This
paper presents a method to synthetically tune atomically precise
megamolecule nanobody–enzyme conjugates for prodrug cancer
therapy. Previous efforts to create heterobifunctional protein conjugates
suffered from heterogeneity in domain stoichiometry, which in part
led to the failure of antibody–enzyme conjugates in clinical
trials. We used the megamolecule approach to synthesize anti-HER2
nanobody–cytosine deaminase conjugates with tunable numbers
of nanobody and enzyme domains in a single, covalent molecule. Linking
two nanobody domains to one enzyme domain improved avidity to a human
cancer cell line by 4-fold but did not increase cytotoxicity significantly
due to lowered enzyme activity. In contrast, a megamolecule composed
of one nanobody and two enzyme domains resulted in an 8-fold improvement
in the catalytic efficiency and increased the cytotoxic effect by
over 5-fold in spheroid culture, indicating that the multimeric structure
allowed for an increase in local drug activation. Our work demonstrates
that the megamolecule strategy can be used to study structure–function
relationships of protein conjugate therapeutics with synthetic control
of protein domain stoichiometry.
Laundry detergent formulations include proteases to digest and clean protein-based stains. Methods that can profile protease activity and specificity in detergents could be important in guiding the development of engineered proteases for these applications. The work reported here uses peptide arrays with analysis by self-assembled monolayers for matrix-assisted laser desorption/ionization (SAMDI) mass spectrometry to analyze protease activity across a 324-peptide library for five commercial laundry detergents. The results showed differences in cleavage activity across different brands. The combination of peptide arrays and SAMDI mass spectrometry provides a rapid and reliable technique for analyzing protease activities in laundry detergents and has the potential to play an important role in the development of new proteases and detergent formulations.
The search for new approaches in cancer therapy requires a mechanistic understanding of cancer vulnerabilities and anti-cancer drug mechanisms of action. Problematically, some effective therapeutics target cancer vulnerabilities that we do not understand and have poorly defined mechanisms of anti-cancer activity. One such drug is decitabine, which is a frontline therapeutic approved for the treatment of high-risk acute myeloid leukemia (AML). Decitabine is thought to kill cancer cells selectively via inhibition of DNA methyltransferase enzymes, but the genes and mechanisms involved remain unclear. Here, we apply an integrated multiomics and CRISPR functional genomics approach to identify genes and processes associated with response to decitabine in AML cells. Our integrated multiomics approach reveals RNA dynamics are key regulators of DNA hypomethylation induced cell death. Specifically, regulation of RNA decapping, splicing and RNA methylation emerge as critical regulators of decitabine killing. Our results provide insights into the mechanisms of decitabine anti-cancer activity in treatment of AML and identify combination therapies which could potentiate decitabine anti-cancer activity.
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