Identification and Evaluation of Novel Acetolactate Synthase Inhibitors as Antifungal Agents ...

33 Pages·2013·5.99 MB·English

AAC Accepts, published online ahead of print on 11 March 2013
Antimicrob. Agents Chemother. doi:10.1128/AAC.01809-12
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
1 Identification and Evaluation of Novel Acetolactate Synthase Inhibitors as Antifungal
2 Agents
3 Running Title: Acetolactate Synthase Inhibitors as Antifungal Agents
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5 Richie Daryl L.1, Thompson Katherine V.1, Studer Christian2, Prindle Vivian1, Aust Thomas2, n
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6 Riedl Ralph2, Estoppey David2, Tao Jianshi1, Sexton Jessica A.1, Zabawa Thomas1, Drumm d
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7 Joseph1, Cotesta Simona2, Eichenberger Jürg2, Schuierer Sven2, Hartmann Nicole2, Movva N. fr
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8 Rao2, Tallarico John A.1, Ryder Neil S.1#, Hoepfner Dominic2#
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10 1Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge MA s
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11 02139, United States of America or
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12 2Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel Switzerland n
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13 #Corresponding authors addresses: a
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14 Dominic Hoepfner, Novartis Institutes for BioMedical Research, Novartis Campus, CH-4056 Basel 2
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15 Switzerland. Phone: [phone], Fax: [phone], Email: [email protected] 9

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16 Neil S. Ryder, Novartis Institutes for BioMedical Research, 500 Technology Square, Cambridge MA g
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17 02139, United States of America. Phone [phone], Fax [phone], Email: st
18 [email protected]
19 Key Words: Acetolactate synthase, Ilv2, Ilv6, HIP HOP, antifungal agents, branched chain
20 amino acid synthesis, target identification, sulfonamide, triazolo-pyrimidine-sulfonamide
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21 ABSTRACT
22 High-throughput phenotypic screening against yeast Saccharomyces cerevisiae revealed a series
23 of triazolo-pyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no
24 significant cytotoxicity, and low protein binding. To elucidate the target of this series we have
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25 applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All o
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26 compounds of the series yielded highly similar profiles that suggested acetolactate synthase lo
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27 (Ilv2p, catalyzes the first common step in branched chain amino acid biosynthesis) as a possible e
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28 target. High correlation to profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided ro
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29 further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae h
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30 identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate //
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31 synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping the .
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32 mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity
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33 and it was possible to dock the triazolo-pyrimidine-sulfonamide compound into this pocket in /

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34 silico. However, fungal growth inhibition could be bypassed through supplementation with J
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35 exogenous branched chain amino acids, or by the addition of serum to the medium in all of the u
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36 fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the 6
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37 identification of triazolo-pyrimidine-sulfonamide as inhibitors of acetolactate synthase but 0
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38 suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo. b
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40 INTRODUCTION
41 Increasing incidence of life-threatening systemic fungal infections, associated with high
42 mortality in immunocompromised patients, poses a significant unmet medical need. As part of a
43 program to identify novel antifungal agents we have run a phenotypic screen using S. cerevisiae
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44 and tested hits for their activity spectrum in a panel of fungal pathogens. Further investigation of o
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45 one such hit led to identification of a series of triazolo-pyrimidine-sulfonamide compounds with lo
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46 broad-spectrum antifungal activity. Yeast chemogenomic profiling indicated that the probable e
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47 target of these compounds was ILV2, encoding acetolactate synthase, a key enzyme in the ro
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48 biosynthesis of branched-chain amino acids. h
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49 Amino acid biosynthesis pathways are potential antifungal targets as they are highly conserved //
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50 in fungi, while humans lack biosynthetic pathways for de novo synthesis of “essential” amino .
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51 acids. The branched-chained amino acid biosynthetic pathway (isoleucine, valine, and leucine) is
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52 of particular interest as auxotrophic mutants in both C. albicans and C. neoformans have been /

