Elsevier Editorial System(tm) for European Journal of Medicinal Chemistry Manuscript Draft Manuscript Number: EJMECH-D-19-02104R2 Title: Identification of a 2,4-diaminopyrimidine scaffold targeting Trypanosoma brucei pteridine reductase 1 from the LIBRA compound library screening campaign. Article Type: Full Paper Keywords: LIBRA compound library high throughput screening anti-parasitic drug discovery pteridine reductase 1 Corresponding Author: Professor Maria Paola Costi, PhD Corresponding Author's Institution: University of Modena and Reggio Emilia First Author: Pasquale Linciano Order of Authors: Pasquale Linciano; Gregorio Cullia; Chiara Borsari; Matteo Santucci; Stefania Ferrari; Gesa Witt; Sheraz Gul; Maria Kuzikov; Bernhard Ellinger; Nuno Santarém; Anabela C da Silva; Paola Conti; Maria Laura Bolognesi; Marinella Roberti; Federica Prati; Francesca Bartoccini; Michele Retini; Giovanni Piersanti; Andrea Cavalli; Luca Goldoni; Sine Mandrup Bertozzi; Fabio Bertozzi; Enzo Brambilla; Vincenzo Rizzo; Daniele Piomelli; Andrea Pinto; Tiziano Bandiera; Maria Paola Costi, PhD Abstract: The LIBRA compound library is a collection of 522 noncommercial molecules contributed by various Italian academic laboratories. These compounds have been designed and synthesized during different medicinal chemistry programs and are hosted by the Italian Institute of Technology. We report the screening of the LIBRA compound library against Trypanosoma brucei and Leishmania major pteridine reductase 1, TbPTR1 and LmPTR1. Nine compounds were active against parasitic PTR1 and were selected for cell-based parasite screening, as single agents and in combination with methotrexate (MTX). The most interesting TbPTR1 inhibitor identified was 4-(benzyloxy)pyrimidine-2,6diamine (LIB_66). Subsequently, six new LIB_66 derivatives were synthesized to explore its Structure-Activity-Relationship (SAR) and absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. The results indicate that PTR1 has a preference to bind inhibitors, which resemble its biopterin/folic acid substrates, such as the 2,4-diaminopyrimidine derivatives. *Cover Letter Modena 31/12/2019 Dear Editor, thank you for reviewing our manuscript “Identification of a 2,4-diaminopyrimidine scaffold targeting Trypanosoma brucei pteridine reductase 1 from the LIBRA compound library screening campaign” submitted for publication in European Journal of Medicinal Chemistry. We thank the Reviewer for the useful comments, which allowed us to improve the paper quality. We have revised our manuscript according to the indications, as described in detail in the “Response to Reviewer letter”. We hope the manuscript is now suitable for publication in your esteemed journal. I take the opportunity to wish happy new year 2020. Maria Paola Costi Prof. Maria Paola Costi Drug Discovery and Biotechnology Lab Department of Life Science University of Modena and Reggio Emilia Via Campi 103, 41125, Modena, Italy Office: +390592058579 Mobile: +393396559131 Skype: costimp *Response to Reviewers Review Journal European Journal of Medicinal Chemistry Manuscript Number EJMECH-D-19-02104 Title Identification of a 2,4-diaminopyrimidine scaffold targeting Trypanosoma brucei pteridine reductase 1 from the LIBRA com-pound library screening campaign. Reviewer’s Comments The enzymes pteridine reductase 1 of Trypanosoma brucei and Leishmania are the centre of this research, for discovering anti-parasitic compounds. The authors have designed and synthesized several sets of compounds of the diamino pyrimidine class. These compounds have been tested for anti-parasitic activity against Trypanosome brucei and Leishmania. In this review only the biological and pharmacological research is addressed. A. We have not designed and synthesized different sets of compounds. Instead, we screened first a library of about 500 compounds, fully characterized. The screening led to the identification of a few hits. Then, on the basis of the identified compounds, we synthesized a few new compounds structurally related to the identified hits. For Authors 1) The introduction refers to drug resistance of the parasites against inhibitors of dehydrofolate reductase (DHFR inhibitors), and notes that the alternative salvage pathway with pteridine reductase would be the cause for this. There are several causes for drug resistance, and this is only one reason (alternative pathways). This raises several questions about the approach as such. A. We thank the reviewer for the suggestion. The function of PTR1 as alternative pathway in folate metabolism as by-pass mechanism for drug resistance has been described better and the following paragraph (and related references) has been added: “Moreover, although PTR1 is mainly involved in the reduction of biopterin to H4B, it is constitutively involved in the reduction of the 10% of folic acid to tetrahydrofolate required by the parasitic cell, overlapping with the activity of DHFR [16,18]. Thus, when parasitic DHFR is inhibited by antifolate drugs, the expression of PTR1 is increased providing the reduced folate necessary for parasite survival, providing a metabolic bypass to alleviate DHFR inhibition and contributing to treatment failure Page 1 of 5 with antifolate drugs [20–23]. Therefore, targeting PTR1, in combination with a classical antifolate drug, is considered a promising strategy for the development of improved therapies [24,25]. Indeed, the combination of PTR1 inhibitors with MTX resulted in a synergic antiparasitic efficacy [26–30].” 2) A diagram of the biosynthetic pathway showing the de novo and salvage pathways for biopterin and folic acid biosynthesis is recommended, with suitable introductions, discussion, references and reviews. A. We thank the reviewer for the suggestion. We introduced in the introduction of the paper a figure describing the enzymatic and metabolic pathway of biopterin and folic acid in T. brucei and L. major, discussing in detail the role and function of PTR1 for parasite survival. The references have been adequately reviewed, uploaded and discussed. 3) The enzymes pteridine reductase 1 of Trypanosoma brucei and Leishmania are the centre of this research, for discovering anti-parasitic compounds. a) These recombinant enzymes have not been adequately described in this manuscript, and there is insufficient reference to the research on these recombinant enzymes in the literature references. A. We thank the reviewer for the suggestion. A brief and referenced description of the structure of PTR1 has been added. b) The enzyme assays have not been adequately described in this manuscript, and there is insufficient reference to these assays in the literature references. A. We thank the reviewer for the suggestion. The protocol adopted for the enzymatic assays was the one developed for the first time by Shanks et al. and already adopted with success in our previous work. The reference to Shanks et al. has been added to the manuscript and the related references revised. 4) The essentiality of the genes in these pathways in cell culture and the animal model of infection needs to be referred to, if this has been researched elsewhere with genetic methods of gene knock-out, gene knock-down or RNA interference. A. We thank the reviewer for the suggestion. We introduced the following paragraph in the main text, supported by the appropriate references. “…there is the evidence that PTR1-null mutants are unable to grow in culture unless supplemented with H2B or H4B [14]. In contrast to the leishmania parasites, knockdown of PTR1 levels in T. brucei by RNA interference is lethal in vitro and cannot be rescued by supplementation with either H2B or H4B [18], unlike L. major PTR1-null mutants[16]. In addition, PTR1 knockdown abolishes infectivity of T. brucei to mice [17]. These results provide convincing evidence that PTR1 is an essential drug target in for the treatment of T. brucei infections”. 5) Enzyme Inhibition; a) the Ki value must be determined. The IC50 value alone is insufficient. Page 2 of 5 A. The Ki values for compounds profiled against TbPTR1 and LmPTR1 have been determined. The manuscript has been updated to reflect these changes. b) the mechanism of inhibition must be determined. A. From the kinetic experiments results, and observing the x-ray crystal structure of AX5 with TbPTR1 and NADP(H), we may conclude that the pyrimidine analogs show a competitive inhibition pattern with respect to the dihydrobiopterine substrate (22a). A similar conclusion is drawn about MTX inhibition. MTX binds in a very similar way into the biopterin active site (22). Therefore Ki (inhibition constant) were obtained from the known equation (22a) 6) The potency of the test compounds in parasite cell culture should be below 1 microMolar. A. We are not sure we understood the meaning of this comment. If the Reviewer means that only compounds showing IC50/EC50 below 1 microMolar can be progressed to further drug discovery studies, we agree on this. However, our study is focused on the early steps of the drug discovery process. We think that the cut-off suggested by the Reviewer is more applicable to lead compounds rather than to hits. Our study is positioned in the early discovery phase, where inhibitors found in the HTS phase are then progressed to a first confirmation step. To this end, the hit was re-synthesized and a few close derivatives were prepared to increase the confidence on compound LIB_066 as a bona fide hit. 7) The mechanism of action of the test compounds within the parasite cell should be elucidated and proven. Which pathway and enzymes are these compounds inhibiting? A. We think that, at this stage of the research, the characterization of the mechanism of action of our confirmed hits is not an essential requirement, because, as stated in the previous point (point 6), we are presenting an early stage drug discovery study that has the aim to identify and confirm the new hits. If we will be able to obtain more active compounds on the basis of the newly identified hits, we will characterize the mechanism of action of the new inhibitors. 