Oligovalent amyloid-binding agents reduce SEVI-mediated enhancement of HIV-1 infection

Article abstract of DOI:10.1021/ja210931b

This paper evaluates the use of oligovalent amyloid-binding molecules as potential agents that can reduce the enhancement of human immunodeficiency virus-1 (HIV-1) infection in cells by semen-derived enhancer of virus infection (SEVI) fibrils. These natur

Full text of DOI:10.1021/ja210931b

Communication
pubs.acs.org/JACS
Oligovalent Amyloid-Binding Agents Reduce SEVI-Mediated
Enhancement of HIV-1 Infection
Christina C. Capule,† Caitlin Brown,‡ Joanna S. Olsen,‡ Stephen Dewhurst,‡,* and Jerry Yang*,†
†Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0358, United States
‡Department of Microbiology and Immunology, University of Rochester, Rochester, New York 14642, United States
S
* Supporting Information
concentration corresponding to 50% inhibition (IC50) of 13
μM.5 We hypothesized that BTA-EG6 forms a protein-resistive
ABSTRACT: This paper evaluates the use of oligovalent
coating on SEVI,5 effectively blocking the interaction of these
amyloid-binding molecules as potential agents that can
reduce the enhancement of human immunodeficiency
virus-1 (HIV-1) infection in cells by semen-derived
enhancer of virus infection (SEVI) fibrils. These naturally
occurring amyloid fibrils found in semen have been
implicated as mediators that can facilitate the attachment
and internalization of HIV-1 virions to immune cells.
Molecules that are capable of reducing the role of SEVI in
HIV-1 infection may, therefore, represent a novel strategy
to reduce the rate of sexual transmission of HIV-1 in
humans. Here, we evaluated a set of synthetic, oligovalent
derivatives of benzothiazole aniline (BTA, a known
amyloid-binding molecule) for their capability to bind
cooperatively to aggregated amyloid peptides and to
neutralize the effects of SEVI in HIV-1 infection. We
demonstrate that these BTA derivatives exhibit a general
trend of increased binding to aggregated amyloids as a
function of increasing valence number of the oligomer.
Importantly, we find that oligomers of BTA show
improved capability to reduce SEVI-mediated infection
of HIV-1 in cells compared to a BTA monomer, with the
pentamer exhibiting a 65-fold improvement in efficacy
compared to a previously reported monomeric BTA
derivative. These results, thus, support the use of
amyloid-targeting molecules as potential supplements for
microbicides to curb the spread of HIV-1 through sexual
contact.
fibrils with HIV-1 virions and cells. A key characteristic of this
approach to reducing HIV transmission compared to more
traditional microbicide candidates is that we target a naturally
abundant mediator (i.e., SEVI) in the cellular attachment of
HIV, rather than targeting the virions themselves. Although
BTA-EG6 was not toxic to cervical cells and did not exhibit any
pro-inflammatory activity at a concentration that was 10 times
the IC50 for efficacy,5 strategies to create more potent
compounds would be desirable for further development of
SEVI-neutralizing agents as supplements to current microbicide
candidates.6−8
Here, we explore whether oligomers of BTA exhibit
improved capability to reduce SEVI-enhanced infection of
HIV in cells compared to a BTA monomer. Multivalent
bindingthat is, the multiple, simultaneous binding of two or
more tethered ligands and receptorsis ubiquitous in nature9
and has been explored as a strategy to increase the affinity of
small molecules to multivalent targets.10−24 Amyloid fibrils
formed from the self-assembly of peptides putatively display
multiple, identical, and periodically spaced binding sites for
small molecules along the fibrillar surface25−29 and, thus,
represent an excellent biological target for multivalent ligand
design (Figure 1).
Lockhart et al.25 and Wu et al.29 had previously reported that
BTA derivatives associate with amyloid fibrils derived from
Alzheimer’s-related amyloid-β (Aβ) peptides, with data
suggesting a spacing between adjacent binding sites of ∼2
nm. Since little information is available regarding the
interaction of small molecules with SEVI, we designed and
synthesized BTA oligomers 2−5 (Figure 1) based on what was
known about the binding of BTA to aggregated Aβ(1−42)
peptides. We designed the BTA monomer 1 to carry a
tetra(ethylene glycol) group terminated with a carboxyl moiety,
which we subsequently used to generate oligovalent BTA
derivatives 2−5 by reaction with commercial oligo-amine
spacers using standard amidation chemistry (see Supporting
Information for details). Although oligo(ethylene glycols) are
quite flexible (which theoretically diminishes the potential gain
in conformational entropy for oligovalent binding9,30−33), we
incorporated them into the design of compounds 2−5 because
of their known minimal interaction with proteins34,35 and for
recent study reported that an abundant peptide found in
A
semen, PAP248−286, forms aggregated amyloid species
(called semen-derived enhancer of virus infection or SEVI) that
can potentially increase infection of human immunodeficiency
virus-1 (HIV-1) in cells by up to 400 000-fold.1 Although the
molecular mechanism of SEVI-mediated transmission of HIV-1
remains poorly understood, evidence suggests that SEVI binds
to both HIV-1 virions and cell membranes, and facilitates viral
infection.1−4 Methods to suppress the effects of such natural
enhancers of HIV-1 transmission may, therefore, significantly
reduce the global spread of HIV through sexual contact among
high-risk populations.
