Discovery and optimization of novel small-molecule HIV-1 entry inhibitors using field-based virtual screening and bioisosteric replacement

Article abstract of DOI:10.1016/j.bmcl.2014.10.027

With the emergence of drug-resistant strains and the cumulative toxicities associated with current therapies, demand remains for new inhibitors of HIV-1 replication. The inhibition of HIV-1 entry is an attractive, yet underexploited therapeutic approach with implications for salvage and preexposure prophylactic regimens, as well as topical microbicides. Using the combination of a field-derived bioactive conformation template to perform virtual screening and iterative bioisosteric replacements, coupled with in silico predictions of absorption, distribution, metabolism, and excretion, we have identified new leads for HIV-1 entry inhibitors.

Full text of DOI:10.1016/j.bmcl.2014.10.027

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5439–5445
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of novel small-molecule HIV-1 entry
inhibitors using field-based virtual screening and bioisosteric
replacement
Marina Tuyishime a, Matt Danish a, Amy Princiotto b, Marie K. Mankowski c, Rae Lawrence d,
Henry-Georges Lombart e, Kirill Esikov e, Joel Berniac e, Kuang Liang f, Ji Jingjing f, Roger G. Ptak c,
Navid Madani b, Simon Cocklin a,
⇑
a Department of Biochemistry & Molecular Biology, Drexel University College of Medicine, Rooms 10302-10306, 245 North 15th Street, Philadelphia, PA, USA
b Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Division of AIDS, Harvard Medical School, Boston, MA, USA
c Department of Infectious Disease Research, Southern Research Institute, 431 Aviation Way, Frederick, MD 21701, USA
d Cresset, New Cambridge House, Bassingbourn Road, Litlington, Cambridgeshire, UK
e AsisChem, Inc., 313 Pleasant St, Watertown, MA 02472, USA
f HD Biosciences Co., Ltd, 590 Ruiqing Road, Zhangjiang East Campus, Pudong, Shanghai 201201, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
With the emergence of drug-resistant strains and the cumulative toxicities associated with current ther-
apies, demand remains for new inhibitors of HIV-1 replication. The inhibition of HIV-1 entry is an attrac-
tive, yet underexploited therapeutic approach with implications for salvage and preexposure
prophylactic regimens, as well as topical microbicides. Using the combination of a field-derived bioactive
conformation template to perform virtual screening and iterative bioisosteric replacements, coupled with
in silico predictions of absorption, distribution, metabolism, and excretion, we have identified new leads
for HIV-1 entry inhibitors.
Received 25 July 2014
Revised 6 October 2014
Accepted 8 October 2014
Available online 16 October 2014
Keywords:
Field-based virtual screening
Three-dimensional virtual screening
HIV-1 Env
Ó 2014 Elsevier Ltd. All rights reserved.
Antiviral
Bioisostere
Entry inhibitor
Field-based scaffold hopping
With the emergence of drug-resistant strains and the cumula-
tive toxicities associated with current therapies, demand remains
for new inhibitors of HIV-1 replication. The inhibition of HIV-1
entry is an attractive, yet underexploited therapeutic approach
with implications for salvage and preexposure prophylactic
regimens, as well as topical microbicides. The entry of HIV-1 into
permissible cells is a complex series of events orchestrated by
the viral envelope glycoprotein complex, the only exposed viral
components on the virion surface. Being the only viral products
accessible to the host cell immune system, the Env glycoproteins
gp120 and gp41 have evolved several strategies to mask function-
ally important regions from the neutralizing antibody response.
