WO2004072276A1 - Structure cristalline de la synthase 2-c-methyle-d-erythritol 4-phosphate en complexe avec des inhibiteurs - Google Patents

Structure cristalline de la synthase 2-c-methyle-d-erythritol 4-phosphate en complexe avec des inhibiteurs Download PDF

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WO2004072276A1
WO2004072276A1 PCT/EP2004/001075 EP2004001075W WO2004072276A1 WO 2004072276 A1 WO2004072276 A1 WO 2004072276A1 EP 2004001075 W EP2004001075 W EP 2004001075W WO 2004072276 A1 WO2004072276 A1 WO 2004072276A1
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inhibitor
synthase
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Adelbert Bacher
Wolfgang Eisenreich
Stefan Hecht
Johannes Kaiser
Felix Rohdich
Robert Huber
Stefan Steinbacher
Stefan Gerhardt
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the present invention relates to a novel crystal form of the protein 2-C-Methyl-D-erythritol 4- phosphate synthase (IspC), notably a crystal form that allows the determination of the three- dimensional structure of IspC with inhibitors.
  • the invention further relates to the three- dimensional structure of IspC in complex with an inhibitor and/or in complex with metal ions. Further, the invention relates to methods of identifying and designing novel inhibitors of IspC using the atomic coordinates of IspC in complex with an inhibitor and/or metal ions.
  • the invention also relates to potential inhibitors identified by these methods that may be used as antimalarial drugs, as antibiotics for pathogenic bacteria, or as herbicides.
  • Malaria represents one of the most threatening diseases especially in developing countries with about 300 million cases every year resulting in more than one million deaths per year (1). Although it had been believed that malaria will be eradicated shortly, the present experience is the contrary since the disease is escalating even in countries with satisfactory medical services. This and the development of multi-resistant variants of other pathogenic organisms defines the urgent need of novel chemotypes of antibiotics that expand the repertoire of presently targeted biochemical pathways (2,3). The availability of the genome sequence of Plasmodium falciparum, the causative agent of malaria, especially the wealth of information on the biochemical pathways used by this organism has opened novel approaches for drug development (4,5).
  • mevalonate-independent "2-C- methyl-D-erythritol 4-phosphate (MEP) pathway (Fig. 1a) that leads to the universal isoprenoid precursors isopentenyl diphosphate (IPP, 4) and dimethylallyl diphosphate (DMAPP, 5) via 2-C-methyl-D-erythritol 4-phosphate (2) and (£)-1-hydroxy-2-methyl-but-2- enyl 4-diphosphate (3) has attracted considerable interest (6,7).
  • the existence of this pathway in certain eubacteria and plants has been demonstrated by pioneering studies of Rohmer (8) and Arigoni (9,10) (reviewed in (11-13)).
  • 2-C-methyl-D-erythritol 4-phosphate (2, MEP) is synthesized from 1-D-deoxyxylulose 5-phosphate (1) by IspC (15) via intramolecular rearrangement of 1-D-deoxyxylulose 5-phosphate (1) (10,16-18) followed by NADPH-dependent reduction of the aldehyde intermediate, 2-C-methyl-D-erythrose 4- phosphate (8) (Fig. 1b).
  • the aldehyde intermediate can be chemically synthesized and converted either into the substrate 1-D-deoxyxylulose 5-phosphate or the product 2-C- methyl-D-erythritol 4-phosphate by IspC and nicotinamide nucleotides depending on the reaction conditions, which demonstrates not only full reversibility of the reaction but also that 2-C-methyl-D-erythrose 4-phosphate is a true intermediate (19).
  • the stereochemical course of the reaction has been studied by isotope labeling experiments and analysis of the resulting compounds by NMR spectroscopy both in vivo (20) and in vitro (21 ,22).
  • the enzymatic properties of IspC from E. coli have been studied in detail (19,23,24).
  • fosmidomycin The enzymes from E. coli, Chlamydomonas, Arabidopsis thallana, and Plasmodium falciparum are susceptible to inhibition by fosmidomycin (9, Fig. 1c) (6,23,25,26). Thus, fosmidomycin and its derivative FR-900098 (10) have also been shown to cure mice infected by Plasmodium vinckei (6). Clinical trials showed that fosmidomycin can be used with a high rate of success for the treatment of malaria (7).
  • the three-dimensional (3D) structure of the E. coli IspC enzyme without ligands is known (27,28). From this structure, however, no structural information on the mode and place of substrate and/or inhibitor binding is available. Further, no metal ions known to be mandatory for enzymatic activity of IspC could be identified and, consequently, no mechanistic role could be assigned to these metal ions. Thus, this structure cannot be used for rational drug design and its usefulness for identifying new inhibitors of IspC is very low.
