Molecular Dynamics of Cobalt Protoporphyrin Antagonism of the Cancer Suppressor REV-Erbβ

molecules

Article Molecular Dynamics of Cobalt Protoporphyrin Antagonism of the Cancer Suppressor REV-ERBβ

Tauﬁk Muhammad Fakih 1,2 , Fransiska Kurniawan 1, Muhammad Yusuf 3 , Mudasir Mudasir 4 and Daryono Hadi Tjahjono 1,*

1 School of Pharmacy, Bandung Institute of Technology, Jalan Ganesha 10, Bandung 40135, Indonesia; tauﬁ[email protected] (T.M.F.); [email protected] (F.K.) 2 Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Universitas Islam Bandung, Jalan Rangga Gading 8, Bandung 40116, Indonesia 3 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jalan Raya Bandung Sumedang Km 21, Sumedang 45363, Indonesia; [email protected] 4 Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada, Sekip Utara BLS 21, Yogyakarta 55281, Indonesia; [email protected] * Correspondence: [email protected]; Tel.: +62-81-222-400120

Abstract: Nuclear receptor REV-ERBβ is an overexpressed oncoprotein that has been used as a target for cancer treatment. The metal-complex nature of its ligand, iron protoporphyrin IX (Heme), enables the REV-ERBβ to be used for multiple therapeutic modalities as a photonuclease, a photosensitizer, or a fluorescence imaging agent. The replacement of iron with cobalt as the metal center of protoporphyrin IX changes the ligand from an agonist to an antagonist of REV-ERBβ. The mechanism behind that phenomenon is still unclear, despite the availability of crystal structures of REV-ERBβ in complex with Heme and cobalt protoporphyrin IX (CoPP). This study used molecular dynamic Citation: Fakih, T.M.; Kurniawan, F.; simulations to compare the effects of REV-ERBβ binding to Heme and CoPP, respectively. The initial Yusuf, M.; Mudasir, M.; Tjahjono, poses of Heme and CoPP in complex with agonist and antagonist forms of REV-ERBβ were predicted D.H. Molecular Dynamics of Cobalt using molecular docking. The binding energies of each ligand were calculated using the MM/PBSA Protoporphyrin Antagonism of the Cancer Suppressor REV-ERBβ. method. The computed binding affinity of Heme to REV-ERBβ was stronger than that of CoPP, Molecules 2021, 26, 3251. https:// in agreement with experimental results. CoPP altered the conformation of the ligand-binding site doi.org/10.3390/molecules26113251 of REV-ERBβ, disrupting the binding site for nuclear receptor corepressor, which is required for REV-ERBβ to regulate the transcription of downstream target genes. Those results suggest that a Academic Editors: subtle change in the metal center of porphyrin can change the behavior of porphyrin in cancer cell Salomé Rodríguez-Morgade, signaling. Therefore, modification of porphyrin-based agents for cancer therapy should be conducted Soji Shimizu and Hai-yang Liu carefully to avoid triggering unfavorable effects.

Received: 6 April 2021 Keywords: breast cancer; REV-ERBβ; porphyrin; nuclear receptor corepressor (NCoR); molecular Accepted: 26 May 2021 dynamics (MD) simulation; porphyrin Published: 28 May 2021

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional afﬁl- iations. Porphyrin-based agents can be an alternative choice for cancer therapy because they have selective cytotoxicity against tumor cells [1]. Porphyrins are a family of organic ring molecules including Heme (Iron protoporphyrin IX), the pigment in red blood cells, an essential molecule for living aerobic organisms, plays a major role in gas exchange, mito- chondrial energy production, antioxidant defense, signal transduction, and they represent Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. one of the oldest and most widely studied chemical structures in nature and in biomedi- This article is an open access article cal applications [2–4]. Moreover, Heme is also contained in the superfamily of enzymes distributed under the terms and Cytochromes P450 (CYPs) which plays a role in supporting oxidative, peroxidative, and re- conditions of the Creative Commons ductive metabolism of endogenous and xenobiotic substrates [5,6]. P450s are endoplasmic Attribution (CC BY) license (https:// reticulum (ER)-anchored hemoproteins which responsible for the metabolism of numer- creativecommons.org/licenses/by/ ous endogenous and foreign compounds. As the prosthetic moiety of all P450s, Heme is 4.0/). responsible for the remarkable and often exquisite, catalytic prowess of these enzymes [7].

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The optimal dose of a porphyrin-based agent is lethal to tumor cells while minimizing damage to adjacent normal tissue [8]. Therefore, multi-modal porphyrin-based agents have great potential for use in cancer imaging and therapy [9]. Porphyrins have been particularly useful in photodynamic therapy and fluorescence imaging of cancer because of their tumor avidity and favorable photophysical properties, such as long wavelength absorption and emission, easy derivatization, high singlet oxygen quantum yield, and low in vivo toxicity [10,11]. In addition, porphyrins are excellent metal chelators that form highly stable metallocomplexes, making them efficient delivery vehicles for radioisotopes [12]. Several studies have investigated the characteristics of various porphyrin-based probes and their clinical applications in cancer imaging and therapy [10]. Porphyrin-based agents can increase the signal-to-background ratio in tumor imaging, allowing better detection of tumor tissues and better preservation of healthy tissues during therapy [13]. The binding of the porphyrin Heme to the nuclear receptor REV-ERBβ leads to decreased proliferation in various cancer cells [14]. REV-ERBβ, along with REV-ERBα, is a member of the REV-ERB family of nuclear receptors. Although REV-ERBβ is structurally and functionally similar to REV-ERBα, it has a unique role in the regulation of circadian rhythms, which in turn affect a variety of diseases including cancer [15]. Moreover, REV- ERBβ is overexpressed in breast cancer cells, accounting for more than 95% of the total REV-ERB mRNA [15]. In addition, REV-ERBβ is dominantly expressed in several cancer cell lines, whereas REV-ERBα is the dominant form in corresponding normal tissues [16]. The binding of Heme to REV-ERBβ (Figure1a) induces recruitment of nuclear receptor corepressors such as NCoR, which in turn regulate diverse pathways ranging from Heme biosynthesis to circadian rhythms [17]. NCoR forms stable complexes with transcription factors that are deregulated in various cancers [18]. Synthetic agonists have been developed that mimic the ability of Heme to induce NCoR recruitment and corepressor-peptide binding to REV-ERBβ [19]. However, previous research could not predict the ability of porphyrin-based REV-ERBβ antagonists to repress NCoR recruitment [20]. Besides Heme, there are other ligands that bind REV-ERBβ, including cobalt protoporphyrin IX (CoPP) and zinc protoporphyrin IX (ZnPP). The only difference between Heme and CoPP and ZnPP is in the coordinated metal ions. CoPP and ZnPP are functional antagonists of REV-ERBβ, whereas Heme is the natural agonist of REV-ERBβ. CoPP and ZnPP block the transcriptional repressive functions of REV-ERBβ by preventing its binding to protoporphyrin IX [21]. The iron metal center of Heme has a good affinity for Histidine (His) and Cysteine (Cys) residues on REV-ERBβ. Cobalt has a lower affinity than iron, but it can still interact with and regulate the properties of REV-ERBβ [22]. Therefore, Heme and CoPP can be used as reference structures for the development of selective porphyrin-based agents for cancer therapy and imaging. Figure1a shows that the addition of small molecules to the REV-ERB β induces enlarge- ment of the ligand-binding domain (purple box) which may affect the recruitment of NCoR (red color). However, the REV-ERBβ-Heme and REV-ERBβ-CoPP have similar crystal structure (Figure1b) but show different biological activity. To develop new porphyrin- based agents for early cancer detection, a preliminary study is needed to compare the molecular dynamics of Heme and CoPP binding to REV-ERBβ. In particular, it is important to determine how iron or cobalt in the center of protoporphyrin IX affects the porphyrin ring conformation, which might determine whether protoporphyrin IX acts as an agonist or an antagonist of REV-ERBβ. Molecules 2021, 26, x FOR PEER REVIEW 3 of 17

