Development of Thiophene Compounds as Potent Chemotherapies for the Treatment of Cutaneous Leishmaniasis Caused by Leishmania major

Leishmania major (L. major) is a protozoan parasite that causes cutaneous leishmaniasis. About 12 million people are currently infected with an annual incidence of 1.3 million cases. The purpose of this study was to synthesize a small library of novel thiophene derivatives, and evaluate its parasitic activity, and potential mechanism of action (MOA). We developed a structure–activity relationship (SAR) study of the thiophene molecule 5A. Overall, eight thiophene derivatives of 5A were synthesized and purified by silica gel column chromatography. Of these eight analogs, the molecule 5D showed the highest in vitro activity against Leishmania major promastigotes (EC50 0.09 ± 0.02 µM), with an inhibition of the proliferation of intracellular amastigotes higher than 75% at only 0.63 µM and an excellent selective index. Moreover, the effect of 5D on L. major promastigotes was associated with generation of reactive oxygen species (ROS), and in silico docking studies suggested that 5D may play a role in inhibiting trypanothione reductase. In summary, the combined SAR study and the in vitro evaluation of 5A derivatives allowed the identification of the novel molecule 5D, which exhibited potent in vitro anti-leishmanial activity resulting in ROS production leading to cell death with no significant cytotoxicity towards mammalian cells.


Introduction
Leishmaniasis is a devastating neglected tropical disease (NTD) [1] caused by the protozoan parasite of the genus Leishmania. The parasite is transmitted from animals to humans through the bite of infected females Lutzomyia or Phlebotomus sand flies [2]. Over 20 species and subspecies of Leishmania infect humans, causing three major clinical forms of the disease: cutaneous (CL), visceral, and mucocutaneous leishmaniasis [3]. The prevalence of CL, the most common form of leishmaniasis, is estimated between 0.7 and 1.3 million new annually cases worldwide [4], and it is commonly caused by Leishmania major (L. major) or L. mexicana. CL presents as singular ulcerative or nodular lesions at the

In Vitro Anti-Leishmanial Activity of Thiophene Derivatives and Their Cytotoxicity
Consequently, to lower the toxicity and increase the parasitic activity, eight new thiophene molecules ( Figure 2) were evaluated. First, the thiophene compounds were tested in the presence of increasing drug concentrations (1.56-12.5 µM), followed by incubation with L. major-luc promastigotes (2 × 10 6 /mL) for 72 h at 28 • C. The experiment was performed using the same conditions as described for the parent drug 5A. As summarized in Table 1, all eight thiophene molecules showed promising antileishmanial activity against L. major promastigotes with an EC 50 ranging from 0.09 to 6.25 µM ( Figure 4A). However, the best thiophene compounds were 5D (EC 50 0.09 ± 0.02 µM) and 5E (EC 50 0.78 ± 0.11 µM) ( Figure 4B and Table 1).
Consequently, cytotoxicity assays were performed by incubating LLC-MK2 or IPΦ with compounds. First, 5D or 5E were incubated with 1 × 10 5 LLC-MK2/mL or 1 × 10 5 IPΦ/mL for 72 and 48 h, respectively. Interestingly, compound 5D did not display perceptible toxicity against LLC-MK2 at concentrations up to 80 µM ( Figure 4C,D). In the case of IPΦ, 5D exhibited a CC 50 value of 27.89 ± 3.19 µM and an excellent S.I. of 310. As 5E compound CC 50 values of 80 ± 4.45 µM in LLC-MK2 cells and 16.59 ± 1.52 µM in IPΦ, with S.I. values of 102.56 and 21.27, respectively. More importantly, we determined that both 5D and 5E displayed lower cytotoxicity to mammalian cells and higher parasitic activity than parent compound 5A (Table 1).

