RESEARCH ARTICLE

Figures and figure supplements

Identification of a novel toxicophore in anti-cancer chemotherapeutics that targets mitochondrial respiratory complex I

Zoe A Stephenson et al

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 1 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

A) B)

Mubritinib (µM) pHER2 (Y1221/1222)

- HER2

p-HER2 (Y1221/1222) p-ACC (S-79)

HER2 ACC

ȕ-tubulin p-RPS6 (S-240/244)

RPS6

ȕ-tubulin

C) D) E)

Untreated Mubritinib 120 125125 140 100 120 100100 80 100 7575 60 80 5050 40 60 HER2 activity HER2activity HER2 activity HER2activity

Kinase activity Kinase activity 40 2525 20

(% relative to untreated) 20

(% relative to untreated) *** 00 0 (% relative to untreated) 0

Mubritinib (µM)

F) G)

Glucose Galactose

120 100 Glucose UntreatedUntreated 100 ns Galactose UntreatedUntreated 75 **** 80 Glucose Mubritinib (2 µ M )

60 *** *** 50 Galactose MubritinibMubritinib (2 µ M ) ****

40 Live (%) cells Cellular ATP Cellular 25 **** **** 20 **** (% relative to untreated) 0 0 0 1 2 4 6 24 0 24 48 72 Mubritinib (hours) Time (hours)

H) I i) I ii)

Glucose Galactose

Glucose Galactose DMSO Mubritinib Antimycin A Rotenone DMSO Mubritinib Antimycin A Rotenone ns **** **** **** **** 150 150 * ** 100 *

*** 0 125 0 125 ** 80 100 100 60 75 75

40 50 50 BPM BPM relative to t BPM relative to t Cellular ATP Cellular 20 25 25 (% relative to untreated) 0 0 0 0 10 30 60 120 0 10 30 60 120

Time (minutes) Time (minutes)

Figure 1. Mubritinib does not inhibit HER2, but inhibits ATP production and beat rate of cardiomyocytes. (A) Western blot analysis of the HER2- overexpressing cell line, BT474, treated with increasing doses of mubritinib. HER2 activity was assessed with antibodies against phosphorylated HER2 Figure 1 continued on next page

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 2 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

Figure 1 continued (Y1221/1222). Cells were treated with the clinically used HER2 inhibitor, lapatinib (10 mM), as a positive control. (B) Western blot analysis of the HER2- overexpressing cell line, BT474, treated with 1 mM of either mubritinib, lapatinib, compound 5 (inactive mubritinib derivative, see Figure 2A), antimycin A or rotenone for 2 hr. (C) Radiometric kinase assays were carried out and the effect of mubritinib (at the concentrations shown) on recombinant human HER2 activity was determined by measuring the incorporation of radioactive 32P-ATP after 15 mins. Activity values are displayed relative to the untreated sample. (D) Radiometric kinase assays were carried out using recombinant human HER2 in the presence of 1 mM mubritinib and lapatinib (DMSO control and lapatinib, n = 3, mubritinib, n = 2). Significance following lapatinib treatment was assessed using the unpaired students t-test (***p<0.001) relative to the DMSO control. (E) Radiometric kinase assays were carried out on recombinant human EGFR, ErbB4, Flt1 and PDFRa in the presence of 2 mM mubritinib. Error bars represent standard deviation (n = 3). (F) A 24 hr time course for loss of ATP from H9c2 cells following treatment with 2 mM mubritinib in media containing either glucose or galactose as the carbon source. Error bars represent standard deviation (n = 3) and significance was assessed using ANOVA with Dunnett’s multiple comparisons test (****p<0.0001, ***p<0.001, ns = not significant). (G) H9c2 cells were treated with 2 mM mubritinib in media containing either glucose or galactose as the carbon source and cell viability was assessed over a 72 hr period using DRAQ7 staining and Annexin-V-FITC labelling. Error bars represent standard deviation (n = 3) and significance at each time point was assessed using ANOVA with Tukey’s multiple comparisons test (****p<0.0001). (H) hESC-cardiomyocytes (CytivaTM Plus, GE Healthcare) were grown in RPMI- 1640 media supplemented with galactose (10 mM) or glucose (11 mM) and treated with 1 mM of mubritinib, or inhibitors of mitochondrial complex III (antimycin A), ATP synthase (oligomycin A) or complex I (rotenone). ATP levels were measured after 2 hr. Error bars represent standard deviation (n = 4) and significance was assessed using the unpaired students t-test (****p<0.0001). (I) hESC-cardiomyocytes (CytivaTM Plus, GE Healthcare) were grown in either galactose (10 mM) or glucose (11 mM) containing media on multi-electrode array plates from which it is possible to assess beat rate. The average beat rates in glucose (i) and galactose (ii) containing media of 52.2 and 30.7 BPM respectively, were set to 100%. Cells were treated with mubritinib (1 mM), antimycin A (1 mM) or rotenone (1 mM). Error bars represent standard deviation (n = 3) and significance was assessed using the unpaired students t-test (*p<0.05, **p<0.01, ****p<0.0001).

