Kinetic characterization of novel HIV-1 entry inhibitors: Discovery of a relationship between off-rate and potency

Article abstract of DOI:10.3390/molecules23081940

The entry of HIV-1 into permissible cells remains an extremely attractive and underexploited therapeutic intervention point. We have previously demonstrated the ability to extend the chemotypes available for optimization in the entry inhibitor class using computational means. Here, we continue this effort, designing and testing three novel compounds with the ability to inhibit HIV-1 entry. We demonstrate that alteration of the core moiety of these entry inhibitors directly influences the potency of the compounds, despite common proximal and distal groups. Moreover, by establishing for the first time a surface plasmon resonance (SPR)-based interaction assay with soluble recombinant SOSIP Env trimers, we demonstrate that the off-rate (k_d) parameter shows the strongest correlation with potency in an antiviral assay. Finally, we establish an underappreciated relationship between the potency of a ligand and its degree of electrostatic complementarity (EC) with its target, the Env complex. These findings not only broaden the chemical space in this inhibitor class, but also establish a rapid and simple assay to evaluate future HIV-1 entry inhibitors.

Full text of DOI:10.3390/molecules23081940

molecules
Article
Kinetic Characterization of Novel HIV-1 Entry
Inhibitors: Discovery of a Relationship
between Off-Rate and Potency
Megan E. Meuser 1, Michael B. Murphy 2, Adel A. Rashad 1 and Simon Cocklin 1,
*
ID
ID
1
Department of Biochemistry & Molecular Biology, Drexel University College of Medicine, Rooms 10307,
10309, and 10315, 245 North 15th Street, Philadelphia, PA 19102, USA; mem484@drexel.edu (M.E.M.);
aaa396@drexel.edu (A.A.R.)
GE Healthcare Life Sciences, 100 Results Way, Marlborough, MA 01752, USA; Michael.B.Murphy@ge.com
Correspondence: sc349@drexel.edu; Tel.: +1-215-762-7234 (Office); +1-215-762-4979 (Lab);
Fax: +1-215-762-4452
2
*
Academic Editor: Mahesh Narayan
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 9 July 2018; Accepted: 1 August 2018; Published: 3 August 2018
Abstract: The entry of HIV-1 into permissible cells remains an extremely attractive and underexploited
therapeutic intervention point. We have previously demonstrated the ability to extend the chemotypes
available for optimization in the entry inhibitor class using computational means. Here, we continue
this effort, designing and testing three novel compounds with the ability to inhibit HIV-1 entry.
We demonstrate that alteration of the core moiety of these entry inhibitors directly influences the
potency of the compounds, despite common proximal and distal groups. Moreover, by establishing
for the first time a surface plasmon resonance (SPR)-based interaction assay with soluble recombinant
SOSIP Env trimers, we demonstrate that the off-rate (kd) parameter shows the strongest correlation
with potency in an antiviral assay. Finally, we establish an underappreciated relationship between
the potency of a ligand and its degree of electrostatic complementarity (EC) with its target, the Env
complex. These findings not only broaden the chemical space in this inhibitor class, but also establish
a rapid and simple assay to evaluate future HIV-1 entry inhibitors.
Keywords: bioisosteres; HIV-1 Env; antiviral; surface plasmon resonance; computer-aided drug design
Highlights:
•
•
•
•
The HIV-1 entry process represents an extremely attractive but underexploited therapeutic
intervention point.
Novel compounds with HIV-1 entry inhibitory activity were designed via field-based computational
methods, synthesized and characterized biologically.
A surface plasmon resonance (SPR) interaction assay was established for the first time for this
class of antivirals, allowing assessment of affinity and kinetic parameters.
The potency of a given entry inhibitor could be correlated with its dissociation rate parameter
(kd) and its degree of electrostatic complementarity (EC) with its target, the Env complex.
1. Introduction
Because of the emergence of drug-resistant strains and the cumulative toxicities associated with
current therapies, demand remains for new inhibitors of HIV-1 replication. An attractive intervention
point for such new inhibitors is viral entry into permissive cells. Only two entry inhibitors are
used in the clinic, enfuvirtide (Fuzeon) and maraviroc (Selzentry), each with their own limitations.
Molecules 2018, 23, 1940; doi:10.3390/molecules23081940
www.mdpi.com/journal/molecules

