Small-Molecule CD4 Mimics Interact
with a Highly Conserved Pocket on HIV-1 gp120
Navid Madani,1,2Arne Scho ¨n,3,8Amy M. Princiotto,1,8Judith M. LaLonde,4Joel R. Courter,5Takahiro Soeta,5
Danny Ng,5Liping Wang,1Evan T. Brower,3Shi-Hua Xiang,1Young Do Kwon,6Chih-chin Huang,6Richard Wyatt,6
Peter D. Kwong,6Ernesto Freire,3Amos B. Smith III,5and Joseph Sodroski1,2,7,*
1Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, JFB 824, Boston, MA 02115, USA
2Department of Pathology, Division of AIDS, Harvard Medical School, Boston, MA 02115, USA
3Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
4Chemistry Department, Bryn Mawr College, Bryn Mawr, PA 19010, USA
5Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA
6Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
7Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA
8These authors contributed equally to this work.
Human immunodeficiency virus (HIV-1) interaction
with the primary receptor, CD4, induces conforma-
tional changes in the viral envelope glycoproteins
that allow binding to the CCR5 second receptor
and virus entry into the host cell. The small molecule
NBD-556 mimics CD4 by binding the gp120 exterior
envelope glycoprotein, moderately inhibiting virus
CCR5 binding and virus entry into CCR5-expressing
cells lacking CD4. Studies of NBD-556 analogs and
gp120 mutants suggest that (1) NBD-556 binds
within the Phe 43 cavity, a highly conserved, func-
tionally important pocket formed as gp120 assumes
the CD4-bound conformation; (2) the NBD-556 phe-
nyl ring projects into the Phe 43 cavity; (3) enhance-
ment of CD4-independent infection by NBD-556
requires the induction of conformational changes in
gp120; and (4) increased affinity of NBD-556 analogs
for gp120 improves antiviral potency during infection
of CD4-expressing cells.
Prevention of human immunodeficiency virus (HIV-1) transmis-
sion requires approaches that interrupt the early phase of retro-
virus infection, before provirus formation. One early event, HIV-1
entry into target cells, involves the viral envelope glycoproteins
(gp120 and gp41) and host cell receptors (CD4 and either the
CCR5 or CXCR4 chemokine receptor; Wyatt and Sodroski,
1998). Binding to CD4 induces conformational changes in the
exterior envelope glycoprotein gp120 that allow CCR5/CXCR4
engagement and that expose elements of the gp41 transmem-
brane glycoprotein (Furuta et al., 1998; Trkola et al., 1996; Wu
et al., 1996). Further conformational changes in gp41 lead to
fusion of the viral and host cell membranes, allowing virus entry.
Eachofthesestepsrepresents apotential target forintervention.
To evade host-antibody responses, HIV-1 has evolved enve-
lope glycoproteins with surface variability, dense glycosylation,
and conformational flexibility (Kwong et al., 2002; Wyatt and
Sodroski, 1998). The unliganded HIV-1 gp120 glycoprotein is
unusually flexible and partially unstructured; binding CD4 locks
gp120 into a rigid conformation (Myszka et al., 2000). This bind-
ing event is characterized by an unusually large and favorable
enthalpy change that is partially countered by a large unfavor-
able entropy change. High-resolution structures of CD4-bound
HIV-1 gp120 have provided insights into these conformational
transitions (Kwong et al., 1998). The conserved gp120 core con-
with a heavily glycosylated surface, and a bridging sheet that
connects these two domains. On the unliganded HIV-1 gp120,
the inner and outer gp120 domains are thought to move with
respect to each other, with the bridging sheet assuming a
conformation different from that seen in the CD4-bound state
(Chen et al., 2005; Kwong et al., 1998).
CD4 primarily contacts the gp120 outer domain and bridging
sheet (Kwong et al., 1998). CD4 binding creates a 153- A˚3cavity
(the Phe 43 cavity) at the interface between gp120 and CD4;
the Phe 43 cavity is bounded by highly conserved residues
from all three gp120 domains and by a single CD4 residue, Phe
43 (Kwong et al., 1998). The contacts made by phenylalanine
43 and arginine 59 of CD4 with gp120 residues in the vestibule
(Arthos et al., 1990; Ashkenazi et al., 1990; Brodsky et al., 1990;
Fontenot et al., 2007; Kwong et al., 1998; Landau et al., 1988;
Olshevsky et al., 1990). Thus, the Phe 43 cavity has been sug-
gested as a desirable target for compounds that could disrupt
gp120-CD4 interactions (Kwong et al., 1998).
NBD-556 is a 337-dalton compound identified in a screen
for inhibitors of the gp120-CD4 interaction (Zhao et al., 2005).
