Structural Analysis of Potent Hybrid HIV-1 Protease Inhibitors Containing Bis-tetrahydrofuran in a Pseudosymmetric Dipeptide Isostere

Article abstract of DOI:10.1021/acs.jmedchem.0c00529

The design, synthesis, and X-ray structural analysis of hybrid HIV-1 protease inhibitors (PIs) containing bis-tetrahydrofuran (bis-THF) in a pseudo-C2-symmetric dipeptide isostere are described. A series of PIs were synthesized by incorporating bis-THF of darunavir on either side of the Phe-Phe isostere of lopinavir in combination with hydrophobic amino acids on the opposite P2/P2′ position. Structure-activity relationship studies indicated that the bis-THF moiety can be attached at either the P2 or P2′ position without significantly affecting potency. However, the group on the opposite P2/P2′ position had a dramatic effect on potency depending on the size and shape of the side chain. Cocrystal structures of inhibitors with wild-type HIV-1 protease revealed that the bis-THF moiety retained similar interactions as observed in the darunavir-protease complex regardless of the position on the Phe-Phe isostere. Analyses of cocrystal structures and molecular dynamics simulations provide insights into optimizing HIV-1 PIs containing bis-THF in non-sulfonamide dipeptide isosteres.

Full text of DOI:10.1021/acs.jmedchem.0c00529

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Article
Structural Analysis of Potent Hybrid HIV-1 Protease Inhibitors Containing
Bis-Tetrahydrofuran in a Pseudo-Symmetric Dipeptide Isostere
Linah N Rusere, Gordon J. Lockbaum, Mina Henes, Sook-Kyung Lee, Ean
Spielvogel, Desaboini Nageswara Rao, Klajdi Kosovrasti, 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.0c00529 • Publication Date (Web): 16 Jul 2020
Downloaded from pubs.acs.org on July 18, 2020
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Structural Analysis of Potent Hybrid HIV-1 Protease
Inhibitors Containing Bis-Tetrahydrofuran in a
Pseudo-Symmetric Dipeptide Isostere
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Linah N. Rusere,†,§ Gordon J. Lockbaum,†,§ Mina Henes,† Sook-
Kyung Lee,‡ Ean Spielvogel,‡ Desaboini Nageswara Rao,† Klajdi
Kosovrasti,† 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
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ABSTRACT
The design, synthesis, and X-ray structural analysis of hybrid
HIV-1 protease inhibitors (PIs) containing bis-tetrahydrofuran
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(bis-THF) in
a
pseudo-C -symmetric dipeptide isostere are
2
described. A series of PIs were synthesized by incorporating bis-
THF of darunavir on either side of the Phe-Phe isostere of
lopinavir in combination with hydrophobic amino acids on the
opposite P2/P2 position. Structure–activity relationship studies
indicated that the bis-THF moiety can be attached at either the P2
or P2 position without significantly affecting potency. However,
the group on the opposite P2/P2 position had a dramatic effect on
potency depending on the size and shape of the side chain.
Cocrystal structures of inhibitors with wild-type HIV-1 protease
revealed that the bis-THF moiety retained similar interactions as
observed in the darunavir-protease complex regardless of position
on the Phe-Phe isostere. Analyses of cocrystal structures and
molecular dynamics simulations provide insights for optimizing
HIV-1 PIs containing bis-THF in non-sulfonamide dipeptide
isosteres.
