Structure-based design, synthesis, and characterization of dual hotspot small-molecule HIV-1 Entry Inhibitors

Article abstract of DOI:10.1021/jm300265j

Cellular infection by HIV-1 is initiated with a binding event between the viral envelope glycoprotein gp120 and the cellular receptor protein CD4. The CD4-gp120 interface is dominated by two hotspots: a hydrophobic gp120 cavity capped by Phe43_CD4 and an electrostatic interaction between residues Arg59_CD4 and Asp368_gp120. The CD4 mimetic small-molecule NBD-556 (1) binds within the gp120 cavity; however, 1 and related congeners demonstrate limited viral neutralization breadth. Herein, we report the design, synthesis, characterization, and X-ray structures of gp120 in complex with small molecules that simultaneously engage both binding hotspots. The compounds specifically inhibit viral infection of 42 tier 2 clades B and C viruses and are shown to be antagonists of entry into CD4-negative cells. Dual hotspot design thus provides both a means to enhance neutralization potency of HIV-1 entry inhibitors and a novel structural paradigm for inhibiting the CD4-gp120 protein-protein interaction.

Full text of DOI:10.1021/jm300265j

Article
pubs.acs.org/jmc
Structure-Based Design, Synthesis, and Characterization of Dual
Hotspot Small-Molecule HIV-1 Entry Inhibitors
Judith M. LaLonde,*,†,∞ Young Do Kwon,‡,∞ David M. Jones,§,∞ Alexander W. Sun,§ Joel R. Courter,§
Takahiro Soeta,§ Toyoharu Kobayashi,§ Amy M. Princiotto,∥ Xueling Wu,‡ Arne Schon,⊥ Ernesto Freire,⊥
̈
Peter D. Kwong,‡ John R. Mascola,‡ Joseph Sodroski,∥,# Navid Madani,∥ and Amos B. Smith, III*,§
†Department of Chemistry, Bryn Mawr College, Bryn Mawr, Pennsylvania 19010, United States
‡Vaccine Research Center, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
20892, United States
§Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
∥Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, 450 Brookline Avenue,
Boston, Massachusetts 02115, United States
⊥Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, United States
#Department of Microbiology and Immunology, Harvard Medical School; Department of Immunology and Infectious Diseases,
Harvard School of Public Health; Ragon Institute of MGH, MIT and Harvard, Boston, Massachusetts 02115, United States
S
* Supporting Information
ABSTRACT: Cellular infection by HIV-1 is initiated with a
binding event between the viral envelope glycoprotein gp120
and the cellular receptor protein CD4. The CD4−gp120
interface is dominated by two hotspots: a hydrophobic gp120
cavity capped by Phe43CD4 and an electrostatic interaction
between residues Arg59CD4 and Asp368gp120. The CD4
mimetic small-molecule NBD-556 (1) binds within the
gp120 cavity; however, 1 and related congeners demonstrate
limited viral neutralization breadth. Herein, we report the
design, synthesis, characterization, and X-ray structures of
gp120 in complex with small molecules that simultaneously engage both binding hotspots. The compounds specifically inhibit
viral infection of 42 tier 2 clades B and C viruses and are shown to be antagonists of entry into CD4-negative cells. Dual hotspot
design thus provides both a means to enhance neutralization potency of HIV-1 entry inhibitors and a novel structural paradigm
for inhibiting the CD4−gp120 protein−protein interaction.
therapeutic agents that target the viral entry process are
approved in the U.S. for the treatment of individuals infected
with HIV-1: maraviroc,21 a small molecule CCR5-antagonist,
and enfuvirtide,22 a 36-amino acid peptide that binds gp41 and
prevents formation of the fusion peptide. Indeed, to date,
limited progress has been made on developing inhibitors that
target the initial step in viral entry, CD4−gp120 binding.23−26
Hence, disruption of the earliest CD4−gp120 protein−protein
interactions at the host cell−viral interface remains an
important, yet difficult strategy for blocking HIV-1 infection.
A number of X-ray crystal structures of the gp120 core
bound to the D1D2 domains of CD4 and the Fab of the
neutralizing antibody 17b, a surrogate for the co-receptor, have
been solved.27−34 The structure of the unbound form of the
simian immunodeficiency virus (SIV) gp120, which has a 35%
sequence identity with HIV-1 gp120, indicates an invariant
INTRODUCTION
■
Infection by the HIV-1 virus and the subsequent progression to
AIDS1,2 begin with protein−protein binding events between
the trimeric envelope glycoprotein spike (Env) and host cell
receptors.3−7 Each trimeric Env spike is composed of three
gp120 envelope glycoproteins and three gp41 transmembrane
proteins.8−10 The HIV-1 viral entry process begins with two
consecutive gp120−protein binding events, each associated
with changes in Env conformation.7,11−13 Attachment of HIV
occurs when gp120 binds to the T-cell CD4 receptor3,14
anchored to the cell membrane. Binding to CD4 then induces a
gp120 conformational change,11,12 which exposes the binding
site for the transmembrane chemokine co-receptor (CCR5 or
CXCR4).15−17 Once co-receptor binding occurs, gp41
rearranges to form the six-helix bundle which inserts into the
host cell membrane, culminating in fusion and viral entry.6,18−20
Inhibition of the initial entry event of the HIV-1 virus into
host cells remains a compelling, yet elusive means to prevent or
treat HIV-1 infection and AIDS. Currently, only two
Received: February 24, 2012
Published: April 12, 2012
© 2012 American Chemical Society
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Figure 1. gp120−CD4 binding interface and crystal structure of gp120−4 complex: (A) X-ray crystal structure of the ternary gp120 (gray)−CD4
(yellow)−17Fab (blue/light blue) complex; (B) dual hotspot interactions observed in the native CD4−gp120 interaction, with Phe43CD4 (yellow)
bound at the entrance to the Phe43 cavity with the adjacent salt bridge between Asp368gp120 and Arg59CD4; (C) crystal structure of 4 (orange)
bound in the Phe43 cavity of gp120(H375S) coree with the CD4 binding footprint highlighted in yellow; (D) 2Fo − Fc electron density map of 4
contoured at 1σ; (E) position of 4 region III relative to Asp368gp120; (F) 4−gp120 interaction map calculated and rendered with MOE ligand
interaction utility;73 (G) symbolic key for the ligand interaction map.
outer domain, with conformational differences in both the
bridging sheet and inner domain.35 In the ternary HIV-1 CD4−
gp120−17b complex a large internal cavity is formed at the
interface of the inner, outer, and bridging sheet domains of
gp120 (Figure 1A).31,32 Furthermore, a recent crystallographic
study of the unliganded HIV-1 gp120 monomeric core
demonstrates the propensity of gp120 to assume the CD4-
bound conformation when not restrained by the presence of
variable loops and interactions with gp41 in the trimer spike.36
At the CD4−gp120 interface, residue Phe43CD4 is located on
the CDR2-like loop and binds within the hydrophobic cavity of
gp120, termed the “Phe43 cavity”, while Arg59CD4 is located on
a neighboring β-strand and forms an electrostatic interaction
with Asp368gp120 (Figure 1B).31,32 Importantly, the amino acids
lining the Phe43 cavity, as well as Asp368gp120, are well-
conserved among primary HIV-1 isolates.37 Upon mutation of
Phe43CD4 to Ala43CD4 a 550-fold reduction of binding affinity
for gp120 was observed.38 Equally significant, the protein−
protein interaction is reduced 9-fold when Arg59CD4 is mutated
to Ala. Thus, the mutagenesis data and high degree of residue
conservation highlight the functionally critical role that both the
Phe43 cavity and Asp368gp120 binding hotspots play in
mediating the HIV-1 viral entry process. Although inhibition
of protein−protein interactions continues to be a significant
challenge,39−41 recent examples indicate that efficient inhibition
can be achieved by targeting surface hotspots.39,42 The large
CD4−gp120 contact surface, the orthogonal arrangement
between the Phe43 cavity and the Asp368gp120 hotspots, and
the therapeutic potential of targeting this interface comprise a
challenging opportunity to combine structure based design and
synthesis to develop novel small molecule antagonists of the
viral attachment and entry process.
