HIV-1 Protease Inhibitors Incorporating Stereochemically Defined P2′ Ligands to Optimize Hydrogen Bonding in the Substrate Envelope

Article abstract of DOI:10.1021/acs.jmedchem.9b00838

A structure-guided design strategy was used to improve the resistance profile of HIV-1 protease inhibitors by optimizing hydrogen bonding and van der Waals interactions with the protease while staying within the substrate envelope. Stereoisomers of 4-(1-hydroxyethyl)benzene and 4-(1,2-dihydroxyethyl)benzene moieties were explored as P2′ ligands providing pairs of diastereoisomers epimeric at P2′, which exhibited distinct potency profiles depending on the configuration of the hydroxyl group and size of the P1′ group. While compounds with the 4-(1-hydroxyethyl)benzene P2′ moiety maintained excellent antiviral potency against a panel of multidrug-resistant HIV-1 strains, analogues with the polar 4-(1,2-dihydroxyethyl)benzene moiety were less potent, and only the (R)-epimer incorporating a larger 2-ethylbutyl P1′ group showed improved potency. Crystal structures of protease-inhibitor complexes revealed strong hydrogen bonding interactions of both (R)- and (S)-stereoisomers of the hydroxyethyl group with Asp30′. Notably, the (R)-dihydroxyethyl group was involved in a unique pattern of direct hydrogen bonding interactions with the backbone amides of Asp29′ and Asp30′. The SAR data and analysis of crystal structures provide insights for optimizing these promising HIV-1 protease inhibitors.

Full text of DOI:10.1021/acs.jmedchem.9b00838

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Article
HIV-1 Protease Inhibitors Incorporating Stereochemically Defined P2#
Ligands to Optimize Hydrogen Bonding in the Substrate Envelope
Linah N Rusere, Gordon J. Lockbaum, Sook-Kyung Lee, Mina Henes, Klajdi Kosovrasti, Ean
Spielvogel, Ellen A. Nalivaika, Ronald Swanstrom, Nese Kurt Yilmaz, Celia A. Schiffer, and Akbar Ali
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00838 • Publication Date (Web): 06 Aug 2019
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HIV-1 Protease Inhibitors Incorporating
Stereochemically Defined P2 Ligands to Optimize
Hydrogen Bonding in the Substrate Envelope
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Linah N. Rusere,†,§ Gordon J. Lockbaum,†,§ Sook-Kyung Lee,‡ Mina
Henes,† Klajdi Kosovrasti,† Ean Spielvogel,‡ Ellen A.
Nalivaika,† Ronald Swanstrom,‡ Nese Kurt Yilmaz,† Celia A.
Schiffer,†,* and Akbar Ali†,*
†Department of Biochemistry and Molecular Pharmacology,
University of Massachusetts Medical School, Worcester,
Massachusetts 01605, United States
‡Department of Biochemistry and Biophysics, and the UNC Center
for AIDS Research, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599, United States
ABSTRACT
A
structure-guided design strategy was used to improve the
resistance profile of HIV-1 protease inhibitors by optimizing
hydrogen bonding and van der Waals interactions with the protease
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while staying within the substrate envelope. Stereoisomers of 4-
(1-hydroxyethyl)benzene
and
4-(1,2-dihydroxyethyl)benzene
moieties were explored as P2 ligands providing pairs of
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diastereoisomers epimeric at P2, which exhibited distinct potency
profiles depending on the configuration of the hydroxyl group and
size of the P1 group. While compounds with the 4-(1-
hydroxyethyl)benzene P2 moiety maintained excellent antiviral
potency against a panel of multidrug-resistant HIV-1 strains,
analogues with the polar 4-(1,2-dihydroxyethyl)benzene moiety were
less potent, and only the (R)-epimer incorporating a larger 2-
ethylbutyl P1 group showed improved potency. Crystal structures
of protease-inhibitor complexes revealed strong hydrogen bonding
interactions
of
both
(R)-
and
(S)-stereoisomers
of
the
hydroxyethyl group with Asp30. Notably, the (R)-dihydroxyethyl
group was involved in a unique pattern of direct hydrogen bonding
interactions with the backbone amides of Asp29 and Asp30. The SAR
data and analysis of crystal structures provide insights for
optimizing these promising HIV-1 protease inhibitors.
