bioRxiv preprint doi: https://doi.org/10.1101/645333; this version posted May 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 Crystal violet structural analogues identified by in silico drug
2 repositioning present anti-Trypanosoma cruzi activity
3 through inhibition of proline transporter TcAAAP069
4 Melisa Sayé1,2, Lucrecia Gauna1,2, Edward Valera-Vera1,2, Chantal Reigada1,2,
5 Mariana R. Miranda1,2, and Claudio A. Pereira1,2,*
6
7 1 Universidad de Buenos Aires, Facultad de Medicina, Instituto de Investigaciones
8 Médicas A. Lanari, Buenos Aires, Argentina.
9 2 Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad de
10 Buenos Aires, Instituto de Investigaciones Médicas (IDIM), Laboratorio de
11 Parasitología Molecular, Buenos Aires, Argentina.
12
13
14 * Corresponding author: E-mail address: [email protected] (CAP)
15
16 Short title: Crystal violet analogues repositioned against Trypanosoma cruzi
17
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18 Abstract
19 Background: Crystal violet (CV) was used for several years in blood banks to
20 eliminate the parasite Trypanosoma cruzi in endemic areas in order to prevent
21 transfusion-transmitted Chagas disease. One mechanism of action described for
22 CV involves inhibition of proline uptake. In T. cruzi, proline is essential for host cell
23 infection and intracellular differentiation among other processes, and can be
24 obtained through the proline permease TcAAAP069.
25 Methodology/Principal Findings: CV inhibited proline transporter TcAAAP069
26 and parasites overexpressing this permease were 47-fold more sensitive to this
27 compound than control parasites. Using CV as reference molecule, loratadine,
28 cyproheptadine, olanzapine and clofazimine were identified as structurally related
29 compounds to CV (structural analogues) by in silico drug repurposing through a
30 similarity-based virtual screening protocol. All these already-approved drugs for
31 clinical use inhibited TcAAAP069 activity with different efficacies and also
32 presented trypanocidal action in epimastigotes, trypomastigotes and amastigotes
33 of the Y strain. Additionally, loratadine, cyproheptadine and clofazimine showed
34 trypanocidal effect on epimastigotes of the CL Brener and DM28c strains. Finally, a
35 synergistic effect between benznidazole and the CV chemical analogues was
36 evidenced by combination and dose-reduction indexes values in epimastigotes of
37 the Y strain.
38 Conclusions/Significance: Loratadine, cyproheptadine and clofazimine inhibit
39 TcAAAP069 proline transporter and also present trypanocidal effect against all T.
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40 cruzi life stages. These CV structural analogues could be a starting point to design
41 therapeutic alternatives to treat Chagas disease by finding new indications for old
42 drugs. This approach, called drug repurposing is a recommended strategy by the
43 World Health Organization to treat neglected diseases, like Chagas disease, and
44 combination therapy may improve the possibility of success of repositioned drugs.
45
46 Author summary
47 Chagas disease, caused by the parasite Trypanosoma cruzi, affects 7 million
48 people worldwide. Despite there are two drugs available since 50 years ago, the
49 therapy present severe side effects and is not effective in the chronic phase of the
50 disease were most of the patients are diagnosed. Crystal violet (CV) was utilized
51 as additive in blood banks to prevent transfusion-transmitted Chagas disease.
52 Proline is involved in many pathways, like infection establishment and life cycle
53 progression. In this work we first demonstrate that CV has the proline permease
54 TcAAAP069 as one of its molecular targets. Then we search in a database of
55 already-approved drugs for compounds that were structurally related to CV under
56 the premise “similar structure, similar activity”. We identified three drugs that inhibit
57 proline transport and present at least the same trypanocidal effect than
58 benznidazole, the current treatment for Chagas disease. Finally we observed a
59 synergistic effect with the multidrug combination therapy. Drug discovery is an
60 expensive and time-consuming process and Chagas disease is associated with
61 poverty. The discovery of new indications to old drugs, called drug repurposing,
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62 can facilitate a rapid and more profitable therapy application since preclinical trials
63 and pharmacokinetic studies are already available.
64
65 Introduction
66 Chagas disease is a neglected disease caused by the protozoan parasite
67 Trypanosoma cruzi that can mainly be acquired through an insect vector, blood
68 transfusion and placental or congenital transmission. Among these mechanisms,
69 blood transfusion is still one of the most important mechanisms of transmission of
70 Chagas disease due to lack of blood bank control and migration of people from
71 endemic to non-endemic countries (1,2). Crystal violet (CV) is a well-known dye
72 common in microbiology and it was used for a long time as an additive in blood
73 banks as an attempt to eliminate Chagas transmission in endemic areas (3).
74 Different trypanocidal mechanisms have been proposed for CV, among them
75 Hoffmann et al. demonstrated that proline and methionine transport were strongly
76 inhibited by this compound (4).
77 Chagas disease affects 7 million people in Latin America and almost 70 million
78 more are at risk of infection (5). The only two approved drugs to treat this disease,
79 benznidazole and nifurtimox, were discovered over 50 years ago and present
80 limited efficacy and several side effects (6). Thus the development of new
81 therapies to treat Chagas disease remains as an urgent need.
82 The first multigenic family of amino acid transporters from T. cruzi (TcAAAP; Amino
83 Acid/Auxin Permeases; TC 2.A.18) was identified by our group (7). Few members
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84 of this family have been characterized in trypanosomatids, including the proline (8)
85 (TcAAAP069), arginine (9,10) (TcAAP3) and lysine (11) (TcAAP7) permeases. A
86 polyamine transporter (12,13) (TcPAT12) has also been characterized in T. cruzi
87 and it probably belongs to the Amino Acid-Polyamine Organocation (APC; TC
88 2.A.3) family, which is phylogenetically very close to the AAAP family. The
89 transporter TcAAAP069 is mono-specific for D- and L-proline and it is involved in
90 resistance mechanisms to trypanocidal drugs and also to reactive oxygen species
91 including hydrogen peroxide and nitric oxide (8). In addition, proline is a relevant
92 amino acid for trypanosomatids since it is used as an alternative carbon and
93 energy source to glucose (14). It has been proved that in T. cruzi proline confers
94 osmotic stress resistance (15) and it also sustains cellular invasion in metacyclic
95 trypomastigotes (16) as well as differentiation of intracellular epimastigotes to
96 trypomastigote forms, which is required for the establishment of infection in
97 mammalian hosts (17).
