Markus Kalesse

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).

The Human Cytomegalovirus UL51 Protein Is Essential for Viral Genome Cleavage-Packaging and Interacts with the Terminase Subunits pUL56 and pUL89

ABSTRACT Cleavage of human cytomegalovirus (HCMV) genomes as well as their packaging into capsids is an enzymatic process mediated by viral proteins and therefore a promising target for antiviral therapy. The HCMV proteins pUL56 and pUL89 form the terminase and play a central role in cleavage-packaging, but several additional viral proteins, including pUL51, had been suggested to contribute to this process, although they remain largely uncharacterized. To study the function of pUL51 in infected cells, we constructed HCMV mutants encoding epitope-tagged versions of pUL51 and used a conditionally replicating virus (HCMV-UL51-ddFKBP), in which pUL51 levels could be regulated by a synthetic ligand. In cells infected with HCMV-UL51-ddFKBP, viral DNA replication was not affected when pUL51 was knocked down. However, no unit-length genomes and no DNA-filled C capsids were found, indicating that cleavage of concatemeric HCMV DNA and genome packaging into capsids did not occur in the absence of pUL51. pUL51 was expressed mainly with late kinetics and was targeted to nuclear replication compartments, where it colocalized with pUL56 and pUL89. Upon pUL51 knockdown, pUL56 and pUL89 were no longer detectable in replication compartments, suggesting that pUL51 is needed for their correct subnuclear localization. Moreover, pUL51 was found in a complex with the terminase subunits pUL56 and pUL89. Our data provide evidence that pUL51 is crucial for HCMV genome cleavage-packaging and may represent a third component of the viral terminase complex. Interference with the interactions between the terminase subunits by antiviral drugs could be a strategy to disrupt the HCMV replication cycle.

Pellasoren: structure elucidation, biosynthesis, and total synthesis of a cytotoxic secondary metabolite from Sorangium cellulosum.

Myxobacteria are efficient producers of numerous secondary metabolites, and the genus Sorangium is frequently described as an proficient source for new, biologically active natural products. We report here the discovery and complete structure elucidation of pellasoren from the myxobacterium Sorangium cellulosum. Identification of the corresponding pel gene cluster from S. cellulosum So ce38 allowed us to establish a model for pellasoren biosynthesis, providing evidence for an unusual route to glycolate extender unit generation. Moreover, we present the first total synthesis of pellasoren and thereby confirm the absolute configuration of this natural product. Pellasoren (1; Scheme 1) was initially isolated from S. cellulosum So ce35 in the course of an activity-guided discovery program. Additionally, we recently identified 1 in relatively high amounts in extracts from the related strain S. cellulosum So ce38 by using LC-MS analysis. Here we determine its cytotoxicity against HCT-116 human colon cancer cells at a concentration of 155 nm (IC50). Full structural elucidation by NMR and ESI-MS analysis was performed and confirmed the identity of pellasoren from both sources (Figures S1–S3 and Table S1 in the Supporting Information). The pellasoren scaffold features an unusual enol ether moiety, also known from a small number of other natural products, which was corroborated by specific HMBC correlations between a methoxy signal and sp-hybridized carbon atoms (Figure S1 in the Supporting Information). The lactone moiety was identified through HMBC correlation between C1 and C5 and characteristic shifts for H-5 and C5 of d = 4.02 and 90.3 ppm, respectively. The position of the amide bond was assigned on the basis of indicative fragments in CID spectra. Efforts were made to establish the molecule s relative configuration by using NOE spectroscopy: ROESY interactions together with molecular modeling suggest an anti configuration of the substituents at C4 and C5, as well as a syn configuration of the methyl groups at C2 and C4 (Figure S2 in the Supporting Information). The stereocenter at C14 maintains the configuration derived from the incorporation of an l-alanine building block during biosynthesis. Stereochemical assignments that could initially not be validated by NOE analysis, such as the configuration at C6, were later established following total synthesis. Additionally, two isomeric pellasorens differing in the configuration of the C10– C11 double bond were isolated from S. cellulosum extracts. Pellasoren A (1 a) represents the all-E configuration while pellasoren B (1b) has a Z-configured double bond at C10– C11 which can be rationalized by photochemical isomerization. Following the structural elucidation of pellasoren, we set out to identify the underlying biosynthetic machinery using fragmentary whole-genome sequence information for strain S. cellulosum So ce38. Assuming that pellasoren is most likely the product of a hybrid PKS/NRPS biosynthetic pathway, the full complement of putative PKR/NRPS-related domains encoded in the So ce38 genome was annotated using bioinformatic tools. Using the presumed incorporation of alanine into pellasoren as a guide, we identified a genomic region roughly 57 500 bp in size, containing seven characteristic PKS modules organized as an apparent operon which also encodes one adenylation (A) domain exhibiting the Scheme 1. Structure of pellasoren A (1a) and B (1b).

