Residue In P1 Position Research Articles

Inhibition of Human and Yeast 20S Proteasome by Analogues of Trypsin Inhibitor SFTI-1

Starting from the primary structure of sunflower trypsin inhibitor SFTI-1, we designed novel non-covalent inhibitors of human and yeast 20S proteasomes. Peptides with Arg residue in P1 position and two basic amino acid residues (Lys or/and Arg) in P2′ and P3′ positions strongly inhibited chymotrypsin-like and caspase-like activities, while trypsin-like activity was poorly modified. We found that some SFTI-1 analogues up-regulated exclusively the chymotrypsin-like activity of latent yeast 20S proteasome.

The Combination of Bortezomib and NPI-0052 Exerts Anti-Tumor Activity in Waldenstrom Macroglobulinemia (WM).

Background: WM is an incurable low-grade lymphoplasmacytic lymphoma. Bortezomib has recently demonstrated about 50% ORR in patients with relapsed WM. We therefore investigated the in vitro effect of the new proteasome inhibitor NPI-0052 (N) alone and in combination with Bortezomib (B).Methods: WM cell lines (BCWM1,WSU-WM) and IgM secreting cell lines (MEK1, Namalwa) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured using MTT assay and [3H]-thymidine uptake. Determination of the synergistic effect [combination index (CI)] of combination was calculated using the CalcuSyn software. Cell signaling and apoptotic pathways were determined by Western Blot. We also tested the effect of N on WM cells in the co-culture with BMSCs. Activity of the 20S proteasome was determined by detecting the release of the fluorophore AMC, after cleavage from the labeled substrates specific for each enzymatic activity.Results: N induced cytotoxicity and inhibition of DNA synthesis (IC50 15nM) in BCWM.1 (48 h). Similar effects were demonstrated in IgM secreting cell lines and primary CD19+ WM cells (IC50 18–30nM). No cytotoxicity was observed on peripheral blood mononuclear cells. The combination of N+B significantly inhibited BCWM.1 proliferation compared to each agent alone: B (5nM) induced cytotoxicity in 8.5%, which increased to 26%, 40% and 53% in the presence of N 2.5nM (CI:0.83), 5nM (CI:0.72) and 10nM (CI:0.7) respectively, indicating synergism. To determine the mechanism of synergy, we investigated the effect of the two agents and their combination on proteasome activity and on signaling pathways, specficially the Akt pathway. Both N and B inhibited the three proteasome activities: the combination of N+B was increased compared to the effect of each agent alone on the caspase-like (C-l) activity of the proteasome. B and N used as single agents induced 29% and 34% inhibition of the C-l activity, respectively, compared to 60% when B and N were used in combination. The C-l activity preferentially cleaves substrates with an aspartic residue in P1 position, like the substrates of the caspases. N induced caspase-8, PARP cleavage and increase of Smac as well as down-modulation of the anti-apoptotic proteins c-IAP, XIAP, survivin, Bcl-2, Mcl-1. The combination of N+B induced a stronger and more significant induction of caspase-8, -PARP cleavage, as well as caspase-3 and -9, which were not affected by using N alone. Similarly, Smac modulation resulted in a more significant induction when cells were exposed to both proteasome inhibitors. N inhibited Akt phosphorylation in BCWM.1 cells (6h) in a dose-dependent manner. GSK3 phosphorylation and ribosomal protein-S6, Akt-downstream target proteins, were also markedly inhibited. Importantly, N inhibited Akt phosphorylation and Akt activity in BCWM.1, even when combined with B, which induced increase of Akt phosphorylation. Lastly, neither exposure to IL-6 nor adherence to BMSCs conferred protection to WM cells against NPI-induced cytotoxicity.Conclusion: NPI-0052 has significant antitumor activity in WM in vitro especially in combination with Bortezomib. These results provide the framework for clinical trials in WM.

