Project goal

A concise in silico workflow was used to design, synthesize, and validate small molecules that disrupt microtubule dynamics, induce G2/M cell cycle arrest, and demonstrate enhanced efficacy in vitro and in vivo compared to osimertinib in a zebrafish GBM model.

Description of Activities (Stages)

• 1.

  Preparation of Candidate Library: Constructed an initial library of fragments and starting compounds, incorporating known binding motifs for microtubule binding sites.

• 2.

  In Situ Fragment Growing: Employed fragment-growing techniques to expand initial scaffolds and assess fit within the target microtubule binding pocket.

• 3.

  Generative Conformer Algorithm: Used generative algorithms to propose molecular conformers optimized for binding to the microtubule structure.

• 4.

  Scoring Function Development: Applied a custom scoring function to evaluate candidate interactions with the binding site and predicted impact on microtubule dynamics.

• 5.

  In Silico Studies: Conducted computational modeling and simulations to pre-select promising candidates without explicit docking.

• 6.

  In Vitro Validation: Performed biological assays on U-251 glioblastoma cell lines to assess G2/M phase arrest, microtubule disruption, and cytotoxicity measurements.

• 7.

  In Vivo Tests (Zebrafish GBM Xenograft Model): Established the model by xenografting U-251 cells into zebrafish and evaluated therapeutic efficacy by comparing three lead compounds to Osimertinib, analyzing survival rates and tumor size reduction.

• 8.

  Results Analysis: Compared LC50 values for test compounds (2.913 µM, 3.538 µM, 3.614 µM) versus Osimertinib (10.72 µM) and selected three top candidates based on potency and safety.

• 9.

  Workflow Validation: Confirmed that in silico predictions correlated with in vitro and in vivo outcomes, validating the design approach.

In Vitro Confirmation

In vitro assays on U-251 cell lines confirmed compound activity, showing G2/M phase arrest and microtubule dynamics disruption and in vivo – GBM xenograft.