biostudies-literatureRebecca VWNCI NIH HHSNIHNIH HHS220-229https://www.ebi.ac.uk/biostudies/studies/S-EPMC6368875biostudies-literatureUnknown9(2)Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy significantly better than hydroxychloroquine (HCQ). Using an <i>in situ</i> photoaffinity pulldown strategy, we identified palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in significant iCancer discoveryPPT1 Promotes Tumor Growth and Is the Molecular Target of Chloroquine Derivatives in Cancer.PMC6368875P50 CA093372R01 CA169134P30 CA016520S10 OD023586P30 CA016672P30 CA010815P01 CA114046R01CA169134P50 CA174523R01 CA198015R50 CA221838Zhang GWinkler JDPiao SHerlyn MMartorella ASpeicher DWRebecca VWNicastri MCNoguera-Ortega EGimotty PAMarmorstein RRonghe AOjha RAmaravadi RKBarber-Rotenberg JSGoldman ARFennelly CMcAfee QChude CILee JJSchuchter LMAlicea GMXu XMcLaughlin NPfalsePPT1 Promotes Tumor Growth and Is the Molecular Target of Chloroquine Derivatives in Cancer.Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy significantly better than hydroxychloroquine (HCQ). Using an <i>in situ</i> photoaffinity pulldown strategy, we identified palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in significant i2019-01-01T00:00:00Z2019 Feb2026-05-07T14:04:08.36Z2020-10-29T11:02:15ZS-EPMC63688753044270910.1158/2159-8290.cd-18-070610.1158/2159-8290.CD-18-0706