International Journal of Molecular Sciences
Communication Chemical Starting Matter for HNF4α Ligand Discovery and Chemogenomics
Isabelle Meijer 1, Sabine Willems 1, Xiaomin Ni 1,2, Jan Heering 3, Apirat Chaikuad 1,2 and Daniel Merk 1,*
1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany; [email protected] (I.M.); [email protected] (S.W.); [email protected] (X.N.); [email protected] (A.C.) 2 Structural Genomics Consortium, BMLS, Goethe-University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt, Germany 3 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch for Translational Medicine and Pharmacology TMP, Theodor-Stern-Kai 7, 60596 Frankfurt, Germany; [email protected] * Correspondence: [email protected]
Received: 14 September 2020; Accepted: 21 October 2020; Published: 24 October 2020
Abstract: Hepatocyte nuclear factor 4α (HNF4α) is a ligand-sensing transcription factor and presents as a potential drug target in metabolic diseases and cancer. In humans, mutations in the HNF4α gene cause maturity-onset diabetes of the young (MODY), and the elevated activity of this protein has been associated with gastrointestinal cancers. Despite the high therapeutic potential, available ligands and structure–activity relationship knowledge for this nuclear receptor are scarce. Here, we disclose a chemically diverse collection of orthogonally validated fragment-like activators as well as inverse agonists, which modulate HNF4α activity in a low micromolar range. These compounds demonstrate the druggability of HNF4α and thus provide a starting point for medicinal chemistry as well as an early tool for chemogenomics.
Keywords: Orphan nuclear receptor; hepatocyte nuclear factor 4α; MODY; type 2 diabetes; fragment-based design; drug discovery
1. Introduction The transcriptional regulator hepatocyte nuclear factor 4α (HNF4α, NR2A1) [1,2] belongs to the protein family of nuclear receptors which are ligand-sensing transcription factors. HNF4α acts as an obligate homodimer [3] and has constitutive transactivation activity [1]. It was initially considered as an orphan nuclear receptor [1,2] prior to the identification of linoleic acid as a potential endogenous ligand, whose binding, however, has been reported to hardly affect the receptor’s transcriptional activity [4]. HNF4α is mainly found in hepatocytes, enterocytes, and pancreatic β-cells [5,6], and exhibits key regulatory roles in intestine [7], liver [8], and pancreas [9]. Its dysregulation has been associated with gastrointestinal and metabolic diseases [5,10,11] as well as gastrointestinal cancers [5,12,13]. Mutations within the HNF4α gene cause the heritable form of type 2 diabetes, maturity-onset diabetes of the young 1 (MODY-1) [10,14–16], highlighting the receptor’s crucial role in metabolic homeostasis. Despite an attractive potential for pharmacological interventions in diabetes or cancer, the collection of ligands that can modulate the transcriptional activity of HNF4α is scarce. Kiselyuk et al. [5] have previously reported antagonists of HNF4α with low micromolar potencies that decreased the receptor levels as well as expression of the targeted genes in cellular settings. In addition, a series of naphthofuranes [17] was found to bind the HNF4α ligand-binding domain (LBD) and enhance the receptor’s transcriptional
Int. J. Mol. Sci. 2020, 21, 7895; doi:10.3390/ijms21217895 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 7895 2 of 11 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 10 activity.the possibility While theseof HNF4 importantα modulation observations with demonstratesmall molecules, the possibility novel HNF4 of HNF4α ligandsα modulation are needed with to smallovercome molecules, the limited novel HNF4potency,α ligands physicochemical are needed tore overcomestrictions, theand limited lack potency,of chemical physicochemical diversity of restrictions,available HNF4 andα lack modulators. of chemical diversity of available HNF4α modulators. To expand the the collection collection of of HNF4 HNF4αα ligands,ligands, we we screened screened a collection a collection of 480 of 480 drug drug fragments fragments for forHNF4 HNF4α modulationα modulation in a in cellular a cellular setting. setting. Dose-response Dose-response profiling, profiling, control control experiments, experiments, effects effects on onHNF4 HNF4α-regulatedα-regulated gene gene expression expression in innative native cellular cellular setting, setting, and and interaction interaction studies studies with with the recombinant HNF4α LBD fully confirmedconfirmed a set of appealingappealing moleculesmolecules as HNF4HNF4α modulatorsmodulators including agonists and inverse agonists. The The most active compounds modulated the orphan nuclear receptor with low micromolar potencies andand modulated mRNA expression of the HNF4α-regulated gene fructose-1,6-bisphosphatase 1 (FBP1) in humanhuman hepatocytes.hepatocytes. For For three HNF4 α modulators, isothermal titration confirmed confirmed direct binding to the recombinantrecombinant HNF4α LBD with micromolar aaffinities.ffinities. This set of chemically diverse HNF4 α ligands will serve as a starting point forfor medicinalmedicinal chemistry, enablingenabling thethe developmentdevelopment of tooltool compoundscompounds for furtherfurther pharmacological studies on the role of HNF4α in diseases.
2. Results 2.1. Primary Screening 2.1. Primary Screening To search for new modulators of HNF4α activity without previous knowledge on the To search for new modulators of HNF4α activity without previous knowledge on the structure– structure–activity relationship of HNF4α ligands, we screened a collection of 480 fragments derived activity relationship of HNF4α ligands, we screened a collection of 480 fragments derived from FDA from FDA approved drugs for their abilities to modulate HNF4α activity in vitro (the fragment approved drugs for their abilities to modulate HNF4α activity in vitro (the fragment structures structures contained in the library and associated primary screening data are provided as Table S1). contained in the library and associated primary screening data are provided as Table S1). This library This library was chemically diverse in terms of feature distribution (Figure1) and sca ffolds, providing was chemically diverse in terms of feature distribution (Figure 1) and scaffolds, providing an an attractive unbiased and economic entry to HNF4α ligand discovery. attractive unbiased and economic entry to HNF4α ligand discovery.
FigureFigure 1. Characteristics 1. Characteristics and featureand feature distribution distribution of the of fragment the fragment screening screening library. library. TPSA—topological TPSA – polar surfacetopological area; HBD—H-bond polar surface donor; area; HBA—H-bondHBD – H-bond acceptor.donor; HBA – H-bond acceptor.
