Alkyl amine bevirimat derivatives are potent and broadly active HIV-1 maturation inhibitors

Article abstract of DOI:10.1128/AAC.02121-15

Concomitant with the release of human immunodeficiency virus type 1 (HIV-1) particles from the infected cell, the viral protease cleaves the Gag polyprotein precursor at a number of sites to trigger virus maturation. We previously reported that a betulinic acid-derived compound, bevirimat (BVM), blocks HIV-1 maturation by disrupting a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. BVM was shown in multiple clinical trials to be safe and effective in reducing viral loads in HIV-1-infected patients. However, naturally occurring polymorphisms in the SP1 region of Gag (e.g., SP1-V7A) led to a variable response in some BVM-treated patients. The reduced susceptibility of SP1-polymorphic HIV-1 to BVM resulted in the discontinuation of its clinical development. To overcome the loss of BVM activity induced by polymorphisms in SP1, we carried out an extensive medicinal chemistry campaign to develop novel maturation inhibitors. In this study, we focused on alkyl amine derivatives modified at the C-28 position of the BVM scaffold. We identified a set of derivatives that are markedly more potent than BVM against an HIV-1 clade B clone (NL4-3) and show robust antiviral activity against a variant of NL4-3 containing the V7A polymorphism in SP1. One of the most potent of these compounds also strongly inhibited a multiclade panel of primary HIV-1 isolates. These data demonstrate that C-28 alkyl amine derivatives of BVM can, to a large extent, overcome the loss of susceptibility imposed by polymorphisms in SP1.

Full text of DOI:10.1128/AAC.02121-15

AAC Accepted Manuscript Posted Online 19 October 2015
Antimicrob. Agents Chemother. doi:10.1128/AAC.02121-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Alkyl Amine Bevirimat Derivatives are Potent and Broadly Active
HIV-1 Maturation Inhibitors
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Emiko Urano1, Sherimay D. Ablan1, Rebecca Mandt1, Gary T. Pauly2, Dina M. Sigano2,
Joel P. Schneider2, David E. Martin3, Theodore J. Nitz3, Carl T. Wild3, and Eric O.
Freed#1
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1Virus-Cell Interaction Section, HIV Dynamics and Replication Program, Center for
Cancer Research, National Cancer Institute, Frederick, MD 21702-1201
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2Chemical Synthesis Group, Chemical Biology Laboratory, Center for Cancer Research,
National Cancer Institute, Frederick, MD 21702-1201
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3DFH Pharma, Gaithersburg, MD
#Corresponding author Email: efreed@nih.gov
Running title: Potent HIV-1 maturation inhibitors
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ABSTRACT
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Concomitant with the release of human immunodeficiency virus type 1 (HIV-1) particles
from the infected cell, the viral protease cleaves the Gag polyprotein precursor at a
number of sites to trigger virus maturation. We previously reported that a betulinic acid-
derived compound, bevirimat (BVM), blocks HIV-1 maturation by disrupting a late step
in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-
SP1) intermediate to mature CA. BVM was shown in multiple clinical trials to be safe
and effective in reducing viral loads in HIV-1-infected patients. However, naturally
occurring polymorphisms in the SP1 region of Gag (e.g., SP1-V7A) led to a variable
response in some BVM-treated patients. The reduced susceptibility of SP1-polymorphic
HIV-1 to BVM resulted in the discontinuation of its clinical development. To overcome
the loss of BVM activity induced by polymorphisms in SP1, we carried out an extensive
medicinal chemistry campaign to develop “second-generation” maturation inhibitors. In
this study, we focused on alkyl amine derivatives modified at the C-28 position of the
BVM scaffold. We identified a set of derivatives that are markedly more potent than
BVM against an HIV-1 clade B clone (NL4-3) and show robust antiviral activity against
a variant of NL4-3 containing the V7A polymorphism in SP1. One of the most potent of
these compounds also strongly inhibited a multi-clade panel of primary HIV-1 isolates.
These data demonstrate that C-28 alkyl amine derivatives of BVM can, to a large extent,
overcome the loss of susceptibility imposed by polymorphisms in SP1.
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INTRODUCTION
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Human immunodeficiency virus type 1 (HIV-1), the primary causative agent of AIDS, is
currently
estimated
to
infect
>33
million
people
worldwide
(http://www.healthline.com/health/hiv-aids/facts-statistics-infographic).
