Ligand and Receptor Dynamics Contribute to the Mechanism of Graded PPARγ Agonism

SummaryLigand binding to proteins is not a static process, but rather involves a number of complex dynamic transitions. A flexible ligand can change conformation upon binding its target. The conformation and dynamics of a protein can change to facilitate ligand binding. The conformation of the ligand, however, is generally presumed to have one primary binding mode, shifting the protein conformational ensemble from one state to another. We report solution nuclear magnetic resonance (NMR) studies that reveal peroxisome proliferator-activated receptor γ (PPARγ) modulators can sample multiple binding modes manifesting in multiple receptor conformations in slow conformational exchange. Our NMR, hydrogen/deuterium exchange and docking studies reveal that ligand-induced receptor stabilization and binding mode occupancy correlate with the graded agonist response of the ligand. Our results suggest that ligand and receptor dynamics affect the graded transcriptional output of PPARγ modulators

Broad Anti-tumor Activity of a Small Molecule that Selectively Targets the Warburg Effect and Lipogenesis

Malignant cells exhibit aerobic glycolysis (the Warburg effect) and become dependent on de novo lipogenesis, which sustains rapid proliferation and resistance to cellular stress. The nuclear receptor liver-X-receptor (LXR) directly regulates expression of key glycolytic and lipogenic genes. To disrupt these oncogenic metabolism pathways, we designed an LXR inverse agonist SR9243 that induces LXR-corepressor interaction. In cancer cells, SR9243 significantly inhibited the Warburg effect and lipogenesis by reducing glycolytic and lipogenic gene expression. SR9243 induced apoptosis in tumors without inducing weight loss, hepatotoxicity, or inflammation. Our results suggest that LXR inverse agonists may be an effective cancer treatment approach

Broad Anti-tumor Activity of a Small Molecule that Selectively Targets the Warburg Effect and Lipogenesis

Malignant cells exhibit aerobic glycolysis (the Warburg effect) and become dependent on de novo lipogenesis, which sustains rapid proliferation and resistance to cellular stress. The nuclear receptor liver-X-receptor (LXR) directly regulates expression of key glycolytic and lipogenic genes. To disrupt these oncogenic metabolism pathways, we designed an LXR inverse agonist SR9243 that induces LXR-corepressor interaction. In cancer cells, SR9243 significantly inhibited the Warburg effect and lipogenesis by reducing glycolytic and lipogenic gene expression. SR9243 induced apoptosis in tumors without inducing weight loss, hepatotoxicity, or inflammation. Our results suggest that LXR inverse agonists may be an effective cancer treatment approach

LXR-Mediated Inhibition of CD4⁺ T Helper Cells

<div><p>T_H17 cells, which require the expression of both retinoic acid receptor-related orphan receptors α and γt (RORαand RORγt) for full differentiation and function, have been implicated as major effectors in the pathogenesis of inflammatory and autoimmune diseases. We recently demonstrated that the Liver X Receptor (LXR) agonist, T0901317 (T09), also displays high-affinity RORα and RORγ inverse activity, potentially explaining its effectiveness in various T_H17-mediated autoimmune disease models. However, recent studies suggest that in conjunction with the RORs, LXR mediates a negative regulatory effect on T_H17 cell differentiation. Since T09 acts on both LXRs and RORs, it presents as a valuable tool to understand how compounds with mixed pharmacology affect potential pathological cell types. Therefore, using T09, we investigated the mechanism by which the LXRs and RORs affect T_H17 cell differentiation and function. Here we demonstrate that T09 activity at RORα and γ, not LXR, is facilitating the inhibition of T_H17 cell differentiation and function. We also demonstrate that LXR activity inhibits the differentiation and function of T_H1, T_H2 and iT_reg cells. Finally, T09 inhibited T cell proliferation and induced cell death. These data help explain much of the efficacy of T09 in inflammatory models and suggest that the generation of synthetic ligands with graded, combined LXR and ROR activity may hold utility in the treatment of inflammatory and autoimmune diseases where targeting both T_H17 and T_H1 cells is required.</p> </div

T09 inhibition of the differentiation and effector function of T_H1, T_H2, and iT_reg cells is a consequence of cell death.

<p><i>A</i>, The effect of T09 (5 µM) on murine T_H1, T_H2, and iT_reg differentiation. Left: Intracellular cytokine staining on splenocytes treated with T09 for the duration of the time course. Middle: Intracellular cytokine staining on splenocytes treated with T09 24 hours prior to the end of the time course. Right: Graphs summarizing the FACS plots. Data was normalized to DMSO controls. <i>B</i>, The effect of T09 (5 µM) on the viability of T_H1, T_H2, T_H17, and iT_reg cells. Cells were FASC analyzed and gated on Annexin V and PI negative cells and normalized to DMSO controls. (<i>n = 3</i>, * p<0.05).</p

T09 inhibits T_H17 cells cytokine expression.

<p><i>A</i>, Realtime - RT PCR analysis of splenocytes cultured under T_H17 polarizing conditions in the presence of T09 (5 µM) for the duration of the time course. (Data are normalized to GAPDH). <i>B</i>, Realtime - RT PCR analysis of splenocytes cultured under T_H17 polarizing conditions for a total of three days (72 hours). T09 (5 µM) was added into the cultures after day 2 (after 48 hours). (Data are normalized to GAPDH). (<i>n = 3</i>, * p<0.05).</p

T09 inhibits IL-17 cytokine production.

<p><i>A</i>, The effect of T09 (5 µM) on IL-17 cytokine expression during murine T_H17 cell differentiation. Left: Intracellular cytokine staining on splenocytes treated with T09 for the duration of the time course (4 days). Middle: Intracellular cytokine staining on splenocytes treated with T09 24 hours prior to the end of the time course. Right: Graphs summarizing the FACS plots. Data was normalized to DMSO controls. <i>B</i>, The effect of T09 treatment (5 µM, 24 hours) on IL-17 cytokine expression on differentiated human peripheral blood mononuclear cells (hPBMCs) using intracellular cytokine staining. <i>C</i>, The effect of T09 (5 µM) on IL-17 cytokine secretion during murine T_H17 cell differentiation. ELISA assay on splenocytes treated with T09 for the duration of the three day time course (T = 0, left) or treated with T09 24 hours prior to the end of the time course (T-24h, right). <i>D</i>, The effect of T09 (5 µM) on IL-17A/F cytokine secretion during murine T_H17 cell differentiation. ELISA assay on splenocytes treated with T09 for the duration of the three day time course (T = 0, left), or on splenocytes treated with T09 24 hours prior to the end of the time course (T-24h, right). (<i>n = 3</i>, * p<0.05).</p

Inhibition of all CD4⁺ T helper lineages is a function of LXR activity.

<p><i>A</i>, Intracellular cytokine staining on splenocytes differentiated under T_H1, T_H2, T_H17, or iT_reg conditions and treated with vehicle (DMSO), T09, GW3965, or SR1001 (5 µM each) for the duration of the time course (4 days). Right: Graphs summarizing the FACS plots. Data was normalized to DMSO controls. <i>B</i>, MTT assays of T_H1, T_H2, T_H17, or iT_reg cells cultured with vehicle (D), T09, GW3965, or SR1001 (5 µM each) for the duration of the time course (4 days). Data was normalized to DMSO controls. (<i>n = 4</i>, * p<0.05).</p