WO2011067715A1 - Structure cristalline d'un complexe de thymidylate synthase (ts) avec un ligand - Google Patents

Structure cristalline d'un complexe de thymidylate synthase (ts) avec un ligand Download PDF

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WO2011067715A1
WO2011067715A1 PCT/IB2010/055503 IB2010055503W WO2011067715A1 WO 2011067715 A1 WO2011067715 A1 WO 2011067715A1 IB 2010055503 W IB2010055503 W IB 2010055503W WO 2011067715 A1 WO2011067715 A1 WO 2011067715A1
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atom
leu
arg
ligand
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Stefano Mangani
Cecilia Pozzi
Maria Paola Costi
Stefania Ferrari
Giambattista Guaitoli
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Universita' Degli Studi Di Siena
Universita' Degli Studi Di Modena E Reggio Emilia
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01045—Thymidylate synthase (2.1.1.45)
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • 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.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/50—Molecular design, e.g. of drugs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment

Definitions

  • the present invention relates to a crystal of the protein thymidylate synthase (TS) , in particular human TS in the presence of a ligand, methods of crystallization thereof and uses thereof.
  • the invention also relates to the three- dimensional structure of a new binding site located in the dimerization interface region of TS protein and applications thereof in the design and obtainment of new inhibitors with pharmacological activity, in particular antitumour activity.
  • thymidylate synthase is a protein of around 60-75 kDa, depending on species, active as a dimer.
  • the TS dimer is made up of identical monomeric units and is characterised by strong stability.
  • the dimerization interface region is highly preserved among the TSs belonging to different species; about 50% of the residues making it up are invariant/fairly invariant.
  • Human thymidylate synthase like that of other species, is a key enzyme for nucleotide metabolism and hence for cell survival. In particular, it represents the only means of adding a methyl group to the 5-position of the pyrimidine ring of thymidine during de novo synthesis.
  • thymidine is the only specific DNA precursor nucleotide
  • the enzyme TS on the basis of the functional role just discussed, represents an ideal candidate for generating antitumour drugs.
  • TS binds its own and other mRNAs in vitro, including c-myc, bcl-2 and p53. The meaning of this interaction is not known; however, it suggests that TS has an additional role besides its catalytic one.
  • experimental evidence has demonstrated that TS protein participates in the regulation of the synthesis of other proteins involved in the cell cycle, in the repair of DNA damage and in transcription.
  • TS performs a dual function: 1 ) as an enzyme, it plays an essential role in the biosynthesis of DNA; 2 ) as a protein, it participates in the regulation of protein synthesis, through a feedback mechanism based on the interaction with mRNA molecules.
  • TS protein in an inactive, unbound form, interacts with its own mRNA and with mRNAs encoding other proteins, acting as a transcriptional repressor. Said model is also able to explain the molecular mechanism at the basis of the resistance to TS inhibitor drugs currently used in antitumour therapies.
  • the inactive dimeric form (AAi) is in equilibrium with the active, unbound form (AAa) and with a ternary complex (AAaL, L stands for Ligand) , which represents the bound form.
  • AAaL, L stands for Ligand
  • Only the active conformation of hTS is capable of binding the substrates and is responsible for catalysis.
  • the structure of active hTS has the catalytic Cys- 195 in the active site and the region 107-128 (catalytic loop) is structured, whereas in the inactive conformation the hTS is bound to four phosphate ions, is not a catalyser and the region 107-128 (catalytic loop), unstructured, appears disorderly in its crystals.
  • the native protein exists in an apparent equilibrium between the conformational states. However, when TS is bound to the molecules of inhibitors it assumes the active conformation and loses the ability to bind mRNA which is characteristic of the native protein.
  • All of the inhibitors presently used in chemotherapy are analogous substrates that bind and stabilize the active conformation of TS, thereby preventing and reducing the enzyme's degradation.
