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Targeting Conformational Plasticity of Protein Kinases Michael Tong, and Markus A Seeliger ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/cb500870a • Publication Date (Web): 08 Dec 2014 Downloaded from http://pubs.acs.org on December 15, 2014

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Targeting Conformational Plasticity of Protein Kinases Michael Tong1, and Markus A. Seeliger1,‡

1

Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11790, USA.

‡ To whom correspondence should be addressed. Markus A. Seeliger E-mail: [email protected] Phone:

(631) 444-3558

Fax:

(631) 444-9749



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ABSTRACT The quest for ever more selective kinase inhibitors as potential future drugs has yielded a large repertoire of chemical probes that are selective for specific kinase conformations. These probes have been useful tools to obtain structural snapshots of kinase conformational plasticity. Similarly, kinetic and thermodynamic inhibitor binding experiments provide glimpses at the timescales and energetics of conformational interconversions. These experimental insights are complemented by computational predictions of conformational energy landscapes, simulations of conformational transitions and of the process of inhibitors binding to the protein kinase domain. A picture emerges in which highly selective inhibitors capitalize on the dynamic nature of kinases.

INTRODUCTION The human genome encodes more than 500 protein kinases which represent one of the largest enzyme families.1 The canonical function of protein kinases is to propagate signaling cascades by modulating the function of substrate proteins via protein phosphorylation. In this process the γ-phosphate from ATP is transferred onto the hydroxyl groups of Ser/Thr or Tyr residues.2 As fundamental mediators of signaling pathways, kinases control many cellular processes. Consequently, their activity must be tightly regulated. Loss of kinase regulation can corrupt normal cellular function and often results in diseases such as cancer, diabetes, and chronic inflammation.3 Therefore, kinases have evolved into highly modular, multi-domain signaling systems that can be regulated and activated by post translational modifications and intra- and inter-molecular interactions.4 Kinase regulation requires plasticity of the kinase domain to switch between ensembles of inactive and active states. Due to their central role in cell signaling, protein kinases are major drug targets with 28 kinase inhibitors approved for clinical use.5-7 Treatment of chronic myeloid leukemia with the BCR-Abl inhibitor, imatinib, is often cited as the proof of principle that targeted kinase therapies can be therapeutically highly successful.8, 9 The majority of FDA-approved small molecule kinase inhibitors target the highly conserved ATP-binding pocket of the catalytic kinase domain. Development of ATP-competitive kinase inhibitors is complicated by the high sequence and structural conservation of the kinase domain, which makes it challenging to achieve high selectivity and potency.10-12 Targeting specific kinase conformations is one of the strategies to obtain more selective inhibitors.13, 14 The rationale is that inactive conformations display greater conformational heterogeneity and therefore potentially provide more structural differences that can be exploited to improve selectivity. Such heterogeneity arises from kinase plasticity and allows to inhibit, or activate the enzymatic kinase activity and to modulate scaffolding functions of kinases.15-18 Serendipitous identification of novel targetable conformations relies in part on high throughput screens of chemical scaffold libraries and structure determination by X-ray crystallography.19-21 Selecting the appropriate kinase construct is also critical for identifying kinase inhibitors because regulatory domains or post translational modifications can alter the stability of the target conformation.22 Computational


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methods can help to predict the dynamic behavior of kinase structures, the existence of novel conformations including transient lowly populated conformations, and mechanisms of regulation and resistance mutations.23-26 Unbiased ligand binding simulations further our understanding of the drug binding process and may assist in the discovery of novel allosteric pockets.27 These computational methods can fill in the structural details and energetics that are difficult to observe experimentally. Additionally, optimizing drug target residence times and understanding the binding process may help to improve drug efficacy.28, 29 Here we review the role of kinase plasticity in kinase regulation, the process of ligand binding and the ability of inhibitors to achieve selectivity. Specifically we will examine conformational states accessible to the kinase domain, targeting of those states with small molecule inhibitors, the stability of these states, their interconversion rates and the relationship of drug binding kinetics and conformational exchange. Furthermore novel methods that can identify ligands targeting distinct states will also be described.

