Title A-loop interactions in Mer give rise to inhibitors with two-step mechanism and long residence time of binding.

Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 Author names and affiliations Alexander Pflug1, Marianne Schimpl1, J. Willem M. Nissink2, Ross C. Overman3, Philip B. Rawlins1, Caroline Truman4, Elizabeth Underwood3, Juli Warwicker3, Jon Winter-Holt2, and William McCoull2 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020

Correspondence: Alexander Pflug ([email protected])

1Structure, Biophysics & Fragment-Based Lead Generation, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK

2Oncology R&D, AstraZeneca, Cambridge, UK

3Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK

4Mechanistic Biology & Profiling, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK

Abstract The activation loop (A-loop) plays a key role in regulating the catalytic activity of kinases. Phosphorylation in this region enhances the phosphoryl transfer rate of the kinase domain and increases its affinity for ATP. Furthermore, the A-loop possesses autoinhibitory functions in some kinases, where it collapses onto the protein surface and blocks substrate binding when unphosphorylated. Due to its flexible nature, the A-loop is usually disordered and untraceable in Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 kinase domain crystal structures. The resulting lack of structural information is regrettable as it impedes the design of drug A-loop contacts, which have proven favourable in multiple cases. Here we characterize the binding with A-loop engagement between type 1.5 kinase inhibitor ‘example 172’ (EX172) and Mer tyrosine kinase (MerTK). With the help of crystal structures and binding Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 kinetics we portray how the recruitment of the A-loop elicits a two-step binding mechanism which results in a drug-target complex characterized by high affinity and long residence time. In addition, the type 1.5 compound possesses excellent kinome selectivity and a remarkable preference for the phosphorylated over the dephosphorylated form of MerTK. We discuss these unique characteristics in the context of known type 1 and type 2 inhibitors and highlight opportunities for future kinase inhibitor design.

Keywords activation loop, activation segment, MerTK, TAM

Introduction MerTK is a member of the TAM family (Tyro3, Axl and MerTK) of receptor tyrosine kinases (RTKs), which act as sensors for apoptotic phosphatidylserine. Once activated, TAM receptors dimerize in the membrane, which enables autophosphorylation of the intracellular kinase domains and triggers downstream signalling events. TAM RTKs are ubiquitously expressed but most abundant in the nervous, vascular, reproductive and immune systems [1]. In line with the broad distribution of the receptors, TAM signalling has pleiotropic functions, with dampening inflammation being the most prominent role [2]. Accordingly, mutant mice lacking the TAM receptors develop autoimmune diseases [3]. Notably, TAM RTKs also exert their immune-inhibitory function in the tumour microenvironment, where they prevent chronic activation of antigen-presenting cells (APCs) and boost apoptotic cell clearance in macrophages; a tumour-promoting process called efferocytosis [2]. On the tumour side, MerTK activity has been directly linked to stimulation of growth and metastasis [4]. TAM RTKs have therefore attracted considerable attention as targets in the immuno-oncology space [5,6].

MerTK crystal structures published so far (as of 09-2020, PDB entries: 2p0c, 3bpr, 3brb, 3tcp, 4m3q, 4mh7, 4mha, 5k0k, 5k0x, 5tc0, 5td2, 5u6c and 6mep) capture the kinase domain in an inactive form with ‘helix-C out’ conformation [for kinase nomenclature consult 7]. In contrast to the active state, helix-C appears rotated and moved away from the DFG motif, thereby breaking the canonical salt bridge between catalytic Lys619 and conserved Glu637. The translocation of helix-C creates a pocket adjacent to the ATP , referred to as ‘allosteric back-pocket’ [8], which can be occupied by type 1.5, type 2 and type 3 inhibitors. Binding to the back pocket allows for contacts between small molecule inhibitor and the kinase activation loop (A-loop). Such interactions have been described for several kinases, including c-Met, MEK1, RIPK1 and RIPK2 [reviewed by 9], and can contribute to both binding potency and kinome selectivity. Experimental Protein Expression & Purification Variations of human MerTK kinase constructs were co-expressed with protein tyrosine phosphatase PTPN1; in accordance with literature precedent [10].

For biochemical activity measurements, the full intracellular domain (R528-M999) with an N-

Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 terminal GST-TEV-6His tag and C-terminal Avi- and FLAG-tags was expressed in S. frugiperda. Protein was purified from cell lysate via immobilized metal affinity chromatography (IMAC) followed by proteolytic cleavage of the GST tag, additional de-phosphorylation with lambda phosphatase and final purification with anion exchange chromatography. Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 For use in SPR measurements, the above protocol was modified by additional co-expression of BirA and supplementation of the growth medium with 60 µM biotin, to afford biotinylation of the Avi-tag. Activated MerTK protein was prepared by in-vitro autophosphorylation. The final buffer contained 20 mM Tris/HCl pH 8.0, 270 mM NaCl, 5% glycerol, 1 mM TCEP. Biotinylation and phosphorylation of all protein batches was verified by LC-MS.

For crystallisation, N-terminally His6-tagged constructs of the kinase domain only (E571-V864) were expressed in E. coli; and purified by IMAC followed by proteolytic cleavage of the affinity tag and size-exclusion chromatography. Protein was concentrated to approximately 10 mg/ml in a final buffer of 20 mM Tris/HCl pH 8.0, 500 mM NaCl, 5% glycerol, 1 mM TCEP.

Surface Plasmon Resonance Dephosphorylated or autophosphorylated biotinylated MerTK tyrosine kinase (R528-M999) were used for surface plasmon resonance (SPR) experiments. A series S streptavidin Biacore chip (#BR100531, Cytiva) was docked onto either a T200 or 8K Biacore instrument (Cytiva). Buffer

consisting of 20 mM Tris (pH 7.7), 150 mM NaCl, 10 mM MgCl2, 0.5 mM TCEP (tris(2- carboxyethyl)phosphine), 0.005% (v/v) Tween-20 (polyoxyethylene(20)sorbitan monolaurate), and 1% (v/v) DMSO (dimethyl sulfoxide) was used for immobilisation and binding experiments. Prior to running each experiment, a desorb method was run and the instrument was primed three times with buffer. All immobilization and binding experiments were equilibrated to 25 °C. MerTK protein was immobilized at a density of between 2000 and 7000 RU. Unbound streptavidin sites were blocked with 50 μM amino-PEG biotin (#21346, ThermoFisher). Compounds were prepared in assay buffer in a 384-well polypropylene microplate (#781201, Greiner) and care was taken to ensure a final concentration of 1% (v/v) DMSO. The ‘Single Cycle Kinetic’ method was used to generate binding sensorgams and a 50% DMSO wash was used between samples. All data was double referenced and solvent (DMSO) corrected. Data was fitted to either a 1:1 or two-state binding model using the Biacore Insight software (Cytiva). Binding parameters were extracted from the Insight analysis software with the exception of the dissociation rate constant for the two-state model which

was defined as koff = k2k4/(k2+k3+k4)).

MerTK Biochemical activity The ability of inhibitors to cause inhibition of MerTK (6His_R528-M999_Thrombin-Avi-Flag, dephosphorylated) in vitro was assessed using Rapidfire LCMS method that quantified Axltide (CKKSRGDYMTMQIG-acid, Cambridge Research Biochemicals) peptide phosphorylation levels. MerTK (1 nM final) in assay buffer (20 mM HEPES pH 7.5, 0.006% Brij-35, 0.5 mM TCEP, 10 mM, 10 mM

Mg(OAc)2, 0.02% Pluoronic-F127) was added to compound plates using a liquid dispenser; Multidrop Combi. Assay plates were equilibrated for 30 min before initiating the reaction by addition of Axltide

(10 μM final) and ATP (at Kmapp; 80 μM final) substrates in assay buffer. The reaction was allowed to progress for 120 min before being stopped with 0.1% formic acid in water. The Axltide peptide substrate and the phosphorylated Axltide phosphopeptide product levels were determined by mass spectrometry (Rapidfire RF360 and Agilent triple quad mass spectrometer system) using aqueous phase; 0.1% formic acid in water and organic phase; 0.1% formic acid in 90% methanol on type E (C8 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 rapidfire cartridge) to elute.

Ratios were plotted to generate concentration response profiles and the dose-response curves were fit to the data using the non-linear regression analysis; 4 parameter logistic smart fit method in the ®

Assay analyser and Condoseo applications of the Genedata Screener software (Genedata, Inc., Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 Basel, Switzerland).

Protein Crystallography and structural biology MerTK protein with surface entropy reduction mutations, MerTK (GSHM_E571-V864, K591R/K693R/K702R/K856R), was crystallized by vapour diffusion at 20 °C in a condition of 4.3 M NaCl, 0.1 M Tris pH 8.5. Reproducibility of crystallisation was substantially improved by micro- seeding. For soaking procedures, crystals were transferred into 1.9 M NaCl, 0.1 M Tris pH 8.5, 20% DMSO, containing 10-20 mM of compound. Soaking was carried out over 1-24 h; the crystals subsequently flash frozen in the soaking solution. The apo crystal was cryo-protected with 1.9 M NaCl, 0.1 M Tris pH 8.5, 30% ethylene glycol, instead.