8) Drug synergy is measured in a different and more comprehensive way. The level of drug synergy seen here is low or moderate. A. We think that, at this stage of the research, the extensive characterization of the drug synergy of our hits is not an essential requirement, because, as stated in the previous points (point 6, 7), we are presenting an early stage discovery study that has the aim to identify and confirm the new hits. If we will be able to develop more active compounds on the basis of the newly identified hits, we will undertake a comprehensive drug synergy study. Page 3 of 5 9) The cyto-toxicity of the test compounds should be above 500 microMolar (or 250 microMolar), and verified in at least five different mammalian cell lines. A. At this stage of the research, we opted for a preliminary characterization of the toxicity in two cell lines, considering that we are dealing with initial hits. Chemical modifications of the hits could affect toxicity. Therefore, we plan to carry out a more extensive characterization once we will obtain new and more potent compounds. 10) Cell based screening of test compounds against Trypanosomes should cover all species and subspecies endemic in the African continent (geographic region). A. We agree with the Reviewer that it will be necessary to extend the Trypanosomes panel evaluation and this will be done in the future, when we will be able to obtain new and more potent compounds . 11) Cell based screening of test compounds against Leishmania should cover all species and subspecies endemic in the southern and western African continent (geographic region). A. We agree with the Reviewer that it will be necessary to extend the Leishmania panel evaluation and this will be done in the future, when we will be able to obtain new and more potent compounds. 12) Structure-based design with x-ray protein crystallographic studies should be done and should not be replaced by “docking studies”. A. We agree with the Reviewer’s suggestions in terms of a general approach to drug discovery, when the x-ray crystal structure or a 3D structure of the target is available. However, we cannot disregard all the advances made in the field of computational studies in which important achievements have been reached in the prediction capacity of docking software, nor we can disregard the fact that many important experiments are costly and time-consuming, in particular in a field where funding is very limited. Our work represents a favorable situation in which the structural similarity between the existing x-ray structure of the complex target-inhibitor-cofactor (PTR1-AX5-NADP(H)) and docking models, where the inhibitor is replaced by our compounds, is very high. Therefore, we opted for a docking study and did not embark in performing costly crystallographic experiments. We wrote in the manuscript: “compound LIB_66 is chemically comparable to some PTR1 inhibitors based on 2,4-diaminopyrimidine scaffold, discovered by Tulloch et al… When compared to the previously published compound 4-(benzylthio)-2,6-diaminopyrimidine (AX5 in Figure 5), LIB_66 differs solely for the oxygen atom in place of the sulphur atom in the linker between the pyrimidine and the side benzyl ring [23]”. The presence of AX5 active PTR1 inhibitor validates both the assay and the selection procedure. Therefore, in presence of a ternary complex TbPTR1-NADPH with a ligand chemically comparable to our hit, we did not proceed with crystallographic studies and preferred to use an alternative and at the same time valuable method, such as docking calculation, to obtain indications on the binding mode of our hit compound. Page 4 of 5 13) The literature references should include the “WHO - fact sheets” for Leishmaniasis and African Trypanosomiasis and text books for clinical medicine for tropical diseases with reference to the chapters on Leishmaniasis and African Trypanosomiasis. A. We thank the reviewer for the suggestions, and we revised the references as suggested. 14) As a word of caution, diamino pyrimidines are not without toxicity issues and cannot be used as medicines for expecting mothers, nursing mothers and children younger than five yrs. A. The suggestion of the reviewer is very important. As stated above, initial toxicity issues will be tackled during the hit to lead and lead optimization phase. More comprehensive toxicity studies, including teratogenicity, are typically performed at the preclinical/early clinical development phase. It has to be considered that the compounds described in our paper are just the starting point of a drug discovery project. In the following steps, i.e. hit to lead and lead optimization, modification of the compounds could lead to molecules with significant structural differences with respect to the hits. This could result in a different toxicity profile of the compounds. We are aware that an initial evaluation of potential toxicities should be part of the selection process of the chemotypes to be chemically expanded in a drug discovery process. In this paper, we only present the data obtained in a HTS of a very small library and the initial follow up of one of the hits by the synthesis of a few analogues. Additional work has to be done before embarking in a full drug discovery project, e.g. expanding the initial SAR of the class. A more extensive characterization of the potential toxicities of the new hit analogues will be part of this work. 15) With the knowledge of the enzyme structure and reaction mechanisms, it is usually possible to discover and design a new class of inhibitors. A. We certainly share the Reviewer’s opinion. Looking for new class of PTR1 inhibitors is one of the main scopes of our research group and platform, and indeed we published in the recent period, and we are going to publish the identification of new chemical scaffolds applying the target-based approach. In parallel, we performed a screening on an already available library of synthetic compounds, which is the scope of this paper, in order to possibly repurpose these molecules on parasitic PTR1 target or to fish out new scaffolds that could be optimize in the future as new antiparasitic drugs. In conclusion, we hope that the revised manuscript will be acceptable for publication in the European Journal of Medicinal Chemistry. Yours sincerely, Maria Paola Costi Page 5 of 5 Tb and LmPTR1 High-throughput Screening 10 HTS Hits In vitro assays Docking simulation Hit validation through new compounds synthesis LIBRA Graphical Abstract (for review) 522 library compounds Click here to download Graphical Abstract (for re PRIVILEGED SCAFFOLD 4-(benzyloxy/benzylamino) -2,6-diaminopyrimidine TbPTR1 IC50 = 0.7 – 4.0 mM Highlights (for review) Highlights • LIBRA is a 522 compounds library, highly characterized. • Compound LB_66 has a 2,4-pyrimidine scaffold emerging as preferred selected HIT with selective inhibition against TbPTR1. • 6 new compounds bearing 2,4-pyrimidine scaffold are confirmed HITS with 1.32.3 potentiation index (P.I.) in combination with methotrexate against Trypanosoma brucei. • PTR1 has a preference to bind inhibitors with 2,4-diaminopyrimidine scaffolds that resemble its biopterin/folic acid substrates. *Revised Manuscript Click here to view linked References Identification of a 2,4-diaminopyrimidine scaffold targeting Trypanosoma brucei pteridine reductase 1 from the LIBRA compound library screening campaign. Pasquale Linciano,1 Gregorio Cullia,2 Chiara Borsari,1† Matteo Santucci,1 Stefania Ferrari,1 Gesa Witt,3 Sheraz Gul,3 Maria Kuzikov,3 Bernhard Ellinger,3 Nuno Santarém,4 Anabela Cordeiro da Silva,4,12 Paola Conti,2 Maria Laura Bolognesi,5 Marinella Roberti,5 Federica Prati,5 Francesca Bartoccini,7 Michele Retini,6 Giovanni Piersanti,6 Andrea Cavalli,5,7 Luca Goldoni,8 Sine Mandrup Bertozzi,8 Fabio Bertozzi,9 Enzo Brambilla,9 Vincenzo Rizzo,9 Daniele Piomelli,10 Andrea Pinto,11,* Tiziano Bandiera9,* and Maria Paola Costi1,* 1 Dipartimento di Scienze della Vita, University of Modena and Reggio Emilia, Via Campi 103, 41125, Modena, Italy. 2 Department of Pharmaceutical Sciences, University of Milan, Via Mangiagalli 25, 20133, Milan, Italy. Fraunhofer Institute for Molecular Biology and Applied Ecology – ScreeningPort, Hamburg, Germany. 4 em Saúde, Universidade do Porto, 4150-180, Porto, Portugal. 5 Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy. 6 Department of Biomolecular Sciences, Section of Chemistry, University of Urbino "Carlo Bo", Piazza Rinascimento 6, 61029 Urbino, Italy. 7 Computational and Chemical Biology, Istituto Italiano di Tecnologia, Via Morego 30, I16163 Genova, Italy. 8 Analytical Chemistry Facility, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy 9 PharmaChemistry Line, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy. 10 Departments of Anatomy and Neurobiology, Pharmacology and Biological Chemistry, University of California, Irvine 92697-4625, USA. 11 Department of Food, Environmental and Nutritional Sciences, University of Milan, Via Celoria 2, 20133 Milan, Italy. 12 Department of Biological Sciences, Faculty of Pharmacy, University of Porto. 3 KEYWORDS LIBRA compound library, high throughput screening, anti-parasitic drug discovery, pteridine reductase 1. 