Synthesis, Antiviral Potency, in Vitro ADMET, and X-ray Structure of Potent CD4 Mimics as Entry Inhibitors That Target the Phe43 Cavity of HIV-1 gp120

Article abstract of DOI:10.1021/acs.jmedchem.7b00179

In our attempt to optimize the lead HIV-1 entry antagonist, NBD-11021, we present in this study the rational design and synthesis of 60 new analogues and determination of their antiviral activity in a single-cycle and a multicycle infection assay to derive a comprehensive structure-activity relationship (SAR). Two of these compounds, NBD-14088 and NBD-14107, showed significant improvement in antiviral activity compared to the lead entry antagonist in a single-cycle assay against a large panel of Env-pseudotyped viruses. The X-ray structure of a similar compound, NBD-14010, confirmed the binding mode of the newly designed compounds. The in vitro ADMET profiles of these compounds are comparable to that of the most potent attachment inhibitor BMS-626529, a prodrug of which is currently undergoing phase III clinical trials. The systematic study presented here is expected to pave the way for improving the potency, toxicity, and ADMET profile of this series of compounds with the potential to be moved to the early preclinical development.

Full text of DOI:10.1021/acs.jmedchem.7b00179

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Article
Synthesis, antiviral potency, in vitro ADMET and X-ray structure of potent
CD4-mimics as entry inhibitors that target the Phe43 cavity of HIV-1 gp120
Francesca Curreli, Young Do Kwon, Dmitry S. Belov, Ranjith R. Ramesh,
Alexander V. Kurkin, Andrea Altieri, Peter D. Kwong, and Asim Kumar Debnath
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00179 • Publication Date (Web): 07 Mar 2017
Downloaded from http://pubs.acs.org on March 9, 2017
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Synthesis, antiviral potency, in vitro ADMET and Xꢀray structure of potent CD4ꢀmimics as
entry inhibitors that target the Phe43 cavity of HIVꢀ1 gp120
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a
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Francesca Curreli , Young Do Kwon , Dmitry S. Belov , Ranjith R. Ramesh , Alexander V.
c
c
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a
Kurkin , Andrea Altieri , Peter D. Kwong & Asim K. Debnath *
a
Laboratory of Molecular Modeling and Drug Design, Lindsey F. Kimball Research Institute, New
York Blood Center, New York, NY 10065, USA.
b
Structural Biology Section, Vaccine Research Center, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
c
EDASA Scientific, Scientific Park, Moscow State University, Leninskie Gory, Bld.75, 77 – 101b;
119992 Moscow ꢀ Russia
*Corresponding author
Keywords
HIVꢀ1, ENVꢀpseudovirus, virus entry antagonist, broad spectrum, structureꢀactivity relationship
(SAR), ADMET
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ABSTRACT
In our attempt to optimize the lead HIVꢀ1 entry antagonist, NBDꢀ11021, we present in this study
the rational design and synthesis of 60 new analogs, determination of their antiviral activity in a
singleꢀcycle and a multiꢀcycle infection assay to derive a comprehensive structureꢀactivity
relationship (SAR). Two of these compounds, NBDꢀ14088 and NBDꢀ14107, showed significant
improvement in antiviral activity compared to the lead entry antagonist in a singleꢀcycle assay
against a large panel of Envꢀpseudotyped viruses. The Xꢀray structure of a similar compound,
NBDꢀ14010 confirmed the binding mode of the newly designed compounds. The in vitro ADMET
profiles of these compounds are comparable to that of the most potent attachment inhibitor BMSꢀ
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626529, a prodrug of which is currently undergoing Phase III clinical trials. The systematic study
presented here is expected to pave the way for improving the potency, toxicity and ADMET
profile of this series of compounds with the potential to be moved to the early preclinical
development.
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INTRODUCTION
Virus attachment and fusion to the host cell membrane are the critical steps for HIVꢀ1 to enter the
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host cells and initiate its lifeꢀcycle by using host cell machinery . Therefore, virus attachment and
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fusion (often collectively termed as “virus entry”) have been the targets for new drug discovery for
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many years now . There are only two drugs currently on the market that target the entry pathway,
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, 6
Fuzeone (Enfuvirtide), which targets the envelope glycoprotein gp41
and Maraviroc, which
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ꢀ9
targets the host cell receptor CCR5 to prevent the entry of the virus . Despite the fact that HIVꢀ1
gp120 is critical for virus entry and has been targeted to discover novel drugs there is no drug yet
approved by the FDA against this target. The most advanced drug in this class, BMSꢀ663068,
which is a prodrug of 91 (1ꢀ(4ꢀbenzoylpiperazinꢀ1ꢀyl)ꢀ2ꢀ(4ꢀmethoxyꢀ7ꢀ(3ꢀmethylꢀ1Hꢀ1,2,4ꢀtriazolꢀ
1
0
1ꢀyl)ꢀ1Hꢀpyrrolo[2,3ꢀc]pyridinꢀ3ꢀyl)ethaneꢀ1,2ꢀdione; BMSꢀ626529 ) and showed its safety and
1
1
efficacy recently in a phase 2b clinical trials, is currently undergoing Phase III clinical trials .
BMSꢀ663068 in 2015 received US FDA breakthrough therapy designation, implicating the
importance of gp120 as a target to develop drugs in preventing virus entry into the host cells.
Our laboratory had focused on developing novel inhibitors targeted to the Phe43 cavity of
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gp120 since 2005 when we first reported the discovery of NBDꢀseries CD4ꢀmimic, NBDꢀ556 .
ꢀ
+
Unfortunately, NBDꢀ556 was demonstrated to enhance HIVꢀ1 infection in CD4 ꢀCCR5 cells,
1
3, 14
therefore, behaving as HIVꢀ1 entry agonist
. Since this finding we and others have striven to
design CD4ꢀmimics with not only higher potency and lower toxicity but also devoid of this
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4ꢀ24
undesirable trait, thereby representing as HIVꢀ1 entry antagonists
. We recently reported our
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first success in converting NBDꢀ556 to a full antagonist 89 (NBDꢀ11021) . However, we
recognize that a concerted optimization effort is needed to improve the antiviral potency and
toxicity of this NBDꢀseries compounds to obtain clinically relevant entry inhibitors. Towards that
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goal, we have coꢀcrystallized the first antagonist, 89 with gp120, which provided us the structural
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details of the binding mode of this molecule in the Phe43 cavity of gp120 . We have utilized the
structural information to design and synthesized a series of next generation gp120 antagonists as
entry inhibitors. Here we report the details of the synthesis, antiviral evaluation, structureꢀactivity
relationship (SAR) analysis of 60 novel analogs. Also, we present the Xꢀray structure
determination of one of this class of new generation compounds, 90 (NBDꢀ14010), published
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recently . Furthermore, we incorporated in vitro absorption, distribution, metabolism, excretion
and toxicity (ADMET) study during the early phase of optimization effort which is recognized as a
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critical step to avoid high attrition rate during the lateꢀstage drug development program
. The
optimization effort yielded valuable information on the key areas of the 89 type antagonist that can
be targeted for further structural optimization to produce nextꢀgeneration NBDꢀseries antagonists
suitable for further preꢀclinical studies.
