WO2004059000A2 - High-throughput screening method for identifying active substances by means of co-cultivation - Google Patents

Abstract

The invention relates to a high-throughput screening method for identifying active substances.

Description

High-throughput suitable screening process for the identification of active substances

The present invention relates to a high-throughput suitable screening method for identifying active substances.

Almost all classic antibiotics were obtained by optimizing substances that showed an antimicrobial effect in whole-cell screening. However, the methods have the disadvantage that they are insensitive and growth inhibitors found often have a cytotoxic effect on humans.

Furthermore, the place of action of found substances is mostly unknown, which makes rational optimization of the antimicrobial substances difficult. It is therefore not surprising that this drug discovery strategy has led to only a few new drugs in recent years.

For this reason, pharmaceutical research has focused on target-oriented technology in the past few years, in which targeted disruption or growth inhibition of the microorganism is achieved by disrupting essential metabolic pathways or structures (“targets”, “targets”).

In target-oriented approaches, substances are searched that specifically inhibit a biochemical reaction or intermolecular interaction. The advantage of such target-oriented approaches is that completely new targets for antibiotics can be determined that have not yet been attacked by antimicrobial substances.

New destinations and mechanisms of action for active substances are particularly necessary because no resistances have yet been developed for them. This has already happened for almost all common antimicrobial substances and they have lost their effectiveness. For example, Candida albicans strains that are caused by changes or overexpression of the site of action or Overexpression of Efflux pumps are resistant to the azole substance class, which is not uncommon.

The raw material for in silico drug target identification represents the sequence information of the genomes in connection with the functional analysis of the discovered genes. As of the filing date of the present invention, more than 60 microbial genomes are completely sequenced and generally accessible.

Selection procedures for drug targets place at least two requirements on a suitable destination. On the one hand, the gene function should be essential for the organism, from which it can be concluded that the inhibition of the associated gene product (protein) by active substances also has a toxic effect on the microorganism.

On the other hand, in order that substances with a broad spectrum of activity against harmful organisms can be found, it is important that the target site (target protein) is as conserved as possible among the harmful organisms (e.g. under bacteria and / or fungi), i.e. that the protein sequences of the target organisms are very similar.

Previous selection procedures only take into account targets for which there is no homologous gene or protein in humans. This increases the probability that an active ingredient found has no negative effects on human cells and is therefore low in side effects. At the same time, this condition drastically restricts the selection of the available targets, especially when looking for new antifungal substances.

This is due to the fact that fungi, like humans, are eukaryotes in which a great number of cellular processes are preserved. In particular, the essential genes of the fungi largely code for basic proteins that are important for cellular processes, to which there are homologous proteins in all eukaryotes and thus also in humans. It could be shown through the work of the applicant that about 80% of all essential S. cerevisiae Proteins have a human homologue and are therefore not suitable as targets according to the above criteria.

The efficiency of screening systems essentially depends on their specificity, i.e. Suitable parameters must be found that minimize the number of false positive results.

In the case of screening systems using the in vitro method (cell-free targeting approaches), the binding of effectors to purified proteins is usually measured. Large substance libraries are used to find interesting lead structures using the high-throughput process (HTS). The functional failure of the target serves as the measurement signal, which usually requires catalytic activity. On the one hand, this severely limits the number of suitable target proteins and, on the other hand, the physiological function of the target protein must already be known.

The advantage of destination-based in vitro approaches is that completely new destinations for antibiotics can be determined that have not yet been attacked by antimicrobial substances. They are more sensitive than whole-cell screenings and weakly inhibiting substances are also found, which can then be rationally optimized since the site of action is known.

A disadvantage of cell-free / biochemical test systems for the identification of new inhibiting substances is that often a very large amount of possible hits ("hits") are detected, which have to be verified in subsequent tests, many of which prove to be false positive. At this point in time, no information is available for the substances found as to whether the enzyme inhibitor found also has an effect on microorganisms in vivo.

So the substances first have to get to their places of action, that is, as a rule, into the cell interior. The cell wall and the cytoplasmic membrane represent selective barriers that not all substances can penetrate in sufficient quantities. Whole cells are used in in vivo test systems. The acquisition of the measurement signal is often a problem here, unless the growth of the cells can be used as a particularly inexpensive and simple parameter. The main problem with screening systems that use growth as measurement parameters is to distinguish the protein-specific toxic effects from a general cytotoxic effects. This is very difficult with essential proteins as targets, since both the protein-specific effect and the cytotoxic effect lead to the same result, cell death.

US6228588B1 describes a method for the identification of essential genes of S. aureus by means of conditionally lethal mutants, sequences of essential S. aureus genes (and their homologues in other bacteria) and the establishment of screening methods by means of conditionally lethal mutants.

It is described among others the use of a collection of conditionally lethal mutants (known mutations in essential genes) to identify the targets of known growth inhibitors. This is possible because in the case of conditionally lethal mutants the gene product is often only partially functional and further influencing this gene product leads to growth inhibition faster than in the wild type.

From WO9526400A1 a yeast cell-based method (reverse two-hybrid) is known, with which it is possible to search for substances that specifically interfere with the interaction between two proteins. It is possible to search for small molecules that specifically disrupt the interaction between two proteins (of any origin, e.g. human, plant, bacterium, fungus). The disorder of the interaction is read out by induction of the expression of a reporter gene caused thereby. However, this method cannot be used to search for growth inhibitors.

WO9957536C2 describes the CAK1 (cdk-activating kinase) gene / protein from C. albicans (among others). Methods of a target-directed protein differential screening ("differential screening format" p.53) for CAK1 are explained, in which the action of substances against a fungal CAK1 is compared with the action against human CAK1.

The object of the invention is to provide a new screening method which combines as many of the advantages of a target-oriented test method as possible with those of an in vivo method.

This object is achieved according to the invention by a screening method for identifying active substances which is suitable for high throughput, characterized in that a) a target organism (target organism) is selected which the active substances to be identified are to inhibit or kill, b) a target gene (target) is selected , which or its gene product is to be deactivated by the active substances to be identified, c) selects an organism to be protected which is to be protected from damage by the target organism, d) selects a test organism which carries a test gene which is functionally homologous to the target gene, e) two test strains constructed a test organism that differ genotypically in exactly two characteristics, namely i. in the target gene, in that one of the test strains tolerates a higher dose of an active ingredient which deactivates the target gene or its gene product than the other test strain and ii. in a gene that codes for a well-detectable property (phenotypic brand), but is not essential for the vitality and proliferation ability of the test organism, f) mixes both test strains and incubates them together, g) adds a potential active ingredient and h) based on this, if necessary, differently strong growth of the two test strains identified an active ingredient

The method according to the invention has considerable advantages over the known prior art: A large number of new targets (target genes) can be tapped without the exact function of the target being known (for example essential ones

Genes / proteins of human pathogenic organisms).

The screening process is HTS-compatible (96 well and 384 well plates) and enables the results to be read very easily.

Target and control strains are mixed and incubated together. When used in the HTS, this leads to a 50% reduction in the total number of wells and thus to significant cost savings.

The sensitivity of the screening process can vary

Inoculation amounts and inoculation ratios of the test strains are modulated and thus shows a very high to very weak as required

Sensitivity.

Even minimally inhibiting drug concentrations can be measured or detected, since the resulting longer doubling times up to the stationary phase lead to an accumulation of the uninhibited strain and thus the phenotypic marker. Thus, a selectively site-specific inhibition, which only results in a 10% slower growing strain, after about 10 doubling of the uninhibited strain leads to twice as many cells of this strain as the inhibited strain in the culture.

The use of microorganisms (e.g. yeast) can make complex

Extracts are examined without affecting the assay.

After the tests have been created, the procedure is quick and inexpensive, since the test material reproduces itself.

Only relevant substances generate a readable signal ("hits"). As a result, only site-specific substances are actually identified and an excessive number of false-positive hits are avoided. This saves a further time-consuming and costly verification / analysis of the hits.

"Hits" are very likely to have "lead potential" because the substance reaches the site of action (can penetrate the cell) and a strong influence on the target is shown in vivo. The target organism is preferably selected from humans and microorganisms, in particular pathogenic organisms, preferably from plant, animal and / or human pathogenic organisms, preferably from human pathogenic fungi or bacteria.

Target organisms are in particular plant pathogens and human or animal pathogens (viruses, pro and eukaryotes), microorganisms and plants (algae) which disrupt industrial production (biofilms, deposits in cooling circuits, etc.) or humans, e.g. for the detection of cytostatics for the treatment of cancer or the search for enzyme inhibitors (HMG-CoA reductase inhibitors for the treatment of arteriosclerosis).