  • the invention provides a novel crystal which comprises the protein 2C-methyl-D-erythritol 4- phosphate synthase (IspC; (EC 1.1.1.267)), namely a monoclinic crystal form.
  • IspC protein 2C-methyl-D-erythritol 4- phosphate synthase
  • the novel crystal comprising IspC is unrelated to that previously described (27,28).
  • the novel crystal of the invention surprisingly allows cocrystallisation with inhibitors, notably with fosmidomycin, as well as soaking in said inhibitors, notably with fosmidomycin or a deprotonated form thereof.
  • the novel crystal of IspC allows the determination of the crystal structure of E.
  • the inhibitor fosmidomycin has been found to bind well-ordered to IspC in the crystal of this invention, thereby identifying the active site of the enzyme and allowing a detailed mapping of active site regions and residues which are responsible for binding certain substrate or inhibitor moieties.
  • the binding site of metal ions known to be essential for catalysis of IspC has been identified in the crystal structure of E. coli IspC in complex with metal ions.
  • Said metal ions are located in the active site of IspC and play a key role in inhibitor binding.
  • Said metal ions are preferably divalent metal ions, and more preferably manganese ions. It is most preferred that the novel crystal comprises an inhibitor and metal ions, notably fosmidomycin and manganese ions.
  • the present invention allows for the first time to perform rational drug design of novel inhibitors of IspC based on an active site conformation as it occurs upon inhibitor binding.
  • the crystals of the invention feature the important advantage that the active site of IspC is easily accessible to substrate and analogs thereof or inhibitors, which allows soaking experiments and the determination of the 3D structure of IspC in complex with low molecular weight organic compounds like inhibitors.
  • Crystallisation may be done by any method known in the art like batch methods or vapour diffusion methods. Hanging- or sitting-drop vapour diffusion methods are preferred.
  • the 3D structure of IspC in complex with an inhibitor may be solved by preparing a crystal containing IspC in complex with the inhibitor. This may be achieved by co-crystallizing IspC with the inhibitor or by soaking a crystal of IspC not containing an inhibitor in mother liquor containing an excess of the inhibitor of interest for a suitable time. Prior to collection of diffraction data, crystals may be frozen according to known methods of cryo-crystallography, preferably after treatment of the crystal with a suitable cryo protectant.
  • the crystal of the invention may be used for the determination of the three-dimensional structure of the protein 2C-methyl-D-erythritol 4-phosphate synthase (IspC) or the three- dimensional structure of said synthase in complex with an inhibitor of said synthase.
  • the 3D structure further contains bound metal ions in the active site of IspC.
  • the 3D structure of IspC, preferably with bound inhibitor, may be solved according to known procedures of protein crystallography.
  • the the 3D structure is solved as described in example 2.
  • the accuracy of the coordinates of a protein crystal structure depends inter alia on the resolution of the diffraction data used in refinement.
  • the resolution should be such that amino acid side chains in well-ordered regions of the protein can be seen in the electron density maps.
  • the resolution should be at least 4 A, preferably better than 3 A, and most preferably at least 2.5 A.
  • the coordinates provided herein contain experimental error and are limited inter alia by the resolution of the diffraction data. Crystallisation conditions may be further improved according to known approaches in protein crystallisation, diffraction data to better resolution or of otherwise better quality may be measured and more accurate coordinates may be obtained. This may e.g. be achieved by using synchrotron radiation, optionally in combination with cryo-crystallography. It has been found that the crystals used herein can easily be frozen by liquid nitrogen.
  • the structure of the synthase may undergo changes. Often, such changes are limited to amino acid side chain conformations but whole groups of amino acids including their peptide back bone may move as well, particularly amino acids in a flexible loop. Such altered conformations are also comprised by this invention as long as the overall fold of the protein remains the same.
  • the three structures disclosed herein provide a framework of conformational states assumable by IspC in the absence and presence of inhibitors and/or substrate analogues. When performing rational drug design or computer modelling, at least the conformation of amino acid side chains will also be varied in the process of finding a potential inhibitor. Preferred and less-preferred side chain conformations (torsion angles) are known in the art.
  • the E. coli protein has been used to determine the structure of IspC.
  • This invention relates also to said synthases from other organisms.
  • orthologous proteins from various organisms may differ considerably in the primary structure, the fold and active site architecture typically remain essentially the same. Active site residues necessary for the function of a protein are conserved.
  • inhibitors found according to the methods of this invention using the 3D structure of IspC from E. coli will also be inhibitors of orthologous synthases from organisms other than E coli.
  • This invention therefore comprises IspC crystal structures having a bound inhibitor and/or metal ion with root mean square deviations from the E.
  • coli synthase structure over protein backbone atoms or better over all non-hydrogen atoms of not more than 3 A, preferably not more than 2 A and most preferably not more than 1 A.
