From the
The human immunodeficiency virus Gag- and Pol-encoded proteins
are expressed as long polypeptide precursors that are proteolytically
cleaved into mature proteins found in infectious virions. This cleavage
is catalyzed by the virus-encoded aspartyl protease and is required for
virus replication(1, 2, 3) . As such, the
viral protease has become a target for HIV ()therapeutics,
resulting in many HIV protease inhibitors reaching clinical trials.
Most of these drugs are substrate-based inhibitors, whose design has
been facilitated by an abundance of crystal structure data for both the
native enzyme and enzyme-inhibitor complexes(4) . Additionally,
there are now extensive biochemical data detailing both the catalytic
mechanism and the molecular basis for substrate
selection(5, 6, 7) .
The primary difficulty encountered while administering HIV therapeutic agents to patients has been the rapid emergence of drug resistance by the virus. Inhibitors of retroviral enzymes have dramatic effects initially, lowering virus load to less than 1% of pretreatment levels and improving the clinical state of the patient(8, 9, 10) . However, virus resistance to the inhibitory effects of the drugs develops rapidly, causing a continued progression of disease symptoms. This has been well documented in many clinical trials using reverse transcriptase inhibitors such as azidothymidine (AZT),(-)-2`-deoxy-3`-thiacytidine (3TC), or nevirapine. Unfortunately, a similar progression is now being observed in patients treated with retroviral protease inhibitors (11) .
The
primary mechanism by which retroviruses develop this resistance has
been thought to be a consequence of the relatively low fidelity of
reverse transcriptase. Because the DNA polymerase does not have a
proofreading function, base mismatch errors are incorporated into viral
DNA before integration into the host cell genome. This proviral DNA
then serves as a template for all new viral transcripts, passing along
any mutations incorporated during the initial reverse transcription
event. Although only one or two reverse transcription products are
integrated successfully into each host cell genome, a large number of
genetic mutations can accumulate in a very short time. This is made
possible by the large pool of infected T-cells that turn over extremely
rapidly. In a report earlier this year in Science, Coffin (12) calculates that at least 10 new cells are
infected each day in a typical HIV-infected patient during the latent
or steady state stages of infection (12) and that every point
mutation that could possibly occur along the length of the viral genome
does occur and at a staggering frequency of between 10
and
10
times daily! In addition to reverse transcriptase
infidelity, mutations may be introduced into the viral genome through
host cell RNA polymerase II transcription errors or through genetic
recombination between homologous regions of the HIV genome and
endogenous viral sequences. Thus, in the presence of a drug selecting
for certain types of mutations in particular proteins, a passive and
relatively random process leads rapidly to the emergence of drug
resistance.
Several groups have sequenced protease genes from
drug-resistant HIV phenotypes isolated from tissue culture
systems(13, 14, 15, 16, 17, 18) .
The protease mutants that have been analyzed contain a similar set of
amino acid changes at very characteristic and predictable positions
within the enzyme molecule. Although many different inhibitor compounds
have been used in these studies, the same regions of the enzyme have
mutated in the drug-resistant strains. Analysis of the data shows that
mutations which produce inhibitor-resistant proteases are concentrated
in regions of the enzyme that have been shown to form the subsites of
the substrate binding pocket. This is best illustrated by the studies
of Colonno and co-workers(18) , who followed the appearance of
drug-resistant viruses in HIV-1-infected cells grown in tissue culture.
Multiple changes in protease amino acid sequence were detected with
time. However, drug resistance emerged only after Val-82 changed to Ala (18) . These mutations affect substrate recognition in the S1
and S1` subsites (Fig. 1). Other key amino acids whose changes
produce resistant phenotypes are at position 32 in the S2 and S2`
subsites, at positions 8, 46, 47, and 48 in the S4 and S4` subsites,
and at position 84 in the S1, S3, S3`, and S1` subsites (Fig. 1). Positions 46, 47, and 48 are in highly flexible and
exposed surface loops. These ``flaps'' cover the substrate
binding cleft and form part of the S4 and S4` subsites. The side chains
of all of these residues, with the exception of Met-46, are orientated
inward toward the inhibitor and contribute directly to its binding. The
Met-46 Leu mutation was shown by molecular dynamic simulations
to decrease the flexibility of the flaps(19) . This may prevent
the flaps from opening as efficiently and thereby limit inhibitor
access to the substrate binding pocket. Most of these residues that are
mutating in these drug-resistant enzymes had been identified previously
as being critical for determining retroviral protease
specificity(7) . Furthermore, it was predicted that these
residues were those likely to play a significant role in developing
drug resistance even before resistance data were
available(7, 20) .
Figure 1:
Stereo view
of the HIV-1 protease with the inhibitor Sequinavir. The structure of
the HIV-1 protease complexed with the inhibitor Ro-8959 (Sequinavir; Fig. S1below) from Krohn et al. (26) now in clinical trials was produced using the MOLSCRIPT
program(27) . The inhibitor, which is in a -sheet
conformation, is shown by the blue lines. Enzyme amino acid
residues that change in developing drug-resistant protease phenotypes
are depicted by the ball and sticks in the red
color. Individual enzyme subunits are shown in yellow and green. The residue numbers for only one of the two identical
subunits are shown. This figure was prepared by Dr. Alex Wlodawer,
NCI-Frederick Cancer Research Center.