1 ABSTRACT The LIBRA compound library is a collection of 522 non-commercial molecules contributed by various Italian academic laboratories. These compounds have been designed and synthesized during different medicinal chemistry programs and are hosted by the Italian Institute of Technology. We report the screening of the LIBRA compound library against Trypanosoma brucei and Leishmania major pteridine reductase 1, TbPTR1 and LmPTR1. Nine compounds were active against parasitic PTR1 and were selected for cell-based parasite screening, as single agents and in combination with methotrexate (MTX). The most interesting TbPTR1 inhibitor identified was 4-(benzyloxy)pyrimidine-2,6-diamine (LIB_66). Subsequently, six new LIB_66 derivatives were synthesized to explore its Structure-ActivityRelationship (SAR) and absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. The results indicate that PTR1 has a preference to bind inhibitors, which resemble its biopterin/folic acid substrates, such as the 2,4-diaminopyrimidine derivatives. 2 1. Introduction Screening in drug discovery allows the rapid testing of compound libraries against biological targets implicated in disease processes to identify hit compounds for further medicinal chemistry optimization in the drug discovery value chain [1]. Several drugs currently in clinical trials originate from hits identified during high throughput screening (HTS) campaigns [2,3]. HTS using target-based assays has been extensively used in the drug discovery process for neglected tropical diseases (NTD) [3]. These diseases mainly affect marginalized and economically disadvantaged communities; in addition, they are the major causes of morbidity and mortality in tropical and subtropical regions [4]. Parasites of the Trypanosomatidae family are the agents of human and animal vector-borne diseases, including human African trypanosomiasis (HAT), which is caused by Trypanosoma brucei, and Leishmaniasis, which is caused by several Leishmania spp. [5]. Currently, NTD control relies extensively on chemotherapy and prophylaxis. The available drugs for these diseases were discovered many decades ago and they suffer from many drawbacks, such as toxicity, poor efficacy, and drug resistance [6–8]. Recently, the combination of nifurtimox and eflornithine [9] as well as the introduction of fexinidazole [10,11], the first oral drug for the treatment of HAT, represent a step forward to an efficacious and safe treatment for this disease. However, drug resistance may still occur, and the existence of animals that represent a reservoir for this infection [12] further justifies research in this area. Amongst the validated parasitic targets, the folate metabolism enzymes represent interesting candidates against trypanosomatidic infections [3]. Trypanosomatidae are auxotrophic for pterins and folates, therefore, inhibiting the enzymes involved in the folate pathways could represent a valuable strategy for the treatment of these trypanosomatidic infections [13,14]. However, although methotrexate (MTX), pyrimethamine (PYR) and trimethoprim (TMP) are nanomolar inhibitors of parasitic dihydrofolate reductase (DHFR) [15], these drugs have reduced activity against Leishmania and Trypanosoma infections and one of the reasons is the pteridine reductase 1 (PTR1) enzyme. PTR1 is a NADPH-dependent short-chain dehydrogenase/reductase (SDR) mainly involved in the reduction of conjugated and unconjugated pterins, such as biopterin, and in their salvage in the parasitic cells. In Leishmania, salvage of extracellular biopterin involves uptake predominantly through the biopterin transporter 1 (BT1) and the fully oxidized pterin is then reduced sequentially to dihydrobiopterin (H2B) and tetrahydrobiopterin (H4B) by PTR1 (Figure 1). H4B is essential for growth of the promastigote stage of L. major and it is mainly involved in the biosynthesis 3 of tyrosine promoted by phenylalanine hydroxylase (PAH) enzyme. PAH enzyme convert H4B to tetrahydrobiopterine-4a-carbinolamine. The regeneration of H4B in L. major is ensured by two other enzymes: PCD (pteridine carbinolamine dehydrogenase) that convert tetrahydrobiopterine-4a-carbinolamine into quinoide dihydrobiopterin (qH2B), and qDPR (quinoide dihydropteridine reductase) which finally reduces qH2B to H4B (Figure 1). In support of the importance of H4B for Leishmania vitality, there is the evidence that PTR1-null mutants are unable to grow in culture unless supplemented with H2B or H4B [14]. In contrast to the leishmania parasites, knockdown of PTR1 levels in T. brucei by RNA interference is lethal in vitro and cannot be rescued by supplementation with either H2B or H4B [16], unlike L. major PTR1-null mutants [17]. In addition, PTR1 knockdown abolishes infectivity of T. brucei to mice [16]. These results provide convincing evidence that PTR1 is an essential drug target for the treatment of T. brucei infections. Moreover, although PTR1 is mainly involved in the reduction of biopterin to H4B, it is constitutively involved in the reduction of the 10% of folic acid to tetrahydrofolate required by the parasitic cell, overlapping with the activity of DHFR [14,16]. Thus, when parasitic DHFR is inhibited by antifolate drugs, the expression of PTR1 is increased providing in toto the reduced folate necessary for parasite survival, providing a metabolic bypass to alleviate DHFR inhibition and contributing to treatment failure with antifolate drugs [18–21]. Therefore, targeting PTR1, in combination with a classical antifolate drug, is considered a promising strategy for the development of improved therapies [22,23]. Indeed, the combination of PTR1 inhibitors with MTX resulted in a synergic antiparasitic efficacy [24–28]. PTR1 is a functional homo-tetramer having the NADPH cofactor bound in each subunit, stabilized by a tight network of conserved H-bonds [18,29]. The substrate pterin moiety binds in a peculiar π-sandwich between the nicotinamide ring of NADPH and the aromatic side chain of Phe97, suggesting an ordered sequential reaction mechanism relying on the substrate accommodation preceded by the cofactor [18,29]. 4 Figure 1. Enzymatic and metabolic pathway of biopterin and folic acid in T. brucei and L. major. Almost all PTR1 inhibitors developed to date were designed as competitive inhibitors of biopterin or folic acid, although few non-competitive inhibitors have been recently identified. A wide variety of scaffolds have been explored. However, these possess a substrate-like structure ascribable to the pyrimidine class [18,23,30–32] or to its bioisosteres [25,28,33,34]. Representative chemical scaffolds are reported in Figure 2. In order to expand the chemical space and the molecular diversity of the PTR1 inhibitors, several HTS campaigns have been published in the field of trypanosomatidic infections, resulting in a promising array of newly identified hits [35,36]. With this in mind, in the present work, we investigated the utility of the LIBRA compound library (composed of 522 non-commercial molecules contributed by various Italian academic laboratories which have been designed and synthesized during various medicinal chemistry programs) to identify new chemical scaffolds targeting PTR1 as 5 potential anti-Trypanosoma or anti-Leishmania agents. The compound library was screened in HTS campaigns against Trypanosoma brucei PTR1 (TbPTR1) and Leishmania major PTR1 (LmPTR1). The hits identified were assessed for their antiparasitic activity in cell-based parasite assays, both alone and in combination with MTX. The prioritization of compounds was based on their potency in the HTS target-based assays, structural features of each chemotype and physicochemical properties. In addition, we carried out docking studies on the hits identified in the primary screening in order to select only the ones able to bind within the PTR1 binding site and to elucidate their binding mode guiding the design of new derivatives. We ascertained that PTR1 has a preference to bind inhibitors, which resemble its biopterin/folic acid substrates, such as the 2,4-diaminopyrimidine derivatives. Figure 2. Chemical scaffolds based on the pyrimidine ring or its bioisosteres used for the development of parasitic PTR1 inhibitors. 2. Results and discussion 2.1. LIBRA compound library characterization The LIBRA compound library consists of 522 non-commercial small molecules synthesized for drug discovery projects in various therapeutic areas (i.e. central nervous system [37–39], antitrypanosomal activity [40] and oncology [41]) and donated to the Italian Institute of Technology (IIT) by several Italian academic research groups. Additional details regarding 6 the main contributors and providers of the compounds forming the LIBRA library are reported in Supporting Information. IIT provided for the assembly and characterization of the library within the LIBRA project. The chemical structures of all the compounds are available as .sdf file which can be downloaded from the LIBRA project website (https://libramolecules.eu/). The synthesis and characterization of the molecules are reported in the respective publications, which are accessible by searching the chemical structure in SciFinder, the most comprehensive database for chemical literature. The identity of each compound was confirmed using NMR and UPLC-MS and their purity was determined by LC-UV-MS. Compounds that were not detectable in the UV trace (215 nm) or did not ionize by electrospray ionization were analyzed by q-NMR. In total, 83% (434 out of 522) and 97% (506 out of 522) of the compounds showed a purity >95% and >85%, respectively (Table 1). The identity and purity analyses confirmed the reliability of the tested molecules. Table 1. Purity of the LIBRA compound library. Purity (%) >95% >90% >85% <85% Number of compounds 434 480 506 16 2.2. Physicochemical properties and chemical space covered by the LIBRA compound library The chemical space covered by the LIBRA compound library was explored and the most representative chemical scaffolds were identified. For each compound, the extendedconnectivity fingerprint 4 (ECFP4) was calculated; the atoms were distinguished by functional type and whether terminal or belonging to a chain or to an aliphatic/aromatic ring; the bonds were distinguished by bond order. A similarity matrix based on the fingerprint was also calculated. When