CONFORMATIONAL PLASTICITY AND THE PROTEIN KINASE DOMAIN Plasticity by definition describes something with the capability of being molded, implying an inherent degree of flexibility. Thus kinase conformational plasticity describes the capability to adopt the many distinct conformations observed in many crystal structures of kinases.16 Structural plasticity is paramount for kinase regulation and enzymatic activity, because different conformations reflect distinct functional states. For example substrate binding, catalysis, and product release, require conformational changes and intrinsic dynamic properties in the kinase domain.15 The kinase domain adopts a highly conserved bilobal fold which comprises of a β-sheet-rich amino terminal N-lobe and an all α-helical carboxy terminal C-lobe. These lobes are connected by the so-called hinge loop which facilitates movement of the lobes relative to one another. Sandwiched between the lobes is the active site where ATP binds (Fig. 1).30 Archetypal structural elements important for catalysis include the glycine-rich phosphate binding P-loop, the catalytic lysine residue, and a conserved glutamate in helix αC, which all help coordinate the ATP phosphate groups. The flexible activation loop facilitates substrate peptide binding and regulates access to the ATP binding site. At the amino-terminal end of the activation loop lies a conserved Asp-Phe-Gly (DFG) tri-peptide motif, necessary for coordinating Mg2+(Fig. 1). Arrangement of these elements determines whether the kinase is catalytically active or inactive.4 The relative stability of the active and inactive kinase conformations determines the overall activity of kinases. Perturbations such as post-translational modifications, binding of regulatory proteins, substrates and ligands can alter the relative stability of conformations, which leads to a shift in their population and therefore a change in overall kinase activity.23 For example, phosphorylation of the activation loop stabilizes the loop in an ordered extended conformation over a collapsed conformation. Upon activation loop phosphorylation, helix αC, the catalytic loop, and activation loop assume a



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catalytically competent conformation. Concomitantly the N-lobe and C-lobe become coupled together, dynamics and conformational heterogeneity are reduced, whilst substrate affinity is also increased.31, 32 Conversely, the inactive conformation of the activation loop is incompatible with activation loop phosphorylation due to charge repulsion. Effectively, activation loop phosphorylation locks the kinase domain in the active conformation. Kinase inhibitors can also perturb the dynamic ensemble of kinase conformations and stabilize specific conformations. Four main types of small molecule kinase inhibitors exist and are defined by their binding mode.5 Type-I and II inhibitors are broadly classed as ATP competitive with type-I typically binding without any discrimination of the DFG conformation, whereas type-II inhibitors target the inactive DFG-Asp-out conformation. Type-III and IV can be either inhibitors or activators and bind to allosteric sites near or remote from the kinase active site, respectively.33

THE ACTIVE CONFORMATION The active conformation which is defined by two structural hallmarks: (i) the orientation of DFG-Asp into the active site (DFG-Asp-in) where it can coordinate Mg2+/ATP, and (ii) a salt bridge between the catalytic lysine and a glutamate in helix αC (Fig. 1). This conformation is compatible with binding ATPMg2+ and substrate peptide in the active site, where it is poised for phosphotransfer. A more extensive analysis of active kinase conformations identified two conserved networks of amino acids which assemble in the so called catalytic (C) and regulatory (R) spine in the active conformation.34, 35 These spines are formed by hydrophobic residues that link both lobes with important regulatory elements.35 Full assembly of the C-spine requires ATP binding because the adenosine completes the catalytic spine. Assembly of the R-spine leads to the hallmarks of the active conformation: the DFG-Asp is facing into the active site, and the salt bridge between the catalytic lysine and a glutamate in helix αC is formed. Due to requirements of the catalytic mechanism, the active kinase conformation is very similar among protein kinases. Therefore, development of selective kinase inhibitors that target the active confirmation would appear to be more challenging but has been achieved in a few cases.36, 37 Several type-I kinase inhibitors that can generally bind to and stabilize the active conformation include dasatinib, gefitinib, and erlotinib to name a few.14 Even though the kinase is in the active conformation when bound to these inhibitors, catalytic activity is inhibited because ATP cannot bind. The recently described structure of the ERK1/2 inhibitor SCH772984 complex illustrates how conformational changes in the kinase can promote inhibitor selectivity even for a state resembling the active conformation (Fig. 2).38 SCH772984 is an ATP competitive inhibitor that shows promising activity against BRAF and MEK inhibitor resistant cancers.24, 39 SCH772984 binds to ERK1/2 with its piperazinephenyl-pyrimidine occupying a novel binding channel (P-loop pocket) which is located between helix αC and the P-loop; adjacent to the ATP binding site.38 Formation of this P-loop pocket was dependent on distortion of the P-loop and it is coupled with tilting of helix αC. Interestingly P-loop distortion causes Tyr36 in the P-loop to flip into the ATP binding site and form a stacking interaction with the inhibitor


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pyrrolidine ring. SCH772984 is 50-400-fold selective for ERK1/2 over the off target kinases haspin and JNK1 which it binds in the typical type-I binding mode, occupying only the adenine pocket but not the Ppocket.38 Importantly, the piperazine-phenyl-pyrimidine moiety, could potentially be added to other type-I scaffolds to create specificity for kinases that possess a P-loop binding pocket similar to ERK1/ERK2. The conformational states of a kinase have different relative stabilities. For example, the activating mutant L858R stabilizes EGFR kinase domain in the active conformation and gefitinib binds to the mutant protein with 20-fold higher affinity than to the wild type.36 This illustrates a case where inhibitor potency is markedly dependent on mutations that shift the equilibrium of inactive and active conformations towards the active conformation. The active EGFR conformation in complex with gefitinib is allosterically stabilized by an asymmetric dimer.36, 37 When this dimer is disrupted by mutation, gefitinib binds to EGFR in the inactive conformation.40 EGFR plasticity thus enables it to bind gefitinib in multiple conformations. Another example of a mutation that activates the kinase domain and affects inhibitor sensitivity is the so-called gatekeeper mutation in Tyr kinases. Typically a threonine residue in wild type kinases, it is located in the hinge region of the kinase domain and regulates access to a hydrophobic pocket (hence “gatekeeper”). Many patients undergoing kinase inhibitory therapy develop resistance mutations against ATP-competitive kinase inhibitors by replacement of this gatekeeper threonine with a more hydrophobic residue (e.g. Abl Thr315Ile, EGFR Thr790Met). The mutation increases the specific activity of the protein kinase and introduces steric clashes with many ATP-competitive kinase inhibitors.41

