Chemical Catalysis Guides Structural Identification for the Major In Vivo Metabolite of the BET Inhibitor JQ1

The bromodomain inhibitor (+)-JQ1 is a highly validated chemical probe; however, it exhibits poor in vivo pharmacokinetics. To guide efforts toward improving its pharmacological properties, we identified the (+)-JQ1 primary metabolite using chemical catalysis methods. Treatment of (+)-JQ1 with tetrabutylammonium decatungstate under photochemical conditions resulted in selective formation of an aldehyde at the 2-position of the thiophene ring [(+)-JQ1-CHO], which was further reduced to the 2-hydroxymethyl analog [(+)-JQ1-OH]. Comparative LC/MS analysis of (+)-JQ1-OH to the product obtained from liver microsomes suggested (+)-JQ1-OH as the major metabolite of (+)-JQ1. The 2-thienyl position was then substituted to generate a trideuterated (−CD3, (+)-JQ1-D) analog having half-lives that were 1.8- and 2.8-fold longer in mouse and human liver microsomes, respectively. This result unambiguously confirmed (+)-JQ1-OH as the major metabolite of (+)-JQ1. These studies demonstrate an efficient process for studying drug metabolism and identifying the metabolic soft spots of bioactive compounds.


Detailed Experimental Procedures
Catalyst preparation: The procedure was carried out on the same scale as described in the literature method 19 Tetrabutylammonium bromide (2.4 g) and sodium tungstate dihydrate (5.0 g) were each separately dissolved in 150 mL of deionized water in an Erlenmeyer flask and kept at 90 °C with vigorous stirring.Concentrated hydrochloric acid was added dropwise to both solutions in order to adjust the pH to 2, at which point the tungstate solution turned slightly greenish yellow in color.The two solutions were then mixed and maintained at 90 °C for 30 min with stirring.A white suspension resulted.The reaction was cooled and the precipitated TBADT was filtered on a Buchner funnel.The solid was washed with water and then dried under high vacuum for 3 hours.Crude TBADT was suspended in dichloromethane (20 mL of solvent per gram of solid) and stirred for 2 hours.Pure TBADT was separated from the yellow supernatant solution by filtration on a Buchner funnel and drying under vacuum.

ALPHA Assay Conditions
The ALPHA Assay was performed as published by Roberts and Bradner 34 with a 40 nM protein concentration, 20 nM Biotin-JQ1 concentration and with nickel acceptor beads and streptavidin donor beads at 25 µg/ml each.
(+)-JQ1 and modified compounds (0.2 μM) were incubated in the mouse or human liver microsomes (0.1 mg protein/mL) at 37 °C.Given the slower rate of metabolism for JQ1-D, we used 0.5 mg protein/ mL.The samples are collected at specific time-points (0, 5, 10, 20, 40, and 60 min.The reactions are terminated by adding equivalent volume of ice-cold MeOH and vortexed.The reaction mixtures are centrifuged at rcf 15,000 for 15 min.Five μL of the supernatant will be analyzed by UHPLC-Q Exactive Orbitrap MS (Thermo Fisher Scientific, San Jose, CA) equipped with 50 mm × 4.6 mm column (XDB C-18, Agilent Technologies, Santa Clara, CA).The column temperature is maintained at 40 °C.The flow rate of is at 0.3 mL/min with a 30% mobile phase (acetonitrile containing 0.1% formic acid).Q Exactive MS is operated in positive or negative mode with electrospray ionization.Ultrapure nitrogen is applied as the sheath (45 arbitrary unit), auxiliary (10 arbitrary unit), sweep (1.0 arbitrary unit) and the collision gas.The capillary gas temperature is set at 275 °C and the capillary voltage was set at 3.7 kV.MS data were acquired from 80 to 1200 Da in profile mode.

SPR binding analysis of (+) JQ1-F analogs
The Surface Plasmon Resonance (SPR) experiment was conducted using a Pioneer FE SPR System (Sartorius) equipped with a SPR HisCap Sensor Chip.The instrument was calibrated and validated prior to the experiment according to the manufacturer's instructions.The chip surface was activated with a mixture of N-hydroxysuccinimide (NHS), 50 mM, and N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC), 200 mM, at a 20 L/min flow rate for 5. minutes.Covalent attachment of his-BRDT to channel 1 was achieved by injecting 150 L of protein at 10 mg/mL at 10 mL/min.Channel 2 of the chip was used as a blank reference and unreacted functional groups of both channels were blocked using ethanolamine.
SPR running buffer consisted of 100 mM Tris-HCl, 200 mM NaCl, and 0.05% Tween-20.The JQ1 analog samples were prepared in a solution that closely mimics the buffer conditions and injected at 40L/min using the OneStep injection system at 0.5, 5, and 50 M concentrations.The real-time binding response was recorded using the instrument software and sensorgrams were analyzed using the QDAT software.The baseline subtraction, referencing, and double-referencing techniques were applied to obtain specific binding responses.Kinetic parameters were calculated by fitting sensograms to OneStep binding models for determining the dissociation rate constant (k d ) for the highest concentration and constraining the lower concentrations to the determined value.All data was locally fitted to a simple 1:1 interaction model.