2017, 17, 271-279.
Thomas, M. and Owen, C. Curr. Opin. Pharmacol. 2008 8, 267-
274.
Berndt, A.; Miller, S.; Williams, O.; Le, D. D.; Houseman, B. T.;
Pacold, J. I.; Gorrec, F.; Hon, W.-C.; Ren, P.; Liu, Y.; Rommel,
C.; Gaillard, P.; Ruckle, T.; Schwarz, M. K.; Shokat, K. M.; Shaw,
J. P.; Williams, R. L. Nat. Chem. Biol. 2010, 6, 117-124.
Shah, A. and Mangaonkar, A. Ann. Pharmacol. 2015, 49, 1162-
1170.
and incubated at ambient temperature for 1 h before quenching
with EDTA. The detection solution (streptavidin-APC with Eu-
labeled anti-GST plus GST-tagged PH-domain) was added and
incubated in the dark for 1 h, followed by measurement of the
HTRF signal with an Envision plate reader (330 nm excitation and
dual emission detection at 620 nm (Eu) and 665 nm (APC)). The
individual kinases were purchased from Upstate (PI3Kα 14-602,
PI3Kβ 14-603, PI3Kγ 14-558 and PI3Kδ 14-604). The assay
format was the same for all four isoforms, the differences lie in the
concentration of enzyme and ATP used. The PI3Kα, PI3Kβ,
PI3Kδ, PI3Kδ assays were run with 0.5, 1, 0.3, 5 nM of enzyme,
respectively. The ATP concentration was 100 μM in the PI3Kα,
PI3Kβ, and PI3Kδ assays and 50 μM in the PI3Kγ assay.
20. High-Throughput (HT) HPLC log D (pH 7.0) determination: The
chromatographic system consists of an Agilent 1200 HPLC
system composed of a G1379B degasser, G1312B binary pump,
G1367C well-plate autosampler, G1316B thermostatted column
compartment, G1315C diode-array UV-vis detector, and
ChemStation software, all from Agilent Technologies, USA. The
separations are carried out on a Supelco Ascentis Express C18, 30
mm x 3.0 mm I.D., 2.7 µm. The mobile phase consists of
phosphate buffered saline at pH 7 (mobile phase A) and
acetonitrile (mobile phase B). The column oven temperature is set
to 30°C. The HPLC analysis consists of a gradient. The injection
volume is 5 µL and the spectrophotometric detection is set to 215,
238 and 254 nm. A 10 mM DMSO stock solution of API is
delivered for analysis. A 100 µM standard solution of API is
generated by diluting 2.5 µL of the 10 mM stock solution with
247.5 µL of diluent (10% DMSO/10% MeCN/80% MeOH, v/v/v).
The chromatographic system is calibrated with a set of standards
with published shake-flask log D (pH 7.0) values. Linear
regression is used to determine the calibration line relating the
retention time to log D for the calibration standards. This line is
then used to determine the HT HPLC log D (pH 7.0) value of API
from the measured retention time by the HPLC/DAD analysis of
the 100 µM standard solution of API.
7.
8.
9.
10. Miller, B. W.; Przepiorka, D.; Claro, R. A.; Lee, K.; Nie,
L.; Simpson, N.; Gudi. R.; Saber, H.; Shord, S.; Bullock,
J.; Marathe, D.; Mehrotra, N.; Hsieh, L.S.; Ghosh, D.; Brown,
J.; Kane, R. C.; Justice, R.; Kaminskas, E.; Farrell, A. T.; Pazdur,
R. Clin. Cancer Res. 2015, 21, 1525.
11. Down, K.; Amour, A.; Baldwin, I. R.; Cooper, A. W. J.; Deakin,
A. M.; Felton, L. M.; Guntrip, S. B.; Hardy, C.; Harrison, Z. A.;
Jones, K. I.; Jones, P.; Keeling, S. E.; Le, J.; Livia, S.; Lucas, F.;
Lunniss, C. J.; Parr, N. J.; Robinson, E.; Rowland, P.; Smith, S.;
Thomas, D. A.; Vitulli, G.; Washio, Y.; Hamblin, J. N. J. Med.