These strategies include the presence of surface-exposed variable
loops on gp120, a high degree of glycosylation, the lability and
defectiveness of many envelope glycoprotein spikes (possible
immunologic decoys), and conformational flexibility.1–4
The Env complex is organized on the virion surface as trimeric
spikes composed of three gp120 molecules noncovalently linked
to three gp41 molecules. The heavily glycosylated surface gp1205
contains a core composed of conserved regions (C1–C5) and hyper-
variable regions that are mostly disulfide-constrained, surface-
exposed loop structures (V1–V5) that retain a large degree of flex-
ibility.4,6–9 The transmembrane glycoprotein gp41 contains the
fusion peptide that is inserted into the membrane of the target
cells,10 as well as two heptad repeat (HR) domains (aminoterminal
Abbreviations: ADME, absorption, distribution, metabolism, and excretion;
AMLV, amphotropic murine leukemia virus; BBB, blood–brain barrier; BIF, bioiso-
stere factor; CNS, central nervous system; DCM, dichloromethane; DiEA, diisopro-
pylethylamine; DIPEA, N,N-diisopropylethylamine; DMF, dimethylformamide;
EtOAc, ethyl acetate; HATU, hexafluorophosphate; hERG, human ether-à-go-go-
related gene; HIA, human intestinal absorption; HPLC, high-performance liquid
chromatography; HR, heptad repeat; IC50, half-maximal inhibitory concentration;
PBMCs, peripheral blood mononuclear cells; Pd/C, palladium on carbon; TC50, half-
maximal toxic concentration; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
⇑
Corresponding author. Tel.: +1 215 762 7234 (Office), +1 215 762 4979 (Lab);
fax: +1 215 762 4452.
E-mail address: scocklin@drexelmed.edu (S. Cocklin).
http://dx.doi.org/10.1016/j.bmcl.2014.10.027
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

5440
M. Tuyishime et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5439–5445
or HR1 and carboxyterminal or HR2) that are implicated in the for-
mation of a six-helix-bundle fusion intermediate via a conforma-
tional change following receptor interaction. HIV-1 infection
usually occurs only after two sequential and specific binding steps:
first, the Env gp120 protein binding to the CD4 antigen present in
CD4+ T cells, monocyte/macrophages, and other immune and non-
immune cells; and second, gp120 binding to a member of the che-
mokine receptor subfamily, within the large G protein–coupled
family of receptors, mainly CCR5 and/or CXCR4.
Advances in knowledge of the molecular mechanisms of HIV-1
entry have allowed the discovery and development of molecules
that target discrete steps in the entry process and have shown suc-
cess in the clinic. Successful examples include maraviroc (Selzen-
try; Pfizer, New York, NY), which binds to CCR5 and blocks the
interaction of the Env complex with the coreceptor, and enfuvirtide
(Fuzeon; Hoffman-La Roche, Nutley, NJ), which binds to gp41 and
stops the fusion of the viral and cell membrane. However, as of
yet, no gp120-targeted therapies have been approved for use in
the clinic.
conserved and available for targeting on the virion and that com-
pounds such as BMS-663068 may have great therapeutic value.
In fact, the entry inhibitor BMS-663068 recently performed favor-
ably in a phase IIb clinical trial (presented at the Conference on
Retroviruses and Opportunistic Infections, March 3–March 6,
2014, Boston, MA).
Although the agents are potent and have a broad therapeutic
spectrum, the piperazine class of entry inhibitors developed by
Bristol–Myers Squibb has been plagued by the problems of low sol-
ubility and poor intrinsic dissolution properties. To circumvent
these issues, a prodrug approach was adopted. BMS-663068 is a
phosphonooxymethyl prodrug of BMS-62652914,16–18 designed to
have increased solubility in the gut. The prodrug is thought to be
cleaved by alkaline phosphatase, located on the luminal surface
of the small intestine brush border membranes, releasing BMS-
626529, which is then rapidly absorbed.19 Despite the success of
this approach, an entry inhibitor candidate with more intrinsic
druglike properties would be preferable.
As a first step towards discovering entry inhibitor candidates
with intrinsic druglike properties, we conducted a series of field-
based three-dimensional similarity virtual screening experiments
using Blaze (Cresset, Litlington, UK) with a high-content field-
based pharmacophore template derived from BMS-62652917,19–21
(Forge/FieldTemplater, Cresset) in order to identify novel scaffolds
that could function as entry inhibitors. To perform a Blaze virtual
screen, an active ligand in its three-dimensional bioactive confor-
mation should be used as a search query. In the absence of struc-
tural information, a binding mode hypothesis may be calculated.
