Featured Article pubs.acs.org/jmc
Mitotic Kinesin Eg5 Overcomes Inhibition to the Phase I/II Clinical Candidate SB743921 by an Allosteric Resistance Mechanism Sandeep K. Talapatra,*,†,§ Nahoum G. Anthony,‡ Simon P. Mackay,*,‡ and Frank Kozielski*,† †
The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BS, Scotland, U.K. Strathclyde Institute of Pharmacy and Biomedical Sciences, 161 Cathedral Street, University of Strathclyde, Glasgow, G4 0RE, Scotland, U.K.
‡
S Supporting Information *
ABSTRACT: Development of drug resistance during cancer chemotherapy is one of the major causes of chemotherapeutic failure for the majority of clinical agents. The aim of this study was to investigate the underlying molecular mechanism of resistance developed by the mitotic kinesin Eg5 against the potent second-generation ispinesib analogue SB743921 (1), a phase I/II clinical candidate. Biochemical and biophysical data demonstrate that point mutations in the inhibitor-binding pocket decrease the efficacy of 1 by several 1000-fold. Surprisingly, the structures of wild-type and mutant Eg5 in complex with 1 display no apparent structural changes in the binding configuration of the drug candidate. Furthermore, ITC and modeling approaches reveal that resistance to 1 is not through conventional steric effects at the binding site but through reduced flexibility and changes in energy fluctuation pathways through the protein that influence its function. This is a phenomenon we have called “resistance by allostery”.
■
INTRODUCTION Certain members of the kinesin superfamily that are involved in various stages of the cell cycle, in particular mitosis and cytokinesis,1 are considered to be potential new antimitotic targets for drug development in cancer chemotherapy. Human Eg5, a member of the kinesin-5 family, is currently under investigation as a prospective cancer drug target. To date, several drug candidates targeting Eg5 have been developed, the most advanced agents being the qinazolinone ispinesib (2, SB715992) currently in multiple phase I and phase II clinical trials,2−9 its more potent chromen-4-one analogue SB743921 (1) in phase I/II trials,10 AZD4877, a structurally similar antimitotic agent,11,12 and ARQ621 (Figure S1A). Eg5 is involved in the formation of the bipolar spindle during prometaphase.13 Through cycles of ATP-binding, ATP hydrolysis, and subsequent release of Pi and ADP, it uses the chemical energy to push antiparallel spindle microtubules (MTs) apart through its plus-end directed motor activity. Under in vitro conditions, 2 interferes with the ATPase cycle by slowing ADP release and subsequently preventing spindle pole separation at the cellular level.14 Inhibitors that target the same allosteric site on Eg5 usually function by a very similar mechanism of action.14,15 In cell-based assays, 1, 2, and another related analogue CK0106023 induce mitotic arrest that leads to apoptotic cell death. These compounds have also shown convincing in vivo antitumor activity in tumor-bearing nude mice.16 Therapeutic inactivation of a drug target in an actively dividing cell population generates a natural selection burden, which can lead tumor cells to evolve mechanisms of resistance. © 2013 American Chemical Society
Researchers at Cytokinetics were able to demonstrate this process by growing the mutagenic colorectal tumor cell line HCT116 in the presence of increasing doses of 2, which through long-term drug exposure generated 2-resistant cell lines.17 Subsequent sequencing revealed that resistance was caused by two point mutations in the Eg5 catalytic domain, D130V and A133D, revealing a possible resistance mechanism.17 These two mutations are located in the loop L5 region of the Eg5 motor (Figure S1B), a particularly long loop inserted into helix α2 that is a unique feature among members of the kinesin superfamily responsible for the high specificity of compounds that bind to this allosteric pocket.18 Although it is now well established that these two loop L5 mutations confer resistance to ispinesib, the mechanistic basis for resistance is still unknown at the molecular level. Here we present our interpretation of biochemical, biophysical, and structural data to explain the development of resistance by Eg5 to 1. We have determined crystal structures of wild-type, single, and double mutants in the absence and presence of 1 and employed molecular dynamics simulations to correlate experimentally determined thermodynamic behavior with structural changes at the molecular level. Our analysis has far-reaching implications for the understanding of the nature of resistance. Received: April 28, 2013 Published: July 22, 2013 6317
dx.doi.org/10.1021/jm4006274 | J. Med. Chem. 2013, 56, 6317−6329
Journal of Medicinal Chemistry
Featured Article
Figure 1. Kinetic data for wild-type and mutant Eg5. (A) Kinetic parameters for wild-type and mutant Eg5 and inhibition of their MT-stimulated ATPase activity by 1 and the Eg5 inhibitor 3. The kcat, K0.5,MTs, KM,ATP, and estimated IC50 values reported represent the mean ± standard error from three experiments. The resistance factor could not be calculated because we did not observe inhibition of Eg5DM: n.i., no inhibition; MIA, maximum inhibition attained; Rf, resistance factor. (B) kcat and KM,ATP values for Eg5WT (black), Eg5A133D (red), Eg5D130V (blue), and Eg5DM (green). (C) kcat and K0.5,MTs values for Eg5WT (black), Eg5D130V (red), Eg5D130V (blue), and Eg5DM (green). (D) Inhibition of Eg5WT (black) by SB743921. (E) Inhibition of Eg5D130V (red), Eg5A133D (blue), and Eg5DM (green) by 1.