A
number of
inhibitors have been developed that suppress HIV-1 replication in infected patients, and
currently more than two dozen anti-HIV-1 drugs are approved for clinical use (1). These
inhibitors, which are administered in combination (combination antiretroviral therapy,
cART), fall into several major classes. Inhibitors of the viral enzymes reverse
transcriptase (RT), protease (PR), and integrase (IN) form the backbone of current cART
regimens. Inhibitors that target fusion and entry are also available (1).
Although current cART is able to suppress viral loads to below the level of
detection in standard commercial assays in the majority of treatment-compliant
individuals, available therapies are not curative and thus require lifelong drug adherence.
Long-term treatment is associated with a variety of issues related to drug toxicity,
unfavorable drug-drug interactions, and patient non-compliance. Multi-drug resistance is
likely to limit treatment options in an increasing number of patients over time,
particularly in resource-limited settings in which viral load testing is not widely available
(2-5). Thus, it is imperative that continued efforts are made to develop novel drugs
targeting steps in the viral replication cycle not affected by current therapies. As an
added benefit, developing inhibitors against novel targets provides a wealth of basic
mechanistic information about fundamental aspects of viral replication.
Maturation of HIV-1 particles, which is triggered by the action of the viral PR,
occurs concomitant with virion release from the infected cell (6-8). PR cleaves a number
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of sites in the Gag polyprotein precursor, Pr55Gag, the major structural protein responsible
for the formation of virus particles. PR-mediated Gag cleavage gives rise to the matrix
(MA), capsid (CA), nucleocapsid (NC), and p6 proteins and two small spacer peptides,
SP1 and SP2, located between CA and NC and between NC and p6, respectively. PR
also cleaves the GagPol polyprotein precursor to generate the mature viral enzymes: PR,
RT, and IN. Cleavage of the Gag and GagPol polyproteins results in a marked change in
virion morphology. In the immature particle, the Gag precursor proteins are arranged
radially around the outer edge of the virus particle, whereas in the mature virion the CA
proteins assemble into a centrally located, conical core (referred to as the capsid) in
which the viral RNA genome and the viral enzymes RT and IN reside. Both Pr55Gag and
mature CA assemble into a largely hexameric lattice, though the unit-to-unit spacing of
the lattice and the intersubunit contacts differ between the immature and mature lattice
(9). The strain of curvature is accommodated in the immature Gag lattice by the presence
of gaps, whereas in the mature capsid the inclusion of a total of 12 pentamers in the
otherwise hexameric capsid lattice allows the capsid to close off at both ends (10-12).
Maturation is critical to particle infectivity (7).
Each processing site within the Gag and GagPol polyprotein precursor is cleaved
by PR with distinct kinetics, largely due to the unique primary amino acid sequences at
each site (13-19). The consequence of these differential rates of cleavage is that Gag and
GagPol processing occurs as a highly ordered cascade of cleavage events. This highly
ordered processing is required for proper maturation. Defects in maturation can affect
both virus entry (20, 21) and subsequent post-entry events. Even partial disruption of
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processing at several sites in Gag leads to severely impaired virus infectivity (22-24),
highlighting the utility of Gag processing as a target for antiretrovirals.
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We and others previously reported that the first-in-class HIV-1 maturation
inhibitor 3-0-(3’-3’-dimethylsuccinyl) betulinic acid (25) (bevirimat or BVM) acts by
blocking cleavage at the CA-SP1 junction (26, 27). We selected a panel of resistant
mutants by propagating HIV-1 in the presence of BVM; the resistance mutations mapped
to the CA-SP1 boundary region (28). Clinical trials showed that BVM was safe and
efficacious. However, polymorphisms present in circulating strains of HIV-1 diminished
the efficacy of BVM. These polymorphisms cluster in a Gln-Val-Thr (QVT) sequence
spanning SP1 residues 6-8. In particular, we observed that in culture, a Val-to-Ala
change at SP1 residue 7 (V7A) almost completely abrogated the ability of BVM to block
CA-SP1 processing and virus replication (29). Our analysis of the structurally distinct
maturation inhibitor PF-46396 (30) demonstrated that this compound retained activity
against a derivative of clade B NL4-3 bearing the V7A change (31). This result, together
with recent reports describing chemically modified BVM derivatives (32-34), suggested
that the insensitivity of SP1 polymorphic HIV-1 to maturation inhibitors could be
overcome.
