Nature | Vol 578 | 13 February 2020 | 307BD2 of BRD4, respectively). Continued optimization of BD2 selectivity,
metabolic stability and physical properties afforded a tertiary alcohol
on the central phenyl ring in place of the ethyl sulfonamide of ABBV-
075, substantially affecting the activity against BD1 of BRD4 (520 nM
for ABBV-744). Addition of a fluorine atom to the phenyl ether resulted
in improved pharmacokinetic properties, leading to the discovery of
ABBV-744 (Fig. 1a).
ABBV-744 potently inhibited the BD2 domain of BET family proteins
with more than 290× selectivity relative to the BD1 domains of BRD2,
BRD3 and BRD4, and more than 95× selectivity compared with BD1
of BRDt using TR-FRET, displayed Kd values of 3,300 nM and 2.1 nM in
surface plasmon resonance experiments and half-maximum inhibi-
tory concentrations (IC 50 ) of 20,700 nM and 27.5 nM using NanoBRET
assays for BD1 and BD2 of BRD4, respectively (Extended Data Fig. 1a,
b). ABBV-744 also lacked significant activity against 75 kinases and
22 bromodomain-containing proteins that represent diverse branches
of the kinome and bromodome (Extended Data Fig. 1c and Supplemen-
tary Tables 1, 2). ABBV-744 is primarily metabolized by CYP3A4 and
shows oral bioavailability, enabling in vivo efficacy and tolerability
studies (Extended Data Fig. 1d, e).
The crystal structures of ABBV-744 complexed with both the BD2 and
BD1 of BRD2 established the binding mode of ABBV-744 that underlies
its BD2 selectivity (Fig. 1b–d and Extended Data Table 1). ABBV-744
maintains all of the important interactions found for canonical DbBi^14 –^16 ,
including binding of the pyrrolopyridone with the conserved Asn156
residue, placement of the N-methyl moiety in the amphipathic water
pocket, and positioning of an aryl ring in the WPF shelf in both BD2 and
BD1 (Fig. 1b, c). The ethyl amide moiety of ABBV-744 exploits the Asp
(BD1) and His433 (BD2) divergence conserved across all bromodomain
BET family members by burying the amide in a channel formed by the
His433, Tyr386 and Pro430 residues of BD2 (Fig. 1b), a binding interac-
tion that is not available in BD1 (Fig. 1c). The 2,6-dimethylphenyl ether
moiety of ABBV-744 targets the subtle size distinction of the Ile162
(BD1) and Val435 (BD2) sequence differences. Thus, incorporation of a
dimethylphenyl ether moiety forces an aryl methyl group to be buried
in the rigid base of the WPF shelf. The smaller BD2 Val435 residue can
accommodate this added methyl group interaction without disrup-
tion of binding, and therefore binding potency is maintained. For the
BD1 protein, however, interaction of this aryl methyl group with the
larger Ile162 residue forces the inhibitor to shift slightly away from
the Ile moiety, causing a subtle change in the placement of both the
aryl group and the hydroxy group of the tertiary alcohol, leading to a
less-optimal binding interaction (overlay in Fig. 1d) and a decrease in
the potency of ABBV-744 with BD1.
We tested ABBV-744 in 59 cancer cell lines that are sensitive to DbBi^17 –^22
and found that ABBV-744 retained robust antiproliferative activity
(IC 50 < 100 nM) mostly—but not exclusively—in acute myeloid leukaemia
cells and a subset of prostate cancer cells that expressed the full-length
AR, but not those expressing AR-V7 or that were AR-negative (Fig. 2a,
Extended Data Table 2 and Supplementary Fig. 1). Similar to ABBV-
075 and the AR antagonist enzalutamide, ABBV-744 induced cell cycle
arrest in G1 followed by senescence in LNCaP cells (Fig. 2b). Narrow
antiproliferative activity was also observed for a structurally distinct
compound, A-083, across 240 cancer cell lines (Extended Data Fig. 2a–c
and Supplementary Table 3). BD2 inhibitor compounds 74, 75 and RVX-
20811 ,^12 displayed a similar albeit weaker antiproliferative trend, prob-
ably owing to their moderate selectivity and weaker binding affinity
(Extended Data Fig. 2d). Relative to ABBV-075, ABBV-744 demonstrated
limited potency in viability assays of megakaryocyte colony forming
units (Mk-CFU) in mice and IEC-6 cells, which are potential surrogate
assays for platelet production and proliferation of normal intestinal
epithelium, respectively^23 (Extended Data Fig. 2e).
In ABBV-744-sensitive LNCaP cells, ABBV-744 elicited far fewer gene
expression changes than ABBV-075 at doses at which BD2 of BRD4 was
similarly inhibited or the reported doses of the DbBi JQ1 and iBET^20 ,^24
(Fig. 2c and Extended Data Fig. 3a). For example, at a BD2-selective
concentration (48 nM), ABBV-744 downregulated ACPP (also known
as ACP3) and MYC but did not affect the ABBV-075-responsive genes
HEXIM1, SPDEF and ZG16B (Fig. 2d and Extended Data Fig. 3b). The
BD2-dependent genes KLK2 and MYC were also partially inhibited by
a potent and selective BD1 inhibitor described in the patent literature
(BD1i)^13 , suggesting that these BD2-dependent genes were in part also
dependent on BD1, and combined blockade of both domains mim-
icked the activity of ABBV-075 (Extended Data Fig. 3c). When used at a
high concentration (6 μM), ABBV-744 probably engaged both BD1 and
BD2, thus recapitulating the activities of ABBV-075 against all genes
(Fig. 2d). Notably, this small set of 241 ABBV-744-regulated genes
is highly enriched in dihydrotestosterone (DHT)-responsive genes
(Fig. 2e and Supplementary Table 4). Gene set enrichment analysis
also revealed common regulation of AR, MYC and E2F1 hallmarks by
ABBV-744, enzalutamide and ABBV-075, similar to reports for JQ1 and
iBET^20 ,^24 (Extended Data Fig. 3d). Although both ABBV-744 and ABBV-075adIle162cAsp104Asn156Ile162 Asp160
ABBV-744
BRD2 BD1 2.03.2
2.9
2.82.8bBRD2 BD2 2.3 ÅABBV-744 WPFshelfAsn429Val435 His433Asp377Water
pocket3.2
2.9
2.92.9O
SNHO O
F FNHNOOSNHNOO ONH
O
OS F FNHNOO ONH
O
OFNHNO
NHOHOABBV-075 1 2 ABBV-744Fig. 1 | ABBV-744 is a potent and highly selective inhibitor of the BD2 domain
of BET family proteins. a, Chemical structure of indicated compounds.
b, Co-crystal structure of ABBV-744 (pink) in complex with BD2 of BRD2.
c, Co-crystal structure of ABBV-744 (blue) in complex with BD1 of BRD2.
d, Overlay of the co-crystal structure of ABBV-744 in complex with BD2 of BRD2
(pink) and with BD1 of BRD2 (blue), displayed on the BRD2 BD1 protein (green).