Inhibition of EphA2 by Dasatinib Suppresses Radiation-Induced Intestinal Injury

Radiation-induced multiorgan dysfunction is thought to result primarily from damage to the endothelial system, leading to a systemic inflammatory response that is mediated by the recruitment of leukocytes. The Eph–ephrin signaling pathway in the vascular system participates in various disease developmental processes, including cancer and inflammation. In this study, we demonstrate that radiation exposure increased intestinal inflammation via endothelial dysfunction, caused by the radiation-induced activation of EphA2, an Eph receptor tyrosine kinase, and its ligand ephrinA1. Barrier dysfunction in endothelial and epithelial cells was aggravated by vascular endothelial–cadherin disruption and leukocyte adhesion in radiation-induced inflammation both in vitro and in vivo. Among all Eph receptors and their ligands, EphA2 and ephrinA1 were required for barrier destabilization and leukocyte adhesion. Knockdown of EphA2 in endothelial cells reduced radiation-induced endothelial dysfunction. Furthermore, pharmacological inhibition of EphA2–ephrinA1 by the tyrosine kinase inhibitor dasatinib attenuated the loss of vascular integrity and leukocyte adhesion in vitro. Mice administered dasatinib exhibited resistance to radiation injury characterized by reduced barrier leakage and decreased leukocyte infiltration into the intestine. Taken together, these data suggest that dasatinib therapy represents a potential approach for the protection of radiation-mediated intestinal damage by targeting the EphA2–ephrinA1 complex.     

Constituents of human meconium: II. Identification of steroidal acids with 21 and 22 carbon atoms

Monohydroxylated acid fraction isolated from human meconium was found to contain, in addition to C₂₀ and C₂₄ acids identified previously, three C₂₂ bile acids-(20S)-3α-hydroxy-23,24-bisnor-5β-cholan-22-oic, (20S)- and (20R)-3β-hydroxy-23,24-bisnor-chol-5-en-22-oic, and one C₂₁ acid-3β-hydroxypregn-5-en-21-oic. These compounds were identified by capillary gas chromatography-mass spectrometry and by comparison with standards. It is postulated that these C₂₂ acids, as well as the two monohydroxylated C₂₄ bile acids (lithocholic and 3β-hydroxychol-5-enoic) are produced in the maternal intestine by microbial flora and transferred to the fetus through the placenta.     

Target Hopping as a Useful Tool for the Identification of Novel EphA2 Protein–Protein Antagonists

Lithocholic acid (LCA), a physiological ligand for the nuclear receptor FXR and the G‐protein‐coupled receptor TGR5, has been recently described as an antagonist of the EphA2 receptor, a key member of the ephrin signalling system involved in tumour growth. Given the ability of LCA to recognize FXR, TGR5, and EphA2 receptors, we hypothesized that the structural requirements for a small molecule to bind each of these receptors might be similar. We therefore selected a set of commercially available FXR or TGR5 ligands and tested them for their ability to inhibit EphA2 by targeting the EphA2‐ephrin‐A1 interface. Among the selected compounds, the stilbene carboxylic acid GW4064 was identified as an effective antagonist of EphA2, being able to block EphA2 activation in prostate carcinoma cells, in the micromolar range. This finding proposes the “target hopping” approach as a new effective strategy to discover new protein–protein interaction inhibitors.     

