Dual and selective inhibitors of pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Leishmania chagasi

Article abstract of DOI:10.1080/14756366.2019.1651311

Leishmaniasis is a tropical disease found in more than 90 countries. The drugs available to treat this disease have nonspecific action and high toxicity. In order to develop novel therapeutic alternatives to fight this ailment, pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHF-TS) have been targeted, once Leishmania is auxotrophic for folates. Although PTR1 and DHFR-TS from other protozoan parasites have been studied, their homologs in Leishmania chagasi have been poorly characterized. Hence, this work describes the optimal conditions to express the recombinant LcPTR1 and LcDHFR-TS enzymes, as well as balanced assay conditions for screening. Last but not the least, we show that 2,4 diaminopyrimidine derivatives are low-micromolar competitive inhibitors of both enzymes (LcPTR1 Ki = 1.50–2.30 μM and LcDHFR Ki = 0.28–3.00 μM) with poor selectivity index. On the other hand, compound 5 (2,4-diaminoquinazoline derivative) is a selective LcPTR1 inhibitor (Ki = 0.47 μM, selectivity index = 20).

Full text of DOI:10.1080/14756366.2019.1651311

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Dual and selective inhibitors of pteridine
reductase 1 (PTR1) and dihydrofolate reductase-
thymidylate synthase (DHFR-TS) from Leishmania
chagasi
Bárbara Velame Ferreira Teixeira, André Lacerda Braga Teles, Suellen
Gonçalves da Silva, Camila Carane Bitencourt Brito, Humberto Fonseca de
Freitas, Acássia Benjamim Leal Pires, Thamires Quadros Froes & Marcelo
Santos Castilho
To cite this article: Bárbara Velame Ferreira Teixeira, André Lacerda Braga Teles, Suellen
Gonçalves da Silva, Camila Carane Bitencourt Brito, Humberto Fonseca de Freitas, Acássia
Benjamim Leal Pires, Thamires Quadros Froes & Marcelo Santos Castilho (2019) Dual and
selective inhibitors of pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate
synthase (DHFR-TS) from Leishmaniaꢀchagasi, Journal of Enzyme Inhibition and Medicinal
Chemistry, 34:1, 1439-1450, DOI: 10.1080/14756366.2019.1651311
To link to this article: https://doi.org/10.1080/14756366.2019.1651311
© 2019 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1439–1450
https://doi.org/10.1080/14756366.2019.1651311
RESEARCH PAPER
Dual and selective inhibitors of pteridine reductase 1 (PTR1) and dihydrofolate
reductase-thymidylate synthase (DHFR-TS) from Leishmania chagasi
a
b,c,d
a
ꢀ
ꢀ
ꢀ
ꢀ
Barbara Velame Ferreira Teixeira , Andre Lacerda Braga Teles
, Suellen Gonc¸alves da Silva ,
ꢀ
Camila Carane Bitencourt Britob, Humberto Fonseca de Freitasa,b, Acassia Benjamim Leal Pires ,
d
Thamires Quadros Froesb and Marcelo Santos Castilhoa,b
a
b
ꢀ
~
ꢀ
ꢀ
~
^
Programa de Pos-Graduac¸ao em Farmacia, Universidade Federal da Bahia, Salvador, BA, Brazil; Programa de Pos-Graduac¸ao em Biotecnologia,
c
ꢀ
~
^
Universidade Estadual de Feira de Santana, Feira de Santana, BA, Brazil; Programa de Pos-Graduac¸ao em Ciencias Farmaceuticas, Universidade
Estadual da Bahia, Salvador, BA, Brazil; Departamento de Ciencias da Vida, Universidade do Estado da Bahia, Salvador, BA, Brazil
d
^
ABSTRACT
ARTICLE HISTORY
Received 31 March 2019
Revised 27 July 2019
Accepted 29 July 2019
Leishmaniasis is a tropical disease found in more than 90 countries. The drugs available to treat this dis-
ease have nonspecific action and high toxicity. In order to develop novel therapeutic alternatives to fight
this ailment, pteridine reductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase (DHF-TS) have
been targeted, once Leishmania is auxotrophic for folates. Although PTR1 and DHFR-TS from other proto-
zoan parasites have been studied, their homologs in Leishmania chagasi have been poorly characterized.
Hence, this work describes the optimal conditions to express the recombinant LcPTR1 and LcDHFR-TS
enzymes, as well as balanced assay conditions for screening. Last but not the least, we show that 2,4 dia-
minopyrimidine derivatives are low-micromolar competitive inhibitors of both enzymes (LcPTR1
Ki ¼ 1.50–2.30 mM and LcDHFR Ki ¼ 0.28–3.00 mM) with poor selectivity index. On the other hand, com-
pound 5 (2,4-diaminoquinazoline derivative) is a selective LcPTR1 inhibitor (Ki¼ 0.47 mM, selectivity
index ¼ 20).
KEYWORDS
Dual inhibitors; pteridine
reductase 1; dihydrofolate
reductase-thymidylate
synthase; Leishmania
chagasi; selective inhibitors
Introduction
the selection of pyrimethamine-resistant Plasmodium falciparum
strains underscores the risks of blocking a single target from this
pathway18. Single point mutations19, as well as biochemical path-
way plasticity17,20, can significantly reduce the drug effectiveness.
In fact, a bypass mechanism explains why Leishmania parasites
have low susceptibility to methotrexate or trimethoprim21–23, des-
pite the fact that DHFR-TS gene knockout is lethal to the
Leishmania parasites24.
Leishmaniasis is an infectious disease caused by several species of
the genus Leishmania, which are responsible for its different epi-
demiological and clinical characteristics1. Visceral leishmaniasis
(VL), the most severe clinical form of leishmaniasis, affects 300,000
people worldwide, 90% of whom would die if no treatment is pro-
vided2. The available drugs to fight this disease have been in the
market for more than 40 years and for that reason, resistant strains
have been selected3. On top of that, N-methyl glucamine
Nare, Luba, and Beverly22 reported that upon dihydrofolate
reductase-thymidylate synthase (DHFR-TS) inhibition, pteridine
reductase 1 (PTR1) provides enough folate to guarantee the para-
site survival. This finding suggested that both enzymes (DHFR-TS
and PTR1) should be targeted to develop useful leishmanicidal
drugs17. This hypothesis is still a matter of debate once the ptr1
gene knockout proved lethal to the parasite, probably because
reduced pterin, which is produced only by PTR1, is essential for
parasite growth and metacyclogenesis22. Accordingly, some
authors consider PTR1 a validated target for drug development
and have pursued its inhibition15,24,25. Despite the advances in the
field, the risk of selecting resistant strains was not addressed in
those papers.
R
(GlucantimeV), the drug of choice to treat patients with leishman-
iasis, may cause nephrotoxicity, anorexia, abdominal pain, lethargy
and cardiotoxicity4 and the second-choice drug (amphotericin B)
shows cardiac and nephron toxicity5. Although two new drug can-
didates to treat VL entered clinical tests in 20176, the search for
new safe, effective, oral, short-course leishmanicidal agents contin-
ues to be a priority. In order to achieve this goal, several efforts
have been made to develop compounds that inhibit carbonic
anhydrase7–9, arginase10, or enzymes that protect the parasite
from the oxidative stress11,12
.