CHEMISTRY
Synthesis of CD4ꢀmimics
We prepared the target compounds in a convergent manner from three fundamental building
blocks: carboxylic acid, protected thiazole and enantiopure imine (Schemes 1ꢀ5). We described the
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synthesis of pyrroleꢀ2ꢀcarboxylic acid 1 in our earlier work . The details of the other carboxylic
acid and their analogs (23, 39, 40, 79, and 80) will be published separately (in preparation).
A set of modified pyrroleꢀ2ꢀcarboxylic acids including Nꢀ, 3ꢀ and 4ꢀmethylated and 4ꢀMeO
substituted compounds (4, 8 and 12) were prepared using known compounds (5, 9 and 10) as per
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the reported procedure
or from commercially available compounds. Aryl imidazole carboxylic
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acids 20 and 22 were also prepared based on literature procedures
. We synthesized thiazoles
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6 and 73 using the method described previously
.
Previously we reported the development of a general route for the synthesis of NBDꢀ
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compounds . An appropriately protected aldehyde (34, 53 and 54) was prepared from readily
available starting materials (see schemes 1ꢀ6 for details). Then aldehyde was treated with Rꢀ or Sꢀ
Ellman auxiliary under dehydration conditions. The resulting enantiopure imines (35, 55, 56, and
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4) were treated with an appropriate thiazolyl lithium derivative to give a 1,2ꢀadditon product
(
Schemes 3, 4 and 5). In all of the 1,2ꢀaddition reactions, only a single stereoisomer was isolated.
In most cases no other products were observed; in some cases, minor products could not be
isolated and characterized. Compounds derived from Sꢀimines were marked as fS (from Sꢀisomer).
Compounds derived from Rꢀimines were marked fR (from Rꢀisomer). The absolute configuration
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of these compounds can be predicted using FelkinꢀAhn model . However, it was not
unambiguously proved by any experiments.
Acidic cleavage of silyl and sulfinyl groups gave suitably protected diamines (38, 59, 60,
1
7
7
7, and 78). Preparation of amine 24 was described earlier . HBTUꢀmediated peptide coupling
with an appropriate acid, followed by Pdꢀcatalyzed deprotection yielded the final compounds.
Reductive amination with formalin was used to obtain two series of dimethylated compounds (44ꢀ
4
6, 67ꢀ69, Schemes 3 and 4). It is important to note that all main building blocks (carboxylic
acids, protected thiazoles, enantiopure imines and protected diamines) were accessible in decagram
scale and benchꢀstable for an extended period (~one year). All of the synthetic procedures are
easily scalable, and all NBDꢀcompounds were prepared on at least 100 mg scale.
RESULTS
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Xꢀray crystal structure of 90 in complex with HIVꢀ1 gp120
The xꢀray structure of 89 with gp120 reported earlier confirmed that the tetramethylpiperidine ring
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4
of the 89 benefited from enabling its nitrogen atom to hydrogen bond with Asp368 of gp120 .
However, much of the hydrophobic portion of the piperidine ring was exposed to solvent, which
may lead to an undesirable thermodynamic penalty. Therefore, we replaced the piperidine ring
with a methylamine group to reduce solvent exposure of hydrophobic regions, while retaining the
amine for a hydrogen bonding with Asp368 of gp120. We also replaced the phenyl ring of the
molecule, which penetrates the Phe43 cavity, with a metaꢀfluoro substituted phenyl ring to enhance
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surface complementarity between the compound and the cavity
. Furthermore, the piperidine
ring in our earlier compound, 89, introduced two chiral centers, which generated four isomers of
this compound. Multiple isomers complicate the separation process after synthesis whereas the
primary amine only has one chiral center and produces only two isomers. The new compound, 90,
showed much improved antiꢀHIVꢀ1 activity, and more importantly, a substantially improved
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cytotoxicity profile compared to that of 89 .
To corroborate our rationale of designs and further understand the mechanism of improved
antiviral activity, we determined the crystal structure of 90 in complex with HIVꢀ1 gp120 at 2.1ꢀÅ
resolution (Figure 1a). This structure revealed the metaꢀfluoro substituted phenyl ring to have
improved surface complementarity with the Phe43 cavity. The surface complementarity by the
fluorine atom has now been demonstrated in Figure S1 using the structure of 90 with and without
fluoro group at the meta position. When superimposed with 89, 90 shared an almost identical mode
of binding to that of 89; the nitrogen in the amine group maintained the hydrogen bond with
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Asp368 as observed in the previous 89ꢀgp120 structure , but with much of the hydrophobic area
present in the tetramethylpiperidine removed (Figure 1b). Notably, the 90 structure revealed five
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ordered water molecules that mediate a hydrogen bonding network, which encompassed the
nitrogen atom of the methylamine group of 90 and gp120 residues Asp368, Gly431, Asn432,
Asn425, and Gly473 (Figure 1c and 1d). The finding of the water molecule networks is a novel
finding and expected to help in guiding further optimization of this series of compounds.
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Antiviral Screening and structureꢀactivity relationship (SAR) analysis
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We recently reported that our second generation compound 90 , besides maintaining the HIVꢀ1
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entry antagonist trait, first reported for 89 also shown better cytotoxicity and improved antiviral
activity compared to 89. Here, we describe the design of a new series of 60 novel NBDꢀseries
compounds in an attempt to expand the series and derive a comprehensive structureꢀactivity
relationship (SAR). We evaluated the HIVꢀ1 inhibitory activity and cytotoxicity (Table 1) of these
molecules. The antiꢀHIVꢀ1 activity was assessed in a singleꢀcycle assay by infecting TZMꢀbl
indicator cells with the pseudovirus HIVꢀ1HXB2 and in a multiꢀcycle assay by infecting the human
Tꢀcell MTꢀ2 with the fullꢀlength labꢀadapted virus HIVꢀ1IIIB. To derive systematic SAR of this
series of NBD derivatives, initially, we concentrated in modifying only the Region I in the phenyl
ring and a primary amine in Region III. When we replaced the chloro at R with fluoro, which
2
resulted in two compounds 25A and 25B, showed a reduction in cytotoxicity compared to 89 in
both assays but the HIVꢀ1 inhibitory activity dropped slightly in the pseudovirus assay but
substantially against MTꢀ2 cellꢀbased assay. This decrease in activity in MTꢀ2 cellꢀbased assay
was somewhat unusual, and we could not find that with other NBD compounds.
