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`OF BIOLOGICAL CHEMISTRY
`THE JOURNAL
`0 2000 by The American Society
`for Biochemistry and Molecular Biology Inc
`
`Vol 275 No 34 Issue of August 25 pp 2626526276
`2000
`Printed in USA
`
`Molecular Recognition of Taxol by Microtubules
`KINETICS AND THERMODYNAMICS OF BINDING OF FLUORESCENT TAXOL DERIVATIVES TO
`AN EXPOSED SITE
`
`Received for publication April 12 2000 and in revised form May 11 2000
`Published JBC Papers in Press May 18 2000 DOI 101074jbcM003120200
`
`J Fernando Diaz Rik Strobel Yves Engelborghsll Andre A Soutor and Jose M Andreu
`Biologicas Consejo Superior de Investigaciones Cientificas C IVelcizquez
`From the $Centro de Investigaciones
`144 28006 Madrid Spain the 1lLaboratory
`of Biomolecular Dynamics Katholieke Universiteit Leuven Celestijnenlaan
`200D B3001 Heverlee Belgium and 17estituto de Quimica Orgcinica Consejo Superior de Investigaciones Cientificas
`C I Juan de la Cierva 3 28006 Madrid Spain
`
`We have determined the kinetic scheme and the reac
`tion rates of binding to microtubules of two fluorescent
`70N4cfluoresceincarbony1LalanyllTaxol
`taxoids
`Flutax1
`and 701N27difluoro4cfluoresceincar
`bony1LalanyllTaxol Flutax2 Flutax1 and Flutax2
`107 M1
`bind to microtubules with high affinity Kr
`37 °C The binding mechanism consists of a fast bimo
`lecular reaction followed by at least two monomolecular
`rearrangements which were
`characterized
`with
`stopped flow techniques The kinetic constants of
`the
`bimolecular reaction were 610 ± 022 x 105 ni1 s1 and
`138 ± 18 x 105 ni1 s1 at 37 °C respectively A second
`slow binding step has been measured employing the
`change of fluorescence anisotropy of the probe The re
`versal of this reaction is the ratelimiting step of disso
`ciation A third step has been detected using small angle
`a 2nm increase in the
`xray scattering and involves
`diameter of microtubules It
`is suggested that the first
`the Taxol moiety and the
`step entails the binding of
`second
`a relative immobilization of
`the fluorescent
`probe The equilibrium and some kinetic measurements
`required the use of stabilized cross linked microtubules
`which preserved taxoid binding The results indicate
`that the Taxol binding site is directly accessible in con
`trast with its location at lumen in the current model of
`microtubules An alternative structural model
`is consid
`ered in which the binding site is located between proto
`filaments accessible from the microtubule surface
`
`are polymers assembled from aptubulin het
`Microtubules
`erodimers Among other functions they form the mitotic spin
`dle which segregates the chromosomes during cell division
`Due to this function they are the target of antimitotic drugs
`treatment 1
`like Vinca alkaloids and taxoids used in cancer
`2 Taxoll is extensively employed in therapy of ovarian cancer
`
`de Madrid
`This work was supported
`by Comunidad Autonoma
`Grant 07B002599 Direccion General de Enserianza Superior
`e Inves
`tigacion Cientifica DGE SIC Grants PB950116 and APC 960071 and
`Fundacion Cientifica de la Asociacion Espanola contra el Cancer
`to
`J M A and DGESIC Grant PB960852 to U Acuna The costs of
`
`publication of this article were defrayed in part by the payment of page
`charges This article must therefore be hereby marked advertisement
`in accordance with 18 USC Section 1734 solely to indicate this fact
`§ To whom correspondence should be addressed Tel 34915611800
`ext 4380 Fax 34915627518 Email ferakiloniacibcsices
`Present address Faculdade de Quimica Universidade Pontificia
`Catolica do Rio Grande do Sul 90619900 Porto Alegre RS Brasil
`1 The abbreviations
`and trivial
`names used are Taxol® Bristol
`410diacetoxy2abenzoyloxy5820
`Myers
`Squibb
`paclitaxel
`353phenylcarbonyl
`epoxy178dihydroxy9oxotax11en13ay12R
`docetaxel Taxotere® Rhone
`amino2hydroxy3phenylpropionate
`
`metastatic breast cancer head and neck cancer and lung can
`cer 35 Taxol arrests cell division by blocking microtubule
`dynamics which is necessary for their function 6 These dy
`namics are normally controlled by the nucleotide content of
`tubulin the heterodimer binds two molecules of guanine nu
`cleotide a nonexchangeable GTP molecule at
`the a subunit
