Hybrid Inhibitors of Malarial Dihydrofolate Reductase with Dual Binding Modes That Can Forestall Resistance

Article abstract of DOI:10.1021/acsmedchemlett.8b00389

The S108N mutation of dihydrofolate reductase (DHFR) renders Plasmodium falciparum malaria parasites resistant to pyrimethamine through steric clash with the rigid side chain of the inhibitor. Inhibitors with flexible side chains can avoid this clash and

Full text of DOI:10.1021/acsmedchemlett.8b00389

Subscriber access provided by Kaohsiung Medical University
Letter
Hybrid Inhibitors of Malarial Dihydrofolate Reductase
with Dual Binding Modes which can Forestall Resistance
Bongkoch Tarnchompoo, Penchit Chitnumsub, Aritsara Jaruwat, Philip J Shaw,
Jarunee Vanichtanankul, Sinothai Poen, Roonglawan Rattanajak, Chayaphat
Wongsombat, Aunchalee Tonsomboon, Sasithorn Decharuangsilp, Tosapol
Anukunwithaya, Uthai Arwon, Sumalee Kamchonwongpaisan, and Yongyuth Yuthavong
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00389 • Publication Date (Web): 07 Nov 2018
Downloaded from http://pubs.acs.org on November 9, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Hybrid Inhibitors of Malarial Dihydrofolate Reductase with Dual
Binding Modes which can Forestall Resistance
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Bongkoch Tarnchompoo,‡ Penchit Chitnumsub,‡ Aritsara Jaruwat, Philip J. Shaw, Jarunee
Vanichtanankul, Sinothai Poen, Roonglawan Rattanajak, Chayaphat Wongsombat, Aunchalee
Tonsomboon,† Sasithorn Decharuangsilp, Tosapol Anukunwithaya, Uthai Arwon, Sumalee
Kamchonwongpaisan, and Yongyuth Yuthavong*
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency,
Pathumthani 12120, Thailand.
KEYWORDS: dual binding, hybrid inhibitors, dihydrofolate reductase, resistance suppression, antimalarials
ABSTRACT: The S108N mutation of dihydrofolate reductase (DHFR) renders Plasmodium falciparum malaria parasites
resistant to pyrimethamine through steric clash with the rigid side chain of the inhibitor. Inhibitors with flexible side chains
can avoid this clash and retain effectiveness against the mutant. However, other mutations such as N108S reversion confer
resistance to flexible inhibitors. We designed and synthesized hybrid inhibitors with two structural types in a single molecule,
which are effective against both wild-type and multiple mutants of P. falciparum through their selective target binding, as
demonstrated by X-ray crystallography. Furthermore, the hybrid inhibitors can forestall the emergence of new resistant
mutants, as shown by selection of mutants resistant to hybrid compound BT1 from a diverse PfDHFR random mutant library
expressed in a surrogate bacterial system. These results show that it is possible to develop effective antifolate antimalarials
to which the range of parasite resistance mutations is greatly reduced.
Antimalarial drugs have been important tools in the fight
against malaria, and will be essential in the current effort to
eliminate the disease.1-4 However, a major problem is the
development of parasite resistance to the drugs, which
threaten their effective use.5-9 Plasmodium falciparum
parasites resistant to antifolate drugs such as
pyrimethamine (Pyr), an inhibitor of parasite dihydrofolate
reductase (PfDHFR), and combinations of antifolate and
sulfa drugs are widespread throughout malaria endemic
regions.10,11 Instead of abandoning PfDHFR as an
antimalarial target, new approaches to develop antifolates
effective against resistant parasites with mutated PfDHFR
could be successful.10,12-15 The crystal structure of PfDHFR,
which is joined with thymidylate synthase as a bifunctional
enzyme, has been solved,16 together with its counterpart in
P. vivax.17 These structures reveal features which explain
the basis for mutation-induced resistance.13,14 The
structures of wild-type and mutant PfDHFRs co-complexes
have been solved with inhibitors namely WR99210, which
can still inhibit mutant enzymes effectively,18 and Pyr, to
which binding has been compromised by PfDHFR
mutations. Analysis of these structures has enabled target-
based design of new inhibitors, some of which have very
high efficacy and selectivity as antimalarial drugs.15,19 These
studies reveal the key S108N mutation in PfDHFR, which
introduces a larger side-chain that sterically interferes with
the binding of inhibitors with a rigid p-Cl-phenyl side chain
such as Pyr and cycloguanil. Antifolate resistance is
amplified when the S108N mutation is accompanied by
additional mutations in the inhibitor binding region of
PfDHFR including N51I, C59R and I164L. Inhibitors such as
WR99210 retain tight binding affinity with mutant PfDHFRs
associated with Pyr resistance and retain efficacy against
parasites with the corresponding mutant PfDHFRs.
