Modeling, substrate docking, and mutational analysis identify residues essential for the function and specificity of a eukaryotic purine-cytosine NCS1 transporter

Background: The purine-cytosine FcyB transporter is a prototype member of the NCS1 family. Results: Using homology modeling, substrate docking, and rational mutational analysis, we identify residues critical for function and specificity. Conclusion: Important aspects concerning the molecular mechanism and evolution of transporter specificity are revealed. Significance: The first systematic approach on structure-function-specificity relationships in a eukaryotic NCS1 member is shown

A substrate translocation trajectory in the monocarboxylate/h+ symporter jen1

Previous mutational analysis of Jen1p, a Saccharomyces cerevisiae monocarboxylate/H+ symporter of the Major Facilitator Superfamily, has suggested that the consensus sequence 379NXX[S/T]HX[S/T]QD387, located in transmembrane segment VII (TMS-VII), is part of the substrate translocation pathway. In this work, we rationally design and analyse novel mutations concerning residues in TMS-V and TMS-XI. Our analysis identifies several residues critical for Jen1p function. Among these, F270 (TMS-V) and Q498 (TMS-XI) function as specificity determinants for the distinction of mono- from di-carboxylates, whereas N501 is irreplaceable for function. Using a novel theoretical model created on the basis of Jen1p similarity with GltP permease, we demonstrate that all polar residues in TMS-VII and TMS-XI, shown previously and herein to be critical for function and/or specificity (N379, H383, D387, Q498, N501), are perfectly aligned in a row along an imaginary axis that lies parallel to a protein pore. The model also predicts that the flexible side-chain of an additional polar residue, R188 in TMSII, faces the pore and subsequent mutational analysis showed that this aminoacid, similar to most polar residues of the pore, is irreplaceable for function. Finally, our model shows that the location of F270 and Q498 could justify their role in substrate specificity. Independent substrate docking approaches reveal a ‘trajectory-like’ displacement of the substrate within the Jen1p pore. In this inward-facing trajectory the flexible side-chain of R188 plays a major dynamic role mediating the orderly relocation of the substrate by subsequent H-bond interactions involving itself and residues H383, N501 and Q498.I.S.S. (SFRH/BPD/22976/2005) and J.S.P. (SFRH/BD/61530/2009) received fellowships from FC

Identification of amino acid residues critical for the substrate translocation in lactate permease JEN1p of saccharomyces cerevisiae

Lactic, acetic and propionic acids have been used for many years in industrial and pharmaceutical companies. In Saccharomyces cerevisiae, Jen1p is a major monocarboxylate:H+ symporter specific primarily for lactate, pyruvate and for acetate (TC # 2.A.1.12.2) (Casal et al., 1999). A phylogenetic tree of ScJen1p homologues (Casal et al., 2008) showed the existence of two main clusters: a Jen1 group (monocarboxylate transporters) and a Jen2-like (dicarboxylate transporters). Structure-function relationships in Jen1p have been approached by using a rational mutational analysis of conserved amino acid residues (Soares-Silva et al., 2007). Analysis of the conserved sequence 379NXX[S/T]HX[S/T]QDXXXT391, located in transmembrane segment seven (TMS-VII), showed that residues N379, H383 or D387 are necessary for function and specificity, while Q386 is important for the kinetics of Jen1p-mediated transport. In this work, we rationally designed and analyzed novel mutations in conserved regions located in TMS-II, TMS-V and TMS-XI of Jen1p, which we predicted to affect Jen1p specificity (distinction between mono and dicarboxylates) and function. Among the residues studied, F270 (TMS-V) and Q498 (TMS-XI) are specificity determinants for the distinction of mono- from dicarboxylates, and N501 (TMS-XI) is critical for function. Using a model based on Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS-VII and TMS-XI are aligned along the protein pore and substrate docking studies reveal a potential substrate translocation trajectory consisting mostly of the polar residues genetically identified as important for function. Overall, our results constitute a first step towards the genetic manipulation of substrate specificity in the lactate/pyruvate:H+ symporter subfamily and a tool for the in silico prediction of the function of Jen1p homologues in other fungi (Soares-Silva et al., 2011).I.S.S. (SFRH/BPD/22976/2005) and J.S.P. (SFRH/BD/61530/2009) received fellowships from FC

Lipophilic Guanylhydrazone Analogues as Promising Trypanocidal Agents: An Extended SAR Study.

