DNA-Encoded Chemical Libraries: A Selection System Based on Endowing Organic Compounds with Amplifiable Information.

The discovery of organic ligands that bind specifically to proteins is a central problem in chemistry, biology, and the biomedical sciences. The encoding of individual organic molecules with distinctive DNA tags, serving as amplifiable identification bar codes, allows the construction and screening of combinatorial libraries of unprecedented size, thus facilitating the discovery of ligands to many different protein targets. Fundamentally, one links powers of genetics and chemical synthesis. After the initial description of DNA-encoded chemical libraries in 1992, several experimental embodiments of the technology have been reduced to practice. This review provides a historical account of important milestones in the development of DNA-encoded chemical libraries, a survey of relevant ongoing research activities, and a glimpse into the future.

Switchable targeting of solid tumors by BsCAR T cells.

The development of chimeric antigen receptor (CAR) T cell therapy has become a critical milestone in modern oncotherapy. Despite the remarkable in vitro effectiveness, the problem of safety and efficacy of CAR T cell therapy against solid tumors is challenged by the lack of tumor-specific antigens required to avoid on-target off-tumor effects. Spatially separating the cytotoxic function of CAR T cells from tumor antigen recognition provided by protein mediators allows for the precise control of CAR T cell cytotoxicity. Here, the high affinity and capability of the bacterial toxin-antitoxin barnase-barstar system were adopted to guide CAR T cells to solid tumors. The complementary modules based on (1) ankyrin repeat (DARPin)-barnase proteins and (2) barstar-based CAR (BsCAR) were designed to provide switchable targeting to tumor cells. The alteration of the DARPin-barnase switches enabled the targeting of different tumor antigens with a single BsCAR. A gradual increase in cytokine release and tunable BsCAR T cell cytotoxicity was achieved by varying DARPin-barnase loads. Switchable BsCAR T cell therapy was able to eradicate the HER2+ ductal carcinoma in vivo. Guiding BsCAR T cells by DARPin-barnase switches provides a universal approach for a controlled multitargeted adoptive immunotherapy.

Christian anthropology-based contributions to the ethics of socially assistive robots in care for older adults.

Our society, in general, and health care, in particular, faces notable challenges due to the emergence of innovative digital technologies. The use of socially assistive robots in aged care is a particular digital application that provokes ethical reflection. The answers we give to the ethical questions associated with socially assistive robots are framed by ontological and anthropological considerations of what constitutes human beings and how the meaning of being human relates to how these robots are conceived. Religious beliefs and secular worldviews, each of which may participate fully in pluralist societies, have an important responsibility in this foundational debate, as anthropological theories can be inspired by religious and secular viewpoints. This article identifies seven anthropological considerations grounded in the synthesis of biblical scriptures, Roman Catholic documents, and recent research literature. We highlight the inspirational quality of these anthropological considerations when dealing with ethical issues regarding the development and use of socially assistive robots in aged care. With this contribution, we aim to foster a global and inclusive dialogue on digitalization in aged care that deeply challenges our basic understanding of what constitutes a human being and how this notion relates to machine artefacts.

Studies on the mechanism of general anesthesia.

Inhaled anesthetics are a chemically diverse collection of hydrophobic molecules that robustly activate TWIK-related K+ channels (TREK-1) and reversibly induce loss of consciousness. For 100 y, anesthetics were speculated to target cellular membranes, yet no plausible mechanism emerged to explain a membrane effect on ion channels. Here we show that inhaled anesthetics (chloroform and isoflurane) activate TREK-1 through disruption of phospholipase D2 (PLD2) localization to lipid rafts and subsequent production of signaling lipid phosphatidic acid (PA). Catalytically dead PLD2 robustly blocks anesthetic TREK-1 currents in whole-cell patch-clamp recordings. Localization of PLD2 renders the TRAAK channel sensitive, a channel that is otherwise anesthetic insensitive. General anesthetics, such as chloroform, isoflurane, diethyl ether, xenon, and propofol, disrupt lipid rafts and activate PLD2. In the whole brain of flies, anesthesia disrupts rafts and PLDnull flies resist anesthesia. Our results establish a membrane-mediated target of inhaled anesthesia and suggest PA helps set thresholds of anesthetic sensitivity in vivo.

