Exploiting the S4–S5 Specificity of Human Neutrophil Proteinase 3 to Improve the Potency of Peptidyl Di(chlorophenyl)-phosphonate Ester Inhibitors: A Kinetic and Molecular Modeling Analysis

Abstract

The neutrophilic serine protease proteinase 3 (PR3) is involved in inflammation and immune response and thus appears as a therapeutic target for a variety of infectious and inflammatory diseases. Here we combined kinetic and molecular docking studies to increase the potency of peptidyl-diphenyl phosphonate PR3 inhibitors. Occupancy of the S1 subsite of PR3 by a nVal residue and of the S4-S5 subsites by a biotinylated Val residue as obtained in biotin-VYDnV(O-CH-4-Cl) enhanced the second-order inhibition constant k/[I] toward PR3 by more than 10 times ( k/[I] = 73000 ± 5000 M s) as compared to the best phosphonate PR3 inhibitor previously reported. This inhibitor shows no significant inhibitory activity toward human neutrophil elastase and resists proteolytic degradation in sputa from cystic fibrosis patients. It also inhibits macaque PR3 but not the PR3 from rodents and can thus be used for in vivo assays in a primate model of inflammation.

Serine proteases at the cutting edge of IBD: Focus on gastrointestinal inflammation

“…The balance between serine proteases and their inhibitors seems to play a crucial role in maintaining the gut homeostasis and may constitute a potential target to develop new therapeutics 8 . In this context, significant efforts have been recently made to design and assess new selective inhibitors for specific serine proteases known to be involved in IBD 168,169 . Further studies on this subject will provide additional mechanistic insights regarding the role of serine protease within the gut.…”

Proteinase 3; a potential target in chronic obstructive pulmonary disease and other chronic inflammatory diseases

“…These specificities are determined by: Amide hydrogens on Gly193 and Ser195 which stabilise charge during catalysis [ 14 ]. 3 charged residues: Lys99, Asp61, Arg143 within the active site region.…”

“… Arg143 (and Pro151) increase the polarity of the S2’ subsite which creates a basic S2’ subsite that binds acidic residues [ 12 , 16 ]. Asp213 (compared to Ala213 in NE) restricts the S1 binding site causing it to preferably bind small hydrophobic residues at P1, which includes alanine, serine, valine, norvaline, and methionine [ 12 , 14 , 16 – 18 ]. Ile217 allows small hydrophobic residues at P4 to bind whilst with Trp218 creating a more hydrophobic S5 subsite [ 12 , 14 , 16 ].…”

“…Asp213 (compared to Ala213 in NE) restricts the S1 binding site causing it to preferably bind small hydrophobic residues at P1, which includes alanine, serine, valine, norvaline, and methionine [ 12 , 14 , 16 – 18 ].…”

“…Ile217 allows small hydrophobic residues at P4 to bind whilst with Trp218 creating a more hydrophobic S5 subsite [ 12 , 14 , 16 ].…”