Novel Allosteric Pathway of Eg5 Regulation Identified through Multivariate Statistical Analysis of Hydrogen-Exchange Mass Spectrometry (HX-MS) Ligand Screening Data

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 β-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. 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 β-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. The kinesin family of molecular nanomotors convert the free energy of nucleotide hydrolysis into coordinated mechanical movement on microtubules (1.Gigant B. Wang W. Dreier B. Jiang Q. Pecqueur L. Pluckthun A. Wang C. Knossow M. Structure of a kinesin-tubulin complex and implications for kinesin motility.Nature Structural & Molecular Biology. 2013; 20: 1001-1007Crossref PubMed Scopus (113) Google Scholar, 2.Kikkawa M. Sablin E.P. Okada Y. Yajima H. Fletterick R.J. Hirokawa N. Switch-based mechanism of kinesin motors.Nature. 2001; 411: 439-445Crossref PubMed Scopus (295) Google Scholar). In cell division, the kinesin Eg5 is an essential element for maintaining proper spindle dynamics and preserving spindle bipolarity. Given its role in dividing cells, Eg5 is an important target for anticancer drugs with the potential to overcome the clinical deficiencies presented by conventional mitotic poisons (3.Waitzman J.S. Rice S.E. Mechanism and regulation of kinesin-5, an essential motor for the mitotic spindle.Biol. Cell. 2014; 106: 1-12Crossref PubMed Scopus (37) Google Scholar, 4.Perez-Melero C. KSP Inhibitors as Antimitotic Agents.Curr Top Med Chem. 2014; 14: 2286-2311Crossref PubMed Scopus (20) Google Scholar). Drug candidates like ispinesib inhibit Eg5 and induce mitotic arrest followed by apoptosis. Continued ligand development is driven in part by the observation of deactivating mutations in the drug binding region, and a dearth of successful monotherapies based on Eg5 inhibition. Ligands target a surface comprised of a dynamic loop (L5) that exerts allosteric control over motor function (5.Wojcik E.J. Buckley R.S. Richard J. Liu L. Huckaba T.M. Kim S. Kinesin-5: cross-bridging mechanism to targeted clinical therapy.Gene. 2013; 531: 133-149Crossref PubMed Scopus (49) Google Scholar). All ligands for which mechanistic information is available engage some element of an integrated allosteric circuit that regulates Eg5's motor function. ATP hydrolysis in the “switch I” domain influences the “switch II” microtubule binding region 10's of angstroms away, and controls the orientation of the neck-linker (NL) 1The abbreviations used are: NL, Neck linker; HCA, Hierarchical clustering analysis; HX-MS, Hydrogen-deuterium exchange mass spectrometry; IC50, Half maximal inhibitory concentration; IPTG, Isopropyl β-d-1-thiogalactopyranoside; MT, Microtubule; MESG, 2-amino-6-mercapto-7-methylpurine ribonucleoside; OPLS-DA, Orthogonal-projection to latent structures discriminant analysis; PCA, Principal component analysis; PMSF, Phenylmethylsulfonyl fluoride; R2, Goodness of fit; Q2, Predictive ability; STLC, S-Trityl-l-Cysteine; VIP, Variable importance to projection. 1The abbreviations used are: NL, Neck linker; HCA, Hierarchical clustering analysis; HX-MS, Hydrogen-deuterium exchange mass spectrometry; IC50, Half maximal inhibitory concentration; IPTG, Isopropyl β-d-1-thiogalactopyranoside; MT, Microtubule; MESG, 2-amino-6-mercapto-7-methylpurine ribonucleoside; OPLS-DA, Orthogonal-projection to latent structures discriminant analysis; PCA, Principal component analysis; PMSF, Phenylmethylsulfonyl fluoride; R2, Goodness of fit; Q2, Predictive ability; STLC, S-Trityl-l-Cysteine; VIP, Variable importance to projection. domain that is involved in the power stroke (5.Wojcik E.J. Buckley R.S. Richard J. Liu L. Huckaba T.M. Kim S. Kinesin-5: cross-bridging mechanism to targeted clinical therapy.Gene. 2013; 531: 133-149Crossref PubMed Scopus (49) Google Scholar). All known L5 inhibitors slow ADP release and preserve an ATP-like state. They induce a conformational change in the switch II region and their study suggests that motor control necessarily involves switch II (6.Yan Y. Sardana V. Xu B. Homnick C. Halczenko W. Buser C.A. Schaber M. Hartman G.D. Huber H.E. Kuo L.C. Inhibition of a mitotic motor protein: where, how, and conformational consequences.J. Mol. Biol. 2004; 335: 547-554Crossref PubMed Scopus (197) Google Scholar, 7.Kaan H.Y. Major J. Tkocz K. Kozielski F. Rosenfeld S.S. “Snapshots” of ispinesib-induced