SummarySexual development in fungi is a complex process involving the generation of new cell types and tissues -an essential step for all eukaryotic life. The characterization of sterile mutants in the ascomycete Sordaria macrospora has led to a number of proteins involved in sexual development, but a link between these proteins is still missing. Using a combined tandem-affinity purification/mass spectrometry approach, we showed in vivo association of developmental protein PRO22 with PRO11, homologue of mammalian striatin, and SmPP2AA, scaffolding subunit of protein phosphatase 2A. Further experiments extended the protein network to the putative kinase activator SmMOB3, known to be involved in sexual development. Extensive yeast two-hybrid studies allowed us to pinpoint functional domains involved in protein-protein interaction. We show for the first time that a number of already known factors together with new components associate in vivo to form a highly conserved multi-subunit complex. Strikingly, a similar complex has been described in humans, but the function of this so-called striatin interacting phosphatase and kinase (STRIPAK) complex is largely unknown. In S. macrospora, truncation of PRO11 and PRO22 leads to distinct defects in sexual development and cell fusion, indicating a role for the fungal STRIPAK complex in both processes.

Diastereomeric norbornapeptides represent globular scaffolds with geometries determined by the chirality of amino acid residues and sharing structural features of β-turns and α-helices.

Unbiased chemoproteomic profiling of small-molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G protein-coupled receptors (GPCRs). These receptors are hardly tractable by affinity pulldown from lysates. We report a capture compound (CC)-based strategy to target and identify GPCRs directly from living cells. We synthesized CCs with sertindole attached to the CC scaffold in different orientations to target the dopamine D2 receptor (DRD2) heterologously expressed in HEK 293 cells. The structure-activity relationship of sertindole for DRD2 binding was reflected in the activities of the sertindole CCs in radioligand displacement, cell-based assays, and capture compound mass spectrometry (CCMS). The activity pattern was rationalized by molecular modelling. The most-active CC showed activities very similar to that of unmodified sertindole. A concentration of DRD2 in living cells well below 100 fmol used as an experimental input was sufficient for unambiguous identification of captured DRD2 by mass spectrometry. Our new CCMS workflow broadens the arsenal of chemoproteomic technologies to close a critical gap for the comprehensive characterization of drug-protein interactions.

Structurally related inhibitors of a shared therapeutic target may differ regarding potential toxicity issues that are caused by different off-target bindings. We devised a differential competition capture compound mass spectrometry (dCCMS) strategy to effectively differentiate off-target profiles. Tolcapone and entacapone are potent inhibitors of catechol-O-methyl transferase (COMT) for the treatment of Parkinson's disease. Tolcapone is also known for its hepatotoxic side effects even though it is therapeutically more potent than entacapone. Here, we identified 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) as a possible toxicity-causing off-target of tolcapone, and this protein is not bound by the less toxic COMT inhibitor entacapone. Moreover, two novel compounds from a focused library synthesized in-house, N(2),N(2),N(3),N(3)-tetraethyl-6,7-dihydroxy-5-nitronaphthalene-2,3-dicarboxamide and 5-(3,4-dihydroxy-5-nitrobenzylidene)-3-ethylthiazolidine-2,4-dione, were utilized to gain insight into the structure-activity relationships in binding to COMT and the novel off-target HIBCH. These compounds, especially N(2),N(2),N(3),N(3)-tetraethyl-6,7-dihydroxy-5-nitronaphthalene-2,3-dicarboxamide, could serve as starting point for the development of improved and more specific COMT inhibitors.

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