Abstract
The prohormone convertases PC1/3 and PC2 are eukaryotic serine proteases involved in the proteolytic maturation of peptide hormone precursors and are implicated in a variety of pathological conditions, including obesity, diabetes, and neurodegenerative diseases. In this work, we screened 45 compounds obtained by derivatization of a 2,5-dideoxystreptamine scaffold with guanidinyl and aryl substitutions for convertase inhibition. We identified four promising PC1/3 competitive inhibitors and three PC2 inhibitors that exhibited various inhibition mechanisms (competitive, noncompetitive, and mixed), with sub- and low micromolar inhibitory potency against a fluorogenic substrate. Low micromolar concentrations of certain compounds blocked the processing of the physiological substrate proglucagon. The best PC2 inhibitor effectively inhibited glucagon synthesis, a known PC2-mediated process, in a pancreatic cell line; no cytotoxicity was observed. We also identified compounds that were able to stimulate both 87 kDa PC1/3 and PC2 activity, behavior related to the presence of aryl groups on the dideoxystreptamine scaffold. By contrast, inhibitory activity was associated with the presence of guanidinyl groups. Molecular modeling revealed interactions of the PC1/3 inhibitors with the active site that suggest structural modifications to further enhance potency. In support of kinetic data suggesting that PC2 inhibition probably occurs via an allosteric mechanism, we identified several possible allosteric binding sites using computational searches. It is noteworthy that one compound was found to both inhibit PC2 and stimulate PC1/3. Because glucagon acts in functional opposition to insulin in blood glucose homeostasis, blocking glucagon formation and enhancing proinsulin cleavage with a single compound could represent an attractive therapeutic approach in diabetes.
Footnotes
This work was supported in part by a grant from the National Institutes of Health National Institute on Drug Abuse [Grant DA050854] (to I.L.); by the State of Florida Executive Officer of the Governor's Office of Tourism, Trade and Economic Development (to K.M-M.); and by the U.S. Department of Defense, U.S. Army Medical Research and Materiel Command [Contract V549P-6073] (to A.J. and G.-S.J.), [Grant OT07-050] (to K.M.-M.).
G.-S.J. is co-corresponding author regarding the inhibitor chemistry (gjiao{at}pantherabio.com).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- PC
- prohormone convertase
- 166369
- 5-(2,4-diguanidino-phenoxy)-2,4-diguanidino-cyclohexyl (4-octylphenyl)carbamate
- 166369a
- N,N′-di-tert-butoxycarbonyl-N″-{4-[2,4-di-(di-tert-butoxycarbonyl-guanidino)-phenoxy]-5-[di-(tert-butoxycarbonyl)-guanidino]-2-hydroxycyclohexyl}-guanidine
- 166550
- N-[2,4-bis(4-guanidino-phenoxy)-5-(4-guanidino-phenylamino)-cyclohexyl]-guanidine
- 166631
- N-[5-guanidino-2,4-bis-(4-guanidino-phenoxy)-cyclohexyl]-guanidine
- 166646
- 5-(2,4-diguanidino-phenoxy)-2,4-diguanidino-cyclohexyl [1,1′-biphenyl]-4-ylcarbamate
- 166691
- N-[5-(4-guanidino-naphthalen-1-ylamino)-2,4-bis-(4-guanidino-naphthalen-1-yloxy)-cyclohexyl]-guanidine
- 166811
- N-[5-guanidino-4-(4-guanidino-naphthalen-1-yloxy)-2-(4-guanidino-phenoxy)-cyclohexyl]-guanidine
- 166812
- N-{2-[2,4-diguanidino-5-(4-guanidino-phenoxy)-cyclohexyloxy]-5-guanidino-phenyl}-guanidine
- 166829
- 5-(2,4-bis(imidazolidin-2-ylideneamino)phenoxy)-2,4-bis(imidazolidin-2-ylideneamino)cyclohexanol
- 166829a
- 2,4-diamino-5-(2,4-diaminophenoxy)cyclohexanol
- 166830
- N1,N3-di(imidazolidin-2-ylidene)-4,6-bis(4 (imidazolidin-2-ylideneamino)phenoxy)cyclohexane-1,3-diamine
- 166830a
- 4,6-bis (4-aminophenoxy)cyclohexane-1,3-diamine
- 1435–6
- (2R)-4-((R)-1-cyclohexyl-3-((S)-2-(((S)-2-(cyclohexylmethyl)piperazin-1-yl)methyl)pyrrolidin-1-yl)propan-2-yl)-2-(cyclohexylmethyl)-1-(2-(4-isobutylphenyl)propyl)piperazine
- TFA
- trifluoroacetic acid
- BSA
- bovine serum albumin
- pERTKR-AMC
- pGlu-Arg-Thr-Lys-Arg-methylcoumarinamide
- PBS
- phosphate-buffered saline
- RIA
- radioimmunoassay.
- Received December 1, 2011.
- Accepted December 14, 2011.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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PC1/3 and PC2 Inhibitors
