U.S. patent application number 10/526207 was filed with the patent office on 2005-10-27 for gtp cyclohydrolase ii as as target for fungicides.
Invention is credited to Althofer, Henning, Freund, Annette, Kaesler, Bruno, Karos, Marvin, Lacour, Thierry, Rohl, Franz.
Application Number | 20050239159 10/526207 |
Document ID | / |
Family ID | 31970279 |
Filed Date | 2005-10-27 |
United States Patent
Application |
20050239159 |
Kind Code |
A1 |
Freund, Annette ; et
al. |
October 27, 2005 |
Gtp cyclohydrolase II as as target for fungicides
Abstract
The present invention relates to the identification of fungal
GTP cyclohydrolase II as a target for fungicides, to a method for
identifying antifungal agents based on fungal GTP cyclohydrolase
II, and also to the use of compounds identified as fungicides via
the abovementioned method.
Inventors: |
Freund, Annette;
(Limburgerhof, DE) ; Rohl, Franz; (Schifferstadt,
DE) ; Althofer, Henning; (Wachenheim, DE) ;
Karos, Marvin; (Neustadt, DE) ; Kaesler, Bruno;
(Ludwigshafen, DE) ; Lacour, Thierry; (Stutensee,
DE) |
Correspondence
Address: |
HUTCHISON & MASON PLLC
PO BOX 31686
RALEIGH
NC
27612
US
|
Family ID: |
31970279 |
Appl. No.: |
10/526207 |
Filed: |
March 4, 2005 |
PCT Filed: |
August 23, 2003 |
PCT NO: |
PCT/EP03/09369 |
Current U.S.
Class: |
435/21 |
Current CPC
Class: |
C12Q 1/34 20130101; G01N
2500/02 20130101; C12Y 305/04025 20130101; A01N 43/90 20130101;
C12N 9/78 20130101; A01N 61/00 20130101 |
Class at
Publication: |
435/021 |
International
Class: |
C12Q 001/42 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 6, 2002 |
EP |
02020051.5 |
Claims
1-19. (canceled)
20. A method for identifying antifungal agents comprising i.
incubating, with at least one candidate compound, a fungal GTP
cyclohydrolase II polypeptide under conditions allowing the binding
of the candidate compound to the fungal GTP cyclohydrolase II; and
ii. selecting, by step ii), at least a candidate compound which
binds to the fungal GTP cyclohydrolase II of step i); or iii.
selecting, by step iii), at least one candidate compound which
reduces or blocks the activity of the fungal GTP cyclohydrolase II
of step i); or iv. selecting, by step iv), at least one candidate
compound which inhibits or decreases transcription, translation or
expression of the fungal GTP cyclohydrolase II of step i), wherein
the GTP cyclohydrolase II activity in steps ii to iv is determined
by a) adding GTP or GTP analog, NAD.sup.+ and formate dehydrogenase
to a sample comprising GTP cyclohydrolase II or I; and b)
determining the NADH content.
21. The method of claim 20, wherein the fungal GTP cyclohydrolase
II is encoded by a nucleic acid sequence comprising a) a nucleic
acid sequence shown in SEQ ID No: 1; or b) a nucleic acid sequence
which, owing to the degeneracy of the genetic code, can be deduced
from the amino acid sequence shown in SEQ ID No: 2 by back
translation; or c) a nucleic acid sequence which, owing to the
degeneracy of the genetic code, can be deduced from a functional
equivalent of the amino acid sequence shown in SEQ ID No: 2, which
has an identity with SEQ ID No: 2 of at least 49%, by back
translation.
22. A nucleic acid having a sequence comprising a) a nucleic acid
sequence shown in SEQ ID No: 4; or b) a nucleic acid sequence
which, owing to the degeneracy of the genetic code, can be deduced
from the amino acid sequence shown in SEQ ID No: 5 by back
translation; or c) a nucleic acid sequence which, owing to the
degeneracy of the genetic code, can be deduced from a functional
equivalent of the amino acid sequence shown in SEQ ID No: 5, which
has an identity with SEQ ID No: 5 of at least 66%, by back
translation.
23. The method of claim 20, wherein the method comprises testing a
candidate compound in a fungal GTP cyclohydrolase II inhibition
assay.
24. The method of claim 23, wherein the method comprises a)
incubating, with a candidate compound, a fungal GTP cyclohydrolase
II in a cell free system; b) selecting, by step b), a candidate
compound which decreases the activity of the fungal GTP
cyclohydrolase II.
25. The method of claim 24, wherein the enzymatic activity of the
fungal GTP cyclohydrolase II is determined in comparison to the
activity of a fungal GTP cyclohydrolase II not incubated with the
candidate compound.
26. A method for determination of GTP cyclohydrolase I or II
activity comprising the steps of a) adding GTP or GTP analog, NAD+
and formate dehydrogenase to a sample comprising GTP cyclohydrolase
II or I; and b) determining the NADH content.
27. A method for identification of inhibitors of GTP cyclohydrolase
I or II comprising the following steps: a) adding GTP or GTP
analog, NAD+ and formate dehydrogenase to a sample comprising GTP
cyclohydrolase I or II; b) adding formate, NAD+ and formate
dehydrogenase to a second sample comprising GTP cyclohydrolase I or
II; c) adding to the sample of step a) and step b) a candidate
compound; d) determining the activity of both samples; e) selecting
candidate compounds that show inhibition in the presence of GTP and
no inhibition in the presence of formic acid.
28. The method of claim 24, wherein inhibitors of fungal GTP
cyclohydrolase II are identified in an inhibition assay comprising
the following steps: a) adding GTP or GTP analog, NAD+ and formate
dehydrogenase to a sample comprising GTP cyclohydrolase I or II; b)
adding formate, NAD+ and formate dehydrogenase to a second sample
comprising GTP cyclohydrolase I or II; c) adding to the sample of
step a) and step b) a candidate compound; d) determining the
activity of both samples; e) selecting candidate compounds that
show inhibition in the presence of GTP and no inhibition in the
presence of formic acid.
29. The method of claim 24, wherein GTP is used as substrate and
the NADH content is determined by monitoring the increase in the
absorption at 340 nm.
30. The method of claim 28, wherein GTP is used as substrate and
the NADH content is determined by monitoring the increase in the
absorption at 340 nm.
31. The method of claim 1, wherein the method comprises the
following steps: a) generating organisms which, following
transformation with a nucleic acid sequence encoding GTP
cyclohydrolase II are capable of overexpressing polypeptide with
GTP cyclohydrolase II activity; b) applying to the organism of step
a) and to an analogous, untransformed organism, a candidate
compound; c) determining the growth, the viability or infectivity
of the transgenic and the untransformed organism following
application of the substance of step b); d) selecting candidate
compounds, which reduces growth, viability or infectivity of the
transgenic and the untransformed organism following application of
the substance of step b).
32. The method of claim 31, wherein the transgenic organism and the
untransformed organism are fungi.
33. The method of claim 20, wherein the substances are identified
in a high-throughput screening.
34. The method of claim 20, wherein the antifungal agent identified
via the method is applied to a phytopathogenic fungus in order to
verify the fungicidal activity.
35. A process for the preparation of a fungicidal composition,
which comprises a) identifying an antifungal agent via a method for
identifying antifungal agents comprising i. incubating, with at
least-one candidate compound, a fungal GTP cyclohydrolase II
polypeptide under conditions allowing the binding of the candidate
compound to the fungal GTP cyclohydrolase II; and ii. selecting, by
step ii), at least a candidate compound which binds to the fungal
GTP cyclohydrolase II of step i) ; or iii. selecting, by step iii),
at least one candidate compound which reduces or blocks the
activity of the fungal GTP cyclohydrolase II of step i); or iv.
selecting, by step iv), at least one candidate compound which
inhibits or decreases transcription, translation or expression of
the fungal GTP cyclohydrolase II of step i), wherein the GTP
cyclohydrolase II activity in steps ii to iv is determined by 1)
adding GTP or GTP analog, NAD+ and formate dehydrogenase to a
sample comprising GTP cyclohydrolase II or I; and 2) determining
the NADH content; and b) formulating the antifungal agent
identified via (a), or an agriculturally useful salt of the active
ingredient identified via (a), with suitable adjuvants.
36. A process for the preparation of a pharmaceutical fungicidal
composition, which comprises a) identifying an antifungal agent via
a method for identifying antifungal agents comprising i.
incubating, with at least-one candidate compound, a fungal GTP
cyclohydrolase II polypeptide under conditions allowing the binding
of the candidate compound to the fungal GTP cyclohydrolase II; and
ii. selecting, by step ii), at least a candidate compound which
binds to the fungal GTP cyclohydrolase II of step i) ; or iii.
selecting, by step iii), at least one candidate compound which
reduces or blocks the activity of the fungal GTP cyclohydrolase II
of step i); or iv. selecting, by step iv), at least one candidate
compound which inhibits or decreases transcription, translation or
expression of the fungal GTP cyclohydrolase II of step i), wherein
the GTP cyclohydrolase II activity in steps ii to iv is determined
by 1) adding GTP or GTP analog, NAD.sup.+ and formate dehydrogenase
to a sample comprising GTP cyclohydrolase II or I; and 2)
determining the NADH content; and b) formulating the antifungal
agent identified via (a), or a pharmaceutically useful salt of the
active ingredient identified via (a), with suitable excipients.
37. A method for culturing plants or plant cells or plant tissues
thereby controlling fungal growth comprising treating said culture
with a fungicide, wherein said fungicide is a compound which is an
inhibitor of fungal GTP cyclohydrolase II.
Description
[0001] The present invention relates to the identification of
fungal GTP cyclohydrolase II as a target for fungicides, to a
method for identifying antifungal agents based on fungal GTP
cyclohydrolase II, and also to the use of compounds identified as
fungicides via the abovementioned method.
[0002] The basic principle of identifying fungicides via inhibition
of a defined enzyme is known (WO 00/3657). With regard to the
increasing problems regarding resistance to fungicides, however,
there exists a great need for detecting enzymes which might
constitute novel targets for fungicides.
[0003] In practice, the detection of targets is extremely
difficult, since often the inhibition of an enzyme participating in
a biochemical pathway does not lead to decreased growth or
infectivity of the pathogenic fungi. A putative reason is the
existence of an alternative, maybe unknown pathway used by the
fungus. Thus, even if the function of the gene itself is known, it
is not possible to predict a fitness for use as a fungicide
target.
[0004] Thus, it is an object of the present invention to identify a
novel fungicide target.
[0005] Surprisingly, we have found that fungal GTP cyclohydrolase
is suitable as a fungicide target. The present invention comprises
the use of a fungal GTP cyclohydrolase as target for the
identification of antifungal agents and methods of identifying
antifungal agents which inhibit fungal GTP cyclohydrolase II
comprising the following steps:
[0006] i. incubating, with at least one candidate compound, a
fungal GTP cyclohydrolase II under conditions allowing the binding
of the candidate compound to the fungal GTP cyclohydrolase II
polypeptide; and
[0007] ii. selecting, by step ii), at least one candidate compound
which binds to the fungal GTP cyclohydrolase II of step i); or
[0008] iii. selecting, by step iii), at least one candidate
compound which reduces or blocks the activity of the fungal GTP
cyclohydrolase II of step i); or
[0009] iv. selecting, by step iv), at least a candidate compound
which inhibits or decreases transcription, translation or
expression of the fungal GTP cyclohydrolase II of step i).
[0010] Some of the terms used in the description are defined at
this point.