  • the 3D structures of such related proteins may easily be obtained by homology modeling using the 3D structures of this invention.
  • other proteins comprised by this invention may be those whose crystal structures can be solved by molecular replacement using the coordinates of the E. coli synthase of this invention.
  • the atomic coordinates may be stored on a computer-readable data storage device for further use.
  • the present invention provides three different crystal structures (cf. table 1 ):
  • MNFOS The coordinates of structures APO, MN, and MNFOS are given in tables 3, 4, and 5, respectively.
  • the three-dimensional structure information disclosed herein allows to perform methods of identifying potential inhibitors of IspC employing computer methods of rational drug design and computer modeling. These methods may be combined with IspC assays using potential inhibitors. Preferably, structural information of active site residues and/or of bound inhibitor and/or inhibitor fragments are used for rational design/computer modelling of potential inhibitors. Potential inhibitors may then be synthesized and their inhibitory potential be tested experimentally. This approach allows the direct design or identification of an inhibitor or reduces the number of compounds which have to be synthesized and to be tested for their inhibitory potential experimentally, since only structures found to be promising in silico are further pursued experimentally.
  • the structures of already existing chemical compounds may be screened in silico using the 3D structures of IspC of the invention and promising potential inhibitors may then be tested experimentally for actual inhibition activity on IspC.
  • An inhibitor obtained by the methods of this invention may be used as antibiotic against bacteria or protozoa, notably the malaria parasite P. falciparum or as herbicides.
  • the invention provides the following methods that can be used for identifying novel inhibitors of IspC:
  • said binding is detected by soaking the monoclinic crystal of the invention with the potential inhibitor or by growing a crystal of said synthase in the presence of the potential inhibitor and determining the three-dimensional structure of the complex comprising the synthase and the potential inhibitor and/or metal ions.
  • a method of identifying a potential inhibitor of 2C-methyl-D-erythritol 4-phosphate synthase by determining binding interactions between the potential inhibitor and a set of binding interaction sites in a binding cavity of said synthase, comprising
  • a potential inhibitor is selected in step (c) that further binds in a bidentate fashion to a metal ion that is bound to said synthase via amino acids Asp150, GIu152 and Glu231.
  • a computer-assisted method for identifying potential inhibitors of the protein 2C-methyl-D- erythritol 4-phosphate synthase using a programmed computer comprising a processor, a data storage system, a data input device, and a data output device, comprising the following steps:
  • a computer-assisted method for identifying potential inhibitors of the protein 2C-methyl ⁇ D- erythritol 4-phosphate synthase using a programmed computer comprising a processor, a data storage system, a data input device, and a data output device, comprising the following steps:
  • a method of identifying a candidate inhibitor capable of binding to and inhibiting the enzymatic activity of 2C-methyl-D-erythritol 4-phosphate synthase comprising the following steps:
  • step (f) determining whether said candidate compound inhibits enzymatic activity of said synthase.
  • said assessing the possibility of bonding of step (d) comprises using information of at least some of the following interactions of the active site with the model inhibitor 3-(N- formyl-N-hydroxy)aminopropylphosphonic acid or a deprotonated form thereof: the divalent metal ion binding the N-formyl oxygen of said model inhibitor, preferably trans to Glu231; the divalent metal ion binding the N-hydroxy oxgen of said model inhibitor, preferably trans to Asp150;
  • Trp212 making hydrophobic interactions with the propylidene moiety of said model inhibitor.
  • compounds are preferably selected that can bind to at least 5 binding sites of said synthase and/or said metal ion.
  • inhibitor-resistant mutants of the synthase may be designed using the 3D structures disclosed herein.
  • IPP (4) and DMAPP (5) are either synthesized from acetyl-CoA (7) via mevalonate (6) or from 1- deoxy-D-xylulose 5-phosphate (1) via 2-C-methyl-D-erythritol 4-phosphate (4) and (E)-1-hy- droxy-2-methyl-but-2-enyl 4-diphosphate (3).
  • Each monomer can be divided into three domains.
  • the N-terminal domain (red) binds NADPH (light green ball-and- stick model) and represents a classical dinucleotide binding fold.
  • the central catalytic domain (yellow) binds the divalent metal (Mg, Mn or Co) and the substrate.
  • Fosmidomycin is depicted as orange ball-and-stick model. It also harbors the flexible active site loop (magenta).
  • a connecting stretch (blue) leads to a C-terminal a-helical domain that supports the catalytic domain.
  • Simulated annealing F 0 -F 0 omit map of bound manganese and fosmidomycin at 2.5 A resolution for both asymmetric monomers.
  • the contour level is 3.5 sigma.
  • the divalent cation (Mn 2+ ) is bound to Asp150, Glu152 and Glu231 that serve as monodentate ligands.
