Structure-based design and synthesis of an HIV-1 entry inhibitor exploiting X-ray and thermodynamic characterization

Article abstract of DOI:10.1021/ml300407y

The design, synthesis, thermodynamic and crystallographic characterization of a potent, broad spectrum, second-generation HIV-1 entry inhibitor that engages conserved carbonyl hydrogen bonds within gp120 has been achieved. The optimized antagonist exhibits a submicromolar binding affinity (110 nM) and inhibits viral entry of clade B and C viruses (IC₅₀ geometric mean titer of 1.7 and 14.0 μM, respectively), without promoting CD4-independent viral entry. The thermodynamic signatures indicate a binding preference for the (R,R)- over the (S,S)-enantiomer. The crystal structure of the small-molecule/gp120 complex reveals the displacement of crystallographic water and the formation of a hydrogen bond with a backbone carbonyl of the bridging sheet. Thus, structure-based design and synthesis targeting the highly conserved and structurally characterized CD4-gp120 interface is an effective tactic to enhance the neutralization potency of small-molecule HIV-1 entry inhibitors.

Full text of DOI:10.1021/ml300407y

Letter
pubs.acs.org/acsmedchemlett
Structure-Based Design and Synthesis of an HIV‑1 Entry Inhibitor
Exploiting X‑ray and Thermodynamic Characterization
Judith M. LaLonde,*,† Matthew Le-Khac,‡,§ David M. Jones,∥ Joel R. Courter,∥ Jongwoo Park,∥
Arne Schon,⊥ Amy M. Princiotto,# Xueling Wu,∇ John R. Mascola,∇ Ernesto Freire,⊥ Joseph Sodroski,#,○
̈
Navid Madani,# Wayne A. Hendrickson,*,§,◆ and Amos B. Smith, III*,∥
†Department of Chemistry, Bryn Mawr College, Bryn Mawr, Pennsylvania 19010, United States
§
‡Department of Pharmacology and Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New
York 10032, United States
∥Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
⊥Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, United States
#Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts
02115, United States
∇Vaccine Research Center, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, United States
○Department of Microbiology and Immunobiology, Harvard Medical School; Department of Immunology and Infectious Diseases,
Harvard School of Public Health; Ragon Institute of MGH, MIT, and Harvard, Boston, Massachusetts 02115, United States
◆Department of Physiology and Cellular Biophysics, Columbia University, New York, New York 10032, United States
S
* Supporting Information
ABSTRACT: The design, synthesis, thermodynamic and crystallographic characterization of a potent, broad spectrum, second-
generation HIV-1 entry inhibitor that engages conserved carbonyl hydrogen bonds within gp120 has been achieved. The
optimized antagonist exhibits a submicromolar binding affinity (110 nM) and inhibits viral entry of clade B and C viruses (IC50
geometric mean titer of 1.7 and 14.0 μM, respectively), without promoting CD4-independent viral entry. The thermodynamic
signatures indicate a binding preference for the (R,R)- over the (S,S)-enantiomer. The crystal structure of the small-molecule/
gp120 complex reveals the displacement of crystallographic water and the formation of a hydrogen bond with a backbone
carbonyl of the bridging sheet. Thus, structure-based design and synthesis targeting the highly conserved and structurally
characterized CD4−gp120 interface is an effective tactic to enhance the neutralization potency of small-molecule HIV-1 entry
inhibitors.
KEYWORDS: HIV, gp120, CD4, entry inhibitor, structure-based drug design, thermodynamics, X-ray crystallography, viral inhibition,
protein−protein interactions
steps of the HIV-1 viral life cycle. Given that the initial step of
viral infection comprises the attachment of gp120,5 the major
surface-exposed protein of the trimeric viral envelope spike,6 to
the human host cell receptor CD4,7 inhibition of this protein−
he prevention of HIV-1 viral infection remains a
1
Tworldwide public health objective. Despite the continued
reduction in AIDS-related deaths, an estimated 2.7 million
adults were newly infected in 2010 with HIV-1.2 Clinical trials
of preventative approaches, including pre-exposure prophylaxis
with oral antiretrovirals3 and/or microbiocide formulations,4
have demonstrated some success and, as such, continue to
motivate the development of inhibitors that target alternative
Received: November 20, 2012
Accepted: February 6, 2013
Published: February 6, 2013
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
protein interaction is attractive and would appear to be a viable
prophylactic strategy to prevent HIV-1 transmission. The viral
attachment process consists of three stages: (1) gp120 binding
to CD4; (2) a conformational change within gp120 induced by
the protein−protein interaction that exposes the binding site
for the secondary transmembrane chemokine receptors CCR5
or CXCR4; and (3) gp120−chemokine receptor binding, which
promotes formation of a six-helix bundle within the gp41
component of the viral envelope spike.5 This cascade of
protein−protein interactions, triggered by conformational
shifts,8 culminates in fusion of the viral and host cell
membranes to permit cellular infection and viral propagation.
The development of small-molecule viral entry antagonists that
target well conserved hot spots within the viral Env complex
and block CD4−gp120 binding continues to hold significant
potential for augmenting pre-exposure prophylactic approaches
to prevent HIV-1 transmission.4
Mutagenesis studies have revealed key hot spots within the
CD4−gp120 interface,9 which were subsequently characterized
structurally by a number of CD4/gp120 cocrystal struc-
tures.10,11 One important hot spot, the internal Phe43 cavity,
is formed at the interface of the three gp120 domains (inner,
outer, and bridging sheet) and is capped by the aromatic side
chain of CD4 residue Phe43. In fact, the crystal structure of the
small molecule NBD-55612 (1, Figure 1), first identified by
binding, manifested by a thermodynamic signature reminiscent
of protein folding (large unfavorable binding entropy and large
favorable binding enthalpy). Not only do 1 and 2 competitively
inhibit CD4 binding, but both compounds also mimic CD4 by
structuring gp120 and triggering the allosteric signal that
enhances coreceptor binding and undesired enhancement of
viral infectivity. Interestingly, thermodynamic analysis of a large
number of NBD-like small molecules revealed a wide
distribution of enthalpic and entropic contributions (ΔH and
−TΔS terms vary by 18.0 kcal/mol), even for compounds
with similar binding affinities.22 These results revealed the
possibility of designing compounds that do not elicit the
unwanted conformational structuring, and delineated a
thermodynamic-based strategy for the optimization of those
compounds. This strategy implies improving binding affinity
(ΔG), while simultaneously minimizing the unfavorable
binding entropy.23 The optimization strategy essentially
combines standard SAR with thermodynamic analysis by
isothermal titration calorimetry (ITC). Importantly, thermody-
namic-guided alanine scanning mutagenesis has recently
demonstrated that gp120 residue contributions to allosteric
structuring are not proportional to binding affinity, providing a
structural foundation to the finding that not all NBD-like
compounds elicit the unwanted allosteric structuring.24
We have previously described the design, synthesis, and
characterization of guanidinium containing trans-1,2-indanes
[(+)-3 and (−)-3; Table 1].21 Both (+)-3 and (−)-3 specifically
inhibit viral infection of 42 Tier 2 clade B and C viruses,
exhibiting geometric mean titers (GMT) IC50 of 7.9 μM and
8.9 μM, for the sensitive strains, respectively. We were pleased
to observe that (+)-3 also displays an improved thermodynamic
signature, with a reduced entropic penalty (−TΔS) upon gp120
binding, relative to 1 (ΔG, ΔH, and −TΔS values of −7.4,
−24.5, and 17.1 kcal/mol, respectively).20 Furthermore, the
undesired property of enhancing CD4 independent viral entry
was essentially eliminated (Table 1). The cocrystal structure of
(+)-3 bound to gp120 revealed specific interactions between
the guanidinium moiety and a water mediated hydrogen-
Figure 1. (A) CD4/gp120/17b structure (1GC1)13 in blue, yellow,
and light gray, respectively. The Phe43 side chain, blue, emanates from
the CD4 β-turn. A second critical CD4−gp120 interaction between
Asp368gp120 and Arg59CD4 is highlighted in red and green, respectively.
