Molecular Targets For Drug Discovery Research Articles

Huntington's disease iPSC models-using human patient cells to understand the pathology caused by expanded CAG repeats.

A major advance in the study of Huntington’s disease (HD) has been the development of human disease models employing induced pluripotent stem cells (iPSCs) derived from patients with HD. Because iPSCs provide an unlimited source of cells and can be obtained from large numbers of HD patients, they are a uniquely valuable tool for investigating disease mechanisms and for discovering potential disease-modifying therapeutics. Here, we summarize some of the important findings in HD pathophysiology that have emerged from studies of patient-derived iPSC lines. Because they retain the genome and actual disease mutations of the patient, they provide a cell source to investigate genetic contributions to the disease. iPSCs provide advantages over other disease models. While iPSC-based technology erases some epigenetic marks, newly developed transdifferentiation methods now let us investigate epigenetic factors that control expression of mutant huntingtin (mHTT). Human HD iPSC lines allow us to investigate how endogenous levels of mHTT affect cell health, in contrast to other models that often rely on overexpressing the protein. iPSCs can be differentiated into neurons and other disease-related cells such as astrocytes from different brain regions to study brain regional differences in the disease process, as well as the cell-cell dependencies involved in HD-associated neurodegeneration. They also serve as a tissue source to investigate factors that impact CAG repeat instability, which is involved in regional differences in neurodegeneration in the HD brain. Human iPSC models can serve as a powerful model system to identify genetic modifiers that may impact disease onset, progression, and symptomatology, providing novel molecular targets for drug discovery.

Management of Congenital Diaphragmatic Hernia (CDH): Role of Molecular Genetics.

Congenital diaphragmatic hernia (CDH) is a relatively common major life-threatening birth defect that results in significant mortality and morbidity depending primarily on lung hypoplasia, persistent pulmonary hypertension, and cardiac dysfunction. Despite its clinical relevance, CDH multifactorial etiology is still not completely understood. We reviewed current knowledge on normal diaphragm development and summarized genetic mutations and related pathways as well as cellular mechanisms involved in CDH. Our literature analysis showed that the discovery of harmful de novo variants in the fetus could constitute an important tool for the medical team during pregnancy, counselling, and childbirth. A better insight into the mechanisms regulating diaphragm development and genetic causes leading to CDH appeared essential to the development of new therapeutic strategies and evidence-based genetic counselling to parents. Integrated sequencing, development, and bioinformatics strategies could direct future functional studies on CDH; could be applied to cohorts and consortia for CDH and other birth defects; and could pave the way for potential therapies by providing molecular targets for drug discovery.

Coronavirus Disease-2019 Treatment Strategies Targeting Interleukin-6 Signaling and Herbal Medicine.

Coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is evolving across the world and new treatments are urgently needed as with vaccines to prevent the illness and stem the contagion. The virus affects not only the lungs but also other tissues, thus lending support to the idea that COVID-19 is a systemic disease. The current vaccine and treatment development strategies ought to consider such systems medicine perspectives rather than a narrower focus on the lung infection only. COVID-19 is associated with elevated levels of the inflammatory cytokines such as interleukin-6 (IL-6), IL-10, and interferon-gamma (IFN-γ). Elevated levels of cytokines and the cytokine storm have been linked to fatal disease. This suggests new therapeutic strategies through blocking the cytokine storm. IL-6 is one of the major cytokines associated with the cytokine storm. IL-6 is also known to display pleiotropic/diverse pathophysiological effects. We suggest the blockage of IL-6 signaling and its downstream mediators such as Janus kinases (JAKs), and signal transducer and activators of transcription (STATs) offer potential hope for the treatment of severe cases of COVID-19. Thus, repurposing of already approved IL-6-JAK-STAT signaling inhibitors as well as other anti-inflammatory drugs, including dexamethasone, is under development for severe COVID-19 cases. We conclude this expert review by highlighting the potential role of precision herbal medicines, for example, the Cannabis sativa, provided that omics technologies can be utilized to build a robust scientific evidence base on their clinical safety and efficacy. Precision herbal medicine buttressed by omics systems science would also help identify new molecular targets for drug discovery against COVID-19.