Particularly suitable human or animal pathogenic target organisms are organisms of the genera Streptococcus, Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Nocardia, Enterobacter, Yersinia, Francisella, Pasturella, Moraxella, Acinetobacter, Erysillelusus, Actobacus, Actinobacella Brucella, Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Klebsiella, Vibrio, Proteus, Borrelia, Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia, Chlamydia, Borrelia, Mycoplasma, Aspergillio, Blidoms, Aspergillio Exophalia, Histoplasma, Pneumocystis, Cryptococcus, Trichosporon, Absidia, Mucor, Rhizomucor, Rhizopus, Members of the species or group Group A Streptococcus, Group B Streptococcus, Group C Streptococcus, Group D Streptococcus, Group G Streptococptoconcus, Streptocogenpt agalactiae, Streptococcus faecalis, Strept ococcus faecium, Streptococcus durans, Neisseria gonorrhoeae, Neisseria meningitidis, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium diphtheriae, Gardnerella vaginalis, Mycobacterium tuberculosis, bovis Mycobacterium, Mycobacterium ulcerans, Mycobacterium leprae, Actinomyces israelii, Listeria monocytogenes, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica , Escherichia coli, Shigella dysenteriae, Haemophilus influenzae, Haemophilus parainfluenzae, Salmonella typhi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris, Yersinia pestis, Klebsiella pneumoniae, Serratia marcescens, Serratia liquefaciens, Vibrio cholerae, Shigella dysenterii, Pseudomonas aeruginosa, Francisella tularensis, Brucella abortis, Bacillus anthracis, Bacillus cereus, Clostridium perfringens, Clostridium tetani, botulinum Clostridium, Treponema pallidum, Rickettsia rickettsii, Chlamydia trachomitis, Aspergillus fumigatus, Blastomyces dermatitidis, Candida dublinensis, Candida glabrata, Candida krusei, Candida albicans, Candida tropicalis, Candida parapsilopsis, Histoplasma capsulatum, Pneumocystis carinii, Cryptococcus neoformans.

Suitable phytopathogenic target organisms are organisms of the genera Alternaria, Gaeumannomyces, Cercospora, Botrytis, Claviceps, Corticium, Colletotrichum, Didymella, Endothia, Exobasidium, Sclerotinia, Erysiphe, Fusarium, Magnaporthe, Plasmopara, Penicillporium, Pythonpora, Pythonoporum, Pythonoporum , Trametes, Ophiostoma, Rhizoctonia, Sphacelotheca, Septoria, Sclerospora, Venturia, Verticillium, Puccinia, Phoma, Tilletia, Ustilago, and Urocystis.

According to the invention, target genes (target genes) or target proteins refer to genes / proteins which are essential for the vegetative growth of the target organism. The disruption, inhibition or inactivation of the genes or the associated gene products (proteins) leads to the target organism being killed or inhibited from growth. This includes in particular all proteins / genes of a target organism which are functional homologs to essential genes / proteins of S. cerevisiae. The following genes are essential genes of the yeast S. cerevisiae:

YER022w, YBR211C, YDR118w, YOR249c, YKL004w, YHR101c, YDL220c,

YLR459w, YMR168c, YER026c, YMR094w, YDR016c, YGR113w, YOL149w,

YJL090c, YMR220w, YBR102c, YKLOβOc, YNL256w, YPR136c, YNL038w,

YJR016c, YER038C, YLR317w, YDR499w, YBR193c, YEL019c, YKL186c, YGR158C, YGR147C, YML031w, YPL124W, YJL039c, YPR168W, YLL004w,

