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The number of patients with heart failure and related deaths is rapidly increasing worldwide, making it a major problem. Cardiac hypertrophy is a crucial preliminary step in heart failure, but its treatment has not yet been fully successful. In this study, we established a system to evaluate cardiomyocyte hypertrophy using a deep learning-based high-throughput screening system and identified drugs that inhibit it. First, primary cultured cardiomyocytes from neonatal rats were stimulated by both angiotensin II and endothelin-1, and cellular images were captured using a phase-contrast microscope. Subsequently, we used a deep learning model for instance segmentation and established a system to automatically and unbiasedly evaluate the cardiomyocyte size and perimeter. Using this system, we screened 100 FDA-approved drugs library and identified 12 drugs that inhibited cardiomyocyte hypertrophy. We focused on ezetimibe, a cholesterol absorption inhibitor, that inhibited cardiomyocyte hypertrophy in a dose-dependent manner in vitro. Additionally, ezetimibe improved the cardiac dysfunction induced by pressure overload in mice. These results suggest that the deep learning-based system is useful for the evaluation of cardiomyocyte hypertrophy and drug screening, leading to the development of new treatments for heart failure.

Computer-aided drug design, an important component of the early stages of the drug discovery pipeline, routinely identifies large numbers of false positive hits that are subsequently confirmed to be experimentally inactive compounds. We have developed a methodology to improve true positive prediction rates in structure-based drug design and have successfully applied the protocol to twenty target systems and identified the top three performing conformers for each of the targets. Receptor performance was evaluated based on the area under the curve of the receiver operating characteristic curve for two independent sets of known actives. For a subset of five diverse cancer-related disease targets, we validated our approach through experimental testing of the top 50 compounds from a blind screening of a small molecule library containing hundreds of thousands of compounds. Our methods of receptor and compound selection resulted in the identification of 22 novel inhibitors in the low μM-nM range, with the most potent being an EGFR inhibitor with an IC

A high-throughput and highly efficient analytical platform for urine drug screening is critical in both clinical and forensic settings. Mass spectrometry (MS) has better sensitivity and specificity than conventional immunoassays (IA); however, not all laboratories have the necessary resources and workforce to operate MS. The goal of this study was to evaluate a multidrug biochip with 20 discrete testing regions (DTRs) for high-throughput urine drug screening (UDS).The Randox DOA Ultra Urine (DOAULT URN) biochip employs chemiluminescent IA to detect various analytes, including stimulants, hallucinogens, sedatives, narcotics, and dextromethorphan. The verification included the evaluation of the limits of detection (LOD), stability of calibrators and controls, cross-reactivity, carryover, interference, and overall performance.LODsquality control low for each DTR. The reconstituted calibrators were stable for up to 2 weeks at -20°C. Controls were stable for 4-6 hours at 22-25°C, with20% within-day and ≤23% between-day imprecision. The accuracy of the controls (%bias) was within ±20% of the target concentration, except for dextromethorphan at -23.8%. No interference was observed with common over-the-counter medications. No carryover was detected in the high-concentration samples. Satisfactory cross-reactivity (≥50%) with known analytes produced presumptive positive results, with readings higher than the proposed decision points. The overall biochip performance of 165 confirmed samples showed 98.0% sensitivity, 96.9% specificity, and 97.5% efficiency.The DOAULT URN biochip is a multidrug analyte IA capable of detecting dozens of parent drugs and their metabolites in urine. It offers clinical and forensic laboratories an alternative UDS tool with LODs comparable to those of MS.

The objective of the pharmaceutical industry is to develop new drugs that are safe for human use. In many cases, the accepted approach codified in guidance from regulatory authorities to assess the nonclinical safety profile of potential pharmaceuticals is to perform toxicity testing in two species. However, the use of a second species to establish the safety of new pharmaceuticals has been the subject of much scrutiny in recent years and the industry has been repeatedly challenged to reduce, refine, or replace some or all of the animals used to establish the safety of these pharmaceutical candidates. Specifically, the value of the dog in this testing paradigm has been questioned. Publications reviewing available data for marketed drugs suggest that for many drugs, the dog does not identify unique toxicities critical to human safety. The weakness of this approach, however, is that many of the cases where the dog (or any other species) has the greatest impact on drug development are cases for which development decisions based on safety concerns are not shared publicly. The European Federation of Pharmaceutical Industries and Associations (EFPIA) Preclinical Development Expert Group (PDEG) decided to share case studies collected from its membership and the literature to illustrate the value of the dog in drug development decision-making and clinical monitoring practices to protect the safety of trial subjects.

Microfluidic drug screening technologies have been extensively explored to evaluate the pharmacology and therapeutic implications of promising chemical compounds in multiplexed physiological microenvironments in vivo. However, conventional poly(dimethylsiloxane) microchips are susceptible to adsorption by hydrophobic molecules on channel surfaces and permeation in the matrix. These can significantly compromise the drug availability and accuracy of dose-dependent quantitative analyses. Here, we prepared a perfluorinated polyether (PFPE) microchip via digital light processing 3D printing as a quantitative drug screening platform for precise concentration-dependent pharmaceutical assays. Cells cultured on PFPE microchips exhibited excellent viability with a spread morphology as well as superior proliferative capability. Importantly, PFPE constructions with a low surface energy significantly prevented the nonspecific molecular adsorption into their surfaces or permeation into the matrix. In particular, the PFPE multibranched channel preserved the concentration of the pharmaceutical drug during the perfusion process and generated a linear concentration gradient, resulting in a dose-dependent chemotherapeutic effect. We suggest that the biocompatible and nonadsorbing PFPE microchannel can provide a cell-based drug screening platform for concentration-dependent quantitative analyses.

The preclinical assessment of efficacy and safety is essential for cardiovascular drug development in order to guarantee effective prevention and treatment of cardiovascular disease and avoid human health endangerment and a huge waste of resources. Rhythmic mechanical beating as one of the crucial cardiomyocyte properties has been exploited to establish a drug assessment biosensing platform. However, the conventional label-free biosensing platforms are difficult to perform high-throughput and high-resolution mechanical beating detection for a single cardiomyocyte, while label-based strategies are limited by pharmacologically adverse effects and phototoxicity. Herein, we propose a biosensing platform involving the multichannel electrode array device and the universal mechanical beating detection system. The platform can determine the optimal characteristic working frequency of different devices and dynamically interrogate the viability of multisite single cardiomyocytes to establish the optimized cell-based model for sensitive drug assessment. The subtle changes of mechanical beating signals induced by cardiovascular drugs can be detected by the platform, thereby demonstrating its high performance in pharmacological assessment. The universal and sensitive drug assessment biosensing platform is believed to be widely applied in cardiology investigating and preclinical drug screening.