WR99210 and related compounds have flexible side-chains
which can avoid steric clash with the S108N side-chain.
However, these flexible inhibitors can be rendered
ineffective by further PfDHFR resistance mutations, as
shown by a surrogate yeast20 or bacterial system,21,22
although the frequencies and the levels of resistance against
WR99210 were lower than those for Pyr. Here we present a
new design of hybrid inhibitors, with both rigid and flexible
side-chains in the same molecule as a combination which
can reduce the emergence of resistance through mutations.
An additional rationale for making such hybrid inhibitors is
that there are conflicting requirements of PfDHFR in
mutations conferring resistance to rigid (Pyr-like) and
flexible (WR99210-like) inhibitors.22
WR99210-resistant mutants isolated using a bacterial
surrogate system are more sensitive to Pyr than Pyr-
resistant mutants owing to the presence of wild-type
PfDHFR residues S108, N51, C59 and I164.21,22 In general,
therefore, PfDHFRs can be divided into two series, namely
the “S108 series” which is sensitive to rigid inhibitors like
Pyr, but resistant to flexible inhibitors in combination with
other mutations, and the “108N series” (or S108N series or
N108 series22), which is sensitive to flexible inhibitors, but
resistant to rigid inhibitors. We hypothesize that the
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 7
combination of the two types of pharmacophore (rigid and
In this study, we designed hybrid inhibitors through
modification of the side chain of a rigid inhibitor at the m-
or p-phenyl substituent, so that they carry a rigid and a
flexible moiety at either end (Figure 1).
1
2
3
4
5
6
7
8
flexible) in a single molecule should be effective against
parasites with both types of PfDHFR. Specifically, the rigid
pharmacophore should inhibit PfDHFRs in the S108 series,
whereas the flexible pharmacophore should inhibit those in
the 108N series. Furthermore, we hypothesize that
mutations conferring resistance to hybrid inhibitors would
be extremely limited owing to constraints on residues in the
substrate/inhibitor binding region of PfDHFR.
In our design approach, hybrid compounds were
prepared
diaminopyrimidines
as
outlined
2a-b
in
Scheme
and the
1.
Rigid
flexible
diaminopyrimidine 3 were prepared according to methods
modified from the previous reports (Scheme 1A).15,24
Mitsunobu reaction between compounds 2a-b and 3
furnished the desired hybrid compounds BT1 and BT2
bearing two DHFR binding sites on opposite ends of the
molecule. BT1 consists of the rigid end linked at the m-
phenyl substituent to the flexible end, whereas BT2 has the
rigid end linked at the p-phenyl substituent to the flexible
end. For a hybrid bearing rigid ends on both sides of the
molecule with a flexible five-atom linker, a condensation
reaction of enol-ether of compound 4 with guanidine was
performed to yield the desired hybrid BT3 (Scheme 1B). In
this study, compounds BT2S and BT3S, which are
hydrochloride salt forms of BT2 and BT3, respectively,
were also prepared to give compounds with greater
solubility for biological testing.
In this paper, we report the synthesis of hybrid inhibitors
capable of inhibiting P. falciparum carrying either wild-type
or multiple-mutant DHFRs, and the modes of binding for
these compounds. The hybrid inhibitors bind with similar
affinities to both types of PfDHFR. Significantly, they bind in
the active site of PfDHFRs in the expected manner, namely,
binding with the rigid end to wild-type (S108) PfDHFR and
with the flexible end to mutant (S108N) PfDHFR. The
inhibitors show a low tendency to induce new antifolate-
resistant mutants. Furthermore, in anticipation of further
potential development as oral formulation, the 2,4-
diaminopyrimidine scaffold was employed to take
advantage of its pKa which is suitable for oral bioavailability
(pKa 6-7), as previously rationalized in P218 development.15
Hence, they should serve as good models for further
development of oral antifolate antimalarials which can
forestall further development of mutation-induced
resistance.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1. Synthesis of hybrid inhibitors: (A) Rigid-
flexible hybrid (B) Rigid-rigid hybrid
RESULTS AND DISCUSSION
Our previous works on the synthesis of DHFR inhibitors
showed that P30 (a rigid diaminopyrimidine; Pyr-like with
m-Cl substituent) and P65 (a flexible diaminopyrimidine;
WR99210-like) are more effective inhibitors than Pyr.15,23
Reagents and conditions: (a) CH2N2, dioxane-MeOH; (b)
o
guanidine, DMSO-MeOH, 90-100 C; (c) cyclohexene, Pd/C, 65
oC; (d) Ph3P, DIAD, DMF, rt; (e) HCl, H2O.