98 The orthologous genes of TcAAAP069 have been characterized in other
99 trypanosomatids. In T. brucei, the orthologous permease TbAAT6 is a low-affinity
100 low-selective transporter for neutral amino acids, including proline (18). The loss of
101 TbAAT6 is sufficient to develop resistance to eflornithine, a drug used to treat
102 human African trypanosomiasis (HAT) (19). Moreover, the importance of amino
103 acid transporters in T. brucei has been demonstrated by RNAi-mediated knock-
104 down of arginine and lysine transporters TbAAT5-3 and TbAAT16-1 from the AAAP
105 family which resulted in growth arrest in bloodstream forms (20). In Leishmania
106 donovani, the transporter LdAAP24 mediates proline and alanine uptake and
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107 regulates the response to osmotic stress and amino acid homeostasis (21). The
108 parasite L. donovani naturally expresses two variants of the proline/alanine
109 transporter, one 18 amino acids shorter than the other. While in T. cruzi no
110 information is available about the determinants of the substrate specificity of
111 TcAAAP transporters, in LdAAP24 these 18 amino acids define its substrate
112 specificity for both amino acids (LdAAP24 long variant) or for proline alone
113 (Δ18LdAAP24 short variant) (22). Recently, it has been reported that LdAAP24
114 also presents a stage-specific expression and it is rapidly degraded when
115 promastigotes are differentiated to amastigotes (23). TcAAAP069 activity and
116 localization have been studied only in epimastigotes overexpressing this proline
117 permease (24), thus there is no information regarding stage-specific expression.
118 Since the transporter TcAAAP069 has many differential features compared to
119 those present in mammals and proline is essential for parasite survival, it could be
120 possible to design a specific inhibitor of this T. cruzi permease with trypanocidal
121 action and non-toxic for the host cells. In fact, a synthetic proline analogue named
122 ITP-1G has been validated as a TcAAAP069 inhibitor which also possesses
123 trypanocidal effect (25). One of the best strategies for the identification of new
124 treatments for neglected diseases is drug repositioning, which is the process of
125 finding new indications for existing drugs. The main benefit of this approach relays
126 on those drugs being ahead in the development and clinical phase I trials,
127 consequently they could reach the patients in approximately half of the time and
128 with a significant lower cost (26). Some successful drugs repositioned for
129 neglected diseases are eflornithine and miltefosine which were originally
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130 discovered for cancer therapy and now are used to treat HAT and leishmaniasis,
131 respectively (27). Considering this information, in this work we performed an in
132 silico screening of a drug database to find compounds structurally related to CV
133 that could also inhibit TcAAAP069 and exert trypanocidal effect with less toxicity.
134
135 Methods
136 Parasites. Epimastigotes of Y, CL Brener and Dm28c strains (5 x106 cells/mL)
137 were cultured at 28 °C in plastic flasks (25 cm2), containing 5 mL of BHT (brain-
138 heart infusion-tryptose) medium supplemented with 10% fetal calf serum (FCS),
139 100 U/mL penicillin, 100 g/mL streptomycin and 20 µg/mL hemin (28).
140 Trypomastigotes were obtained from the extracellular medium of VERO infected
141 cells as previously described (17).
142 Cells. VERO cells (African green monkey Kidney, ATCC® CCL-81™) were
143 cultured in MEM medium supplemented with 10% heat inactivated FCS, 0.15%
144 (w/v) NaHCO3, 100 U/mL penicillin and 100 U/mL streptomycin at 37ºC in 5% CO2.
145 TcAAAP069 overexpressing parasites. The TcAAAP069 overexpressing
146 parasites were available in our laboratory and were previously characterized (25).
147 Briefly, TcAAAP069 (TriTrypDB ID: TcCLB.504069.120) was amplified using
148 genomic T. cruzi DNA as template. Amplification product was subcloned into a
149 modified pTREX expression plasmid called pTREXL (pTREX-069) (29,30).
150 Constructions were transfected into T. cruzi epimastigotes of the Y strain as
151 previously described (31).
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152 Transport assays. Aliquots of epimastigote cultures (107 parasites) were
153 centrifuged at 8,000 xg for 30 s, and washed once with phosphate-buffered saline
154 (PBS). Cells were resuspended in 0.1 mL PBS and the assay started by the
155 addition of 0.1 mL of the transport mixture containing [3H]-proline (PerkinElmer's
156 NEN® Radiochemicals; 0.4 Ci) in the presence of different concentrations of the
157 indicated drug. Following incubation at 28 C, reaction was stopped by adding 1
158 mL of ice-cold PBS. Cells were centrifuged as indicated above, and washed twice
159 with ice-cold PBS. Cell pellets were resuspended in 0.2 mL of water and counted
160 for radioactivity in UltimaGold XR liquid scintillation cocktail (Packard Instrument
161 Co., Meridien CT, USA) (32,33). Cell viability was assessed by direct microscopic
162 examination. Non-specific uptake and carry over were assayed without incubation
163 (T0), or incubated at 4 ºC.
164 Trypanocidal activity assays. Epimastigotes of T. cruzi were cultured as
165 described above, in 24-wells plate at a start density of 0.5 x107 cells/mL in BHT
166 medium. Parasites´ growth was evaluated at different concentrations of BZL, CV
167 and CV structural analogues, and parasite proliferation was determined after 48 h.
168 Trypanocidal activity in trypomastigotes and amastigotes was performed using 1
169 x106 cells/mL in 96-well plates and incubating at 37 ̊C for 24 h in the presence of
170 the corresponding drug. Growth was determined by counting in a Neubauer
171 chamber or by viability assays using “Cell Titer 96® Aqueous One Solution Cell
172 Proliferation Assay (MTS)” (Promega, Madison, WI, USA) according to the
173 manufacturer’s instructions.