Trypanosomatids dependence on host-synthesized purine and
pteridines has long been targeted to fight Chagas disease13,
human African trypanosomiasis14, and leishmaniasis15,16. Among
the enzymes that play a key role in the salvage and folate path-
way, dihydrofolate reductase is the macromolecular target of sev-
Another caveat of previous work is the fact that most of the
biological data reported so far was obtained for PTR1 from
Leishmania major (LmPTR1), which causes cutaneous leishmaniasis,
not the visceral form, as L. chagasi (syn. L. infantum) does. Despite
eral drugs, including those available to fight malaria17. However, the high overall sequence, similarities between LmPTR1 and
ꢀ ~ ꢀ
Programa de Pos-Graduac¸ao em Farmacia, Universidade Federal da Bahia, Salvador, BA, Brazil
CONTACT Marcelo Santos Castilho
Both authors have contributed equally to this work.
castilho@ufba.br
ꢀ
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1440
B. V. F. TEIXEIRA ET AL.
ꢀ
LcPTR1 (90% – Figure S1), the active site of L. chagasi PTR1 is BA262), provided by Dr. Fabio Rocha Formiga (FIOCRUZ - Bahia)
expected to be more flexible, once this enzyme is 100% identical was extracted, as suggested in the UltraClean Tissue & Cells DNA
to L. donovani PTR1, whose X-ray structure shows a disordered Isolation kit protocol (Mobio, Carlsbad, CA, USA), and quantified at
substrate binding site26. As a consequence, LmPTR1 inhibitors may
260 nm wavelength (Nanodrop 200, Thermo Scientific, Waltham,
MA, USA).
The polymerase chain reaction for LcPTR1 gene amplification
not be as effective against LcPTR1.
In order to shed light on this matter, we expressed recombin-
ant DHFR-TS and PTR1 from L. chagasi in Escherichia coli, so that
standardized and balanced kinetic assays could be carried out.
Next, we investigated 2,4-diaminopyrimidine derivatives as poten-
tial dual inhibitors for both enzymes, since derivatives of this
chemical class have already been described as dihydrofolate
reductase from Schistosoma mansoni (SmDHFR)27, LmDHFR-TS and
LmPTR124 inhibitors.
was carried out in the ProFlex 3x32-well thermal cycler (Applied
Biosystems, Waltham, MA, USA). The following steps and condi-
tions were employed: initial step at 95 ꢁC for 3 min, followed by
35 cycles of denaturation step at 95 ꢁC for 30 s, annealing at 43 ꢁC
for 30 s and a final extension step at 72 ꢁC for 110 s. The PCR
product was loaded on a 1% agarose gel for electrophoretic sep-
aration (100 volts for 70 min) and then purified using the kit
R
WizardV SV Gel and PCR Clean-Up System (Promega, Madison, WI,
USA). The purified product was quantified spectrophotometrically
(Nanodrop 200, Thermo Scientific) at 260 nm wavelength.
The pETM11-LIC vector previously linearized by BsaI (NEB)
digestion (60 min at 50 ꢁC) and the insert were treated with T4
DNA polymerase for cohesive ends formation and then incubated
(1:3) for 30 min at 25 ꢁC for annealing. Then, E. coli (DH10b) chem-
ically competent cells were transformed by heat shock28 and the
product of the transformation was grown in Luria Bertani (LB)
medium (tryptone 1%, sodium chloride 1%, yeast extract 0.5%
and bacteriological agar 1.5%), supplemented with 30 lg/mL kana-
mycin, for 16 h at 37 ꢁC.
Materials and methods
Synthesis of 2,4-diaminopyrimidines
The 2,4-diaminopyrimidine derivatives were synthesized as
described by Teles et al.27. Briefly, all compounds were obtained
in moderate yields by the condensation of suitable ketonitriles
and guanidine as described in Figure 1.
Cloning of the enzyme LcPTR1
LcPTR1 cloning was carried out through the ligation independent
cloning (LIC) protocol (https://www.helmholtz-muenchen.de/filead-
min/PEPF/Protocols/LIC-cloning.pdf). The genomic sequence
that codes for PTR1 from L. chagasi, available in Genbank
server (Accession code XM_001465671.1), was employed to pri-
True-positive clones were confirmed by colony PCR as follows:
a colony-forming unit (CFU) was suspended in 100 ll type I ster-
ile-water, which as then boiled for 5 min. The DNA from this sus-
pension was employed as template material for PCR-amplification.
The genetic material was PCR amplified using 1X PCR buffer
R
mers design. The Phusion system (New England BiolabsV) was
R
(InvitrogenV, Madison, WI, USA), MgCl2 1.5 mM, dNTP 0.2 mM,
R
0.2 lL PlatinumV Taq DNA polymerase (Thermo Scientific) and
used to amplify the DNA fragment containing the LcPTR1
sequence using the forward and reverse primers 50-
CAGGGCGCCATGGCTGCTCCGACCGTG-30 and 50-GACCCGACGCG
GTTATCAGGCCCGGGTAAGGCT-3’,
acquired from Exxtend (https://www.exxtend.com.br/site/). The
nucleotide sequence (bold) required for the LIC cloning system
water (q.s. to 20 lL) and the T7 primers (forward: 50-
TAATACGACTCACTATAGGG-30
and
reverse:
50-
TAGTTATTGCTCAGCGGTGG-30) and thermocycling parameters pre-
viously described for LcPTR1 amplification.
respectively,
which
were
The products of this reaction were analyzed on 1% agarose gel
use, was inserted in the sequence of the forward and reverse pri- (100 V/1h) to confirm the presence of the gene’s amplification
mers. The remaining primer sequences hybridize to LcPTR1 gene. band (0.9 kbp) as true-positive construction. Positive clones were
Genomic DNA from Leishmania chagasi cells (MHOM/BR/00/ cultured for 12 h in LB broth, supplemented with kanamycin
Figure 1. Major route steps to obtain 2,4-diaminopyrimidine derivatives 1–5. Chemical structures are shown in the lower panel.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1441
(30 lg/ml), at 37 ꢁC and 150 RPM and then had their expression- chagasi (MHOM/BR/00/BA262) promastigotes was extracted, as
plasmid DNA extracted with the aid of Pureyield Plasmid Miniprep suggested in the UltraClean Tissue & Cells DNA Isolation kit proto-
System kit (Promega – A1223). The extracted plasmid was used col (Mobio) and quantified using Qubit fluorimeter and Qubit
for heat shock transformation28 of the chemically competent dsDNA BR kit (Invitrogen).
E. coli BL21 (DE3) cells. The expression-plasmid containing N-ter-
minal His-tag, TEV cleavage site, and LcPTR1 gene was sequenced
using T7 primers and ABI 3500 Platform (Applied Biosystem) at
Molecular Biology Research Laboratory (Pharmacy college – UFBA,
Salvador, Brazil).
