Dynamic Combinatorial Chemistry to Identify Binders of ThiT, an S-Component of the Energy-Coupling Factor Transporter for Thiamine

Article abstract of DOI:10.1002/cmdc.201700440

We applied dynamic combinatorial chemistry (DCC) to identify ligands of ThiT, the S-component of the energy-coupling factor (ECF) transporter for thiamine in Lactococcus lactis. We used a pre-equilibrated dynamic combinatorial library (DCL) and saturation-transfer difference (STD) NMR spectroscopy to identify ligands of ThiT. This is the first report in which DCC is used for fragment growing to an ill-defined pocket, and one of the first reports for its application with an integral membrane protein as target.

Full text of DOI:10.1002/cmdc.201700440

Accepted Article
Title: Dynamic combinatorial chemistry to identify binders of ThiT,
an S-component of the energy-coupling factor transporter for
thiamine
Authors: Leticia Monjas, Lotteke J. Y. M. Swier, Inda Setyawati, Dirk
Jan Slotboom, and Anna Katharina Herta Hirsch
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To be cited as: ChemMedChem 10.1002/cmdc.201700440
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10.1002/cmdc.201700440
ChemMedChem
Dynamic combinatorial chemistry to identify
binders of ThiT, an S-component of the energy-
coupling factor transporter for thiamine
Leticia Monjas,§a Lotteke J. Y. M. Swier,§b Inda Setyawati,b Dirk J. Slotboom*b and Anna K. H.
Hirsch*a,c
a
Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen,
The Netherlands.
b
Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands. E-mail: d.j.slotboom@rug.nl; Tel:
+31 50 363 4187.
C
Current address: Department of Drug Design and Optimization, Helmholtz Institute for
Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research and
Department of Pharmacy, Medicinal Chemistry, Saarland University, 66123 Saarbrücken,
Germany. E-mail: Anna.Hirsch@helmholtz-hzi.de; Tel: +49 681 98806 2100.
§ These authors contributed equally to this work.
Supporting Information (SI) available: [Figures of STD-NMR spectra and experimental details,
including images of NMR spectra of the compounds].
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10.1002/cmdc.201700440
ChemMedChem
Abstract
We applied dynamic combinatorial chemistry (DCC) to identify ligands of ThiT, the S-
component of the energy-coupling factor (ECF) transporter for thiamine in Lactococcus lactis.
We used a pre-equilibrated dynamic combinatorial library (DCL) and saturation-transfer
difference (STD)-NMR to identify ligands of ThiT. This is the first report in which DCC is used for
fragment growing to an ill-defined pocket, and one of the first reports for its application with an
integral membrane protein as target.
2
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ChemMedChem
Dynamic combinatorial chemistry (DCC) is a powerful tool for hit identification and
optimization. Over the past 20 years, DCC has been successfully applied in medicinal chemistry
[1]–[3]
and chemical biology for the discovery of binders to DNA, RNA and protein targets.
The
DCC technique involves the generation of a library of compounds by reversible reaction of
different building blocks. The main advantage of DCC is that several potential ligands for a
protein can be screened simultaneously, avoiding the individual synthesis, purification and
biochemical evaluation of every member of the dynamic combinatorial library (DCL). Up to date,
there are only a few reports in which DCC has been applied to identify binders of
transmembrane proteins.[4]–[6] Here, we have applied DCC to identify possible binders for ThiT,
the S-component of the energy-coupling factor (ECF) transporter for thiamine (Figure 1A) in
Lactococcus lactis.[7],[8] ECF transporters represent an interesting target for the development of
antibacterial agents with a novel mode of action by blocking vitamin transport.[9]
Figure 1. A) Structures of thiamine and deazathiamine. B) Crystal structure of ThiT in complex with thiamine (PDB
ID: 3RLB):[8] close-up of the thiamine binding pocket, with ThiT shown in surface representation, except for the
Trp34 residue and thiamine that are shown in stick representation to visualize the substrate binding pocket,. Color
code: ThiT in gray, with the residues of the loop L1 (residues Leu26–Ile39) highlighted in darker gray, and thiamine
with the C atoms in green, O in red, N in blue and S in yellow.
In our previous work, we studied binding of thiamine derivatives to ThiT, including compounds
that occupy a subpocket within the substrate binding pocket of ThiT at the hydroxyl end of
thiamine (Figure 1B).[10]–[12] Although we described strong binders (KD values in the nano- and
micromolar range), our predicted KD values did not always correlate very well with the
experimental values. A possible explanation is that when the unliganded substrate binding
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10.1002/cmdc.201700440
ChemMedChem
pocket is ‘open’ to the surrounding solvent (the loop L1 is in a different conformation), this
subpocket adopts a different conformation than the one in the available crystal structure, in
which the loop L1 closes the substrate binding pocket as a lid (PDB ID: 3RLB).[8] This could then
be the reason why structure-based design was unsuccessful in this case.