conformational changes in the mitotic kinesin Eg5.Journal of Biol. Chem. 2013; 288: 18588-18598Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Support for this idea is found in the recent discovery of inhibitors that directly engage switch II (8.Ulaganathan V. Talapatra S.K. Rath O. Pannifer A. Hackney D.D. Kozielski F. Structural Insights into a Unique Inhibitor Binding Pocket in Kinesin Spindle Protein.J Am Chem Soc. 2013; 135: 2263-2272Crossref PubMed Scopus (40) Google Scholar). Terpendole E, the first natural product inhibitor of kinesin-5 to be discovered (9.Nakazawa J. Yajima J. Usui T. Ueki M. Takatsuki A. Imoto M. Toyoshima Y.Y. Osada H. A novel action of terpendole E on the motor activity of mitotic Kinesin Eg5.Chemistry & Biology. 2003; 10: 131-137Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), appears to be a different class of ligand. Although it generates the classical monoastral spindle seen with L5-site ligands, recent work suggests that functional inhibition proceeds through a different mechanism, and perhaps through a novel site (10.Tarui Y. Chinen T. Nagumo Y. Motoyama T. Hayashi T. Hirota H. Muroi M. Ishii Y. Kondo H. Osada H. Usui T. Terpendole E and its derivative inhibit STLC- and GSK-1-resistant Eg5.Chembiochem. 2014; 15: 934-938Crossref PubMed Scopus (20) Google Scholar). Interestingly, Terpendole E inhibits Eg5 mutants that are resistant to loop L5 inhibitors and switch II-directed inhibitors (9.Nakazawa J. Yajima J. Usui T. Ueki M. Takatsuki A. Imoto M. Toyoshima Y.Y. Osada H. A novel action of terpendole E on the motor activity of mitotic Kinesin Eg5.Chemistry & Biology. 2003; 10: 131-137Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 10.Tarui Y. Chinen T. Nagumo Y. Motoyama T. Hayashi T. Hirota H. Muroi M. Ishii Y. Kondo H. Osada H. Usui T. Terpendole E and its derivative inhibit STLC- and GSK-1-resistant Eg5.Chembiochem. 2014; 15: 934-938Crossref PubMed Scopus (20) Google Scholar). To explore this mechanism in further detail and to determine if an alternative regulatory pathway exists, we implemented a large-scale hydrogen deuterium exchange mass spectrometry (HX-MS) method for conformational analysis. HX-MS is a pseudo-structural method that can provide a peptide-level resolution of conformational response data for ligand binding events, in the form of variable mass shifts (11.Chalmers M.J. Busby S.A. Pascal B.D. West G.M. Griffin P.R. Differential hydrogen/deuterium exchange mass spectrometry analysis of protein-ligand interactions.Expert Rev Proteomics. 2011; 8: 43-59Crossref PubMed Scopus (180) Google Scholar, 12.Bennett M.J. Barakat K. Huzil J.T. Tuszynski J. Schriemer D.C. Discovery and characterization of the laulimalide-microtubule binding mode by mass shift perturbation mapping.Chem Biol. 2010; 17: 725-734Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The approach is useful for mechanism-of-action studies (13.Kojetin D.J. Matta-Camacho E. Hughes T.S. Srinivasan S. Nwachukwu J.C. Cavett V. Nowak J. Chalmers M.J. Marciano D.P. Kamenecka T.M. Shulman A.I. Rance M. Griffin P.R. Bruning J.B. Nettles K.W. Structural mechanism for signal transduction in RXR nuclear receptor heterodimers.Nat Commun. 2015; 6: 8013Crossref PubMed Scopus (88) Google Scholar), and is potentially very powerful for driving the development of pharmacophore models. However, current strategies for HX-MS data analysis cannot easily of shift data for protein-ligand where ligand is to a control and are for of a the of in HX-MS data G.M. V. J. Chalmers M.J. Griffin P.R. perturbation of the conformational for the receptor by 2011; Full Text Full Text PDF PubMed Scopus Google Scholar), and clustering a of M.J. Pascal B.D. S. J. Griffin P.R. for the of Differential 2011; PubMed Scopus Google Scholar). However, we that to of an to the data available HX-MS and a in can be by of of variable with and with strategies of the of information the variable of for mass that are the statistical presented by HX-MS and where multivariate have demonstrated B. in 2013; Google Scholar, F. K. A. E. A. as a to of PubMed Scopus Google Scholar). In this we can be used to shift with a to and HX-MS data ligand The was with of nucleotide hydrolysis and to a of Eg5 that Terpendole E a mechanism of which appears to of the L5 binding site region. The motor domain of Eg5 was and a S. L. A. C. Hackney D.D. Kozielski F. of the mitotic inhibitor with kinesin 2003; PubMed Scopus Google Scholar), with some was into E. were with of the was to of with for an of was was with the of were on a were and the in A and with were with a and for and was and and Eg5 was with an in a were with and were to be by a of kinesin inhibitors were in and HX-MS inhibitor, in and inhibitor, in were an in the were by of inhibitor with and on was by to a of for data were for the inhibitor where to was for were and with of active in M. M. Y. Schriemer D.C. for hydrogen/deuterium exchange mass Cell. Proteomics. 