For the primary screening system, we employed a hy hybridbrid Gal4 reporter gene assay in transiently transfected HEK293T cells. Therein, Therein, a Gal4 responsive firefly firefly luciferase construct served as a reporter gene to determine the transcriptional activity of a hybrid receptor composed of the human HNF4α LBD andand thethe DNA-binding DNA-binding domain domain of Gal4of Gal4 from from yeast. yeast. A constitutively A constitutively active active (SV40 (SV40 promoter) promoter) renilla luciferaserenilla luciferase construct construct was additionally was additionally present present to monitor to monitor the transfection the transfection efficiency efficiency and toxicity and toxicity of test compounds.of test compounds. This system This system is advantageous is advantageous as it captures as it captures various characteristicsvarious characteristics of nuclear of receptornuclear modulatorsreceptor modulators including potency,including type potency, of activity, type e ffiofcacy, activity, and cellefficacy, permeability and cell [18 permeability]. In accordance [18]. with In previousaccordance reports with onprevious the behavior reports of on HNF4 the behaviorα [1], the of chimeric HNF4αGal4-HNF4 [1], the chimericα receptor Gal4-HNF4 revealedα receptor marked constitutiverevealed marked transcriptional constitutive activity transcriptiona in the absencel activity of ligands.in the absence of ligands. The entire fragment library was tested for Gal4-HNF4 α modulation using this primary screening assay, whichwhich waswas performedperformed in twotwo biologicallybiologically independent repeats using compounds at 50 µµMM concentration. ReporterReporter activityactivity for for the the entire entire fragment fragment set set was was narrowly narrowly distributed distributed with with a mean a mean of 1.09 of and1.09 aand standard a standard deviation deviation of 0.43. of Fragments0.43. Fragments exhibiting exhibiting a fold a activation fold activation outside outside the mean the meanSD region ± SD ± wereregion evaluated were evaluated as primary as primary hits. Eighteen hits. Eighteen fragments fragments induced induced reporter reporter activity toactivity values to above values 1.52-fold above activation1.52-fold activation (mean + SD) (mean and were+ SD) considered and were as considered potential agonists. as potential Five compoundsagonists. Five suppressed compounds the suppressed the reporter signal to values below 0.67-fold activation (mean - SD) and were considered as potential inverse agonists (Figure 2). The primary hits were then manually curated for toxic
Int. J. Mol. Sci. 2020, 21, 7895 3 of 11
Int.reporter J. Mol. Sci. signal 2020, to21, valuesx FOR PEER below REVIEW 0.67-fold activation (mean - SD) and were considered as potential3 of 10 inverse agonists (Figure2). The primary hits were then manually curated for toxic compounds (as compounds (as observed by effects on constitutively active renilla luciferase) and undesired observed by effects on constitutively active renilla luciferase) and undesired structures (PAINS), leaving structures (PAINS), leaving a primary hit collection of eleven fragments (1-11) for validation (Table a primary hit collection of eleven fragments (1-11) for validation (Table1). This primary hit list 1). This primary hit list comprised eight potential HNF4α activators (1–8) and three inverse agonist comprised eight potential HNF4α activators (1–8) and three inverse agonist candidates (9–11). Int. J. candidatesMol. Sci. 2020 ,( 219–, 11x FOR). PEER REVIEW 3 of 10 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 10 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 10 compounds (as observed by effects on constitutively active renilla luciferase) and undesired compounds (as observed by effects on constitutively active renilla luciferase) and undesired compoundsstructures (PAINS), (as observed leaving by a primary effects hiton collectionconstituti velyof eleven active fragments renilla luciferase)(1-11) for validation and undesired (Table structures (PAINS), leaving a primary hit collection of eleven fragments (1--11)) forfor validationvalidation (Table(Table structures1). This primary (PAINS), hit leavinglist comprised a primary eight hit potential collection HNF4 of elevenα activators fragments (1–8 ()1 and-11) threefor validation inverse agonist (Table 1). This primary hit list comprised eight potential HNF4α activatorsactivators ((1–8)) andand threethree inverseinverse agonistagonist 1).candidates This primary (9–11 ).hit list comprised eight potential HNF4α activators (1–8) and three inverse agonist candidates (9–11). candidates (9–11).
Figure 2. Activity distribution and primary hits in the fragment screening. Red line shows the mean, Figure 2. Activity distribution and primary hits in the fragment screening.ing. RedRed lineline showsshows thethe mean,mean, Figuredotted 2.red Activity lines showdistribution mean ±and SD. prim Eachary point hits in represents the fragment the screenactivitying. of Red one line fragment shows theat 50 mean, µM dotted red lines show mean ± SD. Each point represents the activity of one fragment at 50 µM dottedexpressed red as lines mean show fold meanactivation ± SD. vs. Each 0.2% pointDMSO represents from two thebiologically activity inofdependent one fragment repeats. at 50 Green µM expressed as mean fold activation vs. 0.2% DMSO fromfrom twotwo biologicallybiologically inindependent repeats. Green expressedcrosses refer as tomean HNF4 foldα agonistactivation candidates, vs. 0.2% DMSOyellow crossesfrom two refer biologically to inverse in HNF4dependentα agonist repeats. candidates, Green crosses refer to HNF4α agonistagonist candidates,candidates, yellowyellow crossescrosses referrefer toto inverseinverse HNF4HNF4α agonistagonist candidates,candidates, crossesblue Figuredots refer represent 2. to Activity HNF4 fragmentsα distribution agonist without candidates, and activity prim primary yellowary on hits crossesGal4-HNF4 in the refer fragment α to. inverse screen screening. HNF4ing. αRed Red agonist line line shows showscandidates, the the mean, mean, blue dots represent fragments without activity on Gal4-HNF4α. blue dotteddots represent redred lineslines fragments show show mean mean withoutSD. ± SD. Each activity Each point onpoint represents Gal4-HNF4 repres theentsα activity. the activity of one fragmentof one fragment at 50 µM at expressed 50 µM ± Tableexpressedas 1. mean Primary fold as screening activationmean fold hits vs.activation with 0.2% primary DMSO vs. 0.2% from screening DMSO two biologically datafrom and two control biologically independent experiment independent repeats. on VP16 Green repeats. for crosses non- Green refer Table 1. Primary screening hits with primary screening data and control experiment on VP16 for non- Tablespecificcrossesto 1. HNF4 Primaryactivity. referα agonist screeningtoPrimary HNF4 candidates, α screeninhits agonist withg candidates,yellowprimary data are crosses screening theyellow refermean crossesdata to of inverseand two refer control biologically