Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to beta-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of beta-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin.

Polymer/copper indium sulfide (CIS) nanocomposite solar cells are prepared via a capper free in situ preparation route using copper and indium xanthates as precursors, which decompose and form CIS nanoparticles in the polymer matrix during a mild thermal treatment. The solar cells generate current in a wide range of the solar spectrum and exhibit efficiencies up to 2.8%.

Arylthioindoles (ATIs) that possess a 3-methoxyphenylthio or a 3,5-dimethoxyphenylthio moiety at position 2 of the indole ring were effective tubulin assembly inhibitors, but weak inhibitors of MCF-7 cell growth. ATIs bearing a 3-(3,4,5-trimethoxyphenyl)thio moiety were potent tubulin polymerization inhibitors, with IC(50)s in the 2.0 (35) to 4.5 (37) microM range. They also inhibited MCF-7 cell growth at nanomolar concentrations. The 3,4,5-trimethoxy substituted ATIs showed potencies comparable to those of the reference compounds colchicine and combretastatin A-4 in both tubulin assembly and cell growth inhibition assays. Dynamics simulation studies correlate well with the observed experimental data. Furthermore, from careful analysis of the biological and in silico data, we can now hypothesize a basic pharmacophore for this class of compounds.

Several arylthioindoles had excellent activity as inhibitors both of tubulin polymerization and of the growth of MCF-7 human breast carcinoma cells. Methyl 3-[(3,4,5-trimethoxyphenyl)thio]-5-methoxy-1H-indole-2-carboxylate (21), the most potent derivative, showed IC(50) = 2.0 microM, 1.6 times more active than colchicine and about as active as combretastatin A-4 (CSA4). Compound 21 inhibited the growth of the MCF-7 cells at IC(50) = 13 nM. Colchicine and CSA4 had 13 nM and 17 nM IC(50) values, respectively, with these cells.

A method for phosphopeptide identification by capillary liquid chromatography (muLC) interfaced alternatively to element mass spectrometry (inductively coupled plasma mass spectrometry, ICPMS) and to electrospray ionization mass spectrometry (ESI-MS) is described. ICPMS is used for 31P detection and ESI-MS provides the corresponding molecular weight information. Alignment of the two separate muLC runs is performed using the baseline distortion at the elution front, which shows up in both muLC-ICPMS and muLC-ESI-MS. Both a quadrupole and a magnetic sector field mass analyzer were used in combination with ICP. The detection limit achieved for the muLC-ICP-HRMS runs is approximately 0.1 pmol of phosphopeptide injected. Without any further precautions, contamination by phosphate-containing compounds at this level was found to be uncritical. The method is demonstrated for the analysis of a complex mixture of synthetic phosphopeptides and a set of tryptic digests of three phosphoproteins. These include beta-casein, activated human MAP kinase ERK1, and protein kinase A catalytic subunit. The tryptic phosphopeptides of these proteins could all be detected and identified by our new strategy. Analysis of three fractions of protein kinase A catalytic subunit with different phosphorylation status gives direct access to the order in which the phosphorylation of the four phosphorylation sites occurs. The two most important aspects of using muLC-ICPMS with 31P detection for phosphopeptide identification are (i) that a high selectivity is achieved and (ii) that the signal intensity is independent of the chemical form of phosphorus and directly proportional to the molar amount of 31P in the muLC eluate. Thus, muLC-ICPMS with 31P detection is introduced as a new, robust, and specific method in phosphoproteomics.

The new arylthioindole (ATI) derivatives 10, 14-18, and 21-24, which bear a halogen atom or a small size ether group at position 5 of the indole moiety, were compared with the reference compounds colchicine and combretastatin A-4 for biological activity. Derivatives 10, 11, 16, and 21-24 inhibited MCF-7 cell growth with IC50 values <50 nM. A halogen atom (14-17) at position 5 caused a significant reduction in the free energy of binding of compound to tubulin, with a concomitant reduction in cytotoxicity. In contrast, methyl (21) and methoxy (22) substituents at position 5 caused an increase in cytotoxicity. Compound 16, the most potent antitubulin agent, led to a large increase (56%) in HeLa cells in the G2/M phase at 24 h, and at 48 h, 26% of the cells were hyperploid. Molecular modeling studies showed that, despite the absence of the ester moiety present in the previously examined analogues, most of the compounds bind in the colchicine site in the same orientation as the previously studied ATIs. Binding to beta-tubulin involved formation of a hydrogen bond between the indole and Thr179 and positioning of the trimethoxy phenyl group in a hydrophobic pocket near Cys241.

Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development. However, a lack of emphasis has been placed upon the development and organization of retinal ganglion cells (RGCs) within retinal organoids. Thus, initial efforts were made to characterize RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a complement of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful for cellular replacement in which extensive axonal outgrowth is necessary to reach post-synaptic targets. Organoid-derived RGCs could help to elucidate factors promoting axonal outgrowth, thereby identifying approaches to circumvent a formidable obstacle to RGC replacement. As such, additional efforts demonstrated significant enhancement of neurite outgrowth through modulation of both substrate composition and growth factor signaling. Additionally, organoid-derived RGCs exhibited diverse phenotypes, extending elaborate growth cones and expressing numerous guidance receptors. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing neurite outgrowth from organoid-derived RGCs.

(-)-Dictyostatin is a sponge-derived, 22-member macrolactone natural product shown to cause cells to accumulate in the G2/M phase of the cell cycle, with changes in intracellular microtubules analogous to those observed with paclitaxel treatment. Dictyostatin also induces assembly of purified tubulin more rapidly than does paclitaxel, and nearly as vigorously as does dictyostatin's close structural congener, (+)-discodermolide (Isbrucker et al. (2003), Biochem. Pharmacol. 65, 75-82). We used synthetic (-)-dictyostatin to study its biochemical and cytological activities in greater detail. The antiproliferative activity of dictyostatin did not differ greatly from that of paclitaxel or discodermolide. Like discodermolide, dictyostatin retained antiproliferative activity against human ovarian carcinoma cells resistant to paclitaxel due to beta-tubulin mutations and caused conversion of cellular soluble tubulin pools to microtubules. Detailed comparison of the abilities of dictyostatin and discodermolide to induce tubulin assembly demonstrated that the compounds had similar potencies. Dictyostatin inhibited the binding of radiolabeled discodermolide to microtubules more potently than any other compound examined, and dictyostatin and discodermolide had equivalent activity as inhibitors of the binding of both radiolabeled epothilone B and paclitaxel to microtubules. These results are consistent with the idea that the macrocyclic structure of dictyostatin represents the template for the bioactive conformation of discodermolide.