  • the active conformation is not able to bind the phosphate ions or, presumably, mRNA.
  • hTS human thymidylate synthase
  • this binding site has made it possible to hypothesize a new mechanism of action for a particular class of ligands capable of binding hTS in a site located in the dimerization interface region and, therefore, of inhibiting its enzymatic function while at the same time preserving its ability as a translational repressor.
  • the coordinates of said crystallographic structures can thus be used for the purpose of designing and obtaining molecules capable of interfering with the dimerization process of the TS enzyme while preserving its function as a translational repressor.
  • Said molecules can be used for the treatment of cancer and to overcome the resistance of cancer cells due to use of the current antitumour drugs.
  • the drugs most commonly used at present to treat ovarian cancer e.g. platinum derivatives
  • the inhibitors designed and obtained on the basis of the crystallographic coordinates of the invention can prove useful for restoring the sensitivity of ovarian cancer cells to platinum-derived drugs.
  • Said inhibitors can be administered alone or in combination with other known drugs in order to enhance their therapeutic efficacy.
  • Figure 1 shows the amino acid sequence of histidine- tagged human thymidylate synthase (ht-hTS) ; the histidine tag of 12 residues added to the amino end of the hTS sequence is indicated in grey; the residues involved in binding to the peptide and the catalytic Cys residues are underlined; the sequence of the present construct is shifted by 12 units relative to that of the holoenzyme (hTS) ; the numbering of the sequence illustrated in the present patent application makes reference to the sequence in figure 1, where residue #1 is the Met that begins the sequence MRGSHHH;
  • Figure 2 shows the structure of native hTS (symmetric homodimer) ;
  • Figure 3 shows the structure of the hTS-ligand complex (non-symmetric homodimer) ; the ligand is represented as a darker coloured ribbon;
  • Figure 4 shows the details of the binding site of the peptide SEQ ID NO: 1 on hTS; the 56 aas of the site are shown as lines; the two subunits of hTS are represented in two different shades of grey to highlight the binding site in the interface region; the peptide is represented as a dark ribbon;
  • Figure 5 shows the details of the interactions between the peptide SEQ ID NO: 1 and the interface region of hTS; the two subunits of hTS are represented in two different shades of grey; the peptide is represented as sticks; the polar interactions of the peptide with hTS are represented as dotted lines.
  • Table 1 shows the percentages of identity, homology and diversity of the TS of various organisms compared with human TS;
  • Table 3 shows the amino acid residues involved in forming the dimer interface between the A and B subunits of ht-hTS;
  • Table 4 shows the atomic coordinates of the dimerization interface region; the legend of the columns is the same as described above for Table 2;
  • Table 5 shows the atomic coordinates of the binding site of SEQ ID NO: 1; the legend of the columns is the same as described above for Table 2;
  • Table 6 shows the ht-hTS residues placed at a contact distance ( ⁇ 3.5 A) from SEQ ID NO: 1;
  • Table 7 shows the data collection and refinement statistics of the crystal structures according to the invention .
  • the present invention relates to the three-dimensional structure of thymidylate synthase (TS) complexed with a ligand, preferably a peptide ligand, most preferably the peptide having the sequence LSCQLYQR, identified in the sequence listing with SEQ ID NO: 1.
  • a ligand preferably a peptide ligand, most preferably the peptide having the sequence LSCQLYQR, identified in the sequence listing with SEQ ID NO: 1.
  • the invention also relates to the three-dimensional structure of the TS region in which a peptide ligand binds.
  • the subject matter of the invention is the three- dimensional atomic coordinates associated with the binding site of a peptide ligand.
  • the binding site is located in the dimerization interface region between the A and B subunits of the TS.
  • the crystal structures of the TS-ligand complex and binding site of the invention were obtained using the peptide having SEQ ID NO: 1.
  • analogous crystals of TS can be obtained in the presence of peptide or non-peptide ligands having a sequence or structure analogous to that of peptide SEQ ID NO: 1.