The xꢀray structures of 89 and 90 with gp120 indicate that the methyl group in the thiazole
ring does not contribute to any interactions with gp120 rather it is located at the solventꢀexposed
region outside the Phe43 cavity. Therefore, we decided to remove the methyl group from the
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thiazole ring in Region III. We first synthesized this series with the primary amine (R = NH ). The
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resulting compounds 81A and 81B showed noticeable improvement in both antiviral activity and
cytotoxicity against both singleꢀ and multiꢀcycle assays. Most significantly the selectivity index
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(SI) of 81B improved from 10.7 to 49.4 (~5ꢀfold improvement) in singleꢀcycle assay compared to
89. However, the SI value was slightly worse against multiꢀcycle assay. The reason for this
discrepant result is not apparent at this time. When we added a fluoro at the R position keeping
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chloro in the R position (82A and 82B) we did not find any noticeable improvement in antiviral
2
potency or SI compared to their parent molecules. But when we substituted chloro with a CH3
group at R and kept the fluoro at R the antiviral activity of the resulting compounds [83A and
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83B (NBDꢀ14088)] improved by about 2ꢀfold and the SI values by ~3ꢀ4ꢀfold in singleꢀcycle assay
but marginally enhanced in the multiꢀcycle assay. A further addition of a fluoro at the R position
3
resulted in 84A (NBDꢀ14107) and 84B that showed similar activity as with 83A and 83B.
Although the antiviral activity and cytotoxicity did not improve significantly in these fluoroꢀ
containing molecules, the addition of fluoro is known to help in producing molecules with more
metabolic stability than the unsubstituted ones. The introduction of OCH in R and R resulted in
3
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compounds 85A and 85B that had no antiꢀHIVꢀ1 activity and cytotoxicity at the indicated doses.
Most likely the reduction of antiviral activity was due to the steric effect of the bulky OCH group.
3
Then we modified R by introducing a CH in the primary amine moiety. Some of the resulting
4
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compounds (86A ꢀ88B) showed further improvement in antiviral potency. However, they all turn
out to be more cytotoxic.
We then decided to retain 4ꢀchloro and also use 4ꢀCH at the R position and keep the 3ꢀ
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fluoro unchanged at the R position and introduced a CH in the primary amine group. The
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antiviral activity of all the resulting compounds (41A ꢀ 43B) was similar to 89; however, they
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showed slightly reduced cytotoxicity. The introduction of N(CH ) in the primary amine at R
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yielded compounds 44Aꢀ46B which were not cytotoxic at the doses we tested in both assays but
had no antiviral activity at all. The introduction of CH NH in R produced compounds 61Aꢀ63B
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which did not show improvement in antiꢀHIVꢀ1 activity, but when we substituted Chloro with the
methyl group in R the cytotoxicity reduced slightly. Replacing the CH NH group in R with
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CHN(CH ) (67Aꢀ69B) did not provide any improvement in antiviral activity in both singleꢀcycle
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and multiꢀcycle assays, but unlike in the previous series (44A ꢀ63B), compounds with 4ꢀchloro and
3ꢀfluoro retained similar activity as 89.
In the above SAR study, it was evident that introduction of a fluorine atom, the most
electronegative among halogens) at position R /R (meta) and R (para) in the phenyl ring reduced
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cytotoxicity (25A, 25B) considerably more than 82A and 82B where chloro replaced the fluoro at
R . However, the antiviral potency of compounds with chloro at the R position was ~3ꢀ6ꢀfold
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better. We also observed that when we introduced a methyl group at R , which is electron
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donating, the antiviral potency improved further. We also observed a marginal reduction of
cytotoxicity. Analyzing this series it was evident that the antiviral potency of the compounds
dependent more on the hydrophobic character of the R substituent than its electronegativity. The
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hydrophobic parameter (Π) is very similar for chloro and methyl group. Therefore, the introduction
of Cl/CH at R and fluoro at R /R are critical for obtaining the best antiviral potency and lower
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cytotoxicity in this series of compounds.
We observed that in general, the modifications in region II in the pyrrole ring (26Aꢀ31B),
especially in R position, made the compounds inactive. This observation was not surprising
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because our earlier finding in the xꢀray structure of 89 and also the structure reported here with
gp120 led to the observation that the NH of pyrrole makes an Hꢀbond with the residue Asn425,
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which appears to be critical for the binding of this series of compounds in the Phe43 cavity. The
methylation of this NH (compounds 26A, 26B) may have two detrimental effectsꢀ loss of the Hꢀ
bond donor atom and also possibly a steric effect that interferes with the binding. However,
substituting H at R and R with methyl yielded compounds with somewhat better or similar
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activity as 89 (27A, 27B, 28A and 28B) in the singleꢀcycle assay but slightly poorer activity in the
multiꢀcycle assay. Noticeably, one of the isomers of 28 (28B) showed very high cytotoxicity.
However, the underlying cause of cytotoxicity is not clear at this stage. When we introduced a
methoxy group at the R position (29A and 29B) the antiviral activity was completely lost most
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likely due to steric effect. Then, we moved to find out whether we could modify the pyrrole ring to
imidazole by introducing one additional “N” in the R or R position. These modifications
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resulted in compounds 30Aꢀ31B which, although showing muchꢀreduced cytotoxicity, had no
antiviral activity. The SAR data obtained on pyrrole ring confirmed that modification in the
pyrrole ring was detrimental to the antiviral activity.
Among the 60 new compounds that we tested 83B, and 84A showed the highest SI of ~86
and ~62, respectively in single cycle assay, which represents an enhancement of ~8ꢀ and ~6ꢀfold,
respectively, compared to 89. However, in the multiꢀcycle assay, the SI of those two compounds
were 50 and 38.5, respectively, which represent only ~2 and ~1.3ꢀfold, respectively. Despite
marginal enhancement in activity and selectivity in the multiꢀcycle assay, these norꢀmethyl analogs
bring two distinct advantages over all other compounds reported earlier. 84A/84B and 88A/88B
are expected to be more metabolically stable; and the deletion of the methyl group in the thiazole
ring opened up the opportunity to introduce other groups, especially, solubilizing groups, which is
expected to make these molecules more water soluble and more suitable for further preclinical
studies. Therefore, we selected these two compounds for further research.
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83B and 84A prevent cellꢀtoꢀcell fusion and retain the HIVꢀ1 entry antagonist trait
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Two essential features for the HIVꢀ1 entry inhibitors that target the Phe43 cavity of HIVꢀ1 gp120
are the capability to inhibit HIVꢀ1 Envꢀmediated cellꢀtoꢀcell fusion and have HIVꢀ1 entry
antagonist property. First, we evaluated the inhibitory activity of the two best inhibitors, 83B and
84A, on the HIVꢀ1 Envꢀmediated cellꢀtoꢀcell fusion. To this end, we cocultured the indicator cells
MAGIꢀCCR5 with the Envꢀ and Tatꢀexpressing HL2/3 cells in the presence of escalating doses of
compounds. We used NBDꢀ556 and 89 as a control. Our results (Figure 2A) indicate that both
83B and 84A prevent the HIVꢀ1 Envꢀmediated cellꢀtoꢀcell fusion, but the IC were ~2ꢀ3ꢀfold
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higher than what we observed with NBDꢀ556 and 89 (8.3±0.9 and 13.3±1.2 versus 2.9±0.1 and
.1±0.4, respectively). As next step, we wanted to verify whether 83B and 84A maintained the
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HIVꢀ1 entry antagonist property we previously reported for our lead compound 89 and the
second generation compounds N‐{2‐amino‐1‐[5‐(hydroxymethyl)‐4‐methyl‐1,3‐thiazol‐2‐
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yl]ethyl}‐5‐(4‐chlorophenyl)‐1H‐pyrrole‐2‐carboxamide (NBDꢀ14009) and 90 . To this end, we
infected CD4ꢀnegative and CCR5ꢀpositive cells, Cf2ThꢀCCR5, with recombinant CD4ꢀdependent
HIVꢀ1ADA virus in the presence of the compounds. We used NBDꢀ556 (HIVꢀ1 entry agonist) and
89 (HIVꢀ1 viral entry antagonist) as a negative and a positive control, respectively. As expected,
NBDꢀ556 enhanced the infection of the Cf2ThꢀCCR5 cells, and 83B and 84A did not enhance
HIVꢀ1 infectivity in these cells as was observed for 89, indicating that the HIVꢀ1 entry antagonist
property is maintained in these compounds as well (Figure 2B).