[0011] "Affinity tag": this denotes a peptide or polypeptide whose
coding nucleic acid sequence can be fused to the sequence encoding
the fungal GTP cyclohydrolase II, either directly or using a
linker, by customary cloning techniques. The affinity tag serves to
isolate the recombinant fungal GTP cyclohydrolase II by means of
affinity chromatography. The abovementioned linker can optionally
comprise a protease cleavage site (for example for thrombin or
factor Xa), whereby the affinity tag can be cleaved off from the
fungal GTP cyclohydrolase II, as required. Examples of customary
affinity tags are the "his-tag", for example from Quiagen, Hilden,
"strep-tag", "myc-tag" (Invitrogen, Carlsberg), New England
Biolab's tag which consists of a chitin-binding domain and an
intein, and what is known as the CBD-tag from Novagen.
[0012] "Antifungal agents" are agents against pathogenic fungi such
as human and plant pathogens, preferably plant pathogens.
[0013] "Enzymatic activity/activity assay": the term enzymatic
activity describes the ability of an enzyme to convert a substrate
into a product. In this context, both the natural substrate of the
enzyme and a synthetic modified analog of the natural substrate can
be used. The enzymatic activity can be determined in what is known
as an activity assay via the increase in the product, the decrease
in the starting material, the decrease or increase in a specific
cofactor, or a combination of at least two of the afore-mentioned
parameters as a function of a defined period of time. If the enzyme
catalyzes a reversible reaction, both the starting material and the
product may be employed as substrate in the activity assay in
question.
[0014] "Expression cassette or nucleic acid sequence": an
expression cassette comprising a nucleic acid sequence according to
the invention operatively linked to a promotor and/or terminator
sequence is understood as meaning, for example, a genomic or a
complementary DNA sequence or an RNA sequence and semisynthetic or
fully synthetic analogs of these. These sequences may be present in
linear or circular form, extrachromosomally or integrated into the
genome. The nucleic acid sequences according to the invention can
be generated synthetically or obtained naturally or comprise a
mixture of synthetic and natural DNA components, and be composed of
a variety of heterologous gene segments of various organisms.
[0015] Artificial nucleic acid sequences too are suitable in this
context as long as they make possible the expression of the fungal
GTP cyclohydrolase II in a cell or an organism. For example,
synthetic nucleotide sequences can be generated which were
optimized with regard to the codon usage of the organisms to be
transformed.
[0016] All of the abovementioned nucleotide sequences can be
generated in a manner known per se by chemical synthesis from the
nucleotide units such as, for example, by fragment condensation of
individual overlapping complementary nucleotide units of the double
helix. Oligonucleotides can be synthesized chemically for example
in a known manner using the phosphoamidite method (Voet, Voet, 2nd
Edition, Wiley Press New York, pages 896-897). When preparing an
expression cassette, a variety of DNA fragments can be manipulated
to give rise to a nucleotide sequence which reads in the correct
direction and is in-frame. The nucleic acid fragments are linked to
each other by general cloning techniques as are described, for
example in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1989) and in T. J. Silhavy, M. L. Berman and
L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M.
et al., Current Protocols in Molecular Biology, Greene Publishing
Assoc. and Wiley-Interscience (1994).
[0017] "Gene" describes a nucleic acid sequence which encodes a
protein and which can be transcribed into RNA (mRNA, rRNA, tRNA,
snRNA, sense RNA or antisense RNA) and which can optionally be
associated with regulatory sequences. Examples of
regulatory-sequences are promoter sequences. Other elements which
are optionally present are, for example, introns.
[0018] "Genetic control sequence": the term of the genetic control
sequences (which is equivalent to the term "regulatory sequence")
describes sequences which have an effect on the materialization or
the function of the expression cassette according to the invention
and which ensure for example transcription and, if appropriate,
translation in prokaryotic or eukaryotic organisms. Examples are
promoters or what are known as enhancer sequences. In addition to
these control sequences, or instead of these sequences, the natural
regulation of these sequences before the actual structural genes
may still be present and, if appropriate, may have been modified
genetically in such a way that the natural regulation has been
inactivated and the expression of the fungal GTP cyclohydrolase II
gene increased. The choice of the control sequence depends on the
host organism or starting organism. Genetic control sequences
furthermore also encompass the 5'-untranslated region, introns or
the noncoding 3'-region of genes. Control sequences are furthermore
understood as being those which make possible a homologous
recombination or insertion into the genome of a host organism or
which permit the removal from the genome.
[0019] "Functional equivalents" in the present context describe
nucleic acid sequences which hybridize under standard conditions
with the nucleic acid sequence encoding the GTP cyclohydrolase II
or portions of the nucleic acid sequence encoding the GTP
cyclohydrolase II, and which are capable of bringing about the
expression of an enzymatically active fungal GTP cyclohydrolase II
in a cell or an organism.
[0020] It is advantageous to use short oligonucleotides of a length
between 10 to 50 bp, preferably 15-40 bp, for example of the
conserved or other regions, which can be determined via comparisons
with other related genes in a manner known to the skilled worker
for the hybridization. Alternatively, it is also possible to use
longer fragments of the nucleic acids according to the invention or
the complete sequences for the hybridization. These standard
conditions vary depending on the nucleic acid used, viz.
oligonucleotide, longer fragment or complete sequence, or depending
on which type of nucleic acid, viz. DNA or RNA, is being used for
the hybridization. Thus, for example, the melting temperatures for
DNA:DNA hybrids are approx. 10.degree. C. lower than those of
DNA:RNA hybrids of equal length.
[0021] Standard conditions are understood as meaning, depending on
the nucleic acid, for example temperatures between 42 and
58.degree. C. in an aqueous buffer solution with a concentration of
between 0.1 and 5.times.SSC (1.times.SSC=0.15 M NaCl, 15 mM sodium
citrate, pH 7.2) or additionally in the presence of 50% formamide
such as, for example, 42.degree. C. in 5.times.SSC, 50% formamide.
The hybridization conditions for DNA:DNA hybrids are advantageously
0.1.times.SSC and temperatures of between approximately 20.degree.
C. and 45.degree. C., preferably between approximately 30.degree.
C. and 45.degree. C. The hybridization conditions for DNA:RNA
hybrids are advantageously 0.1.times.SSC and temperatures of
between approximately 30.degree. C. and 55.degree. C., preferably
between approximately 45.degree. C. and 55.degree. C. These
temperatures stated for the hybridization are melting temperature
values which have been calculated by way of example for a nucleic
acid with a length of approx. 100 nucleotides and a G+C content of
50% in the absence of formamide. The experimental conditions for
DNA hybridization are described in specialist textbooks of genetics
such as, for example, Sambrook et al., "Molecular Cloning", Cold
Spring Harbor Laboratory, 1989 and can be calculated using formulae
known to the skilled worker, for example as a function of the
length of the nucleic acids, the type of the hybrids or the G+C
content. The skilled worker can find more information on
hybridization in the following textbooks: Ausubel et al. (eds),
1985, Current Protocols in Molecular Biology, John Wiley &
Sons, New York;. Hames and Higgins (eds), 1985, Nucleic Acids
Hybridization: A Practical Approach, IRL Press at Oxford University
Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A
Practical Approach, IRL Press at Oxford University Press,
Oxford.
[0022] A functional equivalent is furthermore also understood as
meaning, in particular, natural or artificial mutations of the
relevant nucleic acid sequences of the fungal GTP cyclohydrolase II
and their homologs from other organisms which make possible the
expression of the enzymatically active fungal GTP cyclohydrolase II
in a cell or an organism.
[0023] Thus, the scope of the present invention also extends to,
for example, those nucleotide sequences which are obtained by
modification of the nucleic acid sequence of a GTP cyclohydrolase
II. The purpose of such a modification can be, for example, the
insertion of further cleavage sites for restriction enzymes, the
removal of excess DNA, or the addition of further sequences.
Proteins which are encoded via said nucleic acid sequences should
still maintain the desired functions, despite the deviating nucleic
acid sequence.
[0024] The term functional equivalent may also refer to the protein
encoded by the nucleic acid sequence in question. In this case, the
term functional equivalent describes a protein whose amino acid
sequence is up to a specific percentage homologous to or identical
with that of the GTP cyclohydrolase II.
[0025] Functional equivalents thus encompass naturally occurring
variants of the sequences described herein, and also artificial,
for example chemically synthesized, nucleic acid sequences adapted
to the codon usage, or the amino acid sequences derived
therefrom.
[0026] In general, it can be said that functional equivalents
independently of the amino acid sequence in question (encoded by a
corresponding nucleic acid sequence) have in each case the
enzymatic activity of a GTP cyclohydrolase II.
[0027] "GTP cyclohydrolase II activity" denotes the ability of an
enzyme to catalyse a reaction, wherein GTP is metabolized into
2,5-diamino-6-ribosylamino-4(H)-pyrimidinone 5'-monophosphate,
pyrophosphoric acid and formic acid.
[0028] "Homology" between two nucleic acid sequences or polypeptide
sequences is defined by the identity of the nucleic acid
sequence/polypeptide sequence by in each case the entire sequence
length, which is calculated by alignment with the aid of the
program algorithm GAP (Wisconsin Package Version 10.0, University
of Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting
the following parameters:
1 Gap weight: 8 Length weight: 2 Average match: 2,912 Average
mismatch: -2,003 matrix BLOSUM 62
[0029] The term "homology" is used herein as a synonym for
"identity".
[0030] "Mutations" comprise substitutions, additions, deletions,
inversions or insertions of one or more nucleotide residues, which
may also lead to a modification of the corresponding amino acid
sequence of the fungal GTP-cyclohydrolase II by substitution,
insertion or deletion of one or more amino acids.
[0031] "Knock-out transformant" refers to a transgenic organism, in
which a specific gene has been inactivated in a directed fashion by
means of transformation.
[0032] "Natural genetic environment" is understood as meaning the
natural chromosomal locus in the organism of origin or the presence
in a genomic library. In the case of a genomic library, the natural
genetic environment of the nucleic acid sequence is preferably
retained at least in part. The environment flanks the nucleic acid
sequence at least on the 5' or 3' side and has a sequence length of
at least 50 bp, preferably at least 100 bp, especially preferably
at least 500 bp, very especially preferably at least 1000 bp, most
preferably at least 5000 bp.
[0033] "Operative linkage": An operative or else functional linkage
is understood as meaning the sequential arrangement of promoter,
coding sequence, terminator and, if appropriate, further regulatory
elements in such a way that each of the regulatory elements can,
upon expression of the coding sequence, fulfil its function as
intended.
[0034] "Recombinant DNA" describes a combination of DNA sequences
in an arrangement other than their natural arrangement, which can
be generated by recombinant DNA technology, but also DNA comprising
the endogenous and foreign or synthetic DNA, also homologous and
heterologous DNA based on the relatedness of the organisms.
[0035] "Recombinant DNA technology": generally known techniques
from fusing DNA sequences (for example described in Sambrook et
al., 1989, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory
Press).
[0036] "Origins of replication" ensure the amplification of the
expression cassettes or vectors according to the invention in
microorganisms, for example pBR322 ori or P15A ori in E. coli
(Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989).
[0037] "Reporter genes" encode readily quantifiable proteins. Using
these genes, an assessment of transformation efficacy or of the
site or time of expression can be made via growth, fluorescence,
chemoluminescence, bioluminescence or resistance assay or via
photometric measurement (intrinsic color) or enzyme activity. Very
especially preferred in this context are reporter proteins
(Schenborn E, Groskreutz D. Mol. Biotechnol. 1999; 13(1):29-44)
such as the "green fluorescence protein" (GFP) (Gerdes H H and
Kaether C, FEBS Lett. 1996; 389(1):44-47; Chui W L et al., Curr.
Biol. 1996, 6:325-330; Leffel S M et al., Biotechniques.
23(5):912-8, 1997), chloramphenicol acetyl transferase, a
luciferase (Giacomin, Plant Sci. 1996, 116:59-72; Scikantha, J.
Bact. 1996, 178:121; Millar et al., Plant Mol. Biol. Rep. 1992
10:324-414), and luciferase genes, in general .beta.-galactosidase
or .beta.-glucuronidase (Jefferson et al., EMBO J. 1987, 6,
3901-3907), the Ura3 gene, the Ilv2 gene, the
2-desoxyglucose-6-phosphate phosphatase gene, .beta.-lactamase
gene, the neomycin phosphotransferase gene, the hygromycin
phosphotransferase gene, or the BASTA (=gluphosinate) resistance
gene.
[0038] "Selection markers" impart resistance to antibiotics.
Examples which may be mentioned are the
neomycin-phosphotransferase-gen gene, which imparts resistance to
the aminoglyciside antibiotics neomycin (G 418), kanamycin and
paromycin (Deshayes A et al., EMBO J. 4 (1985) 2731-2737), the sul
gene (Guerineau F et al., Plant Mol. Biol. 1990; 15(1):127-136),
the hygromycin B phosphotransferase-Gen (Gen Bank Accession NO: K
01193) and the she-ble gene, which imparts resistance to the
bleomycin antibiotic zeocin. Other examples of selection marker
genes are genes which impart resistance to
2-desoxyglucose-6-phosphate (WO 98/45456) or phosphinothricin and
the like, or those which impart resistance to antimetabolites, for
example the dhfr gene (Reiss, Plant Physiol. (Life Sci. Adv.) 13
(1994) 142-149). Also suitable are genes such as trpB or hisD
(Hartman S C and Mulligan R C, Proc. Natl. Acad. Sci. U S A. 85
(1988.) 8047-8051). Also suitable are the mannose-phosphate
isomerase gene (WO 94/20627), the ODC (ornithin decarboxylase) gene
(McConlogue, 1987 in: Current Communications in Molecular Biology,
Cold Spring Harbor Laboratory, Ed.), or the Aspergillus terreus
deaminase (Tamura K et al., Biosci. Biotechnol. Biochem. 59 (1995)
2336-2338).
[0039] "Significant decrease": referring to the enzymatic activity,
is understood as meaning the decrease in the enzymatic activity of
the enzyme incubated with a candidate compound in comparison with
the activity of an enzyme not incubated with the candidate
compound, which lies outside an error in measurement.
[0040] "Substrate": Substrate is the compound which is recognized
by the enzyme in its original function and which is converted into
a product by means of a reaction catalyzed by the enzyme.
[0041] "Transgenic": Referring to a nucleic acid sequence, an
expression cassette or a vector comprising said nucleic acid
sequence or an organism transformed with said nucleic acid
sequence, expression cassette or vector, transgenic describes all
those constructions generated by recombinant methods in which
either the nucleic acid sequence of the fungal GTP cyclohydrolase
II or a genetic control sequence linked operably to the nucleic
acid sequence of the fungal GTP cyclohydrolase II or a combination
of both of the aforementioned nucleic acid sequences.
[0042] GTP cyclohydrolase II catalyses the first step in the
biosynthesis of riboflavin (vitamin B2), wherein-GTP is metabolized
into 2,5-diamino-6-ribosylamino-4(H)-pyrimidinone 5'-monophosphate,
pyrophosphoric acid and formic acid (Ritz et. al. JBC 2001, 276:
22273-22277). GTP cyclohydrolase II can be used for fermetative
riboflavin synthesis in Ashbya gossypii (WO 95/26406).
[0043] In plants, GTP cyclohydrolase II is used as a target for
herbicides (WO 00/40744). The plant enzyme, however, differs
significantly from the fungal enzyme. The identity between fungal
GTP cyclohydrolase II from Ashbya gossypii (SEQ ID NO:2) and plant
GTP cyclohydrolase II from Arabidopsis thaliana (SWISS-PROT P47924)
is only 31%. The identity between bacterial GTP cyclohydrolase II
from Escherichia coli (SWISS-PROT P25523) and SEQ ID NO:2 is
48%.
[0044] Experiments in the yeast Saccharomyces cerevisiae indicate
that GTP cyclohydrolase II is not essential for yeast
(Saccharomyces Genom Database (SGD);
http://genome-www.stanford.edu/Saccharomyces/see for
http://genome-www4.stanford.edu/cgi-bin/SGD/phenotype/phenotype.pl?feat=R-
IB1&type=locus.
[0045] Surprisingly, it was found that GTP cyclohydrolase II is a
suitable fungicide target by demonstrating the essential role of
GTP cyclohydrolase II for the pathogenic filamentous fungi Ashbya
gossypii. The present invention therefore provides methods of using
a fungal GTP cyclohydrolase II polypeptide (used herein synonymous
to fungal GTP cyclohydrolase II) encoding nucleic acid sequenence
to identify inhibtors thereof, which then can be used as fungicides
to suppress the growth of pathogenic fungi.
[0046] The term "pathogenic fungi" denotes fungi which colonize a
host (a plant or a mammal) and cause disease, e.g. human
pathogenens selected from the group consisting of the genera and
species Candida such as Candida albicans, Candia stettatoidea,
Candiala tropicatis, Candida parapsilosis, Candida krusei, Candida
pseudotropicatis, Candida quittermondii, Candida rugosa,
Aspergillus such as Aspergillus fumigatus, Aspergillus flavus,
Aspergillus niger, Aspergillus nidulans, Aspergillus terreus,
Rhizopus such as Rhizopus arrhizus, Rhizopus oryzae, Absidia such
as Absidia corymbifera, Absidia ramosa and Mucor such as Mucor
pusiltus or phytopathogenic filamentous fungi selected from the
group consisting of the genera and species Ashbya such as Ashbya
gossypii, Alternaria, Podosphaera, Sclerotinia, Physalospora such
as Physalospora canker, Botrytis species such as Botrytis cinerea,
Corynespora such as Corynespora melonis; Colletotrichum;
Diplocarpon such as Diplocarpon rosae; Elsinoe such as Elsinoe
fawcetti, Diaporthe such as Diaporthe citri; Sphaerotheca; Cinula
such as Cinula neccata, Cercospora; Erysiphe such as Erysiphe
cichoracearum and Erysiphe graminis; Sphaerotheca such as
Sphaerotheca fuliginea; Leveillula such as Leveillula taurica;
Magnaporte species such as Magnaporthe (M.) grisea, Mycosphaerella;
Phyllactinia such as Phyllactinia kakicola; Gloesporium such as
Gloesporium kaki; Gymnosporangium such as Gymnosporangium yamadae,
Leptotthrydium such as Leptotthrydium pomi, Podosphaera such as
Podosphaera leucotricha; Pyrenophora (P.) such as P. graminea, P.
hordei, P. japonica, P. teres, P. teres f. maculata, P. teres f.
teres, P. tritici-repentis, Gloedes such as Gloedes pomigena;
Cladosporium such as Cladosporium carpophilum; Phomopsis;
Phytopora; Phytophthora such as Phytophthora infestans;
Verticillium; Glomerella such as Glomerella cingulata; Drechslera;
Bipolaris; Personospora; Phaeoisariopsis such as Phaeoisariopsis
vitis; Spaceloma such as Spaceloma ampelina; Pseudocercosporella
such as Pseudocercosporella herpotrichoides; Pseudoperonospora;
Puccinia; Typhula; Pyricularia such as Pyricularia oryzae;
Rhizoctonia; Stachosporium such as Stachosporium nodorum; Uncinula
such as Uncinula necator; Ustilago such as Ustilago maydis;
Gaeumannomyces species such as Gaeumannomyces graminis and Fusarium
(F.) such as F. dimerium, F. merismoides, F. lateritium, F.
decemcellulare, F. poae, F. tricinctum, F. sporotrichioides, F.
chlamydosporum, F. moniliforme, F. proliferatum, F. anthophilum, F.
subglutinans, F. nygamai, F. oxysporum, F. solani, F. culmorum, F.
sambucinum, F. crookwellense, F. avenaceum ssp. avenaceum, F.
avenaceum ssp. aywerte, F. avenaceum ssp. nurragi, F. hetrosporum,
F. acuminatum ssp. acuminatum, F. aduminatum ssp. armeniacum, F.
longipes, F. compactum, F. equiseti, F. scripi, F. polyphialidicum,
F. semitectum and F. beomiforme, preferably, the term "pathogenic
fungi" denotes filamentous phytopathogenic fungi mentioned
above.
[0047] In one embodiment, the present invention encompasses a
method for identifying antifungal agents comprising the following
steps:
[0048] i. incubating, with at least one candidate compound, a
fungal GTP cyclohydrolase II under conditions allowing the binding
of the candidate compound to the fungal GTP cyclohydrolase II;
and
[0049] ii. selecting, by step ii), at least one candidate compound
which binds to the fungal GTP cyclohydrolase II of step i); or
[0050] iii. selecting, by step iii), at least one candidate
compound which reduces or blocks the activity of the fungal GTP
cyclohydrolase II of, step i); or
[0051] iv. selecting, by step iv), at least a candidate compound
which inhibits or decreases transcription, translation or
expression of the fungal GTP cyclohydrolase II.
[0052] Preferably, the fungal GTP cyclohydrolase II is encoded by a
nucleic acid sequence comprising
[0053] a) a nucleic acid sequence shown in SEQ ID NO:1; or
[0054] b) a nucleic acid sequence which, owing to the degeneracy of
the genetic code, can be deduced from the amino acid sequence shown
in SEQ ID NO: 2 by back translation; or
[0055] c) a nucleic acid sequence which, owing to the degeneracy of
the genetic code, can be deduced from a functional equivalent of
the amino acid sequence shown in SEQ ID NO: 2, which has an
identity with SEQ ID NO:2 of at least 49%, by back translation.
[0056] The functional equivalent of SEQ ID NO:2 set forth in c) has
an identity of at least 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%
preferably at least 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,
67%, 68%, 69%, and 70% more preferably 71%, 72%, 73%, 74%, 75%,
76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% most preferably at
least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%,.98%, 99% homology with the SEQ ID NO:2.
[0057] The nucleic acid sequence originates from a fungus, wherein
the term fungus denotes the above-mentioned pathogenic fungi and
yeast such as Saccharomyces species (e.g. S. cerevisiae), Pichia
species (e.g. P. pastoris, P. methanolica), Schizosaccharomyces
species (e.g. Schizosaccharomyces pombe) and Klyveromyces species
(e.g. K. lactis).
[0058] Within the scope of the present invention also novel nucleic
acid sequences encoding a fungal GTP cyclohydrolase are provided,
whereby said nucleic acid sequences comprise
[0059] a) a nucleic acid sequence shown in SEQ ID NO:4; or
[0060] b) a nucleic acid sequence which, owing to the degeneracy of
the genetic code, can be deduced from the amino acid sequence shown
in SEQ ID NO:5 by back translation; or
[0061] c) a nucleic acid sequence which, owing to the degeneracy of
the genetic code, can be deduced from a functional equivalent of
the amino acid sequence shown in SEQ ID NO:5, which has an identity
with SEQ ID NO:5 of at least 66%, by back translation.
[0062] The functional equivalent of SEQ ID NO:4 set forth in c) has
an identity of at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, preferably at least 77%, 78%, 79%, 80%, 81%, 82%,
83%, more preferably 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% most
preferably at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology
with the SEQ ID NO:5.
[0063] The selection according to step ii) can be based on binding
assays detecting the protein-inhibitor interactions, wherein either
the candidate compound or fungal GTP cyclohydrolase II comprises a
detectable label, such as a fluorescent, radioisotopic,
chemiluminescent, or enzymatic label, such as horseradish
peroxidase, alkaline phosphatase, or luciferase. Detection of a
candidate compound, which is bound to the fungal GTP cyclohydrolase
II can then be accomplished, for example, by direct counting of
radioemmission, by scintillation counting, or by determining
conversion of an appropriate substrate to a detectable product.
