An antibody-recruiting small molecule that targets HIV gp120

Article abstract of DOI:10.1021/ja9057647

(Chemical Equation Presented) HIV/AIDS is a global pandemic for which new treatment strategies are desperately needed. We have designed a novel small molecule, designated as ARM-H, that has the potential to interfere with HIV survival through two mechanis

Full text of DOI:10.1021/ja9057647

Published on Web 10/19/2009
An Antibody-Recruiting Small Molecule That Targets HIV gp120
Christopher G. Parker,† Robert A. Domaoal,‡ Karen S. Anderson,‡ and David A. Spiegel*,†,‡
Department of Chemistry, Yale UniVersity, 225 Prospect Street, P.O. Box 208107, New HaVen, Connecticut
06520-8107, and Department of Pharmacology, Yale UniVersity School of Medicine, 333 Cedar Street, SHM B350B,
New HaVen, Connecticut 06520
Received July 12, 2009; E-mail: david.spiegel@yale.edu
In recent years, antibody-based therapeutics have become
important instruments in treating human disease.1 These approaches
suffer from certain limitations, including severe side effects, lack
of oral bioavailability, and high cost.2 Thus, alternative small-
molecule-based methods that exploit the powerful cytolytic potential
of antibodies already present in the human bloodstream could
address many of these disadvantages.
Here we report that a rationally designed bifunctional small
molecule,3 called “antibody-recruiting molecule targeting HIV”
(ARM-H), is capable of redirecting anti-dinitrophenyl (anti-DNP)
antibodies, a population of antibodies present in high concentra-
tions in the human bloodstream,4 to the HIV gp120 enV gene
product (Figure 1). The Env glycoprotein, a complex between
gp120 and membrane-bound gp41, is expressed on both the
surface of the HIV virus and on virus-infected cells.5 The gp120
component of Env mediates the first step in viral entry into
human cells by binding the protein CD4. We demonstrate here
Figure 1. (A) Schematic depiction of the reported approach to HIV
that a ternary complex formed between anti-DNP antibodies,
ARM-H, and Env-expressing cells can mediate the complement-
dependent destruction of these cells. Further, since ARM-H binds
gp120 competitively with CD4, it also inhibits the entry of live
HIV virus into human T-cells. Thus, ARM-H has the potential
to interfere with the survival of HIV through multiple comple-
mentary mechanisms.
targeting. The synthetic bifunctional small molecule ARM-H is designed
to function through two autonomous mechanisms: (1) by binding the viral
glycoprotein gp120, inhibiting its interaction with CD4, and (2) by recruiting
antibodies to the HIV virus, and to HIV-infected cells, for destruction by
the human immune system. (B) Chemical structures of small molecules
employed in these studies.
Our design of ARM-H began with the small molecule BMS-
378806 (1, Figure 1b), a known inhibitor of the CD4-gp120
interaction.6 On the basis of previously reported structure-activity
relationships in which the carbon atom of the C4 methoxy group
could be replaced with various bulky substituents,7 we hypoth-
esized that it might be possible to derivatize 1 at this site with
a linker attached to DNP without sacrificing the compound’s
ability to inhibit viral entry. This hypothesis was supported by
an analysis of a published computational docking model sug-
gesting that the C4 methoxy group in 1 points toward the solvent
in the complex.8 Thus, we reasoned that BMS-378806 could be
re-engineered to recruit anti-DNP antibodies to gp120-expressing
particles (infected cells or viruses), increasing their “visibility”
to the human immune system. On the basis of this strategy, we
prepared ARM-H (4) in high yield (38% overall) via azide-alkyne
cycloaddition9,10 of 2 and 3, which were derived in turn from
known intermediates (Schemes S2 and S3 in the Supporting
Information).6
Figure 2. ARM-H outcompetes the gp120-CD4 interaction in vitro and
with a live HIV-1 virus assay. (A) Competition ELISA monitoring the
binding of sCD4 to immobilized gp120. (B) HIV-1 viral replication assay.
