Inhibiting Fungal Multidrug Resistance by Disrupting an Activator–Mediator Interaction Joy L

LETTER doi:10.1038/nature16963

Inhibiting fungal multidrug resistance by disrupting an activator–Mediator interaction Joy L. Nishikawa1,2, Andras Boeszoermenyi3, Luis A. Vale-Silva4, Riccardo Torelli5, Brunella Posteraro6, Yoo-Jin Sohn1,2, Fei Ji7,8, Vladimir Gelev9, Dominique Sanglard4, Maurizio Sanguinetti5, Ruslan I. Sadreyev7,10, Goutam Mukherjee11,12, Jayaram Bhyravabhotla11,12,13, Sara J. Buhrlage3,14, Nathanael S. Gray3,14, Gerhard Wagner3, Anders M. Näär1,2 & Haribabu Arthanari3

Eukaryotic transcription activators stimulate the expression of to identify small molecules that block the interaction between the specific sets of target genes through recruitment of co-activators CgGal11A KIX domain and the CgPdr1 AD (Extended Data Fig. 1). such as the RNA polymerase II-interacting Mediator complex1,2. On the basis of the high degree of conservation between S. cerevisiae and Aberrant function of transcription activators has been implicated C. glabrata, we followed up the top hits from the fluorescence polari- in several diseases. However, therapeutic targeting efforts have zation screen with an azole growth inhibition screen in S. cerevisiae to been hampered by a lack of detailed molecular knowledge of the mechanisms of gene activation by disease-associated transcription a b activators. We previously identified an activator-targeted three- In vitro 1.2 uorescence polarization screen helix bundle KIX domain in the human MED15 Mediator subunit (~140,000 compounds) 1.0 that is structurally conserved in Gal11/Med15 Mediator subunits 0.8 YPD 3,4 in fungi . The Gal11/Med15 KIX domain engages pleiotropic In vivo S. cerevisiae 0.6 YPD +KET

viability screen 600 nm drug resistance transcription factor (Pdr1) orthologues, which (352 compounds) A 0.4 iKIX1 are key regulators of the multidrug resistance pathway in 0.2 iKIX1 +KET

Saccharomyces cerevisiae and in the clinically important human Hits with azole-specic 0 5,6 growth inhibition 010 20 pathogen Candida glabrata . The prevalence of C. glabrata is (5 compounds) –1 rising, partly owing to its low intrinsic susceptibility to azoles, [iKIX1] (μg ml ) 7,8 the most widely used antifungal agent . Drug-resistant clinical iKIX1 Cl c iKIX1 isolates of C. glabrata most commonly contain point mutations additional S characterization 9–14 H in Pdr1 that render it constitutively active , suggesting that N N N Cl H H this transcriptional activation pathway represents a linchpin in N C. glabrata multidrug resistance. Here we perform sequential d O biochemical and in vivo high-throughput screens to identify 400 small-molecule inhibitors of the interaction of the C. glabrata Pdr1 300 250 activation domain with the C. glabrata Gal11A KIX domain. The 300 lead compound (iKIX1) inhibits Pdr1-dependent gene activation 200 200 and re-sensitizes drug-resistant C. glabrata to azole antifungals Kd = 0.32 μM 150 Ki = 18 μM in vitro and in animal models for disseminated and urinary tract 100 100 C. glabrata infection. Determining the NMR structure of the 50 C. glabrata Gal11A KIX domain provides a detailed understanding 0 0 Fluorescence anisotropy (a.u.) of the molecular mechanism of Pdr1 gene activation and multidrug 10–5 10–3 10–1 101 103 Fluorescence anisotropy (a.u.) 10–1 100 101 102 103 104 resistance inhibition by iKIX1. We have demonstrated the [CgGal11A KIX domain] (μM) [iKIX1] (μM) feasibility of small-molecule targeting of a transcription factor- Figure 1 | Discovery of inhibitors of the CgGal11A KIX-CgPdr1AD binding site in Mediator as a novel therapeutic strategy in fungal interaction. a, Schematic of the screening process. b, iKIX1 inhibits cell infectious disease. growth in a concentration-dependent manner in the presence of 5 μM On the basis of our previous findings that deletion of the KIX ketoconazole (KET); error bars represent means ±s.d. from duplicate domain of S. cerevisiae GAL11 or C. glabrata GAL11A abrogates Pdr1- plates. c, iKIX1 structure. d, Fluorescence polarization titration curve dependent transcriptional responses and xenobiotic tolerance we showing the interaction of CgGal11A KIX domain with CgPdr1 AD30 fitted to a dissociation constant (Kd) value of 319.7 nM ± 9.5 nM (left). hypothesized that the C. glabrata Pdr1–C. glabrata Gal11A (CgPdr1– iKIX1 competes out CgPdr1 AD30 with an half-maximum inhibitory CgGal11A) interaction interface might serve as a promising target for concentration (IC ) value of 190.2 μM ± 4.1 μM (right). The measured 3 50 novel anti-multidrug resistance compounds . A fluorescently tagged Kd and IC50 values were used to calculate an apparent inhibition constant CgPdr1 activation domain (AD) was used in an in vitro fluorescence (Ki) value of 18.1 μM for iKIX1. Data represent mean of two replicates and polarization screen15 of ~140,000 chemically diverse compounds standard error from the fit is shown.

1Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129, USA. 2Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA. 4Institute of Microbiology, University Hospital Lausanne and University Hospital Center, Lausanne CH-1011, Switzerland. 5Institute of Microbiology, Università Cattolica del Sacro Cuore, Rome 00168, Italy. 6Institute of Public Health, Università Cattolica del Sacro Cuore, Rome 00168, Italy. 7Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA. 8Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. 9Faculty of Chemistry and Pharmacy, Sofia University, Sofia 1164, Bulgaria. 10Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA. 11Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India. 12Supercomputing Facility for Bioinformatics & Computational Biology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India. 13Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India. 14Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA.

a c C. glabrata –3.0 3.0 N N S. cerevisiae N N

F47 K54 K68 d H43 M72

K78 180 K68 L19 iKIX1 R79 C C C C

b N L23 K54 L51 L51 A49 A49 M65 M72 L19 K68 I69 A44 H43 M72 R73 N CgPdr1 AD L23 R73 M24 M40 180 A37 R76 T75 T35 R79 T35 I29 N27 E82 K68 L19 C T36 iKIX1 L23 N30 S83 G31

L23 N M72

L51 L51 C K66 K66 L19 H43 A44 iKIX1 L23 R73 R73 M24 A37 T75 180 T35

I29 T35 E82 N27 C T36 L23 I26 N30 D25 Figure 2 | Elucidation of the CgGal11A KIX domain structure; highlighted in green (L19, L23 and L51) represent important CSPs in the CgPdr1 AD and iKIX1 bind to a similar interface on the CgGal11A side-chain methyl groups of an ILV labelled sample. c, The iKIX1 and KIX domain. a, Backbone representation of the 10 lowest energy NMR CgPdr1 AD target the hydrophobic groove on the CgGal11A KIX domain, structures of CgGal11A KIX domain; backbone r.m.s.d. ~ 0.7 Å, left. which is surrounded by a basic patch. Residues H43, K54, K68, K78, Overlay of CgGal11A (purple) and S. cerevisiae Gal11/Med15 (blue) KIX R79, F47 and M72 present a positive electrostatic surface enclosing the domains, with an overall r.m.s.d. of 2.0 Å (ref. 25), right. b, Chemical binding interface. d, iKIX1 docked to the CgGal11A KIX domain. iKIX1 shift perturbations (CSPs) on the CgGal11A KIX domain in the presence is depicted as red sticks and spheres. Residues near the binding site of of CgPdr1 AD (red) or iKIX1 (blue). Residues coloured in red or blue iKIX1 are depicted as blue sticks. The methyl resonances of L19 and L23 indicate a chemical shift perturbation greater than 2 s.d. Residues experience considerable CSP upon addition of iKIX1. identify hits with in vivo efficacy (Fig. 1a). We identified 5 compounds complements the acidic residues of CgPdr1 AD (Fig. 2c). Residues of that reproducibly inhibited growth in a concentration-dependent the CgGal11A KIX domain that interact with CgPdr1 AD and iKIX1 manner only in the presence of ketoconazole (Extended Data Fig. 1). overlap strongly, suggesting direct competitive binding as the mech- The most potent compound is referred to as iKIX1 (Fig. 1b, c). anism of inhibition. Docking of iKIX1 to the CgGal11A KIX domain In vitro binding studies revealed that the Kd of the CgPdr1 AD for the suggests extensive hydrogen bonding and hydrophobic interactions CgGal11A KIX domain is 0.32 μM and the apparent Ki for iKIX1 is between iKIX1 and KIX domain residues (Fig. 2d and Extended Data 18 μM (Fig. 1d). Fig. 2a), matching the interaction interface mapped by CSP analysis. To facilitate the elucidation of the mechanism of action of iKIX1, we To assess the in vivo effects of iKIX1 on Pdr1-dependent transcrip- determined the high-resolution solution structure of the CgGal11A tion, we initially used a strain in which the two S. cerevisiae PDR1 KIX domain with a backbone root mean square deviation (r.m.s.d.) of orthologues (ScPDR1 and ScPDR3) are deleted and which carries a 0.7 Å (Fig. 2 and Extended Data Table 1; PDB ID 4D7X). The CgGal11A plasmid expressing CgPDR13, and a heterologous luciferase gene driven KIX domain has 51% sequence identity and 61% similarity with the by 3 pleiotropic drug response elements (PDREs). Luciferase activity S. cerevisiae Gal11/Med15 KIX domain3 with an overall r.m.s.d. of 2.0 Å was strongly induced by ketoconazole treatment; iKIX1 co-treatment (Fig. 2a and Extended Data Fig. 2c). The CgGal11A KIX domain forms was able to block this induction in a concentration-dependent manner a three-helix bundle harbouring an extensively hydrophobic core and (Fig. 3a). a short helix at the N terminus (Fig. 2a). We determined interaction A chromatin immunoprecipitation (ChIP) assay was used to interfaces of the CgGal11A KIX domain with the CgPdr1 AD and iKIX1 examine Gal11/Med15 recruitment to Pdr1-regulated target genes in (Fig. 2b) by chemical shift perturbation (CSP) analysis. The CgPdr1 AD S. cerevisiae after iKIX1 treatment. Gal11/Med15 was rapidly recruited and iKIX1 target the same large hydrophobic groove harboured by the to the promoters of the Pdr1 target genes PDR5 and SNQ2 after keto- three helices. Residues from all three helices constitute the interaction conazole addition; in contrast, ketoconazole-induced recruitment of interface, and titration of an isoleucine, leucine, valine (ILV)-methyl Gal11/Med15 was abrogated when the cells were pre-treated with iKIX1 labelled CgGal11A KIX domain reveals large CSPs on the three leucines (Fig. 3b, Extended Data Fig. 3a). iKIX1 did not impede the constitutive (L19, L23 and L51) upon addition of CgPdr1 AD and iKIX1 (Extended occupancy of Pdr1 at the same Pdr1-regulated target genes (Extended Data Fig. 2b). The basic interaction interface on the KIX domain Data Fig. 3b). Consistent with the ChIP data, iKIX1 strongly inhibited

