Chloromethyl ketones protease inhibitors

Peptide chloromethyl ketone inhibitors have been developed for almost every serine protease that has been characterized adequately (30). For example, human leukocyte elastase, due to its involvement in emphysema, has been studied extensively with this class of inhibitor (32). The rate at which peptide chloromethyl ketones inhibit elastase is influenced by their interaction with the primary substrate binding site (Si) of the enzyme and by interactions at other subsites. The most effective chloromethyl ketone elastase inhibitor found thus far is MeO-Suc-Ala-Ala-Pro-ValCH2Cl (MeO-Suc- = CH3OCOCH2CH2CO-). This will not inhibit the other major leukocyte protease, cathepsin G (see Table VI). In contrast, Z-Gly-Leu-Phe-CH2C1 (Z = C6H5CH2OCO-) inhibits cathepsin G, but not elastase. Both enzymes can be inhibited with Ac-Ala-Ala-Pr o-V alCH2Cl. 

This ceramide-mediated apoptosis was shown to be inhibited by the simultaneous addition of PKC activators (Ni et at, 1994 Obeid et al, 1993), implying that PS may activate the ceramide-mediated apoptotic pathway. However, the inhibitors of interleukin-1 converting enzyme (ICE)-like proteases (Caspase), such as tosyl-L-lysine chloromethyl ketone (TLCK), and tosyl-L-phenylalanine chloromethyl ketone (TPCK) which inhibit ceramide-mediated apoptosis, had no effect on PS-induced apoptosis (Figure 4). Thus, PS-induced apoptotic pathway appears to be distinct from that mediated by ceramide. Further studies are required to clarify the molecular mechanisms underlying the PS-induced apoptosis. 

The diazomethyl ketone functional group was first observed to be an affinity label by Buchanan and co-workers who showed that the antibiotic azaserine, an O-diazoacetyl derivative, 9 inhibited an enzyme in the biosynthesis of purine by alkylation of a cysteine residue. 10 The acid protease pepsin was then observed to be inhibited by peptidyl diazomethyl ketones in the presence of copper ions with the resulting esterification of an aspartate residue. 11 Two peptidyl diazomethyl ketones, Z-Phe-CHN2 and Z-Phe-Phe-CHN2, were found to irreversibly inactivate papain, a cysteine protease. 12 Since these reports, many peptidyl diazomethyl ketones have been prepared primarily as inhibitors of various cysteine proteases. 7 Peptidyl diazomethyl ketones are also synthetic intermediates and have been used to prepare chloromethyl ketones (Section 15.1.3), 13 bromomethyl ketones (Section 15.1.3), acyloxymethyl ketones, 14 and (i-peptides. 15 A few peptidyl diazoalkyl ketones have been reported. 16,17 ... 

Note Reactions were performed under the conditions described in the text with either colorimetric peptide pNA4 (250 pM) or fluorogenic peptide F3 (35 pM) as a substrate. The IC50 values represent the inhibitor concentration required to reduce the protease activity by 50% of the control containing no inhibitor. NI, no inhibition was observed at the concentrations indicated. E64, frans-epoxysuccinyl-L-leucylamide-(4-guanidino)-butane PMSF, phenylmethylsulphonyl fluoride TLCK, tosyl-L-lysine-chloromethyl ketone. 

Peptide aldehydes constitute a rather general example of protease inhibitors. The electrophilic carbonyl group is attacked reversibly by the cleaving nucleophile, forming a covalent acetal or thioacetal intermediate. With cysteine proteases the preferred inhibitors are strong electrophiles, for example ketones, chloromethyl ketones, epoxides, or vinyl sulfones. Many cysteine protease inhibitors form an enzyme-inhibitor complex irreversibly these are therefore denoted suicide-inhibitors . 

Peptide Chloromethyl Ketones. Peptide chloromethyl ketone inhibitors have been studied extensively and a fairly detailed picture of the inhibition reaction (see Figure 3) has emerged from numerous chemical and crystallographic studies (30,31). The inhibitor resembles a serine protease substrate with the exception that the scissile peptide bond of the substrate is replaced with a chloromethyl ketone functional group in the inhibitor. The inhibitor binds to the serine protease in the extended substrate binding site and the reactive chloromethyl ketone functional group is placed then in the proper position to alkylate the active-site histidine residue. In addition, the serine OH reacts with the inhibitor carbonyl group to form a hemiketal. 

Figure 3. Reaction of a serine protease with a peptide chloromethyl ketone. The side chain of the Pt residue of the inhibitor is shown interacting with the primary substrate binding subsite (SJ of the enzyme.

Inhibitors of Thiol and Carboxyl Proteases. Thiol proteases are inactivated by peptide chloromethyl ketones (30) and other alkylating agents. Peptide diazomethyl ketones are much more selective reagents since they do not react with serine proteases as do chloromethyl ketones. Diazoketones have been applied to papain and cathepsin B (48) thus far and it appears that they should be applicable to most thiol proteases. Specificity should be obtainable by changing the peptide sequence of the inhibitor to match that of the enzyme being studied. 

