molecules Article Molecular Dynamic Simulations to Probe Stereoselectivity of Tiagabine Binding with Human GAT1 Sadia Zafar and Ishrat Jabeen * Research Center for Modeling and Simulation (RCMS), National University of Sciences and Technology (NUST), Islamabad 44000, Pakistan; [email protected] * Correspondence: [email protected] Academic Editors: Maria Cristina De Rosa and Bernard Maigret Received: 3 September 2020; Accepted: 12 October 2020; Published: 16 October 2020 Abstract: The human gamma aminobutyric acid transporter subtype 1 (hGAT1) located in the nerve terminals is known to catalyze the neuronal function by the electrogenic reuptake of γ-aminobutyric + acid (GABA) with the co-transport of Na and Cl− ions. In the past, there has been a major research drive focused on the dysfunction of hGAT1 in several neurological disorders. Thus, hGAT1 of the GABAergic system has been well established as an attractive target for such diseased conditions. Till date, there are various reports about stereo selectivity of –COOH group of tiagabine, a Food and Drug Administration (FDA)-approved hGAT1-selective antiepileptic drug. However, the effect of the stereochemistry of the protonated –NH group of tiagabine has never been scrutinized. Therefore, in this study, tiagabine has been used to explore the binding hypothesis of different enantiomers of tiagabine. In addition, the impact of axial and equatorial configuration of the–COOH group attached at the meta position of the piperidine ring of tiagabine enantiomers was also investigated. Further, the stability of the finally selected four hGAT1–tiagabine enantiomers namely entries 3, 4, 6, and 9 was evaluated through 100 ns molecular dynamics (MD) simulations for the selection of the best probable tiagabine enantiomer. The results indicate that the protonated –NH group in the R-conformation and the –COOH group of Tiagabine in the equatorial configuration of entry 4 provide maximum strength in terms of interaction within the hGAT1 binding pocket to prevent the change in hGAT1 conformational state, i.e., from open-to-out to open-to-in as compared to other selected tiagabine enantiomers 3, 6, and 9. Keywords: molecular dynamics; GAT1 inhibitors; tiagabine stereoisomers; GABA reuptake 1. Introduction γ-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the mammalian central nervous system (CNS) that contributes to the regulation of several physiological processes such as plasticity, learning, and memory [1]. The major neuronal regulator of GABA i.e., GABA transporter 1 (GAT1), helps in maintaining the extracellular GABA concentration required for the activation of postsynaptic GABA receptors for fast inhibitory neurotransmission [2]. After inhibitory neurotransmission, the major proportion of extra GABA from the synaptic cleft is reverted to presynaptic neurons through the human gamma aminobutyric acid transporter subtype 1 (hGAT1) reuptake process [3,4]. With regard to GAT1’s function in GABA regulation, abnormal hGAT1 neurotransmission has been associated with low GABA levels in the synaptic cleft, which may implicate several neurological disorders such as Alzheimer’s disease [5], schizophrenia [6,7], Parkinson’s disease [8], and epilepsy [9]. Thus, to amplify the GABA-mediated inhibitory neurotransmission/GABA activity in the CNS, the most widely utilized approach is the inhibition of GABA reuptake by locking hGAT1 Molecules 2020, 25, 4745; doi:10.3390/molecules25204745 www.mdpi.com/journal/molecules MoleculesMolecules 20202020,, 2525,, x; 4745 doi: 22 of of 19 18 neurotransmission/GABA activity in the CNS, the most widely utilized approach is the inhibition of GABAin its open-to-out reuptake by conformation locking hGAT1 [4, 10in ].its Hence, open-to-ou amongt conformation all the three [4,10]. distinct Hence, conformations among all ofthe hGAT1 three + distinctduring theconformations translocation of cycle,hGAT1 i.e., during open-to-out the transloc (mediatingation cycle, the co-transport i.e., open-to-out of two (mediating Na and onethe Clco-− transportions along of withtwo Na the+ substrateand one Cl GABA− ions fromalong thewith extracellular the substrate space), GABA occluded-out from the extracellular (representing space), the + occluded-outsealed Na /Cl (representing− ions and GABA the sealed in the Na binding+/Cl− ions pocket and of GABA hGAT1), in the and binding open-to-in pocket (releasing of hGAT1), ions and and open-to-inGABA into (releasing the intracellular ions and space) GABA conformations, into the intracellular the open-to-out space) conformation conformations, is the the most open-to-out targeted conformationconformation is to blockthe most the reuptaketargeted ofconformation extra GABA to from block the the extracellular reuptake