Chem. 2015, 58, 7381-7399.
12. Amour, A.; Barton, N.; Cooper, A. W. J.; Inglis, G.; Jamieson, C.;
Luscombe, C. N.; Morrell, J.; Peace, S.; Perez, D.; Rowland, P.;
Tame, C.; Uddin, S.; Vitulli, G.; Wellaway, N. J. Med. Chem.
2016, 59, 7239-7251.
13. 1) National Institutes of Health. Identifier: NCT03345407 in
ClinicalTrials.gov.
2) Cahn, A.; Hamblin, J. N.; Begg, M.; Wilson, R.; Dunsire, L.;
Sriskantharajah, S.; Montembault, M.; Leemereise, C. N.;
Galinanes-Garcia, L.; Watz, H.; Kirsten, A. M.; Fuhr, R.; Hessel, E.
M. Pulmonary Pharmacology & Therapeutics 2017, 46, 69-77
14. Burris, H. A. III; Flinn, I. W.; Patel, M. R.; Fenske, T. S.; Deng,
C.; Brander, D. M.; Gutierrez, M.; Essell, J. H.; Kuhn, J. G.;
Miskin, H. P.; Sportelli, P.; Weiss, M. S.; Vakkalanka, S.; Savona,
M. R.; O’Connor, O. A. The Lancet Oncology 2018, 19, 486-496.
15. Robak, P.; Robak, T. Expert Opinion on Investigational Drugs
2017, 26(11), 1249-1265.
16. National Institutes of Health. Identifiers: NCT03776864,
NCT03919175, NCT03364231, NCT03801525, NCT03828448,
NCT02535286, NCT03379051, NCT03269669 in
21. Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.;
Greenwood, J. R.; Halgren,T. A.; Sanschagrin, P. C.; Mainz, D.
T. J. Med. Chem.,2006, 49, 6177–6196.
ClinicalTrials.gov
22. Hawkins, P.C.D.; Skillman, A.G. and Nicholls, A. J. Med.
Chem., 2007, 50, 74–82.
8, 975. 2) National Institutes of Health. Identifiers:
23. Human Whole Blood CysLT Assay: Human blood was collected
from healthy volunteers free of medication for 7 days into
vacutainers containing Na-heparin. Basophils were primed for
activation with recombinant human IL-3 (5 ng/mL) (PeproTech)
for 15 minutes at 37 °C in 5% CO2 incubator. 27.5 µL of primed
whole blood was plated with 30 nL compound in 10 point dose
response curves and incubated for 30 minutes in 5% CO2
incubator. 2.5 µL of 600 ng/mL anti-human IgE antibody diluted
in PBS-0.1% BSA (Bethyl Laboratories) was added to primed
blood and incubated for 20 minutes at 37 °C in 5% CO2 incubator
to activate basophils. Samples were then centrifuged at 1,000 g for
5 minutes at 4 °C to separate cells from serum. Maxisorp assay
plates (Nunc) were pre-coated with 100 µL of 5 µg/mL of mouse
IgG (H+L)F(ab)' (KPL) overnight at room temperature and
blocked with 110 µl of 1 X EIA buffer S33 (Cayman) at 4 °C for
24 hours. After aspirating blocking buffer, 25 µL each of 1:10
diluted serum (in 1X EIA buffer, Cayman), 1:1,000 diluted LTC4
alkaline phosphatase conjugate (AssayDesign) and 1:300,000
diluted anti-CysLT (AssayDesign) were added and incubated for
18 hours at room temperature on a plate shaker. Plate was then
washed 5 times with 100 µL washing buffer -0.05% Tween 20
(Cayman) before addition of 60 µL of alkaline phosphatase
substrate pNpp (AssayDesign). Following 2 hour incubation for
color development at room temperature, 15 µL of stop solution
(AssayDesign) was added and plate was read on Envision
(PerkinElmer) at 405 nM absorbance. A 1000 pg/mL to 11.56
pg/mL 2:3 fold serial diluted CysLT standard curve from 4
parameter logistic fitting was generated on each assay plate. The
concentration of CysLT in serum samples was calculated from the
standard curve. Maximum and minimum CysLT release in blood
sample was determined either in the presence or absence of anti-
IgE stimulation and was used to calculate % of inhibition by
compound treatment. Inhibition of CysLT release from IgE
activated basophils in human whole blood was evaluated by pre-
incubation of compounds with IL-3 primed blood for 30 min at 37
°C in a 5% CO2 incubator before stimulation. IC50 was determined
following 10-dose titration and four parameter logistic curve
fitting.