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INTRODUCTION
Combinations of drugs targeting HIV-1 enzymes essential for viral
replication have been highly effective in reducing viral load in
infected individuals and significantly increasing their life
expectancy.1 HIV-1 protease inhibitors (PIs) have played an
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important role as
a
highly potent component of combination
antiretroviral therapy (cART) for people living with HIV-1. There
are nine HIV-1 PIs approved by the US FDA; however, clinical use
of most of these has diminished over the years due to side effects,
unfavorable pharmacokinetics, and drug resistance. Currently only
three PIs, darunavir (DRV), lopinavir (LPV), and atazanavir (ATV)
(Figure 1) – boosted with small doses of ritonavir (RTV) – are
being used in cART as they have comparatively higher potency and
better resistance profiles.2, 3
Most HIV-1 PIs are substrate-based peptidomimetics containing a
hydroxyl group as a transition state mimetic and diverse moieties
that target the S1/S1 and S2/S2 subsites of the enzyme.4, 5 Among
the PIs in clinical use, DRV is the most potent with the highest
barrier to resistance and efficacy against multidrug-resistant
6
HIV-1 strains. The excellent potency and resistance profiles of
DRV are attributed to the bis-tetrahydrofuran (bis-THF) moiety,
which forms strong hydrogen bonding interactions with the main-
chain NH of Asp29 and Asp30 in the S2 subsite of the enzyme.7
Moreover, the extensive van der Waals (vdW) interactions of the
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bis-THF moiety, along with the sulfonamide-based Phe-Leu dipeptide
isostere and the 4-aminobenzene group, also significantly
contribute to the potency of DRV.8 Due to these unique binding
characteristics, the P2 bis-THF moiety of DRV has emerged as a
privileged ligand for targeting the S2 subsite of HIV-1 protease
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especially
when
incorporated
into
the
(R)-
(
hydroxyethylamino)sulfonamide isostere.9
Efforts to improve potency, pharmacokinetics, and resistance
profiles of HIV-1 PIs have led to the discovery of exceptionally
potent compounds exhibiting a wide range of properties.5, 10-14 In
recent years, these efforts have focused mainly on exploring DRV
analogues with similar sulfonamide-based dipeptide isosteres and
P2/P2 moieties. Many analogues of DRV with modifications at P1/P1
and P2/P2 positions have been explored extensively,5, 15-18 including
novel bis-THF analogues showing improved hydrogen bonding and vdW
interactions with the protease.19-22 Moreover, detailed structural
analyses informing structure-based design of inhibitors with the
DRV scaffold have been reported numerous times including recent
structures determined by neutron crystallography.5, 7, 8, 23-25
However, very few inhibitors containing the bis-THF moiety in
non-sulfonamide dipeptide isosteres have been explored.26-28
Previously, Chen and colleagues at Abbott Laboratories explored
PIs with bis-THF on either side of the pseudo-C -symmetric Phe-Phe
2
dipeptide isostere of LPV,26, 27 while Cannizzaro and coworkers at
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Gilead Sciences designed similar analogues using the aza-dipeptide
isostere of ATV.28 Although some of the Phe-Phe isostere-based
compounds, such as 4 and 5 (Figure 1), exhibited significant
antiviral activity against wild-type HIV-1, in the absence of
direct comparison, it is unclear if their potency is similar to
that of DRV.26 Also, due to limited structure-activity relationship
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studies, the S2/S2 subsite preference of the bis-THF moiety in the
context of Phe-Phe isostere remains unclear,26, 27 as is the identity
of an optimal moiety for targeting the opposite S2/S2 subsite.26
Moreover, no cocrystal structures of such hybrid PIs have been
reported, limiting opportunities for structure-guided design and
optimization.
In this study, we investigate HIV-1 PIs containing the bis-THF
moiety of DRV in the Phe-Phe dipeptide isostere of LPV with the
aim to identify compounds with potency similar to that of DRV but
with an alternate non-sulfonamide scaffold. Protease-inhibitor
molecular
interactions
are
characterized
using
X-ray
crystallography and molecular dynamics (MD) simulations to aid
structure-guided optimization of these hybrid PIs. In addition,
interdependence of HIV-1 protease subsites is explored to provide
insight into subsite preferences and influence of different
inhibitor moieties on binding and potency.
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Figure 1. Structures of HIV-1 protease inhibitors darunavir (DRV)
(1), lopinavir (LPV) (2), atazanavir (ATV) (3), previously
reported hybrid compounds 4 and 5, and new hybrid compounds
designed by incorporating the bis-THF moiety of DRV into the
pseudo-symmetric Phe-Phe dipeptide isostere of LPV at the P2 (12a–
h) or P2 position (15a–c), in combination with an ATV-like
hydrophobic amino acid moiety at the P2 or P2 position,
respectively. The canonical nomenclature is used to indicate the
position of each inhibitor moiety.
