Highlights

We used a cellular reporter assay after induction of DNA-damage and application of drugs, or of genome-wide RNA-interference, or CRISPR-Cas9 mediated interference with all known proteasomal proteins, to search for novel targets for NFKB mediated cancer development. As a result  we identified CLK2 and CLK4 as regulators of DNA damage-induced NF-κB and novel small molecule inhibitors of this molecular mechanisms which increase tumor cell apoptosis (Cell Chemical Biology 2023, Mucka et al.).

Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.

Under pathophysiological conditions following nerve injury or diabetic neuropathy, the slightest touch can produce pain, and here STOML3 inhibitors can reverse mechanical hypersensitivity. Thus, small molecules applied locally to the skin can be used to modulate touch and may represent peripherally available drugs to treat tactile-driven pain following neuropathy. Mechanoreceptors can be silenced by local treatment with compound OB-1 which has been identified to interfere with stomatin-like protein-3 (STOML-3) oligomerization in HTS . (a) Inset: electrical search protocol schema. A microelectrode was used to deliver electrical stimuli at two distant points of the saphenous nerve trunk in order to trace reception. Percentage of non-mechanosensitive fibers is shown. We observed an increase in mechanically sensitive Aβ fibers 3 h after local OB-1 treatment; mechanosensitivity recovered 24 h after injection.

Volume-regulated anion channels (VRACs) are important constituents of the cellular response to osmotic swelling. This process occurs as a consequence of aquaporin-mediated water influx into cells under hypotonic conditions. To counteract the volume increase and prevent swelling-induced burst, different ion transport proteins are activated; this enables a controlled efflux of ions, organic osmolytes and consequently water, and allows cells to return to their original unchallenged state.

The group of Thomas Jentsch and the FMP Screening Unit identified the long-sought gene encoding for the channel that helps cells to reduce their volume. For this purpose a human genome-wide RNA-interference library was tested with a cellular reporter system for iodide influx and intracellular quenching of an iodide-sensitive yellow fluorescent protein. The messenger RNA of 21,687 genes was targeted by ~130,000 transfections with interfering RNA. The Volume-Regulated Anion Channel (VRAC) was stimulated by a change of cell culture medium from isotonic to hypotonic conditions. The inhibition of iodide mediated YFP quenching provided a read out for loss of the channel detected by kinetic imaging (FLIPR Tetra-system, Molecular Devices). Eighty-seven genes were identified as candidates and were used in a secondary screen with newly designed silencer RNAs for identification of LRRC8 heteromers as essential components of VRAC. 

Colon cancer cells were genetically manipulated to visualize Wnt signaling activity by linkeage to the expression of a Green Fluorescent Protein.  A heterogeneous expression of the protein becomes visible (GFP low or high) reflecting different Wnt signaling activities within cancer cells. Cells were sorted by FACS due to their different GFP fluorescence and analysed for properties. High expression correlates with properties of cancer stem cells. We identified small molecule inhibitors arresting the cancer cells in cell cycle and preventing the renewal of cancer stem cell populations. Moreover inhibitors drive the stem cells into differentiation and sensitize them for conventional drug therapy. 
Fang L, Zhu Q, Neuenschwander M, Specker E, Wulf-Goldenberg A, Weis WI, Kries von JP, Birchmeier W (2016) A Small-Molecule Antagonist of the β-Catenin/TCF4 Interaction Blocks the Self-Renewal of Cancer Stem Cells and Suppresses Tumorigenesis. Cancer Res. 76, 891–901.

Classical Hodgkin lymphoma (cHL) reflects a clinical challenge when presenting as primary refractory or relapsed disease. cHL is characterized by a virtual lack of gene products whose expression constitutes the B-cell phenotype. Restoring the B-cell phenotype may render cHL susceptible to clinically established antibody therapies, targeting B-cell surface receptors, or small compounds interfering with B-cell receptor signaling. We supported a high-throughput pharmacological screening based on more than 28,000 compounds in cHL cell lines carrying a CD19 reporter to identify drugs that promote re-expression of the B-cell phenotype. Three compounds were identified that robustly enhanced CD19 transcription. Subsequent chromatin immune precipitation-based analyses indicated that the action of two of these compounds was associated with lowered levels of the transcriptionally repressive lysine 9-trimethylated histone H3 mark at the CD19 promoter. Moreover, the anti-leukemia agents all-trans retinoic acid and arsenic trioxide (ATO) were found to reconstitute the silenced B-cell transcriptional program and reduce viability of cHL cell lines. Furthermore, restoration of the B-cell phenotype also rendered cHL cells susceptible to the B-cell non-Hodgkin lymphoma-tailored small-compound inhibitors ibrutinib and idelalisib. In essence, we identified a conceptually novel, redifferentiation-based treatment strategy for cHL.

Du J, Neuenschwander M, Yu Y, Däbritz JHM, Neuendorff N-R, Schleich K, Bittner A, Milanovic M, Beuster G, Radetzki S, Specker E, Reimann M, Rosenbauer F, Mathas S, Lohneis P, Hummel M, Dörken B, von Kries JP, Lee S, Schmitt CA (2017) Pharmacological restoration and therapeutic targeting of the B-cell phenotype in classical Hodgkin lymphoma. Blood 129, 71–81.

N-Acetylmannosamine kinase (MNK) plays a key role in the biosynthesis of sialic acids and glycosylation of proteins. Sialylated glycoconjugates affect a large number of biological processes, including immune modulation and cancer transformation. For the search of effective inhibitors of MNK we applied high-throughput screening of drug-like small molecules. We identified four potential MNK-specific inhibitors with IC50 values in the low micromolar range. Molecular modeling of the inhibitors into the active site of MNK supports their binding to both, either the sugar or the ATP-binding pocket of the enzyme. These compounds are promising to downregulate sialic acid content of glycoconjugates and to study the functional contribution to disease development. 

The Mycobacterium tuberculosis (Mtb) H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many Mtb P450s remain uncharacterized, suggesting their further analysis may provide new insights into Mtb metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explored the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands.