Platform For Drug Development Research Articles

Production of Recombinant Gelonin Using an Automated Liquid Chromatography System.

Advances in recombinant DNA technology have opened up new possibilities of exploiting toxic proteins for therapeutic purposes. Bringing forth these protein toxins from the bench to the bedside strongly depends on the availability of production methods that are reproducible, scalable and comply with good manufacturing practice (GMP). The type I ribosome-inhibiting protein, gelonin, has great potential as an anticancer drug, but is sequestrated in endosomes and lysosomes. This can be overcome by combination with photochemical internalization (PCI), a method for endosomal drug release. The combination of gelonin-based drugs and PCI represents a tumor-targeted therapy with high precision and efficiency. The aim of this study was to produce recombinant gelonin (rGel) at high purity and quantity using an automated liquid chromatography system. The expression and purification process was documented as highly efficient (4.4 mg gelonin per litre induced culture) and reproducible with minimal loss of target protein (~50% overall yield compared to after initial immobilized metal affinity chromatography (IMAC)). The endotoxin level of 0.05–0.09 EU/mg was compatible with current standards for parenteral drug administration. The automated system provided a consistent output with minimal human intervention and close monitoring of each purification step enabled optimization of both yield and purity of the product. rGel was shown to have equivalent biological activity and cytotoxicity, both with and without PCI-mediated delivery, as rGelref produced without an automated system. This study presents a highly refined and automated manufacturing procedure for recombinant gelonin at a quantity and quality sufficient for preclinical evaluation. The methods established in this report are in compliance with high quality standards and compose a solid platform for preclinical development of gelonin-based drugs.

Abstract 1543: Development of a novel ECAD-EmGFP reporter line for pancreatic cancer MET study and drug discovery

Abstract World-wide, metastasis continues to be the leading cause of death in cancer patients1. Although epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) have been implicated in the progression of cancer metastasis and drug resistance, their mechanistic pathway and specific role in disease progression is not fully understood2. As such, the development of a reporter line that enables the real-time monitoring of the changing status of cells will not only aid in dissecting the EMT/MET pathway in the research field, but could also become a robust platform for new cancer drug development. E-cadherin, an adhesion protein expressed in epithelial cells, is upregulated in pancreatic cancer cells during MET and is associated with an increase in tight junctions and apico-basal polarity, as well as a change in morphology3,4. Here, we developed a novel PANC-1 ECAD-EmGFP reporter line using CRISPR/Cas9 genome-editing technology. In this cell line, the emerald green fluorescent protein (EmGFP) reporter was incorporated into the last exon of the endogenous E-cadherin gene, enabling real-time monitoring of MET status in live cells. The ECAD-EmGFP knock-in allele was confirmed at genomic, transcriptional, and translational levels. Functional data revealed that miRNA-200 treatment induced the increased expression of ECAD-EmGFP, and decreased expression of Snail, a transcription factor associated with mesenchymal traits. These changes in marker gene expression, as well as the decrease in invasive capacity upon induction, suggest that cells have undergone MET. This cell line is a valuable tool for dissecting the molecular mechanisms underlying EMT and MET and for evaluating or screening compounds targeting EMT and MET in pancreatic cancer. 1. Dizon DS, et al. Clinical Cancer Advances 2016: Annual Report on Progress Against Cancer from the American Society of Clinical Oncology. J Clin Oncol 34: 987-1011, 2016. 2. Nieto MA, et al. EMT: 2016. Cell 166: 21-45, 2016. 3. Polireddy K, et al, Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation. PLOS ONE 11(10): e0164811, 2016 4. Wong S, et al. E-cadherin: Its dysregulation in carcinogenesis and clinical implications. Critical Reviews in Oncology / Hematology 121: 11- 22, 2018 Citation Format: Diana Douglas, Sangeeta Kumari, Jarkko Huuskonen, Weiguo Shu. Development of a novel ECAD-EmGFP reporter line for pancreatic cancer MET study and drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1543.

Human trabecular meshwork cell behavior is influenced by collagen scaffold pore architecture and glycosaminoglycan composition.