Figure S1: Structure 1
Recent work by Condra et al.(11) describes four patients that developed resistance to the protease inhibitor MK-639. These drug-resistant HIV-1 protease mutants also have at least one mutation in a key amino acid residue positioned directly in one of the enzyme subsites forming the substrate binding pocket. For instance, patients A, B, and C had mutations at Val-82 or Ile-84 in the S1 and S1` binding pockets. Patients A, C, and D had changes in Met-46 at the base of the flaps, and patients C and D had mutations at Val-32, which is a key residue in the S2 and S2` binding pockets. Patient D had an additional flap mutation at Ile-47. Many of these positions were identified earlier by groups looking for drug resistance in tissue culture systems. In fact, many of the exact same mutations were seen in the human isolates. These include I84V, M46I, I47V, and V32I substitutions.
What is the molecular basis for these resistant phenotypes? The substrate binding pocket of protease is formed in a region along the central axis of the symmetric homodimer. Eight individual subsites are formed along the length of the enzyme surface and are designed to accommodate substrate amino acid side chains. Substrate binding in the subsites is governed largely by an ability to form stabilizing van der Waal's interactions between substrate amino acid side chains and enzyme amino acid side chains lining the subsites(20, 21) . The subsites have been shown to act largely independently in the selection of substrate amino acids(22, 23) . For example, mutations can be made in amino acids forming the S2 subsite that change the preference for substrate amino acids at the corresponding P2 position. Protease amino acid residues in the substrate binding cleft can be mutated both singly and in combination to produce mutant proteases with altered substrate specificity(22, 23) . Many of these mutants retain the ability to process the wild-type substrate sequences, albeit at lower rates. Recent work by Erickson and co-workers (24) has shown that the overall catalytic efficiency for each of 11 drug-resistant HIV protease mutants is between 1.2- and 14.8-fold lower than for the wild-type enzyme on a standard reference substrate. However, if drug treatment is continued, near wild-type titers of virus are restored over a period of a year without loss of a drug-resistant mutation(18) . This occurs concomitantly with additional protease mutations, which presumably compensate for the decrease in activity toward the viral polyproteins due to the initial resistance-producing change.
The majority of the drug-resistant
protease mutations that have been identified are relatively
conservative changes involving the gain or loss of a methylene group (Table 1). For example, I84V, V82I, V82A, I47V, and V32I all have
significant effects on inhibitor binding. Other changes that are
observed involve small changes in side chain length or character (Table 1). The only exception is M46F. In the other positions (Table 1), where changes in amino acids probably have little
effect on specificity, more diversity in the substitutions is observed.
It may seem surprising that such seemingly small changes in the
critical regions can effect the IC concentrations by
greater than 30-fold. However, in vitro biochemical studies
using mutant retroviral proteases and altered peptide substrates have
shown that rather modest changes in either enzyme or substrate can
effect catalytic efficiency (k
/K
) by greater
than 10-fold(20, 22) . A particularly vivid example
demonstrating the extent to which retroviral protease specificity is
easily altered by modest changes at the enzyme-substrate interface was
seen in the Rous sarcoma virus protease. This enzyme is structurally
very similar to HIV-1 protease. When the P2 position of an efficiently
cleaved substrate was changed from valine to leucine, the activity was
reduced to 10% of that seen with the unmodified peptide. However,
changing Ile-44 to Val in the enzyme increased the ability to cleave
the P2-modified peptide, restoring relative cleavage to levels
approaching those seen with the unmodified peptide substrate. This
result can be explained using crystallographic data with
computer-modeled substrates. The addition of a methylene group from the
substrate P2 position can be compensated for by the removal of a
methylene group from an amino acid side chain forming part of the S2
subsite. The simple gain or loss of a methylene group from an enzyme
binding pocket can easily lead to 10-fold changes in K
for a given drug. This could render the
drug ineffective by making it impossible to increase drug
concentrations to therapeutic levels. Recent analysis of crystal
structure data for HIV-1 protease complexed with the symmetric
inhibitor A-77003 confirms this conclusion and shows that favorable van
der Waal's interactions in the S1` subsite are disrupted by a
Val-82
Ala substitution, which infers the drug-resistant
phenotype(25) . A similar conclusion with a different inhibitor
complexed to a multidrug-resistant HIV-1 protease has recently been
reported(28) .
It has been realized that drug-resistant
phenotypes selected in the presence of one drug often display
resistance to many other protease inhibitors with different structures.
This is not particularly surprising, for most protease inhibitors are
substrate-based non-hydrolyzable peptide mimetics that are targeted
initially against the wild-type enzyme. Mutating key residues in the
substrate binding pockets alters the ability to form favorable van der
Waal's interactions that stabilize the bound enzyme inhibitor
complex, and the K for the class of
inhibitors increases dramatically. Because tight specific binding
requires maximizing interactions in each of the eight enzyme subsites,
mutations in any of the subsites would be expected to confer resistance
to many inhibitors. The inhibitors used were selected for their ability
to bind tightly to the native wild-type enzyme. This dictates that the
compounds interact favorably with most of the enzyme subsites.
Disrupting this interaction with one compound will likely disrupt the
structurally equivalent interaction with each of the other tightly
binding inhibitors.
Typically, compounds in development that do not inhibit the wild-type enzyme are not examined further. However, it would seem that those in the business of designing HIV protease inhibitors could profit from examining compounds that may not bind especially tightly to the wild-type enzyme. Many of these compounds are likely to exist already and may bind tightly to the mutant proteases. Indeed, by the same arguments described above, an inhibitor designed against the mutant enzymes should not bind tightly to the wild-type protease. While the enzyme clearly demonstrates a remarkable ability to mutate and preserve function, there are almost certainly a limited number of mutations that will alter drug binding while preserving a sufficient level of specific activity on the normal substrates to allow for virus replication. A multidrug approach with protease inhibitors designed against wild-type and mutant form(s) most likely to develop may offer promise as a potentially effective therapy for HIV infection.