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In this study, we synthesized a collection of novel C-28 alkyl amine derivatives of
BVM and tested their ability to block CA-SP1 processing and virus replication using WT
NL4-3 and an V7A variant of NL4-3 that is resistant to BVM. The activity of these
compounds against a multi-clade panel of HIV-1 isolates was also determined. The
results demonstrated that several of these derivatives were markedly more potent and
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more broadly active than the parental BVM, with low-nM antiviral activity against both
WT NL4-3 and the V7A derivative.
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Material and Methods
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Chemical synthesis. Scaffold 7 compounds (Table 1) were synthesized by modification
of C3 and C28 of betulin. Oxidation of C28 allowed acylation followed by deprotection
at C3 to provide the left-hand side of the molecule. Further homologation following by
oxidation and reductive animation at C28 furnished the right-hand side of the molecule
resulting in compounds 7a-7t (Table 1). For additional details, see supplemental
material.
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Cell culture, plasmids, and transfections. The MT-4 T-cell line was maintained in
RPMI-1640 medium supplemented with 10% (vol/vol) fetal bovine serum (FBS), 2 mM
glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin at 37 °C in a humidified 5%
CO2 atmosphere. HeLa and 293T cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 5% (vol/vol) FBS, 2 mM glutamine, 100 U/ml
penicillin and 100 µg/ml streptomycin. Molecular clones used in this study were WT
pNL4-3 (35) and the derivative encoding a Val-to-Ala mutation at SP1 residue 7, referred
to as V7A (29). The MT-4 T-cell line was transfected by using the DEAE dextran-
mediated procedure; HeLa and 293T cells were transfected with linear polyethylenimine
(L-PEI) or Lipofectamine 2000 (Invitrogen) (36, 37).
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CA-SP1 accumulation assay. CA-SP1 accumulation assays were performed as
described previously (28, 31, 37) with some modification. Briefly, HeLa cells were
transfected with WT pNL4-3 or the V7A derivative. At 24 h posttransfection, cells were
starved in Met/Cys-free medium for 30 min and metabolically labeled with [35S]Met/Cys-
Pro-mix (Perkin Elmer) for 2-3 h. Maturation inhibitors were maintained in the cultures
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throughout the transfection and labeling period. Viruses were collected by
ultracentrifugation at 75,000× g for 45–60 min. Virus pellets were resuspended in Triton
X-100 lysis buffer [300 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 10
mM iodoacetamide, and protease inhibitor cocktail tablets (Roche)]. CA and CA-SP1
proteins were separated gels containing between 13.5 and 15% polyacrylamide by SDS-
polyacrylamide gel electrophoresis, exposed to a phosphorimager plate (Fuji) and
quantified by Quantity One software (Bio-Rad).
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Antiviral assays. To produce HIV-1 stocks, 293T cells were transfected with WT
pNL4-3 or the V7A derivative and culture supernatants were collected 24 h post
transfection. RT-normalized virus supernatants were used to infect MT-4 cells at room
temperature for 30 min. Cells were then cultured in the presence of serial dilutions of
each compound. At 4 days post-infection, the culture supernatants were subjected to RT
assay to monitor viral replication as previously described (37). The 50% inhibitory
concentrations (IC50s) were defined as compound concentrations that reduced RT levels
to 50% of those measured in the absence of inhibitor (DMSO only controls) using
GraphPad Prism 6 software (38). The IC50s in primary peripheral blood mononuclear
cells (PBMCs) were determined with a multi-clade panel of HIV-1 isolates by Southern
Research Institute (Frederick, MD) as reported previously (38). PBMCs were purified by
ficoll-hypaque gradient centrifugation from anonymized, healthy blood donors. Cells
were stimulated for three days with phytohemagglutinin and interleukin-2 prior to
infection with HIV-1. IC50s were calculated based on reductions in RT activity on day 6
post-infection as described above.
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Cytotoxicity assays. Cytotoxicity was measured with CellTiter-Blue® Cell Viability
Assay according to the manufacturer’s protocol (Promega). Two cell lines, MT-4 and
HeLa, were used in this assay. Cells were incubated with a serial dilution of compounds
at 37 ˚C for 4 or 8 days and treated with CellTiter-Blue Reagent. The fluorescent signal
was measured at 560 nm Ex /590 nm Em by a plate reader (TECAN, Infinite® M1000
PRO). The 50% cytotoxic concentrations (CC50s) were defined as compound
concentrations at which the fluorescent signals were reduced by 50% relative to the no-
inhibitor (DMSO only) controls using GraphPad Prism 6 software. Cytotoxicity in
PBMCs was determined as described previously (38).
