Crystal structures of HIV-1 reverse transcriptase with picomolar inhibitors reveal key interactions for drug design

Article abstract of DOI:10.1021/ja3092642

X-ray crystal structures at 2.9 ? resolution are reported for two complexes of catechol diethers with HIV-1 reverse transcriptase. The results help elucidate the structural origins of the extreme antiviral activity of the compounds. The possibility of halogen bonding between the inhibitors and Pro95 is addressed. Structural analysis reveals key interactions with conserved residues P95 and W229 of importance for design of inhibitors with high potency and favorable resistance profiles.

Full text of DOI:10.1021/ja3092642

Communication
pubs.acs.org/JACS
Crystal Structures of HIV‑1 Reverse Transcriptase with Picomolar
Inhibitors Reveal Key Interactions for Drug Design
Kathleen M. Frey,‡ Mariela Bollini,† Andrea C. Mislak,‡ Jose A. Cisneros,† Ricardo Gallardo-Macias,†
́
William L. Jorgensen,*,† and Karen S. Anderson*,‡
†Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107, United States
‡Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066, United States
S
* Supporting Information
with 1 and 2. It is of added interest to address the possible
ABSTRACT: X-ray crystal structures at 2.9 Å resolution
are reported for two complexes of catechol diethers with
HIV-1 reverse transcriptase. The results help elucidate the
structural origins of the extreme antiviral activity of the
compounds. The possibility of halogen bonding between
the inhibitors and Pro95 is addressed. Structural analysis
reveals key interactions with conserved residues P95 and
W229 of importance for design of inhibitors with high
potency and favorable resistance profiles.
contribution of halogen bonding to the potency of 1, which
arose from the molecular modeling, in addition to other key
interactions.1
Crystals of recombinant RT52A enzyme in complex with 1
and 2 were prepared using methods similar to those previously
described.2 The best crystals diffracted to amplitudes extending
to a resolution of 2.85 Å for 1 and 2.90 Å for 2. Both structures
were solved by molecular replacement with Phaser3,4 using the
structure of an RT complex with a triazine relative of rilpivirine
as a probe model (PDB code: 1S9E).5 Overall, the electron
density reveals an “open-cleft” conformation as observed in
other RT-NNRTI co-crystal structures (Figure 1).5−10 Root
n a recent report, lead optimization guided by free-energy
Iperturbation (FEP) calculations led to the discovery of
extraordinarily potent anti-HIV agents.1 The new compounds,
which are non-nucleoside inhibitors of HIV-1 reverse tran-
scriptase (NNRTIs), include 1 and 2. Specifically, 1 and 2 yield
EC50 values of 55 and 320 pM for inhibition of replication of
the wild-type virus in infected human T-cells. For comparison,
nevirapine and rilpivirine, well-known approved drugs in the
NNRTI class, are 110 nM and 670 pM inhibitors in this assay
(Table 1). As detailed in the Supporting Information, we have
also confirmed the extreme potency of 1 in cell-free assays for
inhibition of reverse transcription; 1, rilpivirine, and nevirapine
yield IC50 values 3, 38, and 1060 nM, respectively (Table 1). To
help clarify the structural origins of the potency of the catechol
diethers, we also report here co-crystal structures of HIV-RT
Figure 1. Omit Fo − Fc electron density maps at a contour level of
3.0σ showing 1 (A) and 2 (B) in the non-nucleoside binding pocket of
HIV-1 RT. Omit Fo − Fc and 2Fo − Fc electron density maps are
provided as Supporting Information.
