In Silico Strategy for Targeting the Mtor Kinase at Rapamycin Binding Site by Small Molecules

molecules Article In Silico Strategy for Targeting the mTOR Kinase at Rapamycin Binding Site by Small Molecules Serena Vittorio 1 , Rosaria Gitto 1 , Ilenia Adornato 1, Emilio Russo 2 and Laura De Luca 1,* 1 Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale Palatucci 13, I-98168 Messina, Italy; [email protected] (S.V.); [email protected] (R.G.); [email protected] (I.A.) 2 Science of Health Department, School of Medicine, University “Magna Graecia” of Catanzaro, Viale Europa e Germaneto, I-88100 Catanzaro, Italy; [email protected] * Correspondence: [email protected] or [email protected]; Tel.: +39-090-676-6410 Abstract: Computer aided drug-design methods proved to be powerful tools for the identification of new therapeutic agents. We employed a structure-based workflow to identify new inhibitors targeting mTOR kinase at rapamycin binding site. By combining molecular dynamics (MD) simulation and pharmacophore modelling, a simplified structure-based pharmacophore hypothesis was built starting from the FKBP12-rapamycin-FRB ternary complex retrieved from RCSB Protein Data Bank (PDB code 1FAP). Then, the obtained model was used as filter to screen the ZINC biogenic compounds library, containing molecules derived from natural sources or natural-inspired compounds. The resulting hits were clustered according to their similarity; moreover, compounds showing the highest pharmacophore fit-score were chosen from each cluster. The selected molecules were subjected to docking studies to clarify their putative binding mode. The binding free energy of the obtained complexes was calculated by MM/GBSA method and the hits characterized by the lowest DGbind Citation: Vittorio, S.; Gitto, R.; values were identified as potential mTOR inhibitors. Furthermore, the stability of the resulting Adornato, I.; Russo, E.; De Luca, L. In complexes was studied by means of MD simulation which revealed that the selected compounds Silico Strategy for Targeting the were able to form a stable ternary complex with FKBP12 and FRB domain, thus underlining their mTOR Kinase at Rapamycin Binding potential ability to inhibit mTOR with a rapamycin-like mechanism. Site by Small Molecules. Molecules 2021, 26, 1103. https://doi.org/ Keywords: mTOR; molecular dynamics simulation; ternary complex FKBP12-rapamycin-FRB; molec- 10.3390/molecules26041103 ular docking and structure-based virtual screening; pharmacophore modeling Academic Editor: Rita Guedes Received: 23 December 2020 1. Introduction Accepted: 17 February 2021 Published: 19 February 2021 The mammalian target of rapamycin (mTOR) protein is a highly conserved eukary- otic Serine/Threonine (Ser/Thr) protein kinase, which belongs to phosphatidylinositol Publisher’s Note: MDPI stays neutral 3-kinase-related kinase (PI3K) family. mTOR plays a central role in various cellular pro- with regard to jurisdictional claims in cesses including protein biosynthesis by controlling transcription, translation and ribosomal published maps and institutional affil- biosynthesis thus regulating cell growth and size. Since the mTOR inhibition causes cell iations. cycle arrest at G1 phase, the intervention on mTOR pathway might provide a significant direction to new drug development aimed to the identification of anticancer, antiprolifera- tive, anti-restenosis and immunosuppressor agents. In addition, recent evidence suggests that an altered mTOR pathway is implicated in several neurological disorders [1–3]. Copyright: © 2021 by the authors. The mTOR is composed of 2549 amino acidic residues (289 kDa) and organized into Licensee MDPI, Basel, Switzerland. five distinct functional domains, (i) 32 replicates called “Huntington, elongation factor This article is an open access article 3,PR65/A, TOR” (HEAT) (from amino acid residue 16 to 1345); (ii) “FRAP, ATM, TRRAP” distributed under the terms and (FAT) (from amino acid residue 1382 to1982); (iii) “FKBP12 rapamycin-binding” region conditions of the Creative Commons (FRB) (from aminoacid residue 2012 to 2144); (iv) catalytic “kinase domain”(KD) and Attribution (CC BY) license (https:// “regulatory domain” (RD) (from aminoacid residue 2182 to 2516); and (v) “FRAP ATM creativecommons.org/licenses/by/ TRRAP carboxyterminus” (FATC) (from aminoacid residue 2517 to 2549). 