A Nuclear Magnetic Resonance (NMR)- and Mass Spectrometry (MS)-Based Saturation Kinetics Model of a Bryophyllum pinnatum Decoction as a Treatment for Kidney Stones.

Bryophyllum pinnatum (BP) is a medicinal plant used to treat many conditions when taken as a leaf juice, leaves in capsules, as an ethanolic extract, and as herbal tea. These preparations have been chemically analyzed except for decoctions derived from boiled green leaves. In preparation for a clinical trial to validate BP tea as a treatment for kidney stones, we used NMR and MS analyses to characterize the saturation kinetics of the release of metabolites. During boiling of the leaves, (a) the pH decreased to 4.8 within 14 min and then stabilized; (b) regarding organic acids, citric and malic acid were released with maximum release time (tmax) = 35 min; (c) for glycoflavonoids, quercetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (Q-3O-ArRh), myricetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (M-3O-ArRh), kappinatoside, myricitrin, and quercitrin were released with tmax = 5-10 min; and (d) the total phenolic content (TPC) and the total antioxidant capacity (TAC) reached a tmax at 55 min and 61 min, respectively. In summary, 24 g of leaves boiled in 250 mL of water for 61 min ensures a maximal release of key water-soluble metabolites, including organic acids and flavonoids. These metabolites are beneficial for treating kidney stones because they target oxidative stress and inflammation and inhibit stone formation.

Subcellular distribution and mechanism of action of AL906, a novel and potent EGFR inhibitor rationally designed to be green fluorescent.

To enhance the potency of EGFR inhibitors, we developed a novel strategy that seeks to conjugate EGFR to a bioactive moiety leading to a molecule termed "combi-molecule". In order to mimic the penetration of this type of molecules, based upon previously reported structure activity relationship studies, we designed a new molecule containing a quinazoline moiety tethered to a p-nitrobenzoxadiazole (NBD) moiety [molecular weight (MW) 700]. Despite its size, AL906 growth inhibitory activity was superior to that of the clinical drug gefitinib. Furthermore, AL906 retained significant EGFR inhibitory activity and good cellular penetration with abundant distribution in the perinuclear region of the cells. In an isogenic NIH3T3 transfected cell panel, it selectively inhibited the growth  of the NIH3T3-EGFR and HER2 transfectants. Confocal microscopy analysis revealed that it was capable of penetrating multilayer aggregates although to a lesser extent than FD105, a small inhibitor of EGFR inhibitor of the same class (MW 300). Its ability to inhibit EGFR auto-phosphorylation in monolayer culture was stronger than in the aggregates. The results suggest that our strategy did not negatively affect EGFR inhibitory potency, EGFR selectivity and growth inhibition. However, its molecular size may account for its decreased aggregate penetration when compared with a smaller EGFR inhibitor of the quinazoline class.

Molecular Analysis of the Superior Efficacy of a Dual Epidermal Growth Factor Receptor (EGFR)-DNA-Targeting Combi-Molecule in Comparison with Its Putative Prodrugs 6-Mono-Alkylamino- and 6,6-Dialkylaminoquinazoline in a Human Osteosarcoma Xenograft Model.

Background: ZR2002 is a dual EGFR-DNA-targeting combi-molecule that carries a chloroethyl group at the six-position of the quinazoline ring designed to alkylate DNA. Despite its good pharmacokinetics, ZR2002 is metabolized in vivo into dechlorinated metabolites, losing the DNA-alkylating function required to damage DNA. To increase the DNA damage activity in tumor cells in vivo, we compared ZR2002 with two of its 6-N,N-disubstituted analogs: "JS61", with a nitrogen mustard function at the six-position of the quinazoline ring, and "JS84", with an N-methyl group. Methods: Tumor xenografts were performed with the human Saos-2 osteosarcoma cell line expressing EGFR. Mice were treated with ZR2002, JS84 or JS61, and the tumor burden was measured with a caliper and CT/PET imaging. Drug metabolism was analyzed with LC-MS. EGFR and ɣ-H2AX phosphorylation were quantified via Western blot analysis and immunohistochemistry. Results: In vivo analysis showed that significant tumor growth inhibition was only achieved when ZR2002 was administered in its naked form. The metabolic dealkylation of JS61 and JS84 did not release sufficient concentrations of ZR2002 for the intratumoral inhibition of P-EGFR or enhanced levels of P-H2AX. Conclusions: The results in toto suggest that intratumoral concentrations of intact ZR2002 are correlated with the highest inhibition of P-EGFR and induction of DNA damage in vivo. ZR2002 may well represent a good drug candidate for the treatment of EGFR-expressing osteosarcoma.

The larvicide pyriproxyfen blamed during the Zika virus outbreak does not cause microcephaly in zebrafish embryos.

Although the zika virus (ZIKV) has now been strongly correlated with emerging cases of microcephaly in the Americas, suspicions have been raised regarding the use of pyriproxyfen, a larvicide that prevents mosquito development, in drinking water. The effects of this compound on neurodevelopment have not yet been addressed specifically in vertebrates. As a result, we aimed at addressing the effects, if any, of pyriproxyfen on neurodevelopment in the zebrafish embryo as a vertebrate model. Using zebrafish transgenic lines expressing GFP in different cell populations (elavl3 in newborn neurons, gfap and nestin in neural stem cells), we focused on the analysis of whole embryonic brain volume after confocal 3D-reconstruction and the quantification of purified neural stem cells during early neurodevelopment by FACS-cell sorting from whole in vivo embryos. Interestingly, though lethal at very high doses, pyriproxyfen did not cause brain malformation nor any significant changes in the number of observed stem cells in the developing central nervous system. Our data indicate that pyriproxyfen does not affect central nervous system development in zebrafish, suggesting that this larvicide on its own, may not be correlated with the increase in microcephaly cases reported recently.