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 3 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

A) B)

Lapatinib Energy ns 100 sensing 80 * ** ** P 60 HER2 AMP 40

20 ***

HER2 phosphorylation HER2phosphorylation 0 (% relative to untreated)

0 1 PI3K AMPK 10 0.1 0.01

Mubritinib (µM) Lapatinib

P mTOR ACC

p70 S6K

P RPS6

C i) C ii)

p-ACC (S-79) p-RPS6 (S240/244)

** **** 400 120 * ns 100 300 80

200 ns 60 **** 40 100 ns 20 **** ACC phosphorylation ACCphosphorylation RPS6 phosphorylation RPS6phosphorylation (% relative to untreated) (% relative to untreated) 0 0 AMA AMA Lapatinib Lapatinib Rotenone Rotenone Untreated Untreated 1 1 [Mubritinib] 1 [Mubritinib] Compound 5 Compound 5

Figure 1—figure supplement 1. Mubritinib targets complex I in cardiomyocytes. (A) Quantification of HER2 phosphorylation from Figure 1A displayed relative to the untreated sample. Error bars represent standard deviation (n = 3) and significance was assessed using unpaired students t-test (****p<0.0001, **p<0.01, *p<0.05, ns = not significant). (B) Schematic to show the key components in the signalling pathway that is downstream from changes in energy status or inhibition of HER2. Arrows indicate activation and blocked arrows indicate inactivation of the downstream target. (C) Quantification of (i) ACC (S-79) and (ii) RPS6 (S-240/244) phosphorylation from Figure 1B displayed relative to the untreated sample. Error bars represent standard deviation (n = 3) and significance was assessed using the unpaired students t-test (*p<0.05, **p<0.01, ***p<0.001, ns = not significant).

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 4 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

A) B) ****

Mubritinib ns *** 100

80

60

40

20

Phenyl ring substitution Triazole modifications 0 % Basal rot-sensitive % rot-sensitive Basal OCR H9c2 hESC-CM Untreated Oligomycin 1 [Mubritinib]

C) D) Mubritinib Untreated H9c2 hESC-CM ADP ADP Pyr/Mal Succinate 0.5 ȝM 2 ȝM 1 [Mubritinib] PMP Rot 500 10 ȝM Antimycin A 400 Rotenone

300

200 oxidoreduction oxidoreduction activity 2

OCR (pmoles/min) 100 (% relative to untreated) 0 NADH:O

-100 0 20 40 60 80 100 Time (minutes)

E) F) G)

125 125 125 ns ns ns ns ns ns 100 100 ** 100

75 75 75

**** **** 50 50 50 **** oxidoreduction oxidoreduction activity 2 **** **** **** **** Oxidoreduction Oxidoreduction activity 25 25 25 **** (% relative to untreated) (% relative to untreated) **** **** **** **** **** **** ATP(% relative to untreated)

