Novel Allosteric Pathway of Eg5 Regulation Identified through Multivariate Statistical Analysis of Hydrogen-Exchange Mass Spectrometry (HX-MS) Ligand Screening Data.
ABSTRACT: The mitotic kinesin Eg5 is an important target in cancer chemotherapy. A structurally diverse collection of canonical loop L5 inhibitors engage an allosteric pathway that includes elements of its microtubule binding region. However, recent evidence suggests that Eg5 may permit alternative allosteric mechanisms. Terpendole E, a natural-product Eg5 inhibitor, is active against mutants resistant to canonical loop L5 inhibitors and appears to offer a unique mode of inhibition. To investigate the variety of inhibitor responses, the structure-function properties of eighteen kinesin inhibitors were quantified with hydrogen-exchange mass spectrometry (HX-MS), functional analysis and molecular modeling. A unique strategy for high-density data analysis was implemented, based on a scalable multivariate statistical method, as current HX-MS routines have a limited capacity to guide a characterization of ligands when additional functional data is available. Inhibitor evaluation was achieved using orthogonal partial least squares projection to latent structures discriminant analysis (OPLS-DA). The strategy generated a model that identified functionally-significant conformational elements involved in kinesin inhibition, confirming the canonical allosteric pathway and identifying a novel response pathway. Terpendole E is demonstrated to be an atypical L5 site inhibitor, where binding induces an allosteric effect mediated by a destabilization in the ?-sheet core of the molecular motor, an element involved in mechanochemical coupling for structurally-related kinesins. The analysis suggests that a different approach to inhibitor development may be fruitful.
Project description:Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resonance spectroscopy (EPR) to investigate the function of L5 in Eg5. We site-specifically attached EPR probes to ADP, L5, and the neck linker element that docks along the enzymatic head to drive forward motility on microtubules (MTs). Nucleotide-dependent spectral mobility shifts occurred in all of these structural elements, suggesting that they undergo coupled conformational changes. These spectral shifts were altered by deletion of L5 or addition of S-trityl-l-cysteine (STLC), an allosteric inhibitor that binds to L5. In particular, EPR probes attached to the neck linker of MT-bound Eg5 shifted to a more immobilized component in the nucleotide-free state relative to the ADP-bound state, consistent with the neck linker docking upon ADP release. In contrast, after L5 deletion or STLC addition, EPR spectra were highly immobilized in all nucleotide states. We conclude that L5 undergoes a conformational change that enables Eg5 to bind to MTs in a pre-powerstroke state. Deletion or inhibition of L5 with the small-molecule inhibitor STLC blocks this pre-powerstroke state, forcing the Eg5 neck linker to dock regardless of the nucleotide state.
Project description:Eg5 is a mitotic kinesin protein that plays an important role in the formation and maintenance of the bipolar spindle during the mitotic phase. Due to its potentially reduced side effects in cancer therapy, Eg5 is considered to be an attractive target for developing anticancer inhibitors. Herein, we report a computational modeling study involving biphenyl-type inhibitors known to interact with the ?4/?6 allosteric pocket of Eg5. Compared to the well-known ?2/L5/?3 allosteric inhibitors, biphenyl-type inhibitors show a unique activity profile. In the Eg5-PVZB1194 (a biphenyl-type inhibitor) crystal structure, loop L11, which is located in the entrance of the ?4/?6 allosteric-binding pocket, is missing due to crystal-packing effects. To better understand the role of this flexible loop upon biphenyl-type inhibitor-binding, MD simulations were performed to observe the L11 conformations from different states. It was demonstrated that L11 was more stabilized and showed less fluctuation when PVZB1194 was bound to Eg5. Residue Asn287 from L11 forms hydrogen bonding to the sulfone group of PVZB1194, whereby L11 moves inward to the ?4/?6 allosteric pocket and moves away from the pocket in absence of the inhibitor. Pharmacophore, three-dimensional (3D)-QSAR, and ADME studies of biphenyl-type inhibitors of Eg5 were also performed. A best pharmacophore model, DDRRH.6, was generated, having correlation coefficients in the 3D-QSAR study of R2 = 0.81 and Q2 = 0.64. Furthermore, docking studies were carried out to observe the interaction between the remaining biphenyl-type inhibitors with Eg5. In addition, on the basis of fragment docking, a structure-based pharmacophore was generated, which shares good overlap of the DHRR features of the pharmacophore model DDHRR.6. The structure-based pharmacophore also contains extra hydrogen-bond acceptors and hydrophobic groups, features which provide possibilities in developing new or improved series of compounds.
