Discovery of bilirubin as novel P2X7R antagonist with anti-tumor activity.

As a unique ligand gated ion channel in the P2-receptor family, P2X7R is highly expressed in various tumors. The activated P2X7R facilitates tumor growth and metastasis. Hypoxia, inflammation and necrosis in the tumor microenvironment (TME) cause a large amount of adenosine triphosphate (ATP) accumulated in the TME. High concentration of ATP can abnormally activate P2X7R, which induces pore formation and further facilitates the Ca2+ ion influx and non-specific substance intake. Therefore, inhibition of P2X7R activation can be applied as a potential anti-tumor therapy strategy. However, there is currently no FDA approved drugs for this target for anti-tumor treatment. In this study, we identified bilirubin as novel P2X7R antagonist by using structure based virtual screening combined with cell based assays. Molecular docking studies indicated that bilirubin probably interacted with P2X7R by forming hydrogen-π interactions with residues V173, E174 and K311. The compound bilirubin inhibited the P2X7R gated EB intake by cancer cells. Meanwhile, bilirubin was capable to inhibit the cell proliferation and migration of P2X7R expressed HT29 cells. The phosphorylation of mTOR, STAT3 and GSK3ß were significantly decreased when bilirubin was present. Finally, in vivo experiment exhibited the anti-tumor effect of bilirubin in the MC38 bearing mice model, but did not show tissue damage in different organs. In conclusion, bilirubin was identified as a novel P2X7R antagonist and it may have potential for anti-cancer treatment, although various functions of the molecule should be considered.

Bilirubin analogues as model compounds for exciton coupling.

A series of phycobilin analogues have been investigated in terms of coupled excitonic systems. These compounds consist of a monomer, a tetrapyrrole structurally similar to bilirubin (bR), and two conjugated bR analogues. Spectroscopic and computational methods have been used to investigate the degree of interchromophore coupling. We find the synthesised bR analogue shows stronger excitonic coupling than bR, owing to a different molecular geometry. The excitonic coupling in the conjugated molecules can be controlled by modifying the bridge side-group. New computed energy levels for bR using the DFT/MRCI method are also presented, which improve on published values and re-assign the character of excited singlet states.

In Vitro Evaluation of a Novel Synthetic Bilirubin Analog as an Antioxidant and Cytoprotective Agent for Pancreatic Islet Transplantation.

Bilirubin is a natural cytoprotective agent and physiologic doses have proven to be beneficial in various models of organ and cellular transplantation. Recently, we showed that bilirubin has protective effects in models of pancreatic islet transplantation, preventing cell death associated with islet stress and suppressing the release of damage-associated molecular patterns. Despite these promising therapeutic attributes, the natural bilirubin used in these research studies is animal-derived (porcine), making it unsuitable for clinical application. In the current study, we synthesized two bilirubin analogs that can be produced without the use of animal-derived products. Antioxidant activity for the analogs was measured using the ferric-reducing-ability-of-plasma (FRAP) and 2,2V-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) assays. Dose-dependent cytotoxicity and cytoprotective effects were then demonstrated in isolated rat islets. Compound 1 showed similar antioxidant activity to natural bilirubin. Dose-dependent cytotoxicity was seen following treatment with Compound 1 and natural bilirubin at doses >40 µM, resulting in significantly increased cell death when compared to control islets (P < 0.05) or islets treated with doses ≤20 µM (P < 0.05). Following hypoxic challenge, islet cell death was reduced in islets treated with Compound 1 at 10 µM (17.27% ± 0.26%) compared to natural bilirubin at 10 µM (51.36% ± 0.71%; P < 0.0001) or 20 µM (59.02% ± 0.83%; P < 0.0001) and control islets (36.51% ± 0.44%; P < 0.0001). Compound 1 was found to have promising antioxidant and cytoprotective effects, limiting islet cell death in a model of islet transplantation hypoxic stress. Compound 1 may serve as a synthetic drug lead for clinical islet transplantation and further evaluation of this molecule and its analogs is warranted.

Toward an amphiphilic bilirubin: the crystal structure of a bilirubin e-isomer.