  • the TS can be tagged at the amino and/or carboxylic end with a histidine tail (ht-TS) and can be of human origin (ht-hTS) .
  • the human TS of the present invention refers to SEQ ID NO: 2 (see Figure 1) .
  • the TS can be a bacterial TS, preferably chosen from among: TS of Escherichia coli (EcTS) , of Enterobacter faecalis (EfTS) , of Pneumocystis carinii (PcTS) and of Criptococcus neoformans (CnTS) .
  • the atomic coordinates of a three-dimensional structure of the crystal of ht-hTS were determined; its sequence is indicated with SEQ ID NO: 2 and is shown in Figure 1 . Said atomic coordinates are not included in the text of the present patent application.
  • One embodiment of the present invention relates to the three- dimensional structure of the complex between ht-hTS and a ligand, preferably the peptide SEQ ID NO: 1 , and to the atomic coordinates thereof listed in Table 2 .
  • the subunits of the protein are identified as molecule A and molecule B and comprise atoms numbered from 1- 4260 ; the atoms 4261-4521 are oxygen atoms of water molecules; the atoms 4522-4541 belong to four sulphate ions bound to ht-hTS-SEQ ID NO: 1 .
  • the molecule SEQ ID NO: 1 comprises the atoms numbered from 4542-4610 .
  • the atoms 4611- 4615 are sulphide atoms bound to the side chains of the cysteine residues.
  • the crystal of the complex is obtained by co-crystallization of ht-hTS with the peptide SEQ ID NO: 1 .
  • the three-dimensional structure of the crystal of ht-hTS-SEQ ID NO: 1 has the space group P3i .
  • An advantageous characteristic of the structure defined by the atomic coordinates in Table 2 is that it possesses a resolution of around 2 . 3 A.
  • the dimensions of the cell in the two crystals of free ht-hTS and complexed ht-hTS are similar and can be subject to a variability of 5% .
  • the structure of the ht-hTS dimer is described by the coordinates in Table 2.
  • the first 37 residues of the construct which comprise the 12 residues of the histidine tail up to the 25 residues of the amino end of the sequence of hTS, are not visible in the electron density map and are not part of the model.
  • the residues 115-141 are similarly not visible in the electron density map, either in the ht-hTS structure, or in that of ht-hTS-SEQ ID NO: 1 and are thus omitted from the model.
  • the Pro38 residue is the first residue observable in the electron density map and Glu322 is the last.
  • Both the structures of ht-hTS and of the complex ht-hTS-SEQ ID NO: 1 are homodimers correlated, respectively, by a crystallographic axis of order 2 or a non-crystallographic axis of order 2.
  • the two subunits present in each of the two structures adopt the so-called inactive conformation of the loop of the active site (residues 193-209) with the catalytic Cys207 (Cysl95 in the numbering scheme of the wild-type enzyme) which is rotated in a non-productive conformation far from the cavity of the active site.
  • Comparing the structure of the complex ht-hTS-SEQ ID NO: 1 and that of ht-hTS made it possible to identify the binding of the peptide SEQ ID NO: 1 in a region of the hTS enzyme previously not identified as a binding site.
  • the binding region of the peptide SEQ ID NO: 1 in ht-hTS is the principal subject matter of the present invention.
  • the binding region of SEQ ID NO: 1 has the potential of being a binding site also for ligands of various nature.
  • the dimerization interface region between the A and B subunits of ht-hTS comprises the amino acid residues shown in Table 3.
  • LEU 204 PRO 206, CYS 207, LEU 210, GLN 212, TYR 214,
  • TYR 225 ARG 227, SER 228, ILE 261, THR 263, LEU 264,
  • the three-dimensional structure of the dimer interface region is shown in Figure 5.