Previous screening of a small-molecule library for inhibitors
of viral fusion led Debnath and co-workers to identify two
inhibitors of CD4−gp120 binding, NBD-556 (1) and NBD-557
(2) (Table 1).43 Subsequent studies in our laboratories revealed
that while 1 and 2 inhibit HIV-1 viral entry in CD4-positive,
CCR5-expressing T-cells, 1 and 2 actually activate viral
infection in CD4-negative cells (Table 1, column 3).44 Thus,
in the context of CD4-negative cells, these small molecules
both function as surrogates of the CD4 receptor and serve as
agonists by promoting HIV-1 entry. Promotion of HIV-1 entry
by NBD compounds may be possible in CD4-independent
HIV-1 variants;45,46 therefore, the agonistic properties of 1 and
2 must be eliminated for this chemotype. The thermodynamic
signature of 1 binding to gp120 provides further evidence of the
CD4-mimetic properties. For example, soluble CD4 (sCD4)
binding to gp120 exhibits a highly favorable binding enthalpy
balanced with an unfavorable entropy associated with molecular
ordering.47,48 Binding of 1 to the gp120 core is also
characterized by both a favorable change in enthalpy (ΔH)
and a large, unfavorable entropic component (−TΔS) (Table
1).44,49 The similarity in thermodynamic signature for sCD4
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Table 1. Tetramethylpiperidine Analogues
a
Compounds 1, 2, 3, and 4 were assayed in triplicate, and the data are reported as the mean with standard deviation for 1, 1, 1, and 52 experiments,
respectively. The compound concentrations that inhibited 50% of virus infection (IC50) were determined by infecting Cf2Th-CD4/CCR5 cells with
b
10 000 RT units of wild-type HIV-1YU2 virus expressing luciferase with increasing concentrations of the compound. The compound concentrations
that inhibited 50% of virus infection (IC50) when assayed against viruses with the ampotrophic murine leukemia virus (A-MLV) envelope
c
glycoproteins. Activation of viral infectivity was determined by infecting Cf2Th-CCR5 cells with recombinant HIV-1YU2 in the presence of NBD
analogues. The luciferase activity in the target cells incubated with each compound was divided by that in the cells incubated with 2 to obtain the
d
relative activation of infectivity. The dissociation constant (Kd) and the change in enthalpy (ΔH) were determined at 25 °C by isothermal titration
calorimetry using a high-precision VP-ITC titration calorimetric system from MicroCal/GE Healthcare (Northampton, MA, U.S.). The calorimetric
cell (approximately 1.4 mL), containing gp120 from the YU2 strain dissolved in PBS (Roche Diagnostics GmbH, Mannheim, Germany), pH 7.4,
with 2% DMSO, was titrated with the different inhibitors dissolved in the same buffer. The concentration of gp120 was approximately 2 μM, and
e
inhibitor at a concentration of 80−130 μM was added in aliquots of 10 μL until saturation was reached (usually in 20−30 injections). The change in
Gibbs energy (ΔG) was calculated from the affinity according to the relation ΔG = −RT ln Ka, where Ka is the association constant (Ka = 1/Kd), R is
f
g,h
the gas constant (1.987 cal/(K·mol)), and T is the absolute temperature in kelvin. TΔS was calculated from the relation ΔG = ΔH − TΔS. Data
for 1−3 as reported in Schon et al.49 and Madani et al.44
̈
and 1 suggests that the small molecules both induce and
stabilize gp120 in a CD4-bound-like conformation.
RESULTS AND DISCUSSION
■
X-ray Crystallographic Characterization of 4 Bound to
SAR studies identified three pharmacophoric elements that
define the NBD chemotype: region I, a para-halogenated
phenyl ring; region II, the oxalamide linker; region III, a
tetramethylpiperidine ring.44,50−52 Initial optimization of
regions I and II44,51 led to the synthesis of JRC-I-191 (3)
with improved binding affinity.53 Mutagenesis and modeling of
3 revealed that these small molecules bind to the highly
conserved gp120 cavity and compete with CD4 bind-
ing.44,49,51,54 Unexpectedly, we found that 3 inactivates HIV-1
by inducing irreversible conformational changes that result in
an active, yet transient, intermediate state of the Env trimer. If
the metastable trimer does not encounter a target cell within
the lifetime of the activated intermediate, it decays to a state
that is no longer competent for viral entry.55 Further
optimization of 3 led to the synthesis and biological evaluation
of TS-II-224 (4), the most potent inhibitor of CD4−gp120
binding possessing the NBD chemotype.44,51 While analogues 3
and 4 have improved affinities for gp120, as measured by
isothermal titration calorimetry (ITC), they are also more
effective agonists of viral entry (relative to 1) in CD4-negative
cells, clearly an undesired property (Table 1).44,51 The recent
cocrystal structure of 1 bound to gp120 (clade C1086), at a
resolution of 3.0 Å, confirms that 1 binds within the conserved
Phe43 cavity.36 However, 1 does not form specific interactions
with Asp368gp120 at the second binding hotspot. Thus, the
essential Arg59CD4−Asp368gp120 electrostatic interaction on the
CD4−gp120 surface has yet to be integrated in NBD small-
molecule design. Hence, we hypothesized that engaging the
Asp368gp120 binding hotspot would significantly improve viral
inhibition properties while also targeting a residue that is well-
conserved across many HIV-1 subtypes.37 Herein, we describe a
computational approach for the design of region III analogues
that target both the Phe43 cavity and the Asp368gp120 hotspots.
the Core of gp120. Recently, Kwon et al. determined four
structures of the unliganded gp120 “extended core” (coree)
from clade B (YU2 strain), clade C (C1086 and ZM109
strains), and clade A/E (93TH057 strain) primary HIV-1
isolates.36 The gp120 coree includes the N-terminus but
excludes the variable loops and facilitates crystallization of the
unliganded gp120. The clade A/E93TH057 construct of gp120
coree produced the highest resolution structure (1.9 Å).36 This
protein also produced well diffracting crystals in complex with
VRC01-like antibodies.34,56 Therefore, we employed the same
clade A/E gp120 coree as a template for small molecule
cocrystallizations, with the exception that we mutated His375
to Ser within the Phe43 cavity to accommodate ligand binding.
The crystal structure of 4 bound to clade A/E gp120(H375S)
coree was determined at 2.0 Å resolution by molecular
replacement (Figure 1C−E and Table 3). This structure
reveals that 4 binds similarly to 1 in the ligand−gp120
complex,36 with region I bound deep within the Phe43 cavity
and forming aromatic stacking interactions with Phe382gp120
and Trp427gp120, as well as hydrophobic contacts with
Val255gp120 and Ile424gp120. Both amide nitrogens of region II
form hydrogen bonds with the main-chain carbonyls on
opposite sides of the Phe43 cavity (Gly473gp120 from the
outer domain and with Asn425gp120 from the bridging sheet
domain). In the cavity vestibule, one region III gem-dimethyl
moiety forms van der Waals contacts with the bridging sheet
domain residues Trp427gp120, Asn428gp120, and Glu429gp120
,
while the second geminal dimethyl group contacts the outer
domain residues Gln473gp120 and Asn474gp120. This cocrystal
structure was employed as the starting point for our virtual
screening and inhibitor design efforts.
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Structure Based Design. On the basis of the critical role of
the Arg59CD4−Asp368gp120 electrostatic interaction in CD4−
gp120 recognition and binding,38 we hypothesized that
addition of a similar chemotype to region III, directed toward
the Asp368gp120 hotspot, would improve the viral inhibition
properties of 4. The cocrystal structure of 4 bound to the clade
A/E gp120(H375S) coree indicated the proximity of C4 of the
tetramethylpiperidine ring (region III) and the carboxylate side
chain of Asp368gp120 (Figure 1E). Rather than performing
systematic synthetic modifications of region III congeners, we
chose a virtual screening strategy to identify alternative scaffolds
is not a chemically stable entity, we exploited this archetype to
replicate the desired interactions between the small molecule
and gp120. The prototype was assessed with the docking
program GOLD57,58 to provide a three-dimensional model that
incorporated the desired trajectory of the amino group.
Following our previously reported virtual screening paradigm51
employing the ROCS shape-based similarity algorithm,59−61 the
amine prototype was used to search the ZINC database of
commercially available compounds.62,63 Virtual screening
identified several bicyclic primary amines, such as amino-
bicyclononanols, indanols, and diaminoindanes, that displayed
both shape and chemotype similarity to prototype 5 and
directed a hydrogen bond donor toward Asp368gp120 (Table S1
in Supporting Information). In the end, we chose the
synthetically versatile indane scaffold and docked the 1,2- and
1,3-diaminoindane enantiomers with GOLD.57,58 The trans-1,2-
diaminoindane isomers (−)-6 and (+)-6 were predicted to
form weak polar interactions with Asp368gp120 and thus were
selected for synthesis (Figure 2).
that would contain a basic amine oriented toward Asp368gp120
.
Hence, an analogue possessing a primary amine attached to C4
of the 4-aminotetramethylpiperidine was constructed in silico
to afford query structure 5 (Figure 2). While geminal diamine 5
Chemistry. To assess the suitability of the 1,2-diamines as
region III platforms, we began with the synthetic conjugation of
the commercially available cis-1-aminoindan-2-ol (−)-8 to
ethyloxalamide 7 (Figure 3). Amide bond formation was
achieved under thermal conditions to furnish (+)-9. Enan-
tiomer (−)-9 was also prepared employing the same procedure,
beginning with (+)-8. These cis-indanol enantiomers (+)-9 and
(−)-9 exhibited weak inhibition of viral entry (data not shown).