We determined the inhibition constants, anti-Plasmodial
activities and toxicities against mammalian (Vero) cells of
the hybrid inhibitors. Table
1 shows the inhibition
constants (Ki) against PfDHFR enzymes of the hybrid
inhibitors, together with Pyr, P30 and P65 as comparators.
BT1 shows very high affinities for both wild-type and
mutant PfDHFRs, with much higher Ki values for human
DHFR (hDHFR), giving high Ki ratio (selectivity) value, as
compared with P30 and P65. However, BT2 and BT2S show
lower affinities for PfDHFRs and hDHFR, giving lower
selectivity. In contrast, Pyr shows weak binding with the
mutant enzymes, whereas P30 shows stronger binding with
both wild-type and mutant enzymes. However, these
compounds gave very low selectivity values. Unexpectedly,
the bi-rigid BT3S also has high affinities for all PfDHFRs
with high selectivity.
Figure 1. Designs of inhibitors which can automatically
modulate their binding modes to suit the catalytic sites which
they encounter. (A) Structures of representative DHFR
inhibitors. Pyr and P30 are rigid inhibitors whereas P65 is a
flexible inhibitor. (B) Our design of the dual binding mode
DHFR hybrid inhibitors. The rigid part, represented by Pyr
derivative, is joined with a flexible diaminopyrimidine moiety.
(C) Structures of hybrid inhibitors synthesized in this study.
ACS Paragon Plus Environment

Page 3 of 7
ACS Medicinal Chemistry Letters
Table 1. Inhibition constants (Ki) of the compounds against DHFR enzymes
Ki PfDHFR# (nM, ± SD)
1
2
3
4
Selectivity
ratio
(Ki h/Ki QM)
Ki hDHFR
(nM)
Double
mutation
(NRNI)
Triple
mutation 1
(IRNI)
Triple
mutation 2
(NRNL)
Quadruple
mutation (QM;
IRNL)
compd
Wild-type
(NCSI)
5
0.60±0.20a
0.80±0.10a
0.49±0.10
0.44±0.04
0.35±0.02
0.31±0.05
0.40±0.03
53.9±6.5a
1.40±0.00a
3.15±0.13
0.53±0.07
1.03±0.12
1.10±0.19
1.34±0.23
67.1±4.2b
2.10±0.22b
2.61±0.22
0.71±0.14
1.00±0.15
0.74±0.09
1.35±0.27
112±17b
2.71±0.43b
4.21±0.23
1.25±0.10
6.32±0.87
5.72±0.90
2.66±0.35
390±160b
3.30±0.40b
5.59±0.10d
2.32±0.24
6.96±0.91
5.8±1.5
28.2±2.5c
1.60±0.20c
47.6±4.0
22.5±2.9
18.1±1.3
20.2±3.3
33.6±10.6
0.07
0.5
8.5
9.7
2.6
3.5
8.1
6
7
8
9
Pyr
P30
P65
BT1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
BT2*
BT2S^
BT3S^
4.1±1.2
aData from ref.23 bData from ref.25 cData from ref.26 dData from ref.15 #Parentheses refer to PfDHFR haplotypes for residues 51, 59,
108 and 164. *Maximum soluble concentration = 1 µM. ^Salt forms of BT2 and BT3 were used due to their higher solubilities.
Table 2 Anti-plasmodial activity (IC50) against P. falciparum carrying wild-type or mutant DHFR and cytotoxicity
against mammalian (Vero) cells of the compounds
IC50 P. falciparum# (µM, ± SD)
Cytotoxicit
y Vero cells
(µM)
Selectivity
ratio
(Vero/V1/S)
compd
TM4/8.2 (NCSI)
K1CB1 (NRNI)
W2 (IRNI)
CSL-2 (NRNL)
V1/S (IRNL)
Pyr
0.08±0.01a
0.42±0.10a
0.23±0.07d
0.29±0.05
30.9±8.4a
1.31±0.45a
1.92±0.53
0.22±0.06
1.84±0.31
0.33±0.038
73.5±8.5b
3.94±0.80b
2.27±0.65
0.25±0.05
2.35±0.10
0.30±0.03
42±15b
2.53±0.38b
4.4±2.1
>100b
9.1±2.8b
3.5±1.6d
>100
7a
NAc
0.8
1.2
3.4
1.4
7.7
P30
P65
4.03±0.41
5.94±0.65
6.3±1.5
2.53±0.44
BT1
0.51±0.08
3.95±0.36
0.31±0.02
1.72±0.69
4.40±0.71
0.33±0.023
BT2S^
BT3S^
0.063±0.014
0.035±0.0033
aData from ref.23 bData from ref.25 cNot applicable. dData from ref.15 #Parentheses refer to PfDHFR haplotypes in P. falciparum
strains for residues 51, 59, 108 and 164. ^Salt forms of BT2 and BT3 were used due to their higher solubility.
