5. What have we learned towards the design of a PAM targeted therapeutic
When viewed collectively, patterns emerge from the studies of the PHM inhibitors and inactivators. Clearly, PHM prefers a free carboxylate conjugated to a hydrophobic moiety positioned as close as possible to the penultimate amino acid for the glycine-extended peptide substrates. Incorporation of a sulfur-containing group that can coordinate with one of the PHM-bound copper atoms would likely increase binding affinity. Other possibilities to increase binding affinity in future PHM inhibitors/inactivators would be to link mimosine (or a mimosine analog) to a compound that binds at the peptide site to create a bifunctional inhibitor. The identification of a putative mimosine-binding site (Fig.3C ) in conjunction with the published PHM structures (W. A. Francisco, Blackburn, & Klinman, 2003; Prigge et al., 2004) and the wealth of structure-activity data for PHM substrates and inhibitors provide an excellent starting point for in silico modeling of high affinity PHM inhibitors or inactivators. Another possible bifunctional inhibitor could include a copper-chelator linked to a compound that binds at the peptide site in PHM. Inhibitors/inactivators with high affinity unique to PAM could possess a PHM binder linked to the PAL-specific pyruvate-extended amino acids. Without a structure for bifunctional PAM, a series of compounds with different length spacers between the PHM inhibitor and the PAL inhibitor would be required to define the compound with the highest affinity for PAM. The appropriate incorporation of mimosine into a PAM inhibitor could yield a tight-binding trifunctional inhibitor with a group that binds into the mimosine site of PHM, the peptide site of PHM, and the substrate site of PAL.
The development of a high affinity PHM (or PAM) inhibitor will encounter significant hurdles that could hinder clinical use. One concern is delivery. PHM is found within the lumen of the secretory pathway (Kumar, Mains, et al., 2016), a challenging site for drug delivery. As discussed above, the clinical use of a PHM inhibitor is likely dependent upon a molecular zip code for the secretory system or the surface of specific cell types. Perhaps an engineered version of the Shiga toxin might enable the delivery of a high affinity PHM/PAM inhibitor to the secretory system (Luginbuehl, Meier, Kovar, & Rohrer, 2018). Another concern is the diversity of the amidated products produced in vivo by PHM. The inhibition of PHM would produce unselective blocking of the biosynthesis of many amidated peptides and lipids. One solution to this concern is not a PHM inhibitor, but the development of inhibitor that binds selectively and with high affinity to one glycine-extended substrate, which inhibits the amidation of only one PHM substrate (Weiss, McIntyre, McLaughlin, & Merkler, 2006).
The inactivators require at least one trans -olefinic bond positioned β- to a carboxylate for the most efficient inactivation. Inactivators with the highest affinity have the inactivating moiety attached to the C-terminus of a peptide or a hydrophobic group like a phenyl group. The hydrophobic group must be appropriately spaced away from the inactivating species for the highest affinity, exactly as was observed for the glycine-extended substrates and the PHM inhibitors. Again, the available structure-activity data and PHM structures provide an excellent backdrop for the in silico design of high affinity inactivators. The decoration of such high affinity inactivators with the appropriate imaging reagent could yield a PHM-specific imaging reagent, demonstrating that the molecular targeting was in fact successful. Another application would be decoration with biotin to enable the profiling of PHM similar to the activity-based profiling strategies developed by Cravatt et al. (Cravatt, Wright, & Kozarich, 2008). However, additional research is required to unravel uncertainties in the PHM inactivation chemistry to fully exploit the development of a PHM-specific imaging reagent.