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174 Toxicity assays in VERO cells. The toxicity of the compounds was determined in
175 VERO cells by the CV staining assay. The cells (1 x104 cells/well) were incubated
176 in 96-well plates with the indicated compound or DMSO as negative control and
177 maintained at 37° C for 24 h. Then the cells were fixed for 15 min, and stained with
178 0.5% CV. The absorbance was measured at 570 nm. The selectivity index (SI) was
179 calculated for each CV analogue as the ratio between the IC50 obtained in
180 mammalian cells and the IC50 in trypomastigotes. For the combination therapy, the
181 results were presented as percentage of untreated cells.
182 Virtual screening. Similarity screening search was performed using the CV
183 structure as reference (query) compound (ZINC ID: 13763987). Structures (≈
184 10,000) were obtained from the highly curated “Sweetlead” database of world's
185 approved medicines, illegal drugs, and isolates from traditional medicinal herbs
186 (34). The screening was performed using the softwares LiSiCA v1.0 (35) (Ligand
187 Similarity using Clique Algorithm), KCOMBU (36) (K(ch)emical structure
188 COMparison using the BUild-up algorithm), ShaEP (37) (Molecular Overlay Based
189 on Shape and Electrostatic Potential) v 1.1.2, and Shape-it v 1.0.1 (38) (Gaussian
190 volumes as descriptor for molecular shape). For all of them different control
191 structures were used in screening searches in order to check they appear with the
192 higher score in each resulting list, validating the retrieve capabilities of the
193 programs. Then, the screening with CV was performed.
194 Docking simulations and structural alignments. The homology model of
195 TcAAAP069 was constructed using the I-TASSER-server (39) and was based
196 mainly on the structure of the Escherichia coli arginine/agmatine antiporter AdiC
197 (PDB ID: 3LRB), in addition to 9 templates of other proteins showing coverage
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198 between 0.72 and 0.95. The modeled structure had 93.7% residues in the residues
199 in favored region of Ramachandran’s plot, and none of the residues located in non-
200 allowed regions were involved in the active site of the transporter. The model was
201 refined using the software Modeller v7 (40). AutoDock 4.2.6 (41) was employed for
202 docking assays, using a grid that covers the whole transporter molecule to
203 calculate the optimal energy conformations for the ligands interacting with the
204 residues within the permease cavity. The program was run using a Lamarckian
205 Genetic Algorithm 100 times, with a population size of 300, and 2.7 x104 as
206 maximum number of generations. Structures alignments were performed using
207 LigandScout 4.1.10 (42) (Inte:Ligand, Software-Entwicklungs und Consulting
208 GmbH, Austria) whose license was kindly granted to our laboratory by the
209 company. Modeling was performed using the LigandScout default parameters.
210 Analysis of synergism. Calculations were performed using CompuSyn software
211 (http://combosyn.com) which is based on the median-effect equation (43) and its
212 extension, the combination index (44). Results are reported as Combination Index
213 (CI), where CI < 1, CI = 1 and CI > 1 indicate synergism, additive effect and
214 antagonism, respectively. LTD, CPH and CFZ were evaluated as a single drug
215 (LTD-CPH-CFZ) in combinations according to the following scheme: 1/2 IC50, 1/3
216 IC50, 1/4 IC50, 1/5 IC50, 1/10 IC50 and 1/25 IC50, where IC50 refers to the value
217 obtained for each one of the three CV analogues (25 µM, 50 µM and 10 µM of
218 LTD, CPH and CFZ, respectively for epimastigotes). 1/2 IC50 means 42.5 µM (the
219 sum of half of the IC50 value for each CV analogue, 12.5 µM + 25 µM + 5 µM, for
220 LTD, CPH and CFZ, respectively), 1/5 IC50 means 17 µM (5 µM + 10 µM + 2 µM),
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221 1/10 IC50 means 8.5 µM (2.5 µM + 5 µM + 1 µM) and 1/25 IC50 means 3.4 µM (1
222 µM + 2 µM + 0.4 µM). BZL was evaluated at the following concentrations: 10 µM, 5
223 µM, 1 µM and 0.1 µM.
224 Statistics and data analysis. IC50 values were obtained by non-linear regression
225 of dose-response logistic functions, using GraphPad Prism 6.01 for Windows, and
226 synergism was evaluated using the free software CompuSyn. All experiments were
227 performed in triplicate and the results are presented as mean ± standard deviation
228 (SD).
229
230 Results
231 Effect of CV on the proline transporter TcAAAP069. In order to test if the
232 previously reported decrease in proline transport by CV (4) is due to the specific
233 inhibition of the TcAAAP069 proline permease, the effect of CV on this transporter
234 activity was evaluated. After incubation with CV, doses that inhibit 50% of the
235 proline transport (IC50) were calculated for wild type (TcWT) and TcAAAP069
236 overexpressing (Tc069) T. cruzi epimastigotes. The inhibitory effect of CV was
237 significantly decreased in Tc069 parasites with a calculated IC50 2.5-fold higher
238 than control parasites (7.1 µM ± 0.8 and 17.7 µM ± 1.1, respectively; p<0.001) (Fig
239 1a).
240 Fig 1. Validation of crystal violet as proline transporter TcAAAP069 inhibitor.
241 (a) Inhibition of proline transport in wild type parasites (TcWT, ●) and parasites 242 overexpressing TcAAAP069 (Tc069, □). (b) Trypanocidal effect of crystal violet in TcWT 243 (●) and Tc069 (□) parasites. The data is expressed as the mean ± standard deviation and 244 corresponds to one of three independent experiments.