(B) The NBD-556/gp120 structure (3TGS)15 with similar orientation
as part A. The surface of the Phe43 cavity is drawn in yellow with
NBD-556 in orange. (C) NBD-556 (1) and TS-II-224 (2).
bonding network spanning both Asp368gp120 and Met426gp120
.
Thus, we concluded that incorporation of the guanidinium had
converted the NBD congeners into functional antagonists. We
therefore sought to optimize further the interactions between
the guanidinium moiety of (+)-3 based on the cocrystal
structure with residues Asp368gp120 and Met426gp120, an
“affinity hot spot”24, in an effort to improve the functional
antiviral potency.
To improve these interactions, we chose to vary the distance
between the trans indane ring system (region III) and the
guanidinium functionality (region IV; Table 1). Hence, the
binding properties of the methylene and ethylene congeners of
(+)-3 were evaluated by docking (see Supporting Information).
These results led to selection of 4 as an initial synthetic target
(Table 1). Initially, ( )-4 was constructed (see Supporting
Information). When assessed in a single-round viral infection
assay, ( )-4 demonstrated a 2-fold improvement of the IC50
Debnath and co-workers,13 reveals that 1 binds within the
Phe43 cavity. Similarly, chemically derivatized CD4 con-
structs14 and a miniprotein CD4 mimetic possessing a biphenyl
side chain15 form interactions deep within the Phe43 cavity.
Subsequent synthesis and testing of NBD-based compounds by
our group improved the interactions within the Phe43 cavity,
affording the small molecule 2 (Figure 1).16 However, 2 and
other region III congeners did not appear to mimic the second
hot spot, consisting of an electrostatic interaction between
Arg59CD4 and Asp368gp120. Moreover, 1 and 2 functionally
mimic CD4 binding and thus behave as agonists of CD4-
independent viral entry, leading to the enhancement of HIV-1
entry into cells lacking the CD4 receptor (CD4−CCR5+ Cf2Th
cells),17 an undesired trait for the development of viral entry
inhibitors.
value (10.3
3.2 μM) relative to (+)-3 (22.9
2.4 μM).
Titration of gp120 with ( )-4, employing ITC, resulted in a
complex binding curve that suggested more than one binding
event (Figure 2). We reasoned that this observation was related
to one enantiomer having a higher affinity within the mixture of
( )-4.
We turned next to X-ray crystallography to investigate the
interactions between antagonist ( )-4 and gp120 and to define
Measurement of the binding thermodynamics of sCD4,18,19
1, or 220,21 to full-length, monomeric gp120 demonstrates that
gp120 undergoes a large conformational change upon ligand
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ACS Medicinal Chemistry Letters
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Table 1. Antagonists of CD4-gp120 Binding and HIV-1 Entry
a
b
The IC50 was determined in Cf2Th-CD4/CCR5 cells infected with HIV-1 YU2 virus. The IC50 in cells infected with amphotropic murine
c
leukemia virus (A-MLV). The relative activation of viral infectivity in CD4 negative Cf2Th-CCR5 cells infected with HIV-1 YU2 virus normalized
to that of 1. Data for (+)-3 and (−)-3 have been published.22 See experimental details in the Supporting Information.
Scheme 1. Synthetic Route to Single Enantiomer (+)-4
Figure 2. ITC titrations of gp120 with (A) (+)-4 and (B) (−)-4 at 25
°C. The titration with ( )-4 (inset) resulted in a complex binding
curve (see text).
the enantiomer that preferentially binds to the gp120 core. The
formate salt of ( )-4 was soaked into preformed crystals of
gp120 from Clade C1086,12 and diffraction data were obtained
to 2.5 Å Bragg spacings (Supporting Information Table S1).
