively). We hypothesized that a bulkier substituent on the oxadiazole ring might help to improve selectivity. Compound 19, bearing a cyclopropyl group, and compound 20, substituted with an isobutyl group, showed improved potency compared to 3 toward TBK1 in the low ATP assay (IC50 = 58 and 72 nM, respectively); however, both 19 and 20 were inactive in the high ATP assay (see Table 3).
Replacement of the oxadiazole moiety by an N-methylpyrazolyl group (21) resulted in a slight increase in potency in the low ATP assay (IC50 = 55 nM, compared to 129 nM for 3). We next introduced bulkier substituents at a pyrazole nitrogen: cyclopropylmethyl-substituted 23 showed
a 10-fold increased potency toward TBK1 compared to the methyl and ethyl analogues (21/22). Our previous experience that the introduction of a methyl group at the benzylic position increased potency was confirmed with (R)-configured 24 which had a potency of 1 nM (IC50) and 6 nM in the TBK1 low ATP assay and the IKKε assay, respectively, and a very high potency (IC50 = 18 nM) in the TBK1 high ATP assay. Unfortunately, the introduction of a pyrazole had a negative impact on Caco-2 permeability. Compounds 22, 23 and 24 showed lower permeability and higher efflux with Papp A–B = 17 nm/s, Papp B–A = 173 nm/s, ER = 10 for compound 22, Papp A–B = 32 nm/s, Papp B–A = 125 nm/s, ER = 3.9 for 23 and Papp A–B = 16 nm/s, Papp B–A = 118 nm/s, ER = 7.5 for 24.
Table 3. SAR at the 6-position of the benzimidazole core (Targeting the Back Pocket)a
a IC50 values are reported as arithmetic means of several measurements (except stated otherwise); n.d.: not determined. b single measurement. c Kd DiscoverX. d Eurofins IC50 values.
We obtained a co-crystal structure of TBK1 bound to compound 24 (see Supporting Information, Figure S1). As found for 2 (Figure 1), compound 24 adopts an overall similar binding mode within the ATP-binding site, with two hydrogen-bond interactions to residue Cys89 in the hinge region of TBK1. The (R)-configured benzylic methyl group appears to improve the potency by stabilizing the outer piperazine moiety in a conformation which allows both insertion of the trifluoropropionyl amide moiety into a surface indentation above the entrance to the ATP site and formation of a hydrogen bond between a piperazine nitrogen and the backbone carbonyl oxygen of Pro90 (see Supporting Information, Figure S1). This conformation also enables interaction of the propionyl amide with Arg25 and brings one of the fluoro atoms into a beneficial distance to the amide nitrogen of Ile14.
Compound 24 was highly potent in the cellular mechanistic IRF3 phosphorylation assay and showed nanomolar antiproliferative activity in SK-MEL-2 cells but only a micromolar effect on ACHN cells (Table 3). Here as well, introduction of the benzylic substituent enhanced the selectivity profile. However, compared to the unsubstituted benzylic analogue 23 (with lower selectivity), 24 showed a twofold reduction in our SK-MEL-2 antiproliferation assay despite an almost threefold improvement in the cellular mechanistic pIRF3 assay, suggesting that some of
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the antiproliferative effect observed might not be only due to TBK1/IKKε inhibition but rather to inhibition of an as-yet-unidentified off-target kinase.
Compound 24 was further profiled at 100 nM in the DiscoverX KINOMEscan (Figure 3 and Supporting Information Table S2). In this assay, 24 showed an improved selectivity profile compared to 3. Only two other kinases were inhibited more potently than TBK1 (DRAK1 and DRAK2), and two kinases (YSK4 and ULK1) were inhibited as potently as TBK1. Further attempts to improve the selectivity revealed that increasing the steric bulk of the substituent at the pyrazole nitrogen (compound 25) led to a similar profile with improved selectivity toward FLT3 (Table 3).
Figure 3. DiscoverX KINOME trees for compound 3,24 and 34 at 100 nM. The red dots highlight the kinases that show an inhibition of 65 % or more (for more completelist of the kinases see SI Tables S1,S2 and S3).