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245 To further demonstrate that CV specifically interacts with TcAAAP069, the
246 trypanocidal effect of CV was evaluated using the same parasite models (TcWT
247 and Tc069 cultures). The toxicity of CV significantly increased when TcAAAP069
248 was overexpressed as evidenced by a reduction in the IC50 value of 47-fold
249 compared to wild type controls (0.27 µM ± 0.06 and 12.67 µM ± 2.1, respectively;
250 p<0.0001) (Fig 1b). All these evidences demonstrate that proline transporter
251 TcAAAP069 is one of the targets of CV.
252 Similarity virtual screening. In order to identify chemical analogues of CV
253 approved for human administration, the Sweetlead database was analyzed using
254 four different algorithms that rank molecules according to their similarity (LiSICA,
255 ShaEP, KCOMBU and Shape-it). Compounds that complied with the following
256 criteria were selected: 1) being in the top five scored according to at least two
257 different algorithms; 2) have been used in humans, 3) sharing a substructure with
258 CV by 3D alignment, and 4) being easily accessible in the pharmaceutical
259 companies. Only four compounds fitted all the established conditions; these drugs
260 were loratadine (LTD; antihistamine), olanzapine (OLZ; antipsychotic),
261 cyproheptadine (CPH; antihistamine) and clofazimine (CFZ; antibiotic). Dapsone
262 (DPS; antibiotic) is an already-approved drug that shares some substructures with
263 CV, but since it was not in the top scored results it was designated as a control of
264 the selection strategy. The chemical structures of CV and its analogues are
265 presented in the Fig 2. It is worth mentioning that the result with the highest score
266 obtained with the four programs used, was the reference molecule (CV). To identify
267 the common chemical features between CV and the selected chemical analogues,
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268 the LigandScout algorithm was used in order to perform feature-based structure
269 alignments where the similarities are calculated as the number of matched feature
270 pairs (MFP; i.e. aromatic ring, hydrophobic area, hydrogen bond donor or acceptor,
271 negative or positive ionizable atom and metal binding location). The CV possesses
272 eight features that were set as references, which are four hydrophobic interactions,
273 three aromatic rings and one positive ionizable interaction. The chemical features
274 shared between CV and its chemical analogues as well as the alignments are
275 schematized in the S1 Fig. The four CV analogues, CPH, LTD, CFZ and OLZ,
276 possess between 3 and 5 MFPs out of a total of 8 present in the CV. CPH is the
277 compound that shared more chemical features with CV; including two hydrophobic
278 interactions, two aromatic rings and one positive ionizable interaction.
279 Fig 2. Chemical structures of crystal violet, its analogues and negative control.
280 The figure shows the chemical structure of crystal violet (a), the structure of the negative 281 control, dapsone, (b) and the structures of the four crystal violet structural analogues 282 loratadine, cyproheptadine, olanzapine and clofazimine (c).
283 Molecular docking of the TcAAAP069 inhibitors. The possible modes of
284 interaction between LTD, CPH, OLZ, and CFZ with the proline transporter
285 TcAAAP069 were tested by a simulation with AutoDock 4.2.6, using proline and
286 CV as a reference of experimentally identified high affinity binders and to
287 determine if the structure similarity of the selected compounds and the CV
288 translates into a similar mode of interaction with the protein. To check if, as
289 expected, there were not predicted interactions between the transporter and a
290 molecule that does not comply with the selection criteria, a simulation with DPS
291 was also performed. According to the obtained docking models, proline, CV and
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292 the four analogues bind different residues groups, located within the permease
293 channel while DPS does not appear to bind to any pocket in TcAAAP069 (Fig 3a).
294 The lowest score values (ΔG) of the predicted interaction of proline and CV with
295 the transporter are -4.07 and -5.26 kcal.mol-1, respectively. The binding scores of
296 the four CV structural analogues were lower than for proline and CV, with values
297 between -9.60 kcal.mol-1 to -5.81 kcal.mol-1. This suggests that CV and its
298 chemical analogues form a more stable bond to that channel region of TcAAAP069
299 than the natural ligand. All these data are summarized in Table 1.
300 Fig 3. Binding poses suggested by molecular docking between proline, crystal 301 violet and its chemical analogues with proline permease TcAAAP069.
302 (a) Homology modelled TcAAAP069 (represented as cartoon) highlighting the predicted 303 interaction sites of proline (PRO site, green) and crystal violet (CV site, violet; and dark 304 shadowed-violet indicates the hydrophobic “hot-spot”). The predicted binding poses of 305 proline (red), CV (violet), loratadine (blue), cyproheptadine (yellow), olanzapine (dark- 306 grey), clofazimine (green) and dapsone (pink) are shown in the figure. (b) Detail of the 307 residues predicted to interact with CV (right side) and proline (left side).
308 Table 1. Compounds obtained by similarity screening and molecular docking 309 analysis. Compound Compound ΔG all ΔG MPC Abbr ZINC ID (popular name) (brand name) (kcal.mol-1) (kcal.mol-1) CFZ Clofazimine Lamprene® 17953024 -9.60 -9.60
LTD Loratadine Alavert® 537931 -8.01 -7.25
CPH Cyproheptadine Periactin® 968264 -7.80 -7.76
OLZ Olanzapine ZyPREXA® 52957434 -5.81 -5.34
CV Crystal Violet - 13763987 -5.26 -4.54
PRO Proline - 895360 -4.07 -3.60
DPS Dapsone Aczone® 6310 -1.35 -1.08 310 All the drugs obtained by similarity-based screening, in addition to the reference 311 compound (CV) and the natural substrate of TcAAAP069 (PRO), are listed in the table. 312 Columns indicate the compound abbreviation (Abbr.), the popular name and one of the 313 brand names obtained from the website Drugs.com (https://www.drugs.com/), the ZINC
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314 database ID, the lowest binding energy (ΔG all), and the lowest binding energy of the most 315 populated cluster (ΔG MPC). Binding energy values were calculated using the AutoDock 316 software. Dash (-) instead of brand name indicates that the compound is marketed under 317 its chemical (popular) name.