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Enfuvirtide’s clinical application is limited by the fact that it is a peptide, which is not orally bioavailable
and therefore has to be injected, sometimes causing serious local injection reactions. Moreover,
it requires twice-daily dosage at 90 mg per dose, leading to high cost that is not feasible for treatment
in developing countries. Maraviroc, although well tolerated, is only active against R5-tropic viruses as
it blocks HIV-1 entry via targeting the host cell coreceptor CCR5. Ideally, one would hope to be able to
develop virally-targeted, small-molecule HIV-1 entry inhibitors that are orally bioavailable.
Entry of HIV-1 into susceptible cells is orchestrated by the Env complex present on the exterior
of the virion. Env is composed of a heterotrimer of gp41 and gp120 subunits that undergo a series of
interactions and conformational changes that result in the fusion of the viral and cellular membranes.
Over recent years, a great deal has been learnt about the structure and thermodynamics of this process
and which points in this cascade are susceptible to inhibition.
In the HIV-1 entry field, two main small-molecule inhibitor chemotypes, which target the Env
complex, predominate: the NBD-556 analogs [1] and the BMS-378806 analogs [2]. The NBD-556 analogs
are at a very early stage of development, whereas the BMS-378806-type compounds have been entered
into clinical trials a number of times. Although potent across many of the major subtypes of HIV-1,
the piperazine-based BMS-378806 analogs suffer from poor pharmacokinetic properties (primarily
low bioavailability); deficiencies that Bristol-Myers Squibb tried to address using a prodrug approach.
BMS-663068, a phosphonooxymethyl prodrug of BMS-626529 [3], the current lead compound being
developed by ViiV Healthcare under the name fostemsavir, is currently in phase III clinical trials.
Despite encouraging results, fostemsavir is only recommended for highly treatment-experienced
patients and requires dosages of up to 600 mg twice daily.
Our group has been exploring the computationally-driven modification of the BMS-type entry
inhibitors in an overarching effort to bring this class of inhibitors out of the realm of salvage therapy [4,5].
In this study, we demonstrate the continued utility of our computational approach for modification
of inhibitory compounds and subsequent modulation of their drug-like properties. However, most
importantly, we demonstrate a strong correlation between the off-rate values of the compounds for
a soluble recombinant trimer, obtained by a novel surface plasmon resonance (SPR) interaction assay,
and their potency in a single-round infection assay. This new information, along with the novel SPR
assay, will significantly increase the throughput, assessment, and prioritization of future analogs within
our research program.
2. Results and Discussion
2.1. Design of Compounds SC16, SC18, and SC39
Due to the lack of structural information on the bioactive conformation of our inhibitors and
the BMS piperazine-based inhibitors at the time of design, we first used FieldTemplater (Forge,
Cresset) to determine the most likely 3D conformation adopted by BMS-377806 [
6], BMS-488043 [7],
BMS-626529 [ ], and SC11 SC26 (our most potent inhibitors) [ ] when complexed with the HIV-1 Env
8
/
4
complex. The resultant bioactive conformation hypothesis (Figure 1) was then inputted into Spark
(Cresset, UK) to identify nonclassical bioisosteres of the selected region. Given our previous success
in using field-point methods to discover novel analogs of the central core region of our compounds,
we again focused in on this region; that is, the piperazine groups of BMS-377806, BMS-488043, and
BMS-626529, and the dipyrrolodine group of compounds SC11/SC26. From this analysis, three new
core chemotypes were chosen for investigation, due to their calculated likelihood of being able to
span a wide range of potencies. First, a common head group to be used in all of the molecules was
synthesized: compound
6, according to Materials and Methods, Scheme 1. This was subsequently
used in the synthesis of compounds SC16
,
SC18, and SC39 as outlined in Materials and Methods,
Schemes 3–5. The high-affinity compound SC11 was also synthesized, as previously described by
Tuyishime et al. [4], and summarized in Materials and Methods, Scheme 2.

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Figure 1. Bioactive conformation hypothesis and field-point pharmacophore of BMS-377806, BMS-488043,
BMS-626529, and SC11. Blue field points = negatively charged; red = positively charged. Yellow spheres
= van der Waals; gold = hydrophobic. Oxygen atoms are shown in red, nitrogen in blue. The size of the
field point is directly related to the strength of the interaction.
2.2. Potency Determination of Compounds SC11, SC16, SC18, and SC39 against Subtype B
Env Pseudoviruses
Following the synthesis of compounds SC11, SC16, SC18, and SC39, we then tested them for
specificity and potency against HIV-1 using the single-round infection assay [
4,5
]. Single-round
infective HIV-1 were pseudotyped with envelopes from HIV-1 JR-CSF, HIV-1 B41 [
9], or amphotropic
murine leukemia virus (AMLV). HIV-1 pseudoviruses containing the aforementioned envelopes were
produced, normalized to p24 content, and used to infect U87.CD4.CCR5 target cells. Infectivity was
quantified by measuring luciferase activity in cell lysates (Luciferase Assay System, Promega, Madison,
WI, USA) using a microplate luminometer (GloMax, Promega). The specificity of the compounds to
HIV-1 Env was assessed by monitoring their effect upon the infection of AMLV Env pseudotyped
recombinant HIV-1. Lastly, cytotoxicity of the compounds was determined, as outlined in Materials
and Methods, up to a highest test concentration of 50
Table 1.
µM. The results of this analysis are shown in
As can be seen from the Table 1, the individual compounds all had antiviral effects on HIV-1
Env pseudotyped virus in the single-round infection assay. In general, the SC compounds appear
to be more effective against those viruses pseudotyped with the HIV-1JR-CSF. However, the median
IC50 of the HIV-1B41 and HIV-1JR-CSF Env pseudotyped virions are effectively spread through a range
of potencies from low nanomolar to low micromolar. This potency range is perfect for investigating
whether there is a correlation between affinity/kinetics and potency.