setting the Tanimoto coefficient value at 0.85, a total of 54 chemical clusters were generated. For the molecules of each cluster, the common chemical scaffold was determined and the clusters with the same core scaffold were grouped together. The similarity matrix was also used as an input for the iTOL server (http://itol.embl.de/) [42] to visually represent, as a dendrogram, the chemical similarities between molecules (Figure 3). Ten representative chemical clusters were identified: isoxazolines (c1), 1,3-dioxolanes (c4), triazaspiro derivatives (c5), tryptamines (c6), terphenyls (c8), dibenzoannulene (c9), and phenylaminoacetamides (c10). In particular, at least three branches contain a pyrimidine7 based structure, namely c2a (pyrimidines), c2b (purines), and c7 (pyrrolopyrimidones) over the 17 cited core scaffolds. Figure 3. Dendrogram of the LIBRA compound library. Clustering of the library compounds was based on the chemical similarity. Ten clusters were identified; the common chemical scaffold of the compounds of the cluster is reported. For all 522 LIBRA compounds, the important physiochemical properties and drug-likeness were determined in silico and shown to be in accordance with Lipinski’s “rule of five (RO5)” [43]. The molecular weight (MW), clogP, number of H-bond acceptors (HBA), number of Hbond donors (HBD), total polar surface area (TPSA), and number of rotatable bonds were 8 calculated using QikProp [44] under the assumption of standard protonation states at physiological pH conditions. These results are summarized in Table 2. Table 2. Physicochemical parameters of the 522 LIBRA compound library. Minimum Maximum Drug-likeness Average value value criteria MW [g/mol] 115 1081 358 <500 clog P -5.62 9.90 2.31 <5 H-bond acceptors 1 10 5 <10 H-bond donors 0 5 1.2 <5 Total polar surface area (Å2) 12 171 60.5 <140 Rotatable bonds 0 27 5.1 <10 % of compounds passing the RO5 (assuming no more than one violation per compound) Parameter % of compounds satisfying the RO5 89 91 100 100 99 93 94.4 The MW of the compounds ranged between 115 g/mol and 1081 g/mol, with an average of 358 g/mol and a median of 331 g/mol. The calculated lipophilicity (clogP) ranged from −5.62 to 9.90, with a mean value of 2.31. The number of Hydrogen Bond Acceptor (HBA) was 110, with an average of 5; whereas the number of Hydrogen Bond Donor (HBD) varied from 0-5, with an average of 1.2. Extending the RO5 evaluation to include properties associated to favourable bioavailability of small molecules [38], the library showed a Total Polar Surface Area (TPSA) ranged 12 Å2-171 Å2, with a mean value of 60.5 Å2, and between 0-27 rotatable bonds, with a mean of 5.1. Assuming no more than one violation of the rule [39], 94.4% of the compounds were in accordance with the RO5. Since the current therapy for HAT is based on drugs administered parentally, except for fexnidazole, all these treatments require a minimum health infrastructure and personnel, not readily available in the remote areas where this pathology is endemic. Therefore, an oral treatment regimen for the disease could potentially allow quicker and wider access to treatment because distribution and administration of tablets is easier. In addition, because over time the disease invades the central nervous system, the candidate drug for HAT should be able to cross the Blood-Brain Barrier (BBB) in order to be effective against the second stage of the diseases. The predictions for human gastrointestinal absorption and BBB permeations are represented in an intuitive graphical classification model viz. BOILED-Egg diagram as shown in Figure 4. The 95% of the compounds of the library is predicted to be absorbed per os and the 70% is predicted to be able to cross the BBB, indicating an overall good drug-likeness of the library’s components. 9 Figure 4. Boiled-egg diagram for the 522 LIBRA compound library. The white region is the physicochemical space of molecules with highest probability of being absorbed by the gastrointestinal tract, and the yellow region (egg yolk) is the physicochemical space of molecules with highest probability to permeate to the brain. The compounds are color-coded by molecular weight (MW) ranging from 115 Da (red dots) to 1081 Da (blue dots). 2.3. HTS campaigns against TbPTR1 and LmPTR1 and hit identification All 522 compounds of the LIBRA library were initially tested for their inhibitory activity against TbPTR1 and LmPTR1 at 50 µM. The results were reported as a percentage of enzymatic inhibition at 50µM and IC50 when appropriate (Figure 5 and Table SI-1). The HTS was performed in duplicate, and the results yielded Z' > 0.9 for all of the assays indicating their high quality. A total of ten compounds (LIB_66, LIB_133, LIB_135, LIB_136, LIB_138, LIB_143, LIB_146, LIB_162, LIB_190, and LIB_352, Table SI-1) yielded >85% inhibition at 50µM against TbPTR1 (Figure 4, red and green squares). Two compounds were also able to inhibit LmPTR1 >85% (Figure 4, green squares), giving an overall hit rate of 1.92% against TbPTR1. 10 % Inhibition of LmPTR1 at 50 M compound concentration 140 120 100 80 60 40 20 -40 -20 20 40 60 80 100 120 -20 -40 % Inhibition of TbPTR1 at 50 M compound concentration Figure 5. Percentage (%) of inhibition of TbPTR1 and LmPTR1 at 50 µM. The hits selected for the dose-response studies are colored in red (compounds yielding >85% inhibition against TbPTR1) and in green (compounds yielding >85% inhibition against both LmPTR1 and TbPTR1). The screening hits were tested in nine-point dose response experiments ranging from 0.5 µM to 100 µM, in order to confirm and determine their potencies (IC50). The IC50 values were calculated for each compound, which yielded 100% inhibition at the highest compound concentration. Considering the kinetic experiments results and the x-ray crystal structure of AX5 with TbPTR1, we may conclude that the pyrimidine analogs show a competitive inhibition pattern with respect to the dihydrobiopterine substrate. A similar conclusion was drawn about MTX inhibition pattern. MTX binds into the biopterin binding site preventing the substrate binding and acting as PTR1 competive inhibitor [22]. Compounds LIB_136 and LIB_162 (against TbPTR1) and LIB_66, LIB_135, LIB_136, LIB_146, and LIB_190 (against LmPTR1) did not yield 100% inhibition at the highest compound concentration, therefore accurate IC50 values were not calculated and are referred to as IC50 >100 µM (Table SI-2). The Ki for each compound were subsequently calculated assuming a competitive inhibition model [22, 22a] and all data are reported in Table 3. In the dose-response experiments, LIB_136 yielded no inhibition of PTR1 at a concentration >100 µM, therefore was considered to be a false positive. 11 Table 3. Compound identifier, chemical structure, and Ki values towards TbPTR1 and LmPTR1 of the nine hits resulting from the HTS campaign. Compound LIB_66 LIB_133 LIB_135 LIB_138 LIB_143 LIB_146 a b Chemical Structure Compound provider and primary referencea Enzyme inhibition Ki TbPTR1 Ki LmPTR1 (µM) (µM) University of Milan Roth, B. et al. [45] 0.6 >100 University of Bologna, FaBit Bolognesi, M.L. et al. [46] Rosini, M. et al. [47] 1.6 19.2 University of Bologna, FaBit Bolognesi, M.L. et al. [46] Rosini, M. et al. [47] 1.7 >100 University of Bologna, FaBit Bolognesi, M.L. et al. [46] Rosini, M. et al. [47] 2.7 14.8 University of Bologna, FaBit Rosini, M. et al. [47] 0.2 37.2 1.6 >100 University of Bologna, FaBit Rosini, M. et al. [48] LIB_162 University of Bologna, FaBit Pizzirani, D. et al. [41] >100 5.7 LIB_190 University of Bologna, FaBit Cavalli, A. et al. [49] 2.2 >100 LIB_352 University of Urbino “Carlo Bo” Minetti, P. et al. [50] 4.9 12.0 Bibliographic reference where the synthesis of the compound was reported. Standard deviation is within ±10% of the value. 12 Among the nine identified hits from primary screening, eight compounds confirmed the low micromolar activity against TbPTR1 with Ki values ranging from 0.2 to 4.9 uM. In contrast, only five compounds showed measurable Ki against LmPTR1 with values in the 5.7 to 37.2 M range (Table 3). Basically, the compounds resulted from 2.4 to 167-fold more active against TbPTR1 than LmPTR1, with solely LIB_352 (TbPTR1 Ki = 4.9 M and LmPTR1 Ki = 12 M) showing a comparable activity against both enzymes. The only exception was LIB_162 that resulted inactive against TbPTR1 but with an Ki = 5.7 M against LmPTR1. At this stage, the compounds were solely selected on the basis of their potency, rather than the quality/structural features of each chemotype. Thus, to avoid general promiscuity and toxicity issues associated with a high MW and lipophilicity, compounds with a MW >500 g/mol or cLogP >5 were removed [51]. Moreover, the nine compounds resulting from the screening were assessed for pan-assay interference as well as toxicity properties using the in silico tool FAFdrugs4 [52,53]. Accordingly, compounds LIB_133, LIB_135, LIB_138, LIB_143, and LIB_146 were discarded due to their high MW, cLogP, number of rigid bonds and ring size. In particular, for compound LIB_146 the software predicted a high risk associated to the presence of consecutive alkyl chains [54]. LIB_162 and LIB_190 presented only low-risk structural alerts due to the presence of a polyphenolic moiety (for LIB_162) or polyhalogenated rings (for LIB_190) and therefore they were deprioritized. Only LIB_66 (a diaminopyrimidine, cluster C-2a, Figure 3) and LIB_352 (a purine, cluster C-2b, Figure 3) passed the selection criteria. Finally, the two active hits were subjected to a substructure search in literature to identify known chemotypes occurring in approved antiparasitic therapeutics and other published PTR1 inhibitors. To the best of our knowledge, only compound LIB_66 was chemically close to some TbPTR1 inhibitors reported by Tulloch et al. [23] and to a lesser extent to some new L. chagasi PTR1 inhibitors discovered by Teixeira et al. [31], both based on the 2,4-diaminopyrimidines scaffold. When compared to the previously published compound 4-(benzylthio)-2,6-diaminopyrimidine (AX5, Figure 6A), LIB_66 differs from AX5 by an oxygen atom in place of the sulfur atom in the linker between the pyrimidine and the side benzyl ring [23]. The existence of the active PTR1 inhibitor AX5 showing a chemical structure similar to LIB_66 confirmed the accuracy of our processes and suggests that PTR1 has a preference for pyrimidine scaffolds resembling the substrates. 