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The accurate prediction of protein-ligand binding affinity belongs to one of the central goals in computer-based drug design. Molecular dynamics (MD)-based free energy calculations have become increasingly popular in this respect due to their accuracy and solid theoretical basis. Here, we present a combined study which encompasses experimental and computational studies on two series of factor Xa ligands, which enclose a broad chemical space including large modifications of the central scaffold. Using this integrated approach, we identified several new ligands with different heterocyclic scaffolds different from the previously identified indole-2-carboxamides that show superior or similar affinity. Furthermore, the so far underexplored terminal alkyne moiety proved to be a suitable non-classical bioisosteric replacement for the higher halogen-π aryl interactions. With this challenging example, we demonstrated the ability of the MD-based non-equilibrium free energy calculation approach for guiding crucial modifications in the lead optimization process, such as scaffold replacement and single-site modifications at molecular interaction hot spots.

Wnt/β-catenin signaling is a highly conserved pathway essential for embryogenesis and tissue homeostasis. However, deregulation of this pathway can initiate and promote human malignancies, especially of the colon and head and neck. Therefore, Wnt/β-catenin signaling represents an attractive target for cancer therapy. We performed high-throughput screening using AlphaScreen and ELISA techniques to identify small molecules that disrupt the critical interaction between β-catenin and the transcription factor TCF4 required for signal transduction. We found that compound LF3, a 4-thioureido-benzenesulfonamide derivative, robustly inhibited this interaction. Biochemical assays revealed clues that the core structure of LF3 was essential for inhibition. LF3 inhibited Wnt/β-catenin signals in cells with exogenous reporters and in colon cancer cells with endogenously high Wnt activity. LF3 also suppressed features of cancer cells related to Wnt signaling, including high cell motility, cell-cycle progression, and the overexpression of Wnt target genes. However, LF3 did not cause cell death or interfere with cadherin-mediated cell–cell adhesion. Remarkably, the self-renewal capacity of cancer stem cells was blocked by LF3 in concentration-dependent manners, as examined by sphere formation of colon and head and neck cancer stem cells under nonadherent conditions. Finally, LF3 reduced tumor growth and induced differentiation in a mouse xenograft model of colon cancer. Collectively, our results strongly suggest that LF3 is a specific inhibitor of canonical Wnt signaling with anticancer activity that warrants further development for preclinical and clinical studies as a novel cancer therapy. Cancer Res; 76(4); 891–901. ©2015 AACR.

Fig. S5 CSC markers and self-renewal of GFPhigh SW480 cells.

Supplementary figure legends

Fig. S1 Technology and protein production for the primary High Throughput Screen (HTS).

Fig. S7 LF3 reduces tumor growth without showing toxicity in vivo.

Table S3. Gene function clustering using the DAVID bioinformatics resources. Table S1. Gene function clustering using the DAVID bioinformatics resources.

Fig. S6 Influence of LF3 on gene profiling and differentiation of mouse salivary gland CSCs.

Fig. S4 LF3 doesn't cause apoptosis or cell death.

Fig. S3 Inhibition of Wnt signaling by LF3 in colon cancer cells.

Portions of Materials and Methods and Supplementary Tables 1 and 2. Table S1: Primer sequences for ChIP experiment Table S2: Primer sequences for qRT-PCR

Fig. S2 Specific inhibition of Wnt signaling by LF3.

Tryptophan hydroxylases catalyze the first and rate-limiting step in the synthesis of serotonin. Serotonin is a key neurotransmitter in the central nervous system and, in the periphery, functions as a local hormone with multiple physiological functions. Studies in genetically altered mouse models have shown that dysregulation of peripheral serotonin levels leads to metabolic, inflammatory, and fibrotic diseases. Overproduction of serotonin by tumor cells causes severe symptoms typical for the carcinoid syndrome, and tryptophan hydroxylase inhibitors are already in clinical use for patients suffering from this disease. Here, we describe a novel class of potent tryptophan hydroxylase inhibitors, characterized by spanning all active binding sites important for catalysis, specifically those of the cosubstrate pterin, the substrate tryptophan as well as directly chelating the catalytic iron ion. The inhibitors were designed to efficiently reduce serotonin in the periphery while not passing the blood-brain barrier, thus preserving serotonin levels in the brain.

