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WO1999019465A1 - Trihydroxynaphtalene reductase: procedes permettant la determination de sa structure tridimensionnelle et la mise au point rationnelle de ses inhibiteurs - Google Patents

Trihydroxynaphtalene reductase: procedes permettant la determination de sa structure tridimensionnelle et la mise au point rationnelle de ses inhibiteurs Download PDF

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Publication number
WO1999019465A1
WO1999019465A1 PCT/US1998/021550 US9821550W WO9919465A1 WO 1999019465 A1 WO1999019465 A1 WO 1999019465A1 US 9821550 W US9821550 W US 9821550W WO 9919465 A1 WO9919465 A1 WO 9919465A1
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Prior art keywords
thnr
reductase
computer
trihydroxynaphthalene
nadph
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PCT/US1998/021550
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English (en)
Inventor
Arnold Verner Andersson
Gregory Steven Basarab
Douglas Brian Jordan
Der-Ing Liao
Ylva Lindqvist
Gunter Schneider
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E.I. Du Pont De Nemours And Company
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Priority to AU11875/99A priority Critical patent/AU1187599A/en
Publication of WO1999019465A1 publication Critical patent/WO1999019465A1/fr

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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.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)

Definitions

  • the present invention is in the field of three-dimensional protein structure determination, the modeling of new structures, and inhibitor identification and design using three-dimensional protein structures.
  • Trihydroxynaphthalene reductase (THNR, also known as napthol reductase, tetrathydroxynaphthalene reductase, or EC 1.3.1.50) is the biochemical target of plant disease control chemicals such as tricyclazole (CAS RN [41814-78-2]), phthalide(CAS RN [27355-22-2]), and pyroquilon (CAS RN [57369-32-l])which are commercial products in use for the control of blast disease in rice (The Pesticide Manual, tenth edition (1994), Tomlin, C, ed., The Royal Society of Chemistry, Cambridge, UK).
  • THNR is a key enzyme in the biosynthesis of fungal melanin (Wheeler, M. H. (1982) Experimental Mycology 6: 171-179). Fungal melanin biosynthesis is a required component in the infection process whereby certain fungi penetrate host cells (Chumley, F. G. and Valent, B. (1990) Molecular Plant-Microbe Interactions 5:135-143).
  • the primary application of disease control chemicals such as noted above and others which inhibit the physiological activity of THNR is in the prevention of rice blast disease; yet there is the realistic potential for application of appropriate THNR inhibitors to protect other crop plants and fungal pathogens which are implicated in human and other mammalian organisms.
  • Our teachings lead to the design of molecules which inhibit the function of THNR and thereby prevent diseases in host organisms such as crops and mammals.
  • THNR has been studied in some detail with regard to its inhibition by tricyclazole (Thompson, J. E. et al. (1997) Biochemistry 3-5:1852-1860) where it was demonstrated that tricyclazole competes with substrate 1,3,8-trihydoxynaphthalene in the enzyme's active site.
  • a preliminary announcement of the cloning of THNR and the conditions for crystalization of THNR concerning the three-dimensional structure of THNR has been published (Andersson, A. et al. (1996) Proteins: Structure, function and Genetics 24:525-521).
  • a brief description of THNR complexed with NADPH and tricyclazole has been published (Andersson, A. et al. (1996) Structure 4:1161-1170).
  • This invention pertains to a computer readable medium having stored thereon atomic coordinate/X-ray diffraction data defining the three dimensional structure of a ternary complex of trihydroxynaphthalene reductase, NADPH and a ligand that binds to trihydroxynaphthalene reductase or a subunit thereof.
  • the ligand is an active site inhibitor of trihydroxynaphthalene reductase.
  • Preferred inhibitors are tricyclazole, phthalide and pyroquilon.
  • An additional embodiment of the instant invention is a computer readable medium having stored thereon the computer model output data defining the three dimensional structure of a ternary complex of trihydroxynaphthalene reductase, NADPH and a ligand that binds to trihydroxynaphthalene reductase or a subunit thereof.
  • the ligand is an active site inhibitor of trihydroxynaphthalene reductase.
  • Preferred inhibitors are tricyclazole, phthalide and pyroquilon.
  • the computer model output data is derived from analysis of atomic coordinate/X-ray diffraction data defining the three dimensional structure of a ternary complex of trihydroxynaphthalene reductase, NADPH and a ligand that binds to trihydroxynaphthalene reductase or a subunit thereof.
