Viruses 2010, 2, 2509-2535; doi:10.3390/v2112509
Molecular Basis for Drug Resistance in HIV-1 Protease
Akbar Ali 1, Rajintha M. Bandaranayake 1, Yufeng Cai 1, Nancy M. King 1, Madhavi Kolli 1,
Seema Mittal 1, Jennifer F. Murzycki 2, Madhavi N.L. Nalam 1, Ellen A. Nalivaika 1,
Ayşegül Özen 1, Moses M. Prabu-Jeyabalan 3, Kelly Thayer 1 and Celia A. Schiffer 1,*
1 Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical
School, Worcester, Massachusetts 01605, USA; E-Mails: Akbar.Ali@umassmed.edu (A.A.);
Rajintha.Bandaranayake@umassmed.edu (R.M.B.); Yufeng.Cai@umassmed.edu (Y.C.);
Nancy.King@umassmed.edu (N.M.K.); Madhavi.Kolli@umassmed.edu (M.K.);
Seema.Mittal@umassmed.edu (S.M.), Madhavi.Nalam@umassmed.edu (M.N.L.N.);
Ellen.Nalivaika@umassmed.edu (E.A.N.); Aysegul.Ozen@umassmed.edu (A.Ö.);
2 Department of Pediatrics, University of Rochester, Rochester, NY 14627, USA;
3 Division of Basic Sciences, The Commonwealth Medical College, 150 N. Washington Avenue,
Scranton, PA 18503, USA; E-Mail: email@example.com
* Author to whom correspondence should be addressed; E-Mail: Celia.Schiffer@umassmed.edu;
Tel.: +1-508-856-8008; Fax: +1-508-856-6464.
Received: 8 October 2010; in revised form: 22 October 2010 / Accepted: 28 October 2010 /
Published: 12 November 2010
Abstract: HIV-1 protease is one of the major antiviral targets in the treatment of patients
infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed
with extensive use of structure-based drug design, thus the atomic details of how the
inhibitors bind are well characterized. From this structural understanding the molecular
basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in
response to therapy and diversity between clades in HIV-1 protease have altered the shape
of the active site, potentially altered the dynamics and even altered the sequence of the
cleavage sites in the Gag polyprotein. All of these interdependent changes act in synergy to
confer drug resistance while simultaneously maintaining the fitness of the virus. New
strategies, such as incorporation of the substrate envelope constraint to design robust
inhibitors that incorporate details of HIV-1 protease’s function and decrease the probability
Viruses 2010, 2
of drug resistance, are necessary to continue to effectively target this key protein in HIV-1
Keywords: drug resistance; HIV-1 protease; protease inhibitors; substrate envelope;
structure based drug design
According to the recent reports published by UNAIDS, there are about 33.4 million people living
with HIV-AIDS around the globe . Currently, there is no permanent cure or vaccine for AIDS but
there are about 25 drugs that belong to seven classes targeting different stages in the life cycle of
HIV . Although the quality and life expectancy of HIV infected patients has improved since the
introduction of antiviral treatment, low drug adherence, toxicity, and high pill burden, coupled with the
error prone mechanism of HIV reverse transcriptase, have led to the emergence of drug resistance in
HIV infected patients (for recent reviews see [2–5]).
Protease inhibitors (PIs) are one class of drugs that target an essential viral enzyme HIV-1 protease.
Because of its critical role in the processing of Gag and Gag-Pro-Pol polyproteins into individual
proteins necessary for viral maturation , protease is one of the major therapeutic targets for
developing antiviral drugs against HIV-AIDS. In the last two decades, drug discovery efforts aided by
structure-based design have led to the development of nine FDA-approved protease inhibitors (PIs)
(Figure 1): Saquinavir (SQV) , Indinavir (IDV) , Ritonavir (RTV) , Nelfinavir (NFV) ,
Amprenavir (APV) , Lopinavir (LPV) , Atazanavir (ATV) , Tipranavir (TPV) , and
Darunavir (DRV) [15–17]. These inhibitors represent the most potent anti-AIDS drugs reported to date
and are essential components of the highly active antiretroviral therapy (HAART) [18,19]. HAART is
credited with significantly reducing AIDS-related mortality [20,21] and is currently implemented
throughout the world as the standard of care for HIV-AIDS treatment.
Drug resistance to PIs has become a major issue with the failure of HAART. Moreover, newly
infected patients are infected with resistant viruses which are an added challenge in the treatment of
HIV infections. Various strategies have been used to develop new antiviral therapies against
drug-resistant HIV, including increasing the plasma levels of existing PIs by using a boosting
agent  and developing new PIs using structure-based drug design [4,23–25]. Among different
approaches, one design strategy maximizes the number of hydrogen bonds with the protease backbone
and led to the development of highly potent PIs active against drug-resistant HIV [25,26]. PIs with
improved resistance profiles were also developed using a solvent anchoring approach , and
utilizing a new lysine sulfonamide-based molecular core . Another design strategy incorporates
substrate envelope constraints into structure-based design and led to the discovery of novel highly
potent PIs that are less susceptible to drug-resistance . The principles underlying these various
strategies are not necessarily mutually exclusive and all achieved the design of highly potent inhibitors
against drug-resistant HIV.
Viruses 2010, 2
Figure 1. FDA-approved HIV-1 protease inhibitors.
2. FDA-approved HIV-1 Protease Inhibitors
All currently approved HIV-1 PIs are competitive active site inhibitors that bind in the active site of
the protease and, except for TPV, all are peptidomimetics. These PIs were rationally designed based on
the transition state mimetic concept and contain various non-cleavable dipeptide isosteres as core
scaffolds to mimic the transition state of the polyprotein substrates of HIV-1 protease (Figure 2). A key
common feature of these inhibitors is the presence of a secondary hydroxyl group, a surrogate for the
P1 carbonyl moiety of substrates, which makes critical interactions with the catalytic Asp25/25′
residues of the protease and is required for tight inhibitor binding with the protease. Another common
feature in the complexes between peptidomimetic inhibitors and HIV-1 protease is a conserved water
molecule that mediates contacts between the P2/P1′ carbonyl oxygen atoms of the inhibitors and the
amide groups of Ile50/Ile50′ of the enzyme. The development and clinical introduction of HIV-1 PIs is
regarded as a major success of structure-based rational drug design .
Development of the first generation PIs was greatly facilitated by the knowledge of inhibitors of
other aspartic proteases such as renin, and early availability of numerous crystal structures of both
unliganded enzyme and enzyme-ligand complexes [30–32]. Initial designs of inhibitors were based on
pepstatin, a natural transition state mimic, and sequence homology of substrate cleavage sites at the
Gag and Gag-Pro-Pol polyprotein containing a non-cleavable reduced amide dipeptide isostere .
The crystal structures of these early inhibitor-protease complexes provided a wealth of information on
Viruses 2010, 2
the inhibitor-enzyme interactions in the protease active site and led to the optimization of various
SQV, discovered by Roche , was the first HIV-1 PI approved by the FDA in December 1995 for
the treatment of HIV-AIDS. The initial pentapeptide lead was based on the HIV-1 pol substrate
sequence containing the unusual Phe-Pro amide bond at the cleavage site. Lead optimization, including
replacement of the P1-P1′ amide bond with non-cleavable hydroxyethylamine (Figure 2-I) based
dipeptide isostere, replacement of the P1′ proline with a bicyclic decahydroisoquinoline, and
introduction of a quinoline moiety at P3 led to the discovery of SQV. Although SQV is a very potent
(Ki = 0.12 nM) and selective inhibitor of HIV-1 protease, SQV has very poor bioavailability and is
quickly degraded in vivo by cytochrome P450 (CYP-450).
Figure 2. The scissile bond in polyprotein substrate is hydrolyzed by protease through the
transition state intermediate (substrate amino acid residues are marked as...P3, P2, P1, P1′,
P2′, P3′…and the corresponding enzyme binding sites as…S3, S2, S1, S1′, S2′, S3′…).
Transition state mimics I–V used in the design of currently approved drugs.
SQV was soon followed by two structurally distinct PIs, IDV  and RTV . IDV, developed by
Merck, was also optimized from an initial peptide lead in which the P1-P1′ fragment was replaced
with a novel Phe-Gly hydroxyaminopentane dipeptide isostere (Figure 2-II). The other key structural
features of IDV are the aminohydroxyindane moiety at P2′ position and a P1-P2
pyridylmethylpiperazine moiety. IDV has protease inhibitory potency of 0.6 nM, antiviral potency
of 25–100 nM, and has excellent oral bioavailability.
In the discovery of RTV, the Abbott team sought to exploit the C2 symmetry of the HIV-1 protease
and initially designed inhibitors by incorporating a C2 symmetric dihydroxy Phe-Phe isostere core.
During the lead optimization process, they discovered that the second hydroxyl group in the core
isostere could be removed without affecting the potency leading to the development of a
pseudo-symmetric all carbon Phe-Phe hydroxyethylene isostere core (Figure 2-III). RTV potently
inhibits HIV-1 protease (Ki = 0.022 nM) and has moderate antiviral potency (EC50 = 60 nM). Due to
its numerous side effects RTV is no longer used as a PI on its own. However, RTV is a potent inhibitor
Viruses 2010, 2
of CYP-450 3A4 isoform , and, because of this side activity, low dose RTV is currently used as a
boosting agent in HAART therapy with other PIs.
NFV  was developed by truncating the N-terminal moiety in SQV and replacing the P2
asparagine with 3-hydroxy-2-methylbenzamide fragment. These changes in combination with a novel
P1 moiety in the hydroxyethylamine isostere led to NFV (Ki = 2 nM) with significantly reduced
molecular weight and improvement in bioavailability, though NFV is less potent than SQV. Efforts by
Vertex aimed at reducing the molecular weight and peptide character of PIs led to the discovery of
APV , which incorporates a novel hydroxyethylamino-sulfonamide dipeptide isostere (Figure 2-IV).
The 3-hydroxyltetrahydrofuran P2 moiety was designed to mimic the interactions of SQV’s asparagine
side chain with the Asp29 residue. APV, also approved as a prodrug (fosamprenavir), is the smallest of
the 9 currently approved PIs; APV has moderate potency (Ki = 0.6 nM), good bioavailability and long
half-life, allowing twice daily dosing in patients.
