Andreas Brockmeyer

Natural product–inspired cascade synthesis yields modulators of centrosome integrity

In biology-oriented synthesis, the scaffolds of biologically relevant compound classes inspire the synthesis of focused compound collections enriched in bioactivity. This criterion is, in particular, met by the scaffolds of natural products selected in evolution. The synthesis of natural product-inspired compound collections calls for efficient reaction sequences that preferably combine multiple individual transformations in one operation. Here we report the development of a one-pot, twelve-step cascade reaction sequence that includes nine different reactions and two opposing kinds of organocatalysis. The cascade sequence proceeds within 10-30 min and transforms readily available substrates into complex indoloquinolizines that resemble the core tetracyclic scaffold of numerous polycyclic indole alkaloids. Biological investigation of a corresponding focused compound collection revealed modulators of centrosome integrity, termed centrocountins, which caused fragmented and supernumerary centrosomes, chromosome congression defects, multipolar mitotic spindles, acentrosomal spindle poles and multipolar cell division by targeting the centrosome-associated proteins nucleophosmin and Crm1.

Identification of Acyl Protein Thioesterases 1 and 2 as the Cellular Targets of the Ras-Signaling Modulators Palmostatin B and M

Finding the target: activity-based proteomic profiling probes based on the depalmitoylation inhibitors palmostatin B and M have been synthesized and were found to target acyl protein thioesterase 1 (APT1) and 2 (APT2) in cells.

Expanded Click Conjugation of Recombinant Proteins with Ubiquitin‐Like Modifiers Reveals Altered Substrate Preference of SUMO2‐Modified Ubc9

Wrestling with SUMO: the chemical conjugation of proteins with small ubiquitin-like modifiers (SUMO) can be achieved by a copper(I)-catalyzed cycloaddition and unnatural amino acid mutagenesis. This approach overcomes previous restrictions related to the primary sequence of proteins and coupling conditions. Moreover, biochemical data suggests that this triazole linkage presents the modifier in a proper distance and orientation relative to the target protein.

A Natural Product Inspired Tetrahydropyran Collection Yields Mitosis Modulators that Synergistically Target CSE1L and Tubulin

A Prins cyclization between a polymer-bound aldehyde and a homoallylic alcohol served as the key step in the synthesis of tetrahydropyran derivatives. A phenotypic screen led to the identification of compounds that inhibit mitosis (as seen by the accumulation of round cells with condensed DNA and membrane blebs). These compounds were termed tubulexins as they target the CSE1L protein and the vinca alkaloid binding site of tubulin.

Minimally Invasive Mutagenesis Gives Rise to a Biosynthetic Polyketide Library

Not in the public domain: Site-directed mutagenesis of megasynthases was the key to the generation of a library of polyketides in bacteria. Redox derivatizations are used to change the bioactivity profile of the compounds.

The Ras Pathway Modulator Melophlin A Targets Dynamins

The Ras/mitogen-activated protein (MAP) kinase signal transduction pathway regulates numerous biological programs including cell growth and differentiation, and harbors several important anticancer-drug targets. Recent research, in particular inspired by systems biology approaches, revealed the importance of dynamic spatiotemporal regulation of and interplay between the Ras network members and their interaction with other signaling modules for fully functional Ras signaling. Because of their rapid, conditional, and reversible mode of action, small-molecule modulators of protein function are particularly suitable tools for the conditional analysis of such dynamic biological processes, and hold great promise for the study of biological systems. Therefore, the identification of novel small-molecule modulators of signaling through the Ras network and the identification of their molecular targets are of major interest. 6] The naturally occurring tetramic acids melophlin A and B (1 and 2, Scheme 1A) reverse the morphology of HRas-transformed NIH3T3 fibroblasts at a concentration of 5 mgmL 1 (that is, IC50= 14 mm). [7] However, the biological targets of the melophlins and their link to the Ras network have not been identified. Herein, we report the synthesis of a melophlin-inspired compound collection and a subsequent chemical proteomics investigation, which revealed that melo-

Epiblastin A Induces Reprogramming of Epiblast Stem Cells Into Embryonic Stem Cells by Inhibition of Casein Kinase 1.

The discovery of novel small molecules that induce stem cell reprogramming and give efficient access to pluripotent stem cells is of major importance for potential therapeutic applications and may reveal novel insights into the factors controlling pluripotency. Chemical reprogramming of mouse epiblast stem cells (EpiSCs) into cells corresponding to embryonic stem cells (cESCs) is an inefficient process. In order to identify small molecules that promote this cellular transition, we analyzed the LOPAC library in a phenotypic screen monitoring Oct4-GFP expression and identified triamterene (TR) as initial hit. Synthesis of a TR-derived compound collection and investigation for reprogramming of EpiSCs into cESCs identified casein kinases 1 (CK1) α/δ/ɛ as responsible cellular targets of TR and unraveled the structural parameters that determine reprogramming. Delineation of a structure-activity relationship led to the development of Epiblastin A, which engages CK1 isoenzymes in cell lysate and induces efficient conversion of EpiSCs into cESCs.

Identification of Ustilago maydis Aurora kinase as a novel antifungal target.

Infestation of crops by pathogenic fungi has continued to have a major impact by reducing yield and quality, emphasizing the need to identify new targets and develop new agents to improve methods of crop protection. Here we present Aurora kinase from the phytopathogenic fungus Ustilago maydis as a novel target for N-substituted diaminopyrimidines, a class of small-molecule kinase inhibitors. We show that Aurora kinase is essential in U. maydis and that diaminopyrimidines inhibit its activity in vitro. Furthermore, we observed an overall good correlation between in vitro inhibition of Aurora kinase and growth inhibition of diverse fungi in vivo. In vitro inhibition assays with Ustilago and human Aurora kinases indicate that some compounds of the N-substituted diaminopyrimidine class show specificity for the Ustilago enzyme, thus revealing their potential as selective fungicides.

A pull-down procedure for the identification of unknown GEFs for small GTPases

ABSTRACT Members of the family of small GTPases regulate a variety of important cellular functions. In order to accomplish this, tight temporal and spatial regulation is absolutely necessary. The two most important factors for this regulation are GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), the latter being responsible for the activation of the GTPase downstream pathways at the correct location and time. Although a large number of exchange factors have been identified, it is likely that a similarly large number remains unidentified. We have therefore developed a procedure to specifically enrich GEF proteins from biological samples making use of the high affinity binding of GEFs to nucleotide-free GTPases. In order to verify the results of these pull-down experiments, we have additionally developed two simple validation procedures: An in vitro transcription/translation system coupled with a GEF activity assay and a yeast two-hybrid screen for detection of GEFs. Although the procedures were established and tested using the Rab protein Sec4, the similar basic principle of action of all nucleotide exchange factors will allow the method to be used for identification of unknown GEFs of small GTPases in general.

Positional Scanning Synthesis of a Peptoid Library Yields New Inducers of Apoptosis that Target Karyopherins and Tubulin.

We describe the synthesis of a library of 11 638 N‐alkylglycine peptoid trimers in a positional scanning format with adjustment of reaction conditions to account for different reactivities of the monomer building blocks. Evaluation of the library by high‐content phenotypic screening for modulators of the cytoskeleton and mitosis resulted in the identification of two apoptosis‐inducing peptoids, which, despite their structural similarity, target different proteins and cellular mechanisms. Whereas one peptoid binds to karyopherins, which mediate nuclear transport, the other N‐alkylglycine trimer binds tubulin at the vinca alkaloid binding site.