In Vitro Anti-Leishmanial Activity of Thiophene Derivatives and Their Cytotoxicity
Consequently, to lower the toxicity and increase the parasitic activity, eight new thiophene molecules ( Figure 2) were evaluated. First, the thiophene compounds were tested in the presence of increasing drug concentrations (1.56-12.5 µM), followed by incubation with L. major-luc promastigotes (2 × 10 6 /mL) for 72 h at 28 °C. The experiment was performed using the same conditions as described for the parent drug 5A. As summarized in Table 1, all eight thiophene molecules showed promising antileishmanial activity against L. major promastigotes with an EC50 ranging from 0.09 to 6.25 µM ( Figure 4A). However, the best thiophene compounds were 5D (EC50 0.09 ± 0.02 µM) and 5E (EC50 0.78 ± 0.11 µM) ( Figure 4B and Table 1).
Consequently, cytotoxicity assays were performed by incubating LLC-MK2 or IPФ with compounds. First, 5D or 5E were incubated with 1 × 10 5 LLC-MK2/mL or 1 × 10 5 IPФ/mL for 72 and 48 h, respectively. Interestingly, compound 5D did not display perceptible toxicity against LLC-MK2 at concentrations up to 80 µM ( Figure 4C,D). In the case of IPФ, 5D exhibited a CC50 value of 27.89 ± 3.19 µM and an excellent S.I. of 310. As 5E compound CC50 values of 80 ± 4.45 µM in LLC-MK2 cells and 16.59 ± 1.52 µM in IPФ, with S.I. values of 102.56 and 21.27, respectively. More importantly, we determined that both 5D and 5E displayed lower cytotoxicity to mammalian cells and higher parasitic activity than parent compound 5A (Table 1).

In Vitro Efficacy of Thiophene 5D Against Intracellular Amastigotes
Additionally, efficacy of thiophene compounds 5D and 5E was tested against the infectious intracellular amastigote form of L. major, by High-Content Imaging Assay (HCIA) on infected intraperitoneal mouse macrophages. As observed in Figure 5A, in comparison with untreated control and 1% DMSO, 5D and 5E inhibited the proliferation of the intracellular amastigotes by more than 75% and 50%, respectively, at a 0.625 µM concentration. Furthermore, as observed in Figure 5B, a reduced number of infected cells were observed after 5D or 5E treatment (2.5 µM) when compared to control treated with 1% DMSO.

In Vitro Efficacy of Thiophene 5D Against Intracellular Amastigotes
Additionally, efficacy of thiophene compounds 5D and 5E was tested against the infectious intracellular amastigote form of L. major, by High-Content Imaging Assay (HCIA) on infected intraperitoneal mouse macrophages. As observed in Figure 5A, in comparison with untreated control and 1% DMSO, 5D and 5E inhibited the proliferation of the intracellular amastigotes by more than 75% and 50%, respectively, at a 0.625 µM concentration. Furthermore, as observed in Figure 5B, a reduced number of infected cells were observed after 5D or 5E treatment (2.5 µM) when compared to control treated with 1% DMSO. Data are represented as the percentage (%) of infected IPΦ with three or more amastigotes per cell. Note: Data for 5A at concentration 5 and 10 µM were not generated because 5A was cytotoxic for IPΦ at such concentration. (B) Representative monochromatic images of infected IPΦ with L. major after 48 h treatment with 5D or 5E at 2.5 µM, amp B (5 µM), or 1% DMSO.

Molecule 5D Induces ROS in L. major
Based on our previous study [12], it was hypothesized that 5D may induce parasite death through the production of ROS. Thus, 2 × 10 6 L. major promastigotes per mL were incubated with 5D (EC50 0.09 ± 0.02 µM). After 24 h, ROS levels were measured by the addition of 10 µM of the cellpermeable dye H2DCFDA (Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence was monitor for an additional 7 h using a fluorometer. As expected, ROS levels in 5D treated parasites were 14.5-fold higher compared to vehicle control 1% DMSO ( Figure 6).

Molecule 5D Induces ROS in L. major
Based on our previous study [12], it was hypothesized that 5D may induce parasite death through the production of ROS. Thus, 2 × 10 6 L. major promastigotes per mL were incubated with 5D (EC 50 0.09 ± 0.02 µM). After 24 h, ROS levels were measured by the addition of 10 µM of the cell-permeable dye H 2 DCFDA (Thermo Fisher Scientific, Waltham, MA, USA), and fluorescence was monitor for an additional 7 h using a fluorometer. As expected, ROS levels in 5D treated parasites were 14.5-fold higher compared to vehicle control 1% DMSO ( Figure 6).