486 | NATURE | VOL 530 | 25 FEBRUARY 2016 © 2016 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH a b c g Gal11–HA recruitment to ScPDR5_UAS 800 Vehicle Vehicle ) 0.15 –1 30 μM iKIX1 KET 30 600 25 0.10 20 20 1–HA 400 ** 15 ** 10

0.05 ** ux rate (RFU s ** 10 Input (%) * ** ** in GAL1 200 5 *** *** *** 0 Normalized RLU 0 0 Fold change Sc PDR5 0251550 iKIX1 UT 0 5 10 15 0 5 10 15 DMSO 0 DMSO 20 M iKIX1 μM [iKIX1] (μM) μ 20 DMSO +KET Maximum e f Time after KET addition (min) μM iKIX1 +KET –200 20 pdr1Δ F L280 Y584C PDR1+ 1 1 d e f PDR PDR 105 RNA-seq: counts per million reads PDR1L280F PDR1Y584C PDR1T588A PDR1P822L PDR1D1082G 100 10 80 8 8 25 10 60 40 103 20 60 6 PUP1 8 6 30 ** PDR1 CAGL0M09713g 6 40 ** 15 40 4 CDR1 NS 4 ** YOR1 CDR2 20 10 * ** 4 ** ** CAGL0K04301g 20 2 ** 20 2 2 10 * 5 ** 10 YBT1 *** Fold change CgCDR2 Fold change CgCDR1 0 0 MEC3 0 0 0 0 0

CAGL0A01738g CgCDR1 fold change iKIX1 + KET expression

CAGL0B01969g DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO 0.1 10 μ M iKIX1 30 μ M iKIX1 10 μ M iKIX1 30 μ M iKIX1 10 μ M iKIX1 30 μ M iKIX1 10 μ M iKIX1 30 μ M iKIX1 10 μ M iKIX1 30 μ M iKIX1

10 μ M iKIX1 30 μ M iKIX1 10 μ M iKIX1 30 μ M iKIX1 10 μ M iKIX1 30 μ M iKIX1 10 μ M iKIX1 30 μ M iKIX1 0.1 10 103 105 +KET +KET KET expression Figure 3 | iKIX1 blocks Gal11/Med15 recruitment and upregulation of CgCDR2 in a CgPDR1 wild-type strain (SFY114). **P < 0.005. e, RNA- Pdr1 target genes. a, iKIX1 inhibits ketoconazole-induced upregulation seq analysis of a C. glabrata SFY114 (PDR1 wild-type) strain pre-treated of luciferase activity in a dose-responsive manner in a S. cerevisiae with iKIX1 or vehicle alone then induced with ketoconazole (iKIX1 + pdr1Δpdr3Δ strain containing plasmid-borne CgPDR1 and 3XPDRE- KET and KET, respectively). f, iKIX1 inhibits xenobiotic-induced CgPdr1 luciferase. UT, untreated control; **P < 0.001. b, c, iKIX1 prevents the transcription in CgPdr1 gain-of-function mutants (amino acid changes ketoconazole-induced recruitment of Gal11/Med15/Mediator to the indicated). Samples shown were induced with ketoconazole. *P < 0.05 and upstream activating sequences (UAS) of the PDRE-regulated promoter **P < 0.01 as compared to dimethylsulfoxide (DMSO) + ketoconazole ScPDR5 (b), and ScPDR5 induction (c). Representative experiment from control. g, iKIX1 inhibits rhodamine 6 G efflux in C. glabrata as compared two biological replicates (ChIP DNA and RNA from same experiment) is to vehicle control. *P < 0.05, **P < 0.005 as compared to DMSO + shown. Error bars indicate s.d. of technical replicates; ***P < 0.00001, ketoconazole control. a, d, e–g, Data represent the means of three **P < 0.0005 by two-tailed Student’s t-test. d, iKIX1 inhibits biological replicates. Two-tailed Student’s t-test used to determine ketoconazole-induced transcriptional upregulation of CgCDR1 and P values; error bars represent means ±s.d. azole-induced transcription of ScPdr1 target genes (Fig. 3c, Extended set of isogenic strains with gain-of-function CgPDR1 mutations orig- Data Fig. 3a, c). inally identified in azole-resistant C. glabrata clinical isolates9. iKIX1 Next, we determined the effect of iKIX1 on the transcription of reduced azole-induced transcription of CgPdr1 target genes (for exam- C. glabrata Pdr1-regulated genes involved in drug efflux and multi- ple, CgCDR1) in a concentration-dependent manner in all strains tested drug resistance (CgCDR1, CgCDR2 and CgYOR1). CgPdr1 targets (Fig. 3f and Extended Data Fig. 4d). were strongly upregulated after ketoconazole treatment10,12. However, To investigate whether these transcriptional effects translated to pre-treatment with iKIX1 reduced target gene induction in a durable functional effects on drug efflux rates, we used the fluorescent com- and concentration-dependent manner (Fig. 3d and Extended Data pound rhodamine 6 G, a substrate of the CgCdr1 efflux pump17,18. Fig. 4a, b). Treatment with iKIX1 alone did not significantly affect Pdr1- Maximum efflux rates were significantly decreased in PDR1 wild-type target gene induction (Extended Data Fig. 4c, d). or gain-of-function strains pre-treated with iKIX1, as compared to Next-generation RNA sequencing (RNA-seq) was employed to vehicle control (Fig. 3g and Extended Data Fig. 5). query the genome-wide effects of iKIX1 and azole treatments alone Owing to its ability to reduce efflux pump gene expression and and in combination on the transcriptome in both S. cerevisiae and pump activity, we predicted that iKIX1 could restore azole-sensitivity in C. glabrata. In accord with previous reports10,16, azole treatment to CgPDR1 gain-of-function mutant strains. Isogenic C. glabrata strains upregulates Pdr1-dependent genes in both yeasts, such as the drug with wild-type or single gain-of-function alterations across CgPdr1 efflux pumps ScPDR5 and CgCDR1 (Supplementary Tables 1 and 2). (Fig. 4a) were tested for their sensitivity to fluconazole or ketocona- Combined azole and iKIX1 treatment strongly blunted expression of zole on gradient plates with increasing concentrations of iKIX1 or many azole-activated and Pdr1-dependent genes in both S. cerevisiae vehicle. As expected, a CgPDR1 wild-type strain was sensitive to both and C. glabrata (Fig. 3e, Extended Data Fig. 3d and Supplementary fluconazole and ketoconazole, whereas CgPDR1 gain-of-function Tables 1 and 2), consistent with prior data and the proposed mech- mutant strains grew robustly in the presence of azoles. iKIX1 restored anism of action of iKIX1. iKIX1 alone affected very different sets of azole-sensitivity to PDR1 gain-of-function mutant strains in a concen- genes in S. cerevisiae and C. glabrata (Supplementary Tables 1–3). tration-dependent manner (Fig. 4b). CgPDR1 wild-type strains also Treatment of S. cerevisiae and C. glabrata cells with iKIX1 did not exhibited increased growth inhibition in the presence of both iKIX1 significantly alter the expression of PDR1 or GAL11/MED15 after and azole versus single agents alone (Extended Data Fig. 6a, b). azole treatment (Extended Data Fig. 3e). Together, these findings On the basis of the strong combination effect of azoles and iKIX1 in suggest that the primary mechanism of synergistic antifungal effects the CgPDR1L280F mutant we focused follow-up studies on this mutant of iKIX1 with azoles is through blocking the azole-stimulated and strain. To investigate whether azoles and iKIX1 act in a synergistic Pdr1-dependent drug efflux pathway. or additive manner in CgPDR1 wild-type and CgPDR1L280F mutant To ascertain iKIX1 efficacy in azole-resistant C. glabrata strains, we strains, we assessed growth in checkerboard assays with ketocona- examined the effects of iKIX1 on CgPdr1 target gene expression in a zole and iKIX1. In the wild-type CgPDR1 strain, the combination