Remarkably, NBD-556 can also act as a CD4 agonist, inducing
thermodynamic changes in gp120 similar to those observed
upon CD4 binding (Scho ¨n et al., 2006). NBD-556 promotes
CCR5 binding and can enhance HIV-1 entry into CD4-negative
(CD4?) target cells expressing CCR5 (Scho ¨n et al., 2006). Here,
we use gp120 mutants, molecular modeling, and NBD-556 ana-
Structure 16, 1689–1701, November 12, 2008 ª2008 Elsevier Ltd All rights reserved 1689
Phe 43 cavity. The relationship between the ability of NBD-556
analogs to induce changes in gp120 entropy and to stimulate
entry-related conformational changes was examined. The
potency of inhibition of infection of CD4-expressing target cells
was related to the overall affinity of the NBD-556 analogs for
gp120. These studies provide a foundation for understanding
the interaction of small molecules with the CD4-binding site of
NBD-556 Can Replace CD4 in HIV-1 Infection
To examine the ability of NBD-556 to replace CD4 during HIV-1
infection, recombinant HIV-1 expressing firefly luciferase was
pseudotyped with different envelope glycoproteins and incu-
bated with CD4?CCR5+Cf2Th-CCR5 cells in the presence of
different concentrations of NBD-556. The envelope glycopro-
teins were derived from CCR5-using (R5) primary HIV-1 isolates
(YU2 and ADA), dual-tropic (R5X4) primary isolates (89.6 and
KB9), and, as a control, the amphotropic murine leukemia virus
(A-MLV). NBD-556 enhanced infection of the Cf2Th-CCR5 cells
in the following order of efficiency: YU2, ADA > KB9 > 89.6,
A-MLV (Figure 1A). This order corresponds to the affinity of the
gp120 glycoproteins from these HIV-1 isolates for CCR5 (Bab-
cock et al., 2001; Karlsson et al., 1998; Staudinger et al., 2003;
Wu et al., 1996) and suggests that a high coreceptor-binding
affinity is required to achieve CD4-independent infection in this
context. Consistent with this interpretation, NBD-556 did not
into CD4?CXCR4+Cf2Th-CXCR4 cells (data not shown); the
HIV-1 gp120 affinity for CXCR4 is significantly lower than that
for CCR5 (Babcock et al., 2001). Likewise, the envelope glyco-
proteins of the laboratory-adapted CXCR4-using (X4) HIV-1
isolate, HXBc2, bound NBD-556 weakly (see below) and were
not functionally enhanced by NBD-556 for entry into Cf2Th-
CXCR4 cells (data not shown). However, NBD-556 dramatically
enhanced infection of Cf2Th-CCR5 cells by viruses containing
a chimeric HIV-1 envelope glycoprotein [HX(YU2 V3)] in which
the HXBc2 gp120 third variable (V3) loop was replaced by that
protein exhibits a high affinity for the CCR5 coreceptor (Choe
et al., 2003; Sullivan et al., 1998; Xiang et al., 2005). Thus, NBD-
556 can replace CD4 during infection of CCR5+cells by viruses
with a variety of HIV-1 envelope glycoproteins, provided that
the envelope glycoproteins exhibit sufficient affinity for CCR5.
NBD-556 Interacts with the Conserved Core
of the HIV-1 gp120 Glycoprotein
The direct binding of3H-labeled NBD-556 to gp120 glycoprotein
variants was measured (Figure 2A). NBD-556 bound YU2 gp120
efficiently, but HXBc2 gp120 only moderately. The chimeric
HXBc2 gp120 with the substitution of the YU2 V3 loop bound
NBD-556 with the highest efficiency. All three gp120 glycopro-
teins bound the control3H-labeled BMS-806 similarly. These
with theseenvelope glycoproteinstoenhancement byNBD-556.
The gp120 core protein lacks the V1, V2, and V3 hypervariable
loops and the N and C termini of gp120 (Kwong et al., 1998). The
absence and presence of the 17b anti-gp120 antibody; the 17b
antibody preferentially recognizes the CD4-bound conformation
of gp120 and blocks chemokine receptor binding (Thali et al.,
1991; Trkola et al., 1996; Wu et al., 1996). Weak binding of
NBD-556 to the YU2 gp120 core was detected in the absence
of the 17b antibody; NBD-556 binding to both full-length gp120
and the gp120 core was significantly enhanced by the addition
Figure 1. NBD-556 Enhancement of Infection of CD4-Negative Cells
by Viruses with Envelope Glycoproteins from Different HIV-1 Strains
(A) The effect of incubating recombinant, luciferase-expressing HIV-1 bearing
the envelope glycoproteins of the indicated HIV-1 strains with increasing
concentrations of NBD-556 on infection of CD4-negative Cf2Th-CCR5 cells
is shown. Relative virus infectivity represents the amount of infection detected
in the presence of the indicated concentration of compound divided by the
infection detected in the absence of compound. A recombinant HIV-1 with
the A-MLV envelope glycoproteins is included as a control. The values shown
are the mean ± SEM from a single experiment (n = 3). The experiment was
performed three times, with comparable results.
(B) The effect of incubating recombinant HIV-1 bearing the YU2, HXBc2 or
HX(YU2 V3) envelope glycoproteins with increasing concentrations of NBD-
556 on infection of Cf2Th-CCR5 cells is shown. The values shown are the
mean ± SEM from a single experiment (n = 3). The experiment was performed
four times, with similar results.
Small-Molecule CD4 Mimics of HIV-1 Entry
1690 Structure 16, 1689–1701, November 12, 2008 ª2008 Elsevier Ltd All rights reserved
of the 17b antibody (Figure 2B). The thermodynamic cycle of
mined by isothermal titration calorimetry (Figure 2C). NBD-556
bound weakly to the gp120 core (calculated Kd= 40 mM) but
exhibited an approximately 25-fold increase in affinity for the
the 17b antibody preferentially recognized the NBD-556-bound
form of the gp120 core. BIAcore analysis confirmed that NBD-
556 could efficiently recognize the gp120 core-17b complex
Conserved Core of HIV-1 gp120
(A) The binding of [3H]-NBD-556 to the indicated
HIV-1 gp120 envelope glycoprotein variants or to
bovine serum albumin (BSA) is shown (left panel).
The right panel shows binding of the same
gp120 envelope glycoproteins to [3H]-BMS-806
as a control. The values shown are the means ±
SEM from one experiment (n = 3).
(B) The binding of [3H]-NBD-556 to the HIV-1YU2
wild-type (WT) gp120 glycoprotein or to the
gp120 core protein in the absence or presence
of the 17b antibody (1 mg/ml) is shown. Binding
of [3H]-NBD-556 to the BSA control protein is
shown for comparison. The values shown are the
means ± SEM from a single experiment (n = 3).
The experiment was performed three times with
(C) The thermodynamic cycle for the binding of
NBD-556 and the 17b antibody to the HIV-1YU2
gp120 core was studied by titrating the gp120
core with 17b in the presence of a saturating con-
centration of NBD-556. The values associated
with the direct binding of NBD-556 to the gp120
core were calculated by completing the thermo-
dynamic cycle. The structures of the gp120 core
and the 17b Fab fragment (PDB code 1GC1) are
depicted in blue and red, respectively. NBD-556
is represented by the green triangle.
2. Bindingof NBD-556to the
NBD-556 preferentially recognizes the
gp120. Moreover, NBD-556 binds the
relatively conserved portion of gp120
retained in the core molecule.
