Symmetric fluoro-substituted diol-based HIV protease inhibitors
Ortho-fluorinated and meta-fluorinated P1/P1¢-benzyloxy side groups significantly
improve the antiviral activity and preserve binding efficacy
Jimmy Lindberg
1
, David Pyring
2
, Seved Lo¨ wgren
1
,A
˚sa Rosenquist
2
, Guido Zuccarello
2
,
Ingemar Kvarnstro¨m
2
, Hong Zhang
3
, Lotta Vrang
3
, Bjo¨ rn Classon
3,4
, Anders Hallberg
5
,
Bertil Samuelsson
3,4
and Torsten Unge
1
1
Department of Cell and Molecular Biology, BMC, Uppsala University, Sweden;
2
Department of Chemistry, Linko
¨ping University,
Sweden;
3
Medivir AB, Huddinge, Sweden;
4
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, Sweden;
5
Department of Organic Pharmaceutical Chemistry, Uppsala University, BMC, Sweden
HIV-1 protease is a pivotal enzyme in the later stages of the
viral life cycle which is responsible for the processing and
maturation of the virus particle into an infectious virion. As
such, HIV-1 protease has become an important target for the
treatment of AIDS, and efficient drugs have been developed.
However, negative side effects and fast emerging resistance
to the current drugs have necessitated the development of
novel chemical entities in order to exploit different phar-
macokinetic properties as well as new interaction patterns.
We have used X-ray crystallography to decipher the struc-
ture–activity relationship of fluoro-substitution as a strategy
to improve the antiviral activity and the protease inhibition
of C2-symmetric diol-based inhibitors. In total we present six
protease–inhibitor complexes at 1.8–2.3 A
˚resolution, which
have been structurally characterized with respect to their
antiviral and inhibitory activities, in order to evaluate the
effects of different fluoro-substitutions. These C2-symmetric
inhibitors comprise mono- and difluoro-substituted benzyl-
oxy side groups in P1/P1¢and indanoleamine side groups in
P2/P2¢. The ortho- and meta-fluorinated P1/P1¢-benzyloxy
side groups proved to have the most cytopathogenic effects
compared with the nonsubstituted analog and related
C2-symmetric diol-based inhibitors. The different fluoro-
substitutions are well accommodated in the protease S1/S1¢
subsites, as observed by an increase in favorable Van der
Waals contacts and surface area buried by the inhibitors.
These data will be used in the development of potent
inhibitors with different pharmacokinetic profiles towards
resistant protease mutants.
Keywords: AIDS; aspartic protease; crystal structure; fluor-
ine; HIV.
Human immunodeficiency virus 1 (HIV) is the causative
agent of AIDS [1–3]. The single-stranded RNA genome of
HIV encodes a dimeric aspartyl protease (protease) which
processes the viral gag and gag-pol precursor polyproteins
into structural and functional proteins. The HIV protease
has been shown to be essential in the production of mature
and infectious virions [4,5], hence inhibition of this enzyme
has become an attractive target for effective antiviral agents;
several protease inhibitors are currently in clinical trials.
Despite the initial success of the FDA approved protease
inhibitors (saquinavir [6], ritonavir [7], indinavir [8], nelfin-
avir [9], amprenavir [10], lopinavir [11] and atazanavir [12]),
there is an urgent need for improved drugs against HIV
protease because of increasing viral resistance and unfavor-
able pharmacokinetic profiles [13–16].
Our research group has utilized carbohydrates as building
blocks in the design and synthesis of C2-symmetric protease
inhibitors. The applied method of synthesis produces a
symmetry core unit with the C2-symmetry axis in the center
of an asymmetric inhibitor using
L
-mannaric acid as the
building block [17–20]. Subsequent benzylation and coup-
ling with amino acid or amines gave a series of symmetric or
asymmetric diol-based inhibitors which were further opti-
mized on the P1/P1¢and P2/P2¢side groups, providing a
variety of inhibitors with efficient antiviral profiles [21–26].