`which plays a structural stability role 7 and a second ex
`changeable molecule bound to the 0 subunit which can be
`either GTP or GDP This second nucleotide molecule controls
`the activation state of the protein In the GDP bound state the
`protein is in an inactive conformation forming double rings 8
`whereas in the GTP state it
`is active for microtubule assembly
`GTP hydrolysis in the polymers and GDPGTP exchange in the
`the assembled state of tubulin 9
`dimers control
`Taxol drives inactive GDPtubulin into microtubules replac
`of GTP to activate the protein
`ing the need of the yphosphate
`10 It was the first microtubule assembly promoting drug to
`be discovered 11 Taxol stabilizes microtubules
`by binding
`preferentially to assembled purified tubulin with an exact 11
`stoichiometry 10 Thus the cellular microtubule cytoskeleton
`should have a very large number of binding sites for Taxol or
`ligands whose function is unknown Unas
`other endogenous
`sembled tubulin shows insignificant affinity for Taxol 1214
`indicating that the binding site is mostly formed by the polym
`erization process The interaction of microtubules with Taxol
`has been widely studied 10 1225 However
`since Taxol
`binding and microtubule assembly are linked reactions and the
`binding affinity seems to be high 12 14 direct measurements
`In ad
`of the binding thermodynamics
`are extremely difficult
`of the protein and Taxol absorption
`dition the coincidence
`tech
`bands has precluded up to now the use of spectroscopic
`niques to study the kinetics of the reaction
`Taxoid binding affects the microtubular structure 26 27 In
`a previous study 23 kinetic measurements of the interchange
`of radioactive labeled Taxol and docetaxel showed that taxoids
`are able to bind and dissociate freely from microtubules
`in less
`than 3 min The same work demonstrated that the addition of
`to preformed microtubules made of pure tubulin alters
`Taxol
`their structure in less than 2 min Those results indicate that
`the taxoid binding site is accessible in the microtubules This is
`supported by the rapid Taxolinduced
`increase of flexibility
`of
`assembled microtubules 28 and by the fast binding and dis
`of 70 N4fluoresceincarbony1Lalanyl Taxol
`Flutax1 to cellular microtubules 24 All of these character
`istics would be compatible with a taxoid binding site in a zone
`
`sociation
`
`Poulenc Rorer RP56976 4acetoxy2 abenzoyloxy5820epoxy
`178108trihydroxy9oxotax 11 en 13 ay12R3S3 tertbutoxycar
`bonylamino2hydroxy3phenylpropionate
`
`This paper
`
`is available on line at httpwwwjbcorg
`
`26265
`
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`

`

`26266
`
`between the protofilaments 20 26 27 29 However
`in the
`model structure of Taxol stabilized microtubules obtained by
`fitting into an electron microscopy density map 30 the bio
`chemically well supported 3133 high resolution model of the
`complex 34 the Taxol binding site is at one
`tubulinTaxol
`side of the f3tubulin subunit clearly facing the microtubular
`lumen
`
`In order to unveil
`
`the kinetic mechanisms of molecular
`
`rec
`
`ognition of ligands by the Taxol binding site of microtubules
`we have employed two taxoids labeled at position 7 of the
`taxane ring either with fluorescein Flutax1 Refs 24 and 35
`or difluorofluorescein 704N2 7difluoro4fluoresceincar
`bony1LalanyllTaxol Flutax2 The use of these probes which
`retain the microtubule assembly activity has allowed a de
`tailed kinetic characterization
`of the reversible interaction of
`and taxoids The results evidence
`microtubules
`an amazingly
`fast initial binding reaction and provide further insight into the
`interaction of microtubules with Taxol
`
`EXPERIMENTAL PROCEDURES
`
`Tubulin Microtubules and TaxoidsPurified calf brain tubulin and
`chemicals were as described 27 For glycerol induced assembly tubu
`lin was directly equilibrated in 10 mm phosphate 1 mm EDTA 01 mm
`GTP 34 m glycerol pH 67 buffer All tubulin samples were clarified by
`at 50000 rpm 4 °C for 10 min using TL1002 or TL1004
`centrifugation
`rotors in a Beckman Optima TLX centrifuge After centrifugation 6 mm
`MgC12 and up to 1 mm GTP were added to the solution final pH 65
`GAB Microtubules were assembled by raising the temperature to
