Repositioning Salirasib as a new antimalarial agent

Article abstract of DOI:10.1039/c9md00298g

Malaria is a serious tropical disease that kills thousands of people every year, mainly in Africa, due to Plasmodium falciparum infections. Salirasib is a promising cancer drug candidate that interferes with the post-translational modification of Ras. This S-farnesyl thiosalicylate inhibits isoprenylcysteine carboxyl methyltransferase (ICMT), a validated target for cancer drug development. There is a high homology between the human and the parasite enzyme isoforms, in addition to being a druggable target. Looking to repurpose its structure as an antimalarial drug, a collection of S-substituted derivatives of thiosalicylic acid were prepared by introducing 1,2,3-triazole as a diversity entry point or by direct alkylation of the thiol. We further investigated the in vitro toxicity of FTS analogues to Plasmodium falciparum in the asexual stages and in Vero cells. An antiplasmodial activity assay was performed using a simple, high-sensitivity methodology based on nanoluciferase (NLuc)-transfected P. falciparum parasites. The results showed that some of the analogs were active at low micromolar concentration, including Salirasib. The most potent member of the series has S-farnesyl and the 1,2,3-triazole moiety substituted with phytyl. However, the compound substituted with methyl-naphthyl shows promising physicochemical and activity values. The low cytotoxicity in eukaryotic cells of the most active analogs provided good therapeutic indices, being starting-point candidates for future antimalarial drug development.

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Repositioning Salirasib as new antimalarial agent
Exequiel O. J. Porta,a,§ Ignasi Bofill Verdaguer,b,§ Consuelo Perez,c Claudia Banchio,c Mauro Ferreira de
Azevedo,d Alejandro M. Katzin,b,* and Guillermo R. Labadie.a,e,*
Received (in XXX, XXX) XthXXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Malaria is a serious tropical disease that kills thousands of people every year, mainly in Africa, due to
Plasmodium falciparum infections. Salirasib is a promising cancer drug candidate that interferes with the
post-translational modification of Ras. This S-farnesyl thiosalycylate inhibits the isoprenyl carboxyl
methyltransferase (ICMT), a validated target for cancer drug development. There is a high homology
between the human and the parasite enzyme isoforms, in addition to being a druggable target. Looking to
repurpose its structure as antimalarial drug, a collection of S-substituted derivatives of thiosalicylic acid
were prepared, introducing a 1,2,3-triazole as diversity entry point or by direct alkylation of the thiol. We
further investigated the in vitro toxicity of FTS analogues to Plasmodium falciparum in the asexual stages
and in Vero cells. The antiplasmodial activity assay was performed using a simple, high-sensitivity
methodology based on nanoluciferase (NLuc)-transfected P. falciparum parasites. The results showed that
some of the analogs were active at low micromolar concentration, including Salirasib. The most potent
member of the series has S-farnesyl and the 1,2,3-triazole moiety substituted with phytyl. However, the
compound substituted with methyl-naphthyl show promising physicochemical and activity values. The low
cytotoxicity in eukaryotic cells of the most active analogs provided good therapeutic indexes, being a
starting-point candidate for future antimalarial drugs development.
Malaria is one of the most important tropical diseases, and the
World Health Organization describes malaria as one of the top ten
causes of death worldwide.1 In 2016, malaria infected 216 million
and killed more than 400 thousand people; most deaths occurred in
Africa and were due to Plasmodium falciparum infections.
Nowadays, there are no vaccines for malaria immunization.
Therefore, the disease prophylaxis, control and treatment are based
on the existing chemotherapy and vector control.
Currently the WHO suggests the use of combination therapies for
the treatment of malaria.2 Unfortunately, the expanding resistance
puts at risk the ongoing plans to control the disease.3 For these
reasons, new antimalarial compounds effective against
Plasmodium’s asexual stages in humans are required to treat
infected people and for use in collective eradication programs.1,4
To facilitate drug discovery, efforts are being made to develop
more sensitive, robust, easier, and less expensive methods for drug
screening.5,6 Two well-known research strategies for the discovery
of etiological treatments for parasitic protozoan diseases are drug
repositioning and improving drugs’ effectiveness through
structural modifications.7
Salirasib (farnesyl thiosalicylic acid, FTS) is a promising drug
candidate that was first reported more than 20 years ago.8,9 This
simple compound is a potent inhibitor of the isoprenylcysteine
carboxyl methyltransferase (ICMT) in cell-free systems.