Timeꢀdependent HIVꢀ1 inhibitory activity profiles of NBD compounds
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To further understand the mechanism of action of the NBD compounds we performed a time of
addition experiment. We used four known compounds as reference or control. As expected, we
found that all the compounds inhibited HIVꢀ1 when added to the cells at the time of infection
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(Figure 3). The coꢀreceptor CXCR4 inhibitor, AMD3100, and the entry inhibitor BMSꢀ378806
showed a decrease in efficacy when added to the culture 1 h postꢀinfection (p.i.) and their
effectiveness continued dropping to disappear at 2ꢀ4 h p.i completely. Moreover, the efficacy of
the Reverse Transcriptase inhibitor, AZT decreased to about 50% when added 4 h p.i. while we
only detected a slight decline in the efficiency of the Integrase inhibitor, Raltegravir when added 8
h p.i. Finally, the NBD compounds showed some efficacy when added to the culture 4 h p.i. and
they gradually lost efficiency suggesting that 83B and 84A may also target the later phase of the
HIVꢀ1 life cycle. Earlier we reported that 89 inhibits HIVꢀ1 reverse transcriptase (RT) enzyme but
at much higher concentration. Based on that observation, we decided to test these compounds
against HIVꢀ1 RT.
NBD compounds also target HIVꢀ1 Reverse Transcriptase (RT)
The time of addition experiment suggested that 83B and 84A may also have an additional
mechanism of action. Consequently, we decide to evaluate the activity of 83B and 84A against
HIVꢀ1 RT. We used 89, NBDꢀ556, and Nevirapine as controls. As expected, NBDꢀ556 did not
show any activity even at 300 ꢁM and Nevirapine was extremely potent against HIV RT, with IC50
of 0.20 µM (Table 2). 89 inhibited HIVꢀ1 RT with an IC of 47 ꢁM while 83B and 84A had an
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IC50 of 7.2 and 8.4 ꢁM, respectively. Comparing the IC50 values obtained with the NBD
compounds for the entry inhibition assay and the RTꢀinhibition assay we found that the ratio IC ꢀ
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RT/IC ꢀHIVꢀ1 was similar for the three compounds, i.e., 21, 16 and 13 for 89, 83B and 84A,
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Antiviral activity of 83B and 84A against a large panel of HIVꢀ1 Envꢀpseudotyped reference
viruses.
The first and second generation NBD compounds 89 and 90 showed a broad range of antiviral
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activity against a large panel of clinical isolates of different subtype
. In this study, we
evaluated the HIVꢀ1 inhibitory activity of our newly selected compounds, 83B and 84A, against a
large panel of Envꢀpseudotyped viruses and we compared their activity with the 89 (Table 3). We
selected fiftyꢀfive HIVꢀ1 clones of clinical isolates from 9 subtypes including primary and
transmitted/founder HIVꢀ1 isolates and 12 recombinant HIVꢀ1 clones. We found that both these
compounds exhibited improved antiꢀHIVꢀ1 activity compared to 89. For example, we observed
that the overall mean of the IC s determined for 89 against the panel of pseudoviruses was 1.64 ±
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.12 µM (IC s in the range of 0.32ꢀ4.4 µM). On the contrary, we found that the overall means of
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the IC s determined for 83B and 84A were 0.41 ± 0.02 µM (IC s in the range of 0.2ꢀ0.75 µM)
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and 0.49 ± 0.02 µM (IC50s in the range of 0.27ꢀ0.94 µM), respectively. Additionally, we found that
although 84A was equally active against all the pseudoviruses regardless of the subtype, 89 was
slightly less active against the HIVꢀ1 clones of subtype C (mean of 2.16 ± 0.18, n = 16). By
contrast, 83B was ~6ꢀfold more active against subtype C viruses (mean of 0.35 ± 0.03, n = 16)
compared to 89. 83B had similar activity against subtype D (mean of 0.36 ± 0.05, n = 5), subtype
A (mean of 0.43 ± 0.04, n = 12) and subtype B (mean of 0.46 ± 0.04, n = 15) viruses. The data
indicate that both 83B and 84A showed broad spectrum antiviral activity against a large number of
HIVꢀ1 Envꢀpseudotyped viruses representing clinical isolates of diverse subtypes. Finally, both
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compounds 83B and 84A showed much poorer activity against the control pseudovirus VSVꢀG
displaying an IC of ~42.9 µM and ~26 µM respectively versus the ~7.1 µM observed for 89,
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suggesting that the inhibitory activity of these compounds is specific to HIVꢀ1.
In vitro ADMET study of NBDꢀseries compounds
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Early ADMET studies of drug molecules during discovery phase has been recognized as
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critical to reducing the attrition rate in late stage drug development
. Therefore, we decided to
initiate in vitro ADMET studies on 83B and 84A and compare the data with previously published
inhibitor, 89 of this class. We also used 91 as a control, a highly potent attachment inhibitor, the
proꢀdrug of which has been shown to be safe and efficacious and received “breakthrough therapy
designation for investigational HIVꢀ1 attachment inhibitor in 2015 by the US FDA. The in vitro
ADMET data in Table 4 show that the new molecules 83B, 84A, and our earlier reported
compound 89, as well as the control inhibitor 91, have similar aqueous solubility profiles.
However, in the bidirectional Cacoꢀ2 cell permeability assay, which predicts the human intestinal
permeability potential of a molecule required for oral absorption, NBD compounds demonstrated
to have relatively moderate permeability compared to 91. The Cacoꢀ2 cell permeability in the
basolateral to apical side (B2A) was measured principally to calculate the efflux ratio (Mean Papp
B2A / Mean Papp A2B) of these molecules. A value greater than 2 indicates the possibility of these
molecules to be removed out of the basolateral site most likely by the efflux transporter(s).
Since the liver is the major organ where drug metabolism occurs, we decided to study the
metabolic stability of our molecules in human liver microsomes. The data in Table 4 indicate that
83B and 84A showed improved stability compared to 91, whereas 89 has similar stability to 91.
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We can classify all NBD molecules and 91 from their in vitro human intrinsic clearance data
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The NBD compounds were also tested for their human plasma protein binding potential
and compared with the control 91. The mean fraction unbound (fu) data in Table 4 indicate that
6%, 90% and 88.6% of 83B, 84A, and 89, respectively were bound to the plasma which is
9
slightly higher than 91, where 81.5% binding was observed.