Project description:Loop 5 (L5) is a conserved loop that projects from the ?2-helix adjacent to the nucleotide site of all kinesin-family motors. L5 is critical to the function of the mitotic kinesin-5 family motors and is the binding site for several kinesin-5 inhibitors that are currently in clinical trials. Its conformational dynamics and its role in motor function are not fully understood. Our previous work using EPR spectroscopy suggested that L5 alters the nucleotide pocket conformation of the kinesin-5 motor Eg5 (Larson et al., 2010). EPR spectra of a spin-labeled nucleotide analog bound at the nucleotide site of Eg5 display a highly immobilized component that is absent if L5 is shortened or if the inhibitor STLC is added (Larson et al., 2010), which X-ray structures suggest stabilizes an L5 conformation pointing away from the nucleotide site. These data, coupled with the proximity of L5 to the nucleotide site suggest L5 could interact with a bound nucleotide, modulating function. Here we use molecular dynamics (MD) simulations of Eg5 to explore the interaction of L5 with the nucleotide site in greater detail. We performed MD simulations in which the L5-domain of the Eg5·ADP X-ray structure was manually deformed via backbone bond rotations. The L5-domain of Eg5 was sufficiently lengthy that portions of L5 could be located in proximity to bound ADP. The MD simulations evolved to thermodynamically stable structures at 300 K showing that L5 can interact directly with bound nucleotide with significant impingement on the ribose hydroxyls, consistent with the EPR spectroscopy results. Taken together, these data provide support for the hypothesis that L5 modulates Eg5 function via interaction with the nucleotide-binding site.
Project description:Human Kinesin-5 (Eg5) has a large number of known allosteric inhibitors that disrupt its mitotic function. Small-molecule inhibitors of Eg5 are candidate anti-cancer agents and important probes for understanding the cellular function. Here we show that Eg5 is capable of more than one type of microtubule interaction, and these activities can be controlled by allosteric agents. While both monastrol and S-trityl-L-cysteine inhibit Eg5 motility, our data reveal an unexpected ability of these loop5 targeting inhibitors to differentially control a novel Eg5 microtubule depolymerizing activity. Remarkably, small molecule loop5 effectors are able to independently modulate discrete functional interactions between the motor and microtubule track. We establish that motility can be uncoupled from the microtubule depolymerase activity and argue that loop5-targeting inhibitors of Kinesin-5 should not all be considered functionally synonymous. Also, the depolymerizing activity of the motor does not contribute to the genesis of monopolar spindles during allosteric inhibition of motility, but instead reveals a new function. We propose that, in addition to its canonical role in participating in the construction of the three-dimensional mitotic spindle structure, Eg5 also plays a distinct role in regulating the dynamics of individual microtubules, and thereby impacts the density of the mitotic spindle.
Project description:Mitotic kinesin Eg5 is an attractive anticancer drug target. Discovery of Eg5 inhibitors has been focused on targeting the 'monastrol-binding site'. However, acquired drug resistance has been reported for such inhibitors. Therefore, identifying new Eg5 inhibitors which function through a different mechanism(s) could complement current drug candidates and improve drug efficacy. In this study, we explored a novel allosteric site of Eg5 and identified new Eg5 inhibitors through structure-based virtual screening. Experiments with the saturation-transfer difference NMR demonstrated that the identified Eg5 inhibitor SRI35566 binds directly to Eg5 without involving microtubules. Moreover, SRI35566 and its two analogs significantly induced monopolar spindle formation in colorectal cancer HCT116 cells and suppressed cancer cell viability and colony formation. Together, our findings reveal a new allosteric regulation mechanism of Eg5 and a novel drug targeting site for cancer therapy.
Project description:The human Eg5 (HsEg5) protein is unique in its sensitivity to allosteric agents even among phylogenetic kin. For example, S-trityl-l-cysteine (STC) and monastrol are HsEg5 inhibitors that bind to a surface pocket created by the L5 loop, but neither compound inhibits the Drosophila Kinesin-5 homologue (Klp61F). Herein we ask whether or not drug sensitivity can be designed into Klp61F. Two chimeric Klp61F motor domains were engineered, bacterially expressed, and purified to test this idea. We report that effector binding can elicit a robust allosteric response comparable with HsEg5 in both motor domain chimeras. Furthermore, isothermal titration calorimetry confirms that the Klp61F chimeras have de novo binding affinities for both STC and monastrol. These data show that the mechanism of intramolecular communication between the three ligand binding sites is conserved in the Kinesin-5 family, and reconstitution of a drug binding cassette within the L5 pocket is sufficient to restore allosteric inhibition. However, the two compounds were not equivalent in their allosteric inhibition. This surprising disparity in the response between the chimeras to monastrol and STC suggests that there is more than one allosteric communication network for these effectors.
