Drug Discovery Platform Research Articles

Development of high-throughput lacrimal gland organoid platforms for drug discovery in dry eye disease.

Dysfunction and damage of the lacrimal gland (LG) results in ocular discomfort and dry eye disease (DED). Current therapies for DED do not fully replenish the necessary lubrication to rescue optimal vision. New drug discovery for DED has been limited perhaps because in vitro models cannot mimic the biology of the native LG. The existing platforms for LG organoid culture are scarce and still not ready for consistency and scale up production towards drug screening. The magnetic three-dimensional (3D) bioprinting (M3DB) is a novel system for 3D in vitro biofabrication of cellularized tissues using magnetic nanoparticles to bring cells together. M3DB provides a scalable platform for consistent handling of spheroid-like cell cultures facilitating consistent biofabrication of organoids. Previously, we successfully generated innervated secretory epithelial organoids from human dental pulp stem cells with M3DB and found that this platform is feasible for epithelial organoid bioprinting. Research targeting LG organogenesis, drug discovery for DED has extensively used mouse models. However, certain inter-species differences between mouse and human must be considered. Porcine LG appear to have more similarities to human LG than the mouse counterparts. We have conducted preliminary studies with the M3DB for fabricating LG organoids from primary cells isolated from murine and porcine LG, and found that this platform provides robust LG organoids for future potential high-throughput analysis and drug discovery. The LG organoid holds promise to be a functional model of tearing, a platform for drug screening, and may offer clinical applications for DED.

Searching for new therapeutic options for the uncommon pathogen Mycobacterium chimaera: an open drug discovery approach

SummaryBackgroundMycobacterium chimaera is a slowly growing non-tuberculous mycobacterium associated with outbreaks of fatal infections in patients after cardiac surgery, and it is increasingly being detected in patients with chronic lung conditions. M chimaera can cause disseminated disease, osteomyelitis, and chronic skin or soft-tissue infections. We aimed to find new inhibitory compounds and drug repurposing opportunities for M chimaera, as current therapeutic options often result in poor outcomes.MethodsIn an open drug discovery approach, we screened the Medicines for Malaria Venture (MMV) Pathogen Box to assess the in-vitro antimicrobial drug susceptibility of M chimaera compared with the antimicrobial drug susceptibility of the slowly growing, major human pathogen Mycobacterium tuberculosis, and the rapidly growing Mycobacterium abscessus reference strains. Compounds identified from an initial resazurin microtitre cell viability assay screen were further characterised by determining the minimum inhibitory concentration (MIC) of MMV Pathogen Box compounds against M chimaera; and the MICs of a panel of 20 drugs commonly used to treat mycobacterial infections against M tuberculosis, M abscessus, and M chimaera. We also assessed the time-kill kinetics of doxycycline, clarithromycin, ethambutol, and rifabutin against M chimaera.FindingsM chimaera was inhibited by 21 (5%) of 400 compounds in the Pathogen Box. Ten compounds were active against all three mycobacteria. MMV675968, with activity against slowly growing mycobacteria that probably targets folate metabolism, had a mean MIC of 2·22 μM (0·80 μg/mL) against M chimaera. Antimicrobial susceptibility testing showed that oxazolidinones such as linezolid (mean MIC 3·13 μg/mL) were active against M chimaera and that bedaquiline was the most potent compound (mean MIC 0·02 μg/mL). Doxycycline, a broad-spectrum antimicrobial drug with excellent tissue penetration properties, also inhibited M chimaera with a mean MIC of 6·25 μg/mL.InterpretationMolecular diagnostics present an opportunity for more effective, targeted drug therapies—treating bacterial infections at the species level. Using an open drug discovery platform, we identified compounds that inhibit the newly recognised pathogen M chimaera. The existing evidence base is poor and the option for expensive drug discovery is improbable; therefore, we have also found options for drug repurposing. Future in-vivo efficacy studies will reveal whether these findings result in new, targeted treatment regimens for M chimaera.FundingWellcome Trust, National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), and the University of Sussex Junior Research Associate scheme.

Melatonin attenuates dimethyl sulfoxide- and Zika virus-induced degeneration of porcine induced neural stem cells.

