[go: up one dir, main page]

WO1997039143A2 - Procede pour identifier de nouveaux agents antifongiques - Google Patents

Procede pour identifier de nouveaux agents antifongiques Download PDF

Info

Publication number
WO1997039143A2
WO1997039143A2 PCT/US1997/006304 US9706304W WO9739143A2 WO 1997039143 A2 WO1997039143 A2 WO 1997039143A2 US 9706304 W US9706304 W US 9706304W WO 9739143 A2 WO9739143 A2 WO 9739143A2
Authority
WO
WIPO (PCT)
Prior art keywords
kinase
substrate
phosphorylation
fungal
test compound
Prior art date
Application number
PCT/US1997/006304
Other languages
English (en)
Other versions
WO1997039143A3 (fr
Inventor
Jennifer Marion Fostel
Mark Edward Winey
Estelle Marthe Steiner
Francis Conrad Luca
Original Assignee
Abbott Laboratories
University Technology Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Abbott Laboratories, University Technology Corporation filed Critical Abbott Laboratories
Publication of WO1997039143A2 publication Critical patent/WO1997039143A2/fr
Publication of WO1997039143A3 publication Critical patent/WO1997039143A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general

Definitions

  • the present invention relates to methods for the detection of antifungal agents.
  • the invention relates to a new fungal enzyme and the use thereof as a target in whole- cell and purified-enzyme screening assays by means of which agents capable of affecting the function of that enzyme may be identified.
  • antifungal drug discovery efforts have been directed at components of the fungal cell or its metabolism which are unique to fungi, and hence might be used as therapeutic targets of new agents which act on the fungal pathogen without undue toxicity to host cells.
  • potential targets include enzymes critical to fungal cell wall assembly as well as topoisomerases (enzymes required for replication of fungal DNA).
  • a preferred method continues to be the mass screening of compound "libraries" for active agents by exposing cultures of fungal pathogens to the test compounds and assaying for inhibition of growth.
  • a correspondingly large number of fungal cultures must be grown over time periods which are relatively long compared to most bacterial culture times.
  • a compound which is found to inhibit fungal growth in culture may be acting not on the desired target but on a different, less unique fungal component, with the result that the compound may act against host cells as well and thereby produce unacceptable side effects.
  • the present invention comprises the use of a known, unique component of fungal cells, the checkpoint protein kinase Mpslp, as an intracellular target for antifungal drug therapy as well as a target in assays for the identification of agents which act specifically on that protein.
  • Mpslp for "monopole spindle” is a dual-specificity protein kinase encoded for by the MPSl gene of Saccharomyces cerevisiae and essential to two aspects of yeast cell growth, namely, (i) duplication of the spindle pole body ("SPB”) which is the microtuble organizing center for the mitotic spindle and (ii) arrest of the cell cycle at a critical checkpoint in the presence of microtuble damage or loss of spindle integrity.
  • SPB spindle pole body
  • Genetic inactivation of the MPSl gene is lethal, as affected cells first fail to duplicate the SPB leading to an aberrant, monopolar spindle and then fail to arrest their growth cycle, continuing through mitosis to produce non
  • the SPB is a structure which is shared by many species of fungi, but which is markedly different from the centrosome which fulfills the analogous function in higher eukaryotes.
  • searches of available gene databases have failed to identify mammalian homologues to the Mpslp protein. Consequently, it is believed that proteins such as Mpslp kinase and similar fungal kinases will appear widely among fungi but will not have close counterparts in mammalian cells. Inhibitors of Mpslp are therefore expected to have (i) a fungicidal mode of action, (ii) broad-spectrum antifungal activity, and (iii) no specific target in host cells and, consequently, limited potential therapeutic side effects.
  • the present invention includes a method for the identification of antifungal agents, comprising the steps of (a) combining a checkpoint protein kinase with a test compound; (b) catalyzing phosphorylation of a kinase substrate with the combined kinase and test compound under conditions suitable for said phosphorylation; and (c) determining the extent of phosphorylation of the substrate.
