Transition state, protease

Figure 7 Various transition-state protease inhibitors. Bortezomib is an approved drug for the treatment of multiple myeloma. It is a boronic acid analog that inhibits the proteosome, a threonine protease. The boronic acid moiety can adopt a tetrahedral conformation in the active site. Pepstatin is a peptidyl aspartic acid inhibitor. The reactive statine group binds to the catalytic machinery, and the chiral hydroxyl group of the statine mimics the tetrahedral geometry of the transition state. Idinavir is an approved HIV 1 Protease inhibitor that binds to the active site via a hydroxyethylene transition state isostere. Aldehydes are also transition state analogs, which are susceptible to nucleophilic attack. In cysteine, serine and threonine proteases, this results in a covalent, reversible inhibition mechanism.

A Tropsha, J Hermans. Application of free energy simulations to the binding of a transition-state-analogue inhibitor to HIV protease. Protein Eng 51 29-34, 1992. 

X-ray crystallographic studies of serine protease complexes with transition-state analogs have shown how chymotrypsin stabilizes the tetrahedral oxyanion transition states (structures (c) and (g) in Figure 16.24) of the protease reaction. The amide nitrogens of Ser and Gly form an oxyanion hole in which the substrate carbonyl oxygen is hydrogen-bonded to the amide N-H groups. 

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,... 

FIGURE 7.2. Two alternative mechanisms for the catalytic reaction of serine proteases. Route a corresponds to the electrostatic catalysis mechanism while route b corresponds to the double proton transfer (or the charge relay mechanism), gs ts and ti denote ground state, transition state and tetrahedral intermediate, respectively. 

The requirements of protease inhibitors as drugs in terms of potency, pharmacokinetics, and toxicity will vary depending on the nature of the infection and the goals of therapy. At one extreme is treatment of HlV-1, a chroific infection that requires life-long therapy and full suppression of viral replication. At the other extreme is the treatment of human rhinovirus (i.e., the cold virus), where short-term treatment to blunt viremia will likely be sufficient to reduce the unwanted symptoms of a cold. In all cases, viral proteases represent very attractive targets with familiar mechanisms of catalysis that frequently allow for the design of transition state analogs and with distinct specificities from host proteases. 

Catalysis by enzymes that proceeds via a unique reaction mechanism typically occurs when the transition state intermediate forms a covalent bond with the enzyme (covalent catalysis). The catalytic mechanism of the serine protease chymotrypsin (Figure 7-7) illustrates how an enzyme utilizes covalent catalysis to provide a unique reaction pathway. 

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). 

Before our work [39], only one catalytic mechanism for zinc dependent HDACs has been proposed in the literature, which was originated from the crystallographic study of HDLP [47], a histone-deacetylase-like protein that is widely used as a model for class-I HDACs. In the enzyme active site, the catalytic metal zinc is penta-coordinated by two asp residues, one histidine residues as well as the inhibitor [47], Based on their crystal structures, Finnin et al. [47] postulated a catalytic mechanism for HDACs in which the first reaction step is analogous to the hydroxide mechanism for zinc proteases zinc-bound water is a nucleophile and Zn2+ is five-fold coordinated during the reaction process. However, recent experimental studies by Kapustin et al. suggested that the transition state of HDACs may not be analogous to zinc-proteases [48], which cast some doubts on this mechanism. 

The incorporation of an ionizable center, such as an amine or similar function, into a template can bring a number of benefits, including water solubility. A key step in the discovery of the protease inhibitor, indinavir was the incorporation of a basic amine (and a pyridine) into the backbone of hydroxyethylene transition state mimic compounds L-685,434 to enhance solubility (and potency) (Fig. 1.2) [17]. 

Figure 5. The theoretical basis for the diagnostic test given Figure 6. The putative transition state for HIV protease-...

Invirase , Saquinavir, Ro 31-8959 Hoffmann-La Roche HIV protease Transition state analog... 

Asp proteases Transition state isostere Statine, hydroxyethylamine 139, 140... 

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

A. Radzicka, R. Wolfenden, Rates of Uncatalyzed Peptide Bond Hydrolysis in Neutral Solution and the Transition State Affinities of Proteases , J. Am. Chem. Soc. 1996, 118, 6105 - 6109. 

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