Table 2 shows the anti-Plasmodial activities (IC50) and
toxicities against mammalian (Vero) cells of the inhibitors.
As anticipated from the Ki values, BT1 shows good IC50 (< 5
M) against both wild-type and Pyr-resistant mutant P.
falciparum. The corresponding IC50 value is higher against
Vero cells, which translates to moderate to high selectivity.
BT2S shows lower IC50 than BT1; however, it has
comparable IC50 against mutant parasite and selectivity to
P65, but with lower IC50 than BT1. Interestingly, the bi-rigid
inhibitor BT3S shows good IC50 against both wild-type and
mutant parasite with better selectivity than BT1 and BT2S.
In contrast, P30 and P65 show markedly lower selectivity
owing to either poor inhibition of mutant parasite strains,
or similar inhibition of parasites and Vero mammalian cells.
much lower than that of variants resistant to rigid or flexible
antifolates, each with 12 different haplotypes.22 However,
the diversity of PfDHFR resistance mutations in parasites
could be affected by other factors, such as GTP
cyclohydrolase gene copy number27 which are not modeled
in the bacterial surrogate.
Co-crystal structures of the hybrid inhibitors with the
enzymes were investigated. According to the binding modes
of wild-type and mutant PfDHFRs with rigid and flexible
pyrimidine inhibitors,16 the rigid end of BT1 with a m-
phenyl substituent on the pyrimidine nucleus would be
expected to bind preferentially to the active site pocket
containing S108 of the wild-type PfDHFR with no steric
hindrance. However, it was possible that the bulky p-phenyl
substituent of BT2 might encounter steric clash even with
S108, and BT2 might preferentially bind to the wild-type
active site with the flexible end. On the other hand, the
flexible ends of both BT1 and BT2 should bind
preferentially to the active site of mutant PfDHFR
containing the bulky S108N. Notably, BT3 can only bind to
the active sites of wild-type and mutant PfDHFR in the same
way. To investigate the effects and the interaction mode of
the three inhibitors to PfDHFR enzyme, we determined
BT1, BT2 and BT3 co-crystal structures of PfDHFR-TS, both
wild-type and quadruple mutant DHFR
We assessed the potential of PfDHFR variants conferring
resistance to BT1 using a bacterial surrogate system. From
a library of 1.5105 PfDHFR variants, BT1-resistant cells
were obtained. Fourteen independent BT1-resistant
colonies were characterized by DNA sequencing, which
showed novel resistance mutations K97N, S108T and
E199V in addition to the Pyr-resistant mutations N51I,
C59R and I164L. However, all BT1-resistant mutants
shared the same haplotype (combination of mutations) of
IRNTLV, in contrast with the wild-type NCKSIE and
quadruple mutant IRKNLE haplotypes. From these data, we
infer that the diversity of BT1-resistant PfDHFR variants is
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 7
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Inhibition modes of hybrid inhibitors in PfDHFR. Detailed interactions of (A) BT1, (B) BT2 and (C) BT3 with wild-type
PfDHFR, quadruple-mutant PfDHFR and superposition of wild-type and quadruple mutant PfDHFRs. Displacement of NADPH was
observed in quadruple mutant due to steric clash with S108N upon binding of BT3. Inhibitors and key amino acids are shown as a
stick model. Important interactions are highlighted as dashed lines in black and - interaction is drawn as a double dashed line.
domains at 2.2–2.38 and 2.38–2.6
Å
resolutions,
finding is that in the wild-type enzyme, although there is no
steric clash of the rigid end at S108, the p-substituted
phenyl moiety would collide with the hydrophobic pocket
of residues 111–116 if the rigid end of BT2 was docked at
54. For the quadruple-mutant enzyme, the flexible end can
avoid the steric clash with S108N and is preferred to the
rigid end at D54 site, while the rigid end of the molecule
caused some conformational adjustment of the enzyme
(Figure 2B).
respectively (Figure 2 and Table S1 in the Supporting
information). Moreover, BT1 and BT2 co-crystal structures
with hDHFR were determined in order to visualize the
difference in the binding modes of the hybrid inhibitors (see
details in the Supporting information)
As expected, BT1 showed distinctive binding modes
between wild-type and quadruple-mutant PfDHFRs (Figure
2A). It binds to the wild-type enzyme with the rigid end in
the active site, through the pyrimidine moiety interacting
with D54 and the rigid phenyl in the vicinity of S108. In
contrast, the flexible end of BT1 binds to the quadruple-
mutant enzyme at D54, with the flexible alkoxy group
avoiding steric clash with S108N in close proximity (3.4 Å).
