Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas diseaseTrypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (K i * = 18− 350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.

Background:The activity of protein kinase C isoenzymes (PKCs) is regulated by diacylglycerol (DAG). Results: Aromatic residue that tunes DAG affinity influences the recruitment of the conserved region 1 (C1) regulatory domain to a membrane mimic. Conclusion: Membrane pre-association step contributes to the affinity of C1 to diacylglycerol. Significance: This work offers insight into the origin of differential DAG affinities in PKCs.

Ca-dependent conserved-region 2 (C2) domains target their host signaling proteins to anionic membranes. The Ca-binding event is a prerequisite for membrane association. Here, we investigate multiscale metal-ion-dependent dynamics of the C2 domain of protein kinase Cα (C2α) using NMR spectroscopy. Interactions with metal ions attenuate microsecond-timescale motions of the loop regions, indicating that preorganization of the metal-binding loops occurs before membrane insertion. Binding of a full complement of Ca ions has a profound effect on the millisecond-timescale dynamics of the N- and C-terminal regions of C2α. We propose that Ca binding allosterically destabilizes the terminal regions of C2α and thereby facilitates the conformational rearrangement necessary for full membrane insertion and activation of protein kinase Cα.

Conserved homology 1 (C1) domains are peripheral zinc finger domains that are responsible for recruiting their host signaling proteins, including Protein Kinase C (PKC) isoenzymes, to diacylglycerol-containing lipid membranes. In this work, we investigated the reactivity of the C1 structural zinc sites, using the cysteine-rich C1B regulatory region of the PKCα isoform as a paradigm. The choice of Cd2+ as a probe was prompted by previous findings that xenobiotic metal ions modulate PKC activity. Using solution NMR and UV-vis spectroscopy, we found that Cd2+ spontaneously replaced Zn2+ in both structural sites of the C1B domain, with the formation of all-Cd and mixed Zn/Cd protein species. The Cd2+ substitution for Zn2+ preserved the C1B fold and function, as probed by its ability to interact with a potent tumor-promoting agent. Both Cys3His metal-ion sites of C1B have higher affinity to Cd2+ than Zn2+, but are thermodynamically and kinetically inequivalent with respect to the metal ion replacement, despite the identical coordination spheres. We find that even in the presence of the oxygen-rich sites presented by the neighboring peripheral membrane-binding C2 domain, the thiol-rich sites can successfully compete for the available Cd2+. Our results indicate that Cd2+ can target the entire membrane-binding regulatory region of PKCs, and that the competition between the thiol- and oxygen-rich sites will likely determine the activation pattern of PKCs.

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