Design, synthesis, and evaluation of substrate – analogue inhibitors of Trypanosoma cruzi ribose 5-phosphate isomerase type B

Article abstract of DOI:10.1016/j.bmcl.2020.127723

Ribose 5-phosphate isomerase type B (RPI-B) is a key enzyme of the pentose phosphate pathway that catalyzes the isomerization of ribose 5-phosphate (R5P) and ribulose 5-phosphate (Ru5P). Trypanosoma cruzi RPI-B (TcRPI-B) appears to be a suitable drug-target mainly due to: (i) its essentiality (as previously shown in other trypanosomatids), (ii) it does not present a homologue in mammalian genomes sequenced thus far, and (iii) it participates in the production of NADPH and nucleotide/nucleic acid synthesis that are critical for parasite cell survival. In this survey, we report on the competitive inhibition of TcRPI-B by a substrate – analogue inhibitor, Compound B (K_i = 5.5 ± 0.1 μM), by the Dixon method. This compound has an iodoacetamide moiety that is susceptible to nucleophilic attack, particularly by the cysteine thiol group. Compound B was conceived to specifically target Cys-69, an important active site residue. By incubating TcRPI-B with Compound B, a trypsin digestion LC-MS/MS analysis revealed the identification of Compound B covalently bound to Cys-69. This inhibitor also exhibited notable in vitro trypanocidal activity against T. cruzi infective life-stages co-cultured in NIH-3T3 murine host cells (IC₅₀ = 17.40 ± 1.055 μM). The study of Compound B served as a proof-of-concept so that next generation inhibitors can potentially be developed with a focus on using a prodrug group in replacement of the iodoacetamide moiety, thus representing an attractive starting point for the future treatment of Chagas’ disease.

Full text of DOI:10.1016/j.bmcl.2020.127723

Bioorg. Med. Chem. Lett. 32 (2021) 127723
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and evaluation of substrate – analogue inhibitors of
Trypanosoma cruzi ribose 5-phosphate isomerase type B
,
f
Soledad Natalia Gonzaleza , Jonathan J. Millsb,f, Dante Maugeria, Christopher Olayab,
Breana L. Laguerac, Jeffrey R. Endersd, Julian Shermane, Ana Rodrigueze, Joshua G. Pierceb,
a
c,*
´
Juan Jose Cazzulo , Edward L. D’Antonio
a
´
Instituto de Investigaciones Biotecnologicas, Universidad Nacional de General San Martín – CONICET (IIBio-UNSAM), Avenida 25 de Mayo y Francia CP (1650), San
Martín (Buenos Aires), Argentina
b Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Box 8204, Raleigh, NC 27695, USA
c Department of Natural Sciences, University of South Carolina Beaufort, 1 University Boulevard, Bluffton, SC 29909, USA
d Molecular Education, Technology and Research Innovation Center (METRIC), North Carolina State University, Raleigh, NC 27695, USA
e Department of Microbiology, New York University School of Medicine, 430 East 29th Street, New York, NY 10016, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Ribose 5-phosphate isomerase type B (RPI-B) is a key enzyme of the pentose phosphate pathway that catalyzes
the isomerization of ribose 5-phosphate (R5P) and ribulose 5-phosphate (Ru5P). Trypanosoma cruzi RPI-B (TcRPI-
B) appears to be a suitable drug-target mainly due to: (i) its essentiality (as previously shown in other trypa-
nosomatids), (ii) it does not present a homologue in mammalian genomes sequenced thus far, and (iii) it par-
ticipates in the production of NADPH and nucleotide/nucleic acid synthesis that are critical for parasite cell
survival. In this survey, we report on the competitive inhibition of TcRPI-B by a substrate – analogue inhibitor,
Neglected tropical diseases
Chagas’ disease
Trypanosoma cruzi
Amastigote
Trypomastigote
Isomerase
Compound B (Ki = 5.5 ± 0.1 μM), by the Dixon method. This compound has an iodoacetamide moiety that is
Substrate-analogue inhibitors
Competitive inhibition
Trypsin digestion LC-MS/MS
susceptible to nucleophilic attack, particularly by the cysteine thiol group. Compound B was conceived to spe-
cifically target Cys-69, an important active site residue. By incubating TcRPI-B with Compound B, a trypsin
digestion LC-MS/MS analysis revealed the identification of Compound B covalently bound to Cys-69. This in-
hibitor also exhibited notable in vitro trypanocidal activity against T. cruzi infective life-stages co-cultured in NIH-
3T3 murine host cells (IC50 = 17.40 ± 1.055 μM). The study of Compound B served as a proof-of-concept so that
next generation inhibitors can potentially be developed with a focus on using a prodrug group in replacement of
the iodoacetamide moiety, thus representing an attractive starting point for the future treatment of Chagas’
disease.