Y584C a L280F R376W T588A P822L D1082G This example, together with data from iKIX1 analogues and the docked * * * * * * CgPdr1 DBD ID MHR AD structure of iKIX1 to the CgGal11A KIX domain, supports a model where iKIX1 engages the core of the KIX domain using an array of b DMSO DMSO DMSO hydrophobic and hydrogen-bond contacts.

PDR1+ (SFY114) We used two metazoan model systems to evaluate the potential utility PDR1L280F of iKIX1 as a co-therapeutic with fluconazole to treat disseminated PDR1R376W C. glabrata infection. The larvae of the moth Galleria mellonella has PDR1Y584C PDR1T588A been used as a model to test the pathogenicity of a wide variety of PDR1P822L human pathogens19. We used a G. mellonella survival assay to determine PDR1D1082G the virulence of C. glabrata PDR1 wild-type or PDR1L280F strains in the 150 μM FLU 2 μM KET iKIX1 iKIX1iKIX1 presence of fluconazole, iKIX1, or a combination of the two (Fig. 4c). Larvae were injected with C. glabrata and a single injection of flucona- PDR1+ (SFY114) −1 −1 PDR1L280F zole (50 mg kg ), iKIX1 (25 mg kg ), a combination of the two, or PDR1R376W vehicle; survival was monitored every 24 h. G. mellonella injected with PDR1Y584C PDR1T588A wild-type CgPDR1 was sensitive to fluconazole alone, and exhibited PDR1P822L no significant alterations in survival with a fluconazole-iKIX1 combi- PDR1D1082G nation. However, in G. mellonella larvae injected with a CgPDR1L280F 150 μM FLU 2 μM KET strain, whereas the single agents fluconazole or iKIX1 did not signifi- c SFY114: CgPDR1+ SFY115: CgPDR1L280F cantly increase survival compared to vehicle, co-treatment with iKIX1 100 100 and fluconazole significantly increased survival (P < 0.001). *** Non-injected 75 75 PBS + FLU + iKIX1 Prior to mammalian studies, we sought to evaluate the poten- Cg PBS 50 50 tial toxicity of iKIX1 in mammalian cells (Extended Data Fig. 7e, f). * Cg iKIX1 *** Cg FLU Human HepG2 cells treated with iKIX1 revealed toxicity only at high Survival (%) 25 Survival (%) 25 Cg FLU + iKIX1 concentrations of iKIX1 (IC50 ≈ 100 μM). iKIX1 had no effect on the 0 0 04896 144 192 240 04896 144 192 240 transcription of SREBP-target genes at concentrations up to 100 μM, 4 Time (h) Time (h) indicating its specificity for the fungal Gal11/Med15 KIX domain . We

