cRTC

Stages

1. 1.
   Create cell-permeable SNACIP platform(library_design)

   This stage establishes the core SNACIP design needed to overcome limitations of current CIP inducers for endogenous and unligandable targets.

   Selection: Combine chemically induced proximity and chemical nanobody engineering to create cell-permeable small molecule-nanobody conjugates.

2. 2.
   Live-cell functional characterization of cRGT(functional_characterization)

   This stage tests whether the designed inducer works in living cells and can control representative intracellular processes.

   Selection: Assess whether cRGT enters live cells, dimerizes eDHFR and GFP-variants, and enables rapid reversible control of intracellular processes.

3. 3.
   Design latent intracellularly assembled SNACIPs(library_design)

   This stage extends the platform from model dimerization partners to endogenous hard-to-ligand targets.

   Selection: Install small-molecule motifs via post-translational modifications to create latent SNACIPs that assemble inside living cells.

4. 4.
   TPX2-targeted validation in cancer models(confirmatory_validation)

   This stage confirms that the latent SNACIP strategy can modulate an endogenous intrinsically disordered target with disease-relevant consequences.

   Selection: Test whether the TPX2-targeting latent SNACIP inhibits cancer cell proliferation and suppresses tumor growth in vivo.

Steps

1. 1.
   Combine CIP with chemical nanobody engineering to create SNACIPsengineered proximity-inducer platform

   Generate cell-permeable small molecule-nanobody conjugate inducers of proximity.

   The abstract frames this as the foundational design move needed before testing intracellular function.

2. 2.
   Test cRGT for live-cell entry and dimerization of eDHFR and GFP-variantstested inducer

   Establish that the designed inducer functions inside living cells with defined binding partners.

   Demonstrating rapid intracellular entry and dimerization provides an initial live-cell proof of function before extending the platform to endogenous targets.

3. 3.
   Evaluate cRGT control over signaling cascade, cargo transport, and ferroptosistested inducer

   Show that cRGT-mediated proximity can regulate multiple intracellular processes.

   After establishing dimerization, the next step is to demonstrate functional consequences across representative cellular processes.

4. 4.
   Install small-molecule motifs via post-translational modifications to create latent SNACIPs such as cRTClatent intracellularly assembled inducer

   Enable functional assembly inside living cells for endogenous target modulation.

   The abstract presents this as the route for moving beyond model partners to endogenous unligandable targets.

5. 5.
   Validate TPX2-targeting cRTC in cancer cell proliferation and in vivo tumor growth assaysvalidated endogenous-targeting inducer

   Confirm that the latent SNACIP strategy can modulate an endogenous intrinsically disordered target with disease-relevant outcomes.

   This confirmatory validation follows intracellular assembly design to test whether endogenous TPX2 targeting produces cellular and in vivo effects.