1521-0111/89/1/42–52$25.00 http://dx.doi.org/10.1124/mol.115.100693 MOLECULAR PHARMACOLOGY Mol Pharmacol 89:42–52, January 2016 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics Molecular Interactions and Implications of Aldose Reductase Inhibition by PGA1 and Clinically Used Prostaglandins Beatriz Díez-Dacal,1 Francisco J. Sánchez-Gómez,1 Pedro A. Sánchez-Murcia, Ivana Milackova, Tahl Zimmerman, Jana Ballekova, Elena García-Martín, José A. G. Agúndez, Severine Gharbi, Federico Gago, Milan Stefek, and Dolores Pérez-Sala Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain (B.D.-D., F.J.S.-G., T.Z., D.P.-S.); Department of Biomedical Sciences, Universidad de Alcalá, Madrid, Spain (P.A.S.-M., F.G.); Institute of Experimental Pharmacology and Toxicology, Slovak Academy of Sciences, Bratislava, Slovakia (I.M., J.B., M.S.); Department of Pharmacology, University of Extremadura, Cáceres, Spain (E.G.-M., J.A.G.A.); Centro Downloaded from Nacional de Biotecnología, Madrid, Spain (S.G.) Received July 6, 2015; accepted October 19, 2015 ABSTRACT Aldose reductase (AKR1B1) is a critical drug target because of which was supported experimentally. Indeed, loss of biotin molpharm.aspetjournals.org its involvement in diabetic complications, inflammation, and label from the AKR1B1-PGA1-B adduct was favored by tumorigenesis. However, to date, development of clinically glutathione, indicating a retro-Michael reaction, which unveils useful inhibitors has been largely unsuccessful. Cyclopente- new implications of cyPG-protein interaction. PGA1 elicited none prostaglandins (cyPGs) are reactive lipid mediators that only marginal inhibition of aldehyde reductase (AKR1A1), bind covalently to proteins and exert anti-inflammatory and considered responsible for the severe adverse effects of many antiproliferative effects in numerous settings. By pursuing AKR1B1 inhibitors. Interestingly, other prostaglandins (PGs) targets for modification by cyPGs we have found that the inhibited the enzyme, including non-electrophilic PGE1 and cyPG PGA1 binds to and inactivates AKR1B1. A PGA1-AKR1B1 PGE2, currently used in clinical practice. Moreover, both PGA1 adduct was observed, both by matrix-assisted laser desorption/ and PGE1 reduced the formation of sorbitol in an ex-vivo model ionization time-of-flight mass spectrometry and by SDS-PAGE of diabetic cataract to an extent comparable to that attained by at ASPET Journals on October 1, 2021 using biotinylated PGA1 (PGA1-B). Insight into the molecular the known AKR inhibitor epalrestat. Taken together, these interactions between AKR1B1 and PGA1 was advanced by results highlight the role of PGs as AKR1B1 inhibitors and the molecular modeling. This anticipated the addition of PGA1 to interest in PG-related molecules as leads for the development active site Cys298 and the potential reversibility of the adduct, of novel pharmacological tools. Introduction like 4-hydroxy-trans-2-nonenal (Ramana et al., 2006), yielding products that perpetuate inflammation (Srivastava et al., Enzymes of the aldo-keto reductase (AKR) family metab- 2011). AKR1B1 has also been involved in allergic inflamma- olize various aldehydes using NAD(P)(H) as a cofactor. tion and asthma (Yadav et al., 2011; Yadav et al., 2013). Although they play a protective role under physiologic Moreover, AKR1B1 participates in the metabolism of certain situations, their increased activity is considered a patho- prostaglandins (PGs), thus regulating the generation of in- genic factor (Alexiou et al., 2009). AKR1B1 is an important flammatory or vasoactive mediators (Kabututu et al., 2009; mediator of inflammation and diabetic complications Michaud et al., 2014). (Alexiou et al., 2009; Srivastava et al., 2011; Laffin and AKR1B1 metabolizes glucose through the polyol pathway Petrash, 2012). AKR1B1 metabolizes reactive aldehydes, to produce sorbitol. In hyperglycemia this provokes sorbitol accumulation, inducing osmotic stress, favoring epithelial- This work has been funded by grants from the Spanish Ministerio de mesenchymal transition (Ko et al., 1997; Zhang et al., 2012), Economía y Competitividad [SAF2009-11642 (supported in part by FEDER) and SAF2012-36519] and Instituto de Salud Carlos III (RETIC RIRAAF) and contributing to fibrosis, diabetic cataract, and vasculopathy [RD12/0013/0008] to D.P.S.; Instituto de Salud Carlos III (RETIC RIRAAF) (Hashim and Zarina, 2012; Lai et al., 2013). In addition, [RD12/0013/0002] to J.A.G.A.; Slovak Grant Agency [VEGA 2/0041/15] to M.S.; increased AKR1B1 activity depletes NADPH required for the Spanish Ministerio de Economía y Competitividad [SAF2012-39760-C02- 02] and Comunidad Autómoma de Madrid [S2010-BMD-2457] to F.G. The cellular antioxidant defenses and detoxifying systems collaboration and Short Term Scientific Missions between D.P.-S. and M.S. (Pollak et al., 2007b), thus increasing susceptibility to laboratories have been supported by COST Action CM1001. 1B.D.-D. and F.J.S.-G. contributed equally to this work. oxidative stress (Pollak et al., 2007a; Ying, 2008). Moreover, dx.doi.org/10.1124/mol.115.100693. AKR1B1 can elicit a proinflammatory and prothrombotic ABBREVIATIONS: AKR, aldo-keto reductase; cyPG, cyclopentenone prostaglandin; DMSO, dimethyl sulfoxide; GSH, glutathione; GSNO, nitrosoglutathione; HRP, horseradish peroxidase; LC, liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PG, prostaglandin; PGA1-B, biotinylated PGA1; RT, room temperature; TOF, time-of-flight. 42 Prostaglandins Inhibit Aldose Reductase 43 state in diabetes through oxidative stress (Tang et al., 2011) or which incubation mixtures were analyzed on 10% SDS-polyacrylamide Egr-1 hyperacetylation and activation (Vedantham et al., 2014). gels. Gels were transferred to Immobilon P membranes and the Consequently, the search for AKR1B1 inhibitors is an active biotin signal associated with the protein was visualized by incubation – field of research. However, the clinical use of various com- with horseradish peroxidase (HRP) streptavidin and ECL detection. pounds, including tolrestat (Drugbank ID DB02383) and For analysis of the interaction between PGA1 and the AKR1B1 298VCALLSCTSHK307 peptide, a 100 mM solution of the peptide in sorbinil (Drugbank ID DB02712) has been hampered by their water was incubated with 500 mM PGA1 in the presence of 0.5 mM poor effectiveness and severe adverse effects, such as hepatitis dithiothreitol at RT for 16 hours. The integrity of the PG under the or renal toxicity, attributed to inhibition of the related enzyme incubation conditions used was routinely checked by liquid chroma- aldehyde reductase (AKR1A1 or ALR1) (Muthenna et al., tography (LC)-MS at the protein chemistry facility of C.I.B. 2009). Therefore, inhibitor selectivity for AKR1B1 over MS Analysis. For analysis of full-length AKR1B1, 2 ml of the AKR1A1 is preferred over inhibitory potency if it implies incubation mixtures was mixed with 2 ml of 2% trifluoroacetic acid fewer side effects (Suzen and Buyukbingol, 2003). and 2 ml of a suspension of the matrix (2,5-dihydroxy-acetophenone; Cyclopentenone prostaglandins (cyPGs) are electrophilic Bruker Daltonics, Billerica, MA). One microliter of this mixture was compounds that form Michael adducts with proteins, mainly applied to the sample plate (MTP384, 800 mm, AnchorChip; Bruker at cysteine residues located in favorable environments Daltonics) for crystallization at RT before analysis on an Autoflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics) equipped (Garzón et al., 2011; Oeste and Pérez-Sala, 2014). cyPGs with a Smart Beam laser operating in the linear mode. Analysis of the bind to and modulate transcription factors (Cernuda-Morollón purified peptide was carried out by matrix-assisted laser desorption/ Downloaded from et al., 2001; Kim and Surh, 2006; Pérez-Sala, 2011), activate the ionization (MALDI)–time-of-flight (TOF) MS. Peptide fragmentation antioxidant response (Levonen et al., 2004; Martínez et al., was achieved by MALDI-TOF/TOF tandem mass spectrometry 2012), inactivate detoxifying enzymes like glutathione (MS/MS) for sequence analysis and determination of the modifica- transferases (Sánchez-Gómez et al., 2010), and induce anti- tion site. One microliter of peptide was mixed with 1 mlofthematrix inflammatory and antiproliferative effects (Díez-Dacal and (a-ciano-4-hydroxycinnamic acid; Bruker Daltonics) at 3 mg/ml in Pérez-Sala, 2012), for which they have been proposed as 33% acetonitrile (v/v) and 0.1% (v/v) trifluoroacetic acid. One molpharm.aspetjournals.org therapeutic tools (Fukushima et al., 2001; Homem de microliter of this mixture was applied to the sample plate. MALDI-MS – Bittencourt et al., 2007). and MALDI MS/MS data were obtained manually in an Autoflex III MALDI-TOF/TOF mass spectrometer equipped with a LIFT-MS/MS Recently, we identified several AKRs as potential targets device. Data analysis was performed with the FlexAnalysis software for the cyPG PGA1 (Díez-Dacal et al., 2011). Here we have (Bruker Daltonics). Analyses were carried out at the Proteomics and explored the interaction of PGA1 with AKR1B1. Our results Genomics facility of the C.I.B. show that PGA1 inactivates AKR1B1 by interacting at its Animals. Male Wistar rats 8–9 weeks old, weighing 200–230 g, active site, and reduces sorbitol formation in an ex-vivo model were used as organ donors. The animals came