Selectivity screening data of PFI-1 using temperature shift assays. Screened targets are highlighted in bold. Temperature shifts are indicated by red filled circles with increasing radii for higher Tm values as indicated in the figure

Selectivity
Bromodomains
Target	Tm shift °C @ 10 µM
BRD2A 1st Bromodomain	4.6
BRD2 2nd Bromodomain	5.3
BRD3 1st  Bromodomain	5.2
BRD3 2nd Bromodomain	5.5
BRD4 1st Bromodomain	6.5
BRD4 2nd Bromodomain	3.8
BRDT 1st Bromodomain	2.1
CREBBP	2.7
BAZ2B	0.3
LOC93349	0.1
PB1~5	0.8
PCAF	0.8
Other proteins
Invitrogen 50 kinase panel	<20% inhibition @ 1 µM

BRD2 KD=0.123µM 
 

Off Target CREBBP KD=49.5µM
> 350 fold selective against BRDs outside BET

Materials and Methods

Differential Scanning Fluorimetry (DSF)

Thermal melting experiments were carried out using an Mx3005p Real Time PCR machine (Stratagene). Proteins were buffered in 10 mM HEPES pH 7.5, 500 mM NaCl and assayed in a 96-well plate at a final concentration of 2 µM in 20 µl volume. PFI-1 was added at a final concentration of 10 µM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000. Excitation and emission filters for the SYPRO-Orange dye were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3 °C per minute from 25 °C to 96 °C and fluorescence readings were taken at each interval. The temperature dependence of the fluorescence during the protein denaturation process was approximated by the equation where ?u G _(T) is the difference in unfolding free energy between the folded and unfolded state, R is the gas constant and y _F and y _U are the fluorescence intensity of the probe in the presence of completely folded and unfolded protein respectively. The baselines of the denatured and native states were approximated by a linear fit. The observed temperature shifts, ? T _m ^obs, were recorded as the difference between the transition midpoints of sample and reference wells containing protein without ligand in the same plate and determined by non-linear least squares fit.

Isothermal Titration Calorimetry

Experiments were carried out on a VP-ITC titration microcalorimeter from MicroCal ^TM, LLC (Northampton, MA) at 15 °C while stirring at 295 rpm, in ITC buffer (50 mM HEPES pH 7.4 at 25 °C, 150 mM NaCl). The injection syringe (250 µl) was loaded with a solution of the protein sample (300 µM protein, in ITC buffer). All titrations were conducted using an initial injection of 2 µl followed by 34 identical injections of 8 µl with a duration of 16 sec (per injection) and a spacing of 250 sec between injections. Heats of dilution were determined by independent titrations (protein into buffer) and were subtracted from the experimental data. Data analysis was carried out using the MicroCal ^TM Origin software supplied with the instrument to yield enthalpies of binding (?H) and binding constants (K _B). Thermodynamic parameters were calculated (? G = ? H - T? S = -R TlnK _B, where ? G, ? H and ? S are the changes in free energy, enthalpy and entropy of binding respectively). In all cases a single binding site model was employed.

CEREP assay

PFi-1 (10 µm) was screened against a panel of 15 ligand receptors, ion channels and transports using an established and widely utilized commercial assay platform (ExpresSProfile; CEREP, Paris, FRANCE); <50% inhibition was observed.

Alpha screen Assay

All reagents were diluted in 50 mM HEPES, 100 mM NaCl, 0.1 % BSA, pH 7.4 supplemented with 0.05 % CHAPS and allowed to equilibrate to room temperature prior to addition to plates. A 24-point 1:2 serial dilution of the ligands was prepared over the range of 1500 µM and 4 µl transferred to low-volume 384-well plates (ProxiPlateTM-384 Plus, PerkinElmer, USA), followed by 4 µl of HIS-tagged protein (BRD4(1), 50 nM, BRD4(2), 50 nM). Plates were sealed and incubated at room temperature for 30 minutes, before the addition of 4 µl of biotinylated peptide at equimolar concentration to the protein [peptide for BETs H4K5acK8acK12acK16ac, H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH. Plates were sealed and incubated for a further 30 minutes, before the addition of 4 µl of streptavidin-coated donor beads (25 µg/ml) and 4 µl nickel chelate acceptor beads (25 µg/ml) under low light conditions. Plates were foil-sealed to protect from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader (BMG Labtech, Germany) using an AlphaScreen 680 excitation/570 emission filter set. IC50 values were calculated in Prism 5 after normalization against corresponding DMSO controls and are given as the final concentration of compound in the 20 µl reaction volume.

