Inhibitor of protease

Hertogs K, de Bethune MP, Miller V, Ivens T, Schel P, Van Cauwenberge A, Van Den Eynde C, Van Gerwen V, Azijn H, Van Houtte M, Peelers F, Staszewski S, Conant M, Bloor S, Kemp S, Larder B, Pauwels R (1998) A rapid method for simultaneous detection of phenotypic resistance to inhibitors of protease and reverse transcriptase in recombinant human immunodeficiency virus type 1 isolates from patients treated with antiretroviral drugs. Antimicrob Agents Chemother 42 269-276... 

Decodts, G. Wakselman, M. Suicide inhibitors of proteases. Lack of activity of halo-methyl derivatives of some aromatic lactams. Eur. J. Med. Chem. Chim. Ther. 1983, 18, 107-111. 

This approach has been mainly applied to peptide-based inhibitors of proteases, where the inhibitory molecule is a peptide with a transition state isostere appended to it, and the cognate substrate is simply a peptide of the same amino acid sequence, but lacking the isostere functionality. Examples where good correlations between the free energy of inhibitor binding and the free energy of kcJKM have been found, include peptide-trifluoromethyl ketone inhibitors of human leukocyte elastase (Stein et al., 1987) and peptide-phosphonamidate inhibitors of the metalloprotease ther-molysin (Bartlett and Marlowe, 1983). 

Peptidases encoded by many viruses play essential roles at various stages of viral replication, including the coordinated assembly and maturation of virons [7a]. Viral peptidases have become important drug targets in the treatment of viral infections. Of note are inhibitors of proteases of the human immunodeficiency virus (HIV), particularly HIV-1 protease (HIV-1 retropepsin, EC 3.4.23.16) and HIV-2 protease [47-50], Drugs in this class, which include indinavir, ritonavir, and saquinavir, are useful in the treatment of AIDS, especially when administered as a cocktail together with one of the drugs that act on the viral retrotranscriptase (e.g., didanosine, stavudine, and zidovudine (AZT)). 

If the recombinant protein is in the cell pellet, resuspend the pellet in an appropriate lysis buffer containing inhibitors of proteases. 

If the recombinant protein is secreted it will be present in the supernatant. In that case, add an equal volume of an appropriate buffer, containing inhibitors of proteases, to the supernatant and proceed with the purification of the target protein as described for the bacterial system. 

In the literature there are a lot of more or less specific inhibitors of proteases. During daily work with tissue homogenates, a mixture of inhibitors with broad specificity has been proved with success. This mixture is given in Table 8.12. 

Trifluoromethyl ketones are also powerful inhibitors of proteases of the trypsin family (trypsin, thrombin, enzymes of blood coagulation). " ... 

Pathogenesis of systemic abnormalities during pancreatitis is associated with a high concentration of pancreatic proteases in the blood, which reduce activation of the blood proteolytic systems. Alteration of the balance between proteases and their natural inhibitors is a trigger of endogenous intoxication syndrome, which controls disease severity and the clinical outcome. Therefore, drugs which act as inhibitors of proteases are an important component of a complex treatment of acute pancreatitis. 

Inhibitors of proteases have also been developed from polymers. These molecules are as simple as a polymer chain, or much more complex. The mechanisms for protease inhibition are variable depending upon the type of molecule used specific protease inhibitors are available for conjugation to a polymer while other polymeric inhibitors inhibit all divalent cation dependent proteases. The development of these inhibitory polymers and polymer conjugates greatly increase the possibility to protect proteins from degradation in the gastrointestinal tract. 

About 20 families of protein inhibitors of proteases have been described.488 The egg white ovomucoids comprise one family. Turkey ovomucoid is a three-domain protein whose 56-residue third domain is a potent inhibitor of most serine proteases.455 489 The 58-residue pancreatic trypsin inhibitor490 is a member of another family of small proteins. A 36-residue insect (locust) protease inhibitor is even smaller.491... 

The introduction of retro-, retro-inverso-, and PMRI-peptides with free and blocked C-and N-termini has been successful in numerous biological systems such as neurotransmitters, inhibitors of proteases and protein kinases, sweeteners, antimicrobial peptides, hormones, adhesion molecules, antigenic epitopes, immuno-modulators, and immunological probes. Table 1 provides an exhaustive list of retro-, retro-inverso-, PMRI-, and end-group-modified re/ro-mvmo-pseudopeptides derived from bioactive peptides. 