In this report, we extend the SAR analysis of a number of lipophilic guanylhydrazone analogues with respect to in vitro growth inhibition of Trypanosoma brucei and Trypanosoma cruzi. Sleeping sickness and Chagas disease, caused by the tropical parasites T. brucei and T. cruzi, constitute a significant socioeconomic burden in low-income countries of sub-Saharan Africa and Latin America, respectively. Drug development is underfunded. Moreover, current treatments are outdated and difficult to administer, while drug resistance is an emerging concern. The synthesis of adamantane-based compounds that have potential as antitrypanosomal agents is extensively reviewed. The critical role of the adamantane ring was further investigated by synthesizing and testing a number of novel lipophilic guanylhydrazones. The introduction of hydrophobic bulky substituents onto the adamantane ring generated the most active analogues, illustrating the synergistic effect of the lipophilic character of the C1 side chain and guanylhydrazone moiety on trypanocidal activity. The n-decyl C1-substituted compound G8 proved to be the most potent adamantane derivative against T. brucei with activity in the nanomolar range (EC50=90 nM). Molecular simulations were also performed to better understand the structure-activity relationships between the studied guanylhydrazone analogues and their potential enzyme target

Scaffold hybridization strategy towards potent hydroxamate-based inhibitors of Flaviviridae viruses and Trypanosoma species.

Infections with Flaviviridae viruses, such as hepatitis C virus (HCV) and dengue virus (DENV) pose global health threats. Infected individuals are at risk of developing chronic liver failure or haemorrhagic fever respectively, often with a fatal outcome if left untreated. Diseases caused by tropical parasites of the Trypanosoma species, T. brucei and T. cruzi, constitute significant socioeconomic burden in sub-Saharan Africa and continental Latin America, yet drug development is under-funded. Anti-HCV chemotherapy is associated with severe side effects and high cost, while dengue has no clinically approved therapy and antiparasitic drugs are outdated and difficult to administer. Moreover, drug resistance is an emerging concern. Consequently, the need for new revolutionary chemotherapies is urgent. By utilizing a molecular framework combination approach, we combined two distinct chemical entities with proven antiviral and trypanocidal activity into a novel hybrid scaffold attached by an acetohydroxamic acid group (CH2CONHOH), aiming at derivatives with dual activity. The novel spiro-carbocyclic substituted hydantoin analogues were rationally designed, synthesized and evaluated for their potency against three HCV genotypes (1b, 3a, 4a), DENV and two Trypanosoma species (T. brucei, T. cruzi). They exhibited significant EC50 values and remarkable selectivity indices. Several modifications were undertaken to further explore the structure activity relationships (SARs) and confirm the pivotal role of the acetohydroxamic acid metal binding group

Discovery and optimization of a selective ligand for the switch/sucrose nonfermenting-related bromodomains of polybromo protein-1 by the use of virtual screening and hydration analysis

Bromodomains (BRDs) are epigenetic interaction domains currently recognized as emerging drug targets for development of anticancer or anti-inflammatory agents. In this study, development of a selective ligand of the fifth BRD of polybromo protein-1 (PB1(5)) related to switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes is presented. A compound collection was evaluated by consensus virtual screening and a hit was identified. The biophysical study of protein−ligand interactions was performed using X-ray crystallography and isothermal titration calorimetry. Collective data supported the hypothesis that affinity improvement could be achieved by enhancing interactions of the complex with the solvent. The derived SAR along with free energy calculations and a consensus hydration analysis using WaterMap and SZmap algorithms guided rational design of a set of novel analogues. The most potent analogue demonstrated high affinity of 3.3 μM and an excellent selectivity profile, thus comprising a promising lead for the development of chemical probes targeting PB1(5)

