Activity assay based on the immobilized enzyme kallikrein and mass spectrometry (original) (raw)
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Biochemical characterization of a novel high-affinity and specific plasma kallikrein inhibitor
British Journal of Pharmacology, 2011
BACKGROUND AND PURPOSE Kallikrein acts on high molecular weight kininogen (HK) to generate HKa (cleaved HK) and bradykinin (BK). BK exerts its effects by binding to B2 receptors. The activation of B2 receptors leads to the formation of tissue plasminogen activator, nitric oxide (NO) and prostacyclin (PGI2). An elevated kallikrein-dependent pathway has been linked to cardiovascular disease risk. The aim of this study was to investigate whether our novel plasma kallikrein inhibitor abolishes kallikrein-mediated generation of BK from HK and subsequent BK-induced NO and PGI2 formation, thereby influencing endothelial pathophysiology during chronic inflammatory diseases. EXPERIMENTAL APPROACH Kinetic analysis was initially used to determine the potency of PF-04886847. Biochemical ligand binding assays, immunological methods and calcium flux studies were used to determine the selectivity of the kallikrein inhibitor. In addition, the effect of PF-04886847 on BK-induced relaxation of the rat aortic ring was determined in a model of lipopolysaccharideinduced tissue inflammation. KEY RESULTS Evidence was obtained in vitro and in situ, indicating that PF-04886847 is a potent and specific inhibitor of plasma kallikrein. PF-04886847 efficiently blocked calcium influx as well as NO and PGI2 formation mediated through the BK-stimulated B2 receptor signalling pathway. PF-04886847 blocked kallikrein-induced endothelial-dependent relaxation of isolated rat aortic rings pre-contracted with phenylephrine. CONCLUSIONS AND IMPLICATIONS PF-04886847 was shown to be the most potent small molecule inhibitor of plasma kallikrein yet described; it inhibited kallikrein in isolated aortic rings and cultured endothelial cells. Overall, our results indicate that PF-04886847 would be useful for the treatment of kallikrein-mediated inflammatory disorders.
Biochemical Journal, 2003
We have demonstrated that the S 1 and S 2 subsites of human tissue kallikrein (hK1) play determinant roles in the recognition and hydrolysis of substrates. The presence of serine at position P 1 and arginine at P 2 resulted in the best substrate, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, which was derived from the kallistatin reactive-centre loop sequence and quencher groups o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp). Serine and arginine are also the residues at positions P 1 and P 2 in human kininogen, from which hK1 releases Lys-bradykinin. Several peptide analogues of Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, in which the Ser and Arg residues were substituted with various other amino acids, were synthesized and tested as substrates. Most of them were hydrolysed slowly, although they showed significant binding to hK1, as demonstrated by their competitive inhibition constants (K i ). Using this information, six peptides were designed, synthesized and assayed as inhibitors of hK1. Abz-Lys-Phe-Phe-Pro-Arg-Gln-EDDnp, Abz-Lys-Phe-Arg-Pro-Arg-Gln-EDDnp and acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH 2 inhibited hK1 in the range 20-30 nM (letters in italics denote the D-form of the amino acid). The peptide acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH 2 was a weak inhibitor for other serine proteases, as indicated by the higher K i values compared with hK1, but this peptide was a potent inhibitor of human plasma kallikrein, which has a K i value of 8 nM. This result was surprising, since this enzyme is known to be a restricted arginyl-hydrolase. In conclusion, acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH 2 can be used as a leader compound to design specific inhibitors for hK1, plasma kallikrein, or for both at same time, if the inhibition of kinin release is the main goal.
Synthesis of potent and selective inhibitors of human plasma kallikrein
Bioorganic & Medicinal Chemistry Letters, 1999
The synthesis and in vitro enzyme inhibition profile of a series of novel trifluoromethyiketone (TFMK) inhibitors of human plasma kallikrein (PK) are described. We have developed an efficient method for the construction of peptide TFMKs that provides the final product devoid of compromised stereochemieal integrity. Many of these compounds are potent inhibitors of PK and exhibit reduced inhibition of tissue kaUikrein (TK) and plasmin (HP).