Project description:Kinesin spindle protein (KSP), known as Hs Eg5, a member of the kinesin-5 family, plays an important role in the formation and maintenance of the bipolar spindle. We previously reported S-trityl-l-cysteine derivatives as selective KSP inhibitors. Here, we report further optimizations using docking modeling in the L5 allosteric binding site, which led to the discovery of several high affinity derivatives with two fused phenyl rings in the trityl group giving low nanomolar range KSP ATPase inhibition. The representative derivatives potently inhibited cell growth of HCT116 cells in correlation with KSP inhibitory activities and significantly suppressed tumor growth in the xenograft model in vivo.
Project description:The human mitotic kinesin Eg5 represents a novel mitotic spindle target for cancer chemotherapy. We previously identified S-trityl-l-cysteine (STLC) and related analogues as selective potent inhibitors of Eg5. We herein report on the development of a series of 4,4,4-triphenylbutan-1-amine inhibitors derived from the STLC scaffold. This new generation systematically improves on potency: the most potent C-trityl analogues exhibit K(i)(app) ? 10 nM and GI(50) ? 50 nM, comparable to results from the phase II clinical benchmark ispinesib. Crystallographic studies reveal that they adopt the same overall binding configuration as S-trityl analogues at an allosteric site formed by loop L5 of Eg5. Evaluation of their druglike properties reveals favorable profiles for future development and, in the clinical candidate ispinesib, moderate hERG and CYP inhibition. One triphenylbutanamine analogue and ispinesib possess very good bioavailability (51% and 45%, respectively), with the former showing in vivo antitumor growth activity in nude mice xenograft studies.
Project description:Kinesins comprise a superfamily of molecular motors that drive a wide variety of cellular physiologies, from cytoplasmic transport to formation of the bipolar spindle in mitosis. These differing roles are reflected in corresponding polymorphisms in key kinesin structural elements. One of these is a unique loop and stem motif found in all kinesins and referred to as loop 5 (L5). This loop is longest in the mitotic kinesin Eg5 and is the target for a number of small molecule inhibitors, including ispinesib, which is being used in clinical trials in patients with cancer. In this study, we have used x-ray crystallography to identify a new structure of an Eg5-ispinesib complex and have combined this with transient state kinetics to identify a plausible sequence of conformational changes that occur in response to ispinesib binding. Our results demonstrate that ispinesib-induced structural changes in L5 from Eg5 lead to subsequent changes in the conformation of the switch II loop and helix and in the neck linker. We conclude that L5 in Eg5 simultaneously regulates the structure of both the ATP binding site and the motor's mechanical elements that generate force.
Project description:(+)-Morelloflavone (MF) is an antitumor biflavonoid that is found in the Garcinia species. Recently, we reported MF as a novel inhibitor of ATPase and microtubules-gliding activities of the kinesin spindle protein (Eg5) in vitro. Herein, we provide dynamical insights into the inhibitory mechanisms of MF against Eg5, which involves binding of the inhibitor to the loop5/?2/?3 allosteric pocket. Molecular dynamics simulations were carried out for 100 ns on eight complexes: Eg5-Adenosine diphosphate (Eg5-ADP), Eg5-ADP-S-trityl-l-cysteine (Eg5-ADP-STLC), Eg5-ADP-ispinesib, Eg5-ADP-MF, Eg5-Adenosine triphosphate (Eg5-ATP), Eg5-ATP-STLC, Eg5-ATP-ispinesib, and Eg5-ATP-MF complexes. Structural and energetic analyses were done using Umbrella sampling, Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) method, GROMACS analysis toolkit, and virtual molecular dynamics (VMD) utilities. The results were compared with those of the known Eg5 inhibitors; ispinesib, and STLC. Our data strongly support a stable Eg5-MF complex, with significantly low binding energy and reduced flexibility of Eg5 in some regions, including loop5 and switch I. Furthermore, the loop5 Trp127 was trapped in a downward position to keep the allosteric pocket of Eg5 in the so-called "closed conformation", comparable to observations for STLC. Altered structural conformations were also visible within various regions of Eg5, including switch I, switch II, ?2/?3 helices, and the tubulin-binding region, indicating that MF might induce modifications in the Eg5 structure to compromise its ATP/ADP binding and conversion process as well as its interaction with microtubules. The described mechanisms are crucial for understanding Eg5 inhibition by MF.