Inhibition of HIV fusion with multivalent gold nanoparticles

Article abstract of DOI:10.1021/ja710321g

The design and synthesis of a multivalent gold nanoparticle therapeutic is presented. SDC-1721, a fragment of the potent HIV inhibitor TAK-779, was synthesized and conjugated to 2.0 nm diameter gold nanoparticles. Free SDC-1721 had no inhibitory effect on HIV infection; however, the (SDC-1721)-gold nanoparticle conjugates displayed activity comparable to that of TAK-779. This result suggests that multivalent presentation of small molecules on gold nanoparticle surfaces can convert inactive drugs into potent therapeutics. Copyright

Full text of DOI:10.1021/ja710321g

Published on Web 05/13/2008
Inhibition of HIV Fusion with Multivalent Gold Nanoparticles
Mary-Catherine Bowman,† T. Eric Ballard,§ Christopher J. Ackerson, Daniel L. Feldheim,*,
David M. Margolis,*,†,‡ and Christian Melander*,§
Departments of Medicine, Microbiology & Immunology, and Epidemiology, UniVersity of North Carolina at Chapel
Hill, Chapel Hill, North Carolina 27599-7435, Department of Chemistry, North Carolina State UniVersity, Raleigh,
North Carolina 27695, and Department of Chemistry and Biochemistry, UniVersity of Colorado at Boulder,
Boulder, Colorado 80309
Received November 23, 2007; E-mail: daniel.feldheim@colorado.edu; dmargo@med.unc.edu; christian_melander@ncsu.edu
It has been suggested that biological systems exploit multivalency
in the synthesis of high-affinity ligands because they allow an
organism to take advantage of an existing set of monovalent ligands
without the need for evolving completely new molecules for every
required function.1 Multivalent displays are ubiquitous in biology
and can confer dramatically enhanced affinity. For example, binding
between multiple hemagglutinin (HA) ligands of an influenza virus
and sialic acid (SA) surface receptors of an erythrocyte during viral
infection is estimated to occur with an affinity of 1013 M-1, while
the association constant for a single SA-HA interaction is 103
M-1 1
.
In the development of therapeutics, a multivalent binding strategy
could offer many advantages. Synthetic systems that present
multiple low-affinity biomolecule binding ligands may prove to be
more synthetically accessible and could allow one to tune binding
affinity over several orders of magnitude. Moreover, nanoparticles
that are comparable in size to proteins and present multiple protein-
binding ligands may be effective at disrupting protein/protein
interactions that drive disease pathogenesis. Multivalent linear
polymers, dendrimers, proteins, and liposomes have successfully
targeted pathogenic biomolecule targets.1,2 Of particular note are
SA-coated liposomes and dendrimers that bind to HA on the surface
of an influenza virus with affinity enhancements of 102-106.1
Particle-based displays of multiple ligands have the additional
advantage of creating a high local concentration of binding
molecules. Consequently, binding equilibrium between a surface-
bound ligand and receptor favors formation of more ligand-receptor
pairs. For instance, DNA hybridization is thermodynamically
favored by 1 order of magnitude if one of the single-stranded DNA
sequences is conjugated to a 10 nm diameter gold nanoparticle
surface.3,4
Figure 1. TAK-779 and SDC-1721.
The 2.0 nm diameter mercaptobenzoic acid coated gold nano-
particles were conjugated to SDC-1721, a derivative of TAK-779,
a known CCR5 antagonist (Figure 1). CCR5 serves as the principal
entry co-receptor for most commonly transmitted strains of HIV-1
and those that predominate in the early years of infection.6
A
significant amount of structure-activity relationship (SAR) data
has been generated with regard to TAK-779, most notably that the
quaternary ammonium salt is essential for high-affinity binding and
effective inhibition of HIV fusion. TAK-779 homologues that lack
this quaternary ammonium salt are devoid of activity.7 Unfortu-
nately, the quaternary ammonium salt imbues TAK-779 with poor
pharmacological properties, such as significant irritation at the
injection site, which led to an exhaustive search for alternate small
molecule CCR5 antagonists.7 As an alternative approach and proof-
of-concept, we synthesized SDC-1721, a TAK-779 homologue that
lacks the quaternary ammonium salt moiety, to show that it is
possible to conjugate a low-affinity, biologically inactive small
molecule to a gold nanoparticle to create biologically active
multivalent gold nanoparticle therapeutics. The ability to transform
small molecules that are not therapeutically viable into potential
therapeutics by simply conjugating them to a gold nanoparticle
scaffold could greatly accelerate the discovery of effective new drug
formulations.
SDC-1721 was synthesized (Supporting Information) using a
modification of the synthetic approach to TAK-7798 and coupled
to the 2.0 nm diameter gold nanoparticles via a simple ligand
exchange reaction. The average number of SDC-1721 molecules
per particle was determined to be 12, and the particle size was
verified by transmission electron microscopy. Examination of the
micrographs revealed no substantial differences in core size or
dispersion when compared to the 2.0 nm diameter gold nanoparticles
prior to ligand exchange.
The advantages of multivalent therapeutics are illustrated here
with the demonstration that gold nanoparticles transform a weakly
binding and biologically inactive small molecule into a multivalent
conjugate that effectively inhibits HIV-1 fusion to human T cells.
We employed 2.0 nm diameter, mercaptobenzoic acid modified gold
particles as a nanoscale platform to construct our multivalent
therapeutic. These particles have a proposed empirical formula of
[Au144(SC6H4COOH)52] and are related to the [Au102(SC6H4-
COOH)44] monolayer-protected gold nanoparticles that were re-
cently characterized by X-ray crystallography.5 Importantly, this
class of gold particles is similar to proteins and dendrimers in that
they are atomically precise and monodisperse nanoscale molecules.
† Department of Medicine, University of North Carolina at Chapel Hill.
‡ Departments of Microbiology & Immunology, and Epidemiology, University
of North Carolina at Chapel Hill.
§ North Carolina State University.
University of Colorado at Boulder.
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10.1021/ja710321g CCC: $40.75
2008 American Chemical Society

