Assays and reagents for identifying anti-fungal agnets, amd uses related thereto

Abstract

The present invention relates to rapid, reliable and effective assays for screening and identifying pharmaceutically effective compounds that specifically inhibit the biological activity of fungal GTPase proteins, particularly GTPases involved in cell wall integrity, hyphael formation, and/or other cellular functions critical to pathogenesis.

We claim:  
     
         1 . An assay for identifying compounds having potential anti-fungal activity, comprising: 
 (a) forming a reaction mixture including: 
 (i) a fungal geranylgeranyl transferase (GGPTase),  
 (ii) a GGPTase substrate, and  
 (iii) a test compound; and  
   (b) detecting interaction of the GGPTase substrate with the GGPTase,    wherein a statistically significant decrease in the interaction of the GGPTase substrate and GGPTase in the presence of the test compound, relative to the level of interaction in the absence of the test compound, indicates a potential anti-fungal activity for the test compound.    
     
     
         2 . The assay of  claim 1 , wherein GGPTase substrate comprises target polypeptide comprising a fungal Rho-like GTPase, or a polypeptide portion thereof including at least one of (a) a prenylation site which can be enzymatically prenylated by the GGPTase, or (b) a GGPTase binding sequence which specifically binds the GGPTase.  
     
     
         3 . The assay of  claim 1 , wherein the reaction mixture is a prenylation system including an activated geranylgeranyl group, and the step of detecting the interaction of the GGPTase substrate with the GGPTase comprises detecting conjugation of the geranylgeranyl group to the GGPTase substrate.  
     
     
         4 . The assay of any of claims  3  or  23 , wherein at least one of the geranylgeranyl group and the GGPTase substrate comprises a detectable label, and the level of geranylgeranyl group-conjugated to the GGPTase substrate is quantified by detecting the label in at least one of the GGPTase substrate, the geranylgeranyl group, and geranylgeranyl-conjugated GGPTase substrate.  
     
     
         5 . The assay of  claim 1 , wherein the step of detecting the interaction of the GGPTase substrate with the GGPTase comprises detecting the formation of complexes including the GGPTase substrate with the GGPTase.  
     
     
         6 . The assay of  claim 5 , wherein at least one of the GGPTase and the GGPTase substrate comprises a detectable label, and the level of GGPTase/GGPTase substrate complexes formed in the reaction mixture is quantified by detecting the label in at least one of the GGPTase substrate, the GGPTase, and GGPTase/GGPTase substrate complexes.  
     
     
         7 . The method of any of claims  4  or  6 , wherein the label group is selected from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.  
     
     
         8 . The assay of  claim 4 , wherein the substrate target comprises a fluorescent label, the fluorescent characterization of which is altered by the level of prenylation of the substrate target.  
     
     
         9 . The assay of  claim 8 , wherein the substrate target comprises a dansylated peptide substrate of the fungal GGPTase.  
     
     
         10 . The assay of any of claims  3  or  23 , wherein conjugation of the geranylgeranyl group to the GGPTase substrate is detected by an immunoassay.  
     
     
         11 . The assay of  claim 5 , wherein the formation of protein-protein complexes including the GGPTase substrate with the GGPTase is detected by an immunoassay.  
     
     
         12 . The assay of any of claims  1  or  23 , wherein the reaction mixure is reconstituted protein mixture.  
     
     
         13 . The assay of any of claims  1  or  23 , wherein the reaction mixure comprises a cell lysate.  
     
     
         14 . The assay of any of claims  2  or  23 , wherein the fungal Rho-like GPTase is selected from the group consisting of Rho1, Rho2, Rsr1/Bud1 and Cdc42, and homologs thereof.  
     
     
         15 . The assay of  claim 1 , wherein the reaction mixture is a whole cell comprising heterologous nucleic acid recombinantly expressing one or more of the fungal GGPTase subunits and GGPTase substrate.  
     
     
         16 . The assay of  claim 1 , wherein the reaction mixture is a whole cell comprising a heterologous reporter gene construct comprising a reporter gene in operable linkage with a transcriptional regulatory sequence sensitive to intracellular signals transduced by interaction of the GGPTase substrate and GGPTase.  
     
     
         17 . The assay of any of claims  1 ,  23 ,  24 ,  25 , or  26  wherein the assay is repeated for a variegated library of at least 100 different test compounds.  
     
     
         18 . The assay of any of claims  1 ,  23 ,  24 ,  25 , or  26  wherein the test compound is selected from the group consisting of small organic molecules, and natural product extracts.  
     
     
         19 . The assay of any of claims  2  or  23 , wherein one or more of the GGPTase and target polypeptide are derived from a human pathogen which is implicated in mycotic infection.  
     
     
         20 . The assay of  claim 19 , wherein the mycotic infection is a mycosis selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and sporotrichosis.  
     
     
         21 . The assay of  claim 19 , wherein the human pathogen is selected from a group consisting of  Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa , and  Mucor pusillus.    
     
     
         22 . The assay of  claim 19 , wherein the human pathogen is  Pneumocystis carinii.    
     
     
         23 . An assay for identifying compounds having potential anti-fungal activity, comprising: 
 (a) forming a cell-free reaction mixture including: 
 (i) a fungal geranylgeranyl transferase (GGPTase),  
 (ii) a target polypeptide comprising a fungal Rho-like GTPase, or a polypeptide portion thereof including a prenylation site  
 (iii) an activated geranylgeranyl group,  
 (iv) a divalent cation, and  
 (v) a test compound;  
   (b) detecting conjugation of the gernaylgernayl group of the target polypeptide in the reaction mixture,    wherein a statistically significant decrease in the prenylation of the target polypeptide and GGPTase in the presence of the test compound, relative to the level of prenylation in the absence of the test compound, indicates a potential anti-fungal activity for the test compound.    
     
     
         24 . A method of identifying an agent which disrupts the ability of an geranylgeranyl protein transferase (GGPTase) to bind to a fungal GTPase, comprising: 
 i. providing an interaction trap system including 
 (a) a first fusion protein comprising at least a portion of a fungal GGPTase subunit,  
 (b) second fusion protein comprising at least a portion of a fungal GTPase, and  
 (c) a reporter gene, including a transcriptional regulatory sequence sensitive to interactions between the GGPTase portion of the first fusion protein and the GTPase portion of the second polypeptide;  
   ii. contacting the interaction trap system with a candidate agent;    iii. measuring the level of expression of a reporter gene in the presence of the candidate agent; and    iv. comparing the level of expression of the reporter gene in the presence of the candidate agent to a level of expression in the absence of the candidate agent,    wherein a decrease in the level of expression of the reporter gen in the presence of the candidate agent is indicative of an agent that inhibits interaction of the GGPTase and GTPase.    
     
     
         25 . An assay for identifying compounds having potential anti-fungal activity, comprising: 
 (i) providing a first recombinant cell including a first prenylation substrate derived from a Rho-like GTPase which is a substrate for a geranylgeranyl transferase expressed by the cell;    (ii) providing a second recombinant cell including a second prenylation substrate identical to the first prenylation substrate except that it is mutated to be a substrate for a farnesyl transferase expressed by the recombinant cell;    (iii) contacting the first and second cells with a candidate agent; and    (iv) comparing the level of prenylation of the Rho-like GTPases in first and second cells,    wherein a statistically significant decrease in the prenylation of the first prenylation substrate, relative to the level of prenylation of the second prenylation substrate, is indicative of an agent that inhibits interaction of a GGPTase and GTPase.    
     
     
         26 . An assay for screening test compounds to identify agents which modulate the interaction of a fungal geranylgeranyl transferase (GGPTase) with a fungal Rho0-like GTPase, comprising: 
 i. providing a cell expressing a recombinant form of one or more of a fungal GGPTase and a fungal Rho-like GTPase;    ii. contacting the cell with a test compound; and    iii. detecting the level of interaction of the GGPTase and Rho-like GTPase,    wherein a statistically significant change in the level of interaction of the GGPTase and Rho-like GTPase is indicative of an agent that modulates the interaction of those two proteins.    
     
     
         27 . The method of  claim 23 , wherein one or both of a GGPTase subunit or the Rho-like GTPase are fusion proteins.  
     