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Figure 1. (a) Overlay visualization of apo-structure REV-ERBβ (yellow) with bound NCoR (red) andFigureFigure REV-ERB 1. 1.(a()a Overlay)β Overlay in complex visualization visualization form with of of apo-structureHeme apo-structure (green). REV-ERB ( REV-ERBb) Visualizationββ (yellow)(yellow) of withREV-ERB with bound boundβ inNCoR NCoR complex (red) (red) and formandREV-ERB REV-ERBwith Hemeβ inβ in complex (green) complex and form form with with with CoPP Heme Heme (purple). (green). (green). (b ()b Visualization) Visualization of of REV-ERB REV-ERBββin in complex complex form formwith with Heme Heme (green) (green) and and with with CoPP CoPP (purple). (purple). 2. Results 2.2. Results Results 2.1.2.1. Initial Initial Pose Pose of ofthe the Ligand–Protein Ligand–Protein Complex Complex 2.1. Initial Pose of the Ligand–Protein Complex HemeHeme and and CoPP CoPP were were docked docked into intotwo twodifferent different of REV-ERB of REV-ERBβ crystalβ crystal structures: structures: An- Heme and CoPP were docked into two different of REV-ERBβ crystal structures: An- tagonist-REV-ERBAntagonist-REV-ERBβ andβ andAgonist-REV-ERB Agonist-REV-ERBβ. Antagonist-REV-ERBβ. Antagonist-REV-ERBβ (PDBβ (PDB ID: ID: 4N73) 4N73) is isa a tagonist-REV-ERBβ and Agonist-REV-ERBβ. Antagonist-REV-ERBβ (PDB ID: 4N73) is a crystallinecrystalline structure structure of of REV-ERB REV-ERBββ thatthat forms aa complexcomplex with with CoPP, CoPP, whereas whereas Agonist-REV- Agonist- crystalline structure of REV-ERBβ that forms a complex with CoPP, whereas Agonist- REV-ERBERBβ (PDBβ (PDB ID: ID: 3CQV) 3CQV) forms forms a complex a complex with with Heme. Heme. Molecular Molecular docking docking was performedwas per- formedREV-ERBto investigate to βinvestigate (PDB the ID: binding 3CQV)the binding modes forms modes anda complex afﬁnities and affinities with of protoporphyrin Heme. of protoporphyrin Molecular IX docking within IX within the was binding per-the bindingformedpocket pocketto of investigate REV-ERB of REV-ERBβ .the Both bindingβ. crystal Both crystalmodes structures structuresand ofaffinities REV-ERB of REV-ERB ofβ protoporphyrinare shownβ are shown in Figure IX in within Figure2. the 2. binding pocket of REV-ERBβ. Both crystal structures of REV-ERBβ are shown in Figure 2.

(a) (b) (a) (b) FigureFigure 2. 2.(a)( aThe) The crystal crystal structure structure of ofthe the REV-ERB REV-ERBβ thatβ that forms forms a complex a complex with with iron iron protoporphy- protoporphyrin rinFigureIX IX (Agonist-REV-ERB (Agonist-REV-ERB 2. (a) The crystalβ structureβ).). ((bb)) TheThe of crystalcrystal the REV-ERB structurestructureβ that of REV-ERB forms a complexβ that forms forms with a airon complex complex protoporphy- with with co- cobalt baltrinprotoporphyrin IXprotoporphyrin (Agonist-REV-ERB IX IX (Antagonist-REV-ERB (Antagonist-REV-ERBβ). (b) The crystalβ structure).β). of REV-ERBβ that forms a complex with cobalt protoporphyrin IX (Antagonist-REV-ERBβ). TheThe overlay overlay poses poses of ofthe the ligand ligand structure structure during during re-docking re-docking process process and and docking docking simulationsimulationThe overlay are are provided provided poses ofin intheFigure Figure ligand 3.3 .Heme Hemestructure had had duringthe the greatest greatest re-docking negative negative process binding binding and energy energydocking in in simulationboth crystal are structures provided ofin REV-ERBFigure 3. βHeme, with had the bindingthe greatest energy negative values binding of −65.91 energy kJ/mol in (Antagonist-REV-ERBβ) and −71.10 kJ/mol (Agonist-REV-ERBβ; Table1). Those results

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both crystal structures of REV-ERBβ, with the binding energy values of −65.91 kJ/mol (An- tagonist-REV-ERBβ) and −71.10 kJ/mol (Agonist-REV-ERBβ; Table 1). Those results are in agreementare with in agreement the results with of previous the results isothe of previousrmal titration isothermal calorimetry titration (ITC) calorimetry studies that (ITC) stud- found thaties the that binding found of that CoPP the (K bindingD = 2.56 µM) of CoPP was weaker (KD = 2.56 thanµ thatM) wasof Heme weaker (KD than= 353that nM) of Heme [22]. The (resultsKD = 353 reflect nM)[ the22]. atom Theic results radius reﬂect of each the metal atomic bound radius to of the each center metal of bound protopor- to the center phyrin. Ironof protoporphyrin.has an atomic radius Iron of has0.126 an nm, atomic whereas radius cobalt of has 0.126 atomic nM, whereasradius of 0.200 cobalt nm, has atomic indicatingradius that the of 0.200 radius nM, of indicatingthe atom at that the the metal radius center of the plays atom a atrole the in metal determining center plays the a role in affinity ofdetermining the porphyrin the complex afﬁnity offor the REV-ERB porphyrinβ. complex for REV-ERBβ.

Crystal structure = grey Crystal structure = grey Re-docked structure = green Re-docked structure = red

Agonist complex = green Agonist complex = grey Antagonist complex = grey Antagonist complex = red (a) (b)

Figure 3. OverlayFigure 3. structuresOverlay structuresof Heme ( ofa) and Heme CoPP (a) and(b) in CoPP re-docking (b) in re-docking and docking and simulation. docking simulation.

Table 1. TheTable binding 1. The energy binding and energy RMSD and value RMSD of CoPP value and of CoPP Heme and in molecular Heme in molecular docking. docking.

ProtoporphyrinProtoporphyrin Ligand Ligand Re-Docking Re-Docking Docking Docking RMSD = 0.3078 Å RMSD = 0.3703 Å Heme Heme RMSD = 0.3078 Å RMSD = 0.3703 Å ∆G (agonist)∆G = (agonist)−71.10 kJ/mol = −71.10 kJ/mol∆G (antagonist)∆G (antagonist) = −65.91 kJ/mol = −65.91 kJ/mol RMSD = 0.3540 Å RMSD = 0.7110 Å CoPP CoPP RMSD = 0.3540 Å RMSD = 0.7110 Å ∆G (antagonist)∆G (antagonist) = −56.87 kJ/mol = −56.87 kJ/mol∆G (agonist)∆G = (agonist) −56.95 kJ/mol = −56.95 kJ/mol

To investigate the interactions between both forms of protoporphyrin IX and the two To investigate the interactions between both forms of protoporphyrin IX and the two forms of REV-ERBβ, the binding modes of CoPP and Heme within the REV-ERBβ binding forms of REV-ERBβ, the binding modes of CoPP and Heme within the REV-ERBβ binding pocket were observed further. In general, CoPP and Heme had the same interactions with pocket were observed further. In general, CoPP and Heme had the same interactions with the amino acid residues of REV-ERBβ (i.e., Val383, Gly480, and Leu483); however, Heme the amino acid residues of REV-ERBβ (i.e., Val383, Gly480, and Leu483); however, Heme had additional interactions with Cys384 and His568 [21]. Visualization of the docking had additional interactions with Cys384 and His568 [21]. Visualization of the docking pose pose with REV-ERBβ revealed an interaction differences in the bonding of the central with REV-ERBβ revealed an interaction differences in the bonding of the central metal metal atomatom (Figure (Figure 4).4). Those Those differences differences corres correspondpond withwith thethe crystalcrystal structures structures in in that that they were they weredifferent different in in terms terms of of the the interactions interactions with with Cys384 Cys384 and and His568, His568, which which were were coordinated co- by ordinatedthe by metal the metal center center of the of protoporphyrin the protoporphyrin IX ring. IX Thatring. phenomenonThat phenomenon is likely is likely to be the main to be thesource main source of the stronger of the stronger binding afﬁnitybinding of affinity Heme comparedof Heme compared with that ofwith CoPP. that The of effects of CoPP. Theinteractions effects of involvinginteractions the involving central metal the central atom might metal determine atom might the determine agonist and the antagonist agonist andfunctions antagonist of REV-ERB functionsβ. of REV-ERBβ.