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Table 1. Specificity and potency of compounds against HIV-1JR-CSF, HIV-1B41, and AMLV-pseudotyped
HIV-1. N.A. = not active. The chemical structures were drawn with ChemDraw Ultra 15.0.
IC50 AMLV
Median
IC50 (µM)
CC50
(µM)
Compound
IC50 JR-CSF (µM)
IC50 B41 (µM)
(µM)
BMS-626529
Compound 8 (SC11)
0.00006 ± 0.000005 0.00005 ± 0.000006
N.A.
0.000055
0.0006 ± 0.0001
0.002 ± 0.0002
N.A.
0.001
Compound 13 (SC16)
0.029 ± 0.008
0.037 ± 0.005
N.A.
0.033
>50
Compound 18 (SC18)
0.132 ± 0.004
0.360 ± 0.012
N.A.
0.246
Compound 23 (SC39)
0.143 ± 0.052
5.32 ± 0.26
N.A.
2.73

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2.3. Affinity and Kinetic Characterization of SC11, SC16, SC18, and SC39 for Soluble Recombinant
Env Trimer
Having effectively established a set of compounds with a perfect range of potencies for further
correlation analysis, we next sought to characterize their interactions (affinity and kinetics) with
a soluble recombinant Env trimer. To ensure consistency, we chose to utilize the B41 SOSIP.664 gp140
trimer (generously gifted to us by Dr. Andrew Ward, Scripps Institute, La Jolla, CA, USA) and to use
surface plasmon resonance (SPR), so as not to have to label the reagents and to minimize consumption.
Detailed conditions are given in Materials and Methods. Briefly, the B41 SOSIP.664 gp140 trimer
was attached to a Sensor Chip CM5 (GE Healthcare Life Sciences, Bjorkgatan 30, Uppsala, 751 84,
Sweden) via amine coupling chemistry to a final level of approximately 16,000 response units (RU)
on each of eight channels of the BiacoreTM 8K instrument (GE Healthcare). Reference flow cells were
prepared similarly using amine coupling of IgG b12 as a negative control. Compounds were injected
for 160 s at 75 µ
L min−1 and with 600 s of dissociation, using the multicycle kinetics injection mode,
and the surface was regenerated using a 60-s injection of 10 mM glycine pH 1.5 to ensure complete
removal of bound compounds. Association and dissociation rate constants and calculated KD values
for binding were determined using a simple 1:1 binding model in the BiacoreTM 8K Evaluation software
(GE Healthcare Life Sciences, Bjorkgatan 30, Uppsala, 751 84, Sweden) and from quadruplicate runs.
Figure 2 shows overlaid sensorgrams from two such runs for each compound and their corresponding
fits. Table 2 shows the derived parameters from the fitting of all four data sets.
Figure 2. Sensorgrams depicting the interaction of (A) SC11, (B) SC16, (C) SC18, and (D) SC39 with
sensor chip-immobilized B41 SOSIP.664 gp140 trimer. Blue lines represent actual data, whereas black
lines indicate fitting to a 1:1 interaction model. Each panel shows two injections at each concentration
to demonstrate reproducibility.

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Table 2. Kinetics and affinity of BMS-626529 and the SC compounds binding to soluble, cleaved
recombinant Env.
−1 −1
−1
)
Compound
ka (M
s
)
kd (s
KD
−4
−4
−3
−2
−2
3.89 × 104
4.33 × 103
8.62 × 104
1.59 × 104
1.39 × 103
5.9 × 10
BMS-626529 *
SC11
15.2 nM
66.3 nM
69.3 nM
1.56 µM
27.4 µM
2.87 × 10
5.98 × 10
2.48 × 10
3.81 × 10
SC16
SC18
SC39
* The sensorgrams for BMS-626529 binding to soluble, cleaved recombinant Env are shown in Supplementary Materials.
As can be seen from both Figure 2 and Table 2, there is a most striking difference between the
compounds’ interactions within their kinetic profile, in particular for their off-rates. As such, we next
chose to plot these kinetic parameters against the median IC50 to see whether or not we could establish
a relationship that could be exploited. This analysis for the kinetic parameters ka and kd is shown in
Figure 3.
1.0x105
4.0x10-2
3.5x10-2
8.0x104
R2 = 0.99
3.0x10-2
6.0x104
4.0x104
2.0x104
0.0
2.5x10-2
2.0x10-2
1.5x10-2
1.0x10-2
5.0x10-3
0.0
-5.0x10-3
5.0x10-7 1.0x10-6 1.5x10-6 2.0x10-6 2.5x10-6 3.0x10-6
5.0x10-71.0x10-61.5x10-62.0x10-62.5x10-63.0x10-6
[Median IC50](M)
0.0
0.0
[Median IC50](M)
(A)
(B)
Figure 3. Scatter plots showing (A) ka versus median IC50 and (B) kd versus median IC50 for compounds
BMS-626529, SC11, SC16, SC18, and SC39. The red line in panel (B) shows a logarithmic trend line fit to
the data.
2.4. Molecular Modeling and Computational Analysis of Compounds of SC11, SC16, SC18, and SC39
Since the original design of the SC series of compounds, including those included in this study,
the structures of two BMS piperazine-based entry inhibitors complexed with the BG505 SOSIP.664
soluble trimer have been solved by Kwong et al. [10]. Therefore, to gain further insight into the potency
differentials between SC11, SC16, SC18, and SC39, and to inform future design, we performed
molecular modeling of these compounds into the BMS-626529-SOSIP complex structure (PDB: 5U7O)
as outlined in Materials and Methods.
Consistent with their antiviral potency, compounds SC11, SC16, and SC18 align well with
the cocrystallized BMS-626529. The triazole–azaindole moieties of these compounds adopt a very
similar orientation to that of BMS-626529, whereas the terminal benzoyl moieties occupy slightly
different positions (in the case of SC18). The octahydropyrrolo[3,4-c]pyrrole moiety of SC11 effectively
interacts with the
interactions, which may be a reason for the observed potency of SC11 (median IC50 of ~2 nM, predicted
binding energy of 13.23 kcal/mol). For compounds SC16 and SC18, the smaller azetidine may
provide fewer interactions with those residues and hence the weaker activities compared to SC11
SC16 median IC50 = 33 nM, with a predicted binding energy of 12.26 kcal/mol; SC18 median
IC50 = 246 nM, with a predicted binding energy of 12.17 kcal/mol). For the weaker compound SC39
α1-helix residue Trp112 and the β20/21 loop residue Trp427 indole rings via Pi–alkyl
−
(
−
−
,

