WO2004014349A1

WO2004014349A1 - New molecules for modulating drug resistance in fungus

Info

Publication number: WO2004014349A1
Application number: PCT/CA2003/001232
Authority: WO
Inventors: Bernard Turcotte
Original assignee: McGill University
Current assignee: McGill University
Priority date: 2002-08-13
Filing date: 2003-08-13
Publication date: 2004-02-19
Anticipated expiration: 2005-02-13
Also published as: AU2003257330A1

Abstract

The present invention relates to a molecule for reducing resistance to an active agent in a fungus, the molecule reducing the expression of a zinc cluster containing gene in the fungus. The present invention also relates to method of screening a drug capable of preventing resistance to an active agent in a fungus using the molecule of the present invention.

Description

New molecules for modulating drug resistance in fungus

BACKGROUND OF THE INVENTION

(a) Field of the Invention

This invention relates to new molecules for modulating resistance to an active agent in fungus and uses thereof.

(b) Description of Prior Art

Opportunistic fungal infections are becoming more widespread, especially with immunosuppressed patients. Quite often, development of resistance to antifungal drugs is observed. This phenomenon is often due to overexpression of membrane transporters that act as drug efflux pumps resulting in increased resistance to a wide variety of drugs. Saccharomyces cerevisiae (baker's yeast) has been widely used to study multidrug resistance.

The International patent application published under number WO 02/24865 discloses a method for the modulation of secondary metabolite production by fungi through genetic manipulation of such fungi. Also, it discloses commercial processes using ZBC proteins, or variants thereof to increase useful secondary metabolite production. The methods describe in this patent application comprise the expression in a fungus of a ZBC protein or a variant thereof.

Silver and White (The 15^th congress of the International Society for Human and Animal Mycology, San Antonio, Texas, May 25-28 2003, abstract #242) teach that a C. albicans homolog of the S. cerevisiae genes ECM22 and UPC2 have been identified and that deletion of ECM22 results in decreased antifungal drug resistance (in C. albicans).

Talibi and Raymond teach that FCR1 gene behaves as a negative regulator of drug resistance in C. albicans.

It would be highly desirable to be provided with new molecules for modulating resistance to an active agent in fungus that can be used as target molecules for antifungal drugs. SUMMARY OF THE INVENTION

In accordance with the present invention there is provided a molecule for reducing resistance to an active agent in a fungus, the molecule reducing the activity of a zinc cluster protein acting as a positive regulator of the active agent in the fungus. Preferably, the fungus is a pathogen fungus, more preferably a fungus from the family of Candida and Aspergillus. The Candida fungus can be one of, but not limited to, Candida albicans, Candida glabrata, Candida kruser, Candida guillier- mondii and Candida dubliniensis. The Aspergillus fungus can be one of, but not limited to, Aspergillus nidulans, Aspergillus fumigatus and Aspergillus flavus.

In a preferred embodiment of the present invention, the active agent is an antifungal agent, which can be selected from the group of, but not limited to, fluconazole, ketoconazole and brefeldin A.

In a preferred embodiment of the present invention, the gene is UPC2, an homolog thereof or an ortholog thereof.

In accordance with the present invention there is provided a molecule for reducing resistance to an active agent in a fungus, the molecule increasing the activity of a zinc cluster protein acting as a negative regulator of the active agent in the fungus. Preferably, the fungus is a pathogen fungus, more preferably a fungus from the family of Candida and Aspergillus. The Candida fungus can be one of, but not limited to, Candida albicans, Candida glabrata, Candida kruser, Candida guillier- mondii and Candida dubliniensis. The Aspergillus fungus can be one of, but not limited to, Aspergillus nidulans, Aspergillus fumigatus and Aspergillus flavus.

In a preferred embodiment of the present invention, the gene is selected from the group consisting of RDS2, STB5, an homolog thereof or an ortholog thereof. More preferably, the gene is STB5. ln accordance with the present invention, there is provided a composition comprising the molecule of the present invention in association with a pharmaceutically acceptable carrier.

In accordance with the present invention, there is also provided the in vitro use of the molecule of the present invention as a target for determining resistance of the fungus to the active agent.

