Viral macrodomains,
which can bind to and/or hydrolyze adenine
diphosphate ribose (ADP-ribose or ADPr) from proteins, have been suggested
to counteract host immune response and be viable targets for the development
of antiviral drugs. Therefore, developing high-throughput screening
(HTS) techniques for macrodomain inhibitors is of great interest.
Herein, using a novel tracer TAMRA-ADPr, an ADP-ribose
compound conjugated with tetramethylrhodamine, we developed a robust
fluorescence polarization assay for various viral and human macrodomains
including SARS-CoV-2 Macro1, VEEV Macro, CHIKV Macro, human MacroD1,
MacroD2, and PARP9 Macro2. Using this assay, we validated Z8539 (IC50 6.4 μM) and GS441524 (IC50 15.2 μM), two literature-reported small-molecule inhibitors
of SARS-CoV-2 Macro1. Our data suggest that GS441524 is
highly selective for SARS-CoV-2 Macro1 over other human and viral
macrodomains. Furthermore, using this assay, we identified pNP-ADPr (ADP-ribosylated p-nitrophenol, IC50 370 nM) and TFMU-ADPr (ADP-ribosylated trifluoromethyl
umbelliferone, IC50 590 nM) as the most potent SARS-CoV-2
Macro1 binders reported to date. An X-ray crystal structure of SARS-CoV-2
Macro1 in complex with TFMU-ADPr revealed how the TFMU moiety contributes
to the binding affinity. Our data demonstrate that this fluorescence
polarization assay is a useful addition to the HTS methods for the
identification of macrodomain inhibitors.
Maintaining homeostasis of metabolites such as amino
acids is critical
for cell survival. Dysfunction of nutrient balance can result in human
diseases such as diabetes. Much remains to be discovered about how
cells transport, store, and utilize amino acids due to limited research
tools. Here we developed a novel, pan-amino acid fluorescent turn-on
sensor, NS560. It detects 18 of the 20 proteogenic amino acids and
can be visualized in mammalian cells. Using NS560, we identified amino
acids pools in lysosomes, late endosomes, and surrounding the rough
endoplasmic reticulum. Interestingly, we observed amino acid accumulation
in large cellular foci after treatment with chloroquine, but not with
other autophagy inhibitors. Using a biotinylated photo-cross-linking
chloroquine analog and chemical proteomics, we identified Cathepsin
L (CTSL) as the chloroquine target leading to the amino acid accumulation
phenotype. This study establishes NS560 as a useful tool to study
amino acid regulation, identifies new mechanisms of action of chloroquine,
and demonstrates the importance of CTSL regulation of lysosomes.
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