Biguanide pharmacophore interacts with ATP synthase subunit e (ATP5I).

(A) Design of bio-inspired probe biotin functionalized biguanide (BFB) based on the structure of metformin (Met). (B) Immunoblots for the phosphorylation of AMPK (Thr172) and ACC (Ser79) in extracts from KP-4 pancreatic cancer cells treated with 2.5 mM Met or BFB for 16 hours. β-ACTIN was used as loading control. (C) Representative quantification of cell viability and growth with corresponding EC50 values of 3-day treatments with metformin (Met) or biotin functionalized biguanide (BFB) in KP-4 cells. Values represent the mean ± standard deviation of N=3. (D) Representative images of mitochondria and BFB localization in cells as in (B). Cells were treated with 1 mM of metformin (Met) or BFB for 16 hours and mitochondrial signal and BFB localization were analyzed by co-immunofluorescence using Streptavidin fluorophore conjugate and TOMM20 antibody, scale bar= 10 μm. Cells untreated (-) and treated with 1 mM Met were used as negative controls. (E) Colocalization between TOMM20 (TOMM20-568) and Streptavidin (Strep-488) fluorophores was analyzed for the BFB condition from (D) through job plot intensity profile. (F) Pull-down validation experiments with streptavidin beads alone (-), D-biotin (B), BFA and BFB using antibody followed by immunoblot against ATP5I in cells as in in extracts from HEK-293T embryonic kidney cells. The whole cell lysate (WCL) was added as control. (G) Binding interactions studies of BFB with recombinant purified ATP5I (rATP5I) using Surface Plasmons of Resonance (SPR). Representative sensorgrams show affinity kinetics of BFB and rATP5I. BFB was exposed onto streptavidin immobilized sensor chip and several concentrations of rATP5I were added until saturation of the signal. The assay was performed in a running buffer (125 mM NaCl, 5 mM DTT, PBS pH 7.4) at 25°C. RU: Resonance Units. (H) Binding affinity curve obtained from each steady state from (F). KD refers to the dissociation equilibrium constant and Rmax represent the theoretical maximum response.

(A) Synthesis of biotin-NHS (1); (a) NHS, EDC, DMF, room temperature (r.t) overnight (o/n). (B) Synthesis of biotin functionalized biguanide (BFB) chloride salt (3); (a) Dicyandiamide, TMSCl, MeCN, 160°C, 3h, 2: 6-aminohexylbiguanide hydrochloride salt; (b) Biotin-NHS, DIPEA, DMF, r.t, o/n. (C) Synthesis of biotin functionalized amine (BFA) chloride salt (5); (a) N-Boc-1,6-hexanediamine, DMF, r.t, o/n, 4: biotin functionalized N-Boc-amine; (b) HCl/MeOH, MeOH, r.t, o/n. (D) SDS PAGE analysis of recombinant ATP5I (rATP5I) after the purification process. Purity is estimated at ≥ 90 %. MM: molecular weight marker. (E) Gel filtration profile analysis of rATP5I using Superose 12 10/300 GL. DV represents column dead volume. mAU: milli-Absorbance Unit.

ATP5I knockout in pancreatic cancer cells alters the organization of the mitochondrial network.

(A) Immunoblot of five proteins from the OXPHOS complexes in extracts from clones of KP-4 cells expressing a control small guide RNA against GFP (sgGFP: control 1 and control 2) or two clones for each of the two different guides targeting ATP5I (sgATP5I #1: sgATP5I 1 and sgATP5I 2, and sgATP5I #2: sgATP5I 3 and sgATP5I 4). GAPDH antibody was used as loading control. (B) Representative images of mitochondrial morphologies characterized in cells as in (A). Mitochondrial morphologies were analyzed by immunofluorescence using TOMM20 antibody. DAPI is used as a DNA counter stain. Scale bar= 10 μm. (C) Quantification of the percentage of cells exhibiting filamentous, fragmented or punctuated mitochondria from (B). Values represent the mean ± standard deviation of N=3 for each clone, each n= 25-50 cells per clone.

