From the DuPont Merck Pharmaceutical Company, Experimental Station,
Wilmington, Delaware 19880
As long as the threat of human immunodeficiency
virus (HIV) protease drug resistance still exists, there will be a need
for more potent antiretroviral agents. We have therefore determined the
crystal structures of HIV-1 protease in complex with six cyclic urea
inhibitors: XK216, XK263, DMP323, DMP450, XV638, and SD146, in an
attempt to identify 1) the key interactions responsible for their high
potency and 2) new interactions that might improve their therapeutic
benefit. The structures reveal that the preorganized, C2 symmetric scaffolds of the inhibitors are anchored
in the active site of the protease by six hydrogen bonds and that their
P1 and P2 substituents participate in extensive van der Waals
interactions and hydrogen bonds. Because all of our inhibitors possess
benzyl groups at P1 and P1', their relative binding affinities are
modulated by the extent of their P2 interactions, e.g.
XK216, the least potent inhibitor (Ki (inhibition
constant) = 4.70 nM), possesses the smallest P2 and the
lowest number of P2-S2 interactions; whereas SD146, the most potent
inhibitor (Ki = 0.02 nM), contains a
benzimidazolylbenzamide at P2 and participates in fourteen hydrogen
bonds and ~200 van der Waals interactions. This analysis identifies
the strongest interactions between the protease and the inhibitors,
suggests ways to improve potency by building into the S2 subsite, and
reveals how conformational changes and unique features of the viral
protease increase the binding affinity of HIV protease inhibitors.
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INTRODUCTION |
An essential step in the life cycle of the human immunodeficiency
virus (HIV)1 is the
proteolytic cleavage of the viral polyprotein gene products of
gag and gag-pol into active structural and
replicative proteins (1, 2). The finding that a viral-encoded protease
is responsible for processing these precursors, and that its
inactivation produces immature, noninfectious viral particles, elicited
an intense search for synthetic inhibitors. The first competitive
inhibitors of HIV protease (PR) were transition-state analogs
(peptidomimetics) in which the scissile bonds were replaced with
nonhydrolyzable isosteres such as a reduced amide, phosphinate,
hydroxyethylene, dihydroxyethylene, statine, and hydroxyethylamine
(3-5). Recently, the Food and Drug Administration (FDA) has approved
the use of four peptidomimetic protease inhibitors (saquinavir,
ritonavir, indinavir, and nelfinavir) to treat HIV infection. Although
these compounds are potent inhibitors of the wild-type protease, their therapeutic benefit is, in most cases, short-lived because they select
for variants of HIV that have a reduced sensitivity toward inhibitors,
as a result of mutations within the HIV protease sequence (6-10). In
an attempt to delay the onset of drug resistance, the FDA approved the
use of combination therapy, i.e. a mixture of protease and
reverse transcriptase antiretroviral agents. Although multidrug therapy
has reduced the plasma viral load of some HIV-infected individuals to
undetectable levels (11), the daunting ability of the virus to rapidly
mutate suggests an ongoing need for new antiretroviral drugs.
In order to design new and more potent inhibitors of HIV protease, we
must improve our understanding of the principles of molecular
recognition for the protease. So far researchers have identified two
unique features of the viral protease that distinguish it from the
human aspartic proteases pepsin and renin: 1) the active form of the
viral enzyme is a homodimer, in which each monomer contributes equally
to the active site and 2) the presence of a structural water molecular
that bridges linear inhibitors to the flap of the protein via hydrogen
bonds. Although hydroxyethylene isosteres and phosphinates were among
the first C2 symmetric molecules reported to bind HIV PR
(12, 13), C2 symmetric cyclic urea-based inhibitors were
one of the first molecules capable of displacing the structural water
(14). The cyclic urea (CU) scaffold is therefore well suited to
interact with the viral protease and to discriminate against human
proteases. Since these inhibitors were first reported, the number of CU
mimics has rapidly increased, and this class of cyclic compounds may
soon become a viable alternative to the currently available
antiretroviral agents (15-18). In a continuing effort to identify new
interactions that might increase the potency of our inhibitors, and
other members of the cyclic family, we have performed a structural
analysis of HIV-1 protease in complex with a series of CUs, which have
IC90 (concentration of inhibitor required to inhibit viral
replication by 95%) values ranging from 5.1 to 4700 nM.