In accordance with the present invention, there is further provided a method for sςreening a drug capable of preventing resistance to an active agent in a fungus, the method comprising the step of determining reduction of expression of a zinc cluster gene of the fungus caused by a candidate drug, whereby the reduction of the expression of the zinc cluster gene by the candidate drug is indicative that the drug is capable of preventing resistance to the active agent in the fungus.

In accordance with the present invention, there is still further provided a method for decreasing resistance to an active agent in a fungus comprising reducing the expression of a gene encoding a drug efflux pump.

For the purpose of the present invention the following terms are defined below.

The term "active agent" is intended to mean a compound administered to a fungus to enhance or inhibit its reproduction and/or survival rate such as a drug including without limitation antifungal drug.

The term "zinc cluster" is intended to mean a Zn₂Cys₆ binuclear cluster DNA-binding motif having the consensus sequence of CysX₂CysX₆CysX₅.₁₂CysX₂CysX₆.₈Cys (SEQ ID_:NO:1).

The term "drug efflux pump" is intended to mean the mechanism of protection developed by cells to extrude an active agent outside the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A illustrates the homology between S. cerevisiae Rds2 and the gene product of CA2880 present in the genome of Candida albicans;

Fig. 1 B illustrates the homology between S. cerevisiae Rds2 and the putative gene product in contig:4842 in Aspergillus fumigatus; Fig. 1C illustrates the homology between S. cerevisiae Rds2 and the putative gene product in contig1.107 in Aspergillus nidulans;

Fig. 2 illustrates the homology between S. cerevisiae Stb5 and gene product of CA4617 present in the genome of Candida albicans;

Fig. 3 illustrates the homology between S. cerevisiae Upc2 and the gene product of CA3878;

Fig. 4 illustrates the DNA binding domains of CaUpc2 and Fcr1 bound to a DRE in vitro;

Fig. 5 illustrates the DNA binding domain of Candida albicans Rds2 bound to a DRE in vitro;

Fig. 6 is an assay illustrating that the overexpression of CaUpc2 increases resistance to ketoconazole;

Fig. 7 is an assay illustrating that the overexpression of Fcr1 decreases resistance to ketoconazole;

Fig. 8 is an assay illustrating that the overexpression of the DNA binding domain of CaRds2 decreases resistance to ketoconazole;

Fig. 9 is an assay illustrating that CaStbδ decreases resistance to ketoconazole.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there is provided new molecules for modulating resistance to an active agent in fungus and uses thereof.

Expression of the genes encoding multidrug transporters is controlled, in part, by members of the Gal4 family of zinc cluster proteins. These proteins are characterized by a zinc finger which contains the Zn(ll)2Cys6 (or C6 zinc) binuclear cluster DNA-binding motif with . the consensus sequence of CysX₂CysX₆CysX₅-i₂CysX₂CysX₆-sCys (SEQ ID NO:1 ). The cysteines mediate the binding of two zinc atoms, which are necessary for the zinc finger to bind DNA. This type of transcriptional regulator has been identified in fungi and, as a result, constitutes a potential target for drugs specific for fungi. ln S. cerevisiae, the Gal4 family comprises over fifty members that are putative transcriptional regulators. For example, Pdr1 and Pdr3 activate multidrug resistance genes by binding to pleiotropic drug response elements (PDREs) that are short DNA elements found in promoters of target genes such as PDR5 encoding a drug efflux pump (also called an ABC transporter). It was determined herein that additional members of the Gal4 family are involved in conferring resistance or sensitivity to drugs.

A panel of strains carrying deletions of zinc cluster genes was tested in the presence of various drugs. One deletion strain (Δrcfrl ) was resistant to cycloheximide while eight strains showed sensitivity to the antifungal ketoconazole or the translation inhibitor cycloheximide. Unnamed zinc cluster genes were called RDS for regulator of drug sensitivity or RDR for repressor of drug resistance. Promoter activity of multidrug resistance genes was decreased in some deletion strains such as Astbδ. Activity of a reporter containing a PDRE inserted in front of a minimal promoter was also decreased in a Astbδ strain. Moreover, the purified DNA binding domain of Stbδp bound to a PDRE in vitro. Mutations known to affect binding of Pdr1/Pdr3 showed similar effects when assayed with Stb5. These results show that Stb5 is a transcriptional activator of multidrug resistance genes. In addition, it was showed that another zinc cluster protein, Rdr1 , is a transcriptional repressor of the PDR5 gene. Thus, new regulators of drug sensitivity in the family of zinc cluster proteins were identified.

