Teniposide-nonbinding STING double mutant variant

Stages

1. 1.
   High-throughput virtual screening for potential STING ligands(in_silico_filter)

   To identify candidate STING ligands before experimental testing.

   Selection: Potential STING ligand identification

2. 2.
   Biochemical confirmation of direct STING binding(secondary_characterization)

   To experimentally confirm that the selected compound directly interacts with STING.

   Selection: Direct binding of Teniposide to STING's cytosolic domain by ITC

3. 3.
   Mutant-based binding validation(confirmatory_validation)

   To validate that the observed binding depends on a Teniposide-sensitive STING interface.

   Selection: Loss of binding with a STING double mutant unable to bind Teniposide

4. 4.
   Computational binding-mode characterization(functional_characterization)

   To characterize how Teniposide may bind STING after direct interaction was established experimentally.

   Selection: Docking and molecular dynamics characterization of the Teniposide-STING binding mode

5. 5.
   Functional signaling validation(confirmatory_validation)

   To show that direct binding corresponds to pathway activation and to distinguish the mechanism from canonical upstream dsDNA-sensor activation.

   Selection: Activation of the IFN-b2 signaling pathway in a STING-dependent, cGAS/IFI16-independent manner

Steps

1. 1.
   Run high-throughput virtual screening against STING and select Teniposidescreen-selected candidate ligand

   Identify potential STING ligands for downstream validation.

   The source uses virtual screening as the initial candidate-narrowing step before experimental binding and signaling assays.

2. 2.
   Confirm direct Teniposide binding to the STING cytosolic domain by ITCcandidate ligand and binding assay

   Experimentally test whether the selected compound directly binds STING.

   This step follows virtual screening to replace prediction with direct biochemical evidence.

3. 3.
   Validate binding specificity using a STING double mutant unable to bind Teniposidecandidate ligand and negative-control STING construct

   Test whether Teniposide binding depends on a specific STING binding interface.

   The source places mutant validation after direct binding confirmation to strengthen specificity of the interaction claim.

4. 4.
   Model the Teniposide-STING binding mode by docking and molecular dynamicsmodeled ligand

   Characterize the likely binding mode after experimental binding was established.

   The source uses computational analysis after biochemical confirmation to interpret how Teniposide may engage STING.

5. 5.
   Test whether Teniposide activates IFN-b2 signaling in a STING-dependent and cGAS/IFI16-independent mannertested agonist candidate

   Determine whether direct binding corresponds to functional STING pathway activation and whether the mechanism is independent of upstream dsDNA sensors.

   This downstream functional test establishes biological relevance after candidate selection, direct binding confirmation, and binding-mode analysis.