d Kidney Spleen also assessed the in vitro stability and in vivo mouse pharmacokinetics 108 107 of iKIX1 and found that iKIX1 exhibited favourable drug-like proper- * *** ties and in vivo exposure in these studies (Extended Data Fig. 8 g, h). 107 106 * *** ** ** *** To evaluate the therapeutic potential of iKIX1 and azole antifungal *** *** *** 106 105 co-therapy in a mammalian model, we initially turned to an established mouse model of disseminated fungal disease11. Mice were inoculated CFU per g of orga n CFU per g of orga n 105 104 with C. glabrata by tail-vein injection and were dosed peritoneally once- 1 1 daily with 100 mg kg−1 fluconazole (high FLU), 100 mg kg−1 iKIX1, a SFY114 SFY115 SFY114 SFY115 combination of the two, or vehicle alone. After 7 days, mice injected SFY114 iKIX1 SFY115 iKIX1 SFY114 iKIX1 SFY115 iKIX1 SFY114 SFY114low FLU high FLU SFY115 high FLU SFY114 SFY114low FLU high FLU SFY115 high FLU with a CgPDR1 wild-type strain exhibited significantly reduced tissue SFY114 lowSFY114 FLU+iKIX high 1FLU+iKIXSFY115 high FLU+iKIX1 SFY114 lowSFY114 FLU+iKIX high 1FLU+iKIXSFY115 high FLU+iKIX1 fungal burden in the kidney and spleen following fluconazole treatment Figure 4 | iKIX1 as a co-therapeutic in models of C. glabrata alone; iKIX1 co-treatment did not result in further reductions (Fig. 4d). disseminated disease. a, Schematic showing CgPdr1 gain-of-function In contrast, in mice injected with the azole-resistant CgPDR1L280F alterations in relation to putative functional domains. DBD, DNA-binding strain, only co-treatment with iKIX1 and fluconazole resulted in sig- domain; ID, inhibitory domain; MHR, middle homology region; AD, nificant (~tenfold) reductions in fungal burdens in the kidney and activation domain. b, iKIX1 restores the efficacy of azoles towards CgPDR1 spleen (P < 0.0001) (Fig. 4d). Similar results were observed with the gain-of-function mutants. Plates contained increasing concentrations of clinically isolated CgPDR1+ and CgPDR1L280F strains DSY562 and vehicle control (DMSO) or iKIX1 to 150 μM in the absence or presence DSY565 (Extended Data Fig. 8a). Consistent with previous studies9, the of fluconazole (FLU) or ketoconazole (KET). c, iKIX1 in combination L280F with fluconazole but not fluconazole alone significantly extended survival fungal burden in mice infected with the CgPDR1 strain was higher of G. mellonella larvae injected with CgPDR1L280F (SFY115, n = 9). For than those infected with wild-type CgPDR1 strains, suggesting that SFY114, n = 10. *P < 0.05, ***P < 0.001 as compared to PBS vehicle PDR1 mutant strains may be more virulent in vivo. Similar but less pro- control. Statistical differences measured using a log-rank (Mantel–Cox) nounced results were found in mice injected with a CgPDR1P822L strain test. d, iKIX1 combination treatment with 25 mg kg−1 fluconazole (low (Extended Data Fig. 8b). When mice were injected with a CgPDR1 FLU) reduces fungal tissue burden in the kidney or spleen of mice wild-type strain and dosed with 25 mg kg−1 fluconazole (low FLU) injected with CgPDR1 wild-type (SFY114); iKIX1 in combination with −1 alone or in combination with iKIX1, fluconazole alone poorly reduced 100 mg kg fluconazole (high FLU) reduces fungal tissue burden in the tissue burden, whereas combination treatment resulted in significant kidney or spleen of mice injected with CgPDR1L280F (SFY115). n = 5 mice (~tenfold) reductions in fungal burdens in both organs (P < 0.0001) for each treatment condition; *P < 0.05, **P < 0.005 and ***P < 0.0001 as compared to no treatment. Statistical differences measured using a (Fig. 4d). These results suggest that iKIX1 combination treatment with Wilcoxon rank-sum test; error bars represent means ±s.d. azole may be therapeutically desirable even in the absence of CgPDR1 gain-of-function mutations. Mice infected with a Cgpdr1 null strain were more sensitive to iKIX1 alone; unlike mice infected with CgPDR1+ of ketoconazole and iKIX1 was additive (Extended Data Fig. 6c). or CgPDR1L280F strains, low doses of iKIX1 did not further reduce However, the CgPDR1L280F mutant exhibited synergistic growth inhi- fungal burden in Cgpdr1 null infections (Extended Data Fig. 8c, d). bition with iKIX1 and ketoconazole combination treatment, with com- CgPDR1 gain-of-function mutations are also known to control bination indices <1 (Extended Data Fig. 6d), in concordance with the adherence to host cells20. As previously observed20, a PDR1L280F mutant spot-plating assay. increased relative adherence to epithelial cells as compared to a PDR1 We carried out a limited analysis exploring the chemical space wild-type strain. Strikingly, iKIX1 treatment alone reduced adherence around the iKIX1 scaffold using commercial and custom synthesized to levels similar to a PDR1 wild-type strain (Extended Data Fig. 8e). iKIX1 analogues, identifying several compounds that lost activity in Ketoconazole alone or co-treatment with iKIX1 also reduced relative all assays; one analogue (A2) is shown in Extended Data Fig. 7a–d. adherence to levels comparable to a PDR1 wild-type strain. To assess