`of the assembled mi
`37 °C for 30 min The length and morphology
`crotubules were checked
`by negative
`stain electron microscopy
`as
`described 27
`Axonemes from sea urchin Strongylocentrotus
`purpuratus sperm
`tail were kindly provided by Dr Philippe Huitorel Universite Pierre et
`France and diluted a minimum of
`Marie Curie VillefranchesurMer
`100 times in the experimental buffer
`Docetaxel was kindly provided by RhonePoulenc Rorer Antony
`France Flutax1 was synthesized as described 35 Their concentra
`10 24 Flutax2 was syn
`tions were measured spectrophotometrically
`thesized by the reaction of 70alanyl Taxol with Oregon Green 488
`carboxylic acid succinimidyl ester 5 isomer Molecular Probes refer
`ence no 06147 following the described procedures 35 and purified
`by preparative TLC on silica gel with chloroformmethanolacetic acid
`41015 vvv as eluent mass spectrum and NMR data were in ac
`cordance with its structure 58 Flutax2 purity high performance
`Ref 24 was 94 Flutax2 induced the assem
`liquid chromatography
`bly of GDPtubulin similarly to Flutax1 24 except for the critical
`tubulin concentration which was coincident with Taxol Flutax2 con
`centrations were measured in 05 SDS 50 mm sodium phosphate
`M cm at 496 nm
`buffer pH 70 employing an extinction coefficient2 of 49100
`
`1100
`
`to stabilize mi
`Preparation of Cross linked MicrotubulesIn order
`crotubules against disassembly by dilution and low temperatures 50
`tm tubulin in GAB was assembled at 37 °C for 30 min and then 20 mm
`glutaraldehyde EMscope microscopy grade was added to the solution
`which was incubated at 37 °C for 10 min more The remains of
`the
`cross linking agent were quenched by adding 60 mm NaB114 Fluka on
`these conditions 90 of
`ice and the mixture degassed Under
`the
`tubulin was found to be incorporated into the microtubules The mor
`by electron mi
`phology of the cross linked microtubules was checked
`croscopy and found to be normal They were found to be stable against
`dilution and low temperatures The taxoid binding was found to be
`unaffected by the treatment as judged by the stoichiometry and the
`kinetics of the binding reaction which were not altered The number of
`
`active sites was found to decay at a relatively slow rate5 decay in
`
`24 h at 4 °C
`to MicrotubulesThe
`Binding of Fluorescent Taxoids
`binding of
`Flutax1 and Flutax2 to the microtubules was measured using a cen
`trifugation assay Samples of cross linked microtubules were incubated
`The sam
`for 1 h at different temperatures
`and taxoid concentrations
`ples were then centrifuged for 20 min at 50000 rpm in a TL100 rotor
`employing a Beckman Optima TLX ultracentrifuge The supernatants
`were taken and the pellets were resuspended in a 10 mm phosphate
`
`2 J A Evangelio and J M Andreu unpublished observations
`
`Fast Kinetics of Taxoid Binding to Microtubules
`buffer pH 70 containing 1 SDS The pellets and supernatants were
`
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`diluted 15 in the same buffer and their fluorescence was measured
`employing a Shinnadzu RF540
`excitation wave
`spectrofluorometer
`522 nm 5nm excitation
`length 492 nm emission wavelength
`and
`of ligand in the samples was calcu
`emission slits The concentration
`lated using Flutax1 and Flutax2 spectrophotometric
`concentration
`standards
`The percentage of inactive ligand in the stock solutions was obtained
`two different concentrations
`by measuring the titration curves at
`of
`sites 1 and 01 pm The binding constant has to be the same at both
`concentrations of sites and can be calculated from the apparent binding
`constant by discounting the percentage of the inactive ligand from the
`total free ligand The value fitting both curves with the same binding
`constant and the minimal error was calculated using a program based
`on the Marquardt algorithm 36 The percentages found at each exper
`and the effective binding con
`temperature were averaged
`imental
`stants were calculated using the averaged values
`The binding of Flutax1 and Flutax2 can also be monitored by the
`in ligand anisotropy The anisotropy
`change
`of
`the fluorescence of
`Flutax1 and Flutax2 bound and free were measured in a SLM8000D
