Whitesell Supplementary Fig. 1

Whitesell Supplementary Fig. 2

Nature Chemical Biology: doi:10.1038/nchembio.2534

ML316

Flu

Amph

2 1 0.5 0.25 0.12 0.06 0.03 0

10 5 2.5 1.25 0.62 0.31 0.16 0

5 2.5 1.25 0.62 0.31 0.15 0.08 0

(µg/ml)

(µg/ml)

(µM)

2 1 0.5 0.25 0.12 0.06 0.03 0

5 2.5 1.25 0.62 0.31 0.15 0.08 0

30 Degrees C

10 5 2.5 1.25 0.62 0.31 0.16 0

37 Degrees C

Clonogenic survival

Whitesell Supplementary Fig. 3

Proliferation

Flu

ML316

Amph

(µg/ml)

(µM)

(µg/ml)

a)

b)

Nature Chemical Biology: doi:10.1038/nchembio.2534

a WT

R1

R2

R3

173 188 S. cerevisiae FTPILFKQIPYNIAKF C. glabrata FTPILFKQIPYNIAKF C. albicans FTPILFKQIPYNIAKF C. albicans-R FTPILFKQIPYTIAKF A. fumigatus FGPILFKQIPYTMAKF H. sapiens GVAPLWRQIPYTMMKF

b

Whitesell Supplementary Fig. 4

c

Nature Chemical Biology: doi:10.1038/nchembio.2534

10 5 2.5 1.2 0 10 5 2.5 1.2 0 Control 10 µM INZ-5

ML316 (µM)

Control 10 µM INZ-5

ML316 (µM)

a)

b)

10 5 2.5 1.2 0 10 5 2.5 1.2 0

Whitesell Supplementary Fig. 5

Nature Chemical Biology: doi:10.1038/nchembio.2534

SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure 1. Checkerboard analysis of antifungal activity for combination

fluconazole and ML316 shows their interaction to be indifferent. The optical density

measured for each well after 48 hours of growth at 37 °C was standardized to drug-free control

wells and is displayed in heat map format as relative growth (scale bar indicated to the right).

Results shown are the average of 3 independent wells. The fractional inhibitory concentration

index for the combination (ƩFIC) was calculated by summing the FIC for each compound (MIC

of combination/MIC of compound alone). Calculated values and interpretation key are provided

below the figure.

Supplementary Figure 2. ML316 retains highly selective fungicidal activity in serum-free

media. Left: Viability of HepG2 cells was assessed by standard resazurin dye-reduction assay

after 72 hr growth at 37 °C in RPMI supplemented with commercial serum-substitute (B-27

Supplement, Thermo-Fisher) and glucose or galactose (10 mM). Error bars: s.d. Data points

depict the mean of 4 independent wells for each condition tested. Right: The optical density of C.

albicans strain CaCi-2 was determined for each well after growth at 37 °C for 48 hr in serum-

free, B-27-supplemented RPMI containing 10 mM glucose and standardized to drug-free

controls. Error bars: s.d. Data points depict the mean of 3 independent wells.

Supplementary Figure 3. The antifungal activity of ML316 increases with temperature.

(a) Inhibition of proliferation at 30 °C and 37 °C. Fluconazole (Flu), ML316, and amphotericin

B (Amph) were serially diluted two-fold across a 96-well plate in duplicate wells. The optical

Nature Chemical Biology: doi:10.1038/nchembio.2534

density measured for each well at 24 hours was standardized to drug-free control and is

displayed in heat map format as relative growth (scale bar indicated below panels). (b) Inhibition

of clonogenic survival. Aliquots of the cultures described in panel (a) were spotted onto drug-

free agar plates and photographs were obtained after 2 days of additional growth to assess

fungicidal activity. Results from one of two independent experiments are shown.

Supplementary Figure 4. ML316-sensitivity is correlated with MIR1 gene dosage and

mutation status. (a) Recurring mutation (Ca21chr1_C. albicans_SC5314 2236930 A to C,

highlighted in blue) found in MIR1 by sequencing of 3 independent ML316-resistant isolates of

C. albicans (R1-R3). Alignment of sequencing reads is presented using the Integrative Genomics

Viewer. (b) A single amino acid substitution in Mir1 is associated with resistance to ML316.