INTRODUCTION
The HIV-1 protease is a major target for developing antiviral
therapies against HIV-1. Drug discovery efforts, aided by
structure-based drug design, have led to the development of nine
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FDA-approved HIV-1 protease inhibitors (PIs). All approved HIV-1
PIs are competitive inhibitors, and most contain different
2
dipeptide isosteres as transition state mimetics. In addition to
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extensive hydrophobic interactions, PIs mainly rely on a number of
direct and water-mediated hydrogen bonds with the protease for
potency. The clinical efficacy of HIV-1 PIs has significantly
improved since this drug class was first introduced in the mid-
1
990s. The development of second-generation PIs with improved
potency, tolerability, and pharmacokinetic profiles, and the
introduction of low-dose ritonavir as a pharmacokinetic booster in
PI-based antiretroviral therapy (ART) has led to improved clinical
outcomes. Moreover, PIs allow exceedingly high level of viral
inhibition at clinical concentrations due to cooperative dose-
response curves with high slopes.3,4 These transition-state mimetic
inhibitors represent the most potent anti-HIV-1 drugs.
Despite much success, PI-based therapies are associated with
drawbacks that limit their effectiveness, including major side
effects, unfavorable pharmacokinetics, and the acquisition of many
variants.5,6
viable
multidrug-resistant
(MDR)
protease
Drug
resistance remains
a
major issue, as the prevalence of PI
resistance mutations increases with duration of ART and is much
higher among PI-experienced patients.7,8 Even the most effective PI-
based regimens have been reported to select multiple resistance
mutations in the protease, resulting in reduced virologic
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response.7-10
Moreover,
the
prevalence
of
transmitted
drug
resistance continues to increase, which is an added challenge in
the treatment of HIV infections.11,12 The emergence of MDR protease
variants presents a challenge to structure-based drug design
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(SBDD), as the target is not a single protein but an ensemble of
closely related proteases. The new PIs must maintain activity not
only against existing MDR protease variants, but also against
future variants that may emerge. These challenges require
developing new SBDD strategies to optimize PI potency while
avoiding resistance.
The design strategy to maximize interactions in the HIV-1
protease active site, particularly with the protein backbone
atoms, has been quite successful leading to the development of the
FDA-approved drug darunavir.13,14 Darunavir (DRV, 1) is a highly
potent inhibitor of HIV-1 protease with a low pM inhibition
constant (Ki) and a high genetic barrier to resistance. The key
structural feature that distinguishes DRV from previous generation
HIV-1 PIs is the bis-tetrahydrofuran (bis-THF) moiety at the P2
position
(Figure
1)
that
makes
strong
hydrogen
bonding
interactions with the backbone NH of Asp29 and Asp30 in the
protease active site.1,13,15 The application of the design strategy
to maximize interactions with the backbone atoms of HIV-1 protease
has recently led to the discovery of DRV analogues that maintain
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exceptional potency against MDR HIV-1 variants including DRV-
resistant strains.16-19
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Figure 1. Structures of HIV protease inhibitors DRV (1), P2 4-(1-
hydroxymethyl)benzene analogues (2–4) and designed compounds with
stereochemically defined P2 4-(1-hydroxyethyl)benzene and (12–17)
and 4-(1,2-dihydroxyethyl)benzene (22–27) moieties. The canonical
nomenclature for inhibitor moiety position is indicated using DRV.
Among the strategies proposed to rationally design PIs against
drug-resistant HIV-1 variants, the substrate envelope model
provides a more comprehensive framework to incorporate drug
resistance considerations into SBDD.20-22 This structure-guided
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design strategy aims to optimize hydrogen bonding and van der Waals
(vdW) interactions with the protease to improve potency against
MDR protease variants, while constraining inhibitors within the
substrate envelope to avoid resistance.23 We previously used the
substrate envelope-guided design strategy to develop a series of
highly potent DRV analogues that maintained excellent antiviral potencies against
a panel of clinically relevant MDR HIV-1 strains.23 The X-ray
cocrystal structures confirmed that the designed PIs optimally
fill the substrate envelope and make enhanced vdW contacts and
hydrogen bonding interactions with HIV-1 protease.23,24
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Among these PIs, compounds 3 and 4 (Figure 1) with the 4-
(hydroxymethyl)benzene P2 moiety were identified as promising lead
compounds due to distinct polar interactions of the benzylic
hydroxyl group with the protease. Cocrystal structures of PIs 3
and 4 bound to wild-type HIV-1 protease revealed that the P2
benzylic hydroxyl group is positioned close to the backbone NH of
Asp29 and Asp30 in the S2 subsite. The hydroxyl group makes a
direct hydrogen bond only with the backbone NH of Asp30, which is
slightly closer than the NH of Asp29 (Figure S1).23,24 We reasoned
that introduction of a small hydrophobic group such as a methyl at
the benzylic position could shift the resulting secondary hydroxyl
group closer to the backbone NH of Asp29, allowing polar
interactions with both residues, as well as additional vdW contacts
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in the S2 subsite. Similarly, a hydroxymethyl group at the benzylic
position could serve the same purpose, with the primary hydroxyl
group making additional polar interactions in the S2 subsite of
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HIV-1 protease (Figure 1). Optimal polar interactions of the P2
moiety with the backbone NH of Asp29 and Asp30 could potentially
mimic the strong hydrogen bonding interaction of the P2 bis-THF
moiety, further improving potency against MDR HIV-1 variants.
