Discovery of potent and selective inhibitors of toxoplasma gondii thymidylate synthase for opportunistic infections

Article abstract of DOI:10.1021/ml400208v

Infection by the parasite Toxoplasma gondii (tg) can lead to toxoplasmosis in immunocompromised patients such as organ transplant, cancer, and HIV/AIDS patients. The bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) enzyme is crucial for nucleotide synthesis in T. gondii and represents a potential target to combat T. gondii infection. While species selectivity with drugs has been attained for DHFR, TS is much more conserved across species, and specificity is significantly more challenging. We discovered novel substituted-9H-pyrimido[4,5-b]indoles 1-3 with single-digit nanomolar K _i for tgTS, two of which, 2 and 3, are 28- and 122-fold selective over human TS (hTS). The synthesis of these compounds, and their structures in complex with tgTS-DHFR are presented along with binding measurements and cell culture data. These results show, for the very first time, that, in spite of the high degree of conservation of active site residues between hTS and the parasite TS, specificity has been accomplished via novel structures and provides a new target (TS) for selective drug development against parasitic infections.

Full text of DOI:10.1021/ml400208v

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Letter
Discovery of potent and selective inhibitors of Toxoplasma
gondii thymidylate synthase for opportunistic infections
Nilesh Zaware, Hitesh Sharma, Jie Yang, Ravi K. V. Devambatla,
Sherry F. Queener, Karen S. Anderson, and Aleem Gangjee
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml400208v • Publication Date (Web): 04 Oct 2013
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Discovery of potent and selective inhibitors of Toxoplasma
gondii thymidylate synthase for opportunistic infections.
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Nilesh Zaware,a§Hitesh Sharma,b§ Jie Yang,a Ravi Kumar Vyas Devambatla,a Sherry F. Queener,c
Karen S. Andersonb* and Aleem Gangjeea*
aDivision of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes
Avenue, Pittsburgh, PA 15282, United States
bDepartment of Pharmacology, Yale University School of Medicine, New Haven, CT 06511, United StatescDepart-
ment of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, United
States.
§These authors contributed equally to this work.
KEYWORDS. Opportunistic infection; Toxoplasma gondii; Crystal structure; Thymidylate-synthase inhibitors; Active-
site inhibitors.
ABSTRACT: Infection by the parasite Toxoplasma gondii (tg) can lead to toxoplasmosis in immunocompromised patients
such as organ transplant, cancer and HIV/AIDS patients. The bifunctional thymidylate synthase-dihydrofolate reductase
(TS-DHFR) enzyme is crucial for nucleotide synthesis in T. gondii, and represents a potential target to combat T. gondii
infection. While species selectivity with drugs has been attained for DHFR, TS is much more conserved across species and
specificity is significantly more challenging. We discovered novel substituted-9H-pyrimido[4,5-b]indoles 1-3 with single-
digit nanomolar Ki for tgTS, two of which, 2 and 3, are 28- and 122-fold selective over human TS (hTS). The synthesis of
these compounds, and their structures in complex with tgTS-DHFR are presented along with binding measurements and
cell culture data. These results show, for the very first time, that in spite of the high degree of conservation of active site
residues between hTS and the parasite TS, specificity has been accomplished via novel structures and provides a new tar-
get (TS) for selective drug development against parasitic infections.
TEXT
plants and those undergoing cancer chemotherapy are
also highly susceptible to T. gondii infections.
According to the World Health Organization, the para-
site Toxoplasma gondii (T. gondii, tg) has infected up to a
third of the world’s population.1 Though the advent of
antiretroviral therapy (ART) has decreased the incidence
of T. gondii infection in AIDS patients, the infections still
arise because of noncompliance with therapy, discontinu-
ation of therapy because of toxicities with current treat-
ments, and the emergence of drug-resistance strains. Late
diagnosis of HIV and the rise of untreated HIV in devel-
oping countries also contributes to the persistence of T.
