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)