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Figure 4. Visualization of interactions occurring in the binding pocket of REV-ERBβ when docked against Heme (green) and CoPP (red). β 2.2.Figure The 4. Binding Visualization Free Energy of interactions of Agonist occurring occurring and Antagonist in in the the binding binding Ligands pocket pocket against of of REV-ERB REV-ERB REV-ERBβ whenwhenβ Ligand- docked docked against Heme (green) and CoPP (red). Bindingagainst Heme Domain (green) (LBD) and CoPP (red). 2.2. TheThe Binding molecular Free mechanics Energy of Agonist Poisson-Boltzmann and Antagonist surface Ligands area against (MM/PBSA) REV-ERB methodβ Ligand- was usedLigand-BindingBinding to predictDomain Domain the(LBD) binding (LBD) free energy of the four protein-ligand complexes more accurately. The binding free energies of the complexes were more accurate than those observed The molecular mechanicsmechanics Poisson-BoltzmannPoisson-Boltzmann surfacesurface areaarea (MM/PBSA)(MM/PBSA) method was in the docking study (Table 2). Furthermore, the energies that mostly contributed were used toto predictpredict the the binding binding free free energy energy of theof th foure four protein-ligand protein-ligand complexes complexes more more accurately. accu- The binding free energies of the complexes were more accurate than those observed in electrostaticrately. The binding and van free der energies Waals interaction. of the complexes Overall, were all morefour complexesaccurate than had those good observed stability the docking study (Table2). Furthermore, the energies that mostly contributed were inin thethe moleculardocking study dyna mic(Table simulations. 2). Furthermore, the energies that mostly contributed were electrostatic and van der Waals interaction. Overall, all four complexes had good stability electrostatic and van der Waals interaction. Overall, all four complexes had good stability Tablein the 2. molecular The free binding dynamic energies simulations. and their corresponding components for protoporphyrin IX in the molecular dynamic simulations. binding to REV-ERBβ. Table 2. The free binding energies and their corresponding components for protoporphyrin IX Table 2. The free binding energies and their correspon∆Evdw ding∆ componentsEele ∆G forPB protoporphyrin∆GNP ∆ GIXBind Complexβ REV-ERBβ binding to REV-ERB β.. (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) Heme-Agonist-REV-ERBβ ∆E −309.61∆E −208.23∆ G 284.17 ∆G−28.72 ∆−G262.38 Complex REV-ERBβ vdw ∆Evdw ele ∆Eele PB∆GPB ∆NPGNP ∆GBindBind CoPP–Agonist-REV-ERBComplex REV-ERBβ β (kJ/mol) −283.36(kJ/mol) −203.37(kJ/mol) 255.76 (kJ/mol)−27.33 (kJ/mol)−258.30 (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) (kJ/mol) Heme–Antagonist-REV-ERBβ β− −300.41− −170.95 261.51 − −27.24 −−237.10 Heme-Agonist-REV-ERBHeme-Agonist-REV-ERBβ 309.61 −309.61208.23 −208.23 284.17 284.17 28.72−28.72 −262.38262.38 CoPP–Agonist-REV-ERBCoPP–Antagonist-REV-ERBβ β −283.36 −307.76−203.37 −162.81 255.76 257.03 −27.33−27.26 −−258.30240.79 Heme–Antagonist-REV-ERBCoPP–Agonist-REV-ERBβ β −300.41 −283.36−170.95 −203.37 261.51 255.76 −27.24−27.33 −−237.10258.30 Note: ∆Evdw = van der Waals contribution, ∆Eele = electrostatic contribution, ∆GPB = polar contribu- CoPP–Antagonist-REV-ERBHeme–Antagonist-REV-ERBβ β− 307.76 −300.41−162.81 −170.95 257.03 261.51 −27.26−27.24 −−240.79237.10 tion of desolvation, ∆GNP = non-polar contribution of desolvation. Note: ∆ECoPP–Antagonist-REV-ERBvdw = van der Waals contribution,β ∆Eele =−307.76 electrostatic −162.81 contribution, 257.03∆G PB =− polar27.26 contribution −240.79 of desolvation, ∆GNP = non-polar contribution of desolvation. Note: ∆Evdw = van der Waals contribution, ∆Eele = electrostatic contribution, ∆GPB = polar contribu- The graph of the total energy of the system shows the beginning of changes in the tion of desolvation, ∆GNP = non-polar contribution of desolvation. proteinThe conformation graph of the (Figure total energy 5a). The of thetotal system energy shows increased the beginning from 25 ns of to changes 30 ns during in the protein conformation (Figure5a). The total energy increased from 25 ns to 30 ns during formationThe graph of the of Antagonist-REV-ERB the total energy of theβ complex, system showswhereas the it beginning increased offrom changes 0 ns to in 5 the ns duringformation formation of the Antagonist-REV-ERBof the Agonist-REV-ERBβ complex,β complex whereas (Figure it 5b). increased These results from 0 show ns to 5that ns protein conformation (Figure 5a). The totalβ energy increased from 25 ns to 30 ns during theduring antagonist formation ligand of the greatly Agonist-REV-ERB influenced the cocomplexnformational (Figure changes5b). These of the results target show protein. that theformation antagonist of the ligand Antagonist-REV-ERB greatly inﬂuencedβ thecomplex, conformational whereas it changes increased of thefrom target 0 ns protein. to 5 ns during formation of the Agonist-REV-ERBβ complex (Figure 5b). These results show that the antagonist ligand greatly influenced the conformational changes of the target protein.

(a) (b)

Figure 5. Graph of the total system energy from MM/PBSA calculations when (a) Antagonist-REV-ERBβ and (b) Agonist- REV-ERBβ form complexes with Heme(a) (green) and CoPP (red) during molecular dynamic(b) simulations.

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Figure 5. Graph of the total system energy from MM/PBSA calculations when (a) Antagonist-REV-ERBβ and (b) Agonist- Figure 5. Graph of the total system energy from MM/PBSA calculations when (a) Antagonist-REV-ERBβ and (b) Agonist- Molecules 2021, 26, 3251 REV-ERBβ form complexes with Heme (green) and CoPP (red) during molecular dynamic simulations. 6 of 17 REV-ERBβ form complexes with Heme (green) and CoPP (red) during molecular dynamic simulations. 2.3. Structural Analysis from Molecular Dynamics Simulation 2.3. Structural Analysis from Molecular Dynamics Simulation Molecular dynamics (MD) simulation was performed with the four complexes to 2.3. StructuralMolecular Analysis dynamics from (MD) Molecular simulation Dynamics was Simulation performed with the four complexes to gain more structural, dynamical, and energetic information about the stability of Heme gain more structural, dynamical, and energetic information about the stability of Heme Molecularand dynamics CoPP in (MD) the ligand-binding simulation was domain performed (LBD) with of theREV-ERB four complexesβ. Snapshots to gain of the MD trajec- and CoPP in the ligand-binding domain (LBD) of REV-ERBβ. Snapshots of the MD trajec- more structural,tories dynamical, of the four and complexes energetic were information visualized about to theobserve stability the time-dependent of Heme and position of CoPPtories inof thethe ligand-bindingfour complexes domain were visualized (LBD) of REV-ERB to observeβ. Snapshotsthe time-dependent of the MD trajectoriesposition of the ligand and also to analyze the effect of the interaction on the structure of REV-ERBβ. ofthe the ligand four complexesand also to were analyze visualized the effect to observeof the interaction the time-dependent on the structure position of of REV-ERB the ligandβ. Visualization of the complexes during the MD simulations is shown in Figure 6. The poses andVisualization also to analyze of the thecomplexes effect of during the interaction the MD onsimulations the structure is shown of REV-ERB in Figureβ. Visualization 6. The poses of Heme and CoPP in the binding site were generally not different from one another. The of Heme the complexes and CoPP during in the thebinding MD simulationssite were generally is shown not in different Figure6 from. The one poses another. of Heme The central metal atom of protoporphyrin IX was stable and remained in the center of the ring andcentral CoPP metal in theatom binding of protoporphyrin site were generally IX was st notable different and remained from one in another.the center The of the central ring until the end of the simulation. Moreover, the protoporphyrin IX remained planar metaluntil the atom end of protoporphyrinof the simulation. IX was Moreover, stable and the remained protoporphyrin in the center IX of remained the ring until planar the throughout the simulation, indicating its function to deliver the metal atom to the protein endthroughout of the simulation. the simulation, Moreover, indicating the protoporphyrin its function to deliver IX remained the metal planar atom throughout to the protein the target. That is in agreement with the absorption spectral bands of Heme and CoPP, be- simulation,target. Thatindicating is in agreement its function with tothe deliver absorption the metal spectral atom bands to the of protein Heme target. and CoPP, That is be- in cause a nonplanar conformation resulting from repulsive interactions among the periph- agreementcause a nonplanar with the conformation absorption spectral resulting bands from ofrepulsive Heme and interactions CoPP, because among a the nonplanar peripheral substituents would induce dramatic redshifts of the electronic absorption spectral conformationeral substituents resulting would frominduce repulsive dramatic interactions redshifts of among the electronic the peripheral absorption substituents spectral bands [23]. wouldbands [23]. induce dramatic redshifts of the electronic absorption spectral bands [23].

