Pretubulysin derived probes as novel tools for monitoring the microtubule network via activity-based protein profiling and fluorescence microscopy

Microtubules (mt) are highly dynamic polymers composed of alpha- and beta-tubulin monomers that are present in all dividing and non-dividing cells. A broad variety of natural products exists that are known to interfere with the microtubule network, by either stabilizing or de-stabilizing these rope-like polymers. Among those tubulysins represent a new and potent class of cytostatic tetrapeptides originating from myxobacteria. Early studies suggested that tubulysins interact with the eukaryotic cytoskeleton by inhibition of tubulin polymerization with EC50 values in the picomolar range. Recently, pretubulysins have been described to retain the high tubulindegradation activity of their more complex tubulysin relatives and represent an easier synthetic target with an efficient synthesis already in place. Although tubulin has been suggested as the dedicated target of tubulysin a comprehensive molecular target analysis of pretubulysin in the context of the whole proteome has not been carried out so far. Here we utilize synthetic chemistry to develop two pretubulysin photoaffinity probes which were applied in cellular activity-based protein profiling and imaging studies in order to unravel and visualize dedicated targets. Our results clearly show a remarkable selectivity of pretubulysin for beta-tubulin which we independently confirmed by a mass-spectrometry based proteomic profiling platform as well as by tubulin antibody based co-staining on intact cells

A multidisciplinary study of an exceptional prehistoric waste dump in the mountainous inland of Calabria (Italy) : implications for reconstructions of prehistoric land use and vegetation in Southern Italy

The mountainous inland of northern Calabria (Southern Italy) is known for its sparse prehistoric human occupation. Nevertheless, a thorough multidisciplinary approach of field walking, geophysical survey and invasive research led to the discovery of a major archaeological archive. This archive concerns a rich multi-phased dump, spanning about 3000 years (Late Neolithic to Late Imperial Roman Age) and holding two Somma-Vesuvius tephra. Of these, the younger is a distinct layer of juvenile tephra from the Pompeii eruption, while the older concerns reworked tephra from the Bronze Age AP2 eruption (ca. 1700 cal. yr BP). The large dump contains abundant ceramics, faunal remains and charcoal, and most probably originated through long-continued deposition of waste in a former gully like system of depressions. This resulted in an inversed, mound-like relief, whose anthropogenic origin had not been recognized in earlier research. The tephras were found to be important markers that support the reconstruction of the occupational history of the site. The sequence of occupational phases is very similar to that observed in a recent palaeoecological study from nearby situated former lakes (Lago Forano/Fontana Manca). This suggests that this sequence reflects the more regional occupational history of Calabria, which goes back to ca. 3000 BC. Attention is paid to the potential link between this history and Holocene climatic phases, for which no indication was found. The history deviates strongly from histories deduced from the few, but major palaeorecords elsewhere in the inlands of Southern Italy (Lago Grande di Monticchio and Lago Trifoglietti). We conclude that major regional variation occurred in prehistoric land use and its impacts on the vegetation cover of Southern Italy, and studies of additional palaeoarchives are needed to unravel this complex history. Finally, shortcomings of archaeological predictive models are discussed and the advantages of truly integrated multidisciplinary research

Pretubulysin: From Hypothetical Biosynthetic Intermediate to Potential Lead in Tumor Therapy

Pretubulysin is a natural product that is found in strains of myxobacteria in only minute amounts. It represents the first enzyme-free intermediate in the biosynthesis of tubulysins and undergoes post-assembly acylation and oxidation reactions. Pretubulysin inhibits the growth of cultured mammalian cells, as do tubulysins, which are already in advanced preclinical development as anticancer and antiangiogenic agents. The mechanism of action of this highly potent compound class involves the depolymerization of microtubules, thereby inducing mitotic arrest. Supply issues with naturally occurring derivatives can now be circumvented by the total synthesis of pretubulysin, which, in contrast to tubulysin, is synthetically accessible in gram-scale quantities. We show that the simplified precursor is nearly equally potent to the parent compound. Pretubulysin induces apoptosis and inhibits cancer cell migration and tubulin assembly in vitro. Consequently, pretubulysin appears to be an ideal candidate for future development in preclinical trials and is a very promising early lead structure in cancer therapy

Pretubulysin derived probes as novel tools for monitoring the microtubule network via activity-based protein profiling and fluorescence microscopy

Microtubules (mt) are highly dynamic polymers composed of alpha- and beta-tubulin monomers that are present in all dividing and non-dividing cells. A broad variety of natural products exists that are known to interfere with the microtubule network, by either stabilizing or de-stabilizing these rope-like polymers. Among those tubulysins represent a new and potent class of cytostatic tetrapeptides originating from myxobacteria. Early studies suggested that tubulysins interact with the eukaryotic cytoskeleton by inhibition of tubulin polymerization with EC50 values in the picomolar range. Recently, pretubulysins have been described to retain the high tubulindegradation activity of their more complex tubulysin relatives and represent an easier synthetic target with an efficient synthesis already in place. Although tubulin has been suggested as the dedicated target of tubulysin a comprehensive molecular target analysis of pretubulysin in the context of the whole proteome has not been carried out so far. Here we utilize synthetic chemistry to develop two pretubulysin photoaffinity probes which were applied in cellular activity-based protein profiling and imaging studies in order to unravel and visualize dedicated targets. Our results clearly show a remarkable selectivity of pretubulysin for beta-tubulin which we independently confirmed by a mass-spectrometry based proteomic profiling platform as well as by tubulin antibody based co-staining on intact cells