As no structural information is currently available for BMS-
626529 in its target-bound state, we used FieldTemplater (Forge)
to determine a hypothesis for the three-dimensional conformation
adopted in binding to the target using field and shape information
to create a template using compounds BMS-626529, BMS-488043,
and BMS-378806.16,22 The FieldTemplater-derived hypothesis for
the bioactive conformation was then annotated with its calculated
field points, resulting in a three-dimensional field point pattern.
The field point pattern provides a condensed representation of
the compound’s shape, electrostatics, and hydrophobicity. It is well
established that when two diverse structures have conformations
with similar field point patterns, they are experienced by the
In the HIV-1 entry field, two main Env-targeted inhibitor
chemotypes predominate: the NBD-556 analogues11 and the
BMS-378806 analogues (Fig. 1).12 NBD-556 and its analogues bind
to the conserved CD4-binding site in gp120 and block the interac-
tion of the Env complex with cellular CD4.13–15 The binding site for
BMS-378806 and its analogues is poorly understood, and based on
resistance mutation data it may actually be a composite site com-
posed of regions of gp120 and g41.16 The mechanism of action of
BMS-378806 and its derivatives is also under debate, with some
studies claiming a CD4 binding inhibition mechanism and others
describing an allosteric mechanism that prevents the propagation
of the receptor binding signals from gp120 to gp41.17,18 Given
the huge therapeutic potential of inhibiting HIV-1 entry, the devel-
opment of new chemotypes that target viral entry with broad
activities is highly desirable. In this study we describe the use of
high-content field-based pharmacophore screening as a first step
in identifying new chemotypes for this inhibitor class.
To date, the piperazine-based entry inhibitors as first described
by Bristol–Myers Squibb12 are the most broadly acting and potent
HIV-1 entry inhibitors. This indicates that the binding site for these
compounds, although currently not well described, is well
Figure 1. Structures of entry inhibitors developed by Bristol–Myers Squibb and the New York Blood Bank. The chemical structures were drawn with ChemAxon software
(Budapest, Hungary).

M. Tuyishime et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5439–5445
5441
receptor in a similar fashion.23–33 The field-based alignment for the
three selected templating molecules is shown in Figure 2.
Therefore, we investigated whether the piperazine core could be
replaced with another moiety that would function in the same
capacity but be chemically distinct. Pfizer reported a study in
which replacement of an N-methylpiperazine in a lead compound,
JNJ-7777120, with 2-methyloctahydropyrrolo[3,4-c]pyrrole led to
a compound that retained binding affinity and selectivity but also
had improved resistance to metabolism.35 We therefore decided to
determine whether this dipyrrolidine group could successfully
replace the piperazine in compound 11. In silico bioisostere
replacement (scaffold-hopping) experiments using Spark (Cresset,
UK) conducted to assess the potential for this group to substitute
the piperazine in 11 indicated that it may be a viable, if not imper-
fect replacement. This conclusion was based upon the value of the
calculated bioisostere factor (BIF%), a rescaled score, which indi-
cates how much better or worse the replacement is compared with
simply capping the attachment point(s) with hydrogen(s). Positive
BIF% values indicate good bioisosteres; negative values correspond
to replacements where the geometry of the original molecule is
reproduced; however, the fragment is not a good mimic of the
replaced part.
The field point pattern for the hypothesized three-dimensional
bioactive conformation of BMS-626529 was subsequently used to
query a database of approximately 6 million commercially avail-
able compounds using Blaze. The steps involved in the Blaze virtual
screening procedure are shown schematically in Figure 3. The
Blaze procedure resulted in the rank ordering of the top 1000 Blaze
library compounds whose three-dimensional arrangements of field
points had similarities to that of the BMS-626529 template struc-
ture. Fifty compounds were chosen and purchased for biological
testing using the single-round infection assay.34 The structures of
the selected compounds are shown as Supplementary data. Those
compounds found to be active and specific, as judged by their
activity against HIV-1YU-2 Env pseudotyped and amphotropic
murine leukemia virus (AMLV) Env pseudotyped HIV-1 virus, are
shown in Table 1. Five compounds with specific anti-HIV-1 activity
were discovered using this approach with half-maximal inhibitory
concentrations (IC50) in the range of 13–150
was found to be the most potent, inhibiting HIV-1YU-2 with an
IC50 value of 13.1 1.7 M.