Given that the trans-1,2-diaminoindanes exhibited the requisite
spatial arrangement in the docking studies, we next converted
(+)-9 to the corresponding trans-amine (+)-6. Tosylation of
alcohol (+)-9 followed by SN2 displacement with sodium azide
furnished (+)-10. Reduction of the azide employing Lindlar’s
catalyst then provided the desired amine AWS-I-45 [(−)-6]. An
identical synthetic sequence, beginning with (+)-9, was
employed to construct the enantiomer AWS-I-50 [(+)-6].
Although (−)-6 and (+)-6 exhibited nonspecificity in our
standard viral-entry inhibition assays (vide infra), conversion of
the alcohol to the amines demonstrated an increase in binding
affinity [Kd(−)-6 = 1.2 μM; Kd(+)-6 = 1.9 μM] compared to 1
(Kd = 3.7 μM). Encouraged by this result, we sought to mimic
further the Arg59CD4 side chain. Assessment of guanidine
analogues by docking predicted favorable interactions between
Figure 2. Structure-based design strategy: (A) crystal structure of 4
(orange) bound to gp120 and the docked model of the germinal
diamine prototype 5 (green) used as a query in ROCS shape-based
virtual screening; (B) docked conformations of the trans-1,2-
diaminoindane isomers [(−)-6 and (+)-6] (magenta and light blue,
respectively) appended to the regions I and II scaffold of 4.
Figure 3. Synthesis of the guanidinium analogues (−)-12 and (+)-12.
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Table 2. Indane Analogues
a
Compounds (−)-6, (+)-6, (−)-12, and (+)-12 were assayed in triplicate, and the data are reported as the mean with standard deviation for 5, 6, 7,
b
and 32 experiments, respectively. The compound concentrations that inhibited 50% of virus infection (IC50) when assayed against viruses with the
ampotrophic murine leukemia virus (A-MLV) envelope glycoproteins. Activation of viral infectivity was determined by infecting Cf2Th-CCR5 cells
c
with recombinant HIV-1YU2 in the presence of NBD analogues. The luciferase activity in the target cells incubated with each compound was divided
d
by that in the cells incubated with 2 to obtain the relative activation of infectivity. The dissociation constant (Kd) and the change in enthalpy (ΔH)
were determined at 25 °C by isothermal titration calorimetry using a high-precision VP-ITC titration calorimetric system from MicroCal/GE
Healthcare (Northampton, MA, U.S.). The calorimetric cell (approximately 1.4 mL), containing gp120 from the YU2 strain dissolved in PBS (Roche
Diagnostics GmbH, Mannheim, Germany), pH 7.4, with 2% DMSO, was titrated with the different inhibitors dissolved in the same buffer. The
concentration of gp120 was approximately 2 μM, and inhibitor at a concentration of 80−130 μM was added in aliquots of 10 μL until saturation was
e
reached (usually in 20−30 injections). The change in Gibbs energy (ΔG) was calculated from the affinity according to the relation ΔG = −RT ln Ka,
f
where Ka is the association constant (Ka = 1/Kd), R is the gas constant (1.987 cal/(K·mol)), and T is the absolute temperature in kelvin. TΔS was
calculated from the relation ΔG = ΔH − TΔS.
the ligand and Asp368gp120. These analogues were thus selected
for synthesis. Here we employed 1H-pyrazol-1-carboxamidine
monohydrochloride (11) to convert amine (+)-6 and the
corresponding enantiomer (−)-6 to the desired guanidinium
functionality, affording AWS-I-169 [(−)-12] and DMJ-I-228
[(+)-12], respectively. Note that following purification by high
performance liquid chromatography (HPLC), the guanidinium
formate salts of (−)-12 and (+)-12 were isolated and employed
in all biological assays. The synthesis of (+)-12 is illustrated in
Figure 3.
Evaluation of Viral Inhibition. Amines (−)-6 and (+)-6
and the corresponding guanidinium salts (−)-12 and (+)-12
were first tested against monotropic and dual-tropic HIV-1
strains in single-round infection of recombinant HIV-1,
encoding firefly luciferase.64 The recombinant viruses employed
were pseudotyped with HIV-1 envelope glycoproteins derived
from either a CXCR4, laboratory-adapted HXBc2 isolate, or the
CCR5, primary YU2 isolate. To evaluate compound specificity
for HIV-1, the viruses were pseudotyped with the envelope
glycoproteins of the amphotropic murine leukemia virus (A-
MLV), an unrelated retrovirus.53 In both monotropic (HXBc2,
YU2, ADA, JRFL) and dual-tropic (89.6 and KB9) viruses,
(−)-6 and (+)-6 inhibited entry on cells coexpressing CD4 and
CCR5 or CXCR4 with IC50 in the range 50−90 μM but also
exhibited nonspecificity by inhibiting entry of the A-MLV
control virus with a similar IC50 (Table 2). The guanidinium
salts (−)-12 and (+)-12, on the other hand, demonstrated
improved inhibition of viral entry (22.9 and 21.3 μM,
respectively). Moreover, the inhibition proved to be completely
specific to HIV-1, as neither compound was found to inhibit
entry of A-MLV (Table 2 and Figure 4). Assessment of the in
vitro cytotoxicity of (+)-12 in Cf2Th-CD4-CCR5 cells did not
demonstrate measurable inhibition of cell growth (Figure S1 in
Supporting Information). Thus, analogues (−)-12 and (+)-12
possess significantly improved antiviral activities relative to the
starting compounds 1−4.
To characterize further the antiviral breadth, (−)-12 and
(+)-12 were tested against 42 diverse HIV-1 strains (clades B
and C). While the parent compound 1 was found to neutralize
HIV-1 with a geometric mean titer (GMT) IC50 = 29.3 μM,
only 12% of isolates were found to have IC50 < 10 μM (Figure
5 and Table S3 in Supporting Information). In contrast, both
(−)-12 and (+)-12 and were found to display 100%
neutralization breadth among the viral isolates tested.
Compound (−)-12 possessed a GMT IC50 of 8.9 μM with
52% of the viral isolates having IC50 < 10 μM. Similarly, (+)-12
revealed a mean IC50 of 7.9 μM with 57% of the isolates having
IC50 < 10 μM. Compounds (−)-12, (+)-12, and 1 all exhibited
less antiviral activity against clade C viruses compared to clade
B strains. Given the high degree of sequence conservation in
the Phe43 cavity and that the gp120 trimer exhibits a high
degree of conformational diversity in solution,48,65,66 the
decreased potency in clade C is not easily assignable.
Given the broadly neutralizing capabilities of the indane-
based analogues, we asked if (−)-12 and (+)-12 could pose a
therapeutic disadvantage by functionally replacing CD4 and
enhancing cellular infection. We have previously measured the
enhancement of HIV-1 entry in CD4-independent viruses,
demonstrating that 1−4 enhance the entry of HIV-1 YU2
viruses in CD4-negative cells (vide supra).44,51 We found that
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Table 3. Data Collection and Refinement Statistics
4
(+)-6
(−)-12
(+)-12
Data Collection
space group
P21
P21
P21
P212121
cell dimension
a, b, c (Å)
α, β, γ (deg)
resolution (Å)
64.72, 68.90, 94.51
90, 91.23, 90
64.66, 68.48, 94.74
90.0, 91.60, 90
65.44, 68.60, 94.54
90, 91.38, 90
63.74, 67.52, 89.25
90, 90, 90
a
a
a
a
50−2.0 (2.03−2.00)
50−1.80 (1.83−1.80)
50−1.80 (1.83−1.80)
46.3−1.88 (1.9−1.88)
a
a
a
a
Rsym
0.09 (0.49)
0.06 (0.59)
0.08 (0.57)
0.09 (0.37)
a
a
a
a
I /σ(I)
11.5 (1.5)
19.4 (1.1)
17.5 (1.8)
34.7 (2.5)
a
a
a
a
completeness (%)
redundancy
94.1 (74.0)
97.0 (72.9)
93.3 (55.0)
97.4 (72.1)
a
a
a
a
3.1 (1.9)
3.3 (1.9)
6.5 (3.2)
5.2 (2.0)
Refinement
33.7−1.79
74205
resolution (Å)
41−1.98
53979
33.7−1.80
72604
46.3−1.88
29830
no. reflections
R
work/Rfree
no. atoms
protein
20.3/23.8
19.8/22.4
18.8/20.8
19.3/23.3
5308
388
5308
384
5308
392
2654
196
ligand/ion
water
394
479
443
198
B factor
protein
ligand/ion
water
43.9
67.2
42.8
41.0
59.3
42.5
46.0
61.1
45.4
34.0
47.0
39.8
rmsd
bond length (Å)
bond angle (deg)
0.002
0.663
4DKO
0.002
0.699
4DKP
0.002
0.644
4DKR
0.007
0.869
4DKQ
PDB entry
a
Values in parentheses are for the highest-resolution shell.
both amines (−)-6 and (+)-6 enhanced viral entry in CD4-
negative cells at levels comparable to those previously measured
for 1−4 (Table 2). Remarkably, the guanidinum salts (−)-12
and (+)-12 displayed no appreciable entry enhancement of
YU2 viruses into CD4-negative, CCR5-expressing cells (Table
2). We further evaluated whether the lack of viral enhancement
might be related to the unproductive binding of (−)-12−gp120
or (+)-12−gp120 complexes to the CCR5 receptor. Surface
plasmon resonance (SPR) assessment of the CCR5 antibody
surrogate 17b binding to the small molecule−gp120 complexes
indicated that both 4 and the indane congeners [(−)-6, (+)-6,
(−)-12, and (+)-12] enhance binding of 17b to both the
monomeric core and full-length gp120 (Figure S2 in
Supporting Information). Therefore, the enhancement of viral
entry into CD4-negative, CCR5-expressing cells is likely
unrelated to the formation of the 17b binding epitope.