Table 1. Activities for Inhibition of HIV-1 Replication
(EC50) and Reverse Transcription (IC50)
mean square deviations (rmsd) between our structures and
several prior ones range from 1.87 to 3.02 Å (Table S2),
suggesting that the general fold is similar and that there are no
major conformational changes observed upon binding to the
catechol diethers. However, Tyr181 is in the relatively
uncommon “down” conformation as anticipated by the
modeling1 and observed, for example, in the 2BE2 structure
of an iodopyrimidinone NNRTI.7 The 1S9E structure and the
2ZD1 structure for rilpivirine show Tyr181 in the common
“up” conformation.5
compound
EC50 (nM)
IC50 (nM)
1
0.055
0.320
3.2
3.0
ND
10
2
The electron density for the non-nucleoside binding pocket
(NNBP) clearly defines the orientation of 1, 2, and the
3
4
5.2
30
nevirapine
rilpivirine
110
1060
38
Received: September 18, 2012
0.670
© XXXX American Chemical Society
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dx.doi.org/10.1021/ja3092642 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
interacting residues (Figures 1 and S2). Both 1 and 2 are in the
same conformation with only slight variations in flexible regions
of the compounds that include the cyanovinyl group and
ethoxy linker. The catechol diethers adopt an optimal
conformation to make multiple van der Waals interactions
with hydrophobic residues of the NNBP (Figure 2). Previously,
Figure 3. Omit Fo − Fc electron density (contour level at 3.0 σ) for the
RT52A:1 complex. The dashed line represents the interaction distance
between the P95 carbonyl and Cl group, which is 4.72 Å.
region depleted in electron density, a “σ hole”.16 For simple
gas-phase complexes, halogen bonding becomes more favorable
in progressing from Cl to Br to I. There is essentially perfect
agreement between the crystal structure and the predicted
structure for the complex of 1 (Figure 3 in ref 1), including the
conformations of the cyanovinyl and uracilylethoxy side chains
and the hydrogen bonding with K103. However, an issue with
the modeling is that classical force fields, which represent a
carbonyl oxygen and an aryl chlorine as single particles with
partial negative charges, cannot reproduce the attractive
electrostatics of halogen bonds. This was subsequently
remedied for the OPLS force fields by addition of a partial
positive charge to represent the σ hole.17 FEP results then
predicted relative free energies of binding (ΔGb) of 0.0, −3.7,
and −3.5 kcal/mol for 3, 1, and 4 with HIV-RT and of −2.6
kcal/mol for the iodo analogue.17 Thus, the binding and activity
are computed to peak with the chloro analogue 1. Addition of
the partial positive charge on Cl was found to be favorable by
1−1.5 kcal/mol; however, a range of distances (3.6−4.5 Å) for
the Cl−P95 halogen bond was sampled in the Monte Carlo
FEP simulations for 1. This range is greater than the optimal
separation of ca. 3.1 Å for PhCl, PhBr, or PhI interacting with
an amide carbonyl oxygen.17,18
We have now further addressed experimentally the halogen
bonding issue. First, the bromo analogue 4 was synthesized and
its activities were evaluated (Table 1). In general, the EC50 and
IC50 results correlate well; e.g., 1 is more active than rilpivirine
by a factor of 12 in both cases. For the catechol diether series
(3, 1, and 4), the activities are indeed greatest for the chloro
analogue; however, the fluoro analogue 3 is more active than
the bromide 4 in contrast to the expectation from the
computed ΔGb values.16 The issue could be further addressed
with a direct binding assay. However, a notable point is that if
halogen bonding is operative, the I > Br > Cl > F order is not
guaranteed since the heavier halogens do require more space,
which can lead to greater entropic penalties on complexation.
Second, the crystallography has provided a value for the Cl−
P95 O separation of 4.72 Å (Figure 3), with a coordinate
uncertainty of 0.41 Å, which is on the high edge of the range
sampled in the FEP simulations. The C−Cl···O angle of 132° is
also significantly bent from the ideal linear geometry.16−18
While a strong halogen bond is not observed, there may be
some electrostatic benefit from the Cl−P95 O interaction for 1.
In summary, crystal structures for the extraordinarily potent
anti-HIV agents 1 and 2 show striking complementarity
Figure 2. Stereo view of the crystal structure for 1 complexed with
HIV-RT. Multiple contacts with residues in the NNBP are apparent;
the dashed lines highlight the hydrogen bonds with K102 and K103.
