The cysteine protease ATG4B plays a role in key steps of the autophagy process and is of interest as a potential therapeutic target. At an early step, ATG4B cleaves proLC3 isoforms to form LC3-I for subsequent lipidation to form LC3-II and autophagosome membrane insertion. ATG4B also cleaves phosphatidylethanolamine (PE) from LC3-II to regenerate LC3-I, enabling its recycling for further membrane biogenesis. Here, we report several novel assays for monitoring the enzymatic activity of ATG4B. An assay based on mass spectrometric analysis and quantification of cleavage of the substrate protein LC3-B was developed and, while useful for mechanistic studies, was not suitable for high throughput screening (HTS). A doubly fluorescent fluorescence resonance energy transfer (FRET) ligand YFP-LC3B-EmGFP (FRET-LC3) was constructed and shown to be an excellent substrate for ATG4B with rates of cleavage similar to that for LC3B itself. A HTS assay to identify candidate inhibitors of ATG4B utilizing FRET-LC3 as a substrate was developed and validated with a satisfactory Z' factor and high signal-to-noise ratio suitable for screening small molecule libraries. Pilot screens of the 1,280-member library of pharmacologically active compounds (LOPAC(™)) and a 3,481-member library of known drugs (KD2) gave hit rates of 0.6% and 0.5% respectively, and subsequent titrations confirmed ATG4B inhibitory activity for three compounds, both in the FRET and mass spectrometry assays. The FRET- and mass spectrometry-based assays we have developed will allow for both HTS for inhibitors of ATG4B and mechanistic approaches to study inhibition of a major component of the autophagy pathway.

The in vivo hydrolytic pathway of a dual-function bone-targeting EP4 receptor agonist-bisphosphonate pro-drug was deduced from radiolabeling experiments. A (14)C labeled pro-drug was used to monitor liberation of the bisphosphonate and results were compared to parallel studies where the EP4 receptor agonist was labeled with (3)H. The bone-adsorption of the (14)C pro-drug following an IV bolus was about 10% compared to 7.8% for the tritiated pro-drug. The difference in release half-life (5.2 and 19.7 days from (3)H and (14)C experiments, respectively) indicated that, after binding to bone, the initial hydrolysis occurred at the ester moiety of the linker releasing the EP4 agonist. The conjugate was found to concentrate in more porous, high-surface-area regions of the long bones. Both (3)H and (14)C experiments indicated a short circulating half-life (1-2 h) in blood.