In order to obtain new extended thiamine derivatives, we here use DCC for fragment growing,
maintaining the deazathiamine moiety (Figure 1A), and screen various substituents to occupy
the possibly flexible part of the thiamine binding pocket. Fragment growing by DCC has been
done but not into flexible pockets.[13] Identification of ligands for ill-defined pockets is
challenging and DCC is an ideal method to address this challenge. We used an acylhydrazone
motif as a linker, which has been extensively used in DCC,[14],[15],[1]–[3] and selected aldehyde A
and eight hydrazides (H1–H8) to form a small pre-equilibrated dynamic combinatorial library
of eight acylhydrazones (AH1–AH8) (Scheme 1).
Scheme 1. Dynamic combinatorial library (DCL) to afford acylhydrazones as binders of ThiT.
We synthesized aldehyde A as previously described.[10] Hydrazides H1–H8 were obtained from
their corresponding methyl esters (ethyl ester in the case of H6), which were commercially
available or synthesized by esterification of the corresponding carboxylic acid, using HCl in
methanol at reflux, in 91–93% yield. Next, the reaction of the corresponding ester with
hydrazine monohydrate at reflux afforded the hydrazides in 24–97% yield (details of the
synthesis are available in the Supporting Information).
We used saturation-transfer difference (STD)-NMR spectroscopy to identify which of the eight
compounds bound to ThiT. STD-NMR is a powerful technique to study protein-ligand
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ChemMedChem
interactions in solution. Usually, the concentration of ligand(s) is 10–100-fold the concentration
of protein, which allows to work with low protein concentrations (in the micromolar range).[16]
1H-STD-NMR spectroscopy has been successfully applied in combination with DCC in some
studies,[17],[18] and in a few cases to study ligand binding to transmembrane proteins.[19],[20]. In
previous reports in which STD-NMR spectroscopy was applied to transmembrane proteins,
these proteins were embedded in the lipid bilayer of a liposome or in a membrane preparation
derived from cells, and not in detergent solution as in our case. Hence we wanted to show that
DCC can be conveniently analyzed by STD-NMR also for a transmembrane protein in detergent
solution. Using ThiT in a detergent-solubilized state allowed us to work at higher protein
concentrations than if we would have performed the additional reconstitution step into
liposomes. In addition, having the protein solubilized in a detergent micelle eased the buffer-
exchange procedure that we performed to obtain a sample of ThiT in deuterated buffer. For our
target, we first ran a control experiment with ThiT and a known binder (B1) (Figure S1). This
control experiment served two purposes: first, the amounts of ThiT that we can obtain are not
1
enough to record a H-NMR spectrum to identify a suitable irradiation frequency (i.e., a
frequency where only the protein resonates and not the ligands); second, we wanted to
establish whether the conditions we use are optimal and enable us to detect a known binder.
The final concentration of ThiT in 500 μL deuterated buffer (pD 7.0) in the NMR tube was 7.8 μM
. Running the 1H-STD-NMR experiment using a 100-fold excess of ligand, and measuring for 11 h
irradiating at –1 ppm or –2 ppm, resulted in difference spectra featuring peaks corresponding to
our known binder B1, as well as the detergent (n-decyl-β-D-maltopyranoside) that is present in
the buffer (Figure S1). As a result, any of these frequencies are suitable for the experiment with
the DCL. We performed the experiment with our library of compounds using a 100-fold excess
of ligand and irradiation at –1.1 ppm for 11 h. To analyze our DCL, we divided it into two
sublibraries, containing the aldehyde and four hydrazides each as building blocks, selected in a
way that the characteristic NMR signals did not overlap. For the experiment with ThiT, the
building blocks were left to react in a buffer at pD 5.0 with shaking for 24 h. We checked by
UPLC-MS and NMR the DCL before adding the protein, and all the constituents were formed in
about equal amounts. Then, the DCL was added to the solution of ThiT in buffer at pD 7.0, given
than ThiT does not tolerate lower pD and therefore we had to use a static DCL,[21] and the on-
resonance and off-resonance spectra were recorded. For the first library, DCL-A, we included
the aldehyde A and the hydrazides H1, H2, H6 and H7. The second library, DCL-B, consisted of
the same aldehyde A and the hydrazides H3, H4, H5 and H8 (Figure S2). In DCL-A, we observed
that three of the four possible acylhydrazones bind to ThiT. In principle, after 24 h of incubation
1
to form the DCL, all of the aldehyde A was consumed according to H-NMR spectroscopy, but
apparently, a small amount was still in solution, and after many scans during the experiment
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1
with protein we could see it in the H-STD-NMR spectrum. In DCL-B, three of the other four
possible acylhydrazones emerged as binders of ThiT. Taken together, the analysis of both
libraries shows that all the compounds derived from aromatic hydrazides (AH1–AH6) bind to
ThiT, whereas the ones derived from aliphatic hydrazides (AH7 and AH8) do not bind.
