Implementing Fluorescence Anisotropy Screening and Crystallographic Analysis to Define PKA Isoform-Selective Activation by cAMP Analogs

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates many proteins, most notably cAMP-dependent protein kinase (PKA). PKA holoenzymes (comprised of two catalytic (C) and two regulatory (R) subunits) regulate a wide variety of cellular processes, and its functional diversity is amplified by the presence of four R-subunit isoforms, RIα, RIβ, RIIα, and RIIβ. Although these isoforms all respond to cAMP, they are functionally nonredundant and exhibit different biochemical properties. In order to understand the functional differences between these isoforms, we screened cAMP derivatives for their ability to selectively activate RI and RII PKA holoenzymes using a fluorescence anisotropy assay. Our results indicate that RIα holoenzymes are selectively activated by C8-substituted analogs and RIIβ holoenzymes by N6-substituted analogs, where HE33 is the most prominent RII activator. We also solved the crystal structures of both RIα and RIIβ bound to HE33. The RIIβ structure shows the bulky aliphatic substituent of HE33 is fully encompassed by a pocket comprising of hydrophobic residues. RIα lacks this hydrophobic lining in Domain A, and the side chains are displaced to accommodate the HE33 dipropyl groups. Comparison between cAMP-bound structures reveals that RIIβ, but not RIα, contains a cavity near the N6 site. This study suggests that the selective activation of RII over RI isoforms by N6 analogs is driven by the spatial and chemical constraints of Domain A and paves the way for the development of potent noncyclic nucleotide activators to specifically target PKA iso-holoenyzmes

Discovery, Synthesis, And Structure-Based Optimization of a Series of <i>N</i>‑(<i>tert</i>-Butyl)-2‑(<i>N</i>‑arylamido)-2-(pyridin-3-yl) Acetamides (ML188) as Potent Noncovalent Small Molecule Inhibitors of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CL Protease

A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (<b>16-(<i>R</i>)</b>, (<i>R</i>)-<i>N</i>-(4-(<i>tert</i>-butyl)­phenyl)-<i>N</i>-(2-(<i>tert</i>-butylamino)-2-oxo-1-(pyridin-3-yl)­ethyl)­furan-2-carboxamide, Pubchem CID: 46897844). Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, <b>16-(<i>R</i>)</b> is a noncovalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multicomponent Ugi reaction was utilized to rapidly explore structure–activity relationships within S_1′, S₁, and S₂ enzyme binding pockets. The X-ray structure of SARS-CoV 3CLpro bound with <b>16-(<i>R</i>)</b> was instrumental in guiding subsequent rounds of chemistry optimization. <b>16-(<i>R</i>)</b> provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a noncovalent mechanism of action