Conformational Aspects in the Design of Inhibitors for Serine Hydroxymethyltransferase (SHMT): Biphenyl, Aryl Sulfonamide, and Aryl Sulfone Motifs

Article abstract of DOI:10.1002/chem.201703244

Malaria remains a major threat to mankind due to the perpetual emergence of resistance against marketed drugs. Twenty-one pyrazolopyran-based inhibitors bearing terminal biphenyl, aryl sulfonamide, or aryl sulfone motifs were synthesized and tested towards serine hydroxymethyltransferase (SHMT), a key enzyme of the folate cycle. The best ligands inhibited Plasmodium falciparum (Pf) and Arabidopsis thaliana (At) SHMT in target, as well as PfNF54 strains in cell-based assays in the low nanomolar range (18–56 nm). Seven co-crystal structures with P. vivax (Pv) SHMT were solved at 2.2–2.6 ? resolution. We observed an unprecedented influence of the torsion angle of ortho-substituted biphenyl moieties on cell-based efficacy. The peculiar lipophilic character of the sulfonyl moiety was highlighted in the complexes with aryl sulfonamide analogues, which bind in their preferred staggered orientation. The results are discussed within the context of conformational preferences in the ligands.

Full text of DOI:10.1002/chem.201703244

DOI: 10.1002/chem.201703244
Full Paper
&
Drug Design
Conformational Aspects in the Design of Inhibitors for Serine
Hydroxymethyltransferase (SHMT): Biphenyl, Aryl Sulfonamide,
and Aryl Sulfone Motifs
Geoffrey Schwertz,[a] Michelle S. Frei,[a] Matthias C. Witschel,[b] Matthias Rottmann,[c, d]
Ubolsree Leartsakulpanich,[e] Penchit Chitnumsub,[e] Aritsara Jaruwat,[e] Wanwipa Ittarat,[e]
Anja Schꢀfer,[c, d] Raphael A. Aponte,[b] Nils Trapp,[a] Kerstin Mark,[a] Pimchai Chaiyen,[f, g] and
FranÅois Diederich*[a]
Abstract: Malaria remains a major threat to mankind due to
the perpetual emergence of resistance against marketed
drugs. Twenty-one pyrazolopyran-based inhibitors bearing
terminal biphenyl, aryl sulfonamide, or aryl sulfone motifs
were synthesized and tested towards serine hydroxymethyl-
transferase (SHMT), a key enzyme of the folate cycle. The
best ligands inhibited Plasmodium falciparum (Pf) and Arabi-
dopsis thaliana (At) SHMT in target, as well as PfNF54 strains
in cell-based assays in the low nanomolar range (18–56 nm).
Seven co-crystal structures with P. vivax (Pv) SHMT were
solved at 2.2–2.6 ꢁ resolution. We observed an unprecedent-
ed influence of the torsion angle of ortho-substituted bi-
phenyl moieties on cell-based efficacy. The peculiar lipophilic
character of the sulfonyl moiety was highlighted in the com-
plexes with aryl sulfonamide analogues, which bind in their
preferred staggered orientation. The results are discussed
within the context of conformational preferences in the li-
gands.
Introduction
[a] G. Schwertz, M. S. Frei, Dr. N. Trapp, K. Mark, Prof. Dr. F. Diederich
Laboratorium fꢀr Organische Chemie, ETH Zurich
Vladimir-Prelog-Weg 3, 8093 Zurich (Switzerland)
E-mail: diederich@org.chem.ethz.ch
Medicinal chemists enjoy diverse computational tools to sup-
port their drug development efforts, going from pKa or logD
calculations to more sophisticated predictions of preferential
ligand docking modes by molecular modeling or protein–
ligand binding energies by free energy calculations.[1–3] Dock-
ing and modeling of protein–ligand interactions have become
a common routine to prioritize ideas for synthesis. The gain in
Gibbs protein–ligand binding energy becomes enhanced with
increasing degree of preorganization of the ligand; in other
words, the preferred conformations of bound and unbound li-
gands should be similar.[4,5] A difference in conformational
energy between free and bound state of DDGunbound!bound
ꢀ1.4 kcalmolꢁ1 already translates into a tenfold decrease in
Gibbs binding energy.[6,7] Some reports suggest to set the
threshold for the bioactive conformation at 3 kcalmolꢁ1 of the
lowest energy conformation,[6,8] whereas others give a maxi-
[b] Dr. M. C. Witschel, Dr. R. A. Aponte
BASF SE, Carl-Bosch-Strasse 38, 67056 Ludwigshafen (Germany)
[c] Dr. M. Rottmann, A. Schꢁfer
Swiss Tropical and Public Health Institute (SwissTPHI)
Socinstrasse 57, 4051 Basel (Switzerland)
[d] Dr. M. Rottmann, A. Schꢁfer
Universitꢁt Basel, Petersplatz 1, 4003 Basel (Switzerland)
[e] Dr. U. Leartsakulpanich, Dr. P. Chitnumsub, A. Jaruwat, W. Ittarat
National Center for Genetic Engineering and Biotechnology
113 Thailand Science Park, Phahonyothin Road
Pathumthani 12120 (Thailand)
[f] Prof. Dr. P. Chaiyen
Department of Biochemistry and Center for Excellence in Protein and
Enzyme Technology, Faculty of Science Mahidol University
272 Rama VI Road, Bangkok 10400 (Thailand)
[g] Prof. Dr. P. Chaiyen
Department of Biomolecular Science and Engineering
School of Biomolecular Science & Engineering
Vidyasirimedhi Institute of Science and Technology (VISTEC)
Wangchan Valley, Rayong 21210 (Thailand)
mum Gibbs energy difference of 5 kcalmolꢁ1 [1,9]
While the en-
.
ergetics of the conformations of free and bound ligand can be
evaluated by computational methods,[5,10] conformational pref-
erences of small molecules are best extracted from searches in
the Cambridge Structural Database (CSD),[11–14] reaching more
than 875000 entries in 2017.[15,16] The potential of conforma-
tional searches in the CSD was nicely illustrated by Brameld
et al.,[7] who reported a comprehensive study on most of the
structural motifs used in medicinal chemistry. Recently Cottrell
et al. expanded this CSD search to various ring fragments.[17]
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under:
https://doi.org/10.1002/chem.201703244. It contains all synthetic proce-
dures, characterizations of all new compounds, and their H/13C/19F NMR
spectra; detailed biological activities; additional data from the CSD and
PDB searches; theoretical calculations; additional figures on protein-ligand
interactions and design of ligands by modeling; and small-molecule crystal
structures data. The coordinates files of the PvSHMT-ligand complexes were
deposited in the PDB with the following access codes: 5XMP, 5XMQ, 5XMR,
5XMS, 5XMT, 5XMU, and 5XMV.
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Herein, we report two new series of ligands based on a pyra-
zolopyran core that inhibit the enzyme serine hydroxymethyl-
transferase (SHMT) and analyze how their molecular conforma-
tions profoundly affect binding geometry and biological activi-
ty. This enzyme is a crucial component of the folate synthesis
cycle,[18,19] which is an essential pathway for the replication of
Plasmodium parasites causing malaria. SHMT is a pyridoxal 5’-
phosphate (PLP)-dependent enzyme catalyzing the one-
carbon-unit transfer from serine to tetrahydrofolate (H4F)
(Figure 1).[20–24] We previously reported pyrazolopyran-based li-
gands as the first inhibitors of Plasmodium falciparum (Pf)
SHMT with both target affinities IC50 (half maximal inhibitory
concentration) and high in vitro efficacy EC50 (half maximal ef-
fective concentration) against the P. falciparum strain NF54 in
the low nanomolar range, as illustrated for (ꢂ)-1 (Figure 1c).[25]
Lately, we showed that optimized inhibitors targeting SHMT
induce a significant reduction of parasitemia in vivo in a
mouse model.[26] In this optimization program, we also investi-
gated a series of seven ligands bearing an ortho-substituted
terminal phenyl ring, replacing the thiophene ring in (ꢂ)-1. We
report here the influence of the nature of the ortho-substituent
on biological activity. The torsion angles of ortho-substituted
biphenyl motifs were investigated in CSD and Protein Data
Bank (PDB)[27,28] searches, as well as by a computational study,
in order to understand the observed conformational ef-
fects.[29–31] We show that the torsion angle of the biphenyl frag-
ment does not affect target affinity much, but rather influences
cell-based efficacy with a steep structure–activity relationship
(SAR), presumably by impacting cell-penetration and/or trans-
porter-mediated uptake and efflux. In parallel, a second series
of fourteen ligands, incorporating aryl sulfonamide or aryl sul-
fone moieties, was prepared and studied. We aimed at improv-
ing binding affinities to PfSHMT by taking advantage of the
distinct conformational preferences of these fragments[7,32–41]
to establish interactions of lipophilic substituents on the ligand
with the hydrophobic residues lining the channel (Figure 1c),
which hosts the para-aminobenzoate (pABA) side chain of the
natural substrate H4F. However, no significant gain in inhibitory
activity could be measured with those extended ligands. From
new co-crystal structures with Plasmodium vivax (Pv) SHMT, we
document the profound preference of the sulfonyl moiety in
the ligands to bind into hydrophobic, rather than hydrophilic
environments, thereby exposing the hydrophobic substituents
to solvent. Seven new co-crystal structures, paired with small-
molecule X-ray crystallography, also enabled a comparison be-
tween the conformations of the bound and unbound biphenyl,
aryl sulfonamide, and aryl sulfone ligands.