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the kink introduced by the presence of the terminal phenyl–tetrazole pushes the triazole–azaindole
out of the binding site (compared to BMS-626529), resulting in a slightly different binding mode
(Figure 4B), which may be the reason for the reduced activity (median IC50 = 2.73 µM, with a predicted
binding energy of −11.36 kcal/mol).
A
B
Figure 4. Overlay of docked compounds on the cocrystal of BMS-626529; (A) BMS-626529 (cyan),
compound 8 (SC11, green), compound 13 (SC16, yellow), and compound 18 (SC18, magenta). (B) BMS-
626529 (cyan) and compound 23 (SC39, purple).
Having in hand models of compounds bound to their target, we next sought to identify any
other correlations that could inform future design. Therefore, we chose to look at the electrostatic
complementarity between the compounds and the binding site on the BG505 SOSIP.664 protein
using Flare v2.0 (Cresset, Litlington, Cambridgeshire, UK; http://www.cresset-group.com/flare/).
Electrostatic complementarity (EC) maps are based on a calculation of electrostatic potentials
for the ligand and the protein on the surface of the ligand. These potentials are then added
together, normalized, and scaled. Regions of the ligand surface where there is perfect electrostatic
complementarity with the protein are colored green, while the regions where there is a perfect
electrostatic clash are colored red. Flare v2.0 can also compute several electrostatic complementarity
summary scores for quantification and comparison of the EC. Therefore, we performed this analysis
on our derived models for the SC11, SC16, SC18, and SC39 complexes (Figure 5) as well as for
the BMS-626529 complex. We also chose to plot the first computed score for correlation analysis
with potency. This score is effectively the average value of that score over the surface of the ligand
(Figure 5F). As can be seen from this analysis, each ligand has different electrostatic complementarity
with the binding site on Env. Moreover, when quantified and plotted against median IC50, there exists
a reasonable linear correlation between these values; that is, the higher the EC, the higher the potency.
Now identified, this correlation can be integrated seamlessly into our computational design workflow
for future compounds in this inhibitor class.
Figure 5. Cont.

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Figure 5. Electrostatic complementarity (EC) of the binding site in BG505 SOSIP.664 gp140 trimers
and ( ) BMS-626529, ( ) SC11, ( ) SC16, ( ) SC18, and ( ) SC39. ( ) EC score plotted versus
A
B
C
D
E
F
median IC50. Red line shows a linear trend line. Regions of the ligand surface where there is perfect
electrostatic complementarity with the protein are colored green, while the regions where there is
a perfect electrostatic clash are colored red.
3. Material and Methods
3.1. Compounds
BMS-626529 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). SC compounds
were synthesized de novo as outlined below. All available QC data for the SC compounds can be found
in the supplemental data file.
Synthesis of Common Intermediate 6 (Scheme 1)
◦
A solution of compound
1
(30 g, 0.13 mol) in THF (1.5 L) was cooled to
−
78 C. Vinylmagnesium
in THF (508 mL, 1N, 0.5◦1 mol) was added dropwise to the above so◦lution and the temperature was
maintained below
−
40 C. The mixture was stirred for 3 h at
−
78 C before being quenched with
saturated aqueous NH4Cl solution. The layers were separated, the aqueous layer was extracted
with EtOAc (3 500 mL), and the combined organic layer was washed with brine and concentrated.
The crude product was purified by silica gel column chromatography to obtain compound (5 g,
(4.0 g, 17.2 mmol) was dissolved in freshly prepared NaOMe (20 mL). CuI
(3.2 g, 17.2 mmol) was added and the mixture was microwave-irradiated at 110 ◦C and stirred for
3 h. After reaction completion, EtOAc and H2O were added, then the suspension was filtered and
the filtrate was separated. The organic layer was concentrated to give crude product, which was
×
2
17%). Compound
2
purified by column chromatography to give compound
3 (1.8 g, 45%). A mixture of compound 3
(0.7 g, 3.8 mmol), 3-methyl-1,2,4-triazole (6.4 g, 76.8 mmol), copper powder (0.49 g, 7.68 mmol), and

