Drug Screening Research Articles

Engineering G protein-coupled receptors for stabilization.

G protein-coupled receptors (GPCRs) are one of the most important families of targets for drug discovery. One of the limiting steps in the study of GPCRs has been their stability, with significant and time-consuming protein engineering often used to stabilize GPCRs for structural characterization and drug screening. Unfortunately, computational methods developed using globular soluble proteins have translated poorly to the rational engineering of GPCRs. To fill this gap, we propose GPCR-tm, a novel and personalized structurally driven web-based machine learning tool to study the impacts of mutations on GPCR stability. We show that GPCR-tm performs as well as or better than alternative methods, and that it can accurately rank the stability changes of a wide range of mutations occurring in various types of class A GPCRs. GPCR-tm achieved Pearson's correlation coefficients of 0.74 and 0.46 on 10-fold cross-validation and blind test sets, respectively. We observed that the (structural) graph-based signatures were the most important set of features for predicting destabilizing mutations, which points out that these signatures properly describe the changes in the environment where the mutations occur. More specifically, GPCR-tm was able to accurately rank mutations based on their effect on protein stability, guiding their rational stabilization. GPCR-tm is available through a user-friendly web server at https://biosig.lab.uq.edu.au/gpcr_tm/.

An Exploration of Organoid Technology: Present Advancements, Applications, and Obstacles.

Organoids are in vitro models that exhibit a three-dimensional structure and effectively replicate the structural and physiological features of human organs. This work examines the potential applications of organoid technology, as well as the challenges and future directions associated with its implementation. The manuscript was put together by conducting a comprehensive literature review, which involved an in-depth evaluation of globally renowned scientific research databases. The field of organoids has generated significant attention due to its potential applications in tissue development and disease modelling, as well as its implications for personalised medicine, drug screening, and cell-based therapies. The utilisation of organoids has proven to be effective in the examination of various conditions, encompassing genetic disorders, cancer, neurodevelopmental disorders and infectious diseases. The exploration of the wider uses of organoids is still in its early phases. Research shall be conducted to integrate 3D organoid systems as alternatives for current models, potentially improving both fundamental and clinical studies in the future.

Predicting anti-trypanosome effect of carbazole-derived compounds by powerful SVM with novel kernel function and comprehensive learning PSO.

In order to predict the anti-trypanosome effect of carbazole-derived compounds by quantitative structure-activity relationship, five models were established by the linear method, random forest, radial basis kernel function support vector machine, linear combination mix-kernel function support vector machine, and nonlinear combination mix-kernel function support vector machine (NLMIX-SVM). The heuristic method and optimized CatBoost were used to select two different key descriptor sets for building linear and nonlinear models, respectively. Hyperparameters in all nonlinear models were optimized by comprehensive learning particle swarm optimization with low complexity and fast convergence. Furthermore, the models' robustness and reliability underwent rigorous assessment using fivefold and leave-one-out cross-validation, y-randomization, and statistics including concordance correlation coefficient (CCC), [Formula: see text] , [Formula: see text] , and [Formula: see text] . Among all the models, the NLMIX-SVM model, which was established by support vector regression using a nonlinear combination of radial basis kernel function, sigmoid kernel function, and linear kernel function as a new kernel function, demonstrated excellent learning and generalization abilities as well as robustness: [Formula: see text] = 0.9581, mean square error (MSE) = 0.0199 for the training set and [Formula: see text] = 0.9528, MSE = 0.0174 for the test set. [Formula: see text] , [Formula: see text] , CCC, [Formula: see text] , [Formula: see text], and [Formula: see text] are 0.9539, 0.8908, 0.9752, 0.9529, 0.9528, and 0.9633, respectively. The NLMIX-SVM method proved to be a promising way in quantitative structure-activity relationship research. In addition, molecular docking experiments were conducted to analyze the properties of new derivatives, and a new potential candidate drug molecule was ultimately found. In summary, this study will provide help for the design and screening of novel anti-trypanosome drugs.

