Detection and characterization of simvastatin and its metabolites in rat tissues and biological fluids using MALDI high resolution mass spectrometry approach.

Simvastatin (SV) is a hypolipidemic agent, and it is the 2nd most widely prescribed lipid-lowering drug. Here, the detection and characterization of SV and its metabolites was studied in selected organs/tissues (lung, liver, brain, heart and kidney) and biological samples (blood, urine and feces) of rats. MALDI Orbitrap MS was used as a high-resolution mass analyzer. 2,5-Dihydroxybenzoic acid (DHB) and 1,5-diaminonaphthalene (DAN) were used as matrices. Several sample loading methods onto the MALDI plate were attempted and dried droplet method was found to be superior. Two different cell disruption methods, pulverization and homogenization, were also evaluated for the optimum sensitivity in MALDI. Pulverization allowed the detection of more metabolites in all organs except the liver, where homogenization led to the detection of more metabolites. Altogether, 13 metabolites were detected, and one metabolite tentatively identified as a reduced product is being reported for the first time. SV and its metabolites were distributed to all the tissues studied except the brain. Overall, the results implied that the pulverized samples were more uniform and larger in surface area, resulting in their more efficient and complete extraction during sample preparation. As shown in the present study, MALDI Orbitrap MS is a useful tool to study drug and metabolite detection and characterization.

Galeon: A Biologically Active Molecule with In Silico Metabolite Prediction, In Vitro Metabolic Profiling in Rat Liver Microsomes, and In Silico Binding Mechanisms with CYP450 Isoforms.

Galeon, a natural cyclic-diarylheptanoid (CDH), which was first isolated from Myrica gale L., is known to have potent cytotoxicity against A549 cell lines, anti-tubercular activity against Mycobacterium tuberculosis H37Rv, chemo-preventive potential, and moderate topoisomerase inhibitory activity. Here, in silico metabolism and toxicity prediction of galeon by CYP450, in vitro metabolic profiling study in rat liver microsomes (RLMs), and molecular interactions of galeon-CYP450 isoforms were performed. An in silico metabolic prediction study showed demethyl and mono-hydroxy galeon were the metabolites with the highest predictability. Among the predicted metabolites, mono-hydroxy galeon was found to have plausible toxicities such as skin sensitization, thyroid toxicity, chromosome damage, and carcinogenicity. An in vitro metabolism study of galeon, incubated in RLMs, revealed eighteen Phase-I metabolites, nine methoxylamine, and three glutathione conjugates. Identification of possible metabolites and confirmation of their structures were carried out using ion-trap tandem mass spectrometry. In silico docking analysis of galeon demonstrated significant interactions with active site residues of almost all CYP450 isoforms.

Synthesis, Biological Evaluation and Molecular Docking Study of Cyclic Diarylheptanoids as Potential Anticancer Therapeutics.

