BAY-069, a Novel (Trifluoromethyl)pyrimidinedione-Based BCAT1/2 Inhibitor and Chemical Probe

The branched-chain amino acid transaminases (BCATs) are enzymes that catalyze the first reaction of catabolism of the essential branched-chain amino acids to branched-chain keto acids to form glutamate. They are known to play a key role in different cancer types. Here, we report a new structural class of BCAT1/2 inhibitors, (trifluoromethyl)pyrimidinediones, identified by a high-throughput screening campaign and subsequent optimization guided by a series of X-ray crystal structures. Our potent dual BCAT1/2 inhibitor BAY-069 displays high cellular activity and very good selectivity. Along with a negative control (BAY-771), BAY-069 was donated as a chemical probe to the Structural Genomics Consortium.


BCAT1 Protein Production, Crystallization, and Co-Complex Structure Determination
An expression construct comprising human full length BCAT1 (Uniprot entry P5467) with an N-terminal hexa-His tag and a HRV3C cleavage site was transformed into BL21(DE3) cells (8L) for protein expression. Cells were lyzed using a microfluidizer and the supernatant was purified via Nickel-NTA affinity chromatography.
The tag was cleaved using HRV3C Protease (Novagen #71493-3) and the protein further purified via ion exchange (Source Q) and size exclusion chromatography (Superdex200). The final buffer was 10 mM Tris-HCl pH 8.0, 100 mM NaCl, 3 mM DTT. The protein was concentrated to 17 mg/mL, flash-frozen in liquid nitrogen and stored at -80 °C. Data sets were collected at 100 K either at beamline 14.1 at the Helmholtz-Zentrum Berlin (wavelength λ=0.9184 Å) using a PILATUS detector or at beamline P11 at PETRA III at the Deutsches Elektronen-Synchrotron (wavelength 1.000 Å) (see Table S1). The crystals diffracted to a resolution of 1.6 -2.6 Å. Data were processed using the programs XDS 1 and XDSAPP. 2 They belonged either to space group P212121 with one BCAT1 dimer in the asymmetric unit or to space group P21 with two BCAT1 dimers in the asymmetric unit (see Table S1). The structures were solved using molecular replacement (program PHASER 3 from the CCP4 program suite 4 and PDB entry 2ABJ as a search model, refined using REFMAC5 5 and rebuilt using the program COOT. 6 For inhibitor parameterization, 3D models were generated using Discovery Studio (Dassault Systèmes BIOVIA) and parameter files were generated using software PRODRG. 7 The final data collection and refinement statistics are summarized in Table S1.

Structural Biology: Supplementary Results
We determined the co-crystal structure of BCAT1 in complex with Compound A from a BCAT2 inhibitor series from GSK 8 ( Figure 1). The structure confirms the binding mode which was published for a very closely related inhibitor bound to BCAT2 (described as compound 66 by Bertrand et al., 8 PDB accession code 5BWX). A superimposition of the BCAT1 cocrystal structures with BAY-069 and Compound A ( Figure S5) shows that the two inhibitors both insert a hydrophobic group into the substrate binding pocket near the PLP co-factor.
Otherwise, however, they occupy different parts of the BCAT1 active site. While the naphthalene bicycle of BAY-069 occupies a deep groove between Phe 49 and Tyr 193 , this groove is closed by an induced fit in the Xray with compound A. Compound A instead kinks off and places its central scaffold between Thr 260 and Tyr 193 while it inserts its outer dichloro-fluoro-phenyl ring in a pocket between Gln 244 and the Cys 335 -Cys 338 disulfide bond. This subpocket is, in turn, closed in the co-complex structure with BAY-069.

