Optimization of P1–P3 groups in symmetric and asymmetric
HIV-1 protease inhibitors
Hans O. Andersson
1
, Kerstin Fridborg
1
, Seved Lo¨ wgren
1
, Mathias Alterman
2
, Anna Mu¨ hlman
4
,
Magnus Bjo¨ rsne
4
, Neeraj Garg
2
, Ingmar Kvarnstro¨m
3
, Wesley Schaal
2
, Bjo¨ rn Classon
4
,
Anders Karle
´
n
2
, U. Helena Danielsson
5
,Go¨ ran Ahlse

5
Department of Biochemistry, Uppsala University, Sweden;
6
Medivir AB,
Lunastigen 7, Huddinge, Sweden
HIV-1 protease is an important target for treatment of
AIDS, and efficient drugs have been developed. However,
the resistance and negative side effects of the current drugs
has necessitated the development of new compounds with
different binding patterns. In this study, nine C-terminally
duplicated HIV-1 protease inhibitors were cocrystallised
with the enzyme, the crystal structures analysed at 1.8–2.3 A
˚
resolution, and the inhibitory activity of the compounds
characterized in order to evaluate the effects of the individual
modifications. These compounds comprise two central
hydroxy groups that mimic the geminal hydroxy groups of a
cleavage-reaction intermediate. One of the hydroxy groups is
located between the d-oxygen atoms of the two catalytic
aspartic acid residues, and the other in the gauche position
relative to the first. The asymmetric binding of the two
central inhibitory hydroxyls induced a small deviation from
exact C2 symmetry in the whole enzyme–inhibitor complex.
The study shows that the protease molecule could accom-
modate its structure to different sizes of the P2/P2¢ groups.
The structural alterations were, however, relatively conser-
vative and limited. The binding capacity of the S3/S3¢ sites
was exploited by elongation of the compounds with groups
in the P3/P3¢ positions or by extension of the P1/P1¢ groups.
Furthermore, water molecules were shown to be important

bound ligand. This arrangement is advantageous for the
design of inhibitors, because it offers a large number of tight
interactions between the enzyme and the inhibitor. The
active site contains eight C2-symmetric subsites (S4, S3, S2,
S1, S1¢,S2¢,S3¢, and S4¢) [15]. These are the binding sites for
the P4, P3, P2, P1, P1¢,P2¢,P3¢,andP4¢ residues of an
octapeptide substrate [16]. Thus the N-terminal and
C-terminal parts of a bound substrate, or the corresponding
parts of the inhibitor, will interact with structurally similar
subsites. To exploit the C2 symmetry of the protease–
substrate complex, N-terminally or C-terminally duplicated
C2-symmetric inhibitors have been designed [17–20]. The
finding that a point mutation could completely abolish the
inhibitory activity of the symmetric compounds highlights
the weakness of this type of compound [21]. Drug-resistant
Correspondence to T. Unge, Institute of Cell and Molecular Biology,
BMC, Box 590, Uppsala University, SE-751 24, Uppsala, Sweden.
Tel.: + 46 18 471 49 85,
e-mail: [email protected]
Enzyme: HIV-1 protease, POL_HV1B1 (P03366) (EC 3.4.23.16).
Note: The refined co-ordinates of HIV-1 protease in complex with
compounds 1–9 have been deposited in the RCSB Protein Data Bank
under the file names, 1EBW, 1EBY, 1EBZ, 1D4I, 1D4H, 1D4J,
1EC1, 1EC2 and 1EC3.
(Received 9 December 2002, revised 18 February 2003,
accepted 21 February 2003)
Eur. J. Biochem. 270, 1746–1758 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03533.x
forms of the protease have been studied with respect to
kinetic and resistance properties [22]. New generations of
mainly asymmetric compounds have been developed with

(DE3). Bacteria were grown in Luria–Bertani medium
to an D
550
of 1.0 before induction with 0.5 m
M
isopropyl
thio-b-
D
-galactoside. Cells were harvested after 3 h of
induction.
Purification of HIV-1 protease
The chromatographic steps were performed at 5 °C. SDS/
PAGE was used after each chromatographic step to
monitor the purification. Cells were suspended in lysis
buffer (20 m
M
Tris/HCl, pH 7.5, 10 m
M
dithiothreitol,
1m
M
phenylmethanesulfonyl fluoride) and lysed in a
French press. The lysate was centrifuged for 30 min at
12 100 g. The insoluble inclusion body fraction, which
contained more than 90% of the expressed material, was
dissolved in buffer (8
M
urea, 20 m
M
Tris/HCl, pH 8.5,