C O M M U N I C A T I O N S
nanoparticle preparation was unable to inhibit viral replication up
to 300 µM (Supporting Information). There was no difference in
cell viability between samples at these concentrations.
We further verified that inhibition of viral replication was specific
for viral entry. This was assayed by performing single-cycle
infection experiments using the TZM-bl luciferase reporter gene
assay system.12 This cell line is engineered to express high levels
of CCR5 and CD4 and contains a luciferase reporter gene under
control of the HIV-1 promoter, which is inducible in trans by the
viral protein Tat.13 TZM-bl cells were infected with JR-CSF in
the presence or absence of the test compounds. Forty-eight hour
postinfection, luciferase activity was measured as relative lumi-
nescence units (RLU). SDC-1721-modified gold nanoparticles
inhibited viral entry as well as TAK-779 (Figure 2e). At 48 h, viral
production is insignificant, thus establishing inhibition of viral
replication at the stage of viral entry.
In conclusion, we have documented the first application of small-
molecule coated gold nanoparticles as effective inhibitors of HIV
fusion. The results presented here demonstrate that therapeutically
inactive monovalent small organic molecules may be converted into
highly active drugs by simply conjugating them to gold nanoparticles.
Acknowledgment. This research was funded by NCSU (C.M.),
GlaxoSmithKline (T.E.B.), The University of Colorado (D.F.), the
NIH (AI045297 to D.M.M., EY017568 to D.F.), and the UNC
Chapel Hill CFAR (AI50410) to M.C.B. TZM-bl was obtained
through the NIH AIDS Research and Reference Reagent Program,
Division of AIDS, NIAID, NIH, from Dr. John C. Kappes, Dr.
Xiaoyun Wu, and Tranzyme Inc.
Figure 2. (A-D) Multiple-cycle inhibition assays. (A) TAK-779 HIV-1
inhibition; (B) SDC-1721 HIV-1 inhibition; (C) (SDC-1721)-NP HIV-1
inhibition; (D) GSH-NP HIV-1 inhibition; (E) single-cycle HIV-1 infectiv-
ity inhibition with TAK-779 and (SDC-1721)-NP.
Supporting Information Available: Experimental procedures and
characterization for all new compounds; TEM and cell assay procedures.
This material is available free of charge via the Internet at http://
pubs.acs.org.
To evaluate the antiviral activity of the nanoparticle conjugate,
phytohemagglutinin (PHA)-stimulated peripheral blood mono-
nuclear cells (PBMCs) were infected with the CCR5-tropic HIV-1
clone JR-CSF in the presence or absence of the test compounds.
On day 7 postinfection, supernatants were collected and HIV-1
capsid p24 antigen was measured by ELISA. As shown in Figure
2a, TAK-779 inhibited HIV-1 replication with an IC50 of 10 nM.
The IC50 for TAK-779 against four different CCR5-tropic viral
isolates ranged from 1.6 to 3.7 nM.9 However, JR-CSF was not
one of the viruses tested. With a similar virus, JR-FL, TAK-779
revealed an IC50 of 20 nM.10 Sensitivity to CCR5 entry inhibitors
is impacted by receptor expression levels and HIV-1 envelope/
receptor affinity, mediated by both cellular and viral determinants.11
Thus, JR-CSF appears to be inherently less sensitive to TAK-779.
We next tested whether, as had been reported, the quaternary
ammonium group was essential to TAK-779 activity.7 SDC-1721
did not inhibit viral replication (Figure 2b). Conjugation of SDC-
1721 ((SDC-1721)-NP) to gold nanoparticles at an average ratio
of 12:1 restored activity with an IC50 of 10 nM (Figure 2c). To
ensure that the inhibitory effect was not due to the gold particle, a
glutathione-modified gold nanoparticle (GSH-NP) control was
tested. The GSH-NP is unable to inhibit viral replication (Figure
2d). In addition, we verified that multivalent display of SDC-1721
on the nanoparticle surface was required for activity by assaying
(SDC-1721)-NP conjugates with an average ratio of 0.93/1. This
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