  • the (N-formyl-N-hydroxy)amino head group of fosmidomycin (yellow ball-and-stick model) ligands to Mn 2+ , its phosphonate moiety is anchored by a H-bond network including Ser186, Ser222, Asn227 and Lys228.
  • the catalytic loop (magenta) shows only weak electron density that indicates an interaction of Trp212 with a hydrophobic patch formed by His257, Pro274 and Met276. A pronounced hydrophilic cavity behind fosmidomycin harbors Ser151 and Ser254.
  • the asymmetric unit of the IspC crystal of the invention contains an IspC dimer that also represents the solution state of the enzyme (Fig. 2).
  • the crystals showed unisotropic diffraction of x-rays of at least 2.8 to 2.3 A resolution and a mosaicity of better than 1 degree.
  • Based on the merging R-factor of the data and l/sigma(l) criteria for the outermost shell data were included to a limiting resolution of 2.6 A for the APO data set and 2.5 A for the complex data sets with Mn 2+ (MN) and Mn 2 7fosmidomycin (MNFOS), respectively (Table 1 ).
  • MN Mn 2+
  • MNFOS Mn 2 7fosmidomycin
  • the model for the apo form contains residues 1 to 397 excluding the active site loop which is completely disordered (residues Arg208 to Gly215).
  • the catalytic loop remains partially disordered especially for Ser213 and Met214 which are not defined by electron density and very likely adopts more than one conformation.
  • Met214 represents a strictly conserved residue.
  • the complete ordering of the catalytic loop may require the presence of NADPH and/or the substrate 1-D-deoxyxylulose 5-phosphate.
  • IspC forms an elongated homodimer of about 96 x 60 A with a pronounced cleft like structure in each monomer that is covered by a flexible active site loop (Fig 2).
  • Each monomer can be subdivided into basically three domains.
  • the N-terminal domain (residues 1-149) is a member of the classical dinucleotide binding fold and serves as an anchor for NADPH.
  • a central catalytic domain (residues 150 to 285) harbors both the binding site for the divalent metal ion (Mg 2+ , Mn 2+ or Co 2+ ), the phosphate binding site of the substrate and the catalytic loop.
  • the C-terminal, all a-helical domain (residues 312 to 398) is connected to the catalytic domain by a linker region that spans the entire monomer.
  • the C-terminal domain has a structural role in supporting the catalytic domain.
  • the catalytic domain can even be subdivided into the part harboring the substrate binding site and that harboring the active site loop.
  • IspC Binding of manganese and fosmidomycin and implications for anti-malaria drug development IspC was first crystallized in the absence of divalent metal ions, NADP(H) and substrate or the inhibitor fosmidomycin.
  • the NADPH binding site is blocked by the C-terminus of a neighboring molecule in the crystal.
  • the active site is well accessible in our crystals and soaking with either Mn 2+ (resulting in crystal structure MN) or Mn 2+ and fosmidomycin was conducive to the corresponding electron densities (Fig. 3).
  • Mn 2+ is bound to a cluster of the acidic residues Asp150, Glu152 and Glu231.
  • Trp212 from the flexible catalytic loop is in direct vicinity to the bound inhibitor, although not well ordered.
  • Replacement of His257 by glutamine was found to have a drastic effect on enzyme activity (38).
  • the k ca t/ m decreases 27,000 fold and the K m value for NADPH increased by approximately one order of magnitude although it is quite far away from the NADPH binding site.
  • Mutation of His153 that is actually closer to the NADPH binding site does not have a comparable effect. This observation may be explained by an interaction between the His257 site and the nicotinamide moiety that is mediated by the catalytic loop. It appears likely that His257 and Trp212 form a stacking interaction supported by Pro274 in the presence of NADPH that is a prerequisite for completely ordering the active site architecture.
  • This first step is similar to that catalyzed by acetohydroxy acid isomeroreductase involved in the biosynthesis of branched amino acids.
  • a carbonyl group is also further polarized by a Mg 2+ ion inducing a partial positive charge on the carbon atom (reviewed by (40)).
  • 1-D-deoxyxylulose does not act as a substrate of the enzyme (19). Therefore, migration of the C-4 atom to the C-2 carbonyl during the isomerization will likely require a movement of C-2 towards the phosphate binding site. If it is additionally assumed that the oxygen ligands remain bound to the divalent metal ion throughout the reaction a binding mode of the intermediate as illustrated in Figure 5b can be modeled.
  • this geometry not only brings the aldehyde group of the intermediate in close proximity ( ⁇ 3.2 A) to the C-4 position of the NADPH nicotinamide moiety but also results in the observed hydride transfer to the H r ⁇ position at C-1 of the product.
