Deciphering the chemical instability of sphaeropsidin A under physiological conditions - degradation studies and structural elucidation of the major metabolite.

The fungal metabolite sphaeropsidin A (SphA) has been recognised for its promising cytotoxicity, particularly towards apoptosis- and multidrug-resistant cancers. Owing to its intriguing activity, the development of SphA as a potential anticancer agent has been pursued. However, this endeavour is compromised since SphA exhibits poor physicochemical stability under physiological conditions. Herein, SphA's instability in biological media was explored utilizing LC-MS. Notably, the degradation tendency was found to be markedly enhanced in the presence of amino acids in the cell medium utilized. Furthermore, the study investigated the presence of degradation adducts, including the identification, isolation and structural elucidation of a major degradation metabolite, (4R)-4,4',4'-trimethyl-3'-oxo-4-vinyl-4',5',6',7'-tetrahydro-3'H-spiro[cyclohexane-1,1'-isobenzofuran]-2-ene-2-carboxylic acid. Considering the reduced cytotoxic potency of aged SphA solutions, as well as that of the isolated degradation metabolite, the reported antiproliferative activity has been attributed primarily to the parent compound (SphA) and not its degradation species. The fact that SphA continues to exhibit remarkable bioactivity, despite being susceptible to degradation, motivates future research efforts to address the challenges associated with this instability impediment.

Gas-Phase Rearrangement of the O-Glucuronide of Vildagliptin Forms Product-Ion Fragments Suggesting Wrongly an N-Glucuronide.

The O-glucuronide of vildagliptin, a dipeptidyl peptidase 4 inhibitor, is a major metabolite in monkeys and a minor metabolite in humans, rats, and dogs. Its product ion spectrum shows fragments that can be explained only by an N-glucuronide. Biotransformation using rat liver yielded milligram amounts of the O-glucuronide, and its structure was assigned unambiguously by nuclear magnetic resonance. The tandem mass spectra (MS/MS) of this compound was investigated in detail using MSn and accurate mass spectrometers and was identical to the animal metabolite. Thus, the MS/MS fragments suggesting an N-glucuronide had to be formed by gas-phase rearrangement. This gas-phase rearrangement can be observed on quadrupole time-of-flight and ion-trap mass instruments. The literature on gas-phase rearrangements is reviewed.

Diselaginellin B, an Unusual Dimeric Molecule from Selaginella pulvinata, Inhibited Metastasis and Induced Apoptosis of SMMC-7721 Human Hepatocellular Carcinoma Cells.

Two new unusual dimeric selaginellins, diselaginellins A and B (1 and 2), along with two known derivatives, selaginellin (3) and selaginellin B (4), were isolated from Selaginella pulvinata. Their structures were elucidated by extensive NMR and high-resolution ESIMS data analysis. Compound 2 displayed apoptosis-inducing and antimetastatic activities against the human hepatocellular carcinoma cell line SMMC-7721. A microarray analysis revealed that genes related to metabolism, angiogenesis, and metastasis were altered by 2. The up- and down-regulation of the mRNA levels of related genes was confirmed by RT-qPCR. Metabolism modulation and metastasis inhibition might be the mechanisms of the antitumor properties of diselaginellin B (2).

Biocatalytic synthesis and structure elucidation of cyclized metabolites of the deacetylase inhibitor panobinostat (LBH589).

Panobinostat (LBH589) is a novel pan-deacetylase inhibitor that is currently being evaluated in phase III clinical trials for treatment of Hodgkin's lymphoma and multiple myeloma. Under catalysis of recombinant human CYP3A4 and CYP2D6 coexpressed with human cytochrome P450 reductase in Escherichia coli JM109, five metabolites of panobinostat were produced via whole-cell biotransformation. The structures of the metabolites were elucidated with the spectroscopic methods mass spectrometry (MS) and NMR and revealed an oxidative cyclization of the ethyl-amino group to the methylindole moiety. The MS(2) spectrum of the cyclized metabolite showed a base peak, where the closed ring is reopened and that, taken as sole base for structure proposals, would have lead to wrong conclusions. The metabolites were substantially less potent deacetylase inhibitors than the parent compound.