[0064] Preferred examples of these binding assays are fluorescence
correlation spectroscopy (FCS) (Proc. Natl. Acad. Sci. USA (1994)
11753-11575), flurescence polarization (Methods in Enzymology 246
(1995), pp. 283-300) or Fluorescence Energy Transfer (FRET)
(Cytometry 34, 1998, pp. 159-179; homogeneous Time Resolved
Fluorescence (HTRF) is preferred, if FRET is to be used).
[0065] Alternatively, binding of a candidate compound to a fungal
GTP cyclohydrolase II can be determined without labeling either of
the interactants, e.g. by using a microphysiometer to detect
binding of a candidate compound to the fungal GTP cyclohydrolase
II. A microphysiometer (e.g., Cytosensor.TM.) is an analytical
instrument that measures the rate at which a cell acidifies its
environment using a light-addressable potentiometric sensor (LAPS).
Changes in this acidification rate can be used as an indicator of
the interaction between a candidate compound and fungal GTP
cyclohydrolase II (according to McConnell et al., Science 2.57,
19061912, 1992). In addition, determining the ability of a
candidate compound to bind to the fungal GTP cyclohydrolase II can
be accomplished using a technology such as real-time Bimolecular
Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem.
63, 23382345, 1991, and Szabo etal., Curr. Opin. Struct. Biol.
5,699705, 1995), a technology for studying biospecific interactions
in real time, without labeling any of the interactants (e.g.
BIAcore). Changes in the optical phenomenon surface plasmone
resonance can be used as an indication of real-time reactions
between biological molecules. Also surface-enhanced laser
desorption/ionization (SELDI) in combination with a time-of-flight
mass spectrometer (MALDI-TOF) makes the rapid analysis of molecules
on a support possible and can be used for analyzing protein-ligand
interactions (Worral et al., (1998) Anal. Biochem. 70:750-756).
[0066] Alternatively, all of the above-mentioned methods can be
based on a "competition assay", wherein a reference molecule is
replaced by the candidate compound.
[0067] It is also possible to detect further potential antifungal
agents by "molecular modeling" via elucidation of the
three-dimensional structure of the polypeptide according to the
invention using x-ray structure analysis. The preparation of
protein crystals required for x-ray structure analysis, and the
corresponding measurements and subsequent evaluations of said
measurements, as well as the methodology of "molecular modeling'
are known to the skilled worker. In principle, optimization via
"molecular modeling" of the active ingredients identified by the
abovementioned methods is also possible.
[0068] The selection according to steps iii) and iv) preferably
comprises testing a candidate compound in a fungal GTP
cyclohydrolase II inhibition assay.
[0069] By preference, the selection according to step iii), herein
after referred to as "in vitro assay", is based on the following
steps:
[0070] a) incubating, with a candidate compound, the fungal GTP
cyclohydrolase II in a cell free system;
[0071] b) selecting, by step b), a candidate compound which
decreases the activity of fungal GTP cyclohydrolase II.
[0072] The enzymatic activity of the fungal GTP cyclohydrolase II
is preferably determined in comparison to the activity of a fungal
GTP cyclohydrolase II not incubated with the candidate
compound.
[0073] In step (b), candidate compounds are selected which brought
about a significant decrease in the enzymatic activity
corresponding to a reduction of at least 10%, advantageously at
least 20%, preferably at least 30%, especially preferably by at
least 50% and very especially preferably by at least 70%, or a 100%
reduction (blocking) being achieved.
[0074] Suitable substrates added to the reaction mixture in step b)
for determination of enzymatic activity are GTP or GTP comprising a
detectable label, such as a fluorescent, radioisotopic or
chemiluminescent label. These labled derivatives are hereinafter
referred to as "GTP-analogs".
[0075] For determination of enzymatic activity of fungal GTP
cyclohydrolase II in step b) of the in vitro assay, a fungal GTP
cyclohydrolase II comprising mixture (e.g. crude cell extract,
partially or totally purified protein) is incubated with a suitable
substrate and the conversion of the substrate or the increase in
the resultant product is monitored e.g. by HPLC or by measurement
of fluorescence, radioactivity or chemiluminescence of the
respective sample.
[0076] For example, the enzymatic activity can be determined by
HPLC as described in Ritz et. al. Journal of Biological Chemistry
2001, 276: 22273-22277) or by monitoring radioactivity as described
by Foor and Brown (1980, Meth. Enzymol. 66:303-307). For these
methods, GTP cyclohydrolase II is preferably partially purified or
purified to homogeneity.
[0077] Although there are enzymatic activity assays by which GTP
cyclohydrolase II activity can be determined, there is a constant
need for development of new methods for determining enzymatic
activity that are easy to perform and also suitable for high
throughput Screening (HTS).
[0078] Surprisingly, it has been found that GTP cyclohydrolase II
activity can be successfully determined in the presence of the
enzyme formate dehydrogenase (E.C. 1.2.1.2). In this method, the
formic acid formed by GTP cyclohydrolase can be linked to the
reduction of NAD by the combination of both of these enzymes. The
level of formate can be determined by monitoring the formation of
NADH preferaby by spectroscopy as described by Tishkov and Egorov
(SU 1271873).
[0079] This method is not only suitable for fungal GTP
cyclohydrolase II, but also for plant GTP cyclohydrolase II and GTP
cyclohydrolase I [E.C. 3.5.4.16], an enzyme having the same
substrate specificity as GTP cyclohydrolase I but a different
physiological function: GTP cyclohydrolase I that catalyses the
first step in the biosynthesis of tetrahydrofolate and
tetrahydrobiopterin. Within the scope of the present invention,
this method is used preferably for fungal GTP cyclohydrolase
II.
[0080] Thus, the present invention encompasses a method for
determination of GTP cyclohydrolase activity comprising the
following steps:
[0081] a) adding GTP or GTP analog, NAD+ and formate dehydrogenase
to a sample comprising a GTP cyclohydrolase II or I; and
[0082] b) determination of the NADH content.
[0083] If the method is used for an inhibition assay, it can
comprise the following steps to ensure that the candidate compound
inhibits GTP cyclohydrolase II and not formate dehydrogenase:
[0084] a) adding GTP or GTP analog, NAD+ and formate dehydrogenase
to a sample comprising GTP cyclohydrolase I or II;
[0085] b) adding formate, NAD+ and formate dehydrogenase to a
second sample comprising fungal GTP cyclohydrolase II;
[0086] c) adding to the sample of step a) and step b) a candidate
compound;
[0087] d) determining the activity of both samples;
[0088] e) selecting candidate compounds that show inhibition in the
presence of GTP and no inhibition in the presence of formic
acid.
[0089] Thus, in a particularly preferred embodiment, the GTP
cyclohydrolase II activity in step c) of the in vitro assay is
determined in the presence of the enzyme formate dehydrogenase
(E.C. 1.2.1.2) comprising the following steps:
[0090] a) adding GTP or GTP analog, NAD+ and formate dehydrogenase
to a sample comprising fungal GTP cyclohydrolase II; and
[0091] b) determination of the NADH content.
[0092] In another particulary-preferred embodiment, the in vitro
assay comprises the following steps:
[0093] a) adding GTP or GTP analog, NAD+ and formate dehydrogenase
to a sample comprising fungal GTP cyclohydrolase II;
[0094] b) adding formate, NAD+ and formate dehydrogenase to a
second sample comprising fungal GTP cyclohydrolase II;
[0095] c) adding to the sample of step a) and step b) a candidate
compound;
[0096] d) determining the activity of both samples;
[0097] e) selecting candidate compounds that show inhibition in the
presence of GTP and no inhibition in the presence of formic
acid.
[0098] This method is suitable even if unpurified cell extracts
(lysates) are put in the respective assay. Furthermore, this method
is applicable to high throughput screening for inhibitors of fungal
GTP cyclohydrolase II. If lysates or enzyme samples are used in
which both enzymes, GTP cyclohydrolase I and GTP cyclohydrolase II
are present, the selected candidate compounds can be optionally
further tested in another inhibition assay to confirm whether GTP
cyclohydrolase I or GTP cyclohydrolase II is inhibited (e.g.
according to Ritz et. al. Journal of Biological Chemistry 2001,
276: 22273-22277).
[0099] The fungal GTP cyclohydrolase II used for the in vitro test
can be present in the lysate of the fungi or of the transgenic
organism according to the invention. If required, the polypeptide
according to the invention can be purified partially or fully by
customary methods. A general overview of customary techniques for
purification of proteins is given, for example, in Ausubel, F. M.
et al., Current Protocols in Molecular Biology, Greene Publishing
Assoc. and Wiley-Interscience (1994); ISBN 0-87969-309-6. In the
case of recombinant production, purification of the protein fused
to an affinity tag may be effected by affinity chromatography.
[0100] The fungal GTP cyclohydrolase II used for the
above-mentioned in vitro assay can either be expressed in a
transgenic organism transformed with an expression cassette
comprising a nucleic acid sequence encoding a fungal GTP
cyclohydrolase II in enzymatically active form or be obtained by
culturing fungi naturally comprising a GTP cyclohydrolase II.
[0101] How to perform heterologous expression of an enzyme-like
fungal GTP cyclohydrolase II is well known to the skilled artisan.
First, appropriate expression cassettes and/or vectors comprising
the expression cassette have to be prepared, or alternatively,
commercial available vectors can be used. Besides plasmids, vectors
are also understood as meaning all the other vectors known to the
skilled worker such as, for example, phages, viruses such as SV40,
CMV, baculovirus, adenovirus, transposons, IS elements, phasmids,
phagemids, cosmids, linear DNA or circular DNA. These vectors are
capable of being replicated autonomously in the host organism or
replicated chromosomally, chromosomal replication being
preferred.
[0102] Suitable expression cassette comprises fungal GTP
cyclohydrolase II encoding nucleic acid sequence operatively linked
to control elements, which govern the expression of the coding
sequence in the host cell. In accordance with a preferred
embodiment, an expression cassette according to the invention
comprises, at the 5' end of the coding sequence, a promoter and at
the 3' end a transcription/termination signal and, if appropriate,
further genetic control sequences which are linked operably to the
interposed coding sequence of the fungal GTP cyclohydrolase II.
[0103] Also suitable are analogs of the above-described expression
cassettes which can originate, for example, from a combination of
the individual nucleic acid sequences on one polynucleotide
(multiple constructs), more than one polynucleotide in a cell
(cotransformation), or by sequential transformation.
[0104] Advantageous control sequences for the expression cassettes
or vectors according to the invention are present, for example, in
promoters such as the cos, tac, trp, tet, lpp, lac, lacIq, T7, T5,
T3, gal, trc, ara, SP6, 1-PR or in the 1-PL promoter, all of which
can be used for expressing fungal GTP cyclohydrolase II in
Gram-negative bacterial strains.
[0105] Further advantageous control sequences are present for
example in the promoters amy and SPO2, both of which can be used
for expressing fungal GTP cyclohydrolase II in Gram-positive
bacterial strains, and in the yeast or fungal promoters AUG1-, ADC1
GPD-1-, PX6-, TEF-, CUP1-, PGK-, GAP1-, TPI, PHO5-, AOX1,
GAL10/CYC-1, CYC1, OliC-, ADH-, TDH-, Kex2-, MFa-, rp28- or the
NMT-promotor or combinations of the aforementioned promotors
(Degryse et al., Yeast Jun. 15, 1995; 11(7):629-40; Romanos et al.
Yeast June 1992; 8(6):423-88; Benito et al. Eur. J. Plant Pathol.
104, 207-220 (1998); Cregg et al. Biotechnology (N Y) August 1993;
11(8):905-10;
[0106] Luo X., Gene Sep. 22, 1995; 163(1):127-31; Nacken et al.,
Gene Oct. 10 1996; 175(1-2): 253-60; Turgeon et al., Mol Cell Biol
September 1987; 7(9):3297-305) all of which can be used for
expressing fungal GTP cyclohydrolase II in yeast strains. Examples
of suitable terminators are the NMT-, Gcy1-, TrpC-, AOX1-, nos-,
the PGK- or the CYC1-terminator, preferably the nos-terminator
(Degryse et al., Yeast Jun. 15, 1995; 11(7):629-40; Brunelli et al.
