Synthesis, stability, antiviral activity, and protease-bound structures of substrate-mimicking constrained macrocyclic inhibitors of HIV-1 protease

Article abstract of DOI:10.1021/jm000013n

Three new peptidomimetics (1-3) have been developed with highly stable and conformationally constrained macrocyclic components that replace tripeptide segments of protease substrates. Each compound inhibits both HIV-1 protease and viral replication (HIV-1

Full text of DOI:10.1021/jm000013n

J . Med. Chem. 2000, 43, 3495-3504
3495
Articles
Syn th esis, Sta bility, An tivir a l Activity, a n d P r otea se-Bou n d Str u ctu r es of
Su bstr a te-Mim ick in g Con str a in ed Ma cr ocyclic In h ibitor s of HIV-1 P r otea se
J oel D. A. Tyndall,† Robert C. Reid,† David P. Tyssen,§ Darren K. J ardine,§ Belinda Todd,‡ Margaret Passmore,†
Darren R. March,† Leonard K. Pattenden,† Douglas A. Bergman,† Dianne Alewood,† Shu-Hong Hu,†
Paul F. Alewood,† Christopher J . Birch,*,§ J ennifer L. Martin,*,† and David P. Fairlie*,†
Centre for Drug Design and Development, The University of Queensland, Brisbane, Queensland 4072, Australia, Victorian
Infections Diseases Reference Laboratory, Private Bag 815, Carlton South, Victoria 3053, Australia, and Sir Albert Sakzewski
Virus Research Centre, The Royal Children’s Hospital, Herston, Queensland 4029, Australia
Received J anuary 13, 2000
Three new peptidomimetics (1-3) have been developed with highly stable and conformationally
constrained macrocyclic components that replace tripeptide segments of protease substrates.
Each compound inhibits both HIV-1 protease and viral replication (HIV-1, HIV-2) at nanomolar
concentrations without cytotoxicity to uninfected cells below 10 µM. Their activities against
HIV-1 protease (Ki 1.7 nM (1), 0.6 nM (2), 0.3 nM (3)) are 1-2 orders of magnitude greater
than their antiviral potencies against HIV-1-infected primary peripheral blood mononuclear
cells (IC50 45 nM (1), 56 nM (2), 95 nM (3)) or HIV-1-infected MT2 cells (IC50 90 nM (1), 60 nM
(2)), suggesting suboptimal cellular uptake. However their antiviral potencies are similar to
those of indinavir and amprenavir under identical conditions. There were significant differences
in their capacities to inhibit the replication of HIV-1 and HIV-2 in infected MT2 cells, 1 being
ineffective against HIV-2 while 2 was equally effective against both virus types. Evidence is
presented that 1 and 2 inhibit cleavage of the HIV-1 structural protein precursor Pr55gag to
p24 in virions derived from chronically infected cells, consistent with inhibition of the viral
protease in cells. Crystal structures refined to 1.75 Å (1) and 1.85 Å (2) for two of the macrocyclic
inhibitors bound to HIV-1 protease establish structural mimicry of the tripeptides that the
cycles were designed to imitate. Structural comparisons between protease-bound macrocyclic
inhibitors, VX478 (amprenavir), and L-735,524 (indinavir) show that their common acyclic
components share the same space in the active site of the enzyme and make identical
interactions with enzyme residues. This substrate-mimicking minimalist approach to drug
design could have benefits in the context of viral resistance, since mutations which induce
inhibitor resistance may also be those which prevent substrate processing.
In tr od u ction
highly active antiretroviral therapy (HAART).11,12 Clini-
cal trials have shown that many HIV-infected individu-
als undergoing HAART have rapid and profound reduc-
tions in plasma viral load, which can often be sustained
for months or years. Unfortunately, suboptimal inhibitor
concentrations in some patients often result from the
difficulty in adherence to long-term dosing with multiple
antiretroviral drugs or from patient-to-patient differ-
ences in pharmacokinetic drug profiles, inevitably lead-
ing to development of drug resistance. This is particu-
larly problematic with clinical and preclinical protease
inhibitors, with resistance to one often resulting in
cross-resistance to others,13 and inhibitors of other HIV
proteins.14 In reality there is really only a single
protease inhibitor option available to most patients,
highlighting the need for new drugs which either select
for HIV with uncommon resistance-inducing mutations
in the protease or overcome resistance altogether. Novel
strategies for the design of such antiretroviral drugs in
general and protease inhibitors in particular are clearly
needed.
Inhibitors of the human immunodeficiency virus type
1 (HIV-1) protease (HIV PR)1-7 act by blocking the
cleavage of HIV-1 precursor proteins Pr55gag and
Pr160gag-pol. The former is the precursor to the matrix
(p17), capsid (p24), and nucleocapsid (p9) structural
proteins, while the latter is the precursor to the viral
enzymes reverse transcriptase (RT), RNase H, and the
protease itself.8 Although HIV PR inhibitors act late in
the replication cycle of HIV-1 in acutely or chronically
infected cells, they ultimately prevent early replicative
events in newly infected cells, since their activity
ensures that RT and RNase H are not available for the
reverse transcription process and that mature p17 is
not present as part of the preintegration complex.9,10
Combination therapy using inhibitors of both HIV RT
and PR has played an important role in the success of
* Address
correspondence
to D.P.F. (e-mail, d.fairlie@
mailbox.uq.edu.au; fax, +61733651990), J .L.M. (e-mail, j.martin@
mailbox.uq.edu.au), or C.J .B. (e-mail, Chris.Birch@mh.org.au).
† The University of Queensland.
We now report the synthesis, stability, activities, and
structures of new compounds (1-3) containing cyclic
§ Victorian Infections Diseases Reference Laboratory.
‡ The Royal Children’s Hospital.
10.1021/jm000013n CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/25/2000

3496 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Tyndall et al.
replacements for tripeptide sequences. We have dem-
onstrated before in other molecules that such cyclic
components are preorganized in a high-affinity protease-
binding conformation15-17 yet are flexible enough to
potentially adapt to changes in the shape of the inhibi-
tor-binding groove of the protease which occur as a
result of drug-induced mutations. This report is part of
a wider strategy to mimic protease substrates more
closely than do current protease inhibitors and, in so
doing, perhaps minimize the likelihood of viral resis-
tance since only protease mutations that block or
modulate substrate processing are likely to prevent
substrate-mimicking inhibitors from binding to the
protease. As a penultimate step toward this ultimate
goal, we have here appended cyclic tripeptide mimics
to some nonpeptidic moieties that help to confer potent
HIV-1 protease inhibitor activity and antiviral activity
at nanomolar concentrations.
hydroxyaminoindan N-terminus (N-(2(R)-hydroxy-1(S)-
indanyl)-2(R)-(phenylmethyl)-) that has previously been
reported to occupy P2 and P1 in L-735,524 (indinavir).19
Inhibitor 3 is a hexapeptide mimetic,16 with a similar
C-terminal macrocycle as in 2 and with the same
N-terminus as in Ro31-8959 (saquinavir).20
Using our reported syntheses21 for the epoxide de-
rivative 6 of the N-terminal macrocyclic component of
1 (namely 4) and for the amine derivatives21 5 and 9 of
the C-terminal cycle in both 2 and 3, it was a straight-
forward exercise to construct inhibitors 1-3 by append-
ing these to the appropriate acyclic components (Schemes
1, 2). The compounds were purified by reversed-phase
HPLC and fully characterized by 1H NMR spectroscopy
and high-resolution mass spectrometry.
Compounds 1-3 were evaluated as inhibitors of
synthetic HIV-1 PR at pH 6.5 (I ) 0.1 M) as described
in Materials and Methods. They had Ki values of 1.7,
0.6, and 0.3 nM, respectively, versus 1.8 nM for J G36516
and 1.0 nM for DMP32316 under the same conditions.