Inhibitory antibodies identify unique sites of therapeutic vulnerability in rhinovirus and other enteroviruses.

The existence of multiple serotypes renders vaccine development challenging for most viruses in the Enterovirus genus. An alternative and potentially more viable strategy for control of these viruses is to develop broad-spectrum antivirals by targeting highly conserved proteins that are indispensable for the virus life cycle, such as the 3C protease. Previously, two single-chain antibody fragments, YDF and GGVV, were reported to effectively inhibit human rhinovirus 14 proliferation. Here, we found that both single-chain antibody fragments target sites on the 3C protease that are distinct from its known drug site (peptidase active site) and possess different mechanisms of inhibition. YDF does not block the active site but instead noncompetitively inhibits 3C peptidase activity through an allosteric effect that is rarely seen for antibody protease inhibitors. Meanwhile, GGVV antagonizes the less-explored regulatory function of 3C in genome replication. The interaction between 3C and the viral genome 5' noncoding region has been reported to be important for enterovirus genome replication. Here, the interface between human rhinovirus 14 3C and its 5' noncoding region was probed by hydrogen-deuterium exchange coupled mass spectrometry and found to partially overlap with the interface between GGVV and 3C. Consistently, prebinding of GGVV completely abolishes interaction between human rhinovirus 14 3C and its 5' noncoding region. The epitopes of YDF and GGVV, therefore, represent two additional sites of therapeutic vulnerability in rhinovirus. Importantly, the GGVV epitope appears to be conserved across many enteroviruses, suggesting that it is a promising target for pan-enterovirus inhibitor screening and design.

Immunity against cancer cells may promote their proliferation and metastasis.

Herein we present a concept in cancer where an immune response is detrimental rather than helpful. In the cancer setting, the immune system is generally considered to be helpful in curtailing the initiation and progression of tumors. In this work we show that a patient's immune response to their tumor can, in fact, either enhance or inhibit tumor cell growth. Two closely related autoantibodies to the growth factor receptor TrkB were isolated from cancer patients' B cells. Although highly similar in sequence, one antibody was an agonist while the other was an antagonist. The agonist antibody was shown to increase breast cancer cell growth both in vitro and in vivo, whereas the antagonist antibody inhibited growth. From a mechanistic point of view, we showed that binding of the agonist antibody to the TrkB receptor was functional in that it initiated downstream signaling identical to its natural growth factor ligand, brain-derived neurotrophic factor (BDNF). Our study shows that individual autoantibodies may play a role in cancer patients.

Multiscale computation delivers organophosphorus reactivity and stereoselectivity to immunoglobulin scavengers.

Quantum mechanics/molecular mechanics (QM/MM) maturation of an immunoglobulin (Ig) powered by supercomputation delivers novel functionality to this catalytic template and facilitates artificial evolution of biocatalysts. We here employ density functional theory-based (DFT-b) tight binding and funnel metadynamics to advance our earlier QM/MM maturation of A17 Ig-paraoxonase (WTIgP) as a reactibody for organophosphorus toxins. It enables regulation of biocatalytic activity for tyrosine nucleophilic attack on phosphorus. The single amino acid substitution l-Leu47Lys results in 340-fold enhanced reactivity for paraoxon. The computed ground-state complex shows substrate-induced ionization of the nucleophilic l-Tyr37, now H-bonded to l-Lys47, resulting from repositioning of l-Lys47. Multiple antibody structural homologs, selected by phenylphosphonate covalent capture, show contrasting enantioselectivities for a P-chiral phenylphosphonate toxin. That is defined by crystallographic analysis of phenylphosphonylated reaction products for antibodies A5 and WTIgP. DFT-b analysis using QM regions based on these structures identifies transition states for the favored and disfavored reactions with surprising results. This stereoselection analysis is extended by funnel metadynamics to a range of WTIgP variants whose predicted stereoselectivity is endorsed by experimental analysis. The algorithms used here offer prospects for tailored design of highly evolved, genetically encoded organophosphorus scavengers and for broader functionalities of members of the Ig superfamily, including cell surface-exposed receptors.

