Validation Of Novel Drug Targets Research Articles

Ubiquitination and the Proteasome as Drug Targets in Trypanosomatid Diseases.

The eukaryotic pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania are responsible for debilitating diseases that affect millions of people worldwide. The numbers of drugs available to treat these diseases, Human African Trypanosomiasis, Chagas' disease and Leishmaniasis are very limited and existing treatments have substantial shortcomings in delivery method, efficacy and safety. The identification and validation of novel drug targets opens up new opportunities for the discovery of therapeutic drugs with better efficacy and safety profiles. Here, the potential of targeting the ubiquitin-proteasome system in these parasites is reviewed. Ubiquitination is the posttranslational attachment of one or more ubiquitin proteins to substrates, an essential eukaryotic mechanism that regulates a wide variety of cellular processes in many different ways. The best studied of these is the delivery of ubiquitinated substrates for degradation to the proteasome, the major cellular protease. However, ubiquitination can also regulate substrates in proteasome-independent ways, and proteasomes can degrade proteins to some extent in ubiquitin-independent ways. Because of these widespread roles, both ubiquitination and proteasomal degradation are essential for the viability of eukaryotes and the proteins that mediate these processes are therefore attractive drug targets in trypanosomatids. Here, the current understanding of these processes in trypanosomatids is reviewed. Furthermore, significant recent progress in the development of trypanosomatid-selective proteasome inhibitors that cure mouse models of trypanosomatid infections is presented. In addition, the targeting of the key enzyme in ubiquitination, the ubiquitin E1 UBA1, is discussed as an alternative strategy. Important differences between human and trypanosomatid UBA1s in susceptibility to inhibitors predicts that the selective targeting of these enzymes in trypanosomatids may also be feasible. Finally, it is proposed that activating enzymes of the ubiquitin-like proteins SUMO and NEDD8 may represent drug targets in these trypanosomatids as well.

A Biomimetic Tumor Model of Heterogeneous Invasion in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a complex, heterogeneous, and genetically unstable disease. Its tumor microenvironment (TME) is complicated by heterogeneous cancer cell populations and strong desmoplastic stroma. This complex and heterogeneous environment makes it challenging to discover and validate unique therapeutic targets. Reliable and relevant in vitro PDAC tumor models can significantly advance the understanding of the PDAC TME and may enable the discovery and validation of novel drug targets. In this study, an engineered tumor model is developed to mimic the PDAC TME. This biomimetic model, named ductal tumor-microenvironment-on-chip (dT-MOC), permits analysis and experimentation on the epithelial-mesenchymal transition (EMT) and local invasion with intratumoral heterogeneity. This dT-MOC is a microfluidic platform where a duct of murine genetically engineered pancreatic cancer cells is embedded within a collagen matrix. The cancer cells used carry two of the three mutations of KRAS, CDKN2A, and TP53, which are key driver mutations of human PDAC. The intratumoral heterogeneity is mimicked by co-culturing these cancer cells. Using the dT-MOC model, heterogeneous invasion characteristics, and response to transforming growth factor-beta1 are studied. A mechanism of EMT and local invasion caused by the interaction between heterogeneous cancer cell populations is proposed.

The critical importance of basic animal research for neuropsychiatric disorders.

The challenges and critical importance of keeping our thinking about neuropsychiatric disorders mechanisms and classifications up-to-date have prompted a dynamic discourse as to the value, appropriateness, and reliability of the animal models we use and the outcomes we measure. At a time when most major pharmaceutical companies are disbanding their research and development in neuroscience and mental health programs, academia is the remaining bastion for the identification and validation of novel drug targets and the development of novel therapeutic approaches. The 2018 ACNP meeting provided the ideal audience for a discussion centered on this topic, offering a professional setting in which diverse viewpoints could be presented, and allowing voices from all arenas of basic and translational neuroscience to be heard. At its core, there is a growing and healthy discussion about the degree of confidence we have that our preclinical animal models and their behavioral endpoints can predict the success of new drugs in clinical trials. While it is clear that we cannot completely model complex neuropsychiatric and neurodevelopmental disorders in animals, there is certainly agreement across domain criteria and endophenotypes of disease that have proven informative, particularly when multiple levels of outcome measures, including genomics, epigenomics, cellular properties, circuits, network dynamics, and behaviors are included. This issue is now of critical importance with the arrival of big ‘omics data sets that provide novel and informative insights about genetic risk and molecular pathophysiology. How can we best examine these insights into disease risk and resilience using preclinical measures? Similarly, while genome-wide association studies and genome sequencing studies provide valuable clues into the genes and pathways that are associated with disease risk, how can we determine how these loci interact with each other as well as with the environment to assess their importance across developmental and life stages? Most importantly, we seek to identify ways to develop novel therapeutic approaches to improve the lives of those with neuropsychiatric disorders including those of neurodevelopmental origins, a challenge that remains even after decades of intensive research.