Biochemical Journal, 2004
The kininogenase activities of mouse (mK1), rat (rK1) and human (hK1) tissue kallikreins were assayed with the bradykinin-containing synthetic peptides Abz-MTEMARRPPGFSPFRSVTVQ-NH 2 (where Abz stands for o-aminobenzoyl) and Abz-MTS-VIRRPPGFSPFRAPRV-NH 2 , which correspond to fragments Met 374 -Gln 393 and Met 375 -Val 393 of mouse and rat LMWKs (lowmolecular-mass kininogens) with the addition of Abz. Bradykinin was released from these peptides by the mK1-and rK1-mediated hydrolysis of Arg-Arg and Arg-Ser (or Arg-Ala) peptide bonds. However, owing to preferential hydrolysis of Phe-Arg compared with the Arg-Ala bond in the peptide derived from rat LMWK, hK1 released bradykinin only from the mouse LMWK fragment and preferentially released des-[Arg 9 ]bradykinin from the rat LMWK fragment (Abz-MTSVIRRPPGFSPFRAPRV-NH 2 ). The formation of these hydrolysis products was examined in more detail by determining the kinetic parameters for the hydrolysis of synthetic, internally quenched fluorescent peptides containing six N-or C-terminal amino acids of bradykinin added to the five downstream or upstream residues of mouse and rat kininogens respectively. One of these peptides, Abz-GFSPFRAPRVQ-EDDnp (where EDDnp stands for ethylenediamine 2,4-dinitrophenyl), was preferentially hydrolysed at the Phe-Arg bond, confirming the potential des-[Arg 9 ]bradykinin-releasing activity of hK1 on rat kininogen. The proline residue that is two residues upstream of bradykinin in rat kininogen is, in part, responsible for this pattern of hydrolysis, since the peptide Abz-GFSPFRASRVQ-EDDnp was preferentially cleaved at the Arg-Ala bond by hK1. Since this peptidase accepts the arginine or phenylalanine residue at its S 1 subsite, this preference seems to be determined by the prime site of the substrates. These findings also suggested that the effects observed in rats overexpressing hK1 should consider the activation of B1 receptors by des-[Arg 9 ]bradykinin. For further comparison, two short internally quenched fluorescent peptides that bind to hK1 with affinity in the nM range and some inhibitors described previously for hK1 were also assayed with mK1 and rK1.
Activation and enzymatic characterization of recombinant human kallikrein 8
Biological Chemistry, 2006
Human kallikrein 8 (hK8), whose gene was originally cloned as the human ortholog of a mouse brain protease, is known to be associated with diseases such as ovarian cancer and Alzheimer's disease. Recombinant human pro-kallikrein 8 was activated with lysyl endopeptidaseconjugated beads. Amino-terminal sequencing of the activated enzyme demonstrated the cleavage of a 9-aa propeptide from the pro-enzyme. The substrate specificity of activated hK8 was characterized using synthetic fluorescent substrates. hK8 showed trypsin-like specificity, as predicted from sequence analysis and enzymatic characterization of the mouse ortholog. All synthetic substrates tested containing either arginine or lysine at P1 position were cleaved by hK8. The highest k cat /K m value of 20=10 3 M-1 s-1 was observed with Boc-Val-Pro-Arg-7amido-4-methylcoumarin. The activity of hK8 was inhibited by antipain, chymostatin, and leupeptin. The concentration for 50% inhibition by the best inhibitor, antipain, was 0.46 mM. The effect of different metal ions on the enzyme activity was analyzed. Whereas Na q had no effect on hK8 activity, Ni 2q and Zn 2q decreased the activity and Ca 2q , Mg 2q , and K q had a stimulatory effect. Ca 2q was the best activator, with an optimal concentration of approximately 10 mM.