3D printing and bioprinting in the battle against diabetes and its chronic complications

Diabetes is a metabolic disorder characterized by high blood sugar. Uncontrolled blood glucose affects the circulatory system in an organism by intervening blood circulation. The high blood glucose can lead to macrovascular (large blood vessels) and microvascular (small blood vessels) complications. Due to this, the vital organs (notably brain, eyes, feet, heart, kidneys, lungs and nerves) get worsen in diabetic patients if not treated at the earliest. Therefore, acquiring treatment at an appropriate time is very important for managing diabetes and other complications that are caused due to diabetes. The root cause for the occurrence of various health complications in diabetic patients is the uncontrolled blood glucose levels. This review presents a consolidated account of the applications of various types of three-dimensional (3D) printing and bioprinting technologies in treating diabetes as well as the complications caused due to impaired blood glucose levels. Herein, the development of biosensors (for the diagnosis), oral drug formulations, transdermal drug carriers, orthotic insoles and scaffolds (for the treatment) are discussed. Next to this, the fabrication of 3D bioprinted organs and cell-seeded hydrogels (pancreas engineering for producing insulin and bone engineering for managing bone defects) are explained. As the final application, 3D bioprinting of diabetic disease models for high-throughput screening of ant-diabetic drugs are discussed. Lastly, the challenges and future perspective associated with the use of 3D printing and bioprinting technologies against diabetes and its related chronic complications have been put forward.

Generation of Alzheimer’s Disease Model Derived from Induced Pluripotent Stem Cells with APP Gene Mutation

Alzheimer’s disease (AD), the most common cause of dementia, is characterized by disruptions in memory, cognition, and personality, significantly impacting morbidity and mortality rates among older adults. However, the exact pathophysiological mechanism of AD remains unknown, and effective treatment options for AD are still lacking. Human induced pluripotent stem cells (iPSC) are emerging as promising platforms for disease research, offering the ability to model the genetic mutations associated with various conditions. Patient-derived iPSCs are useful for modeling neurodegenerative and neurodevelopmental disorders. In this study, we generated AD iPSCs from peripheral blood mononuclear cells obtained from a 65-year-old patient with AD carrying the E682K mutation in the gene encoding the amyloid precursor protein. Cerebral organoids derived from AD iPSCs recapitulated the AD phenotype, exhibiting significantly increased levels of tau protein. Our analysis revealed that an iPSC disease model of AD is a valuable assessment tool for pathophysiological research and drug screening.

Novel Multifunctional Meta-Surface Plasmon Resonance Chip Microplate for High-Throughput Molecular Screening.

The utilization of surface plasmon resonance (SPR) sensors for real-time label-free molecular interaction analysis is already being employed in the fields of in vitro diagnostics and biomedicine. However, the widespread application of SPR technology is hindered by its limited detection throughput and high cost. To address this issue, our study introduces a novel multifunctional MetaSPR high-throughput microplate biosensor featuring 3D nanocup microarrays, aiming to achieve high-throughput screening at a reduced cost and with enhanced speed. Different types of MetaSPR sensors and analytical detection methods have been developed for accurate subtype identification, epitope binding, affinity determination, antibody collocation, and other applications that have greatly promoted the screening and analysis of early antibody drugs. The MetaSPR platform combined with nano-enhanced particles amplified the detection signal and improved the detection sensitivity, making it more convenient, sensitive, and efficient than traditional ELISA. Our findings demonstrated that the MetaSPR biosensor is a new practical technology detection platform that can improve the efficiency of biomolecular interaction studies with unlimited potential for new drug development. This article is protected by copyright. All rights reserved.