THE INACTIVE CONFORMATIONS While all active kinases resemble each other, they can be inactive in different ways.3 At least two distinct inactive conformations are accessible to the kinase domain and the Tyr kinases Src and Abl exemplify this. Both can adopt the Src/Cdk-like inactive and Abl/c-Kit-like inactive conformations (Fig. 1).8, 42-44 A few select examples of inhibitors which stabilize the inactive conformation are imatinib, lapatinib, sorafenib and the DSA compounds.14, 45 The Src/Cdk-like inactive conformation. In the Src/Cdk-like inactive conformation, helix αC is rotated outwards and the salt bridge between the glutamate in helix αC and the catalytic Lys is broken, causing the loss of kinase activity. In this case, the ability of the DFG-Asp side chain to face into the active site no longer plays a role. The outward rotation of helix αC enlarges the active site and creates a potential binding pocket underneath the β3-αC loop. A recent series of DNA-templated macrocyclic peptides have been identified that potently and selectively bind to Src kinase domain in the Src/Cdk-like inactive conformation, where they compete with ATP and substrate peptide for binding (Fig. 2).46, 47 Surprisingly, this was the first time that this inactive conformation had been observed for the isolated Src kinase domain. These macrocycles occupy the ATP-binding pocket, a hydrophobic pocket underneath β3-αC loop, and an amphipathic pocket that faces the DFG motif. Interestingly, only the outward rotation of



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helix αC opens the pocket underneath the β3-αC loop. This helix αC is movement is coupled to rearrangement of the activation loop into a conformation that is not conducive to substrate peptide binding. Interestingly, the macrocycles exhibited 10-fold greater potency against Src constructs which include the regulatory SH3 and SH2 domains in addition to the kinase domain even though the compounds form no direct interactions with these regulatory domains. This implies that the regulatory domains stabilize the Src/Cdk-like inactive conformation and emphasizes the importance of interdomain interactions in kinase regulation. The 60-80-fold selectivity of the macrocyclic compounds for Src over the closely related Hck kinase can be attributed in part to the hydrophobic pocket that forms as a consequence of the helix αC rotation. In Src a leucine (Leu297) lines the hydrophobic pocket while in Hck a methionine (Met297) reduces the size of the pocket and prevents the macrocycles with bulky substitutions to bind. Intriguingly one of the other two determinants for Src selectivity is residue Gln275, which imparts selectivity for macrocycles by increasing the rigidity of the P-loop via hydrogen bonding to a salt bridge between Lys272 and Glu280. Hck instead possesses an alanine and therefore the same salt bridge is not as stable resulting in a more flexible P-loop. Mutations within the P-loop are common resistance mutations against ATP-competitive kinase inhibitors.48 Likewise the exceptionally selective benzohydroxamate MEK1/2 inhibitors (e.g. PD0325901, PD184352 and PD3180888) also target the Src/Cdk-like inactive conformation.49, 50 PD3180888 occupies a novel pocket in MEK2 adjacent to the ATP binding site even when ATP is bound, thus revealing a non ATP competitive mechanism of inhibition.49 Although ATP was bound, the kinase remained inactive due to PD3180888 stabilizing the Src/Cdk-like inactive conformation. Again the plasticity of structural elements surrounding the pocket were critical in forming the pocket in this inactive state. The activation loop for example also forms a two turn helix in the activation loop which displaces helix αC in a manner similar to what has already been described above for the Src macrocycle example. This inactive structural hallmark is prevalent in many other inactive kinase structures such as EGFR lapatinib.51, 52 Coupling of the activation loop and helix αC conformations illustrates the importance of plasticity in mediating concerted conformational changes that contribute towards pocket formation and influence on inhibitor specificity. The Abl/c-Kit-like inactive conformation. The Abl/c-Kit like inactive conformation is characterized by a 180° crankshaft-like rotation of the DFG-motif relative to the active conformation. As a result, the DFGAsp faces away from the active site, while the DFG-Phe faces into the active site and the R-spine is disassembled.8, 44 Importantly, the selectivity of imatinib binding to Abl kinase is attributed to this conformation.8 The novelty of this conformation is that a hydrophobic pocket becomes available, allowing for a type-II inhibitor to access and form additional interactions and increase selectivity. Another prominent example of a type-II inhibitor is the p38α inhibitor BIRB796.53, 54 The number of kinases which can access this distinct inactive conformation was initially thought to be low although recent investigations to probe this suggest that more than 200 kinases could be sensitive to type-II inhibitors.14 However, the DFG-Asp-out conformation seems to be more prevalent in Tyr kinases.14, 55 Interestingly, specific residues that facilitate the ability to sample the DFG-Asp-out conformation have


