WO1996011398A1 - Procede d'evaluation de la polymerisation de la tubuline - Google Patents

Procede d'evaluation de la polymerisation de la tubuline Download PDF

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Publication number
WO1996011398A1
WO1996011398A1 PCT/US1995/013025 US9513025W WO9611398A1 WO 1996011398 A1 WO1996011398 A1 WO 1996011398A1 US 9513025 W US9513025 W US 9513025W WO 9611398 A1 WO9611398 A1 WO 9611398A1
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WO
WIPO (PCT)
Prior art keywords
tubulin protein
polymerized
test sample
amount
trapped
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Application number
PCT/US1995/013025
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English (en)
Inventor
Daniel M. Bollag
Patricia A. Mcqueney
Catherine M. Woods
Elias Lazarides
Original Assignee
Merck & Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to AU39535/95A priority Critical patent/AU3953595A/en
Publication of WO1996011398A1 publication Critical patent/WO1996011398A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to assessing the effect of biologically active compounds on tubulin protein.
  • Certain anti-cancer agents cause tubulin protein to polymerize when present in concentra ⁇ tions which are equal to the concentrations which cause cytotoxicity in cultured cells.
  • the methods of assessing tubulin protein polymerization in vitro have required significantly higher levels of the compound in question than the methods used for assessing toxicity to cells.
  • the invention requires a lower concentration of tubulin protein than previously described methods. These include analyses carried out by light scattering and by assessing sedimentation (Schiff et al. Nature 277: 665-667 (1979)). Such analyses required levels of the therapeutically useful agent of at least 1 ⁇ M to detect tubulin polymerization.
  • crosslinking agents to reduce tubulin depolymerization.
  • crosslinking agents used in the past include glutaraldehyde and bis [2-(succinimido-oxycarbonyloxy)ethyl]sulfone. No such biologically- irrelevant crosslinking agents are required to perform the present assay.
  • a method of determining tubulin protein polymerization activity in a test sample comprises: combining the test sample with polymerizable tubulin protein; contacting said test sample and polymerizable tubulin protein with a material which traps polymerized tubulin protein; measuring the amount of polymerized tubulin protein trapped by said material, and comparing the amount of polymerized tubulin protein trapped by said material to a standard.
  • a method of determining tubulin protein polymerization inhibition in a test sample comprises: combining said test sample with polymerizable tubulin protein and a pre-determined amount of a compound which induces tubulin protein polymerization to form a mixture; contacting said mixture with a material which traps polymerized tubulin protein; measuring the amount of polymerized tubulin protein trapped by said material, and comparing the amount of polymerized tubulin protein trapped by said material to a standard.
  • FIGURE 1 is an absorbance curve for the compound taxol generated at taxol concentrations ranging up to 1 ⁇ M;
  • FIGURE 2 is an absorbance curve for the compounds Epothilone A and B, generated at concentrations ranging up to 1 ⁇ M, and
  • FIGURE 3 is a bar graph of the inhibition of tubulin protein polymerization caused by 1 mM GTP ("No Addition"), 1 mM vinblastine and vincristine, two known anti-cancer compounds. DETAILED DESCRIPTION OF THE INVENTION
  • tubulin protein is used in the conventional sense, and refers to the heterodimeric protein subunit component of mammalian microtubules.
  • suitable sources include calf, rat, lamb and pig, but other mammalian sources of tubulin protein are acceptable as well.
  • Tubulin protein when non-polymerized is typically in the form of a mixture of dimers (approximately 110,000 Daltons) and other oligomers. Upon polymerization, the molecular weight of the tubulin protein polymer is approximately 2-5 million Daltons.
  • Suitable buffering agents include MES (4-morpholineethanesulfonic acid) (pH 6.7), PIPES (l,4-piperazinebis(ethane sulfonic acid) (pH 6.9) and PG (100 mMol sodium phosphate and 100 mMol sodium glutamate pH 6.75) .
  • MES 4-morpholineethanesulfonic acid
  • PIPES l,4-piperazinebis(ethane sulfonic acid)
  • PG 100 mMol sodium phosphate and 100 mMol sodium glutamate pH 6.75
  • MEM MEM
  • Table 1 A preferred buffer used herein is MEM.
  • Table 1 A preferred buffer used herein is MEM.
  • interaction between the protein and the test compound is conducted with incubation, typically at about 37°C for about 30 minutes.
  • a method of determining tubulin protein polymerization activity in a test sample comprises: combining the test sample with a polymerizable tubulin protein; contacting said test sample and polymerizable tubulin protein with a material which traps polymerized tubulin protein; measuring the amount of polymerized tubulin protein trapped by said material, and comparing the amount of polymerized tubulin protein trapped by said material to a standard.
  • the method further entails combining the test sample with a polymerizable tubulin protein in the essential absence of a crosslinking agent.
  • the material which traps polymerized tubulin protein is comprised of a filter having an average pore size of about 0.22 ⁇ m.
  • Tubulin protein polymerization has been demonstrated to occur upon exposure to one class of anti-cancer compounds.
  • the standard in the process described herein is typically the tubulin protein polymerization caused by this anti-cancer compound.
  • the preferred compound which causes tubulin protein polymerization is taxol, a recognized anti-cancer compound which is of the structural formula A:
  • the detectable concentration range of taxol is about 10 nM to about 1 ⁇ M.
  • the minimum detectable taxol concentration range is from about 10 nM to about 50 nM.
