Author response: Dynamics of human protein kinase Aurora A linked to drug selectivity (2018)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinase Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome. https://doi.org/10.7554/eLife.36656.001 eLife digest Protein kinases are a family of enzymes found in all living organisms. These enzymes help to control many biological processes, including cell division. When particular protein kinases do not work correctly, cells may start to divide uncontrollably, which can lead to cancer. One example is the kinase Aurora A, which is over-active in many common human cancers. As a result, researchers are currently trying to design drugs that reduce the activity of Aurora A in the hope that these could form new anticancer treatments. In general, drugs are designed to be as specific in their action as possible to reduce the risk of harmful side effects to the patient. Designing a drug that affects a single protein kinase, however, is difficult because there are hundreds of different kinases in the body, all with similar structures. Because drugs often work by binding to specific structural features, a drug that targets one protein kinase can often alter the activity of a large number of others too. Gleevec is a successful anti-leukemia drug that specifically works on one target kinase, producing minimal side effects. It was recently discovered that the drug works through a phenomenon called ‘induced fit’. This means that after the drug binds it causes a change in the enzyme’s overall shape that alters the activity of the enzyme. The shape change is complex, and so even small structural differences can change the effect of a particular drug. Do other drugs that target other protein kinases also produce induced fit effects? To find out, Pitsawong, Buosi, Otten, Agafonov et al. studied how three anti-cancer drugs interact with Aurora A: two drugs specifically designed to switch off Aurora A, and Gleevec (which does not target Aurora A). The two drugs that specifically target Aurora A were thought to work by targeting one structural feature of the enzyme. However, the biochemical and biophysical experiments performed by Pitsawong et al. revealed that these drugs instead work through an induced fit effect. By contrast, Gleevec did not trigger an induced fit on Aurora A and so bound less tightly to it. In light of these results, Pitsawong et al. suggest that future efforts to design drugs that target protein kinases should focus on exploiting the induced fit process. This will require more research into the structure of particular kinases. https://doi.org/10.7554/eLife.36656.002 Introduction Protein kinases have become the number one drug target of the 21th century (Cohen, 2002; Hopkins and Groom, 2002), due to their central role in cellular processes and involvement in various types of cancer (Carvajal et al., 2006; Gautschi et al., 2008; Katayama and Sen, 2010). Despite their therapeutic significance, the development of specific kinase inhibitors proves to be extremely challenging because they must discriminate between the very similar active sites of a large number of kinases in human cells. One of the biggest success stories is Gleevec: a highly selective drug that specifically targets Abl kinase, providing an efficient treatment of chronic myelogenous leukemia (CML) and minimizing side effects (Iqbal and Iqbal, 2014). Despite being a multi-billion-dollar cancer drug, the mechanism responsible for its impressive selectivity has been elusive until recently. Seminal work by the Kuriyan lab demonstrated that Gleevec can only bind to an inactive DFG (for Asp-Phe-Gly) loop conformation in the ‘out-conformation’ due to steric clash of the active, DFG-in conformation (Nagar et al., 2002; Schindler et al., 2000; Seeliger et al., 2007). Since then it has long been proposed that the conformational state of the fully-conserved DFG loop (Taylor et al., 2012) dictates the selectivity for Gleevec and other kinase inhibitors (Lovera et al., 2012; Nagar et al., 2002; Schindler et al., 2000; Treiber and Shah, 2013; Xu et al., 1997). The orientation of the DFG-motif and its possible steric clashes is indeed important for the ability of a class of inhibitors to bind to the kinase, but proved insufficient to explain drug selectivity and affinity. Earlier elegant work on Src and Abl recognized this and explored other hypotheses (e.g., differences in drug-binding pocket, energetic changes remote from the binding site and a conformational-selection mechanism) to reconcile the differences in Gleevec binding (Dar et al., 2008; Levinson et al., 2006; Seeliger et al., 2007; 2009), but without conclusive