Inhibitors peptide aldehydes

More than 50 endogenous and exogenous inhibitors of the calpains have been described as either transition-state reversible or irreversible inhibitors. The first transition-state inhibitors were the peptide aldehydes (e.g., leupeptin). Using this compound, new ones were synthesized that exhibited improved membrane permeability and calpain specificity (e.g., calpeptin). Other groups of inhibitors have since been discovered a-dicarbonyls (originally developed as serine protease inhibitors), nonpeptide quinolinecarboxamides,... 

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,... 

Direct thrombin inhibitors such as hirudin, Hirulog, the peptide aldehyde efegatran, and peptidomimetic compound argatroban have undergone clinical trials. Their application in the prevention and treatment of deep vein thrombosis contin-... 

E. Dufour, A. C. Storer, R. Menard, Peptide Aldehydes and Nitriles as Transition State Analog Inhibitors of Cysteine Proteases , Biochemistry 1995, 34, 9136 - 9143. 

Proteasome inhibitors have been instrumental in identifying numerous protein substrates and in elucidating the importance of the proteasome/ubiquitin pathway in many biological processes. Initially, non-specific cell-penetrating peptide aldehydes were used for this purpose. More recently, it became possible to synthesize compounds with increased potency and selectivity (Adams et al. 1998 Elofsson et al. 1999). Furthermore, based on the crystal structure of the yeast and bovine liver CP (Groll et al. 1997 Unno et al. 2002), molecular modeling can now be used to engineer improved inhibitors. 

Following the initial isolation of the Hnl from M. esculenta [33] in which the peptide sequence was established, an overexpressed version of this enzyme (in E. coli) was prepared [41]. This system is not limited for enzyme quantity (as outlined in Sect. 2.3), and can accept a wide range of aromatic, heterocyclic and aliphatic aldehydes, as well as ketones, as substrates. In practical terms, a measure of the degree of enzyme inhibition by substrates is of significant value and for this system this has been quantified for a range of aldehydes, ketones and alcohols [70]. It was deduced that ketones and alcohols are competitive inhibitors, whilst aldehydes are noncompetitive inhibitors. 

Leupeptin is a slow, tight-binding inhibitor of trypsin. Some peptide aldehydes are potent, reversible inhibitors of cysteine proteases forming hemith-ioacetals with the active site cysteine.1 Similarly, peptide nitriles form thioimidate adducts.h... 

Interest in linkers for carbonyl compounds has only slowly emerged in recent years. The main driving force for the development of such linkers was the need for methods to prepare peptide aldehydes and related compounds (e.g. peptide trifluoromethyl ketones), which can be highly specific and valuable enzyme inhibitors [700,701], and are potentially useful for the treatment of various diseases. 

Most peptidyl a,a-difluoroalkyl ketones are actually extended chains based on statone, rather than simple difluoromethyl ketones. The statone derivatives are based on pepstatin, which is an extremely potent peptide inhibitor of aspartic proteases. The difluoro derivatives of statone take advantage of both the electronegativity of fluorine and the potential for additional interactions between the protease and structures on the leaving group side of the inhibitor. 15 This dual nature is part of what makes a,a-difluoroalkyl ketones effective inhibitors of aspartyl proteases as well as serine proteases. There are three main methods of synthesizing peptidyl a,a-difluoroalkyl ketones (1) the Reformatsky reaction with peptide aldehydes (Section 15.1.4.2.1), (2) a modified Dakin-West reaction (Section 15.1.4.2.2), and (3) a Henry nitro-aldol condensation (Section 15.1.4.2.3). 

Evidence for the tetrahedral intermediate includes a Hammett p constant of+2.1 for the deacylation reaction of substituted benzoyl-chymotrypsins and the formation of tetrahedral complexes with many inhibitors, such as boronates, sulfonyl fluorides, peptide aldehydes, and peptidyl trifluoromethyl ketones. In these last the chemical shift of the imidazole proton is 18.9 ppm, indicating a good low-barrier H-bond, and the pJQ of the imidazolium is 12.1, indicating that it is stabilized by 7.3 kcal mol 1 compared to substrate-free chymotrypsin. The imidazole in effect is a much stronger base, facilitating proton removal from the serine. 

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. 

