Selective and brain-penetrant ACSS2 inhibitors target breast cancer brain metastatic cells

Abstract

Breast cancer brain metastasis (BCBM) typically results in an end-stage diagnosis and is hindered by a lack of brain-penetrant drugs. Tumors in the brain rely on the conversion of acetate to acetyl-CoA by the enzyme acetyl-CoA synthetase 2 (ACSS2), a key regulator of fatty acid synthesis and protein acetylation. Here, we used a computational pipeline to identify novel brain-penetrant ACSS2 inhibitors combining pharmacophore-based shape screen methodology with absorption, distribution, metabolism, and excretion (ADME) property predictions. We identified compounds AD-5584 and AD-8007 that were validated for specific binding affinity to ACSS2. Treatment of BCBM cells with AD-5584 and AD-8007 leads to a significant reduction in colony formation, lipid storage, acetyl-CoA levels and cell survival in vitro. In an ex vivo brain-tumor slice model, treatment with AD-8007 and AD-5584 reduced pre-formed tumors and synergized with irradiation in blocking BCBM tumor growth. Treatment with AD-8007 reduced tumor burden and extended survival in vivo. This study identifies selective brain-penetrant ACSS2 inhibitors with efficacy towards breast cancer brain metastasis.

Keywords: ACSS2; acetate; acetyl-CoA; brain metastasis; breast cancer; cancer; computational-aided drug design font: italic formatted: left; metabolism.

FIGURE 1

Computational pipeline and predicted drug-like properties for the discovery of AD-2441 and its analogs. (A) Oral CNS Scoring profile score vs logS of AD-2441 and its analogs, including two currently known control inhibitors, VY-3-249 and VY-3-135. Plot showing the StarDrop V7 (Optibrium, Ltd., Cambridge, United Kingdom)–derived logS versus a multimetric oral CNS profile score. Score composition and importance of each contributor: logS = 0.8, logP = 0.6, logD = 0.6, BBB category = 0.55, BBB log(brain:blood) = 0.55, P-gp category = 0.5, HIA = 0.4, hERG pIC50 = 0.2, 2D6 affinity category = 0.16, 2C9 pKi = 0.16, PPB90 category = 0.1. The size of the circle correlates with the corresponding score or probability. (B) Computational and SPR-based validation pipeline for the discovery of novel ACSS2 inhibitors.

FIGURE 2

Representative sensorgrams and structures for AD-2441 and its analogs binding to ACSS2. (A) Sensorgrams of ACSS2 binders. Colored lines represent collected data from the dilution series, whereas black lines represent a fit to a 1:1 binding model—interaction parameters derived from a triplicate (n = 3) of data given in Supplementary Table S1. (B) Binding isotherms of AD-2441 and analogs. Binding isotherms are derived from panel (A). Experiments were performed in triplicate, and data displayed with standard deviations (SD with n = 3).

FIGURE 3

Fluorescent polarisation-based assay for measurement of inhibition of ATP to AMP conversion of ACSS2 by selected ACSS2 inhibitors. Experiments were performed using the Tecan Spark multimode microplate reader with 100 nM ACSS2 and varying compound concentrations. Experiments were performed in triplicate, and data displayed with standard deviations (SD with n = 3).

FIGURE 4

AD-2441 and its analogs are predicted to bind ACSS2 within the nucleotide-binding pocket and stabilized by various hydrophobic and polar contacts and aromatic stacking interactions. Docking calculations were performed into a homology model (using swissmodel.expasy.org) (Waterhouse et al., 2018; Studer et al., 2020) of ACSS2 based on the crystal structure of Salmonella enterica acetyl-CoA synthetase in complex with cAMP and Coenzyme A (PDB: 5JRH). (A) Homology model of ACSS2 with superimposed CoA (purple) and Adenosine-5′-propylphosphate (yellow, extracted from PDB: 1PG4). (B) Superimposed crystal structure of Adenosine-5′-propylphosphate (yellow, extracted from PDB: 1PG4) and docked (DiffDock and Flare version 5 minimized) Adenosine-5′-propylphosphate pose (blue). (C) All docked compounds are predicted to bind within the Adenosine-5′-propylphosphate site and additionally sterically interfere with CoA (purple) binding indicated by the red helo. (D) Close-up view of binding poses of AD-1363, AD-2441, AD-5584, AD-7346, and AD-8007 between the C- and N-Lobe of ACSS2.