Results and Discussion
Ligand design
Molecular modeling with MOLOC,[42] using the two previously
obtained co-crystal structures with pyrazolopyran-based inhibi-
tors,[25] guided the structural modifications of the ligands. A
schematic representation of the binding mode of (+)-1 at the
active site of PvSHMT (PDB ID code: 4TMR) is depicted in Fig-
ure 1c. Two sub-pockets are conspicuous: the pyrazolopyran
core binds into the pteridine binding pocket, which normally
hosts the pteridine core of H4F (PDB ID code: 4OYT).[24] The
second sub-pocket is the pABA channel, which is occupied by
the pABA side chain of H4F. We designed two new series of li-
gands to establish improved interactions with the hydrophobic
amino acid residues lining this channel. The pyrazolopyran
core and its alkyl substituents (methyl and isopropyl) were
kept intact. The CN moiety of (+)-1 on the phenyl ring depart-
Figure 1. a) The one-carbon-unit transfer reaction catalyzed by SHMT. Dihydrofolate reductase (DHFR), SHMT, and thymidylate synthase (TS) are the three en-
zymes involved in the folate cycle. SHMT converts H4F to 5,10-methylenetetrahydrofolate (5,10-CH2-H4F), a crucial component for the conversion of deoxyuri-
dine monophosphate (dUMP) to the DNA precursor deoxythymidine monophosphate (dTMP) by TS. b) Molecular structure of H4F. c) Schematic depiction of
the binding mode of the initial lead (+)-1 as seen in a co-crystal structure analysis (PDB ID code: 4TMR).[25] Intermolecular hydrogen bonding is indicated by
red dashed lines.
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ing from this core was exchanged for a CF3 substituent, which
was found to considerably improve ligand potency.[26] All other
modifications are reported in the following.
the overall conformation, for instance by sulfur/lone-pair inter-
action (chalcogen bonding) as reported recently.[9]
ortho-Substituted biphenyl analogues (ꢂ)-2–8 (Table 1) were
at first prepared to improve the intermolecular interactions
with hydrophobic residues within the pABA channel, such as
Tyr63, Leu124, Leu130, and Phe134 (Figure 1c). For instance,
modeling of (ꢂ)-8 suggested the establishment of several fa-
vorable contacts with these four residues and additionally with
Phe266 and Pro267 (Supporting Information, Section S5, Fig-
ure S12). Nevertheless, the unsubstituted analogue (ꢂ)-2 was
the most active ligand of the series in the target assay against
PfSHMT (IC50 =111 ꢂ5 nm, Table 1) and introduction of larger
substituents led to weaker binding. IC50 values were also mea-
sured against Arabidopsis thaliana (At) SHMT to refine the SAR,
as the plant enzyme and PfSHMT are highly similar with 45%
of sequence identity.[26,46] Stronger inhibition was obtained on
AtSHMT with IC50 values ranging from 7.3 to 32.0 nm (Table 1).
Overall, a rather flat SAR was revealed in both target-based
assays. In contrast, the nature of the ortho-substituent exerted
a significant influence on the cell-based potency (EC50) and a
steep SAR was obtained. Increase in size of the ortho-substitu-
ent led to considerable decrease in antiparasitic efficacy from
EC50 =18 nm ((ꢂ)-2) to 665 nm ((ꢂ)-8) (Table 1). No direct corre-
lation between cellular potency and lipophilicity (clogP) is dis-
tinguishable.
Synthesis
The same synthetic pathway[25,26] was followed to prepare all
pyrazolopyran-based ligands ((ꢂ)-2–22) reported in this manu-
script (Scheme 1; for the full structures of all ligands, see
Tables 1 and 2 below). Brominated 23 was either converted
into the pinacol boronate ester 24 prior to a Suzuki cross-
coupling[43] (for (ꢂ)-2–20) or directly subjected to a Buchwald–
Hartwig cross-coupling[44] (for (ꢂ)-21 and (ꢂ)-22) leading to in-
termediates 25a–u. A subsequent Knoevenagel condensa-
tion[45] gave access to the dinitrile precursors 26a–u needed
for the one-pot synthesis of the pyrazolopyran core involving a
Michael addition of 3-methyl-1H-pyrazol-5(4H)-one, followed
by an intramolecular cyclization to provide (ꢂ)-2–22 (for de-
tails, see the Supporting Information, Section S1).
To explain these observations, we examined the torsion
angles (t) of biphenyl motifs in the CSD and the PDB.[47] An in-
crease in size of an ortho-substituent directly translates into a
larger torsion angle between the two ring planes (Table 1), sim-
ilarly to the report by Brameld et al.[7] (Figure 2). However, it is
essential to keep in mind that differences may occur between
the solid and solution phase and that unsubstituted biphenyl
is especially susceptible to crystal packing effects.[48] For in-
stance, the torsion angle for biphenyl without any substituent
is located around 358 (Figure 2a) and is slightly lower than the
average torsion angle in the gas phase of 448.[48] By introduc-
ing any substituent, the torsion angle shifts to higher values
located above 508 (Figure 2b and Supporting Information, Sec-
tion S3, Figure S1). A small-molecule X-ray crystal structure re-
corded for (ꢂ)-6 displayed a torsion angle of 57.58, which is
nearly identical to the median value found in the CSD search
(Supporting Information, Section S6.2.1, Figure S24). Not sur-
prisingly, the ortho-CF3 biphenyl possesses the highest torsion
angle, although some caution is advised as only 6 structures
were found in the CSD (Table 1 and Supporting Information,
Section S3, Figure S1).
Scheme 1. Representative synthesis of pyrazolopyran-based ligands (ꢂ)-2–
22. Reagents and conditions: a) bis(pinacolato)diboron, KOAc,
[PdCl2(dppf)]·CH2Cl2 (2.0 mol%), toluene, mw, 1508C, 6 min, 99%; b) 23 and
corresponding aryl boronic acid or 24 and bromoaryl analogue,
[PdCl2(PPh3)2] (5.0 mol%), Na2CO3, THF/H2O 4:1, 608C, 4–16 h; c) 23, corre-
sponding amine, Cs2CO3, [Pd2(dba)3] (2.0 mol%), X-Phos (8.0 mol%), 1,4-diox-
ane, 1108C, 16 h; d) malononitrile, TiCl4, pyridine, CHCl3, 638C, 48–72 h; e) 3-
methyl-1H-pyrazol-5(4H)-one, piperidine, 1,4-dioxane/EtOH 1:1, mw, 65 8C,
3.5 h. dba=dibenzylideneacetone; dppf=1,1’-bis(diphenylphosphino)ferro-
cene; THF=tetrahydrofuran; X-Phos=2-dicyclohexylphosphino-2’,4’,6’-triiso-
propylbiphenyl; mw=microwave irradiaton.
Next, a potential energy scan (PES) at the B3LYP/cc-pVDZ
level of theory (in water with polarizable continuum solvent
model) using Gaussian09[49] was performed for each com-
pound by incremental rotation of the terminal ring around the
biphenyl axis (Supporting Information, Section S4, Figures S8
and S9). The most favorable biphenyl torsion angles calculated
for all compounds (ꢂ)-2–8 range from 36.58 to 62.08, in agree-
ment with the CSD search results. Additionally, the energy gap
between the least stable, co-planar geometry and the most
stable conformations increases with the size of the ortho-
substituent. For example, for (ꢂ)-2 without an ortho-substitu-
Biphenyl series
Biphenyl is the most common motif found in small-molecule
drugs, therefore, it is important to understand its preferred
conformation depending on its substitution pattern.[7] The ge-
ometry of a biphenyl motif can be tuned depending on intra-
molecular interactions. Modification of the substitution pattern
often aims at improving protein–ligand interactions by orient-
ing the two rings and the attached exit vectors in a precise
way. Incorporation of heteroatoms can also strongly influence
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Table 1. Biological activities of biphenyl analogues (ꢂ)-2–8.