The side chain of His-57 is close enough to that of Ser-195 for a hydrogen bond to form between N-3 and the oxygen of the serine hydroxyl group. N-1 is shielded from solvent molecules. N-3 is selectively alkylated by the chloromethyl ketone and bromomethyl ketone inhibitors of serine proteases. These have the structure, R—C—CH2— X, where X... 

K., Neurath, H. and Woodbury, RG. (1985). Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dc skin proteases and human cathepsin G with peptide-4-nitroanilide substrates and with peptide chloromethyl ketone and sulphonyl fluoride inhibitors. Biochemistry 24, 2048-2058. 

Chloromethyl ketones also react with thiol proteases, and could therefore be useful collagenase inhibitors.82... 

Chemical inhibitors of proteases may be used to Inactivate the participating enz5nnes. lodoacetamlde and iodoacetate >prevent processing of virus proteins. Dlisopropyl fluorophosphate, probably acting as an inhibitor of a serine active-site protease, also prevents the processing of poliovirus polyprotein. Finally, chloromethyl ketones of selected amino acids, particularly that of phenylalanine, have been widely used in successful tests to block viral protein processing. The... 

N-Tosyl-L-lysine chloromethyl ketone (3S-l-chloro-3-tosylamino-7-amino-2-heptanone HCl) [4272-74-6] M 369.3, m 150-153 (dec), 156-158 (dec), 165 (dec), [a]g -7.3 (c 2, H2O), pKgst 10.6 (7-NH2). The hydrochloride slowly crystallises from a concentrated solution in absolute EtOH, thinned with EtOH/Et20 for collection and dried in vacuo. It is a suicide enzyme inhibitor of serine proteases, e.g. trypsin and clostripain. [Matsuda et al. Chem Pharm Bull Jpn 30 2512 1982, Shaw et al. Biochemistry 4 2219 7965],... 

A protease inhibitor, L-l-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), specific for chymotrypsin, has been shown to exert a number of effects on platelet processes. This compound also interferes with arachidonic acid metabolism in platelets. The 12-lipoxygenase was inhibited by TPCK, but the main effect was an irreversible inhibition of thromboxane synthetase [157]. 

After collection and dechorionation at room temperature, washed, dechorio-nated embryos should be quick-frozen in liquid N2 and stored at -70 C. Storage for periods of up to 1 year or longer seems to be without adverse effect. Immediately before fractionation, chilled (4°C) extraction buffer (Buffer E) should be prepared Buffer E contains 5 m Af MgC, 50 mM NaCl, 50 m M Tris-HCl, pH 7.5, 250 mM sucrose, 2.5 mM N-ethylmaleimide (NEM), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM L-tosylamide 2-phenyIethyl chloromethyl ketone (TPCK). NEM, PMSF, and TPCK should be included as protease inhibitors. About 20-40 min before fractionation begins, all protease inhibitors are added in solid form to otherwise complete Buffer E in amounts greater or equal to those specified. After addition of protease inhibitors. Buffer E should be stirred continuously at 4°C. Both PMSF and TPCK are incompletely soluble in aqueous solution at the concentrations specified. However, residual undissolved reagents constitute a solid reservoir that becomes completely depleted during embryo fractionation, due to either hydrolysis, protein modification, or both. 

In summary, evidence for a poliovirus gene product participating in the proteolytic cleavage of virus coat protein includes a. kinetics of production b, characteristic pH optimum c. abolition of activity by inhibitors of virus RHA and protein synthesis d. labilization to heat inactivation by amino acid analogs after cellular protein synthesis was abolished e. in similar fashion, recovery of protease activity, after inhibition of virus proteolysis by a leucyl chloromethyl ketone, required protein synthesis f. finally, protease activity co-purified with virus non-structural polypeptide X,... 

The most interesting class of protease inhibitors in viral studies has been the chloromethyl ketone derivatives of amino acids (54j 55) These were designed as affinity labels of serine-type proteases, and react irreversibly with histidine and serine residues in the active sites of proteases. There is a basis for selectivity of the chloromethyl ketones phenylalanyl and lysyl derivatives were synthesized, which had specificity for chymotrypsin and trypsin, respectively (54) This specificity led to studies on inhibition of poliovirus protein cleavage, with positive results (25, 26). 

Amino acid residues of the A.c. are identified by specific labeling with coenzyme, or by reaction with inhibitors or reagents specific for particular side chains. Some widely used irreversible inhibitors of the catalytic center of serine proteases are tosyllysine chloromethyl ketone (TLCK), which reacts with histidine, and diisopropylfluorophosphate (DFF) and phenylmethane sulfonyl fluoride (PMSF), which forms esters with serine residues. 

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