An approach to rapid distributed manufacturing of broad spectrum anti-viral griffithsin using cell-free systems to mitigate pandemics.

This study describes the cell-free biomanufacturing of a broad-spectrum antiviral protein, griffithsin (GRFT) such that it can be produced in microgram quantities with consistent purity and potency in less than 24 h. We demonstrate GRFT production using two independent cell-free systems, one plant and one microbial. Griffithsin purity and quality were verified using standard regulatory metrics. Efficacy was demonstrated in vitro against SARS-CoV-2 and HIV-1 and was nearly identical to that of GRFT expressed in vivo. The proposed production process is efficient and can be readily scaled up and deployed wherever a viral pathogen might emerge. The current emergence of viral variants of SARS-CoV-2 has resulted in frequent updating of existing vaccines and loss of efficacy for front-line monoclonal antibody therapies. Proteins such as GRFT with its efficacious and broad virus neutralizing capability provide a compelling pandemic mitigation strategy to promptly suppress viral emergence at the source of an outbreak.

Preclinical target validation for non-addictive therapeutics development for pain.

INTRODUCTION: The Helping to End Addiction Long-termSM Initiative supports a wide range of programs to develop new or improved prevention and opioid addiction treatment strategies. An essential component of this effort is to accelerate development of non-opioid pain therapeutics. In all fields of medicine, therapeutics development is an arduous process and late-stage translational efforts such as clinical trials to validate targets are particularly complex and costly. While there are plentiful novel targets for pain treatment, successful clinical validation is rare. It is therefore crucial to develop processes whereby therapeutic targets can be reasonably 'de-risked' prior to substantial late-stage validation efforts. Such rigorous validation of novel therapeutic targets in the preclinical space will give potential private sector partners the confidence to pursue clinical validation of promising therapeutic concepts and compounds. AREAS COVERED: In 2020, the National Institutes of Health (NIH) held the Target Validation for Non-Addictive Therapeutics Development for Pain workshop to gather insights from key opinion leaders in academia, industry, and venture-financing. EXPERT OPINION: The result was a roadmap for pain target validation focusing on three modalities: 1) human evidence; 2) assay development in vitro; 3) assay development in vivo.

An approach to rapid distributed manufacturing of broad spectrum anti-viral griffithsin using cell-free systems to mitigate pandemics.

This study describes the cell-free biomanufacturing of a broad-spectrum antiviral protein, griffithsin (GRFT) such that it can be produced with consistent purity and potency in less than 24 hours. We demonstrate GRFT production using two independent cell-free systems, one plant and one microbial. Griffithsin purity and quality were verified using standard regulatory metrics. Efficacy was demonstrated in vitro against SARS-CoV-2 and HIV-1 and was nearly identical to that of GRFT expressed in vivo . The proposed production process is efficient and can be readily scaled up and deployed anywhere in the world where a viral pathogen might emerge. The current emergence of viral variants has resulted in frequent updating of existing vaccines and loss of efficacy for front-line monoclonal antibody therapies. Proteins such as GRFT with its efficacious and broad virus neutralizing capability provide a compelling pandemic mitigation strategy to promptly suppress viral emergence at the source of an outbreak.

Overcoming Wnt-ß-catenin dependent anticancer therapy resistance in leukaemia stem cells.

Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt-ß-catenin and PI3K-Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate ß-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt-ß-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated ß-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, ß-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated ß-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape.

The Opioid Crisis and the Future of Addiction and Pain Therapeutics.

Opioid misuse and addiction are a public health crisis resulting in debilitation, deaths, and significant social and economic impact. Curbing this crisis requires collaboration among academic, government, and industrial partners toward the development of effective nonaddictive pain medications, interventions for opioid overdose, and addiction treatments. A 2-day meeting, The Opioid Crisis and the Future of Addiction and Pain Therapeutics: Opportunities, Tools, and Technologies Symposium, was held at the National Institutes of Health (NIH) to address these concerns and to chart a collaborative path forward. The meeting was supported by the NIH Helping to End Addiction Long-TermSM (HEAL) Initiative, an aggressive, trans-agency effort to speed scientific solutions to stem the national opioid crisis. The event was unique in bringing together two research disciplines, addiction and pain, in order to create a forum for crosscommunication and collaboration. The output from the symposium will be considered by the HEAL Initiative; this article summarizes the scientific presentations and key takeaways. Improved understanding of the etiology of acute and chronic pain will enable the discovery of novel targets and regulatable pain circuits for safe and effective therapeutics, as well as relevant biomarkers to ensure adequate testing in clinical trials. Applications of improved technologies including reagents, assays, model systems, and validated probe compounds will likely increase the delivery of testable hypotheses and therapeutics to enable better health outcomes for patients. The symposium goals were achieved by increasing interdisciplinary collaboration to accelerate solutions for this pressing public health challenge and provide a framework for focused efforts within the research community. SIGNIFICANCE STATEMENT: This article summarizes key messages and discussions resulting from a 2-day symposium focused on challenges and opportunities in developing addiction- and pain-related medications. Speakers and attendees came from 40 states in the United States and 15 countries, bringing perspectives from academia, industry, government, and healthcare by researchers, clinicians, regulatory experts, and patient advocates.

Fully Three-Dimensional Bioprinted Skin Equivalent Constructs with Validated Morphology and Barrier Function.

IMPACT STATEMENT: This article describes a method for the biofabrication of skin tissue equivalents in a multiwell plate format. The technique and results overcome shortcomings of previously published engineering methods, and show good architecture and barrier function from well to well; thus it may be used for compound functional testing and for the development of disease tissue models for screening.

Metastatic Tumor-in-a-Dish, a Novel Multicellular Organoid to Study Lung Colonization and Predict Therapeutic Response.

Metastasis is a major cause of cancer-related deaths. A dearth of preclinical models that recapitulate the metastatic microenvironment has impeded the development of therapeutic agents that are effective against metastatic disease. Because the majority of solid tumors metastasize to the lung, we developed a multicellular lung organoid that mimics the lung microenvironment with air sac-like structures and production of lung surfactant protein. We used these cultures, called primitive lung-in-a-dish (PLiD), to recreate metastatic disease using primary and established cancer cells. The metastatic tumor-in-a-dish (mTiD) cultures resemble the architecture of metastatic tumors in the lung, including angiogenesis. Pretreating PLiD with tumor exosomes enhanced cancer cell colonization. We next tested the response of primary and established cancer cells to current chemotherapeutic agents and an anti-VEGF antibody in mTiD against cancer cells in two-dimensional (2D) or 3D cultures. The response of primary patient-derived colon and ovarian tumor cells to therapy in mTiD cultures matched the response of the patient in the clinic, but not in 2D or single-cell-type 3D cultures. The sensitive mTiD cultures also produced significantly lower circulating markers for cancer similar to that seen in patients who responded to therapy. Thus, we have developed a novel method for lung colonization in vitro, a final stage in tumor metastasis. Moreover, the technique has significant utility in precision/personalized medicine, wherein this phenotypic screen can be coupled with current DNA pharmacogenetics to identify the ideal therapeutic agent, thereby increasing the probability of response to treatment while reducing unnecessary side effects. SIGNIFICANCE: A lung organoid that exhibits characteristics of a normal human lung is developed to study the biology of metastatic disease and therapeutic intervention.

Assay Guidance Manual: Quantitative Biology and Pharmacology in Preclinical Drug Discovery.

The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.

Small-Molecule Screens: A Gateway to Cancer Therapeutic Agents with Case Studies of Food and Drug Administration-Approved Drugs.

High-throughput screening (HTS) of small-molecule libraries accelerates the discovery of chemical leads to serve as starting points for probe or therapeutic development. With this approach, thousands of unique small molecules, representing a diverse chemical space, can be rapidly evaluated by biologically and physiologically relevant assays. The origins of numerous United States Food and Drug Administration-approved cancer drugs are linked to HTS, which emphasizes the value in this methodology. The National Institutes of Health Molecular Libraries Program made HTS accessible to the public sector, enabling the development of chemical probes and drug-repurposing initiatives. In this work, the impact of HTS in the field of oncology is considered among both private and public sectors. Examples are given for the discovery and development of approved cancer drugs. The importance of target validation is discussed, and common assay approaches for screening are reviewed. A rigorous examination of the PubChem database demonstrates that public screening centers are contributing to early-stage drug discovery in oncology by focusing on new targets and developing chemical probes. Several case studies highlight the value of different screening strategies and the potential for drug repurposing.

The translational science training program at NIH: Introducing early career researchers to the science and operation of translation of basic research to medical interventions.

Translational science is an emerging field that holds great promise to accelerate the development of novel medical interventions. As the field grows, so does the demand for highly trained biomedical scientists to fill the positions that are being created. Many graduate and postdoctorate training programs do not provide their trainees with sufficient education to take advantage of this growing employment sector. To help better prepare the trainees at the National Institutes of Health for possible careers in translation, we have created the Translational Science Training Program (TSTP). The TSTP is an intensive 2- to 3-day training program that introduces NIH postdoctoral trainees and graduate students to the science and operation of turning basic research discoveries into a medical therapeutic, device or diagnostic, and also exposes them to the variety of career options in translational science. Through a combination of classroom teaching from practicing experts in the various disciplines of translation and small group interactions with pre-clinical development teams, participants in the TSTP gain knowledge that will aid them in obtaining a career in translational science and building a network to make the transition to the field. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):13-24, 2017.

Novel Phenotypic Outcomes Identified for a Public Collection of Approved Drugs from a Publicly Accessible Panel of Assays.

Phenotypic assays have a proven track record for generating leads that become first-in-class therapies. Whole cell assays that inform on a phenotype or mechanism also possess great potential in drug repositioning studies by illuminating new activities for the existing pharmacopeia. The National Center for Advancing Translational Sciences (NCATS) pharmaceutical collection (NPC) is the largest reported collection of approved small molecule therapeutics that is available for screening in a high-throughput setting. Via a wide-ranging collaborative effort, this library was analyzed in the Open Innovation Drug Discovery (OIDD) phenotypic assay modules publicly offered by Lilly. The results of these tests are publically available online at www.ncats.nih.gov/expertise/preclinical/pd2 and via the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/) (AID 1117321). Phenotypic outcomes for numerous drugs were confirmed, including sulfonylureas as insulin secretagogues and the anti-angiogenesis actions of multikinase inhibitors sorafenib, axitinib and pazopanib. Several novel outcomes were also noted including the Wnt potentiating activities of rotenone and the antifolate class of drugs, and the anti-angiogenic activity of cetaben.

Stretching mechanotransduction from the lung to the lab: approaches and physiological relevance in drug discovery.

Recent years have shown a great deal of interest and research into the understanding of the biological and physiological roles of mechanical forces on cellular behavior. Despite these reports, in vitro screening of new molecular entities for lung ailments is still performed in static cell culture models. Failure to incorporate the effects of mechanical forces during early stages of screening could significantly reduce the success rate of drug candidates in the highly expensive clinical phases of the drug discovery pipeline. The objective of this review is to expand our current understanding of lung mechanotransduction and extend its applicability to cellular physiology and new drug screening paradigms. This review covers early in vivo studies and the importance of mechanical forces in normal lung development, use of different types of bioreactors that simulate in vivo movements in a controlled in vitro cell culture environment, and recent research using dynamic cell culture models. The cells in lungs are subjected to constant stretching (mechanical forces) in regular cycles due to involuntary expansion and contraction during respiration. The effects of stretch on normal and abnormal (disease) lung cells under pathological conditions are discussed. The potential benefits of extending dynamic cell culture models (screening in the presence of forces) and the associated challenges are also discussed in this review. Based on this review, the authors advocate the development of dynamic high throughput screening models that could facilitate the rapid translation of in vitro biology to animal models and clinical efficacy. These concepts are translatable to cardiovascular, digestive, and musculoskeletal tissues and in vitro cell systems employed routinely in drug-screening applications.

Impact of high-throughput screening in biomedical research.

High-throughput screening (HTS) has been postulated in several quarters to be a contributory factor to the decline in productivity in the pharmaceutical industry. Moreover, it has been blamed for stifling the creativity that drug discovery demands. In this article, we aim to dispel these myths and present the case for the use of HTS as part of a proven scientific tool kit, the wider use of which is essential for the discovery of new chemotypes.

Stem cells in drug discovery, tissue engineering, and regenerative medicine: emerging opportunities and challenges.

Stem cells, irrespective of their origin, have emerged as valuable reagents or tools in human health in the past 2 decades. Initially, a research tool to study fundamental aspects of developmental biology is now the central focus of generating transgenic animals, drug discovery, and regenerative medicine to address degenerative diseases of multiple organ systems. This is because stem cells are pluripotent or multipotent cells that can recapitulate developmental paths to repair damaged tissues. However, it is becoming clear that stem cell therapy alone may not be adequate to reverse tissue and organ damage in degenerative diseases. Existing small-molecule drugs and biologicals may be needed as "molecular adjuvants" or enhancers of stem cells administered in therapy or adult stem cells in the diseased tissues. Hence, a combination of stem cell-based, high-throughput screening and 3D tissue engineering approaches is necessary to advance the next wave of tools in preclinical drug discovery. In this review, the authors have attempted to provide a basic account of various stem cells types, as well as their biology and signaling, in the context of research in regenerative medicine. An attempt is made to link stem cells as reagents, pharmacology, and tissue engineering as converging fields of research for the next decade.

A comparison of assay performance measures in screening assays: signal window, Z' factor, and assay variability ratio.

In this article, the authors compare the assay performance measures, signal window, Z' factor, and assay variability ratio. They examine their mathematical formulae for similarities and differences, describe their statistical sampling properties using the results of a computer simulation, and illustrate their use with example data. Based on these results, the authors recommend the Z' factor as a preferred measure of assay performance for screening assays and point out that none of these measures are adequate for characterizing concentration-response assays.