Modeling the NBD-556-gp120
Because NBD-556 preferentially recog-
nizes the CD4-bound conformation of
the HIV-1 gp120 core, we used the avail-
able X-ray crystal structures of gp120-
CD4 complexes (Kwong et al., 1998) to
model the binding of NBD-556 to HIV-1
gp120 in the CD4-bound state. Models
Gold (Jones et al., 1997), and Accelrys
(Kuntz et al., 1994; Luty et al., 1995;
similar binding modes, with the chloro-
phenyl ring of NBD-556 projecting into the Phe 43 cavity of
the phenyl ring of Phe 43 of CD4. Predicted aromatic-aromatic
stacking interactions between the NBD-556 chlorophenyl ring
and Trp 427, Phe 382 and Trp 112 likely stabilize the NBD-566-
Met 475 are within 4 A˚of the NBD-556 chlorophenyl group situ-
Small-Molecule CD4 Mimics of HIV-1 Entry
Structure 16, 1689–1701, November 12, 2008 ª2008 Elsevier Ltd All rights reserved 1691
Figure 3. Interaction of NBD-556, CD4M33 and CD4 with the HIV-1 gp120 Core
In the upper row, the molecular surface of the HIV-1 gp120 core (Kwong et al., 1998) is shown, from the perspective of two-domain CD4, which is depicted as
a yellow ribbon in the right panels. In the second row, the gp120 core has been rotated 90?around the vertical axis. In the middle column, the CD4-mimetic mini-
protein CD4M33 (cyan) is docked onto the gp120 core, based on X-ray crystal structures of the CD4M33:gp120core:17b Fab complex (Huang et al., 2005). In the
left column,thebound NBD-556 hasbeen modeled by the Glide program (Friesner et al.,2004; Halgren etal., 2004).In the third row, aclose-upview of the gp120
region surrounding the Phe 43 cavity is shown. The gp120 structure is shown as a gray ribbon. In the right panel, Phe 43 of CD4 is shown (yellow bonds). The
biphenyl moiety of CD4M33 (cyan bonds) is shown in the middle panel. In the left panel, NBD-556 is shown, with bonds colored according to the atom type
(green = chlorine; orange = carbon; blue = nitrogen; red = oxygen). In the fourth row, the molecular surfaces of NBD-556 (orange), CD4M33 (cyan), and CD4
(yellow) are shown, illustrating the extent to which these ligands fill the Phe 43 cavity (the gp120 surface is shown in gray). In the bottom figure, CD4 Phe 43
(yellow), the CD4M33 biphenyl group (cyan), and the modeled NBD-556 (multicolored) are positioned together in the Phe 43 cavity.
Small-Molecule CD4 Mimics of HIV-1 Entry
1692 Structure 16, 1689–1701, November 12, 2008 ª2008 Elsevier Ltd All rights reserved
gens and gp120 backbone carbonyls in the neck of the cavity.
Scyllatoxin or charybdotoxin scaffolds have been used to
create CD4-mimetic miniproteins (Vita et al., 1999; Zhang
et al., 1999). X-ray crystal structures have shown the similar
ways in which Phe 23 of some scyllatoxin-based miniproteins
and Phe 43 of CD4 contact gp120 (Huang et al., 2005). In one
scyllatoxin derivative, CD4M33, a biphenyl group at residue 23
reaches into the Phe 43 cavity, increasing the affinity for gp120
(Huang et al., 2005; Martin et al., 2003). The predicted position
of the chlorophenyl ring in the bound NBD-556 models is similar
to that of the distal phenyl ring of CD4M33 (Figure 3). However,
the NBD-556 chlorophenyl ring projects slightly deeper into the
Phe 43 cavity than the CD4M33 biphenyl group.
NBD-556 Phenyl Ring/Oxalamide Linker Modification
Themodels predict thatchangesin theNBD-556 phenylringand
oxalamide linker will affect gp120 binding and/or functional
mimicryof CD4.To test this,NBD-556analogs were synthesized
and tested for the ability to bind gp120 and to enhance CCR5
binding and infection of CCR5+cells (Figure 4). NBD-557, which
has a bromo group at the para position of the phenyl ring, bound
gp120 and activated CCR5 binding and entry comparably to
NBD-556. Para substitution of the phenyl ring chloro and bromo
groups with either larger or smaller groups resulted in decreased
enhancement of CCR5 binding and/or HIV-1 entry. The affinity of
gp120binding wassensitive tomodifications attheparaposition
of the phenyl ring; for example, the binding of JRC-I-300, having
only a hydrogen atom in the para position, was too weak to be
determined by isothermal titration calorimetry.
To explore additional options for modifying the interactions of
NBD-556 with the Phe 43 cavity of gp120, different groups were
substituted at the ortho and meta positions of the NBD-556
phenyl ring. NBD-556 was chosen over NBD-557 for these
studies because of better solubility, which resulted in improved
reproducibility in the biological assays. The size and nature of
the group at the meta position significantly affected the affinity
JRC-II-191, with a fluoro group at the meta position, exhibited
the highest affinity for gp120 and the most potent stimulation
of HIV-1 infection of CCR5+cells. Larger groups at the meta
position resulted in decreases in both gp120 binding and viral
enhancement (Figure 5 and data not shown). JRC-II-191 specif-
ically inhibited HIV-1YU2infection of cells expressing CD4 and
Figure 4. Structure-Activity Relationships of NBD-556 Analogs with Different Para-Phenyl Substituents
The values for Kd, DG, DH, and ?TDS associated with the binding of each compound to the WT HIV-1YU2gp120 glycoprotein were determined by isothermal
titration calorimetry (see Methods). The Kdvalues (in mM) are color coded as follows: red = no binding or >8; yellow = 5–8; green = <5. CCR5 binding of
radiolabeled HIV-1YU2gp120 was determined after incubation with 10 mM compound (see Methods). The induction of CCR5 binding by each compound was
normalized to that observed for NBD-556. The relative induction of CCR5 binding is color-coded as follows: red = <0.25; yellow = 0.25–0.7; green = >0.7. To
study compound enhancement of infection of CD4?CCR5+cells, recombinant, luciferase-expressing HIV-1 with the wild-type HIV-1YU2envelope glycoproteins
was incubated with increasing concentrations of the compound and then added to Cf2Th-CCR5 cells. Cells were lysed 48 hr later and luciferase activity
measured (see Methods). The area under the dose-response curve for each compound was calculated and normalized to the value obtained for NBD-556.