Bile acids in tissues: Binding of lithocholic acid to protein

Human liver contains two forms of lithocholic acid. One form is readily extractable by 95% ethanol/0.1% ammonia (soluble lithocholate, SL), while the other remains firmly bound to the residue (tissue-bound lithocholate, TBL). TBL could be hydrolytically released using clostridial cholanoylamino acid hydrolase, suggesting a peptide link between lithocholate and protein. With bovine serum albumin (BSA), lithocholic acid showed spontaneous amino group-modifying activity. When small molecular weight lysine (α-t-BOC-1-lysyl-β-naphthylamide) and arginine peptides (α-CBZ-di-arginyl-β-naphthylamide) were used in place of BSA, lithocholate bound specifically to the lysine peptide. The unusual affinity for lysine suggested that this amino acid might be involved as a residue in TBL. Synthesis of lithocholyl lysines and comparison with products of acid hydrolysis of TBL established ε-lithocholyl lysine as the predominant form in which lithocholic acid is found in tissue bound form. Supported in part by the Gomprecht Hepatitis Fund. The systematic nomenclature of bile acids referred to in this report by trivial names are as follows: Cholanic acid, 5β-cholan-24-oic acid; lithocholic acid, 3α-hydroxy-5β-cholan-24-oic acid; 3-epilithocholic acid, 3β-hydroxy-5β-cholan-24-oic acid; glycolithocholic acid, 3α-hydroxy-5β-cholan-24-oyl glycine; 3-ketocholanic acid, 3-keto-5β-cholan-24-oic acid; 12α-hydroxycholanic acid, 12α-hydroxy-5β-cholan-24-oic acid; chenodeoxycholic acid, 3α, 7α-dihydroxy-5β-cholan-24-oic acid; glycochenodeoxycholic acid, 3α, 7α-dihydroxy-5β-cholan-24-oyl glycine; deoxycholic acid, 3α, 12α-dihydroxy-5β-cholan-24-oic acid; glycodeoxycholic acid, 3α, 12α-dihydroxy-5β-cholan-24-oyl glycine; cholic acid, 3α, 7α, 12α-trihydroxy-5β-cholan-24-oic acid, glycocholic acid, 3α, 7α, 12α-trihydroxy-5β-cholan-24-oyl glycine; dehydrocholic acid, 3, 7, 12-triketo-5β-cholan-24-oic acid.     

氧雄龙的合成

以甲基表雄醇为起始原料,环保高效地合成了氧雄龙。首先采用环保的温和氧化剂2-iodoxybenzoicacid(IBX)在65～70℃,氧化甲基表雄醇直接合成α,β-不饱和羰基甾体-17α-甲基-1-睾酮;经-30～-40℃臭氧化后,用氢氧化钠溶液碱性水解得到17β-羟基-17α-甲基-1-氧代-1,2-开环-A-失碳-5α-雄甾-2-含氧羧酸;最后在0～10℃,经硼氢化钠碱性条件下还原后,调节pH=1～2,内酯化反应合成了目的物。3步操作的产率分别为72%、69%及86%。中间体和目的物经红外光谱、核磁共振氢谱、质谱及元素分析确证了其化学结构。     

Improved Chemical Synthesis,X-Ray Crystallographic Analysis,and NMR Characterization of (22R)-/(22S)-Hydroxy Epimers of Bile Acids

We report an improved synthesis of the (22R)- and (22S)-epimers of 3α,7α,12α,22-tetrahydroxy-5β-cholan-24-oic acid and 3α,7α,22-trihydroxy-5β-cholan-24-oic acid from cholic acid (CA) and chenodeoxycholic acid (CDCA), respectively. The principal reactions involved were as follows: (1) oxidative decarboxylation of the bile acid peracetates with lead tetraacetate, and (2) subsequent Reformatsky reaction of the 23,24-dinor-22-aldehydes with ethyl bromoacetate in the presence of activated Zn as a catalyst with the reaction temperature maintained precisely at 75 °C. The absolute configuration of the chiral center at C-22 of each epimer was established by single-crystal X-ray diffraction data using its ethyl ester-peracetate derivative. The ¹H- and ¹³C-NMR spectra that permit the (22R)- and (22S)-epimers to be distinguished are reported as well as the specific ¹H shift effects induced by C₅D₅N. Bile acids having hydroxyl groups at C-22 are present in a variety of animal biles, previously have been difficult to identify, and are known to have distinctive physicochemical and biological properties.     

耳叶紫菀化学成分及生物活性研究

首次对耳叶紫菀(Aster auriculatus Franch)全草的化学成分及生物活性进行研究,采用柱层析(CC)对植物中的提取物进行分离提纯,并利用现代波谱方法(1H-NMR,13C-NMR,MS等)分析鉴定,确定化合物的结构并对其生物活性进行筛选。从该植物的氯仿部分分离得到8个倍半萜类化合物,它们的结构分别鉴定为:Ilicic acid(1),(7R,10S)-selina-4,11(13)-dien-3-on-12-oic acid(2),2-oxo-isocostic acid(3),1β,5α-diangeloyloxy-eudesm-(15)-ene(4),1β,6α-dihydroxyeudesm-4(15)-ene(5),4(15)-eudesmene-1β,7α-diol(6),Furanoligularenone(7),(4αR,5S,8αR)-4α,5,6,7,8,8α-Hexahydro-8α-hydroxy-3,4α,5-trimethylnaphtho[2,3-b]furan-2(4H)-one(8)。并采用杯碟法对以上8个化合物进行生物活性筛选,通过观测各供试菌抑菌圈的半径,发现化合物2、化合物4对大肠杆菌(Escherichia coli)有较强的抑制作用,所有化合物均为首次从该植物中分离得到。     