HNF4to inverse experimentα agonist HNF4independent on candidates,α agonist VP16 forrepeats. candidates, non- blue dots specificspecific activity.activity. PrimaryPrimary screeninscreening data are the mean of two biologically independent repeats. specificHNF4bluerepresentα follow-upactivity. dots represent fragments Primary data and fragments without screenin VP16 activitycontrol gwithout data ondataare activity Gal4-HNF4 arethe the meanon meanGal4-HNF4α .of ±two SD biologicallyfoldα. activation independent of reporter repeats.activity HNF4α follow-upfollow-up datadata andand VP16VP16 controlcontrol datadata areare thethe meanmean ±± SDSD foldfold activationactivation ofof reporterreporter activityactivity HNF4of at leastα follow-up four biologically data and independentVP16 control repeatsdata are in the duplicates. mean ± SD n.s. fold - not activation significant of reporter(p ≥ 0.05), activity ** p < of at Tableleast four 1. Primary biologically screening independent hits with repeats primary in duplicates. screening datan.s. - and not controlsignificant experiment (p ≥ 0.05),0.05), on ****VP16 p << for of0.01, at Table***least p 1.3 ± 0.2 1.3 ± 0.3 2 1.55 1.3 ± 0.2 1.3 ± 0.3 n.s. 2 1.55 1.3 ±(50 0.2 µM) 1.3 ±(50 0.3 µM) n.s. 2 1.55 1.3(501.3 ±µM) 0.20.2 1.3(501.3 ±µM) 0.30.3 n.s. 2 2 1.551.55 (50 µM) (50 µM) n.s. n.s. (50 µM)±µ (50 µM)±µ (50(50 µM)M) (50(50 µM)M) 1.5 ± 0.2 1.7 ± 0.3 3 2.09 n.s. (50 µM) (50 µM) 1.5 ± 0.2 1.7 ± 0.3 3 2.09 1.5 ± 0.2 1.7 ± 0.3 n.s. 3 2.09 1.51.5 ± 0.20.2 1.71.7 ± 0.30.3 n.s. 3 3 2.092.09 (50 µM) (50 µM) n.s. n.s. 3 2.09 (50(50 µM)µM)±µ4 ± 2 (50(50 µM)µM)0.5±µ ± 0.2 n.s.p = 0.0014 (50(50 µM)M) (50(50 µM)M) 4 5.18 (50 µM) (50 µM) (**) 4 ±2.5 2 ± 0.2 0.5 ±1.1 0.2 ± 0.1 p = 0.0014p < 0.0001 4 5 5.181.80 4 4± 2 2 0.50.5 ± 0.20.2 p == 0.00140.0014 4 4 5.185.18 (504 ±µM)(100 ±2 µM) 0.5(50 ±µM)(100 0.2± µM) p = p(**)0.0014= 0.0014 (***) (**) 4 5.18 (50(50(50 µM)µM)µM) (50(50(50 µM)µM)µM) (**)(**) (50 µM) (50 µM) (**) 2.4 ± 0.5 1.1 ± 0.1 p = 0.0034 6 5.48 2.5 ± (50.2 µM) 1.1 ± (50.1 µM) p < 0.0001(**) 5 1.80 2.52.5 ± 0.20.2 1.11.1 ± 0.10.1 p << 0.00010.0001 5 5 1.801.80 (1002.5 ± µM) ±0.2 (1001.1 ± µM) 0.1± p 2.4 ± 0.5 1.1 ± 0.1 p = 0.0034 6 5.48 2.4 ± 1.10.5 ± 0.2 1.1 ±0.92 0.1 ± 0.07p == 0.00340.0034 6 8 5.482.09 2.4(5 µM)± 0.5 1.1(5 µM)± 0.1 p = (**)0.0034 n.s. 6 5.48 (5 µM)(50 µM) (5 µM)(50 µM) (**) (5 µM) (5 µM) (**) 3.0 ± 0.3 1.4 ± 0.1 p < 0.0001 7 2.36 3.0 ± 0.3 1.4 ± 0.1 p << 0.00010.0001 7 2.36 3.0(50 ±µM) 0.3 1.4(50 ±µM) 0.1 p < (***)0.0001 7 2.36 (50 µM) (50 µM) (***) (50 µM) (50 µM) (***) Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 10 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3 of 10 compounds (as observed by effects on constitutively active renilla luciferase) and undesired structures (PAINS), leaving a primary hit collection of eleven fragments (1-11) for validation (Table 1). This primary hit list comprised eight potential HNF4α activators (1–8) and three inverse agonist candidates (9–11). Figure 2. Activity distribution and primary hits in the fragment screening. Red line shows the mean, dotted red lines show mean ± SD. Each point represents the activity of one fragment at 50 µM expressed as mean fold activation vs. 0.2% DMSO from two biologically independent repeats. Green crosses refer to HNF4α agonist candidates, yellow crosses refer to inverse HNF4α agonist candidates, blue dots represent fragments without activity on Gal4-HNF4α. Table 1. Primary screening hits with primary screening data and control experiment on VP16 for non- specific activity. Primary screening data are the mean of two biologically independent repeats. HNF4α follow-up data and VP16 control data are the mean ± SD fold activation of reporter activity of at least four biologically independent repeats in duplicates. n.s. - not significant (p ≥ 0.05), ** p < 0.01, *** p < 0.001 (t-test). Primary Screen Follow-Up VP16 HNF4α vs. ID Structure (Fold Act., 50 HNF4α Control VP16 µM) 1.1 ± 0.2 0.80 ± 0.07 1 2.48 n.s. (50 µM) (50 µM) 1.3 ± 0.2 1.3 ± 0.3 2 1.55 n.s. (50 µM) (50 µM) 1.5 ± 0.2 1.7 ± 0.3 3 2.09 n.s. (50 µM) (50 µM) 4 ± 2 0.5 ± 0.2 p = 0.0014 4 5.18 (50 µM) (50 µM) (**) Int. J. Mol. Sci. 2020, 21, 7895 4 of 11 Table 1. Cont2.5. ± 0.2 1.1 ± 0.1 p < 0.0001 5 1.80 (100 µM) (100 µM) (***) Primary Screen Follow-up HNF4α vs. ID Structure VP16 Control (Fold Act., 50 µM) HNF4α VP16 2.4 ± 0.5 1.1 ± 0.1 p = 0.0034 6 5.48 2.42.4 ± 0.50.5 1.11.1 ± 0.10.1 p = 0.0034 6 6 5.485.48 (5 µM)± (5 µM)± p(**)= 0.0034 (**) (5(5 µM)µM) (5 (5µM)µM) (**) Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 10 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 10 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 3.03.0 ± 0.30.3 1.41.4 ± 0.10.1 p < 0.00014 of 10 Int. J. Mol.7 7 Sci. 2020, 21, x FOR PEER REVIEW 2.362.36 ± ± p < 0.00014 of (***)10 (50(50 µM)µM) (50(50 µM)µM) (***) 1.1 ± 0.2 0.92 ± 0.07 8 2.09 1.1 ± 0.2 0.92 ± 0.07 n.s. 8 2.09 1.1(50 ± µM) 0.2 0.92(50 ± µM) 0.07 n.s. 8 2.09 1.11.1 ± 0.20.2 0.920.92 ± 0.070.07 n.s. 8 8 2.092.09 (50 µM)± (50 µM)± n.s. n.s. (50(50(50 µM)µM)µM) (50(50(50 µM)µM)µM) 0.6 ± 0.1 1.2 ± 0.2 p < 0.0001 9 0.54 0.6 ± 0.1 1.2 ± 0.2 p < 0.0001 9 0.54 0.60.6(300.6 ± ±µM) 0.10.10.1 1.21.2(301.2 ± ±µM) 0.20.20.2 pp << (***) 0.00010.0001 99 9 0.540.540.54 (30 µM)± (30 µM)± p(***)< 0.0001 (***) (30(30(30 µM)µM)µM) (30(30(30 µM)µM)µM) (***)(***) 0.75 ± 0.05 1.1 ± 0.2 p = 0.0057 10 0.63 0.750.750.75 ± ± 0.05 0.050.05 1.11.11.1 ± ± 0.2 0.20.2 pp = = 0.0057 0.0057 101010 0.630.630.63 0.75(50 ±µM)± 0.05 1.1(50 ±µM) ±0.2 p = p(**)0.0057= 0.0057 (**) 10 0.63 (50(50(50 µM) µM)µM) (50(50(50 µM) µM)µM) (**)(**) (50 µM) (50 µM) (**) 0.21 ± 0.03 0.21 ± 0.02 0.21 0.03 0.21 0.02 1111 0.320.32 0.21 ± 0.03 0.21 ± 0.02 n.s. n.s. 11 0.32 0.210.21(100 ±± µM)± 0.030.03µ 0.210.21(100 ±± µM) 0.02±0.02µ n.s. 1111 0.320.32 (100(100 µM)M) (100(100 µM)M) n.s.n.s. (100(100 µM)µM) (100(100 µM)µM) 2.2. Hit2.2. Validation Hit Validation 2.2. Hit Validation 2.2.2.2. HitHit ValidationValidation HNF4HNF4α modulationα modulation of the of primary the primary screening screening hits 1 hits–111 was–11 wassubsequently subsequently reproduced reproduced with with fresh fresh HNF4α modulation of the primary screening hits 1–11 was subsequently reproduced with fresh materialHNF4materialHNF4 inαα modulation inmodulationfour four biologically biologically ofof thethe primaryprimary independentindependent screeningscreening repeats repeats hitshits in 1 1 duplicates.––in1111 wasduplicates.was subsequentlysubsequently For compoundsFor compounds reproducedreproduced exhibiting withwithexhibiting pronounced freshfresh material in four biologically independent repeats in duplicates. For compounds exhibiting materialmaterialpronouncedtoxicity inin atfourtoxicityfour 50 µbiologicallybiologicallyM, at concentration50 µM, concentrationindependentindependent was reduced wasrepeatsrepeats reduced (6: 5ininµ M;duplicates. duplicates.