The LcDHFR-TS coding region was PCR-amplified using the
genomic DNA, as a template, and the primers described next:
The forward primer includes the NdeI restriction site (underlined)
50-CAATACGCATATGTCCAGGGCAGCTG-30 and the reverse primer
includes the HindIII restriction site (underlined) 50-GCCTCCAAGCTT
TCTTAACGGCCATC-30. The restriction site for the NdeI enzyme was
positioned in the 5’ region of the LcDHFR-TS gene, which allowed
the expression of the enzyme containing the polyhistidine tail in
the N-terminal domain of the protein.
Expression and purification of LcPTR1
A CFU previously grown on LB agar was inoculated into 3 ml of
sterile LB broth supplemented with kanamycin (30 mg/ml) and
kept under constant stirring (250 RPM at 37 ꢁC for 12 h). After this
time, the cell suspension was diluted (1: 100) in sterile LB broth
(10 mg tryptone, 5 mg yeast extract and 5 mg NaCl per ml, pH 7)
supplemented with antibiotics and kept under constant shaking
(180 RPM) at 37 ꢁC until OD600nm ¼ 0.6, at this point, the tempera-
ture was lowered to 25 ꢁC, the culture was kept at constant shak-
ing (180 RPM) for 18 h and multiple combinations of IPTG
concentrations (0.05–1 mM) were evaluated for the soluble expres-
sion of LcPTR1.
The cells were then recovered by centrifugation (16,000 Xg at
4 ꢁC for 15 min in a Sigma 1–14 K centrifuge) and resuspended in
50 mM Tris-HCl buffer pH 8.0 containing 125 mM NaCl and 20 mM
imidazole supplemented by 1 mM phenylmethanesulphonylfluor-
ide (PMSF P7626 Sigma Aldrich, Chicago, IL EUA) to mechanical
lysis by sonication (15 cycles of sonication at 8 Watts for 15 s
each, with 30 s intervals), in an ice-cold bath. The soluble fraction
from lysate was recovered by centrifugation (14.500 Xg at 4 ꢁC for
15 min in a Sigma 1–14 K centrifuge). Next, the soluble fraction
The PCR mixture included 70 ng (1 mL) of genomic DNA, 1ꢂ HF
R
buffer (Thermo ScientificV) buffer, 1.5 mM MgCl2, 0.2 mM dNTP,
0.5 lM each primer and water q.s. to 50 lL. The gene amplification
reaction was carried out in the ProFlex 3x32-well thermal cycler
(Applied Biosystems) using the following parameters: single
denaturation step (94 ꢁC for 2 min), followed by 35 cycles of
denaturation at 94ꢁ C for 30 s, annealing at 56.2 ꢁC for 30 s and
extension at 72ꢁ C for 110 s. PCR included a final extension at
72 ꢁC for 600 s.
The reaction product was loaded on a 1% agarose gel for elec-
trophoretic separation (100 Volts for 70 min). Next, the amplicon
was purified directly from the gel matrix with Gene Jet Gel extract
R
and DNA cleanup kit (Thermo ScientificV) and quantified spectro-
photometrically (Nanodrop 200, Thermo Scientific) at 260 nm
wavelength.
Next, the purified DNA fragment and pET28a vector were indi-
vidually double-digested with NdeI and HindIII FastDigest restric-
tion enzymes (Thermo Scientific) and then ligated (5:1 insert/
vector ratio) in the presence of T4 DNA ligase (Thermo Scientific)
according to manufacturer instructions. E. coli ArticExpress (DE3)
cells were transformed with the recombinant plasmid by heat
shock protocol28. Colonies containing the inserted gene were
selected using the resistance markers to kanamycin and gentamy-
cin. True-positive clones were confirmed by colony PCR (using T7
primers: forward: 50- TAATACGACTCACTATAGGG- 30 and reverse:
50-TAGTTATTGCTCAGCGGTGG-30as described previously. The amp-
lification parameters were set as follows: single initial denaturation
step at 94 ꢁC for 180 s followed by 35 cycles of denaturation at
94 ꢁC for 45 s, annealing at 42.5 ꢁC for 30 s and extension at 72 ꢁC
for 110 s. A final extension step (72 ꢁC for 600 s) was carried out
complete the reaction. The amplification products were analyzed
by 1% agarose gel electrophoresis and those 1718 bp-compatible
amplified DNAs (1569 bp from insert plus 149 bp from the vector)
was submitted to DNA sequencing using T7 primers on ABI 3730
Platform (Applied Biosystem) at Myleus Biotechnology center
(Minas Gerais/Brazil). Colonies transformed with mutation-free
plasmid were employed for the following steps.
was filtered (0.45 lm – Sartorius) and loaded on crude His Trap FF
R
column (GE Healthcare Life SciencesV, Chicago, IL EUA) previously
equilibrated with 10 column volumes (CV) of 50 mM Tris-HCl buf-
fer pH 8.0 containing 200 mM NaCl (buffer A). After the system
was washed with 10 CV of buffer A supplemented with 20 mM
imidazole, followed by 5 CV of buffer A supplemented with
50 mM imidazole. Finally, the LcPTR1 was eluted with 5 CV of buf-
fer A containing 500 mM imidazole.
All steps of the purification were monitored by 12% polyacryl-
amide gel electrophoresis (SDS-PAGE). The fractions containing
the protein were pooled and the imidazole was removed after
successive dilution steps (A 1:10 buffer) and centrifugation at with
Amicon 30 kDa (Millipore, Chicago, IL EUA) a 4 ꢁC e 3500 Xg. Then,
imidazole-free LcPTR1 was incubated with Tobacco Etch Virus pro-
tease (TEV), 1:20 (M/M) ratio, at 4 ꢁC for 12 h to cleavage of N-ter-
minus His tag. The proteolysis product was loaded on a Nickel-
R
sepharose column (GE HealthcareV), previously equilibrated with
buffer A (10 CV).
The cleaved LcPTR1, collected in the void, had its concentration
measured using Bradford reagent29 (595 nm) (Figure S2). All purifi-
cation steps were monitored by 12% SDS-PAGE.
Expression and purification of LcDHFR-TS
A CFU, previously grown on LB agar supplemented with kanamy-
cin (30 mg/mL) and gentamycin (20 mg/mL) was cultivated over-
night in antibiotic-supplemented LB broth (peptone from gelatin
1%, yeast extract 0,5%, sodium chloride 1% adjusted to pH 7,5), at
37 ꢁC and 200 RPM. After that, the cellular suspension (pre-inocu-
lum) was employed to inoculate a freshly prepared sterile anti-
biotic-supplemented LB broth (1:100), which was kept at 37 ꢁC
and 200 RPM until OD600nm ¼ 0.6. At this point, the temperature
Cloning of LcDHFR coding gene into E. coli ArticExpress (DE3)
cells
The coding sequence for Leishmania chagasi strain JPCM5 DHFR-
TS available in Genbank server (Accession code: XM_001463132.2)
plus 23 nucleotides flanking the gene was employed for primer
design and cloning into pET28a vector.