Heterotypic Sam–Sam Association between Odin‐Sam1 and Arap3‐Sam: Binding Affinity and Structural Insights

Arap3 is a phosphatidylinositol 3 kinase effector protein that plays a role as GTPase activator (GAP) for Arf6 and RhoA. Arap3 contains a sterile alpha motif (Sam) domain that has high sequence homology with the Sam domain of the EphA2‐receptor (EphA2‐Sam). Both Arap3‐Sam and EphA2‐Sam are able to associate with the Sam domain of the lipid phosphatase Ship2 (Ship2‐Sam). Recently, we reported a novel interaction between the first Sam domain of Odin (Odin‐Sam1), a protein belonging to the ANKS (ANKyrin repeat and Sam domain containing) family, and EphA2‐Sam. In our latest work, we applied NMR spectroscopy, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize the association between Arap3‐Sam and Odin‐Sam1. We show that these two Sam domains interact with low micromolar affinity. Moreover, by means of molecular docking techniques, supported by NMR data, we demonstrate that Odin‐Sam1 and Arap3‐Sam might bind with a topology that is common to several Sam‐Sam complexes. The revealed structural details form the basis for the design of potential peptide antagonists that could be used as chemical tools to investigate functional aspects related to heterotypic Arap3‐Sam associations.     

The metabolism of lithocholic acid-3α-sulfate by human intestinal microflora

Lithocholic acid-3α-sulfate is metabolized by human intestinal microflora to nonpolar metabolites which have been partially purified by Sephadex LH-20 chromatography. These metabolites were characterized by thin layer and gas liquid chromatography as well as combined gas liquid chromatography-mass spectrometry. The chromatographic properties of one of the metabolites are consistent with those described for a Δ²-or Δ³. The formation of cholenates by the microflora may represent a retoxification of the sulfate ester of lithocholic acid. The following names and abbreviations for chemicals and methods have been used throughout the text: lithocholic acid (LA)=3α-hydroxy-5β-cholan-24-oic acid; isolithocholic acid (ILA)=3β-hydroxy-5β-cholan-24-oic acid; 3-keto=3-keto-5β-cholan-24-oic acid; 5β=5β-cholan-24-oic acid; Δ²-cholenate; = 5β-chol-2-en-24-oate; Δ³-cholenate =5β-chol-3-en-24-oate LASO₄=lithocholic acid-3α-sulfate; methyl lithocholate (MLA)=methyl-3α-hydroxy-5β-cholan-24-oate; methyl isolithocholate (MIL)=methyl-3β-hydroxy-5β-cholan-24-oate; methyl-3-keto (Me-3-keto)=methyl-3-keto-5β-cholan-24-oate; methyl-5β (Me-5β)=methyl-5β-cholan-24-oate; methyl deoxycholate (MEDOXY)=methyl-3α12α-dihydroxy-5β-cholan-24-oate; GLC=gas liquid chromatography; GLC-MS=gas liquid chromatography-mass spectrometry; GFP=glass fiber paper; ITLC-SG=instant thin layer chromatography-silica gel; ITLC-SA=instant thin layer chromatography-silicic acid; BHI=brain heart infusion; MFC=mixed fecal culture.     

Triterpenes from the Outer Bark of Betula nigra

Twelve pentacyclic triterpenes were isolated from the outer bark of river birch, Betula nigra. 3β-Acetoxyolean-11-oxo-12-ene-28-oic acid was isolated for the first time from a Betula species. 3β-Caffeatoxyolean-12-ene-28-oic acid has been spectrally characterized for the first time. 3β-Acetoxyolean-12-ene-28-oic acid and 3β-acetoxyolean-11-oxo-12-ene-28-oic acid have been demonstrated to be active as antifeedants for the Colorado potato beetle, Leptinotarsa decemlineata.     

