Thioamide substitution to probe the hydroxyproline recognition of VHL ligands

Article abstract of DOI:10.1016/j.bmc.2018.03.034

Thioamide substitution influences hydrogen bond and n → π^? interactions involved in the conformational stability of protein secondary structures and oligopeptides. Hydroxyproline is the key recognition element of small molecules targeting the v

Full text of DOI:10.1016/j.bmc.2018.03.034

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Thioamide substitution to probe the hydroxyproline recognition of VHL ligands
Pedro Soares, Xavier Lucas, Alessio Ciulli
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S0968-0896(18)30348-1
https://doi.org/10.1016/j.bmc.2018.03.034
BMC 14272
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Bioorganic & Medicinal Chemistry
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21 March 2018
22 March 2018
Please cite this article as: Soares, P., Lucas, X., Ciulli, A., Thioamide substitution to probe the hydroxyproline
recognition of VHL ligands, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2018.03.034
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Thioamide substitution to probe the hydroxyproline
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Pedro Soares, Xavier Lucas and Alessio Ciulli

Bioorganic & Medicinal Chemistry Letters
Thioamide substitution to probe the hydroxyproline recognition of VHL ligands
Pedro Soaresa, Xavier Lucasa and Alessio Ciullia
a Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, UK.
ARTICLE INFO
ABSTRACT
Article history:
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Thioamide substitution influences hydrogen bond and n→π* interactions involved in the
conformational stability of protein secondary structures and oligopeptides. Hydroxyproline is the
key recognition element of small molecules targeting the von Hippel-Lindau (VHL) E3 ligase,
which are of interest as probes of hypoxia signaling and ligands for PROTAC conjugation. We
hypothesized that VHL ligands could be a privileged model system to evaluate the contribution
of these interactions to protein:ligand complex formation. Herein we report the synthesis of
VHL ligands bearing thioamide substitutions at the central hydroxyproline moiety, and
characterize their binding by fluorescence polarization, isothermal titration calorimetry, X-ray
crystallography and molecular modeling. In spite of a conserved binding mode, the substitution
pattern had a pronounced impact on the ligand affinities. Together the results underscore the role
of hydrogen bond and n→π* interactions in fine-tuning hydroxyproline recognition by VHL.
Keywords:
Protein-ligand interactions
VHL ligands
Thioamides
n→π* interaction
PROTACs
2017 Elsevier Ltd. All rights reserved.
Thioamide is a well-known amide isosteric replacement.1
Despite the subtle differences between the two groups (amide
oxygen converted into a sulfur in thioamides), they present
distinct structural and biological properties.1 The length of the
C=S bond (~1.65 Å) is known to be longer than the C=O (~1.19–
1.25 Å) bond due to the larger size of the sulfur atom.2,3
Additionally in thioamides the N-H group acts as a stronger
hydrogen bond donor than in its amide congener, and vice versa
the sulfur acts as a weaker hydrogen bond acceptor than the
oxygen carbonyl.4 In fact, previous studies revealed that the
hydrogen bond interaction in O is dominated by a charge-charge
attraction, whereas in S the interaction is mainly stabilized by a
weaker charge-quadrupole.5 The O-to-S substitution has been
widely applied to investigate the contribution of hydrogen bond
formation to protein secondary structures.6–11 In contrast,
thioamide replacement to probe protein–ligand interactions
remains understudied.
In recent years a series of potent, selective and cell-active
inhibitors of the von Hippel-Lindau (VHL) E3 ubiquitin ligase
have been designed and optimized around a key hydroxyproline
(Hyp) anchor fragment.18–21 VHL inhibitors act on their own as
chemical probes of hypoxia signaling.22 In addition, VHL ligands
are widely used as E3 ligase ligands for PROTAC (Proteolysis-
targeting chimera) conjugation, a strategy for targeted protein
degradation.23–28 The modified Hyp residue is crucial also for the
recognition of the hypoxia inducible factor 1 alpha subunit (HIF-
1), the natural substrate of VHL.29,30 A closer analysis of the X-
ray co-crystal structure of VHL, elongin B, elongin C (VBC) in
complex with inhibitor VH032 (PDB 4W9H)20 and HIF-1α
peptide (PDB 4AJY)19, suggested a conformation compatible
with an n→π* interaction between the two amide carbonyl
groups around the hydroxyproline core (Fig. 1B). We therefore
hypothesized that this protein–ligand system could be probed
through thioamide substitution.
In addition to affecting hydrogen bonding, thioamide
substitution also modulates the strength of the n→π*
interaction,1,12,13 which has been identified in several systems,
including proteins and small molecules.1,10,12–16 In this interaction
the lone pair (n) electron density of a donor group (carbonyl
oxygen or thiocarbonyl sulfur) overlaps with the antibonding
orbital (π*) of an acceptor group (a second carbonyl or
thiocarbonyl). This overlap is maximized when the donor and
acceptor group form a short contact in the Bürgi–Dunitz
trajectory (~107°, Fig 1A).12,15,17 Conversion of the donor group
from O to a softer base such as S increases the n→π* electronic
delocalization.12 Hence thioamides are better electron-pair
donors, increasing the strength of the interaction.12
Fig. 1. n→π* interaction in prolines and VHL inhibitors. A) Notion of
proline backbone carbonyl-carbonyl n→π* interaction. B) Crystal
structure of VBC (omitted) in complex with inhibitor VH032 (green
carbons) (PDB 4W9H). Possible n→π* interaction in VHL ligands is
shown as a black dashed line.