EC50
PfNF54
[nm]
IC50
IC50
AtSHMT
[nm]
Biphenyl
Median
t [8][b]
Cpd.
PfSHMTꢂSD
clogP[c]
[nm][a]
R
(ꢂ)-
18
20
111 ꢂ5
144ꢂ0
263ꢂ11
165ꢂ4
471ꢂ8
289ꢂ13
330ꢂ22
18.6
7.3
30.3
43.2
55.5
51.3
56.8
46.1
71.8
5.2
4.9
5.3
5.5
5.5
4.7
6.0
2
(ꢂ)-
3
(ꢂ)-
27
30.9
20.9
32.0
26.2
18.5
4
(ꢂ)-
51
5
(ꢂ)-
81
6
(ꢂ)-
356
665
7
(ꢂ)-
8
[a] Standard deviations are given. [b] Derived from the CSD searches in CSD 5.38 (February 2017). [c] Calculated with ACD/Percepta (GALAS prediction
model) from ACD/Labs, release 2016.2.
ent, the energy difference between these two conformations is
2.4 kcalmolꢁ1, whereas it is calculated as 9.4 kcalmolꢁ1 for the
CF3 derivative (ꢂ)-8.
mention a study involving inhibitors of the ABCC2/MRP2 trans-
porters, in which the affinity for the transporter was influenced
by the conformation of a biphenyl moiety.[55] In this study,
larger torsion angles led to improved inhibition.
It is likely that the substituent-dependent conformation of
the biphenyl moiety influences the cell-based efficacy by af-
fecting cell permeation. Cell permeation occurs by means of
passive diffusion, carrier proteins (transporters), and channel
proteins.[50,51] Passive diffusion is thought to be the main con-
tributor to permeation and is less sensitive (or not sensitive at
all) to the stereochemistry of a drug, whereas configuration
and conformation matters with transporters.[50] Transporters
are complex systems to study and in P. falciparum their
number has not been clearly defined yet. Gardner et al. first re-
ported a limited number of transporters, around 50.[52] Later, a
bioinformatics analysis by Martin et al. revealed twice as many
transporters.[53] Carrier proteins can be drug targets, but play
also a role in resistance mechanism. Indeed, the P. falciparum
chloroquine-resistant transporter (PfCRT) is responsible for the
resistance developed towards chloroquine.[54] Regarding our
series of inhibitors (ꢂ)-2–8, it is conceivable that the biphenyl
moiety needs to adopt a conformation close to co-planarity for
the biphenyl to better penetrate into the cell. To our knowl-
edge, such an effect of a torsion angle on cell-based efficacy
has not been observed so far. Nonetheless, it is important to
Disparity in serum albumin binding across this series of li-
gands could be another explanation for the cell-based SAR.
However, our assay was supplemented with only 0.5% of AL-
BUMAXꢃ II and it is therefore rather unlikely that the discrep-
ancies in efficacy arose from protein binding. Finally, it should
be emphasized that cell permeation can be influenced by sev-
eral factors, such as lipophilicity, and that the measured cellular
efficacy is certainly not solely governed by the torsion angle of
the biphenyl motif.
Crystal structure determination of PvSHMT in complex with
(ꢂ)-3, (ꢂ)-4, (ꢂ)-5, and (ꢂ)-7
PvSHMT was crystallized from a ternary complex of PvSHMT,
glycine, and either (ꢂ)-3, (ꢂ)-4, (ꢂ)-5, or (ꢂ)-7 by using the
microbatch method. The co-crystals diffracted to 2.5, 2.4, 2.2,
and 2.6 ꢁ resolution, respectively, and belong to the C2 space
group. The structures were solved by molecular replacement,
using the coordinates of a chain A protomer of PvSHMT (PDB
ID code: 4OYT) as the template.[24] Despite using a racemic
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(+)-5, respectively, are in close proximity to Tyr63 and establish
favorable local direct interactions[56–61] with the phenolic ring.
On the other hand, the respective methyl and cyano moieties
of (+)-4 and (+)-7 point towards Cys364 (Figures 3b and 3d).
The torsion angle of (+)-4 is equal to 628, which sterically pre-
cludes the ortho-methyl group to lie below Tyr63. Indeed, with
such torsion angle, the Me group would be at a sub-van der
Waals distance to Tyr63. Instead of adopting a slightly different
conformation to avoid a steric clash, ring flip occurs thereby
enabling the Me group to interact with Leu124 and Cys364
(Figure 3b). Similarly, in order to avoid any repulsion of the
CꢃN moiety with the phenol ring of Tyr63, this substituent of
(+)-7 also points to Cys364 and lies almost orthogonally to
one CH3 group of Leu124 (Figure 3d). These structural distinc-
tions, however, only have little impact onto target affinities
(Table 1).
In the complexes with (+)-4 and (+)-5, a water molecule
(W1 or W2) solvating Tyr63 was resolved (Figures 3b and 3c).
Additionally, W2 is in the vicinity of the chloroarene ring of
(+)-5 and establishes a weak OꢁH···p interaction.[62] Solvation
of Tyr63 was also observed in a previous co-crystal structure of
a pyrazolopyran-based ligand with PvSHMT (PDB ID code:
5GVN).[26] Superimposition of the three co-crystal structures re-
vealed a quasi-identical positioning of W1, W2, and W3 nearby
Tyr63 (Supporting Information, Section S6.1.1, Figure S18).
Interestingly, in the second active site of the complex with
(+)-7 the cysteine bridge formed by Cys125 and Cys364 was
found to be in its oxidized form (Supporting Information, Sec-
tion S6.1.1, Figure S19). It is likely that the ligand bound to the
active site in its reduced form and after a prolonged seeding
period the disulfide bond was formed. Nevertheless, the bind-
ing mode of (+)-7 remained identical to the one where the di-
sulfide bridge is in its reduced form.
Figure 2. Torsion angle histograms derived from the CSD ligands (blue) and
bound ligands in the PDB (green). a) Biphenyls without any ortho-substitu-
ent. b) ortho-Substituted biphenyls.
mixture of ligands for co-crystallization, only the (+)-(S) enan-
tiomer was present in the structures. Importantly, both active
sites of PvSHMT were populated with a ligand in each complex
(Supporting Information, Section S6.1.1, Figure S15).
Aryl sulfonamide/aryl sulfone series
Binding modes of (+)-3, (+)-4, (+)-5, and (+)-7
Sulfonamide motifs are widely used in chemical research, par-
ticularly in drug discovery, as illustrated with the antimalarial
sulfadoxine,[63] and with the more recently approved drugs
Asunaprevir (treatment of hepatitis C virus), Belinostat (antitu-
mor agent), Glanatecꢃ (treatment of glaucoma and ocular hy-
pertension), or Vonoprazan fumarate (treatment of gastric
ulcer).[64] As elegantly described by Kuhn et al., sulfonamides
can be considered as “molecular chimeras, which are found to
form hydrogen bonds as well as interact with unipolar environ-
ments within proteins”.[8]
The pyrazolopyran core of (+)-3, (+)-4, (+)-5, and (+)-7 occu-
pies the pteridine binding pocket (Supporting Information,
Section S6.1.1, Figure S16), as previously seen in other com-
plexes with wild-type PvSHMT.[25,26] It establishes an array of
polar interactions, through the vinylogous cyanamide and the
pyrazole ring, with key amino acids of PvSHMT (Glu56, Leu124,
Gly128, and Thr357). The measured torsion angles, ranging
from 478 to 628 (Supporting Information, Section S6.1.1, Fig-
ure S17), are in good agreement with the CSD search (Table 1)
and with the optimized structures (B3LYP/cc-pVDZ) (Support-
ing Information, Section S4, Figure S7). Although, it should be
noted that the calculated structure of (+)-5 exhibits a dihedral
of 588, which is markedly different than the 498 measured in
the complex with PvSHMT.