Docking of 5D on TryR from Leishmania
Next, to determine the possible molecular mechanism responsible for the antileishmanial activity of 5D, docking studies on TryR from L. infantum (PDB id: 2JK6) were performed. Using Glide Standard Precision [19] and Extra Precision (XP), we performed Rigid Receptor Docking analysis of control (Quinacrine Mustard) and 5D. The 3D ligand structures were docked against the best potential binding site of 2JK6. Glide SP and XP only accounts for the ligand being dynamic however the protein remains rigid. Docking box coordinates and dimensions remained all at default (20 × 20 × 20 Å). Glide XP gives an output of a docking score, which was analyzed by the lowest number, or whichever is more negative to be the highest scoring ligand. The docking results, summarized in Table 2, showed the control (Quinacrine Mustard) with higher binding affinity than 5D in both SP and XP. However, the XP docking score did not differ by much, indicating more rigorous docking analysis is needed. Thus, both ligands were taken to Schrodinger's Flexible receptor docking.  Schrodinger IFD protocol for all IFD jobs was used [20]. The IFD program makes use of both Glide (for docking) and Prime (for protein structure modeling). The combination of the two software packages allows a more accurate ligand binding calculation. We performed the re-docking with Glide 2.6. Docking of 5D on TryR from Leishmania Next, to determine the possible molecular mechanism responsible for the antileishmanial activity of 5D, docking studies on TryR from L. infantum (PDB id: 2JK6) were performed. Using Glide Standard Precision [19] and Extra Precision (XP), we performed Rigid Receptor Docking analysis of control (Quinacrine Mustard) and 5D. The 3D ligand structures were docked against the best potential binding site of 2JK6. Glide SP and XP only accounts for the ligand being dynamic however the protein remains rigid. Docking box coordinates and dimensions remained all at default (20 × 20 × 20 Å). Glide XP gives an output of a docking score, which was analyzed by the lowest number, or whichever is more negative to be the highest scoring ligand. The docking results, summarized in Table 2, showed the control (Quinacrine Mustard) with higher binding affinity than 5D in both SP and XP. However, the XP docking score did not differ by much, indicating more rigorous docking analysis is needed. Thus, both ligands were taken to Schrodinger's Flexible receptor docking.

Docking of 5D on TryR from Leishmania
Next, to determine the possible molecular mechanism responsible for the antileishmanial activity of 5D, docking studies on TryR from L. infantum (PDB id: 2JK6) were performed. Using Glide Standard Precision [19] and Extra Precision (XP), we performed Rigid Receptor Docking analysis of control (Quinacrine Mustard) and 5D. The 3D ligand structures were docked against the best potential binding site of 2JK6. Glide SP and XP only accounts for the ligand being dynamic however the protein remains rigid. Docking box coordinates and dimensions remained all at default (20 × 20 × 20 Å). Glide XP gives an output of a docking score, which was analyzed by the lowest number, or whichever is more negative to be the highest scoring ligand. The docking results, summarized in Table 2, showed the control (Quinacrine Mustard) with higher binding affinity than 5D in both SP and XP. However, the XP docking score did not differ by much, indicating more rigorous docking analysis is needed. Thus, both ligands were taken to Schrodinger's Flexible receptor docking.  Schrodinger IFD protocol for all IFD jobs was used [20]. The IFD program makes use of both Glide (for docking) and Prime (for protein structure modeling). The combination of the two software packages allows a more accurate ligand binding calculation. We performed the re-docking with Glide

