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

antiviral a nd inhibitory activities, in order to evaluate t he
effects of different fluoro-substitutions. These C 2-symmetric
inhibitors comprise mono- and difluoro-substituted benzyl-
oxy side groups in P 1/P1¢ and indanoleamine side groups in
P2/P2¢. The ortho- an d 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, a s observed by an increase in favorable Van der
Waals c ontacts 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 f unctional 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 b ecome an attractive ta rget for effective a ntiviral agent s;
several protease inhibitors are currently in clinical trials.
Despite t he initial success o f 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-

E-mail:
Enzyme: HIV-1 protease, POL_HV1B1 (P03366) (EC 3.4.23.16).
Note: The refined coo rdinates and assoc iated struc ture factors of
HIV-1 protease in complex with inhibitors 1–6 have been deposited
in the RCSB Protein Data Bank wi th 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) Ó 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 a ccessible v olume 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 bind ing
efficacy t hat w e obser ved 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 b inding

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 c oncentration of 0.3 mgÆmL

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 CentriconÒ Centrifugal
Filter Units to 2 mgÆmL
)1
.
Enzyme activity/inhibition studies
Enzyme activity/inhib ition studies were performed as des-
cribed by Nillroth et al. [34]. Briefly, a fluorimetric assay
was used t o d etermine the effects of the inhibitors on HIV-1
protease. This assay used an internally quen ched fluorescent
peptide substrate, DABSYL- c-Abu-Ser-Gln-Asn-Tyr-Pro-
Ile-Val-Gln-EDANS (Bachem, B ubendorf, 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

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
Ó FEBS 2004 Fluorine substitution of HIV-1 protease inhibitors (Eur. J. Biochem. 271) 4595
Laboratory, D aresbury, 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 s tatistics is
giveninTable2.
Structure refinement
Refinement was performed using the program package
CNS

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 i ndanolamine side
groups in P2/P2¢ [33]. All inhibitors have K
i
values within
the nanomolar to picomolar range, and antiviral activity
expressed as E D
50
values varying from 100 to 20 n
M
(Table 3) .
Table 2. Crystallographic s tructure determination statistics for protease–inhibitor complexes 1–6. Statistics for reflections in h ighest 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

˚
) 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
merge
a
(%) 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 r in
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
cryst
b
(%) 18.1 19.0 19.9 19.9 18.8 18.8
R
free
c
(%) 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

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) Ó 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

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 w ith specific accommoda-
tions of the residues lining the S1/S1¢ subsites, as compared
with the nonsubstituted analog 1. The rmsd of Ca atoms of
S1/S1¢-lining r esidues range from 0.12 to 0.33 A
˚
for the
different protease–inhibitor complexes. I n Fig. 2 an over-
view of the accommodation of S1 subsite lining residues is
presented with r espect 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 re sponse to the fluoro-substitution in
ortho, meta and p ara 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, V al32 and P ro81.
The conformation of the remaining S1-lining residues
remains u naffected by the panel of fluoro-substitutions.
The protease inhibitor complex with inhibitor 6 exhibited
the most pronounced shifts in side-chain position, partic-

No. of
repelling contacts
d
K
i
(n
M
)
ED
50
e
(l
M
)
1 652.7 1394.6 57 10 – 1.2 0.10
2 671.7 1440.1 71 10 – 3.2 0.05
3 671.7 1398.3 78 10 2 7.1 0.06
4 690.7 1435.8 70 11 – 1.6 0.11
5 690.7 1433.5 84 10 2 4.0 0.03
6 690.7 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