Fragment-based screening has evolved as a remarkable approach within the drug discovery process both in the industry and academia. Fragment screening has become a more structure-based approach to inhibitor development, but also towards development of pathway-specific clinical probes. However, it is often witnessed that the availability, immediate and long-term, of a high quality fragment-screening library is still beyond the reach of most academic laboratories. Within iNEXT (Infrastructure for NMR, EM and X-rays for Translational research), a EU-funded Horizon 2020 program, a collection of 782 fragments were assembled utilizing the concept of “poised fragments” with the aim to facilitate downstream synthesis of ligands with high affinity by fragment ligation. Herein, we describe the analytical procedure to assess the quality of this purchased and assembled fragment library by NMR spectroscopy. This quality assessment requires buffer solubility screening, comparison with LC/MS quality control and is supported by state-of-the-art software for high throughput data acquisition and on-the-fly data analysis. Results from the analysis of the library are presented as a prototype of fragment progression through the quality control process. Electronic supplementary material The online version of this article (10.1007/s10858-020-00327-9) contains supplementary material, which is available to authorized users.

Cancer metastasis is the most lethal attribute of cancer, frequently caused by insufficient therapies and limited therapy options. Novel compounds able to interfere with metastasis formation are therefore of tremendous interest. Colorectal Cancer (CRC) is the third most prevalent and second most lethal cancer worldwide. In CRC, metastasis formation is linked to poor patient survival and treatment failure. Up to 90% of CRC related deaths are attributed to metastasis formation. Identification of causative drivers of metastasis represents the basis for effective anti-metastatic therapy. Our lab newly identified the gene MACC1 (Metastasis Associated in Colon Cancer 1). MACC1 is a key causal molecule for tumor progression and metastasis formation. It is a stage independent prognostic marker, predicting the onset of metastasis in stages I, II and III, based on tumor tissue analyses or blood-based tests. MACC1 promotes several cancer hallmark capabilities, providing cells with a malignant phenotype. Further, MACC1 has been established as a prognostic and predictive biomarker for metastasis in CRC and more than 20 other solid cancer entities. We therefore searched for novel compounds targeting MACC1 transcription. A high-throughput screen with more than 118,500 compounds (EMBL, Heidelberg), employing CRC cells stably transfected with a MACC1 promoter-luciferase reporter construct, revealed a Tetrazolo-pyridazine based compound as a promising lead for effective inhibition of MACC1 expression. We demonstrated that several SAR and MedChem-generated analogues of our lead compound effectively inhibit MACC1 gene expression and MACC1 driven cancer cell motility in vitro in CRC and cross entity cell lines. Further, they inhibit MACC1-induced tumor progression and metastasis in vivo in CRC xenograft models in mice. Moreover, ADMET studies were conducted, confirming our compounds are likely to be orally active drug with high stability in human plasma, low plasma protein binding and great permeability with neglectable efflux in MDR1-MDCKII assay. Through RNA-sequencing and subsequent gene set enrichment analysis a hypothesis on the mode of action was formed. An immune pathway has been identified as the most promising signaling pathway targeted by these compounds, which is currently explored through knock down and signaling studies. Taken together, this novel class of small molecules represents promising candidates for anti-metastatic therapy in CRC and other solid cancer patients in a personalized medicine setting. Citation Format: Yan Shixian, Paul Schöpe, Joe Lewis, Kerstin Putzker, Ulrike Uhrig, Edgar Specker, Jens von Kries, Hector E. Sanchez-Ibarra, Anke Unger, Mia Zischinsky, Bert Klebl, Peter Lindemann, Wolfgang Walther, Marc Nazaré, Dennis Kobelt, Ulrike Stein. Novel Tetrazolo-pyridazine based MACC1 inhibitors are promising for anti-metastatic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2471.