A phosphonamidate containing aromatic N-terminal amino group as inhibitor of leucine aminopeptidase—design, synthesis and stability

Fully deprotected phosphonamidate dipeptides, predicted as effective inhibitors of cytosolic leucine aminopeptidase, showed unexpected instability in water solution at pH below 12. Their hydrolysis rate was strictly correlated with basicity of the N-terminal amino group. To improve this feature a phosphonamidate analogue containing less basic, aromatic 2-aminophenylphosphonate residue in P1 position of the inhibitor was designed. The target compound was synthesised starting from diethyl 2-nitrophosphonate in several step procedure. The decrease in basicity of the terminal amino moiety of the modified analogue in fact resulted in satisfactory improvement of hydrolytic stability of the P–N bond. The developed phosphonamidate was proved to be fully resistant to hydrolysis above pH 7. Surprisingly, tested in enzymatic assays towards leucine aminopeptidase (optimum pH 8.5), it did not exhibit inhibition activity up to milimolar concentration. The explanation could be that diminishing the basic character of the terminal amino group may result in a change of its affinity towards the zinc ions.

Engineering d-amino acid containing novel protease inhibitors using catalytic site architecture

The mechanism of proteolysis by serine proteases is a reasonably well-understood process. Typically, a histidine residue acting as a general base deprotonates the catalytic serine residue and the hydrolytic water molecule. We disclose here, the use of an unnatural d-amino acid as a strategic residue in P1 position, designed de novo based on the architecture of the protease catalytic site to impede the catalytic histidine residue at the stage of acyl-enzyme intermediate. Several probe molecules containing d-homoserine or its derivatives at P1 position are evaluated. Compounds 1, 6, and 8–10 produced up to 57% loss of activity against chymotrypsin. More potent and specific inhibitors could be designed with structure optimization as this strategy is completely general and can be used to design inhibitors against any serine or cysteine protease.

The specificity of DP IV for natural substrates is peptide structure determined.

Our results indicate that the substrate properties of peptides are encoded by their own structure. That means, that substrate characteristics depend not only on the primary structure around the catalytic site rather C-terminal located secondary interactions strongly influence the binding and catalysis of the substrates. Such interaction sites seem to force the ligand in a proper orientation to the active site of DP IV. As result of these relations the hydrolysis of peptides with non-proline and non-alanine residues in P1-position (Ser, Val, Gly) becomes possible in longer peptides.

Chromogenic Substrates of Bovine β-Trypsin: The Influence of an Amino Acid Residue in P1 Position on Their Interaction with the Enzyme

The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design and synthesize a series of trypsin chromogenic substrates modified in position P1: Ac-Ala-Val-Abu-Pro-X-pNA, where X = Orn, Lys, Arg, Har, Arg(NO2), Cit, Hci, Phe(p-CN), Phe(p-NH2); pNA = p-nitroanilide. The most active compounds (as determined by specificity constant kcat/Km) were peptides with the Arg and Lys residues in the position discussed. Changes in the length and the decrease of the positive charge of the amino acid residue side chain in position P1 resulted in the decrease or loss of the affinity towards bovine β-trypsin. Among peptides containing amino acid residues with uncharged side chains in position P1, only one with p-cyano-l-Phe revealed activity. These results correspond well with trypsin inhibitory activity of CMTI-III analogues modified in the equivalent position, indicating the same type of interaction between position P1 of the substrate or inhibitor and S1 site specificity of trypsin.

Synthesis and application of sorbents for affinity chromatography of serine proteases

A synthetic peptide derivative,—alanyl-alanyl-leucylmorpholide, (Ala-Ala-Leu-N(CH2CH2)2O), is suggested as a specific ligand for affinity chromatography of subtilisin-like serine proteases that prefer hydrophobic amino acid residue in P1 position of their substrates. Z-Ala-Ala-Leu-N(CH2CH2), a weak and reversible inhibitor of serine proteases, was synthesized by the carbodiimide method. Affinity sorbents were prepared by coupling the synthesized pepitide derivates to CH or AH Sepharose. Serine proteases from different sources were purified up to 17 fold on these sorbents with yields varying from 25% to 100%. Three enzymes (serine protease X, kallikrein and leucine aminopeptidase) were isolated from urine of children with glomerulonephritis with yields of 57, 22 and 55%. Proteolytic enzymes from the dandelion root, Kamchatka crab and culture filtrates of different microorganisms were also purified on the affinity sorbents.