Figure 6. Snapshot of the molecular dynamic simulation trajectories of CoPP and Heme against Figure 6. SnapshotSnapshot of of the the molecular molecular dynamic dynamic simulati simulationon trajectories trajectories of ofCoPP CoPP and and Heme Heme against against Agonist-REV-ERBβ. Agonist-REV-ERBββ.. 2.4. Dynamic Behavior of REV-ERBβ in the Antagonist Form 2.4. Dynamic Behavior of REV-ERBβ inin the the Antagonist Antagonist Form The root-mean-square deviation (RMSD) represents the stability of the molecular The root-mean-square deviation (RMSD) representsrepresents the stability of thethe molecularmolecular structure throughout the simulation. MD simulations of the Heme and CoPP ligands in structure throughout the the simulation. simulation. MD MD simu simulationslations of of the the Heme Heme and and CoPP CoPP ligands ligands in complex with Antagonist-REV-ERBβ were conducted to observe the behavior of both lig- incomplex complex with with Antagonist-REV-ERB Antagonist-REV-ERBβ wereβ were conducted conducted to observe to observe the thebehavior behavior of both of both ligands. Figure 7a shows the RMSD of Antagonist-REV-ERBβ in the presence of Heme and ligands.ands. Figure Figure 7a7 ashows shows the the RMSD RMSD of of Antagonist-REV-ERB Antagonist-REV-ERBββ inin the the presence presence of of Heme andand CoPP. The average RMSD values for Heme and CoPP were 3.49 Å and 2.95 Å, respec- CoPP. TheThe averageaverage RMSD RMSD values values for for Heme Heme and and CoPP CoPP were were 3.49 3.49 Å and Å and 2.95 2.95 Å, respectively. Å, respectively. The small difference between RMSD values of Heme and CoPP in the antagonist Thetively. small The difference small difference between between RMSD valuesRMSD ofvalues Heme of and Heme CoPP and in CoPP the antagonist in the antagonist form of form of the receptor was unable to explain the specific behavior of each ligand. theform receptor of the receptor was unable was to unable explain to theexplai speciﬁcn the behaviorspecific behavior of each ligand.of each ligand.

Figure 7. Plot of the RMSD value of backbone in the active site of (a) Antagonist-REV-ERBβ and (b) Agonist-REV-ERBβ with Heme (green) and CoPP (red) during the molecular dynamic simulation.

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Molecules 2021, 26, 3251 7 of 17 Figure 7. Plot of the RMSD value of backbone in the active site of (a) Antagonist-REV-ERBβ andand ((b) Agonist-REV-ERBβ with Heme (green) and CoPP (red) during the molecular dynamic simulation.

2.5.2.5. Dynamic Dynamic Behavior Behavior of of REV-ERB REV-ERBββ ininin thethe the AgonistAgonist Agonist FormForm Form InIn the the agonist agonist form form of of the the receptor, receptor, the the stability stability of of the the Heme-complex Heme-complex was was better better than than thatthat of of the the CoPP-complex, CoPP-complex, with with average average RMSD RMSD values values of of 4.89 4.89 Å Å and and 6.09 6.09 Å, Å, respectively. respectively. TheThe low low RMSD RMSD value value of of the the Heme-complex Heme-complex repr representsesents the the natural natural dynamics dynamics of ofthe the crystal crys- structure,tal structure, indicating indicating the action the action of the of Heme the Heme agonist agonist on the on receptor the receptor (Figure (Figure 7b). 7Byb). con- By trast,contrast, the antagonistic the antagonistic action action of CoPP of CoPP resulted resulted inin increasesincreases in increases inin thethe in RMSDRMSD the RMSD fromfrom from 4040 nsns 40 toto ns 200200 to ns.200 Furthermore, ns. Furthermore, CoPP, CoPP, but but not not Heme, Heme, appeared appeared tototo changechange change thethe the overalloverall overall conformationconformation conformation ofof Agonist-REV-ERBAgonist-REV-ERBββ (Figure(Figure(Figure8 a,b).8a,b).8a,b). Thus, Thus,Thus, the thethe results resultsresults suggest suggest that thethat antagonist the antagonist ligand ligand might mightinﬂuence influence the function the function of the proteinof the protein target in target the agonist in the formagonist by form altering by thealtering conformation the con- formationof the target. of the target.

FigureFigure 8. 8. ((aa)) The The changes changes in in conformation conformation ofof Agonist-REV-ERBAgonist-REV-ERBββ(red) (red)(red) when whenwhen forming formingforming a aa complex complexcomplex with withCoPP CoPP compared compared with thewith initial the initial simulation simulation pose (white).pose (white). (b) The (b other) The conformationother conformation of Agonist-REV- of Ago- nist-REV-ERBERBβ (green)β when (green)(green) forming whenwhen a formingforming complex aa with complexcomplex Heme withwith compared HemeHeme withcomparedcompared the initial withwith simulation thethe initialinitial posesimulationsimulation (white). pose(c) Graph (white). of RMSD(c) Graph values of RMSD of CoPP values (red) of and CoPP Heme (red) (green) and Heme in the (green) binding in pocketthe binding of Agonist-REV- pocket of Agonist-REV-ERBβduring molecular dynamics simulations. (d) CoPP and Heme conformations at Agonist-REV-ERBERBβduring molecularβduring dynamics molecular simulations. dynamics (simulations.d) CoPP and ( Hemed) CoPP conformations and Heme conformations at the end of theat the end of the simulations. simulations.

TheThe ligand-binding ligand-binding pocket pocket of of REV-ERB REV-ERBββ isisis composedcomposed composed ofof of His381,His381, His381, Leu382,Leu382, Leu382, Val383,Val383, Val383, Cys384,Cys384, Phe443, Phe443, Leu446, Leu446, Phe450, Phe450, Phe454, Phe454, Gl Gly478,y478, Asp481, Asp481, Leu482, Leu482, Leu483, Leu483, and and Ser576. Ser576. TheThe MD MD trajectory trajectory indicated indicated that that CoPP CoPP moved moved away away from from the the binding binding pocket pocket (Figure (Figure 88c),c), whereaswhereas Heme Heme did did not not (Figure (Figure 88d).d). TheThe resultsresults alsoalso revealedrevealed thatthat CoPPCoPP rotatedrotated aboutabout 9090°◦ byby the the end end of of the the simulation. simulation. TheThe movement movement of of CoPP CoPP enlarged enlarged the the cavity cavity of of the the REV-ERB REV-ERBβ ligand-bindingligand-bindingligand-binding pocketpocket pocket (Figure(Figure 99a,b).a,b).POVME POVME 3.0 3.0 was was used used to to calculate calculate the the binding-pocket binding-pocket volume volume during during the MD the MDsimulation. simulation. Figure Figure9c shows 9c shows that thethat binding-pocket the binding-pocket volume volume of Heme of Heme was smallerwas smaller than thanthat ofthat CoPP, of CoPP, with with average average values values of 566.29 of 566.29 A3 and A3 777.39andand 777.39777.39 A3, respectively. AA3,, respectively.respectively.

FigureFigure 9. 9. TheThe cavity cavity of of (a ()a )Heme Heme and and (b ()b CoPP) CoPP on on the the Agonist-REV-ERB Agonist-REV-ERBβ bindingbindingβ binding pocketpocket pocket atat thethe atthe endend end ofof thethe of thesimulation.simulation. simulation. ((c) Plots(c) Plots of cavity of cavity values values of Heme of Heme (green) (green) and CoPP and CoPP(red)(red) inin (red) thethe bindingbinding in the binding sitesite ofof Agonist-REV-ERBAgonist-REV-ERB site of Agonist-REV-ERBβ duringduringβ molecularmolecularduring molecular dynam-dynam- icsicsdynamics simulations.simulations. simulations.

In the ligand-binding pocket, Cys384 and His568 play important roles in stabilizing Heme through axial coordination with the iron atom. Therefore, a change of the metal atom to cobalt might disrupt that interaction and lead to the expansion of the ligand-binding

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Molecules 2021, 26, 3251 In the ligand-binding pocket, Cys384 and His568 play important roles in stabilizing8 of 17 Heme through axial coordination with the iron atom. Therefore, a change of the metal atom to cobalt might disrupt that interaction and lead to the expansion of the ligand-bind- ingpocket. pocket. The The average average distance distance between between cobalt cobalt and and Cys384 Cys384 (4.72 (4.72 Å) was Å) was shorter shorter than than that thatbetween between iron andiron Cys384and Cys384 (5.40 Å).(5.40 By Å). contrast, By contrast, the distance the distance between between cobalt andcobalt His568 and His568was 2.36 was Å longer2.36 Å than longer that than between that between iron and His568iron and (8.96 His568 Å and (8.96 6.60 Å Å, and respectively). 6.60 Å, respec- The tively).interactions The interactions of CoPP and of CoPP Heme and with Heme the twowith residues the two residues are visualized are visualized in Figure in Figure10. In the10. Inreported the reported crystal crystal structures, structures, the distance the dist wasance the was same the from same Cys384 from Cys384 to the metal to the centers metal centersof Heme of andHeme CoPP and (2.34 CoPP Å), (2.34 but Å), the but distances the dist fromances His568 from His568 to the respective to the respective metal centers metal centersdiffered differed by 0.14 Åby [ 210.14]. Nevertheless, Å [21]. Nevertheless, the disruption the disruption of the ligand-binding of the ligand-binding pocket still pocket could stillnot explaincould not the explain antagonistic the antagonistic effect of CoPP effect on of REV-ERB CoPP onβ REV-ERB. β.

FigureFigure 10. 10. TheThe distance distance between between Cysteine Cysteine 384 384 an andd Histidine Histidine 568 568 on the Agonist-REV-ERB β and the themetal metal central central atom atom of Heme of Heme (green) (green) and and CoPP CoPP (red) (red) during during molecular molecular dynamic dynamic simulations. simulations.

TheThe antagonistic effecteffect ofofthe the CoPP CoPP ligand ligand on on REV-ERB REV-ERBβ isβ basedis based on on disruption disruption of the of therecruitment recruitment oftranscriptional of transcriptional corepressors. corepressors. Therefore, Therefore, the the effects effects of Hemeof Heme and and CoPP CoPP on oncorepressor corepressor binding binding were were examined examined further. further.

2.6.2.6. Recruitment Recruitment of of NCoR NCoR by by Protoporphyrin Protoporphyrin IX IX Protein-peptideProtein-peptide docking docking was was conducted conducted to to differentiate differentiate the the NCoR NCoR binding binding to to REV- REV- β ERBERBβ inin the the presence presence of of Heme Heme and and CoPP, CoPP, respectively. respectively. The The binding binding affinity afﬁnity of NCoR of NCoR was was evaluated based on atomic contact energy (ACE) and MM/PBSA binding energy. evaluated based on atomic contact energy (ACE) and MM/PBSA binding energy. The The NCoR structure was taken from a crystal structure with REV-ERBα (PDB ID: 3N00). NCoR structure was taken from a crystal structure with REV-ERBα (PDB ID: 3N00). First, First, the method validation using the PATCH Dock was performed to determine several the method validation using the PATCH Dock was performed to determine several pa- parameters that will be used in the protein-peptide docking simulation. The validation rameters that will be used in the protein-peptide docking simulation. The validation stage stage of this method is carried out using a re-docking approach. In this re-docking process, of this method is carried out using a re-docking approach. In this re-docking process, the the values of RMSD and the active site were observed as well as the possible binding values of RMSD and the active site were observed as well as the possible binding residues residues of the NCoR. of the NCoR. The RMSD value obtained from the re-docking results showed 1.3757 Å (Table3). The RMSD value obtained from the re-docking results showed 1.3757 Å (Table 3). Therefore, the parameters of the method validation results can be used at the protein- Therefore, the parameters of the method validation results can be used at the protein- peptide docking simulation. Table4 shows that the Heme-complex interacting with peptide docking simulation. Table 4 shows that the Heme-complex interacting with NCoR NCoR obtained a high PatchDock score with ACE score and binding energy value of obtained−74.60 kJ/mol a highand PatchDock−13.33 kJ/molscore with, respectively. ACE score Theand CoPP-complexbinding energy interactingvalue of −74.60 with kJ/molNCoR and produced −13.33 lowerkJ/mol, binding respectively. energy The than CoPP-c the Heme-complex,omplex interacting with with ACE NCoR score pro- and ducedbinding lower energy binding value energy of 453.13 than kJ/mol the Heme-complex, and 43.01 kJ/mol, with respectively. ACE score and binding energy value of 453.13 kJ/mol and 43.01 kJ/mol, respectively.

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Table 3. Results of the validation of the protein-peptide docking method. Table 3. Results of the validation of the protein-peptide docking method.

AminoAmino Acid Residue Residue RMSD RMSD

Trp402,Trp402, Val413, Val413, Lys439, Gly441, Gly441, Leu482, Leu482, Leu483,Leu483, Ser499, Ser499, Ser513, Gln529,Gln529, Thr552, Thr552, Arg562, His568, His578 Arg562, His568, His578

1.3757 Å Å CrystalCrystal structurestructure = = red red Re-docked structure = green Re-docked structure = green

TableTable 4. 4. ResultsResults of of protein-peptide protein-peptide docking docking against against nuclear nuclear receptor receptor corepressor corepressor (NCoR). (NCoR).

ResultResult ComplexComplex REV-ERBREV-ERBββ Score ACE (kJ/mol) ∆GBind (kJ/mol) Score ACE (kJ/mol) ∆GBind (kJ/mol) Crystal StructureStructure Agonist-REV-ERBAgonist-REV-ERBββ 1133211332 −−1903.89 −400.43400.43 HemeHeme−−Agonist-REV-ERBβ 98829882 −−74.60 −13.3313.33 CoPPCoPP−−Agonist-REV-ERBβ 1042410424 453.13453.13 43.01