UV absorption at 595 nm, increased by the metabolic action of live MT-2
cells on an assay reagent,13 was monitored as a surrogate for cell viability
in the presence of increasing concentrations of ARM-H alone (white circles)
or ARM-H plus live HIV-1 virus (black circles).
compound BMS-378806 (IC50 ) 1.3 µM). On the basis of this
observation, we then investigated ARM-H’s ability to inhibit the
entry of live HIV-1 virus into the human MT-2 T-cell line (Figure
2b, IC50 ) 6.4 µM). Although in this assay ARM-H once again
proved to be less potent than BMS-378806 (IC50 ) 0.32 µM; Figure
S1),12 it demonstrated potency equivalent to that of d4T, which is
currently a mainstay in HIV pharmacotherapy (IC50 ) 4.2 µM;
Figure S1). Notably, ARM-H demonstrated no observable cyto-
toxicity in control MT-2 cultures lacking HIV virus (Figure 2b,
white circles).
The ability of ARM-H to inhibit CD4 binding to HIV-1 gp120
was assessed first in an enzyme-linked immunosorbent assay
(ELISA) (Figure 2a).11 Here ARM-H was able to out-compete the
CD4-gp120 interaction with a mean inhibitory concentration (IC50)
of ∼8.7 µM, and it was only slightly less potent than the parent
† Yale University.
‡ Yale University School of Medicine.
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C O M M U N I C A T I O N S
Figure 4. ARM-H-mediated killing of live HIV-Env-expressing CHO cells.
Env-expressing CHO cells (+gp120) or control cells lacking Env (-gp120)
were treated with ARM-H (4) or control compound (3) at the indicated
concentrations in the presence (+Ab) or absence (-Ab) of 10% comple-
ment-preserved rabbit serum and rat anti-DNP IgG (50 µg/mL). Values on
the vertical axis correspond to % cell death (vs background). Each data
point depicted represents the mean ( standard deviation of triplicate data
from a representative experiment. The depicted trends were reproduced on
at least three separate occasions.20
Figure 3. Immunofluorescence microscopy. Chinese hamster ovary cells
expressing HIV-1 Envelope (Env) glycoprotein (+gp120) and control cells
lacking Env (-gp120) were stained with Alexa Fluor 488-conjugated anti-
DNP antibodies (15 µg/mL). Microscopy was then performed after treatment
with the following reagents, as indicated: ARM-H (10 µM), sCD4 (4 µg/
mL), BMS-378806 (BMS, 500 µM), or DNP-containing alkyne 2 (50 µM).
Fluorescence signals indicate ARM-H-mediated recruitment of antibodies
to gp120-expressing cells.
observed, suggesting that termolecular complex formation is
necessary for complement-dependent cytotoxicity (CDC) and that
ARM-H itself is not toxic to cells. Positive control experiments
in which cells were directly labeled with 2,4-dinitrobenzene-
sulfonic acid (Figure S4) were found to yield levels of CDC
comparable to those observed for ARM-H, providing a bench-
mark for the assay results depicted in Figure 4. Thus, ARM-H
is capable of recruiting a functional complement-dependent
cytotoxic response against Env-expressing cells.
We next investigated the ability of ARM-H to recruit antibodies
to gp120 both in vitro and in tissue culture. To this end, initial
ELISA experiments (Figure S2) demonstrated a concentration-
dependent increase in anti-DNP antibody binding to the ARM-
H-gp120 complex but not to gp120 alone. Thus, ARM-H is capable
of templating a ternary complex that also includes gp120 and anti-
DNP antibody.