Sch em e 1
Resu lts a n d Discu ssion
P r otea se In h ibitor s. Compounds 1-3 each contain
a cyclic component designed to function as a replace-
ment for a P3-P1 or P1′-P3′ tripeptide segment of a
substrate for HIV PR. Inhibitor 1 is a pentapeptide
mimetic consisting of a 17-membered N-terminal mac-
rocycle15 designed to mimic an N-terminal tripeptide
sequence Xaa-Val-Phe (P3-P1), a hydroxyethylamine
isostere to mimic the transition state of peptide hy-
drolysis, and a nonpeptidic C-terminal N-isobutyl p-
aminobenzenesulfonamide moeity (P1′-P2′) which is an
analogue of the C-terminus of another HIV-1 PR inhibi-
tor VX-478 (amprenavir),18 but with an extra methylene
inserted in the alkyl chain of 1 at P1′. Inhibitor 2 is also
a pentapeptide mimetic, which links a C-terminal 17-
membered macrocycle16 designed to mimic a C-terminal
tripeptide sequence Phe-Ile-Xaa (P1′-P3′) with a hy-
droxyaminopentanamide isostere to mimic the transi-
tion state of peptide hydrolysis and a nonpeptidic
Ma cr ocycle Sta bility. To investigate whether the
cyclic peptide components of these inhibitors were
proteolytically stable toward mammalian proteases that
are likely to be encountered in vivo, compounds 4 and
5 were exposed to various degradative proteases found
in the GI tract, plasma or in cells. The macrocycles were
completely stable in 3 M hydrochloric acid at 40 °C over
5 days with no sign of any decomposition as assessed
by rpHPLC and electrospray MS. When incubated at
37 °C for up to 24 h with either (a) selected gastric,
plasma, or cellular proteases, (b) gastric juice from rat
stomachs, (c) serum (1-h incubation only), or (d) contents

Constrained Macrocyclic Inhibitors of HIV PR
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3497
Ta ble 1. Antiviral Activity and Cytotoxicity of 1-3 in PBMCs
and MT2 Cells Infected with HIV-1
Sch em e 2
drug
cell
CC10a (µM) IC50b (µM) IC90c (µM) SId
1
2
3
CBMCe
PBMCf
MT2f
0.045
0.43
0.09
0.056
0.11
50.0
10.0
0.88
0.22
57
45
CBMCe
PBMCf
MT2f
50
30.0
0.22
0.10
227
300
0.06
CBMCe
CBMCe
0.095
0.110
0.03
0.052
0.056
DMP323
amprenavir MT2f
10.0
10.0
10.0
0.08
125
indinavir
PBMCf
MT2f
0.093
107.5
a
CC10: concentration of inhibitor causing death in 10% or less
b
of cells in the absence of virus. IC50: concentration of inhibitor
producing a 50% decrease in virion-associated RT activity relative
to untreated, infected control. c IC90: concentration of inhibitor
producing a 90% decrease in virion-associated RT activity relative
d
to untreated, infected control. SI (selective index) ) CC10 divided
by IC90.
e HIV-1 strain TC354 (Brisbane). f HIV-1 strain 237288
(Melbourne).
Ta ble 2. Cytotoxicity and Inhibitory Concentrations of 1 and 2
of lysed human lymphocytes, monocytes, or polymor-
phonuclear leukocytes, there were similarly no indica-
tions of decomposition of the macrocycles. Under the
same conditions, control linear peptides (e.g. AcLVF-
{ψ(CHOHCH2)}-FIV-NH2 and YSFKDMQLGR) were
completely hydrolyzed (100%) within minutes.
in MT2 Cells Infected with HIV-2a
drug
CC10b (µM)
IC50c (µM)
IC90d (µM)
SIe
2.0
176
1
2
10.0
30.0
0.50
0.10
5.0
0.17
a
b
Strain of HIV-2 is ROD. CC10
: concentration of inhibitor
causing death in 10% or less of cells in the absence of virus. c IC50
concentration of inhibitor producing a 50% decrease in virion-
associated RT activity relative to untreated, infected control. IC90
:
The results are summarized in Materials and Meth-
ods and indicate that the macrocycles confer important
properties on the peptidomimetics. Although the cycles
contain two amide bonds, they are not susceptible to
hydrolytic/proteolytic cleavage like normal peptide bonds.
An tivir a l Activity. Compounds 1-3 were tested for
antiviral activity by investigators in two different cities,
and each was found to be a potent inhibitor of the
replication of HIV-1 in acutely infected cells (Table 1).
We found that for cord blood mononuclear cells (CBMC)
infected with HIV-1 (strain TC354, SASVRC, Brisbane),
IC50 values were 45 nM (1, n ) 5), 56 nM (2, n ) 5),
and 95 nM (3, n ) 5). Studies were also undertaken
using primary peripheral blood mononuclear cells and
MT2 cells infected with a different strain of HIV-1
(#237288, a laboratory-adapted, syncytial-inducing strain
isolated from an AIDS patient at Fairfield Hospital,
Melbourne). An example of these latter results (Table
1) is the data for MT2 cells in which IC50 was 90 nM
(IC90 0.22 µM) for 1 and 60 nM (IC90 0.10 µM) for 2. No
drug-induced cytotoxicity was observed below 10-50
µM. These compounds thus have significant activity
against HIV-1 at concentrations that are not toxic to
cells.
d
:
concentration of inhibitor producing a 90% decrease in virion-
associated RT activity relative to untreated, infected control. e SI
(selective index) ) CC10 divided by IC90
.
F igu r e 1. Immunoblot of HIV virions produced from H9/
HTLV-IIIB cells after 72-h exposure to either no drug (lane 2)
or 10 µM of either 1 (lane 3) or 2 (lane 4). Standard molecular
weight markers (kD) are present in lane 1. The HIV structural
precursor protein Pr55gag, two of its cleavage intermediates
(of molecular weight 49 and 41 kD), and the product p24 are
indicated.
Complete inhibition of viral replication occurred at
concentrations significantly lower than those which
were cytotoxic, thereby resulting in selective indices
(SIs) of 45 and 57 for 1 in MT2 and PBMCs, respectively,
and 300 and 227 for 2 in the same cell types (Table 1).
These results compare very favorably with those for
indinavir under the same conditions (Table 1, SI ) 107).
However, while compound 2 had comparable activity
against HIV-2 in acutely infected MT2 cells (SI of 176),
compound 1 had only marginal activity against the same
strain of HIV-2 (SI ) 2.0; Table 2).
process, 1 and 2 were examined for inhibition of the
cleavage of the HIV-1 structural protein precursor
Pr55gag in virions derived from chronically infected
HqHTLV-IIIB cells. In the absence of inhibitors, these
cells produced virions containing low levels of the
precursor but substantial amounts of its product p24,
indicating that protease-directed cleavages occur as
expected in these viruses (Figure 1, lane 2). In virions
produced from cells treated with either 1 or 2, there was
evidence of inhibition of protease-directed cleavage
(Figure 1, lanes 3 and 4). Uncleaved Pr55gag was present
in virions derived from both sources. Nevertheless, some
cleavage of Pr55gag did occur in the presence of either
To check that these new macrocyclic compounds were
exerting their antiviral activity via the anticipated
protease inhibition mechanism rather than some other

3498 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Tyndall et al.
F igu r e 2. Stereoview of electron density maps for 1 and 2 bound to HIV-1 PR. The 2Fo - Fc maps are shown, contoured at 1.0σ.
inhibitor, indicated by the increased amounts of 49- and/
or 41-kD intermediates compared to that in virions
derived from untreated cells. However, subsequent
cleavage of these intermediates to the p24 product was
substantially inhibited in virions exposed to either drug,
consistent with protease inhibition being responsible for
their antiviral activity.