lM. Compound 11
l
A BIF% of 57 was obtained for the dipyrrolidine group, and
therefore this replacement for piperazine was investigated. Substi-
tution of the piperazine core in 11 with 2-methyloctahydropyrrol-
o[3,4-c]pyrrole yielded compound SC04 ([5-(1,2-dihydro
The active and specific compounds identified from this study,
despite showing a degree of chemical diversity, all share a pipera-
zine core comparable to the Bristol–Myers Squibb compounds.
Figure 2. (a) Field-based template A containing a single conformation of compounds BMS-378806 (pink), BMS-488043 (lime green), and BMS-626529 (teal green) aligned
based on their three-dimensional field point patterns. Negatively charged field points are shown in blue; positively charged field points are red; van der Waals/shape field
points are displayed in yellow; centers of hydrophobicity are shown in orange. (b) Conformation 69 of BMS-626529 with its associated field points. This conformation was
common to the top three templates identified and was used as the query structure for the Blaze (Cresset, Litlington, UK) field-based virtual screen.
Figure 3. Steps involved in a Blaze (Cresset, Litlington, UK) field-based virtual screen experiment. (1) An active molecule is selected and converted to a relevant bioactive
conformation. (2) Field points are added to this search ligand in the specified conformation to produce the Blaze pharmacophore seed. (3) The Blaze search query consists of
the field point pattern of the pharmacophore seed, which is used to search the Blaze database by alignment of every structure based on field point patterns (of its up to 100
conformations). (4) Top scoring compounds are retrieved as three-dimensional alignments to the search query, along with their score (molecular similarity based on 50%
shape, 50% fields).

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M. Tuyishime et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5439–5445
Table 1
Table 2
Structure and potency of compounds identified within this study with specific activity
against HIV-1 YU-2
Structure and potency of second-generation compounds based on the dipyrrolidine
core scaffold
Compound
IC50 YU-2 (lM)
IC50 AMLV (
lM)
Compound
IC50 (lM)
JR-CSF
HxBc2
AMLV
NA
11
O
N
13.1 1.7
NA
SC04
N
O
100 30
ND
O
N
N
N
O
Cl
O
NH
O
O
O
12
53.5 3.0
NA
N
O
N
N
SC07
0.98 0.06
16.8 6.2
NA
O
N
S
O
N
O
O
N
SC08
28
33.7 4.5
NA
O
O
S
N
NH
N
N
O
S
0.09 0.01
0.19 0.08
NA
O
N
O
O
N
N
32
79.4 11
NA
O
N
N
O
N
N
O
NH
N
N
O
N
S
SC11
0.0008 0.0004 0.001 0.0001
NA
O
N
O
O
34
153 44
NA
N
N
N
O
SC26
N
Chemical structures were drawn with ChemAxon software (Budapest, Hungary).
NA = not active over concentration range tested.
N
N
O
NH
N
O
0.002 0.0001 0.0006 0.00008 NA
acenaphthylene-5-carbonyl)-1,3,3a,4,6,6a-hexahydropyrrolo
[3,4-c]pyrrol-2-yl]-phenyl-methanone]), a novel compound that
retains the properties of 11, albeit with lower potency (IC50
O
N
HIV-1YU-2 = 70
6 lM; IC50 HIV-1JR-CSF = 100 30 lM).