This class of protease inhibitors has previously been
associated with poor absorption profiles in cell assays and
unsatisfactory pharmacokinetics in rats, which led us to
investigate the effect of fluoro-substituted inhibitors on cell
absorption. The substitution of fluorine for hydrogen
introduces a minor increase in molecular mass and minimal
steric changes accompanied by increased lipophilicity
(Table 1) [27–29]. Previously, these properties of fluorine
have been utilized successfully in the development of
receptor-subtype-selective cholinergic and adrenergic drugs
[30–32]. To study these effects of fluorine on symmetric diol-
based protease inhibitors, we synthesized a series of fluoro
inhibitors, with either mono- or di-substituted P1/P1¢-
benzyloxy side groups [33].
Correspondence to T. Unge, Department of Cell and Molecular
Biology, BMC, Box 596, Uppsala University, SE-751 24, Uppsala,
Sweden. Fax: +46 18 530396, Tel.: +46 18 4714985,
E-mail: Torsten.Unge@icm.uu.se
Enzyme: HIV-1 protease, POL_HV1B1 (P03366) (EC 3.4.23.16).
Note: The refined coordinates and associated structure factors of
HIV-1 protease in complex with inhibitors 1–6 have been deposited
in the RCSB Protein Data Bank with accession codes: 1EBY, 1EC0,
1W5V, 1W5W, 1W5X, and 1W5Y.
(Received 20 August 2004, revised 28 September 2004,
accepted 12 October 2004)
Eur. J. Biochem. 271, 4594–4602 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04431.x
Herein we have used X-ray crystallography to decipher
the structure–activity relationship for this series of fluoro
inhibitors. In general, fluoro-substitution results in efficient
utilization of accessible volume in the subsites, associated
with increased number of Van der Waals contacts and
surface area buried by the inhibitors. This is reflected in
moderate to good protease inhibition (K
i
values), albeit
poorer than the nonsubstituted analog. The general reduc-
tion in binding efficacy associated with fluoro-substitution is
contradictory with respect to the increase in number of Van
der Waals contacts and favorable electrostatic contacts. It is
possible that the presence of two binding configurations of
the fluoro-substituted benzyloxy side groups in the S1/S1¢
subsites may account for the general reduction in binding
efficacy that we observed for the fluoro inhibitors compared
with the nonsubstituted analog. Structural and biochemical
data suggest that difluoro-substitutions at the ortho and
meta positions on P1/P1¢-benzyloxy side groups of sym-
metric diol-based protease inhibitors preserve the binding
efficacy and significantly improve the antiviral potency.
Materials and methods
Expression of HIV-1 protease
The expression and purification of HIV-1 protease was
adapted from Andersson et al. [21]. The protease gene was
isolated by PCR with the upstream primer GAACA
TATGGCCGATAGACAAGGAACTGTATCC and the
downstream primer AGGGGATCCCTAAAAATTTAA
AGTGCAACCAATCTG. The annealing site for the
upstream primer corresponds to 12 amino acids before the
protease sequence. These extra amino acids were added to
facilitate the autocatalytic processing of the precursor
protein and thereby ensure a correct N-terminus. Through
the PCR step, the protease DNA fragment was provided
with an NdeI restriction site at the 5¢end and a BamHI site
at the 3¢end. These sites were used for ligation to the
pET11a expression vector. Escherichia coli strains XL-1 and
HB101 were used as hosts for cloning.
Protein expression was performed in E. coli strain
BL21(DE3). Bacteria were grown in Luria–Bertani medium
to an A
550
of 1.0 before induction by the addition of 0.5 m
M
isopropyl b-
D
-thiogalactoside. Cells were harvested after 3 h
of induction.