Glaucoma is a degenerative eye disease in which damage to the optic nerve leads to a characteristic loss of vision. The primary risk factor for glaucoma is an increased intraocular pressure that is caused by an imbalance of aqueous humor generation and subsequent drainage through the trabecular meshwork (TM) drainage system. The small size, donor tissue limitations, and high complexity of the TM make it difficult to research the relationship between the TM cells and their immediate environment. Thus, a biomaterial-based approach may be more appropriate for research manipulations and in vitro drug development platforms. In this work, human TM (hTM) cells were cultured on various collagen scaffolds containing different glycosaminoglycans (GAGs) and different pore architectures to better understand how hTM cells respond to changes in their extracellular environment. Cellular response was measured by quantifying cellular proliferation and expression of an important extracellular matrix protein, fibronectin. The pore architecture of the scaffolds was altered using freeze-casting technique to make both large and small pores that were aligned or with a non-aligned random structure. The composition of the scaffolds was altered with the addition of chondroitin sulfate and/or hyaluronic acid. It was found that the hTM cells grown on large pore scaffolds proliferate more than those grown on small pores. There was an increase in the fibronectin expression with the incorporation of GAGs, and its morphology was changed by the underlying pore architecture. This work will help provide an insight into the behavior of hTM cells when introducing changes in their microenvironment.

Abstract PR11: Dual blockade of BRD4 and the ATR/WEE1 pathway exploits ARID1A loss in clear-cell ovarian cancers

Abstract Purpose: Clear-cell ovarian cancer (CCOC) is often resistant to standard chemotherapy and has limited treatment options in the advanced or recurrent setting. ARID1A is the most prevalent mutation, with approximately 50% of all CCOC harboring this mutation. ARID1A, a member of the SWI/SNF family, regulates transcription and has a major role in the repair of DNA lesions, directly facilitating DNA accessibility on the chromatin or indirectly by facilitating the functions of DNA repair proteins. We propose that CCOCs unique genomic alterations (e.g., ARID1A frameshift or nonsense mutations) will increase dependency on chromatin remodeling and DNA repair (e.g., ATR/CHK1/WEE1) pathways for survival. We hypothesize that combination of low-dose small-molecule inhibitors of the BET family (BRD4i) and the DNA damage repair pathway (ATR/CHK1/WEE1), will especially target ARID1A mutant cancer cells promoting mitotic catastrophe, apoptosis, and tumor regression, sparing normal cells. Method: We compared the sensitivity of BRD4i + ATRi/WEE1i combinations in ARID1A knockdown and knockout cells compared with isogenic parental lines. The effects on transcription, kinase signaling, cell cycle, apoptosis, and DNA damage were evaluated. RNAseq/ATACseq and Reverse Phase Protein Array (RPPA) were also performed. A CCOC preclinical drug development platform was established and BRD4i combinations were evaluated in ARID1A mutant and ARID1A wild-type (ARID1AMUT and ARID1AWT) PDX models. Results: Low-dose BRD4i in combination with DNA damage repair inhibitors, ATRi/WEE1i (BDR4i-ATRi or BRD4i-WEE1i), were synergistic in decreasing survival and colony formation with an increase in apoptosis in ARID1AMUT cells compared to ARID1AWT. BRD4i combinations caused a robust G1 arrest with a decrease pCDC6 levels along with an increase in DNA double-strand breaks in the ARID1AMUT or knockout cells. Combination BRD4i-ATRi showed significant tumor regression and increased overall survival compared to standard chemotherapy or monotherapy in several ARID1AMUT PDX models but not in an ARID1AWT PDX. Among BRD4i-DDRi combinations, BRD4i-ATRi is superior to BRD4i-WEE1i in terms of antitumor effect and drug tolerability. Conclusion: Our studies identify a novel drug combination targeting a genetic alteration (e.g., ARID1A) common in CCOC that is highly effective and tolerable. BRD4i in combination with ATRi or WEE1i was synergistic, decreasing survival, colony formation with an increase in DNA damage and apoptosis in ARID1AMUT models. Using our novel CCOC drug development PDX platform, we demonstrated that BRD4i-ATRi combination therapy is more effective than standard chemotherapy or monotherapy alone with acceptable toxicity in ARID1AMUTPDXs. This abstract is also being presented as Poster B24. Citation Format: Yasuto Kinose, Hyoung Kim, Haineng Xu, Sergey Medvedev, Erin George, Sarah Gitto, Margaret Whicker, Dorothy Hallberg, Lauren Schwartz, Gordon Mills, Victor E. Velculescu, Tian-Li Wang, Ronny Drapkin, Fiona Simpkins. Dual blockade of BRD4 and the ATR/WEE1 pathway exploits ARID1A loss in clear-cell ovarian cancers [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR11.