LDC1267, merestinib and EX172 were co-crystallized with MerTK kinase domain. In each case 1 mM compound was added to the protein from 100 mM DMSO stocks. The complexes were briefly incubated on ice, then subjected to sparse matrix crystallization screens at 20 °C. LDC1267 was co- crystallized with MerTK (GSHM_E571-V864, K591R/K693R/K702R/K856R) in 4.4 M NaCl, 0.05 M Tris pH 7.5 and the crystals flash-frozen in 2.5 M NaCl, 0.05 M Tris pH 8.5, 20% glycerol. Crystals of merestinib-MerTK complex (GSHM_E571-V864, ΔM659-Q662) grew in 23% PEG 6000, 0.75 M LiCl, 0.1 M MES pH 6.2 and were cryo-cooled in reservoir solution supplemented with 20% butane-2,3- diole. MerTK (GS_E571-V864) in complex with EX172 crystallized in 0.1 M HEPES pH 6.6, 3.2M NaCl. The crystals were cryo-cooled in reservoir solution supplemented with 25% ethylene glycol.

Diffraction data were integrated with autoPROC/STARANISO [11–14] and structures solved by molecular replacement with AMoRe or Phaser from CCP4 [15–17]. coordinates plus restraints were generated with GRADE [18], structure coordinates modelled in COOT [19] and refinement carried out in BUSTER [18] and Refmac5 [20]. Structure figures were rendered with PyMOL.

Accession numbers Co-ordinates and structure factors generated during this study are available at (PDB) under following accession numbers: 7AB1, 7AB2, 7AAX, 7AAY, 7AAZ and 7AB0 (see Supplementary Table 1 for details).

Results An improved MerTK crystal system In the course of this work we have optimized the crystallization of the MerTK kinase domain by introducing surface entropy reduction mutations, substituting four lysines with arginines [21]. The modified protein forms the same lattice as in the P21 literature precedent structures [10], however with a centred orthorhombic unit cell (Supplementary Table 1). The novel C2221 crystal form allows Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 for crystallization of apo-protein and subsequent compound soaking at high DMSO concentrations. Crystals generally result in well-resolved (<2 Å) structures, which contain one protein molecule per asymmetric unit.

Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020

Type 1 inhibitors Gilteritinib is a dual Flt3/Axl inhibitor used for the treatment of acute myeloid leukaemia (AML) (Figure 1). The compound was reported to possess double-digit nanomolar off-target activity against MerTK [22], which confirmed in our biochemical and biophysical measurements (Table 1). UNC2025, also known as MRX-6313, was developed for the same indication, however, targets Flt3 and MerTK rather than Axl [23] (Figure 1). The structures of MerTK kinase domain complexed with gilteritinib and UNC2025 depict canonical type 1 binding: the molecules pack against the kinase hinge and gatekeeper Leu671, but do not enter the kinase back pocket or interfere with the conformation of the DFG motif (Figure 1). Each of the inhibitors place bulky, saturated rings in the ribose pocket and possess methylpiperazine solvent tails, which protrude out of the ATP pocket along the kinase hinge. Since their binding does not entail rearrangements of the protein, type 1 inhibitors usually have faster kinetics than types 1.5 and 2. In good agreement with this trend gilteritinib demonstrated residence times below one minute in our SPR measurements (Table 1). The biophysical characterization furthermore discovered a preference of gilteritinib for the active/phosphorylated form of MerTK, to which it binds with increased potency and extended residence time over the non- phosphorylated form (Table 1).

Type 2 inhibitors Merestinib is an inhibitor of c-Met and several other receptor tyrosine oncokinases, including MerTK [24], while LDC1267 was developed as a selective pan-TAM kinase inhibitor [25] (Table 1).The compounds occupy the ATP pocket and cross the DFG motif to displace the sidechain of Phe742; a typical type 2 binding mode with ‘DFG-out’ conformation [7] (Figure 2). Despite the chemical similarity between merestinib and LDC1267, with a common F-aryl amide core, the MerTK kinase domain assumes surprisingly different conformations in the related crystal structures. While the LDC1267-complex formed the same crystals as the unliganded protein (structure 7AB0, Supplementary Table 1), merestinib co-crystallized with MerTK in ‘helix-C in’ conformation. Helix-C moved about 3.4 Å toward the ATP binding site when measured on the backbone carbon of Ser627. The new position enables the formation of the canonical salt bridge between Lys619 and conserved Glu637 on helix-C (Figure 2); one of the requisite interactions leading to a catalytically active kinase conformation. Along with this relocation goes a 50° rotation of the helical N-terminus (helix-A), which packs alongside helix-C now (Figure 2). This is the first report of a structure depicting MerTK in this active-like conformation. However, it is unclear whether the rearrangement of helices A and C happened as a result of merestinib binding, protein engineering or a combination of both. The protein construct co-crystallized with merestinib features a loop-truncation within the N-lobe (Supplementary Table 1), which could have contributed to the movement observed in the structure. The truncation melts the β-sheet formed by strands four and five and thereby makes room for the repositioning of helix-C (Figure 2). Type 2 binding describes compounds that displace the DFG-motif phenylalanine (Phe742 in case of MerTK) from its native pocket to replace it with another hydrophobic group (Figure 2 and Figure 1). This design feature often leads to elevated compound hydrophobicity which, combined with the induced-fit binding mode, usually makes for slower kinetics than seen with type 1 compounds. In line with this general observation, merestinib remains Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 bound to non-phosphorylated MerTK with a residence time of over 120 minutes and LDC1267 about 13 minutes, significantly longer than type 1 compound gilteritinib (Table 1). Despite inducing the ‘DFG-out’ conformation upon binding, the type 2 molecules demonstrate single step kinetics in SPR experiments (Supplementary Figure 2). However, in contrast to gilteritinib, LDC1267 and merestinib exhibit a drop in potency and shortened residence times for the active/phosphorylated form of Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 MerTK compared to the non-phosphorylated form (Table 1).

EX172 – overall binding Example 172 (EX172) is a is a dual MerTK/Axl inhibitor described in patent application US20170066742 [26] (Table 1). The compound was selected from the 458 molecules exemplified in US20170066742 using ‘Frequency of Group’ (FOG) analysis [27]; based on the abundance of chemical moieties, the potency data provided, and predicted physical properties (LogD, solubility).

Structure 7AAZ depicts how EX172 spans from the hinge region in the ATP-pocket to helix-C in the kinase back-pocket, while maintaining a ‘DFG-in’ conformation (Figure 3). This binding mode renders EX172 a type 1.5 kinase inhibitor. The compound fills the space otherwise occupied by Phe742 when in ‘DFG out’ with its cyclopentyl group and further rigidifies the DFG by placing its biphenyl moiety adjacent to it (Figure 3). EX172 features a terminal benzylpiperazine which contacts helix-C; packing against Phe634 and making H-bonds with conserved Glu637 (Figure 2); and interacts closely with the A-loop; packing against Tyr753 and making water-mediated H-bonds with Tyr754 and Gly743 (Figure 3). EX172 could not be soaked into preformed crystals. Instead it co-crystallized with MerTK in a unique conformation where the activation segment (A-loop) and the loop linking β-strand 3 and helix-C (helix-C loop) are stabilized by the compound (residues 744-767 and 621-630, respectively; Figure 3). The A-loop is recruited into forming a binding pocket around EX172 and itself acts as a scaffold for the helix-C loop. Neither of the two loops are usually resolved in MerTK structures due to flexibility and their conformation in EX172-complex 7AAZ is likely stabilised by interactions with the ligand. The terminal benzylpiperazine of EX172 appears crucial for reshaping the protein this way (Figure 3). A detailed analysis of A-loop flexibility in MerTK crystal structures can be found in Supplementary Figure 3.

EX172 binds MerTK with a two-step mechanism Characterization of the interaction between EX172 and the MerTK kinase domain by SPR results in biphasic sensorgrams composed of a rapid initial binding step, followed by a slower second process (Supplementary Figure 2). The observation that complex formation between MerTK and EX172 takes place in sequential events is in good agreement with the induced-fit conformation of MerTK seen in EXP172-complex structure 7AAZ (Figure 3). With the exception of 7AAZ and merestinib-complex 7AAY, all other MerTK structures published appear largely invariant and depict the same overall conformation of the kinase domain, including position of helix-C (Supplementary Figure 3). In contrast to merestinib-complex 7AAY, which shows an inward movement of helix-C (Figure 2), EX172-complex 7AAZ depicts the helix sitting 1.9 Å further out when compared to the ‘default’ MerTK conformation (measured on the backbone carbon of Ile631 on aligned structures 7AAZ and 7AB0). This movement makes way for the bulky phenylpiperazine of EX172 (Figure 3), which clashes with apo-MerTK structure 7AB0 when superimposed. However, apo-MerTK can fit EX172 without structural changes when the benzylpiperazine is slightly rotated outward. Under the assumption that the ‘default’ MerTK conformation seen in more than ten crystal structures is the ground state, we propose a model where the placement of EX172 in the ATP- and back-pocket of MerTK is the fast, Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 initial binding event seen by SPR and the rearrangement of A-loop, helix-C loop and helix-C is the second. Induced-fit binding mechanisms have been reported to augment drug-target residence time in cases where the final complex represents a low-energy state [28]. That such effects can be used for protein kinases has recently been demonstrated in a case study on FAK and PYK2 [29]. The work describes how slow complex dissociation going along with a binding-induced conformational Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 rearrangement of the DFG motif enable to design target selectivity into ATP-competitive inhibitors. It is likely that the conformational rearrangement of the MerTK kinase domain imposes structural barriers which limit compound dissociation and contribute to the comparatively long residence times determined for EX172 (Table 1 and Supplementary Figure 2).