Restriction sites were assessed using the NebCutter server was lowered to 10ꢁ C and IPTG was added (0.5 mM). After 48 h,
(http://nc2.neb.com/NEBcutter2/). Genomic DNA from Leishmania the cells were then recovered by centrifugation (4000 Xg at 4 ꢁC

1442
B. V. F. TEIXEIRA ET AL.
for 10 min) and resuspended in 50 mM phosphate buffer pH 7.0, substrate were determined under a pseudo first-order kinetics
containing 200 mM (NH4)2SO4 supplemented with protease cock- assumption using least-squares non-linear regression method, as
R
tail inhibitors 0,1X (SigmaFASTV - S8830), for mechanical lysis by available in the GraphPad Prism version 5.0 for Windows
R
sonication (15 cycles of sonication at 8 Watts for 30 s each, with (GraphPadV Software, San Diego, CA, USA, www.graphpad.com).
30 s intervals, using Sonics Vibra-Cell Model VCX130PBt), in an
ice bath.
LcPTR1 apparent Km determination
Next, cellular debris and insoluble proteins (sediment) were
separated from the supernatant by centrifugation (14.500 Xg at
4 ꢁC for 30 min in a Sigma 1–14 K centrifuge) and the soluble frac-
tion was loaded on Ni sepharose His-trap fast flow resin (GE
Healthcare) previously equilibrated with 5 CV of buffer A (50 mM
phosphate buffer pH 7.0 containing 200 mM (NH4)2SO4 and 50 mM
imidazole). 200 mM ammonium sulfate was added to the purifica-
tion protocol and stock solution to increase the stability of the
protein30.
After contaminants were eluted with 30 CV of buffer A,
LcDHFR-TS was eluted by linear increase of imidazole concentra-
tion (50–500 mM) within buffer A. The fractions containing the
protein were pooled together and the imidazole was removed by
successive dilution and centrifugation steps (3000 Xg at 4 ꢁC for
30 min) in 50 mM phosphate buffer pH 7.0 containing 200 mM
(NH4)2SO4. Imidazole-free LcDHFR-TS had the N-terminus polyhisti-
dine tag removed by thrombin (Sigma Cat. T7513) digestion at
25 ꢁC for 2.5 h (0.1:5 Thrombin Unit: LcDHFR-TS mg). Cleaved pro-
tein was recovered by loading the digested protein solution onto
Ni sepharose His-trap resin (GE Healthcare), previously equilibrated
with 50 mM phosphate buffer pH 7.0 containing 200 mM of
(NH4)2SO4, and eluting it with 20 ml of the same buffer. All purifi-
cation steps were monitored by 12% SDS-PAGE. The LcDHFR-TS
had its concentration measured using Bradford reagent29 (595 nm)
(Figure S2). The amount of contaminant thrombin present in the
final solution resulting from the LcDHFR-TS purification protocol is
approximately 0.5% in weight (0.0062 mg of thrombin per
LcDHFR-TS mg). Aliquots of the purified enzyme were stored at
-80 ꢁC in the presence of 30% glycerol (v/v) and thawed immedi-
ately before use.
Kinetic measurements were performed according to the
protocol described32 via spectrophotometer (Shimadzu UV-1800)
at pH 4.7 (20 mM sodium acetate). NADPH consumption was
monitored spectrophotometrically (340 nm) at constant tempera-
ture (30 ꢁC). The variation of the absorbance monitored for 60 s
was used to calculate the initial rate of the reaction by linear
regression.
The apparent Km of NADPH was determined by varying its
concentration (6–300 lM), maintaining the biopterin at
saturation concentration (100 lM). Likewise, the apparent Km of
the substrate was determined by saturation of NADPH concentra-
tion (100 lM) and increasing concentrations of biopterin
(3.12–100 lM).
All measurements were performed in triplicate and the
apparent values of Km for the cofactor and substrate were
determined using the non-linear regression method (3-parameter
equation), as available in GraphPad Prism version 5.0 for
R
Windows (GraphPadV Software, San Diego, CA, USA, www.graph-
pad.com).
Single concentration inhibition assays
The in vitro screening of putative inhibitors was carried out in bal-
anced conditions: LcPTR1 0.4 lM, biopterin 25 lM, NADPH 35 lM
in 20 mM sodium acetate buffer (pH 4.7); LcDHFR-TS: 0.2 lM, DHF
5.0 lM, NADPH 5.0 lM in 50 mM sodium phosphate buffer
(pH 7.0).
Comparison of the enzymatic activities in the absence and
presence of the compounds (50 lM), which were dissolved in
DMSO (5% v/v final concentration), was employed to calculate the
percent inhibition (Equation (1))
Kinetic studies with LcDHFR-TS and LcPTR1
% Inhibition ¼ 100 ꢃ ðVi=VcÞ ꢂ 100
where Vi is the initial velocity in the presence of the inhibitor and
(1)
LcDHFR-TS apparent Km determination
Kinetic measurements were carried out using a standard assay31,
at pH 7.0 (50 mM sodium phosphate). Briefly, NADPH consumption
Vc is the reaction rate in the presence of DMSO (5% (v/v)).
R
was monitored spectrophotometrically (340 nM) (ShimadzuV UV-
1800, Columbia MD, USA) at constant temperature (25 ꢁC) and the
absorbance variation within the first 30 s was employed to calcu-
late the initial rate of reaction, by linear regression of the
raw data.
Determination of IC50 values
The compounds that inhibit either LcPTR1 or LcDHFR activity
ꢄ30%, in the single concentration assays, had their IC50 values
determined by making rate measurements for at least five inhibi-
tor concentrations.
The appropriate concentration of enzyme to be employed in
the kinetic assays was evaluated by observing the reaction time
required for substrate depletion under enzyme concentrations of
1.0 and 2.0 lM in the presence of 50 lM of DHF and NADPH.
The effect of the pH (5.0–8.0) over enzymatic activity was
investigated with 0.2 lM of LcDHFR-TS and saturating NADPH and
DHF concentrations (100 lM) (50 mM citrate buffer was used for
pH 5.0 activity assay. 50 mM sodium phosphate buffer was used
for pH range 6.0 to 8.0 activity assay). After the optimal pH was
identified the apparent Km of the cofactor was determined by fol-
Initial velocity measurements (Vi) were employed to calculate
inhibition percentages, whose plot versus log of inhibitor concen-
tration afforded the IC50 values, by nonlinear regression (assuming
Hill constant ¼ 1.0) as available in the GraphPad program Prism
R
version 5.0 (GraphPadV Software, San Diego, CA, USA, www.graph-
pad.com).