2.4. Docking model of hit compounds with TbPTR1 13 The availability of a crystallographic structure of AX5 complexed with TbPTR1 and NADP(H) (PDB ID: 3BMQ) provided the template for a structure-based analysis of the binding of LIB_66 and LIB_352 within the catalytic cavity (Figure 6B). The 2,6diaminopyrimidine scaffold of AX5 binds within the TbPTR1 catalytic pocket forming interactions comparable to those observed between the pteridine moiety of MTX and TbPTR1, in contrast with the canonical pose assumed by the pterin scaffold of the natural PTR1 substrates (i.e., biopterin or folic acid) [23]. The pyrimidine ring is π-π stacked between Phe97 and the nicotinamide ring of NADP(H). The amino group in position 6 is engaged in H-bonding with Tyr174, whereas the second amino group in position 2 establishes a second H-bond with Ser95, anchoring the molecule in the deepest part of the catalytic TbPTR1 active site. The benzylthio side chain in position 4 on the pyrimidine ring of AX5 is instead located in the hydrophobic pocket of the β6-α6 loop, delimitated by Phe97, Pro210, Trp221, and Met213 (Figure 5B). The binding mode of LIB_66 and LIB_352 to the catalytic site of TbPTR1 was investigated by docking studies using Glide [55] and carried out as reported previously on the crystal structure of TbPTR1 (PDB ID: 3JQ7) [21,23]. Docking output revealed that the predicted binding mode of LIB_66 is in accordance with the crystallographic pose observed for AX5 (PDB ID: 3BMQ) (Figure 6C) [23]. The pyrimidine ring of LIB_66 stacks in the active site in a π-sandwich between the nicotinamide of NADP+ and Phe97, whereas the phenyl ring establishes a face-to-edge stacking interaction with the aromatic side chain of Trp221 (Figure 6C). The H-bonding pattern observed in the TbPTR1-NADP(H)AX5 complex was conserved in the predicted binding mode of LIB_66 (Figure 6C). Similarly, the purine ring of LIB_352 stacks in the active site in a π-sandwich between the nicotinamide of NADP+ and Phe97, with the amino group in position 6 H-bonded to the Tyr174 hydroxyl group (Figure 6D). Moreover, the phenyl side chain establishes a face-toedge stacking interaction with the aromatic side chain of Phe97. However, LIB_352 was 8times less active than LIB_66 (Ki = 4.9 and 0.6 µM, respectively). The slightly reduced activity could be due to the lack of a second amino group to reinforce the binding with NADP+ or Ser95, which is only partially compensated by the stronger π-π interactions established by the extended aromatic system of the purine ring (Figure 6D). 14 Figure 6. (A) Chemical structures of AX5, its bioisostere LIB_66, and LIB_352. The chemical differences between LIB_66 and AX5 are highlighted in red. (B) X-ray ternary complex of TbPTR1-NADPH-AX5 (PDB ID: 3BMQ, AX5 and protein in teal sticks and cartoon) [23]. (C) Docking poses of compound LIB_66 (sticks, green carbons) in TbPTR1 (D) Docking poses of compound LIB_352 (sticks, yellow carbons) in TbPTR1. The reference crystal structure used for the docking calculation (PDB ID: 2X9G) of TbPTR1 is shown in gray cartoon; important interacting residues are in stick representation with grey carbons, and NADP+ is stick representation in black carbons). Model atoms except for carbons are colorcoded with protein carbons (teal or gray), oxygen (red), nitrogen (blue), phosphorous (orange), and sulfur (yellow). H-bonds are represented as dotted lines. Based on the binding modes observed for TbPTR1, we evaluated possible binding orientations of the selected hits into LmPTR1 by docking calculation that were performed with the same protocol as for TbPTR1. Our results indicated a comparable binding mode between the diaminopyrimidine ring (in LIB_66) and the purine ring (in LIB_352) with the catalytic pocket of both proteins. LmPTR1 possesses an active site larger than TbPTR1 and with a polar entrance exposed to the solvent (Figure SI-1) [27]. The predicted binding mode 15 of LIB_66 indicated that the benzyl is oriented towards the hydrophilic entrance in LmPTR1, therefore it is not able to form π-π stacking or additional hydrophobic contacts (as in TbPTR1). The missing π-π stacking interaction may explain the observed lower activity of LIB_66 against LmPTR1 (Ki >100 μM) compared with TbPTR1 (Ki : 0.6 μM ). 2.5. LIB_66 hit confirmation, synthesis, and initial structure-activity relationship (SAR) studies Among the two selected hits from the screening, LIB_66 was the most interesting because of its promising TbPTR1 inhibition (TbPTR1 Ki : 0.6 μM) and its synthetic accessibility that allows rapid structure modification. Therefore, six new LIB_66 derivatives were prepared and tested in-vitro (Table 4). These compounds were designed based on visual inspection of the predicted binding mode of LIB_66 within TbPTR1. First, some substituents were introduced exclusively in the para position on the distal benzyl ring in order to capture additional interactions within the two side hydrophobic pockets unique to the TbPTR1 active site (GC_57, FDS_1, FDS_2, and FDS_3). Second, we replaced the oxygen atom in position 4 of the pyrimidine ring of LIB_66 with a nitrogen to investigate the effect of a hydrogen bond acceptor/donor group at this position. Third, with the aim to confirm the ability of the two amino groups in positions 2 and 6 on the pyrimidine ring to form H-bond contacts with key amino acid residues, the amino group at position 6 was capped with a bulky substituent (GC_67). 2.6. Synthesis of LIB_66 derivatives The synthetic approaches for the new compounds are reported in Scheme 1. Compounds GC_57, GC_59, GC_67, and FDS_1–3 were prepared by aromatic nucleophilic substitution of the appropriate benzyl alcohol or benzyl amine on 4-chloro-2,6-diaminopyrimidine. The reaction conditions were adjusted and optimized for each derivative, allowing the desired compounds to be prepared in >70% yield. LIB_66 was prepared by reacting commercially available sodium benzyloxide with 4-chloro-2,6-diaminopyrimidine in benzyl alcohol at 150 °C for 5 h. In contrast, for the synthesis of the analogue GC_57, the nucleophile was directly generated in situ by reacting p-methoxybenzyl alcohol with sodium hydride in DMSO, which was to react with 4-chloro-2,6-diaminopyrimidine at a lower temperature (100 °C) for 16 h. The synthesis of the triaminopyrimidine derivatives GC_67 and FDS_1–3 required microwave irradiation to afford high yields. The use of one equivalent of the appropriate 16 benzylamine allowed the mono-benzyl derivative to be selectively obtained, whereas the use of 2.5 equivalents of amine and a longer reaction time (12 h) led to the dibenzyl pyrimidine GC_67. Scheme 1. Reagents and conditions: a) PhCH2ONa, PhCH2OH, 150 °C, 5 h; b) p-MeOPhCH2OH, NaH, DMSO, 100 °C, 16 h; c) R-NH2 (1 equiv.), EtOH, 150 °C, microwave irradiation, 7 h; d) R-NH2 (2.5 equiv.), EtOH, 150 °C, microwave irradiation, 12 h. 2.7. PTR1 inhibition and structure-activity relationship (SAR) of the newly synthesized compounds All six newly synthesized compounds were assessed for the inhibitory activity against TbPTR1. Compounds GC_57, GC_59, and FDS_1–3 yielded low micromolar inhibition of TbPTR1 (Ki : 0.2 – 3.4 μM) when compared to that of LIB_66 (TbPTR1 Ki = 0.6 μM), FDS_3 being the most potent derivative with Ki of 0.2 μM against TbPTR1 (Table 4). Only compound GC_67 was completely inactive. Table 4. Chemical structure, synthetic procedure, and IC50 values towards TbPTR1 and LmPTR1 of select compounds. Compound X LIB_66 O GC_57 O GC_59 GC_67 FDS_1 NH NH NH FDS_2 NH FDS_3 NH Chemical Structure R2 Synthetic Conditionsa a BnHb (4-MeO)-BnHR1 BnBn(4-F)-Bn- c Enzymatic Activity Ki LmPTR1 (µM) Ki TbPTR1 (µM) 0.6±0.1 >100 0.8±0.1 >100 3.4±1.0 N.I. HBnH- d c 1.1±0.1 >100 >100 >100 (4-MeO)-Bn- H- c 0.3±0.1 41.6±2.1 (4-Ph)-Bn- H- c 0.2±0.1 15.3±1.0 17 a For synthetic procedure and conditions refer to Scheme 1. Standard deviation is within ±10% of the value unless otherwise specified. N.I. No inhibition b The inhibition caused by the synthesized compounds and the binding mode predicted by docking calculation allowed to draw a preliminary SAR around the in-vitro activity of LIB_66. Substitution of the oxygen in position 4 of LIB_66 with a nitrogen atom, as in GC_59, led to a three-fold reduction of inhibitory activity. In contrast, no significative differences in the inhibitor activity were observed when the replacement of the oxygen atom with a nitrogen was applied on the four compounds substituted in position 4 on the side benzyl ring (GC_57 and FDS_1–3). This may be explained by a slightly different binding mode for these four derivatives with respect to the one observed for LIB_66, as suggested by computational docking analysis. The introduction of a substituent on the benzyl moiety in GC_57 and FDS_1–3 prevents the face-to-edge π interaction between the aromatic ring and Trp221, as observed for LIB_66, GC_59, and AX5 (Figure 6) [23]. In order to accommodate these substituents, the 2,4-diaminopyrimidine ring, still stacking between Phe97 and nicotinamide, is slightly rotated with respect to LIB_66. This torsion allows the phenyl ring of the side chain to establish a face-to-face stacking interaction with the aromatic side chain of Trp221 and a face-to-edge interaction with Phe97 (Figure 6). In addition, for compounds GC_57 and FDS_2, the methoxy group in the para position on the distal ring accepts an additional H-bond from Cys168, justifying the slightly higher activity of these two compounds compared with the two analogues without a substituent on the ring (LIB_66 and GC_59, respectively). Compound FDS_3, bearing a 4-phenylbenzylamine moiety linked to the 4-position of the pyrimidine scaffold, was the most active compound (TbPTR1 Ki = 0.2 µM). Notably, the biphenyl adopts a substrate-like orientation with the proximal phenyl ring mimicking the p-aminobenzoic acid moiety of folic acid and the distal phenyl locking the compound in this orientation by spatial restraints and a face-to-edge interaction with Phe171. Only compound GC_67 was completely inactive: the introduction of a benzyl group on the nitrogen in position 4 completely prevents the molecule from being accommodated within the TbPTR1 catalytic pocket and blocks the amino group in position 4 to engage an H-bond with Tyr174. In contrast, all the newly synthesized compounds were less active against LmPTR1 than TbPTR1, and the data are in line with the results achieved for the initial hit LIB_66. Only compounds FDS_2 and FDS_3 showed measurable LmPTR1 Ki of 41.6 and 15.3 M, respectively . 