  • the method is carried out in living fungal cells, i.e., the method comprises whole-cell screening assays in which (a) the kinase is a functional intracellular enzyme, and is combined with the test compound by exposing the fungal cell to the test compound; (b) the substrate is a natural substrate of the kinase, and is phosphorylated intracellularly by culturing the exposed fungal cell under conditions suitable for growth; and (c) the extent of phosphorylation is determined indirectly by observing the ability of the fungal cell to reproduce.
  • the kinase is a functional intracellular enzyme, and is combined with the test compound by exposing the fungal cell to the test compound
  • the substrate is a natural substrate of the kinase, and is phosphorylated intracellularly by culturing the exposed fungal cell under conditions suitable for growth
  • the extent of phosphorylation is determined indirectly by observing the ability of the fungal cell to reproduce.
  • the method of the present invention may be carried out ex vivo, i.e., as a cell-free screening assay in which (a) the kinase is a substantially purified polypeptide; (b) the substrate is substantially purified, and is phosphorylated in a reaction with a substantially purified phosphate source catalyzed by the kinase; and (c) the extent of phosphorylation is determined by measuring the amount of substrate phosphorylated in the reaction.
  • the present invention also includes polypeptides comprising the amino acid sequence shown in Figure 1, below, or an enzymatically active fragment or derivative thereof, as well as fusion proteins comprising such polypeptides which have been fused to an affinity tag.
  • polypeptides and fusion proteins assay targets for the identification of antifungal agents.
  • the invention includes polynucleotides encoding such polypeptides and fusion proteins and DNA plasmids encoding the same, as well as host cells transformed with such plasmids and capable of expressing the polypeptides or fusion proteins.
  • the present invention further includes a method for treating fungal infection in a patient in need of such treatment, comprising administering to said patient an antifungal agent identified according to the above assay method or a pharmaceutically acceptable salt, ester or prodrug thereof. More generally, the invention includes a method for treating fungal infection in a patient in need of such treatment which comprises administering to said patient a specific inhibitor of a fungal checkpoint protein kinase.
  • FIGURE 1 is an amino acid sequence (764 amino acids in length) representing the Saccharomyces cerevisiae checkpoint protein kinase Mpslp;
  • FIGURE 2 is a single-strand nucleotide sequence (2970 bases in length with an open reading frame of 2292 bases) representing the S. cerevisiae gene MPSl which encodes Mpslp as well as 5' and 3' flanking sequences.
  • assays for antifungal agents may be carried out both in whole-cell preparations and in ex vivo, cell-free systems.
  • the assay target is a checkpoint protein kinase essential for cell cycle control, the inhibition of which significantly attenuates or, preferably, causes lethally aberrant cell growth and reproduction.
  • Test compounds which are found to inhibit a target kinase in an assay of the present invention are thus identified as potential antifungal agents.
  • the assay methods of the present invention will be suitable for both small- and large-scale screening of test compounds, as well as in quantitative assays such as serial dilution studies wherein the target kinase is exposed to a range of test compound concentrations.
  • the target kinase is an intracellular enzyme and the entire, living fungal cell is exposed to the test compound under culture conditions normally suitable for growth.
  • Such conditions including essential nutrients, optimal temperatures and other parameters, depend upon the particular fungal strain being used and will be well-known in the art.
  • Inhibition of the kinase, and of phosphorylation of the natural kinase substrate(s) catalyzed by the kinase may be determined by observing the cell culture's growth or lack thereof; such observation may be made visually, by optical densitometric or other light absorption/scattering means, or by yet other suitable means, whether manual or automated.