2013; Full Text Full Text PDF PubMed Scopus (49) Google Scholar). was into a with a and a for drug and control were in with a to an The data was in A of the control was followed by ligand in the was the of analysis for was used to for analysis. a was against a in this study and with a mass for of for and a of was are and were The was into M. V. J. C.A. M. L. 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The for was for were using a statistical in All HX-MS data were on with the A of shift data and was with the HX-MS inhibitor and of The was into for analysis. was and used in component analysis an model of which was for of and based on and and with the to latent structures discriminant analysis model was using binding as class The model was by a with a & Google Scholar, guide to of Eg5 of information for to of of information for to of E Inhibition of information for to of inhibition. in a A Kinesin was used to the activity of the and determine in which microtubules are for the shift the of 2-amino-6-mercapto-7-methylpurine to by in the of generated by the kinesin inhibitor to was with Eg5 and ATP in a and was with a for were the of drug response were to the data in using a least were used to a strategy for different structures of Eg5 the of the to the in known Structure was based on an analysis of the binding the structures and where was as and structures with an were Molecular was for this G.M. W. and with receptor Chem. PubMed Scopus Google Scholar). The binding site was as by and In we different and were to the using the in Molecular the structures were in were the were to the and were using G.M. W. and with receptor Chem. PubMed Scopus Google Scholar). The were as to the binding with of The ligand structures were and an of was generated using was using and the function was used to the G.M. W. and with receptor Chem. PubMed Scopus Google Scholar, G.M. A free energy with Chem. PubMed Scopus Google Scholar). for the were a of maximal and of G.M. R.S. using a and an binding free energy Chem. Scopus Google Scholar). clustering using a of the as the binding the hydrogen a of and a maximal of were and and Terpendole E were and were and were was and were A analysis of the Eg5 motor with and a known inhibitor, STLC, was used to a for studies have that a HX-MS is for analysis of protein-ligand M.J. Busby S.A. Pascal B.D. Griffin P.R. A hydrogen deuterium exchange method for the characterization of Google Scholar). that a in shift for the of Eg5 with deuterium and to A of the was to and which we have Schriemer D.C. of the in and its in of Chem. PubMed Scopus Google Scholar). this was in and which the known binding site studies have that loop L5 by to form an binding site (6.Yan Y. Sardana V. Xu B. Homnick C. Halczenko W. Buser C.A. Schaber M. Hartman G.D. Huber H.E. Kuo L.C. Inhibition of a mitotic motor protein: where, how, and conformational consequences.J. Mol. Biol. 2004; 335: 547-554Crossref PubMed Scopus (197) Google Scholar). The shifts we in this region are by an of the S. S. E. Kozielski F. of the binding region of a inhibitor of the mitotic kinesin 2004; PubMed Scopus Google Scholar). in and the and that the region for the motor S. S. E. Kozielski F. of the binding region of a inhibitor of the mitotic kinesin 2004; PubMed Scopus Google Scholar). The region part of the switch II which conformational changes nucleotide M. Sablin E.P. Okada Y. Yajima H. Fletterick R.J. Hirokawa N. Switch-based mechanism of kinesin motors.Nature. 2001; 411: 439-445Crossref PubMed Scopus (295) Google and ligand binding (6.Yan Y. Sardana V. Xu B. Homnick C. Halczenko W. Buser C.A. Schaber M. Hartman G.D. Huber H.E. Kuo L.C. Inhibition of a mitotic motor protein: where, how, and conformational consequences.J. Mol. Biol. 2004; 335: 547-554Crossref PubMed Scopus (197) Google Scholar). in this region were identified in HX-MS studies with STLC, of S. S. E. Kozielski F. of the binding region of a inhibitor of the mitotic kinesin 2004; PubMed Scopus Google Scholar, S. S. Kozielski F. E. of hydrogen/deuterium exchange mass spectrometry and as a to the binding region of inhibitors the mitotic kinesin Commun. 