Steroids 104 (2015) 220–229
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Steroids journal homepage: www.elsevier.com/locate/steroids
Predicted structures of new Vitamin D Receptor agonists based on available X-ray structures Maura Malinska a,⇑, Andrzej Kutner b, Krzysztof Woz´niak a,⇑ a b
Department of Chemistry, University of Warsaw, 1 Pasteura, 02-093 Warsaw, Poland Pharmaceutical Research Institute, 8 Rydygiera, 01-793 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 28 May 2015 Received in revised form 17 September 2015 Accepted 12 October 2015 Available online 22 October 2015 Keywords: Superagonist Drug design Pseudoatom databank Interaction energy Hydrogen bond Electrostatics
a b s t r a c t Current efforts in the field of vitamin D are to develop 1,25(OH)2D3 analogs that exhibit equal or even increased anti-proliferative activity while possessing a reduced tendency to cause hypercalcemia. The study proposes a new, rational design of vitamin D analogs based on data available in the Protein Data Bank. Undertaken approach was to minimize the electrostatic interaction energies available after the reconstruction of charge density with the aid of the pseudoatom databank, namely the University at Buffalo Pseudoatom Databank (UBDB). Analysis of 24 vitamin D analogs, bearing similar molecular structures complexed with Vitamin D Receptor enabled the design of new agonists forming all advantageous interaction to the receptor, coded TB1, TB2, TB3 and TB4. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction The fundamental actions of the hormonally active form of vitamin D (1,25(OH)2D3) are to maintain calcium and phosphorous homeostasis in vertebrate organisms [1–4]. In addition, 1,25 (OH)2D3 has potent antiproliferative and prodifferentiating actions on various normal as well as cancerous cell types, including breast, prostate, colon, skin, leukemia, and melanoma [5–14]. However, the clinical usefulness of 1,25(OH)2D3 is severely limited by its hypercalcemic and hypercalciuric side effects. Therefore, current efforts in the field are towards development of 1,25(OH)2D3 analogs that exhibit equal or even increased anti-proliferative activity while exhibiting a reduced tendency to cause hypercalcemia. Most if not all, of 1,25(OH)2D3 actions are mediated by Vitamin D Receptor (VDR) which is a member of the nuclear receptor superfamily. Liganded VDR dimerizes with the retinoid X receptor (RXR) [3,15]. These heterodimers recruit and bind vitamin D response elements (VDREs) in target gene promoters and coactivator proteins to modulate target gene transcription. X-ray structure of ligand binding domain (LBD) of VDR revealed the presence of 13 a-helices and three b-sheets in the protein structure [16]. ⇑ Corresponding authors at: Chemistry Department, Warsaw University, ul. Pasteura 1, 02-093 Warszawa, Poland. E-mail addresses: [email protected] (M. Malinska), kwozniak@chem. uw.edu.pl (K. Woz´niak). http://dx.doi.org/10.1016/j.steroids.2015.10.007 0039-128X/Ó 2015 Elsevier Inc. All rights reserved.
Activation of the VDR involves a conformational change within the LBD unit which creates a functional AF-2 domain that serves as a docking site for transcriptional comodulators of VDR action. AF-2 is created by helix H12, along with helixes H3 and H4. The one of the important interaction between helices are charge clamp Lys264. . .Glu420 and hydrogen bonds Ser235. . .Tyr415 and Arg154. . .Leu414. Several residues from H12 make also hydrophobic contacts with the ligand. Crystal structures of complexes of human VDR (hVDR) with 1,25(OH)2D3 (PDBID: 1DB1 (1.8 Å resolution [17]) and one with rat VDR (rVDR, PDBID: 2ZLC, 2.00 Å resolution [18]) were reported in the literature. Active form of vitamin D and analogs occupy the binding site in the b conformation of the A-ring with the 19methylene ‘up’ and the 1a-OH and 3b-OH groups in equatorial and axial orientations, respectively, likewise the plain 1,25 (OH)2D3 [19]. All therapeutic analogues of vitamin D are VDR agonists [20,21]. However, VDR antagonist are also interesting in terms of the mechanism of VDR antagonism and potential application in the therapy of Paget’s disease [22,23]. Many challenges remain in the field of vitamin D biology and, in particular, rational design of vitamin D analogs. Of particular interest is also understanding of the mechanism by which these analogs achieve their differential activity. Here, we are using structural data on VDR agonists, collected over the last several years. Protein Data Bank [24] (PDB) contains around a hundred crystal structures of LBD of VDR crystallized in
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complexes with various ligands (mostly vitamin D analogs [25]). Selected structures from PDB are presented in Table 1 with resolution limit for X-ray measurement and references. Surprisingly, none of the ligands crystalized with VDR imposes a large conformational change in the binding site of the receptor. The other possibility is that the resolution obtained (Table 1), mostly above 1.5 Å, was not high enough to notice some subtle structural differences. Therefore, interactions between ligands and proteins should be analyzed beyond simple geometrical considerations. To perform comprehensive analysis, the charge density of complexes has been reconstructed with the aid of the new version of UBDB [26,27]. On the basis of the modeled charge density, it was subsequently possible to obtain Coulombic interaction energy (electrostatic interaction energy, Ees) using the Exact Potential and Multipole Model (EPMM) method [28]. The dispersion, polarization and exchangerepulsion contribution are important factors contributing to the total energy. However, given the fact that proteins are polar moieties, it is commonly believed that the total interaction energy correlates with the electrostatic interaction energy and the contributions of other terms effectively cancel out. In general, although electrostatic interactions contribute only a part of the interaction energies between macromolecules, unlike dispersion forces they are highly directional and therefore dominate the nature of molecular packing in crystals and in biological complexes. This was already shown for small molecules e.g. sunitinib malate [29] and many other systems [30,31]. Calculation of the other contributions is still a big challenge for protein–ligand complexes and needs significant theoretical advances and also far better quality of experimental data. Nevertheless, binding affinity of ligands is related to change of the Gibbs free energy. It can be divided into two parts: enthalpic and entropic contribution to the binding. Modifications introduced into ligands in design process are generally made with the intent of improving the enthalpy of interaction e.g. the introduction of a hydrogen bond donor. Entropy contribution should be also consider, with the intent of reducing unfavorable entropic contributions to binding e.g. the removal of rotatable bonds or addition of internal ligand constraints. However, real entropic contribution is
Table 1 List of protein complexes with vitamin D analogs with identification code from PDB (PDBID), ligand name, organism, resolution of X-ray measurement and reference. The structural formulae of analyzed ligands are shown in Fig. 1. PDBID
Ligand
VDR organism
Resolution (Å)
Reference
1DB1 2ZlC 3A78 3VRV 3VRW 3VRU 2HB8 2HB7 2HAM 2HAS 2HAR 3AX8 1S19 1S0Z 1IE9 1IE8 3B0T 3CS4 3CS6 3A3Z 3A40 3P8X 4IA2 3AZ1
1,25(OH)2D3 1,25(OH)2D3 3EV YSD YS5 YS3 MVD O1C C33 C3O OCC EIM MC903 EB1089 MC1288 KH1060 MCZ COV 0CO 2MV 23R ZYD GEMINI DS2
Human Rat Human Rat Rat Rat Human Human Human Human Human Human Human Human Human Human Human Human human Human Human Human Zebrafish Human
1.80 2.00 1.90 1.90 2.40 2.00 2.00 1.80 1.90 1.96 1.90 2.60 2.10 2.50 1.40 1.52 1.30 2.00 1.80 1.72 1.45 1.70 2.95 1.50
[17] [18] [37] [37] [37] [35] [35] [35] [35] [35] [58] [39] [39] [39] [39] [46] [46] [47] [47] [59] [60] [50]
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unpredictable at the moment. The goal of presented studies is to understand the energetic differences between vitamin D analogs and explore possible directions in development of new agonists of VDR with all advantageous interaction with the receptor. Ees is presented as a scoring function calculated after reconstruction of charge density using pseudoatom databank [27]. 2. Results and discussion 2.1. Electrostatic interaction energy between 1,25(OH)2D3 and VDR The Ees calculations were performed for selected 29 residues forming the binding pocket of VDR (Fig. 2). Shadows around carbons 1–4, 12, 16, 17, 20–27 and 25-OH indicate contacts shorter than the sum of van der Waals radii between 1,25(OH)2D3 and VDR. In order to better describe the influence of particular parts of vitamin D compound, the molecule was divided into four parts: the A-ring (Frag1), the seco-B ring (Frag2), the CD-rings (Frag3) and the side chain (Frag4) that were analyzed separately (Fig. 3). The most important interactions for binding are hydrogen bonds to the 1-, 3- and 25-hydroxyls of 1,25(OH)2D3. The list of residues and corresponding Ees can be found in Table 2 for interactions of natural ligand and VDR. The 1-hydroxyl (1-OH) is in an equatorial position forming two hydrogen bonds to Ser237 (9 kcal mol1) and Arg274 (20 kcal mol1). The last one is the strongest interaction, probably due to the positive charge of the Arg residue. This explains why 1-OH is the most important group for binding. There are hydrogen bonds between the 3-OH (equatorial) and the Tyr143 (10 kcal mol1) and the Ser278 (9 kcal mol1). Finally, there are two hydrogen bonds between the 25-OH to His305 and His397 with Ees of 12 kcal mol1 and 9 kcal mol1, respectively. The triene system is governed by interactions with Ser275, Trp286 and Leu233. Nonetheless, the Ees with those residues is also significant and has a stabilizing nature. The Ser275 forms a strong C–H. . .O interaction with 3-OH (6 kcal mol1) of similar strength to the standard hydrogen bond with Ser237. Leu233 and Trp286 form C–H. . .p interactions with the seco-B-ring and CDrings with Ees of 2 kcal mol1 and 4 kcal mol1, respectively. The remaining interactions are the van der Waals contacts and here the Ees is negligible, ranging from 1 kcal mol1 to 1 kcal mol1. The Ees results for complexes of 1,25(OH)2D3 with rVDR (PDBID: 2ZLC, 2.00 Å resolution) [18] are summarized in Table 2. The total Ees for 29 residues is 84 kcal mol1 and the equivalent hydrogen bonds and most important contacts do not differ significantly between hVDR and rVDR. 2.2. Electrostatic interaction energy between vitamin D analogs and VDR 2.2.1. Modification of the A-ring Vitamin D and its analogs exhibit many possible conformations. Both forms of A-ring (a and b) coexist in solution [32,33] and in crystal structures [34]. However, in all analyzed VDR complexes, the vitamin D analogs exhibit exclusively the b conformation. The a-conformation would disrupt the hydrogen bonds formed between the hydroxyl and the protein. Analogs modified in the A-ring have been synthesized and crystallized with VDR. The 3epi analog of 1,25(OH)2D3 (3EV, Fig. 1) has the hydroxyl group in the equatorial orientation, as the 1-OH. This configuration disables the formation of a hydrogen bond with Ser278 and, therefore, the Ees of the interaction with this residue is negligible. Moreover, the interaction energy with Tyr143 and Ser275 is significantly lower, resulting in the total Ees for Frag1 equal to 51 kcal mol1 (Table 3). However, the total Ees amounts to 79 kcal mol1, which is very similar to the Ees found for the active form of
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1,25(OH)2D3
3EV
YS3
YS5
YSD
MVD
O1C
C33
C3O
OCC
EIM
MC903
EB1089
MC1288
ZYD
GEMINI
KH1060
MCZ
COV
0CO
Fig. 1. Structural formulae of selected vitamin D analogs with PDBIDs of ligands.
vitamin D. The lack of interaction with Ser278 is compensated by the formation of a larger number of weak contacts e.g. S–H. . .O (Cys288, 3 kcal mol1), C–H. . .O (Tyr147, 2 kcal mol1). This might explain still high affinity of 3-epi-1,25-(OH)2D3 for VDR.