18 2.8. Antiparasitic activity towards T. brucei in-vitro The hits from the screening and the LIB_66 derivatives, which were active against TbPTR1, were assessed for their antiparasitic activity against the bloodstream form of T. brucei. As the compounds yielded modest inhibition of LmPTR1, they were not tested against Leishmania spp. The compounds were tested at 10 µM against T. brucei in a phenotypic screening campaign with the antiparasitic activity expressed as the percentage of parasitic cell growth inhibition at the defined compound concentration. Compounds with antiparasitic activity >85% at 10 µM were followed up in dose-response studies and six compounds (LIB_133, LIB_135, LIB_136, LIB_138, LIB_143, and LIB_190) yielded EC50 against T. brucei in the low/sub micromolar range (0.27-6.1 µM). In contrast, two TbPTR1 inhibitors (LIB_66 and LIB_352) and the derivatives of LIB_66 were inactive against T. brucei. Only FDS_3 showed significant anti-Trypanosoma activity as a single agent, with EC50 3.7 µM (Table 5). Table 5. Antiparasitic activity of the hits from the screening and LIB_66 derivatives against T. brucei. EC50 of the single compound, antiparasitic activity of the compound in combination with 4 µM MTX and the potentiation index (P.I.) are reported. Screening hits Compound Antiparasitic activity against T. brucei % Inhibition + 4 μM MTX[d] 36 ± 3%[b] P.I. LIB_66 N.I. LIB_133 0.56 ± 0.04 41 ± 5%[a] 1.2 ± 0.2 0.61 ± 0.05 [a] 1.4 ± 0.2 [a] 1.1 ± 0.1 [a] 1.1 ± 0.1 [b] 1.3 ± 0.1 [a] 1.1 ± 0.2 [b] LIB_135 LIB_138 LIB_143 LIB_146 LIB_190 Newly synthesized compounds EC50 (µM) 0.58 ± 0.06 0.27 ± 0.03 N.I. 2.45 ± 0.16 43 ± 2% 42 ± 2% 46 ± 3% 51 ± 2% 46 ± 4% 1.9 ± 0.1 LIB_352 N.I. 56 ± 6% 1.7 ± 0.2 GC_57 N.I. 42 ± 0%[b] 2.2 ± 0.6 N.I. [b] 1.3 ± 0.5 [b] 1.8 ± 0.0 [b] 2.2 ± 0.6 GC_59 FDS_1 FDS_2 FDS_3 N.I. N.I. 24 ± 3% 37 ± 6% 41 ± 6% [c] 3.7 ± 0.8 49 ± 7% 2.3 ± 0.1 a b N.I.: No inhibition. P.I.: Potentiation Index. Compound screened at 0.1 µM. Compound screened at 10 µM. c Compound tested at 1 µM. d MTX yielded 19 ± 6 % cell growth inhibition as a single agent at 4 µM. The effective inhibition of parasite growth was expected when both the PTR1 and DHFR enzymes are inhibited [25,28]. On this basis, we studied the effect of the PTR1 inhibitors in 19 combination with MTX against T. brucei to observe the potentiation effect that the PTR1 inhibitors have on MTX parasite growth inhibition. Thus, eight hits resulting from the screening (LIB_66, LIB_133, LIB_135, LIB_138, LIB_143, LIB_146, LIB_190 and LIB_352) and the LIB_66 derivatives were combined with 4 M MTX (corresponding to the EC30 of the DHFR inhibitor against T. brucei) [56]. The compounds were assessed in combination at different concentrations, according to their EC50 values as a single agent. LIB_133, LIB_135, LIB_138, LIB_143, and LIB_190 were tested at 0.1 M. LIB_66, LIB_146, GC_57, GC_59, FDS_1, and FDS_2 were assessed at 10 M because they were inactive as single agent. Only compound FDS_3 was tested at 1 M since it showed an antiparasitic activity >80% at 10 M, which is an inhibition potency too high to allow any potentiation effects in combination to be observed. The antiparasitic activity was expressed as the percentage of cell growth inhibition and is reported in Table 5 and Figure 7. The potency in the combination studies was estimated through determination of a potentiating index (P.I.), given by eq. 1. eq. 1 A P.I. greater than 1.2 reflects the capacity of the compounds to increase the antiparasitic activity of MTX beyond what would be expected by the simple addition of the individual effects. Except for LIB_66 and LIB_352, the combination of each of the eight compounds from the screening with 4 µM MTX resulted in an additive or partial potentiation activity of MTX (P.I. 1.1-1.4). In contrast, LIB_352, LIB_66 and its derivatives (except for GC_59) in combination with 4 µM MTX potentiated the antiparasitic activity of MTX against T. brucei, showing P.I. values 1.8-2.3 (Table 5 and Figure 7). This outcome supported the concept that inhibition of both TbPTR1 and TbDHFR is necessary to exert antiparasitic activity via the folate pathway. 20 Figure 7. Antiparasitic activity against T. brucei of the hits compounds resulting from the screening (LIB_66, LIB_133, LIB_135, LIB_138, LIB_143, LIB_146, LIB_190 and LIB_352) and the LIB_66 derivatives (GC_57, GC_59, FDS_1, FDS_2 and FDS_3) tested as a single agent (in cerulean) and in combination with 4 μM of MTX (dark blue). Compound LIB_66, LIB_146, LIB_352, GC_57, GC_59, FDS_1 and FDS_2 were tested at 10 μM. Compounds LIB_133, LIB_135, LIB_138, LIB_143 and LIB_190 were tested at 0.1 μM. Compound FDS_3 was tested at 1 μM. The antiparasitic activity of MTX as a single agent at 4 μM is reported in stripped cerulean bar. As a quantitative measure of potentiation, a P.I. of the combination was determined (yellow bar). 2.9. In vitro early ADMET and selectivity studies against human THP-1 cell lines Potential early toxicity-related issues for the LIB_66 derivatives were determined using early absorption, distribution, metabolism, excretion and toxicity (ADMET) screening technologies. These studies included inhibition of hERG and CYP2D6 as well as mitochondrial toxicity at compound concentration of 10 µM. In addition, the cytotoxicity against two human cell lines, A549 (human lung adenocarcinoma epithelial cells) and THP-1 (human monocytes) were determined. Pentamidine, the first-line drug for the treatment of HAT, was used as reference compound.[57] The cut-off inhibition for each compound tested in the five assays was established for the assessment of a safety profile of the compounds. The results are reported in Table SI-3 and are organized using a traffic light system for rapid and intuitive visualization (Figure 8) The cut-off for acceptable mitochondrial toxicity and inhibition of hERG and CYP2D6 were set at <30% inhibition at 10 µM; whereas for GI50 and CC50 in the A549 and THP-1 cell-lines, the cut-off was set at >60 µM. With the exception of 21 the initial hit, namely LIB_66 which was associated with a safe profile against the five ADMET targets, all six derivatives showed liabilities against hERG (>50% inhibition in the case of GC_59, GC_67, FDS_1, FDS_2, and FDS_3) and CYP2D6 (30-60% inhibition for GC_59, FDS_1, and FDS_2) and low mitochondrial toxicity was observed. Compound FDS_3, besides its promising anti-T. brucei activity (EC50 of 3.7 µM), showed significant cytotoxicity, with CC50 <25 µM against THP-1 cells and GI50 of 28.6 µM against A549 cells. Figure 8. Heat-map data from the in-vitro early ADMET assays. Compounds were tested at 10 μM for the hERG, CYP2D6, and mitochondrial toxicity assays. For the THP-1 and A549 cell assays, CC50 (<25 µM) and GI50 (28.6 µM) were determined for compound FDS_3. Pentamidine was used as reference drug. Red indicates a significant liability, yellow indicates moderate liability and green indicate the absence of liability. 3. Conclusion In this paper we report the screening of the unique LIBRA compound library against TbPTR1 and LmPTR1 and identified novel compounds, which offer the potential to be progressed as African trypanosomiasis and Leishmaniasis drugs. From the initial screen, LIB_66, characterized by a 4-benzyloxy-2,6-diaminopyrimidine structure, was the most interesting hit targeting TbPTR1 (Ki = 0.6 μM). Docking studies indicated that this compound could bind to PTR1 in a similar manner as its natural substrates; the 2,6diaminopyrimidine scaffold of LIB_66 displays a binding mode comparable to that of the 22 pteridine scaffold of MTX, with the benzyloxy side chain in position4 directed towards the hydrophobic pocket, where usually the p-aminobenzoic acid ring of folic acid or MTX is located. Hit validation and SAR studies for LIB_66 were performed by synthesizing a series of 4-(para-substituted)benzylamino-2,6-diaminopyrimidine derivatives, in which the pyrimidine scaffold was decorated to explore the biopterin and folic acid binding site. The six compounds showed similar and in some cases improved activity relative to the parent compound, with the SAR rationalized by docking studies. The introduction of a substituent on the benzyl ring induces a slight rotation of the 2,4-diaminopyrimidine ring compared to the reported binding mode of AX5. This torsion allows the phenyl ring of the side chain to establish a face-to-face stacking interaction with the aromatic side chain of Trp221 and a face-to-edge interaction with Phe97, whereas the substituents on the benzylic side chain come into contact with the residues forming the distal portion of the TbPTR1 binding pocket, reinforcing binding with the enzyme. Moreover, this different binding mode, induced by the presence of a substituent on the distal aromatic ring, allows the replacement of an H-bond acceptor atom in position 4 (i.e., oxygen or sulfur) with an H-bond donor (i.e., nitrogen), without affecting the activity; these results are in contrast with the preliminary findings observed by Tulloch et al. on the smallest 2,4-diaminopyrimidine derivatives [23]. Furthermore, the compounds designed in this study could potentiate T. brucei growth inhibition in combination with MTX. These results suggest that the 4-benzyloxy/4benzylamino-2,6-diaminopyrimidine scaffold can be considered as an interesting starting point for optimization in the drug discovery value chain to search for anti-T. brucei agents. The pyrimidine scaffold shows an acceptable ADME-Tox profile as illustrated by LIB_66 and is suitable for further progression. Amongst the 522 compounds of the LIBRA compound library, which contains at least 17 different chemical scaffolds, PTR1 was inhibited by pyrimidine and purine derivatives, thus confirming the view that PTR1 enzymes prefer substrate-like compounds over other scaffolds. Considering the high need for new and well-characterized chemical entities and for new candidates to be included in the NID pipeline, this work discovered a new opportunity in the field. 