Selection of human elastase inhibitors from a conformationally constrained combinatorial peptide library

Abstract

A resin-bound cyclic peptide library was constructed based on the sequence of the reactive-site loop of Bowman±Birk inhibitor, a proteinase inhibitor protein. The constrained loop sequence, which incorporates the minimal proteinase-binding motif, was retained throughout the library, but selected residues known to be important for inhibitor specificity were randomised. The approach was used to create a`one bead, one peptide' library with 8000 variants resulting from randomization at three target locations in the sequence (P 4 , P 1 and P 2 H ). This library allows us to examine the degree to which variations in this proteinase-binding motif can redirect activity, as well as providing information about the binding specificity of a proteinase target. Screening this library for binding to human leucocyte elastase identified sequences with a strong consensus, and on resynthesis all were found to act as inhibitors, with K i values as low as 65 nm. Human leucocyte elastase is known to have a substrate preference for small alkyl chains at the P 1 locus, with valine being preferred. However, alanine and not the expected valine was found in 21 out of 23 identified sequences. The remaining two sequences had threonine at P 1 , a finding that would be hard to predict based on substrate specificity alone. Further analysis of resynthesized peptides demonstrated that valine substitution results in an analogue that is hydrolysed far more rapidly than ones having library-selected P 1 residues. Testing of the human leucocyte elastase-selected sequences as inhibitors of porcine pancreatic elastase demonstrates a significant difference in the specificity of the P 4 locus between these two proteinases.Keywords: Bowman±Birk inhibitor; canonical loop; combinatorial chemistry; elastase; peptide library.Elastases, which are serine proteinases capable of cleaving the connective tissue elastin, are considered to play important roles in the tissue destruction associated with pulmonary emphysema, rheumatoid arthritis, cystic fibrosis, adult respiratory distress syndrome, chronic bronchitis, and pancreatitis [1,2]. For some of these pathological conditions an imbalance between the amount of elastase and endogenous proteinaceous inhibitor(s) has been implicated. As a result, there has been considerable interest in the design of inhibitors that may restore this balance and/or clarify the role of these enzymes in the above diseases. In addition to natural or engineered inhibitor proteins, which have high molecular masses, researchers have also pursued both peptide-based and nonpeptide small inhibitor compounds [2].In many natural inhibitor proteins, the portion of the inhibitor protein that interacts with the serine proteinase is an extended or`canonical' loop. This is thought to have a structure similar to that of a productively bound substrate [3,4]. Inhibitors behave as limited proteolysis substrates; residues interacting with proteinase at the reactive site of the inhibitor determine specificity and typically reflect the substrate specificit...

Three-dimensional Structure of a Kunitz-type Inhibitor in Complex with an Elastase-like Enzyme

“…At the bottom of the S1 pocket, the respective residue (Ser 189 in PPE or Gly 189 in HNE) is inaccessible to the P1 side chain of the inhibitor, as it is covered by the hydrophobic parts of Ile 138 and Thr 226 (in PPE) or Val 190 (in HNE). Compared with PPE, the S1 pocket of HNE shows an increased flexibility, enabling the adaptation to a broader variety of P1 side chains (11,23 elastase inhibitory activity determined for wild-type rShPI-1A and for BPTI (18), both of which contain Lys at position P1.…”

“…In contrast, the interaction with PPE is usually very weak or not observed at all (18,21). This elastase specificity could be attributed to a more flexible S1 pocket in HNE that allows accommodation of a broad variety of P1 residues (11,(22)(23)(24). Supporting this concept, the substitution at P1 position with amino acids characterized by medium-sized hydrophobic side chains, such as Val, Ala, and Leu, not only increases the affinity of BPTI for HNE (25)(26)(27) but also converts it into a tight-binding inhibitor of pancreatic elastase with K i values around 10 Ϫ9 M. Affinity selection of a phage-displayed library of BPTI variants against PPE reveals an almost exclusive preference for Leu at the P1 position (28).…”

Tri-domain Bifunctional Inhibitor of Metallocarboxypeptidases A and Serine Proteases Isolated from Marine Annelid Sabellastarte magnifica

“…It therefore seems reasonable to suggest that the SmCI-D1 (domain 1, containing such threonine) could contribute to a lesser extent to inhibiting trypsin and be responsible for elastase inhibition. The additional presence of a hydrophobic residue at P2Ј, able to interact with the S2Ј subsite of the enzyme, could favor elastase inhibition (74) by this first SmCI domain. The inhibition of pancreatic elastase by the rSmCI D1-D2 and its lack by the rSmCI D2-D3 indicates that the D1 domain is probably responsible for the inhibition of this enzyme, also suggesting an important role of the P1 threonine residue in this event.…”

Peptide mimics of the Bowman–Birk inhibitor reactive site loop

“…Screening of this P 4 , P 1 , and P 2 Ј bead library against human leukocyte elastase (HLE) has also been described 49 (Table IX). A total of 23 active binding beads were analyzed in this study, with 21 of these being found to have Ala at P 1 and the remaining two Thr.…”