  • the method may be performed as a paired-cell assay in which each test compound is separately tested against two different fungal cells, the first having a lower kinase activity than that of the second and thereby being more susceptible to inhibition of the enzyme. Compounds which are found to inhibit the first, more susceptible fungal cell but not the second are likely to have acted specifically on the target kinase and not via a different mechanism.
  • One manner of achieving differential susceptibility is by using a first fungal cell which has diminished kinase activity relative to that of a wild-type cell, as for example a mutant strain expressing a modified kinase with reduced catalytic function.
  • a particularly useful strain is one having a temperature-sensitive ("ts") mutation, as a result of which the kinase is more prone than the wild-type enzyme to loss of activity at high temperatures (i.e., temperatures higher than optimal but still permitting growth in wild-type cells).
  • ts temperature-sensitive
  • ts mutations in other fungal enzymes include the yeast gene CDC7, in which a ts mutation (described by Hollingsworth et al. in Genetics 132:53-62, 1992) is known to be correlated with decreased in vivo activity of the corresponding protein kinase, as well as a ts mutation affecting the TOP2 gene encoding a type II DNA topoisomerase (described by Nitiss et al. in Cancer Research 53:89-93, 1993).
  • yeast gene CDC7 in which a ts mutation (described by Hollingsworth et al. in Genetics 132:53-62, 1992) is known to be correlated with decreased in vivo activity of the corresponding protein kinase, as well as a ts mutation affecting the TOP2 gene encoding a type II DNA topoisomerase (described by Nitiss et al. in Cancer Research 53:89-93, 1993).
  • differential susceptibility to kinase inhibitors may be obtained by using a second fungal cell which has increased kinase activity relative to that of a wild-type cell, as for example one which has been genetically manipulated to cause overexpression of the kinase.
  • Such overexpression can be achieved by placing into a wild- type cell a plasmid carrying the gene for the target kinase.
  • the techniques for generating temperature-sensitive mutants, for preparing specific plasmids, and for transforming cell lines with such plasmids are well-known in the art.
  • the whole-cell assay methods of the present invention are those in which the target kinase is an MPSl gene product, and especially Mpslp. Also preferred are those assay methods in which the target kinase is an SPK1 gene product, and especially Spklp.
  • the target kinase is a substantially purified polypeptide
  • the kinase substrate is a substantially purified substrate which in the assay is phosphorylated in a reaction with a substantially purified phosphate source catalyzed by the kinase; and the extent of phosphorylation is determined by measuring the amount of substrate phosphorylated in the reaction.
  • substrates may be used, including the kinase itself in which instance the phosphorylation reaction measured in the assay is autophosphorylation.
  • Exogenous substrates may also be used, including standard protein substrates such as myelin basic protein (MBP); yeast protein substrates; synthetic peptide substrates; and polymer substrates. Of these, MBP and other standard protein substrates may be regarded as preferred. Other substrates may be identified, however, which are superior by way of affinity for the kinase, minimal perturbation of reaction kinetics, possession of single or homogenous reaction sites, ease of handling and post-reaction recovery, potential for strong signal generation, and resistance or inertness to test compounds.
  • MBP myelin basic protein
  • yeast protein substrates yeast protein substrates
  • synthetic peptide substrates synthetic peptide substrates
  • polymer substrates polymer substrates.
  • Other substrates may be identified, however, which are superior by way of affinity for the kinase, minimal perturbation of reaction kinetics, possession of single or homogenous reaction sites, ease of handling and post-reaction recovery, potential for strong signal generation, and resistance or inertness to test compounds.
  • Measurement of the amount of substrate phosphorylated in the ex vivo assay of the invention may be carried out by means of immunoassay, radioassay or other well-known methods.