To demonstrate that this ternary association could form in a
complex cellular milieu, we evaluated the ability of ARM-H to
recruit Alexa Fluor 488-labeled anti-DNP antibodies to HIV-
Env-expressing Chinese hamster ovary cells (CHO-gp120 cells)14
by immunofluorescence microscopy. As depicted in Figure 3, a
strong fluorescence signal was observed when CHO-gp120 cells
were incubated with ARM-H and labeled anti-DNP antibodies
(Figure 3a,b).15 This fluorescence was absent from both CHO-
gp120 cells not treated with ARM-H (Figure 3c,d) and CHO
cells not coding for HIV-enV gene expression (CHO-WT cells,
Figure 3e,f). Furthermore, the intense fluorescence observed in
Figure 3b was considerably diminished in the presence of the
competing ligands soluble CD4 (sCD4, Figure 3g,h), BMS-
378806 (Figure 3i,j), and a DNP-containing alkyne that lacks
the gp120-binding motif (2, Figure 3k,l). Taken together, these
results provide strong evidence that ARM-H is capable of
recruiting anti-DNP antibodies to cells expressing the Env
glycoprotein in a fashion that depends upon its simultaneous
binding to both gp120 and anti-DNP antibodies.
Thus, we have shown that the bifunctional small molecule
ARM-H can both recruit anti-DNP antibodies to gp120-expressing
cells and inhibit the gp120-CD4 interaction. Data supporting these
conclusions include the following: (1) ARM-H binds to gp120
competitively with CD4 and decreases viral infectivity in an MT-2
cell assay. (2) The small molecule can guide the formation of a
ternary complex that includes anti-DNP antibodies and Env-
expressing cells. (3) Antibodies present in this ternary complex can
promote the complement-mediated killing of Env-expressing cells.
Critically, no nonspecific cytotoxicity was observed in either MT-2
or CHO cell lines in response to ARM-H. Also, ARM-H-mediated
inhibition of HIV entry and CDC activity were both operative at
concentrations ranging from 6 to 30 µM, confirming that ARM-H
could function simultaneously through dual mechanisms under
therapeutic conditions.
While a few methods for recruiting naturally occurring
antibodies to cancer cells,21-27 bacteria,28-31 and viruses,32-34
have appeared in the literature, this area remains underexplored.
In the HIV realm, all such approaches have relied upon protein-
or peptide-based antibody targeting constructs. For example,
Shokat and Schultz32 first demonstrated that anti-DNP antibodies
could be redirected to immobilized protein targets (gp120 and
streptavidin) as a therapeutic strategy toward HIV. More recent
work in this vein has employed peptide-R-Gal conjugates to
target human anti-Gal antibodies to HIV-infected cells.33,34 These
peptide conjugates were shown to be effective in killing Env-
expressing cells but were also found to exhibit some nonspecific
cytotoxicity.28 ARM-H is unique in that it represents a small-
molecule-based anti-HIV strategy and targets the virus lifecycle
through mutually reinforcing molecular mechanisms: it both
prevents virus entry and targets Env-expressing cells for immune
recognition and clearance. In general, small molecules have
certain advantages over proteins from a therapeutic standpoint
Finally, we explored whether the ternary complex formed from
anti-DNP antibody, ARM-H, and a live Env-expressing cell could
activate complement proteins and mediate cellular death.
Complement proteins are known to lyse cells by forming pores
in lipid membranes and have been shown to play a critical role
in inactivating HIV in humans.16,17 Thus, rabbit complement
proteins were added to CHO-gp120 cells in the presence of
ARM-H and a fixed concentration anti-DNP antibodies (Figure
4). Substantial cell killing that exhibited a significant dependence
on the ARM-H concentration (data in red) was observed.18,19
Notably, in the absence of anti-DNP antibody and complement-
preserved serum (data in green), in cells lacking the Env
glycoprotein (CHO-WT, data in black), or in the presence of 3,
which lacks the DNP group (data in blue), no cell death was
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C O M M U N I C A T I O N S
(7) Wang, J. S.; Le, N.; Heredia, A.; Song, H. J.; Redfield, R.; Wang, L. X.
