DMD Fast Forward. Published on March 3, 2015 as DOI: 10.1124/dmd.114.062539 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 62539 Towards Predicting Drug-Induced Liver Injury (DILI): Parallel Computational Approaches to Identify MRP4 and BSEP Inhibitors Matthew A. Welch, Kathleen Köck, Thomas J. Urban, Kim L. R. Brouwer, and Peter W. Swaan Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland (M.A.W., Downloaded from P.W.S.); Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (K.K., T.J.U., K.L.R.B.); Center for Human Genome Variation, Duke University Medical Center, Durham, North dmd.aspetjournals.org Carolina (T.J.U.) at ASPET Journals on October 1, 2021 1 DMD Fast Forward. Published on March 3, 2015 as DOI: 10.1124/dmd.114.062539 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 62539 Running Title: Computational Modeling of MRP4 and BSEP to Predict DILI Corresponding author: Peter W. Swaan, Ph.D., Department of Pharmaceutical Sciences, University of Maryland, Baltimore MD 21201. Tel. 410-706-0103. Email [email protected] Number of text pages: 34 Downloaded from Figures: 9 Tables: 2 dmd.aspetjournals.org Number of References: 26 Number of Words in at ASPET Journals on October 1, 2021 Abstract: 249 Introduction: 693 Discussion: 1,356 Abbreviations: BSEP, bile salt export pump; DILI, drug-induced liver injury; PFIC, progressive familial intrahepatic cholestasis; MRP, multidrug resistance protein 2 DMD Fast Forward. Published on March 3, 2015 as DOI: 10.1124/dmd.114.062539 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 62539 Abstract Drug-induced liver injury (DILI) is an important cause of drug toxicity. Inhibition of MRP4, in addition to BSEP, might be a risk factor for the development of cholestatic DILI. Recently, we demonstrated that inhibition of MRP4, in addition to BSEP, may be a risk factor for the development of cholestatic DILI. Here, we aimed to develop computational models to delineate molecular features underlying MRP4 and BSEP inhibition. Models were developed Downloaded from using 257 BSEP and 86 MRP4 inhibitors and non-inhibitors in the training set. Models were externally validated and used to predict the affinity of compounds towards BSEP and MRP4 in the DrugBank database. Compounds with a score above the median fingerprint threshold were dmd.aspetjournals.org considered to have significant inhibitory effects on MRP4 and BSEP. Common feature pharmacophore models were developed for MRP4 and BSEP with LigandScout software using a training set of 9 well-characterized MRP4 inhibitors and 9 potent BSEP inhibitors. Bayesian at ASPET Journals on October 1, 2021 models for BSEP and MRP4 inhibition/non-inhibition were developed with cross-validated Receiver Operator Curve (ROC) values greater than 0.8 for the test sets, indicating robust models with acceptable false positive and false negative prediction rates. Both MRP4 and BSEP inhibitor pharmacophore models were characterized by hydrophobic and hydrogen-bond acceptor features, albeit in distinct spatial arrangements; similar molecular features between MRP4 and BSEP inhibitors may partially explain why various drugs have affinity for both transporters. The Bayesian (BSEP, MRP4) and pharmacophore (MRP4, BSEP) models demonstrated significant classification accuracy and predictability. 3 DMD Fast Forward. Published on March 3, 2015 as DOI: 10.1124/dmd.114.062539 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 62539 Introduction Drug-induced liver injury (DILI) is an important cause of drug toxicity and a major reason for withdrawal of drugs from the market (Abboud and Kaplowitz, 2007) or attrition of drug candidates in late development stages, which can be extremely costly. Unfortunately, current in vitro screens or in vivo preclinical studies cannot accurately predict the potential of compounds to cause hepatotoxicity. DILI remains a major concern in drug discovery and clinical development. This obstacle has necessitated a search for alternative technologies, such as Downloaded from computational approaches to decrease the risk of DILI-associated late-stage failures. Despite extensive research, the underlying mechanisms of DILI are not well understood. However, it is clear that compound-related properties as well as individual patient characteristics dmd.aspetjournals.org affect the occurrence of DILI. Formation of reactive metabolites, mitochondrial impairment, and inhibition of canalicular bile acid transport mediated by the bile salt export pump (BSEP) (e.g. at ASPET Journals on October 1, 2021 troglitazone, bosentan, erythromycin) (Stieger