Peptidyl Chloromethyl Ketones Research Articles

Efficient Synthesis of New Peptidyl Chloromethyl Ketones for the Application of Proteinase K Inhibitors

The synthesis of a new series of peptidyl chloromethyl ketones such as MeOSuc-Ala-Ala-Pro-Ile-CH2Cl (AAPI) and MeOSuc-Ala-Ala-Ala-Pro-Ile-CH2Cl (AAAPI) is described.

Peptide substrate profiling defines fibroblast activation protein as an endopeptidase of strict Gly 2-Pro 1-cleaving specificity

Fibroblast activation protein (FAP) is a serine protease of undefined endopeptidase specificity implicated in tumorigenesis. To characterize FAP’s P 4 – P 2 ′ specificity, we synthesized intramolecularly quenched fluorescent substrate sets based on the FAP cleavage site in α 2-antiplasmin (TSGP-NQ). FAP required substrates with Pro at P 1 and Gly or d-amino acids at P 2 and preferred small, uncharged amino acids at P 3, but tolerated most amino acids at P 4, P 1 ′ and P 2 ′ . These substrate preferences allowed design of peptidyl-chloromethyl ketones that inhibited FAP, but not the related protease, dipeptidyl peptidase-4. Thus, FAP is a narrow specificity endopeptidase and this can be exploited for inhibitor design.

Purification of soluble and membrane-bound proteases with substrate-analogous inhibitors by affinity chromatography

Specific modified substrate-analogous amino acids and peptides have been used as affinity ligands in the affinity chromatography of proteases. Alanine methyl ketone-Sepharose (AMK-Sepharose) is introduced as affinity support for the purification of a bacterial alanyl aminopeptidase (AAP) from a membrane protein extract and Arginine-Agarose as support for the preparation of a membrane-bound proteinase of myeloma cells (MP-1). Peptidyl methyl ketones as affinity ligands have been used to separate subtilisin enzymes and the cysteine proteases cathepsin B, L, and S. As a new type of ligands, spacer-bound peptidyl chloromethyl ketones are presented for a specific and oriented immobilization of proteinases. Oriented-immobilized cathepsin B was used to isolate antibodies against this enzyme.

Characterisation of the antitrypanosomal activity of peptidyl α-aminoalkyl phosphonate diphenyl esters

Two groups of irreversible serine peptidase inhibitors, peptidyl chloromethyl ketones and peptidyl phosphonate diphenyl esters, were examined for antitrypanosomal activity against the bloodstream form of Trypanosoma brucei brucei. Both peptidyl chloromethyl ketones and peptidyl phosphonate diphenyl esters inhibited trypsin-like peptidases of the parasites and exhibited antitrypanosomal activity at micromolar concentrations. In live T. b. brucei, labelled analogues of both of these groups of inhibitors primarily targeted an 80-kDa peptidase, possibly a serine oligopeptidase known as oligopeptidase B. In an in vivo mouse model of infection, one of these inhibitors, carbobenzyloxyglycyl-4-amidinophenylglycine phosphonate diphenyl ester, was curative at 5 mg kg −1 day −1 but appeared toxic at higher doses. There was no significant correlation between the inhibitory potency (as evaluated against purified T. b. brucei oligopeptidase B) and the in vitro antitrypanosomal efficacy of either group of inhibitors, suggesting that these inhibitors were acting on multiple targets within the parasites, or had different cell permeability properties. These findings suggest that serine peptidases may represent novel chemotherapeutic targets in African trypanosomes.

Proteolytic Processing of Marburg Virus Glycoprotein

Processing of the transmembrane glycoprotein (GP) of Marburg virus involved the conversion of an endo H-sensitive, ER-specific form into an endo H-resistant, Golgi-specific precursor that was cleaved into GP1 and GP2. Cleavage was mediated by furin or another subtilisin-like endoprotease with similar substrate specificity as indicated by mutational analysis of the cleavage site and inhibition using peptidyl chloromethylketones. Mature GP consisted of disulfide-linked GP1 and GP2 subunits.

Purification and characterisation of a trypsin-like serine oligopeptidase from Trypanosoma congolense

Trypanosoma brucei contain a serine oligopeptidase (OP-Tb) that is released into (and remains active in) the blood of trypanosome-infected animals. Here a similar enzyme from Trypanosoma congolense is described . This oligopeptidase, called OP-Tc, was purified using three-phase partitioning, and ion-exchange and affinity chromatography. OP-Tc is inhibited by alkylating agents, by serine peptidase-specific inhibitors including 3,4-dichloroisocoumarin, 4-(2-aminoethyl)benzenesulfonylfluoride and diispropylfluoro-phosphate and by other peptidase inhibitors including leupeptin, antipain and peptidyl chloromethyl ketones. Reducing agents such as dithiothreitol enhanced activity as did heparin, spermine and spermidine. The enzyme has trypsin-like specificity since it cleaved fluorogenic peptides that have basic amino acid residues (Arg or Lys) in the P 1 position. Potential substrates without a basic residue in P 1 were not hydrolysed. Although OP-Tc has weak arginine aminopeptidase activity, the enzyme clearly preferred substrates that had amino acids in the P 2 and P 3 positions. Overall, OP-Tc appears to be less efficient than OP-Tb because it usually displayed lower k cat/ K m values for the substrates tested. However, like OP-Tb, the best substrate for OP-Tc was Cbz-Arg-Arg-AMC ( K m=0.72 μM, k cat=96 s −1). OP-Tc preference for amino acids in the P 2 position was (Gly,Lys,Arg)>Phe>Leu>Pro. The results also suggest that the P 3-binding site has hydrophobic characteristics. OP-Tc may not be a naturally immunodominant molecule because neither IgG nor IgM anti- OP-Tc antibodies were detected in the blood of experimentally infected cattle.

Cysteine Proteinase Inhibitors Kill Cultured Bloodstream Forms ofTrypanosoma brucei brucei

Troeberg, L., Morty, R. E., Pike, R. N., Lonsdale-Eccles, J. D., Palmer, J. T., McKerrow, J. H., and Coetzer, T. H. T. 1999. Cysteine proteinase inhibitors kill cultured bloodstream forms ofTrypanosoma brucei brucei. Experimental Parasitology91, 349–355.Trypanosoma brucei bruceiis a causative agent of bovine trypanosomiasis (nagana), a disease of considerable economic significance in much of Africa. Here we report investigations on the effects of various irreversible cysteine proteinase inhibitors, including vinyl sulfones (VS), peptidyl chloromethylketones (CMK), diazomethylketones, and fluoromethyl ketones, on the major lysosomal cysteine proteinase (trypanopain-Tb) ofT. b. bruceiand onin vitro-cultured bloodstream forms of the parasite. Many of the tested inhibitors were trypanocidal at low micromolar concentrations. Methylpiperazine urea-Phe-homoPhe-VS was the most effective trypanocidal agent, killing 50% of test populations at a work ing concentration of 0.11 μM, while carbobenzoxy-Phe-Phe-CMK was the most trypanocidal of the methylketones with an IC50of 3.6 μM. Labelling of live and lysedT. b. bruceiwith biotinylated inhibitor derivatives suggests that trypanopain-Tb is the likely intracellular target for these inhibitors. Kinetic analysis of the inhibition of purified trypanopain-Tb by the inhibitors showed that most hadkassvalues in the 106M−1s−1range. We conclude that cysteine proteinase inhibitors have potential as trypanocidal agents and that a major target of these compounds is the lysosomal enzyme trypanopain-Tb.

Inhibition of 3C Protease from Human Rhinovirus Strain 1B by Peptidyl Bromomethylketonehydrazides

The gene coding for the 3C protease from human rhinovirus strain 1B was efficiently expressed in anEscherichia colistrain which also overexpressed the rareargUtRNA. The protease was isolated from inclusion bodies, refolded, and exhibited akcat/Km= 3280 M−1s−1using an internally quenched peptidyl fluorogenic substrate. This continuous fluorogenic assay was used to measure the kinetics of 3C protease inhibition by several conventional peptidyl chloromethylketones as well as a novel series of compounds, the bromomethylketonehydrazides. Compounds containing the bromomethylketonehydrazide backbone and a glutamine-like side chain at the P1 position were potent, time-dependent inhibitors of rhinovirus 3C protease withkinact/Kinactvalues as high as 23,400 M−1s−1. The inhibitory activity of compounds containing modified P1 side chains suggests that the interactions between the P1 carboxamide group and the 3C protease contributes at least 30-fold to thekinact/Kinactrate constants for bromomethylketonehydrazide inhibition of 3C protease. Electrospray ionization mass spectrometry measurements of the molecular weights of native and inhibited 3C protease have established an inhibitory mechanism involving formation of a covalent adduct between the enzyme and the inhibitor with the loss of a bromide ion from the bromomethylketonehydrazide. Tryptic digestion of bromomethylketonehydrazide-inhibited 3C protease established adduct formation to a peptide corresponding to residues 145–154, a region which contains the active site cysteine-148 residue. The bromomethylketonehydrazides were fairly weak inhibitors of chymotrypsin, human elastase, and cathepsin B and several of these compounds also showed evidence for inhibition of human rhinovirus 1B replication in cell culture.