A similar strategy was used to examine the potential role of a reverse turn as a recognition element adjacent to the cleavage site of substrates of HIV protease.197 A series of inhibitors was prepared, the synthesis of which involved the solution coupling of the statine-like transition state mimic to the (3-turn mimetic to provide 46 (Scheme 22). Subsequent sodium in ammonia reduction provided analogue 47. One of the compounds was a reasonably potent inhibitor of protease activity (IC50=2.6 x 10-8 M) (Table 1). 

The effectiveness of a-aminoboronic acids with neutral side chains as inhibitors of proteases has prompted the preparation of inhibitors with functionalized side chains. These include those with a basic side chain (boroOrn, boroArg, and boroLys), sulfur-containing side chain (boroMet), a pyrrolidine (boroPro), and a 3-cyano-substituted benzyl side chain. The synthesis and properties of the neutral side-chain boronic acids are discussed first followed by descriptions of the preparation of more specialized a-aminoboronic acids. 

Figure 3 Competitive, active site inhibitors of proteases. These inhibitors bind in the active site, but not in a substrate-like manner. Peptide extensions bind in specificity subsites, and sometimes interact with the catalytic residues (rectangle), but not in a catalytically competent manner.

Sometimes called suicide substrates, several protein inhibitors of proteases require proteolytic activity of the enzymes they inhibit, which leads to either covalent modification of the enzyme or releases charged groups that inhibit the catalytic machinery. In either case, this sort of activity-dependent inhibition is powerful and fundamentally different than the competitive mechanisms outlined above the inhibitor acts as a substrate and then uses the enzymes catalytic machinery to trap and then inhibit the enzyme. 

Figure 8 Irreversible inhibitors of proteases. Serine and cysteine proteases can be acylated by aza-peptides, which release an alcohol, but cannot be deacylated due to the relative unreactivity of the (thio) acyl-enzyme intermediate. Reactive carbons, such as the epoxide of E64, can alkylate the thiol of cysteine proteases. Phosphonate inhibitors form covalent bonds with the active site serine of serine proteases. Phosphonates are specific for serine proteases as a result of the rigid and well-defined oxyanion hole of the protease, which can stabilize the resulting negative charge. Mechanism-based inhibitors make two covalent bonds with their target protease. The cephalosporin above inhibits elastase [23]. After an initial acylation event that opens the p-lactam ring, there are a number of isomerization steps that eventually lead to a Michael addition to His57. Therefore, even if the serine is deacylated, the enzyme is completely inactive.

A final group of covalent small-molecule inhibitors of proteases are mechanism-based inhibitors. These inhibitors are enzyme-activated irreversible inhibitors, and they involve a two-hif mechanism that completely inhibits the protease. Some isocoumarins and -lactam derivatives have been shown to be mechanistic inhibitors of serine proteases. A classic example is the inhibition of elastase by several cephalosporin derivatives developed at Merck (Fig. 8). The catalytic serine attacks and opens the -lactam ring of the cephalosporin, which through various isomerization steps, allows for a Michael addition to the active site histidine and the formation of a stable enzyme-inhibitor complex (34). These mechanism-based inhibitors require an initial acylation event to take place before the irreversible inhibitory event. In this way, these small molecules have an analogous mechanism of inhibition to the naturally occurring serpins and a-2-macroglobin, which also act as suicide substrates. 

Maryanoff, B. E. and Costanzo, M. J. (2008) Inhibitors of proteases and amide hydrolases that employ an a-ketoheterocycle as a key enabling functionality. Bioorg. Med. Chem., 16,1562-1595. 

CONCLUSION - The development of inhibitors of proteases must take into account such problems as enzyme and tissue specificity, and toxicity (particularly the inhibitor s ability to alkylate or acylate other important body constituents) although in vivo, toxicity may be diminished by homeostatic resynthesls of enzymes and other protein constituents.9A... 

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... 

Regarding other isothiazole derivatives, different activities have been claimed such as antimicrobial, antibacterial, antifungal, antiviral, antiproliferative and anti-inflammatory activities. They have also been tested as inhibitors of proteases, for the treatment of anxiety and depression, for their action on the 5-HT receptor, and as inhibitors of aldoso reductase. Patented compounds for agrochemical applications are of old interest. This review is concerned with recent results, mainly limited to the last decade. Previous references have been taken into account only if they are of particular relevance or necessary for a better understanding of the text. Furthermore, patents have been included only if they add important information to the existing scientific literature. Articles exclusively dedicated to biological investigations without chemical interest have not been considered. 

Koh Y, Matsumi S, Das D et al (2007) Potent inhibition of HIV-1 replication by novel non-peptidyl small molecule inhibitors of protease dimerization. J Biol Chem 282 28709-28720... 

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