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EUCAST definitive document EDef 7.1: method for the determination of broth dilution MICs of antifungal agents for fermentative yeasts. Clin. Microbiol. Infect. 1. Conaway, R. C. & Conaway, J. W. Function and regulation of the Mediator 14, 398–405 (2008). complex. Curr. Opin. Genet. Dev. 21, 225–230 (2011). 26. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for 2. Poss, Z. C., Ebmeier, C. C. & Taatjes, D. J. The Mediator complex and fast protein structure alignment in three dimensions. Acta Crystallogr. D 60, transcription regulation. Crit. Rev. Biochem. Mol. Biol. 48, 575–608 2256–2268 (2004). (2013). 3. Thakur, J. K. et al. A nuclear receptor-like pathway regulating multidrug Supplementary Information is available in the online version of the paper. resistance in fungi. Nature 452, 604–609 (2008). 4. Yang, F. et al. An ARC/Mediator subunit required for SREBP control of Acknowledgements We are grateful to P. Coote, E. Papadopoulos, R. Oh cholesterol and lipid homeostasis. Nature 442, 700–704 (2006). and R. E. Luna for helpful discussions and advice with data analysis and 5. Paul, S. & Moye-Rowley, W. S. Multidrug resistance in fungi: regulation of manuscript preparation. We acknowledge the ICCB-Longwood Screening transporter-encoding gene expression. Front. Physiol. 5, 143 (2014). Facility at Harvard Medical School for assistance with the high-throughput 6. Prasad, R. & Goffeau, A. Yeast ATP-binding cassette transporters conferring screens and access to the compound libraries, and the MGH Next Gen multidrug resistance. Annu. Rev. Microbiol. 66, 39–63 (2012). sequencing core for RNA-seq library construction. Mouse plasma and 7. Pfaller, M. A. et al. Variation in susceptibility of bloodstream isolates of microsomal stability experiments were carried out at the Scripps Research Candida glabrata to fluconazole according to patient age and geographic Institute and iKIX1 pharmacokinetic parameters were assessed by Sai Life location in the United States in 2001 to 2007. J. Clin. Microbiol. 47, Sciences Limited. We acknowledge support from the National Institute of 3185–3190 (2009). Health (grants GM047467 to G.W. and A.M.N. and EB002026 to G.W.). 8. Pfaller, M. A., Messer, S. A., Moet, G. J., Jones, R. N. & Castanheira, M. Candida J.L.N. was supported by an NSERC fellowship. bloodstream infections: comparison of species distribution and resistance to echinocandin and azole antifungal agents in Intensive Care Unit (ICU) and Author Contributions J.L.N., A.B., G.W., A.M.N. and H.A. conceived and designed non-ICU settings in the SENTRY Antimicrobial Surveillance Program the studies. A.B. and H.A. performed experiments relating to protein structure, (2008–2009). Int. J. Antimicrob. Agents 38, 65–69 (2011). small molecule screening and small molecule-protein interaction and data 9. Ferrari, S. et al. Gain of function mutations in CgPDR1 of Candida glabrata not analysis. J.B. and G.M. performed the docking and free energy calculations. only mediate antifungal resistance but also enhance virulence. PLoS Pathog. V.G., S.J.B. and N.S.G. designed the synthesis for iKIX1 and its analogues. J.L.N. 5, e1000268 (2009). performed the in vivo small molecule screen, luciferase, ChIP, transcription, 10. Ferrari, S., Sanguinetti, M., Torelli, R., Posteraro, B. & Sanglard, D. Contribution efflux, spot plating, combination index and mammalian cell culture (HepG2) of CgPDR1-regulated genes in enhanced virulence of azole-resistant Candida experiments. Y.-J.S. performed transcription and efflux experiments. J.L.N. glabrata. PLoS One 6, e17589 (2011). prepared samples for RNA-seq analysis; bioinformatic analysis was carried 11. Silva, L. V. et al. Milbemycins: more than efflux inhibitors for fungal pathogens. out by F.J. and R.I.S.; L.A.V.-S. and D.S. designed and performed moth survival Antimicrob. Agents Chemother. 57, 873–886 (2013). and adhesion assays. R.T., B.P. and M.S. designed and executed mouse fungal 12. Vermitsky, J. P. et al. Pdr1 regulates multidrug resistance in Candida glabrata: burden and UTI model studies. J.L.N., A.B., G.W., A.M.N. and H.A. wrote the gene disruption and genome-wide expression studies. Mol. Microbiol. 61, manuscript with input from the team. 704–722 (2006). 13. Sanguinetti, M. et al. Mechanisms of azole resistance in clinical isolates of Author Information Coordinates and NMR resonance assignments have been Candida glabrata collected during a hospital survey of antifungal resistance. deposited in the Protein Data Bank (PDB code 4D7X) and Biological Magnetic Antimicrob. Agents Chemother. 49, 668–679 (2005). Resonance Data Bank (BMRB code 25372). RNA-seq data have been deposited 14. Caudle, K. E. et al. Genomewide expression profile analysis of theCandida in the Gene Expression Omnibus (GEO) under accession GSE74361. Reprints glabrata Pdr1 regulon. Eukaryot. Cell 10, 373–383 (2010). and permissions information is available at www.nature.com/reprints. The 15. Roehrl, M. H., Wang, J. Y. & Wagner, G. A general framework for development authors declare no competing financial interests. Readers are welcome to and data analysis of competitive high-throughput screens for small-molecule comment on the online version of the paper. Correspondence and requests for inhibitors of protein-protein interactions by fluorescence polarization. materials should be addressed to G.W. ([email protected]), or Biochemistry 43, 16056–16066 (2004). A.M.N. ([email protected]) or H.A. ([email protected]).

Extended Data Figure 1 | List of compound libraries screened and top that preferentially inhibit growth of S. cerevisiae in a concentration- hits from the secondary in vivo viability screen. Left, table of compound dependent manner in the presence of 5 μM ketoconazole. Top hits from libraries that were screened using a fluorescence polarization assay at the the screen are shown; A600 nm values are the average of values from Institute of Chemistry & Cell Biology (ICCB) facility at Harvard Medical duplicate plates. Error bars denote mean ± s.d. School. Right, an S. cerevisiae viability screen identifies small molecules

Extended Data Figure 2 | Mapping the interaction interface of iKIX1 showing ILV methyl resonances of CgGal11A KIX domain in presence and CgPdr1 AD on the CgGal11A KIX domain and molecular details (brown) and absence (teal) of CgPdr1 AD (twofold excess). Right, of iKIX1 interaction with the CgGal11A KIX domain based on targeted 1H-13C HSQC spectrum showing ILV methyl resonances of CgGal11A docking. a, 2-dimensional representation of the H-bonding network KIX domain in presence (purple) and in absence (teal) of iKIX1 (fourfold between the CgGal11A KIX domain and iKIX1 based on docking studies. excess). Three leucines (L19, L23, L51) show significant CSPs in both b, Chemical shift perturbations (CSPs) of ILV methyl resonances. Left, spectra. c, Sequence alignment of the C. glabrata Gal11A and S. cerevisiae 1H-13C heteronuclear single quantum coherence (HSQC) spectrum Gal11/Med15 KIX domains24.

Extended Data Figure 3 | iKIX1 blocks Gal11/Med15 recruitment and biological replicates is shown. Error bars represent mean ±s.d. of technical upregulation of Pdr1 target genes. a, iKIX1 prevents the ketoconazole- replicates; *P < 0.05, **P < 0.01 and ***P < 0.001 as calculated by two- induced recruitment of ScGal11/Med15/Mediator to the upstream tailed Student’s t-test. d, RNA-seq analysis of a wild-type S. cerevisiae activating sequences (UAS) of the PDRE-regulated promoter ScSNQ2 and strain (BY4741) pre-treated with iKIX1 or vehicle alone then induced with transcriptional upregulation of ScSNQ2. b, Haemagglutinin (HA)–tagged ketoconazole (iKIX1 + KET and KET, respectively) demonstrate a blunted Pdr1 occupies PDRE-regulated promoters of ScPDR5 and ScSNQ2 in the induction of Pdr1 target genes following iKIX1 pre-treatment. Data presence of 20 μM iKIX1 or vehicle (DMSO) control before and following represent means of three biological replicates. e, iKIX1 pre-treatment does ketoconazole addition. c, 20 μM iKIX1 inhibits ketoconazole-induced not significantly alter the transcript levels of PDR1 or GAL11/GAL11A in upregulation of ScPdr1 target genes ScPDR5 and ScSNQ2 in the HA–Pdr1 S. cerevisiae or C. glabrata after azole induction. Cells were pre-incubated strain. RNA was harvested concurrently with representative chromatin with vehicle (DMSO) or iKIX1 and then induced with 40 μM ketoconazole immunoprecipitation experiment shown in b at t = 0 min (DMSO, (+KET) for 15 min before harvest. Average value of three biological 20 μM iKIX1) and t = 15 min after ketoconazole induction (DMSO + replicates is shown and error bars represent mean ±s.d.; *P < 0.05, KET, 20 μM iKIX1 + KET). Transcripts are normalized to ScSCR1 and **P < 0.001 as compared to DMSO or DMSO+KET control, calculated by un-induced DMSO control. a–c, Representative experiment from two two-tailed Student’s t-test.

Extended Data Figure 4 | iKIX1 pre-treatment durably represses does not have significant effects on CgPdr1 target gene induction either in azole-induced CgPdr1-dependent transcription but not basal levels the presence of wild-type (SFY114) or gain-of-function mutant CgPDR1 of CgPdr1-dependent transcripts. a, b, With iKIX1 pre-treatment, (amino acid alterations indicated). d, Table of average CgCDR1 delta Cp CgPdr1-dependent transcription of (a) CgCDR1 and (b) CgYOR1 remains values (CpCgCDR1 – CpCgRDN25-1) and corresponding standard deviation for repressed 120 min after ketoconazole induction. SFY114 (PDR1 wild- quantitative real-time PCR experiments shown in Fig. 3 f and Extended type) cells were pre-incubated with vehicle (DMSO) or iKIX1 and then Data Fig. 4c. a–d, For all panels of Extended Data Fig. 4, average value induced with 40 μM ketoconazole (+KET). Transcript levels were assessed of three biological replicates is shown and error bars represent ±s.d.; by quantitative RT–PCR before and for 120 min following ketoconazole *P < 0.05, **P < 0.01, and ***P < 0.005 as compared to vehicle induction. Transcript levels are normalized to CgRDN25-1 and + ketoconazole control. P values calculated using two-tailed Student’s t-test. un-induced vehicle control (DMSO) at t = 0. c, iKIX1 treatment alone

Extended Data Figure 5 | iKIX1 inhibits efflux of rhodamine 6 G in PDR1 wild-type, PDR1L280F and PDR1Y584C strains. Data points indicate mean of three biological replicates and error bars represent mean ±s.d.

