title: Synthesis and evaluation of diphenyl phosphonate esters as inhibitors of the trypsin-like granzymes A and K and mast cell tryptase.
abstract: Thirty-six new amino acid and peptidyl diphenyl phosphonate esters were synthesized and evaluated to identify potent and selective inhibitors for four trypsin-like proteases: lymphocyte granzymes A and K, human mast cell tryptase, and pancreatic trypsin. Among five Cbz derivatives of Lys and Arg homologues, Z-(4-AmPhe)P(OPh)2 is the most potent inhibitor for granzyme A, and Z-LysP(OPh)2 is the best inhibitor for granzyme K, mast tryptase, and trypsin. The amidino P1 residue D,L-(4-AmPhGly)P(OPh)2 was utilized in a series of compounds with several different N-protecting groups and systematic substitutions at P2 in Cbz-AA derivatives and at P3 in Cbz-AA-Ala derivatives. Generally, these phosphonates inhibit granzyme A and trypsin more potently than granzyme K and tryptase. The P2 Thr and Ala dipeptide phosphonates, Cbz-AA-(4-AmPhGly)P(OPh)2, are the most potent inhibitors for granzyme A, and Cbz-Thr-(4-AmPhGly)P(OPh)2 (kobs/[I] = 2220 M-1 s-1) was quite specific with much lower inhibition rates for granzyme K and trypsin (kobs/[I] = 3 and 97 M-1 s-1, respectively) and no inhibition with tryptase. The most effective inhibitor of granzyme A was Ph-SO2-Gly-Pro-(4-AmPhGly)P(OPh)2 with a second-order rate constant of 3650 M-1 s-1. The most potent inhibitor for granzyme K was 3, 3-diphenylpropanoyl-Pro-(4-AmPhGly)P(OPh)2 with a kobs/[I] = 1830 M-1 s-1; all other phosphonates inhibited granzyme K weakly (kobs/[I] < 60 M-1 s-1). Human mast cell tryptase was inhibited slowly by these phosphonates with Cbz-LysP(OPh)2 as the best inhibitor (kobs/[I] = 89 M-1 s-1). The overall results suggest that scaffolds of Phe-Thr-(4-AmPhe) and Phe-Pro-Lys will be useful to create selective phosphonate inhibitors for granzymes A and K, respectively, and that P4 substituents offer opportunities to further enhance selectivity and reactivity.
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400, USA.
authors.forename: D S
authors.initials: DS
authors.lastname: Jackson
authors.forename: S A
authors.initials: SA
authors.lastname: Fraser
authors.forename: L M
authors.initials: LM
authors.lastname: Ni
authors.forename: C M
authors.initials: CM
authors.lastname: Kam
authors.forename: U
authors.initials: U
authors.lastname: Winkler
authors.forename: D A
authors.initials: DA
authors.lastname: Johnson
authors.forename: C J
authors.initials: CJ
authors.lastname: Froelich
authors.forename: D
authors.initials: D
authors.lastname: Hudig
authors.forename: J C
authors.initials: JC
authors.lastname: Powers
mesh_terms: D000596:Amino Acids / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D053818:Chymases; D053804:Granzymes; D006801:Humans; D007700:Kinetics; D008407:Mast Cells / Q000201:enzymology; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D063065:Organophosphonates / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D053802:Tryptases
chemical_list: D000596:Amino Acids; D009842:Oligopeptides; D063065:Organophosphonates; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D053804:Granzymes; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D014357:Trypsin; C509223:Tpsab1 protein, rat; C509221:Tpsb2 protein, rat; D053802:Tryptases; C509212:GZMA protein, human