Potent and selective bivalent inhibitors of BET bromodomains.

Waring MJ, Chen H, Rabow AA, Walker G, Bobby R, Boiko S, Bradbury RH, Callis R, Clark E, Dale I, Daniels DL, Dulak A, Flavell L, Holdgate G, Jowitt TA, Kikhney A, McAlister M, Méndez J, Ogg D, Patel J, Petteruti P, Robb GR, Robers MB, Saif S, Stratton N, Svergun DI, Wang W, Whittaker D, Wilson DM, Yao Y, Nat Chem Biol 12(12):1097-1104 (2016) Europe PMC

SASDBA6 – Compound 1:BRD4 (2:1)

Bromodomain-containing protein 4
MWI(0) 38 kDa
MWexpected 56 kDa
log I(s) 4.38×101 4.38×100 4.38×10-1 4.38×10-2
Bromodomain-containing protein 4 small angle scattering data  s, nm-1
ln I(s)
Bromodomain-containing protein 4 Guinier plot ln 4.38×101 Rg: 6.3 nm 0 (6.3 nm)-2 s2
(sRg)2I(s)/I(0)
Bromodomain-containing protein 4 Kratky plot 1.104 0 √3 sRg
p(r)
Bromodomain-containing protein 4 pair distance distribution function Rg: 7 nm 0 Dmax: 24.5 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Bromodomain-containing protein 4 DAMMIF model
log I(s)
 s, nm-1
Bromodomain-containing protein 4 EOM/RANCH model
Synchrotron SAXS data from solutions of Compound 1:BRD4 (2:1) in 20mM Hepes, 100mM NaCl, 1mM Tris(2-carboxyethyl)phosphine hydrochloride, pH 7.4 were collected on the BM29 beam line at the ESRF storage ring (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.9 m and at a wavelength of λ = 0.0992 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). Solute concentrations ranging between 1 and 10 mg/ml were measured . 50 successive 0.200 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted. The low angle data collected at lower concentrations were extrapolated to infinite dilution and merged with the higher concentration data to yield the final composite scattering curve.

A pool of 10000 models comprising two rigid bromodomains (residues 42-168, PDB ID: 2oss and 349-458, PDB ID: 2yem) connected by a flexible linker, N-terminal tag and flexible N- and C-termini were generated by the program RANCH (EOM). The scattering from each model from the pool was calculated with the program CRYSOL. For the 100 models best fitting the experimental scattering the histograms of the distances between the centres of the two bromodomains were computed; the average distance was 15±1 nm, the average Rg was 7.4±0.5 nm.

Bromodomain-containing protein 4 (BRD4)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   55.8 kDa
 
UniProt   O60885 (1-477)
Sequence   FASTA
 
PDB ID   2OSS
 
PDB ID   2YEM