  • an antibody such as a goat or mouse anti-phosphotyrosine antibody
  • an antibody such as a goat or mouse anti-phosphotyrosine antibody
  • the phosphotyrosine- antibody complex would itself be detected by a further antibody linked to a label capable of developing a measurable signal (as for example a flourescent or radioactive label).
  • the phosphate source may be radiolabelled with an isotope such as 32p 0 r 33p, and the amount of substrate phosphorylation may be measured by determining the amount of radiolabel incorporated into the substrate during the reaction; commercially available scintillant- containing plates, beads and reagents are readily available with which radioactive substrate may be detected, using a beta-counter, after adsorbtion to a filter or a microtiter well surface, or by photometric means after binding to a scintillation proximity assay bead or scintillant plate. (References describing such methods include Oude Weernink and Kijksen, J. Biochem. Biophys.
  • the substrate is attached to a solid support surface by means of non-specific or, preferably, specific binding. Such attachment permits separation of the phosphorylated subtrate from unincorporated, labeled phosphate source (such as adenosine triphosphate) prior to signal detection.
  • the substrate may be physically immobilized prior to reaction, as through the use of NuncTM high protein binding plates (described in Hanke et ai, J. Biol. Chem. 271:695, 1996) or Wallac ScintiStripTM plates (described in Braunwalder et al., Anal. Biochem. 234:23, 1996).
  • Substrate may also be immobilized after reaction by capture on, for example, P81 phosphocellulose (for basic peptides), PEI/acidic molybdate resin or DEAE, or TCA precipitation onto WhatmanTM 3MM paper (described in Tiganis et al., Arch. Biochem. Biophys. 325:289, 1996; Morawetz et al., Mol. Gen. Genet. 250:17, 1996; Budde et ai, Int. J. Pharmacognosy 33:27, 1995; and Casnellie, Meth. Enz. 200: 115, 1991).
  • P81 phosphocellulose for basic peptides
  • PEI/acidic molybdate resin or DEAE or TCA precipitation onto WhatmanTM 3MM paper
  • affinity tags such as glutathione-S-transferase (GST), biotin, His6, myc and hemagglutinin.
  • GST glutathione-S-transferase
  • Such tags are capable of directly or indirectly binding the substrate to the support surface, as by conjugation with binding partners such as glutathione and streptavidin (in the case of GST and biotin, respectively) which have been attached to the support, or via antibodies specific for the tags which are likewise attached to the support.
  • checkpoint protein kinases will be identified which are relatively unique to fungi and, accordingly, are suitable for use in the assay methods of the present invention and also as therapeutic targets for antifungal agents.
  • Preferred, however, among the ex vivo assay methods are those in which the target kinase is an MPSl gene product, and particulary Mpslp or an enzymatically active fragment or derivative thereof, as well as homologs thereof obtained from fungi other than 5. cerevisiae (including Candida albicans or other Candida species).
  • the target kinase is an SPK1 gene product, and particularly Spklp, an enzymatically active fragment or derivative thereof, or a suitable homolog thereof.
  • fusion proteins comprising the respective Mpslp, Spklp or homologous kinase, or an enzymatically active fragment or derivative thereof, linked to an affinity tag such as GST.
  • affinity tag such as GST.
  • Such fusion proteins are readily expressible in yeast culture (as described in Mitchell et al., Yeast 9:715-723, 1993), and are easily purified by means of affinity chromatography.
  • the bound peptides may be enzymatically cleaved to release the enzyme portion of the chimera; alternatively, the assay may be conducted using the entire, bound fusion protein.
  • both the kinase and the substrate may be linked to affinity tags and bound to adjacent or even the same solid support.
  • polypeptides of the present invention are proteins having the amino acid sequence of Figure 1 as well as enzymatically active fragments or derivatives thereof. It is expected that, because the target kinases used in the assay methods of the invention comprise multiple domains, not all of the kinase is essential to kinase activity or autophosphorylation, and that polypeptides obtained by significant truncation and/or re ⁇ arrangement of the native enzyme amino acid sequence may be used.
