WO2000012543A2 - Compositions cristallines de farnesyl proteine transferase et procedes d'utilisation - Google Patents

Compositions cristallines de farnesyl proteine transferase et procedes d'utilisation Download PDF

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WO2000012543A2
WO2000012543A2 PCT/US1999/018819 US9918819W WO0012543A2 WO 2000012543 A2 WO2000012543 A2 WO 2000012543A2 US 9918819 W US9918819 W US 9918819W WO 0012543 A2 WO0012543 A2 WO 0012543A2
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Corey Strickland
Patricia C. Weber
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Schering Corporation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention relates to crystalline farnesyl protein transferase (FPT) and FPT in complex with substrates or inhibitors. This invention also relates to methods of using the structure coordinates of FPT to solve the structure of similar or homologous proteins or protein substrate or inhibitor complexes.
  • FPT farnesyl protein transferase
  • Ras oncogene The biological significance of the Ras oncogene, and the role of both Ras and the enzyme known as farnesyl protein transferase ("FPT") in the conversion of normal cells to cancer cells, are described in PCT International Publication Nos. WO95/00497 and WO95/10516.
  • FPT farnesyl protein transferase
  • the precursor of the Ras oncoprotein must undergo farnesylation of the cysteine residue located in a carboxyl-terminal tetrapeptide.
  • Farnesyl protein transferase catalyzes this modification.
  • Inhibitors of this enzyme have therefore been suggested as anticancer agents for tumors in which RAS contributes to transformation.
  • FPT inhibitors Drug discovery efforts directed toward FPT inhibitors have been hampered by the lack of adequate structural information about FPT and its complex with substrates and inhibitors.
  • the structure of FPT was first determined at Duke University using a crystalline form where the active site was blocked by the carboxy terminus of an adjacent molecule in the crystalline lattice. Beese et al., 1997, Science 275:1800-1804. These crystals are not suited for drug discovery because, as reported therein, the active site is blocked by part of the crystal lattice.
  • Another crystalline form of FPT was also reported in Dunten et al., 1998, Biochemistry 37(22):7907-7912. These crystals grow at pH 4.4 and are reported to only diffract to 2.8 A resolution.
  • Applicants have solved this problem by providing crystalline compositions comprising a farnesyl protein transferase (FPT) complexed with its natural substrates and with molecules that mimic its natural substrates.
  • FPT farnesyl protein transferase
  • the invention also provides the structure coordinates of these complexes. Further provided are methods for soaking these crystalline complexes in the presence of an inhibitor, thereby efficiently forming new crystalline enzyme: inhibitor complexes.
  • the invention also provides a method for determining at least a portion of the three- dimensional structure of molecules or molecular complexes which contain at least some structurally similar features to an FPT: substrate or FPT: inhibitor complex.
  • Figure 1 depicts stereo ribbon diagrams of an FPT: ⁇ HFP:Ac-CVIM-COOH complex. The view is into the active site cavity of the enzyme.
  • Figure 2 shows a diagram of a system used to carry out the instructions encoded by the storage medium of Figure 3 and 4.
  • Figure 3 shows a cross section of a magnetic storage medium.
  • Figure 4 shows a cross section of a optically-readable data storage medium.
  • FPT-mediated prenylation of Ras involves formation of a ternary complex comprising farnesyl protein transferase (FPT), farnesyl diphosphate (FPP) and Ras (FPT:FPP:Ras), followed by transfer of the 15-carbon isoprenoid from FPP to a cysteine sidechain of Ras.
  • the cysteine residue is part of a conserved carboxyl terminal Ca t a X sequence where X is a serine residue in H-Ras and methionine in N-Ras, K-Ras 4a, and K-Ras 4b isoforms.
  • FPT farnesyl protein transferase
  • the FPT-like polypeptide portion of the complex is any polypeptide that has the activity of naturally occurring FPT (Reiss et al, 1990, Cell 62:81-88), particularly the ability to cause farnesylation of the cysteine residue located in a carboxyl-terminal tetrapeptide of the precursor of the Ras oncoprotein.
  • FPT and “FPT-like polypeptide” are used interchangeably herein and include FPT and polypeptides that differ from FPT by having amino acid deletions, substitutions, and additions, but which retain the activity of FPT.