318 The PRO site refers to the groove occupied by the proline pose, comprised by the
319 sequential group of residues 82-87 (GNVGSA), and the CV site refers to the
320 pocket occupied by the CV pose that includes the residues 264-265 (MM), 397-404
321 (SLIGALPA), 407 (M), and 462-474 (IMVVGTLSMVKGL) (Fig 3b). All the docked
322 poses of CV analogues are located within the CV site (S2 Fig). There is a "hot-
323 spot" rich in A, M and V residues in the CV pocket, where the hydrophobic rings of
324 LTD, CPH, CFZ, and CV appear to converge. OLZ is not near this hot-spot and it
325 binds to the V and A residues of the PRO site, as well as to the residue I-399 of the
326 CV site. As mentioned, DPS predicted poses did not occupy any site inside the
327 protein channel. All these results explain the most probable interaction between the
328 CV, CV analogues and TcAAAP069; however they were not taken as a criterion for
329 the selection of the compounds to be used in the subsequent in vitro tests.
330 Inhibition of proline transport by CV chemical analogues. The four selected
331 drugs that were structurally similar to CV and the negative control (DPS) were
332 tested as TcAAAP069 inhibitors. Preliminarily, proline transport assays were
333 performed using drug concentrations of 25 µM and 100 µM. All the CV analogues
334 produced a significant proline transport inhibition at both concentrations in
335 epimastigotes, except for OLZ which only inhibited proline transport at 100 µM (S3
336 Fig). DPS did not inhibit the proline transport in any concentration assayed. Next,
337 the concentration required to inhibit 50% of the proline transport activity (IC50) was
338 calculated for the four CV structural analogues. The IC50s obtained were 7.1 µM ±
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339 0.8, 23.2 µM ± 3.8, 71.9 µM ± 11.2 and 4.3 µM ± 0.8 for CV, LTD, CPH, and CFZ,
340 respectively (Fig 4). It was not possible to calculate an IC50 for OLZ since this
341 compound produced the death of parasites in the lapse time of the assay in
342 concentrations over 300 µM.
343 Fig 4. Calculation of CV structural analogues concentrations required to inhibit 50%
344 of proline transport activity (IC50).
345 The IC50s were calculated for three of the four CV analogues. It was not possible to
346 calculate an IC50 for OLZ since this compound produced the death of parasites in the lapse 347 time of the assay in concentrations over 300 µM. The data is expressed as the mean ± 348 standard deviation and corresponds to one of three independent experiments. CV, crystal 349 violet. LTD, loratadine. CPH, cyproheptadine. OLZ, olanzapine. CFZ, clofazimine. N/A, not 350 available.
351 Trypanocidal effect of CV structural analogues. Since the four CV analogues
352 tested showed a significant inhibition of proline transport through TcAAAP069
353 permease, all of them were also evaluated for trypanocidal activity on T. cruzi
354 epimastigotes. Although the IC50 of OLZ for proline transport inhibition could not be
355 calculated, it was included it in the trypanocidal activity assays because it produced
356 the death of parasites in the proline transport inhibition assay. Parasites were
357 incubated with different concentration of each compound during 48 h and the
358 concentration required to inhibit 50% of parasite growth (IC50) was calculated. All
359 the compounds showed trypanocidal activity on epimastigotes, being CFZ the
360 compound with the lowest IC50 (9.3 µM ± 0.6) (Table 2 and S4 Fig). Moreover, CFZ
361 presented a significantly lower IC50 than the control drug benznidazole (BZL,
362 p<0.001) while the other CV analogues, LTD, CPH and OLZ, were less effective
363 than BZL (p<0.0001 for all of them). Subsequently, the drugs were tested on
364 trypomastigotes, one of the mammalian stages of T. cruzi, and all of them proved
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365 to have trypanocidal action. CFZ was also the compound that presented the
366 highest effect (2.8 µM ± 0.1) and, together with CPH, showed an IC50 value
367 significantly lower than BZL (p<0.0001 and p<0.01, respectively) (Table 2 and S4
368 Fig). On the contrary, LTD presented a similar IC50 value than BZL while OLZ was
369 less effective (p<0.0001). In concordance with the proline transport inhibition
370 assays, OLZ was discarded for further studies due to its elevated IC50s (IC50 > 100
371 µM in epimastigotes and IC50 > 50 µM in trypomastigotes). The remaining CV
372 analogues were also evaluated on amastigotes, the intracellular mammalian stage
373 of T. cruzi, and all of them presented trypanocidal effect with similar (LTD and
374 CPH) or significantly lower IC50 values than BZL (CFZ, 1.1 µM ± 0.1, p<0.0001)
375 (Table 2 and S4 Fig). Finally the toxicity of LTD, CPH and CFZ was assessed in
376 VERO cells in order to calculate the selectivity index (SI) and all the CV analogues
377 assayed were more selective to parasite cells with SI values between 5.5 and 36.1
378 (Table 2).
379 Table 2. Trypanocidal effect of CV chemical analogues in T. cruzi Y strain. Selectivity Compound Epimastigotes Trypomastigotes Amastigotes Index
Benznidazole 20.9 µM ± 1.4 14.9 µM ± 1.5 11.4 µM ± 0.5 -
Crystal Violet 12.7 µM ± 2.1 0.4 µM ± 0.0 0.3 µM ± 0.1 -
Loratadine 26.4 µM ± 1.0 12.9 µM ± 0.8 13.2 µM ± 2.4 5.5
Cyproheptadine 52.6 µM ± 2.9 11.3 µM ± 0.6 10.7 µM ± 1.3 9.1
Olanzapine 120.5 µM ± 11.4 54.1 µM ± 2.9 N/A N/A
Clofazimine 9.3 µM ± 0.6 2.8 µM ± 0.09 1.1 µM ± 0.1 36.1 380 N/A: Data not available
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381 Altogether, the results indicate that CFZ is the only CV analogue tested that
382 presents lower IC50 values than BZL in all the T. cruzi stages assayed, while LTD
383 and CPH have trypanocidal activity against amastigotes and trypomastigotes with
384 at least similar effect to BZL.