gondii infections as a clinical issue.2 T. gondii has been
defined by the Centers for Disease Control and Preven-
tion as an AIDS defining infection and a major cause of
morbidity and indeed mortality in HIV patients as recent-
ly as 20093 and 2013.4 Toxoplasma encephalitis (TE) is the
principal opportunistic infection (OI) caused by T. gondii
in AIDS patients. In addition to AIDS, other immune
compromised patients such as those with organ trans-
Sulfadiazine (SDZ) with pyrimethamine (PM) (Fig. 1) is
the principal drug combination for T. gondii infection
along with folinic acid for rescue of host bone marrow
toxicity from PM.2 Alternate first line therapy with sulfa-
methoxazole (SMX) and trimethoprim (TMP) (Fig. 1) is
also used.2 Problems in chemoprophylaxis, treatment fail-
ures, high rates of adverse drug reactions and drug intol-
erance to these very similar first-line agents, particularly
PM and the sulfonamide component,5 indicate an urgent
need for new agents. The most clinically useful combina-
tions, PM/SDZ (with folinic acid rescue) or TMP/SMX, act
synergistically.6 The sulfonamide component inhibits di-
hydropteroate synthase (DHPS) which catalyzes an early
step in the synthesis of folic acid. DHPS catalyzes the syn-
thesis of de novo folate biosynthesis, a pathway absent in
the human host. PM and TMP inhibit dihydrofolate re-
ductase (DHFR). DHFR catalyzes the synthesis of tetra-
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hydrofolate (THF) from folic acid as well as dihydrofolate tionally, hydrogen bonds are formed between the N9 of 2
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(DHF). Thus the most successful drugs for T. gondii inhib-
it folate biosynthesis and metabolism at two separate
steps to afford inhibition of purine and pyrimidine syn-
thesis. Since Toxoplasma cannot salvage thymidine from
the host,7 indirect inhibition of thymidylate synthase (TS)
via DHFR inhibition is possible, but the direct inhibition
of TS, the enzyme that catalyzes the conversion of dUMP
to dTMP for DNA synthesis would be most logical. How-
ever, no selective inhibitors of T. gondii TS (tgTS) are
known, up to now, that do not also potently inhibit hu-
man TS (hTS) as well.8 Unlike DHFR, where the human
and parasite enzymes are somewhat divergent in amino
acid sequence and structure allowing for selective inhibi-
tion of the parasite DHFR by 100-fold or greater, such
divergence is much less in TS from parasites and hu-
mans.9 There is significant homology between the active
site residues of tgTS and hTS. The significance of selective
inhibition of tgTS is that this would inhibit the replication
of T. gondii cells since T. gondii, unlike human, lacks sal-
vage of thymidine thus TS function in T. gondii is obliga-
tory for survival.10 Such selective inhibitors could be used
alone or in combination with PM, TMP, SMX and other
sulfonamides.
and carboxyl of Asn406, the Asp513 carboxylic oxygen
with N3 and the 2-NH2 group. The 2-NH2 group forms
additional hydrogen bond with the backbone oxygen of
Ala609. The scaffold is also stabilized by hydrophobic
interactions of the C-ring with Trp403. The phenyl ring is
oriented almost at a right angle to the tricyclic scaffold
and interacts with Ile402, Leu516, Phe520 and Met608.
Compound 3 retains the interactions of the tricyclic scaf-
fold and the hydrogen bonds with Asp513, Ala609 and
Asn409. Similar to 2, the 1 -naphthyl ring of 3 is oriented
almost at right angle to the tricyclic scaffold and interacts
with Ile402, Leu516, Phe520 and Met608.
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The structure of 3 in the TS active site suggested that
the 1 -naphthalene moiety of 3 must be a key determinant
of higher selectivity. The position of the compound in the
active site orients the 1 -naphthalene moiety close to the
C-terminal tail of the T. gondii TS domain. In fact, the
residues of the C-terminal tail are well ordered only in T.
gondii and not in the known X-ray crystal structures of
human TS. The 1 -naphthalene substituent of 3 also
makes hydrophobic interactions with M608 only in tgTS,
since the C-terminal tail shields the active site from sol-
vent, the tail could be a possible structural determinant
that facilitates additional hydrophobic interactions with
tgTS conferring selectivity for the parasite (Fig. 2).
Based on the potent bicyclic hTS inhibitor nolatrexed
reported by Webber et al.,11 we designed tricyclic com-
pounds 1-3 using as a novel scaffold for hTS inhibition
and as potential anticancer agents. Compounds 1-3 (Fig-
ure 1) contain a tricyclic pyrimido[4,5-b]indole scaffold
connected to a phenyl or a naphthalene moiety via a sul-
fur bridge. Compounds 1-3 were synthesized using
Scheme 1. Compound 4 was prepared by a method devel-
oped by Gangjee et al.12 Ullmann coupling13 of 4 with aryl
thiols 5-7 in a microwave apparatus (Initiator® from Bio-
tage) in the presence of potassium carbonate provided
target compounds 1-3, respectively, in yields of 28-40%.