The nicotinamide moiety of NADPH was not perturbed by
binding of BT1 and the pyrimidine core of the rigid end
formed - interaction with F116. BT1 with m-phenyl
substitution is therefore a potentially good template for
inhibitors which would forestall further resistance
mutations in both the S108 and 108N series.
As for compound BT3, comprising rigid moieties on both
ends, its good binding to quadruple-mutant as well as wild-
type PfDHFR was at first surprising. However, the high
affinity binding can be explained by the X-ray structures of
BT3 complexed with both wild-type and quadruple-mutant
PfDHFR, which showed well adapted accommodation of the
active sites to BT3 (Figure 2C). The side chain of S108N
greatly swung away from the active pocket to make room
for accommodation of BT3 as compared to BT1 and BT2.
Interestingly, the binding geometry of BT3 is very similar
for both wild-type and quadruple mutant PfDHFR; however,
in the PfDHFR quadruple mutant, BT3 binding causes a
significant displacement of the nicotinamide moiety,
resulting from a severe steric clash (Figure 3). From these
Interestingly, in contrast to the BT1 structures, BT2
binds to both wild-type and quadruple-mutant PfDHFR
with its flexible ends (Figure 2B). An explanation for this
ACS Paragon Plus Environment

Page 5 of 7
ACS Medicinal Chemistry Letters
Accession Codes
data, we infer that the enhanced inhibition of BT3 in
PfDHFR quadruple mutant occurs through displacement of
NADPH cofactor, which is insignificant in the wild-type
enzyme. A high temperature factor of NADPH was observed
in monomer A of quadruple mutant PfDHFR, whereas in
monomer B, NADPH could not be fully presented owing to
very weak electron density. It is noteworthy that no or very
little NADPH perturbation was observed where the flexible
part of the compounds was docked at D54. Therefore, the
displacement of the nicotinamide ring of NADPH is
important for inhibition of the PfDHFR quadruple mutant
by BT3.
1
2
3
4
5
6
7
8
The atomic coordinates and structure factor amplitudes have
been deposited in the Protein Data Bank, PfDHFR-TS with
accession codes 6A2K (TM4-BT1), 6A2L (V1/S-BT1), 6A2M
(TM4-BT2), 6A2N (V1/S-BT2), 6A2O (TM4-BT3) and 6A2P
(V1/S-BT3), and hDHFR with accession codes 6A7C (BT1) and
6A7E (BT2).
AUTHOR INFORMATION
Corresponding Author
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
*Tel. +6681 005 9293, Email: yongyuth@biotec.or.th
ORCID
Bongkoch Tarnchompoo: 0000-0002-8296-8976
Penchit Chitnumsub: 0000-0001-5920-3708
Philip J. Shaw: 0000-0003-3913-3057
Jarunee Vanichtanankul: 0000-0003-3431-1467
Sumalee Kamchonwongpaisan: 0000-0002-4723-5232
Yongyuth Yuthavong: 0000-0002-3582-8401
Present Addresses
Figure 3. Superposition of co-complex BT1, BT2 and BT3
structures of PfDHFRs. (A) wild-type PfDHFR. (B) quadruple-
mutant PfDHFR. The nicotinamide ring of NADPH was
perturbed by the binding of the hybrid inhibitors to different
levels in quadruple mutant PfDHFR structures while
insignificant displacement of NADPH occurred in wild-type.
The most severe perturbation of the NADPH was observed with
the binding of BT3, which coincided with the displacement of
S108N side chain.
†Faculty of Medical Technology, Rangsit University,
Pathumthani 12000, Thailand
Author Contributions
‡These authors contributed equally. The manuscript was
written by YY, BT, PC, PJS, JV and SK with contributions of all
authors.
Funding Sources
We gratefully acknowledge the financial support from Grand
Challenges Explorations - The Bill & Melinda Gates Foundation
(Grant ID # 52992). Protein crystallography was financially
supported by grants from Cluster Program and Management
Office, National Science and Technology development Agency,
Thailand (CPMO-P-13-00835 & P-14-50883), Synchrotron
Light Research Institute, Thailand (P-10-10168).