The
active
site
of
HIV-1
protease
exhibits
subsite
interdependence in substrate recognition and inhibitor binding,29,
30
but this feature of the protease has rarely been considered in
inhibitor design. While identifying optimal moieties for targeting
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each of the subsites in the protease active site is important,
understanding the subsite interdependence and selection of the
best combination of moieties are also crucial to achieving higher
potency especially against drug-resistant variants.19 The bis-THF
moiety potentially can be incorporated in diverse dipeptide
isosteres in combination with suitable ligands targeting the other
subsites. Thus, a series of hybrid HIV-1 PIs were synthesized by
incorporating the bis-THF moiety of DRV on either side of the
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pseudo-C -symmetric
Phe-Phe
dipeptide
isostere
of
LPV
in
2
combination with ATV-like hydrophobic P2/P2 ligands of varying
size and shape (Figure 1). The hybrid PIs were tested for
inhibitory activity against wild-type and two drug-resistant
variants and for antiviral activity in cellular assays. We also
determined high-resolution X-ray crystal structures of all
designed hybrid compounds and previously reported PI 4 bound to
wild-type
HIV-1
protease
and
characterized
the
binding
interactions of bis-THF in the context of Phe-Phe isostere. In
addition, molecular dynamics simulations were utilized to assess
the stability of protease-inhibitor interactions in solution. The
comprehensive structural and dynamic analyses of hybrid PIs have
provided insights into inhibitor binding and interdependence of
protease subsites.
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CHEMISTRY
The synthesis of hybrid PIs incorporating the bis-THF moiety at
either the P2 or P2 position is outlined in Scheme 1. The required
Phe-Phe dipeptide isostere core intermediate 7 was synthesized
from phenylalanine following previously reported methods with
minor modifications (see Supporting Information for details)
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(
Haight, OPRD, 1999).31 Reaction of the dipeptide isostere 7 with
bis-THF activated carbonate 8 in the presence of DIEA provided
intermediate 9. Debenzylation of 9 using ammonium formate and 10%
palladium on activated carbon in methanol gave the corresponding
deprotected amine 10, which was subsequently coupled with the amino
acid derivatives 11a–h using HATU and DIEA to provide the target
compounds 12a–h with bis-THF at the P2 position. While N-
(methoxycarbonyl)-protected amino acid derivatives 11a and 11e
were commercially available, intermediates 11b–d and 11f–h were
prepared from the corresponding amino acids by reaction with methyl
chloroformate under basic conditions following
procedure.32
a
reported
The PIs 15a–c with the bis-THF at the P2 position were
synthesized using a similar reaction sequence. Coupling of the
dipeptide isostere 7 with acids 11a–c under HATU peptide coupling
conditions gave the corresponding intermediates 13a–c. Catalytic
debenzylation of compounds 13a–c followed by coupling with bis-
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THF activated carbonate 8 in the presence of DIEA provided the
target compounds 15a–c. A pair of previously reported PIs 4 and 5
containing the dimethylphenoxy acetate and bis-THF moieties as the
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P2 or P2 ligands in the Phe-Phe isostere were also synthesized
using the same reaction sequences.
Scheme 1. Synthesis of Protease Inhibitors Incorporating Bis-
Tetrahydrofurn in a Pseudo-Symmetric Phe-Phe Dipeptide Isostere
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Reagents and conditions: (a) K CO , KOH, BnCl, H O, 100 °C, 5 h;
2
3
2
(
1
b) NaNH , CH CN, MTBE, 0 °C, 2 h, then BnMgCl (2 M in THF), RT,
2 3
4 h; (c) NaBH , MsOH, i-PrOH, EGDME, 0 °C, 12 h; (d) NaBH , TEA,
4
4
DMA, 0–15 °C, 2 h; (e) DIEA, CH CN, RT, 24 h; (f) Pd/C, HCO NH ,
3
2
4
MeOH, 50 °C, 12 h; (g) N-(methoxycarbonyl)-capped amino acid, HATU,
DIEA, DMF, RT, 4 h.
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RESULTS AND DISCUSSION
We explored hybrid HIV-1 PIs containing the bis-THF moiety of DRV
in the Phe-Phe isostere of LPV, with the goal to characterize the
binding interactions of this P2 ligand in the context of a pseudo-
C2-symmetric dipeptide isostere. The bis-THF moiety was used in
combination with diverse N-(methoxycarbonyl)-capped amino acid
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derivatives similar to the P2/P2 moiety of ATV to optimize hydrogen
bonding and vdW interactions in both the S2 and S2 subsites of
HIV-1 protease. To explore subsite preference for the bis-THF
moiety, pairs of compounds were synthesized with the bis-THF moiety
attached at either the P2 (12a–c) or P2 (15a–c) position with
respect to the central hydroxyl group of the Phe-Phe isostere.