(6:9 5: 30µM;µ M).9 : ForFor30 Fragments µM). compoundscompounds Fragments5 and exhibitingexhibiting 115 andrevealed 11 a pronounced toxicity at 50 µM, concentration was reduced (6: 5 µM; 9: 30 µM). Fragments 5 and 11 pronouncedpronouncedrevealedtendency a tendency toxicitytoxicity for HNF4 atatfor α5050 modulationHNF4 µM,µM, concentrationαconcentration modulation at 50 µM andwasatwas 50 were reducedreduced µM subsequently and (( 66were:: 55 µM;µM; subsequently characterized 99:: 3030 µM).µM). FragmentscharacterizedFragments at 100 µM. 55 Additionally, andand at 100 1111 revealed a tendency for HNF4α modulation at 50 µM and were subsequently characterized at 100 revealedrevealedµM. theAdditionally, primary aa tendencytendency hits the were forfor primary HNF4HNF4 furtherαα modulation validatedhitsmodulation were infurther control atat 5050 µMµMvalidated experiments andand werewere in involving control subsequentlysubsequently experiments the potent, characterizedcharacterized ligand-independent involving atat 100100the µM. Additionally, the primary hits were further validated in control experiments involving the µM.µM.potent, transcriptionalAdditionally,Additionally, ligand-independent thethe activator primaryprimary Gal4–VP16transcriptional hitshits werewere to furtherfurther replace activator validated Gal4–HNF4validated Gal4–VP16 inin α controlcontrolwith to otherwise replaceexperimentsexperiments Gal4–HNF4 identical involvinginvolving assayα with the settingsthe potent, ligand-independent transcriptional activator Gal4–VP16 to replace Gal4–HNF4α with potent,potent,otherwiseand ligand-independentligand-independent test identical compound assay concentrations settings transcriptionaltranscriptional and test (Table compound activatoractivator1). Like concentrations Gal4–HNF4Gal4–VP16Gal4–VP16 toαto, (Table Gal4–VP16replacereplace 1). Gal4–HNF4 Gal4–HNF4Like has Gal4–HNF4 highα constitutiveα withwithα, otherwise identical assay settings and test compound concentrations (Table 1). Like Gal4–HNF4α, otherwiseotherwiseGal4–VP16transcriptional identicalidentical has high assay inducerassay constitutive settingssettings activity andtranscriptionaland but testtest is notcompoundcompound responsive indu cerconcentrationsconcentrations toactivity ligands. but Thus, (Table(Tableis not fragments 1).responsive1). LikeLike Gal4–HNF4Gal4–HNF4 aff toecting ligands. reporterαα,, Gal4–VP16 has high constitutive transcriptional inducer activity but is not responsive to ligands. Gal4–VP16Gal4–VP16Thus,activity fragments hashas in thehighhigh affecting Gal4–VP16 constitutiveconstitutive reporter setting transcriptionaltranscriptional activity likely in modulate the induindu Gal4–VP16cercer non-specific activityactivity setting butbut cellular isislikely notnot responsive processes.modulateresponsive non-specific to Fragmentsto ligands.ligands. that Thus, fragments affecting reporter activity in the Gal4–VP16 setting likely modulate non-specific Thus,Thus,cellularexhibited fragmentsfragments processes. HNF4 affectingaffecting Fragmentsα modulation reporterreporter that exhibited activity inactivity this initial inHNF4in thethe follow-up αGal4–VP16Gal4–VP16 modulation and settingsetting in had this nolikelylikely initial effects modulatemodulate follow-up on Gal4–VP16-induced non-specificnon-specific and had no cellular processes. Fragments that exhibited HNF4α modulation in this initial follow-up and had no cellularcellulareffectsreporter processes.onprocesses. Gal4–VP16-induced activity FragmentsFragments were further thatthat reporter exhibited studiedexhibited by activity HNF4HNF4 full dose-responseαα modulationweremodulation further in characterization.in thisthisstudied initialinitial by follow-upfollow-up full1– 3,dose-response8 , andand and hadhad11 showed nono effects on Gal4–VP16-induced reporter activity were further studied by full dose-response effectseffectscharacterization.no on statisticallyon Gal4–VP16-inducedGal4–VP16-induced 1–3 significant, 8, and 11 activityshowed reporterreporter inno theactivitystatisticallyactivity Gal4–HNF4 werewere significant furtherαfurtherassay activitystudied comparedstudied in by bythe to fullGal4–HNF4full the Gal4–VP16dose-responsedose-responseα assay control, characterization. 1–3, 8, and 11 showed no statistically significant activity in the Gal4–HNF4α assay characterization.characterization.comparedsuggesting to the unspecific 1Gal4–VP161––33,, 88,, andand eff 11ects.control,11 showedshowed Fragments sugg nonoesting statisticallystatistically4–7 unspecificwere confirmed significantsignificant effects. as activity activityFragments HNF4 α ininagonists thethe 4– 7 Gal4–HNF4Gal4–HNF4 were and confirmed fragmentsαα assayassay as9 and compared to the Gal4–VP16 control, suggesting unspecific effects. Fragments 4–7 were confirmed as comparedcomparedHNF410αexhibited agonists toto thethe Gal4–VP16 Gal4–VP16 preliminarilyand fragments control,control, validated 9 and suggsugg 10 inverseexhibitedestingesting unspecificunspecific HNF4 preliminarilyα agonism. effects.effects. validated FragmentsFragments inverse 44––77 werewere HNF4 confirmedconfirmedα agonism. asas HNF4α agonists and fragments 9 and 10 exhibited preliminarily validated inverse HNF4α agonism. HNF4HNF4Dose-responseαα agonistsagonistsDose-response andand profiling fragmentsfragments profiling (Table 99 andand (Table 2, 1010 Figure 2 exhibitedexhibited, Figure 3) revealed3 ) preliminarilypreliminarily revealed 6 as 6the as validatedvalidated strongest the strongest inverseinverse and andmost HNF4HNF4 most potentαα agonism. potentagonism. HNF HNFα α Dose-responseDose-response profiling profiling (Table (Table 2, 2, Figure Figure 3) 3) revealed revealed 6 6 as as the the strongest strongest and and most most potent potent HNF HNFαα agonistagonistDose-response with with an EC an50 profilingEC value50 value of (Table5.8 of µM 5.8 2, andµ FigureM anda high 3) a high revealed6.1-fold 6.1-fold maximum6 as maximum the strongest activation activation and efficacy. most effi cacy.potent Fragments Fragments HNF α4 4 agonist with an EC50 value of 5.8 µM and a high 6.1-fold maximum activation efficacy. Fragments 4 agonist50 = with an EC50 value of 5.8 µM and50 a high 6.1-fold maximum activation efficacy. Fragments 4 agonist(EC (EC 15with50 =µM, 15an µ5.6-fold ECM,50 5.6-fold value act.) of act.)and 5.8 µM7 and (EC and7 (EC = a 3150high =µM,31 6.1-fold µ3.8-foldM, 3.8-fold maximum act.) act.)comprised activation comprised slightly efficacy. slightly weaker Fragments weaker agonist agonist 4 (EC50 = 15 µM, 5.6-fold act.) and 7 (EC50 = 31 µM, 3.8-fold act.) comprised slightly weaker agonist (EC50 = 15 µM, 5.6-fold act.) and 7 (EC50 = 31 µM, 3.8-fold act.) comprised slightly weaker agonist (ECpotencies50potencies = 15 thanµM, than 65.6-fold, while6, while fragmentact.) fragment and 57 (EC (EC5 50(EC50 > = 10050 31> µM)µM,100 µ was3.8-foldM) considerably was considerablyact.) comprised less active. less slightly active. The inverse Theweaker inverse agonists agonist agonists potencies than 6, while fragment 5 (EC50 > 100 µM) was considerably less active. The inverse agonists potenciespotencies9 (IC50 = 8 thanthan µM) 66 ,and, whilewhile 10 fragmentfragment(IC50 = 24 5 5µM) (EC(EC 5050demonstrated >> 100100 µM)µM) waswas low considerablyconsiderably micromolar lessless activity, active.active. and TheThe both inverseinverse diminished agonistsagonists 9 (IC50 = 8 µM) and 10 (IC50 = 24 µM) demonstrated low micromolar activity, and both diminished 99remaining (IC(IC5050 == 88 µM) µM)HNF4 andandα activity 1010 (IC(IC5050 by == 24approximately24 µM)µM) demonstrateddemonstrated half. lowlow micromolarmicromolar activity,activity, andand bothboth diminisheddiminished remaining HNF4α activity by approximately half. remainingremaining HNF4HNF4αα activityactivity byby approximatelyapproximately half.half. Table 2. Validated HNF4α ligands with control experiment HNF4α modulatory activity and binding Table 2. Validated HNF4α ligands with control experiment HNF4α modulatory activity and binding TableTableaffinity 2.2. ValidatedtoValidated the recombinant HNF4HNF4αα ligandsligands HNF4 withwithα ligand-binding controlcontrol experimentexperiment domain HNF4 HNF4 (LBD).