TheThe ACE ACE score score is is an an atomic atomic desolvation desolvation energy, energy, which which is is defined deﬁned as as the the energy energy of of replacingreplacing a a protein-atom–water protein-atom–water contact contact with with a aprotein-atom–protein-atom protein-atom–protein-atom contact contact [24]. [24]. MM/PBSAMM/PBSA calculations calculations were were performed performed to evaluate to evaluate the therelative relative stability stability of each of each complex com- resultingplex resulting from fromprotein-peptide protein-peptide docking. docking. TheThe results results of ofprotein-peptide protein-peptide docking docking and and MM/PBSAMM/PBSA showed showed that that the the Heme-complex Heme-complex had had a better a better ability ability to recruit to recruit NCoR NCoR than thanthe CoPP-complex.the CoPP-complex. That phenomenon That phenomenon appears appears to be toa unique be a unique feature feature of Heme, of Heme, as synthetic as syn- agoniststhetic agonists that induce that induceNCoR recruitment NCoR recruitment in a cellular in a cellular context context cause an cause increase an increase in core- in pressor-peptidecorepressor-peptide binding binding [21]. [21]. TheThe interaction interaction between between the the Heme-complex Heme-complex and and NCoR NCoR consisted consisted of of three three hydrogen hydrogen bondsbonds (with (with Gly441, Gly441, Ser499, Ser499, and and Gln529, Gln529, respectively), respectively), one one electrostatic electrostatic interaction interaction (with (with Thr552),Thr552), and sevenseven hydrophobichydrophobic interactions interactions (with (with Trp402, Trp402, Val413, Val413, Leu482, Leu482, Leu483, Leu483, His568, His568,and His578, and His578, respectively). respectively). The interaction The interaction between between the CoPP-complex the CoPP-complex and NCoR and consisted NCoR consistedof six hydrogen of six hydrogen bonds (with bonds Gly441, (with Ser513, Gly441 Ser499,, Ser513, Gln529, Ser499, and Gln529, His578, and respectively) His578, respec- and tively)four hydrophobic and four hydrophobic interactions interactions (with Val413, (with Lys439, Val413, Leu483, Lys439, and Arg562,Leu483, respectively).and Arg562, re- For spectively).that reason, For it canthat be reason, predicted it can that be predicted the positive that ACE the positive score and ACE MM/PBSA score and value MM/PBSA for the valueCoPP-complex for the CoPP-complex were the result were of the an absenceresult of of an electrostatic absence of interactions.electrostatic interactions. TheThe end end of of Helix-11 Helix-11 is is a astructural structural area area that that is is part part of of the the NCoR NCoR binding binding site site [25,26]. [25,26]. ComparedCompared with with the the crystal crystal structure, structure, the the Co CoPP-complexPP-complex undergoes undergoes many many changes changes in in the the conformationconformation of of Helix-11 Helix-11 [21]. [21]. There There were were 13 13 amino amino acid acid residues residues that that turned turned into into loops loops atat the the end end of of the the simulation, simulation, including including Ser5 Ser563,63, Leu564, Leu564, Asn565, Asn565, Asn566, Asn566, His568, His568, Ser569, Ser569, Glu570,Glu570, Glu571, Glu571, Leu572, Leu572, Leu573, Leu573, Ala574, Ala574, Phe575, Phe575, and and Lys576 Lys576 (Figure (Figure 11b). 11 b).In contrast In contrast to CoPP,to CoPP, that thatpart partof Helix-11 of Helix-11 in the in Heme-complex the Heme-complex retained retained similarities similarities to the crystal to the crystalstruc- ture,structure, with changes with changes at only at onlya few a fewamino amino acid acid residues residues located located in inthe the middle middle of of Helix-11 Helix-11 (Pro599,(Pro599, Asp560, Asp560, Leu561, Arg562,Arg562, Ser563, Ser563, Leu564, Leu564, Asn565, Asn565, Asn566, Asn566, and and His His 568; 568;Figure Figure 11c). 11c).Therefore, Therefore, the Heme-complexthe Heme-complex was was better better able able to recruit to recruit nuclear nuclear receptor receptor corepressors corepressors and repress the transcription of downstream genes. and repress the transcription of downstream genes.

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((aa)) ((bb)) ((cc)) Figure 11. The visualization of Helix-11 (yellow) in Agonist-REV-ERBβ on the crystal structure (a), FigureFigure 11. 11. TheThe visualization visualization of ofHelix-11 Helix-11 (yellow) (yellow) in inAgonist-REV-ERB Agonist-REV-ERBβ onβ on the the crystal crystal structure structure (a), (a ), after molecular dynamic simulation in complex with CoPP (b) and in complex with Heme (c). afterafter molecular molecular dynamic dynamic simulation simulation in in complex complex with with CoPP CoPP (b ()b and) and in in complex complex with with Heme Heme (c ().c ).

BasedBasedBased on on on the the the visualization, visualization, visualization, it it could itcould could be be beobse obse observedrvedrved that that that non-polar non-polar non-polar peptides peptides peptides derived derived derived from from from NCoRNCoRNCoR bound bound bound to to toa a hydrophobic ahydrophobic hydrophobic patch patch patch on on on the the the LBD LBD LBD of of of REV-ERB REV-ERB REV-ERBββ. ..Compared Compared Compared with with the thethe crys- crystalcrys- taltalstructure, structure, structure, therethere there waswas was aa a difference difference in in the thethe bind bindingbindinging area area of of the the the corepressor corepressor corepressor (Figure (Figure (Figure 12). 12). 12 ).The The The changeschangeschanges inin in conformationconformation conformation atat atthethe the endend end ofof of HeliHeli Helix-11x-11x-11 causedcaused caused byby by thethe the enlargedenlarged enlarged CoPP–Agonist-CoPP–Agonist- CoPP–Agonist- REV-ERBREV-ERBREV-ERBββ-LBD-LBDβ-LBD (Figure (Figure (Figure 13) 13) 13 might might) might weaken weaken weaken and and and negati negati negativelyvelyvely affect affect affect corepressor corepressor corepressor recruitment. recruitment. recruitment. Thus,Thus,Thus, the the the ligands ligands ligands might might might control control control the the the switch switch switch between between between open open open and and and closed closed closed conformations conformations conformations of of ofthe the the hydrophobichydrophobichydrophobic surface surface surface on on on the the the LBD LBD LBD for for for corepressor corepressor corepressor recruitment recruitment recruitment [27,28]. [27,28]. [27,28 ].

((aa)) ((bb))

((cc)) ((dd)) Figure 12. Visualization of the peptide-docking results between the nuclear receptor corepressor FigureFigure 12. 12. VisualizationVisualization of ofthe the peptide-docking peptide-docking results results between between the thenuclear nuclear receptor receptor corepressor corepressor (NCoR) and (a) the crystal structure, (b) the CoPP-complex, (c) the Heme-complex, and (d) overlay (NCoR) and (a) the crystal structure, (b) the CoPP-complex, (c) the Heme-complex, and (d) overlay NCoR(NCoR) in crystal and (a structure) the crystal (yellow), structure, CoPP-c (b) theomplex CoPP-complex, (green), and ( cHeme-complex) the Heme-complex, (red). and (d) overlay NCoR in crystal structure (yellow), CoPP-complex (green), and Heme-complex (red). NCoR in crystal structure (yellow), CoPP-complex (green), and Heme-complex (red).

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(CoPP-complex) (Apo-form) (Heme-complex)