Domestic pigs have become increasingly popular as a model for human diseases such as neurological diseases. Drug discovery platforms have increasingly been used to identify novel compounds that combat neurodegeneration. Currently, bioactive molecules such as melatonin have been demonstrated to offer a neuroprotective effect in several studies. However, a neurodegenerative platform to study novel compounds in a porcine model has not been fully established. In this study, characterized porcine induced neural stem cells (iNSCs) were used for evaluation of the protective effect of melatonin against chemical and pathogenic stimulation. First, the effects of different concentrations of melatonin on the proliferation of porcine iNSCs were studied. Second, porcine iNSCs were treated with the appropriate concentration of melatonin prior to induced degeneration with dimethyl sulfoxide or Zika virus (ZIKV). The results demonstrated that the percentages of Ki67 expression in porcine iNSCs cultured in 0.1, 1, and 10 nM melatonin were not significantly different from that in the control groups. Melatonin at 1 nM protected porcine iNSCs from DMSO-induced degeneration, as confirmed by a dead cell exclusion assay and mitochondrial membrane potential (ΔΨm) analysis. In addition, pretreatment with melatonin reduced the percentage of dead porcine iNSCs after ZIKV infection. Melatonin increased the ΔΨm, resulting in a decrease in cell degeneration. However, pretreatment with melatonin was unable to suppress ZIKV replication in porcine iNSCs. In conclusion, the present study demonstrated the anti-degenerative effect of melatonin against DMSO- and ZIKV-induced degeneration in porcine iNSCs.

Sold on the new machine

Artificial intelligence has yet to produce a drug from scratch. But the technology is well advanced in pharmaceutical industry laboratories, despite initial pushback at the research bench. Major drug companies are designing their own technology and partnering with AI platform developers, many of which are also drug discovery companies, for support in target identification, drug design, and clinical trial analysis. The goal is to connect research and development from end to end. Two leading AI technology companies claim they’re ready to provide the full connection, but some research managers question the effectiveness of AI at various points on the route from early discovery to the still-elusive AI-derived drug. In December, Roche announced a deal with Recursion, an artificial intelligence technology developer with its own drug discovery program. Roche will access Recursion’s AI-driven drug discovery platform to identify and develop up to 40 new medications for cancer and neurological conditions. Hailed

Pyrazine and Phenazine Heterocycles: Platforms for Total Synthesis and Drug Discovery.

There are numerous pyrazine and phenazine compounds that demonstrate biological activities relevant to the treatment of disease. In this review, we discuss pyrazine and phenazine agents that have shown potential therapeutic value, including several clinically used agents. In addition, we cover some basic science related to pyrazine and phenazine heterocycles, which possess interesting reactivity profiles that have been on display in numerous cases of innovative total synthesis approaches, synthetic methodologies, drug discovery efforts, and medicinal chemistry programs. The majority of this review is focused on presenting instructive total synthesis and medicinal chemistry efforts of select pyrazine and phenazine compounds, and we believe these incredible heterocycles offer promise in medicine.

A human breast cancer-derived xenograft and organoid platform for drug discovery and precision oncology

Models that recapitulate the complexity of human tumors are urgently needed to develop more effective cancer therapies. We report a bank of human patient-derived xenografts (PDXs) and matched organoid cultures from tumors that represent the greatest unmet need: endocrine-resistant, treatment-refractory and metastatic breast cancers. We leverage matched PDXs and PDX-derived organoids (PDxO) for drug screening that is feasible and cost-effective with in vivo validation. Moreover, we demonstrate the feasibility of using these models for precision oncology in real time with clinical care in a case of triple-negative breast cancer (TNBC) with early metastatic recurrence. Our results uncovered a Food and Drug Administration (FDA)-approved drug with high efficacy against the models. Treatment with this therapy resulted in a complete response for the individual and a progression-free survival (PFS) period more than three times longer than their previous therapies. This work provides valuable methods and resources for functional precision medicine and drug development for human breast cancer.