The observed electron density for each of the two 4/gp120
complexes in the asymmetric unit clearly revealed preferential
binding of the (R,R)-4 enantiomer to gp120 during the soaking
process (Supporting Information Figure S1). Interestingly, the
(R,R)-4/gp120 crystal structure revealed that the guanidinium
primary amine via oxidation to the aldehyde, followed by
reductive animation, proved unsuccessful, we were pleased to
find that mildly acidic conditions led to epimerization of the α
stereocenter. Subsequent reduction with sodium borohydride
established the desired trans stereochemical relationship [cf.
(+)-12]. A three-step sequence involving mesylation, displace-
ment of the mesylate with sodium azide, and reduction of the
azide led to amine (+)-13. Finally, installation of the
guanidinium functionality employing 1H-pyrazol-1-carboxami-
dine monohydrochloride (14) furnished (+)-4 (99% ee by
chiral SFC).21 An identical synthetic sequence was employed to
furnish (−)-4 following opening of the β-lactam (+)-8.
moiety did not directly interact with Asp368gp120
.
With the crystal structure suggesting that the (R,R)-4
enantiomer possesses higher affinity for gp120, a synthetic
route to the single (R,R)-enantiomer was developed (Scheme
1). Toward this end, [2 + 2] cycloaddition of indene (6) and
chlorosulfonyl isocyanate (7) furnished the racemic β-lactam
( )-8. A kinetic resolution employing lipase B was carried out
to yield a separable mixture of β-amino acid (−)-9 and β-lactam
(+)-8, both in 99% ee.25−27 The enantiomeric excess was
determined by supercritical fluid chromatography (SFC), as
described in the Supporting Information. The β-amino acid
(−)-9 was next converted to the cis indanol (+)-11 in two
steps.21 Although the initial synthetic plan to incorporate a
Antiviral assays revealed that (+)-4 inhibits viral entry of the
YU-2 primary HIV-1 isolate with an IC50 value of 3.1 0.6 μM,
while the (−)-4 antipode exhibits a 10-fold reduction in
antiviral activity, with an observed IC50 = 37.9
22.7 μM
(Table 1). To assess further the HIV-1 neutralization breadth
and potency, we assayed 1, (+)-3, and (+)-4 against 42 diverse
strains of clades B and C Env-pseudoviruses (see Table S2 in
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ACS Medicinal Chemistry Letters
Letter
the Supporting Information). As previously observed for 1 and
(+)-3,21 (+)-4 also neutralized clade B viruses better than clade
C viruses, with 100% breadth and an IC50 GMT of 1.7 μM
against clade B viruses, compared to 59% breadth and an IC50
GMT of 14.0 μM against the sensitive clade C viruses.
Moreover, (+)-4 demonstrated a 60% improvement over (+)-3
based on IC50 titers in clade B viruses and a 1.5-fold
improvement based on IC80 titers. In addition, ITC measure-
ments found that (+)-4 binds full-length gp120 with Kd = 110
nM (Table 1 and Figure 2). In contrast, (−)-4 has a
significantly reduced binding affinity of 6200 nM. These results
are consistent with the gp120-bound cocrystal structure derived
from ( )-4, suggesting the (R,R)-enantiomer preferentially
binds to the monomer gp120 core.