While compounds 26 and 27, bearing a pyridyl substituent at the 6-position, retained potency toward TBK1, their selectivity was lower, especially regarding CDK9 and FLT3 (Table 3). Relative to 26, pyrimidinyl-substituted 28 displayed a moderate increase in potency in the
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biochemical TBK1 assay (low ATP). This compound, however, showed a different and improved selectivity toward CDK9 (IC50 = 320 nM vs 71 nM for 26). Again, the introduction of an (R)- configured benzylic methyl group improved potency and selectivity: compound 29 showed high potency toward both our targets, TBK1 and IKKε (IC50 = 2 nM, low ATP assay, and 4 nM, respectively), and a potency of 140 nM (IC50) in the mechanistic pIRF3 assay. As this was the first time that selectivity toward ULK1 (50-fold) was observed, we investigated a broader set of pyrimidine derivatives. Introduction of a bulkier substituent on the pyrimidine moiety (compounds 30 and 31) resulted in very high potency in our biochemical assays as well as in the pIRF3 assay, along with some antiproliferative effect on SK-MEL-2 cells. As seen before, however, the potency increase achieved did not translate into an antiproliferative effect on ACHN cells therefore challenging the hypothesis that the antiproliferative effect observed was due to TBK1/IKKε inhibition. Despite the good selectivity profile of these compounds, they were still active toward FLT3 and DRAK1. We observed as well that the change from pyrazole to pyrimidines didn’t improve Caco-2 permeability with for example compound 30 showing low permeability and strong efflux (Papp A–B = 14 nm/s, Papp B–A = 218 nm/s, ER = 15). The introduction of an amino substituent on the pyrimidine moiety (compounds 32 and 33) led to improved selectivity toward ULK1. The dimethylamino substituent (33) seemed to have a strong influence on FLT3 and DRAK1 potencies. Combining this substituent with an (R)-methyl group at the benzylic position led to the identification of BAY-985 (34). Compound 34 showed high potency toward TBK1 and IKKε (IC50 = 2 nM, low ATP assay; 30 nM, high ATP assay), as well as high potency in the mechanistic pIRF3 assay (IC50 = 74 nM), and an antiproliferative effect on SK-MEL-2 cells (IC50 = 900 nM). Biochemically, 34 also displayed good selectivity toward FLT3 (60×), RSK4 (138×), DRAK1 (155×), and ULK1 (~4000×) (Table 3). To confirm the improved selectivity of 34, it was
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further profiled at 100 nM in the DiscoverX KINOMEscan (see Figure 3 and Supporting Information Table S3). Compound 34 showed high selectivity with respect to all kinases tested in the panel, except YSK4. Compound 34 still showed low permeability and strong efflux in the Caco-2 assay (Papp A–B = 9 nm/s, Papp B–A = 146 nm/s, ER = 17).
Further investigations on the pyrimidine family allowed us to also identify compound 35, a close derivative of 34 in which the pyrimidine moiety is substituted with an isopropoxy group (Table 3). This compound showed high potency toward TBK1, but reduced selectivity, highlighting the importance of the dimethylamino substituent for the selectivity. Compound 36 Bearing a Cl on the pyrimidine ring turned out to be a lot less active. We were able to obtain a co-crystal structure of compound 35 with TBK1 (Figure 4). In addition to the double hinge interaction of the aminobenzimidazole moiety to Cys89 of the hinge region, the new pyrimidine moiety enables formation of a hydrogen bond to the conserved lysine residue (Lys38) at the back wall of the ATP site. As observed for compound 24 (Supporting Information, Figure S1), the (R)-configured benzylic methyl group helps to lock the outer piperazine moiety in a conformation which enables insertion of the trifluoropropionyl amide moiety into a surface pocket above the entrance to the ATP site, with hydrogen bonds to the side chain of Arg25, the backbone nitrogen of Ile14, and the carbonyl group of Pro90. Thus, compound 35 successfully combines both SAR insights from the previous optimization steps: the pyrimidine substituent is bulky enough to fully occupy the back pocket and push the Met86 side chain in a similar way as seen with compound 2, which induces selectivity toward kinases with more bulky and rigid gatekeeper residues, such as CDKs; additionally, the pyrimidine is able to form a hydrogen bond to Lys38 in the back pocket (Figure 4). Although compound 35 retains some residual CDK9 activity (IC50 = 824 nM), the TBK1
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affinity was further improved with respect to compound 2 (IC50 = 2 nM vs 93 nM in lw ATP assay).
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Figure 4. Crystal structure of TBK1 in complex with 35 (PDB accession code 6RST). Left, view into the ATP site of TBK1, with the protein shown in surface representation (transparent). Right, view rotated and surface omitted for clarity. mFo-DFc electron density omit map contoured at 2 shown around the ligand. Hydrogen-bond interactions between the inhibitor 35 and TBK1 shown as yellow dotted lines.
Pharmacokinetics. The in vitro metabolic clearance profile [CLb,in vitro(rat hepatocytes) = 1.7 L/h/kg, CLb,in vitro(human liver microsomes) = 0.50 L/h/kg] and CYP450 inhibition potential (CYP1A2, CYP2D6, CYP3A4; IC50 >20 µM) of compound 34 encouraged further in vivo evaluation. To this end, 34 was dosed iv/po at 0.30/0.30 mg/kg to male Wistar rats. Compound 34 demonstrated a high clearance (CLb = 4.0 L/h/kg, ca. 95% hepatic extraction) which was higher than predicted from both rat hepatocytes and rat liver microsomes (CLb,in vitro = 1.7 L/h/kg). The volume of distribution at steady state was large (Vss = 2.9 L/kg) with a short terminal half-life (t1/2 = 0.79 h). Following oral administration, 34 showed low bioavailability (F = 11%), consistent with in vivo blood clearance (Figure 5).
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Figure 5. Overall profile for BAY-985 including pharmacological and physicochemical properties as well as in vitro DMPK properties.
Following administration of a single oral dose of 34 at 100 mg/kg to female NMRI nude mice the exposure was low to moderate, without hints of accumulation after repeated q.d. (once daily) administration. Biochemical IC50,u(TBK1) and cell-based IC50,u(TBK1/IKKε) were covered by plasma exposure for 10 hours and 11 hours, respectively. However, proliferative IC50,u(SK-MEL- 2) was ca. threefold higher than Cmax,u.
In Vivo Antitumor Efficacy Study. The in vivo efficacy and tolerability of 34 in the cell line derived SK-MEL-2 human melanoma xenograft model in female NMRI nude mice was investigated. The maximal tolerated dose of 34 was 200 mg/kg applied twice daily (b.i.d.) continuously. Treatment resulted in weak antitumor efficacy with a T/Crel.area ratio of 0.8 and T/Ctumor weight ratio of 0.6, as determined at the study termination (day 111 after tumor
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inoculation) after 35 treatment days (Figure 5A and 5B). The treatment was well tolerated, with a maximum body weight loss of less than 10% (Figure 5C).
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Figure 5. Antitumor efficacy of 34 as monotherapy in the SK-MEL-2 human melanoma xenograft model in female NMRI nude mice. Treatment schedule: 34 (200 mg/kg) applied twice daily (b.i.d.)
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for 35 days. (A) Tumor area (n = 10 mice/group). (B) Tumor weight at study end (day 111). Tumor areas and weights given as mean ± SD. (C) Body weight change (defined as the percentage change of the actual mean body weight compared to the maximal mean body weight after start of treatment).
Synthesis of 34 (BAY-985). The synthesis of 34 started with a Suzuki coupling between pinacol ester 36 (available in one step from the corresponding bromide) and chloropyrimidine 37 (available in one step from 4,6-dichloropyrimidine) using tetrakis(triphenylphosphine)palladium(0) (Scheme 1). The resulting product 38 was converted into benzimidazole 40 in a two-step procedure: 38 was first reacted with 1,1′-thiocarbonyldiimidazole (TCDI) and amine 39 (available in five steps from commercially available reagents, see the Supporting Information) to provide a thiourea intermediate that was cyclized using N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide (EDCI). Acid deprotection of the Boc group, followed by amide coupling with 3,3,3-trifluoropropanoic acid (using HATU), led to 34.
Scheme 1. Synthesis of 34a
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aReagents and conditions: (a) Na2CO3, Pd(PPh3)4, 1,4-dioxane, 90 °C; (b) 1H-imidazole, TCDI, DCM, 0 °C; (c) EDCI, DCM; (d) HCl, 1,4-dioxane, DCM; (e) 3,3,3-trifluoropropanoic acid, HATU, NaHCO3, DMF.
CONCLUSION
Starting from a HTS campaign, we identified a family of aryl-substituted benzimidazoles as highly potent TBK1/IKKε inhibitors. Early in our investigations, we observed that achieving a very high kinase selectivity will be challenging within this compound class. Using X-ray crystallography, we could confirm the aminobenzimidazole interaction at the hinge as well as identify the hydrogen bond interaction with the protein from the oxygen of the piperazine amide. With the help of computational chemistry, we hypothesized that bulky substituents addressing the back pocket of the protein could improve overall kinase selectivity.
Addressing the back-pocket of the protein with different aromatic substituents turned out to be key in order to improve the selectivity profile of our compounds. Pyrazole-substituted benzimidazoles such as compound 24 displayed an improved overall kinase profile with TBK1 and IKKε being the most potently inhibited targets and with a limited number of off-targets inhibited. Finally, pyrimidine-substituted benzimidazoles appeared to have a superior selectivity profile. This observation resulted in the identification of compound 34 (BAY-985), a highly potent and selective ATP-competitive dual inhibitor of TBK1 and IKKε. Compound 34 was active in our cellular mechanistic assay and showed anti-proliferative activity in a few cancer cell lines. However, we noticed that the antiproliferative activity observed on ACHN cells did not correlate with the potency on our targets and appeared to be weaker for compounds showing a high selectivity. This result highlighted the fact that the antiproliferative activity observed early in our
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investigations might not be a consequence of TBK1/IKKε inhibition but is more likely resulting from off-targets effects.
The pharmacokinetic profile of compound 34 was then investigated and a efflux ratio of 17 fold was observed in the Caco-2 permeability assay.
In vivo, compound 34 showed high clearance (CLb = 4.0 L/h/kg, ca. 95% hepatic extraction), large volume of distribution at steady state (Vss = 2.9 L/kg) and a short terminal half-life (t1/2 = 0.79 h).
Despite the high potency of compound 34, we observed only weak antitumor efficacy in the SK- MEL-2 human melanoma xenograft model, presumably because we didn’t reach sufficient exposure to cover the antiproliferative concentration of SK-MEL-2 cells. It is possible that we haven’t identified the appropriate cell line system characterized by specific oncogenic aberrations or certain biomarkers related to sensitization to TBK1/IKK inhibition. Possibly, the use of combination treatments might be beneficial. Therefore, we have not devalidated TBK1/IKKε as anti-cancer targets but simply have not provided full (in vivo) evidence for it.
Compound 34 was recently accepted by the Structural Genomics Consortium as a dual TBK1/IKKε probe (along with the negative control BAY-440) and is now available to the scientific community via the SGC35–43 as a tool compound to aid the understanding of the pharmacology and potential therapeutic use of TBK1/IKK pathway inhibition in tumors.
EXPERIMENTAL SECTION
Chemistry.
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General methods and materials. Commercially available reagents and anhydrous solvents were used as supplied, without further purification. All air- and moisture-sensitive reactions were carried under an inert atmosphere of argon. Reactions were monitored by TLC and UPLC analysis with a Waters Acquity UPLC MS Single Quad system (see SI for methods details). Flash chromatography was carried out using a Biotage® Isolera™ One system with 200–400 nm variable detector. Preparative HPLC was carried out with a Waters AutoPurification MS Single Quad system; column: Waters XBridge C18 5 µm, 100 × 30 mm; basic conditions: eluent A: H2O + 0.2 vol% aq NH3 (32%), eluent B: MeCN; gradient: 0–0.5 min 5% B, flow: 25 mL/min; 0.51–5.50 min 10–100% B, flow: 70 mL/min; 5.51–6.5 min 100% B, flow: 70 mL/min; acidic conditions: eluent A: H2O + 0.1 vol% formic acid (99%), eluent B: MeCN; gradient: 0–0.5 min 5% B, flow: 25 mL/min; 0.51–5.50 min 10–100% B, flow: 70 mL/min; 5.51–6.5 min 100% B, flow: 70 mL/min; temperature: 25 °C; DAD scan: 210–400 nm. NMR spectra were recorded at ambient temperature (22 ± 1 °C), unless otherwise noted, on Bruker Avance III HD spectrometers. 1H NMR spectra were obtained at 300, 400, 500 or 600 MHz, and referenced to the residual solvent signal (2.50 ppm for [D6]DMSO). 13C NMR spectra were obtained at 125 MHz and also referenced to the residual solvent signal (39.52 ppm for [D6]DMSO). 1H NMR data are reported as follows: chemical shift (δ) in ppm, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet) and integration. Low-resolution mass spectra (electrospray ionization) were obtained via HPLC-MS (ESI) using a Waters Acquity UPLC system equipped with an SQ 3100 Mass Detector; column: Acquity UPLC BEH C18 1.7 µm, 50 × 2.1 mm; eluent A: H2O + 0.05% formic acid (99%), eluent B: MeCN + 0.05% formic acid (99%); gradient: 0–0.5 min 5% B, 0.5– 2.5 min 5–100% B, 2.5–4.5 min 100% B; total run time: 5 min; flow: 0.5 mL/min. Melting points were determined with a Büchi B-540 melting point apparatus. Optical rotations were recorded on
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a JASCO P-2000 polarimeter. The purity of all target compounds was at least 95%, as determined by 1H NMR spectroscopy and UPLC analysis. Compound names were generated using ICS software.