Formation of Stable Vascular Networks by 3D Coaxial Printing and Schiff-Based Reaction

Vascularized organs hold potential for various applications, such as organ transplantation, drug screening, and pathological model establishment. Nevertheless, the in vitro construction of such organs encounters many challenges, including the incorporation of intricate vascular networks, the regulation of blood vessel connectivity, and the degree of endothelialization within the inner cavities. Natural polymeric hydrogels, such as gelatin and alginate, have been widely used in three-dimensional (3D) bioprinting since 2005. However, a significant disparity exists between the mechanical properties of the hydrogel materials and those of human soft tissues, necessitating the enhancement of their mechanical properties through modifications or crosslinking. In this study, we aim to enhance the structural stability of gelatin–alginate hydrogels by crosslinking gelatin molecules with oxidized pullulan (i.e., a polysaccharide) and alginate molecules with calcium chloride (CaCl2). A continuous small-diameter vascular network with an average outer diameter of 1 mm and an endothelialized inner surface is constructed by printing the cell-laden hydrogels as bioinks using a coaxial 3D bioprinter. The findings demonstrate that the single oxidized pullulan crosslinked gelatin and oxidized pullulan/CaCl2 double-crosslinked gelatin–alginate hydrogels both exhibit a superior structural stability compared to their origins and CaCl2 solely crosslinked gelatin–alginate hydrogels. Moreover, the innovative gelatin and gelatin–alginate hydrogels, which have excellent biocompatibilities and very low prices compared with other hydrogels, can be used directly for tissue/organ construction, tissue/organ repairment, and cell/drug transportation.

Application and challenge of pancreatic organoids in therapeutic research

The in-vivo non-human primate animal and in-vitro cell disease models play a crucial part in the study of the mechanisms underlying the occurrence and development of pancreatic diseases, but with increasingly prominent limitations with in-depth research. Organoids derived from human pluripotent and adult stem cells resemble human in-vivo organs in their cellular composition, spatial tissue structure and physiological function, making them as an advantageous research tool. Up until now, numerous human organoids, including pancreas, have been effectively developed, demonstrating significant potential for research in organ development, disease modeling, drug screening, and regenerative medicine. However, different from intestine, liver and other organs, the pancreas is the only special organ in the human body, consisting of an exocrine gland and an endocrine gland. Thus, the development of pancreatic organoid technology faces greater challenges, and how to construct a composite pancreatic organoid with exocrine and endocrine gland is still difficult in current research. By reviewing the fundamental architecture and physiological role of the human pancreas, along with the swiftly developing domain of pancreatic organoids, we summarize the method and characteristics of human pancreatic organoids, and its application in modeling pancreatic diseases, as a platform for individualized drug screening and in regenerative medicine study. As the first comprehensive review that focus on the pharmacological study of human pancreatic organoid, the review hopes to help scholars to have a deeper understanding in the study of pancreatic organoid.

A TEMPERATURE-CONTROLLED ON-CHIP HIGH-THROUGHPUT SINGLE-CELL CULTURE PLATFORM

Modern lab-on-a-chip and microfluidic technologies offer opportunities in developing portable cell culture platforms for growing and analyzing the behavior of living cells. We have proposed the magnetomicrofluidic chips, as a novel single-cell analysis tool, composed of microfluidic microchambers and microchannels overlaid on magnetic thin films designed to precisely transport magnetized single cells and magnetic particles with the help of hybrid hydrodynamic and magnetic forces. In order to provide the optimal temperature for the cells to grow on the chip, here we propose an on-chip microincubator at the single-cell level, without the need for a conventional incubator. An electronic circuit is used to read the chip temperature and control it with a feedback system. We perform finite element methods analysis to study the chip temperature profile, based on which the final chip is designed. We show that experimental results agree well with the simulation results. Using the proposed chip and the on-chip incubator, we transport thousands of single cells into the microchambers, culture them, and demonstrate their proper proliferation. The introduced chip can be used in crucial applications, such as drug screening, in which long-term study of the cells under a microscope, at the single-cell level, is required.

Three-dimensional cardiac models: a pre-clinical testing platform.

Major advancements in human pluripotent stem cell (hPSC) technology over recent years have yielded valuable tools for cardiovascular research. Multi-cell type 3-dimensional (3D) cardiac models in particular, are providing complementary approaches to animal studies that are better representatives than simple 2-dimensional (2D) cultures of differentiated hPSCs. These human 3D cardiac models can be broadly divided into two categories; namely those generated through aggregating pre-differentiated cells and those that form self-organizing structures during their in vitro differentiation from hPSCs. These models can either replicate aspects of cardiac development or enable the examination of interactions among constituent cell types, with some of these models showing increased maturity compared with 2D systems. Both groups have already emerged as physiologically relevant pre-clinical platforms for studying heart disease mechanisms, exhibiting key functional attributes of the human heart. In this review, we describe the different cardiac organoid models derived from hPSCs, their generation methods, applications in cardiovascular disease research and use in drug screening. We also address their current limitations and challenges as pre-clinical testing platforms and propose potential improvements to enhance their efficacy in cardiac drug discovery.