FIGURE 5

In Vitro effect of ACSS2 inhibitors on MDA-MB-231BR cells cell survival. (A) Representative images of MDA-MB-231BR cells treated with ACSS2 inhibitors for 48 h at 100 μM and seeded in clonogenic cells survival assay stained with crystal violet. Average colony formation quantified and presented as average from three independent experiments. Two-way ANOVA reported as mean ± SEM. ***p < 0.001, ****p < 0.0001. (B) Representative images of MDA-MB-231BR cells treated with ACSS2 inhibitors for 48 h and seeded clonogenic cells survival assay stained with crystal violet. Average colony formation quantified and presented as average from three independent experiments. One-way ANOVA reported as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Quantification of cell death as measured by Propidum Idodine (PI)+ cells detected by flow cytometry analysis of MDA-MB-231BR cells treated with ACSS2 inhibitors at 100 μM for 48 h. One-way ANOVA reported as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

In Vitro effect of ACSS2 inhibitors on MDA-MB-231BR cells lipid content. (A) Acetyl-CoA levels were quantified by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) in MDA-MB-231BR cells treated with Control (DMSO) or ACSS2 inhibitors (VY-3-135, AD-5584, AD-8007) at 100 μM for 48 h (n = 3). One-way ANOVA reported as mean ± SEM. *p-value <0.05, **p < 0.01. (B) Representative images of MDA-MB-231BR cells stained with BOPIDY following 48 h treatment with ACSS2 at 100 μM and presented as average lipid droplet per cell from three independent experiments (left). Quanitification of lipid content presented (right) as One-way ANOVA reported as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. (C) Immunoblot analysis of MDA-MB-231BR cells with shRNA against scramble or ACSS2 with the indiciated antibodies. (D) Immunoblot analysis of MDA-MB-231BR cells treated with control (DMSO) or ACSS2 inhibitors (VY-3-249, AD-5584, AD-8007) for 24 h with the indiciated antibodies.

FIGURE 7

Ex vivo and BBB permeability of ACSS2 inhibitors. (A) Representative images of ex vivo tumor-bearing brain slices were obtained from Nu/Nu mice injected with luciferase-tagged MDA-MB-231BR cells treated with ACSS2 inhibitors at 100 μM for 6 days. Quantified graph of relative bioluminescence signal at indicated day (n = 3) Two-way ANOVA reported as mean ± SEM. *p-value ***p < 0.001 (B) Representative images of ex vivo tumor bearing brain slices derived from nu/nu mice injected with luciferase tagged MDA-MB-231BR cells exposed to no irradiation (control) or one dose of 6Gy treated with ACSS2 inhibitors at 20 μM for 6 days (n = 3). ANOVA reported as mean.*p < 0.05. (C) Quantification of LC-MS peaks of ACSS2 inhibitor present in the brain over blood following intraperitoneal delivery of 50 mg/kg of drug for 30 min (AD-5584) or 1 h (AD-8007, VY-3–135) (n = 3). Blood extraction via intracardiac injection, perfusion, and brain retrieval. Student’s paired t-test reported as mean ± SEM; *p < 0.05, **p < 0.01.

FIGURE 8

Effects of AD-8007 on BCBM growth in vivo. (A) Schematic workflow for in vivo studies of AD-8007 treatment. (B) Representative images of bioluminescent detection of tumors from Nu/Nu mice injected with luciferase tagged MDA-MB-231BR cells at Day 0 (prior to drug treatment) and at 14 days post-drug treatment. Data are quantified and presented as average Relative Bioluminescence signal from mice injected with MDA-MB-231BR cells treated with Vehicle (n = 3) or AD-8007 treated mice (n = 3) (right). Student’s t-test reported as mean ± SEM; *p < 0.05. (C) Representative images of brain sections stained for H&E and Ki67 at 14 days-post treatment. Top: 4x magnification. Bottom: 10x magnification. (D) Kaplan Meyer survival analysis quantifying survival of mice injected with MDA-MB-231BR cells and treated with vehicle (n = 5) or AD-8007 (n = 5), *p < 0.05. (E) Quantification of weights (grams) of mice injected with MDA-MB-231BR cells and treated with vehicle (n = 3) or AD-8007 (n = 3) for 14 days, analyzed with two-way ANOVA, n. s.