Structural Basis for Ligand Activity in Vitamin D Receptor

C H A P T E R

11 Structural Basis for Ligand Activity in Vitamin D Receptor Anna Y. Belorusova, Natacha Rochel Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)/Institut National de Santé et de Recherche Médicale (INSERM) U964/Centre National de Recherche Scientifique (CNRS) UMR 7104/Université de Strasbourg, Illkirch, France

O U T L I N E Introduction189 1α,25-Dihydroxyvitamin D3 Recognition by Vitamin D Receptor191 Crystal Structure of Human Vitamin D Receptor Ligand-Binding Domain in Complex With 1α,25-Dihydroxyvitamin D3191 Rat and Zebrafish Vitamin D Receptor Ligand-Binding Domain–1α,25-Dihydroxyvitamin D3 Complexes 192 Insights Into the apo Vitamin D Receptor Ligand-Binding Domain Structure 192 Structure of the Full Human Retinoid X ReceptorVitamin D Receptor Complex 193 Natural Metabolites

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Other Vitamin D Receptor Natural Ligands 194 Crystal Structures of Vitamin D Receptor Ligand-Binding Domain With Lithocholic Acid 194 An Alternative Lithocholic Acid-Binding Site Revealed by the Zebrafish Vitamin D Receptor Ligand-Binding Domain–Lithocholic Acid Crystal Structure 194

INTRODUCTION The vitamin D receptor (VDR, NR1I1) is a member of the nuclear receptor (NR) superfamily that classically acts as a heterodimer with one of the three retinoid X receptor (RXR) isotypes (RXRα, NR2B1; RXRβ, NR2B2; and RXRγ, NR2B3) (see other chapters in this section). VDR binds with high affinity to its natural ligand 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3,

Vitamin D, Volume 1: Biochemistry, Physiology and Diagnostics, Fourth Edition http://dx.doi.org/10.1016/B978-0-12-809965-0.00011-2

Secosteroidal Analogs of 1α,25-Dihydroxyvitamin D3195 A-Ring Modified Analogs 196 Analogs With Modifications in C/D Rings 196 Side Chain Modifications 197 Structures of Vitamin D Receptor With Analogs That Induce Structural Rearrangements Gemini Analogs 22-Alkyl Derivatives Adamantyl-Containing Compounds

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Synthetic Mimics of 1α,25-Dihydroxyvitamin D3203 Derivatives of LG190178 203 Bis- and Tris-Aromatic Compounds 203 Compounds With p-Carborane Core 205 Conclusions and Perspectives

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Acknowledgments205 References206

calcitriol) (Fig. 11.1). VDR and its ligand play important roles in calcium metabolism, cell growth, differentiation, antiproliferation, apoptosis, and adaptive/innate immune responses [1–4]. VDR is a promiscuous NR, found in the prostate, ovary, breast, and the skin, and also in the brain, heart, pancreas, kidney, intestine, and the colon. Consequently, deregulation of VDR function may lead to severe diseases such as cancers, psoriasis, rickets, renal osteodystrophy, and autoimmunity disorders

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(multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases, type I diabetes) [5–8]. Various VDR ligands have been developed and some of them are used in clinical applications for the standard topical treatment of psoriasis, in secondary hyperparathyroidism or osteoporosis [9,10]. A new 1,25(OH)2D3 derivative, inecalcitol, is being developed for prostate cancers and chronic leukemia [11]. In addition, 1,25(OH)2D3 and its analogs have potential use in the treatment of neurodegenerative and autoimmune diseases [12]. VDR mediates the biological effects of its ligand by regulating the transcription of target genes through binding to specific DNA motifs of controlled genes. Recent genome-wide studies on VDR binding have revealed that only few dozens of vitamin D response elements are located within 10 kb of the transcription start site (TSS) of 1,25(OH)2D3 target genes [13–15], whereas thousands of additional VDR loci are located at distal sites over the entire genome [16,17]. In a ligand-dependent manner, the DNA-bound heterodimer recruits various coregulators of transcription: typically, in the absence of ligands or in the presence of antagonists, corepressors are recruited to the target genes, while agonist ligands induce a change in the structure of the NR that allows interaction with coactivators. Recruitment of coactivators results first in histone acetylation, which prepares target gene promoters through decondensation of the chromatin and, further, provides a link to the basal transcriptional machinery. Numerous proteins with large structural and functional diversities have been identified as VDR coregulators [18]. VDR shares the main structural characteristics of NRs: it consists of a highly conserved DNA-binding domain (DBD), a ligand-binding domain (LBD), and a hinge region

FIGURE 11.1  Chemical structure of 1α,25-dihydroxyvitamin D3.

connecting the DBD to the LBD (Fig. 11. 2). Other NRs also contain a variable N-terminal domain (NTD) displaying a ligandindependent activation function; however, the NTD domain of VDR is very short (23 AA in human VDR (hVDR)) and its function is not completely understood. The DBD contains two zinc fingers, each comprising a zinc atom coordinated by four cysteine residues in a tetrahedral arrangement [19]. The LBD of the NRs harbors a ligand-dependent activation function, or AF-2, a major interface for homo- and heterodimerization and an interface for coactivators as well as corepressors. Since the first crystal structures of NR LBDs described in 1995, the crystal structures of most of the 48 human NRs have now been solved. X-ray crystallography has been the most powerful technique for unraveling NR–LBD structures [20]. Despite giving only static snapshots of NRs, X-ray crystallography has provided invaluable mechanistic insights into the flexibility and adaptability of NRs. The prototypical LBD consists of 10–12 helices and 1 to 2 short β turns arranged in a three-layer helical sandwich. Functionally, ligands act as switches for coregulator binding by triggering major movements of the C-terminal helix (H12) also referred to as the activating domain of the AF-2 function. Different conformational changes of H12 have been observed on the binding of diverse ligands. In an agonist conformation with an active LBD, H12 is approximately perpendicular to H3 and H5, and helix H12 seals the binding cavity of classical endocrine NR-endogenous ligand complexes and stabilizes ligand binding by contributing to the hydrophobic environment, making, in some cases, additional contacts with the ligand. The agonist conformation of H12 allows interactions with coactivators from the p160 family of steroid receptor coactivators (SRCs) that remodel chromatin [21], or of the vitamin D receptor interacting protein (DRIP)/thyroid hormone receptor associated protein (TRAP)/mediator complexes that interact with the basal transcriptional machinery and help recruit the RNA polymerase to the TSS [22]. Only one agonist active conformation is observed for all NRs agonist complexes that permits coactivator binding while several other conformations have been shown allowing corepressor binding [20]. In the absence of ligand, helix H12 can adopt a conformation leading to an autorepressed conformation of the NR, which blocks coactivator interaction, or to a conformation where H12 is freely moving in solution. To bind to corepressors, NRs differentially associate with amino acid sequences that resemble the coactivator motifs (LxxI/HIxxxI/L) on NRs docking surfaces that do overlap with coactivators binding surfaces but are distinct [23]. The first crystal structure of human VDR LBD, LBD1,25(OH)2D3 [24], revealed a similar fold as the other NRs. Because of the high pharmaceutical potential of VDR ligands,

FIGURE 11.2  Modular organization of vitamin D receptor. AF-2, activation function-2; DBD, DNA-binding domain; LBD, ligand-binding domain.

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1α,25-DIHYDROXYVITAMIN D3 RECOGNITION BY VITAMIN D RECEPTOR

the mechanism of the receptor functioning has been intensively investigated, and structural details of the VDR signaling are now available for various VDR ligands (reviewed in Refs. [18,25–27]). These crystal structures explain most features of VDR ligand binding, superagonism, mutual adaptability of the ligands and of the pocket, provide clues for the antagonism mechanism, and indicate the presence of an alternative binding site in the VDR LBD. Here, we will discuss the structural information on recognition of the natural ligands and some of the synthetic analogs by VDR. We will focus primarily on structural data obtained by X-ray crystallography, but will also highlight important new insights into the functional potential of VDR ligands provided by other methods.