Purification of HIV-1 protease
Cells were suspended in lysis buffer (20 m
M
Tris/HCl,
pH 7.5, 10 m
M
dithiothreitol, 1 m
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, 10 m
M
NaCl, 10 m
M
dithiothreitol,
1m
M
EDTA) and incubated for 1 h at room temperature
followed by centrifugation for 20 min at 48 200 g.
The chromatographic steps were performed at 5 C.
ThesupernatantwasappliedtoaPOROSQ
TM column
(Perspective Biosystems, Cambridge, CA, USA). The flow-
through fraction was collected and diluted to a final
protein concentration of 0.3 mgÆmL
)1
. Refolding was
performed by dialysis against 20 m
M
sodium phosphate
buffer, pH 6.5, containing 10 m
M
dithiothreitol and 1 m
M
EDTA, at room temperature for 60 min. The refolded
protein was diluted with an equal volume of buffer (50 m
M
MES, pH 6.5, 1 m
M
dithiothreitol, 1 m
M
EDTA), applied
to a POROS HS column (Centricon, Billerica, CA, USA),
and eluted with a linear gradient of 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-mercaptoetha-
nol, and 1 m
M
EDTA. The solution was desalted on a
PD-10 column (AP Biotech AB, Uppsala, Sweden) and
concentrated by ultrafiltration with CentriconCentrifugal
Filter Units to 2 mgÆmL
)1
.
Enzyme activity/inhibition studies
Enzyme activity/inhibition studies were performed as des-
cribed by Nillroth et al. [34]. Briefly, a fluorimetric assay
was used to determine the effects of the inhibitors on HIV-1
protease. This assay used an internally quenched fluorescent
peptide substrate, DABSYL-c-Abu-Ser-Gln-Asn-Tyr-Pro-
Ile-Val-Gln-EDANS (Bachem, Bubendorf, Switzerland).
The measurements were performed in 96-well plates with a
Fluoroscan plate reader (Labsystems, Helsinki, Finland).
Excitation and emission wavelengths were 355 nm and
500 nm, respectively.
Anti-HIV activity was assayed in vitro in MT4 cells with
the vital dye XTT (Sigma-Aldrich, Steinheim, Germany) to
monitor the cytopathogenic effects [35].
Crystallization
Crystallization was performed at 4 C with the hanging-
drop vapour-diffusion method. Drops were prepared by
mixing 5 lL protein inhibitor solution with an equal volume
of reservoir solution. The protein inhibitor solution con-
tained 2 mgÆmL
)1
protein in 50 m
M
MES, pH 6.5, con-
taining 10 m
M
dithiothreitol and 1 m
M
EDTA, and 7 m
M
inhibitor in 10% (v/v) dimethyl sulfoxide. The reservoir
solution contained 50 m
M
MES, pH 5.5, and 0.5
M
NaCl.
The drops were microseeded after 2 days. Crystals appeared
after 1 week, and grew to the 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 the Daresbury
Table 1. Physicochemical properties of the carbon–fluorine bond.
Values used in the evaluation of intermolecular contacts among the
protease–inhibitor complexes.
Element
Electro-
negativity
Bond length
(CH
2
X, A
˚)
Van der Waals
radius (A
˚)
H 2.1 1.09 1.2
F 4.0 1.39 1.4
C 2.5 1.42 1.7
O (OH) 3.5 1.43 1.6
FEBS 2004 Fluorine substitution of HIV-1 protease inhibitors (Eur. J. Biochem. 271) 4595
Laboratory, Daresbury, Cheshire, 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 [36,37]. A summary of data collection statistics is
giveninTable2.
Structure refinement
Refinement was performed using the program package
CNS
[38]. The protease model coordinates from 1EBW
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 showed the position and orientation of
inhibitor together with many water molecules. The
inhibitor was built into the electron density with the
molecular visualization program
O
[39]. Water molecules
were added to the structures determined from the
difference Fourier maps at chemically acceptable sites.