■
RESULTS Kinetic Analysis of Eg5WT and Mutant Activity. We initially investigated the kinetic parameters for the MTstimulated ATPase activity in the absence and presence of inhibitors for the two single and the double point mutations Eg5D130V, Eg5A133D, and Eg5DM and compared them to those of wild-type Eg5 (Eg5WT) (Figure 1A). The kcat values of the mutants had similar rates of ATP turnover to wild-type Eg5 (5.0 ± 0.1 s−1) with a maximal 1.9-fold reduction for the double mutant (2.7 ± 0.1 s−1). The K0.5,MT and KM,ATP values were also very similar to wild-type Eg5 with a maximal 1.7-fold increase and a 1.6-fold decrease, respectively (Figure 1B,C). The kinetic parameters for the MT-stimulated ATPase activity of the wildtype and mutant Eg5 therefore display no significant differences. For the inhibition of the MT-stimulated ATPase activity by 1, the IC50 estimate is 0.14 ± 0.001 nM for Eg5WT, which increases to 607 ± 18.5 and 484 ± 26.9 nM for Eg5D130V and Eg5A133D, respectively (Figure 1D,E). This corresponds to resistance factors of ∼4300 and ∼3500 for the single mutants, respectively. For the double mutant, only partial inhibition by 1 was observed in this assay, indicating that the mutations are synergistic rather than additive. We were similarly able to demonstrate that the single and double mutants fully abolish the inhibition of Eg5 by another antimitotic inhibitor S-trityl-Lcysteine (STLC, 3), indicating that these mutations confer resistance not only to 1, 2, and monastrol (4)19 but also to other agents that target the same allosteric site. When measuring the inhibition of the basal ATPase activities of
wild-type and mutant Eg5, we obtained comparable results (data not shown). Calorimetric Analysis of SB743921 Binding to Eg5WT and Mutants. To independently assess 1 binding to the mutants while gaining insights into the thermodynamics of the process, we employed isothermal titration calorimetry (ITC) binding studies. Parts A−D of Figure 2 show the calorimetric titrations of 1 with Eg5WT, Eg5D130V, Eg5A133D, and Eg5DM, respectively, while Figure 2E summarizes the measured binding parameters. In all cases, the stoichiometry is approximately 1, indicating binding of one inhibitor molecule per catalytic domain. Analysis of the enthalpy change versus molar ratio of 1 revealed an apparent Kd for 1 of 3 kcal/mol each) to binding than in the wild-type complex. Only Glu118 was more favorable in wild-type Eg5 by ∼1 kcal/mol. Significantly, the mutated residue A133D itself made little difference to the overall enthalpy in the two complexes (WT Ala133, −0.45
although the distances are slightly different compared to the Eg5WT. As in the wild-type complex, the distance between the primary amine of 1 and the carboxylate side chain of Glu116 (2.64 Å) is compatible with hydrogen bond formation. We observed two water molecules interacting with the inhibitor: one with the amide oxygen atom and the other with the primary amine. Molecular Dynamics Simulations of Eg5WT−1 and EgA133D−1 Complexes: Correlating Biochemical, Thermodynamic, and Structural Data. The biophysical and biochemical measurements of 1 binding to Eg5WT and Eg5A133D mutant clearly show that inhibition was more effective in the former, yet the crystal structures of the two complexes are remarkably similar. The thermodynamic profiles are also significantly different: the mutant has a much more favorable enthalpic contribution to binding than wild-type Eg5 but also a much greater entropic penalty which negates the enthalpic gain. To examine kinetic systems, molecular dynamics (MD) provides an excellent tool to dissect and analyze movements of fully solvated proteins over real timeframes. To understand how the subtle structural changes could convey such emphatic differences in inhibitory effect, we investigated the two complexes using extended MD simulations to extract structural and thermodynamic binding parameters. Simulations were performed on solvated structures of the Eg5WT and Eg5A133D complexes for 50 and 100 ns, respectively, and the MM-GBSA methodology used to calculate binding 6321
dx.doi.org/10.1021/jm4006274 | J. Med. Chem. 2013, 56, 6317−6329
Journal of Medicinal Chemistry
Featured Article
