VirtuaDrug Ltd.

Budapest, Hungary

VirtuaDrug Ltd.

Budapest, Hungary
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Poguntke M.,Friedrich - Alexander - University, Erlangen - Nuremberg | Hazai E.,Virtua Drug Ltd | Fromm M.F.,Friedrich - Alexander - University, Erlangen - Nuremberg | Zolk O.,Friedrich - Alexander - University, Erlangen - Nuremberg
Expert Opinion on Drug Metabolism and Toxicology | Year: 2010

Importance of the field: The ATP-binding cassette transporter ABCG2 is a well-known major mediator of multi-drug resistance in cancers. Beyond multi-drug resistance, experimental and recent clinical studies demonstrate a role for ABCG2 as a determinant of drug pharmacokinetic, safety and efficacy profiles. Areas covered in this review: The clinical evidence of the role of ABCG2 in pharmacokinetics and pharmacodynamics is reviewed. Key questions that arise from the perspective of preclinical drug evaluation are addressed, including the structure of ABCG2 and mechanisms of drugtransporter interactions, mechanisms responsible for the polyspecificity of ABCG2, methods suitable for studying drugABCG2 interactions in vitro and in silico prediction of ABCG2 substrates and inhibitors. What the reader will gain: An update on current knowledge of the importance of ABCG2 in drug disposition with special emphasis on drug development. Take home message: The field of drugABCG2 interaction is rapidly advancing and beginning to expand into clinical practice. However, the structural understanding of drug binding and transport by ABCG2 is still incomplete. Incorporation of novel concepts of drugtransporter interactions such as electrostatic funneling might help explain the multispecificity of ABCG2 and enable in silico predictions. © 2010 Informa UK, Ltd.


Li L.,University of Minnesota | Sham Y.Y.,University of Minnesota | Bikadi Z.,Virtua Drug Ltd. | Elmquist W.F.,University of Minnesota
Drug Metabolism and Disposition | Year: 2011

Breast cancer resistance protein (BCRP), an ATP-dependent efflux transporter, confers drug resistance to many chemotherapy agents. BCRP is overexpressed in tumors exposed to an acidic environment; therefore, it is important to establish the effect of low pH on BCRP transport activity. It has recently been reported that BCRP transports substrates more efficiently in an acidic microenvironment. In the study presented here, we examine the pH dependence of BCRP using methothrexate (MTX), pemetrexed (PMX), and estrone sulfate (ES) as model substrates. Our study revealed an increase of approximately 40-fold in the BCRP-mediated transport of PMX and MTX when the pH was decreased from 7.4 to 5.5. In contrast, only a 2-fold increase was observed for ES. These results indicate a mechanism of transport that is directly dependent on the effective ionization state of the substrates and BCRP. For ES, which retains a constant ionization state throughout the applied pH, the observed mild increase in activity is attributable to the overall changes in the effective ionization state and conformation of BCRP. For MTX and PMX, the marked increase in BCRP transport activity was likely due to the change in ionization state of MTX and PMX at lowered pH and their intermolecular interactions with BCRP. To further rationalize the molecular basis of the pH dependence, molecular modeling and docking studies were carried out using a homology model of BCRP, which has previously been closely examined in structural and site-directed mutagenesis studies (Am J Physiol Cell Physiol 299:C1100-C1109, 2010). On the basis of docking studies, all model compounds were found to associate with arginine 482 (Arg482) by direct salt-bridge interactions via their negatively charged carboxylate or sulfate groups. However, at lower pH, protonated MTX and PMX formed an additional salt-bridge interaction between their positively charged moieties and the nearby negatively charged aspartic acid 477 (Asp477) carboxylate side chain. The formation of this "salt-bridge triad" is expected to increase the overall electrostatic interactions between MTX and PMX with BCRP, which can form a rational basis for the pH dependence of the observed enhanced binding selectivity and transport activity. Removal of Arg482 in site-directed mutagenesis studies eliminated this pH dependence, which lends further support to our binding model. These results shed light on the importance of electrostatic interactions in transport activity and may have important implications in the design of ionizable chemotherapeutics intended for tumors in the acidic microenvironment. Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics.


