Development and pharmacological evaluation of a new chemical series of potent pan-ERR agonists, identification of SLU-PP-915

Abstract

Estrogen-related receptors (ERR) are an orphan nuclear receptor sub-family that play a critical role in regulating gene transcription for several physiological processes including mitochondrial function, cellular energy utilization and homeostasis. They have also been implicated to play a role in several pathological conditions. Herein, we report the identification, synthesis, structure-activity relationships and pharmacological evaluation of a new chemical series of potent pan-ERR agonists. This template was designed for ERRγ starting from the known acyl hydrazide template and compounds such as agonist GSK-4716 employing a structure-based drug design approach. This led to the preparation of a series of 2,5-disubstituted thiophenes from which several were found to be potent agonists of ERRγ in cell-based co-transfection assays. Additionally, direct binding to ERRγ was established through ¹H NMR protein-ligand binding experiments. Compound optimization revealed that the phenolic or aniline groups could be replaced with a boronic acid moiety, which was able to maintain activity and demonstrated improved metabolic stability in microsomal in vitro assays. Further pharmacological evaluation of these compounds showed that they had roughly equivalent agonist activity on ERR isoforms α and β representing an ERR pan-agonist profile. One potent agonist, SLU-PP-915 (10s), which contained a boronic acid moiety was profiled in gene expression assays and found to significantly upregulate the expression of ERR target genes such as peroxisome-proliferator activated receptor γ co-activators-1α, lactate dehydrogenase A, DNA damage inducible transcript 4 and pyruvate dehydrogenase kinase 4 both in vitro and in vivo.

Keywords: 2 5-disubstituted thiophenes; Boronic acid; Estrogen-related receptor; Nuclear receptor; Pan-agonist.

Figure 1.

Representative ERR synthetic ligands: ERRγ inverse agonists 4-OHT(1) and DES(2), ERRβ/γ agonist GSK-4716 (3), ERRγ agonist 4 and ERRα agonist 5

Figure 2.

Structure and ERR co-transfection data for Pan-ERR agonist SLU-PP-332 (6)

Figure 3.

Two-dimensional representation of GSK-4716 (3) bound in the LBD of ERRγ, PDB:2GPP

Figure 4.

General structure-based design strategy to develop new ERR agonists

Figure 5.

1H-NMR methyl region spectra of ERRγ with 0.5% DMSO-d₆. where the blue line represents vehicle alone and the red line is protein + 1.05:1 molar equivalence of ligand. A) protein + SR-9238 B) protein + 6 C) Protein + 10s D) Protein + 12e

Figure 6.

Molecular modeling of key compounds docked in the ligand binding domain of ERRγ obtained from the X-ray structure of 3 (PGB:2GPP). Compounds are shown in green. A) 9b docked in the anticipated compound 6 like orientation B) 9b docked in the ‘flipped’ conformation C) Compound 10s in the compound 6 like orientation. D) 10s in the ‘flipped’ conformation

Figure 7.

Gene expression in C2C12 myoblast cells treated with several compounds at 5uM for 24hrs. A. Relative gene expression levels for PGC1α vs control B. Relative gene expression levels for PDK4 vs control C. Relative gene expression levels for LDHA vs control. n=3 per condition, * p<0.05, ** p<0.01.

Figure 8:

ERR agonist SLU-PP-915 (10s) induces expression of ERR target genes in vivo: A) Gene expression from muscle from mice treated with SLU-PP-915 (20mg/kg, I.P) for 1h (n=3 per group). B. Running time and distance from mice 1h after being treated with SLU-PP-915 (20mg/kg, I.P). * p<0.05, ** p<0.01.

Scheme 1.

Synthesis of C(5)-hydroxyphenyl thiophene analogs^a Reaction conditions: (a) RNH₂, DIPEA, TBTU, DMF (b) 4-hydroxyphenyl boronic acid, Pd(PPh₃)₄; K₂CO₃, DMF, 90 °C

Scheme 2.

Synthesis of 2,5-disubstituted thiophene analogs^a Reaction conditions: (a) Boronic acid, Pd(PPh₃)₄; K₂CO₃, EtOH or DMF, heat or Boronic acid, Pd(dppf)CI₂-CH₂CI₂; Na₂CO₃, dioxane, 80°C

Scheme 3

Synthesis of 2,5-disubstituted thiophene analogs with the HBD group in the amide substituent ^{a a} Reaction conditions: (a) RNH₂, DIPEA, TBTU, DMF (b) Boronic acid, Pd(PPh₃)₄; K₂CO₃, DMF, heat