active site. The 2F
o
–F
c
difference electron-density m ap unambiguously
shows a unique orientation of the inhibitor and the fluorine substitu-
ents on the P1/P1¢ side groups. The electron density maps were ca l-
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]
( and 3D-rendered with
POV
-
RAY
( />Ó FEBS 2004 Fluorine substitution of HIV-1 protease inhibitors (Eur. J. Biochem. 271) 4597
sition of the two inhibitors on the nonsubstituted a nalog
revealed that the position of 2-fluoro; 2 and 3-fluoro; 3
benzyloxy side g roups in the S1/S1¢ subsites are similar, and
slightly closer to Arg8/Arg8¢ than in the non-substituted

carbon. Notably, the 5-fluoro s ubstituents are observed
within dipole–dipole interaction range of the partially
charged C f carbon of the arginines. The presence of an
electrostatic interaction is supported by quantum mechan-
ical calculat ions of the partial charges for Cf carbon
(+ 0.3 4) a nd the 5-fluoro substituents ()0.11) in vacuu m
(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. Fu rthermore, the crystal structure of inhibitor
6 reveals increased flexibility (higher B values) and reduced
quality of the electron density for the isoleucine side chains
compared with the i nhibitor 2 complex. 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 a s proton acceptors in two
hydrogen bonds to the nitrogen atoms of Arg8/Arg8¢ side
chains, and the 2-fluoro substituen ts 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

of 3.3 n
M
compared with the reference compound 1 with an
ED
50
of 0.1 l
M
and a K
i
of 1.2 n
M
. However, the most
convincing e vidence on the ability of fluorine substitution to
enhance antiviral activity in cell assay was observed for
inhibitor 3 which 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 p lotted pairwise. The expansion of the S 1 subsite is most apparent

i
values indicate inefficient accom-
modation of fluoro-substituted P1/P1¢ benzyloxy side
groups in the respective subsites. However, the orientation
of the fluoro inhibitors in the active site showed an increased
number of Van der Waals contacts and favorable electro-
static contacts to the p rotease s ubsites, which is evidence for
an improvement i n protease i nhibition. These contradictory
results may mean that the asymmetrically fluoro-substituted
benzyloxy side groups have two binding configurations,
differing by a 180° rotation, with two distinct affinities for
the S1/S1¢ subsites. However, i n the structures of the
complexed forms of the protease, only one configuration is
trapped in the crystal lattices and observed at full occu-
pancy. Computer modeling of the 2- and 3-fluorobenzyloxy
side groups showed that the 180°-rotated configurations
were equally possible; the fluoro substituents filled t he space
in the vicinity of Gly27/Gly27¢ and Leu23/Leu23¢ without
need for s ide-chain adaptation. However, the modeling also
revealed repelling contacts between the fluoro substituents
and the back bone carbonyl o xygen of G ly27/Gly27¢ and
Leu23/Leu23¢, making that confi guration highly unfavora-
ble. Extending the modeling to the disubstituted inhibitors
4–6 showed similar unfavorable binding properties of the
180°-shifted configurations. This is also reflected in a
reduction in binding efficacy (increased K
i
values) for
Table 4. Effect of P1/P1¢ fluoro-substitution on interatomic d istances between inhibitor side groups and subsite-lining residues. Dista nces presented in
bold represent f avorable Van de r Waals and electrostatic contacts, whereas dista nces in italics represent unfavorable charge r epulsions. Hydrogen

