Mutation goals in the vitamin D receptor predicted by computational methods.

The mechanism through which nuclear receptors respond differentially to structurally distinct agonists is a poorly understood process. We present a computational method that identifies nuclear receptor amino acids that are likely involved in biological responses triggered by ligand binding. The method involves tracing how structural changes spread from the ligand binding pocket to the sites on the receptor surface, which makes it a good tool for studying allosteric effects. We employ the method to the vitamin D receptor and verify that the identified amino acids are biologically relevant using a broad range of experimental data and a genome browser. We infer that surface vitamin D receptor residues K141, R252, I260, T280, T287 and L417 are likely involved in cell differentiation and antiproliferation, whereas P122, D149, K321, E353 and Q385 are linked to carcinogenesis.

13,13-Dimethyl-des-C,D analogues of (20S)-1α,25-dihydroxy-2-methylene-19-norvitamin D3 (2MD): total synthesis, docking to the VDR, and biological evaluation.

As a continuation of our studies focused on the vitamin D compounds lacking the C,D-hydrindane system, 13,13-dimethyl-des-C,D analogues of (20S)-1α,25-dihydroxy-2-methylene-19-norvitamin D(3) (2, 2MD) were prepared by total synthesis. The known cyclohexanone 30, a precursor of the desired A-ring phosphine oxide 11, was synthesized starting with the keto acetal 13, whereas the aldehyde 12, constituting an acyclic 'upper' building block, was obtained from the isomeric esters 34, prepared previously in our laboratory. The commercial 1,4-cyclohexanedione monoethylene ketal (13) was enantioselectively α-hydroxylated utilizing the α-aminoxylation process catalyzed by l-proline, and the introduced hydroxy group was protected as a TBS, TPDPS, and SEM ether. Then the keto group in the obtained compounds 15-17 was methylenated and the allylic hydroxylation was performed with selenium dioxide and pyridine N-oxide. After separation of the isomers, the newly introduced hydroxy group was protected and the ketal group hydrolyzed to yield the corresponding protected (3R,5R)-3,5-dihydroxycyclohexanones 30-32. The esters 34, starting compounds for the C,D-fragment 12, were first α-methylated, then reduced and the resulted primary alcohols 36 were deoxygenated using the Barton-McCombie protocol. Primary hydroxy group in the obtained diether 38 was deprotected and oxidized to furnish the aldehyde 12. The Wittig-Horner coupling of the latter with the anion of the phosphine oxide 11, followed by hydroxyl deprotection furnished two isomeric 13,13-dimethyl-des-C,D analogues of 2MD (compounds 10 and 42) differing in configuration of their 7,8-double bond. Pure vitamin D analogues were isolated by HPLC and their biological activity was examined. The in vitro tests indicated that, compared to the analogue 7, unsubstituted at C-13, the synthesized vitamin D analogue 10 showed markedly improved VDR binding ability, significantly enhanced HL-60 differentiation activity as well as increased transcriptional potency. Docking simulations provided a rational explanation for the observed binding affinity of these ligands to the VDR. Biological in vivo tests proved that des-C,D compound 10 retained some intestinal activity. Its geometrical isomer 42 was devoid of any biological activity.

MSITE: a new computational tool for comparison of homological proteins in holo form.

The mechanism by which nuclear receptors respond differentially to structurally distinct agonists is not a well understood process. However, it is now obvious that transcriptional activity of nuclear receptors is a function of their interactions with co-activators. Recently, we released a new computational tool, CCOMP, for comparing side chain conformations in crystal structures of homologous protein complexes. Application of the CCOMP program revealed that 20-epi-1alpha,25-(OH)2D3 changes the side chain conformation of vitamin D receptor amino acids residing mostly far away from the ligand-receptor contacts. This strongly suggests that the ligand-co-activator signaling pathway involves indirect interactions between amino acids lining the binding pocket and outer surface residues that could attract co-activators. To facilitate identification of amino acids transmitting the subtle receptor changes upon ligand/modulator binding we developed another simple tool, MSITE. The program automatically lists the nearest neighbors of a given amino acid (for example neighbors of residues that are in contact with a ligand or reorient their side chains in the presence of a co-factor) in an arbitrary number of compared complexes. Comparison of seven binary vitamin D receptor complexes holding as ligands the analogs of 1alpha,25-(OH)2D3 with inverted configuration at carbon 14 or 20, or with incorporated oxolane ring bridging carbons 20 and 23, is reported.

Structural changes of vitamin D receptor induced by 20-epi-1alpha,25-(OH)2D3: an insight from a computational analysis.