Only solvent molecules with Bvalues less than 50 A
˚
2
were accepted. Several cycles of minimization, simulated
annealing, and B factor refinement were performed for
each complex, accompanied with manual rebuilding. The
R
cryst
and R
free
factors were used to monitor the
refinement [40]. The refinement statistics are shown in
Table 2.
Results
Inhibitor properties
The linear C2-symmetric inhibitors in this study encompass
a six-carbon chiral center derived from
L
-mannaric acid.
The five P1/P1¢fluoro-substituted C2-symmetric inhibitors
2–6 were synthesized based on the nonsubstituted analog; 1
with benzyloxy side groups in P1/P1¢and indanolamine side
groups in P2/P2¢[33]. All inhibitors have K
i
values within
the nanomolar to picomolar range, and antiviral activity
expressed as ED
50
values varying from 100 to 20 n
M
(Table 3).
Table 2. Crystallographic structure determination statistics for protease–inhibitor complexes 1–6. Statistics for reflections in highest resolution shells
are indicated in parentheses.
123456
PDB accession number 1EBY 1EC0 1W5V 1W5W 1W5X 1W5Y
Data collection details
Space group P2
1
2
1
2P2
1
2
1
2P2
1
2
1
2P2
1
2
1
2P2
1
2
1
2P2
1
2
1
2
Wavelength (A
˚) 1.386 1.386 0.976 0.976 0.976 0.976
No of crystals 156444
Cell dimensions (A
˚)
a¼59.2 58.4 58.5 58.3 58.5 59.2
b¼86.9 86.3 86.1 85.9 86.3 87.0
c¼47.2 46.8 46.6 46.8 46.6 47.2
d
min
(A
˚) 2.3 1.8 1.8 1.8 1.8 1.9
No. of observations 28849 52852 97169 102338 86063 49671
No. of unique reflections 10685 21005 21224 21819 21258 16080
Completeness (%) 93.8 (93.0) 88.2 (84.1) 94.1 (90.8) 97.7 (89.3) 95.3 (95.0) 82.9 (83.0)
R
mergea
(%) 4.6 (22.2) 12.6 (31.2) 3.4 (16.0) 7.0 (25.2) 4.8 (23.0) 11.4 (31.9)
Reflections I > 2 r(%)847686889090
Reflections I > 2 rin
highest resolution shell (%)
74 50 66 64 61 78
Bin resolution (A
˚) 2.40–2.30 2.00–1.80 1.90–1.80 1.83–1.80 1.83–1.80 2.02–1.90
Refinement statistics
Resolution range (A
˚) 24.0–2.3 25.0–1.8 25.0–1.8 28–1.8 25.0–1.8 30.0–1.9
R
crystb
(%) 18.1 19.0 19.9 19.9 18.8 18.8
R
freec
(%) 20.0 22.0 21.8 21.8 20.7 21.8
No. of atoms 1662 1668 1684 1691 1690 1669
Mean B factor (A
˚
2
)
All 20.2 19.1 21.9 21.3 20.1 22.3
Solvent 34.5 42.0 33.4 34.9 35.4 46.0
Deviation from ideality
d
Bond lengths (A
˚) 0.008 0.007 0.005 0.005 0.006 0.006
Angles () 1.2 1.3 1.2 1.2 1.2 1.2
Dihedrals () 25.4 25.3 25.2 25.1 25.1 25.2
Impropers () 0.77 0.69 0.70 0.69 0.70 0.90
a
R
merge
¼S|I
i
)<I>|/SI
i
, where I
i
is an observation of the intensity of an individual reflection and <I> is the average intensity over
symmetry equivalents.
b
R
cryst
¼S||F
o
|)|F
c
||/S|F
o
|, where F
o
and F
c
are the observed and calculated structure factor amplitudes, respect-
ively.
c
R
free
is equivalent to R
cryst
but calculated for a randomly chosen set of reflections that were omitted from the refinement process.
d
Ideal parameters are those defined by Engh & Huber [47].