The relative enhancement of infection of CCR5-expressing cells is color coded as follows: red = 0–0.24; yellow = 0.25–0.7; green = >0.7.
Small-Molecule CD4 Mimics of HIV-1 Entry
Structure 16, 1689–1701, November 12, 2008 ª2008 Elsevier Ltd All rights reserved 1693
CCR5 (Figure 5); this contrasts with several of the other meta-
substituted analogs, which exhibited only nonspecific inhibitory
effects on infection of viruses with HIV-1 and A-MLV envelope
glycoproteins. Ortho substitutions invariably resulted in loss of
gp120 binding and viral enhancement (data not shown). Like-
wise, replacement of the phenyl ring and/or oxalamide linker
resulted in compounds that did not detectably bind gp120
and exhibited no specific enhancement or inhibition of HIV-1
infection (data not shown). These findings are consistent with
modeling predictions in which the Phe 43 cavity constrains the
nature and size of the phenyl and oxalamide substituents that
can be tolerated in functionally active compounds.
Binding thermodynamics were analyzed for the subset of
NBD-556 analogs that exhibited detectable gp120 affinity. As
expected for compounds with only modest differences in affinity
change (DH) and the entropy change (?TDS) associated with
gp120 binding was observed (Figure 6A). The enthalpy changes
associated with the binding of all of these phenyl ring variants
tions of the compounds with the gp120protein. Moreover, differ-
ences in ?TDS values up to 11 kcal/mol were observed for the
binding of closely related compounds. The observed entropy
changes originate from conformational and solvation changes.
is negative (Scho ¨n et al., 2006) and consistent with a favorable
desolvation entropy (Luque and Freire, 1998). That the overall
entropy changes associated with compound-gp120 binding are
unfavorable indicates that desolvation does not compensate
for the unfavorable conformational entropy changes. It has
been estimated that binding of NBD-556 induces the structuring
of 67 residues of gp120 on average (Scho ¨n et al., 2006). Appar-
ently, the NBD-556 analogs structure the gp120 glycoprotein to
The relationship between thermodynamic parameters and
the enhancement of HIV-1 entry into CCR5+cells was examined
(Figure 6B). NBD-556 analogs that stimulated HIV-1 infection
exhibited both high affinities for gp120 and large unfavorable
entropy changes upon gp120 binding. Thus, high affinity for
gp120 is necessary but not sufficient for the ability of NBD-556
analogs to replace CD4 in the HIV-1 entry process. The confor-
mational fixation of gp120, reflected in the large entropic change
observed during compound-gp120 binding, apparently contrib-
utes to CD4 mimicry.
One of the para-substituted analogs (DN-3186), which bound
gp120 efficiently but minimally stimulated HIV-1 infection, in-
hibited the enhancement of HIV-1 infection by NBD-556 (data
not shown). This observation, together with the thermodynamic
data, supports a model in which these analogs bind, with varying
degrees of CD4 mimicry, to the same general region of gp120.
Effects of gp120 Changes Near the Phe 43 Cavity
To test the models further, the interaction of mutants of the
HIV-1YU2gp120 glycoprotein with NBD-556 and the analogs
was examined. Substitution of gp120 Ser 375 with a tryptophan
residue fills the Phe 43 cavity but does not disrupt CD4 binding
(Xiang et al., 2002). Compared with the wild-type (WT) HIV-1YU2
gp120, the S375W mutant bound radiolabeled NBD-556 ineffi-
ciently (Figure 7A). The binding of the S375W gp120 to Cf2Th-
CCR5 cells was induced by a soluble form of CD4 but not by
NBD-556 (Figure 7B). A control compound, BMS-806, which
ing of either WT or S375W gp120 to CCR5+cells. The infection of
viruses with the S375W mutant envelope glycoproteins was not
observed for viruses with the WT envelope glycoproteins (Fig-
ure 7C). These findings suggest that filling the Phe 43 cavity with
the indole side chain of tryptophan prevents NBD-556 binding.
Figure 5. Structure-Activity Relationships of NBD-556 Analogs with Different Meta-Phenyl Substituents
Values for Kd, thermodynamic parameters, CCR5 binding, and enhancement of virus infection of CD4?CCR5+cells were obtained as described in the Figure 4
legend. In the CCR5-binding experiments, the compounds were used at a concentration of 1 mM. The 50% inhibitory concentration (IC50) is shown for the
compound’s inhibition of infection of Cf2Th-CD4/CCR5 cells by recombinant HIV-1 with the WT HIV-1YU2envelope glycoproteins or with the control A-MLV
envelope glycoproteins. In the first column, the values for NBD-556 are shown for comparison. The values are color coded as in Figure 4.
Small-Molecule CD4 Mimics of HIV-1 Entry
1694 Structure 16, 1689–1701, November 12, 2008 ª2008 Elsevier Ltd All rights reserved
We examined the effect of changes in several gp120 residues
that line the Phe 43 cavity on the sensitivity of HIV-1 to enhance-
ment by a panel of NBD-556 analogs (Figure 8). Two analogs,
NBD-557 and JRC-II-191, that efficiently enhanced infection by
the entry of viruses with the S375W envelope glycoproteins. The
replacement of gp120 Ser 375 with glycine dramatically reduced
(Figures 7C and 8), suggesting that some element of the Ser 375
structures of the CD4-bound gp120 (1G9M; Kwong et al., 2000),
this water molecule, which was not displaced in docking NBD-
556with Gold, likely affects the shape and flexibility of the cavity.
Viruses bearing envelope glycoproteins with Ser 375 changed to
alanine exhibited greater enhancement by NBD-556 than the
viruses with WT envelope glycoproteins (Figures 7C and 8).