385 Trypanocidal effect of CV analogues on other strains. The results presented so
386 far were obtained from T. cruzi parasites from Y strain (discrete typing unit –DTU–
387 TcII). The trypanocidal action of LTD, CPH and CFZ was also evaluated on
388 epimastigotes of two other DTU strains, such as Dm28c (DTU TcI) and CL Brener
389 (DTU TcVI) since variations in trypanocidal treatment response have been reported
390 for different T. cruzi strains (45). BZL and CV were assayed as well in order to
391 compare the trypanocidal effect between strains. The Y parasites were 1.6- and
392 1.3-fold more sensitive to BZL treatment than Dm28c and CL Brener
393 epimastigotes, respectively (p<0.0001, Table 3). Dm28c was 1.2-fold more
394 resistant to BZL than CL Brener (p<0.01). On the contrary, Dm28c and CL Brener
395 parasites presented similar response to CV treatment and were 31.8- and 4.0-fold,
396 respectively, more sensitive to this compound than Y epimastigotes (p<0.0001). In
397 addition, the results indicate that the three CV chemical analogues are effective in
398 other T. cruzi strains, including DTUs of clinical relevance (46) (Table 3 and S5
399 Fig). Dm28c parasites were significantly more sensitive to LTD and CPH than the
400 epimastigotes of Y (p<0.01 and p<0.0001, respectively) and CL Brener strains
401 (p<0.0001, for both drugs). However, no differences between strains were
402 observed in the treatment with CFZ.
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403 Table 3. Trypanocidal effect of CV structural analogues in epimastigotes from 404 different strains of T. cruzi.
T. cruzi strain Compound Y Dm28c CL Brener
Benznidazole 20.9 µM ± 1.4 32.5 µM ± 1.9 27.8 µM ± 1.8
Crystal Violet 12.7 µM ± 2.1 0.4 µM ± 0.0 3.2 µM ± 1.1
Loratadine 26.4 µM ± 1.0 21.0 µM ± 1.2 28.6 µM ± 2.0
Cyproheptadine 52.6 µM ± 2.9 37.6 µM ± 0.7 50.6 µM ± 2.2
Olanzapine 120.5 µM ± 11.4 N/A N/A
Clofazimine 9.3 µM ± 0.6 7.5 µM ± 0.8 9.0 µM ± 0.5 405 N/A: Data not available
406 Synergism between CV structural analogues and benznidazole. The
407 synergistic action between BZL, LTD, CPH, CFZ and a combination of these three
408 CV analogues (LTD-CPH-CFZ) was evaluated in T. cruzi. Drug interactions were
409 classified according to the combination index (CI) recognized as the standard
410 measure of combination effect based on the Chou-Talalay method (44). CI < 1
411 means synergism, CI = 1 indicates additive effect, and CI > 1 is interpreted as
412 antagonism. The IC50 calculated for BZL treatment alone was 18.57 µM ± 1.07 and
413 for the LTD-CPH-CFZ treatment was 18.05 µM ± 0.96. While each compound by
414 itself did not enhance the BZL trypanocidal activity (data not shown), the combined
415 administration of LTD-CPH-CFZ and BZL produced a synergistic effect. All
416 combinations between BZL and LTD-CPH-CFZ presented a CI < 1 with different
417 synergism degree, from strong to slight synergism (Fig 4a). The exceptions were
418 the combination of 1/2 IC50 LTD-CPH-CFZ with 0.1 and 1 µM BZL, which
419 presented a nearly additive effect and moderate antagonism, respectively. The
420 Chou-Talalay plot shows the CI and the effect (Fa, fraction affected) produced by
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421 each combination point, where 0 means no effect and 1.0 means maximum
422 trypanocidal effect (Fig 4b). Dose-reduction index (DRI) represents the dose
423 reduction that is allowed in combination for a given degree of effect as compared
424 with the dose of each drug alone. DRI > 1 indicates favorable dose-reduction.
425 Except for the two combinations that did not present a synergistic effect, the DRI
426 values were higher than 1 for all the combinations assayed with values between
427 2.5 and 374.2 for BZL and between 1.2 and 10.4 for LTD-CPH-CFZ (S1 Table).
428 Altogether, these results suggest that combination therapy improves the
429 trypanocidal action of the CV analogues together with BZL.
430 Fig 5. Synergism between benznidazole and the combination of the crystal violet 431 analogues in T. cruzi epimastigotes.
432 (a) Drug combinations between BZL and CV analogues (LTD-CPH-CFZ). Combination 433 index (CI) value for each combination point is presented under the corresponding graded 434 symbol. Graded symbols mean strong synergism (++++, CI between 0.1-0.3), synergism 435 (+++, CI between 0.3-0.7), moderate synergism (++, CI between 0.7-0.85), nearly additive 436 effect (±, CI between 0.9-1.1), and moderate antagonism (- -, CI between 1.2-1.45) (47). 437 The boxes coloured with light-grey correspond to the combination points where no 438 synergism was observed. (b) Chou-Talalay plot. Representation of CI vs effect (Fa, 439 fraction affected), where CI > 1, CI = 1 (dotted line) and CI < 1 indicate antagonism, 440 additive effect and synergism, respectively. For each data series BZL concentrations 441 increase from left to right (from 0.1 to 20 µM). The data is expressed as the mean ± 442 standard deviation and corresponds to one of three independent experiments. All 443 calculations were performed with CompuSyn software. LTD, loratadine. CPH, 444 cyproheptadine. CFZ, clofazimine. BZL, benznidazole. IC50 LTD = 25 µM. IC50 CPH = 50 445 µM. IC50 CFZ = 10 µM. LTD-CPH-CFZ, combination of the three crystal violet analogues 446 as a single drug. 1/2 IC50, refers to the sum of half of each IC50, 12.5 µM + 25 µM + 5 µM 447 = 42.5 µM. 1/5 IC50, 5 µM + 10 µM + 2 µM = 17 µM. 1/10 IC50, 2.5 µM + 5 µM + 1 µM = 448 8.5 µM. 1/25 IC50, 1 µM + 2 µM + 0.4 µM = 3.4 µM.