Human plasma kallikrein processing: proteolysis as an alternative control
Agents and actions. Supplements, 1992
Human plasma kallikrein (HuPK) was detected in normal non-activated and dilution-activated plasma by immunoblotting, using polyclonal antibodies. In non-activated plasma, the predominatly detected protein corresponds to prokallikrein (Mr 80,000-90,000). Activated plasma, besides kallikrein, contains larger proteins (Mr > 130,000) that possibly represent complexes between kallikrein and proteinase inhibitors. Plasma also contains species (Mr 43,000) which corresponds to kallikrein heavy chain. In activated plasma, monoclonal antibodies against kallikrein heavy chain detected, besides these same bands described above, two additional bands (Mr 30,000 and 20,000) possibly correspondent to fragments of kallikrein heavy chain.
European Journal of Medicinal Chemistry, 2013
Netherton syndrome is caused by loss-of-function mutations in SPINK5 encoding the Kazal-type inhibitor LEKTI-1 leading to dysregulation of proteolytic cascades involving several kallikreins. We used both structure-based and ligand-based virtual screening computations to identify commercially available noncovalent inhibitors of human kallikrein 5 (hK5), a serine protease (trypsin-like) that plays a central role in the initiation of the molecular cascades leading to the Netherton syndrome phenotype. The efficacy and mechanism of inhibition of the identified new families of organic compounds were analyzed not only for hK5 but also on other proteases implicated in the cascades (hK7, hK14 and matriptase). These inhibitors are nontoxic on healthy human keratinocytes and are structurally different from traditional serine protease inhibitors validating their potential utility as initial hits to control proteolytic disorders observed in dermatological pathologies such as Netherton syndrome.
Highly sensitive automated chemiluminometric assay for measuring free human glandular kallikrein-2
Clinical chemistry, 1999
Human glandular kallikrein (hK2) is a serine protease that has 79% amino acid identity with prostate-specific antigen (PSA). Both free hK2 and hK2 complexed to alpha1-antichymotrypsin (ACT) are present in the blood in low concentrations. We wished to measure hK2 in serum with limited contribution from hK2-ACT for the results. We developed an automated assay for hK2 with use of a select pair of monoclonal antibodies. The prototype assay was implemented on a Beckman Coulter ACCESS(R) analyzer. The detection limit of the assay was 1.5 ng/L, the "functional sensitivity" (day-to-day CV <15%) was <4 ng/L, cross-reactivity with PSA and PSA-ACT was negligible, and cross-reactivity with hK2-ACT was 2%. After surgical removal of prostate glands, serum hK2 was <7 ng/L and was <15 ng/L in most healthy women. The median serum concentration of hK2 in healthy men without prostate cancer was 26 ng/L. The median concentration of hK2 was 72 ng/L for men having prostate cancer wi...
Journal of Protein Chemistry, 2000
Human plasma kallikrein (huPK) is a proteinase that participates in several biological processes. Although various inhibitors control its activity, members of the Kazal family have not been identified as huPK inhibitors. In order to map the enzyme active site, we synthesized peptides based on the reactive site (PRILSPV) of a natural Kazal-type inhibitor found in Cayman plasma, which is not an huPK inhibitor. As expected, the leader peptide (Abz-SAPRILSPVQ-EDDnp) was not cleaved by huPK. Modifications to the leader peptide at P 1 , P 3 and P 4 positions were made according to the sequence of a phage display-generated recombinant Kazal inhibitor (PYTLKWV) that presented huPK-binding ability. Novel peptides were identified as substrates for huPK and related enzymes. Both porcine pancreatic and human plasma kallikreins cleaved peptides at Arg or Lys bonds, whereas human pancreatic kallikrein cleaved bonds involving Arg or a pair of hydrophobic amino acid residues. Peptide hydrolysis by pancreatic kallikrein was not significantly altered by amino acid replacements. The peptide Abz-SAPRILSWVQ-EDDnp was the best substrate and a competitive inhibitor for huPK, indicating that Trp residue at the P 4 position is important for enzyme action.