Validation Of Assay Research Articles

Liquiritin exerts psoriasis therapy and prevention by regulating the YY1/RBP3 axis

BackgroundPsoriasis (PSO) poses a global health threat. The current research challenge in PSO is relapse. Liquiritin (LIQ), a major active compound from Glycyrrhiza inflata Batalin, has multiple pharmacological properties, including anti-inflammatory and anti-proliferative. Nonetheless, the precise mechanisms underlying LIQ's therapeutic actions in PSO and prevention abilities remain elusive. PurposeThe present study aimed to delve into the potential to treat and prevent PSO and the mechanism of LIQ. MethodsThe anti-inflammatory and anti-proliferative effects of LIQ were studied in vitro with the HaCaT cell line. Then, Transcriptional analysis and bioinformatic analysis were used to determine the internal associations of the target set. Subsequently, functional experiment, luciferase report assay, ChIP-PCR, and immunohistochemical validation of clinical samples were performed to investigate the mechanism of LIQ. Finally, the anti-psoriatic effects and prevention abilities of LIQ were verified in vivo with imiquimod (IMQ)-induced PSO-like mouse models. ResultsHere, we identified differentially expressed genes in LIQ-stimulated HaCaT cells and Retinol-Binding Protein 3 (RBP3) as the core target, whereas YY1 was a predicted upstream transcription factor of RBP3. The YY1/RBP3 axis was obviously altered after administering LIQ at optimal doses of 20 μM in vitro and 100 µg/ml in vivo. LIQ can significantly inhibit the progression of PSO in vivo. Notably, LIQ also prevented the relapse of psoriatic lesions induced by the second round of low-dose IMQ. Mechanistically, we observed that LIQ could increase the promotion of YY1 for RBP3 by enhancing the binding affinity between them. ConclusionThese findings revealed that the YY1/RBP3 axis is a potential psoriatic target, and LIQ is a promising and innovative therapeutic candidate for the treatment and prevention of PSO.

CHD4 R975H mutant activates tumorigenic pathways and promotes stemness and M2-like macrophage polarization in endometrial cancer

Endometrial cancer (EC), one of the most prevalent carcinomas in females, is associated with increasing mortality. We identified the CHD4 R975H mutation as a high-frequency occurrence in EC patients through a comprehensive survey of EC databases. Computational predictions suggest that this mutation profoundly impacts the structural and functional integrity of CHD4. Functional assays revealed that the CHD4 R975H mutation enhances EC cell invasion, proliferation, and colony formation, promoting a cancer stem cell (CSC)-like phenotype. RNA-seq analysis of cells expressing CHD4 R975H mutant revealed a transcriptomic landscape marked by the activation of several cancer-promoting signaling pathways, including TNF-α signaling via NF-κB, KRAS, P53, mTOR, TGF-β, EGFR, Myc and growth factor signaling. Validation assays confirmed the activation of these pathways, further demonstrating that CHD4 R975H mutation induces stemness in EC cells and M2-like polarization of tumor-associated macrophages (TAMs). Our study elucidated the oncogenic role of CHD4 R975H mutation, highlighting its dual impact on facilitating cancer stemness and transforming TAMs into an immunosuppressive subtype. These findings contribute valuable insights into the molecular mechanisms driving EC progression and open avenues for targeted therapeutic interventions.

Transcriptomic Analysis of Vitrified-Warmed vs. Fresh Mouse Blastocysts: Cryo-Induced Physiological Mechanisms and Implantation Impact.

