A total of 53 N-benzoylated phenoxazines and phenothiazines, including their S-oxidized analogues, were synthesized and evaluated for antiproliferative activity, interaction with tubulin, and cell cycle effects. Potent inhibitors of multiple cancer cell lines emerged with the 10-(4-methoxybenzoyl)-10H-phenoxazine-3-carbonitrile (33b, IC(50) values in the range of 2-15 nM) and the isovanillic analogue 33c. Seventeen compounds strongly inhibited tubulin polymerization with activities higher than or comparable to those of the reference compounds such as colchicine. Concentration-dependent flow cytometric studies revealed that inhibition of K562 cell growth was associated with an arrest in the G2/M phases of the cell cycle, indicative of mitotic blockade. Structure-activity relationship studies showed that best potencies were obtained with agents bearing a methoxy group placed para at the terminal phenyl ring and a 3-cyano group in the phenoxazine. A series of analogues highlight not only the phenoxazine but also the phenothiazine structural scaffold as valuable pharmacophores for potent tubulin polymerization inhibitors, worthy of further investigation.

SummaryThe green alga Volvox represents the simplest kind of multicellular organism: it is composed of only two cell types, somatic and reproductive, making it suitable as a model system. The sexual development of males and females of Volvox carteri is triggered by a sex-inducing pheromone at a concentration of < 10 ±16 M. Early biochemical responses to the pheromone involve structural modi®cations within the extracellular matrix (ECM). By differential screenings of cDNA libraries made from mRNAs of pheromonetreated Volvox, four novel genes were identi®ed that encode four closely related Volvox metalloproteinases that we use to de®ne a new protein family, the VMPs. The existence of several features common to matrix glycoproteins, such as signal peptides, a (hydroxy)proline content of 12±25%, and Ser(Pro) 2±4 repeats, suggest an extracellular localization of the VMPs within the ECM. Synthesis of VMP cDNAs is triggered not only by the sex-inducing pheromone, but also by wounding, and is restricted to the somatic cell type. Sequence comparisons suggest that the VMPs are members of the MB clan of zinc-dependent matrix metalloproteinases, although the putative zinc binding site of all VMPs is QEXXHXXGXXH rather than HEXXHXXGXXH. The presence of glutamine instead of histidine in the zinc binding motif suggests a novel family, or even clan, of peptidases. Like the matrixin family of human collagenases, Volvox VMPs exhibit a modular structure: they possess a metalloproteinase homology domain and a (hydroxy)proline-rich domain, and one of them, VMP4, also has two additional domains. Metalloproteinases seem to be crucial for biochemical modi®cations of the ECM during development or after wounding in the lower eukaryote Volvox with only two cell types, just as in higher organisms.

The sex-inducing pheromone of the multicellular green alga Volvox carteri is a glycoprotein that triggers development of males and females at a concentration Ͻ 10 ؊ 16 M. By differential screening of a cDNA library, two novel genes were identified that are transcribed under the control of this pheromone. Unexpectedly, one gene product was characterized as a lysozyme/chitinase, and the other gene product was shown to encode a polypeptide with a striking modular composition. This polypeptide has a cysteine protease domain separated by an extensin-like module from three repeats of a chitin binding domain. In higher plants, similar protein families are known to play an important role in defense against fungi. Indeed, we found that the same set of genes triggered by the sexual pheromone was also inducible in V. carteri by wounding. INTRODUCTIONThe sex-inducing pheromone (sex inducer) of the multicellular green alga Volvox carteri is a glycoprotein that is probably among the most potent biological effector molecules known. At a concentration of Ͻ 10 Ϫ 16 M, the pheromone converts asexually growing males and females to the sexual pathway, producing in the next generation sperm-and eggcontaining organisms, respectively (Starr and Jaenicke, 1974;Tschochner et al., 1987;Mages et al., 1988).The asexually growing organism of V. carteri is composed of only two cell types exhibiting a complete division of labor: 2000 to 4000 biflagellate Chlamydomonas -like somatic cells are arranged in a monolayer at the surface of a hollow sphere (Starr, 1969(Starr, , 1970, and 16 much larger reproductive cells ("gonidia") lie just below the somatic cell sheet. Eleven or 12 rapid and synchronous cleavage divisions of a gonidium generate all of the cells of an adult organism. Volvox cells are surrounded and held together by a glycoproteinrich extracellular matrix (ECM) (for review, see Sumper and Hallmann, 1998).Many lines of evidence indicate that the ECM plays a key role in the sexual induction process. The earliest biochemical responses to the pheromone detected thus far are structural modifications within the ECM Sumper, 1982, 1986;Gilles et al., 1983). In particular, the volvocacean ECM contains members of the newly described pherophorin family (Sumper et al., 1993;Godl et al., 1995Godl et al., , 1997. Pherophorins are glycoproteins that contain a C-terminal domain with homology to the sex-inducing pheromone. Some pherophorins are incorporated into the ECM constitutively, but under the influence of the pheromone, synthesis of other members of the pherophorin family is initiated, and from a subset of pherophorins (pherophorin II), this pheromone-like domain becomes proteolytically liberated from the parent glycoproteins. It has been proposed that this modification and processing of the ECM are part of the signal amplification process required to achieve the exquisite sensitivity observed for this sexual induction system. A molecular-genetic approach was initiated to detect additional genes that might be induced under the influence of t...

The extracellular matrix (ECM) of Volvox is modified during development or in response to external stimuli, like the sex-inducing pheromone. It has recently been demonstrated that a number of genes triggered by the sex-inducing pheromone are also inducible by wounding. By differential screening of a cDNA library, a novel gene was identified that is transcribed in response to the pheromone. Its gene product was characterized as an ECM glycoprotein with a striking feature: it exhibits a hydroxyproline content of 68% and therefore is an extreme member of the family of hydroxyproline-rich glycoproteins (HRGPs). HRGPs are known as constituents of higher plant ECMs and seem to function as structural barriers in defense responses. The Volvox HRGP is also found to be inducible by wounding. This indicates that the wound response scenarios of higher plants and multicellular green algae may be evolutionary related.

A novel DSN-depletion method allows elimination of selected sequences from full-length-enriched cDNA libraries. Depleted cDNA can be applied for subsequent EST sequencing, expression cloning, and functional screening approaches. The method employs specific features of the kamchatka crab duplex-specific nuclease (DSN). This thermostable enzyme is specific for double-stranded (ds) DNA, and is thus used for selective degradation of ds DNA in complex nucleic acids. DSN depletion is performed prior to library cloning, and includes the following steps: target cDNA is mixed with excess driver DNA (representing fragments of the genes to be eliminated), denatured, and allowed to hybridize. During hybridization, driver molecules form hybrids with the target sequences, leading to their removal from the ss DNA fraction. Next, the ds DNA fraction is hydrolyzed by DSN, and the ss fraction is amplified using long-distance PCR. DSN depletion has been tested in model experiments.