  • FPT is complexed with farnesyl diphosphate (FPP) and an inhibitor. These crystals diffract to at least 2.2 A resolution. At this resolution, most atoms of the FPT: FPP: inhibitor complex can be visualized using x-ray crystallographic methods.
  • FPP farnesyl diphosphate
  • the pre-formed crystals of FPT are grown by first complexing FPT with molecules that mimic the natural substrates.
  • FPT is complexed with an FPP analog and a tetrapeptide substrate based on the carboxy terminus of Ras referred to herein as a Caja X peptide.
  • the FPP analog used is ⁇ -hydroxyphosphonic acid ( ⁇ HFP) (Calbiochem-Novabiochem Corp.)
  • the Caja 2 X peptide is N-acetyl-Cys-Val-Ile-Met- COOH (CVIM) (SynPep Inc.).
  • FPP analogs and other Caja X peptides may be used in these complexes.
  • methylene-farnesyl diphosphate [Davisson et al, 1986, J. Org. Chem. 51:4768-4779] can also be used in place of ⁇ HFP as an FPP analog, however any analog of FPP could be used.
  • Caja X peptides may be used.
  • X is serine, methionine, cysteine, alanine or glutamine. See Reiss et al, 1991, PNAS 88:732-36.
  • the Ca ⁇ a 2 X peptide is Ac-CVLS-COOH (SynPep Inc.), Ac-CVIM(Se)-COOH (AnaSpec Inc.), or Ac- CVIM-COOH (SynPep Inc.).
  • the pre-formed FPT:FPP analog peptide crystals of this invention can be soaked in the presence of an inhibitor, thereby forming new protein/compound complexes and obviating the need to crystallize each individual protein/compound complex.
  • Crystalline complexes of FPT with FPP and an inhibitor can also be soaked in the presence of another inhibitor so that the original inhibitor is displaced and a new crystalline FPT: FPP inhibitor complex is formed.
  • the ability to form FPT:FPP:inhibitor complexes in the crystalline state allows very rapid turnaround of structural information.
  • an FPT : ⁇ HFP :C VIM complex is crystallized from a well-defined solution.
  • a solution for stabilizing the crystals artificial mother liquor
  • the crystals of FPT: ⁇ HFP:CVIM are soaked in solutions of artificial mother liquor containing inhibitor and farnesyl diphosphate (FPP).
  • FPP farnesyl diphosphate
  • the present invention provides the three-dimensional structure of (1) an FPT: ⁇ HFP:CVIM peptide complex at 2.4 A resolution, (2) an FPT:FPP:SCH66381 complex at 2.2 A resolution, (3) an FPT:FPP:SCH66701 complex at 2.9 A resolution, and (4) an FPT:FPP:SCH69132 complex at 2.5 A resolution.
  • the crystalline structures of the present invention provide information about the shape and structure of the FPT active site containing both an isoprenoid and a peptide or inhibitor.
  • structure coordinates refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of FPT:FPP/FPP analog:Peptide/ Inhibitor complexes in crystal form.
  • the diffraction data are used to calculate an electron density map of the repeating unit of the crystal.
  • the electron density maps are then used to establish the positions of the individual atoms of the FPT:FPP/FPP analog: Peptide/Inhibitor complexes.
  • a set of structure coordinates for an enzyme or an enzyme-complex or a portion thereof is a relative set of points that define a shape in three dimensions.
  • an entirely different set of coordinates could define a similar or identical shape.
  • slight variations in the individual coordinates will have little effect on overall shape.
  • the variations in coordinates discussed above may be generated because of mathematical manipulations of the structure coordinates.
  • the structure coordinates set forth in Figures 2-5 could be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, rotation of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
  • modifications in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also account for variations in structure coordinates. If such variations are within an acceptable standard error as compared to the original coordinates, the resulting three-dimensional shape is considered to be the same.
  • the Molecular Similarity application (Molecular Simulations Inco ⁇ orated) permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure.
  • the procedure used in Molecular Similarity to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results.
  • Each structure is identified by a name.
  • One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures). Since atom equivalency within QUANTA is defined by user input, for the pu ⁇ ose of this invention we will define equivalent atoms as protein backbone atoms (N, Ca, C and O) for all conserved residues between the two structures being compared.