Given that inclusion of the methylene spacer between
regions III and IV led to significant improvements in both
binding affinity and functional antagonism of HIV-1 viral entry
into target cells, we constructed ( )-5, containing an additional
methylene spacer between the indane scaffold and the
guanidinium moiety (see Supporting Information). Semi-
preparative chiral SFC furnished samples of (+)-5 and (−)-5
for biological evaluation. Assessment of the functional
antagonist activity of (+)-5 and (−)-5 revealed that both
were less potent than (+)-4 (Table 1). Evaluation of
compounds (+)-4, (−)-4, (+)-5, and (−)-5 by ITC (Table 1
and Figure 2) when compared to (+)-3 and (−)-3
demonstrates that (+)-4 exhibits the best submicromolar
binding affinity observed thus far for a small-molecule/gp120
complex. Moreover, (+)-3, (+)-4, and (+)-5 all exhibit
improved binding affinities when compared to the correspond-
ing (−)-antipodes. Examination of the thermodynamic
signatures (Table 1) reveals that (−)-3, (−)-4, and (−)-5
exhibit a larger favorable enthalpy coupled to a larger
compensatory unfavorable entropy, suggesting that these
compounds trigger a more significant structuring in gp120
than the (+)-3, (+)-4, and (+)-5 compounds. The smaller
entropic penalty for (+)-3, (+)-4, and (+)-5 indicates that the
(R,R)-configuration possesses the greatest potential for
improving enthalpic contributions to binding that are not
related to gp120 structuring. In general, improving inhibitor−
gp120 interactions should be reflected in better enthalpic
interactions not correlated to the structuring of gp120.22
Crystallography was once more employed to ascertain the
specific binding interactions between gp120 and (+)-4.
Cocrystallization of (+)-4 with the clade A/E93TH057
extended gp120(H375S) core12 produced crystals that diffracted
to 2.5 Å spacings (crystallographic data are summarized in
Table S1 in the Supporting Information). There are two
complexes in the asymmetric unit of these crystals, and each
(+)-4 molecule in both complexes has similar conformations
that closely resemble those observed in the (R,R)-4/gp120
structure obtained from ( )-4 (Supporting Information Figure
S1 and Table S3). As expected, the previously observed
hydrogen bonds between the oxalamide linker and the
Asn425gp120 and Gly473gp120 amide nitrogen atoms12,21 are
preserved in the (+)-4/gp120 complex (Figure 3). Surprisingly,
as noted for (R,R)-4/gp120 (vide supra), the guanidinium
moiety did not directly interact with Asp368gp120. Instead, a
hydrogen bond is formed between one guanidinium nitrogen
Figure 3. Comparison of the structures of (A) (+)-3/gp120
(4DKQ)21 and (B) (+)-4/gp120 (Clade E, copy A) indicates that
(+)-3 interacts with Met426gp120 via a network of water molecules
whereas the guanidinium group of (+)-4 hydrogen bonds directly to
the backbone carbonyl of Met426gp120. Coloring is by atom type:
nitrogen (blue), oxygen (red), chlorine (neon-green), fluorine (neon-
yellow-green), and carbon [magenta for (+)-3 and sky blue for (+)-4].
Hydrogen bonds are represented by dashed lines.
(+)-4/gp120 (Figure 3), allowing for direct hydrogen bonding
to the carbonyl of Met426gp120. The direct hydrogen bond and
the displacement of crystallographic water molecules, as well as
the improved binding affinity of (+)-4 over the previous
described (+)-3, provide additional motivation to modify
region III moieties. Further modification of the guanidinium-
Asp368gp120 contact, as previously observed in the (+)-3/gp120
complex with the newly revealed guanidinum−Met426gp120
interaction exhibited in the (+)-4/gp120 structure, will in the
future yield even more potent inhibitors of viral entry.
In summary, we have employed the crystal structures of
antagonist/gp120 complexes to achieve an increase of both
binding affinity and antiviral potency for HIV-1 entry inhibitors.
The crystal structure of this new compound, (+)-4, bound to
gp120, reveals a newly formed hydrogen bond to Met426gp120
and the displacement of crystallographic water derived from the
extension of the guanidinium group via a methylene spacer.
Whereas the parent compounds (−)-3 and (+)-3 had
comparable potency and bound alternatively to gp120, the
(R,R)-enantiomer (+)-4 is strongly preferred and binds
uniquely. On the basis of the antiviral potency and binding
affinity, the methylene spacer between regions III and IV of
(+)-4 is favored compared to the ethylene spacer of (+)-5.