Role of Cytochrome P450 in Prostate Cancer and its Therapy

Prostate cancer has become a global health concern as it is one of the leading causes of mortality in males. With the emerging drug resistance to conventional therapies, it is imperative to unravel new molecular targets for disease prevention. Cytochrome P450 (P450s or CYPs) represents a unique class of mixed-function oxidases which catalyses a wide array of biosynthetic and metabolic functions including steroidogenesis and cholesterol metabolism. Several studies have reported the overexpression of the genes encoding CYPs in prostate cancer cells and how they can be used as molecular targets for drug discovery. But due to functional redundancy and overlapping expression of CYPs in several other metabolic pathways there are several impediments in the clinical efficacy of the novel drugs reported till now. Here we review the most crucial P450 enzymes which are involved in prostate cancer and how they can be used as molecular targets for drug discovery along with the clinical limitations of the currently existing CYP inhibitors.

Proteomics of Select Neglected Tropical Diseases.

Technological advances in mass spectrometry have enabled the extensive identification, characterization, and quantification of proteins in any biological system. In disease processes proteins are often altered in response to external stimuli; therefore, proteomics, the large-scale study of proteins and their functions, represents an invaluable tool for understanding the molecular basis of disease. This review highlights the use of mass spectrometry-based proteomics to study the pathogenesis, etiology, and pathology of several neglected tropical diseases (NTDs), a diverse group of disabling diseases primarily associated with poverty in tropical and subtropical regions of the world. While numerous NTDs have been the subject of proteomic studies, this review focuses on Buruli ulcer, dengue, leishmaniasis, and snakebite envenoming. The proteomic studies highlighted provide substantial information on the pathogenic mechanisms driving these diseases; they also identify molecular targets for drug discovery and development and uncover promising biomarkers that can assist in early diagnosis.

Structure-Based and Molecular Modeling Studies for the Discovery of Cyclic Imides as Reversible Cruzain Inhibitors With Potent Anti-Trypanosoma cruzi Activity.

Chagas disease causes ~10,000 deaths each year, mainly in Latin America, where it is endemic. The currently available chemotherapeutic agents are ineffective in the chronic stage of the disease, and the lack of pharmaceutical innovation for Chagas disease highlights the urgent need for the development of new drugs. The enzyme cruzain, the main cysteine protease of Trypanosoma cruzi, has been explored as a validated molecular target for drug discovery. Herein, the design, molecular modeling studies, synthesis, and biological evaluation of cyclic imides as cruzain inhibitors are described. Starting with a micromolar-range cruzain inhibitor (3a, IC50 = 2.2 μM), this molecular optimization strategy resulted in the nanomolar-range inhibitor 10j (IC50 = 0.6 μM), which is highly active against T. cruzi intracellular amastigotes (IC50 = 1.0 μM). Moreover, most compounds were selective toward T. cruzi over human fibroblasts, which were used as host cells, and are less toxic to hepatic cells than the marketed drug benznidazole. This study enabled the discovery of novel chemical diversity and established robust structure-activity relationships to guide the design of optimized cruzain inhibitors as new trypanocidal agents.

Dissecting Alzheimer's Disease Molecular Substrates by Proteomics and Discovery of Novel Post-translational Modifications.