     
         28 . The method of  claim 23 , wherein the level of interaction of the GGPTase and Rho-like GTPase is detected by detecting prenylation of the Rho-like GTPase.  
     
     
         29 . The method of  claim 25 , wherein the Rho-like GTPase is a fusion protein further comprising a transcriptional regulatory protein, and level of prenylation of the Rho-like GTPase is detected by measuring the level of expression of a reporter gene construct which is sensitive to the transcriptional regulatory protein portion of the fusion protein, wherein inhibition of prenylation of the fusion protein results in loss of membrane partitioning of the fusion protein and increases expression of the reporter gene construct.  
     
     
         30 . The assay of any of claims  1 ,  20 ,  21 ,  22 , or  23 , which comprises a further step of preparing a pharmaceutical preparation of one or more compounds identified as having potential antifungal activity.  
     
     
         31 . An assay for identifying compounds having potential antifungal activity, comprising: 
 i. forming a reaction mixture including a fungal Rho-like GTPase, a fungal protein kinase C (PKC), and a test compound; and    ii. detecting interaction of the Rho-like GTPase and PKC, wherein a statistically significant decrease in the interaction of the Rho-like GTPase and PKC in the presence of the test compound, relative to the level of interaction in the absence of the test compound, indicates a potential antifungal activity for the test compound.    
     
     
         32 . The assay of  claim 31 , wherein the reaction mixture is a kinase system including ATP and a PKC substrate, and the step of detecting interaction of the GTPase and PKC comprises detecting phosphorylation of the PKC substrate by a PKC/GTPase complex.  
     
     
         33 . The assay of  claim 32 , wherein at least one of the PKC substrate and ATP comprises a detectable label, and the level of phosphorylation of the PKC substrate is quantified by detecting the label in at least one of the PKC substrate or ATP.  
     
     
         34 . The assay of  claim 31 , wherein the step of detecting the interaction of the GTPase with the PKC comprises detecting the formation of protein-protein complexes including the GTPase and PKC.  
     
     
         35 . The assay of  claim 34 , wherein at least one of the PKC and GTPase comprises a detectable label, and the level of PKC/GTPase complexes formed in the reaction mixture is quantified by detecting the label in at least one of the GTPase, the PKC, and PKC/GTPase complexes.  
     
     
         36 . The method of any of claims  33  or  35 , wherein the label group is selected from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.  
     
     
         37 . The assay of  claim 36 , wherein the detectable label is a protein having a measurable activity, and one of the PKC or GTPase is fusion protein including the detectable label.  
     
     
         38 . The assay of  claim 33 , wherein the PKC substrate comprises a fluorescent label, the fluorescent characterization of which is altered by the level of phosphorylation of the PKC substrate.  
     
     
         39 . The assay of  claim 33 , wherein phosphorylation of the PKC substrate is detected by immunoassay.  
     
     
         40 . The assay of  claim 34 , wherein the formation of protein-protein complexes including the GTPase and PKC is detected by an immunoassay.  
     
     
         41 . The assay of  claim 31 , wherein the reaction mixure is reconstituted protein mixture.  
     
     
         42 . The assay of  claim 31 , wherein the reaction mixure comprises a cell lysate.  
     
     
         43 . The assay of  claim 31 , wherein the GPTase is selected from the group consisting of Rho1, Rho2, Rsr1/Bud1 and Cdc42, and fungal homologs thereof.  
     
     
         44 . The assay of  claim 31 , wherein the reaction mixture is a whole cell comprising heterologous nucleic acid recombinantly expressing one or more of the PKC and GTPase.  
     
     
         45 . The assay of  claim 31 , wherein the reaction mixture is a whole cell comprising a heterologous reporter gene construct comprising a reporter gene in operable linkage with a transcriptional regulatory sequence sensitive to intracellular signals transduced by interaction of the GTPase and PKC.  
     
     
         46 . The assay of  claim 31 , wherein the assay is repeated for a variegated library of at least 100 different test compounds.  
     
     
         47 . The assay of  claim 31 , wherein the test compound is selected from the group consisting of small organic molecules, and natural product extracts.  
     
     
         48 . The assay of  claim 31 , wherein one or more of the PKC and GTPase are derived from a human pathogen which is implicated in mycotic infection.  
     
     
         49 . The assay of  claim 48 , wherein the mycotic infection is a mycosis selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and sporotrichosis.  
     
     
         50 . The assay of  claim 48 , wherein the human pathogen is selected from a group consisting of  Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillusfumigatus, Aspergillusflavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa , and  Mucor pusillus.    
     
     
         51 . The assay of  claim 48 , wherein the human pathogen is  Pneumocystis carinii.    
     
     
         52 . The assay of  claim 31 , which comprises a further step of preparing a pharmaceutical preparation of one or more compounds identified as having potential antifungal activity.  
     
     
         53 . An assay for identifying compounds having potential antifungal activity, comprising: 
 i. forming a reaction mixture including a fungal Rho-like GTPase, a fungal glucan synthase complex or subunit thereof (GS protein), and a test compound; and    ii. detecting interaction of the Rho-like GTPase and GS protein, wherein a statistically significant decrease in the interaction of the Rho-like GTPase and GS protein in the presence of the test compound, relative to the level of interaction in the absence of the test compound, indicates a potential antifungal activity for the test compound.    
     
     
         54 . The assay of  claim 53 , wherein the reaction mixture is a glucan synthesis system including a GTP and a UDP-glucose, and the step of detecting interaction of the GTPase and GS protein comprises detecting formation of glucan polymers in the reaction mixture.  
     
     
         55 . The assay of  claim 54 , wherein the UDP-glucose comprises a detectable label, and the level of glucan polymer formation is quantified by detecting the labelled glucan polymers.  
     
     
         56 . The assay of  claim 53 , wherein the step of detecting the interaction of the GTPase with the GS protein comprises detecting the formation of protein-protein complexes including the GTPase and GS protein.  
     
     
         57 . The assay of  claim 56 , wherein at least one of the GS protein and GTPase comprises a detectable label, and the level of GS protein/GTPase complexes formed in the reaction mixture is quantified by detecting the label in at least one of the GTPase, the GS protein, and GS protein/GTPase complexes.  
     
     
         58 . The method of any of claims  53  or  57 , wherein the label group is selected from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.  
     
     
         59 . The assay of  claim 56 , wherein the formation of protein-protein complexes including the GTPase and GS protein is detected by an immunoassay.  
     
     
         60 . The assay of  claim 53 , wherein the reaction mixure is reconstituted protein mixture.  
     
     
         61 . The assay of  claim 53 , wherein the reaction mixure comprises a cell lysate.  
     
     
         62 . The assay of  claim 53 , wherein the GPTase is selected from the group consisting of Rho1, Rho2, Rsr1/Bud1 and Cdc42, and fungal homologs thereof.  
     
     
         63 . The assay of  claim 53 , wherein the reaction mixture is a whole cell comprising heterologous nucleic acid recombinantly expressing one or more of the GS protein and GTPase.  
     
     
         64 . The assay of  claim 53 , wherein the assay is repeated for a variegated library of at least 100 different test compounds.  
     
     
         65 . The assay of  claim 53 , wherein the test compound is selected from the group consisting of small organic molecules, and natural product extracts.  
     
     
         66 . The assay of  claim 53 , wherein one or more of the GS protein and GTPase are derived from a human pathogen which is implicated in mycotic infection.  
     
     
         67 . The assay of  claim 66 , wherein the mycotic infection is a mycosis selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and sporotrichosis.  
     
     
         68 . The assay of  claim 66 , wherein the human pathogen is selected from a group consisting of  Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramrosa , and  Mucor pusillus.    
     
     
         69 . The assay of  claim 66 , wherein the human pathogen is  Pneumocystis carinii.    
     
     
         70 . The assay of  claim 53 , which comprises a further step of preparing a pharmaceutical preparation of one or more compounds identified as having potential antifungal activity.  
     
     
         71 . A recombinant cell comprising (i) exogenous nucleic acid encoding one or more subunits of a fungal geranylgeranyl protein transferase (GGPTase), and (ii) exogenous nucleic acid encoding a fungal Rho-like GTPase or a fragment thereof including at least one of (a) a prenylation site which can be enzymatically prenylated by the GGPTase, or (b) a GGPTase binding sequence which specifically binds the GGPTase.  
     