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KOH (0.43 g, 7.68 mmol) was heated to melt at 170–175 ◦C while being protected by N2 atmosphere.
After consumption of the starting material, EtOAc and H2O were added to the mixture, and the
suspension was filtered and the filtrate was collected. The organic layer was concentrated to give the
crude product, which was purified by column chromatography to give compound
4 (0.23 g, 26%).
◦
A solution of compound
3.5 mmol) was added dropwise at
4
(0.23 g, 1 mmol) in THF (12 mL) was cooled to
−
10 C, and EtMgBr (0.47 g,
−
10 ◦C, followed by the addition of pyridine (0.3 mL). The slurry
was cooled to
−
45 ◦C, and the ethyl 2-chloro-2-oxoacetate (0.55 g, 4 mmol) was added dropwise at
◦
◦
−
45 C. The temperature was allowed to rise to
−
10 C and the mixture was stirred for 1h. After
reaction completion, the mixture was quenched with IPA (2 mL) and H2O (20 mL). The mixture was
extracted with EtOAc, and the organic layer was washed with brine and concentrated to the give
crude product which was purified by column chromatography to give compound
5 (0.17 g, 52%).
Compound
5 (0.17 g, 0.52 mmol) was dissolved in MeOH (5 mL). NaOH (0.041 g, 1.03 mmol) and H2O
(2 mL) were added and the mixture was stirred at 25 ◦C for 6 h. HCl (1N) was added to adjust the pH
to 6, and the mixture was concentrated to give compound 6 (0.29 g) as crude product.
◦
◦
40 C; (b) NaOMe, CuI,
Scheme 1. Reagents and conditions: (a) vinylmagnesium bromide, THF,
−78 C to
−
◦
◦
MeOH, microwave, 110 C; (c) neat copper powder, KOH, N2 atmosphere, 170–175 C; (d) ethylmagnesium
bromide, ethyl-2-chloro-2-oxoacetate, pyridine, THF, −45 C to −10 C; (e) NaOH, MeOH, H2O.
◦
◦
Synthesis of Compound 8 (SC11, Scheme 2)
Compound
8 (SC11) was synthesized following the procedures described by Tuyishime et al. [4].
Scheme 2. Reagents and conditions: (a) DEPBT, DIEA, DMF room temperature, 12 h.
Synthesis of Compound 13 (SC16, Scheme 3)
Compound
9
(2 g, 10 mmol) was dissolved in DCM (30 mL). NMM (2 g, 20 mmol) was added,
◦
the mixture was cooled to 0 C, and MsCl (1.37 g, 12 mmol) was added dropwise. After addition,

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the mixture was stirred at 25 ◦C for 16 h. After reaction completion, H2O was added, and the mixture
was extracted with DCM, the combined DCM layers were combined and evaporated, and the residue
was purified by silica gel column chromatography to obtain compound 10 (1.8 g, 68%). Compound 10
(1.8 g, 6.79 mmol) was dissolved in DMF (10 mL), K2CO3 (1.87 g, 14 mmol), and benzothioic S-acid
(1.12 g, 8.15 mmol) were added. The mixture was heated to 80 ◦C for 2 h. After reaction completion,
H2O was added, and the mixture was extracted with EtOAc. The combined EtOAc layers were
combined and washed with H2O and brine, then concentrated under vacuum to give crude product
which was purified by silica gel column chromatography to obtain compound 11 (2 g, 96%). TFA
(3 mL) in DCM (20 mL) was added to compound 11 (2 g, 6.5 mmol) and the mixture was stirred at
◦
25 C for 3 h, and the mixture was concentrated to give compound 12 (2.3 g, crude) as a TFA salt.
To a solution of compound 12 (0.83 g, 0.4 mmol) in DMF (3 mL), DIEA (0.085 g, 0.66 mmol) was
added, followed by the intermediate compound
6 (0.1 g, 0.33 mmol) and HATU (0.15 g, 0.4 mmol).
The mixture was stirred at 25 ◦C for 2 h. The reaction mixture was then concentrated under reduced
pressure to remove DMF. The residue was partitioned between with EtOAc (10 mL) and saturated
NaHCO3 (10 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated.
The crude product was purified by preparative HPLC to give compound 13 (SC16) (0.032 g, 20%).
Scheme 3. Reagents and conditions: ◦(a) MsCl, NMM, DCM, 0–25 ◦C; (b) benzothioic S-acid, K2CO3,
◦
◦
DMF, 80 C, 2 h; (c) TFA, DCM, 0–25 C, 3 h; (d) HATU, DIEA, DMF, 20 C, 18 h.
Synthesis of Compound 18 (SC18, Scheme 4)
Compound 14, 2 g (0.018 mol), was dissolved in 30 mL of THF/H2O (2:1) and cooled to 0 ◦C.
To this solution, 6.06 g (0.043 mol) of K2CO3 was added, and a solution of (2.823 g, 0.0198 mol) of
benzoyl chloride in 5 mL of dry THF was added dropwise. The reaction mixture was stirred at room
temperature for 2 h and poured into 100 mL of a 5% solution of NaHCO3. The mixture was extracted
with DCM (2
×
30 mL), and the organic layer was dried with Na2SO4 and concentrated to yield
1.2 g (40%) of compound 15 as colorless crystals. Compound 15, 1 g (0.0054 mol), was dissolved in
10 mL of THF and 0.877 (0.0085 mol) of TEA was added. The mixture was cooled to 0◦ C, and 0.909 g
(0.0077 mol) of mesyl chloride was added dropwise. The reaction mixture was stirred for 1 h at room
temperature and poured into 100 mL of water. The mixture was extracted with DCM (3 × 30 mL), and
the combined organic layers were washed with 20 mL of brine, dried over Na2SO4, and concentrated
to yield 1.2 g (80%) of compound 16 as white crystals. Compound 16 (1.2 g, 0.0048 mol) was dissolved
in 10 mL of IPA, then 7.5 g of 40% aqueous methylamine solution was added and the obtained mixture
was stirred and heated in a sealed tube for 48 h at 130◦ C. The reaction mixture was poured into
100 mL water and extracted with DCM (5
×
20 mL). Combined extracts were dried over Na2SO4