We employ a new computational tool CCOMP for the comparison of side chain (SC) conformations between crystal structures of homologous protein complexes. The program is applied to the vitamin D receptor (VDR) liganded with 1alpha,25-(OH)(2)D(3) (in 1DB1) or its 20-epi (in 1IE9) analog with an inverted C-20 configuration. This modification yields no detectable changes in the backbone configuration or ligand topology in the receptor binding cavity, yet it dramatically increases transcription, differentiation and antiproliferation activity of the VDR. We applied very stringent criteria during the comparison process. To eliminate errors arising from the different packing of investigated crystals and the thermal flexibility of atoms, we studied complexes belonging to the same space group, having a low R value (0.2) and a B-factor below 40 for compared residues. We find that 20-epi-1alpha,25-(OH)(2)D(3) changes side chain conformation of amino acids residing far away from direct ligand-VDR contacts. We further verify that a number of the reoriented residues were identified in mutational experiments as important for interaction with SRC-1, GRIP, TAFs co-activators and VDR-RXR heterodimerization. Thus, CCOMP analysis of protein complexes may be used for identifying amino acids that could serve as targets for genetic engineering, such as mutagenesis.

Computational analysis of the active sites in binary and ternary complexes of the vitamin D receptor.

We have developed a program CCOMP that compares overlapping fragments of two protein complexes and identifies differently oriented amino acids. CCOMP initially performs a sequence alignment of the analyzed receptors, then superimposes the corresponding aligned residues, and finally calculates the root mean square deviation (RMSD) of individual atoms, every amino acid and the entire complex. Thus, amino acids important for functional differences between both complexes can be detected. Application of CCOMP to 1alpha,25-(OH)(2)D(3)-hVDR (1DB1) [Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 5491] and 1alpha,25-(OH)(2)D(3)-rVDR-peptide (1RK3) [Biochemistry 43 (2004) 4101] complexes revealed that the peptide (KNHPMLMNLLKDN) mimicking a co-activator sequence significantly changes the side chain conformation of 35 amino acids. Four of these residues (K242, I256, K260, E416) actually contact the peptide, but all of them are essential for biological activity. Only two (L309 and L400) of the 35 differently oriented amino acids contact the ligand. Interestingly, when the peptide is present (1RK3) leucine 400 shifts closer (0.7A) to the vitamin D 26-methyl group. Applying the CCOMP and DSSP programs to binary and ternary VDR complexes also resulted in establishing that seven amino acids (I238, S252, I256, L413, L415, E416, V417) exhibit significant differences in solvent accessibility and are capable of interacting with co-activators.

NMR assignments of tryptophan residue in apo and holo LBD-rVDR.

Binding sites in the full-length, ligand-binding domain of rat vitamin D receptor (LBD-rVDR) for an active hormone derived from vitamin D (1alpha,25-dihydroxyvitamin D(3)) and three of its C-2 substituted analogs were compared by nuclear magnetic resonance (NMR) spectroscopy. Specific residue labeled with [UL]-(15)N(2) Trp allowed assignment of the side-chain H(epsilon1) and N(epsilon1) resonances of the single tryptophan residue at position 282 in LBD-rVDR. Comparison of (1)H[(15)N] Heteronuclear Single Quantum Correlation (HSQC) spectra of apo and holo LBD-rVDR revealed that the position of the Trp282 H(epsilon1) and N(epsilon1) signals are sensitive to the presence of the ligand in the receptor cavity. Binding of the ligands to LBD-rVDR results in a shift of both Trp H(epsilon1) and N(epsilon1) resonances to lower frequencies. The results indicate that the interaction between the ligands and Trp282 is not responsible for differences in calcemic activity observed in vitamin D analogs.

2-Methylene analogs of 1alpha-hydroxy-19-norvitamin D3: synthesis, biological activities and docking to the ligand binding domain of the rat vitamin D receptor.

In continuing efforts towards the synthesis of biologically active vitamin D compounds of potential therapeutic value, new 2-methylene-1alpha-hydroxy-19-norvitamin D(3) analogs 3 and 4 with modified alkyl side chains have been synthesized. The key synthetic step involved Lythgoe-type Wittig-Horner coupling of Windaus-Grundmann type ketones 9, possessing different 17beta-alkyl substituents, with the phosphine oxide 10 prepared from (-)-quinic acid. The prepared vitamins 3 and 4 were ca. eight times less potent than 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) (1) in binding to the rat intestinal vitamin D receptor (VDR). In comparison with the hormone 1 they exhibited slightly lower cellular HL-60 differentiation activity. When tested in vivo; the analog 3 was characterized by very high bone calcium mobilizing potency and intestinal calcium transport activity. Unexpectedly, the 25-methyl compound 4 showed marked calcemic activity in both assays. Computational docking of the vitamin 3 into the binding pocket of the rat vitamin D receptor is also reported.