In conclusion, hybrid compounds such as BT1 with rigid
and flexible pharmacophores in the same molecule can bind
with high affinity to either wild-type/S108 series or
quadruple-mutant/108N series PfDHFR in the expected
manner, consequently with favorable IC50 and selectivity.
Significantly, the diversity of mutants resistant to BT1 is
extremely limited, in accordance with our prediction from
our earlier finding22 that there are conflicting requirements
for resistance mutations against rigid and flexible inhibitors
which tightly constrain resistance mutations against hybrid
compounds. It is noted that another hybrid compound, BT2,
binds both wild-type and mutant PfDHFR with the flexible
end. Yet another compound, BT3, which is rigid at both
ends, binds both wild-type and mutant PfDHFR with good
affinities. These apparent anomalies are, however, fully
explained by the X-ray structures of the complexes, which
show conformational changes of the enzyme to
accommodate the inhibitor. The high antimalarial activity of
BT1 and, importantly, its ability to forestall resistance
mutations, makes it a good template for further design and
synthesis of antifolates which are less vulnerable to
resistance. This family of rigid-flexible hybrid compounds
can be added to the list of new antifolates such as P218 with
good activity against pyrimethamine-resistant P.
falciparum.15
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank the experimental facility and the technical services at
beamline 13B1 provided by the “Synchrotron Radiation
Protein Crystallography 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 Technology, Taiwan, ROC. We
gratefully acknowledge the Synchrotron Light Research
Institute, Thailand for the experimental facility and technical
services at beamline 7.2W.
ABBREVIATIONS
PfDHFR, Plasmodium falciparum dihydrofolate reductase;
hDHFR, human dihydrofolate reductase; PfDHFR-TS,
Plasmodium falciparum dihydrofolate reductase-thymidylate
synthase;
azodicarboxylate
Pyr,
Pyrimethamine;
DIAD,
Diisopropyl
ASSOCIATED CONTENT
Supporting Information
REFERENCES
(1) Diagana, T. T. Supporting malaria elimination with 21st
century antimalarial agent drug discovery. Drug Discov. Today
2015, 20, 1265-1270.
The Supporting Information is available free of charge on the
ACS Publications website.
Result of co-crystal structures of hybrid inhibitors with hDHFR;
Materials and methods; Figure S1, Table S1 and Table S2 (PDF).
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 7
(2) Rabinovich, R. N.; Drakeley, C.; Djimde, A. A.; Hall, B. F.; Hay,
against a resistance-compromised target. Proc. Natl. Acad. Sci.
U.S.A. 2012, 109, 16823-16828.
S. I.; Hemingway, J.; Kaslow, D. C.; Noor, A.; Okumu, F.; Steketee, R.;
Tanner, M.; Wells, T. N. C.; Whittaker, M. A.; Winzeler, E. A.; Wirth,
D. F.; Whitfield, K.; Alonso, P. L. malERA: An updated research
agenda for malaria elimination and eradication. PLoS Med. 2017,
14, e1002456.
(3) Okombo, J.; Chibale, K. Recent updates in the discovery and
development of novel antimalarial drug candidates. Med. Chem.
Commun. 2018, 9, 437-453.
1
2
3
4
5
6
7
8
(16) Yuvaniyama, J.; Chitnumsub, P.; Kamchonwongpaisan, S.;
Vanichtanankul, J.; Sirawaraporn, W.; Taylor, P.; Walkinshaw, M. D.;
Yuthavong, Y. Insights into antifolate resistance from malarial
DHFR-TS structures. Nat. Struct. Biol. 2003, 10, 357-365.
(17) Kongsaeree, P.; Khongsuk, P.; Leartsakulpanich, U.;
Chitnumsub, P.; Tarnchompoo, B.; Walkinshaw, M. D.; Yuthavong,
Y. Crystal structure of dihydrofolate reductase from Plasmodium
vivax: pyrimethamine displacement linked with mutation-induced
resistance. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 13046-13051.
(18) Canfield, C. J.; Milhous, W. K.; Ager, A. L.; Rossan, R. N.;
Sweeney, T. R.; Lewis, N. J.; Jacobus, D. P. PS-15: a potent, orally
active antimalarial from a new class of folic acid antagonists. Am. J.
Trop. Med. Hyg. 1993, 49, 121-126.
