Synthesis and biological characterization of an orally bioavailable lactate dehydrogenase-A inhibitor against pancreatic cancer

Abstract

Lactate dehydrogenase-A (LDHA) is the major isoform of lactate dehydrogenases (LDH) that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma (PDAC). Despite some progress, current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development. The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif. Compounds 6 and 21 are structurally related analogs that demonstrated potent inhibition of LDHA with IC₅₀s of 46 nM and 72 nM, respectively. We solved cocrystal structures of compound 21-bound to LDHA that showed that the compound binds to a distinct allosteric site between the two subunits of the LDHA tetramer. Inhibition of LDHA correlated with reduced lactate production and reduction of glycolysis in MIA PaCa-2 pancreatic cancer cells. The lead compounds inhibit the proliferation of human pancreatic cancer cell lines and patient-derived 3D organoids and exhibit a synergistic cytotoxic effect with the OXPHOS inhibitor phenformin. Unlike current LDHA inhibitors, 6 and 21 have appropriate pharmacokinetics and ligand efficiency metrics, exhibit up to 73% oral bioavailability, and a cumulative half-life greater than 4 h in mice.

Keywords: Lactate; Lactate Dehydrogenase-A; OXPHOS; Pancreatic cancer; Pharmacokinetics; Small molecule inhibitor.

Fig. 1.

Chemical structures of known Lactate Dehydrogenase inhibitors.

Fig. 2.

Rational design of succinic acid monoamide derivatives as novel LDHA inhibitors.

Fig. 3.

Crystal structure of compound 21-A1P1 (a) and 21-A1P2 (b). Color code: C, gray; N, blue; O, red; H, white and F, light green.

Fig. 4.

General structure of compounds synthesized for SAR.

Fig. 5.

Compounds 6 and 21 LDHA dose-response curves. Data are shown as mean ± SD from n = 3, three technical and three biological replicates.

Fig. 6.

Structure of the LDHA complex with compound 21. (A) LDHA tetramer colored magenta (subunit A), cyan (subunit B), green (subunit C) and gold (subunit D). The inhibitor, 21, is rendered at spheres. (B) Electron density (Fo-Fc) omit map (green mesh) contoured at 3σ for the inhibitor. (C) Hydrogen contacts (dashed lines) between LDI56 and subunits A and D. (D) Electrostatic surface rendering showing the binding mode of 21 (cylinders and yellow surface) to LDHA.

Fig. 7.

Orientation of the methyl-imidazole ring of compound 21 near LDHA residues related by crystallographic symmetry (coral).

Fig. 8.

Structure of the LDHA-compound 21-o complex. (A) Electron density (Fo-Fc) omit map (green mesh) contoured at 3σ for the inhibitor and hydrogen bonds (dashed lines) between 21 and subunit D. (B) Orientation of compound 21 trifluoromethyl aryl ring near LDHA residues related by crystallographic symmetry (coral and light blue). (C) Superposition of LDHA complexed with 21 (gray) with the NADH bound structure (PDB 5ZJD).

Fig. 9.

(A) and (B) compound 21 reduced the extracellular lactate secreted in the culture media after 6 h of treatment in MIA PaCa-2 cells compared to the untreated control. (A) cells were cultured in DMEM media comprising 25 mM glucose, 2 mM glutamine, and 1 mM pyruvate. (B) cells were cultured in DMEM media comprising 10 mM glucose, 2 mM glutamine without pyruvate. (C) compound 21 reduced intracellular lactate levels after 6 h and 24 h of treatment in MIA PaCa-2 cells as compared to the untreated control in the metabolomics assay. (D) Effect of compound 21 on the intracellular pyruvate levels in MIA PaCa-2 cells after 6 h and 24 h of treatment. Data is represented as mean ± SEM from n = 3 (*p < 0.05, ***p < 0.001, one-way ANOVA).

Fig. 10.

Seahorse glycolytic rate assay. (A) MIA PaCa-2 cells were treated with 21 for 6 h, and the changes in the glyco PER were measured before and after the addition of drugs, Rotenone + Antimycin and 2-DG, until over 60 min. Compound 21 reduces (B) basal and (C) compensatory glycolysis compared to the untreated control. Data represent mean ± SEM (n = 3) (*p < 0.05, **p < 0.01, one-way ANOVA).

Fig. 11.

Effect of compound 21 on ATP production. (A) In the Seahorse ATP rate assay, 21 reduced the total ATP production rate, including glycolysis ATP production rate, in MIA PaCa-2 cells after 24 h compared to the untreated control. (B) in the CellTiter-Glo assay, 21 reduced the intracellular ATP levels relative to untreated control after 48 h of treatment. Data represent mean ± SEM (n = 3) (*p < 0.05, one-way ANOVA).

Fig. 12.

Compounds 6 and 21 inhibit the proliferation of MIA PaCa-2 cells. Cells were treated with varying concentrations of compounds for 48 h and cell count were determined using Operetta image cytometer (n = 3).

Fig. 13.

Compound 21 (20 μM) showed synergy with phenformin (PF) (500 μM) in the proliferation assay in MIA PaCa-2 cells after 48 h. Synergy was determined using the Chou-Talalay method with a combination index (CI) < 1 (n = 3).

Fig. 14.

Effect of compounds 6 and 21 on pancreatic cancer organoids (PANC137). (A) Dose-response curve for 6 and 21 showing the antiproliferative effect and inhibition of the growth of pancreatic cancer organoids; (B) Suppression of growth of pancreatic cancer organoids (PANC137B) shown by bright-field image at 200x magnification after treatment of compounds 6 and 21 at indicated concentrations for 48 h.

Fig. 15.

Metabolic profiling of compound 21 investigated in mice.

Scheme 1.

(a) MeOH, H₂SO₄, rt, 2 h, 84.2%–90.1% yield; (b) BuLi, diisopropyl amine, THF, tert-butyl bromoacetate, −78 °C- rt, 6 h, 45.9%–89.5% yield; (c) TFA, DCM, rt, 4 h, 62.8% - quantitative yield; (d) method A: Oxalyl chloride, DMF, DCM 0°C-rt, 2 h, then R₂NH₂, Et₃N, DCM, rt, overnight; Method B: HATU, Et₃N R₂NH₂, DCM, 24 h–72 h, 24.2%–90.7% yield; (e) LiOH, H₂O, THF, rt, 3 h, 22.9%–74.3%.

Scheme 2.

(a) Malonic acid, ammonium nitrate, methanol, reflux, overnight, 47.9% yield; (b) Thionyl chloride, ethanol, 65 °C, 6 h, 81.3% yield; (c) Acid chloride, pyridine, THF, DMF, rt, 24 h or (method B) Acid, HATU, Et₃N, DCM, rt, 24 h, 66.07%–93.4%; (d) LiOH, H₂O, THF, rt, 3 h, 10.8%–49.7%.