Boron cluster-based development of potent nonsecosteroidal vitamin D receptor ligands: direct observation of hydrophobic interaction between protein surface and carborane

We report here the design and synthesis of a novel vitamin D receptor (VDR) agonist whose hydrophobic core structure is p-carborane (1,12-dicarba-closo-dodecaborane, an icosahedral carbon-containing boron cluster having remarkable thermal and chemical stability and a characteristically hydrophobic B-H surface). This carborane-based VDR ligand exhibited moderate vitamin D activity, comparable to that of the natural hormone, despite its simple and flexible structure. X-ray structure analysis provided direct evidence that the carborane cage binds to the hydrophobic surface of the ligand-binding pocket of the receptor, promoting transition to the active conformation. These results indicate that the spherical B-H surface of carborane can function efficiently as a hydrophobic anchor in binding to the receptor surface, thereby allowing induced fitting of the three essential hydroxyl groups on the alkyl chains to the appropriate positions for interaction with the VDR binding site, despite the entropic disadvantage of the flexible structure. We suggest that carborane structure is a promising option in the design of novel drug candidates.     

New C15-substituted active vitamin D3

C15-Substituted 1α,25-dihydroxyvitamin D(3) analogs were synthesized for the first time to investigate the effects of the modified CD-ring on biological activity concerning the agonistic positioning of helix-3 and helix-12 of the vitamin D receptor (VDR). X-ray cocrystallographic analysis proved that 0.6 ? shifts of the CD-ring and shrinking of the side chain were necessary to maintain the position of the 25-hydroxy group for proper interaction with helix-12. The 15-hydroxy-16-ene derivative showed higher binding affinity for hVDR than the natural hormone.     

Interaction of graphene with antipsychotic drugs: Is there any charge transfer process?

Antipsychotics represent an effective therapy for schizophrenia (a chronic mental disorder). Their benefits are related to the interaction of the drugs with dopamine D2 receptor (D2R). Antipsychotics are classified as agonists or antagonists. One of the working hypotheses is that there is a charge transfer process between the drugs and the receptors, which is different for agonists and antagonists. To have more insight into the nature of the interaction of these molecules and the differences between agonists and antagonists, we analyze the interaction of graphene with three molecules: dopamine, pramipexole (an agonist of dopamine), and risperidone (an antagonist of dopamine). The idea is to use graphene as a simple model to analyze the charge transfer process of these three drugs. Optimized structures, atomic charges, and Density of States results indicate that global charges of dopamine and pramipexole are similar, while for risperidone, it is more than double. Pramipexole is an agonist, and the charge transfer process is similar to that of dopamine. Risperidone is an antagonist, and the charge transfer process is different from dopamine. The charge transfer is more significant with risperidone than with dopamine, and this could be related to the mechanism of action. This is in agreement with the working hypotheses that establish that it is possible to distinguish between agonists and antagonists since they have different capacity to transfer charge.     

New mu-opioid receptor agonists with phenoxyacetic acid moiety

New muq-opioid receptor (MOR) agonists containing 4-hydroxypiperidine, piperidine and piperazine moieties were synthesized and evaluated to find a peripheral opioid analgesic. Among the synthesized compounds, 12-[1-[3-(N,N-dimethylcarbamoyl)-3,3-diphenylpropyl]-4-hydroxypiperidin-4-yl]phenoxy]acetic acid (8: SS620) having phenoxyacetic acid and 4-hydroxypiperidine moieties showed the highest agonist potency on the MOR in an isolated guinea-pig ileum preparation, and it also had selectivity to the human MOR expressed in Chinese hamster ovary (CHO)-K1 cells compared with the same types of delta- and kappa-opioid receptors (DOR and KOR). In addition, compound 8 showed a 10 times more potent MOR agonist activity than loperamide. Furthermore, compound 8 showed a peripheral analgesic activity in vivo screening on rat.     

