Structure-activity relationship studies of indole-based compounds as small molecule HIV-1 fusion inhibitors targeting glycoprotein 41

Article abstract of DOI:10.1021/jm500344y

We previously described indole-containing compounds with the potential to inhibit HIV-1 fusion by targeting the hydrophobic pocket of transmembrane glycoprotein gp41. Here we report optimization and structure-activity relationship studies on the basic scaffold, defining the role of shape, contact surface area, and molecular properties. Thirty new compounds were evaluated in binding, cell-cell fusion, and viral replication assays. Below a 1 μM threshold, correlation between binding and biological activity was diminished, indicating an amphipathic requirement for activity in cells. The most active inhibitor 6j exhibited 0.6 μM binding affinity and 0.2 μM EC₅₀ against cell-cell fusion and live virus replication and was active against T20 resistant strains. Twenty-two compounds with the same connectivity displayed a consensus pose in docking calculations, with rank order matching the biological activity. The work provides insight into requirements for small molecule inhibition of HIV-1 fusion and demonstrates a potent low molecular weight fusion inhibitor.

Full text of DOI:10.1021/jm500344y

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Article
Structure-Activity Relationship Studies of Indole-based Compounds
as Small Molecule HIV-1 Fusion Inhibitors Targeting Glycoprotein-41
Guangyan Zhou, Vladimir Sofiyev, Hardeep Kaur, Beth A Snyder,
Marie K Mankowski, Priscilla Hogan, Roger G. Ptak, and Miriam Gochin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500344y • Publication Date (Web): 24 May 2014
Downloaded from http://pubs.acs.org on June 2, 2014
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StructureꢀActivity Relationship Studies of Indoleꢀbased Compounds as
Small Molecule HIVꢀ1 Fusion Inhibitors Targeting Glycoproteinꢀ41
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Guangyan Zhoua , Vladimir Sofiyeva, Hardeep Kaura, Beth A. Snyderc, Marie K.
Mankowskic , Priscilla A. Hoganc, Roger G. Ptakc, and Miriam Gochina,b *
a Department of Basic Sciences, Touro University-California ,Vallejo,CA 94592
b Department of Pharmaceutical Chemistry, University of California San Francisco, CA
94143
c Southern Research Institute, 431 Aviation Way, Frederick, Maryland 21701
* M. Gochin: Touro University ꢀ California, 1310 Club Drive, Mare Island, Vallejo,
CA 94592. Tel. (707) 638ꢀ5463; FAX: (707) 638ꢀ5255; eꢀmail:
miriam.gochin@tu.edu
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Abstract
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We previously described indoleꢀcontaining compounds with the potential to inhibit HIVꢀ
1 fusion by targeting the hydrophobic pocket of transmembrane glycoprotein gp41. Here
we report optimization and structure activity relationship studies on the basic scaffold,
defining the role of shape, contact surface area and molecular properties. Thirty new
compounds were evaluated in binding, cellꢀcell fusion and viral replication assays. Below
a 1µM threshold, correlation between binding and biological activity was diminished,
indicating an amphiphilic requirement for activity in cells. The most active inhibitor 6j
exhibited 0.6 µM binding affinity and 0.2 µM EC50 against cellꢀcell fusion and live virus
replication, and was active against T20 resistant strains. Twenty two compounds with the
same connectivity displayed a consensus pose in docking calculations, with rank order
matching the biological activity. The work provides insight into requirements for small
molecule inhibition of HIVꢀ1 fusion and demonstrates a potent low molecular weight
fusion inhibitor.
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Key Words: gp41, small molecule, fusion inhibitor, bisindole, docking.
Abbreviations: HIV, human immunodeficiency virus; CHR, Cꢀterminal heptad repeat;
NHR, Nꢀterminal heptad repeat; CCF, cellꢀcell fusion; VCF, virusꢀcell fusion; 6ꢀHB, sixꢀ
helix bundle; HP, hydrophobic pocket; EC50, effective concentration for 50% inhibition;
TC50, concentration causing 50% cytotoxicity; DMSO, dimethylsulfoxide; DMEM,
Dulbecco's Modified Eagle Medium; FBS, fetal bovine serum; SAR, StructureꢀActivity
Relationship; POPC, 1ꢀpalmitoylꢀ2ꢀoleoylꢀsnꢀglyceroꢀ3ꢀphosphocholine.
Introduction
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Fusion of Human Immunodeficiency Virus Type 1 (HIVꢀ1) with host cells is mediated by
viral envelope glycoproteinꢀ41 (gp41) through a series of conformational rearrangements
culminating in the formation of a sixꢀhelix bundle (6ꢀHB) between Nꢀheptad repeat
(NHR) and Cꢀheptad repeat (CHR) regions of the extracellular domain of gp41 trimer.1ꢀ3
Any chemical entity that disrupts 6ꢀHB formation has the potential to inhibit the fusion
process, thereby blocking HIVꢀ1 entry into the target cells.4 Cꢀpeptide inhibitors derived
from the CHR have been shown to be potent inhibitors of HIV fusion,5 and time of
addition experiments have revealed that they typically remain active for 30–90 minutes
after initiation of viral infection.1, 6 Thus it appears that a fairly longꢀlived intermediate
conformation of gp41 exists in which the NHR coiled coil in the gp41 trimer is exposed
and susceptible to inhibition. Peptides directed against different regions of gp41 have
been developed into drugs or drug candidates.7ꢀ9 They are useful in salvage therapy
against HIV strains resistant to HAART therapy or can be used as vaccine antigens10 or
microbicides,11 but suffer from the usual limitations of peptide drugs, including shelf life,
stability, bioavailability and cost.
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Multiple studies have shown that Dꢀpeptides and small molecules targeting a conserved
hydrophobic pocket on the coiled coil12ꢀ14 have fusion inhibitory properties.11, 15ꢀ20
Antiviral activity at low nM concentrations was described for some of the small
molecules, although they had IC50’s of several µM against cell –cell fusion.18, 20 Low
molecular weight compounds able to potently inhibit HIVꢀ1 fusion remain elusive,
notwithstanding the important role they could play in countering multidrug resistance,
viral latency and the cell–to–cell route of transmission thought to be responsible for rapid
spread and resurgence of the virus.21 We recently described the development of a series
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of indole compounds as hydrophobic pocket binding fusion inhibitors.22 Two benzylꢀ
substituted bisindole compounds containing 4 aromatic ring systems demonstrated ~0.9
µM activity against cell–cell and virus–cell fusion (Figure 1). In this work, we have
performed lead optimization based on this scaffold, where we describe SAR studies
examining isomeric forms, alternative benzyl ring and other substituents, and compounds
containing benzimidazoles. The work has resulted in several 6–6’ linked bisindole
compounds with subꢀµM activity against cell–cell and virus–cell fusion, including 6j
with EC50 = 200 nM, and has provided an assessment of molecular properties associated
with potency. The two most active compounds 6j and 6k were tested and found to be
effective against multiple strains of HIV, including a strain that is resistant to T20.23
These compounds are a promising advancement in fusion inhibitor design.