Multiplexed drug testing of tumor slices using a microfluidic platform

Current methods to assess the drug response of individual human cancers are often inaccurate, costly, or slow. Functional approaches that rapidly and directly assess the response of patient cancer tissue to drugs or small molecules offer a promising way to improve drug testing, and have the potential to identify the best therapy for individual patients. We developed a digitally manufactured microfluidic platform for multiplexed drug testing of intact cancer slice cultures, and demonstrate the use of this platform to evaluate drug responses in slice cultures from human glioma xenografts and patient tumor biopsies. This approach retains much of the tissue microenvironment and can provide results rapidly enough, within days of surgery, to guide the choice of effective initial therapies. Our results establish a useful preclinical platform for cancer drug testing and development with the potential to improve cancer personalized medicine.

Call for Special Issue Papers: Drug Delivery Research.

ASSAY and Drug Development TechnologiesVol. 18, No. 4 Calls for PapersFree AccessCall for Special Issue Papers: Drug Delivery ResearchDeadline for Manuscript Submission: August 1, 2020Guest Editor: Harsh ChauhaGuest Editor: Harsh ChauhaSpecial Editor of Drug Delivery Research, ASSAY and Drug Development TechnologiesSearch for more papers by this authorPublished Online:21 May 2020https://doi.org/10.1089/adt.2020.29094.cfp2AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail Advent of in silico screening techniques and high throughput technologies have resulted in synthesis of highly potent compounds with challenging physicochemical properties. Also, there are several issues related to the formulation of current marketed and repurposed drugs. For these challenging compounds, delivery systems are designed to enhance solubility/ stability, target specific sites in the human body, optimize drug therapy, improve PK-PD profile and reduce side/ adverse effects. The special issue focuses on recent advancement in the area of formulation and drug delivery technologies (solutions, semi-solids, nanoparticles, liposomes, emulsions, amorphous dispersions etc.) for challenging drugs.Special Issue Editor Dr. Harsh Chauhan will consider articles from the full breadth of research in the field from original research papers, reviews, and methods papers, to perspectives, profiles, commentaries, and exemplary front matter.Areas of interest include, but are not limited to: Advancement in novel drug delivery technologies.Formulation, characterization, in vitro/in vivo studies of nanoparticles.Solubilization and delivery of poorly soluble compounds.Targeted drug delivery systems.Advancement in topical/ transdermal drug delivery systems.Contributions will receive prompt and thorough peer review. Please refer to our Instructions for Authors at www.liebertpub.com/adt before submitting your manuscript for consideration. Authors are encouraged to vet ideas directly with Dr. Chauhan via e-mail (harshchauhan@creighton.edu) before submitting.ASSAY and Drug Development Technologies provides access to novel techniques and robust tools that enable critical advances in early-stage screening. This research published in the Journal leads to important therapeutics and platforms for drug discovery and development. This reputable peer-reviewed journal features original papers application-oriented technology reviews, topical issues on novel and burgeoning areas of research, and reports in methodology and technology application.Manuscript Submission Deadline: August 1, 2020Visit the Instructions for Authors:www.liebertpub.com/adtSubmit your paper for peer review online:https://mc.manuscriptcentral.com/adtFiguresReferencesRelatedDetails Volume 18Issue 4May 2020 InformationCopyright 2020, Mary Ann Liebert, Inc., publishersTo cite this article:Guest Editor: Harsh Chauha.Call for Special Issue Papers: Drug Delivery Research.ASSAY and Drug Development Technologies.May 2020.155-155.http://doi.org/10.1089/adt.2020.29094.cfp2Published in Volume: 18 Issue 4: May 21, 2020Online Ahead of Print:April 20, 2020PDF download

Molecular targeting of FATP4 transporter for oral delivery of therapeutic peptide.