N
Having identified a new scaffold, we focused on redesigning
compound SC04 to improve its potency and to remove any poten-
tial toxophores. The head region of SC04, like compound 11, is an
acenaphthene group, which may be carcinogenic owing to its
potential to intercalate into DNA sequences. Moreover, several
studies by Bristol–Myers Squib have demonstrated that this head
region greatly influences the potency of compounds in the pipera-
zine class. Subsequently, in order to improve the potency of SC04,
we downloaded from PubChem 453 compounds that displayed
similarity to BMS-488043. These compounds were exhaustively
fragmented (Spark DBGen, Cresset, UK), and we used the resulting
fragments in iterative Spark experiments, seeking fragments that
could function as bioisosteric substitutions for the acenaphthene
O
Chemical structures were drawn with ChemAxon software (Budapest, Hungary).
AMLV = amphotropic murine leukemia virus; NA = not active over concentration
range tested; ND = not determined.
moiety but with greater potential for hydrogen bonding interac-
tions. This resulted in the identification of two head groups,
7-chloro-4-methoxy-1H-pyrrolo[2,3-c]pyridine and 4,7-dime-
thoxy-1H-pyrrolo[2,3-c]pyridine, with BIF% values (compared with
acenaphthene) of 57 and 64, respectively. Two compounds bearing
these head groups were synthesized (SC07 and SC08) and assessed

M. Tuyishime et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5439–5445
5443
in the single-round infection assay. Owing to the relative decreased
sensitivity of HIV-1JR-CSF to SC04, as compared with HIV-1YU-2, this
isolate was selected for use in potency optimization. Both com-
pounds were specific to HIV-1 (no inhibition of AMLV-pseudotyped
HIV-1) and inhibited HIV-1JR-CSF pseudotyped HIV-1 virus with IC50
this compound and compare them with those of the BMS pipera-
zine-based entry inhibitors (Fig. 4). To accomplish this comparison,
we used a combination of computationally guided bioisosteric
replacement using Spark (focusing on the terminal phenyl group
of SC11) and in silico prediction of druglike metrics of the results
as implemented in the oral non-central nervous system (CNS) drug
profile in StarDrop 5.5 (Optibrium, Ltd, Cambridge, UK).40
Optibrium’s oral non-CNS drug profile is composed of the fol-
lowing metrics: logS (intrinsic aqueous solubility); classification
for human intestinal absorption; logP (octanol/water); hERG
(human ether-à-go-go-related gene) pIC50 (mammalian cells);
cytochrome P450 CYP2D6 classification; cytochrome P450 CYP2C9
pKi values; classification of P-glycoprotein transport; classification
of blood–brain barrier (BBB) penetration; and predicted BBB pene-
tration value. The models and their respective importance to the
profile are shown in Figure 4a.
values of 0.98 0.06
lM and 0.09 0.01 lM, for SC07 and SC08,
respectively (Table 2).
The head group of SC08 is the same as in BMS-488043.36,37
Replacement of the methoxy in the 7 position on the azaindole ring
with a methyltriazole greatly improved the potency of the com-
pound, resulting in the creation of BMS-626529.38,39 Therefore,
we explored this substitution in SC08 to determine whether it
would result in a similar enhancement of potency (SC11). SC11
was synthesized and tested for specificity and activity against
HIV-1JR-CSF using the single-round infection assay. SC11 displayed
greatly enhanced potency as compared with SC08, retaining HIV-
1 specificity and inhibiting HIV-1JR-CSF with an IC50 value of
Details for the specific models are provided in the StarDrop Ref-
erence Guide from Optibrium and are online at the StarDrop FAQs
(http://www.optibrium.com/community/faq/adme-qsar-models).
A probabilistic scoring algorithm41 is then used to combine the
model predictions in the oral non-CNS drug profile into an overall
score.
0.0008 0.0004 lM (Table 2).
Synthesis and assessment of SC11 revealed that the dipyrroli-
dine core can support compounds of nanomolar potency that pre-
vent HIV-1 entry. The next step was to analyze the predicted ADME
properties (absorption, distribution, metabolism, and excretion) of
Figure 4. (a) The individual models that comprise the oral non-CNS (central nervous system) drug profile and their respective importance to the profile. HIA = human
intestinal absorption; hERG (human ether-à-go-go-related gene); IC50 = half-maximal inhibitory concentration; BBB = blood–brain barrier. (b) Plot showing the StarDrop
(Optibrium, Ltd, Cambridge, UK)—derived logS versus the score from a multimetric oral non-CNS profile for the BMS and SC compounds.