Yeast Dec. 9, 1993 (12): 1309-18; Frisch et al., Plant Mol. Biol.
27 (2), 405-409 (1995); Scorer et al., Biotechnology (N.Y.) 12 (2),
181-184 (1994), Genbank acc. number Z46232; Zhao et al. Genbank acc
number: AF049064; Punt et al., (1987) Gene 56(1), 117-124).
[0107] Control elements which may be mentioned as being suitable
for expression in insect cells are, for example, the polyhedrin
promoter and the p10 promoter (Luckow, V. A. and Summers, M. D.
(1988) Bio/Techn. 6, 47-55).
[0108] Examples of advantageous control sequences for expressing
fungal GTP cyclohydrolase II in cell culture are, besides
polyadenylation sequences, the following eukaryotic promoters of
viral origin, such as, for example, promoters of the polyoma virus,
adenovirus 2, cytomegalovirus or simian virus 40.
[0109] Further prokaryotic and eukaryotic expression systems are
mentioned in Chapters 16 and 17 in Sambrook et al., Molecular
Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0110] The expression cassettes according to the invention and the
vectors derived from them may also comprise further functional
elements, in addition to the abovementioned promoters. The
following may be mentioned by way of example, but not by
limitation: reporter genes, origins of replication, selection
markers and/or affinity tags, fused to fungal GTP cyclohydrolase II
either directly or by means of a linker optionally comprising a
protease cleavage site.
[0111] The expression cassette and the vectors derived from them
can be employed for the transformation of bacteria, cyanobacteria,
yeasts, filamentous fungi and algae and eukaryotic cells (for
example insect cells) with the purpose of recombinantly producing
fungal GTP cyclohydrolase II, the generation of a suitable
expression cassette depending on the organism in which the gene is
to be expressed.
[0112] The nucleic acid encoding a fungal GTP cyclohydrolase II may
advantageously also be introduced into the organisms in the form of
a linear DNA and integrated into the genome of the host organism
via heterologous or homologous recombination. This linear DNA may
consist of a linearized plasmid or else only of the nucleic acid
construct as vector or the nucleic acid sequences used. In a
further advantageous embodiment, the nucleic acid sequences used in
the method according to the invention may also be introduced into
an organism by themselves. If, besides the nucleic acid sequences,
further genes are to be introduced into the organism, all the genes
may be introduced together into the organism in a single vector or
each individual gene may be introduced into the organism in one
vector each, it being possible to introduce the various vectors
simultaneously or in succession.
[0113] The transgenic organisms generated by transformation can be
used for recombinant expression of fungal GTP cyclohydrolase
II.
[0114] Other preferred microorganisms for the recombinant
expression are, besides bacteria, yeasts and fungi, and eukaryotic
cell lines.
[0115] Preferred within the bacteria are bacteria of the genus
Escherichia, Erwinia, Flavobacterium, Alcaligenes or cyanobacteria,
for example of the genus Synechocystis or Anabena.
[0116] Preferred yeasts are Candida, Saccharomyces, Hansenula or
Pichia.
[0117] Preferred fungi are Aspergillus, Trichoderma, Ashbya,
Neurospora, Fusarium, Beauveria, Mortierella, Saprolegnia, Pythium,
or other fungi described in Indian Chem Engr. Section B. Vol 37, No
1, 2 (1995).
[0118] In principle, transgenic animals are also suitable as host
organisms, for example C. elegans.
[0119] As aforementioned, also preferred is the use of expression
systems and vectors which are publicly accessible or commercially
available.
[0120] The typical advantageous commercially available fusion and
expression vectors may be mentioned in this context: pGEX
[Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene
67:31-40], pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which comprises glutathion
S-transferase (GST), Maltose binding protein, or protein A, the
pTrc vectors (Amann et al., (1988) Gene 69:301-315), the "pKK233-2"
from CLONTECH, Palo Alto, Calif. and the "pET" and "pBAD" vector
series from Stratagene, La Jolla.
[0121] Further advantageous vectors for use in yeast are pYepSec1
(Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and
Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987)
Gene 54:113-123), and pYES derivatives, PGAPZ derivatives, pPICZ
derivatives, and the vectors of the "Pichia expression kit"
(Invitrogen Corporation, San Diego, Calif.). Vectors for use in
filamentous fungi are described in: van den Hondel, C. A. M. J. J.
& Punt, P. J. (1991) "Gene transfer systems and vector
development for filamentous fungi, in: Applied Molecular Genetics
of Fungi, J. F. Peberdy, et al., eds., p. 1-28, Cambridge
University Press: Cambridge.
[0122] As an alternative, insect cell expression vectors may also
be used advantageously, for example for expression in Sf 9 cells.
These are for example the vectors of the pAc series (Smith et al.
(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and
Summers (1989) Virology 170:31-39). Others which may be mentioned
are the baculovirus expression systems "MaxBac 2.0 kit" from
Invitrogen, Carlsbad, or "BacPAK baculovirus expression system"
from CLONTECH, Palo Alto, Calif.
[0123] Moreover, the fungal GTP cyclohydrolase II can be expressed
in mammalian cells. Examples of such expression vectors are pCDM8
and pMT2P, which are mentioned in: Seed, B. (1987) Nature 329:840
or Kaufman et al. (1987) EMBO J. 6:187-195). In this complex,
promoters to be used by preference are of viral origin such as, for
example, promoters of polyoma virus, adenovirus 2, cytomegalovirus
or simian virus 40. Further prokaryotic and eukaryotic expression
systems are mentioned in Chapters 16 and 17 in Sambrook et al.,
Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0124] All above-mentioned organisms transformed with at least one
of the above-mentioned expression cassettes or vectors are herein
below termed as "transgenic organism according to the
invention".
[0125] The fungal GTP cyclohydrolase II can be isolated from an
organism naturally comprising a fungal GTP cyclohydrolase II, for
example from the pathogenic fungi mentioned above and for example
from fungi selected from the group consisting of the genera and
species, e.g. Pichia such as Pichia pastoris and Pichia
methanolica, Saccharomyces such as Saccharomyces cerevisiae,
Hansenula such as Hansenula poymorpha; Trichoderma, Ashbya such as
Ashbya gossipii, Neurospora such as Neurospora crassa, Beauveria,
Mortierella, Saprolegnia, Pythium, or other fungi described in
Indian Chem Engr. Section B. Vol 37, No 1, 2 (1995).
[0126] The selection according to step iv) is based on an in vivo
assay. In a preferred embodiment this comprises the following
steps:
[0127] a) the generation of a transgenic organism according to the
invention which, following transformation with a nucleic acid
sequence encoding a fungal GTP cyclohydrolase II, is capable of
overexpressing polypeptide with GTP cyclohydrolase II activity;
[0128] b) the application, to the fungi of step a) and to an
analogous, untransformed fungi, of a candidate compound;
[0129] c) the determination of the growth, the viability or
infectivity of the transgenic and the untransformed organism
following application of the substance of step b); and
[0130] d) the selection of candidate compounds, which reduces
growth, viability or infectivity of the transgenic and the
untransformed fungi following application of the substance of step
b).
[0131] In this step (c), compounds are selected which brought about
a reduction in growth, viability or infectivity of at least 10%,
advantageously at least 20%, preferably at least 30%, especially
preferably by at least 50% and very especially preferably by at
least 70%, or a 100% reduction (blocking) being achieved.
[0132] An analogous untransformed organism is to be understood as
the fungi which has been used for generating the transgenic
organism according to the invention in step a).
[0133] Suitable organisms are the fungi defined above, preferably
those, which can be easily genetically manipulated by the skilled
artisan, e.g. Saccharomyces species, Pichia species, Fusarium
species, Ashbya species, Schizosaccharomyces species, Magnaporte
species, Ustilago species, Neurospora species and Klyveromyces
species.
[0134] When a sample comprising-an antifungal agent has been
identified by the method according to the invention, it is either
possible to isolate the substance directly from the original
samples, or else the sample can be divided into different groups,
for example when it consists of a multiplicity of different
components, in order to reduce the number of different substances
per sample and then to repeat the method according to the invention
with such a "subsample" of the original sample. Depending on the
complexity of the sample, the above-described steps can be repeated
several times, preferably until the sample identified in accordance
with the method according to the invention only encompasses a small
number of substances or only one substance. The substance
identified in accordance with the method according to the
invention, or derivatives thereof, is preferably formulated further
so that it is suitable for use in plant breeding or in plant cell
or plant tissue culture.
[0135] All of the antifungal agents identified by the
abovementioned methods can subsequently be tested for their
fungicidal action in a further in-vivo activity test. Here, the
substance in question is incubated with a culture of a pathogenic
fungus, preferably a culture of a phytopathogenic fungus,
especially preferably a culture of a filamentous phytopathogenic
fungus, it being possible to determine the fungicidal action for
example on the basis of limited growth.
[0136] The above-mentioned embodiments of the method for
identifying antifungal agents are preferably realized in a high
throughput screening. Using high throughput screening, many
discrete compounds can be tested in parallel so that large numbers
of candidate compounds can be quickly screened.
[0137] The most widely established techniques utilize 96-well,
384-well and 1536-well microtiter plates. The wells of the
microtiter plates typically require assay volumes that range from
50 to 500 .mu.l, preferably 200 .mu.l. In addition to the plates,
many instruments, materials, pipettors, robotics, plate washers,
and plate readers are commercially available to fit the respective
well format.
[0138] Alternatively, free format assays or assays that have no
physical barrier between samples, can be used as described in
Jayaickreme et al. (Proc. Natl. Acad. Sci U.S.A. 19 (1994) 161418),
Chelsky ("Strategies for Screening Combinatorial Libaries", First
Annual Conference of The Society for Biomolecular Screening in
Philadelphia, Pa. (November 710, 1995)) and Salmon et al.
(Molecular Diversity 2 (1996), 5763). Additionally, a high
throughput screening method as described in U.S. Pat. No. 5,976,813
can be used based on a porous matrix, in which test samples are
placed; one or more assay components are then placed within, on top
of, or at the bottom of a matrix such as a gel, a plastic sheet, a
filter, or other form of easily manipulated solid support. When
samples are introduced to the porous matrix they diffuse
sufficiently slowly, such that the assays can be performed without
the test samples running together.
[0139] It may be desirable for HTS to immobilize either the fungal
GTP cyclohydrolase II or the candidate compound to facilitate
separation of bound and unbound forms of one or both of the
interactants, as well as to accommodate automation of the assay.
Thus, either the fungal GTP cyclohydrolase II or the candidate
compound is preferably bound to a solid support. Suitable solid
supports include, but are not limited to, glass or plastic slides,
tissue culture plates, microtiter wells, tubes, silicon chips, or
particles such as beads (including, but not limited to, latex,
polystyrene, or glass beads). Any method known in the art can be
used to attach the fungal GTP cyclohydrolase II or candidate
compound to a solid support, including the use of covalent and
non-covalent linkages, passive absorption, or pairs of binding
moieties attached respectively to the fungal GTP cyclohydrolase II
or candidate compound and the solid support. Candidate compounds
are preferably bound to the solid support in an array, so that the
location of individual candidate compounds can be tracked. Binding
of a candidate compound to a fungal GTP cyclohydrolase II can be
accomplished in any vessel suitable for containing the reactants.
Examples of such vessels include microtiter plates, test tubes, and
microcentrifuge tubes.
[0140] All of the antifungal agents identified by the
abovementioned methods further designated as "identified compounds"
are subject matter of the present invention. Preferably, they have
a molecular weight below 1000 g/mol, preferably 500 g/mol, more
preferably 400 g/mol and most preferably 300 g/mol. The identified
compounds further exhibit a Ki value below 1 mM, preferably 1
.mu.M, more preferably 0.1 .mu.M and most preferably 0.01
.mu.M.