Several crystal structures of LBD of VDR bound to selected C-2a substituted analogs have been described [18,35,36]. The crystal structures of hVDR in complexes with C-2a substituted analogs bearing methyl (MVD, Fig. 1), propyl (C33, Fig. 1), propoxy (C3O,
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2MV
23R
LG190178
DS2
Fig. 1 (continued)
Fig. 1), hydroxypropyl (O1C, Fig. 1), and hydroxypropoxy (OCC, Fig. 1) groups on C2a position were solved and refined [35]. These specific replacements do not modify the structure of the protein or the conformation of ligands. The methyl, propyl and propoxy substituents provide additional van der Waals contacts and enhance the strength of the contact with Arg274, resulting in the high total Ees for Frag1. While the hydroxypropyl (O1C) and hydroxypropoxy (OCC) groups maintain essential interactions also form a second hydrogen bond with the side chain of the Arg274 residue. The Ees with Arg274 is 48 and 46 kcal mol1, respectively. However, our calculation does not account for water molecules present in the position close to Arg274. The difference in energy between 1,25 (OH)2D3 and O1C, OCC with the described residue would be easily compensated for if the water molecules were taken into account. The C-2a substitutions are energetically preferred in the case of A-ring modification, however the difference between 1,25 (OH)2D3 and C-2a substituted analogs may be overestimated in our calculations. Therefore in the study we assume that the favorable Ees with Farg1 of analogs with the long chain at C2a position would be compensated by interaction with water molecules. In 19-nor analogs [37,38] the methylene group is formally transferred from C-10 to C-2 (YS3, YS5, YSD, Fig. 1). However, the modification of the A-ring does not significantly influence the interaction with the selected residues in comparison with the natural hormone bound to rVDR. The methylene group in contrast to methyl group does not stabilize the interaction with Arg274. 2.2.2. Modification of the CD-ring The EIM ligand with methoxy group at C-15 (Fig. 1) is the only one example of a vitamin D analog with an altered CD-ring fragment. The EIM ligand exhibits analogous interactions as 1,25 (OH)2D3 and additionally stronger Ees interactions between Frag3 and Met272 (2 kcal mol1) and Leu313 (3 kcal mol1), resulting in stabilization of interaction between Frag3 and VDR (8 kcal mol1). 2.2.3. Modification of the side chain In the crystal structures of VDR complexed with several agonists, the ligand is tightly bound to the receptor around the A-, seco-B- and CD-rings. In contrast, the aliphatic side chain is less constrained, thus allowing a number of alternative conformations. Therefore, the vitamin D analogs with different side chains have been intensively studied. Moreover, the binding affinities of vitamins D for proteins involved in metabolism of vitamin D are different. Therefore, the molecules with unsaturated bonds in the side chain were studied e.g. the calcipotriol (MC903, Fig. 1) and the EB1089 analog (Fig. 1). Despite the shorter side chain of MC903 (PDBID: 1S19) [39], a shift of 0.4 Å of His305 is sufficient to maintain the hydrogen bond with the hydroxyl group. Another feature of calcipotriol compared to 1,25(OH)2D3 is the absence of a direct contact with H12 resulting in a significantly smaller Ees for Frag4
(19 kcal mol1). In the case of the EB1089 analog [39], the side chain is elongated in comparison to 1,25(OH)2D3. Nevertheless, Ees for the side chain of for EB1089 is very similar (21 kcal mol1) to that of 1,25(OH)2D3. Replacement of the C-18 methyl group with the di-homologated side chain, as in ZYD analog (Fig. 1), resulted in a similar Ees of 86 kcal mol1 (Table 3). Also Gemini analog with the two identical side chains at C-20 in a natural and a 20-epi orientation does not form advantageous interaction. The total Ees for this analogue is even smaller that of 1,25(OH)2D3 (Table3), explaining the result that Gemini binds to VDR with 38% of that of 1,25(OH)2D3 [40]. Of the top interest are vitamin D analogs that are superagonists of VDR. Superagonist KH1060 [41] (Fig. 1) is a member of 20-epi family which exhibits properties similar to that of 1,25(OH)2D3 with decreased calcemic side effects [42–45]. The reason for the differences in biological activity of 1,25(OH)2D3 and the 20-epi analogs are not yet understood. The 20-epi analogs induce transcription and this correlates with the ability of these compounds to promote coactivator interaction [45]. From the geometrical point of view these molecules form contacts very similar to those of 1,25(OH)2D3, nonetheless the interaction energy for KH1060 is 20 kcal mol1 lower than for the natural ligand (Table 2). KH1060 forms tighter contacts with His305 and His397 (22 kcal mol1 and 17 kcal mol1, respectively), and generally stronger ligand–protein contacts. The MC1288 analog [41] is also a superagonist (PDBID: 1IE9), but the total Ees is similar to that of 1,25(OH)2D3. However, the geometry of MC1288 ligand is poorly resolved in this protein–ligand structure. Based on structural information of 20-epi analogs (KH1060 mainly), novel ligands were designed to maximize the number of protein–ligand contacts. The new ligands have the 20-epi configuration and oxygen at position 22. To avoid entropy loss with the new ligands, the side chain was designed with the oxolane ring. Due to the additional chirality at C-23, two epimers (0CO and COV, Fig. 1) with opposite stereochemistry at C-23 of the oxolane moiety were generated [46]. The 2a-methyl group was also incorporated in these analogs (2MV and 23R, Fig. 1) to provide additional van der Waals contacts compared to those obtained in the complex of hVDR with 1,25(OH)2D3 [47]. All 6 hydrogen bonds are formed with similar Ees to the one of 1,25(OH)2D3. However, only COV and 2MV have the superagonist properties. The 19-nor analogs form the same characteristic intermolecular interaction patterns as the natural hormone, but the total Ees is at least 10 kcal mol1 lower. This difference is a result of a modification of the side chain, suggesting, once more, that the side chain interactions are responsible for specific activation of VDR. The additional side chain at C-22 enforces the formation of a butyl pocket (YS5) [37]. This change enables formation of additional contacts, but rather of dispersive character. The higher interaction energy for YSD for Frag4 is similar to that of the KH1060 ligand.
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Fig. 2. (a) Binding pocket for 1,25(OH)2D3 in VDR structure (PDBID: 1DB1 and 2ZLC). Ligands and the most important residues drawn as sticks. VDR (PDBID: 1DB1) drawn in magenta cartoon representation. (b) Schematic visualization of the positions of 29 amino acid residues forming VDR binding pocket. 1,25(OH)2D3 drawn in lines. Drawing obtained using Maestro program (Schrödinger LLC, 2014).
This confirms that the side chains with two ethyl groups at C25 form the most advantageous contacts by stabilizing the hydrogen bonds with His305 and His397. Generally, the analogues with less flexible Frag4 as unsaturated bonds (MC903, EB1089) or bulky side chains (YS5, Gemini) result in a smaller total Ees. The result of Ees for YS5 analogue is less pronounced, because of its advantageous ethyl groups at C-25 carbon atom in 24-nor side chain.
One modification planned during design of the vitamin D analogues was the replacement of the C-22 carbon atom by an oxygen atom to form preferred interaction with Val300 (e.g. KH1060, COV, 23R, OCO, 2MV, MCZ (Fig. 1). However the Ees of interaction with Val300 is similar to the one of 1,25(OH)2D3 (SI). Therefore analogues without this modification can be similarly effective in activation of VDR.
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enabling the formation of numerous close contacts with VDR [50]. The DS2 ligand forms four standard hydrogen bonds with Tyr143, Ser278, His305 and His397 with Ees equal to 15, 18, 21 and 14 kcal mol1, respectively. The hydrogen bond to Arg274 is absent, but the close distance of positive and negative charge has a stabilizing nature (5 kcal mol1). The DS2 analog does not form any strong contacts with Ser273, however the C–H. . .O contacts to Tyr147 (4 kcal mol1), Leu233 (4 kcal mol1), Ser275 (7 kcal mol1), and p. . .H–C to Trp286 (4 kcal mol1) result in a total Ees of 96 kcal mol1. The total Ees for Frag4 amounts to 41 kcal mol1 and this is one of the lowest value. 2.3. Influence of ligands on the stability of H12
Fig. 3. 1,25(OH)2D3 divided into four fragments: A-ring (Frag1), the triene system (seco-B ring, Frag2), CD-rings (Frag3), and the side chain (Frag4). The 1,25(OH)2D3 shown as an example.
2.2.4. Interaction between VDR and nonsteroidal ligands A variety of vitamin D analogs reported previously possess the same secosteroidal skeleton, identical to 1,25(OH)2D3. The studies of compounds with different structures were also reported. The first proposed analog was LG190178 (Fig. 1) with diphenyl subunit, which exhibits potent transcriptional activity in vitro [48,49]. Structure–activity relationship studies were done by Kashiwagi et al., who obtained the ligand DS2 with a molecular structure
To test the influence of the ligand on the strength of the interaction governing the position of helix H12 the Ees calculation was performed for the Lys264. . .Glu420 salt bridge and the Ser235. . .Tyr415, the Arg154. . .Leu414 hydrogen bonds. Additionally, the Ees calculation for close contact with the Arg154 residue was performed (Arg154. . .Asp232, Arg154. . .Met412). Final results are presented in SI. The highest values of Ees for the Lys264. . .Glu420 in rVDR (PDBID: 3VRW, 3VRV and 3VRU) result from these compounds crystallized with short peptide (mimics the coactivator interactions) and therefore the salt bridge is broken. The Ees values for the charge clamp amount to 90 kcal mol1 for the complex between 1,25(OH)2D3 and VDR (PDBID: 1DB1). The superagonist ligands KH1060, COV, MC1288 and 2MV do not differ significantly from the other agonists. Moreover, none of the tested agonist influences meaningfully the strength of interaction responsible for the positioning of the H12 helix. These results might suggest that the position of H12 is maintained irrespectively to the ligand occupying binding pocket of VDR and is influenced by other interactions. 2.4. Design of new vitamin D analogs
Table 2 Ees [kcal mol1] for selected 29 residues for 1,25(OH)2D3 calculated with hVDR (PDBID: 1DB1) and rVDR (PDBID: 2ZLC). Residue TYR TYR PHE LEU LEU LEU VAL TYR SER ILE ILE MET ARG SER SER TRP CYS TYR VAL ALA HIS LEU LEU HIS TYR LEU LEU VAL PHE
143 147 150 227 230 233 234 236 237 268 271 272 274 275 278 286 288 295 300 303 305 309 313 397 401 404 414 418 422 P
1DB1
2ZLC
10 1 0 0 0 2 0 0 9 1 1 1 20 6 9 4 1 0 1 1 12 0 0 9 1 0 0 0 0
10 1 1 0 0 0 0 0 14 0 0 1 14 5 13 4 1 0 0 0 10 0 0 10 2 0 0 0 0
81
84
The table summarizing all values found in literature concerning the binding affinity of analyzed vitamin D analogues can be found in SI. Different types and conditions of measurements do not allow
Table 3 Summary of Ees [kcal mol1] calculated for selected vitamin D analogs. Protein
Ligand
Frag1
Frag2
Frag3
Frag4
Frag(1–4)
2HAR 1IE8 2HB7 3AX8 3AZ1 3VRW 2HAM 2HAS 3VRU 3VRV 3CS4 2HB8 3P8X 3A40 2CS6 3B0T 3A3Z 1IE9 1DB1 1S0Z 3A78 4IA2 1S19
OCC KH1060 O1C EIM DS2 YS5 C33 C3O YS3 YSD COV MVD ZYD 23R 0CO MCZ 2MV MC1288 1,25(OH)2D3 EB1089 3EV GEMINI MC903
95 61 86 66 61 62 72 75 67 50 66 70 66 62 61 59 61 62 62 60 51 61 42
1 4 3 3 6 4 4 4 3 5 4 4 4 2 3 3 2 4 4 4 4 4 2
13 7 8 8 12 3 10 8 5 5 10 5 4 11 9 9 12 6 5 3 4 7 11
18 46 19 24 41 32 28 23 28 41 32 21 19 32 30 31 31 22 20 21 28 16 19
111 103 101 100 96 95 95 94 93 91 91 90 86 85 85 83 83 82 81 81 79 75 53
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for a general comparison and conclusions. Therefore, only the values from the same publication have been plotted against calculated Ees (SI). There are some promising cases of correlation, however, it is difficult to judge if they are significant. Correlation between Ees and Gibbs energy was tested for 8 complexes measured at the same condition between aminoglycosides and RNA [31] with the determination coefficient R2 > 80% for least square method. Both findings support our attempt to use Ees as a scoring function. The discussed ligands do not impose a substantial structural change in VDR. All presented ligands form similar interactions considering the geometrical aspects of binding. However, the Ees are in the range 53 kcal mol1 to 111 kcal mol1 (Ees for 1,25 (OH)2D3 is 81 kcal mol1). The vitamin D analogs with the lowest total Ees are OCC, KH1060, O1C and EIM. It is subsequently possible to build the ligand with the lowest Ees with Frag1, Frag2, Frag3 and Frag4 (Fig. 4). Theoretically, it should have a molecular structure which enables the formation of all favorable interactions with VDR, at least from the electrostatic point of view. The TB1 was reconstructed from the Frag1, Frag2, Frag3 and Frag4 with the lowest Ees as a test case. To gain the lowest possible Ees new agonist should have the side chain of DS2 or KH1060 and the methyl substitution at C2a (TB2, TB3 and TB4). The methyl group at C-2 occupies the small cavity and can stabilize the hydrogen bond with Arg274. Moreover two ethyl groups at C25 stabilize the hydrogen bonds with His305 and His394. Therefore the 2MV ligand can be further develop into TB4 (Fig. 4). Naturally, all of them have the three hydroxyls responsible for formation of 6 hydrogen bonds.
