Human neutrophil proteinase 3: Mapping of the substrate binding site using peptidyl thiobenzyl esters

doi:10.1016/0006-291X(92)91375-Z

Biochemical and Biophysical Research Communications

Volume 188, Issue 3, 16 November 1992, Pages 1318-1324

https://doi.org/10.1016/0006-291X(92)91375-Z Get rights and content

Abstract

A series of peptidyl thiobenzyl esters was used to map the active site of human leukocyte proteinase 3. The steady-state kinetics parameters reveal the following features regarding the substrate specificity of proteinase 3 and its putative active site: (a) the preferred P₁ residue is a small hydrophobic amino acid such as aminobutyric acid, norvaline, valine or alanine (in decreasing order of preferences (b) the enzyme has an extended active site, and (c) its active site is similar to that of the related serine proteinases leukocyte elastase and leukocyte cathepsin G.

References (15)

N.V. Rao et al.
J. Biol. Chem
(1991)
J.C. Jennette et al.
Blood
(1990)
O. Wiedow et al.
Biochem. Biophys. Res. Comm
(1991)
R.L. Stein
Arch. Biochem. Biophys
(1985)
L.C. Kao et al.
J. Clin. Invest
(1988)
W.C. Groutas et al.
J. Med. Chem
(1990)
D. Campanelli et al.
J. Exp. Med
(1990)

There are more references available in the full text version of this article.

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The inhibitory activity of isocumarines, chloromethyl ketones and peptidyl phosphonates against PR3 was reported two years later [27]. At that moment most substrates and inhibitors which were tested on PR3 were primarily designed for HNE or porcine pancreatic elastase (PPE) and PR3 was considered less important protease of low significance [27–29,42]. Although the peptidyl sequence of α-aminoalkylphosphonate diphenyl esters spanned up to four residues with the Boc/MeOSuc N-terminal group, the analyzed positions were limited mainly to Val/nVal and Ala/Pro at P1 and P2 positions, respectively (Table 2).
Neutrophils are one of the most important military services of the armed forces of the immune system, a crucial line of defense against bacterial or fungal onslaughts. One of their mechanisms of action relies on the production of serine proteases. One of these enzymes is proteinase 3 (PR3), which is engaged in the processing of pro-inflammatory cytokines, receptors, heat shock proteins and in the generation of antibacterial peptides. Despite its protective function, uncontrolled activity of PR3 has been associated with the progression of inflammation and tissue injury.
Although PR3 was characterized at the beginning of 90's of the last century for the first time, the research on the development of its inhibitors is barely noticeable. Here we present the recent findings on the design, synthesis and the activity of phosphonic PR3 inhibitors together with the historical perspective.
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The action of neutrophil serine proteases on elastin and its precursor
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Previous studies of their crystal structures have indicated that, in general, S1 pockets of different NSPs show similar geometries but differ slightly in the size, flexibility, polarity and bonding arrangements between residues [43–45]. HLE and PR3 each show hemispheric binding sites of hydrophobic character that are able to comfortably accommodate residues with a two- or three-carbon side chain in the S1 pocket [44,46]. Compared to HLE, the binding sites of PR3 are slightly more polar and restrictive in size; in particular the S1 pocket is smaller [43].
This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P₁, which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P₁ in tropoelastin, whereas Lys was not identified at P₁ in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P₂ and P₄′. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG.
The new fluorogenic substrates of neutrophil proteinase 3 optimized in prime site region
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Citation Excerpt :
Hydrophobic amino acid residues are not likely to be accepted in this position, resulting in the two orders of magnitude lower activity (kcat/KM = 1000 M−1 s−1). That is in good agreement with crystallography [24] and specificity [25] data describing PR3 substrate preference. It is strongly suggested that the prime site of this enzyme (P1′–P3′ substrate’s binding site) possesses hydrophilic character.
Previously selected by the combinatorial chemistry approach, potent fluorogenic substrate of proteinase 3 was used as the starting structure to design new substrates. The general formula of the synthesized peptides is as follows: ABZ-Tyr-Tyr-Abu-ANB-X-NH₂, where ANB (5-amino-2-nitrobenzoic acid) served as a chromophore and an acceptor of fluorescence, ABZ (aminobenzoic acid) is a donor of fluorescence in these fluorescence resonance energy transfer (FRET) peptides, and X is a proteinogenic amino acid (except Cys). The introduced modifications influenced substrate activity of the synthesized peptides. The highest value of specificity constant for proteinase 3 was obtained for the single peptide with Gln in the discussed position (k_cat/K_M= 275,000 M⁻¹ s⁻¹), which was nearly twice as active as the reference compound (lacking a substituent in the X position). In addition, more efficient energy transfer was observed, due mainly to the bathochromic effect for the introduced modification. This approach opens a new possibility to design potent and highly specific substrates of proteinase 3 and other proteinases optimized in the prime site region.
Utilization of the 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide scaffold in the design of potential inhibitors of human neutrophil proteinase 3
2010, Bioorganic and Medicinal Chemistry
The S′ subsites of human neutrophil proteinase 3 (Pr 3) were probed by constructing diverse libraries of compounds based on the 1,2,3,5-thiatriazolidin-3-one 1,1-dioxide using combinational and click chemistry methods. The multiple points of diversity embodied in the heterocyclic scaffold render it well-suited to the exploration of the S′ subsites of Pr 3. Molecular modeling studies suggest that further exploration of the S′ subsites of Pr 3 using the aforementioned heterocyclic scaffold may lead to the identification of highly selective, reversible competitive inhibitors of Pr 3.

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Human neutrophil proteinase 3: Mapping of the substrate binding site using peptidyl thiobenzyl esters

Abstract

J. Biol. Chem

Blood

Biochem. Biophys. Res. Comm

Arch. Biochem. Biophys

J. Clin. Invest

J. Med. Chem

J. Exp. Med