Subcellular distribution of a fluorescence-labeled combi-molecule designed to block epidermal growth factor receptor tyrosine kinase and damage DNA with a green fluorescent species.

To monitor the subcellular distribution of mixed epidermal growth factor (EGF) receptor (EGFR)-DNA targeting drugs termed combi-molecules, we designed AL237, a fluorescent prototype, to degrade into a green fluorescent DNA damaging species and FD105, a blue fluorescent EGFR inhibitor. Here we showed that AL237 damaged DNA in the 12.5 to 50 mumol/L range. Despite its size, it blocked EGFR phosphorylation in an enzyme assay (IC(50) = 0.27 mumol/L) and in MDA-MB468 breast cancer cells in the same concentration range as for DNA damage. This translated into inhibition of extracellular signal-regulated kinase 1/2 or BAD phosphorylation and downregulation of DNA repair proteins (XRCC1, ERCC1). Having shown that AL237 was a balanced EGFR-DNA targeting molecule, it was used as an imaging probe to show that (a) green and blue colors were primarily colocalized in the perinuclear and partially in the nucleus in EGFR- or ErbB2-expressing cells, (b) the blue fluorescence associated with FD105, but not the green, was colocalized with anti-EGFR red-labeled antibody, (c) the green fluorescence of nuclei was significantly more intense in NIH 3T3 cells expressing EGFR or ErbB2 than in their wild-type counterparts (P < 0.05). Similarly, the growth inhibitory potency of AL237 was selectively stronger in the transfectants. In summary, the results suggest that AL237 diffuses into the cells and localizes abundantly in the perinuclear region and partially in the nucleus where it degrades into EGFR and DNA targeting species. This bystander-like effect translates into high levels of DNA damage in the nucleus. Sufficient quinazoline levels are released in the cells to block EGF-induced activation of downstream signaling. Mol Cancer Ther; 9(4); 869-82. (c)2010 AACR.

Molecular analysis of the in vivo metabolism and biodistribution of metabolically and non-metabolically activated combi-molecules of the triazene class.

Combi-molecules are novel agents designed to be hydrolyzed into two bioactive species: an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor + a DNA alkylating agent. With the purpose of enhancing the tumour concentration of the bioactive species, we synthesized and compared the activities of RB107, a quinazolinotriazene designed to generate the bioactive BJ2000 upon hydrolysis, ZRDM and RB107ZR that require metabolic activation to generate BJ2000. The results showed that RB107 released the highest level of BJ2000 and its degradation product FD105 in vivo and high levels of the DNA alkylating methyl diazonium ion in the brain, kidney, liver and the DU145 tumours as confirmed by (14)C-labeling. The results in toto suggest that RB107 was stable enough to deliver the bioactive species to the tumour site and for optimal tumour distribution of the bioactive species, combi-molecules of the triazene class must be designed to be primarily degraded by hydrolytic cleavage and not by metabolic activation.

Synthesis of water soluble bis-triazenoquinazolines: an unusual predicted mode of binding to the epidermal growth factor receptor tyrosine kinase.

A novel type of 3,3-disubstituted bis-triazenes containing an ethylaminoethyl linker flanked by two identical anilinoquinazoline ring was synthesized. These model molecules contained an N-ethylaminomorpholine moiety designed to enhance water solubility. Despite their significant bulkiness, they blocked epidermal growth factor receptor (EGFR) tyrosine kinase in a dose-dependent manner with IC(50) values in low micromolar range. Molecular modeling to predict the interactions of the molecule with the ATP binding site of EGFR suggests that the N-ethylaminomorpholine side chain plays a binding role.

Optimization of novel combi-molecules: identification of balanced and mixed bcr-abl/DNA targeting properties.

Steps toward the identification of combi-molecules with strong abl tyrosine kinase (TK) inhibitory property and significant DNA damaging potential are described. The optimized combi-molecule 13a was shown to induce approximately twofold stronger abl TK inhibitory activity than Gleevec and high levels of DNA damage in chronic myelogenous leukemic cells.

Novel C2-C3' N-peptide linked macrocyclic taxoids. Part 2: synthesis and biological activities of docetaxel analogues with a peptide side chain at C2 and their macrocyclic derivatives.

The synthesis of a series of novel docetaxel analogues possessing a peptide side chain at the C2 position as well as peptide macrocyclic taxoids is described. These compounds were designed to mimic a region of the alpha-tubulin loop equivalent to the paclitaxel binding pocket of beta-tubulin. Fifteen new peptide taxoids were obtained and evaluated as inhibitors of microtubule disassembly as well as cell proliferation. The relationships between these new taxoids and the tau protein motif interacting with microtubules are discussed.

Novel C2-C3' N-peptide linked macrocyclic taxoids. Part 1: Synthesis and biological activities of docetaxel analogues with a peptide side chain at C3'.

A series of novel docetaxel analogues possessing a peptide side chain at the C3'-N position was synthesized. These compounds were designed to mimic a region of the alpha-tubulin loop that is equivalent to the paclitaxel binding pocket in beta-tubulin. Eight new peptidic taxoids were obtained and evaluated as inhibitors of microtubule disassembly, as well as for their cytotoxicity.