Protease, HIV protease

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... 

Human immunodeficiency virus (HIV) is the etiological agent of acquired immune deficiency syndrome (AIDS). The replication of the HIV requires a viral aspartyl protease (HIV protease) to process the virally encoded gag and gag-pol polyproteins. These cleavage events release enzymes and structural proteins that are essential for the assembly of infectious viral particles. Inhibition of HIV protease activity in infected cells thus leads to the production of an immature, noninfectious virus. HIV protease (PR) differs from monomeric aspartyl proteases in that the active site is formed by the assembly of two 99 amino acid polypeptides into a functional homodimer. 

In 1989, researchers discovered the structure of a molecule called HIV-protease. HIV-protease is a protein (a class of biological molecules) synthesized by the human immxmodeficiency virus (HIV), which causes AIDS. HIV-protease is crucial to the virus s ability to replicate itself. Without HIV-protease, HIV could not spread in the human body because the virus could not copy itself, and AIDS would not develop. 

There are several types of proteases. HIV protease is an example of aspartic acid proteases. The structure of HIV protease as determined by X-ray crystallography is shown in Fig. 21.11. How the peptide bond is cleaved is shown schematically in Fig. 7.3. The set of two aspartic acid residues shown catalyzes the addition of water molecule to gag-pol protein (a). The result is the formation of an intermediate shown as (b) in the figure, (b) is known to decompose into two separate entities as shown in (c). This completes the hydrolysis. The region in the gag-pol protein where this splitting occurs has an amino acid sequence of -Leu-Asn-Phe-Pro-lle- (Asn=aparagine, He=isoleucine, Leu=leucine, Phe=phenyl alanine, Pro=proline). 

There are, indeed, many biological implications that have been triggered by the advent of fullerenes. They range from potential inhibition of HIV-1 protease, synthesis of dmgs for photodynamic therapy and free radical scavenging (antioxidants), to participation in photo-induced DNA scission processes [156, 157, 158, 159, 160, 161, 162 and 163]. These examples unequivocally demonstrate the particular importance of water-soluble fullerenes and are summarized in a few excellent reviews [141, 1751. 

Friedman S H, DeCamp D L, Si]besma R, Srdanov G and WudI F 1993 Inhibition of HIV-1 protease by fullerene derivatives model building studies and experimental verifioation J. Am. Chem. See. 115 6506-9... 

Hanessian and Devasthale, 1996] Hanessian, S., and Devasthale, P. Design and synthesis of novel, pseudo C2 symmetric inhibitors of HIV protease. Bioorg. Med. Chem. Lett. 6 (1996) 2201-2206... 

Lebon et al., 1996] Lebon, F., Vinals, C., Feytmans, E., and Durant, F. Computational drug design of new HIV-1 protease inhibitors. Arch. Phys. Biochem. 104 (1996) B44. 

Structure-based Design Methods to Design HiV-1 Protease Inhibitors... 

An impressive example of the application of structure-based methods was the design of a inhibitor of the HIV protease by a group of scientists at DuPont Merck [Lam et al. 1994 This enzyme is crucial to the replication of the HIV virus, and inhibitors have bee shown to have therapeutic value as components of anti-AIDS treatment regimes. The star1 ing point for their work was a series of X-ray crystal structures of the enzyme with number of inhibitors boimd. Their objective was to discover potent, novel leads whid were orally available. Many of the previously reported inhibitors of this enzyme possessei substantial peptide character, and so were biologically unstable, poorly absorbed am rapidly metabolised. 

Flow chart showing the design of novel orally active HIV-1 protease inhibitor. (Figure adapted from Lam P K ]adhav, C E Eyermann, C N Hodge, Y Ru, L T Bacheler, ] L Meek, M ] Otto, M M Rayner, Y N V /ong, ang, P C Weber, D A Jackson, T R Sharpe and S Erickson-Viitanen 1994. Rational Design of Potent, able. Nonpeptide Cyclic Ureas as HIV Protease Inhibitors. Science 263 380-384.)... 

Priestle J P, A Fassler, J Rosel, M Tintelnog-Blomley, P Strop and M G Gruetter 1995. Comparati Analysis of The X-Ray Structures of HIV-l and HIV-2 Proteases in Complex with a Nov Pseudosymmetric Inhibitor. Structure (London) 3 381-389. 

SG Deeks, M Smith, M Holdniy, JO Kahn. HIV-1 protease inhibitors A review for clinicians. J Am Med Assoc 277 145-153, 1997. 

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. 

DM Perguson, RJ Radmer, PA Kollman. Determination of the relative binding free energies of peptide inhibitors to the HIV-1 protease. J Med Chem 34 2654-2659, 1991. 

The most recent advance in treating HIV infections has been to simultaneously attack the virus on a second front using a protease inhibitor. Recall from Section 27.10 that proteases are enzymes that catalyze the hydrolysis of proteins at specific points. When HIV uses a cell s DNA to synthesize its own proteins, the initial product is a long polypeptide that contains several different proteins joined together. To be useful, the individual proteins must be separated from the aggregate by protease-catalyzed hydrolysis of peptide bonds. Protease inhibitors prevent this hydrolysis and, in combination with reverse transcriptase inhibitors, slow the reproduction of HIV. Dramatic reductions in the viral load in HIV-infected patients have been achieved with this approach. 

HIV-protease AIDS virus Processing of AIDS virus proteins... 

FIGURE 16.25 Structures of (a) HIV-1 protease, a dimer, and (b) pepsin (a monomer). Pepsin s N-terminal half is shown in red C-ter-minal half is shown in blue. 

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. 

FIGURE 16.28 HIV mRNA provides the genetic information for synthesis of a polyprotein. Proteolytic cleavage of this polyprotein by HIV protease produces the individnal proteins required for viral growth and cellular infection. 

FIGURE 16.29 (left) HIV-1 protease com-plexed with the inhibitor Crixivan (red) made by Merck. The flaps (residues 46-55 from each snbnnit) covering the active site are shown in green and the active site aspartate residues involved in catalysis are shown in white. 

FIGURE 16.30 A mechanism for the incorporation of from H9 0 into peptide substrates in the HIV protease reaction. (Adapted from Hyland, ., et ai, 1991. BiochemisU y 30 8441-8453.)... 

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. 

A final but important consideration is viral mutation. Certain mutant HIV strains are resistant to one or more of the protease inhibitors, and even for patients who respond initially to protease inhibitors it is possible that mutant viral forms may eventually thrive in the infected individual. The search for new and more effective protease inhibitors is ongoing. 

The pH dependence of HIV-1 protease has been assessed by measuring the apparent inhibition constant for a synthetic substrate analog (b). The data are consistent with the catalytic involvement of ionizable groups with pK values of 3.3 and 5.3. Maximal enzymatic activity occurs in the pH range between these two values. On the basis of the accumulated kinetic and structural data on HIV-1 protease, these pK values have been ascribed to the... 

Chen, Z., Li, Y, Schock, H. B., et al., 1995. Three-dimensional structure of a mutant HIV-1 protease displaying cros.s-resi.stance to all protease inhibitors in clinical txiAs. Journal of Biological Chemistry 270 21433-21436. 

Wang, Y X., Freedberg, D. I., Yamazaki, T, et al., 1997. Soludon NMR evidence diat the HIV-1 protease catalytic aspartyl groups have different ionization. states in the complex formed with die a.symmetric drug KNI-272. 

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