Drug efflux pumps similar to Pdr5 are found in C. albicans. For example, CDR1 and CDR2 {Candida drug resistance 1 and 2) encode ABC transporters similar to S. cerevisiae Pdr5. Drug response elements (DREs) that are DNA sequences important for the expression of CDR1 and CDR2 (and hence the production of the transporters) have been identified in the promoters of these genes. C. albicans strains that are resistant to antifungals usually show increased levels of multidrug transporters and their corresponding mRNAs. In S. cerevisiae, drug resistance is almost always due to mutations that render the transcriptional activators Pdr1 or Pdr3 hyperactive resulting in greatly increased expression of genes encoding drug efflux pumps. However, there is no obvious ortholog ("homologs") of Pdr1 and Pdr3 in C. albicans. Highly conserved homologs of Rds2, Stb5, and Upc2/Ecm22 are found in various species of the gender Saccharomyces such as S. mikatae, S. bayanus, S. catellii, S. kudravzevii and S. kluyve . Sequence of the genome of the pathogenic fungi Candida albicans has recently been completed. Investigation was performed to see if zinc cluster proteins similar to those identified in the screen with S. cerevisiae are found in C. albicans as well as other pathogenic fungi. Thus, a database search was performed to identify homologs of the newly identified zinc cluster proteins involved in conferring drug resistance/sensitivity in S. cerevisiae. Results show that the genome of C. albicans has a homolog of RDS2 (hereafter named CaRDS2) (Fig. 1A) (whereas in Fig. 1A RDS2 (SEQ ID NO:2) is compared with RDS2CA (SEQ ID NO:3) and the consensus sequence is illustrated (SEQ ID NO:4)). RDS2 homologs are also found in Aspergillus fumigatus (Fig. 1 B) (whereas in Fig. 1B RDS2 (SEQ ID NO:2) is compared with RDS2 pro fimiga (SEQ ID NO:5) and the consensus sequence is illustrated (SEQ ID NO:6)) and Aspergillus nidulans (Fig. 1 C) (whereas is Fig. 1 C RDS2 (SEQ ID NO:2) is compared with RDS2 Aspergillus (SEQ ID NO:7) and the consensus sequence is illustrated (SEQ ID NO:8)). Similarly, STB5 homologs are found in C. albicans (Fig. 2) (whereas STB5 (SEQ ID NO:9) is compared with STB5CA (SEQ ID NO: 10) and the consensus sequence is illustrated (SEQ ID NO:11 )) as well as in Aspergillus fumigatus and Aspergillus nidulans. Moreover, the two highly related S. cerevisiae zinc cluster proteins Upc2 and Ecm22 have a homolog in C. albicans (Fig. 3) (whereas in Fig. 3 UPC2 (SEQ ID NO: 12) is compared with CaUPC2 (SEQ ID NO:13) and the consensus sequence is illustrated (SEQ ID NO: 14)). Comparison between RDS2 and CaRDS2 shows that the two proteins are related. In Figs. 1A, 1 B, 1C, 2 and 3, rectangles correspond to identical amino acids. For example, the N- terminus of CaRds2 contains 6 cysteines that are characteristic of zinc cluster proteins. Moreover, there is a high degree of amino acid identity between Rds2 and CaRds2 at the C-terminus (Fig. 1A).