We evaluated the NBD compounds and 91 against five CYP450 isoforms (CYP2C19,
CYP3A4, CYP2D6, CYP2C9, and CYP1A), which metabolizes more than 80% of the drugs to
identify any possible drugꢀdrug interactions. All NBD compounds tested and the control 91
showed no inhibition of any of the isoforms tested. The only inhibition observed was for 83B
against CYP1A, with an IC50 of 8.01 µM, implicating a moderate inhibition potential of this
particular molecule.
We also evaluated the potential of these molecules to inhibit hERG channel, known to be a
predictor of the development of acquired long QT syndrome that may lead to fatal ventricular
arrhythmia. All NBD compounds were tested along with the control 91. 84A and 91 showed weak
or no inhibition of the hERG channel. However, 89 and 83B showed moderate inhibition of hERG
channel.
DISCUSSION
The HIVꢀ1 gp120 binding site on the human primary cell receptor CD4 has been long recognized
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as one of the major sites for developing entry inhibitors . The major success came with the
discovery of the first attachment inhibitor BMSꢀ378806 with high potency, which, however, could
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not proceed further for development due to a deficiency in its bioavailability that leads to the
development of next generation BMS analogs with improved pharmacokinetics profiles. One such
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13, 18ꢀ20, 22ꢀ24, 34, 35, 37ꢀ40
drug, BMSꢀ663068 is currently in phase III clinical trials. We
and others
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have validated Phe43 cavity of HIVꢀ1 gp120 as a target for discovering a new class of entry
inhibitor drugs. Our success in converting a full agonist (NBDꢀ556) to an antagonist (89)
motivated us to optimize this molecule further to more potent and less toxic inhibitors that
maintain the antagonist trait. Towards that goal, we recently reported the Xꢀray structure of 89
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with HIVꢀ1 gp120 . An inꢀdepth analysis of the structure indicated that despite the introduction of
a piperidine ring which enabled an Hꢀbond interaction with Asp368 of gp120, the ring had a
significant hydrophobic component which was located in the solvent exposed area, which may be
undesirable for binding to gp120 due to high thermodynamic penalty. Therefore, to avoid such
adverse effect, we recently reported the synthesis of a few new molecules by replacing piperidine
17
ring with primary amines . One of these molecules 90 showed improvement in antiviral potency
against a large panel of Envꢀpseudotyped viruses. The xꢀray structure of 90, reported in this study,
confirmed that this modification still could maintain the critical Hꢀbond interaction with Asp368.
This confirmation prompted us to synthesize a large number of analogs to derive a comprehensive
SAR to guide in further optimization of this class of inhibitors. We have initially tested these
molecules in a singleꢀcycle and a multiꢀcycle HIVꢀ1 inhibition assay and identified two molecules,
83B and 84A with improved antiviral activity and selectivity index. A further test with a large
panel of Envꢀpseudotyped HIVꢀ1 representing clinical isolates of different subtypes showed their
broadꢀspectrum antiviral potency. The improvement of the mean IC values of 83B and 84A when
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compared with 89 among the same set of Envꢀpseudoviruses, was ~4ꢀfold. Most significantly,
these two molecules showed much improved antiviral potency across multiple subtypes (AꢀD)
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tested unlike BMSꢀ378806, which despite its low nM antiviral potency against subtype B, it
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showed relatively weak potency against A, C and D subtypes . The cytotoxicity values of the
NBD series molecules presented here showed an only marginal reduction in our best compounds.
However, in the assay against a large panel of pseudoviruses containing the envelope from clinical
isolates we see substantial improvement in antiviral activity, which leads to the belief that further
modifications will result in compounds with higher potency, thereby, with higher selectivity index.
The time of addition data with 83B and 84A showed that there might be additional
inhibitory activity besides inhibiting entry because these compounds show some activity even after
adding beyond 2 h, indicative of some other target. Earlier we reported that some next generation
NBD compounds, such as, 89 showed activity against HIVꢀ1 RT enzyme. We also confirmed in
this study that like 89, 83B and 84A also target RT at higher doses. Despite the fact that the
activity against HIVꢀ1 RT of these two molecules was more potent than 89, the relative ratio of
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inhibitors. It is worth noting that the new generation compounds, such as 83B and 84A, are
severalꢀfold more potent entry inhibitors against HIVꢀ1 than 89.
Poor physicochemical properties, pharmacokinetics, and toxicity are the principal cause of
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lateꢀstage failure in drug development . Use of in vitro evaluation of ADMET of drugs is
especially attractive in this phase due to high cost with in vivo procedures involving an animal. We
have decided to address this critical issue in our early optimization phase of developing these entry
inhibitors as future clinical candidates. Published report demonstrated that addition of a fluoro
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we observed mixed results in our study . However, it is wellꢀestablished that fluorine in medicinal
chemistry has been used quite extensively to improve physicochemical properties and metabolic
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profiles of drugs
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vulnerable positions in the phenyl ring of the NBDꢀseries of compounds. Therefore, it was
imperative for us to evaluate the effect on physicochemical and pharmacokinetic profiles of the
two most active compounds in this study by in vitro ADMET studies. The in vitro ADMET data in
Table 4 indicate that NBDꢀseries compounds have similar ADMET profiles to that of the clinical
candidate, 91, except Cacoꢀ2 permeability which is slightly lower. The Cacoꢀ2 permeability
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ꢀ1
ꢀ6
(expressed as P , 10ꢀ x cms ) of 83B, 84A and 89 have P values of 2.56, 1.17 and 2.31 x 10
app
aap
ꢀ1
ꢀ6
ꢀ1
cms , respectively compared to 7.62 x 10 cms of 91. However, the Cacoꢀ2 permeability value
ꢀ6
ꢀ1
(
Papp of >1 x 10 cms ) of a molecule is considered to have an acceptable permeability in drug
4
5
development program . The oral drug, atenolol, used as a control in our experiment, has a P of
app
ꢀ6
ꢀ1
0
.220 x 10 cms . The biꢀdirectional Cacoꢀ2 cell permeability data also indicate that all the NBD
compounds and 91 have high efflux ratio demonstrating that all these molecules may be subject to
Pꢀgp mediated efflux. However, the role of other transporters, such as BCRP, MRP2, etc could not
46
be ruled out . The results on metabolic stability in human microsomes indicate that all the NBD
compounds are stable up to the 45 min limit of the experimental protocol. The intrinsic clearance
data (CL , µL/min/mg protein) also indicate that these compounds have low clearance potential. It
int
is worthwhile to mention that drugs with high clearance are considered unfavorable because they
may be cleared rapidly in vivo and the drugs may have a short duration of action and require
multiple/repeat dosing.
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The protein binding data in Table 4, expressed as mean fraction unbound (fu), indicate that
all NBD compounds and 91 bind strongly to plasma protein. However, Smith et al. from Pfizer
Global Research, in 2010 convincingly illustrated that we need to be cautious in interpreting the
plasma binding data and optimizing molecules based on those data because the efficacy of a drug
is often dependent on the free drug concentration surrounding the drug target rather than the free
8
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drug fraction .