αα modulatorymodulatory EC50 and activityactivity IC50 andvaluesand bindingbinding were affinity to the recombinant HNF4α ligand-binding domain (LBD). EC50 and IC50 values were 50 50 affinityaffinitycalculated toto fromthethe recombinantdose–responserecombinant HNF4HNF4 curvesαα andligand-bindingligand-binding are the mean domaindomain ± SD of at(LBD).(LBD). least fourECEC50 biologically andand ICIC50 values independentvalues werewere calculated from dose–response curves and are the mean ± SD of at least four biologically independent calculatedcalculatedrepeats in from duplicates.from dose–response dose–response Fold and curves curvesremaining and and are(rem.)are the the actimean meanvation ± ± SD SD (act.) of of at at referleast least four tofour the biologically biologically maximum independentindependentfold increase repeats in duplicates. Fold and remaining (rem.) activation (act.) refer to the maximum fold increase repeatsrepeatsor decrease inin duplicates.duplicates. in reporter FoldFold activity andand remainingremaining relative (rem.)to(rem.) 0.1% actiacti DMSO.vationvation (act.)Binding(act.) referrefer was toto thethedetermined maximummaximum by foldfold isothermal increaseincrease or decrease in reporter activity relative to 0.1% DMSO. Binding was determined by isothermal orortitration decreasedecrease calorimetry inin reporterreporter (ITC). activityactivity n.d. – relativenotrelative determined toto 0.1%0.1% DMSO.DMSO. BindingBinding waswas determineddetermined byby isothermalisothermal titration calorimetry (ITC). n.d. – not determined titrationtitration calorimetrycalorimetry (ITC).(ITC). n.d.n.d. –– notnot determineddetermined HNF4α Validated HNF4α HNF4α LBD ID Structure HNF4α Validated HNF4α HNF4α LBD ID Structure LigandHNF4HNF4 Typeαα ValidatedValidatedModulation HNF4HNF4 αα HNF4HNF4Bindingαα LBDLBD IDID StructureStructure Ligand Type Modulation Binding LigandLigand TypeType ModulationModulation BindingBinding Int. J. Mol. Sci. 2020, 21, 7895 5 of 11 9 (IC50 = 8 µM) and 10 (IC50 = 24 µM) demonstrated low micromolar activity, and both diminished remaining HNF4α activity by approximately half. Table 2. Validated HNF4α ligands with control experiment HNF4α modulatory activity and binding affinity to the recombinant HNF4α ligand-binding domain (LBD). EC50 and IC50 values were calculated from dose–response curves and are the mean SD of at least four biologically independent repeats in ± duplicates. Fold and remaining (rem.) activation (act.) refer to the maximum fold increase or decrease in reporter activity relative to 0.1% DMSO. Binding was determined by isothermal titration calorimetry Int.(ITC). J. Mol. n.d.—not Sci. 2020, determined21, x FOR PEER REVIEW 5 of 10 Int.Int. J.J. Mol.Mol. Sci.Sci. 20202020,, 2121,, xx FORFOR PEERPEER REVIEWREVIEW 55 ofof 1010 Int.Int. J.J. Mol.Mol. Sci.Sci. 2020,, 21,, xx FORFOR PEERPEER REVIEWREVIEW 5 of 10 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEWHNF4 α Ligand Validated HNF4α HNF4α LBD 5 of 10 ID Structure Type Modulation Binding 50 EC 50 15 ± 1 µM 4 Agonist ECEC50 1515 ±± 11 µMµM no/weak binding 4 Agonist EC50 15 ± 1 µM no/weak binding 4 Agonist 5.65.6EC ±±50 0.3-fold0.3-fold 15 ± 1 µM act.act. no/weak binding 4 Agonist 5.6EC ±50 0.3-fold15 ± 1 µµM Mact. no/weak binding 4 4 AgonistAgonist 5.6 ±50 0.3-fold± act. no/weakno/weak binding binding 5.65.6 ± 0.3-fold0.3-fold act.act. ± 50 5 Agonist EC 50 > 100 µM n.d. 55 AgonistAgonist ECEC50 >> 100100 µMµM n.d. n.d. 50 5 Agonist EC 50 >> 100100 µMµM n.d. n.d. 5 5 AgonistAgonist EC 5050 > 100100 µµMM n.d. n.d. 50 EC 50 5.8 ± 0.6 µM 6 Agonist ECEC50 5.85.8 ±± 0.60.6 µMµM no binding 6 Agonist 6.1EC 50± 5.80.7-fold ± 0.6 act.µM no binding 6 Agonist 6.1EC 50± 5.80.7-fold ± 0.6 µact.µMM no binding 6 6 AgonistAgonist EC6.150 ± 5.8 0.7-fold ± 0.6 µMact. no bindingno binding 6 Agonist 6.16.1 ± 0.7-fold0.7-fold act.act. no binding 6.1 ± 0.7-fold act. 50 EC 50 31 ± 8 µM 7 Agonist ECEC50 3131 ±± 88 µMµM Kd 7 µM 7 Agonist 50 µ Kd 7 µM 7 Agonist 3.8ECEC ±5050 0.6-fold 3131 ± 8 µM act.M Kd 7 µM 7 7 AgonistAgonist 3.8EC ±50 0.6-fold 31 ±± 8 µM act. Kd 7KµdM 7 µM 7 Agonist 3.8EC ± 0.6-fold31 ± 8 µM act. act. Kd 7 µM 7 Agonist 3.8 ± 0.6-fold act. Kd 7 µM 3.8 ± 0.6-fold act. Inverse IC50 8 ± 2 µM Inverse IC50 8 ± 2 µM 9 Inverse ICIC50 88 ± 22 µµMM Kd 0.3 µM 9 50 Kd 0.3 µM 9 9 InverseagonistInverse agonist 0.57IC ± 500.04 8 ± rem.2 µM act. Kd 0.3Kdµ 0.3M µM agonistInverse 0.57IC ±50 0.04 8 ± rem.2 µM act. d 9 Inverseagonist 0.57IC ± 0.04 8 ± rem.2rem. µM act. act. Kd 0.30.3 µMµM 9 agonist 0.57 ± 0.04 rem. act. Kd 0.3 µM agonist 0.57 ± 0.04 rem. act. Inverse IC50 24 ± 5 µM IC50 24 5 µM 10 InverseInverse ICIC5050 2424 ±± 55 µMµM Kd 1.7 µM 1010 Inverse agonist 50 ± Kd 1.7Kdµ 1.7M µM 10 agonistInverse 0.65IC ±50 0.05 24 ± rem. 5 µM act. Kd 1.7 µM agonistInverse 0.650.65IC ±50 0.05 0.0524 ± rem. 5 µM act.act. d 10 Inverseagonist 0.65IC ±± 0.0524 ± rem.5 µM act. Kd 1.7 µM 10 agonist 0.65 ± 0.05 rem. act. Kd 1.7 µM agonist 0.65 ± 0.05 rem. act. To capture HNF4α modulation by 4, 6, 7, 9, and 10 in a more physiological setting, we treated HNF4α-expressingFigure 3. In [vitro19] human characterization hepatocytes of HNF4 (HepG2)α modulators with the HNF44, 6, 7, α9, modulatorsand 10. (a) Dose–response and then determined curves FigureFigure 3.3. InIn vitrovitro characterizationcharacterization ofof HNF4HNF4αα modulatorsmodulators 44,, 66,, 77,, 99,, andand 1010.. ((aa)) Dose–responseDose–response curvescurves the mRNAofFigure 4 expression, 6, 73., 9InIn, and vitrovitro levels 10 characterizationcharacterization onof Gal4-HNF4 the HNF4 α ofofα (mean -regulated HNF4HNF4 ±α S.E.M.;modulatorsmodulators gene n fructose-1,6-bisphosphatase≥ 4). 4, , ( 6b,,) 7 Effects,, 9,, andand of 10 4.., ((6a,)) 7 Dose–responseDose–response, 9, and 1 (FBP1)10 on mRNA curvescurves [19] by ofFigureof 44,, 66,, 73.7,, 9In9,, andvitroand 1010 characterization onon Gal4-HNF4Gal4-HNF4 αofα (mean (meanHNF4 ±α± S.E.M.;modulatorsS.E.M.; nn ≥≥ 4).4). 