Low oral bioavailability of peptide drugs has limited their application to parenteral administration, which suffers from poor patient compliance. Here, we show that molecular targeting of the FATP4 transporter is an effective approach to specifically transport long-chain fatty acid (LCFA)-conjugated peptides across the enterocytic membrane and, thus, enables oral delivery of drug peptides. We packaged LCFA-conjugated exendin-4 (LCFA-Ex4) into liposomes and coated with chitosan nanoparticles to form an orally deliverable Ex4 (OraEx4). OraEx4 protected LCFA-Ex4 from damage by the gastric fluid and released LCFA-Ex4 in the intestinal cavity, where LCFA-Ex4 was transported across the enterocyte membrane by the FAPT4 transporter. OraEx4 had a high bioavailability of 24.8% with respect to subcutaneous injection and exhibited a substantial hypoglycemic effect in murine models of diabetes mellitus. Thus, molecular targeting of the FATP4 transporter enhances oral absorption of therapeutic peptides and provides a platform for oral peptide drug development.

Vitis Vinifera L.: A new bioactive anticandidal agent to combat oral candidal carriage in Down syndrome patients.

Down syndrome (DS) patients have a high risk of opportunistic infections from Candida species. Therefore, the purpose of this research was to assess the prevalence of Candida species in oral cavity of DS patients and investigating of grape seed extract (GSE) as a potential anticandidal agent. Our study included fifty patients with DS and fifty healthy children. Oral tongue swabs were taken from both groups and isolated Candida species were identified. The anticandidal activity of GSE against isolated Candida was studied using agar well diffusion method. Scanning electron microscope (SEM) was used to determine the major alterations in Candida after GSE treatment. GSE was subjected to GC-MS analysis and HPLC analysis. Forty six samples showed growth of Candida in DS group, where as ten samples in control group. The most common pathogenic Candida isolated from DS patients was C.tropicalis (65.2%). GSE showed significant inhibitory activity against C.tropicalis (MIC 30 mg/ml). The SEM showed complete alterations in Candida cell morphology. GC-MS chromatogram of GSE revealed 30 compounds, the major one is (9, 12-Octadecadienoic acid) which confirmed by HPLC chromatogram with a peak at RT 20.311 min. GSE could be a promising anticandidal agent and it may form a primary platform for further toxicological assessment and development of new drugs.

3D optical/CT as a preclinical companion imaging platform for glioblastoma drug development

Multimodality 3D Optical Imaging (OI)/CT has the potential to play a major role in drug development for glioblastomas (GBM), as it is an accessible preclinical method. To demonstrate the potential of 3D OI/CT to visualize orthotopic GBM implantation, we labeled GBM cells with Cy7 and imaged their location using 3D OI/CT. To confirm the accuracy of the spatial localization and demonstrate the ability to image locoregionally delivered therapies, we labeled mouse albumin with Cy7 (Cy7ALB) and delivered it via locoregional infusion 1 mm or 3 mm into the brain and demonstrated correlation of signal between the 3D OI/CT and post necropsy brain slices. In addition, we demonstrated the potential of systemically delivered Cy7ALB contrast to detect blood–brain barrier (BBB) permeability caused by orthotopic GBMs using 3D OI/CT. We also tested the potential of 3D OI/CT to assess focal BBB permeability induced by high intensity focused ultrasound (HIFU), a methodology being used in clinical trials to noninvasively permeabilize the BBB for systemic therapeutic delivery to GBM. We demonstrated the ability of systemic Cy7ALB contrast together with 3D OI/CT to accurately assess real-time HIFU-induced BBB permeability, which correlated to post necropsy imaging of brains. Furthermore, we demonstrated that 3D OI/CT can also image the therapeutic distribution of a Cy7-labeled anti-PD-1 antibody, a prototype translational antibody therapy. We successfully imaged real-time antibody distribution after HIFU-induced BBB permeability, which correlated with post necropsy Cy7 signal and translational PET imaging after injection of [89Zr] anti-PD-1 antibody. Thus, we demonstrated the broad potential of using 3D OI/CT as an accessible preclinical tool to develop anti-GBM therapies.