Only the synthesis of compound 34 (BAY-985) is described in this section. For all other compounds please see the supplementary information.
Synthesis of 34 (BAY-985).
6-Chloro-N,N-dimethylpyrimidin-4-amine (38)
4,6-Dichloropyrimidine (50.0 g, 336 mmol), dimethylamine (180 mL, 2.0 M in THF, 369 mmol), and K2CO3 (51.0 g, 369 mmol) were stirred in 1,4-dioxane (400 mL) for 14 h at reflux. Then, the reaction mixture was cooled to rt and diluted with water. The aqueous phase was extracted with DCM. The organic layer was dried over a silicone filter and concentrated under reduced pressure to provide 38 (53.7 g), which was used without further purification. 1H NMR (400 MHz, DMSO- d6): δ = 3.06 (br s, 6H), 6.75 (d, J = 1.01 Hz, 1H), 8.31 (d, J = 1.01 Hz, 1H). LC-MS (method 2, see Supporting Information): tR (min) = 0.76. MS (ESI+): m/z = 158 [M + H]+.
4-[6-(Dimethylamino)pyrimidin-4-yl]benzene-1,2-diamine (39)
4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)benzene-1,2-diamine (37; 20.0 g, 85.4 mmol) and crude chloropyrimidine 38 (16.2 g) were dissolved in 1,4-dioxane (500 mL), and Na2CO3 (130 mL, 2.0 M in water, 260 mmol) and Pd(PPh3)4 (9.87 g, 8.54 mmol) were added. The mixture was stirred for 2 h at 90 °C, then additional Pd(PPh3)4 (2.0 g, 1.73 mmol) was added and stirring continued overnight at 90 °C. The mixture was cooled to rt and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by flash chromatography (hexane/EtOAcEtOAc/EtOH) to give 39. Yield 15.4 g (74% yield). 1H NMR (400 MHz, DMSO-d6): δ = 3.09 (s, 6H), 4.55 (s, 2H), 4.92 (s, 2H), 6.54 (d, J = 8.36 Hz, 1H), 6.79 (d, J = 1.01
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Hz, 1H), 7.24 (dd, J = 8.11, 2.03 Hz, 1H), 7.37 (d, J = 2.03 Hz, 1H), 8.41 (d, J = 1.01 Hz, 1H). LC-MS (method 2, see Supporting Information): tR (min) = 0.68. MS (ESI+): m/z = 230 [M + H]+.
tert-Butyl 4-{(1R)-1-[2-({6-[6-(Dimethylamino)pyrimidin-4-yl]-1H-benzimidazol-2-yl}- amino)pyridin-4-yl]ethyl}piperazine-1-carboxylate (41)
Step 1: 1H-Imidazole (989 mg, 14.5 mmol), TCDI (15.5 g, 87.2 mmol), and tert-butyl 4-[(1R)- 1-(2-aminopyridin-4-yl)ethyl]piperazine-1-carboxylate (40; 24.5 g, 79.9 mmol) were dissolved in DCM (836 mL) and the mixture was stirred at 0 °C for 30 min, then stored in a freezer at –20 °C for 36 h. Benzene-1,2-diamine 39 (16.6 g, 72.6 mmol) dissolved in DCM (100 mL) was added, and the mixture was stirred at rt for 2 h. Then, it was diluted with water and extracted with DCM (3 ×). The combined organic layers were washed with water and brine, dried (MgSO4), and concentrated under reduced pressure.