Previous efforts to identify an optimal P2 moiety have only been
partially successful, and none of the reported P2 moieties make
direct hydrogen bonding interactions with the backbone atoms of
both Asp29 and Asp30.16,25-27
On the basis of insights from the structural analysis and
modeling, modifications of the P2 moiety of PIs 3 and 4, in
combination with variations at the P1 group, were explored to
enhance inhibitor interactions in the S1 and S2 subsites. Here,
we describe the substrate envelope-guided design, synthesis,
evaluation of biochemical and antiviral potency, and crystal
structure analysis of a series of novel HIV-1 PIs. The inhibitors
were designed by incorporating stereoisomers of the 4-(1-
hydroxyethyl)benzene and 4-(1,2-dihydroxyethyl)benzene moieties
as novel P2 ligands to enhance hydrogen bonding interactions in
the S2 subsite of HIV-1 protease. Structure-activity relationship
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(
SAR) studies identified a number of compounds with improved
potency profiles compared to DRV and the parent compounds against
highly drug-resistant HIV-1 strains representing the spectrum of
clinically relevant MDR viruses. We also report high-resolution
crystal structures of all new compounds bound to wild-type HIV-1
protease revealing key interactions of the chiral 4-(1-
hydroxyethyl)benzene and 4-(1,2-dihydroxyethyl)benzene moieties
in the protease active site. A unique pattern of hydrogen bonding
interactions was observed for compounds incorporating the (R)-4-
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(1,2-dihydroxyethyl)benzene as P2 ligand, where both P2 and P2
moieties make direct hydrogen bonds with the backbone NH of
residues Asp29/29 and Asp30/30.
CHEMISTRY
The synthesis of HIV-1 PIs with the (S)- and (R)-(1-
hydroxyethyl)benzene moieties at the P2 position is outlined in
Scheme 1. We envisioned generating both stereoisomers of the
hydroxyethyl group at the P2 moiety from the corresponding 4-
acetylbenzene derivatives by enantioselective reduction of the
acetyl group using the Corey–Bakshi–Shibata (CBS) catalyst.28 The
4-acetylbenzenesulfonamide intermediates 8a–c were prepared from
the commercially available epoxide 5 in two steps. Ring opening of
the chiral epoxide 5 with selected amines provided the amino
alcohols 6a–c, which were reacted with 4-acetylbenzenesulfonyl
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chloride 7 using Na CO as a base under biphasic conditions to
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afford the 4-acetylbenzenesulfonamide intermediates 8a–c in
excellent yield. Reduction of the acetyl group using BH -THF in
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the presence of the chiral catalyst (R)-CBS-Me provided the
required intermediates with the (S)-(1-hydroxyethyl)benzene moiety
9a–c with excellent enantioselectivity. After purification by
flash chromatography, the products were recrystallized from a
mixture of EtOAc and hexanes to ensure chiral purity. Removal of
the Boc protecting group with TFA and reaction of the resulting
amine salts with the bis-THF activated carbonate 11 provided the
target
compounds
12,
14
and
16
with
the
(S)-4-(1-
hydroxyethyl)benzene moiety at the P2 position. Inhibitors with
the corresponding (R)-4-(1-hydroxyethyl)benzene at the P2 position
were prepared similarly using the (S)-CBS-Me as the catalyst during
the stereoselective reduction step and provided the desired
inhibitors 13, 15 and 17.
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Scheme 1. Synthesis of protease inhibitors incorporating (S)- and
(R)-4-(1-hydroxyethyl)benzene as P2 ligands
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Reagents and conditions: (a) RNH , EtOH, 70 °C, 3 h, 79–91%; (b)
2
Na CO , EtOAc, H O, RT, 18 h, 98–100%; (c) R-CBS-Me, BH -THF (1 M),
2
3
2
3
THF, 0 °C to RT, 3 h, 57–85%; (d) TFA, CH Cl , RT, 1 h; (e) DIEA,
2
2
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CH CN, RT, 24 h, 73–91%; (f) S-CBS-Me, BH -THF (1 M), THF, 0 °C to
RT, 3 h, 47–87%.