  • the working structure is translated and rotated to obtain an optimum fit with the target structure.
  • the fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA.
  • any molecule or molecular complex that has a root mean square deviation of conserved residue backbone atoms (N, Ca, C, O) of less than 3 A when superimposed on the relevant backbone atoms described by structure coordinates listed in any one of Tables 2-5 are considered identical. More preferably, the root mean square deviation is less than 1.0 A.
  • root mean square deviation means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object.
  • root mean square deviation defines the variation in the backbone of a protein or protein complex from the relevant portion of the backbone of the FPT-like polypeptide portion of the complex as defined by the structure coordinates described herein.
  • the structure coordinates of an FPT: FPP/ FPP analog: peptide/Inhibitor complex and portions thereof are stored in a machine- readable storage medium.
  • Such data may be used for a variety of pu ⁇ oses, such as drug discovery and X-ray crystallographic analysis of protein crystals.
  • a machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Table 2, 3, 4 or 5.
  • System 10 includes a computer 11 comprising a central processing unit ("CPU") 20, a working memory 22 which may be, e.g. RAM (random-access memory) or “core” memory, mass storage memory 24 (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals 26, one or more keyboards 28, one or more input lines 30, and one or more output lines 40, all of which are interconnected by a conventional bi-directional system bus 50.
  • CPU central processing unit
  • working memory 22 which may be, e.g. RAM (random-access memory) or “core” memory
  • mass storage memory 24 such as one or more disk drives or CD-ROM drives
  • CRT cathode-ray tube
  • Input hardware 36 coupled to computer 11 by input lines 30, may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems 32 connected by a telephone line or dedicated data line 34. Alternatively or additionally, the input hardware 36 may comprise CD-ROM drives or disk drives 24. In conjunction with display terminal 26, keyboard 28 may also be used as an input device.
  • Output hardware 46 coupled to computer 11 by output lines 40, may similarly be implemented by conventional devices.
  • output hardware 46 may include CRT display terminal 26 for displaying a graphical representation of a binding pocket of this invention using a program such as INSIGHT as described herein.
  • Output hardware might also include a printer 42, so that hard copy output may be produced, or a disk drive 24, to store system output for later use.
  • CPU 20 coordinates the use of the various input and output devices 36, 46, coordinates data accesses from mass storage 24 and accesses to and from working memory 22, and determines the sequence of data processing steps.
  • a number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of hardware system 10 are included as appropriate throughout the following description of the data storage medium.
  • Figure 3 shows a cross section of a magnetic data storage medium 100 which can be encoded with a machine-readable data that can be carried out by a system such as system 10 of Figure 2.
  • Medium 100 can be a conventional floppy diskette or hard disk, having a suitable substrate 101, which may be conventional, and a suitable coating 102, which may be conventional, on one or both sides, containing magnetic domains (not visible) whose polarity or orientation can be altered magnetically.
  • Medium 100 may also have an opening (not shown) for receiving the spindle of a disk drive or other data storage device 24.
  • the magnetic domains of coating 102 of medium 100 are polarized or oriented so as to encode in manner which may be conventional, machine readable data such as that described herein, for execution by a system such as system 10 of Figure 2.
  • Figure 4 shows a cross section of an optically-readable data storage medium 110 which also can be encoded with such a machine-readable data, or set of instructions, which can be carried out by a system such as system 10 of Figure 2.
  • Medium 110 can be a conventional compact disk read only memory (CD-ROM) or a rewritable medium such as a magneto-optical disk which is optically readable and magneto-optically writable.
  • Medium 100 preferably has a suitable coating 112, which may be conventional, usually of one side of substrate 111. In the case of CD-ROM, as is well known, coating 112 is reflective and is impressed with a plurality of pits 113 to encode the machine-readable data. The arrangement of pits is read by reflecting laser light off the surface of coating 112.
  • a protective coating 114 which preferably is substantially transparent, is provided on top of coating 112.
  • coating 112 has no pits 113, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser (not shown).
  • the orientation of the domains can be read by measuring the polarization of laser light reflected from coating 112.
  • the arrangement of the domains encodes the data as described above.
