Lysosomes are ubiquitous membrane-bound organelles found in all eukaryotic cells. Outside of their well-known degradative function, lysosomes are integral in maintaining cellular homeostasis. Growing evidence has shown that lysosomal dysfunction plays an important role not only in the rare group of lysosomal storage diseases but also in a host of others, including common neurodegenerative disorders, such as Alzheimer disease and Parkinson disease. New technological advances have significantly increased our ability to rapidly isolate lysosomes from cells in recent years. The development of the Lyso-IP approach and similar methods now allow for lysosomal purification within ten minutes. Multiple studies using the Lyso-IP approach have revealed novel insights into the pathogenic mechanisms of lysosomal disorders, including Niemann-Pick type C disease, showing the immense potential for this technique. Future applications of rapid lysosomal isolation techniques are likely to greatly enhance our understanding of lysosomal dysfunction in rare and common neurodegeneration causes.

No abstract

e15102 Background: KRAS G12C mutation is an oncogenic driver most frequently found in non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and other tumor types. Covalent small molecule inhibitors such as sotorasib, recently FDA-approved, and others in clinical trials that target G12C-altered protein by irreversibly binding to the GDP-bound inactive form of KRAS are clinically active in NSCLC patients carrying this somatic mutation. However, in in vitro assays we have developed to evaluate cellular GTP-bound KRAS G12C level, these molecules did not achieve maximal KRAS G12C target inhibition at the minimum concentration (Cmin) of their respective clinically doses. We therefore hypothesize that the potency of KRAS G12C inhibitors may be further improved through medicinal chemistry optimization. Methods: A series of in vitro and in vivo studies were conducted to assess the preclinical pharmacological activity of D3S-001. The biochemical potency of D3S-001 was evaluated by SPR binding analysis. The kinetics of KRAS G12C target engagement was measured by ELISA method. The inhibitor effect of D3S-001 on phospho-ERK1/2 was determined by HTRF assay, and the anti-proliferation effect on cancer cell lines was evaluated by CTG luminescence assay. For in vivo efficacy studies, several human cancer xenograft models and a mouse syngeneic model were employed. Results: D3S-001 demonstrated an exceptionally high Kinact/KI value of 1.58^106 M-1 s-1 by SPR binding analysis. In target engagement assays, D3S-001 achieved rapid and near complete (> 95%) KRAS G12C inhibition within 2 hours at 5 nM. This rapid and effective target engagement correlated with its substantially enhanced cellular potency in both in vitro and in vivo efficacy studies. D3S-001 demonstrated single-digit nanomolar IC50 values in inhibiting cellular phospho-ERK1/2 and cell proliferation in a panel of cancer cell lines harboring the KRAS G12C mutation. D3S-001 was also high selective with no anti-proliferation effects on non-KRAS G12C mutant cell lines. In in vivo studies, D3S-001 demonstrated robust anti-tumor effects as monotherapy in KRAS G12C xenograft models with tumor regression observed at 10 mg/kg and a 30% durable complete remission (CR) rate in CT26, a KRAS G12C syngeneic model, at 30 mg/kg. When combined with an anti-PD-1 antibody, a 70% durable CR rate was achieved with no tumor re-growth observed in CR mice after rechallenging with CT26 tumor, suggesting a memory T cell-induced response. Conclusions: D3S-001 is a highly potent, selective KRAS G12C inhibitor that is differentiated by its rapid and effective target engagement. Human PK modeling predicts > 95% constant target blockage using a 200 mg QD dosing regimen, which may be important and relevant to the induction of deeper and more durable clinical response. D3S-001 will enter a first-in-human Phase 1 clinical trial in 2022.