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449
450 Discussion
451 New alternatives to current treatments against Chagas disease have been
452 explored in the last decade, including new trypanocidal drugs, such as the
453 posaconazole, and the use of BZL in the Chagas cardiomyopathy (BENEFIT trial)
454 (48–51). Unfortunately none of them were successful, and there is still an urgent
455 need for the development of new therapeutic substitutes for conventional
456 treatments. Drug repurposing or repositioning consist of finding new indications for
457 drugs already approved for its use in humans. This approach is a recommended
458 strategy by the World Health Organization (WHO) to fight neglected diseases, like
459 Chagas disease, since costs and development time are significantly reduced for its
460 application in therapy.
461 There are some recent examples of drug repositioning by virtual screening that use
462 the T. cruzi polyamine transporter TcPAT12 as main target; triclabendazole, an
463 antiparasitic, sertaconazole, a broad-spectrum antifungal, paroxetine, an
464 antidepressant agent, isotretinoin, a well-known medication used to treat severe
465 acne, and cisapride, a gastroprokinetic agent. All these compounds proved to be
466 effective as TcPAT12 inhibitors that also have trypanocidal action (52–54).
467 Regarding proline transport as therapeutic target, to our knowledge there are no
468 previous reports of drug repositioning although some proline analogues have been
469 studied as trypanocidal compounds. For example, the proline analogue L-
470 thiazolidine-4-carboxylic acid (T4C) has been reported as a proline transporter
471 inhibitor with trypanocidal effect (55), and more recently, another proline analogue
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472 synthetized by rational design and called ITP-1G has been validated as a
473 TcAAAP069 inhibitor that also possesses trypanocidal activity (25).
474 Other compounds have been identified by drug repositioning as possible
475 trypanocidal drugs using different targets; such as the cruzipain (56–59), some T.
476 cruzi kinases (60–62) and the trans-sialidase (63), among other examples. Other
477 drug repurposing approaches have inferred the possible trypanocidal activity of
478 already-approved drugs based on the reported effect or mechanism of action of
479 some compounds in other organisms (64–69).
480 In this work, a combined strategy involving similarity-based virtual screening
481 followed by in vitro assays was applied in order to obtain new trypanocidal
482 compounds from a database that contains already-approved drugs. CV was
483 selected as a reference molecule since it has a reported trypanocidal action and
484 one of its mechanisms of action involved proline transport inhibition (4). The aim
485 was to obtain structurally related compounds which may have similar biological
486 activity but with no (or less) toxicity toward the host cells since they are already in
487 use to treat human diseases. The proline transporter TcAAAP069 was selected as
488 the probable main target because: a) proline is a relevant amino acid for T. cruzi
489 biology, including host cell invasion and life cycle progression (16,17), b)
490 TcAAAP069 mediates proline uptake and it can also be used as a way of entry
491 drugs into the parasite and, c) there is evidence of compounds that inhibit
492 TcAAAP069 and also have trypanocidal activity, like the proline analogues T4C
493 and ITP-1G (25,55).
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494 Once CV was validated as a TcAAAP069 inhibitor, the similarity-based virtual
495 screening was performed and four drugs already-approved for clinical use were
496 obtained for further analysis. All these CV chemical analogues retrieved by the
497 virtual screening have already been tested as antiparisitic agents in protozoans.
498 LTD was evaluated in Leishmania (L.) infantum as an antileishmanial compound
499 and even though it presented an intermediate effect on promastigotes it did not
500 have effect on intracellular amastigotes (70). CPH was assayed in T. evansi in
501 order to evaluate the ability of this compound to reverse drug resistance to some
502 trypanocidal drugs, like suramin, and the initial experiments showed the intrinsic
503 toxic effect of this compound (71). OLZ was tested in Toxoplasma gondii due to the
504 strong link demonstrated between toxoplasmosis and psychiatric disorders but it
505 presented a low anti-toxoplasmic activity (72). Finally, CFZ was recently identified
506 as a cruzipain inhibitor in T. cruzi with trypanocidal action, but it was also reported
507 that cruzipain is not the only target for this compound (73).
508 LTD, CPH and CFZ were all able to inhibit proline transport and also proved to
509 have trypanocidal effect in epimastigotes, trypomastigotes and amastigotes of the
510 parasite T. cruzi. The IC50s obtained for these CV analogues were in the range of
511 those reported with BZL in epimastigotes for strains from different DTUs (IC50
512 values between 7-30 µM) (45). Moreover, the trypanocidal effect of CFZ was
513 significantly higher than the observed with BZL in trypomastigotes and amastigotes
514 while the other two CV analogues showed at least similar effect to the BZL. In
515 contrast, OLZ presented an elevated IC50 both for epimastigotes and
516 trypomastigotes, and it was discarded for further analysis. The molecular docking
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517 predicted two hydrophobic sites where CV and proline could stably bind to the
518 transporter TcAAAP069 and, interestingly, all the CV analogues clustered inside
519 the TcAAAP069 channel, mainly in the CV site as expected for structural
520 analogues. These results could explain the observed inhibition of proline transport
521 by the CV structural analogues.
522 The fact that LTD, CPH and CFZ were also effective as trypanocidal compounds
523 on epimastigotes of CL Brener and Dm28c strains is interesting because not all T.
524 cruzi strains present the same response to current treatments (45), and even drug
525 resistance has been strain-associated (74). Indeed there were differences on the
526 treatment response to BZL and CV between the three strains evaluated in this
527 work. The response observed to CV was particularly striking, with IC50 values 4.0-
528 and 31.8-fold lower for CL Brener and Dm28c epimastigotes, respectively, than the
529 IC50 obtained for the Y strain. These results indicate the importance of evaluate
530 trypanocidal compounds in several strains in order to prevent different and
531 unexpected responses. Regarding the CV analogues, CFZ presented the same
532 effect on the three strains evaluated, while LTD and CPH proved to be significantly
533 more effective on Dm28c strain.