Docking of 5D on TryR from Leishmania
Next, to determine the possible molecular mechanism responsible for the antileishmanial activity of 5D, docking studies on TryR from L. infantum (PDB id: 2JK6) were performed. Using Glide Standard Precision [19] and Extra Precision (XP), we performed Rigid Receptor Docking analysis of control (Quinacrine Mustard) and 5D. The 3D ligand structures were docked against the best potential binding site of 2JK6. Glide SP and XP only accounts for the ligand being dynamic however the protein remains rigid. Docking box coordinates and dimensions remained all at default (20 × 20 × 20 Å). Glide XP gives an output of a docking score, which was analyzed by the lowest number, or whichever is more negative to be the highest scoring ligand. The docking results, summarized in Table 2, showed the control (Quinacrine Mustard) with higher binding affinity than 5D in both SP and XP. However, the XP docking score did not differ by much, indicating more rigorous docking analysis is needed. Thus, both ligands were taken to Schrodinger's Flexible receptor docking.  Schrodinger IFD protocol for all IFD jobs was used [20]. The IFD program makes use of both Glide (for docking) and Prime (for protein structure modeling). The combination of the two software packages allows a more accurate ligand binding calculation. We performed the re-docking with Glide Schrodinger IFD protocol for all IFD jobs was used [20]. The IFD program makes use of both Glide (for docking) and Prime (for protein structure modeling). The combination of the two software packages allows a more accurate ligand binding calculation. We performed the re-docking with Glide XP for the refined docking results [21]. The IFD data presented in Table 2 show that our lead molecule 5D had a better binding affinity. Docking scores from Rigid Receptor Docking and Flexible Receptor Docking differed significantly. This is accounted for the protein dynamic movement during drug binding in IFD. Furthermore, Figure 7A,B presents the IFD binding pocket of the protein-ligand complex. Figure 7A shows our lead molecule 5D which displays hydrogen bond interactions with SER 1632, ARG 287, VAL 55, and also with CYS 57. Compound 5D also exhibits π-cation interaction with residue ARG 287. Quinacrine Mustard interacted with a new set of residues and showed only two hydrogen bonds between MET 333 and ALA 365 ( Figure 7B). The control also formed salt bridges with ASP 327 as well as GLU 202. π-π and π-cation interaction was also shown between TYR 198 and LYS 60, respectively. These results provided evidence that the possible MOA of 5D may be through the inhibition of TryR, an essential enzyme to the thiol metabolism of the parasite [22,23], and promising chemotherapeutic target against leishmaniasis [24]. XP for the refined docking results [21]. The IFD data presented in Table 2 show that our lead molecule 5D had a better binding affinity. Docking scores from Rigid Receptor Docking and Flexible Receptor Docking differed significantly. This is accounted for the protein dynamic movement during drug binding in IFD. Furthermore, Figure 7A,B presents the IFD binding pocket of the protein-ligand complex. Figure 7A shows our lead molecule 5D which displays hydrogen bond interactions with SER 1632, ARG 287, VAL 55, and also with CYS 57. Compound 5D also exhibits π-cation interaction with residue ARG 287. Quinacrine Mustard interacted with a new set of residues and showed only two hydrogen bonds between MET 333 and ALA 365 ( Figure 7B). The control also formed salt bridges with ASP 327 as well as GLU 202. π-π and π-cation interaction was also shown between TYR 198 and LYS 60, respectively. These results provided evidence that the possible MOA of 5D may be through the inhibition of TryR, an essential enzyme to the thiol metabolism of the parasite [22,23], and promising chemotherapeutic target against leishmaniasis [24].

Discussion
There is an urgent need for new therapeutics that are more effective and less toxic than conventional treatments used to treat infectious diseases, including leishmaniasis [25]. Thiophenes derivatives are known for their therapeutic applications and have shown promising results to treat different types of cancer, degenerative diseases, HIV, and malaria [26][27][28][29][30][31][32][33]. Thus, we evaluated the anti-leishmania activity and selectivity of nine thiophene derivatives against L. major, and potential MOA was elucidated for our best candidate, 5D.
Thiophene derivatives 5A, 5D and 5E exhibited potent parasitic activity against L. major promastigotes (Table 1). Experimental models involving macrophages are ideal to study leishmaniasis since they are the major host cell for Leishmania spp. [34]. Thus, our three best candidates were further evaluated against the most important form of the parasite, intracellular

