(GSK-3) as a druggable target for the human protozoan parasite Leishmania [23]; and cruzipain enzyme, a sulfated glycoprotein acting as primary cysteine peptidase of Trypanosoma cruzi, playing an important role in Chagas illness [24]. The screening from the exact same compound library against a lot of various target organisms and proteins involved in many illnesses might be conceptually linked for the repurposing approach and can bring about the identification of novel chemical structures (core structures) against the chosen targets [25,26]. Within this work, we sought to exploit 3 anti-kinetoplastid chemical boxes (Kinetoboxes, namely LEISH-, CHAGAS- and HAT-box) for the identification of new selective inhibitors of PTR1 showing core structures unique from the recognized folate/pyrimidine ones [21]. Every single Kinetobox was clustered in the original GSK collection like 1.8 million compounds, based on whole-cell assays performed for the three kinetoplastids (Leishmania donovani, T. cruzi and Trypanosoma brucei). A total of 592 compounds have been identified as the most active: 192 were active against L. JAK3 web donovani (LEISH-box), 222 against T. cruzi (CHAGAS-box) and 192 against T. brucei (HAT-box). Interestingly, 88 of your selected chemical collection was not previously published along with the remaining 12 showed an activity profile unrelated to the activity against Leishmania or Trypanosoma. Additionally, the three boxes did not contain structural analogs of drugs currently applied in the clinic for Leishmaniasis, Chagas illness or HAT. To determine inhibitors of Leishmania main (Lm) and T. brucei (Tb) PTR1, we firstly analyzed the chemical tructural attributes on the compound library and after that performed a medium-throughput screening (MTS) assay against Lm and Tb forms of DHFR-TS and PTR1 enzymes. Some active compounds possessing an antifolate scaffold have been identified, with a few of them showing a pan-inhibitor DDR2 site character, when they inhibit PTR1 from each Lm ad Tb kinetoplastids, and other people displaying dual inhibitors, when they inhibit both PTR1 and DHFR-TS of no less than 1 parasitic species. Interestingly, some novel structures different from the folate core had been also identified. Compound TCMDC-143249, a benzenesulfonamide derivative, getting an in vivo efficacy towards both parasites in the low micromolar variety, was in a position to selectively inhibit in vitro each LmPTR1 and TbPTR1, but not the corresponding DHFR-TSs. Molecular modelling studies showed that the inhibitor mimics the substrate pose in each TbPTR1 and LmPTR1, though it will not fulfill active site binding requirements in TbDHFR-TS nor in LmDHFR-TS, hence giving a structural basis for the differential activity ofPharmaceuticals 2021, 14,three ofTCMDC-143249 compound in PTR1 and DHFR-TS. The homology model of LmDHFR-TS was obtained through computational studies for docking purposes. The present study also proposes a novel core structure that can be exploited for the improvement of new anti-parasitic compounds. 2. Outcomes and Discussion two.1. In Silico Evaluation of Drug-Likeness Properties and Hierarchical Clustering analysis A chemoinformatic in silico analysis was firstly performed to characterize the druglikeness properties and chemical space of your Kinetobox collection, aiming to determine its most representative chemical core structure employing the QikProp descriptor tool (Canvas software-Schr inger) [27,28]. For each and every compound, molecular weight (MW), alogP, quantity of H-bond acceptors (HBA) and H-bond donors (HBD), total p