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

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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. 25 FEBRUARY 2016 | VOL 530 | NATURE | 485 © 2016 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER 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.
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