Discovery and characterization of bromodomain 2-specific inhibitors of BRDT

Abstract

Bromodomain testis (BRDT), a member of the bromodomain and extraterminal (BET) subfamily that includes the cancer targets BRD2, BRD3, and BRD4, is a validated contraceptive target. All BET subfamily members have two tandem bromodomains (BD1 and BD2). Knockout mice lacking BRDT-BD1 or both bromodomains are infertile. Treatment of mice with JQ1, a BET BD1/BD2 nonselective inhibitor with the highest affinity for BRD4, disrupts spermatogenesis and reduces sperm number and motility. To assess the contribution of each BRDT bromodomain, we screened our collection of DNA-encoded chemical libraries for BRDT-BD1 and BRDT-BD2 binders. High-enrichment hits were identified and resynthesized off-DNA and examined for their ability to compete with JQ1 in BRDT and BRD4 bromodomain AlphaScreen assays. These studies identified CDD-1102 as a selective BRDT-BD2 inhibitor with low nanomolar potency and >1,000-fold selectivity over BRDT-BD1. Structure-activity relationship studies of CDD-1102 produced a series of additional BRDT-BD2/BRD4-BD2 selective inhibitors, including CDD-1302, a truncated analog of CDD-1102 with similar activity, and CDD-1349, an analog with sixfold selectivity for BRDT-BD2 versus BRD4-BD2. BROMOscan bromodomain profiling confirmed the great affinity and selectivity of CDD-1102 and CDD-1302 on all BET BD2 versus BD1 with the highest affinity for BRDT-BD2. Cocrystals of BRDT-BD2 with CDD-1102 and CDD-1302 were determined at 2.27 and 1.90 Å resolution, respectively, and revealed BRDT-BD2 specific contacts that explain the high affinity and selectivity of these compounds. These BD2-specific compounds and their binding to BRDT-BD2 are unique compared with recent reports and enable further evaluation of their nonhormonal contraceptive potential in vitro and in vivo.

Keywords: BET bromodomains; DNA-encoded chemistry; male contraceptive; small-molecule inhibitors.

Fig. 1.

Comparisons of normalized enrichment from parallel DEC-Tec screens against the isolated bromodomains of BRDT. Selected library members are highlighted which showed significant and selective enrichment for BRDT-BD2 compared with BRDT-BD1 (Left) and strong dependence on the presence of JQ1 (Right). For each highlighted library member, the building blocks for cycles 1, 2, and 3 are shown from top to bottom.

Scheme 1.

Syntheses of DEC-Tec selection hits 3 to 12. Reagents and conditions: step a, 4-(methylcarbamoyl)benzoic acid, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), N,N-diisopropylethylamine (DIEA), N,N-dimethylformamide (DMF), room temperature (rt), 16 h; step b, Zn, AcOH, CH₃OH, rt, 1 h; step c, 3-iodo-1-methyl-1H-indazole-5-carboxylic acid, HATU, DIEA, DMF, rt, 16 h, 27% for 2a and 35% for 2b (three steps); step d, boronic acid derivative, [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (Pd(dppf)Cl₂·CH₂Cl₂) (10 mol %), 1,2-dimethoxyethane (DME)/Na₂CO_3(sat) (vol/vol, 1:1), microwave 85 to 110 °C, 1 h; 24 to 94%.

Fig. 2.

CDD-1102 is a potent and selective inhibitor of the second bromodomain of BRDT and BRD4. Competitive inhibition of human BET bromodomain (BRDT-BD1, BRDT-BD2, BRD4-BD1, and BRD4-BD2) binding to biotinylated JQ1 by CDD-1102 and (+)−JQ1 using proximity detection assays. GraphPad Prism software was used to generate inhibition fitting curves and to determine IC₅₀ values.

Scheme 2.