Determination of the inhibition mechanism
lowing a decrease in absorbance (340 nm) at growing up the con- The type of inhibition was determined under the same reaction
centration of NADPH (0.39 to 50 lM) at saturating concentration conditions described above for different inhibitor concentrations
of DHF (100 lM). Likewise, substrate apparent Km was determined (0.0–20.0 lM) at five varying substrate concentrations (biopterin:
at saturating NADPH concentration (50 lM) and varying DHF con- 6.25, 12.5, 25, 50 and 100 lM; DHF 0.78, 1.56, 3.12, 6.25, 12.5 lM),
centrations (0.20–12 lM). All the measurements were carried out whereas the cofactor concentration was at saturating conditions
in triplicate and the apparent Km values for the cofactor and (LcPTR-NADPH ¼ 100 lM; LcDHFR-TS-NADPH ¼ 50 lM).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1443
Next, the kinetic measurements were carried out in saturating the inhibition assay and Km is the apparent Michaelis constant for
substrate concentrations (biopterin: 100 lM – DHF: 12.5 lM) and the substrate in the absence of the inhibitor, at saturating NADPH
varying the concentration of NADPH (6.25, 12.5, 25, 50 and concentration.
100 lM). Apparent Km values, calculated by non-linear regression,
were compared by T-test, available in GraphPad Prism version 5.0,
and considered significantly different for p<.05. The results of this
analysis were plotted in double-reciprocal graphs for visual ana-
lysis purposes only. The dissociation constant (Ki) of the active
Results and discussion
Cloning, expression, and purification of LcPTR1
derivatives against both enzymes was obtained through The complete coding sequence for LcPTR1 from L. chagasi plus
Cheng–Prussov equation for competitive inhibitors33, as follows:
eight nucleotides flaking the gene was employed for primer
design, which began with the identification of restriction enzymes
that would not cleave the LcPTR1 gene.
ꢀ
ꢁ
½ ꢅ
S
Km
IC50 ¼ Ki 1 þ
(2)
The amplification reaction afforded a single product of 867 bp
where IC50 is the inhibitor concentration required to reduce (Figure 2(A)), which, following purification, was cloned into the
enzyme activity by 50%, [S] is the substrate concentration used in pET-M11 vector using the LIC system.
Figure 2. Cloning, expression, purification, and kinetic characterization results for LcPTR1. (A) Cloning steps to insert LcPTR1 gene into pETM11 expression vector moni-
tored by 1% agarose gel. A1: (1) Marker 1 kb DNA Ladder, A1: (2) PCR amplification product with base pairs (bp) equivalent to LcPTR1 gene; A2: (1) Tridye 100pb lad-
der (NEB); A2: (2) Product of PCR reaction employing E. coli CFU resulting from the transformation protocol. (B) Expression and purification of LcPTR1. SDS/page 12%.
(B1) Protein expression profile of LcPTR1 in E. coli BL21 (DE3). After expression induction at 25ꢁC (18h), in the presence of different IPTG concentrations (0.05, 0.5, and
1.0mM). NI: the absence of IPTG; P: pellet: S: supernatant; MM: LMW-SDS GE molecular weight standard (kDa). B2: (1) molecular weight standard (kDa); (2) purification
supernatant; (3) fraction 20 mM imidazole; (4–6) 50 mM imidazole; (7–9) 500mM imidazole. (10) LcPTR1 after cleavage. (C) Activity of LcPTR1 in different pH ranges.
Values represent median and interquartile range of % activity when compared to the highest median of activity obtained, at pH 4, 7 (N ¼ 3). (D) Apparent Km deter-
mination for recombinant LcPTR1.

1444
B. V. F. TEIXEIRA ET AL.
Figure 3. Cloning, expression, purification and kinetic characterization results for LcDHFR-TS. (A) Cloning steps of the LcDHFR-TS gene in the pET28a vector, monitored
by 1% agarose gel. A1, A2: (1) Marker 1 kb DNA Ladder; A1: (2) Product of the amplification reaction of the LcDHFR-TS gene from the genomic DNA; A2: (2) Product
of PCR reaction with primer T7 employing CFU from the transformation protocol. (B) Expression and purification of LcDHFR-TS. Gel SDS-page 12%. (1) Molecular weight
standard; (2–3) insoluble and soluble fraction of the lysate; (4–5) contaminants eluted in low imidazole concentration; (6–7) Wash with buffer containing 500 mM imid-
azole; (8–10) Column eluate with imidazole-free buffer after application of the dialyzed material and incubated with thrombin. (C) Activity of LcDHFR-TS in different pH
ranges. Values represent median and interquartile range of % activity when compared to the highest median of activity obtained, at pH 7.0 (N ¼ 3). (D) Apparent Km
determination for LcDHFR-TS.
This strategy allows the expression LcPTR1 fused to a N-ter- protocol affords 5-fold more protein (15 mg.Lꢃ1 of bacterial cul-
ture) than the one described for LdPTR134 and a similar yield as
minal His-tag. A similar approach was employed to obtain the
PTR1 from Leishmania donovani in the soluble form34. Following
the transformation of competent E. coli BL21 (DE3) cells and
the one described for LmPTR136.
“colony PCR” screening,
a positive and sequence verified
Cloning, expression and purification of LcDHFR-TS
(Figure S3) colony was selected for protein expression studies.
After expression of LcPTR1 was carried out at 25 ꢁC, 18 h,
0.5 mM IPTG, a single-step purification, using affinity chromatog-
raphy, that affords heterologous protein with high purity
(Figure 2(B)), once this tag can influence protein stability and/or
catalytic properties35.
The migration profile of the PCR product shows a band that is
consistent with the expected amplicon containing the LcDHFR-TS
gene (1563 bp), plus the extension-primers (23 bp) (Figure 3(A)).
After purification of the PCR product, the amplified insert (1.2 lg
(46 ng/ll)) was cloned into the pET28a expression vector using
the restriction site for the NdeI enzyme positioned in the 5’ region
TEV protease was employed to remove it and allow LcPTR1 to
be obtained, after another round of affinity chromatography. This of the LcDHFR-TS gene.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1445
This strategy allows the expression of the recombinant protein increase Vmax49, this is not observed for DHFR-TS cases men-
fused to His-tag in the N-terminal domain to facilitate its purifica- tioned above, probably due to structural factors.
Once reaction conditions were standardized, we proceeded to
tion through nickel resin affinity chromatography. The same N-ter-
minal His-tag fusion strategy was successfully employed in the LcDHFR-TS kinetic characterization. Determination of kinetic
cloning, expression, and purification of Leishmania (Vianna) brazil- parameters is crucial to the standardization of assay conditions for
iensis37, and T. cruzi DHFR-TS38.
inhibitors screening50 since cofactor and substrate concentrations
must be close to the Km apparent in the assay to allow identifica-
tion of different inhibitors modalities: competitive, noncompeti-
tive, and uncompetitive43. As an ordered bi-random mechanism is
described to the formation of DHFR ꢆ NADPH ꢆ DHF complex51,
the simplifications described by Michaelian kinetics can be applied
for the LcDHFR-TS by maintaining saturating conditions of one
substrate to allow kinetic characterization of the other33.
Finally, heat shock transformation protocol allowed to isolate
sequence verified (Figure S4) colony-forming units containing the
gene that codes for LcDHFR-TS (Figure 3(A)). Initial LcDHFR-TS
expression tests using E. coli BL21 (DE3) showed that the recom-
binant protein was observed predominantly in the insoluble frac-
tion of the cell lysate (Figure S5). Low expression profile was
reported for L. major DHFR-TS when BL21 strain was employed
39
.
The values obtained for Kmapp and Vmaxapp for DHF were
1.0 0.2 lM and 2.0 0.11 nmol.minꢃ1.mLꢃ1, respectively, whereas
for NADPH, Km and Vmax values were 2.3 0.5 lM and
1.6 0.1 nmol.minꢃ1.mLꢃ1, respectively (Figure 3(D)). The Kmapp
values of LcDHFR-TS are close to the described for DHFR-TS of
other species of Leishmania genus such as L. tropica, with 1.5 lM
and 2.7 lM39, and L. major, 1.6 lM and 0.45 lM31 for DHF and
NADPH, respectively, what are already expected since the differen-
ces in its catalytic regions of the sequences are punctual. Both
data obtained here for L. chagasi and those reported for L. tropica
DHFR-TS shows lower affinity values of cofactor relative to the
substrate for the enzyme, such a feature can be exploited by
designing inhibitors directed to bind in the cofactor site, compet-
ing with NADPH, since its lower affinity would imply in lower con-
centration of the inhibitor needed to displace it from the
enzyme site.
In order to increase the yield of a soluble and correctly folded
recombinant protein that might be obtained, the Arctic Express E.
coli (DE3) strain was employed40. This strategy proved successful
and allowed us to obtain soluble LcDHFR-TS when the cells were
grown at 10 ꢁC, for 48 h, in the presence of IPTG 0.5 mM
(Figure 3(B), lanes 2 and 3). Several DHFT-TS are susceptible to
protease action (T. brucei DHFR-TS41,42, L. tropica DHFR-TS31, and
L. major DHFR-TS39).
Likewise, proteolytic cleavage of LcDHFR-TS was also observed
in the present work (Figure 3(B), lane 7), even upon addition of
protease inhibitors cocktail (SigmaFAST) to the cell lysis buffer.
Nevertheless, chromatographic purification by affinity made it pos-
sible to obtain the partially purified protein (Figure 3(B), lanes
6–7), which has its His-tag cleaved with thrombin. Non-cleaved
LcDHFR-TS was removed by a second elution on nickel sepharose
resin (Figure 3(B), lanes 8–10), as described for LcPTR1. Hence, the
ꢃ1
overall yield of LcDHFR-TS is 10.0 mg.L
of bacterial culture.
When we consider the number of catalytic turnover events
(kcat)52, LcDHFR-TS presents a kcat of 0.033 sꢃ1. Among DHFR-TS
from other organisms, L. major31 and T. brucei42 kcat constants,
27 sꢃ1 and 26 sꢃ1, respectively, are higher than that of the L. cha-
gasi enzyme (0.033 sꢃ1).
Kinetic characterization of LcDHFR-TS and LcPTR1
Catalytic activity of LcDHFR-TS
Absorbance decrease at 340 nm over time (350 s) after the start of
the reaction showed a linear profile up to 300 s when used 1.0 lM
of the enzyme, indicating the steady-state condition is reached in
the assays for at least the initial 30 s of the assay.
In addition, in order to determine optimum assays conditions
for LcDHFR-TS kinetic characterization and inhibition, it was neces-
sary to evaluate the effect of pH on the catalytic activity, since
medium pH affects protonation states of amino acids related to
catalysis and/or intermolecular interactions43.
The results obtained showed that catalytic activity of the DHFR
domain of LcDHFR-TS is influenced by pH in the range of 5–8.
Among the pH ranges evaluated, the maximum catalytic activity
was observed at pH 7.0 (Figure 3(C)), that explains why the next
steps were carried out on this pH.
Intrategumental pH studies of L. donovani observed that the
parasite maintains neutral intracellular pH44, even under acidity or
basicity medium conditions, thus demonstrating that the assay
has biological significance as it approaches the enzyme natural
habitat. Studies with DHFR-TS from different organisms agree with
this data when describing the optimum pH at 7.0 for L. major45
and Plasmodium vivax46 enzymes. Other studies indicate that the
optimum pH for the DHFR-TS of Trypanosoma brucei41 and
Plasmodium berghei47 is in the pH range of 6.5–8.0. On the other
hand, some studies show that for vertebrates and certain bacteria,
catalytic activity is promoted at lower pH values48.
Catalytic activity of LcPTR1
The enzymatic reaction monitored through the decrease in
absorbance (340 nm) showed a linear profile for up to 600 s when
using 0.4 lM of LcPTR1 which indicates steady-state conditions in
the assays for at least 60 initial seconds of the assay.
Next, in order to determine optimum assays conditions for
LcPTR1 activity, its dependence on pH variation was examined.
Significant variation of LcPTR1 activity was observed in the pH
range between 2.7 and 5.7 (Figure 2(C)). As a marked peak of
activity was observed at pH 4.7, this was then considered as the
optimum pH for LcPTR1 catalytic activity. Similarly, the pH of 4.7
was determined as optimal for LmPTR122 and for LdPTR1, the opti-
mum pH is 4.853.
In addition to the reaction medium pH, the substrate and
cofactor concentrations are critical for the establishment of bal-
anced assay conditions in kinetic assays employed for potency
and mechanism of inhibition determinations. Such conditions are
achieved when the kinetic reaction medium presents substrate
and cofactor concentrations close to the Km, since these concen-
trations can influence the identification of competitive, non-com-
petitive, or uncompetitive inhibitors52.
Accordingly, as PTR1 presents an ordered mechanism of
PTR1ꢆNADPHꢆBPT complex formation23, in which the substrate
(BPT) only binds the enzyme after formation of PTR1ꢆNADPH, it is
Although the decrease in the pH of the medium tends to pro-
mote catalytic activity by facilitating the transfer of the hydride to possible for the use of the Michaelian simplifications for the kin-
the nitrogen 5 of the DHF pteridine ring and consequently, etic characterization of LcPTR1 by maintaining saturating

1446
B. V. F. TEIXEIRA ET AL.
conditions for BPT to enable the determination of cofactor Km, this case, IC50 values are suitable only to compare the compoundsꢀ
and vice versa33.
potency for one specific target, either LcPTR1 or LcDHFR-TS. For
instance, compounds 1, 2, 3 show a discrete variation in IC50 val-
ues against LcPTR1 (Figure 4(B)).
The values obtained from Kmapp and Vmaxapp for BPT were
25 1.2 lM and 13.0 0.4 nmol.min-1.mL-1, respectively, whereas
for NADPH, the values of Km and Vmax were 37.48 2.1 lM and
15.4 0.6 nmol.min-1.mL-1 (Figure 2(D)), respectively. The following
values were reported for LdPTR1: BPT Kmapp¼5.8 lM; NADPH
Kmapp¼18.5 lM54. It is worth mentioning that these values were
obtained through the double-reciprocal plot, which according to
Copeland33 can infer errors of up to 20%, in addition, the protein
used to determine the kinetic constants were fused with His-tag.