Hunting for Novel Routes in Anticancer Drug Discovery: Peptides against Sam-Sam Interactions

Among the diverse protein binding modules, Sam (Sterile alpha motif) domains attract attention due to their versatility. They are present in different organisms and play many functions in physiological and pathological processes by binding multiple partners. The EphA2 receptor contains a Sam domain at the C-terminus (EphA2-Sam) that is able to engage protein regulators of receptor stability (including the lipid phosphatase Ship2 and the adaptor Odin). Ship2 and Odin are recruited by EphA2-Sam through heterotypic Sam-Sam interactions. Ship2 decreases EphA2 endocytosis and consequent degradation, producing chiefly pro-oncogenic outcomes in a cellular milieu. Odin, through its Sam domains, contributes to receptor stability by possibly interfering with ubiquitination. As EphA2 is upregulated in many types of tumors, peptide inhibitors of Sam-Sam interactions by hindering receptor stability could function as anticancer therapeutics. This review describes EphA2-Sam and its interactome from a structural and functional perspective. The diverse design strategies that have thus far been employed to obtain peptides targeting EphA2-mediated Sam-Sam interactions are summarized as well. The generated peptides represent good initial lead compounds, but surely many efforts need to be devoted in the close future to improve interaction affinities towards Sam domains and consequently validate their anticancer properties.     

Development of improved PPARβ/δ inhibitors

GSK0660 (1) is the first peroxisome proliferator-activated receptor (PPAR) β/δ-selective inhibitory ligand described in the literature. Based on its structure, we designed and synthesized a series of modified compounds to establish preliminary structure-activity relationships. Most beneficial for increased binding affinity towards the PPARβ/δ ligand binding domain was the replacement of the 4'-aminophenyl substituent by medium-length n-alkyl chains, such as n-butyl or iso-pentyl. These compounds show activity down to the one-digit nanomolar range, thus possessing up to a tenfold higher binding affinity compared with GSK0660. Additionally, the subtype-specific inhibition of PPARβ/δ was confirmed in a cell-based assay making these compounds invaluable tools for the further exploration of the functions of PPARβ/δ.     

Discovery of Tyrosine Kinase Inhibitors by Docking into an Inactive Kinase Conformation Generated by Molecular Dynamics

Several small molecules that bind to the inactive DFG‐out conformation of tyrosine kinases (called type II inhibitors) have shown a good selectivity profile over other kinase targets. To obtain a set of DFG‐out structures, we performed an explicit solvent molecular dynamics (MD) simulation of the complex of the catalytic domain of a tyrosine kinase receptor, ephrin type‐A receptor 3 (EphA3), and a manually docked type II inhibitor. Automatic docking of four previously reported type II inhibitors was used to select a single snapshot from the MD trajectory for virtual screening. High‐throughput docking of a pharmacophore‐tailored library of 175 000 molecules resulted in about 4 million poses, which were further filtered by van der Waals efficiency and ranked according to a force‐field‐based energy function. Notably, around 20 % of the compounds with predicted binding energy smaller than ?10 kcal mol^?1 are known type II inhibitors. Moreover, a series of 5‐(piperazine‐1‐yl)isoquinoline derivatives was identified as a novel class of low‐micromolar inhibitors of EphA3 and unphosphorylated Abelson tyrosine kinase (Abl1). The in silico predicted binding mode of the new inhibitors suggested a similar affinity to the gatekeeper mutant T315I of Abl1, which was verified in vitro by using a competition binding assay. Additional evidence for the type II binding mode was obtained by two 300 ns MD simulations of the complex between N‐(3‐chloro‐4‐(difluoromethoxy)phenyl)‐2‐(4‐(8‐nitroisoquinolin‐5‐yl)piperazin‐1‐yl)acetamide and EphA3.     

