ATP-competitive inhibitors of the mitotic kinesin KSP that function via an allosteric mechanism

Luo, Liang; Parrish, Cynthia A.; Nevins, Neysa; McNulty, Dean E.; Chaudhari, Amita M.; Carson, Jeffery D; Sudakin, Valery; Shaw, Antony N.; Lehr, Ruth; Zhao, Huizhen; Sweitzer, Sharon; Lad, Latesh; Wood, Kenneth W.; Sakowicz, Roman; Annan, Roland S.; Huang, Pearl S.; Jackson, Jeffrey R.; Dhanak, Dashyant; Copeland, Robert A.; Auger, Kurt R.

doi:10.1038/nchembio.2007.34

Cited by 97 publications

(119 citation statements)

References 26 publications

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“…The ability to confer drug resistance by introducing mutations in the induced fit binding pocket of Eg5 raises the possibility that such mutations may be encountered in tumors during chemotherapy if Eg5 inhibitors were to enter the clinic. In support of our results it has been reported that Ispinesib-resistant cancerous cells were found to harbor a D130V substitution located in loop L5 of Eg5 [46]. …”

Section: Monastrol and Stlc Have Been Characterized As Eg5 Specific Isupporting

confidence: 92%

Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl-l-cysteine in tumor derived cell lines

Tcherniuk

Lis

Kozielski

et al. 2010

Biochemical Pharmacology

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. Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-Trityl-L-Cysteine in tumor derived cell lines.. Biochemical Pharmacology, Elsevier, 2010, 79 (6) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. confirms the target specificity of monastrol and STLC for Eg5. These data also suggest a possible mechanism by which drug resistance may occur in tumors treated with agents targeting Eg5.

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Section: Monastrol and Stlc Have Been Characterized As Eg5 Specific Isupporting

confidence: 92%

Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl-l-cysteine in tumor derived cell lines

Tcherniuk

Lis

Kozielski

et al. 2010

Biochemical Pharmacology

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“…Only the E116R and D130K motor proteins displayed differing levels of inhibition between the two compounds used, the former being more sensitive to monastrol and the latter to STC. Of the C-terminal L5 mutants, D130V (open blue triangles) and A133D (filled red triangles) were the most insensitive to either inhibitor, as anticipated from studies on ispinesib (16). For the N-terminal L5 substitutions, loss of allosteric compound sensitivity was more marked in samples with aliphatic residue substitutions at position 116, compared with Eg5 proteins with charged residues at these positions.…”

Section: Preservation Of N-terminal Local Structure and C-terminal Chmentioning

confidence: 82%

“…1B). However, we note that the E116V and E118N substitutions are present in the Drosophila Kinesin-5 protein that is insensitive to allosteric inhibitors and that the D130V and A133D mutations were found in human cell lines resistant to Eg5 allosteric inhibitors (16).…”

Section: Substitutions Of L5 Residues Results In Altered Sds-pagementioning

confidence: 99%

“…In line with this conclusion, the clustering of the Glu-116 mutant samples in PCA argues that a large 1646 cm Ϫ1 component is strongly correlated with drug resistance. Thus, loss of the 3 10 helix in the L5 loop unilaterally prohibits binding of small molecules to the allosteric site, as shown by the D130V mutation that displays a large 1646 cm Ϫ1 component and confers resistance in Eg5 to the clinical trial drugs in purified form, in tumor cell culture, and in xenograft model systems (16).…”

Section: Distortions In ␤-Sheets Are a Conserved Means Of Mechanochemmentioning

confidence: 99%

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Allosteric Drug Discrimination Is Coupled to Mechanochemical Changes in the Kinesin-5 Motor Core