Chagas’ disease is a vector borne disease of mammals caused by the
parasitic protozoan Trypanosoma cruzi and constitutes a major health
problem that is estimated to affect 6 to 7 million people worldwide.1
Despite the fact that Chagas’ disease was first described more than a
century ago, chemotherapy is presently based only on two drugs,
nifurtimox and benznidazole. These drugs are effective for acute phase
infections, congenital infections, reactivated infections, and early
chronic disease.2 However, their efficacy during the chronic phase in
adult patients is controversial.3 These treatments are also prolonged and
result in serious side effects that compromise the treatment continu-
ity,4,5 additionally, parasite resistance has been reported due to varia-
tions in the susceptibility of T. cruzi strains 6,7. Another anti-infective
that is effective at countering T. cruzi parasite growth is Amphotericin
B. This compound is mainly used as an anti-Leishmanial agent,1 but it
has had a restricted clinical use for Chagas’ disease. Therefore, the
development of new chemotherapeutic agents against Chagas’ disease
has become an urgent need.
The pentose phosphate pathway (PPP) is a metabolic route that starts
Abbreviations: G6P, glucose 6-phosphate; PPP, pentose phosphate pathway; R5P, ribose 5-phosphate; RPI, ribose 5-phosphate isomerase; Ru5P, ribulose 5-
phosphate; TcRPI-B, Trypanosoma cruzi, ribose 5-phosphate isomerase type B.
* Corresponding author.
E-mail address: edantonio@uscb.edu (E.L. D’Antonio).
f
Authors contributed equally to this work.
https://doi.org/10.1016/j.bmcl.2020.127723
Received 22 September 2020; Received in revised form 17 November 2020; Accepted 23 November 2020
Available online 26 November 2020
0960-894X/© 2020 Elsevier Ltd. All rights reserved.

S.N. Gonzalez et al.
Bioorganic & Medicinal Chemistry Letters 32 (2021) 127723
with glucose 6-phosphate (G6P), the first glycolytic intermediate, and is
classically divided into two branches: an oxidative branch from G6P to
ribulose 5-phosphate (Ru5P), with the reduction of two molecules of
NADP+; and a non-oxidative branch, which ultimately, when func-
tioning as a cycle, leads back to glycolytic intermediates. The PPP plays
two major roles in T. cruzi metabolism: (a) it provides NADPH, a coen-
zyme necessary for biosynthetic reactions and a key molecule for
parasite protection against oxidative stress8 and (b) it provides ribose 5-
phosphate (R5P), which is used as a precursor for nucleotide biosyn-
thesis.9 Ribose 5-phosphate isomerase (RPI, EC 5.3.1.6) is an enzyme of
the non-oxidative branch of the PPP and it catalyzes the reversible
aldose – ketose isomerization between R5P and Ru5P (Fig. 1). At the
present time, there are two isoenzymes capable of catalyzing this reac-
tion and they are known as RPI type A (RPI-A) and RPI type B (RPI-B).