Based on the first generation PI RTV, Abbott developed a highly potent second generation
inhibitor, LPV , which is also active against RTV-resistant protease variants. Significant efforts
directed at replacing the bulky (2-isopropylthiazolyl)methyl P3′ moiety with smaller groups led to the
discovery of cyclic urea as a high affinity P3′ moiety. The P2 thiazolylmethyl moiety was also replaced
with a more lipophilic 2-(2,6-dimethylphenoxy)acetamide resulting in an exceedingly potent PI
with 10-fold better potency than RTV. Although LPV has poor bioavailability and pharmacokinetic
profile, its plasma levels could be significantly enhanced by adding low dose RTV ; a combination
of LPV/RTV (Kaletra) is one of the most widely used PI therapies.
ATV , approved in 2003, incorporates
aza-hydroxyethylamine dipeptide isostere (Figure 2-V), an extended 4-(2-pyridinyl)phenylmethyl
moiety and a methylcarbamate capped tert-leucine moiety at both P2/P3 and P2′/P3′ positions.
Compared to the hydroxyethylene core of LPV, the P1-P2 aza-linkage eliminates one of the three
chiral centers allowing easier large-scale synthesis. ATV has high antiviral potency and oral
bioavailability, and is the only PI that allows once daily dosing.
TPV  is the only non-peptidomimetic PI developed from lead compounds 4-hydroxycoumarin
and 4-hydroxy-2-pyranone, identified by high throughput screening. Unlike other PIs, TPV is not a
transition state mimetic, and instead binds to the protease in a distinct fashion replacing the conserved
flap water. The phenolic hydroxyl group of the central 4-hydroxy-2-pyranone moiety makes hydrogen
bond interactions with the Asp25/25’ in the floor of the active site and the carbonyl group, unlike
peptidomimetic inhibitors, makes direct hydrogen bond interactions with Ile50/50’ in the flap region of
the protease. TPV potently inhibits multidrug-resistance protease variants and the replication of viruses
that are resistant to most other PIs. TPV, due to its unique binding mode with the protease, a resistance
profile different from other drugs, and a higher barrier to resistance requiring multiple mutations, is
recommended for therapy with patients containing preexisting protease resistance.
DRV [15–17], the latest protease inhibitor approved by the FDA, incorporates the same
hydroxyethylamino-sulfonamdie isostere present in APV. In fact, both compounds are very similar
with the only difference being a condensed bis-tetrahydrofuranyl (bis-THF) moiety at P2 present in
darunavir instead of a single tetrahydrofuranyl (THF) ring of APV. DRV was developed by both
academic and industrial research efforts based on the crystal structures of HIV-1 protease bound to
APV, SQV and its analogues bearing the bis-THF moiety at P2 position. These crystal structures
a novel (hydroxyethyl)hydrazine or
Viruses 2010, 2
revealed that the oxygen atoms of the THF/bis-THF moieties make extensive hydrogen bond
interactions with the Asp29/Asp30 residues of the protease enzyme. The critical interactions of the
bis-THF moiety in the S2 binding pocket of the protease enzyme are largely responsible for the
exceptionally high inhibitory and antiviral potency of darunavir (Ki = 15 pM; EC50 = 1–4 nM). DRV is
the most potent antiviral protease inhibitor approved to date and is also highly effective against most of
the multi-drug resistant HIV-1 variants.
The enzyme inhibitory activities of all FDA approved HIV PIs against wild-type (WT) protease and
three drug-resistant variants and their cellular antiviral potencies against wild-type HIV are provided in
Table 1 for comparison. The first generation PIs, RTV, SQV, IDV, NFV, lose significant activity
against drug-resistant protease variants, however, recently approved drugs TPV and DRV retain low
picomolar (pM) inhibitory activities.
Table 1. Binding affinity [29,35] and antiviral potency  of FDA approved HIV-1
3. Interdependency of Drug Resistance
3.1. Substrate Envelope Hypotheses
Within the Gag and Gag-Pro-Pol, HIV-1 protease cleavage sites are non-homologous and
asymmetric, both in charge and size. These characteristics begged the question as to how a symmetric
protease could recognize and cleave an asymmetric substrate. Structural studies have shown that the
various cleavage site peptides adopt a conserved shape/volume, which was hypothesized as the basis
for recognition of substrate sites by the HIV-1 protease . This overlapping volume of the majority
of the substrates within the active site of the protease defines the conserved shape or the “substrate
envelope” (Figure 3A). The P1-P3 region of the substrates forms a toroid, which is thought to be
important for specificity, whereas the numerous backbone-to-backbone interactions of the protease and
the substrates facilitate binding . The substrate envelope not only explains specificity of the
substrates but also the development of resistance to various PIs and substrate co-evolution .
Viruses 2010, 2
Figure 3. (A) Substrate envelope of HIV protease. PyMOL model generated from
overlapping van der Waals volume of substrate peptides. Red: matrix capsid, green:
capsid-p2, blue: p2-nucleocapsid, cyan: p1-p6, magenta: reverse transcriptase-
ribonucleaseH, yellow: ribnucleaseH-integrase. (B) The inhibitor envelope in red, within
the active site of HIV-1 protease, calculated from overlapping van der Waals volume of
five or more of eight inhibitor complexes. (C) Superimposition of the substrate consensus
volume (blue) with the inhibitor consensus volume (red). Residues that contact with the
inhibitors where the inhibitors extend beyond the substrate volume and confer drug
resistance when they mutate are labeled (Figures 3A-C, modified from King et al. ).
Crystallographic studies of the wild-type protease bound to inhibitor molecules have shown that
most of the PIs occupy a similar volume (defined as the inhibitor envelope, Figure 3B) and contact
Viruses 2010, 2
similar residues within the active site of the protease. Drug resistance occurs where inhibitor atoms
protrude beyond the substrate envelope and contact protease residues (Figure 3C) . Thus,
mutations at these sites would specifically impact inhibitor binding while substrate recognition and
cleavage remains relatively unaffected. The fact that most of the sites of drug resistant mutations in the
active site do not contact the substrates led to the development of the substrate envelope hypothesis:
Inhibitors that fit well within the substrate envelope would be less susceptible to drug resistance, as a
mutation that affects inhibitor binding would simultaneously impact the recognition and processing of
the majority of the substrates . Of the currently prescribed inhibitors the most efficacious is DRV
and although not designed using the substrate envelope constraint, DRV fits well within this
volume [39,40]. These studies also suggested that if the substrate atoms that protrude out of the
substrate envelope contact the very same residues in the active site of the protease that mutate to
prevent inhibitor binding, it could lead to impaired substrate recognition and cleavage resulting in the
co-evolution of compensatory mutations within the protease cleavage sites .
3.2. Drug Resistance—A Change in Molecular Recognition at the Active Site
The development of drug resistance is a major factor for the failure of protease inhibitor therapy.
The virus evolves to accumulate a multitude of mutations within the protease that prevent PIs from
binding to the protease. More than half the residues within the protease mutate in different
combinations and lead to drug resistance [41,42]. Drug resistance is a subtle change in the balance of
recognition events: The protease is still able to recognize and process the natural substrate sites in the
Gag and Gag-Pro-Pol polyprotein, while no longer being effectively inhibited by competitive drug
molecules. This hints that as drug resistance emerges, the interactions of the protease with an inhibitor
should significantly be altered to facilitate the reduced affinity of the protease to the inhibitors while
the interactions with a natural substrate should be maintained as in the wild-type structures.
As the functional HIV-1 protease is a symmetric dimer, both monomers contribute to substrate
binding. The active site region is primarily formed by residues 25–32, 47–53 and 80–84. Mutations
occurring anywhere else in the protease are referred to as the non-active site mutations.
Under protease inhibitor therapy, a majority of initial mutations arise within the active site of the
enzyme, directly affecting inhibitor binding and are the primary cause of resistance to PIs. Typical
primary mutations include D30N, G48V, I50L/V, V82A/F/T, I84V and L90M . Several primary PI
resistance mutations have been described that are a signature of particular PIs. For example, patients
failing NFV therapy develop the D30N protease mutation , while the I50V and I50L mutations are
selected in patients failing APV/DRV and ATV therapy, respectively [45,46]. Mutations at protease
residue 82 are observed in patients treated with RTV and SQV, and the G48V mutation results in
resistance to SQV and ATV [47,48]. The I84V mutation is one of the severe primary resistance
mutations causing cross-resistance to most PIs . Thus, a range of primary resistance mutations are
selected, some of which are unique to a single PI, whereas others confer resistance to two or more PIs.
Mutations in HIV-1 protease, either within or outside the active site, can decrease the binding
affinity of inhibitor molecules in a complex, interdependent and cooperative manner. When a protease
variant binds to an inhibitor, the structure of the protease adjusts to accommodate the inhibitor by
rearranging the interactions not only at the mutated residue but also throughout the protein [50–52].
Viruses 2010, 2
Analysis of protease inhibitor complexes has shown that the structure of HIV protease is highly
plastic [4,35,51]. The conformational change observed in the mutant protease is not always just around
the vicinity of the mutation. Various conformations found in crystal structures are probably the
combined effect of the nature of the inhibitor and the combination of mutations present in the protease.
Whether there is a major conformational change in the protease backbone or not, the drug resistant
mutation(s) does have an impact on the binding affinity to the inhibitor.
The rearrangement of the backbone can be observed either in the entire protease or in some parts of
the protease, as in flap region or P1 loop region, or just locally around the mutated residue .
Previous studies involving the drug resistant inactive variant of protease (D25N and V82A) with the
inhibitors SQV and RTV showed that the binding of the inhibitor is compromised because of the drug
resistant mutation, V82A . In addition to the direct loss of van der Waals contacts between the
inhibitors and the protease as a result of the V82A mutation, the mutant protease also undergoes
conformational changes as observed by the large shifts in the Cα backbone compared to the wild-type
structure. In another study , the binding of inhibitors, APV, DRV, ATV and SQV to the protease
variant containing L10I, G48V, I54V and V82A mutations has shown large changes in the flap regions
of the protease. In this case, the changes in the flap region are attributed to the two mutations present in
the flap (G48V and I54V), which may have locked the conformation of the flaps. The study by Munshi
et al.  revealed that the 80’s loop is intrinsically flexible and that mutations in this loop are not
necessary to result in conformational changes. Conformation of the P1 loops in the inhibitor-protease
complex depends mainly upon the nature of the bound inhibitor and may be influenced by mutations in
the protease . This means that the rearrangement of the protease also depends on the relative shifts
and tilts in the bound inhibitor. For instance, in the study  involving the V82T/I84V protease
variant bound to APV and DRV, minor changes in the backbone of the protease were observed
compared to the wild type. The P1 loop of only one monomer is shifted in the mutant structure
corresponding to the shift and tilt of DRV whereas, P1 loops of both the monomers of the protease are
shifted in the APV mutant protease structure corresponding to the shift and tilt of APV. Additionally,
there are minor backbone rearrangements in the crystal structures of the V82T/I84V protease variant
with ATV and SQV  where the shifts and tilts of the inhibitors account for the altered interactions
and hence, to the reduced affinity of the inhibitor. In a study by Konvalinka et al. , the impaired
binding of the inhibitor to the drug resistant protease is explained by a change in hydrogen bonding
pattern due to a substantial shift of the aminophenyl moiety of DRV.
