The Discovery and Development of Eg5 Inhibitors for the Clinic

  • James A. D. GoodEmail author
  • Giacomo Berretta
  • Nahoum G. Anthony
  • Simon P. MackayEmail author


The mitotic kinesin Eg5 (also known as kinesin spindle protein, KSP, Kif11, a member of the kinesin-5 family) represents an attractive oncology drug target in the ongoing development of anti-mitotic drugs that selectively block mitosis through disruption to the mitotic spindle. In this state-of-the-art review, we outline the progress that has been made in the development of Eg5 inhibitors for clinical use. We evaluate the preclinical development and attributes of key Eg5 inhibitors that have undergone clinical evaluation or extensive preclinical optimisation, and discuss the medicinal chemistry strategies utilised in their design to overcome the challenges encountered during lead optimisation. We critically analyse the progress that has been made towards delivering clinical benefits, and the wider implications this has in the utility of mitotic kinesin inhibitors as prospective oncology drugs.


Anti-mitotic Drug discovery Multiple myeloma Kinesins Eg5 

Abbreviations and Definitions


α-1-acid glycoprotein


Acute myeloid leukemia

Basal Eg5 inhibition

Inhibition of the basal ATPase activity of Eg5


Clinical benefit rate


Cytochrome P450


Drug metabolism and pharmacokinetics




Fraction unbound


Human ether-a-go-go-related gene




High-throughput screening




Estimated apparent K i value


Antiapoptotic protein myeloid cell leukemia 1


Multidrug resistance


Multiple myeloma



MT Eg5 inhibition

Inhibition of the microtubule stimulated ATPase activity of Eg5


Maximum tolerated dose


National Cancer Institute


No inhibition


Overall response rate






Relapsed/refractory multiple myeloma


Structure activity relationship


S-trityl L-cysteine



We apologise to authors whose work we were unable to include due to limitations of space. We thank Prof. Frank Kozielski for helpful comments on the manuscript. We are grateful to Cancer Research UK for supporting the STLC programme and funding the postdoctoral positions of NGA and GB on the Small Molecule Drug Discovery Programme, in association with Prostate Cancer UK. JADG thanks the Umeå Centre for Microbial Research for funding his postdoctoral research at Umeå University.

Copyright Acknowledgements: Excerpts from this chapter appeared previously in the doctoral thesis of James A. D. Good [76]. The data appearing in Tables 2.1, 2.4 and 2.6 was adapted with permission from the cited references and is copyright American Chemical Society [15, 21, 25, 28, 58, 67]. The date appearing in Tables 2.2, 2.3 and 2.5 was adapted with permission from the cited references and is copyright Elsevier [26, 31, 33, 34, 35, 36, 37, 38, 39].