[0141] The identified compounds may be: expression libraries, for
example cDNA expression libraries, peptides, proteins, nucleic
acids, antibodies, small organic substances, hormones, PNA(s) or
the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell. 83
(1995), 237-245; Gibbs, Cell. 79 (1994), 193-198 and references
cited therein). They may be chemically synthesized substances or
substances produced by microorganisms and can be present for
example in cell extracts or, for example, plants, animals or
microorganisms. The reaction mixture can be a cell-free extract or
comprise a cell or cell culture. Suitable methods are known to the
skilled worker and are described generally for example in Alberts,
Molecular Biology the cell, 3.sup.rd Edition (1994), for example
Chapter 17. For example, the substances mentioned can be added to
the reaction mixture or the culture medium or injected into the
cells or sprayed onto a plant.
[0142] The identified compounds may also be present in the form of
their agriculturally useful salts. Suitable salts among
agriculturally useful salts are mainly the salts of those cations
or the acid addition salts of those acids whose cations, or anions,
respectively, do not adversely affect the fungicidal action of the
identified compound.
[0143] All of the identified compounds--if they comprise
asymmetrically substituted .alpha.-carbon atoms--exist either as
racemates, enantiomer mixtures or as pure enantiomers and--if they
have chiral substituents--may also exist as diastereomer mixtures.
They are suitable for controlling the pathogenic fungi mentioned at
the outset.
[0144] The invention therefore furthermore relates to processes for
the preparation of the fungicidal composition, which comprises
[0145] a) selection of an identified compound; and
[0146] b) formulating the identified compound, or an agriculturally
useful salt of the identified compound identified via (a), with
suitable adjuvants.
[0147] The identified compounds according to the invention in step
a) can be formulated for example in the form of directly sprayable
aqueous solutions, powders, suspensions, also highly concentrated
aqueous, oily or other suspensions or suspoemulsions or
dispersions, emulsions, oil dispersions, pastes, dusts,
compositions for spreading, or granules, and applied by spraying,
atomizing, dusting, spreading or pouring. The use forms depend on
the intended purposes and the nature of the identified compound
used; in any case, they should ensure the finest possible
distribution of the identified compounds according to the
invention.
[0148] For the preparation of emulsions, pastes or aqueous or
oil-containing dispersions, the identified compounds as such can be
dissolved or dispersed in an oil or solvent, it being possible to
add further formulation auxiliaries for homogenization. However, it
is also possible to prepare liquid or solid concentrates which are
composed of identified compound and, if appropriate, solvent or oil
and optionally further auxiliaries, and these concentrates are
suitable for dilution with water. Materials which may be mentioned
in this context are emulsion concentrates (EC, EW), suspensions
(SC), soluble concentrates (SL), pastes, pellets, wettable powders
or granules, it being possible for the solid formulations to be
either soluble or dispersible (wettable) in water. Moreover, such
powders or granules or tablets may additionally be provided with a
solid coating which prevents abrasion or an unduly early release of
the identified compound.
[0149] The term auxiliaries is understood as meaning, in principle,
the following classes of substances: antifoams, thickeners,
wetters, stickers, dispersants or emulsifiers, bactericides and
thixotropic agents. The skilled worked is familiar with the meaning
of the abovementioned agents.
[0150] SLs, EWs and ECs can be prepared by simply mixing the
constituents in question; powders can be prepared via mixing or
grinding in specific types of mills (for example hammer mills). SCs
and SEs are usually prepared by wet milling, it being possible to
prepare an SE from an SC by adding an organic phase comprising
further auxiliaries or identified compounds. The preparation is
known. Granules, for example coating granules, impregnated granules
and homogeneous granules, can be prepared by binding the identified
compounds to solid carriers. The skilled worker is familiar with a
multiplicity of solid carriers which are suitable for granules
according to the invention, for example mineral earths such as
silicas, silica gels, silicates, talc, kaolin, limestone, lime,
chalk, bole, loess, clay, dolomite, diatomaceous earth, calcium
sulfate, magnesium sulfate, magnesium oxide, ground synthetic
materials, fertilizers such as ammonium sulfate, ammonium
phosphate, ammonium nitrate, ureas, and products of vegetable
origin such as cereal meal, tree bark meal, wood meal and nutshell
meal, cellulose powders or other solid carriers. The skilled worker
is familiar with details of the preparation; they are stated, for
example, in the following publications: U.S. Pat. No. 3,060,084,
EP-A 707445 (for liquid concentrates), Browning, "Agglomeration",
Chemical Engineering, Dec. 4, 1967, 147-48, Perry's Chemical
Engineer's Handbook, 4th Ed., McGraw-Hill, New York, 1963, pages
8-57 and ff. WO 91/13546, U.S. Pat. No. 4,172,714, U.S. Pat. No.
4,144,050, U.S. Pat. No. 3,920,442, U.S. Pat. No. 5,180,587, U.S.
Pat. No. 5,232,701, U.S. Pat. No. 5,208,030, GB 2,095,558, U.S.
Pat. No. 3,299,566, Klingman, Weed Control as a Science, John Wiley
and Sons, Inc., New York, 1961, Hance et al., Weed Control
Handbook, 8th Ed., Blackwell Scientific Publications, Oxford, 1989,
and Mollet, H., Grubemann, A., Formulation technology, Wiley VCH
Verlag GmbH, Weinheim (Federal Republic of Germany), 2001.
[0151] The skilled worker is familiar with a multiplicity of inert
liquid and/or solid carriers which are suitable for the
formulations according to the invention, such as, for example,
liquid additives such as mineral oil fractions of medium to high
boiling point, such as kerosene or diesel oil, furthermore coal tar
oils and oils of vegetable or animal origin, aliphatic, cyclic and
aromatic hydrocarbons, for example paraffin, tetrahydronaphthalene,
alkylated naphthalenes or their derivatives, alkylated benzenes or
their derivatives, alcohols such as methanol, ethanol, propanol,
butanol, cyclohexanol, ketones such as cyclohexanone, or strongly
polar solvents, for example amines such as N-methylpyrrolidone, or
water.
[0152] The skilled worker is familiar with a multiplicity of
surface-active substances (surfactants) which are suitable for the
formulations according to the invention, such as, for example, the
alkali, alkaline earth or ammonium salts of aromatic sulfonic
acids, for example lignin sulfonic acid, phenol sulfonic acid,
naphthalene sulfonic acid and dibutylnaphthalenesulfonic acid, and
of fatty acids, alkyl sulfonates, alkylaryl sulfonates, alkyl
sulfates, lauryl ether sulfates and fatty alcohol sulfates, and
salts of sulfated hexadecanols, heptadecanols and octadecanols, and
of fatty alcohol glycol ethers; condensates of sulfonated
naphthalene and its derivatives with formaldehyde, condensates of
naphthalene or of the naphthalene sulfonic acids with phenol and
formaldehyde, polyoxyethylene octylphenol ether, ethoxylated
isooctylphenol, octylphenol or nonylphenol, alkylphenyl or
tributylphenyl polyglycol ether, alkylaryl polyether alcohols,
isotridecyl alcohol, fatty alcohol/ethylene oxide condensates,
ethoxylated castor oil, polyoxyethylene alkyl ethers or
polyoxypropylenealkyl ethers, lauryl alcohol polyglycol ether
acetate, sorbitol esters, lignin-sulfite waste liquors or
methylcellulose.
[0153] Powders, dusts and materials for spreading, being solid
carriers, can be prepared advantageously by mixing or concomitantly
grinding the active substances with a solid carrier.
[0154] The concentrations of the identified compounds in the
ready-to-use preparations can be varied within wide limits and
depend on the nature of the formulation in question.
[0155] The fungicidal compositions, or the identified compounds,
can be applied in curative.
[0156] The applications of identified compounds (=substances and/or
compositions) amount to from 0.001 to 3.0, preferably 0.01 to 1.0
kg/ha active substance, depending on the aim of the control
measures, the season, the target plants and the stage of
growth.
[0157] The present invention furthermore relates to a method of
controlling harmful fungi, which comprises treating the fungi or
the materials, plants, soils or seeds to be protected from fungal
infection, with an effective amount of an antifungal agent
according to the invention or of a fungicidal composition according
to the invention. Harmful fungi are understood as meaning the
pathogenic fungi mentioned at the outset.
[0158] Another object of the present invention is the use of the
identified compounds for preparation of drugs, preferably
pharmaceutical compositions comprising at least an identified
compound. The identified compounds according to the invention can
be administered orally or parenterally (subcutaneously,
intravenously, intramuscularly and intraperitoneally) in a
conventional way. Administration may also take place with vapors or
sprays through the nasopharyngeal space.
[0159] The dosage depends on the age, condition and weight of the
patient and on the mode of administration. As a rule, the daily
dose of active substance is about 0.5-50 mg/kg of bodyweight on
oral administration and about 0.1-10 mg/kg of bodyweight on
parenteral administration.
[0160] The identified compounds can be used in conventional solid
or liquid pharmaceutical forms, eg. as uncoated or (film-) coated
tablets, capsules, powders, granules, suppositories, solutions,
ointments, creams or sprays. These are produced in a conventional
way. For this purpose, the active substances can be processed with
conventional pharmaceutical excipients such as tablet binders,
bulking agents, preservatives, tablet disintegrants, flow
regulators, plasticizers, wetting agents, dispersants, emulsifiers,
solvents, release-slowing agents, antioxidants and/or propellant
gases (cf. H. Sucker et al.: Pharmazeutische Technologie,
Thieme-Verlag, Stuttgart, 1991). The administration forms obtained
in this way normally contain from 0.1 to 90% by weight of active
substance.
[0161] The invention is now illustrated by the examples which
follow, but are not limited thereto.
[0162] The recombinant methods on which the exemplary embodiments
which follow are based are now described briefly:
[0163] A: General Methods
[0164] Cloning methods such as, for example, restriction cleavages,
DNA isolation, agarose gel electrophoresis, purification of DNA
fragments, transfer of nucleic acids to nitrocellulose and nylon
membranes, linking of DNA fragments, transformation of E. coli
cells, bacterial cultures, sequence analysis of recombinant DNA and
Southern and Western Blots were carried out as described by
Sambrook et al., Cold Spring Harbor Laboratory Press (1989) and
Ausubel, F. M. et al., Current Protocols in Molecular Biology,
Greene Publishing Assoc. and Wiley-Interscience (1994); ISBN
0-87969-309-6.
[0165] The bacterial strains used hereinbelow (E. coli XL1-blue)
were obtained from BRL Gibco or Invitrogen, Carlsberg, Calif. The
Ashbya gossypii wild-type strain has the ATTC number ATTC
10895.
[0166] B: Sequence Analysis of Recombinant DNA
[0167] Recombinant DNA molecules were sequenced using an ABI laser
fluorescence DNA sequencer following the method of Sanger (Sanger
et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467(1977)). Fragments
resulting from a polymerase chain reaction were sequenced and
verified in order to avoid polymerase errors in constructs to be
expressed.
[0168] C: Materials Used
[0169] Unless otherwise specified in the text, all of the chemicals
used were obtained in analytical grade quality from Fluka
(Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg)
and Sigma (Deisenhofen). Solutions were prepared using pure
pyrogen-free water, referred to in the following text as H.sub.2O,
from a Milli-Q water system purification unit (Millipore,
Eschborn). Restriction enzymes, DNA-modifying enzymes and
molecular-biological kits were obtained from AGS (Heidelberg),
Amersham (Brunswick), Biometra (Gottingen), Roche (Mannheim),
Genomed (Bad Oeynnhausen), New England Biolabs (Schwalbach/Taunus),
Novagen (Madison, Wis., USA), Perkin-Elmer (Weiterstadt), Pharmacia
(Freiburg), Qiagen (Hilden) and Stratagene (Heidelberg). Unless
otherwise specified, they were used following the manufacturer's
instructions.