The kinetic parameters found for LcPTR1 substrate and cofactor
are not similar to the values reported for other species of
Leishmania: for L. tropica PTR155, was found for BPT Km ¼ 3.5 mM
and for NADPH Km ¼ 19.0 mM, while L. major PTR122 were found
Km of 4.6 and 6.7 lM for BPT and NADPH, respectively.
This result suggests that structural variations at position 6 of
the 2,4-diaminopyrimidine nucleus (Figure 4(B)) appear not to
influence the potency of these compounds against this target. On
the other hand, the activity profile of compounds 1–3 against
LcDHFR-TS (Figure 5(B)) suggests that the presence of an aromatic
ring at least two carbons away from the 2,4 diaminopyrimidine
nucleus is crucial for LcDHFR-TS inhibition (see IC50 of compounds
1 vs. 2).
Although the IC50 values of compound 1 are lower for LcDHFR-
TS than LcPTR1, one cannot say it is more potent against the first,
since substrate concentration, reaction pH, temperature and Km
of each enzyme can influence the value of IC5057,58. In order to
overcome this limitation, Ki values calculated with the Cheng-
Prussov equations might be employed, as long as the compoundsꢀ
mode of inhibition was known. Consequently, we undertake a
careful investigation of compounds inhibition mode. Once com-
pounds 1–4 possess high structural similarity, it is reasonable to
assume that they share the same mechanism of inhibition. Thus,
just results for compound 1 and 5 are discussed next.
However, the values found for LcPTR1 agree with the crystallo-
graphic data of LdPTR1, since a disordered active site must have
lower affinity for its ligands26. Such PTR1 kinetic data show a
lower affinity for the cofactor relative to the substrate, this pattern
is also observed for L. tarantoleae PTR1 (BPT/NADPH Kmapp¼3.5/
56
19)55 and L. major (BPT/NADPH Kmapp¼4.6/6.7)
.
Comparing LcPTR1 and LcDHFR kinetic characterization data,
both enzymes show lower affinity for the cofactor relative to the
substrate, which is evidenced by higher Kmapp values for NADPH
(LcDHFR: DHF/NADPH Kmapp¼1.0/2.3; for LcPTR1: BPT/NADPH Mechanism of inhibition assays
Kmapp¼25.0/37.5). When we consider the number of catalytic turn-
Kinetic parameters like Kmapp and Vmaxapp under different inhibi-
over events per time (kcat)50, the LcPTR1 presents a kcat of 0.54 s-1.
Among PTR1 from other organisms, L. major PTR156 kcat is similar
to the L. chagasi enzyme (0.44 versus 0.54 sꢃ1, respectively),
whereas the for T. brucei PTR132 kcat is higher than that of L. cha-
gasi (4.3 versus 0.54 sꢃ1, respectively).
tor concentrations were analyzed in order to determine the mech-
anism of action of compound 1 over LcPTR1. Results obtained
with saturating concentration of NADPH (Figure 4(C)), show a set
of lines that intercept the Y-axis at the same point (1/Vmaxapp
and cross the X-axis (-1/Kmaxapp) at different points.
)
Although the kcat values are higher for LcPTR1 with respect to
LcDHFR-TS (0.54 versus 0.033 sꢃ1, respectively), when the catalytic
efficiency is considered (kcat/km ratio), the LcDHFR-TS presents
higher constant value (3.3 ꢂ 104 versus 2.1 ꢂ 104Mꢃ1sꢃ1, respect-
ively). This pattern is also observed for L. major (PTR1 5.8ꢂ vs
DHFR-TS 21 ꢂ 106Mꢃ1sꢃ1, respectively)31,56 and T. brucei (PTR1
4.3 ꢂ 105 vs. DHFR-TS 6.8 ꢂ 106, respectively)32,42. Hence, our data
suggest that LcDHFR-TS recognizes its substrate more efficiently
than LcPTR1.
This behavior suggests a competitive mechanism of inhibition,
but the fact that PTR has a bi-bi ordered catalytic mechanism23
requires that compound 1 also shows an uncompetitive mechan-
ism of inhibition to the cofactor.
In order to confirm this hypothesis, enzymatic inhibition experi-
ments were carried out at a saturating concentration of biopterin,
while the NADPH concentration was varied (Figure 4(C)). As
expected, the values of Kmapp and Vmaxapp decrease proportion-
ally, as the concentration of compound 1 increases. Thus, the
graph of the reciprocal double shows parallel lines, typical of an
inhibitor uncompetitive concerning the cofactor.
LcPTR1 and LcDHFR inhibition assays
As the structure of compound 5 differs substantially from the
other compounds, we decided to perform experiments to deter-
mine the mechanism of inhibition to this compound as described
above.
Although PTR1 and DHFR are not related at the primary sequence
level, both enzymes are inhibited by compounds containing the
2,4-diaminopyrimidines skeleton24 which can be explained by the
chemical similarity between the natural substrate of PTR1 and
DHFR-TS and the 2,4-diaminopyrimidine nucleus.
Once the inhibition of both enzymes may have a synergistic
detrimental effect over the parasite survival17, and prevent the
selection of resistant strains, we decided to assay diaminopyrimi-
dine derivatives, which have already been described as S. mansoni
DHFR-TS inhibitors27, against LcPTR1 and LcDHFR-TS. Single-dose
assays (Figure 4(A) and 5(A)) suggest that compounds 1 and 3
Thus, when the assays were performed with saturating concen-
tration of NADPH and different concentrations of biopterin, it was
observed that the lines intersect the Y-axis at the same point,
while the intersection with the X-axis occurs at different points
(Figure 4 (D)). Therefore, compound 5 exhibits the same behavior
observed for compound 1 (competitive mechanism with bio-
pterin). In fact, when different concentrations of NADPH were
used, reduced values of Kmapp and Vmaxapp were observed
(Figure 4 (D)). These results confirm that compound 5 has an
affinity for the PTR1-NADPH complex (mechanism of inhibition
uncompetitive with the cofactor).
are active against both enzymes (e.g. compound
1
ꢂ
LcPTR1 ¼ 90 1% vs. LcDHFR-Ts ¼ 100 1.17%), whereas com-
pounds 2 and 4 are more active against LcPTR1 than LcDHFR
(LcPTR1 inhibition ¼ 40 1%, vs. LcDHFR inhibition ¼ 20 2.4%)
and compound 5 seems to inhibit LcPTR1 (98 0.8% inhibition at
50 mM) but not LcDHFR (20 2.4% inhibition at 50 mM). However,
When the assay was carried out with LcDHFR-TS, it was
observed that DHF Kmapp increases linearly as compound 1 con-
this preliminary analysis might be misleading once the catalytic centration (Compound 1¼0 mM – Km ¼ 1.0 0.2 mM; 1¼2 mM –
efficiency of LcPTR1 and LcDHFR-TS are significantly different. In Km ¼ 7.8 1.2 mM; 1¼5 mM – Km ¼ 15.0 4.8 mM), whereas its

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1447
Figure 4. Kinetic inhibition assays results for LcPTR1. (A) Single concentration (50lM) inhibition assay. (B) Dose-response curves for compounds 1–5 against LcPTR1.