Virtual Screening in Hepatitis B Virus Drug Discovery: Current Stateof- the-Art and Future Perspectives.

Hepatitis B Virus (HBV) is a major global health burden. Interferon alpha and nucleos(t)ide analogues are currently the standard-of-care for chronic HBV infection. However, these antiviral agents have limited efficacy and do not result in a sustained virological response in the majority of infected patients. Virtual Screening (VS) strategies have now a strong impact on drug discovery, the strength of this research field has been corroborated by recent contributions in the development of novel drug candidates which are in clinical trials or which are already available in the clinics. In this context, different VS strategies have been applied to HBV in order to discover novel inhibitors. In this review, we summarize the VS efforts to identify and design novel HBV interventions. We believe that the combination of in silico and in vitro tools can lead to faster validation of novel drug targets which could accelerate the HBV drug discovery and development efforts.

Combining Zebrafish and CRISPR/Cas9: Toward a More Efficient Drug Discovery Pipeline.

The use of zebrafish larvae in basic and applied research has grown exponentially during the last 20 years. The reasons for this success lay in its specific experimental advantages: on the one hand, the small size, the large number of progeny and the fast life cycle greatly facilitate large-scale approaches while maintaining 3Rs amenability; on the other hand, high genetic and physiological homology with humans and ease of genetic manipulation make zebrafish larvae a highly robust model for understanding human disease. Together, these advantages allow using zebrafish larvae for performing high-throughput research, both in terms of chemical and genetic phenotypic screenings. Therefore, the zebrafish larva as an animal model is placed between more reductionist in vitro high-throughput screenings and informative but low-throughput preclinical assays using mammals. However, despite its biological advantages and growing translational validation, zebrafish remains scarcely used in current drug discovery pipelines. In a context in which the pharmaceutical industry is facing a productivity crisis in bringing new drugs to the market, the combined advantages of zebrafish and the CRISPR/Cas9 system, the most powerful technology for genomic editing to date, has the potential to become a valuable tool for streamlining the generation of models mimicking human disease, the validation of novel drug targets and the discovery of new therapeutics. This review will focus on the most recent advances on CRISPR/Cas9 implementation in zebrafish and all their potential uses in biomedical research and drug discovery.

CRISPR/Cas9-The ultimate weapon to battle infectious diseases?

Infectious diseases are a leading cause of death worldwide. Novel therapeutics are urgently required to treat multidrug-resistant organisms such as Mycobacterium tuberculosis and to mitigate morbidity and mortality caused by acute infections such as malaria and dengue fever virus as well as chronic infections such as human immunodeficiency virus-1 and hepatitis B virus. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, which has revolutionized biomedical research, holds great promise for the identification and validation of novel drug targets. Since its discovery as an adaptive immune system in prokaryotes, the CRISPR/Cas9 system has been developed into a multi-faceted genetic modification tool, which can now be used to induce gene deletions or specific gene insertions, such as conditional alleles or endogenous reporters in virtually any organism. The generation of CRISPR/Cas9 libraries that can be used to perform phenotypic whole genome screens provides an important new tool that will aid in the identification of critical host factors involved in the pathogenesis of infectious diseases. In this review, we will discuss the development and recent applications of the CRISPR/Cas9 system used to identify novel regulators, which might become important in the fight against infectious diseases.

Abstract 202: Depletion of PTP4A3 phosphatase disrupts colorectal cancer cell adhesion and extracellular matrix interactions

