Aldehydic inhibitors

Another PDF inhibitor series derived from comparisons with known metalloprotease inhibitor classes has been reported by Merck [68]. Their study investigated a small set of peptide aldehyde inhibitors, postulating that the aldehyde might bind to the metal centre in the form of a hydrate,... 

The state of the prior art. Numerous studies with small molecule inhibitors of BACE have been published and some studies with peptide inhibitors have also been disclosed. However, with regard to peptide aldehyde inhibitors of BACE, very little is known. Accordingly, the state of the prior art with regard to claims 1 is relatively undeveloped. 

DJ Durand, GB Gordon, JF O Connell, SK Grant. Peptide aldehyde inhibitors of bacterial peptide deformylases. Arch Biochem Biophys 367 297-302, 1999. 

Another example are naturally occurring peptide aldehyde inhibitors, discovered in microorganisms, such as antipain (14), chymostatin (15), leupeptin (IS) and elasti-nal (17)31 33). The discovery of the inhibitors stimulated the synthetic work of peptide aldehyde analogs, and a large number of peptide aldehydes have been prepared 34). 

Trimethylammonium trifluoroacetophenone (19) was found to be a highly effective inhibitor of acetylcholinesterase 37 f The ketone activated by an electron-withdrawing trifluoroacetyl group will enhance the tendency to add a nucleophile (the hydroxyl group of the catalytic serine residue of acetylcholinesterase) to form a tetrahedral adduct as an aldehyde inhibitor. 

Peptide aldehydes were the first proteasome inhibitors to be developed and are still the most widely used inhibitors [41, 42]. Aldehyde inhibitors are slow-binding... 

Several structures of proteasome inhibitor complexes have been determined by X-ray crystal analysis [22, 50, 51]. Calpain inhibitor I (acetyl-L-L-norleudnal) was covalently bound to Thr-1 of the /91, pi and y95 subunits, respectively. This peptide aldehyde inhibitor is attacked by the threonyl Oy to form a hemiacetal. The nor-leucine side chain projects into a pocket with an opening in its side towards a tunnel leading to the particle surface. The leucine side chain at P2 is not in contact with the protein, whilst the leucine side chain at P3 is in contact with the adjacent / -type subunit. 

Westrik and Wolfenden (65) have recently reported that aldehydes, with side chains similar to those comprising the acyl portion of substrates which papain effectively hydrolyzes, were very potent inhibitors of this enzyme. Umezawa and his associates (66) have also recently reported that certain microorganisms (actinomyces) produced an aldehyde, acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin), which inhibits papain. The structures of some of the more effective aldehyde inhibitors of papain are shown in Figure 4. 

Figure 14, Analogy of formation of thiol adduct with aldehyde inhibitors to formation of tetrahedral intermediate in papain catalysis...

As for the proteases described above, a number of spectrophotometric and fluorescent methods are available, but typically AMC conjugates are used (Stennicke and Salvesen, 2000). Aldehyde inhibitors can be used in order to verify specificity of the assays (Bedner et al., 2000), and labelled inhibitor can also be used for direct cell staining (Bidle and Bender, 2008). 

Another class of transition-state inhibitors is the peptide aldehyde inhibitors (Fig. 7). Aldehydes inhibit cysteine, serine, and threonine proteases via a covalent, reversible mechanism, and metaUoproteases using an analogous but nonco-valent mechanism. Aldehydes were discovered in screens for protease inhibitors from microorganisms and generally consist of a peptidyl moiety that binds in the non-prime specificity sites with a C-terminal aldehyde group. These inhibitors are... 

An aldehyde inhibitor. Elastase is specifically inhibited by an aldehyde derivative of one of its substrates ... 

Of particular interest is the study of the biological mechanisms associated with enzyme stereoselectivity and enantioselectivity. For example, MD simulations have been successful in explaining the different affinities of trypsin and acetylcholinesterase to the diastereomers of soman inhibitors [154] and the ability of subtilisin Carlsberg and a-chymotrypsin to discriminate between R-and S- configurations of chiral aldehyde inhibitors [155, 156]. 

Leupeptin, (6-1) (Table 2.6), the first peptide aldehyde inhibitor of serine and cysteine proteinases was isolated in 1969 from several species of Streptomyces [119]. It was only weakly active against trypsin and was inactive against elastases, but its discovery spurred continued screening of... 

Simultaneous with this work, the first synthetic aldehyde inhibitors of elastases were independently conceived by Thompson [122]. He had predicted that aldehydes, such as (6-3), would be transition-state inhibitors of serine proteinases (PPE). The finding that aldehyde (6-3) was approximately 1000-fold more potent as an inhibitor of PPE than the corresponding alcohol and that it had approximately 5000-fold higher affinity for PPE than the corresponding primary amide substrate (6-4) provided strong evidence in support of his argument. Also, as was described for aj-PI, the importance of the Ser-195 hydroxyl to the activity of these aldehydes was shown in a study wherein the dissociation constant between (6-3) and anhydro-PPE increased 5000-fold as compared to its potency as an inhibitor of PPE [123]. 

In the laboratory of R. Bihovsky, a series of peptide mimetic aldehyde inhibitors of calpain I was prepared in which the P2 and P3 amino acids were replaced with substituted 3,4-dihydro-1,2-benzothiazine-3-carboxylate-1,1-dioxides. The synthesis began with the diazotization of the substituted aniline substrate using sodium nitrite and hydrochloric acid. The aqueous solution of the corresponding diazonium chloride product was added dropwise to the solution of acrylonitrile in a water-acetone mixture, which contained catalytic amounts of copper(ll) chloride. This Meerwein arylation step afforded the chloronitrile derivative, which was subjected to sulfonation with chlorosulfonic acid, and the resulting sulfonyl chloride was treated with the solution of ammonia in dioxane to give the desired 3,4-dihydro-1,2-benzothiazine-2-carboxamide. 

Shokhen et al. [56] analyzed possible mechanisms for the reversible formation of the complex between papain, a prototype enzyme of cysteine proteases, and pep-tidy 1 aldehyde inhibitors, using the quantum mechanical (DFT)/self consistent reaction field (virtual solvent) approach. 

Once an aldehyde inhibitor is bound to luciferase, the enzyme can no longer bind the substrate. 

The dissociation of the aldehyde inhibitor from luciferase is slower than the autooxidation of... 

Chemical modifications of the aC106 (not shown in Figure 136.1) result in luciferase inactivation. This residue is beheved to be at or near the aldehyde inhibitor site but is not essential to the binding of FMNHj or decanaP or catalysis. ... 

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