Ribosomally synthesized thiopeptide antibiotics targeting elongation factor Tu.

We identified the thiomuracins, a novel family of thiopeptides produced by a rare-actinomycete bacterium typed as a Nonomuraea species, via a screen for inhibition of growth of the bacterial pathogen Staphylococcus aureus. Thiopeptides are a class of macrocyclic, highly modified peptides that are decorated by thiazoles and defined by a central six-membered heterocyclic ring system. Mining the genomes of thiopeptide-producing strains revealed the elusive biosynthetic route for this class of antibiotics. The thiopeptides are chromosomally encoded, ribosomally synthesized proteins, and isolation of gene clusters for production of thiomuracin and the related thiopeptide GE2270A revealed the post-translational machinery required for maturation. The target of the thiomuracins was identified as bacterial Elongation Factor Tu (EF-Tu). In addition to potently inhibiting a target that is unexploited by marketed human therapeutics, the thiomuracins have a low propensity for selecting for antibiotic resistance and confer no measurable cross-resistance to antibiotics in clinical use.

Identification and mechanistic characterization of low-molecular-weight inhibitors for HuR.

Careful regulation of mRNA half-lives is a fundamental mechanism allowing cells to quickly respond to changing environmental conditions. The mRNA-binding Hu proteins are important for stabilization of short-lived mRNAs. Here we describe the identification and mechanistic characterization of the first low-molecular-weight inhibitors for Hu protein R (HuR) from microbial broths (Actinomyces sp.): dehydromutactin (1), MS-444 (2) and okicenone (3). These compounds interfere with HuR RNA binding, HuR trafficking, cytokine expression and T-cell activation. A mathematical and experimental analysis of the compounds' mode of action suggests that HuR homodimerizes before RNA binding and that the compounds interfere with the formation of HuR dimers. Our results demonstrate the chemical drugability of HuR; to our knowledge HuR is the first example of a drugable protein within the Hu family. MS-444, dehydromutactin and okicenone may become valuable tools for studying HuR function. An assessment of HuR inhibition as a central node in malignant processes might open up new conceptual routes toward combatting cancer.

Sanglifehrin-cyclophilin interaction: degradation work, synthetic macrocyclic analogues, X-ray crystal structure, and binding data.

Sanglifehrin A (SFA) is a novel immunosuppressive natural product isolated from Streptomyces sp. A92-308110. SFA has a very strong affinity for cyclophilin A (IC(50) = 6.9 +/- 0.9 nM) but is structurally different from cyclosporin A (CsA) and exerts its immunosuppressive activity via a novel mechanism. SFA has a complex molecular structure consisting of a 22-membered macrocycle, bearing in position 23 a nine-carbon tether terminated by a highly substituted spirobicyclic moiety. Selective oxidative cleavage of the C(26)=C(27) exocyclic double bond affords the spirolactam containing fragment 1 and macrolide 2. The affinity of 2 for cyclophilin (IC(50) = 29 +/- 2.1 nM) is essentially identical to SFA, which indicates that the interaction between SFA and cyclophilin A is mediated exclusively by the macrocyclic portion of the molecule. This observation was confirmed by the X-ray crystal structure resolved at 2.1 A of cyclophilin A complexed to macrolide 16, a close analogue of 2. The X-ray crystal structure showed that macrolide 16 binds to the same deep hydrophobic pocket of cyclophilin A as CsA. Additional valuable details of the structure-activity relationship were obtained by two different chemical approaches: (1) degradation work on macrolide 2 or (2) synthesis of a library of macrolide analogues using the ring-closing metathesis reaction as the key step. Altogether, it appears that the complex macrocyclic fragment of SFA is a highly optimized combination of multiple functionalities including an (E,E)-diene, a short polypropionate fragment, and an unusual tripeptide unit, which together provide an extremely strong affinity for cyclophilin A.