2.4.1. Docking results-proposed ligands with VDR To verify the hypothesis that the designed new ligands will form all advantageous interactions with VDR, the docking procedure in the AutoDock4.2 program was performed. All designed ligands bind to hVDR in the same position as previously described analogs, forming at least 6 hydrogen bonds with corresponding residues (Fig. 5). The Gibbs free energy (DG) calculated by AutoDock for TB1, TB2, TB3 and TB4 is shown in Table 4. The lowest DG, 15.53 kcal mol1, is obtained for TB1, followed by TB2 with Eint 15.25 kcal mol1. Additionally, these results were therefore
TB1
TB2
Fig. 5. Binding pocket for VDR obtained in docking procedure ligands and the most important residues drawn as sticks TB1 in green, TB2 in blue, TB3 in orange and TB4 in magenta. VDR (PDBID: 1IE9) drawn in gray cartoon representation. For the clarity hydrogen bond shown only for the TB4 agonist.
checked by performing EPMM calculations for complexes with geometries obtained by the docking procedure (Table 4). The total Ees with the 29 selected residues for TB1, TB2, TB3 and TB4 were 153, 112, 118 and 116 kcal mol1, respectively. These Ees values are the lowest among all vitamin D analogs studied. The Ees values for TB2, TB3 and TB4 can be additionally lowered after incorporating of the methoxy group at C15 to the molecular structure, similarly to the EIM analog. In order to verify our computations and our new way of designing of VDR agonists a partial synthesis of analogs TB1–TB4 might be envisaged. Analogs TB1–TB4 could be accessible by a convergent approach starting from the pre-formed structural fragments. The A-ring of new agonists might be obtained from the two available precursors, quinic acid and homo-chiral carvone, to retain all the natural absolute configurations. The A-ring fragment for agonist TB1 might be converted into 2a-methyl derivative for the synthesis of agonists TB2–TB4. The CD-ring fragment will be obtained by degrading oxidations from vitamin D2 to give the Inhoffen
TB3
TB4
Fig. 4. Structural formulae of newly designed agonists of VDR.
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Table 4 Docking results for 1,25(OH)2D3, OCC, KH1060 and designed ligands TB1, TB2, TB3 and TB4 to hVDR (PDBID: 1IE9). DG stands for free Gibbs energy obtained in AutoDock4 [kcal mol1], Eint (FF) stands for interaction energy obtained with AutoDock4 based on the force field method [kcal mol1] and Ees(EPMM) – electrostatic interaction energy calculated on the basis of reconstructed charge density [kcal mol1]. Method
1,25(OH)2D3
0CC
KH1060
TB1
TB2
TB3
TB4
DG Eint (FF) Ees (EPMM)
10.77 13.45
12.61 19.61
14.49 18.07
15.53 20.01 153
15.25 18.17 112
14.47 18.05 118
12.61 17.85 116
Lythgoe diol. Side-chains of TB1–TB4 will be prepared from natural precursors by the methods well established in vitamin D chemistry. Coupling of the respective structural fragments by Julia modified olefination will give the respective ACD-ring synthons. These might be condensed conveniently with the respective side-chain fragments by palladium-catalyzed Suzuki–Miyaura cross coupling.
protein with PDBID: 1IE9 were used as a docking protein. The protein is complete (no missing residues or side chains) and the data resolution is the highest among selected structures. The results of docking can be found in Table 4 with Gibbs free energy and interaction energy for selected ligands. The docking algorithm found the proper position of the ligands. The RMSD between the ligand with the lowest energy obtained from docking and the crystal structure was below 1 Å.
3. Methods 3.1. Alignment Alignment of the selected VDR protein structures was performed to find the residues forming the binding pocket. The first step was the sequence alignment, performed in the Muscle program [51,52]. The structural alignment was done in the Theseus program [53,54] only for Ca atoms. 3.2. Preparation of protein structures For all the analyzed PDB structures, the hydrogen atoms were added using the Reduce program [55], and then water molecules were removed. The orientations of OH, SH, NH3+, Met-CH3 and the Asn, Gln and His side chains were optimized. The Arg, Lys, Asp and Glu residues were treated as ionized. X–H bond lengths were extended to the standard neutron diffraction values [56] and fixed in the case of all performed calculations. 3.3. Electrostatic interaction calculation Pseudoatom databanks allow reconstruction of the electron density of macromolecular systems for which experimentally derived geometries are available. In this study we used the UBDB [27] together with the LSDB program [26] to transfer the multipole parameters of the atom types stored in UBDB to the studied. None of the 29 selected residues discussed in the presented paper is disordered. The EPMM method was used to compute electrostatic interactions. The Buckingham approximation was employed in order to estimate the electrostatic potential at distances greater than 4.5 Å. The individual contribution of 29 selected residues for all ligands can be found in SI. 3.4. Docking AutoDock4.2 program [57] was used to obtain the geometry of complexes with selected known and new proposed ligands. AutoDock uses a grid-based method to allow rapid rating of the binding energy of trial conformations. The AutoDock program changes the conformation of the ligand by definition of movable torsion angle and docks some trial conformation. Movable bonds were defined only for single bonds in the side chain and the torsion angles for –OH groups. Firstly, the docking procedure was checked by docking the ligands with the solved crystal structure of complex of VDR with 1,25(OH)2D3, KH1060 and OCC. The coordination of
3.4.1. EPMM calculation after docking procedure It was necessary to modify the PDB output files from the AutoDock program to make the EPMM calculations feasible. First, polar hydrogen atom were deleted. Than all hydrogen atoms were added with the Reduce program. Transfer of multipole parameters from the UBDB was done as previously described. Finally the calculation of Ees in XDPROP was performed. 4. Conclusions Analysis of the crystal structures of vitamin D analogs bearing similar molecular structures with a complex of a VDR enabled the design of new agonists. For the first time the electrostatic interaction energies available after the reconstruction of charge density with the aid of the pseudoatom databank (UBDB) was used as a tool in ligand design process. Electrostatic interaction energies obtained for series of ligands gave an opportunity to understand interactions between the natural ligand, vitamin D analogs and the VDR. The structural analysis of the available VDR – ligand complexes, together with alignment techniques, revealed that there are 29 amino acid residues which contact the ligand. Trp286, which is specific to VDR among the representatives of the nuclear receptor family, plays the crucial role of positioning the ligand. Trp286 forms dispersive interactions, mostly C–H. . .p, with an average strength of 4 kcal mol1 indicating its very strong character. Ligands curve around the helix H3 of the VDR, with its A-ring interacting with the H5 helix and the 24/25-hydroxyl group with residues of helices H7 and H11. The ligand binding pocket is primarily composed of hydrophobic residues, however there are 6 hydrogen bonds, which is characteristic for most ligands studied. The 1-hydroxyl forms two hydrogen bonds with Ser237 (H3) and Arg274 (H5), whereas the 3-hydroxyl forms two hydrogen bonds with Ser278 (H5) and Tyr143. Their electrostatic interaction energies strongly contribute to the total interaction energy with binding pocket, with an average strengths of 8 kcal mol1, 19 kcal mol1, 11 kcal mol1 and 12 kcal mol1, respectively. A water channel is observed, with water molecules hydrogen bonded to Arg274 leading to the solvent. The analogs with a long chain at the C2 disturb the water channel, therefore only the 2methyl derivatives contribute to the formation of favorable interactions. Moreover the 2-methyl derivatives stabilize the hydrogen bond with Arg274. The 25-hydroxyl is hydrogen bonded to His305 (which lies in the loop connecting H6 and H7) and His397 (which lies in helix H11) with average electrostatic interaction energies of 13 and 11 kcal mol1. The agonists (KH1060 and YSD) with
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ethyl groups at the C-24 atom have much stronger interaction with these histidine residues. Unfortunately, the electrostatic interaction energy cannot distinguish between the agonist and superagonist of VDR. Superagonists KH1060, MC1288, COV and 2MV form contacts with similar energy and do not change the strength of interaction governing the position of the H12 helix. Only KH1060 forms somewhat stronger contacts with residues of the binding pockets. These results highlight once more the importance of studying the dynamics of the proteins in its natural environment. Nonetheless, the electrostatic interaction energy for all studied vitamin D analogs are in the range 53 kcal mol1 to 111 kcal mol1 depending on the change in molecular structure of the ligand. The vitamin D analogs with the lowest electrostatic interaction energies are OCC, KH1060, O1C and EIM. Substitution at the C2a and C24 atoms strongly influence final electrostatic interaction, which stabilizes close hydrogen bonds with Arg274 and His305, His394 residues, respectively. The substitution at C15 with methoxy group allow for a formation of more favorable contacts with CD-rings (e.g. EIM). On the other hand, the non secosteroidal ligand (DS2) gave one of the lowest electrostatic interaction energy for the interactions with the side chain. Generally our analysis suggests that analogs with flexible side chain have the preference in the binding to VDR. Four new ligands of VDR (TB1, TB2, TB3 and TB4) have been proposed as potential agonist of VDR better than 1,25(OH)2D3 in terms of electrostatic interaction energy between agonists and residues forming binding pocket of VDR. The geometries of the designed ligands complexed with VDR were obtained by the docking procedure followed by electrostatic interaction energy calculations. The electrostatic interaction energy for the ligands are the smallest among all vitamin D analogs studied, due to formation of all the beneficial interactions with VDR. Acknowledgment MM thanks the Polish NCN for financial support within the Etiuda scholarship UMO-2013/08/T/ST4/00494. KW acknowledges the Polish NCN Council MAESTRO grant decision number DEC2012/04/A/ST5/00609. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.steroids.2015.10. 007. References [1] L.A. Plum, H.F. DeLuca, Vitamin D, disease and therapeutic opportunities, Nat. Rev. Drug. Discov. 9 (2010) 941–955, http://dx.doi.org/10.1038/nrd3318. [2] D.D. Bikle, D. Vitamin, Metabolism, mechanism of action, and clinical applications, Chem. Biol. 21 (2014) 319–329, http://dx.doi.org/10.1016/j. chembiol.2013.12.016. [3] V.C. Yu, C. Delsert, B. Andersen, J.M. Holloway, O.V. Devary, A.M. Näär, et al., RXR?: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements, Cell 67 (1991) 1251–1266, http://dx.doi.org/10.1016/0092-8674(91)90301-E. [4] X. Zhang, B. Hoffmann, P.B.-V. Tran, G. Graupner, M. Pfahl, Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors, Nature 355 (1992) 441–446, http://dx.doi.org/10.1038/355441a0. [5] A.V. Krishnan, D. Feldman, Mechanisms of the anti-cancer and antiinflammatory actions of vitamin D, Annu. Rev. Pharmacol. Toxicol. 51 (2011) 311–336, http://dx.doi.org/10.1146/annurev-pharmtox-010510-100611. [6] A.S. Dusso, A.J. Brown, E. Slatopolsky, Vitamin D, Am. J. Physiol. Renal Physiol. 289 (2005) F8–F28, http://dx.doi.org/10.1152/ajprenal.00336.2004. [7] M. Sebag, J. Henderson, J. Rhim, R. Kremer, Relative resistance to 1,25dihydroxyvitamin D3 in a keratinocyte model of tumor progression, J. Biol. Chem. 267 (1992) 12162–12167.