  • the assay described herein can also be conducted as an inhibition assay.
  • the inhibition of tubulin protein polymerization is determined.
  • this method comprises: combining the test sample with a polymerizable tubulin protein and a pre-determined amount of a compound which causes tubulin protein polymerization, forming a mixture; contacting the mixture with a material which traps polymerized tubulin protein; measuring the amount of polymerized tubulin protein trapped by said material, and comparing the amount of polymerized tubulin protein trapped by said material to a standard.
  • test sample can be incubated with the polymerizable tubulin protein and the compound which causes tubulin protein polymerization, at about 37 °C for about 30 minutes.
  • any test sample containing a compound which causes a decrease in the amount of tubulin protein polymerization is identified.
  • the standard compound which causes tubulin protein poly ⁇ merization for use in the inhibition assay is guanosine-5'-triphosphate (GTP).
  • GTP guanosine-5'-triphosphate
  • the guanosine-5'-triphosphate is present in a concentra ⁇ tion ranging from about 50 ⁇ M to about 2.5 mM, and preferably is about 1 mM.
  • the material which is used to trap polymerized tubulin protein is typically comprised of a filter having an average pore size of about 0.22 ⁇ m. As noted above, the polymerized tubulin protein is preferably trapped under vacuum pressure.
  • R 2 represents H or methyl.
  • the preferred detection method involves direct staining of the trapped polymerized tubulin protein.
  • One preferred staining solution used is Amido black solution.
  • the formula for Amido black stain solution is shown below in Table 1. Evaluation of the Amido black stain from the filter can be conducted in accordance with the methods of Schaffner and Weissman Anal. Biochem. 56: 502-514 (1973) the procedure of which is incorporated herein by reference.
  • One advantage of the present invention is that essentially only polymerized tubulin protein is trapped by the material and stained. Any unpolymerized tubulin is removed via a washing step.
  • Another advantage of the present invention is that depolymerization is not required to determine the extent of tubulin polymerization.
  • Tubulin protein is prepared from bovine brains as described by Tiwari, et al. Anal. Biochem. 215: 96-103 (1993), which is incorporated by reference.
  • the microtubule pellets from the second cycle of polymerization are frozen in liquid nitrogen and stored at -80 °C.
  • a microtubule pellet (5-1 Omg) is resuspended in MEM buffer (100 mM MES-NaOH, ImM EGTA, 1 mM MgCb, pH 6.75) on ice and centrifuged at 100,000 x g for 20 minutes. The supernatant of this centrifugation comprises tubulin protein.
  • test material is dissolved in dimethyl sulfoxide (0.5 mL). The mixture is sonicated for 20 minutes and mixed to ensure maximal solubility.
  • the tubulin solution (0.25 ml, 0.3 mg/ml) from Example 1 is placed into an assay tube.
  • a solution of taxol (2.5 ⁇ l, final concentra ⁇ tions of 10 nM, 20 nM, 50 nM, 100 nM, 250 nM, 500 nM and 1 ⁇ M) is introduced into the assay tube and mixed. The mixture is incubated at 37°C for 30 minutes.
  • the 0.22 ⁇ m pore size 96 well filter plates (Millipore, Cat. No. MAGVN2250) (under 15 inches Hg vacuum) are washed with MEM buffer (200 ⁇ l). 150 ml of the sample mixture is applied to the filter plate. The filter is washed with MEM buffer (200 ⁇ l). The vacuum is removed and amido black stain solution (50 ⁇ l) is applied to the filter. After two minutes of incubation, the vacuum is reapplied, and the filter is washed twice with AB destain solution (200 ⁇ l).
  • the filter is then washed with water (200 ⁇ l), and allowed to dry for at least two hours at room temperature.
  • the elution solution (200 ⁇ l) from Schaffner, et al. Anal. Biochem. 56: 502-514 (1973) is applied to the filter plate well with no vacuum.
  • the plate is incubated at room temperature for 10 minutes and the absorbance of the solution is measured as described in Schaff er, et al. An absorbance curve is shown in Figure I.
  • Epothilone A is a compound of formula B wherein R 2 represents H.
  • Epothilone B is a compound of formula B wherein R 2 represents methyl.
  • GTP is added to a tubulin solution (0.25 ml, 0.3 mg/mL) to a final GTP concentration of ImM.
  • the test solution is applied, and the procedure of Example 3 is used.
  • the control sample containing GTP but no test compound produces a significant absorbance reading as shown in Figure 3.
  • the test compounds shown in Figure 3 are evaluated based upon the inhibition of tubulin polymerization, as determined in comparison to the absorbance reading observed with GTP alone.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Procédé de détermination de l'activité de polymérisation de la tubuline dans un échantillon d'essai. Le procédé consiste à combiner l'échantillon d'essai à une tubuline polymérisable; à mettre ledit échantillon et la tubuline polymérisable en contact avec une substance qui piège la tubuline; à mesurer le volume de tubuline piégé par ladite substance; à comparer le volume de tubuline piégé par ladite substance à un étalon. Un procédé de détermination de l'inhibition de la polymérisation de la tubuline dans un échantillon d'essai est également décrit. Il consiste à combiner ledit échantillon à de la tubuline polymérisable et à un volume prédéterminé d'un composé induisant la polymérisation de la tubuline pour former un mélange; à mettre ledit mélange en contact avec une substance qui piège la tubuline polymérisée; à mesurer le volume de tubuline polymérisée piégée par ladite substance, et à comparer le volume de tubuline polymérisée piégée par ladite substance à un étalon.
PCT/US1995/013025 1994-10-07 1995-10-03 Procede d'evaluation de la polymerisation de la tubuline WO1996011398A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU39535/95A AU3953595A (en) 1994-10-07 1995-10-03 Process for assessing tubulin protein polymerization