YCL052C, YNL282w, YGR03ÖC, YBR167c, YBL018c, YGR075c, YFR029w,

YJL173c, YBR256c, YOR095c, YMR200w, YBL093C, YML091c, YML043c,

YLR141W, YBLO c, YJL025w, YMR270c, YCR035c, YDR303c, YLR033w, YDR041W, YER147C, YNR026c, YDR498c, YMR059w, YPL083C, YLL003w, YGL113W, YIL147C, YDR478w, YDL098C, YDR240c, YDR201W, YBR253W, YHR178W, YDR082W, YIROHc, YJL035c, YPL128c, YGR099W, YLR010C, YAL001C, YDR362C, YPL007c, YJL054w, YNL131w, YBR126c, YJL087c, YDR407C, YDR472W, YEL035c, YAL035C-A, YBL073W, YBL077W, YBR168W, YBR190W, YCL041C, YCR013C, YDL016C, YDL163W, YDL196W, YDL221W, YDR053W, YDR187C, YDR327W, YDR355c, YDR367w, YDR396w, YDR413C, YDR526C, Yfr042w, YGL069C, YGL074C, YGL102C, YGL239C, YGL247W, YGR046W, YGR073C, YGR114C, YGR128c, YGR190C, YGR251W, YGR265W, YGR277C, YHR083W, YHR196w, YJL009W, YJL010C, YJL015C, YJL018W, YJL032W, YJL086C, YJL091C, YJL195C, YJL202C, YJR012C, YJR023c, YJR041C, YJR046W, YJR136c, YKLO c, YKL036c, YKL083W, YKL111C, YKL153W, YKR081C, YKR083C, YLL037w, YLR007w, YLR076c, YLR101c, YLR112W, YLR132C, YLRUOw, YLRUδw, YLR198c, YLR230W, YLR339C, YLR379W , YLR440C, YLR458W, YML023C, YML127W, YMR134W, YMR290w-a, YMR298w, YNL114C, YNL149C, YNL150W, YNL158W, YNL194C, YNL260c, YNL306W, YNL310C, YOL026C, YOL134C, YOR060C, YOR102W, YOR146W, YOR169C, YOR203W, YOR218C, YOR282W, YDL235c, YPL044C, YPL142C, YPL233W, YPL238C, YPL251W, YPR085C, YPR142C, YPR177C, YHR148w, YFL039C, YER133W, YFL037w, YDR064w, YGL048c, YLR229c, YLR293c, YLR167W, YER148W, YEL026W, YPRO c, YML085c, YER025w, YOR117w, YDL007W, YPL235W, YDR190c, YMR043w, YLR029c, YBR109C, YDL126c, YER070W, YOR259C, YBR143c, YER043c, YKL145w, YDR021w, YKL049c , YOR210W, YHL015W, YDR091c, YKL013c, YOR063w, YDR394w, YDL143w, YOR145C, YJL026W, YPL204w, YJL034w, YPR082c, YIL142w, YDL047w, YBL026w, YGL030W, YJR045c, YJR064W, YPR043W, YDR212w, YOL094c, YFR004W, YOR257W, YCL059c, YBR160w , YPR103w, YDL029W, YGL120c, YLR075W, YLR359W, YJR123w, YPR165w, YMR29ÖC, YJL111w, YHR174w, YNL244C, YDL084W, YHR183w, YOL127w, YJLOUw, YDR331w, YOL038w, YLR147C, YHR165C, YOL133w, YPL266w, YCR012W, YLR175W, YER136w, YNL178w , YDLOδδc, YGR267c, YDLOUw, YOR074C, YMR26ÖC, YIL003W, YKR068c, YJLOOlw, YDR339C, YOL077c, YPL211W, YGL103W, YDR373w, YLR340W, YOL040C, YER094c, YGL091c, YLR008c, YLR275w, YDL064w, YDR188W, YHR065C, YKL035w, YHR005c-a, YGR253c, YHR1474W, YLR378C, YGR185C, YLR259c, YFL017w-a, YHR143w-a, YHR068W, YCL017c, YOR151C, YFLOOδw, YOL097c, YBL092w, YJR007w, YIL143c, YDR037w, YDL208W, YKR038C, YGL011c, YGL123w, YBL076c, YER171w, YNR053C, YOR157c, YNL189W, YKL210w, YOL120C, YOL066c, YOR159c, YKL024c, YBR196C, YBR135W, YPR094W, YIL078w, YBL105c, YPR176c, YNL132w, YPL218W, YNL290W, YPL151c, YER159c, YBR011c, YDR454c, YHR072w-a, YLR009W, YBL04OC, YDR172w, YHR02ÖW, YLL018c, YJR058c, YBR252w, YJLOδOw, YGL022W, YOR244w, YMR314w, YMR301c, YJROβδc, YOR046c, YDR047W, YDR510W, YJR068w, YKL180W, YDLUOc, YOR362C, YNL075w, YIL126W, YHR019C, YOR261C, YPR182w, YHR122w, YFL022c, YAL025c, YGR180C, YGR024C, YKL152c, YGR264c, YBR202w, YOLOOδc, YER013w, YOL100W, YLR197W, YIL048w, YER048W-A, YDROδOc, YLR243W, YLR186w, YAL038W, YOR103C, YPL131w, YOR262W, YML092c, YHROOδc, YDR062w, Y0R116C, YGL137W, YPR11ÖC, YOL123w, YOR310c, YDL108w, YMR288w, YLR298C, YKR002W, YGR029w, YGR094w, YER036c, YDR044w, YGR218w, YPR088c, YOR33δc, YLR208w, YPR181c, Y PL117c, YPL028w, YIL106w, YKL196C, YPR019W, YJR0δ7w, YDL102w, YLL034c, YNL113w, YPL2δ2c, YBR080C, YDR002W, YJL143w, YHR17ÖW, YDR086c, YML126C, YIR022w, YPL093W, YER012W, YJR072c, YDR397c, YPR033c, YDL097c, YKR086w, YNL267w, YLR336C, YDL147w, YMLOIδc, YJL008c, YPL17δw, YMR213w, YBR1δ4c, YER165W, YLR163c, YNL232w, YPR107c, YFROδOc, YCR072c, YMR146C, YGL201C, YEL032w, YNL222w, YDL166C, YJL167W, YBL041w, YNLOOβw, YLR022C, YHR089c, YFL009w, YLR274w, YGL24ÖW, YOR204w, YHR190W, YDR378C, YPROlOc, YKL193c, YPL143w, YBR087w, YDR238c, YER172C, YDL031W, YDR328c, YOR020c, YLL011w, YPL160w, YNL061w, YLR314c, YIL021w, YNR011c, YBR121c, YER029c, YLR438c-a, YGL106w, YPR187W, YGL171W, YHR088w, YDLUδc, YDR341c, YBR088c, YNR016c, YNR043W, YLR397C, YLR276c, YBR247c, YKL0δ8w, YDL028c, YLROβOw, YDR4δ7w, YBR091C, YDR460w, YDR404c, YPLOlOw, YLR1δ3c, YCR0δ7c, YAR019C, YOL010W, YBL023c, YLR066w, YDR023W, YGR211w, YLR212c, YPL209C, YNL247W, YLL031c, YAL003w, YER082C, YKL088W, YNL088w, YHR102W, YER0 09W, YJL09δw, YKL203c, YEL020w-a, YOR048c, YDR0δ4c, YDR04δc, YJR017c, YLR19δc, YDRUδw, YGR172C, YDR412w, YNL262w, YEL0δ8w, YALKLL33dc, YALKL03D3dc YHR169W, YNL048W, YGL068W, YOR326w, YMR128w, YDR267C, YLR277c, YLR117C, YPL063W, YLLOδOc, YLR268W, YGLOδδw, YFR031c, YIL07δc, YOL021C, YNL317W, YJL104w, YNL207W, YDR28ÖW, YHR107c, YJL167w, YHR024c, YKR026C, YOR232w, YML098W, YPR108W, YPL237w, YBR192w, YOR319W, YBR243C, YER003c, YOL139C, YLR086w, YBR029c, YOR224c, YDL103c, YGL238W, YER021w, YHR027c, YGR19δw, YGLOβδc, YKL16δc, YNL110C, YDR0δ2c, YOR294w, YLL008W, YLROOδw, YHR069c, YHR072w, YBLOδOw, YOR207c, YBR123c, YJL033w, YJR022w, YJL12δc, YJL074c, YLR21δC, Y0R119C, YFL038c, YML12δC, YPL094c, YPR113W, YLR129w, YKL04δw, YDL087C, YMR236w, YGL029w, YBR234c, YNL007c, YGLOOlc, YDRδ27W, YNL163C, YGL003c, YNL221c, YIL083C, YKL189w, YOR287C, YNL287W, YGR048W, YBR017c, YNL263c, YPL122c, YHR007c, YBR17ÜC, YIL004C, YGL018C, YPR041w, YKL022c, YPR180w, YDR449C, YGR145W, YDL207W, YML064C, YDR189w, YDL008w, YKL172w, YPR086w, YNL280c, YGR091W, YGR17δc, YOR077w, YAR007c, YLR321c, YPL217c, YGROβOw, YDR292C, YPL082C, YOR168w, YGL207w, YKL099C, YNR0δ4C, YIR008c, YPR161C, YPL242C, YJR013W, YDL017w, YML077w, YER023w, YGL112c, YFL008w, YDR196C, YGL116w, YDR243c, YBR070c, YMR208w, YOL142w, YBR1δ9w, YCL031C, YGR24δc, YDR36δc, YFR0δ2w, YBR142w, YNL2δ8c, YFR028C, YLR347C, YOL102c, YBR110w, YLR323C, YPR178w, YLR291c, YDR167W, YDR013W, YMR131c, YOR361c, YDL1Ö2W, YIROIδw, YIL109c, YKL012w, YHR070W, YBR237w, YIL061c, YML069w, YER12δw, YKL173w, YDR429C, YIL046W, YGR083c, YDRδ31W, YDR390c, YDR208w, YMR277w, Y0L13δc, YBR2δ4c, YFL002c, YGR216c, YKL09δw, YMR23δc, YML130c, YGR278W, YNR003C, YGROOδc, YDL111c, YMROOlc, YKL082C, YER008c, YPR183W, YBR198C, YHR164c, YOR341w, YHR042w, YNL102w, YLROδlc, YOR281C, YOR0δ7w, YNL002c, YDR170c, YDR437W, YOR206W, YOR148c, YNL245C, YDR236C, YBR236c, YJL097W, YFROδlc, YDLOβOw, YNR038w, YDL132W, YCL004W, YNL039w, YKR063c, YMR093W, YKL006c-a, YKL019w, YHR062C, YDLOIδc, YDR376w, YOR004W, YLR222C, YKR008w, YLR166c, YPR048W, YDR398W, YBR079C, YMR308c, YNL0δ9c, YNL026w, YGL073w, YLR1 16W, YOR174W, YPL190c, YPR143W, YGR2δδc, YDR211w, YMR112C, YMR239C, YJL072C, YFL017c, YMR079w, YPL020C, YPROδβw, YPL1δ3c, YGR246c, YIL1622, YIL1622 YOL0δ2c, YIL091C, YCL043c, YJL031c, YGR167w, YPR112C, YMR296c, YLR002C, YPR144C, YDR141c, YGR074w, YGR09δc, YLR43ÖW, YMR197c, YMR061W, YDL04δc, YER006W, YJROOβw, YPR190c, YOL034W, YAL034w-a, YNL1δ1c, YDR087C, YHR166c, YMR24ÖC, YNR017w, YBR2δ7w, YLR026c, YBL020W, YMLOWw, YDR416w, YJR002W, YNR046W, YGL111W, YJR112w, YDR300C, YDL141W, YIL019W, YBRIδδw, YOR194c, YLR4δ7c, YER112w, YLR310C, YGL047W, YMR281w, YLRIOδc, YDR361c, YJR067c, YNL126W, YJL002C, YELOδδc, YDR302w, YHR08δw, YJL109C, YHR188c, YPL228w, YGL008C, YDR3δ3w, YDL217c, YJL069C, YGL098W, YMLOβδw, YDR473c, YML093W, YGR047C, YLR409C, YGR002C, YDR228c, YDR168w, YGR274c, YFR037C, YMROOδw, YJLOOδw, YGL044c, YJL061w, YLR383w, YMR013c, YDL139C, YDR468c, YHR197w, YGR098c, YNL181W, YHR099w, YOR2δ0c, YIL171W, YMR309C, YNL240c, YIL104C, YPR137w, YDR060W, YOR340c, YMR028W, YDR301W, YCL0δ4w, YDL0δ8w, YGR1δ6w, YOR37ÖC, YMR091c, YJL081C, YKL018W, YDR308c, YBL084c, YPR186c, YDL088c, YOR122c, YHR0δ2w, YNL308c, YDL148c, Y LR103c, YFR002w, YMR049c, YKL19δW, YDR164C, YHR0δ8c, YDL193W, YAL043c, YLR088w, YLR272c, YMR211w, YBROδδc, YLR30δc, YIROlOw, YER168c, YKR02δw, YLR106c, YHR186c, YJL011C, YOROδβC, YOL022C, YFROOδc, YGL097w, YMR203w, YOR143c, YCR093W, YLL03δw, YAL033w, YER018c, YLR249w, YBL034c, YKL021c, YDR283C, YBR136W, YDR489W, YPR17δw, YOR149c, YOR2δ4c, YBL03δc, YGR280C, YOR260w, YGL142c, YBR049c, YPL012w, YAL032c, YEL002c, YKL108W, YKR079C, YLRWOw, YNL172w, YAL047C, YDR081c, YMR117c, YIL026C, YJL194W, YGR Ow, YGR116w, YDR246W, YFR003C, YNL312w, YDL043C, YCR042C, YBROβOc, YGL07δc, YDL03ÖW, YML049c, YLR316c, YNL313C, YGL108C, YKL0δ9c, YKL028w, YJL203W, YGR009c, YKL1δ4w, YDR3δ6w , YPL231W, YMR227c, YPL043w, YGR179C, YKR037c, YKL182w, YDR324C, YLL036C, YGL13ÖW, YHR090C, YKR062w, YGR119c, YFL029c, YDR23δw, YAR008w, YAL041w, YPR02δc, YPL2δδw, YNL062c, YOR110w, YNL2δ1c, YFR027W, YER127W, YJL012c, YGLIδδw , YIL144W, YHR040w, YNL216W, YBL004W, YKL144c, YPL076w, YDR32δW, YLR04δc, YDR288W, YILIδOc, YOL069W, YKL12δw, YHR036w, YML02δC, YMR268c, YKR071C, YPL210C, YBR26δw, YDR381w, YMR229c, YGL233W, YMR149w, YOL146W11, YOL146WLL, YOL146WL, YOL146WL, YOL146WL YNL124W, YIL068C, YJR141w, YDL19δw, YLR078c, YGL172W, YPL011c, YLR127c, YKL042W, YIR012w, YNL261w, YGR013w, YGR090W, YDR299w, YNL188W, YIL11δc, YDL1δ3c, YGR11δC, YJL019W, YOR2δ6C, YBL097W, YDL003W, YMR218C, YDLWδw, YGR191w, YGL092W, YMR076C, YOR07δw, YPROδδw, YJR093C, YNL272c, YDR180w, YDR432w, YNL182C, YML104c, YPRIOδC, YKL0δ2c, YFL024c, YML046w, YGL093W, YGL22δw, YKR022C, YDR434W, YDLIδOw, YDR311w, YPL08δw, YPL169c, YPR034w, YOR372c, YOR373W, YML114C, YPR169W, YHR118c, YGLUδw, YGR186w, YDR088c, YDL209C, YOL078W, YLR424W, YNL1Ö2W, YJL041w, YGR198W, YPL126w, YNL118C, YKL033W, YMR033w, YOL13ÖW, YDR420w, YJL08δw, YDR207c, YKL089W, YDR166C, YDR182w, YHR172w, YDR464w, YOL144W, YJR089W, YIROOβc, YCR0δ2w, YLR223c, YOR329c, YBR1δ6c, YJL042W, YDR113c, YGL122C, YMR113W, YPR03δW, YHR026W, YJR139C, YBR1δ3W, YHR066W, YOR272W, YJL1δ3C, YDR120C, YDR329C, YFL04ÖC, YGL033W, YLR18ÖW, YHR20δW, YPR133C, YDR066C, YLR136C, YLR064w, YLR143w, YCR0δ4C, YBL074C, YBL030C, YBR002c, Y BR004c, YBR089w, YBR118w, YBR124w, YBR14ÖC, YCR064C, YDL212w, YDLWδw, YDL092w, YDL004w, YDR160w, YDR177W, YDR224C, YDR232w, YDR427w, YDR487c, YEL034w, YER093c, YER104W, YER107c, YER126c, YER1δ7w, YGL169w, YGLIδOc, YGL040c, YGROβδc, YGR082W, YGR120c, YHR023w, YHR084w, YIL118w, YIL063c, YIL062C, YIL033C, YIL031W, YJL174w, YJLIδβc, YJR042w, YJR076c, YKL209c, YKL192C, YKL181W, YKL141w, YKL122c, YKL104c, YLR3δδc, YMLIOδc, YMR047C, YMR108W, YMR16δc, YMR217w, YNL161w, YNL137c, YNL112w, YNR03δc, YOR098c, YOR160w, YOR176w, YOR181w, YOR236w, YOR278w, YPL07δw, YPL016W, YCL0δ3c, YFL0N04L, YFL0L04L, YFL0N04C0