4
8
Cytochrome P450 enzymes (CYP450) are essential for the metabolism of many drugs .
However, inhibition of these enzymes may lead to drugꢀdrug interaction when two or more drugs
are coꢀadministered through inhibition of CYP450 enzymes specific for one or other drugs.
Inhibition of metabolism may lead to increased level of drug concentration in the system which
may lead to toxicity. We have selected five isoforms (CYP2C19, CYP3A4, CYP2D6, CYP2C9
and CYP1A) responsible for the metabolism of the majority drugs. All NBD compounds and 91
showed a low level of inhibition of all five isoforms tested (IC50 >25 µM except 83B has IC of
50
8.01 µM against CYP1A) indicating that these compounds are less likely to have toxicity due to
49
drugꢀdrugꢀinteractions associated with drug metabolism .
Several drugs either had to be withdrawn or imposed restricted use due to cardiac toxicity.
Many drugs are known to induce acquired long QT syndrome by prolonging QT interval and
cardiac action potential due to inhibition of human etherꢀaꢀgoꢀgoꢀrelated gene (hERG) that encode
5
0
the inward rectifying voltageꢀgated potassium channel . Therefore, it was prudent for us to
evaluate the inhibitory potential of the NBD compounds against hERG and compare that with the
control, 91. The data in Table 4 demonstrate that all these compounds have µM IC values. 84A
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inhibited hERG with similar potency as the 91 control. 89 showed the highest inhibition of hERG
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but our next generation compounds 83B and 84A showed some improvement. It is worthwhile to
mention that the drugs that had to be either taken out of the market or imposed restriction in use all
had nM level inhibitory activity. It was evident from the above data that the best NBD compounds
in this study have very similar ADMET profiles to that of the control 91.
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CONCLUSIONS
In our continuous effort to optimize the first HIVꢀ1 entry antagonist, 89, we have synthesized 60
new analogs, determined their antiviral potency and experimentally evaluated in vitro ADMET
profile of two of the most active compounds. 83B and 84A showed measurable progress in
improving the antiviral potency and the selectivity index compared to 89. The xꢀray structure
confirms the binding mode of this class of molecules as well as provides structural details, which is
expected to provide additional guidance in further optimizing these new molecules. The data
presented here show that these molecules have the broadꢀspectrum potency against a large panel of
Envꢀpseudotyped viruses representing clinical isolates of a wide variety of subtypes. These
molecules also have comparable in vitro ADMET profiles to that of the clinical attachment
inhibitor, 91. The systematic study presented here is expected to pave the way for improving
further the potency, toxicity and ADMET profile of this series of compounds with the potential to
be moved to the early preclinical development.
EXPERIMENTAL SECTION
Cells and viruses
51
52
53
54
55
MTꢀ2 cells , TZMꢀbl cells , `MAGIꢀCCR5 cells , U87CD4+CXCR4+ cells and HL2/3 cells
were obtained through the NIH ARP. HEK 293T cells were purchased from ATCC. CD4ꢀnegative
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Cf2ThꢀCCR5+ cells and Env expression vector pSVIIIenvꢀADA were kindly provided by Dr. J. G.
56
Sodroski . HIVꢀ1 Env molecular clone expression vector pHXB2ꢀenv (X4) DNA was also
5
7
obtained through the NIH ARP . HIVꢀ1 Env molecular clones of gp160 genes for HIVꢀ1 Env
pseudovirus production were obtained as follows: clones representing the standard panels A, A/D,
A2/D, D and C (QB099.391M.Env.B1) were obtained through the NIH ARP from Dr. J.
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8, 59
Overbaugh
. The HIVꢀ1 Env molecular clones panel of subtype A/G and A/E (CRF01_AE
clone 269) Env clones were obtained through the NIH ARP from Drs. D. Ellenberger, B. Li, M.
6
0
Callahan and S. Butera . The AE clone AA058 was kindly provided by Drs. R. J. McLinden and
A. L. Chenine from US Military HIV Program, Henry M. Jackson Foundation (Silver Spring,
MD). The HIVꢀ1 Env panel of standard reference subtype B Env clones was obtained through the
NIH ARP from Drs. D. Montefiori, F. Gao and M. Li (PVO, clone 4 (SVPB11); TRO, Clone 11
(SVPB12); AC10.0, clone 29 (SVPB13); QH0692, clone 42 (SVPB6); SC422661, clone B
(SVPB8)); from Drs. B. H. Hahn and J. F. SalazarꢀGonzalez (pREJO4541, clone 67 (SVPB16);
pRHPA4259, clone 7 (SVPB14)); from Drs. B. H. Hahn and D. L. Kothe (pTHRO4156 clone 18
6
1, 62
(SVPB15), pCAAN5342 clone A2 (SVPB19))
. The subtype B clones pWEAUd15.410.5017,
p1058_11.B11.1550, p1054.TC4.1499, p9014_01.TB1.4769 and p9021_14.B2.4571 were obtained
6
3
through the NIH ARP from Drs. B. H. Hahn, B. F. Keele and G. M. Shaw . The subtype C HIVꢀ1
reference panel of Env clones were also obtained through the NIH ARP from Drs. D. Montefiori,
F. Gao, S. A. Karim and G. Ramjee (Du 156.12; Du172.17); from Drs. D. Montefiori, F. Gao, C.
Williamson and S. A. Karim (Du422.1), from Drs. B. H. Hahn, Y. Li and J. F. SalazarꢀGonzalez
(ZM197M.PB7; ZM249M.PL1); from Drs. E. Hunter and C. Derdeyn (ZM53M.PB12;
ZM135M.PL10a; ZM109F.PB4); from Drs. L. Morris, K. Mlisana and D. Montefiori,
6
4ꢀ66
(CAP45.2.00.G3; CAP210.2.00.E8)
. The HIVꢀ1 Subtype C Panel of Indian gp160 Env Clones
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HIVꢀ16055ꢀ2 clone 3, HIVꢀ16936ꢀ2 clone 21, HIVꢀ25711ꢀ2 clone 4, HIVꢀ225925ꢀ2 clone 22 and
HIVꢀ26191ꢀ2, clone 48 were obtained through the NIH ARP from Drs. R. Paranjape, S. Kulkarni
6
0
and D. Montefiori . HIVꢀ1 Env molecular clones MF535.W0M.Env.C1 and BF535.W6M.Env.A1
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of subtype D/A were obtained through the NIH ARP from Dr. J. Overbaugh . The Envꢀ
pseudotyped genes of BG505.T332N, KNH1144 and B41 were kindly provided by Dr. J. P. Moore
of the Weil Cornell Medical College, NY.
env
52,
The Envꢀdeleted proꢀviral backbone plasmid and pSG3ꢂ DNA (Drs. J. C. Kappes and X. Wu)
6
2
was obtained through the NIH ARP. MLV gagꢀpolꢀexpressing vector pVPackꢀGP, Envꢀ
expressing vector pVPackꢀVSVꢀG and a pFBꢀluc vector were obtained from Stratagene (La Jolla,
CA).