These enzymes do not share a common ancestor and they exhibit dif-
ferences amongst their 1◦ – 3◦ protein structures.10 The RPI-As are the
most widely distributed in the three kingdoms of life, including most
eukaryotic organisms, fungi, and some bacteria. Most RPI-Bs are found
in prokaryotic organisms with a few exceptions in lower eukaryotes,
such as the trypanosomatids, some fungi, and in the insect Anopheles
gambiae.11
studies, and from this, we reasoned that substrate – analogue inhibitors
could be used to target Cys-69 (Fig. 2b).17 Interestingly, Cys-69 is
positioned well into the active site cavity and since iodoacetamide
groups are well known to react with cysteine sulfhydryl groups from
peptide mapping experiments (Fig. 3b), we proposed that compounds
containing a halogen group, such as compounds B – E (Fig. 2b) would
irreversibly bind in the active site of TcRPI-B through the same reaction
by cysteine. A model for the inhibitor was constructed in reference to the
known crystal structure of the TcRPI-B – R5P complex using the program
WinCoot 0.8.9. Compounds B – E were predicted to act as potent in-
hibitors and Fig. 3c shows a predicted 3-dimensional model for any of
the “reacted” compounds B – E that would have lost their corresponding
halogen atoms.
In the present study, a series of R5P (open-chain form) substrate –
analogue derivatives termed Compounds A – E were conceived, and we
set out to determine if those compounds were competitive inhibitors of
TcRPI-B. We determined that Compound B was the most potent inhibitor
yet tested against TcRPI-B (Ki = 5.5 ± 0.1
μ
M), and it exhibited a notable
in vitro trypanocidal activity (IC50 of 17.40 ± 1.055
μ
M). Thus, Com-
pound B might represent an attractive starting point for the develop-
ment of novel drugs for future treatment options of Chagas’ disease,
either alone or in combination with other inhibitors.
The essentiality of the RPIs was already shown for some organisms
like Escherichia coli, Saccharomyces cerevisiae, and Leishmania
infantum.12–15 Moreover, although the essentiality was not completely
proven in Trypanosoma brucei, silencing of the RPI-B gene led to the
reduction of parasite growth and lower parasitemia of infected mice.16
T. cruzi only expresses an RPI type B (TcRPI-B), that was cloned,
expressed, and characterized showing that residue Cys-69 was essential
for the isomerization, and that His-102 was required for the opening of
the furanose ring of R5P.17 TcRPI-B performs a vital role in all phases of
the T. cruzi life cycle8 and is absent from all mammalian genomes
sequenced thus far. Interestingly, mammalian organisms have a struc-
turally unrelated RPI-A. Since TcRPI-B and Homo sapiens RPI-A (HsRPI-
A) are considered analogous enzymes and their active sites are
completely different,10 the design of highly selective inhibitors should
be possible. Such inhibitors would serve the long-term goal in drug
discovery by reducing adverse side effects in humans and by having a
reference point to design newer inhibitors with enhanced anti-T. cruzi
effectiveness.18,19 Taken together, the crucial role of TcRPI-B for the
T. cruzi cell cycle, its essentiality (previously shown in other trypano-
somatids), and the fact that it is non-homologous in comparison to other
host mammalian RPIs, makes TcRPI-B appear to be a suitable drug-
target for the development of new chemotherapeutic agents against
the parasite. This is particularly important since there have only been a
limited number of inhibitors for RPIs that typically achieved inhibition
constants (Ki) observed in the low millimolar range.20,21
Compounds A – E (Fig. 2b) were tested for the possible inhibition of
TcRPI-B. Although Compound A was not part of the haloacetamide se-
ries, we included it as a derivative to explore if it could possibly inhibit
the enzyme as a simple test. Compounds B – E are indeed hal-
oacetamides that differ from each other just by having a different
halogen X group. As a first step at inhibition testing, it was necessary to
check if the compounds inhibited the enzymes used in the coupled assay
employed to determine RPI activity. To this end, the activities of both
TcRPI-B and the first coupled enzyme, T. cruzi ribulose 5-phosphate
epimerase (TcRPE-1), were assayed as described (see Supplementary
Information). Testing the TcRPE-1 activity was enough, since an inhib-
itory effect on any of the other coupled enzymes would also be detected.