3
3
The
protease
inhibitors
with
the
(R)-
and
(S)-4-(1,2-
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dihydroxyethyl)benzene moieties at the P2 position were prepared
from the corresponding styrene derivatives by Sharpless asymmetric
dihydroxylation using the AD-mix catalyst29,30 as outlined in Scheme
2.
Briefly,
reactions
of
amino
alcohols
6a–c
with
4-
vinylbenzenesulfonyl chloride 18 using Na CO as a base under
2
3
biphasic
conditions
provided
the
4-vinylbenzenesulfonamide
intermediates 19a–c. The asymmetric dihydroxylation reaction using
AD-mix-β as a chiral catalyst proceeded smoothly and provided the
required intermediates with the (R)-4-(1,2-dihydroxyethyl)benzene
moiety 20a–c. In all cases the (S)-epimer was not detected by 1H
NMR; however, the products were recrystallized to provide
enantiomerically pure intermediates 20a–c. Analogous to the
compound series with the 4-(1-hydroxyethyl)benzene P2 moiety, Boc
deprotection and the reaction of the resulting amine salts with
the bis-THF carbonate 11 provided the target compounds 22, 24 and
26 with the (R)-4-(1,2-dihydroxyethyl)benzene moiety at the P2
position. Inhibitors with the corresponding (S)-4-(1,2-
dihydroxyethyl)benzene moiety at the P2 position were prepared in
a similar fashion using the AD-mix-α as the chiral catalyst in the
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asymmetric dihydroxylation reaction and provided the target
inhibitors 23, 25 and 27.
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Scheme 2. Synthesis of protease inhibitors incorporating (S)- and
(R)-4-(1,2-dihydroxyethyl)benzene as P2 ligands
1
1
1
1
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Reagents and conditions: (a) Na CO , EtOAc, H O, RT, 18 h, 75–94%;
2
3
2
(
b) AD-mix-β, t-BuOH, H O, RT, 4 h, 68–87%; (c) TFA, CH Cl , RT, 1
2 2 2
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h; (d) DIEA, CH CN, RT, 24 h, 33–79%; (e) AD-mix-α, t-BuOH, H O,
RT, 4 h, 47–86%.
3
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RESULTS AND DISCUSSION
Our goal was to improve potency of PIs against MDR HIV-1 variants
by optimizing hydrogen bonding interactions in the S2 subsite of
HIV-1 protease. Specifically, we explored the possibility of
additional hydrogen bonding interactions between the hydroxyl
1
1
1
1
1
1
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1
1
1
2
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0
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8
9
0
group of the P2 moiety and the backbone amide NH of Asp29 and
Asp30 on the basis of an analysis of 3- and 4-bound HIV-1 protease
structures and molecular modeling. Addition of a methyl group to
the hydroxymethyl substituent was expected to shift the hydroxyl
group closer to the backbone NH of Asp29, while increasing vdW
interactions with residues in the S2 subsite. Similarly,
replacement of the hydroxymethyl group with a more polar 1,2-
dihydroxyethyl was intended to enhance polar interactions;
particularly the second hydroxyl group was expected to replace the
water-mediated interactions between the P2 moiety and the Asp30
side chain. However, due to the free rotation of the hydroxyethyl
and dihydroxyethyl groups, modeling did not provide clear
distinction which of the two stereoisomers would provide optimal
polar and vdW interactions. Thus, both (R)- and (S)-stereoisomers
of 4-(1-hydroxyethyl)benzene and 4-(1,2-dihydroxyethyl)benzene
moieties were explored as P2 ligands in combination with three P1
groups of varying size and hydrophobicity.
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5
6
Enzyme inhibition assays. The binding affinity of HIV-1 PIs has
reached a level where assessing the inhibition of wild-type
protease using standard FRET-based assays is quite difficult.
Accurate measurement of inhibition constants in the low pM range
0
1
2
3
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0
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9
0
remains
a
challenge even with the recently reported highly
sensitive fluorogenic assay.31 Due to these limitations, two drug-
resistant protease variants, I84V and I50V/A71V were selected to
assess the potency of new PIs. I84V is a common drug resistance
mutation that reduces susceptibility to all FDA-approved PIs,
while I50V/A71V mutations arise in response to therapy with APV
and DRV-based regimens, resulting in reduced susceptibility to
these PIs. The enzyme inhibition constants (Ki) were determined
against the I84V and I50V/A71V protease variants using the highly
31
sensitive FRET assay ; DRV was used as a control in all assays
(Table 1).