  • modified proteins also may be expressed as fusion proteins or chimera in which they are fused to an affinity tag such as GST or biotin, of which GST is the more preferred.
  • a polynucleotide encoding a polypeptide of the invention is operably linked with a promoter sequence capable of directing expression of the polynucleotide in a suitable host cell.
  • the transformed host cell When inserted into the host cell, the transformed host cell may be cultured under suitable conditions until sufficient levels of the polypeptide are achieved, after which the cells may be lysed and the polypeptide isolated.
  • the polynucleotide itself may be isolated from fungal cells using familiar techniques for the fragmentation and sequence analysis of genetic material, including the use of oligonucleotide probes and PCR/LCR amplification techniques to identify known kinase sequences or suspected sequences having substantial sequence homology with known kinase genes.
  • a specific inhibitor of a fungal checkpoint protein kinase is administered to a patient in need of such treatment to suppress or prevent fungal infection; preferred are those agents which are specific inhibitors of the protein kinases Mpslp or Spklp.
  • the compound so administered may be an antifungal agent identified according to the assay methods of the invention or a pharmaceutically acceptable salt, ester or prodrug thereof.
  • the compound When used in a treatment, the compound may be employed in pure form or administered as a pharmaceutical composition containing the compound of interest in combination with one or more pharmaceutically acceptable excipients.
  • a therapeutically effective amount of the inhibiting compound may be given for such time as is necessary to achieve the desired result.
  • a “therapeutically effective amount” of the compound of the invention is meant a sufficient amount of the compound to treat or prevent pathogenic or opportunistic fungal infection at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgement.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than required for to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • Activity in connection with a protein kinase of the invention or other enzyme refers to both the quantity and the catalytic potential of the enzyme; accordingly, decreased activity can result both from reduction in the amount of enzyme or attenuation of its catalytic strength, as by mutation or denaturation.
  • Antifungal agent refers to a compound found to be active against a target checkpoint kinase of the present invention and therefore more likely to inhibit fungal growth by inhibiting activity of that kinase in vivo.
  • Checkpoint protein kinase refers to a fungal enzyme which functions at one of the cell cycle control points to assist in the regulation and timing of cell growth and replication, and triggers arrest of the cell cycle upon one of a variety of events. Numerous checkpoints exist in yeast and other cells; some produce a lethal arrest while others merely place the cell in a dormant state which is ended upon reversal of the triggering condition.
  • yeast checkpoints include a DNA integrity checkpoint, responsive to DNA damage and/or a blocking of DNA replication, which causes arrest in the Gl, S and G2 phases (depending on when the damage occurs); a spindle integrity checkpoint, responsive to microtubule damage or spindle malfunction, which causes arrest in the G2 phase (just prior to mitosis); a successful division checkpoint, responsive to normal post-anaphase, which causes arrest prior to exit from the M phase and resumption of Gl; and the START checkpoint, responsive to cell size, nutrient levels and mating pheromones, which produces a minimally- lethal arrest in the Gl phase.
  • Preferred checkpoints in the context of the assays and methods of treatment of the present invention are those which produce a deleterious or lethal arrest of the cell cycle, and which have no close homologs in mammalian cells.
  • DNA plasmid refers to an extrachromosomal, independently reproducing genetic element capable of acting as a vector for the cloning and/or expression of a gene of interest.