Compounds 1-3 have also been evaluated against T.
gondii cells in culture (Table 2) and 3, at 5 µM, was found
to be equivalent to PM at 3 µM indicating a significant
effect on T. gondii in culture comparable to a clinically
used agent PM. The culture model requires that the ex-
perimental compounds rapidly kill exposed tachyzoites as
they are released from cells and/or that the experimental
compounds penetrate the cell and the vacuole containing
tachyzoites where the compounds may kill the organism
or prevent its replication.
Inhibition assays were performed on isolated T. gondii
TS-DHFR and hTS. Compounds 1-3 exhibited single-digit
nanomolar Ki values (Table 1). Surprisingly compounds 2
and 3 showed unprecedented selectivity for tgTS over hTS
of 28- and 122-fold respectively.
We have discovered, for the first time, highly potent
(single-digit nM) tgTS inhibitors 1-3 with up to a remark-
able 122-fold selectivity for tgTS over hTS (Ki in Table 1).
This level of selectivity is unprecedented for any parasite
with regard to TS. For comparison, TMP selectively inhib-
its tgDHFR and is used extensively with SMX to treat
Toxoplasma infection, but has an IC50 = 2.7 µM and a se-
lectivity of only 49-fold for tgDHFR over hDHFR.15 Fur-
ther PM, a widely used gold standard tgDHFR inhibitor
for TE and other T. gondii infections, only has a potency
of IC50 = 3.9 µM and a selectivity of only 5.9 for tgDHFR
over mammalian DHFR. In addition, we have also, for the
very first time, solved the X-ray crystal structure of the
bifunctional tgTS-DHFR enzyme with compounds 2 and
3.16 In cell cuture study, compound 3 showed similar po-
tency to PM, a highly effective standard drug used as a
control in these studies. We have thus demonstrated with
2 and 3 that structure variation affords increased selectivi-
ty while maintaining potency (compared to 1). Supple-
mentary Figure 3 shows dUMP and compound 3 and the
superposition of key active site residues (using tg num-
To determine the structural basis for the observed ac-
tivity and selectivity, we determined the crystal structures
of inhibitors 2 and 3 with T. gondii TS-DHFR (Figure 2).
By superimposing the tgTS and hTS (Figure S3), almost all
of the residues in the active site were identical. It was
surprising that a remarkable difference of 28- and 122-fold
in the Ki values of 2 and 3 respectively were observed in
spite of almost identical architectures of the active sites of
TS of the two organisms. As shown in Fig. 2, the crystal
structure of the tgTS-DHFR with 2 and 3 revealed exten-
sive base stacking interactions between the inhibitor and
the nucleotide dUMP as expected. Using co-crystal struc-
tures of 2 and 3 with tgTS-DHFR, we generated ligand
interactions using MOE 2011.10.14 Compound 2 is bound to
the active site by aromatic stacking of the pyrimido[4,5-
b]indole scaffold and the dUMP pyrimidine ring. Addi-
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ACS Medicinal Chemistry Letters
bering) in both human and tgTS. While all residues are
SCHEMES
1
2
3
4
5
6
7
8
identical between the two species, only M608 is present
in the tgTS structure. Future studies will be directed to-
ward solving the structure of compound 3 with human TS
to more fully discern possible differences. TS is a com-
pletely new and novel target for T. gondii. There is no
report, to our knowledge, of selective and potent inhibi-
tors of tgTS as anti-OI agents. The ability to provide
agents that are selective for tgTS on the basis of structure-
based rational design is a major step towards providing
novel clinically useful agents with tgTS inhibition.
Scheme 1. Synthesis of 1-3
Ar
Cl
O
S
O
N
CuI, K2CO3, DMF
ArSH, µW,
HN
H2N
HN
H2N
200 °C, 30 min
5 Ar = 2-naphthyl
6 Ar = phenyl
N
N
N
H
H
4
1 Ar = 2'-naphthyl, 30%
2 Ar = phenyl, 40%
7
Ar = 1-naphthyl
3
Ar = 1'-naphthyl, 28%
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TABLES
FIGURES
Ar
S
Table 1. Kinetic Evaluation of 1-3 Reveals Species Selectiv-
itya
Cl
5
O
4
NH2
N
NH2
OMe
OMe
HN
N
N
IC50 (nM)
Ki (nM)
tgTS
Selectivi-
ty (hTS
Ki/ tgTS
Ki)
2
9
N
H2N
H2N
N
H2N
N
H
Cmpd
OMe
1 Ar = 2'-naphthyl
2 Ar = phenyl
Trimethoprim
TMP
Pyrimethamine
PM
tgTS
hTS
hTS
(
)
(
)
3
Ar = 1'-naphthyl
Figure 1. DHFR inhibitors (PM and TMP) in the clinic for T.
gondii infections and compounds 1-3.