Alzheimer's disease (AD) is a common complex disease and a major public health burden in both developed and developing countries. Postgenomic technologies such as proteomics and intelligent mining of multi-omics Big Data offer new prospects for diagnostics and therapeutics innovation for AD. In this context, it is noteworthy that mass spectrometry (MS) data are often searched against proteomics databases to unravel the identity of protein biomarkers. In contrast, only a fraction of the MS data can be matched to known proteins, while a large portion of such raw data remains underutilized. Furthermore, the spectral data can be mined for multiple high-confidence post-translational modifications (PTMs) without a priori enrichment. Thus, AD research stands to gain by greater attention to the biological mechanisms regulated by PTMs. Protein modifications may serve as diagnostic biomarkers or as novel molecular targets for drug discovery. We report here novel PTMs discovered in relation to the AD from MS/MS-based proteomic datasets. Publicly available label-free proteomics data were searched for select PTMs using SEQUEST-HT. Only high-confidence PTMs were analyzed using bioinformatics analysis. We identified 4961 unique modified peptides corresponding to 1856 proteins from AD datasets. Of these, 52 proteins were known to be involved in Alzheimer's pathway. Importantly, 3164 PTMs reported in this study are novel in the context of AD. Furthermore, protein quantification revealed expression of 13 high-abundant secretary proteins across multiple studies, which can be potentially harnessed in the future to develop biomarkers. In summary, this study identifies novel PTMs which might help develop new insights on the molecular substrates of AD and thus inform future development of novel diagnostics and treatments for this highly prevalent disease.

Molecular docking and simulation studies of Phyllanthus amarus phytocompounds against structural and nucleocapsid proteins of white spot syndrome virus.

White spot disease caused by white spot syndrome virus (WSSV) is a lethal disease for shrimp. Envelope structural proteins play a major role in viral attachment and are believed to be the initial molecules to interact with the host cell. Thus, the envelope proteins have been preferred as a potential molecular target for drug discovery. In the present investigation, molecular docking and simulation analysis were performed to predict the binding efficiency of phytocompounds identified from Phyllanthus amarus with major envelope proteins, VP26, VP28, and VP110, and a nucleocapsid protein VP664 of WSSV. The docking result reveals that the compounds 2H-1-benzopyran-6-ol, 3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-acetate and 1,4-benzenediamine, N,N'-diphenyl exhibited highest binding energy with the envelope proteins. The mobility of protein-ligand binding complex at various time intervals was validated by molecular dynamics and simulation study. Therefore, P. amarus phytocompounds were found to be most suitable inhibitors for the antiviral treatment for WSSV infection.

Current Techniques for Studying Oligomer Formations of G-Protein-Coupled Receptors Using Mammalian and Yeast Cells.

G-protein-coupled receptors (GPCRs) are physiologically important transmembrane proteins that sense signaling molecules such as hormones, neurotransmitters, and various sensory stimuli; GPCRs represent major molecular targets for drug discovery. Although GPCRs traditionally have been thought to function as monomers or homomers, in the recent years these proteins have also been shown to function as heteromers. Heteromerization among GPCRs is expected to generate potentially large functional and physiological diversity and to provide new opportunities for drug discovery. However, due to the existence of numerous combinations, the larger universe of possible GPCR heteromers is unknown, and thus its functional significance is still poorly understood. The oligomerization of GPCRs in living cells now has been demonstrated in mammalian cells and in native tissues by using genetic, biochemical, and physiological approaches, as well as various resonance energy transfer (RET) technologies. In addition, the yeast Saccharomyces cerevisiae, which can serve as a biosensor for monitoring eukaryotic biological processes, can also be used for the identification of functionally significant heteromer pairs of GPCRs. In this review, we focus on studies of GPCR oligomers, and summarize the technologies used to evaluate GPCR oligomerization. We additionally consider the potential limitations of these methods at present, and envision the possible future applications of these techniques.

Functional Expression of a Ca(2+)-activated Cl(-) Channel Modulator Involved in Ion Transport and Epithelial Cell Differentiation