The Pax6 master control gene initiates spontaneous retinal development via a self-organising Turing network.

ABSTRACTUnderstanding how complex organ systems are assembled from simple embryonic tissues is a major challenge. Across the animal kingdom a great diversity of visual organs are initiated by a ‘master control gene’ called Pax6, which is both necessary and sufficient for eye development. Yet precisely how Pax6 achieves this deeply homologous function is poorly understood. Using the chick as a model organism, we show that vertebrate Pax6 interacts with a pair of morphogen-coding genes, Tgfb2 and Fst, to form a putative Turing network, which we have computationally modelled. Computer simulations suggest that this gene network is sufficient to spontaneously polarise the developing retina, establishing the first organisational axis of the eye and prefiguring its further development. Our findings reveal how retinal self-organisation may be initiated independently of the highly ordered tissue interactions that help to assemble the eye in vivo. These results help to explain how stem cell aggregates spontaneously self-organise into functional eye-cups in vitro. We anticipate these findings will help to underpin retinal organoid technology, which holds much promise as a platform for disease modelling, drug development and regenerative therapies.

Structural Insights into Electrophile Irritant Sensing by the Human TRPA1 Channel

Transient receptor potential channel subfamily A member 1 (TRPA1) is a Ca2+-permeable cation channel that serves as one of the primary sensors of environmental irritants and noxious substances. Many TRPA1 agonists are electrophiles that are recognized by TRPA1 via covalent bond modifications of specific cysteine residues located in the cytoplasmic domains. However, a mechanistic understanding of electrophile sensing by TRPA1 has been limited due to a lack of high-resolution structural information. Here, we present the cryoelectron microscopy (cryo-EM) structures of nanodisc-reconstituted ligand-free TRPA1 and TRPA1 in complex with the covalent agonists JT010 and BITC at 2.8, 2.9, and 3.1Å, respectively. Our structural and functional studies provide the molecular basis for electrophile recognition by the extraordinarily reactive C621 in TRPA1 and mechanistic insights into electrophile-dependent conformational changes in TRPA1. Thiswork also provides a platform for future drug development targeting TRPA1.

Thermosensitive Nanosystems Associated with Hyperthermia for Cancer Treatment.

Conventional chemotherapy regimens have limitations due to serious adverse effects. Targeted drug delivery systems to reduce systemic toxicity are a powerful drug development platform. Encapsulation of antitumor drug(s) in thermosensitive nanocarriers is an emerging approach with a promise to improve uptake and increase therapeutic efficacy, as they can be activated by hyperthermia selectively at the tumor site. In this review, we focus on thermosensitive nanosystems associated with hyperthermia for the treatment of cancer, in preclinical and clinical use.

Cardiomyocyte functional screening: interrogating comparative electrophysiology of high-throughput model cell systems.