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been mapped to the gatekeeper and the residue preceding the DFG motif.56 Such knowledge could potentially help predict or sensitize kinases as type-II binders. While some type-II inhibitors are exceptionally selective, other type-II inhibitors are less so. For example, sorafenib, a clinically approved type-II inhibitor targets multiple kinase targets with similar affinity and demonstrates broad spectrum kinase inhibition in various types of cancers.57 Sorafenib illustrates that many kinases can access the DFG-Asp-out conformation required for binding of type-II inhibitors. Sorafenib may be less specific than other type-II inhibitors because of a different balance of specificitygenerating hydrogen bonds and less specific hydrophobic contacts.58 Additionally, sorafenib is slightly smaller than other type-II inhibitors such as imatinib and may therefore be sterically less demanding.

IDENTIFYING LIGAND STABILIZED DISTINCT CONFORMATIONS Identifying ligands that target novel inactive conformations is difficult and requires often structural studies. A recently developed method dubbed FLiK (Fluorescent labels in kinases) helps to address this problem.59-61 In this method a fluorophore that is sensitive to its local polar environment is used to report on kinase conformational changes. The flurophore is conjugated to a Cys residue that has been engineered into a site on the kinase where monitoring a conformational change near the site is desired. For example activation loop or P-loop labeling in has helped to discriminate ligands that stabilized either DFG-Asp-in or DFG-Asp-out conformations. Even type-I inhibitors suggesting non canonical targeting of the DFG-Asp out conformation were identified.60 FLiK is also applicable to identifying type-IV binders, which has been demonstrated for the p38α C-lobe lipid binding pocket and Abl myristate pocket.62, 63 The method has been extended further with iFLiK (interface-Fluorescent Labels in Kinases) in which allosteric inhibitors targeting interdomain interfaces can be identified. Using Akt as a model system, Fang and coworkers characterized and discovered inhibitors novel inhibitor scaffolds which bound at the Akt kinase domain and PH domain interface and stabilized Akt in an autoinhibited closed inactive state.64 One of the advantages with this binding assay is that inactive protein kinase states can be used because the readout for binding is a measure of the ratiometric fluorescence at two distinct wavelengths. Other methods to determine the binding modes of ligands to protein kinases are kinetic ligand binding assays (see below) and surface plasmon resonance studies 65, 66. Other spectroscopic methods such as optical second-harmonic generation could be promising tools to study conformational changes associated with ligand binding. 67-69

CONFORMATIONAL PLASTICITY WITHIN DEFINED STATES Research by Chen and coworkers represents an example where plasticity of the FGFR inactive state plays a role in basal activity. Like most protein kinases, FGFR possesses intrinsic low basal activity in an unphosphorylated state and almost 30-fold greater activity in a phosphorylated fully active state. Based