Surprisingly, its activity did not correlate in intact cells, being a
poor methylation inhibitor. It has been shown that Salirasib acts on
the Ras signaling cascade, which is required for cell proliferation
and differentiation. This process starts with the removal of all Ras
isoforms from their membrane anchoring sites.10 Since its
discovery, different clinical trials have been conducted with
Salirasib for pancreatic, colon, lung and breast cancers.11
§ Both authors contributed equally to this work
aInstituto de Química Rosario, UNR, CONICET, Suipacha 531,
S2002LRK, Rosario, Argentina. Tel-Fax: +54-341-4370477
E-mail: labadie@iquir-conicet.gov.ar
bDepartamento de Parasitología, Instituto de Ciências
Biomédicas, Universidade de São Paulo, São Paulo, Brazil
E-mail: amkatzin@icb.usp.br
cInstituto de Biología Molecular y Celular (IBR-CONICET-UNR),
Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad
Nacional de Rosario. Suipacha 531, S2002LRK, Rosario,
Argentina.
dDepartamento de Biociências, Universidade Federal de São
Paulo, Santos, Brazil.
eDepartamento de Química Orgánica, Facultad de Ciencias
Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario,
Suipacha 531, S2002LRK, Rosario, Argentina.
One possible target for the development of potential antimalarial
drugs is the isoprenoid biosynthesis.12
† Electronic Supplementary Information (ESI) available: detailed
experimental procedures. Copies of 1H and13C NMR spectra of all
new products. See DOI: XX.XXXX/XXXXXXXX/
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Isoprenoid are synthesized in P. falciparum, plants plastids and
most bacteria (among other organisms) by the 2-C-methyl-D-
erythritol-4-phosphate (MEP) pathway.12 In contrast, most animal 60 a collection of thioethers and additionally, further diversity was
portion in our design and introduce modifications on the thiol. The
diversity was introduced by direct alkylation of the thiol, preparing
cells, several eubacteria, some archaea and fungi synthesize
isoprenoid precursors only by the mevalonate pathway. In 2004,
Goulart et al.13 showed that, for P. falciparum strain 3D7 cultured
in vitro, FTS possesses a dose-response growth inhibition effect
with a half maximum inhibitory concentration (IC50) of 14 μM
(confidence interval: 13.61-19.18 μM). The greatest lethal effects
added by 1,2,3-triazol formation by click chemistry (Figure 1).
5
10 were observed between the schizont stage and the formation of
rings.13 The same authors showed that FTS specifically decreased
radioactive uptake in immunoprecipitated Ras isoprenylated
proteins radiolabeled with [1-(n)-3H] farnesyl pyrophosphate
triammonium salt.13 This effect was only observed in the schizont
15 stage, for which the authors suggested a higher Ras protein
expression.13 It was also suggested that, as it happens in other cell
systems, Ras proteins could be involved in cell signaling or
proliferation in the parasite.14,15 Based on those precedents, we
envisioned FTS as a promising scaffold to develop new
20 antimalarial agents. To explore this hypothesis, a collection of
thiosalicylic acid derivatives were prepared and assayed against P.
falciparum.
Figure 1. Proposed Salirasib analogs
65
First, to prepare the thioether collection the selective thiol
alkylation was optimized. The propargyl bromide was selected as
alkylating agent for that purpose because it was the key
intermediate for the 1,2,3-triazole library preparation. (Scheme 1)
70 Starting from commercial thiosalicylic acid, different reactions
were assayed using different bases and solvents.
The attempts using potassium hydroxide and potassium carbonate
in water were unsuccessful (Table 1, entries 1-2).
