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Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

Published on: November 15, 2013

Nuclear receptor RORγ inverse agonists/antagonists display tissue- and gene-context selectivity through distinct

Hongye Zou¹, Yatian Yang¹, Zhenrui Shi²

¹Department of Biochemistry and Molecular Medicine, University of California, Davis, School of Medicine, Sacramento, CA, USA.

Pharmacological Research

|June 24, 2022

Abstract:

The nuclear receptor RORγ is a major driver of autoimmune diseases and certain types of cancer due to its aberrant function in T helper 17 (Th17) cell differentiation and tumor cholesterol metabolism, respectively. Compound screening using the classic receptor-coactivator interaction perturbation scheme led to identification of many small-molecule modulators of RORγ(t). We report here that inverse agonists/antagonists of RORγ such as VTP-43742 derivative VTP-23 and TAK828F, which can potently inhibit the inflammatory gene program in Th17 cells, unexpectedly lack high potency in inhibiting the growth of TNBC tumor cells. In contrast, antagonists such as XY018 and GSK805 that strongly suppress tumor cell growth and survival display only modest activities in reducing Th17-related cytokine expression. Unexpectedly, we found that VTP-23 significantly induces the cholesterol biosynthesis program in TNBC cells. Our further mechanistic analyses revealed that VTP-23 enhances the local chromatin accessibility, H3K27ac mark and the cholesterol master regulator SREBP2 recruitment at the RORγ binding sites, whereas XY018 exerts the opposite activities. Yet, they display similar inhibitory effects on circadian rhythm program. Similar distinctions and contrasting activities between TAK828F and SR2211 in their effects on local chromatin structure at Il17 genes were also observed. Together, our study shows for the first-time that structurally distinct RORγ antagonists possess different or even contrasting activities in tissue/cell-specific manner. Our findings also highlight that the activities at natural chromatin are key determinants of RORγ modulators' tissue selectivity.

Insights

RORγ modulators show cell-specific activities. Some inhibit inflammation but not cancer, while others suppress tumors but not Th17 cells, revealing tissue selectivity determinants.

Area of Science:

Molecular Biology
Immunology
Oncology

Background:

Nuclear receptor RORγ drives autoimmune diseases and cancer via Th17 cells and tumor metabolism.
Small-molecule modulators of RORγ have been identified through compound screening.
RORγ(t) plays a critical role in T helper 17 (Th17) cell differentiation and tumor cholesterol metabolism.

Purpose of the Study:

To investigate the differential activities of RORγ modulators in Th17 cells and triple-negative breast cancer (TNBC) cells.
To elucidate the mechanisms underlying the tissue-specific effects of RORγ antagonists.
To identify determinants of RORγ modulators' tissue selectivity.

Main Methods:

Screening of small-molecule modulators targeting RORγ(t).
Assessing inhibitory effects of compounds on Th17 cell inflammatory gene programs and TNBC cell growth.
Mechanistic analyses including chromatin accessibility, H3K27ac marks, and transcription factor recruitment (SREBP2).

Main Results:

Inverse agonists/antagonents like VTP-23 and TAK828F potently inhibit Th17 inflammation but not TNBC growth.
Antagonists like XY018 and GSK805 suppress TNBC growth but show modest effects on Th17 cytokines.
VTP-23 induces cholesterol biosynthesis in TNBC cells by enhancing chromatin accessibility and SREBP2 recruitment at RORγ sites, while XY018 has opposite effects.
Distinct RORγ antagonists exhibit contrasting activities on local chromatin structure at Il17 genes.

Conclusions:

Structurally distinct RORγ antagonists display cell- and tissue-specific activities.
RORγ modulators' effects on chromatin are key determinants of their tissue selectivity.
Understanding these differential activities is crucial for developing targeted therapies for autoimmune diseases and cancer.

Read full analysis

Keywords:

Breast cancer Cholesterol biosynthesis Chromatin accessibility RORγt Tissue-selective modulator

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