A new bilirubinoid analog (1) with two methoxy beta-substituents on the lactam ring of each dipyrrinone was synthesized and examined spectroscopically. It is more soluble in CH3OH and CHCl3 than bilirubin, which is insoluble in CH3OH but soluble in CHCl3. The solubility of 1 is approximately 10 microg/mL in CH3OH (vs < or =1 microg/mL for bilirubin) and approximately 3 mg/mL in CHCl3 (vs approximately 0.6 mg/mL for bilirubin). Vapor pressure osmometry indicates that 1, like bilirubin, is monomeric in CHCl3, and NMR studies show that the most stable structure has the syn-4Z,syn-15Z configuration, with the pigment's dipyrrinones engaged in intramolecular hydrogen bonding to the propionic acid carboxyl groups. And, like bilirubin, Z,Z-1 adopts a conformation that is bent in the middle into a ridge-tile shape. For the first time, a crystal structure of a bilirubin E-isomer has been obtained. Crystallization of 1 under dim room lighting gave an X-ray quality crystal of the anti-4E,syn-15Z-(photo) isomer, in which only the Z-dipyrrinone half is engaged in intramolecular hydrogen bonding to a propionic acid. Hydrogen bonding is nearly completely disengaged in the E-dipyrrinone half; yet, the ridge-tile conformation persists.

Influence of conformation and intramolecular hydrogen bonding on the acyl glucuronidation and biliary excretion of acetylenic bis-dipyrrinones related to bilirubin.

Glucuronidation and transporter-mediated efflux into bile are important in the elimination of xeno- and endobiotics, including the natural biladienone pigment bilirubin. The mechanisms of these processes and the structural factors that dictate whether cholephilic compounds are excreted directly in bile or require prior glucuronidation are poorly understood. To investigate effects of molecular shape and intramolecular hydrogen bonding on the interplay between direct excretion and glucuronidation in the liver, we studied a series of novel synthetic exploded and homologated bilirubin analogues. These include dicarboxylic mono- and diacetylenic tetrapyrroles with linear shapes that are unable to adopt the folded ridge-tile conformations that are crucially important in bilirubin metabolism. Intramolecular hydrogen bonding was varied by adjusting the alkyl chain lengths of the pendent carboxyl groups, and preferred conformations were predicted by molecular dynamics calculations. Metabolism studies were done in rats, including Gunn rats, congenitally deficient in UGT1 glucuronosyl tranferases, and TR- rats, deficient in the canalicular transporter Mrp2 (Abcc2). The results show strikingly that minor, seemingly inconsequential, changes in constitution, amplified by their influence on hydrogen bonding and molecular conformation, can profoundly influence competing clearance pathways in the liver, an effect that is unlikely to be restricted to bis-dipyrrinone carboxylic acids. Exposed carboxyl groups seem to favor the direct route of elimination, whereas the potential for carboxyl infolding by hydrogen bonding seems to favor glucuronidation. The results also show that molecular shape is less important in the hepatic glucuronidation and biliary excretion of bilirubin and of this series of acids than the capacity for intramolecular hydrogen bonding.

Novel benzoic acid congeners of bilirubin.

Three regioisomeric bilirubins and biliverdins with propionic acids replaced by benzoic acids were synthesized from the corresponding xanthobilirubic acids by oxidative coupling. The rubins were found to exhibit widely varying polarity, spectroscopic properties, and stereochemistry. The isomer with ortho benzoic acids (1o) was much less polar than either the meta (1m) or para (1p) because 1o (but not 1m and 1p) can adopt a folded conformation with both carboxylic acids intramolecularly hydrogen bonded to the opposing dipyrrinones. The consequences of such conformational differentiation are found in the varying 1H NMR, UV-vis, and circular dichroism spectral properties.

Synthesis and spectroscopic properties of a new class of strongly fluorescent dipyrrinones.

A new, highly fluorescent (Phi(F) > or = 0.8) chromophore has been synthesized in one step from dipyrrinones by reaction with N,N-carbonyldiimidazole to form the 3H,5H-dipyrrolo[1,2-c:2',1'-f]pyrimidine-3,5-dione nucleus.

