Hepatology, VOL. 73, NO. 1, 2021 AUTOIMMUNE, CHOLESTATIC AND BILIARY DISEASE Synthetic Conjugates of Ursodeoxycholic Acid Inhibit Cystogenesis in Experimental Models of Polycystic Liver Disease Francisco J. Caballero-Camino,1,2 Ivan Rivilla ,1 Elisa Herraez ,3,4 Oscar Briz ,3,4 Alvaro Santos-Laso ,2 Laura Izquierdo-Sanchez,2,4 Pui Y. Lee-Law,2 Pedro M. Rodrigues ,2 Patricia Munoz-Garrido,2 Sujeong Jin,5,6 Estanislao Peixoto ,5,6 Seth Richard,5,6 Sergio A. Gradilone,5,6 Maria J. Perugorria ,2,4 Manel Esteller ,7-10 2,4 3,4 2,4,11 1 Luis Bujanda , Jose J.G. Marin , Jesus M. Banales , * and Fernando P. Cossío * BACKGROUND AND AIMS: Polycystic liver diseases with UDCA-HDAC6i #1 reduced their hepatomegaly and (PLDs) are genetic disorders characterized by progressive de- cystogenesis, increased UDCA concentration, and inhibited velopment of symptomatic biliary cysts. Current surgical and HDAC6 activity in liver. In cystic cholangiocytes UDCA- pharmacological approaches are ineffective, and liver trans- HDAC6i #1 restored primary cilium length and exhibited plantation represents the only curative option. Ursodeoxycholic potent antiproliferative activity. UDCA-HDAC6i #1 was ac- acid (UDCA) and histone deacetylase 6 inhibitors (HDAC6is) tively transported into cells through BA and organic cation have arisen as promising therapeutic strategies, but with par- transporters. tial benefits. CONCLUSIONS: These UDCA-HDAC6i conjugates open a APPROACH AND RESULTS: Here, we tested an ap- therapeutic avenue for PLDs. (Hepatology 2021;73:186-203). proach based on the design, synthesis, and validation of a P family of UDCA synthetic conjugates with selective HDAC6i olycystic liver diseases (PLDs) constitute a capacity (UDCA-HDAC6i). Four UDCA-HDAC6i conju- gates presented selective HDAC6i activity, UDCA-HDAC6i heterogeneous group of genetic disorders #1 being the most promising candidate. UDCA orientation characterized by progressive development of within the UDCA-HDAC6i structure was determinant for multiple fluid-filled biliary cysts (>10), which are HDAC6i activity and selectivity. Treatment of polycystic rats the main cause of morbidity.(1,2) PLDs are inherited Abbreviations: 2D, two-dimensional; 3D, three-dimensional; ADPKD, autosomal-dominant polycystic kidney disease; BA, bile acid; ERK, extracellular signal-regulated kinase; H3K9, lysine 9 of histone 3; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor; HDAC1, histone deacetylase 1; HDAC6, histone deacetylase 6; HDAC6is, histone deacetylase 6 inhibitors; IC50, half maximal inhibitory concentration; OATP, organic anionic transporting polypeptide; OCT, organic cation transporter; pERK1/2, phosphorylated extracellular signal-regulated kinase 1 and 2; PKD, polycystic kidney disease; PKHD1, polycystic kidney and hepatic disease 1; PLDs, polycystic liver diseases; UDCA, ursodeoxycholic acid; UDCA- HDAC6i, synthetic conjugate of ursodeoxycholic acid; WT, wild type. Received October 18, 2019; accepted February 23, 2020. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.31216/suppinfo. Supported by the Spanish Carlos III Health Institute (ISCIII; J.M. Banales: FIS PI15/01132, PI18/01075 and Miguel Servet Program CON14/00129; M.J. Perugorria: PI14/00399, PI17/00022; J.J.G. Marin: FIS PI16/00598) cof inanced by “Fondo Europeo de Desarrollo Regional” (FEDER); CIBERehd (ISCIII): J.M. Banales, M.J. Perugorria, L. Bujanda, and J.J.G. Marin; Spanish Ministry of Economy and Competitiveness (M.J. Perugorria: Ramon y Cajal Program RYC-2015-17755); IKERBASQUE, Basque foundation for Science (M.J. Perugorria and J.M. Banales), Spain; “Junta de Castilla y Leon” ( J.J.G. Marin: SA06P17); “Diputación Foral Gipuzkoa” ( J.M. Banales: DFG15/010, DFG16/004; M.J. Perugorria: DFG18/114, DFG19/081), BIOEF (Basque Foundation for Innovation and Health Research: EiTB Maratoia BIO15/CA/016/ BD to J.M. Banales), Department of Health of the Basque Country ( J.M. Banales: 2017111010; M.J. Perugorria: 2019111024), and Euskadi RIS3 ( J.M. Banales: 2016222001, 2017222014, and 2018222029; 2019222054); La Caixa Scientif ic Foundation ( J.M. Banales: HR17-00601); “Fundación Científica de la Asociación Española Contra el Cáncer” (AECC Scientif ic Foundation, to J.M. Banales and J.J.G. Marin); and “Centro Internacional sobre el Envejecimiento”, Spain ( J.J.G. Marin: OLD-HEPAMARKER, 0348-CIE-6-E). F.J. Caballero-Camino was funded by the Spanish Ministry of Science and Innovation (BES-2014-069148), A. Santos-Laso by the Basque Government (PRE_2018_2_0195), and Pui Y. Lee-Law by the European Association for the Study of the Liver (EASL; Sheila Sherlock Award). The Spanish Ministry of Science and Innovation supported F.P. Cossío: (CTQ2016-80375-P and CTQ2014-51912-REDC) as well as the Basque Government (F.P. Cossío: IT-324-07). I. Rivilla had a postdoctoral contract from the Donostia International Physics Center. *Both authors share co-seniorship. 186 Hepatology, Vol. 73, No. 1, 2021 CABALLERO-CAMINO ET AL. as dominant (i.e., autosomal-dominant polycystic molecular mechanisms of pathogenesis are required. liver disease [~1:100,000 prevalence] and autoso- Cystogenesis in PLDs is characterized by diverse mal-dominant polycystic kidney disease [ADPKD; functional alterations in the bile duct cells (i.e., chol- ~1:1,000 prevalence]) or recessive (i.e., autosomal- angiocytes),(1,2) which include hyperproliferation and recessive polycystic kidney disease [ARPKD; -secretion, increased matrix metalloproteolytic activ- ~1:20,000 prevalence]) forms. Depending on the gene ity, changes in microRNA expression patterns, and affected and the type of mutation, cysts arise exclu- morphological and functional alterations of the pri- sively in liver (i.e., protein kinase C substrate 80K-H, mary cilium, a sensory organelle. These pathological translocation protein SEC63 homolog, low-density events are intracellularly mediated by increased levels lipoprotein receptor-related protein 5, neutral alpha- of cAMP and decreased Ca2+, their regulation being glucosidase AB [GANAB], protein transport protein postulated as a potentially therapeutic target.(1-3) Sec61 subunit beta, and asparagine-linked glycosyla- The use of somatostatin analogues aimed to tion 8 genes) or also in kidneys (polycystic kidney down-regulate the characteristic increased intra- disease [PKD]; PKD1, PKD2, GANAB, and polycys- cellular cAMP levels in cystic cholangiocytes, and tic kidney and hepatic disease 1 [PKHD1] genes).(1,2) their resultant hyperproliferation and -secretion, is Available treatments are mainly surgical resection the only current pharmacological option with lim- of symptomatic cysts and systemic treatment with ited therapeutic benefits and several side effects. somatostatin analogues, which show short-term and Ursodeoxycholic acid (UDCA), an endogenous bile modest benefits. Liver transplantation represents acid (BA) with choleretic and hepatoprotective prop- the only curative option.