  • the methylgroup at C-2 is predicted to remain exposed to Trp212 of the catalytic loop. It has been shown that this methylgroup is essential for the affinity of the aldehyde intermediate to the enzyme as D-erythrose 4-phosphate binds significantly weaker (19). The hydride transfer may occur very rapidly, perhaps concomitantly with the isomerization reaction which is compatible with the fact that the putative intermediate can not be isolated or trapped.
  • the binding mode of fosmidomycin to IspC indicates that it is a competitive inhibitor of the synthase.
  • Other potential inhibitors may be identified using the structural information and the methods provided herein.
  • potential inhibitors are selected by their potential of binding to the active site. Compounds which bind to the active site can be expected to compete with binding of the substrate, thus functioning as competitive inhibitors of IspC.
  • the atomic coordinates of the 3D structure of the synthase preferably structure MN or more preferably structure MNFOS, is loaded from a data storage device into a computer memory and may be displayed (generated) on a computer screen using a suitable computer program.
  • a subset of interest of the coordinates of the whole structure of the synthase is loaded in the computer memory or displayed on the computer screen.
  • This subset of interest may comprise the coordinates of active site residues and/or those which make up the binding cavity where fosmidomycin and the above-mentioned metal ion was found.
  • This subset may be called a criteria data set; this subset of atoms may be used for designing an inhibitor. It may contain amino acid residues mentioned in claim 15. Further interactions that may be used are shown in table 2.
  • a potential inhibitor may then be designed de novo by rational drug design in conjunction with computer modelling.
  • Models of chemical structures or molecule fragments may be generated on a computer screen using information derived from known low-molecular weight organic chemical structures stored in a computer data base or are built using the general knowledge of an organic chemist regarding bonding types, conformations etc. Suitable computer programs may aid in this process in order to build chemical structures of realistic geometries.
  • Chemical structures or molecule fragments may be selected and/or used to construct a potential inhibitor such that favorable interactions to said subset or criteria data set become possible. The more favorable interactions become possible, the stronger the potential inhibitor will bind to the synthase.
  • at least one favorable interaction with said metal ion and favourable interactions with at least two amino acid residues should become possible.
  • Favorable interactions are any non-covalent attractive forces which may exist between chemical structures such as hydrophobic or van-der-Waals interactions and polar interactions such as hydrogen bonding, salt-bridges, metal ion-ligand interaction etc.
  • Unfavorable interactions such as hydrophobic-hydrophilic interactions should be avoided but may be accepted if they are weaker than the sum of the attractive forces.
  • Steric interference such as clashes or overlaps of portions of the inhibitor being selected or constructed with protein moieties will prevent binding unless resolvable by conformational changes.
  • the binding strength of a potential inhibitor thus created may be assessed by comparing favorable and unfavorable interactions on the computer screen or by using computational methods implemented in commercial computer programs.
  • Conformational freedom of the potential inhibitor and amino acid side chains of the synthase should be taken into account.
  • Accessible conformations of a potential inhibitor may be determined using known rules of molecular geometry, notably torsion angles, or computationally using computer programs having implemented procedures of molecular mechanics and/or dynamics or quantum mechanics or combinations thereof.
  • a potential inhibitor is at least partially complementary to at least a portion of the active site of the synthase in terms of shape and in terms of hydrophilic or hydrophobic properties.
  • Databases of chemical structures e.g. Cambridge Structural Database or from Chemical Abstracts Service; for a review see: Rusinko (1993) Chem. Des. Auto. News 8, 44-47
  • all structures in a data base may be compared to the active site or to the binding pockets of the synthase for complementarity and lack of steric interference computationally using the processor of the computer and a suitable computer program.
  • computer modeling which comprises manual user interaction at a computer screen may not be necessary.
  • molecular fragments may be selected from a data base and assembled or constructed on a computer screen e.g. manually.
  • the ratio of automation to manual interaction by a person skilled in the art in the process of selecting may vary a lot. As computer programs for drug design and docking of molecules to each other become better, the need for manual interaction decreases.
  • a preferred approach of selecting or identifying potential inhibitors of the synthase makes use of structure MNFOS of the invention.
  • the atomic coordinates of fosmidomycin may be removed if desired. However, the conformations of amino acid side chains of IspC then still reflect a bound inhibitor.
  • Fosmidomycin may be used as a lead structure for other inhibitors of the invention.
  • a potential inhibitor may be found based on the phosphonate group and/or based on the (N-formyl-N-hydroxy)amino head group of fosmidomycin.
  • chemical structures or fragments thereof may be selected from data bases or constructed based on similarity of these groups of fosmidomycin.
  • These groups of fosmidomycin may be exchanged by such selected or constructed fragments or chemical structures in order to improve the inhibitory function and/or in order to improve the suitability of the potential inhibitor thus obtained for pharmaceutical purposes.