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RESULTS
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C-28 BVM analogs inhibit CA-SP1 processing of both WT HIV-1NL4-3 and the SP1-
V7A derivative. Previous results from this laboratory indicated that compounds
containing scaffold 7 possessed some activity against V7A (data not shown). To identify
BVM analogs with increased potency and breadth of activity, we synthesized a panel of
related C-28 homologated amines (Materials and Methods; Supplemental Information;
Table 1). We then tested the ability of these derivatives to inhibit CA-SP1 processing of
a clade B viral clone, NL4-3, and the BVM-resistant V7A derivative (29). HeLa cells
transfected with pNL4-3 or the V7A derivative were left untreated or were treated with
100 nM of each compound 7a-7j and were metabolically radiolabeled with [35S]Met/Cys.
Released virus particles were concentrated by ultracentrifugation and lysates were
subjected to SDS-PAGE and fluorography (see Materials and Methods). This approach
evaluates the ability of a maturation inhibitor to block CA-SP1 processing in a
quantitative manner by measuring the percentage of CA-SP1 accumulation. As indicated
in Fig. 1A, treatment with BVM or with analogs 7a-7j led to a marked accumulation
(~50-80%) of CA-SP1 for WT NL4-3. In contrast, and consistent with our previous
report (29), for samples transfected with the V7A mutant BVM treatment resulted in only
~ 10% accumulation of CA-SP1, which is comparable to the DMSO control (Fig. 1B).
For the remaining compounds in this set, levels of CA-SP1 accumulation ranged from
~15-55%, with compounds 7h and 7j, which incorporate an additional amino grouping
into the C-28 side chain, showing the greatest potency in disrupting CA-SP1 processing.
Based on these results, we synthesized a new set of compounds (7k-7t) and evaluated
their potency in the CA-SP1 accumulation assay. Compared to BVM, compounds 7k-7t
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were considerably more effective against WT NL4-3 (Fig. 1C) and showed a high degree
of potency against the V7A mutant (Fig. 1D) with V7A CA-SP1 accumulation in the
60% range for many of these compounds (Fig. 1D).
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To refine our analysis of CA-SP1 accumulation, we focused on compounds with
the lowest IC50 values against V7A, namely 7m, 7n, 7r, and 7s, and performed a dose-
response analysis. Virus-producer cells were treated with increasing concentrations of
compound from 0.8 to 100 nM. WT NL4-3 showed a dose-dependent accumulation of
CA-SP1 when cells were treated with BVM while SP1-V7A was resistant to all
concentrations used (Fig. 2A and B). In contrast, compounds 7m, 7n, 7r, and 7s showed
a strong dose-responsive accumulation of CA-SP1 for both WT NL4-3 (Fig. 2A) and the
V7A mutant (Fig. 2B).
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C-28 BVM analogs display low-nanomolar antiviral activity against WT NL4-3 and
the SP1-V7A derivative. Our previous studies established a close correlation between
the inhibition of CA-SP1 processing imposed by BVM and the compound’s antiviral
activity (28, 29, 31). To determine whether this correlation extends to the BVM analogs
being studied here, we used a virus replication assay based on spreading HIV-1 infection
in the MT-4 T-cell line to measure the IC50 of these compounds. HIV-1-infected MT-4
cells were cultured for four days in the presence of a serial dilution of each compound.
Antiviral activity was monitored by reductions in RT activity in culture supernatants. As
shown in Table 2, the IC50s of the first series of compounds against WT NL4-3 were
mainly in the 10-20 nM range. Against V7A, compound 7h and 7j had IC50s of 76 and 39
nM, respectively. The significantly improved antiviral activity of 7h and 7j against V7A
parallels the increased ability of these compounds to inhibit CA-SP1 processing relative
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to BVM (Fig. 1B). Most of the second series of compounds (7k-7t) displayed single-
digit nM IC50 values against WT and predominantly sub-20 nM IC50 values against V7A.