Gyimesi M.,Momentum | Harami G.M.,Momentum | Sarlos K.,Momentum | Hazai E.,VirtuaDrug Ltd. | And 2 more authors.
Nucleic Acids Research | Year: 2012

Bloom's syndrome DNA helicase (BLM), a member of the RecQ family, is a key player in homologous recombination (HR)-based error-free DNA repair processes. During HR, BLM exerts various biochemical activities including single-stranded (ss) DNA translocation, separation and annealing of complementary DNA strands, disruption of complex DNA structures (e.g. displacement loops) and contributes to quality control of HR via clearance of Rad51 nucleoprotein filaments. We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module. Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn2+-binding domain (residues 642-1077) can efficiently perform all mentioned HR-related activities. The results demonstrate that the Zn2+-binding domain is necessary for functional interaction with DNA. We show that the extensions of this core, including the winged-helix domain and the strand separation hairpin identified therein in other RecQ-family helicases, are not required for mechanochemical activity per se and may instead play modulatory roles and mediate protein-protein interactions. © 2012 The Author(s).


Ni Z.,University of Washington | Bikadi Z.,University of Manchester | Shuster D.L.,University of Washington | Zhao C.,University of Washington | And 3 more authors.
Biochemistry | Year: 2011

The human breast cancer resistance protein (BCRP/ABCG2) confers multidrug resistance and mediates the active efflux of drugs and xenobiotics. BCRP contains one nucleotide-binding domain (NBD) followed by one membrane-spanning domain (MSD). We investigated whether prolines in or near the transmembrane helices are essential for BCRP function. Six proline residues were substituted with alanine individually, and the mutants were stably expressed in Flp-In TM-293 cells at levels comparable to that of wild-type BCRP and predominantly localized on the plasma membrane of the cells. While P392A showed a significant reduction (35-50%) in the efflux activity of mitoxantrone, BODIPY-prazosin, and Hoechst 33342, P485A exhibited a significant decrease of approximately 70% in the efflux activity of only BODIPY-prazosin. Other mutants had no significant changes in the efflux activities of these substrates. Drug resistance profiles of the cells expressing the mutants correlated well with the efflux data. ATPase activity was not substantially affected for P392A or P485A compared to that of wild-type BCRP. These results strongly suggest Pro 392 and Pro 485 are important in determining the overall transport activity and substrate selectivity of BCRP, respectively. Prazosin differentially affected the binding of 5D3, a conformation-sensitive antibody, to wild-type BCRP, P392A, or P485A in a concentration-dependent manner. In contrast, mitoxantrone had no significant effect on 5D3 binding. Homology modeling indicates that Pro 392 may play an important role in the communication between the MSD and NBD as it is predicted to be located at the interface between the two functional domains, and Pro 485 induces flexible hinges that may be essential for the broad substrate specificity of BCRP. © 2011 American Chemical Society.


Ni Z.,University of Washington | Bikadi Z.,University of Washington | Bikadi Z.,Virtua Drug Ltd. | Cai X.,University of Washington | And 2 more authors.
American Journal of Physiology - Cell Physiology | Year: 2010

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics. In this study, we investigated the role of polar residues within or near the predicted transmembrane α-helices 1 and 6 of BCRP in drug transport. We substituted Asn 387, Gln 398, Asn 629, and Thr 642 with Ala, Thr 402 with Ala and Arg, and Tyr 645 with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Immunoblotting and confocal microscopy analysis revealed that all of the mutants were well expressed and predominantly targeted to the plasma membrane. While T402A and T402R showed a significant global reduction in the efflux of mitoxantrone, Hoechst 33342, and BODIPY-prazosin, N629A exhibited significantly increased efflux activities for all of the substrates. N387A and Q398A displayed significantly impaired efflux for mitoxantrone and Hoechst 33342, but not for BODIPY-prazosin. In contrast, T642A and Y645F showed a moderate reduction in Hoechst 33342 efflux only. Drug resistance profiles of human embryonic kidney-293 cells expressing the mutants generally correlated with the efflux data. Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Mutations of some of the polar residues may cause conformational changes, as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin. The inward-facing homology model of BCRP indicated that Thr 402 within transmembrane 1 may be important for helical interactions, and Asn 629 may be involved in BCRP-substrate interaction. In conclusion, we have demonstrated the functional importance of some of these polar residues in BCRP activity. Copyright © 2010 the American Physiological Society.