Abstract Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the United States and the world and the 5-year survival rate in the United States of ∼60% highlights the need for better treatment options. The central challenge in the design and development of drugs to treat CRC has been the validation of novel drug targets and the generation of new therapies aimed at those targets. The goal of this project is to better understand the role of one such potential CRC molecular target. Protein Tyrosine Phosphatase 4A3 (PTP4A3) is currently among the most consistently up-regulated proteins seen in colorectal tumors but it suffers from an incomplete understanding of its substrate(s) and of its participation in tumor formation and progression. To clarify how PTP4A3 expression impacts cell behavior, we isolated primary colon tumor epithelial cells from Ptp4a3fl/fl mice. PTP4A3 was deleted in vitro by adenoviral infection of Ptp4a3fl/fl cells with Cre recombinase accompanied by a green fluorescent protein (GFP) marker. As a control, the Ptp4a3fl/fl cells were infected with GFP alone. Virally infected cells were selected by fluorescence activated cell sorting. In the sorted cells, the expression of Cre resulted in a complete lack of PTP4A3 protein by western blotting and undetectable Ptp4a3 mRNA as determine by quantitative RT-PCR. Analysis of these genetically similar cells revealed that Ptp4a3−/− tumor cells had significantly reduced colony formation and migration as compared to Ptp4a3fl/fl cells. Notably, the growth rate of Ptp4a3fl/fl and Ptp4a3−/− cells was similar when cultured on plastic in a 2D monolayer. In contrast, Ptp4a3fl/fl spheroids grew significantly faster than the Ptp4a3−/− cells when cultured in Matrigel. Moreover, while the Ptp4a3fl/fl cells were able to form cohesive spheroids when cultured in cell repellent plates, there was a striking inability of the Ptp4a3−/− cells to form these 3D structures. In addition, treatment of the Ptp4a3fl/fl cells with a potent and selective PTP4A3 phosphatase inhibitor, thienopyridone, phenocopied the appearance of the Ptp4a3−/− cells grown in cell repellent plates. These results suggest PTP4A3 expression-dependent changes in tumor cell behavior may be rooted in a disruption in the adhesion and extracellular matrix-signaling axis. This hypothesis was supported by significant changes in the gene expression profile of adhesion and extracellular matrix proteins in the Ptp4a3−/− CRC cells. The isogenic Ptp4a3fl/fl and Ptp4a3−/− CRC cells provide an invaluable tool to elucidate further the function of PTP4A3 and its link to adhesion and the extracellular matrix interactions. Citation Format: Kelley E. McQueeney, Paula Pekic, Joseph M. Salamoun, Jennifer Ahn, Elizabeth R. Sharlow, Peter Wipf, John S. Lazo. Depletion of PTP4A3 phosphatase disrupts colorectal cancer cell adhesion and extracellular matrix interactions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 202.

Abstract A078: Computational discovery and experimental validation of novel drug targets in immuno-oncology

Abstract The past few years have witnessed a renaissance in the field of immuno-oncology largely due to the clinical success in targeting the immune checkpoints CTLA-4 and PD-1. Towards identification of novel immune checkpoint drug targets we developed a dedicated predictive discovery platform. The B7/CD28 discovery platform is a predictive model based on genomic and protein features along with expression patterns of known B7/CD28 proteins. The platform has been tested and validated extensively and has demonstrated its validity by identifying non-novel immune checkpoints such as TIGIT and VISTA, which were not used in the design stage. The B7/CD28 predictive platform was employed to identify 11 new immune checkpoint candidates which are currently in different validation stages. In this poster, we will describe our discovery approach as well as our validation path. In addition, we will present experimental data demonstrating the immuno-modulatory function and expression patterns of several of our novel immune checkpoints. These experimental results serve as an additional confirmation to the accuracy of our B7/CD28 predictive discovery platform and shed light on the therapeutic potential of the novel immune checkpoints identified using this unique discovery approach. Citation Format: Arthur Machlenkin, Ofer Levy, Galit Rotman. Computational discovery and experimental validation of novel drug targets in immuno-oncology. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A078.

Computational discovery and experimental validation of novel drug targets in immuno-oncology

The past few years have witnessed a renaissance in the field of immuno-oncology largely due to the clinical success in targeting the immune checkpoints CTLA-4 and PD-1. Towards identification of novel immune checkpoint drug targets we developed a dedicated predictive discovery platform. The B7/CD28 discovery platform is a predictive model based on genomic and protein features along with expression patterns of known B7/CD28 proteins. The platform has been tested and validated extensively and has demonstrated its validity by identifying non-novel immune checkpoints such as TIGIT and VISTA, which were not used in the design stage. The B7/CD28 predictive platform was employed to identify several novel immune checkpoint candidates which are currently in different validation stages. In this poster, we will describe our discovery approach as well as our validation path. In addition, we will present experimental data demonstrating the immuno-modulatory function and expression patterns of several of our novel immune checkpoints. These experimental results serve as an additional confirmation to the accuracy of our B7/CD28 predictive discovery platform and shed light on the therapeutic potential of the novel immune checkpoints identified using this unique discovery approach.

The application of tetracyclineregulated gene expression systems in the validation of novel drug targets in Mycobacterium tuberculosis.

