Synthetic Small-Molecule Prohormone Convertase 2 Inhibitors

Abstract

The proprotein convertases are believed to be responsible for the proteolytic maturation of a large number of peptide hormone precursors. Although potent furin inhibitors have been identified, thus far, no small-molecule prohormone convertase 1/3 or prohormone convertase 2 (PC2) inhibitors have been described. After screening 38 small-molecule positional scanning libraries against recombinant mouse PC2, two promising chemical scaffolds were identified: bicyclic guanidines, and pyrrolidine bis-piperazines. A set of individual compounds was designed from each library and tested against PC2. Pyrrolidine bis-piperazines were irreversible, time-dependent inhibitors of PC2, exhibiting noncompetitive inhibition kinetics; the most potent inhibitor exhibited a K i value for PC2 of 0.54 M. In contrast, the most potent bicyclic guanidine inhibitor exhibited a K i value of 3.3 M. Cross-reactivity with other convertases was limited: pyrrolidine bis-piperazines exhibited K i values greater than 25 M for PC1/3 or furin, whereas the K i values of bicyclic guanidines for these other convertases were more than 15 M. We conclude that pyrrolidine bis-piperazines and bicyclic guanidines represent promising initial leads for the optimization of therapeutically active PC2 inhibitors. PC2-specific inhibitors may be useful in the pharmacological blockade of PC2-dependent cleavage events, such as glucagon production in the pancreas and ectopic peptide production in small-cell carcinoma, and to study PC2-dependent proteolytic events, such as opioid peptide production.

Inhibition of Prohormone Convertases PC1/3 and PC2 by 2,5-Dideoxystreptamine Derivatives

“…These differences agree with results obtained for fluorogenic substrate cleavage, confirming that the ranking of the best PC2 inhibitors is 166830 Ͼ 166369 Ͼ 166829 for both proglucagon cleavages to immunoreactive glucagon and for pERTKR-AMC hydrolysis. We used the same assay to compare the pyrrolidine bis-piperazine 1435-6 compound, previously identified by our group as a potent PC2 inhibitor (Kowalska et al, 2009), with the present compounds. We found that the potency of compound 1435-6 in blocking PC2-mediated generation of immunoreactive glucagon from recombinant proglucagon was much weaker than that of 166830, 166369, and 166829 (Fig.…”

“…PC2 is known to have a potent natural inhibitor in the form of the 7B2 carboxyl-terminal peptide (Apletalina et al, 2000a); it is also inhibited by the cystatin-related epididymal spermatogenic protein (Cornwall et al, 2003). The only previous report of small molecule PC2 inhibitors are certain pyrrolidine bis-piperazines and bicyclic guanidines, which inhibit this enzyme with micromolar potency (Kowalska et al, 2009). …”

“…Inhibition constants were determined using different equations, depending on the inhibition mechanism (Copeland, 2005). The K m values of PC1/3 and PC2 for pERTKR-AMC were 11 and 42 M, respectively, as described previously (Cameron et al, 2000a;Kowalska et al, 2009).…”

“…The presence of multiple aryl groups, such as those present on compounds 166691 and 166646, and probably their spatial distribution on the scaffold, seem to be associated with stimulation. We have previously reported that L-polyarginines (Cameron et al, 2000a) and bicyclic guanidines are also able to activate PC2 at very low concentrations, though pyrrolidine bis-piperazine-based inhibitors do not (Kowalska et al, 2009). We speculate that bicyclic guanidines and the aryl-derivatized dideoxystreptamine compounds described here may allosterically bind an exosite, effecting a conformational change that enhances enzyme activity.…”

“…Nonprotein, nonpeptide convertase inhibitors reported thus far are the natural products of the andrographalide family and their succinoyl ester derivatives (Basak et al, 1999); certain metal complexes (Podsiadlo et al, 2004); dicoumarol and its derivatives (Komiyama et al, 2009); and the bicyclic guanidine and pyrrolidine bis-piperazine derivatives we previously identified as PC2 inhibitors (Kowalska et al, 2009). Nonpeptide furin inhibitors based on 2,5-dideoxystreptamine have also been described (Jiao et al, 2006b).…”