A Portable Device for in Situ Noninvasive Monitoring of Cell Secretions and Communications with Fluorescence and Nanochannel Electrochemistry.

In situ monitoring of cell secretions and communications plays a fundamental role in screening of disease diagnostic biomarkers and drugs. Quantitative detection of cell secretions and monitoring of intercellular communication have been separately reported, which often rely on target labeling or complex pretreatment steps, inevitably causing damage to the target. Simultaneous in situ noninvasive detection of cell secretions and monitoring of intercellular communication are challenging and have never been reported. Herein, we smartly developed a portable device for in situ label-free monitoring of cell secretions and communications with fluorescence and ion-transport-based nanochannel electrochemistry. Based on the dual signal mode, a series of nonelectroactive secretions were sensitively and accurately quantified. The detection limits for VEGF, MUC1, and ATP were 3.84 pg/mL, 32.7 pg/mL, and 47.4 fM (3σ/S), which were 1/3.9, 1/1.1, and 1/41 of those of commercial ELISA kits, respectively. More interestingly, under the released secretions, the gradual opening of the nanochannel connected the two cells in the left and right chambers of the device; thus, the secretion mediated intercellular communication can be monitored. The proposed platform may provide a promising tool for understanding the mechanism of intercellular communication and discovering new therapeutic targets.

Drug screening on digital microfluidics for cancer precision medicine

Drug screening based on in-vitro primary tumor cell culture has demonstrated potential in personalized cancer diagnosis. However, the limited number of tumor cells, especially from patients with early stage cancer, has hindered the widespread application of this technique. Hence, we developed a digital microfluidic system for drug screening using primary tumor cells and established a working protocol for precision medicine. Smart control logic was developed to increase the throughput of the system and decrease its footprint to parallelly screen three drugs on a 4 × 4 cm2 chip in a device measuring 23 × 16 × 3.5 cm3. We validated this method in an MDA-MB-231 breast cancer xenograft mouse model and liver cancer specimens from patients, demonstrating tumor suppression in mice/patients treated with drugs that were screened to be effective on individual primary tumor cells. Mice treated with drugs screened on-chip as ineffective exhibited similar results to those in the control groups. The effective drug identified through on-chip screening demonstrated consistency with the absence of mutations in their related genes determined via exome sequencing of individual tumors, further validating this protocol. Therefore, this technique and system may promote advances in precision medicine for cancer treatment and, eventually, for any disease.

Label-free active single-cell encapsulation enabled by microvalve-based on-demand droplet generation and real-time image processing

Droplet microfluidics-based single-cell encapsulation is a critical technology that enables large-scale parallel single-cell analysis by capturing and processing thousands of individual cells. As the efficiency of passive single-cell encapsulation is limited by Poisson distribution, active single-cell encapsulation has been developed to theoretically ensure that each droplet contains one cell. However, existing active single-cell encapsulation technologies still face issues related to fluorescence labeling and low throughput. Here, we present an active single-cell encapsulation technique by using microvalve-based drop-on-demand technology and real-time image processing to encapsulate single cells with high throughput in a label-free manner. Our experiments demonstrated that the single-cell encapsulation system can encapsulate individual polystyrene beads with 96.3% efficiency and HeLa cells with 94.9% efficiency. The flow speed of cells in this system can reach 150 mm/s, resulting in a corresponding theoretical encapsulation throughput of 150 Hz. This technology has significant potential in various biomedical applications, including single-cell omics, secretion detection, and drug screening.

Interferometric Biosensor for High Sensitive Label-Free Recording of HiPS Cardiomyocytes Contraction in Vitro.

