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.

Granzyme K Displays Highly Restricted Substrate Specificity That Only Partially Overlaps with Granzyme A

“…Likewise, Mahrus and Craik (10) used combinatorial libraries of 4-amino acid peptides to show that P4-P2 subsite specificities of both GrK and GrA appear broad but distinct, and unlike GrB and GrM, no clear P4-P2 consensus sequence could be identified. In agreement with this, GrA and GrK cleave oligo-peptide synthetic substrates only slightly better than single amino acid (Arg or Lys) substrates (27,28). The lack of a clear consensus sequences around the P1 scissile bond further is consistent with known cleavage sites in macromolecular substrates of GrA (7,20) or GrK (␤-tubulin; Fig.…”

“…The relatively small number of granzyme-induced cleavage events detected in a tumor cell proteome indicates that GrK and GrA display restricted macromolecular substrate specificity. Remarkably, whereas GrB requires a tripeptide synthetic substrate to observe cleavage and is maximal with an idealized tetrapeptide, GrA and GrK readily cleave single amino acid substrates (data not shown) (27,28). This indicates that the macromolecular substrate specificities of GrA and GrK are not restricted to the active site and fully depend on (i) extended binding sites close to the active site cleft (subsites) that interact with residues around P1 in the substrate and/or (ii) interactions between exosites of the substrate and the enzyme.…”

Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics

“…This kinetic advantage is a clear demonstration that the complementary matching of the shape and chemical reactivity of substrate 1b with the selected reactibody exceeds the performance of an enzyme such as BChE with a classical phosphorylating agent. Enzyme selectivity for the phosphonate structure may also distort these comparisons (33)(34)(35)(36). Reactive phosphoesters diisopropyl fluorophosphate (DFP) (2), paraoxon (5), and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) (3) inhibited A.17 reaction with 1a, whereas echothiophate (4) had no effect at concentrations up to 10 mM (Fig.…”

The 2.2-Å Crystal Structure of Human Pro-granzyme K Reveals a Rigid Zymogen with Unusual Features

“…GzmK cleaves this oligopeptide between basic residues, most rapidly after position 6 and 9 and more slowly after position 7 and 8 (11). The best synthetic inhibitors of GzmK known are 3,3-diphenylpropanoyl-Pro-4-amidinophenylglycyldiphenylphosphonate esters (12) and D-Phe-Pro-Arg-chloromethyl ketone (13). The most efficient natural inhibitor is inter-␣-trypsin inhibitor, a heterotrimeric protein complex containing bikunin.…”