Stretch-induced Hypertrophic Growth of Cardiocytes and Processing of Brain-type Natriuretic Peptide Are Controlled by Proprotein-processing Endoprotease Furin

When hypertrophic growth is induced in neonatal rat cardiocytes by stretching, the cardiocytes express high levels of brain-type natriuretic peptide (BNP) and the proprotein-processing enzyme furin. A BNP precursor, gammaBNP, possesses a furin-cleavable Arg-X-X-Arg motif, which is cleaved when gammaBNP is processed to form BNP-45. The Arg-X-X-Arg motif is found in many precursors of growth factors and growth-related proteins. To determine if furin converts gammaBNP to BNP-45 as well as other unidentified growth-promoting protein precursors to their active form that may induce hypertrophic growth in cardiocytes, we used two protease inhibitor systems, synthetic peptidyl chloromethyl ketones (CMK) (dec-Arg-Val-Lys-Arg-CMK and dec-Phe-Ala-Lys-Arg-CMK; where dec is decanoyl) and vaccinia vector-integrated native and variant alpha1-antitrypsins. The furin-specific inhibitors, dec-Arg-Val-Lys-Arg-CMK and variant alpha1-antitrypsin with the inhibitory determinant Arg-X-X-Arg, suppressed the stretch-induced hypertrophic growth of cardiocytes as well as the processing of gammaBNP to BNP-45. The other serine protease inhibitors and variant alpha1-antitrypsin against elastase, or thrombin, however, neither suppressed the hypertrophic growth nor prevented the processing of gammaBNP to BNP-45. Thus, we suggest that furin catalyzes the conversion of gammaBNP to BNP-45 as well as growth-promoting proproteins to their active form, which might induce hypertrophic growth in cardiocytes.

Amino Acids and Peptides. Part 48.1 Studies on the Structure of an Unexpected Reaction Product from Dipeptidyl Chloromethyl Ketone during Acid Hydrolysis 2

T. Yokoi, H. Taguchi, Y. Nishiyama, K. Igarashi, H. T. Y. N. K. I. F. K. Y. O., F. Kasuya and Y. Okada, J. Chem. Res. (S), 1997, 10 DOI: 10.1039/A606548A

Labeled proteinase inhibitors: Versatile tools for the characterization of serine proteinases in solid-phase assays

Peptidyl chloromethyl ketones were used for the specific labeling of proteinases by attaching a biotin group to the N-terminal end of the peptide. Such labeled peptide inhibitors allowed the detection and quantitation of proteolytic enzymes immobilized on the plastic surface of a microtiter plate, as well as on nitrocellulose. The validity of these solid-phase assays was demonstrated using subtilisin Carlsberg as a model enzyme and biotinyl-ε-aminocaproyl-L-alanyl-L-alanyl-L-prolyl-L-p henylalanyl-chloromethyl ketone as a specific reagent. In addition to being usable for the screening of a particular proteinase in a large number of samples, these assays can be adapted for the analysis of specific proteolytic enzyme present in complex mixtures.

Zymogen/enzyme discrimination using peptide chloromethyl ketones

Glutamylglycinylarginyl chloromethyl ketone, tyrosylglycinylarginyl chloromethyl ketone, and phenylalanylprolylarginyl chloromethyl ketone have been labeled at their amino termini using fluorescein, rhodamine-X, lissamine-rhodamine, pyrene, and the 1,5-, 2,5-, and 2,6-dimethylaminonaphthalene-1-sulfonyl moieties. These peptidyl chloromethyl ketones have also been modified by incorporation of biotin and epsilon-amino caproyl biotin. The ability of these various chloromethyl ketones to be incorporated into a collection of zymogen-enzyme pairs has been evaluated using a variety of coagulation and fibrinolytic proteins. All labeled chloromethyl ketones were efficiently incorporated into the proteases tested, with the exception of urokinase which was refractory to inhibition by phenylalanylprolylarginyl chloromethyl ketone derivatives. No modification of any zymogen species was observed even under conditions designed to detect minimal reactivity. When enzymes were modified using chloromethyl ketones labeled with epsilon-amino caproyl biotin, the modified proteins readily reacted with avidin under a variety of different conditions. The observed reactivity with avidin was used in enzyme "blotting" following electrophoretic resolution of polypeptide chains and to remove active enzyme present in enzyme-zymogen mixtures. These reagents have been used to evaluate the potential for active site expression by the single-chain human factor VII molecule. Studies conducted with tissue factor, phospholipids, and calcium using factor X as substrate demonstrate that no activity can be obtained without initial activation of either factor X to factor Xa or factor VII to factor VIIa by an external source. We thus conclude that factor VII is a true zymogen, inert in the blood clotting process prior to its cleavage to factor VIIa.