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EXPERIMENTAL PROCEDURES |
Inhibitors and Ki Measurements--
The inhibitors
XK216, XK263, DMP323, DMP450, XV638, and SD146 were synthesized as
reported (14, 19-22), and their Ki values were
measured as described previously (23).
HIV-1 Protease Preparation and Purification--
The protease
was mutated at a single position (Cys95
Ala), expressed
in Escherichia coli BL21 (DE-3) (24), purified from inclusion bodies, and refolded using a hydrophobic interaction column
(25).
Crystallization and Data Collection--
Frozen aliquots (~120
ml) of the protease (32 µg/ml) in 50 mM sodium acetate
buffer (pH 5.5), 1 mM dithiothreitol, 1 mM
EDTA, 10% glycerol, 5% ethylene glycol, and 350 mM NaCl
were thawed and immediately mixed with one of the six inhibitors at a
concentration equal to a 1000-5000-fold molar excess over its
Ki value. The protein was then concentrated to 150 µg/ml, using an Amicon-stirred cell equipped with a YM3 membrane, and
exchanged by diafiltration into 17.4 mM acetic acid, 5 mM dithiothreitol, and an inhibitor concentration equal to
a 1000-5000-fold molar excess over its Ki value.
Finally, the protein was concentrated to 5 mg/ml, using a YM10
membrane, and crystallized at 18 °C in hanging drops by vapor
diffusion (12). Hexagonal rods (0.35 × 0.08 × 0.08 mm) grew
within 7 days in 4-µl drops, which contained 1 mg/ml protease, 250 mM acetate buffer (pH 4.8-5.6) and 80-240 mM
ammonium sulfate.
Diffraction data were collected at room temperature with a R-AXIS II
imaging plate mounted on a Rigaku RU200 rotating anode generator
operating at 50 kV/100 mA (CuK
radiation), equipped with
a 0.3-mm cathode and a graphite crystal monochromator. Full data sets
were obtained from a single crystal by collecting 30-50 oscillation
images, at 2° intervals for 60 min. The unit cell parameters were
determined from four still frames (15° intervals) using the RAXIS
processing software. All protease-CU complexes crystallized in the
space group P61 with a dimer in the asymmetry unit and the
following unit cell parameters: a = b = 62.9 Å and c = 83.5 Å.
Structure Refinement--
The protein model from the HIV
PR-A74704 complex (12) (Protein Data Bank file 9HVP) was used as the
starting model to refine the first CU complex, HIV PR-XK216, which was
then used as the protein model in all subsequent refinements. The
structures were refined by performing several cycles of simulated
annealing followed by positional and restrained B-factor refinements
(26). The conformations of the protein and inhibitors were adjusted
using simulated annealing omit maps. The data collection and refinement statistics are tabulated in Table I. The
refined coordinates for HIV-1 protease in complex with XK216, XK263,
DMP323, DMP450, XV638, and SD146 have been deposited in the Protein
Data Bank under the file names 1HWR, 1HVR, 1QBS, 1DMP, 1QBR, and 1QBT,
respectively.
Binding Energies--
The energetic contributions of the vdw
interactions and hydrogen bonds to the binding energies of CU
inhibitors were calculated using the X-PLOR program: the vdw energy was
approximated by the Lennard-Jones potential energy function and the
hydrogen bond energy was calculated based on the donor-acceptor
distance and the donor-hydrogen-acceptor angle (27). Although the two
energy terms, by themselves, do not provide an estimate of the total binding energy, we believe they can help us gain some insights into the
relative binding energies of the inhibitors. Because the inhibitors
presented in this report have very similar structures (only differing
by their P2 substituents) and bound conformations, variations in their
P2 vdw interactions and hydrogen bonds to the protease must be in part
responsible for the differences in their Ki values.
In addition, we believe these two terms can be used to help identify
which moieties of our inhibitors interact favorably with the
protease.
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RESULTS |
To study the interactions between CU inhibitors and HIV-1 PR, we
have solved the crystal structures of the protease in complex with six
inhibitors: XK216, XK263, DMP323, DMP450, XV638, and SD146. These CUs
are symmetric molecules that possess a common central structural unit:
a seven-membered heterocyclic ring, a urea moiety, and diols; and their
P1(P1') and P2(P2') substituents are attached to C3(C6) (atoms adjacent
to the diols) and the urea nitrogen atoms of the ring, respectively
(Table II).