Compound 7m, 7n, 7r, and 7s showed low-nM activity against SP1-V7A (~9, 14, 10, and
8 nM, respectively). As seen with 7s and 7t, additional substitution on the second amine
grouping is tolerated and will allow for continued exploration of the structure-activity
relationship for this series of analogs. We also evaluated the cytotoxicity of these BVM
analogs by using the CellTiter-Blue cell viability assay in HeLa and MT-4 cells (see
Materials and Methods). As shown in Table 3, although many of the highly potent
compounds displayed increased cytotoxicity in HeLa and MT-4 cells relative to BVM,
the CC50 values for all the BVM analogs were considerably higher than the
concentrations required for maximal (plateau) antiviral activity.
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C-28 BVM analogs are broadly active against multiple clades of HIV-1.
Polymorphisms in SP1 (e.g., V7A) reduce susceptibility of HIV-1 to BVM both in in
vitro assays and in vivo in HIV-1-infected patients. To assess the breadth of antiviral
activity displayed by our novel panel of BVM analogs, we extended our analyses to a
panel of primary HIV-1 isolates that includes representative viruses from HIV-1 subtypes
A, B, C, D, E, F, and G. This panel of isolates was selected in part because of their
highly diverse SP1 sequences (Fig. 3). Compound 7r was selected for this analysis
because of its high level of potency against V7A. Antiviral activity was measured in
PBMCs by quantifying the reduction in RT activity in the culture supernatant six days
post-infection. BVM displayed low-nM IC50s against only some of these isolates (Table
4). In contrast, compound 7r was active across all of the primary isolates except for the
subtype A isolate 92UG031. This virus contains both CA-V230I and SP1-V7A
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polymorphisms (Fig. 3). Our preliminary resistance data suggest that the combination of
the V230I and V7A polymorphisms confers resistance to BVM analogs (data not shown).
However, the frequency of both of these polymorphisms occurring together is very low,
fewer than 1% of more than 30,000 viral sequences compiled in the Los Alamos HIV-1
sequence database (http://www.hiv.lanl.gov/content/index). Cytotoxicity was limited in
PBMCs, with the CC50 for BVM >10,000 nM and no toxicity observed for compound 7r
up to 1000 nM (data not shown). These data clearly demonstrate that, relative to parental
BVM, the C-28 alkyl amine derivatives of BVM display a marked improvement in
potency and breadth of activity.
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DISCUSSION
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In this study, we conducted an extensive medicinal chemistry campaign to
develop second-generation maturation inhibitors that not only demonstrate increased
potency against WT HIV-1, but also are active against SP1 polymorphic HIV-1 isolates
in the low-nM concentration range. The lack of antiviral activity against HIV-1 isolates
with polymorphisms near the CA-SP1 cleavage site, particularly in SP1 residues 6-8, was
a major reason for the discontinuation of BVM clinical development. Herein, we
synthesized a series of BVM analogs with C-28 modifications, with the top candidates
potently inhibiting the processing of CA-SP1 and viral replication of a representative
polymorphic virus, V7A. These compounds were also active against a multi-clade panel
of HIV-1 isolates in PBMCs. While several of the most potent compounds exhibited
increased cytotoxicity in laboratory cell lines relative to BVM, cytotoxicity was not
observed up to 1000 nM in PBMC.
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Recently, several other groups reported the synthesis and antiviral activity of
BVM analogs. Most of these analogs have C-28 modifications and demonstrated slightly
improved antiviral activity compared to BVM against prototypic clade B HIV-1 (34, 39,
40). Interestingly, some of these BVM analogs reportedly inhibited both viral maturation
and viral entry (32, 41). In our study, the antiviral activity correlated well with CA-SP1
accumulation, and infection with VSV-G-pseudotyped virus was potently inhibited by the
BVM derivatives in single-cycle assays. In addition, the infectivity of virions bearing the
HIV-1 Env glycoprotein was not affected by the inhibitors when they were present at the
time of infection (data not shown). Thus, the compounds synthesized here act as
maturation inhibitors, with no evidence of an entry-based inhibitory activity. In addition
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to C-28 modifications, it was reported that position C-3 can be derivatized without losing
antiviral activity (39). More recently, Dang et al. identified a C-28 BVM derivative that
exhibited 20-fold increased activity against virus bearing the V7A polymorphism (32). A
series of macrocyclized betulinic acid derivatives was also reported; however, these
compounds were not able to overcome the resistance conferred by the V7A
polymorphism (40). The most potent compounds in our study demonstrated IC50 values
against the V7A polymorph that were >50-fold lower than that of parental BVM. Thus,
these compounds significantly contribute to the available information concerning the
structure-activity relationship of HIV-1 maturation inhibitors.