  • An additional embodiment of the instant invention is a method for identifying a ligand of trihydroxynaphthalene reductase or a subunit thereof, the method comprising: (a) providing a computer readable medium having stored thereon computer model output data defining the three dimensional structure of a ternary complex of trihydroxynaphthalene reductase, NADPH and a ligand that binds to trihydroxynaphthalene reductase or a subunit thereof; (b) providing a computer readable medium having stored thereon computer model output data defining the three dimensional structure of a potential ligand that binds to trihydroxynaphthalene reductase or a subunit thereof; (c) providing a computer system comprising a computer and a computer algorithm, the computer system capable of processing the computer model output data of step (a) and step (b); (d) processing the computer model output data of step (a) and step (b) using the computer system of step (c) wherein the processing calculates the
  • Figure 1 presents the atomic coordinates derived from X-ray diffraction data (atomic coordinate/X-ray diffraction data) defining the three dimensional structure of a ternary complex of trihydroxynaphthalene reductase complexed with NADPH and tricyclazole.
  • Figure 2 presents the modeled atomic coordinates predictive of the three dimensional structure of a ternary complex of trihydroxynaphthalene reductase complexed with NADPH and phthalide.
  • Figure 3 presents the atomic coordinates derived from X-ray diffraction data (atomic coordinate/X-ray diffraction data) defining the three dimensional structure of a ternary complex of trihydroxynaphthalene reductase complexed with NADPH and phthalide.
  • Figure 4 presents the modeled atomic coordinates predictive of the three dimensional structure of a ternary complex of trihydroxynaphthalene reductase complexed with NADPH and pyroquilon.
  • Figure 5 presents the comparison of the sequences of the THNR and verl proteins as determined with the program "BESTFIT”.
  • Figure 6 presents the modeled atomic coordinates for the homology model of the verl protein.
  • Figure 7 presents the modeled atomic coordinates predictive of the three dimensional structure of a ternary complex of trihydroxynaphthalene reductase complexed with NADPH and pyroquilon as determined by molecular replacement.
  • SEQ ID NO:l presents the amino acid sequence of trihydroxynaphthalene reductase from Magnaporthe grisea. The N-terminal methionine from the precursor has been removed.
  • SEQ ID NO:2 presents the amino acid sequence of the verl protein (versicolorin A dehydrogenase) from Aspergillus par asiticus. (GenBank accession M91369). DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides methods for expressing, purifying and crystallizing trihydroxynaphthalene reductase (THNR), where the crystals diffract x-rays with sufficiently high resolution to allow determination of the three-dimensional structure of the THNR, or a portion or subdomain thereof.
  • the three-dimensional structure e.g., as provided on computer readable media of the present invention
  • Such ligands can be synthesized or recombinantly produced and are useful as diagnostic agents or drugs for diagnosing, treating, inhibiting the activity of THNR or preventing diseases of plants or animals caused by microorganisms producing THNR.
  • the determined structure is made using the THNR amino acid sequences and/or atomic coordinate/x-ray diffraction data, which are analyzed to provide atomic model output data corresponding to the three-dimensional structure, e.g., as provided on computer readable media.
  • the computer analysis of the atomic coordinate/x-ray diffraction data and/or the amino acid sequence allows the calculation of the secondary, tertiary and/or quaternary structures, domains, and/or subdomains of the protein. These domains are combined and refined by additional calculations using suitable computer subroutines to determine the most probable or actual three-dimensional structure of the THNR, including potential or actual active sites, binding sites or other structural or functional domains or subdomains of the protein.
  • the resulting three-dimensional structure is represented as atomic model output data on the computer readable media.
  • Structure determination methods are also provided by the present invention for rational design of THNR ligands. Such design uses computer modeling programs that calculate different molecules expected to interact with the determined active sites, binding sites, or other structural or functional domains or subdomains of a THNR. These ligands can then be produced and screened for activity in modulating or binding to a THNR, according to methods and compositions of the present invention.
  • the actual THNR-ligand complexes can optionally be crystallized and analyzed using x-ray diffraction techniques.
  • the diffraction patterns obtained are similarly used to calculate the three-dimensional interaction of the ligand and the THNR, to confirm that the ligand binds to, or changes the conformation of, particular domain(s) or subdomain(s) of the THNR.
  • screening methods are selected from assays for at least one biological activity of a THNR.
  • the resulting ligands provided by methods of the present invention, modulate or bind at least one THNR and are useful for treating or preventing THNR-related pathologies in animals, such as humans, or plants, such as rice.
  • Ligands of a particular THNR can similarly modulate other THNRs from other sources such as other eukaryotes.
  • a THNR is also provided as a crystallized protein suitable for x-ray diffraction analysis.