  1. 1.
    Mayer TU et al (1999) Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286:971–974CrossRefPubMedGoogle Scholar
  2. 2.
    Zhu C et al (2005) Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference. Mol Biol Cell 16:3187–3199CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Maliga Z, Kapoor TM, Mitchison TJ (2002) Evidence that monastrol is an allosteric inhibitor of the mitotic kinesin Eg5. Chem Biol 9:989–996CrossRefPubMedGoogle Scholar
  4. 4.
    Yan Y et al (2004) Inhibition of a mitotic motor protein: where, how, and conformational consequences. J Mol Biol 335:547–554CrossRefPubMedGoogle Scholar
  5. 5.
    Bergnes G, Brejc K, Belmont L (2005) Mitotic kinesins: prospects for antimitotic drug discovery. Curr Top Med Chem 5:127–145CrossRefPubMedGoogle Scholar
  6. 6.
    Knight SD, Parrish CA (2008) Recent progress in the identification and clinical evaluation of inhibitors of the mitotic kinesin KSP. Curr Top Med Chem 8:888–904CrossRefPubMedGoogle Scholar
  7. 7.
    Jiang C, You Q (2013) Kinesin spindle protein inhibitors in cancer: a patent review (2008 – present). Expert Opin Ther Pat 23:1547–1560CrossRefPubMedGoogle Scholar
  8. 8.
    Sakowicz R et al (2004) Antitumor activity of a kinesin inhibitor. Cancer Res 64:3276–3280CrossRefPubMedGoogle Scholar
  9. 9.
    Johnson RK et al (2002) SB-715992, a potent and selective inhibitor of the mitotic kinesin KSP, demonstrates broad-spectrum activity in advanced murine tumors and human tumor xenografts. Proc Annu Meet Am Assoc Cancer Res 43:269Google Scholar
  10. 10.
    Chu Q et al (2003) A phase I study to determine the safety and pharmacokinetics of IV administered SB-715992, a novel kinesin spindle protein (KSP) inhibitor, in patients with solid tumors. Proc Am Soc Clin Oncol 22:525Google Scholar
  11. 11.
    Lad L et al (2008) Mechanism of inhibition of human KSP by Ispinesib. Biochemistry 47:3576–3585CrossRefPubMedGoogle Scholar
  12. 12.
    Zhang B, Liu J-F, Xu Y, Ng S-C (2008) Crystal structure of HsEg5 in complex with clinical candidate CK0238273 provides insight into inhibitory mechanism, potency, and specificity. Biochem Biophys Res Commun 372:565–570CrossRefPubMedGoogle Scholar
  13. 13.
    Talapatra SK, Schuttelkopf AW, Kozielski F (2012) The structure of the ternary Eg5-ADP-ispinesib complex. Acta Crystallogr D 68:1311–1319CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Carol H et al (2009) Initial testing (stage 1) of the kinesin spindle protein inhibitor ispinesib by the pediatric preclinical testing program. Pediatr Blood Cancer 53:1255–1263CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Good JAD et al (2013) Optimized S-trityl-L-cysteine-based inhibitors of kinesin spindle protein with potent in vivo antitumor activity in lung cancer xenograft models. J Med Chem 56:1878–1893CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Rath O, Kozielski F (2012) Kinesins and cancer. Nat Rev Cancer 12:527–539CrossRefPubMedGoogle Scholar
  17. 17.
    Matsuno K, Sawada J, Asai A (2008) Therapeutic potential of mitotic kinesin inhibitors in cancer. Expert Opin Ther Pat 18:253–274CrossRefGoogle Scholar
  18. 18.
    Bergnes G et al (2002) Mitotic kinesin-targeted antitumor agents: discovery, lead optimization and anti-tumor activity of a series of novel quinazolinones as inhibitors of kinesin spindle protein (KSP). Abstr Pap Am Chem Soc 223:B140Google Scholar
  19. 19.
    Holland JP, Jones MW, Cohrs S, Schibli R, Fischer E (2013) Fluorinated quinazolinones as potential radiotracers for imaging kinesin spindle protein expression. Bioorg Med Chem 21:496–507CrossRefPubMedGoogle Scholar
  20. 20.
    Bergnes G et al (2002) Mitotic kinesin-targeted antitumor agents: discovery, lead optimization and anti-tumor activity of a series of novel quinazolinones as inhibitors of kinesin spindle protein (KSP). Proc Annu Meet Am Assoc Cancer Res 43:736Google Scholar
  21. 21.
    Wang F et al (2012) Triphenylbutanamines: kinesin spindle protein inhibitors with in vivo antitumor activity. J Med Chem 55:1511–1525CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Ansbro MR, Shukla S, Ambudkar SV, Yuspa SH, Li L (2013) Screening compounds with a novel high-throughput ABCB1-mediated efflux assay identifies drugs with known therapeutic targets at risk for multidrug resistance interference. PLoS One 8:e60334CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Jackson JR et al (2006) A second generation KSP inhibitor, SB-743921, is a highly potent and active therapeutic in preclinical models of cancer. AACR Meet Abstr 2006:B11Google Scholar
  24. 24.
    Holen K et al (2011) A first in human study of SB-743921, a kinesin spindle protein inhibitor, to determine pharmacokinetics, biologic effects and establish a recommended phase II dose. Cancer Chemother Pharmacol 67:447–454CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Cox CD et al (2008) Kinesin spindle protein (KSP) inhibitors. 9. Discovery of (2S)-4-(2,5-difluorophenyl)-N-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide (MK-0731) for the treatment of taxane-refractory cancer. J Med Chem 51:4239–4252CrossRefPubMedGoogle Scholar
  26. 26.
    Schiemann K et al (2010) The discovery and optimization of hexahydro-2H-pyrano[3,2-c]quinolines (HHPQs) as potent and selective inhibitors of the mitotic kinesin-5. Bioorg Med Chem Lett 20:1491–1495CrossRefPubMedGoogle Scholar
  27. 27.
    Kim ED et al (2010) Allosteric drug discrimination is coupled to mechanochemical changes in the kinesin-5 motor core. J Biol Chem 285:18650–18661PubMedCentralPubMedGoogle Scholar
  28. 28.
    Theoclitou M-E et al (2011) Discovery of (+)-N-(3-Aminopropyl)-N-[1-(5-benzyl-3-methyl-4-oxo-[1,2]thiazolo[5,4-d]pyrimidin-6-yl)-2-methylpropyl]-4-methylbenzamide (AZD4877), a kinesin spindle protein inhibitor and potential anticancer agent. J Med Chem 54:6734–6750CrossRefPubMedGoogle Scholar
  29. 29.
    Kantarjian H et al (2012) Phase I/II multicenter study to assess the safety, tolerability, pharmacokinetics and pharmacodynamics of AZD4877 in patients with refractory acute myeloid leukemia. Invest New Drugs 30:1107–1115CrossRefPubMedCentralPubMedGoogle Scholar
  30. 30.
    Cox CD, Garbaccio RM (2010) Discovery of allosteric inhibitors of kinesin spindle protein (KSP) for the treatment of taxane-refractory cancer: MK-0731 and analogs. Anticancer Agents Med Chem 10:697–712CrossRefPubMedGoogle Scholar
  31. 31.
    Cox CD et al (2005) Kinesin spindle protein (KSP) inhibitors. Part 1: the discovery of 3,5-diaryl-4,5-dihydropyrazoles as potent and selective inhibitors of the mitotic kinesin KSP. Bioorg Med Chem Lett 15:2041–2045CrossRefPubMedGoogle Scholar
  32. 32.
    Bissantz C, Kuhn B, Stahl M (2010) A medicinal chemist’s guide to molecular interactions. J Med Chem 53:5061–5084CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Fraley ME et al (2006) Kinesin spindle protein (KSP) inhibitors. Part 2: The design, synthesis, and characterization of 2,4-diaryl-2,5-dihydropyrrole inhibitors of the mitotic kinesin KSP. Bioorg Med Chem Lett 16:1775–1779CrossRefPubMedGoogle Scholar
  34. 34.
    Garbaccio RM et al (2006) Kinesin spindle protein (KSP) inhibitors. Part 3: Synthesis and evaluation of phenolic 2,4-diaryl-2,5-dihydropyrroles with reduced hERG binding and employment of a phosphate prodrug strategy for aqueous solubility. Bioorg Med Chem Lett 16:1780–1783CrossRefPubMedGoogle Scholar
  35. 35.
    Cox CD et al (2006) Kinesin spindle protein (KSP) inhibitors. Part 4: Structure-based design of 5-alkylamino-3,5-diaryl-4,5-dihydropyrazoles as potent, water-soluble inhibitors of the mitotic kinesin KSP. Bioorg Med Chem Lett 16:3175–3179CrossRefPubMedGoogle Scholar
  36. 36.
    Cox CD et al (2007) Kinesin spindle protein (KSP) inhibitors. Part V: Discovery of 2-propylamino-2,4-diaryl-2,5-dihydropyrroles as potent, water-soluble KSP inhibitors, and modulation of their basicity by β-fluorination to overcome cellular efflux by P-glycoprotein. Bioorg Med Chem Lett 17:2697–2702CrossRefPubMedGoogle Scholar
  37. 37.
    Coleman PJ et al (2007) Kinesin spindle protein (KSP) inhibitors. Part 6: Design and synthesis of 3,5-diaryl-4,5-dihydropyrazole amides as potent inhibitors of the mitotic kinesin KSP. Bioorg Med Chem Lett 17:5390–5395CrossRefPubMedGoogle Scholar
