Angelika Ullrich

Pretubulysin, a Potent and Chemically Accessible Tubulysin Precursor from Angiococcus disciformis

Simplify, simplify, simplify! Pretubulysin (structure without the green substituents), a simplified tubulysin was prepared in the laboratory and also found in a natural myxobacterial source. This biosynthetic precursor of the tubulysins is not as active as tubulysins A and D but is still effective in picomolar concentrations against cancer cell lines.

Discovery of 23 Natural Tubulysins from Angiococcus disciformis An d48 and Cystobacter SBCb004

The tubulysins are a family of complex peptides with promising cytotoxic activity against multi-drug-resistant tumors. To date, ten tubulysins have been described from the myxobacterial strains Angiococcus disciformis An d48 and Archangium gephyra Ar 315. We report here a third producing strain, Cystobacter sp. SBCb004. Comparison of the tubulysin biosynthetic gene clusters in SBCb004 and An d48 reveals a conserved architecture, allowing the assignment of cluster boundaries. A SBCb004 strain containing a mutant in the putative cyclodeaminase gene tubZ accumulates pretubulysin A, the proposed first enzyme-free intermediate in the pathway, whose structure we confirm by NMR. We further show, using a combination of feeding studies and structure elucidation by NMR and high-resolution tandem mass spectrometry, that SBCb004 and An d48 together biosynthesize 22 additional tubulysin derivatives. These data reveal the inherently diversity-oriented nature of the tubulysin biosynthetic pathway.

Biosynthesis of 2‐Alkyl‐4(1H)‐Quinolones in Pseudomonas aeruginosa: Potential for Therapeutic Interference with Pathogenicity

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium capable of surviving in a broad range of natural environments and known to be involved in infectious diseases of various hosts. In humans, the opportunistic pathogen is one of the leading causes for nosocomial infections in immuno-compromised patients and is responsible for chronic lung infections in the majority of cystic fibrosis patients. The ability of P. aeruginosa to adapt to different environments and lifestyles is closely related to its ability to coordinate the survival strategy of a population by so-called quorum-sensing (QS) systems. QS is based on the production and release of small signaling molecules, called autoinducers, that increase in concentration as a function of cell density and activate corresponding transcriptional regulators after a threshold concentration has been reached. Three different QS systems are known from P. aeruginosa. The las and rhl systems use acyl-homoserine lactone (AHL) autoinducers and belong to the LuxI/LuxR-type systems that are widespread among Gram-negative bacteria. The third QS system is rather unique and restricted to particular Pseudomonas and Burkholderia strains. Therein 2-alkyl-4(1H)quinolones (AQ) autoinducers such as 2-heptyl-3-hydroxy4(1H)-quinolone (the Pseudomonas quinolone signal : PQS) and its direct precursor 2-heptyl-4(1H)-quinolone (HHQ) are used (see Scheme 1). The pqs system is involved in the regulation of P. aeruginosa virulence such as pyocyanin biosynthesis, biofilm formation and maturation, the production of exoproducts like elastase, alkaline proteases, rhamnolipids, and hydrogen cyanide, and the expression of efflux pumps. Further, PQS itself can down-regulate the host-innate immune response. The sum of these effects makes the pqs system a highly attractive target for drug development to interfere with P. aeruginosa pathogenicity and biofilm formation. The general validity of this approach is supported by the results of several infection models in which PQS-deficient mutants show a reduced pathogenicity compared to P. aeruginosa wild type. 10] A reduced pathogenicity was also observed in a mouse infection model when animals infected with the wild-type strain were treated with halogenated anthranilic acid derivatives that inhibit PQS biosynthesis. This treatment led to a significant increase in survival in comparison to the control group. To further explore this target it is necessary to understand the details of PQS formation in the pathogen. It is known that HHQ biosynthesis absolutely requires the genes pqsA–D, encoding an anthranilate:coenzyme A (CoA) ligase (pqsA) and three b-ketoacyl-acyl carrier protein synthase III (KAS III) homologues. An additional gene (pqsH), located apart from pqsA– D, is responsible for the hydroxylation of HHQ to form PQS. Feeding studies in vivo have demonstrated that HHQ most likely arises from “head-to-head” condensation of an anthraniloyl precursor and a b-keto fatty acid derivative (Scheme 1). However, the details of the enzymatic mechanism of this reaction and the nature of the b-keto fatty acid remained elusive. Furthermore, it has been shown in vitro that PqsA and PqsD catalyze the formation of 2,4-dihydroxyquinoline (DHQ), another secondary metabolite of P. aeruginosa. In DHQ biosynthesis PqsA activates anthranilic acid to anthraniloyl-CoA which is loaded to the active-site cysteine (C112) of PqsD, which itself catalyzes the decarboxylative Claisen condensation with malonyl-CoA. Based on the structural similarity between DHQ and HHQ, we reasoned that PqsD might be involved in a similar condensation reaction in HHQ biosynthesis. To prove this hypothesis, we heterologously expressed PqsD from strain PA14 in Escherichia coli and purified the enzyme for biochemical characterization in vitro. Anthraniloyl-CoA and three potential b-keto acid derivatives were chemically synthesized as substrates, including b-ketodecanoic acid (1), b-ketodecanoyl-CoA (2), and b-ketodecanoyl-N-acetylcysteamine thioester (3) as mimics of the hypothetical ACP-bound substrate (for details see the Supporting Information). The in vitro reaction contained recombinant PqsD, anthraniloyl-CoA, and one of the Scheme 1. Biosynthetic pathways to DHQ, HHQ, and PQS. The nature of the accepted b-ketodecanoyl moiety is unknown to date. Possible substrates are b-ketodecanoic acid (1), b-ketodecanoyl-CoA (2) and b-ketodecanoyl-ACP.