`fluorometer using an excitation wavelength of 470 nm and an emission
`wavelength of 560 nm with 2nm excitation
`and emission slits The
`binding of Flutax1 and Flutax2 to the microtubules was measured
`using a ligand anisotropy assay Samples of taxoids were incubated for
`and concentrations
`15 min at different
`of cross linked
`temperatures
`the samples were measured in a Po
`microtubules The anisotropy of
`lastarGalaxy BMG Labtechnologies
`plate reader using the 485P
`excitation filter and the 520P emission filter Since with this method
`the binding constants are determined
`of
`using the free concentration
`sites total measured binding sites minus bound ligand measured they
`are not influenced by the percentage of inactive ligand
`Kinetics of Binding and Dissociation of Fluorescent Taxoids to Mi
`crotubulesThe kinetics of Flutax1 and Flutax2 binding to and dis
`sociation from microtubules were measured by following the change of
`the fluorescence of the probe employing a High Tech Sci
`intensity of
`entific SS 51 stopped flow device equipped with a fluorescence detec
`of 492 nm 2nm slit
`tion system A wavelength
`in the excitation
`pathway and a filter with a cutoff of 530 nm in the emission pathway
`were used The dead time of the instrument was determined using the
`of Nbromosuccinimide with Nacetyltryptophanamide 37
`reaction
`and was found to be 2 ms With these conditions both Flutax1 and
`Flutax2 are photostable within the time of the measurement
`The kinetics of the binding of Flutax1 and Flutax2 to microtubules
`were also measured by the change in the fluorescence anisotropy of the
`probe in a Spex spectrofluorometer Fluorolog 1691 excitation wave
`length 492 nm emission wavelength 570 nm 16nm slits a cutoff filter
`of 550 nm was employed in the emission pathway
`to eliminate any
`contribution due to scattered light The device was equipped with a
`of Biomo
`stopped flow module designed and built at
`the Laboratory
`these
`
`lecular Dynamics K U Leuven Flutax2 is photostable under
`photoquenching 1 of
`
`conditions while Flutax1 shows appreciable
`the total intensity per second during the measurement The dead time
`of the instrument was measured as described above and was found to be
`10 ms A minimum of 8 curves were averaged for each measurement
`The slower dissociation of Flutax1 and Flutax2 from microtubules was
`measured by the decrease in fluorescence anisotropy in the SLM8000D
`fluorometer equipped with a home built mixing device consisting of two
`1 ml syringes fixed to a thermostated aluminum block in such a way
`to a threeway Hamil
`that they moved simultaneously and connected
`ton valve which acted as a mixing chamber The output of the valve was
`to the cuvette The dead time of
`the complete system was
`connected
`2 s comparable with the 1s time constant employed in the
`fluorometer
`curves was done using a nonlinear least
`The fitting of
`squares fitting program based on the Marquardt algorithm 36 when
`pseudo first order conditions were used otherwise the FITSIM pack
`age 38 was employed
`Xray Scattering
`SolutionsMeasurements
`were
`by Microtubule
`made at station 21 of the Daresbury Laboratory Synchrotron Radiation
`and
`and processing
`Source Instruments employed data acquisition
`the microtubule xray scattering were as described
`interpretation of
`previously 23
`
`about
`
`the kinetic
`
`RESULTS
`
`to monitor the binding of the
`In order to determine the signal
`taxoids the spectroscopic properties of the bound
`fluorescent
`and free Flutax1 and Flutax2 were investigated Fig 1 shows
`
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`

`

`Fast Kinetics of Taxoid Binding to Microtubules
`
`26267
`
`TABLE I
`
`Anisotropy of the fluorescence emission of the fluorescent
`1 iim in GAB at 37 °C A = 470 nm
`= 560 nm
`Bound a
`
`Ligand
`
`Free
`
`Displaced
`
`taxoids
`
`Flutaxl
`
`Flutax2
`
`005
`
`004
`
`002
`
`001
`
`024
`
`026
`
`002
`
`002
`
`005
`
`008
`
`002
`
`001
`
`assembled from 20 pm pure tubulin in GAB
`a Bound to microtubules
`or displaced by the addition of 50 pm docetaxel
`
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`10
`
`9
`
`8
`
`7
`
`6
`
`5
`
`Log Flutax
`
`Log Sites
`
`FIG 2 A solid line and circles titration curve of 1 jM taxoid sites in