The leading cause of cancer related deaths is the formation of metastasis, frequently caused by insufficient therapies and limited therapy options. Novel compounds able to interfere with metastasis formation are therefore of tremendous interest. Colorectal Cancer (CRC) is the third most prevalent and second most lethal cancer worldwide. In CRC, metastasis formation is linked to poor patient survival and treatment failure. Up to 90% of CRC related deaths are attributed to metastasis formation. Identification of causative drivers of metastasis represents the basis for effective anti-metastatic therapy. Our lab newly identified the gene MACC1 (Metastasis Associated in Colon Cancer 1) in 2009. Since then, MACC1 has been established as a key causal molecule for tumor progression and metastasis formation. It was shown that MACC1 can function as a stage independent prognostic marker, predicting the onset of metastasis in stages I, II and III, based on tumor tissue analyses or through a blood based test with an accuracy of up to 85%. MACC1 promotes several cancer hallmark capabilitites, providing cells with a malignant phenotype. Further, MACC1 has been established as a prognostic and predictive biomarker for metastasis in CRC and more than 20 other solid cancer entities. We therefore searched for novel compounds targeting MACC1 transcription. A high-throughput screen employing HCT116 cells stably transfected with a MACC1 promoter-luciferase reporter construct with more than 118,500 compounds was conducted at the EMBL in Heidelberg. The screen revealed a Tetrazolo-pyridazine based compound as a promising lead for effective inhibition of MACC1 expression. We demonstrated that several SAR and Medchem-generated analogues of our lead compound effectively inhibit MACC1 gene expression and MACC1 driven cancer cell motility in vitro in CRC and cross entity cell lines. Further, they inhibit MACC1-induced tumor progression and metastasis in vivo in a CRC xenograft model in mice. Moreover, ADMET studies were conducted, confirming our compounds are likely to be orally active drugs with high stability in human plasma, low plasma protein binding and great permeability with neglectable efflux in MDR1-MDCKII assay. Through RNA-sequencing and subsequent gene set enrichment analysis a first hypothesis on the mode of action was shaped. An immune pathway has been identified as the most promising signaling pathway targeted by these compounds and is currently explored through knock down and signaling studies. Taken together, this novel class of small molecules represents promising candidates for anti-metastatic therapy in CRC and other solid cancer patients in a personalized medicine setting. Funding: This study is adviced and financed by the SPARK BIH Program Citation Format: Paul Curtis Schöpe, Shixian Yan, Dennis Kobelt, Joe Lewis, Kerstin Putzker, Ulrike Uhrig, Edgar Specker, Jens Peter von Kries, Mathias Dahlmann, Hector E Sanchez-Ibarra, Anke Unger, Mia-Lisa Zischinsky, Bert Klebl, Peter Lindemann, Wolfgang Walther, Marc Nazaré, Ulrike Stein. Novel tetrazolo-pyridazine based MACC1 transcriptional inhibitors as promising anti-metastatic therapy [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A039.

Jumonji domain-containing lysine demethylase (KDM) enzymes are encoded by genes of the KDM superfamily. Activities of the KDM4 subfamily promote aggressive phenotypes associated with prostate cancer (PCa). Previously, we discovered a benzimidazole pyrazole molecule that inhibited KDM4 isoforms with properties tractable for development. Here, we demonstrate that a benzyl-substituted variant of this inhibitor exhibits improved potency in biochemical assays, is cell-permeable, and kills PCa cells at low micromolar concentrations. By X-ray crystallography and kinetics-based assays, we demonstrate that the mechanism of inhibition is complex, proceeding via competition with the enzyme for binding of active-site Fe(2+) and by populating a distal site on the enzyme surface. Furthermore, we provide evidence that the inhibitor's cytostatic properties arise from direct intracellular inhibition of KDM4 enzymes. PCa cells treated with the inhibitor exhibit reduced expression of genes regulated by the androgen receptor, an outcome accompanied by epigenetic maintenance of a heterochromatic state.

Fragment-based drug discovery is a powerful complement to conventional high-throughput screening, especially for difficult targets. Screening low-molecular-weight fragments usually requires highly sensitive biophysical methods, because of the generally low affinity of the identified ligands. Here, we developed a cell-based fragment screening assay (cellFy) that allows sensitive identification of fragment hits in a physiologically more relevant environment, in contrast to isolated target screenings in solution. For this, a fluorescently labeled multivalent reporter was employed, enabling direct measurement of displacement by low-molecular-weight fragments without requiring enzymatic reactions or receptor activation. We applied this technique to identify hits against two challenging targets of the C-type lectin receptor (CLR) family: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin (DC-SIGN) and Langerin. Both receptors are involved in pathogen recognition and initiation of an immune response, which renders them attractive targets for immune modulation. Because of their shallow and hydrophilic primary binding site, hit identification for CLRs is challenging and druglike ligands for CLRs are sparse. Screening of a fragment library followed by hit validation identified several promising candidates for further fragment evolution for DC-SIGN. In addition, a multiplexed assay format was developed for simultaneous screening against multiple CLRs, allowing a selectivity counterscreening. Overall, this sensitive cell-based fragment screening assay provides a powerful tool for rapid identification of bioactive fragments, even for difficult targets.