We were interested in improving the binding affinities in our
class of inhibitors by harvesting favorable nonpolar contacts
with residues such as Lys139, Val141, Ser263, and Pro267 at
the exit of the pABA channel (Supporting Information, Sec-
tion S5, Figures S13 and S14). We implemented a sulfonamide
group into a series of extended derivatives (ꢂ)-9–17 to proper-
ly orient the terminal lipophilic moieties. In the modeling pro-
cess, care was taken to consider the preferred orientation of
aryl sulfonamide and aryl sulfone moieties. Previous studies
showed that the sulfonamide conformation can markedly influ-
ence target affinity as in the development of factor Xa inhibi-
The main difference between those four co-crystal structures
lies in the positioning of the ortho-substituent of the terminal
phenyl ring (Figure 3). On the one hand, the halo ligands (+)-3
and (+)-5 adopt an anti-parallel alignment of their CꢁF/CꢁCl
bond dipole relative to the CꢁO dipole of Tyr63 (Figures 3a
and 3c). The fluoride and chloride substituents in (+)-3 and
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Figure 3. Co-crystal structures showing the protein-ligand interactions of PvSHMT (grey) and pyrazolopyran ligands: a) (+)-3 (orange; PDB ID code: 5XMS,
2.5 ꢁ); b) (+)-4 (lime; PDB ID code: 5XMU, 2.4 ꢁ); c) (+)-5 (gold; PDB ID code: 5XMV, 2.2 ꢁ); d) (+)-7 (cyan; PDB ID code: 5XMT, 2.6 ꢁ). The surface spans the
volume of the pABA channel. Water molecules (W1 and W2) are represented as red spheres. PLP is omitted for clarity. Distances are given in ꢁ. Color code:
Cprotein grey, C(+)-3 orange, C(+)-4 lime, C(+)-5 gold, C(+)-7 cyan, Cl green, F light cyan, N blue, O red, S yellow.
tors.[38,39] There are two distinct dihedral angles in aryl sulfona-
mides and one in aryl sulfones that determine the conforma-
tional preferences of these fragments.
ed around 908, highlighting the conformation in which the p
orbital of the ipso-carbon atom bisects the SO2 angle. This fa-
vored conformation for aryl sulfonamides and aryl sulfones
was also computed by density functional theory (DFT) (Fig-
ure 4b and Supporting Information, Section S4, Figure S10),
which is in agreement with calculations performed in 2006.[37]
Comparable results were obtained when searching in the PDB
(Supporting Information, Section S3, Figure S2). However, a
broader distribution was observed due to the lower accuracy
in the determination of small-molecule conformation preferen-
ces in macromolecular X-ray structures, as several ligand poses
can be fitted to electron densities.[65]
The first is the torsion angle about the Csp2ꢁS bond in both
moieties. An extensive CSD search by Brameld et al. pinpointed
the favored conformation of aryl sulfonamides and aryl sul-
fones, in which the p orbital of the ipso-carbon atom bisects
the SO2 angle,[7] a conformation described for the first time in
1986 by Beddoes et al. in an X-ray crystallographic analysis.[33]
Since the number of structures deposited in the CSD increases
exponentially over time, we performed a new search to update
the results obtained by Brameld et al. (Figure 4a). In both
motifs the torsion angles converge towards a maximum locat-
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Figure 4. a) Torsion histogram derived from CSD ligands for aryl sulfona-
mides (blue) and aryl sulfones (green). b) Relative energy while driving the
dihedral angle t(C1-C2-S-N) of an aryl sulfonamide from 0–1808. DFT-B3LYP/
cc-pVDZ calculations carried out in water (with polarizable continuum sol-
vent model) using Gaussian09.[49]
Figure 6. a) Scatterplot for N,N-disubstituted sulfonamides derived from the
molecules in the CSD. The distance between N and the plane of its three
substituents is plotted against the Csp2-S-N-Csp3 torsion angle. The absolute
value of the larger of the two alternative Csp2-S-N-Csp3 torsion angles was
chosen. b) ORTEP plot at the 50% probability level of the small-molecule
crystal structure of (ꢂ)-9 (for clarity hydrogen atoms are omitted and only
(+)-9 is shown). c) Front view of the sulfonamide from (+)-9 showing the N-
lone pair bisecting the O-S-O angle. d) Front view of the sulfonamide from
(+)-9 showing the p orbital of the ipso-carbon atom bisecting the O-S-O
angle.
The second dihedral angle of interest in aryl sulfonamides is
the Csp2-S-N-Csp3 angle. This angle can adopt either an eclipsed
or a staggered conformation (Figure 5). Early ab initio calcula-
tions on N-methylmethanesulfonamide predicted the eclipsed
conformation to be the more stable with an energy barrier
ranging from 1.46 kcalmolꢁ1 (at the RHF/6-31G* level of
theory)[34] to 2.63 kcalmolꢁ1 (at the MP2/6-31G* level of
theory).[35] Later, those results were contrasted by the crystal
structure of N-methylmethanesulfonamide found in the stag-
gered conformation only.[36]
ure 6a and Supporting Information, Section S3, Figure S3). This
conformational preference goes in parallel with decreased N-
pyramidalization. The retrieved distances (<0.40 ꢁ) between
the nitrogen atom and the plane of its three substituents are
characteristics of sulfonamides. The nitrogen atoms are gener-
ally slightly less pyramidal than in acyclic tertiary amines
(d(N···plane)ꢀ0.45 ꢁ).[7] In addition, PES at the B3LYP/cc-pVDZ
and B3LYP/cc-pVTZ levels of theory (in water with polarizable
continuum solvent model) for the fragments PhSO2NH2,
PhSO2NHMe, and PhSO2NMe2 (Supporting Information, Sec-
tion S4, Figure S11), are in good agreement with the CSD and
PDB searches. At both levels of theory, the staggered confor-
mation was found to be favored over the eclipsed one. The
preference for this conformation can be explained by stereo-
electronic and steric effects. The nitrogen lone pair interacts in
an antiperiplanar orientation with the s* orbital of the weakest
bond, which is the Csp2ꢁS bond. Steric repulsion might also
In most of the molecules in the CSD and the PDB ligands,
the Csp2-S-N-Csp3 torsion angles are in a range between 608 and
908 meaning a clear preference for the staggered conforma-
tion with the nitrogen lone pair bisecting the SO2 angle (Fig-
Figure 5. a) Stable conformation of aryl sulfonamides/aryl sulfones relative
to the Csp2ꢁS bond. b) and c) Two energetically stable conformations of sul-
fonamides relative to the NꢁS bond.
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favor the staggered over the eclipsed conformation, in particu-
lar if larger substituents are attached to the nitrogen atom.
The preference of the aryl sulfonamide moiety to adopt the
staggered conformation is nicely illustrated by the small-mole-
cule X-ray crystal structures recorded for (ꢂ)-9, (ꢂ)-11, and (ꢂ)-
16 (Figure 6b and Supporting Information, Sections S6.2.2,
S6.2.3, and S6.2.4). The measured Csp2-Csp2-S-N torsion angles
are equal to 96.78, 90.98 and 79.98, respectively, matching
nicely the CSD search. In the three structures, the nitrogen
lone pair perfectly bisects the SO2 angle (Figure 6c and Sup-
porting Information, Sections S6.2.2, S6.2.3, and S6.2.4).
sensitive strain PfNF54 (Table 2). All nitrogen substituents were
selected based on MOLOC modeling to generate favorable in-
teractions with the lipophilic residues at the exit of the pABA
channel. In the modeling, the more polar sulfonamide moiety
in the staggered conformation was oriented towards the sol-
vent. The outcome, however, was disappointing (Table 2).
While the IC50 values against AtSHMT were in the lower nano-
molar range from 22.8 to 87.7 nm, a meaningful SAR was not
recognizable (Table 2 and Supporting Information, Section S2,
Table S1). Similarly, mixed results were obtained in the cell-
based assay on PfNF54 (Table 2). The biological activities sug-
gested that no appreciable additional favorable contacts were
harvested by the lipophilic nitrogen substituents. This was
A series of nine aryl sulfonamide analogues ((ꢂ)-9–17) was
synthesized and tested on Pf- and AtSHMT, as well as on the
Table 2. Biological activities of sulfonamides and sulfones (ꢂ)-9–22.
EC50
IC50
EC50
IC50
Cpd.
PfNF54
[nm]
AtSHMT
[nm]
Cpd.
PfNF54
[nm]
AtSHMT
[nm]
R
R
(ꢂ)-
114
210
22.8
74.4
(ꢂ)-17
(+)-17
104
56
73.9
9
(ꢂ)-
n.d.[a]
10
(ꢂ)-
657
400
43.0
39.1
(ꢁ)-17
(ꢂ)-18
1584
91
n.d.
11
(ꢂ)-
16.8
12
(ꢂ)-
391
374
29.0
87.7
(ꢂ)-19
(ꢂ)-20
1399
557
n.d.