Cardiac arrhythmias of both atrial and ventricular origin are an important feature of cardiovascular disease. Novel antiarrhythmic therapies are required to overcome current drug limitations related to effectiveness and pro-arrhythmia risk in some contexts. Cardiomyocyte culture models provide a high-throughput platform for screening antiarrhythmic compounds, but comparative information about electrophysiological properties of commonly used types of cardiomyocyte preparations is lacking. Standardization of cultured cardiomyocyte microelectrode array (MEA) experimentation is required for its application as a high-throughput platform for antiarrhythmic drug development. The aim of this study was to directly compare the electrophysiological properties and responses to isoproterenol of three commonly used cardiac cultures. Neonatal rat ventricular myocytes (NRVMs), immortalized atrial HL-1 cells, and custom-generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured on microelectrode arrays for 48-120 h. Extracellular field potentials were recorded, and conduction velocity was mapped in the presence/absence of the β-adrenoceptor agonist isoproterenol (1 µM). Field potential amplitude and conduction velocity were greatest in NRVMs and did not differ in cardiomyocytes isolated from male/female hearts. Both NRVMs and hiPSC-CMs exhibited longer field potential durations with rate dependence and were responsive to isoproterenol. In contrast, HL-1 cells exhibited slower conduction and shorter field potential durations and did not respond to 1 µM isoproterenol. This is the first study to compare the intrinsic electrophysiologic properties of cultured cardiomyocyte preparations commonly used for in vitro electrophysiology assessment. These findings offer important comparative data to inform methodological approaches in the use of MEA and other techniques relating to cardiomyocyte functional screening investigations of particular relevance to arrhythmogenesis.

Copper (II) complexes modified with water-soluble porphyrin and various small molecules ligand for DNA binding and potential selectivity antitumor agents

Many strategies have been used in treatment of cancer, but currently the chemotherapy is still the main force, unfortunately, general agents exhibit inevitable toxicity and inferior selectivity. Therefore, less toxic and high selectivity anticancer reagents have been extensively sought and developed. Here we report a series of Copper (II) complexes CuP1~CuP5 which have good biological activities. Due to the influence of the molecular size, CuP1 displayed the strongest bound with ct-DNA. All of the complexes show positive anticancer activities toward A549, H1975, HepG2 and T47D cancer cells. Additionally, flow cytometry experiments indicated that complexe CuP2 arrest cell cycles at S and G2 phase and mitochondrial membrane potential tests were also performed to explore the mechanism of action. Our findings disclosed that this type of complex represents a potent platform for development of metal anticancer drugs.

Targeting CYP4A attenuates hepatic steatosis in a novel multicellular organotypic liver model

BackgroundNon-alcoholic fatty liver disease (NAFLD) begins as simple hepatic steatosis, but further progress to chronic liver diseases results in severe liver damage and hepatic failure. However, therapeutic options are scarce due to the lack of reliable human in vitro liver models for understanding disease progression mechanisms and developing therapies.ResultsWe describe here a novel method for generating 3D hepatic spheroids using HepaRG cells, vascular endothelial cells, and mesenchymal stem cells cultured on a thick layer of soft matrix in a narrow conical tube; this method improved self-organization efficiency and functional competence. We further developed a 3D hepatic steatosis model with excess glucose and palmitate, accurately recapitulating steatosis phenotypes such as neutral lipid accumulation, enhanced expression of lipogenesis and gluconeogenesis markers, increased intracellular triglyceride content, and reduced glucose uptake. The expression and activity of cytochrome P450 4A (CYP4A), a hepatic glucose and lipid homeostasis enzyme, that is highly expressed in liver tissues from NAFLD patients, was induced in our in vitro steatosis model, and inhibiting CYP4A with the selective inhibitor HET0016 or a specific siRNA ameliorated steatosis-related pathology through reduced ER stress and improved insulin signaling.ConclusionsWe provide here a novel 3D human cell-based hepatic model that can be easily generated and reliably simulate hepatic steatosis pathology. We have experimentally validated its potential for target validation and drug evaluation by focusing on CYP4A, which may serve as a translational platform for drug development.

Engineered skeletal muscles for disease modeling and drug discovery

Skeletal muscle is the largest organ of human body with several important roles in everyday movement and metabolic homeostasis. The limited ability of small animal models of muscle disease to accurately predict drug efficacy and toxicity in humans has prompted the development in vitro models of human skeletal muscle that fatefully recapitulate cell and tissue level functions and drug responses. We first review methods for development of three-dimensional engineered muscle tissues and organ-on-a-chip microphysiological systems and discuss their potential utility in drug discovery research and development of new regenerative therapies. Furthermore, we describe strategies to increase the functional maturation of engineered muscle, and motivate the importance of incorporating multiple tissue types on the same chip to model organ cross-talk and generate more predictive drug development platforms. Finally, we review the ability of available in vitro systems to model diseases such as type II diabetes, Duchenne muscular dystrophy, Pompe disease, and dysferlinopathy.

