Research Article For reprint orders, please contact [email protected]

Integration of methods in cheminformatics and biocalorimetry for the design of trypanosomatid enzyme inhibitors Background: The enzyme GAPDH, which acts in the glycolytic pathway, is seen as a potential target for pharmaceutical intervention of Chagas disease. Results: Herein, we report the discovery of new Trypanosoma cruzi GAPDH (TcGAPDH) inhibitors from target- and ligand-based virtual screening protocols using isothermal titration calorimetry (ITC) and molecular dynamics. Molecular dynamics simulations were used to gain insight on the binding poses of newly identified inhibitors acting at the TcGAPDH substrate (G3P) site. Conclusion: Nequimed125, the most potent inhibitor to act upon TcGAPDH so far, which sits on the G3P site without any contact with the co-factor (NAD+) site, underpins the result obtained by ITC that it is a G3P-competitive inhibitor. Molecular dynamics simulation provides biding poses of TcGAPDH inhibitors that correlate with mechanisms of inhibition observed by ITC. Overall, a new class of dihydroindole compounds that act upon TcGAPDH through a competitive mechanism of inhibition as proven by ITC measurements also kills T. cruzi. Chagas disease, also known as American tryp­ anosomiasis, along with its causative agent Trypanosoma (Schizotrypanum) cruzi, already existed in the Americas as an enzootic disease for millions of years before any human occu­ pancy. Chagas disease is named as a tribute to a Brazilian researcher, the sanitary physician Carlos Chagas, who described its entire etiol­ ogy in 1909. Despite efforts over more than a century to reduce the parasite transmission, the WHO estimates approximately 7–8 million individuals are infected in Latin America and 25 million individuals at risk of becoming ill. In 2010 alone [1], there were over 10,000 deaths from complications of this disease [101]. Chagas disease is endemic in Latin America, however, it is also found in Canada, the USA [2], Europe (mainly Spain and Portugal), Japan and Australia [3,4]. T. cruzi has a complex heteroxenous life cycle, encompassing one phase of intracellular multi­plication in the vertebrate host (mam­ mals, including humans) and an extracellular one in the insect vector (Triatominae). T. cruzi is described in three main stages of development, which are morphologically distinct: trypomasti­ gote (non-replicative infective form, eliminated by the invertebrate vector, also present in the blood of the host, i.e., human), amastigote and epimastigote (two replicative forms, found in the tissues of the host and the intestinal tract of the insect vector, respectively) [5].

The treatment of Chagas disease is still ineffective. There are two drugs currently in use: nifurtimox (Lampit®) and benznidazole (Rochagan®), which are effective for curing the acute phase of infection with up to 80% success, but with a sharp decline in effectiveness to only 20% in the chronic phase. Both drugs exhibit strong side effects such as anorexia, weight loss, psychological changes, excitability, muscle trem­ ors, drowsiness, hallucinations, nausea, vomit­ ing, abdominal pain, diarrhea and convulsions [6,7]. Nifurtimox is no longer used in Brazil, Argentina, Chile and Uruguay due to the low susceptibility of the drug to the parasite strains present in chagasic patients, and also because it is poorly tolerated among adults with chronic Chagas disease [8]. Chagas disease is considered a neglected disease according to the WHO. It is a tropical infection, which has no specific treat­ ment and is considered a disease of poverty. The search for safer and more effective drugs is, therefore, eagerly awaited. There are several ongoing studies to iden­ tify chemical substances aiming to eliminate T. cruzi. Compounds containing azoles rep­ resent the greatest breakthrough in antifungal therapy, among which imidazoles and triazoles are known inhibitors of the sterol biosynthe­ sis [9]. T. cruzi contains ergosterol and for this reason the antifungal agents have been tested against the parasite: miconazole and econazole showed high potency against parasite growth,

Igor M Prokopczyk1, Jean FR Ribeiro1, Geraldo R Sartori1, Renata Sesti-Costa 2 , João S Silva2 , Renato F Freitas1, Andrei Leitão1 & Carlos A Montanari*1

