Bax-responsive genes for drug target identification in yeast and fungi

Abstract

The invention describes the use of nucleic acids and polypeptides which are involved in a pathway eventually leading to programmed cell death of yeast or fungi for the preparation of a medicament for treating diseases associated with yeast or fungi or for the treatment of proliferative disorders or for preventing apoptosis in certain diseases. Methods are provided to identify compounds which selectively modulate the expression or functionality of said polypeptides in the same or a parallel pathway. Also provided are compounds as well as pharmaceutical compositions, medicaments and vaccines. The invention also comprises new nucleic acid sequences, probes and primers derived thereof, expression vectors and host cells transformed with said vectors, polypeptides and antibodies raised against said polypeptides.

1 - 79 . (canceled)  
     
     
         80 . A method for identifying Bax-resistant yeast or fungi comprising the steps of: 
 (a) providing (a) genetically modified yeast or fungi, said genetically modified yeast or fungi is transformed, transfected or infected with a nucleic acid as represented by SEQ ID NO:1, or a sequence as represented by SEQ ID NO:1 as a fusion with a carrier gene,    (b) treating said genetically modified yeast or fungi with a mutagen,    (c) isolating resistant yeast or fungal cells, and,    (d) optionally identifying and/or characterizing mutated genes in said resistant yeast or fungal cells.    
     
     
         81 . A method for identifying  Candida  spp. sequences which are differentially expressed in a pathway eventually leading to programmed cell death using a nucleic acid as represented by SEQ ID NO:1, or a sequence as represented by SEQ ID NO:1 as a fusion with a carrier gene.  
     
     
         82 . A method for obtaining and identifying  Candida  spp. sequences involved in a pathway eventually leading to programmed cell death comprising the steps of: 
 (a) providing a two hybrid system wherein a polypeptide encoded by a nucleic acid as represented by SEQ ID NO:1, or a sequence as represented by SEQ ID NO: 1 as a fusion with a carrier gene as a bait and a  Candida  spp. cDNA library as a prey are expressed,    (b) detecting an interaction between said polypeptide and a  Candida  spp. polypeptide encoded by said cDNA library, and    (c) identifying said  Candida  spp. polypeptide or cDNA.    
     
     
         83 . A method for identifying inhibitors (or inhibitor sequences) of Bax-induced cell death comprising the steps of: 
 (a) providing a genetically modified organism transfected or infected with a nucleic acid as represented by SEQ ID NO:1, or a sequence as represented by SEQ ID NO:1 as a fusion with a carrier gene,    (b) expressing a cDNA library in said genetically modified organism, and,    (c) identifying a polypeptide or a cDNA which expression has a beneficial effect on the survival and/or growth of said genetically modified organism.    
     
     
         84 . An isolated polypeptide which is involved in a pathway for programmed cell death of  Candida  spp. and encoded by a nucleic acid, identifiable by a method according to any of claims  80 - 83  wherein said polypeptide is selected from: 
 (a) a polypeptide having an amino acid sequence as represented in any of SEQ ID NOs 434, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 688, 718, 720, 722, 724, 726, 728, 730 and 732, or encoding a functional equivalent, derivative or bioprecursor of said protein;    (b) a polypeptide having an amino acid sequence which is more than 70% similar to any of the amino acid sequences as represented by any of SEQ ID NOs 434, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 688, 718, 720, 722, 724, 726, 728, 730 and 732,    (c) a polypeptide having an amino acid sequence which is more than 70% identical to any of the amino acid sequences as represented by any of SEQ ID NOs 434, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 688, 718, 720, 722, 724, 726, 728, 730 and 732, and    (d) a functional fragment of any of said polypeptides as defined in a) to c).    
     
     
         85 . A polypeptide according to  claim 84  for use as a medicament.  
     
     
         86 . An antibody capable of specifically binding to a polypeptide of  claim 84  or to a specific epitope of said polypeptide.  
     
     
         87 . An antibody according to  claim 86  for use as a medicament.  
     
     
         88 . A pharmaceutical composition comprising an antibody of  claim 86 .  
     
     
         89 . A pharmaceutical or fungicidal composition comprising a polypeptide as defined in  claim 84  together with a pharmaceutically acceptable carrier diluent or excipient therefor.  
     
     
         90 . A vaccine for immunizing a mammal against yeast or fungal infections comprising at least one polypeptide as defined in  claim 84  in a pharmaceutically acceptable carrier.  
     
     
         91 . A genetically modified yeast or fungus in which modification results in the overexpression or underexpression of at least one of the polypeptides as defined in  claim 84 , which overexpression or underexpression of said polypeptide prevents, delays or sensitizes for apoptosis of said genetically modified yeast or fungus.  
     
     
         92 . A method of identifying compounds which selectively modulate expression or functionality of polypeptides involved in a pathway eventually leading to programmed cell death of yeast or fungi or in metabolic pathways in which said polypeptides are involved, which method comprises: 
 (a) contacting a compound to be tested with a genetically modified yeast or fungus according to  claim 91 , in addition to contacting wild type cells with said compound,    (b) monitoring the growth and/or death rate and/or activity of said genetically modified cells compared to said wild type cells; wherein differential growth or activity of said genetically modified yeast or fungi cells is indicative of selective action of said compound on a polypeptide in the same or a parallel pathway,    (c) alternatively monitoring the growth and/or death rate and/or activity of said genetically modified cells compared to genetically modified cells which were not contacted with the compound to be tested, wherein differential growth or activity of said genetically modified yeast of fungi cells is indicative of selective action of said compound on a polypeptide in the same or a parallel pathway,    (d) alternatively monitoring changes in morphologic and/or functional properties of components in said genetically modified cells caused by the addition of the compound to be tested, and,    (e) identifying the compound.    
     
     
         93 . A method of identifying compounds or polypeptides which bind to or modulate the properties of polypeptides which are involved in a pathway eventually leading to programmed cell death of yeast or fungi, which method comprises: 
 (a) contacting a compound or polypeptides to be tested with at least one of the polypeptides as defined in  claim 84 ,    (b) detecting the complex formed between the compound or polypeptide to be tested and said polypeptide,    (c) alternatively, examining the diminution of complex formation between said polypeptide and a binding partner, caused by the addition of the compound or polypeptide being tested,    (d) alternatively, examining the alteration in the functional activity of the polypeptide, caused by the addition of the compound or polypeptide being tested, and,    (e) identifying the compound or protein.    
     
     
         94 . A method for identifying compounds interacting with a polypeptide involved in a pathway eventually leading to programmed cell death of yeast and fungi comprising the steps of: 
 (a) providing a two-hybrid screening system wherein an interacting polypeptide obtainable by a method of  claim 92 , are expressed,    (b) interacting said compound with the complex formed by the expressed proteins as defined in a),    (c) detecting a second complex, wherein the presence of said second complex identifies a compound which specifically binds to one of said polypeptide or to said second complex, and,    (d) identifying the compound.    
     
     
         95 . Use of a polypeptide selected from any of the polypeptides as defined in  claim 84  or a human homologue thereof for treating and/or preventing and/or alleviating proliferative disorders or for the prevention of apoptosis in certain diseases.  
     
     
         96 . Use of an antibody capable of specifically binding to at least one of the polypeptides as defined in  claim 84  or to a human homologue thereof or to a specific epitope of said polypeptide or said human homologue, for the preparation of a medicament for treating proliferative disorders or for the prevention of apoptosis in certain diseases.