1α,25-DIHYDROXYVITAMIN D3 RECOGNITION BY VITAMIN D RECEPTOR Crystal Structure of Human Vitamin D Receptor Ligand-Binding Domain in Complex With 1α,25Dihydroxyvitamin D3 Detailed information on the molecular mechanism of action of 1,25(OH)2D3 has been initially obtained by the elucidation of the crystal structure of its complex with the human VDR LBD (protein data bank identifier (PDB ID): 1DB1) [24] that has revealed, as previously mentioned, a conformation similar to other agonist-bound NR LBDs crystal structures. The general fold of the VDR LBD consists of a three-layered α-helical sandwich composed of 12 helices (H1 to H12) and a three-stranded β-sheet (Fig. 11.3A). The ligand-binding pocket (LBP) is located on the bottom of the LBD and is surrounded by helices H2, H3, H5, H6, H7, H10, and H12. The residues of each of β-sheet strands also form contacts with a ligand. For the crystallization of the hVDR1,25(OH)2D3 complex, a truncated form of the hVDR LBD was used: 50 amino acid residues from a large insertion domain at

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the N-terminal part of the LBD connecting helices H1 to H3 was deleted. This region is characterized by poor sequence conservation between VDR family members and predicted disordered state. No clear biological function has been assigned to the insertion region [28], although this has never been examined in vivo. Helix 12 is in the agonist position and stabilized by two interactions with the ligand. Helix 12 is also stabilized by several hydrophobic contacts with residues of H3, H5, and H11 and two polar interactions with residues of H3 and H4. Some of these residues contact the ligand, thus indicating an additional indirect ligand control of the position of helix H12. In the hVDR structure, a strong crystalline contact is observed between helix H3n and helices H3, H4, and H12 of a symmetrically related molecule, with H3n mimicking coactivator peptide contacts. In this crystal structure, the A-ring of 1,25(OH)2D3 in its VDR LBP, adopts a chair B conformation with the 19-methylene “up” and the 1α-OH and 3β-OH groups in equatorial and axial orientations, respectively. The aliphatic chain at position 17 of the D-ring adopts an extended conformation (Fig. 11.3B). The conformation of the ligand is maintained through the interactions of two types: first, with the hydrophobic residues lining the LBP and, second, through the hydrogen bonds formed between polar residues and three hydroxyl groups of the ligand. The main anchoring interaction points are: for the 1-OH group with Ser237 (H3) and Arg274 (H5), for the 3-OH group with Ser278 (H5) and Tyr143 (loop H1–H2), and for the 25-OH group with His305 (loop H6–H7) and His397 (H11) (Fig. 11.3C). The triene connecting the A- and C-ring is tightly sandwiched in a hydrophobic channel between Ser275 (loop H5-β) and Trp286 (β1) on one side and Leu233 (H3) on the other side. The C ring contacts Trp286 while the C18 methyl group points toward Val234 (H3). The side chain is surrounded by hydrophobic residues (Fig. 11.3C). The ligand-binding cavity of hVDR is large (697 Å3) with the 1,25(OH)2D3 occupying only 56% of its volume. A channel of water molecules near the position 2 of the A-ring creates an extra cavity; additional space around the aliphatic chain is also observed.

FIGURE 11.3 1α,25-dihydroxyvitamin D3 recognition by vitamin D receptor (VDR). (A) Overall structure of the VDR ligand-binding domain (LBD). The VDR LBD bound to 1,25(OH)2D3 is composed of 13 helices (H1,2, 2n,3–12) (protein data bank identifier: 1DB1). (B) Conformation of 1,25(OH)2D3 in the ligand-binding pocket (LBP). (C) Binding mode of 1,25(OH)2D3 in the VDR LBP. The volume of the LBP is shown as a grey surface. Specific H-bonds anchoring the three hydroxyl-groups of the 1,25(OH)2D3 are shown as dash lines.

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Rat and Zebrafish Vitamin D Receptor LigandBinding Domain–1α,25-Dihydroxyvitamin D3 Complexes VDR LBDs from two other species, Rattus norvegicus, r [29] and Danio rerio, z [30,31] have been also crystallized. In case of rat VDR (rVDR) LBD (PDB ID: 1RK3), the same truncation of the large insertion region connecting helices H1 to H3 as for hVDR was applied. For the zebrafish VDR (zVDR) LBD (PDB ID: 2HC4), a wild-type protein was used; however, the insertion region was not visible in the electron density maps indicating its high flexibility resulting in a static disorder. The differences observed between the structures of h, r, and z VDR LBDs are small and primarily involve the loops. The root mean square deviations of the superimposed structures rVDR and hVDR LBDs and of the zVDR and hVDR LBDs, are of 0.53 Å and 0.72 Å over 236 main chain atoms, respectively. The LBP is conserved both in sequence and structure, and the ligand conformation and interactions observed in all crystal structures are very similar. Species-specific difference will not be discussed. In contrast to hVDR LBD cocrystallized with ligand in the absence of coactivator peptide, rVDR and the zVDR LBDs were crystallized in presence of additional coactivator peptides that were added to stabilize the complexes and promote crystal growth. For the rVDR complexes the second LXXLL motif of DRIP205/Med1 has been used [29] and for the zVDR the second NR box of SRC-1 or SRC-2 [30,32]. The LXXLL peptides observed in the crystal structures of z and rVDR are bound to a groove formed by helices H3, H5, and H12 of the LBD (Fig. 11.4A). For the LXXLL motif of DRIP205/Med1 (625–636 KNHPMLMNLLKD) this interaction buries about 507 Å2 of the receptor’s surface. The side chains of Leu630, 633, and 634 of a peptide are buried within the pocket and surrounded by hydrophobic residues, while Met631 and Asn632 are oriented outward from the solvent (Fig. 11.4B). Coactivator peptide is additionally

locked through the hydrogen bonds between Glu416 and H12 (hGlu420) with the backbone amide nitrogen of Leu630 and Leu633. At the other end, Lys242 from H3 (hLys246) forms a hydrogen bond to the main chain oxygen of Leu634. The same binding is observed for the zVDR LBD-peptide–ligand complex. The interactions of coactivator peptides to VDR are similar to those described for other NRs [33–35].

Insights Into the apo Vitamin D Receptor LigandBinding Domain Structure No apo VDR crystal structure is currently available. However, other structural methods as nuclear magnetic resonance (NMR) and hydrogen-deuterium exchange coupled to mass spectrometry (HDX–MS) have been used to get structural insight into the apo VDR structure [36,37]. Both methods have suggested that the process of ligand binding is a dynamic process, and have provided particularly important insights into the disordered state of the region around helix 12 in the apo form. HDX–MS analysis has been used to probe the conformational dynamics on ligand binding to VDR. Within the apo VDR LBD, the entire C-terminal part has been shown to be very dynamic with 80% of amide hydrogen exchange [36]. The region forming the LBP also showed high exchange rate, while the central layer of the α-helical sandwich and helix H10 appeared protected. As expected, binding of 1,25(OH)2D3 or the synthetic analog ED-71 led to a significant protection from hydrogen amide exchange. Impaired protection from the amide hydrogen exchange was observed for the VDR complex with alfacalcidol (a precursor of 1,25(OH)2D3 lacking a 25-OH group). In case of “weak” ligands helices H7 and H12 stayed significantly more mobile, explaining partial agonism as a result of formation of a less stable complex with VDR. Another type of experiment monitoring the structural changes within the LBD on ligand binding in solution is NMR.

FIGURE 11.4  Binding mode of the vitamin D receptor interacting protein 205 coactivator (CoA) peptide LXXLL (protein data bank identifier: 1RK3). (A) Coactivator peptide binding surface (mesh representation) formed by helices H3, H5, and H12 of the ligand-binding domain. (B) Details of interaction between vitamin D receptor and coactivator peptide. Amino acids are colored based on the hydrophobicity scale as defined by Eisenberg D, Schwarz E, Komarony M, Wall RJ. Mol Biol 1984;179:125–42. II.  MECHANISM OF ACTION

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Natural Metabolites

Singarapu et al. reported assigned NMR chemical shifts of rVDR–LBD in presence of either one of two agonists used (1,25(OH)2D3 and 2MD) or an antagonist (OU-72) [37]. The comparison of apo- and holoforms of VDR LBD revealed significant conformational changes on the ligand binding for the exception of OU-72 complex where the AF-2 function remained structurally dynamic. The fact that the antagonist did not stabilize the active VDR conformation was further supported by comparison of ternary complexes between rVDR LBD, ligands, and LXXLL motif of DRIP205: binding of this peptide to rVDR LBD complexed with 1,25(OH)2D3 or 2MD resulted in significant chemical shift changes, whereas the rVDR LBD complex with OU-72 failed to exhibit chemical shift changes in the same conditions.