HIVꢀ1IIIB laboratory adapted strain was obtained through the NIH ARP. Fullꢀlength clinical
isolates HIVꢀ1 clones were obtained through the NIH ARP.
Pseudovirus preparation
Pseudoviruses capable of single cycle infection were prepared as previously described with a
1
5, 17
6
minor modification
. Briefly, 5 x 10 HEK293T cells were transfected in a 15 ml volume of a
ꢂenv
solution containing 8 µg of an HIVꢀ1 Envꢀdeleted proꢀviral backbone plasmid pSG3 , and 8 µg
of an HIVꢀ1 Envꢀexpression plasmid with FuGENE 6 (Roche). VSVꢀG pseudovirus was prepared
by transfecting the HEK293T cells with a combination of the Envꢀexpressing plasmid pVPackꢀ
VSVꢀG, the MLV gagꢀpolꢀexpressing plasmid pVPackꢀGP, the pFBꢀluc plasmid and FuGENE 6.
Pseudovirusꢀcontaining supernatants were collected two days after transfection, filtered, tittered
and stored in aliquots at −80 °C.
Measurement of antiviral activity
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Singleꢀcycle infection assay in TZMꢀbl cells. The NBD compounds were tested against the
pseudovirus HIVꢀ1 expressing the Env from the labꢀadapted HIVꢀ1HXBꢀ2 (X4) in a singleꢀcycle
infection assay. Moreover, the small molecules 89, 83B, and 84A were also tested against a large
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group of HIVꢀ1 pseudotyped viruses expressing the Env from the panel of clinical isolates as
15, 17
previously described
. Briefly, TZMꢀbl cells were plated in a 96ꢀwell tissue culture plate at 1 x
4
10 / well and cultured at 37 °C. Following overnight incubation, aliquots of HIVꢀ1 pseudovirus
preꢀtreated with graded concentrations of the small molecules for 30 min were added to the cells
and incubated for 3 days. Cells were washed and lysed with 50 µl of lysis buffer (Promega). 20 µl
of the lysates were transferred to a white plate and mixed with the luciferase assay reagent
(Promega). The luciferase activity was measured immediately with a Tecan Infinite M1000 reader,
and the percent inhibition by the compounds and IC (the half maximal inhibitory concentration)
50
values were calculated using the GraphPad Prism software.
Singleꢀcycle infection assay in U87ꢀCD4ꢀCXCR4 cells. The small molecules 89, 83B and 84A
were also tested against control pseudovirus VSVꢀG, obtained as described above. Briefly, U87ꢀ
4
CD4ꢀCXCR4 cells were plated in a 96ꢀwell tissue culture plate at 1 x 10 / well and cultured
overnight. The following day, aliquots of VSVꢀG pseudovirus preꢀtreated with graded
concentrations of the small molecules for 30 min were added to the cells and incubated for three
days. Cells were washed and lysed with 40 µl of lysis buffer. The lysates were then transferred to a
white plate and mixed with the luciferase assay reagent. The luciferase activity was immediately
measured to calculate the percent of inhibition and IC50 values by using the GraphPad Prism
software.
Multiꢀcycle infection assay in MTꢀ2 cells. The NBD small molecules were evaluated against the
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fullꢀlength laboratoryꢀadapted HIVꢀ1IIIB as previously described . Briefly, MTꢀ2 cells were
infected with HIVꢀ1IIIB at 100 TCID and preꢀincubated with graded concentrations of compounds
50
for 30 min. The following day, the culture supernatants were replaced with fresh media. Four days
postꢀinfection, the supernatants were collected and mixed with an equal volume of 5 % Triton Xꢀ
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00 and tested for p24 antigen by sandwichꢀELISA. The percent inhibition of p24 production and
IC values were calculated by the GraphPad Prism software.
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Evaluation of cytotoxicity
TZMꢀbl cells. The cytotoxicity of the small molecules in TZMꢀbl cells was measured by the
1
4
colorimetric XTT method as previously described . Briefly, TZMꢀbl cells were plated in a 96ꢀwell
4
tissue culture plate at 1 x 10 / well and cultured at 37 °C. Following overnight incubation, the cells
were incubated with 100 µl of the compounds at graded concentrations and cultured for three days.
The XTT solution was added to the cells and four h later the soluble intracellular formazan was
quantitated at 450 nm. The percent of cytotoxicity and the CC50 (the concentration for 50 %
cytotoxicity) values were calculated as above.
MTꢀ2 cells. The cytotoxicity of the small molecules in MTꢀ2 cells which ran parallel to the
neutralization assay was also measured with the colorimetrical XTT method as previously
6
8
described . Briefly, 100 µl of a small molecule at graded concentrations was added to an equal
5
volume of cells (10 cells/ml) in 96ꢀwell plates. The following day, the culture supernatants were
replaced with fresh media and incubated for four days. Four hours after the addition of XTT the
soluble intracellular formazan was quantitated, and the percent of cytotoxicity and the CC values
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HIVꢀ1 mediated cellꢀtoꢀcell fusion inhibition assay.
14, 69, 70
The HIVꢀ1 mediated cellꢀtoꢀcell fusion assay was performed as previously described
with
some minor modifications. We used the indicator cells MAGIꢀCCR5, a HeLa cell line expressing
CD4, CXCR4, and CCR5, and HIVꢀLTRꢀβꢀgal under control of HIVꢀ1 Tat as target cells. And
HL2/3 cells, a HeLa cell line which expresses HIVꢀ1HXB2 Env on the surface and Tat, Gag, Rev
and Nef proteins in the cytoplasm but does not produce mature virions, as effector cells. Briefly,
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MAGIꢀCCR5 cells were plated in a 96ꢀwell 1.5 × 10 / well and cultured for four h at 37 °C. Then,
the cells were incubated for one h with escalating concentrations of NBDꢀcompounds. HL2/3 cells
3
were then added to the culture at 7.5 x 10 cells/well and cultured for 24 h. βꢀgal expression was
quantified with the BetaꢀGlo Assay system (Promega) following the manufacturer’s instructions.
The percent inhibition and the IC values were calculated using the GraphPad Prism software
50
Assay in Cf2ThꢀCCR5 cells
3
CD4ꢀnegative Cf2ThꢀCCR5 cells were plated at 6 × 10 cells/well in a 96ꢀwell tissue culture plate
and incubated overnight. The cells were infected with the luciferase expressing recombinant CD4ꢀ
1
3
dependent pseudovirus HIVꢀ1ADA as previously described . Briefly, following overnight
incubation, aliquots of HIVꢀ1ADA pseudovirus preꢀtreated with graded concentrations of the small
molecules for 30 min were added to the cells and cultured for 48 h. Cells were washed with PBS
and lysed with 40 µl of cell lysis reagent. Lysates were transferred to a white 96ꢀwell plate and
mixed with 100 µl of luciferase assay reagent. The luciferase activity was immediately measured
to obtain the relative infection compared to the untreated control. The Relative virus infectivity
indicates the amount of infection detected in the presence of the compounds divided by the amount
of infection detected in the absence of the compounds.