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The mechanism by which C-28 BVM derivatives potently and broadly inhibit
HIV-1 replication relative to the parental compound remains to be determined. The
modifications may allow the C-28 side chain of the compound to interact more tightly
with the putative Gag substrate. Although the binding site for BVM and its analogs has
not been structurally characterized, several lines of evidence support the hypothesis that
contacts are made between these compounds and two regions of Gag: the CA-SP1
boundary region and the major homology region (MHR) of CA. An early study
demonstrated that BVM is specifically incorporated into immature but not mature
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particles (42). Association of H-labeled BVM with immature particles was reduced by
resistance-conferring mutations at the CA-SP1 cleavage site (43). Nguyen et al.
demonstrated that a photoaffinity BVM analog interacted with residues around the CA-
SP1 cleavage site and the CA MHR (44). In a previous study (31), we demonstrated that
resistance to a structurally distinct maturation inhibitor, PF-46396, mapped not only to
the vicinity of the CA-SP1 cleavage site (where resistance mutations against BVM map)
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but also to the MHR. The MHR mutants were strongly compound-dependent, and could
revert in culture by acquiring a mutation at residue 8 of SP1 (SP1-T8I). The ability of an
SP1 mutation to rescue the replication defect conferred by an MHR substitution
suggested functional cross-talk between the MHR and SP1. It is interesting to note that
residue 8 of SP1 falls within the so-called ‘QVT’ motif (SP1 residues 6-8) previously
shown to influence BVM activity (29). Poor solubility of BVM and its analogs has
hampered structural characterization of BVM binding, although a BVM derivative with
improved solubility has been reported and shown by NMR to bind a CA-SP1-NC peptide
(45). Adding to the challenging nature of defining the maturation inhibitor-binding site is
the fact that the structure of the CA-SP1 boundary region of Gag is poorly defined.
Although recent progress has been made in delineating the structure of the CA domain in
the immature Gag lattice, the high level of flexibility of SP1 has thus far precluded an
atomic-resolution structure of SP1 in the immature particle (9). Binding of maturation
inhibitors to Gag may stabilize SP1 (46, 47) thus potentially facilitating structural studies
of this region. Mutations in SP1 may also limit the conformation flexibility of this
peptide, providing another useful tool for structural studies.
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The second-generation HIV-1 maturation inhibitors developed in this study
provide insights into the structure-activity relationship of this class of compounds.
Ongoing studies are aimed at further optimizing the potency and breadth of activity of
these inhibitors.
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ACKNOWLEDGEMENTS
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We thank members of the Freed laboratory for helpful discussion and critical review of
the manuscript and Wei Shau (National Cancer Institute) for HIV-1 sequence analyses.
We gratefully acknowledge Southern Research Institute, Frederick, MD for the
multiclade analysis shown in Table 4 and Ioannis Kagiampakis (National Cancer
Institute) for assistance with graphpad statistical analyses. Work in the Freed laboratory
is supported by the Intramural Research Program of the Center for Cancer Research,
National Cancer Institute, NIH, and by the Intramural AIDS Targeted Antiviral Program.
This research was supported by Public Health Service grant 1R41AI108457-1 to C.T.W.
and contract N01-AI-70041. This project was conducted in part by Southern Research
Institute using federal funds from the Division of AIDS, National Institute of Allergy and
Infectious Diseases, National Institutes of Health under contract HHSN272200700041C
entitled “Confirmatory In Vitro Evaluations of HIV Therapeutics”.
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21

480
FIGURE LEGENDS
481
482
483
484
485
486
487
488
489
Fig. 1. C-28 alkyl amine BVM derivatives potently disrupt CA-SP1 processing. HeLa
cells were transfected with WT pNL4-3 (A and C) or the pNL4-3 derivative encoding the
V7A polymorph (B and D). Cells were treated with 100 nM compounds 7a-7j (A and
B) or 7k-7t (C and D) and were metabolically labeled with [35S]Met/Cys in the presence
of the compounds. Released virions were collected by ultracentrifugation and virion-
associated CA and CA-SP1 were analyzed by SDS-PAGE and quantified by
phosphorimager analysis. A representative gel image is shown on the top and
quantification of the %CA-SP1 relative to total CA + CA-SP1 is presented in the graphs.
Error bars indicate standard deviations from three independent experiments.
490
491
492
493
494
495
496
497
498
Fig. 2. Dose-dependent inhibition of CA-SP1 processing by the top compounds.