We began by designing a series of thioamide derivatives of
the model VHL ligand 1,21 bearing single as well as double O-to-
S substitutions (Fig. 2). The general procedure for the synthesis
of thioamide derivatives 2–4 is summarized in Scheme 1 and
briefly described in the following paragraph (see Supporting
information for detailed synthesis protocols).
labeled high-affinity HIF peptide (Table 1). The results from both
assays revealed an excellent agreement on the compounds
affinity rank (Fig. 3A and B). The highest affinity compound 3,
which bears a single thioamide conversion at the left-hand side,
revealed comparable binding to the unmodified ligand 1, with
only a small two-fold loss in binding affinity, as measured by
both techniques. In contrast, ligand 2 (single right-hand side
thioamide) showed a greater loss in binding affinity (10-fold loss
by FP, and 20-fold loss by ITC). Double substitution in ligand 4
resulted in the weakest binder of this series, with Kd > 10 M.
We next evaluated the impact of thioamide substitutions to the
n→π* interaction in the ligand free state in solution. This
interaction is only possible when the Hyp (thio)amide group is in
the trans isomer conformation (Fig. 1A). Thus, the ratio between
the trans and cis isomers at equilibrium can be used to infer the
strength of the n→π* interaction.13 Since both cis and trans
isomers of proline backbone amides are populated at room
temperature, they can be monitored by NMR spectroscopy due to
their slow interconversion, as demonstrated by Newberry et. al.13
The NMR results (Table 1) revealed a small decrease on the trans
isomer when the acceptor group was substituted by a thioamide
(2) and vice versa a small increase of this isomer was observed
when the substitution was made on the donor group (3). This
results support the stronger n→π* donor character of thioamides
when compared with amides.12,13 However, in contrast to what is
observed for simple proline models13,14, only modest differences
in trans isomer stabilization in compounds 2 and 3 could be
observed (between ± 2–3%).
We performed high-level density functional theory (DFT)
calculations of the n→π* contribution in model compounds 10–
13, in which the substituents at the right- and left-hand side of
inhibitors 1–4 were omitted for simplicity (see Supp. Fig. 6 and
7). The results, summarized in Table 1 (see Supp. Table 2 for full
details), show that the donor sulfur compound 12 presents an
n→π* stabilization energy (En→π*) of 2.6 kcal/mol, higher than
the parent derivatives 10 and 11 and in good agreement with the
NMR results. The n→π* contribution in compound 13, in which
both carbonyls are converted to thiocarbonyls, is further
increased to 3.4 kcal/mol, indicating a better overlap of the
corresponding molecular orbitals in this compound, and
consistent with previous calculations by Newberry et al.13 Those
results would suggest that 4 should have the highest population
of trans isomer among the studied compounds. Conversely, full
thioamide replacement in compound 1 to yield compound 4 led
to an unexpected 2% decrease on the trans isomer as observed by
NMR. Therefore, the conformational preferences of larger
compounds 1–4 cannot be reliably studied using surrogate model
compounds 10–13. Importantly, the trans:cis ratios as observed
by NMR did not correlate with binding affinities. This motivated
us to instead carry out a detailed crystallographic analysis of the
Fig. 2. Chemical structure of inhibitor 1 and thioamide derivatives 2–4.
The thioamide derivatives 2–4 were obtained from
intermediate 5, which was synthesized as previously described.20
Boc deprotection of 5 followed by amide coupling with
thioamide derivative
A
((S)-tert-Butyl (1-(6-nitro-1H-
benzo[d][1,2,3]triazol-1-yl)-1-thioxopropan-2-yl)carbamate,
prepared according to literature31) led to intermediate 7 (71%).
Boc deprotection of this intermediate followed by acylation
yielded the final compound 3 (76%) bearing a single thioamide at
the left-hand side. To introduce the O-to-S substitution at the
right-hand side, the Hyp hydroxyl group of intermediate 5 was
first protected with TBSCl, followed by thioamide conversion to
give intermediate 6 (68%). Subsequent Boc deprotection of 6 and
HATU (1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium 3-oxid hexafluorophosphate)-assisted amide
coupling with Boc-protected alanine led to 8 (75%). Boc
deprotection of 8 followed by acylation yielded the final
thioamide 2 (69%). To obtain the final doubly substituted
compound, deprotected intermediate
6 was reacted with
thioamide derivative A to give intermediate 9 (49%). Boc
deprotection of this intermediate followed by acylation led to the
final thioamide derivative 4 (64%).
We also attempted to introduce the thioamide substitution on a
more potent analogue of compound 1, the VHL inhibitor VH032,
which bears a tert-leucine moiety instead of an alanine.20–22
However the conversion of the amide on the left-hand side of the
hydroxyproline core was not accomplished. In fact, as observed
by Engel-Andreasen et al.,32 the presence of the hindered tert-
butyl group near a carbonyl could prevent its conversion to
thiocarbonyl.
The binding affinity of the newly synthesized compounds to
VHL was evaluated by two orthogonal assays: a direct binding
assay using isothermal titration calorimetry (ITC) and a
competition assay using fluorescence polarization (FP), which
monitored the compound’s ability to displace a fluorescently-
Scheme 1. Synthesis of thioamide compounds 2–4. (i) TFA:DCM (3:7), r.t., 1h; (ii) activated thioamide (A), DIPEA, DMF, r.t., 3h; (iii) acetic anhydride,
Et3N, DCM, r.t., 3h; (iv) TBSCl, imidazole, DMF, r.t., o.n.; (v) ammonium O,O'-diethyl dithiophosphate, toluene, reflux, o.n.; (vi) TFA:DCM (1:9), r.t., 2h;
(vii) Boc-Ala-OH, HATU, DIPEA, DMF, r.t., 2h.