[0170] All of the media and buffers used for the genetic
engineering experiments were sterilized either by filter
sterilization or by heating in the autoclave.
EXAMPLE 1
A) Preparation of the Knock-Out Plasmid
[0171] The KO plasmid pDeltarib1G418 was obtained from pJR765
(WO95/26406) by inserting the G418R expression cassette (Degryse et
al., Yeast 1995, 11(7):629-40) into pJR765 so that the GTP
cyclohydrolase II gene (rib 1) that is set forth in SEQ ID NO:3 is
deleted from 220 bp upstream the ATG codon to 739 bp downstream the
ATG codon. 6 mg plasmid DNA of pDeltarib1G418 were linearized with
the restriction enzyme Asp 700 and purified by classical gel
elution for subsequent transformation.
B) Transformation of Asbya goosypii
[0172] A. goosypii spores were cultured for 12 h in MA2 medium
(peptone 10 g/l, yeast extract 1 g/l, myoinositol 0.3 g/l, pH7) at
28.degree. C. and 250 rpm. The cells were pelleted by classical
centrifugation and suspended with phosphate buffer 50 mM, DTT 25 mM
and incubated at 28.degree. C. with low agitation for 30 min. The
cells were collected by centrifugation and suspended with 25 ml
cold phosphate buffer 50 mM pH 7.5; 150 .mu.l of the cell
suspension were mixed with 6 .mu.g pDeltarib1G418 treated with Asp
700. The mixture was electroporated with a Gene Pulser II
electroporator (Bio-Rad; parameters: 200 ohms; 1.5 Kv; 25
.mu.F)
[0173] Immediately after the electric pulse, the cells were mixed
with 1 ml MA2 medium and spread on fresh Petri dishes containing
MA2-agar supplemented with 200 mg vitamin b2. The plates were
incubated at 28.degree. C. for 6 h. Then, a fine layer of Top-agar
(1% LMP agarose) containing G418 antibiotic 50 mg/ml was loaded at
the top of the plates. The incubation was further conducted at
28.degree. C. for 5 days. Several colonies capable of growth on
selective medium were isolated for subsequent characterization.
C) Characterization of the KO Mutants
[0174] The KO mutants were grown on Petri dishes containing MA2
agar, G418 50 mg/ml and vitamin b2 200 mg/ml and then replicated on
the same medium in the absence of vitamin b2. In the latter case,
the KO mutants were not able to grow at all. This convincingly
demonstrates that GTP cyclohydrolase II is essential for the life
of the fungi.
EXAMPLE 2
[0175] cl) Enzyme Preparation
[0176] Ashbya gossypii cells may be obtained after 2 days from a
culture in 3% (w/v) malt extract+0.3% (w/v) difco-soyton, pH 5.6 at
28.degree. C. A cell-free extract can be obtained by mechanical
breakage of the cells with a kitchen blender in 50 mM imidazole
buffer pH 7 containing 1 mM EDTA-Na salt, 5 mM MgSO4, 10 mM KCl, 5
mM dithiothreitol and 30% (v/v) glycerol and separation of unbroken
cells and debris by centrifugation.
B) Activity Assay
[0177] The assay is performed in a suitable buffer e.g. Tris/HCl,
pH 7.8 including 1 mM Mg.Cl.sub.2, 1 mM DTT, 0.5 mM NAD, 2 units/ml
formate dehydrogenase and 2.5 mM GTP Li salt. After the addition of
the GTP cyclohydrolase comprising cell free extracts of step A),
the reaction was monitored by measuring the absorption at 340
nm.
[0178] After dissolving the respective candidate compounds in a
suitable solvent e.g. DMSO, an aliquot of the afore made solution
is added to the reaction mixture. The enzyme activity of this
sample is compared with the activity of a control comprising the
pure solvent instead of the candidate compound. The resulting
relative activity was calculated as percent inhibition in relation
to the sample without the candidate compound.
[0179] In FIG. 1, the GTP dependant formation of NADH in the
reaction mixture and inhibition by
3-Butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]-
quinoline-2,4-dione is shown.
[0180] To distinguish between the inhibition of GTP cyclohydrolase
and formate dehydrogenase in the above-mentioned assay, the
subtrate GTP is replaced with formic acid. An inhibitor of GTP
cyclohydrolase shows inhibition in the presence of GTP and no
inhibition in the presence of formic acid (see e.g. FIG. 2).
C) Determination of fungicidal activity of
3-butyl-10-(3-chlorophenyl)-10H-
-pyrimido[4,5-b]quinoline-2,4-dione
[0181] A stock solution of
3-butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]-
quinoline-2,4-dione is prepared in DMSO at a concentration of
10,000 ppm a.i. For the test, this is diluted with sterile
deionized water to a concentration of 125 ppm; the DMSO
concentration is constant at all test concentrations.
[0182] Spore suspensions of the fungi employed in the test
(Phytophthora infestans, Pyricularia oryzae and Septoria tritici)
are made in double strength growth medium (4% (w/v) malt extract in
water).
[0183] For each fungal species, 3 (three) wells are prepared: 50
.mu.l of compound solution are pipetted to each well, were to this
are added 50 .mu.l of spore suspension. A water/DMSO blank serves
as the 100% growth control (=0 ppm); a well without added fungal
inoculum but with the growth medium serves as medium blank.
[0184] The prepared plates are then incubated at 18.degree. C. for
7 days after which the density of developed fungal mycelium is
measured in a photometer at a wavelength of 405 nm.
[0185] After subtracting the medium blank readings and setting, the
100% growth control reading, the measurements from the
concentration series are converted into "% growth compared with the
0 ppm control". The data set forth in table 1 clearly show that
fungal growth was significantly inhibited by
3-butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]quinoline-2,4-
-dione.
2TABLE 1 Pyricularia oryzae*) Phytophthora infestans*) Septoria
tritici*) 47.6% 39.9% 0% *)[% growth compared with the 0 ppm
control]
BRIEF DESCRIPTION OF FIGURES
[0186] FIG. 1 shows the GTP dependent formation of NADH in the
reaction mixture and inhibition by
3-butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]-
quinoline-2,4-dione. In this figure, .diamond-solid. designates
values measured in the presence of GTP, .box-solid. values measured
without GTP and .tangle-solidup. values measured in the presence of
GTP and
3-butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]quinoline-2,4-dione.
[0187] FIG. 2 shows the specificity of the inhibition by
3-butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]quinoline-2,4-dione.
No inhibition of formate dehydrogenase alone is observed.
Therefore, the inhibition of the GTP dependent NADH formation is a
consequence of the inhibition of GTP cyclohydrolase. In this
figure, .diamond-solid. designates values measured in the presence
of formate, *.box-solid. values measured without formate and values
measured in the presence of formate and
3-butyl-10-(3-chlorophenyl)-10H-pyrimido[4,5-b]quinoline-2,4--
dione.
Sequence CWU 1
1
5 1 903 DNA Ashbya gossipii CDS (1)..(903) 1 atg act gaa tac aca
gtg cca gaa gtg acc tgt gtc gca cgc gcg cgc 48 Met Thr Glu Tyr Thr
Val Pro Glu Val Thr Cys Val Ala Arg Ala Arg 1 5 10 15 ata ccg acg
gta cag ggc acc gat gtc ttc ctc cat cta tac cac aac 96 Ile Pro Thr
Val Gln Gly Thr Asp Val Phe Leu His Leu Tyr His Asn 20 25 30 tcg
atc gac agc aag gaa cac cta gcg att gtc ttc ggc gag aac ata 144 Ser
Ile Asp Ser Lys Glu His Leu Ala Ile Val Phe Gly Glu Asn Ile 35 40
45 cgc tcg cgg agt ctg ttc cgg tac cgg aaa gac gac acg cag cag gcg
192 Arg Ser Arg Ser Leu Phe Arg Tyr Arg Lys Asp Asp Thr Gln Gln Ala
50 55 60 cgg atg gtg cgg ggc gcc tac gtg ggc cag ctg tac ccc ggg
cgg acc 240 Arg Met Val Arg Gly Ala Tyr Val Gly Gln Leu Tyr Pro Gly
Arg Thr 65 70 75 80 gag gca gac gcg gat cgg cgt cag ggc ctg gag ctg
cgg ttt gat gag 288 Glu Ala Asp Ala Asp Arg Arg Gln Gly Leu Glu Leu
Arg Phe Asp Glu 85 90 95 aca ggg cag ctg gtg gtg gag cgg gcg acg
acg tgg acc agg gag ccg 336 Thr Gly Gln Leu Val Val Glu Arg Ala Thr
Thr Trp Thr Arg Glu Pro 100 105 110 aca ctg gtg cgg ctg cac tcg gag
tgt tac acg ggc gag acg gcg tgg 384 Thr Leu Val Arg Leu His Ser Glu
Cys Tyr Thr Gly Glu Thr Ala Trp 115 120 125 agc gcg cgg tgc gac tgc
ggg gag cag ttc gac cag gcg ggt aag ctg 432 Ser Ala Arg Cys Asp Cys
Gly Glu Gln Phe Asp Gln Ala Gly Lys Leu 130 135 140 atg gct gcg gcg
aca gag ggc gag gtg gtt ggc ggt gcg ggg cac ggc 480 Met Ala Ala Ala
Thr Glu Gly Glu Val Val Gly Gly Ala Gly His Gly 145 150 155 160 gtg
atc gtg tac ctg cgg cag gag ggc cgc ggc atc ggg cta ggc gag 528 Val
Ile Val Tyr Leu Arg Gln Glu Gly Arg Gly Ile Gly Leu Gly Glu 165 170