Moreover, several compounds (DN-3186, JRC-II-75, and JRC-
II-11) stimulated the entry of viruses with the S375A change
more efficiently than they enhanced WT virus infection (Figure 8).
These findings suggest that the hydroxyl group of Ser 375 is
detrimental to the binding and/or activity of some NBD-556
analogs that contain large para-phenyl substituents.
Alteration of gp120 Asp 368 to alanine reduced the basal level
of replication of HIV-1 in cells expressing CD4 and CCR5 (data
not shown), consistent with the importance of this residue for
CD4 binding (Kwong et al., 1998; Olshevsky et al., 1990; Wyatt
and Sodroski, 1998). The infectivity of the D368A mutant viruses
in CD4?CCR5+target cells was enhanced by incubation with
NBD-556, JRC-II-191, and JRC-II-11 (Figure 8). In the presence
of the first two compounds, the entry of viruses with the D368A
envelope glycoproteins into Cf2Th-CCR5 cells was similar to
the levels achieved by WT viruses. Viruses with the D368A enve-
lope glycoproteins were not enhanced by NBD-557, in contrast
to WT viruses (Figure 8); this suggests that NBD-556 and NBD-
557 differ in their sensitivity to changes in Asp 368 of gp120.
Changes in other gp120 residues lining the Phe 43 cavity or
creased the enhancement of virus infection byNBD-556 analogs
Phe43vestibule,it formsasaltbridgewith Lys121andthusmay
help to stabilize the CD4-bound conformation of gp120 (Kwong
et al., 1998). Replacement of Glu429 with a lysine residue, which
disrupts this salt bridge, eliminated the activation of infection for
NBD-556, NBD-557, and JRC-II-191. Substitution of an alanine
residue for Glu 429 was less disruptive of NBD-556-induced
virus activation, although the virus-enhancing activities of
NBD-557 and JRC-II-191 were significantly diminished by this
change. Thus, alteration of several gp120 residues that line the
Phe 43 pocket or reside in the vestibule leading into the pocket
can affect, in a positive or negative manner, the CD4-mimetic
effects of NBD-556 or the analogs on HIV-1 infection.
Binding of NBD-556 to HIV-1 gp120 Mutants
The binding of NBD-556 to selected HIV-1YU2gp120 mutants
was studied by isothermal titration calorimetry. Compared with
the WT gp120 glycoprotein, both the D368A and S375A gp120
mutants bound NBD-556 and JRC-II-191 with higher affinity
agree with measurements of radiolabeled NBD-556 binding to
these gp120 glycoproteins (data not shown). Several gp120
mutants (S375W, I424A, W427A, and M475A) with changes in
residues that contact the Phe 43 cavity did not detectably bind
NBD-556 in the isothermal titration calorimetric analyses
Figure 6. Thermodynamic Parameters Associated with the Binding
of NBD-556 Analogs to HIV-1 gp120
(A) The relationship between the entropy change (?TDS) and enthalpy change
(DH) associated with the binding of NBD-556 analogs to HIV-1YU2gp120 is
shown. The least-squares regression line is depicted, along with the R2value.
(B) The relationship is shown between the ability of an NBD-556 analog to
enhance HIV-1 infection of CCR5+cells and the dissociation constant (Kd)
and entropy change (-TDS) associated with compound-gp120 binding. The
symbols associated with the compounds are colored according to the ability
of the compound to promote HIV-1 infection of CD4-CCR5+cells. The color
code is the same as that used in Figures 4 and 5, from red (none/low) to green
(high level of enhancement).
Small-Molecule CD4 Mimics of HIV-1 Entry
Structure 16, 1689–1701, November 12, 2008 ª2008 Elsevier Ltd All rights reserved 1695
(Table 1). These findings support the importance of gp120 resi-
and lend credence to the docked binding mode. Figure 7D
illustrates the location of gp120 residues in which changes affect
the binding and/or virus-enhancing ability of NBD-556 analogs,
Figure 7. Interaction of NBD-556 with HIV-1 gp120 Mutants
(A) The binding of [3H]-NBD-556 to equivalent amounts of the WT or S375W HIV-1YU2gp120 glycoprotein or bovine serum albumin (BSA) is indicated. The means
from a single experiment, with the standard errors derived from triplicate samples, are shown. The experiment was performed three times with comparable
(B) The amount of radiolabeled WT or S375W mutant HIV-1YU2gp120 bound to Cf2Th-CCR5 cells is shown, after incubation without (0) or with CD4-Ig (1 mg/ml),
NBD-556 (100 mM), or BMS-806 (10 mM). Numbers indicate molecular weight (in kD).
(C) Recombinant viruses bearing the WT or indicated mutant HIV-1YU2envelope glycoproteins were incubated with Cf2Th-CCR5 cells in the presence of increas-
ing concentrations of NBD-556. Luciferase activity in the Cf2Th-CCR5 cells was measured, and the relative virus infectivity, compared with that observed in the
absence of added NBD-556, is shown. The mean values and standard errors (from triplicate samples) are shown from a typical experiment.
(D) The location of HIV-1YU2gp120 residues in which changes affect NBD-556 binding and/or entry enhancement is shown. The modeled NBD-556, with bonds
colored according to the identity of the atoms (see Figure 3 legend), is shown penetrating into the Phe 43 cavity of gp120. The gp120 core is depicted as a Ca
ribbon and transparent molecular surface. The HIV-1YU2gp120 residues that, when changed, affect NBD-556 binding and/or enhancement of HIV-1 entry into
Cf2Th-CCR5 cells are labeled and highlighted in arbitrary colors, with side chain bonds shown.
Small-Molecule CD4 Mimics of HIV-1 Entry
1696 Structure 16, 1689–1701, November 12, 2008 ª2008 Elsevier Ltd All rights reserved
Compound-gp120 Affinity and HIV-1 Inhibition
Because natural target cells for HIV-1 in vivo all express CD4,
future efforts to create antiviral agents directed against the Phe
43 cavity of gp120 would benefit from an understanding of the
properties of these compounds that correlate with inhibition of
HIV-1 infection of CD4+CCR5+cells. We used gp120 variants
and NBD-556 analogs exhibiting a range of affinities to examine
the contribution of this parameter to virus inhibition. Infection of
(50% inhibitory concentration [IC50] = 100 mM; Figure 9A).