  • the atomic coordinates of the residues involved in the interface are shown in Table 4. Thanks precisely to the comparison between the atomic coordinates of the protein crystal and the crystal of the complex, it was possible to identify a new binding site in the dimerization interface region of the ht-hTS homodimer.
  • the specific atomic coordinates of the binding site are shown in Table 5 and form a preferred object of the present invention .
  • the cleft comprises Cysl92 and it is close to the catalytic Cys207.
  • the difference Fourier maps reveal a continuous electron density that extends from the Sy atom of Cysl92 B, which can be interpreted as the peptide SEQ ID NO: 1 covalently bound via a disulphide bridge to Cysl92B through the Cys3 residue of SEQ ID NO: 1.
  • the residues 1-8 of the peptide SEQ ID NO: 1 were modelled and are shown in Table 2.
  • the 4 amino acids at the amino end of the peptide SEQ ID NO: 1 (Leul - Gln4) adopt an extended conformation and, in addition to the covalent bond formed between Cys3 and Cysl92B, they interact with hTS through three hydrogen bonds that involve NH or CO groups present in the skeleton of the peptide.
  • Hydrophobic contacts occur between Leul of SEQ ID NO: 1 and et202 of the A subunits of hTS.
  • the loops between the residues 153-172 and residues 194-207 of ht-hTS, which contact with the peptide SEQ ID NO: 1, show root mean square deviations of between 1.2 A and 2.0 A. Larger deviations can be observed for some residues of the side chains in this region, such as, for example, Phel54 and Leu204, which deviate respectively by 2.5 A and 3.2 A between the two structures.
  • Table 7 shows the ht-hTS residues which are at a contact distance from the peptide SEQ ID NO: 1.
  • the atomic coordinates shown in the tables and the numberings of the amino acid residues used in the present patent application refer to the hTS sequence tagged with histidine (12-residues) added at the amino end of the sequence of hTS.
  • the residue number 1 is the Met which begins the histidine tag.
  • the coordinates of tables 2, 4 and 5 provide a measure of the atomic position in Angstroms, given to 3 decimal places.
  • the coordinates are a relative set of positions which define a structure in three dimensions.
  • a person skilled in the art will be aware of the fact that a set of completely different coordinates, having a different origin and/or axes, can define a similar or identical form.
  • the relative positions of the atoms of the structure or selected coordinates are diversified so that the root mean square deviation of the resulting varied structure is less than 1.5 A when superimposed on the coordinates provided in tables 2, 4 and 5, and/or when the number, and/or positions of the water molecules and/or the ligand molecules are changed.
  • the method used to obtain the ht-hTS crystal or the crystal of ht-hTS-SEQ ID NO: 1 illustrated in the present invention can be used to obtain an ht-hTS crystal or crystal of ht-hTS- SEQ ID NO: 1 characterised by a resolution of 2 to 3 A, preferably of around 3.0 A or greater.
  • Human thymidylate synthase was purified from E. Coli BL21 strain DH5a transformed with the vector pQE80L, which contains the complete sequence encoding the human thymidylate synthase (hTS) tagged with a histidine tail.
  • the purification involved sequential chromatography on a column with Ni Sepharose Fast Flow resin and a HiTrap desalting column, both by GE Healthcare.
  • the thymidylate synthase of E. Coli was purified by ammonium sulphate precipitation and sequential chromatography using DEAE Sephacel and Phenylsepharose CL-4B resins.
  • the enzymes showed a pattern with a single band when submitted to SDS- polyacrylamide gel electrophoresis.
  • the purified protein was stored at -80°C after the concentration had been determined by measuring absorbance at 280 nm.
  • the activity of the TS was spectrophotometrically determined by monitoring the increase in absorbance at 340nm in the presence of dUMP and mTHF.