Blastocyst vitrification has significantly improved embryo transfer methods, leading to higher implantation success rates and better pregnancy outcomes in subsequent frozen embryo transfer cycles. This study aimed to simulate the transcriptional changes caused by vitrifying human blastocysts using mouse blastocysts as a model and to further investigate these changes' effects. Utilizing a human vitrification protocol, we implanted both vitrified and fresh embryos into mice. We observed the implantation success rates and performed transcriptomic analysis on the blastocysts. To validate the results from messenger RNA sequencing, we conducted reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) to measure the expression levels of specific genes. Based on mRNA profiling, we predicted the microRNAs responsible for the regulation and used qPCR basic microRNA assays for validation. Our observations revealed a higher implantation success rate for vitrified embryos than fresh embryos. Transcriptomic analysis showed that vitrified-warmed blastocysts exhibited differentially expressed genes (DEGs) primarily associated with thermogenesis, chemical carcinogenesis-reactive oxygen species, oxidative phosphorylation, immune response, and MAPK-related signaling pathways. RT-qPCR confirmed increased expression of genes such as Cdk6 and Nfat2, and decreased expression of genes such as Dkk3 and Mapk10. Additionally, gene-microRNA interaction predictions and microRNA expression analysis identified twelve microRNAs with expression patterns consistent with the predicted results, suggesting potential roles in uterine epithelial cell adhesion, trophectoderm development, invasive capacity, and immune responses. Our findings suggest that vitrification induces transcriptomic changes in mouse blastocysts, and even small changes in gene expression can enhance implantation success. These results highlight the importance of understanding the molecular mechanisms underlying vitrification to optimize embryo transfer techniques and improve pregnancy outcomes.

Development and validation of a rapid loop-mediated isothermal amplification assay for the detection of Chrysomyxa and characterization of Chrysomyxa woroninii overwintering on Picea in China

Chrysomyxa rusts cause significant damage to spruce in both natural forests and plantations. Particularly, Three Chrysomyxa species, Chrysomyxa deformans, Chrysomyxa qilianensis, and Chrysomyxa rhododendri, listed as National Forest Dangerous Pests in China, have severely affected many economically and ecologically important spruce native species in China. Also, Chrysomyxa arctostaphyli, an important plant quarantine fungus, causes a damaging broom rust disease on spruce. Therefore, rapid, and efficient detection tools are urgently needed for proper rust disease detection and management. In this study, a sensitive, genus-specific loop-mediated isothermal amplification (LAMP) assay targeting the ITS-28S rRNA region was developed to detect the presence of Chrysomyxa in spruce needle and bud samples. After optimization and validation, the LAMP assay was found to be sensitive to detect as low as 5.2 fg/µL DNA, making it suitable for rapid on-site testing for rust infection. The assay was also specific to Chrysomyxa species, with no positive signals from other rust genus/species. The application of LAMP in the early detection of rust infections in spruce needles and buds was investigated, and spatial colonization profiles as well as the means of overwintering of Chrysomyxa woroninii in infected buds and branches were verified using the LAMP assay. This LAMP detection method will facilitate further studies on the characteristics of the life cycle and inoculation of other systemic rusts.

Multiplex Real-Time Polymerase Chain Reaction Assay To Detect Acanthamoeba spp., Vermamoeba vermiformis, Naegleria fowleri, and Balamuthia mandrillaris in Different Water Sources.

Free-living amoebae (FLA) are widely distributed in the environment. Among these, Acanthamoeba spp., Naegleria fowleri, Balamuthia mandrillaris, and Vermamoeba vermiformis have been reported as human pathogens with health effects ranging from lethal encephalitis to different epithelial disorders. Despite this, FLA still present many diagnostic challenges. The aim of this study was to develop a rapid and efficient multiplex real-time quantitative polymerase chain reaction (qPCR) to simultaneously detect Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis in different water sources. For the validation of the qPCR assay, 38 samples (19 tap water and 19 stagnant water sources) were analyzed. The qPCR assay accurately identified the four types of FLA with no cross-reactivity. Considering water samples with results subsequently confirmed by conventional PCR, the multiplex qPCR assay detected 18/38 (47.4%) positive samples (Acanthamoeba spp. in 44.7% and V. vermiformis in 31.6%) and growth in nonnutritive agar (NNA) cultures identified 7/38 (18.4%) positive samples. Of the tap water samples analyzed, 26.3% of samples positive for FLA were detected by growth in NNA culture whereas 31.6% were identified by qPCR. In addition, FLA were detected in 2/19 stagnant water samples (10.5%) by growth in NNA culture and in 12/19 stagnant water samples (63.2%) by qPCR. Neither N. fowleri nor B. mandrillaris was detected in the water samples analyzed. In conclusion, the qPCR developed showed its potential as a rapid tool for detection of Acanthamoeba spp., N. fowleri, B. mandrillaris, and V. vermiformis. Moreover, FLA species were detected in half of the water sources evaluated, suggesting the importance of the surveillance of these potential infectious agents.

BD Vaginal Panel assay results on the high-throughput BD COR system compared to the BD MAX system.