R
IC50 values calculated by non-linear regression in GraphPad PrismV 5.0 software. (C) Effect of compound 1 over NADPH and BPT kinetic constants (Kmapp and
Vmaxapp). Statistical difference of results were considered when p < .05 (Kruskal–Wallis ANOVA). (D) Effect of compound 5 over NADPH and BPT kinetic constants
ꢀ
(Kmapp and Vmaxapp
ꢀ
) Statistical difference of results were considered when p < .05 (Kruskal–Wallis ANOVA).
Vmaxapp is not affected by increasing concentrations of com- 2 mM
–
Vmax
¼
1.0 0.1 nmol.minꢃ1.mLꢃ1
, 5 mM – Vmax
pound 1 (Vmax ¼ 1.9 0.1 nmol.minꢃ1.mLꢃ1).
¼ 0.5 0.05 nmol.minꢃ1.mLꢃ1) (Figure 5(C)).
On the other hand, NADPH Kmapp remains the same (Km
The competitive mechanism related to substrate observed for
¼ 2.1 0.3 mM) but its Vmaxapp decreases linearly when increasing compound 1 is consistent with the data described for 2,4-diami-
concentrations of compound
1
are added to the catalytic nopyrimidine inhibition of L. major enzyme59. The same pattern
reaction (Compound 1¼0 mM – Vmax ¼ 1.6 0.1 nmol.minꢃ1.mLꢃ1
;
was also observed for Bacillus anthracis60 and Schistosoma

1448
B. V. F. TEIXEIRA ET AL.
Figure 5. Kinetic inhibition assays results for LcDHFR-TS. (A) Inhibition profile against in 50 lM inhibitor single concentration assay. (B) Dose-response curves for com-
R
pounds 1–3 against LcDHFR-TS. IC50 values calculated by non-linear regression in GraphPad PrismV 5.0 software. (C) Effect of compound 1 over NADPH and DHF kinetic
ꢀ
constants (Kmapp and Vmaxapp). Statistical difference of results were considered when p < .05 (Kruskal–Wallis ANOVA).
Table 1. Selectivity ratio for the 2,4-diaminopyrimidine derivates active against LcDHFR-TS and LcPTR1.
LcDHFR-TS
LcPTR1
Compounds
IC50 (mM)
Ki (mM)a
IC50 (mM)
Ki (mM)a
Selectivity KiLcPTR1/ KiLcDHFR-TS
1
2
3
5
1.65
18.00
4.20
0.28
3.00
3.00
4.10
4.60
0.94
1.50
2.05
2.30
0.47
5.45
0.68
3.29
0.70
>50b
>8.33b
<0.05
aObtained from Cheng-Prussov equation50
.
bData from single-concentration assay.
mansoni27 enzymes. On the other hand, although the noncompe- however one must bear in mind that these macromolecules have
Km values that differ by more than 15 fold (substrate 25ꢂ (LcPTR1
titive inhibition related to cofactor is also observed against S.
mansoni27 and Gallus gallus59 DHFR, uncompetitive interaction is
25 2.1 mM vs. LcDHFR-TS 1.0 0.2 mM); cofactor 16ꢂ (LcPTR1
observed for E. coli DHFR-TS59.
37.2 3.7 mM vs. LcDHFR-TS 2.3 0.5 mM)). In addition, IC50 values
change according to substrate or enzyme concentration. For
instance, when kinetic data is recorded at [S]¼Km, the IC50/Ki ratio
for noncompetitive inhibitors is equal to 1/2. If higher concentra-
Selective and dual inhibitors
tions of the substrate are employed, the ratio changes.
The design of dual-acting inhibitors depends on the identification
of compounds that have similar affinity for LcPTR1 and LcDHFR-TS, kinetic assays for LcDHFR-TS were recorded at S/KM ¼ 5.0 for the
All kinetic data for LcPTR1 was recorded at S/KM ffi 1.0, but the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1449
substrate (S/KM¼2.0 for the cofactor). Under these circumstances,
the direct comparison of IC50 values is misleading. In order to cir-
cumvent this dilemma, the affinity of the compounds to their tar-
gets was assessed by their dissociation constant (Ki), calculated with
the Cheng–Prussov equation for competitive inhibitors33 (Table 1).
This approach shows that compounds 1 and 3 have moderate
selectivity for LcDHFR-TS (KiLcPTR1/KiLcDHFR-TS ratio ¼ 5.4 and 3.3-
fold selectivity, respectively), whereas compound 2 has similar
affinity to both targets (KiLcPTR1/KiLcDHFR-TS ratio ¼ 0.68). Two out of
three 2,4-diaminopyrimidine derivatives described are active
against LmDHFR-TS and LmPTR1 have selectivity profile similar to
compounds 1 and 3: 6 to 13-fold selectivity for LmDHFR-TS over
LmPTR124. On the other hand, compound 5 is highly selective for
LcPTR1.
Despite the promising results presented so far, ADMET liabil-
ities, such as high toxicity, might pose a severe hurdle to their
advance as lead compounds. In order to evaluate this potential
limitation, their predictive cardiac toxicity (hERG receptor inhib-
ition model), mutagenicity (AMES test model) and carcinogenicity
(Three-class model) was evaluated through admetSAR server
(http://lmmd.ecust.edu.cn/admetsar1). These in silico models
underscore no red light alerts and suggests oral acute toxicity
doses higher than 500 mg/Kg for in vivo evaluation of all com-
pounds (Figure S6).
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Conclusions
~
Although DHFR-TS and PTR1 from L. major have long been explored 12. Martin-Montes A, Santivanez-Veliz M, Moreno-Viguri E, et al.
for drug development purposes, the same cannot be said about their
counterparts in L. chagasi. This work sheds some light on this subject
by reporting not only the optimal conditions to obtain LcDHFR-TS
and LcPTR1, but also the balanced conditions for in vitro screening.
This assay shows that 2,4 diaminopyrimidine derivatives, substituted
at position 6, are competitive inhibitors of both enzymes whereas
the 2,4-diaminoquinazoline derivative 5 is a selective inhibitor of
LcPTR1. This information shall guide the development of potent
inhibitors with dissimilar selectivity profiles.
In vitro antileishmanial activity and iron superoxide dismu-
tase inhibition of arylamine Mannich base derivatives.
Parasitology 2017;114:1783–90.
ꢀ
13. Martınez VC, Vera M. Therapeutic potential of pteridine deriva-
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Disclosure statement
No potential conflict of interest was reported by the authors.
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16. Romero AH, Rodrıguez N, Oviedo H. 2-Aryl-quinazolin-4 (3 H)
Funding
-ones as an inhibitor of leishmania folate pathway: in vitro
biological evaluation, mechanism studies and molecular
docking. Bioorganic Chemistry 2019;83:145–53.
This work was supported by Conselho Nacional de
Desenvolvimento Cientıfico e Tecnologico; Fundac¸ao de Amparo a
Pesquisa do Estado da Bahia.
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~
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ORCID
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Marcelo Santos Castilho
http://orcid.org/0000-0002-9563-5679
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