  • Another preferred approach is the design of mechanism-based inhibitors like suicide inhibitors which modify the synthase when turning over with the inhibitor.
  • Programs usable for computer modelling include Quanta (Molecular Simulations, Inc.) and Sibyl (Tripos Associates). Other useful programs are Autodock (Scripps Research Institute, La Jolla, described in Goodsell and Olsen (1990) Proteins: Structure, Function and Genetics, 8, 195-201), Dock (University of California, San Francisco, described in: Kuntz et al. (1982) J. Mol. Biol. 161 , 269-288. Experimental assessment of potential inhibitors
  • Potential inhibitors may be assessed experimentally for binding to IspC and/or for their inhibitory action on the catalytic activity of IspC.
  • the potential inhibitors can be synthesized according to the methods or organic chemistry. Preferably, compounds from a database have been selected without remodelling, since their synthesis may already be known or they may even be available on the market. In any event, the synthetic effort needed to find an inhibitor is greatly reduced by the achievements of this invention due to the preselection of promising inhibitors by the above methods.
  • Binding of a potential inhibitor may be determined after contacting the potential inhibitor with IspC. This may be done crystallographically by soaking a crystal of the synthase with the potential inhibitor or by cocrystallisation and determining the crystal structure of the complex. Preferably, binding may be measured in solution according to methods known in the art. More preferably, inhibition of the catalytic activity of the synthase by the inhibitor is determined e.g. using the assays described in the examples section.
  • the activity of the synthase may be measured by determining the consumption of 1- deoxyxylulose- 5-phosphate or NADPH and/or the formation of 2C-methyl-D-erythritol 4- phosphate.
  • the measurement may be carried out either directly with the reaction mixture or after the separation of the reaction mixture by chromatography, such as HPLC.
  • the reaction should preferably be carried out in the presence of Mn 2+ or in the presence of other divalent cations like Mg 2+ .
  • the reactions is followed photometrically around 340 nm by the consumption of the co-substrate NADPH.
  • the start of this reaction can be timed by the addition of the last of the essential components e.g. the synthase or substrate.
  • the reaction can be stopped, if necessary, by methanol, chelating agents, like EDTA or acids like trichloro acetic acid.
  • the activity of the synthase may be expressed as the amount of substrate comsumed or product produced in a specified period of time and under specified conditions.
  • Inhibition by potential inhibitor may be determined by repeating an activity assay in the presence of a predetermined concentration of a potential inhibitor and comparing the obtained activities of the synthase. An inhibitor may be tested at several different concentrations. Further, the type of inhibition e.g. competitive, non-competitive, un-competitive or irreversible may be determined according to known methods. Assays of IspC are known from Ref. 15; see also example 3. Examples
  • the ispC gene of Escherichia coli was cloned, overexpressed and the corresponding gene product was purified as described previously (29).
  • the protein obtained according to Ref. 29 was dialysed against 100 mM Tris/HCI, pH 8.0 with 2 mM dithioerythrol and 0.02 % (m/v) NaN 3 and concentrated to 10 mg/ml.
  • 15 ⁇ l of protein solution were mixed with 15 ⁇ l of buffer solution containing 8 % (w/v) PEG 4000 and 100 mM Na acetate, 110 mM Hepes, 100 mM glycine, 100 mM guanidinium chloride, 10 mM EDTA, 12 mM DTT, pH 5.8 and equilibrated over 900 ⁇ l of the same buffer without EDTA in Linbro plates at 21 °C.
  • the protein had a strong tendency to precipitate irreversibly. Diffraction quality crystals appeared rather irregularly after about one week and grew to a final size of about 0.45 x 0.2 x 0.2 mm 3 .
  • Data sets of the substrate free crystals and of the complexes were measured at 100 K using an Oxford cryo stream. Prior to flash-freezing or soaking the crystals were gradually transferred in about ten steps into a buffer solution containing the mother liquor without EDTA supplemented with 30% (v/v) PEG 400 during a time period of about 30 minutes. For soaking either 5 mM MnCI 2 (data set MN) or 5 mM MnCI 2 together with 2 mM fosmidomycin (data set MNFOS) were added to the cryo solution. Crystals were soaked for 2 hours. Fosmidomycin was purchased from Molecular Probes (Eugene, Origon, USA).
  • X-ray diffraction data were collected on a MarResearch imaging plate detector mounted on a Rigaku rotating anode X-ray generator, operating at 50 kV and 100 mA. Diffraction data were evaluated with the HKL suite (30) and CCP4 program suite (31). The crystal structure of the apo E. coli IspC (PDB entry 1 K5H) (27) of space group C222(1 ) with three monomers in the asymmetric unit provided the starting set of coordinates. Model building was done with the program MAIN (32). The structures were refined with CNS (33) using a test set of 5 % of the reflections for cross-validation.
