The ENL Inhibitors

A New Generation of Oral Drugs Revolutionizing Acute Myeloid Leukemia Treatment

Why AML Needs a Revolution

Acute myeloid leukemia (AML) strikes with terrifying speed—this aggressive blood cancer progresses rapidly, often requiring immediate intervention. With a dismal five-year survival rate of just 25-30% in adults, AML accounts for nearly half of all leukemia deaths globally ¹ .

For decades, treatment has relied on brutal chemotherapy regimens like the "7+3" protocol (7 days of cytarabine + 3 days of anthracycline), which ravage healthy cells alongside cancerous ones. Elderly patients, who represent most AML cases, frequently can't withstand this assault.

Figure: AML cells under microscope

A new class of targeted therapies—Eleven-Nineteen-Leukemia (ENL) inhibitors—now offers unprecedented hope by attacking a critical molecular weak spot in AML ¹ ³ .

The NanoBRET Breakthrough: Accelerating Drug Development

Rationale: The Cellular Permeability Problem

Developing ENL inhibitors faced a major hurdle: confirming that drug candidates could effectively penetrate cells and engage their target. Traditional AML cell proliferation assays take 1-2 weeks, creating bottlenecks in compound screening ¹ .

Key Discovery

Researchers adapted NanoBRET technology—a real-time cellular monitoring system that acts like a molecular surveillance camera ¹ ² .

Methodology: Lighting Up ENL Interactions

The experimental design was elegantly precise ¹ ² :

Engineered Sensor Cells

HEK293T cells were genetically modified to stably express a fusion protein—NanoLuc luciferase (NLuc) tagged to the ENL YEATS domain. This created consistent "reporters".

Tracer Design

Inspired by a potent ENL inhibitor (SR-0813), scientists synthesized a fluorescent "tracer" (Tracer 2) by attaching BODIPY590 dye via a flexible butyl linker.

BRET Signal Detection

When the tracer binds NLuc-ENL, it comes within 10 nm of furimazine (a luciferase substrate). Energy transfers between them emit red light (BRET signal).

Competition Assay

Test compounds displace Tracer 2, reducing BRET signal. The faster the drop, the stronger the compound's binding affinity and cellular penetration.

Table 1: Key Reagents in the NanoBRET ENL Engagement Assay
Reagent	Role	Function
NLuc-ENL fusion protein	Target reporter	Generates luminescence when bound by furimazine
Tracer 2 (BODIPY590-linked inhibitor)	Molecular beacon	Binds ENL, producing BRET signal when near NLuc
Furimazine	Luciferase substrate	Chemical energy source for NLuc luminescence
HEK293T cell line	Cellular environment	Hosts stable NLuc-ENL expression for consistent assays

Results: Identifying a Champion Compound

Screening revealed Inhibitor 13 as exceptional. It displaced Tracer 2 with high efficiency (IC₅₀ = 46 nM), confirming potent intracellular ENL binding within just 2 hours—versus weeks for traditional assays ¹ .

Metabolic stability: Half-life >120 mins in liver microsomes (vs. 9.3 mins for earlier SR-0813)
Antiproliferative activity: CC₅₀ = 1.25 μM (MOLM-13 cells) and 0.81 μM (MV4-11 cells)
In vivo efficacy: Significantly extended survival in AML-xenografted mice ¹ ²

Table 2: Performance Profile of Inhibitor 13 vs. Predecessors
Parameter	Inhibitor 13	SR-0813	SGC-iMLLT
ENL Binding (KD)	18 nM	30 nM	129 nM
Metabolic Stability (t₁/₂)	>120 min	9.3 min	45 min
MOLM-13 CC₅₀	1.25 ± 0.18 μM	3.4 μM	5.1 μM
MV4-11 CC₅₀	0.81 ± 0.15 μM	2.7 μM	4.3 μM

From Inhibitor 13 to SR-C-107 (R): The Making of an Oral Drug

Optimization of Inhibitor 13 produced SR-C-107 (R)—a compound designed for oral bioavailability and enhanced potency. In mouse models:

Reduced leukemia burden by >80% at 25 mg/kg/day
Extended median survival from 28 days (untreated) to 65 days
Showed no significant toxicity to normal blood cells ¹ ²

SR-C-107 (R)'s success lies in its benzimidazole-amide scaffold, which snugly fits ENL's hydrophobic reader pocket while resisting metabolic breakdown ¹ . Crucially, it achieves >28-fold selectivity for ENL over the similar AF9 YEATS domain—minimizing off-target effects ⁵ .

Oral Drug Advantages

Improved patient compliance
Outpatient treatment possible
Better quality of life
Reduced hospital visits

The Expanding Arsenal of ENL-Targeting Therapies

Beyond traditional inhibitors, innovative strategies are emerging:

PROTAC Degraders

Compounds like PROTAC-1 hijack cellular waste disposal systems to eliminate ENL entirely (DC₅₀ = 37 nM). This approach suppresses cancer genes more durably than simple inhibition ⁶ .

Dual-Target Inhibitors

Galecto's GB3226 simultaneously blocks ENL and FLT3—a common AML mutation. Preclinical data shows synergy against resistant AML ⁴ .

Orally Available Candidates

TDI-11055 replaces benzimidazole with pyrrolopyridine, boosting oral absorption while maintaining potency against MLL-rearranged and NPM1-mutant AML ³ ⁷ .

Table 3: Next-Generation ENL-Targeting Agents in Development
Agent	Mechanism	Key Advantage	Stage
SR-C-107 (R)	ENL-YEATS inhibitor	Optimized oral bioavailability	Preclinical
TDI-11055	Chromatin-displacing inhibitor	Activity against NPM1-mutant AML	Preclinical
PROTAC-1	ENL degrader	Sustained oncogene suppression	Research
GB3226	Dual ENL/FLT3 inhibitor	Overcomes resistance mutations	IND-enabling

The Scientist's Toolkit: Core Technologies Powering ENL Research

Research Technologies

NanoBRET Systems: Real-time target engagement sensors for rapid inhibitor screening ¹ .
MLL-Fusion Cell Lines: Essential for evaluating antiproliferative effects ¹ ⁶ .
SuFEx Chemistry: Sulfur(VI) fluoride exchange enables rapid synthesis of inhibitor libraries ¹ .

Experimental Models

MOLM-13: (MLL-AF9 translocation)
MV4-11: (MLL-AF4 translocation)
Patient-Derived Xenografts (PDX): Mice implanted with human AML tumors predict clinical efficacy ³ ⁶ .
CRISPR Mutagenesis Screens: Validate on-target effects by identifying resistance-conferring ENL mutations ⁷ .

The Future: Precision Medicine for AML

ENL inhibitors could transform AML treatment paradigms:

Oral administration replaces intravenous chemo, enabling outpatient care.
Molecular targeting benefits elderly/unfit patients who tolerate chemotherapy poorly .
Combination potential with venetoclax or FLT3 inhibitors may deepen responses ⁴ .

Ongoing challenges include overcoming resistance mutations and confirming clinical safety. With Galecto aiming for clinical trials of GB3226 by 2026 and SR-C-107 (R) advancing, the first ENL-targeted therapies could soon reach patients ⁴ .

"It's really exciting to have first-in-class ENL inhibitors, but we still have a long way to go before we can make a drug to cure patients." — Dr. Xiang David Li, University of Hong Kong ⁵

Clinical Development Timeline

2023-2024

Preclinical optimization of lead compounds

2025

IND submission for first candidates

2026

Phase I clinical trials begin