Bioinformatics studies of the molecular target
25 In order to position the ICMT as a possible molecular target for
repositioning drugs, multiple sequence alignments and homology
modelling were refined (Figure S1 Supplementary Information).16
The primary human and infectious apicomplexans ICMT
sequences (in particular, Toxoplasma and Plasmodium genus)
30 were analyzed. All sequences correspond to functional proteins
belonging to the ICMT superfamily containing the conserved the
C-terminal substrate-binding site, (particularly, between amino
acids 160-279 in H. sapiens). There is also complete in the active
site amino acids conservation and those interacting with the S-
35 adenosyl methionine cofactor. Particularly, there is a 36% identity
and 54% similarity between Hs-ICMT and Pf-ICMT. These values
are considerably higher when the domains involved in the catalytic
site are analyzed. Expanding the sequence analysis towards
different species, allows us to conclude that the catalytic domain
40 of the enzyme is highly conserved throughout evolution (Figure
S2 Supplementary Information). In addition, the TDR Targets
database17 indicates that ICMT would be a druggable target and is
potentially essential in P. falciparum. Promisingly, TDR target
predicts that Salirasib would be a potential inhibitory drug for Pf-
45 ICMT. Based on these facts, ICMT is an interesting molecular
target and Salirasib an attractive starting-point for repositioning
drug toward P. falciparum and liable to consequent reengineering.
75
Scheme 1. Alkylation of thiosalicylic acid
When those bases were used, but the solvent was exchanged by
MeOH, KOH produced complete product decomposition and with
K2CO3 alkylated ester product was obtained (Table 1, entries 3-4).
Fortunately, when using guanidinium carbonate as base in acetone,
80 a complete selectivity was achieved obtaining the S-propagyl
thiosalicylic acid 1 as the only product (Table 1, entries 5).
Table 1. Optimization of thiosalicylic S-alkylation.
Entry
Reagentsa
Results
NR
1
2
3
4
KOH (1 eq), H2O
K2CO3 (1 eq) H2O
KOH (1 eq), MeOH
NR
Decomposition
S-propargyl propargyl
thiosalicylate
K2CO3 (1 eq), MeOH
5
S-propagyl thiosalicylic
acid
Guanidinium carbonate (1 eq.)
acetone, r.t.
81% yield
85
a) thiosalicylic acid (1 eq), propargyl bromide (1eq), reflux, 12 h.
With those conditions optimized, we moved to prepare a collection
of analogs. Alkylating agents were selected aiming to cover a wide
spectrum of steric and lipophilic demand. Most of the required
90 alkylating agents were commercial. For those not commercially
accessible, the bromides were easily prepared from the alcohol
precursor by substitution with phosphorous tribromide in Et2O.
The isoprenols (geraniol, farnesol, and phytol) were transformed
on the respective chloride through the Corey-Kim modified
95 conditions,19 using N-chlorosuccinimide, DMS in DCM.
Design and synthesis
50 Salirasib activity has been undoubtedly linked to the presence of
the thiosalicylic acid on the structure.9 The simplicity of its
structure only allowed minimal structural modifications to explore
a structure-activity relationship and improve the activity. It has
been observed that a free carboxyl group is absolutely required for
55 its activity. Introduction of halogenic substitution of the benzene
ring implies a decrease or a loss of activity on the target.18
Therefore, we decided to preserve the 2-mercaptobenzoic acid
The final collection of the alkylated thiosalicylic acid was prepared
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by S-alkylation (Table 1, entry 5), using the optimized conditions
providing twelve analogs with an average yield of 79% after
purification by column chromatography (Scheme 2, Table 2,
entries 1 to 11).
were cultured with FTS or its analogues at 200 μM concentration.
Growth was quantified daily by NLuc assay and compared to
parasites cultured with the solvent control ethanol. Because most
compounds are soluble in ethanol, 200 μM is approximately the
50 highest assayable drug concentration that does not produce
significant effects due to ethanol toxicity (0.5-1% at maximum).
The inhibition percentage caused by FTS and its analogues at the
200 μM concentration (as compared to an untreated control) was
calculated three times. We also performed quality controls with
55 antimalarial drugs, including 200 μM of clindamycin (which is a
delayed-effect antimalarial compound), 200 nM of both artesunate
and chloroquine (as supralethal controls) and 3.125 nM of
artesunate (as a sublethal control). Compounds 2k and 2m showed
haemolysis at 200 μM but not at 100 μM; for this reason, the
60 inhibition percentage study was performed at 100 μM. The results
showed that clindamycin, as well as FTS and most of its
derivatives, had higher inhibitory effect at 72 h than after one or
two days (Figure S3 Supplementary Information). On the other
hand, inhibition by either sub- or supralethal artesunate
65 concentrations was similar from 1-3 days. Considering the assays
initiated with ring stage parasites, the compounds likely act on late
stage parasites as previously reported for FTS.
