Discovery of potent BET bromodomain 1 stereoselective inhibitors using DNA-encoded chemical library selections
Ram K Modukuri1, Zhifeng Yu1, Zhi Tan1
1Center for Drug Discovery, Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX 77030.
Abstract:
BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structure–activity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1–specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.
Insights
Researchers developed highly selective bromodomain and extraterminal (BET) BD1 inhibitors, CDD-787 and CDD-956, showing potent anti-leukemic activity and unique structural insights into chirality for BET specificity.
Area of Science:
- Medicinal Chemistry
- Molecular Biology
- Cancer Research
Background:
- Bromodomain and extraterminal (BET) proteins (BRDT, BRD2, BRD3, BRD4) possess tandem bromodomains (BD1 and BD2).
- Selective inhibition of BET BD1 mimics effects of tandem BET inhibition in cancer and testes.
- Targeting BET BD1 offers a promising therapeutic strategy.
Purpose of the Study:
- To discover novel, selective inhibitors of BET bromodomain 1 (BD1).
- To characterize the structure-activity relationships and selectivity of identified compounds.
- To evaluate the anti-leukemic potential of selective BET BD1 inhibitors.
Main Methods:
- Screening of a DNA-encoded chemical library (>4.5 billion molecules) against BRDT-BD1 and BRDT-BD2.
- Resynthesis and medicinal chemistry optimization of enriched compounds.
- Stereoisomer separation and activity testing.
- Biochemical and cellular assays (BROMOscan, NanoBRET) for affinity and selectivity.
- X-ray crystallography for structural elucidation.
Main Results:
- Identification of CDD-724, a potent and selective BRDT-BD1 inhibitor (>2,000-fold selectivity over BD2).
- Chiral optimization led to CDD-787 (R-enantiomer), 50-fold more potent than CDD-786 (S-enantiomer).
- CDD-956 demonstrated picomolar potency, high selectivity, improved metabolic stability, and confirmed pan-BET BD1 affinity.
- Cocrystal structure revealed specific interactions driving BD1 selectivity.
- CDD-787 and CDD-956 exhibited potent anti-leukemic activity in cell lines.
Conclusions:
- Novel, structurally unique BET BD1-specific inhibitors (CDD-787, CDD-956) were discovered.
- Chirality is crucial for achieving high specificity and potency in BET inhibitors.
- These compounds provide valuable tools for studying BET proteins and potential therapeutics for acute myeloid leukemia.
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