Glioblastoma (GBM) is the most aggressive primary brain tumor with poor patient survival that is at least partly caused by malignant and therapy-resistant glioma stem-like cells (GSLCs) that are protected in GSLC niches. Previously, we have shown that the chemo-attractant stromal-derived factor-1α (SDF-1α), its C-X-C receptor type 4 (CXCR4) and the cysteine protease cathepsin K (CatK) are localized in GSLC niches in glioblastoma. Here, we investigated whether SDF-1α is a niche factor that through its interactions with CXCR4 and/or its second receptor CXCR7 on GSLCs facilitates their homing to niches. Furthermore, we aimed to prove that SDF-1α cleavage by CatK inactivates SDF-1α and inhibits the invasion of GSLCs. We performed mass spectrometric analysis of cleavage products of SDF-1α after proteolysis by CatK. We demonstrated that CatK cleaves SDF-1α at 3 sites in the N-terminus, which is the region of SDF-1α that binds to its receptors. Confocal imaging of human GBM tissue sections confirmed co-localization of SDF-1α and CatK in GSLC niches. In accordance, 2D and 3D invasion experiments using CXCR4/CXCR7-expressing GSLCs and GBM cells showed that SDF-1α had chemotactic activity whereas CatK cleavage products of SDF-1α did not. Besides, CXCR4 inhibitor plerixafor inhibited invasion of CXCR4/CXCR7-expressing GSLCs. In conclusion, CatK can cleave and inactivate SDF-1α. This implies that CatK activity facilitates migration of GSLCs out of niches. We propose that activation of CatK may be a promising strategy to prevent homing of GSLCs in niches and thus render these cells sensitive to chemotherapy and radiation.

Just like the drugs themselves, their metabolites have to be evaluated to succeed in a drug development and approval process. It is therefore essential to be able to predict drug metabolism and to synthesise sufficient metabolite quantities for further pharmacological testing. This study evaluates the possibility of using in vitro biotransformations to solve both these challenges in the case of testosterone as a representative component for steroids. The application of cells of Pichia pastoris with expressed membraneassociated human liver cytochrome P450 enzyme (P450) 3A4 in two cycles of a preparative-scale bioreactor experiment enabled the isolation of the common metabolites 6β-hydroxytestosterone and 6β-hydroxyandrostenedione on a 100 mg scale. Side-product formation caused by enzymes intrinsic to P. pastoris was reduced. In addition more polar testosterone metabolites formed by a P450 3A4-catalysed bioconversion, than the known mono-hydroxylated ones, are reported and 6-dehydro-15β-hydroxytestosterone as well as the di-hydroxylated steroids 6β,16β-dihydroxytestosterone, 6β,17β-dihydroxy-4-androstene-3,16-dione and 6β,12β-dihydroxyandrostenedione were isolated and verified by NMR analysis. Their respective biological significance remains to be investigated. Whole-cell P450 catalysts expressed in P. pastoris qualify as a tool for the preparative-scale synthesis of human metabolites. Biotransformation processes in combination with standard chemical procedures allow the isolation and characterisation even of minor drug metabolite products.

Efficient synthetic techniques for the diversification of natural products are incremental for drug discovery processes of the pharmaceutical industry because these complex bioactive compounds often require an adjustment of properties. Human liver P450 3A4, key player of the body's detoxification system and decisive factor of a drug's metabolic fate, is renowned for its broad substrate scope including many natural products. In this study, we investigated the synthetic potential of human P450 3A4 for the diversification of natural product classes and isolated the produced metabolites of six selected natural products at a preparative 100-mg scale. Aided by efficient expression levels in P. pastoris, this whole-cell biocatalyst was found to be highly effective at the intended job allowing the identification of a total of 31 authentic human metabolites, many of them for the first time. By revealing an unprecedented degree of diversification, this study extends the synthetic repertoire for efficient enzymatic natural product modification in a one-step fashion and adds a completely new view to an old enzyme traditionally used for inhibition and toxicology studies.

Polycyclic aromatic hydrocarbons (PAHs) and their N- and O-containing derivatives (N-/O-PAHs) are environmental pollutants and synthetically attractive building blocks in pharmaceuticals. Functionalization of PAHs can be achieved via C-H activation by cytochrome P450 enzymes (e.g., P450 CYP3A4) in an environmentally friendly manner. Despite its broad substrate scope, the contribution of CYP3A4 to metabolize common PAHs in humans was found to be small. We recently showcased the potential of CYP3A4 in whole-cell biocatalysis with recombinant yeast Komagataella phaffii (Pichia pastoris) catalysts for the preparative-scale synthesis of naturally occurring metabolites in humans. In this study, we aimed at exploring the substrate scope of CYP3A4 towards (N-/O)-PAHs and conducted a bioconversion experiment at 10 L scale to validate the synthetic potential of CYP3A4 for the preparative-scale production of functionalized PAH metabolites. Hydroxylated products were purified and characterized using HPLC and NMR analysis. In total, 237 mg of fluorenol and 48 mg of fluorenone were produced from 498 mg of fluorene, with peak productivities of 27.7 μmol/L/h for fluorenol and 5.9 μmol/L/h for fluorenone; the latter confirmed that CYP3A4 is an excellent whole-cell biocatalyst for producing authentic human metabolites.

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