4.0/). Molecules 2021, 26, 1103. https://doi.org/10.3390/molecules26041103 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 14 Molecules 2021, 26, 1103 (FRB) (from aminoacid residue 2012 to 2144); (iv) catalytic “kinase domain”(KD) and “reg-2 of 13 ulatory domain” (RD) (from aminoacid residue 2182 to 2516); and (v) “FRAP ATM TRRAP carboxyterminus” (FATC) (from aminoacid residue 2517 to 2549) FromFrom a functional a functional point point of of view, view, mTOR existsexists inin two two distinct distinct multi-protein multi-protein complexes com- plexescommonly commonly known known as mTOR as complexmTOR 1complex (mTORC1) 1 and(mTORC1) mTOR complex and mTOR 2 (mTORC2) complex located 2 (mTORC2)in different located cellular in compartments.different cellular It has comp beenartments. well established It has been that well the rapamycin-sensitive established that themTORC1 rapamycin-sensitive complex transmits mTORC1 signals complex of nutrient transmits availability signals of to nutrient control availability various cellular to controlfunctions, various while cellular the rapamycin-insensitivefunctions, while the rapamycin-insensitive mTORC2 complex regulatesmTORC2 thecomplex assembly reg- of ulatesthe cytoskeletonthe assembly byof actin.the cytoskeleton by actin. It isIt well-known is well-known that that for for the the microbial-derived microbial-derived macrolide macrolide rapamycin rapamycin (sirolimus) (sirolimus) and and its itsanalogues analogues (Scheme (Scheme 1) 1the) the inhibition inhibition mechanism mechanism of ofmTOR mTOR pathway pathway occurs occurs through through the the bindingbinding with with FKBP12 FKBP12 domain domain to to form form a a binary binary complex; inin turn,turn, this this complex complex binds binds to to FRB FRBdomain domain of of mTOR mTOR only only when when is associatedis associated to otherto other mTORC1 mTORC1 components; components; this this association asso- ciationinduces induces changes changes in the in mTOR the mTOR conformation, conformation, thus thus inhibiting inhibiting the crucial the crucial kinase kinase activity ac- of tivitythe of mTORC1 the mTORC1 [4,5]. Experimental [4,5]. Experimental studies studies highlighted highlighted that in absencethat in absence of FKBP12, of FKBP12, rapamycin rapamycinpossesses possesses a modest a affinitymodestfor affinity the FRB for domain,the FRB domain, suggesting suggesting that the FKBP12-rapamycinthat the FKBP12- rapamycincomplex complex plays a pivotal plays a role pivotal for the role binding for the tobinding mTOR to [6 mTOR]. [6]. Scheme 1. Chemical structures of Rapamycin and rapalogs as well-established mTOR/FKBP12 ligands. Scheme 1. Chemical structures of Rapamycin and rapalogs as well-established mTOR/FKBP12 ligands. Rapamycin is 31-membered macrocyclic lactone produced by Streptomyces hygro- scopicusRapamycinwhich is was 31-membered developed asmacr anocyclic immunosuppressant lactone produced agent by as Streptomyces allosteric inhibitor hygro- of scopicusmTORC1. which The was crystal developed structure as an of theimmunosuppressant FKBP12–rapamycin–mTOR agent as allosteric ternary complexinhibitor(PDB of mTORC1.code 1FAP) The crystal unveiled structure the protein of the interactions. FKBP12–rapamycin–mTOR It has been found ternary that complex the pipecolyl (PDB α- codeketoamide 1FAP) unveiled of rapamycin the protein anchored interactions it into the. It proline-bindinghas been found pocket,that thewhereas pipecolyl the α-ke- triene toamidesystem of was rapamycin exposed anchored for interactions it into withthe pr mTOR.oline-binding Rapamycin pocket, displays whereas low the water-solubility triene sys- temand was poor exposed stability, for sointeractions that rapamycin with analoguesmTOR. Rapamycin (also named displays rapalogs) low withwater-solubility improved bio- andpharmaceutical poor stability, properties so that rapamycin have been analogues developed [(also7,8] andnamed approved rapalogs) by FDA with (see improved Scheme 1) biopharmaceuticalas the first-generation properties of mTOR have inhibitors been developed to fight cancer [7,8] malignanciesand approved and by other FDA diseases. (see SchemeApart 1) from as the the first-generation weakness in poor of mTOR druglike inhibitors properties, to fight the rapalogscancer malignancies possess a complex and otherchemical diseases. structure Apart [from5]; therefore, the weakness the structural in poor druglike modifications properties of macrolide, the rapalogs ring were possess gener- a complexally limited. chemical structure [5]; therefore, the structural modifications of macrolide ring were generallyFurther limited. allosteric mTOR inhibitors belonging to rapalog series are modified at C- 7,Further C-22, C-27 allosteric and C-42 mTOR positions inhibitors as well belonging as the to C-1/C4 rapalog fragment. series are A modified carefully at analysis C-7, C-22,of structure-activityC-27 and C-42 positions relationships as well as of the rapalogs C-1/C4 hasfragment. been recentlyA carefully reported analysis [5 of]; struc- the best ture-activityresults were relationships obtained for of structural rapalogs has optimization been recently carried reported out addressing

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