Aspartic protease

Novel Polymer and Linker Reagents for the Preparation of Protease-inhibitor Libraries 

Despite the current success and popularity of polymer reagents the severe limitations of the available resins have become obvious during recent years. For many synthetically important transformations reliable reagents are not available and polymer-assisted synthesis (Box 14) was usually restricted to small scale applications and suffered from the inherent limitations of the standard support material, cross-linked polystyrene, for example solvent intolerance, undesired adsorption of reagents, or the chemical reactivity of the resin backbone [1], 

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. 

To gain access to a broader spectrum of natural products including protease inhibitors, C-C-bond formation is often required in central steps. For this purpose polymer-supported carbanion equivalents have been investigated (Chapter 4). Several strategies leading to supported acyl anion equivalents are presented these have been employed for general synthesis of protease inhibitors containing the a-hydroxy-jS-amino motif. 

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Mammals, fungi, and higher plants produce a family of proteolytic enzymes known as aspartic proteases. These enzymes are active at acidic (or sometimes neutral) pH, and each possesses two aspartic acid residues at the active site. Aspartic proteases carry out a variety of functions (Table 16.3), including digestion pepsin and ehymosin), lysosomal protein degradation eathepsin D and E), and regulation of blood pressure renin is an aspartic protease involved in the production of an otensin, a hormone that stimulates smooth muscle contraction and reduces excretion of salts and fluid). The aspartic proteases display a variety of substrate specificities, but normally they are most active in the cleavage of peptide bonds between two hydrophobic amino acid residues. The preferred substrates of pepsin, for example, contain aromatic residues on both sides of the peptide bond to be cleaved. 

Most aspartic proteases are composed of 323 to 340 amino acid residues, with molecular weights near 35,000. Aspartic protease polypeptides consist of... 

FIGURE 16.26 Acyl-enzyme and amino-enzyme intermediates originally proposed for aspartic proteases were modeled after the acyl-enzyme intermediate of the serine proteases. 

FIGURE 16.27 A mechanism for the aspartic proteases. In the first step, two concerted proton transfers facilitate nucleophilic attack of water on the substrate carbonyl carbon. In the third step, one aspartate residue (Asp" " in pepsin) accepts a proton from one of the hydroxyl groups of the amine dihydrate, and the other aspartate (Asp" ) donates a proton to the nitrogen of the departing amine. 

The HIV-l protease is a remarkable viral imitation of mammalian aspartic proteases It is a dimer of identical subunits that mimics the two-lobed monomeric structure of pepsin and other aspartic proteases. The HIV-l protease subunits are 99-residue polypeptides that are homologous with the individual domains of the monomeric proteases. Structures determined by X-ray diffraction studies reveal that the active site of HIV-l protease is formed at the interface of the homodimer and consists of two aspartate residues, designated Asp and Asp one contributed by each subunit (Figure 16.29). In the homodimer, the active site is covered by two identical flaps, one from each subunit, in contrast to the monomeric aspartic proteases, which possess only a single active-site flap. 

Candidate protease inhibitor drugs must be relatively specific for the HIV-1 protease. Many other aspartic proteases exist in the human body and are essential to a variety of body functions, including digestion of food and processing of hormones. An ideal drug thus must strongly inhibit the HIV-1 protease, must be delivered effectively to the lymphocytes where the protease must be blocked, and should not adversely affect the activities of the essential human aspartic proteases. 

The first hint that two active-site carboxyl groups—one proto-nated and one ionized—might be involved in the catalytic activity of the aspartic proteases came from studies of the pH dependence of enzymatic activity. If an ionizable group in an enzyme active site is essential for activity, a plot of enzyme activity versus pH may look like one of the plots at right. 

Pepstatin (see page 531) is an extremely potent inhibitor of the monomeric aspartic proteases, with A) valnes of less than 1 nM. 

While catalysis by aspartic proteases involves the direct hydrolytic attack of water on a peptide bond, catalysis... 

Figure 7-6. Mechanism for catalysis by an aspartic protease such as HIV protease. Curved arrows Indicate directions of electron movement. Aspartate X acts as a base to activate a water molecule by abstracting a proton. The activated water molecule attacks the peptide bond, forming a transient tetrahedral Intermediate. Aspartate Y acts as an acid to facilitate breakdown of the tetrahedral intermediate and release of the split products by donating a proton to the newly formed amino group. Subsequent shuttling of the proton on Asp X to Asp Y restores the protease to its initial state.

It is worth noting here that inhibitors that interact with enzyme active site functionalities in ways that mimic the structure of covalent intermediates of catalysis can bind with very high affinity. This was seen in Chapter 1 with the example of statine-and hydroxyethylene-based inhibitors of aspartic proteases other examples of this inhibitor design strategy will be seen in subsequent chapters of this text. 

Longbottom, D., Redmond, D.L., Russell, M., Liddell, S., Smith, W.D. and Knox, D.P. (1997) Molecular cloning and characterisation of an aspartate protease associated with a highly protective gut membrane protein complex from adult Haemonchus contortus. Molecular and Biochemical Parasitology 88, 63-72. 

D. H. Rich, M. S. Bematowicz, N. S. Agarwal, M. Kawai, F. G. Salituro, and P. G. Schmidt, Inhibition of aspartic proteases by pepstatin and 3-methylstatine derivatives of pepstatin. Evidence for collected-substrate enzyme inhibition, Biochemistry 24 3165... 

Meek, Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease, Proc. Natl. Acad. Sci. 86 9752 (1989). 

Vassar, R., Bennett, B. D., Babu-Khan, S. et al. P-secretase cleavage of Alzheimer s amyloid precusor protein by the transmembrane aspartic protease BACE. Science 286 735-741, 1999. 

Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E. A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., Luo, Y., Fisher, S., et al. (1999). Beta-secretase cleavage of Alzheimer s amyloid precursor protein by the transmembrane aspartic protease BAGE. Science 286, 735—741. 

In apoptosis a series of events takes place in an orderly sequence involving the activation of various proteases which are called caspases, for cysteine and aspartate proteases. Several distinct caspases act in a cascade vaguely reminiscent of the blood-clotting cascade of complement proteins. If one wishes to interfere with the apoptotic process, then one strategy would be to develop drugs that inhibit various caspases, a current effort underway in the pharmaceutical industry. 

Potent inhibitor of add proteases, including pepsin, renin and cathepsin D and many microbial aspartic proteases... 

Weihofen, A., Lemberg, M.K., Friedmann, E., et al. (2003) Targeting presenilin-type aspartic protease signal peptide peptidase with y-secretase inhibitors. J. Biol. Chem., 278, 16528-16533. 

Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, Luo Y, Fisher S, Fuller J, Edenson S, File J, Jarosinski MA, Biere AL, Curran E, Burgess T, Louis JC, Collins F, Treanor J, Rogers G Citron M. (1999) Beta-secretase cleavage of Alzheimer s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286 735-741. 

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