Cr ysta l Str u ctu r es. Inhibitors 1 and 2 were cocrys-
tallized with synthetic HIV-1 PR containing the muta-
tions Q7K, L33I, C37Aba, and C95Aba (Aba ) R-ami-
nobutyric acid). The cysteine residues were replaced for
easier synthesis and handling, while the two autocleav-
age sites (Q7, L33) were mutated to reduce autolysis.22
The synthetic mutant [Lys7,Ile33,Aba67,95]-HIV-1 PR is
designated HIVKI, and its enzymatic properties have
been fully characterized elsewhere.9,23 Both complexes
crystallized in the space group P212121 in a crystal form
isomorphous with the HIV-1 PR:J G-365 complex.24 The
inhibitor electron density observed in both structures
(Figure 2) was in excellent agreement with the composi-
tion of inhibitors 1 and 2.
The crystal structure of 1 shows that carbonyl oxy-
gens and NH groups of both amides in the macrocycle
form hydrogen bonds with the enzyme (Figure 3), and
these hydrogen bonds are equivalent to those observed
in acyclic peptidic inhibitor complexes. Also, the aro-
F igu r e 3. Hydrogen-bonding interactions between HIV-1 PR
and 1 (top), with the corresponding inhibitor-numbering
system below.
matic ring (P1) and isopropyl (P2) and pentyl (P3)
groups of the macrocycle in 1 occupy similar positions
in the enzyme as the side chains of Phe/Tyr, Val/Asn,
and Nle, respectively, from acyclic peptidic inhibitors
that complex in the active site of HIV-1 PR. The
aliphatic pentyl chain in the macrocycle of 1 (C1-C5)
is more disordered than the rest of the inhibitor with
an average B factor of ∼21 Å2 for the five carbon atoms
compared to ∼15 Å2 for other inhibitor atoms. The
methylene group (C2) of the aliphatic chain appears to
make an unfavorable contact (3.3 Å) with Arg108 Nη2,
while at the opposite end of the active site the corre-
sponding Arg8 Nη2 also approaches closely (within 3.9
Å) to C26 of the flexible P1′ isoamyl group of 1.
The hydroxyl group of 1 forms hydrogen bonds with
both catalytic aspartates (Figure 3), as observed for most
protease inhibitors containing this transition-state isos-
tere. The C-terminal N-alkylsulfonamide makes inter-
actions with the enzyme that are similar to those
observed for the alkylsulfonamide of VX-478.18 Specif-
ically, a sulfonyl oxygen (O5) interacts with Wat301
which in turn hydrogen-bonds to the amide nitrogens
of flap residues Ile50 and150. The other sulfonyl oxygen

Constrained Macrocyclic Inhibitors of HIV PR
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3499
Ta ble 3. Crystallographic Parameters and Data Processing
Statistics
1
2
resolution (Å)
a (Å)
1.75
51.4
1.85
51.8
b (Å)
58.8
58.9
c (Å)
Rsym
(in top shell)
61.7
61.9
a
0.083
(0.302)
94.5
(84.6)
9.5
0.051
(0.277)
92.7
(87.9)
12.0
completeness (%)
(in top shell (%))
I/σI
(in top shell)
(2.1)
(2.9)
no. obsd (I/σI > 1)
no. unique reflns
top resolution shell (Å)
59019
18470
1.83-1.75
51287
15546
1.92-1.85
a
Rsym ) Σh(Ih - Ih )/Σ Ih
.
as for L-735,524. The extra methylene in the transition-
state isostere of 2 compared with that in 1 imparts
added flexibility to this region, permitting two alterna-
tive isostere conformations to be modeled in the crystal
structure. Both conformations enable the hydroxyl
group to interact with both enzyme catalytic aspartates.
The extra methylene also causes a C-terminal transla-
tion of the inhibitor along the active site groove com-
pared with 1, so that the aliphatic hydrocarbon linker
in the macrocycle becomes solvent accessible and some-
what disordered. This is in contrast to other inhibitors
that incorporate an hydroxyethylamine isostere linked
to a C-terminal macrocycle, which are all translated
toward the N-terminus and have well-ordered macro-
cycles.22 However, the HIV PR:2 complex retains the
interaction between the protonated secondary amine
(N2) and catalytic aspartate (2.9 Å), as well as hydrogen
bonds with the Phe amide CO and Ile CO and NH atoms
of 2 (Figure 4), as observed in crystal structures of other
F igu r e 4. Hydrogen-bonding interactions between HIV-1 PR
and 2 (top), with the corresponding inhibitor-numbering
system below.
(O6) is close to side chains of Ile50 (Cδ 3.5 Å) and Ile184
(Cδ 3.5 Å). The isoamyl group of P1′ is flexible (two
conformations were modeled) and interacts with side
chains of Leu23, Pro81, Val82, and Ile184. The para-
amino substituent is within hydrogen-bonding distance
of the amide nitrogen and oxygen of Asp130.
The crystal structure of 2 bound to HIV-1 PR shows
that the nonpeptidic N-terminal end and transition-
state isostere interact with the enzyme in the same way
F igu r e 5. Superimposition of (top) 1 (green) and 2 (red), (middle) 1 and VX-478 (blue), and (bottom) 2 and L-735,524 (gray).

3500 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Tyndall et al.
Ta ble 4. Refinement Statistics for the HIV-1 PR:Inhibitor
inhibitors with C-terminal macrocycles bound to HIV-1
PR.
Complexes
1
2
The isoleucine side chain of 2 is flexible, permitting
two inhibitor conformations to be modeled, and has
hydrophobic interactions with Ile50, Ala128, Val132,
and Ile184 side chains in the S2′ pocket. As for the
complex with 1, unfavorable contacts occur between
Arg8 Νη1 and 2 (macrocycle C40, 3.0 Å; benzyl ring C28,
3.1 Å). A third unfavorable interaction occurs between
Arg108 Nη1 and C17 of the phenylalanyl substituent of
2. Although Wat301 forms the usual hydrogen bonds
between the inhibitor and the flap nitrogens in the HIV
PR:2 complex, it is less ordered than in other HIV PR
complexes. The B-factor of Wat301 in the HIV PR:2
complex is high (28 Å2) compared with the correspond-
ing water in the complex with 1 (∼9 Å2) and the two
waters are ∼0.5 Å apart. In the active site of the
protease three of the four hydrogen bonds of Wat301
are lengthened (0.2-0.4 Å) in the complex with 2. The
two equivalent acceptor atoms that interact with Wat301
in the P1′ site (O6 of 1 and O4 of 2) are 2.2 Å apart.
There may be several reasons for this. The constrained
cycle of 2, in conjunction with the isoleucyl residue at
S2′, forces the tyrosinyl group deep into the S1′ pocket
(Figure 5, red, top). Thus the nature of the substituents
in the S1′ and S2′ sites dictate the position of these
acceptor atoms. The sulfonyl oxygen (O5), 1, is closer
to Wat301 (by 0.4 Å) than the corresponding carbonyl
donor of 2 (O4), due to the sulfonamide group puckering
toward the flap region. However, this puckering also has
the effect of pushing the enzyme flaps into a more open
position. In both crystal structures reported here the
main chain atoms of Ile150 and Gly151 are modeled
with two alternate conformations. This observation has
been described previously.22 One difference in these two
structures is that the corresponding residues in the
opposite flap, Ile50 and Gly51, have only one conforma-
tion. Furthermore, the conformation observed in the
complex with 1 is opposite to that observed in the
complex with 2.
Figure 5 compares in stereo the bound conformations
of 1 and 2 with that of their corresponding acyclic inhib-
itors, VX-478 (middle, rmsd 0.55 Å) and L-735,524
(bottom, rmsd 0.47 Å), respectively. Both macrocyclic
inhibitors bind in a fashion similar to the equivalent
parts of the acyclic drugs. The isosteric hydroxyl groups
of 1 and 2 are separated from the hydroxyls of the
acyclic inhibitors by 0.5 Å (middle) and 0.2 Å (or 0.7 Å
for the alternate conformation, bottom), respectively.
The tertiary nitrogen of the sulfonamide group, 1, is
shifted by ∼1 Å compared with VX-478 due to the
addition of an extra methylene group in the alkyl chain
(middle). Tables 5 and 6 also demonstrate that the
hydrogen-bonding distances between analogous acyclic
portions of the inhibitor-enzyme complexes for 1 versus
amprenavir (Table 5) and 2 verus indinavir (Table 6)
are similar. Clearly the binding modes of these nonpep-
tidic acyclic portions are very similar.
resolution range (Å)
no. protein atomsa
no. inhibitor atomsa
no. waters
8-1.75
1542
8-1.85
1530
45
121
58
89
no. sulfate atoms
reflections (F >0σ (F))
R-factorb
3 × 5
18244
0.189
(0.288)
0.231
(0.323)
0.009
1.48
2 × 5
15323
0.212
(0.288)
0.259
(0.312)
0.005
1.16
(top shell)c
R-freed
(top shell)c
rms ideal bond length (Å)
rms ideal bond angle (deg)
coordinate error (Å)e
B all atoms (Å2)f
B inhibitor atoms (Å2)f
0.20-0.25
0.24-0.28
19.5
15.3
24.5
22.3
Including alternative conformations. b R ) Σ|Fo - Fc/ΣFo. c Top
a
d
resolution shell (Å): 1.83-1.75, 1; 1.93-1.85, 2. Cross-validation
R-factor using 10% of data. e Ref 42. B : average B-factor.
f
Ta ble 5. Corresponding Hydrogen-Bonding Interactions
between Acyclic Components of Inhibitors 1 and Amprenavir
with HIV-1 PR (cutoff 3.3 Å)
distances (Å)
inhibitor atom
protein residue
1
amprenavir
O4H
O4H
O4H
O4H
O5
Asp25 Oδ1
Asp25 Oδ2
Asp125 Oδ1
Asp125 Oδ2
Wat301
3.1
2.8
2.8
2.7
2.7
3.1
3.3
3.1
2.6
2.8
2.7
2.8
3.2
3.2
N4H
N4H
Asp130 Oδ
Asp130 NH
Ta ble 6. Corresponding Hydrogen-Bonding Interactions
between Acyclic Components of Inhibitors 2 and Indinavir with
HIV-1 PR (cutoff 3.3 Å)
distance (Å)
atom
protein residue
2
indinavir
O1H
O1H
O1H
N1H
O2
O3H
O3H
O3H
O3H
Wat321
3.1
3.0
3.3
3.3
3.0
2.8
3.1
3.2
3.0
2.8
2.6
3.0
2.9
2.8
Asp29 O
Asp29 NH
Gly27 CdO
Wat301
Asp25 Oδ1
Asp25 Oδ2
Asp125 Oδ1
Asp125 Oδ2
2.8 (2.8)a
3.1 (3.0)a
3.3 (2.7)a
3.0 (2.9)a
a
Denotes second conformation.
in HIV-1-infected cells, and (c) replication of two dif-
ferent viral strains of HIV-1 in infected primary and
cultured lymphocytes. The macrocycles confer a number
of valuable properties. They are structural mimics of
tripeptides fixed in the extended strand conformation
that is now known25,40 to be crucial for recognition by
all proteases, including HIV-1 PR. The crystal struc-
tures show that the cycles position their two amides
precisely to form the same hydrogen-bonding patterns
as acyclic peptides. The macrocycles also permit orien-
tation of side chains into the same three-dimensional
space occupied by the corresponding side chains of
acyclic peptides leading to identical protease-inhibitor
interactions. Together with previous results,15-17,22 it is
now clear that this novel class of inhibitors can be
translated along the active site through modification of
the length and charge state of the transition-state
isostere.
Su m m a r y
Three new compounds (1-3) containing 15-17-
membered macrocycles have been found to potently
inhibit (a) processing of a synthetic peptide by HIV-1
PR, (b) intracellular processing of a gag-pol polypeptide
Unlike the tripeptide segments that they mimic, the

Constrained Macrocyclic Inhibitors of HIV PR
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3501
in d a n ea m id e (2). A solution of the macrocyclic amine 9 (220
mg, 0.61 mmol, 2.4 equiv) and the chiral, optically active
epoxide 8 (93 mg, 0.25 mmol) in 2-propanol (3 mL) and
diisopropylethylamine (200 mL) was stirred and refluxed for
14 h (Scheme 2). Hydrochloric acid (2 M, 2 mL) and MeOH (5
mL) were added and heating was continued for 30 min. The
solution was evaporated to dryness and the residue was
purified by RP HPLC (A: 0.1% TFA in H2O, B: 0.1% TFA in
90/10 MeCN/H2O 30-min gradient to 40:60% B), retention time
30 min, giving 2 as a white powder (130 mg, 75%) after
lyophilization: 1H NMR (500 MHz, CD3OH) δ 7.83 (d, J ) 8.5
Hz, 1H, NH), 7.78 (m, 1H, CH2NH), 7.70 (d, J ) 7.5 Hz, NH),
7.33-7.15 (m, 9H, Ar), 7.05 (m, 2H), 6.83 (m, 2H), 5.25 (dd, J
) 8.5, 5.0 Hz, 1H), 4.36 (m, 1H), 4.31-4.25 (m, 1H), 4.20 (dd,
J ) 11.6, 5.7 Hz, 1H), 4.14 (m, 1H), 3.96 (m, 1H), 3.87 (dd, J
) 7.5, 5.5 Hz, 1H), 3.58-3.49 (m, 1H), 3.28 (dd, J ) 12.7, 5.5
Hz, 1H), 3.12 2.66 (m, 10H), 1.90 (m, 1H), 1.82-1.65 (m, 2H),
1.59-1.33 (m, 6H), 1.32-1.21 (m, 1H), 1.05 0.94 (m, 1H), 0.86
(t, J ) 7.4 Hz, Ile-γCH3), 0.81 (d, J ) 6.8 Hz, Ile-âCH3); 13C
NMR (CD3OD) δ 177.4, 171.0, 167.1, 159.4, 142.2, 141.8, 140.3,
131.4, 130.2, 129.6, 128.9, 127.9, 127.6, 126.9, 126.2, 125.3,
117.2, 74.0, 67.9, 66.4, 63.5, 59.2, 58.7, 53.4, 46.1, 40.8, 40.6,
40.2, 39.2, 39.0, 38.0, 36.4, 29.5, 29.3, 26.6, 23.7, 15.2, 12.1;
HRMS m/e 698.4059 calcd for C41H54N4O6 698.4043.
macrocycles are stable for at least 24 h at 37 °C to 3 M
HCl and to gastric, plasma, and cellular proteases under
conditions where linear peptides are completely de-
graded within minutes. This greater stability suggests
that the macrocycles should survive all proteolytic
conditions encountered during oral delivery, plasma
circulation, and cellular localization. The two crystal
structures reported here, together with others for this
class of inhibitor, also support the idea that nonpeptidic
units can be attached to the macrocycles without loss
of protease activity. Indeed this may be a useful means
of regulating antiviral activity, membrane permeability,
oral bioavailability, and pharmacokinetic and toxicologi-
cal properties of these drugs.
Our results show that the antiviral activity of these
macrocyclic peptidomimetics against HIV-1 involves a
mechanism characteristic of inhibition of HIV-1 PR. All
three compounds inhibit replication of HIV-1 to a
similar extent as does indinavir. Compound 2 was also
active in preventing replication of HIV-2, contrasting
with the poor activity of 1 against the same virus. In
light of the development of clinical resistance to HIV
PR inhibitors and the issue of cross-resistance, the
availability of novel inhibitors of HIV PR is becoming
increasingly important. Although not reported here, we
have had difficulty in generating resistance to 1 and 2
in vitro and are examining the efficacy of these drugs
against clinical isolates of HIV-1 known to be resistant
to HIV-1 PR inhibitors that have been approved for
human use. In view of its structural similarity to peptide
substrates, this macrocyclic class of HIV PR inhibitor
is very promising because HIV PR mutations that
reduce inhibitor affinity may also be expected to reduce
substrate processing.
Compound 3 was prepared and characterized as described
elsewhere.16
2. P r otea se In h ibition . [Aba67,95]HIV-1 PR (SF2 sequence)
was chemically synthesized by a solid-phase method as
described26 and is kinetically characterized elsewhere using a
fluorometric assay27 to quantify its activity. Cleavage of the
substrate 2-aminobenzoyl-Thr-Ile-Nle-(p-nitroPhe)-Gln-Arg-
NH2 releases a fluorescent product (2-aminobenzoyl-Thr-Ile-
Nle). Using appropriate buffer, substrate, ionic strength,
diluents and pH, protease inhibition was determined by
fluorescence using a J asco FP-770 spectrofluorimeter.
3. Ma cr ocycle Sta bility. Macrocycles 4 and 5 (50 µL of
1.0 mg in 1.0 mL of DMSO) were incubated for up to 24 h at
37 °C with 1 mL of aqueous buffer containing either pepsin,
gastricsin, trypsin or chymotrypsin (0.3 mg in 3 mL; pH 2, 3,
8, 8, respectively). Aliquots (0.3 mL) were sampled at 1, 4, 24
h and quenched with aqueous base (10 µL of 1 M NaOH) before
analysis by reversed-phase HPLC (50% acetontrile/50% water/
0.1% TFA for 30 min then 100% acetonitrile 5 min) versus
buffer alone. Fractions were monitored for peak identification
by electrospray MS. There was no evidence for decomposition
of the macrocycles under these conditions, whereas the linear
peptide was completely degraded within 5 min.
The compounds were also separately incubated with gastric
juice from stomachs of three Dark Agouti rats euthanized with
nembutal. The stomach and gut were removed and washed
with saline solution. A saline suspension of the GI tract was
macerated and refrigerated (-20 °C) overnight to lyse the cells
and release their contents. The solution was centrifuged (400g)
and the supernatant diluted 1:3 into separate aqueous buffers
(pH 2.2, glycine‚HCl; pH 4.0, Mes, acetic acid; pH 6.0, NaMES).
To each gastric/buffer solution (1 mL) was added 50 µL of
compound stock from above or control linear peptide, and after
incubation at 37 °C aliquots were taken at 1, 5, 24 h before
adding 10 µL of 1 M NaOH and analysis by rpHLC and MS
as above. Results indicate that both compounds were com-
pletely stable for at least 24 h in simulated conditions of the
digestive tract.
Blood (3 mL) was collected by vena puncture and layered
at room temperature onto a Ficoll density gradient solution
1077 (Histopaque-1077) in a ratio 1:1 in a heparinized 10-mL
conical centrifuge tube. Histopaque-1077 is a mixture of
polysucrose and sodium diatrizoate adjusted to a density of
1.077 ( 0.001 g/mL and facilitates rapid recovery of viable
mononuclear cells from blood. After centrifugation (400g, 30
min, 22 °C), the upper plasma layer was aspirated with a
Pasteur pipet to within 0.5 cm of the opaque interface
containing mononuclear cells and discarded. The opaque
interface was transferred to a clean tube, diluted with 10 mL
of phosphate-buffered saline (CaCl2 0.9 mM, MgCl2 0.5 mM,
Ma ter ia ls a n d Meth od s
1. In h ibitor Syn th esis a n d Ch a r a cter iza tion . (10S,-
13S,1′R)-13-[1′-H yd r oxy-2′-(N-p -a m in ob en zen esu lfon yl-
1′′-a m in o-3′′-m eth ylbu tyl)eth yl]-8,11-d ioxo-10-isop r op yl-
2-o x a -9,12-d i a z a b i c y c lo [13.2.2]n o n a d e c a -15,17,18-
tr ien e (1). The amine 7 and 4-acetamidobenzenesulfonyl
chloride were dissolved in THF:H2O (4:1) and diisopropylethy-
lamine was added (Scheme 1). After 10 min the solution was
diluted with EtOAc and washed with 2 M HCl. The solvent
was evaporated and the residue was dissolved in MeOH and
2 M HCl and refluxed for 2 h. The solution was basified with
10% K2CO3 and extracted with EtOAc. The EtOAc extracts
were washed with brine and dried over MgSO4 and evaporated.
The residue was purified by flash chromatography (SiO2, 100%
EtOAc) giving 1 as a white powder: Rf 0.42 (100% EtOAc);
1H NMR (300 MHz, CD3OH) δ 7.92 (d, J ) 9.7 Hz, 1H, Tyr-
NH), 7.51 (m, 2H, J AX + J AX′ ) 8.7 Hz, ortho to SO2), 7.19 (d,
J ) 9.2 Hz, 1H, Val-NH), 7.07 (m, 2H, J AX + J AX′ ) 8.5 Hz,
ortho to CH2), 6.77 (m, 2H, J AX + J AX′ ) 8.5 Hz, ortho to O),
6.73 (m, 2H, J AX + J AX′ ) 8.7 Hz, ortho to NH2), 4.27-4.05
(m, 3H, Tyr-RH and H-3), 4.07 (dd, J ) 9.2, 6.0 Hz, 1H, Val-
RH), 3.75 (m, 1H, H-1′), 3.40 (dd, J ) 14.3, 4.0 Hz, H-2′), 3.35-
3.02 (m, 2H, H-4′), 3.18 (dd, J ) 13.6, 3.7 Hz, 1H, H-14), 2.93
(dd, J ) 14.3, 8.6 Hz, H-2′), 2.41 (dd, J ) 13.2, 12.5 Hz, 1H,
H-14), 2.20-2.11 (m, 2H, H-7), 1.86 (m, 1H, Val-âCH), 1.75-
1.60 (m, 2H), 1.60-1.10 (m, 7H), 0.87 (d, J ) 6.4 Hz, 9H, Val-
γCH3, H-7′, H-8′), 0.76 (d, J ) 6.8 Hz, 3H, Val-γCH3); 13C NMR
(CD3OD) δ 174.8, 172.6, 158.3, 154.3, 131.4, 130.3, 117.4, 114.5,
74.2, 68.8, 58.6, 55.0, 53.1, 38.2, 36.1, 36.0, 33.5, 30.7, 27.1,
26.1, 22.9, 22.8, 20.0, 18.2; HRMS m/e 616.3285 calcd for
C
32H48N4O6S 616.3295.
N -13-[(10S ,13S )-9,12-Dioxo-10-(2-b u t yl)-2-oxa -8,11-
d ia za bicyclo[13.2.2]n on a d eca -15,17,18-tr ien yl]-(2R)-ben -
zyl-(4S)-h yd r oxy-5-a m in op en t a n oic-(1R)-h yd r oxy-(2S)-

3502 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Tyndall et al.
KCl 2.7 mM, KH2PO4 1.5 mM, NaCl 136.9 mM, Na2HPO4 3.2
mM), and aspirated by pipet. The solution was centrifuged
(250g, 10 min), the supernatant discarded, the cell pellet
resuspended in PBS (5 mL), and this washing procedure twice
repeated. Using a Rapid Dif Kit, a thin film of cell solution
was smeared onto microscope slides and treating with MeOH
(5× to fix the cells), xanthene dye (5×), thiazene dye (1×),
before examined cell types by microscopy. Cells were then
successfully lysed by sonication (3 × 18 s) and centrifuged
(2000g, 15 min) and the supernatant was collected.
PMNs were prepared from the original centrifugation with
Histopaque-1077; the bottom layer containing the red cells was
removed and exposed to hypotonic shock with cold distilled
water (40s) to lyse the red blood cells, after which tonicity was
restored with 10 N PBS. Cells were centrifuged (700g, 20 min)
and the pellet was again exposed to hypotonic shock to remove
residual red cells. Tonicity was restored, cells were centrifuged
(700g, 20 min) and the pellet was resuspended in PBS. The
cells were then lysed by sonication (3 × 18 s) and centrifuged
and the supernatant was collected.