Step 2: The crude material (44.8 g) was dissolved in DCM (700 mL), and EDCI hydrochloride (14.9 g, 77.6 mmol) was added. The mixture was stirred for 2 d under argon atmosphere, then concentrated under reduced pressure. The crude product was purified by flash chromatography (EtOAc/EtOH) to give 41. Yield 16.25 g (42%). 1H NMR (400 MHz, DMSO-d6): δ = 1.29 (d, J = 6.59 Hz, 3H), 1.38 (s, 9H), 2.25–2.31 (m, 2H), 2.36–2.43 (m, 2H), 3.15 (s, 6H), 3.27–3.32 (m, 4H), 3.40–3.48 (m, 1H), 6.95 (br s, 1H), 7.03–7.13 (m, 1H), 7.17 (s, 1H), 7.36–7.55 (m, 1H), 7.84– 7.93 (m, 1H), 8.14–8.33 (m, 2H), 8.52 (d, J = 1.01 Hz, 1H), 10.68 (br s, 1H), 12.09–12.33 (br m, 1H). LC-MS (method 2, see Supporting Information): tR (min) = 1.24. MS (ESI+): m/z = 544 [M + H]+.
6-[6-(Dimethylamino)pyrimidin-4-yl]-N-{4-[(1R)-1-(piperazin-1-yl)ethyl]pyridin-2-yl}- 1H-benzimidazol-2-amine Hydrochloride (42)
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Boc-Protected benzimidazole 41 (21.5 g, 39.6 mmol) was dissolved in DCM (300 mL) and MeOH (150 mL), and HCl (149 mL, 4.0 M in 1,4-dioxane, 594 mmol) was added. The mixture was stirred for 4 h at rt, then concentrated to give hydrochloride 42 (25.3 g), which was used without further purification. LC-MS (method 2, see Supporting Information): tR (min) = 0.92. MS (ESI+): m/z = 444 [M + H]+.
1-(4-{(1R)-1-[2-({6-[6-(Dimethylamino)pyrimidin-4-yl]-1H-benzimidazol-2-yl}amino)- pyridin-4-yl]ethyl}piperazin-1-yl)-3,3,3-trifluoropropan-1-one (34, BAY-985)
Hydrochloride 42 (19.3 g, crude material) was dissolved in DMF (310 mL), and 3,3,3- trifluoropropanoic acid (4.6 mL, 52 mmol), NaHCO3 (17.6 g, 209 mmol), and HATU (19.9 g, 52.4 mmol) were added. The mixture was stirred for 2 h at rt, then filtered. The filtrate was poured into half-saturated NaHCO3 solution. The suspension was filtered, and the solids were washed with water and dried under reduced pressure at 60 °C to give 34. Yield 11.89 g (100% purity by UPLC- MS). 1H NMR (400 MHz, DMSO-d6): δ = 1.30 (d, J = 6.59 Hz, 3H), 2.32–2.47 (m, 4H), 3.15 (s, 6H), 3.41–3.52 (m, 5H), 3.56–3.67 (m, 2H), 6.95 (br d, J = 4.82 Hz, 1H), 7.03–7.13 (m, 1H), 7.18 (s, 1H), 7.37–7.59 (m, 1H), 7.85–7.93 (m, 1H), 8.15 (br s, 0.5H), 8.27 (d, J = 5.32 Hz, 1H), 8.31 (br s, 0.5H), 8.52 (d, J = 1.01 Hz, 1H), 10.70 (br d, J = 12.93 Hz, 1H), 12.22 (br d, J = 17.24 Hz, 1H). 13C NMR (151 MHz, DMSO-d6): δ = 162.5, 161.8, 157.6, 154.4, 153.7, 147.0, 132.9, 129.0, 125.1, 115.5, 115.0, 109.7, 96.6, 62.6, 49.9, 43.4, 36.9, 36.7, 18.1. LC-MS (method 2, see Supporting Information): tR (min) = 1.06. MS (ESI+): m/z = 554 [M + H]+. HRMS (ESI+): m/z = 554.2607 [M + H] (calc. m/z : 553.2525 [M])
ASSOCIATED CONTENT
Supporting Information.