The inhibition attained with four concentrations of the compounds (0.2,
0.5, 1.0, and 2.0 mM) on the activities of TcRPI-B and TcRPE-1 are
shown in Tables 1 and 2, respectively. Compounds A and D had a similar
low inhibitory effect on both enzymes at all of the concentrations
assayed, indicating that TcRPI-B was not inhibited. Compound C
inhibited both TcRPI-B (82%) and TcRPE-1 (35%) at 2.0 mM. Since the
inhibition of TcRPI-B was not high, considering the inhibitor concen-
trations tested, and the inhibition of TcRPE-1 (or any other of the
coupled enzymes) was certainly not negligible, it would be very difficult
to perform a kinetic study of this effect. Surprisingly, Compound E
showed an unexpected behavior, since the activity of TcRPI-B, but not
that of TcRPE-1, was very much increased (32-fold at 1.0 mM).
The substrate of TcRPI-B is R5P (Fig. 1) and previous designs of
TcRPI-B competitive inhibitors have been identified, such as D-allose 6-
phosphate22 and 4-phospho-D-erythronohydroxamic acid17 (Fig. 2a).
Moreover, a high-resolution X-ray crystal structure of the TcRPI-B – R5P
complex was previously solved and R5P is shown bound in the TcRPI-B
active site (PDB entry 3K7S; Fig. 3a).22 Stern and colleagues determined
that Cys-69 was essential for catalysis by site-directed mutagenic
Compound B caused complete inhibition of TcRPI-B (98.8%) at 1.0
mM, and at this concentration TcRPE-1 was not inhibited. Therefore,
Compound B was chosen for further kinetic studies. We determined the
value of Ki for this compound, by performing a Dixon plot of 1/V as a
function of [Compound B] (Fig. 4a) at three concentrations of R5P (0.4,
0.8, and 2 mM). The value calculated from the extrapolation of the
intersection of the three lines with the X-axis was 5.5 ± 0.1 μM (mean of
four independent experiments). The plot of [R5P]/V vs. [Compound B],
at the same substrate concentrations assayed for the Ki determination is
shown in Fig. 4b; since the three lines were parallel, the inhibition was
determined to be competitive, as shown by the Cornish-Bowden
method.23
Fig. 3 shows the substrate or inhibitor interactions in the active site
of TcRPI-B. To test the possibility of a covalent bond being formed be-
tween the inhibitor and Cys-69, TcRPI-B was incubated with Compound
B in the absence of substrate, and was subjected to trypsin digestion
afterwards. The resultant enzymatic digestion was analyzed by LC-MS/
MS. Protein segment [V66-R86] (Fig. 5) showed an MS13+ mass spec-
trum, which included the inhibitor, Compound B, covalently bound to
Cys-69 (color-coded red). The portion of the inhibitor that linked onto
Fig. 1. Isomerization reaction between R5P and Ru5P as catalyzed by TcRPI-B.
2

S.N. Gonzalez et al.
Bioorganic & Medicinal Chemistry Letters 32 (2021) 127723
Fig. 2. TcRPI-B inhibitors. (a) Open-chain forms of inhibitors D-allose 6-phosphate (All6P, Ki = 15 mM) and 4-phospho-D-erythronohydroxamic acid (4PEH, Ki = 1.2
mM). (b) Compounds A – E as substrate – analogue inhibitors conceived by using an inhibitor model closely related to R5P/Ru5P.