Structural studies of the interactions between kinases ans small molecule inhibitors

The present study focuses on the implementation of various methodologies for the discovery and rational development of novel protein kinase inhibitors. Protein kinases constitute a superfamily of highly important and emerging drug targets involved in the pathology of many diseases. The subject of the study is the interaction between representative disease-relevant kinases like GSK-3β, the CDKs, the Auroras as well as DYRK1α and DYRK2 with small molecules that are potential inhibitors of those kinases. In the first part, a theoretical model of the interaction between GSK-3β and several inhibitors belonging to the indirubin scaffold is created using docking-scoring calculations. This model is utilized to gain insight to the phenomenon of inhibitor selectivity and then is used to design modifications for optimizing the inhibitors in terms of their inhibitory potency as well as their physicochemical profile. A similar model is created for the Aurora-indirubin interaction and several factors are determined that are critical for the creation of an accurate model like the presence of explicit structural water molecules. In the second part a comparative study of virtual and physical screening is presented and the evaluation of two compound collections towards the beforementioned kinases is performed. The two collections are the NCI repositoty and the collection of the NKUA School of Pharmacy. Several active compounds that can constitute novel leads for the development of highly original kinase inhibitors are recovered. Moreover, a comparison between the two methods is presented and the favourable contribution of virtual screening in the drug discovery process is discussed. The third part begins with the structural study of the interaction of DYRK2 with a selective indirubin-based inhibitor. The study is enhanced by the determination of the apoenzyme structure of DYRK2. On the basis of the crystallographic data and by the utilization of theoretical models the reproduction of the experimental structure through modeling is achieved and the structural determinants for selective DYRK inhibition are elucidated.Η παρούσα διατριβή εστιάζει στην εφαρμογή διαφορετικών μεθοδολογιών με σκοπό την ανακάλυψη και ορθολογική ανάπτυξη νέων αναστολέων πρωτεϊνικών κινασών. Οι πρωτεϊνικές κινάσες αποτελούν φαρμακολογικούς στόχους υψηλής σημασίας, οι οποίοι σχετίζονται με τη θεραπευτική ενός μεγάλου αριθμού ασθενειών. Στην παρούσα εργασία μελετάται η αλληλεπίδραση μεταξύ επιλεγμένων αντιπροσώπων της υπεροικογένειας των κινασών όπως η GSK-3β, οι CDK1, 2 και 5, οι κινάσες Aurora καθώς και οι DYRK1α και DYRK2 με μόρια τα οποία έχουν εν δυνάμει ανασταλτική δράση έναντι των συγκεκριμένων πρωτεϊνών. Στο πρώτο μέρος της εργασίας, μέσω υπολογισμών πρόσδεσης και αποτίμησης κατασκευάζεται ένα θεωρητικό μοντέλο της αλληλεπίδρασης μεταξύ της κινάσης GSK-3β και αναστολέων οι οποίοι ανήκουν στο μοριακό σκελετό της ιντιρουμπίνης. Το μοντέλο που προκύπτει χρησιμοποιείται αρχικά για την κατανόηση της εκλεκτικότητας των μορίων αυτών και στη συνέχεια για το σχεδιασμό νέων μορίων με βελτιστοποιημένη βιολογική δράση και φυσικοχημικές ιδιότητες. Στη συνέχεια, η κατασκευή αντίστοιχου μοντέλου για την περίπτωση των κινασών Aurora οδηγεί σε προσδιορισμό των μεθοδολογικών παραγόντων εκείνων οι οποίοι είναι κρίσιμοι για την πιστή προσομοίωση παρομοίων συστημάτων, με βασικότερο παράγοντα την παρουσία επιλεγμένων διακριτών μορίων νερού στο ενεργό κέντρο των υπό μελέτη πρωτεϊνών. Στο δεύτερο μέρος παρουσιάζεται η παράλληλη εφαρμογή πειραμάτων εικονικής και φυσικής φαρμακολογικής αξιολόγησης μορίων ως υποψηφίων αναστολέων κινασών. Στη συνέχεια αποτιμώνται δύο συλλογές μικρών μορίων ως προς την ανασταλτική τους δράση έναντι των υπό μελέτη πρωτεϊνικών στόχων. Αξιολογείται η συλλογή του ιδρύματος NCI και η χημειοθήκη της Φαρμακευτικής σχολής και εντοπίζεται ένας σημαντικός αριθμός βιοδραστικών μορίων, τα οποία αποτελούν ενώσεις-οδηγούς με δυνατότητα μελλοντικής συνθετικής ανάπτυξης προς νέες κατηγορίες εκλεκτικών αναστολέων κινασών. Στο μέρος αυτό παρουσιάζεται επίσης η σύγκριση μεταξύ των δύο μεθόδων και στη συνέχεια ποσοτικοποιείται η συνεισφορά της μεθοδολογίας εικονικής αξιολόγησης στη διαδικασία ανακάλυψης νέων βιοδραστικών ενώσεων-οδηγών. Στο τρίτο μέρος παρουσιάζεται η κρυσταλλογραφική μελέτη της αλληλεπίδρασης μεταξύ της κινάσης DYRK2 και ενός εκλεκτικού αναστολέα της οικογένειας των ιντιρουμπινών. Η δομική μελέτη εμπλουτίζεται με τη δομή της DYRK2 στην αποενζυμική της μορφή. Με βάση τα κρυσταλλογραφικά δεδομένα και τη συνεισφορά θεωρητικών μοντέλων προκύπτουν παρατηρήσεις σχετικά με τη σχέση μεταξύ της δομής του αναστολέα και της δραστικότητας προς τη συγκεκριμένη οικογένεια κινασών. Επιπλέον καθορίζονται οι κρίσιμες παραμέτροι για την πιστή αναπαραγωγή των κρυσταλλογραφικών δεδομένων μέσω θεωρητικών υπολογισμών και τέλος, η ερμηνεία των δεδομένων οδηγεί σε προτάσεις σχετικά με το σχεδιασμό νέων εκλεκτικών αναστολέων των κινασών DYRK1α και DYRK2