`stabilized microtubules with Flutax1 at 27 °C measured by centrifu
`gation data are corrected for the percentage of inactive ligand Dashed
`line and open circles uncorrected data and fit A small part of the ligand
`remained in the supernatant
`in use was found to be inactive since it
`at
`very high concentrations of microtubules disturbing the measurement
`of the free ligand concentration and artifactually decreasing the appar
`ent binding constant measured This effect was corrected as described
`under Experimental Procedures This percentage was found to be 4
`
`1 in the case of Flutax1 and 6 1 in the case of Flutax2 Solid
`squares and dotted line titration curve of 25 nm Flutax1 with taxoid
`sites in stabilized microtubules measured by anisotropy B solid line
`jM taxoid sites in stabilized microtu
`and circles titration curve of 1
`bules with Flutax2 at 27 °C corrected for the percentage of inactive
`ligand Dashed line and open circles uncorrected data Solid squares
`and dotted line titration curve of 25 nm Flutax2 with stabilized micro
`tubules measured by anisotropy
`
`centrifugation
`
`NI
`
`Flutax1 and Flutax2 to microtubules
`assembled from pure
`tubulin in GAB were measured by sedimentation
`and anisot
`ropy using diluted cross linked microtubules This was neces
`sary since in preliminary experiments using nonstabilized mi
`crotubules the reaction was essentially displaced toward the
`bound ligand state due to the high concentration
`of binding
`sites The cross linking employed did not affect
`the binding
`stoichiometry or the kinetics of binding described below
`The titration curves corrected for
`the small percentage of
`inactive ligand in the experiments in the case of the sedimen
`tation assays are shown in Fig 2 The stoichiometry was found
`to be 11 099 ± 007 mol of Flutax2mol of assembled tubu
`lin and the affinity was high K = 66 ± 10 X 107 NI 1
`1 anisotropy for
`or 60 ± 02 X 107
`Flutax1 and lc = 61 ± 17 X 107 NI 1 centrifugation
`59 ± 03 X 107 NI 1 anisotropy for Flutax2 at 27 °C The
`affinity constants determined
`
`or
`
`at different
`
`temperatures
`
`are
`
`100
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20 V
`
`10
`
`0
`
`90
`
`80
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`FluorescenceArbitraryunits
`
`500
`
`520
`
`540
`
`560
`
`580
`
`600
`
`Wavelength
`
`FIG 1 A fluorescence emission spectra of
`1 pm Flutax1 in GAB
`assembled from 20 pm tubulin before
`buffer in presence of microtubules
`Amax = 521 nm solid line and after Amax = 522 nm dashed line being
`displaced by 50 off docetaxel B 1 tm Flutax2 in GAB buffer
`in the
`assembled from 20 pm tubulin before Amax =
`presence of microtubules
`520 nm solid line and after Amax = 524 nm dashed line being dis
`The spectra were taken in a Shimadzu
`placed by 50 off docetaxel
`RF540 spectrofluorometer the excitation wavelength was 480 nm and
`and emission slits was 5 nm The
`the bandwidth
`chemical structure of both probes is shown in A
`
`the excitation
`
`of
`
`the fluorescence emission spectra of the bound and free forms
`of Flutax1 and Flutax2 To match the scattering contribution
`of microtubules the spectrum of the free species was obtained
`by displacing the bound form with a large excess of the non
`taxoid docetaxel This closely related compound
`fluorescent
`binds to the Taxol binding site and has a larger solubility 10
`confirmed
`Centrifugation of the solution after displacement
`taxoids were dissociated from the micro
`that the fluorescent
`tubules It can be seen clearly that the fluorescence intensity of
`the bound form of Flutax1 is 50 larger than that of the free
`toward the blue The
`form The spectrum shows a small shift
`emission spectrum of bound Flutax2 shows a larger shift
`to
`ward the blue but a smaller change in fluorescence intensity
`The intensity change is due to the shift of the ionization equi
`librium of the fluorescein group upon binding 24 ie the
`bound group is mostly in the dianionic form which has a larger
`quantum yield than the monoanion By choosing adequately
`the pH of the buffer it was possible to maximize the change of
`intensity of fluorescence upon binding which was found to be
`optimum at pH 65 Since the pK value for the ionization of the
