Protease HIV

3 Structure-Based Design From Renin to HTV-I Protease 

Prior to the determination of the X-ray crystal structure of HIV-1 protease, a model of the enzyme was derived from the homologous RS V protease [56]. Within a short amount of time, the first crystal structures of the native HIV-1 protease were solved [57,58], followed by numerous enzyme/inhibitor crystal complexes [59]. These experimental results not only confirmed the accuracy of the model with regard to the substrate binding residues, but provided 

The HIV-1 protease, as a member of the aspartic protease family of enzymes, contains an aspartic acid dyad at the catalytic site. The enzyme is active as a homodimer, with each subunit containing 99 amino acids (the numbering system is 1-99 for the first monomers and 101-199 for the second). This differs from the monomeric structure of renin, which is composed of 340 residues. The identical monomers in HIV-1 protease produce a symmetric 

Hybrid potentials have been used to understand the mechanism of the human immunodeficiency virus (HIV) protease with the ultimate aim of being able to help in the design of inhibitors which could be useful as AIDS therapies. This enzyme, which catalyzes the hydrolysis of peptide bonds, is a homodimer. Its active center is at the interface of the two chains and consists of two catalytic aspartic acid residues from identical positions in each of the two chains. Although the aspartates are equivalent in the sequence, they are not equivalent when the substrate is present. It is known that when the enzyme is active one of the aspartic residues is protonated and it is thought that there is a lytic water molecule that is also involved in the catalysis. 

Several mechanisms have been proposed, hi their study, van Gunsteren et al investigated two general acid-general base catalytic mechanisms in which one or the other of the two aspartates was protonated. The difference between the two is that in one case the initial proton transfer is to the carbonyl oxygen of the scissile peptide whereas in the other it is to the nitrogen. They performed molecular dynamics umbrella sampling simulations with a PM3/MM potential to 

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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... 

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... 

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.)... 

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

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. 

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. 

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. 

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. 

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. 

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]. 

Efficient coupling between a chiral 3-phenylpropionamide enolate and (S)-glyci-dyl tosylate was achieved in a practical route to the HIV-1 protease inhibitor L-735-524 [68b]. 

J ,3J ,4J ,5J )-2,5-bis(benzyloxy)-3,4-dihydroxy-Nd -bis (lS)-2-methyl-l-[(methylamino)carbonyl]propyl hexanediamide is a C2-symmetric HIV-1 protease inhibitor [29]. Derivatization in the para positions of the benzyl-oxy groups via microwave-assisted Stille reaction on the corresponding di-brominated inhibitor smoothly yielded the desired heteroarylated derivatives (Scheme 10). Interestingly, the 1,3-thiazole derivative showed a higher antiviral activity on the wild type virus than the lead compound. The activity remained at the same level in the presence of seriun. Unfortimately, a low activity was observed on mutants. 

Other types of HIV-1 protease inhibitors have also been prepared using microwave-promoted Suzuki reaction [37]. The symmetric cyclic sulfamide (3K,4S,5S,6it)-3,6-bis(phenoxymethyl)-2,7-bis[4-(2-thienyl)benzyl]-l,2,7-thi-adiazepane-4,5-diol 1,1-dioxide, for instance, was synthesized via cross-couphng of (3aS,4R,8it,8aS) - 5,7 - bis(4 - bromobenzyl) - 2,2 - dimethyl - 4,8 - bis-(phenoxymethyl) hexahydro [1,3] dioxolo [4,5 - d] [ 1,2,7 ] - thiadiazepine 6,6 - dioxide with 2-thienylboronic acid for 3 min at 45 W (Scheme 19). 

As a direct appUcation a potent C2-symmetric HIV-1 protease inhibitor (with two tetrazoles as carboxyl group bioisosteres) was prepared in one pot [77]. The process involved microwave-promoted cyanation followed by conversion of the nitrile group in a tetrazole with azide (Scheme 64). It is notable that the fimctionahzation was achieved so smoothly without side reactions such as the ehmination of water. 

The Structural and Mechanistic Basis of HIV-1 Protease Inhibitor Resistance. 93... 

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. 

Fig. 1 A ribbon diagram of the crystal structure of a substrate complex of the homo-dimer HIV-1 protease (lkj7) (Prabu-Jeyabalan et al. 2002), Each monomer is shown in cyan and pink the substrate is shown in green, and the catalytic aspartic acids are highlighted in yellow...

Crystallographic studies imply that although little sequence homology exists between the different protease cleavage sites, what is conserved is the shape that they adopt within the active site of the enzyme (Prabu-Jeyabalan et al. 2002). This shape has been termed the substrate envelope and represents the consensus volume overlapping the majority of the substrates. Most likely, HIV-1 protease recognizes a particular peptide sequence as being a substrate by a combination of accessibility and the shape the sequence can adopt. 

Allaire M, Chernaia MM, Malcolm BA, James MN (1994) Picomaviral 3C cysteine proteinases have a fold similar to chymotrypsin-Kke serine proteinases. Nature 369 72-76 Altman MD, Nalivaika EA, Prabu-Jeyabalan M, Schiffer CA, Tidor B (2008) Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease. Proteins 70 678-694... 

Danner SA, Carr A, Leonard JM, Lehman LM, Gudiol F, Gonzales J, Raventos A, Rubio R, Bouza E, Pintado V et al (1995) A short-term study of the safety, pharmacokinetics, and efficacy of ritonavir, an inhibitor of HIV-1 protease, European-Australian Collaborative Ritonavir Study Group, N Engl J Med 333 1528-1533... 

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