Discussion
There is an urgent need for new therapeutics that are more effective and less toxic than conventional treatments used to treat infectious diseases, including leishmaniasis [25]. Thiophenes derivatives are known for their therapeutic applications and have shown promising results to treat different types of cancer, degenerative diseases, HIV, and malaria [26][27][28][29][30][31][32][33]. Thus, we evaluated the anti-leishmania activity and selectivity of nine thiophene derivatives against L. major, and potential MOA was elucidated for our best candidate, 5D.
Thiophene derivatives 5A, 5D and 5E exhibited potent parasitic activity against L. major promastigotes (Table 1). Experimental models involving macrophages are ideal to study leishmaniasis since they are the major host cell for Leishmania spp. [34]. Thus, our three best candidates were further evaluated against the most important form of the parasite, intracellular amastigotes, in an in vitro infection model of murine macrophages. In this case, 5D presented the best anti-leishmanial activity by decreasing the proliferation of the parasite by 80%.
The in vitro toxicity of 5D and 5E was evaluated towards IPΦ and LCC-MK2 cells. Our best two compounds were safer for the two cytotoxic models than the reference drug, amphotericin B, which is already known for its cytotoxic effects [35]. Even though amphotericin B presented similar activity as 5D against promastigotes and amastigotes, this result further supports the application of 5D and 5E as anti-leishmanial agents. Furthermore, the selectivity presented by 5D was remarkably higher than the parent compound 5A (10-fold higher), demonstrating the success to increase the anti-leishmanial activity and reduced cytotoxicity effects when compared to our previously reported arylalkylamine type-compound [12].
Next, we studied the potential MOA of derivative 5D. ROS can be generated in response to some drugs, resulting in destruction of cellular macromolecular components inducing cell death by affecting parasite mitochondrial function [36,37]. Here, we observed that 5D induced ROS production in L. major promastigotes after 31 h of treatment. The redox homeostasis in Leishmania is achieved through the activity of several superoxide dismutases, heme peroxidases, as well as of a series of thiol-containing proteins that directly or indirectly depend on trypanothione reductase [23,38]. In this regard, the trypanothione metabolism is unique to trypanosomatids and its main detoxification pathway [39]. This pathway protects parasites from oxidative stress and participates in several cellular processes that are carried out by glutathione in other organisms. Moreover, there are several trypanothione-dependent pathways that include enzymes such as tryparedoxin peroxidase (detoxication of hydroperoxide), ascorbate peroxidase (homeostasis of ascorbate), ribonucleotide reductase (synthesis of DNA precursors), and others [40,41]. With this idea on mind, we decided to explore in silico docking analysis to assess the possibility of L. major TryR as the target of 5D. Moreover, our results suggested that TryR interacts with 5D, however we do not exclude the possibility that other redox metabolism enzymes could be also targeted by compound.
In conclusion, to discover new chemotherapy agents against leishmaniasis, we efficiently synthetized nine thiophene type-compounds including 5A, following a two-step synthesis from low-priced commercially available starting materials. We then showed that our novel thiophene type-compounds possess high in vitro antileishmanial activity. Based on our SAR study, 5D analog was selected as the most promising lead compound among this library with excellent antiparasitic and S.I. Furthermore, 5D may act against trypanothione metabolism, followed by the production of ROS in the parasite; nevertheless, biological studies with recombinant TryR enzyme needs to be performed to further support this assumption. Overall, 5D represents a potential chemotherapeutic agent for the treatment of leishmaniasis, and further evaluation in a pre-clinical mouse model of cutaneous leishmaniasis is currently in progress in our laboratory.

General
Unless otherwise noted, all commercial reagents were used as purchased from Aldrich, St. Louis, MO, USA. All the reactions were monitored by thin-layer chromatography (TLC) that was performed on silica gel plates GF254. Compounds were visualized under a UV lamp. Flash chromatography was performed using silica gel (200-300 mesh) with various ratios of Dichloromethane: Methanol solvents as indicated in the text. Spectroscopy: 1 H and 13 C-NMR spectra were obtained on a Bruker DPX 400 MHz. Chemical shifts (δ) are quoted in parts per million (ppm), to the nearest 0.01 ppm and internally referenced relative to the solvent nuclei. 1 H-NMR spectral data are reported with their chemical shift in parts per million (ppm). The multiplicity in 1 H-NMR is abbreviated as follows: brs: broad; s: singlet; d: doublet; t: triplet; q: quartet; quint: quintet; sext: sextet; m: multiplet; or as a combination (e.g., dd, dt, etc.). The coupling constant (J) in hertz, integration and proton count were determined.

Luciferase Assay-Viability of Leishmania Major promastigotes
The antiparasitic activity of the 9 thiophene compounds was determined by adding the analogs together with 2 × 10 6 L. major-luc promastigotes per mL in 96-well NUNC white microplates (Thermo Fisher Scientific, Waltham, MA, USA) followed by incubation for 72 h at 28 • C. Then, parasite survival was measured by luciferase activity with the addition of the substrate 5 -fluoroluciferin (ONE-Glo luciferase assay system; Promega, Madison, WI, USA), using a luminometer (Luminoskan; Thermo Fisher Scientific, Waltham, MA, USA). The luminescence intensity was a direct measure of the parasite survival, and 50% effective concentration (EC 50 ) was determined for each drug and summarized in Table 1.