Considering potential subsite interdependence, incorporating a
relatively small and conformationally flexible moiety at the other
P2/P2 position was expected to allow the bis-THF moiety to
optimally interact with the backbone atoms in the S2/S2 subsite,
likely improving overall inhibitor binding with the protease.
Structure-activity relationships were explored by incorporating
hydrophobic amino acids of varying size and shape to identify an
optimal ligand for targeting the other S2/S2 subsite.
Enzyme Inhibition and Antiviral Assays. The potency of PIs was
assessed using biochemical and antiviral assays. The enzyme
inhibition constants (Ki) were determined against wild-type
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5
5
5
5
5
5
6
protease and two drug-resistant variants (I84V and I50V/A71V)
using a highly sensitive fluorogenic assay (Table 1).33 Both I50V
and I84V are major protease inhibitor resistance mutations that
reduce susceptibility to LPV and DRV. For a subset of compounds,
antiviral potencies (EC ) were determined against wild-type HIV-
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
50
1
(NL4-3 strain) using a cell-based antiviral assay. LPV and DRV
were used as controls in all assays.
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Table 1. Enzyme Inhibitory Activity and Antiviral Potency of Hybrid HIV-1 Protease Inhibitors
Ki (nM)a
EC50 (nM)b
6
7
8
9
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
Inhibitor
Structure
WT
I84V
I50V/A71V
WT
1
1
2a
5a
0.018 ± 0.003 0.208 ± 0.014 0.273 ± 0.043
0.013 ± 0.001 0.372 ± 0.021 0.072 ± 0.005
11
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
39
1
1
2b
5b
46 ± 3
236 ± 39
191 ± 30
484 ± 70
40 ± 3
NT
NT
7.3 ± 0.5
1
1
2c
5c
0.235 ± 0.016
0.161 ± 0.011
0.71 ± 0.10
1.18 ± 0.26
2.35 ± 0.39
1.39 ± 0.12
NT
NT
1
2d
0.019 ± 0.004 0.069 ± 0.013 0.073 ± 0.003
0.074 ± 0.006 0.302 ± 0.022 0.295 ± 0.082
66
55
1
2e
1
2f
0.571 ± 0.076
1.82 ± 1.50
1.66 ± 0.48
NT
1
2g
0.055 ± 0.004 0.198 ± 0.014 0.560 ± 0.094
0.027 ± 0.004 0.091 ± 0.013 0.029 ± 0.003
70
12h
240
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2
2
2
2
2
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4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
4
5
0.017 ± 0.002 0.069 ± 0.005 0.072 ± 0.002
38
16
12
0.037 ± 0.005 0.259 ± 0.055
NT
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
1
2
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
LPV
< 0.005
< 0.005
0.051 ± 0.004 0.061 ± 0.011
DRV
0.025 ± 0.006 0.075 ± 0.006
5
a
Enzyme inhibition constants against wild-type HIV-1 protease and
b
drug resistant variants; Antiviral potency against wild-type HIV-
1; NT = Not Tested
Compounds 12a and 15a, with the bis-THF moiety at the P2 and P2
position, respectively, and the N-(methoxycarbonyl)-tert-leucine
at the opposite P2/P2 position, showed excellent inhibitory
potencies against wild-type protease and the drug resistant
variants. Compared to DRV and LPV, both compounds were less active,
particularly against the I84V variant. Interestingly, compounds
12a and 15a exhibited similar Ki values, though the latter was 4-
fold more active against the I50V/A71V variant, indicating no clear
S2/S2 subsite preference for the bis-THF moiety. However, in
antiviral assays compound 12a was 3-fold more potent than 15a
against wild-type HIV-1. The compound 12a, with the bis-THF moiety
at the P2 position, exhibited excellent antiviral potency (EC50
=
11 nM) comparable to that of LPV (EC50 = 12 nM) and DRV (EC50 = 5
nM). A similar potency profile was observed for previously reported
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compounds 4 and 5 containing the bis-THF and the dimethylphenoxy
acetate moieties as the P2 and P2 ligands. Compound 5 exhibited
2-fold better antiviral potency than 4, indicating a reverse
subsite preference for the bis-THF moiety relative to the 12a/15a
pair. The small variation in the antiviral potencies of compounds
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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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
12a/15a and 4/5 indicate that the position of the bis-THF moiety
with respect to the central hydroxyl group of the Phe-Phe isostere,
though not critical for binding, may affect potency in cellular
assays.