  • the x-ray diffraction patterns obtained by the x-ray analysis are of moderate, to moderately high, to high resolution, e.g., 30-10, 10-3.5 or 1.5-3.5 A, respectively, with the higher resolutions included. These diffraction patterns are suitable and useful for three- dimensional structure determination of a THNR, domain or subdomain thereof.
  • the determination of the three-dimensional structure of a THNR has a broad-based utility. Significant sequence identity and conservation of important structural elements are expected to exist among different THNRs and other homologs. Therefore, the three- dimensional structure from one or few THNRs can be used to identify ligands that have diagnostic or therapeutic value for at least one THNR-related pathology that may involve THNRs or homologs having different amino acid sequences. Isolated THNR Polypeptides
  • a THNR polypeptide can refer to any subset of a THNR as a domain, subdomain, fragment, consensus sequence or repeating unit thereof.
  • a THNR polypeptide of the present invention can be prepared by any of the following methods:
  • THNR chemical peptide synthesis methods well-known in the art; and/or (d) by any other method capable of producing a THNR polypeptide and having a conformation similar to a structural or functional subdomain of a THNR.
  • a biological activity of THNR can be screened according to known screening assays.
  • the minimum peptide sequence to have activity is based on the smallest unit containing or comprising a particular domain, subdomain, fragment, region, consensus sequence, or repeating unit thereof, having at least one biological activity of a THNR, such as protecting activity, inhibiting activity or enzyme activity.
  • a THNR polypeptide of the invention can have at least 80% homology or sequence identity, such as 80-100% overall homology or identity, with one or more corresponding THNR subdomains or fragments as described herein, such as amino acids 5-282 of SEQ ID NO: 1.
  • the above configurations of subdomains are provided as part of a THNR polypeptide of the invention, when expressed in a suitable host cell, or otherwise synthesized, to provide at least one structural or functional feature of a native THNR, such as at least one THNR-related biological activity. Such activities can be assayed using a suitable assay, to establish at least one THNR biological activity of one or more THNR of the invention.
  • a THNR polypeptide of the invention is not naturally occurring or is naturally occurring but is in a purified isolated form which does not occur in nature. Examples of THNR assays are found in Thompson, J. E. et al. ((1997) Biochemistry 3(5:1852-1860). Percent homology or identity can be determined, for example, by comparing sequence information using the GAP or BESTFIT computer programs (version 9.0-OpenVMS, Genetics Computer Group (GCG)). The GAP program utilizes the alignment method of Needleman and Wunsch ((1970) J. Mol. Biol. 45:443) and performs the comparison across the entire length of the sequences. The BESTFIT program uses the local homology program of Smith and Waterman ((1981) Adv.
  • Non-limiting examples of substitutions of a THNR domains or polypeptide of the invention are those in which at least one amino acid residue in the protein molecule has been removed and a different residue added in its place.
  • the types of substitutions which can be made in the protein or peptide molecule of the invention can be based on analysis of the frequencies of amino acid changes between a homologous protein of different species. Based on such an analysis, alternative substitutions are defined herein as exchanges within one of the following five groups:
  • An amino acid sequence of a THNR and/or atomic coordinate/x-ray diffraction data useful for computer structure determination of a THNR or a portion thereof, can be "provided” in a variety of mediums to facilitate use thereof.
  • provided refers to a manufacture, which contains a THNR amino acid sequence and/or atomic coordinate/x-ray diffraction data of the present invention, e.g., the amino sequence provided in SEQ ID NO:l, a representative fragment thereof, or an amino acid sequence having at least 80-100% overall identity to amino acids 5-282 of SEQ ID NO: 1.
  • Such a method provides the amino acid sequence and/or atomic coordinate/x-ray diffraction data in a form which allows a skilled artisan to analyze and determine the three-dimensional structure of a THNR or a subdomain thereof.
  • THNR amino acid sequence and/or atomic coordinate/x-ray diffraction data of the present invention is recorded on computer readable media.
  • computer readable media refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • magnetic storage media such as floppy discs, hard disc storage medium, and magnetic tape
  • optical storage media such as optical discs or CD-ROM
  • electrical storage media such as RAM and ROM
  • hybrids of these categories such as magnetic/optical storage media.
  • recorded refers to a process for storing information on computer readable medium.
  • a skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising an amino acid sequence and/or atomic coordinate/x-ray diffraction data information of the present invention.
  • a variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon an amino acid sequence and/or atomic coordinate/x-ray diffraction data of the present invention.
  • the choice of the data storage structure will generally be based on the means chosen to access the stored information.
  • a variety of data processor programs and formats can be used to store the amino acid sequence and/or atomic coordinate/x-ray diffraction data of the present invention on computer readable medium.
  • the amino acid sequence information can be represented in a word processing text file, formatted in commercially-available, word processing software, or represented in the form of an ASCII file, or stored in a database application.
