Potent and Selective Oxidatively Labile Ether-Based Prodrugs through Late-Stage Boronate Incorporation.

This manuscript describes a new strategy for prodrug synthesis in which a relatively inert ether group is introduced at an early stage in a synthetic sequence and functionalized in the final step to introduce a prodrug-activating group through a chemoselective process. Boryl allyloxy (BAO) ether groups are synthesized through several metal-mediated processes to form entities that are readily cleaved under oxidative conditions commonly found in cancer cells. The high cleavage propensity of the BAO group allows for ether cleavage, making these compounds substantially more hydrolytically stable in comparison to acyl-linked prodrugs while retaining the ability to release alcohols. We report the preparation of prodrug analogues of the natural products camptothecin and pederin from acetal precursors that serve as protecting groups in their synthetic sequences. The BAO acetal groups cleave in the presence of hydrogen peroxide to release the cytotoxic agents. The pederin-based prodrug shows dramatically greater cytotoxicity than negative controls and outstanding selectivity and potency toward cancer cell lines in comparison to non-cancerous cell lines. This late-stage functionalization approach to prodrug synthesis should be applicable to numerous systems that can be accessed through chemoselective processes.

DNA Logic Gates for Small Molecule Activation Circuits in Cells.

DNA-based devices such as DNA logic gates self-assemble into supramolecular structures, as dictated by the sequences of the constituent oligonucleotides and their predictable Watson-Crick base pairing interactions. The programmable nature of DNA-based devices permits the design and implementation of DNA circuits that interact in a dynamic and sequential manner capable of spatially arranging disparate DNA species. Here, we report the application of an activatable fluorescence reporter based on a proximity-driven inverse electron demand Diels-Alder (IEDDA) reaction and its robust integration with DNA strand displacement circuits. In response to specific DNA input patterns, sequential strand displacement reactions are initiated and culminate in the hybridization of two modified DNA strands carrying probes capable of undergoing an IEDDA reaction between a vinyl-ether-caged fluorophore and its reactive partner tetrazine, leading to the activation of fluorescence. This approach provides a major advantage for DNA computing in mammalian cells since circuit degradation does not induce fluorescence, in contrast to traditional fluorophore-quencher designs. We demonstrate the robustness and sensitivity of the reporter by testing its ability to serve as a readout for DNA logic circuits of varying complexity inside cells.

Direct Activation of Nucleobases with Small Molecules for the Conditional Control of Antisense Function.

Conditionally controlled antisense oligonucleotides provide precise interrogation of gene function at different developmental stages in animal models. Only one example of small molecule-induced activation of antisense function exist. This has been restricted to cyclic caged morpholinos that, based on sequence, can have significant background activity in the absence of the trigger. Here, we provide a new approach using azido-caged nucleobases that are site-specifically introduced into antisense morpholinos. The caging group design is a simple azidomethylene (Azm) group that, despite its very small size, efficiently blocks Watson-Crick base pairing in a programmable fashion. Furthermore, it undergoes facile decaging via Staudinger reduction when exposed to a small molecule phosphine, generating the native antisense oligonucleotide under conditions compatible with biological environments. We demonstrated small molecule-induced gene knockdown in mammalian cells, zebrafish embryos, and frog embryos. We validated the general applicability of this approach by targeting three different genes.

Expanding the Genetic Code of Xenopus laevis Embryos.

The incorporation of unnatural amino acids into proteins through genetic code expansion has been successfully adapted to African claw-toed frog embryos. Six unique unnatural amino acids are incorporated site-specifically into proteins and demonstrate robust and reliable protein expression. Of these amino acids, several are caged analogues that can be used to establish conditional control over enzymatic activity. Using light or small molecule triggers, we exhibit activation and tunability of protein functions in live embryos. This approach was then applied to optical control over the activity of a RASopathy mutant of NRAS, taking advantage of generating explant cultures from Xenopus. Taken together, genetic code expansion is a robust approach in the Xenopus model to incorporate novel chemical functionalities into proteins of interest to study their function and role in a complex biological setting.

Dissection of MKK6 and p38 Signaling Using Light-Activated Protein Kinases.