(1,2) Therefore, therapeutic erties, is able to halt hepatic cystogenesis in exper- approaches based on a deeper understanding of the imental models (in vitro and in vivo in PCK rats) © 2020 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases. This is an open access article under the terms of the Creat​ive Commo​ns Attri​butio​n-NonCo​mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.31216 Potential conflict of interest: Nothing to report. ARTICLE INFORMATION: From the 1 Department of Organic Chemistry I, Center of Innovation in Advanced Chemistry (ORFEO-CINQA), University of the Basque Country/Euskal Herriko Unibertsitatea (UPV/EHU), Donostia International Physics Center (DIPC), Donostia-San Sebastian, Spain; 2 Department of Liver and Gastrointestinal Diseases, Biodonostia Health Research Institute, Donostia University Hospital, UPV/EHU, Donostia-San Sebastian, Spain; 3 Experimental Hepatology and Drug Targeting (HEVEFARM), Biomedical Research Institute of Salamanca (IBSAL), University of Salamanca, Salamanca, Spain; 4 National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd), Carlos III National Institute of Health, Madrid, Spain; 5 The Hormel Institute, University of Minnesota, Austin, MN; 6 Masonic Cancer Center, University of Minnesota, Minneapolis, MN; 7 Josep Carreras Leukaemia Research Institute (IJC), Barcelona, Spain; 8 Centro de Investigacion Biomedica en Red Cancer (CIBERONC), Madrid, Spain; 9 Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain; 10 Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB), Barcelona, Spain; 11 IKERBASQUE, Basque Foundation for Science, Bilbao, Spain. ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Jesus M. Banales, Ph.D. Fernando P. Cossío, Ph.D. Department of Liver and Gastrointestinal Diseases, Biodonostia Department of Organic Chemistry I and Center of Innovation Health Research Institute–Donostia University Hospital in Advanced Chemistry (ORFEO-CINQA), University of the Paseo del Dr. Begiristain s/n Basque Country/Euskal Herriko Unibertsitatea (UPV/EHU) E-20014, Donostia-San Sebastian, Spain Donostia International Physics Center (DIPC) E-mail: jesus.banales@biodonostia.org Manuel Lardizabal Ibilbidea 3 Tel.: +34943006067 E-20018, Donostia/San Sebastian, Spain or E-mail: fp.cossio@ehu.es Tel.: +34943015442 187 CABALLERO-CAMINO ET AL. Hepatology, January 2021 and patients with advanced ADPKD, by normaliz- ing intracellular calcium levels in cystic cholangio- Materials and Methods cytes and by clearing the liver from accumulation of DOCKING AND HOMOLOGY cytotoxic and promitotic BAs.(4,5) Of note, UDCA treatment improved symptomatology in both PCK MODELING rats and patients with ADPKD.(4,5) On the other The X-ray structure of the human HDAC6 catalytic hand, the therapeutic potential of histone deacety- domain 2 was obtained from the RCSB Protein Data lase (HDAC) 6 (HDAC6) inhibitors (HDAC6is), Bank (PDB entry: 5EDU). The protein structure was such as tubastatin-A and ACY1215, for treat- optimized and minimized using the protein prepa- ment of both PLDs and PKDs has been described ration wizard software included in the Maestro 10.4 in vitro(6-8) and in vivo,(9) given that cystogenesis package (Schrödinger, LLC, New York, NY). Ligands is characterized by cytoplasmic overexpression of were represented with a Maestro graphic interface HDAC6, which promotes the deacetylation of α- and optimized with the LigPrep tool included in the tubulin, one of the main structural proteins of the Maestro 10.4 package. Flexible docking experiments primary cilium.(9) Consequently, decreased acetyl- were carried out using Glide software included in the ation of α-tubulin results in morphological and Maestro 10.4 package, using extra precision mode to functional abnormalities of the primary cilium and maximize accuracy of the obtained results. Obtained promotes cell proliferation.(9) structures and binding-predicted energies were ana- The hypothesis of the present study was that lyzed using Maestro and Pymol graphic interfaces. UDCA, owing to its particular structural features The homology model of rat HDAC6 catalytic domain and ability to conjugate taurine or glycine, could be 2 (Uniprot entry: A0A0G2QC41) was generated an excellent external van der Waals cap molecule based on human HDAC6 catalytic domain 2 (PDB for the design of HDAC6is (i.e., UDCA-HDAC6i entry: 5EDU) with Prime software, also included in [synthetic conjugate of ursodeoxycholic acid]). It is the Maestro 10.4 package (Schrödinger, LLC). For known that preorganized macrocyclic systems can docking studies, the same procedure as for the human exhibit high noncovalent binding constants with the HDAC6 catalytic domain 2 was followed. area close to the rim that connects the active sites of HDACs with the surface of these enzymes.(10,11) We HDAC ACTIVITY ASSAYS reasoned that a polycyclic molecule such as UDCA could be a suitable conformationally restricted ana- HDAC activity assays were performed using logue to these macrocyclic systems. This polycyclic acetylated peptide substrates labeled with 7-amino- moiety should be covalently bound to a spacer able 4-methylcoumarin at Reaction Biology Corporation to fill the tube that connects the active site of the (Malvern, PA). All the enzymes and substrates used enzyme with the rim.(12) Finally, this kind of HDAC are summarized in Supporting Table S9, and the inhibitor (HDACi) should include a chelating group details of the assays and protocols are described in the able to bind the Zn2+ present in the active site.(13) The Supporting Information. presence of UDCA in the UDCA-HDAC6i structure could also improve the hepatotropism of these mol- TREATMENT OF PCK RATS WITH ecules and their targeting toward cystic cholangio- HDAC6i-UDCA #1 cytes.(14-16) Moreover, UDCA-HDAC6i could retain some of the previously observed therapeutic benefits The PCK rat (Charles River Laboratories Inc, of UDCA on PLDs, and the abundance of UDCA in Wilmington, MA) is an animal model of ARPKD, the BA pool could be increased because of intestinal presenting a mutation in the human orthologue Pkhd1 reuptake after UDCA-HDAC6i hydrolysis by the gut gene and spontaneously developing both hepatic and microbiota.(17) In contrast to conventional HDAC6i, renal cysts. Both wild-type (WT; n = 12) and PCK UDCA-HDAC6i biotransformation is not expected rats (n = 20) were 8 weeks old at the beginning of to result in toxic intermediate molecules. Therefore, the experiment. PCK rats were randomized into this strategy would provide a single-molecule–based HDAC6i-UDCA #1–treated (n = 8; 15 mg/kg/d by combination therapy. oral gavage for 5 months) and nontreated (n = 12) 188 Hepatology, Vol. 73, No. 1, 2021 CABALLERO-CAMINO ET AL. groups. After treatment, animals were euthanized and PRIMARY CILIA ANALYSIS blood was collected, and serum was further isolated to determine biochemical markers of liver and kidney Primary cilia length and number of ciliated chol- disease. Liver and kidneys were collected, weighted, angiocytes were analyzed by immunofluorescence as and either fixed in 4% paraformaldehyde or stored at described in the Supporting Information. −80°C for further analysis. All in vivo experimental procedures were performed according to the Guide CELL PROLIFERATION for the Care and Use of Laboratory Animals and Evaluation of proliferation in cystic cholangiocytes approved by the Ethics Committee of Biodonostia was carried out as described in the Supporting Data. Health Research Institute. BA MEASUREMENT STATISTICAL ANALYSIS GraphPad Prism software was applied for sta- Concentrations of BA species in liver, bile, periph- tistical analysis (version 6; GraphPad Software Inc., eral, and portal blood were measured after methanol La Jolla, CA). Data are expressed as mean ± SEM. precipitation/extraction by high-performance liquid Unless otherwise indicated, for comparisons between chromatography with tandem mass spectrometry two groups, a two-tailed parametric Student t test or using a modification(18) of a described method.(19) nonparametric Mann-Whitney U test were used. In all cases, measurements were taken from distinct sam- CELLS AND CULTURE ples (i.e., cell cultures, tissue samples, and individual CONDITIONS cystic cholangioids). Statistically significant data, rep- Primary cultures of normal and cystic human chol- resented as asterisks (*, **, and ***), denote a P value angiocytes, previously isolated and characterized by <0.05, <0.01, and <0.001, respectively. our group,(20) were used for in vitro assays. Culture conditions, and the three-dimensional (3D) cul- turing of cystic cholangiocytes, are described in the Results Supporting Information. SYNTHETIC UDCA CONJUGATES EXPERIMENTAL SELECTIVELY INHIBIT HDAC6 OVEREXPRESSION OF HUMAN ACTIVITY TRANSPORTERS IN CELLS AND We applied our previously described HDAC inhi- TRANSPORT ASSAYS bition model(21) to the X-ray structure of the catalytic domain 2 of human HDAC6 (PDB code: 5EDU)(22) Experimental overexpression of the human trans- (Fig. 1A). HDAC6 active sites can be schematically porters in cells and transport assays were performed as described as an ensemble of external shallow cavi- described in the Supporting Information. ties connected through a hydrophobic channel with a Zn2+-containing catalytic site. Following this general qPCR model, we designed a family of synthetic UDCA con- Total RNA extraction and retrotranscription and jugates, in which the steroid skeleton of UDCA acted further qPCRs were performed as described in the as the external van der Waals cap of the inhibitor, fol- Supporting Information. All the primer sequences are lowed by a number of different hydrophobic spacers summarized in Supporting Table S11. ended on a Zn2+ chelating group (Fig. 1A). Chemical synthesis of the UDCA-HDAC6i mol- ecules (Fig. 1B) started with the coupling reaction IMMUNOBLOTTING of UDCA (1) with amines (2) to yield amide esters Analysis of protein expression was performed as (3). Cleavage of the methoxy groups of adducts (3) described in the Supporting Information and using and in situ reaction with hydroxylamine permitted the the antibodies listed in Supporting Table S12. isolation and characterization of UDCA-hydroxamic 189 CABALLERO-CAMINO ET AL. Hepatology, January 2021 A B FIG. 1. Design, synthesis, in silico prediction, and in vitro characterization of the HDACi activity of UDCA synthetic conjugates. (A) Scheme of the designing of UDCA-HDAC6i molecules based on HDAC6 structure. (B) Chemical synthesis of the 10 different UDCA-HDAC6i molecules. (C) Docking studies. Graphical representation of the biding modes of UDCA-HDAC6 is (yellow #9, acids #1,UDCA-HDAC6i green #1, #6, blue #2, and grey #4, #10, #3-6, and #5, and #3) #10, to the which UDCA-HDAC6i active site of human HDAC6 CD2. #7 Dashed and #8. Thelines yellow spacer between represent the hydrogen possess bonds different between hydroxyllengths groups ofand UDCAstereoelectronic and amino acidsfea- chelating of the protein group surface. and Table the UDCA displays component the predicted was elon- binding energies for UDCA-HDAC6is. (D) Representative immunoblotting of acetylation (Ac) levels of α-tubulin and H3K9 tures. Alternatively, hydrolysis of esters (3) permitted gated by means of an additional hydrolysis-coupling after 24 hours of treatment with 2 μM of each compound. (E) Experimental validation of the in silico model. Correlation between the predicted biding energies the preparation of acid (4). Subsequent coupling with sequence (5), thus yielding esters (6) whose cleav- (HDAC6i) and levels (%) of acetylated α-tubulin in PLD cells treated with each UDCA-HDAC6i. Abbreviations: Asp567, aspartate; orthophenylenediamines DMF, N,N-dimethylformamide; (7)His, yielded amide histidine; candidates Leu, leucine; ageTBTU, Ser, serine; and N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium reaction with hydroxylamine permitted the tetrafluoroborate; Trp, tryptophan; XP Gscore, XP GlideScore. 190 Hepatology, Vol. 73, No. 1, 2021 CABALLERO-CAMINO ET AL. C D E FIG. 1. Continued. 191 CABALLERO-CAMINO ET AL. Hepatology, January 2021 isolation and characterization of candidates UDCA- fluorogenic acetylated peptide (Fig. 2A). UDCA- HDAC6i #2 and #9. HDAC6i #1, #2, #6, and #9 displayed low half max- The potential HDAC6 inhibitory capacity of this imal inhibitory concentration (IC50) values (58, 56.1, family of UDCA synthetic conjugates was first evalu- 82.1, and 4.26 nM, respectively) for HDAC6 inhi- ated in silico with a docking model (Fig. 1C). Significant bition, concomitantly with a high degree of selec- binding affinities to HDAC6 were predicted for eight tivity, particularly in the case of compounds #1, #2, of these compounds, with UDCA-HDAC6i #1, #2, and #9 (Fig. 2A). As expected, UDCA-HDAC6i and #9 being the ones that displayed the highest #8 showed both high inhibitory activity and selec- energy scores (−8.3, −7.9, and −9.4 kcal/mol, respec- tivity for HDAC1 (IC50 = 140 nM; Fig. 2A). tively; Fig. 1C). Interestingly, these three compounds Importantly, UDCA itself did not exhibit any direct oriented the hydrocarbon skeleton of UDCA toward HDAC inhibitory activity; however, its formal cou- the same region of the protein surface (Fig. 1C). We pling with the separate spacer-chelating component, decomposed the total binding energy of each synthetic 4-(aminomethyl)-N-hydroxybenzamide hydrochloride derivative component into the contribution of the dif- [Fig. 2B, chelating-spacer (8)], to form UDCA- ferent descriptors (i.e., hydrogen bonds, van der Waals HDAC6i #1 significantly increased (~12-fold) the interactions) to the overall score (Supporting Tables S1 HDAC6 inhibitory capacity of the chemical entity and S2A,B). Importantly, the contribution of UDCA (IC50 = 689 vs. 58 nM; Fig. 2B), highlighting the to the final binding energy of UDCA-HDAC6i #1, active contribution of UDCA to the inhibitory activ- #2, and #9 was approximately one-third of the total ity of the UDCA-HDAC6is (Fig. 2B and Supporting value, and was mainly integrated by van der Waals Fig. S1). In addition, the effect of UDCA-HDAC6i interactions, and hydrogen bonds established by the #1 and #2 was also evaluated on purified HDAC2-5 two hydroxyl groups of the steroid skeleton of the BA and HDAC7-11 enzymes, showing quite similar (Fig. 1C; Supporting Tables S1 and S2A,B). inhibitory capacity to HDAC1 and significantly less Given that cytoplasmic levels of acetylated compared to HDAC6 (Supporting Table S3). Of note, α-tubulin in cholangiocytes are directly linked to the the 3D structural alignment of the catalytic domains degree of HDAC6 expression and activity,(9,23) cystic of human HDAC1, 2, 3, 4, 7, and 8 and zebrafish human cholangiocytes in culture were incubated with HDAC10 (PDB entries: 5ICN, 6G3O, 4A69, 4CBT, UDCA-HDAC6i and protein expression of acetyl- 3ZNR, 5VI6, and 5TD7) with our docking model of ated α-tubulin was measured by immunoblotting. human HDAC6 CD2 revealed that although all the In addition, acetylation levels of lysine 9 of nuclear analyzed HDAC active