Several peptidic aldehydes have been reported to be inhibitors for either y-secretase or yS-secretase or both. Common to both series are lipophilic di- and tripeptides with bulky N-terminal protection, e.g. Z-LLL-CHO (MG132), Z-YIL-CHO, and Boc-GW-CHO. The general lack of specificity of these aldehydes and their simultaneous inhibition of serine and cysteine proteases makes interpretation of data rather cumbersome. Indirect mechanisms through general protease inhibition result in complex concentration activity observations. Z-LLL-CHO (MG132), in fact, blocks maturation of the amyloid precursor protein. Some of these drawbacks were avoided by difluoro ketones as pioneered by Merryl Dow (Scheme 3.5.6), which... 

In this article we will focus on two types of novel polymer reagent useful for preparation of protease inhibitor libraries. Oxidizing polymers have been developed for synthesis of amino and peptide aldehydes (Chapter 3) which are an important class of protease inhibitors by themselves and can also be used as reactive electrophiles in subsequent transformations. 

For many of the prominent inhibitor families (Fig. 3.6.1, Boxes 12 and 13), the available synthetic strategies do not fulfil these requirements. In the synthesis of peptide aldehydes, e.g., the parallel synthesis of variable structures without race-mization is an unsolved problem. The same applies to the synthesis of many peptide isosteres such as the norstatines or the l,3-diamino-2-propanols. Typically, combinatorial variation of the N-terminal and C-terminal positions can be easily attained. The side chain of the isosteric building block itself and its stereochemistry can, however, currently be varied only by a synthetic effort this excludes versatile variation of the central inhibitory element. 

Oxidizing polymers and polymer-supported carbanion equivalents, in particular, have been employed successfully for preparation of protease inhibitor collections as a practically relevant example. With peptide aldehydes and differently substituted a-hydroxy-jS-amino carbonyl moieties two inhibitory motifs have been prepared with hitherto unprecedented ease and efficiency. Now it remains to be demonstrated that the synthetic methodology as introduced here is capable of assisting the identification of novel biologically active structures. 

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 . 

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). 

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... 

Peptide boronates are much more potent proteasome inhibitors than aldehydes [43[. Boronate-proteasome adducts have much more slower dissociation rates than proteasome-aldehyde adducts, and although boronates are considered reversible inhibitors, the inhibition is practically irreversible over a period of hours. In addition, PS-341 inhibits serine proteases 1000-fold weaker than it does the proteasome [43[. This combination of potency, selectivity and metabolic stability makes the peptide boronates better drug candidates than other classes of proteasome inhibitors. In vitro and mouse xenograft studies of PS-341 have shown anti-tumor activity in a variety of tumor types. DFLB and PS-273 are usefiil fluorescent active site probes binding of these inhibitors enhances the fluorescence of their environment-sensitive dansyl and naphthyl moieties [44]. 

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. 

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... 

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... 

The peptide aldehyde series demonstrated the potential for carbonyl derivatives to be potent inhibitors of serine proteinases. The 200-fold increase in values which was found in going from an aldehyde (7-1) Table 2.7) to its corresponding methyl ketone (7-2) Table 2.7) [129] implied that the physical properties of the carbonyl group were an important factor in determining the potency of the inhibitor. As a means of increasing both the stability and potency of peptide ketone derivatives Trainor and co-workers [130, 131] and Imperiali and Abeles [132] independently prepared the first peptide trifluoromethyl ketone (TFMK) inhibitors of serine proteinases in... 

Since peptide aldehydes were recognized very early as efficient inhibitors of the proteasome [62], the X-ray structure of the 20 S proteasome from Thermoplasma acido-philum [50] and subsequently from Sac-charomyces cerevisiae [34] were resolved with the proteases complexed with the cal-pain I inhibitor - that is, the tripeptide aldehyde Ac-Leu-Leu-Nle-H (1). 

With this type of inhibitor, the basic principle of multivalency was applied in a new version where specific recognition of peptide aldehydes led to a covalent grafting near the active site and thus to an increase of their in-loco concentration to values that make the inhibition practically irreversible. However, such bifunctional inhibitors are of limited application in cell biology because of the high intracellular... 

Peptide aldehydes of microbial origin, including leupeptin, chymo-statin, antipain, and elastatinal, which have been used extensively, are potent inhibitors of both thiol and serine proteinases (67). The extent of inhibition depends mainly on the amino acid composition and the binding specificity of the proteinase. 

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