4596 J. Lindberg et al.(Eur. J. Biochem. 271)FEBS 2004
Structure of the complexes
The crystal structures of the six protease–inhibitor com-
plexes have been solved and refined down to 1.8–2.3 A
˚
resolution with R
cryst
and R
free
of 18.1–19.9% and 20.0–
22.5%, respectively (Table 2). All complexes were crystal-
lized in the orthorhombic space group P2
1
2
1
2withthe
complete protease molecule in the asymmetric unit. This
twofold symmetry arrangement of the crystal packing does
not impose symmetry restraints on the protease dimer–
inhibitor complex. All six inhibitors bind in an asymmetric
manner to the protease, with the hydroxyls of the chiral
center in staggered and gauche positions with respect to the
catalytic aspartates and the strong interaction with one of
the hydroxyls, which involves short distance hydrogen
bonds with strong polar components. The geometric
restraints of the hydroxyls cause the asymmetric binding.
The asymmetry of the central hydroxyls is present in all
inhibitor structures, propagating significant differences in
the distances between the inhibitor side groups and
respective subsites. The characteristic structural water
molecule is bridging the main-chain amino groups of
Ile50/Ile50¢to the carbonyls of the inhibitor and is observed
in all complexes. The difference Fourier maps, 2F
o
–F
c
and
F
o
–F
c
, unambiguously indicate the conformation of the
inhibitors and the position of the fluorine substitutions in
P1/P1¢(Fig. 1).
Structural accommodations in response to ortho-, meta-
and para-fluoro-substituted P1/P1¢-benzyloxy side groups
Overall. The mono- and di-substituted inhibitors 2–6 bind
to the active site of the protease with specific accommoda-
tions of the residues lining the S1/S1¢subsites, as compared
with the nonsubstituted analog 1. The rmsd of Caatoms of
S1/S1¢-lining residues range from 0.12 to 0.33 A
˚for the
different protease–inhibitor complexes. In Fig. 2 an over-
view of the accommodation of S1 subsite lining residues is
presented with respect to mono- and difluoro-substituted
benzyloxy side groups. The rmsd of all atoms from residue
side chains that are within 3.9 A
˚of the P1-benzyloxy side
groups (Arg8, Leu23, Gly48, Gly49, Ile50, Val32, Pro81 and
Ile84) are plotted pairwise for the protease–inhibitors
complexes 1–6. Generally, the most pronounced side-chain
accommodations in response to the fluoro-substitution in
ortho, meta and para positions on the P1-benzyloxy side
groups are in the range of 0.3–0.4 A
˚, and are mainly
observed for residues Arg8, Leu23, Gly48, Val32 and Pro81.
The conformation of the remaining S1-lining residues
remains unaffected by the panel of fluoro-substitutions.
The protease inhibitor complex with inhibitor 6exhibited
the most pronounced shifts in side-chain position, partic-
ularly for residues Arg8 and Leu23, which are displaced
0.5 A
˚with respect to inhibitors 1–5.
2- and 3-Fluorobenzyloxy side groups. The mono-substi-
tuted inhibitors 2and 3are fluoro-substituted in the ortho
(2-fluoro) and meta (3-fluoro) positions of the benzyloxy
side groups, respectively. Table 4 summarizes the binding
characteristics of the fluoro inhibitors. For the P1/P1¢
mono-substituted inhibitors, 2and 3, the accessible volume
of the subsites is utilized more efficiently in relation to
the surface area buried by the inhibitors compared with the
nonsubstituted analog, as evidenced by an increase in the
number of favorable Van der Waals contacts. Superimpo-
Table 3. Binding characteristics of the fluoro inhibitors to the protease active site.