10.4155/FMC.13.185 © 2014 Future Science Ltd

Future Med. Chem. (2014) 6(1), 17–33

ISSN 1756-8919

Grupo de Química Medicinal do Instituto de Química de São Carlos da Universidade de São Paulo Av. Trabalhador Sancarlense, 400, 13566–590, São Carlos/SP, Brazil 2 Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, 14049–900, Ribeirão Preto/SP, Brazil Tel:. +55 16 3373 9986 Fax: +55 16 3373 9985 *Author for correspondence: E-mail: [email protected] 1

17

Research Article | Prokopczyk, Ribeiro, Sartori et al. Key Terms Mechanism of action:

Specific biochemical interaction of ligands to a molecular target (Trypanosoma cruzi GAPDH) that involves inhibition of the enzyme, thereby leading to the biological outcome (i.e., killing Trypanosoma cruzi in our case). Medicinal chemists in general seek for ligands that competitively inhibit target enzymes.

Target- and ligand-based virtual screening: Integrating computational techniques to navigate chemical space of small molecules that can bind to a protein receptor or enzyme. For ligands, common features are explored to help focusing their docking to targets.

Isothermal titration calorimetry: Biophysical

technique used to determine the thermodynamic and kinetic parameters of interactions between ligands and targets in solution.

Enthalpy of the reaction:

Enthalpy change for the reaction between the substrate and its target molecule used to measure the kinetic affinity of ligands binding to target enzyme.

with concomitant decrease in the amount of 5,7-diene sterol. Subsequent studies demon­ strated that ketoconazole and other potent antifungal azoles were also active in protect­ ing mice from lethal infection with T. cruzi, by inhibiting intracellular multiplication of the parasite and blocking sterol biosynthesis. Imidazoles, such as ketoconazole and itra­ conazole, are inhibitors of cytochrome P450dependent lanosterol 14a-demethylase, and inhibition of this enzyme results in the accu­ mulation of 14a-methylsterol and decreased availability of ergosterol. Although this enzyme is present in mammalian cells, it is less sensi­ tive to the action of these compounds as com­ pared with fungi and trypanosomatidae [10]. Compounds with this mechanism of action are the most advanced antichagasics in the drugdevelopment pipeline. The antifungal posacon­ azole is in Phase II clinical trials in Spain; and the compound E12–24 (Esai, Japan) has com­ pleted Phase II clinical trials, with Phase II clinical trials to be pursued in Bolivia with potential extension to Spain. The compound Tak-187 (Takeda, Japan) has been evaluated in Phase I clinical trials [11]. Another important antichagasic target is the glycolytic pathway. In this biochemical process, glucose is involved in a series of biochemical reactions to produce ATP in the parasite, includ­ ing one specific glucose transporter. Inhibition of the pathway could then block the main energy source of the parasite, leading to cell death. In this pathway, GAPDH stands out as one of the main targets, as it is a key enzyme in this process [12]. Humans also possess GAPDH that could be at least partially inhibited by non-selective compounds [13]. However, it is claimed that no toxic effect in human cells would be observed even when more than 95% of human GAPDH (hGAPDH) is inhibited [14]. To date, there are other reports using the T. cruzi GAPDH (TcGAPDH) G3P site as target for T. cruzi intervention [15–17], and also the NAD+ site [18,19]. This study aims to identify new TcGAPDH inhibitors by cheminformatics methods and calorimetric assays that could be selective toward the parasite in cell-based studies using T. cruzi trypomastigote infective form and mouse spleen. The role of cheminformatics in the identification & optimization of ligands The exploration of the chemical space to properly define the chemical–biological space is nowadays feasible by applying in silico database-mining