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and concentrated. The crude material was purified by column chromatography using a mixture of
DCM/MeOH (98:2), then 97:3 as the eluent and fractions with Rf = 0.15 (DCM/MeOH 95:5) were
collected to yield 370 mg (40%) of 17 as a colorless oil. A solution of acid
6 (69 mg, 0.23 mmol), HATU
(261 mg, 0.68 mmol), and DIPEA (118 mg, 0.91 mmol) in dry DMF (10 mL) was stirred for 1 h. Then,
amine 17 (87 mg, 0.45 mmol) was added. The reaction mixture was stirred at 20 ◦C for 18 h. Then,
water (100 mL) with saturated NaHCO3 (10 mL) was added. The mixture was with EtOAc (3 × 30 mL).
Combined extracts were dried over Na2SO4 and concentrated. The crude material was purified by
column chromatography using the mixture DCM/MeOH (98:2) as the eluent to yield 27 mg (25%) of
compound 18 (SC18) as a pale-yellow solid.
Scheme 4. Re◦agents and conditions: (a) benzoyl chlo◦ride, K2CO3, H2O, THF, 0 ◦C, 2 h, r.◦t.; (b) MsCl,
Et3N, THF, 0 C, 1 h, r.t.; (c) H2O, (CH3)2CHOH, 130 C, 48 h; (d) HATU, DIEA, DMF, 20 C, 18 h.
Synthesis of Compound 23 (SC39, Scheme 5)
A mixture of aldehyde 19 (1.1 g 2.98 mmol) and hydrazine 20 (0.42 g, 2.38 mmol) in 20 mL of
methanol was refluxed for 2 h, concentrated, and purified with column chromatography (DCM/MeOH
99:1) to give 214 mg (26%) of compound 21 as a white solid. To 5 mL of a 2 M solution of HCl in dry
dioxane, compound 21 (140 mg, 0.39 mmol) was added at room temperature. The reaction mixture
was stirred overnight at room temperature, then 20 mL of dry ether was added, and the precipitated
solid was filtered off and washed with dry ether (2
×
10 mL). The amine 22 HCl salt (64 mg, 57%) was
obtained as a white solid. A mixture of acid (101 mg, 0.33 mmol), amine hydrochloride 22 (98 mg,
6
0.33 mmol), DIPEA (217 mg, 1.66 mmol), and HATU (382 mg, 0.99 mmol) in 5 mL of dry DMF was
stirred for 18 h at room temperature. The reaction mixture was poured into 100 mL of water and
extracted with DCM (3
×
30 mL), and the combined organic layers were washed with brine, dried with
Na2SO4, and concentrated. The residue was suspended in water and was filtered off and recrystallized
from 3 mL of EtOH to give compound 23 (SC39; 16 mg, 9%) as a pale-yellow solid.
Scheme 5. Reagents and conditions: (a) MeOH, reflux, 2 h; (b) 2 M HCl in dioxane, r.t.; (c) HATU,
DIEA, DMF, r.t., 18 h.