The putative DNA binding domains of CaRDS2, CaUPC2 and FCR1 were expressed in E. coli and the polypeptides were purified for an electrophoretic mobility shift assay (EMSA). As a probe for EMSA, a drug response element (DRE) consisting of an imperfect direct repeat containing two CGG triplets (CGGN4CGG) was used. The DRE has been shown to increase the expression of the C. albicans genes CDR7 and CDR2 encoding transporters involved in drug resistance. Results show that the DNA binding domains of both CaUPC2 and FCR1 specifically bind to the DRE (Fig. 4, probe "A" (SEQ ID NO: 15 and complementary sequence SEQ I D NO: 16)). Since CGG triplets have been shown to be important for DNA recognition by many zinc cluster proteins in S. cerevisiae, the importance of these triplets was tested by using DREs containing a mutation in either CGG triplet (probes "B" (SEQ ID NO: 17 and complementary sequence SEQ ID NO: 18) and "C" (SEQ ID NO:19 and complementary sequence SEQ ID NO:20), Fig. 4). Binding of CaUpc2 or Fcr1 was nearly abolished when tested with these mutant probes. Other mutations in between the CGG triplets had little effect on binding of CaUpc2 or Fcr1 (Fig. 4, probes "D" (SEQ ID NO:21 and complementary sequence SEQ ID NO:22), "E" (SEQ ID NO: 23 and complementary sequence SEQ ID N0.24), "F" (SEQ ID NO:25 and complementary sequence SEQ ID NO:26) and "G" (SEQ ID NO:27 and complementary sequence SEQ ID NO:28)). Finally, a shorter DRE reduced binding of CaUpc2 but not Fcr1 (Fig. 4, DRE "H" (SEQ ID NO: 29 and complementary sequence SEQ ID NO:30)). A second DRE (called DRE1 B) has been shown to mediate expression of CDR1. DRE1B consists of a direct repeat containing two CGG triplets spaced by 15 bp (CGGN15CGG). This DNA element was also recognized by CaUpc2 and Fcr1. Finally, the purified DNA binding domain of CaRds2 also bound to a DRE as shown in Fig. 5. Thus, in vitro data obtained show that DNA elements known to modulate expression of genes encoding drug efflux pumps are recognized in vitro by the DNA binding domains of CaUpc2, Fcr1 and CaRds2.

Tests were made to verify if overproduction of CaUpc2, Fcr1 and CaRds2 would result in an increase of the sensitivity to the antifungal drug ketoconazole. Expression vectors (under the control of the MET3 promoter) for the proteins of interest were constructed and stably integrated in the genome of Candida albicans (at the RP10 locus). An empty expression vector was also integrated as a negative control. Strains containing an expression vector for CaUpc2 (or an empty expression vector) were grown overnight in rich medium, serially diluted and spotted on plates containing or not ketoconazole. Surprisingly, overexpression of the DNA binding domain of CaUpc2 (Fig. 6) and CaRDS2 (Fig. 8) increased resistance (lowers sensitivity) to the antifungal. These results show that CaUpc2 and CaRDS2 are involved in conferring drug resistance. On the other hand, overexpression of Fcr1 rather decreased resistance to ketoconazole as compared to an empty expression vector (Fig. 7, top). No decreased resistance to ketoconazole was observed with a mutant of FCR1 carrying a frameshift mutation at the 5' part of the open reading frame (Fig. 7, middle). Overexpression of the DNA binding domain of Fcr1 also decreased resistance to ketoconazole (Fig. 7, bottom). These results show that the DNA binding domain of Fcr1 is sufficient to alter drug sensitivity and that Fcr1 is a negative regulator of drug resistance genes.

The DNA binding domain of CaUPC2 act as a dominant positive regulator of drug resistance genes while Fcr1 is a negative regulator of these genes as already known.

Moreover, appearance of C. albicans resistant strains is due to mutations in the CaUPC2 gene or other related genes. Inactivation of CaUpc2 and other related transcription factors results in cells showing high sensitivity to drugs. Thus, CaUpc2 (and other related proteins) is a target for antifungal drugs. For example, a compound that disrupt the zinc finger inactivate the protein leading to cells hypersensitive to classical antifungals. Moreover, a drug targeting zinc cluster proteins in general is lethal to fungi but not to other eukaryotes since they do not possess zinc cluster motifs.

METHODS

Strains

Genomic DNA for amplification by PCR was isolated from Candida albicans strain CAI4 or SGY243. In vivo assays were performed with strain SGY243.