3.3. Contribution of Protease Mutations outside the Active Site
Structural analyses of inhibitor complexes have been useful in the elucidation of the mechanism by
which active site mutations confer resistance to PIs [37,38,51,56]. Notably, the substrate envelope
hypothesis has helped explain the change in molecular recognition in resistant protease, where the
enzyme evolves to resist inhibitor binding but continues to recognize and bind its natural
substrates . However, the protease mutates extensively in the regions beyond active site, and these
non-active site mutations have been known to greatly contribute to drug resistance. The mechanism by
which the mutations outside the active site confer resistance remains elusive. Some of these mutations
Viruses 2010, 2
are primary drug resistant mutations and others have been suggested to contribute to drug resistance
when present along with other major mutations.
Of the 99 positions in each monomer, nearly 37 are known to be invariant (with mutation
frequencies <0.5%) and 17 positions are sites of non-treatment related polymorphisms [41,42,57].
Nearly 45 positions in each monomer have been implicated in drug resistance. Of these 45 positions,
mutations at 26 positions have been shown to significantly decrease susceptibility to one or more PIs
and the others are polymorphic mutations that occur more frequently when associated with inhibitor
therapy [42,58]. Furthermore, almost 60% of these 26 positions fall outside the active site region
(Table 2). Thus, excluding the invariant positions and including the polymorphic sites associated
with drug resistance, almost 40–45% of the protease sequence is implicated in contributing to
drug resistance [41,42,57], and a staggering 60–63% of the sequence has been known to vary in
Table 2. The major non-active site mutation positions which cause decreased susceptibility
to one or more PIs . The known polymorphisms are listed for subtype B .
10 L FI
Various groups, in the past, have studied thermodynamic, structural and kinetic parameters of
various combinations of the major drug resistant mutations and contributory or associated non-active
site secondary mutations in recombinant protease system [59–64]. Almost all these studies have shown
that the effect of major drug resistance mutations is highly diminished in the absence of paired
secondary non-active site mutations. Although the mechanism by which these diversely placed
non-active site residues orchestrate altered inhibitor-binding remains largely unknown, some
residue-specific explanations and suggestions have been put forth [65,66]. One of the reasons for this
altered binding has been suggested to lie in the internal dynamics and inherent plasticity of HIV-1
protease [60,67,68]. Some of these mutations may induce conformational perturbations in the enzyme,
Viruses 2010, 2
altering binding of the inhibitors. Kinetic studies conducted on various permutations and combinations
of active and non-active site protease mutants have shown that many of these protease variants have
decreased catalytic efficiencies, resulting from either increased KM values or reduced turnover rates or
a combination of both [60,69]. Some mutations, e.g., L90M, have been shown to make protease a
better enzyme for one substrate over the other in a clade specific manner [52,70].
3.4. Impact of the Co-evolution of Protease Cleavage Sites on Resistance
Following accumulation of resistance mutations within the protease, mutations also develop within
the substrate cleavage sites in Gag and Gag-Pro-Pol [71,72]. Mutations were first reported within the
NC-p1 and p1-p6 cleavage sites [71,73,74]. Additionally, associations between specific mutations in
the protease and the cleavage sites have been reported previously, and were demonstrated to alter
susceptibility to various PIs [71,73–76]. The A431V mutation within the NC-p1 cleavage site and
L449F in the p1-p6 cleavage site selected during the evolution of PI resistance were observed to
correlate with V82A and I50V protease resistance mutations, respectively [71,76].
Gag processing is enhanced by the A431V and I437V mutations within the NC-p1 cleavage
site [77,78]. In fact, there were clear structural changes that increased binding of the A431V NC-p1
site with the V82A protease . Recently though, both A431V and I437V have been shown to
directly increase resistance, possibly as a result of this enhanced Gag processing [78,80]. Similarly, the
L449F mutation within the p1-p6 cleavage site has been shown to increase processing at this cleavage
site [76,77,81]. Likely, the change from a smaller amino acid to a larger Phe improves van der Waals
contacts contributing improved Gag processing. These studies revealed that the p1-p6 cleavage site
mutations are associated with the NFV-resistant D30N/N88D protease mutations. In addition to these,
several other correlations between the NC-p1 and p1-p6 cleavage site mutations and primary drug
resistant mutations were observed . These cleavage site mutations have been demonstrated to be
compensatory in nature by improving replicative capacity and/or Gag processing [77,79]. Other
cleavage site mutations, including I437V and P453R, have now been well documented and are
associated with several major protease resistance mutations [76,82,83]. This suggests a mechanism
whereby decreased interactions between cleavage sites and mutant protease can be offset by
compensatory mutations within the cleavage sites leading to improved binding and processing. This
implies that with prolonged PI therapy, evolution of protease cleavage sites could be a fairly frequent
mechanism for maintaining viral fitness even as the virus evolves resistance to PIs.
Studies have shown that co-evolution of substrate cleavage sites and protease mutations also
contribute to PI resistance [78,82]. Primary PI resistance mutations, especially in the active site, reduce
both protease catalytic efficiency and viral replicative capacity (RC) [84–87]. Several studies have
demonstrated that the evolution of compensatory mutations within cleavage sites leads to improved
viral fitness compensating for the loss in fitness resulting from the protease resistance
mutations [71,72,74,88]. However, significant differences were not observed in viral fitness with
protease resistance mutations in the presence and absence of mutations within the Gag cleavage
sites . More recently, Larrouy et al. observed that baseline cleavage site mutations, in treatment-
naïve patients, were significantly linked to virological outcomes . More specifically, mutations at
Gag 128 within the MA-CA and Gag 449 within the p1-p6gag cleavage sites were associated with low
Viruses 2010, 2
virological response whereas mutations at Gag-Pol 437 within the TFP-p6pol were frequent in patients
achieving virological response . In a recent study, Parry et al. demonstrated that mutations in the
matrix and partial capsid in the N-terminal regions of Gag fully restore RC to WT levels and thus play
a key role in fitness . However, these mutations significantly enhanced resistance to PIs even in the
absence of PI resistance mutations in the protease . Thus, the evolution of mutations within the
cleavage sites and outside play an important role in the development of resistance and affect
virological response during therapy.
Statistical analysis on the effect of the observed correlations on phenotypic susceptibilities to
various PIs showed that these correlations were observed to significantly affect PI susceptibilities. In
most instances, a significant decrease in phenotypic susceptibility to particular PIs was observed.
Although mutations at either Gag 431 or Gag 437 were not associated with D30N/N88D protease
mutations, significantly lower PI susceptibilities were observed. A similar trend was also observed
with Gag A431V and the L90M protease mutation. Mutations at either of these residues within the
NC-p1 cleavage site likely directly enhance resistance to PIs, as was observed and demonstrated
previously [78,91]. At least in the case of the Gag A431V mutation, this is likely due to enhanced Gag
processing at this site as demonstrated by Nijhuis et al. . Thus, Gag cleavage site mutations
enhance resistance to PIs in combination with primary drug resistance mutations in the protease. A
detailed review of the role Gag cleavage sites on protease inhibitor resistance by Clavel and Mammano
is included in this issue .
4. Altered Pathways to Drug Resistance between the HIV-1 Clades
Based on genomic diversity, HIV-1 has been classified into nine clades (A, B, C, D, F, G, H, J,
and K) and 43 circulating recombinant forms (CRFs) [93,94]. The protease amino acid sequences
between clades vary up to about 10%. A number of these amino acid variations have been associated
with PI resistance in clade B (Table 3). With the exception of clade G, which has an active site amino
acid substitution when compared to clade B, all sequence variations within other clades map to
positions outside the active site (Figure 4). While currently available PIs are effective against different
HIV-1 clades very few studies have been carried out to understand the effect of clade specific
sequence variations on the emergence of drug resistance.
Despite the lack of data on pathways to resistance on non-B clade proteases, a number of studies
focusing on sequence polymorphisms in protease have highlighted differences in biochemical and
structural profiles as well as viral replication in non-B clade viruses when compared to clade B.
Enzyme kinetics studies show higher KM values, 1.4-fold, for clade A and lower KM values, 2.6-fold
and 3.4-fold, for clade C and G protease when compared to clade B and indicates that affinity for
substrates might be different between clades . Studies carried out on CRF01_AE have shown that
while KM values were comparable to that of clade B the catalytic turnover rates (kcat) were significantly
lower in CRF01_AE protease . Crystal structures of the AE protease indicate that the flap hinge
region of the protease is less flexible when compared to clade B protease that might lead to the lower
turnover rates observed in the AE protease. Thus, currently available data suggest that despite the fact
that sequence variations in non-B proteases map outside the active site, they play a role in modulating
Viruses 2010, 2
In vitro studies carried out by Holguín and colleagues have shown that M36I, a polymorphism
found in most non-B clade proteases, increased viral replicative capacity in the absence of drug
pressure while both K20I and M36I increased viral replication under drug pressure . This suggests
that the replicative advantage resulting from sequence polymorphisms could allow non-B clade
variants to spread even under drug pressure.
Table 3. Protease positions that differ between HIV-1 clades. The line highlighted in orange
shows amino acid substitutions that are associated with inhibitor resistance in clade B.
Position 10 12 13 14 15 20 35 36 41 57 61 69 82 89 93
L T I K I K E M
Mutations in clade B
R R Q H V L I
I V I/R D I A M L
D I/V K
I K K
Figure 4. HIV-1 protease is a homodimer with the catalytic active site formed at the
dimeric interface. The majority of residues that differ between various HIV-1 clades map
to positions that are outside the active site. Red spheres represent amino acid positions and
are indicated only on one monomer for clarity.