13
(ꢂ)-
24.5
14
(ꢂ)-
872
838
79.1
85.6
(ꢂ)-21
(ꢂ)-22
200
186
22.4
19.2
15
(ꢂ)-
16
[a] n.d.=Not determined.
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later confirmed by co-crystal structures analyses, as shown
below.
protein–ligand complexes, both active sites were found popu-
lated by a ligand (Supporting Information, Section 6.1.2, Fig-
ure S20).
The two enantiomers of the benzyl sulfonamide (ꢂ)-17 were
separated by chiral-phase HPLC to afford pure (+)-17 and
(ꢁ)-17. As already observed with other pyrazolopyran-based li-
gands,[25,26] there is a high level of chiral recognition at the
active site of PfSHMT and large discrepancies in biological ac-
tivity were measured for (+)-17 and (ꢁ)-17. Ligand (+)-17 in-
hibited PfSHMT (IC50 =150 nm) much more efficiently than
(ꢁ)-17 (only 38% inhibition at 250 mm) (Supporting Informa-
tion, Section S2, Table S1). The preference for the (+)-enantio-
mer is not surprising when considering the binding mode of
pyrazolopyran-based ligands. Indeed, for all analogues co-crys-
tallized with PvSHMT in this program, exclusively the (+)-enan-
tiomers were found to be bound to the enzyme. Cell-based ef-
ficacy confirmed this trend, as (+)-17 was again much more
potent than (ꢁ)-17 (EC50 =56 and 1584 nm, respectively)
(Table 2).
Binding mode of (+)-11, (+)-17, and (+)-20
Gratifyingly, the three co-crystal structures provided an explan-
ation for the lacking additional gain in affinity by the lipophilic
residues at the termini of the aryl sulfonamides and aryl sul-
fones.
The binding mode of the pyrazolopyran core was found to
be the same as in all other co-crystals with wild-type PvSHMT
solved so far, with a strong hydrogen-bonding network an-
choring this scaffold into the pteridine binding pocket (Fig-
ure 7a and Supporting Information, Section S6.1.2, Figures
S21a and S22a). The sulfonamide moieties in (+)-11 and
(+)-17 and the sulfone in (+)-20 are nicely found in their
favored conformation with Csp2-Csp2-S-N (or Csp2-Csp2-S-Csp3) tor-
sion angles of 748, 868, and 988, respectively. However, the ori-
entation of the sulfonamide moiety in (+)-11 (Supporting In-
formation, Section S6.1.2, Figure S21b) and (+)-17 (Figure 7b),
and the sulfone moiety in (+)-20 (Supporting Information, Sec-
tion S6.1.2, Figure S22b) was found to be opposite to that pre-
dicted by modeling (Supporting Information, Section S5, Fig-
ures S13 and S14). The SO2 moiety is pointing towards the hy-
drophobic residues at the exit of the pABA channel, while the
hydrophobic terminal substituents of the ligands are all point-
ing towards solvent instead of interacting with the protein.
Indeed, the sulfonamide moiety of (+)-17 is at close distances
to Val141 (d(O···HꢁCVal141)=2.8 and 3.3 ꢁ) and Pro267 (d(O···Hꢁ
Three ligands containing an aryl sulfone moiety ((ꢂ)-18–20)
and two reverse sulfonamides ((ꢂ)-21 and (ꢂ)-22) were also
prepared, yielding target- and cell-based activities in a similar
range to the aryl sulfonamide ligands (Table 2 and Supporting
Information, Section S2, Table S1). Crystals of the reverse sulfo-
namide (ꢂ)-21 also featured a staggered conformation with a
Csp3-N-S-Csp3 torsion angle of ꢁ71.88 (Supporting Information,
Section 6.2.5, Figure S32). This moiety is twisted almost orthog-
onally with respect to the phenyl ring with a Csp2-Csp2-N-S tor-
sion angle of 77.68 (Supporting Information, Section 6.2.5, Fig-
ure S31).
Although several ligands of both biphenyl and aryl sulfona-
mide/aryl sulfone series proved to be highly potent in our in
vitro assays, they were not studied further due to their limited
metabolic stability in human liver microsomes (t1/2 <10 min)
(Supporting Information, Section S2, Table S2). The terminal
fragments on the phenyl ring departing from the core are pre-
sumably responsible for this intrinsic instability and not the
pyrazolopyran core. Indeed, we recently reported a series of
pyrazolopyran-based ligands with half-lives up to 4 h.[26]
CPro267)=3.2 ꢁ), establishing several van der Waals interactions
with apolar atoms and forming weak hydrogen-bond-type
contacts to aliphatic CꢁH moieties (Figure 7b).[41] Very similar
interactions were found for (+)-11 and (+)-20 with contacts
ranging from 2.8 to 3.4 ꢁ (Supporting Information, Sec-
tion S6.1.2, Figures S21b and S22b). Those three C364A-
PvSHMT–ligand complexes highlight the low hydrophilicity of
the SO2 group, which resembles the low hydrophilicity of the
nitro group[66] that also prefers pointing into hydrophobic
pockets rather than into solvent. We performed a search in
both CSD and PDB for hydrogen bonding from strong hydro-
gen-bond donors (OꢁH and NꢁH, but excluding CꢁH) to the
SO2 group in aryl sulfonamides and aryl sulfones (Supporting
Information, Section S3, Table S3). Of 7856 hits in the CSD,
6617 sulfonamides formed no hydrogen bond and 1239 one
hydrogen bond. A higher frequency of single hydrogen bonds
was retrieved from the PDB, as 847 out of 1410 hits formed
one hydrogen bond. Cases of two hydrogen bonds were rarer
in both the CSD and PDB, with 37 and 496 hits, respectively.
Regarding structures containing a sulfone moiety, only 344 hits
out of the 2352 in the CSD established one hydrogen bond,
and approximately half of the hits in the PDB. Only a limited
number of sulfone formed two hydrogen bonds (Supporting
Information, Section S3, Table S3). The distances of the hydro-
gen bonds to the SO2 group are in a range of 2.8 to 3.5 ꢁ (Sup-
porting Information, Section S3, Figure S4) and no specific
directionality was observed as the hydrogen bonds cover the
Crystal structure determination of C364A-PvSHMT in com-
plex with (ꢂ)-11, (ꢂ)-17, and (ꢂ)-20
Several attempts made to co-crystallize either (ꢂ)-11, (ꢂ)-17,
or (ꢂ)-20 with wild-type PvSHMT showed no electron density
of the bound ligands. Instead, a partial or full formation of the
disulfide bridge between Cys125 and Cys364 was observed. To
circumvent the cysteine oxidation and prevent the disulfide
bridge formation, which might be linked to the binding of the
ligands, Cys364 was mutated to Ala364. Subsequently, co-crys-
tal structures of (+)-11, (+)-17, or (+)-20 with the C364A-
PvSHMT mutant were obtained. The co-crystals diffracted to
2.4, 2.2, and 2.6 ꢁ resolution, respectively, and belong to the
C2 space group. The structures were solved by molecular re-
placement using the coordinates of a chain A protomer of
PvSHMT (PDB ID code: 4OYT) as the template.[24] Despite using
a racemic mixture of ligands for co-crystallization, only the
(+)-(S) enantiomer was present in all structures. In the three
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thesized, and tested on targets At- and PfSHMT, and in vitro on
the PfNF54 strain. The aim of this study was to introduce lipo-
philic moieties on pyrazolopyran-based ligands to improve
their affinities by enhancing the lipophilic contacts with the
pABA channel of the enzyme.
Regarding the seven biphenyl analogues ((ꢂ)-2–8), similar
target affinities across the series were measured on AtSHMT
and PfSHMT. However, the cell-based potency of those
compounds was dramatically impacted by the nature of the
ortho-substituent with differences in EC50 values up to 37-fold.
To explain these results, we performed CSD and PDB searches,
as well as a potential energy scan (PES) of the considered bi-
phenyls. We found that the loss of cellular efficacy correlates
with the increase of the torsion angle of the biphenyl, which
depends on the size of its ortho-substituent. We postulate that
cell permeation was impacted by the conformational changes
of the biphenyls and this was accounted for the disparity be-
tween the efficacies on PfNF54. A total of four PvSHMT–ligand
co-crystal structures were solved for this series of molecules.
Interestingly, in the complexes with (+)-3 and (+)-5 the ortho-
halo substituent is turned towards Tyr63, undergoing antiparal-
lel dipolar CꢁX···CꢁO interactions with the CꢁO bond of Tyr63
and local direct electrostatic interactions with the aromatic
ring. In contrast, in the co-crystal structures with (+)-4 and
(+)-7, their respective methyl and cyano substituents point to
Cys364 to avoid steric repulsion with Tyr63.