inhibitors fluoro-su bstituted a symmetrically on the benzyl-
oxy side groups compared with symmetric analogs, such as
the nonsubstituted inhibitor (1), 2,6- and 3,5-difluoro-
substituted analogs [33]. However, it cannot be excluded
that other factors are involved in the reduced binding
efficacy f o r the fluoro inhibitors, including decreased
entropy and increased solvation energies.
Modeling of the two possible orientations of the side
groups indicates that the trapped configurations observed in
the X-ray structures should have the highest binding efficacy
compared with the 180°-rotated configurations. Thus, o n
the basis of the physicochemical properties of the fluorine–
carbon bonds (Table 1), the effect from individual fluori-
nations on binding efficacy c ould be discerned by e valuating
the intermolecular contacts among the protease–inhibitor
complexes. The m onosubstituted 2-fluoro s ide groups are
accommodated differently in the S1/S1¢ subsites compared
with the 3-fluoro and nonsubstituted side groups. The
2-fluoro inhibitor utilize s the accessible volume in the
subsites more efficiently, which is reflected as a gain of two
Van der Waals contacts to Ile50/Ile50¢ side chains, contacts
that are not present in the case of the 3 -fluoro inhibitor.
Interestingly, the 1 80°-rotated configuration i s not ob served
in the X-ray structure, but modeling reveals that the
benzyloxy side groups need to adapt their configuration to
prevent steric clashes with Arg8/Arg8¢, which results in lost
contacts to the Ile50/Ile50¢ side chains. This underlines the
importance of p reserved Van der Waals contacts between
the 2-fluoro substituents and the isoleucine side chains. The
lower B values and improved electron-density map quality

accounts for the t wofold improvement in protease inhibition
(K
i
1.6 n
M
) compared with the 2-fluoro-substituted analog
(K
i
3.2 n
M
). The 2-fluoro inhibitor 2 and 2 ,5-difluoro
inhibitor 6 were equipotent in terms of protease inhibition
despite the 2,5-difluorobenzyloxy side group repositioning.
This is at tributed to the contacts g ained to Arg8/Arg8¢ by
the 5-fluoro substituents and to contacts lost to Ile50/Ile50¢
by the 2-fluoro substituents. Hence, t he K
i
values are
influenced not only by the number of fluorine substituents
but also by the position of the fluorine on t he benzyloxy s ide
groups.
Antiviral activity, ED
50
Our monofluoro- and difluoro-substituted inhibitors exhibit
significant improvements in antiviral activities in MT4 cell
culture assay co mpared with the nonsubstituted analog 1.
Fig. 4. Superimposition o f the 2,4-difluoro-substituted inhibitor on the
nonsubstituted analog in the S1¢ subsite. Th e 2,4-difluoro-substitution o f
inhibitor 4 fills the accessible volume o f the S1 ¢ subsite more efficiently
than the nonsubstituted a nalog. The 2 -fluoro substituent is in Van der

tration, distribution, and toxicity, properties that have been
discussed in a number of different reviews [13,16,41]. It is
noteworthy, however, that ortho-, meta- and fluoro-substi-
tuted benzyloxy side group s had markedly improved ED
50
values than the nonsubstituted analog and related
C2-symmetric diol-based protease inhibitors [23,26]. These
values were comparable to those for the reference drugs
ritonavir, indinavir, saquinavir, and nelfinavir. This ten-
dency can be attributed in part to the higher lipophilicity of
the fluoro-substituted i nhibitors [42]. Fluorine contributes
to overall pharmacological activity by enh ancing bioavail-
ability and retarding metabolic degradation. It thereby
extends the c linical applications for several different drug
candidates [43,44]. In our series of fluoro inhibitors, the
fluorine substitution with most enhanced antiviral activity i n
a cell assay w as observed for inhibitor 3 which had an ED
50
of 0.06 l
M
and a K
i
as high as 7.1 n
M
. In addition to the
improved antiviral effect, fluorination offers pharmacologi-
cal alternatives to co mbat resistance mutations of HIV-1
protease, because it extends th e cont act surf ace and has
polarity d ifferences. This is documented by the threefold
improvement in activity of inhibitors 2 and 6 against the