  • the ht-hTS crystals were grown at 4°C, using the sitting-drop method, in a solution containing 200 ⁇ ht-hTS in 0.1 M HEPES at pH 7.5 and 20 mM ⁇ -mercaptoethanol . 2-3 ⁇ , of said solution was mixed with an equal volume of a precipitating solution containing a solution obtained by diluting a saturated solution (in water) of (NH ) 2 S0 4 , 20 mM ⁇ - mercaptoethanol and 0.1 M Tris-HCl to 20-25% (v/v) at pH 8.3.
  • the ht-hTS crystals grew in about a week, reaching a final size of approximately 100 x 200 x 200 um.
  • the crystals of the complex of ht-hTS with the peptide SEQ ID NO: 1 were grown in the same ht-hTS solution as described above and incubated for 2 hours at 4°C with a 1 mM solution of the peptide SEQ ID NO: 1 in water.
  • the sitting-drop plates were prepared by adding 3 ⁇ , of the solution of ht-hTS-SEQ ID NO: 1 with an equal volume of precipitating solution consisting in a solution obtained by diluting a saturated solution (in water) of (NH ) 2 S0 , 20 mM ⁇ -mercaptoethanol and 0.1 M Tris-HCl to 20-25% (v/v) at pH 8.3.
  • the crystals of the complex ht-hTS-SEQ ID NO: 1 appeared in 3-5 days and grew until reaching a final size similar to that of native crystals in three weeks.
  • the crystals of ht-hTS and of the complex ht-hTS-SEQ ID NO: 1 demonstrate the same behaviour and belong to the trigonal system.
  • Diffraction data were collected on crystals frozen at 100 K under cold nitrogen flow. 20% glycerol was added to the crystal mother liquor as a cryoprotectant .
  • the data were collected at ESRF (Grenoble) on the beamlines ID 14-1 and ID 23-1 equipped with ADSC Q210 CCD detectors.
  • the crystals demonstrated to be stable to X-ray exposure throughout the data collection time (about 25 ' ) . A single crystal was used for each dataset.
  • the final statistics of the data collection are reported in Table 7 .
  • La ht-hTS structure was resolved in trigonal space group ⁇ 3 21 using the molecular replacement technique.
  • the model used was that of a subunit of human TS crystallized under low-salinity conditions (PDB 1YPV) with all of the water molecules omitted.
  • the content of the asymmetric unit consists in a subunit of the ht-hTS dimer which occupies a special position having a symmetry of point 2 with a molecular axis of order 2 coinciding with the crystallographic axis of order 2 present in the cell.
  • the correct orientation and the translation of the molecule inside the crystallographic unit cell was determined by analyzing the Patterson function, as implemented in the OLREP software program.
  • the program provided an evident solution for the positioning of the content of the asymmetric unit.
  • the first 37 N- terminal residues of ht-hTS present in the construct were not visible in the electron density map and are not part of the model; the residues from 120 to 141 were not visible in the electron density map and were omitted from the model, as were the last three residues of the C-terminal part.
  • the XtalView and Coot programs were used to manually reconstruct the model.
  • the crystal structure of the complex ht-hTS-SEQ ID NO: 1 was resolved using MOLREP molecular replacement and ht-hTS as a model.
  • the formation of a 1 : 1 complex between the ht-hTS dimer and the peptide breaks the binary symmetry of the molecule and lowers the symmetry of the space group from P3i21 to P3i , as subsequently confirmed by refinement of the structure.
  • the content of the asymmetric unit consists in the entire dimer and the peptide SEQ ID NO: 1.
  • the initial molecular replacement models were submitted to an initial rigid body refinement cycle followed by numerous conventional refinement cycles using the method which exploits the maximum likelihood function as implemented in REF AC5. Between the refinement cycles, the model was submitted to a manual reconstruction using XtalView and Coot.
  • the Coot program was used to model the peptide SEQ ID NO: 1. Water molecules were added in both cases using the standard procedure in the ARP/wARP suite.
  • the refined model of ht-hTS consists of 263 amino acids, 91 water molecules and 2 sulphate anions originating from the crystallization solution.