Although the potency of the hybrid compounds was not particularly
sensitive to the position of the bis-THF moiety, compounds with
different hydrophobic amino acid derivatives at the opposite P2/P2
position showed markedly different inhibitory potencies. The
compounds with the cycloleucine moiety, 12b and 15b, were
significantly less active (>2500-fold and 560-fold, respectively)
than the corresponding tert-leucine analogues 12a and 15a. The
dramatic loss of potency is likely due to the altered shape and
flexibility of the hydrophobic amino acid moiety. This was further
supported by the fact that the corresponding cyclopentylglycine
analogues 12c and 15c, though also an order of magnitude less
active than 12a and 15a, still exhibited sub-nanomolar to low nM
potency against wild-type protease and drug-resistant variants.
Together these data confirm that the hydrophobic amino acid moiety
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2
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2
2
2
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4
4
4
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5
5
5
5
5
5
5
5
5
6
at the opposite P2/P2 position significantly influence potency in
both biochemical and antiviral assays.
Since potency of the compounds with bis-THF moiety attached to
the Phe-Phe isostere largely depended on the hydrophobic amino
acid moiety at the opposite side of the core scaffold, we
investigated size and shape requirements for optimally targeting
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
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9
0
1
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9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
the S2 subsite. Analogues of compound 12a were synthesized with
diverse hydrophobic amino acid derivatives as P2 ligands while
keeping the bis-THF moiety at the P2 position. The alanine analogue
12d exhibited excellent inhibitory potency with Ki values against
resistant variants comparable to that of LPV and DRV. The
corresponding valine analogue 12e was 4-fold less active against
wild-type protease but maintained similar inhibitory potency as
12a against the resistant variants. However, compared to 12a
compounds 12d and 12e were 6- and 5-fold less active in antiviral
assays, respectively, indicating that factors other than binding
affinity are responsible for lower antiviral potency. The
cyclopropyl analogue 12f was considerably less active than 12a,
with Ki values similar to the cyclopentyl analogue 12c, strongly
suggesting that cyclic moieties at the P2/P2 position are
unfavorable. Replacement of tert-leucine with an isoleucine
(compound 12g) or alloisoleucine (compound 12h) moiety at the P2
position also resulted in slightly lower activity against wild-
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type protease but similar or better potency against resistant
variants. This further confirmed earlier findings that the P2/P2
group opposite to the bis-THF moiety on the Phe-Phe core has a
significant impact on potency. These results are in contrast to
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
the sulfonamide-based dipeptide isostere of DRV, where diverse P2
6
modifications result in relatively minor changes in potency. The
identity of the P2/P2 moiety opposite to the bis-THF on the Phe-
Phe core considerably impacts potency in both biochemical and
antiviral assays.
Analysis of Protease-Inhibitor Complexes. To characterize
protease-inhibitor molecular interactions, twelve high-resolution
(1.8–2.0 Å) crystal structures of hybrid PIs and compound 4 bound
to wild-type HIV-1 protease of the NL4-3 strain were determined.
A cocrystal structure of LPV was also determined bound to the same
protease enzyme for direct comparison. The crystallographic data
collection and refinement statistics are summarized in Table S1.
Following convention, protease chains were assigned chain A (non-
prime) or chain B (prime) depending on the interactions between
the central hydroxyl group of the Phe-Phe isostere and the
catalytic aspartates Asp25/Asp25.
