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 EC 50 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.

The syntheses of new tubulysin analogues are described, in which the central amino acid, tubuvaline, is replaced by the rather simple building blocks phenyltubuvaline and phenoxytubuvaline. These analogues can be obtained in only two to three steps from easily accessible starting materials. Although the new derivatives are less active than the tubulysins, their activities towards U‐2 OS tumor cells are still in the nanomolar range.

Cushing's disease, characterized by elevated plasma cortisol levels, can be controlled by inhibition of 11β-hydroxylase (CYP11B1). The previously identified selective and potent CYP11B1 inhibitor 5-((5-methylpyridin-3-yl)methyl)-2-phenylpyridine Ref 7 (IC= 2 nM) exhibited promutagenic potential as well as very low oral bioavailability in rats (F = 2%) and was therefore modified to overcome these drawbacks. Successful lead optimization resulted in similarly potent and selective 5-((5-methoxypyridin-3-yl)methyl)-3-phenylisoxazole 25 (IC = 2 nM, 14-fold selectivity over CYP11B2), exhibiting a superior pharmacological profile with no mutagenic potential. Furthermore, compound 25 inhibited rat CYP11B1 (IC = 2 μM) and showed a high oral bioavailability (F = 50%) and sufficient plasma concentrations in rats, providing an excellent starting point for a proof-of-principle study.

Tubulin binding agents are a potent group of cancer chemotherapeutics. Most of these substances are naturally derived compounds. A novel substance class of destabilizing agents is the group of tubulysins. The tubulysins and their derivative pretubulysin have shown high efficacy in vitro and in vivo. Due to their complex chemical structures, one major bottleneck of the tubulysins is their accessibility. Biotechnological as well as chemical production is challenging, especially on larger scales. Thus, the synthesis of chemically simplified structures is needed with retained or improved biological activity. Herein is presented the biological evaluation of two pretubulysin derivatives [2-desmethylpretubulysin AU816 (1) and phenylpretubulysin JB337 (2)] in comparison to pretubulysin. Both 1 and 2 display a simplification in chemical synthesis. It was shown that both compounds exhibited potent biological activity against cancer cells. These simplified compounds inhibited tubulin polymerization in the nanomolar range. The cytotoxic effects of 1 and 2 were in a similar range, when compared with pretubulysin [IC50 (nM): pretubulysin: 0.6; 1: 10; 2: 100]. Furthermore, it was shown that cell cycle arrest is induced and migration is hampered in MDA-MB-231 breast cancer cells. In conclusion, 1 was shown to be about 10-fold more active than 2 and as potent as pretubulysin.

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The [3+2]-cycloaddition of an azido tripeptide, corresponding to the left hand side of pretubulysin, with a range of alkynes, such as propiolic acid amides and propargyl ethers, allows the straightforward syntheses of libraries of tubulysin derivatives. Via this click approach, a chimera of pretubulysin and dolastatin 10, both highly potent antimitotic drug candidates, also becomes accessible.

Diverse malfunctions in the expression and regulation of matrix metalloproteinases (MMPs) are often the cause of severe human diseases, bringing the identification of specific MMP inhibitors into major focus, particularly in anticancer treatment. Here, we describe a novel bioassay based on recombinant yeast cells (Pichia pastoris) that express, deliver, and incorporate biologically active human MMP-2 and MMP-9 at the yeast cell surface. Using Sed1p for cell wall targeting and covalent anchorage, a highly efficient bioassay was established that allows high-throughput screening and subsequent validation of novel MMP inhibitors as potential anticancer drugs. In addition, we developed a straightforward synthesis of a new aspartate-derived MMP inhibitor active in the nM range and bearing an amino functionality that should allow the introduction of a wide range of side chains to modify the properties of these compounds.Diverse malfunctions in the regulation of human matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, play a key role in the development of a wide range of diseases, including diabetes mellitus, arthritis, cardiovascular diseases, and, most of all, cancer (5, 16). Recent oncology research is therefore focusing on the specific inhibition of distinct MMPs as potential therapeutic targets (3). Most of these inhibitors are peptidic succinates with a hydroxamate functionality, binding to the zinc ion at the active site of the proteolytic enzyme (1, 7, 10). For example, hydroxamate 1 (Fig. 1, structure 1) shows significant selectivity toward MMP-2, -8, and -9 relative to other MMPs (17), which can be explained by the interaction of the nonpolar phenylpropyl side chain with the deep, tunnellike binding pockets of these enzymes (20). The introduction of polar substituents (e.g., R ϭ OH) onto the ␣-position of the hydroxamate group in general results in higher solubility and oral bioavailability (23,27).Nevertheless, early generations of MMP inhibitors (MMPIs) did not meet the high expectations for them in clinical trials, as poor inhibitor specificities caused massive side effects due to the inhibition of non-MMP targets (5,14,16,24). Thus, identification of more specific inhibitors is currently a major focus in MMPI development. Until now, the design and validation of novel inhibitors have often been hampered by costly and timeconsuming MMP purification from human tumor cell lines or primary fibroblasts (9), as well as by the lack of a suitable high-throughput bioassay for comprehensive MMP inhibitor screening. To bypass such a limitation, the major objective of the present study was to immobilize biologically active human MMPs on the surface of yeast (Pichia pastoris) and to establish a cell-based bioassay for MMP inhibitor screening. In addition, we developed a straightforward synthesis of a potential inhibitor of these MMPs based on structure 1 (Fig. 1), bearing an amino functionality at the ␣ position, which should allow the introduction of a wide range of side chains to modify the properties of th...

A short, four-step synthesis of Boc-protected (S)-dolaphenine is described, starting from protected phenylalanyl glycine. The key step is the cyclization of an endothiodipeptide to give a thiazolyl triflate, which can be subjected to a palladium-catalyzed reduction with formic acid.