534 The combination of two or more drugs with different mechanisms of action is an
535 alternative approach to increase the success rate of drug repositioning and,
536 although multidrug therapy may be difficult to implement, combination of drugs has
537 been used to treat other neglected diseases, for example the leprosy multidrug
538 treatment recommended and supplied by the WHO involves rifampicin, clofazimine
539 and dapsone (MDT-COMBI®). The combination of already-approved drugs
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540 enables dosage reduction and diminishes toxicity, in addition to an improved
541 efficacy and reduced drug resistance emergence. The final experiment herein
542 presented showed positive interactions between the well-known drugs LTD, CPH
543 and CFZ, and BZL in epimastigotes of T. cruzi. Most of the combination points of
544 BZL with LTD-CPH-CFZ presented a synergistic effect, with CI values from 0.27 to
545 0.85 and DRI values from 2.5 to 374.2 for BZL and between 1.2 and 10.4 for LTD-
546 CPH-CFZ. For example, the combination of 1 µM BZL with 1/5 IC50 LTD-CPH-CFZ
547 (17 µM, CI = 0.70) produced an effect of 74% with 54-fold less BZL and 1.5-fold
548 less LTD-CPH-CFZ than each drug alone needed to achieve the same effect.
549 These results suggest that the combination therapy would make possible to
550 diminish the dosage of BZL and thus, the toxicity and side effects reported (6).
551 However, future studies will need to focus on the effect of the combination therapy
552 between BZL and the CV structural analogues in trypomastigotes and in infected
553 cells.
554 The final goal would be to identify new treatments based on drug repositioning
555 and/or combination therapy that are more effective, better tolerated, and simpler to
556 administrate than current therapies for treating Chagas disease. Taking into
557 account the urgent need for the development of new drugs for Chagas disease, we
558 can conclude that CV chemical analogues could be a starting point to design
559 therapeutic alternatives either alone or in combination with BZL to treat this
560 neglected disease.
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561 Acknowledgments
562 Special thanks to Lic. Fabio di Girolamo (IDIM, UBA-CONICET), Dr. Patricia
563 Bustos and Dr. Alina Perrone (Instituto Nacional de Parasitología "Dr. Mario Fatala
564 Chaben") for technical support, and to Dr. Viviana Amparo Ybarra and Dr.
565 Guillermo Buiyi Alonso (INGEBI-CONICET) for their collaboration. Also we would
566 like to thank Thomas Lemker from "Inte:Ligand Software Development &
567 Consulting" for his help with the license of the LigandScout and to Richmond
568 Laboratories for olanzapine supply. CAP and MRM are members of the career of
569 scientific investigator; MS, EVV and CR are research fellows from CONICET, LG is
570 a undergraduate student fellow
571
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818
819
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820 Supporting Information
821
822 S1 Fig. Chemical features shared between crystal violet and the selected 823 compounds.
824 The LigandScout software was used to identify the common chemical features between 825 crystal violet (CV) and its chemical analogues. This algorithm performs feature-based 826 structure alignments where the similarities are calculated as the number of matched 827 feature pairs (MFP). (a) In order to perform the structure comparisons, the 8 CV features 828 were set as references. (b) The LigandScout similarity score and the number of MFP 829 obtained for each compound are listed. (c) The features shared between CV and each 830 compound are shown and the structure alignments with the van der Waals surfaces are 831 schematized. N/A, not available. AR, aromatic ring (purple circles). H, hydrophobic area 832 (yellow remarks). PI, positive ionizable atom (purple lines).
833 S2 Fig. Predicted poses by molecular docking of the crystal violet structural 834 analogues and the proline permease TcAAAP069.
835 Residues corresponding to the PRO and CV sites in TcAAAP069 are indicated in green 836 and violet, respectively. Detail of the TcAAAP069 residues predicted to interact with (a) 837 clofazimine, (b) loratadine, (c) cyproheptadine and (d) olanzapine.
838 S3 Fig. Inhibition of proline transport by crystal violet chemical analogues in wild 839 type parasites (TcWT).
840 The crystal violet analogues and dapsone were evaluated as potential proline transport 841 inhibitors at two concentrations, 25 and 100 µM. Control, no treatment. The data is 842 expressed as the mean ± standard deviation and corresponds to one of three independent 843 experiments. DPS, dapsone. LTD, loratadine. CPH, cyproheptadine. OLZ, olanzapine. 844 CFZ, clofazimine. *, p< 0.05; ***, p< 0.001; ****, p<0.0001. ns, not significant.
845 S4 Fig. Trypanocidal effect of CV structural analogues in (a) epimastigotes, (b) 846 trypomastigotes and (c) amastigotes of T. cruzi Y strain.
847 The concentrations required to inhibit 50% of parasite growth or parasite survival were 848 calculated for the four CV chemical analogues. The data is expressed as the mean ± 849 standard deviation and corresponds to one of three independent experiments. BZL, 850 benznidazole. CV, crystal violet. LTD, loratadine. CPH, cyproheptadine. OLZ, olanzapine. 851 CFZ, clofazimine. N/A, not available.
852 S5 Fig. Trypanocidal effect of CV structural analogues concentrations in 853 epimastigotes of (a) T. cruzi DM28c and (b) CL Brener strains.
854 The concentrations required to inhibit 50% of parasite growth were calculated for three CV
855 chemical analogues. OLZ was not tested in these strains because of the high IC50s values 856 obtained in trypomastigotes and epimastigotes of the Y strain. The data is expressed as 857 the mean ± standard deviation and corresponds to one of three independent experiments.
33 bioRxiv preprint doi: https://doi.org/10.1101/645333; this version posted May 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
858 BZL, benznidazole. CV, crystal violet. LTD, loratadine. CPH, cyproheptadine. OLZ, 859 olanzapine. CFZ, clofazimine. N/A, not available.
860 S1 Table. Dose-reduction index for drug combination therapy with benznidazole and 861 crystal violet chemical analogues in wild type epimastigotes of T. cruzi Y strain.
34 bioRxiv preprint doi: https://doi.org/10.1101/645333; this version posted May 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/645333; this version posted May 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/645333; this version posted May 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/645333; this version posted May 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/645333; this version posted May 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.