CU Conformations: Unbound Versus Bound--
Several design
features of CUs were confirmed by determining the small molecule
crystal structures of XK263 and DMP323. For example, the seven-membered
ring adopts a twisted chair conformation, the urea group is planar, and
the configurations of the chiral ring atoms C3, C4, C5, and C6 are
R, S, S, and R,
respectively. Furthermore, the C2 symmetry of these
inhibitors was confirmed by the presence of a noncrystallographic
2-fold axis that passes through the urea carbonyl bond and bisects the
bond between the hydroxyl-bearing carbon atoms, C4 and C5. The only
deviations from C2 symmetry were caused by the different
crystal packing environments around P2 and P2', e.g. the
torsion angles of the P2 and P2' groups of XK263 differ by 25°, and
those of the P2 and P2' hydroxyls of DMP323 differ by 145° (Fig.
1).

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Fig. 1.
Induced fit of cyclic urea inhibitors.
Stereo drawings of the bound (dark blue) and unbound
(red) states of XK263 (top) and DMP323
(bottom). The superpositions of the inhibitors were
performed by minimizing the r.m.s. deviations between the scaffolds in
the two states; note the different P2 conformations. The protease is
drawn with light blue bonds, and dashed lines
correspond to hydrogen bonds between the P2/P2' (OH) and amides of
Asp29(30)(29')(30'). The crystal structures of XK263
and DMP323 were determined by J. Calabrese
(unpublished data).
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When XK263 and DMP323 are complexed to the protease, the conformations
of their scaffolds and P1, P1', and P2' groups are very similar to
their uncomplexed states; only P2 reorganizes to alleviate steric
strain, e.g. the P2 naphthyl of XK263 rotates 20° toward
the flap to avoid steric interactions with Ala28,
Asp29, and Asp30; and the hydroxymethylbenzyl
and P2-hydroxyl of DMP323 rotate 20 and 85°, respectively, away from
Ile84 (Fig. 1). Overall, the similarities between the bound
and unbound structures of the CUs indicate that the inhibitors are
preorganized for binding. The structures also confirm that the size and
C2 symmetry of the scaffold and the stereochemical
arrangement of the chiral ring atoms are responsible for placing the
urea oxygen near the flaps of the protease, the diols between the
catalytic aspartates, and correctly projecting the substituents into
the subsites. Finally, the 2-fold axis of each inhibitor coincides with
that of the homodimer (Fig. 2), which
means that CUs interact with symmetry related residues, e.g.
the P2 and P2' hydroxyls of DMP323 hydrogen bond to the amides of
Asp29 and Asp30 and their symmetry-related
residues (Fig. 1 and Table
III).

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Fig. 2.
C2 symmetry of HIV-1 PR and
CUs. Homodimer of HIV-1 PR complexed with XK216 (dark
blue), XK263 (black), DMP450 (green), DMP323
(pink), XV638 (light blue), and SD146
(red); note the similar conformations of the inhibitors and
the coincident pseudo 2-fold symmetry axes of the dimer and
inhibitors.
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Table III
Hydrogen bonds between HIV-1 PR and cyclic urea inhibitors
Hydrogen bonds were identified using the following cutoff criteria:
2.5-3.6 Å for the donor-acceptor distance and a maximum angular
deviation of 60° from donor-hydrogen-acceptor angle of 180°.
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CU-induced Changes in HIV-1 PR--
All CUs induce very
similar conformational changes in the protease; the r.m.s. (root mean
square) deviations between the C
atoms of any two
complexes is less than 0.40 Å. The most notable change induced by CU
binding is the well documented 7-Å shift in the position of the flap,
which closes over the active site in the complexes (28). Although
cyclic ureas induce flap closure by directly interacting with the
backbone atoms of the flaps, peptidomimetics induce closure with the
aid of a bridging water molecule (Fig.
3). A superposition of CU and linear
inhibitor complexes confirms that the urea oxygen displaces the
structural water, as the distances between O1 of XK216 and the
structural water molecules in HIV PR-P9941,2 PR-A77003,
PR-MK639, PR-VX478, and PR-A74704 (Protein Data Bank files 1HSG, 1HVI,
1HPV, and 9HVP), are only 0.16, 0.34, 0.54, 0.56, and 0.64 Å,
respectively. Other conformational changes are more subtle and unique
for each inhibitor. For example, Asp29, Asp30,
and the C-terminal helix move out of the active site by ~0.5 Å to
accommodate the P2-hydroxyl of DMP323 (29), and Gly48 of
the flap moves deeper into the active site by ~0.5 Å when complexed
with SD146 and XV638 (Fig. 4). Although
these structural shifts are relatively small compared with flap
closure, they appear to have a significant impact on the binding
affinity of the protease. For example, the shift at Asp30
reduces steric contacts to DMP323 and permits the formation of a
hydrogen bond between Asp30 (NH) and O26 of the inhibitor,
and the shift at Gly48 strengthens the hydrogen bonds
between Gly48 (CO and NH) and SD146 (NH25 and
N28), respectively, by reducing the donor-acceptor distance by ~0.5
Å.

