Dirk Menche

Argyrin A Reveals a Critical Role for the Tumor Suppressor Protein p27kip1 in Mediating Antitumor Activities in Response to Proteasome Inhibition

A reduction in the cellular levels of the cyclin kinase inhibitor p27(kip1) is frequently found in many human cancers and correlates directly with patient prognosis. In this work, we identify argyrin A, a cyclical peptide derived from the myxobacterium Archangium gephyra, as a potent antitumoral drug. All antitumoral activities of argyrin A depend on the prevention of p27(kip1) destruction, as loss of p27(kip1) expression confers resistance to this compound. We find that argyrin A exerts its effects through a potent inhibition of the proteasome. By comparing the cellular responses exerted by argyrin A with siRNA-mediated knockdown of proteasomal subunits, we find that the biological effects of proteasome inhibition per se depend on the expression of p27(kip1).

Stereochemical Determination and Complex Biosynthetic Assembly of Etnangien, a Highly Potent RNA Polymerase Inhibitor from the Myxobacterium Sorangium cellulosum

A potent novel analogue of the natural macrolide antibiotic etnangien, a structurally unique RNA polymerase inhibitor from myxobacteria, is reported. It may be readily obtained from fermentation broths of Sorangium cellulosum and shows high antibiotic activity, comparable to that of etnangien. However, it is much more readily available than the notoriously labile authentic natural product itself. Importantly, it is stable under neutral conditions, allowing for elaborate NMR measurements for assignment of the 12 hydroxyl- and methyl-bearing stereogenic centers. The full absolute and relative stereochemistries of these complex polyketides were determined by a combination of extensive high-field NMR studies, including J-based configuration analysis, molecular modeling, and synthetic derivatization in combination with an innovative method based on biosynthetic studies of this polyketide which is also presented here. A first look into the solution conformation and 3D structure of these promising macrolide antibiotics is reported. Finally, the complete biosynthetic gene cluster was analyzed in detail, revealing a highly unusual and complex trans-AT type polyketide biosynthesis, which does not follow colinearity rules, most likely performs programmed iteration as well as module skipping, and exhibits HMG-CoA box-directed methylation.

Stereoselective Total Synthesis of Etnangien and Etnangien Methyl Ester

A highly stereoselective joint total synthesis of the potent polyketide macrolide antibiotics etnangien and etnangien methyl ester was accomplished by a convergent strategy and proceeds in 23 steps (longest linear sequence). Notable synthetic features include a sequence of highly stereoselective substrate-controlled aldol reactions to set the characteristic assembly of methyl- and hydroxyl-bearing stereogenic centers of the propionate portions, an efficient diastereoselective Heck macrocyclization of a deliberately conformationally biased precursor, and a late-stage introduction of the labile side chain by means of a high-yielding Stille coupling of protective-group-free precursors. Along the way, an improved, reliable protocol for a Z-selective Stork-Zhao-Wittig olefination of aldehydes was developed, and an effective protocol for a 1,3-syn reduction of sterically particularly hindered beta-hydroxy ketones was devised. Within the synthetic campaign, a more detailed understanding of the intrinsic isomerization pathways of these labile natural products was elaborated. The expedient and flexible strategy of the etnangiens should be amenable to designed analogues of these RNA-polymerase inhibitors, thus enabling further exploration of the promising biological potential of these macrolide antibiotics.

New methods for stereochemical determination of complex polyketides: configurational assignment of novel metabolites from myxobacteria

An overview on recently developed methods for the stereochemical determination of complex polyketides is given and NMR-spectroscopic, computational, biosynthetic and synthetic methods are discussed. These methods are presented in their applications to structurally novel polyketide classes from myxobacteria.

Simultaneous determination of the conformation and relative configuration of archazolide a by using nuclear overhauser effects, J couplings, and residual dipolar couplings.

Natural products with promising drug activity must undergo a long configuration and conformation characterization before their target-binding mode can be investigated and exploited. One prime candidate, the polyketide archazolide A (ArcA, Figure 1), is a unique 24-membered-macrolide molecule isolated from the myxobacterium Archangium gephyra. This compound and its derivatives are of interest because they exhibit very potent cytotoxic activity by inhibiting the growth of numerous cell lines at concentrations down to the subnanomolar range. Their activity is known to involve a selective interaction with the membrane-bound portion of the vacuolar-type adenosine triphosphatase (V-ATPase), a proton-translocating enzyme that has been linked to various diseases such as osteoporosis, renal acidosis, and various cancers. This strong interaction has stimulated the investigation of the binding mechanism of ArcA at a molecular level. For the determination of the receptor-bound structure of ligands, it is essential to elucidate the configuration at all stereogenic centers. For ArcA, this has been achieved by a demanding procedure based on combined chemical derivatization and NMR spectroscopy methods. The success of this analysis on ArcA has led to the design of a synthetic route, which has allowed the confirmation of the configuration, given access to more advantageous yields, and enabled strategic derivatization to modulate the drug activity.