  • a skilled artisan can readily adapt any number of data-processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the information of the present invention.
  • THNR sequence and/or atomic coordinates derived from x-ray diffraction data By providing on computer readable media having stored therein a THNR sequence and/or atomic coordinates derived from x-ray diffraction data, a skilled artisan can routinely access the sequence and atomic coordinates or x-ray diffraction data to model a three dimensional structure THNR, a subdomain thereof, or a ligand thereof.
  • Computer algorithms are publicly and commercially available which allow a skilled artisan to access this data provided on a computer readable medium and analyze it for structure determination and/or rational inhibitor design. See, e.g. Biotechnology Software Directory, Mary Ann Liebert Publ., New York (1995).
  • the present invention further provides systems, particularly computer-based systems, which contain the amino acid sequence and/or atomic coordinate/x-ray diffraction described herein.
  • Such systems are designed to do structure determination and rational design for a THNR or at least one subdomain thereof.
  • Non-limiting examples are microcomputer workstations available from Silicon Graphics Incorporated and Sun Microsystems running Unix based, Windows NT or IBM OS/2 operating systems.
  • a computer-based system refers to the hardware means, software means, and data storage means used to analyze the amino acid sequence and/or atomic coordinate/x-ray diffraction of the present invention.
  • the minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means.
  • CPU central processing unit
  • a monitor is optionally provided to visualize structure data.
  • the computer-based systems of the present invention comprise a data storage means having stored therein a THNR or fragment amino acid sequence and/or atomic coordinate/x-ray diffraction data of the present invention and the necessary hardware means and software means for supporting and implementing an analysis means.
  • data storage means refers to memory which can store amnio acid sequence or atomic coordinate/x-ray diffraction data of the present invention, or a memory access means which can access manufactures having recorded thereon the amnio acid sequence or atomic coordinate/x-ray diffraction data of the present invention.
  • search means or analysis means refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the amnio acid sequence or atomic coordinate/x-ray diffraction data stored within the data storage means. Search means are used to identify fragments or regions of a THNR which match a particular target sequence or target motif.
  • search means are used to identify fragments or regions of a THNR which match a particular target sequence or target motif.
  • a variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting computer analyses that can be adapted for use in the present computer-based systems.
  • a target structural motif refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration or electron density map which is formed upon the folding of the target motif.
  • target motifs include, but are not limited to, enzymic active sites, structural subdomains, epitopes, functional domains and signal sequences.
  • a variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention.
  • comparing means can be used to compare a target sequence or target motif with the data storage means to identify structural motifs or interpret electron density maps derived in part from the atomic coordinate/x-ray diffraction data.
  • a skilled artisan can readily recognize that any one of the publicly available computer modeling programs can be used as the search means for the computer-based systems of the present invention.
  • the structure of the THNR-NADPH-tricyclazole ternary complex was determined by multiple isomorphous replacement using two mercury derivatives.
  • the refined model includes 270 of the 283 amino acid residues; electron density for the first 13 residues of the polypeptide chain is missing. Except for the side chains of the residues Glu65, Glu68, Asn229 and Glu233, located at the surface of the protein, all side chains were fitted into the electron density.
  • the model has been refined using all data between 8.0 and 2.8 A to a crystallographic R factor of 22.3%
  • the model has good stereochemistry, reflected in the root mean square (rms) deviation from standard values for bond lengths of 0.009 A and angles of 1.5°; 89.0% of all non-glycine residues have their ( ⁇ , ⁇ ) angles in the most favored region of the Ramachandran plot.
  • the positions of the six heavy-atom ions were confirmed by a difference Fourier map, calculated with phases from the protein model after the crystallographic refinement was concluded. All mercury ions in the two heavy-atom derivatives bind to cysteine residues, and the three heavy metal binding sites in the subunit are formed by residues Cys83, Cysl91 and Cys282, respectively.
  • the THNR subunit is composed of one domain, with dimensions 35X50X30 A.
  • the subunit contains seven ⁇ strands, forming a parallel ⁇ sheet, eight ⁇ helices and a number of loops of varying length. Flanking the parallel ⁇ sheet on one side are ⁇ helices D, E and F and on the other side ⁇ helices C, B and G.
  • the short ⁇ helix, ⁇ 2 is positioned nearly perpendicular to the ⁇ sheet, at its top edge.
  • the subunit structure contains the 'Rossmann fold', comprising strands ⁇ A- ⁇ F, often found in dinucleotide-binding enzymes.
  • the substrate and coenzyme need to access the catalytic site, which is buried below the ⁇ l-loop- ⁇ 2 region.