Heart disease remains a leading cause of global mortality, underscoring the need for advanced technologies to study cardiovascular diseases and develop effective treatments. We introduce an innovative interferometric biosensor for high-sensitivity and label-free recording of human induced pluripotent stem cell (hiPSC) cardiomyocyte contraction in vitro. Using an optical cavity, our device captures interference patterns caused by the contraction-induced displacement of a thin flexible membrane. First, we demonstrate the capability to quantify spontaneous contractions and discriminate between contraction and relaxation phases. We calculate a contraction-induced vertical membrane displacement close to 40 nm, which implies a traction stress of 34 ± 4 mN/mm2. Finally, we investigate the effects of a drug compound on contractility amplitude, revealing a significant reduction in contractile forces. The label-free and high-throughput nature of our biosensor may enhance drug screening processes and drug development for cardiac treatments. Our interferometric biosensor offers a novel approach for noninvasive and real-time assessment of cardiomyocyte contraction.

Tracking Plasma Membrane Damage Using a Ruthenium(II) Complex Phosphorescent Indicator Paired with Cholesterol.

Long-term in situ plasma membrane-targeted imaging is highly significant for investigating specific biological processes and functions, especially for the imaging and tracking of apoptosis processes of cells. However, currently developed membrane probes are rarely utilized to monitor the in situ damage of the plasma membrane. Herein, a transition-metal complex phosphorescent indicator, Ru-Chol, effectively paired with cholesterol, exhibits excellent properties on staining the plasma membrane, with excellent antipermeability, good photostability, large Stokes shift, and long luminescence lifetime. In addition, Ru-Chol not only has the potential to differentiate cancerous cells from normal cells but also tracks in real time the entire progression of cisplatin-induced plasma membrane damage and cell apoptosis. Therefore, Ru-Chol can serve as an efficient tool for the monitoring of morphological and physiological changes in the plasma membrane, providing assistance for drug screening and early diagnosis and treatment of diseases, such as immunodeficiency, diabetes, cirrhosis, and tumors.

Fluorescence-Guided Spatial Drug Screening in 3D Colorectal Cancer Spheroids.

The limited recapitulation of critical cancer features in two-dimensional (2D) cultures causes poor translatability of preclinical results from in vitro assays to in vivo tumor models. This contributes to slow drug development with a low success rate. Three-dimensional (3D) cultures better recapitulate the tumor microenvironment, enabling more accurate predictions when screening drug candidates and improving the development of chemotherapeutics. Platinum (Pt) (IV) compounds are promising prodrugs designed to reduce the severe systemic toxicity of widely-used Food and Drug Administration (FDA)-approved Pt(II) drugs such as cisplatin. Here, we present spatiotemporal evaluations in 3D colorectal cancer (CRC) spheroids of mitochondria-targeting Pt(IV) complexes. CRC spheroids provide a greater pathophysiological recapitulation of in vivo tumors than 2D cultures by a marked upregulation of the ABCG2 chemoresistance marker expression. Furthermore, we introduce new 3D-staining protocols to evaluate the real-time decrease in mitochondria membrane potential (ΔΨ) in CRC spheroids, and a Pt-sensing dye to quantify the Pt mitochondrial accumulation. Finally, we demonstrate a correlation between in vitro results and the efficacy of the compounds in vivo. Overall, the CRC spheroids represent a fast and cost-effective model to assess the behavior of Pt compounds in vitro and predict their translational potential in CRC treatment. This article is protected by copyright. All rights reserved.

GAS-Luc2 Reporter Cell Lines for Immune Checkpoint Drug Screening in Solid Tumors

GAS-Luc2 Reporter Cell Lines for Immune Checkpoint Drug Screening in Solid Tumors

Immune micro-environment analysis and drug screening for ovarian endometriosis.

Patients of ovary endometriosis have an abnormal immune micro-environment, leading to endometrial tissue that from retrograde menstruation evade immune surveillance and subsequently develop into ectopic lesions. This study aims to elucidate the crucial immune cells and molecular pathways that are associated with an aberrant immune micro-environment of endometriosis. In this study, we identified differentially expressed genes between ovarian ectopic endometrial tissue (OVE) and eutopic endometrial tissue from patients with endometriosis (PE) and non-endometriosis patients (CON) by analyzing the mRNA sequencing data. Additionally, we used WGCNA(Weighted Gene Co-expression Network Analysis) to screen for key genes related to immune cell infiltration and compared the sub-types of infiltrating immune cells using CIBERSORT(cell-type identification by estimating relative subsets of RNA transcript). Subsequently, we conducted a single-cell analysis on the identified key genes. Furthermore, we analyzed potential drugs suitable for ovarian endometriosis treatment using pRRophertic. Seven key genes associated with immune cell infiltration were screened out. The expression of these genes in OVE was significantly lower than that in PE and CON. These key genes were mainly enriched in the NK cell-mediated cytotoxicity pathway, especially for CD16 + CD56dim NK. Moreover, NK cells infiltration in ovarian endometriosis was significantly reduced compared with PE and CON, while M2 macrophage shown the opposite. Results of the single-cell analysis showed that the expression of the seven key genes in NK cells and monocyte-macrophages in OVE was significantly lower than that in PE or CON. Additionally, we identified potential drugs suitable for ovarian endometriosis treatment. The decreased infiltration of NK cells and increased infiltration of M2 macrophages contribute to the evasion of immune surveillance against endometrial tissue, promoting the progression of OVE. Therefore, potential strategies for the treatment of OVE include increasing NK cell activation and decreasing M2 macrophage polarization.

A Flexible Fibrin‐Based Platform for Driving One‐Day Self‐Assembly of Millimeter‐Sized Cell‐Rich Spheroids and Their Applications: An Old Material with New Tricks

AbstractCell‐rich spheroids have gained increasing applications due to their ability to mimic the 3D microenvironment of real tissues. Given human organ dimensions, millimeter‐sized (>1 mm) spheroids are more clinically valuable than micro‐sized ones (<500 µm). However, constructing millimeter‐sized spheroids faces the primary challenge of core necrosis due to insufficient nutrient diffusion during long‐term in vitro cultivation (>3 days). Developing rapid spheroid fabrication strategies can address the issue but remains challenging. Here, a flexible fibrin‐based platform for rapid fabrication of the spheroids is developed using a one‐step manufacturing technology. Cell‐laden fibrin hydrogels are polymerized from cell‐containing fibrinogen by thrombin‐catalyzed reactions within minutes and cultured in an agarose‐coated plate. Benefiting from the unique viscoelasticity, softness, and cell adhesion natures of fibrin, the expression levels of several cellular junction proteins are significantly upregulated compared to the scaffold‐free spheroids. As a result, cells rapidly self‐assemble into 1–15 mm‐sized spheroids within 6–24 h, accommodating high cell density (6 × 104 cells µL−1) without leakage. The spheroid formation platform is also flexible for various cell densities and cell types. Moreover, the spheroids effectively mimic the 3D tissue niches, showing promising potential in tumor drug screening with MCF‐7 spheroids and liver regeneration with HepG2 spheroids.

Organoids as a new approach for improving pediatric cancer research

A key challenge in cancer research is the meticulous development of models that faithfully emulates the intricacies of the patient scenario, with emphasis on preserving intra-tumoral heterogeneity and the dynamic milieu of the tumor microenvironment (TME). Organoids emerge as promising tool in new drug development, drug screening and precision medicine. Despite advances in the diagnoses and treatment of pediatric cancers, certain tumor subtypes persist in yielding unfavorable prognoses. Moreover, the prognosis for a significant portion of children experiencing disease relapse is dismal. To improve pediatric outcome many groups are focusing on the development of precision medicine approach. In this review, we summarize the current knowledge about using organoid system as model in preclinical and clinical solid-pediatric cancer. Since organoids retain the pivotal characteristics of primary parent tumors, they exert great potential in discovering novel tumor biomarkers, exploring drug-resistance mechanism and predicting tumor responses to chemotherapy, targeted therapy and immunotherapies. We also examine both the potential opportunities and existing challenges inherent organoids, hoping to point out the direction for future organoid development.