Three-dimensional (3D) cell arrays provide an

Spheroidal cancer microtissues are highly advantageous for a wide range of biomedical applications, including high-throughput drug screening, multiplexed target validation, mechanistic investigation of tumor-extracellular matrix (ECM) interactions, among others. Current techniques for spheroidal tissue formation rely heavily on self-aggregation of single cancer cells and have substantial limitations in terms of cell-type-specific heterogeneities, uniformity, ease of production and handling, and most importantly, mimicking the complex native tumor microenvironmental conditions in simplistic models. These constraints can be overcome by using engineered tunable hydrogels that closely mimic the tumor ECM and elucidate pathologically relevant cell behavior, coupled with microfluidics-based high-throughput fabrication technologies to encapsulate cells and create cancer microtissues. In this study, we employ biosynthetic hybrid hydrogels composed of poly(ethylene glycol diacrylate) (PEGDA) covalently conjugated to natural protein (fibrinogen) (PEG-fibrinogen, PF) to create monodisperse microspheres encapsulating breast cancer cells for 3D culture and tumorigenic characterization. A previously developed droplet-based microfluidic system is used for rapid, facile, and reproducible fabrication of uniform cancer microspheres with either MCF7 or MDA-MB-231 (metastatic) breast cancer cells. Cancer cell-type-dependent variations in cell viability, metabolic activity, and 3D morphology, as well as microsphere stiffness, are quantified over time. Particularly, MCF7 cells grew as tight cellular clusters in the PF microspheres, characteristic of their epithelial morphology, while MDA-MB-231 cells displayed elongated and invasive morphology, characteristic of their mesenchymal and metastatic nature. Finally, the translational potential of the cancer microsphere platform toward high-throughput drug screening is also demonstrated. With high uniformity, scalability, and control over engineered microenvironments, the established cancer microsphere model can be potentially used for mechanistic studies, fabrication of modular cancer microtissues, and future drug-testing applications.

Prion diseases are a group of rare and lethal, rapidly progressive neurodegenerative diseases arising due to conversion of the physiological cellular prion protein into its pathological counterparts, denoted as 'prions.' These agents are resistant to inactivation by standard decontamination procedures and can be transmitted between individuals, consequently driving the irreversible brain damage typical of the diseases.Since its infancy, prion research has mainly depended on animal models for untangling the pathogenesis of the disease as well as for the drug development studies. With the advent of prion-infected cell lines, relevant animal models have been complemented by a variety of cell-based models presenting a much faster, ethically acceptable alternative.To date, there are still either no effective prophylactic regimens or therapies for human prion diseases. Therefore, there is an urgent need for more relevant cellular models that best approximate

Recent developments in applied developmental physiology have provided well-defined methodologies for producing human stem cell derived cardiomyocytes. The cardiomyocytes produced have become commonplace as cardiac physiology research models. Accessibility has also allowed for the development of tissue engineered human heart constructs for drug screening, surgical intervention, and investigating cardiac pathogenesis. However, cardiac tissue engineering is an interdisciplinary field that involves complex engineering and physiological concepts, which limits its accessibility. Our review provides a readable, broad reaching, and thorough discussion of major factors to consider for the development of cardiovascular tissues from stem cell derived cardiomyocytes. In this study, our review will examine important considerations in undertaking a cardiovascular tissue engineering project and will present, interpret, and summarize some of the recent advancements in this field. Throughout, we review different forms of tissue engineered constructs, a discussion on cardiomyocyte sources, and an in-depth discussion of the fabrication and maturation procedures for tissue engineered heart constructs. Impact statement With advancements in cardiac differentiation protocols, the production of human induced pluripotent stem cell derived cardiomyocytes is becoming cost effective and routine in the laboratory setting. Monolayer based culture methods are rapidly being replaced by three-dimensional (3D) tissue engineered constructs, which are more representative of the heart geometry. In the review presented, we delve into important concepts and tissue engineering principles that should be considered when generating 3D cardiac constructs, interpreting data acquired from, and embarking on a 3D cardiac tissue-based research project.

Protein networks can be assembled in vitro for basic biochemistry research, drug screening, and the creation of artificial cells. Two standard methodologies are used: manual pipetting and pipetting robots. Manual pipetting has limited throughput in the number of input reagents and the combination of reagents in a single sample. While pipetting robots are evident in improving pipetting efficiency and saving hands-on time, their liquid handling volume usually ranges from a few to hundreds of microliters. Microfluidic methods have been developed to minimize the reagent consumption and speed up screening but are challenging in multifactorial protein studies due to their reliance on complex structures and labeling dyes. Here, we engineered a new impact-printing-based methodology to generate printed microdroplet arrays containing water-in-oil droplets. The printed droplet volume was linearly proportional (

Before advancing to clinical trials, new drugs are screened for their pro-arrhythmic potential using a method that is overly conservative and provides limited mechanistic insight. The shortcomings of this approach can lead to the mis-classification of beneficial drugs as pro-arrhythmic.An in silico-in vitro pipeline was developed to circumvent these shortcomings. A computational human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model was used as part of a genetic algorithm to design experiments, specifically electrophysiological voltage clamp (VC) protocols, to identify which of several cardiac ion channels were blocked during in vitro drug studies. Such VC data, along with dynamically clamped action potentials (AP), were acquired from iPSC-CMs before and after treatment with a control solution or a low- (verapamil), intermediate- (cisapride or quinine) or high-risk (quinidine) drug.Significant AP prolongation (a pro-arrhythmia marker) was seen in response to quinidine and quinine. The VC protocol identified block of IWe developed an in silico-in vitro pipeline that simultaneously identifies pro-arrhythmia risk and mechanism of ion channel-blocking drugs. The approach offers a new tool for evaluating cardiotoxicity during preclinical drug screening.

In the current pandemic, scenario the world is facing a huge shortage of effective drugs and other prophylactic medicine to treat patients which created havoc in several countries with poor resources. With limited demand and supply of effective drugs, researchers rushed to repurpose the existing approved drugs for the treatment of COVID-19. The process of drug screening and testing is very costly and requires several steps for validation and treatment efficacy evaluation ranging from in-vitro to in-vivo setups. After these steps, a clinical trial is mandatory for the evaluation of treatment efficacy and side effects in humans. These processes enhance the overall cost and sometimes the lead molecule show adverse effects in humans and the trial ends up in the final stages. Recently with the advent of three-dimensional (3D) organoid culture which mimics the human tissue exactly the process of drug screening and testing can be done in a faster and cost-effective manner. Further 3D organoids prepared from stems cells taken from individuals can be beneficial for personalized drug therapy which could save millions of lives. This review discussed approaches and techniques for the synthesis of 3D-printed human organoids for drug screening. The key findings of the usage of organoids for personalized medicine for the treatment of COVID-19 have been discussed. In the end, the key challenges for the wide applicability of human organoids for drug screening with prospects of future orientation have been included.