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on structural studies it was assumed that the unphosphorylated state has only basal levels of activity, because its activation loop can interconvert with the phosphorylated active state only at a lower frequency.70 Here, a set of pathogenic gain of function mutations located in the activation loop (K650T, K650Q, K650N, K650M, K650E) each conferred a graded increase in intrinsic activity relative to the wild type unphosphorylated state. Crystal structures of these mutants showed activation loop conformations similar to that of the active phosphorylated state. NMR chemical shift changes showed that the mutant proteins were in fast chemical exchange between a conformation that resembled the unphosphorylated state and a conformation that resembled the phosphorylated state. Individual mutations shifted the populations of these conformations towards the conformation that resembles the phosphorylated state. The extent of population shift towards the conformation resembling the phosphorylated conformation corresponded to an increase in kinase activity upon mutation. Minimal and maximal population of active kinase corresponding to unphosphorylated and phosphorylated kinase, respectively. Ultimately the plasticity of the FGFR inactive state activation loop was responsible for the intrinsic basal activity. Similarly the Ser/Thr p38 MAP kinase apo state is also inherently flexible around the DFG motif. The DFG-Phe (F169) was broadened out and absent from NMR spectra of apo p38 because it was undergoing conformational exchange on an intermediate NMR times scale. Upon titration with BIRB796, a potent inhibitor type-II p38 inhibitor, Phe169 was perturbed into an observable resonance suggesting that the pre-existing conformational exchange of the DFG motif in the apo state was suppressed and stabilized in the DFG-out conformation.71 In the same study, crystal structures of the apo state revealed that the DFG motif occupied both DFG-in and out conformations equally implying that both states pre-existed in solution with similar relative stabilities.71 BIRB796 binds to p38 with a Kd of 0.1 nM.53 These NMR experiments on the solution dynamics of the p38 kinase domain show that the slow rates of BIRB796 binding and release can be attributed to the slow interconversion of the DFG motif.53 The importance of kinase flexibility is emphasized in a recent study which showed a correlation between dynamics of the hinge connecting the N-lobe and C-lobe and kinase activity.72 The idea is that specific activation loop phosphorylation increases the relative motions of the lobes to promote the assembly of the active conformation within the catalytic site of the kinase. Subsequent NMR studies showed that the domain motions are in fact constrained at the hinge region unless the kinase is activated by phosphorylation. Phosphorylation alters the rates of interconversion between the two states and it dramatically shifts the relative populations - leading to activation of the kinase. 73 Similarly, inactive and active conformations of Abl bound to inhibitors were investigated structurally and dynamically.74 Here, Vajpai and coworkers demonstrate by NMR that the solution conformations of Abl in complex with type-II inhibitors imatinib, nilotinib, and type-I inhibitor dasatinib are consistent with their respective inactive and active conformations in crystal structures.74 Chemical shift perturbation analysis indicated that Abl-imatinib and nilotinib complexes were similar in terms of specific residues perturbed and magnitude of perturbation, which was no surprise since both are type-II inhibitors. As expected, perturbations induced by the type-I-inhibitor, dasatinib, differed by the affected amino acids and by the magnitude of perturbation. Similarly, NMR residual dipolar coupling (RDC) were used to determine the relative domain orientation and overall fold of the kinase. The experimentally determined


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RDC corresponded very well with the RDCs predicted from the respective crystal structures. This indicates that the solution conformations were similar to the conformations observed in the crystal structures. Interestingly, Abl•dasatinib showed more pronounced broadening of NMR resonances than the other two state. Broadening was associated with the P-loop and activation loop and indicative of interconverting conformations.74 These observations indicated that the Abl•dasatinib state was more dynamic in terms of conformational exchange. The role of plasticity in the active conformation has recently been addressed for protein kinase A (PKA) using NMR. Here the apo, binary and ternary states were probed in terms of their backbone dynamics.15 Masterson and co-workers found that the apo state displayed minimal conformational exchange dynamics on the microsecond to millisecond timescale, but the complex of PKA with a non-hydrolysable ATP-analog showed increased dynamics on the picosecond to nanosecond timescales around the ATP binding site particularly for the P-loop and around the DFG motif. An increase in conformational exchange dynamics was also apparent for key active site structural elements with a notable differential increase in helix αC residues orientated towards the active site versus those facing away from the active site. This was interpreted as a conformational change whereby the helix αC assumes the active conformation. Formation of the ternary complex (PKA•ATP•peptide) by saturation with a substrate peptide continued to illustrate differential dynamics. There was now a redistribution of the slow dynamics in which conformational exchange of the helix αC dynamics became quenched. However, the P-loop and activation loop increased their conformational exchange. Consequently this was interpreted as reflecting conformational changes associated with product release. The residues which exhibited conformational exchange were part of the same active site cleft opening and closing conformational exchange process.15 Flexibility was also observed for the substrate peptide. Thus both kinase and substrate are flexible as they engage each other. Moreover it indicated that the active state demonstrates plasticity and that there is a preexisting equilibrium of open and closed states in the binary complex with nucleotide bound. This finding reinforced the notion that conformational selection controls ligand recognition in kinases.

THE ROLE OF KINASE PLASTICITY IN PROTEIN ASSEMBLIES Plasticity of the kinase domain plays an important role in the formation of kinase oligomers and in allosteric regulation of protein kinases. This is illustrated by the multidomain Ser/Thr kinase IRE1α, which features both a canonical kinase domain and a RNase domain. Upon dimerization of the kinase domain, two halves of the RNase domain can complement each other to form an active RNase (Fig. 3). The RNase creates an alternative splice product of the XBP1 mRNA which promotes the unfolded protein response.75 Binding of conformationally selective kinase inhibitors can modulate RNase activity of IRE1α.76, 77 The type-I inhibitor, APY24, paradoxically actives IRE1α RNase activity.76 Conversely, the kinase inhibitor, KIRA6 (kinase inhibiting RNase Attenuators), inhibits IRE1α RNase activity. 76, 78 Neither inhibitor binds to the RNase domain. The kinase inhibitors seem to stabilize kinase conformations which promote RNase dimerization (APY24) or prevent RNase dimerization (KIRA6). IRE1α can form either