Synthesis of vitamin D(3) and calcitriol dimers as potential chemical inducers of vitamin D receptor dimerization

The design and synthesis of vitamin D(3) dimers 2 and 3 and 1 alpha, 25-dihydroxyvitamin D(3) (calcitriol) dimers 4 and 5 are described. The dimers were designed with a view to doubly binding the vitamin D receptor (VDR) and inducing the receptor homodimerization. In the dimers the units are linked through the C-11 position in ring C by an alkyl side chain of six or 10 carbon atoms, far from the hydroxy groups responsible for the VDR binding. The linker is formed by olefin metathesis of an olefinic side chain at the C-11 position introduced by stereoselective cuprate addition. The synthesis, which is both short and convergent, uses the Wittig-Horner approach to construct the vitamin D triene system and allows the preparation of dimers with a linker of modulated length with the purpose of optimizing the vitamin D(3)-VDR interaction.     

Synthesis of N1-phenethyl substituted indole derivatives as new melatoninergic agonists and antagonists

The potency of new indolic N1-phenethyl substituted melatoninergic ligands with and without methyl groups in the alpha and beta position of the alkanamidoethyl side chain was examined using the pigment aggregation response in a clonal line of Xenopus laevis melanophores. The non 5-OMe substituted compounds, 8a--e, are all weak antagonists while introduction of the 5-OMe group, 9a--e, increases both agonist and antagonist activity except for 9c (R=C3H7), which is only an agonist and 9e (R=c-C4H7), which is only an antagonist. Introduction of an alpha-methyl group into the 5-OMe derivatives, 14a-e, reduces the agonist potency while introduction of a beta-methyl group has only a small effect on either the agonist or antagonist potency. Double beta-methyl substitution of the 5-OMe derivatives, 20a--e, generally increases the agonist potential (20c, R=C3H7 is the most potent agonist of the compounds described) and decreases the antagonist potency, except for 20a (R=CH3), which is the most potent antagonist of this series of compounds.     

Synthesis and serotonin 2 (5-HT2) receptor antagonist activity of 5-aminoalkyl-substituted pyrrolo[3,2-c]azepines and related compounds

A series of 5-aminoalkylpyrrolo[3,2-c]azepine derivatives was synthesized and their serotonin 2 (5-HT2) receptor antagonist and antiplatelet aggregation activities were evaluated. 5-HT2 receptor antagonist activity was largely determined by the nature of the substituent at the 8-position as well as the aminoalkyl group at the 5-position of the pyrrolo[3,2-c]azepine ring. Compound 18a, 5-[3-[4-(4-fluorophenyl)piperazin-1-yl]propyl]-8-hydroxy-1-methyl- 1,4,5,6,7,8-hexahydropyrrolo[3,2-c]azepin-4-one, was recognized as having potent 5-HT2 receptor antagonist activity with weak alpha1 adrenoceptor blocking activity and no significant D2 receptor binding affinity, while the corresponding isomeric pyrrolo[3,4-c]azepine derivative (22) displayed only weak 5-HT2 receptor antagonist activity. After racemic 18a was resolved directly via diastereomeric salt formation, each enantiomer was evaluated precisely. The 5-HT2 receptor antagonist activity of 18a was found to reside primarily in (-)-18a (which was about 14-fold more potent than (+)-18a in isolated guinea pig arteries). Consequently, (S)-(-)-18a (SUN C5174) displayed the overall best profile with potent 5-HT2 receptor antagonist activity (pA2=8.98+/-0.06) and high selectivity versus other receptors. SUN C5174 showed a marked inhibitory effect on the platelet aggregation induced by serotonin in combination with collagen and adenosine diphosphate (ADP) in canine or human platelet-rich plasma (IC50=6.5 to 16 nM). Moreover, this compound significantly inhibited the mortality rate in mouse acute pulmonary thromboembolytic death induced by collagen and serotonin at oral doses of 0.3 mg/kg or higher. SUN C5174 is currently undergoing clinical evaluation.     

