Small molecule Screening at Helmholtz Zentrum München – From Biology to Molecules

225 (PB105) - Discovery of novel small molecules that recruit DCAF11 for selective degradation of BRD4

The Society of Biomolecular Imaging and Informatics First Annual Conference

Screening for Inhibitors of Low-Affinity Epigenetic Peptide-Protein Interactions: An AlphaScreen™-Based Assay for Antagonists of Methyl-Lysine Binding Proteins

Epiblastin A Induces Reprogramming of Epiblast Stem Cells Into Embryonic Stem Cells by Inhibition of Casein Kinase 1

Soil microbial communities are pivotal to plant health and nutrient acquisition. It is becoming increasingly clear that many interactions, both among and between microbes and plants, are governed by small bioactive molecules or "secondary metabolites" that can aid in communication, competition, and nutrient uptake. Yet, secondary metabolite biogeography - who makes what, where, and why-is in its infancy. Further, secondary metabolite biosynthesis genes are often silent or weakly expressed under standard laboratory conditions, making it incredibly difficult to study these small molecules. To begin to address these dual challenges, we focused on redox-active metabolites (RAMs), a specific class of small molecules, and took advantage of recent findings that many RAMs aid in acquiring phosphorus and that their production is frequently stimulated by stress for this macronutrient. We developed a screen for RAM-producing bacteria that leverages phosphorus limitation to stimulate metabolite biosynthesis and uses a colorimetric (ferrozine) iron-reduction assay to identify redox activity. We isolated 557 root-associated bacteria from grasses collected at sites across the United States (Santa Rita Experimental Range [AZ], Konza Prairie Biological Station [KS], and Harvard Forest [MA]) and from commercial tomato plants and screened them for RAM production. We identified 128 soil isolates of at least 19 genera across Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes that produced RAMs under phosphorus stress. Our work reveals that the production of RAMs under phosphorus stress is common across diverse soil bacteria and provides an approach to screen for these small molecules rapidly.IMPORTANCEBy secreting secondary metabolites, bacteria at the plant root can defend against diseases and help acquire essential nutrients. However, the genes that synthesize secondary metabolites are typically inactive or are weakly expressed under standard laboratory conditions. This fact makes it difficult to study these small molecules and hinders the discovery of novel small molecules that may play crucial roles in agricultural and biomedical settings. Here, we focus on redox-active metabolites (RAMs), a class of secondary metabolites that can help bacteria solubilize phosphorus and are often produced when phosphorus is limited. We developed a screen that rapidly identifies RAM-producing bacteria by utilizing a colorimetric iron-reduction assay in combination with phosphorus limitation to stimulate biosynthesis. The screen reveals that RAM-producing bacteria are far more prevalent in soil than previously appreciated and that this approach can be used to identify RAM producers.

An In Vivo Platform for Rapid High-Throughput Antitubercular Drug Discovery

Skeletal muscle progenitor stem cells (referred to as satellite cells) represent the primary pool of stem cells in adult skeletal muscle responsible for the generation of new skeletal muscle in response to injury. Satellite cells derived from aged muscle display a significant reduction in regenerative capacity to form functional muscle. This decrease in functional recovery has been attributed to a decrease in proliferative capacity of satellite cells. Hence, agents that enhance the proliferative abilities of satellite cells may hold promise as therapies for a variety of pathological settings, including repair of injured muscle and age- or disease-associated muscle wasting. Through phenotypic screening of isolated murine satellite cells, we identified a series of 2,4-diaminopyrimidines (e.g., 2) that increased satellite cell proliferation. Importantly, compound 2 was effective in accelerating repair of damaged skeletal muscle in an in vivo mouse model of skeletal muscle injury. While these compounds were originally prepared as c-Jun N-terminal kinase 1 (JNK-1) inhibitors, structure-activity analyses indicated JNK-1 inhibition does not correlate with satellite cell activity. Screening against a broad panel of kinases did not result in identification of an obvious molecular target, so we conducted cell-based proteomics experiments in an attempt to identify the molecular target(s) responsible for the potentiation of the satellite cell proliferation. These data provide the foundation for future efforts to design improved small molecules as potential therapeutics for muscle repair and regeneration.

Discovery of small molecules targeting GRP78 for antiangiogenic and anticancer therapy