et al., 2000; Fattinger et al., 2001; Kostrubsky et al., 2003) are known risk factors for the development of DILI in humans. This has been substantiated by large scale in vitro screening studies revealing that drugs that cause cholestatic DILI have higher potencies as well as frequencies of BSEP inhibition compared to drugs that are not liver toxic or that cause hepatocellular DILI. (Morgan et al., 2010; Dawson et al., 2012) BSEP is located at the canalicular membrane of the hepatocyte where it is involved in the excretion of bile acids into bile under physiological conditions. (Noe et al., 2002) The importance of this protein in bile acid homeostasis is emphasized by the observation that mutations in the BSEP gene ABCB11 have been associated with progressive familial intrahepatic cholestasis type 2 (PFIC 2). Although BSEP inhibition may explain bile acid-mediated DILI liability for a large proportion of compounds, a subset of hepatotoxic drugs remains that cannot be explained by BSEP inhibition alone. 4 DMD Fast Forward. Published on March 3, 2015 as DOI: 10.1124/dmd.114.062539 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 62539 In addition to canalicular BSEP, multidrug resistance protein 4 (MRP4) is a bile acid efflux protein localized at the basolateral membrane of hepatocytes. While hepatic expression is low under normal physiological conditions, MRP4 up-regulation has been demonstrated under cholestatic conditions. MRP4 is hypothesized to serve as a back-up system for bile acid efflux from hepatocytes into sinusoidal blood when the normal vectorial transport of bile acids from hepatocytes into bile is compromised. (Scheffer et al., 2002; Teng and Piquette-Miller, 2007; Gradhand et al., 2008; Chai et al., 2012) Recently, we screened 88 drugs (BSEP inhibitors and Downloaded from non-inhibitors) for inhibition of MRP4-mediated transport of the prototypical substrate [3H]- dehydroepiandrosterone sulfate (DHEAS) and discovered potent MRP4 inhibition among dmd.aspetjournals.org cholestatic BSEP non-inhibitors. A statistically significant relationship was observed between the potency of MRP4 inhibition and the probability of cholestatic classification: for each 1% increase in MRP4 inhibition, the probability that a drug was cholestatic increased by 3.1%. at ASPET Journals on October 1, 2021 Interestingly, many BSEP inhibitors also were MRP4 inhibitors. These data suggested that MRP4 inhibition may serve as a confounding factor in BSEP-mediated DILI, or in some cases lead to DILI in the absence of BSEP inhibition. Thus, MRP4 inhibition may be an additional risk factor for the development of cholestatic DILI. The role of hepatic bile acid transport inhibition in the etiology of DILI emphasizes the urgent need to develop screening tools to accurately predict drug-bile acid transporter interactions. While in vitro membrane vesicle assays have been developed for BSEP and MRP4 screening, use of these assays early in drug development is time consuming, labor- and resource- intensive, and requires the physical availability of compounds (including metabolites) for testing. An alternative approach to in vitro testing is the use of computational models to predict drug-bile acid transporter interactions and aid in identifying transporter-associated DILI early in the drug 5 DMD Fast Forward. Published on March 3, 2015 as DOI: 10.1124/dmd.114.062539 This article has not been copyedited and formatted. The final version may differ from this version. DMD # 62539 discovery process. For example, pharmacophore models have been used in ligand-based drug design to define the key structural characteristics that a molecule must possess in order to bind to the biological target (Ekins et al., 2012). Since models for BSEP have been reported recently (Pedersen et al., 2013; Ritschel et al., 2014), the aim of the current study was to develop a comprehensive model for MRP4 inhibition and evaluate its predictive ability. In addition, we developed Bayesian models to delineate molecular features underlying both MRP4 and BSEP inhibition. These in silico models were used to identify potential novel MRP4 inhibitors by Downloaded from virtual screening of an existing database, and to classify drugs as BSEP and MRP4 inhibitors in an effort to correlate these features with DILI incidence. dmd.aspetjournals.org at ASPET Journals on October 1, 2021 6 DMD Fast Forward. Published on March 3, 2015 as DOI: 10.1124/dmd.114.062539 This article has not been copyedited and formatted. The final version may differ from this version.