4, (6(bb, ))7 Effects,Effects 9, and of of10 44., , ( 6a6,), 7Dose–response7,, 99,, andand 1010 onon mRNA mRNAcurves quantitativeexpressionof 4,, real-time 6,, 7,, 9 ,of, andand fructose-1,6-bisphosphatase PCR 10 on (qRT-PCR).on Gal4-HNF4Gal4-HNF4 Inα accordance (mean(mean 1 (FBP1)±± S.E.M.;S.E.M.; with in humann its ≥ activity 4).4). ( (hepatocytesb)) EffectsEffects in the ofof Gal4-HNF4 (HepG2). 4,, 6,, 7,, 9,, HNF4andandα assay, 10α ononactivatorsthe mRNAmRNA most expressionofexpression 4, 6, 7, 9 ,of ofand fructose-1,6-bisphosphatase fructose-1,6-bisphosphatase 10 on Gal4-HNF4α (mean 11 (FBP1)±(FBP1) S.E.M.; inin n humanhuman ≥ 4). ( b hepatocyteshepatocytes) Effects of (HepG2). 4(HepG2)., 6, 7, 9, HNF4andHNF4 10αα on activators activators mRNA active HNF44expression, 6, andα activator 7 promotedof fructose-1,6-bisphosphatase6 significantly FBP1 expression, promoted while 1 (FBP1)(FBP1) the FBP1 inverse inin human expressionhuman HNF4 hepatocyteshepatocytesα inagonists HepG2 (HepG2).(HepG2). 9 and cells. 10 HNF4HNF4 decreased Theα less activatorsactivators potentFBP1 4expression4,, 66,, andand 77 promotedofpromoted fructose-1,6-bisphosphatase FBP1FBP1 expression,expression, whilewhile 1 (FBP1) thethe inverse inversein human HNF4HNF4 hepatocytesαα agonistsagonists (HepG2). 99 andand 1010 HNF4 decreaseddecreasedα activators FBP1FBP1 agonistsmRNA44, and6, and 7levels. revealed7 promoted Data aare tendency FBP1 the mean expression, to ± S.E.M. enhanced while mRNA FBP1the le inversevels mRNA determined HNF4 levels.α agonists by The qRT-PCR inverse 9 and and 10 HNF4 analyzeddecreasedα agonists by FBP1 the 9 mRNA4, 6, and levels. 7 promoted Data are FBP1 the mean expression, ± S.E.M. while mRNA the le inversevels determined HNF4α agonists by qRT-PCR 9 and and 10 decreasedanalyzed by FBP1 the mRNA-ΔΔCt levels. Data are the mean ± S.E.M. mRNA levels determined by qRT-PCR and analyzed by the 2mRNA-ΔΔCt method; levels. nData = 3. are (c) theControl mean experiments ± S.E.M. mRNA on Gal4-VP16 levels determined (boxplots by show qRT-PCR mean, and min.-max.; analyzed nby ≥ the4). and 10 robustly2mRNA-ΔΔCt method; levels. decreased nData = 3. FBP1are (c) theControl expression, mean experiments ± S.E.M. confirming mRNA on Gal4-VP16 levels their determined activity (boxplots observed by show qRT-PCR mean, in the and min.-max.; screening analyzed systemnby ≥ the4). mRNA2-ΔΔCt method; levels. Datan = 3. are (c )the Control mean experiments± S.E.M. mRNA on Gal4-VP16levels determined (boxplots by showqRT-PCR mean, and min.-max.; analyzed byn ≥ the 4). 2 -ΔΔCt method; n = 3. (c) Control experiments on Gal4-VP16 (boxplots show mean, min.-max.; n ≥ 4). as well. These(2d-ΔΔ) ChemicalCt method; results structures furthern = 3. (c validate) ofControl 4, 6, 7cellular ,experiments 9, and 10 HNF4. * p on <α 0.05, Gal4-VP16modulation ** p < 0.01, (boxplots by ***4 ,p6 <, show0.0017, 9, and(mean,t-test).10 min.-max.; and demonstrate n ≥ 4). (2(dd)) ChemicalChemical method; structuresstructures n = 3. (c) ofControlof 44,, 66,, 77 ,experiments, 99,, andand 1010.. ** ppon << 0.05,0.05,Gal4-VP16 **** pp << 0.01, 0.01,(boxplots ****** pp < Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 5 of 10 Int. J. Mol. Sci. 2020, 21, 7895 6 of 11 IC50 8 ± 2 µM 9 Inverse agonist Kd 0.3 µM efficiency (SILE). Fragment 6, due to its exceptional polarity,0.57 ± 0.04 was rem. superior act. regarding lipophilic ligand efficiency (LLE). Both inverse HNF4α agonists 9 and 10 revealed acceptable lipophilicity with predicted logP values of 3–4. LE, LLE, and SILE were comparableIC for50 both24 ± 5 compounds µM and favorably high for 10 Inverse agonist Kd 1.7 µM fragment-like screening hits. 0.65 ± 0.05 rem. act. Figure 3. In vitro characterization of HNF4α modulators 4, 6, 7, 9,, andand 1010.(. (a) Dose–responseDose–response curves of 4, 6, 7,, 9,, andand 1010 onon Gal4-HNF4Gal4-HNF4α (mean(mean ± S.E.M.; n ≥ 4).4). ( (bb))E Effectsffects of of 44,, 66,, 77,, 9,, and and 10 on mRNA ± ≥ expression of fructose-1,6-bisphosphatase 11 (FBP1) in human hepatocytes (HepG2). HNF4 α activators 4, 6, and 7 promoted FBP1 expression, while the inverse HNF4 α agonists 9 and 10 decreased FBP1 mRNA levels. Data are the mean ± S.E.M.S.E.M. mRNA mRNA le levelsvels determined determined by by qRT-PCR qRT-PCR and analyzed by the ± 2-ΔΔ∆∆CtCt method;method; n n= =3.3. (c ()c Control) Control experiments experiments on on Gal4-VP16 Gal4-VP16 (boxplots (boxplots show show mean, mean, min.-max.; min.-max.; nn ≥ 4).4). − ≥ (d) Chemical structures ofof 4,, 6,, 7,, 99,, andand 1010.*. * p < 0.05,0.05, ** ** pp << 0.01,0.01, *** *** pp << 0.0010.001 (t (-test).t-test). α TableTo capture 3. Calculated HNF4α lipophilicity modulation and by ligand 4, 6, e7ffi, 9ciency, and metrics10 in a ofmore HNF4 physiologicalmodulators. setting, LogP and we logS treated were retrieved from the ALOGPS 2.1 resource [20]. Ligand efficiency (LE), lipophilic ligand efficiency HNF4α-expressing [19] human hepatocytes (HepG2) with the HNF4α modulators and then (LLE), and size-independent ligand efficiency (SILE) were calculated as described in Reference [21]. determined the mRNA expression levels of the HNF4α-regulated gene fructose-1,6-bisphosphatase 1 (FBP1) [19] by quantitativeID HNF4 real-timeα Activity PCR LogP (qRT-PCR). LogS In accordance LE LLE with its SILE activity in the Gal4- HNF4α assay, the most4 activepEC HNF44.8α activator 4.13 6 significantly3.72 0.39 promoted 0.69 FBP12.1 expression in HepG2 50 − cells. The less potent5 agonistspEC 4 and< 4 7 revealed 4.84 a tendency5.50 < to0.32 enhanced<0 FBP1<1.7 mRNA levels. The 50 − inverse HNF4α agonists6 9 pECand 105.2 robustly 2.45decreased1.30 FBP1 0.42 expression, 7.7 confirming 2.2 their activity 50 − − 7 pEC 4.5 0.19 0.95 1.03 4.7 2.6 observed in the screening system50 as well. Th−ese results− further validate cellular HNF4α modulation 9 pIC 5.1 3.77 2.85 0.44 1.3 2.2 by 4, 6, 7, 9, and 10 and demonstrate50 that these compounds− can also be used as initial tools to study 10 pIC 4.6 3.01 3.65 0.40 1.6 2.0 HNF4α function in native settings.50 − For a preliminary quality assessment of this validated HNF4α ligand collection as a starting matterTo for orthogonally medicinal confirmchemistry, direct we interaction calculated between key physicochemical HNF4α and the features identified and hits, ligand-efficiency we determined metricsthe binding [20,21] of (Table4, 6, 7 ,3).9 ,As and feasibly10 to theexpe recombinantcted from their HNF4 chemicalα LBD structures, protein by the isothermal HNF4α agonists titration 6 andcalorimetry 7 comprised (ITC). favorably In line with low the lipophilicity nuclear receptor’s with low mode predicted of action logP [3 ],values. we observed Fragment dimerization 7 possessed of Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 6 of 10 the highest ligand efficiency (LE) owing to its small size but also a preferable size-independent ligand efficiency (SILE). Fragment 6, due to its exceptional polarity, was superior regarding lipophilic ligand efficiency (LLE). Both inverse HNF4α agonists 9 and 10 revealed acceptable lipophilicity with predicted logP values of 3–4. LE, LLE, and SILE were comparable for both compounds and favorably high for