(19) Yuthavong, Y.; Vilaivan, T.; Kamchonwongpaisan, S.;
Tarnchompoo, B.; Thongpanchang, C.; Chitnumsub, P.;
Yuvaniyama, J.; Matthews, D.; Charman, W.; Charman, S.; Vivas, L.;
Katiyar, S. B. Antimalarial Compounds with Flexible Side-Chains.
U.S. Patent 8,503,491. September 10, 2013.
(20) Ferlan, J. T.; Mookherjee, S.; Okezie, I. N.; Fulgence, L.; Sibley,
C. H. Mutagenesis of dihydrofolate reductase from Plasmodium
falciparum: analysis in Saccharomyces cerevisiae of triple mutant
alleles resistant to pyrimethamine or WR99210. Mol. Biochem.
Parasitol. 2001, 113, 139-150.
(21) Chusacultanachai, S.; Thiensathit, P.; Tarnchompoo, B.;
Sirawaraporn, W.; Yuthavong, Y. Novel antifolate resistant
mutations of Plasmodium falciparum dihydrofolate reductase
selected in Escherichia coli. Mol. Biochem. Parasitol. 2002, 120, 61-
72.
(22) Japrung, D.; Leartsakulpanich, U.; Chusacultanachai, S.;
Yuthavong, Y. Conflicting requirements of Plasmodium falciparum
dihydrofolate reductase mutations conferring resistance to
pyrimethamine-WR99210 combination. Antimicrob. Agents
Chemother. 2007, 51, 4356-4360.
(23) Tarnchompoo, B.; Sirichaiwat, C.; Phupong, W.; Intaraudom,
C.; Sirawaraporn, W.; Kamchonwongpaisan, S.; Vanichtanankul, J.;
Thebtaranonth, Y.; Yuthavong, Y. Development of 2,4-
diaminopyrimidines as antimalarials based on inhibition of the
S108N and C59R+S108N mutants of dihydrofolate reductase from
pyrimethamine-resistant Plasmodium falciparum. J. Med. Chem.
2002, 45, 1244-1252.
(24) Holsworth, D. D.; Jeremy, M. S.; Edmunds, J.; He, W.; Place,
S.; Maiti, S. An Expeditious Synthesis of 6-Alkyl-5-(4′-amino-
phenyl)-pyrimidine-2,4-diamines. Synth. Commun. 2003, 33, 3467-
3475.
(4) Hooft van Huijsduijnen, R.; Wells, T. N. The antimalarial
pipeline. Curr. Opin. Pharmacol. 2018, 42, 1-6.
9
(5) Imwong, M.; Suwannasin, K.; Kunasol, C.; Sutawong, K.;
Mayxay, M.; Rekol, H.; Smithuis, F. M.; Hlaing, T. M.; Tun, K. M.; van
der Pluijm, R. W.; Tripura, R.; Miotto, O.; Menard, D.; Dhorda, M.;
Day, N. P. J.; White, N. J.; Dondorp, A. M. The spread of artemisinin-
resistant Plasmodium falciparum in the Greater Mekong subregion:
a molecular epidemiology observational study. Lancet Infect. Dis.
2017, 17, 491-497.
(6) Straimer, J.; Gnadig, N. F.; Witkowski, B.; Amaratunga, C.;
Duru, V.; Ramadani, A. P.; Dacheux, M.; Khim, N.; Zhang, L.; Lam, S.;
Gregory, P. D.; Urnov, F. D.; Mercereau-Puijalon, O.; Benoit-Vical, F.;
Fairhurst, R. M.; Menard, D.; Fidock, D. A. Drug resistance. K13-
propeller mutations confer artemisinin resistance in Plasmodium
falciparum clinical isolates. Science 2015, 347, 428-431.
(7) The malERA Refresh Consultative Panel on Insecticide and
Drug Resistance. malERA: An updated research agenda for
insecticide and drug resistance in malaria elimination and
eradication. PLoS Med. 2017, 14, e1002450.
(8) Woodrow, C. J.; Dahlstrom, S.; Cooksey, R.; Flegg, J. A.; Le
Nagard, H.; Mentre, F.; Murillo, C.; Menard, D.; Nosten, F.; Sriprawat,
K.; Musset, L.; Quashie, N. B.; Lim, P.; Fairhurst, R. M.; Nsobya, S. L.;
Sinou, V.; Noedl, H.; Pradines, B.; Johnson, J. D.; Guerin, P. J.; Sibley,
C. H.; Le Bras, J. High-throughput analysis of antimalarial
susceptibility data by the WorldWide Antimalarial Resistance
Network (WWARN) in vitro analysis and reporting tool.