  • An inspection of the difference Fourier maps showed that four of the five cysteine residues present in ht-hTS reacted with ⁇ -mercaptoethanol (BME) to give a covalent adduct.
  • BME ⁇ -mercaptoethanol
  • a whole molecule of BME was visible in the electron density map only for Cys211, whereas in the case of Cys55, Cysl92 and Cys207 only a sulphur atom was identified and fitted into the model.
  • the tendency of cysteine residues of hTS to react with ⁇ -mercaptoethanol has been observed previously.
  • the two sulphate ions are bound to the dimerization interface region by means of Argl87 and Argl97 hydrogen bonds in the same anion recognition site where the substrate dUMP is bound.
  • the occupation of the peptide was maintained at 1.00 in the case of P3i, though the refinement ended with high temperature factors (50-70% greater than the atoms of ht-hTS) , indicating an occupation slightly less than 1.0 for the peptide SEQ ID NO: 1.
  • the final R-cryst and R-free factors converged at 0.24 - 0.28 for the structure in P3i21, as compared to values of 0.20 - 0.25 obtained for the structure in space group P3i at the same refinement steps.
  • the lower values of the R-cryst and R-free parameters indicate the correctness of space group P3i for the complex ht-hTS-SEQ ID NO: 1.
  • the stereochemical quality of the refinement model was checked using the PROCHECK program.
  • the crystal structures obtained according to the present invention can be used in different ways for drug design.
  • the design of ligands that are selective for the new binding site identified by the present invention thanks to the co-crystallization of the TS protein with the ligand SEQ ID NO: 1 can be achieved by making reference to the coordinates reported in Table 2, optionally varied by a root mean square deviation of less than 1.5 A.
  • the selected coordinates which are preferred for the purposes of the invention are reported respectively in tables 4 and 5, and may be optionally varied by a root mean square deviation of less than 1.5 A.
  • the selected coordinates in Table 5 are particularly preferred in that they represent the coordinates of the new binding site located in the dimerization interface region of TS.
  • the ligands that bind to the new binding site will be capable of interfering with the dimerization process of the enzyme TS while preserving its activity as a translational repressor. Therefore, they can be used as antitumour drugs, in particular for the treatment of ovarian cancer. Said ligands can also be used to restore the sensitivity of tumour cells, in particular ovarian cancer cells, to the antitumour drugs commonly used.
  • ligand refers to TS "modulators", i.e. peptide (or non-peptide) compounds capable of causing a change (i.e. a modulation) in the level of biological activity of the TS enzyme.
  • modulators i.e. peptide (or non-peptide) compounds capable of causing a change (i.e. a modulation) in the level of biological activity of the TS enzyme.
  • a modulation may include physiological changes that bring about an increase (activation) or a decrease (inhibition) in the activity of TS.
  • the modulation could originate directly as a result of the binding with the new binding site, or it could be an indirect modulation (e.g. through the binding of the ligand to any binding site of TS such that the activity of TS or its interactions with other biomolecules (e.g. mRNA) are compromised) .
  • an indirect modulation e.g. through the binding of the ligand to any binding site of TS such that the activity of TS or its interactions with other biomolecules (e.g. mRNA) are compromised
  • the modulation could imply the over- or under- expression of TS caused by these mechanisms, as well as hyper- or hypo-activity due to the binding of the ligand to the new binding site.
  • information about the orientation of the bond of a ligand in the binding site can be determined by co- crystallization, soaking or computational docketing of the ligand. This will suggest some specific modifications to be applied to the chemical structure of the ligand in order to improve it and render interaction with the protein more specific.
  • the determination of the three-dimensional structure of TS and of the TS-ligand complex provides a basis for the design of new ligands, e.g. chemical compounds that interact with TS enzymes .
  • the invention provides a method of determining the structure of a potential ligand of human TS or bacterial TS protein.