Table 1. Protease inhibitory activity of PIs 12–17 and 22–27
against drug-resistant variants
Inhibi
tor
Ki (nM)
Structure
I84V
I50V/A71V
0.048 ±
0.004
0.057 ±
0.008
1
2
3
0.109 ±
0.092 ±
0.008
1
0.007
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0
0
.111 ±
.008
0.093 ±
0.013
1
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
0
.095 ±
.005
0.132 ±
0.014
15
16
0
0
.020 ±
.002
0.102 ±
0.010
0
0
.028 ±
.003
0.107 ±
0.014
17
0
0
.098 ±
.008
0.090 ±
0.008
22
0.283 ±
0.024
23
NT
NT
0
0
0
.216 ±
0
24
25
.011
.504 ±
.039
NT
0
.057 ±
.004
0.074 ±
0.006
26
0
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1
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1
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2
2
2
2
2
2
2
2
2
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3
4
4
4
4
4
4
4
4
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4
5
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5
5
5
5
5
5
5
6
0.134 ±
.007
0.220 ±
0.011
2
7
0
0
1
2
3
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5
6
7
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9
0
1
2
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0
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0
1
2
3
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5
6
7
8
9
0
0.010 ±
0.003
0.080 ±
0.006
3
0.005 ±
0.055 ±
0.004
4
0.002
0.025 ±
0.006
0.075 ±
0.006
DRV
NT: not tested
DRV retained excellent potency against the I84V and I50V/A71V
protease variants (Ki = 25 pM and 75 pM, respectively) compared to
that reported for wild-type protease (Ki = 5–16 pM).13,23,31 Compound
3
, which incorporates a 4-(hydroxymethyl)benzene moiety as P2
ligand and (S)-2-methylbutyl group at the P1 position, showed
better potency against the I84V protease but was equipotent to DRV
against the I50V/A71V variant. Analogue 4 with a larger 2-
ethylbutyl group at the P1 position was more active than DRV
against both the I84V and I50V/A71V protease variants. The
corresponding analogue 2 with the isobutyl P1 group was previously
reported which was equipotent to DRV against wild-type protease
but exhibited relatively lower antiviral potency, presumably due
to reduced hydrophobicity.13,32 Thus, as we have previously shown,
PIs with 4-(hydroxymethyl)benzene as the P2 moiety in combination
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with larger, more hydrophobic (S)-2-methylbutyl and 2-ethylbutyl
P1 groups maintained potency against drug-resistant protease
variants.
1
1
1
1
1
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1
2
2
2
2
2
2
2
2
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2
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Replacement of the 4-(hydroxymethyl)benzene P2 moiety of PIs 2–
4
with (S)- and (R)-4-(1-hydroxyethyl)benzene resulted in pairs of
diastereoisomers epimeric at P2. The P2 hydroxyethyl analogues
2–17 showed excellent inhibitory potencies against the I84V and
I50V/A71V protease variants with Ki values ranging between 20 to
32 pM. In each case, both diastereoisomers were relatively less
1
1
potent than the corresponding parent hydroxymethyl compound,
particularly against the I84V protease variant. However, no major
difference in inhibitory potency was observed between P2 epimeric
compounds. Only compounds with the isobutyl P1 group showed minor
differences in inhibitory potencies between the P2 epimers, as
diastereoisomer 12 with (S)-configuration of the hydroxyethyl
group was slightly more potent than the corresponding (R)-epimer
13. While this work was underway, Ghosh et al. reported the
synthesis of PIs 12 and 13 using a much longer reaction sequence;
both compounds were tested against wild-type HIV-1 protease, and
only compound 13 was evaluated for antiviral activity.33
Diastereoisomers 14 and 15 with the (S)-2-methylbutyl P1 group
showed similar potencies against the I84V and I50V/A71V protease
variants, and both were less potent than the parent 3. Similarly,
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6
no difference in inhibitory potency was observed between
diastereoisomers 16 and 17 incorporating the 2-ethylbutyl P1
group. However, analogues 16 and 17 were more potent against the
I84V protease (Ki = 20 and 28 pM, respectively) and exhibited
overall potency profiles comparable to that of DRV. Together, these
0
1
2
3
4
5
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8
9
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9
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data demonstrate that minor modifications to the P1 and P2
moieties result in distinct potency profiles.
In contrast to the 4-(1-hydroxyethyl)benzene P2 series,
compounds with the 4-(1,2-dihydroxyethyl)benzene P2 moiety showed
clear differences in inhibitory potency between the P2-epimers.