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RNase inactive face-face dimers or RNase active back-back dimers or higher oligomers.75, 76, 78, 79 The physiological mechanism of selection and stabilization of the active kinase conformation occurs in part via trans autophosphorylation of the kinase activation loop leading to RNase activation. Inhibitors therefore exploit the plasticity within IRE1α to mimic this regulatory process. Similarly, binding of ATP competitive inhibitors to MAPK ERK2 or p38α can control the non-catalytic functions of the kinases.80, 81 MAPK can allosterically activate regulatory phosphatases such as DUSP6/10. While inhibitors that stabilized the DFG-Asp-in conformation (SB203580) inhibited the phosphatase, inhibitors that stabilized the DFG-Asp-out conformation increased the activity of phosphatase. This is another example where conformationally selective inhibition of kinase activity can have unexpected effects on signaling due to non-enzymatic, allosteric events. The conformational plasticity in a multidomain kinase environment and its relationship in synergistic drug binding is illustrated by Abl kinase. Studies have shown that using both imatinib and the allosteric inhibitor GNF-5 can provide therapeutic benefits against the imatinib resistant Abl T334I gatekeeper mutant when using in vivo systems.82 However the molecular mechanism behind this synergistic inhibition strategy was unknown until recently. NMR and small angle X-ray scattering (SAXS) experiments were used to probe the solution conformations of the multidomain Abl encompassing the SH3-SH2 and kinase domain (SH3-SH2-KD).83 In their experiments, experimental RDCs were determined for different Abl states including Abl-imatinib, Abl-GNF-5, and Abl-imatinib-GNF-5. These RDCs allowed the determination of the relative orientation of the domains to each other. Basically the Abl-imatinib state displayed a flexible open inhibited state, with open defined as disengagement of the SH3-SH2 domain from the kinase domain representing the disassembled inactive state. NMR dynamic measurements of the protein backbone for the Abl-imatinib state also indicated increased tumbling of the SH3, SH2 domains for this state compare to the other states, thereby implying that Abl possessed greater flexibility when only bound to imatinib and that the SH3/SH2 domains dissociated from the kinase domain. The Abl•GNF5, and Abl•imatinib•GNF5 states adopted closed inhibited states corresponding to the assembled inactive conformation. Moreover, SAXS which reports global changes in the shape of the protein, corroborated their NMR observations. SAXS revealed that the ternary state of Abl•imatinib•GNF-5, was equivalent to apo Abl, and Abl•GNF-5 in terms of distance distribution but Abl•imatinib was noticeably different. The Inherent plasticity of the multidomain Abl enables it to exist in an equilibrium of multiple inactive states, simplified as the assembled inactive, dissembled inactive and disassembled active state. Because of their independent modes of action, GNF5 and imatinib synergistically inhibit Abl kinase. Given that the assembled conformation of SH3-SH2-KD kinases often are more consistent with type-I inhibitor binding, it is tempting to speculate whether type-I inhibitors would synergize more with GNF5.


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THE PROCESS OF INHIBITOR BINDING The process of ligand binding to proteins in general and to protein kinases specifically remains poorly understood. This lack of kinetic information is surprising given that drug binding and dissociation rates have emerged as powerful predictors of drug efficacy and specificity independent of drug affinity.28 Especially for growth inhibition of bacteria residence time of antibiotics on their targets correlates better with growth inhibition than inhibitor affinity. In a simple two-state system the equilibrium dissociation constant (KD) is determined by the ratio of dissociation rate (koff) and association rate (kon) of ligand and complex. This means that protein•ligand complexes can have the same affinity but vary largely in their association and dissociation kinetics. The rationale for the pharmacological benefit of long residence time inhibitors is as follows: while the binding affinity is typically measured under equilibrium conditions, drug molecules in the cellular context are continuously metabolized and excreted. Once the cellular concentration of free drug decreases, drugs with slow binding and dissociation kinetics inhibit their target protein longer than fast binding/dissociating drugs. An example for an inhibitor with extremely long residence time would be inhibitors that bind covalently to the target protein. Examples of covalent kinase inhibitors are the BTK inhibitor PCI-32765 and the EGFR/Her inhibitors BIBW-2992 and HKI-272 – all of which are in phase III clinical trials.84-87 Covalent inhibitors inhibit their target protein, even when the concentration of free inhibitor is well below the dissociation constant. Similarly, a ligand can have different binding kinetics to different proteins, affecting its cellular specificity. A recent example would be the slow dissociation kinetics of SCH772984 from its target ERK1/2 compared to the fast dissociation kinetics from the off-targets haspin and JNK2.38 What is the molecular basis for different binding kinetics? Conformational changes of the protein that precede ligand binding, can slow down association of ligand and receptor. For example, binding of imatinib to Abl kinase and Src kinase requires protonation of the DFG-Asp for the DFG-flip to occur.88 In this process of conformational selection only “flipped” kinases can bind to the inhibitor and binding rates become pH-dependent (Fig. 4); the affinity of the complex does not change with pH. Mutation of the DFG-Asp to Gln simulates the protonated state and leads to pH-independent, slow binding of imatinib. Similarly, replacement of the Asp with Ala, leads to pH-independent, fast binding of imatinib. Binding kinetics of the conformationally less selective drug dasatinib seemed to be independent of conformational pre-equilibria. Similarly slow binding and release kinetics have been observed for a number of kinases: Cdk2, JAK2, ERK1/2 and p38 MAP kinase.38, 54, 89 Dissociation of BIRB796 from p38 MAP kinase occurs extremely slowly with residence times of tens of hours.54 Other factors apart from protein flexibility that determine ligand binding kinetics include molecular size of the ligand, accessibility of the ligand binding site 90 and flexibility of the ligand. Electrostatic steering of ligands to the binding site can dramatically change the rates of protein-protein interactions as well as protein-ligand interactions.91 Removal of water molecules that occupy the ligand binding site can also affect ligand binding kinetics.92 Overall, these factors can be taken into account for the design of kinase inhibitors with preferred binding kinetics and potentially improved pharmacodynamics.



