Screening Unit

Inhibition of mechanical hypersensitivity (G. Lewin, Nature Neuroscience 2017)

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.

Identification of the gene encoding for the volume regulated ion channel (VRAC 8) by genome-wide silencer-RNA screening (T. Jentsch, Science 2014)

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. 

Blocking self-renewal of cancer stem cells (W. Birchmeier, Cancer Research 2016)

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.

Restoration of B-cell phenotype in cHodgkin´s lymphoma (C. Schmitt, Blood 2017)

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.

Small molecules targeting human N-acetylmannosamine kinase (C. Hackenberger, ChemBioChem 2017)

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. 

Drug Mediated Rescue of Heart Development (S. Seyfried)

Frontal views of 48 hpf zebrafish hearts of the myocardial reporter line Tg[myl7:GFP]twu34 reveals that cardiac expansion is rescued in mutants after 24 hours of treatment with a pharmacological compound. The heart becomes visible by specific expression of Green Fluorescent Protein, GFP. The small size (~ 1 mm) and transparency of embryos enables to analyse organ development by simple microscopic observation (Salim Seyfried, MDC & Screening Unit, AG von Kries, FMP).

Complex Structure of Inhibitors with Mycobacterium tuberculosis cytochrome P450 CYP126A1 (AW Munro, University Manchester, UK; JBC 2016)

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. 

Chenge, J.T., Le, D.V., Swami, S., McLean, K.J., Kavanagh, M.E., Coyne, A.G., Rigby, S.E.J., Cheesman, M.R., Girvan, H.M., Levy, C.W., et al. (2016). Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1. J. Biol. Chem. 292, 1310-29.

DNA Damage Induced NFKB Signalling (C. Scheidereit)

A cell line stably expressing a fusion of NFKB and GFP (green fluorescent protein) allows to monitor NFKB activation (translocation to nucleus) by compound induced DNA damage. The aim of the project is to identify and use inhibitory compounds to characterise the molecular mechanism of this activation process (ECRC funded project, Claus Scheidereit, MDC & Screening Unit, AG von Kries, FMP)

Genome-Wide RNA-Interference & High Content Imaging: Aquaporin Translocation (E. Klussmann, submitted)

High content screening combined with genome-wide RNA-Interference allows analyzing the contribution of a single gene towards cellular trafficking of aquaporin-2 (AQP2) in renal principal cells. Upon rise of cAMP, AQP2 redistributes from intracellular vesicles to the plasma membrane. Analysis of the very complex distribution pattern (not all cells respond similar due to neighbor effects, therefore no black/white output generated for analysis) of AQP2 requires the development of robust automated object identification routines (Doerte Faust, Walter Rosenthal and Enno Klussmann, MDC & Screening Unit, AG von Kries, FMP)

Tankyrase Inhibitor Reduces Tumor Growth in APCMin mice (S. Kraus, Oslo, Cancer Research 2012)

A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice.

Left image shows untreated SW480 colon cancer cells. beta-Catenin staining by antibodies shows nuclear accumulation of the protein due to dysfunction of APC for destruction of cellular beta-Catenin caused by corresponding mutations in the APC gene. Application of the compound to SW-480 cells results in increased AXIN2 stability thereby inducing beta-Catenin degradation.

The initial hit compound has been optimised for metabolic stability and reduced tumor formation in  APCMin mice as a model for colon cancer development. The compound demonstrates a very specific interaction profile with less then three protein interactions with total proteins of cells.

Small molecule inhibitors of clathrin function in endocytosis (V. Haucke, Cell 2011)

Infectious diseases begin when viruses, bacteria, or parasites enter cells; many other health conditions are caused by defects in cells, for example when they receive faulty signals that change their fates. Both processes depend on a complex transport system that acts as a cellular post office, moving molecules and substances to their proper sites. Volker Haucke of the Free University of Berlin, in collaboration with researchers in Australia and at the FMP, has just developed a new method of sealing off cells to prevent the entry of both pathogens such as HIV and some undesirable signals.

The plasma membrane, which is the outermost border of the cell, acts as a first line of defense when things go wrong, and it can bar entry to most unwanted parasites or substances. Some make it through, though, by taking advantage of an intricate system that usually transports healthy molecules through cells. Their delivery relies on envelopes made of membranes, address labels made of proteins, motor molecules that drive the packages around, and many other players.