Rosenberg M.F.,University of Manchester | Bikadi Z.,University of Washington | Bikadi Z.,Virtua Drug Ltd. | Chan J.,University of Manchester | And 5 more authors.
Structure | Year: 2010

BCRP/ABCG2 mediates efflux of drugs and xenobiotics. BCRP was expressed in Pichia pastoris, purified to > 90% homogeneity, and subjected to two-dimensional (2D) crystallization. The 2D crystals showed a p121 symmetry and projection maps were determined at 5 Å resolution by cryo-electron microscopy. Two crystal forms with and without mitoxantrone were observed with unit cell dimensions of a = 55.4 Å, b = 81.4 Å, γ = 89.8°, and a = 57.3 Å, b = 88.0 Å, γ = 89.7°, respectively. The projection map without mitoxantrone revealed an asymmetric structure with ring-shaped density features probably corresponding to a bundle of transmembrane α helices, and appeared more open and less symmetric than the map with mitroxantrone. The open and closed inward-facing forms of BCRP were generated by homology modeling, representing the substrate-free and substrate-bound conformations in the absence of nucleotide, respectively. These models are consistent with the experimentally observed conformational change upon substrate binding. © 2010 Elsevier Ltd.


Zsila F.,Institute of Biomolecular Chemistry | Bikadi Z.,Virtua Drug Ltd. | Malik D.,Delta Services Ltd. | Hari P.,Delta Services Ltd. | And 3 more authors.
Bioinformatics | Year: 2011

Motivation: Human serum albumin (HSA), the most abundant plasma protein is well known for its extraordinary binding capacity for both endogenous and exogenous substances, including a wide range of drugs. Interaction with the two principal binding sites of HSA in subdomain IIA (site 1) and in subdomain IIIA (site 2) controls the free, active concentration of a drug, provides a reservoir for a long duration of action and ultimately affects the ADME (absorption, distribution, metabolism, and excretion) profile. Due to the continuous demand to investigate HSA binding properties of novel drugs, drug candidates and drug-like compounds, a support vector machine (SVM) model was developed that efficiently predicts albumin binding. Our SVM model was integrated to a free, web-based prediction platform (http://albumin.althotas.com). Automated molecular docking calculations for prediction of complex geometry are also integrated into the web service. The platform enables the users (i) to predict if albumin binds the query ligand, (ii) to determine the probable ligand binding site (site 1 or site 2), (iii) to select the albumin X-ray structure which is complexed with the most similar ligand and (iv) to calculate complex geometry using molecular docking calculations. Our SVM model and the potential offered by the combined use of in silico calculation methods and experimental binding data is illustrated. © The Author 2011. Published by Oxford University Press. All rights reserved.


Ni Z.,University of Washington | Bikadi Z.,Virtua Drug Ltd. | Rosenberg M.F.,University of Manchester | Mao Q.,University of Washington
Current Drug Metabolism | Year: 2010

The human breast cancer resistance protein (BCRP/ABCG2) is the second member of the G subfamily of the large ATP-binding cassette (ABC) transporter superfamily. BCRP was initially discovered in multidrug resistant breast cancer cell lines where it confers resistance to chemotherapeutic agents such as mitoxantrone, topotecan and methotrexate by extruding these compounds out of the cell. BCRP is capable of transporting non-chemotherapy drugs and xenobiotiocs as well, including nitrofurantoin, prazosin, glyburide, and 2-amino-1-methyl-6- phenylimidazo {4,5-b]pyridine. BCRP is frequently detected at high levels in stem cells, likely providing xenobiotic protection. BCRP is also highly expressed in normal human tissues including the small intestine, liver, brain endothelium, and placenta. Therefore, BCRP has been increasingly recognized for its important role in the absorption, elimination, and tissue distribution of drugs and xenobiotics. At present, little is known about the transport mechanism of BCRP, particularly how it recognizes and transports a large number of structurally and chemically unrelated drugs and xenobiotics. Here, we review current knowledge of structure and function of this medically important ABC efflux drug transporter. © 2010 Bentham Science Publishers Ltd.


Bikadi Z.,Virtua Drug Ltd. | Hazai I.,Virtua Drug Ltd. | Malik D.,Virtua Drug Ltd. | Jemnitz K.,Hungarian Academy of Sciences | And 7 more authors.
PLoS ONE | Year: 2011

Human P-glycoprotein (P-gp) is an ATP-binding cassette multidrug transporter that confers resistance to a wide range of chemotherapeutic agents in cancer cells by active efflux of the drugs from cells. P-gp also plays a key role in limiting oral absorption and brain penetration and in facilitating biliary and renal elimination of structurally diverse drugs. Thus, identification of drugs or new molecular entities to be P-gp substrates is of vital importance for predicting the pharmacokinetics, efficacy, safety, or tissue levels of drugs or drug candidates. At present, publicly available, reliable in silico models predicting P-gp substrates are scarce. In this study, a support vector machine (SVM) method was developed to predict P-gp substrates and P-gp-substrate interactions, based on a training data set of 197 known P-gp substrates and non-substrates collected from the literature. We showed that the SVM method had a prediction accuracy of approximately 80% on an independent external validation data set of 32 compounds. A homology model of human P-gp based on the X-ray structure of mouse P-gp as a template has been constructed. We showed that molecular docking to the P-gp structures successfully predicted the geometry of P-gp-ligand complexes. Our SVM prediction and the molecular docking methods have been integrated into a free web server (http://pgp.althotas.com), which allows the users to predict whether a given compound is a P-gp substrate and how it binds to and interacts with P-gp. Utilization of such a web server may prove valuable for both rational drug design and screening. © 2011 Bikadi et al.


Hazai E.,Virtua Drug Ltd. | Hazai I.,Virtua Drug Ltd. | Ragueneau-Majlessi I.,University of Washington | Chung S.P.,University of Washington | And 2 more authors.
BMC Bioinformatics | Year: 2013

Background: Human breast cancer resistance protein (BCRP) is an ATP-binding cassette (ABC) efflux transporter that confers multidrug resistance in cancers and also plays an important role in the absorption, distribution and elimination of drugs. Prediction as to if drugs or new molecular entities are BCRP substrates should afford a cost-effective means that can help evaluate the pharmacokinetic properties, efficacy, and safety of these drugs or drug candidates. At present, limited studies have been done to develop in silico prediction models for BCRP substrates.In this study, we developed support vector machine (SVM) models to predict wild-type BCRP substrates based on a total of 263 known BCRP substrates and non-substrates collected from literature. The final SVM model was integrated to a free web server.Results: We showed that the final SVM model had an overall prediction accuracy of ~73% for an independent external validation data set of 40 compounds. The prediction accuracy for wild-type BCRP substrates was ~76%, which is higher than that for non-substrates. The free web server (http://bcrp.althotas.com) allows the users to predict whether a query compound is a wild-type BCRP substrate and calculate its physicochemical properties such as molecular weight, logP value, and polarizability.Conclusions: We have developed an SVM prediction model for wild-type BCRP substrates based on a relatively large number of known wild-type BCRP substrates and non-substrates. This model may prove valuable for screening substrates and non-substrates of BCRP, a clinically important ABC efflux drug transporter. © 2013 Hazai et al.; licensee BioMed Central Ltd.

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