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Recent ab initio simulations of the entire ligand binding process showed distinct pathways of ligand binding as well as the presence of novel secondary ligand binding sites.93 In the case of the small molecule inhibitors PP1 and dasatinib binding to Src kinase domain, the process could be roughly separated into 4 phases: (1) After diffusion through the solvent, the ligand adsorbs non-specifically to the protein surface and diffuses across it in a reduced dimensionality search (2). The ligand often resides in secondary, low affinity binding sites that may constitute intermediates in the binding process (3). Binding of the ligand to the primary high affinity binding site (4) is, for some conformational selective inhibitors like imatinib, limited by the rates of conformational changes of the kinase (Fig. 4).27

COMPUTATIONAL STUDIES OF KINASE PLASTICITY AND ITS RELATIONSHIP TO INHIBITOR BINDING. Computational studies are powerful complements to experimental studies into the regulation of protein kinaes. A comprehensive review is far beyond the scope of this review and we are only able to illustrate the important contribution of computational studies on a few select examples. Src and Abl kinase have been the object of intense computational studies to understand their differences in conformational plasticity and potential hints at why these two kinases have such different affinities for imatinib. Roux and coworkers have performed all molecular dynamics simulations and found that the imatinib binding competent conformation is overall less stable and therefore less populated in a conformational selection process 94-96. It is important to point out that high affinity of imatinib binding requires both the DFG-Asp out conformation (Type-II) as well as the protection of the pyridine/pyrimidine rings from solvent. In Abl kinase the P-loop kinks towards the C-lobe of the kinase and the Tyr at the tip forms a hydrogen bond with the backbone of the hinge region. It appears that a similar kink in the P-loop of Src would be energetically highly unfavorable. Type-II inhibitors with more hydrophilic or more sterically demanding groups instead of imatinib’s pyrimidine/pyridine, do not require the kink of the P-loop and bind to Src and Abl kinase equipotently.45, 97 Similar simulations have shown the considerable flexibility of the protein kinase domain in the unphosphorylated state and the stabilization of the active conformation in the activation loop phosphorylated state.25 In a recent simulation of the activation pathway of Src kinase domain from the Src-like inactive conformation to the active conformation, multiple stable intermediates were predicted including one that contains a potentially targetable binding pocket adjacent to helix αC.25 Intriguingly, a similar binding pocket has been observed experimentally in CDK2 bound to the fluorescent dye ANS and has subsequently been used to develop allosteric inhibitors of CDK2.98, 99 Protein kinases are allosteric signaling hubs that integrate multiple input signals (e.g. activation loop phosphorylation, substrate binding, inhibitory phosphorylations and binding of regulators) to phosphorylate substrates as their output signal. The mechanistic basis of long range allosteric coupling in Tyr kinases has recently been studied computationally.100 In Src kinase, a network of amino acids changes structural parameters collaboratively, in molecular dynamics transitions from the active to the Src-like inactive conformation. Interestingly, mutation of members within this network distant from the


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active site can change the observed negative cooperativity of substrate binding in the active site. This negative cooperativity and the experimentally observed effect of mutation can explain the activating effect of resistance mutations to ATP competitive drugs: while the affinity for the common and overly abundant substrate, ATP, is reduced, the affinity for the limiting substrate peptide is increased; resulting in a net increase in kinase activity.41 While the computational studies are already powerful contributors to our understanding of kinase regulation we can expect a number of further improvements within the near future: broader access to hardware that allows the extremely long time scale simulations and more precise quantification of the conformational energy landscape of protein kinases. These improvements may allow for a kinome-wide comparison of conformational states, the simulation of larger kinase constructs including regulatory domains and multiprotein complexes, precise predictions of the thermodynamic stability of kinase conformations and their interconversion rates.