Genes or proteins of target organisms are of particular interest if the similarity of the derived amino acid sequences is at least 30% to the derived amino acid sequences of the S. cerevisae essential genes or if complementation has shown that the gene / protein of the target organism is the corresponding gene / protein from S. cerevisiae. Particularly suitable targets are those for which there are no homologous representatives in higher eukaryotes (eg humans). These are, in particular, homologous genes / proteins to the following essential S. cerevisiae genes or their gene products:

YER022W, YBR211c, YDR118w, YOR249c, YKL004w, YHR101c, YDL220c, YLR4δ9w, YMR168c, YER026c, YMR094w, YDR016c, YGR113W, YOL149w, YJL090C, YMR220w, YBR102c, YKLOβOc, YNL2δ6w, YPR136C, YNL038w, YJR016C, YER038C, YLR317w, YDR499w, YBR193c, YEL019C, YKL186c, YGR1δ8c, YGR147C, YML031w, YPL124w, YJL039c, YPR168W, YLL004w, YCL0δ2c, YNL282W, YGR030c, YBR167c, YBL018c, YGR07δc, YFR029w, YJL173C, YBR2δ6c, YMR20ÖW, YML091c, YML043c, YLR141w, YBLOUc, YJL02δw, YMR270c, YCR03δc, YDR303c, YLR033w, YDR041W, YER147c, YNR026C, YDR498C, YMR0δ9w, YPL083c, YLL003w, YGL113W, YIL147c, YDR478W, YDL098c, YDR24ÖC, YDR201w, YBR2δ3w, YHR178W, YDR082w, YIR011C, YJL03δc, YPL128c, YGR099w, YLR010C, YAL001c, YDR362c, YPL007C, YJL0δ4w, YNL131w, YBR126c, YJL087c, YDR407C, YDR472w, YEL03δc, YAL03δC-A, YBL073W, YBL077W, YBR168w, YBR190w, YCL041C, YCR013C, YDL016C, YDL163W, YDL196W, YDL221W, YDR0δ3w, YDR187C, YDR327W, YDR3δδc, YDR367w, YDR396w, YDR413C, YDRÖ26C, YFR042W, YGL069C, YGL074C, YGL102C, YGL239 C, YGL247W, YGR046W, YGR073C, YGR114C, YGR128C, YGR190C, YGR2δ1W, YGR26δW, YGR277C, YHR083w, YHR196W, YJL009W, YJL010C, YJLOIδC, YJL032W, YJL086C, YJL091C, YJL19δC, YJL202C, YJR012C, YJR023c, YJR041c, YJR046w, YJR136c, YKLOUc, YKL036C, YKL083W, YKL111C, YKL1δ3W, YKR081C, YKR083C, YLL037W, YLR007W, YLR076c, YLR101c, YLR112w, YLR132c, YLRUOw, YLRUδw, YLR198C, YLR230W, YLR339C, YLR379W, YLR440C, YLR4Ö8W, YML023C, YML127W, YMR134W, YMR290w- a, YMR298w, YNL114C, YNL149C, YNLIδOW, YNL1Ö8W, YNL260c, YOL026C, YOL134C, YOR060C, YOR102W, YOR146W, YOR169C, YOR203W, YOR218C, YOR282W, YDL23δc, YPL044C, YPL142C, YPL233W, YPL238C, YPL2Ö1W, YPR08δC, YPR142C, YPR177C, YLR336C, YMLOIδc, YNL232W, YER029c, YLR438c-a, YBR091c, YCR0δ4C, YDR412W, YDR0δ2c, YBR123c, YJR022w, YDRδ27W, YNL221c, YIL004c, YCLL22CYYLL22C, YCL24 YER008c, YOR148C, YNL039w, YKR063c, YKR008w, YLR166c, YDR398w, YOR174w, YPL190C, YMR112C, YMR239c, YOR3δ3c, YER104w, YPR19ÖC, YAL034w-a, YNL1δ1c, YJR112w, YIL019W, YOR194c, YLR4δ7c, YLRWδc, YJR067C, YELOδδc, YJL109C, YDR3δ3w, YGL098W, YGR047c, YLR409C, YDR228c, YDR168W, YMROOδw, YJL061w, YDL139c, YDR468c, YHR197w, YOR340c, YGR1δ6w, YDR308c, YOR122c, YHR0δ2w, YMR049c, YKL19δW, YAL043C, YMR211w, YIROWw, YER168c, YKR02δw, YLL03δw, YAL033w, YER018c, YBL034C, YKL108W, YDR081c, YMR117c, YGRUOw, YFR003C, YGL07δc, YLR316C, YKL0δ9c, YGR009c, YPL231w, YGR179C, YKL182w, YKR062W, YDR23δw, YPL2δδw, YOR110W, YNL2δ1c, YER127w, YHR040w, YNL216w, YKL144C, YPL076w, YLR04δc, YDR288W, YHR036w, YML02δC, YMR149w, YOL146W, YGL061c, YLR071c, YJRUIw, YLR078c, YPL011c, YKL042W, YGR013W, YGR090W, YNL188w, YGR11δC, YDL003W, YMR218c, YOR07δw, YNL272C, YPRIOδC, YKL0δ2c, YFL024c, YML046W, YGL093w, YGL22δw, YKR022C, YPL08δw, YOR372c, YML114c, YPR169W, YHR118c, YGLUδw, YGR186W, YDL209C, YN L1Ö2W, YGR198W, YPL126w, YMR033w, YOL130w, YKL089W, YDR166c, YDR182W, YDR464w, YOL144W, YJR089W, YDR113C, YGL122c