  • Enzymatically active fragments or derivatives refers, in the context of proteins or DNA, to polypeptides or polynucleotides generated by truncation, substitution or rearrangement of portions of an original sequence so as to substantially retain the characteristic biological properties of the original sequence.
  • yeast cells refers to yeasts and other fungi including, but not limited to, Saccharomyces cerevisiae, Pichia species and Candida albicans.
  • Saccharomyces cerevisiae In the context of fungal cells used to express the target kinase and/or in whole-cell screening assays, S. cerevisiae is preferred.
  • Candida, Histoplasma and Aspergillus species are more representative.
  • Fusion protein refers to the expression product of two or more genes or gene fragments joined in a contiguous open reading frame and expressed as a single polypeptide.
  • Gene product refers to an expressed polypeptide comprising some or all of the amino acid sequence encoded by a gene or a mutant or derivative thereof, whether or not modified during or after transcription or translation, and optionally fused to other terminal or intervening amino acid sequence.
  • An example of a gene product obtained from the MPSl gene is native Mpslp protein kinase which, following expression, must be partially phosphorylated before becoming enzymatically active.
  • “Host cell” refers, in connection with expression of cloned genes, to culturable cells capable of replicating and expressing those genes. For expression of yeast kinases, yeast host cells are preferred.
  • Identification of antifungal agents refers to the determination that a particular test compound is a more rather than less likely candidate for use in antifungal therapy.
  • Kinase substrate refers to such compounds as are capapable of phosphorylation by a protein kinase of the present invention, and includes both natural substrates (i.e., those present in the living cell and acted upon by the kinase) and artificial substrates (such as standard protein substrates, synthetic peptide substrates and yeast protein substrates, defined below).
  • the kinase itself may be a kinase substrate, as in the case of autophosphorylation.
  • “Pharmaceutically acceptable salts, esters and prodrugs” refers to those derivative compounds which are, within the scope of sound medical judgement, suitable for use in contact with with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use. Discussions of salts and prodrugs are found in Berge et al., "Pharmaceutical Salts,” J. Pharm. Sci. 66:1-19, 1977; Higuchi and Stella, "Pro-drugs as Novel Delivery Systems", Vol 14 of the A.C.S. Symposium Series; and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.
  • Polymer substrates refers to amino acid polymers including, but not limited to poly(arg-ser) n 3:1, poly(glu-thr) n 4.4:1, and poly(glu-tyr) n 4: 1, described by Racker (Meth. Enz. 200:107, 1991) and available commercially.
  • Polypeptide refers to a polymer of amino acids covalently joined by amide linkages between the carboxyl group of one amino acid and the alpha-amino group of the next, in which the amino acids are taken from the 20 standard (naturally-occurring) amino acids.
  • Solid support surface refers to an inert surface upon which certain reagents used in the assay methods of the present invention may by temporarily or permanently immobilized, including but not limited to beads, microtitre wells and filter disks.
  • Standard protein substrates refers to proteins commonly used as non-native kinase substrates including, but not limited to, myelin basic protein (MBP), acid-denatured enolase and hi stone.
  • Substantially purified refers to protein kinases, substrates or phosphate sources which are sufficiently free of contaminants as to permit their use in the assay methods of the present invention without undue degradation of result.
  • Synthetic peptide substrates refers to non-naturally occurring kinase substrates including, but not hmited to, commercial peptides designed for use as kinase substrates, derivatives obtained by modification of known substrates, and other suitable peptides selected from a combinatorial peptide library.
  • Test compound refers to a chemical compound or mixture of compounds to be evaluated according to the assay method of the present invention.
  • yeast protein substrates refers to native yeast proteins which are not the primary substrate of a protein kinase but which interact with the kinase and are potentially phosphorylated by it, as for example Spklp which interacts with and is a potential substrate of Mpslp.