     
         72 . The cell of  claim 71 , wherein one or more of the nucleic acids encoding the GGPTase and GTPase are derived from a human pathogen which is implicated in mycotic infection.  
     
     
         73 . The cell of  claim 72 , wherein the mycotic infection is a mycosis selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and sporotrichosis.  
     
     
         74 . The cell of  claim 72 , wherein the human pathogen is selected from a group consisting of  Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa , and  Mucor pusillus.    
     
     
         75 . The cell of  claim 72 , wherein the human pathogen is  Pneumocystis carinii.    
     
     
         76 . The cell of  claim 71 , which cell is a recombinantly manipulate yeast cell selected from the group consisting of Kluyverei spp, Schizosaccharomyces spp, Ustilaqo spp and Saccharomyces spp.  
     
     
         77 . The cell of  claim 71 , which cell is a recombinantly manipulate  Schizosaccharomyces cerivisae  cell.  
     
     
         78 . The cell of  claim 71 , which cell is constitutively or inducibly defective for an endogenous activity corresponding to one or more of the GGPTase and GTPase encoded by the exogenous nucleic acids.  
     
     
         79 . A reconstituted protein mixture or a cell lysate mixture comprising (i) a recombinant fungal geranylgeranyl protein transferase (GGPTase), and (ii) a recombinant fungal Rho-like GTPase or a fragment thereof including at least one of (a) a prenylation site which can be enzymatically prenylated by the GGPTase, or (b) a GGPTase binding sequence which specifically binds the GGPTase.  
     
     
         80 . The mixture of  claim 79 , wherein one or more of the recombinant GGPTase and GTPase are derived from a human pathogen which is implicated in mycotic infection.  
     
     
         81 . The mixture of  claim 80 , wherein the mycotic infection is a mycosis selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocaidiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, monoliasis, para-actinomycosis, and sporotrichosis.  
     
     
         82 . The mixture of  claim 80 , wherein the human pathogen is selected from a group consisting of  Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramrosa , and  Mucor pusillus.    
     
     
         83 . The mixture of  claim 80 , wherein the human pathogen is  Pneumocystis carinii.    
     
     
         84 . A recombinant cell comprising (i) exogenous nucleic acid encoding a fungal Rho-like GTPase, and (ii) exogenous nucleic acid encoding a fungal protein selected from the group consisting of a fungal protein kinase C (PKC) or one or more subunits of a fungal glucan synthase.  
     
     
         85 . A reconstituted protein mixture or a cell lysate mixture comprising (i) a recombinant fungal Rho-like GTPase, and (ii) a recombinant fungal protein selected from the group consisting of a fungal protein kinase C (PKC) or a fungal glucan synthase.  
     
     
         86 . A recombinant cell comprising exogenous nucleic acid encoding one or more subunits of a geranylgeranyl protein transferase (GGPTase) cloned from a human fungal pathogen.  
     
     
         87 . A recombinant cell comprising exogenous nucleic acid encoding a Rho-like GTPase cloned from a human fungal pathogen.  
     
     
         88 . A recombinant cell comprising exogenous nucleic acid encoding one or more subunits of a glucan synthase cloned from a human fungal pathogen.  
     
     
         89 . A recombinant cell comprising exogenous nucleic acid encoding a protein kinase C cloned from a human fungal pathogen.  
     
     
         90 . A reconstituted protein mixture or a cell lysate mixture comprising one or more subunits of a recombinant geranylgeranyl protein transferase (GGPTase) cloned from a human fungal pathogen.  
     
     
         91 . A reconstituted protein mixture or a cell lysate mixture comprising a recombinant Rho-like GTPase cloned from a human fungal pathogen.  
     
     
         92 . Areconstituted protein mixture or a cell lysate mixture comprising one or more recombinantn subunits of a glucan synthase cloned from a human fungal pathogen.  
     
     
         93 . A reconstituted protein mixture or a cell lysate mixture comprising a recombinant protein kinase C cloned from a human fungal pathogen.  
     
     
         94 . The cell of any of claims  86 - 93  wherein the human pathogen is selected from a group consisting of  Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa , and  Mucor pusillus.