[8] K.E. Abdaimi, V. Papavasiliou, D. Goltzman, R. Kremer, Expression and regulation of parathyroid hormone-related peptide in normal and malignant melanocytes, Am. J. Physiol. Cell Physiol. 279 (2000) C1230– C1238. [9] E. Abe, C. Miyaura, H. Sakagami, M. Takeda, K. Konno, T. Yamazaki, et al., Differentiation of mouse myeloid leukemia cells induced by 1 alpha, 25dihydroxyvitamin D3, Proc. Natl. Acad. Sci. 78 (1981) 4990–4994. [10] K.W. Colston, C.M. Hansen, Mechanisms implicated in the growth regulatory effects of vitamin D in breast cancer, Endocr. Relat. Cancer 9 (2002) 45–59, http://dx.doi.org/10.1677/erc.0.0090045. [11] T.C. Polek, N.L. Weigel, Vitamin D and prostate cancer, J. Androl. 23 (2002) 9– 17, http://dx.doi.org/10.1002/j.1939-4640.2002.tb02596.x. [12] M.J. Wargovich, P.H. Lointier, Calcium and vitamin D modulate mouse colon epithelial proliferation and growth characteristics of a human colon tumor cell line, Can. J. Physiol. Pharmacol. 65 (1987) 472–477, http://dx.doi.org/10.1139/ y87-081. [13] J. Yu, V. Papavasiliou, J. Rhim, D. Goltzman, R. Kremer, Vitamin D analogs: new therapeutic agents for the treatment of squamous cancer and its associated hypercalcemia, Anticancer Drugs 6 (1995). [14] K. Colston, M.J. Colston, D. Feldman, 1,25-dihydroxyvitamin D3 and malignant melanoma: the presence of receptors and inhibition of cell growth in culture, Endocrinology 108 (1981) 1083–1086, http://dx.doi.org/10.1210/endo-108-31083. [15] M. Leid, P. Kastner, R. Lyons, H. Nakshatri, M. Saunders, T. Zacharewski, et al., Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently, Cell 68 (1992) 377– 395. [16] Y. Brélivet, N. Rochel, D. Moras, Structural analysis of nuclear receptors: from isolated domains to integral proteins, Nucl. Recept. Struct. Dyn. Funct. 348 (2012) 466–473, http://dx.doi.org/10.1016/j.mce.2011.08.015. [17] N. Rochel, J.M. Wurtz, A. Mitschler, B. Klaholz, D. Moras, The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand, Mol. Cell 5 (2000) 173–179, http://dx.doi.org/10.1016/S1097-2765(00)80413-X. [18] M. Shimizu, Y. Miyamoto, H. Takaku, M. Matsuo, M. Nakabayashi, H. Masuno, et al., 2-Substituted-16-ene-22-thia-1,25-dihydroxy-26,27-dimethyl-19norvitamin D3 analogs: synthesis, biological evaluation, and crystal structure, Bioorg. Med. Chem. 16 (2008) 6949–6964, http://dx.doi.org/ 10.1016/j.bmc.2008.05.043. [19] K. Suwin´ska, A. Kutner, Crystal and molecular structure of 1,25dihydroxycholecalciferol, Acta Crystallogr. Sect. B 52 (1996) 550–554, http:// dx.doi.org/10.1107/S0108768195017071. [20] N. Kubodera, Pharmaceutical Studies on Vitamin D Derivatives and Practical Synthesis of Six Commercially Available Vitamin D Derivatives that Contribute to Current Clinical Practice, 2010. [21] S. Nadkarni, M. Chodyn´ski, A. Corcoran, E. Marcinkowska, G. Brown, A. Kutner, Double point modified analogs of vitamin D as potent activators of vitamin D receptor, Curr. Pharm. Des. 21 (2015) 1741–1763, http://dx.doi.org/10.2174/ 1381612821666141205125113. [22] S. Ishizuka, N. Kurihara, D. Miura, K. Takenouchi, J. Cornish, T. Cundy, et al., Vitamin D antagonist, TEI-9647, inhibits osteoclast formation induced by 1a,25-dihydroxyvitamin D3 from pagetic bone marrow cells, in: Proc 12th Workshop Vitam D 2004, 89–90, pp. 331–334. doi:http://dx.doi.org/10.1016/j. jsbmb.2004.03.025. [23] H. Saito, K. Takagi, K. Horie, S. Kakuda, M. Takimoto-Kamimura, E. Ochiai, et al., Synthesis of novel C-2 substituted vitamin D derivatives having ringed side chains and their biological evaluation on bone, J. Steroid Biochem. Mol. Biol. (2013) 3–8, http://dx.doi.org/10.1016/j.jsbmb.2013.02.004. [24] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, et al., The Protein Data Bank, Nucleic Acids Res. 28 (2000) 235–242, http://dx.doi. org/10.1093/nar/28.1.235. [25] C. Carlberg, F. Molnár, A. Mouriño, Vitamin D receptor ligands: the impact of crystal structures, Expert Opin. Ther. Pat. 22 (2012) 417–435, http://dx.doi. org/10.1517/13543776.2012.673590. [26] A. Volkov, X. Li, T. Koritsanszky, P. Coppens, Ab initio quality electrostatic atomic and molecular properties including intermolecular energies from a transferable theoretical pseudoatom databank, J. Phys. Chem. A 108 (2004) 4283–4300, http://dx.doi.org/10.1021/jp0379796. [27] K.N. Jarzembska, P.M. Dominiak, New version of the theoretical databank of transferable aspherical pseudoatoms, UBDB2011 – towards nucleic acid modelling, Acta Crystallogr. Sect. A 68 (2012) 139–147, http://dx.doi.org/ 10.1107/S0108767311042176. [28] A. Volkov, T. Koritsanszky, P. Coppens, Combination of the exact potential and multipole methods (EP/MM) for evaluation of intermolecular electrostatic interaction energies with pseudoatom representation of molecular electron densities, Chem. Phys. Lett. 391 (2004) 170–175, http://dx.doi.org/10.1016/j. cplett.2004.04.097. [29] M. Malin´ska, K.N. Jarzembska, A.M. Goral, A. Kutner, K. Woz´niak, P.M. Dominiak, Sunitinib: from charge-density studies to interaction with proteins, Acta Crystallogr. Sect. D 70 (2014) 1257–1270, http://dx.doi.org/ 10.1107/S1399004714002351. [30] P.M. Dominiak, A. Volkov, A.P. Dominiak, K.N. Jarzembska, P. Coppens, Combining crystallographic information and an aspherical-atom data bank in the evaluation of the electrostatic interaction energy in an enzyme–substrate complex: influenza neuraminidase inhibition, Acta Crystallogr. Sect. D 65 (2009) 485–499, http://dx.doi.org/10.1107/ S0907444909009433.
M. Malinska et al. / Steroids 104 (2015) 220–229 [31] M. Kulik, A.M. Goral, M. Jasin´ski, P.M. Dominiak, J. Trylska, Electrostatic interactions in aminoglycoside-RNA complexes, Biophys. J. 108 (2015) 655– 665, http://dx.doi.org/10.1016/j.bpj.2014.12.020. [32] W. Okamura, A. Norman, R. Wing, Vitamin D: concerning the relationship between molecular topology and biological function, Proc. Natl. Acad. Sci. U.S. A. 71 (1974) 4194–4197. [33] M.D. Mizhiritskii, L.E. Konstantinovskii, R. Vishkautsan, 2D NMR study of solution conformations and complete 1H and 13C chemical shifts assignments of vitamin D metabolites and analogs, Tetrahedron 52 (1996) 1239–1252, http://dx.doi.org/10.1016/0040-4020(95)00954-X. [34] A. Pietraszek, M. Malin´ska, M. Chodyn´ski, J. Martynow, M. Krupa, W. Maruszak, et al., Synthesis and crystal structure of anhydrous analog of 1,25dihydroxyvitamin D3, J. Pharm. Sci. 102 (2013) 3925–3931, http://dx.doi. org/10.1002/jps.23701. [35] S. Hourai, T. Fujishima, A. Kittaka, Y. Suhara, H. Takayama, N. Rochel, et al., Probing a water channel near the A-ring of receptor-bound 1alpha,25dihydroxyvitamin D3 with selected 2alpha-substituted analogues, J. Med. Chem. 49 (2006) 5199–5205, http://dx.doi.org/10.1021/jm0604070. [36] J.L. Vanhooke, M.M. Benning, C.B. Bauer, J.W. Pike, H.F. DeLuca, Molecular structure of the rat vitamin D receptor ligand binding domain complexed with 2-carbon-substituted vitamin d3 hormone analogues and a LXXLL-containing coactivator peptide, Biochemistry (Mosc) 43 (2004) 4101–4110, http://dx.doi. org/10.1021/bi036056y. [37] N. Yoshimoto, Y. Sakamaki, M. Haeta, A. Kato, Y. Inaba, T. Itoh, et al., Butyl pocket formation in the vitamin D receptor strongly affects the agonistic or antagonistic behavior of ligands, J. Med. Chem. 55 (2012) 4373–4381, http:// dx.doi.org/10.1021/jm300230a. [38] I.K. Sibilska, M. Szybinski, R.R. Sicinski, L.A. Plum, H.F. DeLuca, Highly potent 2methylene analogs of 1a,25-dihydroxyvitamin D3: synthesis and biological evaluation, SI Vitam Workshop 136 (2013) 9–13, http://dx.doi.org/10.1016/j. jsbmb.2013.02.001. [39] G. Tocchini-Valentini, N. Rochel, J.-M. Wurtz, D. Moras, Crystal structures of the vitamin D nuclear receptor liganded with the vitamin D side chain analogues calcipotriol and seocalcitol, receptor agonists of clinical importance. insights into a structural basis for the switching of calcipotriol to a receptor antagonist by further side chain modification, J. Med. Chem. 47 (2004) 1956– 1961, http://dx.doi.org/10.1021/jm0310582. [40] Y. Bury, M. Herdick, M.R. Uskokovic, C. Carlberg, Gene regulatory potential of 1a,25-dihydroxyvitamin D3 analogues with two side chains, J. Cell. Biochem. 81 (2001) 179–190, http://dx.doi.org/10.1002/jcb.1082. [41] G. Tocchini-Valentini, N. Rochel, J.M. Wurtz, A. Mitschler, D. Moras, Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands, Proc. Natl. Acad. Sci. 98 (2001) 5491–5496, http://dx.doi.org/10.1073/ pnas.091018698. [42] L. Binderup, S. Latini, E. Binderup, C. Bretting, M. Calverley, K. Hansen, 20-EPIvitamin D3 analogues: a novel class of potent regulators of cell growth and immune responses, Biochem. Pharmacol. 42 (1991) 1569–1575, http://dx.doi. org/10.1016/0006-2952(91)90426-6. [43] S. Peleg, M. Sastry, E.D. Collins, J.E. Bishop, A.W. Norman, Distinct conformational changes induced by 20-epi analogues of 1,25DIhydroxyvitamin D Are associated with enhanced activation of the vitamin D receptor, J. Biol. Chem. 270 (1995) 10551–10558, http://dx.doi.org/10.1074/ jbc.270.18.10551. [44] W. Yang, L.P. Freedman, 20-epi analogues of 1,25-dihydroxyvitamin D3 are highly potent inducers of DRIP coactivator complex binding to the vitamin D3 receptor, J. Biol. Chem. 274 (1999) 16838–16845, http://dx.doi.org/10.1074/ jbc.274.24.16838.
229
[45] Y.-Y. Liu, E.D. Collins, A.W. Norman, S. Peleg, Differential interaction of 1alpha,25-dihydroxyvitamin D3 analogues and their 20-epi homologues with the vitamin D receptor, J. Biol. Chem. 272 (1997) 3336–3345, http://dx.doi.org/ 10.1074/jbc.272.6.3336. [46] S. Hourai, L.C. Rodrigues, P. Antony, B. Reina-San-Martin, F. Ciesielski, B.C. Magnier, et al., Structure-based design of a superagonist ligand for the vitamin D nuclear receptor, Chem. Biol. 15 (2008) 383–392, http://dx.doi.org/10.1016/ j.chembiol.2008.03.016. [47] P. Antony, R. Sigüeiro, T. Huet, Y. Sato, N. Ramalanjaona, L.C. Rodrigues, et al., Structure-function relationships and crystal structures of the vitamin D receptor bound 2alpha-methyl-(20S,23S)- and 2alpha-methyl-(20S,23R)epoxymethano-1alpha,25-dihydroxyvitamin D3, J. Med. Chem. 53 (2010) 1159–1171, http://dx.doi.org/10.1021/jm9014636. [48] W. Hakamata, Y. Sato, H. Okuda, S. Honzawa, N. Saito, S. Kishimoto, et al., (2S,20 R)-analogue of LG190178 is a major active isomer, Bioorg. Med. Chem. Lett. 18 (2008) 120–123, http://dx.doi.org/10.1016/j.bmcl.2007.11.007. [49] S. Kakuda, K. Okada, H. Eguchi, K. Takenouchi, W. Hakamata, M. Kurihara, et al., Structure of the ligand-binding domain of rat VDR in complex with the nonsecosteroidal vitamin D3 analogue YR301, Acta Crystallogr. Sect. F 64 (2008) 970–973, http://dx.doi.org/10.1107/S1744309108026754. [50] H. Kashiwagi, Y. Ono, K. Shimizu, T. Haneishi, S. Ito, S. Iijima, et al., Novel nonsecosteroidal vitamin D3 carboxylic acid analogs for osteoporosis, and SAR analysis, Bioorg. Med. Chem. 19 (2011) 4721–4729, http://dx.doi.org/10.1016/ j.bmc.2011.07.001. [51] R. Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic Acids Res. 32 (2004) 1792–1797. [52] R. Edgar, MUSCLE: a multiple sequence alignment method with reduced time and space complexity, BMC Bioinformatics 5 (2004) 113. [53] D.L. Theobald, P.A. Steindel, Optimal simultaneous superpositioning of multiple structures with missing data, Bioinformatics 28 (2012) 1972–1979, http://dx.doi.org/10.1093/bioinformatics/bts243. [54] D.L. Theobald, D.S. Wuttke, Accurate structural correlations from maximum likelihood superpositions, PLoS Comput. Biol. 4 (2008) e43, http://dx.doi.org/ 10.1371/journal.pcbi.0040043. [55] J.M. Word, S.C. Lovell, J.S. Richardson, D.C. Richardson, Asparagine and glutamine: using hydrogen atom contacts in the choice of side-chain amide orientation, J. Mol. Biol. 285 (1999) 1735–1747, http://dx.doi.org/10.1006/ jmbi.1998.2401. [56] F.H. Allen, I.J. Bruno, Bond lengths in organic and metal-organic compounds revisited: X-H bond lengths from neutron diffraction data, Acta Crystallogr. Sect. B 66 (2010) 380–386, http://dx.doi.org/10.1107/S0108768110012048. [57] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, et al., AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility, J. Comput. Chem. 30 (2009) 2785–2791, http://dx.doi.org/ 10.1002/jcc.21256. [58] K. Shindo, G. Kumagai, M. Takano, D. Sawada, N. Saito, H. Saito, et al., New C15substituted active vitamin D3, Org. Lett. 13 (2011) 2852–2855, http://dx.doi. org/10.1021/ol200828s. [59] L. Verlinden, A. Verstuyf, G. Eelen, R. Bouillon, P. Ordonez-Moran, M.J. Larriba, et al., Synthesis, structure, and biological activity of des-side chain analogues of 1alpha,25-dihydroxyvitamin D3 with substituents at C18, ChemMedChem 6 (2011) 788–793, http://dx.doi.org/10.1002/cmdc.201100021. [60] H. Maehr, N. Rochel, H.J. Lee, N. Suh, M.R. Uskokovic, Diastereotopic and deuterium effects in gemini, J. Med. Chem. 56 (2013) 3878–3888, http://dx. doi.org/10.1021/jm400032t.
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Steroids journal homepage: www.elsevier.com/locate/steroids
Predicted structures of new Vitamin D Receptor agonists based on available X-ray structures Maura Malinska a,⇑, Andrzej Kutner b, Krzysztof Woz´niak a,⇑ a b
Department of Chemistry, University of Warsaw, 1 Pasteura, 02-093 Warsaw, Poland Pharmaceutical Research Institute, 8 Rydygiera, 01-793 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 28 May 2015 Received in revised form 17 September 2015 Accepted 12 October 2015 Available online 22 October 2015 Keywords: Superagonist Drug design Pseudoatom databank Interaction energy Hydrogen bond Electrostatics
a b s t r a c t Current efforts in the field of vitamin D are to develop 1,25(OH)2D3 analogs that exhibit equal or even increased anti-proliferative activity while possessing a reduced tendency to cause hypercalcemia. The study proposes a new, rational design of vitamin D analogs based on data available in the Protein Data Bank. Undertaken approach was to minimize the electrostatic interaction energies available after the reconstruction of charge density with the aid of the pseudoatom databank, namely the University at Buffalo Pseudoatom Databank (UBDB). Analysis of 24 vitamin D analogs, bearing similar molecular structures complexed with Vitamin D Receptor enabled the design of new agonists forming all advantageous interaction to the receptor, coded TB1, TB2, TB3 and TB4. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction The fundamental actions of the hormonally active form of vitamin D (1,25(OH)2D3) are to maintain calcium and phosphorous homeostasis in vertebrate organisms [1–4]. In addition, 1,25 (OH)2D3 has potent antiproliferative and prodifferentiating actions on various normal as well as cancerous cell types, including breast, prostate, colon, skin, leukemia, and melanoma [5–14]. However, the clinical usefulness of 1,25(OH)2D3 is severely limited by its hypercalcemic and hypercalciuric side effects. Therefore, current efforts in the field are towards development of 1,25(OH)2D3 analogs that exhibit equal or even increased anti-proliferative activity while exhibiting a reduced tendency to cause hypercalcemia. Most if not all, of 1,25(OH)2D3 actions are mediated by Vitamin D Receptor (VDR) which is a member of the nuclear receptor superfamily. Liganded VDR dimerizes with the retinoid X receptor (RXR) [3,15]. These heterodimers recruit and bind vitamin D response elements (VDREs) in target gene promoters and coactivator proteins to modulate target gene transcription. X-ray structure of ligand binding domain (LBD) of VDR revealed the presence of 13 a-helices and three b-sheets in the protein structure [16]. ⇑ Corresponding authors at: Chemistry Department, Warsaw University, ul. Pasteura 1, 02-093 Warszawa, Poland. E-mail addresses: [email protected] (M. Malinska), kwozniak@chem. uw.edu.pl (K. Woz´niak). http://dx.doi.org/10.1016/j.steroids.2015.10.007 0039-128X/Ó 2015 Elsevier Inc. All rights reserved.
Activation of the VDR involves a conformational change within the LBD unit which creates a functional AF-2 domain that serves as a docking site for transcriptional comodulators of VDR action. AF-2 is created by helix H12, along with helixes H3 and H4. The one of the important interaction between helices are charge clamp Lys264. . .Glu420 and hydrogen bonds Ser235. . .Tyr415 and Arg154. . .Leu414. Several residues from H12 make also hydrophobic contacts with the ligand. Crystal structures of complexes of human VDR (hVDR) with 1,25(OH)2D3 (PDBID: 1DB1 (1.8 Å resolution [17]) and one with rat VDR (rVDR, PDBID: 2ZLC, 2.00 Å resolution [18]) were reported in the literature. Active form of vitamin D and analogs occupy the binding site in the b conformation of the A-ring with the 19methylene ‘up’ and the 1a-OH and 3b-OH groups in equatorial and axial orientations, respectively, likewise the plain 1,25 (OH)2D3 [19]. All therapeutic analogues of vitamin D are VDR agonists [20,21]. However, VDR antagonist are also interesting in terms of the mechanism of VDR antagonism and potential application in the therapy of Paget’s disease [22,23]. Many challenges remain in the field of vitamin D biology and, in particular, rational design of vitamin D analogs. Of particular interest is also understanding of the mechanism by which these analogs achieve their differential activity. Here, we are using structural data on VDR agonists, collected over the last several years. Protein Data Bank [24] (PDB) contains around a hundred crystal structures of LBD of VDR crystallized in
M. Malinska et al. / Steroids 104 (2015) 220–229
complexes with various ligands (mostly vitamin D analogs [25]). Selected structures from PDB are presented in Table 1 with resolution limit for X-ray measurement and references. Surprisingly, none of the ligands crystalized with VDR imposes a large conformational change in the binding site of the receptor. The other possibility is that the resolution obtained (Table 1), mostly above 1.5 Å, was not high enough to notice some subtle structural differences. Therefore, interactions between ligands and proteins should be analyzed beyond simple geometrical considerations. To perform comprehensive analysis, the charge density of complexes has been reconstructed with the aid of the new version of UBDB [26,27]. On the basis of the modeled charge density, it was subsequently possible to obtain Coulombic interaction energy (electrostatic interaction energy, Ees) using the Exact Potential and Multipole Model (EPMM) method [28]. The dispersion, polarization and exchangerepulsion contribution are important factors contributing to the total energy. However, given the fact that proteins are polar moieties, it is commonly believed that the total interaction energy correlates with the electrostatic interaction energy and the contributions of other terms effectively cancel out. In general, although electrostatic interactions contribute only a part of the interaction energies between macromolecules, unlike dispersion forces they are highly directional and therefore dominate the nature of molecular packing in crystals and in biological complexes. This was already shown for small molecules e.g. sunitinib malate [29] and many other systems [30,31]. Calculation of the other contributions is still a big challenge for protein–ligand complexes and needs significant theoretical advances and also far better quality of experimental data. Nevertheless, binding affinity of ligands is related to change of the Gibbs free energy. It can be divided into two parts: enthalpic and entropic contribution to the binding. Modifications introduced into ligands in design process are generally made with the intent of improving the enthalpy of interaction e.g. the introduction of a hydrogen bond donor. Entropy contribution should be also consider, with the intent of reducing unfavorable entropic contributions to binding e.g. the removal of rotatable bonds or addition of internal ligand constraints. However, real entropic contribution is
Table 1 List of protein complexes with vitamin D analogs with identification code from PDB (PDBID), ligand name, organism, resolution of X-ray measurement and reference. The structural formulae of analyzed ligands are shown in Fig. 1. PDBID
Ligand
VDR organism
Resolution (Å)
Reference
1DB1 2ZlC 3A78 3VRV 3VRW 3VRU 2HB8 2HB7 2HAM 2HAS 2HAR 3AX8 1S19 1S0Z 1IE9 1IE8 3B0T 3CS4 3CS6 3A3Z 3A40 3P8X 4IA2 3AZ1
1,25(OH)2D3 1,25(OH)2D3 3EV YSD YS5 YS3 MVD O1C C33 C3O OCC EIM MC903 EB1089 MC1288 KH1060 MCZ COV 0CO 2MV 23R ZYD GEMINI DS2
Human Rat Human Rat Rat Rat Human Human Human Human Human Human Human Human Human Human Human Human human Human Human Human Zebrafish Human
1.80 2.00 1.90 1.90 2.40 2.00 2.00 1.80 1.90 1.96 1.90 2.60 2.10 2.50 1.40 1.52 1.30 2.00 1.80 1.72 1.45 1.70 2.95 1.50
[17] [18] [37] [37] [37] [35] [35] [35] [35] [35] [58] [39] [39] [39] [39] [46] [46] [47] [47] [59] [60] [50]
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unpredictable at the moment. The goal of presented studies is to understand the energetic differences between vitamin D analogs and explore possible directions in development of new agonists of VDR with all advantageous interaction with the receptor. Ees is presented as a scoring function calculated after reconstruction of charge density using pseudoatom databank [27]. 2. Results and discussion 2.1. Electrostatic interaction energy between 1,25(OH)2D3 and VDR The Ees calculations were performed for selected 29 residues forming the binding pocket of VDR (Fig. 2). Shadows around carbons 1–4, 12, 16, 17, 20–27 and 25-OH indicate contacts shorter than the sum of van der Waals radii between 1,25(OH)2D3 and VDR. In order to better describe the influence of particular parts of vitamin D compound, the molecule was divided into four parts: the A-ring (Frag1), the seco-B ring (Frag2), the CD-rings (Frag3) and the side chain (Frag4) that were analyzed separately (Fig. 3). The most important interactions for binding are hydrogen bonds to the 1-, 3- and 25-hydroxyls of 1,25(OH)2D3. The list of residues and corresponding Ees can be found in Table 2 for interactions of natural ligand and VDR. The 1-hydroxyl (1-OH) is in an equatorial position forming two hydrogen bonds to Ser237 (9 kcal mol1) and Arg274 (20 kcal mol1). The last one is the strongest interaction, probably due to the positive charge of the Arg residue. This explains why 1-OH is the most important group for binding. There are hydrogen bonds between the 3-OH (equatorial) and the Tyr143 (10 kcal mol1) and the Ser278 (9 kcal mol1). Finally, there are two hydrogen bonds between the 25-OH to His305 and His397 with Ees of 12 kcal mol1 and 9 kcal mol1, respectively. The triene system is governed by interactions with Ser275, Trp286 and Leu233. Nonetheless, the Ees with those residues is also significant and has a stabilizing nature. The Ser275 forms a strong C–H. . .O interaction with 3-OH (6 kcal mol1) of similar strength to the standard hydrogen bond with Ser237. Leu233 and Trp286 form C–H. . .p interactions with the seco-B-ring and CDrings with Ees of 2 kcal mol1 and 4 kcal mol1, respectively. The remaining interactions are the van der Waals contacts and here the Ees is negligible, ranging from 1 kcal mol1 to 1 kcal mol1. The Ees results for complexes of 1,25(OH)2D3 with rVDR (PDBID: 2ZLC, 2.00 Å resolution) [18] are summarized in Table 2. The total Ees for 29 residues is 84 kcal mol1 and the equivalent hydrogen bonds and most important contacts do not differ significantly between hVDR and rVDR. 2.2. Electrostatic interaction energy between vitamin D analogs and VDR 2.2.1. Modification of the A-ring Vitamin D and its analogs exhibit many possible conformations. Both forms of A-ring (a and b) coexist in solution [32,33] and in crystal structures [34]. However, in all analyzed VDR complexes, the vitamin D analogs exhibit exclusively the b conformation. The a-conformation would disrupt the hydrogen bonds formed between the hydroxyl and the protein. Analogs modified in the A-ring have been synthesized and crystallized with VDR. The 3epi analog of 1,25(OH)2D3 (3EV, Fig. 1) has the hydroxyl group in the equatorial orientation, as the 1-OH. This configuration disables the formation of a hydrogen bond with Ser278 and, therefore, the Ees of the interaction with this residue is negligible. Moreover, the interaction energy with Tyr143 and Ser275 is significantly lower, resulting in the total Ees for Frag1 equal to 51 kcal mol1 (Table 3). However, the total Ees amounts to 79 kcal mol1, which is very similar to the Ees found for the active form of
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1,25(OH)2D3
3EV
YS3
YS5
YSD
MVD
O1C
C33
C3O
OCC
EIM
MC903
EB1089
MC1288
ZYD
GEMINI
KH1060
MCZ
COV
0CO
Fig. 1. Structural formulae of selected vitamin D analogs with PDBIDs of ligands.
vitamin D. The lack of interaction with Ser278 is compensated by the formation of a larger number of weak contacts e.g. S–H. . .O (Cys288, 3 kcal mol1), C–H. . .O (Tyr147, 2 kcal mol1). This might explain still high affinity of 3-epi-1,25-(OH)2D3 for VDR.
Several crystal structures of LBD of VDR bound to selected C-2a substituted analogs have been described [18,35,36]. The crystal structures of hVDR in complexes with C-2a substituted analogs bearing methyl (MVD, Fig. 1), propyl (C33, Fig. 1), propoxy (C3O,
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2MV
23R
LG190178
DS2
Fig. 1 (continued)
Fig. 1), hydroxypropyl (O1C, Fig. 1), and hydroxypropoxy (OCC, Fig. 1) groups on C2a position were solved and refined [35]. These specific replacements do not modify the structure of the protein or the conformation of ligands. The methyl, propyl and propoxy substituents provide additional van der Waals contacts and enhance the strength of the contact with Arg274, resulting in the high total Ees for Frag1. While the hydroxypropyl (O1C) and hydroxypropoxy (OCC) groups maintain essential interactions also form a second hydrogen bond with the side chain of the Arg274 residue. The Ees with Arg274 is 48 and 46 kcal mol1, respectively. However, our calculation does not account for water molecules present in the position close to Arg274. The difference in energy between 1,25 (OH)2D3 and O1C, OCC with the described residue would be easily compensated for if the water molecules were taken into account. The C-2a substitutions are energetically preferred in the case of A-ring modification, however the difference between 1,25 (OH)2D3 and C-2a substituted analogs may be overestimated in our calculations. Therefore in the study we assume that the favorable Ees with Farg1 of analogs with the long chain at C2a position would be compensated by interaction with water molecules. In 19-nor analogs [37,38] the methylene group is formally transferred from C-10 to C-2 (YS3, YS5, YSD, Fig. 1). However, the modification of the A-ring does not significantly influence the interaction with the selected residues in comparison with the natural hormone bound to rVDR. The methylene group in contrast to methyl group does not stabilize the interaction with Arg274. 2.2.2. Modification of the CD-ring The EIM ligand with methoxy group at C-15 (Fig. 1) is the only one example of a vitamin D analog with an altered CD-ring fragment. The EIM ligand exhibits analogous interactions as 1,25 (OH)2D3 and additionally stronger Ees interactions between Frag3 and Met272 (2 kcal mol1) and Leu313 (3 kcal mol1), resulting in stabilization of interaction between Frag3 and VDR (8 kcal mol1). 2.2.3. Modification of the side chain In the crystal structures of VDR complexed with several agonists, the ligand is tightly bound to the receptor around the A-, seco-B- and CD-rings. In contrast, the aliphatic side chain is less constrained, thus allowing a number of alternative conformations. Therefore, the vitamin D analogs with different side chains have been intensively studied. Moreover, the binding affinities of vitamins D for proteins involved in metabolism of vitamin D are different. Therefore, the molecules with unsaturated bonds in the side chain were studied e.g. the calcipotriol (MC903, Fig. 1) and the EB1089 analog (Fig. 1). Despite the shorter side chain of MC903 (PDBID: 1S19) [39], a shift of 0.4 Å of His305 is sufficient to maintain the hydrogen bond with the hydroxyl group. Another feature of calcipotriol compared to 1,25(OH)2D3 is the absence of a direct contact with H12 resulting in a significantly smaller Ees for Frag4
(19 kcal mol1). In the case of the EB1089 analog [39], the side chain is elongated in comparison to 1,25(OH)2D3. Nevertheless, Ees for the side chain of for EB1089 is very similar (21 kcal mol1) to that of 1,25(OH)2D3. Replacement of the C-18 methyl group with the di-homologated side chain, as in ZYD analog (Fig. 1), resulted in a similar Ees of 86 kcal mol1 (Table 3). Also Gemini analog with the two identical side chains at C-20 in a natural and a 20-epi orientation does not form advantageous interaction. The total Ees for this analogue is even smaller that of 1,25(OH)2D3 (Table3), explaining the result that Gemini binds to VDR with 38% of that of 1,25(OH)2D3 [40]. Of the top interest are vitamin D analogs that are superagonists of VDR. Superagonist KH1060 [41] (Fig. 1) is a member of 20-epi family which exhibits properties similar to that of 1,25(OH)2D3 with decreased calcemic side effects [42–45]. The reason for the differences in biological activity of 1,25(OH)2D3 and the 20-epi analogs are not yet understood. The 20-epi analogs induce transcription and this correlates with the ability of these compounds to promote coactivator interaction [45]. From the geometrical point of view these molecules form contacts very similar to those of 1,25(OH)2D3, nonetheless the interaction energy for KH1060 is 20 kcal mol1 lower than for the natural ligand (Table 2). KH1060 forms tighter contacts with His305 and His397 (22 kcal mol1 and 17 kcal mol1, respectively), and generally stronger ligand–protein contacts. The MC1288 analog [41] is also a superagonist (PDBID: 1IE9), but the total Ees is similar to that of 1,25(OH)2D3. However, the geometry of MC1288 ligand is poorly resolved in this protein–ligand structure. Based on structural information of 20-epi analogs (KH1060 mainly), novel ligands were designed to maximize the number of protein–ligand contacts. The new ligands have the 20-epi configuration and oxygen at position 22. To avoid entropy loss with the new ligands, the side chain was designed with the oxolane ring. Due to the additional chirality at C-23, two epimers (0CO and COV, Fig. 1) with opposite stereochemistry at C-23 of the oxolane moiety were generated [46]. The 2a-methyl group was also incorporated in these analogs (2MV and 23R, Fig. 1) to provide additional van der Waals contacts compared to those obtained in the complex of hVDR with 1,25(OH)2D3 [47]. All 6 hydrogen bonds are formed with similar Ees to the one of 1,25(OH)2D3. However, only COV and 2MV have the superagonist properties. The 19-nor analogs form the same characteristic intermolecular interaction patterns as the natural hormone, but the total Ees is at least 10 kcal mol1 lower. This difference is a result of a modification of the side chain, suggesting, once more, that the side chain interactions are responsible for specific activation of VDR. The additional side chain at C-22 enforces the formation of a butyl pocket (YS5) [37]. This change enables formation of additional contacts, but rather of dispersive character. The higher interaction energy for YSD for Frag4 is similar to that of the KH1060 ligand.
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Fig. 2. (a) Binding pocket for 1,25(OH)2D3 in VDR structure (PDBID: 1DB1 and 2ZLC). Ligands and the most important residues drawn as sticks. VDR (PDBID: 1DB1) drawn in magenta cartoon representation. (b) Schematic visualization of the positions of 29 amino acid residues forming VDR binding pocket. 1,25(OH)2D3 drawn in lines. Drawing obtained using Maestro program (Schrödinger LLC, 2014).
This confirms that the side chains with two ethyl groups at C25 form the most advantageous contacts by stabilizing the hydrogen bonds with His305 and His397. Generally, the analogues with less flexible Frag4 as unsaturated bonds (MC903, EB1089) or bulky side chains (YS5, Gemini) result in a smaller total Ees. The result of Ees for YS5 analogue is less pronounced, because of its advantageous ethyl groups at C-25 carbon atom in 24-nor side chain.
One modification planned during design of the vitamin D analogues was the replacement of the C-22 carbon atom by an oxygen atom to form preferred interaction with Val300 (e.g. KH1060, COV, 23R, OCO, 2MV, MCZ (Fig. 1). However the Ees of interaction with Val300 is similar to the one of 1,25(OH)2D3 (SI). Therefore analogues without this modification can be similarly effective in activation of VDR.
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enabling the formation of numerous close contacts with VDR [50]. The DS2 ligand forms four standard hydrogen bonds with Tyr143, Ser278, His305 and His397 with Ees equal to 15, 18, 21 and 14 kcal mol1, respectively. The hydrogen bond to Arg274 is absent, but the close distance of positive and negative charge has a stabilizing nature (5 kcal mol1). The DS2 analog does not form any strong contacts with Ser273, however the C–H. . .O contacts to Tyr147 (4 kcal mol1), Leu233 (4 kcal mol1), Ser275 (7 kcal mol1), and p. . .H–C to Trp286 (4 kcal mol1) result in a total Ees of 96 kcal mol1. The total Ees for Frag4 amounts to 41 kcal mol1 and this is one of the lowest value. 2.3. Influence of ligands on the stability of H12
Fig. 3. 1,25(OH)2D3 divided into four fragments: A-ring (Frag1), the triene system (seco-B ring, Frag2), CD-rings (Frag3), and the side chain (Frag4). The 1,25(OH)2D3 shown as an example.
2.2.4. Interaction between VDR and nonsteroidal ligands A variety of vitamin D analogs reported previously possess the same secosteroidal skeleton, identical to 1,25(OH)2D3. The studies of compounds with different structures were also reported. The first proposed analog was LG190178 (Fig. 1) with diphenyl subunit, which exhibits potent transcriptional activity in vitro [48,49]. Structure–activity relationship studies were done by Kashiwagi et al., who obtained the ligand DS2 with a molecular structure
To test the influence of the ligand on the strength of the interaction governing the position of helix H12 the Ees calculation was performed for the Lys264. . .Glu420 salt bridge and the Ser235. . .Tyr415, the Arg154. . .Leu414 hydrogen bonds. Additionally, the Ees calculation for close contact with the Arg154 residue was performed (Arg154. . .Asp232, Arg154. . .Met412). Final results are presented in SI. The highest values of Ees for the Lys264. . .Glu420 in rVDR (PDBID: 3VRW, 3VRV and 3VRU) result from these compounds crystallized with short peptide (mimics the coactivator interactions) and therefore the salt bridge is broken. The Ees values for the charge clamp amount to 90 kcal mol1 for the complex between 1,25(OH)2D3 and VDR (PDBID: 1DB1). The superagonist ligands KH1060, COV, MC1288 and 2MV do not differ significantly from the other agonists. Moreover, none of the tested agonist influences meaningfully the strength of interaction responsible for the positioning of the H12 helix. These results might suggest that the position of H12 is maintained irrespectively to the ligand occupying binding pocket of VDR and is influenced by other interactions. 2.4. Design of new vitamin D analogs
Table 2 Ees [kcal mol1] for selected 29 residues for 1,25(OH)2D3 calculated with hVDR (PDBID: 1DB1) and rVDR (PDBID: 2ZLC). Residue TYR TYR PHE LEU LEU LEU VAL TYR SER ILE ILE MET ARG SER SER TRP CYS TYR VAL ALA HIS LEU LEU HIS TYR LEU LEU VAL PHE
143 147 150 227 230 233 234 236 237 268 271 272 274 275 278 286 288 295 300 303 305 309 313 397 401 404 414 418 422 P
1DB1
2ZLC
10 1 0 0 0 2 0 0 9 1 1 1 20 6 9 4 1 0 1 1 12 0 0 9 1 0 0 0 0
10 1 1 0 0 0 0 0 14 0 0 1 14 5 13 4 1 0 0 0 10 0 0 10 2 0 0 0 0
81
84
The table summarizing all values found in literature concerning the binding affinity of analyzed vitamin D analogues can be found in SI. Different types and conditions of measurements do not allow
Table 3 Summary of Ees [kcal mol1] calculated for selected vitamin D analogs. Protein
Ligand
Frag1
Frag2
Frag3
Frag4
Frag(1–4)
2HAR 1IE8 2HB7 3AX8 3AZ1 3VRW 2HAM 2HAS 3VRU 3VRV 3CS4 2HB8 3P8X 3A40 2CS6 3B0T 3A3Z 1IE9 1DB1 1S0Z 3A78 4IA2 1S19
OCC KH1060 O1C EIM DS2 YS5 C33 C3O YS3 YSD COV MVD ZYD 23R 0CO MCZ 2MV MC1288 1,25(OH)2D3 EB1089 3EV GEMINI MC903
95 61 86 66 61 62 72 75 67 50 66 70 66 62 61 59 61 62 62 60 51 61 42
1 4 3 3 6 4 4 4 3 5 4 4 4 2 3 3 2 4 4 4 4 4 2
13 7 8 8 12 3 10 8 5 5 10 5 4 11 9 9 12 6 5 3 4 7 11
18 46 19 24 41 32 28 23 28 41 32 21 19 32 30 31 31 22 20 21 28 16 19
111 103 101 100 96 95 95 94 93 91 91 90 86 85 85 83 83 82 81 81 79 75 53
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for a general comparison and conclusions. Therefore, only the values from the same publication have been plotted against calculated Ees (SI). There are some promising cases of correlation, however, it is difficult to judge if they are significant. Correlation between Ees and Gibbs energy was tested for 8 complexes measured at the same condition between aminoglycosides and RNA [31] with the determination coefficient R2 > 80% for least square method. Both findings support our attempt to use Ees as a scoring function. The discussed ligands do not impose a substantial structural change in VDR. All presented ligands form similar interactions considering the geometrical aspects of binding. However, the Ees are in the range 53 kcal mol1 to 111 kcal mol1 (Ees for 1,25 (OH)2D3 is 81 kcal mol1). The vitamin D analogs with the lowest total Ees are OCC, KH1060, O1C and EIM. It is subsequently possible to build the ligand with the lowest Ees with Frag1, Frag2, Frag3 and Frag4 (Fig. 4). Theoretically, it should have a molecular structure which enables the formation of all favorable interactions with VDR, at least from the electrostatic point of view. The TB1 was reconstructed from the Frag1, Frag2, Frag3 and Frag4 with the lowest Ees as a test case. To gain the lowest possible Ees new agonist should have the side chain of DS2 or KH1060 and the methyl substitution at C2a (TB2, TB3 and TB4). The methyl group at C-2 occupies the small cavity and can stabilize the hydrogen bond with Arg274. Moreover two ethyl groups at C25 stabilize the hydrogen bonds with His305 and His394. Therefore the 2MV ligand can be further develop into TB4 (Fig. 4). Naturally, all of them have the three hydroxyls responsible for formation of 6 hydrogen bonds.