  • These two helices are stabilized by interhelical hydrogen bonds.
  • the first multiple hydrogen bond is formed between Lys212 and Tyr216, from ⁇ l, and Asp236 from ⁇ 2.
  • Another hydrogen bond is formed from the side chain of Arg221, in ⁇ l, to the peptide oxygens of Leu230 and Asn232, in ⁇ 2.
  • native THNR subunits assemble into a homotetramer. In the crystal asymmetric unit, two subunits of the tetramer are related by a local twofold symmetry axis.
  • the tetrameric molecule is generated from this dimer by the crystallographic twofold symmetry.
  • the size of the tetramer is 65X70X80 A and each subunit makes contacts with two neighboring subunits.
  • Each individual subunit has a solvent-accessible surface of 12900 A 2 whereas the tetramer has a solvent accessible surface of 35300 A 2 . Therefore, upon tetramer formation, approximately 4000 A 2 of the solvent-accessible surface is buried per subunit.
  • the tetramer displays 222 symmetry with two different interfaces between subunits about three orthogonal molecular axes, called P, Q and R, where the Q axis corresponds to the crystallographic twofold axis.
  • subunits are numbered 1, 2, 3 and 4. Contacts across the P axis are between subunits 1 and 2 (and between 3 and 4), contacts across the R axis are between subunits 1 and 3 (2 and 4) and contacts across the Q axis between subunits 1 and 4 (2 and 3).
  • the interface between subunit 1 and 2 is formed by contacts between ⁇ G 1 and ⁇ G 2 in the form of Phe261 1 -Phe261 2 stacking and an Asp266 1 -Arg52 2 salt bridge. Closer to the center of the tetramer, the two ⁇ sheets are positioned antiparallel to each other and form a bowl-shaped 14-stranded ⁇ sheet. At the interface close to ⁇ G 1 and ⁇ G 2 , a salt bridge is formed between ASP278 1 and Lys273 2 . Between the two interacting ⁇ helices and the ⁇ sheet there is a large hydrophobic region spanning both subunits.
  • two salt bridges, Argl40 1 -Aspl32 4 and Aspl98 1 -Hisl22 4 are present. In total, 1700 A of solvent-accessible surface is buried per subunit in this interface.
  • the dinucleotide, NADPH, in the enzyme complex has an extended conformation, and binds at the C-terminal side of the parallel ⁇ sheet.
  • the nicotinamide ring of NADPH is buried in the protein interior, close to the loop ⁇ F- ⁇ l and not accessible to the bulk solvent.
  • the nicotinamide mononucleotide ribose moiety has the CT-endo conformation and is located between ⁇ strands ⁇ D and ⁇ E.
  • the adenosine part is positioned in a pocket made up of ⁇ strands ⁇ B, ⁇ C and ⁇ D; the ribose ring adopts the CT-endo conformation, and is bound at the switch point of the ⁇ sheet between the C-terminal ends of strands ⁇ A and ⁇ D.
  • the adenine ring binds in a pocket formed by side chains Ala ⁇ l, Asn87, Val88, Ser 115 and He 137, Hydrogen bonds are formed between NI of the adenine ring and the peptide nitrogen of Val88 and also between N3 and the side chain of Serll5.
  • One oxygen atom of the 2' ribose phosphate group forms hydrogen bonds to the peptide NH groups of Ala ⁇ l, Asn62 and Ser63 at the N terminus of helix ⁇ C.
  • a second oxygen atom of the 2' ribose phosphate group forms hydrogen bonds to the side chains of Ser63 and Arg39 and the third oxygen atom hydrogen bonds to the side chain of Asn62.
  • the pyrophosphate moiety of the NADPH molecule interacts with the N termini of helices ⁇ B and ⁇ l, through hydrogen bonds with the peptide NH group of residue Ile41 and the side chain of Thr213, Arg39 is located 3.4 A from one of the oxygen atoms of the pyrophosphate moiety, in part compensating for the negatively charged oxygen atoms.
  • Interactions made by the nicotinamide mononucleotide ribose hydroxyl groups include hydrogen bonds to the side chains of Tyrl78, Lysl82 and the peptide oxygen of Asnll4.
  • the nicotinamide ring has the syn conformation and has its B face oriented towards the inhibitor. It binds in a pocket formed by the side chains of Ile41, Metl62, Tyrl78, Pro208, Thr213, Met215 and Tyr216.
  • Specific hydrogen bonds position the nicotinamide ring in its binding site; the carboxamide group forms hydrogen bonds with the side chain of Thr213 and the peptide nitrogen of Ile211.