Viruses 2010, 2
Binding studies carried out on clade A, C and G by Velazquez-Campoy and colleagues  and on
CRF01_AE by Bandaranayake and colleagues  show that the wild type non-B clade proteases have
an inherent weaker affinity for a number of currently available FDA approved PIs. Though these
observations are indicative that background polymorphisms observed in non-B clade protease can
affect inhibitor binding, clinical data suggest that currently available PIs can be just as effective against
non-B clade variants as they are against clade B. However, the weaker affinity for inhibitors observed
may make resistance easier to occur for non-B clade viruses against the current regime of PIs. This
idea has been further strengthened by the observation of altered PI resistance pathways in some non-B
clade proteases. Two distinct examples of altered resistance pathways in non-B clade variants have
been in clade C, which develops L90M, and in CRF01_AE, which develops N88S, in response to NFV
therapy whereas clade B develops D30N, N88D [98,99]. Work carried out on CRF01_AE suggests that
the protease has an inherent weaker affinity for NFV and thus, the reduced affinity for NFV might
allow the CRF01_AE protease to confer resistance through N88S, non-active site mutation, whereas
clade B protease which has a higher affinity for NFV requires a combination of an active site and
non active site mutations, D30N and N88D, in order to effectively disrupt NFV binding.
While currently available PIs are highly effective in treating all clades, different clades might vary
in how they respond to PI therapy. Resistance to PIs remains to be a major challenge in the effective
treatment of HIV-1 and becomes even more relevant in geographic locations where administering
optimal treatment regimens is difficult. Given that non-B clade HIV-1 variants are more prevalent
across the world continued studies on non-B clade proteases are important to elucidate how sequence
variations influence protease activity and the emergence of resistance mutations. Such studies would
add to our current understanding of drug resistance and help formulate effective global
5. The Atomic Energetics of Drug Resistance
At the roots of the molecular basis for drug resistance are the alterations in the atomic interactions
between the PI and the resistant variant of HIV-1 protease. Free energy calculation and decomposition
techniques are providing new insights into protein-ligand interactions [100–106]. Specifically, the
MM-PB/GBSA method [107,108] has been applied in several cases to study the molecular mechanism
of HIV-1 protease drug resistance [109–112]. Compared to the classic free energy perturbation and
thermodynamic integration methods [100,102,113,114], MM-PB/GBSA is computationally less
demanding and a more practical solution for scanning the chemical compound library to discover lead
compounds for potential new inhibitors . The MM-PB/GBSA method combines molecular
mechanism energies and solvation energies to estimate the absolute protein-ligand binding energy,
allowing for the elucidation of which interactions contribute the most to the binding energy. Most of
the interactions are calculated by the atom pairs allowing decomposition of the interaction energy to
the residues of the protease or the functional groups of inhibitors [116,117]. Such decomposition helps
to elucidate the protease drug resistance mechanism on an atomic level and generates valuable
suggestions on modification of the current inhibitors for improvement.
Wang et al.  calculated the binding energy between the wild-type protease and the inhibitors
APV, SQV, RTV, IDV, NFV and a substrate of eight amino acid residues. By comparing energy
Viruses 2010, 2
profiles and the differences at each protease residue, it was suggested that the drug resistant mutations
are more likely to occur at protease residues that interact more favorably with inhibitors than the
substrate. They proposed that a strategy for new inhibitor design is to develop compounds that interact
most favorably with the well conserved protease residues. By considering a residue’s energy
contribution to the binding and the site’s sequence variability, Wang et al. defined an empirical
parameter to identify the drug resistant mutations. In a study of protease binding with seven cyclic
ureas , Mardis et al. reproduced the U-shaped trend of binding free energy as a function of
aliphatic chain length of the inhibitors. Their results also demonstrated that in treating the desolvated
system such as the protein binding site, the finite difference Poisson-Boltzmann model  are more
accurate than the generalized Born method. Recently, Hou et al. calculated the binding affinities
between APV, TMC-126, DRV (with the WT protease and a multi-drug resistant variant
(V82F/I84V) . Stoica et al. calculated the binding affinities between SQV with wild-type protease
and three different drug resistant variants (G48V, L90M, G48V/L90M) . Cai et al. calculated the
binding affinities between DRV with wild-type protease and two multi-drug resistant variants
(L10I/G48V/I54V/V82A, V82T/I84V) . The largest uncertainty came from the evaluation of the
vibrational entropy. Hou et al.  showed that by excluding the entropy terms, the predicted binding
free energies were in better correlation with the experimental energies. In these applications of the
MM-PB/GBSA methods to the energetic features of protease binding with inhibitors, the predicted
absolute binding free energy were in good agreement with the experimental results. They predicted the
ranking of the binding affinities correctly. The more rigorous thermodynamic integrations method
showed better prediction on the relative binding energy .
Overall, by free energy decomposition analysis, the drug-resistant mutations were found to distort
the geometry of the binding site and hence weakened the binding affinity of the inhibitors [110,112].
Van der Waals interaction has been found to have the biggest contribution to the protease-inhibitor
binding affinity [109–112]. Modification of current inhibitors to design more robust inhibitors can be
attained by evaluating changes in van der Waals interaction energy between the protease and each
atom of the inhibitors . The electrostatic energy becomes less important than the van der Waals
because a more favorable coulombic interaction was usually associated with a higher penalty for the
solvation energy [109–112]. Charge optimization studies have been carried out to find the best balance
between the coulombic interaction energy and the polar solvation energy to generate compounds with
highest electrostatic interactions energy with the protease [120–122].
6. Incorporating the Substrate Envelope Constraint in Structure Based Drug Design
Developing robust HIV-1 PIs that avoid drug resistance has proven a challenging task, and the
substrate envelope hypothesis provides an approach to solving this problem. A survey of five approved
drugs using quantitative measures of the bound inhibitor outside the substrate envelope indicated that
the exterior volume of the inhibitors correlated with the loss of affinity to mutant proteases .
A recent study of the inhibitor R01 suggested that individual mutations did not confer drug resistance,
but when multiple sites protrude beyond the envelope collectively, resistance may occur . The
drug DRV, which is structurally similar to APV, demonstrated improved potency with the resistant
Viruses 2010, 2
mutants which is attributed to both DRV’s high binding affinity and that DRV lies within the substrate
The ability of the substrate envelope to correlate with resistance mutations prompted the use of
substrate envelope constraints in the design of new inhibitors [24,29,35,125,126]. Inhibitors were
designed by varying different groups on the hydroxyethylamine scaffold using three different
methodologies: Two computational methods incorporated structural constraints of the substrate
envelope as an a priori consideration during the design stage of the inhibitors while the third method,
structure activity relationship (SAR), did not include the substrate envelope constraint explicitly in its
design. The first computational design  based on optimized docking resulted in two good
candidates exhibiting flat affinity profiles against multi-drug resistant mutants. But these inhibitors have
binding affinity in the nM range. The second computational study systematically explored the
combinatorial space for three constituent R groups on the hydroxyethylamine scaffold  in two rounds
of inverse drug design, synthesis, testing, and retrospective structural analysis. The first round produced
compounds with Ki in the range of 26 M–30 nM, which was improved to Ki of 4.1 nM–14 pM in the
second round compounds. Majority of these inhibitors, whether they are nanomolar or picomolar
inhibitors, have flatter resistance profiles against drug resistant variants. Although the inhibitors
designed using SAR approach  resulted in inhibitors with picomolar affinity to the wild-type
protease they all lose significant affinity while binding to the drug resistant protease variants. These
studies validated the use of the substrate envelope hypothesis  for the development of therapeutics
with low susceptibility to resistance mutations in HIV-1 protease and have yielded several leads for
potential new drugs.
Application of the substrate envelope hypothesis to development of therapeutics to other quickly
evolving drug targets is beginning to emerge. In a recent study , the hypothesis has been applied
to five prospective drug targets from a diverse set of diseases, and the volume of inhibitors protruding
beyond the native substrate specified envelope correlates with average mutation sensitivity. This
suggests that inhibitor design for these enzymes would benefit from a similar reverse engineering
strategy as was implemented in the case of HIV-1 protease. The substrate envelope model has also
been applied in the development of tenofovir, a reverse transcriptase inhibitor . Similar to the
case of HIV-1 protease, the drugs AZT and 3TC protrude beyond the consensus volume, creating an
opportunity for the reverse transcriptase to develop resistant mutations. The newer drug, lacking such
protrusions, is expected to evade resistance mutations as an improvement over its predecessors. Thus
the substrate envelope hypothesis appears to be a valid general strategy for avoiding drug resistance.
Drug resistance in HIV protease is a subtle change in the balance of recognition events between the
relative affinity of the HIV protease to bind inhibitors and its ability to bind and cleave substrates.
Viral maturation involves the cleavage of Gag and Gag-Pro-Pol polyproteins by the viral protease in a
complex, interdependent, and order-specific series of recognition and processing events. Mutations
that confer resistance while balancing viral fitness have long been identified, both within and outside
the active site of the enzyme, although their direct mechanism of action is not always well understood.
Most changes confer resistance not only by altering a direct contact with a protease inhibitor, but also
Viruses 2010, 2
by conferring subtle changes in the structure and energetics throughout the active site. As many
mutations occur simultaneously in complex combinations within a single protease variant, they are
most likely altering both the structure and dynamics of this enzyme. Recent data also implicate that
mutations at the protease cleavage sites as well as remote sites within Gag contribute to HIV protease
drug resistance, possibly without altering viral fitness. The mechanism by which these changes confer
resistance is likely an alteration in the balance of recognition events of the entire viral system and how
the virus interacts within the host cell. Subtle changes between viral clades also alter this balance.
Taken together, all these changes necessitate taking a comprehensive systems approach to
understanding the molecular basis for drug resistance in the highly interdependent molecular
system of HIV.
HIV-1 protease, with its ability to recognize and cleave diverse substrate sequences, has proved to
be a resilient drug target. If targeted optimally in a manner that is evolutionarily constrained, the
protease may be less susceptible to resistance. The substrate envelope hypothesis described a structure
based drug design approach that decreases the probability of drug resistance by understanding the
functional complexes of the HIV protease bound to its cleavage sites. The substrate envelope was then
used as an added constraint in optimizing existing inhibitor scaffolds and designing novel robust
inhibitors. Other strategies, such as focusing on main chain interactions, also may lead to similar
results. A robust inhibitor is one that successfully inhibits a resilient target and does not quickly lose
effectiveness due to resistance. Such an inhibitor may bind only to critical regions within the target that
would be essential for function and thus intolerant to change. Of the currently prescribed PIs, DRV is
the closest to being such a robust inhibitor. However, with the continuous evolution of HIV strains,
development of other potent and robust HIV-1 protease inhibitors is highly warranted. In addition to
drug resistance, other factors such as bioavailability, in vivo stability, and toxicity must also be taken
into consideration when selecting a drug candidate for development.
This work was supported by the National Institutes of Health Grants (P01-GM66524 and
References and Notes
The Joint United Nations Program on HIV/AIDS (UNAIDS). 2008 Report on the Global AIDS
Epidemic; UNAIDS/08.25E/JC1510E; UNAIDS: Geneva, Switzerland, 2008.
Menendez-Arias, L. Molecular basis of human immunodeficiency virus drug resistance: An
update. Antivir. Res. 2010, 85, 210–231.
Martinez-Cajas, J.L.; Wainberg, M.A. Protease inhibitor resistance in HIV-infected patients:
Molecular and clinical perspectives. Antivir. Res. 2007, 76, 203–221.
Wensing, A.M.; van Maarseveen, N.M.; Nijhuis, M. Fifteen years of HIV Protease Inhibitors:
Raising the barrier to resistance. Antivir. Res. 2010, 85, 59–74.
Mehellou, Y.; De Clercq, E. Twenty-six years of anti-HIV drug discovery: Where do we stand
and where do we go? J. Med. Chem. 2010, 53, 521–538.
Viruses 2010, 2
6. Kohl, N.E.; Emini, E.A.; Schleif, W.A.; Davis, L.J.; Heimbach, J.C.; Dixon, R.A.; Scolnick,
E.M.; Sigal, I.S. Active human immunodeficiency virus protease is required for viral infectivity.
Proc. Natl. Acad. Sci. U. S. A. 1988, 85, 4686–4690.
Roberts, N.A.; Martin, J.A.; Kinchington, D.; Broadhurst, A.V.; Craig, J.C.; Duncan, I.B.; Galpin,
S.A.; Handa, B.K.; Kay, J.; Krohn, A.; Lambert, R.W.; Merrett, J.H.; Mills, J.S.; Parkes, K.E.B.;
Redshaw, S.; Ritchie, A.J.; Taylor, D.L.; Thomas, G.J.; Machin, P.J. Rational design of peptide-
based HIV proteinase inhibitors. Science 1990, 248, 358–361.
Dorsey, B.D.; Levin, R.B.; McDaniel, S.L.; Vacca, J.P.; Guare, J.P.; Darke, P.L.; Zugay, J.A.;
Emini, E.A.; Schleif, W.A.; Quintero, J.C.; Lin, J.H.; Chen, I.-W.; Holloway, M.K.; Fitzgerald,
P.M.D.; Axel, M.G.; Ostovic, D.; Anderson, P.S.; Huff, J.R. L-735,524: The design of a potent
and orally bioavailable HIV protease inhibitor. J. Med. Chem. 1994, 37, 3443–3451.
Kempf, D.J.; Marsh, K.C.; Denissen, J.F.; McDonald, E.; Vasavanonda, S.; Flentge, C.A.; Green,
B.E.; Fino, L.; Park, C.H.; Kong, X.P.; Wideburg, N.E.; Saldivar, A.; Ruiz, L.; Kati, W.M.; Sham,
H.L.; Robins, T.; Stewart, K.D.; Hsu, A.; Plattner, J.J.; Leonard, J.M.; Norbeck, D.W. ABT-538 is
a potent inhibitor of human immunodeficiency virus protease and has high oral bioavailability in
humans. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 2484–2488.
10. Kaldor, S.W.; Kalish, V.J.; Davies, J.F., 2nd; Shetty, B.V.; Fritz, J.E.; Appelt, K.; Burgess, J.A.;
Campanale, K.M.; Chirgadze, N.Y.; Clawson, D.K.; Dressman, B.A.; Hatch, S.D.; Khalil, D.A.;
Kosa, M.B.; Lubbehusen, P.P.; Muesing, M.A.; Patick, A.K.; Reich, S.H.; Su, K.S.; Tatlock, J.H.
Viracept (nelfinavir mesylate, AG1343): A potent, orally bioavailable inhibitor of HIV-1 protease.
J. Med. Chem. 1997, 40, 3979–3985.
11. Kim, E.E.; Baker, C.T.; Dwyer, M.D.; Murcko, M.A.; Rao, B.G.; Tung, R.D.; Navia, M.A.
Crystal structure of HIV-1 protease in complex with VX-478, a potent and orally bioavailable
inhibitor of the enzyme. J. Am. Chem. Soc. 1995, 117, 1181–1182.
12. Sham, H.L.; Kempf, D.J.; Molla, A.; Marsh, K.C.; Kumar, G.N.; Chen, C.M.; Kati, W.; Stewart, K.;
Lal, R.; Hsu, A.; Betebenner, D.; Korneyeva, M.; Vasavanonda, S.; McDonald, E.; Saldivar, A.;
Wideburg, N.; Chen, X.; Niu, P.; Park, C.; Jayanti, V.; Grabowski, B.; Granneman, G.R.; Sun, E.;
Japour, A.J.; Leonard, J.M.; Plattner, J.J.; Norbeck, D.W. ABT-378, a highly potent inhibitor of the
human immunodeficiency virus protease. Antimicrob. Agents Chemother. 1998, 42, 3218–3224.
13. Robinson, B.S.; Riccardi, K.A.; Gong, Y.F.; Guo, Q.; Stock, D.A.; Blair, W.S.; Terry, B.J.;
Deminie, C.A.; Djang, F.; Colonno, R.J.; Lin, P.F. BMS-232632, a highly potent human
immunodeficiency virus protease inhibitor that can be used in combination with other available
antiretroviral agents. Antimicrob. Agents Chemother. 2000, 44, 2093–2099.
14. Turner, S.R.; Strohbach, J.W.; Tommasi, R.A.; Aristoff, P.A.; Johnson, P.D.; Skulnick, H.I.;
Dolak, L.A.; Seest, E.P.; Tomich, P.K.; Bohanon, M.J.; Horng, M.M.; Lynn, J.C.; Chong, K.T.;
Hinshaw, R.R.; Watenpaugh, K.D.; Janakiraman, M.N.; Thaisrivongs, S. Tipranavir (PNU-
140690): A potent, orally bioavailable nonpeptidic HIV protease inhibitor of the 5,6-dihydro-4-
hydroxy-2-pyrone sulfonamide class. J. Med. Chem. 1998, 41, 3467–3476.
15. De Meyer, S.; Azijn, H.; Surleraux, D.; Jochmans, D.; Tahri, A.; Pauwels, R.; Wigerinck, P.; de
Bethune, M.P. TMC114, a novel human immunodeficiency virus type 1 protease inhibitor active
against protease inhibitor-resistant viruses, including a broad range of clinical isolates.
Antimicrob. Agents Chemother. 2005, 49, 2314–2321.
Viruses 2010, 2
16. Koh, Y.; Nakata, H.; Maeda, K.; Ogata, H.; Bilcer, G.; Devasamudram, T.; Kincaid, J.F.; Boross,
P.; Wang, Y.F.; Tie, Y.; Volarath, P.; Gaddis, L.; Harrison, R.W.; Weber, I.T.; Ghosh, A.K.;
Mitsuya, H. Novel bis-tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (PI)
UIC-94017 (TMC114) with potent activity against multi-PI-resistant human immunodeficiency
virus in vitro. Antimicrob. Agents Chemother. 2003, 47, 3123–3129.
17. Surleraux, D.L.; Tahri, A.; Verschueren, W.G.; Pille, G.M.; de Kock, H.A.; Jonckers, T.H.;
Peeters, A.; De Meyer, S.; Azijn, H.; Pauwels, R.; de Bethune, M.P.; King, N.M.; Prabu-
Jeyabalan, M.; Schiffer, C.A.; Wigerinck, P.B. Discovery and selection of TMC114, a next
generation HIV-1 protease inhibitor. J. Med. Chem. 2005, 48, 1813–1822.
18. Gulick, R.M.; Mellors, J.W.; Havlir, D.; Eron, J.J.; Meibohm, A.; Condra, J.H.; Valentine, F.T.;
McMahon, D.; Gonzalez, C.; Jonas, L.; Emini, E.A.; Chodakewitz, J.A.; Isaacs, R.; Richman,
D.D. 3-Year suppression of HIV viremia with indinavir, zidovudine, and lamivudine. Ann. Intern.
Med. 2000, 133, 35–39.
19. Bartlett, J.A.; DeMasi, R.; Quinn, J.; Moxham, C.; Rousseau, F. Overview of the effectiveness of
triple combination therapy in antiretroviral-naive HIV-1 infected adults. AIDS 2001, 15, 1369–1377.
20. Palella, F.J.; Delaney, K.M.; Moorman, A.C.; Loveless, M.O.; Fuhrer, J.; Satten, G.A.; Aschman,
D.J.; Holmberg, S.D.; The, H.I.V.O.S.I. Declining morbidity and mortality among patients with
advanced human immunodeficiency virus infection. N. Engl. J. Med. 1998, 338, 853–860.
21. Hogg, R.S.; Heath, K.V.; Yip, B.; Craib, K.J.P.; O'Shaughnessy, M.V.; Schechter, M.T.;
Montaner, J.S.G. Improved survival among HIV-infected individuals following initiation of
antiretroviral therapy. JAMA 1998, 279, 450–454.
22. Zeldin, R.K.; Petruschke, R.A. Pharmacological and therapeutic properties of ritonavir-boosted
protease inhibitor therapy in HIV-infected patients. J. Antimicrob. Chemother. 2004, 53, 4–9.
23. Gulnik, S.V.; Eissenstat, M. Approaches to the design of HIV protease inhibitors with improved
resistance profiles. Curr. Opin. HIV AIDS 2008, 3, 633–641.
24. Nalam, M.N.L.; Schiffer, C.A. New approaches to HIV protease inhibitor drug design II: Testing
the substrate envelope hypothesis to avoid drug resistance and discover robust inhibitors. Curr.
Opin. HIV AIDS 2008, 3, 642–646.
25. Ghosh, A.K.; Chapsal, B.D.; Weber, I.T.; Mitsuya, H. Design of HIV protease inhibitors targeting
protein backbone: An effective strategy for combating drug resistance. Acc. Chem. Res. 2008, 41,
26. Ghosh, A.K.; Leshchenko-Yashchuk, S.; Anderson, D.D.; Baldridge, A.; Noetzel, M.; Miller,
H.B.; Tie, Y.; Wang, Y.F.; Koh, Y.; Weber, I.T.; Mitsuya, H. Design of HIV-1 protease inhibitors
with pyrrolidinones and oxazolidinones as novel P1'-ligands to enhance backbone-binding
interactions with protease: Synthesis, biological evaluation, and protein-ligand X-ray studies.