`group of Flutax2 is much lower than that of
`difluorofluorescein
`change in fluorescence is observed upon binding
`
`Flutax1 little
`to its site
`In addition the immobilization of the fluorescent group that
`results from the binding reaction produces a large increase in
`the fluorescence anisotropy of Flutax1 and Flutax2 The ani
`sotropy values of these probes under different conditions are
`shown in Table I
`
`Equilibrium Binding of Fluorescent Taxoids to Cross linked
`MicrotubulesThe
`equilibrium constants
`of binding of
`
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`

`

`42C
`
`NDb
`15
`
`ND
`
`18
`
`01
`
`02
`
`Flutax1
`37 °C
`
`62
`12
`08
`
`26
`5
`06
`
`26268
`
`Fast Kinetics of Taxoid Binding to Microtubules
`
`Ligand
`
`Flutax1
`Flutax1c
`Flutax2
`Flutax2c
`
`22C
`
`21
`
`35
`
`122
`ND
`108
`ND
`
`TABLE II
`
`Equilibrium constants of fluorescent
`taxoids binding to microtubules
`As determined by equilibrium measurements Kr X 107 m1
`32C
`37C
`
`27C
`
`66
`60
`61
`59
`
`10
`02
`17
`03
`
`46
`43
`40
`42
`
`15
`04
`04
`02
`
`26
`29
`20
`22
`
`07
`03
`07
`02
`
`40C
`
`15
`21
`10
`20
`
`03
`02
`02
`01
`
`As calculated from the kinetic measurements
`
`Flutax2
`
`35 °C
`
`81
`109
`89
`
`61
`50
`61
`
`37C
`
`84
`123
`104
`
`12
`
`45
`55
`
`39C
`
`68
`98
`68
`
`41
`44
`44
`
`10
`
`0
`
`200
`
`150 1
`
`100 I
`
`arbitraryunits
`
`32 °C
`
`K x 105 Aff1
`K2
`KovR X107 M1
`Data from centrifugation measurements
`b ND not determined
`Data from anisotropy measurements
`
`139
`134
`188
`
`81
`44
`82
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`a c
`
`t 10
`
`000 025 050 075
`
`time s b
`
`50 H
`
`AFluorescence
`
`0
`
`000
`
`025
`
`075
`
`100
`
`050
`
`time s
`
`70
`
`60
`
`50
`
`52 400
`
`o2
`
`30
`
`20
`
`10
`
`0
`
`20
`
`40
`
`60
`
`80
`
`Sites11M
`
`FIG 3 A kinetics of binding of Flutax1 to microtubules
`at 35 C In
`the stopped flow device a 1 µM solution of Flutax1 was mixed with 25
`pm pure tubulin assembled into microtubules
`of sites 20
`concentration
`pm final concentrations
`of 500 nm Flutax and 10 pm sites in the
`absence a and presence b of 50 pm docetaxel Curve a is fitted to an
`exponential decay Inset residues between the experimental and theo
`retical curves B dependence
`of sites of the ob
`on the concentration
`served rate constants of binding of Flutax1 500 nm at 32 °C trian
`gles 35 °C diamonds 37 °C circles 39 °C inverted triangles and
`assembled from pure tubulin in GAB
`42 °C squares to microtubules
`The solid lines are the best fittings to the experimental
`data Inset
`for the binding of Flutax1 50
`on the sites concentration
`dependence
`nm to cross linked microtubules
`
`employing cross linked microtu
`concentrations were achieved
`bules in this way the concentration
`of sites can be decreased
`and the value of k 1 can be properly determined The results of
`these experiments are shown in an inset of Fig 3B The values
`obtained with cross linked microtubules were very sim
`of le +1
`to those obtained with normal microtubules Table III
`ilar
`Measuring the rate constants of the binding of Flutax2 to
`
`summarized in Table II The reactions were exothermic and
`characterized by negative entropy changes Table IV
`Kinetics of Binding of Flutax1 and Flutax2 to Microtubules
`Followed by Fluorescence IntensityThe mechanism of Taxol
`binding to microtubules was studied with kinetic methods The
`binding reaction was found to be very fast requiring stopped
`before and
`The length of the microtubules
`flow techniques
`after passing through the stopped flow system was checked
`using electron microscopy average length 485 ± 215 nm and
`370 ± 210 nm respectively and their morphology was found
`to be normal
`Fig 3A curve a shows the time course of binding of Flutax1
`have been
`to a 20 fold excess of microtubule sites Microtubules
`assembled from pure tubulin in GAB at 37 °C To check that
`the change of fluorescence is due to the specific binding of the
`probe to the Taxol site on the microtubules the same experi
`saturated with a non
`ment was performed with microtubules
`taxoid docetaxel
`fluorescent
`in this case no change of fluo
`rescence can be observed Fig 3A curve b The kinetics of the
`reaction can be expressed as one single exponential a fitting to