Model of three-dimensional structure of VDR bound with Vitamin D3 analogs substituted at carbon-2.

All Vitamin D analogs possessing the A ring modified at C-2 and showing calcemic activities nest themselves in the VDR binding pocket, oriented towards Tyr 143. Such topology resembles the position of the Vitamin D hormone in hVDRmt [Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 5491]. Conversely, inactive 2beta-methyl-19-nor-analogs anchor the receptor cavity in a distinguishably different manner, namely by their side chain. Moreover, these inactive vitamins have a different conformation around C(6)-C(7) bond. Topology of modeled complexes suggests that a Vitamin D analog will be biologically active if its intercyclic 5,7-diene moiety assumes parallel position to tryptophan aromatic rings; such orientation allows for creating pi-pi interactions. The broad comparison of calcemic activities of the analogs, and their interactions with VDR, revealed that specific hydrophobic contacts are involved in bone calcium mobilization (BCM). These contacts occur between 21-methyl group and a few amino acids (V296, L305 and L309), conserved in the nuclear receptor superfamily. In the inactive 2beta-methyl-19-nor analogs such contacts do not exist. We speculate that two hydrophobic receptor patches, being in close contact with ligand methyl groups, might influence interaction with co-modulators involved in calcium homeostasis.

Solvent polarity and hydrogen-bonding effects on the nitrogen NMR shieldings of N-nitrosamines and DFT calculations of the shieldings of C-, N-, and O-nitroso systems.

High-precision nitrogen NMR shieldings, bulk susceptibility corrected, are reported for dimethyl-N-nitrosamine (I) and diethyl-N-nitrosamine (II) in a variety of solvents which represent a wide range of solvent properties from the point of view of polarity as well as hydrogen bond donor and acceptor strength. The observed range of solvent-induced nitrogen shielding variations of (I) and (II) is significant for the amino-type nitrogens, up to about 16 ppm, and originates essentially from the deshielding effect of the increasing polarity of solvent. On the other side, the nitroso nitrogen shieldings reveal an even stronger response to solvent effects, within about 20 ppm, but in this case the increasing polarity and hydrogen bond donor strength of solvent produce enhanced shielding. DFT quantum-mechanical calculations using the GIAO/B3PW91/6-311++G** approach and geometry optimizations employing the same basis set and hybrid density functionals show an excellent correlation with the experimental data on C-, N-, and O-nitroso moieties and reproduce not only major changes but also most of the subtle variations in the experimental nitrogen shieldings of the nitroso systems as a whole. A combination of the calculations involving the corresponding N and O-protonated species and the trends observed in the solvent-induced nitrogen shielding variations shows clearly that the prime acceptor site for hydrogen bonding is the nitroso oxygen atom.

2-Ethyl and 2-ethylidene analogues of 1alpha,25-dihydroxy-19-norvitamin D(3): synthesis, conformational analysis, biological activities, and docking to the modeled rVDR ligand binding domain.

Novel 19-nor analogues of 1alpha,25-dihydroxyvitamin D(3) were prepared and substituted at C-2 with an ethylidene group. The synthetic pathway was via Wittig-Horner coupling of the corresponding A-ring phosphine oxides with the protected 25-hydroxy Grundmann's ketones. Selective catalytic hydrogenation of 2-ethylidene analogues provided the 2alpha- and 2beta-ethyl compounds. The 2-ethylidene-19-nor compounds with a methyl group from the ethylidene moiety in a trans relationship to the C(6)-C(7) bond (E-isomers) were more potent than the corresponding Z-isomers and the natural hormone in binding to the vitamin D receptor. Both geometrical isomers (E and Z) of (20S)-2-ethylidene-19-norvitamin D(3) and both 2alpha-ethyl-19-norvitamins (in the 20R- and 20S-series) have much higher HL-60 differentiation activity than does 1alpha,25-(OH)(2)D(3). Both E-isomers (20R and 20S) of 2-ethylidene vitamins are characterized by very high calcemic activity in rats. The three-dimensional structure model of the rat vitamin D receptor and the computational docking of four synthesized (20R)-19-norvitamin D(3) analogues into its binding pocket are also reported.