3D cell cultures are being utilized for drug discovery and development. However, there are still challenges to implementing them generally in quantitative high-throughput screening (HTS) due to the complexity of the 3D architecture, the time- and labor-consuming process, and the lack of compatibility with traditional screening protocols. Therefore, there is a great need for the integration of microfabrication techniques, automation systems, and high-throughput analytical tools that reveal the pharmacological and toxicological effects of therapeutics using 3D cultures. We first review the current advances in 3D culture models and discuss their key challenges in HTS. Last, we review recent progress and breakthroughs in the automation and high-throughput imaging of 3D culture models, which can be integrated with machine-learning (ML) tools to aid quantitative HTS for drug discovery and development.

Clinical research has been desperately looking for effective treatments for tumors. As the molecular mechanisms of tumors have not been thoroughly defined, and the problem of anti-tumor drug resistance and clinical drug screening are not effective, new ways are currently needed to explore and improve. Given the limitations of traditional cell culture models and preclinical animal models, we review the recently developed tumor chip, a micro-physiological platform based on microfluidic technology that maximizes replication of the human TEM. Here, we focus on the design basis of the tumor microarray and its application in urology oncology, summarizing the challenges facing future development in the field. In conclusion, this review demonstrates the wide range and application of tumor chips and the potential of these systems for the future treatment and management of urological tumors.

The IQ Consortium NHP Reuse Working Group (WG) comprises members from 15 pharmaceutical and biotechnology companies. In 2020, the WG developed and distributed a detailed questionnaire on protein non-naïve NHP reuse to the WG member companies. The WG received responses from key stakeholders including principal investigators, facility managers, animal welfare officers and research scientists. This paper’s content reflects the consolidated opinion of the WG members and the questionnaire responses on the subject of NHP reuse within nonclinical programs at all stages of research and development. Many of the pharmaceutical companies represented in the working group or participating in the questionnaire have already achieved some level of NHP reuse in their nonclinical programs, but the survey results suggested that there is significant potential to increase NHP reuse further and a need to understand the considerations involved in reuse more clearly. The WG has also focused carefully on the inherent concerns and risks of implementing protein non-naive NHP reuse and has evaluated the best methods of risk assessment and decision-making. This paper presents a discussion on the challenges and opportunities surrounding protein non-naïve NHP reuse and aims to stimulate further industry dialogue on the subject and provide guidance for pharmaceutical companies to establish roadmaps and decision trees enabling increased protein non-naïve NHP reuse. In addition, this paper represents a solid basis for collaborative engagement between pharmaceutical and biotechnology companies with contract research organizations (CROs) to discuss how the availability of protein non-naïve NHP within CROs can be better leveraged for their use within nonclinical studies.

Isolated marijuana use is frequently used as an indication for urine drug screening in labor and delivery units. We aimed to identify the results of urine drug screening in a labor and delivery unit for isolated marijuana use. This retrospective cohort study reviewed data from patients admitted for delivery at an urban academic center from January 1, 2020, to December 31, 2020. Patients undergoing urine drug screening for isolated marijuana use were more likely to be younger (median age 25 vs 29 years, P.001), more often Black (adjusted odds ratio [aOR] 2.58, 95% CI 1.94-3.41), and more likely to have public insurance (aOR 1.54, 95% CI 1.21-1.95). A few (5/338, 1.5%) urine drug screening tests performed for isolated marijuana use were positive for substances besides marijuana. Most patients (177/197, 89.8%) with a urine drug screening test result that was positive for marijuana were reported to the state child abuse hotline. The utility of isolated marijuana use as a criterion for urine drug screening thus appears limited in benefit but rife with inequitable potential to harm.

The global health burden of stroke is significant and few therapeutic treatment options currently exist for patients. Pre-clinical research relies heavily on rodent stroke models but the limitations associated with using these systems alone has meant translation of drug compounds to the clinic has not been greatly successful to date. Zebrafish disease modeling offers a potentially complementary platform for pre-clinical compound screening to aid the drug discovery process for translational stroke research.In this review, the authors introduce stroke and describe the issues associated with the current pre-clinical drug development pipeline and the advantages that zebrafish disease modeling can offer. Existing zebrafish models of ischemic and hemorrhagic stroke are reviewed. Examples of how zebrafish models have been utilized for drug discovery in other disease disciplines are also discussed.Zebrafish disease modeling holds the capacity and potential to significantly enhance the stroke drug development pipeline. However, for this system to be more widely accepted and incorporated into translational stroke research, continued improvement of the existing zebrafish stroke models, as well as focussed collaboration between zebrafish and stroke researchers, is essential.

This study is designed to systematically review the accessible researches regarding influence of Coriandrum sativum L. on inflammatory mediators including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α). Databases Scopus, PubMed, WOS, ProQuest, and a Google Scholar were searched until February 2022 and search alerts were turned on to find papers published following the primary search. There was not any restriction in language and/or date. No human study was gained; thus, animal and in vitro researches were considered. The references of related papers were reviewed to access plausible researches. Twenty-four papers were entered in review. Inflammatory factors IL-1β, IL-6, and TNF-α considerably had a descending direction following C. sativum consumption. In other words, the pooled direction of influences was consistently lower for inflammatory mediators in 7 of 9 in vitro and 10 of 16 animal investigations. These results demonstrated the potential of C. sativum in reducing IL-1β, IL-6, and TNF-α. C. sativum is hopeful but not yet a confirmed natural ingredient to reduce systemic inflammation in subjects with inflammation-prone disorders. Additional investigations are required to concentrate on assessing the impact of C. sativum on inflammatory factors that are not exceedingly fluctuating and the clinical consequences of inflammation-linked diseases.

Conventional approaches to study ligand-receptor interactions using solution-state NMR often involve laborious sample preparation, isotopic labeling, and receptor reconstitution. Each of these steps remains challenging for membrane proteins such as G protein-coupled receptors (GPCRs). Here we introduce a combinational approach integrating NMR and homogenized membrane nano-discs preparation to characterize the ligand-GPCR interactions. The approach will have a great potential for drug screening as it benefits from minimal receptor preparation, minimizing non-specific binding. In addition, the approach maintains receptor structural heterogeneity essential for functional diversity, making it feasible for probing a more reliable ligand-GPCR interaction that is vital for faithful ligand discovery.