`a sum of two or more exponentials
`does not
`improve the resid
`uals and yields practically identical
`rate constants indicating
`that a single process is observed Fig 3B shows the dependence
`of the observed rate constant on the concentration
`of sites at
`temperatures The observed rate constant depends
`of sites calculated for each ex
`linearly on the concentration
`tubulin concentration measured as
`periment from the critical
`described in Ref 10 indicating a simple mechanism for the
`binding of the ligand to its site
`
`different
`
`k+i
`Flutax +site=> Flutaxsite
`
`SCHEME
`
`1
`
`With such a model the observed rate constant under pseudo
`on the rate constants le± and
`first order conditions depends
`k
`as follows
`
`1
`
`kobs = k isites + k1
`
`Eq 1
`
`in Fig 3B it
`From the representation
`is possible to deter
`mine k±1 from the slope of the regression and k1 from the
`of the regression to concentration zero The value
`extrapolation
`can be obtained with sufficient precision But in order to
`of k
`+1
`obtain the value of the dissociation constant with enough pre
`1e ±isites has to be low enough
`cision the value of the product
`so that k
`has a significant weight
`in hobs In order to do that
`1
`the concentration
`of tubulin has to be 1 order of magnitude
`lower than the one necessary to assemble microtubules These
`
`Abraxis EX2059
`Cipla Ltd. v. Abraxis Bioscience, LLC
`IPR2018-00162; IPR2018-00163; IPR2018-00164
`Page 4 of 13
`
`

`

`Fast Kinetics of Taxoid Binding to Microtubules
`
`26269
`
`Kinetic constants of binding k k and dissociation k k of fluorescent
`
`TABLE III
`
`taxoids from microtubules
`
`32 °C
`
`35 °C
`
`37C
`
`39C
`
`42C
`
`Flutax1k x105
`k x105 ms 1b
`s 1b
`k2 sIc
`Flutax2k x105
`sl
`
`k
`
`k
`
`s 1b
`
`s 1a
`
`s 1b
`
`029
`365
`343
`015
`004
`016
`NDa
`
`415
`030
`19
`
`070
`018
`05
`
`ND
`
`538
`612
`045
`
`566
`070
`21
`
`026
`022
`018
`
`050
`041
`08
`
`610
`743
`099
`03
`
`1383
`163
`27
`
`022
`026
`027
`01
`
`018
`018
`08
`
`936
`983
`121
`
`ND
`
`011
`014
`042
`
`1235
`181
`26
`
`090
`067
`10
`
`1265
`1174
`155
`ND
`
`025
`010
`033
`
`1772
`238
`ND
`
`072
`049
`
`20 °C
`
`25 °C
`
`30 °C
`
`35C
`
`37C
`
`40C
`
`42C
`
`Downloaded
`
`from
`
`httpwwwjbcorg
`
`by
`
`guest
`
`on
`
`May
`
`12
`
`2017
`
`179
`213
`
`192
`
`007
`013
`
`010
`
`243
`261
`
`007
`012
`
`361
`
`020
`
`453
`
`010
`
`ND
`
`ND
`
`220
`
`012
`
`271
`
`013
`
`343
`
`021
`
`from purified tubulin consist of a mixture of microtubules with
`a different number of protofilaments typically ranging from 11
`to 15 26 and have a certain percentage of open microtubules
`If microtubuleorganizing centers are used to nucleate micro
`tubule growth the number of protofilaments in microtubules
`assembled from pure tubulin is fixed at 13 and the fraction of
`is reduced 39 40 Microtubules were as
`open microtubules
`sembled from pure tubulin in GAB in the presence of 1 2 and
`5 ww axonemes from sea urchin sperm it was checked by
`
`electron microscopy that most of the microtubules were at
`tached to the axonemes The observed kinetics of Flutax1
`or absence of axon
`binding were not affected by the presence
`at 37 °C was 627 ± 051 NI 1 s1 with 1 axonemes
`638 ± 043 NI 1 s1 with 2 axonemes 636 ± 038 NI 1 s1
`with 5 axonemes 639 ± 049 NI 1 s1 This indicates that
`
`ernes le +1
`
`Followed by Fluorescence AnisotropyA large change
`
`taxoid were not sig
`the kinetics of binding of the fluorescent
`nificantly modified by the heterogeneity of in vitro assembled
`microtubules
`Kinetics of Binding of Flutax1 and Flutax2 to Microtubules
`in the
`fluorescence anisotropy of the probe is observed upon binding
`Table I The process was studied with stopped flow anisotropy
`measurements
`to obtain more information about
`in order
`the
`binding reaction Fig 6A shows the time course of the binding
`of Flutax1 to microtubules
`followed both by the change
`in
`intensity inset and anisotropy of the fluorescence It can be
`seen that the change in fluorescence anisotropy is much slower
`than the change in intensity The apparent
`rate constant of the
`change upon binding is very weakly dependent on
`anisotropy
`the concentration of sites hobs with 5 tod sites 022 ± 007 s1