4. Materials and Methods 4.1. LIBRA compound library The compounds are available at the PharmaChemistry Line, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova, Italy. Requests should be addressed to F. Bertozzi: 23 Fabio.bertozzi@iit.it. Details about LIBRA are reported at the address: https://libramolecules.eu/. 4.1.1. Characterization of the LIBRA compound library Compounds (solid or oil) were provided in 1.4 mL barcoded MATRIX microtubes. A weighed aliquot of each compound, enough to make about 1 mL of a 10 mM stock solution, was transferred into another microtube and dissolved with the addition of the proper volume of DMSO-d6. Vigorous shaking ensured dissolution and homogeneity within the microtube. The entire process was automated with a dedicated Hamilton Robotics Microlab Star workstation that also performed transfer of microtube plates in and out of the storage refrigerator, Hamilton Robotics Active Sample Manager, where pure compounds and frozen solutions were stored under nitrogen at 4 °C. The workstation was also capable of detecting partial solubilization; in this case, the solution was manually filtered, and the concentration was determined by quantitative 1H-NMR as described below. 4.1.2. UPLC-MS characterization of the LIBRA compound library Each compound was submitted to characterization for identity and purity by means of LCUV-MS. A Waters Acquity instrument equipped with Photodiode Array detector and single quadrupole ESI, equipped with 2.1 x 100 mm UPLC columns (1.7 µm), was used to this purpose. Chromatographic methods and dilution media were adapted to compound polarity, as estimated by cLogP as follows. The most polar compounds (cLogP <1) were diluted with water/acetonitrile (95/5) and chromatographed on a Waters HSS T3 column with a gradient from 100% to 50% water. Compounds with intermediate polarity (<1 cLogP <5) were dissolved in water/acetonitrile (1/1) and chromatographed on a Waters BEH column with a gradient from 90% to 10% water. Less polar compounds (cLogP >5) were dissolved in acetonitrile and chromatographed on a Waters BEH column with a gradient from 50% to 0% water. The mobile phase was buffered with 10 mM ammonium acetate, pH 5, and acetonitrile was always used as strong solvent. Flow (0.5 mL/min), gradient duration and column reequilibration were adapted to UPLC requirements and full method execution did not exceed 10 min per compound. Both positive and negative ion traces were collected and identity was verified for the main chromatographic peak by intense [M+H]+ or [M+Na]+ ions in the positive trace, or [M-H]– in the negative one. Purity was assessed from the relative main peak 24 area of the UV trace at 215 nm. Compounds that were not detectable in the 215 nm UV trace or did not ionize in ESI were analyzed by quantitative 1H-NMR. 4.1.3. NMR characterization of the LIBRA compound library Stock solutions of the compounds in 10 mM DMSO-d6 were submitted to quantitative 1H NMR (q-NMR) experiments, which were performed using the PULCON (PUlse Length Based CONcentration Determination) procedure [58]. This procedure does not require the addition of an internal standard as it relies on external standard calibration (performed only once in several months), thus allowing the sample to be used for further analyses (e.g., UPLCMS) and for biological assays. This method returns both the measured concentration of the compound of interest in solution and its absolute purity by comparison with its theoretical concentration. Structure identities were confirmed by 1H-1H COSY and 1H-13C HSQC experiments. Among the analyzed compounds, the reported structure was confirmed in 81% (138) of cases. Moreover, structure elucidation was performed in more than half of the cases in which the NMR spectra were not in accordance with the reported structure (14 compounds). All the NMR spectra were acquired at 300.0 K on a Bruker Avance III 400 MHz spectrometer equipped with a Broad Band Inverse (BBI) probe. For each compound, 150 µL of 10 mM DMSO-d6 stock solution was transferred into a 3 mm disposable tube (Bruker), which was inserted in the NMR probe using the Sample Jet automatic sampler. Before each acquisition, an automatic matching and tuning were run, and the homogeneity automatically adjusted. In the quantitative 1H-NMR (q-NMR) experiments, using the PULCON method, the 1 H 90° pulse was optimized on each sample tube by an automatic pulse calculation routine [59]. Then, 16 transients were accumulated over a spectral width of 20.55 ppm (with a transmitter frequency offset at 6.175 ppm), at a fixed receiver gain (64), using 30s of interpulses delay and no steady state scans. Spectra were manually phased and automatically baseline corrected. The integrated peaks intensity of the compound under analysis, normalized with respect to proton numbers, was compared to the integrated peak intensity of a 10 mM DMSO-d6 solution of a reference material (maleic acid, Sigma Aldrich NMR standard) whose spectrum was acquired under the same experimental parameters. The PULCON formula returns the measured concentration of the compound under analysis in solution. Absolute (percent) NMR purity was calculated by the ratio between the concentration measured by PULCON and the theoretical concentration (10 mM), multiplied by 100. This is a direct measurement, independent from the detectability of the impurities. 2D experiments for 25 structure identity or structure elucidation were acquired as follows. 1H-1H COSY (COrrelation SpectroscopY): 4 transients, 2048 data points, 128 increments, with an automatic spectral width and transmitter frequency offset optimization. 1H-13C HSQC (multiplicity edited Heteronuclear Single Quantum Coherence): 8 transients, 1024 data points, 256 increments, with an automatic spectral width and transmitter frequency offset optimization for proton dimension, and with a spectral with of 165.6 ppm and transmitter frequency offsets at 74.6 ppm for carbon dimension. 4.1.4. Determination of kinetic solubility of the LIBRA compound library The aqueous kinetic solubility (Skin) was determined from a 10 mM DMSO stock solution of test compound in Phosphate Buffered Saline (PBS) at pH 7.4. The study was performed by incubation of an aliquot of 10 mM DMSO stock solution in PBS (pH 7.4) at a target concentration of 250 µM resulting in a final concentration of 2.5% DMSO (v/v). The incubation was carried out under shaking at 25°C for 24 h followed by centrifugation at 14.800 rpm (21100 g) for 30 min. The supernatant was analyzed by UPLC-MS for the quantification of dissolved compound (in µM) by UV at a specific wavelength (215 nm). Compounds were grouped into low (Skin <10 μM), medium/moderate (<10 Skin <100 μM), and high solubility (Skin >100 μM). 4.2. Synthesis Solvents and chemicals purchased from commercial sources were of analytical grade or better and used without further purification. 1H NMR and 13 C NMR spectra were recorded on a Varian Mercury 300 (300 MHz) spectrometer and on a Bruker Avance 400 (400 MHz) spectrometer. Chemical shifts (δ) are expressed in ppm and coupling constants (J) are expressed in Hz. TLC analyses were performed on commercial silica gel 60 F254 aluminum sheets; spots were further evidenced by spraying with a dilute alkaline potassium permanganate solution. Melting points were determined on a model B 540 B i apparatus and are uncorrected. MS analyses were performed on a Varian 320-MS triple quadrupole mass spectrometer with ESI source. HRMS was obtained on a 6520 Accurate-Mass Q-TOF LC/MS. Compound purity was determined by means of UPLC-MS as described for LIBRA compounds library. All the compounds showed a level of purity above 95%. The compounds passed the check for PAINS performed with the in-silico tool FAFdrugs4 [52,53]. 26 4.2.1. 4-Benzyloxy-2,6-diaminopyrimidine (LIB_66) The synthetic procedure, the spectroscopic data and physicochemical properties are in accordance with literature [60]. 4.2.2. 9-Methyl-2-phenethyl-9H-purin-6-amine (LIB_352) CF3SO3H (0.37 mL, 3.3 mmol) was added dropwise to a solution of dibenzyl-(9-methyl-2phenethyl-9H-purin-6-yl)amine (143 mg, 0.33 mmol) in anhydrous CH2Cl2 (3 mL), under nitrogen at 0 °C. The mixture was refluxed for 6 h, then cooled and diluted with water, basified to pH 10 with 30% NaOH and extracted with CH2Cl2. The organic phase was dried over anhydrous Na2SO4 and concentrated. The residue was purified by flash chromatography (cyclohexane-ethyl acetate 3:7) to give a yellow solid that was crystallized from ethanol. Yellow solid (33% yield). M.p.