Cys-69 had a molecular mass of 178.9984 g/mol; additionally, that
portion of the inhibitor lacked the iodine atom. The MS13+ peak
appeared in the mass spectrum at 727.0285, which included residue
segment V66-R86 and the bound inhibitor portion. Other protein seg-
ments appeared in the mass spectrum analysis that did not include the
bound inhibitor, such as the y4+ [protein segment: P83-R86], y+5 [protein
segment: V82-R86], y+9 [protein segment: A78-R86], and y1+1 [protein
segment: S76-R86]. The following masses were detected: 428.2621 for
competing with its natural substrate R5P with a Ki of 5.5 ± 0.1 μM
(Fig. 4a). To the best of our knowledge, Compound B is the most potent
inhibitor tested against TcRPI-B. This finding is very interesting since
only a limited number of inhibitors of RPIs have been reported in the
literature.20,21 All of the reported inhibitors are analogues of the inter-
mediate cis-enediolate (5-phospho-D-ribonohydroxamic acid) of the
isomerization reaction, with Ki values of 1.2 mM or higher.25 Moreover,
for the majority of these compounds, only the IC50 values were deter-
mined.17 From the above mentioned series of inhibitors, the more
promising one, 4PEH, which achieved a Ki in the low millimolar range
(Ki of 1.2 mM), was also described to be a potent competitive inhibitor
(Ki of 0.029 ± 0.003 mM) of the spinach RPI-A21 enhancing the occur-
rence of an off-target effect over human RPI-A if it were employed as
part of a Chagas’ disease chemotherapy. Surprisingly, we observed that
TcRPI-B in the presence of Compound E revealed a substantial increase
in activity with substrate R5P (Table 1), functioning as an enzyme
activator instead of an inhibitor, despite its similarity with Compounds B
– D. This striking behavior resulted in a very interesting observation and
we will certainly go further into this matter in future surveys, since it
was not the purpose of the present work.
y+4 , 527.3288 for y+5 , 911.5163 for y9+, and 1111.6832 for y+11
.
In order to evaluate the trypanocidal activity of the compounds, a
trypomastigote mammalian host cell infection assay was carried out
using a transgenic T. cruzi strain expressing the reporter enzyme
β-galactosidase (see Supplementary Information). Through these assays,
β-galactosidase activity was considered proportional to T. cruzi amasti-
gote – trypomastigote viability. Results illustrated in Fig. 6a show that
Compound B was found to be selectively active against T. cruzi infective
life-stages co-cultured in NIH-3T3 murine host cells, displaying a mod-
erate micromolar inhibition of T. cruzi in vitro, with an IC50 of 17.40 ±
1.055
is due to not screening to higher concentrations of Compound B beyond
50 M. The other compounds, Compounds A and C – E, were ineffective
μM. The reason why Fig. 6a does not show a plateau in its IC50 plot
μ
To further study the enzyme – inhibitor interaction, we first
attempted to obtain the X-ray crystal structure of the TcRPI-B – Com-
pound B complex, that would have included a more thorough structural
understanding of this interaction. However, it was not possible at the
present time to obtain good X-ray diffraction quality protein crystals. In
the inhibitor soaking experiments of inhibitorless TcRPI-B crystals with
Compounds A – E dissolved in mother liquor, the method seemed to be
quite harsh to yield crystals having a suitable integrity for X-ray
diffraction. Even after several attempts of crystal soaking, or even co-
crystallization trials, inhibitorless TcRPI-B crystals would substantially
degrade or they simply would not afford crystal growth, respectively. In
order to study this interaction through another experimental method,
we incubated intact TcRPI-B with Compound B for 30 min followed by a
trypsin digestion. The sample was then tested by LC-MS/MS to see if the
sulfhydryl sulfur-atom of Cys-69 would make a covalent bond with a
carbon-atom of the iodoacetamide portion of the inhibitor. Fig. 5 dis-
plays a convincing MS13+ peak with the added molecular mass of
178.9984 added to protein segment VLACGSGIGMSIAANKVPGVR
(bolded Cys-69) totaling a mass of 727.0285 with the inhibitor bound
after covalent attachment. This result showcases that Compound B
reacted with TcRPI-B at site Cys-69 with the mass of the iodine atom not
being present, as expected. More specifically, a carbon from the iodoa-
cetamide moiety of the inhibitor underwent a nucleophilic attack from
the sulfhydryl group of Cys-69 at the enzyme substrate binding site. In
summary, we demonstrated a competitive inhibition mediated by
Compound B, in the presence of R5P, while in its absence, the results of
our study indicated a mechanism – based inactivation. The difference
observed in both approaches could be due to the protective effect of the
substrate on the active center.
