Prioritization of Eleven-Nineteen-Leukemia Inhibitors as Orally Available Drug Candidates for Acute Myeloid Leukemia

Abstract

Acute myeloid leukemia (AML) is the second most prevalent and fatal form of leukemia. The growth of AML cells harboring oncogenic MLL rearrangements relies on the YEATS domain-containing protein ENL. Many small molecule inhibitors targeting ENL have been developed. To prioritize these inhibitors for in vivo studies, a NanoBRET system was introduced to evaluate their cellular permeability and potency. This screening identified inhibitor 13 as a promising candidate. This inhibitor has remarkable metabolic stability and potent antiproliferative effects on MLL-fusion leukemia cell lines. In AML-xenografted mice, inhibitor 13 significantly improved survival. Subsequent optimization efforts led to the development of SR-C-107 (R), which exhibited strong activity against AML both at the cellular level (CC_{50 (MOLM-13)}: 1.25 ± 0.18 μM; CC_{50 (MV4-11)}: 0.81 ± 0.15 μM) and in vivo. These findings establish SR-C-107 (R) as a compelling candidate for AML treatment and lay the groundwork for the development of next-generation AML inhibitors.

Figure 1

ENL YEATs domain and its representative inhibitors. (a) The structure of the ENL YEATS domain bound to an H3K9ac peptide, derived from PDB entry 5J9S. The ENL YEATS domain is depicted in gray in cartoon representation. Key residues that form the acetyl-lysine (Kac) binding pocket are shown in stick representation and highlighted in orange. The K3K9ac ligand is represented in hot pink, with the Kac moiety displayed in stick form. Two hydrogen bonds between Kac and residues S58 and Y78 are highlighted in yellow. (b) Representative chemical probes for the ENL YEATS domain in the literature.

Figure 2

ENL-NanoBRET assay and its applications in assessing cellular potencies of ENL inhibitors. (a) Schematic representation of intracellular target engagement in a designed ENL-NanoBRET assay. A cell-permeable fluorescence tracer binds dynamically to its intracellular target protein, ENL YEATS, which is fused to NLuc, resulting in a measurable BRET signal. (b) Structure of SR-0813 and its derived Tracer 2, in which the SR-0813 core and the BODIPY 590 moiety are shown in blue and red, respectively, with the 4-aminobutyric acid linker depicted in orange. (c) Apparent affinity of Tracer 2 for the NLuc-ENL YEATS fusion protein in HEK293T cells. (d) Normalized NanoBRET signal response of ENL inhibitors in HEK293T cells expressing NLuc-ENL YEATS. (e) IC₅₀ and logD values, along with the structures of corresponding inhibitors are presented as the mean ± SD, n = 3.

Scheme 1. Synthesis of Intermediates for Two Enantiomers of Inhibitor 13 and SR-C-107

Reagents and conditions: (a) NaBH₄, MeOH, −10 °C, 30 min; (b) OBHA*HCl, Al (CH₃)₃, DCM, 0 °C—rt, 16 h; (c) TPP, CCl₄, TEA, ACN, 0 °C—rt, 24 h; (d) Raney Ni, MeOH, rt, 16 h; (e) LAH, TMS-Cl, THF, 0 °C—rt, 48 h, 2 M HCl.

Scheme 2. Synthesis of Target Compounds

Reagents and conditions: (a) R = amine, NaBH(OAc)₃, DCE, cat. AcOH, 16 h; (b) SEM-Cl, NaH, 0 °C-rt, 2 h; (c) BINAP/Pd₂(dba)₃, NaOtBu, toluene, 110 °C, 4 h; (d) HCl, THF/H₂O, 16 h; (e) [1,2,4]triazolo[4,3-a]pyridine-6-carboxylic acid, EDCI, pyridine, 50 °C, 12 h; (f) TFA, DCM, 12 h.