FigureFigure 13. 13. VisualizationVisualization of the of the changes changes in conformation in conformation at the at end the endof Helix of Helix 11 (purple 11 (purple box) that box) that causedcaused by by the the enlarged enlarged ligand-Agonist-REV-ERB ligand-Agonist-REV-ERBβ-LBDβ-LBD (red (red box). box). The The visualization visualization of REV-ERB of REV-ERB apo-structuredapo-structured was was shown shown as as comparison comparison and and the the NCoR NCoR structure structure was was omitted omitted to to make clear im- image. age. 3. Discussion 3. DiscussionIt is difficult to describe changes in protein structure that accompany ligand–protein interactionsIt is difficult in order to describe to distinguish changes structural in protein changes structure that that occur accompany when agonists ligand–protein and antag- interactionsonists interact in order with to the distinguish same protein structural [29]. Even changes experimental that occur data when in theagonists form ofand crystal an- tagonistsstructures interact are unable with tothe distinguish same protein the [29]. actions Even of experimental ligand agonists data and in antagonists the form of on crystal target structuresproteins [are30]. unable Nuclear to receptordistinguish REV-ERB the actionsβ has of two ligand forms agonists of crystal and structure,antagonists an on agonist tar- getform proteins (complexed [30]. Nuclear with Heme) receptor and REV-ERB an antagonistβ has formtwo forms (complexed of crystal with structure, CoPP). Heme an ago- and nistCoPP form are (complexed protoporphyrin with Heme) IX ligands and thatan anta havegonist affinity form for (complexed REV-ERBβ .with The onlyCoPP). difference Heme andbetween CoPP themare protoporphyrin is in the metal atomsIX ligands in the that middle have ofaffinity the protoporphyrin for REV-ERBβ IX. The ligand. only Heme dif- ferenceis a prosthetic between group them thatis in consiststhe metal of atoms a heterocyclic in the middle protoporphyrin of the protoporphyrin IX ring with IX an ligand. iron ion Hemeas the is metal a prosthetic center. group Heme that is an consists essential of componenta heterocyclic of manyprotoporphyrin proteins, including IX ring with oxygen an irontransport ion as the proteins metal suchcenter. as Heme hemoglobin is an essential and myoglobin component as wellof many as the proteins, cytochrome including p450 oxygenenzymes, transport in which proteins the Heme such moiety as hemoglobin carries out and electron myoglobin transport as well [31 as,32 the]. Beyond cytochrome Heme, p450an array enzymes, of other in which protoporphyrin the Heme IXmoiety compounds carries haveout electron been synthesized transport and/or[31,32]. areBeyond found Heme,naturally an array in cells, of other depending protoporphyrin on the physiological IX compounds availability have been of differentsynthesized metal and/or atoms are in foundtissues. naturally Instead in of cells, mimicking depending the agonist on the actionphysiological of Heme, availability CoPP functions of different as an antagonistmetal at- omsof REV-ERBin tissues.β functionInstead of [21 mimicking]. That phenomenon the agonist was action unexpected, of Heme, because CoPP functions the only distinc-as an antagonisttion between of REV-ERB the twoβ ligands function is the[21]. coordinated That phenomen metalon ion. was Therefore, unexpected, subtle because changes the in onlythe distinction porphyrin metalbetween center the andtwo ringligands conformation is the coordinated appear to metal influence ion. Therefore, the agonist subtle versus changesantagonist in the actions porphyrin of protoporphyrin metal center and IX. ring conformation appear to influence the agonist versusIn general, antagonist protoporphyrin actions of protoporphyrin IX ligands have IX. bonds with Val, Gly, and Leu residues β in REV-ERBIn general,. protoporphyrin Heme has several IX ligands additional have bonds bonds withwith CysVal, andGly, Hisand residuesLeu residues in REV- in β REV-ERBERB . Inβ. theHeme Fe(III) has state,several a six-coordinate additional bonds Heme-receptor with Cys and complex His residues is formed, in REV-ERB involvingβ. Cys and His residues. Upon reduction to Fe(II), the Cys ligand is replaced by another In the Fe(III) state, a six-coordinate Heme-receptor complex is formed, involving Cys and neutral-donor ligand to form a six-coordinate species, or else the Heme can exist as a His residues. Upon reduction to Fe(II), the Cys ligand is replaced by another neutral-do- five-coordinate species with only the His ligand [21]. That phenomenon allows Heme to be nor ligand to form a six-coordinate species, or else the Heme can exist as a five-coordinate a suitable ligand for REV-ERBβ. In contrast to Heme, CoPP has no interaction with Cys species with only the His ligand [21]. That phenomenon allows Heme to be a suitable and His residues and therefore has a lower binding energy than Heme. CoPP displays a ligand for REV-ERBβ. In contrast to Heme, CoPP has no interaction with Cys and His complex pharmacology, which includes an ability to induce Heme oxygenase 1 (HMOX-1) residues and therefore has a lower binding energy than Heme. CoPP displays a complex expression. The induction of HMOX-1 has been suggested as the mechanism by which pharmacology, which includes an ability to induce Heme oxygenase 1 (HMOX-1) expres- CoPP displays its anti-obesity activity [33]. sion. The induction of HMOX-1 has been suggested as the mechanism by which CoPP There are no changes known to be typical of Antagonist-REV-ERBβ, so the differential displays its anti-obesity activity [33]. characteristics of ligand agonists and antagonists cannot be observed for that species. Heme ligandsThere can are stabilize no changes target known receptors. to be Such typical stabilization of Antagonist-REV-ERB is observed in Helix-3β, so the and differen- Helix-11 tialof characteristics REV-ERBβ, which of ligand play agonists a role in and binding antagonists to nuclear cannot receptor be observed corepressors for that [species.20,21,34 ]. HemeREV-ERBs ligands function can stabilize as transcriptional target receptors. repressors Such stabilization by recruiting is NCoR–HDAC3 observed in Helix-3 complexes and Helix-11to REV-ERB of REV-ERB responseβ, elementswhich play in enhancersa role in binding and promoters to nuclear of target receptor genes. corepressors REV-ERBβ [20,21,34].agonists, REV-ERBs such as endogenous function as Hemetranscriptional or synthetic repressors agonists, by recruiting function by NCoR–HDAC3 increasing the complexestranscriptional to REV-ERB repression response of REV-ERB elementsβ target in enhancers genes [15 and,19, 21promoters,35]. As with of target Heme, genes. CoPP REV-ERBdoes notβ cause agonists, damage such to as the endogenous active site ofHeme Antagonist-REV-ERB or synthetic agonists,β, allowing function NCoR by increas- binding ingto the REV-ERB transcriptionalβ-LBD.The repression presence of REV-ERB of both ligandsβ target is genes expected [15,19,21,35]. to not change As with the Heme, ability

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of Antagonist-REV-ERBβ to recruit nuclear receptor corepressors and thus repress the transcription of downstream genes [21]. The presence of CoPP in Agonist-REV-ERBβ changes the end part of Helix-11, causing a decrease in the ability of REV-ERBβ to recruit NCoR. In addition, the regions around Helix- 11 display the highest B-factors in Agonist-REV-ERBβ. In the CoPP-complex, Helix-11 moves slightly out of the ligand-binding pocket (open conformation), therefore modulating the degree to which the corepressor-binding surface is expanded. Thus, by opening the LBD of REV-ERBβ, CoPP can reduce the ability of NCoR to interact with the active site of REV-ERBβ. NCoR docking simulations revealed that the CoPP-complex has lower energy than the crystal structure of REV-ERBβ. Corepressors require areas with high hydrophobicity. That is reﬂected in the non-polar nature of the NCoR, which binds the binding pocket of Agonist-REV-ERBβ [21,34,36]. Compared with the CoPP-complex, the Heme-complex has a more compact corepressor-binding surface, which enhances the recruitment of corepressors. The difference in corepressor-binding surface occurs because Helix-3 and Helix-11 in the Heme-complex remain intact as in the crystal structure, with only a slight change in some amino acid residues located in the middle of Helix-11. Thus, the end of the Helix remains intact, and the ability of Heme to recruit nuclear receptor corepressors is not affected. The apparent constitutive repressor effect previously noted for REV-ERBβ is most likely due to the fact that all cells have some level of intracellular Heme [21]. The results of this study can explain the behaviors of agonist and antagonist ligands that bind to REV-ERBβ to recruit nuclear receptor (NR) corepressors. MD simulations showed that Heme and CoPP caused identical changes to Helix-11 of REV-ERBβ, which is one of the key structural elements for ligand binding and corepressor recruitment in REV-ERBβ. It is also evident that subtle changes in the protoporphyrin IX metal center and ring conformation can inﬂuence the agonist versus antagonist actions of protoporphyrin IX. That agrees with other studies suggesting that a ligand binding to the iron metal center in Heme drives alterations in REV-ERBβ activity. This study provides new information about the interactions of porphyrins with the cancer suppressor REV-ERBβ and the effects of those interactions on the ability of REV-ERBβ to regulate transcription. Those results will be useful in efforts to develop novel porphyrin-based agents for cancer therapy.

4. Materials and Methods 4.1. Macromolecule Preparation Two crystalline structures of nuclear receptor REV-ERBβ were used as target molecules in this study. Both receptor structures were downloaded from the Protein Data Bank with PDB ID 3CQV (Agonist-REV-ERBβ)[20] and 4N73 (Antagonist-REV-ERBβ)[21], respectively. The preparations of macromolecules were conducted by removing the water molecules and natural ligand, adding polar hydrogen atoms, and calculating the Koll- man charges.

4.2. Ligand Preparation The ligands used in this study were natural ligands bound to each REV-ERBβ crystal structure. Iron protoporphyrin IX (Heme) was used as an agonist ligand, whereas cobalt protoporphyrin IX (CoPP) was used as an antagonist ligand. Both structures were used as inputs for molecular docking studies (Figure 14). Molecules 2021, 26, 3251 13 of 17 Molecules 2021, 26, x FOR PEER REVIEW 13 of 17

(a) (b)

FigureFigure 14. 14. (a()a )The The structure structure of of iron iron pr protoporphyrinotoporphyrin IX IX (Heme). (Heme). (b (b) )The The structure structure of of cobalt cobalt proto- protopor- porphyrinphyrin IX IX (CoPP). (CoPP).