J. Med. Chem. 2009, 52, 3902–3914.
27. Cihlar, T.; He, G.X.; Liu, X.; Chen, J.M.; Hatada, M.; Swaminathan, S.; McDermott, M.J.; Yang,
Z.Y.; Mulato, A.S.; Chen, X.; Leavitt, S.A.; Stray, K.M.; Lee, W.A. Suppression of HIV-1
protease inhibitor resistance by phosphonate-mediated solvent anchoring. J. Mol. Biol. 2006, 363,
Viruses 2010, 2
28. Stranix, B.R.; Sauve, G.; Bouzide, A.; Cote, A.; Sevigny, G.; Yelle, J. Lysine sulfonamides as
novel HIV-protease inhibitors: Optimization of the Nepsilon-acyl-phenyl spacer. Bioorg. Med.
Chem. Lett. 2003, 13, 4289–4292.
29. Altman, M.D.; Ali, A.; Reddy, G.S.K.K.; Nalam, M.N.L.; Anjum, S.G.; Cao, H.; Chellappan, S.;
Kairys, V.; Fernandes, M.X.; Gilson, M.K.; Schiffer, C.A.; Rana, T.M.; Tidor, B. HIV-1 Protease
inhibitors from inverse design in the substrate envelope exhibit subnanomolar binding to drug-
resistant variants. J. Am. Chem. Soc. 2008, 130, 6099–6113.
30. Wlodawer, A.; Vondrasek, J. Inhibitors of HIV-1 protease: A major success of structure-assisted
drug design. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 249–284.
31. Navia, M.A.; Fitzgerald, P.M.D.; McKeever, B.M.; Leu, C.-T.; Heimbach, J.C.; Herber, W.K.;
Sigal, I.S.; Darke, P.L.; Springer, J.P. Three-dimensional structure of aspartyl protease from
human immunodeficiency virus HIV-1. Nature 1989, 337, 615–620.
32. Wlodawer, A.; Miller, M.; Jaskolski, M.; Sathyanarayana, B.K.; Baldwin, E.; Weber, I.T.; Selk,
L.M.; Clawson, L.; Schneider, J.; Kent, S.B. Conserved folding in retroviral proteases: Crystal
structure of a synthetic HIV-1 protease. Science 1989, 245, 616–621.
33. Miller, M.; Schneider, J.; Sathyanarayana, B.K.; Toth, M.V.; Marshall, G.R.; Clawson, L.; Selk,
L.; Kent, S.B.; Wlodawer, A. Structure of complex of synthetic HIV-1 protease with a substrate-
based inhibitor at 2.3 A resolution. Science 1989, 246, 1149–1152.
34. Kempf, D.J.; Marsh, K.C.; Kumar, G.; Rodrigues, A.D.; Denissen, J.F.; McDonald, E.; Kukulka,
M.J.; Hsu, A.; Granneman, G.R.; Baroldi, P.A.; Sun, E.; Pizzuti, D.; Plattner, J.J.; Norbeck, D.W.;
Leonard, J.M. Pharmacokinetic enhancement of inhibitors of the human immunodeficiency virus
protease by coadministration with ritonavir. Antimicrob. Agents Chemother. 1997, 41, 654–660.
35. Nalam, M.N.L.; Ali, A.; Altman, M.D.; Reddy, G.S.K.K.; Chellappan, S.; Kairys, V.; Ozen, A.;
Cao, H.; Gilson, M.K.; Tidor, B.; Rana, T.M.; Schiffer, C.A. Evaluating the substrate-envelope
hypothesis: Structural analysis of novel HIV-1 protease inhibitors designed to be robust against
drug resistance. J. Virol. 2010, 84, 5368–5378.
36. Surleraux, D.L.; de Kock, H.A.; Verschueren, W.G.; Pille, G.M.; Maes, L.J.; Peeters, A.;
Vendeville, S.; De Meyer, S.; Azijn, H.; Pauwels, R.; de Bethune, M.P.; King, N.M.; Prabu-
Jeyabalan, M.; Schiffer, C.A.; Wigerinck, P.B. Design of HIV-1 protease inhibitors active on
multidrug-resistant virus. J. Med. Chem. 2005, 48, 1965–1973.
37. Prabu-Jeyabalan, M.; Nalivaika, E.A.; Schiffer, C.A. Substrate shape determines specificity of
recognition for HIV-1 protease: Analysis of crystal structures of six substrate complexes.
Structure 2002, 10, 369–381.
38. King, N.M.; Prabu-Jeyabalan, M.; Nalivaika, E.A.; Schiffer, C.A. Combating susceptibility to
drug resistance: Lessons from HIV-1 protease. Chem. Biol. 2004, 11, 1333–1338.
39. King, N. M.; Prabu-Jeyabalan, M.; Nalivaika, E. A.; Wigerinck, P.; de Bethune, M. P.; Schiffer,
C. A. Structural and thermodynamic basis for the binding of TMC114, a next-generation human
immunodeficiency virus type 1 protease inhibitor. J. Virol. 2004, 78, 12012–12021.
40. Lefebvre, E.; Schiffer, C.A. Resilience to resistance of HIV-1 protease inhibitors: Profile of
darunavir. AIDS Rev. 2008, 10, 131–142.
41. Stanford HIV Drug Resistance Database. Available online: http://hivdb.Stanford.edu (accessed on
20 October 2010).
Viruses 2010, 2
42. Wu, T.D.; Schiffer, C.A.; Gonzales, M.J.; Taylor, J.; Kantor, R.; Chou, S.; Israelski, D.; Zolopa,
A.R.; Fessel, W.J.; Shafer, R.W. Mutation patterns and structural correlates in human
immunodeficiency virus type 1 protease following different protease inhibitor treatments. J. Virol.
2003, 77, 4836–4847.
43. Gulnik, S.V.; Suvorov, L.I.; Liu, B.; Yu, B.; Anderson, B.; Mitsuya, H.; Erickson, J.W. Kinetic
characterization and cross-resistance patterns of HIV-1 protease mutants selected under drug
pressure. Biochemistry 1995, 34, 9282–9287.
44. Patick, A.; Duran, M.; Cao, Y.; Shugarts, D.; Keller, M.; Mazabel, E.; Knowles, M.; Chapman, S.;
Kuritzkes, D.; Markowitz, M. Genotypic and phenotypic characterization of human
immunodeficiency virus type 1 variants isolated from patients treated with the protease inhibitor
nelfinavir. Antimicrob. Agents Chemother. 1998, 42, 2637–2644.
45. Mahalingam, B.; Louis, J.; Reed, C.; Adomat, J.; Krouse, J.; Wang, Y.; Harrison, R.; Weber, I.
Structural and kinetic analysis of drug resistant mutants of HIV-1 protease. Eur. J. Biochem.
1999, 263, 238–245.
46. Colonno, R.; Rose, R.; McLaren, C.; Thiry, A.; Parkin, N.; Friborg, J. Identification of I50L as the
signature atazanavir (ATV)-resistance mutation in treatment-naive HIV-1-infected patients
receiving ATV-containing regimens. J. Infect. Dis. 2004, 189, 1802–1810.
47. Deeks, S.G.; Grant, R.M.; Beatty, G.W.; Horton, C.; Detmer, J.; Eastman, S. Activity of a
ritonavir plus saquinavir-containing regimen in patients with virologic evidence of indinavir or
ritonavir failure. AIDS Res. Hum. Retroviruses 1998, 12, F97–F102.
48. Molla, A.; Korneyeva, M.; Gao, Q.; Vasavanonda, S.; Schipper, P.J.; Mo, H.M.; Markowitz, M.;
Chernyavskiy, T.; Niu, P.; Lyons, N.; Hsu, A.; Granneman, G.R.; Ho, D.D.; Boucher, C.A.;
Leonard, J.M.; Norbeck, D.W.; Kempf, D.J. Ordered accumulation of mutations in HIV protease
confers resistance to ritonavir. Nat. Med. 1996, 2, 760–766.
49. Zolopa, A.R.; Shafer, R.W.; Warford, A.; Montoya, J.G.; Hsu, P.; Katzenstein, D.; Merigan, T.C.;
Efron, B. HIV-1 genotypic resistance patterns predict response to saquinavir-ritonavir therapy in
patients in whom previous protease inhibitor therapy had failed. Ann. Intern. Med. 1999, 131,
50. Munshi, S.; Chen, Z.; Yan, Y.; Li, Y.; Olsen, D.B.; Schock, H.B.; Galvin, B.B.; Dorsey, B.; Kuo,
L.C. An alternate binding site for the P1-P3 group of a class of potent HIV-1 protease inhibitors
as a result of concerted structural change in the 80s loop of the protease. Acta Crystallogr. D Biol.
Crystallogr. 2000, 56, 381–388.
51. Prabu-Jeyabalan, M.; Nalivaika, E.A.; King, N.M.; Schiffer, C.A. Viability of a drug-resistant
human immunodeficiency virus type 1 protease variant: Structural insights for better antiviral
therapy. J. Virol. 2003, 77, 1306–1315.
52. Shen, C.H.; Wang, Y.F.; Kovalevsky, A.Y.; Harrison, R.W.; Weber, I.T. Amprenavir complexes
with HIV-1 protease and its drug-resistant mutants altering hydrophobic clusters. FEBS J. 2010,
53. Saskova, K.G.; Kozisek, M.; Lepsik, M.; Brynda, J.; Rezacova, P.; Vaclavikova, J.; Kagan, R.M.;
Machala, L.; Konvalinka, J. Enzymatic and structural analysis of the I47A mutation contributing to
the reduced susceptibility to HIV protease inhibitor lopinavir. Protein Sci. 2008, 17, 1555–1564.
Viruses 2010, 2
54. Schiffer, C.A. University of Massachusetts Medical School, Worcester, MA, USA. Unpublished work,
55. Saskova, K.G.; Kozisek, M.; Rezacova, P.; Brynda, J.; Yashina, T.; Kagan, R.M.; Konvalinka, J.
Molecular characterization of clinical isolates of human immunodeficiency virus resistant to the
protease inhibitor darunavir. J. Virol. 2009, 83, 8810–8818.
56. Tie, Y.; Boross, P.I.; Wang, Y.F.; Gaddis, L.; Liu, F.; Chen, X.; Tozser, J.; Harrison, R.W.;
Weber, I.T. Molecular basis for substrate recognition and drug resistance from 1.1 to 1.6
angstroms resolution crystal structures of HIV-1 protease mutants with substrate analogs. FEBS J.