Human lysine demethylase (KDM) enzymes (KDM1-7) constitute an emerging class of therapeutic targets, with activities that support growth and development of metastatic disease. By interacting with and co-activating the androgen receptor, the KDM4 subfamily (KDM4A-E) promotes aggressive phenotypes of prostate cancer (PCa). Knockdown of KDM4 expression or inhibition of KDM4 enzyme activity reduces the proliferation of PCa cell lines and highlights inhibition of lysine demethylation as a possible therapeutic method for PCa treatment. To address this possibility, we screened the ChemBioNet small molecule library for inhibitors of the human KDM4E isoform and identified several compounds with IC50 values in the low micromolar range. Two hits, validated as active by an orthogonal enzyme-linked immunosorbent assay, displayed moderate selectivity toward the KDM4 subfamily and exhibited antiproliferative effects in cellular models of PCa. These compounds were further characterized by their ability to maintain the transcriptionally silent histone H3 tri-methyl K9 epigenetic mark at subcytotoxic concentrations. Taken together, these efforts identify and validate a hydroxyquinoline scaffold and a novel benzimidazole pyrazolone scaffold as tractable for entry into hit-to-lead chemical optimization campaigns.

Wnt/β-catenin signaling is a highly conserved pathway essential for embryogenesis and tissue homeostasis. However, deregulation of this pathway can initiate and promote human malignancies, especially of the colon and head and neck. Therefore, Wnt/β-catenin signaling represents an attractive target for cancer therapy. We performed high-throughput screening using AlphaScreen and ELISA techniques to identify small molecules that disrupt the critical interaction between β-catenin and the transcription factor TCF4 required for signal transduction. We found that compound LF3, a 4-thioureido-benzenesulfonamide derivative, robustly inhibited this interaction. Biochemical assays revealed clues that the core structure of LF3 was essential for inhibition. LF3 inhibited Wnt/β-catenin signals in cells with exogenous reporters and in colon cancer cells with endogenously high Wnt activity. LF3 also suppressed features of cancer cells related to Wnt signaling, including high cell motility, cell-cycle progression, and the overexpression of Wnt target genes. However, LF3 did not cause cell death or interfere with cadherin-mediated cell–cell adhesion. Remarkably, the self-renewal capacity of cancer stem cells was blocked by LF3 in concentration-dependent manners, as examined by sphere formation of colon and head and neck cancer stem cells under nonadherent conditions. Finally, LF3 reduced tumor growth and induced differentiation in a mouse xenograft model of colon cancer. Collectively, our results strongly suggest that LF3 is a specific inhibitor of canonical Wnt signaling with anticancer activity that warrants further development for preclinical and clinical studies as a novel cancer therapy. Cancer Res; 76(4); 891–901. ©2015 AACR.