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Future Med. Chem. (2014) 6(1)

tools that are amenable for high-throughput virtual screening. This could be covered by two main approaches [20]. Ligand-based virtual screening uses the chemical structure of known bioactive molecules as reference to undergo the definition of the chemical space that is going to be dealt with. On the other hand, target-based virtual screening relies upon the elucidation of the macromolecular 3D structure. In this paper, virtual screening methods are going to be discussed for the identification of novel inhibitors for T. cruzi GAPDH. Besides the parasite, humans also possess GAPDH; hence, it is necessary to describe the selectivity of the new compounds toward the parasite. To accom­ plish that, target- and ligand-based virtual screening approaches were applied to increase the chemical diversity of inhibitors in an effort to characterize the chemical–biological space [21,22]. „„Glycolytic

pathway The intracellular amastigote form of T. cruzi is dependent on the glycolytic pathway to produce energy (ATP) to survive and GAPDH is one promising target of this biochemical cascade [23]. GAPDH catalyses the oxidative phosphorylation of glyceraldehyde 3-phosphate to 1,3-diphos­ phoglycerate using inorganic phosphate and the NAD+ coenzyme. The catalytic and coenzyme sites are at the vicinity of each other and could be targeted by many different chemical archetypes. The analysis of TcGAPDH (1K3T [24] and 1QXS [25]) and hGAPDH (1U8F) [26] x-ray crystallographic structures from the PDB pro­ vided many hints regarding the properties of the targets that would be useful for the selec­ tion of novel compounds. Comparison between 1QXS and 1U8F sequences using Align97 pro­ vided an identity of 50.3% and a similarity of 66.7%, and the Ca root-mean-square devia­ tion is 0.793 Å, according to UCSF Chimera (version 1.5; CA, USA) [27]. When amino acids up to 15 Å away from the ligand in 1QXS are examined, the identity and similarity increase to 68.2 and 80.8%, respectively. Despite the high similarity between hGAPDH and TcGAPDH, four amino acids could be hot spots of selectivity: the corresponding residues for Ser247, Ser224, Asp210 and Gly213 from the TcGAPDH are Ala232, Ala209, Leu195 and Asp198 at the hGAPDH (Figure 1). This demonstrates that the human enzyme is more lipophilic than the one from the parasite. In the TcGAPDH 1QXS structure, the ligand (S )-(3 -hydrox y-2-oxo- 4 -(phosphonoox y) future science group

Integration of methods in cheminformatics & biocalorimetry butyl)phosphonic acid interacts at many dif­ ferent sites with the hydrophilic active pocket of the enzyme, namely hydrogen bonding with Thr197, Thr199, Thr167, Ser247 and salt bridge with Arg249, depicted in Figure 2 . Isothermal titration calorimetry to measure kinetic parameters The isothermal titration calorimetry (ITC) is a technique used for the thermodynamic charact­ erization of interactions between biomolecules, which is important for understanding the pro­ cess of molecular recognition. A small molecule interacting with proteins is also of utmost inter­ est when enzyme kinetics and thermodynamic signatures are used in the drug-discovery pro­ cess. In a chemical reaction, heat exchange is proportional to the velocity and, therefore, it is possible, through the ITC, to determine the kinetics of reaction systems with enzymatic catalysis, since the amount of heat exchanged is detectable. Wiggers et al. [28] following on from Todd and Gomez [29], demonstrated that the amount of energy produced by the reaction is proportional to the heat released and, therefore, a Michaelis–Menten curve is obtained and the kinetic constants kcat and K M are readily deter­ mined by nonlinear regression of these values ​​in Equation 1 [30]. v=

| Research Article

Ser224 Asp210

Ser247

Gly213 Cys166

Figure 1. 3D alignment of 1QXS (cyan) and 1U8F (gray) structures pinpointing the residues that could lead to selective compounds. Illustration generated using UCSF Chimera. This figure can be viewed in full color at: www.future-science.com/doi/ full/10.4155/FMC.13.185

solutions of the compound in an aprotic solvent are prepared a priori. As the tolerance of enzymes towards the addition of co-solvents changes from one enzyme to another, the determination of the

Thr192

Thr226

Ser247

Thr167

Vmax [S] K M [S]