Figure 3

Newly developed inhibitors and their in vitro characteristics. (a) AlphaScreen analysis (top) showing the inhibition of ENL YEATS binding to the ENL-S1 peptide by various molecules. IC₅₀ values for corresponding inhibitor structures are given as the mean ± SD, n = 3 (bottom). Binding kinetics of (b) SR-C-107 (top) and (c) SR-C-107 (R) (bottom) to the ENL YEATS domain were characterized using BLI with varying concentrations of inhibitors. (d) Normalized NanoBRET signal response of newly developed ENL inhibitors in HEK293T cells expressing NLuc-ENL YEATS. (e) Determined characteristics of newly developed ENL inhibitors.

Figure 4

Metabolic stability of ENL inhibitors. Tests done in (a) human plasma and (b) HLM. (c) Corresponding parameters given as the mean ± SD, n = 3.

Figure 5

In vitro antitumor potencies of ENL inhibitors. (a) MOLM-13, (b) MV4-11, and (c) Jurkat cell viability post 8 day treatment with previously reported ENL inhibitors. (d) MOLM-13, (e) MV4-11 and (f) Jurkat cell viability post 8 day treatment with newly designed ENL inhibitors. Proliferation of (g) MOLM-13, (h) MV4-11, and (i) Jurkat cells in the presence of 1 μM of ENL inhibitors. (j) Half-maximum cytotoxic concentration (CC₅₀) values for all compounds across each cell line are shown (n = 3). (k) Comparison of the percentage of cells in the G1 phase in various cell lines following 72 h treatment with different concentration of SR-C-107 (R). (l) CETSAs of ENL in MOLM-13 (top) and MV4-11 (bottom) cells treated with 10 μM of SR-C-107 (R) (+) or DMSO control (−) at indicated temperatures, with GAPDH as a loading control. (m) qRT-PCR analysis of HOXA9, HOXA10, MEIS1, MYB, and MYC gene expression in MOLM-13 (left) and MV4-11 (right) cells post 72 h treatment with SR-C-107 (R) or the DMSO negative control. *P < 0.05, **P < 0.01, not significant (n.s.) P > 0.05.

Figure 6

In vivo PK and antitumor analysis of inhibitor 13. (a) In vivo PK profile of inhibitor 13. (b) Quantification of bioluminescence levels (mean ± SEM) in MOML-13 xenografted mice and (c) body weight of mice (mean ± SD) on the indicated days post treatment with inhibitor 13 or vehicle, tumors were allowed to grow for 7 days prior to treatment (day 0 marked the start of treatment). (d) Kaplan–Meier survival curves of NSG mice transplanted with MOLM-13 cells, treated with either vehicle or inhibitor 13 (n = 5). (e) Bioluminescent imaging of NSG mice.

Figure 7

In vivo PK and antitumor analysis of SR-C-107 (R). (a) In vivo PK profile of SR-C-107 (R). (b) Quantification of bioluminescence levels (mean ± SEM) in MOML-13 xenografted mice and (c) body weight of mice (mean ± SD) on the indicated days post treatment with SR-C-107 (R) or vehicle, tumors were allowed to grow for 7 days prior to treatment (day 0 marked the start of treatment). (d) Kaplan–Meier survival curves of NSG mice transplanted with MOLM-13 cells, treated with either vehicle or inhibitor 13 (n = 5). (e) Bioluminescent imaging of NSG mice.

Scheme 3. Synthesis of Fluorescence Tracers

Reagents and conditions: (a) (Boc)₂O, Et₃N, ACN, 16 h; (b) LiAlH₄, dry THF, 0 °C-rt, 4 h; (c) [1,2,4]triazolo[4,3-a]pyridine-6-carboxylic acid, EDC, DIPEA, DMF, rt, 16 h; (d) 4 M HCl, 1,4-dioxane, 2 h; (e) 19, DMAP, EDC, DMF, 36 h; (f) 4-((tert-butoxycarbonyl)amino)butanoic acid, HATU, DIPEA, DMF, rt, 16 h.