0 NADH:O 0 0 2 2 NADH:dQ NADH:O2 NADH:O NADH:dQ NADH:FeCN NADH:APAD Succinate:O2 NADH:FeCN Succinate:O NADH:APAD

Membranes Isolated complex I

Figure 2. Mubritinib is an inhibitor of mitochondrial complex I. (A) Variants of mubritinib were synthesised with alterations to the trifluoromethylphenyl group (2, 3 and 4) or the triazole (5, 6, 7 and 8). Mubritinib (1) and mubritinib synthesised ‘in house’ were used as positive controls. (B) Rotenone- sensitive oxygen consumption rates (OCRs) of H9c2 or hESC-CM cells in glucose containing media were measured after the addition of FCCP in the presence of either 1 mM mubritinib or 1 mM oligomycin A. OCR values are represented relative to untreated cells and error bars represent standard deviation (n = 3). Significance was assessed using the unpaired students t-test (*** p = <0.001, **** p = <0.0001, ns = not significant). (C) OCR was measured in cells pre-treated with 0.5 mM, 2 mM and 10 mM mubritinib, 1 mM rotenone or 10 mM antimycin A using a Seahorse XF Analyzer. PMP was Figure 2 continued on next page

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 5 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

Figure 2 continued added to permeabilise the plasma membrane, followed by pyruvate, malate and ADP to drive complex I linked respiration. Then, rotenone was added to abolish complex I respiration followed by ADP and succinate to drive complex II linked respiration. Error bars represent standard deviation (n = 3). (D) Mubritinib was incubated with mitochondrial membranes from bovine heart at the concentrations shown, then the rate of NADH was measured Hill slope spectrophotometrically. Error bars represent standard error of the mean (n = 3). Data were fit using activity (%) = 100/(1 + (IC50 / [inhibitor]) and yielded an IC50 value of 19.2 nM. (E) Relative rates of NADH or succinate oxidation by mitochondrial membranes or complex I isolated from bovine + heart using O2, dQ, APAD , or FeCN as the electron acceptor in the presence of 500 nM mubritinib. Error bars represent standard deviation (n = 3) and significance was assessed using ANOVA with Dunnett’s multiple comparisons test (****p<0.0001, ns = not significant). (F) Mubritinib and the variants from (A) were incubated with mitochondrial membranes at 500 nM. The rate of NADH oxidation was measured spectrophotometrically. The activity is expressed relative to the DMSO control, set to 100%. Error bars represent standard deviation (n = 3) and significance was assessed using ANOVA with Dunnett’s multiple comparisons test (****p<0.0001, **p<0.01). Activities were inter/extrapolated from measured data points for compounds 7 and 8. (G) H9c2 cells were treated with mubritinib and all compound variants (10 mM) in galactose containing media for 24 hr and ATP levels were measured. Error bars represent standard deviation (n = 3) and significance was assessed using ANOVA with Dunnett’s multiple comparisons test (****p<0.0001, ns = not significant).

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 6 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

A) B)

Drug FCCP Rot Drug: 2200 0 0 Drug oligomycin FCCP Rot Drug: Untreated 3003 0 0 Untreated Oligomycin 1150 5 0 1 [Mubritinib] 1 [Mubritinib] 2002 0 0 1100 0 0

1001 0 0 550 0 % Basal rot-sensitive % rot-sensitive Basal OCR % rot-sensitive Basal OCR

00 0

0 0 404 0 808 0 120 160 0 40 80 80 120 120 160 160 1 2 0 1 6 0 Time (minutes) Time (minutes)

Figure 2—figure supplement 1. Mubritinib inhibits OCR in H9c2 and hESC-CM. (A and B) Representative seahorse trace (for Figure 2B) from (A) H9c2 or (B) hESC-CM cells in glucose containing media and treated with mubritinib (1 mM) or oligomycin A (1 mM).