Hence, the addition of the guanidinium functionality reduces
the undesired enhancement of viral infection observed for 1−4,
(−)-6, and (+)-6. We therefore conclude that (−)-12 and
(+)-12 function as viral entry antagonists in the context of
CD4-negative cells expressing the CCR5 co-receptor.
Thermodynamic Signature of Small Molecule Antag-
onist Binding. The binding of the novel analogues to full-
length gp120 from the YU2 strain was next characterized by
ITC. The measured binding affinities of (−)-12 (Kd = 0.30
μM) and (+)-12 (Kd = 0.25 μM) demonstrate that
incorporation of the guanidinium functionality restores potency
comparable to that of parental compound 4 (Kd = 0.30 μM)
(Table 2). We also note that the observed discrepancies
between measured IC50 and Kd values (Table 2) reflect the
differences in small molecule binding to the full Env trimer and
the gp120 monomer in viral and ITC binding assays,
respectively. The binding efficiency index (BEI) is defined as
the binding affinity (Kd) divided by molecular weight, while the
surface efficiency index (SEI) is defined as the binding affinity
(Kd) divided by the molecular polar surface area.67 Both (−)-12
and (+)-12, with identical affinity and structural properties,
yield the same calculated BEI and SEI, 16.8 and 6.4,
respectively. When compared to 22 known inhibitors of
protein−protein interactions (BEI = 11.7
2.4 and SEI =
7.2 3.0)41,68 and 92 marketed drugs, (BEI = 25.8 7.9 and
SEI = 14.5 8.7), the values for (−)-12 and (+)-12 fall within
the lower bounds of binding efficiency and surface efficiency
space for marketed drugs.67
We next assessed the enthalpic and entropic contributions to
binding compared to sCD4 binding (Table 2 and Figure 6A).
The binding of sCD4 to gp120 at 25 °C is associated with a
large enthalpy change (ΔH = −34.5 kcal/mol) that is partially
negated by a large unfavorable entropy change (−TΔS = 23.6
kcal/mol) and is associated with a large negative heat capacity
(ΔCp) of −1800 cal/(K·mol).49 Such a binding event suggests a
large molecular reordering of gp120 upon CD4 binding.48,49
Compounds 1−4 also exhibit a large unfavorable entropy upon
binding to gp120.49 Thus, these small molecules bind and
stabilize a gp120 conformation similar to that observed in the
CD4 bound state. Characterization of analogues (−)-6 and
(−)-12 (Table 2) indicated a thermodynamic signature
resembling that of 4 (Figure 6B and Table 2). Conversely,
the antipodes (+)-6 and (+)-12 exhibited decreased unfavor-
able entropy contributions (−TΔS = 7.6 and 5.9 kcal/mol,
respectively) compared to 1−4, (−)-6, and (−)-12. Therefore,
the (+)-6 and (+)-12 enantiomers, as shown in Figure 6, have a
distinct and preferable thermodynamic signature, with gp120
incurring a lower entropic penalty upon binding. The
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Figure 5. Comparison of neutralization by 1, (−)-12, and (+)-12
against HIV-1 Env-pseudoviruses. Dendrograms are made by the
neighbor-joining method, showing the protein distance of gp160
sequences from 42 HIV-1 clades B and C isolates. The clade B
reference strain HXBc2 was used to root the tree, and the amino acid
distance scale is indicated with a value of 1% distance as shown.
Neutralization potency of 1, (−)-12, and (+)-12 is indicated by the
color of the branch for each virus. The data under the dendrograms
show the percent of viruses neutralized with IC50 < 100 μM and IC50
<
10 μM and the IC50 geometric mean titer (GMT) for viruses
neutralized with IC50 < 100 μM. Data are included in Table S3 in
Supporting Information.
conserved Phe43 cavity in the context of the functional
trimeric Env spike. Cocrystallization experiments with com-
pounds (+)-6, (−)-12, and (+)-12 bound to the monomeric A/
E gp120(H375S) coree were employed to define further the
binding interactions of these small molecule ligands with
gp120.
Figure 4. Virologic assessment of guanidinium analogues (−)-12 and
(+)-12: (A) effect of (−)-12 on the infection of Cf2Th-CD4-CCR5
cells by recombinant luciferase-expressing HIV-1 envelope glycopro-
teins of the YU2 strain of HIV-1 or the amphotropic murine leukemia
virus (AMLV); (B) effect of (+)-12 on the infection of Cf2Th-CD4-
CCR5 cells by recombinant luciferase-expressing HIV-1 envelope
glycoproteins of the YU2 strain of HIV-1 or AMLV. Virus infection is
expressed as the percentage of infection (measured by luciferase
activity in the target cells) observed in the presence of increasing
concentrations of (−)-12 or (+)-12 relative to the level of infection
observed in the absence of compound. The results are representative
of 17 independent experiments. Additional data are provided in Tables
S2 and S3 in Supporting Information.
Structural Characterization of Small Molecule−gp120
Interactions by X-ray Crystallography. Amine (+)-6 and
guaninidium salts (−)-12 and (+)-12 were cocrystallized in
complex with the clade A/E gp120(H375S) coree and yielded high
resolution X-ray cocrystal structures at 1.8, 1.8, and 1.9 Å,
respectively (Figures 7, 8, and 9 and Table 3).72 In the three
crystal structures, regions I and II maintain similar interactions,
as observed with 4, binding deep within the Phe43 cavity of
gp120 (Figures 7, 8, and 9). The mean rmsd values of the
backbone and side chain atoms for residues lining the Phe43
cavity are 0.39 and 0.68 Å, respectively, indicating structural
conservation in the Phe43 hotspot among the ligand−gp120
complexes. Moreover, comparison of the (+)-6−gp120,
(−)-12−gp120, or (+)-12−gp120 complex with the unliganded
gp120 structure36 or the CD4−gp120 structure (PDB code
1G9M)31 shows that the Asp368gp120 side chain has the same
orientation upon small molecule binding. Inspection of region
III in the (+)-6−gp120 complex (Figure 7) reveals that the 2-
amino group does not interact with the Asp368gp120 hotspot.
Nonetheless, the indane scaffold of (+)-6 occupies a similar
position to the tetramethylpiperidine ring of 4 (Figure S3A in
Supporting Information). The five-membered ring of (+)-6
overlaps with the piperidine ring, while the aromatic ring
overlaps with one of the gem-dimethyl groups of 4 (Figure S3A
in Supporting Information). The aromatic ring of (+)-6 also
forms extensive contacts with outer domain residues
Gly473gp120 and Asn474gp120. Superposition of the (+)-6−
associated changes in heat capacity for the binding of 4, (−)-12,
and (+)-12, calculated from the temperature dependence of the
binding enthalpies, are −738 36, −817 15, and −398
5
cal/(K·mol), respectively (Figure 6B). The change in the heat
capacity for (+)-12 approaches the expected value for the burial
of a small hydrophobic molecule.69,70 Thus, enantiomer (+)-12
displays chemical properties and a thermodynamic signature
resembling small molecule drug binding.
To determine if the antiviral and thermodynamic properties
of (−)-12 and (+)-12 were exerted through direct binding to
the Phe43 cavity hotspot, we measured viral inhibition in the
Phe43 cavity filling mutant S375Wgp120 YU2 virus, wherein the
tryptophan side chain restricts access to the Phe43 cavity.71
Unlike the wild-type virus, the YU2 S375Wgp120 was completely
resistant to (−)-12 and (+)-12 (Table S2 in Supporting
Information), supporting the claim that these new compounds
exhibit their antiviral effects through binding within the
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chain atoms, while the five-membered ring overlaps with the
main-chain atoms of Ser42CD4 and Phe43CD4 (Figure S3A and
S3B in Supporting Information). Therefore, the indane ring
effectively mimics the CD4 β-turn spanning the interface of the
outer domain and bridging sheet of gp120.