2005, 272, 5265–5277.
57. Rhee, S.Y.; Taylor, J.; Fessel, W.J.; Kaufman, D.; Towner, W.; Troia, P.; Ruane, P.; Hellinger, J.;
Shirvani, V.; Zolopa, A.; Shafer, R.W. HIV-1 Protease Mutations and Protease Inhibitor Cross
Resistance. Antimicrob. Agents Chemother. 2010, 54, 4253–4261.
58. Velazquez-Campoy, A.; Vega, S.; Freire, E. Amplification of the effects of drug resistance
mutations by background polymorphisms in HIV-1 protease from African subtypes. Biochemistry
2002, 41, 8613–8619.
59. Liu, F.; Boross, P.I.; Wang, Y.F.; Tozser, J.; Louis, J.M.; Harrison, R.W.; Weber, I.T. Kinetic,
stability, and structural changes in high-resolution crystal structures of HIV-1 protease with drug-
resistant mutations L24I, I50V, and G73S. J. Mol. Biol. 2005, 354, 789–800.
60. Clemente, J.C.; Moose, R.E.; Hemrajani, R.; Whitford, L.R.; Govindasamy, L.; Reutzel, R.;
McKenna, R.; Agbandje-McKenna, M.; Goodenow, M.M.; Dunn, B.M. Comparing the
accumulation of active- and nonactive-site mutations in the HIV-1 protease. Biochemistry 2004,
61. Svicher, V.; Ceccherini-Silberstein, F.; Erba, F.; Santoro, M.; Gori, C.; Bellocchi, M.C.;
Giannella, S.; Trotta, M.P.; Monforte, A.; Antinori, A.; Perno, C.F. Novel human
immunodeficiency virus type 1 protease mutations potentially involved in resistance to protease
inhibitors. Antimicrob. Agents Chemother. 2005, 49, 2015–2025.
62. Luque, I.; Todd, M.J.; Gomez, J.; Semo, N.; Freire, E. Molecular basis of resistance to HIV-1
protease inhibition: A plausible hypothesis. Biochemistry 1998, 37, 5791–5797.
63. Mahalingam, B.; Louis, J.M.; Hung, J.; Harrision, R.W.; Weber, I.T. Structural implications of
drug-resistant mutants of HIV-1 protease: High-resolution crystal structures of the mutant
protease/substrate analogue complexes. Proteins 2001, 43, 455–464.
64. Mahalingam, B.; Boross, P.; Wang, Y.F.; Louis, J.M.; Fischer, C.C.; Tozser, J.; Harrison, R.W.;
Weber, I.T. Combining mutations in HIV-1 protease to understand mechanisms of resistance.
Proteins 2002, 48, 107–116.
65. Johnston, E.; Winters, M.A.; Rhee, S.Y.; Merigan, T.C.; Schiffer, C.A.; Shafer, R.W. Association
of a novel human immunodeficiency virus type 1 protease substrate cleft mutation, L23I, with
protease inhibitor therapy and in vitro drug resistance. Antimicrob. Agents Chemother. 2004, 48,
66. Skalova, T.; Dohnalek, J.; Duskova, J.; Petrokova, H.; Hradilek, M.; Soucek, M.; Konvalinka, J.;
Hasek, J. HIV-1 protease mutations and inhibitor modifications monitored on a series of
complexes. Structural basis for the effect of the A71V mutation on the active site. J. Med. Chem.
2006, 49, 5777–5784.
Viruses 2010, 2
67. Piana, S.; Carloni, P.; Rothlisberger, U. Drug resistance in HIV-1 protease: Flexibility-assisted
mechanism of compensatory mutations. Protein Sci. 2002, 11, 2393–2402.
68. Foulkes-Murzycki, J.E.; Scout, W.R.P.; Schiffer, C.A. Hydrophobic sliding: A possible
mechanism for drug resistance in human immunodeficiency virus type 1 protease. Structure 2007,
69. Shuman, C.F.; Markgren, P.O.; Hamalainen, M.; Danielson, U.H. Elucidation of HIV-1 protease
resistance by characterization of interaction kinetics between inhibitors and enzyme variants.
Antivir. Res. 2003, 58, 235–242.
70. Coman, R.M.; Robbins, A.H.; Fernandez, M.A.; Gilliland, C.T.; Sochet, A.A.; Goodenow, M.M.;
McKenna, R.; Dunn, B.M. The contribution of naturally occurring polymorphisms in altering the
biochemical and structural characteristics of HIV-1 subtype C protease. Biochemistry 2008, 47,
71. Zhang, Y.M.; Imamichi, H.; Imamichi, T.; Lane, H.C.; Falloon, J.; Vasudevachari, M.B.;
Salzman, N.P. Drug resistance during indinavir therapy is caused by mutations in the protease
gene and in its Gag substrate cleavage sites. J. Virol. 1997, 71, 6662–6670.
72. Doyon, L.; Croteau, G.; Thibeault, D.; Poulin, F.; Pilote, L.; Lamarre, D. Second locus involved
in human immunodeficiency virus type 1 resistance to protease inhibitors. J. Virol. 1996, 70,
73. Bally, F.; Martinez, R.; Peters, S.; Sudre, P.; Telenti, A. Polymorphism of HIV type 1 gag p7/p1
and p1/p6 cleavage sites: Clinical significance and implications for resistance to protease. AIDS
Res. Hum. Retroviruses 2000, 16, 1209–1213.
74. Mammano, F.; Petit, C.; Clavel, F. Resistance-associated loss of viral fitness in human
immunodeficiency virus type 1: Phenotypic analysis of protease and gag coevolution in protease
inhibitor-treated patients. J. Virol. 1998, 72, 7632–7637.
75. Doyon, L.; Payant, C.; Brakier-Gingras, L.; Lamarre, D. Novel Gag-Pol frameshift site in
human immunodeficiency virus type 1 variants resistant to protease inhibitors. J. Virol. 1998, 72,
76. Maguire, M.F.; Guinea, R.; Griffin, P.; Macmanus, S.; Elston, R.C.; Wolfram, J.; Richards, N.;
Hanlon, M.H.; Porter, D.J.; Wrin, T.; Parkin, N.; Tisdale, M.; Furfine, E.; Petropoulos, C.;
Snowden, B.W.; Kleim, J.P. Changes in human immunodeficiency virus type 1 Gag at positions
L449 and P453 are linked to I50V protease mutants in vivo and cause reduction of sensitivity to
amprenavir and improved viral fitness in vitro. J. Virol. 2002, 76, 7398–7406.
77. Feher, A.; Weber, I.T.; Bagossi, P.; Baross, P.; Mahalingam, B.; Louis, J.M.; Copeland, T.D.;
Yorshin, I.Y.; Harrison, R.W.; Tozser, J. Effect of sequence polymorphism and drug resistance on
two HIV-1 Gag processing sites. J. Biochem. 2002, 269, 4114–4120.
78. Dam, E.; Quercia, R.; Glass, B.; Descamps, D.; Launay, O.; Duval, X.; Krausslich, H.G.; Hance,
A.J.; Clavel, F. Gag mutations strongly contribute to HIV-1 resistance to protease inhibitors in
highly drug-experienced patients besides compensating for fitness loss. PLoS Pathog. 2009, 5,
79. Prabu-Jeyabalan, M.; Nalivaika, E.A.; King, N.M.; Schiffer, C.A. Structural basis for coevolution
of a human immunodeficiency virus type 1 nucleocapsid-p1 cleavage site with a V82A drug-
resistant mutation in viral protease. J. Virol. 2004, 78, 12446–12454.
Viruses 2010, 2
80. Nijhuis, M.; van Maarseveen, N.M.; Lastere, S.; Schipper, P.; Coakley, E.; Glass, B.; Rovenska,
M.; de Jong, D.; Chappey, C.; Goedegebuure, I.W.; Heilek-Snyder, G.; Dulude, D.; Cammack,
N.; Brakier-Gingras, L.; Konvalinka, J.; Parkin, N.; Krausslich, H.G.; Brun-Vezinet, F.; Boucher,
C.A. A novel substrate-based HIV-1 protease inhibitor drug resistance mechanism. PLoS Med.
2007, 4, e36.
81. Kolli, M.; Lastere, S.; Schiffer, C.A. Co-evolution of nelfinavir-resistant HIV-1 protease and the
p1-p6 substrate. Virology 2006, 347, 405–409.
82. Kolli, M.; Stawiski, E.; Chappey, C.; Schiffer, C.A. Human immunodeficiency virus type 1
protease-correlated cleavage site mutations enhance inhibitor resistance. J. Virol. 2009, 83,
83. Nijhuis, M.; Schuurman, R.; de Jong, D.; Erickson, J.; Gustchina, E.; Albert, J.; Schipper, P.;
Gulnik, S.; Boucher, C.A. Increased fitness of drug resistant HIV-1 protease as a result of
acquisition of compensatory mutations during suboptimal therapy. AIDS 1999, 13, 2349–2359.
84. Martinez-Picado, J.; Savara, A.V.; Shi, L.; Sutton, L.; D'Aquila, R.T. Fitness of human
immunodeficiency virus type 1 protease inhibitor-selected single mutants. Virology 2000, 275,
85. Martinez-Picado, J.; Savara, A.V.; Sutton, L.; D'Aquila, R.T. Replicative fitness of protease
inhibitor-resistant mutants of human immunodeficiency virus type 1. J. Virol. 1999, 73, 3744–3752.
86. Croteau, G.; Doyon, L.; Thibeault, D.; McKercher, G.; Pilote, L.; Lamarre, D. Impaired fitness of
human immunodeficiency virus type 1 variants with high-level resistance to protease inhibitors.
J. Virol. 1997, 71, 1089–1096.
87. Bleiber, G.; Munoz, M.; Ciuffi, A.; Meylan, P.; Telenti, A. Individual contributions of mutant
protease and reverse transcriptase to viral infectivity, replication, and protein maturation of
antiretroviral drug-resistant human immunodeficiency virus type 1. J. Virol. 2001, 75, 3291–3300.
88. Robinson, L.H.; Myers, R.E.; Snowden, B.W.; Tisdale, M.; Blair, E.D. HIV type 1 protease
cleavage site mutations and viral fitness: Implications for drug susceptibility phenotyping assays.
AIDS Res. Hum. Retroviruses 2000, 16, 1149–1156.