However, JRC-II-191 inhibited WT virus infection with an IC50of
54 mM, consistent with the higher affinity of this analog for the
HIV-1YU2gp120 glycoprotein. Viruses with the S375A envelope
glycoproteins, which exhibit a higher affinity for NBD-556 and
JRC-II-191, were inhibited by both compounds with IC50values
of 10–20 mM (Figure 9A). There is a close relationship between
the Kdof the compound for the particular envelope glycoprotein
variant and the IC50(Figure 9B). The Kdvalues are less than the
tially influence the inhibition of virus infection are not expected to
affect the binding of the compounds to gp120 monomers. None-
theless, it is apparent that increasing the binding affinity of
a small-molecule CD4 mimic results in improved antiviral effect.
The binding of CD4 and some neutralizing antibodies to the
HIV-1 gp120 glycoprotein (or to the gp120 core) is accompanied
by unfavorable changes in entropy that are unusually large for
protein-protein interactions (Kwong et al., 2002; Myszka et al.,
2000). These unfavorable entropic changes, reflecting the intro-
duction of order into the conformationally flexible gp120, result
from the formation of intramolecular interactions within gp120,
reflected in favorable enthalpic changes. That the binding of
a small molecule like NBD-556 to gp120 also involves such
dramatic changes in enthalpy and entropy is remarkable (Scho ¨n
et al., 2006). The observed binding thermodynamics imply that
NBD-556, like CD4 and some antibodies (Kwong et al., 2002;
Myszka et al., 2000), fixes gp120 into a limited subset of confor-
mations. The NBD-556-induced conformation(s) must resemble
the CD4-bound state, given the ability of NBD-556 to stimulate
Figure 8. NBD-556 Analog-Mediated Enhancement of Infection of CD4-Negative, CCR5-Expressing Cells by Viruses Containing Mutant
As described in the Figure 4 legend, the area under the dose-response curve (in arbitrary units) was determined for each compound incubated with the viruses
bearing the indicated HIV-1YU2envelope glycoprotein mutant (or A-MLV envelope glycoproteins). The value for this area was normalized to the value calculated
for NBD-556 and the viruses with WT YU2 envelope glycoproteins in the same experiment. The numbers reported represent the mean values and standard
deviations obtained from at least three independent experiments. The relative enhancement of infection of Cf2Th-CCR5 cells is color coded as follows: red =
0–0.24; yellow = 0.25–0.7; green = >0.7.
Table 1. Binding Affinities of NBD-556 and JRC-II-191 to
Different gp120 Variants
HIV-1 YU2 gp120 glycoprotein
S375W NB NB
The binding experiments were carried out by isothermal titration calorim-
Small-Molecule CD4 Mimics of HIV-1 Entry
Structure 16, 1689–1701, November 12, 2008 ª2008 Elsevier Ltd All rights reserved 1697
both CCR5 binding and HIV-1 infection of CD4?CCR5+cells
(Scho ¨n et al., 2006). The thermodynamic and mechanistic simi-
larities of NBD-556 and CD4 justify the use of the CD4-bound
structure of HIV-1 gp120 (Kwong et al., 1998, 2000) to construct
models of the NBD-556-gp120 complex. The consistency of the
NBD-556 binding modes predicted by independent modeling
approaches attests to the enthalpic benefits of interacting with
the Phe 43 pocket. The predicted orientation of the bound
NBD-556, with the phenyl ring projecting into the Phe 43 cavity,
is necessitated by the steric bulk of the tetramethyl-piperidine
ring at the other end of the molecule.
Studies with a panel of HIV-1 gp120 mutants supported the
importance of gp120 residues near the Phe 43 cavity to NBD-
556 binding. Filling the Phe 43 cavity with the indole ring of tryp-
tophan in the S375W mutant resulted in loss of NBD-556 binding
Figure 9. Inhibition of HIV-1 Infection of
CD4-Expressing Cells by NBD-556 and
(A) Recombinant viruses bearing the WT or S375A
viruses with the A-MLV envelope glycoproteins
were incubated with increasing concentrations of
NBD-556 (open symbols) or JRC-II-191 (filled
symbols) and then added to Cf2Th-CD4/CCR5
cells. The level of infection was determined by
luciferase assay. The percentage of infection
relative to that seen in the absence of added com-
pound is shown. The means and standard errors
from a single experiment, performed with triplicate
samples, are shown. The experiment was re-
(B) The concentrations (IC50) of NBD-556 and
JRC-II-191 inhibiting 50% of infection of Cf2Th-
CD4/CCR5 cells by viruses bearing the indicated
HIV-1 envelope glycoproteins
against the Kdfor the binding of the compounds
to the indicated HIV-1 gp120 variants. The IC50
values represent the means and standard devia-
tions of values obtained infive independent exper-
iments, with each experiment containing triplicate
and activity, even though CD4 binding
was preserved. These observations sup-
port the modeling predictions that NBD-
556 inserts more deeply into the Phe 43
pocket than CD4. Alteration of several
other gp120 residues near the Phe 43
cavity resulted in increased or decreased
NBD-556 binding and/or activity. Nota-
bly, substitution of Ser 375 with alanine
resulted in a gp120 glycoprotein that
bound NBD-556 analogs better than the
WT glycoprotein. The S375A viruses
were also more sensitive to the enhanc-
ing effects of NBD-556 analogs (in
CD4?CCR5+cells) and to the inhibitory
cells). Substitution of alanine for Asp 368
reduced CD4 binding and HIV-1 replica-
tion, but resulted in tighter NBD-556 binding and increased
NBD-556 enhancement of infection of CD4?target cells.