  • the inhibitor tricyclazole
  • Tricyclazole is enclosed by the C terminus of strand ⁇ F, the ⁇ F- ⁇ l loop and the ⁇ l helix, and is completely shielded from the outside solution.
  • Tricyclazole is bound in a rather large pocket (with a volume of 57 A 3 ), formed by residues Valll8, Serl64, Ilel65, Tyrl78, Gly210, Met215, Val219, Cys220, Tyr223, Trp243 and Met283.
  • the N2 and N3 atoms of the inhibitor form hydrogen bonds with the phenolic hydroxyl group of Tyr 178 and the hydroxyl group of Serl64, respectively.
  • the inhibitor is stacked between the side chain of Tyr223 on one side and to a small extent the nicotinamide ring of NAPDH on the other side, with stacking distances of about 3.5 A.
  • the observed binding mode of tricyclazole at the active site of the enzyme is consistent with its behavior as a competitive inhibitor.
  • the inhibitor and part of NADPH are buried in the protein and their binding and release might be controlled by conformational changes of the enzyme. These changes could involve helix ⁇ l, the connecting loop to ⁇ 2, and possibly helix ⁇ 2, which by shifting their position could open the way into the active site.
  • This part of the subunit is one of the most variable in structure when compared to other similar enzymes, such as 3 ⁇ ,20 ⁇ -hydroxysteroid dehydrogenase and dihydropteridine reductase (see below).
  • the three dimensional structural model obtained for THNR clearly identifies the enzyme as a member of the short chain dehydrogenase/reductase (SDR) family.
  • This fold consists of a single domain subunit made up of a central ⁇ sheet (seven or eight ⁇ strands), which is flanked on both sides by ⁇ helices.
  • the fold shows a common nucleotide-binding site characterized by a Gly-X-X-X-Gly-X-Gly fingerprint.
  • a strictly conserved lysine residue located four residues downstream (Lys 182) are both implicated as part of the catalytic machinery.
  • THNR shows amino acid sequence identity (between 20-39%) to several members of the SDR family, including glucose- 1 -dehydrogenase, 7 ⁇ -hydroxysteroid dehydrogenase, corticosteroid ll ⁇ -dehydrogenase and alcohol dehydrogenase.
  • glucose- 1 -dehydrogenase 7 ⁇ -hydroxysteroid dehydrogenase
  • corticosteroid ll ⁇ -dehydrogenase corticosteroid ll ⁇ -dehydrogenase
  • alcohol dehydrogenase Three-dimensional structures of six members of the SDR family are known,
  • the coordinate sets of three members of the SDR family are available from the PDB database: 3 ⁇ ,20 ⁇ -hydroxysteroid dehydrogenase; dihydropteridine reductase; and enoyl acyl carrier protein reductase.
  • MLCR mouse lung carbonyl reductase
  • a general pattern responsible for the determination of nucleotide specificity in the SDR family was proposed.
  • the determinants comprise two basic residues, a lysine (or an arginine) and an arginine residue.
  • the first basic residue is Arg39 (coincidentally the same sequence number as the second basic residue in MLCR), found close to the 2 '-phosphate group, but there is no second arginine.
  • a sequence alignment shows no second arginine close to the expected position, instead an alanine is found at that position in the THNR sequence. This observation suggests that one basic residue is sufficient to confer NADPH specificity.
  • Enzymes of the SDR family contain a highly conserved Ser-Tyr-Lys triad at the active site and mechanistic proposals have focused on the central role of these residues in catalysis.
  • Common to these proposals is the suggestion that the invariant tyrosine residue polarizes the carbonyl oxygen of the substrate, thereby increasing the electrophilicity of the carbonyl carbon atom and facilitating hydride transfer from NADPH.
  • the tyrosine residue donates its phenolic proton to the substrate hydroxyl group.
  • the resulting negative charge of the side chain of the tyrosine residue is stabilized by the lysine residue of the catalytic triad, either directly through a hydrogen bond or through electrostatic interactions.
  • the serine residue of the triad was suggested to be involved in binding of the substrate, reaction intermediates and products.
  • the active site of THNR contains a catalytic triad similar to other members of this family which suggests that they share mechanistic features.
  • the position of the substrate, trihydroxynaphthalene, has been modelled in the active site of THNR based on the position of the inhibitor.
  • trihydroxynaphthalene accepts a hydride from NADPH at the C3 position.
  • the keto tautomer with a carbonyl group at the C3 position has to be generated at the active site. Formation of this less polar species of the substrate is favored by the predominantly apolar environment of the active site of THNR.
  • the third residue of the triad, Lys 182 forms a bifurcated hydrogen bond to the 2'-hydroxyl and 3 '-hydroxyl groups of the nicotinamide mononucleotide ribose ring, stabilizing the position and orientation of the nicotinamide ring.