sites fitted well to the general histone 3 (H3K9) were assessed as a selectivity test model previously mentioned (Fig. 1A), they possessed (negative control). The results (Fig. 1D) revealed that, important differences in the architectures of their at low concentration (2 μM), UDCA-HDAC6i #1, outer rims, hydrophobic channels, and catalytic sites #2, #6, and #9 increased acetylated α-tubulin levels in (Fig. 2C). In this regard, we found particularly rel- cystic cholangiocytes, without affecting H3K9 acetyl- evant the high degree of sequence disparity between ation, thus indicating a selective inhibitory activity on HDAC6 and the rest of the isoforms in the surface HDAC6. In contrast, UDCA-HDAC6i #8 increased area occupied by UDCA in the three most active/ H3K9 acetylation, without affecting α-tubulin acetyl- selective inhibitors (Supporting Fig. S2), resulting in ation. Of note, the predicted binding affinity (i.e., the absence of this cavity in the other HDAC iso- energy values by docking) of the aforementioned forms, whereas the other two cavities occupied by UDCA-HDAC6i for HDAC6 and their effect on less active/selective UDCA-HDAC6is (e.g., UDCA- HDAC6-dependent acetylated α-tubulin levels in HDAC6i #6) remained almost intact (Fig. 2D and cystic cholangiocytes showed significant correlation Supporting Fig. S2). (r2 = 0.8031), supporting the accuracy of the compu- Once the affinity and selectivity for HDAC6 tational approach used here (Fig. 1E). inhibition was confirmed in four of the synthesized Next, inhibitory capacity of UDCA-HDAC6i #1, UDCA-HDAC6i molecules, we generated a homol- #2, #6, #8, and #9 on purified histone deacetylase 1 ogy model of the catalytic domain 2 of rat HDAC6 (HDAC1) and HDAC6 enzymes was measured by in order to predict the inhibitory capacity of these monitoring the dose-dependent deacetylation of a compounds between species (Supporting Fig. S3). 192 Hepatology, Vol. 73, No. 1, 2021 CABALLERO-CAMINO ET AL. A C D B FIG. 2. In vitro and in silico characterization of the critical role of UDCA for the HDAC6 inhibitory activity and selectivity of UDCA- HDAC6is. (A) Dose-response curves of the inhibitory activity of UDCA-HDAC6i #1 (n = 9), #2 (n = 9), #6 (n = 3), #8 (n = 3), and #9 (n = 3) on HDAC6 and HDAC1. For curve fitting and IC50 calculation, log (inhibitor) versus normalized response with variable slope equation was used in GraphPad software (version 6; GraphPad Software Inc., La Jolla, CA). Extended information about HDAC IC50 values is provided in Supporting Table S3. (B) Comparison of the IC50 values of UDCA-HDAC6i #1 and 4-(aminomethyl)-N-hydroxybenzamide hydrochloride (8) for HDAC6 and graphical representation of the main interactions taking place in the binding of UDCA-HDAC6i #1 to the catalytic domain 2 of HDAC6 (hydrogen bonds in purple and π-π stacking interactions in green). Black arrows point to the two hydroxyl groups of UDCA within UDCA-HDAC6i #1 and the hydrogen bonds established between these and tryptophan 496 and histidine 560 of the catalytic domain 2 of human HDAC6. (C) Cross-sectional view of 3D-aligned active sites of different HDAC isoforms (HDAC1, 2, 3, 4, 6, 7, and 8 and zebrafish HDAC10 [PDB entries: 5ICN, 6G3O, 4A69, 4CBT, 5EDU, 3ZNR, 5VI6, and 5TD7]). (D) 3D structural alignments of the docking model for the catalytic domain 2 of human HDAC6 with the catalytic domains of different HDAC isoforms. In blue, surface region of each isoform aligned to the cavity in which the three most active/selective UDCA-HDAC6is accommodate UDCA in HDAC6. Abbreviations: Gly, glycine; His, histidine; ND, not determined; Phe, phenylalanine; Ser, serine; Trp, tryptophan. 193 CABALLERO-CAMINO ET AL. Hepatology, January 2021 Sequence alignment of the catalytic domains of both acetylated α-tubulin levels was found in both liver species revealed an identity of 88.4%. Importantly, as and kidney of animals receiving UDCA-HDAC6i #1 for the human HDAC6, UDCA-HDAC6i #1, #2, compared to untreated PCK rats (Fig. 3D). and #9 were the compounds with the highest bind- ing energies in the rat homology model (Supporting UDCA-HDAC6i #1 MODULATES Fig. S3), suggesting that their inhibitory potential THE BA POOL AND INCREASES would be maintained in the rat. This points to a direct correspondence between our results and those expected LEVELS OF UNCONJUGATED UDCA in humans. IN PCK RATS Next, UDCA-HDAC6i #1 was selected for further BA concentrations and pool composition were in vivo analysis (Supporting Table S4). In particular, determined in liver, bile, and both portal and periph- we hypothesized that the UDCA-HDAC6i molecules eral blood. Consistent with previous reports,(4,5) total could benefit from the intrinsic hepatotropic features BA concentrations in both liver and peripheral blood of UDCA, concentrating them preferentially into the were higher in PCK rats than in normal animals. liver and in cystic cholangiocytes after oral administra- These values were not modified in PCK rats treated tion and gastrointestinal absorption. In addition, these with UDCA-HDAC6i #1 (Fig. 4). Moreover, total molecules could also be reabsorbed through the entero- BA concentrations in portal blood and bile were simi- hepatic/bile circulation. In this regard, we applied the lar in treated and untreated groups (Fig. 4). However, QikProp software included in the Maestro package 10.4 some differences in composition of the BA pool were (Schrödinger, LLC) to in silico predict several pharmaco- found. Thus, the described(4,5) decreased UDCA lev- kinetic parameters for all UDCA-HDAC6i molecules, els in liver of PCK rats were restored upon UDCA- obtaining considerably better values for UDCA- HDAC6i #1 treatment (Fig. 4). Similarly, increased HDAC6i #1 than for the other three active candidates levels of UDCA were found in portal and peripheral in terms of oral absorption. UDCA-HDAC6i #1 dis- blood, as well as in bile, of PCK rats treated with played values within the recommended range for all the UDCA-HDAC6i #1 compared to both normal and parameters analyzed, whereas UDCA-HDAC6i #2 and PCK untreated animals (Fig. 4). Likewise, a normal- #9 presented several potential limitations. ization of major unconjugated bile acids (i.e., cholic acid [CA], α/β murocholic acid, and sulpholithocholic in liver; CA and hyodeoxycholic acid in bile) in UDCA-HDAC6i #1 HALTS treated PCK animals was found (Fig. 4; Supporting HEPATO- AND NEPHROMEGALY Tables S6 and S7). IN PCK RATS The therapeutic value of UDCA-HDAC6i #1 UDCA-HDAC6i #1 HALTS LIVER was evaluated in PCK rats (Pkhd1 mutant), an ani- CYSTOGENESIS IN VIVO, mal model for hepatorenal cystogenesis (Fig. 3).