Oncology immunotherapy has gained significant advances in recent years and benefits cancer patients with superior efficacy and superior clinical responses. Currently over ten immune checkpoint antibodies targeting CTLA-4 and PD-1/PD-L1 have received regulatory approval worldwide and over thousands are under active clinical trials. However, compared to the rapid advance of Monoclonal Antibody (mAb), studies on immunotherapeutic small molecules have far lagged behind. Small molecule immunotherapy not only can target immunosuppressive mechanisms similar to mAbs, but also can stimulate intracellular pathways downstream of checkpoint proteins in innate or adaptive immune cells that mAbs are unable to access. Therefore, small molecule immunotherapy can provide an alternative treatment modality either alone or complementary to or synergistic with extracellular checkpoint mAbs to address low clinical response and drug resistance. Fortunately, remarkable progress has achieved recently in the pursuit of small molecule immunotherapy. This review intends to provide a timely highlight on those clinically investigated small molecules targeting PD-1/PD-L1, IDO1, and STING. The most advanced IDO1 inhibitor epacadostat have been aggressively progressed into multiple clinical testings. Small molecule PD-1/PD-L1 inhibitors and STING activators are still in a premature state and their decisive application needs to wait for the ongoing clinical outcomes. Since no small molecule immunotherapy has been approved yet, the future research should continue to focus on discovery of novel small molecules with distinct chemo-types and higher potency, identification of biomarkers to precisely stratify patients, as well as validation of many other immune-therapeutic targets, such as LAG3, KIRs, TIM-3, VISTA, B7-H3, and TIGIT.

Background:Capsular contracture is a devastating complication that occurs in patients undergoing implant-based breast reconstruction. Ionizing radiation drives and exacerbates capsular contracture in part by activating cytokines, including transforming growth factor-beta (TGF-β). TGF-β promotes myofibroblast differentiation and proliferation, leading to excessive contractile scar formation. Therefore, targeting the TGF-β pathway may attenuate capsular contracture.Methods:A 20,000 small molecule library was screened for anti-TGF-β activity. Structurally diverse anti-TGF-β agents were identified and then tested on primary human capsular fibroblasts. Fibroblasts were irradiated or not, and then treated with both TGF-β and candidate molecules. Resulting cells were then analyzed for myofibroblast activity using myofibroblast markers including alpha-smooth muscle actin, collagen I, Thy1, and periostin, using Western Blot, quantitative real-time polymerase chain reaction, and immunofluorescence.Results:Human capsular fibroblasts treated with TGF-β showed a significant increase in alpha-smooth muscle actin, collagen I, and periostin levels (protein and/or mRNA). Interestingly, fibroblasts treated with latent TGF-β and 10 Gy radiation also showed significantly increased levels of myofibroblast markers. Cells that were treated with the novel small molecules showed a significant reduction in myofibroblast activation, even in the presence of radiation.Conclusions:Several novel small molecules with anti-TGF-β activity can effectively prevent human capsular fibroblast to myofibroblast differentiation in vitro, even in the presence of radiation. These results highlight novel therapeutic options that may be utilized in the future to prevent radiation-induced capsular contracture.

Discovery and characterization of selective small molecule inhibitors of the mammalian mitochondrial division dynamin, DRP1

Finding new drugs to enhance anion secretion in cystic fibrosis: Toward suitable systems for better drug screening. Report on the pre-conference meeting to the 12th ECFS Basic Science Conference, Albufeira, 25–28 March 2015

Chapter 23 - Designing Zebrafish Chemical Screens

Origin of cancer is strongly related to the unusual epigenetic regulation of gene function as indicated by recent reports. The covalent modifications to DNA or histones without affecting genomes that finally lead to phenotypical changes in cells or organisms are referred as "Epigenetics." The possibility to reprogram the epigenetics in the cancer epigenome is the most important target for cancer treatment and drug resistance. The development of epigenetic drugs holds a great potential for the current cancer therapeutic approaches. Nevertheless, targeting cancer epigenetic pathways is still exciting due to the lack of selective and effective small molecule compounds or drug molecules. Therefore, the current book chapter highlights epigenetic pathways for cancer and potential small molecule inhibitors and epidrugs targeting DNA methyltransferase, histone modification, and more new therapies with nanomaterials and imaging to improve the effectiveness of cancer treatment. The structural aspects on discovery of novel small molecules or drugs targeting epigenetic pathways in cancer exploration as promising strategies will be also discussed.

Refractory hepatocellular carcinoma (HCC) perpetuates metastasis or recurrence through anti-cancer drug resistance, necessitating more effective and reliable therapeutic strategies. We propose a new therapeutic approach involving the discovery of novel small molecules through target identification and validation in a patient-derived metastatic HCC model. We showed that calcium/calmodulin-dependent protein kinase 2 alpha (CaMK2α)-mediated enhancement of sarco/endoplasmic reticulum (ER) calcium ATPase 1 (SERCA1) expression level was pivotal events under anti-cancer drug treated conditions in patient-derived metastatic HCC cells. Increased SERCA1 was regulates to overloaded free calcium. SERCA is widely recognized as a key regulator of cytosolic free calcium under severe ER stress conditions. Though a cardiac dysfunction was unavoidable in vivo because of non-specific inhibition of SERCA isoforms by standard SERCA inhibitors. Based on the molecular structure of SERCA1, we discovered and synthesized two SERCA1-specific inhibitors, candidate 56 and 62. These compounds significantly reduced tumor size in the metastatic HCC xenograft tumor model without cardiac contractile dysfunction. This study first showed survival mechanism of patient-derived metastatic HCC cell, and propose a new therapeutic approach by the new small molecules, candidate 56 and 62, which are SERCA1 isoform-specific inhibitors without cardiac dysfunction by SERCA1 selectively inhibition.