  38. 38.
    Garbaccio RM et al (2007) Kinesin spindle protein (KSP) inhibitors. Part 7: Design and synthesis of 3,3-disubstituted dihydropyrazolobenzoxazines as potent inhibitors of the mitotic kinesin KSP. Bioorg Med Chem Lett 17:5671–5676CrossRefPubMedGoogle Scholar
  39. 39.
    Roecker AJ et al (2007) Kinesin spindle protein (KSP) inhibitors. Part 8: Design and synthesis of 1,4-diaryl-4,5-dihydropyrazoles as potent inhibitors of the mitotic kinesin KSP. Bioorg Med Chem Lett 17:5677–5682CrossRefPubMedGoogle Scholar
  40. 40.
    Cerny MA, Hanzlik RP (2005) Cyclopropylamine inactivation of cytochromes P450: Role of metabolic intermediate complexes. Arch Biochem Biophys 436:265–275CrossRefPubMedGoogle Scholar
  41. 41.
    Goncharov NV, Jenkins RO, Radilov AS (2006) Toxicology of fluoroacetate: a review, with possible directions for therapy research. J Appl Toxicol 26:148–161CrossRefPubMedGoogle Scholar
  42. 42.
    Holen K et al (2012) A phase I trial of MK-0731, a kinesin spindle protein (KSP) inhibitor, in patients with solid tumors. Invest New Drugs 30:1088–1095CrossRefPubMedCentralPubMedGoogle Scholar
  43. 43.
    Allen S et al (2012) The discovery and optimization of kinesin spindle protein (KSP) inhibitors: path to ARRY-520. Cambridge Healthtech Institute conference, 4 June 2012.
  44. 44.
    Woessner R et al (2009) ARRY-520, a novel KSP inhibitor with potent activity in hematological and taxane-resistant tumor models. Anticancer Res 29:4373–4380PubMedGoogle Scholar
  45. 45.
    Waring MJ (2010) Lipophilicity in drug discovery. Expert Opin Drug Discov 5:235–248CrossRefPubMedGoogle Scholar
  46. 46.
    Lemieux C et al (2007) ARRY-520, a novel, highly selective KSP inhibitor with potent anti-proliferative activity. Proc Am Assoc Cancer Res 48:5590Google Scholar
  47. 47.
    Tunquist BJ, Woessner RD, Walker DH (2010) Mcl-1 stability determines mitotic cell fate of human multiple myeloma tumor cells treated with the kinesin spindle protein inhibitor ARRY-520. Mol Cancer Ther 9:2046–2056CrossRefPubMedGoogle Scholar
  48. 48.
    Lonial S et al (2013) Prolonged survival and improved response rates with ARRY-520 in relapsed/refractory multiple myeloma (RRMM) patients with low α-1 acid glycoprotein (AAG) levels: results from a phase 2 study. ASH Annu Meet Abstr 122:285Google Scholar
  49. 49.
    Shah JJ et al (2013) Phase 1 study of the novel kinesin spindle protein inhibitor ARRY-520 + carfilzomib (car) in patients with relapsed and/or refractory multiple myeloma (RRMM). ASH Annu Meet Abstr 122:1982Google Scholar
  50. 50.
    Chari A et al (2013) A phase 1 study of ARRY-520 with bortezomib (BTZ) and dexamethasone (dex) in relapsed or refractory multiple myeloma (RRMM). ASH Annu Meet Abstr 122:1938Google Scholar
  51. 51.
    Owens B (2013) Kinesin inhibitor marches toward first-in-class pivotal trial. Nat Med 19:1550CrossRefPubMedGoogle Scholar
  52. 52.
    Hollebecque A et al (2013) A phase I, dose-escalation study of the Eg5-inhibitor EMD 534085 in patients with advanced solid tumors or lymphoma. Invest New Drugs 31:1530–1538Google Scholar
  53. 53.
    Weisberger AS, Levine B (1954) Incorporation of radioactive L-cystine by normal and leukemic leukocytes in vivo. Blood 9:1082–1094PubMedGoogle Scholar
  54. 54.
    Goodman L, Ross LO, Baker BR (1958) Potential anticancer agents. V. Some sulfur-substituted derivatives of cysteine. J Organ Chem 23:1251–1257CrossRefGoogle Scholar
  55. 55.
    Theodoropoulos D (1959) Synthesis of certain S-substituted L-cysteines. Acta Chem Scand 13:383–384CrossRefGoogle Scholar
  56. 56.
    Zee-Cheng K-Y, Cheng C-C (1970) Experimental antileukemic agents. Preparation and structure-activity study of S-tritylcysteine and related compounds. J Med Chem 13:414–418CrossRefPubMedGoogle Scholar
  57. 57.
    DeBonis S et al (2004) In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities. Mol Cancer Ther 3:1079–1090PubMedGoogle Scholar
  58. 58.
