Targeting the Transcription Factor Upc2 for Antifungal ... › wp-content › uploads ›...
Transcript of Targeting the Transcription Factor Upc2 for Antifungal ... › wp-content › uploads ›...
ABSTRACT
RATIONALE
RESULTS
CONCLUSIONS & FUTURE DIRECTIONS
RESULTS
Targeting the Transcription Factor Upc2 for Antifungal Drug Discovery
Ilana L. Stroke, Melissa T. Manners, Robert N. Swanson, Denise Dimitrov, Christina Gallo-Ebert, and Joseph Nickels
Venenum Biodesign, LLC, Hamilton, NJ, 08690
Infection by Candida albicans and related fungal pathogens poses a serious health problem for immunocompromised patients. Azole drugs, the most common agents used to combat these infections, target the sterol biosynthesis pathway. Resistance to azole therapy develops as stressed cells compensate by upregulating several genes in the pathway, a process mediated by a “master regulator” transcription factor, Upc2. Upc2 is conserved among yeast species and binds to DNA motifs known as sterol response elements (SRE’s). Historically, targeting transcription factors for drug discovery has proved to be a challenge, and generally the initial hits from compound screens are only weakly active. We have therefore taken a two-pronged approach to identify compounds that interfere with the function of Upc2. We have implemented a high-throughput assay for inhibitors of C. albicans Upc2 DNA binding, along with a parallel cell-based screen for specific inhibitors of the induction of sterol biosynthesis gene transcription by Upc2 in response to azole drugs. The latter screen is designed to identify not only DNA binding inhibitors, but also compounds impeding the activation of Upc2 by drug treatment. Our data show a comparison of screening results using the two very different but complementary approaches. Additionally, we present data showing cross-species DNA binding, and that a novel, non-consensus SRE sequence functions as a Upc2 binding site. (Supported by the Genesis Biotechnology Group).
• Biochemical and cell-based screens for small molecule inhibitors of Upc2 yielded non-overlapping sets of hits.
• Compounds identified in the cell-based screen did not inhibit DNA binding.
• Studies are underway to identify mechanism of action and to confirm Upc2 as the molecular target. Additionally, compounds thought to be specific for Upc2 or the Upc2 activation pathway will be tested for synergy with azole drugs at a panel of yeast species and mutant strains.
• Goal: identification of compounds increasing sensitivity to azole drugs, for treatment of Candida infections
• The transcription factor Upc2 upregulates several ergosterol biosynthesis genes in response to antifungal drug treatment
• Redundant function of ECM22 and UPC2 in S. cerevisiae• C. albicans has UPC2 only
• Binding sequence conserved in S. cerevisae and C. albicans• 7 bp sterol response element (SRE)
• Yeast strains• Wild type S. cerevisiae with UPC2-LacZ reporter
• S. cerevisiae ecm22Δ
• Wild type C. albicans
Candida albicans Upc2 DNA binding assay
C. albicans Upc2 DNA binding domain: Alpha Screen assay for binding to WT SRE
in double-stranded C. albicans ERG2 oligonucleotide
Compe&&on by unlabeled SRE DNA’s
Alpha Screen DNA binding assay competition with unbiotinylated versions of double-stranded SRE oligonucleotides * demonstrates novel SRE motif is functional in binding Upc2 ** as previously shown by EMSA (S. MacPherson et al., 2005. Antimicrob. Agents Chemother. 49:1745)
Prestwick drug library: non-‐overlapping hits from DNA binding vs. cell-‐based assays
5 intercalators/ topoisomerase inhibitors,
e.g., anthracyclines, propidium iodide,
mitoxantrone
7 hits @ IC50 < 20 µM
C. albicans DNA-binding assay
S. cerevisiae cell-based assay
7 selective hits @ IC50 < 10 µM
other
AlphaScreen artifact test
C. albicans azole sensitivity assay
inhibitors
Prestwick drug library: UPC2-‐specific hits in the S. cerevisae cell-‐based assay
n≥2 unless specified n=2
76,000-‐compound diversity library screen: representa&ve UPC2-‐specific hits from the S. cerevisae cell-‐based assay
compound UPC2-LacZ IC50 (n=4)*
PGK1-LacZ IC50 (n=2)
DNA binding % control at 30 µM
VB00075177 0.29 +/- 0.07 µM no inhibition at 30 µM 99
VB00075853 0.63 +/- 0.06 µM > 20 µM 51
VB00075845 0.92 +/- 0.10 µM > 20 µM 51
VB00049027 1.5 +/- 1.0 µM no inhibition at 60 µM 83
VB00008679 2.5 +/- 0.5 µM no inhibition at 60 µM 90
VB00073293 2.6 +/- 0.7 µM no inhibition at 30 µM 94
VB00059166 3.3 +/- 0.5 µM no inhibition at 60 µM 84
VB00068594 4.2 +/- 0.7 µM no inhibition at 30 µM 69
* mean +/- SEM
Reproducibility of a uHTS UPC2-‐LacZ reporter library screen
• Prestwick FDA-approved drug library (1120 compounds) • well-by-well comparison • 4 µL assay volume in 1536-well format
enhanced expression
inhibition
test #1, % control
test #2, % control
SRE-‐containing oligonucleo&des
• 1: Vik and Rine (2001). Mol. Cell. Biol. 21:6395. • 2: S. MacPherson et al., 2005 Antimicrob. Agents Chemother. 49:1745.
Top strand of duplex DNA is shown.
Consensus SRE motif1 5’-TCGTATA-3’
C. albicans ERG2 modified to novel SRE motif 5’-TCGATGTCGTTTAAAA-3’
C. albicans WT ERG2 SRE2 5’-TCGATGTCGTATAAAA-3’
C. albicans mutant ERG2 SRE2 5’-TCGATGTCAGATAAAA-3’
Drugs targe&ng the sterol biosynthesis pathway
Promoters activated by Upc2 include ERG2, ERG7, ERG11, and ERG25, as well as UPC2 itself.
Screening strategy C. albicans
Upc2 DNA binding screen
S. cerevisiae UPC2-LacZ cell-based
screen
S. cerevisiae housekeeping promoter-
LacZ counterscreen
AlphaScreen artifact counterscreen
DNA intercalator counterscreen
C. albicans azole sensitivity
Synergism?
UPC2-‐LacZ reporter gene induc&on by azole drugs in S.cerevisiae
EC50 = 12 ± 1 µg/mL (n=5)
VENENUM BiodesignHamilton, NJ 08691
Toll Free:877-950-1032 • Fax:609-587-1768www.venenumbiodesign.com
Drugs up-‐regula&ng UPC2-‐LacZ expression
20 most potent enhancers identified from Prestwick FDA-approved drug library, in order of decreasing potency
Tomatidine Econazole Fluvastatin
Clotrimazole Isoconazole Halofantrine Miconazole Tolnaftate Lovastatin
Benzethonium chloride Butaconazole Clofazimine Sulconazole Merbromin
Methyl benzethonium chloride Sertaconazole
Naftifine Meclozine
Enilconazole Simvastatin
Azoles (lanosterol 14 α-demethylase inhibitors) Allylamines (squalene epoxidase inhibitor) Statins (HMG-CoA reductase inhibitors) Steroidal alkaloid (ergosterol biosynthesis inhibitor)
Drugs up-‐regula&ng UPC2-‐LacZ expression
20 most potent enhancers identified from Prestwick FDA-approved drug library, in order of decreasing potency
Tomatidine Econazole Fluvastatin
Clotrimazole Isoconazole Halofantrine Miconazole Tolnaftate Lovastatin
Benzethonium chloride Butaconazole Clofazimine Sulconazole Merbromin
Methyl benzethonium chloride Sertaconazole
Naftifine Meclozine
Enilconazole Simvastatin
Azoles (lanosterol 14 α-demethylase inhibitors) Allylamines (squalene epoxidase inhibitor) Statins (HMG-CoA reductase inhibitors) Steroidal alkaloid (ergosterol biosynthesis inhibitor)
TomatidineEconazoleFluvastatin
ClotrimazoleIsoconazoleHalofantrineMiconazoleTolnaftateLovastatin
Benzethonium chlorideButaconazoleClofazimineSulconazoleMerbromin
Methyl benzethonium chlorideSertaconazole
NaftifineMeclozine
EnilconazoleSimvastatin
Azoles (lanosterol 14 α-demethylase inhibitors)Allylamines (squalene epoxidase inhibitor)Statins (HMG-CoA reductase inhibitors)Steroidal alkaloid (ergosterol biosynthesis inhibitor)