We recently reported that targeting SEVI fibrils with the
amyloid-binding molecule BTA-EG6, a hexa(ethylene glycol)
derivative of benzothiazole aniline (BTA), reduced SEVI- and
semen-mediated enhancement of HIV infection in T cells at a
Received: November 21, 2011
Published: December 27, 2011
© 2011 American Chemical Society
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Journal of the American Chemical Society
Communication
units in 2 to the fibrillar surface. Surprisingly, BTA trimer 3 and
tetramer 4 bound only with similar Kd values to Aβ(1−42)
fibrils compared to dimer 2. One possible explanation for this
result is that the structures of 3 and 4 make it preferable for
these molecules to bind divalently to the amyloid surface.
Alternatively, it may also be possible that 3 and 4 bind to
amyloid fibrils with a valency greater than 2 but incur a
significant loss in expected binding energy due to partial
docking of the BTA units to their respective binding sites. For
BTA pentamer 5, the measured apparent Kd value was an
additional 10-fold lower than that of tetramer 4 and was 117-
fold lower than the Kd value for monomer 1. If we assume that
all five BTA moieties in 5 participate in binding to the Aβ
fibrils, we can approximate a Kd value per BTA moiety of 10
nM. Although the effects of multivalent binding to Aβ fibrils are
modest for compounds 2−5, there appears to be a general
trend of improved binding from monomer to pentamer (most
notably from monomer to dimer and from tetramer to
pentamer) within this series of compounds.
When we examined the apparent Kd values of compounds
1−5 to SEVI fibrils, we found a similar trend for increased
binding of the oligomeric BTA compounds as we observed for
their binding to Aβ(1−42) fibrils (Table 1). We, again,
observed the greatest improvement in binding as a function of
increasing valence number in the oligomer when we compared
the monomer to dimer and the tetramer to pentamer. We
found that BTA pentamer 5 had a Kd value of 0.4 nM for
binding to aggregated SEVI peptides (or 2 nM per BTA moiety
if we assume that all five BTA moieties in 5 participate in
binding to the SEVI fibrils), which was 590-fold lower than the
Kd value of monomer 1.
Figure 1. Structures of monovalent and oligovalent amyloid-binding
molecules. (A) Cartoon depicting the monovalent (left) or oligovalent
(right) binding of molecules to amyloid fibrils. (B) Chemical
structures of monovalent (1) and oligovalent (2−5) derivatives of
benzothiazole aniline (BTA). A rudimentary estimate of the length (in
fully extended conformation) of the flexible group attached to BTA
was calculated using ChemBio3D Ultra 12.0 software.
their water solubilizing properties.36,37 We estimated that the
flexible spacer on each BTA monomer could span a length of
∼2.5 nm when modeled in a fully extended conformation
(Figure 1), suggesting that the BTA units on oligomers 2−5
could easily span the expected ∼2 nm distance between binding
sites on Aβ fibrils.38 Additionally, dimers of Thioflavin T (ThT,
a BTA-based histological amyloid staining agent), where the
ThT moieties were linked by 2−5 ethylene glycol units, have
recently been reported to associate with Aβ fibrils with
increased affinity compared to ThT alone.39 These studies
suggest that BTA oligomers 2−5 could also bind oligovalently
to amyloid targets.
In order to investigate whether the improved binding of BTA
oligomers 2−5 to SEVI fibrils compared to monomer 1 would
translate into improved efficacy for blocking SEVI-mediated
HIV-1 infection (Figure 2A), we evaluated compounds 1−5 for
their capability to inhibit SEVI-enhanced infection of HIV-1IIIB
in TZM-bl cells. TZM-bl cells are a HeLa-derived cell line that
express high levels of the CD4 receptor as well as CCR5 and
CXCR4 coreceptors and contain the HIV-1 long terminal
repeat (LTR)-driven luciferase cassette.42,43 Since HIV-1 LTR
is a weak transcriptional regulator in the absence of its cognate
activator protein, Tat, the expression levels of luciferase in these
cells are directly proportional to the extent of HIV-1 infection.