(A) Relative qPCR quantification of the mRNAs encoding proteins representative of the five OXPHOS complexes (in Figure 2A) in clones of KP-4 cells expressing a control small guide RNA against GFP (sgGFP: control 1 and control 2) or two clones for each of the two different guides targeting the knockout of ATP5I (sgATP5I #1: sgATP5I 1 and sgATP5I 2, and sgATP5I #2: sgATP5I 3 and sgATP5I 4). Values represent the mean ± standard deviation of three biological replicates. (B) qPCR quantification of mitochondrial genomic DNA (Mt) over cellular nuclear genomic DNA (Nu) in cells as in (A). Values represent the mean ± standard deviation of N=3. (C) Representative mitochondrial morphologies of cells exhibiting filamentous, fragmented or punctuated mitochondrial network phenotypes in KP-4 cells of cells as in (A). Mitochondrial morphologies were detected by immunofluorescence using TOMM20 antibody, scale bar= 10 μm.

ATP5I knockout desensitizes pancreatic cancer cells to biguanides.

(A) Quantification of NAD+/NADH ratio in KP-4 cells expressing a control small guide RNA against GFP (sgGFP) or a representative clone of two different guides targeting ATP5I (sgATP5I #1 or sgATP5I #2). Values represent the mean ± standard deviation of N=3. (***) P < 0.001 using an unpaired Student’s t-test. (B) Relative quantification of oxygen consumption rate (OCR) over extracellular acidification rate (ECAR) by Seahorse analysis in cells as in (A). Values represent the mean ± standard deviation of at least N=3. (***) P < 0.001 using a paired Student’s t-test. (C) Immunoblot for total and phosphorylated levels of AMPK (Thr172) protein in extracts from cells as in (A). ATP5I confirms loss of expression in KO and GAPDH was used as loading control. (D) Growth curves of cells as in (A) measuring the relative number of cells over 6 days. Media was changed every two days. (E) Representative kinetic curves of OCR in cells as in (A) treated with 5 mM of metformin (Met) relative to control treated cells (dashed line) using Seahorse. (F) Representative kinetic curves of ECAR in cells as in (A) treated with 5 mM metformin (Met) relative to control treated cells (dashed line) using Seahorse. (G) Quantification of OCR/ECAR ratio fold change at 3 and at 6 hours from kinetic curves (E-F). Values represent the mean ± standard deviation of N=3. (ns) not significative, (*) P < 0.05, (**) P < 0.01, (****) P < 0.0001 using a repeated measures (RM) one-way ANOVA with Sidak’s multiple comparison test. (H) Representative growth of cells as in (A) exposed to different concentrations of metformin (Met) for three days with corresponding EC50 values of metformin. Values represent the mean ± standard deviation of N=3. (***) P < 0.001 and (****) P < 0.0001 using an unpaired Student’s t-test. (I) Representative kinetic curves of OCR in cells as in (A) treated with 100 μM of phenformin (Phen) relative to control treated cells (dashed line) using Seahorse. (J) Representative kinetic curves of ECAR in cells as in (A) treated with 100 μM of phenformin (Phen) relative to control treated cells (dashed line) using Seahorse. (K) Quantification of OCR/ECAR ratio fold change at 3 and at 6 hours from kinetic curves (I-J). Values represent the mean ± standard deviation of at least three biological replicates. ns: not significative, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001 using a RM one-way ANOVA with Sidak’s multiple comparison test. (L) Representative growth of cells as in (A) exposed to different concentrations of phenformin (Phen) for three days with corresponding EC50 values. Values represent the mean ± standard deviation of N=3. (****) P < 0.0001 using an unpaired Student’s t-test.