Compounds incorporating the (R)-4-(1,2-dihydroxyethyl)benzene P2
moiety were 2–3-fold more potent than the corresponding (S)-
epimers. Diastereoisomer 22, with the isobutyl group at the P1
position and (R)-configuration of the dihydroxyethyl group,
exhibited excellent inhibitory potency against the I84V and
I50V/A71V proteases, while the corresponding (R)-epimer 23 was
about 3-fold less active against the I84V protease. A similar trend
was observed for diastereoisomers 24 and 25 incorporating the (S)-
2-methylbutyl P1 group, though both compounds were 2-fold less
active against the I84V protease than the corresponding isobutyl
P1 analogues 23 and 24. The analogue 26 incorporating the larger
2-ethylbutyl P1 group and (R)-configuration of the dihydroxyethyl
group was the most potent in this series, with enzyme potency
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against the I50V/A71V protease variant comparable to that of DRV.
Again, the corresponding P2 (S)-epimer 27 was 2–3-fold less potent
against the protease variants tested. Thus, shape of the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hydrophobic
group
at
P1
and
the
configuration
of
the
dihydroxyethyl group at P2 affected inhibitor potency against
drug-resistant protease variants.
Antiviral assays. The potency and resistance profiles of PIs were
evaluated using a cell-based antiviral assay against wild-type
HIV-1 and three representative MDR variants selected from a panel
of clinically relevant multi-PI resistant recombinant clones
(
Table 2, Table S1).34 Relative to the wild-type HIV-1 strain NL4-3
(with a near consensus protease sequence), the MDR variants contain
9, 20 and 24 amino acid substitutions in the protease and are
1
named SLK19, VSL20, and KY24, respectively. All three variants
show high-level resistance and cross-resistance to multiple PIs
including DRV, as determined by the Monogram Biosciences (South
San Francisco, CA) PhenoSense assay.34 Accordingly, in antiviral
assays, the MDR variants exhibited increasingly high-level
resistance to DRV: compared to wild-type HIV-1, DRV was 6-fold
less potent against SLK19, 58-fold less potent against VSL20, and
210-fold less potent against KY24. Thus, the three selected MDR
HIV-1 strains represent clinically relevant MDR viruses with a
wide spectrum of DRV resistance.
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. Antiviral potency of PIs 12–17 and 22–27 against WT HIV-1
and drug-resistant variants
Antiviral EC50 (nM) (fold change)b
cLogPa
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
0
1
2
3
4
5
6
7
8
9
0
Inhibitor
WT
SLK19
VSL20
KY24
12
2.885
2.885
3.414
3.414
3.943
3.943
1.673
1.673
2.202
2.202
2.731
2.731
3.105
3.634
2.387
4.7
31 (7)
117 (25)
352 (75)
1
3
4
7.5
4.6
4.2
4.4
5.1
176
189
80
100 (13)
49 (11)
91 (22)
26 (6)
32 (6)
NT
148 (20)
360 (48)
1
281 (61) 490 (107)
15
291 (69)
112 (25)
420 (82)
NT
342 (81)
196 (45)
345 (68)
NT
16
17
22
23
NT
NT
NT
24
NT
NT
NT
2
5
6
92
NT
NT
NT
2
40
116 (3)
151 (4)
52 (9)
36 (9)
33 (6)
100 (3)
507 (13)
27
3
41
417 (10) 1154 (28)
087
1
5.7
4.2
5.5
310 (54)
(191)
426 (101) 495 (118)
157
4
1
DRV
320 (58)
(210)
a
b
cLogP values were calculated using ChemDraw 18. The multidrug-
resistant HIV-1variants SLK19, VSL20 and KY24 contain the
following amino acid substitutions in HIV-1 protease compared to
the WT NL4-3 strain: SLK19 (L10I, V11I, I13V, K14R, A22V, E35D,
M36I, N37D, G48M, F53L, I54V, I62V, L63P, A71V, T74S, V82A, I84V,
T91S, Q92K); VSL20 (L10I, K20R, L33F, E34A, E35D, M36I, N37D, K43T,
M46I, G48V, I50V, I54T, I62V, L63P, A71V, I72V, V77I, V82A, I85V,
I93L); KY24 (L10V, T12V, I13V, I15V, K20M, V32I, L33F, K43T, M46I,
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I47V, I54M, D60E, Q61N, I62V, L63P, C67Y, H69K, A71I, I72L, G73S,
V77I, V82A, L89V, L90M). NT: not tested.