  • the present invention permits the use of structure-based or rational drug design techniques to design, select, and synthesize chemical entities, including inhibitory compounds that are capable of binding to the active site of FPT, or any portion thereof.
  • Iterative drug design is a method for optimizing associations between a protein and a compound by determining and evaluating the three-dimensional structures of successive sets of FPT: FPP or FPP analog: Peptide or Inhibitor complexes.
  • binding pocket refers to a region of a molecule or molecular complex, that, as a result of its shape, favorably associates with another chemical entity or compound.
  • many drugs exert their biological effects through association with the binding pockets of receptors and enzymes. Such associations may occur with all or any parts of the binding pockets. An understanding of such associations will help lead to the design of drugs having more favorable associations with their target receptor or enzyme, and thus, improved biological effects. Therefore, this information is valuable in designing potential ligands or inhibitors of receptors or enzymes, such as inhibitors of FPT.
  • association refers to a condition of proximity between chemical entities or compounds, or portions thereof.
  • the association may be non-covalent ⁇ wherein the juxtaposition is energetically favored by exclusively or a combination of hydrogen bonding or van der Waals and/or electrostatic interactions — or it may be covalent.
  • crystals of a series of protein/compound complexes are obtained and then the three-dimensional structure of each complex is solved.
  • Such an approach provides insight into the association between the proteins and compounds of each complex. This is accomplished by selecting compounds with inhibitory activity, obtaining crystals of this new protein/compound complex, solving the three-dimensional structure of the complex, and comparing the associations between the new protein/compound complex and previously solved protein/compound complexes. By observing how changes in the compound affected the protein/compound associations, these associations may be optimized.
  • iterative drug design is carried out by forming successive protein- compound complexes and then crystallizing each new complex.
  • a pre-formed protein crystal is soaked in the presence of an inhibitor, thereby forming a protein/compound complex and obviating the need to crystallize each individual protein/compound complex.
  • the FPT:FPP/FPP analog:Peptide/Inhibitor complex crystals, and in particular the FPT: ⁇ HFP:CVIM crystals, provided by this invention may be soaked in the presence of a compound or compounds, to provide FPT:FPP:inhibitor crystal complexes.
  • the term "soaked" refers to a process in which the crystal is transferred to a solution containing the compound of interest.
  • the structure coordinates set forth in Tables 2-5 can also be used to aid in obtaining structural information about another crystallized molecule or molecular complex. This may be achieved by any of a number of well-known techniques, including molecular replacement.
  • the structure coordinates set forth in Tables 2-5 can also be used for determining at least a portion of the three-dimensional structure of molecules or molecular complexes which contain at least some structurally similar features to FPT.
  • structural information about another crystallized molecule or molecular complex may be obtained. This may be achieved by any of a number of well-known techniques, including molecular replacement.
  • this invention provides a method of utilizing molecular replacement to obtain structural information about a crystallized molecule or molecular complex whose structure is unknown comprising the steps of: a) generating an X-ray diffraction pattern from said crystallized molecule or molecular complex; and b) applying at least a portion of the structure coordinates set forth in any one of Tables 2-5 to the X-ray diffraction pattern to generate a three-dimensional electron density map of the molecule or molecular complex whose structure is unknown.
  • Molecular replacement provides an accurate estimation of the phases for an unknown structure. Phases are a factor in equations used to solve crystal structures that can not be determined directly. Obtaining accurate values for the phases, by methods other than molecular replacement, is a time-consuming process that involves iterative cycles of approximations and refinements and greatly slows the solution of crystal structures. However, when the crystal structure of a protein containing at least a homologous portion has been solved, the phases from the known structure provide a satisfactory estimate of the phases for the unknown structure.
  • this method involves generating a preliminary model of a molecule or molecular complex whose structure coordinates are unknown, by orienting and positioning the relevant portion of the complex according to any one of Tables 2-5 within the unit cell of the crystal of the unknown molecule or molecular complex so as best to account for the observed X-ray diffraction pattern of the crystal of the molecule or molecular complex whose structure is unknown. Phases can then be calculated from this model and combined with the observed X-ray diffraction pattern amplitudes to generate an electron density map of the structure whose coordinates are unknown. This, in turn, can be subjected to any well-known model building and structure refinement techniques to provide a final, accurate structure of the unknown crystallized molecule or molecular complex (E.