(A) Quantification of NAD+ concentration in KP-4 cells expressing a control small guide RNA against GFP (sgGFP) or a representative clone of two different guides targeting ATP5I (sgATP5I #1 or sgATP5I #2). Values represent the mean ± standard deviation of N=3. (***) P < 0.001, (****), P < 0.0001 using an unpaired Student’s t-test. (B) Quantification of NADH concentration in cells as in (A). Values represent the mean ± standard deviation of N=3. (ns) not significative, (**) P < 0.01 using an unpaired Student’s t-test. (C) Relative quantification of oxygen consumption rate (OCR) by Seahorse analysis in cells as in (A). Values represent the mean ± standard deviation of at least N=3. (ns) not significative, (*) P < 0.05 using a paired Student’s t-test. (D) Relative quantification of extracellular acidification rate (ECAR) by Seahorse analysis in cells as in (A). Values represent the mean ± standard deviation of at least N=3. (***) P < 0.001 using a paired Student’s t-test. (E) Representative cell growth of cells as in (A) treated with different concentrations of 2-D-deoxyglucose with corresponding EC50 values of 2-D-deoxyglucose treatment in cells as in (A). Values represent the mean ± standard deviation of N=3. (**) P < 0.01 using an unpaired Student’s t-test. (F) Representative cell viability curves with corresponding EC50 values of treatments of 2-D-deoxyglucose in combination without (-) and with different concentrations (1, 2.5 and 5 mM) of metformin (Met) in KP-4 cells expressing control sgGFP. Values represent the mean ± standard deviation of three biological replicates. (**) P < 0.01 using an unpaired Student’s t-test. (G) Total and phosphorylated levels of AMPK protein extracts from cells as in (A) treated with 2.5 mM or 5 mM of Met for 16 hours. β-ACTIN antibody was used for loading control. (H) Growth curves of cells as in (A) supplemented with 100 μg/mL sodium pyruvate (Pyr) and 50 μg/mL uridine (Uri) by measuring the percentage of confluency over 6 days. Media was changed every two days.

Exogenous ATP5I enable the reorganization of mitochondrial network in ATP5I knockout pancreatic cancer cells.

(A) A representative immunoblot of the five OXPHOS complexes in KP-4 cells expressing exogenous ATP5I (exoATP5I: +) in control cells expressing a small guide RNA against GFP (sgGFP) or in ATP5I KO cells (clones of two different small guide RNAs: sgATP5I #1 sgATP5I #2) compared with the same cell lines without expression of exogenous ATP5I (-). GAPDH antibody was used as loading control. (B) Representative images of mitochondrial morphologies characterized in cells as in (A). Mitochondrial morphologies were analysed by immunofluorescence using TOMM20 antibody, scale bar= 10 μm. DAPI was used as DNA counterstain. (C) Quantification of the percentage of cells exhibiting filamentous, fragmented or punctuated mitochondria from (B). Values represent the mean ± standard deviation of N=3 for each clone, n= 50-100 cells per clone.

(A) Representative images of mitochondrial localization of ATP5I in KP-4 cells expressing exogenous ATP5I (exoATP5I: +) in control (clones with sgGFP) or a representative clone of each of two small guide RNA against ATP5I (sgATP5I #1 or sgATP5I #2) compared with the same cell lines without expression of exogenous ATP5I (-). Mitochondrial localization was analyzed by co-immunofluorescence using ATP5I and TOMM20 antibodies, scale bar= 10 μm. DAPI was used as a DNA counter stain. (B) Colocalization between TOMM20 (TOMM20-568) and ATP5I (ATP5I-488) signals was analyzed with job plot intensity profile.

Re-expression of ATP5I rescues metabolic profile and resensitizes ATP5I knockout pancreatic cancer cells to biguanides.

(A) Quantification of NAD+/NADH ratio in KP-4 cells expressing exogenous ATP5I (exoATP5I: +) in control sgGFP or a representative clone of two different small guide RNAs (sgATP5I #1 and sgATP5I #2) compared with the same cell lines without expression of exogenous ATP5I (-). Values represent the mean ± standard deviation of three biological replicates. (ns) not significative, (*) P < 0.05, (**) P < 0.01, (***) P < 0.001 using an ordinary one-way ANOVA with Sidak’s multiple comparison test. (B) Relative quantification of oxygen consumption rate (OCR) over extracellular acidification rate (ECAR) by Seahorse analysis in cells as in (A). Values represent the mean ± standard deviation of at least three biological replicates. (ns) not significative, (*) P < 0.05, (***) P < 0.001, (****) P < 0.0001 using a repeated measures (RM) one-way ANOVA with Sidak’s multiple comparison test. (C) Immunoblot of total and phosphorylated levels of AMPK (Thr172) protein in extracts from cells as in (A). GAPDH antibody was used as loading control. (D) Growth curves of cells as in (A) by measuring the relative number of cells over 6 days. Media was changed every two days. (E) EC50 values of metformin (Met) treatments in cells as in (A). Values represent the mean ± standard deviation of N=3. (ns) not significative, (**) P < 0.01, (****) P < 0.0001 using an ordinary one-way ANOVA with Sidak’s multiple comparison test. (F) EC50 values of phenformin (Phen) treatment in cells as in (A). Values represent the mean ± standard deviation of three biological replicates. (ns) not significative, (*) P < 0.05, (***) P < 0.001, (****) P < 0.0001 using an ordinary one-way ANOVA with Sidak’s multiple comparison test.

