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Published on: November 9, 2020
Identification of a novel Azaspirooxindolinone-based PROTAC for selective BTK degradation and enhanced anticancer
Naveen Kumar Rampeesa1, Rambabu Gundla2, Gopal Mudasani1
1Department of Chemistry, School of Science, GITAM University, Hyderabad 502102, Telangana, India; Aragen Life Sciences Ltd, Medicinal Chemistry Laboratory Division, Survey, No: 125(Part) & 126, IDA Mallapur, Hyderabad 500076, India.
Abstract:
Bruton's Tyrosine Kinase (BTK) is a key driver of hematological malignancies, autoimmune disorders, and neuroinflammation, making it an attractive therapeutic target. Proteolysis targeting chimeras (PROTACs) offer a novel strategy for BTK degradation via the E3 ubiquitin ligase pathway. Here, we evaluated nine azaspirooxindolinone-based PROTAC derivatives for their cytotoxicity and BTK-targeting activity. Several compounds exhibited potent cytotoxicity against BTK-high RAMOS lymphoma cells without affecting non-cancer fibroblasts or normal T/B-cell lymphocytes. Among them, PROTAC 25 emerged as the most effective degraded, achieving a Dmax of 72.84 % and DC50 of 0.27 μM in a proteasome-dependent manner. Although PROTAC 25 was cytotoxic to IL-2-inducible T cell Kinase (ITK)-positive cells, ITK protein levels remained unaffected. Furthermore, kinase assays revealed that PROTAC 25 inhibited BTK kinase activity (IC₅₀ = 0.44 μM) with moderate selectivity over ITK (IC₅₀ = 2.16 μM). Notably, PROTAC 25 suppressed BTK-mediated downstream signaling in RAMOS cells, as evidenced by reduced phosphorylation of BTK and its downstream effector, p38 MAPK. These findings highlight PROTAC 25 as a promising BTK degrader with therapeutic potential and underscore the value of azaspirooxindolinone-based PROTACs in targeting BTK-driven diseases.
Insights
New PROTACs effectively degrade Bruton's Tyrosine Kinase (BTK) in cancer cells. PROTAC 25 shows potent BTK degradation and inhibition, offering therapeutic potential for BTK-driven diseases.
Area of Science:
- Biochemistry
- Pharmacology
- Oncology
Background:
- Bruton's Tyrosine Kinase (BTK) is a critical target in hematological malignancies and autoimmune diseases.
- Proteolysis targeting chimeras (PROTACs) represent a novel therapeutic strategy for targeted protein degradation.
Purpose of the Study:
- To evaluate novel azaspirooxindolinone-based PROTAC derivatives for BTK degradation.
- To assess the cytotoxicity and selectivity of these PROTACs against cancer cells and normal lymphocytes.
Main Methods:
- Synthesis and evaluation of nine azaspirooxindolinone-based PROTACs.
- Cytotoxicity assays on RAMOS lymphoma cells, fibroblasts, and normal T/B cells.
- Western blotting to assess protein degradation (BTK, ITK) and signaling pathways (p38 MAPK).
- Kinase inhibition assays to determine IC50 values for BTK and ITK.
Main Results:
- Several PROTACs demonstrated potent cytotoxicity against BTK-high RAMOS cells, sparing normal cells.
- PROTAC 25 achieved significant BTK degradation (Dmax 72.84%, DC50 0.27 μM) in a proteasome-dependent manner.
- PROTAC 25 inhibited BTK kinase activity (IC50 = 0.44 μM) with moderate selectivity over ITK (IC50 = 2.16 μM) and suppressed downstream signaling.
Conclusions:
- Azaspirooxindolinone-based PROTACs are effective for BTK degradation.
- PROTAC 25 is a promising candidate for targeting BTK-driven hematological malignancies and other diseases.
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