0–0.6
M
NaCl in Mes buffer. The pooled fractions were
precipitated with (NH
4
)
2
SO
4
. The precipitate was collected
by low-speed centrifugation and dissolved in 50 m
M
Mes,
pH 6.5, containing 10 m
M
dithiothreitol, 100 m
M
2-mercap-
toethanol and 1 m
M
EDTA. The solution was desalted on a
PD-10 column (AP Biotech AB, Uppsala, Sweden) and
concentrated by ultrafiltration with Centricon Centrifugal
Filter Units to 2 mgÆmL
)1
.
Enzyme activity and inhibition studies
Enzyme activity/inhibition studies were performed as des-
cribed by Nillroth et al. [28]. The method includes active-site
titrations. Briefly, a fluorimetric assay was used to determine

after 2 days with seeds from protease/inhibitor crystals
belonging to space group P2
1
2
1
2. Crystals appeared after
1 week, and grew to a final size of 0.3 · 0.3 · 0.05 mm in
3–4 weeks.
Data collection and processing
X-ray data were recorded on MAR-imaging plates on the
synchrotron beam lines 9.5 DRAL at Daresbury, UK,
DL41 and DW32 at Lure, France, and I711 at MAX-lab
Lund, Sweden. The programs
DENZO
and
SCALEPACK
were
used for processing and scaling [30,31]. A summary of data
collection statistics is given in Table 1.
Structure refinement
Refinement was performed using the program package
CNS
[32]. The protease model co-ordinates from 1AJV were used
for molecular replacement calculations. The starting model
was refined with rigid-body refinement and simulated
annealing. The difference Fourier map (F
o
–F
c
) clearly

-
RAY
(http://www.povray.org/).
Results and Discussion
Inhibitor properties
The linear C-terminally duplicated inhibitors in this study
encompass a central six-carbon skeleton derived from
L
-mannaric acid (Table 2). Five of the inhibitors [1,2,7–9]
are chemically C2 symmetric. Seven of these nine
compounds exhibit K
i
values in the nanomolar or low-
nanomolar range, with antiviral effects (ED
50
) ranging from
>75 l
M
(compound 6) to 0.04 l
M
[7].
Crystallographic calculations
The crystal structures of the nine inhibitors in complex
withHIV-1proteaseweredeterminedtohighresolution
(Table 1). All the complexes were crystallized in the
orthorhombic space group P2
1
2
1
2. The asymmetric unit

j)jF
c
jj/SjF
o
j,whereFoandFc are the observed and
calculated structure factor amplitudes, respectively. R
free
is equivalent to R
crystal
but calculated for a randomly chosen set of reflections that were
omitted from the refinement process. Ideal parameters are those defined by Engh & Huber [55].
123456789
Data collection details
Space group P2
1
2
1
2P2
1
2
1
2P2
1
2
1
2P2
1
2
1
2P2

d
min
(A
˚
) 1.80 2.30 2.00 1.81 1.81 1.81 2.10 2.00 1.90
No. of observations 42993 28849 55336 95666 61625 92684 35374 44926 62391
No. of unique reflections 16613 10685 15626 21735 20047 21687 13120 15612 18784
Completeness (%) 69.7 93.8 95.9 97.5 90.5 97.2 93.1 92.9 95.7
R
merge
(%) 9.4 4.6 7.5 11.1 8.9 11.2 7.5 11.6 8.1
Reflections I >2 r (%) 63.0 84.1 83.4 84.6 79.7 80.5 77.1 72.5 90.4
Reflections I >2 r in
highest resolution shell (%)
9.4 74.2 70.1 60.8 52.5 51.1 63.1 61.0 79.2
Bin resolution (A
˚
) 1.86–1.80 2.40–2.30 2.07–2.00 1.87–1.81 1.87–1.81 1.87–1.81 2.18–2.10 2.07–2.00 1.97–1.90
Refinement statistics
Resolution range (A
˚
) 24.6–1.81 24.0–2.30 24.6–2.01 27.9–1.81 22.6–1.81 15.0–1.81 24.3–2.10 24.7–2.00 24.3–2.10
R
cryst
(%) 19.0 18.1 18.0 18.8 19.4 20.1 20.4 20.7 20.4
R
free
(%) 21.8 20.0 20.3 222.4 23.4 23.1 23.7 23.9 23.7
No. of atoms 1677 1662 1647 1697 1694 1655 1676 1688 1688
Mean B factors (A