  • the method of molecular replacement is utilized to obtain structural information about a molecule or molecular complex, wherein the complex comprises an FPT:FPP/FPP analog :Peptide/Inhibitor complex.
  • the structure coordinates provided by this invention are particularly useful to solve the structure of crystals of FPT:FPP/FPP analog:Peptide/Inhibitor complexes, particularly FPT:FPP, co-complexed with a variety of chemical entities. This approach enables the determination of the optimal sites for interaction between chemical entities, including interaction of candidate FPT inhibitors with FPT or the FPT:FPP complex.
  • All of the complexes referred to above may be studied using well-known X-ray diffraction techniques and may be refined versus 1.5-3 A resolution X-ray data to an R value of about 0.25 or less using computer software, such as XPLOR [Yale University, ⁇ 1992; see, e.g., Blundell & Johnson, supra; Meth. Enzymol., vol. 114 and 115, H. W. Wyckoff et al, eds., Academic Press (1985)]. This information may thus be used to optimize known FPT inhibitors, and more importantly, to design new FPT inhibitors.
  • Rat FPT ⁇ and ⁇ subunit cDNAs in pUC13 were obtained from ATCC (ATCC clone numbers 63134 and 63127 respectively).
  • the rat ⁇ subunit cDNA was excised from pUC13 with Eco RI (5') and Hind III (3') and ligated downstream of the polyhedron promoter in p2Bac (Invitrogen) cut with Eco RI and Hind III to produce pRatA-2B.
  • the ATG start codon in the Nco I site upstream of the rat ⁇ subunit in pRatA-2B was removed by cutting with Nco I, removing the single stranded ends with mung bean nuclease, and religating the blunt ends together to produce pRatAN-2B.
  • the rat ⁇ subunit was excised from pUC13 with Xma I (5') and Nhe I (3') and ligated downstream of the P10 promoter in pRatAN-2B cut with Xma I (5') and Xba I (3') to produce pRABN-Bac.
  • pRABN-Bac was co-transfected with Baculovirus Gold DNA (Pharmingen) into Sf9 cells following the manufacturer's protocol. Recombinant baculoviruses were plaque purified and individual clones were tested for expression of FPT activity by SPA (Amersham) and for protein expression by Western blot (Santa Cruz anti-rat FPT antibodies). The highest expressing virus was amplified to high titre (>10 9 /ml) in Sf9 cells (infecting at MOI ⁇ 0.1 to retard amplification of defective viruses).
  • Protein purification was conducted at 4 °C. At each stage of the purification eluted proteins were detected by absorbance at 280 nm and active enzyme fractions were assayed by the SPA assay as described below.
  • Western blotting during protein purification was carried out according to a standard protocol using a BCIP/NBT kit (Kirkegaard & Perry Laboratories, Gaitherburg, MD) with an anti- ⁇ subunit polyclonal antibody raised against two peptide sequences of FPT ⁇ subunit and an anti- ⁇ subunit polyclonal antibody obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
  • the molecular weight of FPT was determined by MALDI mass spectroscopy with sinapinic acid as the matrix on a Voyager DE TOF mass spectrometer (PerSeptive Biosystem, Framingham, MA). FPT concentrations were determined either by the method of Bradford using a Bio-Rad assay kit with bovine serum albumin (BSA) as a standard or using UV abso ⁇ tion at 280 nm with a molar extinction coefficient of 1.53 x 10 5 M "1 cm "1 .
  • BSA bovine serum albumin
  • the cell pellet was resuspended in 300 ml homogenization buffer containing 50 mM Tris-HCl, pH 7.5, 1 ⁇ g/ml E-64, 2 ⁇ g/ml aprotinin, 0.7 ⁇ g/ml pepstatin, 0.1 mM leupeptin, 1 mM Pefabloc ® SC, 1 mM EDTA, 1 mM EGTA and 1 mM DTT (inhibitors from Boehringer Mannheim, DTT from Calbiochem).
  • the resuspended cells were disrupted by pressure using a microfluidizer (Microfluidics Co ⁇ oration, Newton, MA).