In parallel, fourteen ligands bearing an aryl sulfonamide or
an aryl sulfone moiety ((ꢂ)-9–22) were examined. By CSD and
PDB searches, as well as by theoretical calculations, we demon-
strated that aryl sulfonamides and aryl sulfones preferentially
adopt a conformation in which the p orbital of the ipso-carbon
atom bisects the O-S-O angle.[7] We also showed the prefer-
ence for the nitrogen lone pair of sulfonamides to bisect the
O-S-O angle, resulting in a characteristic staggered conforma-
tion. Small-molecule X-ray crystal structures of pyrazolopyran-
based inhibitors nicely illustrated these favored conformations.
No significant activity gain was measured, regardless of the
size of the terminal apolar moiety grafted onto the sulfona-
mide/sulfone. This could be rationalized with the help of three
co-crystal structures of (+)-11, (+)-17, and (+)-20 with a
C364A-PvSHMT mutant, in which the respective sulfonyl moiet-
ies were found pointing towards the hydrophobic residues
lining the pABA channel and establishing several short van der
Waals interactions. That way, the terminal apolar groups graft-
ed onto the sulfonamide point into the bulk and cannot inter-
act with the protein to improve the binding affinity. These co-
crystal structures, complemented by CSD and PDB searches,
highlight the low hydrophilicity of the SO2 moiety, which
prefers to point to hydrophobic environments rather than into
polar ones. Effective chiral recognition at the active site was
shown, with the target binding of enantiopure ligand (+)-17
being largely preferred over (ꢁ)-17. Taken together, this inves-
tigation provided valuable input, not only regarding the devel-
opment of SHMT inhibitors, but also for the general design of
drug-like molecules that incorporate the discussed functional
groups.
Figure 7. Co-crystal structure (PDB ID code: 5XMQ, 2.2 ꢁ) showing the pro-
tein–ligand interactions of pyrazolopyran (+)-17 (lime) and C364A-PvSHMT
mutant (grey). a) Polar interactions between (+)-17 and the protein. b) Inter-
actions, largely dipole-dipole type, involving the sulfonamide moiety in the
pABA channel. The surface spans the volume of the pABA channel. The
water molecule (W1) is represented as a red sphere. PLP is omitted for clarity
in b). Distances are given in ꢁ. Color code: Cprotein grey, C(+)-17 lime, CPLP gold,
F light cyan, N blue, O red, P orange, S yellow.
hemisphere around one of the S=O bonds (Supporting Infor-
mation, Section S3, Figures S5 and S6).
Conclusions
Based on X-ray co-crystal structures information, two series of
SHMT inhibitors featuring widely used chemical motifs, namely
biphenyl and aryl sulfonamide/aryl sulfone moieties, were syn-
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tant. X-ray diffraction data were collected at 100 K at wavelength
of 1 ꢁ using ADSC Quantum-315 CCD detector at beamline 13B1,
NSRRC (Taiwan). Data were processed using the HKL2000 package.
X-ray diffraction data and refinement statistics are listed in Sup-
porting Information, Section S6.1.1, Table S4 and Section S6.1.2,
Table S5. The structure of PvSHMT (or C364A-PvSHMT) was deter-
mined by molecular replacement using Phaser in CCP4 suite with a
chain A protomer of PvSHMT coordinate (PDB ID code: 4OYT) as
the template. Model building and structure refinement were car-
ried out using Coot and Refmac5. The ligand structure was pre-
pared using HYPERCHEM.
Experimental Section
Chemical synthesis: All synthetic protocols and analytical data of
the preparation of ligands (ꢂ)-2–22 and their respective intermedi-
ates are detailed in the Supporting Information, Sections S1, S7,
and S8.
In vitro antimalarial activity: The Plasmodium falciparum drug-
sensitive NF54 strain was cultivated in a variation of the medium
previously described, consisting of RPMI 1640 supplemented with
0.5% ALBUMAXꢃ II, 25 mm Hepes, 25 mm NaHCO3 buffer (pH 7.3),
0.36 mm hypoxanthine, and 100 mgmLꢁ1 neomycin.[67,68] Human er-
ythrocytes served as host cells. Cultures were maintained in an at-
mosphere of 3% O2, 4% CO2, and 93% N2 in modular chambers at
378C. Compounds were dissolved in DMSO (10 mgmLꢁ1), diluted
in hypoxanthine-free culture medium, and titrated in duplicates
over a 64-fold range in 96 well plates. Infected erythrocytes (1.25%
final hematocrit and 0.3% final parasitemia) were added into the
wells. After 48 h incubation, 0.25 mCi of [3H]hypoxanthine was
added and plates were incubated for an additional 24 h. Parasites
were harvested onto glass-fiber filters, and radioactivity was count-
ed using a MicroBeta2 plate liquid scintillation counter (PerkinElm-
er). The results were recorded and expressed as a percentage of
the untreated controls. Fifty percent effective concentrations (EC50)
were estimated by linear interpolation.[69]
Acknowledgements
Work at ETH Zurich was supported by a grant from the ETH
Research Council (ETH-01 13-2). At ETH Zurich, we thank Dr.
Bruno Bernet for help with the compound characterizations
and Michael Solar for recording the small-molecule X-ray struc-
tures. We thank the MMV foundation, especially Dr. Jeremy
Burrows and Dr. Roger Bonnert, for their support, for giving us
access to their assay network, and their great expertise. We
also acknowledge Dr. Karen White and Dr. Susan Charman at
CDCO for performing microsomal stability assays. We are grate-
ful to Abhijit Kundu and Surajit Sadhukhan at TCG Lifesciences
for providing free-of-charge several key building blocks for the
synthesis. Protein–ligand studies including Plasmodium
enzyme inhibition and protein crystallography were financially
supported by grants from Cluster Program and Management
Office, National Science and Technology development Agency,
Thailand (CPMO-P-13-00835), National Center for Genetic Engi-
neering and Biotechnology (Platform P-14-50241), the Thailand
Research Fund (RTA5980001), and Mahidol University. We
gratefully acknowledge the National Synchrotron Radiation Re-
search Center (Taiwan) for the beamline 13B1 and the operat-
ing staff. We thank the experimental facility and the technical
services provided by the “Synchrotron Radiation Protein Crys-
tallography Facility of the National Core Facility Program for
Biotechnology, Ministry of Science and Technology” and the
“National Synchrotron Radiation Research Center”, a national
user facility supported by the Ministry of Science and Technol-
ogy, Taiwan, ROC.
Enzymatic PfSHMT assay: Assay reactions (200 mL total volume)
contained SHMT (ꢀ0.5 mM), l-serine (2 mm), (6S)-H4F (0.4 mm), b-
NADP+ (0.25 mm), and the coupling enzyme methylene tetrahy-
drofolate dehydrogenase (FolD, 5 mM) in 50 mm HEPES pH 7.0 con-
taining 1 mm DTT and 0.5 mm EDTA. To this mixture, inhibitors
(1 mL) with various concentrations were added and initial rates of
the reaction monitored to measure the activity of the enzyme. The
inhibitors were dissolved in DMSO, and the control assays without
inhibitor but in the presence of 0.5% DMSO (final concentration)
were also carried out.
Enzymatic AtSHMT assay: Assay reactions (200 mL total volume)
contained SHMT (1 mg), l-serine (20 mm), (6S)-H4F (0.3 mm), b-
NAD+ (2 mm) and the coupling enzyme methylene tetrahydrofo-
late dehydrogenase (FolD, 20 mg) in 50 mm potassium phosphate
buffer pH 7.4, containing 7.5 mm DTT. To this mixture inhibitors
(10 mL) with various concentrations (final concentrations from 1–
1000 nm) were added and initial rates of the reaction monitored to
measure the amount of non-inhibited enzyme. The inhibitors were
dissolved in 80% DMSO, and the control assays without inhibitor
in the presence of 1% DMSO (final concentration) were also carried
out. The accumulation of NADH was followed for 20 minutes at
340 nm using a BioTek Synergy HTX plate reader.
Crystallization of recombinant PvSHMT and compounds (ꢂ)-3,
(ꢂ)-4, (ꢂ)-5, (ꢂ)-7, (ꢂ)-11, (ꢂ)-17, and (ꢂ)-20: PvSHMT (or C364A-
PvSHMT) was crystallized using a microbatch method in a 60-well Conflict of interest
plate (Ø 1 mm at bottom of each well) covered with baby oil
(6 mL; Johnson; a mixture of mineral oil, olive oil, and vitamin E,
The authors declare no conflict of interest.