values for reference substances tested in the same
assay: ritonavir (ED
50
0.06 l
M
), indinavir (ED
50
0.06 l
M
), saquinavir (ED
50
0.01 l
M
) and nelfinavir (ED
50
0.04 l
M
). Compound 4 was synthesized
with only one central hydroxy group.
Compound no. A B C K
i
(n
M
)ED
50
(l
M
)
1
0.90 1.24

˚
. The significance of this discrepancy is apparent
from a comparison with the HIV-1 protease–inhibitor
structure 4phv, which contains an inhibitor homologous to
compound 2 but with only one central hydroxy group and
which is also shorter by one carbon in the central part of the
inhibitor [41]. In this structure, the distances between the P1/
P1¢ benzyl groups and Arg8/Arg108 were 4.0 and 3.9,
respectively. Thus, like the natural protein substrate, the
inhibitors, including the symmetric ones, tend to bind in an
asymmetric fashion [40,42]. Compounds 3, 5 and 6 were
made in order to exploit the asymmetric binding.
Electron density maps revealed a preference for the same
arrangement of the central hydroxyls for compounds 1, 3
and 5–8 (see Figs 1, 2 and 4). Compound 4, with only one
central hydroxyl, had it arranged as in compounds 1,3 and
5–8. Preference for the opposite arrangement was found for
compound 9 (see Fig. 6). However, the central hydroxyls
were not completely uniquely arranged in any of the
structures of compounds 1–3 or 5–9. Degrees of uniqueness
varied from 75 to 90%. For the asymmetric compound 3,
the density indicated a 90% unique orientation (Fig. 1).
Small but significant deviations from exact C2 symmetry
were also observed in parts of the protease structure
connected with movement of the flaps. Application of a
Fig. 1. Orientation of the inhibitor in the active site and arrangement of the central vicinal hydroxyls (stereo view). The figure shows the structure of the
asymmetric compound 3 as it is arranged in the HIV-1 protease active site. The electron density map indicates a unique orientation of the inhibitor
and the whole protease–inhibitor complex in the crystal lattice. The density also indicates an  90% unique orientation of the central vicinal
hydroxy groups in the complex with this compound. The Fo–Fc electron density map was calculated at 2.0 A
˚

distances between 2.63 and 2.89 A
˚
(Fig. 2, Table 3). This
position is 1.4 A
˚
away from the position of the catalytic
water as suggested by the structure of the hydrated
difluoroketone inhibitor A79285 (1DIF) [39]. The active
site of the inhibitor complex is rich in hydrogen bonds. In
addition to the abundant hydrogen-bond network involving
the inhibitor, the main-chain amide nitrogen atoms of
Gly27 and Gly127 are hydrogen-bonded to the carboxylate
oxygens Asp25 OD1 and Asp125 OD1, respectively, at
distances of 2.8–2.9 A
˚
. The position and orientation of
G27/G127 is to a large extent determined by the stabilizing
hydrogen-bond network, which involves the second mem-
ber of the catalytic triad T26/T126 [43]. The close packing of
the central hydroxy group between the aspartic carboxylates
is a common property of not only the compounds in this
series but also of other linear inhibitors [44]. The positioning
of the inhibitor in the active site results in a tight interaction
between the inhibitor’s hydroxy group and the catalytic
residues. This close positioning of the inhibitor’s hydroxyl to
the carboxylate oxygens is not only caused by the attraction
between these groups, but also by the interactions between
the inhibitor’s side chains and the S1-S3 site residues. This
was revealed by a comparison with the position of a
homologous inhibitor that lacked the co-ordinating central