Addition of the guanidinium group to afford (−)-12 and
(+)-12 was envisioned to strengthen the ligand interactions
with the Asp368gp120 hotspot to resemble more closely the
native protein−protein interaction. The 1.8 Å cocrystal
structure of the (−)-12−gp120 complex reveals that the indane
ring is rotated 90° in the cavity vestibule (relative to region II),
leaving the plane of the ring tilted away from the gp120 surface
(Figure 8). Hence, the arene ring of (−)-12 does not form
contacts with the outer domain as observed in the (+)-6−
gp120 complex (Figure 8). Furthermore, the altered orientation
imposed by the stereochemical configuration of (−)-12 dictates
that the guanidinium functionality approach Asp368gp120 near
the outer domain rather than near the bridging sheet as
observed with (+)-6. Interestingly, the guanidinium moiety of
(−)-12 does not form a salt bridge with Asp368gp120. Instead,
the guandinium group forms hydrogen bonds with a network of
ordered water molecules (Wat601−Wat603) and forms a single
ionic interaction with Asp368gp120 (Figure 8). Therefore, in the
(−)-12−gp120 complex, the guanidinum moiety resides at the
center of a hydrogen-bonded network between ordered
watered and the outer domain residues Gly472gp120 and
Asp368gp120
.
Further analysis of the (+)-12−gp120 cocrystal structure
indicates that the guanidinium group of (+)-12 approaches
Asp368gp120 between the bridging sheet and outer domains
(Figure 9). The guanidinium moiety of (+)-12 also forms a
single ionic interaction with Asp368gp120 and in addition forms
an intramolecular hydrogen bond to the region II oxalamide
and a hydrogen bond to crystallographic water Wat501.
Interestingly, Wat501 occupies a position equivalent to that
of Arg59CD4 observed in the CD4−gp120 complex (Figure S3B
in Supporting Information).31 Wat501 is at the center of a
water mediated hydrogen-bonding network that connects
(+)-12 with both the outer domain (Asp368gp120) and the
bridging sheet (Met426gp120). Thus, between the two
complexes [(+)-12 versus (−)-12], a unique pattern of
hydrogen bonding exists between the guanidinium moiety,
ordered water molecules, and the gp120 outer and bridging
sheet domains. As judged by the dissociation constant of
Figure 6. Thermodynamic characterization of indanes. (A) Gibbs
energy (ΔG) and the enthalpic (ΔH) and entropic (−TΔS)
contributions are compared for the binding of 4, (−)-6, (+)-6,
(−)-12, and (+)-12 to full-length monomeric gp120 at 25 °C. (B) The
temperature dependence of ΔH for 4 (circles), (−)-12 (squares), and
(+)-12 (triangles) is shown. The changes in heat capacity, calculated
from linear regression of the slopes, are −738 36 cal/(K·mol) for 4
(solid line), −817 15 cal/(K·mol) for (−)-12 (short dashed line),
and −398 5 cal/(K·mol) for (+)-12 (dashed line).
gp120 structure with a CD4−gp120 structure indicates that the
indane arene ring overlaps with Asn40CD4 and Gly41CD4 main-
Figure 7. Crystal structure of (+)-6 bound to gp120: (A) 2Fo − Fc electron density map of (+)-6 bound within the Phe43 cavity of gp120(H375S)
coree contoured at 1σ; (B) position of (+)-6 region III relative to Asp368gp120; (C) (+)-6−gp120−ligand interaction map, as described in Figure 1.
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Figure 8. Crystal structure of (−)-12 bound to gp120: (A) 2Fo − Fc electron density map of (−)-12 (pink) and four crystallographic water
molecules (red spheres) bound within the Phe43 cavity of gp120(H375S) coree contoured at 1.2σ; (B) position of (−)-12 region III relative to
Asp368gp120, illustrating the water-mediated hydrogen bonding network; (C) (−)-12−gp120 ligand interaction map, as described in Figure 1.
Figure 9. Crystal structure of (+)-12 bound to gp120: (A) 2Fo − Fc electron density map of (+)-12 (magenta) and four crystallographic water
molecules (red spheres) bound within the Phe43 cavity of gp120(H375S) coree contoured at 1.2σ; (B) position of (+)-12 region III relative to
Asp368gp120 illustrating the water-mediated hydrogen bonding network; (C) (+)-12−gp120 ligand interaction map, as described in Figure 1.
(+)-12 (Kd = 0.25 μM) and (−)-12 (Kd = 0.30 μM) compared
to (+)-6 (Kd = 1.9 μM), the guanidinium−Asp368gp120
interaction and water mediated hydrogen bonding contributes
favorably to the binding affinity of both (+)-12 and (−)-12 to
gp120. Furthermore, the distinct thermodynamic signatures of
gp120 binding between the (+)-12 and (−)-12 enantiomers are
consistent with the different binding modes revealed in the
crystal structures of these enantiomeric compounds bound to
gp120.
We subsequently demonstrated that (−)-12 and (+)-12
specifically inhibit HIV-1 entry through interaction with the
Env spike by virtue of the incorporation of a guandinium group.
Both (−)-12 and (+)-12 have improved and broad antiviral
potency and, importantly, do not enhance viral infection in
CD4-negative cells. The wide neutralization breadth against
diverse strains of clades B and C viral isolates validates the
importance of considering strain variability when evaluating
binding hotspots appropriate for inhibitor design. Furthermore,
in the context of CD4-negative cells, amines (−)-6 and (+)-6
function as agonists, promoting the CD4-independent viral
entry process. Incorporation of the guanidinium functionality to
afford (−)-12 and (+)-12 converts these agonists to antagonists
of viral entry and eliminates the undesired property of
promoting CD4-independent viral entry. The ability to
suppress the undesired agonist properties demonstrates that
the Phe43 cavity of gp120 and the NBD compound class
remains an attractive target for the development of HIV-1 entry
inhibitors.
CONCLUSIONS
■
We have targeted the highly conserved Phe43 cavity and
Asp368gp120 hotspots at the protein−protein interface of the
CD4−gp120 complex to improve the antiviral potency and
breadth of 1−4. In this work a modified ROCS “scaffold
hopping”,60,61 virtual screening strategy was employed to
develop inhibitors that target both the Phe43gp120 and
Asp368gp120 hotspots. A hypothetical 1,1-diamine molecule
(5), encompassing the desired chemotype and spatial features,
was employed to search chemical space to identify a lead
compound possessing a basic amine directed toward the
Asp368 hotspot. This strategy, in conjunction with molecular
design and synthesis, has led to the identification of two novel
analogues that employ the trans-1,2-disubstituted indane
scaffold [e.g., (−)-12 and (+)-12] to direct a guanidinium
group toward the Asp368gp120 hotspot.
The crystal structures described herein further confirm our
design criteria and represent the highest resolution structures,
to date, of a small molecule entry antagonist bound to the
gp120 core. Importantly, the guanidinium groups of (−)-12
and (+)-12 form an electrostatic interaction with Asp368gp120
and reveal two distinct water-mediated hydrogen-bonding
networks between the antagonist guanidinium group and
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and carbon-13 NMR spectra were recorded on a Bruker AM-500 at
305 K unless otherwise noted. Chemical shifts are reported relative to
chloroform (δ 7.26), methanol (δ 3.31), or dimethyl sulfoxide (δ 2.50)
for 1H NMR and chloroform (δ 77.0), methanol (δ 49.2), or dimethyl
sulfoxide (δ 39.4) for 13C NMR. High-resolution mass spectra were
recorded at the University of Pennsylvania Mass Spectroscopy Service
Center on either a VG Micromass 70/70H or VG ZAB-E
spectrometer. Analytical HPLC was preformed with a Waters
HPLC−MS system, consisting of a 515 pump and Sunfire C18
reverse phase column (20 μL injection volume, 5 μm packing material,
4.5 mm × 50 mm column dimensions) with detection accomplished
by a Micromass ZQ mass spectrometer and 2996 photodiode array
detector. Preparative HPLC was preformed with a Gilson 333/334
preparative pump system equipped with a 5 mL injection loop, Sunfire
C18 OBD column (5 μm packing material, 19 mm × 100 mm column
dimensions) equipped with a UV−vis dual wavelength detector (210
and 254 nm) and 215 liquid handling module. Solvent systems
employed were based on the following buffers: buffer A consisting of
H2O containing 0.05% formic acid; buffer B consisting of MeCN
containing 0.05% formic acid. The purity of new compounds was
judged by NMR and HPLC−MS to be in excess of 95%.