20: PubMed Scopus Google Scholar). analysis that this HX-MS provide the to of inhibitor the binding its properties and domain for of elements of the Eg5 motor domain involved in nucleotide binding the binding and the microtubule binding region M. Sablin E.P. Okada Y. Yajima H. Fletterick R.J. Hirokawa N. Switch-based mechanism of kinesin motors.Nature. 2001; 411: 439-445Crossref PubMed Scopus (295) Google Scholar, A. J. A. E. of kinesin and Cell. PubMed Scopus Google Scholar). of Eg5 to ADP and are to the in of Eg5 with with an of are based on the The study was to a of kinesin directly Eg5 and for kinesins. The Eg5 inhibitors a of as in an ATP using the Eg5 motor domain HX-MS data for protein-ligand inhibitor, was in using which a and of the Eg5 motor domain The shift data for ligand to the was using M. V. J. C.A. M. L. Schriemer D.C. for 2014; Full Text Full Text PDF PubMed Scopus Google Scholar), which provide a of against the However, the approach is for a of the of HX-MS this we a multivariate data analysis which is in HX-MS data is with functional evidence to the development of a multivariate statistical which ligands to a functional a of that the and a functional In to the of we used with is an method that to data to like B. in 2013; Google Scholar). is a approach that the to of known class information as an additional Hierarchical to an Scopus Google Scholar, M. M. O. E. J. discriminant the of and 20: Scopus Google Scholar). information is used to the in the into that are of class M. M. O. E. J. discriminant the of and 20: Scopus Google Scholar). In this it to the statistical method to HX-MS the functional data with the the to form a in have used very in the data structures in a B. in 2013; Google Scholar, K. least a for the analysis of 8: PubMed Scopus Google Scholar). They class which when the of is limited M. M. O. E. J. discriminant the of and 20: Scopus Google Scholar). is the natural found in the HX-MS the of a can to a be by its a the can be the of the orthogonal signal in for which in the of HX-MS the information in the functional HX-MS to and To HX-MS data by and OPLS-DA, of inhibitor were the data a strategy of data used in we that the in a of and that to data cannot be with The additional of the data involved was for a in multivariate as HX-MS data is to the of M.J. and the information of PubMed Scopus Google Scholar). The model of a component and orthogonal A which the of by the of the and a were using for and models. that the model was and for the of in is in H. the Predictive of a Scopus Google Scholar). model demonstrated binding and a that analysis with a this with the Eg5 inhibition was to the in presented as a The analysis was based on a a of the data as ligands and with on Eg5 activity the used as a are and the of an change in In the of ligands, the of inhibition to the orthogonal The which to can be used for further of the in the L5 region to the The the of the shifts for that the binding site (6.Yan Y. Sardana V. Xu B. Homnick C. Halczenko W. Buser C.A. Schaber M. Hartman G.D. Huber H.E. Kuo L.C. Inhibition of a mitotic motor protein: where, how, and conformational consequences.J. Mol. Biol. 2004; 335: 547-554Crossref PubMed Scopus (197) Google Scholar, 7.Kaan H.Y. Major J. Tkocz K. Kozielski F. Rosenfeld S.S. “Snapshots” of ispinesib-induced conformational changes in the mitotic kinesin Eg5.Journal of Biol. Chem. 2013; 288: 18588-18598Abstract Full Text Full Text PDF PubMed Scopus (24) Google and be an of the for However, the are to when of an are to to the ligand and the statistical for identifying important changes over the is by an is to the in to that to the In a can be generated the model which that a variable is identified in the the can be by of the shift to the control offer an evaluation of the of the on a L5 inhibitors like in of as as in the and the of the binding site and L5 ligands a conformational response the switch II site H.Y. Major J. Tkocz K. Kozielski F. Rosenfeld S.S. “Snapshots” of ispinesib-induced conformational changes in the mitotic kinesin Eg5.Journal of Biol. Chem. 2013; 288: 18588-18598Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), which is in HX-MS as in the and and identified this region as a to the of