82879 induces LXRα-dependent upregulation of ABCA1 mRNA in MEFs whereas apoE expression is stimulated through LXR-dependent and LXR-independent activities.

<p>LXRα-/β-, and LXRα expressing (LXRα+/β-) MEF cells were treated for 24 h withDMSO alone, GW3965, or compound 82879 at the indicated concentrations. Differential mRNA expression levels of (<i>A</i>) apoE and (<i>B</i>) ABCA1 were measured. Data are expressed as fold-change relative to the respective control group (dashed line) within each genotype. The responses to drug treatment compared to the DMSO control within each genotype (***p <0.001) were compared using a Linear Mixed Model with a Sidak's multiple-comparison test on ΔCT values obtained by qRT-PCR. The magnitudes of drug effect between two genotypes were compared using one-way ANOVA with a blocking factor (Experiment) on ΔΔCT values (# p<0.05, ## p<0.01, ### p<0.001). Data are expressed as mean and 95% CI from at least 6 independent experiments for GW3965 treatment and 3 experiments for 82879 treatments.</p

Compound 82879 upregulates apoE and ABCA1 mRNA in primary human astrocytes.

<p>Primary human astrocytes from three donors genotyped for <i>APOE</i> (D1: <i>E3/E4</i>, D2: <i>E3/E4</i>, D3: <i>E3/E3</i>) were treated with DMSO alone, GW3965, or compound 82879 for 96 h in two independent experiments. (<i>A-C</i>) ApoE mRNA and (<i>D-F</i>) ABCA1 mRNA levels were measured. Graphs represent mean and 95% CI of fold change relative to DMSO treatment (which corresponds to 1 on the Y-axis, dashed line). Statistics were determined by a one-way ANOVA with a blocking factor (Experiment) and a Dunnett’s post-test (* p<0.05, ** p<0.01, *** p< 0.001).</p

Compound 82879 upregulates LXR target gene expression in CCF-STTG1 cells.

<p>CCF-STTG1 (CCF) astrocytoma cells were treated for 48 h with DMSO alone, GW3965, or compound 82879. Following treatment, the mRNA levels for (<i>A</i>) apoE, (<i>B</i>) ABCA1, (<i>C</i>) LXRα, (<i>D</i>) IDOL and (E) SREBP-1c were determined in whole cell lysates. Data are expressed as fold-change relative to DMSO treatment (which corresponds to 1 on the Y-axis, dashed line). Graphs represent mean and 95% CI (N = 3). Statistics were determined by a one-way ANOVA with a blocking factor (Experiment) and a Dunnett’s post-test (*** p< 0.001).</p

Compound 82879 upregulates cellular ABCA1 protein levels in primary human astrocytes.

<p>Primary human astrocytes from three donors genotyped for <i>APOE</i> (<i>A</i>) D1: <i>E3/E4</i>, (<i>B</i>) D2: <i>E3/E4</i>, (<i>C</i>) D3: <i>E3/E3</i>) were treated with DMSO alone, GW3965, or compound 82879 for 96 h in two independent experiments. Cellular ABCA1 protein levels were determined by a capillary western system (representative pictures shown). Graphs represent mean and 95% CI of fold change relative to DMSO treatment (which corresponds to 1 on the Y-axis, dashed line). Statistics were determined by a one-way ANOVA with a blocking factor (Experiment) and a Dunnett’s post-test (* p<0.05, ** p<0.01, *** p< 0.001).</p

Response of LXR target genes to compound 82879 in various cell lines.

<p>Response of LXR target genes to compound 82879 in various cell lines.</p

Compound 82879 upregulates apoE secretion in primary human astrocytes.

<p>Primary human astrocytes from three donors genotyped for <i>APOE</i> (<i>A</i>) D1: <i>E3/E4</i>, (<i>B</i>) D2: <i>E3/E4</i>, (<i>C</i>) D3: <i>E3/E3</i>) were treated with DMSO alone, GW3965, or compound 82879 for 96 h in two independent experiments and apoE secretion levels were measured by ELISA. Graphs represent mean and SD of media apoE concentration normalized against total cellular protein content. Statistics were determined by a one-way ANOVA with a blocking factor (Experiment) and a Dunnett’s post-test (* p<0.05, *** p< 0.001).</p

82879 regulates LXR target genes differentially in primary human macrophages and hepatocytes.

<p>Primary human macrophages (hMacrophage) and hepatocytes (hHepatocytes) were treated for 48h with DMSO alone, GW3965, or compound 82879. Following treatment, the mRNA levels for (<i>A</i>) apoE, (<i>B</i>) ABCA1, (<i>C</i>) SREBP-1c, and (<i>D</i>) LXRα were determined in whole cell lysates. Data are expressed as fold-change relative to DMSO treatment (which corresponds to 1 on the Y-axis, dashed line). Graphs represent mean and 95% CI of 3 independent experiments. Statistics were determined by a one-way ANOVA with a blocking factor (Experiment) and a Dunnett’s post-test (* p<0.05, ** p<0.01, *** p< 0.001).</p

Quantitative RT-PCR primer sequences.

<p>Quantitative RT-PCR primer sequences.</p

Structural analogues of 82879 do not increase secreted apoE in CCF-STTG1 cells.

<p>(<i>A</i>) Planar structure of compound 82879. (<i>B</i>) Structural analogues from the small molecule library used for the high throughput screen were tested for their effects on apoE secretion. Media apoE was measured following 96 h of treatment (0.1 to 30 μM) using the initial screen assay conditions. Data are expressed as fold-change relative to DMSO treatment (N = 1). (<i>C</i>) Additional structural analogues were tested at a concentration of 10 μM and apoE secretion was measured following 48 h of CCF-STTG1 (CCF) cell treatment using the validation assay conditions (N = 3). Graphs represent mean and SD of apoE concentration. Statistics were determined by one-way ANOVA with blocking factor (Experiment) and a Dunnett’s post-test (*** p<0.001).</p

Discovery of VU0467485/AZ13713945: An M₄ PAM Evaluated as a Preclinical Candidate for the Treatment of Schizophrenia

Herein, we report the structure–activity relationships within a series of potent, selective, and orally bioavailable muscarinic acetylcholine receptor 4 (M₄) positive allosteric modulators (PAMs). Compound <b>6c</b> (VU0467485) possesses robust <i>in vitro</i> M₄ PAM potency across species and <i>in vivo</i> efficacy in preclinical models of schizophrenia. Coupled with an attractive DMPK profile and suitable predicted human PK, <b>6c</b> (VU0467485) was evaluated as a preclinical development candidate