Cl(-)-permeable channels and transporters expressed on the cell membranes of various mammalian cell types play pivotal roles in the transport of electrolytes and water, pH regulation, cell volume and membrane excitability, and are therefore expected to be useful molecular targets for drug discovery. Both TMEM16A (a possible candidate for Ca(2+)-regulated Cl(-) channels recently identified) and cystic fibrosis transmembrane conductance regulator (CFTR) (or cAMP-regulated Cl(-) channels) have been known to be involved in Cl(-) secretion and reabsorption in the rat salivary gland. Crosstalk between two types of regulatory pathways through these two types of channels has also been described. Previously, we demonstrated that CLCA, a Ca(2+)-activated Cl(-) channel modulator, was involved in Cl(-) absorption in rat salivary ducts. In addition to Ca(2+), basal NF-κB activity in a mouse keratinocyte line was shown to be involved in the transcriptional regulation of CLCA. Conversely, a truncated isoform of CLCA was found in undifferentiated epithelial cells present in the rat epidermal basal layers. Under regulation by Ca(2+) and PKC, the surface expression of β1-integrin and cell adhesion were decreased in the CLCA-overexpressing cells. Knockdown of this isoform elevated the expression of β1-integrin in rat epidermis in vivo. These results indicate that the specific differentiation-dependent localization of CLCA, and transcriptional regulation through Ca(2+), are likely to affect ion permeability and the adhesive potential of epithelial cells. In summary, these types of Cl(-) channels and their modulators may function in a coordinated manner in regulating the functions of epithelial cells under different physiological conditions.

Small molecule discoidin domain receptor kinase inhibitors and potential medical applications.

Discoidin domain receptors (DDRs) are members of the transmembrane receptor tyrosine kinase (RTK) superfamily which are distinguished from others by the presence of a discoidin motif in the extracellular domain and their utilization of collagens as internal ligands. Two types of DDRs, DDR1 and DDR2, have been identified with distinct expression profiles and ligand specificities. These DDRs play important roles in the regulation of fundamental cellular process, such as proliferation, survival, differentiation, adhesion, and matrix remodeling. They have also been closely linked to a number of human diseases, including various fibrotic disorders, atherosclerosis, and cancer. As a consequence, DDRs have been considered as novel potential molecular targets for drug discovery and increasing efforts are being devoted to the identification of new small molecule inhibitors targeting the receptors. In this review, we offer a contemporary overview on the discovery of DDRs inhibitors and their potential medical application for the treatment of cancer and inflammation related disorders.

Bioinformatics in Retina.

Bioinformatics, a word coined for the applications of computer science in biology, is now promising as a major constituent in modern biology and biomedical research. Bioinformatics plays an important role for the integration of broad disciplines of biology to understand the complex mechanisms of the cell. Bioinformatics also aids the way in which biomedical investigators use the information in their testing. Development and implementation of this novel field enable efficient access and management of different types of biological information including those at the genomic, proteomic, and metabolomic level to understand about disease mechanisms and identify new molecular targets for drug discovery. Bioinformatics has expanded its wings in exploring out different important contributions in relation with medical sciences such as neurology, parasitology, hematology, and pathology including ophthalmology. Many bioinformatics-oriented studies have contributed a lot in ophthalmology and given birth to new avenues of occuloinformatics, hence, a new coined term, occuloinformatics: a new approach of research and diagnostics related to ocular disorders with significant inputs of bioinformatics. In this current review, we tried to focus on current avenues and significant contributions of bioinformatics with special reference to retinal disorders.

Search for small molecule activators of latent HIV

Reservoirs of HIV that persist during ART represent barriers to eradication of this virus. One well documented reservoir of latent HIV is found in memory CD4+ T-cells. Identifying means to safely eliminate latently infected memory CD4+ T-cells is an important goal that may contribute to a cure for HIV. One approach toward this end is to activate latent proviruses with the premise that viral particles emanating from these cells will cause a cytopathic effect leading to the demise of the host cell. We have optimized and automated a primary cell-based HIV latency assay that can be used for high throughput screening of small molecule libraries in search of HIV activators. Using this assay, we have identified novel histone deacetylase (HDAC) inhibitors fromGilead’s compound collection that activate latent HIV. Analysis of these inhibitors revealed that the magnitude of HIV expression correlated with the breadth of cellular HDAC inhibition. In addition, we have identified a variety of other compounds that activate latent HIV such as kinase inhibitors which may point to novel mechanisms that govern HIV latency. This screening assay has the potential to identify novel molecular targets for drug discovery and new chemical classes that could be optimized to create new drugs to eliminate reservoirs of latent HIV.