The IC50 was determined for FTS analogues which inhibited
parasites growth ˃90% after 72 h, an effect like the observed
70 inhibition of antimalarial control drugs at supralethal
concentrations (Tables 2, Figure S4 Supplementary Information).
The IC50s of chloroquine and artesunate were determined as
positive and quality controls. The IC50 was calculated at 72 h
because produced the best antimalarial effect and to be comparable
75 with previous FTS. We used an initial lethal maximum
concentration of 200 μM for FTS (100 μM for compounds 2k and
2m); and its analogues, or 200 nM for chloroquine and artesunate,
in addition to several smaller concentrations prepared by serial
dilutions.
5
Scheme 2. Alkylated thiosalisylic acid library prepared.
Once the S-alkylated thiosalisylic acid collection was finished, we
10 moved to synthesize the 1,2,3-triazole collection. A pool of azides
were selected, following the same chemotype used for the
alkylated products, and applied to prepare the new derivatives.
Azides were mostly prepared by direct substitution of the bromide
with sodium azide in DMF. For the isoprenyl azides, the isoprenol
15 was transformed in one-pot reaction to the azides, whose
regioisomers are in equilibrium.20 The triazoles were synthesized
by reaction of S-propargyl thiosalicylate 1 with the azide under
conventional conditions using copper sulphate, sodium ascorbate
in tBuOH:H2O (1:1).21 The collection of sixteen new products was
20 obtained with an average yield of 81% after purification. (Scheme
3, Table 2 entries 12 to 28)
Biological activity
In vitro activity against P. falciparum
The NanoLuc Luciferase (NLuc) method for drug screening was
25 applied and validated with results previously obtained6 by the
Malaria SYBR Green I Fluorescence assay (MSF). While the
fluorescence-based method relied on quantifying fluorescence on
parasite cultures previously incubated with the DNA intercalating
dye, the most sensitive and affordable NLuc method involves
30 quantifying bioluminescence in NLuc expressing transgenic
parasites.22 In order to confirm that IC50s determined by both
methods are similar and that the transgenic NLuc expressing
parasites are not more resistant to known antimalarials, wild type
3D7 strain and parasites transfected with the pEF-NLuc expressing
35 vector22 were cultured in serially diluted concentrations of
artesunate for 48h and growth was quantified by MSF and/or NLuc
assay. Non-linear regression of growth values generated by both
methods produced similar IC50 values suggesting results are
comparable (Table S1 Supplementary Information)
80 We also checked all the compounds for which the calculated IC50
values indicated that they may have been directly interfering with
nLuc luminescence emission (Figure S3 Supplementary
Information). For this purpose, we added a 200 μM concentration
of each prepared analog to 100 μL of parasite lysate prepared as
85 previously described from unsynchronized cultures (parasitaemia
1%, haematocrit 2%); we then counted the emitted luminescence
with respect to a control without added experimental compounds.
The results showed few luminescence modifications which is
comparable to those results obtained by other authors’ homologous
90 assays for antimalarial drug screenings.4,5 Again, in this assay all
samples had Z ˃ 0.5.
40
In vitro antimalarial activity of the collection of S-alkylated
thiosalicylic acid (Compounds 1a-k) is shown on the Table 2
(entries 1-11). Most of the derivatives of this collection were
95 inactive at the maximum concentration tested of 200 M, including
the key intermediate S-propargyl thiosalicylic acid 1.
Scheme 3. 1,2,3-triazolyl thiosalicylate derivatives.
One NLuc assay was validated an initial screening was performed
45 to select the most effective compounds. For this purpose, parasites
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Table 2. In vitro antimalarial activity and yield of alkylated thiosalicylic acid derivatives and of 1,2,3-triazolyl thiosalicylic
acid derivatives.
P. falciparum *
Yield
(%)
Entry Cmpd
R
Z value
R2
IC50 M
1
2
1
Propargyl
Cyclohexyl
Benzyl
81
87
73
75
68
83
81
78
72
88
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
1a
1b
1c
1d
1e
1f
3
4
3-phenyl-propyl
Cinnamyl
Octyl
5
6
7
Decyl
8
1g
1h
1i
CH2CH2CO2CH3CH3
Prenyl
9
10
Geranyl
11
12
1j
Farnesyl (Salirasib)
Phytyl
79
85
20.34±5.52
13.40±1.53
0.93±0.06 0.95±0.02
0.93±0.06 0.98±0.02
1k
13
14
15
2a
2b
2c
Cyclohexyl
Benzyl
84
80
88
> 200
> 200
> 200
3-phenyl-propyl
16
2d
Cinnamyl
86
65.33±10.71
0.89±0.09 0.87±0.01
17
18
19
20
21
2e
2f
Octyl
Decyl
78
88
91
89
84
> 200
> 200
> 200
> 200
> 200
2g
2h
2i
CH2CH2CO2CH2CH3
Prenyl
Geranyl
22
2j
Farnesyl
90
25.97±1.32
0.94±0.06 0.96±0.02
0.92±0.06 0.98±0.01
23
24
25
2k
2l
Phytyl
CH2CO2CH2CH3
Cetyl
83
92
81
9.75±1.97
> 200
2m
14.44±5.43
0.93±0.06 0.97±0.01
0.88±0.08 0.97±0.02
26
27
28
2n
2o
2p
Tridecanyl
2-phenyl-ethyl
Naphthyl
55
94
33
19.83±5.14
> 200
20.54±5.45
0.92±0.06 0.96±0.04
0.75±0.18 0.96±0.02
0.86±0.11 0.96±0.03
CQ
8.15±1.83 nM
1.64±0.08 nM
ART
CQ=Chloroquine, ART=Artesunate, *evaluated by NanoLuc Luciferase method
5
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Interestingly the only active compounds of the series were the
Table 3. Cytotoxicity of selected analogs
prenylated analogs 1j and 1k. Compound 1j (R=E,E-farnesyl,
Salirasib) and 1k (R=phytyl) has IC50 of 20.34 and 13.40 M,
respectively. The derivatives with smaller prenyl chains (1h
R=prenyl and 1i R=geranyl) were inactive at 200 M linking the
activity with the number of isoprene units. That is not surprising
having in mind that early studies of thiosalicylic as prenylated
protein methyl transferase inhibitors shown to be very sensitive to
nature of the prenyl chain, being inactive for geranylated
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Vero cells
SI
Entry
Cmpd
Family / R
IC50 µM
5
1
2
3
4
5
6
7
8
1j
1k
2d
2j
S-Alk/farnesyl
S-Alk/phytyl
123T/cinnamyl
123T/farnesyl
123T/phytyl
210 ± 5
52 ± 4
> 200
10.3
3.87
> 3.06
> 7.70
3.67
10 analogs.18,23
> 200
On the other hand, it is interesting to note that only the most
voluminous molecules of this family that contain fatty acid chain
(1j and 1k) are active in P. falciparum. Members with shorter
chains are inactive (1e and 1f). Finally, the remaining inactive
15 analogs that has aromatic, carbocyclic or alkyl esters put some
limits to possible stereo-electronic variations on the structure.
In vitro antimalarial activity of the collection of the 1,2,3-triazolyl
thiosalicylic acid derivatives (Compounds 2a-p) are presented on
the Table 2 (entries 13-28). The behavior of this collection is
20 similar to the S-alkylated analogs with long fatty substituents
providing better activity. A detailed look on the activities shown
that compounds 2a, 2b, 2c, 2e, 2f, 2g, 2h, 2i, 2l and 2o did not
display activity on P. falciparum at the maximum concentration
tested (200 M).
25 The remaining six analogs displayed IC50s in the range of 65.33
M, for the cinnamyl derivative 2d, to 9.75 M for the phytyl
analog 2k, the most active compounds of both collections (twice
as active as Salirasib). A detailed look on the nature of the
substituent that provide better activities revealed again that
30 farnesyl (2j), phytyl (2k), and long alkyl linear chains (2m=cetyl
and 2n=tridecanyl) are between the most active. But in this case,
the aromatic substituted derivatives 2d (cinnamyl) and 2p (methyl
naphthyl) also appeared between the most active, while
compounds 2b (benzyl), 2c (3-phenyl propyl) and 2o (2-phenyl
35 ethyl) are not active at concentrations below at 200 M.
2k
2m
2n
2p
34 ± 1
27 ± 4
59 ± 4
143 ± 6
123T/cetyl
1.86
123T/tridecanyl
123T/naphthyl
2.99
6.95
65 SI= Selectivity index. Calculated as IC50Vero cells / IC50P. falciparum
ADME-Tox calculations
An oral drug requires good bioavailability than can be achieved by
balancing their partitioning and solubility properties. Those
70 properties are difficult to achieve on any drug development
pipeline. Indeed, one third of new chemical entities have poor
pharmacokinetics properties and have not been able to reach the
clinical trial phase. To avoid these problems an early estimation of
ADME-Tox properties seems mandatory in any drug discovery
75 program. The in silico ADME-Tox gave substantial information
for the feasible and pharmacotherapeutic use of the chemical
library.
To rationalize the profile of our analogs computational studies of
all the synthesized compounds were performed to predict their
80 absorption, distribution, metabolism and excretion (ADME)
properties, Lipinski's rule of five, toxicity liabilities and drug
likeness. The calculation was performed using the web-based
software Molinspiration,25 Osiris,26 ChemAxon,27 and
SwissADME.28 The Molinspiration platform uses a sum of
85 fragment-based contributions and correction factors being able to
calculate parameters of most of the organic and even
organometallic molecules. The analysis of the collection revealed
that only two compounds have a MW > 500. Almost half of those
(13 analogs) violated the rule with logP > 5. However, our library
90 contains ionizable groups that are charged at physiological pH,
which make logD a better descriptor of the lipophilicity of these
molecules. The calculated logD at physiological pH (7.4) of the
collection shows that 93% of the library being ≤ 5 (-1.62 to 6.31).
Finally, all compounds fulfill the restrictions on the number of
95 hydrogen donors and acceptors. The physicochemical profile and
distribution of the prepared compounds based on molecular
weight, polar surface area, logD and octanol/water partition
coefficient are displayed on Figure 2.
In vitro cytotoxicity assay in Vero cells.
The collection prepared was assayed against Vero cells (ATCC
CCL-81, already available in our laboratory) to determine the
40 selectivity against the P. falciparum. The initial screening showed
that all the compounds were non-cytotoxic at the maximum
concentration tested of 4.75 g/mL. Then, a selection of the most
active analogs was submitted to a full analysis to determine the
IC50.
45 Compounds 1j, 1k, 2d, 2j, 2k, 2m, 2n and 2p were assayed
showing different degree of cytotoxicity as was expected because
they have an anticancer origin (Table 3). The most toxic
compound, analog 2l, has an IC50 of 34 M where the less toxic
analogs, compounds 1j, 2d and 2j, were inactive at the maximum
50 concentration tested of 200 M. A detailed analysis on the
activities shows that compounds with long aliphatic or prenyl
chains are the most cytotoxic derivatives. That is the case for
analogs 1k, 2k, 2m, 2n and 2p that has IC50s below 60 M, but in
no case the selectivtivy index is lower than 1.8. Taking into
55 consideration those values, the selected hits of the series were
compounds 1j, 2j and 2p that have therapeutic indexes of 10.3,
>7.70 and 6.95, respectively. According with those values, the
analogues can be considered as promising starting-point
candidates24 to move to further in vivo studies.
The OSIRIS Property Explorer platform was used to perform a
100 toxicology analysis also providing the drug likeness and a drug
score. The results revealed that none of the products prepared are
potentially mutagenic, irritant, teratogenic or toxic for sexual
reproduction. The solubility is a critical property which aids in the
circulation of a drug after the administration and into the
105 bloodstream. The calculated drug likeness shows a variable profile
with thirteen derivatives with a poor profile (<-3), but the
remaining derivatives fall between -3 and 0. The combination of
solubility, logP, MW and toxicity risk are used to calculate the drug
score, that were from moderate to good compared with a standard
110 drug (0.13-0.61).
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35
Figure 3. Top: Correlation between the in vitro antimalarial
activity and the logD. Bottom: Expansion of “high activity” area
and tendency.
Figure 2. Distribution of physicochemical parameters of the
chemical library.