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Chemistry
The synthesis of compounds containing four aromatic systems is described in Schemes
1–3. Derivatives of 1a with alternative substitutions on ring D or different indole linkages
were synthesized as shown in Scheme 1 (compounds 6aꢀm). The synthesis has been
improved since our previous paper,22 taking advantage of the common intermediate 5 that
is accessed through SuzukiꢀMiyaura crossꢀcoupling to form the bisindole scaffold.24, 25
Alternative linkage through the 5ꢀposition of the indole (6oꢀq) was explored using a
similar synthetic strategy with 5ꢀbromoindole or indoleꢀ5ꢀboronic acid as the starting
material (Scheme S1). For the benzimidazole series 10aꢀd (Scheme 2), alkylation of 6ꢀ
bromobenzimidazole formed both 5ꢀ (8c or 8d) and 6ꢀbromobenzimidazole (8a or 8b)
intermediates in one pot. The two isomers were separated by flash chromatography and
their structures were verified by ROESY correlations between benzimidazole 6ꢀ
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membered ring protons and the methylene of the attached benzyl moiety (Supporting
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Information Figure S1).
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Compounds 14a, 14b, 14c and 16 were made having one or both indoles substituted at
the 3ꢀposition (Scheme 3) to take advantage of a potential NH interaction in the
hydrophobic pocket. The compounds in this series were prepared using Zn(OTf)2
mediated alkylation of the 3ꢀposition with benzyl bromides.26 All potential combinations
were prepared including the 1,3’ꢀ, 3,1’ꢀ, and 3,3’ꢀsubstituted bisindoles to complement
the previously prepared 1,1’ꢀsubstituted 6b. In addition, a 5–5’ linked bisindole 16 was
made that is 3,3’ꢀsubstituted and has a complementary shape to 6j. Truncated compounds
with 3 aromatic ring systems were included in the SAR analysis to gauge the effect of
compound size (Scheme 4), some of which were obtained by saponification of
corresponding methyl ester intermediates (18b, 20aꢀb, 23).
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Results and Discussion
The most active previously synthesized compounds 1a, 1b22 with four aromatic systems
labeled A–D (Figure 1) had virtually identical binding affinity and in vitro potency.
Binding affinity (KI) to the hydrophobic pocket was on the order of 1–2 µM and the
compounds exhibited comparable activity against cell–cell fusion (EC50CCF = 0.5–1.0 µM)
VCF
and viral infectivity (EC50
= 0.9 µM). The following aromatic system substitutions
were made: indole B was replaced with benzimidazole (10a, 10c, 20a, 20b); the benzoic
acid substituent was moved to the 3ꢀposition (14b, 14c, 16, 18a, 18b); indole C was
replaced with benzimidazole (10b, 10d), benzene (21a, 21b) or quinoline (22a, 22b); ring
D was moved to the 3ꢀposition (14a); ring D was replaced with a proton (18a, 18b) or
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methyl group (19), or substituted with a variety of polar and nonꢀpolar groups (6a–m).
The connectivity between B and C was also varied (6oꢀq). These substitutions allowed us
to explore variation of shape, charge, hydrophobic contact surface area and polar
interactions. These studies were intended to verify that the hydrophobic and electrostatic
interactions of the inhibitors are specific, rather than being due to amorphous long range
electrostatic or nonꢀspecific hydrophobic effects,27 and to test the potential of the
compounds to form additional hydrogen bonds. Many of the isomers were also expected
to have improved drugꢀlike properties including a lower lipophilicity. The results are
shown in Table 1 and detailed below.
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Molecular Shape
The linkage between indoles B and C was examined as a means of
changing the overall shape of the molecule. Rings B and C in 1a are linked through the
6–6’ carbons. Compounds with 5–6’ (6o), 6–5' (6p) and 5–5’ (6q) linkages were
synthesized and evaluated. The activity data indicated that all three compounds had
reduced activity compared to 1a, not only in hydrophobic pocket binding affinity (3–4
times less active), but also in cell–cell fusion and viral replication assays (4–20 times less
active) (Table 1). Conformational space sampled by the isomeric forms 1a, 6oꢀq was
examined by generating 20 low energy conformers of each compound using Omega2ꢀ
2.5.1.4 (OpenEye, Inc).28, 29 A sample of three conformers of each compound is shown in
Figure S2 (Supporting Information) to illustrate accessible shapes of the molecules. The
6–6’ꢀlinked bisindole scaffold was able to adopt the most compact shape overall. The 5–
5’ isomer adopted the most extended solution conformations, and was also the worst
performing of the 4 isomers. Continued synthetic exploration was restricted to 6–6’
linked indoles, which apparently better conform to the shape of the hydrophobic pocket.
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Hydrophobic Surface Area Since the hydrophobic pocket is located on the long grove
formed along the NHR, we next tested the effect of increasing van der Waal’s contacts by
introducing additional hydrophobic substituents to ring D. Compounds 6e, 6f and 6g all
contain an additional phenyl group attached to ring D and compound 6h has ring D
replaced with a benzotriazole (Table 1). Activity did not improve, leading us to believe
that optimization should focus on specific interactions in the hydrophobic pocket rather
than on extending the contact surface area. We also could not rule out whether the
compounds might exhibit lower potency due to lower solubility under the conditions of in
vitro assays.
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We then tested the effect of reduced compound size by eliminating ring D, which
automatically reduces total surface area. Compounds 18aꢀb, 19, 20aꢀb, 23 (Scheme 4)
and four previously studied compounds 21a, 21b, 22a, 22b22 all contain 3 aromatic ring
systems (Table 2). Compound 23, the precursor of 6aꢀm, demonstrated that ring D is
essential for antiviral potency; it had an EC50 of 60 µM against HIVꢀ1 BaꢀL, while our
best 4ꢀaromatic system compound 6j had 0.2 µM potency. Two of the compounds had
subꢀµM binding affinity: 18b with a 3ꢀsubstituted indole B and two free indole NH
protons as well as 19 containing NꢀMe (Scheme 4), but this did not translate into subꢀµM
antiviral activity. The discrepancy between binding and fusion of compounds 18b and 19
suggests that factors other than HP binding may play a role in inhibition in the biological
milieu, either by a change in pocket shape or accessibility or by contribution of a
secondary interaction.
In the context of three aromatic system compounds, the character of rings B and C were
important, and binding affinity decreased in the order indole ≥ quinoline > phenyl >
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benzimidazole. Further examination was confined to compounds with four aromatic
systems, given the inability of smaller compounds to yield subꢀµM inhibition against
viral fusion.