Structure of the Full Human Retinoid X ReceptorVitamin D Receptor Complex The structure of the full-length hVDR complex with its heterodimeric partner, RXR, ligands, and DNA has been revealed by several structural methods in solution, small angle X-ray scattering [38], cryo-electron microscopy [39], and HDX–MS [40]. Comparison of the LBD and DBD domains of this multidomain structure with those of previously solved individual domain structures showed structural conservation. The structure of VDR–RXR DNA-bound heterodimer revealed a relative orientation of the LBDs and DBDs: the LBDs are situated in an asymmetric manner on the 5’-end of the response element, shifted away from the center of the two half-sites (Fig. 11.5) [38,39]. This structure reveals that the hinge region stabilizes the whole complex, thus facilitating the positioning of the LBD and the surface to be accessible by the coregulators. However, even with the relatively separated positioning, there is an evidence of long-range allosteric connections between the domains [40]. These studies indicated cooperative effects between the VDR DBD and LBD to fine tune transcriptional regulation by the ligands and the DNA. On 1,25(OH)2D3 binding to full VDR–RXR, the HDX profile was very similar to the binding to VDR LBD alone with a stabilization of VDR H12. Interestingly, an increase of solvent exchange in the DBD of VDR was also observed on ligand binding, indicating that the

ligand impact on the DBD conformation. On ligand binding, a stabilization of the heterodimer interface was also observed. The HDX profile of 1,25(OH)2D3 binding in the presence of RXR ligand was similar to that of 1,25(OH)2D3 alone. The effect of DNA binding was also analyzed that indicate that DNA binding modulates both DBD–DBD and LBD–LBD interactions and influences coactivator recognition and binding.

NATURAL METABOLITES 1,25(OH)2D3 is subjected to enzymatic inactivation via two major pathways leading to C24 and C23 hydroxylated metabolites in various tissues [4]. The 24-hydroxylation is generally considered as the first step in the degradation pathway of 1,25(OH)2D3 [41]. However, some data have demonstrated that both 1,25(OH)2D3 and the 24-hydroxylated metabolite 1α,24R,25-(OH)3D3 induce gene transcription [42]. Thus, not only 1,25(OH)2D3 but also the presumed 24-hydroxylated “degradation” products have the potential to stimulate differentiation of human osteoblasts through VDR activation. In addition, other natural metabolite, 1β,25(OH)2D3, acts in bone as an agonist and directly stimulates mineralization in a nuclear VDR-dependent way [42,43]. Another metabolite modified at the A-ring is 1,25(OH)23epi-D3, which has been shown to retain significant biological activity compared to the natural hormone [44,45]. 1,25(OH)23epi-D3 was initially identified in cultured human neonatal keratinocytes. 1,25(OH)2-3epi-D3 has been shown to exhibit tissue-specific activities comparable to 1,25(OH)2D3 [44,45]. The structural analysis showed that VDR displays specific adaptation of the LBP (Fig. 11.6) [44]. Although the two diastereomers differ only in the position of the C3–OH group, the 1,25(OH)2-3epi-D3 takes a slightly more compact conformation in the LBP. While the C1–OH and C25–OH display the canonical hydrogen bonds, the 3-epi-OH of 1,25(OH)2-3epi-D3 interacts through hydrogen bonding only with Tyr143 instead of interacting with both Tyr143 and Ser278 (Fig. 11.6). A significant feature of the 1,25(OH)2-3epi-D3 is the compensation of the loss of interaction with Ser278 by a water-mediated hydrogen bond.

FIGURE 11.5  Solution structures of the vitamin D receptor–retinoid X receptor alpha (VDR–RXRα) heterodimer bound to canonical DR3 response element. cryo-EM, cryo-electron microscopy; DBD, DNA-binding domain; LBD, ligand-binding domain; SAXS, small angle X-ray scattering. II.  MECHANISM OF ACTION

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W W

C1

C25

W

Tyr147

C3 Tyr143 Ser278

Glu277

FIGURE 11.6  Overlay of human vitamin D receptor–ligand-binding domain complexes with 1,25(OH)2-3epi-D3 (protein data bank identifier (PDB ID): 3A78) and 1α,25-dihydroxyvitamin D3 (PDB ID: 1DB1; light grey).

OTHER VITAMIN D RECEPTOR NATURAL LIGANDS The evolution proximity of VDR to pregnane X receptor (PXR) and farnesoid X receptor (FXR), NRs involved in detoxification and bile acid metabolism, supported an idea that these receptors might recognize similar ligands. Indeed, it is now well established that, together with PXR and FXR, VDR is weakly activated by the lithocholic acid (LCA), which was identified as a selective low-potency agonist for VDR among several secondary bile acids over a decade ago [46,47] (see Chapter 88 (vol. 2 of this book) and others in Section IX). Interestingly, LCA activates VDR in target organs such as the intestine and the kidney without causing hypercalcemia [48]. However, due to its low potency, concentrations that might induce hypercalcemica in vivo were never tested. Several nutritional lipophilic compounds directly compete with tritiated 1,25(OH)2D3 for VDR binding and therefore have been identified as potential VDR ligands. The first class of such molecules is represented by curcumin, an epigenetically active polyphenol that is included in the composition of curry [49,50]. The second class of nutritional lipids that has been evaluated as potential ligands for VDR are the polyunsaturated fatty acids, including eicosapentaenoic acid, docosahexaenoic acid, linoleic acid, and arachidonic acid [49]. All these compounds have been shown to bind VDR with low affinity.

Crystal Structures of Vitamin D Receptor LigandBinding Domain With Lithocholic Acid Secondary bile acids such as LCA are converted from bile acids by bacteria in the intestine. When present in high concentrations, they increase cytotoxicity as cancer promoters and carcinogen for colon cancer [51]. By binding to LCA and its derivatives, VDR acts as a secondary bile acid sensor in addition to its role as an endocrine receptor. As the scaffold of LCA is significantly different from that of 1,25(OH)2D3, it was important to obtain structural information on the mechanism of selective activation of VDR by LCA. In 2013, the crystal structures of the rVDR LBD with LCA and its derivatives were described [52]. The overall structures are similar to that

of the agonist conformation of the 1,25(OH)2D3 complexes. The coactivator peptide Med1 is similarly bound to VDR–LCA complexes. Interestingly, LCA and its derivatives adopt an orientation opposite to 1,25(OH)2D3 with their A-ring facing H12, the 24-carboxyl group pointing toward the β-turns, and the β-region of the steroid toward the H6-7/11 region. LCA forms similar H-bond networks and hydrophobic interactions as 1,25(OH)2D3. Only two direct H-bonds are formed between rTyr143 (H1) and rSer274 (H5) and the 24-carboxyl group of the LCA. The other anchoring residues of 1,25(OH)2D3, rSer233 (H3), and rArg270 (H5), interact with the 24-carboxyl group of LCA through water molecules. Residues rHis301 (loop 6–7) and rHis393 (H11) interact with the hydroxyl group on the A-ring of LCA (Fig. 11.7A). The shorter length of LCA over 1,25(OH)2D3 (14 Å vs. 15.1 Å) leads to weakening of interactions with the C-terminal part of VDR. That, and water-mediated anchoring hydrogen bonds, explain the weaker agonistic properties of this ligand. LCA and its 3-substituents derivatives (3-keto, acetate, and propionate) that differ in their substituents at the C3 position of the A-ring, form similar interactions with rVDR LBD. Although through water-mediated H-bonds for the LCA and 3-ketoLCA and direct H-bonds for the two other derivatives, with rHis301 (loop 6–7) and rHis393 (H11) [52]. In the complex with LCA acetate, the oxygen atom of the acetyl group directly forms a hydrogen bond with rHis301. In the complex with the LCA propionate, the oxygen atom of the propionyl group forms two directed hydrogen bonds with the nitrogen atoms of the imidazole rings of rHis301 and rHis393. In addition, the alkyl parts of the LCA acetate and LCA propionate interact with residues of H11 (rTyr397) and of H12 (rLeu410, rVal414, and rPhe418). These additional interactions notably for the longest alkyl part of the LCA propionate and the direct H-bonds explain the increased agonist activity of this ligand [48].