  • the Ramachandran plot (35) showed only 0.0 % (APO), 1.3 % (MN) or 0.8% (MNFOS) in the generously allowed region and 0.3% (APO) and 0.0% (MN and MNFOS) in the disallowed regions.
  • Figures were prepared with BOBSCRIPT (36).
  • Assay mixtures contain 100 mM Tris hydrochloride, pH 8.0, 1 mM MnCI 2 , 0.2 mM NADPH, 0.2 mM 1-deoxy-D-xylulose 5-phosphate, and 50 ⁇ l protein in a total volume of 1 ml.
  • the reaction is initiated by adding protein to the complete assay mixture and the mixture is incubated at 37 °C.
  • the oxidation of NADPH is monitored photometrically at 340 nm.
  • the extinction coefficient ( ⁇ ) of NADPH at 340 nm is 6.2 x10 3 M _1 cm " 1.
  • Table 2 Selected distances of fosmidomycin with active site atoms of IspC. The nomenclature of fosmidomycin atoms is as shown at the end of the table.
  • ATOM 90 CDI ILE 13 -2. .834 -0. .573 36, .582 1. .00 57, .28 AAAA
  • ATOM 110 CA THR 17 -6 .508 6 .410 32 .352 1, .00 51 .50 AAAA
  • ATOM 158 CA HIS 23 -5, .856 16, .360 29, .369 1. .00 62. .71 AAAA
  • ATOM 213 CA ARG 29 -15. ,020 10, .741 26, .559 1, .00 56. ,07 AAAA
  • ATOM 238 CA ALA 32 -19 .318 4 .284 31 .830 1 .00 50 .45 AAAA
  • ATOM 258 CA ALA 35 -15. ,581 0. ,022 40. .269 1. ,00 52. ,46 AAAA
  • ATOM 260 C ALA 35 -16. ,086 -1. ,051 41. ,229 1. ,00 53. ,34 AAAA
  • ATOM 263 CA GLY 36 -15. .857 -2. ,207 43. ,310 1. ,00 51. ,73 AAAA
  • ATOM 276 CA ASN 38 -14, .796 2, .509 46, .721 1, .00 58, .51 AAAA
  • ATOM 342 CA CYS 46 -18 581 10 .608 38 266 1 00 61 55 AAAA
  • ATOM 356 CA GLU 48 -15 .732 15 .156 36 .919 1 .00 66 .04 AAAA
  • ATOM 382 CD PRO 51 -19. .455 9. ,851 34. ,682 1. ,00 61. ,82 AAAA
  • ATOM 400 CA TYR 53 -23 .554 4 .947 33, .900 1, .00 59, .33 AAAA
  • ATOM 406 CE2 TYR 53 -27 .072 2 .961 31 .407 1 .00 69, .58 AAAA
  • ATOM 457 CA ALA 60 -25, .053 -1, .507 49 .319 1, .00 64, .76 AAAA
  • ATOM 458 CB ALA 60 -24, .311 -2, .319 50, .390 1. .00 60, .99 AAAA
  • ATOM 468 CA ALA 62 -24 .551 1 .284 44 .738 1 .00 57 .67 AAAA
  • ATOM 530 CA GLN 70 -28, .291 12, .563 44 .094 1, .00 73, .74 AAAA
  • ATOM 542 CD GLN 71 -25 .858 13 .909 49 .994 1 .00 84 .84 AAAA
  • ATOM 570 CD ARG 75 -29. ,524 13. .981 33. .994 1. ,00 93. ,32 AAAA
  • ATOM 578 CA THR 76 -25. .152 9, .324 36, .287 1, .00 67, .68 AAAA
  • ATOM 615 CA GLY 81 23. 461 -6. 560 40. 033 1. 00 56. 79 AAAA
  • ATOM 679 CA LEU 91 -22. 429 -0. ,794 25. ,444 1. ,00 63. ,60 AAAA
  • ATOM 687 CA GLU 92 -23. ,236 -0. ,174 21. ,760 1. ,00 70. ,00 AAAA
  • ATOM 743 CA ALA 99 -9. 975 -3. ,830 31. .254 1. ,00 47. ,24 AAAA
  • ATOM 748 CA ALA 100 -8. ,327 -2. ,927 34. ,562 1. ,00 48. ,61 AAAA
  • ATOM 750 C ALA 100 -8. ,755 -3. ,604 35. .849 1. ,00 50. ,55 AAAA
  • ATOM 753 CA ILE 101 -10. ,308 -5. .179 36. .888 1. ,00 53. ,85 AAAA
  • ATOM 768 CA GLY 103 -8. ,690 -11, .302 39, .439 1, .00 57. .98 AAAA
  • ATOM 772 CA ALA 104 -11, .608 -13, .574 38, .648 1, .00 57, .77 AAAA
  • ATOM 824 CA ALA 112 17 862 -9 425 27 226 1 00 54 62 AAAA
  • ATOM 834 CA ILE 114 ⁇ 14 .213 -9 .023 23 .542 1 .00 51 .56 AAAA
  • ATOM 842 CA ARG 115 -17 .289 -10 .852 22 .266 1 .00 50 .54 AAAA
  • ATOM 858 CA GLY 117 -15.628 -6.598 18.961 1.00 48.48 AAAA
  • ATOM 862 CA LYS 118 -14.527 -3.615 21.061 1.00 50.52 AAAA

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Abstract

L'invention concerne un nouveau cristal contenant la synthase protéinique 2C-méthyle-D-érythritol 4-phosphate (IspC). La présente invention porte également sur la structure cristalline de IspC en complexe avec des ions de métal et un inhibiteur de liaison, et sur des procédés pour identifier de nouveaux inhibiteurs de IspC.