Development of a Metabolically Stable Neurotensin Receptor 2 (NTS2) Ligand

Subtype‐selective neurotensin receptor 2 (NTS2) ligands can be used as molecular probes to investigate the physiological role of neurotensinergic systems and serve as lead compounds to initiate the development of drugs for the treatment of tonic pain. Starting from our recently described NTS2 ligand 1 , structural variants of type 2 were synthesized to further improve binding affinity and selectivity to gain metabolic stability. The peptide–peptoid hybrid 2 b showed excellent NTS2 binding affinity (K_i=2.8 nM ) and 22 000‐fold selectivity over NTS1, as well as metabolic stability over 32 h in a serum degradation assay. Employing a MAPK‐driven luciferase reporter gene assay and an IP accumulation assay, the neurotensin mimetic 2 b displayed respective inhibitions of constitutive activity exceeding 4.3‐ and 3.9‐fold that of the inverse agonist activity of the endogenous ligand neurotensin.     

Chemical Synthesis of Rare Natural Bile Acids: 11α‐Hydroxy Derivatives of Lithocholic and Chenodeoxycholic Acids

A method for the preparation of 11α‐hydroxy derivatives of lithocholic and chenodeoxycholic acids, recently discovered to be natural bile acids, is described. The principal reactions involved were (1) elimination of the 12α‐mesyloxy group of the methyl esters of 3α‐acetate‐12α‐mesylate and 3α,7α‐diacetate‐12α‐mesylate derivatives of deoxycholic acid and cholic acid with potassium acetate/hexamethylphosphoramide; (2) simultaneous reduction/hydrolysis of the resulting △¹¹‐3α‐acetoxy and △¹¹‐3α,7α‐diacetoxy methyl esters with lithium aluminum hydride; (3) stereoselective 11α‐hydroxylation of the △¹¹‐3α,24‐diol and △¹¹‐3α,7α,24‐triol intermediates with B₂H₆/tetrahydrofuran (THF); and (4) selective oxidation at C‐24 of the resulting 3α,11α,24‐triol and 3α,7α,11α,24‐tetrol to the corresponding C‐24 carboxylic acids with NaClO₂ catalyzed by 2,2,6,6‐tetramethylpiperidine 1‐oxyl free radical (TEMPO) and NaClO. In summary, 3α,11α‐dihydroxy‐5β‐cholan‐24‐oic acid and 3α,7α,11α‐trihydroxy‐5β‐cholan‐24‐oic acid have been synthesized and their nuclear magnetic resonance (NMR) spectra characterized. These compounds are now available as reference standards to be used in biliary bile acid analysis.     

Synthesis, structure, and biological activity of des-side chain analogues of 1α,25-dihydroxyvitamin D3 with substituents at C18

An improved synthetic route to 1α,25‐dihydroxyvitamin D₃ des‐side chain analogues 2 a and 2 b with substituents at C18 is reported, along with their biological activity. These analogues display significant antiproliferative effects toward MCF‐7 breast cancer cells and prodifferentiation activity toward SW480‐ADH colon cancer cells; they are also characterized by a greatly decreased calcemic profile. The crystal structure of the human vitamin D receptor (hVDR) complexed to one of these analogues, 20(17→18)‐abeo‐1α,25‐dihydroxy‐22‐homo‐21‐norvitamin D₃ ( 2 a ) reveals that the side chain introduced at position C18 adopts the same orientation in the ligand binding pocket as the side chain of 1α,25‐dihydroxyvitamin D₃.     

Targeting EphA2‐Sam and Its Interactome: Design and Evaluation of Helical Peptides Enriched in Charged Residues

The EphA2 receptor controls diverse physiological and pathological conditions and its levels are often upregulated in cancer. Targeting receptor overexpression, through modulation of endocytosis and consequent degradation, appears to be an appealing strategy for attacking tumor malignancy. In this scenario, the Sam domain of EphA2 plays a pivotal role because it is the site where protein regulators of endocytosis and stability are recruited by means of heterotypic Sam–Sam interactions. Because EphA2‐Sam heterotypic complexes are largely based on electrostatic contacts, we have investigated the possibility of attacking these interactions with helical peptides enriched in charged residues. Several peptide sequences with high predicted helical propensities were designed, and detailed conformational analyses were conducted by diverse techniques including NMR, CD, and molecular dynamics (MD) simulations. Interaction studies were also performed by NMR, surface plasmon resonance (SPR), and microscale thermophoresis (MST) and led to the identification of two peptides capable of binding to the first Sam domain of Odin. These molecules represent early candidates for the generation of efficient Sam domain binders and antagonists of Sam–Sam interactions involving EphA2.     

