Acyl Glucuronide Metabolites of 6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic Acid (PF-06409577) and Related Indole-3-carboxylic Acid Derivatives are Direct Activators of Adenosine Monophosphate-Activated Protein Kinase (AMPK)

Abstract

Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) α1β1γ1 isoform have culminated in the identification of PF-06409577 (1), PF-06885249 (2), and PF-06679142 (3) as potential clinical candidates. Compounds 1-3 are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of 1-3. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of β1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Cocrystallization of the AMPK α1β1γ1 isoform with 1-3 and M1-M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates.

10-Gingerol, a natural AMPK agonist, suppresses neointimal hyperplasia and inhibits vascular smooth muscle cell proliferation

“…Interestingly, these amino acid residues are considered the binding domains of AMPK agonists. 44,45 When 10-G binds to AMPKα and β, it increases the interaction etween…”

6-Chloro-5-[4-(1-Hydroxycyclobutyl)Phenyl]-1H-Indole-3-Carboxylic Acid is a Highly Selective Substrate for Glucuronidation by UGT1A1, Relative toβ-Estradiol

“…PF-06409577 is commercially available from Sigma Aldrich (St. Louis, MO). The biosynthetic methodology (scale-up of UDPGA-supplemented HLM incubations of PF-06409577) for preparation of the acyl glucuronide M1 has been previously described (Ryder et al, 2018). Examination of the degradation kinetics of the biochemically isolated M1 in deuteriated phosphate buffer (pH ;7.4) at 37°C using a NMR method developed to monitor the disappearance of the anomeric proton (d;5.7-5.8 ppm) in b-O-1-acyl glucuronides revealed that M1 was inert toward hydrolysis or acyl migration (half-life .…”

“…Examination of the degradation kinetics of the biochemically isolated M1 in deuteriated phosphate buffer (pH ;7.4) at 37°C using a NMR method developed to monitor the disappearance of the anomeric proton (d;5.7-5.8 ppm) in b-O-1-acyl glucuronides revealed that M1 was inert toward hydrolysis or acyl migration (half-life . 21 hours) (Ryder et al, 2018). As a general precaution, solid material corresponding to parent (PF-06409577) and acyl glucuronide (M1) was stored in sealed vials under argon.…”

“…PF-06409577 is resistant toward oxidative modifications by cytochrome P450 enzymes in liver microsomes and/or hepatocytes from preclinical species and human. Recently, we described the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugate of PF-06409577-(2S,3S,4S,5R,6S)-6-((6-chloro-5-(4-(1-hydroxycyclobutyl)phenyl)-1H-indole-3-carbonyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (M1)-obtained from incubations of the parent compound in uridine 59-diphosphoglucuronic acid (UDPGA)-supplemented human liver microsomes (HLM) (Ryder et al, 2018). A combination of chemical derivatization and spectral characterization studies on purified M1 indicated that the metabolite was derived from the glucuronidation of the carboxylic acid moiety in PF-06409577 ( Fig.…”

“…revealed that M1 was an active metabolite, and retained selective activation of the human b1-containing AMPK isoforms. Cocrystallization of the human AMPK a1b1g1 isoform with PF-06409577 and M1 provided molecular insights into the structural basis for AMPK activation by the phase 2 conjugation product (Ryder et al, 2018).…”