  • the distance between the hydroxyl oxygen atom of Tyr 178 and the N ⁇ atom of the side chain of Lys 182 is approximately 4.5 A, therefore, a hydrogen bond between these two residues is unlikely.
  • Lys 182 might influence Tyrl78 through electrostatic interactions, as suggested previously.
  • the coordinates in Examples 1-4, and 6 define the hydrogen bonding network between the inhibitors and side chains of ser 164 and tyr 178, and Van der Waals contacts between the inhibitors, NADPH, and amino acids within the active site of THNR.
  • the inhibitor binding region of the active site as defined in these examples is relatively small (approximately 250 cubic angstroms) and this provides a valuable constraint on inhibitor design.
  • the models can be used for visualizing the orientations and interactions of amino acids within the active site with tricyclazole, phthalide, and pyroquilon for the purpose of designing novel inhibitors of THNR for use as fungicides.
  • Sybyl® (TRIPOS) inhibitor molecules may be visualized by using the Build/Edit algorithms to make and break bonds and to add or delete atoms to aid in the design of novel inhibitors.
  • the models allow for the visualization of designed or other inhibitors in three dimensions within the active site (after removal of the inhibitor structures from the models in the four Examples) by using the docking routine within Sybyl® or other such programs to manually position such inhibitors within the active site. After manually docking the inhibitors the THNR-NADPH-Inhibitor structures may be minimized by using the minimization procedures within Sybyl® in order to improve the models.
  • DOCK® written by Paul McCloskey, University of California; a WWW site for the DOCK® program may be found at the URL http://www.cmpharm.ucsf.edu/kuntz/dock.html
  • UNITY® TRIPOS
  • Such programs apply constraints imposed by the enzyme active site and other constraints imposed by the user for computer generation of three dimensional pharmacophores which are useful for searching through three dimensional data bases.
  • the models lacking inhibitors may be applied to computer programs such as Leapfrog® (TRIPOS) for building virtual molecules within the active site from small three dimensional molecular fragments for the purpose of discovering new inhibitors of the enzyme.
  • Sybyl®, DOCK®, UNITY®, Leapfrog® and other such computer programs can calculate an approximate binding energy for each of the molecules docked thus allowing the user to select favorable molecules for synthesis and inhibition analysis against the activity of the enzyme (Thompson et al.).
  • Inhibitors of THNR discovered by these enablements may be evaluated for their ability to control fungal diseases.
  • Inhibitors of verl may be discovered by using similar procedures as described above for discovering inhibitors of THNR.
  • the docking programs such as DOCK® and UNITY® and the molecule-building programs such as Leapfrog can be applied to verl alone or verl complexed with NADPH.
  • the later complex is obtained by using the Sybyl® Build/Edit routines to extract NADPH from one of the THNR-NADPH-Inhibitor models in Examples 1-4 and to merge it into the verl structure.
  • Inhibitors of verl would have utility in preventing aflatoxin contamination of the food supply.
  • Example 5 Similar to the homology model of Example 5 there is the potential utility of using the coordinates of THNR-NADPH-Inhibitor to build homology models of other proteins having commercial importance as long as the other proteins have 40% or greater amino acid sequence identity to THNR so that adequate homology models can be built. Inhibitor discovery on such potential targets would proceed as described above for verl .
  • THNR-NADPH-Tricyclazole has an hydrogen bonding network between serl64 and tyrl78 (donors) and two of the ring nitrogens (acceptors) of tricyclazole. This hydrogen bonding network is likely to be an important contributor to the binding of tricyclazole and future design of inhibitors should retain appropriate hydrogen bond acceptors. Tricyclazole is sandwiched between the nicotinamide ring of NADPH and tyr223. The rather small binding region (which can be visualized by creating a solvent-accessibility contour surface by using the MOLCAD algorithm within Sybyl®) limits the size of molecules to consider for designing new inhibitors of THNR. EXAMPLE 2
  • Hydrogen atoms were added to the protein using the BIOPOLYMER module in Sybyl®. Valencies on the NADPH ligand were filled with hydrogen atoms. The torsions of carbon-oxygen single bonds of Serl64 and Tyr 178 were adjusted such that the closest contact between N3 and N2, respectively, of tricyclazole and the hydrogen atoms on the oxygen atoms was obtained. The tricyclazole molecule was replaced with a molecule of phthalide such that 014 and O were situated approximately 2 A from the hydrogen atoms on the hydroxyls of Ser 164 and Tyr 178 respectively. Optimization was carried out using the Tripos Force Field and charges as generated by the Gasteiger- Marsili algorithm (Gasteiger, J.