Swedish N ational Board for Industrial and Technical Development
(NUTEK), the Swedish Research Council for Engineering Sciences
(TFR), and Medivir AB, Huddinge, Sweden.
References
1. Gallo, R .C., Salahuddin, S.Z., Popovic, M., Shearer, G.M.,
Kaplan, M., Haynes, B.F., Palker, T.J., Redfield, R., Oleske, J.,
Safai, B. et al. (1984) Frequent detection and isolation of cyto-
pathic retroviruses (HTLV-III) from patients with AIDS and at
risk for AIDS. Science 224, 500–503.
2. Gallo, R.C., Sarin, P.S., Gelmann, E.P., Robert-Guroff, M.,
Richardson, E., Kalyanaraman, V.S., Mann, D., Sidhu, G.D.,
Stahl, R.E., Zolla-Pazner, S., Leibowitch, J. & Popovic, M. (1983)
Isolation of human T-cell leukemiavirusinacquiredimmune
deficiency synd rome ( AIDS). Science 220, 865–867.
3.Barre-Sinoussi,F.,Chermann,J.C.,Rey,F.,Nugeyre,M.T.,
Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-
Brun, F., Rouzioux, C., Rozenbaum, W. & Montagnier, L. (1983)
Isolation of a T-lymphotropic retrovirus from a p atient at risk f or
acquired immune deficiency syndrome (AIDS). Science 220,868–
871.
4. Peng, C ., Ho, B.K., Chang, T.W. & Chang, N .T. (1989) Role
of human immunodeficiency virus type 1-specific protease in
core protein maturation and viral infectivity. J. Virol. 63, 2550–
2556.
5. Kohl, N.E., Emini, E.A., Schleif, W.A., Davis, L.J., Heimbach,
J.C., Dixon, R.A., Scolnick, E.M. & Sigal, I.S. (1988) Active
human immunodeficiency virus protease is required for viral
infectivity. Proc. Natl Acad. Sci. USA 85, 4686–4690.
6. Roberts, N.A., Martin, J.A., K inchington, D ., Broadh urst, A.V.,
Craig, J.C., Duncan, I.B., G alpin, S.A., Handa, B.K., Kay, J.,

14. Flexner, C. (1998) HIV-protease inhibito rs. N.Engl.J.Med.338,
1281–1292.
15. Molla, A., Granneman, G.R., Sun, E. & Kempf, D.J. (1998)
Recent developments in HIV protease inhibitor therapy. Anti-viral
Res. 39, 1–23.
16. Moyle, G. (2002) Overcoming obsta cles to t he success o f protease
inhibitors in highly active antiretroviral therapy regimens. AIDS
Patient Care STDS 16, 585–597.
Ó FEBS 2004 Fluorine substitution of HIV-1 protease inhibitors (Eur. J. Biochem. 271) 4601
17. Erickson, J. & Ke mpf, D. (1994) Structure-based design of
symmetric inhibitors of HIV-1 protease. Arch. Virol. Suppl. 9,
19–29.
18. Kempf, D.J. (1994) Design of symmetry-based, peptidomimetic
inhibitors of human immunodeficien cy virus protease. Methods
Enzymol. 241, 334–354.
19. Kempf, D.J., Norbeck, D.W., Codacovi, L., Wang, X.C., Kohl-
brenner, W.E., Wideburg, N.E., Paul, D.A., Knigge, M.F.,
Vasavanonda, S., Craig-Kennard, A. et al. (1990) Structure-
based, C2 symmetric inhibitors of HIV protease. J. Med. Chem.
33, 2687–2689.
20. Erickson, J., Neidhart, D.J., VanDrie, J., Kempf, D .J., Wang,
X.C., Norbeck, D.W., Plattner, J.J., Rittenhouse, J.W., Turon,
M., Wideburg, N. et al. (1990) De sign, activity, and 2 .8 A crystal
structure of a C2 symmetric inhibitor complexed to HIV-1 pro-
tease. Science 249, 527–533.
21. Andersson, H.O., Fridborg, K., Lo
¨
wgren, S., Alterman, M.,
Muhlman, A., Bjo
¨