Three dimensional (3D) bioprinting technology has been making a progressive advancement in the field of tissue engineering to produce tissue constructs that mimic the shape, framework, and microenvironment of an organ. The technology has not only paved the way to organ development but has been widely studied for its application in drug and cosmetic testing using 3D bioprinted constructs. However, not much has been explored on the utilization of bioprinting technology for the development of tumor models to test anti-cancer drug efficacy. The conventional methodology involves a two dimensional (2D) monolayer model to test cellular drug response which has multiple limitations owing to its inability to mimic the natural tissue environment. The choice of bioink for 3D bioprinting is critical as cell morphology and proliferation depend greatly on the property of bioink. In this study, we developed a multicomponent bioink composed of alginate, diethylaminoethyl cellulose, gelatin, and collagen peptide to generate a 3D bioprinted construct. The bioink has been characterised and validated for its printability, shape fidelity and biocompatibility to be used for generating tumor models. Further, a bioprinted tumor model was developed using lung cancer cell line and the efficacy of 3D printed construct for drug screening application was established.

Proposing efficient prophylactic and therapeutic strategies for coronavirus 2019 (COVID-19) requires precise knowledge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis. An array of platforms, including organoids and microfluidic devices, have provided a basis for studies of SARS-CoV-2. Here, we summarize available models as well as novel drug screening approaches, from simple to more advanced platforms. Notably, organoids and microfluidic devices offer promising perspectives for the clinical translation of basic science, such as screening therapeutics candidates. Overall, modifying these advanced micro and macro 3D platforms for disease modeling and combining them with recent advances in drug screening has significant potential for the discovery of novel potent drugs against COVID-19.

The management of multiple myeloma (MM) is challenging: An assortment of available drug combinations adds complexity to treatment selection, and treatment resistance frequently develops. Given the heterogeneous nature of MM, personalized testing tools are required to identify drug sensitivities. To identify drug sensitivities in MM cells, we established a drug testing pipeline to examine ex vivo drug responses. MM cells from 44 patients were screened against 30 clinically relevant single agents and 44 double- and triple-drug combinations. We observed variability in responses across samples. The presence of gain(1q21) was associated with low sensitivity to venetoclax, and decreased ex vivo responses to dexamethasone reflected the drug resistance observed in patients. Less heterogeneity and higher efficacy was detected with many combinations compared to the corresponding single agents. We identified new synergistic effects of melflufen plus panobinostat using low concentrations (0.1-10 nm and 8 nm, respectively). In agreement with clinical studies, clinically approved combinations, such as triple combination of selinexor plus bortezomib plus dexamethasone, acted synergistically, and synergies required low drug concentrations (0.1 nm bortezomib, 10 nm selinexor and 4 nm dexamethasone). In summary, our drug screening provided results within a clinically actionable 5-day time frame and identified synergistic drug efficacies in patient-derived MM cells that may aid future therapy choices.

UV-4 (N-(9-methoxynonyl)-1-deoxynojirimycin) is a broad-spectrum antiviral drug candidate with demonstrated activity in vitro and in vivo against multiple, diverse viruses. Nonclinical safety pharmacology studies were conducted to support the filing of an Investigational New Drug (IND) application. Preliminary in vitro pharmacology testing evaluating potential for binding to “off-target” receptors and enzymes indicated no significant liability for advanced development of UV-4. The safety pharmacology of UV-4 was evaluated in the in vitro human ether-à-go-go-related gene (hERG) assay, in a central nervous system (CNS) study in the mouse (modified Irwin test), in a respiratory safety study in conscious mice using whole body plethysmography, and in a cardiovascular safety study in conscious, radiotelemetry-instrumented beagle dogs. There were no observed adverse treatment-related effects following administration of UV-4 as the hydrochloride salt in the hERG potassium channel assay, on respiratory function, in the CNS study, or in the cardiovascular assessment. Treatment-related cardiovascular effect of decreased arterial pulse pressure after 50 or 200 mg of UV-4/kg was the only change outside the normal range, and all hemodynamic parameters returned to control levels by the end of the telemetry recording period. These nonclinical safety pharmacology assessments support the evaluation of this host-targeted broad-spectrum antiviral drug candidate in clinical studies.

Japanese encephalitis (JE) caused by the Japanese encephalitis virus (JEV) is one of Asia's most common viral encephalitis. JEV is a flavivirus, common in rural and sub-urban regions of Asian countries. Although only 1% of JEV-infected individuals develop JE, there is a 20–30% chance of death among these individuals and possible neurological sequelae post-infection. No licensed anti-JE drugs are currently available, despite extensive efforts to develop them. Literature search was performed using databases such as PubMed Central, Google Scholar, Wiley Online Library, etc. using keywords such as Japanese encephalitis virus, antiviral drugs, antiviral drug screening, antiviral drug targets, etc. From around 230 papers/abstracts and research reviews retrieved and reviewed for this study, approximately 180 most relevant and important ones have been cited. Different approaches in drug testing and various antiviral drug targets explored so far have been thoroughly searched from the literature and compiled, besides addressing the future perspectives of the antiviral drug development strategies. Although the development of effective anti-JE drugs is an urgent issue, only supportive care is currently available. Recent advancements in understanding the biology of infection and new drug targets have been promising improvements. Despite hindrances such as the unavailability of a proper drug delivery system or a treatment regimen irrespective of the stage of infection, several promising anti-JE candidate molecules are in different phases of clinical trials. Nonetheless, efficient therapy against JEV is expected to be achieved with drug combinations and a highly targeted drug delivery system soon. Graphical abstract

The SARS-CoV-2 virus has infected more than 261 million people and has led to more than 5 million deaths in the past year and a half

Coronavirus disease 2019 (COVID-19) pandemic, a global health threat, was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 papain-like cysteine protease (PLpro) was recognized as a promising drug target because of multiple functions in virus maturation and antiviral immune responses. Inhibitor GRL0617 occupied the interferon-stimulated gene 15 (ISG15) C-terminus-binding pocket and showed an effective antiviral inhibition. Here, we described a novel peptide–drug conjugate (PDC), in which GRL0617 was linked to a sulfonium-tethered peptide derived from PLpro-specific substrate LRGG. The EM-C and EC-M PDCs showed a promising in vitro IC50 of 7.40 ± 0.37 and 8.63 ± 0.55 μM, respectively. EC-M could covalently label PLpro active site C111 and display anti-ISGylation activities in cellular assays. The results represent the first attempt to design PDCs composed of stabilized peptide inhibitors and GRL0617 to inhibit PLpro. These novel PDCs provide promising opportunities for antiviral drug design.