Other particularly suitable essential genes of a target organism are those genes or proteins which have functional homologues in higher eukaryotes, but in which differences (ideally maximum) in the protein structure between the protein consist of higher eukaryotes (control protein) and the target protein (in primary , and / or secondary, and / or tertiary, and / or quaternary structure). The control gene or protein can in particular come from a mammal (e.g. human) or a plant. Particularly suitable target genes / proteins of the human pathogenic target organism Candida albicans are homologous genes / proteins to the essential S. cerevisiae genes / proteins YDR236c (FMN1), YBR26δw (TSC10), YPR113W (PIS1), YOR122c (PFY1) and YMR197c (VTI1).

The organism to be protected is preferably selected from plants, animals and humans, in particular humans. Test organisms, test strains or cultures that can be used here are basically all organisms or cell cultures that are easy to cultivate (e.g. bacteria in a test tube / microtiter plate or cell cultures in cell culture bottles / microtiter plates).

These can be of prokaryotic as well as eukaryotic origin. Organisms that are genetically accessible and in which genes can be heterologously expressed are preferably used here.

Both gram-positive and gram-negative bacteria are suitable among the prokaryotic test organisms. Exemplary, but not exclusive, test organisms from the prokaryote kingdom are, for example, species of the genera Bacillus (B. subtilis), Escherichia (E. coli), Klebsiella [K. planticola), Lactobacillus (L. delbruckii, L. lactis), Pseudomonas (P. aeroginosa, P. fluorescens) Salmonella (S. typhimurium) Serratia (S. marcescens) Streptococcus (S. lactis, S. mutans, S. pyogenes) , Staphylococcus (S. aureus, S. epidermidis) Vibrio (V. cholerae) Yersinia (Y. ruckeri).

Test organisms for which a range of molecular biological tools (e.g. vectors) are available, such as Escherichia coli and Bacillus subtilis, are particularly suitable.

Eukaryotic unicellular organisms (protozoa, slime molds, fungi, etc.) and eukaryotic cell cultures are suitable for the eukaryotic test organisms. Yeasts are particularly suitable among the eukaryotic unicellular organisms. Species from the genera Saccharomyces, Schizosaccharomyces, Candida, Kluyveromyces, Yarrowia, Ashbya, Hansenula, Pichia are particularly suitable among the yeasts. Among these, Saccharomyces cerevisae, Schizosaccharomyces pombe and Candida albicans are particularly suitable.

Saccharomyces cerevisiae strains are particularly suitable, preferably the strains CEN.PK2, BY4743, BMA46 (W303), FY1679 and their haploid derivatives. The method according to the invention is particularly suitable for high-throughput screening, preferably by parallelization, particularly preferably by performing it in a plurality of parallel batches, very particularly preferably in microtiter plates or other devices known to the person skilled in the art, which allow batches to be parallelized.

According to the invention, different genotypic characteristics represent the differential expression of the target gene in the test strains (different "gene dose"), e.g. caused by the deletion of a target gene allele (for test organisms with ploidy> 1), introduction of additional target gene copies into one of the test strains or differently regulated expression of the target genes in the test strains.

Cells that express a target gene more strongly and thus produce more target protein tolerate a much higher concentration of active substance that are directed against exactly this taget protein. Another distinguishing feature is a genetic change in the target gene in one of the test strains. This can be caused by conditional mutations (point mutations, deletions or truncation).

Such genetic changes often lead to partially functional proteins that can be more easily inhibited by active substances than wild-type proteins. The test strains can also differ due to the substitution of essential genes by functionally homologous proteins from other organisms (eubacteria, archaea, eukaryotic viruses). Depending on the goal, tests can be created with a wide variety of test master combinations.

In addition to this genotypic characteristic, the strains differ in another characteristic, since one of the two test strains is given a phenotypic brand. Phenotypic brands refer to phenotypic characteristics that have little or no influence on the growth of the test strain. This can be, for example, a deletion in a further gene locus, which has the consequence that the cells accumulate a colored pigment or thereby special biochemical properties are given that are easy to read. For example, the deletion of the ADE2 gene locus in yeasts (e.g. Saccharomyces cerevisiae, Schizosaccharomyces pombe or Candida albicans) leads to the accumulation of phosphoribosylaminoimidazole in the vacuole, which is converted into a red pigment by oxygen. The Zeil sediment of such a culture is red and can easily be seen with the eye. Thus, the> ADE2 deletion basically offers itself as a phenotypic brand, but unfortunately the accumulation of the red pigment leads to a deterioration in the growth of the deletion strain compared to the wild-type strain (Ugolini and Bruschi, 1996), which is very disadvantageous for use in the below described method is. By cleverly arranging the test or process sequence, the accumulation of the growth-inhibiting pigment is only induced after the cells have been grown, so that the ADE2 deletion can still be used and even represents a particularly suitable phenotypic brand. Other phenotypic brands are the expression of the green fluorescent protein (GFP) in one of the strains or the expression of other reporter genes such as, for example, the β-galactosidase, the luciferase or the like.

The two test strains (target and control strains) which differ only in the target gene / protein and the phenotypic brand are grown and mixed. Fresh medium is inoculated with a low cell count of this stock mixture. After application of test substances, the cultures are grown until the stationary growth phase. The final composition of the resulting mixed cultures can now be determined very easily by quantifying the characteristic phenotypic brand. If, for example, one of the two strains is specifically or more strongly inhibited by a substance in growth, this leads to an intensification or weakening of the corresponding phenotypic brand, which in turn is read out through stronger / weaker fluorescence or more intense / weaker coloring or stronger / weaker biochemical reaction can be. Another object of the present invention is a test kit for identifying active substances, which comprises means for performing the method according to the invention.

Another object of the present invention is a method for producing a cosmetic or pharmaceutical preparation, characterized in that a) the method according to the invention is carried out and an active ingredient is thus determined and b) active ingredients found to be effective with cosmetically and / or pharmacologically suitable and compatible carriers mixed.

Another object of the present invention is the use of the method according to the invention for finding active substances.

The following examples illustrate the invention without, however, restricting it thereto:

Example 1 :

Target selection for the identification of antibiotic substances in the substitution process of the protein difference screening

Targets for the PDS substitution procedure for the identification of antibiotic substances are essential for the vegetative growth of the pathogen under full medium conditions and are preserved under pathogens. For the targets, a functionally homologous gene / protein must exist in the test organism, which is also an essential gene / protein for the test organism. The raw material for the target selection represents the sequence information of the genomes in connection with the functional analysis of the discovered genes.

In many cases, proteins that perform the same function (usually the enzyme-catalytic function is meant) in humans (or animals) and in the infection germ (functionally homologous proteins) differ in molecular structural areas (protein domains). It can be assumed that the disruption of these specific protein domains through the binding of active substances can also lead to the functional failure of the entire protein. Such active substances would thus be specific for the protein of the infection germ in their inhibitory function, without the function of the isofunctional control protein (the human or plant cell) being affected. Accordingly, ideal targets for the method described here only show slight sequence matches with their control proteins with simultaneous functional identity.