Using deep mutational scanning to identify the determinants of antibiotic resistance

Current drug discovery platforms have proven incapable of supplying enough new antibiotic types to meet the challenge of rapidly developing clinical resistances. New techniques that specifically incorporate potential resistance adaptations during design are necessary. In cases of known specific resistance mechanisms, a biophysical approach may allow rational development of existing drugs that escape resistance. In this work, we use deep mutational scanning (DMS), a next-generation sequencing-based technique that reports on the site-specific fitness effects of mutations across an entire protein, to investigate the contribution of Virginiamycin acetyltransferase (VatD) to several streptogramin analogues.

FRET assays for RyR-targeted drug discovery platforms

Using fluorescence lifetime FRET-based high-throughput screening (HTS) assays, we have identified compounds that modulate the sarcoplasmic reticulum's (SR) Ca2+ gatekeeper, the ryanodine receptor (RyR) channel. Intracellular Ca2+ regulation is critical for striated muscle function, and SR Ca2+ release via opening of RyR is essential for triggering muscle contraction. Under cellular rest, increased propensity of channel opening due to RyR dysregulation is associated with severe cardiac and skeletal myopathies, and neurodegenerative diseases.

Current Status and Challenges of Human Induced Pluripotent Stem Cell-Derived Liver Models in Drug Discovery.

The pharmaceutical industry is in high need of efficient and relevant in vitro liver models, which can be incorporated in their drug discovery pipelines to identify potential drugs and their toxicity profiles. Current liver models often rely on cancer cell lines or primary cells, which both have major limitations. However, the development of human induced pluripotent stem cells (hiPSCs) has created a new opportunity for liver disease modeling, drug discovery and liver toxicity research. hiPSCs can be differentiated to any cell of interest, which makes them good candidates for disease modeling and drug discovery. Moreover, hiPSCs, unlike primary cells, can be easily genome-edited, allowing the creation of reporter lines or isogenic controls for patient-derived hiPSCs. Unfortunately, even though liver progeny from hiPSCs has characteristics similar to their in vivo counterparts, the differentiation of iPSCs to fully mature progeny remains highly challenging and is a major obstacle for the full exploitation of these models by pharmaceutical industries. In this review, we discuss current liver-cell differentiation protocols and in vitro iPSC-based liver models that could be used for disease modeling and drug discovery. Furthermore, we will discuss the challenges that still need to be overcome to allow for the successful implementation of these models into pharmaceutical drug discovery platforms.

Oligonucleotide Therapeutics: From Discovery and Development to Patentability.

Following the first proof of concept of using small nucleic acids to modulate gene expression, a long period of maturation led, at the end of the last century, to the first marketing authorization of an oligonucleotide-based therapy. Since then, 12 more compounds have hit the market and many more are in late clinical development. Many companies were founded to exploit their therapeutic potential and Big Pharma was quickly convinced that oligonucleotides could represent credible alternatives to protein-targeting products. Many technologies have been developed to improve oligonucleotide pharmacokinetics and pharmacodynamics. Initially targeting rare diseases and niche markets, oligonucleotides are now able to benefit large patient populations. However, there is still room for oligonucleotide improvement and further breakthroughs are likely to emerge in the coming years. In this review we provide an overview of therapeutic oligonucleotides. We present in particular the different types of oligonucleotides and their modes of action, the tissues they target and the routes by which they are administered to patients, and the therapeutic areas in which they are used. In addition, we present the different ways of patenting oligonucleotides. We finally discuss future challenges and opportunities for this drug-discovery platform.

Small Molecules Enhance Reprogramming of Adult Human Dermal Fibroblasts to Dorsal Forebrain Precursor Cells.

The development of human cell-based platforms for disease modeling, drug discovery, and regenerative therapy relies on robust and practical methods to derive high yields of relevant neuronal subtypes. Direct reprogramming strategies have sought to provide a means of deriving human neurons that mitigate the low conversion efficiencies, and protracted timing of human embryonic stem cell and induced pluripotent stem cell-derived neuron specification in vitro. However, few studies have demonstrated the direct conversion of adult human fibroblasts into multipotent neural precursors with the capacity to differentiate into cortical neurons with high efficiency. In this study, we demonstrate a reprogramming strategy using chemically modified mRNA encoding the proneural genes SOX2 and PAX6 coupled with small molecule supplementation to enhance the derivation of human-induced dorsal forebrain precursors directly from adult human dermal fibroblasts (aHDFs). Through transcriptional and phenotypic analysis of lineage-specific precursor and cortical neuron markers, we have demonstrated that this combined strategy significantly enhances the direct derivation of dorsal forebrain precursors from aHDFs, which, after timely exposure to defined differentiation media, gives rise to high yields of functional glutamatergic neurons. We propose that this combined strategy provides a highly tractable and efficient human cell-based platform for disease modeling and drug discovery.