Inhibitor
Molecular
mass (Da)
Buried surface
area (A
˚
2
)
a
No. of inhibitor–
protease contacts
b
No. of
hydrogen bonds
c
No. of
repelling contacts
d
K
i
(n
M
)
ED
50e
(l
M
)
1652.7 1394.6 57 10 – 1.2 0.10
2671.7 1440.1 71 10 – 3.2 0.05
3671.7 1398.3 78 10 2 7.1 0.06
4690.7 1435.8 70 11 – 1.6 0.11
5690.7 1433.5 84 10 2 4.0 0.03
6690.7 1456.3 69 10 – 3.3 0.02
a
Buried surface area was calculated with programs in the
CNS
package [38].
b
An atom-pair distance of less than 3.9 A
˚was used as criterion
for a close contact.
c
Hydrogen bonds were calculated with a maximum distance of 3.2 A
˚between acceptors and donors.
d
An atom-pair
distance of less then 3.5 A
˚between atom with same polarity was used as a criterion for a repelling contact.
e
ED
50
for reference substances
tested in the same assay: ritonavir (ED
50
0.06 l
M
), indinavir (ED
50
0.06 l
M
), saqinavir (ED
50
0.01 l
M
), nelfinavir (ED
50
0.04 l
M
).
Fig. 1. Conformation of the C2-symmetric inhibitor 4 in the protease
active site. The 2F
o
–F
c
difference electron-density map unambiguously
shows a unique orientation of the inhibitor and the fluorine substitu-
ents on the P1/P1¢side groups. The electron density maps were cal-
culated at 1.8 A
˚resolution with the inhibitor omitted, employing the
omit option in
CNS
[38]. Map contouring is at 0.4 eÆA
˚
3)1
(1 r).
The figure was drawn with the program
SWISS
-
PDBVIEWER
[45]
(http://www.expasy.ch/spdbv/) and 3D-rendered with
POV
-
RAY
(http://www.povray.org/).
FEBS 2004 Fluorine substitution of HIV-1 protease inhibitors (Eur. J. Biochem. 271) 4597
sition of the two inhibitors on the nonsubstituted analog
revealed that the position of 2-fluoro; 2and 3-fluoro; 3
benzyloxy side groups in the S1/S1¢subsites are similar, and
slightly closer to Arg8/Arg8¢than in the non-substituted
analog. For inhibitor 2, this position prevents steric clashes
between the 2-fluoro substituents and Ile50/Ile50¢side
chains and puts the 2-fluoro in range of Van der Waals
contacts to the Cc1 carbons. In contrast, the 3-fluoro-
benzyloxy side groups of inhibitor 3have no contacts with
the isoleucines. Instead, there are Van der Waals contacts to
Gly48/Gly48¢and Pro81/Pro81¢where the former contacts
display unfavorable electrostatic interactions between the 3-
fluoro atoms and the glycine backbone carbonyls (Table 4).
2,3-, 2,4- and 2,5-Difluorobenzyloxy side groups. The
changed size and chemical character of the difluoro-
substituted benzyloxy side groups is accommodated for by
the S1/S1¢subsites. It is evident by slight changes in
position of the P1/P1¢side groups and residue side-chain
adaptations. In Fig. 3 inhibitors 2and 6are superimposed
on to the nonsubstituted analog to show the difference in
position of the benzyloxy side groups associated with
2-fluoro and 2,5-difluoro-substitutions compared with the
nonsubstituted analog. The 2,5-difluoro-substitution in
inhibitor 6changes the position of the benzyloxy side
groups 0.8 A
˚towards the Cf-carbon of Arg8/Arg8¢,
accompanied by 0.3/0.4 A
˚shifts in side-chain positions
compared with inhibitors 1–5. The conformations of the
arginine side chains are stabilized by hydrogen bonds from
Asp29/Asp29¢resulting in a restrained repositioning of the
2,5-difluorobenzyloxy side groups in proximity of the Cf
carbon. Notably, the 5-fluoro substituents are observed
within dipole–dipole interaction range of the partially
charged Cfcarbon of the arginines. The presence of an
electrostatic interaction is supported by quantum mechan-
ical calculations of the partial charges for Cfcarbon
(+ 0.34) and the 5-fluoro substituents ()0.11) in vacuum
(unpublished observations). In addition, the repositioning
of the benzyloxy side groups results in lost Van der Waals
contacts between the 2-fluoro substituents and Ile50/Ile50¢
side chains. Furthermore, the crystal structure of inhibitor
6reveals increased flexibility (higher Bvalues) and reduced
quality of the electron density for the isoleucine side chains
compared with the inhibitor 2complex. In Fig. 4,
inhibitors 4(2,4-difluoro) and 2(2-fluoro) are superim-
posed on the nonsubstituted analog 1. In contrast with the
structural adaptation required for the 2,5-difluoro-substi-
tutions, the 2,4-difluoro-substituted benzyloxy side groups
(4) accommodate well in the S1/S1¢subsites. Thus, the
4-fluoro substituents act as proton acceptors in two
hydrogen bonds to the nitrogen atoms of Arg8/Arg8¢side
chains, and the 2-fluoro substituents are within Van der
Waals distance of Ile50/Ile50¢.
