Proteases active site

Figure 6. An example of inter-family target hopping between human and viral aspartyl proteases. The aspartyl protease active site is located at a homodimer interface in HIV and within a single domain in Cathepsin D, so sequence and structure alignments between these proteins cannot be constructed. By using an approach independent of sequence or structure homology to directly align the sites, SiteSorter finds that the HIV protease and Cathepsin D substrate sites are highly similar (identical chemical groups within 1 A are colored dark blue). It has been verified experimentally that Cathepsin D is susceptible to inhibition by HIV-protease inhibitors. ...

Pharmacology Indinavir is an inhibitor of the HIV protease. HIV protease is an enzyme required for the proteolytic cleavage of the viral polyprotein precursors into the individual functional proteins found in infectious HIV. Indinavir binds to the protease active site and inhibits the activity of the enzyme. This inhibition prevents cleavage of the viral polyproteins resulting in the formation of immature... 

Indinavir binds to the protease active site and inhibits the activity of the enzyme HIV protease preventing cleavage of the viral polyproteins resulting in the formation of immature noninfectious viral particles. 

Fig. 6. A symmetrical inhibitor designed to fit the symmetrical HIV protease active site.

HIV protease active site filled with skeletal structures... 

Ca2+ which helps to tie these proteins to the phospholipids of platelet surfaces. In factors VII, IX, X, and protein C this Ca2+-binding domain is followed by two epidermal growth factor (EGF)-like domains, each containing one residue of en/Firo-P-hydroxyaspartate or hydroxyasparagine formed by hydroxylation of an aspartate or asparagine residue in the first EGF-like domain.540,540a,b The C-terminal catalytic domain of each enzyme contains the protease active site. 

Peptide a-oxo acids 1 (R4=H), a-oxo esters 1 (R4= alkyl or substituted alkyl), and a-oxo-amides 2 (R5=R6=H, alkyl, substituted alkyl, aryl, and/or heteroaryl) are potent reversible inhibitors for cysteine and serine proteases (Scheme 1).[1 9 Their inhibitory potency is the result of their enhanced electrophilic a-carbonyl functional group that can better compete with the substrate in the formation of a tetrahedral adduct with the cysteine or serine residue at the protease active site. In the case of peptide a-oxo esters and a-oxoamides, the extension in PI and beyond gives the inhibitors additional interactions with the protease at the corresponding sites. 

Higaki, J.N., Evnin, L.B., and Craik, C.S. 1989. Introduction of a cysteine protease active site into trypsin. Biochemistry 28, 9256—9263. 

While a few very potent non-peptide protease inhibitors (Pis) have been isolated from plants many plant protease inhibitor proteins (PIPs) have evolved to have protease interaction Kj values in the nanomolar and picomolar range. These extraordinary affinities derive from the matching of the PI protein amino sequence about the scissile peptide bond (Pl-Pl ) and evolution of adjacent sequences to fit and interact appropriately within the target protease active site [1, 120, 121]. The structure and function of the different classes of PI proteins from plants are succinctly but comprehensively reviewed below. 

Figure 15. 3- and 4-point multiple pharmacophore overlaps for the thrombin ligand MQPA and the serine protease active-site derived pharmacophores the left-side arrow indicates the incorrect indication of factor Xa selectivity from the 3-point figures, and the right-side arrow the observed activity and the increased resolution of selectivity using the 4-point relative pharmacophores. 

Eco is a powerful tool for defining the active sites of serine protease due to the extended substrate-like interaction that it makes with the protease. The three-dimensional structure of a complex with eco has many advantages that a structure of a protease alone or bound to a small molecule inhibitor does not have. Eco can be used to take a molecular impression of the serine protease active site and reveal features that determine substrate preference. These features are used to design specific inhibitors with therapeutic prospects. Often, a small molecule inhibitor is used to define a protease active site cleft, but the resulting structures have particular drawbacks. Typically, a small molecule inhibitor lacks the prime side interac-... 

H-bond acceptor (C=0), acid (COj), base (NH and lipophilic (aromatic CH). Figure 4.7 illustrates the contours and the atoms which were added (with associated pharmacophore features) for the Factor Xa serine protease active site. 

Zhou GW, Guo J, Huang W, Hetterick, RJ, Scanlan TS. Crystal stracture of a catalytic antibody with a serine protease active site. Science 1994 265(5175) 1059-1064. 

Figure 1 Diagram of a protease active site. A protease cieaves a peptide at the scissiie bond, and has a number of specificity subsites, which determine protease specificity. Substrates bind to a protease with their non-prime residues on the N-terminai side of the scissiie bond and their prime-side residues C-terminal to the scissiie bond. The cataiytic residues determine the ciass of protease. Serine, cysteine, and threonine proteases hydroiyze a peptide bond via a covalent acyl-enzyme intermediate, and aspartic, giutamic and metaiioproteases activate a water moiecuie to hydroiyze the peptide bond in a non-covalent manner.

Several protease inhibitors are competitive, and they bind in the protease active site, but also they have secondary binding sites outside the active site, which are critical to inhibition. Exosite binding provides two major benefits 1) It increases the surface area of the interaction, which leads to a greater affinity, and 2) it can provide a greatly increased amount of specificity. 

Transition-state inhibitors stably mimic the transition state of the enzymatic reaction, and thereby interact with the substrate-bin-ding and catalytic machinery of the enzyme in a low-energy conformation. Transition-state analogs are competitive, reversible inhibitors, although some have extremely low Kj s and very slow off-rates. All proteases activate a nucleophile to attack a carbonyl, which leads to the formation of a tetrahedral intermediate that then collapses to form the enzyme products—two peptides. Thus, synthetic small molecules that mimic the tetrahedral intermediate of the protease reaction are attractive transition-state analogs. A classic class of protease transition-state inhibitors uses a boronic acid scaffold (4, 10). Boronic acid adopts a stable tetrahedral conformation in the protease active site that is resistant to nucleophilic attack. Boronic acid inhibitors, which are derivatized with different specificity elements, have been developed against every class of protease... 

Phosphonates (Fig. 8) and sulfonates represent a third class of covalent irreversible inhibitors. These inhibitors adopt a stable tetrahedral geometry and are covalently bound transition-state analogs. They often have a peptide-like specificity element, and the electrophilicity of the leaving groups can be modified to mne the reactivity of the inhibitor. These inhibitors are specific for serine proteases, because the serine protease active site has a well-defined oxyanion hole, which stabilizes the transition-state mimic. 

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