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Polarity of the Indole Core Benzimidazole replacement of either indole B or C was
introduced (Scheme 2) as a way to increase polarity in the core of the scaffold without
altering the shape of compounds with four aromatic systems. In addition, coupling of
rings A and/or D to the 3’ position of the indoles was tested (Scheme 3), which leaves a
free indole NH proton that could potentially make hydrogen bonds to residues of the
protein.
Compounds 14aꢀc and 16 with at least one free indole NH proton had low to subꢀµM
KI’s, and moderate in vitro EC50’s. Compound 16, having shape equivalent to 6j but with
two free indole NH protons, forms a compact structure and is the most active of these
variants. While binding affinity is similar, bioactivity is lower than for 6j, and while it
appears that 3ꢀsubstitution does not significantly affect KI, it is detrimental in the local
environment of in situ gp41. By virtue of the 3ꢀsubstituted indoles, compounds 14a, 14b
and 14c have extended shapes similar to 6p, 6o, and 6q respectively, and have similar
activity. Replacement of indole groups with benzimidazole increased KI and practically
abrogated antiꢀfusion activity entirely.
Substitution at Ring D
It is clear from Table 1 that addition of ring D to
compound 23 (KI = 3µM) had a significant effect on compound potency, leading to KI
values as low as 0.6 µM or as high as 12 µM, and over two orders of magnitude variation
in fusion inhibitory activity.
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Varying small non-polar substituents on ring D had a small effect on compound activity
We observed that mꢀmethoxy (6b) or pꢀCF3 (6d) substitution had no effect on
binding affinity or potency, while a 3,5ꢀdimethoxy substitution on ring D (6c) caused a
barely perceptible decrease in biological activity, but no change in binding affinity.
Compound 6a with unsubstituted ring D showed a 5ꢀfold decrease in activity. Bulky
additions to ring D, discussed earlier, compromised binding and antiviral potency
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compared to 1 (6eꢀh). We observed an overall agreement between cellꢀcell fusion and
CCF
viral infectivity EC50’s and, with the exception of compound 6e, between KI and EC50
This suggests that a hydrophobic pocket binding mechanism is central to the potency of
the compounds.
.
CCF
Polar substituents on ring D had a marked effect in decoupling KI from EC50
and
VCF
EC50
In all cases, a carboxylate, an amide or an ester as a meta substituent on
ring D improved binding affinity to mostly subꢀµM levels. However, carboxylic acid and
amide groups in 6i and 6m30 invariably resulted in significantly reduced activity against
fusion. Conversely, retaining the methyl or ethyl ester resulted in enhanced fusion
inhibitory activity that exceeded the KI in potency by a factor of 2–3. This result suggests
that the characteristics required for hydrophobic pocket binding in solution begin to
deviate from those required for fusion inhibition below a 1 µM threshold of potency. It is
possible that the proximity of the membrane to the hydrophobic pocket of gp41 in situ
modifies the requirements for a compound to bind effectively and inhibit fusion. In this
scenario, a charge on ring D would have a deleterious effect on the ability of the
compound to reach the site of action, while uncharged groups such as esters can still
approach and bind tightly. We have observed that the indole inhibitors can partition into
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POPC:cholesterol liposomes, cells and virus particles, with a reduced interaction for 6i
compared to 6b (unpublished). Furthermore, the relationship between membrane
partitioning and efficacy has been established for gp41 Cꢀpeptide inhibitors.31
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Acid Esters on ring D yielded 200 nM inhibition of viral replication and also inhibited
T20-resistant virus
The best performing compounds 6j and 6k inhibited cellꢀcell fusion with EC50CCF = 200–
VCF
700 nM and inhibited viral replication with EC50
= 200–300 nM. We measured the
antiviral activity of the compounds against various primary strains of HIVꢀ1 in PBMCs,
finding that they retained high potency even against a T20 resistant strain (Table 3). The
methyl ester containing compound 6j gave EC50’s in the range 250–460 nM, tested
against 6 different strains of HIVꢀ1, including a strain with T20 EC50 > 2000 nM. For the
ethyl ester 6k, EC50’s varied from 350–420 nM. SubꢀµM EC90’s were typically obtained.
For two viral strains with partial insensitivity to T20, EC50’s for 6j and 6k were only
three times higher than for T20. Retention of activity against T20 resistant strains is an
expected property of compounds targeting the hydrophobic pocket, since they do not
overlap the binding surface of T20. It suggests that the hydrophobic pocket binders could
be useful in combination therapy with T20. The mechanism of inhibition of HIV entry
was further confirmed by studies showing that 6j and 6k had minimal effect in a MAGI
attachment assay (Supporting Information Table S1 and Figure S3), which is performed
with a washout of compound and unbound virus following a short term incubation that
coincides with the virus attachment and fusion steps of virus infection. Coupled with the
results observed in the other assays, loss of compound activity in this assay indicates the
virus is able to attach to cells in the presence of the compounds and continue with fusion
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and subsequent steps of virus infection and replication after the compounds are washed
out of the assay.
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Interpretation of SAR on the Ring Systems
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The diversity of the compound set has allowed us to obtain an informative SAR data set
and to evaluate potential binding modes of the compounds in the pocket. The low binding
and fusion inhibitory properties of 20a and 20b, mirrored also in 10a and 10c, in which
ring B is benzimidazole, suggests that an indole group is desirable for ring B, which is
likely to be buried in a hydrophobic part of the pocket. A lone pairꢀcontaining nitrogen
atom at the 3 position cannot be accommodated, in contrast to an indole NH that was
tolerated (14b, 14c, 16). Similar functionality was required for ring C, suggesting that the
bisindole core might anchor the compounds in the pocket. Variation of a factor of 20 in
VCF
KI and a factor of 250 in EC50
was observed for different ring D substituents, and
methyl and ethyl esters were clearly favored. This fact points to an important role of ring
D in conferring activity. Ring D was required for subꢀµM in vitro potency against fusion
although it was not essential for subꢀµM hydrophobic pocket binding.
Based on previous reports of the importance of saltꢀbridge interactions between peptide
and small molecule inhibitors and pocket Lys574,32, 33 we evaluated the binding affinity
of select compounds to a mutant receptor in which Lys574 was replaced with norleucine
(Nle). Nle contains the hydrophobic side chain of a Lys but is missing the εꢀamino group.