An Alternative Lithocholic Acid-Binding Site Revealed by the Zebrafish Vitamin D Receptor Ligand-Binding Domain–Lithocholic Acid Crystal Structure LCA was also crystallized in complex with zVDR LBD [53]. In this structure, in addition to the first ligand molecule

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(A)

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(B)

(C) W zGln267 zAsp181 zTyr264 zLeu443

zSer263

zPhe185

zArg184

zAsp260

FIGURE 11.7  Crystal structures of vitamin D receptor–ligand-binding domain (VDR–LBD) complexes with lithocholic acid (LCA). (A) Binding mode of LCA in the canonical rat VDR (rVDR) ligand-binding pocket (LBP) (protein data bank identifier (PDB ID): 3W5P). (B) Overall structure of zebrafish VDR (zVDR)–LBD complex with LCA showing the location of the alternative binding site within LBD (PDB ID: 4Q0A). (C) Details of interactions in the second LCA binding site.

occupying the classical LBP similarly as in the rVDR structure, a second LCA molecule was observed that was not fully buried but was rather weakly attached to the surface of VDR close to the loop 1–3 and H3 (Fig. 11.7B). This second LCA molecule is primarily stabilized by nonpolar interactions with zAsp181, zArg184, zPhe185 (H2), zAsp260, zSer263, zTyr264, zGln267 (H3), and zLeu443 (H12). The hydroxyl group on the A-ring forms two direct H-bonds with zSer263 (H3) and zGln267 (H3) (Fig. 11.7C). The side chain is more loosely positioned, and its 24-carboxyl group interacts through a water molecule with zAsp181 (H2′) and zLys268 (H3). Biophysical characterization confirmed a multiple LCA binding by the VDR LBD in solution, while the structural analysis and molecular dynamics simulation suggest that the binding of an additional ligand has a stabilizing effect on active receptor conformation, and, importantly, on a bound coactivator peptide. Binding residues forming both the canonical LBP and the second binding site are conserved between VDR family members, suggesting a common mechanism of their activation by LCA. This crystal structure was the first one that revealed two ligands bound to VDR. At the same time, other NRs have

been shown to be able to bind two ligands: estrogen receptor β (ERβ) [54], thyroid receptor (TR) [55], androgen receptor (AR) [56], and peroxisome proliferator-activated receptors alpha [57] and gamma [58]. The rising number of reports on multiple ligand binding events clearly indicates that ligand recognition by NRs is far more complex than had initially been postulated. It is clear that the cooperativity between ligand binding sites needs to be further investigated to better understand the mechanism of specific actions of diverse NR modulators.

SECOSTEROIDAL ANALOGS OF 1α,25DIHYDROXYVITAMIN D3 1,25(OH)2D3 is a highly flexible molecule and a very large number of analogs have been synthesized by industry and academia in an attempt to provide beneficial therapeutic compounds with low calcemic activity (see also additional chapters in Section IX). The VDR LBP that is only 56% occupied by 1,25(OH)2D3 provides additional space for fitting modified moieties of the hormone, particularly at the position of the A-ring and aliphatic chain. Chemical modifications of

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every portion of the 1,25(OH)2D3 molecule, the A-ring, the seco-B ring, the central C/D rings, and the 17β-aliphatic side chain, have been reported. The most interesting analogs of 1,25(OH)2D3 result from a combination of several of these modifications. Most of these analogs are based on a trial and error approach. However, the crystal structures of VDR–LBD have provided significant information for understanding structure–function relationships of 1,25(OH)2D3 and synthetic analogs and for the design of structure-based analogs. Most of the analogs are VDR agonists or partial agonists, and few of them are antagonists. For hundreds of 1,25(OH)2D3 analogs, the crystal structures of the VDR LBD complexes have been reported. All compounds are anchored by the same residues in the LBP, with the hydroxyls of the A-ring and of the side chain located in identical positions and forming the same hydrogen bonds. Mutual adaptability and flexibility of the ligand and the amino acids lining the LBP have been observed in several crystal structures. Several modifications such as 19nor, 16ene, 23yne, or 20epi were shown to lower the calcemic effects and to enhance their antiproliferative properties compared to 1,25(OH)2D3. The mechanisms of action of these analogs can be explained by a combination of effects, differences in pharmacokinetics with a lower affinity for vitamin D-binding protein (DBP) resulting in differences in half-lives, differences in the intracellular metabolism of the compounds, and stabilization of the interactions within the VDR LBP through subtle differences in contacts. Current understanding of the relationship between selected stereochemical modifications of key structural regions on A-ring, CD-ring, and side chain of the 1,25(OH)2D3 and their effects on biological potency and selectivity will next be described.

A-Ring Modified Analogs Several water molecules forming a channel near the position 2 of the A-ring can be observed in the different crystal structures of VDR in complex with 1,25(OH)2D3 (Fig. 11.8A). The space surrounding the water molecules channel is considered as an extra cavity that can accommodate ligands with additional moieties at position 2. Indeed, the fourfold increase in binding affinity of the 2α-methyl analogs is in agreement with this observation [59]. Several crystal structures of the VDR LBD bound to selected C2α-substituted analogs that fill this water channel have been described [29,60]. In case of C2α-substituted analogs bearing methyl, propyl, propoxy, hydroxypropyl, and hydroxypropoxy groups at C2 position, the water molecules are displaced or their location is affected (Fig. 11.8B). The exception here is a methyl substituent, which is small enough not to affect the water molecules network while providing additional van der Waals contacts that explain the higher binding affinity of this analog [60] (Fig. 11.8C). The affinities of the C2-substituted analogs for VDR result from the balance between the loss of water-mediated H-bonds, additional van der Waals contacts, and entropic effect. In a series of analogs bearing various groups at 2β-position, the compound with a 3-hydroxypropoxy showed the strongest potential [61]. The 1α,25-dihydroxy-2β-(3-hydroxypropoxy)

vitamin D3 or edelcalcitol has been shown to be very effective in osteoporosis treatment [62]. Its binding mode to VDR has been investigated by X-ray crystallography that has revealed that the terminal hydroxyl group forms H-bonds with Arg278 and Thr142 while the alkyl chain of the 3-hydroxypropoxy group forms a CH–π interaction with Tyr143, stabilizing the eldecalcitol–VDR complex [62]. Several C2-substitutients bearing additional modifications act as VDR superagonists: 2MD which presents a 19nor configuration, a methylene moiety on C2α, and a 20-epi configuration on C20 [29]; 20S analog bearing 2β-hydroxyethoxy group together with other modifications (16ene–22thia–19nor and C26 and C27 methylation) [63] and others (reviewed in Ref. [64]). The 2β-hydroxyethoxy group in the 20S complex shows stabilizing interactions via hydrogen bonds between the terminal OH moiety of the 2-substituent and both rArg270 (hArg274) and a water molecule (Fig. 11.8D). These interactions may stabilize the active receptor conformation and explain the increased potency of the ligand. In addition, potency in cell culture is also due to a ligand’s absence of interaction with DBP in the serum. Another type of the A-ring modification commonly used for the synthesis of 1,25(OH)2D3 analogs is removal of the C19 methylene group. The 19-nor analogs have the advantage of enhanced chemical stability due to the absence of the triene function. Interestingly, binding of 19-nor–1,25(OH)2D3 to the VDR has been shown to be only 30% of that of 1,25(OH)2D3 while the effects on HL60 differentiation were similar and calcemic effects were reduced [65,66]. Although numerous 19-nor–1,25(OH)2D3 analogs have been synthesized and their crystal structures solved (reviewed in Ref. [27]), there is no structural comparison of agonists differing only by the presence or absence of the C19 methylene group.

Analogs With Modifications in C/D Rings Analogs with modified CD-region comprise the sixmembered C-ring analogs (lacking the D-ring) [67,68], fivemembered D-ring analogs (lacking the C-ring) [69,70], and six-membered D-ring analogs (lacking the C-ring and possessing an enlarged D-ring) [71,72]. Biological characterization of C/D-ring 1,25(OH)2D3 modified analogs have demonstrated that full C/D-rings are not required for the biological activity of the compound as C/D-ring replacements in many cases retain full or modulatory 1,25(OH)2D3 VDR mediatedactivities. The D-ring analogs (CD578, WU515, and WY1113 in which the C-ring has been deleted) have a significantly more potent prodifferentiating action on human SW480-ADH colon cancer cells, a stronger transactivating potency and a stronger induction of the interaction between the VDR and some coactivators [73]. These ligands are protected from CYP24 degradation [73]. The only structural data for such compounds are the crystal structure of CD578 analog in complex with zVDR LBD [73] (Fig. 11.9A). Some adaptations of residues within the VDR LBP occur due to the absence of the C-ring, such as the indole group of zTrp314 shifted by 0.6 Å closer to the ligand to fill the space created by the absence of the C ring. Some differential contacts with zLeu258 and zLeu341 are observed. The CD578

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(A)

(B) hTyr236

hTyr236

hSer237

hSer237 W

hArg274

W

C2

W

hArg274 W

W

C2

hTyr143

hTyr143

hSer278

hSer278

(C)

(D) hTyr236

hTyr236

hSer237

hSer237 W

hArg274

hArg274

W

W C2

W

C2

hTyr143

hTyr143

hSer278

hSer278

FIGURE 11.8  Effect of C2-substituted analogs on a water channel formation. (A) human vitamin D receptor ligand binding domain (hVDR LBD) complex with 1α,25-dihydroxyvitamin D3 (protein data bank identifier (PDB ID): 1DB1). (B) hVDR LBD complex with C2α-propyl analog (PDB ID: 2HAM). (C) hVDR LBD complex with C2α-methyl analog (PDB ID: 2HB8). (D) rat VDR LBD complex with the 20S analog bearing a 2β-hydroxyethoxy group (PDB ID: 2ZLA).

differs also by fluorine atoms in the side chain. These fluorine atoms form interactions with zLeu440, zVal444, and zPhe448 and thus stabilize the agonist conformation of the complex. As a consequence the coactivator SRC-1 peptide makes additional interactions compared to zVDR–1,25(OH)2D3 complex. These structural data provide an explanation for the increased prodifferentiating action of these analogs not only by protecting the analogs from metabolic degradation but also by increasing the stability of the agonist conformation of VDR. Few C/D-ring 1,25(OH)2D3 modified analogs exhibit an additional branched group or a different positioning of the side chain as, for example, analogs bearing a side chain at C12 instead of at C17 [74] or C18 substituted analogs in which side chains homologous to that of the natural hormone are linked to C18 through a C–C bond and that have no substituents on C17 (des-side chain analog) [75]. The crystal structure of one of these compounds in complex with VDR LBD revealed that the 7-membered side chain introduced at position C18

adopted the same orientation in the LBP as the side chain of 1,25(OH)2D3 with maintenance of the hydrogen bonds that form the anchoring points of the ligand (Fig. 11.9B). Despite the fact that the analog fitted well in the VDR binding pocket, it displayed a weak binding to VDR.

Side Chain Modifications Large number of 1,25(OH)2D3 analogs with a variety of side chain modifications have been synthetized. Such modifications provide a diversity of functional ligand activities ranging from superagonist to antagonist. The crystal structures of some of such analogs in complex with VDR revealed important information about their mechanism of action. As a general rule, a full side chain together with a 25-OH group is required for maximal ligand activity. For various modifications the orientation of the side chain within the LBP also has a significant influence on biological activity. For example, for active

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FIGURE 11.9  Crystal structures of vitamin D receptor–ligand-binding domain (VDR–LBD) complexes with CD-ring modified analogs. (A) Overlay of VDR–LBD complexes with the D-ring analog CD578 (protein data bank identifier (PDB ID): 3DR1) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) (PDB ID: 1DB1; light grey). Specific for CD578 hydrophobic contacts are shown at 4.5 Å cutoff. (B) Overlay of VDR–LBD complexes with C-18 substituted analog (PDB ID: 3P8X) and 1,25(OH)2D3 (PDB ID: 1DB1; light grey).

VDR agonists numerous novel or strengthened interactions are observed, notably with the C-terminal end of the receptor, explaining elevated properties of these analogs. Based on the knowledge of the VDR crystal structures, new side chain analogs with improved activity have been designed. For some of them, the structural analysis will now be discussed. One of the best studied classes of 1,25(OH)2D3 side chain modifications are 20-epi VDR superagonists that have an inverted stereochemistry at position C20 in the flexible aliphatic chain. 20-epi compounds induce VDR-mediated transcription at concentrations of at least 100-fold lower than the natural ligand and provoke antiproliferative activity several orders of magnitude higher than 1,25(OH)2D3 [76–78]. These analogs do not bind DBP, which accounts for their 2-log increased potency in cell culture. Biochemical data and cellular assays suggested that the enhanced transactivation ability of 20-epi analogs correlated with the ability of these compounds to promote coactivator interaction [79]. The crystal structures of the hVDR LBD in complex with MC1288 and KH1060 have shown that, contrary to a belief that synthetic analogs would induce a different active conformation, the overall organization

and especially the position of helix H12 are strictly maintained in all VDR-ligand complexes [80]. However, subtle differences are observed with tighter interactions of the ligand with H12 that stabilize the active conformation and coactivator interactions. The specific interactions observed in the ligand-protein complexes with MC1288 and KH1060 involve the hydrophobic contacts of the 17β-aliphatic chains. For the MC1288, the main difference is the positioning of the methyl group C21, which results in different contacts with Val300, Leu230, and His397 (Fig. 11.10A). In the case of KH1060, the major observed differences are tighter and more numerous ligand–protein contacts. The methyl groups C26a and C27a, specific to KH1060, form additional contacts with H3, loop 6–7, H11, and H12 (Fig. 11.10B). Additional interactions through a different conformation of the side chain and additional methyl groups of 20-epi compounds have been shown to stabilize the active conformation of VDR, therefore, explaining the superagonistic nature of KH1060. A longer half-life of VDR-superagonist complexes is responsible for a more potent complex (acting at lower ligand concentration) of the receptor with coactivator and subsequent transcriptional activation. As was observed from the crystal structures of VDR complexes, the aliphatic side chain of ligand is not particularly constrained in the LBP, allowing alternative conformations of the side chain for the 1,25(OH)2D3 and the 20-epi analogs as described previously. Superimposition of the side chain conformations of the VDR complexes with KH1060 and 1,25(OH)2D3 shows that the two lateral side chains exactly coincide, except at the C17 and C23 positions, where the two carbon orientations form a virtual heterocycle. Based on this structural information, analogs with an oxolane group incorporated into the side chain were designed to minimize the entropic loss and to maximize the number of protein–ligand contacts [81]. In these analogs, the oxolane ring is positioned at position C20 of 1,25(OH)2D3. The crystal structures of those complexes, notably with stereoisomers, AMCR277A and AMCR277B, revealed that some specific contacts of the aliphatic side chains are made with the VDR LBP with Val300, His305, His397, and Val418, and a van der Waals contact of O21 located in the oxolane group with Val300 (Fig. 11.10C). Biological assays have shown that the oxolane containing analogs were more active than 1,25(OH)2D3 [81,82]. The superagonistic activity of these analogs can be explained by a combination of enthalpic effects (additional and tighter intermolecular contacts due to the higher fraction of LBP being occupied) and entropic effects (energetically favorable preformed conformations), resulting in an overall gain both in binding energy and kinetics. All these factors contribute to a better specificity of the ligand for VDR. Superagonist compounds AMCR277A and AMCR277B were further improved by introduction of a 2α-methyl group on the A-ring [82] that significantly improved the affinity to the receptor. Following a similar strategy, analogs that incorporated an aromatic furan in the side chains have been developed [83]. These analogs exhibit significant prodifferentiation effects and transactivation potency. The crystal structure of one of the furan side chains analogs in complex with hVDR LBD revealed that the aromatic furan group is less tightly bound

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SECOSTEROIDAL ANALOGS OF 1α,25-DIHYDROXYVITAMIN D3

199

FIGURE 11.10  Crystal structures of vitamin D receptor–ligand-binding domain (VDR–LBD) complexes with analogs bearing side chain modifications. Compounds are shown in overlay with 1,25(OH)2D3 (protein data bank identifier (PDB ID): 1DB1; light grey). Specific hydrophobic interactions are presented as grey dash lines, hydrogen bonds as red dash lines. (A) human VDR (hVDR) LBD complex with 20-epi analog MC1288 (PDB ID: 1IE9). (B) hVDR LBD complex with 20-epi analog KH1060 (PDB ID: 1IE8). (C) hVDR LBD complex with an oxolane-containing superagonist AMCR277 A (PDB ID: 3CS6). (D) zebrafish VDR (zVDR) LBD complex with a locked side chain compound (PDB ID: 2HBH). (E) zVDR LBD complex with an o-carborane compound (PDB ID: 5E7V).

