2. Academic Validation
  3. Identification of a novel Azaspirooxindolinone-based PROTAC for selective BTK degradation and enhanced anticancer activity

Identification of a novel Azaspirooxindolinone-based PROTAC for selective BTK degradation and enhanced anticancer activity

  • Bioorg Chem. 2025 Apr:157:108316. doi: 10.1016/j.bioorg.2025.108316.
Naveen Kumar Rampeesa 1,  Rambabu Gundla 2,  Gopal Mudasani 1,  Sudhakar Tangallapalli 3,  Sreenivasa Reddy Anugu 4,  Soňa Gurská 5,  Juan Bautista De Sanctis 5,  Petr Džubák 5,  Marián Hajdúch 5,  Viswanath Das 6
Affiliations

Affiliations

  • 1 Department 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.
  • 2 Department of Chemistry, School of Science, GITAM University, Hyderabad 502102, Telangana, India. Electronic address: rgundla@gitam.edu.
  • 3 Department of Chemistry, School of Science, GITAM University, Hyderabad 502102, Telangana, India.
  • 4 Aragen Life Sciences Ltd, Medicinal Chemistry Laboratory Division, Survey, No: 125(Part) & 126, IDA Mallapur, Hyderabad 500076, India.
  • 5 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University and University Hospital Olomouc, Hněvotínská 1333/5, 779 00 Olomouc, Czech Republic; Institute of Molecular and Translational Medicine, Czech Advanced Technologies and Research Institute, Palacký University Olomouc, Křížkovského 511/8, 779 00, Olomouc, Czech Republic.
  • 6 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University and University Hospital Olomouc, Hněvotínská 1333/5, 779 00 Olomouc, Czech Republic; Institute of Molecular and Translational Medicine, Czech Advanced Technologies and Research Institute, Palacký University Olomouc, Křížkovského 511/8, 779 00, Olomouc, Czech Republic. Electronic address: viswanath.das@upol.cz.
PMID: 40037026 DOI: 10.1016/j.bioorg.2025.108316
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.

Keywords

Antiproliferative activity; Azaspirooxindolinone; Bruton's tyrosine kinase; IL-2-inducible T-cell kinase; Jurkat; PROTAC; Ramos.

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