Molecular-based assays demonstrate excellent sensitivity for the detection of vaginitis causes. Here, the high-throughput BD Vaginal Panel for BD COR System (VP-COR) performance was compared to that of the predicate, BD MAX Vaginal Panel for BD MAX System (VP-MAX). Clinical or contrived samples were used to determine the agreement between VP-COR and VP-MAX. Acceptance criteria for VP-COR agreement were as follows: bacterial vaginosis (BV) required a positive percent agreement (PPA) point estimate of ≥95% and a negative percent agreement (NPA) point estimate of ≥98%; Candida group, Candida glabrata, Candida krusei, and Trichomonas vaginalis (TV) required a PPA and NPA point estimate of ≥95% [with lower bound of 95% confidence interval (95% CI) ≥90%]. PPA was 99.5% (95% CI: 97.5-100) and 97.9% (95% CI: 96.5-98.8) for BV contrived (n = 516) and BV clinical (n = 1,050) specimens, respectively. For the Candida group (clinical; n = 724), C. glabrata (contrived; n = 544), C. krusei (contrived; n = 522), and TV (clinical; n = 702), PPA was 99.4% (95% CI: 98.0-99.9), 100% (95% CI: 97.9-100), 100% (95% CI: 97.6-100), and 99.7% (95% CI: 98.3-100), respectively; the lowest lower bound CI value was 97.6%. NPA was >95% for BV contrived and BV clinical specimens. For the Candida group, C. glabrata, C. krusei, and TV, NPA was ≥98.9%; the lowest lower bound CI value was 97.3%. These results demonstrate the equivalent performance of the VP-COR assay when compared to VP-MAX.IMPORTANCEVaginitis is common among women of reproductive age, resulting in around 10 million office visits a year. Diagnosis is often difficult due to its multiple causes-including bacterial vaginosis, vulvovaginal candidiasis, and trichomoniasis-as well as variation in symptom presentation. Typically, cases are identified with a combination of symptomology, medical history, physical examination, and office- or laboratory-based testing. These traditional techniques involve subjective elements and demonstrate varying sensitivity and specificity. Inaccurate or delayed diagnosis leads to continued symptoms, repeat visits, inappropriate treatment, and unnecessary costs. Alternatively, the use of molecular-based assays increases sensitivity for the detection of vaginitis causes. With the validation of the vaginal panel molecular assay on COR (a high-throughput platform), a workflow can be streamlined in high-demand laboratories while providing high sensitivity for vaginitis detection.

Deciphering the interplay of HPV infection, MHC-II expression, and CXCL13+ CD4+ T cell activation in oropharyngeal cancer: implications for immunotherapy

BackgroundHuman papillomavirus (HPV) infection has become an important etiological driver of oropharyngeal squamous cell carcinoma (OPSCC), leading to unique tumor characteristics. However, the interplay between HPV-associated tumor cells and tumor microenvironment (TME) remains an enigma.MethodsWe performed a single-cell RNA-sequencing (scRNA-seq) on HPV-positive (HPV+) and HPV-negative (HPV‒) OPSCC tumors, each for three samples, and one normal tonsil tissue. Ex vivo validation assays including immunofluorescence staining, cell line co-culture, and flow cytometry analysis were used to test specific subtypes of HPV+ tumor cells and their communications with T cells.ResultsThrough a comprehensive single-cell transcriptome analysis, we uncover the distinct transcriptional signatures between HPV+ and HPV‒ OPSCC. Specifically, HPV+ OPSCC tumor cells manifest an enhanced interferon response and elevated expression of the major histocompatibility complex II (MHC-II), potentially bolstering tumor recognition and immune response. Furthermore, we identify a CXCL13+CD4+ T cell subset that exhibits dual features of both follicular and pro-inflammatory helper T cells. Noteworthily, HPV+ OPSCC tumor cells embrace extensive intercellular communications with CXCL13+CD4+ T cells. Interaction with HPV+ OPSCC tumor cells amplifies CXCL13 and IFNγ release in CD4+T cells, fostering a pro-inflammatory TME. Additionally, HPV+ tumor cells expressing high MHC-II and CXCL13+CD4+ T cell prevalence are indicative of favorable overall survival rates in OPSCC patients.ConclusionsTogether, our study underscores a synergistic inflammatory immune response orchestrated by highly immunogenic tumor cells and CXCL13+CD4+ T cells in HPV+ OPSCC, offering useful insights into strategy development for patient stratification and effective immunotherapy in OPSCC.