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 7 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

A i) A ii)

A iii) A iv)

A v) A vi)

A vii) A viii)

Figure 2—figure supplement 2. Complex I inhibition by mubritinib and the synthesised variant compounds. (A) Mubritinib and the compound variants (i-viii) were incubated with mitochondrial membranes at the concentrations shown. The rate of NADH oxidation was measured spectrophotometrically, and data were fit to the standard dose-effect relationship (activity (%) = 100/(1 + (IC50 / [inhibitor])Hill slope). The activity is expressed relative to the DMSO control, set to 100%. For each compound the IC50 values (nM) measured on mitochondrial membranes are shown with standard errors (n = 3).

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 8 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

A)

9 Carboxyamidotriazole [CAI] 10 11 12

B) C i) C ii) H9c2 Galactose Basal OCR Max OCR *** **** *** 120 120 *** 120 **** ns ns ns ns **** 100 100 * 100 * * *** 80 80 80 ** 60 60 OCR OCR 60

Cellular ATP Cellular 40 *** *** 40 40 20 **** 20 (% relative to untreated) (% relative to untreated) (% relative to untreated) 20 0 0 0 10 11 12 10 11 12 10 11 12 10 11 12 10 11 12 10 11 12 9 9 [CAI] 9 9 [CAI] 9 9 [CAI] 9 9 [CAI] 9 9 [CAI] 9 [CAI] "inhouse" CAI "inhouse" CAI "inhouse" CAI "inhouse" CAI in in houseCAI in houseCAI ȝ M ȝ M ȝ M ȝ M ȝ M ȝ M

D) E)

A549 Galactose Basal OCR Max OCR **** **** **** 120 100 **** **** **** **** 100 80 **** **** 80 60 60 **** 40

Cellular ATP Cellular 40 20 20 (% relative to untreated)

0 OCR (% relative to untreated) 0 10 11 10 11 10 11 10 11 10 11 9 9 [CAI] 9 [CAI] 9 9 [CAI] 9 [CAI] 9 [CAI]

 ȝ M)  ȝ M)  ȝ M) Untreated Untreated 1 1 [Mubritinib] 1 [Mubritinib]

F) G) **** ADP ADP Pyr/Mal Succinate ns ns Piericidin A 100 2502 5 0 UntreatedU ntreated 9 [CAI][C AI] 80 2002 0 0 101 0 111 1 60 1501 5 0 1 [mubritinib][Mubritinib] AntimycinAntim ycin A 40 **** **** 1 0 0 oxidoreduction activity 100 PiericidinP iericidin A 2 20 (% relative to untreated) OCR (% baseline) 505 0 ****

NADH:O 0

00 10 11 12 00 10 20 30 40 DMSO 9 9 [CAI] Time (minutes) "inhouse" CAI

Figure 3. The toxicophore present in carboxyamidotriazole inhibits mitochondrial complex I. (A) Chemical structure of carboxyamidotriazole (CAI) (9) and three variants whereby the core triazole ring was replaced with either a pyrazole (10 and 11) or imidazole (12). For the pyrazoles, 11 is an analogue Figure 3 continued on next page

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 9 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