SUMMARY Plasticity has been established as an intrinsic property of kinases from primary to quarternary structures. It is a prerequisite for their catalytic function and regulation because these process require conformational changes. A rich repertoire of three dimensional high resolutions kinase structures is available and together with conformationally selective kinase inhibitors give insight into the structural details of kinase conformational states. Solution NMR, SAXS, HD exchange and inhibitor binding experiments are beginning to put the structural snapshots into their dynamic context. How regulatory domains, membrane localization or complex formation affects these dynamics may soon be possible to study. The staggering increase in computational power to simulate structures on the biologically important micro- to millisecond timescales has the power to fill in the structural details for dynamic processes that experimental methods cannot offer. Beyond the value that these computational results have in their own right – they provide hypotheses that can be experimentally tested to get a better understanding of the biological process but also to validate computational methodology. The computational methods can also provide quantitative insights into the energetics and kinetics of conformational changes. These quantitative approaches will be important for rational identification of druggable states and kinase•drug interactions. Small molecule kinase inhibitors already exploit small conformational changes in kinases to achieve specificity. With increased computational power it may soon be possible to computationally screen ligand libraries against flexible protein structures. Conformational changes underlie the non-enzymatic signaling function of protein kinases. We are gradually gaining the ability to control these conformational signaling functions with small molecule kinase ligands. Currently, this often comes at the price of inhibiting the enzymatic kinase function by using kinase inhibitors as conformational regulators. With a better understanding of the conformational



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ensembles of the kinases, we may soon be able to develop allosteric kinase ligands that control conformational kinase signaling independent of enzymatic kinase function.


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Figure Legends

Figure 1. Overview of tyrosine kinase structure and conformational states. (A) The active conformation of the Src kinase domain is defined by (I) a salt bridge between the catalytic Lys295 and Glu310 in helix aC (orange) and (II) Asp404 of the conserved Asp-Phe-Gly (DFG) motif at the beginning of the activation loop (blue) facing into the ATP binding pocket underneath the glycine rich phosphate binding P-loop (red). (B) Disruption of the active conformation through an outward rotation of helix αC defines the inactive (DFG Asp-In) conformation. A 180° rotation of the DFG motif (DFG flip) results in the inactive (DFG-Asp-out) conformation. Inhibitors of Src and Abl kinase (Imatinib, DSA, Macrocyles and Dasatinib) bind these conformations specifically. Phosphorylation, binding of regulatory domains, ligands or substrates stabilize the specific conformations.

Figure 2. Helix αC rotation creates pockets for selective inhibitor binding. (A) The macrocycle MC4b binds to the ATP binding site of Src kinase domain and occupies a binding pocket underneath the β3-αC loop (PDB-entry 3U4W).47 Outward rotation of helix αC in the Cdk/Src-like inactive conformation is required for formation of this binding pocket (bottom). (B) The MAPK inhibitor SCH779284 binds to a hydrophobic pocket of ERK2 between helix αC and the P-loop (PDB-entry 4QTA).38 ERK2 is in the active conformation and the salt bridge between the catalytic lysine and the glutamate in helix αC is intact. (C) SCH7792984 binds with a strikingly different conformation and lower affinity to the off-target kinase haspin (PDB-entry 4QTC).38

Figure 3. Conformational signaling of Ire1α α. (A) The Ire1a kinase domain (grey) is fused to a C-terminal RNase domain (green), that is active when the kinase domain forms a back-to-back dimer (top, PDBentry 2RIO, yeast Ire1α).101 Formation of the face-to-face kinase dimer prevents formation of the active RNase dimer (bottom, PDB-entry: 3P23, human Ire1α).75 Regulatory elements of the kinase domain are colored red (P-loop), orange (helix αC) and blue (activation loop). (B) Schematic view on the N-lobe of the kinase in the back-to-back dimer from the direction of the arrow in panel A (top). The face-to-face dimer is aligned on back-to-back dimer (bottom). The outward rotation of helix αC observed in the faceto-face dimer would create a clash in the back-to-back dimer.

Figure 4. Kinetics of ligand binding to Src kinases. (A) Results of unbiased ligand binding simulations of PP1 to Src 27. PP1 adsorbs onto the protein, scans the surface, resides in transient sites and binds to the high affinity site. PP1 is colored in a spectrum from red (0 µsec) to blue (20 µsec) based on the timepoint of the simulation. (B) Conformational selection of the type-II inhibitor DSA1 binding to Src kinase domain.45 The pH-dependent curvature of observed rate constant of binding at higher ligand



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concentrations is due to a conformational change in the protein that limits the maximum binding rate. Adapted from [Seeliger 2009]. (C) Schematic overview of inhibitor binding to Src kinase domain as observed in MD simulations. 27 After diffusion through the solvent (1), the inhibitor adsorbs nonspecifically to the protein surface and diffuses across it (2). The inhibitor often resides in secondary, low affinity binding sites that constitute intermediates in the binding process (3). Binding of the inhibitor to the primary high affinity binding site (4) is, for some inhibitors, accompanied by conformational changes in the protein.

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Targeting Conformational Plasticity of Protein Kinases - American

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