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Fig. 3.
Displacing the structural water
molecule. Overlay of HIV-1 protease (light blue)
complexed with XK216 (dark blue) and the election density
(pink) corresponding to the structural water molecule found
in the linear inhibitor complex HIV PR-P9941. Note the urea oxygen (O1)
of XK216 displaces the structural water molecule and hydrogen bonds
directly to the flaps. Electron density (|Fo Fc|) was contoured at 1.5 ; dashed
lines correspond to hydrogen bonds between the urea oxygen and the
amides of Ile50(50') and between the diols and carboxylates
of Asp25(25').
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Fig. 4.
CU-induced changes in the protease.
Overlay of the C atoms of HIV PR-DMP323 (red)
and HIV PR-SD146 (blue). Note the contraction of the active
site at Asp30 and Gly48 by ~0.5 Å when
complexed with SD146 (green); dashed lines indicate hydrogen
bonds to Asp30(30') and Gly48(48'). DMP323 was
omitted for clarity.
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Finally, CU binding reduces the crystallographic 2-fold symmetry
of the uncomplexed enzyme to pseudo symmetry; the r.m.s. deviations for
the backbone atoms of the symmetry-related monomers in the XK216,
DMP450, XV638, XK263, SD146, and DMP323 complexes are 0.45, 0.36, 0.30, 0.28, 0.26, and 0.23 Å, respectively, and only 0.16 Å for the core
(excluding the surface loops) C
atoms of HIV PR-DMP323.
Unfortunately, the induced asymmetry cannot be directly linked to
inhibitor binding, because the observed structural deviations from
C2 symmetry are located in regions involved in crystal
packing, i.e. the flap, C and N termini, and surface
loops.
Interactions between CUs and HIV-1 PR--
The interactions
between CUs and the protease can be divided into two groups: those that
anchor the scaffold in the active site and those that fix the
substituents in their target subsites. The most important interactions
in the first group are 1) two hydrogen bonds between the urea oxygen
and the amides of Ile50 and Ile50', which
contribute a total of 2.3-3.2 kcal/mol to binding, and 2) four
hydrogen bonds between the diols and the carboxylates of the catalytic
aspartates (Fig. 3). Although we did not attempt to estimate the
energetic contributions of the later interactions because the catalytic
aspartates are protonated and probably form several nearly isoenergetic
hydrogen bonding networks with the diols (30), we believe the existence
of alternate networks should provide even greater stability. The
scaffold is therefore firmly held in place by ~six hydrogen bonds, as
evidenced by the lack of lateral movement in the active site when CUs
with different P2 substituents are superimposed (Fig. 2).
The most important P2-S2 interactions are vdw contacts and hydrogen
bonds. XK216 is the least potent inhibitor (Ki = 4.7 nM) in this series and its P2(P2') allyls participate in only 10 vdw interactions. The fact that XK216 possesses the lowest number of P2-S2 vdw contacts and no P2 hydrogen bonds is consistent with its Ki rank-order in this series (Tables II and III). By building larger P2 groups such as hydroxymethylbenzyl, aniline, and naphthyl, the Ki values decreased by
15-fold. The higher relative affinities for DMP323 and DMP450 are
consistent with the formation of 35 new vdw contacts, which increase
Evdw (energy associated with vdw
interactions) by ~10 kcal/mol, and to a lesser extent, four
additional hydrogen bonds, the strongest of which are between the
backbone amides of Asp30(Asp30') and O26(O26')
of DMP323 and between the carboxylates of
Asp30(Asp30') and N-anilines of
DMP450, contributing a total of ~0.5 and 2.0 kcal/mol, respectively.
The 15-fold decrease for XK263, however, appears to be in part due to a
greater increase in vdw interactions (Table II).