EX172 binds to MerTK with phospho-selectivity The engagement of the A-loop adds a second particular aspect to the binding between EX172 and MerTK, in addition to the two-step mechanism discussed above. Two of the A-loop residues which shape a pocket around the compound, Tyr753 and Tyr754 (Figure 3), have been reported as activity- modulating autophosphorylation sites [30]. Interestingly, phosphorylation of these residues does not impede binding of EX172, but instead increases the compounds binding affinity about 16-fold (Table 1). Since structural characterization of phosphorylated MerTK protein failed, phosphorylation of Tyr753 and Tyr754 was simulated in a model (Figure 3). In brief: with the help of CCG MOE phosphate groups were added and the water molecule adjacent to Tyr754 removed; the structure was then minimized in an Amber force-field (resulting coordinates available in the supplemental material). The modifications were accommodated in the model without significant structural rearrangements when compared with the input coordinates. Tyr753 places its phosphate at the centre of the helix-C loop (Figure 3) without forming H-bonds, while phospho-Tyr754 engages into numerous polar contacts with the piperazine of EX172, an adjacent water molecule and the sidechain of Arg758 (Figure 3). The additional polar contacts rationalize the gain in potency of EX172 upon the phosphorylation of Mer. Hence, the structural model gives some confidence that the induced-fit conformation of non-phosphorylated MerTK seen in EX172-complex 7AAZ is also relevant in the context of phosphorylation on tyrosines 753 and 754 (Figure 3).

Kinome selectivity of EX172 EX172 was screened biochemically against a large set of human kinases to establish selectivity against the human kinome (ThermoFisher SelectScreen Kinase Profiling Services) (Figure 4, Supplementary Table 1). At a concentration of 0.1 μM the compound inhibited only three protein kinases to more than 35%; MerTK (99.5% inhibition), Axl (97.0%) and Chk2 (55.2%). Follow up dose

response assays gave IC50 (nM) values at 0.87 (Mer), 4.3 (Axl) and 102 (Chk2) showing that EX172 was 117-fold selective for MerTK and 24-fold selective for Axl compared to the next most inhibited kinase Chk2. Noteworthy, the third TAM family member Tyro3 was inhibited to 31.4% by 0.1 μM EX172, with a concentration response assay resulting an IC50 of 532 nM. This result renders EX172 a dual MerTK/Axl inhibitor with a particularly clean kinome profile. The similar potency of EX172 for MerTK and Axl is in good agreement with the importance of A-loop interactions discussed above. The kinase domains of MerTK (E571-V864) and Axl (E520-A813) share 69% sequence identity overall and differ only in one sidechain on the A-loop; S750 in MerTK which corresponds to N699 in Axl (Supplementary Figure 1).

Discussion Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 A-loop interactions remain a curiosity among kinase inhibitors but have been reported in some cases and the potential for target selectivity discussed [9]. Here we demonstrate that stabilisation of A- loop interactions may give rise to both exquisite selectivity, as well as a specific kinetic profile.

Furthermore, it could be envisioned that specific compounds enable selectivity for a particular Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 phosphorylation state of the target, when interacting favourably with a phosphorylated residue. The lock-in binding mechanism seen between the A-loop of MerTK and EX172 results in residence times which compare well with the type 2 molecules tested. With slow enough dissociation times, the lifetime of the drug-target complex might surpass the compound’s pharmacokinetic lifetime in circulation and positively affect pharmacodynamics, along the lines seen for covalent binders [31].

A-loop interactions benefit kinome-selectivity The A-loop is part of the kinase domain peptide-binding cleft, a region of lower sequence conservation, adjacent to the ATP-binding site [32]. Engaging into contacts with the loop therefore represents an opportunity to augment the target selectivity of ATP-pocket binders, when desired. The feasibility of this approach has been demonstrated with Necrostatins directed against RIPK1 [33]. This family of type 3 inhibitors binds in the kinase back pocket, stabilizing an inactive ‘helix-C out’ and ‘DLG-out’ conformation (RIPK1 belongs to a subset of kinases where the phenylalanine of the DFG motif is substituted by a leucine). Much like compound EX172 characterized here, Necrostatin analogue Nec-1 recruits the A-loop into forming a binding pocket and forms an H-bond with one of the A-loop phospho-acceptor residues, Ser161 [34]. The contact is productive as Nec-1 shows reduced binding to Ser161Ala substituted protein [34]. Nec-1 displayed exclusive selectivity towards RIPK1 in a biochemical screen of over 400 human kinases [35], a similar degree of selectivity as EX172 has demonstrated here (Figure 4). It is reasonable to assume that this selectivity is partially attributed to the sequence-specific interaction between the compounds and the kinase A-loop.

A-loop interactions enable phospho-selectivity A key observation is that EX172 shows selectivity for the active/phosphorylated state over the unphosphorylated form of the protein (Table 1). The other examples of A-loop interacting compounds discussed here show the opposite effect. Type 2 compounds LDC1267 and merestinib bind the active MerTK kinase domain with reduced affinity and faster kinetics (Table 1), and affinity of Nec-1 for RIPK1 drops about 10 fold upon phosphorylation of Ser161, mimicked by a glutamate residue [34]. In contrast to aforementioned inhibitors, the binding mode of EX172 can accommodate phosphorylation of the MerTK A-loop and benefits from additional polar contacts, resulting in higher potency than the other compounds (Figure 3, Table 1). This increased binding affinity of EX172 for

active MerTK measured by SPR agrees well with the IC50 determined in a kinase activity assay (Table 1). The data on EX172 demonstrates that it is feasible to develop selective kinase inhibitors, tuned to favour a defined phosphorylation state of a kinase. This opens opportunities to modulate the drug phenotype, or simply to avoid large in-vitro to in-vivo potency drops by addressing the most biologically relevant form.

The first structure of MerTK in ‘helix-C in’ conformation It is not clear whether the ‘helix-C out’ conformation is a common feature all kinases share. However, structural characterisation of the kinome suggests that kinases have varying propensities to adopt the conformation (Figure 2). The back-pocket ligand binding space made available by ‘helix-

Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 C out’ can therefore be utilized to design target selectivity into kinase inhibitors. In case of MerTK, it appears that the kinase domain easily assumes or even favours the ‘helix-C out’ conformation. Regardless, with merestinib-complex 7AAY we present a first structure depicting MerTK in an active- like form with ‘helix-C in’. Where desired, the structure can be utilized to design type 2 inhibitors for

MerTK which are agnostic to the conformational state of helix-C. Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020

Competing Interests All authors are currently or have been (R.C.O and J.W.) employees and shareholders of AstraZeneca at the time of the study.

Funding The study was funded solely by AstraZeneca.

Acknowledgements We thank Dr Jason Breed for crystallographic data collection, and the beamline scientists at DLS I02, I03 and I04 for technical assistance.

Abbreviations A-loop, activation loop / activation segment; EX172, compound example 172; IMAC, immobilized metal affinity chromatography; helix-C loop, loop linking β-strand 3 and helix-C; MerTK, Mer tyrosine kinase; RTK, ; SEM, standard error of the mean

Figure 1. Comparison of unliganded MerTK with type 1 complexes. (A) Superposition of apo-MerTK structure (PDB entry 7AB0, yellow), and complexes with gilteritinib (cyan, PDB entry 7AB1) and UNC2025 (red, PDB entry 7AB2). The compounds do not affect the overall conformation of the MerTK kinase domain or the position of the DFG motif. (B and C) Gilteritinib and UNC2025 bind to the kinase hinge without entering the back pocket. H-bonds are represented as yellow dashes with distances in Å indicated.

Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 Figure 2. MerTK complexes with type 2 kinase inhibitors. LDC1267-complex 7AAX is rendered in magenta, merestinib-complex 7AAY in purple. Alignment of the whole structures, using the CE algorithm (‘cealign’ in PyMOL). (A) Top and side view on the kinase domains. Alpha helices A and C (αA, αC) and beta strands 1-5 (β1- β5) of the kinase N-lobe are labelled. The merestinib structure depicts MerTK in ‘helix-C in’ conformation. Helix-C is repositioned 3.4 Å inward the ATP-pocket (measured on atom CA of Ser627 at the extremity of the helix), thereby enabling formation of the canonical salt bridge between Lys619 and Glu637 (yellow dashes, distance in Å Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 indicated). (B) Typical for type 2 compounds, both merestinib and LDC1267 displace Phe742 of the DFG-motif.

Figure 3. MerTK in complex with EX172 (PDB entry 7AAZ). The kinase domain N-lobe is rendered in white, the C-lobe in grey and the A-loop in orange. (A) Structure overview. EX172 packs its methylpiperazine against helix-C and stabilizes the A-loop. (B) The binding pocket in detail. Residues within 5 Å of the ligand are rendered as wires and the cavity they define as surface. H-bonds are depicted as yellow dashes with distances in Å indicated. (C) Model of the structure with A-loop tyrosine 754 phosphorylated. A phosphate would replace a water molecule which is found between tyrosine hydroxyl and EX172 piperazine in the original structure 7AAZ. A phosphate would directly contact the compound, an adjacent water and Arg758.

Figure 4. EX172 is a selective MerTK/Axl inhibitor. The compound was tested at a concentration of 0.1 μM in a biochemical screen against a panel of 395 human kinases (ThermoFisher SelectScreen Kinase Profiling Services). (A) Inhibition of all kinases tested. (B) Kinases with >35% inhibition in detail. The raw data is listed in Supplementary Table 2.