Sodium etiobilirubin-IVgamma-C10-sulfonate: a highly solvated bile pigment structure containing two different non-ridge-tile conformers in the unit cell.

The crystal structure of the title compound is the first example of a bilirubin existing in both extended and cyclic conformations and the first bile pigment structure showing two markedly different conformations in the unit cell. In contrast to previous rubin structures the dipyrrinone rings are twisted out of planarity in both conformers. Because of numerous hydrogen-bonding and ionic interactions a highly complex tetrameric structure is observed in which each extended conformer is held pincer-like by another.

Synthesis and metabolism of the first thia-bilirubin.

A symmetrical C(10)-thiabilirubin analogue, 8,12-bis(2-carboxyethyl)-2,3,17,18-tetraethyl-7,13-dimethyl-10-thia-(21H,23H,24H)-bilin-1,19-dione (1), was synthesized from 8-(2-carboxyethyl)-2,3-diethyl-7-methyl-10H-dipyrrin-1-one in one step by reaction with sulfur dichloride. The thia-rubin exhibited the expected IR, UV-vis, and NMR spectroscopic properties, which are rather similar to those of mesobilirubin-XIIIalpha. Like bilirubin and mesobilirubin, 1 adopts an intramolecularly hydrogen-bonded conformation, shaped like a ridge-tile but with a steeper pitch. The longer C-S bond lengths and smaller bond angles at C-S-C, as compared to C-CH(2)-C, lead to an interplanar angle between the two dipyrrinones of only 74 degrees -or considerably less than that of bilirubin (approximately 100 degrees). On normal- and reversed-phase chromatography, 1 is substantially less polar than bilirubin. Despite this conformational distortion, 1 is metabolized in normal rats to acyl glucuronides, which are secreted into bile. In mutant (Gunn) rats lacking bilirubin glucuronosyl transferase, 1 (like bilirubin) was not excreted in bile.

Effects of serum-isolated vs synthetic bilirubin-albumin complexes on dye-binding methods for estimating serum albumin.

Bush and Reed reported (Clin Chem 1987;33:821-3) that the reaction of albumin with bromcresol purple but not with bromcresol green underestimated the concentration of albumin in synthetically obtained bilirubin-albumin (Bd) by 29%. Their unproven assumption was that chemically synthesized Bd behaved in a manner indistinguishable from the natural Bd in icteric serum. Here we verify that Bd, whether synthetically obtained or isolated from serum, causes an underestimation of albumin in the bromcresol purple but not in the bromcresol green method. The molar ratio of Bd from either source to underestimated albumin approximates 1.0, suggesting that one molecule of Bd would react equivalently to a molecule of albumin in the bromcresol purple method. This underestimation might falsely suggest hypoalbuminemia in patients with increased serum Bd.

Synthesis, chromatographic purification, and analysis of isomers of biliverdin IX and bilirubin IX.

Neutral solvent systems were developed to isolate the alpha, beta, gamma, and delta isomers of biliverdin IX dimethyl ester by TLC. The individual free acids of biliverdin IX were obtained by saponification of the corresponding dimethyl esters. The bilirubin IX isomers were prepared by reducing the corresponding biliverdin IX isomers with NaBH3CN. Starting from a pure biliverdin IX dimethyl ester, the corresponding free acid of biliverdin IX or bilirubin IX was available within 3-4 h. Preparation of spectrally pure bile pigment required final TLC on acid-cleaned neutral TLC plates. The absorption spectra of the free acids and dimethyl esters of biliverdin IX in methanol showed a broad band at about 650 nm and a sharp band at about 375 nm. The long-wave-length band was extremely sensitive to the presence of strong acid. A 10-fold molar excess of HCl caused a 35- to 50-nm shift of the absorption maximum to longer wavelengths and near doubling of the maximum absorption. The molar absorption coefficients of biliverdins were identical for each free acid and dimethyl ester pair. In each case, Beer's law was followed in both methanol and acidified methanol. Methanol also proved to be a suitable solvent for spectroscopic determination of the non-alpha isomers of bilirubin IX. The wavelength of maximum absorption and molar absorption coefficient of each dipyrrolic ethyl anthranilate azo pigment derived from the various bilirubin IX isomers are also reported.