Org. Biomol. Chem. 2005, 3, 1781–1786.
because of their low propensity for immunogenicity, high
metabolic stability, ready large-scale production, and relatively
low cost. Small-molecule antibody-recruiting therapeutics such
as ARM-H would have additional benefits over available
treatment approaches to HIV. For example, directing HIV-
infected cells and virus particles to Fcγ receptors on antigen-
presenting cells could enhance the presentation of viral antigens
on MHC proteins and contribute to long-lasting anti-HIV
immunity.26,35 Furthermore, because anti-DNP antibodies are
already present in the human bloodstream, no pre-vaccination
would be necessary for ARM-H activity. Also, the binding of
bifunctional small-molecule targeting agents to antibodies should
prolong their plasma half-life, thus increasing their effective-
ness.27 Elucidation of the molecular details governing the
interactions among ARM-H, gp120, and anti-DNP antibodies
will assist in optimization efforts as well as in the evaluation of
this strategy in more complex biological models for HIV
infection. These and other investigations are currently ongoing
in our laboratories.
(8) Kong, R.; Tan, J.; Ma, X.; Chen, W.; Wang, C. Biochim. Biophys. Acta
2006, 1764, 766–772.
(9) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596–2599.
(10) Tornøe, C.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057–
3064.
(11) Ho, H.; et al. J. Virol. 2006, 80, 4017–4025.
(12) The increase in potency of BMS-378806 in MT-2 cells versus ELISAs
may be the result of a cooperative enhancement in binding to viral envelope
gp120, which exists as a trimer (ELISA studies were performed using
monomeric gp120). The steric bulk of ARM-H due to the C4 tether may
impede binding of more than one ARM-H molecule per gp120 trimer.
(13) More details regarding assay conditions and troubleshooting can be found
in the Supporting Information.
(14) Weiss, C.; White, J. J. Virol. 1993, 67, 7060–7066.
(15) Because it was necessary to permeabilize the cells prior to labeling,
intracellular gp120 can also be observed in these micrographs.
(16) Aasa-Chapman, M. M. I.; Holuigue, S.; Aubin, K.; Wong, M.; Jones, N. A.;
Cornforth, D.; Pellegrino, P.; Newton, P.; Williams, I.; Borrow, P.;
Mcknight, A. J. Virol. 2005, 79, 2823–2830.
(17) Gerencer, M.; Burek, V.; Crowe, B. A.; Barrett, N. P.; Dorner, F. Microb.
Pathog. 1998, 25, 253–266.
(18) The modest levels of complement-dependent cytotoxicity observed for
ARM-H in Figure 4 are similar to values reported in other systems (see
ref 34) and may result from the low levels of Env expression on the CHO-
gp120 cells (see ref 14). Also, because of assay incompatibilities, CDC
data corresponding to ARM-H concentrations greater than 30 µM could
not be obtained. More details can be found in the Supporting Information.
(19) Characteristic autoinhibition of ternary complex formation at high levels
of bifunctional molecule, arising from excess free bifunctional material
that drives the equilibria toward formation of binary complexes, was
not reliably observed in these assays (see the Supporting Information
for more details). For more information on autoinhibitory behavior in
ternary complexes, see: Mack, E. T.; Perez-Castillejos, R.; Suo, Z.;
Whitesides, G. M. Anal. Chem. 2008, 80, 5550–5555, and references
contained therein.
Acknowledgment. The authors thank Jacob Appelbaum and
Phillip Lichtor for helpful suggestions, Caitlin Sengelaub for
technical assistance, and Professor Guido Ferrari (Duke Uni-
versity) for helpful discussions. This work was funded by the
National Institutes of Health through the NIH Director’s New
Innovator Award Program (Grant DP22OD002913 to D.A.S.).
K.S.A. acknowledges NIH Grant GM49551 for funding support.
The following reagents were obtained through the AIDS
Research and Reference Reagent Program, Division of AIDS,
NIAID, NIH: CHO-WT (here called CHO-gp120) from Dr. Carol
Weiss and Dr. Judith White and MT-2 cells, catalog no. 237,
from Douglas Richman.
(20) More details regarding assay conditions and troubleshooting can be found
in the Supporting Information.