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3.2. Cells
Human embryonic kidney 293T cells (a gift from Dr. Irwin Chaiken, Drexel University, Philadelphia,
PA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), 10% FBS, 100 U/mL penicillin,
100 µg/mL streptomycin, and 2 mM L-glutamine. Human astroglioma U87 cells stably expressing
CD4/CCR5 or CD4/CXCR4 (obtained from Prof. Hongkui Deng, Peking University, and Prof. Dan
Littman, New York University, USA, through the AIDS Research and Reference Reagent Program,
Division of AIDS, NIAID, NIH [11,12]) were cultured in DMEM supplemented with 10% FBS, 100 U/mL
penicillin, and 100 g/mL streptomycin and 2 mM L-glutamine, 300
µ
µg/mL G418 (Thermo Scientific,
Waltham, MA, USA), and 1 µg/mL puromycin (Thermo Scientific). Cells were incubated continuously,
unless otherwise stated, at 37 ◦C in a humidified 5% CO2/95% air environment.
3.3. Proteins
The soluble cleaved trimer B41 SOSIP.664 gp140 was a generous gift from Dr. Andrew Ward
(Scripps Institute, La Jolla, CA, USA); IgG b12 anti-HIV-1 gp120 was obtained through the NIH AIDS
Reagent Program, Division of AIDS, NIAID, NIH; anti-HIV-1 gp120 monoclonal antibody (IgG1 b12)
was obtained from Dr. Dennis Burton and Carlos Barbas. The overproduction and purification of
p24 (HIV-1 capsid [CA]) were performed as previously described [13]. Briefly, a vector containing
C-terminally His-tagged HIV-1NL4-3 CA (a gift from Dr. Eric Barklis, Oregon Health and Science
University, Portland, OR, USA) was transformed into BL21-Codon Plus (DE3)-RIL Competent Cells
(Agilent Technologies, Wilmington, DE, USA) and grown up in autoinduction ZYP-5052 medium
overnight with shaking (225 rpm) at 30 ◦C [14]. Bacterial cultures were spun down at 7000 rpm, and
the supernatant was discarded. Cell pellets were resuspended in 40 mM Tris–HCl pH 8.0, 500 mM
NaCl, and 3 mM imidazole and lysed via sonication. The resultant supernatant was clarified and
immediately applied to a Talon cobalt resin affinity column (Clonetech Laboratories, Mountain View,
CA, USA). Bound CA was washed and eluted using increasing concentrations of imidazole. Purified
CA-H6 was dialyzed overnight into 20 mM Tris–HCl pH 8.0 at 4 ◦C, concentrated to 120
µM, flash
frozen in liquid nitrogen, aliquoted, and stored at −80 ◦C.
3.4. Production of Pseudotyped Viruses
Single-round infectious envelope-pseudotyped luciferase-reporter viruses were produced by dual
transfection of two vectors (3 g of vector 1 and 4 g of vector 2) in 6-well plated 293T cells (1
106
µ
µ
×
cells/well) [12]. Vector 1 is an envelope-deficient HIV-1 pNL4-3-Luc+R-E plasmid which carries the
luciferase reporter gene [15]. Vector 2 is a plasmid expressing either the HIV-1 gp160 Env, vesicular
stomatitis virus glycoprotein (VSV-G), or amphotropic murine leukemia virus Env (AMLV) [16,17].
Transfections of these vectors were carried out via calcium phosphate (ProFection Mammalian
Transfection System, Promega, Madison, WI, USA) for 5 h. Following the 5 h transfection incubation,
DNA-containing medium was removed and cells were washed with DMEM and replenished with fresh
culture media. Supernatants containing pseudovirus were collected 72 h post-transfection, clarified,
filtered, aliquoted, and stored at −80 ◦C.
3.5. ELISA-Based Quantification of p24 Content
ELISA plate was coated with 50 ng of mouse anti-p24 (Abcam, ab9071, Cambridge, MA, USA)
◦
for overnight at 4 C, blocked with 3% BSA for 2 h at room temperature, and washed with 0.5%
Tween in PBS. Pseudoviral stocks were lysed with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis,
◦
◦
MO, USA) at 37 C for 1 h and added to the plate overnight at 4 C. Simultaneously, p24 protein
(produced, purified, and validated as described above) was added for the generation of a standard
curve. Following the overnight incubation, the plate was washed with PBST and 1:5000 dilution of
rabbit anti-p24 (Abcam, ab63913) was added for 2 h at room temperature. After washing the unbound
rabbit anti-p24 off the plate with PBST, goat anti-rabbit-HRP at a 1:5000 dilution was added for 1 h

Molecules 2018, 23, 1940
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at room temperature. The plate was then extensively washed with PBST. Subsequently, a solution of
0.4 mg/mL O-phenylenediamine in a phosphate–citrate buffer with sodium perborate (Sigma-Aldrich)
was added and incubated in the dark for 30 min. Optical densities were then obtained at 450 nm in
a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific).
3.6. Single-Round Infection Assay
The details of the single-round HIV-1 infection assay have been published previously [15
,18,19].
Briefly, U87.CD4.CCR5/CXCR4 (1.2
compatible tissue culture plates (Greiner Bio-One, Monroe, NC, USA). After 24 h, compound, DMSO
(vehicle control for compounds, Sigma) was mixed with pseudotyped viruses (normalized to p24
×
104 cells/well) target cells were seeded in 96-well luminometer-
◦
content), and the mixture was added to the target cells and incubated for 48 h at 37 C. Following this,
the media was removed from each well, and the cells were lysed by the addition of 50
µL of luciferase
lysis buffer (Promega) and one freeze–thaw cycle. A GloMax 96 microplate luminometer (Promega)
was used to measure the luciferase activity of each well after the addition of 50
substrate (Promega).
µL of luciferase assay
3.7. Cellular Toxicity
The viability of the U87.CD4.CCR5/CXCR4 cells was determined by the Cell Counting Kit-8 cell
proliferation and cytotoxicity assay (Dojindo Molecular Technologies, Rockville, MD, USA) as per the
manufacturer’s instructions. U87.CD4.CCR5/CXCR4 cells were seeded at a density of 1.2
×
104 cells/well
in 96-well tissue culture plates (Olympus plastics) 24 h before treatment. Cells were treated with compound
◦
(1 mM–0.1
µ
M) or DMSO (Sigma) for 48 h at 37 C. Subsequently, 10
µ
L of the CCK-8 solution was
added to each well and allowed to incubate at 37 ◦C for 4 h before absorbance at 450 nm was measured
in a Multiskan GO Microplate Spectrophotometer (Thermo Scientific). Untreated cells were used as
™
a medium control and 0.1% SDS-treated cells served as a positive control. The 50% cytotoxicity (CC50)
value was defined as the concentration of the compounds that reduced the absorbance of treated cells by
50% as compared with control cells.
3.8. SPR Direct Interaction Analysis
Binding kinetics and affinity of the compound inhibitors were measured using SPR-based
binding assays that were performed with a BiacoreTM 8K instrument (GE Healthcare). The B41
SOSIP.664 gp140 trimer was attached to a Sensor Chip CM5 (GE Healthcare) via amine coupling
chemistry. SOSIP Env trimer was diluted into 10 mM sodium acetate pH 5.0 to a concentration of
50 µ
g mL−1 and injected for 10 min over the active flow cell of an EDC/NHS (400 mM N-ethyl-N-(3-
dimethylaminopropyl)-carbodiimide hydrochloride and 100 mM N-hydroxysuccinimide) reactive
surface to a final level of approximately 16,000 RU on each of 8 channels. Non-crosslinked reactive
esters were blocked with 1 M ethanolamine. Reference flow cells were prepared similarly using amine
coupling of IgG b12 as a negative control. The running buffer used was PBS-P+ (20 mM phosphate
buffer at pH 7.4, with 27 mM KCl, 137 mM NaCl, and 0.05% surfactant P20) for amine coupling,
with the addition of 3% DMSO for compound binding assays. Compounds were injected for 160 s
at 75 µ
L min−1, and 600 s of dissociation, using the multicycle kinetics injection mode. Data was
collected at a rate of 10 Hz. The surface was regenerated using a 60-s injection of 10 mM glycine pH
1.5 to ensure complete removal of bound compounds. BiacoreTM 8K Evaluation software was used for
kinetic analysis. Raw sensorgram data were reference-subtracted and blank-buffer-subtracted before
kinetic and affinity analysis to account for nonspecific binding and injection artifacts. Association and
dissociation rate constants and calculated KD values for binding were determined using a simple 1:1
binding model from quadruplicate runs.