Applications Claiming Priority (2)

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US32009594A 1994-10-07 1994-10-07
US320,095 1994-10-07

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WO1996011398A1 true WO1996011398A1 (fr) 1996-04-18

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0843018A1 (fr) * 1996-11-14 1998-05-20 Rohm And Haas Company Utilisation de certains amides commes sondes pour la détection d'activité antitubuline et de surveillance de la résistance
WO2001013121A1 (fr) * 1999-08-13 2001-02-22 Biovitrum Ab Procedes d'identification de composes stimulant la secretion d'insuline
US7125899B2 (en) 1997-07-08 2006-10-24 Bristol-Myers Squibb Company Epothilone derivatives

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5242842A (en) * 1989-05-11 1993-09-07 Axis Research As Glycosylated haemoglobin assay

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5242842A (en) * 1989-05-11 1993-09-07 Axis Research As Glycosylated haemoglobin assay

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANALYTICAL BIOCHEMISTRY, Volume 215, issued 1993, S.C. TIWARI et al., "A pH- and Temperature-Dependent Cycling Method That Doubles the Yield of Microtubule Protein", pages 96-103. *
JOURNAL OF MOLECULAR BIOLOGY, Volume 89, issued 1974, F. GASKIN et al., "Turbidimetric Studies of the in Vitro Assembly and Disassembly of Porcine Neurotubules", pages 737-758. *
NATURE, Volume 277, issued 22 February 1979, P.B. SCHIFF et al., "Promotion of Microtubule Assembly in Vitro by Taxol", pages 665-667. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0843018A1 (fr) * 1996-11-14 1998-05-20 Rohm And Haas Company Utilisation de certains amides commes sondes pour la détection d'activité antitubuline et de surveillance de la résistance
US7125899B2 (en) 1997-07-08 2006-10-24 Bristol-Myers Squibb Company Epothilone derivatives
US7241755B2 (en) 1997-07-08 2007-07-10 Bristol-Myers Squibb Company Epothilone derivatives
USRE41895E1 (en) 1997-07-08 2010-10-26 Bristol-Myers Squibb Company Epothilone derivatives
USRE41893E1 (en) 1997-07-08 2010-10-26 Bristol-Myers Squibb Company Epothilone derivatives
USRE41911E1 (en) 1997-07-08 2010-11-02 Bristol-Myers Squibb Company Epothilone derivatives
USRE43003E1 (en) 1997-07-08 2011-12-06 Bristol-Myers Squibb Company Epothilone derivatives
US8536327B2 (en) 1997-07-08 2013-09-17 Bristol-Myers Squibb Company Epothilone derivatives
US8921542B2 (en) 1997-07-08 2014-12-30 Bristol-Myers Squibb Company Epothilone derivatives
WO2001013121A1 (fr) * 1999-08-13 2001-02-22 Biovitrum Ab Procedes d'identification de composes stimulant la secretion d'insuline

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Publication number Publication date
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