The corresponding illustration for 2 is nearly identical (Figure S3).
the NNBP has been described to feature three channels
designated as the entrance, groove, and tunnel.11 The cyanovinyl
phenyl fragment interacts with P95, L100, V108, Y188, W229,
and L234 and is positioned beneath W229, projecting into the
tunnel region. The catechol ring is proximal to V106, V108, and
Y181, and the Cl or F on C5 protrudes into the entrance
channel near K103. The ethoxy linker interacts with L100,
V106, F227, and Y318, positioning the terminal uracil ring in
the groove near P236. Notably, the C2 carbonyl and adjacent
NH of the uracil form hydrogen bonds with the K103 backbone
NH and O. K102 adopts a unique rotamer conformation in
+
which the NH3 atom forms a hydrogen bond with the uracil
O4 atom. The observed N−O distances for the hydrogen
bonds are ca. 3.50 Å or less (Table S3). The multiple
interactions with the uracil are consistent with the diminished
activity observed for analogues with alternative heterocycles.1
It may also be noted that in spite of the differences in the
core structures of 1/2 and rilpivirine, the positioning of the
cyanovinylphenyl groups is the same, yielding beneficial
contacts with W229. The effect is evident in comparing the
activity of 1 in the T-cell assay (55 pM) versus that of its
analogue with chlorine replacing the cyanovinyl group (20
nM).1 The high potency of 1 and 2 undoubtedly also includes
contributions from the hydrogen bonds with K102 and K103.
Most significantly, the halocyanovinylphenyl (HCVP) group
forms numerous van der Waals contacts in the tunnel region
with P95 and W229, both conserved residues near or part of
the primer grip.12,13 Based on in vitro mutational and clinical
studies, P95 and W229 are immutable residues in the NNBP
essential for preserving reverse transcriptase activity.13−15 Thus,
the HCVP group may be an advantageous moiety compensat-
ing for potential lost interactions, with NNBP mutations
conferring resistance.
Another intriguing notion raised by the modeling1 was the
possible contribution of halogen bonding between the carbonyl
oxygen of P95 and the chlorine atom in the terminal phenyl
ring of 1 (Figure 3). Halogen bonding can occur between Lewis
bases and chlorine, bromine, and iodine atoms.16 It features a
favorable electrostatic interaction between the Lewis base, e.g.,
a carbonyl oxygen, and the backside of the halogen, which has a
B
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Journal of the American Chemical Society
Communication
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between the conformation of the inhibitors and the NNBP with
substantial extensions into the three channels that characterize
the binding site. The activity of the catechol diethers has been
further assessed by determination of the inhibition of the
activity of HIV-RT, which was found to parallel the cell-based
results and to confirm the extreme potency of 1. The
contribution to the activity of 1 from halogen bonding has
received attention from the structural analyses and addition of
data for the bromo analogue 4. The crystallography shows that
the halogen bonding is not optimal for the present compounds,
which raises the intriguing possibility of designing even more
potent agents with favorable resistance profiles. From this
analysis, a design strategy emerges to improve interactions with
P95 and maintain interactions with W229, both residues
important for the assembly and activity of HIV-RT and thus
not prone to mutation.13−15
(16) For reviews, see: (a) Politzer, P.; Murray, J. S.; Clark, T. Phys.
Chem. Chem. Phys. 2010, 12, 7748. (b) Metrangolo, P.; Murray, J. S.;
Pilati, T.; Politzer, P.; Resnati, G.; Terraneo, G. Cryst. Growth Des.
2011, 11, 4238.
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ASSOCIATED CONTENT
* Supporting Information
Full details on the crystallography, synthesis and character-
ization of 4, and enzymatic assays. This material is available free
of charge via the Internet at http://pubs.acs.org. Data for 1 and
2 have been deposited in the RCSB Protein Data Bank with
codes 4H4M and 4H4O.
■
S
AUTHOR INFORMATION
Corresponding Author
william.jorgensen@yale.edu; karen.anderson@yale.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Gratitude is expressed to the National Institutes of Health
(AI44616, GM32136, GM49551) for support and to Prof.
Eddy Arnold for discussions.
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dx.doi.org/10.1021/ja3092642 | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX