| Method for sorting antifungal molecules acting on the glucanosyltransferase activity -> Monitor Keywords |
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Method for sorting antifungal molecules acting on the glucanosyltransferase activityRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidMethod for sorting antifungal molecules acting on the glucanosyltransferase activity description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060211013, Method for sorting antifungal molecules acting on the glucanosyltransferase activity. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This is a division of application Ser. No. 10/347,252, filed Jan. 21, 2003, which is a division of application Ser. No. 09/242,913, filed Oct. 13, 1999, now U.S. Pat. No. 6,551,811, which is a Section 371 application of PCT/FR97/01540, filed Aug. 29, 1997, and further claims the benefit of U.S. Provisional Application No. 60/024,910, filed Aug. 30, 1996, the disclosures of all of which are incorporated herein by reference. [0002] This invention relates to a protein with an activity of the glucanosyltransferase type, and more especially a .beta.-(1-3)-glucanosyltransferase activity. [0003] This invention also relates to oligonucleotides coding for this protein having an enzymatic activity. [0004] It also relates to molecules having an effect on the activity of this enzyme. [0005] Opportunistic fungal infections due to Candida, Aspergillus, Cryptococcus and Pneumocystis are responsible for the increase in morbidity and mortality among patients suffering from AIDS and other patients with clinically compromised immunity. In addition, the yeast Candida and the dermatophytes today remain a major medical problem amongst patients with adequate immunity. Despite the increase in the number of infections due to pathogenic and opportunistic fungi, therapy against mycoses has not improved in recent years. Two families of drugs are used: the azoles and Amphotericin B. These drugs have some disadvantages since treatment based on Amphotericin B is associated with nephrotoxicity and that based on azole is more fungistatic than fungicidal. [0006] Fungi are microorganisms of the eukaryotic type which share the majority of their biochemical pathways with their hosts, with one important exception: the biosynthesis of the cell wall. The cell wall is a rigid envelope which protects the cell against the environment and mechanical stresses, but is also a dynamic structure which is involved in the transport of ions and macromolecules and in the localization of enzymes involved in fungal growth. In consequence, disorganization of the organization of the cell wall should be detrimental to fungi. [0007] The skeleton of the fungal cell wall is mainly composed of polymers of the polysaccharide type (.beta.(1-3) glucans, mannans, chitin) which are not found in humans. For this reason, the biosynthesis of the cell wall has been a target for research into new antifungal drugs. The penicillins and cephalosporins, which are both inhibitors of the bacterial cell wall, and potential antibiotics lend support to this hypothesis. Moreover, many molecules which inhibit the development of the fungal cell wall have antifungal properties (Debono and Gordee, 1994, Annu. Rev. Microbiol, 48, 471-497). Among these are: [0008] 1) The families of the echinocandin lipopeptides and the palulacandin glycopeptides which are non-competitive inhibitors of the glucan synthetase complex. [0009] 2) The polyoxins and nikkomycins which are analogs of UDP-GlcNac and potential competitive inhibitors of chitin synthetase, and [0010] 3) The pradimycins binding mannan and the benanomycins. [0011] The synthesis of .beta.