    DeBonis S et al (2008) Structure–activity relationship of S-trityl-L-cysteine analogues as inhibitors of the human mitotic kinesin Eg5. J Med Chem 51:1115–1125CrossRefPubMedGoogle Scholar
  59. 59.
    Wiltshire C et al (2010) Docetaxel-resistant prostate cancer cells remain sensitive to S-trityl-l-cysteine–mediated Eg5 inhibition. Mol Cancer Ther 9:1730–1739CrossRefPubMedGoogle Scholar
  60. 60.
    Zee-Cheng KY, Cheng CC (1972) Structural modification of S-trityl-L-cysteine. Preparation of some S-(substituted trityl)-L-cysteines and dipeptides of S-trityl-L-cysteine. J Med Chem 15:13–16CrossRefPubMedGoogle Scholar
  61. 61.
    Kozielski F et al (2008) Proteome analysis of apoptosis signaling by S-trityl-L-cysteine, a potent reversible inhibitor of human mitotic kinesin Eg5. Proteomics 8:289–300CrossRefPubMedGoogle Scholar
  62. 62.
    Ogo N et al (2007) Synthesis and biological evaluation of l-cysteine derivatives as mitotic kinesin Eg5 inhibitors. Bioorg Med Chem Lett 17:3921–3924CrossRefPubMedGoogle Scholar
  63. 63.
    Kaan HYK, Ulaganathan V, Hackney DD, Kozielski F (2010) An allosteric transition trapped in an intermediate state of a new kinesin–inhibitor complex. Biochem J 425:55–60CrossRefGoogle Scholar
  64. 64.
    Basso AD et al (2010) SCH 2047069, a novel oral kinesin spindle protein inhibitor, shows single-agent antitumor activity and enhances the efficacy of chemotherapeutics. Mol Cancer Ther 9:2993–3002CrossRefPubMedGoogle Scholar
  65. 65.
    Abualhasan MN et al (2012) Doing the methylene shuffle – further insights into the inhibition of mitotic kinesin Eg5 with S-trityl L-cysteine. Eur J Med Chem 54:483–498CrossRefPubMedGoogle Scholar
  66. 66.
    Skoufias DA et al (2006) S-Trityl-L-cysteine is a reversible, tight binding inhibitor of the human kinesin Eg5 that specifically blocks mitotic progression. J Biol Chem 281:17559–17569CrossRefPubMedGoogle Scholar
  67. 67.
    Kaan HYK et al (2011) Structure – activity relationship and multidrug resistance study of new S-trityl-L-cysteine derivatives as inhibitors of Eg5. J Med Chem 54:1576–1586CrossRefPubMedGoogle Scholar
  68. 68.
    Arrowsmith J, Miller P (2013) Trial watch: phase II and phase III attrition rates 2011–2012. Nat Rev Drug Discov 12:569CrossRefPubMedGoogle Scholar
  69. 69.
    Orloff J et al (2009) The future of drug development: advancing clinical trial design. Nat Rev Drug Discov 8:949–957PubMedGoogle Scholar
  70. 70.
    Komlodi-Pasztor E, Sackett DL, Fojo AT (2012) Inhibitors targeting mitosis: tales of how great drugs against a promising target were brought down by a flawed rationale. Clin Cancer Res 18:51–63CrossRefPubMedGoogle Scholar
  71. 71.
    Mitchison TJ (2012) The proliferation rate paradox in antimitotic chemotherapy. Mol Biol Cell 23:1–6CrossRefPubMedCentralPubMedGoogle Scholar
  72. 72.
    Tanenbaum ME et al (2009) Kif15 cooperates with Eg5 to promote bipolar spindle assembly. Curr Biol 19:1703–1711CrossRefPubMedGoogle Scholar
  73. 73.
    Voskoglou-Nomikos T, Pater JL, Seymour L (2003) Clinical predictive value of the in vitro cell line, human xenograft, and mouse allograft preclinical cancer models. Clin Cancer Res 9:4227–4239PubMedGoogle Scholar
  74. 74.
    Humphries M et al (2012) Abstract 1782: human tumor explants are better predictors of clinical trial outcome than cell line xenografts for the KSP inhibitor ARRY-520. Cancer Res 72:1782CrossRefGoogle Scholar
  75. 75.
    Morgan P et al (2012) Can the flow of medicines be improved? Fundamental pharmacokinetic and pharmacological principles toward improving phase II survival. Drug Discov Today 17:419–424CrossRefPubMedGoogle Scholar
  76. 76.
    Good JAD (2012) The development of S-trityl L-cysteine based inhibitors of Eg5 as anticancer chemotherapeutics. PhD thesis, The Beatson Institute for Cancer Research, University of Glasgow, GlasgowGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  1. 1.Department of ChemistryUmeå UniversityUmeåSweden
  2. 2.Umeå Centre for Microbial ResearchUmeå UniversityUmeåSweden
  3. 3.Strathclyde Institute of Pharmacy and Biomedical SciencesUniversity of StrathclydeGlasgowUK

Personalised recommendations