cellular thermal shift assay

Stages

1. 1.
   Cellular target engagement assessment(functional_characterization)

   This stage tests whether Kyn-CKA engages Keap1 in cells before deeper mechanistic interpretation.

   Selection: Thermal stabilization of Keap1-mCherry but not free mCherry after Kyn-CKA exposure.

2. 2.
   Residue-specific biochemical reactivity test(secondary_characterization)

   This stage tests whether the proposed Keap1 engagement is specifically directed to C151 and favored by the Keap1 protein environment.

   Selection: Differential reactivity of Kyn-CKA with wild-type versus C151S mutant Keap1 BTB domain and comparison against N-acetyl cysteine.

3. 3.
   Cellular genotype-dependence confirmation(confirmatory_validation)

   This stage confirms that residue-specific biochemical reactivity translates into cellular Nrf2 pathway activation.

   Selection: Comparison of Nrf2 abundance and NQO1 induction in wild-type Keap1 versus C151S-Keap1 mutant MEFs.

4. 4.
   Primary-cell pathway attribution of anti-inflammatory activity(confirmatory_validation)

   This stage determines which signaling pathway is required for the macrophage phenotype after Kyn-CKA treatment.

   Selection: Comparison of acute anti-inflammatory activity in WT, AhR-knockout, and Nrf2-knockout primary murine bone marrow-derived macrophages.

Steps

1. 1.
   Measure thermal stabilization of Keap1-mCherry after Kyn-CKA exposurecompound under test and target-engagement assay

   Test whether Kyn-CKA engages Keap1 in cells.

   The campaign first establishes cellular target engagement before assigning residue specificity or downstream pathway causality.

2. 2.
   Compare Kyn-CKA reactivity with wild-type and C151S mutant Keap1 BTB domaincompound under biochemical reactivity test

   Determine whether Kyn-CKA reactivity depends on Keap1 C151.

   After showing cellular engagement, the workflow tests whether the interaction is specifically directed to the proposed sensor residue.

3. 3.
   Benchmark Kyn-CKA reactivity against N-acetyl cysteinecompound under comparator reactivity test

   Assess whether Kyn-CKA reacts faster with Keap1 than with a small-molecule thiol comparator.

   This comparator helps argue that the surrounding protein environment targets Kyn-CKA toward C151 rather than reflecting generic thiol reactivity.

4. 4.
   Compare Nrf2 and NQO1 induction in wild-type versus C151S-Keap1 MEFscompound under cellular genotype-dependence test

   Confirm that C151 dependence observed biochemically translates into cellular Nrf2 pathway induction.

   This step follows biochemical residue mapping to test whether the same residue controls functional signaling in cells.

5. 5.
   Test acute anti-inflammatory activity in WT, AhR-knockout, and Nrf2-knockout macrophagescompound under pathway-requirement test

   Determine whether the macrophage anti-inflammatory phenotype requires Nrf2 or AhR.

   After establishing Keap1 C151-dependent Nrf2 activation, the workflow asks which downstream pathway explains the primary-cell phenotype.