Carlo Gallina

2 Å X-ray structure of adamalysin II complexed with a peptide phosphonate inhibitor adopting a retro-binding mode

The search of reprolysin inhibitors offers the possibility of intervention against both matrixins and ADAMs. Here we report the crystal structure of the complex between adamalysin II, a member of the reprolysin family, and a phosphonate inhibitor modeled on an endogenous venom tripeptide. The inhibitor occupies the primed region of the cleavage site adopting a retro‐binding mode. The phosphonate group ligates the zinc ion in an asymmetric bidentate mode and the adjacent Trp indole system partly fills the primary specificity subsite S1′. An adamalysin‐based model of tumor necrosis factor‐α‐converting enzyme (TACE) reveals a smaller S1′ pocket for this enzyme.

Peptidyl and azapeptidyl methylketones as substrate analog inhibitors of papain and cathepsin B

Summary Peptidyl methylketones containing Phe, Tyr, Tyr(I), Tyr(I 2 ), Leu and Ile in P 2 were synthesized and tested as substrate analog reversible inhibitors of papain and bovine spleen cathepsin B. The most effective cathepsin B inhibitor contained Tyr(I 2 ) and displayed an inhibition constant of 4.7 μM at pH 6.8 and 25°C, while Leu or Ile gave practically inert analogs. Replacement of the amino acids in P 2 with the analogous α-azaamino acids, as well as the glycine in P 1 with α-azaglycine, led to complete loss of inhibiting activity. Introducing alkoxy substituents at the methyl adjacent to the ketone group generally resulted in more effective inhibitors, with inhibition constants in the micromolar range for both papain and cathepsin B.

INHIBITION OF ADAMALYSIN II AND MMPS BY PHOSPHONATE ANALOGUES OF SNAKE VENOM PEPTIDES

Phosphonate analogues of the peptidomimetic N-(Furan-2-yl)carbonyl-Leu-Trp-OH were prepared with the goal of evaluating the effect of phosphonate for carboxylate replacement on binding with snake venom metalloproteinases and MMPs. N-(Furan-2-yl)carbonyl-Leu-L-Trp(P)-(OH)2 showed a 75-fold increase of the inhibiting activity against adamalysin II, a snake venom metalloproteinase structurally related to MMPs and TACE. Both the phosphonate and carboxylate peptidomimetics fit into the active site adopting a retrobinding mode and provide the structural base for a new class of metalloproteinases inhibitors.

Irreversible inactivation of papain and cathepsin B by epoxidic substrate analogues

Abstract Epoxidic substrate analogues related to allylamine (4a–4e) and allyl alcohol (5a–5f) were synthesized and tested as models of cysteine-protease inhibitors. They proved to be irreversible inhibitors of papain and cathepsin B with pseudo-first-order inactivation rates ranging from 0.3 to 33 M−1 min−1. The most active of the studied oxiranes 4a bears N-acetyl- l -Phe as peptidyl unity. Most of the inhibitory activity was retained when the recognising moiety was extensively modified, provided that a phenyl group ∂ or the trapping epoxide e was present. Specificity of peptidylepoxides for cysteine-proteases was confirmed, since no inhibitory activity was displayed toward serine or metallo-proteases.

Synthesis and inhibiting activity of peptidylketones as substrate analogues of papain

Abstract Four representative peptidylketones were synthesized and evaluated as models of cysteine—protease inhibitors. They were shown to be slow-binding, reversible inhibitors of papain with K i ranging from 8.4–;260 μM. The effectiveness of the inhibitor was enhanced by the introduction of 3 chlorine atoms α to the keto group while a small decrease was observed with analogous substitution by fluorine. The structure—activity relationships are discussed.

Computational study of the catalytic domain of human neutrophil collagenase. specific role of the S3 and S'3 subsites in the interaction with a phosphonate inhibitor.

Human neutrophil collagenase (HNC, MMP-8) is one of the target enzymes for drug treatment of pathologic extracellular matrix degradation. Peptidomimetic inhibitors bind in the S′-side of the enzyme active site occupying the S′1primary specificity pocket by their large hydrophobic side-chains. The crystal structure of the complex between the catalytic domain of MMP-8 and Pro-Leu-l-TrpP(OH)2(PLTP) showed that this phosphonate inhibitor binds in the S side of the active site. This finding was unexpected since it represents the first example of accommodation of the bulky Trp indolyl chain in the S1rather than in the S′1subsite. Dynamical and structural factors favouring this uncommon mode of binding were therefore investigated.MD simulations performed on the uncomplexed enzyme show that its structure in aqueous solution is only slightly different from the crystal structure found in the complex with PLTP. ED analysis of the MD simulations, performed on PLTP alternatively interacting with the S- or S′-side of the active site, shows that the enzyme fluctuation increases in both cases. The main contribution to the overall enzyme fluctuation is given by the loop 164–173. The fluctuation of this loop is spread over more degrees of freedom when PLTP interacts with the S-side. This dynamical factor can enhance the preference of PLTP for the S subsites of MMP-8. MD simulations also show that ligation of PLTP in the S subsites is further favoured by better zinc chelation, a cation-π interaction at the S3subsite and unstrained binding conformations. The role of the S3, S′3and S′1subsites in determining the inhibitor binding is discussed.