BACKGROUND: Cancer is one of the leading causes of mortality globally. To cope with cancer, it is necessary to develop anticancer drugs. Bioactive natural products, i.e. diarylheptanoids, have gained significant attention of researchers owing to their intriguing structures and potent biological activities. In this article, considering the development of anticancer drugs with enhanced selectivity towards cancerous cells, a series of Cyclic Diarylheptanoids (CDHs) are designed, synthesized and evaluated their biological activity. OBJECTIVE: To establish an easy route for the synthesis of diarylheptanoids, and evaluate their antiproliferative, and topoisomerase-I & -IIα inhibitory activities, for developing potential anticancer drugs among CDHs. METHODS: Diarylheptanoids were synthesized from reported linear diarylheptanoids using the classical Ullmann reaction. Antibacterial activity was evaluated by the filter paper disc diffusion method. Cell viability was assessed by measuring mitochondrial dehydrogenase activity with a Cell Counting Kit (CCK-8). Topoisomerases I and II (topo-I and -IIα) inhibitory activity was measured by the assessment of relaxation of supercoiled pBR322 plasmid DNA. IFD protocol of Schrodinger Maestro v11.1 was used to characterize the binding pattern of studied compounds with the ATPase domain of the human topo-IIα. RESULTS: The synthesized CDHs were evaluated for their biological activities (antibacterial, antiproliferative, and topoisomerase-I & -IIα inhibitory activities, respectively). Leading to obtain a series of anticancer agents with the least inhibitory activities against different microbes, improving their selectivity for cancer cells. In brief, most of the synthesized CDHs had excellent antiproliferative activity against T47D (human breast cancer cell line). Pterocarine possessed the strongest activity (2i; IC50 = 0.63µM) against T47D. The cyclic diarylheptanoid 2b induced 30% inhibition of topoisomerase-IIα activity at 100µM compared with the reference of etoposide, which induced 72% inhibition. Among the tested compounds, galeon (2h) displayed very low activity against four bacterial strains. Compounds 2b, 2h, and 2i formed hydrogen bonds with Thr215, Asn91, Asn120, Ala167, Lys168 and Ile141 residues, which are important for binding of ligand compound to the ATPase binding site of topoisomerase IIα by acting as ATP competitive molecule validated by docking study. In silico Absorption, Distribution, Metabolism and Excretion (ADME) analysis revealed the predicted ADME parameters of the studied compounds which showed recommended values. CONCLUSION: A series of CDHs were synthesized and evaluated for their antibacterial, antiproliferative, and topo-I & -IIα inhibitory activities. SARs study, molecular docking study and in silico ADME analysis were conducted. Five compounds exhibited excellent and selective antiproliferative activity against the human breast cancer cell line (T47D). Among them, a compound 2h showed topo-IIα activity by 30% at 100µM, which represented a moderate intensity of inhibition compared with etoposide. Three of them formed hydrogen bonds with Thr215, Asn91, Asn120, and Ala167 residues, which are considered as crucial residues for binding to the ATPase domain of topoisomerase IIα. According to in silico drug-likeness property analysis, three compounds are expected to show superiority over etoposide in case of absorption, distribution, metabolism and excretion.

In-vitro metabolic profiling study of potential topoisomerase inhibitors 'pyrazolines' in RLMs by mass spectrometry.

Taking into consideration of the cytotoxicity and topo-IIα inhibitory activity of pyrazoline derivatives (1-3) against HCT15 cells, and known topo-IIα inhibitor, etoposide, respectively, the compounds were biotransformed in rat liver microsomes. LC-MS/MS and MALDI mass spectrometric techniques has been used for analysis. All three compounds were biotransformed into demethylated metabolites. Among three compounds, compounds 1 and 2 were biotransformed into mono-hydroxylated metabolites and compound 3 biotransformed into reduced and epoxidized metabolites. Reduced and reduced along with demethylation metabolites were identified from MALDI Orbitrap spectrometric analysis. Without NADPH or microsomes no compounds (1-3) were generated metabolites, it shows CYP450 enzymes involvement in the presence of NADPH in the metabolisms.

Linear diarylheptanoids as potential anticancer therapeutics: synthesis, biological evaluation, and structure-activity relationship studies.

In efforts to develop effective anticancer therapeutics with greater selectivity toward cancerous cell and reduced side-effects, such as emetic effects due to detrimental action of the drug toward the intestinal flora, a series of linear diarylheptanoids (LDHs) were designed and synthesized in 7 steps with good-to-moderate yields. All synthesized compounds were evaluated for their antibacterial, antiproliferative, and topoisomerase-I and -IIα inhibitory activity. Overall, all compounds showed little to no activity against the bacterial strains tested. Most of the synthesized compounds showed good antiproliferative activity against human breast cancer cell lines (T47D); specifically, the IC50 values of compounds 6a, 6d, 7j, and 7e were 0.09, 0.64, 0.67, and 0.99 µM, respectively. Among the tested compounds, 7b inhibited topo-I by 9.3% (camptothecin 68.8%), 7e and 7h inhibited topo-IIα by 38.4 and 47.4% (etoposide 76.9%), respectively, at the concentration of 100 µM. These results suggest that a set of promising anticancer agents can be obtained by reducing inhibitory actions on different microbes to provide enhanced selectivity against cancerous cells.