PZ Johnson, Thailand). Protein–ligand complexes were prepared by
mixing purified PvSHMT protein (60 mL; 20–25 mgmLꢁ1) with 1 mm
PLP, 60 mm b-mercaptoethanol, 90 mm of glycine, and 5.7 mm (ꢂ)-
3, or (ꢂ)-4, or (ꢂ)-5, or (ꢂ)-7, or (ꢂ)-11, or (ꢂ)-17, or (ꢂ)-20. The
mixture was equilibrated on ice for 30 min to allow complete com-
plex formation. The crystallization drop was composed of 1 mL
each of a crystallization solution and the protein complex. Protein
crystals of PvSHMT were grown at 293 K in 20–24% w/v PEG4000,
0.06–0.12m NaCl, 0.1m Tris-HCl buffer pH 8.5.
Keywords: co-crystal structures · conformation analysis · drug
design · plasmodium · serine hydroxymethyltransferase
[1] J. A. Feng, I. Aliagas, P. Bergeron, J. M. Blaney, E. K. Bradley, M. F. T. Koeh-
ler, M.-L. Lee, D. F. Ortwine, V. Tsui, J. Wu, A. Gobbi, J. Comput.-Aided
Mol. Des. 2015, 29, 511–523.
[2] A. Hillisch, N. Heinrich, H. Wild, ChemMedChem 2015, 10, 1958–1962.
PvSHMT crystal structure data collection, structure determina-
tion and refinement: A single crystal was flash-vitrified in liquid ni-
trogen using 20% glycerol in crystallizing agent as a cryoprotec-
´
[3] B. Kuhn, M. Tichy, L. Wang, S. Robinson, R. E. Martin, A. Kuglstatter, J.
Benz, M. Giroud, T. Schirmeister, R. Abel, F. Diederich, J. Hert, J. Med.
Chem. 2017, 60, 2485–2497.
Chem. Eur. J. 2017, 23, 1 – 14
www.chemeurj.org
11
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[4] C. Lerner, A. Ruf, V. Gramlich, B. Masjost, G. Zꢄrcher, R. Jakob-Roetne, E.
Borroni, F. Diederich, Angew. Chem. Int. Ed. 2001, 40, 4040–4042;
Angew. Chem. 2001, 113, 4164–4166.
[5] M. Giroud, J. Ivkovic, M. Martignoni, M. Fleuti, N. Trapp, W. Haap, A.
Kuglstatter, J. Benz, B. Kuhn, T. Schirmeister, F. Diederich, ChemMedChem
2017, 12, 257–270.
[33] R. L. Beddoes, L. Dalton, J. A. Joule, O. S. Mills, J. D. Street, C. I. F. Watt, J.
Chem. Soc. Perkin Trans. 2 1986, 787–797.
[34] J. B. Nicholas, R. Vance, E. Martin, B. J. Burke, A. J. Hopfinger, J. Phys.
Chem. 1991, 95, 9803–9811.
[35] G. Liang, J. P. Bays, J. P. Bowen, J. Mol. Struct.: THEOCHEM 1997, 401,
165–179.
[6] J. Bostrçm, P.-O. Norrby, T. Liljefors, J. Comput.-Aided Mol. Des. 1998, 12,
383–396.
[36] T. C. Higgs, A. Parkin, S. Parsons, P. A. Tasker, Acta Crystallogr. Sect. E
2002, 58, o523–o525.
[7] K. A. Brameld, B. Kuhn, D. C. Reuter, M. Stahl, J. Chem. Inf. Model. 2008,
48, 1–24.
[8] B. Kuhn, W. Guba, J. Hert, D. Banner, C. Bissantz, S. Ceccarelli, W. Haap,
M. Kçrner, A. Kuglstatter, C. Lerner, P. Mattei, W. Neidhart, E. Pinard,
M. G. Rudolph, T. Schulz-Gasch, T. Woltering, M. Stahl, J. Med. Chem.
2016, 59, 4087–4102.
[37] F. Hof, A. Schꢄtz, C. Fꢀh, S. Meyer, D. Bur, J. Liu, D. E. Goldberg, F. Die-
derich, Angew. Chem. Int. Ed. 2006, 45, 2138–2141; Angew. Chem. 2006,
118, 2193–2196.
[38] S. Senger, M. A. Convery, C. Chan, N. S. Watson, Bioorg. Med. Chem. Lett.
2006, 16, 5731–5735.
[39] S. Senger, C. Chan, M. A. Convery, J. A. Hubbard, G. P. Shah, N. S.
Watson, R. J. Young, Bioorg. Med. Chem. Lett. 2007, 17, 2931–2934.
[40] A. K. H. Hirsch, M. S. Alphey, S. Lauw, M. Seet, L. Barandun, W. Eisenreich,
F. Rohdich, W. N. Hunter, A. Bacher, F. Diederich, Org. Biomol. Chem.
2008, 6, 2719–2730.
[41] C. Bissantz, B. Kuhn, M. Stahl, J. Med. Chem. 2010, 53, 5061–5084.
[42] P. R. Gerber, K. Mꢄller, J. Comput.-Aided Mol. Des. 1995, 9, 251–268.
[43] S. Kotha, K. Lahari, D. Kashinath, Tetrahedron 2002, 58, 9633–9695.
[44] A. R. Muci, S. L. Buchwald, Top. Curr. Chem. 2002, 219, 131–209.
[45] G. Jones, Org. React. 1967, 15, 204–599.
[46] M. Witschel, F. Stelzer, J. Hutzler, T. Qu, T. Mietzner, K. Kreuz, K. Gross-
mann, R. Aponte, H. W. Hçffken, F. Calo, T. Ehrhardt, A. Simon, L. Parra
Rapado, WO 2013/182472 A1, 2013.
[47] CSD search: CSD version 5.38, February 2017, containing about 875 000
entries. All searches were performed with ConQuest 1.19 for structures
with R-factor <10% (no error, no partial disorder), excluding polymeric,
powder, or metalorganic compounds. PDB search: Searches were per-
formed using Relibase+ v3.2.0. All searches were limited to resolution
of protein crystal structures ꢄ2.5 ꢁ.
[9] B. M. Hudson, E. Nguyen, D. J. Tantillo, Org. Biomol. Chem. 2016, 14,
3975–3980.
[10] I.-J. Chen, N. Foloppe, Drug Dev. Res. 2011, 72, 85–94.
[11] R. Taylor, Acta Crystallogr. Sect. D 2002, 58, 879–888.
[12] F. H. Allen, R. Taylor, Chem. Soc. Rev. 2004, 33, 463–475.
[13] G. M. Battle, G. M. Ferrence, F. H. Allen, J. Appl. Crystallogr. 2010, 43,
1208–1223.
[14] E. Pidcock, J. A. Chisholm, P. A. Wood, P. T. A. Galek, L. Fꢅbiꢅn, O. Korb,
A. J. Cruz-Cabeza, J. W. Liebeschuetz, C. R. Groom, F. H. Allen in Supra-
molecular Chemistry: From Molecules to Nanomaterials Vol. 6 (Eds. J. W.
Steed, P. A. Gale), Wiley, Chichester, 2012, pp. 2927–2946.
[15] CCDC Website database, Accessed April 25, 2017. http://www.ccdc.
cam.ac.uk/solutions/csd-system/components/csd/
[16] C. R. Groom, F. H. Allen, Angew. Chem. Int. Ed. 2014, 53, 662–671;
Angew. Chem. 2014, 126, 675–684.
[17] S. J. Cottrell, T. S. G. Olsson, R. Taylor, J. C. Cole, J. W. Liebeschuetz, J.
Chem. Inf. Model. 2012, 52, 956–962.
[18] J. E. Salcedo-Sora, S. A. Ward, Mol. Biochem. Parasitol. 2013, 188, 51–62.
[19] I. B. Mꢄller, J. E. Hyde, Mol. Biochem. Parasitol. 2013, 188, 63–77.
[20] V. Schirch, D. M. E. Szebenyi, Curr. Opin. Chem. Biol. 2005, 9, 482–487.
[21] S. Maenpuen, K. Sopitthummakhun, Y. Yuthavong, P. Chaiyen, U. Leart-
sakulpanich, Mol. Biochem. Parasitol. 2009, 168, 63–73.
[22] C. K. T. Pang, J. H. Hunter, R. Gujjar, R. Podutoori, J. Bowman, D. G. Mu-
deppa, P. K. Rathod, Mol. Biochem. Parasitol. 2009, 168, 74–83.