Iodo and diiodotyrosine epoxysuccinyl derivatives as selective inhibitors of cathepsin B

Eight new analogs of l-trans-epoxysuccinyl-l-leucylamido(3-methyl)butane (E-64-c) containing Phe, Tyr, Tyr(1) or Tyr(I2) in place of Leu, were synthesized and tested as inhibitors of papain, bovine spleen cathepsin B, calpain I and II from porcine red cells and porcine kidney, respectively. By use of kinetic methods, the new E-64 analogs proved to irreversibly inactivate both papain and cathepsin B via reversible enzyme-inhibitor intermediates EI. Second-order rate constants for inactivation were in the range 3500-55 100 M−1s−1 for papain and 650–105 000 M−1s−1 for cathepsin B. For the inactivation of calpain I and II they ranged between 250 and 2000 M−1s−1 and were similar to those of the known E-64-c. The effectiveness of the amino acid contained in the inhibitors tested increased in the order Tyr(I) ≈ Tyr(I2) < Tyr < Phe < Leu for papain and Phe < Tyr < Tyr(I) < Leu < Tyr(I2) for cathepsin B inactivation. Replacement of the l with the d-trans-epoxysuccinyl unit caused a 10–100-fold decrease in inhibitor potencies.

N-Haloacetyl-amino-acid amides as active-site-directed inhibitors of papain and cathepsin B

Abstract A series of N -haloacetyl-amino-acid amides were synthesized and tested as models of cysteine-protease inhibitors. They irreversibly inactivated papain and cathepsin B via a reversible enzyme-inhibitor intermediate. Apparent second-order rate constants of inactivation ranging from 65 to 16 700 M −1 s −1 were observed. Reactivity against papain, as compared to glutathione, was increased 16 400-fold for N -bromoacetyl-leucine isopentylamide and 25 700-fold for the corresponding iodoacetyl derivative; these increases are probably due to proximity effects. No inhibition of trypsin, chymotrypsin and porcine pancreatic elastase was observed. Haloacetamides represent an interesting class of easily synthesized, efficient, irreversible inhibitors of cysteine proteases, which have low non-specific alkylating properties.

Synthesis and inhibiting activities of 1-peptidyl-2-haloacetyl hydrazines toward cathepsin B and calpains

Abstract Twenty-four 1-peptidyl-2-haloacetyl hydrazines which can be considered azapeptide halomethanes were synthesized and tested as models of cathepsin B, calpain I and calpain II inhibitors. Reagents designed for cathepsin B inactivation include Z-Tyr, Z-Tyr(I) and Z-Leu-Leu attached to an α-azaglycine or α-azaalanine unit in P 1 . By use of kinetic analysis, they proved to irreversibly inactivate cathepsin B via a reversible enzyme-inhibitor intermediate. Second-order rate constants in the range 725-306 000 M −1 s −1 were found for cathepsin B inactivation, with no more than 7 500 M −1 s −1 for calpain II. K 1 for the reversible EI adducts ranged from 11 to 0.06 μM for cathepsin B. Structure of the possible reversible EI complex is proposed and used to discuss the effects of structural variation of the inhibitors on the kinetic parameters of inactivation. 1-Peptidyl-2-haloacetyl hydrazines designed for calpain inactivation include Boc-Val-(N ϵ -carbomethoxy)Lys-Leu, Boc-Val-Lys(N ϵ -Z)-Leu, Boc-Val-Lys(N ϵ -Tos)-Leu and Z-Leu-Leu attached to an α-azatyrosine unit in P 1 . They gave poor results. Title compounds proved to be selective for cysteine proteases, since no inhibiting activity could be detected toward trypsin, chymotrypsin and porcine pancreatic elastase at 0.1 mM concentration. Relatively low aspecific alkylating properties were also demonstrated in tests using glutathione as the nucleophile.

Functional residues at the active site of horse liver phosphopantothenoylcysteine decarboxylase

Horse liver phosphopantothenoylcysteine decarboxylase (EC 4.1.1.36) is rapidly inactivated by N‐acetoacetylation with diketene following a pseudo‐first‐order kinetics: the presence of substrate quantitatively protects against this inactivation. Histidine photo‐oxidation with methylene blue or rose bengal brings about the total loss of activity. These results indicate the presence of functional lysyl and histidyl groups at the active site of the enzyme. The substrate sulphydryl group is essential for enzyme activity. Enzymatic decarboxylation is proposed to result from a combined action of the keto group of the enzyme‐bound pyruvate protonated by an essential histidine and a protonated amino group of a lysine.