  • the Saccharomyces cerevisiae MPS 1 gene was isolated and characterized as described in Lauze et al. in The EMBO Journal 14(8): 1655-1663, 1995, by identification and examination of a plasmid which complemented the mpsl-1 temperature sensitive mutation. Further fragmenting and analysis of the plasmid demonstrated that the isolated gene, designated MPSl, was that responsible for SPB formation and related checkpoint control.
  • a pEG(KT) plasmid prepared as described in Mitchell et al. (Yeast 9:715-723, 1993) and carrying the MPSl gene as a fusion to GST, was expressed in FLY 14 cells (BJ2168 [a leu2 trpl ura3-52 prbl-1122 pep4-3 prcl-407 gal2] with barl). Transformed cells were grown under selection with 2% raffinose carbon source to late log phase; the culture was then diluted 100-fold and grown overnight. After further dilution to 0.5 OD and addition of sucrose-free galactose to 4%, the culture was grown another 5-6 hours.
  • the cells were coUected, washed with sorbitol break buffer (0.3 M sorbitol, 100 mM NaCl, 5 mM MgC12 10 mM Tris pH 7.4, 0.1 to IX protease inhibitor cocktail ("PI", Boehringer Mannheim), 5 mM DTT, 1 mM PMSF, 25 mM glycerol phosphate), frozen on dry ice and stored at -80° C.
  • sorbitol break buffer 0.3 M sorbitol, 100 mM NaCl, 5 mM MgC12 10 mM Tris pH 7.4, 0.1 to IX protease inhibitor cocktail ("PI", Boehringer Mannheim), 5 mM DTT, 1 mM PMSF, 25 mM glycerol phosphate
  • the cells were then thawed, suspended in sorbitol break buffer with fresh PI/DTT/PMSF, and disrupted with three passes in a French press at 900 psi, and the supernatant was clarified by centrifugation. Fresh PI/DTT/PMSF was added and the suspension incubated on a LabquakeTM mixer with glutathione-Sepharose (Pharmacia) for 1 hour. The resin was recovered and the adsorption was repeated an additional two times. The resin was then washed 4X in phosphate buffered saline and PI/DTT/PMSF and stored in aliquots at -80°C.
  • the Mpslp beads prepared in Example 2 were added to a reaction cocktail containing 50 mM Tris pH 6.8, 10 mM MgC12, 5 ⁇ M ATP, 1 ⁇ Ci ⁇ - 32 P-ATP, 1 mM PMSF, and 0.5 mM DTT for 30 minutes at 30°C. 4 ⁇ g of heterologous substrate myelin basic protein was also be added. To each mixture was added one of a series of test compounds at one of a series of concentrations. The reactions were stopped with 2X Laemmli buffer, boiled for 5 minutes, and analyzed by 10-18% SDS-PAGE and autoradiography to assess the amount of radiolabel incorporated into different substrates.
  • Temperature- sensitive, wild-type and MPSZ-overexpressing S. cerevisiae cells were prepared using standard transformation protocols and were grown at 23-26°C under auxotrophic selection in rrtinimal medium to approximately IO 7 cells/ml, each aliquot being mixed with a vortex mixer prior to counting to disperse clumps.
  • the ceUs were then diluted to a concentration of 2xl0 5 cells/ml in minimal medium, and aliquots of 200 ⁇ l per well were dispensed into a flat-bottomed microtiter plate containing approximately 50 suspected kinase inhibitors dissolved in DMSO such that ts, wild-type and overexpressing ceUs were paired in aU possible combinations.
  • the plates were incubated at 23-26°C until growth of the ceUs in wells without test compound was confluent, about 6 to 9 days. Growth of cells was scored both by eye and by measuring absorbance at 595 nm.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des méthodes de criblage permettant d'identifier des agents antifongiques. Dans lesdites méthodes (a) on combine une protéine kinase de point de contrôle appropriée à un composé test; (b) on catalyse la phosphorylation d'un substrat à base de kinase, la kinase et le composé test étant dans des conditions appropriées pour ladite phosphorylation; et (c) on détermine le degré de phosphorylation du substrat. L'invention concerne également des polypeptides et des protéines de fusion utiles pour lesdites méthodes, ainsi que des ADN plasmidiques et des cellules hôtes transformées utiles pour la préparation desdits polypeptides et protéines de fusion.
PCT/US1997/006304 1996-04-17 1997-04-17 Procede pour identifier de nouveaux agents antifongiques WO1997039143A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US63361796A 1996-04-17 1996-04-17
US08/633,617 1996-04-17