Binding affinity was slightly impaired, by a factor of 2–3 (Supporting Information Table
S2), which is within the range of experimental error of measurement of KI, but was
consistently observed for almost all the compounds measured. It appears likely that a
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specific salt bridge between Lys574 and the ligand carboxylate, if it exists, does not
contribute substantially to potency. We have seen this previously with peptides and other
small molecules.27, 34
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Prediction of Bound Conformation
Evaluation of binding modes is complicated by the variability of the gp41 hydrophobic
pocket in the available protein data bank (PDB) structures. Pockets containing small
molecules, peptides or antibodies show significant side chain variations,35 and it is likely
that binding of the indole compounds would result in induced fits that are difficult to
predict a priori. Interestingly, most gp41 structures in the PDB have a pocket structure
that is not large enough to accommodate compounds with 4 aromatic systems fully within
the groove. The exception to this is the structure 2XRA, which has an antibody HK20
bound to it.36 We investigated the poses and calculated affinities of the compounds listed
in Tables 1 and 2 using FRED 2.2.5 OEDocking software (OpenEye, Inc.), which
includes options to analyze poses using a variety of scoring functions. We used scoring
functions OEChemscore, Shapegauss, Piecewise Linear Potential, Zapbind, Screenscore
and Chemgauss3 to evaluate the lowest energy pose and 20 alternative conformations of
each ligand. These energy functions are described in detail in FRED documentation, and
are briefly explained in the Supporting Information. Due to the much lower dynamic
CCF
range of KI and EC50
data compared to EC50VCF, we used the latter as a more
discriminating data set to distinguish between different poses and structures.
The lowest energy consensus pose of 6–6’ linked bisindoles in 2XRA showed excellent
correlation to experimental data
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Extensive HK20 side chain interactions in and surrounding the hydrophobic pocket create
a large core cavity for accommodating a ligand in 2XRA. In this structure we obtained a
highly consistent lowest energy consensus pose for almost all 6–6’ 4 ring compounds,
shown in Figure 2A. We consistently found the best correlation to experimental data
using OEChemscore. OEChemscore is a modification of Chemscore,37 using simple
contact terms to define lipophilic interactions, an explicit term for hydrogen bonds and a
penalty for clashes between protein and ligand. This scoring function sums over the pair
wise interactions between ligand and protein and is likely to reflect the total contact area
between them. Figure 3 shows the calculated OEChemscore values for the lowest energy
conformer, plotted against pEC50VCF. Compounds were separated into three categories
based on the observed results: 1) 6–6’ 4 ring systems not containing benzimidazole, and
all 3 ring systems; 2) benzimidazoleꢀcontaining compounds and alternativelyꢀlinked 4
ring compounds; and 3) two outliers 6f and 6i not included in the previous two categories.
A remarkable correlation coefficient R2 = 0.844 was obtained for category 1, which
included most of the compounds in our data set (23 compounds). Category 2 is a
miscellaneous combination of 11 compounds with different shapes and charge
distribution, as a result of which they gave a variety of different bound poses and there
was insufficient sampling within each subgroup to reach detailed conclusions. It is clear,
however, that these compounds are predicted to be more active than what was found
experimentally, which could be a result of bioavailability considerations or a flexible
receptor structure. In this context, the poor activity of benzimidazole derivatives is not
understood.
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Observed KI and antiviral activity of 6i and 6m, showed the greatest discrepancy of all
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compounds, and this was mirrored in the calculation for 6i, which adopts the same pose
as 6j (Figures 2 and 3). Interestingly, a similar observation of low antiviral activity for
compounds containing two carboxylates, and much improved activity for the methyl ester,
was obtained in a recent study of elongated pyrroleꢀthiazolidinyl containing
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compounds.19 Carboxymethyl substituents on a terminal benzyl ring were at least ten
CCF
times more active (EC50
= 1.8 µM) than the corresponding free carboxylic acid
(EC50CCF = 21–68 µM). It is possible that the loss of potency is due to the local lipophilic
environment of in situ gp41, since 6i binds strongly to gp41 in solution and is not an
outlier in correlations with pKI. Compound 6m formed an internal Hꢀbond in the
calculations and could not assume the extended pose shown in Figure 2A. The compact
pose made far fewer contacts with the protein surface, consequently resulting in poor
calculated activity, which correlated with its poor VCF activity. The remaining outlier is
6f, a 5ꢀring compound in which the extensive contact area with the protein is not matched
by experimental efficacy. With such compounds, solubility could play a role in the
diminished potency. A few additional outliers were found in the pKI correlation
(Supporting Information Table S3 and Figure S4), but this is not considered as reliable
due to the available low dynamic range.
In the bound pose in 2XRA in Figure 2B, compounds 6j and 6k were the top ranked
compounds and they are clearly also the most potent compounds experimentally. They
ranked at positions 1 and 3 in the data set of 36 compounds. In fact, several scoring
functions ranked these compounds at or near to top of the list. Table S3 shows the
calculated correlation coefficients for all scoring functions and the rank of the top
compounds. Five of the six scoring functions yielded correlation coefficients R2 > 0.53
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with VCF data and 6j was ranked as the top scoring compound using OEChemscore,
Shapegauss, Piecewise Linear Potential and Chemgauss3; 6k ranked 3rd, 2nd, 9th and 6th
respectively, out of 36 compounds. This encourages us to believe that the pose found in
2XRA (Figure 2B) is independent of scoring function and may well represent an
accurate pose.
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Conclusions
In this study, we designed, synthesized and tested a series of indole derivatives as entry
inhibitors against gp41ꢀmediated HIVꢀ1 fusion. SAR studies revealed a defined shape,
charge and hydrophobic contact surface required to fit into the hydrophobic pocket.
Optimization resulted in improved binding affinity, viral replication and cellꢀcell fusion
inhibition, with several compounds having subꢀµM activity and the most active
compound 6j exhibiting binding affinity with KI of 0.6 µM and EC50 of 0.2 µM in cellꢀ
cell fusion inhibition and viral replication inhibition. A computational pose was obtained
that explained the observed SAR.
We found that subꢀµM inhibition constants for binding to the hydrophobic pocket on the
gp41 coiled coil were a necessary but not sufficient condition for observing subꢀµM
inhibition of cell–cell and virus–cell fusion. Below KI = 1µM, the correlation between
KI and EC50 was diminished, indicating a factor other than hydrophobic pocket binding
required for fusion inhibition. Polar and nonꢀpolar groups at opposite ends of the
elongated molecules were a dominant feature of subꢀµM fusion inhibitors, but
compounds containing polar groups at both ends were poor fusion inhibitors, while
equally potent HP binders. We surmise that proximity of the target to lipid membranes
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may influence accessibility of the pocket and bioavailability of the inhibitors, given the
lipophilic character of the best inhibitors. Future work will focus on improving the
therapeutic index and understanding the mechanism that yields subꢀµM activity in cellꢀ
culture.
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Experimental Section
Chemistry
Starting materials and solvents were purchased from commercial sources
and used as is. HPLC was performed on a Waters Breeze HPLC with multi λ
fluorescence detector and dual λ UV detector, using a water/acetonitrile or
water/methanol gradient with 0.05% TFA. Mass spectra were obtained on LCMS
(Finnigan LCQ Duo, H2O/methanol gradient with 0.1% formic acid) by ESI, or by highꢀ
1
resolution mass spectrometry (HRMS) on a ThermoFisher Orbitrap XL (UC Davis). H
NMR and 13C NMR spectra were collected on a Bruker spectrometer at 400MHz and
100MHz respectively in d6ꢀDMSO unless otherwise stated and calibrated using the
residual solvent signal. Multiplicities are abbreviated as follows: s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, and br = broad. Where consistent coupling
constants have been observed in the NMR spectrum, the apparent multiplicity of the
proton signal concerned is reported.