Interaction between vitamin D receptor and vitamin D ligands: two-dimensional alanine scanning mutational analysis

We present a new method to investigate the details of interaction between vitamin D nuclear receptor (VDR) and various ligands, namely a two-dimensional alanine scanning mutational analysis. In this method, the transactivation of various ligands is studied in conjunction with a series of alanine scanning mutations of the residues lining the ligand binding pocket (LBP) of VDR, and the complete set of results is profiled in a patch table. We investigated examples from four structurally diverse groups of known VDR ligands: the native vitamin D hormone and two compounds with the same side chain configuration; four 20-epi compounds; three 19-nor compounds; and two nonsecosteroids. The patch table of the results indicates characteristics of each group in terms of its interaction with 18 LBP residues. We demonstrate the validity of this approach by application to docking studies of the two nonsecosteroids.     

Efficient synthesis of 2-modified 1alpha,25-dihydroxy-19-norvitamin D3 with Julia olefination: high potency in induction of differentiation on HL-60 cells

Six novel 2-substituted analogues of 1alpha,25-dihydroxy-19-norvitamin D(3), 6a,b-8a,b, were efficiently synthesized utilizing (-)-quinic acid as the A-ring precursor. The C2-modified A-rings were prepared as 4-alkylated (3R,5R)-3,5-dihydroxycyclohexanones 12-15 from (-)-quinic acid based on radical allylation at the C4 position of methyl (-)-quinicate. The new type of the CD-ring coupling partner 23 was synthesized from 25-hydroxy Grundmann's ketone 19 to apply to the modified Julia olefination to construct a diene unit between the A-ring and the CD-ring. The coupling yields, including a deprotection step, were 47-62%. After the separation of the diastereomers based on C2 stereochemistry, the structure (2alpha or 2beta) was determined by (1)H NMR experiments and compared to DeLuca's 2-methyl- and 2-ethyl-1alpha,25-dihydroxy-19-norvitamin D(3). Thus, the synthesized 2alpha-(3-hydroxypropyl)-1alpha,25-dihydroxy-19-norvitamin D(3) (8a) showed almost the same potency in binding to the bovine thymus vitamin D receptor (VDR) as the natural hormone 1, while its beta-isomer 8b had only a 3% affinity. Both 2alpha-allyl- and 2alpha-propyl-1alpha,25-dihydroxy-19-norvitamin D(3) (6a and 7a) and their 2beta-analogues (6b and 7b) possessed a weak affinity for the VDR. The strong VDR ligand 8a was ca. 36-fold more potent in induction of HL-60 cell differentiation than 1, and interestingly, even the weaker ligand 8b showed a 6.7-fold higher potency in the cell differentiation activity than that of 1.     

Chimeric protein probes for C5a receptors through fusion of the anaphylatoxin C5a core region with a small-molecule antagonist

Blockade of the interaction of anaphylatoxin C5a with its receptor C5aR1 has been actively studied as a potential treatment for many inflammatory diseases; but current C5a antagonists exhibit inadequate potency and poor species cross-reactivity, and novel biochemical tools are needed to investigate whether the core region of C5a contains important interaction epitopes that can explain these limitations. Herein, we report the development of chimeric protein C5a probes containing both the complete core region of rat or human C5a, and the small-molecule antagonist PMX53-1. These probes were chemically synthesized through hydrazide-based native chemical ligation of a linear peptide hydrazide with the requisite cyclopeptidic antagonist, both of which were made by solid-phase synthesis. Quasi-racemic X-ray crystallography established that attachment of PMX53-1 did not affect the structure of the core region of C5a. Subsequent C5aR1 activity assays demonstrated the probes can provide valuable insights into the development of C5a antagonists; for example, they exhibited significantly better binding affinity and much improved species cross-reactivity than PMX53-1, supporting the notion that the effect of some epitopes outside the C-terminus of C5a should be taken into consideration when designing better C5a antagonists. Surprisingly, the core region of C5a was found to partially agonize C5aR1, suggesting the presence of more than one agonistic interaction in the binding of C5a to C5aR1. This study exemplifies the value of chemical protein synthesis in developing novel receptor probes for drug discovery research.     