fragment-like screening hits. Table 3. Calculated lipophilicity and ligand efficiency metrics of HNF4α modulators. LogP and logS were retrieved from the ALOGPS 2.1 resource [20]. Ligand efficiency (LE), lipophilic ligand efficiency (LLE), and size-independent ligand efficiency (SILE) were calculated as described in Reference [21]. ID HNF4α Activity LogP LogS LE LLE SILE 4 pEC50 4.8 4.13 −3.72 0.39 0.69 2.1 5 pEC50 < 4 4.84 −5.50 <0.32 <0 <1.7 6 pEC50 5.2 −2.45 −1.30 0.42 7.7 2.2 7 pEC50 4.5 −0.19 −0.95 1.03 4.7 2.6 9 pIC50 5.1 3.77 −2.85 0.44 1.3 2.2 10 pIC50 4.6 3.01 −3.65 0.40 1.6 2.0 Int. J.To Mol. orthogonally Sci. 2020, 21, 7895 confirm direct interaction between HNF4α and the identified hits,7 ofwe 11 determined the binding of 4, 6, 7, 9, and 10 to the recombinant HNF4α LBD protein by isothermal titration calorimetry (ITC). In line with the nuclear receptor’s mode of action [3], we observed the HNF4α LBD protein and dimer dissociation upon dilution, which hindered inverse ITC experiments. dimerization of the HNF4α LBD protein and dimer dissociation upon dilution, which hindered Fragments 4, 6, 7, 9, and 10 were, therefore, titrated to the HNF4α LBD (Figure4). Binding of 5 was not inverse ITC experiments. Fragments 4, 6, 7, 9, and 10 were, therefore, titrated to the HNF4α LBD studied due to its weak potency observed in the cell-based assay. The ITC results indicated very weak (Figure 4). Binding of 5 was not studied due to its weak potency observed in the cell-based assay. The HNF4α binding of 4 and revealed no proper binding isotherm for 6. According to this observation, ITC results indicated very weak HNF4α binding of 4 and revealed no proper binding isotherm for 6. the effects of 6 on HNF4α activity in the Gal4-HNF4α assay and in HepG2 cells seem to be mediated According to this observation, the effects of 6 on HNF4α activity in the Gal4-HNF4α assay and in by indirect pathways and might, for example, involve HNF4α activation by phosphorylation. Since HepG2 cells seem to be mediated by indirect pathways and might, for example, involve HNF4α the control experiments on Gal4-VP16 revealed no unspecific effects of 6 and since 6 caused marked activation by phosphorylation. Since the control experiments on Gal4-VP16 revealed no unspecific upregulation of the HNF4α-regulated FBP1 in hepatocytes, its activity still appears HNF4α-mediated. effects of 6 and since 6 caused marked upregulation of the HNF4α-regulated FBP1 in hepatocytes, its Current evidence, however, does not support direct HNF4α agonism of 6, and further studies are activity still appears HNF4α-mediated. Current evidence, however, does not support direct HNF4α needed to elucidate the mechanism by which the compound promotes HNF4α activity. For fragments agonism of 6, and further studies are needed to elucidate the mechanism by which the compound 7, 9, and 10, ITC experiments clearly demonstrated their binding with micromolar binding affinities promotes HNF4α activity. For fragments 7, 9, and 10, ITC experiments clearly demonstrated their (7:Kd ~7 µM; 9:Kd ~0.3 µM; 10:Kd ~1.7 µM), orthogonally validating their direct effect on HNF4α binding with micromolar binding affinities (7: Kd ~7 µM; 9: Kd ~0.3 µM; 10: Kd ~1.7 µM), orthogonally modulation. In addition, fragment 4 likely activated the nuclear receptor through interaction with validating their direct effect on HNF4α modulation. In addition, fragment 4 likely activated the the LBD despite weak binding affinity and 5 was another weak HNF4α activator according to the nuclear receptor through interaction with the LBD despite weak binding affinity and 5 was another cellular experiments. weak HNF4α activator according to the cellular experiments. Figure 4.4. IsothermalIsothermal titration titration calorimetry calorimetry (ITC) (ITC) demonstrated demonstrated binding binding of 7, 9, andof 710, 9to, theand recombinant 10 to the recombinantHNF4α LBD HNF4 protein,α LBD confirming protein, their confirming HNF4α -mediatedtheir HNF4 activity.α-mediated Recombinant activity. Recombinant HNF4α LBD HNF4 proteinα LBD(10–20 proteinµM) was (10–20 titrated µM) withwas titrated ligands with (50–200 ligandsµM). (50–200 µM). 3. Discussion Discussion Our systematic screening yielded a a considerable hi hit-rate,t-rate, leading leading to to the the discovery discovery of of three three fully fully α validated HNF4 HNF4α ligandsligands (7 (,7 9,,9 10, 10) exhibiting) exhibiting low low micromolar micromolar potencies potencies and andbinding binding affinities affinities as well as aswell two as additional two additional weaker weaker HNF4 HNF4α modulatorsα modulators (4, 5). (4 These, 5). These hits were hits were highly highly chemically chemically diverse. diverse. We We observed no preference for certain chemotypes or functional groups and, surprisingly, only a single carboxylate was discovered as HNF4α ligand, although the nuclear receptor is known to bind fatty acids [4] and the screening library comprised 47 (10%) carboxylic acid-containing fragments. In line with the high constitutive transcriptional inducer activity of HNF4α, we observed bidirectional modulation of the nuclear receptor in the screening Gal4–HNF4α assay and in the native cellular setting in HepG2 cells. The active fragments exhibited different types of activity including agonism and inverse agonism, and direct interaction with the HNF4α LBD was confirmed for 7, 9, and 10. The most active compounds 7, 9, and 10, therefore, present as appealing starting points for medicinal chemistry as they offer favorable ligand efficiencies, low molecular weights, and low lipophilicity. Their scaffolds are simple and common, allowing rapid diversification and structure–activity relationship studies. The ligand-activated transcription factor HNF4α has been found to involve in metabolic balance and cancer development, but, to date, knowledge on its ligands is very limited. Potent modulators for the nuclear receptor are urgently needed to validate its promising therapeutic potential in metabolic diseases and oncology. We disclose three orthogonally validated HNF4α ligands (7, 9, and 10) as high-quality chemical starting matter for medicinal chemistry. Additionally, our results provide further evidence that activity of the poorly studied nuclear receptor HNF4α can be controlled by small-molecule ligands in a bidirectional fashion thus offering great potential as a molecular drug target. Modulatory effects of 4, 6, 7, 9, and 10 on expression levels of the HNF4α-regulated gene FBP1 Int. J. Mol. Sci. 2020, 21, 7895 8 of 11 in a native cellular setting indicate that this set of HNF4α modulators can also serve as an initial chemogenomic tool for early functional and phenotypic studies. Still, further efforts are needed to develop highly optimized HNF4α ligands as probes to question the therapeutic potential of the nuclear receptor in depth. 