(24,25) RESTORES PRIMARY CILIUM Eight-week-old PCK rats were treated with UDCA- LENGTH, AND INHIBITS HDAC6i #1 (15 mg/kg/d by oral gavage) for 5 months. CYSTIC CHOLANGIOCYTE Remarkably, the characteristic hepato- and nephromeg- PROLIFERATION aly that PCK rats develop over time was significantly reduced after treatment with UDCA-HDAC6i #1. Histological analysis of the cholangiocyte marker, Liver- and kidney-to-body weight ratios were also cytokeratin 19 (CK19), revealed a reduction in mag- reduced compared to untreated PCK rats (Fig. 3A,B, nitude of cystic areas in liver of UDCA-HDAC6i respectively). These effects were associated with an #1–treated animals compared to PCK untreated rats improvement of both liver and kidney functions, as (Fig. 5A). HDAC6 inhibitory activity of UDCA- reflected by increased serum albumin and reduced urea HDAC6i #1 increased (64%) the primary cilium levels, respectively (Fig. 3A,B). At the end of treat- length of cystic cholangiocytes and the number of ment, UDCA-HDAC6i #1 was detected in liver, bile, ciliated cholangiocytes (182%) in liver of the treated and portal and peripheral blood, but was particularly animals (Fig. 5B); similarly, in vitro, UDCA- abundant in bile (Fig. 3C). Moreover, an increase in HDAC6i #1 increased levels of acetylated α-tubulin 194 Hepatology, Vol. 73, No. 1, 2021 CABALLERO-CAMINO ET AL. A B D C FIG. 3. Effect of UDCA-HDAC6i #1 on hepatorenal cystogenesis in vivo. (A) Representative liver images from a normal rat, as well as nontreated and UDCA-HDAC6i #1–treated PCK rats. Graphical representation of relative comparisons of liver weight, total body weight, liver/body weight ratio, and serum albumin concentration in the three experimental groups. A statistical unpaired two-tailed t-test analysis was applied to determine significance in all cases, with the exception of PCK treated versus nontreated liver/body weight in which an unpaired one-tailed t-test analysis was applied. (B) Representative kidney images from a normal rat, as well as nontreated and UDCA-HDAC6i #1–treated PCK rats. Graphical representation of relative comparisons of kidney weight, kidney/body weight ratio, and serum urea concentration in the three experimental groups. A statistical unpaired two-tailed t test was applied to determine significance. (C) Analysis of UDCA-HDAC6i #1 pharmacokinetics. Graph represents the bioavailability profile of UDCA-HDAC6i #1 in blood (n = 8), liver (n = 8), bile (n = 7), and peripheral blood (n = 8) during chronic oral administration. An unpaired two-tailed t test was applied to assess for significance. (D) Representative immunoblotting of the acetylated (Ac) α-tubulin levels in liver and kidney of treated (n = 8) and nontreated (n = 12) PCK rats. Graph represents the relative amount of acetylated α-tubulin in liver and kidney using total tubulin as normalizing control. For statistical significance assessment, an unpaired two-tailed t test was used. Abbreviation: ns, not significant. 195 CABALLERO-CAMINO ET AL. Hepatology, January 2021 FIG. 4. BA pool in portal blood, liver, bile, and peripheral blood. Bar graphs represent total BA concentration, unconjugated UDCA concentration, and relative abundance of conjugated BAs in each biological sample. Quantitative and statistical information about the relative abundancy of single BAs is depicted in Supporting Tables S5-S8. A statistical unpaired two-tailed t test was applied to determine significance. Abbreviation: ns, not significant. (Fig. 5C) and restored the primary cilium length analyzed the effect of UDCA-HDAC6i #1 on pro- of cystic cholangiocytes (~40% increase; Fig. 5D), liferation/expansion of cystic cholangiocytes. UDCA- but induced no changes in normal cholangiocytes HDAC6i #1 inhibited the 3D growth of cystic (Fig. 5D). cholangioids compared to control conditions (Fig. 6A). Given that the primary cilium promotes epithelial Similarly, UDCA-HDAC6i #1 inhibited two-di- cell polarity and prevents cell-cycle initiation,(26,27) we mensional (2D) cystic cholangiocyte proliferation in 196 Hepatology, Vol. 73, No. 1, 2021 CABALLERO-CAMINO ET AL. A B C D FIG. 5. Effect of UDCA-HDAC6i #1 on liver cystogenesis and primary cilium length. (A) Representative images of hematoxylin/eosin staining (scale bar = 250 µm) and CK19 immunohistochemistry (scale bar = 100 µm). Graph represents the cystic area relative to total tissue area of individual hepatic lobes (three for each animal). A statistical unpaired two-tailed t test was applied to determine significance. (B) Representative images of liver sections from untreated and UDCA-HDAC6i #1–treated PCK rats. Cilia are labeled with antibodies against acetylated (Ac) α-tubulin (green) and nuclei are stained (blue) with DAPI. Bar graphs represent length of primary cilia and percentage of ciliated cholangiocytes. White asterisks indicate cyst lumen. Cysts were randomly selected from untreated (n = 6) and treated rats (n = 9). A statistical unpaired two-tailed t test was applied to determine significance. Data are expressed as mean ± SEM. (C) Representative immunoblotting of the effect of increasing doses of UDCA-HDAC6i #1 in levels of acetylated α-tubulin in cultured PLD cholangiocytes. GAPDH was used as normalizing control. (D) Representative immunofluorescence images of acetylated α-tubulin in cholangiocytes (primary cilium in green and nucleus in blue stained with DAPI). Bar graph represents the mean cilia length of normal (nontreated [n = 23] and 2 µM of UDCA-HDAC6i #1 treated [n = 21]) and PLD (nontreated [n = 30] and 2 µM of UDCA-HDAC6i #1 treated [n = 27]) human cholangiocytes. A statistical one-way ANOVA was used. Abbreviations: ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2- phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NHC, normal human cholangiocytes; ns, not significant. vitro in a dose-dependent manner (Fig. 6B), which (Fig. 6C). Importantly, this antiproliferative effect was was associated with phosphorylated extracellular sig- significantly higher than that observed upon com- nal-regulated kinase (ERK) 1/2 (pERK1/2) regulation bined treatment with UDCA plus the separate spacer- 197 CABALLERO-CAMINO ET AL. Hepatology, January 2021 chelating component (8) (Fig. 6B), demonstrating was measured in cystic cholangiocytes exposed to that UDCA-HDAC6i #1 has intrinsic properties. UDCA-HDAC6i #1 at 4°C or 37°C, respectively. Importantly, significant differences in uptake ratios HEPATOTROPIC PROPERTIES OF at both temperatures were observed, suggesting that an important part of UDCA-HDAC6i #1 internal- UDCA-HDAC6i #1 ization occurs through plasma membrane proteins To investigate the mechanism of UDCA- (37°C), rather than by passive diffusion across the HDAC6i #1 uptake by cystic cholangiocytes (i.e., lipid bilayer (4°C; Fig. 7A). Therefore, in order passive diffusion or membrane protein trans- to assess the specific transporters involved in the port), time-dependent compound internalization UDCA-HDAC6i #1 internalization process, we A B C FIG. 6. Effect of UDCA-HDAC6i #1 on proliferation of 2D- and 3D-cultured PLD cholangiocytes. (A) 3D culture in collagen type I of bile ducts/cysts isolated from PCK rats. Representative images and graph representing the relative circumferential area of nontreated (n = 20) and UDCA-HDAC6i #1–treated (n = 20; 10 µM) cystic cholangioids along time. (B) Proliferation of PLD cholangiocytes by flow cytometry. Bar graph represents the percentage of cell proliferation after 48 hours of treatment with 10 µM of UDCA (n = 7), 100 µM of UDCA (n = 22), 2 µM of UDCA-HDAC6i #1 (n = 16), 10 µM of UDCA-HDAC6i #1 (n = 23), 10 µM of spacer chelating component (8) (n = 23), or combined treatment with 10 µM of UDCA + 10 µM of spacer chelating component (8) (n = 7) relative to control PLD cholangiocytes (n = 23) without treatment. (C) Representative immunoblotting of phosphorylated and total ERK1/2 protein levels from cell lysates of nontreated and UDCA-HDAC6i #1–treated PLD cholangiocytes. Graph represents the relative abundance of phosphorylated ERK1/2 in baseline (n = 5) and 2 µM (n = 5) or 10 µM (n = 5) of UDCA-HDAC6i #1–treated PLD cholangiocytes using total ERK as normalizing control. In all cases, a statistical unpaired two-tailed t test was applied to determine significance. Abbreviation: ns, not significant. 