`10 pod sites 026 ± 004 s1 20 tod sites 028 ± 005 s1
`37 °C indicating that a monomolecular
`reaction is observed
`The change in anisotropy
`can be expressed with a single expo
`indicating that a single process
`is being observed
`nential
`within the experimental limitations due to the technical diffi
`culty of the measurement see Experimental Procedures The
`is blocked by docetaxel Since photosta
`change
`in anisotropy
`bility controls showed that Flutax1 suffers photolysis about
`17 of the total
`intensity during the experiment measure
`ments were done mainly with Flutax2 which is devoid of this
`effect Fig 6B shows the time course of Flutax2 binding to
`is much slower than
`microtubules The observed rate constant
`that measured by the fluorescence intensity change and shows
`a weak dependence
`on the concentration of binding sites hobs 5
`
`029
`
`003
`
`ND
`
`043
`
`003
`
`059
`050
`
`071
`
`001
`005
`
`004
`
`105
`116
`
`005
`007
`
`136
`
`003
`
`Flutax1
`k2 X102 s
`k2 X102 s
`Flutax2
`k2 X102 s
`a Data obtained with non cross linked microtubules
`b Data obtained with cross linked microtubules
`Data obtained by measuring the change
`in fluorescence anisotropy
`a ND not determined
`e Data obtained by measuring the change
`
`in fluorescence intensity
`
`1
`
`1
`
`is more difficult
`microtubules
`change in fluo
`since very little
`rescence intensity is observed upon binding to its site Never
`is possible to follow the reaction due to the shift
`theless it
`to
`the blue of the spectrum Fig 1B By using a 530nm cutoff
`filter coincident with the isosbestic point
`the part of the
`spectrum at which the intensity of the bound form is smaller
`than that of the free form can be selected It
`is possible then to
`monitor
`of the
`the binding reaction by the small decrease
`observed fluorescence intensity Fig 4A In this way the val
`and k
`for the binding of Flutax2 to the taxoid
`ues of 1e
`binding site can be determined As in the case of Flutax1 the
`binding reaction is monophasic within the experimental inde
`termination due to the much lower signalnoise ratio and is
`The observed kinetic
`lin
`blocked by docetaxel
`rate depends
`of sites Fig 4B The kinetic con
`early on the concentration
`stants of the binding of Flutax1 and Flutax2 to its site are
`summarized in Table III
`Kinetic Homogeneity of the Binding Sites Since the change
`in fluorescence occurs in the ligand and not in the protein the
`order conditions
`to achieve
`in excess
`reactant
`pseudo first
`needs to be the assembled tubulin The use of microtubules
`as
`the ligand in excess implies the assumption that all of the sites
`in micro
`in the microtubule
`are equal Since the site of Taxol
`tubules has been mapped in the lumen of the tube using elec
`tron diffraction electron microscopy and docking methods 30
`34 it might be possible that the drug has to diffuse through
`the ends of the tube or through openings in the tube wall
`in
`to gain access to the site In this way the sites closer to
`order
`the outer solution would be more accessible
`than the more
`internal sites When those sites are in excess over
`the drug
`under the pseudo first order conditions it might be possible
`that only the outer ones become filled To evaluate this possi
`experiments were performed at equimolecular
`concen
`bility
`trations 5 10 and 20 tod of Flutax1 and sites and the
`resulting kinetic curves were analyzed using the proposed ki
`netic model Scheme 1 with the kinetic
`package
`analysis
`FITSIM 38 Fig 5 The data fit
`the predicted bimolecular
`reaction scheme with the same kinetic
`constants within the
`experimental error as those determined under pseudo first or
`der conditions thus demonstrating that all taxoid binding sites
`are equal from the kinetic point of view
`in the microtubule
`On the other hand structural differences like changes in the
`number of protofilaments or microtubule
`openings might af
`the kinetics of binding Microtubule
`solutions assembled
`fect
`
`Abraxis EX2059
`Cipla Ltd. v. Abraxis Bioscience, LLC
`IPR2018-00162; IPR2018-00163; IPR2018-00164
`Page 5 of 13
`
`

`

`26270
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`Fast Kinetics of Taxoid Binding to Microtubules
`
`Downloaded
`
`from
`
`a
`
`to
`
`Co