Flash column chromatography was carried out with Silicycle SiliaFlash® P60 40ꢀ63 µm
silica gel. Reactions and chromatography fractions were monitored with Sorbent
Technologies silica gel 60 F254 TLC plates and visualized using UV light. Reactions were
carried out under inert gas atmosphere in ovenꢀdried glassware and reaction solutions
were magnetically stirred. All other reagents and solvents were used without further
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purification from commercial sources unless otherwise noted. All target compounds were
purified by preparative HPLC or analyzed by HPLC, to ensure ≥ 95% purity.
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The organic synthesis was carried out according to Schemes 1–4 and Scheme S1 in the
Supporting Information. Representative synthetic procedures for the preparation of
compounds 4, 5, 6b, 6i, 6j, 6k, 6m, 11a, 12a, 19, and 23 are described below.
Characterization data for other target compounds and synthetic intermediates are
provided in the Supporting Information.
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Synthesis of methyl 3ꢀ[(6ꢀbromoindolꢀ1ꢀyl)methyl]benzoate (4). To a solution
of methyl 3ꢀ(bromomethyl)benzoate 3a (458 mg, 2.00 mmol) and 6ꢀbromoindole 2a (382
mg, 1.95 mmol) in dry DMF (5.0 mL) was added K2CO3 (1.38 g, 10.0 mmol). The
mixture was stirred at room temperature overnight. TLC indicated no starting material
remained; the reaction was quenched by adding 25 mL water. The solution was extracted
with ethyl acetate (30 mL×3). The organic solvent was combined and evaporated, the
product was purified by silica gel chromatography using EtOAc/hexane (15:85) as eluent.
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655.2 mg target compound achieved as pale yellow oil, 95%. H NMR δ 7.86 (d, J = 7.6
Hz, 1H), 7. 79 (s, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.27 (t, J = 8.0 Hz, 1H),
7.14–7.06 (m, 2H), 7.00 (d, J = 3.2 Hz, 1H), 6.45 (d, J = 3.2 Hz, 1H), 5.20 (s, 2H), 3.81
(s, 3H); 13C NMR δ 166.99, 137.74, 137.32, 131.41, 131.08, 129.40, 129.11, 128.20,
127.91, 123.32, 122.61, 115.81, 114.25, 112.81, 102.66, 52.58, 50.09.
Synthesis of methyl 3ꢀ[[6ꢀ(1Hꢀindolꢀ6ꢀyl)indolꢀ1ꢀyl]methyl] benzoate (5). To a
solution of indoleꢀ6ꢀboronic acid (274 mg, 1.7 mmol) and compound 4 (516 mg, 1.5
mmol) in THF (15 mL) was added Pd(PPh3)4 (87.2 mg, 0.075 mmol), followed by K2CO3
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(2.0 M, 3.0 mL). The mixture was stirred and heated to reflux under Argon for about 4
hours. After the reaction was completed, the mixture was cooled to room temperature,
water (20.0 mL) was added and the product was extracted with ethyl acetate (20 mL×3).
The organic solvent was combined, dried over anhydrous Na2SO4, filtered and
evaporated. The crude product was purified by silica gel chromatography (20%
EtOAc/hexanes). 380.1 mg target compound was obtained as pale yellow oil, 67%. MS
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calculated for C25H20N2O2: 380.2; found: 381.1 (M+H)+; H NMR δ 8.19 (br, 1H), 7.97
(s, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.68 (dd, J =12.8 Hz, J = 8.2 Hz, 2H), 7.58 (s, 1H), 7.49
(s, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 7.25
(br, 1H), 7.20 (t, J = 2.4 Hz, 1H), 7.14 (d, J = 3.0 Hz, 1H), 6.59 (d, J = 3.0 Hz, 1H), 6.56
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(br, 1H), 5.40 (s, 2H), 3.89 (s, 3H); C NMR δ 167.14, 138.32, 137.16, 137.01, 136.94,
136.79, 131.64, 130.97, 129.36, 129.22, 128.82, 128.39, 127.98, 127.69, 127.12, 123.32,
122.61, 115.81, 114.25, 110.16, 108.56, 102.68, 102.27, 52.56, 50.07.
Synthesis of 3ꢀ[[6ꢀ[1ꢀ[(3ꢀmethoxyphenyl)methyl]indolꢀ6ꢀyl]indolꢀ1ꢀyl]methyl]benzoic
acid (6b). To a stirred solution of compound 5 (19 mg, 0.05 mmol) in anhydrous DMF
(3.0 mL) at 0°C was added sodium hydride in oil (60%, 15 mg), and the reaction mixture
was allowed to warm up to rt over 60 min. 3ꢀMethoxybenzyl bromide (10.5 mg, 0.052
mmol) was added, and the mixture was stirred at room temperature overnight. The
reaction was quenched by adding 10 mL water. The solution was extracted with ethyl
acetate (15 mL×3). The organic solvent was combined and evaporated; the crude product
(22.5 mg, 90%) was directly used for saponification by adding 5.0 ml of 4.0M NaOH and
stirring for 1 hour, and neutralized by adding 2.0 M HCl to pH 3.0. The solution was
extracted with ethyl acetate (15 mL×3), organic solvent was combined and concentrated.
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8.5 mg purified target compound was obtained by HPLC as a grey powder. Data for 6b:
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MS calculated for C32H26N2O3: 486.2; found: 485.1 (M–H)–; H NMR δ 7.82 (d, J = 8.5
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Hz, 2H), 7.68 (d, J = 7.8 Hz, 2H), 7.60 (t, J = 7.8 Hz, 2H), 7.52 (d, J = 2.9 Hz, 2H), 7.46
(m, 2H), 7.35 (m, 2H), 7.20 (t, J = 7.3 Hz, 1H), 6.78 (m, 3H), 6.51 (d, J = 2.8 Hz, 1H),
6.48 (d, J = 2.8 Hz, 1H), 5.58 (s, 2H), 5.45 (s, 2H), 3.67 (s, 3H).