Kim

Buckley

Learman

et al. 2010

Journal of Biological Chemistry

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Essential in mitosis, the human Kinesin-5 protein is a target for >80 classes of allosteric compounds that bind to a surfaceexposed site formed by the L5 loop. Not established is why there are differing efficacies in drug inhibition. Here we compare the ligand-bound states of two L5-directed inhibitors against 15 Kinesin-5 mutants by ATPase assays and IR spectroscopy. Biochemical kinetics uncovers functional differences between individual residues at the N or C termini of the L5 loop. Infrared evaluation of solution structures and multivariate analysis of the vibrational spectra reveal that mutation and/or ligand binding not only can remodel the allosteric binding surface but also can transmit long range effects. Changes in L5-localized 3 10 helix and disordered content, regardless of substitution or drug potency, are experimentally detected. Principal component analysis couples these local structural events to two types of rearrangements in ␤-sheet hydrogen bonding. These transformations in ␤-sheet contacts are correlated with inhibitory drug response and are corroborated by wild type Kinesin-5 crystal structures. Despite considerable evolutionary divergence, our data directly support a theorized conserved element for long distance mechanochemical coupling in kinesin, myosin, and F 1 -ATPase. These findings also suggest that these relatively rapid IR approaches can provide structural biomarkers for clinical determination of drug sensitivity and drug efficacy in nucleotide triphosphatases.Allostery is important in controlled catalysis, signal transduction, and apoptosis (1). The classic view of proteins demonstrating this property (2) asserts that binding of a ligand at one site provokes conformational changes at a remote, second site. Recent studies (3) evaluating underlying mechanisms of allostery alternatively suggest that ligand binding results in selection of preexisting conformational substates. Implicit in the latter model is the principle that interactions between the orthosteric and allosteric sites are tightly linked through structure and thermodynamics (4). Active challenges in structural biology, which are central to this work, are deciphering the chemical nature of the ligand-protein interactions as well as how energy is transduced through protein structures to transmit allosteric events.Our experimental model, the human Kinesin-5 motor protein (Eg5 or KSP), plays key roles in bipolar mitotic spindle formation and is a protein target for allosteric compounds (5-7) that alter catalytic ATPase activity of the protein (8, 9). Biochemical studies demonstrate a wide concentration range of inhibition by these compounds (10 -12); there may be differences in the kinetic mechanism of allostery (13-15), and even allosteric activation (16) is possible. The best characterized inhibitors, monastrol (10) and S-trityl-L-cysteine (STC)2 (11), were uncovered from independent chemical screens.Interest in these allosteric compounds has been acute because they are potential anticancer agents. Additionally, these compo...

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“…Interestingly, a distinct, but related, effect has recently been described in a mutant of the mitotic kinesin KSP (also called Eg5). The KSP mutant was discovered in a laboratory screen and confers drug resistance by an allosteric mechanism involving enhanced affinity for ATP (33).…”

Section: Discussionmentioning

confidence: 99%

The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP

Yun¹,

Mengwasser²,

Toms³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

1,742

1,480

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Lung cancers caused by activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to small molecule tyrosine kinase inhibitors (TKIs), but the efficacy of these agents is often limited because of the emergence of drug resistance conferred by a second mutation, T790M. Threonine 790 is the ''gatekeeper'' residue, an important determinant of inhibitor specificity in the ATP binding pocket. The T790M mutation has been thought to cause resistance by sterically blocking binding of TKIs such as gefitinib and erlotinib, but this explanation is difficult to reconcile with the fact that it remains sensitive to structurally similar irreversible inhibitors. Here, we show by using a direct binding assay that T790M mutants retain low-nanomolar affinity for gefitinib. Furthermore, we show that the T790M mutation activates WT EGFR and that introduction of the T790M mutation increases the ATP affinity of the oncogenic L858R mutant by more than an order of magnitude. The increased ATP affinity is the primary mechanism by which the T790M mutation confers drug resistance. Crystallographic analysis of the T790M mutant shows how it can adapt to accommodate tight binding of diverse inhibitors, including the irreversible inhibitor HKI-272, and also suggests a structural mechanism for catalytic activation. We conclude that the T790M mutation is a ''generic'' resistance mutation that will reduce the potency of any ATP-competitive kinase inhibitor and that irreversible inhibitors overcome this resistance simply through covalent binding, not as a result of an alternative binding mode. lung cancer ͉ tyrosine kinase ͉ x-ray crystallography

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ATP-competitive inhibitors of the mitotic kinesin KSP that function via an allosteric mechanism

Cited by 97 publications

References 26 publications

Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl-l-cysteine in tumor derived cell lines

Mutations in the human kinesin Eg5 that confer resistance to monastrol and S-trityl-l-cysteine in tumor derived cell lines

Allosteric Drug Discrimination Is Coupled to Mechanochemical Changes in the Kinesin-5 Motor Core

The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP

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