Supplementary Figure 1. MerTK and Axl activation loops are conserved in their sequences. Sequence alignment of Axl and MerTK kinase domains, generated with UniProtKB sequences P30530 and Q12866, using the program ALINE [36]. The secondary structure of apo-MerTK PDB entry 7AB0 is superimposed on top of the sequences. Secondary structure elements are labelled according to Fabbro et al. [8]; with α-helices depicted as tubes, β-strands as arrows, 310 helices as spirals and disordered regions as dashed lines. The position of DFG-motif, APE-motif and A-loop are indicated, variant residues are highlighted in red.

Supplementary Figure 2. Binding analysis for MerTK by surface plasmon resonance. Sensorgrams are presented for gilteritinib (A, B), LDC1267 (C, D), merestinib (E, F) and EX172 (G, H) for binding to dephosphorylated MerTK (A, C, E, G) and autophosphorylated MerTK (B, D, F, H).

Supplementary Figure 3. Disorder of the activation loop in MerTK crystal structures. (A) The superposition of all MerTK PDB entries and apo structure 7AB0 highlights the conformational conformity of the kinase domain and the general absence of the A-loop. Sequences of all residues included in the coordinates were extracted and aligned, with A-loop residues 736-770 depicted in (B). (B) PDB ID and model chain are indicated. For clarity a reference sequence of MerTK was added at the bottom of the alignment, and the location of DFG-motif, APE-motif and A-loop highlighted.

Table 1. Biochemical inhibition of MerTK kinase domain and biophysical binding by surface plasmon resonance (SPR). a b Unless stated otherwise, all data reported are a mean of at least three independent measurements. pIC50 SEM < 0.30. pKd c SEM < 0.21. pKd SEM < 0.20. *SPR data could not be reliably determined for UNC2025 / MRX-6313.

Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 biochem SPR – dephosphorylated MerTK SPR – phosphorylated MerTK compound a b c pIC50 pKD t1/2 (min) pKD t1/2 (min)

7.5 7.8 0.3 8.3 0.8 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020

gilteritinib (type 1)

8.3 *N/A *N/A *N/A *N/A

UNC2025 / MRX-6313 (type 1)

8.1 7.9 13 7.2 2.5

LDC1267 (type 2)

8.7 8.5 124 8.3 45

merestinib (type 2)

9.1 8.2 37 9.4 42

example 172 / EX172 (type 1.5)

Supplementary Table 1. Details and refinement statistics of the crystal structures presented. Values in parentheses are for the highest-resolution shell.

PDB entry 7AB1 7AB2 7AAX 7AAY 7AAZ 7AB0 MerTK construct GSHM_E571- GSHM_E571- GSHM_E571- GSHM_E571- GS_E571-V864 GSHM_E571- V864, V864, V864, V864, ΔM659- V864,

Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 K591R/K693R/ K591R/K693R/ K591R/K693R/ Q662 K591R/K693R/ K702R/K856R K702R/K856R K702R/K856R K702R/K856R ligand gilteritinib UNC2025 LDC1267 merestinib EX172 apo Complex formation soak soak co-cryst. co-cryst. co-cryst. N/A Data Collection Beamline DLS i03 DLS i04 DLS i03 DLS i02 DLS i03 DLS i04 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 Date 2020-01-23 2020-02-07 2016-07-22 2016-08-02 2017-08-11 2019-12-05 Detector Eiger 16M Eiger 16M PILATUS3 6M PILATUS 6M-F PILATUS3 6M Eiger 16M Wavelength (Å) 0.976254 0.979499 0.97623 0.97949 0.97627 0.97950 AutoPROC / STARANISO Space group C 2 2 21 C 2 2 21 C 2 2 21 C 2 2 21 P 43 21 2 C 2 2 21 Cell: a b c (Å) 93.3 94.1 70.7 91.9 92.0 71.6 92.7 92.8 71.0 93.1 131.0 54.2 78.6 78.6 137.5 91.6 91.6 71.6 Cell: α β γ (°) 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 90.0 Resolution (Å) 46.66-1.93 48.14-1.78 65.59-1.76 46.56-1.86 68.25-1.86 48.04-1.74 (2.05-1.93) (1.96-1.78) (1.89-1.76) (2.08-1.86) (1.97-1.86) (1.98-1.74) Rmeas (%) 12.5 (126.9) 4.0 (118.1) 4.2 (81.1) 11.8 (91.0) 13.8 (227.8) 5.5 (98.4) Reflections, total 100656 (4179) 119488 (5299) 139432 (4651) 120285 (3353) 403632 (18945) 114741 (5092) Reflections, unique 18953 (949) 18168 (908) 23363 (1169) 19081 (954) 32643 (1634) 17430 (872) Mean(I)/sd(I) 9.8 (1.5) 21.0 (1.5) 19.0 (1.5) 10.6 (1.4) 13.1 (1.4) 16.3 (1.7) Completeness, 88.3 (56.3) 93.8 (64.0) 89.1 (42.9) 93.8 (63.6) 95.7 (59.4) 93.2 (67.3) ellipsoidal (%) Multiplicity 5.3 (4.4) 6.6 (5.8) 6.0 (4.0) 6.3 (3.5) 12.4 (11.6) 6.6 (5.8) CC(1/2) (%) 99.6 (80.8) 100.0 (63.5) 100.0 (70.2) 99.8 (58.2) 99.9 (53.8) 100.0 (70.9) Model Structure molecular molecular molecular molecular molecular molecular Determination replacement replacement replacement replacement replacement replacement molecules / AU 1 1 1 1 1 1 REFMAC5 Rwork (%) / Rfree (%) 23.4 / 29.8 19.3/ 25.0 19.9 / 25.3 19.4 / 22.4 18.8 / 21.9 20.7 / 24.7 B value (Å2), protein 55.0 49.7 48.5 37.1 40.5 43.0 B value (Å2), ligand 65.3 82.6 46.9 28.9 30.6 NA B value (Å2), water 51.8 48.0 46.5 34.6 44.5 44.0 B value (Å2), solvent 72.1 65.5 46.7 33.0 40.8 45.2 N° of atoms, protein 2075 2138 2154 2162 2279 2147 N° of atoms, ligand 40 35 41 41 43 NA N° of atoms, water 54 89 87 117 132 132 N° of atoms, solvent 8 12 3 3 14 5 rmsd 0.012 0.013 0.012 0.009 0.012 0.009 bond lengths (Å) rmsd 1.54 1.52 1.50 1.30 1.47 1.32 bond angles (°) MOL Probity [37] Ramachandran 95.5 97.0 97.0 97.4 97.6 98.5 favored (%) Ramachandran outliers 1.1 0.0 0.0 0.0 0.4 0.0 (%)

Supplementary Table 2. Biochemical inhibition of 395 human kinases by EX172. Inhibition was determined at a compound concentration of 0.1 μM (ThermoFisher SelectScreen Kinase Profiling Services). The table is sorted by mean inhibition.

Mean Kinase Name Inhibition Molecular Target (%) MERTK (cMER) 101 c-mer Proto-Oncogene Tyrosine Kinase

Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 MERTK (cMER) 98 c-mer Proto-Oncogene Tyrosine Kinase AXL 97 AXL Receptor Tyrosine Kinase CHEK2 (CHK2) 55 Checkpoint Kinase 2 NTRK1 (TRKA) 32 Neurotrophic Receptor Tyrosine Kinase Type 1 TYRO3 (RSE) 31 TYRO3 Protein Tyrosine Kinase MST4 29 Mammalian STE20-like 4 STK38L (NDR2) 28 Serine/Threonine Kinase 38 like Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 MAP2K4 (MEK4) 28 Mitogen-Activated Protein Kinase Kinase 4 SGKL (SGK3) 24 Serum/glucocorticoid Regulated Kinase 3 MAPK9 (JNK2) 22 Mitogen Activated Protein Kinase 9 CAMKK1 (CAMKKA) 21 Calmodulin Dependent Kinase Kinase Alpha MAP2K1 (MEK1) 21 Mitogen Activated Protein Kinase Kinase 1 RPS6KA2 (RSK3) 21 Ribosomal Protein S6 Kinase polypeptide 2 PKN2 (PRK2) 20 Protein Kinase C Related Kinase 2 MELK 20 Maternal Embryonic Leucine Zipper Kinase NLK 18 Nemo-like Kinase CDK17/cyclin Y 18 PCTAIRE Protein Kinase 2 DNA-PK 18 DNA-dependent Protein Kinase SNF1LK2 17 SNF1-like Kinase 2 PRKCG (PKC gamma) 17 Protein Kinase C gamma LATS1 17 LATS, large tumor suppressor, homolog 1 (Drosophila) AMPK (A1/B2/G3) 16 AMP-activated Protein Kinase? FLT3 15 fms-Related Tyrosine Kinase 3 RPS6KB2 (p70S6Kb) 15 Ribosomal Protein S6 Kinase, 70kDa, polypeptide 2 STK22D (TSSK1) 14 Testis-specific Serine Kinase 1B PRKCA (PKC alpha) 14 Protein Kinase C alpha ZAP70 14 Zeta Chain Associated Protein Kinase STK17B (DRAK2) 14 Serine/Threonine Kinase 17b SRC 13 v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog MAP4K1 (HPK1) 13 Mitogen-Activated Protein Kinase Kinase Kinase Kinase 1 STK24 (MST3) 13 Serine/Threonine Kinase 24 (STE20 homolog, yeast) ACVR1B (ALK4) 13 Activin A Receptor type IB MAPK10 (JNK3) 13 Mitogen-activated protein kinase 10 MAP2K1 (MEK1) 13 Mitogen Activated Protein Kinase Kinase 1 TAOK2 (TAO1) 12 TAO Kinase 2 CDC42 BPG (MRCKG) 12 CDC42 Binding Protein Kinase gamma (DMPK-like) CDC42 BPA (MRCKA) 12 CDC42 Binding Protein Kinase alpha (DMPK-like) STK38 (NDR) 12 Serine/Threonine kinase 38 PAK3 12 p21 (CDKN1A)-activated Kinase 3 CSNK2A1 (CK2 alpha 1) 12 Casein Kinase 2, alpha 1 polypeptide IRAK3 12 -1 Receptor-Associated Kinase 3 DAPK2 12 Death-Associated Protein Kinase 2 TBK1 12 TANK-binding Kinase 1 PRKCE (PKC epsilon) 11 Protein Kinase C epsilon MAP2K2 (MEK2) 11 Mitogen-Activated Protein Kinase Kinase 2 LYN B 11 v-yes-1 Yamaguchi sarcoma viral related oncogene homolog STK25 (YSK1) 11 Serine/Threonine Kinase 25 (STE20 homolog, yeast) PRKCN (PKD3) 11 Protein Kinase D3 CSNK1G1 (CK1 gamma 1) 11 Casein Kinase 1 gamma 1 PIK3CG (p110 gamma) 11 Phosphoinositide 3 Kinase Gamma (PI3K Gamma) VRK2 11 Vaccinia Related Kinase 2 EPHA4 11 EPH Receptor A4 PHKG1 11 Phosphorylase Kinase, gamma 1 (muscle) IKBKE (IKK epsilon) 11 I Kappa B Kinase Epsilon SRMS (Srm) 11 Src-related Kinase Lacking C-terminal Regulatory Tyrosine and N-terminal Myristylation Sites CDK7/cyclin H/MNAT1 11 Cyclin Dependent Kinase 6:Cyclin H:MNAT1 DYRK1A 11 Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A SIK3 11 SIK Family Kinase 3 SBK1 10 SH3-Binding Domain Kinase 1 MAPK14 (p38 alpha) Direct 10 Mitogen-Activated Protein Kinase 14 MAPK7 (ERK5) 10 Mitogen-Activated Protein Kinase 7 NEK6 10 NIMA (never in mitosis a)-related kinase 6 PRKCB2 (PKC beta II) 10 Protein Kinase C beta 1 BRAF 10 v-raf Murine Sarcoma Viral Oncogene Homolog B1 RAF1 (cRAF) Y340D Y341D 10 v-raf murine leukemia viral oncogene homolog 1 TAOK3 (JIK) 10 TAO Kinase 3 SGK2 9 Serum/glucocorticoid Regulated Kinase 2 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 PHKG2 9 Phosphorylase Kinase gamma 2 PKN1 (PRK1) 9 Protein Kinase N1 TEK (Tie2) 9 Tie2 receptor tyrosine kinase CSNK2A2 (CK2 alpha 2) 9 Casein Kinase 2 alpha prime polypeptide MUSK 9 Muscle Skeletal Receptor Tyrosine Kinase CSNK1A1 (CK1 alpha 1) 9 Casein Kinase 1, alpha 1 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 KDR (VEGFR2) 9 Kinase Insert Domain Receptor PIK3C2A (PI3K-C2 alpha) 9 PIK3C2A phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2 alpha MAPK11 (p38 beta) 9 Stress Activated Protein Kinase 2b CDKL5 9 Cyclin-Dependent Kinase-like 5 DCAMKL1 (DCLK1) 9 Doublecortin-like Kinase 1 CSNK1G2 (CK1 gamma 2) 9 Casein Kinase 1 gamma 2 MAP2K6 (MKK6) 9 Mitogen Activated Protein Kinase Kinase 6 KSR2 9 Kinase Suppressor of Ras 2 PAK1 9 P21 Activated Kinase 1 TTK 8 TTK Protein Kinase PIK3C2G (PI3K-C2 gamma) 8 Phosphoinositide-3-Kinase, class 2, gamma polypeptide MAPKAPK3 8 Mitogen-Activated Protein Kinase-Activated Protein Kinase 3 PRKD2 (PKD2) 8 Protein Kinase D2 PRKACG (PRKAC gamma) 8 Protein Kinase, cAMP-dependent, catalytic, gamma AMPK (A2/B1/G3) 8 AMP-activated Protein Kinase? PAK2 (PAK65) 8 p21 (CDKN1A)-activated kinase 2 FGFR4 8 Fibroblast 4 FYN 8 FYN oncogene related to SRC, FGR, YES INSRR (IRR) 8 Receptor-Related Receptor MAP4K3 (GLK) 8 Mitogen-Activated Protein Kinase Kinase Kinase Kinase 3 MAP4K5 (KHS1) 8 Mitogen-Activated Protein Kinase Kinase Kinase Kinase 5 CAMK4 (CaMKIV) 8 Calcium/calmodulin-Dependent Protein Kinase IV ULK3 8 Unc-51-like kinase 3 CAMK2D (CaMKII delta) 8 Calcium/calmodulin-Dependent Protein Kinase (CaM kinase) II delta PDK1 8 3-Phosphoinositide Dependent Protein Kinase-1 CDK5 (Inactive) 8 Cyclin Dependent Kinase 5 ROCK2 8 Rho Associated, Coiled Coil Containing Protein Kinase 2 MAPKAPK2 8 MAPK Activated Protein Kinase-2 IRAK1 8 Interleukin-1 Receptor-associated Kinase 1 PRKCQ (PKC theta) 8 Protein Kinase C theta PAK6 8 p21 Activated Kinase 6 FRK (PTK5) 8 Fyn-related Kinase TNIK 8 TRAF2 and NCK Interacting Kinase STK32B (YANK2) 7 Serine/Threonine Kinase 32B AMPK A2/B1/G1 7 AMP-activated Protein Kinase? LYN A 7 v-yes-1 Yamaguchi sarcoma viral related oncogene homolog MARK4 7 MAP/microtubule Affinity-Regulating Kinase 4 CSK 7 C-Src Kinase PRKG1 7 Protein Kinase, cGMP-dependant, type 1 ROCK1 7 Rho Associated, Coiled Coil Containing Protein Kinase 1 CLK1 7 CDC-like Kinase 1 MASTL 7 Microtubule Associated Serine/threonine kinase like TNK1 7 Tyrosine Kinase, non-Receptor, 1 TEC 7 Tec protein tyrosine kinase LATS2 7 LATS, large tumor suppressor, homolog 2 (Drosophila) ACVRL1 (ALK1) 7 Activin A Receptor Type II-like 1 SLK 7 STE20-like kinase HIPK4 7 Homeodomain Interacting Protein Kinase 4 HCK 7 Hemopoietic Cell Kinase RET V804L 7 Ret Proto-oncogene (multiple endocrine neoplasia and medullary thyroid carcinoma 1, Hirschsprung disease) ULK1 7 Unc-51-like Kinase 1 (C. elegans) 6 Lymphocyte-Specific Protein Tyrosine Kinase GRK6 6 G protein-coupled receptor kinase 6 CAMKK2 (CaMKK beta) 6 Calmodulin Dependent Kinase Kinase Beta RPS6KB1 (p70S6K) 6 P70S6 Kinase ERN1 6 Endoplasmic Reticulum to Nucleus Signaling 1 CAMK2G (CaMKII gamma) 6 Calcium/calmodulin-Dependent Protein Kinase (CaM kinase) II gamma MAP4K4 (HGK) 6 Mitogen