Identification of the substrate recognition and transport pathway in a eukaryotic member of the nucleobase-ascorbate transporter (NAT) family.

Using the crystal structure of the uracil transporter UraA of Escherichia coli, we constructed a 3D model of the Aspergillus nidulans uric acid-xanthine/H(+) symporter UapA, which is a prototype member of the Nucleobase-Ascorbate Transporter (NAT) family. The model consists of 14 transmembrane segments (TMSs) divided into a core and a gate domain, the later being distinctly different from that of UraA. By implementing Molecular Mechanics (MM) simulations and quantitative structure-activity relationship (SAR) approaches, we propose a model for the xanthine-UapA complex where the substrate binding site is formed by the polar side chains of residues E356 (TMS8) and Q408 (TMS10) and the backbones of A407 (TMS10) and F155 (TMS3). In addition, our model shows several polar interactions between TMS1-TMS10, TMS1-TMS3, TMS8-TMS10, which seem critical for UapA transport activity. Using extensive docking calculations we identify a cytoplasm-facing substrate trajectory (D360, A363, G411, T416, R417, V463 and A469) connecting the proposed substrate binding site with the cytoplasm, as well as, a possible outward-facing gate leading towards the substrate major binding site. Most importantly, re-evaluation of the plethora of available and analysis of a number of herein constructed UapA mutations strongly supports the UapA structural model. Furthermore, modeling and docking approaches with mammalian NAT homologues provided a molecular rationale on how specificity in this family of carriers might be determined, and further support the importance of selectivity gates acting independently from the major central substrate binding site