General synthetic routes to analogs 13 to 36. Reagents and conditions: step a, 3-iodo-1-methyl-1H-indazole-5-carboxylic acid, HATU, DIEA, DMF, rt, 16 h, 71 to 95%; step b, boronic acid derivative, Pd(dppf)Cl₂·CH₂Cl₂ (10 mol%), DME/Na₂CO_3(sat) (vol/vol, 1:1), microwave 90 to 110 °C, 1 h, 29 to 95%; step c, for ester only: LiOH·H₂O, THF, H₂O, 45 °C, 16 h; step d, RNH₂, HATU, DIEA, DMF, rt, 16 h; step e, for N-Boc deprotection only: 4 N HCl, 1,4-dioxane, rt, 1 h, 14 to 91% (three steps).

Fig. 3.

BROMOscan bromodomain profiling of JQ1, CDD-1102, and CDD-1302 on various bromodomains. Phylogenetic tree of bromodomains demonstrating preferential compound binding of CDD-1102 and CDD-1302 for the BET subfamily BD2 domains using the BROMOscan bromodomain competition binding assay performed by the Eurofins DiscoverX Corporation (Top). BromoKdELECT dose–response curves and the calculated binding constants (K_d) confirmed that CDD-1102 and CDD-1302 are strong binders to the BET subfamily BD2 domains, with the highest affinity for BRDT-BD2 (Middle and Bottom). JQ1 binding to both BET bromodomains served as a control in these assays.

Fig. 4.

Detailed BRDT-BD2 inhibitor interactions. (A) BRDT-BD2/CDD-1102 complex. The electrostatic potential surface is shown with the secondary structure on the Left and a zoom-in view of detailed interactions on the Right. The ZA loop is colored in red, the BC loop is in magenta, and the rest are in wheat. CDD-1102 is shown as sticks and colored by atom type, with carbon in yellow, nitrogen in blue, and oxygen in red. The ordered waters are shown by red spheres. The hydrogen bonds are shown by dotted lines. (B) BRDT-BD2/CDD-1302 complex with one CDD-1302 bound. The electrostatic potential surface is shown with the secondary structure on the Left and a zoom-in view of detailed interactions on the Right. CDD-1302 is colored by atom type using the same color scheme as in A. (C) BRDT-BD2/CDD-1302 complex with two CDD-1302 bound. The electrostatic potential surface is shown with the secondary structure in the cartoon in the Center. Zoom-in views of detailed interactions for CDD-1302 bound at the KAc pocket are shown on the Left, and the second site is on the Right.

Fig. 5.

The H bonding interactions between the KAc binding site residues of BRDT-BD2 and the small-molecule inhibitors (A) CDD-1102 (yellow) and (B) CDD-1302 (cyan). To better show these, the cocrystal structures of CDD-1102 and CDD-1302 were subjected to a restrained geometry optimization after addition of hydrogens, estimation of protonation states, and optimization of hydrogen bond networks via the Schrodinger suite of programs. The residues interacting with the ligand are shown in stick representation and labeled. The water molecules are shown as red dots. The black dashed lines represent H bonds. The point of view angle was adjusted to display the H bonding interactions as clearly as possible.

Fig. 6.

Structure comparisons. (A) Superposition of the BRDT-BD2/CDD-1102 and BRDT-BD2/CDD-1302 complexes. The BD2/CDD-1102 complex is superimposed with the BD2/CDD-1302 complex. Only the electrostatic potential surface belonging to the BD2/CDD-1302 complex is shown. CDD-1102 and CDD-1302 are colored using the same color scheme as in Fig. 4 except for carbon atoms of CDD-1302 (cyan). (B) Superposition with the BRDT-BD1/JQ1 complex. The BRDT-BD2/CDD-1102 complex (PDB ID code 7L9A) is superimposed with the BRDT-BD1/JQ1 complex (PDB ID code 4FLP). For the BRDT-BD2/CDD-1102 complex, the ZA loop is colored in red, the BC loop is in magenta, and the rest is in wheat. BRDT-BD1 is colored in black. Key interacting residues are shown as sticks. BD2-specific contact residues and corresponding BD1 residues are labeled. JQ1 is colored by atom type using the same color scheme except for carbon (black), chloride (green), and sulfur (yellow).