Multifunctional amino acids empowering bifunctional biosensing platform for depression study

Poor availability of objective approaches hinders effective diagnosis and treatment for depression. Biosensors provide a promising platform for the development of quantitative and practical methods for disease detection, as well as for drug discovery. Here, we developed an electrochemical biosensor has been established with the ability to simply and accurately detect the trace glucocorticoid receptor alpha (GRα), as a key biomarker of depression, in both hippocampus and blood cells. The integration of amino-ion graphene oxide (IL-rGO) and amino acid-coated gold nanoparticles (AA-AuNPs) via green synthesis remarkably magnifies the electrochemical signals, where amino acids play multiple roles as reducing agents, stabilizers, and bridging agents. After the optimization among AA-AuNPs@IL-rGO nanocomposites based on five typical amino acids, a biosensing surface has been constructed to implement analysis in real samples as a bifunctional platform. The obtained biosensor exhibited a remarkably low limit of detection (0.283 pg mL−1) and could thus sensitively identify the GRα differences in healthy and depressive rats with and without fluoxetine. The electrochemical biosensor developed herein was not only outstandingly sensitive but also simple to use and labor-saving, making it a promising all-in-one platform for depression diagnosis and drug discovery.

Generation of hiPSC line UMi030-A from an individual with the hearing loss-related GJB2 mutation c.109G>A.

Genetic variants in the GJB2 gene which encodes for the Connexin 26 protein account for∼60% of cases of genetic hearing loss. A novel hiPSC line was generated from an individual with the hearing loss-related variant c.109G>A in GJB2 leading to the p.V37I alteration in the Connexin26 protein. These cells will help to delineate the role of GJB2 in hearing loss pathogenesis and serve as a platform for drug discovery and development.

Stage-Specific Generation of Human Pluripotent Stem Cell Derived Lung Models to Measure CFTR Function.

Human embryonic stem cells (ES) and induced pluripotent stem cells (iPSC) are powerful tools that have the potential to generate in vitro human lung epithelial cells. However, challenges in efficiency and reproducibility remain in utilizing the cells for therapy discovery platforms. Here, we optimize our previously published protocols to efficiently generate three developmental stages of the lung model (fetal lung epithelial progenitors, fLEP; immature airway epithelial spheroid, AES; air-liquid interface culture, ALI), and demonstrate its potential for cystic fibrosis (CF) drug discovery platforms. The stepwise approach directs differentiation from hPSC to definitive endoderm, anterior ventral foregut endoderm, and fetal lung progenitor cells. The article also describes the generation of immature airway epithelial spheroids in Matrigel with epithelial cells sorted by a magnetic-activated cell sorting system, and the generation of adult-like airway epithelia through air-liquid interface conditions. We demonstrate that this optimized procedure generates remarkably higher cystic fibrosis transmembrane conductance regulator (CFTR) expression and function than our previous method, and thus is uniquely suitable for CF research applications. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: hESC/hiPSC differentiation to fetal lung progenitors Basic Protocol 2: Formation of airway epithelial spheroids Alternate Protocol 1: Cryopreservation of airway epithelial spheroids Basic Protocol 3: Differentiation and maturation in air-liquid interface culture Alternate Protocol 2: Differentiation and maturation of epithelial progenitors from airway epithelial spheroids in ALI culture.

