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, 10 (24), 2355-2368

Novel N,N-dialkyl Cyanocinnamic Acids as Monocarboxylate Transporter 1 and 4 Inhibitors

Affiliations

Novel N,N-dialkyl Cyanocinnamic Acids as Monocarboxylate Transporter 1 and 4 Inhibitors

Shirisha Jonnalagadda et al. Oncotarget.

Abstract

Potent and dual monocarboxylate transporter (MCT) 1 and 4 inhibitors have been developed for the first time as potential anticancer agents based on α-cyanocinnamic acid structural template. Candidate inhibitors 1-9 have been evaluated for in vitro cell proliferation against MCT1 and MCT4 expressing cancer cell lines. Potential MCT1 and MCT4 binding interactions of the lead compound 9 have been studied through homology modeling and molecular docking prediction. In vitro effects on extracellular flux via glycolysis and mitochondrial stress tests suggest that candidate compounds 3 and 9 disrupt glycolysis and OxPhos efficiently in MCT1 expressing colorectal adenocarcinoma WiDr and MCT4 expressing triple negative breast cancer MDA-MB-231 cells. Fluorescence microscopy analyses in these cells also indicate that compound 9 is internalized and concentrated near mitochondria. In vivo tumor growth inhibition studies in WiDr and MDA-MB-231 xenograft tumor models in mice indicate that the candidate compound 9 exhibits a significant single agent activity.

Keywords: 2-alkoxy-N,N-dialkyl cyanocinnamic acid; cancer; metabolism; monocarboxylate transporter 1 inhibitor; monocarboxylate transporter 4 inhibitor.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest. A patent has been issued to the University of Minnesota.

Figures

Figure 1
Figure 1. MCT1 and MCT4 lactate uptake inhibition
(A) Chemical structures of 2-methoxy-4-N,N-dialkyl cyanocinnamic acids 1–9. Bar graphs of (B) MCT1 inhibition and (C) MCT4 inhibition using lactate uptake study with compounds 1–9 in comparison to CHC. The final average ± sem of at least three independent experimental values were calculated. Repeated measures one-way ANOVA was used to calculate statistical significance (P < 0.05) between test compounds and CHC. ****P < 0001.
Figure 2
Figure 2. Glycolysis and mitochondrial stress tests of compound 9, CHC, and AZD3965
(A–C) represent the parameters from glycolysis stress test: (A) glycolysis, (B) glycolytic capacity, and (C) glycolytic reserve of compounds at 30 μM concentration in MCT1 expressing WiDr and MCT4 expressing MDA-MB-231 cells. (D–G) represent the parameters from mitochondrial stress test: (D) maximal respiration, (E) ATP production, (F) proton leak, and (G) spare respiratory capacity in WiDr and MDA-MB-231 cells. The ECAR and OCR values of were calculated using wave software. The average + SEM values of at least three independent experimental values were calculated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 3
Figure 3. Glycolysis and mitochondrial stress tests of compounds 3 and 9
(A–C) represent the parameters from glycolysis stress test: (A) glycolysis, (B) glycolytic capacity, and (C) glycolytic reserve of compounds at 30 μM concentration in WiDr and MDA-MB-231 cells. (D–G) represent the parameters from mitochondrial stress test: (D) maximal respiration, (E) ATP production, (F) proton leak, and (G) spare respiratory capacity in WiDr and MDA-MB-231 cells. The ECAR and OCR values of were calculated using wave software. The average + SEM values of at least three independent experimental values were calculated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 4
Figure 4. Mitotracker red staining in compound 9 treated MDA-MB-231 and WiDr cell lines
Representative pictures of (A) MDA-MB-231 and (B) WiDr cells after exposure to compound 9 (green) for 1 hour and MitoTracker red (MTR) for 15 minutes. Compound 9 is localized in regions of higher mitochondrial density in WiDr cell line. MTR-Pseudo images show the MTR signal pseudocolored using the Rainbow RGB LUT of the FIJI software program, to demonstrate mitochondrial hyperpolarization after addition of compound 9. (C, D) Compound 9 localizes to granular regions of MDA-MB-231 cells. Compound 9 localizes to regions near to, but does not overlap with, most mitochondria (red) in both (E) WiDr and (F) MDA-MB-231 cells. Images are representative of multiple fields of view from three independent experiments. Scale bar, 25 μm.
Figure 5
Figure 5. Homology model of human MCT1 and MCT4 docked with compound 9
Most favorable compound 9 binding pose to human MCT1 and MCT4 were represented. (A) Cα ribbon homology structure of MCT1 with docked compound 9 (yellow) and binding site residues within 4.5 Å shown. (B) Cα ribbon homology structure of MCT4 with docked compound 9 (yellow) and binding site residues within 4.5 Å shown. (C) Overlay of MCT1 and MCT4 homology models and their respective best compound 9 docking pose. (D) Compound 9 (yellow) and residue forming its binding site in MCT1, all residues within 4.5 Å are shown. (E) Compound 9 (yellow) and residue forming its binding site in MCT1, all residues within 4.5 Å are shown. (F) Overlay of most favorable binding pose of compound 9 for MCT1 and MCT4 and all residues within 4.5 Å. Models were displayed with Chimera.
Figure 6
Figure 6. In vivo xenograft studies in WiDr and MDA-MB-231 tumor models
(A) WiDr tumor xenograft study of compound 3 and compound 9. Mice (n = 8) were treated with 8 mg/kg of compound 3, intraperitoneally, two times a day. (B) Tumor growth inhibition study with compound 9 in MDA-MB-231 tumor xenograft model (n = 6). Mice were treated with compound 9 (70 mg/kg, ip, bid until day-4; qd from day-5), a combination of 9 and doxorubicin (0.5 mg/kg, ip, five days a week), and doxorubicin. (C) Tumor growth inhibition based on isolated tumor mass. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Schematic representation of (D) untreated tumor cells and (E) inhibition of MCT1 and MCT4 and decreased glycolysis and mitochondrial OxPhos in compound 9 treated tumor cells. Upward hollowed arrow indicates “increase” in function/amount and downward hollowed arrow indicates “decrease” in function/amount.

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