Although efforts to identify novel therapies for the treatment of tuberculosis have led to the identification of several promising drug candidates, the identification of high-quality hits from conventional whole-cell screens remains disappointingly low. The elucidation of the genome sequence of Mycobacterium tuberculosis (Mtb) facilitated a shift to target-based approaches to drug design but these efforts have proven largely unsuccessful. More recently, regulated gene expression systems that enable dose-dependent modulation of gene expression have been applied in target validation to evaluate the requirement of individual genes for the growth of Mtb both in vitro and in vivo. Notably, these systems can also provide a measure of the extent to which putative targets must be depleted in order to manifest a growth inhibitory phenotype. Additionally, the successful implementation of Mtb strains engineered to under-express specific molecular targets in whole-cell screens has enabled the simultaneous identification of cell-permeant inhibitors with defined mechanisms of action. Here, we review the application of tetracycline-regulated gene expression systems in the validation of novel drug targets in Mtb, highlighting both the strengths and limitations associated with this approach to target validation.

RNA interference in adult Ascaris suum – an opportunity for the development of a functional genomics platform that supports organism-, tissue- and cell-based biology in a nematode parasite

The sustainable control of animal parasitic nematodes requires the development of efficient functional genomics platforms to facilitate target validation and enhance anthelmintic discovery. Unfortunately, the utility of RNA interference (RNAi) for the validation of novel drug targets in nematode parasites remains problematic. Ascaris suum is an important veterinary parasite and a zoonotic pathogen. Here we show that adult A. suum is RNAi competent, and highlight the induction, spread and consistency of RNAi across multiple tissue types. This platform provides a new opportunity to undertake whole organism-, tissue- and cell-level gene function studies to enhance target validation processes for nematode parasites of veterinary/medical significance.

How can We Discover Safer Drugs?

How can We Discover Safer Drugs?

Functional Human 5-HT6 Receptor Assay for High Throughput Screening of Chemical Ligands and Binding Proteins

Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT(6) receptor (5-HT(6)R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT(6)R are poorly understood due to the lack of selective 5-HT(6)R ligands. In the present study, we examined functional coupling of the human 5-HT(6)R, 5-HT(7A)R, or 5-HT(7B)R with various Galpha-proteins (Galpha(15), Galpha(qs5), or Galpha(qG66Ds5)) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT(6)R with Galpha(qG66Ds5) produced the highest levels of Ca(2+) signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT(6)R HTS system (Z'-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT(6)R, we screened approximately 500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC(50) value of 6.3 microM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT(6)R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT(6)R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT(6)R interacting protein. Taken together, these results suggest that the 5-HT(6)R/Galpha(qG66Ds5) FDSS6000 system can be utilized to screen for selective 5-HT(6)R ligands and to examine any functional relationships between 5-HT(6)R and its binding proteins.

Identification of Glucose-Dependant Insulin Secretion Targets in Pancreatic β Cells by Combining Defined-Mechanism Compound Library Screening and siRNA Gene Silencing

Identification and validation of novel drug targets continues to be a major bottleneck in drug development, particularly for polygenic complex diseases such as type 2 diabetes. Here, the authors describe an approach that allows researchers to rapidly identify and validate potential drug targets by combining chemical tools and RNA interference technology. As a proof-of-concept study, the known mechanism Sigma LOPAC library was used to screen for glucose-dependent insulin secretion (GDIS) in INS-1 832/13 cells. In addition to several mechanisms that are known to regulate GDIS (such as cyclic adenosine monophosphate-specific phosphodiesterases, adrenoceptors, and Ca(2+) channels), the authors find that several of the dopamine receptor (DRD) antagonists significantly enhance GDIS, whereas DRD agonists profoundly inhibit GDIS. Subsequent siRNA studies in the same cell line indicate that knockdown of DRD2 enhanced GDIS. Furthermore, selective DRD2 antagonists and agonists also enhance or suppress, respectively, GDIS in isolated rat islets. The data support that the approach described here offers a rapid and effective way for target identification and validation.

Proteomics-based discovery of biomarkers and therapeutic targets in neurodegenerative diseases: perspective of microglia and neuroinflammation

Proteomics is a field of study that characterises a complete profile of proteins present in a given cell, tissue or biological system. Recent advances in proteomics research made possible the identification of novel biomarkers and therapeutic targets in neurodegenerative diseases. The field of neurodegenerative diseases is particularly well suited for proteomic analysis, given the multifactorial nature of the diseases and complexity of the nervous system. As increasing evidence indicates that microglia-associated chronic neuroinflammation plays an important role in the pathophysiology of neurodegenerative diseases, microglial activation may be a potential drug target in CNS disorders. On the basis of both the neuroprotective and neurotoxic role of microglia, the proteomic analysis of microglial signalling and neuroinflammation may thus be used for the identification and validation of novel drug targets as well as for the discovery of useful biomarkers of disease diagnosis and progression in the CNS.