RIKEN Program for Drug Discovery and Medical Technology Platforms Overview and Future Prospects

RIKEN Program for Drug Discovery and Medical Technology Platforms Overview and Future Prospects

Establishment of reconstituted depolarization-induced Ca<sup>2+</sup> release platform for drug discovery of skeletal muscle diseases

Establishment of reconstituted depolarization-induced Ca<sup>2+</sup> release platform for drug discovery of skeletal muscle diseases

Anticancer effect of marine bivalves derived polysaccharides against human cancer cells

In recent decades, studies on cancer prophylactics and therapeutics with development of novel anticancer drugs have garnered interest on a global scale. The diverse marine environment is a major source of biocompounds and has been acknowledged as an important platform for drug discovery. A wide variety of novel agents in the form of protein, polysaccharide, polypeptide and steroid from marine microbes, plants and animals are under preclinical and clinical evaluation as potential anticancer drugs. Polysaccharides, with their manifold structures and side groups, have been extensively investigated in biomedical and pharmaceutical fields. The present study investigates the potential of polysaccharides extracted by enzyme hydrolysis from five marine bivalves to inhibit human cancer cells. Cytotoxicity of crude polysaccharides was analyzed using a normal cell line (vero). Antiproliferative effect of polysaccharides on the breast (MDA-MB-231), cervical (HeLa), liver (HepG2) and colon (HT-29) cancer was evaluated by 3-(4, 5 dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide assay (MTT). The polysaccharides from different bivalve species showed varied results on different cell lines but highest inhibitory activity was observed in the polysaccharide of Donax variabilis with IC50 at the concentration 350 μg/mL in MDA-MB-231 and HeLa cells, 400 μg/mL in HepG2 cells and 200 μg/mL in HT-29 cells. Apoptosis-related characteristics were observed by cell morphological observation and nuclear morphological analysis by propidium iodide staining. The late stages of apoptosis were detected by dual acridine orange/ethidium bromide staining and confirmed by DNA fragmentation assay and MMP using Rhodamine 123 stainings. The results obtained substantiate that novel polysaccharides from marine bivalves are potent antiproliferative agents and further studies might unveil a promising anticancer drug.

Precision oncology using ex vivo technology: a step towards individualised cancer care?

Despite advances in cancer genomics and the increased use of genomic medicine, metastatic cancer is still mostly an incurable and fatal disease. With diminishing returns from traditional drug discovery strategies, and high clinical failure rates, more emphasis is being placed on alternative drug discovery platforms, such as ex vivo approaches. Ex vivo approaches aim to embed biological relevance and inter-patient variability at an earlier stage of drug discovery, and to offer more precise treatment stratification for patients. However, these techniques also have a high potential to offer personalised therapies to patients, complementing and enhancing genomic medicine. Although an array of approaches are available to researchers, only a minority of techniques have made it through to direct patient treatment within robust clinical trials. Within this review, we discuss the current challenges to ex vivo approaches within clinical practice and summarise the contemporary literature which has directed patient treatment. Finally, we map out how ex vivo approaches could transition from a small-scale, predominantly research based technology to a robust and validated predictive tool. In future, these pre-clinical approaches may be integrated into clinical cancer pathways to assist in the personalisation of therapy choices and to hopefully improve patient experiences and outcomes.

Cancer Stem Cells: Innovative Approach for Testable Alternatives against Therapy Resistant Cancer

Chemo-resistant cancer stem cells represent a sub-population of cancer initiating phenotype in primary cancer. These cells evolve in to a metastatic phenotype via activation of multiple cell signaling pathways for cancer cell survival and epithelial-mesenchymal transition. Reliable cancer stem cell models represent a valuable experimental approach for drug discovery platform to identify efficacious testable alternatives against therapy-resistant cancer. Present commentary provides a systematic discussion of relevant conceptual and technical aspects of cancer stem cell biology and its significance for therapeutic alternatives.

Chemically induced senescence in human stem cell-derived neurons promotes phenotypic presentation of neurodegeneration.

Modeling age‐related neurodegenerative disorders with human stem cells are difficult due to the embryonic nature of stem cell‐derived neurons. We developed a chemical cocktail to induce senescence of iPSC‐derived neurons to address this challenge. We first screened small molecules that induce embryonic fibroblasts to exhibit features characteristic of aged fibroblasts. We then optimized a cocktail of small molecules that induced senescence in fibroblasts and cortical neurons without causing DNA damage. The utility of the “senescence cocktail” was validated in motor neurons derived from ALS patient iPSCs which exhibited protein aggregation and axonal degeneration substantially earlier than those without cocktail treatment. Our “senescence cocktail” will likely enhance the manifestation of disease‐related phenotypes in neurons derived from iPSCs, enabling the generation of reliable drug discovery platforms.