success. Recent quantitative binding kinetics combined with ancestral sequence reconstruction put forward a mechanism where an induced-fit step after drug binding is the key determinant for Gleevec’s selectivity (Agafonov et al., 2014; Wilson et al., 2015), and fully recapitulates the binding affinities. Here we ask the question whether this fundamentally different mechanism is a more general principle for drug efficacy and selectivity not only for Tyr kinases such as Abl, but also for Ser/Thr kinases. To this end, we chose the Ser/Thr kinase Aurora A and investigated the binding kinetics of three distinct kinase drugs: Danusertib, AT9283, and Gleevec. Aurora A kinase is one of the key regulators of mitotic events, including mitotic entry, centrosome maturation and spindle formation (Fu et al., 2007; Lukasiewicz and Lingle, 2009; Marumoto et al., 2005), as well as assisting in neuronal migration (Nikonova et al., 2013). Aurora A has attracted significant attention for the development of targeted agents for cancer because it is overexpressed in a wide range of tumors, including breast, colon, ovary and skin malignancies (Carvajal et al., 2006; Gautschi et al., 2008; Katayama and Sen, 2010; Lok et al., 2010; Marzo and Naval, 2013). The focus was mainly on ATP-competitive inhibitors, but more recently inhibition by allosteric compounds has also been pursued with the aim of achieving higher selectivity (Asteriti et al., 2017; Bayliss et al., 2017; Burgess et al., 2016; Janeček et al., 2016; McIntyre et al., 2017). So far, only the clinical significance of Aurora A inhibition by ATP-competitive drugs has been established (Bavetsias and Linardopoulos, 2015; Borisa and Bhatt, 2017), but little is known about their binding mechanisms. Many high-resolution X-ray structures of Aurora A kinase bound to different inhibitors have been solved (Bavetsias et al., 2015; Dodson et al., 2010; Fancelli et al., 2006; Ferguson et al., 2017; Heron et al., 2006; Howard et al., 2009; Kilchmann et al., 2016; Martin et al., 2012; Zhao et al., 2008), but the selectivity profile of those kinase inhibitors remains very difficult to explain. The drugs used in this study are small, ATP-competitive inhibitors. Danusertib (PHA739358) and AT9283 were developed for Aurora kinases, whereas Gleevec is selective for the Tyr kinase Abl. Danusertib inhibits all members of the Aurora family with low nanomolar IC50 values (13, 79 and 61 nM for Aurora A, B and C, respectively) (Carpinelli et al., 2007; Fraedrich et al., 2012) and was one of the first Aurora kinase inhibitors to enter phase I and II clinical trials (Kollareddy et al., 2012; Steeghs et al., 2009). A crystal structure of Danusertib bound to Aurora A kinase shows an inactive kinase with the DFG-loop in the out conformation (Fancelli et al., 2006). AT9283 inhibits both Aurora A and B with an IC50 of 3 nM (Howard et al., 2009) and has also entered several clinical trials (Borisa and Bhatt, 2017). Interestingly, the crystal structure of Aurora A with AT9283 shows that this drug binds to the DFG-in, active conformation of the kinase (Howard et al., 2009). Both drugs are high-affinity binders that reportedly bind to a discrete kinase conformation and would allow us to probe for a conformational-selection step. Lastly, we selected Gleevec as a drug that is not selective for Aurora A and should, therefore, have a weaker binding affinity. We reasoned that this choice of inhibitors could reveal general mechanisms underlying drug selectivity and affinity. The combination of X-ray crystallography, NMR spectroscopy and comprehensive analysis of drug binding and release kinetics delivered a general mechanistic view. Differential drug affinity is not rooted in the overwhelmingly favored paradigm of the DFG-conformation, but instead in the dynamic personality of the kinase that is manifested in conformational changes after drug binding. Notably, such conformational changes have evolved for its natural substrates, and the drugs take advantage of this built-in protein dynamics. Results Dephosphorylated Aurora A samples both an inactive and active structure A plethora of X-ray structures and functional assays led to the general notion that dephosphorylated Aurora A and, more universally, Ser/Thr kinases are in an inactive conformation and that phosphorylation or activator binding induces the active structure. A comparison of many X-ray structures of inactive and active forms of Ser/Thr kinases resulted in an elegant proposal of the structural hallmarks for the active state by Taylor and collaborators: the completion of both the regulatory and catalytic spines spanning the N- and C-terminal domains, including the orientation of the DFG-motif (Kornev and Taylor, 2010; 2015). X-ray structures, however, provide merely static snapshots of possible kinase conformations that do not necessarily reflect the situation in solution. In fact, recent experimental data postulate that phosphorylation of Aurora A does not ‘lock’ the kinase in the active conformation, and that the activation-loop still exhibits conformational dynamics (Gilburt et al., 2017; Ruff et al., 2018). On the other hand, X-ray crystallography provides high-resolution structural data that cannot readily be obtained from FRET or EPR and IR spectroscopy. Two crystals from the same crystallization well capture both the inactive and active conformations of dephosphorylated Aurora A bound with AMPPCP (Figure 1A,B). As anticipated, the first structure (PDB 4C3R [Zorba et al., 2014]) superimposes with the well-known inactive, dephosphorylated Aurora A structure (PDB 1MUO [Cheetham et al., 2002]) and the activation loop is not visible as commonly observed for kinases lacking phosphorylation of the activation loop (Zorba et al., 2014). The second structure (PDB 6CPF; Table 1) adopts the same conformation as the previously published phosphorylated, active structure (PDB 1OL7 [Bayliss et al., 2003]) (Figure 1C) and the first part of the activation loop could be built, although the B-factors are high. Every hallmark of an active state is seen for this dephosphorylated protein, including the DFG-in conformation that is essential for completing the regulatory spine. In contrast, the DFG-loop is in the out position for the inactive form of Aurora A (Figure 1D, cyan). In the active, non-phosphorylated structure, electron density is seen in the canonical tighter Mg2+-binding site, where the metal ion is coordinated to the α- and β-phosphates of AMPPCP and Asp274. The presence of the metal is supported by the CheckMyMetal (Zheng et al., 2017) validation, except that the coordination is incomplete. We surmise that two water molecules, not visible in our data, complete the coordination sphere as is seen in several higher-resolution structures. In the inactive structure, no electron density for Mg2+ can be identified possibly due to the fact that Asp274 is rotated to the DFG-out position and is, therefore, lost as coordination partner. Furthermore, sampling of the active conformation does not depend on AMPPCP binding as dephosphorylated, apo Aurora A also crystallizes in the active form (PDB 6CPE; Figure 1E,F and Table 1). Our results are consistent with other crystallographic studies on wild-type, dephosphorylated Aurora A in its apo or nucleotide bound state, where the kinase was also found in the active conformation (Gustafson et al., 2014; Janeček et al., 2016; Nowakowski et al., 2002). Figure 1 Download asset Open asset Dephosphorylated Aurora A samples both the active and inactive conformation. (A) Superposition of X-ray structures of dephosphorylated Aurora A (residues 122–403) with Mg2+·AMPPCP (AMPPCP in gray sticks and magnesium as yellow sphere) in the inactive (cyan, PDB 4C3R [Zorba et al., 2014]) and active (orange, PDB 6CPF) state, solved from crystals of the same crystallization well. (B) Zoom-in of (A) to visualize the nucleotide binding region (K162, D274, and E181), the R-spine (L196, Q185, F275, H254, and D311) and the activation loop region (D256, K258, and T292). (C) Same zoom-in as in (B), but dephosphorylated Aurora A in active state (orange) is superimposed with phosphorylated Aurora A (red, PDB 1OL7 [Bayliss et al., 2003]). (D) Superposition of the DFG(W) motif in the three states shown in (B) and (C). (E) Superposition of phosphorylated Aurora A in active conformation (red) and apo, dephosphorylated Aurora A also in the active conformation (yellow, PDB 6CPE). (F) Zoom-in of (E) showing the same region as in (B). https://doi.org/10.7554/eLife.36656.003 Table 1 Data collection and refinement statistics for dephosphorylated Aurora A (122-403). https://doi.org/10.7554/eLife.36656.004 apo-Aurora A (6CPE)Aurora A + AMPPCP (6CPF)Aurora A + Mb + AT9283 (6CPG)Data collectionSpace groupP 61 2 2P 61 2 2P 21 21 21Cell dimensionsa, b, c (Å)80.55, 80.55, 169.7981.75, 81.75, 172.8763.86, 69.7, 175.56α, β, γ (°)90, 90, 12090, 90, 12090, 90, 90Resolution (Å)84.90–2.45 (2.55–2.45)*86.44–2.30 (2.39–2.30)*43.14–2.80 (2.87–2.80)*Rmeas0.073 (1.308)0.113 (2.260)0.189 (1.268)I/σ(I)15.0 (1.6)10.3 (1.3)8.9 (1.1)CC1/20.998 (0.711)0.997 (0.465)0.986 (0.625)Completeness (%)99.9 (100)100 (100)99.2 (98.8)Redundancy7.6 (6.3)9.7 (7.8)5.4 (5.3)RefinementResolution (Å)64.52–2.4554.79–2.3036.17–2.80No. reflections12617 (1224)15756 (1527)19556 (1845)Rwork/Rfree0.2151/0.25280.2179/0.25870.2792/0.3350No. atomsProtein203520555122Ligand/ion113256Water46B