Proteomics: Opportunities and Challenges

‘‘Proteomics’’, is the emerging technology leading to high-throughput identification and understanding of proteins. Proteomics is the protein equivalent of genomics and has captured the imagination of biomolecular scientists, worldwide. Because proteome reveals more accurately the dynamic state of a cell, tissue, or organism, much is expected from proteomics to indicate better disease markers for diagnosis and therapy monitoring.
 Proteomics is expected to play a major role in biomedical research, and it will have a significant impact on the development of diagnostics and therapeutics for cancer, heart ailments and infectious diseases, in future. Proteomics research leads to the identification of new protein markers for diagnostic purposes and novel molecular targets for drug discovery. 
 Though the potential is great, many challenges and issues remain to be solved, such as gene expression, peptides, generation of low abundant proteins, analytical tools, drug target discovery and cost. A systematic and efficient analysis of vast genomic and proteomic data sets is a major challenge for researchers, today. Nevertheless, proteomics is the groundwork for constructing and extracting useful comprehension to biomedical research. This review article covers some opportunities and challenges offered by proteomics.

Development of cationic colloidal silica‐coated magnetic nanospheres for highly selective and rapid enrichment of plasma membrane fractions for proteomics analysis

PM (plasma membrane) proteins play critical roles in many biological processes and are often used as molecular targets for drug discovery. In PM proteome research, fast and highly selective methods for PM preparation are highly desirable for efficient PM protein identification. In the present study, an improved PM isolation technique involving coating intact cells with synthesized cationic silica-coated magnetic nanospheres was developed and applied to the proteomic analysis of the PM from human erythroleukaemia K562 cells. Western blotting characterization and protein identification of the prepared PM indicated that the PM enrichment method using the prepared magnetic nanospheres is a fast and inexpensive strategy with high specificity. Our results demonstrate the potential of these cationic silica-coated magnetic nanospheres for high-throughput identification of PM proteins from cells.

Exploiting apoptosis pathways for the treatment of pediatric cancers

Resistance to apoptosis (programmed cell death) is a characteristic feature of human cancers including childhood malignancies. Further, evasion of apoptosis is a frequent cause of treatment resistance, since most anti-cancer therapies, for example chemo- or radiotherapy act primarily by inducing apoptosis in cancer cells. Over the last two decades, the dissection of apoptosis pathways in pediatric tumors has resulted in the identification of many key molecules that may serve as molecular targets for drug discovery. Accordingly, components of the apoptotic cascade are currently exploited for the development of rationally designed molecular targeted therapies. This approach is expected to open new and more effective approaches for the treatment of childhood cancers.

Opportunities and Challenges in the Discovery of New Central Nervous System Drugs

There have been relatively few new mechanism-based approvals for nervous system relevant drugs over the past 5 years, despite the increasing budgets of pharmaceutical and biotechnology companies. The genomic revolution has provided scientists with many molecular targets for drug discovery and research advances in chemistry, drug metabolism, pharmacology, and toxicology have provided much insight into understanding the pitfalls of the drug discovery and development process. Herein is provided a perspective on both the opportunities and challenges in the discovery and development of novel medicines for the treatment of human CNS disorders.

Abstract 3944: Converged Signaling of β-Catenin and Notch Induces Differentiation of Arterial Endothelial Cells from VEGFR2+ Vascular Progenitors