Samuelsson, B. (2001) Synt hesis of novel, potent, diol-based HIV-
1 protease inhibitors via intermolecular pinacol homocoupling of
(2S)-2-benzyloxymethyl-4-phenylbutanal. J. Med. Chem. 44,
3407–3416.
25. Muhlman, A., Classon, B., Hallberg, A. & S amuelsson, B. (2001)
Synthesis of p otent C(2)-sym metric, d iol-based h iv-1 protease
inhibitors. Investigation of thioalkyl and thioaryl P1/P1¢ substit-
uents. J. Med. Ch em. 44 , 3402–3406.
26. Oscarsson, K., Lahmann, M., Lindberg, J., Kangasmetsa, J.,
Unge, T ., Oscarson, S., Hallberg, A. & Samuelsson, B. (2003)
Design and synthesis of HIV-1 protease inhibitors. Novel tetra-
hydrofuran P2/P2¢-group s interacting with Asp29/30 of the
HIV-1 protease. Determination of binding from X-ray crystal
structure of inhibitor protease complex. Bioorg. Med. Chem. 11,
1107–1115.
27. Lee, K., Jung, W.H., H wang, S.Y. & Lee, S.H. (1999) Fluor-
obenzamidrazone thrombin inhibitors: influ ence o f fluorine on
enhancing oral absorption. Bioorg. Med. Chem. Lett. 9, 2483–
2486.
28. Park, B.K. & Kitteringham, N.R. (1994) Effects of fluorine sub-
stitution on drug metabolism: pharmacological and toxicological
implications. Drug Metab. Rev. 26, 605–643.
29. Park, B.K., Kitteringham, N.R. & O’Neill, P.M. (2001) Metabo-
lism of fluorine-containing drugs. An nu. Rev. Pharmacol. Toxicol.
41, 443–470.
30. Lu, S.F., H erbert, B., Haufe, G., Laue, K.W., Padgett, W.L.,
Oshunleti, O., Daly, J.W. & Kirk, K.L. (2000) Syntheses of (R)-
and (S)-2- and 6 -fluoronore pinephrine and ( R)- and (S)-2- and
6-fluoroepinephrine: effect of stereochemistry on fl uorine-indu ced
adrenergic selectivities. J. Med. Chem. 43, 1611–1619.

Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T. & Warren,
G.L. (1998) Crystallography & NMR system: a new software suite
for macromolecular structure determination. Acta Crystallogr. D
Biol. Crystallogr. 54, 905–921.
39. Jones, T.A., Zou, J Y., Cowan, S.W. & Kjeldgaard, M. (1991)
Improved methods for b uilding protein models in electron d ensity
maps and the location of errors in th ese models. Acta Crystallogr.
A 47 , 110–119.
40. Kleywegt, G.J. & Brunger, A.T. (1996) Checking your imagina-
tion: applications of the free R value . Structure 4, 897–904.
45. Guex, N. & Peitsch, M .C. (1997) SWISS-MODEL and the Swiss-
PdbViewer: an environment for comparative p rotein modeling.
Electrophoresis 18, 2714–2723.
41. Moyle, G.J. & Back, D. (2001) Principles and practice of
HIV-protease inhibitor pharmacoenhancement. HIV Med. 2,
105–113.
42. Welch, J. (1987) Advances in the preparation of biologically active
fluorinated compoun ds. Tetrahedron 43, 3123–3197.
43. Resnati, G. (1990) Aspects of the medicinal chemistry of fluoro-
organic compounds. Part I. Farmaco. 45, 1043–1066.
44. Bryskier, A. & Chantot, J.F. (1995) Classification and structure-
activity relationships of fluoroquinolon es. Drugs 49, 16–28.
46. Kleywegt, G.J. & Jones, T.A. (1997) Detecting folding m otifs and
similarities in protein structures. Methods Enzymol. 525–545.
47. Engh, R.A. & Huber, R. (1991) Accurate bond and angle para-
meters for X-ray p rotein structure refinement. Acta Crystallogr.
A47, 392–400.
4602 J. Lindberg et al.(Eur. J. Biochem. 271) Ó FEBS 2004