Experimental validation of novel drug targets

Experimental validation of novel drug targets

Adaptation of the Folts and electrolytic methods of arterial thrombosis for the study of anti-thrombotic molecules in small animals

Introduction Animal preparations of arterial thrombosis play a crucial role in the discovery and validation of novel drug targets in vivo, aiding in the selection of new drugs for clinical evaluation. The Folts and electrolytic methods of arterial thrombosis are two of the most commonly used techniques to investigate drugs with novel anti-thrombotic potential. However, these techniques often involved the use of large animals such as dogs, and their application to small animals was limited. The aim of the present study was to adapt the Folts and electrolytic methods previously described in large animals to create highly reproducible, quantitative models of arterial thrombosis in mice, rats and rabbits. Methods Carotid artery blood flow was measured in anaesthetised mice, rats and rabbits. In the Folts-like method, a silk suture was tied around one carotid artery distal to a flow probe and tightened to cause a concentric stenosis sufficient to decrease blood flow by 50%. Intimal damage was induced by pinching the artery at the site of stenosis using forceps. The sequential formation and mechanical dislodgement of the resultant platelet-rich occlusive thrombus caused cyclic carotid artery flow reductions. In the electrolytic method in mice, a platinum hook electrode was placed distal to a flow probe on one carotid artery. The artery was clamped distally to the electrode to cause stasis and an electrical current (4 mA for 1.25 min) was applied before clamp release. This induced vascular injury resulting in occlusive thrombus (platelet- and fibrin-rich) formation. Conclusion The Folts-like method of arterial thrombosis was successfully adapted for use in mice, rats and rabbits, and the electrolytic technique for use in mice. Compared with larger animals, these methods are highly reproducible and ideal for pre-clinical, cost-effective, low-cost routine screening of novel anti-thrombotic drugs.

Identification and Validation of Novel Drug Targets in Tuberculosis

There is an urgent need for new antimycobacterial drugs, and in particular for novel agents that will shorten the duration of tuberculosis chemotherapy, or overcome drug-resistant strains of the causative organism, Mycobacterium tuberculosis. Our knowledge of the tubercle bacillus and its complex interaction with the human host has improved dramatically in recent years, particularly with the determination of its complete genome sequence. New genome-scale tools are being applied to aid in drug target identification, alongside traditional approaches aimed at understanding the basic biology of M. tuberculosis. Many potential drug targets have been identified, but very few have been validated by showing that they are essential for growth or survival of the bacterium. In this review, the landscape of potential drug targets is surveyed.Structural Bioinformatic Approaches to the Discovery of New Antimycobacterial Drugs.

Prospects for the Xenopus Embryo Model in Therapeutics Technologies

Access to suitable animal models is essential in the field of therapeutics technologies. Recently, lower vertebrates have emerged as attractive low-cost animal models which offer new exciting applications in functional genomics and therapeutics technologies. Amphibian embryos of the genus Xenopus have long served as important models for the study of vertebrate development. Xenopus is evolutionary significantly less distant to humans than fish models, which suggests that experimental findings gained with Xenopus will more accurately predict human biology. Numerous experimental advantages, including external development, large size, identifiable blastomeres, and their ability to withstand extensive surgical intervention and culture in vitro, have favored the use of the Xenopus model in the past. More recently, the introduction of a simple efficient method to disrupt gene functions and the rapid development of genomic resources has further increased the attractiveness of this low-cost, high-throughput model for the analysis of vertebrate gene functions. Using the Xenopus embryo as the primary animal model, our research in the field of therapeutics technologies has focused on the identification and validation of novel drug targets by employing genomic and transcriptomic information in the analysis of the molecular and cellular processes underlying kidney organogenesis and vascular development. Furthermore, our research on signaling pathways controlling cellular differentiation of embryonic tissues provides important insights that may ultimately lead to the development of novel cell-based therapies in regenerative medicine. Finally, we are exploring the possibility of employing the Xenopus embryos in chemical library screens to identify novel chemical modulators of organogenesis.

The Metastasis Suppressor Gene KiSS-1 Encodes Kisspeptins, the Natural Ligands of the Orphan G Protein-coupled Receptor GPR54

Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta. These 54-, 14,- and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP(2) hydrolysis, Ca(2+) mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed this hypothesis.