Assessment of Thiophene Compound Mammalian Cell Cytotoxicity
The potential cytotoxicity of 5A was tested by alamarBlue TM Cell Viability Assay (Thermo Fisher Scientific, Waltham, MA, USA) as previously described [18]. Briefly, 1 × 10 6 /mL rhesus monkey kidney epithelial cells (LLC-MK2), and 1 × 10 6 /mL BALB/c IPΦ were seeded in a 96-well clear bottom black microplate (BD Biosciences, Franklin Lakes, NJ, USA). Cells were incubated in the presence of increasing drug concentrations for 72 h (LLC-MK2) or 48 h (IPΦ) at 37 • C, 5% CO 2 , followed by addition of alamarBlue TM . Fluorescence was measured using a fluorometer (Fluoroskan; Thermo Fisher Scientific, Waltham, MA, USA). Compound 5D or 5E were incubated with 1 × 10 5 cells/mL (LLC-MK and IPΦ) for 72 or 48 h, respectively, at 37 • C, 5% CO 2 . After the incubation period, a dilution of 20:1000 in PBS from a stock at 1 mg/mL of Propidium Iodide (PI) and Hoechst 33342 (Thermo Scientific, Waltham, MA, USA) were added for survival discrimination as previously described [44]. Analysis was performed by High-Content Imaging Assay (HCIA) using an IN Cell 2000 Analyzer Bioimaging System (GE Healthcare, Chicago, IL, USA) for LLC-MK2 cells, and BD Pathway 855 High-resolution Bioimager System (BD Biosciences, Franklin Lakes, NJ, USA) for IPΦ. The 50% cytotoxic concentration (CC 50 ) and selective index (S.I.) was determined and summarized in Table 1.

High-Content Imaging Assay-Proliferation Experiments
BALB/c IPΦ were acquired and seeded at a density of 1 × 10 5 cells/mL for 2 h at 37 • C, 5% CO 2 . After adherence, IPΦ were infected with 1 × 10 6 /mL metacyclic promastigotes of L. major-luc, at a ratio of 10:1 parasites per macrophage. Subsequently, infected IPΦ were incubated with derivatives 5A, 5D and 5E at increasing concentrations (0.625 to 10 µM) for 48 h treatment. Afterwards, cells were fixed with 4% paraformaldehyde, and stained with (1.25:100) Alexa Fluor™ 488 Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) and (1:1000) DAPI (Sigma Aldrich, St. Louis, MO, USA). Then, the numbers of infected cells and amastigotes were determined by HCIA using an IN Cell 2000 Analyzer Bioimaging System (GE Healthcare, Chicago, IL, USA). Parameters were set for the excitation and emission spectra of Alexa Fluor™ 488 Phalloidin and DAPI, and a constraint of 3 or more parasites per macrophage was set as previously reported [44,45]. All graphs, EC 50 and CC 50 values were produced using Graph Pad Prism 7 Software (GraphPad Software, Inc., La Jolla, CA, USA).

Docking Studies-Pre-Docking Preparation
The structure of trypanothione reductase bound to Flavin adenine dinucleotide (PDB ID: 2JK6) [46] were obtained from the Protein Data Bank. The Protein Preparation Wizard in Maestro was used to minimize the protein structure, add hydrogens and charges, and find any missing residues. The two-dimensional structures of 5D and Quinacrine Mustard, a recently experimentally approved drug as a control, were drawn using the molecular structure editor ChemDraw Software (PerkinElmer, Waltham, MA, USA) and processed by LigPrep Schrödinger (Schrödinger, LLC., New York, NY, USA) to generate the 3D structures.

Binding Site Analysis
Maestro's SiteMap tool (Schrödinger, LLC., New York, NY, USA) was used to predict the likely binding sites of trypanothione reductase. The SiteMap tool uses a series of algorithm that generates a map of hydrophobic and hydrophilic surfaces on the protein surface [47]. Hydrophilic surface maps are divided into donor, acceptor, and metal-binding regions. Five potential binding sites were identified with at least 15 site points. However, the top SiteMap was chosen to be the receptor grid.

Conflicts of Interest:
The authors declare no conflict of interest.