Computational Studies
All quantum mechanical simulation were carried out using the General Atomic and Molecular Electronic Structure System 9 (GAMESS-US).
All molecular sketching, 3D structure building, manipulation, and visualisation in preparation of the FMO calculations was carried out using the Molecular Operating Environment (MOE). 10 The calculation of interaction energies between each ligand and the protein target was carried out as described in; 11 the process can be summarised as follows. The protein is represented by the set of residues having close contacts with the ligand, as defined by MOE's 'near ligand' option. Non-hydrogen geometries are taken directly from available crystal structures; hydrogen atoms are added in using MOE. A quantum mechanical simulation is carried out of each residue in this set, and of the ligand, in isolation. These simulations produce energies and atomic charges (derived via the CHelpG scheme 12 ). A second set of quantum mechanical simulations is performed, of each pairwise system formed by the ligand and one of the residues set. The pairwise system in each of these simulations is embedded in the set of charges derived from all the neglected residues. For example, in a system consisting of a ligand molecule plus five closecontacted protein residues, five quantum mechanical calculations would be carried out: the ligand plus residue one, embedded in the atomic charges calculated for residues 2-5; then the ligand plus residue two, embedded in the atomic charges calculated for residues 1 and 3-5; and so on. The individual energies calculated for the isolated ligand and residues are then subtracted from the energies calculated for the pairwise system, to give an interaction energy for each pair. These are summed to give an overall interaction energy for the ligand with the protein. All quantum mechanical simulations for FMO were carried out at MP2 level of theory with a 6-31G* basis set.
Using this method, the binding energy of 34 was calculated to be 0.9 kcal mol -1 stronger than that of 12 (ΔE = -0.9 kcal mol -1 ).
For purposes of torsion angle scanning, an initial geometry was obtained by sketching and 3D building in MOE. QM simulations were then carried out at the HF level of theory using a 3-21G* basis set. The initial molecule was subjected to unconstrained quantum mechanical local energy minimisation / geometry optimisation in GAMESS-US. The indicated dihedral angle θ was then altered in steps of 10° up to a full rotation of 360°. At each step, constrained energy minimisation was carried out, with the dihedral θ fixed at the chosen value and the rest of the molecular geometry subject to minimisation. The energy barrier to rotation was calculated as the maximum molecular energy calculated for any step in this dihedral scan, minus the minimum molecular energy.

S 11
Supplementary Figure S4. Example biphenyl molecule with scanned dihedral angle indicated.

Supplementary Figure S6
Query setup for the Cavbase query using X-ray structure of BCAT1 in complex with compound 35. Within an envelope of 4Å radius around the cocrystallized ligand all pseudocentres (donor/acceptor/pi/aromatic/aliphatic) were picked.

Ethyl 4-chloro-4,4-difluoro-3-oxobutanoate (46)
To a solution of ethyl acetate (2.20 g, 25.2 mmol) in tetrahydrofuran (50 mL) was added lithium diisopropylamide (9.5 mL, 2 mol/L in THF) at -78 °C and the resulting mixture was stirred at this temperature for 30 min. Then ethyl 2-chloro-2,2-difluoroacetate (2.0 g, 12.6 mmol) was added to the above mixture at -78 °C and the resulting solution was stirred at RT for overnight under nitrogen atmosphere. Upon completion of the reaction, aq. ammonium chloride solution was added at 0 °C and the resulting mixture was extracted with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate and the solvent was removed in vacuo to give the title compound (3.00 g, 59% yield, 50% purity) as light red oil. LCMS (ESIneg):

4-Amino-5-bromo-2-(2-methylphenoxy)benzonitrile (52)
To a solution of 4-amino-2-(2-methylphenoxy)benzonitrile (55, 5.0 g, 19.6 mmol, 88% purity) in AcOH (100 mL) was added N-bromosuccinimide (3.50 g, 19.6 mmol) and the resulting mixture was stirred at room temperature for overnight. Upon completion of the reaction, the solvent was removed in vacuo and the residue was re-dissolved with water. The resulting mixture was extracted with ethyl acetate and the combined organic layer was dried over anhydrous sodium sulfate. The solvent was removed in vacuo and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 3: 1) to give 5.5 g (80% yield) of the product as light-yellow oil.
The reaction mixture was stirred for 16 h at reflux temperature. After completion of the reaction, the solvent

S 29
was evaporated under vacuum, and the thus obtained solid was acidified with 20% aq. HCl (up to 6.5-7 pH).
The solid was filtered off and washed with water (50 mL) and dried under vacuum to give the title compound (6.8 g) as an off-white solid. 1  All carbamates were synthesized following to the general procedure GP1 as in the example 65 below.

General Procedure 1: Formation of the carbamate
To an ice-cooled solution of the aniline (1.0 eq.) in pyridine (1.5 mL/mmol), was added dropwise the commercially available either ethyl chloroformiate (CAS: 541-41-3, 1.2 eq.) or methyl carbonochloridoate (CAS: 79-22-1). After complete addition, the corresponding suspension was stirred at 0 °C for 2 h, or until the reaction was complete. Upon reaction completion, the reaction mixture was diluted with an aq. solution of 1 M HCl (10 mL/mmol). The resulting precipitate (carbamate) was collected by filtration and washed with water and dried under vacuum overnight and used without any further purification step in the next reaction step.