89. Larrouy, L.; Chazallon, C.; Landman, R.; Capitant, C.; Peytavin, G.; Collin, G.; Charpentier, C.;
Storto, A.; Pialoux, G.; Katlama, C.; Girard, P.M.; Yeni, P.; Aboulker, J.P.; Brun-Vezinet, F.;
Descamps, D. Gag mutations can impact virological response to dual-boosted protease inhibitor
combinations in antiretroviral-naive HIV-infected patients. Antimicrob. Agents Chemother. 2010,
90. Parry, C.M.; Kohli, A.; Boinett, C.J.; Towers, G.J.; McCormick, A.L.; Pillay, D. Gag
determinants of fitness and drug susceptibility in protease inhibitor-resistant human
immunodeficiency virus type 1. J. Virol. 2009, 83, 9094–9101.
91. Parkin, N.; Chappey, C.; Lam, E.; Petropoulos, C. Reduced susceptibility to protease inhibitors
(PI) in the absence of primary PI resistance-associated mutations. Antivir. Ther. 2005, 10, S118.
92. Clavel, F.; Mammano, F. Role of Gag in HIV Resistance to Protease Inhibitors. Viruses 2010, 2,
Viruses 2010, 2
93. Robertson, D.L.; Anderson, J.P.; Bradac, J.A.; Carr, J.K.; Foley, B.; Funkhouser, R.K.; Gao, F.;
Hahn, B.H.; Kalish, M.L.; Kuiken, C.; Learn, G.H.; Leitner, T.; McCutchan, F.; Osmanov, S.;
Peeters, M.; Pieniazek, D.; Salminen, M.; Sharp, P.M.; Wolinsky, S.; Korber, B. HIV-1
nomenclature proposal. Science 2000, 288, 55–56.
94. HIV Sequence Database. Available online: http://www.hiv.lanl.gov/ (accessed on 20 October 2010).
95. Velazquez-Campoy, A.; Vega, S.; Fleming, E.; Bacha, U.; Sayed, Y.; Dirr, H.W.; Freire, E.
Protease inhibition in African subtypes of HIV-1. AIDS Rev. 2003, 5, 165–171.
96. Bandaranayake, R.M.; Kolli, M.; King, N.M.; Nalivaika, E.; Heroux, A.; Kakizawa, J.; Sugiura,
W.; Schiffer, C.A. The Effect of Clade Specific Sequence Polymorphisms on HIV-1 Protease
Activity and Inhibitor Resistance Pathways. J. Virol. 2010, 84, 9995–10003.
97. Holguin, A.; Sune, C.; Hamy, F.; Soriano, V.; Klimkait, T. Natural polymorphisms in the protease
gene modulate the replicative capacity of non-B HIV-1 variants in the absence of drug pressure.
J. Clin. Virol. 2006, 36, 264–271.
98. Grossman, Z.; Paxinos, E.E.; Averbuch, D.; Maayan, S.; Parkin, N.T.; Engelhard, D.; Lorber, M.;
Istomin, V.; Shaked, Y.; Mendelson, E.; Ram, D.; Petropoulos, C.J.; Schapiro, J.M. Mutation
D30N is not preferentially selected by human immunodeficiency virus type 1 subtype C in the
development of resistance to nelfinavir. Antimicrob. Agents Chemother. 2004, 48, 2159–2165.
99. Ariyoshi, K.; Matsuda, M.; Miura, H.; Tateishi, S.; Yamada, K.; Sugiura, W. Patterns of point
mutations associated with antiretroviral drug treatment failure in CRF01_AE (subtype E)
infection differ from subtype B infection. J. Acquir. Immune Defic. Syndr. 2003, 33, 336–342.
100. Lau, F.T.; Karplus, M. Molecular recognition in proteins. Simulation analysis of substrate binding
by a tyrosyl-tRNA synthetase mutant. J. Mol. Biol. 1994, 236, 1049–1066.
101. Bash, P.A.; Singh, U.C.; Langridge, R.; Kollman, P.A. Free energy calculations by computer
simulation. Science 1987, 287, 564–567.
102. Gao, J.; Kuczera, K.; Tidor, B.; Karplus, M. Hidden thermodynamics of mutant proteins: A
molecular dynamics analysis. Science 1989, 244, 1069–1072.
103. Wong, C.F.; McCammon, J.A. Dynamics and Design of Enzymes and Inhibitors. J. Am. Chem.
Soc. 1986, 108, 3830–3832.
104. Bash, P.A.; Singh, U.C.; Brown, F.K.; Langridge, R.; Kollman, P.A. Calculation of the relative
change in binding free energy of a protein-inhibitor complex. Science 1987, 235, 574–576.
105. Kollman, P.A. Free energy calculations: Applications to chemical and biochemical phenomena.
Chem . Rev. 1993, 93, 2395–2417.
106. Purohit, R.; Sethumadhavan, R. Structural basis for the resilience of Darunavir (TMC114)
resistance major flap mutations of HIV-1 protease. Interdiscip. Sci. 2009, 1, 320–328.
107. Massova, I.; Kollman, P.A. Combined molecular mechanical and continuum solvent approach
(MM-PBSA/GBSA) to predict ligand binding. Perspect. Drug Discovery Des. 1999, 18, 113–135.
108. Swanson, J.M.; Henchman, R.H.; McCammon, J.A. Revisiting free energy calculations: A
theoretical connection to MM/PBSA and direct calculation of the association free energy.
Biophys. J. 2004, 86, 67–74.
109. Stoica, I.; Sadiq, S.K.; Coveney, P.V. Rapid and accurate prediction of binding free energies for
saquinavir-bound HIV-1 proteases. J. Am. Chem. Soc. 2008, 130, 2639–2648.
Viruses 2010, 2
110. Hou, T.; Yu, R. Molecular dynamics and free energy studies on the wild-type and double mutant
HIV-1 protease complexed with amprenavir and two amprenavir-related inhibitors: Mechanism
for binding and drug resistance. J. Med. Chem. 2007, 50, 1177–1188.
111. Wang, W.; Kollman, P.A. Computational study of protein specificity: The molecular basis of
HIV-1 protease drug resistance. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 14937–14942.
112. Cai, Y.; Schiffer, C. Decomposing the Energetic Impact of Drug Resistant Mutations in HIV-1
Protease on Binding DRV. J. Chem. Theory Comput. 2010, 6, 1358–1368.
113. Singh, U.C.; Benkovic, S.J. A free-energy perturbation study of the binding of methotrexate to
mutants of dihydrofolate reductase. Proc. Natl. Acad. Sci. U. S. A. 1988, 85, 9519–9523.
114. Blondel, A. Ensemble variance in free energy calculations by thermodynamic integration: Theory,
optimal "Alchemical" path, and practical solutions. J. Comput. Chem. 2004, 25, 985–993.
115. Wlodawer, A.; Erickson, J.W. Structure-based inhibitors of HIV-1 protease. Annu. Rev. Biochem.
1993, 62, 543–585.
116. Massova, I.; Kollman, P.A. Computational alanine scanning to probe protein-protein interactions:
A novel approach to evaluate binding free energies. J. Am. Chem. Soc. 1999, 121, 8133–8143.
117. Archontis, G.; Simonson, T.; Karplus, M. Binding free energies and free energy components from
molecular dynamics and Poisson-Boltzmann calculations. Application to amino acid recognition
by aspartyl-tRNA synthetase. J. Mol. Biol. 2001, 306, 307–327.
118. Mardis, K.L.; Luo, R.; Gilson, M.K. Interpreting trends in the binding of cyclic ureas to HIV-1
protease. J. Mol. Biol. 2001, 309, 507–517.
119. Luo, R.; David, L.; Gilson, M.K. Accelerated Poisson-Boltzmann calculations for static and
dynamic systems. J. Comput. Chem. 2002, 23, 1244–1253.
120. Altman, M.D.; Nalivaika, E.A.; Prabu-Jeyabalan, M.; Schiffer, C.A.; Tidor, B. Computational
design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1
protease. Proteins 2008, 70, 678–694.
121. Huggins, D.J.; Altman, M.D.; Tidor, B. Evaluation of an inverse molecular design algorithm in a
model binding site. Proteins 2009, 75, 168–186.
122. Sherman, W.; Tidor, B. Novel method for probing the specificity binding profile of ligands:
Applications to HIV protease. Chem. Biol. Drug Des. 2008, 71, 387–407.
123. Chellappan, S.; Kairys, V.; Fernandes, M.X.; Schiffer, C.; Gilson, M.K. Evaluation of the
substrate envelope hypothesis for inhibitors of HIV-1 protease. Proteins 2007, 68, 561–567.
124. Prabu-Jeyabalan, M.; King, N.M.; Nalivaika, E.A.; Heilek-Snyder, G.; Cammack, N.; Schiffer, C.A.
Substrate envelope and drug resistance: Crystal structure of RO1 in complex with wild-type human
immunodeficiency virus type 1 protease. Antimicrob. Agents Chemother. 2006, 50, 1518–1521.
125. Ali, A.; Reddy, G.S.K.K.; Cao, H.; Anjum, S.G.; Nalam, M.N.L.; Schiffer, C.A.; Rana, T.M.
Discovery of HIV-1 protease inhibitors with picomolar affinities incorporating N-aryl-
oxazolidinone-5-carboxamides as novel P2 ligands. J. Med. Chem. 2006, 49, 7342–7356.
126. Chellappan, S.; Reddy, G.S.K.K.; Ali, A.; Nalam, M.N.L.; Anjum, S.G.; Cao, H.; Kairys, V.;
Fernandes, M.X.; Altman, M.D.; Tidor, B.; Rana, T.M.; Schiffer, C.A.; Gilson, M.K. Design of
mutation-resistant HIV protease inhibitors with the substrate envelope hypothesis. Chem. Biol.
Drug Des. 2007, 69, 298–313.
Viruses 2010, 2 Download full-text
127. Kairys, V.; Gilson, M.K.; Lather, V.; Schiffer, C.A.; Fernandes, M.X. Toward the design of
mutation-resistant enzyme inhibitors: Further evaluation of the substrate envelope hypothesis.
Chem. Biol. Drug Des. 2009, 74, 234–245.
128. Tuske, S.; Sarafianos, S.G.; Clark, A.D., Jr.; Ding, J.; Naeger, L.K.; White, K.L.; Miller, M.D.;
Gibbs, C.S.; Boyer, P.L.; Clark, P.; Wang, G.; Gaffney, B.L.; Jones, R.A.; Jerina, D.M.; Hughes,
S.H.; Arnold, E. Structures of HIV-1 RT-DNA complexes before and after incorporation of the
anti-AIDS drug tenofovir. Nat. Struct. Mol. Biol. 2004, 11, 469–474.
© 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license