We have been investigating molecular mechanisms of vascular development and diversification using embryonic stem (ES) cells. Previously, we established an ES cell differentiation system that reproduces early vascular development using vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells as common vascular progenitors (Nature, 2000). Endothelial cells (ECs) were induced from VEGFR2 + progenitor cells with VEGF. VEGF alone mainly induced venous ECs, which are negative for arterial EC markers, ephrinB2 and CXCR4. Interestingly, addition of 8bromo-cAMP with VEGF activated Notch signaling in ECs and substantially induced arterial ECs. Nevertheless, activation of Notch using notch intracellular domain (NICD)-estrogen receptor fusion protein failed to induce arterial ECs (Arterioscler Thromb Vasc Biol, 2006). These results indicate that Notch activation was not sufficient to induce arterial ECs from vascular progenitors. Then we further investigated the downstream of cAMP, which would suffice signals for constructive induction of arterial ECs. We found that phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, completely inhibited cAMP-induced arterial EC differentiation as well as Notch activation. GSK3β inhibitor, Bio, partially reversed the inhibitory effects of LY294002. Whereas activation of β-catenin signaling alone using Doxycycline-induced constitutive active form of β-catenin expression showed only a weak effect on arterial EC induction, simultaneous activation of β-catenin and Notch signaling completely restored arterial EC induction even in the absence of cAMP. Thus, cAMP-PI3K-GSK3β-β-catenin pathway plays a critical role in arterial EC differentiation. Furthermore, chromatin immunoprecipitation assay on the two RBP-J binding sites of mouse EphrinB2 revealed that RBP-J, NICD, and β-catenin formed a protein complex on these two sites only in arterial ECs, but not in venous ECs. These findings suggest that Notch and β-catenin signaling converged to form a novel arterial EC-specific protein complex to induce arterial ECs. This study would provide novel understandings of cellular and molecular mechanisms for arterial-venous specification and novel molecular targets for drug discovery.

Thioredoxin and Thioredoxin Reductase As Redox-Sensitive Molecular Targets for Cancer Therapy

Tumor cell proliferation, de-differentiation, and progression depend on a complex combination of altered intracellular processes including cell cycle regulation, excessive growth factor pathway activation, and decreased apoptosis. Metabolites from these processes result in significant cellular oxidative stress that must be buffered to prevent permanent cell damage and cell death. Tumor cells depend on a complex set of respiratory pathways to generate the necessary energy as well as redox-sensitive pro-survival signaling pathways and factors to cope with and defend against the detrimental effects of oxidative stress. It has been hypothesized that redox-sensitive signaling factors such as thioredoxin reductase-1 (TR) and thioredoxin (TRX) may represent central pro-survival factors that would allow tumor cells to evade the damaging and potentially cytotoxic effects of endogenous and exogenous agents that induce oxidative stress. The overarching theme of this review is an extension of the hypothesis that tumor cells use these redox sensitive pro-survival signaling pathways/factors, which are up-regulated due to increased tumor cell respiration, to evade the cytotoxic effects of anticancer agents. These observations suggest that redox-sensitive signaling factors may be potential novel molecular targets for drug discovery.

Development of Novel Nuclear Receptor Ligands Based on Receptor-Folding Inhibition Hypothesis

Nuclear receptors are ligand-inducible transcriptional factors, and regulate various significant biological phenomena such as cell differentiation, proliferation, metabolism, and homeostasis. By the elucidation of the physiological functions of nuclear receptors, they have become one of the most significant molecular targets for drug discovery in the fields of cancer, autoimmune diseases, and metabolic syndrome. In this study, several novel nuclear receptor ligands have been developed, based on the receptor-folding inhibition hypothesis is discussed. In this hypothesis, the antagonists for nuclear receptors are classified into two types, the misfolding inducers and the folding inhibitors, related to the helix 12 (AF-2 region) conformation of the receptor that is significant for the receptor activation. Then, in order to overcome the resistance in the treatment of prostate tumors with androgen antagonists, the novel folding-inhibitor type antagonists such as isoxazole and pyrrolecarboxamide derivatives were designed and synthesized. Some of them exhibited the androgen antagonistic activities in LNCaP cells with mutated androgen receptor in which conventional antagonists such as flutamide and RU56187 were inactive. The folding-inhibitor type vitamin D3 antagonists (DLAM series) are similarly developed. Further, novel non-seco-steroidal vitamin D3 analogs were designed and synthesized by using a 3,3-diphenylpropane derivative, LG190178 as lead compound. The aza analogs exhibited both potent vitamin D agonistic and androgen antagonistic activities. The results indicate the drug design based on the receptor-folding inhibition hypothesis is efficient in medicinal chemistry of nuclear receptors.