The Ki values for SD146 and XV638 are 0.03 and 0.02 nM, respectively, which correspond to approximately a
200-fold decrease compared with XK216 and a 10-fold reduction relative to DMP323 and DMP450. The greater potency of these CUs was achieved by
further increasing the number of P2 hydrogen bonds and vdw interactions
(Tables II and III). Not only do the latter inhibitors have the highest
Evdw (50 and 55 kcal/mol, respectively), they participate in 12 and 14 hydrogen bonds, respectively. In both inhibitor complexes, the strongest hydrogen bonds are between Gly48(48') (CO) and NH25(25'), contributing a
total of 3.6 kcal/mol to binding. The number of interactions is so
extensive that all of the inhibitors' electronegative atoms (except
for the sulfur atom in XV638) are hydrogen-bonded, and the only
accessible protein atoms that are not hydrogen-bonded to the inhibitors
are Gly27 (CO), Asp29 (OD and NH),
Asp30 (CO), and the guanidino group of Arg8,
but these functional groups interact with other protein residues, e.g. Asp29' forms a salt bridge with
Arg8. Therefore, XV638 and SD146 are the most potent
inhibitors in this series due in part to the following factors: 1) a
large buried surface area, in which no unpaired polar/charged atoms
(protein or inhibitor) are trapped in a hydrophobic environment, 2)
strong P2 hydrogen bonds, and 3) extensive vdw interactions.
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DISCUSSION |
CUs are nanomolar competitive inhibitors of HIV-1 PR (Table II).
Their high potency can be attributed primarily to the design of their
scaffold and the high degree of complementary surfaces between their P1
and P2 substituents and the corresponding S1 and S2 subsites. The CU
scaffold evolved from a methoxyphenyl ring in which the methoxy group
was first replaced with a ketone and then with a urea moiety to improve
the hydrogen bonding character of the carbonyl oxygen, and the ring was
enlarged to seven members to position diols between the catalytic
aspartates (14). These modifications created two hydrogen bonds to the
flaps and four to the catalytic aspartates, interactions needed to
anchor the ring in the active site (Fig. 3 and Table III). A cyclic
scaffold was also chosen in an attempt to reduce the entropy penalty
associated with complex formation of flexible ligands. Fortunately, the
CU scaffold exists predominantly in a single pseudo chair conformation, whether it is bound to the protease or not, because of steric strain
between its P1 and P2 substituents (19). An important consequence of
the ring's lack of flexibility is the requirement for RR,
derived from D-phenylalanines, and SS
configurations of the chiral ring atoms C3 C6 and C4 C5, respectively,
to optimally project the substituents into the subsites.
The P1 and P2 substituents of CUs interact extensively with the
protease through a combination of vdw contacts and hydrogen bonds.
While the S1 subsite clearly prefers hydrophobic substituents, the
specificity of S2 is much broader, accommodating the naphthyl group of
XK263 and the electronegative atoms of XV638 and SD146. Because the
scaffolds and P1(P1') benzyls participate in a similar number of
interactions (~30 vdw contacts) in every complex, the observed
Ki values for the different CUs are in part modulated by the extent of P2 interactions (Table II). The
Ki values for DMP323 (DMP450) and SD146 (XV638), for
example, are 15- and 200-fold lower than that for XK216, which is
consistent with the fact that an allyl group is too small to fill the
S2 subsite (Evdw is only
29 kcal/mol). The
most potent inhibitor, SD146 (Ki = 0.02 nM), participates in ~200 vdw interactions and 14 hydrogen bonds, which are evenly distributed around the binding pocket:
six to the flaps, four to the catalytic aspartates, and four more to
the base of the pocket. Hydrogen bonds, however, are not always
required for high potency. For example, XK263, which does not hydrogen
bond to the S2 subsite, is just as potent as DMP323 and DMP450
because it has a larger Edvw (
47 kcal/mol) (Table
II).
Another important feature of CUs is the displacement of the intervening
water molecule by the urea oxygen atom. In the XK263 complex, the urea
oxygen (O1) clearly occupies the same site as the structural water
found in the linear inhibitor complex HIV PR-P9941 (Fig. 3). An
independent NMR study also concludes that DMP323 displaces the
long-lived water molecule (31). Displacing a conformationally
restrained water increases the entropy of the system, and direct
interaction with the flaps may help to stabilize the closed state of
the protease. Many other cyclic compounds now displace the structural
water (15-18).