LPV and DRV achieve low picomolar affinity to wild-type HIV-1
protease by making a number of hydrogen bonds and vdW interactions
with the active site residues (Figure S1). The interactions of the
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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
transition state mimetic secondary hydroxyl group with the
catalytic aspartates, together with the vdW interactions of the
adjacent P1/P1 moieties, are critical for overall inhibitor
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
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7
8
9
0
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
binding. Together these interactions determine the positions of
the P2/P2 moieties in the active site, which in turn determine
the patterns of hydrogen bonding and vdW interactions of these
moieties with the protease. DRV contains a sulfonamide-based Phe-
Leu dipeptide isostere core with the (R)-configuration of the
secondary hydroxyl group, whereas LPV has a pseudo-C -symmetric
2
Phe-Phe dipeptide isostere with the (S)-configuration of the
hydroxyl group. Though chemically quite distinct, the core
scaffolds of LPV and DRV make largely similar hydrogen bonding and
vdW interactions with the protease. These inhibitors, however,
differ in their interactions with the protease in the S2 and S2
subsites.
The bis-THF moiety of DRV forms direct hydrogen bonds with the
main-chain NH of Asp29 and Asp30 in the S2 subsite, while the 4-
aminobenzene makes a direct hydrogen bond with the main-chain
carbonyl of Asp30 as well as a water-mediated interactions with
the side-chain carboxylate of the same residue in the S2 subsite
(Figure S1). The cyclic urea moiety of LPV makes direct hydrogen
bonds with the main-chain NH and side-chain carboxylate of Asp29.
The same moiety also makes water-mediated interactions with the
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main-chain carbonyl of Gly27 and the side-chain carboxylate of
Asp29 in the S2 subsite. However, in contrast to the P2 moiety of
DRV, the dimethylphenoxy acetate moiety of LPV only makes
hydrophobic interactions in the S2 subsite (Figure S1). The hybrid
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
compounds combine the pseudo-C -symmetric Phe-Phe isostere of LPV
2
with the bis-THF moiety of DRV and an ATV-like amino acid moiety
to enhance direct hydrogen bonding and vdW interactions in the S2
and S2 subsites.
The cocrystal structures of hybrid compounds revealed similar
binding conformations of the Phe-Phe isostere and the bis-THF
moiety as observed in the protease complexes with LPV and DRV,
respectively (Figure 2). Importantly, the bis-THF moiety is
positioned to interact with the main-chain NH of residues Asp29
and Asp30, mimicking the binding interactions of DRV (Figure 3).
This binding conformation is maintained in all hybrid compounds
including 4 regardless of the position of the bis-THF moiety. As
a result, within each compound series (with the bis-THF moiety at
the P2 and P2 position respectively), the structures aligned well
with variations occurring only at the hydrophobic amino acid moiety
(Figure 4).
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5
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6
LPV
DRV
Compound 12a
Compound 15a
Compound 4
P2
P2
P2′
P2′
0
1
2
3
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7
8
9
0
1
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0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Overlays of protease cocrystal structures bound to LPV
(PDB 6PJB) (grey, top) and DRV (6DGX) (brown, bottom) with
compounds 12a (bis-THF at P2; PDB 6PJD) (green), 15a (bis-THF at
P2′; PDB 6PJM) (orange), and 4 (PDB 6PJC) (salmon) revealed similar
binding conformations of the Phe-Phe isostere and the bis-THF
moiety as observed in the protease complexes with LPV and DRV,
respectively. Note that DRV is flipped 180º to compare with bis-
THF of 15a at the P2′ position.
In all complexes, the central hydroxyl group of the Phe-Phe core
maintains the key hydrogen bonds with the side-chain carboxylates
of the catalytic residues Asp25/Asp25. Nevertheless, subtle
differences in hydrogen bonding and vdW interactions were observed
between the Phe-Phe cores in LPV and the hybrid compounds. Notably,
one of the four water-mediated interactions with the flap residue
Ile50 is consistently longer (2.9–3.4 Å) than what is observed in
the LPV structure (2.7 Å) (Table S2). The two amide NH groups on
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each side of the Phe-Phe core could potentially interact with the
backbone carbonyl of Gly27 and Gly27′, but in the LPV structure
only the Gly27 is close enough to form a hydrogen bond (2.9 Å
versus 3.9 Å) (Figure 3). This phenomenon is also observed in the
protease complex with compound 4 (2.9 Å versus 3.7 Å) indicating
that the large hydrophobic P2′ moiety prevents the additional
hydrogen bond from occurring with Gly27′. In contrast, in all hybrid
compound structures the core NH groups are within 3.5 Å from the
backbone carbonyls of Gly27 and Gly27′ (Table S2), although
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
simultaneous interactions with both Gly27/Gly27 may not be stable.