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Time of addition assay
4
TZMꢀbl cells were plated in a 96ꢀwell tissue culture plate at 1 x 10 / well and cultured at 37 °C
overnight. On the day of the infection, aliquots of the pseudovirus HIV1HXB2 were added to the
cells. The plates were immediately spun for 30 min at 2090 g at 4 ºC and subsequently incubated at
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37 ºC for 90 min. Afterward, the medium was removed to eliminate unbound virus, and the cells
were washed one time with PBS. Fresh medium was added to the cells. The cells were treated
41
with 10ꢀ15 times their respective IC values as previously described Lin PF. Et al. at the time of
5
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infection (Time “0”) or 30 min, 1, 2, 4, 6 and 8 h postinfection (p.i.). As a control, cells were
treated with AMD3100 (coꢀreceptor CXCR4 inhibitor), BMSꢀ378806 (entry inhibitor) Zidovudine
(AZT, Nucleoside Reverse Transcriptase Inhibitor (NRTI)) and Raltegravir (Integrase inhibitor).
Fortyꢀeight hours postꢀinfection, the cells were washed two times with PBS and lysed. The
luciferase activity was immediately measured as described above. Two independent experiments
were performed in triplicate, and the graph is representative of one experiment.
Quantitative determination of HIVꢀ1 Reverse Transcriptase activity.
The inhibitory effect of the NBD compounds on the HIVꢀ1 Reverse Transcriptase activity was
determined by using the Colorimetric Reverse Transcriptase Assay (Roche) by following the
manufacturer’s instructions. NBDꢀ556 and Nevirapine (Nonꢀnucleoside Reverse Transcriptase
Inhibitor (NNRTI)) were used as a control.
Crystallization, structure determination and refinement
Clade A/E gp120 core H375S was produced by transient expression and purified as described
e
7
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previously . The protein was concentrated to ~10 mg/ml in 5 mM TrisꢀHCl pH 7.5, 150 mM
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NaCl, and 0.02% NaN and mixed with 5 mM 90 in 50% DMSO in 10 to 1 ratio prior to
3
crystallization. The protein and 90 mixture was then set up for crystallization by mixing 0.5 ꢁl of
the mixture and 0.5 ꢁl of the reservoir solution containing 10ꢀ16% PEG 3350, 5% isopropanol, and
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0.1M HEPES 7.5 by hanging drop vapor diffusion. Good diffracting quality crystals grew at 14%
PEG3350, 5% isopropanol, and 0.1M HEPES 7.5. Crystals were flashꢀfrozen with cryoprotectant
containing 30% ethylene glycol, 14% PEG3350, 5% isopropanol, and 0.1M HEPES 7.5 for data
collection at synchrotron beamline (SERꢀCAT ID22, Advanced Photon Source, Argonne National
7
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Laboratory). The diffraction data were processed and scaled with HKL2000 and the structure
7
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was determined by molecular replacement using Phaser . The molecule was built with Coot and
73
refined with PHENIX . The refinement statistics were summarized in Table S1. Figures were
generated using PyMOL (The PyMOL Molecular Graphics System, Version 2.0, Schrödinger,
LLC).
Turbidimetric Aqueous Solubility
Test compound (10 mM in DMSO) was serially diluted to give solutions of 0.1, 0.3, 1 and 3 mM
in DMSO. Each test compound concentration was then further diluted 1 in 100 in buffer (typically
0.01 M phosphate buffered saline pH 7.4) so that the final DMSO concentration was 1 % and the
final test compound concentrations were 1, 3, 10, 30 and 100 µM. The experiment was performed
at 37 °C and each concentration was incubated in 7 replicate wells. The plates were incubated for
2
hr at 37 °C before the absorbance was measured at 620 nm. Nicardipine and pyrene were
included as control compounds. The solubility of nicardipine is pH dependent whereas the
solubility of pyrene is pH independent.
Data Analysis
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The solubility was estimated from the concentration of test compound that produced an increase in
absorbance above vehicle control (i.e., 1 % DMSO in the buffer).
Cacoꢀ2 Permeability (Biꢀdirectional)
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Cacoꢀ2 cells obtained from the ATCC were used between passage numbers 40 ꢀ 60. Cells were
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seeded onto Millipore Multiscreen Transwell plates at 1 x 10 cells/cm . The cells were cultured in
DMEM and media was changed every two or three days. On day 20 the permeability study was
performed. Cell culture and assay incubations were carried out at 37 ºC in an atmosphere of 5 %
CO with a relative humidity of 95 %. On the day of the assay, the monolayers were prepared by
2
rinsing both basolateral and apical surfaces twice with Hanks Balanced Salt Solution (HBSS) at
the desired pH warmed to 37 °C. Cells were then incubated with HBSS at the desired pH in both
apical and basolateral compartments for 40 min to stabilize physiological parameters.
The dosing solutions were prepared by diluting test compound with assay buffer to give a
final test compound concentration of 10 µM (final DMSO concentration of 1 % v/v). The
fluorescent integrity marker lucifer yellow was also included in the dosing solution. Analytical
standards were prepared from test compound DMSO dilutions and transferred to buffer,
maintaining a 1 % v/v DMSO concentration.
Assay buffer was composed of supplemented HBSS pH 7.4.
For assessment of AꢀB permeability, HBSS was removed from the apical compartment and
replaced with test compound dosing solution. The apical compartment insert was then placed into
a companion plate containing a fresh buffer (containing 1 % v/v DMSO). For assessment of BꢀA
permeability, HBSS was removed from the companion plate and replaced with test compound
dosing solution. Fresh buffer (containing 1 % v/v DMSO) was added to the apical compartment
insert, which is then placed into the companion plate.
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At 120 min the apical compartment inserts and the companion plates were separated and
apical and basolateral samples diluted for analysis. Test compound permeability was assessed in
duplicate. Compounds of known permeability characteristics were run as controls on each assay
plate.
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Test and control compounds were quantified by LCꢀMS/MS cassette analysis using an
8ꢀpoint calibration with an appropriate dilution of the samples. Cyprotex generic analytical
conditions were used. The starting concentration (C ) was determined from the dosing solution
0
and the experimental recovery calculated from C and both apical and basolateral compartment
0
concentrations.
The integrity of the monolayer throughout the experiment was checked by monitoring lucifer
yellow permeation using fluorimetric analysis. Lucifer yellow permeation is high if monolayers
have been damaged.
Data Analysis
The permeability coefficient (P ) for each compound was calculated from the following equation:
app
dQ/dt
Pꢀꢁꢁ =ꢂꢃ
ꢅ
Cꢄꢂ × A
Where dQ/dt is the rate of permeation of the drug across the cells, C is the donor compartment
0
concentration at time zero and A is the area of the cell monolayer. C was obtained from analysis
0
of the dosing solution.
For biꢀdirectional experiments, an efflux ratio (ER) was calculated from mean AꢀB and BꢀA data.
This is derived from:
Pꢀꢁꢁ(ꢆꢇꢈ)
ER =ꢂ
Pꢀꢁꢁ(ꢈꢇꢆ)
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