Superagonistic fluorinated vitamin D3 analogs stabilize helix 12 of the vitamin D receptor

Side chain fluorination is often used to make analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant to degradation by 24-hydroxylase. The fluorinated nonsteroidal analogs CD578, WU515, and WY1113 have an increased prodifferentiating action on SW480-ADH colon cancer cells, which correlated with stronger induction of vitamin D receptor (VDR)-coactivator interactions and stronger repression of beta-catenin/TCF activity. Cocrystallization of analog CD578 with the zebrafish (z)VDR and an SRC-1 coactivator peptide showed that the fluorine atoms of CD578 make additional contacts with Val444 and Phe448 of activation helix 12 (H12) of the zVDR and with Leu440 of the H11-H12 loop. Consequently, the SRC-1 peptide makes more contacts with the VDR-CD578 complex than with the VDR-1,25(OH)2D3 complex. These data show that fluorination not only affects degradation of an analog but can also have direct effects on H12 stabilization.     

24,24-Dimethylvitamin D3-26,23-lactones and their 2α-functionalized analogues as highly potent VDR antagonists

Novel vitamin D receptor (VDR) antagonists, 24,24-dimethyl-1α-hydroxyvitamin D₃-26,23-lactones (8 and 9) and their C2α functionalized analogues (8a-c and 9a-c) were efficiently synthesized and their biological activities were evaluated. The construction of vitamin D₃ triene skeleton was achieved by palladium-catalyzed alkenylative cyclization of A-ring precursor enyne (22 and 22a-c) with CD-ring bromoolefin having a 24,24-dimethyl-α-methylene-γ-lactone unit on the side chain (13 and 14). The CD-ring precursors 13 and 14 were prepared by using chromium-mediated allylation of the aldehyde 10 derived from vitamin D₂. On the other hand, the A-ring enyne having 2α-(3-hydroxypropyl) group (22b) was newly synthesized from epoxide 15 using regio- and stereoselective alkylation methodology. The potency of the antagonistic activity of the newly designed analogues (8 and 9) increased up to 12 times that of TEI-9647 (2). Furthermore, introduction of the three motifs, that is, a methyl (8a and 9a), an ω-hydroxypropyl (8b and 9b) or an ω-hydroxypropoxyl group (8c and 9c) into the C2α position of 8 and 9, respectively, resulted in remarkable enhancement, up to 89 times, of the antagonistic activity on VDR.     

hCB2 ligand-interaction landscape: cysteine residues critical to biarylpyrazole antagonist binding motif and receptor modulation

The human cannabinoid 2 GPCR (hCB2) is a prime therapeutic target. To define potential cysteine-related binding motifs critical to hCB2-ligand interaction, a library of hCB2 cysteine-substitution mutants and a novel, high-affinity biarylpyrazole hCB2 antagonist/inverse agonist (AM1336) functionalized to serve as a covalent affinity probe to target cysteine residues within (or in the microenvironment of) its hCB2 binding pocket were generated. The data provide direct experimental demonstration that both hCB2 TMH7 cysteines [i.e., C7.38(284) and C7.42(288)] are critical to optimal hCB2-AM1336 binding interaction and AM1336 pharmacological activity in a cell-based functional assay (cAMP formation). Elongating the AM1336 aliphatic side chain generated another novel?hCB2 inverse agonist that binds covalently and selectively to C7.42(288) only. Identification of specific cysteine residues critical to hCB2 ligand interaction and function informs the structure-based design of hCB2-targeted medicines.     

Real time differentiation of G-protein coupled receptor (GPCR) agonist and antagonist by two photon fluorescence laser microscopy

Receptor-based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors (GPCR) is the largest and most ubiquitous of the receptor-mediated processes. Desensitization of G-protein-coupled receptors is a fundamental mechanism regulating the cellular response to agonists. We have recently studied the agonist and antagonist of the human melanocortin receptors (hMC1, hMC3, hMC4, and hMC5 receptors), the human delta opioid receptor, and the human gluacagon receptor with the help of synthetic fluorescent labeled ligands and fluorescent protein-labeled beta-arrestin-receptors that shed new insight on cellular signaling and rapid screening of drugs in real time. It was demonstrated that stimulation of these receptors by the cognate agonist triggers the rapid internalization of ligand-receptor complexes, while the interaction of the receptor with antagonists does not follow this pathway. Furthermore, receptor internalization is dependent upon beta-arrestin, which has been shown to be responsible for the rapid desensitization of cAMP-signaling processes.     

Modulation effects of zinc on the formation of vitamin D receptor and retinoid X receptor alpha-DNA transcription complexes: analysis by microelectrospray mass spectrometry

The vitamin D receptor (VDR) binds zinc, and the activity of vitamin D dependent genes in cells is influenced by intracellular zinc concentrations. To determine whether zinc influences vitamin D action in cells by modulating the formation of VDR and retinoid x receptor alpha (RXR alpha) heterodimer-DNA complexes, we used microelectrospray ionization mass spectrometry (microESI-MS) to assess receptor-DNA interactions in the presence of varying amounts of zinc. In the absence of DNA, VDR and RXR alpha proteins were primarily monomeric with small amounts of protein homodimers also observed. Zn(2+) (up to 300 microM) did not change VDR or RXR alpha monomer/homodimer ratios. Mass spectra of VDR combined with RXR alpha were a sum of individual protein spectral data. Zn(2+) had no effect on the interactions of receptors. With increasing amounts of Zn(2+), additional Zn(2+) ions were detected bound to VDR and RXR alpha. microESI-MS analyses of RXR alpha in the presence of an osteopontin vitamin D DNA response element (OP-VDRE) showed RXR alpha homodimer/OP-VDRE complexes. DNA-protein complex formation increased on addition of Zn(2+) up to 200 microM; at 300 microM, Zn(2+) dissociation of the RXR alpha homodimer/OP-VDRE complexes occurred, coincident with the appearance of RXR alpha monomeric protein. When microESI-MS analyses were carried out with VDR and OP-VDRE, VDR homodimer/OP-VDRE complexes were not detected. Addition of Zn(2+) did not result in VDR/OP-VDRE complex formation. Heterodimeric VDR/RXR alpha complexes with OP-VDRE were detected by microESI-MS. Addition of 300 microM Zn(2+) resulted in dissociation of the heterodimeric VDR/RXR alpha/OP-VDRE complex. Addition of Mg(2+) in place of Zn(2+) did not alter protein/OP-VDRE complexes. Our results show that zinc modulates steroid hormone receptor-DNA interactions.     

Interaction between a Novel Oligopeptide Fragment of the Human Neurotrophin Receptor TrkB Ectodomain D5 and the C-Terminal Fragment of Tetanus Neurotoxin

This article presents experimental evidence and computed molecular models of a potential interaction between receptor domain D5 of TrkB with the carboxyl-terminal domain of tetanus neurotoxin (Hc-TeNT). Computational simulations of a novel small cyclic oligopeptide are designed, synthesized, and tested for possible tetanus neurotoxin-D5 interaction. A hot spot of this protein-protein interaction is identified in analogy to the hitherto known crystal structures of the complex between neurotrophin and D5. Hc-TeNT activates the neurotrophin receptors, as well as its downstream signaling pathways, inducing neuroprotection in different stress cellular models. Based on these premises, we propose the Trk receptor family as potential proteic affinity receptors for TeNT. In vitro, Hc-TeNT binds to a synthetic TrkB-derived peptide and acts similar to an agonist ligand for TrkB, resulting in phosphorylation of the receptor. These properties are weakened by the mutagenesis of three residues of the predicted interaction region in Hc-TeNT. It also competes with Brain-derived neurotrophic factor, a native binder to human TrkB, for the binding to neural membranes, and for uptake in TrkB-positive vesicles. In addition, both molecules are located together in vivo at neuromuscular junctions and in motor neurons.