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 tubulin-degradation 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.

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.

Anti-angiogenic effects of the tubulysin precursor pretubulysin and of simplified pretubulysin derivatives

BACKGROUND AND PURPOSE The use of tubulin‐binding compounds, which act in part by inhibiting tumour angiogenesis, has become an integral strategy of tumour therapy. Recently, tubulysins were identified as a novel class of natural compounds of myxobacterial origin, which inhibit tubulin polymerization. As these compounds are structurally highly complex, the search for simplified precursors [e.g. pretubulysin (Prt)] and their derivatives is mandatory to overcome supply problems hampering clinical development. We tested the anti‐angiogenic efficacy of Prt and seven of its derivatives in comparison to tubulysin A (TubA).

Development of catalysts for the stereoselective hydrogenation of α,β-unsaturated ketones.

Iridium phosphinitoxazoline complexes were found to be new efficient catalysts for the asymmetric hydrogenation of arylated α,β-unsaturated ketones. Linear as well as cyclic substrates are hydrogenated with similar success, giving selectivities of up to 99.7% ee.

Simplified Pretubulysin Derivatives and Their Biological Effects on Cancer Cells

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.

Synthetic Approaches Towards Tubulysins and Derivatives Thereof

Tubulysins, linear tetrapeptides produced by several strains of myxobacteria, show an extremely high toxicity towards a wide range of cancer cell lines, with IC50 values in the nano or even picomolar range. Therefore, tubulysins and their derivatives might be suitable candidates for the development of antitumor drugs. Several synthetic approaches for tubulysins and derivates have been developed, which will be discussed in the review.

Novel Tubulin Antagonist Pretubulysin Displays Antivascular Properties In Vitro and In Vivo

Objective—Pretubulysin (PT) is a novel, synthetically accessible myxobacterial compound that acts as a tubulin-depolymerizing agent and inhibits cancer cell growth in vitro and in vivo. Moreover, PT was found to attenuate tumor angiogenesis. Here, we hypothesized that PT could exert antivascular activities on existing tumor vessels. Approach and Results—We aimed to characterize the antivascular effects of PT and to elucidate the underlying mechanisms in endothelial cells. In vitro, PT rapidly induced endothelial hyperpermeability and a concentration-dependent disassembly of established endothelial tubes on Matrigel and in an ex vivo aortic ring model. It disrupted endothelial cell junctions and triggered F-actin stress fiber formation and cell contraction by the RhoA/Rho-associated protein kinase pathway without causing cell death. In vivo, using a hamster dorsal skinfold chamber preparation, PT significantly decreased blood flow and vessel diameter in hamster A-Mel-3 amelanotic melanoma tumors but not in the neighboring healthy tissue. In a second tumor model using mice with subcutaneous murine B16 melanoma tumors, a single dose of PT (10 mg/kg) caused a shut down of tumor blood flow and a strong central tumor cell necrosis within 24 hours. Repeated PT administration significantly decelerates tumor growth and seems to be well tolerated. Conclusions—In summary, we could show for the first time that the antitumor effect of PT is, at least in part, mediated via its antivascular activities on existing tumor vessels.