Compound screening in biological assays and subsequent optimization of hits is indispensable for the development of new molecular research tools and drug candidates. To facilitate such discoveries, the European Research Infrastructure EU-OPENSCREEN was founded recently with the support of its member countries and the European Commission. Its distributed character harnesses complementary knowledge, expertise, and instrumentation in the discipline of chemical biology from 20 European partners, and its open working model ensures that academia and industry can readily access EU-OPENSCREEN’s compound collection, equipment, and generated data. To demonstrate the power of this collaborative approach, this perspective article highlights recent projects from EU-OPENSCREEN partner institutions. These studies yielded (1) 2-aminoquinazolin-4(3H)-ones as potential lead structures for new antimalarial drugs, (2) a novel lipodepsipeptide specifically inducing apoptosis in cells deficient for the pVHL tumor suppressor, (3) small-molecule-based ROCK inhibitors that induce definitive endoderm formation and can potentially be used for regenerative medicine, (4) potential pharmacological chaperones for inborn errors of metabolism and a familiar form of acute myeloid leukemia (AML), and (5) novel tankyrase inhibitors that entered a lead-to-candidate program. Collectively, these findings highlight the benefits of small-molecule screening, the plethora of assay designs, and the close connection between screening and medicinal chemistry within EU-OPENSCREEN. The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: NOR-OPENSCREEN, funded by the Research Council of Norway (RCN) and the K.G. Jebsen Centre for Neuropsychiatric Disorders, for financial support (to A.M.). The Danish Research Infrastructure for Chemical Biology, DK-OPENSCREEN, acknowledges financial support from the Ministry of Higher Education and Science (grant case no. 5072-00019B), the Technical University of Denmark, and the other contributing universities. The Latvian Institute of Organic Synthesis acknowledges the European Regional Development Fund (agreement no. 1.1.1.1/16/A/290) for financial support. L.L. acknowledges the Academy of Finland (grant nos. 287063 and 294085) and the Jane and Aatos Erkko Foundation for funding. EU-OPENSCREEN acknowledges its member and observer states for funding and support. P. Bartunek acknowledges MEYS (LO1220 and LM2015063) for funding. P.G. and H.L. acknowledge funding from the German Federal Ministry of Education and Research. J.L.K. and R.P. acknowledge financial support from the Polish Ministry of Science and Higher Education (KNOW program and POL-OPENSCREEN project number 401086). M.J.V. acknowledges the European Regional Development Fund and the Conselleria de Sanitat Universal i Salut Pública (Generalitat Valenciana, GVA) SI

The thyrotropin receptor (TSHR) is the target for autoimmune thyroid stimulating antibodies (TSAb) triggering hyperthyroidism. Whereas elevated thyroid hormone synthesis by the thyroid in Graves' disease can be treated by antithyroid agents, for the pathogenic activation of TSHR in retro-orbital fibroblasts of the eye, leading to Graves' orbitopathy (GO), no causal TSHR directed therapy is available.Due to the therapeutic gap for severe GO, TSHR inhibitors were identified by high-throughput screening in Chinese hamster ovary cells expressing the TSHR. Stereo-selective synthesis of the screening hits led to the molecule S37, which contains seven chiral centers. Enantiomeric separation of the molecule S37 resulted in the enantiopure molecule S37a-a micro-molar antagonist of thyrotropin-induced cyclic adenosine monophosphate accumulation in HEK 293 cells expressing the TSHR.The unique rigid bent shape of molecule S37a may mediate the observed high TSHR selectivity. Most importantly, the closely related follitropin and lutropin receptors were not affected by this compound. S37a not only inhibits the TSHR activation by thyrotropin itself but also activation by monoclonal TSAb M22 (human), KSAb1 (murine), and the allosteric small-molecule agonist C2. Disease-related ex vivo studies in HEK 293 cells expressing the TSHR showed that S37a also inhibits cyclic adenosine monophosphate formation by oligoclonal TSAb, which are highly enriched in GO patients' sera. Initial in vivo pharmacokinetic studies revealed no toxicity of S37a and a remarkable 53% oral bioavailability in mice.In summary, a novel highly selective inhibitor for the TSHR is presented, which has promising potential for further development for the treatment of GO.

The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs. ispartof: PLOS ONE vol:13 issue:12 ispartof: location:United States status: published

This work describes a collaborative effort to define and apply a protocol for the rational selection of a general-purpose screening library, to be used by the screening platforms affiliated with the EU-OPENSCREEN initiative. It is designed as a standard source of compounds for primary screening against novel biological targets, at the request of research partners. Given the general nature of the potential applications of this compound collection, the focus of the selection strategy lies on ensuring chemical stability, absence of reactive compounds, screening-compliant physicochemical properties, loose compliance to drug-likeness criteria (as drug design is a major, but not exclusive application), and maximal diversity/coverage of chemical space, aimed at providing hits for a wide spectrum of drugable targets. Finally, practical availability/cost issues cannot be avoided. The main goal of this publication is to inform potential future users of this library about its conception, sources, and characteristics. The outline of the selection procedure, notably of the filtering rules designed by a large committee of European medicinal chemists and chemoinformaticians, may be of general methodological interest for the screening/medicinal chemistry community. The selection task of 200K molecules out of a pre-filtered set of 1.4M candidates was shared by five independent European research groups, each picking a subset of 40K compounds according to their own in-house methodology and expertise. An in-depth analysis of chemical space coverage of the library serves not only to characterize the collection, but also to compare the various chemoinformatics-driven selection procedures of maximal diversity sets. Compound selections contributed by various participating groups were mapped onto general-purpose self-organizing maps (SOMs) built on the basis of marketed drugs and bioactive reference molecules. In this way, the occupancy of chemical space by the EU-OPENSCREEN library could be directly compared with distributions of known bioactives of various classes. This mapping highlights the relevance of the selection and shows how the consensus reached by merging the five different 40K selections contributes to achieve this relevance. The approach also allows one to readily identify subsets of target- or target-class-oriented compounds from the EU-OPENSCREEN library to suit the needs of the diverse range of potential users. The final EU-OPENSCREEN library, assembled by merging five independent selections of 40K compounds from various expert groups, represents an excellent example of a Europe-wide collaborative effort toward the common objective of building best-in-class European open screening platforms.

The layout of multipurpose screening libraries must address criteria for the compounds such as novelty, diversity potential, innovative design, and last but not least synthetic tractability. While academic compound collections are often innovative, novel, and highly divers, synthesis of analogs or larger substance quantities is often hampered by complex multistep syntheses with low overall yields. In addition, covalently binding compounds and interaction motifs designed to bind metal ions were discriminated against by the paradigm that these interaction types must almost inevitably lead to toxic effects. We would like to challenge this hypothesis. The lack of such interactions could be a reason for frequent failure in the disclosure of hits for hitherto undruggable target proteins using commercially available screening collections. Thus, easily synthesizable screening candidates equipped to bind covalently to nucleophiles or to metalloenzymes by chelation are under-represented in public access screening libraries. Within this work, we present the synthesis and deposition of 88 compounds with five distinct functional classes, each of which features under-represented screening motifs, for example, metal ion complexation, reversible covalent binding, or halogen bonding. The collection includes acetohydrazides, acylhydrazones, propylene glycol ethers, 2-cyanoacetamides, and 2-cyanoacrylamides. The rational for the synthesis of most of the compounds was recently published by our group and is now supplemented by additional compounds reported here for the first time. The public access disposition enables academic research groups to collectively expand the druggable space and interdisciplinary collaborate within the scientific field.

Phosphorylation and dephosphorylation of starch and glycogen are important for their physicochemical properties and also their physiological functions. It is therefore desirable to reliably determine the phosphorylation sites. Heteronuclear multidimensional NMR-spectroscopy is in principle a straightforward analytical approach even for complex carbohydrate molecules. With heterogeneous samples from natural sources, however, the task becomes more difficult because a full assignment of the resonances of the carbohydrates is impossible to obtain. Here, we show that the combination of heteronuclear (1) H,(13) C and (1) H,(13) C,(31) P techniques and information derived from spectra of a set of reference compounds can lead to an unambiguous determination of the phosphorylation sites even in heterogeneous samples.

International audience; The skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. Indeed, some mechanoreceptors can detect even nanometer-scale movements. Movement is transformed into electrical signals via the gating of mechanically activated ion channels at sensory endings in the skin. The sensitivity of Piezo mechanically gated ion channels is controlled by stomatin-like protein-3 (STOML3), which is required for normal mechanoreceptor function. Here we identify small-molecule inhibitors of STOML3 oligomerization that reversibly reduce the sensitivity of mechanically gated currents in sensory neurons and silence mechanoreceptors in vivo. STOML3 inhibitors in the skin also reversibly attenuate fine touch perception in normal mice. 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.

Dynamic nuclear polarization exploits electron spin polarization to boost signal-to-noise in magic-angle-spinning (MAS) NMR, creating new opportunities in materials science, structural biology, and metabolomics studies. Since protein NMR spectra recorded under DNP conditions can show improved spectral resolution at 180-200 K compared to 110 K, we investigate the effects of AMUPol and various deuterated TOTAPOL isotopologues on sensitivity and spectral resolution at these temperatures, using proline and reproducibly prepared SH3 domain samples. The TOTAPOL deuteration pattern is optimized for protein DNP MAS NMR, and signal-to-noise per unit time measurements demonstrate the high value of TOTAPOL isotopologues for Protein DNP MAS NMR at 180-200 K. The combined effects of enhancement, depolarization, and proton longitudinal relaxation are surprisingly sample-specific. At 200 K, DNP on SH3 domain standard samples yields a 15-fold increase in signal-to-noise over a sample without radicals. 2D and 3D NCACX/NCOCX spectra were recorded at 200 K within 1 and 13 hours, respectively. Decreasing enhancements with increasing (2)H-content at the CH2 sites of the TEMPO rings in CD3-TOTAPOL highlight the importance of protons in a sphere of 4-6 Å around the nitroxyl group, presumably for polarization pickup from electron spins.