  • the invention provides a method of evaluating the ability of a potential ligand to bind to TS protein, in particular to at least one amino acid in a binding site located in the dimer interface region of TS. The method comprises the following steps:
  • the complex of human TS or bacterial TS can be obtained by co-crystallization with the following procedure: samples of purified protein are incubated for a period of time (usually > 1 hour) with a potential ligand. The solution of the protein-ligand complex can be assayed to determine the conditions of crystallization.
  • step b) indicated above it is possible to back-soak the crystal of the complex of said potential ligand with human TS or bacterial TS where the ligand is bound to binding site located in the dimer interface region of TS, in order to form a complex between the TS and another potential ligand, which will be analysed as indicated in steps c) and d) .
  • the protein crystals complexed with a ligand can be back-soaked to remove the ligand by placing the crystals in a stabilizing solution in which the potential ligand is not present. The resulting crystals are then transferred into a second solution containing the potential ligand.
  • Any ligand identified with this method will be an inhibitor of TS protein and may have antitumour action.
  • a blend of compounds can be used for the soaking procedure (step b) or co-crystallized with TS to obtain in any case crystals of the TS-ligand complex. In this way one can determine which of the compounds present in the blend is an inhibitor of TS .
  • the electron density maps can be computed using programs such as those featured in the CCP4 computational suite (Collaborative Computational Project 4.
  • the CCP4 Suite Programs for Protein Crystallography, Acta Crystallographica, D50, (1994), 760-763).
  • Programs like Coot can be used to display the map and construct the model.
  • the invention comprises a method of resolving the structure of a TS complexed with a ligand that binds to at least one amino acid in a binding site located in the dimer interface region of TS, comprising the steps of: - collecting the X-ray diffraction data of a crystal of TS complexed with a ligand;
  • ATOM 292 or ALA A 75 -58.579 2.080 5.282 1.00 25.50 o
  • ATOM 580 CA ASN A 109 -32. 581 17.105 -5.312 1.00 93.37 C
  • ATOM 842 CA SER A 163 -23.067 10.512 -6.104 1.00107.97 c

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Abstract

L'invention concerne: un cristal de thymidylate synthase (TS), en particulier de TS humaine, en présence d'un ligand; ses méthodes de cristallisation; et ses utilisations. L'invention concerne également la structure en 3D d'un nouveau site de fixation situé dans la région de l'interface de dimérisation de la TS, et ses applications pour la conception et l'obtention de nouveaux inhibiteurs à activité pharmacologique, et en particulier à activité anti-tumorale.
PCT/IB2010/055503 2009-12-01 2010-11-30 Structure cristalline d'un complexe de thymidylate synthase (ts) avec un ligand WO2011067715A1 (fr)

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IT002117A ITMI20092117A1 (it) 2009-12-01 2009-12-01 Struttura del cristallo del complesso di timidilato sintetasi (ts) con un ligando
ITMI2009A002117 2009-12-01

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002072753A2 (fr) 2001-03-07 2002-09-19 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Peptides de thymidylate synthase se fixant a l'arnm de la thymidylate synthase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002072753A2 (fr) 2001-03-07 2002-09-19 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Peptides de thymidylate synthase se fixant a l'arnm de la thymidylate synthase

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ALLEGRA C. ET AL., BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 297, 2002, pages 24 - 31
BERGER S H ET AL: "Effects of ligand binding and conformational switching on intracellular stability of human thymidylate synthase", BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - PROTEINS & PROTEOMICS, ELSEVIER LNKD- DOI:10.1016/J.BBAPAP.2003.09.005, vol. 1696, no. 1, 14 January 2004 (2004-01-14), pages 15 - 22, XP004483641, ISSN: 1570-9639 *
BERGER S. H. ET AL., EFFECTS OF LIGAND BINDING AND CONFORMATIONAL SWITCHING ON INTRACELLULAR STABILITY OF HUMAN THYMIDYLATE SYNTHASE. BBA-2004
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