Ser165 Cys166 Equation 1

Furthermore, the inhibition constant (K i) could be obtained from such experiments. A control experiment was carried out and the Michaelis–Menten constants were determined, then the experiment in the presence of the inhibitor was carried out and the data were graphically adjusted to the velocity in an equa­ tion corresponding to the type of inhibition observed. Therefore, the K i and the mechanism of inhibition could be determined. determination of the apparent enthalpy The apparent enthalpy of the reaction for sub­ strate phosphorylation in TcGAPDH was deter­ mined by isothermal titration calorimetry and the value of molar DH APP was -5.47 ± 0.91. Poor solubility and poor permeability are a challenge when assaying compounds in buffered solutions. Therefore, for organic compounds that generally possess low solubility in water, stock

Hid194 Arg249

S70 804

Thr197 Thr199 NAD 862

„„Experimental

future science group

Figure 2. Main hydrophilic interactions between the ligand (S)-(3hydroxy-2-oxo-4-(phosphonooxy)butyl)phosphonic by hydrogen bonding with Thr197, Thr199, Thr167, Ser247, NAD + co-factor and salt bridge with Arg249.

www.future-science.com

19

Research Article | Prokopczyk, Ribeiro, Sartori et al. Key Term 20 18 16 14 12 10 8 6 4 2 0

20 Database (%)

Quantification of cooperative binding if a ligand is already bound to the target.

Database (%)

Hill coefficient:

10 5

0

100

200 300 MW (Da)

400

0

500

25

0

1

2 3 XLogP

4

5

40 Database (%)

Database (%)

15

20

30

15

20

10

10

5 0 0

1

2 3 4 5 6 7 8 Hydrogen bond acceptor

9

10

0

0

1

2

3 4 5 6 7 8 Hydrogen bond donor

9 10

Figure 3. Distribution of compounds within the database after the application of the filter. (A) MW, (B) calculated log P, (C) number of hydrogen bond acceptors and (D) number of hydrogen bond donors.

concentration of non-aqueous solvent (which does not detrimentally modify the activity of the enzyme) is a critical step when developing a protocol for testing enzyme inhibitors. One of

Figure 4. Assessment of docking poses. (A) Representative results of the docking of compounds (gray) in the presence of NAD + (orange). (B) Docking pose of Neq125, within the G3P site but not in contact with NAD +. This figure can be viewed in full color at: www.future-science.com/doi/ full/10.4155/FMC.13.185

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Future Med. Chem. (2014) 6(1)

the most used aprotic solvents is dimethylsulfox­ ide (DMSO). In our previous work, we assessed the effect of co-solvents on the enzymatic activ­ ity of GAPDH and it was established that addi­ tion of 5% DMSO had a beneficial effect on the activity of the enzyme [28]. The apparent reaction molar enthalpy for TcGAPDH was determined in the presence of 5% DMSO and the value of -4.60 ± 0.37 kcal/mol was obtained. Comparing this value with the one obtained without the use of DMSO, 1.16 kcal/mol was observed as the incremental enthalpy value due to the presence of 5% DMSO in the medium. Application of ITC & molecular dynamic simulations to discover new TcGAPDH inhibitors In our previous work, a possible region for selectivity between TcGAPDH and hGAPDH was identified [15]. We first built up the database from the ZINC module ‘drug-like in stock’. The initial database contained approximately 3 million structures that were filtered using the program FILTER (version 2.1.1, OpenEye future science group

Integration of methods in cheminformatics & biocalorimetry Scientific Software; NM, USA). The drug-like filter was used to remove from the collection of molecules those that did not fall in line with the molecular characteristics shown in Figure 3, as defined by Lipinski [31] and Veber [32]. Solubility is another critical factor, particularly in the ini­ tial tests and, therefore, such information was taken into account when filtering compounds. To this end, the following molecular param­ eters were used within the drug-like filter: MW = 200–500 Da (Figure 3A); XLogP = -1–5 (F igure  3B) ; hydrogen bond acceptor = 1–10 (F igure   3C) ; hydrogen bond donor = 1–5 (Figure  3D). After these steps, the number of compounds of the database was reduced to 100,000 molecules. Then, this database was docked in the G3P binding site and those mole­ cules having the best score, complementarity to the binding site, and interactions with the key amino acids were selected for a visual analysis, as exemplified in Figure 4. The following criteria, as exemplified for compounds Neq104, Neq111 and Neq125 (F igure   5) , were used for visual inspection: the compound Neq111 was allocated, with the methyl sulfonamide group, in a region with hydrophobic amino acids (Gly227, Ser165 and Pro136). The dihydro-pyrazole ring was at a distance of 3.72 Å from Cys166. The third portion of the compound, the nitro-benzene ring, showed interaction with the Thr199 and Arg249 and was close to the nicotinamide group of NAD +. The compound Neq104, unlike Neq111, did not explore the hydro­ phobic amino acids region, but explored the region of Asp210. The sulfone group has a distant 4.38 Å distance from Cys166 and the hydrophobic group, in this case with a toluenyl moiety, is oriented towards the nicotinamide group of NAD+. Neq125 occupied the outer­ most region of the site, positioning itself near Lys230 and making a hydrophobic interaction with the Pro126. The sulfonamide shows two interactions with the amino acids used as con­ straint for docking (Ser247 and Thr167) and the His194, which is an amino acid that takes part in catalysis. It kept a distance of 3.92 Å from Cys166 and showed no interactions or proximity with respect to NAD+. Based on this visual analysis, we selected 25 commercially available compounds to be assayed via ITC, with the structures shown in Figure 5. The chosen TcGAPDH structure 1QXS is co-crystallized with an analog of the prod­ uct 1,3-BPG (S70) [25], therefore we also future science group

| Research Article

considered the occupancy of the desired region at the active site by a ligand when the co-factor is bound to its respective site. The docking pro­ cedures were performed using the docking pro­ gram Glide (version 5.8, Schröedinger, LLC; NY, USA) employing our concepts of molec­ ular docking that were recently put forward within the guidelines published elsewhere by Sherman et al. [33]. Table 1 displays the scores, which were used along with other tools, such as poses in the G3P site (Figure 4) and molecular dynamics (MD) simulation (Figures 6 & 7), to select ligands by visual inspection. The aim of this work was to identify TcGAPDH inhibitors by ITC, using previ­ ously published procedures [15,28]. Out of 25 assayed compounds, five were active against TcGAPDH with concentration values lower than 300 µM and three of them (Neq104, Neq111 and Neq125) showed K iapp values below 100 µM (Table 2). Assays were carried out using the G3P site as the competitive one, while keep­ ing the NAD+ site fully occupied, as previously described [15]. For compounds with K iapp values less than 100 µM, the experiments were conducted at two concentrations of inhibitors (50 and 100 µM), and in the absence of inhibitor (0 µM). Tests with multiple concentrations allowed us to describe the mechanism of inhi­ bition for three compounds. The competitive inhibition mechanism is illustrated for Neq125 by the non-linear fit from the data obtained (F igure   8A) and the Lineweaver–Burk plot (Figure 8B). It is known that TcGAPDH is an enzyme with two important sites, G3P and the NAD+. This unique archetype is driving us to artifi­ cially force a pseudo first-order reaction, either when studying the interaction with the sub­ strate or co-factor. For instance, we used a high concentration of NAD+ to ensure that the site was always occupied. In this manner, forced experimental conditions led to the observa­ tion of Michaelian kinetics behavior towards TcGAPDH. Therefore, for such a study, it is necessary to determine the Hill coefficient (nH) and classify it as positive (n H > 1), negative (nH 500 >500

>14 >21

Trypanosoma cruzi Tulahuen LacZ strain. Our previous IC50 (µM) value for benznidazole acting against T. cruzi Tulahuen strain is 64.3 ± 12.3 [37].

† ‡

28

Future Med. Chem. (2014) 6(1)

future science group

Integration of methods in cheminformatics & biocalorimetry

2.5 2.0 1.5 1.0 0.5 2.5 2.0 1.5 1.0 0.5

125B A = 849.678

Neq125 + 100 mM potassium phosphate buffer + NADPH 125C

A = 821.728

Neq125 + 100 mM potassium phosphate buffer + microsomes 125D

1.5 Neq125 + 100 mM potassium 1.0 phosphate buffer + 0.5 microsomes + NADPH 0 1 2 3 4 5 6

A = 611.425

7

125A

Neq125 + 100 mM potassium phosphate buffer

40 30 20 10

125B

Neq125 + 100 mM potassium phosphate buffer + NADPH

50 40 30 20 10

125C

40 30 20 10

A = 994.579

Intensity (arbitrary units)

Intensity x104 (arbitrary units)

125A 2.5 2.0 Neq125 + 100 mM potassium 1.5 phosphate buffer 1.0 0.5

8

Time (min)

| Research Article

500 400 300 200 100 0

Neq125 + 100 mM potassium phosphate buffer + microsomes

125D Neq125 + 100 mM potassium phosphate buffer + microsomes + NADPH 1

2

3

4

5

6

7

8

Time (min)

Figure 10. Coupling of LC–MS-ESI-QqTOF to identify Neq125 metabolites in rat liver microsomes. (A) Extracted ion chromatograms of m/z 375 in mass spectrometry mode of operation of the controls and the microsomal incubation study for the biotransformation of compound Neq125. (B) Identification of the metabolite m/z 347 [M-28] +. „„In vitro

assays In vitro trypanocidal activity of the compounds was evaluated against amastigote forms of Tulahuen strain that was genetically modi­ fied to express the β-galactosidase gene from Escherichia coli (lacZ). A monkey kidney cell strain (LLC-MK2; ATCC) was resuspended in RPMI medium without phenol red (GibcoBRL Life Technologies, NY, USA) containing 10% fetal bovine serum (Life Technologies Inc., MD, USA), and antibiotics (Sigma Chemical Co., MO, USA) 2 × 103 cells per well were cul­ tured in 96-well plates for 24 h. The cells were infected with 104 trypomastigote forms of T  cruzi Tulahuen strain, and after 24 h compounds were added in serial dilutions (250, 125, 62.5, 31.25, 15.6, 7.8, 3.9 and 1.95 µM). After 4 days of culture, 50 µl of phosphate buffered saline solution (PBS) containing 0.5% of Triton X-100 and 100 µM chlorophenol red-β-d-galactoside (Sigma) were added. Plates were incubated at 37°C for 4 h and absorbance was read at 570 nm. Benznidazole (N-benzyl-2-nitro-1-imidazolacetamide) was used as a reference trypanocidal drug (positive control), in the same concentrations as above [41]. The experiments were performed in duplicate. Spleen cells from C57BL/6 mice were iso­ lated by mechanical dissociation and assayed as future science group

previously described [42]. After the incubation in 96-well plates, compounds were tested in dupli­ cate using 5 × 106 cells/ml for 24 h at 37°C with 5% CO2. After cells were harvested, propidium iodide was added to each well at a concentration of 10 µg/ml followed by incubation. Data acqui­ sition was performed using a FACSCanto™ ll flow cytometer. „„Metabolic

profiling Wistar rats, of 6–8 weeks old, were maintained in an appropriate room at 20–23°C, with rela­ tive humidity of approximately 50%, light and dark cycles of 12 h, free of pathogens, in clean air and constant supply of water. The livers of rats were thawed, weighed, minced and subjected to extraction of microsomal fractions. The pieces of chopped liver were homogenized in an ultrasound (Ultra Turraz; Janke and Kunkel, Germany) in cold PBS buffer solution. The fractions were obtained by a series of centrifugations. The first centrifugation lasted for 5 min at 600 g, and the supernatant was collected and centrifuged again at 6500 g for 10 min. The supernatant was collected and once again subjected to another centrifugation for 20 min, now on a rotation 12,000 g. All centrifugations were carried out at a temperature of 4°C. Finally, to obtain the www.future-science.com

29

Research Article | Prokopczyk, Ribeiro, Sartori et al. Key Term Metabolic profiling:

Composed by small-molecule metabolites and their intermediates as a dynamic response for chemical reactivity in biological systems.

microsomal fractions, the sample was subjected to ultracentrifugation with a rotation of 126,000 g for 60 min at 4°C. The deposited material (pellet) was removed from the ultracentrifuge tube and transferred to a polypropylene tube in PBS buffer (pH = 7.4). The solution was homogenized using an ultrasound. The samples were transferred to cryogenic tubes and stored in a freezer at -80°C. For each microsomal extract, the determination of the total protein concentration was performed using the Bradford method. In vitro assays were performed in test tubes and in triplicate. Compounds were initially pre­ pared as 10 mM stock solutions in DMSO. The microsomes were thawed and adjusted to a con­ centration of 20 mg/ml microsomal protein. A solution was obtained by adding 181 µl of potas­ sium phosphate buffer (100 mM, pH 8.0), 2 µl of a solution of 20 mM NADPH (to a final con­ centration of 1 mM) and 5 µl of microsomes (to a final concentration of 0.5 mg/ml). Following the pre-incubation of microsomes, buffer and test compound (2 µl of stock solution to a final concentration of 100 µM) were incubated in a water bath at 37°C for 5 min. The percentage of DMSO in the assay was 0.01%. The reaction was initiated by addition of 10 µl of 20 mM NADPH followed by incubation for 3 h at 37°C under smooth agitation. The reaction was terminated by the addition of 200 µl ethyl acetate. The mixture was brought to the vortex and then centrifuged at 9000 rpm for 5 min. The organic fraction was collected and dried at room temperature using a SpeedVac (Savant Instruments; NY, USA). The resulting dried material was redissolved in mobile phase and frozen at -20°C until analysis by LC–MS. In this assay, the following controls were added: n Test compound dissolved in methanol at a concentration of 100 µM; Test compound solution in 100 µM concentration, dissolved in PBS (100 mM pH 8.0) 0.01% DMSO;

n

Test compound solution in 100 µM concentration, dissolved in PBS (100 mM pH 8.0), 0.01% DMSO, and the concentration of 1 mM NADPH;

n

Test compound solution in 100 µM concentra­ tion, dissolved in PBS (100 mM pH 8.0), 0.01% DMSO and rat liver microsomes at a concentration of 0.05 mg/ml microsomal protein.

n

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Future Med. Chem. (2014) 6(1)

„„LC–TOF

analyses Positive ion mode ESI mass spectrum of Neq125 was measured using a microTOF II instrument (Bruker Daltonics, Wissembourg, France) with N2 as a spray gas, at 180°C (drying tempera­ ture), 8.5 l/min (gas flow) and nebulizer pres­ sure of 2 bar. The flow rate was 0.2 ml/min. Resolution parameters were adjusted to achieve optimal mass resolution in the 50–800 amu mass range. Mass spectra were recorded with an acquisition frequency of 20 Hz. Data were acquired and processed using the MicroTOFControl and Data-Analysis software (Bruker Daltonics). Conclusion The challenge in cheminformatics applied to drug discovery is to find plausible regions containing bioactive compounds, within the chemical–biological space. In this sense, cheminformatic methods as virtual screening and design based on target structures were essential for the development of this work. The application of these tools allowed carry­ ing out searches of large collections of com­ pounds for winnowing to a limited number of compounds that presented high probability of being active in experimental trials against GAPDH enzyme of T. cruzi. These methods helped the reduction of the chemical space to be investigated, therefore allowing the identi­ fication of three compounds that inhibit the TcGAPDH, which can be taken to the next step in the drug design pipeline, one of them being a new trypanocidal agent. The ITC is shown to be extremely sensi­ tive, monitoring the kinetics of the reaction and obtaining the values​ of K iapp so precise and accurate to allow the identification of the first low micromolar TcGAPDH inhibitors. Moreover, this method is not destructive and is universal for any enzymatic reaction where there is heat flow. In addition, ITC does not require that substrates contain chromophores or fluorophores, and opaque solutions can be throughout used. Overall, the new trypanocidal scaffold exemplified by Neq125 presented good in vitro potency against the trypomastigote T. cruzi Tulahuen strain, being comparable with the reference compound benznidazole. As far as we know, this is the first time that a potent low molecular mass G3P-driven small inhibitor of GAPDH is described. Moreover, together with its metabolic profiling on CYP450, our future science group

Integration of methods in cheminformatics & biocalorimetry results pinpoint Neq125 as a promising com­ pound for further optimization once it is a leadlike compound having a comparable potency/ cytotoxicity (i.e., selectivity ratio) towards the parasite in relation to the benznidazole drug. Future perspective The results described show good consistency. The good correlation between the experimen­ tal mechanism of action and the predicted binding poses further proved the reliability of the constructed models and experiments. It is noteworthy that we employed a workflow from in silico to in vitro studies, and the results showed that investigations can be carried out concomi­ tantly to search for synergies between in silico and ITC technologies. Our results suggest that the in silico model of TcGAPDH can be used in target-based virtual screening for the design of novel structurally related TcGAPDH inhibi­ tors. This is remarkably important considering that old drugs (nifurtimox and benznidazole), being used in non-efficacious treatment, have to be replaced by new and innovative ones to alleviate chagasic individuals of terrible suf­ fering from a centennial disease. To highlight such possibilities, our best compound inhibits a key glycolytic enzyme (TcGAPDH) and kills T. cruzi in a dose-dependent manner, therefore acting as a new trypanocidal agent that is equi­ potent to the drug benznidazole currently in use to treat chagasic people. In addition to this, Neq125 is not cytotoxic but metabolically safe. Accordingly, we envisage that the new trypano­ cidal agent that discloses a new chemical scaf­ fold will be amenable for furthering medicinal chemists’ interest. However, in order to fulfill this we have to take into consideration that Chagas disease is analogous to three most widespread diseases – HIV, tuberculosis and malaria, but has lim­ ited treatment and lower research funding.

| Research Article

Although these are more deadly diseases, Chagas disease is achingly affecting sufferers daily to disability-adjusted life year standards that are outrageous for a centennial disease. To end this negligence, effective and collaborative public–private partnership is envisaged as the pivotal goal, which is driven by a high level of unmet patient needs. If so, new mechanisms of action, such as the one presented here, will pave the way to offer new medicines to treat Chagas disease in 10 years time. Acknowledgements The authors would like to thank F Rosini and GM Titato for their help in enzyme purification and operation of the mass spectrometer, respectively. Special thanks go to RV Oliveira and NM Cassiano, from Federal University of Sao Carlos, for their valuable assistance in the development of this work.

Financial & competing interests disclosure The Brazilian granting agencies CAPES (PROCAD NF2009, 666/2010), CNPq and FAPESP (grant #2011/01893-3) are acknowledged for supporting this research. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Ethical disclosure The Ethics Committee on Animal Experimentation of the Faculty of Medicine of Ribeirão Preto – University of Sao Paulo approved the cytotoxicity assays (approval no. 076/2010). This Committee adheres to Conselho Nacional de Controle de Experimentação Animal – CONCEA, created by Brazilian Law number 11794 of 8 October 2008. Assays were run according to the guidelines of the Ministry of Science, Technology and Innovation of Brazil.

Executive summary „„

Chagas disease is a disease for which there is no safe and efficacious treatment, and which has been described by the WHO as a ‘disease of poverty’. Currently, there are only two drugs to treat Chagas disease, which are more than 40 years old. Adhesion to the treatment regime is low.

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Integrating technologies for target-based screening are promising in the search for new drug candidates to treat Chagas disease.

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GAPDH is an important enzyme in the glycolytic pathway that controls the energy production of the Trypanosoma cruzi and it is a target for antiparasitic compounds.

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In this study five out of 25 selected compounds were active against TcGAPDH when assayed using isothermal titration calorimetry, which is an extremely high enrichment factor (with concentration values lower than 300 µM).

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Neq125 had a good inhibitory profile in biochemical assays and is equipotent to the reference drug benznidazole in trypomastigote cells.

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Neq125 displays a selectivity ratio in the same range of benznidazole, having a good metabolic profile in liver microsomal assays.

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