Modulation of Host-Parasite Interactions with Small Molecules Targeting Schistosoma mansoni microRNAs.

Parasites use different strategies of communication with their hosts. One communication channel that has been studied in recent years is the use of vesicle microRNAs to influence the host immune system by trematodes. sma-microRNA-10, secreted from Schistosoma mansoni, has been shown to influence the fate of host T-cells through manipulation of the NF-κB pathway. We have identified low molecular weight tool compounds that can interfere with this microRNA-mediated manipulation of the host immune system. We used a fragment-based screening approach by means of nuclear magnetic resonance (NMR) to identify binders to the precursor of the parasite sma-microRNA-10 present in their extracellular vesicles. The small fragments identified were used to select larger molecules. These molecules were shown to counteract the inhibition of NF-κB activity by sma-microRNA-10 in cell-based assays.

Discovery of Small Molecule Drugs Targeting the Biogenesis of microRNA-155 for the Treatment of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease that often leads to functional disorder in multiple organs, most often with symptoms related to skin lesions, cardiovascular disease and kidney damage. Although significant efforts have been made to find efficient therapies, it still remains uncured. Furthermore, the current therapy is often associated with adverse side effects and leads to a high economic burden for society. At Saverna Therapeutics, in collaboration with our partners, we initiated a lead discovery program that aims to modulate the biogenesis of miR-155. This non-coding RNA is upregulated in SLE patients and SLE mouse models. We used our drug discovery platform based on iterative fragment-based screening by nuclear magnetic resonance (NMR) and machine learning to identify ligands of pre-miR-155. After several iterations and chemical modifications, we have identified compounds that show structure-activity relationships in cellular assays. These inhibitors reduced the level of miR-155 as well as its associated inflammatory protein TNF α whereas the cells remained viable during exposure of the compounds.

Identification of SPPL2a Inhibitors by Multiparametric Analysis of a High-Content Ultra-High-Throughput Screen.

The intramembrane protease signal peptide peptidase-like 2a (SPPL2a) is a potential drug target for the treatment of autoimmune diseases due to an essential role in B cells and dendritic cells. To screen a library of 1.4 million compounds for inhibitors of SPPL2a, we developed an imaging assay detecting nuclear translocation of the proteolytically released cytosolic substrate fragment. The state-of-the-art hit calling approach based on nuclear translocation resulted in numerous false-positive hits, mainly interrupting intracellular protein trafficking. To filter the false positives, we extracted 340 image-based readouts and developed a novel multiparametric analysis method that successfully triaged the primary hit list. The identified scaffolds were validated by demonstrating activity on endogenous SPPL2a and substrate CD74/p8 in B cells. The multiparametric analysis discovered diverse cellular phenotypes and provided profiles for the whole library. The principle of the presented imaging assay, the screening strategy, and multiparametric analysis are potentially applicable in future screening campaigns.

Discovery of Novel Allosteric Non-Bisphosphonate Inhibitors of Farnesyl Pyrophosphate Synthase by Integrated Lead Finding.

Farnesyl pyrophosphate synthase (FPPS) is an established target for the treatment of bone diseases, but also shows promise as an anticancer and anti-infective drug target. Currently available anti-FPPS drugs are active-site-directed bisphosphonate inhibitors, the peculiar pharmacological profile of which is inadequate for therapeutic indications beyond bone diseases. The recent discovery of an allosteric binding site has paved the way toward the development of novel non-bisphosphonate FPPS inhibitors with broader therapeutic potential, notably as immunomodulators in oncology. Herein we report the discovery, by an integrated lead finding approach, of two new chemical classes of allosteric FPPS inhibitors that belong to the salicylic acid and quinoline chemotypes. We present their synthesis, biochemical and cellular activities, structure-activity relationships, and provide X-ray structures of several representative FPPS complexes. These novel allosteric FPPS inhibitors are devoid of any affinity for bone mineral and could serve as leads to evaluate their potential in none-bone diseases.

Differentiation and visualization of diverse cellular phenotypic responses in primary high-content screening.

