ABSTRACT
Inhibition of the nuclear receptor Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) is a promising strategy for the treatment of autoimmune diseases. In this paper, we describe a series of allosteric, cysteine-dependent, inverse agonists of RORγt. Site-directed mutagenesis and molecular dynamics simulations are supportive of a mechanism of action through specific binding to Cys476 on alpha helix 11 of the ligand binding domain (LBD). Representative compounds in the series selectively inhibit RORγt, potently suppress interleukin-17A (IL-17A) production by human CD4+ T cells, and inhibit T helper 17 (Th17) differentiation from human naïve CD4+ T cells. The advanced compound 13 is orally bioavailable and active at a dose of 3 mg/kg in a murine collagen-induced model of rheumatoid arthritis. Collectively, these data are supportive of the development of compound 13 in autoimmune diseases.
Subject(s)
Arthritis, Rheumatoid/drug therapy , Cysteine/chemistry , Nuclear Receptor Subfamily 1, Group F, Member 3/agonists , Pyrimidines/chemistry , Allosteric Site , Animals , Cell Differentiation/drug effects , Collagen/metabolism , Disease Models, Animal , Humans , Hydroxycholesterols/metabolism , Interleukin-17/metabolism , Ligands , Mice , Molecular Dynamics Simulation , Mutagenesis/drug effects , Protein Binding , Protein Conformation , Pyrimidines/metabolism , Pyrimidines/pharmacology , Structure-Activity Relationship , Th17 CellsABSTRACT
BACKGROUND: Semi-synthetic oleanane triterpenoid antioxidant inflammation modulators (tpAIMs) are small molecules that interact with KEAP1 cysteine residue 151 (C151) and activate NRF2. Exploration of the structure-activity relationship between the tpAIMs and KEAP1 is limited by the predominantly hydrocarbon nature of the oleanane triterpenoid pentacyclic ring structure. Therefore, we used novel, chemically-tractable, synthetic antioxidant inflammation modulators (sAIMs) to probe the stereoselectivity of the ligand-protein interaction. METHODS: We measured several parameters of NRF2 activation to assess the potency of sAIM enantiomers with natural (tpAIM-like) 4(S),5(S),10(R) or unnatural 4(R),5(R),10(S) configurations. Additionally, we determined the crystal structure of the KEAP1 BTB domain in complex with two different sAIMs. RESULTS: We found that the potencies of sAIM enantiomers in the natural configuration were similar to those of the tpAIM, RTA 405. Strikingly, sAIM enantiomers in the unnatural configuration were 10- to 40-fold less potent than their natural counterparts. Crystallographic studies of sAIMs in complex with the KEAP1 BTB domain demonstrated that these ligands form a covalent bond with C151 and revealed the presence of additional hydrogen bonds, Van der Waals interactions, and pi-stacking interactions. CONCLUSIONS: Although KEAP1 C151 is required for NRF2 activation by tpAIMs and sAIMs, interactions with other KEAP1 residues are critical for the stereospecific recognition and potency of these ligands. GENERAL SIGNIFICANCE: This work demonstrates that reversible cyanoenone Michael acceptors, such as the tpAIMs and sAIMs, can be specifically tuned to regulate redox sensitive cysteine residues on key signaling molecules, an approach with significant promise for innovative drug development.
Subject(s)
Antioxidants/pharmacology , Kelch-Like ECH-Associated Protein 1/metabolism , NF-E2-Related Factor 2/metabolism , Oleanolic Acid/chemistry , Quantitative Structure-Activity Relationship , Small Molecule Libraries/pharmacology , Animals , Antioxidants/chemistry , Binding Sites , HEK293 Cells , Humans , Kelch-Like ECH-Associated Protein 1/chemistry , Mice , Molecular Docking Simulation , NF-E2-Related Factor 2/chemistry , Small Molecule Libraries/chemistryABSTRACT
To explore more potent N-acylimidazole analogues of CDDO than CDDO-Im, which is one of the most potent compounds in several widely used bioassays related to protection against inflammation and carcinogenesis; we have synthesized and evaluated five new N-acyl(acetylenic)imidazole analogues. Among them, 4-ethynylimidazole 4 is nearly equivalent to CDDO-Im in potency in these bioassays. Remarkably, the solid form of 4 is more stable than that of CDDO-Im. These findings suggest that 4 is a very promising anti-inflammatory and cytoprotective agent and its further preclinical evaluation is warranted.
Subject(s)
Anti-Inflammatory Agents/chemical synthesis , Imidazoles/chemistry , Imidazoles/chemical synthesis , Oleanolic Acid/analogs & derivatives , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Cell Line, Tumor , Cytoprotection/drug effects , Heme Oxygenase-1/metabolism , Imidazoles/pharmacology , Interferon-gamma/metabolism , Mice , NAD(P)H Dehydrogenase (Quinone)/metabolism , Nitric Oxide/metabolism , Oleanolic Acid/chemical synthesis , Oleanolic Acid/chemistry , Oleanolic Acid/pharmacologyABSTRACT
2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride (CDDO anhydride) has been synthesized, which is the first example of an oleanane triterpenoid anhydride. CDDO anhydride shows potency similar to or higher than the corresponding acid (CDDO) in various in vitro and in vivo assays related to inflammation and carcinogenesis. Notably, preliminary phamacokinetics studies show that CDDO anhydride levels are higher than CDDO levels in mouse tissues and blood. Further evaluation of CDDO anhydride is in progress.
Subject(s)
Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Nitric Oxide/antagonists & inhibitors , Oleanolic Acid/chemistry , Oleanolic Acid/pharmacology , Anhydrides/chemistry , Anhydrides/pharmacokinetics , Anhydrides/pharmacology , Animals , Anti-Inflammatory Agents/pharmacokinetics , Cell Line, Tumor , Cytoprotection/drug effects , Heme Oxygenase-1/metabolism , Mice , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/metabolism , Oleanolic Acid/pharmacokinetics , Triterpenes/chemistry , Triterpenes/pharmacokinetics , Triterpenes/pharmacologyABSTRACT
The Type 1 PI3Kinases comprise a family of enzymes, which primarily phosphorylate PIP2 to give the second messenger PIP3, a key player in many intracellular signaling processes [Science, 2002, 296, 1655; Trends Pharmacol. Sci.2003, 24, 366]. Of the four type 1 PI3Ks, the gamma-isoform, which is expressed almost exclusively in leukocytes [Curr. Biol., 1997, 7, R470], is of particular interest with respect to its role in inflammatory diseases such as rheumatoid arthritis (RA) and chronic obstructive pulmonary disease (COPD) [Mol. Med. Today, 2000, 6, 347]. Investigation of a series of 4,6-disubstituted-4H-benzo[1,4]oxazin-3-ones has led to the identification of single-digit nanomolar inhibitors of PI3Kgamma, several of which had good cell based activity and were shown to be active in vivo in an aspectic peritonitis model of inflammatory cell migration.