Our findings provide insights into the surprising ability of small
molecules to structure the conformationally flexible HIV-1 gp120
glycoprotein. The favorable enthalpic changes (up to 24.5 kcal/
mol) observed upon binding of a series of NBD-556 analogs are
much larger than those values expected from the interaction of
the compounds with gp120, as delineated in the docking model.
The large enthalpy changes, which are partially compensated by
large unfavorable entropy changes, are reminiscent of those
changes observed during protein folding (Robertson and Mur-
phy, 1997). The magnitude of the enthalpic change is consistent
with the formation of a significant network of interactions within
gp120 upon compound binding. Moreover, some NBD-556
analogs that vary in the phenyl ring substituents have similar
Small-Molecule CD4 Mimics of HIV-1 Entry
1698 Structure 16, 1689–1701, November 12, 2008 ª2008 Elsevier Ltd All rights reserved
affinities for gp120, yet exhibit enthalpy changes upon gp120
binding that differ by more than 10 kcal/mol. These observations
hintthattheformation ofnewinteractions within gp120accounts
for the major portion of the favorable enthalpy changes associ-
ated with the binding of some NBD-556 analogs, as has been
previously suggested for CD4 binding (Myszka et al., 2000).
NBD-556 contacts with gp120 presumably contribute only
a small fraction of the favorable enthalpic changes that occur
upon binding. Modeling predicts that these interactions involve
aromatic-aromatic stacking interactions in the base of the Phe
43 cavity and hydrogen bonds between the NBD-556 oxalamide
and gp120 backbone carbonyls in the neck of the cavity. All
known gp120 protein ligands that induce large entropic changes
bind to at least two of the three gp120 core domains (Huang
et al., 2005; Kwong et al., 2000, 2002; Myszka et al., 2000; Vita
et al., 1999; Zhang et al., 1999), implying that interdomain flexi-
bility likely contributes to the high entropy of unliganded gp120.
Binding in the Phe 43 cavity, at the nexus of all three gp120
domains, provides an appealing explanation for the ability of a
small molecule like NBD-556 to decrease gp120 conformational
flexibility. Modeling of NBD-556 binding predicts that the para-
phenyl substituents are situated adjacent to strand b16 (gp120
outer domain residues 374–379) and loop B (gp120 residues
255–257), which links strands b8 in the inner domain and b9 in
the outer domain (Kwong et al., 1998). The differences in the en-
tropy changes associated with the binding of analogs with subtly
different para-phenyl substitutions indicate the importance of
interactions with these two gp120 elements in inducing or stabi-
lizing the CD4-bound conformation. Studies with chemically
modified soluble CD4 or CD4-mimetic miniproteins have
suggested that extending ligand interactions deeper into the
Phe 43 cavity than those made by the Phe 43 ring of CD4 can in-
crease affinity and antiviral potency (Van Herrewege et al., 2008;
Xie et al., 2007; Zhang et al., 1999).
Our analysis of structure-activity relationships for a series of
NBD-556-like compounds is consistent with the predicted con-
straints on the size and polarity of the phenyl ring and oxalamide
than 90 NBD-556 analogs with alterations in these moieties were
exhibited better gp120 binding than NBD-556 or NBD-557. Both
JRC-II-191 and JRC-II-192 have halogen groups at the para
position and at one of the meta positions of the phenyl ring, sug-
43 cavity is allowed and even favored. Asymmetric binding is
supported by docking calculations, which suggest that the
metahalogens inJRC-II-191andJRC-II-192areoriented toward
gp120 loop B and b16, rather than toward the water channel that
opens onto the opposing surface of the Phe 43 cavity (Kwong
et al., 1998).
Achieving adequate affinity for gp120 is essential but not
sufficient for the ability of NBD-556-like compounds to promote
CCR5 binding and HIV-1 infection of CD4-CCR5+cells. A series
of NBD-556 analogs with various substituents in the para posi-
tion of the phenyl ring bound comparably to gp120 yet exhibited
a wide range of abilities to activate CCR5 binding or virus infec-
tion. Only by taking into account both the binding affinity and
entropy changes could functional activation of the HIV-1 enve-
lope glycoproteins be accounted for. Likewise, JRC-II-191 and
JRC-II-192, which differ in the meta-phenyl substituent, ex-
hibited similar affinities for gp120 and shared predicted binding
modes (see previous text); nonetheless, only JRC-II-191 acti-
vated HIV-1 infection of CD4?target cells. The greater magni-
tudes of the unfavorable entropy change and favorable enthalpy
change associated with JRC-II-191 binding are consistent with
the importance of conformational fixation of gp120 to the
CD4-mimetic effects of these compounds.
The evolutionary requirement to evade the binding of neutral-
izing antibodies has resulted in several unusual features of the
HIV-1 envelope glycoproteins: surface variability, a high degree
of glycosylation, and conformational flexibility (Kwong et al.,
2002; Wyatt and Sodroski, 1998). Although these features are
most effective in abrogating the binding of large molecules like
antibodies, they can also influence the interaction of gp120
a formidable barrier to the development of antiviral agents that
are able to inhibit a broad range of natural HIV-1 variants. The
Phe 43 pocket and surrounding vestibule represent one of the
few well-conserved, accessible surfaces on the HIV-1 envelope
glycoprotein trimer (Kwong et al., 1998; Wyatt et al., 1998; Wyatt
and Sodroski, 1998). Our findings indicate that small molecules
binding in the Phe 43 pocket can interact with several different
gp120 glycoproteins from distinct HIV-1 strains. Thus, the com-
pounds that most closely mimic CD4 can take advantage of
the requirement for HIV-1 to conserve the gp120 region that
mediates CD4 binding.
The conformational flexibility of HIV-1 gp120 is thought to
protect the receptor-binding sites from neutralizing antibodies
(Kwong et al., 2002). Presumably, however, HIV-1 gp120 has
also maintained throughout evolution a natural propensity to
make the transition into the CD4-bound conformation. The bind-
ingof CD4and CD4mimicstriggers acascadeof cooperative in-
teractions within gp120 that result in the structuring of previously
disordered regions (Huang et al., 2005; Kwong et al., 1998;
Myszka et al., 2000; Scho ¨n et al., 2006; Zhang et al., 1999).
