Acid/aspartyl proteases

The HIV-1 protease, like other retroviral proteases, is a homodimeric aspartyl protease (see Fig. 1). The active site is formed at the dimer interface, with the two aspartic acids located at the base of the active site. The enzymatic mechanism is thought to be a classic acid-base catalysis involving a water molecule and what is called a push-pull mechanism. The water molecule is thought to transfer a proton to the dyad of the carboxyl groups of the aspartic acids, and then a proton from the dyad is transferred to the peptide bond that is being cleaved. In this mechanism, a tetrahedral intermediate transiently exists, which is nonconvalent and which is mimicked in most of the currently used FDA approved inhibitors. 

The carboxyl proteases are so called because they have two catalytically essential aspartate residues. They were formerly called acid proteases because most of them are active at low pH. The best-known member of the family is pepsin, which has the distinction of being the first enzyme to be named (in 1825, by T. Schwann). Other members are chymosin (rennin) cathepsin D Rhizopus-pepsin (from Rhizopus chinensis) penicillinopepsin (from Penicillium janthinel-lum) the enzyme from Endothia parasitica and renin, which is involved in the regulation of blood pressure. These constitute a homologous family, and all have an Mr of about 35 000. The aspartyl proteases have been thrown into prominence by the discovery of a retroviral subfamily, including one from HIV that is the target of therapy for AIDS. These are homodimers of subunits of about 100 residues.156,157 All the aspartyl proteases contain the two essential aspartyl residues. Their reaction mechanism is the most obscure of all the proteases, and there are no simple chemical models for guidance. 

The pepsin, activated by cleavage of a proenzyme, has two putative active site domains comprising hydrophobic-hydrophobic-Asp-Thr-Gly amino acids, is potentially glycosylated and has a free cysteine residue which may allow it to form dimers, as in the case of human and Plasmodium falciparum-derived aspartyl proteases (Longbottom et al., 1997). However,... 

The human retrovirus HIV can be controlled using chemotherapy directed at the reverse transcriptase and aspartyl protease encoded by the viral genome as with other microbial pathogens, however, resistance to drug therapy becomes a major problem. Figure 7.3 shows a crystal structure (PDB 1HXW) of the HIV protease, where mutated amino acids (shown in cyan) lead to disrupted binding of the clinically effective inhibitor ritonavir [24]. 

Figure 7. Potent aspartyl protease inhibitors synthesized from simple Grignard, amine, acid, sulfonyl chloride, and isocyanate building blocks.

Figure 1. Schematic representation of the relationships between proposed catalytic and inhibitory mechanisms. A. Postulated general acid-general base catalyzed mechanism for substrate hydrolysis by an aspartyl protease. The water molecule indicated is extensively hydrogen bonded to both aspartic acid residues plus other sites in the active site (see Reference 16 for details). Hydrogen bonds to water are omitted here. B. Kinetic events associated with the inhibition of pepsin by pepstatin. The pro-S hydroxyl group of statine displaces the enzyme immobilized water molecule shown in Figure lA. Variable aspartyl sequence numbers refer to penicillopepsin (pepsin, Rhizopus pepsin), respectively.

These data unambiguously establish that a gem diol species is formed in the active site of pepsin when the ketone analogs 6 and 10 are added to the aspartyl protease as shown in Figure 6 and exclude the formation of a covalent intermediate. Our data strongly support the general acid-general base catalysis mechanism for aspartyl proteases that is illustrated schematically in Figure 1. 

The acidic character of fluorinated alcohols, and consequently the excellent ability to donate hydrogen bonds, justifies their interest as central peptidomimetic units in inhibitors of serine and aspartyl proteases [2, p. 59], An enhancement of... 

Renin is an aspartyl protease that specifically catalyzes the hydrolytic release of the decapeptide Ang I from angiotensinogen. It is synthesized as a preprohormone that is processed to prorenin, which is inactive, and then to active renin, a glycoprotein consisting of 340 amino acids. 

Additionally, 1,2-dihydroxyethylene dipeptide analogues without the C-terminal carboxylic acid have been used to obtain aspartyl proteases inhibitors.[641 These efforts include stereoselective alkylation of imines, one-pot reductive amination of epoxy ketones, ring opening of epoxides with sodium azide, diastereoselective dihydroxylation of allylic amines, and enzymatic resolution and stereocontrolled intramolecular amidation. 

Potent inhibitors of aspartyl proteases have been prepared by incorporating hydroxy-methylene transition-state analogues modified in the a-position. The a-methylene moiety is either functionalized by a heteroatom 16,71 72 or it is dihalogenated)15,73,74 Alkyl substituents in that position are found in several residues present in compounds of natural origin, such as dolaproine (8) in dolastatin 10 and 4-amino-3-hydroxy-2-methylpentanoic acid (9) in bleomycin D (Scheme 1). This position is also substituted in synthetic, conformationally constrained six-membered-ring analogues)75 77 ... 

Figure 16.7 Mechanism of aspartyl proteases involving general acid-base catalysis and the formation of a protonated terahedral intermediate. Bottom Proposal by T. J. Rodriguez. T. A. Angeles, and T. D. Meek, Biochemistry 32, 12380 (1993), that the first step is peptide bond isomerization. This accounts for the observed inverse 15N/14N kinetic isotope effect, which implies that bonding with the N atom becomes stiffer in the transition state.

The imidoyl chloride moiety of 5-chloro-l-alkyl-1,4-benzodiazepin-2-ones participates in Pd-catalyzed, Suzuki crosscoupling reactions, reacting with a range of functionalized aromatic boronic acids to provide an efficient and versatile approach to 5-aryl and 5-heteroaryl compounds (Scheme 19) <2003JOC2844>. This chemistry readily extends to 3-amino-substituted compounds that are orally bioavailable inhibitors of the aspartyl protease 7-secretase <2003BML4143>. 

Fermentation by microorganisms accounts for a two-thirds share of commercial protease production in the world (Kumar, 1999). Depending on their pH optimum and active-site characteristics, microbial proteases are classified as aspartyl proteases (E.C. 3.4.23, acidic), metalloproteases (E.C. 3.4.24, neutral), cysteine or sulf-hydryl proteases (E.C. 3.4.22, alkaline), or serine proteases (E.C. 3.4.21, also alkaline) (Kalisz 1988 Rao, 1998). Their commercial uses are listed in Table 10.4. 

BACE1 is the sole identified (3-secretase and is an aspartyl protease produced with a 46 amino acid pro-domain. This pro-domain must be removed for efficient... 

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