Calcium Transient Assays for Compound Screening with Human iPSC-derived Cardiomyocytes: Evaluating New Tools

Calcium (Ca2+) plays a central role in regulating many biological processes in the cell from muscle contraction to neurotransmitter release. The need for reliable fluorescent calcium indicator dyes is of vast importance for studying many aspects of cell biology as well as screening compounds using phenotypic high throughput assays. We have assessed two of the latest generation of calcium indicator dyes, FLIPR Calcium 6 and Cal-520 AM for studying calcium transients (CaTs) in induced pluripotent stem cell (iPSC) -derived human cardiomyocytes. FLIPR Calcium 6 and Cal-520 dyes both displayed robust CaTs with a high signal-to-noise ratio (SNR) and were non-toxic to the cells. The analysis showed that CaT amplitudes were stable between measurements, but CaT duration was more variable and tended to increase between reads. Two methods were compared for drug-screening hit-selection; difference in average (unstandardized) and standardized difference. The unstandardized difference was better for assessing CaT amplitude, whereas standardized difference was equal to or better for assessing CaT duration. In summary, FLIPR Calcium 6 and Cal-520 are suitable dyes for drug-screening using iPSC-derived human cardiomyocytes

BIS-cyclic guanidine compound compositions, methods of use and treatment thereof

The present disclosure provides compositions including a bis-cyclic guanidine compound, pharmaceutical compositions including a bis-cyclic guanidine compound, methods of treatment of a condition {e.g., bacterial infection) or disease, methods of treatment using compositions or pharmaceutical compositions, and the like

Effectiveness and Safety of Tranexamic Acid Use in Acute Traumatic Injury in the Prehospital and In-hospital Settings: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Background and Objectives: This systematic review and meta-analysis of randomized controlled trials (RCTs) aims to assess efficacy and safety of tranexamic acid (TXA) use in acute traumatic injuries. Methods: PubMed and Cochrane libraries were searched for relevant RCTs published between January 2011 and January 3, 2021. Cohen’s Q Test for heterogeneous effects was used to determine the appropriateness of fixed versus random effects models. Results: Twenty-two studies met inclusion criteria. Meta-analysis of relative risk of mortality between treatment and placebo groups in the in-hospital, and perioperative settings was not significant. However, the risk of mortality is significantly lower in the treatment versus placebo group when TXA was given as loading dose only. Ten of the 11 studies evaluating perioperative use of TXA included in systematic review found significantly lower blood loss in the treatment compared with placebo groups, but results of meta-analysis showed no significant difference. Results of meta-analysis indicate that the risk of venous thromboembolism (VTE) in the in-hospital treatment group is greater than that of the placebo. In subset analysis of studies using only a single loading dose, there were no significant differences in VTE. Conclusions: Systematic review supports TXA benefits are most evident when given shortly after injury and meta-analysis supports TXA reduces mortality as a single loading dose. Systematic review supports perioperative use of TXA when large volume blood loss is anticipated. Meta-results showed no significant difference in risk of thromboembolism in single-dose TXA treatment compared with placebo. These findings suggest that TXA is safe and effective for control of traumatic bleeding. Mini-abstract: This systematic review indicates that tranexamic acid (TXA) is most beneficial when given shortly postinjury and TXA given preoperatively reduces intraoperative blood loss. Meta-analysis reveals single-dose TXA was associated with improved mortality with no significant difference in VTE risk or complications. Cohesive guidelines are needed

Identification of Protein Palmitoylation Inhibitors from a Scaffold Ranking Library

