Biomedical Engineering Reference
In-Depth Information
polypeptide chains of 99 amino acids, each contributing a single catalytic aspartate residue within
the active site that lies at the dimer interface. This active site is covered by two symmetric l aps
whose dynamic motions allow entry and exit of polypeptide substrates. For each of the different
substrates, three to four amino acids on each side of the scissile bond are thought to be involved in
binding to the substrate cavity. Since there is little similarity in the primary sequence of the cleavage
sites of each of the protease substrates, binding specii city is thought to be driven by the conserva-
tion in the secondary structure surrounding the cleavage sites. All of the inhibitors currently used to
treat HIV infection are competitive in nature and bind to the protease active site.
Saquinavir was the i rst HIV protease inhibitor available for the treatment of AIDS, and its
design was based on a strategy using a transition state mimetic. A distinguishing feature of HIV
PR is its ability to cleave Tyr-Pro and Phe-Pro sequences found in the viral substrates, as mamma-
lian endopeptidases are unable to cleave peptide bonds followed by a proline. A rational inhibitor
design approach based on this property offered hopes of identifying inhibitors selective for the viral
enzyme. Since reduced amides and hydroxyethylamine isosteres most readily accommodate the
amino acid moiety of Tyr-Pro and Phe-Pro in the HIV substrates, they were chosen for further inter-
rogation. Systematic substitutions were explored on a minimum peptidic pharmacophore, and one
compound containing an (
S
,
S
,
S
)-decahydro-isoquinoline-3-carbonyl (DIQ) replacement for proline
exhibited a
K
i
value of 0.12 nM at pH 5.5 for HIV-1 PR and <0.1 nM for HIV-2 PR. The interactions
of this compound, later named saquinavir, with HIV-1 PR were studied crystallographically (Figure
11.10). The compound was shown to bind to the enzyme in an extended conformation with the carbonyl
of the DIQ group binding to a water molecule that connects the inhibitor with the l ap regions. These
studies shed much light on the binding mode of the i rst HIV PR inhibitors and set the stage for
further exploration of novel compounds with improved properties.
The availability of new HIV protease inhibitors represented a great triumph in the i ght against
AIDS, but it was only a matter of time before the selective pressure of antiretroviral therapy led to
the emergence of HIV strains harboring drug-resistant mutations against protease inhibitors. One
of the primary mutations i rst noted in protease inhibitor-resistant strains was in Val82 of HIV-1
PR. Crystallographic and modeling studies suggested that the binding of protease inhibitors like
ritonavir might be compromised due to the loss of hydrophobic interactions between the isopropyl
side chain of Val 82 of the enzyme and the isopropyl substituent projecting from the 2 position of
the P3 thiazolyl group of ritonavir. This functionality was substituted to identify an inhibitor whose
activity was less dependent on interaction with Val 82, and the optimization that was supported by
modeling studies led to the identii cation of ABT-378, later named lopinavir. This novel inhibitor
had extraordinary potency against wild-type and mutant HIV PR (
K
i
= 1.3-3.6 pM)
in vitro
, and
maintained activity against ritonavir-resistant mutants of HIV-1. Lopinavir, as a combination drug
FIGURE 11.10
The crystal structure of HIV-1 protease in complex with the inhibitor saquinavir (PDB 1hxb).