Bio-Layer Interferometry

Kinetic measurements were done using OctetRed384 instrument (ForteBio Inc, CA, USA). Biotinylated protein was immobilized on Super Streptavidin Biosensors at 2µg/ml concentration. Association and dissociation measurements were done in 50 mM HEPES, 100 mM NaCl, pH 7.4 buffer supplemented with 0.01 % Tween. Experiments were performed at 25°C with association and dissociation times of 240 sec. Compounds were prepared as one in two dilutions starting from 128µM. Binding to the reference sensors (no protein attached) was subtracted before calculations. Binding constants were calculated using ForteBio Analysis software.

b

Fluorescence Recovery After Photobleaching (FRAP)

FRAP studies were performed on U2OS cells transfected (Fugene HD; Roche) with mammalian overexpression constructs encoding GFP fused to the N-terminus of full length BRD4. Compounds were added as indicated 16 h post transfection.  The FRAP and imaging system consisted of a Zeiss LSM 710 scanhead (Zeiss GmbH, Jena, Germany) coupled to an inverted Zeiss Axio Observer.Z1 microscope equipped with a high-numerical-aperture (N. A. 1.3) 40x oil immersion objective (Zeiss GmbH, Jena, Germany). Samples were placed in an incubator chamber capable of maintaining temperature (37 °C) and humidity. FRAP and GFP fluorescence imaging were carried out with an argon-ion laser (488nm) and with a piezomultiplier tube (PMT) detector set to detect fluorescence between 500-550nm. Once an initial scan had been taken, a region of interest corresponding to approximately 50% of the entire GFP positive nucleus was empirically selected for bleaching. A time lapse series was then taken to record GFP recovery using 1% of the power used for bleaching. The image datasets and fluorescence recovery data were exported from ZEN 2009, the microscope control software, into Microsoft Excel to determine the average half-time for full recovery for 10-20 cells per treatment point. The average intensity at each imaging time was normalized to an independent region of interest before bleaching.

PFI-1 shows considerably accelerated FRAP recovery at 1 µM

Fluorescent Recovery After Photo-bleaching (FRAP) experiments using full-length GFP-BRD4. Nuclei of U2OS cells transfected with GFP-BRD4 were treated with the DMSO control (upper panel),  1 mM or 5 mM  PFI-1 (middle and lower panel). Photographs were taken at 0, 10, 20 and 30 s after photo-bleaching of half the nucleus

Half recovery time measured when  treating cells with PFI-1 (1 μM, 5 μM) show faster recovery than the DMSO control. (a) Half times of fluorescence recovery of DMSO and PFI-1 (1, 5 μM) treated cells. (b) Time dependence of fluorescent recovery in the bleached area for DMSO  (black  box and line) and PFI-1 1 μM and 5 μM (red box and line, blue box and line, respectively)  treated cells.

Antiproliferative effects of PFI-1 on MLL tumour cells

Differentiation of mll-AF9 leukemic blasts induced by PFI-1. Shown are murine mll-AF9 transformed blast cells in the presence of DMSO (left) and 5 mM PFI-1 after 48h exposure.

Dose-response of the anti-proliferative effects of PFI-1 on leukaemic cell lines

Identification of a chemical probe for BET bromodomain inhibition through optimization of a fragment-derived hit
Paul Fish, Panagis Filippakopoulos, Gerwyn Bish, Paul Brennan, Mark Edward Bunnage, Andrew Cook, Oleg Fedorov, Brian S Gerstenberger, Hannah Jones, Stefan Knapp, Brian Marsden, Karl Nocka, Dafydd R Owen, Martin Philpott, Sarah Picaud, Michael Primiano, Michael Ralph, Nunzio Sciammetta, and John Trzupek
J. Med. Chem., 2012, 55 (22), 9831–9837

PFI-1 - A highly Selective Protein Interaction Inhibitor Targeting BET bromodomains
Sarah Picaud, David Da Costa, Angeliki Thanasopoulou, Panagis Filippakopoulos, Paul V. Fish, Martin Philpott, Oleg Fedorov, Paul Brennan, Mark E. Bunnage, Dafydd R. Owen, James E. Bradner, Philippe Taniere, Brendan O’Sullivan, Susanne Müller-Knapp, Juerg Schwaller, Tatjana Stankovic, Stefan Knapp
Cancer Res, 2013, 73, 3336-3346

(a)

(b)

(a) PFI-1 bound to BRD4 ε-N-acetylated lysine (Kac) binding pocket; (b) PFI-1 forms polar contacts with the conserved asparagine and interacts with many hydrophobic and aromatic residues in the BET Kac binding pocket. PDB ID: 4E96