The addition of palmitoyl moieties to proteins regulates their membrane targeting, subcellular localization, and stability. Dysregulation of the enzymes which catalyzed the palmitoyl addition and/or the substrates of these enzymes have been linked to cancer, cardiovascular, and neurological disorders, implying these enzymes and substrates are valid targets for pharmaceutical intervention. However, current chemical modulators of zDHHC PAT enzymes lack specificity and affinity, underscoring the need for screening campaigns to identify new specific, high affinity modulators. This report describes a mixture based screening approach to identify inhibitors of Erf2 activity. Erf2 is the Saccharomyces cerevisiae PAT responsible for catalyzing the palmitoylation of Ras2, an ortholog of the human Ras oncogene proteins. A chemical library developed by the Torrey Pines Institute for Molecular Studies consists of more than 30 million compounds designed around 68 molecular scaffolds that are systematically arranged into positional scanning and scaffold ranking formats. We have used this approach to identify and characterize several scaffold backbones and R-groups that reduce or eliminate the activity of Erf2 in vitro. Here, we present the analysis of one of the scaffold backbones, bis-cyclic piperazine. We identified compounds that inhibited Erf2 auto-palmitoylation activity using a fluorescence-based, coupled assay in a high throughput screening (HTS) format and validated the hits utilizing an orthogonal gel-based assay. Finally, we examined the effects of the compounds on cell growth in a yeast cell-based assay. Based on our results, we have identified specific, high affinity palmitoyl transferase inhibitors that will serve as a foundation for future compound design

Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis

Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNg ELISPOT assay is a well-standardized and validated method for the determination of functional IFNg-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNg ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNg ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning

The Mathematics of a Successful Deconvolution: A Quantitative Assessment of Mixture-Based Combinatorial Libraries Screened Against Two Formylpeptide Receptors

In the past 20 years, synthetic combinatorial methods have fundamentally advanced the ability to synthesize and screen large numbers of compounds for drug discovery and basic research. Mixture-based libraries and positional scanning deconvolution combine two approaches for the rapid identification of specific scaffolds and active ligands. Here we present a quantitative assessment of the screening of 32 positional scanning libraries in the identification of highly specific and selective ligands for two formylpeptide receptors. We also compare and contrast two mixture-based library approaches using a mathematical model to facilitate the selection of active scaffolds and libraries to be pursued for further evaluation. The flexibility demonstrated in the differently formatted mixture-based libraries allows for their screening in a wide range of assays

Antigen Discovery for the Identification of Vaccine Candidates and Biomarkers Using a T Cell Driven Approach in Combination with Positional Scanning Peptide Libraries

The prevention and treatment of infectious diseases is highly dependent on the availability of reliable diagnostic tests and protective or therapeutic vaccines. There also exists an urgent need to develop reliable biomarkers to monitor treatment success and to predict disease progression from asymptomatic to symptomatic disease in several disease scenarios. The elucidation of the disease-relevant antigens that elicit the protective immune responses is critical and required for the development of biomarkers, diagnostics, and vaccines. However; one of the main obstacles to the study of antigen specificity in human T cells is their low frequency in PBMC samples. To overcome this problem we have implemented strategies to generate memory T cell libraries and clones specific to the pathogen of interest. Due to the fact that memory T cells represent a repository of the human T cell response to infection, examination of their antigen specificity can efficiently reveal immunogenic and relevant antigens involved in the in vivo response to infection or vaccines. To examine the specificity of the memory T cells we use an unbiased collection of antigens together with an in silico analysis, namely positional scanning based biometrical analysis. Here we present a summary of our approach and ongoing work on the development of strategies for the culture of memory T cells from patients with Chagas disease. While most studies focus on the identification of vaccine candidates using preselected immunogenic proteins derived from animal models or by or bioinformatics prediction, here we present an innovative approach that directly examines the specificity of the memory response following infection or immunization in humans

Methodological approach to the ex vivo expansion and detection of T. cruzi-specific T cells from chronic Chagas disease patients