28.8 ±
21
1.8 ±
1.1
3.3 ±
2.4
1
2
3
21 ± 13
1.83
23 ±
6.6
481 ±
162
2.0 ±
0.6
55.7 ±
18.8
27.9
121.9
35.8 ± 3250 ±
2.9 184
3.1 ±
0.25
378 ±
21
Compound 2
aT. gondii TS-DHFR (25 nM) and human TS (50 nM) were
preincubated with 100 µM dUMP and inhibitor. The reac-
tion was initiated with 100 µM methyleneTHF. Due to
the concentrations of enzyme required for assay and the limits
of detection, for those compounds having Ki values in the
lower nanomolar range, the values reported represent an upper
limit.
b
Table 2. Toxoplasma Cell Culture Study
F374
Compound 3
Estimated
IC50 value,
µMa
T. gondii
Condition in media
at Day 4
T. gondii in
cells at Day
4
F520
L516
I402
No drug
1 (5 µM)
2 (5 µM)
3 (5 µM)
3710000
2350000
100000
<25000
1140000
340000
140000
<2500
D513
W403
2.6
3.7
M608
L486
Y553
N406
0.6
0.57
Figure 2. T. gondii TS active site with key residues high-
lighted in red. The inhibitors 2 and 3 and dUMP are colored
yellow.
PM (3 µM) <25000
<2500
aDose response curves using data from the three concen-
trations of test compounds gave estimates of IC50 values.
ACKNOWLEDGMENTS
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This work was supported, in part, by NIH grants CA152316 (to
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ASSOCIATED CONTENT
Supporting Information. Synthetic procedures, compound
characterization, biological assays and X-ray structure de-
termination. This material is available free of charge via the
Internet at http://pubs.acs.org.
9
Accession Codes
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The atomic coordinates of T.gondii TS-DHFR/Compound 2
and T.gondii TS-DHFR/Compound 3 described here have
been deposited in the RCSB Protein Data Bank
(www.pdb.org)
with
accession
codes
4KY4
and
4KYA respectively.
AUTHOR INFORMATION
Corresponding Authors
*These authors contributed equally to this work: (A.G.) Tel.:
+1-412-396-6070, fax: +1-412-396-5593, e-mail:
gangjee@duq.edu; (K.S.A.) Tel.: +1-203-785-4526, fax: +1-203-
785-7670, e-mail: karen.anderson@yale.edu.
12. Gangjee, A.; Zaware, N.; Raghavan, S.; Ihnat, M.; Shenoy, S.;
Kisluik, R. L. Single Agents with Designed Combination
Chemotherapy Potential: Synthesis and Evaluation of Sub-
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and Thymidylate Synthase Inhibitors and as Antitumor
Agents. J. Med. Chem. 2010, 53, 1563–1578.
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G. Ligand-Free Copper-Catalyzed C-S Coupling of Aryl Io-
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14. Molecular Operating Environment (MOE 2011.10); Chemical
Computing Group, Inc.: Montreal, Quebec, Canada, 2008;
www.chemcomp.com.
15. Piper, J. R.; Johnson, C. A.; Krauth, C. A.; Carter, R. L.; Hos-
mer, C. A.; Queener, S. F.; Borotz, S. E.; Pfefferkorn, E. R.
Lipophilic Antifolates as Agents against Opportunistic In-
fections. 1. Agents Superior to Trimetrexate and Piritrexim
against Toxoplasma gondii and Pneumocystis carinii in in
vitro Evaluations. J. Med. Chem. 1996, 39, 1271–1280.
16. Sharma, H.; Landau, M. J.; Vargo, M. A.; Spasov, K. A.; An-
derson, K. S. First Three-Dimensional Structure of Toxo-
plasma gondii Thymidylate Synthase-Dihydrofolate Reduc-
tase: Insights for Catalysis, Interdomain Interactions, and
Substrate Channeling. Biochemistry 2013, Submitted.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
tg, T. gondii; TS, thymidylate synthase; DHFR, dihydrofolate
reductase; TE, toxoplasma encephalitis; OI, opportunistic
infection; SDZ, sulfadiazine; PM, pyrimethamine; SMX, sul-
famethoxazole; TMP, trimethoprim.
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