www.sciencemag.org/cgi/content/full/316/5832/1759/DC1

Supporting Online Material for

An Antifungal Agent Inhibits an Aminoacyl-tRNA Synthetase by Trapping tRNA in the Editing Site

Fernando L. Rock, Weimin Mao, Anya Yaremchuk, Mikhail Tukalo, Thibaut Crepin, Huchen Zhou, Yong-Kang Zhang, Vincent Hernandez, Tsutomu Akama, Stephen J.

Baker, Jacob J. Plattner, Lucy Shapiro, Susan A. Martinis, Stephen J. Benkovic, Stephen Cusack, M. R. K. Alley*

*To whom correspondence should be addressed. E-mail: dalley@anacor.com

Published 22 June 2007, Science 316, 1759 (2007)

DOI: 10.1126/science.1142189

This PDF file includes:

Materials and Methods SOM Text Figs. S1 to S9 Tables S1 to S3 References

Supporting Online Material For

An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site

Fernando L Rock1¶, Weimin Mao1¶, Anya Yaremchuk2,3, Mikhail Tukalo2,3, Thibaut

Crepin2, Huchen Zhou1,4, Yong-Kang Zhang1, Vincent Hernandez1, Tsutomu Akama1, Stephen J. Baker1, Jacob J Plattner1, Lucy Shapiro5, Susan A Martinis6, Stephen J

Benkovic7, Stephen Cusack2, M R K Alley1* ¶These authors contributed equally to this work *To whom correspondence should be addressed. E-mail: dalley@anacor.com This Supporting Online Material contains Materials and Methods Figs. S1, S2, S3, S4, S5, S6, S7, S8, S9 Tables S1, S2, S3 References

2

Materials and Methods Chemicals Compound C (4-fluoro-2-methyl-phenylboronic acid) and compound D (4-fluoro-phenylboronic acid) were purchased from Sigma-Aldrich. AN2690 and compound A were synthesized as previously outlined (S1).

Compound B (4-fluoro-2-(methoxymethyl)phenylboronic acid) was synthesized as follows. To a solution of (2-bromo-5-fluorophenyl)methanol (5 g, 24.4 mmol) in 75 mL of N,N-dimethylformamide (DMF) was added sodium hydride [60% suspension in mineral oil](1.27 g, 31.7 mmol) in portions. The mixture was stirred at room temperature for 10 minutes before the addition of methyl iodide (1.98 mL, 31.7 mmol) and then stirred for a further 3 hrs before water was added to quench the reaction. The reaction mixture was then extracted with ethyl acetate, washed with brine, dried over sodium sulfate and concentrated in vacuo to give 1-bromo-4-fluoro-2-(methoxymethyl)benzene (4.77 g, 89%) as a yellow oil. 1H NMR 300 MHz (DMSO-d6) δ 7.60-7.65 (dd, J=5.4, J=8.4 Hz, 1H), 7.22-7.27 (dd, J=3.0, J=9.3 Hz, 1H) 7.08-7.15 (m, 1H), 4.40 (s, 2H), 3.36 (s, 3H). To a solution of 1-bromo-4-fluoro-2-(methoxymethyl)benzene (4 g, 18.3 mmol) in 100 mL of tetrahydrofuran (THF) we added triisopropyl borate (6.3 mL, 27.4 mmol). The solution was cooled to -78oC before the addition of n-butyl lithium (1.6M in hexanes, 1.98 mL, 31.7 mmol). This reaction mixture was stirred and allowed to slowly warm to room temperature. Water was then added to quench the reaction. The reaction mixture was concentrated in vacuo and partitioned between ethyl acetate and 3N HCl. The organic phase was washed with brine, dried over sodium sulfate and concentrated in vacuo until a white precipitate was formed. This solid was filtered and air dried to give compound B, 4-fluoro-2-(methoxymethyl)phenylboronic acid (1.84 g, 54%). m.p. 127-136oC. 1H NMR 300 MHz (DMSO-d6 w/D2O) δ 7.50-7.55 (dd, J=6.6, J=8.1 Hz, 1H), 7.00-7.11 (m, 2H), 4.53 (s, 2H), 3.26 (s, 3H). MS (ESI) m/z [M-H]-: calculated 183.0; observed 183.0.