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53 reported to show reduced virulence in mouse models (1,2). Acetolactate synthase (ALS, also J
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54 termed acetohydroxyacid synthase) catalyzes the first common step in branched-chained amino u
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55 acid synthesis in fungi as well as in plants and bacteria (3,4). Interestingly, several classes of 6
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56 commercially used herbicides, including sulfonylureas, imidazolinones, sulfonanilides and some 0
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57 others, target this pathway in plants through the inhibition of ALS (4,5). The b
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58 triazolopyrimidine/sulfonanilide herbicides were shown to be sub-micromolar inhibitors of ALS u
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59 from both plants and yeast (6,7). We report here the identification and characterization of a
60 structurally similar series of putative ALS inhibitors and their evaluation as potential leads for
61 antifungal drug discovery.
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62 MATERIALS and METHODS
63 Fungal strains and media conditions
64 Fungal strains used in this study were as follows: Saccharomyces cerevisiae BY4741 (Open
65 Biosystems Cat # YSC1056), Saccharomyces cerevisiae (ATCC 9763), Candida albicans
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66 (ATCC 24433), Aspergillus fumigatus (ATCC MYA-3627), Rhizopus oryzae (ATCC 4621), and
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67 Cryptococcus neoformans (ATCC 36556). Strains used in the expanded MIC panel are described n
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68 in Supplemental Table S1. For analysis of the amino acid rescue using defibrinated sheep blood de
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69 (DSB), 5,000 conidia were spotted on to AMM (Aspergillus Minimal Medium, minus fr
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70 supplement solution) agar (8)or water-agarose (1% agarose in distilled water) supplemented with h
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71 5% DSB (Remel, R54012) containing the indicated concentrations of Compound 1. The plates :/
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72 were incubated at 35°C, for 3 days before being photographed. Compounds were dissolved in c
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73 90% DMSO and stored at 4°C for up to 8 weeks. Further scientific studies with the chemical and m
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74 biological material published in this study are encouraged and can be obtained under MTA. g
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75 Primary screen and antifungal susceptibility testing Ja
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76 The primary compound screen against S. cerevisiae was performed as described previously (9). ry

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77 Antifungal susceptibility testing was performed according to the Clinical and Laboratory 2
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78 Standards Institute (CLSI) guidelines for broth microdilution M27-A3 and M38-A2 (10,11). 9

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79 Testing was performed in RPMI-1640 (HyClone, SH30011.03) with 2.05 mM glutamine, phenol g
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80 red, without bicarbonate and buffered with 0.165 mol/l 3-(N-morpholino) propanesulfonic acid s
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81 (MOPS, AppliChem, A1076,0250). The medium was adjusted to pH 7.0 and filtered-sterilized.
82 Where indicated, assay medium was supplemented as described: 10% human serum (Sigma,
83 S7023, 115K89091), 10% fetal calf serum (Remel, R92157), 10% fetal bovine serum (FBS,
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84 Gibco, 10082-139), 10% bovine serum albumin (BSA, Fisher, BP671-10), leucine (Acros
85 Organics, 328-39-2), isoleucine (Acros Organics, 73-32-5), and/or valine (Acros Organics, 72-
86 18-4). Two endpoints were recorded: MIC-0 (complete suppression of visible fungal growth) and
87 MIC-2 (prominent, >50% growth inhibition). All experiments were repeated at least 3 times with
88 similar results. D
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89 Assay for mammalian cell toxicity n
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90 The WST-1 cytotoxicity/proliferation assay was performed according to manufacturer’s d
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91 instructions (Roche Cat # 11 644 807 001) to test compounds for growth inhibition over 72 h in 2 f
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92 mammalian cell lines: K562 (CRL-1573), and HEPG2 (HB-8065).
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93 Chemogenomic profiling :
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94 The growth-inhibitory potency of test substances was determined using wild-type S. cerevisiae ac
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95 BY4743. OD600 values of exponentially growing S. cerevisiae cultures in rich medium were m
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96 recorded with a robotic system. Twelve point serial dilutions were assayed in 96 well plates with rg
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97 a reaction volume of 150 µl. Solutions containing dimethyl sulfoxide (DMSO) were normalized n

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98 to 2%. IC30 values were calculated using logistic regression curve fits generated by TIBCO nu
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99 Spotfire v3.2.1 (TIBCO Software Inc). ry

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100 The Saccharomyces haploinsufficiency profiling (HIP) and homozygous profiling (HOP) 0
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101 and microarray analysis was performed as published previously (12). The basic concept behind b
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102 this assay is that HIP identifies genes where one functional copy, compared to two, confers u
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103 hypersensitivity to inhibition by the compound. This indicates pathways directly affected by the
104 compound. HOP (both gene copies deleted) indicates synthetic lethality and identifies
105 compensating pathways to those directly affected by the compound. Thus genome wide hetero-
106 and homozygous deletion libraries of S. cerevisiae strains were purchased, (OpenBiosystems,
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107 Cat # YSC1056 and YSC1055) and pools were constructed as published previously (12). Each
108 HIP strain is heterozygous and each HOP strain completely null for one gene (and each strain
109 being identified by a unique DNA sequence, called “bar-code” or “tag” inserted into the deleted
110 gene). For each HIP and HOP experiment, each test substance was assayed in duplicate (2 wells)
111 at its IC30 concentration, in 24 well plates (Greiner 662102), with 1600 µl /well YPD. DMSO D
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112 was normalized to 2%. At the onset of the HIP experiment ~250 yeast cells/strain (100 µl of a n
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113 1.5 OD600/ml culture) from an overnight log phase pre-culture where added to the compound and d
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114 DMSO containing wells. Plates were incubated for 16 hours in a robotic shaking incubator at f
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115 30°C/550 RPM allowing for ~5 doublings. ~250 yeast cells/strain (120 µl of a 1.2 OD600/ml h
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116 culture) were subsequently transferred into a new 24 well plate. Once inoculated, the new plate :
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117 was incubated at 30°C/550 RPM to allow the next 5 yeast generations (generation 6-10). This ac
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118 procedure was repeated 2x more until the final plate containing the yeast with ~20 generations m
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119 were stored at 4°C. The HOP assay was performed similar to the HIP experiment but the rg
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120 duration was reduced to 16 hours/ ~5 doublings and thus no back-dilutions were necessary. n