The amino acid substitution N184T (highlighted in red) that arose upon ML316 selection in

culture (C. albicans-R) is the native allele in humans and other fungi that are insensitive to the

compound. (c) Antifungal susceptibility testing with ML316 was performed using wild type and

genetically modified S. cerevisiae strains. Cells were grown in synthetic media containing 2%

glycerol as the sole carbon source for 72hr. Relative growth as monitored by OD600 was

normalized to untreated control for each strain and is displayed in heat map format. Results are

the mean of two independent experiments.

Supplementary Figure 5. INZ-5, an inhibitor of the bc1 complex which disrupts

mitochondrial electron transport rescues the cidality of ML316. (a) Antifungal susceptibility

testing of C. albicans grown at 30 °C in the presence of DMSO or INZ-5. ML316 was diluted

two fold across the plate in triplicate wells. Optical density of the cultures after 24 hours was

Nature Chemical Biology: doi:10.1038/nchembio.2534

standardized to drug-free control wells and is displayed in heat map format as relative growth

(scale bar to right). (b) INZ-5 blocks the fungicidal activity of ML316. Aliquots of the cultures

described in panel (a) were spotted onto drug-free agar plates and photographs obtained after 2

days of additional growth to assess fungicidal activity. Results are presented for one of two

independent experiments; each experiment performed using triplicate wells for each condition

tested.

Nature Chemical Biology: doi:10.1038/nchembio.2534

Supplementary Table 1

Minimal Inhibitory Concentration (MIC) measured or previously reported for ML316 and fluconazole

across a diverse panel of fluconazole-sensitive and fluconazole-resistant fungal strains.

MIC values in μg/mL

C. albicans SC5314 WT

CaCi-2

CaCi-8

CaCi-12

CaCi-15

CaCi-17

Ca3107

Ca3281

Ca5044

Ca5052

Ca1649

Ca3034

C. krusei

C. glabrata*

C. dubliniensis

C. tropicalis

SC5314 cox4Δ/Δ

SC5314 mir1 N184T

C. auris 381

C. auris 382

C. auris 383

C. auris 384

A. fumigatus

A. terreus

ML316

MIC80

0.016

0.063

0.063

0.125

0.125

0.250

0.016

0.125

0.063

0.063

0.016

0.125

4

>4

0.016

2

>4

4

0.043

0.130

1.156

1.156

>16

>16

ML316

MIC50

0.016

0.063

0.063

0.063

0.063

0.250

0.008

0.125

0.031

0.008

0.016

0.063

1

>4

0.016

1

>4

4

0.005

0.043

0.385

0.385

>16

>16

Flu

MIC80

0.5

2

8

16

128

128

4

64

8

32

1

4

32

32

0.25

128

1

0.5

Flu

MIC50

0.25

1

8

8

32

64

4

32

8

32

0.25

2

32

32

0.25

1

1

0.25

Flu

MIC Rep

4

16

128

128

Source

ATCC MYA-2876

Ref. 1

Ref. 1

Ref. 1

Ref. 1

Ref. 1

Ref. 2

Ref. 2

Ref. 2

Ref. 2

Ref. 2

Ref. 2

ATCC 200917

ATCC 200918

ATCC MYA-577

ATCC MYA-3404

Ref. 3

This manuscript

AR-BANK#0381

AR-BANK#0382

AR-BANK#0383

AR-BANK#0384

Clinical Isolate

ATCC 10029

All testing was performed in RPMI (glucose) at 37 °C for 24 hrs. Shading indicates serial strains isolated from the same individual ordered with respect to fluconazole from sensitive to resistant. See methods for strain information and references. “MIC Rep” is the MIC reported by the CDC for the C. auris strains. * ML316 IC50 in glycerol for this organism = 0.003 ug/mL (https://www.ncbi.nlm.nih.gov/books/NBK143544/)

Nature Chemical Biology: doi:10.1038/nchembio.2534

Supplementary Table 2

Alterations in fungal citrate and succinate levels distinguish ML316 from classical

inhibitors of mitochondrial function.

Experiment 1 Rotenone Antimycin KCN 2,4 DNP FCCP FT-350

Experiment 2 Rotenone Antimycin KCN 2,4 DNP FCCP FT-350

Citrate 2.234 2.638 2.141 0.974 0.447 7.376

Citrate 1.96

3.071 2.357 1.441 0.873 7.044

Succinate 0.56

0.429 0.347 0.366 0.209 0.262

Succinate 0.486 0.328 0.244 0.428 0.159 0.213

Values indicate fold-change induced by the specified compound compared to DMSO-treated controls.

Nature Chemical Biology: doi:10.1038/nchembio.2534