Organ-on-e-chip: Three-dimensional self-rolled biosensor array for electrical interrogations of human electrogenic spheroids.

Cell-cell communication plays a pivotal role in coordination and function of biological systems. Three-dimensional (3D) spheroids provide venues to explore cellular communication for tissue development and drug discovery, as their 3D architecture mimics native in vivo microenvironments. Cellular electrophysiology is a prevalent signaling paradigm for studying electroactive cells. Currently, electrophysiological studies do not provide direct, multisite, simultaneous investigation of tissues in 3D. In this study, 3D self-rolled biosensor arrays (3D-SR-BAs) of either active field-effect transistors or passive microelectrodes were implemented to interface human cardiac spheroids in 3D. The arrays provided continuous and stable multiplexed recordings of field potentials with high sensitivity and spatiotemporal resolution, supported with simultaneous calcium imaging. Our approach enables electrophysiological investigation and monitoring of the complex signal transduction in 3D cellular assemblies toward an organ-on-an-electronic-chip (organ-on-e-chip) platform for tissue maturation investigations and development of drugs for disease treatment, such as arrhythmias.

Abstract 2083: Human normal and tumor colon organoids: a 3D high content screening platform for anticancer drug development

Abstract Background: Recent advances in establishing organoids from human specimens have enabled further development of advanced 3D cell culture models such as normal and tumor colon organoids derived from the same patient. These organoid cultures increase the scope for predicting treatment responses in patients, enabling the testing of cancer (immune)therapies, discriminating different drug responses and flagging off-target effects. Here we present a high throughput 3D human intestinal organoid culture platform combined with high content image-based analysis. This platform allows visualization and quantification of various treatment effects on healthy and diseased gut epithelium. Methods: Intestinal organoids, obtained from HUB Organoid Technology, are cultured in natural extracellular matrix scaffolds and show gene expression patterns, differentiation and functional characteristics that closely resemble the in vivo biology. To investigate the effect of standard-of-care (SoC) treatments like 5-FU, Oxaliplatin and Irinotecan and/or (in combination with) novel targeting antibodies or other small molecules on the function, formation and integrity of the gut epithelium, we set up the following intestinal organoid test systems: 1. Colorectal cancer organoids with a broad heterogeneity of mutations for sensitive and high throughput phenotypic screening that enable accurate compound profiling and 2. Human normal vs. tumor organoid pair assays for off-target effect studies. Results: High content 3D image analysis of the organoid models enables sensitive detection of treatment-induced and compound-specific morphological changes such as (inhibition of) growth, development, lumen formation, epithelial integrity and cell death. This enables distinction between mechanisms of action and determining compound synergy. The image-based measurements can be complemented with detection of secreted factors (e.g. cytokines, chemokines) or expressed genes in response to various therapeutic compounds. Conclusion: OcellO’s advanced 3D image analysis of in vitro cultured organoids represents a rapid and biologically relevant approach to test various candidate compounds (e.g. antibodies, antibody-drug conjugates and small molecules) that can treat intestinal disorders such as Inflammatory Bowel Disease and colon cancer. This organoid technology also allows identifying potential adverse effects that can occur during in-vivo studies. Therefore, our intestinal organoid screening platform represents a significant advance on conventional in vitro models and helps bridge the translational gap between in vivo studies. Note: This abstract was not presented at the meeting. Citation Format: Mariusz Madej, Jara Garcia Mateos, Kuan Yan, Lidia Daszkiewicz, Bram Herpers, Leo S. Price. Human normal and tumor colon organoids: a 3D high content screening platform for anticancer drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2083.