[23] P. Chitnumsub, W. Ittarat, A. Jaruwat, K. Noytanom, W. Amornwatchara-
pong, W. Pornthanakasem, P. Chaiyen, Y. Yuthavong, U. Leartsakulpa-
nich, Acta Crystallogr. Sect. D 2014, 70, 1517–1527.
[24] P. Chitnumsub, A. Jaruwat, P. Riangrungroj, W. Ittarat, K. Noytanom, W.
Oonanant, J. Vanichthanankul, P. Chuankhayan, S. Maenpuen, C.-J.
Chen, P. Chaiyen, Y. Yuthavong, U. Leartsakulpanich, Acta Crystallogr.
Sect. D 2014, 70, 3177–3186.
[25] M. C. Witschel, M. Rottmann, A. Schwab, U. Leartsakulpanich, P. Chit-
numsub, M. Seet, S. Tonazzi, G. Schwertz, F. Stelzer, T. Mietzner, C. McNa-
mara, F. Thater, C. Freymond, A. Jaruwat, C. Pinthong, P. Riangrungroj,
M. Oufir, M. Hamburger, P. Mꢀser, L. M. Sanz-Alonso, S. Charman, S. Wit-
tlin, Y. Yuthavong, P. Chaiyen, F. Diederich, J. Med. Chem. 2015, 58,
3117–3130.
[48] C. P. Brock, R. P. Minton, J. Am. Chem. Soc. 1989, 111, 4586–4593.
[49] Gaussian09, Revision A.02, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E.
Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, G. A. Pe-
tersson, H. Nakatsuji, M. Caricato, A. Marenich, J. Bloino, B. G. Janesko,
R. Gomperts, B. Menucci, H. P. Hratchian, J. V. Ortiz, A. F. Izmaylov, J. L.
Sonnenberg, D. Williams-Young, F. Ding, F. Lipparini, F. Egidi, J. Goings,
B. Peng, A. Petrone, T. Henderson, D. Ranasinghe, V. G. Zakrzewski, J.
Gao, N. Rega, G. Zheng, W. Liang, M. Hada, M. Ehara, K. Toyota, R.
Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H.
Nakai, T. Vreven, K. Throssell, J. A. Montgomery, Jr., J. E. Peralta, F.
Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov,
T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C.
Burant, S. S. Iyengar, J. Tomasi, M. Cossi, J. M. Millam, M. Klene, C.
Adamo, R. Cammi, J. W. Ochterski, R. L. Martin, K. Morokuma, ꢆ. Farkas,
J. B. Foresman, D. J. Fox, Gaussian, Inc., Wallingford CT, 2016.
[50] K. Sugano, M. Kansy, P. Artursson, A. Avdeef, S. Bendels, L. Di, G. F. Ecker,
B. Faller, H. Fischer, G. Gerebtzoff, H. Lennernaes, F. Senner, Nat. Rev.
Drug Discovery 2010, 9, 597–614.
[51] N. Noinaj, S. K. Buchanan, Curr. Opin. Struct. Biol. 2014, 27, 8–15.
[52] M. J. Gardner, et. al., Nature 2002, 419, 498–511.
[53] R. E. Martin, R. I. Henry, J. L. Abbey, J. D. Clements, K. Kirk, Genome Biol.
2005, 6, R26.
[54] A. Ecker, A. M. Lehane, J. Clain, D. A. Fidock, Trends Parasitol. 2012, 28,
504–514.
[26] G. Schwertz, M. C. Witschel, M. Rottmann, R. Bonnert, U. Leartsakulpa-
nich, P. Chitnumsub, A. Jaruwat, W. Ittarat, A. Schꢀfer, R. A. Aponte, S. A.
Charman, K. L. White, A. Kundu, S. Sadhukhan, M. Lloyd, G. M. Freiberg,
M. Srikumaran, M. Siggel, A. Zwyssig, P. Chaiyen, F. Diederich, J. Med.
Chem. 2017, 60, 4840–4860.
[27] H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig,
I. N. Shindyalov, P. E. Bourne, Nucleic Acids Res. 2000, 28, 235–242.
[28] H. Berman, K. Henrick, H. Nakamura, Nat. Struct. Biol. 2003, 10, 980.
[29] E. L. Eliel, S. H. Wilen, L. N. Mander in Stereochemistry of Organic Com-
pounds, Wiley, New York, 1994, pp. 1142–1155.
[30] D. Vonlanthen, A. Mishchenko, M. Elbing, M. Neuburger, T. Wandlowski,
M. Mayor, Angew. Chem. Int. Ed. 2009, 48, 8886–8890; Angew. Chem.
2009, 121, 9048–9052.
[31] E. Masson, Org. Biomol. Chem. 2013, 11, 2859–2871.
[32] While several earlier papers provided evidence for the distinct confor-
mational preferences of aryl sulfonamides and aryl sulfones, they were
brought to the attention to the broader medicinal and organic chemis-
try communities by Dr. Klaus Mꢄller (F. Hoffman-La Roche), in seminar
series worldwide, such as at ETH Zurich in 2004.
[55] Y. Lai, L. Xing, G. I. Poda, Y. Hu, Drug Metab. Dispos. 2007, 35, 937–945.
[56] S. E. Wheeler, K. N. Houk, J. Am. Chem. Soc. 2008, 130, 10854–10855.
[57] S. E. Wheeler, J. Am. Chem. Soc. 2011, 133, 10262–10274.
[58] S. E. Wheeler, Acc. Chem. Res. 2013, 46, 1029–1038.
[59] R. M. Parrish, C. D. Sherrill, J. Am. Chem. Soc. 2014, 136, 17386–17389.
[60] M. Harder, M. A. Carnero Corrales, N. Trapp, B. Kuhn, F. Diederich, Chem.
Eur. J. 2015, 21, 8455–8463.
[61] L.-J. Riwar, N. Trapp, B. Kuhn, F. Diederich, Angew. Chem. Int. Ed. 2017,
56, 11252–11257; Angew. Chem. 2017, 129, 11405–11410.
[62] L. M. Salonen, M. Ellermann, F. Diederich, Angew. Chem. Int. Ed. 2011, 50,
4808–4842; Angew. Chem. 2011, 123, 4908–4944.
[63] A. Nzila, J. Antimicrob. Chemother. 2006, 57, 1043–1054.
[64] A. C. Flick, H. X. Ding, C. A. Leverett, R. E. Kyne, Jr., K. K.-C. Liu, S. J. Fink,
C. J. O’Donnell, Bioorg. Med. Chem. 2016, 24, 1937–1980.
&
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12
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Full Paper
[65] G. L. Warren, T. D. Do, B. P. Kelley, A. Nicholls, S. D. Warren, Drug Discov-
ery Today 2012, 17, 1270–1281.
[69] W. Huber, J. C. Koella, Acta Tropica 1993, 55, 257–261.
[66] N. Sagawa, T. Shikata, Phys. Chem. Chem. Phys. 2014, 16, 13262–13270.
[67] A. Dorn, R. Stoffel, H. Matile, A. Bubendorf, R. G. Ridley, Nature 1995,
374, 269–271.
Manuscript received: July 13, 2017
Revised manuscript received: August 15, 2017
Version of record online: && &&, 0000
[68] W. Trager, J. B. Jensen, Science 1976, 193, 673–675.
Chem. Eur. J. 2017, 23, 1 – 14
www.chemeurj.org
13
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FULL PAPER
Mind the conformation! Two potent
series of pyrazolopyran-based inhibitors
of the enzyme serine hydroxymethyl-
transferase (SHMT), bearing either termi-
nal biphenyl, aryl sulfonamide, or aryl
sulfone motif are reported. The substan-
tial influence of the torsion angle of the
biphenyl moiety on the cell-based effi-
cacy is discussed. Additionally, the pre-
ferred conformations of aryl sulfona-
mide/aryl sulfone moieties and their lip-
ophilic character in the complexes with
P. vivax SHMT were analyzed.
&
Drug Design
G. Schwertz, M. S. Frei, M. C. Witschel,
M. Rottmann, U. Leartsakulpanich,
P. Chitnumsub, A. Jaruwat, W. Ittarat,
A. Schꢁfer, R. A. Aponte, N. Trapp,
K. Mark, P. Chaiyen, F. Diederich*
&& – &&
Conformational Aspects in the Design
of Inhibitors for Serine
Hydroxymethyltransferase (SHMT):
Biphenyl, Aryl Sulfonamide, and Aryl
Sulfone Motifs
&
&
Chem. Eur. J. 2017, 23, 1 – 14
www.chemeurj.org
14
ꢂ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!