The target selected in the example is bacterial dihydrofolate reductase. In a two-step reaction, dihydrofolate reductase catalyzes the reduction of folic acid by NADPH to tetrahydrofolic acid, an important coenzyme for the transfer of C1 units. This enzyme is essential for the vegetative growth of the infection germ. Furthermore, homologous enzymes exist both in the test organism S. cerevisiae and in humans. The human protein used as a control protein in the method has a 47% sequence similarity and a 27% sequence identity to the dihydrofolate reductase (FolA) of E. coli. Examples of further targets of this target category, ie bacterial targets with homologous proteins in fungi (S. cerevisiae) and in humans are functionally homologous proteins to the following essential proteins of the yeast S. cerevisiae:

Acs2p (Acetyl-Coenzyme A Synthetase), Alalp (Alanyl-tRNA Synthetase), Hsp60p (heat shock protein; 60kD), Gualp (GMP synthetase), Kar2p (nuclear fusion protein), Ilv2p (Acetolactate Synthase), Gnd1 (6-phosphogluconate Dehydrogenase), Thsl p (Threonyl tRNA Synthetase), Nfslp (NifS-like protein), Gln4p (Glutaminyl-tRNA Synthetase), Prp22p (Helicase-like protein), Prp43p (involved in spliceosome disassembly), Eno2p (Enolase), Ssdp ( mitochondrial heat shock protein 70-related protein), Vaslp (Valyl-tRNA Synthetase), Sgvlp (ser / thr protein kinase), Rrp46p (involved in rRNA processing), Krslp (lysyl-tRNA synthetase), Prp16p (RNA-dependent ATPase) , Cdc19p (pyruvate kinase), Kre30p (strong similarity to members of the ABC transporter family), Dhr2p (RNA helicase, involved in ribosomal RNA maturation), llslp (cytoplasmic isoleucyl-tRNA synthetase), Prp2p (RNA splicing factor RNA-dependent NTPase with DEAD-box motif), Ygl245wp (glutamyl-tRNA synthetase), Pro2p (gamma-glutamyl phosphate reductase ), Tn p (thioredoxin reductase), Erg10 (acetyl-CoA C-acetyltransferase), Gfalp (glutamine-fructose-6-phosphate aminotransferase), Hem13p (coproporphyrinogen III oxidase), Rrp3p (required for maturation of the 35S primary transcript), Ydr341cp (arginyl-tRNA synthetase), Hem12p (uroporphyrinogen decarboxylase), Gpmlp (phosphoglycerate mutase), Pgklp (3-phosphoglycerate kinase), Hiplp (histidine permease), Meslp (methionyl tRNA synthetase), Pmalp (H + ATPase transporting ), Dedlp (ATP-dependent RNA helicase), FaMp (involved in maturation of 18S rRNA), Rnrl p (ribonucleoside-diphosphate reductase), Dbp2p (ATP-dependent RNA helicase of the DEAD-box family), Drslp (RNA helicase of the DEAD box family), Atmlp (ATP-binding cassette transporter protein), Acdp (acetyl-CoA carboxylase), Sub2p (RNA helicase), Prp28 (pre-mRNA splicing factor RNA helicase of DEAD box family), Heml p (5- aminolevulinate synthase), Prolp (glutamate 5-kinase), Tpil p (triosephosphate isomerase), Hem2p (Porphobili nogen synthase), Ynl247wp (cysteinyl-tRNA synthetase), Hem3p (phorphobilinogen deaminase), Ded81p (asparaginyl-tRNA synthetase), Seslp (seryl-tRNA synthetase), Pmi40p (mannose-6-phosphate isomerase), Lcb2p (serine C-palmitoyltransferase), Guk1 p (guanylate kinase), Prslp (phosphoribosylpyrophosphate synthetase), Fol3p (dihydrofolate synthetase), Rer2p (cis-prenyltransferase), Fol2 Dpslp (aspartyl-tRNA synthetase), Frs2p (phenylalanyl-tRNA synthetase), Lcblp (serine C-palmitoyltransferase), Pro3p (delta 1-pyrroline-5-carboxylate reductase), Ura6p (uridine-monophosphate kinase), Hem15p (Ferrochelat Rib2p (DRAP deaminase), Htslp (histidine-tRNA ligase), Rkilp (Ribose-5-phosphate ketol isomerase), Glnlp (glutamine synthetase).

Also of interest are bacterial targets for which there are no sequence-homologous proteins in humans, but certainly in fungi (e.g. the yeast S. cerevisiae). The bacterial homolog can serve as the target protein and the protein of the fungus as the control protein. Hits achieved in such an approach are either interesting fungicides and / or antibiotics.

Particularly interesting examples of this target category are homologs of the S. cerevisae proteins Fba1 p (fructose-bisphosphate aldolase), Ilv3p (dihydroxyacid dehydratase), Tpslp (alpha, alpha-trehalose-phosphate synthase), Ssyl p (regulator of transporters), Rib3p (3,4-Dihydroxy-2-butanone 4-phosphate synthase), Homδp (homoserine dehydrogenase) Ergδp (phosphomevalonate kinase), Ilv5p (ketol-acid reductoisomerase), Fohp (dihydroneopterin aldolase) and Ribδp (riboflavin synthase).

With functional homology, ideal targets for protein difference screening in the substitution process show a maximum difference in the structure of the two homologous proteins (target and control protein).

Homologs to the following essential proteins of the yeast S. cerevisiae are therefore of particular interest for the identification of fungicidal substances: ThiδOp (thiamine pyrophosphokinase), Pfylp (profilin), Prolp (glutamate 5-kinase), Vtilp (v-SNARE: involved in Golgi retrograde protein traffic), Ero1 p (required for protein disulfide bond formation in the ER), Pro3p (delta 1-pyrroline-5-carboxylate reductase), Cdslp (CDP-diacylglycerol synthase), Olelp (delta-9-fatty acid desaturase), Ergδp (phosphomevalonate kinase), Fbal p (fructose bisphosphate aldolase), TsdOp (3-ketosphinganine reductase), Gnalp (glucosamine-phosphate N-acetyltransferase), Fmnlp (Riboflavin kinase), Fadlp (flavin adenine dinucleotide synthetase), Pgs1 p (phosphatidylglycerol phosphate synthase), Fol3 CDP diacylglycerol-inositol 3-phosphatidyl transferase), Lcblp (serine C-palmitoyl transferase).

Example 2:

Target validation

If it is not yet known whether the selected target is an essential gene / protein of the infection germ, this has to be confirmed. This is also the case when putative targets are selected on the basis of the known essential function of a homologous protein in a related organism. For example, one knows all the essential genes of the baker's yeast S. cerevisiae (approx. 1100) and can therefore conjecture that the homologous proteins in related pathogenic fungi are also essential proteins and thus represent interesting drug targets. However, this still has to be proven, for example by gene deletion of the putative target gene in the infection germ with subsequent phenotype analysis.

In the case of dihydrofolate reductase, it is known that it is an essential enzyme of E. coli.

Example 3:

Cloning of target and control genes in test organism expression vectors.

To construct the test strains, the target gene, the bacterial dihydrofolate reductase (E. coli folA) and the control gene (human DHFR) had to be cloned into expression vectors suitable for the test organism. The baker's yeast S. cerevisea, which is known to be dihydrofolate reductase (encoded by DFR1), is selected as the test organism and is an essential protein in the organism. This test organism is furthermore well suited for such analyzes, since a large selection of molecular biological tools such as expression vectors and controllable promoters for Available. The expression vector used is pDE95, a centromer-based vector which carries the HIS3 gene as a selection marker and which cloned genes are expressed by means of the yeast MET2δ promoter. For the cloning of E. coli folA, the gene from genomic E. coli DNA was amplified and cloned into pDE95 using the primer combination ecfolA_5 'GGA AAT CGA TAT GAT CAG TCT GAT TGC GG and ecfolA_3' TTC TCT CGA GAA TTA CCG CCG CTC CAG AAT C. (using molecular biological methods that reflect the state of the art). The resulting vector is named pDE95-ecfolA. A cDNA of the human DHFR was amplified from the cDNA gene bank using the primer combination HDFR_5 'CGC TAT CGA TAT GGT TGG TTC GCT AAA CTG and HDFR_3' ACA CCT CGA GAT TAA TCA TTC TA TCAT AC and was also cloned into pDE95 as described above. This vector has the name pDE95-HDFR.

Example 4:

Production of the test strains for the PDS substitution procedure.

S. cerevisiae was chosen as the test organism. Per test, i.e. Two S. cerevisiae strains must be produced for each target to be examined, one target strain which lacks its own dihydrofolate reductase (due to a deletion of the endogenous DFR1 gene), but which contains the gene for the bacterial dihydrofolate reductase and a control strain which also contains the own dihydrofolate reductase is missing, but it expresses the gene for human dihydrofolate reductase.

One of the two test strains is additionally provided with a phenotypic brand, for which the \ DE2 deletion was selected. Deletion of this gene locus in S. cerevisiae leads to the accumulation of a red intermediate (Dorfman, 1969) in the vacuole. This pigment has fluorescent properties and can be excited with light of the wavelength 488 nm (blue). The emission maximum of the pigment is in the red light range at 569 nm (Brushi and Chuba, 1988).

The starting point for the production of the test strains was a diploid S. cerevisiae laboratory strain (BY4743). One of the two alleles of the DFR1- Genes deleted. This is done by means of a PCR-mediated gene deletion with short homologous flanks, as described, for example, in patent no. WO 99/55907 is described and represents the prior art. The plasmid pDE95-ecfolA and the plasmid pDE95-HDFR were introduced into this diploid strain, which was now heterozygous for the DFR gene locus. Both resulting strains were subjected to a tetrad analysis, in which a reduction division is induced with 4 haploid spores as a result. Two of the spores contained the DFR1 deletion and could only grow because they expressed the functionally homologous protein / gene from E. coli (folA) or human (HDFR), which now took over the function of Dfrlp.

In order to label one of the two strains with the phenotypic label, an allele of the ADE2 gene was deleted in the S. cerevisiae laboratory strain (BY4743) using the method described above. Sporulation was induced in the resulting strain. Two of the spores now bore the ADE2 deletion. One of the> 4DE2 mutants was crossed with the test strains produced and sporulation was induced again in the resulting strains. In the haploid strains generated in this way, searches were carried out for those which carried both the DFR1 and 4DE2 deletion.

After the test strains described here had been prepared, the following test strain combinations were available for further screening:

Strain combination A

Target strain: BY4743 Adfrl + pDE95-ecfolA

Control strain: BY4743 Adfrl Aade2 + pDE95-HDFR

and

Strain combination B

Target strain: BY4743 Adfrl Aade2 + pDE95-ecfolA

Control strain: BY4743 Adfrl + pDE95-HDFR Example 5:

Analysis of target-specific inhibitors by the PDS substitution method (test organism: S. cerevisiae).

It is known that diamino-benzylpyrimidines specifically inhibit bacterial dihydrofolate reductase and have an affinity for dihydrofolate reductases of mammals that is several orders of magnitude lower. An active ingredient in this class of substances is trimethoprim, which has long been used in medicine to treat bacterial infections. This substance offered the possibility of analyzing the PDS in terms of its functionality and sensitivity in the substitution process.

For this purpose, the strains from Example 4 (strain combination A and strain combination B) were grown overnight and in each case cavities of a 96-well microtiter plate which contained 100 μl fresh medium with strain combination A or strain combination B in a 1: 2000 dilution inoculated. Then 1.5 μl trimethoprim solution of different concentrations (500 mM, 250 mM 100 mM, 50 mM, 25 mM, 10 mM, 5 mM, 2.5 mM, 1 mM, 0.5 mM, 0.25 mM, 0.1 mM, 0.05 mM - all dissolved in DMSO) pipetted into the cavities.

The microtiter plate was then incubated standing at 30 ° C. (ie not shaking). The cells settle quickly and the growth takes place exclusively at the bottom of the individual cavities. Almost anaerobic conditions exist here, which prevents the formation of the toxic red pigment in the ADE2 deletion strain. After 2 days of incubation, the cell mixtures were in the stationary growth phase and the conversion of the non-toxic phosphoribosylaminoimidazole to a red pigment could be induced. For this purpose, the medium in each cavity was drawn down to a few μl, then the Zeil sediment was resuspended in the residual medium by shaking and this suspension was incubated for 1 h at room temperature. Now there was an oxidation of the accumulated colorless intermediate product phosphoribosylaminoimidazole, which was mediated by atmospheric oxygen, and it was converted into an unspecified colored end product. The evaluation could now be carried out without further evaluation devices, since the phenotypic marker colored the sediment of the cultures red, which was clearly visible to the eye. All cavities to which nothing or only DMSO was added consistently showed a weak pink color. In the concentration range 500 mM - 2.5 mM, the sediments of the mixed cultures with strain combination A showed a clear red color (final concentrations 2.1 mg / ml - 10.5 μg / ml). In the concentration range 1 mM - 0.1 mM, the sediments of strain combination A showed a gradually weakening red color towards pink, which was, however, even more intense and therefore clearly distinguishable from the pink color of the controls (final concentrations 4.2 μg / ml - 0.42 μg / ml). At the concentration of 0.05 mM, the sediment from strain combination A showed no discernible difference to the sediments of the untreated cultures or the DMSO applied cultures (controls).

In the concentration range 500 mM - 0.5 mM, the cavities of strain combination B showed uniform white sediments (final concentrations 2.1 mg / ml - 2.1 μg / ml).

In the concentration range 0.5 mM - 0.05 mM, the sediments of strain combination B showed no discernible difference to the sediments of the untreated cultures or the cultures applied to DMSO (controls).

A highly sensitive assay is thus available, with the aid of which substances are identified which are directed against targets from selected organisms without excessively inhibiting a control protein from other organisms (e.g. humans). The assay works over a wide range of concentrations so that both very specific and less specific substances can be identified.

The sensitivity of the test can also be modified by selecting different starting dilutions of the parent combinations. The more diluted the two test strains are inoculated, the more strongly a differential inhibition of the target / control strains becomes noticeable (readable by the phenotypic marker).

Example 6:

Transfer of the assays into the 384 well format for the search for target-specific inhibitors in high-throughput screening (HTS)

The test used in Example 5 (strain combination A) was transferred to 384 well format microtiter plates. These microtiter plates are a common format for searching for interesting active substances in substance libraries using the high-throughput method. With the help of the new assay, it is possible to search for active substances which specifically inhibit the bacterial target protein without unduly interfering with the function of the human control protein.

The test strains were grown in full medium overnight and the cell density of the cultures was adjusted to identical values. The cultures were then diluted approximately 5000 times in fresh medium and combined. The cell suspension obtained in this way was mixed well and the cavities of 384 well microtiter plates (100 μl) were filled with it. After adding 0.8 μl of trimethoprim solution of different concentrations (5 mM, 1 mM 0.25 mM, 0.1 mM), the plates were incubated for 2 days at 30 ° C. and then treated as described in Example 5. The color evaluation showed that in the concentration range from 5 mM to 0.25 mM the sediments of the mixed cultures were clearly colored red and clearly differed from the untreated cultures.

If substance libraries are used, this is already included in the microtiter plates or is added using a pipetting robot. In addition to pure substances, complex extracts can also be examined for effectiveness, since yeast cells and thus the screening process are very robust with regard to interference from solvents or other substances and there are no false positive hits or unwanted "background noise". Signals are only generated when the target and / or the control protein are hit and the corresponding strain is inhibited. This saves time-consuming and costly cleaning up of complex extracts. The plates are now incubated at 30 ° C for 2 days and then evaluated. Fluorimeters (due to the fluorescent properties of the accumulated pigment) or systems that can measure color intensities (CCD cameras) are suitable for evaluation.

With the help of a FACS device, the numerical composition of the individual cavity cultures can even be determined, which allows a very precise statement about the specific inhibitory potential of a substance.

In order to rule out false positives, all substances that have generated a signal ("hits") are checked to see whether they also generate a signal with a parent combination B. For this purpose, the strains of this strain combination are grown overnight in full medium and the cell density of the cultures is adjusted to identical values. The cultures are then diluted approximately 5,000 times in fresh medium and combined in a ratio of 80% target strain (BY4743 Adfrl Aade2 + pDE95-ecfolA) to 20% control strain (BY4743 Adfrl + pDE95-HDFR). The cell suspension obtained in this way is mixed well and the cavities of 384 well microtiter plates (50 μl) are filled again. Now the hit substances of the first screening run (screening with strain combination A) are added.

The plates are incubated for 2 days at 30 ° C and then evaluated. The sediments from the controls, ie cavity cultures to which nothing or only DMSO was added, show a clear red color. Substances that specifically inhibit bacterial dihydrofolate reductase lead to a decrease in red color intensity (compared to the control) to white sediments. Substances that do not specifically inhibit the strain with the bacterial dihydrofolate reductase but may have generated a signal in the first screening run because they give the control strain (BY4743 Adfrl Aade2 + pDE95-HDFR) a selection advantage due to the / 4DE2 deletion are not able to shift the color of the sediment from red to white, but also lead to a color intensification of the red sediment.

Thus, after these two screening runs, substances are identified that specifically target dihydrofolate reductase at the site of action and at the same time selectively inhibit the bacterial enzyme more strongly than the human control enzyme.

Example 7:

Analysis of target-specific inhibitors by the PDS method (test organism: E. coli)

For the same target (dihydrofolate reductase), a bacterial test organism (e.g. Escherichia coli) can also be used to search for growth site-specific growth inhibitors.

For this purpose, the gene coding for human dihydrofolate reductase is cloned into an E. coli expression vector. Both adjustable (lac, tac, trp, 17, araB, phoA) and constitutive promoters can be used as promoters. This plasmid is introduced into the E. coli control strain by transformation (e.g. electroporation). The target strain contains no plasmid or only an empty vector. Thus, the control and target strains differ only in one genetic trait, namely in the additional expression of the human dihydrofolate reductase in the control strain. If a substance specifically inhibits bacterial dihydrofolate reductase (folA), this leads to a specific growth inhibition of the target strain.

In addition, the control strain (alternatively the target strain) also contains a plasmid which ensures the constitutive expression of the green fluorescent protein (GFP). The control and target strains are grown, mixed and the wells of a microtiter plate (filled with fresh medium) are inoculated thinly. Now substances (or whole substance libraries) are added and then incubated at 37 ° C. After reaching the stationary growth phase (overnight incubation), the composition of the cavity cultures with control and target strain is determined by means of the GFP fluorescence of each individual cavity using a fluorimeter. Cavities that show increased fluorescence contain more cells from the control strain, which indicates a selective inhibition of the Taget strain (and thus the bacterial dihydrofolate reductase).

Example 8:

PDS haploinsufficiency screening

In diploid organisms, a copy of a gene is usually sufficient for the normal growth of the organism under optimal conditions (e.g. full media for microorganisms).

However, if the growth conditions are changed, e.g. Haplo insufficiency is often induced by adding active substances that are specifically directed against the gene product (protein / enzyme) of the remaining alley, since the primary effect of the active substance causes a further impairment of the function of the remaining protein. Now the amount and thus the activity of the protein (e.g. enzyme-catalytic activity) in organisms that only have one allele of the corresponding gene is no longer sufficient to ensure normal cell growth. Growth phenotypes occur, whereas wild-type cells (2 alleles of the gene) are influenced less or not at all with the same concentration of active substance (gene dose effect).

Haploinsufficiency, ie the occurrence of phenotypes when an alley of approximately 1100 essential genes fails, is very rarely observed, particularly in the test organism S. cerevisiae. However, haplo insufficiency can be induced very simply by adding growth site-specific growth inhibitors. This fact has already been used to characterize the sites of action of various substances in S. cerevisae (Giaever et al., 1999). For this purpose, numerous 2n heterozygous strains, in which one allele of an essential gene has been deleted, are mixed. The culture was divided, half was made with an active ingredient which has a toxic effect on the yeast in higher concentrations, while the other half remained untreated. If the substance now acts in a site-specific manner on one of the essential genes / proteins, the strain which is haploid for this gene is more strongly inhibited than the other strains of the mixture.

Previous methods have taken advantage of the fact that each deletion cassette with which an allele in S. cerevisiae was deleted contains a gene-specific 20 bp long sequence. With the help of these sequences, each gene deletion strain can be identified using molecular biological methods (PCR). Chromosomal DNA is now isolated from both total cultures, the specific sequences of all strains are amplified by means of PCR and the representation of the individual strains in the cultures is finally determined using the PCR products obtained in this way via DNA array analyzes. Information about which strains are selectively inhibited in the culture with the active ingredient can ultimately be used to make statements about the mechanism of action, and ideally the target can be identified.

This method is very interesting in order to identify places of action and mechanisms of action of cytotoxic extracts. This knowledge enables the potential to be e.g. antifungal agents can be estimated. However, this method has so far only been feasible with the help of complex and technically demanding DNA array analyzes and is completely unsuitable for investigating substances in HTS.

The procedure is massively simplified with the help of PDS haploinsufficiency screening. A collection of 2n heterozygous strains, in which one allele of an essential gene was deleted (Aess. Gen / ESS.GEN), was greatly diluted and placed in two cavities of a microtiter plate. A diploid strain in which both alleles of the ADE2 gene were deleted (Aade2 / Aade2) was diluted with fresh medium and 100 μl each was added to the cavities to the presented strains. The two strains of a cavity were present in the same dilution. Now half of the cavities (each Stock mixture once) with a sublethal dose of ketoconazole (1.5 μl of a 0.05 mg ketoconazole / ml DMSO), the other half with DMSO.

The plates were incubated for two days at 30 ° C., the formation of the red color pigment was induced (as described in Example 5) and then evaluated. Ketoconazole is an antifungal substance that specifically inhibits the activity of Lanosterol Demethylase (Erg11p). Accordingly, PDS haploinsufficiency screening showed a clear red signal in the cavity with the parent combination Aade2 / Aade2 + Aerg11 / ERG11, to which ketoconazole was added, compared to the control with DMSO. This different staining behavior is specific for this strain combination, all other strain combinations showed no different staining between the active ingredient and the control cavity.

With the help of this method, the DNA array analysis described above has been massively simplified and the mechanism of action for a large number of antifungal substances can now be characterized in more detail by simple means, by comparing their differential effect on the wild type with the effect on the 2n heterozygotes Deletion strain is examined.

Claims

claims: 1. High-throughput suitable screening method for identifying active substances, characterized in that a) a target organism is selected which the active substances to be identified are to inhibit or kill, b) a target gene is selected which, or the gene product thereof, is deactivated by the active substances to be identified c) selects an organism to be protected that is to be protected from damage by the target organism, d) selects a test organism that carries a test gene that is functionally homologous to the target gene, e) constructs two test strains of a test organism that genotypically have exactly two characteristics distinguish, namely i. in the target gene, in that one of the test strains tolerates a higher dose of an active ingredient which deactivates the target gene or its gene product than the other test strain and ii. in a gene that codes for a well-detectable property, but is not essential for the vitality and proliferation ability of the test organism, f) mixes and incubates both test strains, g) adds a potential active ingredient and h) based on the possibly differently strong growth of the two Test strains identified an active ingredient 2. The method according to claim 1, characterized in that the target organism is selected from humans and microorganisms, in particular pathogenic organisms, preferably from plant, animal and / or human pathogenic organisms, preferably from human pathogenic fungi or bacteria. 3. The method according to claim 1 or 2, characterized in that the target gene is selected from genes which are essential for the vegetative growth of The target organism, in particular all target genes of a target organism, are the functional homologues to essential genes of S. cerevisiae. 4. The method according to any one of the preceding claims, characterized in that the organism to be protected is selected from animals, plants and humans. 5. The method according to any one of the preceding claims, characterized in that the test organism is selected from prokaryotes, such as species of the genera Bacillus (B. subtilis), Escherichia (E. coli), Klebsiella [K. planticola), Lactobacillus (L. delbruckii, L. lactis), Pseudomonas (P. aeroginosa, P. fluorescens) Salmonella (S. typhimurium) Serratia (S. marcescens) Streptococcus (S. lactis, S. mutans, S. pyogenes) , Staphylococcus (S. aureus, S. epidermidis) Vibrio (V. cholerae) Yersinia (Y. ruckeri), in particular Escherichia coli and Bacillus subtilis and eukaryotes, such as yeasts, in particular species from the genera Saccharomyces, Schizosaccharomyces, Candida, Kluyveromyces, Yarrowia , Ashbya, Hansenula, Pichia, particularly preferably Saccharomyces cerevisae, Schizosaccharomyces pombe or Candida albicans, very particularly preferably Saccharomyces cerevisiae strains, preferably the strains CEN.PK2, BY4743, BMA46 (W303), FY1679 and their haploid derivatives. 6. The method according to any one of the preceding claims, characterized in that it is carried out in a plurality of parallel approaches. 7. Test kit for identifying active substances, which comprises means for carrying out the method according to one of the preceding claims. 8. A method for producing a cosmetic or pharmaceutical preparation, characterized in that c) the method according to one of claims 1 to 6 is carried out and an active ingredient is thus determined and d) active ingredients found to be effective are mixed with cosmetically and / or pharmacologically suitable and compatible carriers. 9. Use of the method according to one of claims 1 to 6 or the test kit according to claim 7 for finding active ingredients.