Nevertheless, the combination of bis-THF and
a
flexible,
hydrophobic amino acid moiety allow both core NH groups to interact
with the main-chain carbonyls of residues Gly27/Gly27′.
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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
A
B
F53
F53
F53′
F53′
G48
G48′
G48
G48′
I47
I47′
I47
I47′
I50′
I50
I50′
I50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
1
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
P2
P2′
P2
P2′
D25
D25′
D25
D25′
D30
D30′ D30
D30′
G27′
D29′
F53′
G27′
D29′
D29
G27
D29
G27
C
D
F53
F53
F53′
G48
G48
G48′
G48′
I47
I47′
I47′
I47
I50′
I50
I50′
I50
P2
P2′
P2
P2′
D25 D25′
D25 D25′
D30
D30′ D30
D30′
D29
G27
G27′
D29′
D29
G27
G27′
D29′
Figure 3. Crystal structures of wild-type HIV-1 protease in complex
with LPV (PDB 6PJB) (A) and hybrid compounds 4 (PDB 6PJC) (B), 12a
(PDB 6PJD) (C), and 15a (PDB 6PJM) (D). The bis-THF moiety
maintains the hydrogen bonding interactions with the main-chain NH
of Asp29/Asp29 and Asp30/Asp30 in the S2/S2 subsites regardless
of the its position on the Phe-Phe core. Hydrogen bonds and water-
mediated interactions as determined by PyMol are shown as black
dashed lines with waters shown as red spheres.
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A
B
P1′
P1′
P2
P2
P2′
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
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P2′
P1
Figure 4. Superposition of HIV-1 protease complexes with compounds
2a–12h (A) and compounds 15a–15c (B), showing similar binding
P1
1
conformations of the bis-THF moiety regardless of the position and
minor differences at the Phe-Phe isostere, but major differences
at the P2/P2 amino acid moiety. The protease active site is shown
in a ribbon representation, with bound inhibitors depicted as
sticks.
The Phe-Phe isostere in hybrid compounds, in addition to
maintaining key hydrogen bonding interactions with the protease,
makes a number of favorable vdW interactions with the hydrophobic
residues that make up the S1/S1 subsites. Despite the same
phenylalanine side chains at the P1 and P1 positions, there are a
few notable differences in vdW contacts with the protease (Figure
S2). Compared to LPV, the P1/P1 phenyl group adjacent to the amino
acid moiety is shifted towards Val82 resulting in improved vdW
contacts with that residue, particularly for compounds with larger
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P2/P2 amino acid moieties. These localized gains in vdW contacts
are slightly lower for compounds 15a–c with bis-THF at the P2
position than their corresponding analogues 12a–c. The shift of
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the P1/P1 phenyl group towards Val82, creating more vdW contacts,
resulted in a nearly equal and opposite loss of contacts with Ile50
in the same subsite. The packing of the phenyl group adjacent to
the bis-THF moiety resulted in a small increase in vdW contacts
with the 80’s loop (residues 81, 82 and 84) but decrease in
contacts with flap residue Gly49. For compounds 15a–c, gains in
vdW contacts are smaller and the losses are greater. Overall,
despite localized losses and gains in vdW interactions for both
series of compounds, the packing of the P1 and P1 groups remain
generally similar to LPV.
The cocrystal structures of hybrid compounds (and 4) also
revealed similar binding interactions of the bis-THF moiety as in
DRV-protease complex regardless of the position, with the two ring
oxygen atoms positioned to form hydrogen bonds with the main-chain
NH of Asp29 and Asp30. However, there are subtle differences in
the position and puckering of the bis-THF rings, causing the
carbamate-linked THF ring oxygen atom to slightly shift away from
the backbone NH of Asp30 compared to DRV (3.1–3.4 Å versus 2.9 Å)
(Figure 5). Whereas the oxygen atom of the other THF ring maintains
a similar distance to the backbone NH of Asp29 (2.7–2.9 Å versus
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.8 Å) (Table S2). For compounds 12a–c, these subtle changes in
the bis-THF moiety arise from the differences in the binding of
core isosteres, where the P1 benzyl group and the carbamate are
shifted towards the B-chain compared to their position in DRV.
