title: A novel class of tubulin inhibitors that exhibit potent antiproliferation and in vitro vessel-disrupting activity.
abstract: OBJECTIVE
  Since anticancer agents that interfere with microtubule function are in widespread use and have a broad spectrum of activity against both hematological malignancies and solid tumors, there is an urgent need to develop novel tubulin inhibitors with broader activities and avoiding drug resistance.
  
  
  RESULTS
  In this study, we describe the characterization of select lead compounds from a novel class of indazole-based tubulin inhibitors. Three lead compounds, TH-337, TH-482 and TH-494, exhibit potent antiproliferative activity against cell lines derived from human pancreatic carcinoma, human breast adenocarcinoma and human colorectal adenocarcinoma cells. The three compounds were also tested for cytotoxicity against a panel of clinically relevant drug resistant cancer cell lines that either overexpress the drug resistance pumps MDR-1, MRP-1 and BCRP-1 or have altered Topoisomerase II activity. TH-482 and -494 retained cytotoxic activities against all of the resistant cell lines tested; however, TH-337 exhibited decreased cytotoxicity in the cell line overexpressing BCRP-1, indicating that TH-337 is a substrate of that pump. We show that TH-482's antiproliferative activity is due to cell cycle arrest at the G(2)/M phase. We demonstrate that TH-482 binds specifically to the colchicine site of tubulin and that it inhibits tubulin polymerization in vitro in a concentration-dependent manner. The in vitro anti-vascular activities of TH-482 were assessed using the HUVEC-C cell line. TH-482 inhibits in vitro neovessel formation and disrupts pre-established vessels using HUVEC-C cells. TH-482 also increases permeability of vascular endothelial cells in a concentration- and time-dependent manner.
  
  
  CONCLUSIONS
  TH-482 demonstrates potent in vitro efficacy as a novel tubulin-targeted anti-proliferative and anti-vascular agent and notably is more potent in antiproliferative assays than the benchmark compound combretastatin A-4. These results identify TH-482 as a potent tubulin inhibitor, and support the investigation of its in vivo efficacy and pharmacokinetic properties as the prototype of a new class of anti-tubulin agents.
authors.affiliation: Threshold Pharmaceuticals, Inc., 1300 Seaport Blvd, Redwood City, CA 94063, USA. fmeng@thresholdpharm.com
authors.forename: Fanying
authors.initials: F
authors.lastname: Meng
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Cai
authors.forename: Jianxin
authors.initials: J
authors.lastname: Duan
authors.forename: Mark G
authors.initials: MG
authors.lastname: Matteucci
authors.forename: Charles P
authors.initials: CP
authors.lastname: Hart
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D002152:Calorimetry, Differential Scanning; D002199:Capillary Permeability / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005452:Fluoresceins; D005456:Fluorescent Dyes; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D008856:Microscopy, Fluorescence; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D020533:Angiogenesis Inhibitors; D004250:DNA Topoisomerases, Type II; D005452:Fluoresceins; D005456:Fluorescent Dyes; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D014404:Tubulin; D050257:Tubulin Modulators; C116614:ABCG2 protein, human; C085925:calcein AM; C421109:multidrug resistance-associated protein 1