HeLa cells were transfected with WT pNL4-3 (A) or the pNL4-3 derivative encoding the
V7A polymorph (B). Cells were treated with a range of concentrations (0.8, 4, 20, and
100 nM) of compounds 7m, n, r, or s and were metabolically labeled with [35S]Met/Cys
in the presence of the compounds. Released virions were collected by ultracentrifugation
and virion-associated CA and CA-SP1 were analyzed by SDS-PAGE and quantified by
phosphorimager analysis. Quantification of the %CA-SP1 relative to total CA + CA-SP1
is presented in the graphs. Error bars indicate standard deviations from at least three
independent experiments.
499
500
Fig. 3. Amino acid sequences of the CA-SP1 boundary region from the multi-clade panel
of HIV-1 isolates tested in Table 4. Asterisks indicate amino acid sequence identity with
22

501
502
WT NL4-3 (top line); gaps are indicated by hyphens. The QVT motif spanning SP1
residues 6-8 is highlighted in bold.
503
504
23

Table 1. Analogs Prepared and Characterized for This Study
Scaffold 7
Compound
Molecular
Formula
Molecular
Weight
R1
H
R2
#
7a
-CH2CH2OH
-CH2CH2OH
C40H67NO5
C42H71NO6
C42H69NO5
C41H69NO5
C44H75NO6
C42H71NO5
C43H72N2O4
C45H76N2O4
C41H67NO4
641.96
686.02
668.02
656.00
714.07
670.02
681.04
709.09
637.98
7b
7c
7d
7e
7f
-CH2CH2OH
-CH2CH2OCH2CH2-
H
-CH2CH2OCH3
-CH2CH2OCH3
-C(CH3)2CH2OH
-CH2CH2OCH3
H
7g
7h
7i
-CH2CH2N(CH3)CH2CH2-
H
H
-cyclopropyl
7j
7k
7l
H
C44H74N2O4
C40H67NO4
C42H71NO4
C42H72N2O4
C43H74N2O4
C44H76N2O4
695.07
625.96
654.02
669.03
683.06
697.09
H
-CH2CH3
-CH2CH3
-CH2CH3
7m
7n
7o
H
H
H
-CH2CH2N(CH3)2
-CH2CH2CH2N(CH3)2
-CH2CH2CH2CH2N(CH3)2

7p
7q
7r
H
H
H
C44H74N2O4
C46H78N2O4
C45H76N2O4
695.07
723.12
709.10
7s
7t
H
H
C45H76N2O5
C46H78N2O5
725.10
739.12

Table 2. Antiviral activity of C-28 alkyl amine derivatives against
WT and V7A in MT-4 cellsα
IC50 (nM)
Compound
WT
136 ± 30
33 ± 26
19 ± 6
98 ± 44
16 ± 7
49 ± 32
9 ± 4
V7A
>500
BVM
7a
7b
7c
7d
7e
7f
>500
>500
>500
>500
>500
>500
7g
7h
7i
32 ± 16
27 ± 3
22 ± 14
16 ± 0.4
3 ± 1
>500
76 ± 15
>500
7j
39 ± 10
145 ± 29
201 ± 44
9 ± 2
7k
7l
14 ± 7
3 ± 0.3
7 ± 3
7m
7n
7o
7p
7q
7r
14 ± 4
22 ± 5
26 ± 6.4
22 ± 7
10 ± 3
8 ± 2
9 ± 3
3 ± 0.4
5 ± 1
5 ± 3
7s
21 ± 19
2 ± 0.5
7t
19 ± 6
αThe IC50 values represent the mean ± SEM from four independent experiments.

Table 3. Cytotoxicity of C-28 alkyl amine derivatives in MT-4 and HeLa
cellsα
CC50 (nM)
Compound
MT-4
>1000
HeLa
>1000
BVM
7a
7b
7c
7d
7e
7f
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
7g
7h
7i
>1000
>1000
911 ± 35
>1000
>1000
>1000
7j
>1000
>1000
7k
7l
>1000
>1000
>1000
927 ± 232
>1000
7m
7n
7o
7p
7q
7r
425 ± 25
210 ± 17
201 ± 21
377 ± 54
381 ± 48
195 ± 13
366 ± 44
406 ± 48
381 ± 101
305 ± 13
614 ± 33
291 ± 40
877 ± 287
>1000
7s
7t
551 ± 83
αThe CC50 values represent the mean ± SEM from three independent experiments.