Publications (2)

Publication Number Publication Date
WO1997039143A2 true WO1997039143A2 (fr) 1997-10-23
WO1997039143A3 WO1997039143A3 (fr) 1998-05-14

Family

ID=24540386

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/006304 WO1997039143A2 (fr) 1996-04-17 1997-04-17 Procede pour identifier de nouveaux agents antifongiques

Country Status (1)

Country Link
WO (1) WO1997039143A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998044148A1 (fr) * 1997-03-28 1998-10-08 Mycotox, Inc. Dosage d'histidine kinase
US6670167B1 (en) 1999-11-01 2003-12-30 Agouron Pharmaceuticals, Inc. Catalytic domain of the human effector cell cycle checkpoint protein kinase materials and methods for identification of inhibitors thereof
WO2005054457A1 (fr) * 2003-12-03 2005-06-16 Bayer Cropscience Aktiengesellschaft Procede pour identifier des composes fongicides a partir de pyruvate kinases de champignons

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0446798B1 (fr) * 1990-03-13 1994-11-23 Hoechst-Roussel Pharmaceuticals Incorporated Alkyl-1, alcényl-1 et alkynylaryl-1 amino-2 propanediols-1,3 et composés apparentés, procédé et produits intermédiaires pour leur préparation et leur application comme médicaments
US5418144A (en) * 1993-05-25 1995-05-23 American Cyanamid Company Spindle pole body screen for fungicides
US6348310B1 (en) * 1994-03-04 2002-02-19 Promega Corporation Quantitation of individual protein kinase activity

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998044148A1 (fr) * 1997-03-28 1998-10-08 Mycotox, Inc. Dosage d'histidine kinase
US6670167B1 (en) 1999-11-01 2003-12-30 Agouron Pharmaceuticals, Inc. Catalytic domain of the human effector cell cycle checkpoint protein kinase materials and methods for identification of inhibitors thereof
WO2005054457A1 (fr) * 2003-12-03 2005-06-16 Bayer Cropscience Aktiengesellschaft Procede pour identifier des composes fongicides a partir de pyruvate kinases de champignons

Also Published As

Publication number Publication date
WO1997039143A3 (fr) 1998-05-14

Similar Documents

Publication Publication Date Title
Tyers et al. The Cln3‐Cdc28 kinase complex of S. cerevisiae is regulated by proteolysis and phosphorylation.
CA2171982C (fr) Proteine de liaison de medicament
Michaud et al. 14-3-3 is not essential for Raf-1 function: identification of Raf-1 proteins that are biologically activated in a 14-3-3-and Ras-independent manner
JP3091769B2 (ja) プロテインキナーゼ
Winston et al. The SCFβ-TRCP–ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IκBα and β-catenin and stimulates IκBα ubiquitination in vitro
Shiozaki et al. Heat stress activates fission yeast Spc1/StyI MAPK by a MEKK-independent mechanism
Roelants et al. Differential roles of PDK1-and PDK2-phosphorylation sites in the yeast AGC kinases Ypk1, Pkc1 and Sch9
Lanker et al. Rapid degradation of the G1 cyclin Cln2 induced by CDK-dependent phosphorylation
Alessi et al. Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines
Casamayor et al. Functional counterparts of mammalian protein kinases PDK1 and SGK in budding yeast
Takegawa et al. Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology
Lu et al. The interferon-induced double-stranded RNA-activated protein kinase PKR will phosphorylate serine, threonine, or tyrosine at residue 51 in eukaryotic initiation factor 2α
US20100105064A1 (en) Method of screening test substances for treating or preventing a disease mediated by plasma cells
NZ503983A (en) An isolated nucleic acid encoding human checkpoint kinase, HCDS1 for use in making medicaments treating cancer or proliferative diseases
EP1281770A2 (fr) Phosphodiesterase du cerveau humain et procédé de criblage
US7132278B2 (en) PAS kinase
US6207393B1 (en) Inhibition of intracellular signal transduction by 14-3-3-binding peptides
Pitluk et al. Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis
EP0889971B1 (fr) Modulateur de la phosphatase
WO1997039143A2 (fr) Procede pour identifier de nouveaux agents antifongiques
US6383753B1 (en) Yeast mammalian regulators of cell proliferation
Zelada et al. Interactions between regulatory and catalytic subunits of the Candida albicans cAMP-dependent protein kinase are modulated by autophosphorylation of the regulatory subunit
US20100055719A1 (en) Screening method for identifying new aminoacyl-tRNA synthetase inhibitors
US7252944B2 (en) Methods and compositions for modulating cell proliferation
RU2252256C2 (ru) ГОМОЛОГИ (ATR) ПОЛИПЕПТИДА rad3, ПОЛИНУКЛЕОТИДЫ И СВЯЗАННЫЕ С НИМИ СПОСОБЫ И МАТЕРИАЛЫ

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): CA JP MX

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): CA JP MX

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 97537333

Format of ref document f/p: F

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA