Journal of Medicinal Chemistry
Article
respectively. The final model contains residues 3−157 and 1−106,
109−157 for molecules A and B, respectively, with 424 solvent
molecules and no residues in disallowed regions of the Ramachandran
plot as assessed by Rampage.36 Data collection and refinement
statistics are summarized in Table S1. PDB ID 7KH8.
the co-crystal structure with 5d, details of the PK
SMILES molecular formula strings (CSV)
Accession Codes
AKT Phosphorylation in HepG2 Cells. Human HepG2 cells
(American Type Culture Collection [ATCC] catalogue #HB-8065)
were cultured in Eagle’s minimal essential medium (ATCC)
containing 10% fetal bovine serum (FBS), 100 U/mL penicillin,
and 100 μg/mL streptomycin. Cells were treated with DMSO or 500
nM 6g in serum starvation media (0.1% FBS) overnight, following
which cells were stimulated with 10 nM bovine insulin (Sigma) for 5
min at 37 °C in the presence of DMSO or 500 nM 6g. For detection
of AKT phosphorylation by western blotting, cells were lysed in 1×
cell lysis buffer (Cell Signaling Technology; CST) with 1 mM
phenylmethylsulfonyl fluoride (PMSF). Lysates were sonicated at 4
°C for 15 intervals of 10−15 s. Insoluble fractions were cleared by
centrifugation. Prior to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, protein concentrations of lysates were assessed using
the Pierce BCA Protein Assay Kit (Thermo Scientific). Detection of
AKT phosphorylation by western blotting was assessed using anti-
phospho-AKT (pAKT) Thr308 (#4056) and anti-panAKT (#4691)
antibodies (CST). Western blotting quantification was performed
using ImageJ. Photo editing was performed using Adobe Photoshop.
Diabetes Assessment in DIO Mice. Animal experiments were
conducted in accordance with the Animal Care and Use Committee-
approved protocol at the University of California, San Diego
(#S16098). B6 mice were purchased from Jackson Laboratory (JAX
#000664) and bred in-house. To generate DIO mice, male littermates
were fed HFD chow containing 60 kcal % fat (Research Diets) for 3
months starting at 1−2 months of age. DIO littermates weighing ≥35
g were assigned to treatment (HFD formulated with 0.03% w/w 6g)
or control (HFD alone) groups. After 2 weeks of treatment, mice
were fasted overnight for 13 h, and intraperitoneal glucose tolerance
test (IPGTT) was performed by administering 1 g glucose/kg body
weight by IP injection. Tail ends were snipped 1 h before glucose
injection. Blood glucose levels were obtained from a small drop of
blood from tail snip right before glucose injection and at the indicated
time points after glucose injection using a OneTouch glucometer. For
insulin levels, mice were fasted overnight for 13 h and blood was
collected from the facial vein. Plasma insulin levels were assessed
using the Ultra-Sensitive Mouse Insulin enzyme-linked immunosorb-
ent assay (ELISA) kit (Crystal Chem). To assess liver insulin
signaling, fasted mice were injected IP with 10 U insulin (Eli Lilly)/kg
body weight, and after 10 min mouse livers were harvested, flash-
frozen, and homogenized in 1× cell lysis buffer with 1 mM PMSF.
Homogenates were sonicated at 4 °C for 10 intervals of 30 s.
Insoluble fractions were cleared by centrifugation. IR tyrosine
phosphorylation was assessed using the PathScan phospho-IR (pIR)
β (Tyr1150/Tyr1151) Sandwich ELISA Kit (#7258, CST). AKT
Thr308 phosphorylation was assessed by western blotting as
described above.
The coordinates and diffraction data for the co-crystal
structure described in this study have been deposited to the
will release the atomic coordinates upon article publication.
AUTHOR INFORMATION
Corresponding Authors
■
Stephanie M. Stanford − Department of Medicine, University
of California, San Diego, La Jolla, California 92037, United
Nunzio Bottini − Department of Medicine, University of
California, San Diego, La Jolla, California 92037, United
States; Phone: 01-858-246-2398; Email: nbottini@
Anthony B. Pinkerton − Conrad Prebys Center for Chemical
Genomics, Sanford Burnham Prebys Medical Discovery
Institute, La Jolla, California 92037, United States;
Authors
Michael A. Diaz − Department of Medicine, University of
California, San Diego, La Jolla, California 92037, United
States
Robert J. Ardecky − Conrad Prebys Center for Chemical
Genomics, Sanford Burnham Prebys Medical Discovery
Institute, La Jolla, California 92037, United States
Jiwen Zou − Conrad Prebys Center for Chemical Genomics,
Sanford Burnham Prebys Medical Discovery Institute, La
Jolla, California 92037, United States
Tarmo Roosild − Conrad Prebys Center for Chemical
Genomics, Sanford Burnham Prebys Medical Discovery
Institute, La Jolla, California 92037, United States
Zachary J. Holmes − Department of Medicine, University of
California, San Diego, La Jolla, California 92037, United
States
Tiffany P. Nguyen − Department of Medicine, University of
California, San Diego, La Jolla, California 92037, United
States
Michael P. Hedrick − Conrad Prebys Center for Chemical
Genomics, Sanford Burnham Prebys Medical Discovery
Institute, La Jolla, California 92037, United States
Socorro Rodiles − Conrad Prebys Center for Chemical
Genomics, Sanford Burnham Prebys Medical Discovery
Institute, La Jolla, California 92037, United States
April Guan − Conrad Prebys Center for Chemical Genomics,
Sanford Burnham Prebys Medical Discovery Institute, La
Jolla, California 92037, United States
Stefan Grotegut − Conrad Prebys Center for Chemical
Genomics, Sanford Burnham Prebys Medical Discovery
Institute, La Jolla, California 92037, United States
Eugenio Santelli − Department of Medicine, University of
California, San Diego, La Jolla, California 92037, United
States
Statistical Analysis. All linear regressions, non-linear data fitting,
and statistical analyses were performed using GraphPad Prism
software. α values were determined by fitting of data to a mixed
model of inhibition. K′i values were determined by fitting of data to an
uncompetitive model of inhibition. The two-way analysis of variance
(ANOVA), unpaired t-test with Welch’s correction, and Mann−
Whitney test were performed where appropriate as reported in the
figure legends. A comparison was considered significant if p was less
than 0.05.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
Thomas D. Y. Chung − Conrad Prebys Center for Chemical
Genomics, Sanford Burnham Prebys Medical Discovery
Institute, La Jolla, California 92037, United States
Experimental procedures and spectroscopic data for
selected compounds, selectivity of 3 and 5d, details of
5651
J. Med. Chem. 2021, 64, 5645−5653