Activated Protein Kinase Kinase Kinase Kinase 4 PRKG2 (PKG2) 6 Protein Kinase, cGMP-dependent, type II NEK1 6 NIMA (never in mitosis gene a)-related Kinase 1 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 ACVR2B 6 Activin A Receptor, type IIB GSG2 (Haspin) 6 germ cell associated 2 haspin IGF1R 6 Insulin Like Growth Factor Receptor 1 EGFR (ErbB1) 6 Receptor PRKCZ (PKC zeta) 6 Protein Kinase C zeta CDK18/cyclin Y 6 PCTAIRE Protein Kinase 3 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 PEAK1 6 Pseudopodium Enriched Atypical kinase 1 PRKD1 (PKC mu) 6 Protein Kinase D1 CDK2/cyclin O 6 Cyclin Dependent Kinase 2:Cyclin O TNK2 (ACK) 6 Tyrosine Kinase, non-Receptor, 2 DMPK 6 Dystrophia Myotonica-protein Kinase RPS6KA4 (MSK2) 6 Ribosomal Protein S6 Kinase polypeptide 4 TGFBR2 6 Transforming Growth Factor - Beta Receptor Type 2 AKT3 (PKB gamma) 6 v-akt murine thymoma viral oncogene homolog 3 MAPK8 (JNK1) 6 Mitogen Activated Protein Kinase 8 GRK5 6 G protein-coupled receptor kinase 5 FGFR3 5 Receptor 3 CAMK1G (CAMKI gamma) 5 Calcium/Calmodulin-dependent Protein Kinase IG PKMYT1 5 Protein Kinase, Membrane Associated Tyrosine/Threonine 1 PIM1 5 PIM 1 oncogene CSNK1E (CK1 epsilon) 5 Casein Kinase 1, epsilon NEK7 5 NIMA (never in mitosis gene a)-Related Kinase 7 PRKACB (PRKAC beta) 5 Protein Kinase, cAMP-dependent, catalytic, beta AURKA (Aurora A) 5 Aurora Kinase A AURKC (Aurora C) 5 Aurora Kinase C CDK9/cyclin T1 5 Cyclin Dependent Kinase 9:Cyclin T PLK2 5 Polo-like Kinase 2 MAPK13 (p38 delta) 5 Mitogen Activated Protein Kinase 13 CDK8/cyclin C 5 Cyclin Dependent Kinase 8:Cyclin C TLK2 5 Tousled-like Kinase 2 STK17A (DRAK1) 5 Serine/Threonine Kinase 17a (apoptosis-inducing) YES1 5 v-yes-1 Yamaguchi Sarcoma Viral Oncogene Homolog 1 DDR2 5 Discoidin Domain Receptor Family Member 2 EPHA5 5 EPH Receptor A5 CSNK1A1L 4 Casein Kinase 1, alpha 1-like MAPK3 (ERK1) 4 Mitogen Activated Protein Kinase 3 DYRK3 4 Dual Specificity Tyrosine Phosphorylated Regulated Kinase 3 RPS6KA1 (RSK1) 4 MAPK Activated Protein Kinase-1a MKNK2 (MNK2) 4 MAP Kinase Interacting Serine/Threonine Kinase 2 BMX 4 BMX Non-Receptor Tyrosine Kinase PDGFRB (PDGFR beta) 4 Platelet Derived Growth Factor Receptor Beta LIMK1 4 LIM Domain Kinase 1 JAK2 JH1 JH2 4 2 AMPK (A1/B1/G2) 4 AMP-activated Protein Kinase? PI4K2A (PI4K2 alpha) 4 Phosphatidylinositol 4-kinase type 2 alpha PDK1 Direct 4 3-Phosphoinositide Dependent Protein Kinase-1 DYRK2 4 Dual Specificity Tyrosine Phosphorylation Regulated Kinase 2 CSF1R (FMS) 4 Colony Stimulating Factor 1 Receptor BRAF 4 v-raf Murine Sarcoma Viral Oncogene Homolog B1 FLT4 (VEGFR3) 4 Vascular endothelial growth factor receptor 3 NEK8 4 NIMA (never in mitosis gene a)- related Kinase 8 EPHA3 4 EPH Receptor A3 STK23 (MSSK1) 4 Serine/Threonine Kinase 23 CDK2/cyclin A 4 Cyclin Dependent Kinase 2:Cyclin A GRK7 4 G Protein-coupled Receptor Kinase 7 AMPK (A2/B2/G2) 4 AMP-activated Protein Kinase? PTK2B (FAK2) 4 PTK2B Protein beta MAPK12 (p38 gamma) 4 Mitogen-Activated Protein Kinase 12 MARK3 4 MAP / Microtubule Affinity-Regulating Kinase 3 ACVR2A 4 Activin A Receptor, type IIA CAMK2B (CaMKII beta) 3 Calcium/calmodulin-Dependent Protein Kinase (CaM kinase) II beta MYO3B (MYO3 beta) 3 Myosin IIIB MAPK14 (p38 alpha) Direct 3 Mitogen-Activated Protein Kinase 14 STK3 (MST2) 3 Serine/Threonine Kinase 3 (STE20 homolog, yeast) STK22B (TSSK2) 3 Testis-specific Serine Kinase 2 PRKX 3 Protein Kinase, X-linked AAK1 3 AP2 Associated Kinase 1 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 MAPKAPK2 3 MAPK Activated Protein Kinase-2 CHUK (IKK alpha) 3 I Kappa B Kinase 1 MAP2K6 (MKK6) 3 Mitogen Activated Protein Kinase Kinase 6 FLT1 (VEGFR1) 3 Vascular Endothelial Growth Factor Receptor 1 NEK4 3 NIMA (never in mitosis gene a)- related Kinase 4 CLK2 3 CDC-like kinase 2 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 PTK6 (Brk) 3 Protein Tyrosine Kinase 6 RPS6KA3 (RSK2) 3 MAPK Activated Protein Kinase-1b MAP3K7/MAP3K7IP1 3 Mitogen-Activated Protein Kinase Kinase Kinase 7 (TAK1-TAB1) TGFBR1 (ALK5) 3 Transforming growth factor, beta receptor I EPHB2 3 EPH Receptor B2 MARK2 3 MAP/microtubule Affinity-Regulating Kinase 2 MARK1 (MARK) 3 MAP/microtubule Affinity-Regulating Kinase 1 TYK2 3 Tyrosine Kinase 2 NEK9 3 NIMA (never in mitosis gene a)- related Kinase 9 ROS1 3 v-ros UR2 Sarcoma Virus Oncogene Homolog 1 PTK2 (FAK) 3 Focal Adhesion Kinase 1 FGFR1 V561M 3 Fibroblast Growth Factor Receptor 1 PDGFRA (PDGFR alpha) 3 Platelet Derived Growth Factor Receptor Alpha EPHA7 3 EPH Receptor A7 TXK 3 TXK Tyrosine Kinase HIPK3 (YAK1) 3 Homeodomain-interacting Protein Kinase 3 WEE1 3 WEE1 homolog (S. pombe) PIM3 2 PIM 3 oncogene ADRBK1 (GRK2) 2 Adrenergic, Beta Receptor Kinase 1 PAK7 (KIAA1264) 2 p21 Activated Kinase 5 HIPK1 (Myak) 2 Homeodomain Interacting Protein Kinase 1 EPHB1 2 EPH Receptor B1 ICK 2 Lymphocyte-Specific Protein Tyrosine Kinase DCAMKL2 (DCK2) 2 Doublecortin and CaM Kinase-like 2 CDK5/p35 2 Cyclin Dependent Kinase 5 ERN2 2 Endoplasmic Reticulum to Nucleus signaling 2 MAP4K2 (GCK) 2 Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 FES (FPS) 2 FES proto-oncogene, tyrosine kinase IKBKB (IKK beta) 2 I Kappa B Kinase 2 ERBB4 (HER4) 2 v-erb-a erythroblastic leukemia viral oncogene homolog 4 FGFR1 2 Fibroblast Growth Factor Receptor 1 SGK (SGK1) 2 Serum Glucocorticoid-regulated Kinase 1 PRKCB1 (PKC beta I) 2 Protein Kinase C beta 1 ABL2 (Arg) 2 v- Abelson Murine Leukemia Viral oncogene homolog 2 MAPK9 (JNK2) 2 Mitogen Activated Protein Kinase 9 CDK14 (PFTK1)/cyclin Y 2 PFTAIRE Protein Kinase 1 NEK2 2 NIMA (never in mitosis gene a)-related Kinase 2 GSK3A (GSK3 alpha) 2 Glycogen Synthase Kinase-3 alpha CDK1/cyclin A2 2 Cyclin-Dependent kinase 1:Cyclin A2 EPHB3 2 EPH Receptor B3 PRKCH (PKC eta) 2 Protein Kinase C eta MYLK (MLCK) 1 Myosin, Light chain Kinase DYRK4 1 Dual-Specificity Tyrosine Phosphorylation Regulated kinase 4 HIPK2 1 Homeodomain-interacting Protein Kinase 2 EPHA1 1 EPH Receptor A1 CDC7/DBF4 1 Cell Division Cycle 7 Homolog (S. cerevisiae) ULK2 1 Unc-51-Like Kinase 2 PASK 1 PAS Domain Containing Serine/threonine Kinase MAPKAPK5 (PRAK) 1 p38 Regulated Activated Kinase BTK 1 Bruton agammaglobulinemia Tyrosine Kinase RPS6KA5 (MSK1) 1 Mitogen and Stress Activated Protein Kinase 1 CDK9 (Inactive) 1 Cyclin-Dependent Kinase 9 (CDC2-related kinase) CDK11 (Inactive) 1 Cyclin-Dependent Kinase 19 DAPK3 (ZIPK) 1 Death-Associated Protein Kinase 3 CDK1/cyclin B 1 Cyclin Dependent Kinase 1:Cyclin B SRPK1 1 SFRS Protein Kinase 1 EPHA2 1 EPH Receptor A2 CSNK1D (CK1 delta) 1 Casein Kinase 1 delta GSK3B (GSK3 beta) 1 Glycogen Synthase Kinase-3 beta NTRK3 (TRKC) 1 Neurotrophic Tyrosine Kinase, Receptor, type 3 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 