Figure 3 continued of 9 with one of the triazole nitrogens removed, whereas 10 also removes one of the triazole nitrogen atoms whilst additionally shifting the trichlorobenzophenonemethyl moiety to the 2-position equivalent. (B) H9c2 cells were treated with 3, 6 or 12 mM of CAI (9), 10, 11, 12 or the ‘in house’ synthesised CAI in galactose containing media and after 24 hr ATP levels were measured and data shown are relative to the untreated control (n = 3). Significance was assessed using ANOVA with Dunnett’s multiple comparisons test (****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns = not significant). (C) Basal (i) and maximum (ii) oxygen consumption rates were measured using a Seahorse XF Analyzer in H9c2 cells treated with 3 and 10 mM of either CAI (9), 10, 11, 12 or the ‘in house’ synthesised CAI. Error bars represent standard deviation (n = 3) and significance was assessed using ANOVA with Tukey’s multiple comparisons test (****p<0.0001, ***p<0.001, *p<0.05). (D) A549 cells were treated with 3, 6 or 12 mM of CAI (9) or 10 and 11 in galactose containing media and after 24 hr ATP levels were measured and normalised to the untreated control. Error bars represent standard deviation (n = 3) and significance was assessed using ANOVA with Tukey’s multiple comparisons test (****p<0.0001). (E) Basal and maximum oxygen consumption rates were measured using a Seahorse XF Analyzer in A549 cells treated with 5 mM of either CAI (9), 10, 11; or 5 mM mubritinib. Error bars represent standard deviation (n = 3) and significance was assessed using ANOVA with Tukey’s multiple comparisons test (****p<0.0001). (F) A549 cells were pre-treated with either 5 mM CAI (9), 10, 11, mubritinib, antimycin A or piericidin A (complex I inhibitor) and the OCR was measured over the times indicated. PMP was added to permeabilise the plasma membranes followed by addition of pyruvate, malate and ADP to drive complex I respiration and OCR determined. Finally, piericidin A was added to abolish complex I respiration followed by ADP and succinate to drive complex II respiration and OCR was again measured. Representative trace from three independent experiments. (G) CAI (9), 10, 11, 12 and the ‘in house’ synthesised CAI were incubated with mitochondrial membranes at 500 nM. The rate of NADH oxidation was measured spectrophotometrically. The activity is expressed relative to the DMSO control, set to 100%. Error bars represent standard deviation (n = 3) and significance was assessed using ANOVA with Tukey’s multiple comparisons test (****p<0.0001, ns = not significant). Activities were interpolated from measured data points for compounds 10, 11, 12 and ‘in house’ CAI.

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 10 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

A) B) H9c2 3 uM H9c2 10 uM

FCCP FCCP Oligomycin Antimycin 2004 0 0 Untreated 2004 0 0 Oligomycin Antimycin Untreated 9 [CAI] 9 [CAI] 1503 0 0 10 1503 0 0 10 11 11 1002 0 0 1002 0 0 13 13 “in-house” CAI “in-house” CAI 150 0 0 150 0 0

00 OCR (% relative to untreated) 00 0 50 100 150 200 OCR (% relative to untreated) 0 50 100 150 200 0 50 100 150 200

Time (minutes) Time (minutes)

C) D) A549 A549 Glucose

120 FCCP

2002 0 0 Oligomycin Antimycin 100 Untreated 9 [CAI] 80 1501 5 0 10 11 60 1 [ Mubritinib] 1001 0 0 40 Cellular ATP Cellular

20 505 0 (% relative to untreated)

0 OCR (% relative to untreated) 0 0

10 11 10 11 0 50 100 150 150 200

9 9 [CAI] 9 [CAI] Time (minutes)  ȝ M)  ȝ M)

Figure 3—figure supplement 1. The toxicophore in CAI inhibits mitochondrial complex I. (A and B) Representative seahorse trace (for Figure 3C) of H9c2 cells grown in glucose containing media and treated with either (A) 3 mM or (B) 10 mM of the indicated compounds. (C) A549 cells were treated with 6 or 12 mM of CAI (9) or compound 10 and compound 11 in glucose containing media and after 24 hr ATP levels were measured and normalised to the untreated control. Error bars represent standard deviation (n = 3). (D) Representative seahorse trace (for Figure 3E) of A549 cells grown in glucose containing media and treated with 5 mM of either CAI (9), 10, 11; or 5 mM mubritinib.