Electrophoretic mobility shift assay (EMSA)

A DNA fragment encoding the DNA-binding domain of CaRDS2 (a.a. 1- 144) was amplified by PCR using the oligos CGGGATCCAT GGATGGTCCC AATTTTGC (SEQ ID NO:31) and GGAATTCCTT GGTACGTCTT GGGGCTC (SEQ ID NO:32) and genomic DNA from C. albicans as a template. The PCR product was digested with BamHI and EcoRI and subcloned into plasmid pGEX-F cut with the same enzymes to give pGST-CaRDS2. An expression vector for the DNA binding domain of CaUpc2 (amino acids 1 to 148) was constructed in a similar way using the oligonucleotides CGGGATCCAT GATGATGACA GTGAAACA (SEQ ID NO:33) and GGAATTCTTA AATCACCGGC TGAGTTTTGA (SEQ ID N0:34). For the DNA binding domain of Fcr1 (a.a. 1-1 37), oligonucleotides ATCTAGAGAT CTATGTCTGA CGATCATTCA AT (SEQ ID NO:35) and GTAGAATTCT GTTCTTCAAC (SEQ ID NO: 36) were used for PCR amplification. The PCR product was cut with Bglll and EcoRI for subcloning into pGEX-F cut with BamHI and EcoRI.

The DNA-binding domains of zinc cluster proteins of interest fused to GST were expressed in E. coli and purified according to standard procedures. The GST moiety was removed by thrombin cleavage. EMSA was performed on a 4% polyacrylamide gel in 1X TBE buffer. The probe used in the EMSA corresponds to a DRE found in the CDR1 gene. The probe consists of two complementary oligonucleotides TCGATGTTAT TCAATTCACG GAAATCGGAT ATTTTTTTTT GTT (SEQ ID NO:37) and TCGAAACAAA AAAAAATATC CGATTTCCGT GAATTGAATA ACA (SEQ ID NO: 38) that were annealed and filled-in with Klenow and dGTP, dTTP, dATP and [32P]dCTP. Sequences of mutant DREs are shown in Fig. 4. Probe for DRE1 B was obtained using the oligonucleotides TCGAAGGGAT CGGATAGTGG GAGCTCAACG GAAAATT (SEQ ID NO:39) and TCGAAATTTT CCGTTGAGCT CCCACTATCC GATCCCT (SEQ ID N0.4O)

Expression vectors for Candida albicans

The open reading frame of CaUPC2 was amplified by PCR using the oligonucleotides CGGGATCCAT GATGATGACA GTGAAACA (SEQ ID N0:41 ) and AGATTACTCG AGCTATTTCA TATTCATAAA CCCAT (SEQ ID NO:42) using genomic DNA from Candida albicans. The PCR product was cut with BamHI and Xhol and subcloned into a modified pCaEXP expression vector (pCaEXP-MCS) cut with the same enzymes (Care, J. et al., Molecular Microbiology (1999) 34(4), 792-798). The CaUPC2 open reading frame is under the control of the MET3 promoter and carries a URA3 selection marker and RP10 sequences for targeted integration. An expression vector for Fcr1 was constructed as described above using the oligonucleotides ATCTAGAGAT CTATGTCTGA CGATCATTCA AT (SEQ ID NO:43) and AGATTACTCG AGATTGAAGA AAGGATCCAA TG (SEQ ID NO:44) except that the PCR product and the expression vector were cut with Bglll and Xhol. Oligonucleotides ATCTAGAGAT CTATGTCTGA CGATCATTCA AT (SEQ ID NO:45) and GTAGAATTCT GTTCTTCAAC (SEQ ID NO:46) were used for the construction of the expression vector for the DNA binding domain of FCR1 (amino acids 1 to 137). For expression of the DNA binding domain of RDS2, oligonucleotides CGGGATCCAT GTCTACCATG AGTACTCA (SEQ ID NO:47) and AACTGCAAGA ATTCTCCGTT T (SEQ ID NO:48) were used to amplify sequences encoding the DNA binding domain of CaRDS2. The PCR product was cut with BamHI and EcoRI for subcloning into pCaEXP-MCS cut with the same enzymes.