2.4.1. Docking results-proposed ligands with VDR To verify the hypothesis that the designed new ligands will form all advantageous interactions with VDR, the docking procedure in the AutoDock4.2 program was performed. All designed ligands bind to hVDR in the same position as previously described analogs, forming at least 6 hydrogen bonds with corresponding residues (Fig. 5). The Gibbs free energy (DG) calculated by AutoDock for TB1, TB2, TB3 and TB4 is shown in Table 4. The lowest DG, 15.53 kcal mol1, is obtained for TB1, followed by TB2 with Eint 15.25 kcal mol1. Additionally, these results were therefore
TB1
TB2
Fig. 5. Binding pocket for VDR obtained in docking procedure ligands and the most important residues drawn as sticks TB1 in green, TB2 in blue, TB3 in orange and TB4 in magenta. VDR (PDBID: 1IE9) drawn in gray cartoon representation. For the clarity hydrogen bond shown only for the TB4 agonist.
checked by performing EPMM calculations for complexes with geometries obtained by the docking procedure (Table 4). The total Ees with the 29 selected residues for TB1, TB2, TB3 and TB4 were 153, 112, 118 and 116 kcal mol1, respectively. These Ees values are the lowest among all vitamin D analogs studied. The Ees values for TB2, TB3 and TB4 can be additionally lowered after incorporating of the methoxy group at C15 to the molecular structure, similarly to the EIM analog. In order to verify our computations and our new way of designing of VDR agonists a partial synthesis of analogs TB1–TB4 might be envisaged. Analogs TB1–TB4 could be accessible by a convergent approach starting from the pre-formed structural fragments. The A-ring of new agonists might be obtained from the two available precursors, quinic acid and homo-chiral carvone, to retain all the natural absolute configurations. The A-ring fragment for agonist TB1 might be converted into 2a-methyl derivative for the synthesis of agonists TB2–TB4. The CD-ring fragment will be obtained by degrading oxidations from vitamin D2 to give the Inhoffen
TB3
TB4
Fig. 4. Structural formulae of newly designed agonists of VDR.
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Table 4 Docking results for 1,25(OH)2D3, OCC, KH1060 and designed ligands TB1, TB2, TB3 and TB4 to hVDR (PDBID: 1IE9). DG stands for free Gibbs energy obtained in AutoDock4 [kcal mol1], Eint (FF) stands for interaction energy obtained with AutoDock4 based on the force field method [kcal mol1] and Ees(EPMM) – electrostatic interaction energy calculated on the basis of reconstructed charge density [kcal mol1]. Method
1,25(OH)2D3
0CC
KH1060
TB1
TB2
TB3
TB4
DG Eint (FF) Ees (EPMM)
10.77 13.45
12.61 19.61
14.49 18.07
15.53 20.01 153
15.25 18.17 112
14.47 18.05 118
12.61 17.85 116
Lythgoe diol. Side-chains of TB1–TB4 will be prepared from natural precursors by the methods well established in vitamin D chemistry. Coupling of the respective structural fragments by Julia modified olefination will give the respective ACD-ring synthons. These might be condensed conveniently with the respective side-chain fragments by palladium-catalyzed Suzuki–Miyaura cross coupling.
protein with PDBID: 1IE9 were used as a docking protein. The protein is complete (no missing residues or side chains) and the data resolution is the highest among selected structures. The results of docking can be found in Table 4 with Gibbs free energy and interaction energy for selected ligands. The docking algorithm found the proper position of the ligands. The RMSD between the ligand with the lowest energy obtained from docking and the crystal structure was below 1 Å.
3. Methods 3.1. Alignment Alignment of the selected VDR protein structures was performed to find the residues forming the binding pocket. The first step was the sequence alignment, performed in the Muscle program [51,52]. The structural alignment was done in the Theseus program [53,54] only for Ca atoms. 3.2. Preparation of protein structures For all the analyzed PDB structures, the hydrogen atoms were added using the Reduce program [55], and then water molecules were removed. The orientations of OH, SH, NH3+, Met-CH3 and the Asn, Gln and His side chains were optimized. The Arg, Lys, Asp and Glu residues were treated as ionized. X–H bond lengths were extended to the standard neutron diffraction values [56] and fixed in the case of all performed calculations. 3.3. Electrostatic interaction calculation Pseudoatom databanks allow reconstruction of the electron density of macromolecular systems for which experimentally derived geometries are available. In this study we used the UBDB [27] together with the LSDB program [26] to transfer the multipole parameters of the atom types stored in UBDB to the studied. None of the 29 selected residues discussed in the presented paper is disordered. The EPMM method was used to compute electrostatic interactions. The Buckingham approximation was employed in order to estimate the electrostatic potential at distances greater than 4.5 Å. The individual contribution of 29 selected residues for all ligands can be found in SI. 3.4. Docking AutoDock4.2 program [57] was used to obtain the geometry of complexes with selected known and new proposed ligands. AutoDock uses a grid-based method to allow rapid rating of the binding energy of trial conformations. The AutoDock program changes the conformation of the ligand by definition of movable torsion angle and docks some trial conformation. Movable bonds were defined only for single bonds in the side chain and the torsion angles for –OH groups. Firstly, the docking procedure was checked by docking the ligands with the solved crystal structure of complex of VDR with 1,25(OH)2D3, KH1060 and OCC. The coordination of
3.4.1. EPMM calculation after docking procedure It was necessary to modify the PDB output files from the AutoDock program to make the EPMM calculations feasible. First, polar hydrogen atom were deleted. Than all hydrogen atoms were added with the Reduce program. Transfer of multipole parameters from the UBDB was done as previously described. Finally the calculation of Ees in XDPROP was performed. 4. Conclusions Analysis of the crystal structures of vitamin D analogs bearing similar molecular structures with a complex of a VDR enabled the design of new agonists. For the first time the electrostatic interaction energies available after the reconstruction of charge density with the aid of the pseudoatom databank (UBDB) was used as a tool in ligand design process. Electrostatic interaction energies obtained for series of ligands gave an opportunity to understand interactions between the natural ligand, vitamin D analogs and the VDR. The structural analysis of the available VDR – ligand complexes, together with alignment techniques, revealed that there are 29 amino acid residues which contact the ligand. Trp286, which is specific to VDR among the representatives of the nuclear receptor family, plays the crucial role of positioning the ligand. Trp286 forms dispersive interactions, mostly C–H. . .p, with an average strength of 4 kcal mol1 indicating its very strong character. Ligands curve around the helix H3 of the VDR, with its A-ring interacting with the H5 helix and the 24/25-hydroxyl group with residues of helices H7 and H11. The ligand binding pocket is primarily composed of hydrophobic residues, however there are 6 hydrogen bonds, which is characteristic for most ligands studied. The 1-hydroxyl forms two hydrogen bonds with Ser237 (H3) and Arg274 (H5), whereas the 3-hydroxyl forms two hydrogen bonds with Ser278 (H5) and Tyr143. Their electrostatic interaction energies strongly contribute to the total interaction energy with binding pocket, with an average strengths of 8 kcal mol1, 19 kcal mol1, 11 kcal mol1 and 12 kcal mol1, respectively. A water channel is observed, with water molecules hydrogen bonded to Arg274 leading to the solvent. The analogs with a long chain at the C2 disturb the water channel, therefore only the 2methyl derivatives contribute to the formation of favorable interactions. Moreover the 2-methyl derivatives stabilize the hydrogen bond with Arg274. The 25-hydroxyl is hydrogen bonded to His305 (which lies in the loop connecting H6 and H7) and His397 (which lies in helix H11) with average electrostatic interaction energies of 13 and 11 kcal mol1. The agonists (KH1060 and YSD) with
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ethyl groups at the C-24 atom have much stronger interaction with these histidine residues. Unfortunately, the electrostatic interaction energy cannot distinguish between the agonist and superagonist of VDR. Superagonists KH1060, MC1288, COV and 2MV form contacts with similar energy and do not change the strength of interaction governing the position of the H12 helix. Only KH1060 forms somewhat stronger contacts with residues of the binding pockets. These results highlight once more the importance of studying the dynamics of the proteins in its natural environment. Nonetheless, the electrostatic interaction energy for all studied vitamin D analogs are in the range 53 kcal mol1 to 111 kcal mol1 depending on the change in molecular structure of the ligand. The vitamin D analogs with the lowest electrostatic interaction energies are OCC, KH1060, O1C and EIM. Substitution at the C2a and C24 atoms strongly influence final electrostatic interaction, which stabilizes close hydrogen bonds with Arg274 and His305, His394 residues, respectively. The substitution at C15 with methoxy group allow for a formation of more favorable contacts with CD-rings (e.g. EIM). On the other hand, the non secosteroidal ligand (DS2) gave one of the lowest electrostatic interaction energy for the interactions with the side chain. Generally our analysis suggests that analogs with flexible side chain have the preference in the binding to VDR. Four new ligands of VDR (TB1, TB2, TB3 and TB4) have been proposed as potential agonist of VDR better than 1,25(OH)2D3 in terms of electrostatic interaction energy between agonists and residues forming binding pocket of VDR. The geometries of the designed ligands complexed with VDR were obtained by the docking procedure followed by electrostatic interaction energy calculations. The electrostatic interaction energy for the ligands are the smallest among all vitamin D analogs studied, due to formation of all the beneficial interactions with VDR. Acknowledgment MM thanks the Polish NCN for financial support within the Etiuda scholarship UMO-2013/08/T/ST4/00494. KW acknowledges the Polish NCN Council MAESTRO grant decision number DEC2012/04/A/ST5/00609. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.steroids.2015.10. 007. References [1] L.A. Plum, H.F. DeLuca, Vitamin D, disease and therapeutic opportunities, Nat. Rev. Drug. Discov. 9 (2010) 941–955, http://dx.doi.org/10.1038/nrd3318. [2] D.D. Bikle, D. Vitamin, Metabolism, mechanism of action, and clinical applications, Chem. Biol. 21 (2014) 319–329, http://dx.doi.org/10.1016/j. chembiol.2013.12.016. [3] V.C. Yu, C. Delsert, B. Andersen, J.M. Holloway, O.V. Devary, A.M. Näär, et al., RXR?: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements, Cell 67 (1991) 1251–1266, http://dx.doi.org/10.1016/0092-8674(91)90301-E. [4] X. Zhang, B. Hoffmann, P.B.-V. Tran, G. Graupner, M. Pfahl, Retinoid X receptor is an auxiliary protein for thyroid hormone and retinoic acid receptors, Nature 355 (1992) 441–446, http://dx.doi.org/10.1038/355441a0. [5] A.V. Krishnan, D. Feldman, Mechanisms of the anti-cancer and antiinflammatory actions of vitamin D, Annu. Rev. Pharmacol. Toxicol. 51 (2011) 311–336, http://dx.doi.org/10.1146/annurev-pharmtox-010510-100611. [6] A.S. Dusso, A.J. Brown, E. Slatopolsky, Vitamin D, Am. J. Physiol. Renal Physiol. 289 (2005) F8–F28, http://dx.doi.org/10.1152/ajprenal.00336.2004. [7] M. Sebag, J. Henderson, J. Rhim, R. Kremer, Relative resistance to 1,25dihydroxyvitamin D3 in a keratinocyte model of tumor progression, J. Biol. Chem. 267 (1992) 12162–12167.