Classical Hodgkin's lymphoma (cHL), although originating from B-cells, is characterized by the virtual lack of gene products whose expression constitutes the B-cell phenotype. Epigenetic repression of B-cell-specific genes via promoter hypermethylation and histone deacetylation as well as compromised expression of B-cell-committed transcription factors were previously reported to contribute to the lost B-cell phenotype in cHL. Restoring the B-cell phenotype may not only correct a central malignant property, but render cHL susceptible to clinically established antibody therapies targeting B-cell surface receptors or small compounds interfering with B-cell receptor signaling. We conducted now 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 chemicals were retrieved that robustly enhanced CD19 transcription. Subsequent chromatin immunoprecipitation-based analyses indicated that 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. When applied in combination with a screening-identified chemical, ATO evoked re-expression of the CD20 antigen, which could be further therapeutically exploited by enabling CD20 antibody-mediated apoptosis of cHL cells. Furthermore, restoration of the B-cell phenotype also rendered cHL cells susceptible to the B-cell Non-Hodgkin's lymphoma-tailored small compound inhibitors Ibrutinib and Idelalisib. In essence, we report here a conceptually novel, re-differentiation-based treatment strategy for cHL.

Based on the iso-peptidase activity of human plasma FXIII, a novel fluorometric assay that determines FXIII concentrations in human plasma below 0.05 IU/ml is introduced. We considered a peptide sequence derived from alpha(2)-antiplasmin (n =12) to yield high sensitivity. Peptide Abz-NE(Cad-Dnp)EQVSPLTLLK exhibits a K(m) value of 19.8+/-2.8 microM and is used in a concentration of 50 microM. The assay design is suitable for measurements in cuvettes (1 ml volume) as well as for the microtiter plate (MTP) format (0.2 ml volume). It provides linear dose-response curves over a wide range of FXIII concentrations (0.05-8.8 IU/ml). The assay was validated with respect to precision, detection and quantitation limits, accuracy/specificity, linearity, and range. A comparison of the fluorometric assay with the photometric assay for FXIII determinations in plasma pools as well as single donor plasma revealed suitability of the fluorometric assay for FXIII determination in plasma of healthy individuals. FXIII concentrations in plasma samples of patients with severe FXIII deficiency are discussed in the context of FXIII antigen levels. These assays correlate well in the critical range below 0.1 IU/ml, whereas the photometric assay may overestimate residual FXIII activity in severe FXIII-deficient patients.

At present nine FDA-approved HIV protease inhibitors have been launched to market, however rapid drug resistance arising under antiviral therapy calls upon novel concepts. Possible strategies are the development of ligands with less peptide-like character or the stabilization of a new and unexpected binding-competent conformation of the protein through a novel ligand-binding mode. Our rational design of pyrrolidinedimethylene diamines was inspired by the idea to incorporate key structural elements from classical peptidomimetics with a non-peptidic heterocyclic core comprising an endocyclic amino function to address the catalytic aspartic acid side chains of Asp 25 and 25'. The basic scaffolds were decorated by side chains already optimized for the recognition pockets of HIV protease or cathepsin D. A multistep synthesis has been established to produce the central heterocycle and to give flexible access to side chain decorations. Depending on the substitution pattern of the pyrrolidine moiety, single-digit micromolar inhibition of HIV-1 protease and cathepsin D has been achieved. Successful design is suggested in agreement with our modelling concepts. The subsequently determined crystal structure with HIV protease shows that the pyrrolidine moiety binds as expected to the pivotal position between both aspartic acid side chains. However, even though the inhibitors have been equipped symmetrically by polar acceptor groups to address the flap water molecule, it is repelled from the complex, and only one direct hydrogen bond is formed to the flap. A strong distortion of the flap region is detected, leading to a novel hydrogen bond which cross-links the flap loops. Furthermore, the inhibitor addresses only three of the four available recognition pockets. It achieves only an incomplete desolvation compared with the similarly decorated amprenavir. Taking these considerations into account it is surprising that the produced pyrrolidine derivatives achieve micromolar inhibition and it suggests extraordinary potency of the new compound class. Most likely, the protonated pyrrolidine moiety experiences strong enthalpic interactions with the enzyme through the formation of two salt bridges to the aspartic acid side chains. This might provide challenging opportunities to combat resistance of the rapidly mutating virus.