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3.9. Molecular Modeling
3.9.1. Bioactive Conformation Hypothesis Prediction
Generation of a hypothesis for the conformation adopted by BMS-377806, BMS488043, BMS-
626529, and SC11 was achieved using FieldTemplator in Forge, version 10.5 (Cresset®, Litlington,
Cambridgeshire, UK, http://www.cresset-group.com/forge/). Briefly, each of the four compounds
was conformationally populated using Cresset’s Xedex conformational search method (embedded
within Forge). The 3D field point pattern for each conformation of each template molecule was
then calculated and used to cross-compare to each other in field/shape space, with no dependence
on chemical structure. Using 3D field point patterns, the conformations of the compounds were
exhaustively compared in a pairwise fashion until field point patterns common to all four molecules
were identified. The resulting templates (i.e., binding mode hypotheses) each consist of one
conformation of each of the four molecules (quartets). The quartets from the FieldTemplater experiment
using the compounds were visually inspected, and one conformation of BMS-626529 was selected as
the query molecule for the Spark bioisosteric replacement experiments (Spark, version 10.5, Cresset®,
Litlington, Cambridgeshire, UK; http://www.cresset-group.com/spark/). The selected conformation
of BMS-626529 appeared in several of the top-scoring templates.
3.9.2. Docking Simulations
BMS-626529, SC11, SC16, SC18, and SC39 were prepared and then energy-minimized using
the MMFF94 Force Field (ChemBio3D Ultra 15.0) with a root mean squared (RMS) gradient of 0.01
and number of alterations of 10,000. The minimized structures were then saved as PDB files for the
docking simulations. The Autodock tools graphical interface (MGtools 1.5.7rc1) [15
to prepare the compounds for docking.
,16] was then used
The crystal structure of BMS-626529 bound to BG505 SOSIP.664 trimer (PDB code: 5U7O) [10
]
was downloaded and prepared by the Autodock tools graphical interface (MGtools 1.5.7rc1), where
nonpolar hydrogens were merged, Kollman charges were added, and Gasteiger charges were
calculated. The grid box for the docking search was centered on the cocrystalized ligand BMS-626529
and set to 40, 56, and 40 points for the x, y, and z dimensions, respectively, with a spacing grid of
0.375 Å. The AutoGrid 4.2 algorithm [20,21] was used to evaluate the binding energies between the
compounds and the protein and to generate the energy maps for the docking run. For high-accuracy
mode docking of the compounds with the protein, the maximum number of evaluations (25 Å~106)
was used. Fifty runs were generated for each compound by using the Autodock 4.2 Lamarckian
genetic algorithm [20,21] for the searches. Cluster analysis was performed on docked results, with
a root mean square tolerance of 0.5 Å. The docking protocol was validated by docking the prepared
BMS-626529 structure, and this generated only one stable cluster of poses. The lowest energy pose
was very similar to the cocrystalized BMS-626529, with an RMS value of 0.67. The calculated binding
energy (∆G binding) and predicted Ki values for the compound−protein complexes were recorded.
4. Conclusions
In this study, we have generated a series of four compounds with ranges of antiviral potency
against two subtype B Env pseudotyped HIV-1 viruses in the low nanomolar to the low micromolar
range. We have established a novel SPR assay to determine the kinetic parameters and correlated
the off-rates with antiviral potency. Lastly, we have identified a relationship between electrostatic
complementarity of the ligands with their respective binding site on Env and their potency. These
relationships can now be incorporated into our computational design and experimental workflows to
greatly facilitate our entry inhibitor design efforts and fulfill our overarching goal.
Supplementary Materials: The following are available online, SPR supplemental methods and Figure S1: QC data
for the SC compounds.

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Author Contributions: Conceptualization, S.C. and M.E.M.; Methodology, S.C., M.E.M., A.A.R.; Formal Analysis,
S.C., M.B.M., A.A.R., M.E.M.; Investigation, M.E.M., M.B.M., A.A.R.; Writing-Original Draft Preparation, S.C.;
Writing-Review & Editing, S.C., M.B.M., A.A.R., M.E.M.; Supervision, S.C.; Funding Acquisition, S.C.
Funding: This work was supported by NIH/NIAID grant AI118415 (Cocklin, PI).
Acknowledgments: We would like to thank Cresset (UK; http://www.cresset-group.com/) for access to their
software via an academic license.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).