Redefining pseudokinases: A look at the untapped enzymatic potential of pseudokinases.

Catalytically inactive kinases, known as pseudokinases, are conserved in all three domains of life. Due to the lack of catalytic residues, pseudokinases are considered to act as allosteric regulators and scaffolding proteins with no enzymatic function. However, since these "dead" kinases are conserved along with their active counterparts, a role for pseudokinases may have been overlooked. In this review, we will discuss the recently characterized pseudokinases Selenoprotein O, Legionella effector SidJ, and the SARS-CoV2 protein nsp12 which catalyze AMPylation, glutamylation, and RNAylation, respectively. These studies provide structural and mechanistic insight into the versatility and diversity of the kinase fold.

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD.

The covalent transfer of the AMP portion of ATP onto a target protein-termed adenylylation or AMPylation-by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore-Fl-ATP-we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE's enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.