Furthermore, the thermodynamic signatures indicate that the
(R,R)-stereochemical configuration encounters less entropic
penalty than the (S,S) one when binding to gp120. The design,
development, and synthesis of small-molecule viral entry
antagonist (+)-4 thus validates the application of static
structures of ligands bound to the conserved monomeric core
of gp120 to improve antagonist potency. Structural information
from small-molecule/gp120 complexes, however, must be
combined with thermodynamic information, as allosteric
structuring and solvation effects are not revealed in the X-ray
structures. Moreover, the improved antiviral potency and
structural characterization of (+)-4 provides an important
contribution to small-molecule viral entry antagonists for
prophylactic strategies for the prevention of HIV-1 trans-
mission.
and the bridging sheet backbone carbonyl of Met426gp120
.
Importantly, the crystallographic water molecules necessary for
the indirect interaction with Met426gp120 in the (+)-3/gp120
structure are now displaced by the extended guanidinium of the
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ASSOCIATED CONTENT
* Supporting Information
Synthesis, experimental methods, and crystallographic data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
■
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Accession Codes
Coordinates and structure factors have been deposited in the
Protein Data Bank with the following accession numbers: 4I53
and 4I54.
AUTHOR INFORMATION
Corresponding Author
*J.M.L.: e-mail, jlalonde@brynmawr.edu; phone, 610-526-
5679. WA.H. e-mail, wayne@xtl.cumc.columbia.edu; phone,
212-305-3456. A.B.S.: e-mail, smithab@sas.upenn.edu; phone,
215-898-4860.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
Funding was provided by NIH GM 56550 to J.M.L., E.F.,
W.A.H., A.B.S., and J.S. and by NIH Intramural IATAP and
NIAID programs to J.R.M. and J.S. Funding to N.M. was
provided by NIH AI090682-01. N.M. and J.S. were also
supported by Ragon Institute of MGH, MIT, and Harvard.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Irwin Chaiken and all the members of the PO1
Consortium Structure-Based Antagonism of HIV-1 Envelope
Function in Cell Entry. J.M.L. thanks the Pittsburgh Super-
computing Center for an allocation for computing resources
#MCB090108. M.L. and W.A.H. thank Young Do Kwon and
Peter Kwong of the Vaccine Research Center of NIAID for
transferring clade C and clade A/E gp120 crystallization
technology.
ABBREVIATIONS
■
(HIV-1), Human immunodeficiency virus type 1; (SIV), simian
immunodeficiency virus; (sCD4), soluble CD4; (ITC),
isothermal titration calorimetry; (A-MLV), amphotropic
murine leukemia virus; (GMT), geometric mean titer; (GA),
genetic algorithm; (HRMS), high-resolution mass spectrosco-
py; (DMEM), Dulbecco’s Modified Eagle Medium; (TsCl),
tosyl chloride; (DMAP), 4-dimethylaminopyridine
(14) Xie, H.; Ng, D.; Savinov, S. N.; Dey, B.; Kwong, P. D.; Wyatt,
R.; Smith, A. B., 3rd; Hendrickson, W. A. Structure−activity
relationships in the binding of chemically derivatized CD4 to gp120
from human immunodeficiency virus. J. Med. Chem. 2007, 50, 4898−
908.
(15) Huang, C. C.; Stricher, F.; Martin, L.; Decker, J. M.; Majeed, S.;
Barthe, P.; Hendrickson, W. A.; Robinson, J.; Roumestand, C.;
Sodroski, J.; Wyatt, R.; Shaw, G. M.; Vita, C.; Kwong, P. D. Scorpion-
toxin mimics of CD4 in complex with human immunodeficiency virus
gp120 crystal structures, molecular mimicry, and neutralization
breadth. Structure 2005, 13, 755−768.
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