Asp368gp120. Interestingly, (−)-12 and (+)-12 also display
distinct thermodynamic signatures. The smaller unfavorable
entropy for (+)-12 binding to gp120, along with the favorable
binding efficiency and surface efficiency indices, may make this
enantiomer a more suitable candidate for continued opti-
mization. The modest IC50 = 20 μM exhibited by (−)-12 and
(+)-12 indicates that additional optimization will be required to
make this class of molecules more effective. In region I, the
surface complementarity of the halogenated phenyl ring could
be improved, and in region III, the guaninidium−Asp368gp120
interactions could be strengthened and the adjacent bound
solvent incorporated in compound design. Therefore, the high-
resolution crystal structures of these antagonists, with
demonstrated improvement in antiviral potency and breadth,
provide a novel structural paradigm to facilitate future cycles of
design, synthesis, and biological evaluation to develop this class
of small molecule inhibitors of HIV-1 entry.
EXPERIMENTAL SECTION
■
N1-(4-Chloro-3-fluorophenyl)-N2-[(1S,2R)-2-hydroxy-2,3-di-
hydro-1H-inden-1-yl]oxalamide [(+)-9]. To a solution containing
ethyl 2-(4-chloro-3-fluorophenylamino)-2-oxoacetate (7) (1.87 g, 7.62
mmol) in 20 mL of EtOH was added (1S,2R)-1-amino-2,3 dihydro-
1H-inden-2-ol [(−)-8, 1.19 g, 7.98 mmol] in one portion. The
reaction mixture was stirred and heated to 150 °C in a sealed tube for
1 h, after which the suspension was allowed to cool to room
temperature. The precipitate was collected by vacuum filtration and
washed with small portions of dichloromethane to provide 2.22 g
(83%) of analytically pure TK-II-52 (+)-9 as a white semicrystalline
solid. [α]2D9 +103.5° (c 0.56, DMSO); 1H NMR (500 MHz, DMSO-d6)
δ 11.19 (s, 1H), 8.36 (d, J = 8.7 Hz, 1H), 7.97, (dd, J = 2, 11.7 Hz,
1H), 7.77, (d, J = 8.8 Hz, 1H), 7.59, (t, J = 8.7 Hz, 1H), 7.28−7.18,
(m, 4H), 5.46, (d, J = 4.9 Hz, 1H), 5.25, (dd, J = 5.2, 8.6 Hz, 1H),
4.52, (dd, J = 4.6, 8.6 Hz, 1H), 3.14, (dd, J = 4.9, 16.2 Hz, 1H), 2.88,
(d, J = 16.1 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 159.3, 158.8,
156.8, (d, JCF = 242.5 Hz), 141.0, 140.8, 138.2, (d, JCF = 10 Hz), 131.6,
127.7, 126.5, 125.0, 124.1, 117.5 (d, JCF = 2.9 Hz), 114.5 (d, JCF = 17.5
Hz), 108.6, (d, JCF = 25 Hz), 71.6, 56.9. HRMS (ES+) m/z 371.0572
[(M + Na)+; calcd for C17H14ClFN2O3, 371.0575].
Small Molecule Modeling. Molecules were constructed in
MOE73 and ionized using MOE’s WashMDB function, and hydrogens
were added. The small molecule conformation was minimized to a
gradient of 0.01 with the MMFF94x74,75 using a distance-dependent
dielectric constant of 1.
Protein Modeling. Molprobity76,77 was used to add hydrogen
atoms and determine the tautomeric states and orientation of Asn,
Gln, and His in the X-ray crystal structure of 4 bound to HIV-1 clade
A/E gp120(H375S) coree. Hydrogens were added to crystallographic
waters using MOE (2010).73 The OPSLAA78 force field in MOE was
used, and all hydrogen atoms were minimized to an rms gradient of
0.01, holding the remaining heavy atoms fixed. A stepwise
minimization followed for all atoms, using a quadratic force constant
(100) to tether the atoms to their starting geometries. For each
subsequent minimization, the force constant was reduced by a half
until 0.25. This was followed by a final cycle of unrestrained
minimization.
Docking. GOLD (version 4.0.1)57,58 was used for docking, and the
binding site was defined by using the crystallographic position of 4.
One-hundred genetic algorithm (GA) docking runs were performed
with the following parameters: initial_virtual_pt_match_max=3.5,
diverse_solutions=1, divsol_cluster_size=1, and divsol_rmsd=1.5. All
other parameters were set to defaults.
N1-(4-Chloro-3-fluorophenyl)-N2-[(1S,2S)-2-azido-2,3-dihy-
dro-1H-inden-1-yl]oxalamide [(+)-10]. To a solution containing
(+)-9 (2.20 g, 6.31 mmol) in a mixture of dichloroethane (50 mL) and
THF (10 mL) was added p-toluenesulfonyl chloride (3.60 g, 18.88
mmol), followed by NEt3 (2.64 mL, 18.91 mmol), DMAP (0.77 g,
2.82 mmol), and a stir bar. A reflux condenser was attached, and the
solution was heated to 60 °C and stirred for 2 h. After cooling, the
reaction mixture was quenched with 100 mL of saturated NH4Cl
solution, followed by extraction with DCM/EtOAc (3:1, 3 × 100 mL).
The combined organic fractions were washed with brine and dried
over Na2SO4, filtered, and concentrated in vacuo. The crude product
was sonicated in 50 mL of DCM/hexanes (1:1) for 30 min and then
filtered. The filtrate was then washed with DCM/hexanes (1:1, 2 × 25
mL) to provide 2.21 g (70%) of the desired tosylate [(+)-SI-1] as a
white flakey solid. [α]D25 +10.4° (c 0.29, CH2Cl2); 1H NMR (500 MHz,
DMSO-d6) δ 10.97 (s), 8.84 (d, J = 9.0 Hz, 1H), 8.00 (dd, J = 2.5, 12.0
Hz, 1H), 7.79 (dd, J = 2.0, 9.0 Hz, 1H), 7.75 (d, J = 8 Hz, 2H), 7.62 (t,
J = 8.5 Hz, 1H), 7.33−7.22 (m, 6H), 5.49 (dd, J = 5.0, 8.5 Hz, 1H),
5.24 (dt, J = 1.5, 5.0 Hz, 1H), 3.34 (m, overlap with water, 1H), 3.13
(d, J = 16.0 Hz, 1H), 2.33 (s, 3H); 13C NMR (125 MHz, DMSO-d6) δ
159.4, 157.9, 156.8 (d, JCF = 243.8 Hz), 144.8, 138.7, 138.6, 138.2, (d,
JCF = 10 Hz), 132.7, 130.6, 129.9, 128.3, 127.5, 127.0, 124.8, 123.9,
117.3, (d, JCF = 2.7 Hz), 114.4, (d, JCF = 17.5 Hz), 108.4, (d, JCF = 25.0
Hz), 82.7, 55.6, 37.6, 20.9. HRMS (ES+) m/z 503.0862 [(M + H)+;
calcd for C24H20ClFN2O5S, 503.0844].
ROCS Virtual Screening. Flipper from Open Eye was used to
expand compounds with unspecified chirality prior to generation of
conformers. Using Omega (version 2.2.1)79 from Open Eye with
default parameters, a maximum of 50 low energy conformers for all
compounds in the ZINC database (version 7)62 were generated and
stored in sd files of approximately 10 000 molecules. ROCS60,61
searches were run using 3D coordinates from the docked binding
mode of the amine containing the tetramethylpiperidine prototype.
The implicit Mills−Dean80 force field was used to match chemotypes
as well as shape. Hits were ranked by a combination of the Tanimoto
coefficient and the scaled color score (ComboScore). Primary amines
with ComboScore > 1.0 were selected from the set of hits, filtered for
currently commercial availability, and conjugated in silico and were
docked with GOLD57,58 and scored with a mass-corrected Goldscore.
Synthesis Procedures. All reactions were conducted in oven-
dried glassware under an inert atmosphere of nitrogen or argon unless
otherwise stated. All solvents were reagent or high performance liquid
chromatography (HPLC) grade. Anhydrous CH2Cl2 and THF were
obtained from the Pure Solve PS-400 system under an argon
atmosphere. All reagents were purchased from commercially available
sources and used as received. Mixtures were magnetically stirred under
a nitrogen atmosphere and reactions monitored by either thin layer
chromatography (TLC) with 0.25 mm E. Merck precoated silica gel
plates or analytical HPLC. Yields refer to chromatographically and
spectroscopically pure compounds. Optical rotations were measured
on a JASCO P-2000 polarimeter within the specified solvent. Proton
To a solution of (+)-SI-1 (2.20 g, 4.38 mmol), prepared above, in 8
mL of DMSO were added NaN3 (2.86 g, 43.99 mmol) and a stir bar.
The solution was heated to 50 °C and stirred for 2.5 h. After the
mixture was cooled to room temperature, the reaction was quenched
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with H2O (50 mL) followed by extraction with DCM/EtOAc (3:1, 3
× 100 mL). The combined organic fractions were washed with brine
(3 × 100 mL), dried over Na2SO4, filtered, and concentrated in vacuo.