These events may involve gp120 structures distant from the
binding site, creating the possibility of altering the binding of
CD4 mimics by changes outside the gp120 residues that directly
contact the ligand. For example, we observed a difference in the
efficiency with which NBD-556 bound the gp120 glycoproteins
of the YU2 and HXBc2 HIV-1 strains, even though the gp120 res-
idues predicted to contact NBD-556 are identical in these two
variants. Presumably, envelope glycoprotein differences outside
the NBD-556 binding site influence the affinity of gp120 for the
compound in this instance. The propensity of HIV-1 to escape
small-molecule CD4 mimics by this mechanism is balanced by
the viral requirements to maintain efficient CD4 binding.
The most effective CD4 mimics can allow HIV-1 to circumvent
the requirement for CD4 on target cells overexpressing CCR5.
The degree to which this activating effect might extend the tro-
pism of HIV-1 to CD4?cells in vivo, which express much lower
levels of CCR5, requires further investigation. This activating
effect of CD4 mimetic compounds will be balanced by virus-
inhibitory effects. Thelatter includecompetitionfor CD4onnatu-
ral target cells and the premature triggering of metastable states
The working model for specific gp120-NBD-556 interactions
Small-Molecule CD4 Mimics of HIV-1 Entry
Structure 16, 1689–1701, November 12, 2008 ª2008 Elsevier Ltd All rights reserved 1699
relationships and guide efforts to improve these compounds
further. Our findings suggest that increasing the affinity of
NBD-556 for gp120 should be a high priority in seeking to
improve antiviral efficacy. The relatively low molecular weight
of JRC-II-191, which is optimized for insertion into the Phe 43
cavity, allows future manipulation of the piperidine ring to
achieve greater antiviral potency. CD4 mimetic drugs could
eventually be combined with modalities that recognize the
highly conserved chemokine receptor-binding surface of gp120
and interfere with CCR5 binding. Further understanding of the
HIV-1 receptor-binding regions should assist the development
of rational, targeted therapeutic or prophylactic interventions.
NBD-556 analogs were synthesized and stored as described previously
(Scho ¨n et al., 2006). Additional details of synthesis are available from the
authors on request.
Cell lines were maintained as described in the Supplemental Data.
Plasmids Expressing HIV-1 Envelope Glycoproteins
The WT and mutant HIV-1 envelope glycoproteins were expressed from the
pSVIIIenv vector (Olshevsky et al., 1990; Sullivan et al., 1998). Full description
of the preparation of mutant plasmids is provided in the Supplemental Data.
Effects of Compounds on Virus Infectivity
Recombinant, luciferase-expressing viruses were made as described previ-
ously (Scho ¨n et al., 2006) and in the Supplemental Data. Cf2Th-CD4-CCR5
or Cf2Th-CCR5 target cells were seeded at a density of 6 3 103cells per
well in 96-well luminometer-compatible tissue culture plates (Perkin Elmer;
Waltham, MA) 24 hr before infection. On the day of infection, NBD-556 or
one of its analogs (1–100 mM) was added to recombinant viruses (10,000 re-
verse transcriptase units) in a final volume of 50 ml and incubated at 37?C for
30 min. The medium was removed from the target cells, which were then incu-
bated with the virus-drug mixture for 48 hr at 37?C. The medium was removed
from each well, and the cells were lysed in 30 ml of passive lysis buffer (Prom-
ega; Madison, WI) by three freeze-thaw cycles. An EG&G Berthold Microplate
Luminometer LB 96V was used to measure luciferase activity in each well after
the addition of 100 ml of luciferin buffer (15 mM MgSO4, 15 mM KPO4[pH 7.8],
1 mM ATP, 1 mM dithiothreitol) and 50 ml of 1 mM D-luciferin potassium salt
(BD Biosciences Pharmingen; Franklin Lakes, VT).
The production of gp120 and assays measuring the binding of gp120 to CCR5
and radiolabeled NBD-556 are described herein (Scho ¨n et al., 2006) as well as
in the Supplemental Data.
Isothermal Titration Calorimetry
Isothermal titration calorimetric experiments were performed as described
herein (Scho ¨n et al., 2006) and in the Supplemental Data.
Modeling the Binding of NBD-556 and Its Analogs to HIV-1 gp120
For the docking calculations, small molecule and protein preparation used
standard protocols, as described in the Supplemental Data. For Glide (4.018)
based on the positions of CD4 Phe 43 and the isopropanol molecule from the
1G9M crystal structure. The Glide grids were computed with a box center at
28.10, ?12.35, 81.57, and an inner and outer box range of 14 A˚and 36 A˚,
respectively. Docking calculations were performed in standard sampling
mode with maxkeep 5,000 and maxref 1,000. Docking was repeated using
tion of NBD-556 produced with Glide. Docking calculations were performed
with three crystallographic water molecules (HOH 343, HOH 6, and HOH 327)
residing inthe cavity, as described in theSupplemental Data.Docking calcula-
tions were conducted with default parameters with the following exceptions:
100 genetic algorithm (GA) docking runs were performed using an initial_
virtual_pt_match_max = 3.5, diverse_solutions = 1, divsol_cluster_size = 1,
and divsol_rmsd = 1.5 A˚.
core using Insight II/Discover Software (Accelrys; San Diego), as described in
the Supplemental Data.
Coordinates of the CD4-bound gp120 core are available in the Protein Data
Bank under ID codes 1GC1 and 1G9M.
Supplemental Data include Supplemental Experimental Procedures and
Supplemental References and can be found with this article online at http://
We thank Wayne Hendrickson and Irwin Chaiken for valuable discussion. We
thank Yvette McLaughlin and Elizabeth Carpelan for manuscript preparation,
and Jonathan Stuckey for preparation of figures. This study was supported
by the National Institutes of Health (Grants GM56550, AI24755, and AI60354),
the International AIDS Vaccine Initiative, and William F. McCarty-Cooper.
Received: August 8, 2008
Revised: September 16, 2008
Accepted: September 18, 2008
Published: November 11, 2008
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