198 Hepatology, Vol. 73, No. 1, 2021 CABALLERO-CAMINO ET AL. A B C D FIG. 7. Mechanisms of UDCA-HDAC6i #1 uptake into cells. (A) Time-course analysis of UDCA-HDAC6i #1 uptake in cystic cholangiocytes incubated with 100 µM of UDCA-HDAC6i #1 at 37°C or 4°C. A statistical unpaired two-tailed t test between both conditions at each time point was applied to determine significance. Data are expressed as mean ± SEM. (B) Bar graphs representing UDCA-HDAC6i #1 uptake in HepG2 or CHO Mock cells or stably expressing OCT1 (n = 4) or OCT3 (n = 5), respectively, in the presence or absence of quinine as a competing substrate. The paired two-tailed t test was used to assess statistical significance. (C) RT-qPCR analysis of mRNA expression of OCT1 and OCT3 transporters in normal (n = 6) and PLD (n = 6) human cholangiocytes. The RPL22 gene was used as the normalizing control. An unpaired two-tailed t test was applied to determine statistical significance. (D) Immunohistochemical analysis of OCT1 and OCT3 in liver sections of PCK rats. Abbreviations: ns, not significant; RPL22, 60S ribosomal protein L22. next used cell models experimentally overexpressing (Fig. 7C). Furthermore, we confirmed by immuno- plasma membrane transporters in substrate compe- histochemistry the expression (plasma membrane tition assays (Supporting Fig. S4). The results indi- and cytoplasm) of OCT1 and OCT3 transport- cated that UDCA-HDAC6i #1 was transported by ers in cyst-lining cholangiocytes (and hepatocytes; the organic cation transporters (OCTs), OCT1 and Fig. 7D) of PCK rat liver tissue. OCT3, (Fig. 7B) as well as by the sodium-dependent BA transporter (Na+-taurocholate cotransporting polypeptide), commonly expressed in hepatocytes (Supporting Fig. S5). In contrast, neither the apical Discussion sodium-dependent bile salt transporter expressed in Oral administration of UDCA is well tolerated, cholangiocytes nor any of the assayed organic anion safe, and is currently the first-line therapy interna- transporters (i.e., organic anionic transporting poly- tionally approved for the treatment of primary bili- peptide [OATP] 1A2, OATP1B1, OATP1B3, and ary cholangitis.(28) Chronic treatment with UDCA OATP2B1) mediated UDCA-HDAC6i #1 uptake inhibits hepatic cyst growth in experimental models (Supporting Fig. S5). Interestingly, the analysis of and also in a subset of patients with advanced PLD expression (RT-qPCR) of these transporters marked (CURSOR [Ursodeoxycholic Acid as Treatment up-regulation of OCT1 and OCT3 in cystic for Polycystic Liver Disease] phase II clinical trial), human cholangiocytes compared to normal controls improving their symptomatology.(4,5) On the other 199 CABALLERO-CAMINO ET AL. Hepatology, January 2021 hand, inhibition of HDAC6 activity has been pro- Data of the present study showed that UDCA posed as a therapeutic strategy for PLD. has no HDAC inhibitory activity. However, its for- The currently available HDAC6is do not show mal coupling to the spacer-chelating component [Fig. preferential targeting to the liver and, in particular, 2B, chelating-spacer (8)] (i.e., UDCA-HDAC6i #1) to biliary cells. Most HDAC6is developed to date reduced 1 order of magnitude the IC50 value of [Fig. comprise on their structure a hydrophobic linker 2B, chelating-spacer (8)] for HDAC6 inhibition. Of ended on a Zn2+ chelating group that enters into the note, the three UDCA-HDAC6i molecules displaying catalytic sites of HDAC6 and an external cap that the highest binding affinities to HDAC6 (i.e., UDCA- accommodates on the surface cavities.(21) HDAC6i HDAC6i #1, #2, and #9) were those containing one molecules usually contain aromatic peptide deriva- or two 1,4-phenylene spacers and hydroxamates as tives and hydrocarbons acting as the closing cap of chelating groups. These three molecules oriented the the inhibitor,(29-32) which are prone to be released hydrocarbon skeleton of UDCA toward the same into systemic circulation after their hepatic metabo- region of the protein surface; indeed, the difference lism. These residues have been largely demonstrated in binding energies with the other compounds, which to be potentially toxic in humans. Most frequently accommodated UDCA in other surface cavities, was observed manifestations of toxicity in clinical trials are >3 kcal/mol. Importantly, predicted inhibitory activity fatigue, nausea, diarrhea, and cardiotoxicity, including of the UDCA-HDAC6i molecules on HDAC6 was ventricular arrhythmia, being the most worrisome further validated in both cystic cholangiocytes (i.e., adverse effects.(33,34) Considering these limitations acetylated α-tubulin expression) and enzymatic assays for combination therapies,(35) and taking advantage of (specific HDAC6 vs. HDAC1 inhibitory activity) our experience in the designing of HDACis through in vitro. Of note, replacement of the hydroxamic acid innovative in silico docking analyses,(21) we hypoth- moiety of UDCA-HDAC6i #1 by the ortho-phenyl- esized that UDCA could be an appropriate external enediamine group present in UDCA-HDAC6i #8 cap to reinforce the HDAC6 inhibitory capacity of resulted in a complete switch in both the inhibitory the chelating groups, improve their hepatotropism, potential and selectivity toward nuclear HDACs decrease their toxicity, and provide some of the afore- instead of HDAC6. These data highlight the accu- mentioned UDCA benefits, because: (1) Our in sil- racy of our in silico predictive model for molecules ico docking experiments indicate that UDCA may to inhibit HDAC6. In this regard, the importance actively contribute to reinforce the inhibitory poten- of certain regions of the HDAC6 CD2 surface to tial of the hydrophobic linker + Zn2+ chelating group determine inhibitor selectivity has been recently through the establishment of van der Waals interac- reported.(39) In accordance, structure alignment of tions and hydrogen bonds between the two hydroxyl different HDAC isoforms with our docking model groups present in the UDCA structure and the side revealed that the surface cavity occupied by UDCA in chains of several amino acid residues located on the the three most HDAC6 active/selective compounds is external cavities of HDAC6 close to the catalytic not present in the other HDAC isoforms, whereas the domains; (2) UDCA would provide hepatotropic cavity occupied by a still active, but much less selec- features to these new UDCA-HDAC6i entities by tive, compound (i.e., UDCA-HDAC6i #6) remained promoting its enterohepatic transport through BA fully accessible in all isoforms. All these data highlight and/or OCTs; (3) UDCA is usually amidated with the critical role that UDCA plays in dictating both taurine (tauroursodeoxycholic acid) or glycine (gly- activity and selectivity of UDCA-HDAC6is. coursodeoxycholic acid) by hepatocytes,(36,37) so this Once the inhibitory potential of the three best can- residue could be used for the formal conjugation to didates was confirmed in human and rat HDAC6 by the spacer-chelating group; and (4) the gut microbi- using a homology model of the catalytic domain 2, ome deconjugates