Phosphorylation and dephosphorylation of starch and glycogen are important for their physicochemical properties and also their physiological functions. It is therefore desirable to reliably determine the phosphorylation sites. Heteronuclear multidimensional NMR-spectroscopy is in principle a straightforward analytical approach even for complex carbohydrate molecules. With heterogeneous samples from natural sources, however, the task becomes more difficult because a full assignment of the resonances of the carbohydrates is impossible to obtain. Here, we show that the combination of heteronuclear (1) H,(13) C and (1) H,(13) C,(31) P techniques and information derived from spectra of a set of reference compounds can lead to an unambiguous determination of the phosphorylation sites even in heterogeneous samples.

A sub-library of 88 information-rich lead-like purine derivatives were prepared and deposited in an open access academic screening facility. The rationale for the synthesis of these rigid low complexity structures was the privileged character of the purine heterocycle associated with its inherent probability of interactions with multiple adenine-related targets. Although generally expected to be weak binders in many assays, such fragment-like compounds are estimated to match diverse binding sites. It is suggested that heterocycles with many anchor points for hydrogen bonds can be anticipated to undergo very specific interactions to produce more negative enthalpies and thus provide superior starting points for lead optimization than compounds that owe their activity to entropic effects. The in vitro cytotoxicity of the small compounds on a panel of human cancer cell lines has been investigated and some of them showed marked unselective or selective toxicity. This data may be useful if these fragments are to be incorporated into drug-like structures via metabolically cleavable connections. The sub-library will be implemented as part of the ChemBioNet ( www.chembionet.info ) library, and it is open to screening campaigns of academic research groups striving for a fragment-based approach in their biological assays.

Methods enabling structural studies of membrane-integrated receptor systems without the necessity of purification provide an attractive perspective in membrane protein structural and molecular biology. This has become feasible in principle since the advent of dynamic nuclear polarization (DNP) magic-angle-spinning NMR spectroscopy, which delivers the required sensitivity. In this pilot study, we observed well-resolved solid-state NMR spectra of extensively (13)C-labeled neurotoxin II bound to the nicotinic acetylcholine receptor (nAChR) in native membranes. We show that TOTAPOL, a biradical required for DNP, is localized at membrane and protein surfaces. The concentration of active, membrane-attached biradical decreases with time, probably because of reactive components of the membrane preparation. An optimal distribution of active biradical has strong effects on the NMR data. The presence of inactive TOTAPOL in membrane-proximal situations but active biradical in the surrounding water/glycerol "glass" leads to well-resolved spectra, yet a considerable enhancement (e = 12) is observed. The resulting spectra of a protein ligand bound to its receptor are paving the way for further DNP investigations of proteins embedded in native membrane patches.

Hydroxyethylene sulfones were developed as novel scaffolds against aspartyl proteases. A diastereoselective synthesis has been established to introduce the required side chain decoration with desired stereochemistry. Depending on the substitution of the hydroxyethylene sulfone core, micro- to submicromolar inhibition of HIV-1 protease is achieved for the S-configuration at P1 and R-configuration at the hydroxy-group-bearing backbone atom. This stereochemical preference is consistent with the S,R configuration of amprenavir. The racemic mixture of the most potent derivative (K(i) = 80 nM) was separated by chiral HPLC, revealing the S,R,S-enantiomer to be more active (K(i) = 45 nM). Docking studies suggested this isomer as the more active one. The subsequently determined crystal structure with HIV-1 protease, cocrystallized from a racemic mixture, exclusively reveals the S,R,S-enantiomer accommodated to the binding pocket. The transition state mimicking hydroxy group of the inhibitor is centered between both catalytic aspartates, while either its carbonyl or sulfonyl group forms H-bonds to the structurally conserved water mediating interactions between ligand and Ile50NH/Ile50NH' of both flaps. Biological testing of the stereoisomeric hydroxyethylene sulfones against cathepsin D and beta-secretase did not reveal significant inhibition. Most likely, the latter proteases require inverted configuration at the hydroxy group.