Phenotypic selection with an intrabody library reveals an anti-apoptotic function of PKM2 requiring Mitofusin-1.

Bcl-2 family proteins control a decisive apoptotic event: mitochondrial outer membrane permeabilization (MOMP). To discover MOMP-regulating proteins, we expressed a library of intracellular single-chain variable fragments (scFvs) ("intrabodies") and selected for those rescuing cells from apoptosis induced by BimS (the short isoform of Bim). One anti-apoptotic intrabody, intrabody 5 (IB5), recognized pyruvate kinase M2 (PKM2), which is expressed in cancer cells. PKM2 deletion ablated this clonogenic rescue; thus, IB5 activated a latent cytoprotective function of PKM2. This resulted not from pyruvate kinase activity per se but rather from the formation of an active tetrameric conformation of PKM2. A stably tetrameric PKM2 mutant, K422R, promoted cell survival even in the absence of IB5, and IB5 further increased survival. Mitochondria isolated from IB5-expressing cells were relatively resistant to MOMP in vitro. In cells, IB5 expression up-regulated Mitofusin-1 (Mfn1) and increased mitochondrial length. Importantly, Mfn1 deficiency abrogated IB5's cytoprotective effect. PKM2's anti-apoptotic function could help explain its preferential expression in human cancer.

A cell-cell interaction format for selection of high-affinity antibodies to membrane proteins.

Generating and improving antibodies and peptides that bind specifically to membrane protein targets such as ion channels and G protein-coupled receptors (GPCRs) can be challenging using established selection methods. Current strategies are often limited by difficulties in the presentation of the antigen or the efficiency of the selection process. Here, we report a method for obtaining antibodies specific for whole cell membrane-associated antigens which combines a cell-cell interaction format based on yeast display technology with fluorescence-activated cell sorting of dual fluorescent complexes. Using this method, we were able to direct the affinity maturation of an antagonist antibody specific for the proton-gated ion channel ASIC1a and showed that both the affinity and potency were improved. We were also able to use this method to do kinetic selections to generate clones with better dissociation profiles. In addition, this method was employed successfully to handle the difficult problem of selecting antibodies specific to a GPCR target, the mu-opioid receptor.

Antigen-Specific Stimulation and Expansion of CAR-T Cells Using Membrane Vesicles as Target Cell Surrogates.

Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. This step is challenging, especially if non-viral low-efficiency delivery protocols are used to generate CAR-T cells. Modern protocols for CAR-T cell expansion are imperfect since non-specific stimulation results in rapid outgrowth of CAR-negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. To develop a specific and improved protocol for CAR-T cell expansion, cell-derived membrane vesicles are taken advantage of, and the simple structural demands of the CAR-antigen interaction. This novel approach is to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens, and then use them for stimulation and expansion of CAR-T cells. The data presented in this article clearly demonstrate that this protocol produced antigen-specific vesicles with the capacity to induce stronger stimulation, proliferation, and functional activity of CAR-T cells than is possible with existing protocols. It is predicted that this new methodology will significantly advance the ability to obtain improved populations of functional CAR-T cells for therapy.

DNA-Encoded Libraries: Hydrazide as a Pluripotent Precursor for On-DNA Synthesis of Various Azole Derivatives.

DNA-encoded combinatorial chemical library (DEL) technology, an approach that combines the power of genetics and chemistry, has emerged as an invaluable tool in drug discovery. Skeletal diversity plays a fundamental importance in DEL applications, and relies heavily on novel DNA-compatible chemical reactions. We report herein a phylogenic chemical transformation strategy using DNA-conjugated benzoyl hydrazine as a common versatile precursor in azole chemical expansion of DELs. DNA-compatible reactions deriving from the common benzoyl hydrazine precursor showed excellent functional group tolerance with exceptional efficiency in the synthesis of various azoles, including oxadiazoles, thiadiazoles, and triazoles, under mild reaction conditions. The phylogenic chemical transformation strategy provides DELs a facile way to expand into various unique chemical spaces with privileged scaffolds and pharmacophores.