VHL:inhibitor interactions to provide structural insights on the
observed differences in binding affinities.
and is consistent with the thioamide group being a weaker
hydrogen bond acceptor compared to the amide group.33
Together, our data reveal a key role of the Tyr98 side chain
hydrogen bond in VHL ligand binding.
We obtained high-resolution X-ray crystal structures of
compounds 2–4 in complex with VBC (Supporting information
Fig. 4, see Table 1 from Supporting information for data
collection and refinement statistics). The omit difference electron
density (Fo–Fc) observed unambiguously identified the inhibitor
bound at the expected VHL site (Fig. 4A and Supporting
information Fig. 2). The new structures revealed that the
noncovalent interaction network between the compounds and
VHL residues were fully conserved and consistent with those
observed with previous inhibitors (Fig. 4B).20 Superposition of
the crystal structures of VBC in complex with compounds 1–4
also revealed that thioamide conversion induced a slight change
in the position of the Tyr112 side chain (Fig. 4C). The presence
of the thioamide on the left-hand side of Hyp resulted in Tyr112
bending slightly to accommodate the bulkier group. Additionally
we measured the hydrogen bond distances between the
compounds right-hand side O/S acceptor groups and the side
chain hydroxyl of Tyr98. An increase in hydrogen bond distances
was observed for compounds 2 and 4 (3.0±0.1 and 2.95±0.05 Å
for O–H···S=C groups, respectively) compared to 1 and 3
(2.52±0.05 Å for O–H···O=C groups, on both compounds). This
trend could be explained by the increased van der Walls radius
and decreased electronegativity of sulfur compared to oxygen5,
We next performed molecular mechanics calculations on each
of the ligand:VHL complexes using the generalized-Born surface
area (MM-GBSA) approximation and estimated interaction
energies (EMM-GBSA) between the ligands and the protein. The
predicted EMM-GBSA values are in excellent agreement with
experimental enthalpic contributions to binding (∆H, Fig. 3C),
and in good agreement with Gibbs free energies (∆G, Supporting
information Fig. 8). We therefore evaluated the interaction
energy of each protein amino acid in the complexes with its
surrounding to determine differences in relative EMM-GBSA
compared to reference inhibitor 1. The results, shown in Table 1,
indicate that the loss in binding affinity observed in compounds 2
and 4 is largely contributed by destabilization of Tyr98 and, to a
much lesser extent, of Tyr112, especially in compound 4. Thus,
the molecular modeling calculations provided
a
solid
computational model to predict the impact of O-to-S substitutions
of amides in binding affinity and enabled quantification of the
subtle structural changes observed in the crystal structures of
VHL in complex with the inhibitors.
Table 1. SAR results and computational data of compounds 1–4. FP back calculated Kds, ITC measured Kds and ∆H; % of observed
trans and cis isomers in solution measured by NMR; stabilization energy of the n→π* interaction quantified by DFT calculations; and
estimation of interaction energies of the VHL:compound complexes and destabilization of pocket residues Tyr98 and Tyr112 upon
binding the thioamide derivatives, as quantified by MM-GBSA calculations.
Rel. EMM-GBSA Rel. EMM-GBSA
Kd FP
(μM)
Kd ITC
(μM)
∆H
(kcal/mol)
trans:cis
(%)
En→π*
EMM-GBSA
(kcal/mol) (kcal/mol)
Compound
of Tyr98
of Tyr112
(kcal/mol)
(kcal/mol)
1
2
3
4
0.69±0.03
7.07±0.10
1.0±0.4
> 13
0.44±0.04
9.4±0.3
-9.12±0.07
-5.37±0.05
92:8
89:11
94:6
2.1
2.0
2.6
3.4
-110.9
-103.7
-104.9
-98.4
-
-
2.3
0.1
3.3
0.0
0.1
0.8
0.76±0.02 -6.79±0.013
21.6 ±0.8 -2.56±0.012
90:10
Fig. 3. Biophysical characterization of compounds 1–4 binding to VHL. A) Competitive fluorescence polarization binding assay curve of
compounds displacing a 20-mer FAM-labeled HIF-1α peptide bound to VBC (Kd = 3 nM). B) ITC direct titration of compounds into VBC
protein complex. C) Predicted EMM-GBSA versus experimental ΔH for compounds 1–4 binding to VBC.