The 4-(hydroxymethyl)benzene P2 analogues 3 and 4 were equipotent
to DRV against wild-type HIV-1 with EC50 values of 5.7 and 4.2 nM,
respectively. The two analogues retained potent activity against
the SLK19 variant but were significantly less potent against the
VSL20 variant, with EC50 values similar to that of DRV. Compared
to DRV and compound 3, both of which showed similar potencies
against the three MDR variants, analogue 4 retained 2-fold better
potency against the KY24 variant. The slightly improved resistance
profile of compound 4 is in agreement with previous results and
1
1
1
1
1
1
1
1
1
1
2
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3
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4
4
4
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4
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0
indicates that optimizing vdW interactions in the S1 pocket can
improve potency against MDR viruses.23
All compounds incorporating the 4-(1-hydroxyethyl)benzene P2
moieties (12–17) potently inhibited wild-type HIV-1 with EC50
values similar to that of DRV and parent compounds 3 and 4.
However,
the
three
MDR
HIV-1
variants
showed
distinct
susceptibilities to each of these compounds, depending on the P1
group and the configuration of the hydroxyethyl group at the P2
moiety. Compound 12, with the isobutyl P1 group and the (S)-4-(1-
hydroxyethyl)benzene P2 moiety, was equipotent to DRV against
SLK19 but exhibited 2–3-fold better potency against the highly
resistant MDR variants VSL20 and KY24, resulting in an overall
improved resistance profile. The corresponding epimer 13,
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6
incorporating the (R)-4-(1-hydroxyethyl)benzene P2 moiety, while
showing a 3-fold lower potency against SLK19, retained similar
potency as 12 against VSL20 and KY24. Diastereoisomers 14 and 15
0
1
2
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0
with the (S)-2-methylbutyl P1 group exhibited similar potency
profiles as the corresponding analogues 12 and 13, except for a 2-
fold loss in potency against VSL20. Further improvement in overall
potency profile was realized with analogue 16 incorporating a
larger 2-ethylbutyl P1 group and the (S)-4-(1-hydroxyethyl)benzene
P2 moiety, which maintained similar potency against SLK19 as DRV
but demonstrated about 3- and 6-fold increase in potency against
VSL20 and KY24, respectively. Moreover, compared to the parent
compound 4, analogue 16 demonstrated 4- and 2-fold improved potency
against VSL20 and KY24. Compared to 16, the (R)-epimer 17
maintained similar potency against SLK19 but exhibited lower
potency against VSL20 and KY24. While all compounds with the 4-
(1-hydroxyethyl)benzene as P2 ligands maintained excellent potency
against MDR HIV-1 variants, each exhibited distinct resistance
profile depending on the configuration of the hydroxyethyl group
and size of the P1 group.
The introduction of a more polar 4-(1,2-dihydroxyethyl)benzene
moiety as the P2 ligand resulted in significantly reduced potency
against wild-type HIV-1, despite compounds 22–27 showing pM
inhibitory activities in biochemical assays. The observed loss of
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potency in cellular assays is likely due to reduced hydrophobicity,
as indicated by lower calculated partition coefficient (LogP)
values compared to DRV (Table 1). The cellular potency of HIV-1
protease inhibitors has been shown to strongly correlate with the
hydrophobicity descriptor LogP, suggesting that membrane transport
is a key factor affecting antiviral potency.35
1
1
1
1
1
1
1
1
1
1
2
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0
The
observed
antiviral
potencies
of
the
4-(1,2-
dihydroxyethyl)benzene containing compounds indeed correlate with
their cLogP values (Figure S2). In all cases both diastereoisomers
exhibited similar antiviral potencies against wild-type HIV-1.
Compounds 22 and 23 incorporating the isobutyl P1 group showed
significantly lower potency than DRV. Replacement of the isobutyl
P1 group with a slightly more hydrophobic (S)-2-methylbutyl group,
providing analogues 24 and 25, resulted in further improvement in
antiviral potency. Compounds 26 and 27 incorporating a larger 2-
ethylbutyl P1 group further increased hydrophobicity and resulted
in a 2-fold improvement in potency compared to the (S)-2-methylbuty
P1 analogues 24 and 25. However, both diastereoisomers 26 and 27
were 8-fold less active than DRV against wild-type HIV-1 despite
26
exhibiting similar potency in enzyme inhibition assays.
Compared to DRV the P2 (R)-epimer 26 maintained better potency
against MDR variants VSL20 and KY24 and exhibited much lower fold
potency losses. The corresponding (R)-epimer 27, though less
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active against SLK19, showed similar potency as DRV against VSL20
and KY24. Thus, the combination of a more hydrophobic 2-ethylbutyl
P1 group and the (R)-4-(1,2-dihydroxyethyl)benzene P2 moiety
provided compounds with improved potency profiles.