The discovery of T cell epitopes is essential not only for gaining knowledge about host response to infectious disease but also for the development of immune-intervention strategies. In Chagas disease, given the size and complexity of the Trypanosoma cruzi proteome and its interaction with the host’s immune system, the fine specificity of T cells has not been extensively studied yet, and this is particularly true for the CD4+ T cell compartment. The aim of the present work was to optimize a protocol for the generation of parasite-specific memory T cell lines, representative of their in vivo precursor populations and capable of responding to parasite antigens after long-term culture. Accordingly, peripheral blood mononuclear cells (PBMC) from both chronic asymptomatic and cardiac patients, and from non-infected individuals, underwent different in vitro culture and stimulation conditions. Subsequently, cells were tested for their capacity to respond against T. cruzi lysate by measuring [3H]-thymidine incorporation and interferon-γ and GM-CSF secretion. Results allowed us to adjust initial T. cruzi lysate incubation time as well as the number of expansions with phytohemagglutinin (PHA) and irradiated allogeneic PBMC prior to specificity evaluation. Moreover, our data demonstrated that parasite specific T cells displayed a clear and strong activation by using T. cruzi lysate pulsed, Epstein-Barr virus (EBV)-transformed human B lymphocytes (B-LCL), as autologous antigen presenting cells. Under these culture conditions, we generated a clone from an asymptomatic patient’s memory CD4+ T cells which responded against epimastigote and trypomastigote protein lysate. Our results describe a culture method for isolating T. cruzispecific T cell clones from patients with Chagas disease, which enable the acquisition of information on functionality and specificity of individual T cells

Discovery of an enzyme and substrate selective inhibitor of ADAM10 using an exosite-binding glycosylated substrate

ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive breast cancer in response to trastuzumab. The majority of ADAM10 and ADAM17 inhibitor development has been focused on the discovery of compounds that bind the active site zinc, however, in recent years, there has been a shift from active site to secondary substrate binding site (exosite) inhibitor discovery in order to identify non-zinc-binding molecules. In the present work a glycosylated, exosite-binding substrate of ADAM10 and ADAM17 was utilized to screen 370,276 compounds from the MLPCN collection. As a result of this uHTS effort, a selective, time-dependent, non-zinc-binding inhibitor of ADAM10 with Ki = 883 nM was discovered. This compound exhibited low cell toxicity and was able to selectively inhibit shedding of known ADAM10 substrates in several cell-based models. We hypothesize that differential glycosylation of these cognate substrates is the source of selectivity of our novel inhibitor. The data indicate that this novel inhibitor can be used as an in vitro and, potentially, in vivo, probe of ADAM10 activity. Additionally, results of the present and prior studies strongly suggest that glycosylated substrate are applicable as screening agents for discovery of selective ADAM probes and therapeutics

Identification of Tetrapeptides from a Mixture Based Positional Scanning Library That Can Restore nM Full Agonist Function of the L106P, I69T, I102S, A219V, C271Y, and C271R Human Melanocortin-4 Polymorphic Receptors (hMC4Rs)

Human obesity has been linked to genetic factors and single nucleotide polymorphisms (SNPs). Melanocortin-4 receptor (MC4R) SNPs have been associated with up to 6% frequency in morbidly obese children and adults. A potential therapy for individuals possessing such genetic modifications is the identification of molecules that can restore proper receptor signaling and function. These compounds could serve as personalized medications improving quality of life issues as well as alleviating diseases symptoms associated with obesity including type 2 diabetes. Several hMC4 SNP receptors have been pharmacologically characterized in vitro to have a decreased, or a lack of response, to endogenous agonists such as α-, β-, and γ2-melanocyte stimulating hormones (MSH) and adrenocorticotropin hormone (ACTH). Herein we report the use of a mixture based positional scanning combinatorial tetrapeptide library to discover molecules with nM full agonist potency and efficacy to the L106P, I69T, I102S, A219V, C271Y, and C271R hMC4Rs. The most potent compounds at all these hMC4R SNPs include Ac-His-(pI)DPhe-Tic-(pNO2)DPhe-NH2, Ac-His-(pCl)DPhe-Tic-(pNO2)DPhe-NH2, Ac-His-(pCl)DPhe-Arg-(pI)Phe-NH2, and Ac-Arg-(pCl)DPhe-Tic-(pNO2)DPhe-NH2, revealing new ligand pharmacophore models for melanocortin receptor drug design strategies