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M. Tuyishime et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5439–5445
Table 3
profile. Like the BMS piperazine-based entry inhibitors, the new
dipyrrolidine scaffold displayed varying degrees of potency against
HIV-1 isolates from different subtypes, probably reflecting isolate-
specific differences in the binding site on Env. Further investigation
and determination of the exact binding site of these class of entry
inhibitors will undoubtedly be invaluable in the redesign and
optimization of such compounds, while illuminating a new site
of vulnerability in the Env complex of HIV-1. Taken as a whole,
these results extend the chemotypes for this class of HIV-1 inhib-
itor and validate the use of multiparameter optimization using
high-content three-dimensional field-based models, bioisosteric
replacement, and consideration of druglike metrics in drug design.
Therapeutic spectrum of SC26 against highly prevalent HIV-1 subtypes and HIV-2
Virus isolate
Subtype
IC50
(
l
M)
TC50
M)
Antiviral index
(TC50/IC50)
(
l
92UG037
91US004
94US_33931N
98US_MSC5016
99UG_A07412M1
HIV-2 CDC310319
A
B
B
C
D
NA
25.2 6.9
0.195 0.012
0.0006 0.0005
3.9 0.8
0.49 0.06
>100
>100
>3.97
>513
>166,666
>25.6
>204
NA
IC50 = half-maximal inhibitory concentration; NA = not applicable; TC50 = half-
maximal toxic concentration.
Acknowledgments
This combined Spark/StarDrop analysis suggested that a simple
replacement of phenyl with cyclohexene may be a good transfor-
mation to perform in order to improve ADME properties while
retaining the potency associated with the dipyrrolidine scaffold.
StarDrop analysis of the cyclohexene variant of SC11 (designated
SC26) versus BMS-663068 using the oral non-CNS drug profile pro-
vided scores for SC26 and BMS-663068 of 0.3815 0.2323 and
0.0799 0.1014, respectively (Fig. 4b). For reference, scores range
from 0 to 1, with 0 suggesting extremely non-druglike, and 1 sug-
gesting the perfect drug.
This work was supported in part by NIH/NIAID grant
1R21AI104354-01A1 (Cocklin, PI) and W. W. Smith Charitable
Trust (Four Falls Corporate Center, Gladwyne, PA 19035, USA)
grant #A1301 (Cocklin, PI). We thank Diana Winters (Academic
Publishing Services, Drexel University College of Medicine) for
proofreading the manuscript.
Supplementary data
Given two oral non-CNS drug profile scores with errors corre-
sponding to 1 standard deviation:
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.10.
027.
X1 ꢀ r1
X2 ꢀ r2
;
and assuming that the error is normally distributed, the difference
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Because it demonstrated good potency against two isolates in
the single-round infection assay as well as specificity to HIV-1,
we next sought to quantify the potency of SC26 against the fully
infectious virus. Moreover, this assay was performed using healthy,
primary cells to gain further insight into potential toxicities of the
compound towards natural target cells. Additionally, as a key issue
in the development of novel HIV drugs is their ability to inhibit the
replication of genetically diverse isolates (especially isolates from
the most globally prevalent subtypes A, B, C, and D), we chose to
assess the potency of SC26 to inhibit the replication of isolates
from subtypes A, B, C, and D in primary human peripheral blood
mononuclear cells (PBMCs).42–44 Concomitantly, we assessed the
toxicity of SC26 to the PBMCs using an MTS assay. The results of
these analyses are summarized in Table 3. As can be observed from
the IC50 values, the potency of SC26 against different isolates
ranges quite dramatically, most likely reflecting differences in the
binding site between subtypes. Moreover, SC26 had no effect on
the replication of an HIV-2 isolate, mirroring the specificity analy-
ses using the AMLV pseudotyped recombinant HIV-1 virus, and
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