0
1
2
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0
Analysis of protease-inhibitor complexes. To explore molecular
interactions of the chiral 4-(1-hydroxyethyl)benzene and 4-(1,2-
dihydroxyethyl)benzene
P2
moieties,
we
determined
crystal
structures of PIs 12–17 and 22–27 bound to wild-type HIV-1 protease
of the NL4-3 strain. The cocrystal structures of PIs 2–4 were also
determined with the same protease enzyme, as the previously
reported cocrystal structures of PIs 3 and 4 were with the wild-
type protease of SF-2 sequence, which is slightly different from
NL4-3. The two variants differ by four amino acids which caused
minor structural differences at the distal loops, but the active
sites were nearly identical, including inhibitor binding and
crystallographic waters. The crystallographic data collection and
refinement statistics are summarized in Table S2. All 15 high-
resolution (1.86–2.03 Å) cocrystal structures were solved in the
same P2 2 2 space group with one protease homodimer in the
1
1 1
asymmetric unit, and only one orientation of the bound inhibitor
in the protease active site, which was crucial for direct
comparison and analysis.
The overall binding conformations of all PIs incorporating the
4-(hydroxymethyl)benzene (2–4), 4-(1-hydroxyethyl)benzene (12–
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1
7), and 4-(1,2-dihydroxyethyl)benzene (22–27) P2 moieties are
similar to that of DRV (Figure 2A). However, clear differences in
contacts were observed in the binding of the P2 moieties depending
on the substituent at the 4-position of the benzene ring and the
configuration of the hydroxyl group (Figure 2B, 2D). Compared to
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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0
DRV, all PIs with modified P2 ligands maintain similar hydrogen
bonding interactions with the protease, except in the S2 subsite
(
Figures 3 and 4). The cocrystal structures of PIs 2–4 in complex
with protease showed minor changes in the conformation of the 4-
hydroxymethyl)benzene moiety compared to the 4-aminobenzene of
(
DRV in the S2 subsite (Figure S3). In the DRV-bound protease
structure, the amino group of the 4-aminobenzene P2 moiety is
involved in hydrogen bonding interactions with the main chain
carbonyl of Asp30 and water-mediated interactions with the side-
chain carboxylate of Asp30 (Figure S1). The primary hydroxyl group
of the 4-(hydroxymethyl)benzene P2 moiety in compounds 2–4 is
oriented toward Asp29 and Asp30 backbone and makes a direct
hydrogen bond with the backbone NH of Asp30. Moreover, the hydroxyl
group interacts with the backbone NH of Asp29 and the side chain
of Asp30 through water-mediated hydrogen bonds. This network of
hydrogen bonding interactions in the S2 subsite likely underlie
the improved potency of compounds 2–4 compared to DRV (Figure S1).
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A
B
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C
D
Figure 2. Comparison of binding modes of protease inhibitors with
4-(1-hydroxyethyl)benzene and 4-(1,2-dihydroxyethyl)benzene P2
moieties in the active site of wild-type HIV-1 protease. The two
protease monomers are in cyan (denoted as non-prime) and magenta
(denoted as prime). Superposition of protease complexes with DRV
(
1), parent compounds (2–4), and new analogues (12–17 and 22–27).
The inhibitors are shown as sticks and HIV protease dimers are
shown as ribbons. (B) Zoomed-in active site of superimposed
complexes. The inhibitors bind to wild-type HIV protease in similar
conformations except minor variations in the S2 subsite. (C) Fit
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of inhibitors within the substrate envelope. The substrate
envelope is in blue space filling representation, and the
superimposed inhibitors are displayed as sticks. There is minimal
protrusion of inhibitors outside the active site. (D) Variations
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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in the binding mode of the inhibitors’ P2 moieties.
A
B
F53
F53’
F53
F53’
G48’
G48’
G48
G48
I50’
I50’
I50
I50
I47
I47’
I47
I47’
D30
D30
D25
D25’
D25
D25’
D30’
D30’
D29
G27
G27’
D29’
D29
G27
G27’
D29’
C
D
F53
F53’
F53
F53’
G48’
G48’
G48
G48
I50’
I50’
I50
I50
I47
I47’
I47
I47’
D30
D30
D25
D25’
D25
D25’
D30’
D30’
D29
G27
G27’
D29’
D29
G27
G27’
D29’
Figure 3. Crystal structures of wild-type HIV-1 protease in complex
with inhibitors (A) 16, (B) 17, (C) 26, and (D) 27. Both (R)- and
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