(21) Carlson, C.; Mowery, P.; Owen, R.; Dykhuizen, E. C.; Kiessling, L. ACS
Chem. Biol. 2007, 2, 119–127.
(22) Owen, R.; Carlson, C.; Xu, J.; Mowery, P.; Fasella, E.; Kiessling, L.
ChemBioChem 2007, 8, 68–82.
(23) Popkov, M.; Gonzalez, B.; Sinha, S.; Barbas, C. Proc. Natl. Acad. Sci.
U.S.A. 2009, 106, 4378–4383.
(24) Popkov, M.; Rader, C.; Gonzalez, B.; Sinha, S.; Barbas, C. Int. J. Cancer
2006, 119, 1194–1207.
Supporting Information Available: Detailed experimental pro-
cedures, compound characterization, and complete author lists for
refs 6 and 11. This material is available free of charge via the Internet
at http://pubs.acs.org.
(25) Low, P.; Henne, W.; Doorneweerd, D. Acc. Chem. Res. 2008, 41, 120–
129.
(26) Lu, Y.; You, F.; Vlahov, I.; Westrick, E.; Fan, M.; Low, P. S.; Leamon,
C. P. Mol. Pharm. 2007, 4, 695–706.
(27) Rader, C.; Sinha, S. C.; Popkov, M.; Lerner, R. A.; Barbas, C. F. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 5396–5400.
(28) Bertozzi, C. R.; Bednarski, M. D. J. Am. Chem. Soc. 1992, 114, 5543–
5546.
References
(29) Bertozzi, C. R.; Bednarski, M. D. J. Am. Chem. Soc. 1992, 114, 2242–
2245.
(1) Brekke, O. H.; Sandlie, I. Nat. ReV. Drug DiscoVery 2003, 2, 52–62.
(2) Allen, T. M. Nat. ReV. Cancer 2002, 2, 750–763.
(30) Li, J.; Zacharek, S.; Chen, X.; Wang, J. Q.; Zhang, W.; Janczuk, A.; Wang,
P. G. Bioorg. Med. Chem. 1999, 7, 1549–1558.
(3) Corson, T. W.; Aberle, N.; Crews, C. M. ACS Chem. Biol. 2008, 3, 677–
(31) Krishnamurthy, V. M.; Quinton, L. J.; Estroff, L. A.; Metallo, S. J.; Isaacs,
J. M.; Mizgerd, J. P.; Whitesides, G. M. Biomaterials 2006, 27, 3663–
3674.
692.
(4) Antibodies recognizing the DNP epitope have been estimated to constitute
1% of circulating IgM and 0.8% of circulating IgG. See: (a) Karjalainen,
K.; Makela, O. Eur. J. Immunol. 1976, 6, 88–93. (b) Farah, F. S.
Immunology 1973, 25, 217–226. The prevalence of anti-DNP antibodies
has been estimated at between 18 and 90% of humans. See: (c) Ortega,
E.; Kostovetzky, M.; Larralde, C. Mol. Immunol. 1984, 21, 883–888. (d)
Jormalainen, S.; Makela, O. Eur. J. Immunol. 1971, 1, 471–478.
(5) Miranda, L. R.; Schaefer, B. C.; Kupfer, A.; Hu, Z. X.; Franzusoff, A.
Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 8031–8036.
(32) Shokat, K. M.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 1861–1862.
(33) Naicker, K. P.; Li, H.; Heredia, A.; Song, H.; Wang, L. Org. Biomol. Chem.
2004, 2, 660–664.
(34) Perdomo, M. F.; Levi, M.; Ilberg, M. S.; Vahlne, A. Proc. Natl. Acad. Sci.
U.S.A. 2008, 105, 6.
(35) Rawool, D. B.; Bitsaktsis, C.; Li, Y.; Gosselin, D. R.; Lin, Y.; Kurkure,
N. V.; Metzger, D. W.; Gosselin, E. J. J. Immunol. 2008, 180, 5548–5557.
(6) Wang, T.; et al. J. Med. Chem. 2003, 46, 4236–4239.
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