  • THNR-NADPH-Phthalide predicts that hydrogen bond donors ser 164 and tyr 178 are hydrogen bonded to the inhibitor's hydrogen bond acceptors carbonyl oxygen and ring oxygen, respectively, and that the inhibitor is sandwiched between the nicotinamide ring of NADPH and tyr223.
  • THNR-NADPH-Phthalide The X-ray crystal structure of THNR-NADPH-Phthalide confirms the prediction of Example 2 in that hydrogen bond donors ser 164 and tyr 178 are hydrogen bonded to the inhibitor's hydrogen bond acceptors carbonyl oxygen and ring oxygen, respectively, and that phthalide has an overall very similar orientation in the active site of THNR as predicted by Example 2.
  • Valencies on the NADPH ligand were filled with hydrogen atoms.
  • the torsions of carbon-oxygen single bonds of Serl64 and Tyr 178 were adjusted such that the closest contact between N3 and N2, respectively, of tricyclazole and the hydrogen atoms on the oxygens was obtained.
  • the tricyclazole molecule was replaced with a molecule of pyroquilon such that the carbonyl oxygen atom was situated approximately 2 A from the hydrogen atoms on the hydroxyls of both Serl64 and Tyrl78. Optimization was carried out using the Tripos Force Field and charges as generated by the Gasteiger-Marsili algorithm.
  • the protein and NADPH ligand were treated as an aggregate.
  • THNR-NADPH-Pyroquilon predicts that ser 164 and tyrl 78 both donate hydrogen bonds to the inhibitor's carbonyl and that pyroquilon is sandwiched between the nicotinamide ring of NADPH and tyr223.
  • a three-dimensional model of verl protein (versicolorin A dehydrogenase) of the aflatoxin biosynthesis pathway was constructed based on the three-dimensional atomic coordinates of THNR listed in Figure 1.
  • the amino acid sequence of verl from Aspergillus parasiticus was obtained from GenBank (accession M91369) and is presented in SED ID NO:2.
  • the amino acid sequence of this protein was found to be approximately 60% identical to that of THNR when compared with the BESTFIT program (GCG), as shown in Figure 5.
  • Atomic coordinates encoding a single subunit of the tetramer of THNR in Figure 1 were extracted by using the computer program Sybyl®.
  • the model of verl may be used for inhibitor design by docking NADPH into the active site and applying one of several methods for docking potential inhibitors within the constraints of the active site defined by the model. Inhibitors of this enzyme could be used for preventing aflatoxin contamination of the food supply.

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Abstract

L'invention concerne les coordonnées atomiques/les données de diffraction des rayons X définissant la structure tridimensionnelle d'un complexe ternaire constitué de la trihydroxynaphtalène réductase (THNR), du NADPH et d'un ligand qui se lie à la THNR ou à l'une de ses sous-unités. L'invention concerne également des procédés permettant d'identifier d'autres ligands qui se lient à la THNR. Ces ligands, qui peuvent être utiles comme inhibiteurs des sites actifs de l'activité trihydroxynaphtalène réductase, peuvent être utilisés pour diagnostiquer ou pour traiter les maladies des végétaux et des animaux provoquées par des micro-organismes produisant de la THNR.
PCT/US1998/021550 1997-10-14 1998-10-13 Trihydroxynaphtalene reductase: procedes permettant la determination de sa structure tridimensionnelle et la mise au point rationnelle de ses inhibiteurs WO1999019465A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997015588A1 (fr) * 1995-10-26 1997-05-01 St. Jude Children's Research Hospital Proteine/cathepsine a protectrice et precurseur: cristallisation, diffraction des rayons x, determination de structure tridimensionnelle et elaboration rationnelle de substances therapeutiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997015588A1 (fr) * 1995-10-26 1997-05-01 St. Jude Children's Research Hospital Proteine/cathepsine a protectrice et precurseur: cristallisation, diffraction des rayons x, determination de structure tridimensionnelle et elaboration rationnelle de substances therapeutiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANDERSSON A ET AL: "A flexible lid controls access to the active site in 1,3,8- trihydroxynaphthalene reductase.", FEBS LETTERS, (1997 JAN 3) 400 (2) 173-6. JOURNAL CODE: EUH. ISSN: 0014-5793., Netherlands, XP002095246 *
ARNOLD ANDERSSON ET AL.: "Crystal structure of the ternary complex of 1,3,8-trihydroxy-naphthalene reductase from Magnaporthe grisea with NADPH and an active site inhibitor", STRUCTURE, vol. 4, no. 10, 1996, pages 1161 - 1170, XP002095245 *

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