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3ꢀ[[6ꢀ[1ꢀ[(3ꢀcarboxyphenyl)methyl]indolꢀ6ꢀyl]indolꢀ1ꢀyl]methyl]benzoic acid (6i). To
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a solution of compound 23 (19 mg, 0.05 mmol) in anhydrous DMF (3.0 mL) at 0 C was
added sodium hydride in oil (60%, 15 mg), and the reaction mixture was allowed to
warm up to rt over 60 min. Methyl 3ꢀ(bromomethyl)benzoate (11.9 mg, 0.052 mmol) was
added, then the mixture was stirred at room temperature overnight. Saponification was
achieved by adding 5.0 ml of 4.0 M NaOH and stirring for 1 hour. The reaction was
neutralized by adding 2.0 M HCl to pH 3.0, and then extracted with ethyl acetate (15
mL×3). The organic solvent was combined and evaporated; the crude product (23.0 mg,
92%) was purified by HPLC, 15.5 mg target compound was obtained as a yellow powder.
Data for 6i: MS (HRMS) calculated for C32H24N2O4 (M–H): 499.1652; found: 499.1648
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(MꢀH)ꢀ; H NMR δ 12.85 (s, 2H), 7.82 (br, 4H), 7.68 (s, 2H), 7.60 (d, J = 8.5 Hz, 2H),
7.52 (s, 2H), 7.45 (m, 4H), 7.36 (d, J = 8.0 Hz, 2H), 6.51 (s, 2H), 5.57 (s, 4H); 13C NMR
δ 167.0, 138.8, 136.4, 135.0, 131.3, 131.2, 129.5, 128.8, 128.2, 127.8, 127.2, 120.7, 119.0,
108.0, 101.0, 48.6.
3ꢀ[[6ꢀ[1ꢀ[(3ꢀmethoxycarbonylphenyl)methyl]indolꢀ6ꢀyl]indolꢀ1ꢀyl]methyl]benzoic
acid (6j). To a stirred solution of 23 (232 mg, 0.632 mmol) in DMF (7.0 mL) at 0 oC was
added NaH (60% in mineral oil, 61.5 mg, 1.54 mmol) and the reaction mixture was
allowed to warm up to rt over 30 min. Methyl 3ꢀ(bromomethyl)benzoate (145 mg, 0.633
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mmol) was added and the mixture was stirred at room temperature overnight. The
reaction mixture was quenched by adding 2.0 M HCl (20 mL), extracted with DCM (30
mL × 3), and the combined organic fractions were concentrated. Purification by silica gel
chromatography (40%ꢀ50%ꢀ60% EtOAc/hexanes) afforded 6j (249 mg, 76%) as a
lightꢀbrown oil. Data for 6j: Rf 0.13 (40% EtOAc/hexanes); MS (HRMS) calculated for
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C33H25N2O4 (M–H)–: 513.1809; found: 513.1803; H NMR (CDCl3) δ 10.68 (br, 1H),
7.99 (m, 3H), 7.91 (d, 1H, J = 7.7 Hz), 7.69 (d, 2H, J = 8.3 Hz), 7.46 (s, 2H), 7.41 (d, 2H,
J = 8.3 Hz), 7.34–7.18 (m, 4H), 7.11 (d, 1H, J = 3.0 Hz), 7.09 (d, 1H, J = 3.0 Hz), 6.57
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(m, 2H), 5.35 (s, 2H), 5.33 (s, 2H), 3.86 (s, 3H); C NMR (CDCl3) δ (5 pairs of signals
are overlapping) 171.7, 167.0, 138.4, 138.2, 137.0, 136.9, 136.8, 132.3, 131.5, 130.7,
130.0, 129.6, 129.3, 129.1, 129.0, 128.8, 128.7, 128.3, 127.9, 121.3, 120.31, 120.29,
108.4, 102.2, 102.1, 52.4, 49.8, 49.7.
3ꢀ[[6ꢀ[1ꢀ[(3ꢀethoxycarbonylphenyl)methyl]indolꢀ6ꢀyl]indolꢀ1ꢀyl]methyl]benzoic acid
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(6k). To a stirred solution of 23 (107 mg, 0.292 mmol) in DMF (3.0 mL) at 0 C was
added NaH (60% in mineral oil, 30.2 mg, 0.755 mmol) and the reaction mixture was
allowed to warm up to rt over 30 min. Ethyl 3ꢀ(bromomethyl)benzoate (73 mg, 0.30
mmol) was added and the mixture was stirred at room temperature overnight. The
reaction mixture was quenched by adding 2.0 M HCl (10 mL), extracted with DCM (30
mL × 3), and the combined organic fractions were concentrated. Purification by silica gel
chromatography (40%ꢀ50% EtOAc/hexanes) afforded 6k (128 mg, 83%) as a lightꢀ
brown oil. Data for 6k: Rf 0.48 (50% EtOAc/hexanes); MS (ESI) calculated for
C34H26N2O4 (M–H)–: 527.1965; found: 527.1963; 1H NMR (CDCl3) δ 8.04 (m, 3H), 7.97
(d, 1H, J = 7.8 Hz), 7.73 (m, 2H), 7.50 (s, 2H), 7.44 (d, 2H, J = 8.0 Hz), 7.41–7.30 (m,
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3H), 7.23 (d, 1H, J = 7.9 Hz), 7.17 (d, 1H, J = 3.1 Hz), 7.15 (d, 1H, J = 3.1 Hz), 6.61 (m,
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2H) 5.43 (s, 2H), 5.41 (s, 2H), 4.38 (q, 2H, J = 7.2 Hz), 1.39 (t, 3H, J = 7.2 Hz); C
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NMR (CDCl3) δ (2 pairs of signals are overlapping) 171.6, 166.6, 138.4, 138.1, 137.01,
136.99, 136.93, 136.8, 132.3, 131.4, 131.1, 130.0, 129.7, 129.3, 129.1, 129.0, 128.8,
128.70, 128.67, 128.9, 127.9, 121.35, 121.33, 120.36, 120.31, 108.5, 102.2, 102.1, 61.4,
49.88, 49.85, 14.5.
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Synthesis of 3ꢀ[[6ꢀ[1ꢀ[(3ꢀcarbamoylphenyl)methyl]indolꢀ6ꢀyl]indolꢀ1ꢀyl]methyl]
benzoic acid (6m). To a stirred solution of compound 6l (4.8 mg, 0.01mmol) in DMSO
(1.0 mL) cooled in an iceꢀwater bath was added H2O2 (30%, 20.0 uL) and K2CO3 (10.0
mg). The mixture was allowed to warm up to room temperature. Then distilled water was
added, extracted with DCM (10 mL×3). Purified by HPLC. After lyophilization, 2.4 mg
compound achieved as offꢀwhite powder. MS calculated for C32H25N3O3: 499.2; LCMS:
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3H), 7.33–7.26 (m, 5H), 6.49 (s, 1H), 6.45 (s, 1H), 5.55 (s, 2H), 5.51 (s, 2H).