PCT/EP2004/001075 2003-02-14 2004-02-05 Structure cristalline de la synthase 2-c-methyle-d-erythritol 4-phosphate en complexe avec des inhibiteurs WO2004072276A1 (fr)

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DE10308594A DE10308594A1 (de) 2003-02-14 2003-02-27 Struktur der 2-C-Methyl-D-erythritol-4-phosphat-Synthase im Komplex mit Inhibitoren

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002092800A2 (fr) * 2001-05-15 2002-11-21 Max-Planck-Gesellschaft Structure cristalline de 2c-methyl-d-erythritol 2,4-cyclodiphosphate synthase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002092800A2 (fr) * 2001-05-15 2002-11-21 Max-Planck-Gesellschaft Structure cristalline de 2c-methyl-d-erythritol 2,4-cyclodiphosphate synthase

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ALTINCICEK B ET AL: "Tools for discovery of inhibitors of the 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase: An approach with enzymes from the pathogenic bacterium Pseudomonas aeruginosa", FEMS MICROBIOLOGY LETTERS, AMSTERDAM, NL, vol. 190, no. 2, 15 September 2000 (2000-09-15), pages 329 - 333, XP002266370, ISSN: 0378-1097 *
HECHT S ET AL: "Biosynthesis of terpenoids: efficient multistep biotransformation procedures affording isotope-labeled 2C-methyl-D-erythritol 4-phosphate using recombinant 2C-methyl-D-erythritol 4-phosphate synthase.", THE JOURNAL OF ORGANIC CHEMISTRY. 16 NOV 2001, vol. 66, no. 23, 16 November 2001 (2001-11-16), pages 7770 - 7775, XP002279401, ISSN: 0022-3263 *
HOEFFLER JEAN-FRANÇOIS ET AL: "Isoprenoid biosynthesis via the methylerythritol phosphate pathway. Mechanistic investigations of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase.", EUROPEAN JOURNAL OF BIOCHEMISTRY / FEBS. SEP 2002, vol. 269, no. 18, September 2002 (2002-09-01), pages 4446 - 4457, XP002279402, ISSN: 0014-2956 *
KOPPISCH ANDREW T ET AL: "E. coli MEP synthase: Steady-state kinetic analysis and substrate binding", BIOCHEMISTRY, vol. 41, no. 1, 8 January 2002 (2002-01-08), pages 236 - 243, XP001181239, ISSN: 0006-2960 *
LYBRAND T P: "LIGAND-PROTEIN DOCKING AND RATIONAL DRUG DESIGN", CURRENT OPINION IN STRUCTURAL BIOLOGY, CURRENT BIOLOGY LTD., LONDON, GB, vol. 5, 1995, pages 224 - 228, XP000764926, ISSN: 0959-440X *
REUTER KLAUS ET AL: "Crystal structure of 1-deoxy-D-xylulose-5-phosphate reductoisomerase, a crucial enzyme in the non-mevalonate pathway of isoprenoid biosynthesis", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 7, 15 February 2002 (2002-02-15), pages 5378 - 5384, XP001181241, ISSN: 0021-9258 *
STEINBACHER STEFAN ET AL: "Structural basis of fosmidomycin action revealed by the complex with 2-C-methyl-D-erythritol 4-phosphate synthase (IspC). Implications for the catalytic mechanism and anti-malaria drug development.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 278, no. 20, 16 May 2003 (2003-05-16), pages 18401 - 18407, XP001181240, ISSN: 0021-9258 *
TAKAHASHI S ET AL: "A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosysnthesis", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 95, August 1998 (1998-08-01), pages 9879 - 9884, XP002136183, ISSN: 0027-8424 *

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