We synthesized the six identified binders by reaction of aldehyde A and the corresponding
hydrazides H1–H6 at reflux in MeOH, affording the desired products AH1–AH6 in 32–86% yield
(Scheme 2).
Scheme 2. Synthesis of the acylhydrazones identified as binders of ThiT.
We determined the binding affinity of the synthesized acylhydrazones by isothermal titration
calorimetry (ITC), and tested the compounds as mixtures of E/Z isomers using ThiT stabilized
with detergent as we performed the STD-NMR experiments (Table 1). We observed that ThiT
has a higher binding affinity for acylhydrazones AH1, AH4 and AH5 than for acylhydrazones
AH2, AH3 and AH6.
Table 1. Binding affinities of ThiT for thiamine, deazathiamine and the acylhydrazones AH1–AH6 determined by ITC
with the errors indicated as standard deviations.
Compound
Thiamine[7]
Deazathiamine[10]
AH1
KD ± S.D. (μM)
(0.122 ± 0.013) x 10-3
(4.23 ± 1.69) x 10-3
5.30 ± 1.19a
AH2
28.8 ± 6.89b
AH3
20.9 ± 10.5a
AH4
3.02 ± 0.172a
AH5
8.41 ± 5.54c
AH6
44.5 ± 8.06a
a–c The error represents the standard deviation obtained from a4, b6 or c3 experiments.
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ChemMedChem
In conclusion, we successfully applied DCC in combination with STD-NMR spectroscopy to
identify binders of ThiT. The advantage of this method is that it requires a low concentration of
unlabeled protein (in the micromolar range), which is particularly advantageous for proteins
that are difficult or expensive to produce, such as integral membrane proteins. The
disadvantages are the limited size of the library and the requirement to determine the 1H-NMR
reference spectrum of each individual product for comparison with the 1H-STD-NMR spectrum.
This study is the first example in which DCC is applied to a challenging target for fragment
1
growing to an ill-defined pocket. In addition, it is one of the first applications of H-STD-NMR
spectroscopy to transmembrane proteins, and the first one that uses detergents instead of
1
liposomes to embed the protein. The acylhydrazones identified by H-STD-NMR spectroscopy
bind to ThiT with KD values in the micromolar range. Comparing their binding affinities with
similar compounds,[12] in which most of the compounds show KD values in the nanomolar range,
the acylhydrazones are weaker binders. We need to take into account, however, that the KD
values were determined for mixtures of E/Z isomers, and probably only one of them binds with
high affinity to ThiT. Furthermore, the linker required for acylhydrazone formation makes these
compounds longer and more rigid than those previously reported. As a result, the compounds
may not be able to adopt a favorable conformation to interact with ThiT, explaining the
decrease in binding affinity. Even if the acylhydrazones are not better binders than the
compounds reported previously, it is remarkable that DCC enables fragment growing into
flexible pockets that cannot be addressed by structure-based design, opening up opportunities
in medicinal chemistry.
Acknowledgments
The authors acknowledge Pieter van der Meulen for useful suggestions and technical support of
STD-NMR spectroscopy, and Dr. Milon Mondal for useful discussions about DCC. The research
leading to these results has received funding from the European Community's Seventh
Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement no. 283570),
the Ministry of Education, Culture and Science (Gravitation program 024.001.035), the
Netherlands Organisation for Scientific Research (NWO) (NWO ChemThem grant 728.011.104,
NWO Vidi grant 723.014.008 and NWO Vici grant 865.11.001) and the European Research
Council (ERC) (ERC Starting Grant 282083).
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Keywords:
Energy-coupling factor transporter – transmembrane protein – dynamic combinatorial
chemistry – saturation-transfer difference NMR spectroscopy– flexible protein
pocket
Table of contents text
1
We applied DCC and H-STD-NMR spectroscopy with detergent to identify binders for
the ECF transporter for thiamine that target the ill-defined pocket of this
transmembrane protein.
9
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