  • the active fractions were adjusted to 0.75 M NaCl using a 5 M NaCl stock solution. This sample was loaded onto a Phenyl Sepharose CL-4B column (Pharmacia), washed with the same starting buffer and the protein was eluted using a 0.75 M - 0 M NaCl gradient. The protein eluted at the end of the gradient - 0 M NaCl and was - 50 - 70 % pure.
  • the pooled rat FPT fractions were adjusted to 0.1 M NaCl and loaded onto a Mono Q HRlO/10 column (Pharmacia), washed with the same buffer and eluted with a gradient of 0.1 - 0.5 M NaCl.
  • the active fractions eluted ⁇ 0.3 M NaCl and were 60 - 80 % pure.
  • the active fractions were pooled and the buffer was adjusted to 0.75 M NaCl, 20 mM Tris, 10 ⁇ M ZnCl 2 , 1 mM DTT, pH 7.7. This sample was loaded onto a Phenyl Superose HR10/10 column (Pharmacia), washed with the starting buffer and sample eluted using a 0.75 - 0 M NaCl gradient.
  • FPT fractions (eluting ⁇ 0 M NaCl) that were > 95 % pure were pooled and dialyzed into a storage buffer consisting of 20 mM Tris- HCl, 20 mM KC1, 10 ⁇ M ZnCl 2 , 1 mM DTT, pH 7.7. The resulting protein solution was stored at -80 °C until use.
  • FPT Activity Assay FPT fractions were assayed using the Amersham Scintillation Proximity Assay (SPA) kit. Assays were performed at 23 °C in 200 ⁇ L with biotinylated human K-Ras-4B C-terminal peptide KKSKTKCVIM (100 nM) and [1- 3 H]-FPP (90 nM) as substrates in a buffer of 50 mM Tris, pH 7.7, 5 mM MgCl 2 , 5 ⁇ M ZnCl 2 , 0.01% Triton X- 100, 0.2 mg/ml BSA and 2 mM DTT. Reactions were initiated by adding FPT fractions (25 to 250 ng) to the substrate mixture.
  • SPA Amersham Scintillation Proximity Assay
  • reaction mixture was incubated for 30 min before it was terminated by 3 reaction volumes of a quench solution containing 5 mg/mL scintillation beads, 500 mM EDTA, pH 8.0, and 0.5% BSA. After rocking the quenched solution at room temperature for 30 min, the radioactivity inco ⁇ orated into the product was directly counted in a 96-well plate format with a Wallac 1204 Betaplate BS liquid scintillation counter.
  • rat FPT Purified rat FPT was dialyzed against 20 mM Tris pH 7.7, 1 mM DTT, 20 mM KC1, 10 ⁇ M ZnCl 2 and concentrated to 0.27 mM (25 mg/mL) and stored at -30°C prior to crystallization.
  • the Ac-Cys-Val-Ile-selenoMet-COOH: ⁇ HFP:FPT(CVIM: ⁇ HFP:FPT) ternary complex was prepared by incubating 108 ⁇ M FPT (10 mg/ml) with 300 ⁇ M ⁇ HFP (Calbiochem-Novabiochem Co ⁇ oration) for about 2 hours prior to adding 300 ⁇ M Ac-Cys- Val-Ile-Met(Se)-COOH (AnaSpec Inc.). ⁇ HFP was added from a 23.6mM solution in ethanol, yielding 0.6% (v/v) ethanol in the final solution. The peptide was added from a 50mM solution in DMSO, resulting in a 0.3% (v/v) final DMSO concentration.
  • the ternary mixture was incubated at 4°C for approximately 4 hours.
  • Vapor diffusion crystallization experiments were conducted using the hanging drop method. Potential crystallization conditions were screened by sparse matrix sampling (Jancarik et al., 1991, J Appl. Cryst. 24:409-411) and systematic grid screens (Cox et al, 1998, J. Cryst. Growth 90:318-324) using standard solutions (Hampton Research Inc.). Crystals formed when the reservoir contained polyethylene glycol (PEG) and sodium acetate.