Integration of expression vectors in the Candida albicans genome

The expression vectors were linearized with Stul and transformed into Candida albicans strain SGY243 for integration at the RP10 locus. Transformants were selected on minimal plates lacking uridine. At least two independent clones for each expression vector were used for the drug sensitivity assays.

Assay for sensitivity to ketoconazole

Strains carrying integrated expression vectors were grown overnight in rich medium. Cells were then serially diluted and spotted on minimal plates lacking uridine and containing or not ketoconazole. Cells were grown for one to three days at 30°C.

Assay for resistance to ketoconazole

An assay for the resistance was performed as described in the method section. As illustrated in Fig. 9, the overexpression of CaStbδ decreases resistance to ketoconazole. In Fig. 9, the concentration of ketoconazole is indicated on the left. Each assay was performed with two independent clones. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A molecule for reducing resistance to an active agent in a fungus, said molecule reducing the activity of a zinc cluster protein acting as a positive regulator of said active agent in said fungus.

2. The molecule of claim 1 , wherein said fungus is a pathogen fungus.

3. The molecule of claim 2, wherein said fungus is selected from the group consisting of Candida and Aspergillus.

4. The molecule of claim 3, wherein said Candida is selected from the group consisting of Candida albicans, Candida glabrata, Candida kruser, Candida guillier-mondii and Candida dubliniensis.

5. The molecule of claim 3, wherein said Aspergillus is selected from the group consisting of Aspergillus nidulans, Aspergillus fumigatus and Aspergillus flavus.

6. The molecule of claim 1 , wherein said active agent is an antifungal agent.

7. The molecule of claim 6, wherein said antifungal agent is selected from the group consisting of fluconazole, ketoconazole and brefeldin A.

8. The molecule of claim 1 , wherein said gene is UPC2, an homolog thereof or an ortholog thereof.

9. A molecule for reducing resistance to an active agent in a fungus, said molecule increasing the activity of a zinc cluster protein acting as a negative regulator of said active agent in said fungus.

10. The molecule of claim 9, wherein said fungus is a pathogen fungus.

11. The molecule of claim 10, wherein said fungus is selected from the group consisting of Candida and Aspergillus.

12. The molecule of claim 11 , wherein said Candida is selected from the group consisting of Candida albicans, Candida glabrata, Candida kruser, Candida guillier-mondii and Candida dubliniensis.

13. The molecule of claim 11 , wherein said Aspergillus is selected from the group consisting of Aspergillus nidulans, Aspergillus fumigatus and Aspergillus flavus.

14. The molecule of claim 9, wherein said active agent is an antifungal agent.

15. The molecule of claim 14, wherein said antifungal agent is selected from the group consisting of fluconazole, ketoconazole and brefeldin A.

16. The molecule of claim 9, wherein said gene is RDS2, STB5, an homolog thereof or an ortholog thereof.

17. The molecule of claim 9, wherein said gene is STB5, an homolog thereof or an ortholog thereof.

18. A composition comprising the molecule of any one of claims 1- 17 in association with a pharmaceutically acceptable carrier.

19. In vitro use of the molecule of anyone of claims 1-17 as a target for determining resistance of said fungus to said active agent.

20. A method for screening a drug capable of preventing resistance to an active agent in a fungus, said method comprising the step of determining reduction of activity of a zinc cluster protein of said fungus caused by a candidate drug, whereby reduction of activity of said zinc cluster protein by said candidate drug is indicative of said drug being capable of preventing resistance to said active agent in said fungus.

21. The method of claim 20, wherein said drug is an antifungal agent.

22. A method for decreasing resistance to an active agent in a fungus comprising reducing the expression of a gene encoding a drug efflux pump.

23. The method of claim 22, wherein said gene comprises a zinc- cluster.

24. The method of claim 22, wherein said gene is UPC2, an homolog thereof or an ortholog thereof.

25. A method for decreasing resistance to an active agent in a fungus comprising increasing the expression of a gene encoding a drug efflux pump.

26. The method of claim 25, wherein said gene comprises a zinc- cluster.

27. The method of claim 26, wherein said gene is RDS2, STB5, an homolog thereof or an ortholog thereof.

28. The method of claim 26, wherein said gene is STB5, an homolog thereof or an ortholog thereof.