Synthesis of unsaturated lactone moieties by asymmetric hetero Diels–Alder reactions with binaphthol-titanium complexes

Natural products like ratjadone and callystatin A contain an α,β-unsaturated lactone moiety which adds to the biological activity of these compounds. Here we report a rapid and practical access to lactone precursor 3 by an asymmetric hetero Diels–Alder reaction as the key step and its subsequent transformation into a suitable building block 4.

Total Synthesis of Chivosazole F

The first synthesis of the highly biologically active chivosazole F is described. It features an intramolecular Stille coupling for the macrolactone formation and thereby circumvents the problem of isomerization associated with the tetraene segment. Additionally, the synthesis confirms the structure which has been proposed based solely on a combination of NMR/computational methods and genetic analysis.

The Total Synthesis of (+)‐Tedanolide—A Macrocyclic Polyketide from Marine Sponge Tedania ignis

Tedanolide, which was isolated by Schmitz in 1984 from the marine sponge Tedania ignis, is a highly cytotoxic macrolide leading to strong growth inhibition of P338 tumor cells in bioassays. A unique structural feature of the known tedanolides is the primary hydroxyl group incorporated in the macrolactone. This unusual motif for macrolactones originated from PKS biosynthesis might arise through lactonizations others than those derived by the thioesterase reaction. First experimental data that support this hypothesis and reflect the inherent preference of PKS-induced macrolactonization were obtained during this synthesis. The inherent preference for the formation of a 14-membered macrocyclization is discussed together with the pivotal steps in the synthesis.

Vinylogous Mukaiyama aldol reactions with triarylboranes

The first vinylogous Mukaiyama aldol reactions with tris(pentafluorophenyl)borane and triphenylborane are described. Both Lewis acids diastereoselectively generate the C19–C21 all-syn stereo triad of ratjadone, and in the case of tris(pentafluorophenyl)borane the reaction proceeds with substoichiometric amounts.

Configurational assignment of secondary hydroxyl groups and methyl branches in polyketide natural products through bioinformatic analysis of the ketoreductase domain.

Profile hidden Markov models (HMMs) were used to predict the configuration of secondary alcohols and α‐methyl branches of modular polyketides. Based on the configurations of two chiral centers in these polyketides, 78 ketoreductases were classified into four different types of polyketide producers. The identification of positions that discriminate between these protein families was followed by fitting six profile HMMs to the data set and the corresponding subsets, to model the conserved regions of the protein types. Ultimately, the profile HMMs described herein predict protein subtypes based on the complete information‐rich region; consequently, slight changes in a multiple sequence alignment do not significantly alter the outcome of this classification method. Additionally, Viterbi scores can be used to assess the reliability of the classification.

Mode of action of epoxyphomalins A and B and characterization of related metabolites from the marine-derived fungus Paraconiothyrium sp.

Epoxyphomalins A (1) and B (2) are highly potent cytotoxic fungal metabolites. During the course of purifying larger quantities of 1 and 2 from Paraconiothyrium sp. fermentation extracts, three new epoxyphomalins (3-5) were isolated and characterized, showing modifications to the oxidation pattern of the cyclohexene moiety or of C-9 of the decalin system. IC(50) values for cytotoxicity against a panel of 36 human tumor cell lines were determined for all new compounds. Compound 4 was found to be cytotoxic particularly toward prostate PC3M (IC(50) = 0.72 μM) and bladder BXF 1218 L (IC(50) = 1.43 μM) cancer cell lines. In addition, inhibition of chymotrypsin-, caspase-, and trypsin-like activity of purified 20S proteasomes was determined for epoxyphomalins A (1) and B (2). The results indicate that compounds 1 and 2 exert their cytotoxic effect through potent inhibition of the 20S proteasome.

The Total Synthesis of Corallopyronin A and Myxopyronin B

Leading the way: the synthesis of natural products with new biological targets is one of the driving forces for the development of new antibiotics. The synthesis of the two secondary metabolites corallopyronin and myxopyronin have been achieved, which are prominent leads for the inhibition of bacterial RNA polymerase.