factorsProtein71.8363.6878.84Ligand/ion75.7776.4481.05Water52.5245.84R.m.s. deviationsBond lengths (Å)0.0050.0040.003Bond angles (°)0.980.970.98 The number of crystals for each structure is one for apo-Aurora A and Aurora A + AMPPCP and two crystals for Aurora A + Mb + AT9283. *Values in parentheses are for highest-resolution shell. We note that in Aurora kinase sequences a tryptophan residue, Trp277, is immediately following the DFG motif and displays a drastically different orientation whether Aurora A is in an active (DFG-in) or inactive (DFG-out) conformation (Figure 1D). This Trp moiety is unique for the Aurora kinase family in the Ser/Thr kinome and its position is suggested to be important for tuning the substrate specificity (Chen et al., 2014). We used this Trp residue as probe to monitor the DFG flip and drug binding in real time as described below. The fact that the inactive and active states are seen in the crystal implies that both are sampled; however, it does not deliver information about the relative populations or interconversion rates. Therefore, we set out to monitor the conformational exchange of the DFG-in/out flip in solution. Owing to the reported importance of the DFG flip for activity, regulation and drug design, there have been extensive efforts to characterize this conformational equilibrium by computation (Badrinarayan and Sastry, 2014; Barakat et al., 2013; Meng et al., 2015; 2017; Sarvagalla and Coumar, 2015; Shukla et al., 2014). The general notion of these computational studies is that in the absence of phosphorylation the inactive form of the kinase is most favored, in agreement with experimental evidence. Nevertheless, short-lived excursions to the active state are observed. As an experimental approach, NMR spectroscopy is an obvious choice; however efforts on several Ser/Thr and Tyr kinases led to the general conclusion that the activation loop, including the DFG motif and most of the active-site residues, cannot be detected due to exchange broadening, and at best can only be seen after binding of drugs that stabilize conformations (Campos-Olivas et al., 2011; Langer et al., 2004; Vajpai et al., 2008; Vogtherr et al., 2006). [1H-15N]-TROSY-HSQC experiments on uniformly 15N-labeled samples of Aurora A proved to be no exception: many peaks are missing and only three out of four tryptophan side chain indole signals are seen in the 2D spectra of a [15N]-Trp labeled sample (Figure 2A,B). Therefore, we sought a strategy to overcome this general problem of exchange broadening that hampers the detection of the DFG equilibrium. Aurora A was produced containing 5-fluoro-tryptophan residues to allow for one-dimensional 19F spectroscopy to deal with exchange broadening while providing sensitivity close to proton NMR (Kitevski-LeBlanc and Prosser, 2012). Now, we observe as expected four peaks in our NMR spectra for apo- and AMPPCP-bound wild-type Aurora A (Figure 2C). A deconvolution of the spectrum yields almost identical integral values for all four peaks, whereas the linewidth of one resonance is approximately 5-fold larger (Figure 2D, purple signal). This broad peak is a prime candidate to originate from Trp277, directly adjacent to the DFG-loop. The W277L mutation confirmed our hypothesis (Figure 2C), and the extensive line broadening of this signal in a one-dimensional spectrum is consistent with its absence in the [1H,15N]-TROSY-HSQC spectrum. Of note, the W277L mutant is still active, as confirmed by a kinase assay, most likely because this Trp is not conserved in Ser/Thr kinases, where a Leu residue is found at that position for several Ser/Thr family members. Mutating any of the other, more conserved Trp residues resulted in insoluble proteins. The broad line shape for the peak at exchange broadening in the of the DFG-loop and is consistent with the B-factors for and its residues observed in all crystal structures described of relative populations and of interconversion is not possible from this data, but this missing of information was obtained by kinetics of drug binding. Figure 2 Download asset Open asset NMR spectra extensive dynamics of the DFG-loop. (A) The four tryptophan residues in Aurora A are shown on the structure (PDB 4C3R [Zorba et al., 2014]) in in the is in (B) of [1H-15N]-TROSY-HSQC spectra of dephosphorylated Aurora A in its and AMPPCP-bound three instead of the four expected peaks for tryptophan side are (C) 19F NMR spectra of labeled dephosphorylated wild-type Aurora A in and AMPPCP-bound in and the W277L Aurora A mutant bound to AMPPCP The of following the DFG-loop is (D) 19F spectrum of wild-type Aurora A bound to AMPPCP (red) with its deconvolution into four line the overall fit is shown as a The for all four signals are but the linewidth for is approximately 5-fold Gleevec binding to Aurora A conformational selection induced-fit mechanisms experiments on the Tyr kinases Abl and the concept of drug selectivity on the DFG-loop conformation has attention in kinase drug (Lovera et al., 2012; Treiber and Shah, 2013). A recent provides for such conformational but an induced-fit step after drug binding as the for Gleevec selectivity Abl to Src (Agafonov et al., 2014). Here, we ask the obvious question this mechanism of Gleevec binding to Abl a more general mechanism for kinase inhibitors. To which control drug affinity and selectivity, we first studied the binding kinetics for Gleevec to Aurora A by spectroscopy tryptophan to reduce the binding of Gleevec to Aurora A was to be and, therefore, experiments were performed at kinetics of Gleevec to Aurora A (Figure The phase is by a in the (Figure with an observed with Gleevec (Figure The to the of Gleevec binding to Aurora A and the of Gleevec is from the 2 (Figure We note that the for the binding step are to the obtained for Gleevec binding to Abl and at (Agafonov et al., 2014). The phase exhibits an in (Figure with the observed with Gleevec (Figure The provides of conformational where its of interconversion is the of The values of and can be by the data to 1 and are and (Figure These the conformational change from DFG-in to and Gleevec is a DFG-out selective inhibitor due to steric (Nagar et al., 2002; Schindler et al., 2000; Seeliger et al., 2007). Figure 3 Download asset Open asset of Gleevec binding to Aurora A at by Trp to all binding (A) after Aurora A with Gleevec is with a and a in (B) The in due to the binding phase was (C) of binding phase were of Gleevec 2 from the (D) The in of phase (A) is to conformational The of of this phase Gleevec was fit to 1 and yields and (E) kinetics of with Aurora A and Gleevec by after an of the yields the 2 (F) The of Gleevec binding to Aurora A by Gleevec as binding to Aurora A to a binding conformational selection by the binding step. The obtained from the fit and overall equilibrium and are are the of at and and in the in the In to more the data and the all time of the changes were fit the as values (Figure to the in Figure where also the are The of a conformational after drug binding induced-fit in Aurora A should drug affinity in comparison to Abl. Gleevec binds to Aurora A with a of (Figure to the low nM affinity to Abl (Agafonov et al., 2014). Two of that there is indeed no induced-fit step in Gleevec binding to Aurora A: the from the is in agreement with the Figure and and the observed from the (Figure with the of the binding 2 in Figure In the of an induced-fit step for Gleevec binding to Aurora A is the major for Gleevec’s and not the DFG-loop conformation or drug-binding consistent with our results et al., 2015). Figure Download asset Open asset of Gleevec and to Aurora A at of of the of Aurora A with different Gleevec at two and and kinetics were performed the with the binding in Figure the results of the fit to the experimental data in of Danusertib binding to Aurora A: kinetics with conformational selection and an induced-fit step we to light on Danusertib, binds very tightly to Aurora A. A high-resolution X-ray structure shows Danusertib bound to Aurora active site with its DFG-loop in the out conformation (Figure (Fancelli et al., and to affinity we the kinetics of Danusertib binding to Aurora A directly by experiments at in was observed at all Danusertib and (Figure The of the two observed on drug is for one of (Figure and for the other with an at approximately 2 (Figure The step with inhibitor to the binding whereas a at protein conformational a of the observed with substrate one cannot a between a conformational selection and an induced fit However, conformational selection drug and the DFG-in to DFG-out interconversion in Aurora A revealed by Gleevec binding (Figure therefore, be Since the of 2 (Figure is two of it can only reflect an induced-fit step Figure with 2 all Download asset Open asset of Danusertib binding to Aurora A at (A) Danusertib bound to the DFG-out conformation of Aurora A is shown important active-site residues in (PDB et al., (B) The in Danusertib binding is to a (C) of the of Danusertib for the phase yields and 3 and the for the phase (D) a 2 (E) of Danusertib from Aurora A at after a of the Aurora by and with single a of (F) time of 1 Danusertib to Aurora A by long step by a with was by to the of by Aurora A was with various of Danusertib nM nM nM nM nM nM nM and nM and was observed until of equilibrium. A