within the wild-type complex, particularly in regions 55−62 (loop L2), 172−181 (loop L7), 198−202 (loop L8), 205−209 (strand β5), 222−235 (helix α3), and 273−282 (loop L11). This is in contrast to the Eg5A133D mutant, which only shows greater flexibility in region 247−257 (strand β6 and loop 10). From a thermodynamic perspective, a less flexible, conformationally restrained mutant complex with 1 represents a greater entropic penalty than the wild-type complex. Consequently, despite the mutant having more favorable contacts with 1 in enthalpic terms, its reduced configurational entropy reduces the total binding free energy compared to Eg5WT. To examine whether the source of this reduced conformational flexibility in the mutant arises at the structural level, we calculated contact maps22,36 (Figure S3) for each average simulated structure. Such maps highlight the inter-residue contacts that stabilize and restrain complexes during an MD trajectory and can identify how mutations or binding events alter protein structure. Analysis of the contact maps for each average simulated structure revealed that there are significant interactions that serve to restrain the movement of secondary structural motifs in the mutant complex that do not appear in Eg5WT and can explain the differences in conformational flexibility. This reduced fluctuation is a consequence of local changes at the molecular level around the mutation (Figure 6B,C), which induce more far-reaching effects through the rearrangement of the A133D hydrogen bond network that are transmitted through this simulated movement (Figure 7). All these extra interactions serve to dampen the movement of the mutated protein bound to 1 when compared with the wild-type Eg5. In entropic terms, a more restrained mutant complex has greater order and could account for the improved enthalpy while producing an entropic penalty on the binding free energy of the complex.
■
DISCUSSION A major challenge in cancer chemotherapy is the emergence of resistance. Tumour cells may take different routes to circumvent inhibition of a primary target, which include up-regulating alternative pathways, increasing expression of drug efflux pumps, or producing drug-resistant variants of the targeted protein, for example, through point mutations in or close to the inhibitor binding pocket. These processes are evolutionary adaptations to the natural selection pressure exerted by intervention with a chemotherapeutic agent. Most of the information on the mechanisms that lead to the development of resistance is gained from experiments conducted in tumor cell lines, and only sparse information is available for resistance mechanisms in tumors, since this requires tumor biopsies after relapse. In the most favorable cases, understanding the underlaying mechanism that leads to resistance during cancer treatment can lead to the development of second generation treatments, as observed in the treatment of chronic myelogenous leukemia (CML). Targeting of proteins involved in mitosis is an important strategy for cancer treatment. Within this target class, inhibiting Eg5 has received considerable attention and a number of drug candidates are in phase I or phase II clinical trials. When tumor cells lines were exposed to prolonged treatment with 2, resistance developed that coincided with mutations in the loop L5 region of Eg5. We have employed a combination of methods including kinetics assays, calorimetry, X-ray crystallography, and molecular dynamics simulations to understand how resistance is conveyed against the second-generation clinical
Figure 5. Simulated thermodynamic parameters extracted from MD simulations of Eg5WT and Eg5A133D in complex with 1. Complexes were solvated and 1000 structures sampled from the fully equilibrated production phases (last 43 ns of the wild-type complex, last 35 ns of the mutant). (A) Calculated total binding free energies (kcal/mol) using the MM-GBSA approach (eq 1) on 1000 snapshots once solvent and counterions had been removed. (B) Decomposition energies (kcal/mol) showing the individual enthalpic contributions of key residues within the complex to the overall binding enthalpy of 1 with Eg5WT (white) and Eg5A133D (blue). (C) Actual values are tabulated.
kcal/mol; mutant Asp133, −0.79 kcal/mol). In general, more favorable enthalpic contacts were made in the mutant, as the experimentally determined ΔH values demonstrated. Reproducing entropic contributions by simulation is more challenging, particularly water entropy, which plays an important role in the binding event through the hydrophobic effect on the ligand moving from bulk solvent to the complex. However, because the ligand is invariant in both systems, a major entropic contribution must be mediated through the complex itself. Protein conformational entropy in binding can be assessed by analyzing differences in the dynamic flexibility of the complexes throughout their trajectories. To explore the relative conformational flexibility of the two complexes, we performed residual fluctuation analysis by comparing the average minimized structure of each complex with all of the structures taken from the sampled phase of the trajectory (Figure 6A). There is clearly greater fluctuation 6322
dx.doi.org/10.1021/jm4006274 | J. Med. Chem. 2013, 56, 6317−6329
Journal of Medicinal Chemistry
Featured Article
Figure 6. Residual fluctuations of the Eg5WT−1 and Eg5A133D−1 complexes that arise during MD simulations. (A) Residual fluctuations (Å) for Eg5WT−1 (red), Eg5A133D−1 (blue) complexes and the difference between them (green). Values of >0 represent greater flexibility in the wild-type complex, whereas values of