Conclusions
5
Even though there are diverse routes of drug administration, oral
dosing is highly preferred, especially for tropical diseases. Early
Drug reposition has emerged as a valuable strategy for the
estimation of oral bioavailability (for example, the fraction of the 40 development of new treatment against tropical diseases including
dose that reaches the bloodstream after oral administration) is very
useful in drug development pipeline. Bioavailability is highly
10 multifactorial but is primarily driven by gastrointestinal
absorption. SwissADME use the BOILED-Egg method to predict
malaria. Drug repurpose and reengineering have been shown as
efficient strategies to reduce the cost and time of drug
development. Almost 40% of drugs registered by the US FDA
found new uses in the treatment of several human conditions.29
gastrointestinal absorption and brain penetration of small 45 Based on that premise, we selected Salirasib as a starting point to
molecules. In this point, among all the active compounds, the one
that stands out the most is compound 2p.
15 A good proportion of the analogs did not comply the logP
requirement. However, the analogs contain ionizable groups, so
reengineer the anticancer drug as a new antimalarial agent,
preparing a library of new 28 thiosalycilic acid derivatives. The
whole collection was tested against P. falciparum, the etiological
agent of malaria. The selectivity towards P. falciparum in
the logD calculated at physiological pH are more adequate. We 50 comparison with Vero cells could point Salirasib as an example of
wanted to find a correlation between the compound activity and its
partition coefficient. A chart correlating the P. falciparum IC50 vs.
20 logD presented on Figure 3 (on top) showed a clear pattern. An
interesting outcome of this chart is the location of the most active
drug repositioning. Moreover, an island of high activity was found
for derivatives substituted with long alkyl chains or the farnesyl
terpene derivative. Compound 2k is the most active structure
against P. falciparum (IC50 9.75 M, SI 3.67). Some compounds
compounds. On the one hand, a region of “high activity” can be 55 have high logP values breaking Lipinsky´s rules, but logD
defined being all the compounds with logD > 3. On the other hand,
the remaining analogs were inactive except for the methyl-
25 naphthyl derivative 2p. The tendency shows that the highest
activity for the library is concentrated for the analogs with logD
calculated at physiological pH seems to be a better descriptor,
based ionizable groups on the prepared analogs. The naphthyl
derivative 2p has an antimalarial activity profile similar to
Salirasib, good ADME-T parameters and excellent drug score
between 4.7 to 6.5, with a maximum value in 6 (Figure 3, bottom). 60 being the best candidate as antiplasmodial agent. The lack of
The analog 2p seems to conjugate good activity, excellent
ADMETox values and proper physicochemical parameters.
30 Likewise, the difference in the physicochemical properties with
respect to the other active molecules of the family suggest that the
compound 2p would exert a different mechanism of action.
toxicity of 2p (predicted in silico and validated on Vero cells)
together with its structural simplicity, prompted us to point out this
new analog as a starting-point candidate for future antimalarial
drug development. The most active members of the collection have
65 activity values are comparable to other examples of drug
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Chen, G.J. Crowther, M.A. Doyle, C. Hertz-Fowler, A.L.
Hopkins, G. McAllister, S. Nwaka, J.P. Overington, A. Pain, G.V.
Paolini, U. Pieper, S.A. Ralph, A. Riechers, D.S. Roos, A. Sali, D.
Shanmugam, T. Suzuki, W.C. Van Voorhis, C.L. Verlinde, Nat
Rev Drug Discov. 2008, 7 900–907.
repositioning towards malaria, such as verteporfin, amlodipine
besylate, triamterene, rocuronium bromide, or apoptozole.30,31
Same conclusion can be reached if examples of repositioning
towards other parasitic diseases are analyzed.32,33 However, in
some cases the selectivity index is low, or they break more than
one of the Lipinsky’s rules. Finally, further optimization would be
required for series to become a serious contender for future drug
development.
65
70
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Conflicts of interest
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10 There are no conflicts to declare.
Acknowledgements
This work was supported in part by grants from National Research
Council of Argentina, CONICET (PIP 2009-11/0796 and 2012-
14/0448 awarded to G.R.L.); Agencia Nacional de Promoción
15 Científica y Tecnológica, ANPCyT- Argentina (PICT 2011/0589
awarded to G.R.L.), Universidad Nacional de Rosario and
Fundación Josefina Prats; Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP 2017/22452-1 awarded to A.M.K.
and 2015/19316-3 awarded to M.F.A.) Brasil; Conselho
20 Desenvolvimento Científico e Tecnológico (CNPq) Brasil. GRL
and CB are members of the scientific staff of CONICET-
Argentina. E.O.J.P. thanks CONICET for the award of a
Fellowship. I.B.V. is a fellow from the FAPESP.
80
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