the taurine or glycine residues of UDCA-HDAC6 #1 was selected for further in vivo UDCA,(38) and, consequently, UDCA-HDAC6i pres- and in vitro evaluation based on comparative analy- ent in bile and entering the gut could be deconjugated sis of its selective HDAC6 inhibitory capacity (IC50), by this process, resulting in a secondary increase of the potential pharmacokinetics, and synthetic cost. Oral enterohepatic levels of UDCA that would additionally administration of UDCA-HDAC6i #1 to PCK rats inhibit PLD progression. considerably reduced the hepato- and nephromegaly 200 Hepatology, Vol. 73, No. 1, 2021 CABALLERO-CAMINO ET AL. characteristic on these animals, as well as hepatic UDCA-HDAC6i #1–treated and nontreated animals. cystogenesis. Of note, this new molecule partially However, changes in certain BA species in bile, and to normalized the decreased serum levels of albumin, a lesser extent in liver, were found. Of note, increased suggesting an improvement of liver function of these levels of free UDCA in liver, bile, and serum were animals. In addition, levels of urea in serum were found in PCK rats treated with UDCA-HDAC6i #1, reduced upon treatment, indicating an improvement reinforcing the idea that biliary UDCA-HDAC6i #1 in renal function. On the other hand, no signs of tox- could be further metabolized in the gut, leading to icity were observed at biochemical and histological excision of the amide bond and thus resulting in the levels. The therapeutic effects of UDCA-HDAC6i release of free UDCA that may enter the enterohe- #1 in the liver seem to be comparable (i.e., hepatic patic circulation in a second round and induce addi- cystogenesis) or even superior (i.e., hepatomegaly) tional therapeutic benefit on hepatic cystogenesis. to the ones previously reported for UDCA(4,5) in the As indicated above, two important limitations of same animal model, even at substantially lower doses currently available HDAC6is are their lack of selective (i.e., 15 vs. 25 mg/kg/d for 5 months, respectively; affinity toward hepatic tissue as well as the presence of Supporting Fig. S6). Moreover, in contrast to UDCA, aromatic hydrocarbons or heterocycles in their struc- UDCA-HDAC6i #1 also provides therapeutic effects tures,(29-32) which can be released as potentially toxic on nephromegaly and renal function of PCK rats metabolites. Our data reveal that, after oral admin- (Supporting Fig. S6). istration, UDCA-HDAC6i #1 accumulates preferen- At the histological level, increased acetylated tially in liver and bile, rather than in peripheral blood, α-tubulin was obtained in both liver and kidney of indicating a superior targeting to the liver and poste- treated PCK rats compared to untreated animals, sup- rior secretion into bile, thus potentially diminishing porting that UDCA-HDAC6i #1 is properly absorbed the off-target effects in other tissues. This preferential by the gut and is able to reach both the liver and kid- biodistribution to the liver could be favored by specific neys, leading to HDAC6 inhibition. Importantly, this transport activities driven by transport proteins located effect was mirrored by a significant increase in the at the plasma membrane of cystic cholangiocytes. number of ciliated cholangiocytes and primary cilium In summary, we have designed, synthesized, and length in treated animals, providing important insights functionally analyzed a family of UDCA synthetic into the biological mechanisms behind the therapeutic conjugates with selective HDAC6 inhibitory activity. effects. In line with this result, UDCA-HDAC6i #1 UDCA-HDAC6i #1 inhibited hepatorenal cystogen- restored the primary cilium length of cystic cholan- esis in vivo, improved ciliogenesis in cystic cholan- giocytes in an acetylated α-tubulin–dependent man- giocytes, and inhibited their hyperproliferation. These ner, whereas it had no effect on primary cilium length therapeutic effects are superior to the simple sum of of normal cholangiocytes in vitro. Moreover, UDCA- the individual or combined effects of its constitut- HDAC6i #1 inhibited the proliferation of cystic chol- ing pharmacologically active elements. These UDCA angiocytes cultured in 2D and 3D conditions, and this synthetic conjugates have preferential targeting to the was more significant than the combined treatment liver through specific BA and organic cation trans- with its forming components (i.e., UDCA + (8)) at port properties and are highly concentrated into the the same concentration, indicating that UDCA rein- enterohepatic circulation. These particular features forces the HDAC6 inhibitory activity of the spacer- make UDCA-HDAC6i molecules especially promis- chelating component. This observation was also ing for the treatment of hepatic and gastrointestinal accompanied by a significant reduction of ERK1/2 disorders where HDAC6 inhibition is considered a phosphorylation, which mediates cystic cholangio- therapeutic target, such as acute liver failure, hepa- cyte hyperproliferation.(3) All these data are consistent tocellular carcinoma, cholangiocarcinoma, pancreatic with the fact that the primary cilium acts as a check- cancer, or colon cancer, among others,(23,41-45) high- point inhibitor of the cell cycle, its restoration in cys- lighting the high degree of translational potential of tic cholangiocytes being a therapeutic strategy.(26,27,40) this family of chemical entities. Regarding BA metabolism, and similarly to our previous data with UDCA,(4) no significant changes Author Contributions: F.J.C.-C., I.R., E.H., O.B., in total BA concentration were observed between A.S.-L., L.I.-S., P.Y.L.-L., P.M.R., P.M.-G., S.J., E.P., 201 CABALLERO-CAMINO ET AL. Hepatology, January 2021 S.R., S.G., M.J.P., M.E., L.B., J.J.G.M., J.M.B., F.P.C.: 16) Dominguez MF, Macias RI, Izco-Basurko I, de La Fuente A, study concept and design, acquisition of data, statisti- Pascual MJ, Criado JM, et al. Low in vivo toxicity of a novel cal analysis, analysis and interpretation of data, draft- cisplatin-ursodeoxycholic derivative (Bamet-UD2) with enhanced cytostatic activity versus liver tumors. J Pharmacol Exp Ther ing of the manuscript. L.B., J.J.G.M., J.M.B., F.P.C.: 2001;297:1106-1112. obtained funding. 17) Wahlstrom A, Sayin SI, Marschall HU, Backhed F. Intestinal crosstalk between bile acids and microbiota and its impact on host metabolism. Cell Metab 2016;24:41-50. REFERENCES 18) Nytofte NS, Serrano MA, Monte MJ, Gonzalez-Sanchez E, Tumer Z, Ladefoged K, et al. A homozygous nonsense mutation 1) Gevers TJ, Drenth JP. Diagnosis and management of polycystic (c.214C->A) in the biliverdin reductase alpha gene (BLVRA) re- liver disease. Nat Rev Gastroenterol Hepatol 2013;10:101-108. sults in accumulation of biliverdin during episodes of cholestasis. 2) Perugorria MJ, Masyuk TV, Marin JJ, Marzioni M, Bujanda L, J Med Genet 2011;48:219-225. LaRusso NF, et al. Polycystic liver diseases: advanced insights 19) Steiner C, von Eckardstein A, Rentsch KM. Quantification of the into the molecular mechanisms. Nat Rev Gastroenterol Hepatol 15 major human bile acids and their precursor 7alpha-hydroxy-4- 2014;11:750-761. cholesten-3-one in serum by liquid chromatography-tandem mass 3) Banales JM, Masyuk TV, Gradilone SA, Masyuk AI, Medina spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci JF, LaRusso NF. The cAMP effectors Epac and protein kinase 2010;878:2870-2880. a (PKA) are involved in the hepatic cystogenesis of an animal 20) Urribarri AD, Munoz-Garrido P, Perugorria MJ, Erice O, model of autosomal recessive polycystic kidney disease (ARPKD). Merino-Azpitarte M, Arbelaiz A, et al. Inhibition of metallopro- Hepatology 2009;49:160-174. tease hyperactivity in cystic cholangiocytes halts the development 4) Munoz-Garrido P, Marin JJ, Perugorria MJ, Urribarri AD, Erice of polycystic liver diseases. Gut 2014;63:1658-1667. O, Saez E, et al. Ursodeoxycholic acid inhibits hepatic cystogen- 21) Zubia A, Ropero S, Otaegui D, Ballestar E, Fraga MF, Boix- esis in experimental models of polycystic liver disease. J Hepatol Chornet M, et al. Identification of (1H)-pyrroles as histone 2015;63:952-961. deacetylase inhibitors with antitumoral activity. Oncogene 5) D’Agnolo HM, Kievit W, Takkenberg B, Riaño I, Bujanda L, 2009;28:1477-1484. Neijenhuis MK, et al. Ursodeoxycholic acid in advanced polycystic 22) Hai Y, Christianson DW. Histone deacetylase 6 structure and liver disease: an international multicenter randomized controlled molecular basis of catalysis and inhibition. Nat Chem Biol phase 2 trial. J Hepatol 2016;65:601-607. 2016;12:741-747. 6) Wu M, Mei C. Histone deacetylases 6 increases the cyclic adenos- 23) Gradilone SA, Radtke BN, Bogert PS, Huang BQ, Gajdos GB, ine monophosphate level and promotes renal cyst growth. Kidney LaRusso NF. HDAC6 inhibition restores ciliary expression and Int 2016;90:20-22. decreases tumor growth. Cancer Res 2013;73:2259-2270. 7) Yanda MK, Liu Q, Cebotaru L. An inhibitor of histone deacety- 24) Mason SB, Liang Y, Sinders RM, Miller CA, Eggleston-Gulyas lase 6 activity, ACY-1215, reduces cAMP and cyst growth in T, Crisler-Roberts R, et al. Disease stage characterization of hepa- polycystic kidney disease. Am J Physiol Renal Physiol 2017;313: torenal fibrocystic pathology in the PCK rat model of ARPKD. F997-F1004. Anat Rec (Hoboken) 2010;293:1279-1288. 8) Lorenzo Pisarello M, Masyuk TV, Gradilone SA, Masyuk AI, 25) Masyuk TV, Huang BQ, Masyuk AI, Ritman EL, Torres VE, Ding JF, Lee PY, et al. Combination of a histone deacetylase 6 in- Wang X, et al. Biliary dysgenesis in the PCK rat, an orthologous hibitor and a somatostatin receptor agonist synergistically reduces model of autosomal recessive polycystic kidney disease. Am J hepatorenal cystogenesis in an animal model of polycystic liver Pathol 2004;165:1719-1730. disease. Am J Pathol 2018;188:981-994. 26) Mansini AP, Peixoto E, Thelen KM, Gaspari C, Jin S, Gradilone 9) Gradilone SA, Habringer S, Masyuk TV, Howard BN, Masyuk SA. The cholangiocyte primary cilium in health and disease. AI, Larusso NF. HDAC6 is overexpressed in cystic cholan- Biochim Biophys Acta 2018;1864:1245-1253. giocytes and its inhibition reduces cystogenesis. Am J Pathol 27) Pan J, Snell W. The primary cilium: keeper of the key to cell divi- 2014;184:600-608. sion. Cell 2007;129:1255-1257. 10) Cheng K, Li S, Liao C. Progress in the discovery of macrocyclic 28) Beuers U, Trauner M, Jansen P, Poupon R. New paradigms in the histone deacetylase inhibitors for the treatment of cancer. Curr treatment of hepatic cholestasis: from UDCA to FXR, PXR and Med Chem 2017;24:4166-4179. beyond. J Hepatol 2015;62(1 Suppl.):S25-S37. 11) Mwakwari SC, Patil V, Guerrant W, Oyelere AK. Macrocyclic 29) Butler KV, Kalin J, Brochier C, Vistoli G, Langley B, Kozikowski histone deacetylase inhibitors. Curr Top Med Chem 2010;10: AP. Rational design and simple chemistry yield a superior, neu- 1423-1440. roprotective HDAC6 inhibitor, tubastatin A. J Am Chem Soc 12) Miller TA, Witter DJ, Belvedere S. Histone deacetylase inhibitors. 2010;132:10842-10846. J Med Chem 2003;46:5097-5116. 30) Haggarty SJ, Koeller KM, Wong JC, Grozinger CM, Schreiber 13) Hodawadekar SC, Marmorstein R. Chemistry of acetyl transfer SL. Domain-selective small-molecule inhibitor of histone by histone modifying enzymes: structure, mechanism and implica- deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc tions for effector design. Oncogene 2007;26:5528-5540. Natl Acad Sci U S A 2003;100:4389-4394. 14) Monte MJ, Dominguez S, Palomero MF, Macias RI, Marin 31) Santo L, Hideshima T, Kung AL, Tseng JC, Tamang D, Yang M, JJ. Further evidence of the usefulness of bile acids as molecules et al. Preclinical activity, pharmacodynamic, and pharmacokinetic for shuttling cytostatic drugs toward liver tumors. J Hepatol properties of a selective HDAC6 inhibitor, ACY-1215, in com- 1999;31:521-528. bination with bortezomib in multiple myeloma. Blood 2012;119: 15) Lozano E, Monte MJ, Briz O, Hernandez-Hernandez A, Banales 2579-2589. JM, Marin JJ, et al. Enhanced antitumour drug delivery to chol- 32) Dallavalle S, Pisano C, Zunino F. Development and therapeu- angiocarcinoma through the apical sodium-dependent bile acid tic impact of HDAC6-selective inhibitors. Biochem Pharmacol transporter (ASBT). J Control Release 2015;216:93-102. 2012;84:756-765. 202 Hepatology, Vol. 73, No. 1, 2021 CABALLERO-CAMINO ET AL. 33) Shah MH, Binkley P, Chan K, Xiao J, Arbogast D, Collamore M, progression by inhibiting P53 transcriptional activity. FEBS et al. Cardiotoxicity of histone deacetylase inhibitor depsipeptide Lett 2013;587:880-886. in patients with metastatic neuroendocrine tumors. Clin Cancer 42) Zhang WB, Zhang HY, Jiao FZ, Wang LW, Zhang H, Gong Res 2006;12:3997-4003. ZJ. Histone deacetylase 6 inhibitor ACY-1215 protects against 34) Lane AA, Chabner BA. Histone deacetylase inhibitors in cancer experimental acute liver failure by regulating the TLR4-MAPK/ therapy. J Clin Oncol 2009;27:5459-5468. NF-κB pathway. Biomed Pharmacother 2018;97:818-824. 35) Suraweera A, O’Byrne KJ, Richard DJ. Combination therapy 43) Li D, Sun X, Zhang L, Yan B, Xie S, Liu R, et al. Histone with histone deacetylase inhibitors (HDACi) for the treatment of deacetylase 6 and cytoplasmic linker protein 170 function together cancer: achieving the full therapeutic potential of HDACi. Front to regulate the motility of pancreatic cancer cells. Protein Cell Oncol 2018;8:92. 2014;5:214-223. 36) Hofmann AF. Pharmacology of ursodeoxycholic acid, an entero- 44) Arlt A, Muerkoster SS, Schafer H. Targeting apoptosis pathways hepatic drug. Scand J Gastroenterol Suppl 1994;204:1-15. in pancreatic cancer. Cancer Lett 2013;332:346-358. 37) Hofmann AF, Hagey LR. Bile acids: chemistry, pathochemis- 45) Kaliszczak M, Trousil S, Aberg O, Perumal M, Nguyen QD, try, biology, pathobiology, and therapeutics. Cell Mol Life Sci Aboagye EO. A novel small molecule hydroxamate preferen- 2008;65:2461-2483. tially inhibits HDAC6 activity and tumour growth. Br J Cancer 38) Chiang JYL. Bile acid metabolism and signaling in liver disease 2013;108:342-350. and therapy. Liver Res 2017;1:3-9. Author names in bold designate shared co-first authorship. 39) Miyake Y, Keusch JJ, Wang L, Saito M, Hess D, Wang X, et al. Structural insights into HDAC6 tubulin deacetylation and its se- lective inhibition. Nat Chem Biol 2016;12:748-754. 40) Masyuk TV, Masyuk AI, LaRusso NF. Therapeutic targets in polycystic liver disease. Curr Drug Targets 2017;18:950-957. Supporting Information 41) Ding G, Liu HD, Huang Q, Liang HX, Ding ZH, Liao ZJ, et al. HDAC6 promotes hepatocellular carcinoma Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.31216/suppinfo. 203