The development of novel therapeutics is associated with high rates of attrition, with unexpected adverse events being a major cause of failure. Serious adverse events have led to organ failure, cancer development and deaths that were not expected outcomes in clinical trials. These life-threatening events were not identified during therapeutic development due to the lack of preclinical safety tests that faithfully represented human physiology. We highlight the successful application of several novel technologies, including high-throughput screening, organs-on-chips, microbiome-containing drug-testing platforms and humanised mouse models, for mechanistic studies and prediction of toxicity. We propose the incorporation of similar preclinical tests into future drug development to reduce the likelihood of hazardous therapeutics entering later-stage clinical trials.

Introduction Abnormal extravillous trophoblast (EVT) function is closely related to preeclampsia (PE) and may be caused by inadequate autophagy, apoptosis, and senescence. Cyclosporin A (CsA) is an effective immunosuppressant that has been reported to stimulate autophagy and exert benign biological effects on EVTs. Therefore, we hypothesized that CsA may display therapeutic efficacy against PE by activating autophagy. Methods We established the nitro- l -arginine methyl ester ( l -NAME)-induced preeclamptic mice model and a hypoxia‐reoxygenation (H/R) model in vitro. The effects of CsA on autophagy were evaluated by western blotting (WB). The effects of CsA on apoptosis were analyzed by Hematoxylin-eosin (H&E) staining, cell apoptosis assay and WB. Senescence‐associated β‐galactosidase (SA‐β‐gal) staining, RT-qPCR and WB were used to examine the senescence level. RT-qPCR were used to detect the senescence-associated secretory phenotype (SASP) level. DCFH-DA fluorescent probe, dihydroethidium (DHE) staining and mitochondrial membrane potential (ΔΨm) were used to detect senescence-associated mitochondrial dysfunction (SAMD). Results CsA alleviated PE-like symptoms and reduced placental necrosis and senescence in mice injected with l -NAME. CsA ameliorated placental SASP and SAMD level induced by l -NAME. CsA also upregulated the expression of autophagic proteins in mouse placentas disrupted using l -NAME. In vitro, we found that CsA reversed H/R-induced apoptosis and senescence, as well as decreasing SASP and SAMD levels and upregulating autophagic proteins levels. Notably, 3-methyladenine (3-MA), an early phase inhibitor of autophagosome formation, abolished the protective effects of CsA against H/R. Discussion CsA may display some therapeutic effects against PE by activating autophagy in vivo and in vitro.

Osteoarthritis (OA) is a prevalent debilitating age-related joint degenerative disease. It is a leading cause of pain and functional disability in older adults. Unfortunately, there is no cure for OA once the damage is established. Therefore, it promotes an urgent need for early detection and intervention of OA. Theranostics, combining therapy and diagnosis, emerges as a promising approach for OA management. However, OA theranostics is still in its infancy. Three fundamental needs have to be firstly fulfilled: i) a reliable OA model for disease pathogenesis investigation and drug screening, ii) an effective and precise diagnostic platform, and iii) an advanced fabrication approach for drug delivery and therapy. Meanwhile, microfluidics emerges as a versatile technology to address each of the needs and eventually boost the development of OA theranostics. Therefore, this review focuses on the applications of microfluidics, from benchtop to bedside, for OA modelling and drug screening, early diagnosis, and clinical therapy. We first introduce the basic pathophysiology of OA and point out the major unfilled research gaps in current OA management including lack of disease modelling and drug screening platforms, early diagnostic modalities and disease-modifying drugs and delivery approaches. Accordingly, we then summarize the state-of-the-art microfluidics technology for OA management from in vitro modelling and diagnosis to therapy. Given the existing promising results, we further discuss the future development of microfluidic platforms towards clinical translation at the crossroad of engineering and biomedicine.

High content screening (HCS) plays an important role in biological applications, and drug development. Existing techniques fail to simultaneously meet multiple needs of throughput, efficiency in sample and chemical consumption, real-time imaging of a large view at high resolution. Leveraging advances in microfluidics and imaging technology, we setup a new paradigm of large-scale, high-content drug screening solutions for rapid biological processes, like cardiotoxicity. The designed microfluidic chips enable 10 types of drugs each with 5 concentrations to be assayed simultaneously. The imaging system RUSH has 30 Hz video rate for a centimeter filed-of-view at 0.8 m resolution. Using the HCS system, we assayed 12 small molecules through their effects on the Ca2+ ion signal of cardiomyocytes. Experimental results demonstrated the unparallelized capability of the system in revealing the spatiotemporal patterns of Ca2+ imaging of cardiomyocytes, and validated the cardiotoxicity of certain molecules. We envision this new HCS paradigm and cutting-edge platform as the most advanced alternative to well-plate based methods.

The absence of in vitro platforms for human pulmonary toxicology studies is becoming an increasingly serious concern. The respiratory system has a dynamic mechanical structure that extends from the airways to the alveolar region. In addition, the epithelial, endothelial, stromal, and immune cells are highly organized in each region and interact with each other to function synergistically. These cells of varied lineage, particularly epithelial cells, have been difficult to use for long-term culture in vitro, thus limiting the development of useful experimental tools. This limitation has set a large distance between the bench and the bedside for analyzing the pathogenic mechanisms, the efficacy of candidate therapeutic agents, and the toxicity of compounds. Several researchers have proposed solutions to these problems by reporting on methods for generating human lung epithelial cells derived from pluripotent stem cells (PSCs). Moreover, the use of organoid culture, organ-on-a-chip, and material-based techniques have enabled the maintenance of functional PSC-derived lung epithelial cells as well as primary cells. The aforementioned technological advances have facilitated the in vitro recapitulation of genetic lung diseases and the detection of ameliorating or worsening effects of genetic and chemical interventions, thus indicating the future possibility of more sophisticated preclinical compound assessments in vitro. In this review, we will update the recent advances in lung cell culture methods, principally focusing on human PSC-derived lung epithelial organoid culture systems with the hope of their future application in toxicology studies.

Soybean (Glycine max) harbours high quality proteins which have been evident to exhibit therapeutic properties in alleviating many diseases including but not limited to diabetes and its related metabolic complications. Since diabetes is often manifested with hyperglycemia, impaired energy homeostasis and even low-grade chronic inflammation, plenty of information has raised the suggestion for soy protein supplementation in preventing and controlling these abnormalities. Moreover, clinical intervention studies have established a noteworthy correlation between soy protein intake and lower prevalence of diabetes. Besides soy protein, various soy-derived peptides also have been found to trigger antidiabetic response in different in vitro and in vivo models. Molecular mechanisms underlying the antidiabetic actions of soy protein and peptide have been predicted in many literatures. Results demonstrate that components of soy protein can act in diversified ways and modulate various cell signaling pathways to bring energy homeostasis and to regulate inflammatory parameters associated with diabetic pathophysiology. The main objective of the present review lies in a systemic understanding of antidiabetic role of soy protein and peptide in the context of impaired glucose and lipid metabolism, and inflammation.