Enzyme-instructed self-assembly with photo-responses for the photo-regulation of cancer cells.

Using a short peptide precursor modified by the biaryltetrazole with intramolecular photo-click reactivity, we realized the photo-regulation of the pericellular nanofibers formed by the enzyme-instructed self-assembly on the cell membrane. Upon light irradiation, the fluorescence of nanofibers could be turned on to monitor both enzyme-instructed self-assembly and photo-induced disassembly processes. Moreover, the cell fate could be controlled through the photo-regulation.

In vitro investigation of metabolic profiling of newly developed topoisomerase inhibitors (ethyl fluorescein hydrazones, EtFLHs) in RLMs by LC-MS/MS.

Metabolic profiling of newly reported five topoisomerase inhibitors namely ethyl fluorescein hydrazones (EtFLHs) were studied in rat liver microsomes (RLMs) and the data were acquired in a liquid chromatography (LC) ion trap mass spectrometry. Hydroxyl group containing EtFLHs derivatives (1-3) were bio-transformed into hydrolyzed, mono-hydroxylated and hydrolyzed together with mono-hydroxylated metabolites. On the other hand, nitro and methoxy groups containing EtFLHs derivatives (4-5) were bio-transformed into hydrolyzed, hydrolyzed together with mono-hydroxylated and azo-reductive metabolites in the presence of NADPH. No metabolites were observed in the absence of either NADPH or microsomes for the compounds (1-5), indicating a likely involvement of CYP450 enzymes and cofactor NADPH in the metabolisms.

In vitro Investigation of Metabolic Profiling of a Potent Topoisomerase Inhibitors Fluorescein Hydrazones (FLHs) in RLMs by LC-MS/MS.

Metabolic profiling of newly reported two topoisomerase inhibitors namely fluorescein hydrazones (FLHs) were studied in rat liver microsomes (RLMs) and the data were acquired in a liquid chromatography (LC) ion trap mass spectrometry. (E)-3',6'-dihydroxy-2-((2-nitrobenzylidene)amino)spiro[isoindoline-1,9'-xanthen]-3-one (1) was bio-transformed into two mono-hydroxylated, one double hydroxylated and a tetra hydroxylated metabolites, on the other hand, (Z)-3',6'-dihydroxy-2-((2-oxoindolin-3-ylidene)amino)spiro[isoindoline-1,9'-xanthen]-3-one (2) was bio-transformed into eight possible metabolites by cleavage reduction, hydroxylation, cleavage reduction together with hydroxylated and azo-reduction in RLMs in presence of NADPH-generating system. No metabolites were generated in the absence of either NADPH or microsomes for the compounds (1 and 2), indicating a likely involvement of CYP450 enzymes and the cofactor NADPH in the metabolisms. The structures of those metabolite were elucidated by MS/MS fragmentation study.

A photo-degradable supramolecular hydrogel for selective delivery of microRNA into 3D-cultured cells.

Multi-functional supramolecular hydrogels have emerged as smart biomaterials for diverse biomedical applications. Here we report a multi-functional supramolecular hydrogel formed by the conjugate of the bioactive GRGDS peptide with biaryltetrazole that is the substrate of photo-click reaction. The hydrogel was used as a biocompatible matrix to encapsulate live cells for 3D culture. The presence of the RGD epitope in the hydrogelator enhanced the interaction of the nanofiber with integrin over-expressing cells, which resulted in the selective enhancement in the miRNA delivery into the encapsulated U87 cells. The intramolecular photo-click reaction of the biaryltetrazole moiety in the hydrogelator leads to a sensitive photo-response of the hydrogel, which allowed photo-degradation of the hydrogel for release of the encapsulated live cells for further bio-assay of the intracellular species.