Modular Total Synthesis of Archazolid A and B

A modular total synthesis of the potent V-ATPase inhibitors archazolid A and B is reported. The convergent preparation was accomplished by late-stage diversification of joint intermediates. Key synthetic steps involve asymmetric boron-mediated aldol reactions, two consecutive Still-Gennari olefinations to set the characteristic (Z,Z)-diene system, a Brown crotyboration, and a diastereoselective aldol condensation of highly elaborate intermediates. For macrocyclization, both an HWE reaction and a Heck coupling were successfully employed to close the 24-membered macrolactone. During the synthetic campaign, a generally useful protocol for an E-selective Heck reaction of nonactivated alkenes and a method for the direct nucleophilic displacement of the Abiko-Masamune auxiliary with sterically hindered nucleophiles were developed. The expedient and flexible strategy will enable further SAR studies of the archazolids and more detailed evaluations of target-inhibitor interactions.

Stereodivergent Synthesis of 1,3-syn- and -anti-Tetrahydropyrimidinones

An efficient protocol for the stereoselective synthesis of 1,3-syn and -anti-tetrahydropyrimidinones (syn- and anti-11a) is reported. The modular procedure is based on a stereodivergent cyclization of readily available urea-type substrates (10a) by intramolecular allylic substitution. The cyclization proceeds with excellent yield (up to 99%) and selectivity (up to dr > 20:1), purely based on substrate control. The product pyrimidines can be readily transformed into the corresponding free syn- and anti-amines.

Total Synthesis of Etnangien

The first total synthesis of the potent RNA-polymerase inhibitor etnangien is described, which establishes unequivocally the relative and absolute configuration of this sensitive macrolide antibiotic. Key features of the expedient and modular synthesis include stereoselective substrate-controlled boron- and tin-mediated aldol couplings to set the characteristic sequences of methyl and hydroxyl bearing stereogenic centers with high degrees of stereoselectivity and yield, an efficient Heck macrocyclization of a conformationally restricted substrate, and a late-stage introduction of the labile side chain. The convergent approach should be amenable to designed analogues.

Full Stereochemical Determination of Ajudazols A and B by Bioinformatics Gene Cluster Analysis and Total Synthesis of Ajudazol B by an Asymmetric Ortholithiation Strategy

The stereochemical determination of the potent respiratory chain inhibitors ajudazols A and B and the total synthesis of ajudazol B are reported. Configurational assignment was exclusively based on biosynthetic gene cluster analysis of both ketoreductase domains for hydroxyl-bearing stereocenters and one of the first predictive enoylreductase alignments for methyl-bearing stereocenters. The expedient total synthesis resulting in unambiguous proof of the predicted stereochemistry involves a short stereoselective approach to the challenging isochromanone stereotriad by an innovative asymmetric ortholithiation strategy, a modular oxazole formation, and a late-stage Z,Z-selective Suzuki coupling.

Archazolid A Binds to the Equatorial Region of the c-Ring of the Vacuolar H+-ATPase

The macrolactone archazolid is a novel, highly specific V-ATPase inhibitor with an IC50 value in the low nanomolar range. The binding site of archazolid is presumed to overlap with the binding site of the established plecomacrolide V-ATPase inhibitors bafilomycin and concanamycin in subunit c of the membrane-integral VO complex. Using a semi-synthetic derivative of archazolid for photoaffinity labeling of the V1VO holoenzyme we confirmed binding of archazolid to the VO subunit c. For the plecomacrolide binding site a model has been published based on mutagenesis studies of the c subunit of Neurospora crassa, revealing 11 amino acids that are part of the binding pocket at the interface of two adjacent c subunits (Bowman, B. J., McCall, M. E., Baertsch, R., and Bowman, E. J. (2006) J. Biol. Chem. 281, 31885–31893). To investigate the contribution of these amino acids to the binding of archazolid, we established in Saccharomyces cerevisiae mutations that in N. crassa had changed the IC50 value for bafilomycin 10-fold or more and showed that out of the amino acids forming the plecomacrolide binding pocket only one amino acid (tyrosine 142) contributes to the binding of archazolid. Using a fluorescent derivative of N,N′-dicyclohexylcarbodiimide, we found that the binding site for archazolid comprises the essential glutamate within helix 4 of subunit c. In conclusion the archazolid binding site resides within the equatorial region of the VO rotor subunit c. This hypothesis was supported by an additional subset of mutations within helix 4 that revealed that leucine 144 plays a role in archazolid binding.