Extended Data Figure 6 | iKIX1 increases the sensitivity of Cg strains on the growth of a CgPDR1 wild-type strain. d, iKIX1 and ketoconazole bearing wild-type CgPDR1 to azole treatment and synergistically synergistically inhibit the growth of the CgPDR1L280F mutant. c, d, The enhances azole sensitivity in a CgPDR1L280F mutant strain. a, b, Two EUCAST broth microdilution method25 was used to assess the effects strains bearing wild-type CgPDR1 alleles (SFY114, DSY759) were plated of iKIX1 and ketoconazole combination treatment. Growth, as assessed at concentrations differing by tenfold (10×, 1×) on plates containing by A540 nm, was normalized to no drug control. All combination indices increasing concentrations of iKIX1 to 300 μM in the presence or absence (CI) for the CgPDR1L280F mutant were less than 1, indicating synergy. of 1 μM ketoconazole (a) or iKIX1 to 250 μM in the presence or absence A representative of three biological replicates is shown and the red line of 50 μM fluconazole (b). c, iKIX1 and ketoconazole have additive effects indicates a combination index of 1.

Extended Data Figure 7 | iKIX1 exhibits favourable drug-like t-test. e, iKIX1 inhibits viability of HepG2 cells at concentrations >50 μM. properties and an inactive analogue highlights the importance of The mean of 3 biological replicates is shown; error bars represent means the electron withdrawing chloride groups on the aromatic ring of ±s.d. f, iKIX1 exhibits no effect on transcription of SREBP-target genes iKIX1. a–d, A structurally similar iKIX1 analogue (A2) lacking electron- in HepG2 cells at concentrations up to 100 μM. Biological duplicates were withdrawing groups increases the IC50 in the fluorescence polarization assessed; representative experiment is shown and error bars represent assay (a) and abolishes activity in the S. cerevisiae luciferase reporter assay means ±s.d. of technical (real-time PCR) replicates. g, Mouse plasma (b), repression of CgCDR1 expression (c), and synergistic C. glabrata cell stability of iKIX1 and mouse and human microsomal stability of iKIX1, growth inhibition with azoles (d). Error bars in b, c indicate mean ±s.d. of n = 1. h, In vivo pharmacokinetic parameters of iKIX1, n = 3 mice per technical replicates (reads/real-time PCR reactions, respectively). time point. **P < 0.005; statistical differences calculated using two-tailed Student’s

Extended Data Figure 8 | iKIX1-fluconazole combination treatment **P < 0.005, ***P < 0.0001. d, Mice infected with SFY114 (PDR1+), reduces fungal tissue burden by CgPDR1+ or CgPDR1 gain-of-function SFY115 (PDR1L280F) or SFY93 (pdr1Δ) were treated with low (10 mg kg−1 mutant strains in disseminated disease models; iKIX1 alone reduces day−1) iKIX1, low fluconazole (low FLU; 25 mg kg−1 day−1), fluconazole adherence and fungal burden in a UTI model. a, Clinical isolates at 100 mg kg−1 day−1 or combination with the two. iKIX1 did not confer DSY562/DSY565 (azole sensitive and PDR1L280F azole-resistant strains, additional reductions in CFU per g of kidney with SFY93 infection. + respectively) behave similarly to SFY114/SFY115 (isogenic PDR1 and n = 10 mice for each treatment condition. ***P < 0.0005. e, iKIX1 and PDR1L280F strains, shown in Fig. 4d) in the mouse infection model. n = 10 ketoconazole reduce adherence of CgPDR1L280F (SFY115) to CHO-Lec2 mice for each treatment condition; *P < 0.01, **P < 0.005 and cells. Adherence is normalized to SFY114 DMSO control; each column ***P < 0.0001. b, iKIX1 combination treatment with fluconazole reduces represents the average of 4 biological replicates. *P < 0.05 as compared fungal tissue burdens in the spleen or kidney of mice injected with to SFY114 DMSO control. f, iKIX1 (100 mg kg−1 day−1) or fluconazole C. glabrata PDR1P822L (SFY116). n = 5 mice for each treatment condition; significantly reduces fungal burden in the bladder and kidney in a urinary **P < 0.01 and *P < 0.05. c, 100 mg kg−1 day−1 iKIX1 (high iKIX1) tract infection model in mice. n = 15 mice were infected in each group and treatment of mice infected with SFY93 (pdr1Δ) significantly reduces points at 0 log10 CFU per g of organ fell below the detection limit of the fungal burden in a mouse infection model (colony forming units (CFU) method (50 CFU per g of organ). *P < 0.05, **P < 0.005. a–f, Statistical per g of kidney) alone as compared to SFY114 (PDR1+) or SFY115 differences were measured using a Mann–Whitney/Wilcoxon rank-sum (PDR1L280F). n = 10 mice for each treatment condition; *P < 0.01, test as compared to no treatment control; error bars represent means ±s.d.

Extended Data Figure 9 | Model of iKIX1 function. iKIX1 functions as a co-therapeutic in combination with an azole, blocking the azole-induced recruitment of Gal11/Med15-Mediator to Pdr1 target genes upon azole-treatment and preventing the upregulation of Pdr1 target genes, including those which encode drug efflux pumps. This figure has been adapted from ref. 3.

Extended Data Table 1 | Summary of quality statistics for the ensemble of 10 structures calculated with AMBER explicit water refinement and list of experimental restraints