A European Bioanalysis Forum recommendation for requiring a context-of-use statement for successful development and validation of biomarker assays.

The European Bioanalysis Forum, alongside key industry stakeholders, has been driving the discussions around the implementation of context-of use for biomarker assays to ensure that these assays are validated appropriately depending on their purpose. Insights into understanding why the implementation of context-of-use in assay strategies has also shown that the key stakeholder, or requester for the biomarker data, is responsible for providing the context-of-use statement for all biomarker assay requests. Expertsfrom across the industry haves repeatedly soughta cross-industry recommended format in which the context-of-use statement could be provided. In this manuscript, the European Bioanalysis Forum suggests a format for this.

Abstract We103: Integration of deep learning assisted high-content screening and deep tissue-phenotyping to identify cardioprotective compounds in dilated cardiomyopathy

With a prevalence of 4:10,000, dilated cardiomyopathy (DCM) is the most common hereditary heart disease, characterized by dilatation of one or both ventricles and impaired systolic and diastolic function. Numerous mutations have been identified to cause DCM. TITIN-truncating variants (TTNtv) are commonly observed in 20% of the genetic DCM cases. Progress in drug discovery for DCM has been hampered by the lack of robust high-throughput/high-content analysis pipelines of disease-specific cell and tissue platforms for deep structural and functional phenotyping. To address this challenge, we have developed a two-tiered human induced pluripotent stem cell (iPSC)-based screening approach, comprised of high-content cardiomyocyte imaging with artificial intelligence (AI)-driven image and data analyses as well as deep tissue phenotyping in engineered heart muscle (EHM) models. As a DCM use case, we have developed several isogenic human iPSC-lines with and without a previously reported and pathologically relevant A-band-TTNtv mutation (c.70692_70693insAT; p.T23565SfsX5; Gerull et al. 11788824, Gramlich et al. 25759365, Fomin et al. 34731013) Using fixed weights from a convolutional deep neural network trained on ImageNet, we generated unbiased deep embeddings from each 2D cell model and applied these to train machine learning (ML) models to detect morphological disease phenotypes. Our ML model is able to confidently separate healthy controls from TTNtv cells with high accuracy (>93%), specificity (WT >79%) across different batches/plate layouts and generate concentration-response curves, demonstrating platform robustness and sensitivity. Building upon this work, we will present our latest efforts applying the platform technology for a chemogenomic screening campaign to identify novel cardioprotective drugs using a library of 7,100 bioactive compounds and validation assays, including measuring functional changes in EHMs. The phenotypic profiling platform, as presented here, can be readily adapted to other disease-relevant cell types and pathologies. Our results demonstrate the power of combining iPSC-CMs with cytological profiling and deep learning as well as tissue-level phenotyping to accelerate drug discovery for patients with DCM.

Performance Characteristics of Next-Generation Sequencing–Based Engraftment Monitoring and Microchimerism Detection in Allogeneic Hematopoietic Cell Transplantation: A Practical Approach for Clinical Assay Validation

Chimerism analysis by next-generation sequencing (NGS) is an emerging method for engraftment monitoring after allogeneic hematopoietic cell transplantation. A high-sensitivity method is required for the detection of microchimerism (<1% chimerism), which may have clinical utility in early relapse detection, allograft monitoring in organ transplantation, and other allogeneic cellular therapies (such as microtransplantations). As more clinical laboratories adopt this method, a thorough assessment of performance is needed. This study evaluated one such NGS-based assay that uses both single-nucleotide polymorphisms and insertions/deletions as genetic markers. An assessment of accuracy, linearity, sensitivity, and reproducibility was performed. Analytical sensitivity was 0.2% donor for single donor and 0.5% donors for double donors. The assay showed a high degree of reproducibility over a full range of chimerism. Comparison to short-tandem-repeat (STR) PCR showed high concordance; yet <5% chimerism was consistently detected by NGS, but not by STR-PCR. Comparison to real-time quantitative PCR showedhigh concordance, but with lower correlation in the midrange (40% to 60% chimerism). Overall, the assay showed consistent performance with high sensitivity and accuracy compared with STR-PCR and real-time quantitative PCR across a full range of chimerism in the setting of single-donor and multidonor transplantations. In addition, criteria for quality metrics were established for sequencing performance and data analysis and considerations made for clinical laboratory validation of NGS-based chimerism assay andanalysis software.