group could be exchanged for a hydrogen atom without any
major effects on the positioning of the inhibitor in the active
site. However, the modification had a negative effect on the
K
i
value, which was seven times higher for compound 4
(1.4 n
M
) than for compound 2 (0.2 n
M
). Thus, the gauche
hydroxy group contributes significantly to the binding
capability. The contribution, which is complex, includes
hydrogen-bonding to Asp25/Asp125, van der Waals
Table 3. Hydrogen bonds between the protease and the inhibitor com-
pounds 1, 2, 3 and 5.
Residue Atom Atom
Protease–inhibitor
distance (A
˚
)
Compound 1
25 Asp Od1 O27 2.71
25 Asp Od2 O27 2.89
27 Gly O N18 3.13
29 Asp N O24 2.88
48 Gly O N25 2.93
125 Asp Od1 O27 2.74
125 Asp Od2 O27 2.68
125 Asp Od2 O28 2.70

125 Asp Od2 O02 2.66
125 Asp Od2 O32 2.63
127 Gly O N14 2.97
129 Asp N O24 3.10
129 Asp Od2 O24 2.97
Ó FEBS 2003 Optimization of HIV-1 protease inhibitors (Eur. J. Biochem. 270) 1751
interactions with Ala28, as well as energy contributions
from restriction of the rotational freedom around the C4
carbon.
Overall hydrogen-bond pattern between the inhibitors
and the protease
The compounds in this series contain several hydrogen-
bond donors and acceptors (Fig. 2 and Table 3). The
number of hydrogen bonds between the protease and the
inhibitors varies between 7 and 12 (Table 4). The fact that
these inhibitors are all based on the same skeleton is
reflected in the conserved hydrogen-bond pattern between
the different compounds (Table 3). The asymmetric substi-
tutions in the P2 positions do not significantly alter the
pattern of the retained groups. All polar groups in the
inhibitors, except the ether link in P1/P1¢, are involved in
hydrogen bonding to the protein, either directly or indirectly
through water molecules. Compared with the substrate-like
peptide bond in the P2/P3 positions of compounds 1, 3 and
7–9, substitutions with the indanyl and benzyl groups in P2
led to loss of the hydrogen bond to the carbonyls of Gly48.
As expected, because of its position close to the entrance of
the binding site, a water molecule co-ordinates Gly48 in
these complexes (Fig. 2B). According to a study by Ala
et al. [45], hydrogen bonds contribute less than hydrophobic

only weak interaction with the protein. The closest atoms
are the Od2 atoms of Asp25/Asp125 at distances of 3.4/
3.7 A
˚
.TheP1/P1¢ benzyloxy side groups are within a 3.7-A
˚
radius of the S1/S1¢ site atoms O of Gly27/Gly127, CG of
Table 4. Summary of the inhibitor/protease interactions. Buried surface area was calculated with programs within the CNS package [32]. Hydrogen
bonds were calculated with a maximum distance of 3.5 A
˚
between acceptors and donors. An atom/pair distance of less than 3.9 A
˚
was used as
criterion for a contact.
Compound
Molecular
mass (Da)
Buried surface
area (A
˚
2
)
No. of hydrogen
bonds
No. of inhibitor/
protease contacts K
i
(n
M
)

[16]. The P2/P2¢ side chains of the compounds studied here
are manly hydrophobic. Thus, the lipophilic groups valinyl
(the side chain of valine) [3,7–9], isoleucinyl (the side chain
of isoleucine) [1], indanyl [2–6], benzyl [5] and 2-chloro-6-
fluorobenzyl [6] were explored (Table 2). The interacting
S2/S2¢ ligands were Ala28/Ala128, Asp30/Asp130, Val32/
Val132, Ile47/Ile147 and Ile84/Ile184. Even though the sizes
of the P2/P2¢ side chains differ significantly and penetrate
the binding site with a difference of 2.1 A
˚
, the positions of
the contacting S2/S2¢ amino acids are relatively conserved
except for those in the 30s and 80s loops. The side chain of
Asp30 and Val32 moves as much as 2.0 A
˚
and 0.6 A
˚
,
respectively, to accommodate the benzyl and 2-chloro-6-
fluorobenzyl groups of compounds 5 and 6 (Fig. 4). Fig. 5
shows a summary of the shifts in the Ca positions. In
addition to the shifts around amino-acid position 30,
significant shifts of the order of 0.2–0.7 A
˚
are observed
around positions 18, 67 and 81. Only the peptide chain
harbouring the S2 site was used in the calculations.
The change in position of Asp30 leads to small changes
in the hydrogen-bond networks involving this residue. A
comparison with compounds 1, 2 and 3 indicate how the

˚
, respectively,
compared with their positions with the smaller P2¢ groups.
The electronegative fluorine is within van der Waals radius
(3.3 A
˚
) of the similarly electronegative carbonyl oxygen of
Gly84. The most serious problems with this P2 group are
the breaking of the hydrogen bond and repulsion between
the dipoles.
In compound 5, one of the indanyl groups was exchanged
for a benzyl group to investigate the requirements for
optimal asymmetric binding to the S2/S2¢ site. The benzyl
group is coplanar with the six-carbon ring of the indanyl
group but its position is shifted by  0.3 A
˚
. Thus, the
hydroxy group is not necessary for the orientation of the
plane of the aromatic moiety. A bound water molecule
(B ¼ 39.6) positioned in contact with the phenyl planes of
the P1¢ and P2¢ side chains and 0.5 A
˚
from the position of
the indanyl oxygen atom replaces the function of the
indanyl hydroxy group as hydrogen-bond donor (Fig. 6).
Interestingly, compounds 5 and 2 have about the same K
i
values, which indicates the value of the bridging water
molecule for specific and efficient interactions between
ligand and enzyme.

i
¼ 0.1 n
M
) than compounds 7 and 9 (1.2 n
M
and
0.92 n
M
, respectively) (Table 2). The electron density of
the thienyl ring of compound 7 was low, which indicated
undefined binding, whereas the densities for the pyridyl
rings of compounds 8 and 9 were well defined. The
significantly lower K
i
value for compound 8 is explained
by the van der Waals interactions of the pyridyl rings with
Phe53/Phe153, Pro181/Pro81 and Gly48/Gly148, where
Pro packs against the plane of the pyridyl ring. Further-
more, the pyridyl nitrogens are co-ordinated to Arg8/108
via water molecules (Fig. 7). The pyridyl ring of com-
pound 9 interacts with Gly48/148 and Arg8/108. How-
ever, the orientations of the two pyridyl rings as well as
their binding patterns are different. There are not, as for
compound 8, any apparent co-ordinating ligands to the
pyridyl nitrogens. Only in one side of the S3 sites is it
possible to find water molecules within binding distances.
Loosely bound water molecules at the entrance to the S3/
S3¢ sites are displaced by the extending pyridyl and thienyl
rings.
Water molecules located in the active site

movable parts in a protein molecule, increase the promis-
cuity of the interactions, and add to the enthalpy energy
term by contributing additional bonds to the protein–
inhibitor complex [52–54]. In support of this role of water
molecules in enzyme–ligand complexes is the finding that
compounds 2 and 5 inhibit the protease activity with similar
efficacy although one of the indanyl groups of compound 2
was exchanged for a benzyl and a co-ordinating water
molecule in compound 5 (Fig. 6 and Table 2).
Conclusions
We have exploited the technique of structure-aided drug
design to improve the inhibitory efficacy of HIV-1 protease
lead compounds. The flexibility of the target molecule
complicates prediction of the effect of a modification of the
inhibitor and necessitates structural analysis of each
complex. In this case, the HIV-1 protease, the flexibility
was relatively conservative. The asymmetric binding of the
two central inhibitor hydroxyls to the active-site aspartates
induced a small deviation from the exact C2 symmetry in
the whole enzyme–inhibitor complex. This study shows
that, even without changing the chemistry of the inhibitor
scaffold but with modifications limited to the side groups,
several potent compounds can be designed. The most
active compounds in this series had the highest number of
contacts (bonds) between the protease and the inhibitor
Fig. 6. Value of a water molecule in the inter-
face between inhibitor and protein (stereo view).
A bound water molecule in the compound 5
protease complex (A) fulfils the function of the
indanyl hydroxyl in compound 2 (B) The two

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