PI4KA (PI4K alpha) 1 Phosphatidylinositol 4-kinase type III alpha TAOK1 1 TAO kinase 1 CDC42 BPB (MRCKB) 0 CDC42 Binding Protein Kinase beta (DMPK-like) MLCK (MLCK2) 0 Myosin Light Chain Kinase 3 ZAK 0 and Leucine Zipper containing Kinase AZK BLK 0 B Lymphoid Tyrosine Kinase Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 FER 0 Fer (fps/fes related) Tyrosine Kinase phosphoprotein NCP94 AMPK A2/B1/G1 0 AMP-activated Protein Kinase? PAK4 0 P21 Activated Kinase 4 NTRK2 (TRKB) 0 Neurotrophic Receptor Tyrosine Kinase Type 2 AMPK (A1/B1/G3) 0 AMP-activated Protein Kinase? NIM1K 0 Serine/threonine-protein Kinase NIM1 CLK3 0 CDC-like Kinase 3 CDK9/cyclin K 0 Cyclin-Dependent Kinase 9 (CDC2-related kinase) AMPK (A2/B1/G2) 0 AMP-activated Protein Kinase? PLK1 0 Polo-like Kinase 1 RET 0 Ret Proto-oncogene (multiple endocrine neoplasia and medullary thyroid carcinoma 1, Hirschsprung disease) PI4K2B (PI4K2 beta) 0 Phosphatidylinositol 4 Kinase? MAPK15 (ERK7) 0 Mitogen-Activated Protein Kinase 15 ALK 0 Anaplastic Lymphoma Kinase PLK4 0 Polo-like Kinase 4 (Drosophila) AKT1 (PKB alpha) 0 v-akt murine thymoma viral oncogene homolog 1 SIK1 0 Salt-Inducible kinase 1 INSR 0 TLK1 0 Tousled-like Kinase 1 SYK 0 Spleen tyrosine kinase MLK4 0 Mixed Lineage Kinase 4 AMPK (A1/B2/G1) 0 AMP-activated Protein Kinase? MAP3K10 (MLK2) -1 Mitogen-Activated Protein Kinase Kinase Kinase 10 ABL1 -1 v-abl Abelson Murine Leukemia Viral oncogene homolog 1 GAK -1 Cyclin G Associated Kinase CAMK2A (CaMKII alpha) -1 Calcium/calmodulin-Dependent Protein Kinase (CaM kinase) II,alpha PIK3CB/PIK3R1 (p110 -1 Phosphoinositide 3 Kinase Beta (PI3K Beta) beta/p85 alpha) RIPK2 -1 Receptor-Interacting serine/threonine Kinase 2 MAPK1 (ERK2) -1 Mitogen Activated Protein Kinase 1 MAP3K11 (MLK3) -1 Mitogen-Activated Protein Kinase Kinase Kinase 11 LTK (TYK1) -1 Leukocyte Tyrosine Kinase FGR -1 Gardner-Rasheed feline sarcoma viral (v-) oncogene homolog EPHB4 -1 EPH receptor B4 CLK4 -2 CDC-like Kinase 4 EIF2AK2 (PKR) -2 Eukaryotic Translation Initiation Factor 2-alpha Kinase 2 AMPK (A2/B2/G3) -2 AMP-activated Protein Kinase? MYO3A (MYO3 alpha) -2 Myosin IIIA STK33 -2 Serine/Threonine Kinase 33 PLK3 -2 Polo-like Kinase 3 MAP3K14 (NIK) -2 Mitogen-Activated Protein Kinase Kinase Kinase 14 LRRK2 FL -2 Leucine-rich Repeat Kinase 2 CDK16 (PCTK1)/cyclin Y -2 PCTAIRE Protein Kinase 1 MAP3K19 (YSK4) -2 Yeast Sps1/Ste20-Related Kinase 4 (S. cerevisiae) EGFR (ErbB1) T790M L858R -2 Epidermal Growth Factor Receptor MAPK10 (JNK3) -2 Mitogen-activated protein kinase 10 CSNK1G3 (CK1 gamma 3) -2 Casein Kinase 1 gamma 3 BRSK2 -2 BR Serine/Threonine Kinase 2 MAP2K2 (MEK2) -2 Mitogen-Activated Protein Kinase Kinase 2 NUAK2 -2 NUAK Family, SNF1-like Kinase, 2 MET (cMet) -2 c-met Tyrosine Kinase (MTK) BMPR2 -3 Bone Morphogenetic Protein Receptor, type II (Serine/Threonine Kinase) CDK5/p25 -3 Cyclin Dependent Kinase 5 MAP3K8 (COT) -3 Mitogen-Activated Protein Kinase Kinase Kinase 8 ADRBK2 (GRK3) -3 Adrenergic, Beta Receptor kinase 2 CAMK1D (CaMKI delta) -3 Calcium/calmodulin-Dependent Protein Kinase I delta SPHK2 -3 Sphingosine Kinase 2 RET V804M -3 Ret Proto-oncogene (multiple endocrine neoplasia and medullary thyroid carcinoma 1, Hirschsprung disease) ADCK3 -3 Chaperone, ABC1 activity of bc1 complex homolog (S. pombe) MAP2K5 (MEK5) -3 Mitogen-activated Protein Kinase Kinase 5 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 AMPK (A2/B2/G1) -3 AMP-activated Protein Kinase? MAP3K2 (MEKK2) -3 Mitogen-Activated Protein Kinase Kinase Kinase 2 FRAP1 (mTOR) -3 FK506 binding protein 12-rapamycin associated protein 1 MAP3K5 (ASK1) -4 Mitogen-Activated Protein Kinase Kinase Kinase 5 PRKCD (PKC delta) -4 Protein Kinase C delta MKNK1 (MNK1) -4 MAP Kinase Interacting Serine/Threonine Kinase 1 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 EPHA8 -4 EPH Receptor A8 MYLK2 (skMLCK) -4 Myosin Light Chain Kinase 2 GRK1 -4 G Protein-coupled Receptor Kinase 1 STK4 (MST1) -4 Serine/Threonine Kinase 4 CDK2/cyclin A1 -4 Cyclin Dependent Kinase 2:Cyclin A SRPK2 -5 SFRS Protein Kinase 2 PIK3C2B (PI3K-C2 beta) -5 Phosphoinositide-3-Kinase, class 2, beta polypeptide MATK (HYL) -5 Megakaryocyte-associated Tyrosine Kinase ITK -5 IL2 Inducible T Cell Kinase DDR1 -5 Discoidin Domain Receptor family, member 1 ACVR1 (ALK2) -5 Activin A Receptor, type I PIK3C3 (hVPS34) -5 Phosphoinositide-3-kinase, Class 3 CAMK1 (CaMK1) -6 Calcium/Calmodulin-Dependent Protein Kinase 1 BMPR1A (ALK3) -6 Bone Morphogenetic Protein Receptor, type IA EPHA6 -6 EPH Receptor A6 PRKCI (PKC iota) -6 Protein Kinase C iota DYRK1B -6 Dual-specificity Tyrosine Phosphorylation Regulated Kinase 1B RIPK3 -6 Receptor-Interacting Serine-Threonine Kinase 3 JAK2 -7 RPS6KA6 (RSK4) -7 Ribosomal Protein S6 Kinase polypeptide 6 SPHK1 -7 Sphingosine Kinase 1 STK16 (PKL12) -7 Serine/Threonine Kinase 16 CHEK1 (CHK1) -7 Checkpoint Kinase I MINK1 -7 Misshapen-like Kinase 1 ERBB2 (HER2) -7 Erythroblastic leukemia viral oncogene homolog 2Erythroblastic leukemia viral oncogene homolog 2 TESK1 -7 Testis-Specific Kinase 1 MAP3K9 (MLK1) -8 Mitogen-Activated Protein Kinase Kinase Kinase 9 FGFR2 -8 Fibroblast Growth Factor Receptor 2 BRSK1 (SAD1) -8 BR Serine/threonine Kinase 1 GRK4 -8 G Protein-coupled Receptor Kinase 4 AKT2 (PKB beta) -8 v-akt murine thymoma viral oncogene homolog 2 LIMK2 -8 LIM domain Kinase 2 PRKACA (PKA) -8 Protein Kinase, cAMP-dependent, catalytic, alpha LRRK2 -9 Leucine-rich Repeat Kinase 2 IRAK4 -9 Interleukin-1 Receptor-Associated Kinase 4 STK32C (YANK3) -9 Serine/Threonine Kinase 32C DAPK1 -9 Death-Associated Protein Kinase 1 JAK3 -10 JAK1 -10 STK39 (STLK3) -10 Serine Threonine Kinase 39 (STE20/SPS1 homolog, yeast) MAP3K3 (MEKK3) -10 Mitogen-Activated Protein Kinase Kinase Kinase 3 WNK2 -10 WNK lysine deficient Protein Kinase 2 CDK3/cyclin E1 -10 Cyclin Dependent Kinase 3:Cyclin E1 PIK3CA/PIK3R1 (p110 -11 Phosphoinositide-3-kinase, catalytic, alpha Polypeptide:phosphoinositide-3-kinase, alpha/p85 alpha) regulatory subunit 1 (alpha) Complex MST1R (RON) -11 Macrophage Stimulating 1 Receptor (c-met-related tyrosine kinase) PI4KB (PI4K beta) -13 Phosphatidylinositol 4-kinase type III beta CASK -13 Calcium/calmodulin-dependent Serine Protein Kinase KIT -13 cKIT AURKB (Aurora B) -13 Aurora Kinase B PIK3CD/PIK3R1 (p110 -15 Phosphoinositide 3 Kinase Delta (PI3K Delta) delta/p85 alpha) FYN A -15 FYN oncogene related to SRC, FGR, YES CDK2/cyclin E1 -17 Cyclin Dependent Kinase 2:Cyclin E MYLK4 -17 Myosin Light Chain Kinase Family, member 4 NUAK1 (ARK5) -17 AMPK-Related Protein Kinase 5 WNK3 -18 WNK lysine deficient Protein Kinase 3 HUNK -19 Hormonally Upregulated Neu-associated Kinase PIM2 -19 PIM 2 oncogene EEF2K -21 Eukaryotic Elongation Factor 2 Kinase

Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735

References 1 Lemke, G. and Rothlin, C. V. (2008) Immunobiology of the TAM receptors. Nat Rev Immunol 8, Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 327–336. 2 Paolino, M. and Penninger, J. M. (2016) The Role of TAM Family Receptors in Immune Cell Function: Implications for Cancer Therapy. Cancers (Basel) 8. 3 Lu, Q. and Lemke, G. (2001) Homeostatic Regulation of the Immune System by Receptor Tyrosine Kinases of the Tyro 3 Family. Science 293, 306–311. 4 Cook, R. S., Jacobsen, K. M., Wofford, A. M., DeRyckere, D., Stanford, J., Prieto, A. L., Redente, E., Sandahl, M., Hunter, D. M., Strunk, K. E., et al. (2013) MerTK inhibition in tumor leukocytes decreases tumor growth and metastasis. J Clin Invest 123, 3231–3242. 5 Myers, K. V., Amend, S. R. and Pienta, K. J. (2019) Targeting Tyro3, Axl and MerTK (TAM receptors): implications for macrophages in the tumor microenvironment. Molecular Cancer 18, 94. 6 Huelse, J. M., Fridlyand, D. M., Earp, S., DeRyckere, D. and Graham, D. K. (2020) MERTK in cancer therapy: Targeting the receptor tyrosine kinase in tumor cells and the immune system. Pharmacology & Therapeutics 213, 107577. 7 Modi, V. and Dunbrack, R. L. (2019) Defining a new nomenclature for the structures of active and inactive kinases. PNAS 116, 6818–6827. 8 Fabbro, D., Cowan-Jacob, S. W. and Moebitz, H. (2015) Ten things you should know about protein kinases: IUPHAR Review 14. Br J Pharmacol 172, 2675–2700. 9 Suebsuwong, C., Pinkas, D. M., Ray, S. S., Bufton, J. C., Dai, B., Bullock, A. N., Degterev, A. and Cuny, G. D. (2018) Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors. Bioorg Med Chem Lett 28, 577–583. 10 Huang, X., Finerty, P., Walker, J. R., Butler-Cole, C., Vedadi, M., Schapira, M., Parker, S. A., Turk, B. E., Thompson, D. A. and Dhe-Paganon, S. (2009) Structural insights into the inhibited states of the Mer receptor tyrosine kinase. Journal of Structural Biology 165, 88–96. 11 Tickle, I. J., Flensburg, C., Keller, P., Paciorek, W., Sharff, A., Vonrhein, C. and Bricogne, G. (2018) STARANISO. Cambridge, United Kingdom: Global Phasing Ltd. 12 Kabsch, W. (2010) XDS. Acta Crystallogr D Biol Crystallogr 66, 125–132. 13 Vonrhein, C., Flensburg, C., Keller, P., Sharff, A., Smart, O., Paciorek, W., Womack, T. and Bricogne, G. (2011) Data processing and analysis with the autoPROC toolbox. Acta Cryst D 67, 293–302. 14 Evans, P. R. and Murshudov, G. N. (2013) How good are my data and what is the resolution? Acta Crystallogr D Biol Crystallogr 69, 1204–1214. 15 McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. and Read, R. J. (2007) Phaser crystallographic software. J Appl Cryst 40, 658–674. 16 Navaza, J. (1994) AMoRe: an automated package for molecular replacement. Acta Cryst A 50, 157–163. 17 Winn, M. D., Ballard, C. C., Cowtan, K. D., Dodson, E. J., Emsley, P., Evans, P. R., Keegan, R. M., Krissinel, E. B., Leslie, A. G. W., McCoy, A., et al. (2011) Overview of the CCP4 suite and current developments. Acta Cryst D 67, 235–242. 18 Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, O. S., Vonrhein, C., et al. (2017) BUSTER version 2.11.7. Cambridge, United Kingdom: Global Phasing Ltd. 19 Emsley, P., Lohkamp, B., Scott, W. G. and Cowtan, K. (2010) Features and development of Coot. Acta Cryst D 66, 486–501. 20 Murshudov, G. N., Vagin, A. A. and Dodson, E. J. (1997) Refinement of Macromolecular

Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 Structures by the Maximum-Likelihood Method. Acta Cryst D 53, 240–255. 21 Derewenda, Z. S. (2004) Rational Protein Crystallization by Mutational Surface Engineering. Structure 12, 529–535. 22 Mori, M., Kaneko, N., Ueno, Y., Yamada, M., Tanaka, R., Saito, R., Shimada, I., Mori, K. and

Kuromitsu, S. (2017) Gilteritinib, a FLT3/AXL inhibitor, shows antileukemic activity in mouse Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 models of FLT3 mutated acute myeloid leukemia. Invest New Drugs 35, 556–565. 23 Zhang, W., DeRyckere, D., Hunter, D., Liu, J., Stashko, M. A., Minson, K. A., Cummings, C. T., Lee, M., Glaros, T. G., Newton, D. L., et al. (2014) UNC2025, a Potent and Orally Bioavailable MER/FLT3 Dual Inhibitor. J. Med. Chem. 57, 7031–7041. 24 Yan, S. B., Peek, V. L., Ajamie, R., Buchanan, S. G., Graff, J. R., Heidler, S. A., Hui, Y.-H., Huss, K. L., Konicek, B. W., Manro, J. R., et al. (2013) LY2801653 is an orally bioavailable multi-kinase inhibitor with potent activity against MET, MST1R, and other oncoproteins, and displays anti- tumor activities in mouse xenograft models. Invest New Drugs 31, 833–844. 25 Paolino, M., Choidas, A., Wallner, S., Pranjic, B., Uribesalgo, I., Loeser, S., Jamieson, A. M., Langdon, W. Y., Ikeda, F., Fededa, J. P., et al. (2014) The E3 Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells. Nature 507, 508–512. 26 Kim, M.-S., Kim, S., Kim, J. K., Kim, H., Ryu, K. M., Park, S. J., Park, T., Sheen, J.-H., Yoon, T. and Jang, M. Y. (2017, March 9) Heteroaryl compounds and their use as therapeutic drugs. 27 Tyrchan, C., Boström, J., Giordanetto, F., Winter, J. and Muresan, S. (2012) Exploiting Structural Information in Patent Specifications for Key Compound Prediction. J. Chem. Inf. Model. 52, 1480–1489. 28 Copeland, R. A. (2016) The drug–target residence time model: a 10-year retrospective. Nat Rev Drug Discov 15, 87–95. 29 Berger, B.-T., Amaral, M., Kokh, D. B., Nunes-Alves, A., Musil, D., Heinrich, T., Schröder, M., Neil, R., Wang, J., Navratilova, I., et al. (2020) Structure-Kinetic-Relationship Reveals the Mechanism of Selectivity of FAK Inhibitors Over PYK2, SSRN Scholarly Paper, Social Science Research Network, Rochester, NY. 30 Ling, L., Templeton, D. and Kung, H. J. (1996) Identification of the major autophosphorylation sites of Nyk/Mer, an NCAM-related receptor tyrosine kinase. J. Biol. Chem. 271, 18355–18362. 31 Bradshaw, J. M., McFarland, J. M., Paavilainen, V. O., Bisconte, A., Tam, D., Phan, V. T., Romanov, S., Finkle, D., Shu, J., Patel, V., et al. (2015) Prolonged and tunable residence time using reversible covalent kinase inhibitors. Nat Chem Biol 11, 525–531. 32 Knight, J. D. R., Qian, B., Baker, D. and Kothary, R. (2007) Conservation, Variability and the Modeling of Active Protein Kinases. PLOS ONE 2, e982. 33 Degterev, A., Hitomi, J., Germscheid, M., Ch’en, I. L., Korkina, O., Teng, X., Abbott, D., Cuny, G. D., Yuan, C., Wagner, G., et al. (2008) Identification of RIP1 kinase as a specific cellular target of necrostatins. Nature Chemical Biology 4, 313–321. 34 Xie, T., Peng, W., Liu, Y., Yan, C., Maki, J., Degterev, A., Yuan, J. and Shi, Y. (2013) Structural Basis of RIP1 Inhibition by Necrostatins. Structure 21, 493–499. 35 Christofferson, D. E., Li, Y., Hitomi, J., Zhou, W., Upperman, C., Zhu, H., Gerber, S. A., Gygi, S. and Yuan, J. (2012) A novel role for RIP1 kinase in mediating TNF α production. Cell Death Dis 3, e320–e320. 36 Bond, C. S. and Schüttelkopf, A. W. (2009) ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Cryst D 65, 510–512. 37 Williams, C. J., Headd, J. J., Moriarty, N. W., Prisant, M. G., Videau, L. L., Deis, L. N., Verma, V., Keedy, D. A., Hintze, B. J., Chen, V. B., et al. (2018) MolProbity: More and better reference data for improved all-atom structure validation. Protein Science 27, 293–315.

Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735

Figure 1 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020

B 2.9 2.8 A 2.7 hinge

N-lobe

Leu671 hinge Phe742 ('DFG in') A-loop, disordered C 3.0 2.8 C-lobe hinge Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735

Figure 2 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020

A 50° rotation of helix-A B 5 A 1 4 2 3 A C

3.4 Å shift of helix-C 90° Phe742 ('DFG out')

2.8

Lys619-Glu637 salt bridge Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735

Figure 3 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020

A Phe643 helix-C loop B Tyr753

2.6 3.3

3.4 2.5 Tyr754 2.6 Phe742

3.0 3.1

C 2.8 2.8 2.8 3.4 2.5 2.9 3.1

AArg758rg758 pp-Tyr754-Tyr754 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version available at https://doi.org/10.1042/BCJ20200735 Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200735/896152/bcj-2020-0735.pdf by UK user on 04 November 2020

Figure 4

A B 100 100

80 80

60 60 40

40 20 Mean Inhibition (%)

0 20

-20 0 kinases, sorted by inhibition MerTK Axl Chk2