Validation of a triple-color pseudovirion-based neutralization assay for immunogenicity assessment of a 14-valent recombinant human papillomavirus vaccine.

Validation of bioanalytical methods is crucial, especially in the pharmaceutical industry, to determine their suitability for specific purposes and the accuracy of analytical results. The pseudovirion-based neutralization assay (PBNA) is considered the gold standard for detecting and quantifying neutralizing antibodies against human papillomavirus in vaccine development for disease prevention. This paper introduces an improved triple-color PBNA method, capable of simultaneous detection of two or three human papillomavirus (HPVtypes for use in the development of a 14-valent HPV vaccine candidate. The primary objective was to comprehensively validate the triple-color PBNA method for general vaccine immunogenicity assays. Results show that the method has good specificity, accuracy, precision, linearity, robustness, and applicability. This innovative triple-color PBNA offers an improved approach for large-scale immunogenicity assessment in vaccine development. This study lays a solid foundation that can serve as a guiding paradigm for assessing vaccine responses in preclinical and clinical phases, providing valuable insights to the field.

A Data Driven Strategy and Case Study for Implementation of Singlicate Analysis in Ligand Binding Assays Used for PK Quantitation.

Duplicate analysis has been a conventional practice in the industry for ligand-binding assays (LBA), particularly for plate-based platforms like Enzyme-linked immunosorbent assay (ELISA) and Meso Scale Discovery (MSD) assays. Recent whitepapers and guidance have opened a door to exploring the implementation of single-well (singlicate) analysis approach for LBAs. Although the bioanalytical industry has actively investigated the suitability of singlicate analysis, applications in supporting regulated LBA bioanalysis are limited. The primary reason for this limitation is the absence of appropriate strategy to facilitate the transition from duplicate to singlicate analysis. In this paper we present the first case study with our data-driven approach to implement singlicate analysis in a clinical pharmacokinetics (PK) plate based LBA assay with ISR data. The central aspect of this strategy is a head-to-head comparison with Precision and Accuracy assessment in both duplicate and singlicate formats as the initial stage of assay validation. Subsequently, statistical analysis is conducted to evaluate method variability in both precision and accuracy. The results of our study indicated that there was no impactful difference between duplicate vs singlicate, affirming the suitability of singlicate analysis for the remaining steps of PK assay validation. The validation results obtained through singlicate analysis demonstrated acceptable assay performance characteristics across all validation parameters, aligning with regulatory guidance. The validated PK assay in singlicate has been employed to support a Phase I study. The appropriateness of singlicate analyses is further supported by initial Incurred Sample Reanalysis (ISR) data in which 90.1% of ISR samples fall within the acceptable criteria.

HMGA1 promotes the progression of esophageal squamous cell carcinoma by elevating TKT-mediated upregulation of pentose phosphate pathway

Esophageal squamous cell carcinoma (ESCC) possesses a poor prognosis and treatment outcome. Dysregulated metabolism contributes to unrestricted growth of multiple cancers. However, abnormal metabolism, such as highly activated pentose phosphate pathway (PPP) in the progression of ESCC remains largely unknown. Herein, we report that high-mobility group AT-hook 1 (HMGA1), a structural transcriptional factor involved in chromatin remodeling, promoted the development of ESCC by upregulating the PPP. We found that HMGA1 was highly expressed in ESCC. Elevated HMGA1 promoted the malignant phenotype of ESCC cells. Conditional knockout of HMGA1 markedly reduced 4-nitroquinoline-1-oxide (4NQO)-induced esophageal tumorigenesis in mice. Through the metabolomic analysis and the validation assay, we found that HMGA1 upregulated the non-oxidative PPP. With the transcriptome sequencing, we identified that HMGA1 upregulated the expression of transketolase (TKT), which catalyzes the reversible reaction in non-oxidative PPP to exchange metabolites with glycolytic pathway. HMGA1 knockdown suppressed the PPP by downregulating TKT, resulting in the reduction of nucleotides in ESCC cells. Overexpression of HMGA1 upregulated PPP and promoted the survival of ESCC cells by activating TKT. We further characterized that HMGA1 promoted the transcription of TKT by interacting with and enhancing the binding of transcription factor SP1 to the promoter of TKT. Therapeutics targeting TKT with an inhibitor, oxythiamine, reduced HMGA1-induced ESCC cell proliferation and tumor growth. Together, in this study, we identified a new role of HMGA1 in ESCCs by upregulating TKT-mediated activation of PPP. Our results provided a new insight into the role of HMGA1/TKT/PPP in ESCC tumorigenesis and targeted therapy.

