pharmaceuticals Article Repurposing the Trypanosomatidic GSK Kinetobox for the Inhibition of Parasitic Pteridine and Dihydrofolate Reductases Matteo Santucci 1,† , Rosaria Luciani 1,† , Eleonora Gianquinto 2,† , Cecilia Pozzi 3 , Flavio di Pisa 3 , Lucia dello Iacono 3 , Giacomo Landi 3 , Lorenzo Tagliazucchi 1 , Stefano Mangani 3 , Francesca Spyrakis 2 and Maria Paola Costi 1, *   Citation: Santucci, M.; Luciani, R.; Gianquinto, E.; Pozzi, C.; Pisa, F.d.; dello Iacono, L.; Landi, G.; Tagliazucchi, L.; Mangani, S.; Spyrakis, F.; et al. Repurposing the Trypanosomatidic GSK Kinetobox for the Inhibition of Parasitic Pteridine and Dihydrofolate Reductases. Pharmaceuticals 2021, 14, 1246. https://doi.org/10.3390/ph14121246 Academic Editor: Christophe Dardonville Received: 31 October 2021 Accepted: 25 November 2021 Published: 30 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Life Science, University of Modena and Reggio Emilia, Via Campi 103, 41125 Modena, Italy; matteo.santucci86@gmail.com (M.S.); rosaria.luciani@libero.it (R.L.); lorenzo.tagliazucchi@unimore.it (L.T.) 2 Department of Drug Science and Technology, University of Turin, Via Giuria 9, 10125 Turin, Italy; eleonora.gianquinto@unito.it (E.G.); francesca.spyrakis@unito.it (F.S.) 3 Department of Biotechnology, Chemistry and Pharmacy—Department of Excellence 2018–2020, University of Siena, Via Aldo Moro 2, 53100 Siena, Italy; pozzi4@unisi.it (C.P.); dipisa2@unisi.it (F.d.P.); delloiacono3@unisi.it (L.d.I.); landi31@unisi.it (G.L.); stefano.mangani@unisi.it (S.M.) * Correspondence: mariapaola.costi@unimore.it † These Authors contribute equally to the work. Abstract: Three open-source anti-kinetoplastid chemical boxes derived from a whole-cell phenotypic screening by GlaxoSmithKline (Tres Cantos Anti-Kinetoplastid Screening, TCAKS) were exploited for the discovery of a novel core structure inspiring new treatments of parasitic diseases targeting the trypansosmatidic pteridine reductase 1 (PTR1) and dihydrofolate reductase (DHFR) enzymes. In total, 592 compounds were tested through medium-throughput screening assays. A subset of 14 compounds successfully inhibited the enzyme activity in the low micromolar range of at least one of the enzymes from both Trypanosoma brucei and Lesihmania major parasites (pan-inhibitors), or from both PTR1 and DHFR-TS of the same parasite (dual inhibitors). Molecular docking studies of the protein–ligand interaction focused on new scaffolds not reproducing the well-known antifolate core clearly explaining the experimental data. TCMDC-143249, classified as a benzenesulfonamide derivative by the QikProp descriptor tool, showed selective inhibition of PTR1 and growth inhibition of the kinetoplastid parasites in the 5 μM range. In our work, we enlarged the biological profile of the GSK Kinetobox and identified new core structures inhibiting selectively PTR1, effective against the kinetoplastid infectious protozoans. In perspective, we foresee the development of selective PTR1 and DHFR inhibitors for studies of drug combinations. Keywords: GSK Kinetobox; PTR1; DHFR-TS; Leishmaniasis; trypanosomiasis; drug discovery; molecular modelling; medium throughput screening 1. Introduction Neglected tropical diseases (NTDs) are a diverse set of 20 diseases that cause a dev- astating human, social and economic burden on more than 1 billion people worldwide, predominantly in tropical and subtropical areas [1]. Trypanosomatids are single-celled pro- tozoan parasites, which cause various diseases such as Leishmaniasis, Chagas disease and human African trypanosomiasis (HAT), all known as vector borne parasitic diseases [2,3]. The little or no prospects of financial gain has made the pharmaceutical industry show low interest in developing new drugs for NTDs [4]. The treatment with currently available drugs, discovered decades ago, presents many drawbacks, such as high toxicity, poor efficacy, difficulties in administration and drug resistance [5–9]. Thus, there is an urgent need to discover new, improved and affordable drugs as well as promising drug targets for the design of new antiparasitic compounds. Pharmaceuticals 2021, 14, 1246. https://doi.org/10.3390/ph14121246 https://www.mdpi.com/journal/pharmaceuticals