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121 Before the experiment, aliquots of the HOP pool were thawed and recovered for 3 hours in YPD, n
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122 then ~320 yeast cells/strain (110 µl of a 1.5 OD600/ml culture) were transferred into each well. ry

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123 The cell material from the final HIP and HOP plates was harvested, the gDNA extracted and the 2
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124 tags amplified as published previously (12). Then relative abundance of each strain in the 9

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125 compound treated wells was compared to eight no-drug control samples that were produced in g
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126 the same experiment. st
127 For the experimental analysis we used the same computation of normalized tag intensities,
128 outlier masking and saturation correction as published previously (12). Sensitivity was computed
129 as Median absolute deviation logarithmic (MADL) score for each compound/concentration
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130 combination, then gene-wise z-scores (across all experiments) which are based on a robust
131 parametric estimation of gene variability allowing for up to 15% outliers were computed as
132 described in detail in (Hoepfner et al., submitted for publication). Similarity of the obtained HOP
133 profiles was compared by calculating the Pearson correlation coefficients of a compound HOP
134 profile with all other compound profiles. In order to assess the significance of the correlation D
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135 coefficients we applied the Fisher z´ transformation and fitted a normal distribution to the n
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136 transformed values. This empirical normal distribution allowed us to assign a p-value to each d
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137 correlation coefficient. The (negative) logarithm of the false discovery rate (FDR) corrected p- f
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138 value is then displayed. The complete dataset for all compounds presented in this study is
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139 provided in the online supplemental materials (Supplemental Material S1). The raw microarray :
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140 data will be provided upon request. ac
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141 Selection of drug-resistant S. cerevisiae
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142 Strain BY4743∆8, derived from BY4741 but deleted for eight genes involved in drug resistance o
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143 (efflux pumps: SNQ2, PDR5, YOR1; transcription factors: PDR1, PDR2, PDR3, YAP1, YRM1) a
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144 was incubated in 2.5% ethylmethanesulfonate until only 50% of the cells formed colonies. A r
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145 total of 2 x 107 mutagenized cells were plated on two 14 cm dishes with synthetic complete ,
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146 medium (0.7 g/l Difco Yeast Nitrogen Base w/o amino acids, 0.79 g/l MPbio CSM amino acid 19

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147 mixture, 2% Glucose) containing 100 nM compound 1. After four days 13 resistant colonies with y
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148 mutations in ILV2 (as identified by direct sequencing) could be isolated. Resistance by mutated es
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149 ILV2 was confirmed by cloning the corresponding mutations into fresh BY4743∆8 cells and
150 recording dose-response curves in YPD medium with serial dilutions of compound 1,
151 chlorimuron-ethyl and sulfometuron-methyl (PS1081, 34224, Sigma-Aldrich) at 200 µM
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152 maximum concentration, and 11 serial dilutions. DMSO was normalized to 2%. Curves were
153 calculated by taking the 22 h OD600 measurements and applying a logistic regression curve fit.
154 In silico docking
155 The published S. cerevisiae Ilv2-chlorimuron-ethyl co-crystal structure (13) was used to perform
156 the molecular docking of compound 1. The docking was performed using ICM (Molsoft, version D
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157 3.6-1) using default parameters. The obtained docking mode has been further minimized in the n
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158 binding pocket using Macromodel (implemented in Maestro, version 9.3) using OPLS_2005 d
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159 force field and implicit water. The digital file is available on the Dryad website f
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160 (http://dx.doi.org/10.5061/dryad.qb753).
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161 Amino acid rescue experiments :
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162 OD600 values of exponentially growing S. cerevisiae cultures in YPD medium were recorded ac
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163 with a robotic system. Twelve point serial dilutions were assayed in 96 well plates with a m
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164 reaction volume of 150 µl. Solutions containing DMSO were normalized to 2%. Logistic rg
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165 regression curve fits were used to generate dose-response curves using TIBCO Spotfire v3.2.1 n

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166 (TIBCO Software Inc). Cells were grown in YPD medium in absence or presence of additional n
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167 branched-chain amino acids (50 mM Valine, 50 mM Leucine, 50 mM Isoleucine), and the Erg11 ry

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168 inhibitor voriconazole (PZ0005, Sigma-Aldrich) was used as a control. 2
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169 RESULTS
170 Triazolo-pyrimidine-sulfonamides have broad spectrum in vitro activity and minimal
171 cytotoxicity
172 To identify novel compounds with antifungal activity, high-throughput screening was performed
173 using the Novartis compound archive against S. cerevisiae cells in a miniaturized 1536 well plate D
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174 assay (9). Compound 1 showed complete growth inhibition at the screening concentration of 20 n
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175 μM. Using compound 1 as a lead tool molecule, similarity searches revealed 7 structurally d
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176 similar substances of the triazolo-pyrimidine-sulfonamide class (Figure 1), which had not been fr
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177 part of the primary screening collection. All compounds displayed low µM IC30s on S. cerevisiae h
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178 (Figure 1) and significant broad-spectrum antifungal activity in vitro as shown in Table 1. In :/
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179 most cases, the compounds achieved a complete suppression of fungal growth (MIC-0). c
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180 Robustness of the obtained MIC values was confirmed when we repeated testing on a larger m
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181 panel of strains, mostly clinical isolates (for details see Supplemental Table S1 and g
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182 Supplemental Table S2). Additionally, the WST-1 cytotoxicity/proliferation assay
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183 demonstrated that all 8 compounds had no significant cytotoxicity activity against two common u
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184 cell lines used for measuring the cytotoxicity and antiproliferative properties of xenobiotics
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185 (Table 2). These characteristics, coupled with the observation that the substances fall nicely 0
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186 within the drug-like chemical space as predicted by Lipinski (not more than 5 hydrogen bond b
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187 donors, not more than 10 hydrogen bond acceptors, a molecular mass below 500 Daltons, and a u
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188 octanol-water partition coefficient logP not greater than 5) (14), suggested an attractive starting t
189 point for an antifungal program.
190 Chemogenomic profiling identifies acetolactate synthase Ilv2p as potential target
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191 While target identification is not absolutely essential for drug development, it facilitates the
192 optimization of a compound’s inhibitory activity. To investigate the mechanism of action we
193 subjected all 8 compounds of the series to chemogenomic profiling using the S. cerevisiae
194 heterozygous and homozygous deletion collections. Haploinsufficiency profiling (HIP) and
195 homozygous profiling (HOP) is a gene-dosage dependent method that assesses the effect of D
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196 compounds against potential targets encoded by the S. cerevisiae genome (15). HIP indicates n
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197 pathways directly affected by the compound. HOP (both gene copies deleted) indicates synthetic d
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198 lethality and identifies compensating pathways to those directly affected by the compound. f
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199 Compound 1 was tested in two independent experiments at slightly different concentrations (3 h
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200 and 4 μM) and both experiments yielded very similar results (Figure 2A and 2B). The :/
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201 heterozygous ILV6/ilv6 strain showed the most pronounced hypersensitivity against compound 1 c.
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202 at both concentrations tested. ILV6 codes for the regulatory subunit of ALS(16). Interestingly,
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203 ILV6 was also the most pronounced hit in the HOP experiments. As in the HOP strains the genes g/

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204 are completely deleted, this indicates that Ilv6p cannot be the target of compound 1 as the protein
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205 is not present in the ilv6/ilv6 HOP strain. But it indicates that ILV6 scores synthetic genetic u
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206 interactions with the compound target. Such interactions are published for the ILV6/ILV2 pair: 6
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207 Ilv6p regulates activity of the catalytic subunit of ALS, encoded by ILV2 (16,17). The ILV2 HIP 0
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208 strain did not score as hypersensitive as ILV6, but its sensitivity was scored as relevant by the b
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209 applied z-score (z-score <-5). Profiling the established Ilv2p sulfonylurea inhibitors chlorimuron- u
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210 ethyl and sulfometuron-methyl (Figure 2C and 2D) provided further evidence for Ilv2p being
211 the primary target of compound 1 as both substances produced HIP HOP profiles that were very
212 similar to those of compound 1 with ILV6 scoring in HIP and HOP and ILV2 scoring by the
213 applied z-score in HIP (Figure 2E and 2F). Extending testing to the other 7 compounds resulted
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