Compound E (5-fluoro-phthalide) was prepared as follows, to a solution of AN2690 (1 g, 6.6 mmol) in 20 mL of methanol, Pd(OAc)2 (1.48 g, 6.6 mmol) was added. The mixture was stirred under 1 atm carbon monoxide at room temperature for 2hrs. The reaction mixture was extracted by ethyl acetate from water, washed with brine, dried over sodium sulfate and concentrated in vacuo. The crude compound was purified by column chromatography using a mobile phase of hexane : ethyl ether (2:1) to give a mixture of compound E and its ring-opened form. A 5 mL volume of 6M hydrochloric acid was added to the mixture and then stirred at room temperature for 30 minutes. Compound E was extracted by ethyl acetate from water, then recrystallized from hexane and ethyl acetate to give compound E (5-fluoro-phthalide) as a white crystal (240 mg, 24%). 1H-NMR (300 MHz, DMSO-d6� ppm 7.90 (dd, J = 8.4, 5.1 Hz, 1H), 7.54 (dd, J = 8.4, 1.5 Hz, 1H), 7.42 (td, J = 8.7, 2.4 Hz, 1H), 5.39 (s, 2H). m.p. 104-106 oC. ESI-MS m/z [M+H]+: calculated 153.0; observed 153.1.

Compound F (4-fluoro-6-hydroxymethylbenzaldehyde) was prepared as follows, to a solution of 2-bromo-5-fluorobenzaldehyde (10 g, 49 mmol) in methanol (75 mL) and THF (10 mL) in an ice bath, sodium boron hydride (1.4 g, 37 mmol) was slowly added. The mixture was stirred at room temperature for 30 minutes. Water was added to quench the reaction. The reaction mixture was then extracted with ethyl acetate, washed

3

with brine, dried over sodium sulfate and concentrated in vacuo to give 2-bromo-5-fluorobenzyl alcohol (10 g, 100%). A mixture of 2-bromo-5-fluorobenzyl alcohol (10 g, 49 mmol), 3,4-dihydro-2H-pyran (6.7 mL, 73.5 mmol) and (1S)-(+)-10-camphorsulfonic acid (232 mg, 1 mmol) in dichloromethane was stirred overnight at room temperature. The reaction was quenched by addition of sodium bicarbonate aqueous solution folowed by an ethyl acetate extraction, washed with brine, dried over sodium sulfate and concentrate in vacuo. The crude compound was subject to column chromatography (dichloromethane) to give 4-Fluoro-2-[(tetrahydro-2H-pyran-2-yl)oxy] methylbromobenzene (14.6 g, 100%). A solution of 4-fluoro-2-[(tetrahydro-2H-pyran-2-yl)oxy] methylbromobenzene (10 g, 34.6 mmol) in anhydrous THF (75 mL) was cooled to -78oC under a nitrogen atmosphere. To the above solution 1.6 M n-butyl lithium in hexane (25.9 mL, 41.5 mmol) was added dropwise. The reaction was stirred for 5 minutes before the addition of anhydrous DMF (5.4 mL, 69.2 mmol) at -78oC and then the reaction mixture was warmed to room temperature. After stirring at room temperature for 15 minutes, the reaction was quenched by addition of water, then extracted with THF, washed with brine, dried over sodium sulfate and concentrated in vacuo. The crude compound was subject to column chromatography will a mobile phase of hexane:ethyl aceteate (10:1) to give 4-fluoro-2-[(tetrahydro-2H-pyran-2-yl)oxy]methylbenzaldehyde (6.2 g, 75%). To a solution of 4-fluoro-2-[(tetrahydro-2H-pyran-2-yl)oxy]methylbenzaldehyde (500 mg, 2.1 mmol) in THF (2 mL) was added 6M hydrochloric acid (1 mL, 6.3 mmol). The mixture was then stirred at room temperature for 1.5 hrs followed by the addition of 1 M NaOH to raise the pH above 7. The mixture was extracted with ethyl acetate, washed with brine, dried over sodium sulfate and concentrated in vacuo. The crude compound was subject to column chromatography (hexane : ethyl acetate= 4 :1; with 0.5% triethyl amine) to give compound F which formed a white solid (112 mg, 35%). Compound F exists in both aldehyde and hemi-acetal forms in solution (1 :1.5 in DMSO-d6). 1H-NMR (300 MHz, DMSO-d6)� ppm aldehyde: 10.12 (s, 1H), 7.99 (dd, J = 8.4, 5.7 Hz, 1H), 7.00-7.50 (m, 2H), 5.54 (t, J = 5.4 Hz, 1H), 4.91 (d, J = 5.4 Hz, 2H); hemi-acetal 7.00-7.50 (m, 3H), 6.76 (d, J = 7.5 Hz, 1H), 6.29 (dd, J = 7.5, 2.1 Hz, 1H), 4.84-5.08 (m, 2H). m.p. 45-60 oC. ESI-MS m/z [M-OH]+: calculated 137.04; observed 137.1. Strains and plasmids

All Saccharomyces cerevisiae strains and shuttle vectors were obtained from ATCC. Wild type haploid S. cerevisiae strain ATCC 201388 (MATa his3Δ1 Leu2Δ0 met5Δ0 ura3Δ0) was used in the selection of AN2690 spontaneous resistant mutants, and ATCC 200901 (MATα Leu2Δ0 lys2Δ0 ura3Δ0) was used to create diploids to show that AN2690 resistant mutations were dominant. The yeast-Escherichia coli centromere-based shuttle vector pRS315 (ATCC 77144) was used to construct the AN2690 resistant mutant libraries. While pRS425 (ATCC 77106), which is a multicopy 2 µM based shuttle vector, was used to show that additional copies of CDC60+ (S. cerevisiae gene encoding cytoplasmic leucyl-tRNA synthetase) conferred an eight-fold increased resistance to AN2690 (S2). Determination of minimum inhibitory concentrations (MIC)

4

The determination of the minimum inhibitory concentrations (MIC) were performed following the CLSI guidelines M27-A2 (S3) with the exception that for S. cerevisiae growth YPD (1% yeast extract, 2% Bacto™Peptone, 2% glucose) was used instead of RPMI-MOPS media. Isolation of resistant mutants The haploid S. cerevisiae strain ATCC 201388 was grown overnight in YPD broth (BD) at 300C to about 1.0x108 cells/mL. Cells were concentrated 10-fold in YPD broth, and 100 µL was plated out onto 30 YPD agar (YPD broth plus 1.5% Bacto™Agar) plates containing 1.6 µg/mL of AN2690. Resistant mutants appeared after 2 days of incubation at 300C. The frequency of resistance was determined by dividing the number of the mutants by the total number of cells plated. To determine the dominant nature of these AN2690 resistant mutants, they were mated to an isogenic MATα strain ATCC 200901 and diploid heterozygotes were selected on synthetic defined media minus lysine, methionine and histidine (S4). The MIC values of the resulting diploid heterozygotes were determined in YPD media using the microbroth dilution method (S3). A 300 µL aliquot of ethyl methanesulfonate (EMS) was added to 10ml of approximately 5x107 cells/mL of S. cerevisiae ATCC 201388 in 50mM potassium phosphate buffer, pH 7.0 and then incubated for 30 minutes at 300C with shaking (S4). The EMS was inactivated by the addition of 10% (w/v) sodium thiosulfate (Sigma-Aldrich). The cells were washed twice with sterile water and plated out on YPD agar plates containing 1.6 µg/mL of AN2690. Construction of plasmid genomic DNA library and identification of the AN2690 resistance gene

Genomic DNA was isolated from ANA327, ANA326 and ANA322 (Table S2) using a Qiagen Genomic tip. DNA from these three mutants were partially digested with Mbo I (Fermentas) and size fractionated into 4-10 kb DNA fragments by agarose gel electrophoresis. The DNA was gel purified using a Wizard®SV Gel kit (Promega), and then ligated into Bam HI (Fermentas) digested pRS315. The ligation reactions were dialyzed against deionized and reverse-osmosis treated water for 30 minutes using VSWP02500 filters (Millipore). The dialyzed DNA was used to transform E. cloni 10G SUPREME Electrocompetent Cells (Lucigene) via electroporation. The Plasmid DNA was extracted from pools of E. coli colonies using a Qiagen miniprep kit and S. cerevisiae ATCC 201388 was transformed (S4) and selected for resistance to 1 µg/mL of AN2690 and leucine prototrophy on synthethic defined media minus leucine. DNA was extracted from the AN2690 resistant colonies using the Qiagen DNeasy kit and used to transform E. coli. DNA was extracted from the carbenicillin resistant E. coli colonies and retested by transforming S. cerevisiae ATCC 201388 to ensure the plasmid contained the AN2690 resistant gene. If the plasmid conferred AN2690 resistance on S. cerevisiae ATCC 201388, the plasmid was sequenced by Sequetech Corporation. Additional AN2690 resistance mutations were mapped by amplifying the CDC60 DNA sequence for the editing domain using the following primers, 5'-GAT TCA AGC TCA GAT CTT GAC AAA TCC-3' and 5'-CAG GTT TAA TGC CGT AGT ATT CAG GTT-3', and sequencing those PCR products that conferred resistance on S. cerevisiae ATCC 201388 upon transformation.

5

Cloning and over-expressing wild type CDC60 in S. cerevisiae and E.coli To increase the copy number of CDC60 in S. cerevisiae we amplified the 4.0kb Bam HI-Sal I DNA fragment containing the whole CDC60 open reading frame (ORF) and 700bp of upstream sequence from S. cerevisiae genomic DNA (Novagen) using the following primers; 5'-GAG GGA TCC GGT TAG TTT TAG TTC GCG AGT GAC CTG-3' (Operon), 5'-GAG GTC GAC GAT TTC TGG TTG CTG TTT ATT GAT CTT-3' (Operon). This DNA fragment was digested with Bam HI and Sal I then cloned into the Bam HI-Sal I sites of pRS425 (ATCC 77106) and the resulting construct was used to transform S. cerevisiae ATCC 201388. For our biochemical studies we over-expressed in E. coli an N-terminal six histidine-tagged Cdc60p, which was made by amplifying the CDC60 ORF from the S. cerevisiae genomic DNA (Novagen) using the following primers; 5'-GAG AGC TAG CCA TCA TCA TCA TCA TCA CAT GTC TTC TGG TTT GGT CTT AGA AAA CAC-3' (Operon), 5'-AGA GCT CGA GAA TAT TTT GGA AGA CAA CAC CTG GGT TAC-3' (Operon) and a proof-reading DNA polymerase KOD (Novagen). The amplified DNA fragment was digested with Nhe I and Xho I and cloned into the corresponding sites in pET21b to create pWT-CDC60N. The correct plasmid construct was confirmed by DNA sequencing (Sequetech) and then transformed into Rosetta 2(DE3) cells (Novagen). The N-terminal six histidine-tagged Cdc60p was over-expressed and purified according to Novagen’s commercial protocol with the exception that mid-log Rosetta 2(DE3) cells containing pWT-CDC60N were induced with 0.1 mM IPTG and allowed to grow overnight at room temperature. Protein synthesis inhibition.

A 50 µL aliquot of mid-log S. cerevisiae S288C (ATCC 204508) grown in synthetic defined media minus Leucine (SDM-Leu) was added to 50 µL of SDM-Leu with 0.25 µCi of [14C]-Leucine (GE Healthcare) containing different concentrations of antifungal compounds. The cells were incubated at 300C for 15 minutes, then precipitated by the addition of 100 µL of ice cold 10% (w/v) tricloroacetic acid (TCA). This mixture was incubated for a further 15 minutes at 40C. The TCA precipitate was filtered through a PerkinElmer Unifilter®-96, GF/B® filter using a PerkinElmer FilterMate™ cell harvester. The TCA precipitate was washed with a further 400 µL ice cold 5% (w/v) TCA. The filters were dried and 40 µL of OptiPhase ‘Supermix’ scintillation cocktail (PerkinElmer) was added to the filters and the radioactivity was measured in a Wallac MicroBeta Trilux model 1450 liquid scintillation counter.

Norvaline sensitivity test

The relevant S. cerevisiae strain was grown overnight in synthetic define media (SDM) including Leucine at 100 µg/mL. A 200 µL aliquot of S. cerevisae was added to 3 mL of SDM containing 0.7% (w/v) Bacto™Agar. The agar was quickly poured on top of a SDM 100 mm Petri-plate and allowed to solidify. Then a ¼ inch 740-E filter disc (Schleicher & Schuell) was placed on top of the agar and a 20 µL of norvaline (40 mg/mL) was quickly added on top of the filter. The plates were then incubated right side up for 24-48 hrs at 300C. The control S. cerevisiae strain was ATCC 201388. The images of the agar plates were captured using a UVP Biochemi Image acquisition system

6

and exported as TIFF files, which were then cropped in Adobe Photoshop v8 and placed in a Adobe Illustrator V11 file. Synthesis of mischarged [3H]-Ile-tRNALeu

The mischarged [3H]-Ile-tRNALeu was synthesized by incubating 1 µM of S. cerevisiae editing defective Cdc60p (C326F) in 500 µL of 50mM Tris-HCl (pH 8.0), 60mM MgCl2, 4mM ATP, 1mM dithiothreitol (DTT), 0.02% (w/v) bovine serum albumin (BSA), 0.2 mg/mL brewer’s yeast tRNA (Roche), 0.1mM isoleucine and 5 mCi L-[3H]isoleucine (100Ci/mmole, GE Healthcare) and 20% (v/v) dimethylsulfoxide (DMSO) for 1 hour at 300C. The reaction was stopped by adding 10 µl of 10% (v/v) acetic acid followed by two acidic phenol (Sigma) extractions (S5). The aqueous phase was precipitated by adding 1/10 volume of 3M sodium acetate pH 5.2 and two volumes of 96% (v/v) ethanol and the pellet was washed twice with 70% (v/v) ethanol. The tRNA pellet containing the mischarged [3H]-Ile-tRNALeu was resuspended in 50 mM potassium phosphate buffer pH 5.2. Post-transfer editing assay

Mischarged [3H]IletRNALeu substrate, 40nM, was added to 50mM HEPES-KOH pH 8.0, 30mM KCl, 30mM MgCl2, 0.02% (w/v) BSA, 1mM DTT, 2.4 nM S. cerevisiae Cdc60p at 300C to start the reaction. At indicated time points 20 µL aliquots were taken and were added to ice cold 200 µL 10% (w/v) TCA. The TCA precipitates were washed twice with 200 µL ice cold 5% (w/v) TCA and filtered through a Multiscreen HTS HA filter (Millipore). Optiphase (Perkin Elmer) scintillation cocktail was added to the filters and the radioactivity of the filtered TCA precipitates were measured in a Wallac MicroBeta Trilux model 1450 liquid scintillation counter. Aminoacylation assay.

Experiments were performed in 500 µL reaction mixtures containing 50 mM HEPES-KOH (pH 8.0), 30 mM MgCl2 and 30 mM KCl, 20 µM [14C]leucine (306 mCi/mmol, Perkin-Elmer), 0.2 mg/mL brewer’s yeast tRNA (Roche), 0.02% (w/v) BSA, 1 mM DTT, 2 nM Cdc60p and 4 mM ATP at 300C. Reactions were started by the addition of 4 mM ATP. At specific time intervals, three 20 µL aliquots were added to 150 µL of 10% (w/v) TCA to separate wells in a 96-well nitrocellulose membrane filter plate (Millipore Multiscreen HTS, MSHAN4B50). Each well was then washed three times with 100 µL of 5% TCA. Filter plates were then dried under a heat lamp and the precipitated [14C]leucine tRNALeu were quantified by liquid scintillation counting using a Wallac MicroBeta Trilux model 1450 liquid scintillation counter. Unless started otherwise, the inhibitory effects for each compound were determined by incubating Cdc60p with the compound, tRNA and leucine for 20 minutes prior to the addition of ATP to starting the aminoacylation reaction. Equilibrium dialysis

Equilibrium dialysis experiments were performed in 50 mM HEPES-KOH (pH 8.0), 30 mM MgCl2 and 30 mM KCl using a DispoEquilibrium Dialyzer apparatus (5k MWCO, Harvard Apparatus). On one side of the dialysis membrane (side A), [14C]AN2690, 2.04 GBq/ mmol (S6) was added at concentrations ranging from 1 to 200

7

µM in a 20 µL sample volume. On the opposite side of the membrane (side B), 30 µM recombinant Cdc60p and 10 mM AMP (Adenosine 5′-monophosphate, Sigma) was added in a 20 µL sample volume. Samples were incubated at room temperature (210 C) while shaking for 4.5 hrs to establish equilibrium of AN2690 across the membrane. At equilibrium, AN2690 on each side of the dialysis membrane was quantified in triplicate by scintillation counting using a Wallac MicroBeta Trilux model 1450 liquid scintillation counter and the amount of AN2690 bound to Cdc60p was determined by subtracting the amount on side A from side B. Crystallization, data collection and structure determination

Crystals of T. thermophilus LeuRS were grown with or without the sulphamoyl-analogue of leucyl-adenylate (LeuAMS) at pH 6.5 using ammonium sulphate as precipitant as described (S7). To the crystallization drops 2 mM AMP and 1 mM AN2690 were added. Crystals were cryoprotected with 22% glycerol, rapidly frozen in a nitrogen stream at 100K and diffraction data measured on beamline ID14-4 at the European Synchrotron Radiation Facility (ESRF) using an ADSC Q315r CCD detector. Pre-grown crystals of the T. thermophilus LeuRS-tRNALeu complex in the post-transfer editing conformation (S8) were soaked with 2.5 mM AN2690, cryoprotected with 20% glycerol and measured frozen on ESRF beamline ID14-4. Diffraction data were integrated with XDS (S9). Structures were solved by straightforward molecular replacement using the relevant known T. thermophilus LeuRS structures (pdb entries 1H3N or 2BYT) followed by manual adjustment, inclusion of the AMP-AN2690 or tRNA-AN2690 adduct and refinement with REFMAC5 (S10). Data collection and refinement statistics are given in Table S3. The co-ordinates and structure factors of T. thermophilus LeuRS with bound LeuAMS and AMP-AN2690 at 1.85Å is available from the PDB with entry 2VOC and those of T. thermophilus LeuRS complexed with tRNALeu-AN2690 at 3.5Å resolution with entry 2VOG.

T314M L315VWT

C326F G405VC326R

R316I

N415D

T319I

S416L

Fig. S1. The norvaline sensitivity of the S. cerevisiae AN2690 resistant mutants. The parental strain S. cerevisiae ATCC 201388 was labelled WT. The other strains are listed in Table S2 and were labelled by their respective mutations in the editing domain of CDC60. The norvaline sensitivity of the AN2690 resistant mutants was determined as described in the materials and methods.

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Amphotericin B

}Cyclohexim

ide

}Cerulenin

}AN2690

} MIC

[14C

]Leu

cine

inco

rpor

atio

n (%

)

0%

20%

40%

60%

80%

100%

120%

Leu-

tRN

ALe

u (pm

ole

s)

Time (minutes)

0 M AN2690

2 M AN2690

A

B

4 M AN2690

10 M AN2690

0 1 2 3 4 5 6 7 80.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

Fig. S2. AN2690 inhibits aminoacylation in vitro and in vivo protein synthesis. (A) Aminoacylation of total brewer’s yeast tRNA by Cdc60p. Cdc60p was incubated with total brewer’s yeast tRNA, Leucine and AN2690 for 20 minutes, then the aminoacylation reaction was initiated by the addition of 4 mM ATP. Cdc60p was treated with 10 µM AN2690 (red diamond), 4 µM AN2690 (green triangles), 2 µM AN2690 (purple circles) and no compound (blue squares). All reactions were performed in triplicate and the mean values were plotted. (B) AN2690 inhibits in vivo protein synthesis. The incorporation of [14C]leucine was measured in the presence of antifungal compounds that exceed their MIC. The antifungals amphotericin B and cerulenin were used as negative controls. Cycloheximide, a known protein synthesis inhibitor, was the positive control. The percent of [14C]leucine incorporation was obtained by comparing the incorporation [14C]leucine in untreated cells to cells treated with the respective antifungal compounds. These experiments were repeated twice and the average was graphed. The MICs for S. cerevisiae in synthetic defined media minus leucine was 1 µg/mL for amphotericin B, 0.5 µg/mL for cycloheximide, 0.5 µg/mL for cerulenin and 0.06 µg/mL for AN2690.

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A

B

2- 1- 0 1 2 3 4

5.00.15.10.25.20.35.30.45.4

Mm ,]PTA[/1 1-

1/v

01.0- 50.0- 00.0 50.0 01.0 51.0 02.0

5.0

0.1

5.1

0.2

5.2

1/[Leucine], mM-1

1/v

Fig. S3. AN2690 is a non-competitive inhibitor with respect to ATP and leucine. A Lineweaver-Burke analysis of the rates of tRNA leucylation from different concentrations of ATP (A) and leucine (B) in the absence of AN2690 (blue squares), and the presence of 0.66 µM AN2690 (green triangles) and 2 µM AN2690 (red triangles). Cdc60p, total brewer’s yeast tRNA and leucine were incubated with AN2690 for 20 minutes prior to initiating the aminoacylation reaction with ATP. The Km for ATP was 1 mM, while the Km for leucine was 17.4 µM. The Ki was measured by plotting the y-intercepts from the Lineweaver-Burke plot against AN2690. Varying ATP and leucine yielded Ki values of 1.75 µM and 1.94 µM, respectively. The average of these values is 1.85 ± 0.1 µM and this was used as the Ki for AN2690. The Ki for the 2 minute incubation experiments was determined similarly to obtain a value of 31.4 ± 2.8 µM for AN2690. In contrast mupirocin, which binds to the synthetic site and is a competitive inhibitor with respect to ATP and isoleucine (S12, S13). Furthermore, most mutations that confer resistance to mupirocin either affect the Rossmann-fold domain forming the synthetic site or lie directly in this active site (S14-S17).

10

0.4

0.3

0.2

0.1

0.00 4 8 10 16

AN2690 ( M)

k obs

(min

-1)

Fig. S4. The first-order rates from each concentration in figure 2A were plotted against the AN2690 concentra-tion and the best fit was obtained by a linear regression. The slope of the plot was 0.0211 ± 0.0014 min-1 µM-1. Definitive evidence for saturation by AN2690 could not be obtained because at concentrations higher than 16.5 µM the rate of inactivation was too fast to measure accurately.

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E..tRNA + AN2690 E..tRNA..AN2690

kobs = kinactivation ( [AN2690] )

E..tRNA..AN2690**kinactivationKi

[AN2690] + Ki

kobs = kinactivation ( [AN2690] ) Ki

kobs = kinactivation

Ki [AN2690]Slope =

(eq 1)

(eq 2)

(eq 3)

Fig. S5. A scheme for the inactivation of Cdc60p by AN2690 and the derivation of the kinetic equations to determine the rate of inactivation (kinactivation). Equation 1 can be simplified to equation 2 because no evidence of AN2690 saturation was observed when the kobs was plotted against [AN2690]. Therefore the slope from Fig. S4 can be used in equation 3 to determine the rate of inactivation (kinactivation). Using the Ki value derived from the 2 minute incubation experiment of 31.4 ± 2.8 µM, we obtained a rate of inactivation (kinactivation) of 0.66 ± 0.10 min-1.

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ATP 2'dATP

3'dATPddATP

Rel

ativ

e b

ind

ing

(%)

100

75

50

25

0

-25None

ATP AMP

ADP AdeNone

Rel

ativ

e b

ind

ing

(%)

100

75

50

25

0

-25

B

A

CAN2690 ( M)

Frac

tio

n C

dc6

0p w

ith

AN

2690

0 20 40 60 80 100

1.0

0.8

0.6

0.4

0.2

0.0Bound ( M)

Bo

und

/Fre

e0.5

0.00

1.5

1.0

10 20 30

Fig. S6. AN2690 binding to Cdc60p requires an adenosine. (A) Adenosine can effect AN2690 binding to Cdc60p. The binding of [14C]AN2690 to Cdc60p was determined by equilibrium dialysis in the presence of 5mM adenosine 5'-triphosphate (ATP), 5 mM adenosine 5'-diphosphate (ADP), 5 mM adenosine 5'-monophosphate (AMP) and 5 mM adenosine (Ade) or in the absence of adenosine (None). The percent of relative binding of [14C]AN2690 to Cdc60p was determined in comparison to binding in the presence of ATP. (B) Affinity of AN2690 to Cdc60p in the presence of AMP. The fraction of Cdc60p with bound [14C]AN2690 was plotted against increasing AN2690 concentra-tions. The data was fitted to a one-site binding hyperbola by Prism® (Graphpad). These data were transformed to a Scatchard plot (inset) to calculate a Kd for AN2690 in the presence of AMP of 20.8 ± 1.5 µM. All reactions were performed in triplicate and the mean values were plotted. (C) The cis-diols on adenosine are required for AN2690 binding. Different adenosine analogs were used to determine whether AN2690 required the 2',3'-hydroxy groups on adenosine for binding to Cdc60p. The binding of [14C]AN2690 to Cdc60p was determined by equilibrium dialysis in the presence of 5mM adenosine 5'-triphosphate (ATP), 5mM 2'-deoxyadenosine 5'-triphosphate (2’dATP), 5mM 3'-deoxyadenosine 5'-triphosphate (3'dATP) and 5 mM 2'3'-dideoxyadenosine 5’-triphosphate (ddATP). The percent of relative binding of [14C]-AN2690 to Cdc60p was determined in comparison to binding in the presence of ATP. All reactions were performed in triplicate and the mean values were used plotted.

13

Adenosine 76

Cytosine 75

Fig. S7. Unbiased difference map (3.5 Å resolution) for the AN2690 adduct and the last two nucleotides of tRNALeu in the editing site of LeuRS. Green colored atom is fluorine, mauve is boron, yellow is carbon, purple is phosphate, light blue is nitrogen and red is oxygen.

Fluorine

2',3'-oxygen atoms of A76

Boron

14

Fig. S8. The AN2690 inhibition cycle. (1) ATP and leucine are bound in the synthetic site of LeuRS, which synthesizes the leucyl-adenylate releasing pyrophosphate (PPi) in the process prior to tRNALeu binding (2). (3) The 3' terminal adenosine (A76) of the tRNALeu can be bound either by the editing or synthetic sites, which are represented by the black holes. (4) If A76 is bound by the editing site the boron atom in AN2690 will bond to the 2',3'-hydroxy groups on A76 trapping tRNALeu in the editing conformation with a half-life of 424 minutes. This half-life is significantly longer than the generation time for S. cerevisiae. (5) If A76 enters the synthetic site then it will be charged with leucine and leucyl-tRNALeu will be released (6) for binding to eEF1A, thus allowing a new tRNALeu to bind to LeuRS to reinitiate the aminoacylation/inhibition cycle.

15

OO

O OH

FOB

N

NN

N

NH2

PO

OO--

tRNA

H2O

H2O

OO

HO O

FOB

N

NN

N

NH2

PO

OO--

tRNA

OO

O OB--O

F

N

NN

N

NH2

H3O+

PO

OO--

tRNA

OO

HO OH

FOB

HO

N

NN

N

NH2

+

PO

OO--

tRNA

AN2690

A76 (tRNALeu )

tRNALeu -AN2690adduct

Fig. S9. A putative chemical reaction scheme for the formation of the AN2690-adenosine adduct (the terminal A76 of tRNALeu). In scheme 1 the boron attacks the 3'OH of the tRNA terminus first, while in scheme 2 the boron attacks the 2'OH of the ribose of A76 first. We propose that boron in the tRNA-AN2690 adduct is negatively charged and is stabilized by a protonated water molecule. There are two water molecules within 3.72 Å and 3.31 Å, respectively, of the boron atom (Fig. 3C) and one of these could theoretically participate in our proposed schema for oxaborole adduct formation

16

17

18

Table S2List of AN2690 resistant mutants

µµATCC201388 0.125 NoneANA320 4 N415DANA321 16 R316IANA322 16 C326FANA323 16 G405VANA324 16 C326RANA326 16 L315VANA327 32 T314MANA358 32 S416LANA360 32 T319IMinimal inhibitory concentrations (MIC) were determined according to the M27-A2CLSI guidelines for testing of yeasts with the exception that YPD media was used ( ).The mutants ANA320-327 were selected spontaneously and ANA358 and 360 wereinduced by EMS.

19

Table S3. X-ray data collection and refinement statistics.

Space group C222(1) C2Cell dimensions

, , (Å) 101.8 154.2 174.9 202.1 125.8 173.2, , (°) 90.0, 90.0, 90.0 90.0, 118.7, 90.0

Resolution (Å) 50-1.85 (1.9-1.85)* 50-3.5 (3.6-3.5)*sym 0.065 (0.551)* 0.116 (0.452)*/ 13.86 (2.42)* 9.02 (2.69)*

Completeness (%) 99.3 (91.2)* 94.9 (96.8)*Redundancy 4.07 (3.67)* 3.00 (2.95)*

Resolution (Å) 50-1.85 (1.90-1.85) 50-3.5 (3.5-3.59)No. reflections work (free) 112717 (3477) 43437 (2324)work 18.0 (25.4) 24.5 (31.9)free 20.2 (29.0) 31.1 (35.1)

Total non-hydrogen atoms 7283 17690**Protein 6608 14244**tRNA - 3352**Ligand/ion

SubstrateZnHgInhibitor

31 (LeuAMS)2-33 (AN2690-AMP)

18** (Leucine)4**2**20** (AN2690)

Solvent moleculesWaterSulphate

6845

-10

Average -factor (Å2) 21.1 66.0RMS deviationsBond lengths (Å) 0.014 0.009Bond angles (°) 1.494 1.288

*Highest resolution shell is shown in parenthesis.

20

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