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than the oxolane group and the side chain furan ring adopts two conformations with the LBP. Similarly, a synthetic analog with a locked side chain containing two triple bonds (21-nor-calcitriol-20(22),23-diyne) has been synthesized to gain in entropy energy with a predefined active side chain conformation [84]. The rigidity of the side chain leads to the systematic extension and thus weakening of the hydrogen bonds formed between hydroxyl groups of the ligand and the LBP (Fig. 11.10D). This weakening of interactions is consistent with the observed lower binding affinity. The latter is one order of magnitude lower for the synthetic analog, compared to the natural ligand. The loss in hydrogen bond energy resulting in a difference of 0.3 Å in the distance between the hydrogen and the acceptor atoms is sufficient to explain the experimental observation. Another difference observed for the locked side chain analog when compared to VDR–1,25(OH)2D3 structure is the absence of interaction with zLeu337, that also would contribute to the loss of energy. At the same time, an entropic gain from an energetically favorable preformed conformation of the ligand may result in an overall kinetics profit and compensate the loss of interactions, as reflected by the biological activity of this ligand, which is similar to that of the natural ligand. On the basis of the crystal structures of the hVDR LBD complexes, a secosteroidal analog with an o-carborane group introduced in the side chain were also reported [85]. This analog efficiently binds to the VDR LBD and is as transcriptionally active as 1,25(OH)2D3 in terms of potency and efficacy on vitamin D target genes, but is significantly less hypercalcemic than 1,25(OH)2D3. The ligand adopts the canonical active conformation in which the rings and the triene system of the new ligand have similar positions as the natural hormone [85]. Remarkably, the carborane moiety binds by unusual dihydrogen bonding with the His305 and His397 residues and form addition hydrophobic interactions with the C-terminal end of the VDR LBD (Fig. 11.10E).

STRUCTURES OF VITAMIN D RECEPTOR WITH ANALOGS THAT INDUCE STRUCTURAL REARRANGEMENTS Some of the 1,25(OH)2D3 analogs include bulky groups in the side chain (additional or branched side chains, aromatic rings, heterocyclic, and adamantyl groups) and induce structural rearrangement of the amino acid residues lining the LBP. These analogs formed an additional cavity in the LBP for accommodation of the bulky groups and thus altered the structure of the LBP and changed the VDR LBP–ligand interactions. Interestingly, the ligands showed agonistic, partial agonistic, or antagonistic activity depending on the structure of the side chain. These ligands alter the pocket structure and open a new perspective for the design of VDR ligands exhibiting a specific biological profile.

Gemini Analogs Gemini (1,25-dihydroxy-21-(3-hydroxy-3methylbutyl)vitamin D3) exhibits two identical side chains branching at carbon 20. Gemini binds less efficiently to VDR than 1,25(OH)2D3, however, its transactivation potency is similar to that of 1,25(OH)2D3 in rat osteoblastic cells [86] and 10-fold higher in Hela and monkey fibroblast-like cells [87]. It has also been shown that in presence of an excess of corepressor, the VDR– gemini complex shifts from an agonist to an inverse agonist conformation through the recruitment of N-Cor, and mediates repression [87]. The crystal structure of zVDR LBD– gemini reveals its binding mode. A new pocket is created by the combined effect of a backbone shift and a side-chain conformational reorientation to adapt the second side chain (Fig. 11.11A) [31]. The increase of the LBP volume is caused by the repositioning of zLeu337 (hLeu309) at the beginning of H7 (Fig. 11.11B). The hydrogen bonds anchoring gemini are similar to those formed with 1,25(OH)2D3 with an additional stabilization effect from zHis333, which interacts with

FIGURE 11.11  Crystal structure of zebrafish vitamin D receptor–ligand-binding domain (zVDR–LBD) complex with Gemini (protein data bank identifier (PDB ID): 2HCD). (A) Expansion of the ligand-binding pocket by the second side chain of the compound. (B) Conformational change of the zLeu337. Overlay of zVDR–LBD complex with gemini and human VDR (hVDR)–LBD complex with 1α,25-dihydroxyvitamin D3 (PDB ID: 1DB1; light grey). II.  MECHANISM OF ACTION

Structures of Vitamin D Receptor With Analogs That Induce Structural Rearrangements

hydroxyl groups of both side chains. New gemini compounds with increased agonistic properties and resistance to metabolic degradation have been developed [32,88]. 19-nor compounds Gemini-0072 and Gemini-0097 bear modifications on the side chains: deuterated geminal methyl groups, trifluoromethyl groups introduced to the second side chain, and unsaturated C23. The side-chain fluorine atoms stabilize helices H3, H11, and H12, therefore, explaining the superagonistic properties of these gemini derivatives. Between the two gemini analogs that differ only by the hydrogens in the geminal methyl groups substituted by deuteriums, the pocket is more compact [88].

22-Alkyl Derivatives Similarly to gemini and its derivatives, 22S- and 22R-alkyl analogs cause the formation of an extra cavity within the LBP (Fig. 11.12A) (reviewed in Ref. [89]). The crystal structures of rVDR LBD in complexes with Med1 coactivator peptide and the potent agonist ligand, 22S-butyl-20-epi-25,26,27-trinor-1,25(OH)2D3 or the antagonist 22S-butyl-25,26,­ 27-trinor-1,25(OH)2D3 are

201

similar to that of VDR–1,25(OH)2D3 complex [90]. The 22-butyl group of the agonist ligand is oriented toward H12, and the 24-hydroxyl group toward H6 forming a hydrogen bond with Val296 (hVal300) on H6. Consequently, some adaptations of some side chains of the VDR LBP are observed, notably of residues on H6, loop 6–7 and loop 11–12 (Fig. 11.12A and B). Thus the pocket is slightly expanded in the terminal region of H11. In the antagonist bound VDR crystal structure, the 22-butyl group of the ligand induces a side chain reorientation of Leu305 (hLeu309). A novel cavity is produced in the region surrounded by H6, loop 6–7, the N-terminal of H7, and H11 (Fig. 11.12A and C). These changes in the LBP cavity to accommodate the branched moiety of the antagonist’s side chain are similar to what has been seen for the gemini compounds that act as VDR agonists. In the case of compounds of this type, the synergy between the pocket expansion and the ligand interaction with the VDR C-terminus determines whether the compound demonstrates agonistic/partial agonistic or antagonistic properties [91,92]. For example, 22-alkyl compounds with terminal methyl groups

(A)

(B)

(C)

FIGURE 11.12  Crystal structures of rat vitamin D receptor–ligand-binding domain (rVDR–LBD) complexes with 22-alkyl substituted analogs. (A) Crystal structures of rVDR LBD in complexes with Med1 coactivator peptide and the potent agonist ligand, 22S-butyl-20-epi-25,26,27-trinor-1,25(OH)2D3 (left; pink) or the antagonist 22S-butyl-25,26,27-trinor-1,25(OH)2D3 (right; blue) in comparison to that of VDR-1,25(OH)2D3 complex (middle; grey). (B–C) Adaptations of some side chains of the VDR LBP upon binding to 22S-butyl-20-epi-25,26,27-trinor-1,25(OH)2D3 (B) or to the antagonist 22S-butyl25,26,27-trinor-1,25(OH)2D3 (C). 1,25(OH)2D3; 1α,25-dihydroxyvitamin D3. II.  MECHANISM OF ACTION

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FIGURE 11.13  Crystal structure of rat vitamin D receptor–ligand-binding domain (rVDR–LBD) complexes with adamantyl compounds. (A) Left: overlay of rVDR–LBD complex with 25-AD-25-hydroxyl (ADTT) (protein data bank identifier (PDB ID): 2ZMI) and human VDR (hVDR)–LBD complex with 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) (PDB ID: 1DB1; light grey). Middle: overlay of rVDR–LBD complex with ADNY (PDB ID: 2ZMH) and hVDR–LBD-1,25(OH)2D3. Right: oberlay of rVDR–LBD complex with ADMI4 (PDB ID: 2ZMJ) and hVDR–LBD-1,25(OH)2D3. (B) Adamantyl moiety of ADTK-1 compound (PDB ID: 3VTB) forms hydrophobic interactions with Phe418 and Val414, residues of H12 of the rVDR–LBD. ADMI4, 26-AD-25hydroxyl; ADNY, 24-AD-24-hydroxyl.

would more likely behave as VDR agonists than their 25,26,27nor counterparts. On the other hand, the size of alkyl moieties would also influence agonistic nature of ligand. As a general rule, 22S-ethyl and 22S-butyl moieties function as VDR partial agonists and antagonists, respectively, showing that the formation of a profound pocket is likely to cause antagonistic properties of the ligand [93]. However, recently reported 22S-hexyl derivative showed partial agonistic but not antagonistic activity when bound to VDR [94]. Indeed, for this compound the presence of mixed populations of bound agonist and antagonists conformations were observed in the crystal structure. As described above for the 22S-butyl-20-epi-25,26,27-trinor-1,25(OH)2D3, in the agonist conformation 22S-hexyl group adopts position toward the C-terminus of the protein, whereas in the antagonist form it occupies the extra cavity. Similarly, crystal structure analysis revealed a mixed population of binding agonist and antagonist conformers for some of the 22R-alkyl derivatives, explaining their partial agonism [91].

Adamantyl-Containing Compounds The 1,25(OH)2D3 derivatives with adamantyl group and 19-nor-modification have a bulky side chain that expands the

LBP near the C-terminus of H11, loop 11–12, the N-terminus of H3, and loop 6–7 (Fig. 11.13A) [95]. As a result of the LBD secondary structure perturbation, adamantyl analogs demonstrate weak agonistic or antagonistic properties, although the VDR LBD adopts the active conformation in all cocrystallized complexes. The crystal structures of the rVDR–LBD in complex with three different adamantyl containing analogs with various lengths of the side chain 25-AD-25-hydroxyl (ADTT), 24-AD-24-hydroxyl (ADNY), and 26-AD-25-hydroxyl (ADMI4) display adaptability of the LBP to ligands of different size: for shorter ADTT and ADNY no global perturbation is observed but the ADMI4 complex shows a displacement of rLeu400 by 3 Å from its original position and thereby widening of the LBP. In addition, the position of the two anchoring histidines, rHis301, and rHis393 is shifted away from the analog preventing the formation of strong hydrogen bonds with the 25-OH group thus explaining the only partial agonist activities. Newly synthesized adamantyl analogs with higher VDR agonistic activities have been recently described [96]. Compounds ADTK1–4 have more rigid side chains due to the presence of a triple bond. When bound to VDR, the side chain of the adamantyl compounds is significantly repositioned in comparison with that of the natural hormone. In this way, the adamantyl group takes the place of the 26,27-dimethyl group of

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SYNTHETIC MIMICS OF 1α,25-DIHYDROXYVITAMIN D3

1,25(OH)2D3. Importantly, the adamantyl moiety forms hydrophobic interactions with Phe418 and Val414, residues of H12 of the rVDR LBD, and these contacts are tighter than those observed with the 1,25(OH)2D3 (Fig. 11.13B). One of the main advantages of these compounds is their cell-selective VDR modulation activity, which explains their further investigation and derivatization.

SYNTHETIC MIMICS OF 1α,25DIHYDROXYVITAMIN D3 Most of the 1,25(OH)2D3 analogs carry modifications of the natural hormone skeleton. However, as already described above, there are also semisteroidal analogs, with significant changes in all part of the ligand such as CD-ring deletion. In addition to the secosteroidal analogs of 1,25(OH)2D3, another type of ligands are the nonsecosteroidal molecules that mimic 1,25(OH)2D3 without being structurally related to it, and hundreds of them have already been described (reviewed in Refs. [26,27]). There are currently three classes of synthetic nonsecosteroidal mimics of 1,25(OH)2D3: diphenylmethane LG190178 and its derivatives [97–102]; bis- and tris-aromatic derivatives [103,104]; and compounds with a p-carborane group [105]. Only some of the diphenylmethane derivatives and of the bis- and tris-aromatic derivatives have been shown to be potent VDR agonists. Some of these ligands possess less calcium mobilization activity and are attractive therapeutics against psoriasis, osteoporosis, and cancer. Nonsteroidal compounds for other steroid NRs are currently used in cancer treatment such as raloxifene for ER [106] or flutamide for AR [107]. In case of all nonsecosteroidal ligands cocrystallized with the VDR LBD, these compounds take a similar position and a conformation to that of the natural ligand.

Derivatives of LG190178 Nonsecosteroidal VDR ligand LG190178 was one of the first improved modifications of the hit bis-phenyl derivative LG190090 identified by screening compound libraries in cotransfection–cotransactivation assay. LG190178 is a mixture of four diastereomers at C2 and C2′, and it was shown to mimic various activities of 1,25(OH)2D3 in vitro and in vivo, such as VDR binding, VDR-dependent transcriptional activation, inhibition of proliferation, and differentiation of various cell types [98]. The YR301 compound (Fig. 11.14A) which is a (2S,2′R)- stereoisomer of LG190178 has been crystallized in complex with the rVDR LBD [100]. This compound has been shown to exhibit potent transcriptional activity in vitro. The X-ray crystallographic analysis revealed that the 2-hydroxy-3,3-dimethylbutyl group acts as a mimic for the side-chain of 1,25(OH)2D3, while the 2,3-hydroxypropyl group replaces the A-ring and its diethylmethyl-group takes the same spatial position as the CD-rings of the natural compound. YR301 possesses two functional hydroxyl-groups: the first forms hydrogen bonds with rHis301 and rHis393 (C2); and the second with rSer233 and rArg270 (C2′). C2 and C2′ groups replace the 1-OH and 25-OH groups of the natural ligand, respectively. The terminal hydroxyl group of YR301 forms two direct hydrogen bonds with rArg270 and

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interacts with rTyr232 and rAsp144 via water molecules (Fig. 11.14B). Additional derivatization with a hydroxyl group in YR335 allows this hydroxyl group to form H-bonds with rSer274 and rTyr139, similarly to the 3-OH group of 1,25(OH)2D3, but without improving its transcriptional potency [101]. To combine VDR agonistic and histone deacetylase activities and on the basis of the LG190178 skeleton, bifunctional diphenylmethane derivatives containing a hydroxamic acid moiety in place of the A-ring mimic diol of LG190178 have been designed [102] (see Chapter 89 (vol. 2 of this book)). The crystal structures of two enantiomers of bifunctional derivatives JF-C71 and JF-C72 ((R)- and (S)-isomers, respectively) in complex with the zVDR LBD were solved. Both ligands adopt the same orientation as YR301 (Fig. 11.14C) [100]. The 2-hydroxy-3,3-dimethylbutyl side chains of the two enantiomers adopt different conformation that result in a similar position of the hydroxyl group interacting through hydrogen bonds with zHis333 and zHis423. The diethyl groups occupy the space filled by the CD-rings of the natural ligand with the hydroxamate groups pointing toward helix H1 of the zVDR LBD and occupying the 1,25D A-ring binding pocket. The hydroxy group interacts through an H-bond with zArg302 (2.6 Å for JF-C71, 3.0 Å for JF-C72). This interaction directly mimics that of the C1–OH of 1,25(OH)2D3 in the LBP [31]. In addition, the NH forms a hydrogen bond with zSer265 (2.5–2.6 Å). Van der Waals interactions of the OH and carbonyl group are observed with zTyr264, zArg302, and zTyr175. These structural studies demonstrate that the hydroxamic acid moiety is a viable surrogate for the A-ring of 1,25(OH)2D3, and confirm the remarkable structural flexibility in conversion of 1,25(OH)2D3 analogs into fully integrated bifunctional molecules. Several hybrids with modifications to the diarylpentane core and to the aliphatic spacer unit were developed to provide molecules with increased inhibition potency towards Histone Deacetylases while maintaining VDR agonist activity, notably, hybrid DK-366 [108].