spaceof extra thereby GABA normalizing from the extracellularthe synaptic space GABA thereby concentration normalizing [11–13 th].e synaptic GABA concentration [11–13]. TheThe determination determination of of the the crystal crystal structures structures of of AquifexAquifex aeolicus’ aeolicus’ leucineleucine transporter transporter ( (AaAaLeuT)LeuT) [14] [14] andand DrosophilaDrosophila melanogaster’s melanogaster’s dopaminedopamine transporter transporter (dDAT) (dDAT) [15] [15] provided provided the the possibility possibility of of the the first first structure-basedstructure-based ligand ligand docking docking and and simulation simulation in in hGAT1. hGAT1. Since Since 1950s, 1950s, seve severalral different different functional functional groupsgroups have beenbeen introducedintroduced to to the the hGAT1 hGAT1 inhibitors inhibitors in order in order to improve to improve their selectivitytheir selectivity and affi andnity. affinity.Until now, Until nipecotic now, nipecotic acid (polar, acid zwitterionic (polar, zwitterionic GABA analog) GABA and analog) its subsequent and its subsequent synthesized synthesized derivatives derivativesare employed are to employed inhibit in vitroto inhibithGAT1 in activityvitro hGAT [16–181 ].activity The general [16–18]. architecture The general of hGAT1 architecture inhibitors of hGAT1has a common inhibitors pattern has a common of attachment pattern of of lipophilic attachment chain of lipophilic to the parent chai moleculen to the parent (e.g., molecule nipecotic (e.g., acid) nipecoticfollowed acid) by the followed substitution by the ofsubstitution aromatic moieties of aromat asic inmoieties case of as two in case well-known of two well-known hGAT1 inhibitors hGAT1 inhibitorstiagabine [tiagabine19] and SKF-89976A [19] and SKF-89976A [20], of which [20], tiagabine of which is thetiagabine only approved is the only antiepileptic approved FDA antiepileptic drug [21]. FDAPrevious drug studies [21]. Previous illustrate studies that the illustrateR-enantiomers that the of R hGAT1-enantiomers inhibitors, of hGAT1 e.g., R -nipecoticinhibitors, acide.g., and R- nipecoticR-SK&F-89976A acid and (Figure R-SK&F-89976A1), are known (Figure to have1), are better known hGAT1 to have inhibitory better hGAT1 activity inhibitory in comparison activity toin comparisonS-enantiomers to S [22-enantiomers]. [22]. FigureFigure 1. 1. ChemicalChemical structures structures of of well-known well-known inhibitors inhibitors of of the the human human gamma gamma aminobutyric aminobutyric acid acid transportertransporter subtype subtype 1 1 (hGAT1). (hGAT1). However,However, the impactimpact ofof stereoisomerismstereoisomerism ( R(R/S/Sconfiguration) configuration) of of the the protonated protonated –NH –NH group group of theof thepolar polar moiety moiety (e.g., (e.g., piperidine, piperidine, pyrrolidine, pyrrolidine, or azetidine or azetidine ring)along ring) withalong the with orientation the orientation of aromatic of moieties attached to the linker chain of hGAT1 inhibitors on biological activity (IC ) has not been aromatic moieties attached to the linker chain of hGAT1 inhibitors on biological activity50 (IC50) has not beendetermined determined yet. Therefore, yet. Therefore, the current the study current explores study the explores binding hypothesisthe binding of Rhypothesis/S conformations of R/ ofS conformationsthe –NH group of that the may –NH provide group athat starting may pointprovide for a the starting design point of a new for setthe ofdesign selective of a inhibitors new set of of selectivehGAT1 in inhibitors neurological of hGAT1 disorders. in neurological The finally selected disorders. binding The hypothesisfinally selected of tiagabine binding distereoisomers hypothesis of tiagabinewas further distereoisomers cross validated was with further the stereoisomers cross validated of another with knownthe stereoisomers inhibitor of GAT1,of another i.e., NNC-711 known inhibitor(Figure1). of GAT1, i.e., NNC-711 (Figure 1). 2. Results and Discussion 2. Results and Discussion 2.1. Docking and Clustering of R- and S-enantiomers of Tiagabine in hGAT1 2.1. Docking and Clustering of R- and S-enantiomers of Tiagabine in hGAT1 Overall, binding solutions of both R- and S-enantiomers of tiagabine obtained in docking protocol I sharedOverall, similar binding binding solutions positions of at both transmembrane R- and S-enantiomers (TM) segments of tiagabine 1a, 1b, 6a, obtained 6b, and 10in of docking hGAT1. protocolHowever, I shared binding similar conformations binding positions of the –NH at transmembrane group, –COOH (TM) group, segments and thiophene 1a, 1b, 6a, rings 6b, and of R -10 and of hGAT1. However, binding conformations of the –NH group, –COOH group, and thiophene