The crude product was then dissolved in a minimal amount of hot
EtOAc, followed by addition of an equal portion of hot hexane. The
obtained precipitate was collected, the mother liquor concentrated,
and the residual solid recrystallized via the same procedure. The
isolated solids were combined to provide 1.30 g (79%) of the azide
(+)-10 as a white flakey solid. [α]2D5 +40.7° (c 0.51, EtOAc); 1H NMR
(500 MHz, DMSO-d6) δ 11.14 (s, 1H), 9.66 (d, J = 9.0 Hz, 1H), 7.98
(dd, J = 2.0, 11.5 Hz, 1H), 7.77 (dd, J = 1.5, 8.5 Hz, 1H), 7.60 (t, J =
8.5 Hz, 1H), 7.28−7.22 (m, 3H), 7.15 (d, J = 7.0 Hz, 1H), 5.34 (t, J =
8.0 Hz, 1H), 4.54 (q, J = 8.0 Hz, 1H), 3.34 (dd, J = 7.5, 15.5 Hz, 1H),
2.88 (dd, J = 8.5, 15.5 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ
stored at 10 mM at −20 °C. The compounds were diluted in
Dulbecco's modified Eagle medium (DMEM, Invitrogen) to create 1
mM solutions before use. Soluble CD4 (sCD4) was purchased from
ImmunoDiagnostics (Woburn, MA). Human 293T embryonic kidney
and canine Cf2Th thymocytes (ATCC) were grown at 37 °C and 5%
CO2 in DMEM (Invitrogen) containing 10% fetal bovine serum
(Sigma) and 100 μg/mL penicillin−streptomycin (Meditech, Inc.).
Cf2Th cells stably expressing human CD4 and either CCR5 or
CXCR481,82 were grown in medium supplemented with 0.4 mg/mL
G418 (Invitrogen) and 0.20 mg/mL hygromycin B (Roche
Diagnostics). By use of the Effectene transfection reagent (Qiagen),
293T human embryonic kidney cells were co-transfected with plasmids
expressing the pCMVΔP1ΔenvpA HIV-1 Gag-Pol packaging con-
struct, the wild-type or mutant HIV-1YU2 envelope glycoproteins or the
envelope glycoproteins of the control amphotropic murine leukemia
virus (A-MLV), and the firefly luciferase-expressing vector at a DNA
ratio of 1:1:3 μg. For the production of viruses pseudotyped with the
A-MLV glycoprotein, a rev-expressing plasmid was added. The single-
round, replication-defective viruses in the supernatants were harvested
24−30 h after transfection, filtered (0.45 μm), aliquoted, and frozen at
−80 °C until further use. The reverse transcriptase (RT) activities of
all viruses were measured as described previously.83
Assay of Virus Infectivity and Inhibitor Sensitivity. Cf2Th/
CD4-CCR5 or Cf2Th/CD4-CXCR4 target cells were seeded at a
density of 6 × 103 cells/well in 96-well luminometer-compatible tissue
culture plates (Perkin-Elmer) 24 h before infection. On the day of
infection, 1−100 μM was added to recombinant viruses (10 000
reverse transcriptase units) in a final volume of 50 μL and incubated at
37 °C for 30 min. The medium was removed from the target cells,
which were then incubated with the virus−drug mixture for 2−4 h at
37 °C. At the end of this time point, complete medium was added to a
final volume of 150 μL and incubated for 48 h at 37 °C. The medium
was removed from each well, and the cells were lysed with 30 μL of
passive lysis buffer (Promega) by three freeze−thaw cycles. An EG&G
Berthold microplate luminometer LB 96V was used to measure
luciferase activity in each well after the addition of 100 μL of luciferin
buffer (15 mM MgSO4, 15 mM potassium phosphate buffer [pH 7.8, 1
mM ATP, 1 mM dithiothreitol) and 50 μL of 1 mM D-luciferin
potassium salt (BD Pharmingen).
Luminescent Cell-Based Viability Assay. Compounds were
dissolved in dimethyl sulfoxide (DMSO) and stored at 10 mM at −20
°C. The compounds were diluted in Dulbecco's modified Eagle
medium (DMEM, Invitrogen) to create 1 mM solutions before use. In
addition, DMSO (Sigma) was also diluted in DMEM and used as
negative control. Cytotoxicity assay was done in parallel with the cell-
based infection assay as described in the methods section of this article
using Cf2Th cells stably expressing human CD4 and CCR5. Briefly,
Cf2Th/CD4-CCR5 target cells were seeded at a density of 6 × 103
cells/well in 96-well luminometer-compatible tissue culture plates
(Perkin-Elmer) 24 h before infection. On the day of infection, medium
from target cells was removed and 1−100 μM (+)-12 or DMSO to 2%
(equivalent to concentration of DMSO in 100 μM (+)-12) was added
to target cells at a final volume of 50 μL and incubated at 37 °C for 2−
4 h. At the end of this time point, complete medium was added to a
final volume of 150 μL and incubated for 48 h at 37 °C. After 48 h, the
plates were equilibrated to room temperature for 30 min and 30 μL of
substrate reagent, CellTiter-Glo (Promega), was added to each well
and incubated at room temperature for 5−10 min. An EG&G Berthold
microplate luminometer LB 96V was used to measure luciferase
activity in each well for 5 s.
Isothermal Titration Calorimetry. Isothermal titration calori-
metric experiments were performed using a high-precision VP-ITC
titration calorimetric system from MicroCal LLC (Northampton,
MA). The calorimetric cell (∼1.4 mL), containing gp120 at about 2
μM dissolved in PBS, pH 7.4 (Roche Diagnostics GmbH), with 2%
DMSO, was titrated with the different compounds dissolved in the
same buffer at 80−130 μM. The compound solution was added in
aliquots of 10 μL at preset intervals. All solutions were degassed to
avoid any formation of bubbles in the calorimeter during stirring. All
experiments were performed at 25 °C. The heat evolved upon
160.1, 158.5, 156.8, (d, JCF = 242.5 Hz), 139.9, 138.8, 138.2 (d, JCF
=
10 Hz), 130.5, 128.2, 127.1, 124.7, 123.4, 117.4, (d, JCF = 3.0 Hz),
114.4, (d, JCF =17.8 Hz), 108.5, (d, JCF = 25.6 Hz), 65.7, 59.4, 35.3.
HRMS (ES+) m/z 372.0652 [(M − H)−; calcd for C17H12ClFN5O2,
372.0664].
N1-(4-Chloro-3-fluorophenyl)-N2-[(1S,2S)-2-amino-2,3-dihy-
dro-1H-inden-1-yl]oxalamide [AWS-I-50, (+)-6]. To a solution of
(+)-10 (1.30 g, 3.48 mmol) in 35 mL of MeOH were added Lindlar’s
catalyst (5% Pd/CaCO3, poisoned with lead, 650 mg) and a stir bar.
Hydrogen was bubbled through the solution, after which the reaction
mixture was stirred for 2 h under a hydrogen atmosphere at room
temperature. The reaction mixture was then filtered through a plug of
Celite and the filter cake washed with additional methanol. The filtrate
was concentrated in vacuo to obtain the crude product, which was
then purified by silica gel chromatography utilizing a gradient solvent
system (DCM/MeOH/NH4OH, 100:1:0.1 to 50:1:0.1 to 20:1:0.1 to
10:1:0.1) to afford 1.06 g (87%) of amine (+)-6 as a white flakey solid.
[α]2D5 +93.0° (c 0.14, MeOH); H NMR (500 MHz, CDCl3): δ 11.07
1
(s, 1H), 9.22 (d, J = 9.0 Hz, 1H), 7.98 (dd, J = 2.4, 11.9 Hz, 1H), 7.77
(ddd, J = 1.0, 2.5, 8.9 Hz, 1H), 7.60 (t, J = 8.7 Hz, 1H), 7.20−7.14 (m,
3H), 7.06 (d, J = 7.2 Hz, 1H), 5.00 (t, J = 8.5 Hz, 1H), 3.69 (q, J = 8.5
Hz, 1H), 3.08 (dd, J = 7.5 Hz, 1H), 2.64 (dd, J = 9.2 Hz, 1H); 13C
NMR (125 MHz, DMSO-d6) δ 160.3, 159.0, 156.8, (d, JCF = 242.5
Hz), 142.0, 140.6, 138.3 (d, JCF = 10 Hz), 130.6, 127.4, 126.4, 124.4,
123.3, 117.3, (d, JCF = 3.75 Hz), 114.2, (d, JCF = 17.5 Hz), 108.4 (d,
JCF = 25 Hz), 62.6, 59.6, 48.6. HRMS (ES+) m/z 348.0911 [(M +
H)+; calcd for C17H15ClFN3O2, 348.0915].
N1-(4-Chloro-3-fluorophenyl)-N2-[(1S,2S)-2-guanidino-2,3-
dihydro-1H-inden-1-yl]oxalamide Formate [DMJ-I-228, (+)-12].