Migration-based selections of antibodies that convert bone marrow into trafficking microglia-like cells that reduce brain amyloid ß.

One goal of regenerative medicine is to repair damaged tissue. This requires not only generating new cells of the proper phenotype, but also selecting for those that properly integrate into sites of injury. In our laboratory we are using a cell-migration-based in vivo selection system to generate antibodies that induce cells to both differentiate and selectively localize to different tissues. Here we describe an antibody that induces bone marrow stem cells to differentiate into microglia-like cells that traffic to the brain where they organize into typical networks. Interestingly, in the APP/PS1 Alzheimer's disease mouse model, these induced microglia-like cells are found at sites of plaque formation and significantly reduce their number. These results raise the intriguing question as to whether one can use such antibody-induced differentiation of stem cells to essentially recapitulate embryogenesis in adults to discover cells that can regenerate damaged organ systems.

Antibody selection using clonal cocultivation of Escherichia coli and eukaryotic cells in miniecosystems.

We describe a method for the rapid selection of functional antibodies. The method depends on the cocultivation of Escherichia coli that produce phage with target eukaryotic cells in very small volumes. The antibodies on phage induce selectable phenotypes in the target cells, and the nature of the antibody is determined by gene sequencing of the phage genome. To select functional antibodies from the diverse antibody repertoire, we devised a selection platform that contains millions of picoliter-sized droplet ecosystems. In each miniecosystem, the bacteria produce phage displaying unique members of the antibody repertoire. These phage interact only with eukaryotic cells in the same miniecosystem, making phage available directly for activity-based antibody selection in biological systems.

Fully human agonist antibodies to TrkB using autocrine cell-based selection from a combinatorial antibody library.

The diverse physiological roles of the neurotrophin family have long prompted exploration of their potential as therapeutic agents for nerve injury and neurodegenerative diseases. To date, clinical trials of one family member, brain-derived neurotrophic factor (BDNF), have disappointingly failed to meet desired endpoints. Contributing to these failures is the fact that BDNF is pharmaceutically a nonideal biologic drug candidate. It is a highly charged, yet is a net hydrophobic molecule with a low molecular weight that confers a short t1/2 in man. To circumvent these shortcomings of BDNF as a drug candidate, we have employed a function-based cellular screening assay to select activating antibodies of the BDNF receptor TrkB from a combinatorial human short-chain variable fragment antibody library. We report here the successful selection of several potent TrkB agonist antibodies and detailed biochemical and physiological characterization of one such antibody, ZEB85. By using a human TrkB reporter cell line and BDNF-responsive GABAergic neurons derived from human ES cells, we demonstrate that ZEB85 is a full agonist of TrkB, comparable in potency to BDNF toward human neurons in activation of TrkB phosphorylation, canonical signal transduction, and mRNA transcriptional regulation.

Selection of an ASIC1a-blocking combinatorial antibody that protects cells from ischemic death.

Acid-sensing ion channels (ASICs) have emerged as important, albeit challenging therapeutic targets for pain, stroke, etc. One approach to developing therapeutic agents could involve the generation of functional antibodies against these channels. To select such antibodies, we used channels assembled in nanodiscs, such that the target ASIC1a has a configuration as close as possible to its natural state in the plasma membrane. This methodology allowed selection of functional antibodies that inhibit acid-induced opening of the channel in a dose-dependent way. In addition to regulation of pH, these antibodies block the transport of cations, including calcium, thereby preventing acid-induced cell death in vitro and in vivo. As proof of concept for the use of these antibodies to modulate ion channels in vivo, we showed that they potently protect brain cells from death after an ischemic stroke. Thus, the methodology described here should be general, thereby allowing selection of antibodies to other important ASICs, such as those involved in pain, neurodegeneration, and other conditions.