in the dose range tested (up to 50 μM). None of the compounds tested
were toxic to 3T3 cells before significantly affecting the growth of par-
asites. Under these assay conditions, the IC50 of amphotericin B used as a
positive control was found to be 0.23 ± 0.02 μM (Fig. 6b), comparable
with the value of 0.125 µM reported by Horvath and Zierdt.24
Currently available drugs for Chagas’ disease treatment are effective
for acute phase infections, congenital infections, reactivated infections,
and the early chronic phase disease,2 but have a controversial efficacy
during the chronic phase in adult patients.3 The chronic phase is a
category where a majority of people are currently infected with T. cruzi.
Furthermore, these drugs also seem to be inadequate for their toxicity,
leading to discontinuation of treatment for about 30% of patients4,5 and
for the resistance developed by some strains of the parasite.6,7 As such,
there is a need for the development of new therapeutic agents against
Chagas’ disease capable of targeting all stages of the disease in a safe and
efficacious manner. For all the reasons stated in the Introduction, TcRPI-
B appears to be a suitable drug-target. We tested five potential TcRPI-B
substrate – analogue inhibitors that gave rise to a series of R5P (open
chain form) derivatives (Compounds A – E), most of them predicted to
react with the sulfhydryl group of a cysteine sidechain in the active site
of the enzyme. Although haloacetamides are quite reactive compounds
and are probably not the most practical types of inhibitors to be used as
drugs because of cross reactivity, our study investigated on a proof-of-
concept purpose to ascertain the anti-T. cruzi effect and the effective-
ness of these compounds as substrate – analogue inhibitors against
TcRPI-B. From all of the compounds tested, it was only Compound B that
caused complete inhibition of TcRPI-B (98.8%) at 1.0 mM concentration
(Table 1), at which TcRPE-1 was not inhibited (Table 2). Kinetic studies
showed that Compound B acted as a competitive inhibitor of TcRPI-B,
For the biological evaluation of all compounds, we tested for
3

S.N. Gonzalez et al.
Bioorganic & Medicinal Chemistry Letters 32 (2021) 127723
Fig. 3. Substrate or inhibitor interactions in the
active site of TcRPI-B. (a) X-ray crystal structure of the
TcRPI-B – R5P complex (PDB entry 3K7S) in the active
site. Carbon atoms color-coded blue and orange
represent different subunits. (b) Proposed reaction
mechanism for the sulfur – carbon bond formation
between the sulfhydryl group of TcRPI-B (Cys-69) and
an iodoacetamide moiety from Compound B. (c)
Proposed irreversible complex of Compound B bound
in the active site of TcRPI-B.
Table 1
Enzymatic inhibition of TcRPI-B by Compounds A – E.a
Inhibitor (mM)
% of TcRPI-B activity
A
B
C
D
E
0.2
0.5
1.0
2.0
98.1 ± 3.2
97.4 ± 0.5
95.3 ± 4.2
89.2 ± 3.5
25.2 ± 3.5
7.5 ± 3.9
1.2 ± 0.3
0.0 ± 0.0
78.5 ± 4.8
62.7 ± 5.9
41.3 ± 2.7
18.3 ± 2.5
107 ± 9
994 ± 107
2042 ± 686
3194 ± 921
2718 ± 269
105 ± 7
98.5 ± 2.1
83.7 ± 8.9
NOTE: a Effect of compounds over TcRPI-B activity. Inhibition attained with four concentrations (0.2, 0.5, 1.0, and 2.0 mM) of Compounds A – E on the activity of
TcRPI-B. The percentages of activity shown are the mean of three independent determinations ± SEM.
Table 2
Enzymatic inhibition of TcRPE-1 by Compounds A – E.a.