(1-3) glucan and chitin is under the control of enzyme complexes (glucan synthetase and chitin synthetase) which are localized in the plasma membrane. Once the polymers have been released into the periplasmic space, cross-links are created between the polymers and it is these which are responsible for the rigidity of the cell wall. The proteins and genes of the glucan and chitin synthetases are beginning to be fairly well understood. [0012] However, the inhibition of the glucan and chitin synthetases by a molecule requires three steps: its transfer across the cell wall, crossing of the plasma membrane and transfer inside the cell to the target, each step representing a potential barrier for the enzymatic inhibitor from being an effective antifungal drug or a potential source of resistant strains against the drug. [0013] The transferases which are responsible for creating the covalent bonds between the different polymers of the wall have been very little studied up till now. [0014] These enzymes represent a better target than the chitin and glucan synthetase complexes since they are more easily accessible for a putative antifungal drug. [0015] Nuoffer et al. (1991, Mol. Cell. Bio., 11, 27-37) have described a glycoprotein, named Gas1p, exposed on the surface of the yeast Saccharomyces cerevisiae. The genes coding for this protein have been cloned. The function of the Gaslp protein is not essential for the viability of the cell, and has not been determined. [0016] Saporito-Irwing et al. (1995, Mol. Cell. Biol., 15, 601-613) have isolated a gene originating from the yeast Candida albicans, designated PHR1. The amino acid sequence determined for this protein PHR1 was 56% identical to that of the protein Gas1. The gene was regulated in response to the pH of the culture medium. As for the protein gas1p, no function has been determined. [0017] It clearly emerges from this analysis of the prior art that there has been a problem in obtaining molecules with effective antifungal activity. [0018] The inventors have solved this problem. [0019] They have shown that the introduction of mutations into a glucanosyltransferase originating from Aspergillus fumigatus interferes with the development of this microorganism. [0020] They have also determined the sequences of several of these enzymes. [0021] The present invention thus relates to a first protein with an activity of the .beta.(1-3)-glucanosyltransferase type characterized in that it has at least 50%, preferably 60%, and even more preferably 85% homology with proteins having the sequences, or a part of the sequences SEQ ID NO. 2 or SEQ ID NO. 3 as follows: TABLE-US-00001 SEQ ID NO:2: Met Lys Ala Ser Ala Val Thr Ala Ala Leu Ala Val Gly Ala Ser Thr Val Leu Ala Ala Pro Ser Ile Lys Ala Arg Asp Asp Val Thr Pro Ile Thr Val Lys Gly Asn Ala Phe Phe Lys Gly Asp Glu Arg Phe Tyr Ile Arg Gly Vai Asp Tyr Gln Pro Gly Gly Ser Ser Asp Leu Ala Asp Pro Ile Ala Asp Ala Asp Gly Cys Lys Arg Asp Ile Ala Lys Phe Lys Glu Leu Gly Leu Asn Thr Ile Arg Val Tyr Ser Val Asp Asn Ser Lys Asn His Asp Glu Cys Met Asn Thr Leu Ala Asp Ala Gly Ile Tyr Leu Val Leu Asp Val Asp Thr Pro Lys Tyr Ser Ile Asn Arg Ala Lys Pro Lys Glu Ser Tyr Asn Asp Val Tyr Leu Gln Tyr Ile Phe Ala Thr Val Asp Ala Phe Ala Gly Tyr Lys Asn Thr Leu Ala Phe Phe Ser Gly Asn Glu Val Ile Asn Asp Gly Pro Ser Ser Ser Ala Ala Pro Tyr Val Lys Ala Val Thr Arg Asp Leu Arg Gln Tyr Ile Arg Ser Arg Lys Tyr Arg Glu Ile Pro Val Gly Tyr Ser Ala Ala Asp Ile Asp Thr Asn Arg Leu Gln Met Ala Gln Tyr Met Asn Cys Gly Ser Asp Asp Glu Arg Ser Asp Phe Phe Ala Phe Asn Asp Tyr Ser Trp Cys Asp Pro Ser Ser Phe Lys Thr Ser Gly Trp Asp Gln Lys Val Lys Asn Phe Thr Gly Tyr Gly Leu Pro Leu Phe Leu Ser Glu Tyr Gly Cys Asn Thr Asn Lys Arg Gln Phe Gln Glu Val Ser Ser Leu Tyr Ser Thr Asp Met Thr Gly Val Tyr Ser Gly Gly Leu Val Tyr Glu Tyr Ser Gln Glu Ala Ser Asn Tyr Gly Leu Val Glu Ile Ser Gly Asn Asn Val Lys Glu Leu Pro Asp Phe Asp Ala Leu Lys Thr Ala Phe Glu Lys Thr Ser Asn Pro Ser Gly Asp Gly Asn Tyr Asn Lys Thr Gly Gly Ala Asn Pro Cys Pro Ala Lys Asp Ala Pro Asn Trp Asp Val Asp Asn Asp Ala Leu Pro Ala Ile Pro Glu Pro Ala Lys Lys Tyr Met Thr Glu Gly Ala Gly Lys Gly Pro Gly Phe Ala Gly Pro Gly Ser Gln Asp Arg Gly Thr Gln Ser Thr Ala Thr Ala Glu Pro Gly Ser Gly Ser Ala Thr Gly Ser Ser Ser Ser Gly Thr Ser Thr Ser Ser Lys Gly Ala Ala Ala Gly Leu Thr Val Pro Ser Leu Thr Met Ala Pro Val Val Val Gly Ala Val Thr Leu Leu Ser Thr Val Phe Gly Ala Gly Leu Val Leu Leu [0022] TABLE-US-00002 SEQ ID NO: 3: (BGT2) Asp Asp Val Thr Pro Ile Thr Val Lys Gly Asn Ala Phe Phe Lys Gly Asp Glu Arg Phe Tyr Ile Arg Gly Val Asp Tyr Gln Pro Gly Gly Ser Ser Asp Leu Ala Asp Pro Ile Ala Asp Ala Asp Gly Cys Lys Arg Asp Ile Ala Lys Phe Lys Glu Leu Gly Leu Asn Thr Ile Arg Val Tyr Ser Val Asp Asn Ser Lys Asn His Asp Glu Cys Met Asn Thr Leu Ala Asp Ala Gly Ile Tyr Leu Val Leu Asp Val Asn Thr Pro Lys Tyr Ser Ile Asn Arg Ala Lys Pro Lys Glu Ser Tyr Asn Asp Val Tyr Leu Gln Tyr Ile Phe Ala Thr Val Asp Ala Phe Ala Gly Tyr Lys Asn Thr Leu Ala Phe Phe Ser Gly Asn Glu Val Ile Asn Asp Gly Pro Ser Ser Ser Ala Ala Pro Tyr Val Lys Ala Val Thr Arg Asp Leu Arg Gln Tyr Ile Arg Ser Arg Lys Tyr Arg Glu Ile Pro Val Gly Tyr Ser Ala Ala Asp Ile Asp Thr Asn Arg Leu Gln Met Ala Gln Tyr Met Asn Cys Gly Ser Asp Asp Glu Arg Ser Asp Phe Phe Ala Phe Asn Asp Tyr Ser Trp Cys Asp Pro Ser Ser Phe Lys Thr Ser Gly Trp Asp Gln Lys Val Lys Asn Phe Thr Gly Tyr Gly Leu Pro Leu Phe Leu Ser Glu Tyr Gly Cys Asn Thr Asn Lys Arg Gln Phe Gln Glu Val Ser Ser Leu Tyr Ser Thr Asp Met Thr Gly Val Tyr Ser Gly Gly Leu Val Tyr Glu Tyr Ser Gln Glu Ala Ser Asn Tyr Gly Leu Val Glu Ile Ser Gly Asn Asn Val Lys Glu Leu Pro Asp Phe Asp Ala Leu Lys Thr Ala Phe Glu Lys Thr Ser Asn Pro Ser Gly Asp Gly Asn Tyr Asn Lys Thr Gly Gly Ala Asn Pro Cys Pro Ala Lys Asp Ala Pro Asn Trp Asp Val Asp Asn Asp Ala Leu Pro Ala Ile Pro Glu Pro Ala Lys Lys Tyr Met Thr Glu Gly Ala Gly Lys Gly Pro Gly Phe Ala Gly Pro Gly Ser Gln Asp Arg Gly Thr Gln Ser Thr Ala Thr Ala Glu Pro Gly Ser Gly Ser Ala Thr Gly Ser Ser Ser Ser Gly Thr Ser Thr Ser Ser Lys Gly Ala Ala Ala Gly Leu Thr Val Pro Ser Leu Thr Met Ala Pro Val Val Val Gly Ala Val Thr Leu Leu Ser Thr Val Phe Gly Ala Gly [0023] This protein preferably has a molecular weight of about 44 kD, or of about 49 kD if it carries at least one residue of the N-glycosyl type. [0024] The present invention also relates to proteins with .beta.-(1-3)-glucanosyltransferase activity characterized in that they have at least 50%, preferably 60% and even more preferably 85% homology with proteins having the sequences, or a part of the sequences SEQ ID NO. 10 or SEQ ID NO. 12 as follows: TABLE-US-00003 SEQ ID NO 10: Gly Phe Phe Ala Gly Asn Glu Val Ile Asn Glu Gln Ser Val Lys Asn Val Pro Thr Tyr Val Arg Val Cys His Pro Ser Pro Gln Leu Thr Ile Ala Cys Pro Leu [0025] TABLE-US-00004 SEQ ID NO 12 (BGT4) Gly Phe Phe Ala Gly Asn Glu Val Val Asn Gln Ala Asn Gln Ser Ala Gly Ala Ala Phe Val Lys Ala Ala Ala Arg Asp Met Lys Ala Tyr Ile Lys Thr Lys Gly Tyr Arg Gln Ser Leu Ala Ile Gly Tyr Ala Thr Thr Asp Asn Pro Glu Ile Arg Leu Pro Leu Ser Asp Tyr Leu Asn Cys Gly Asp Gln Ala Asp Ala Val Asp Phe Phe Gly Tyr Asn Ile Tyr Glu Trp Cys Gly Asp Gln Thr Phe Gln Thr Ser Gly Tyr Gln Asn Arg Thr Glu Glu Tyr Lys Asp Tyr Ser Ile Pro Ile Phe Ile Ser Glu Tyr Gly Cys Asn [0026] The present invention also relates to fragments of these proteins. [0027] Said invention is not limited to the proteins having the sequences SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 10 or SEQ ID NO. 12 but extends to any protein having sequences similar to those having the sequences SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 10 or SEQ ID NO. 12 and in particular having certain amino acid substitutions in which an amino acid is replaced by another amino acid having essentially the same physico-chemical properties. Lehninger's biochemistry manual (Flammarion Medecine-Science, 1977, or one of its more recent editions) distinguishes four groups of amino acids, based on their physico-chemical behavior: those with a non-polar or hydrophobic side-chain, those with an uncharged polar side-chain, those with a negatively charged side-chain, and those with a positively charged side-chain. Continue reading about Method for sorting antifungal molecules acting on the glucanosyltransferase activity... 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