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 11 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

A i) A ii)

A iii) A iv)

A v)

Figure 3—figure supplement 2. Complex I inhibition by mubritinib and the synthesised variant compounds. (A) CAI and the compound variants (i–v) were incubated with mitochondrial membranes at the concentrations shown. The rate of NADH oxidation was measured spectrophotometrically, and data were fit to the standard dose-effect relationship (activity (%) = 100/(1 + (IC50 / [inhibitor])Hill slope). The activity is expressed relative to the DMSO control, set to 100%. For each compound the IC50 values (nM) measured on mitochondrial membranes are shown with standard errors (n = 3).

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 12 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

A) B i) B ii)

p-ACC (S-79) p-RPS6 (S240/244)

250 120

100 200

80 150 p-ACC (S-79) 60 100 ACC 40 ACC phosphorylation ACCphosphorylation RPS6 phosphorylation RPS6phosphorylation (% relative to untreated)

(% relative to untreated) 50 p-RPS6 (S-240/244) 20

RPS6 0 0 Ct Ct ȕ-tubulin CAI CAI Cpd11 Cpd11 Rotenone Rotenone Bepridilhydrochloride Bepridilhydrochloride

Figure 3—figure supplement 3. CAI inhibits signalling pathways responsive to changes in energy status. (A) Western blot analysis of A549 cells treated with 5 mM of CAI (9), 5 mM of 11 (inactive CAI derivative), 2.5 mM bepridil hydrochloride (non-specific calcium channel inhibitor) or 1 mM rotenone (complex I inhibitor) for 24 hr. (B) Quantification of (i) ACC (S-79) and (ii) RPS6 (S-240/244) phosphorylation from (E) displayed relative to the untreated sample. Error bars represent standard deviation (n = 3).

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 13 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

A) B) HL60 Galactose 72 hr HL60 Glucose 72 hr

ns ns 100 ns 100 ns 75 75 **** 50 50 **** Live Live (%) Cells Live Live (%) Cells 25 25 **** **** 0 0

C) D) M059K Galactose 72 hr M059K Glucose 72 hr

100 ns 100 ns 75 75

50 **** 50 Live Live (%) Cells Live Live (%) Cells 25 25 **** 0 0

E) F)

A549 Galactose 72 hr A549 Glucose 72 hr

100 ns ns 100

75 75

50 50 Live Live (%) Cells Live Live (%) Cells 25 **** 25 **** 0 0

G) H)

4 4 88

3 3 66

2 2 44 Cell Index CellIndex (CI) CellIndex (CI) 1 1 22

0 0 00 00 24 24 48 48 72 72 96 0 24 24 48 72 72 96 96 Time (hours) Time (hours)

UntreatedU ntreated 11 [Mubritinib] [M ubritinib] 55 UntreatedU ntreated 99 [CAI][C AI] 111 1

Figure 4. The toxicophore present in mubritinib and CAI is required for efficacy as an anti-cancer agent. (A and B) HL60 cells grown in media containing galactose (A) or glucose (B) as an energy source treated with mubritinib (1) Figure 4 continued on next page

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 14 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

Figure 4 continued (2 mM), CAI (9) (5 mM) or the inactive derivatives 5 (2 mM) or 11 (5 mM) for 72 hr. The percentage of live cells was assessed by DRAQ7 staining and Annexin-V-FITC labelling. Error bars represent standard deviation (n = 3) and significance relative to the untreated control sample was assessed using ANOVA with Dunnett’s multiple comparisons test (****p<0.0001, ns = not significant). (C and D) M059K cells grown in media containing galactose (C) or glucose (D) as an energy source treated with mubritinib (1) (2 mM), CAI (9) (5 mM) or the inactive derivatives 5 (2 mM) or 11 (5 mM) for 72 hr. The percentage of live cells was assessed by DRAQ7 staining and Annexin-V-FITC labelling. Error bars represent standard deviation (n = 3) and significance relative to the untreated control sample was assessed using ANOVA with Dunnett’s multiple comparisons test (****p<0.0001, ns = not significant). (E and F) A549 cells grown in media containing galactose (E) or glucose (F) as an energy source treated with mubritinib (1) (2 mM), CAI (9) (5 mM) or the inactive derivatives 5 (2 mM) or 11 (5 mM) for 72 hr. The percentage of live cells was assessed by DRAQ7 staining and Annexin-V-FITC labelling. Error bars represent standard deviation (n = 3) and significance relative to the untreated control sample was assessed using ANOVA with Dunnett’s multiple comparisons test (****p<0.0001, ns = not significant). (G) Cell proliferation profiles from xCELLigence RTCA DP instrument. BT474 cells grown in glucose containing media were seeded in an E-plate 16 and after 24 hr were treated with either 5 mM mubritinib (1) or the derivative compound five which contains a modified triazole ring. The cell index was monitored for 96 hr to determine cell proliferation rates. Error bars represent standard deviation (n = 3). (H) Cell proliferation profiles from xCELLigence RTCA DP instrument. A549 cells grown in glucose containing media were seeded in an E-plate 16 and after 20 hr were treated with either 5 mM CAI (9) or the derivative compound 11 which contains a modified triazole ring. The cell index was monitored for 120 hr to determine cell proliferation rates. Error bars represent standard deviation (n = 3).

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 15 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

A) B) HL60 Galactose HL60 Glucose HL60 Galactose HL60 Glucose

1100 0 0 1100 0 0 UntreatedU ntreated UntreatedU ntreated

11 [Mubritinib][M ubritinib] 11 [Mubritinib] [M ubritinib] 775 5 757 5 55 55

99 [CAI][C AI] 99 [CAI][C AI] 550 0 505 0 111 1 111 1 Live Live (%) Cells Live (%) Cells Live Cells (%) 225 5 Live Cells 252(%) 5

0 0 0 24 48 72 0 24 48 72 TimTime e (hours) TimTime e (hours) C) D) M0569K Galactose M0569K Glucose M059K Galactose M059K Glucose

1100 0 0 1001 0 0 UntreatedU ntreated UntreatedU ntreated

11 [Mubritinib][M ubritinib] 11 [Mubritinib] [M ubritinib] 775 5 757 5 55 55

99 [CAI][C AI] 99 [CAI] [C AI] 550 0 505 0 111 1 111 1 Live Live (%) Cells Live Live (%) Cells Live Cells (%) 225 5 Live Cells25 2(%) 5

0 0 0 24 48 48 72 0 24 48 72 72 TimTime e (hours) TimeTim e (hours) E) F) A549 Galactose A549 Glucose A549 Galactose A549 Glucose

1100 0 0 1100 0 0 UntreatedU ntreated UntreatedU ntreated

11 [Mubritinib] [M ubritinib] 11 [Mubritinib] [M ubritinib] 757 5 775 5 55 55

99 [CAI] [C AI] 99 [CAI][C AI] 505 0 550 0 111 1 111 1 Live Live (%) Cells Live (%) Cells Live Cells (%) Live Cells25 2(%) 5 225 5

0 0 0 24 48 72 0 24 48 72 TimTime e (hours) TimTime e (hours) G i) G ii)

U2OS Glucose HeLa Glucose U2OS Glucose HeLa Glucose

1100 0 0 1100 0 0 UntreatedU ntreated UntreatedU ntreated

11 [Mubritinib] [M ubritinib] 11 [Mubritinib][M ubritinib] 757 5 775 5 55 55

99 [CAI] [C AI] 99 [CAI][C AI] 505 0 550 0 111 1 111 1 Live Live (%) Cells Live (%) Cells Live Cells (%) 252 5 Live Cells 225(%) 5

0 0 0 24 48 72 0 24 48 72 H i) TimTime e (hours) H ii) TimTime e (hours)

U2OS Galactose HeLa Galactose U2OS Galactose HeLa Galactose

1100 0 0 1100 0 0 UntreatedU ntreated UntreatedU ntreated

11 [Mubritinib] [M ubritinib] 11 [Mubritinib][M ubritinib] 757 5 775 5 55 55

99 [CAI] [C AI] 99 [CAI][C AI] 505 0 550 0 111 1 111 1 Live Live (%) Cells Live (%) Cells Live Cells (%) 252 5 Live Cells 225(%) 5

0 0 0 24 48 72 0 24 48 72 TimTime e (hours) TimTime e (hours)

Figure 4—figure supplement 1. CAI and mubritinib inhibit cell growth and induce apoptosis in glycolytic-deficient tumour cell lines. (A and B) HL60 cells grown in media containing galactose (A) or glucose (B) as an energy source treated with mubritinib (1) (2 mM), CAI (9) (5 mM) or the inactive Figure 4—figure supplement 1 continued on next page

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Figure 4—figure supplement 1 continued derivatives 5 (2 mM) or 11 (5 mM). Cells were harvested at the indicated time points and the percentage of live cells assessed by DRAQ7staining and Annexin-V-FITC labelling. Error bars represent standard deviation (n = 3). (C and D) M059K cells grown in media containing galactose (C) or glucose (D) as an energy source treated with mubritinib (1) (2 mM), CAI (9) (5 mM) or the inactive derivatives 5 (2 mM) or 11 (5 mM). Cells were harvested at the time points shown and the percentage of live cells assessed by DRAQ7staining and Annexin-V-FITC labelling. Error bars represent standard deviation (n = 3). (E and F) A549 cells grown in media containing galactose (E) or glucose (F) as an energy source treated with mubritinib (1) (2 mM), CAI (9) (5 mM) or the inactive derivatives 5 (2 mM) or 11 (5 mM). Cells were harvested at the time points shown and the percentage of live cells assessed by DRAQ7staining and Annexin-V-FITC labelling. Error bars represent standard deviation (n = 3). (G) U-20S (i) cells and HeLa (ii) cells were treated with mubritinib (1) (2 mM), CAI (9) (5 mM) or the inactive derivatives compound 5 (2 mM) or compound 11 (5 mM) in media containing glucose as an energy source. Cells were harvested at the time points shown and the percentage of live cells assessed by DRAQ7staining and Annexin-V-FITC labelling. (H) U- 20S (i) cells and HeLa (ii) cells were treated with mubritinib (1) (2 mM), CAI (9) (5 mM) or the inactive derivatives compound 5 (2 mM) or compound 11 (5 mM) in media containing galactose as an energy source. Cells were harvested at the time points shown and the percentage of live cells assessed by DRAQ7staining and Annexin-V-FITC labelling.

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 17 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

Compounds that contain a heterocyclic 1,3 nitrogen motif

Figure 5. Schematic diagram to show the cell-wide effect of ETC complex I inhibition by the toxicophore. Chemical inhibition of mitochondrial respiratory complex I with mubritinib and CAI leads to a decrease in cellular ATP and the subsequent activation of the energy sensor AMPK. Importantly, AMPK has been shown to phosphorylate and inhibit HER2 (Jhaveri et al., 2015) suggesting how mubritinib has been misattributed as a HER2 inhibitor, as well to negatively regulate protein synthesis via the mTOR axis to inhibit tumour cell growth. Moreover, cancer cells that are dependent on oxidative phosphorylation for ATP production, such as in AML, will be more sensitive to compounds that inhibit complex I. However, the decrease in ATP levels following treatment with these compounds will also have a particularly toxic effect on tissues with a high energy demand, such as cardiac tissue, and thus impact on heart function.

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 18 of 20 Research article Biochemistry and Chemical Biology Cancer Biology

Appendix 1—figure 1. Mubritinib analogues.

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Appendix 1—figure 2. Carboxyamidotriazole analogues.

Stephenson et al. eLife 2020;9:e55845. DOI: https://doi.org/10.7554/eLife.55845 20 of 20