(A) Quantification of NAD+ concentration in KP-4 cells expressing exogenous ATP5I (exoATP5I: +) in control sgGFP clones or a representative clone of two different small guide RNAs against ATP5I (sgATP5I #1 and sgATP5I #2) compared with the same cell lines without expression of exogenous ATP5I (-). Values represent the mean ± standard deviation of three biological replicates. (ns) not significative, (*) P < 0.05, (****) P < 0.0001 using an ordinary one-way ANOVA with Sidak’s multiple comparison test. (B) Quantification of NADH concentration in cells as in (A). Values represent the mean ± standard deviation of three biological replicates. (ns) not significative using an ordinary one-way ANOVA with Sidak’s multiple comparison test. (C) Relative quantification of oxygen consumption rate (OCR) by Seahorse analysis in cells as in (A). Values represent the mean ± standard deviation of at least three biological replicates. (ns) not significative, (**) P < 0.01 using a repeated measures (RM) one-way ANOVA with Sidak’s multiple comparison test. (D) Relative extracellular acidification rate (ECAR) by Seahorse analysis in cells as in (A). Values represent the mean ± standard deviation of at least N=3. (ns) not significative, (*) P < 0.05, (****) P < 0.0001 using a RM one-way ANOVA with Sidak’s multiple comparison test. (E) EC50 values of 2-D-deoxyglucose treatment in cells as in (A). Values represent the mean ± standard deviation of N=3. (**) P < 0.01, (***) P < 0.001 using an ordinary one-way ANOVA with Sidak’s multiple comparison test.

(A) Representative kinetic curves of oxygen consumption rate (OCR) fold change in KP-4 cells expressing exogenous ATP5I (exoATP5I: +) in control sgGFP clones or a representative clone of each of two small guide RNA against ATP5I (sgATP5I #1 and sgATP5I #2) compared with the same cell lines without expression of exogenous ATP5I (-) treated with 5 mM metformin (Met) using Seahorse. (b) Representative kinetic curves of extracellular acidification rate (ECAR) fold change in cells as in (A) treated with 5 mM Met using Seahorse. (C) Quantification of OCR/ECAR ratio fold change at 3 and at 6 hours from kinetic curves in (A) and (B). Values represent the mean ± standard deviation of at least three biological replicates. (ns) not significative, (*) P < 0.05, (**) P < 0.01, (***) P < 0.001 using a repeated measures (RM) one-way ANOVA with Sidak’s multiple comparison test. (D) Representative kinetic curves of OCR fold change in cells as in (A) but treated with 100 μM phenformin (Phen) using Seahorse. (E) Representative kinetic curves of ECAR fold change in cells as in (A) but treated with 100 μM Phen using Seahorse. (F) Quantification of OCR/ECAR ratio fold change at 3 and at 6 hours from kinetic curves (D) and (E). Values represent the mean ± standard deviation of at least three biological replicates. (ns) not significative, (****) P < 0.0001 using a RM one-way ANOVA with Sidak’s multiple comparison test.

Chemogenomic screening of metformin reveals an imprint on F1Fo-ATP synthase.

A) Results of the pooled genome-wide CRISPR/Cas9 KO screen made in NALM-6 cells treated with 16 mM metformin or control. Data are represented as a Volcano plot of gene enrichment/depletion scores vs p-values from using the CRANKS algorithm. Some genes of interest are labeled. Enhancers of metformin growth inhibition with negative CRANKS scores below 2.5 (dashed line) are labeled blue, while suppressors with positive CRANKS scores above 2.5 (dashed line) are labeled red. B-C) Pairwise comparison of gene CRANKS scores obtained from screening metformin 16 mM in NALM-6 cells against that from screening either 70 nM rotenone or (C) 2 μM oligomycin A. D) Model for metformin action triggering the OMA1-DELE1-HRI pathwat.