= 140-141 °C. 1H NMR (CDCl3, 400 MHz) δ: 7.73 (s, 1H), 7.29-7.19 (m, 5H), 5.84 (br s, 2H), 3.82 (s, 3H), 3.15 (t, 4H, J = 7.0 Hz). 13C NMR (DMSO, 100 MHz) δ: 158.1, 150.9, 144.8, 140.7, 128.9, 128.8, 126.7, 116.8, 36.8, 33.1, 30.5. ESIHRMS calcd. for C14H16N5 [M+H]+ 254.1406, found 254.1413. 4.2.3. 4-(4-Methoxy-benzyloxy-)2,6-diamino methoxybenzyloxypyrimidine (GC-57) 72 mg of NaH (60% on mineral oil, 1.3 eq.) were slowly added to a solution of (4methoxyphenyl)methanol (171 µL, 1.0 eq.) and 4-chloro-2,6-diaminopyrimidine (200 mg, 1.38 mmol) in DMSO (1.0 mL). The reaction mixture was heated at 100 °C for 16 h. After cooling at room temperature, CH2Cl2 (20 mL) was added and the mixture was washed with water (2 x 10 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated. The crude was purified by flash chromatography (ethyl acetate 100%) to afford GC_57 as a yellow solid (70% yield). Rf= 0.37 (ethyl acetate 100%). M.p.= 166.2-167.0 °C. 1H-NMR (DMSO-d6, 300 MHz) δ: 7.30 (d, J= 8.7 Hz, 2H), 6.89 (d, J= 8.7 Hz, 2H), 5.98 (s, 2H), 5.86 (s, 2H), 5.10 (s, 2H), 5.03 (s, 1H), 3.72 (s, 3H). 13 C-NMR (DMSO-d6, 75 MHz) δ: 170.3, 166.5, 163.3, 159.3, 130.1, 129.9, 114.1, 76.7, 66.1, 55.5. MS (ESI): m/z calcd. for C12H14N4O2: 246.1; found: 246.9 [M+H]+. 4.2.4. General procedure for the synthesis of 4-benzylamino-2,6-diaminopyrimidine derivatives The appropriate amine (0.5 mmol) was added to a solution of 4-chloro-2,6-diaminopyrimidine (0.5 mmol) in EtOH (1 mL) in a sealed vial and heated by microwave irradiation at 150 °C for 27 7 h. After cooling at room temperature, the solvent was evaporated under vacuum the crude material was purified by flash chromatography (CH2Cl2/MeOH 9:1) to afford the desired compound. 4.2.4.1. 4-Benzylamino-2,6-diaminopyrimidine (GC-59) The spectroscopic data and physicochemical properties are in accordance with literature [61]. 4.2.4.2. 4-(4-Fluoro-benzylamino-)2,6-diaminopyrimidine (FDS_1) White solid (78% yield). Rf= 0.20 (CH2Cl2/MeOH 9:1). M.p.= 125.2-127.5 °C. 1H-NMR (DMSO-d6, 300 MHz) δ: 7.32-7.24 (m, 2H), 7.14-7.04 (m, 2H), 6.56 (t, J= 6.1 Hz, 1H), 5.50 (s, 2H), 5.33 (s, 2H), 4.82 (s, 1H), 4.30 (d, J= 6.1 Hz, 2H). 13C-NMR (DMSO-d6, 75 MHz) δ: 164.8, 164.3, 163.3, 161.4 (d, JC-F= 240.4 Hz), 137.5 (d, JC-F= 2.9 Hz), 129.2 (d, JC-F= 8.0 Hz), 115.2 (d, JC-F= 20.5 Hz), 74.7, 43.3. MS (ESI): m/z calcd for C11H12FN5: 233.1; found: 234.1 [M+H]+. 4.2.4.3. 4-(4-Methoxy-benzylamino-)2,6-diaminopyrimidine (FDS_2) Yellow solid (83% yield). Rf= 0.28 (CH2Cl2/MeOH 9:1). M.p.= dec > 128 °C. 1H-NMR (DMSO-d6, 300 MHz) δ: 7.17 (d, J= 8.4 Hz, 2H), 6.84 (d, J= 8.4 Hz, 2H), 6.58 (t, J= 5.7 Hz, 1H), 5.60 (s, 2H), 5.45 (s, 2H), 4.82 (s, 1H), 4.25 (d, J= 5.7 Hz, 2H), 3.70 (s, 3H). 13C-NMR (DMSO-d6, 75 MHz) δ: 164.3, 164.2, 162.8, 158.4, 133.0, 128.6, 114.0, 74.5, 55.5, 43.5. MS (ESI): m/z calcd for C12H15N5O: 245.1; found: 246.2 [M+H]+. 4.2.4.4. 4-(4-Phenyl-benzylamino-)2,6-diaminopyrimidine (FDS_3) Yellow solid (75% yield). Rf= 0.30 (CH2Cl2/MeOH 9:1). M.p.= dec. > 141 °C. 1H-NMR (DMSO-d6, 300 MHz) δ: 7.65-7.54 (m, 4H), 7.42 (t, J= 7.7 Hz, 2H), 7.37-7.30 (m, 3H), 6.61 (t, J= 6.3 Hz, 1H), 5.52 (s, 2H), 5.35 (s, 2H), 4.84 (s, 1H), 4.37 (d, J= 6.3 Hz, 2H). 13C-NMR (DMSO-d6, 75 MHz) δ: 164.8, 164.3, 163.3, 140.6, 140.5, 138.8, 129.3, 127.9, 127.7, 127.0, 126.9, 74.6, 43.7. MS (ESI): m/z calcd for C17H17N5: 291.1; found: 292.1 [M+H]+. 4.2.4.5. 4,6-Benzylamino-2-aminopyrimidine (GC_67) 4- Benzyl amine (2.5 eq. 1.73 mmol) was added to a solution of 4-chloro-2,6diaminopyrimidine (0.69 mmol) in EtOH (1 mL) in a sealed vial and heated by microwave irradiation at 150 °C for 12 h. After cooling at room temperature, the solvent was evaporated, 28 and the crude purified by flash chromatography (CH2Cl2/MeOH 9:1) to afford GC_67 as a yellow solid (73% yield). Rf= 0.23 (CH2Cl2/MeOH 95:5). M.p.= dec. > 129 °C. 1H-NMR (DMSO-d6, 300 MHz) δ: 7.31-7.15 (m, 10H), 6.60 (t, J= 5.7 Hz, 2H), 5.43 (s, 2H), 4.83 (s, 1H), 4.31 (d, J= 5.7 Hz, 4H). 13 C-NMR (DMSO-d6, 75 MHz) δ: 164.1, 163.1, 141.3, 128.6, 127.4, 126.8, 74.0, 44.1. MS (ESI): m/z calcd for C18H19N5: 305.2; found: 306.0 [M+H]+. 4.3. Molecular modelling Docking was performed using Glide and carried out as previously reported [25,27]. X-ray structures were downloaded from the Protein Data Bank (http://www.rcsb.og) and prepared using prepWizard to assig bond orders and add hydrogen atoms. The 3D structures of the compounds were created from SMILE string and optimized with the OPLS_2005 force field using Maestro [62]. Ionization states and tautomers were generated at pH 7.0±0.5 using Epik [63]. Up to eight stereoisomers and one low-energy ring conformation were generated per compound. For TbPTR1 important structural water sites were previously identified and considered conserved [27]. Refinement was carried out with Prime for residues within 5 Å of the ligand. Up to 20 final docking solutions were reported. 4.4. In-vitro biological assays 4.4.1. TbPTR1 and LmPTR1 protein purification, kinetic characterization purification, screening and IC50 determination The TbPTR1 and LmPTR1 proteins were purified as reported previously [23]. The in-vitro coupled enzyme assays for each enzyme have also been reported in the literature and have independently been shown to be suitable for screening purposes [25,27,36,57,64–67]. For each compound IC50, pIC50 values and Hill Slope were determined using a 4-parameter logistic fit in the XE module of ActivityBase (IDBS). The Ki for each compound were calculated using a competitive inhibition model (Ki = IC50/((S/Km)+1)) [22a]. 4.4.2. Evaluation of activity against T. brucei The antiparasitic activity of compounds against T. brucei bloodstream forms were evaluated using a modified resazurin-based assay previously described in the literature [68]. The antitrypanosomatid effect was evaluated by the determination of the EC50 value (concentration required to inhibit growth by 50%) and calculated by nonlinear regression analysis using GraphPad Prism, Version 5.00 for Windows, GraphPad Software, San Diego California USA (www.graphpad.com). For potentiation experiments, the synergy index was 29 calculated as the ratio between the activity of the combination (activity of the tested compound in the presence of 4 µM MTX) and the added activity of the components of the combination alone (activity of the compounds + activity of 4 µM MTX). 4.5. In-vitro early ADMET assays 4.5.1. hERG inhibition, CYP2D6 inhibition and mitochondrial toxicity These assays made use of Invitrogen’s Predictor hERG fluorescence polarization assay, Promega P450-Glo assay platform and MitoTracker Red chloromethyl-X-rosamine (CMXRos) uptake and high content imaging to monitor compound-mediated mitochondrial toxicity in the 786-O (renal carcinoma) cell-line respectively as previously described [29]. 4.5.2. Cytotoxicity against A549 cell line The assays were performed using the Cell Titer-Glo assay from Promega. The assay detects cellular ATP content with the amount of ATP being directly proportional to the number of cells present. The A549 cell-line was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and was grown in DMEM with FCS (10% v/v), streptomycin (100 µg/mL), and penicillin G (100 U/mL) [27]. 4.5.3. Cytotoxicity against THP-1 macrophages The cytotoxicity of THP-1-derived macrophages was assessed by the colorimetric (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay [27]. ASSOCIATED CONTENT Supporting Information. Chemical structures of the 522 compounds; Predicted binding mode on LmPTR1; Inhibitory activity of compounds against TbPTR1 and LmPTR1 of the entire LIBRA library; Inhibition of hERG, CYP2D6, mitochondrial toxicity, and cytotoxicity against A549 and THP-1 cell-lines of LIB_66 and selected analogues. AUTHOR INFORMATION Corresponding Authors. *A.P. University of Milan, Via Celoria 2, 20133 Milan, Italy; *T.B. Istituto Italiano di Tecnologia; *M.P.C. University of Modena and Reggio Emilia, Via Campi 103, 41125, Modena, Italy. AUTHOR CONTRIBUTIONS 30 M.P.C. S.F. and T.B. conceived the work and led the experimental design. M.P.C., P.L. and S.G. wrote the first draft paper and all Authors contributed to manuscript writing. M.L.B., A.P, G.P. M.N., P.C., M.R., F.P., F.B. and M.R., performed the synthetic medicinal chemistry. P.L performed statistical, computational, chemionformatic analyses. T.B., A.C. D.P., E.B. and V.R. contributed to the library collection and maintenance. S.G., G.W, M.K. and B.E, performed the early ADMET assays. A.C. and N.S. performed the anti-parasitic assays. The authors declare no competing financial interest. Present Addresses † Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland. ACKNOWLEDGMENT This project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement n° 603240 (NMTrypI - New Medicines for Trypanosomatidic Infections). 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Drugs Drug Resist. 2 (2012) 262–270. doi:10.1016/j.ijpddr.2012.02.002. 40 GRAPHICAL ABSTRACT LIBRA Tb and LmPTR1 High-throughput Screening 10 HTS Hits In vitro assays Docking simulation Hi t va lidation through new compounds synthesis 522 l i brary compounds PRIVILEGED SCAFFOLD 4-(benzyloxy/benzylamino) -2,6-diaminopyri midine TbPTR1 IC50 = 0.7 – 4.0 M 41 Supplementary Material - For Publication Online Click here to download Supplementary Material - For Publication Online: LIBRA_SI_MPC.pdf 3D molecular models (.PDB, .PSE, .MOL, .MOL2) Click here to download 3D molecular models (.PDB, .PSE, .MOL, .MOL2): TbPTR1_LIB66.pdb 3D molecular models (.PDB, .PSE, .MOL, .MOL2) Click here to download 3D molecular models (.PDB, .PSE, .MOL, .MOL2): TbPTR1_LIB352.pdb Chemical Compounds (.mol) Click here to download Chemical Compounds (.mol): libra_library.sdf *Declaration of Interest Statement Declaration of interests ☐ X The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nothing to declare In faith Prof. Maria Paola Costi