Fig. 4. Co-crystal structures of compounds 1–4 in complex with VBC. A) The omit difference electron density (Fo-Fc)
superimposed around 3 is shown in blue contoured at 3σ and with a 2.0 Å carve radius. B) Detailed binding interactions of
compound 3 (yellow carbons) with VHL pocket residues. VHL residues forming the binding pocket are shown as orange stick
representations. Water forming hydrogen bond with the compound is shown as a red sphere. Hydrogen bond interactions
between compound, bound waters and VHL pocket residues are shown as black dashed lines. C) Superposition of VBC
structures in complex with compounds 1 (green carbons), 2 (light blue carbons), 3 (yellow carbons) and 4 (purple carbons)
showing details of VHL binding pocket. Residues around hydroxyproline core are presented with the same color as the
respective bound compounds. Hydrogen bond interaction between compounds and Tyr98 is shown as black dashed lines.
In summary we describe the synthesis and biophysical
characterization of a series of thioamide derivatives of a potent
VHL inhibitor. In spite of a fully conserved binding mode, the
pattern of substitution had markedly varying effects on binding
affinities. Substitution at the left-hand side amide was tolerated,
while substitution at the right-hand side had the most detrimental
effect, highlighting the prevalent role of the Tyr98 hydrogen
bond in molecular recognition. Molecular modeling calculations
could recapitulate the trends in binding affinities observed
experimentally and provided a theoretical framework for
understanding the subtle structural changes observed
crystallographically. The results of this study could prove useful
to future drug design of VHL inhibitors for PROTACs. More
generally, we provide a combined biophysical, structural and
modeling characterization cascade that could be applied to study
the role of thioamide substitutions in other protein–ligand
interaction systems.
Source for beamtime (BAG proposal MX10071) and beamline
support at beamline I04-1.
Supplementary Material
Inhibitors synthesis protocols and NMR data, FP displacement
assay protocol and data, ITC protocol and data, crystallographic
protocol and refinement statistics, and computational methods
and data are presented in the supporting information.
Accession codes
PDB accession codes of VBC in complex with 2–4 are 6FMI,
6FMJ and 6FMK, respectively. Authors will release the atomic
coordinates upon article publication.
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Acknowledgments
This work was supported by the European Research Council
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