Discovery of I-BRD9, a Selective Cell Active Chemical Probe for Bromodomain Containing Protein 9 Inhibition

Abstract

Acetylation of histone lysine residues is one of the most well-studied post-translational modifications of chromatin, selectively recognized by bromodomain "reader" modules. Inhibitors of the bromodomain and extra terminal domain (BET) family of bromodomains have shown profound anticancer and anti-inflammatory properties, generating much interest in targeting other bromodomain-containing proteins for disease treatment. Herein, we report the discovery of I-BRD9, the first selective cellular chemical probe for bromodomain-containing protein 9 (BRD9). I-BRD9 was identified through structure-based design, leading to greater than 700-fold selectivity over the BET family and 200-fold over the highly homologous bromodomain-containing protein 7 (BRD7). I-BRD9 was used to identify genes regulated by BRD9 in Kasumi-1 cells involved in oncology and immune response pathways and to the best of our knowledge, represents the first selective tool compound available to elucidate the cellular phenotype of BRD9 bromodomain inhibition.

Figure 1

Chemical structures for selected bromodomain inhibitors.

Figure 2

X-ray crystal structures of (A) BRD4 BD1 bromodomain (orange) with I-BET762 (1) (cyan); (B) BRD9 bromodomain (blue); (C) BRD4 BD1 surface, and (D) BRD9 surface. In each, the conserved asparagine residue is colored green (BRD4 Asn140 / BRD9 Asn100) and the gatekeeper residue is colored blue (BRD4 Ile146 / BRD9 Tyr106). BRD4 Leu94 and BRD9 Ala54 are colored red

Figure 3

Bromodomain phylogenetic tree

Figure 4

(A) pIC₅₀ values for compound 17 in the bromodomains of BRD9 and BRD4 BD1 as determined by TR-FRET assays. (B) Differences between X-ray crystal structures of 17 in complex with the bromodomain of BRD9 (green) and BRD4 BD1 (blue). (C) Binding mode of 17 in BRD9. Hydrogen-bonds are shown as grey dashed lines and the four conserved water molecules as red spheres. Unfavorable interactions are marked as yellow dashed lines. (D) Binding mode of 17 in BRD4 BD1 (blue) with the position of acetyl-lysine (orange, bromodomains superimposed, from protein data bank entry 2wp2). (E,F) Two different orientations of the complex of 17 with BRD9. (G,H) The complex of 17 with BRD4 BD1.

Figure 5

(A) X-ray crystal structures of BRD9 with 32 (orange) and 17 (green). (B) X-ray crystal structure of 32 in the bromodomains of BRD9 (orange) and BRD4 BD1 (blue). Hydrogen bonds are shown as grey dashed lines and water molecules as red spheres.

Figure 6

X-ray structure of (A) 32 (orange) and 44 (green) in BRD9, showing the rotation around the thiophene-amidine bond of 44. The significant difference in conformation of the ZA loop that brings the carbonyl group of Ile53 close to the amidine of 44 is also visible. (B) 32 (orange) and 44 (green) in BRD4 BD1 with the 3.6Å distance between the amidine nitrogen and Leu94 shown as a yellow dashed line.

Figure 7

(A) Superimposed X-ray crystal structures of the bromodomain of BRD9 complexed with compound 45 (magenta) and 44 (green), showing the subtle movement of Phe45 in response to the N1-methyl/ethyl difference between the two compounds. (B) BROMOscan data for compound 45 (generated at DiscoveRx Corp.)

Figure 8

(A) Dose-response binding of compound 45 (I-BRD9) for endogenous BRD9 and BRD3 from HuT-78 cell lysates, measured in a chemoproteomic competition binding assay followed by Western blot analysis. (B) BRD9 bromodomain cellular NanoBRET dose-response curve of compound 45 (I-BRD9). (C) qPCR validation of CLEC1, DUSP6, FES and SAMSN1 genes selectively regulated by compound 45 (I-BRD9) (10 μM), but not by I-BET151 (1 μM) mean+/-SD; n=3. Genes were previously identified by full gene transcriptomics in Kasumi-1 cells.

Scheme 1

Synthesis of amides 17 and 27–29 & 32 (a) n-BuLi, THF, −78 °C, then DMF, THF, −60 °C to rt, 65%; (b) NBS, THF, rt, 79%; (c) MeI, Cs₂CO₃, THF, rt, 71%; (d) NaClO₂, NaH₂PO₄, H₂O₂, DMSO, H₂O, rt, 42–72%; (e) HATU, amine, DIPEA, DMF, rt, 13–79%; (f) (3,4-dimethoxyphenyl)boronic acid, PdCl₂(PPh₃)₂, K₂CO₃, DME, H₂O, microwave, 120 °C, 24–62%.

Scheme 2

Synthesis of amidines 38–44^{a a}Reagents and conditions: (a) MeI, Cs₂CO₃, THF, rt, quant.; (b) Zn(CN)₂, Pd(PPh₃)₄, DMF, microwave, 115 °C, 68%; (c) NBS, THF, rt, 86%; (d) NaOMe, MeOH, 75 °C, then amine hydrochloride salt, 87%–quant.; (e) R²B(OH)₂, K₂CO₃, PEPPSI-ⁱPr, IPA, H₂O, microwave, 120 °C, 4–52%.