Melanosomes are intracellularly transported from the perinuclear region to the cell periphery and then to neighboring keratinocytes. We recently reported that the flavonoid rhamnazin suppresses melanosomal transport within pigment cells, yet the action mechanism remained unclear.Our aim was to elucidate how rhamnazin influences the intracellular transport of melanosomes.A melanosome distribution assay and immunostaining were performed using B16F10 mouse melanoma cells and normal human epidermal melanocytes, respectively. Expression levels of melanosome transport-related proteins, including melanophilin (MLPH), RAB27A, and myosin VA (MYO5A), were analyzed by immunoblotting. Ubiquitinated MLPH was detected using a commercial ubiquitin detection kit. To investigate the interaction between rhamnazin and MLPH, we prepared rhamnazin conjugated with magnetic FG beads.Immunoblotting analysis revealed that rhamnazin specifically reduces the expression of MLPH but not RAB27A or MYO5A proteins. The ubiquitin detection assay, which made use of a proteasome inhibitor, showed that MLPH accumulated as a polyubiquitinated protein after treatment with rhamnazin. We speculated that the affinity of rhamnazin for the components of the melanosome transport-related tripartite complex may alter the stability of the formation of the tripartite assembly. By using affinity-based techniques with B16F10 whole cell lysates or recombinant MLPH and RAB27A proteins, we revealed the interaction of rhamnazin with the components of the tripartite complex.We found that rhamnazin inhibits intracellular transport of melanosomes through proteasomal degradation of MLPH. Our results suggest that topical application of rhamnazin may provide a new approach for treating skin pigmentation disorders.

Drug-induced vascular injury (DIVI) in preclinical animal models often leads to candidate compound termination during drug development. DIVI has not been documented in human clinical trials with drugs that cause DIVI in preclinical animals. A robust human preclinical assay for DIVI is needed as an early vascular injury screen. A human vascular wall microfluidic tissue chip was developed with a human umbilical vein endothelial cell (HUVEC)—umbilical artery smooth muscle cell (vascular smooth muscle cell, VSMC) bilayer matured under physiological shear stress. Optimized temporal flow profiles produced HUVEC-VSMC bilayers with quiescent endothelial cell (EC) monolayers, EC tight junctions, and contractile VSMC morphology. Dose–response testing (3–30 μM concentration) was conducted with minoxidil and tadalafil vasodilators. Both drugs have demonstrated preclinical DIVI but lack clinical evidence. The permeability of severely damaged engineered bilayers (30 μM tadalafil) was 4.1 times that of the untreated controls. Immunohistochemical protein assays revealed contrasting perspectives on tadalafil and minoxidil-induced damage. Tadalafil impacted the endothelial monolayer with minor injury to the contractile VSMCs, whereas minoxidil demonstrated minor EC barrier injury but damaged VSMCs and activated ECs in a dose–response manner. This proof-of-concept human vascular wall bilayer model of DIVI is a critical step toward developing a preclinical human screening assay for drug development. IMPACT STATEMENT: More than 90% of drug candidates fail during clinical trials due to human efficacy and toxicity concerns. Preclinical studies rely heavily on animal models, although animal toxicity and drug metabolism responses often differ from humans. During the drug development process, perfused in vitro human tissue chips could model the clinical drug response and potential toxicity of candidate compounds. Our long-term objective is to develop a human vascular wall tissue chip to screen for drug-induced vascular injury. Its application could ultimately reduce drug development delays and costs, and improve patient safety.

Whole-organism phenotype screening and complex in vitro model technology increase the likelihood in identifying successful lead compounds and lower drug attrition rates at later and more expensive stages of the drug discovery process.

Affinity selection-mass spectrometry, which includes magnetic microbead affinity selection-screening (MagMASS), is ideal for the discovery of ligands in complex mixtures that bind to pharmacological targets. Therapeutic agents are needed to prevent or treat COVID-19, which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection of human cells by SARS-CoV-2 involves binding of the virus spike protein subunit 1 (S1) to the human cell receptor angiotensin converting enzyme-2 (ACE2). Like antibodies, small molecules have the potential to block the interaction of the viral S1 protein with human ACE2 and prevent SARS-CoV-2 infection. Therefore, a MagMASS assay was developed for the discovery of ligands to the S1 protein. Unlike previous MagMASS approaches, this new assay used robotics for five-fold enhancement of throughput and sensitivity. The assay was validated using the SBP-1 peptide, which is identical to the ACE2 amino acid sequence recognized by the S1 protein, and then applied to the discovery of natural ligands from botanical extracts. Small molecule ligands to the S1 protein were discovered in extracts of the licorice species, Glycyrrhiza inflata. In particular, the licorice ligand licochalcone A was identified through dereplication and comparison with standards using HPLC with high-resolution tandem mass spectrometry.

In preclinical drug development, ex vivo and in vitro permeability studies are a decisive element for specifying subsequent development steps. In this context, reliability, physiological alignment and appropriate in vivo correlation are mandatory for predictivity regarding drug absorption. Especially in oromucosal drug delivery, these prerequisites are not adequately met, which hinders its progressive development and results in the continuous need for animal experiments. To address current limitations, an innovative, standardized, and controlled ex vivo permeation model was applied. It is based on Kerski diffusion cells embedded in automated sampling and coupled to mass spectrometric quantification under physiologically relevant conditions. This study aimed to evaluate the predictivity of the developed model using porcine mucosa (ex vivo) in relation to data of sublingual propranolol absorption (in vivo). In addition, the usefulness of biomimetic barriers (in vitro) as a replacement for porcine mucosa was investigated. Therefore, solubility and permeability studies considering microenvironmental conditions were conducted and achieved good predictivity (R2 = 0.997) for pH-dependent permeability. A multiple level C correlation (R2 ≥ 0.860) between obtained permeability and reported pharmacokinetic animal data (AUC, Cmax) was revealed. Furthermore, a point-to-point correlation was demonstrated for several sublingual formulations. The successful IVIVC confirms the standardized ex vivo model as a viable alternative to animal testing for estimating the in vivo absorption behavior of oromucosal pharmaceuticals.