An Engineered Biocatalyst for the Synthesis of Glycoconjugates: Utilization of β1,3‐N‐Acetyl‐D‐glucosaminyltransferase from Streptococcus agalactiae Type Ia Expressed in Escherichia coli as a Fusion with Maltose‐Binding Protein

A fusion protein composed of β1,3‐N‐acetyl‐D ‐glucosaminyltransferase (β1,3‐GlcNAcT) from Streptococcus agalactiae type Ia and maltose‐binding protein (MBP) was produced in Escherichia coli as a soluble and highly active form. Although this fusion protein (MBP‐β1,3‐GlcNAcT) did not show any sugar‐elongation activity to some simple low‐molecular weight acceptor substrates such as galactose, Galβ(1→4)Glc (lactose), Galβ(1→4)GlcNAc (N‐acetyllactosamine), Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)Glc (lacto‐N‐tetraose), and Galβ(1→4)GlcβCer (lactosylceramide, LacCer), the multivalent glycopolymer having LacCer‐mimic branches (LacCer mimic polymer, LacCer primer) was found to be an excellent acceptor substrate for the introduction of a β‐GlcNAc residue at the O‐3 position of the non‐reducing galactose moiety by this engineered enzyme. Subsequently, the polymer having GlcNAcβ(1→3)Galβ(1→4)Glc was subjected to further enzymatic modifications by using recombinant β1,4‐D ‐galactosyltransferase (β1,4‐GalT), α2,3‐sialyltransferase (α2,3‐SiaT), α1,3‐L ‐fucosyltransferase (α1,3‐FucT), and ceramide glycanase (CGase) to afford a biologically important ganglioside; Neu5Aα(2→3)Galβ(1→4)[Fucα(1→3)]GlcNAcβ(1→3)Galβ(1→4)GlcCerα(IV3Neu5Acα,III3Fucα‐nLc4Cer) in 40% yield (4 steps). Interestingly, it was suggested that MBP‐β1,3‐GlcNAcT could also catalyze a glycosylation reaction of the LacCer mimic polymer with N‐acetyl‐D ‐galactosamine served from UDP‐GalNAc to afford a polymer carrying trisaccharide branches, GalNAcβ(1→3)Galβ(1→4)Glc. The versatility of the MBP‐β1,3‐GlcNAcT in the practical synthesis was preliminarily demonstrated by applying this fusion protein as an immobilized biocatalyst displayed on the amylose resin which is known as a solid support showing potent binding‐affinity with MBP.     

The metabolism of lithocholic acid and lithocholic acid-3-α-sulfate by human fecal bacteria

Both lithocholic acid and lithocholic acid-3α-sulfate are metabolized by mixed fecal bacteria and by pure strains of the genusClostridium. Mixed fecal bacteria metabolized lithocholic acid to 3-ketolithocholic acid; lithocholic acid-3α-sulfate was metabolized to isolithocholic acid, 5β-cholanic acid and Δ³-cholenic acid under both aerobic and anaerobic conditions. The results indicate that a specific genus, theClostridium, has a primary role in the metabolism of lithocholic acid-3α-sulfate to Δ³-cholenic acid     

An indole-binding site is a major determinant of the ligand specificity of the GABA type A receptor-associated protein GABARAP

The role of tryptophan as a key residue for ligand binding to the ubiquitin-like modifier GABA(A) receptor associated protein (GABARAP) was investigated. Two tryptophan-binding hydrophobic patches were identified on the conserved face of the GABARAP structure by NMR spectroscopy and molecular docking. GABARAP binding of indole and indole derivatives, including the free amino acid tryptophan was quantified. The two tryptophan binding sites can be clearly distinguished by mapping the NMR spectroscopy-derived residue-specific apparent dissociation constant, K(d), onto the three-dimensional structure of GABARAP. The biological relevance of tryptophan-binding pockets of GABARAP was supported by a highly conserved tryptophan residue in the GABARAP binding region of calreticulin, clathrin heavy chain, and the gamma2 subunit of the GABA(A) receptor. Replacement of tryptophan by alanine abolished ligand binding to GABARAP.