Assay Development and Validation for Innovative Antiviral Development Targeting the N-Terminal Autoprocessing of SARS-CoV-2 Main Protease Precursors.

The SARS-CoV-2 main protease (Mpro) is initially synthesized as part of polyprotein precursors that undergo autoproteolysis to release the free mature Mpro. To investigate the autoprocessing mechanism in transfected mammalian cells, we examined several fusion precursors, with the mature SARS-CoV-2 Mpro along with the flanking amino acids (to keep the native substrate sequences) sandwiched between different tags. Our analyses revealed differential proteolysis kinetics at the N- and C-terminal cleavage sites. Particularly, N-terminal processing is differentially influenced by various upstream fusion tags (GST, sGST, CD63, and Nsp4) and amino acid variations at the N-terminal P1 position, suggesting that precursor catalysis is flexible and subject to complex regulation. Mutating Q to E at the N-terminal P1 position altered both precursor catalysis and the properties of the released Mpro. Interestingly, the wild-type precursors exhibited different enzymatic activities compared to those of the released Mpro, displaying much lower susceptibility to known inhibitors targeting the mature form. These findings suggest the precursors as alternative targets for antiviral development. Accordingly, we developed and validated a high-throughput screening (HTS)-compatible platform for functional screening of compounds targeting either the N-terminal processing of the SARS-CoV-2 Mpro precursor autoprocessing or the released mature Mpro through different mechanisms of action.

Using Laponite to Deliver BMP‐2 for Bone Tissue Engineering – In Vitro, Chorioallantoic Membrane Assay and Murine Subcutaneous Model Validation

AbstractFracture non‐union occurs due to various factors, leading to the development of potentially substantial bone defects. While autograft and allograft are the current gold standards for non‐union fractures, challenges related to availability and immune rejection highlight the need for improved treatments. A strategy in bone tissue engineering is to harness growth factors to induce an effect on cells to change their phenotype, behavior and initiate signaling pathways which lead to increased matrix deposition and tissue formation. Bone morphogenetic protein‐2 (BMP‐2) is a potent osteogenic growth factor however, given its rapid clearance time in vivo, there is a specific therapeutic window for efficacy while avoiding potential deleterious side‐effects. It is demonstrated that a Laponite nanoclay coating on a 3D printable and bioresorbable poly(caprolactone) trimethacrylate‐based resin enables binding of BMP‐2, decreases the rate of release, enabling reduced concentrations to be used while enhancing osteoinduction in both in vitro and in vivo models.

Recommendations for Development and Validation of a Fit-For-Purpose Biomarker Assays Using Western Blotting; An-AAPS Sponsored Initiative to Harmonize Industry Practices.

Western blot (WB) assays are routinely used for detection and quantification of biomarkers. Although assay validation to measure biomarkers in complex matrices has become a mainstay process for ligand binding assays (LBA) and mass spectrometry (MS), no guidelines exist yet validate biomarker methods using WB techniques. In this cross-industry white paper, we outlined in detail the key steps for development and for validation of WB assays for protein biomarkers under different contexts of use (COU). In addition, we described how to determine the level of assay validation needed for biomarker assays using Western blotting. For simplicity, we described two paths of WB assay validation. The first path (Path 1) is for biomarkers being analyzed for exploratory research or for internal go- or no/go- decision making. The second path (Path 2) is for clinical decision making such as dose determination or drug response that need to be run in a regulated environment. This work is supported through AAPS Biomarkers and Precision Medicine subteam and represents AAPS members opinion.