NCT02435173, NCT02859727, NCT02775916 in
ClinicalTrials.gov
18. (a) Sutherlin, D. P.; Baker, S.; Bisconte, A.; Blaney, P. M.;
Brown, A.; Chan, B. K.; Chantry, D.; Castanedo, G.; DePledge,
P.; Goldsmith, P.; Goldstein, D. M.; Hancox, T.; Kaur, J.;
Knowles, D.; Kondru, R.; Lenick, J.; Lucas, M. C.; Lewis, C.;
Murray, J.; Nadin, A. J.; Nonomiya, J.; Pang, J.; Pegg, N.; Price,
S.; Reif, K.; Safina, B. S.; Salphati, L.; Staben, S.; Seward, E. W.;
Shuttleworth, S.; Sohal, S.; Sweeney, Z. K.; Ultsch, M.;
Waszkowycz, B.; Wei, B. Bioorg. Med. Chem. Lett. 2012, 22,
4296-4302. (b) Safina, B. S.; Baker, S.; Baumgardner, M.;
Blaney, P. M.; Chan, B. K.; Chen, Y. H.; Cartwright, M. W.;
Castanedo, G.; Chabot, C.; Cheguillaume, A. J.; Goldsmith, P.;
Goldstein, D. M.; Goyal, B.; Hancox, T.; Handa, R. K.; Iyer, P.
S.; Kaur, J.; Kondru, R.; Kenny, J. R.; Krintel, S. L.; Li, J.;
Lesnick, J.; Lucas, M. C.; Lewis, C.; Mukadam, S.; Murray, J.;
Nadin, A. J.; Nonomiya, J.; Padilla, F.; Palmer, W. S.; Pang, J.;
Pegg, N.; Price, S.; Reif, K.; Salphati, L.; Savy, P. A.; Seward,
E. M.; Shuttleworth, S.; Sohal, S.; Sweeney, Z. K.; Tay, S.;
Tivitmahaisoon, P.; Waszkowycz, B.; Wei, B.; Yue, Q.; Zhang,
C.; Sutherlin, D. P. J Med Chem 2012, 55, 5887-900.
19. PI3K Biochemical HTRF (Homogeneous Time-Resolved
Fluorescence) Assay. PI3K family biochemical potencies in the
phosphorylation of PIP2 (phosphatidylinositol (4,5)-bisphosphate)
to PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) were
measured using an HTRF assay. PI3K biochemical assays were
optimized from an Upstate (Millipore) HTRF kit. Briefly,
compounds were serially diluted (3-fold in 100% DMSO) for a
10-concentration dose response. PI3K reaction buffer was
prepared by dilution of stock with DI water, then treated with
DTT, PIP2 and Biotin-PIP3 at a final concentration of 5 μM, 5 μM
and 25 nM, respectively. Enzyme and the compounds were added
at ambient temperature for a 15 min preincubation. Reactions
were initiated by addition of substrate solution (PIP2 and ATP)