Disrupting a single vital component could close down the entire system. Volker and his colleagues focused on a protein called clathrin, which made a good candidate because it carries out several important tasks. First, it picks the site where a package will be assembled, by creating a pit-like depression in the membrane. The well forms a bubble-like shape containing the cargo which clathrin now pinches off, creating a compartment called a vesicle that is released and sent on its way.
Stopping this process required finding something that could block clathrin. The screening for compounds, which inhibit protein binding to clathrin, at the FMP produced a list of "hits" to focus on in further experiments with living cells. This posed a challenge because some of the most interesting candidates from the initial screen with purified clathrin couldn't pass through the cell membrane, meaning they couldn't reach clathrin. The institutes' chemists began tinkering with the substances to enable them to do so and to make them more potent.
This approach resulted in the discovery of two compounds that are named "pitstops," because of their effect on clathrin's ability to form pits in the membrane. Within minutes of their introduction into cells, the substances caused a general "strike" in the transport system. Since this is the mechanism that HIV, undesirable signaling molecules, and other intruders take advantage of, the intervention successfully blocked their entry into cells.

Figure left: Pitstop 2 reversibly inhibits Tf uptake. After 15 min preincubation HeLa cells were incubated with Alexa568-Tf in the presence of DMSO or 30 μM pitstop 2 for 15 min. Tf uptake is seen to resume after washout of the drug for 1 h. Scale bar, 10 mm.

von Kleist, L., Stahlschmidt, W., Bulut, H., Gromova, K., Puchkov, D., Robertson, MJ., MacGregor, KA., Tomlin, N., Pechstein, A., Chau, N., Chircop, M., Sakoff, J., von Kries, JP., Saenger, W., Kräusslich, H-G., Shupliakov, O., Robinson,PJ., McCluskey, A., and Haucke V. Essential Role of the Clathrin Terminal Domain in Regulating Coated Pit Dynamics Revealed by Small Molecule Inhibition. Cell 2011, 146 (3):p 335-488).


Specific Inhibition of MARK2 (E. Mandelkow, JBC 2011)

The expression of active MARK in CHO cells leads to the disruption of the microtubule (MT) network, subsequently the cells shrink, round up and finally die. The effect is explained by the destabilization of microtubules following the phosphorylation of MAPs, it can be counteracted by microtubule stabilizers (taxol) or by increasing the effective concentration of MAPs. Consistent with this, in the present experiments, the MARK2-dependent toxicity was rescued by treatment of the cells with the inhibitor 39621 (Fig. right) which suppressed MARK2 activity, phosphorylation of MAPs, and thus prevented MT breakdown as shown by almost normal cell size. As a control, the transfection of an inactive mutant of MARK2 had no effect on the microtubule network and toxicity, confirming that the effect was indeed due to phosphorylation by MARK2.

Timm, T., von Kries, JP., Li, X., Mandelkow, E. und Mandelkow, E-V. Microtubule affinity regulating kinase (MARK) activity in living neurons examined by a genetically encoded FRET/FLIM based biosensor: Inhibitors with therapeutic potential J. Biol. Chem. 2011 jbc.M111.257865. First Published on October 7, 2011, doi:10.1074/jbc.M111.257865

Novel inhibitors for CYP51 of Mycobacterium tuberculosis (L. Podust, UCSF)

Mycobacterium tuberculosislanosterol demethylase inhibitor complex2cib

Binding of the inhibitor allowed for the first time to characterize the complete structure of the enzyme from tuberculosis bacteria and to identify the molecular mechanism of enzyme function.

Nasser Eddine A, von Kries JP, Podust MV, Warrier T, Kaufmann SH, Podust LM. X-ray structure of 4,4'-dihydroxybenzophenone mimicking sterol substrate in the active site of sterol 14alpha -demethylase (CYP51). J Biol Chem. 2008 May 30;283(22):15152-9. Epub 2008 Mar 26

Inhibitors of ptp-Shp2 block metastasis (W. Birchmeier, PNAS 2008)

Inhibitors of HGF/SF induced cell scattering of MDCK colonies in 384well MTP´s:

Cells are stained for actin with phalloidinB (red) , for total cells with CMFDA (green, live cell tracker)  and for nuclei with Hoechst 3342 (blue) . Images are documented and analyzed with the ArrayScan VTi. In absence of HGF/SF (-SF, left) cells build colonies, after addition of Hepatocyte Growth Factor (+SF, 3 units, 24 hrs) cells in colonies detach from each other and get motile. This process resembles metasasis of tumor cells in cancer development. In presence of Shp2 inhibitors this process is blocked.

K. Hellmuth, S. Grosskopf, C. T. Lum, M. Würtele, N. Röder, J. P. von Kries, M. Rosario, J. Rademann, W. Birchmeier. (2008) Proc. Nat. Acad. Sci. USA , 105, 7275-7280.

Minister of Science PR China visits Open Access Platform

Prof. Wang Gang, Minister of Science and Technology

Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
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