Inhibitor (mM)
% of TcRPE-1 activity
A
B
C
D
E
0.2
0.5
1.0
2.0
100 ± 5
89.5 ± 4
89.5 ± 4
103 ± 5
91.1 ± 5.1
85.8 ± 4.4
77.3 ± 7.5
65.1 ± 5.2
100 ± 6
111 ± 10
103 ± 7
91.9 ± 4.3
87.4 ± 3.1
85.9 ± 4.4
103 ± 5
95.5 ± 7.9
93.2 ± 11.1
57.4 ± 0.4
82.6 ± 12
83.5 ± 9.7
NOTE: aEffect of compounds over TcRPE-1 activity. Inhibition attained with four concentrations (0.2, 0.5, 1.0, and 2.0 mM) of Compounds A – E on the activity of
TcRPE-1. The percentages of activity shown are the mean of three independent determinations ± SEM.
trypanocidal activity against T. cruzi infecting mammalian cells. Since
T. cruzi trypomastigotes are incubated with the host cells at the same
time as the test compounds, the assay determines the activity of the
compounds against trypomastigote infection of host cells and/or
amastigote proliferation after host cell invasion. The use of the trypo-
mastigote form and the amastigote form is relevant for drug discovery
studies since they are the relevant parasite stages of mammalian host
infection.26,27 Additionally, the amastigote form is responsible for the
chronic phase of Chagas’ disease, where the available treatments are
only partially beneficial.28 Through this approach, we found that while
none of the compounds tested were toxic to 3T3 cells before significantly
affecting the growth of parasites, only Compound B was capable of
exerting a moderate trypanocidal activity in vitro (Fig. 6) with an anti-
T. cruzi effect, which was very close to that of benznidazole.29 However,
while the use of nitroimidazoles draws in concern over cytotoxicity,
mutagenicity, and genotoxicity apparently related to DNA damage by
the bioreduction products of the nitro group,30 a substrate – analogue
inhibitor such as Compound B might avoid these types of issues related
to adverse effects during treatment.
observe such close IC50 values between assays, which most likely cor-
relates to low off-target effects. However, we expect Compound B to
have a higher degree of cross-reactivity if introduced into in vivo assays.
Treatment of a parasitic disease has the challenge where a drug requires
good biological stability, in particular, being able to not react with
anything in the bloodstream of the mammalian host. As such, it would
be strategic to re-design Compound B into a prodrug where it would
have a masking group on the iodoacetamide moiety. Once the prodrug
makes entry into the parasite cell by passive diffusion and activates by
either enzymatic or chemical cleavage, Compound B could quickly react
with Cys-69 in the active site of TcRPI-B. Ruda and co-workers have
examined six prodrug designs that have mostly been successful in
T. brucei parasites where they created a masking group for the phosphate
component of their inhibitors of 6-phosphogluconate dehydroge-
nase.33,34 In creating prodrug designs for Compound B that in turn target
TcRPI-B, could, in the long-term lead to novel therapeutic agents that
may well be employed as an alternative treatment for the chronic phase
of Chagas’ disease either alone or in combination with other inhibitors.
Although this path is still very long, the identification of novel promising
lead compounds for drug development in Chagas’ disease is an impor-
tant first step towards this.
The IC50 observed for Compound B vs. T. cruzi infective life-stages
was 17.40 ± 1.055
μM and is moderately potent. In comparison of
this assay to the TcRPI-B enzyme inhibition assay, where Compound B
was determined to have a Ki of 5.5 ± 0.1 M (Fig. 4), the IC50 value is
The work described herein was centered on the design and synthesis
of substrate – analogue inhibitors of TcRPI-B, a promising drug-target in
the search for a new alternative to the current treatment of Chagas’
disease. The inhibitors developed (Compounds A – E) have hal-
oacetamide functional groups (principally) for the purpose of forming a
μ
predicted to be 11 μM (e.g. IC50 = 2Ki for the case of competitive inhi-
bition and when [S] = KM.31,32 Due to the high reactivity of the iodoa-
cetamide moiety with sulfhydryl functional groups, it was surprising to
4

S.N. Gonzalez et al.
Bioorganic & Medicinal Chemistry Letters 32 (2021) 127723
Fig. 4. Kinetics of inhibition: TcRPI-B and Compound B. (a) Dixon plots of 1/V as a function of [Compound B] to determine the value of Ki for Compound B. The
value calculated from the extrapolation of the intersection of the three lines with the X-axis was 5.5 ± 0.1 M (mean of four independent experiments). (b) Plot of
[R5P]/V vs. [Compound B], to determine the type of inhibition for Compound B. Since the three lines were observed to be parallel, the inhibition was concluded as
μ
competitive inhibition. The inhibitor concentrations assayed were 0, 10, 20, 40, 60, and 80 μM, at three concentrations of R5P (0.4, 0.8, and 2 mM). In panels (a) and
(b), one of four independent experiments are shown as an example. The experimental points shown are the mean of two determinations.
Fig. 5. The tandem mass spectrum of the peptide from TcRPI-B (residues V66-R86) that is labeled to Cys-69 by Compound B (left panel). The MS1-ion that is labeled
by the inhibitor lacking the iodine atom (+178.9984 Da) is colored-coded in red with mass shown (middle panel). The unlabeled y-ions are colored-coded in red and
masses are shown (right panel). All unlabeled y-ions have a + 1 charge and the MS1-ion has a + 3 charge.
Fig. 6. Dose-response curves expressed as the percentage of parasite growth inhibition as a function of inhibitor concentration of (a) the TcRPI-B competitive in-
hibitor, Compound B, activity and (b) amphotericin B activity on T. cruzi (Tulahuen strain) intracellular amastigote growth inhibition in NIH-3T3 fibroblasts. The
IC50 determinations were made through GraphPad Prism. All IC50 measurements were performed in triplicate.
covalent bond with an internal cysteine thiol group located in the active
site. The study followed by means of a biochemical and biological
evaluation that revealed Compound B to bind as a competitive inhibitor
carbon-atom of the iodoacetamide portion of Compound B less the
iodine atom. Furthermore, Compound B revealed substantial trypano-
cidal activity with an IC50 of 17.40 ± 1.055 μM. Although the com-
in the active site of TcRPI-B with a Ki of 5.5 ± 0.1
μ
M and that the
pounds were designed as a proof-of-concept, in which a substrate –
analogue inhibitor class could be demonstrated for TcRPI-B, the
sulfhydryl sulfur atom of Cys-69 forms a covalent attachment with a
5

S.N. Gonzalez et al.
Bioorganic & Medicinal Chemistry Letters 32 (2021) 127723
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altering the iodoacetamide moiety to include a temporary inert prodrug
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Declaration of Competing Interest
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15 Faria J, Loureiro I, Santarem N, et al. Disclosing the essentiality of ribose-5-
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
phosphate isomerase B in trypanosomatids. Sci Rep. 2016;6:1–16.
16 Loureiro I, Faria J, Clayton C, et al. Ribose 5-phosphate isomerase B knockdown
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Acknowledgments
The work was supported by an Advanced Support for Innovative
Research Excellence: Track I (ASPIRE-I) grant to E.L.D. and an under-
graduate research grant through the Magellan program to B.L.L. that
were provided by the University of South Carolina, Office of the Vice
President for Research. This work was additionally supported by the
National Agency for Promotion of Scientific and Technological Research
to J.J.C. from the Argentinian Ministry of Science and Technology
(ANPCyT, MinCyT), grant PICT 2013-0969. We thank Zachary J. Taylor
for technical support.
19 Cavasotto CN, Phatak SS. Homology modeling in drug discovery: current trends and
applications. Drug Discovery Today. 2009;14:676–683.
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.
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25 Sinatti, V. d. V. C., Baptista, L. P. R., Alves-Ferreira, M., Dardenne, L., Silva, J. H. M.,
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