Bis- and Tris-Aromatic Compounds The second class of 1,25(OH)2D3 mimics are the bis- and tris-aromatic derivatives. A dibenzyl alcohol is a common moiety in these ligands. The two phenyl groups are either linked by an ether (CD3938, CD4720, CD4742, and CD4802) or an alkyl (CD4528 and CD4849) chain [103]. For the bis-aromatic derivatives, a dienyl alcohol is branched to the second phenyl while the methyl moieties of the dienyl alcohol in CD4528 and CD4420 are replaced by tri-fluoromethyls. In the tris-aromatic derivatives, a third phenyl group is included in the branched side chain replacing the dienyl alcohol of the bis-aromatic analogs. Like 1,25(OH)2D3, bis- and tris-aromatic derivatives stimulate transcription at nanomolar concentrations [104]. Examination of the protein–ligand interactions reveals that these compounds have a position and an H-bond network within the LBP similar to the natural ligand (Fig. 11.14D). Bisand tris-aromatic analogs are better accommodated within the VDR LBP than the other nonsecosteroidal ligands as the intrinsic hydrophobic van der Waals contacts of the VDR– 1,25(OH)2D3 complex are maintained in these new structures. In case of CD4528, additional interactions are mediated

II.  MECHANISM OF ACTION

FIGURE 11.14  Crystal structures of vitamin D receptor–ligand-binding domain (VDR–LBD) complexes with synthetic mimics of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). (A) Chemical structures of 1,25(OH)2D3 (grey) and YR301 (blue), a derivative of LG190178. (B) Hydrogen-bonding network between YR301 and rat VDR (rVDR)–LBD (protein data bank identifier (PDB ID): 2ZFX). (C) Hydrogen bonding-network formed by two enantiomers of bifunctional derivatives JF-C71 and JF-C72 in complex with the zebrafish VDR (zVDR) LBD (PDB IDs: 4FHI and 4FHH). (D) Hydrogen-bonding network between zVDR–LBD and the bis-aromatic derivative CD4528 (PDB ID: 4G2H). (E) Crystal structure of zVDR–LBD complex with the tris-aromatic compound CD4720 (PDB ID: 4G1D). Specific hydrophobic interactions are presented as grey dash lines, hydrogen bonds as red dash lines. (F) Crystal structure of rVDR–LBD complex with 10S carborane derivative (PDB ID: 3VJS). H-bonds are shown in dashed lines.

Conclusions and Perspectives

through the trifluoromethyl groups. For the tris-aromatic analogs (Fig. 11.14E), a larger fraction of the LBP is occupied by the ligand that results in the formation of additional stabilizing contacts. The length of the alkyl group attached to the second phenyl ring induces some rearrangement of the VDR LBP in the region of helix H6 without affecting the agonistic behavior of the complex. These extended pockets may be important to achieve selectivity and dissociated biological profiles without affecting the agonistic activity of the ligands. The nature of the linker (oxygen vs. carbon) between the two first phenyl rings plays a crucial role in VDR binding affinity through the desolvation cost of the ether linker on VDR binding.

Compounds With p-Carborane Core The third class of 1,25(OH)2D3 mimics consists of ligands with p-carborane (1,12-diarba-closo-dodecarborane) as hydrophobic core structure and flexible acyclic triols. Although first reported carborane ligands have a low binding affinity to VDR, they induce the active receptor conformation [105]. The carborane group is located at the same place as the CD rings of the natural ligand and functions as a hydrophobic anchor in binding to the VDR. Three hydroxyl groups of the flexible acyclic triol allow its suitable positioning in the LBP to form hydrogen bonds similarly to the natural ligand (Fig. 11.14E). The same authors reported design and synthesis of ω-hydroxyalkoxy derivatives of the carborane-containing compound with improved affinity to VDR and activity [109]. The structural studies on these different nonsecosteroidal analogs have provided the molecular basis for their transcriptional activity. A significant effect of the nonsecosteroidal ligands on VDR activity confirms the fact that the secosteroid backbone of 1,25(OH)2D3 is less important than the positioning of the three anchoring hydroxyl-groups. Structural scaffolds of nonsecosteroidal ligands different from that of the natural ligand and their potent VDR agonistic activities with low calcemic actions make these mimics potential drugs for clinical applications.

CONCLUSIONS AND PERSPECTIVES Since the first crystal structure of the hVDR LBD in complex with 1,25(OH)2D3 was published [24], more than 100 structures of the VDR LBD in complex with various synthetic analogs of the natural hormone have been deposited in the PDB, 70% of them in the last 5 years. The interest in the development of synthetic compounds, which would be able to selectively modulate VDR activity remains very high. This can be explained by the major role of VDR in the regulation of a broad range of important physiological processes, including regulation of immune response, cell proliferation, and apoptosis, etc. Because VDR is considered as an important drug target, numerous synthetic analogs of 1,25(OH)2D3 have been developed with the aim of improving biological properties of the parental hormone. Most of the synthetic analogs have been designed as modifications of the parental compound and some of them mimic the

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1,25(OH)2D3 scaffold without being structurally related to it. Nevertheless, their implication in systemic treatment is limited as they either are not active or, very often in the case of superagonists, induce hypercalcemia, a major side effect of calcitriol. There is still a need of novel selective modulators of the receptor that would be as active as the natural hormone and display reduced hypercalcemic side effects. From the early point of structural studies it became clear that there is no uniform correlation between the structure of a ligand and its displayed in vivo biological activities. However, the information coming from the analysis of crystal structures of VDR–ligand complexes remains one of the most powerful tools to explain and validate the properties of the compounds at the level of atomic interactions with the receptor. X-ray crystallography also brings new rationales for the design of new compounds. One of the important implications of the VDR ligands structural analysis is the combination of ligand development with innovative chemistry. For example, fluorination of VDR ligands often leads to improvement of the interaction with the receptor through numerous novel contacts, as well as to enhanced metabolic activity. Another example is carborane drug discovery: carboranes have been used as unique pharmacophores in VDR ligands that could be combined with boron neutron capture therapy of cancer with the objective to selectively destroy cancer cells as an alternative to conventional radiation therapy. While the X-ray crystallography provides the atomic details of VDR–ligand interactions, other techniques such as HDX–MS and NMR are classically used for exploring the conformational dynamics of protein on ligand binding in solution. A combination of these methods, as well as compound metabolism and kinetic information, should be more extensively applied to the investigation of detailed mechanism of ligand mode of action, and for the rational design of new selective modulators of VDR activity. Going even further, it would be of high interest to integrate structural analysis of VDR ligands with large-scale ChIP-seq, transcriptomics, and proteomics data. Such studies would significantly move forward the development of cell- and coregulator-specific modulators targeting the receptor and coregulators. Finally, comparative analysis of various ligands, their structure, and function in the context of full-length VDR-coregulator complexes would also bring new important insights into ligand selectivity. There are several reports on high-throughput screening of the small molecules selectively modulating VDR-coregulator interactions; however, they are mostly limited to the VDR LBD and coregulator peptides [110,111]. It is likely that applying similar approaches to the full-length proteins would increase chances to identify compounds with enhanced selectivity to specific coregulators or those targeting alternative sites.

Acknowledgments Grants from the Agence Nationale de Recherche (ANR), the Fondation ARC pour la Recherche sur le Cancer and the Fondation pour la Recherche Médicale (FRM).

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II.  MECHANISM OF ACTION