Methyl mꢀ[(6ꢀbromoꢀ1Hꢀindolꢀ3ꢀyl)methyl]benzoate (11a). To a vigorously stirred
solution of 6ꢀbromoindole (2b) (390 mg, 1.99 mmol) in toluene (8.0 mL) was added
tetrabutylammonium iodide (367 mg, 0.994 mmol), Zn(OTf)2 (450 mg, 1.24 mmol),
followed by DIPEA (0.36 mL, 2.1 mmol). After the suspension was stirred for 20 min,
methyl 3ꢀbromomethylbenzoate (231 mg, 1.01 mmol) was added and the reaction
mixture was stirred overnight. Reaction mixture was diluted with EtOAc (50 mL), water
(10 mL), and saturated aqueous NH4Cl (30 mL). The organic phase was separated and
washed with water (30 mL) and brine (10 mL) then concentrated. Purification by silica
gel chromatography (15% ꢀ 25% EtOAc/hexanes) afforded 11a (338 mg, 97%) as a
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pink oil. Data for 11a: Rf 0.07 (15% EtOAc/hexanes); MS calculated for C17H1379BrNO2
(M–H)–: 342.01, found: 342.01; 1H NMR (CDCl3) δ 8.39 (br s, 1H), 8.04 (s, 1H), 7.95 (d,
1H, J = 7.6 Hz), 7.47 (m, 2H), 7.38 (d, 1H, J = 7.6 Hz), 7.35 (d, 1H, J = 8.1 Hz), 7.20
(dd, 1H, J1 = 8.4 Hz, J2 = 1.7 Hz), 6.86 (d, 1H, J = 2.2 Hz), 4.12 (s, 2H), 3.93 (s, 3H); 13C
NMR (CDCl3) δ 167.6, 141.5, 137.3, 133.4, 130.2, 129.8, 128.6, 127.4, 126.2, 123.4,
122.6, 120.2, 115.6, 115.0, 114.3, 52.2, 31.3.
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tertꢀButyl 6ꢀbromoꢀ3ꢀ[(mꢀmethoxycarbonylphenyl)methyl]ꢀ1Hꢀindoleꢀ1ꢀcarboxylate
(12a). To a stirred solution of 11a (338 mg, 0.982 mmol) in THF (9.0 mL) was added
Boc2O (0.24 mL, 1.04 mmol) and DMAP (10.0 mg) and the mixture stirred overnight.
Concentration and purification by silica gel chromatography (10% EtOAc/hexanes)
afforded 12a (429 mg, 98%) as a colorless oil. Data for 12a: Rf 0.32 (10%
EtOAc/hexanes); MS (ESI) calculated for C22H2379BrNO4 (M+H)+: 444.08, found:
1
444.08; H NMR (CDCl3) δ 8.34 (br s, 1H), 7.96 (s, 1H), 7.89 (d, 1H, J = 7.8 Hz), 7.43
(d, 1H, J = 7.6 Hz), 7.34 (m, 2H), 7.26 (dd, 1H, J1 = 8.4 Hz, J2 = 1.9 Hz), 7.18 (d, 1H, J =
8.4 Hz), 4.03 (s, 2H), 3.89 (s, 3H), 1.67 (s, 9H); 13C NMR (CDCl3) δ 167.1, 149.4, 139.9,
136.5, 133.2, 130.5, 129.8, 129.1, 128.7, 127.8, 125.8, 124.2, 120.5, 119.4, 118.6, 118.3,
84.2, 52.2, 31.2, 28.2.
3ꢀ[[6ꢀ(1ꢀmethylindolꢀ6ꢀyl)indolꢀ1ꢀyl]methyl]benzoic acid (19). To compound 5 (19 mg,
0.05 mmol) in anhydrous DMF (2.0 mL) was added iodomethane (35 mg, 0.25mmol) and
NaOH (20.0 mg). The mixture was stirred overnight, quenched by H2O (10.0 mL) then
adjusted to pH 3.0 using HCl (2.0 M). The solution was extracted with CH2Cl2 (10
mL×3). The organic solvent was combined and dried, then evaporated. Part of the crude
product (16.2 mg, 84 %) was purified by HPLC; 2.4 mg target compound was obtained as
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yellow powder. MS calculated for C25H20N2O2: 380.2; LCMS: 379.2 (M–H)–; 1H NMR δ
7.85 (s, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.77 (s, 1H ), 7.64ꢀ7.61 (m,2H), 7.57 (d, J = 8.0
Hz, 1H), 7.52 (d, J = 3.2 Hz, 1H), 7.46ꢀ7.41 (m,3H), 7.35 (dd, J = 8.0 Hz, J = 1.6 Hz,
1H), 7.30 (d, J = 2.8 Hz, 1H), 6.52 (d, J = 2.8 Hz, 1H), 6.41 (d, J = 2.8 Hz, 1H), 5.59 (s,
2H), 3.84 (s, 3H).
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3ꢀ[[6ꢀ(1Hꢀindolꢀ6ꢀyl)indolꢀ1ꢀyl]methyl]benzoic acid (23). To a vigorously stirred
solution of compound 5 (444 mg, 1.17 mmol) in THF/MeOH (4:1, 20.0 mL) was added
aq. NaOH (25%, 5.0 mL). The mixture was stirred at room temperature for 3 h, then
quenched with 3.0 M HCl (24 mL), extracted with DCM (50 mL x 3), dried, filtered, and
concentrated. Purification by silica gel chromatography (10:1 DCM:MeOH) afforded 23
(400 mg, 93%) as an offꢀwhite solid. Data for 23: Rf 0.13 (40% EtOAc/hexanes); MS
1
(ESI) calculated for C24H17N2O2 (M–H)–: 365.13; found: 365.13; H NMR (d6ꢀDMSO) δ
12.87 (s, 1H), 11.02 (s, 1H), 7.75 (m, 2H), 7.66 (s, 1H), 7.55 (m, 3H), 7.47 (d, J = 4.0 Hz,
13
1H), 7.40 (m, 2H), 7.28 (m, 3H), 6.46 (d, J = 3.1 Hz, 1H), 6.36 (s, 1H), 5.54 (s, 2H); C
NMR (d6ꢀDMSO) δ 167.1, 139.1, 136.6, 136.5, 135.5, 134.7, 131.4, 131.1, 129.5, 128.9,
128.2, 127.7, 127.2, 126.6, 125.8, 120.8, 120.2, 119.0, 118.8, 109.4, 108.1, 101.1, 100.9,
48.6.
Binding Affinity Assay
Inhibition constants Ki for binding in the hydrophobic
pocket were determined using a fluorescence intensity assay as previously described.38, 39
Briefly, FeII(env2.0)3 was used to mimic the hydrophobic pocket in the gp41 NHR coiled
coil.