[8] K.E. Abdaimi, V. Papavasiliou, D. Goltzman, R. Kremer, Expression and regulation of parathyroid hormone-related peptide in normal and malignant melanocytes, Am. J. Physiol. Cell Physiol. 279 (2000) C1230– C1238. [9] E. Abe, C. Miyaura, H. Sakagami, M. Takeda, K. Konno, T. Yamazaki, et al., Differentiation of mouse myeloid leukemia cells induced by 1 alpha, 25dihydroxyvitamin D3, Proc. Natl. Acad. Sci. 78 (1981) 4990–4994. [10] K.W. Colston, C.M. Hansen, Mechanisms implicated in the growth regulatory effects of vitamin D in breast cancer, Endocr. Relat. Cancer 9 (2002) 45–59, http://dx.doi.org/10.1677/erc.0.0090045. [11] T.C. Polek, N.L. Weigel, Vitamin D and prostate cancer, J. Androl. 23 (2002) 9– 17, http://dx.doi.org/10.1002/j.1939-4640.2002.tb02596.x. [12] M.J. Wargovich, P.H. Lointier, Calcium and vitamin D modulate mouse colon epithelial proliferation and growth characteristics of a human colon tumor cell line, Can. J. Physiol. Pharmacol. 65 (1987) 472–477, http://dx.doi.org/10.1139/ y87-081. [13] J. Yu, V. Papavasiliou, J. Rhim, D. Goltzman, R. Kremer, Vitamin D analogs: new therapeutic agents for the treatment of squamous cancer and its associated hypercalcemia, Anticancer Drugs 6 (1995). [14] K. Colston, M.J. Colston, D. Feldman, 1,25-dihydroxyvitamin D3 and malignant melanoma: the presence of receptors and inhibition of cell growth in culture, Endocrinology 108 (1981) 1083–1086, http://dx.doi.org/10.1210/endo-108-31083. [15] M. Leid, P. Kastner, R. Lyons, H. Nakshatri, M. Saunders, T. Zacharewski, et al., Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently, Cell 68 (1992) 377– 395. [16] Y. Brélivet, N. Rochel, D. Moras, Structural analysis of nuclear receptors: from isolated domains to integral proteins, Nucl. Recept. Struct. Dyn. Funct. 348 (2012) 466–473, http://dx.doi.org/10.1016/j.mce.2011.08.015. [17] N. Rochel, J.M. Wurtz, A. Mitschler, B. Klaholz, D. Moras, The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand, Mol. Cell 5 (2000) 173–179, http://dx.doi.org/10.1016/S1097-2765(00)80413-X. [18] M. Shimizu, Y. Miyamoto, H. Takaku, M. Matsuo, M. Nakabayashi, H. Masuno, et al., 2-Substituted-16-ene-22-thia-1,25-dihydroxy-26,27-dimethyl-19norvitamin D3 analogs: synthesis, biological evaluation, and crystal structure, Bioorg. Med. Chem. 16 (2008) 6949–6964, http://dx.doi.org/ 10.1016/j.bmc.2008.05.043. [19] K. Suwin´ska, A. Kutner, Crystal and molecular structure of 1,25dihydroxycholecalciferol, Acta Crystallogr. Sect. B 52 (1996) 550–554, http:// dx.doi.org/10.1107/S0108768195017071. [20] N. Kubodera, Pharmaceutical Studies on Vitamin D Derivatives and Practical Synthesis of Six Commercially Available Vitamin D Derivatives that Contribute to Current Clinical Practice, 2010. [21] S. Nadkarni, M. Chodyn´ski, A. Corcoran, E. Marcinkowska, G. Brown, A. Kutner, Double point modified analogs of vitamin D as potent activators of vitamin D receptor, Curr. Pharm. Des. 21 (2015) 1741–1763, http://dx.doi.org/10.2174/ 1381612821666141205125113. [22] S. Ishizuka, N. Kurihara, D. Miura, K. Takenouchi, J. Cornish, T. Cundy, et al., Vitamin D antagonist, TEI-9647, inhibits osteoclast formation induced by 1a,25-dihydroxyvitamin D3 from pagetic bone marrow cells, in: Proc 12th Workshop Vitam D 2004, 89–90, pp. 331–334. doi:http://dx.doi.org/10.1016/j. jsbmb.2004.03.025. [23] H. Saito, K. Takagi, K. Horie, S. Kakuda, M. Takimoto-Kamimura, E. Ochiai, et al., Synthesis of novel C-2 substituted vitamin D derivatives having ringed side chains and their biological evaluation on bone, J. Steroid Biochem. Mol. Biol. (2013) 3–8, http://dx.doi.org/10.1016/j.jsbmb.2013.02.004. [24] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, et al., The Protein Data Bank, Nucleic Acids Res. 28 (2000) 235–242, http://dx.doi. org/10.1093/nar/28.1.235. [25] C. Carlberg, F. Molnár, A. Mouriño, Vitamin D receptor ligands: the impact of crystal structures, Expert Opin. Ther. Pat. 22 (2012) 417–435, http://dx.doi. org/10.1517/13543776.2012.673590. [26] A. Volkov, X. Li, T. Koritsanszky, P. Coppens, Ab initio quality electrostatic atomic and molecular properties including intermolecular energies from a transferable theoretical pseudoatom databank, J. Phys. Chem. A 108 (2004) 4283–4300, http://dx.doi.org/10.1021/jp0379796. [27] K.N. Jarzembska, P.M. Dominiak, New version of the theoretical databank of transferable aspherical pseudoatoms, UBDB2011 – towards nucleic acid modelling, Acta Crystallogr. Sect. A 68 (2012) 139–147, http://dx.doi.org/ 10.1107/S0108767311042176. [28] A. Volkov, T. Koritsanszky, P. Coppens, Combination of the exact potential and multipole methods (EP/MM) for evaluation of intermolecular electrostatic interaction energies with pseudoatom representation of molecular electron densities, Chem. Phys. Lett. 391 (2004) 170–175, http://dx.doi.org/10.1016/j. cplett.2004.04.097. [29] M. Malin´ska, K.N. Jarzembska, A.M. Goral, A. Kutner, K. Woz´niak, P.M. Dominiak, Sunitinib: from charge-density studies to interaction with proteins, Acta Crystallogr. Sect. D 70 (2014) 1257–1270, http://dx.doi.org/ 10.1107/S1399004714002351. [30] P.M. Dominiak, A. Volkov, A.P. Dominiak, K.N. Jarzembska, P. Coppens, Combining crystallographic information and an aspherical-atom data bank in the evaluation of the electrostatic interaction energy in an enzyme–substrate complex: influenza neuraminidase inhibition, Acta Crystallogr. Sect. D 65 (2009) 485–499, http://dx.doi.org/10.1107/ S0907444909009433.
M. Malinska et al. / Steroids 104 (2015) 220–229 [31] M. Kulik, A.M. Goral, M. Jasin´ski, P.M. Dominiak, J. Trylska, Electrostatic interactions in aminoglycoside-RNA complexes, Biophys. J. 108 (2015) 655– 665, http://dx.doi.org/10.1016/j.bpj.2014.12.020. [32] W. Okamura, A. Norman, R. Wing, Vitamin D: concerning the relationship between molecular topology and biological function, Proc. Natl. Acad. Sci. U.S. A. 71 (1974) 4194–4197. [33] M.D. Mizhiritskii, L.E. Konstantinovskii, R. Vishkautsan, 2D NMR study of solution conformations and complete 1H and 13C chemical shifts assignments of vitamin D metabolites and analogs, Tetrahedron 52 (1996) 1239–1252, http://dx.doi.org/10.1016/0040-4020(95)00954-X. [34] A. Pietraszek, M. Malin´ska, M. Chodyn´ski, J. Martynow, M. Krupa, W. Maruszak, et al., Synthesis and crystal structure of anhydrous analog of 1,25dihydroxyvitamin D3, J. Pharm. Sci. 102 (2013) 3925–3931, http://dx.doi. org/10.1002/jps.23701. [35] S. Hourai, T. Fujishima, A. Kittaka, Y. Suhara, H. Takayama, N. Rochel, et al., Probing a water channel near the A-ring of receptor-bound 1alpha,25dihydroxyvitamin D3 with selected 2alpha-substituted analogues, J. Med. Chem. 49 (2006) 5199–5205, http://dx.doi.org/10.1021/jm0604070. [36] J.L. Vanhooke, M.M. Benning, C.B. Bauer, J.W. Pike, H.F. DeLuca, Molecular structure of the rat vitamin D receptor ligand binding domain complexed with 2-carbon-substituted vitamin d3 hormone analogues and a LXXLL-containing coactivator peptide, Biochemistry (Mosc) 43 (2004) 4101–4110, http://dx.doi. org/10.1021/bi036056y. [37] N. Yoshimoto, Y. Sakamaki, M. Haeta, A. Kato, Y. Inaba, T. Itoh, et al., Butyl pocket formation in the vitamin D receptor strongly affects the agonistic or antagonistic behavior of ligands, J. Med. Chem. 55 (2012) 4373–4381, http:// dx.doi.org/10.1021/jm300230a. [38] I.K. Sibilska, M. Szybinski, R.R. Sicinski, L.A. Plum, H.F. DeLuca, Highly potent 2methylene analogs of 1a,25-dihydroxyvitamin D3: synthesis and biological evaluation, SI Vitam Workshop 136 (2013) 9–13, http://dx.doi.org/10.1016/j. jsbmb.2013.02.001. [39] G. Tocchini-Valentini, N. Rochel, J.-M. Wurtz, D. Moras, Crystal structures of the vitamin D nuclear receptor liganded with the vitamin D side chain analogues calcipotriol and seocalcitol, receptor agonists of clinical importance. insights into a structural basis for the switching of calcipotriol to a receptor antagonist by further side chain modification, J. Med. Chem. 47 (2004) 1956– 1961, http://dx.doi.org/10.1021/jm0310582. [40] Y. Bury, M. Herdick, M.R. Uskokovic, C. Carlberg, Gene regulatory potential of 1a,25-dihydroxyvitamin D3 analogues with two side chains, J. Cell. Biochem. 81 (2001) 179–190, http://dx.doi.org/10.1002/jcb.1082. [41] G. Tocchini-Valentini, N. Rochel, J.M. Wurtz, A. Mitschler, D. Moras, Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands, Proc. Natl. Acad. Sci. 98 (2001) 5491–5496, http://dx.doi.org/10.1073/ pnas.091018698. [42] L. Binderup, S. Latini, E. Binderup, C. Bretting, M. Calverley, K. Hansen, 20-EPIvitamin D3 analogues: a novel class of potent regulators of cell growth and immune responses, Biochem. Pharmacol. 42 (1991) 1569–1575, http://dx.doi. org/10.1016/0006-2952(91)90426-6. [43] S. Peleg, M. Sastry, E.D. Collins, J.E. Bishop, A.W. Norman, Distinct conformational changes induced by 20-epi analogues of 1,25DIhydroxyvitamin D Are associated with enhanced activation of the vitamin D receptor, J. Biol. Chem. 270 (1995) 10551–10558, http://dx.doi.org/10.1074/ jbc.270.18.10551. [44] W. Yang, L.P. Freedman, 20-epi analogues of 1,25-dihydroxyvitamin D3 are highly potent inducers of DRIP coactivator complex binding to the vitamin D3 receptor, J. Biol. Chem. 274 (1999) 16838–16845, http://dx.doi.org/10.1074/ jbc.274.24.16838.
229
[45] Y.-Y. Liu, E.D. Collins, A.W. Norman, S. Peleg, Differential interaction of 1alpha,25-dihydroxyvitamin D3 analogues and their 20-epi homologues with the vitamin D receptor, J. Biol. Chem. 272 (1997) 3336–3345, http://dx.doi.org/ 10.1074/jbc.272.6.3336. [46] S. Hourai, L.C. Rodrigues, P. Antony, B. Reina-San-Martin, F. Ciesielski, B.C. Magnier, et al., Structure-based design of a superagonist ligand for the vitamin D nuclear receptor, Chem. Biol. 15 (2008) 383–392, http://dx.doi.org/10.1016/ j.chembiol.2008.03.016. [47] P. Antony, R. Sigüeiro, T. Huet, Y. Sato, N. Ramalanjaona, L.C. Rodrigues, et al., Structure-function relationships and crystal structures of the vitamin D receptor bound 2alpha-methyl-(20S,23S)- and 2alpha-methyl-(20S,23R)epoxymethano-1alpha,25-dihydroxyvitamin D3, J. Med. Chem. 53 (2010) 1159–1171, http://dx.doi.org/10.1021/jm9014636. [48] W. Hakamata, Y. Sato, H. Okuda, S. Honzawa, N. Saito, S. Kishimoto, et al., (2S,20 R)-analogue of LG190178 is a major active isomer, Bioorg. Med. Chem. Lett. 18 (2008) 120–123, http://dx.doi.org/10.1016/j.bmcl.2007.11.007. [49] S. Kakuda, K. Okada, H. Eguchi, K. Takenouchi, W. Hakamata, M. Kurihara, et al., Structure of the ligand-binding domain of rat VDR in complex with the nonsecosteroidal vitamin D3 analogue YR301, Acta Crystallogr. Sect. F 64 (2008) 970–973, http://dx.doi.org/10.1107/S1744309108026754. [50] H. Kashiwagi, Y. Ono, K. Shimizu, T. Haneishi, S. Ito, S. Iijima, et al., Novel nonsecosteroidal vitamin D3 carboxylic acid analogs for osteoporosis, and SAR analysis, Bioorg. Med. Chem. 19 (2011) 4721–4729, http://dx.doi.org/10.1016/ j.bmc.2011.07.001. [51] R. Edgar, MUSCLE: multiple sequence alignment with high accuracy and high throughput, Nucleic Acids Res. 32 (2004) 1792–1797. [52] R. Edgar, MUSCLE: a multiple sequence alignment method with reduced time and space complexity, BMC Bioinformatics 5 (2004) 113. [53] D.L. Theobald, P.A. Steindel, Optimal simultaneous superpositioning of multiple structures with missing data, Bioinformatics 28 (2012) 1972–1979, http://dx.doi.org/10.1093/bioinformatics/bts243. [54] D.L. Theobald, D.S. Wuttke, Accurate structural correlations from maximum likelihood superpositions, PLoS Comput. Biol. 4 (2008) e43, http://dx.doi.org/ 10.1371/journal.pcbi.0040043. [55] J.M. Word, S.C. Lovell, J.S. Richardson, D.C. Richardson, Asparagine and glutamine: using hydrogen atom contacts in the choice of side-chain amide orientation, J. Mol. Biol. 285 (1999) 1735–1747, http://dx.doi.org/10.1006/ jmbi.1998.2401. [56] F.H. Allen, I.J. Bruno, Bond lengths in organic and metal-organic compounds revisited: X-H bond lengths from neutron diffraction data, Acta Crystallogr. Sect. B 66 (2010) 380–386, http://dx.doi.org/10.1107/S0108768110012048. [57] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, et al., AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility, J. Comput. Chem. 30 (2009) 2785–2791, http://dx.doi.org/ 10.1002/jcc.21256. [58] K. Shindo, G. Kumagai, M. Takano, D. Sawada, N. Saito, H. Saito, et al., New C15substituted active vitamin D3, Org. Lett. 13 (2011) 2852–2855, http://dx.doi. org/10.1021/ol200828s. [59] L. Verlinden, A. Verstuyf, G. Eelen, R. Bouillon, P. Ordonez-Moran, M.J. Larriba, et al., Synthesis, structure, and biological activity of des-side chain analogues of 1alpha,25-dihydroxyvitamin D3 with substituents at C18, ChemMedChem 6 (2011) 788–793, http://dx.doi.org/10.1002/cmdc.201100021. [60] H. Maehr, N. Rochel, H.J. Lee, N. Suh, M.R. Uskokovic, Diastereotopic and deuterium effects in gemini, J. Med. Chem. 56 (2013) 3878–3888, http://dx. doi.org/10.1021/jm400032t.