Development and validation of an enzyme-linked immunosorbent assay for the quantification of a recombinant humanized anti-IL-4Rα monoclonal antibody CM310 in serum and its application to pharmacokinetic study in Sprague-Dawley Rats

CM310 is a recombinant humanized monoclonal antibody targeting Interleukin (IL)-4 receptor alpha (IL-4Rα). IL-4Rα blockade prevents IL-4 and IL-13 from binding to their receptor, thereby inhibiting downstream signaling pathways that drive Type 2 helper T-cell (Th2) inflammation. CM310 holds potential for treating Th2-related inflammatory diseases, such as asthma, atopic dermatitis and chronic sinusitis with nasal polyposis. In this study, a direct enzyme-linked immunosorbent assay (ELISA) was developed to measure the concentrations of CM310 in rat serum. Seven calibration standards (ranging from 25 to 1600 ng/mL) and three quality controls (70, 500 and 1250 ng/mL) were defined. The limit of detection (LOD), lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) were 13, 25 and 1600 ng/mL, respectively. The method exhibited excellent precision and accuracy and successfully applied to in vitro serum stability and pharmacokinetic (PK) studies. In conclusion, we have developed and validated a highly sensitive and selective method for measuring CM310 in Sprague-Dawley rats. The development and validation ELISA method met the acceptable criteria, which suggested that these can be applied to quantify CM310, as well as in PK studies.

Implementation of flow cytometry testing on rare matrix samples: Special considerations and best practices when the sample is unique or difficult to obtain.

The publication of Clinical and Laboratory Standards Institute's guideline H62 has provided the flow cytometry community with much-needed guidance on development and validation of flow cytometric assays (CLSI, 2021). It has also paved the way for additional exploration of certain topics requiring additional guidance. Flow cytometric analysis of rare matrices, or unique and/or less frequently encountered specimen types, is one such topic and is the focus of this manuscript. This document is the result of a collaboration subject matter experts from a diverse range of backgrounds and seeks to provide best practice consensus guidance regarding these types of specimens. Herein, we define rare matrix samples in the setting of flow cytometric analysis, address validation implications and challenges with these samples, and describe important considerations of using these samples in both clinical and research settings.

Identification of Potent ADCK3 Inhibitors through Structure-Based Virtual Screening.

ADCK3 is a member of the UbiB family of atypical protein kinases in humans, with homologues in archaea, bacteria, and eukaryotes. In lieu of protein kinase activity, ADCK3 plays a role in the biosynthesis of coenzyme Q10 (CoQ10), and inactivating mutations can cause a CoQ10 deficiency and ataxia. However, the exact functions of ADCK3 are still unclear, and small-molecule inhibitors could be useful as chemical probes to elucidate its molecular mechanisms. In this study, we applied structure-based virtual screening (VS) to discover a novel chemical series of ADCK3 inhibitors. Through extensive structural analysis of the active-site residues, we developed a pharmacophore model and applied it to a large-scale VS. Out of ∼170,000 compounds virtually screened, 800 top-ranking candidate compounds were selected and tested in both ADCK3 and p38 biochemical assays for hit validation. In total, 129 compounds were confirmed as ADCK3 inhibitors, and among them, 114 compounds are selective against p38, which was used as a counter-target. Molecular dynamics (MD) simulations were then conducted to predict the binding modes of the most potent compounds within the ADCK3 active site. Through metadynamics analysis, we successfully detected the key amino acid residues that govern intermolecular interactions. The findings provided in this study can serve as a promising starting point for drug development.

Discovery of Two Highly Selective Structurally Orthogonal Chemical Probes for Activin Receptor-like Kinases 1 and 2.

Activin receptor-like kinases 1-7 (ALK1-7) regulate a complex network of SMAD-independent as well as SMAD-dependent signaling pathways. One of the widely used inhibitors for functional investigations of these processes, in particular for bone morphogenetic protein (BMP) signaling, is LDN-193189. However, LDN-193189 has insufficient kinome-wide selectivity complicating its use in cellular target validation assays. Herein, we report the identification and comprehensive characterization of two chemically distinct highly selective inhibitors of ALK1 and ALK2, M4K2234 and MU1700, along with their negative controls. We show that both MU1700 and M4K2234 efficiently block the BMP pathway via selective in cellulo inhibition of ALK1/2 kinases and exhibit favorable in vivo profiles in mice. MU1700 is highly brain penetrant and shows remarkably high accumulation in the brain. These high-quality orthogonal chemical probes offer the selectivity required to become widely used tools for in vitro and in vivo investigation of BMP signaling.