Iodination of peptidyl chloromethyl ketones for protease affinity labels

The specificity of peptidyl chloromethyl ketones has been used to label proteases in complex biological systems by incorporating tyrosine into the structure for eventual radioiodination. Contrary to results with iodination of proteins, a mild reagent, that is, one which iodinates at neutrality, was unsuitable, giving complex mixtures with poor reproducibility, apparently because of side reactions at the chloromethyl ketone group. On the other hand, iodine monochloride in acetic acid provided clean products. In the cases examined where a tyrosine residue was not appropriate for the specificity of the target protease, this residue was located well displaced from the primary specificity site. The resultant diiodotyrosine-containing derivatives were generally highly active as protease inhibitors. The p-aminobenzoyl group was used as an alternative to tyrosine as an iodinatable component.

The inactivation of one- and two-chain forms of tissue plasminogen activator by a series of peptidyl chloromethyl ketones

The inhibition of one- and two-chain forms of tissue-type plasminogen activator by a series of peptide derivatives of arginine chloromethyl ketone was studied. Both forms of the enzyme were inhibited by such reagents at varying rates, dependent on the peptide sequence of the inhibitor. The order of relative effectiveness of the inhibitors was the same for both one- and two-chain tissue plasminogen activator. However, an approximately ten to twenty-fold higher concentration of reagent was required with the one-chain activator to produce similar rates of inactivation. It appears that the specificity of the active sites of the one and two chain forms of tissue plasminogen activator are very similar. It is suggested that the differences in enzymatic properties of the two forms are due mainly to effects on binding (Ki) rather than on the rate of alkylation and inhibition (k2).

Active site mapping of human and rat urinary kallikreins by peptidyl chloromethyl ketones

The reactivity of human and rat urinary kallikrein has been determined with peptides of arginine and lysine chloromethyl ketone. Pro-Phe-ArgCH 2Cl, the reagent corresponding to the sequence of kininogen hydrolyzed by kallikrein, was considerably more effective than reagents containing other substituents in the P 1 and P 2 positions (the Arg and Phe binding sites, respectively). Pro-Phe-ArgCH 2Cl inactivates the human enzyme at the 10 −5 m level ( K i 45 μ m, k 2 0.36 min −1) and the rat enzyme at the 10 −6 m level ( K i 4.8 μ m, k 2 0.26 min −1). More effective reagents were obtained by substitution of d-Phe for the P 3 Pro and addition of a dansyl residue in the P 4 position, yielding reagents effective at the 10 −7 m level for both kallikreins. Expansion of the sequence of kininogen to accomodate the P 4 and P 5 binding sites of kallikrein resulted in a reagent, Phe-Ser-Pro-Phe-ArgCH 2Cl, which is approximately 6-fold more reactive than the corresponding tripeptide analog for human kallikrein, while for the rat kallikrein, the tri- and pentapeptide analogs are comparable in reactivity. The importance of Arg in the P 1 position and Phe in the P 2 positions in the sequence of kallikrein's physiological substrate in determining specificity was shown by comparison of the reactivities of the proteases with Ala-Phe-ArgCH 2Cl and Ala-Phe-LysCH 2Cl and with Pro-Phe-ArgCH 2Cl and Pro-Gly-ArgCH 2Cl. Substitution of Lys for the P 1 Arg and substitution of Gly for the P 2 Phe decreased the reactivity of the reagent 10- and 150-fold, respectively, for the human kallikrein and 200- and 250-fold, respectively, for the rat kallikrein. Substitution of l-amino acid residues for the P 3 Pro had little effect on the reactivity of human kallikrein with the affinity labels and decreased the reactivity of the rat enzyme with the affinity labels from 3- to 6-fold.