Antimicrob. Agents Chemother. 2013, 57, 3121-3130.
(9) Corey, V. C.; Lukens, A. K.; Istvan, E. S.; Lee, M. C.; Franco, V.;
Magistrado, P.; Coburn-Flynn, O.; Sakata-Kato, T.; Fuchs, O.; Gnadig,
N. F.; Goldgof, G.; Linares, M.; Gomez-Lorenzo, M. G.; De Cozar, C.;
Lafuente-Monasterio, M. J.; Prats, S.; Meister, S.; Tanaseichuk, O.;
Wree, M.; Zhou, Y.; Willis, P. A.; Gamo, F. J.; Goldberg, D. E.; Fidock,
D. A.; Wirth, D. F.; Winzeler, E. A., A broad analysis of resistance
development in the malaria parasite. Nat. Commun. published
online June 15, 2016; DOI: 10.1038/ncomms11901.
(10) Gregson, A.; Plowe, C. V. Mechanisms of resistance of
malaria parasites to antifolates. Pharmacol. Rev. 2005, 57, 117-
145.
(11) Venkatesan, M.; Alifrangis, M.; Roper, C.; Plowe, C. V.
Monitoring antifolate resistance in intermittent preventive
therapy for malaria. Trends Parasitol. 2013, 29, 497-504.
(12) Nzila, A. The past, present and future of antifolates in the
treatment of Plasmodium falciparum infection. J. Antimicrob.
Chemother. 2006, 57, 1043-1054.
(13) Yuthavong, Y.; Kamchonwongpaisan, S.; Leartsakulpanich,
U.; Chitnumsub, P. Folate metabolism as a source of molecular
targets for antimalarials. Future Microbiol. 2006, 1, 113-125.
(14) Yuthavong, Y.; Yuvaniyama, J.; Chitnumsub, P.;
Vanichtanankul, J.; Chusacultanachai, S.; Tarnchompoo, B.;
Vilaivan, T.; Kamchonwongpaisan, S. Malarial (Plasmodium
falciparum) dihydrofolate reductase-thymidylate synthase:
structural basis for antifolate resistance and development of
effective inhibitors. Parasitology 2005, 130, 249-259.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(25) Kamchonwongpaisan, S.; Quarrell, R.; Charoensetakul, N.;
Ponsinet, R.; Vilaivan, T.; Vanichtanankul, J.; Tarnchompoo, B.;
Sirawaraporn, W.; Lowe, G.; Yuthavong, Y. Inhibitors of multiple
mutants of Plasmodium falciparum dihydrofolate reductase and
their antimalarial activities. J. Med. Chem. 2004, 47, 673-680.
(26) Kamchonwongpaisan, S.; Vanichtanankul, J.; Tarnchompoo,
B.; Yuvaniyama, J.; Taweechai, S.; Yuthavong, Y. Stoichiometric
selection of tight-binding inhibitors by wild-type and mutant forms
of malarial (Plasmodium falciparum) dihydrofolate reductase. Anal.
Chem. 2005, 77, 1222-1227.
(27) Heinberg, A.; Kirkman, L. The molecular basis of antifolate
resistance in Plasmodium falciparum: looking beyond point
mutations. Ann. N. Y. Acad. Sci. 2015, 1342, 10-18.
(15) Yuthavong, Y.; Tarnchompoo, B.; Vilaivan, T.; Chitnumsub,
P.; Kamchonwongpaisan, S.; Charman, S. A.; McLennan, D. N.;
White, K. L.; Vivas, L.; Bongard, E.; Thongphanchang, C.; Taweechai,
S.; Vanichtanankul, J.; Rattanajak, R.; Arwon, U.; Fantauzzi, P.;
Yuvaniyama, J.; Charman, W. N.; Matthews, D. Malarial
dihydrofolate reductase as a paradigm for drug development
ACS Paragon Plus Environment

Page 7 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Hybrid Inhibitors of Malarial Dihydrofolate Reductase with Dual
Binding Modes which can Forestall Resistance
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Bongkoch Tarnchompoo,‡ Penchit Chitnumsub,‡ Aritsara Jaruwat, Philip J. Shaw, Jarunee
Vanichtanankul, Sinothai Poen, Roonglawan Rattanajak, Chayaphat Wongsombat, Aunchalee
Tonsomboon,† Sasithorn Decharuangsilp, Tosapol Anukunwithaya, Uthai Arwon, Sumalee
Kamchonwongpaisan, and Yongyuth Yuthavong*
For Table of Contents Use Only
7
ACS Paragon Plus Environment