Abstract 294: ChemoINTEL: A high-throughput, multi-parametric compound screening platform for intelligent lead compound and therapeutic combination identification

Abstract Pierian Biosciences has developed the ChemoINTEL platform for high-throughput, semi-automated quantification of cellular responses to chemotherapeutics. ChemoINTEL relies on a microscopy-based process that captures and analyzes single cell behavior to reflect responses within a population. The platform is customizable to a variety of small molecule panels, either specific to certain tumor types or for titration screening of small molecules in development. Automated imaging of each well of a 384-well plate over time provides real-time kinetic response data under a variety of treatment conditions. Brightfield and fluorescent images are used to determine quantitative changes in morphology and molecular metrics reflecting induced apoptosis and cell death. Unique to ChemoINTEL as a drug screening platform is the quantification of intensity values, at the single cell level versus whole well fluorescence, for each fluorescent probe at each timepoint through a high-throughput analysis routine. These data are further processed through an internally developed algorithm to compare a treated population’s response relative to an untreated control and reports a sensitivity score. By combining different treatment conditions, the platform provides intelligent design of single agent or combination treatment approaches. By analyzing individual cells in the population, ChemoINTEL aids in better understanding response of tumor cell sub-populations to different chemotherapeutic agents. As a compound library screening tool or potential clinical diagnostic, Pierian Biosciences’ quality-controlled processes ensure all its equipment, reagents and processes follow ISO17025 guidelines to ensure the quality of all data generated. Each assay is internally controlled to include a standard cell line, whose response is monitored for accuracy of the reported results. In collaboration with several biorepositories and through the development of a standardized dissociation approach resulting in purification of viable primary tumor cells, the ChemoINTEL platform is also under development to assess chemotherapeutic response within patient populations. In conjunction with the ChemoINTEL platform, Pierian Biosciences has also developed a sophisticated multi-color flow cytometry platform (ImmunoINTEL) that when used in conjunction with ChemoINTEL provides information on the purity of isolated primary tumor cells prior to plating, as well as information on the cell populations present within the tumor microenvironment. These two platforms can be further complexed to evaluate immunotherapies in combination with front-line therapies. In summary, Pierian Biosciences has developed a high-throughput, multi-parametric platform for both drug development and diagnostic applications. Note: This abstract was not presented at the meeting. Citation Format: Kellye C. Kirkbride, Kevin J. Polach, Samantha J. Braxton, Megan D. Hoeksema, Dustin C. Rogers, Patricia Ladd-Ward, Durdica Vojnic Zelic, Santosh Putta, Matt Westfall, Norman Purvis. ChemoINTEL: A high-throughput, multi-parametric compound screening platform for intelligent lead compound and therapeutic combination identification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 294.

Atomistic Simulations of Membrane Ion Channel Conduction, Gating, and Modulation.

Membrane ion channels are the fundamental electrical components in the nervous system. Recent developments in X-ray crystallography and cryo-EM microscopy have revealed what these proteins look like in atomic detail but do not tell us how they function. Molecular dynamics simulations have progressed to the point that we can now simulate realistic molecular assemblies to produce quantitative calculations of the thermodynamic and kinetic quantities that control function. In this review, we summarize the state of atomistic simulation methods for ion channels to understand their conduction, activation, and drug modulation mechanisms. We are at a crossroads in atomistic simulation, where long time scale observation can provide unbiased exploration of mechanisms, supplemented by biased free energy methodologies. We illustrate the use of these approaches to describe ion conduction and selectivity in voltage-gated sodium and acid-sensing ion channels. Studies of channel gating present a significant challenge, as activation occurs on longer time scales. Enhanced sampling approaches can ensure convergence on minimum free energy pathways for activation, as illustrated here for pentameric ligand-gated ion channels that are principal to nervous system function and the actions of general anesthetics. We also examine recent studies of local anesthetic and antiepileptic drug binding to a sodium channel, revealing sites and pathways that may offer new targets for drug development. Modern simulations thus offer a range of molecular-level insights into ion channel function and modulation as a learning platform for mechanistic discovery and drug development.