Positive Youth Development in 2020: Theory, Research, Programs, and the Promotion of Social Justice.

We use Hamilton's (1999) tripartite conception of the positive youth development (PYD) literature - that is, PYD as a theoretical construct, PYD as a frame for program design, and PYD as an instance of specific youth development programs - as a framework for reviewing scholarship involved in the PYD field across the second decade of the 21st century. Advances were made in all three domains and, as well, new issues emerged; chief among them was a focus on the promotion of social justice. We discuss ways in which social justice issues are being addressed within each of these domains and we present a vision for enhancing the PYD-social justice relation in future scholarship involving theory, research, program design, and community-based PYD programs.

Antibody Libraries as Tools to Discover Functional Antibodies and Receptor Pleiotropism.

Most antibodies currently in use have been selected based on their binding affinity. However, nowadays, antibodies that can not only bind but can also alter the function of cell surface signaling components are increasingly sought after as therapeutic drugs. Therefore, the identification of such functional antibodies from a large antibody library is the subject of intensive research. New methods applied to combinatorial antibody libraries now allow the isolation of functional antibodies in the cellular environment. These selected agonist antibodies have provided new insights into important issues of signal transduction. Notably, when certain antibodies bind to a given receptor, the cell fate induced by them may be the same or different from that induced by natural agonists. In addition, combined with phenotypic screening, this platform allows us to discover unexpected experimental results and explore various phenomena in cell biology, such as those associated with stem cells and cancer cells.

gem-Difluoromethylene Alkyne-Enabled Diverse C-H Functionalization and Application to the on-DNA Synthesis of Difluorinated Isocoumarins.

Using gem-difluoromethylene alkynes as effectors, unprecedented diverse C-H activation/[4+2] annulations of simple benzoic acids are reported. The chemodivergent reaction outcomes are well-tuned by Rh/Ir-catalyzed system; in the RhIII catalysis, 3-alkenyl-1H-isochromen-1-one and 3,4-dialkylideneisochroman-1-one skeletons are afforded in a solvent-dependent manner whereas difluoromethylene-substituted 1H-isochromen-1-ones are generated under the IrIII -catalyzed system. Mechanistic studies revealed that unusually double ß-F eliminations and fluorine effect-induced regioselective reductive elimination are independently involved to enable distinct reaction modes for divergent product formations. Besides, synthetic application in both the derivatization of obtained diene products and the on-DNA synthesis of DNA-tagged difluorinated isocoumarin have been demonstrated, which manifested great potential for synthetic utility of the developed protocols.

Reflections on DNA-encoded chemical libraries.

In this short Editorial, we reflect on certain milestones that have led to the development of DNA-Encoded Chemical Libraries, while also providing a glimpse to future challenges and opportunities.

A DNA-encoded library for the identification of natural product binders that modulate poly (ADP-ribose) polymerase 1, a validated anti-cancer target.

Natural products have been an invaluable source of drug discovery, but their targets remain largely unknown. Natural products enriched DNA-encoded chemical libraries (nDELs) empower the researchers to rapidly and economically screen numerous natural products against various protein targets, and therefore promote the elucidation of the molecular mechanisms. In this work, we used poly (ADP-ribose) polymerase 1 (PARP1), as an example to explore the usage of nDEL for the functional natural products selection. We used late-stage modification approach to label three positive binders with unique DNA barcodes, whose dissociation constants range from sub-micromolar to micromolar. The selection criterion was set up according to the enrichment of these controls. Five natural products selected by this criterion directly bind to PARP1 in SPR, among which luteolin exhibits the highest inhibitory activity against PARP1. Moreover, luteolin selectively induces accumulation of DNA double-strand breaks and G2/M phase arrest in BRCA-deficient cells. All the findings from these investigations on luteolin support that PARP1 inhibition is one of the mechanisms for its anti-cancer activity.