4. Materials and Methods 4.1. Hybrid Gal4-HNF4α Reporter Gene Assay Plasmids: The Gal4-fusion receptor plasmid pFA-CMV-hHNF4α-LBD coding for the hinge region and LBD of the canonical isoform of HNF4α (uniprot entry: P41235-1, residues 142-377) was constructed by integrating cDNA fragments obtained from PCR amplification using natural cDNA (purchased as cDNA clone IRCBp5005M2212Q from Source BioScience, Nottingham, UK) as template between the BamHI cleavage site of the pFA-CMV vector (Stratagene, La Jolla, CA, USA) and an afore inserted KpnI cleavage site as described previously [22–24]. The frame and sequence of the fusion plasmid were verified by sequencing. pFR-Luc (Stratagene) was used as reporter plasmid and pRL-SV40 (Promega, Madison, WI, USA) served for normalization of transfection efficiency and cell growth. The Gal4-VP16 [25] expressed from plasmid pECE-SV40-Gal4-VP16 [26] (Addgene, entry 71728, Watertown, MA, USA) was used as a ligand-independent transcriptional inducer for control experiments. Assay Procedure: HEK293T cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) high glucose, supplemented with 10% Fetal Bovine Serum (FCS), sodium pyruvate (1 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 ◦C and 5% CO2. The day before transfection, HEK293T cells were seeded in 96-well plates (3.0 104 cells/well). Before transfection, · the medium was changed to Opti-MEM without supplements. Transient transfection was carried out using Lipofectamine LTX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol with pFR-Luc (Stratagene), pRL-SV40 (Promega), and the pFA-CMV-hHNF4α-LBD clone or pECE-SV40-Gal4-VP16 for control experiments. Five hours after transfection, the medium was changed to Opti-MEM supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), now additionally containing 0.1% DMSO (0.2% in the primary screen) and the respective test compounds or 0.1% DMSO (0.2% in the primary screen) alone as the untreated control. In the primary screening, the core set of the Prestwick Drug Fragment Library (Prestwick Chemical, Illkirch, France) was tested at 50 µM concentration in two biologically independent repeats. In the follow-up and dose-response profiling, each concentration was tested in duplicates in at least three biologically independent repeats. Following overnight (12 14 h) incubation with the test compounds, cells were assayed for luciferase activity using − Dual-Glo™ luciferase assay system (Promega) according to the manufacturer’s protocol. Luminescence was measured with a Tecan Spark 10M luminometer (Tecan Group Ltd., Männedorf, Switzerland). Normalization of transfection efficiency and cell growth was done by division of firefly luciferase data by renilla luciferase data and multiplying the value by 1000, resulting in relative light units (RLU). Fold activation was obtained by dividing the mean RLU of test compounds at a respective concentration by the mean RLU of untreated control. 4.2. Recombinant HNF4α LBD Expression and Purification The LDB domain of HNF4α (aa. 148–377) subcloned into pNIC28-Bsa4 was expressed in E. coli Rosetta. The recombinant protein harboring an N-terminal His6 tag was initially purified by Ni2+-affinity chromatography. Size exclusion chromatography was performed using Superdex S75 column (GE Healthcare, Chicago, IL, USA.), and the pure protein was stored in a buffer containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 200 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), and 5% w/v glycerol. Int. J. Mol. Sci. 2020, 21, 7895 9 of 11 4.3. Quantification of Human FBP1 mRNA Expression in HepG2 Cells by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) HepG2 cells were grown in 12-well plates in DMEM high glucose, supplemented with 10% FCS, sodium pyruvate (1 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 ◦C and 5% CO2 to approximately 60% confluence. Before incubation with test compounds, cells were kept in MEM supplemented with 1% charcoal-stripped FCS, penicillin (100 U/mL), and streptomycin (100 µg/mL) for 24 h [27]. For gene expression analysis, cells were incubated with 4 (50 µM), 6 (10 µM), 7 (50 µM), 9 (50 µM), 10 (50 µM), or 0.1% DMSO as untreated control in the same medium for 8 h. The cells were then harvested and directly used for RNA extraction by the E.Z.N.A. Total RNA Kit I (R6834-02, Omega Bio-Tek, Inc., Norcross, GA, USA). Three micrograms of total RNA extracted from HepG2 cells were reverse-transcribed into cDNA using the High-Capacity RNA-to-cDNA Kit (4387406, Thermo Fischer Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol. HNF4α-regulated FBP1 expression was evaluated by qRT-PCR analysis with a StepOnePlus System (Life Technologies, Carlsbad, CA, USA) using Power SYBR Green (Life Technologies; 12.5 µL/well). Each sample was set up in duplicates and repeated in three independent experiments. Data were analyzed by the comparative ∆∆Ct 2− method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference gene. The following PCR primers were used: hGAPDH [28]: 50-ATA TGA TTC CAC CCA TGG CA (fw), 50-GAT GAT GAC CCT TTT GGC TC (rev) and hFBP1 [19]: 50-AGC CTT CTG AGA AGG ATG CTC (fw), 50-GTC CAG CAT GAA GCA GTT GAC (rev). 4.4. Isothermal Titration Calorimetry Isothermal titration calorimetry was performed on an Affinity ITC (TA Instruments, New Castle, DE, USA). The instrument was equilibrated to 25 ◦C and the stirring rate was set to 75 rpm. HNF4α buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 0.5 mM TCEP, 5% w/v glycerol) with up to 1% DMSO (final concentration) was used for ITC. The recombinant HNF4α LBD protein (10–20 µM, 172 µL) was titrated with fragments (4, 6, 7, 9, 10; 50–200 µM; dissolved in the same buffer). A total of 20-30 injections with a volume of 2.5–4 µL and with an interval of 200–300 s were performed. As a control experiment, HNF4α protein was titrated with buffer, and the buffer was titrated with test compound to detect dissolution artifacts with otherwise identical experimental parameters. Data analysis was performed with NanoAnalyzeTM Software (TA Instruments, New Castle, DE, USA.) using an independent binding model. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/21/ 7895/s1. Table S1. All molecular structures of library compounds with associated primary screening data. Author Contributions: I.M. and S.W. performed the experiments; X.N., J.H., and A.C. contributed materials; D.M. conceptualized and supervised the project and wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 875510. The JU receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA and Ontario Institute for Cancer Research, Royal Institution for the Advancement of Learning McGill University, Kungliga Tekniska Hoegskolan, Diamond Light Source Limited. Acknowledgments: D.M. is grateful for financial support from the Aventis Foundation. The SGC is a registered charity (no: 1097737) that receives funds from AbbVie, Bayer AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], EU/EFPIA/OICR/McGill/KTH/Diamond, Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Merck & Co (aka MSD outside Canada and US), Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome [106169/ZZ14/Z]. Conflicts of Interest: The authors declare no conflict of interest. Int. J. Mol. 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