The lymphocyte and PMN solutions (1 mL) were incubated
with 4, 5 or control peptides (50 µL of 1 mg/mL DMSO stock
solution) at 37 °C for 24 h and aliquots taken at 1, 24 h for
analysis by RP HPLC and ESMS. Human serum and test
compounds were similarly incubated and after 1 h the serum
proteins were precipitated by addition of cold acetonitrile (1
mL). The samples were vortexed (30 s) and centrifuged
(10000g, 5min), and the supernatant was analyzed as above.
4. Vir ology. Cells: Cord blood mononuclear cells (CBMC)
were isolated by centrifugation of heparinized umbilical cord
blood (Royal Brisbane Hospital) over Ficoll-paque. Phytohe-
magglutinin (PHA)-stimulated CBMC were prepared by cul-
ture of CBMC in RPMI 1640 supplemented with 20% heat-
inactivated fetal calf serum (FCS), 2 mM glutamine, 100 U/mL
penicillin and 100 µg/mL streptomycin (20FC/RPMI), with the
addition of 0.2% (v/v) PHA for 3 days. PHA-CBMC were
washed twice with 20FC/RPMI immediately prior to use.
HIV-1 strain TC354 was propagated in PHA-CBMC in 20FC/
RPMI supplemented with 20 U/mL recombinant interleukin-
2.
MT2 and H9/HTLV-IIIB cells were obtained from the
National Institutes of Health AIDS Research and Reference
Program. PBMCs from individuals not infected with HIV were
obtained from blood packs supplied by the Australian Red
Cross Blood Bank, Melbourne. The medium used for passage
and maintenance of MT2 and H9/HTLV-IIIB cells was RF10,
an RPMI-1640-based medium.28 RF10 supplemented with
interleukin-2 (RF10/IL-2)28 was used for experiments involving
the use of PBMCs.
An t i-HIV a ct ivit y of 1-3 in a cu t ely in fect ed CBMC
lym p h ocytes: In these Brisbane studies PHA-CBMC were
inoculated with HIV-1 (TC354) in the presence of 20 µg/mL
diethylaminoethyl-dextran (DEAE-dextran) at a multiplicity
of infection of 1 and allowed to adsorb for 2 h at 37 °C.
Uninoculated PHA-CBMC were held as negative controls.
Unbound virus was removed by washing three times with
20FC/RPMI. 105 Cells were placed in microtiter wells to which
the appropriate dilutions of each test compound (in DMSO)
or DMSO controls were added in triplicate. Culture medium
(20FC/RPMI supplemented with 20 U/mL rIL-2) was added
to make up the volume to 200 µL. Cultures were placed in an
incubator at 37 °C (5% CO2) for 3 days.
Virus replication was assessed by monitoring Day 3/4
culture supernatant for the presence of reverse transcriptase
activity. 50 µL of reaction cocktail (20 µCi/mL 3H-thymidine
triphosphate, 50 mM Tris, ph 7.8, 75 mM KCl, 5 mM MgCl2,
2 mM dithioreitol (DTT), 5 µg/mL poly-rA, 80 µg/mL dATP,
1.5 ng/mL oligo-dT, 0.05% Triton X-100) and 10 µL of culture
supernatant were added to the appropriate wells of a micro-
titer plate and mixed gently and the plate was incubated (37
°C, 2 h). DE81 paper was marked with a labeled grid of
rectangles measuring 0.9 cm × 3 cm and 10 µL of reaction
mixture from each well was carefully spotted onto the corre-
sponding rectangle on the paper and allowed to dry. The paper
was washed with agitation (4 × 5 min in 2× SSC) at room
temperature, washed twice with EtOH (5 min), and dried.
Rectangles were cut out and placed into scintillation files, 3
mL of scintillation fluid was added to each tube and counts
were made for 1 min on a â counter (LKB Rackbeta), counting
was controlled by a computer program GENTERM which
averaged the mean of the control reaction cocktail blanks and
subtracted this background from the test specimen counts.
Corrected results were given as counts/minute and a factor of
600 was used to adjust results to cpm/mL. Based on sigmoidal
plots of RT activity (cpm) versus inhibitor concentration, the
IC50 values were determined for each compound.
An ti-HIV a ctivity of 1 a n d 2 in a cu tely in fected MT2
or P BMC lym p h ocytes: In these Melbourne studies the
degree of inhibition of HIV-1 and HIV-2 by 1 and 2 in acutely
infected MT2 cells or PBMCs was determined by measuring
the extent by which they reduced HIV-specific cytopathic
effects (CPE) at noncytotoxic concentrations. This inhibition
was confirmed by measurement of virion-associated reverse
transcriptase (RT) activity in cell supernatants. All tests were
performed in 48-well tissue culture plates (Costar). Each drug
concentration and all controls were tested in duplicate. Virus
inoculum was added immediately after the addition of the
drugs to the cells.
MT2 cells or PBMCs were counted and resuspended at a
concentration of 5 × 104/0.25 mL of RF10 medium for MT2-
based assays or 3.75 × 105/0.25 mL of RF10/IL-2 medium for
PBMC-based assays. Compounds 1 and 2 dilutions at twice
the final concentration required in the test were prepared in
the appropriate medium from a 20 mM stock of the drugs
prepared in 60% v/v ethanol. 0.5 mL of each dilution was
pipetted into duplicate test and cytotoxicity control wells,
followed by the addition of 0.25 mL of cell suspension to all
wells. 0.5 mL of RF10 or RF10/IL-2 medium was added to the
virus-only controls, 0.75 mL to the cell-only controls and 0.25
mL to the cytotoxicity controls. A volume of 0.25 mL of strain
#237288 or HIV-2ROD diluted in the appropriate medium to
contain 200 TCID50 (for MT2 assays) or 300 TCID50 (PBMC
assays) was then immediately added to virus-only control wells
and the test wells. The plates were then incubated at 37 °C in
5% CO2. All wells in the test were examined by light micros-
copy and harvested after 6-7 days incubation, at which time
viable cell numbers were determined in cell-only and cytotox-
icity control wells using trypan blue dye exclusion counting.
Drug dilutions producing cell counts that were less than 90%
of the cell-only control were considered to have some degree
of toxicity and excluded from calculation of the selective index
(SI) of the drug (see below).
The cell-only, virus-only and test cultures were harvested
by removing 0.8 mL of supernatant from each well to separate
microtubes. The virus present in each supernate was then
precipitated using poly(ethylene glycol) and assayed for virion-
associated RT activity expressed as counts/minute (cpm) on a
liquid scintillation counter as previously described.29 The
percentage (%) inhibition associated with 1 and 2 was calcu-
lated by expressing the RT activity at each dilution as a % of
the cpm in virus-only controls. The % inhibition was this value
subtracted from 100. The SI, which was used as a measure of
the relative antiviral activity of 1 and 2, was calculated using
the formula SI ) highest noncytotoxic drug concentration
(CC10) divided by the lowest inhibitory concentration (the IC90
,
defined as the drug concentration producing 90% inhibition
of virion-associated RT activity in drug-treated, infected cells
relative to infected, untreated cells).
An ti-HIV a ctivity of 1 a n d 2 in lym p h ocytes ch r on i-
ca lly in fected w ith HIV-1: In these Melbourne studies H9/
HTLV-IIIB cells were incubated in the presence of either 10
µM 1 or 2 or in the absence of drug at 37 °C for 72 h.