  • PEG polyethylene glycol
  • Crystals most suitable for structure determination grew when the droplet contained 4 ⁇ l of the ⁇ HFP:CVIM:FPT complex, 4 ⁇ l of the reservoir solution (7% PEG 4000, 0.1 M sodium acetate, pH 5.7) and 1 ⁇ l of 200 mM DTT. Crystallization trays were incubated at 22°C and after 2-3 weeks, hexagonal rods (0.1 mm X 0.3 mm) appeared. Crystals can also be grown with other combinations of FPP analogs and Caja X peptides such as the following:
  • FPT:FPP inhibitor complexes were grown as described above by substituting FPP at 150 ⁇ M for ⁇ HFP and the inhibitor at 150 ⁇ M for the CVIM peptide.
  • FPT FPP:Inhibitor Complexes by Soaking Preformed Co-Crystals
  • the cocrystals of FPT with ⁇ HFP and the CVIM peptide or with FPP and an inhibitor as described above in Examples 2 and 3 can be used to soak FPT inhibitors. Crystals are harvested into the reservoir solution at 4° C. For each new inhibitor, crystals are transferred into reservoir solution supplemented with 10 ⁇ M ZnCl 2 , 2 mM DTT, 100 ⁇ M FPP and 100 ⁇ M inhibitor. The complex is allowed to form for about 24 hours at 4°C. In the presence of FPP and inhibitors, the FPP analog and peptide or original inhibitor are displaced, allowing structure determination of the new inhibitor:FPP:FPT complex.
  • X-ray diffraction data were collected on FPT complexes formed by co-crystallization. Prior to data collection, crystals were taken from the crystallization droplet and flash frozen in liquid propane using a cryoprotectant consisting of the reservoir solution supplemented with 25%o (v/v) glycerol. Crystals belong to space group P6j.
  • RES refers to the amino acid, FPP, FPP analog, peptide or inhibitor that the atom belongs to.
  • Atom refers to the element whose coordinates have been determined. Elements are defined by the first letter in the column except for zinc which is defined by the letters "ZIN”, bromine which is defined by the letters "BR” and chlorine which is defined by the letters "CL”. "#” is a number to identify the amino acid, FPP, FPP analog, peptide or inhibitor.
  • X, Y, Z is the crystailographically determined atomic position determined for each atom.
  • B is a thermal factor that measures movement of the atom around its atomic center.
  • C refers to the part of the complex the atom belongs to.
  • A alpha subunit of FPT.
  • B beta subunit of FPT.
  • Z zinc atom.
  • TYR CA 63 191 .3 157 .7 19 .1 39
  • a ARG CZ 69 187 .6 154 .6 15 .1 35
  • TRP CA 72 180 7 154 9 10 3 61 A ASP CG 82 173 2 141 8 28 4 53 A
  • TYR CD1 100 183 .2 144 .7 22 .1 31 A
  • GLU C 111 180 3 142 7 6 0 48 A
  • ARG NE 112 180 3 137 0 4 5 46 A ASP N 122 189 8 145 7 16 5 31 A
  • ARG CZ 112 180 0 135 8 4 0 46 A ASP CA 122 189 7 146 4 17 8 29 A
  • GLU CD 114 190 3 146 2 4 7 50 A ILE CG2 124 191 8 140 1 18 8 24 A
  • GLU OE1 114 190 9 146 2 5 8 50 A ILE CGI 124 189 7 139 8 17 4 23 A
  • GLU OE2 114 190 4 145 2 3 9 51 A ILE CD1 124 190 4 139 5 16 1 23 A
  • GLU N 150 200 5 138 3 6 0 35 A
  • GLU CA 150 200 4 139 6 6 7 35 A
  • GLU CB 160 204 0 138 1 21 5 24 A
  • GLU CB 150 199 7 140 5 5 8 38 A
  • GLU OEl 150 197 9 142 3 4 7 51 A
  • GLU OE2 160 206 7 136 0 20 0 27 A
  • GLU OE2 150 198 4 143 9 6 1 52 A
  • TRP CD2 169 199 3 129 0 20 5 18 A
  • TRP CE2 169 200 0 129 6 21 7 18 A
  • TRP CE3 169 200 1 128 6 19 5 16 A
  • TRP CD1 169 197 8 129 6 22 0 18 A
  • TRP NE1 169 199 0 130 0 22 5 19
  • TRP CD2 178 192 8 130 1 6 0 24 A
