Discovery of Nonlipogenic ABCA1 Inducing Compounds with Potential in Alzheimer's Disease and Type 2 Diabetes

Abstract

Selective liver X receptor (LXR) agonists have been extensively pursued as therapeutics for Alzheimer's disease and related dementia (ADRD) and, for comorbidities such as type 2 diabetes (T2D) and cerebrovascular disease (CVD), disorders with underlying impaired insulin signaling, glucose metabolism, and cholesterol mobilization. The failure of the LXR-focused approach led us to pursue a novel strategy to discover nonlipogenic ATP-binding cassette transporter A1 (ABCA1) inducers (NLAIs): screening for ABCA1-luciferase activation in astrocytoma cells and counterscreening against lipogenic gene upregulation in hepatocarcinoma cells. Beneficial effects of LXRβ agonists mediated by ABCA1 include the following: control of cholesterol and phospholipid efflux to lipid-poor apolipoproteins forming beneficial peripheral HDL and HDL-like particles in the brain and attenuation of inflammation. While rare, ABCA1 variants reduce plasma HDL and correlate with an increased risk of ADRD and CVD. In secondary assays, NLAI hits enhanced cholesterol mobilization and positively impacted in vitro biomarkers associated with insulin signaling, inflammatory response, and biogenic properties. In vivo target engagement was demonstrated after oral administration of NLAIs in (i) mice fed a high-fat diet, a model for obesity-linked T2D, (ii) mice administered LPS, and (iii) mice with accelerated oxidative stress. The lack of adverse effects on lipogenesis and positive effects on multiple biomarkers associated with T2D and ADRD supports this novel phenotypic approach to NLAIs as a platform for T2D and ADRD drug discovery.

Scheme 1. Chemical Structures and Abbreviations of Chemical Probes and NLAI Hits

Figure 1

NLAIs increased ABCA1 without affecting lipogenic gene induction. Response of primary screen (A: CCF-ABCA1) and counterscreen (B: HepG2-SREBP1c) to the NLAIs F4 and M2 was compared to benchmark LXR agonist T0 and C) concentration–response for T0 alone. D, E) Response, normalized to the vehicle, of primary screen (D) and counterscreen (E) to HTS hits (F4, M2; 10 μM), RXR agonist (BEX; 10 μM), endogenous LXR agonist (24S-OHC; 10 μM), and exogenous LXR agonists (T0, GW3965; 10 μM), alone or in combination, with significant increases observed in treatment groups versus the vehicle. F) Comparison of induction of ABCA1 (solid bars) and SREBP1c (open bars) by exogenous hits and benchmark compounds, normalized to the combination of T0+BEX. G) Modulation of mRNA for ABCA1 in CCF cells and for FAS and SREBP1c in HepG2 cells after treatment by T0, Bex, and hits F4 and M2. Data analyzed by one-way ANOVA with Dunnett’s multicomparison analysis: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 2

NLAIs enhanced cholesterol efflux and expression of cholesterol transport genes. A) Representative images of BODIPY-cholesterol in CCF cells treated with T0, Bex, and F4, before and after addition of acceptor ApoAI (also see Figure S2F). B) Quantitation of BODIPY-cholesterol efflux to ApoAI for benchmark LXR and RXR agonists and hits F4, M2, and L7, showing significance relative to the DMSO vehicle control. C) ABCA1, ABCG1, and APOE mRNA levels measured by RT-PCR in primary human astrocytes after treatment with validated hits or benchmark agonists (all at 5 μM), relative to the DMSO vehicle control. All fold changes are significantly different from the vehicle (p < 0.05) except those marked by ns. D) Comparison of hits F4 and M2 with T0 (all at 5 μM) in glial cell cultures from mice expressing human APOE4 showing significance of induction of ABCA1, ABCG1, APOE, and PGC1A mRNA relative to the DMSO vehicle control. Data analyzed by one-way ANOVA with Dunnett’s multicomparison analysis relative to the DMSO vehicle control: *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 3

Target identification for NLAIs at various nuclear receptors. A) LXRβ, PPARγ, and PPARα transactivation by NLAI hits: F4, L7, M2, and L5 displayed LXRβ agonistic activity in the luciferase reporter assay, whereas other NLAIs (see Figure S5) displayed PPARγ agonist activity. B) Recruitment of coactivator TRAP220/DRIP2 to human LXRα-LBD by NLAI hits, T0, or GW, as measured by coregulator recruitment TR-FRET (CRT). C) Recruitment of coactivator D22 to human LXRβ-LBD by NLAI hits, T0, or GW, as measured by CRT. CRT data expressed as the mean ratio of the emission signal at 520 nm and the signal at 495 nm, averaged across n = 3 independent experiments, and normalized to maximal coactivator recruitment. D) Tabulation of calculated potency and efficacy from CRT data: (*) to calculate LXRα potency for NLAI hits L5 and L7, maximal efficacy was constrained at 100%.

Figure 4

In vivo pharmacodynamic readouts revealed target engagement without increased triglyceride levels. Mice were dosed daily (p.o.) for 7 days with the vehicle or the indicated doses of F4, M2, or T0. A,B) Fold induction of ABCA1 and ABCG1 mRNA relative to the vehicle control was analyzed in brain tissues (A) and liver (B). C) Fold induction of lipogenic gene mRNA relative to the vehicle control was measured in liver. D) Plasma and liver triglycerides were quantified after oral dosing in response to T0 and F4 treatment. Data analyzed by one-way ANOVA with Dunnett’s multicomparison analysis: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 5

Response to F4 treatment in a choline-deficient high-fat diet mouse model. Male C57BL/6 mice were fed choline-deficient HFD for 8 weeks, with F4 or T0 (p.o. 30 mg/kg/day), or the vehicle administered from weeks 5–8 of diet. A) Weight gain was significantly attenuated at 8 weeks in the F4 group versus T0 group. B) Liver weight and plasma triglycerides were significantly increased in the T0 group but not in the F4 treatment group, with overt steatohepatitis observed in the T0 treatment group. C) Decreased HDL-cholesterol due to HFD was significantly restored by F4 and T0 treatment, while LDL and total cholesterol increased only in the T0 treatment group. D–F) Well-known paradoxical decreases in lipogenic genes in the liver caused by HFD were observed, with significant increases in FASN and SCD in the T0 treatment group. G) Brain levels of ABCA1 and APOE were significantly upregulated by F4 and T0 treatment relative to the vehicle. H) Liver levels of ABCA1 and APOE were significantly downregulated by HFD, with F4 treatment significantly restoring levels relative to T0. Data analyzed by one-way ANOVA with Dunnett’s or Tukey’s multicomparison analysis: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05.

Figure 6

F4 suppressed inflammation in two mouse models of acute (LPS) and chronic (HFD) inflammation. A–C) WT and ALDH2^–/– (KO) mice were administered LPS to induce expression of pro-inflammatory markers and chemokines in the brain, with responses significantly attenuated by pretreatment with F4. D–F) HFD increased brain expression of pro-inflammatory genes, which was reversed by F4 treatment. G) KO mice administered HFD showed attenuation of pro-inflammatory gene expression when cotreated with F4. H) Discrimination Index of KO mice and WT littermates in NOR task before initiating HFD (Pre) and after 1 and 2 weeks on HFD showing a decline in performance and rescue by F4. Data analyzed by one-way ANOVA with Dunnett’s or Tukey’s multicomparison analysis: **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05.