High-throughput screening, based on subcellular imaging, has become a powerful tool in lead discovery. Through the generation of high-quality images, not only the specific target signal can be analyzed but also phenotypic changes of the whole cell are recorded. Yet analysis strategies for the exploration of high-content screening results, in a manner that is independent from predefined control phenotypes, are largely missing. The approach presented here is based on a well-established modeling technique, self-organizing maps (SOMs), which uses multiparametric results to group treatments that create similar morphological effects. This report describes a novel visualization of the SOM clustering by using an image of the cells from each node, with the most representative cell highlighted to deploy the phenotype described by each node. The approach has the potential to identify both expected hits and novel cellular phenotypes. Moreover, different chemotypes, which cause the same phenotypic effects, are identified, thus facilitating "scaffold hopping."

High-content screening to distinguish between attachment and post-attachment steps of human cytomegalovirus entry into fibroblasts and epithelial cells.

Human cytomegalovirus (HCMV) enters cells through a complex pathway involving the interaction of multiple viral glycoproteins and cellular receptors. While HCMV clinical isolates enter a wide range of cell types, entry has historically been studied using a laboratory strain of virus that can only infect fibroblasts. Herein, we have constructed a HCMV reporter strain that contains GFP fused to the abundant tegument protein pp65 to allow for the direct visualization of virus attachment and entry. Furthermore, the UL131 gene of this strain was restored to clinical isolate sequence to expand our studies of entry into physiologically relevant epithelial cell types. Using the HCMV-GFP reporter virus, we developed an image-based assay and screened a library containing 65,000 compounds for the inhibition of virus entry into fibroblasts. In addition to assessing the effect on virus entry, automated image analysis provided information on compound toxicity and whether the compounds acted as attachment or post-attachment inhibitors. To identify therapeutically viable inhibitors capable of blocking entry in multiple cell types, the inhibitors were screened further for their ability to inhibit virus entry into epithelial cells. Compounds were identified that were able to inhibit virus entry into both cell types at either attachment or post-attachment steps.

HCA-vision: Automated neurite outgrowth analysis.

Automating the analysis of neurons in culture represents a key aspect of the search for neuroactive compounds. A number of commercial neurite analysis software packages tend to measure some basic features such as total neurite length and number of branching points. However, with only these measurements, some differences between neurite morphologies that are clear to a human observer cannot be identified. The authors have developed a suite of image analysis tools that will allow researchers to produce quality analyses at primary screening rates. The suite provides sensitive and information-rich measurements of neurons and neurites. It can discriminate subtle changes in complex neurite arborization even when neurons and neurites are dense. This allows users to selectively screen for compounds triggering different types of neurite outgrowth behavior. In mixed cell populations, neurons can be filtered and separated from other brain cell types so that neurite analysis can be performed only on neurons. It supports batch processing with a built-in database to store the batch-processing results, a batch result viewer, and an ad hoc query builder for users to retrieve features of interest. The suite of tools has been deployed into a software package called HCA-Vision. The free version of the software package is available at http://www.hca-vision.com.

Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.

An imaging assay to analyze primary neurons for cellular neurotoxicity.

The development of high-content screening technologies including automated immunostaining, automated image acquisition and automated image analysis have enabled higher throughput of cellular imaging-based assays. Here we used high-content imaging to thoroughly characterize the cultures of primary rat cerebellar granule neurons (CGNs). We describe procedures to isolate and cultivate the CGNs in 96-well and 384-well format, as well as a procedure to freeze and thaw the CGNs. These methods allow the use of CGNs in 96-well format analyzing 2500 samples per experiment using freshly isolated cells. Down-scaling to 384-well format and freezing and thawing of the CGNs allow even higher throughput. A cellular assay with rat CGN cultures was established to study the neurotoxicity of compounds in order to filter out toxic compounds at an early phase of drug development. The imaging-based toxicity assay was able to reveal adverse effects of compounds on primary neurons which were not detected in neuroblastoma or other cell lines tested.