Discovery and Characterization of BAY-184: A New Potent and Selective Acylsulfonamide-Benzofuran In Vivo-Active KAT6AB Inhibitor

Abstract

KAT6A and KAT6B genes are two closely related lysine acetyltransferases that transfer an acetyl group from acetyl coenzyme A (AcCoA) to lysine residues of target histone substrates, hence playing a key role in chromatin regulation. KAT6A and KAT6B genes are frequently amplified in various cancer types. In breast cancer, the 8p11-p12 amplicon occurs in 12-15% of cases, resulting in elevated copy numbers and expression levels of chromatin modifiers like KAT6A. Here, we report the discovery of a new acylsulfonamide-benzofuran series as a novel structural class for KAT6A/B inhibition. These compounds were identified through high-throughput screening and subsequently optimized using molecular modeling and cocrystal structure determination. The final tool compound, BAY-184 (29), was successfully validated in an in vivo proof-of-concept study.

Figure 1

Selected examples of known KAT6AB inhibitors. Reported IC₅₀ values for WM-1119 are from Priebbenow et al. IC₅₀ values for Example 41 are from Morrow et al.K_i for CTx-648/PF-9363 are from Sharma et al.

Figure 2

High throughput screening (HTS) strategy, hit validation, and prioritization.

Figure 3

(A) Glide docking mode of compound 1 in the AcCoA binding site of KAT6A (2OZU.pdb). (B) Comparison of the diphosphate group of AcCoA and the different polar moieties observed in KAT6A inhibitors, i.e., WM-1119, CTx-648 (Figure 1), and compound 1. The KAT6A ligand binding site accommodates the diphosphate group of AcCoA in a protein region with high H-bond donor density stemming from five closely located backbone NHs (of the loop from Arg⁶⁵⁵ to Arg⁶⁶⁰) and from the side chain of Ser⁶⁹⁰ (see Table, light green: 1 H-bond, dark green: 2 or more H-bonds). Predicted pK_a was calculated using ADMET predictor by software Simulations Plus (Version 11).^,

Figure 4

Binding mode of compound 29 (BAY-184) in the cocrystal structure with KAT6A (PDB entry 9FKR). (A) Overall view of KAT6A (chain A) in the cocrystal structure with 29. The KAT6A protein is shown as a ribbon model in gray, the inhibitor 29 as a stick model (carbon atoms in yellow), and a structural Zn ion as a purple sphere. To illustrate the position of the binding site of the cofactor AcCoA, the crystal structure of KAT6A in complex with AcCoA (PDB entry 2OZU) was superimposed onto the structure with 29 (for clarity, only the cofactor is shown, with carbon atoms in blue). (B) Same orientation as in (A), with KAT6A in surface representation to show the buried nature of this binding pocket. (C) View into the binding pocket of 29 (chain A): inhibitor as a stick model with carbon atoms in yellow. Protein residues lining the binding pocket are depicted as stick models with carbon atoms in gray and labeled using the one-letter code for amino acids. (D) KAT6A with AcCoA (PDB entry 2OZU) superimposed on chain A of KAT6A in complex with 29 to illustrate which part of the AcCoA binding pocket is occupied by 29. AcCoA is shown with carbon atoms in cyan.

Figure 5

Expansion of initial benzofurane-N-sulfonyl-carboxamides hit series in the underlying drug discovery project. Activity in the ER target gene reporter assay in the MVLN cells versus biochemical activity on the his-tagged HAT domain of KAT6A (TR-FRET assay) is shown. Optimization of cellular activity led to a larger series of 4-[H/F]-6-dimethylamino-substituted benzofurans (green) with generally improved cellular activity compared to the original hit series of 4,6-dimethyl- (yellow), 4,6-dimethoxy- (orange), and 4-methyl-6-methoxy-substituted benzofurans (red, Table 1).

Figure 6

Consequences of KAT6A inhibition: (A) Western Blot showing time-resolved reduction of H3K23ac and ERα level in ZR-75-1 cells treated with BAY-184 (29). Concentration series of BAY-184 (29) and its negative control BAY-644 (40) were utilized in the (B) biochemical TR-FRET assay, (C) H3K23 acetylation HTRF, (D) ER target gene reporter assay (MVLN), and (E) ZR-75-1 proliferation assay.

Figure 7

(A) Volume of ZR-75-1 xenografts after treatment with BAY-184 (29). Female estradiol-primed NMRI nude mice were subcutaneously inoculated with 5 × 10⁶ ZR-75-1 cells suspended in 50% Matrigel in a volume of 100 μL. Starting on day 15 after implantation at a tumor volume of ca. 146 mm³, BAY-184 (29) was administered by oral gavage 2 qd at doses of 50 (■), 100 (▲), and 150 mg/kg (▼) solved in PEG/EtOH 90/10 compared to vehicle alone (•). Data are presented as mean + standard deviation. The asterisk indicates statistically significant differences compared to the control on day 19. p < 0.0001. n = 11 (ONE WAY ANOVA). BAY-184 (29) inhibited the growth of ZR-75-1 xenografts in NMRI nude mice significantly at all doses with complete growth inhibition at a dose of 150 mg/kg. (B) Body weight changes in percent compared to the body weight at the start of treatment. The body weight further increased while mice were treated (n = 11). (C) Examples of immunohistochemistry (IHC) staining. Samples were stained with an anti-H3K23ac antibody and quantified with QuPath software (D). H3AcetylK23 was quantified as H-score [0–300]; Ns = not significant, ** = p-value ≤0.01; * = p-value ≤0.5.

Scheme 1. Synthetic Routes for BAY-184 (29) and Its Sodium Salt 43

Reagents and conditions: (a) ethyl chloroacetate, K₂CO₃ DMF, 160 °C, 2 h, 70%; (b) LiOH, THF/H₂O, rt, overnight, 30%; (c) NMe₂ (1.1 equiv), Xphos Pd G2 (0.1 equiv), Cs₂CO₃ (2.5 equiv), dioxane, (d) LiOH, THF/H₂O, rt, overnight, 30% (2 steps); (e) [1,1′-biphenyl]-2-sulfonamide, CDI, DBU, THF, rt, 3 h, 72% or [1,1′-biphenyl]-2-sulfonamide, pyBOP, DIPEA, CH₂Cl₂, 38%; (f) i. 1 M NaOH, ii. EtOH, H₂O, rt, overnight, 58%. (g) pyBOP, DIPEA, CH₂Cl₂, 52%; (h) NMe₂ (1.2 equiv), Xphos Pd G2 (0.1 equiv), Cs₂CO₃ (2.5 equiv), DMF, 54%.

Scheme 2. Synthetic Route for BAY-644 (40)

Reagents and conditions: (a) NBS, TFA, 1,2-dichlorethan, 3%; (b) 1,1′-bis(diphenylphosphino)ferrocene-palladium, methanboronic acid, K₂CO₃, dioxane, H₂O, 130 °C, 1 h, 4%.