  • HIS CB 170 192 3 127 8 17 3 24 A
  • TRP C 178 196 7 127 6 3 7 31 A
  • HIS CD2 170 190 4 127 5 15 6 25 A
  • HIS ND1 170 191 8 125 8 15 8 25 A
  • HIS ND1 171 192 1 133 6 13 2 21
  • LYS CG 180 198 9 123 4 2 4 43 A
  • HIS CE1 171 192 3 134 6 12 2 20
  • LYS CD 180 198 0 124 4 1 6 46 A
  • GLU N 187 210 3 128 1 15 2 31 A ALA CA 197 209 0 127 1 29 0 21 A
  • TRP CE3 208 199 7 122 8 13 6 22 A
  • TRP CZ3 208 199 5 123 6 12 4 21 A ASP CB 217 215 4 116 0 11 4 47 A
  • GLU CB 212 200 7 118 3 10 5 42 A LEU O 220 216 8 116 7 21 9 36 A
  • GLU CD 212 198 .5 117 .2 10 5 51 A
  • GLU OE2 212 198 .4 116 .0 11 .1 53 A GLN CG 221 219 8 117 7 18 0 51 A
  • PHE CE2 213 202 .5 121 .9 6 .6 37 A TYR CD1 222 214 .4 123 .7 21 .1 30 A TYR CEl 222 213 7 124 8 21 6 31 A ARG CG 232 213 5 120 1 38 4 31 A

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Abstract

L'invention concerne des compositions cristallines comprenant des polypeptides de type farnésyl protéine transférase en complexe avec des substrats et inhibiteurs. L'invention concerne également des conditions de cristallisation pour ces compositions et leur utilisation pour la détermination structurale de complexes FPT:FPP/analogues de FPP:peptide/inhibiteur.
PCT/US1999/018819 1998-08-28 1999-08-26 Compositions cristallines de farnesyl proteine transferase et procedes d'utilisation WO2000012543A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU56787/99A AU5678799A (en) 1998-08-28 1999-08-26 Crystalline farnesyl protein transferase compositions and methods for use

Applications Claiming Priority (2)

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US14165198A 1998-08-28 1998-08-28
US09/141,651 1998-08-28

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WO2000012543A2 true WO2000012543A2 (fr) 2000-03-09
WO2000012543A3 WO2000012543A3 (fr) 2000-06-15

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5525479A (en) * 1992-06-17 1996-06-11 Merck & Co., Inc. Fluorescence assay of Ras farnesyl protein transferase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5525479A (en) * 1992-06-17 1996-06-11 Merck & Co., Inc. Fluorescence assay of Ras farnesyl protein transferase

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DUNTEN P ET AL: "Protein farnesyltransferase: structure and implications for substrate binding." BIOCHEMISTRY, (1998 JUN 2) 37 (22) 7907-12. , XP000872369 *
LONG S B ET AL: "Cocrystal structure of protein farnesyltransferase complexed with a farnesyl diphosphate substrate." BIOCHEMISTRY, (1998 JUL 7) 37 (27) 9612-8. , XP000872368 *
PARK H W ET AL: "Crystal structure of protein farnesyltransferase at 2.25 angstrom resolution Äsee commentsÜ Äpublished erratum appears in Science 1997 Apr 4;276(5309):21Ü." SCIENCE, (1997 MAR 21) 275 (5307) 1800-4. , XP000872421 *
PARK H W ET AL: "Protein farnesyltransferase." CURRENT OPINION IN STRUCTURAL BIOLOGY, (1997 DEC) 7 (6) 873-80. REF: 65 , XP000872382 *
STRICKLAND C L ET AL: "Crystal structure of farnesyl protein transferase complexed wit a CaaX peptide and farnesyl diphosphate analogue." BIOCHEMISTRY, (1998 NOV 24) 37 (47) 16601-11. , XP000872402 *
WU Z ET AL: "Farnesyl protein transferase: identification of K164 alpha and Y300 beta as catalytic residues by mutagenesis and kinetic studies." BIOCHEMISTRY, (1999 AUG 31) 38 (35) 11239-49. , XP000872405 *

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AU5678799A (en) 2000-03-21

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