binding and the first component The conformational change we in the switch II region to a of an inhibitor with a IC50, some the a effect on the conformational of the of L5 appears that partial L5 it is by a conformational change in and is to the switch II region and However, this allosteric pathway is by ligands with the of Terpendole E. Terpendole E is very in the model L5 ligands of L5 and very of a effect on the region of the binding site this is the known binding site for canonical we a molecular analysis of Terpendole E on the region, to determine if site provide an for the different for we generated a analysis of the Eg5 inhibitors used in this against a diverse of Eg5 and the known binding this Terpendole E was to in the binding site in a that the inhibitors The model a different binding through a hydrogen and an orientation that is L5 and analysis suggests the conformational of the was in the HX-MS the a analysis. that the model a recent observation that Terpendole E active against a in L5 (10.Tarui Y. Chinen T. Nagumo Y. Motoyama T. Hayashi T. Hirota H. Muroi M. Ishii Y. Kondo H. Osada H. Usui T. Terpendole E and its derivative inhibit STLC- and GSK-1-resistant Eg5.Chembiochem. 2014; 15: 934-938Crossref PubMed Scopus (20) Google Scholar). is Terpendole E to ligands, and it is to inhibition by Terpendole E to allosteric control over the switch II region. conformational changes are in cannot be to as Terpendole E an in the of ligands into the of Terpendole E the of the inhibitors using a to additional that the of this the Although the the inhibitor and microtubule binding the β-sheet core are A unique conformational response to this inhibitor is in the core β-sheet of the motor domain and where an in the and The change is in in the of the β-sheet The appears as are very slow in the that Terpendole E motor inhibition using an alternative the β-sheet in some To on the microtubule a on the neck-linker (NL) is a element of kinesin motor function. inhibitors the the motor by a conformational change in the switch II region S.K. Kozielski F. The of the Biol. PubMed Scopus Google Scholar, T. Kozielski F. of the motor domain of the Mol. Biol. 2004; PubMed Scopus Google Scholar). changes are and the release of The switch II region, and the is a element of the microtubule binding domain in inhibitors the which may to a motor J.C. T.M. inhibition of the mitotic kinesin Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, an mode of Biol. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar). The HX-MS data we for inhibitors with this mechanism with Terpendole E we conformational effect on the switch II region, and of Terpendole E on microtubule binding in a A role for the core β-sheet in motor activity is of in A. structures essential of J. 2004; PubMed Scopus Google Scholar, S.A. by kinesin and motor 2013; PubMed Scopus Google Scholar). In hydrolysis product release to a of the core data a effect (1.Gigant B. Wang W. Dreier B. Jiang Q. Pecqueur L. Pluckthun A. Wang C. Knossow M. Structure of a kinesin-tubulin complex and implications for kinesin motility.Nature Structural & Molecular Biology. 2013; 20: 1001-1007Crossref PubMed Scopus (113) Google Scholar), studies of Eg5 a role for and β-sheet conformational of the to the β-sheet core is essential for of the motor in the kinesin the Buckley S. Richard J. C. E.J. Kim S. drug is to mechanochemical changes in the kinesin-5 motor Biol. Chem. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). was in this study that Eg5 to inhibitors can be if is a in as in a resistant work be to determine if the pathway influences motor the of and of its in ADP is to and ligands in binding based on their HX-MS and this with activity data to the that with the functional Although of drug are in multivariate of the we have are to and of response in of They and offer a useful to data into the are as HX-MS to in and of ligands different on statistical routines the that we in the of HX-MS the of a mass shift in a drug when it is with and over of ligands, it can a the of the data In of the in this study can be to a of the can be as for of in HX-MS E. J. C. S. strategy for and in 2015; PubMed Scopus Google Scholar, A. Y. Wang H. T. Y. M. N. H. H. W. analysis for discovery and of in with Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar, Schriemer D.C. for shift in 2011; Scopus Google Scholar).

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