Env2.0 contains 20 hydrophobic pocket
–
associated residues
(565LLQLTVWGIKQLQARILAVE584). It is Nꢀterminally capped by 2,2ꢀbipyridineꢀ5ꢀ
carboxylate, a bidentate ferrous iron chelator that assures the trimeric structure of the
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NHR upon metal binding. A fluorescein (FL)ꢀlabeled pocketꢀbinding Cꢀpeptide C18FL
was used to probe inhibitor binding. Quenching of probe fluorescence occurred in the
presence of FeII(env2.0)3. KI was determined by measuring the dose dependent
fluorescence recovery in the presence of a competitive inhibitor. A modified
metallopeptide construct with K574 replaced with norleucine was employed to measure
the effect of the lysine side chain amino group on inhibitor binding affinity.
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Cellꢀcell Fusion Assay
Cellꢀcell fusion was measured as previously published
and using cell lines obtained through the NIH AIDS Research and Reference Reagent
Program, Division of AIDS, NIAID, NIH. Target cells were TZMꢀbl cells (#8129,
contributed by J.C. Kappes, X. Wu and Tranzyme Inc.) expressing CD4, CCR5 and
CXCR4,40 and containing an integrated reporter gene for firefly luciferase under control
of HIVꢀ1 LTR.41 Effector cells were HL2/3 (#1294, contributed by B.K. Felber and G.N.
Pavlakis) which produce HXB2 Env, Tat and Rev.42 Serially diluted inhibitors were
added to 96 well plates containing 25,000 TZMꢀbl cells per well cultured overnight.
50,000 HL2/3 cells were added per well, and fusion allowed to proceed for 6 hours in
reduced serum medium (Gibco) with a final concentration of 1% DMSO. Luciferase
expression was measured using Luciferase Assay Reagent (Promega) according to the
manufacturer’s instructions. Controls containing 1µl DMSO with and without HL2/3
cells were measured for each compound, and experiments were performed in triplicate.
Viral Replication and Attachment Assays Inhibition of HIVꢀ1 replication was
determined in CCR5ꢀ and CXCR4ꢀtropic MAGI antiviral assays and inhibition of HIVꢀ1
attachment/entry was determined in CCR5ꢀtropic MAGI attachment assays as previously
described.43, 44 HIVꢀ1 isolates and cells were obtained from the NIH AIDS Research and
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Reference Reagent Program, Division of AIDS, NIAID, NIH, as follows: HIVꢀ1 BaꢀL
from Suzanne Gartner, Mikulas Popovic, and Robert Gallo.44, 45 HIVꢀ1 IIIB from Robert
C. Gallo.44, 46 MAGIꢀCCR5 cells from Dr. Julie Overbaugh.47, 48 For MAGI antiviral
assays, MAGIꢀCCR5 cells were grown overnight in 96 well plates in DMEM
supplemented with 10% FBS, using 10,000 cells per well. The following day the
medium was removed and compounds diluted in medium were added (6 dilutions in
triplicate at each dilution), followed by the addition of either HIVꢀ1 BaꢀL (CCR5ꢀtropic
assay) or HIVꢀ1 IIIB (CXCR4ꢀtropic assay) at approximately ten 50% tissue culture
infective doses per well (~10 TCID50/well). Assay plates were incubated for 48 hours,
after which medium was removed and HIVꢀ1 Tatꢀinduced βꢀGal enzyme expression was
determined by chemiluminescence using Tropix GalꢀScreen (Applied Biosystems)
according to the manufacturer’s instructions. MAGI attachment assays were performed
similarly, but with a washout of unbound virus and compounds three (3) hours postꢀ
infection. Assays were conducted at a serum concentration of 2%.
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HIVꢀ1 PBMC Assay Testing of compounds against HIVꢀ1 in PBMCs was performed as
previously described.49 PBMCs were isolated from blood of screened donors (Biological
Specialty Corp., Colmar, PA) by Ficoll gradient purification and stimulated using 4
µg/mL phytohemagglutinin and subsequently maintained in media containing 20 U/mL
of ILꢀ2. Cells from two donors were mixed, adjusted to 1x106 cells/mL and added to 96ꢀ
well roundꢀbottom plates at 50 µL/well (5x104 cells/well). Test compound dilutions
(nine total serial dilutions) were prepared in media at 2X concentrations and added to
triplicate wells at 100 µL/well followed by the addition of a predetermined dilution of
virus stock at 50 µL/well (final multiplicity of infection [MOI] of ~0.1). Antiviral
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efficacy was determined 6 days later based on supernatant reverse transcriptase (RT)
activity using a microtiter plateꢀbased RT reaction.50 The following virus isolates were
obtained from the NIH AIDS Research and Reference Reagent Program, Division of
AIDS, NIAID, NIH: HIVꢀ1 isolates 92BR014 (Cat# 1753) and 92UG001 (Cat# 1647)
from The UNAIDS Network for HIV Isolation and Characterization.51 HIVꢀ1 isolates
CAM0005BBY(Cat# 11278), CAM0015BBY (Cat# 11282), and D26830M4 (Cat#
11264) from Dr. Victoria Polonis;52 HIVꢀ1 91DJ263 (Cat# 7685) from Dr. Nelson
Michael;53 and HIVꢀ1 BaꢀL as noted above.
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Cytotoxicity Assay The cytotoxic effect of the compounds was determined using the
identical cell culture procedure to that described above for viral replication or cellꢀcell
fusion, but measuring cell viability, using 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(3ꢀ
carboxymethoxyphenyl)ꢀ2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀtetrazolium (MTS, CellTiter 96 reagent;
Promega; used for MAGI and PBMC assays) or using a resazurin cell viability reagent
(Alamar Blue or Presto Blue, Life Technologies; used for cellꢀcell fusion assays)
following the manufacturers’ protocols.
Cheminformatics and Computational Docking The OpenEye software suite
(OpenEye Scientific Software, Santa Fe, NM. http://www.eyesopen.com) was used for
ligand analysis, conformer predictions, compound overlays and computational docking.
Charges and 3D coordinates of each ligand were generated from SMILES strings using
OMEGA2 v2.5.1.428, 29. Compound overlays were created using ROCS v3.1.2. 54, 55
Multiconformer representations of each ligand were docked into the receptor 2xra
(Protein Data Bank) using the OEDocking utility and FRED v2.2.556. 20 alternate
docked poses were retained for each ligand.
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Acknowledgments
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This work was supported by NIH grants AI093243 and GM087998 to MG and internal
funds from Southern Research Institute. We thank S. Chu for assistance with running
ROESY spectra of benzimidazole compounds. Molecular graphics images were
produced using the UCSF Chimera package from the Resource for Biocomputing,
Visualization, and Informatics at the University of California, San Francisco (supported
by NIH P41 RRꢀ01081) or using Vida v.4.2.1 (OpenEye Scientific Software, Inc.).
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Supporting Information Available: 1) Synthetic procedures and compound
characterization not described in Materials and Methods; 2) Sample solution
conformations of bisindole compounds; 3) MAGI attachment assay results, binding
affinities to mutant receptor L574Nle, data and correlation coefficients for calculated vs.
observed activity. This material is available free of charge via the Internet at
http://pubs.acs.org.
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