Functional Mapping of APOL1 G1 Reveals Key Regions Modulating Cytotoxicity and Inhibitor Sensitivity

Background: Two variants in APOL1 (G1 and G2) are known to increase the risk of developing kidney disease. APOL1 is predicted to form a cytotoxic cation pore, though its full structure remains unresolved. Multiple small molecules inhibit APOL1 pore function and reduce toxicity in overexpression systems, trypanolysis assays, and kidney disease models. We used systematic alanine scanning to identify regions that regulate toxicity and alter responses to structurally distinct inhibitors, providing important insights into APOL1 biology. Methods: We performed an alanine scan of APOL1 G1, substituting each residue to identify positions that influence toxicity and inhibitor sensitivity. Variants were introduced into HEK293 cells via a landing pad system for consistent genomic integration. Expression was induced with doxycycline, and cells were treated with APOL1 inhibitors, or vehicle at IC90, IC60, and IC30 concentrations. Cell viability was assessed by CellTiter-Glo 72 hours post-induction. Variants of interest were further analyzed with compound dose–response titrations and APOL1 protein level assessments. Results: Comprehensive alanine scanning identified structurally relevant patterns of APOL1 toxicity. Multiple N-terminal variants increased APOL1 toxicity, while loss-of-function mutations primarily localized to the pore-forming domain. APOL1 protein levels were unchanged, indicating that differences in toxicity or compound rescue reflect functional changes. Compound profiling revealed distinct reversion patterns in inhibitor efficacy. Conclusion: Our study identified key functional regions of APOL1 that influence its cytotoxicity and response to inhibitors. N-terminal variants that increased APOL1 toxicity suggest an autoinhibitory function in this region. These findings indicate that APOL1’s toxicity is finely tuned, likely to balance trypanolytic function and minimize host cell damage. Loss-of-function mutations localized to the pore-forming domain, which is essential for APOL1-induced cytotoxicity, underscoring the pore's vital role in cellular damage and the potential of pore inhibitors as therapeutic agents. Differential reversal of APOL1 variant-induced toxicity suggests that, despite belonging to the same compound class, these molecules engage APOL1 through distinct mechanisms. Funding: Commercial Support - Maze Therapeutics