CUs Versus Linear Inhibitors--
The four FDA-approved
inhibitors, saquinavir, ritonavir, indinavir, and nelfinavir, are
linear, asymmetric compounds with mono-ol functionality (32-34),
whereas CUs are cyclic, C2 symmetric diols. Because both
classes of compounds are potent inhibitors of the wild-type protease,
it is not evident which features are more desirable. Further studies
are needed to quantitate the gain in binding energy associated with the
rigid CU scaffold; the use of diols, which form a network of hydrogen
bonds with the catalytic aspartates, and C2 symmetry, which
allows CUs to interact symmetrically with both monomers. The benefit of
displacing the structural water molecule, however, is more evident,
because human proteases do not use an intervening water to bind
substrates or inhibitors, i.e. their flaps interact directly
with ligands. This different binding requirement allows CUs to easily
discriminate between human and viral proteases. For example, the large
CU scaffold can only fit in pepsin's active site if the flap opens by
~2.0 Å, such a displacement would enlarge the binding pocket and
reduce the overall number of interactions to the inhibitor (Fig.
5). This is consistent with the fact that
a concentration of DMP323 that inhibits HIV-1 PR by 50% only inhibits
pepsin by 1% (23). The templates of linear inhibitors, on the other
hand, are more flexible and can probably more easily reorganize to fit
into the active sites of several different proteases. Therefore, in
practice, most linear inhibitors discriminate against human proteases
based only on their P1-S1 and P2-S2 interactions, whereas CUs
discriminate with their substituents as well as their scaffolds. The
rigid CU scaffold therefore not only reduces the entropy penalty
associated with complex formation but also plays an important role in
discriminating against human proteases.

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Fig. 5.
CU specificity. Stereoview of pepsin
(light blue) complexed with pepstatin (dark blue)
(Ref. 34; Protein Data Bank file 1PSO). DMP323 (red) was
positioned in the active site by optimizing the interactions between
its diols and the catalytic aspartates; note the steric strain between
the urea moiety and the flap: the O1 of CU is only ~1 Å away from
Gly76. Hydrogen bonds are indicated as dashed
lines.
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Implications for Drug Design--
The design of effective
antiretroviral agents requires a detailed understanding of the target,
for inhibitors that do not interact directly with residues involved in
substrate binding or catalytic activity will likely select for
drug-resistant variants. The CU scaffold addresses this issue by
displacing the structural water molecule and interacting with the
catalytic aspartates. These are important design elements, for mutants
that displace the structural water with a large side chain will not
bind substrates as efficiently, and those that replace the catalytic
aspartates with any other residue will be inactive (35). The CU
scaffold is therefore well suited to inhibit mutant proteases.
To investigate the structure-activity relationships of the P2
substituents, we built various P2-substituted inhibitors (Table II). In
general, the small allyl group of XK216 simply does not interact
sufficiently with the subsite to achieve a subnanomolar Ki value. A significant improvement was only
observed when we increased the size and the hydrogen bonding character of the substituent, e.g. XK263, DMP323, and DMP450. Further
attempts at increasing EHBOND (energy associated
with hydrogen bonds) generated XV638, one of the first CUs to hydrogen
bond to Gly48 of the flap. The thiazole ring of XV638 was
later replaced with a benzimidazole to pick up an extra hydrogen bond
to Asp30 (OD) (Table III). Apparently, the S2 subsite can
accommodate a large variety of substituents, which rely in part on vdw
interactions and hydrogen bonds for tight binding. Unfortunately, our
most potent inhibitor (20 pM), SD146, is not a development
candidate because it is too lipophilic. Based on our structures,
however, we might be able to improve the solubility of SD146 by
replacing the benzyl moiety of the benzimidazole with a hydrophilic
group or simply removing it altogether; either modification should not significantly affect its potency because the benzyl is partially exposed to the solvent and thus does not interact extensively with the
protease. Finally, further attempts to increase CU potency might
include building off the P1 benzyl to hydrogen bond with the carbonyl
oxygen of Gly27 and the guanidino group of
Arg8', and off P2, to interact with Asp29 (NH
and COO).
We thank Ru Yu for the synthesis of
XK216, XK263, and DMP323; Paul Aldrich for DMP450; and Ronald Klabe for
Ki measurements. We also thank Drs. John Erickson
and Alexander Wlodawer for providing the coordinates of HIV-1 PR-A74704
prior to their public release and Drs. Stephen Brenner, David Jackson,
and Paul Anderson for supporting this project.