Supernatant fluid was then removed, clarified at 700g for 10
min and virions pelleted by ultracentrifugation at 160000g for
1 h at 4 °C in an SW41 rotor using a Beckman L8 ultracen-
trifuge. Virus pellets were resuspended in saline, then 5×
Laemmli sample buffer30 was added and the samples were
stored at -70 °C. For immunoblotting, samples were electro-

Constrained Macrocyclic Inhibitors of HIV PR
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3503
phoresed through 10% polyacrylamide gels and transferred to
nitrocellulose. After blocking with 3% casein in TBST (50 mM
Tris-HCl, pH 7.5, 50 mM NaCl, 0.5% v/v Tween 20) overnight
at 4 °C, the blot was stained with a 1 in 1000 dilution of
monoclonal antibodies specific for HIV Pr55gag and p24 (re-
agent ADP313, kindly supplied by Drs. Ferns and Tedder,
British Medical Research Council AIDS Reagent Project) for
2 h at room temperature, washed four times with TBST, then
incubated for 1 h at room temperature with a 1 in 2000 dilution
of anti-mouse IgG conjugated to horseradish peroxidase.
Protein bands were detected by chemiluminescence (ECL
Western Blot Analysis System, Amersham).
lian Research Council. J .L.M. is an ARC Senior Re-
search Fellow.
Refer en ces
(1) Wlodawer, A.; Erickson, J . W. Structure-based inhibitors of
HIV-1 protease. Annu. Rev. Biochem. 1993, 62, 543-85.
(2) Darke, P. L.; Huff, J . R. HIV protease as an inhibitor target for
the treatment of AIDS. Adv. Pharmacol. 1994, 25, 399-454.
(3) West, M. L.; Fairlie, D. P. Targeting HIV-1 protease: a test of
drug-design methodologies. Trends Pharmacol. Sci. 1995, 16,
67-75.
(4) Kempf, D. J .; Sham, H. L. HIV protease inhibitors. Curr. Pharm.
Des. 1996, 2, 225-46.
(5) March, D. R.; Fairlie, D. P. Designing new antiviral drugs for
AIDS: HIV-1 Protease and its inhibitors; Wise, R., Ed.; R. G.
Landes Co.: Austin, TX, 1996.
5. Cr ysta llogr a p h y. Inhibitors 1 and 2 were cocrystallized
with synthetic HIV PR. The enzyme was mutated for ease of
synthesis and preparation22 (mutations Q7K, L33I, C37Aba,
and C95Aba, where Aba ) R-aminobutyric acid). Crystalliza-
tion was by the hanging drop-vapor diffusion method. The
protein was concentrated to 5 mg/mL and a solution of the
inhibitor in DMSO was diluted into the protein sample to give
an inhibitor:protein molar ratio of 10:1. Protein-inhibitor
mixtures were incubated at 4 °C for 24 h prior to crystalliza-
tion. Reservoir conditions were 0.1 M acetate buffer (pH 5.5)
and 35-60% saturated ammonium sulfate. 2-5-µL drops were
incubated at 20 °C. Rod shaped crystals appeared within a
week and grew over 2-6 weeks.
Da ta collection a n d d a ta p r ocessin g: Crystallographic
data were measured at 16 °C using an R-AXIS IIC imaging
plate system with Cu KR X-radiation generated from a Rigaku
RU-200 rotating anode generator. Data collection parameters
were similar to those described previously.22 Diffraction data
were integrated and scaled using DENZO and SCALEPACK.31
Statistics from data processing are given in Table 1.
(6) Wlodawer, A.; Vondrasek, J . Inhibitors of HIV-1 protease:
a
major success of structure-assisted drug design. Annu. Rev.
Biophys. Biomol. Struct. 1998, 27, 249-84.
(7) Misson, J .; Clark, W.; Kendall, M. J . Therapeutic advances:
protease inhibitors for the treatment of HIV-1 infection. J . Clin.
Pharm. Ther. 1997, 22, 109-17.
(8) Hahn, B. H. Viral genes and their products. In Textbook of AIDS
Medicine; Broder, S., Merigan, T. C., Bolognesi, D., Eds.;
Williams and Wilkins: Baltimore, MD, 1994; pp 21-43.
(9) Mildner, A. M.; Rothrock, D. J .; Leone, J . W.; Bannow, C. A.;
Lull, J . M.; Reardon, I. M.; Sarcich, J . L.; Howe, W. J .; Tomich,
C. S.; Smith, C. W.; et al. The HIV-1 protease as enzyme and
substrate: mutagenesis of autolysis sites and generation of a
stable mutant with retained kinetic properties. Biochemistry
1994, 33, 9405-13.
(10) Bukrinsky, M.; Haggerty, S.; Dempsey, M. P.; Sharova, N.;
Adhubei, A.; Spitz, P.; Goldfarb, D.; Emerman, M.; Stevenson,
M. A nuclear localization signal within HIV-1 matrix protein
that governs infection of nondividing cells. J . Virol. 1993, 67,
6387-94.
(11) Palella, F.; Delaney, K. M.; Moorman, A. C.; Loveless, M. O.;
Fuhrer, J .; Satten, G. A.; Aschman, D. J .; Holmberg, S. D.
Declining morbidity and mortality among patients with ad-
vanced human immunodeficiency virus infection. N. Engl. J .
Med. 1998, 338, 853-60.
(12) Hammer, S. M.; Squires, K. E.; Hughes, M. D.; Grimes, J . M.;
Demeter, L. M.; Currier, J . S.; Eron, J . J .; Feinberg, J . E.;
Balfour, H. H.; Deyton, L. R.; Chodakewitz, J . A.; Fischl, M. A.
A controlled trial of two nucleoside analogues plus indinavir in
persons with human immunodeficiency virus infection and CD4
cell counts of 200 per cubic millimeter or less. N. Engl. J . Med.
1997, 337, 725-33.
(13) Condra, J .; Schlief, W.; Blahy, O.; Gabryelski, L.; Graham, D.;
Quintero, J .; Rhodes, A.; Robbins, H.; Roth, E.; Shivaprakash,
M.; Titus, E.; Yang, T.; Teppler, H.; Squires, K.; Deutsch, P.;
Emini, E. In vivo emergence of HIV-1 variants resistant to
multiple protease inhibitors. Nature 1995, 375, 569-71.
(14) Palmer, S.; Schafer, R. W.; Merigan, T. C. Highly drug-resistant
HIV-1 clinical isolates are cross-resistant to many antiretroviral
compounds in current clinical development. AIDS 1999, 13, 661-
7.
(15) Abbenante, G.; March, D.; Bergman, D.; Hunt, P. A.; Garnham,
B.; Dancer, R. J .; Martin, J . L.; Fairlie, D. P. Regioselective
Structural and Functional Mimicry Of Peptides: Design Of
Hydrolytically Stable Cyclic Peptidomimetic Inhibitors Of Hiv
1 Protease. J . Am. Chem. Soc. 1995, 117, 10220-6.
(16) March, D.; Abbenante, G.; Bergman, D.; Brinkworth, R. I.;
Wickramasinghe, W.; Begun, J .; Martin, J . L.; Fairlie, D. P.
Substrate Based Cyclic Peptidomimetics Of Phe Ile Val That
Inhibit Hiv 1 Protease Using a Novel Enzyme Binding Mode. J .
Am. Chem. Soc. 1996, 118, 3375-9.
(17) Reid, R. C.; March, D. R.; Dooley, M. J .; Bergman, D. A.;
Abbenante, G.; Fairlie, D. P. A novel bicyclic enzyme inhibitor
as a consensus peptidomimetic for the receptor-bound conforma-
tions of 12 peptidic inhibitors of HIV-1 protease. J . Am. Chem.
Soc. 1996, 118, 8511-7.
(18) Kim, E. E.; Baker, C. T.; Dwyer, M. D.; Murcko, M. A.; Rao, B.
G.; Tung, R. D.; Navia, M. A. Crystal Structure of HIV-1 protease
in complex with VX-478, a Potent and Orally Bioavailable
Inhibitor of the enzyme J . Am. Chem. Soc. 1995, 117, 1181-2.
(19) Chen, Z.; Li, Y.; Chen, E.; Hall, D. L.; Darke, P. L.; Culberson,
C.; Shafer, J . A.; Kuo, L. C. Crystal structure at 1.9-A resolution
of human immunodeficiency virus (HIV) II protease complexed
with L-735,524, an orally bioavailable inhibitor of the HIV
proteases. J . Biol. Chem. 1994, 269, 26344-8.