Innovative technologies have been designed to improve efficacy and safety of chemical UV filters. Encapsulation can enhance efficacy and reduce transdermal permeation and systemic exposure. The aims of this work were (i) to determine the cutaneous biodistribution of avobenzone (AVO), oxybenzone (OXY), and octyl methoxycinnamate (OMC) incorporated in mesoporous silica SBA-15 and (ii) to perform preclinical (in vitro) and (iii) clinical safety studies to demonstrate their innocuity and to evaluate sun protection factor (SPF) in humans. Skin penetration studies showed that deposition of OXY and AVO in porcine and human skin after application of stick formulation with incorporated filters (stick incorporated filters) was significantly lower than from a marketed (non-encapsulated) stick. Cutaneous deposition and transdermal permeation of OXY in and across human skin were 3.8-and 13.4- fold lower, respectively, after application of stick entrapped filters. Biodistribution results showed that encapsulation in SBA-15 decreased AVO and OXY penetration reaching porcine and human dermis. Greater deposition (and permeation) of OXY in porcine skin than in human skin, pointed to the role of follicular transport. Stick incorporated filters had good biocompatibility in vivo and safety profiles, even under sun-exposed conditions. Entrapment of UV filters improved the SPF by 26% and produced the same SPF profile as a marketed stick. Overall, the results showed that SBA-15 enabled safety and efficacy of UV filters to be increased.

In this work, we investigated in vitro the antioxidant, cytotoxic and anti-leishmanial activities of a lignin extracted from the leaves of Morinda citrifolia. Initially, an analysis of the composition of the sheets was performed, then the lignin was obtained by alkaline delignification and characterized by different techniques: elemental analysis, FT-R, UV–vis, HSQC-NMR, thermal analysis, Py-GC/MS and by GPC. The results showed that the leaves had in their composition cellulose (31.29%), hemicellulose (25.01%), lignin (18.34%), extractives (14.39%) and ash (10.03%). The lignin extraction yield was 89.8%. The lignin obtained is of the GSH type with the following contents 79.39%, 13.58% and 7.03% respectively. Furthermore, it is low molecular weight and thermally stable. It had a phenolic content of 93.3 mg GAE/g and low antioxidant activity. In macrophage cytotoxicity assays, it presented a CC50 of 31.0 μg/mL, showing less toxicity than amphotericin B. In assays against the promastigote forms of Leishmania amazonensis, lignin presented an IC50 of 29.56 μg/mL, a less effective concentration than amphotericin B (IC50 = 0.14 μg/mL). However, it was able to promote inhibition of the parasites, a fact confirmed by structural changes. These findings reinforce that M. citrifolia lignin is a promising macromolecule for use as an antiparasitic and antioxidant agent.

Despite the prevalence of treatment-resistant depression, many pharmaceutical companies have abandoned the development of new antidepressants. Experts have attributed this, in part, to the low quality of preclinical tests available in this field, often citing over-reliance on animal behavioral screens, such as the forced swim test (FST). This retrospective review assessed whether compounds tested in the FST by major pharmaceutical companies were shown to have antidepressant effects in humans. Of 109 compounds identified, only 28% had been explored for antidepressant effects in humans. Of these, there were only three for which the FST appeared to positively predict antidepressant efficacy, but none are currently approved to treat any type of depression. With such poor accuracy for identifying novel antidepressants, the FST might not be a useful screening tool for this purpose.

MTargeting METTL3 has opened a new window in the development of novel inhibitors/drugs.In this study, commercially available natural compounds were randomly screened to avoid the bias of screening small molecules on the basis of structural similarity. From 810 compounds that were screened, 80 commercially available compounds were showing better score when compared with the existing substrate/substrate-analogue and the inhibitor bound crystal structures in terms of docking score and binding energy calculation.Among this pool of compounds, the best seven small molecules have been selected and further validated by different computational tools like binding energy calculation, molecular dynamics simulation, ADME analysis, and toxicity prediction. The novel hits found in this study can function as lead compounds which can be developed into inhibitors as well as drugs, specific against METTL3.

Mifepristone treatment for advanced cancer has demonstrated considerable improvement in both length and quality of life in patients who no longer have any other treatment options. The target is the progesterone induced blocking factor (PIBF), which helps the tumor to invade the normal tissue and proliferate and suppress cellular immunity. Most of the benefit has been observed in cancers not associated with the classical nuclear progesterone receptor (nPR). There are data showing that the presence of a nPR may be associated with a better prognosis. Membrane PRs seem to be responsible for PIBF secretion. Mifepristone, possibly fails to block another P associated protein that enables the tumor to proliferate, e.g., the progesterone receptor membrane component-1 (PGRMC-1) protein. One hypothesis is that the nPR helps to inhibit tumor production of PGRMC-1 protein. Thus, mifepristone may inhibit tumor spread by suppressing PIBF, but this may be negated by blocking the nPR, allowing PGRMC-1 levels to increase.

The safety windows and toxicity of clinically available known drugs allow drug repurposing to be a popular treatment strategy for several diseases, including cancers. Several common drugs, e.g., metformin, statin, and aspirin are on clinical trials for repurposing in oncology treatment. Most of repurposed drugs, however, cannot be used as single agents and some do not exert any clinically significant effects. The limitations and possible biases from observational studies and preclinical models to repurpose these drugs are debatable. In this article, the limitations and probability of using metformin, one of the most repurposed drugs for cancer treatment and in oncological practice, are discussed.

Acute myocardial infarction (AMI) has been a preclinical and clinical concern due to high hospitalization rate and mortality. This study was aimed at evaluating the effectiveness and safety of Shexiang Baoxin Pill (SBP) for AMI and exploring the possible mechanism of oxidative stress. Six databases were searched on March 26, 2021. Twenty-four studies were included and accessed by the RoB 2.0 or SYRCLE tool. Compared with routine treatment (RT), SBP showed the effectiveness in the clinical efficacy ( RR = 1.15 , 95% CI [1.06, 1.25]), left ventricular ejection fraction (LVEF) ( SMD = 0.73 , 95% CI [0.62, 0.95]), glutathione (GSH) ( SMD = 2.07 , 95% CI [1.51, 2.64]), superoxide dismutase (SOD) ( SMD = 0.92 , 95% CI [0.58, 1.26]), malondialdehyde (MDA) ( SMD = − 4.23 , 95% CI [-5.80, -2.66]), creatine kinase-myocardial band (CK-MB) ( SMD = − 4.98 , 95% CI [-5.64, -4.33]), cardiac troponin I (cTnI) ( SMD = − 2.17 , 95% CI [-2.57, -1.76]), high-sensitivity C-reactive protein (Hs-CRP) ( SMD = − 1.34 , 95% CI [-1.56, -1.12]), interleukin-6 (IL-6) ( SMD = − 0.99 , 95% CI [-1.26, -0.71]), triglycerides (TG) ( SMD = − 0.52 , 95% CI [-0.83, -0.22]), flow-mediated dilation (FMD) ( SMD = 1.39 , 95% CI [1.06, 1.72]), von Willebrand Factor (vWF) ( SMD = − 1.77 , 95% CI [-2.39, -1.15]), nitric oxide (NO) ( SMD = 0.89 , 95% CI [0.65, 1.13]), and recurrent rate ( RR = 0.30 , 95% CI [0.15, 0.59]). But SBP adjunctive to RT plus PCI had no improvements in almost pooled outcomes except for the Hs-CRP ( SMD = − 1.19 , 95% CI [-1.44, -0.94]) and TG ( SMD = − 0.25 , 95% CI [-0.48, -0.02]). Laboratory findings showed that SBP enhanced the endothelial nitric oxide synthase (eNOS) activity and regulated laboratory indexes especially for homocysteine. In conclusion, SBP has adjunctive effects on AMI via the mechanism of antioxidative stress. The current evidence supports the use of SBP for mild and moderate AMI patients.

