Protease Discovery

The restricted applicability of commercially available proteases for peptide coupling reactions warrants the continuous importance of mining natural resources for enzymes with novel properties, such as a broad substrate range, acceptance of nonproteinogenic amino acids, high coupling efficiency, and robustness xmder reaction conditions. 

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Today, 3D databases, which provide the means for storing and searching for 3D information of compounds, are proven to be useful tools in drug discovery programs. This is well exemplified with the recent discovery of novel nonpeptide HIV-1 protease inhibitors using pharmacophore searches of the National Cancer Institute 3D structural database [13-15]. 

Hsu JT, Wang HC, Chen GW et al (2006) Antiviral drug discovery targeting to viral proteases. Curr Pharm Des 12 1301-1314... 

Thaisrivongs S, Strohbach JW (1999) Structure-based discovery of Tipranavir disodium (PNU-140690E) a potent, orally bioavadable, nonpeptidic HIV protease inhibitor. Biopolymers 51 51-58... 

This type of analysis is now well established and has been used in many drug discovery projects over the past fifteen years. Examples include HIV protease inhibitors [13], anti-influenza drugs [14], isoform-selective ligands for the estrogen receptor [15], and many more. 

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 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]. 

Studies using free energy calculations for the design and analysis of potential drug candidates are reviewed in section five. The chapters in this section cover drug discovery programs targeting fructose 1,6-bisphosphatase (diabetes), COX-2 (inflammation), SRC SH2 domain (osteoporosis and cancer), HIV reverse transcriptase (AIDS), HIV-1 protease (AIDS), thymidylate synthase (cancer), dihydrofolate reductase (cancer) and adenosine deaminase (immunosuppression, myocardial ischemia). 

The first protease inhibitor studied in human clinical trials was ciluprevir (BILN-2061), whose discovery was predicated on earlier studies that identified 54, a 6 amino acid N-terminal cleavage product of an NS5A/5B substrate, as a competitive inhibitor of NS3 [103,104], Although the development of ciluprevir was halted due to cardiotoxicity in animals, it demonstrated that a potent... 

M. Whittaker, Discovery of Protease Inhibitors Using Targeted Libraries , Curr. Opin. Chem. Biol 1998, 2, 386-396. 

FIGURE 1 Selection of the HIV protease as a drug discovery target. Demonstration that inactivation of the HIV protease interferes with HIV infectiv-ity. Immunoblot of HIV proteins in mock-transfected cells (lane 1), cells transfected with HIV containing active protease (lanes 2 and 4), and cells transfected with HIV in which the protease was inactivated. Reprinted with permission from ref. 8 (copyright Kohl et al). 

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