Str u ctu r e r efin em en t: The two crystal structures were
solved by difference Fourier analysis. The starting model was
the HIV-1 PR structure from an isomorphous inhibitor complex
(PDB code: 1cpi)15,22 with the inhibitor and solvent molecules
removed. X-PLOR 3.85132 was used for refinement. Cross-
validation was performed using R-free from 10% of the
reflections.33 Simulated annealing molecular dynamics was
performed (temperature ) 3000 K), to decouple R and R-free.
Both positional and individual B-factor refinements were
carried out for several rounds. Protein modeling and rebuilding
was performed using O.34 Parameters for protein refinement
were those of Engh and Huber.35 Water molecules were added
where peaks were found in both |Fo| - |Fc| (3σ) and 2|Fo| -
|Fc| (1σ) maps and where at least one stereochemically reason-
able hydrogen bond could be formed. Parameters for the
inhibitors were based on equivalent features of peptides
modified accordingly, e.g. parameters for the indanyl moiety,
2, were based on the ring structure of tryptophan. A low
resolution cutoff of 8.0 Å and structure factor amplitude cutoff
of 0 σF were used during refinement. Refinement statistics
are given in Table 2.
In a number of cases where there is poor density for protein
side chains, residues have been modeled as alanine or with
half-occupancy for side chain atoms. A further group of protein
residues have been modeled with two alternative conforma-
tions. The majority of these residues are on the surface of the
protein. In addition the enzyme main chain atoms of residues
Ile150 and Gly150 (located in the flap region above the active
site) have been modeled with two alternate conformations.
Parts of both inhibitors have also been modeled with either
half-occupancy or two conformations including the isosteric
hydroxyl of compound 2.
The programs PROCHECK,36 WHATCHECK37 and In-
sightII 97.0 (MSI) were used. Figure 2 was generated using
SETOR.38 Atomic coordinates and structure factors have been
deposited in the Protein Data Bank39 and HIV data bank41
(accession codes: 1d4l (1), 1d4k (2)).
(20) Krohn, A.; Redshaw, S.; Ritchie, J . C.; Graves, B. J .; Hatada,
M. H. Novel binding mode of highly potent HIV-proteinase
inhibitors incorporating the (R)-hydroxyethylamine isostere. J .
Med. Chem. 1991, 34, 3340-2.
Ack n ow led gm en t. We gratefully acknowledge par-
tial financial support from the National Health and
Medical Research Council of Australia and the Austra-

3504 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Tyndall et al.
(21) Reid, R. C.; Scanlon, M. J .; Kelso, M. J .; Fairlie, D. P. Synthesis
of Conformationally Restrained Cyclic Mimetics Of HIV-1 Pro-
tease Substrates. Templates for Combinatorial Development of
Protease Inhibitors. J . Org. Chem. 2000, submitted for publica-
tion.
(22) Martin, J . L.; Begun, J .; Schindeler, A.; Wickramasinghe, W.
A.; Alewood, D.; Alewood, P. F.; Bergman, D. A.; Brinkworth,
R. I.; Abbenante, G.; March, D.; Reid, R. C.; Hunt, P. A.; Fairlie,
D. P. Molecular Recognition of Macrocyclic Peptidomimetic
inhibitors by HIV-1 Protease. Biochemistry 1999, 38, 7978-88.
(23) Rose, J . R.; Salto, R.; Craik, C. S. Regulation of autoproteolysis
of the HIV-1 and HIV-2 proteases with engineered amino acid
substitutions. J . Biol. Chem. 1993, 268, 11939-45.
(32) Bru¨nger, A. T. X-PLOR (Version 3.1) Manual; Yale University:
New Haven, CT, 1992.
(33) Bru¨nger, A. T. Assessment of phase accuracy by cross valida-
tion: the free R value. Methods and applications. Acta Crystal-
logr. D 1993, D46, 24-36.
(34) J ones, T. A.; Zou, J . Y.; Cowan, S. W.; Kjeldgaard, M. Improved
methods for building protein models in electron density maps
and the location of errors in these models. Acta Crystallogr. A
1991, 47, 110-9.
(35) Engh, R. A.; Huber, R. Accurate bond and angle parameters for
X-ray protein structure refinement. Acta Crystallogr. 1991, A47,
392-400.
(24) Swain, A. L.; Miller, M. M.; Green, J .; Rich, D. H.; Schneider,
J .; Kent, S. B.; Wlodawer, A. X-ray crystallographic structure
of a complex between a synthetic protease of human immuno-
deficiency virus 1 and a substrate-based hydroxyethylamine
inhibitor. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 8805-9.
(25) Tyndall, J . D. A.; Fairlie, D. P. Conformational Homogeneity in
Molecular Recognition by Proteolytic Enzymes. J . Mol. Recognit.
1999, 12, 1-8.
(36) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J .
M. PROCHECK: A program to check the stereochemical quality
of protein structures. J . Appl. Crystallogr. 1993, 26, 283-91.
(37) Hooft, R. W. W.; Vriend, G.; Sander, C.; Abola., E. E.
WHAT_CHECK (verification routines from WHAT IF). Errors
in protein structures. Nature 1996, 381, 272.
(38) Evans, S. V. SETOR: hardware-lighted three-dimensional solid
model representations of macromolecules. J . Mol. Graph. 1993,
11, 134-8, 127-8.
(39) Berman, H. M.; Westbrook, J .; Feng, Z.; Gilliand, G.; Bhat, T.
N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data
Bank. Nucleic Acids Res. 2000, 28, 235-242.
(40) Fairlie, D. P.; Tyndall, J . D. A.; Reid, R. C.; Wong, A. K.;
Abbenante, G.; Scanlon, M. J .; March, D. R.; Bergman, D. A.;
Chai, C. L. L.; Burkett, B. A. Conformational Selection of
Inhibitors and Substrates by Proteolytic Enzymes: Implications
for Drug Design and Polypeptide Processing. J . Med. Chem.
2000, 43, 1271-1281.
(26) Schneider, J .; Kent, S. B. Enzymatic activity of a synthetic 99
residue protein corresponding to the putative HIV-1 protease.
Cell 1988, 54, 363-8.
(27) Bergman, D. A.; Alewood, D.; Alewood, P. F.; Andrews, J . L.;
Brinkworth, R. I.; Englebretsen, D. R.; Kent, S. B. H. Kinetic-
properties of HIV-1 Protease Produced by Total Chemical
Synthesis with Cysteine Residues Replaced by Isosteric L-alpha-
amino-n-butyric Acid. Lett. Pept. Sci. 1995, 2, 99-107.
(28) Tachedjian, G.; Tyssen, D.; Locarnini, S.; Gust, I.; Birch, C.
Investigation of topoisomerase inhibitors for activity against
human immunodeficiency virus: inhibition by coumermycin A1.
Antivir. Chem. Chemother. 1990, 1, 131-8.
(29) Neate, E. V.; Pringle, R. C.; J owett, J . B. M.; Healey, D. S.; Gust,
I. D. Isolation of HIV from Australian patients with AIDS, AIDS-
related conditions and healthy antibody positive individuals.
Aust. New Zealand J . Med. 1987, 17, 461-6.
(30) Laemmli, U. K. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 1970, 227,
680-5.
(31) Otwinski, Z.; Minor, W. Processing of X-ray diffraction data
collected in oscillation mode. Methods Enzymol. 1997, 276, 307-
26.
(41) Vondrasek, J .; van Buskirk, C. P.; Wlodawer, A. Database of
three-dimensional structures of HIV proteinases Nat. Struct.
Biol. 1997, 4, 8.
(42) Luzzati, V. Traitment statistique des erreurs dans la determi-
nation des structures cristallines. Acta Crystallogr. 1952, 5,
802-810.
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