This is likely due to the additional carbon in the core or to
1
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accommodate the larger P1 group. In compounds 15a–c, the core
scaffold from the central hydroxyl to the bis-THF moiety is
identical. However, the opposite stereochemistry of the central
hydroxyl causes a subtle shift preventing the bis-THF moiety from
optimally interacting with Asp30′ (Figure 5). Despite these minor
differences in the position of the bis-THF moiety, the hybrid
compounds make similar hydrogen bonding interactions with the
protease in the S2/S2′ subsite compared to DRV.
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DRV
DRV
/
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D30
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D29
D29
DRV
12a
12a
15a
/
/15a
3.1
2.8
3.2
2.9
3.4
3
.2
D30
D30′
D29
D29′
DRV
12b
15b
/
/
12b
15b
3.1
2.9
3.1
2.9
3
.4
3.3
D30
D30′
D29
D29′
Figure 5. Comparison of the binding conformations of the bis-THF
moiety and protease-inhibitor intramolecular distances with the
main-chain NH of Asp29 and Asp30 in the S2/S2′ subsite of HIV-1
protease. As observed in the DRV-protease complex (PDB 6DGX), the
bis-THF moiety retained similar interactions with the protease in
complexes with compound 4 (PDB 6PJC) (top), the most potent hybrid
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compounds 12a (PDB 6PJD) and 15a (PBD 6PJM) (middle), and the least
potent hybrid compounds 12b (PDB 6PJE) and 15b (PDB 6PJN) (bottom),
irrespective of position on the Phe-Phe core.
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The bis-THF moiety of DRV is critical to its potency, not only
for the hydrogen bonding interactions with Asp29 and Asp30, but
also vdW packing with residues in the S2 subsite, particularly
Val32 and Ile47. In hybrid compounds, the minor change in the
position and puckering of the bis-THF moiety causes reduced vdW
interactions with Ala28 and Asp30, but increased interactions with
Ile47 and Gly48 (Figure S3). The main-chain carbonyl of Gly48 is
oriented towards the hydrophobic region of bis-THF, so enhanced
contacts with this residue may be unfavorable. In contrast,
compounds 4, 15a and 15b do not make these unfavorable contacts
with Gly48. With the exception of enhanced interactions with Ile47,
the bis-THF moiety in most hybrid compounds make less optimal vdW
interactions with the protease compared to those of DRV.
The major difference between the hybrid compounds and the
previously reported Phe-Phe core-based bis-THF containing PIs 4
and 5 is the introduction of an ATV-like amino acid moiety to
target the opposite S2/S2 subsite. The amino acid side chain
interacts with hydrophobic protease residues, while the N-
(methoxycarbonyl)-capping group allows for additional hydrogen
bonding interactions with the protease. As already noted, LPV’s
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P2′ moiety does not form any hydrogen bonds, and DRV’s P2′ moiety
makes one direct and one water-mediated interactions in the S2′
subsite (Figure S1). In most hybrid compounds, the carbonyl oxygen
of the carbamate makes one direct hydrogen bond with the backbone
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NH of Asp29 (Asp29), and the carbamate NH makes another direct
hydrogen bond with the backbone carbonyl of Gly48 (Gly48) in the
flaps (Figure 6). There are also a number of coordinated water
molecules that allow for additional water-mediated interactions.
The carbonyl oxygen of the carbamate coordinates the same water-
mediated interactions as the cyclic urea moiety of LPV, with the
backbone carbonyl of Gly27 (Gly27) and the side-chain carboxylate
of Asp29 (Asp29) (Figure 6). This network of polar interactions
is maintained by all amino acid moieties in both the S2′ and S2
subsites except the cycloleucine in compound 12b.
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A
B
G48
G48
P2
P2
LPV
ATV
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G27
D29
D29
G27
G48′
C
D
G48
12a P2′
15a
P2
G27
G27′
G48′
D29′
D29
E
G48
F
15b
12b
P2
P2′
G27
G27′
D29
D29′
Figure 6. Comparison of protease-inhibitor direct hydrogen bonds
and water-mediated interactions in the S2/S2 subsites for the
cyclic urea moiety of LPV (PDB 6PJB) (A), N-(methoxycarbonyl)-
tert-leucine of ATV (PDB 3EKY) (B), and the N-(methoxycarbonyl)-
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