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The Supporting Information is available free of charge on the ACS Publications website
Detailed descriptions of methods for the HTS; all biochemical and cellular assays, pharmacokinetics assays; the synthesis of compounds 1 to 36; crystallization and structure determination; tables containing IC50 values with standard deviation; kinase selectivity results; crystallographic data collection and refinement statistics; additional crystallography results figure (PDF); Molecular formula strings are also available (CSV).
ACCESSION CODES
The coordinates and structure factors for the described crystal structures have been deposited with the Protein Data Bank (PDB). The PDB accession codes are 6RSR (compound 2), 6RST (compound 24), and 6RSU (compound 35).
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Phone: (+49)30468-194612.
ORCID
Julien Lefranc: 0000-0002-2116-9425
Volker Klaus Schulze: 0000-0002-0158-1789 Roman Christian Hillig: 0000-0001-6267-7250
Jozsef Balint: 0000-0002-9726-0401 Srinivasan Rengachari: 0000-0003-4237-8258 Carl Friedrich Nising: 0000-0002-9931-7096
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Franz von Nussbaum: 0000-0001-7188-7364 Daniel Panne: 0000-0001-9158-5507 Present Address
†Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, Göttingen, Germany
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Notes
The authors declare the following competing financial interest(s): J.L., V.K.S., R.C.H., H.B., F.P., A.M., T.H., H.I., D.S., U.B., B.B., S.N.G., C.F.N., F.v.N., D.M., and A.M.W. are or have been employees and stockholders of Bayer AG.
ACKNOWLEDGMENT
We would like to thank S. Böttcher, L. Ehresmann, J. Fischer, M. Keifler, A. Klinner, C. Kröber, S. Osterberg, V. Raschke, S. Schulze, P. Stollberg, G. Piechowiak, F. Probst, and D. Wolleh for technical support, the staff at beamline ID30A-1 at the European Synchrotron Radiation Facility (ESRF) in Grenoble for access to synchrotron radiation and support during data collection, and DiscoverX and Eurofins for performing the kinase selectivity studies. We would like to thank K. Greenfield for support with writing the manuscript as well as J. Fischer and K. Deja for their
help with compiling the experimental part.
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ABBREVIATIONS
TBK1: TANK-binding kinase 1; IKKε: Inhibitor of nuclear factor kappa-B kinase subunit epsilon; CDK: cyclin-dependent kinase; CK1γ: Caseine kinase 1; JNK3: c-Jun N-terminals kinase; ER: efflux ratio (in Caco-2 assay); SAR: structure activity relationship; RSK4: ribosomal S6 kinase 4; DRAK1: death-associated protein kinase-related apoptosis-inducing kinase 1; YSK4: yeast Sps1/Ste20-related kinase 4 (also called MAP3K19); FLT3: fms like tyrosine kinase 3; CYP: cytochrome P450; CLb: blood clearance; HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; Boc : tert-butoxy carbonyl.
REFERENCES
(1)Fitzgerald, K. A.; McWhirter, S. M.; Faia, K. L.; Rowe, D. C.; Latz, E.; Golenbock, D. T.; Coyle, A. J.; Liao, S.-M.; Maniatis, T. IKKε and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 2003, 4, 491–496.
(2)Sharma, S.; tenOever, B. R.; Grandvaux, N.; Zhou, G.-P.; Lin, R.; Hiscott, J. Triggering the interferon antiviral response through an IKK-related pathway. Science 2003, 300, 1148–1151.
(3)Newman, A. C.; Scholefield, C. L.; Kemp, A. J.; Newman, M.; McIver, E. G.; Kamal, A.; Wilkinson, S. TBK1 kinase addiction in lung cancer cells is mediated via autophagy of Tax1bp1/Ndp52 and non-canonical NF-κB signalling. PLoS One 2012, 7, e50672.
(4)Pilli, M.; Arko-Mensah, J.; Ponpuak, M.; Roberts, E.; Master, S.; Mandell, M. A.; Dupont, N.; Ornatowski, W.; Jiang, S.; Bradfute, S. B.; Bruun, J. A.; Hansen, T. E.; Johansen, T.; Deretic,
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GRAPHICAL ABSTRACT
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