Drug-induced liver injury is a leading cause of compound attrition during both preclinical and clinical drug development, and early strategies are in place to tackle this recurring problem. Human-relevant in vitro models that are more predictive of hepatotoxicity hazard identification, and that could be employed earlier in the drug discovery process, would improve the quality of drug candidate selection and help reduce attrition. We present an evaluation of four human hepatocyte in vitro models of increasing culture complexity (i.e., two-dimensional (2D) HepG2 monolayers, hepatocyte sandwich cultures, three-dimensional (3D) hepatocyte spheroids, and precision-cut liver slices), using the same tool compounds, viability end points, and culture time points. Having established the improved prediction potential of the 3D hepatocyte spheroid model, we describe implementing this model into an industrial screening setting, where the challenge was matching the complexity of the culture system with the scale and throughput required. Following further qualification and miniaturization into a 384-well, high-throughput screening format, data was generated on 199 compounds. This clearly demonstrated the ability to capture a greater number of severe hepatotoxins versus the current routine 2D HepG2 monolayer assay while continuing to flag no false-positive compounds. The industrialization and miniaturization of the 3D hepatocyte spheroid complex in vitro model demonstrates a significant step toward reducing drug attrition and improving the quality and safety of drugs, while retaining the flexibility for future improvements, and has replaced the routine use of the 2D HepG2 monolayer assay at GlaxoSmithKline.

Highly fluorinated candidates containing anticancer pharmacophores like thiosemicarbazone (5a-e) and its cyclic analogues hydrazineylidenethiazolidine (6a-e), 2-aminothiadiazole (7a-e), and 2-hydrazineylidenethiazolidin-4-one (8a-e) were synthesized, and their cytotoxic activity was assayed against 60 tumor cell lines. Compounds 6c, 7b, and 8b displayed the most potent activity with lower toxic effects on MCF-10a. In vitro phosphatidylinositol 3-kinase (PI3K) enzyme inhibition was performed. Compound 6c displayed half-maximal inhibitory concentration (IC50, μM) values of 5.8, 2.3, and 7.9; compound 7b displayed IC50 values of 19.4, 30.7, and 73.7; and compound 8b displayed IC50 values of 77.5, 53.5, and 121.3 for PI3Kα, β, and δ, respectively. Moreover, cell cycle progression caused cell cycle arrest at the S phase for compounds 6c and 8b and at G1/S for compound 7b, while apoptosis was induced. In silico studies; molecular docking; physicochemical parameters; and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis were performed. The results showed that compound 6c is the most potent one with a selectivity index (SI) of 39 and is considered as a latent lead for further optimization of anticancer agents.

Since severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific T cells have been found to play essential roles in host immune protection and pathology in patients with coronavirus disease 2019 (COVID-19), this study focused on the functional validation of T cell epitopes and the development of vaccines that induce specific T cell responses. A total of 120 CD8+T cell epitopes from the E, M, N, S, and RdRp proteins were functionally validated. Among these, 110, 15, 6, 14, and 12 epitopes were highly homologous with SARS-CoV, OC43, NL63, HKU1, and 229E, respectively; in addition, four epitopes from the S protein displayed one amino acid that was distinct from the current SARS-CoV-2 variants. Then, 31 epitopes restricted by the HLA-A2 molecule were used to generate peptide cocktail vaccines in combination with Poly(I:C), R848 or poly (lactic-co-glycolic acid) nanoparticles, and these vaccines elicited robust and specific CD8+T cell responses in HLA-A2/DR1 transgenic mice as well as wild-type mice. In contrast to previous research, this study established a modified DC-peptide-PBL cell coculture system using healthy donor PBMCs to validate the in silico predicted epitopes, provided an epitope library restricted by nine of the most prevalent HLA-A allotypes covering broad Asian populations, and identified the HLA-A restrictions of these validated epitopes using competitive peptide binding experiments with HMy2.CIR cell lines expressing the indicated HLA-A allotype, which initially confirmed the in vivo feasibility of 9- or 10-mer peptide cocktail vaccines against SARS-CoV-2. These data will facilitate the design and development of vaccines that induce antiviral CD8+T cell responses in COVID-19 patients.

Background: Diabetes mellitus is a serious global health issue, currently affecting 425 million people and is set to affect over 690 million people by 2045. It is a chronic disease characterized by hyperglycemia due to relative or absolute insulin hormone deficiency. Dipeptidyl peptidase- IV (DPP-IV) inhibitors are hypoglycemic agents augmenting the action of the incretin hormones that stimulate insulin secretion from the pancreatic beta cells. Objectives: In this study, synthesis and biological evaluation of seven piperazine derivatives 3a-g was carried out. Methods: The synthesized molecules were characterized using proton-nuclear magnetic resonance, carbon-nuclear magnetic resonance, infrared spectroscopy and mass spectrometry. Results: In vitro biological evaluation study showed comparable DPP-IV inhibitory activity for the targeted compounds ranging from 19%-30% at 100 μM concentration. Furthermore, the in vivo hypoglycemic activity of 3d was evaluated using streptozotocin-induced diabetic mice. It was found that compound 3d significantly decreased the blood glucose level when the diabetic group treated with 3d was compared to the control diabetic group. Quantum–Polarized Ligand Docking (QPLD) studies demonstrate that 3a-g fit the binding site of DPP-IV enzyme and form H-bonding with the backbones of R125, E205, E206, K554, W629, Y631, Y662, R669, and Y752. Conclusion: Piperazine derivatives were successfully found to be new scaffolds as potential DPP-IV inhibitors.