175 aag ctg aag gcg tac aac ctg cag gac ctg ggc gcg gac acg gtg cag
576 Lys Leu Lys Ala Tyr Asn Leu Gln Asp Leu Gly Ala Asp Thr Val Gln
180 185 190 gcg aac gag ctg ctc aac cac cct gcg gac gcg cgc gac ttc
tcg ttg 624 Ala Asn Glu Leu Leu Asn His Pro Ala Asp Ala Arg Asp Phe
Ser Leu 195 200 205 ggg cgc gca atc cta ctg gac ctc ggt atc gag gac
atc cgg ttg ctc 672 Gly Arg Ala Ile Leu Leu Asp Leu Gly Ile Glu Asp
Ile Arg Leu Leu 210 215 220 acg aat aac ccc gac aag gtg cag cag gtg
cac tgt ccg ccg gcg cta 720 Thr Asn Asn Pro Asp Lys Val Gln Gln Val
His Cys Pro Pro Ala Leu 225 230 235 240 cgc tgc atc gag cgg gtg ccc
atg gtg ccg ctt tca tgg act cag ccc 768 Arg Cys Ile Glu Arg Val Pro
Met Val Pro Leu Ser Trp Thr Gln Pro 245 250 255 aca cag ggc gtg cgc
tcg cgc gag ctg gac ggc tac ctg cgc gcc aag 816 Thr Gln Gly Val Arg
Ser Arg Glu Leu Asp Gly Tyr Leu Arg Ala Lys 260 265 270 gtc gag cgc
atg ggg cac atg ctg cag cgg ccg ctg gtg ctg cac acg 864 Val Glu Arg
Met Gly His Met Leu Gln Arg Pro Leu Val Leu His Thr 275 280 285 tct
gcg gcg gcc gag ctc ccc cgc gcc aac aca cac ata 903 Ser Ala Ala Ala
Glu Leu Pro Arg Ala Asn Thr His Ile 290 295 300 2 301 PRT Ashbya
gossipii 2 Met Thr Glu Tyr Thr Val Pro Glu Val Thr Cys Val Ala Arg
Ala Arg 1 5 10 15 Ile Pro Thr Val Gln Gly Thr Asp Val Phe Leu His
Leu Tyr His Asn 20 25 30 Ser Ile Asp Ser Lys Glu His Leu Ala Ile
Val Phe Gly Glu Asn Ile 35 40 45 Arg Ser Arg Ser Leu Phe Arg Tyr
Arg Lys Asp Asp Thr Gln Gln Ala 50 55 60 Arg Met Val Arg Gly Ala
Tyr Val Gly Gln Leu Tyr Pro Gly Arg Thr 65 70 75 80 Glu Ala Asp Ala
Asp Arg Arg Gln Gly Leu Glu Leu Arg Phe Asp Glu 85 90 95 Thr Gly
Gln Leu Val Val Glu Arg Ala Thr Thr Trp Thr Arg Glu Pro 100 105 110
Thr Leu Val Arg Leu His Ser Glu Cys Tyr Thr Gly Glu Thr Ala Trp 115
120 125 Ser Ala Arg Cys Asp Cys Gly Glu Gln Phe Asp Gln Ala Gly Lys
Leu 130 135 140 Met Ala Ala Ala Thr Glu Gly Glu Val Val Gly Gly Ala
Gly His Gly 145 150 155 160 Val Ile Val Tyr Leu Arg Gln Glu Gly Arg
Gly Ile Gly Leu Gly Glu 165 170 175 Lys Leu Lys Ala Tyr Asn Leu Gln
Asp Leu Gly Ala Asp Thr Val Gln 180 185 190 Ala Asn Glu Leu Leu Asn
His Pro Ala Asp Ala Arg Asp Phe Ser Leu 195 200 205 Gly Arg Ala Ile
Leu Leu Asp Leu Gly Ile Glu Asp Ile Arg Leu Leu 210 215 220 Thr Asn
Asn Pro Asp Lys Val Gln Gln Val His Cys Pro Pro Ala Leu 225 230 235
240 Arg Cys Ile Glu Arg Val Pro Met Val Pro Leu Ser Trp Thr Gln Pro
245 250 255 Thr Gln Gly Val Arg Ser Arg Glu Leu Asp Gly Tyr Leu Arg
Ala Lys 260 265 270 Val Glu Arg Met Gly His Met Leu Gln Arg Pro Leu
Val Leu His Thr 275 280 285 Ser Ala Ala Ala Glu Leu Pro Arg Ala Asn
Thr His Ile 290 295 300 3 2528 DNA Ashbya gossipii 3 atccgccgca
caagggacgc catgctgctc acctccggcg agtcctcgcg ttgtcccgta 60
atgtccacat ccaccacgat cagctccgac gtcaccgtgt ggtccaccac cttgctcttg
120 acgctcacca gcgcctcgct cccgtcgctg gtaattatcc gcgcagaccc
gtttgagtta 180 ggtaagaaat caaccgccac atccaagggg cggaactgcg
ctgccgcccg cccgtctgcg 240 cgaatcggtg gtatcgcctt cagtgaatca
atcagataca gctgctcggt cactgatagc 300 atcatggcta atttctgtcc
gcatacttca tatgctcatc gcacattgat aatgtacatt 360 cgaaaaattt
caagattagc ctccgtgaac agcgatttac cttaggcaaa agtaacaaaa 420
ggcttttccg taggtgcttt gtcattcaac aatccacgtc ggaattggcg actatatagt
480 gtagggccca taaagcagta gtcggtgttg atagctgtgt cagaccaact
ctttgttaat 540 tactgaagct gatatgactg aatacacagt gccagaagtg
acctgtgtcg cacgcgcgcg 600 cataccgacg gtacagggca ccgatgtctt
cctccatcta taccacaact cgatcgacag 660 caaggaacac ctagcgattg
tcttcggcga gaacatacgc tcgcggagtc tgttccggta 720 ccggaaagac
gacacgcagc aggcgcggat ggtgcggggc gcctacgtgg gccagctgta 780
ccccgggcgg accgaggcag acgcggatcg gcgtcagggc ctggagctgc ggtttgatga
840 gacagggcag ctggtggtgg agcgggcgac gacgtggacc agggagccga
cactggtgcg 900 gctgcactcg gagtgttaca cgggcgagac ggcgtggagc
gcgcggtgcg actgcgggga 960 gcagttcgac caggcgggta agctgatggc
tgcggcgaca gagggcgagg tggttggcgg 1020 tgcggggcac ggcgtgatcg
tgtacctgcg gcaggagggc cgcggcatcg ggctaggcga 1080 gaagctgaag
gcgtacaacc tgcaggacct gggcgcggac acggtgcagg cgaacgagct 1140
gctcaaccac cctgcggacg cgcgcgactt ctcgttgggg cgcgcaatcc tactggacct
1200 cggtatcgag gacatccggt tgctcacgaa taaccccgac aaggtgcagc
aggtgcactg 1260 tccgccggcg ctacgctgca tcgagcgggt gcccatggtg
ccgctttcat ggactcagcc 1320 cacacagggc gtgcgctcgc gcgagctgga
cggctacctg cgcgccaagg tcgagcgcat 1380 ggggcacatg ctgcagcggc
cgctggtgct gcacacgtct gcggcggccg agctcccccg 1440 cgccaacaca
cacatataat ctttgctata ttaaaactct ataaacgtat gccacacggc 1500
gcccgcgggc tgccacacgc tgctcacggg ctgccgaaca gttctaacaa gtaatcgcgc
1560 gcctcgccag tgatcgtggc gagcaccttg tcgtccatca tcacatatcc
tcggctacag 1620 tcgtcgttga agagcgtcga cgtgcgcttc gacttgtgcg
atttaaggaa gtcgttgtat 1680 ccgttgaccg tggttagctc gaccggcgcg
ctaacgagaa acgatcctgt ggaacccgtg 1740 gactcggacg actggaattg
cgattggttc ttaagcttgt atagggtctg catcttctgt 1800 gttcagcttg
gggatcgcgg acggttttgt cacccacggt ctagtagtcg catttatata 1860
ctagcgtact agccgcccct agctggtccc gggaggggga gcgtcgccat cggttacggg
1920 tcacgtggtt ttggtcgaag gcaatcgaag cgtcagggga gattctatgt
gatgtctggg 1980 tatttgtacg gctgacgcac gtgactggcg gcataagtgt
cagcacgcca gacgtgacgc 2040 gagccgcacg agccgtgcgg cactgactgc
tgcgattggc gcgcatctca accacggatg 2100 aggggtccgc ttatggtcat
gagcttagta aacttctgat tatattaaag aatcatactc 2160 atataacatt
caacgatata tcattctatt taaccactca agaataaacc tctaagtata 2220
ttacagaggt catatacata ttagattata caacagatta gtgtatttct tatctcacgt
2280 ataaacaaat aagtagattg gaggattcat atcagatatt aatgtaagac
tcatattaaa 2340 ttcttagttc cttacaagtt taaacttcta agtatattga
agaggtcata cttgaattaa 2400 actatacgat agatgatact cttttctttc
tcttcgatta tattaaatga ttagtatatt 2460 atatgccatt tgataaaatg
attgatatat tacaatatta tctttaatat tttaataata 2520 acaaatat 2528 4
582 DNA Fusarium graminearum CDS (1)..(582) 4 act ctc ccg gag gtg
gaa tgc atc gtt cgt gcc cgt atc ccc acg gtt 48 Thr Leu Pro Glu Val
Glu Cys Ile Val Arg Ala Arg Ile Pro Thr Val 1 5 10 15 gca gga acc
gag atg ttc ttg cac ctg tac acc aac aat gtg gac aac 96 Ala Gly Thr
Glu Met Phe Leu His Leu Tyr Thr Asn Asn Val Asp Asn 20 25 30 aag
gag cac ctc gcc atc gtg ttt ggc aaa aat atc cga agc aag agt 144 Lys
Glu His Leu Ala Ile Val Phe Gly Lys Asn Ile Arg Ser Lys Ser 35 40
45 cta gat gct gtc cgg gag ggt gag acc gag atg gac cgc atg gtg cgc
192 Leu Asp Ala Val Arg Glu Gly Glu Thr Glu Met Asp Arg Met Val Arg
50 55 60 ggc gca tac aca gga agg ctg ttc ccc ggt cgc aca acc agt
ggc atc 240 Gly Ala Tyr Thr Gly Arg Leu Phe Pro Gly Arg Thr Thr Ser
Gly Ile 65 70 75 80 ggt cca gcg acc cct cag gag gaa cag cca ccg cag
ccg tcg gat gag 288 Gly Pro Ala Thr Pro Gln Glu Glu Gln Pro Pro Gln
Pro Ser Asp Glu 85 90 95 cct cct ctg gtg agg att cat tcc gag tgc
tac aca ggt gag acg gcg 336 Pro Pro Leu Val Arg Ile His Ser Glu Cys
Tyr Thr Gly Glu Thr Ala 100 105 110 tgg tca gcg cga tgc gac tgc ggc
gag cag ctc gat gaa gca gcg cgc 384 Trp Ser Ala Arg Cys Asp Cys Gly
Glu Gln Leu Asp Glu Ala Ala Arg 115 120 125 ctg atg agt ctg cca ggc
aac aag gcc ggc ggc atc atc atc tac ctg 432 Leu Met Ser Leu Pro Gly
Asn Lys Ala Gly Gly Ile Ile Ile Tyr Leu 130 135 140 cga caa gag ggt
cgt ggt atc ggt ctg gga gag aag ctc aag gcg tac 480 Arg Gln Glu Gly
Arg Gly Ile Gly Leu Gly Glu Lys Leu Lys Ala Tyr 145 150 155 160 aat
ctt cag gat ctg ggg tct gat act gtc gag gcg aat ttg ctt ttg 528 Asn
Leu Gln Asp Leu Gly Ser Asp Thr Val Glu Ala Asn Leu Leu Leu 165 170
175 cgc cat cct gcc gat gct cga agc tac ggt ctt gct acc gct atg ctg
576 Arg His Pro Ala Asp Ala Arg Ser Tyr Gly Leu Ala Thr Ala Met Leu
180 185 190 ctg gat 582 Leu Asp 5 194 PRT Fusarium graminearum 5
Thr Leu Pro Glu Val Glu Cys Ile Val Arg Ala Arg Ile Pro Thr Val 1 5
10 15 Ala Gly Thr Glu Met Phe Leu His Leu Tyr Thr Asn Asn Val Asp
Asn 20 25 30 Lys Glu His Leu Ala Ile Val Phe Gly Lys Asn Ile Arg
Ser Lys Ser 35 40 45 Leu Asp Ala Val Arg Glu Gly Glu Thr Glu Met
Asp Arg Met Val Arg 50 55 60 Gly Ala Tyr Thr Gly Arg Leu Phe Pro
Gly Arg Thr Thr Ser Gly Ile 65 70 75 80 Gly Pro Ala Thr Pro Gln Glu
Glu Gln Pro Pro Gln Pro Ser Asp Glu 85 90 95 Pro Pro Leu Val Arg
Ile His Ser Glu Cys Tyr Thr Gly Glu Thr Ala 100 105 110 Trp Ser Ala
Arg Cys Asp Cys Gly Glu Gln Leu Asp Glu Ala Ala Arg 115 120 125 Leu
Met Ser Leu Pro Gly Asn Lys Ala Gly Gly Ile Ile Ile Tyr Leu 130 135
140 Arg Gln Glu Gly Arg Gly Ile Gly Leu Gly Glu Lys Leu Lys Ala Tyr
145 150 155 160 Asn Leu Gln Asp Leu Gly Ser Asp Thr Val Glu Ala Asn
Leu Leu Leu 165 170 175 Arg His Pro Ala Asp Ala Arg Ser Tyr Gly Leu
Ala Thr Ala Met Leu 180 185 190 Leu Asp
* * * * *
References