EDTA solutions for the dissolution of calcium bilirubinate stones.

The calcium chelating of different EDTA solutions was investigated after addition of 5.49% calcium bilirubinate powder. Calcium chelating seems to be an essential step in disaggregation of pigment stones. We found that Ca chelating is pH dependent and that the system must be buffered in order to prevent pH shifting during the dissolution process. When an alkaline (pH 9.4), buffered 1% EDTA solution is mixed with bile in a ratio of 70:30, dissolution of Ca will decrease to 60% as compared to investigations without bile. Furthermore, we could demonstrate that the EDTA solution would not only dissolve Ca bilirubinate powder, but complete bovine pigment stones of homogeneous structure and mostly inhomogenously composed stones of man as well.

Photobilirubin II.

An improved preparation of photobilirubin II in ammoniacal methanol is described. Evidence is presented which distinguishes between the two structures proposed earlier for photobilirubin II in favour of the cycloheptadienyl structure. Nuclear-Overhauser-enhancement measurements with bilirubin IX alpha and photobilirubin II in dimethyl sulphoxide are complicated by the occurrence of negative and zero effects. The partition coefficient of photobilirubin II between chloroform and phosphate buffer (pH 7.4) is 0.67.

Synthesis of biliverdin and bilirubin 1-O-acyl-beta-D-glucopyranuronic acids.

Biliverdin and bilirubin mono- and di-beta-glucuronides were prepared by nucleophilic substitution of the 1-O-mesyl derivative of alpha-ethoxyethyl-protected glucuronic acid (compound II) with the tetrabutylammonium salts of biliverdin and bilirubin. Removal of the acetal-protecting groups by mild acid treatment yielded biliverdin glucuronides, which were reduced to bilirubin glucuronides. Depending on reaction conditions the pure beta-anomers or mixtures highly enriched in the beta-anomers were obtained. The biliverdin and bilirubin glucuronides were identical with pigments derived from bile. They were characterized as the IX alpha isomers and the beta-anomers by alkaline hydrolysis, n.m.r. spectroscopy, hydrolysis with beta-glucuronidase and conversion into dipyrrolic azopigments. Model reactions of the 1-O-mesylate (II) with other nucleophiles also were performed, i.e. the acetate anion and various alcohols.

Synthesis and separation by thin-layer chromatography of bilirubin-IX isomers. Their identification as tetrapyrroles and dipyrrolic ethyl anthranilate azo derivatives.

Procedures for the synthesis, separation and determination of structure of the bilirubin-IX isomers are described. 1. The four biliverdin-IX isomers were prepared by oxidative cleavage of haemin and were separated as their dimethyl esters. The individual esters were reduced with NaBH4, and the bilirubin esters obtained were subjected to alkaline hydrolysis yielding the corresponding bilirubin-IX isomers. 2. The bilirubin-IX isomers were structurally characterized (a) at the tetrapyrrolic stage by mass spectrometry of their trimethylsilyl derivatives and (b) by formation and structural analysis of their dipyrrolic ethyl anthranilate azo derivatives. 3. The absorption spectrum of bilirubin-IX alpha differed strikingly from the spectra of the other isomers. The presence of a pronounced shoulder around 453 nm in the spectrum of bilirubin-IXbeta allows easy differentiation from bilirubin-IXdelta. Methylation of the carboxyl groups largely eliminates the spectral differences between the IXalpha- and non-alpha isomers. 4. The bilirubin-IX isomers are conveniently separated by t.l.c. Detection and unequivocal identification is possible on a micro-scale by (a) t.l.c. with respect to reference compounds and (b) subsequent formation and t.l.c. of the more stable ethyl anthranilate azopigments. 5. Pronounced differences in polarity, i.e. solvent distribution, between the bilirubin-IX isomers indicate that a re-evaluation of conclusions reached previously with regard to the presence in, or absence from, biological fluids of some isomers and their relative amounts is needed.