To a solution containing (+)-6 (1.06 g, 3.05 mmol) in 35 mL of DMF
were added 1H-pyrazole-1-carboxamidine hydrochloride (11, 879 mg,
6.00 mmol), N,N-diisopropylethylamine (2.10 mL, 12.06 mmol), and
a stir bar. The solution was heated to 110 °C for 16 h and then allowed
to cool. The light-red reaction mixture was concentrated to
approximately 5 mL and diluted with 20 mL MeCN/H2O (1:1),
and the solution was subjected to purification via HPLC to give 350
mg (27%) of (+)-12 as a white fluffy solid after lyophilization. [α]D26
1
+5.8° (c 0.72, MeOH); H NMR (500 MHz, CD3OD): δ 8.3 (br s,
1Hformate), 7.84 (dd J = 2.5, 11.5 Hz, 1H), 7.50 (d, J = 8.75 Hz, 1H),
7.43 (t, J = 8.5 Hz, 1H)), 7.34−7.29 (m, 4 H), 5.27 (d, J = 4.5 Hz,
1H), 4.30 (m, 1H), 3.52 (dd, J = 7.5 Hz,1H), 2.93 (dd, J = 5, 16.5 Hz,
1H); 13C NMR (125 MHz, CD3OD) δ 162.8, 159.4, 159.3, (d, JCF
=
244.4 Hz), 159.0, 141.9, 139.7, 139.1 (d, JCF = 9.8 Hz), 131.9, 130.6,
129.0, 126.4, 126.3, 118.3 (d, JCF = 3.3 Hz), 117.4 (d, JCF = 17.8 Hz),
110.0 (d, JCF = 26.3 Hz), 62.3, 59.7, 37.8. HRMS (ES+) m/z 390.1132
[(M + H)+; calcd for C18H18ClFN5O2, 390.1133]. The formate
counterion was not observed under the HRMS conditions.
The enantiomeric compounds (−)-6 and (−)-12 were prepared
using the procedures described above, starting with commercially
available aminoindanol (+)-8. The intermediates and final compounds
possess identical 1H and 13C NMR spectra and HRMS data. The
observed optical rotations were the following: (−)-9, [α]2D9 −112.1° (c
0.48, DMSO); (−)-10, [α]D25 −34.9° (c 0.30, EtOAc); (−)-6, [α]2D5
−83.2° (c 0.16, MeOH); (−)-12, [α]2D5 −3.2° (c 0.72, MeOH).
Cell-Based Infectivity Assays: General Considerations.
Compounds were dissolved in dimethyl sulfoxide (DMSO) and
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Journal of Medicinal Chemistry
Article
injection of compound was obtained from the integral of the
calorimetric signal. The heat associated with the binding reaction
was obtained by subtracting the heat of dilution from the heat of
reaction. The individual binding heats were plotted against the molar
ratio, and the values for the enthalpy change (ΔH) and association
constant, Ka (Kd = 1/Ka), were obtained by nonlinear regression of the
data.
were added to all atoms within a 4.5 Å radius of the small molecule
ligand and were minimized in the Merck molecular force field.74,75
ASSOCIATED CONTENT
* Supporting Information
■
S
SPR, mutation, and neutralization data and NMR spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Viral Breadth Studies. HIV-1 Env-pseudoviruses were prepared
by transfecting 293T cells with 10 μg of rev/Env expression plasmid
and 30 μg of an Env-deficient HIV-1 backbone vector (pSG3Δenv),
using FuGENE 6 transfection reagents (Invitrogen). Pseudovirus-
containing culture supernatants were harvested 2 days after trans-
fection, filtered (0.45 μm), and stored at −80 °C or in the vapor phase
of liquid nitrogen. Neutralization was measured using HIV-1 Env-
pseudoviruses to infect TZM-bl cells as described previously84−87 with
minor modifications. Briefly, the test reagent [(+)-12, (−)-12, 1, or
CD4-Ig] was diluted in complete medium containing 10% DMSO.
Then 40 μL of virus was added to 10 μL of serial diluted test reagent
in duplicate wells of a 96-well flat bottom culture plate, and the virus−
reagent mix was incubated for 30 min at 37 °C. To keep assay
conditions constant, sham medium containing 10% DMSO was used
in place of test reagent in specified control wells. The virus input was
set at a multiplicity of infection of approximately 0.01−0.1, which
generally results in 100000−400000 relative light units (RLUs) in a
luciferase assay (Promega, Madison, WI). The test reagent
concentrations were defined at the point of incubation with virus
supernatant. Neutralization curves were fit by nonlinear regression
using a five-parameter Hill slope equation as previously described.86
The 50% or 80% inhibitory concentrations (IC50 or IC80) were
reported as the reagent concentrations required to inhibit infection by
50% or 80%.
Accession Codes
Coordinates and structure factors have been deposited in the
Protein Data Bank with the following accession numbers:
4DKO, 4DKP, 4DKQ, and 4DKR.
AUTHOR INFORMATION
Corresponding Author
■
*For J.M.L.: phone, 610-526-5679; e-mail, jlalonde@
brynmawr.edu. For A.B.S.: phone, 215-898-4860; e-mail,
smithab@sas.upenn.edu.
Author Contributions
∞These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Irwin Chaiken and Wayne Hendrickson and all the
members of the PO1 Consortium “Structure-Based Antago-
nism of HIV-1 Envelope Function in Cell Entry”. We also
thank Jonathan Stuckey for assistance with figures, and
members of the Structural Biology Section, Vaccine Research
Center, NIAID for comments on the manuscript. Funding was
provided by NIH Grant GM 56550 to J.M.L., E.F., A.B.S., by
NIH Intramural IATAP and NIAID programs to Y.D.K.,
P.D.K., J.R.M., and J.S. J.M.L. thanks the Pittsburgh Super-
computing Center for an allocation for computing resources,
Grant MCB090108.
Construction of the HIV-1 Envelope Sequence Phylogenetic
Trees. The HIV-1 gp160 protein sequences of isolates used in the
neutralization assays were aligned using MUSCLE (multiple sequence
comparison by log expectation).88,89 The protein distance matrix was
calculated by “protdist” using the Jones−Taylor−Thornton model,90
and the dendrogram was constructed using the neighbor-joining
method91 by “Neighbor”. The analysis was performed at the NIAID
Biocluster (https://niaid-biocluster.niaid.nih.gov/). The trees were
displayed with Dendroscope.92
Plasmids. A point mutation was introduced to pVRC8400-HIV-1
ABBREVIATIONS USED
clade A/E
ΔV123 expression vector to generate clade A/
■
93TH057
E93TH057 gp120 coree (H375S) expressing plasmid. The plasmid
construct was verified by DNA sequencing.
HIV-1, human immunodeficiency virus type 1; SIV, simian
immunodeficiency virus; sCD4, soluble CD4; ITC, isothermal
titration calorimetry; A-MLV, amphotropic murine leukemia
virus; GMT, geometric mean titer; SPR, surface plasmon
resonance; BEI, binding efficiency index; SEI, surface efficiency
index; GA, genetic algorithm; HRMS, high-resolution mass
spectrometry; DMEM, Dulbecco’s modified Eagle medium;
TsCl, tosyl chloride; DMAP, 4-dimethylaminopyridine
Crystallization, Data Collection, Structure Determination,
and Refinement. Clade A/E93TH057 gp120(H375S) coree was purified as
described.34 Small molecules in 100% DMSO were incorporated in the
purified gp120 to make a final concentration of 100 μM. Then the
small molecule−gp120 complexes were set up for crystallization using
vapor diffusion at 20 °C. Crystals grew in a mixture of 0.5 μL of
protein−small molecule complex and 0.5 μL of reservoir solution
containing 8−10% (v/v) PEG 8000, 5% isopropanol, 0.1 M HEPES
(pH 7.5). Crystals were soaked in cryoprotection solution containing
30% ethylene glycol, 12% PEG 8000, and 0.1 M HEPES (pH 7.5) and
were flash frozen in liquid nitrogen. Data were collected on beamline
SERCAT ID-22 at the Advanced Photon Source and processed with
HKL2000.93 The structures were solved by molecular replacement
with PHENIX94 using the coordinates of unliganded clade A/E93TH057
gp120 coree (PDB code 3TGT). The initial Fo − Fc map generated
after a rigid body refinement clearly indicated the electron densities of
(−)-12 and (+)-12 and allowed us to place them into the densities
manually using COOT.95 The initial densities of 4 and (+)-6, however,
were not as clear as those found in (−)-12 and (+)-12, specifically in
the region III. After several rounds of refinement using PHENIX,94 the
Rwork/Rfree values for (−)-12 and (+)-12 converged to 18.8/20.8% and
19.3/23.3%, respectively (Table 3). The geometry of the refined
model was checked with Molprobity.96 Figures 1, 2, and 7−9 were
generated by PyMOL.97 To facilitate analysis of protein−ligand
interactions in the context of the crystal structures, hydrogen atoms
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