4.3.4.3. Molecular Molecular Docking Docking Study Study TheThe docking docking study study was was performed performed using using AutoDock AutoDock 4.2 4.2 with with MGLTools MGLTools 1.5.6 1.5.6 [37,38]. [37,38]. AllAll ligands ligands used used in in the the simulations simulations were were set set with with maximum maximum torsion torsion [39]. [39]. The The grid grid spacing spacing waswas changed changed from from 0.375 0.375 nm, nM, and and the the cubic cubic grid grid map map was was 64 64 ×× 6060 ×× 6060 Å Åtoward toward the the REV- REV- ERBERBββ bindingbinding site. site. The The docking docking parameters parameters were were set set as as follows: follows: the the number number of of Genetic Genetic AlgorithmAlgorithm (GA) (GA) Runs Runs was was set as 100, populationpopulation sizesize waswasset set as as 150, 150, the the maximum maximum number num- berof evaluationsof evaluations was was set asset 2,500,000, as 2,500,000, and 100and runs 100 wereruns performed.were performed. Then, specialThen, special parameters pa- rameterswere prepared were prepared for iron (Fe)for iron and (Fe) cobalt and (Co) coba aslt the (Co) central as the metal central atom metal of protoporphyrinatom of proto- porphyrinIX, respectively, IX, respectively, and all other and parametersall other para weremeters set aswere the set default as the values. default Thevalues. docking The dockingprocess process was performed was performed as follows. as follows. First, molecular First, molecular docking docking was performed was performed to evaluate to evaluatethe docking the docking poses. Then, poses. defined Then, defined docking docking was conducted was conducted on the binding on the binding pocket. Threepocket. to Threesix independent to six independent docking docking calculations calculations were conducted. were conducted. The corresponding The corresponding lowest bindinglowest energies and predicted inhibition constants (pKi) were obtained from the docking log files binding energies and predicted inhibition constants (pKi) were obtained from the docking (dlg) [40]. AutoDock Tools and Discovery Studio 2016 were used to visualize the docking log files (dlg) [40]. AutoDock Tools and Discovery Studio 2016 were used to visualize the results. Surface representation images showing the binding pocket of REV-ERBβ were docking results. Surface representation images showing the binding pocket of REV-ERBβ generated using Discovery Studio 2016 software [41]. were generated using Discovery Studio 2016 software [41]. 4.4. Molecular Dynamics Simulation 4.4. Molecular Dynamics Simulation Molecular dynamic simulations were performed for both ligands against each nuclear receptorMolecular REV-ERB dynamicβ. The simulations simulations were were perfor performedmed for using both Gromacs2016ligands against [42 each–46], nu- and clearthe analysesreceptor wereREV-ERB performedβ. The simulations with visual were molecular perfor dynamicsmed using (VMD, Gromacs2016 Theoretical [42–46], and andComputational the analyses Biophysicswere performed group with at the visual Beckman molecular Institute, dynamics University (VMD, of Illinois Theoretical at Urbana- and ComputationalChampaign) [ 47Biophysics]. AMBER99SB-ILDN group at the forceBeckman field Institute, [48,49] was University used to parameterizeof Illinois at Ur- the bana-Champaign)protein. The ligand [47]. was AMBER99SB-ILDN parameterized using force twofield software [48,49] was programs: used to AnteChamberparameterize thePython protein. Parser The interfacE ligand was (ACPYPE) parameterized for protoporphyrin using two software IX [50] and programs: ForceGen AnteChamber for the central Pythonmetal atomsParser iron interfacE (Fe) and (ACPYP cobaltE) (Co) for [protoporphyrin51]. Long-range IX electrostatic [50] and ForceGen force was for determined the cen- tralby metal the Particle atoms Meshiron (Fe) Ewald and method cobalt (Co) [52]. [51]. Neutralization Long-range ofelectr theostatic system force was was performed deter- minedby adding by the Na+ Particle and Cl Mesh− ions. Ewald The method cubic of [52]. TIP3P Neutralization water model of was the used system to solvate was per- the formedsystem. by Berendsen adding Na+ thermostats and Cl− ions. and The barostats cubic areof TIP3P used duringwater model the heating was used stage to withsolvate the thepressure system. being Berendsen maintained thermostats at 1 bar. and The barostat stepss of are the used simulation during includedthe heating minimization stage with theuntil pressure 500,000 being steps, maintained heating until at 3101 bar. K, The temperature steps of the equilibration simulation (NVT) included in 500 minimization ps, pressure untilequilibration 500,000 steps, (NPT) heating in 500 until ps, and 310 a K, production temperature run equilibration with a 2 fs timestep (NVT) in for 500 200 ps, ns. pres- The surestability equilibration of the system (NPT) was in 500 verified ps, and by analysisa production of the run energy, with temperature,a 2 fs timestep pressure, for 200 andns. Theroot-mean-square stability of the deviationsystem was (RMSD). verified Analyses by analysis of the of binding-pocketthe energy, temperature, analysis and pressure, cavities andwere root-mean-square carried out using deviation POVME 3.0(RMSD). [53]. Analyses of the binding-pocket analysis and cavities were carried out using POVME 3.0 [53].

Molecules 2021, 26, 3251 14 of 17

4.5. Protein-Peptide Docking Protein-peptide docking was carried out between the nuclear receptor corepressor (NCoR) and the structures of the CoPP and Heme complexes obtained from molecular dynamics simulations to observe the ability of each ligand to recruit the corepressor. The NCoR structure that formed in crystals with REV-ERBα was downloaded from Protein Data Bank (PDB ID 3N00) [54] and then separated. PATCH Dock software was used to observe protein–protein interactions based on molecular docking. That software uses the shape complementarity of soft molecular surfaces to generate the best starting candidate solution [55,56]. The default clustering RMSD 2.0 Å was used, and the complex type was chosen to be protein–small ligand. Connolly dot surface representations of the molecules as different components such as convex, concave, and flat patches were generated using the PATCH Dock algorithm [57,58]. Then, the PATCH Dock solutions were optimized for small-size molecules, provides a list of refined complexes, reshuffled the molecule’s relative orientation, and the side-chains interface of the top 10 candidate solutions were rescored. The orientation of the relative molecules were amended by confining the flexibility of the side-chains of the interacting surface and allowing movements of small rigid bodies [59]. The results of the protein-peptide docking simulation were identified based on the PatchDock score, atomic contact energy (ACE) score, and MM/PBSA binding energy. The interactions and binding in the docked conformations in PDB format were visualized using the Discovery Studio 2016 software.

4.6. MM/PBSA Calculation MM/PBSA calculation was performed using the g_mmpbsa package [60] integrated in the Gromacs software. Polar desolvation energy was calculated with the Poisson- Boltzmann equation with a grid size of 0.5 Å. The dielectric constant of the solvent was set to 80 to represent water as the solvent [61,62]. Non-polar contribution was determined by calculation of the solvent-accessible surface area with the radii of the solvent as 1.4 Å [63]. The binding free energy of the complex was determined based on 200 snapshots taken from the beginning to the end of the molecular dynamic simulation trajectories of the complex.

5. Conclusions The mechanism of action of the agonist of iron protoporphyrin IX (Heme) and the antagonist of cobalt protoporphyrin IX (CoPP) against the REV-ERBβ nuclear receptor can be predicted based on the results of molecular dynamics simulations. The replacement of the central metal atom has been shown to affect the afﬁnity of the two porphyrins to the REV-ERBβ receptor and changes to the protein conformation during molecular dynamics simulations. This phenomenon will affect the ability of the REV-ERBβ receptor to bind to the NCoR corepressor which plays a role in regulating cancer cell signals to suppress target gene transcription. Therefore, in silico studies are very important in designing and developing porphyrin derivatives, especially for the therapy of cancer.

Author Contributions: D.H.T. conceived and designed the experiments (methodology), analyzed the data, and corrected manuscript; T.M.F. performed the experiments, collected the data, and drafted the manuscript; F.K. and M.Y. analyzed the data and corrected manuscript; and M.M. analyzed the data and corrected the manuscript. D.H.T. also contributed with resources, funding acquisition, and supervision. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Bandung Institute of Technology, grant number 7526/I1.B04/ PL/2018 (Riset & Inovasi) and 021.1/SK/I1.C03/KP/2019 (P3MI). The APC was funded by The Center for Research and Community Services-Bandung Institute of Technology. Conﬂicts of Interest: The authors declare that they have no conﬂict of interest related to the contents of this article. Molecules 2021, 26, 3251 15 of 17

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