In these HIV-1 infectivity experiments, we chose concen-
trations of compounds 1−5 that maintained a 10 or 1 μM
concentration of the BTA moiety in all samples of monomer
and oligomers (e.g., since there are 2 BTA moieties in dimer 2,
we used a 5 μM concentration of dimer compared to a 10 μM
concentration of monomer to afford the same total
concentration of BTA). We expected that this experimental
design would highlight any multivalent enhancement of efficacy
from the oligomers compared to the monomer. Figure 2B
shows that all of the oligomers were more effective at reducing
SEVI-mediated enhancement of HIV-1 infection compared to
monomer 1. As a control, compounds 1−5, at all
concentrations tested, did not have any significant effect on
HIV infection in these cells in the absence of SEVI (see Figure
S3 in the Supporting Information). We found that 5 μM dimer,
3.3 μM trimer, 2.5 μM tetramer, or 2 μM pentamer all reduced
SEVI-mediated HIV-1 infectivity almost completely to the level
of infection observed in the absence of SEVI (Figure 2B). This
level of activity from all oligomers 2−5 is superior to the
activity of monomer 1 and to the activity reported previously
Table 1 lists the apparent Kd values for compounds 1−5 to
fibrils formed from Aβ(1−42) peptides, as estimated using a
Table 1. Table of Apparent Kd Values Obtained for
Compounds 1−5 for Binding to Fibrils Formed from
a
Synthetic Aβ(1-42) or SEVI Fibrils
Kd(nM)
to Aβ(1−42) fibrils
Kd (nM)
to SEVI fibrils
Compound
1
2
3
4
5
235 75
236 90
29
26
20
4
6
5
69
40
59
1
6
6
2.0 0.4
0.4 0.2
a
These values were estimated using a previously reported fluorescence
binding assay.26
known fluorescence binding assay.5,26,40,41 As expected, BTA
dimer 2 bound more strongly than monomer 1 to Aβ(1−42)
fibrils, albeit with only a modest 8-fold lower Kd value. The
flexibility of the oligo(ethylene glycol) groups presumably
attenuates the degree of cooperative binding of the two BTA
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Journal of the American Chemical Society
Communication
to use polymeric9,16,45 and more rigid water-soluble46 and
biocompatible scaffolds47−49 to generate multivalent amyloid-
targeting agents with improved efficacy for reducing SEVI-
mediated infection of HIV are currently underway.
ASSOCIATED CONTENT
* Supporting Information
■
S
Details for the synthesis and characterization of compounds 1−
5, experimental protocol for the fluorescence-based binding and
HIV infection assay. Complete ref 1. This information is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
jerryyang@ucsd.edu; Stephen_Dewhurst@URMC.Rochester.
edu
■
ACKNOWLEDGMENTS
■
This work was supported, in part, by NIH Grants
R21AI094511, T32GM007356 (to J.S.O.), T32DA007232 (to
J.S.O.), a California HIV/AIDS Research Program grant (ID10-
SD-034), and the University of Rochester Developmental
Center for AIDS Research (NIH P30AI078498). We would
also like to acknowledge the NIH for support of the Mass
Spectrometry facility at UCSD (1S10RR025636-01A1)
Figure 2. Inhibition of SEVI-mediated enhancement of HIV-1
infection by compounds 1−5. (A) Schematic illustration showing
the proposed coating of SEVI fibrils with amyloid-binding oligomers.
These coatings prevent the direct interaction of HIV-1 with SEVI
fibrils and, thus, prevent SEVI-mediated enhancement of viral infection
in cells. (B) Graph showing the reduction of SEVI-mediated
enhancement of HIV-1IIIB infection in TZM-bl cells in the presence
of compounds 1−5 (10 μM total BTA moiety), as estimated by
cellular luciferase expression levels. (C) Graph showing the reduction
of SEVI-mediated enhancement of HIV-1IIIB infection in TZM-bl cells
in the presence of 1−5 (1 μM total BTA moiety), as estimated by
cellular luciferase expression levels. RLUs = relative luciferase units. All
data are presented as normalized values relative to the luciferase
expression in cells that were exposed to HIV-1IIIB+SEVI. A p-value of
<0.05 was considered statistically significantly different compared to
cells treated with HIV-1IIIB+SEVI as determined by one-way ANOVA
with Tukey’s post test.
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