Enzyme activity/inhibition studies
The present series of fluoro-substituted inhibitors shows
satisfactory protease inhibition with K
i
values in the
picomolar to nanomolar range, albeit poorer than the
nonsubstituted analog 1. Notably, for P1/P1¢fluoro-substi-
tutions, the antiviral activity (as measured by ED
50
values)
were markedly improved compared with the nonsubstituted
analog and other related C2-symmetric diol-based protease
inhibitors [23,26], and comparable to the reference com-
pounds indinavir, ritonavir, nelfinavir, and saquinavir. The
inhibitory efficacy (as measured by K
i
values) on enzyme
activity of the P1/P1¢-fluorinated analogs differs depending
on the position of fluoro-substitution on the benzyloxy side
group, para being greater than ortho and ortho greater than
meta. Among the fluoro-substituted inhibitors, the most
potent was the disubstituted inhibitor 4(2,4-difluoro).
However, the effect on the antiviral activity is more complex
(Table 3).
Benzyloxy side groups disubstituted in the ortho and
meta position exhibit the highest antiviral potency
(Table 3). Among the disubstituted analogs, the most
potent was inhibitor 6(2,5-difluoro), but the difference in
antiviral activity among the mono- and di-substituted fluoro
inhibitors was minor. However, not surprisingly, the
significant increase in volume of the P1/P1¢side group
affects the inhibitory efficacy. For example, inhibitor 6(2,5-
difluoro) has an ED
50
of 0.02 l
M
, albeit it has a moderate K
i
of 3.3 n
M
compared with the reference compound 1with an
ED
50
of 0.1 l
M
and a K
i
of 1.2 n
M
. However, the most
convincing evidence on the ability of fluorine substitution to
enhance antiviral activity in cell assay was observed for
inhibitor 3which has an ED
50
of 0.06 l
M
and a K
i
of
7.1 n
M
.
Fig. 2. Accommodation of S1-lining residues as a result of P1-benzyloxy
fluorination. The root-mean-square deviation (RMSD) of all side chain
atoms within 3.9 A
˚of the P1 benzyloxy side groups of inhibitors 1–6
are plotted pairwise. The expansion of the S1 subsite is most apparent
for residues Leu23, Gly48 Val32 and Pro81, which show the most
significant accommodations on P1-benzyloxy fluorination compared
with the nonsubstituted analog 1. Inhibitor-specific side-chain
accommodations are most pronounced for inhibitor 6where Arg8 and
Leu23 are displaced 0.5 A
˚with respect to inhibitors 2–5.The
S1-subsite residues Ile50 and Ile84 display high flexibility with large
atomic displacements, which correlates with Bvalues above average.
The RMSD values were calculated using
LSQMAN
[46].
4598 J. Lindberg et al.(Eur. J. Biochem. 271)FEBS 2004