Stress-activated signaling pathways orchestrate cellular behaviors and fates. Studying the precise role(s) of stress-activated protein kinases is challenging, because stress conditions induce adaptation and impose selection pressure. To meet this challenge, we have applied an optogenetic system with a single plasmid to express light-activated p38α or its upstream activator, MKK6, in conjunction with live-cell fluorescence microscopy. In starved cells, decaging of constitutively active p38α or MKK6 by brief exposure to UV light elicits rapid p38-mediated signaling, release of cytochrome c from mitochondria, and apoptosis with different kinetics. In parallel, light activation of p38α also suppresses autophagosome formation, similarly to stimulation with growth factors that activate PI3K/Akt/mTORC1 signaling. Active MKK6 negatively regulates serum-induced ERK activity, which is p38-independent as previously reported. Here, we reproduce that result with the one plasmid system and show that although decaging active p38α does not reduce basal ERK activity in our cells, it can block growth factor-stimulated ERK signaling in serum-starved cells. These results clarify the roles of MKK6 and p38α in dynamic signaling programs, which act in concert to actuate apoptotic death while suppressing cell survival mechanisms.

Development of a light-activated STING agonist.

The STING pathway is critical to innate immunity and is being investigated as a potential therapeutic target. Existing agents targeting STING suffer from several undesirable effects, particularly the possibility of systematic activation, which increases the risk of autoimmune disorders. In this proof-of-concept study, we report the development of a light-activated STING agonist, based on the potent compound SR-717. We first screened the activity of the non-caged agonist toward 5 human STING variants to identify the most viable target. A photocaged agonist was designed and synthesized in order to block an essential interaction between the carboxy acid group of the ligand with the R238 residue of the STING protein. We then investigated the selective activation of STING with the photocaged agonist, demonstrating an irradiation-dependent response. The development and characterization of this selective agonist expands the growing toolbox of conditionally controlled STING agonists to avoid systematic immune activation.

Visible Light-Induced Specific Protein Reaction Delineates Early Stages of Cell Adhesion.

Light is well-established for control of bond breakage but not for control of specific bond formation in complex environments. We previously engineered the diffusion-limited reactivity of the SpyTag003 peptide with its protein partner SpyCatcher003 through spontaneous isopeptide bond formation. This system enables precise and irreversible assembly of biological building blocks with applications from biomaterials to vaccines. Here we establish a system for the rapid control of this amide bond formation with visible light. We have generated a caged SpyCatcher003, which allows light triggering of covalent bond formation to SpyTag003 in mammalian cells. Photocaging is achieved through site-specific incorporation of an unnatural coumarin-lysine at the reactive site of SpyCatcher003. We showed a uniform specific reaction in cell lysate upon light activation. We then used the spatiotemporal precision of a 405 nm confocal laser for uncaging in seconds, probing the earliest events in mechanotransduction by talin, the key force sensor between the cytoskeleton and the extracellular matrix. Reconstituting talin induced rapid biphasic extension of lamellipodia, revealing the kinetics of talin-regulated cell spreading and polarization. Thereafter we determined the hierarchy of the recruitment of key components for cell adhesion. Precise control over site-specific protein reaction with visible light creates diverse opportunities for cell biology and nanoassembly.

Conditional Control of Universal CAR T Cells by Cleavable OFF-Switch Adaptors.

As living drugs, engineered T cell therapies are revolutionizing disease treatment with their unique functional capabilities. However, they suffer from limitations of potentially unpredictable behavior, toxicities, and nontraditional pharmacokinetics. Engineering conditional control mechanisms responsive to tractable stimuli such as small molecules or light is thus highly desirable. We and others previously developed "universal" chimeric antigen receptors (CARs) that interact with coadministered antibody adaptors to direct target cell killing and T cell activation. Universal CARs are of high therapeutic interest due to their ability to simultaneously target multiple antigens on the same disease or different diseases by combining with adaptors to different antigens. Here, we further enhance the programmability and potential safety of universal CAR T cells by engineering OFF-switch adaptors that can conditionally control CAR activity, including T cell activation, target cell lysis, and transgene expression, in response to a small molecule or light stimulus. Moreover, in adaptor combination assays, OFF-switch adaptors were capable of orthogonal conditional targeting of multiple antigens simultaneously, following Boolean logic. OFF-switch adaptors represent a robust new approach for the precision targeting of universal CAR T cells with potential for enhanced safety.

Isoform-specific optical activation of kinase function reveals p38-ERK signaling crosstalk.

Evolution has diversified the mammalian proteome by the generation of protein isoforms that originate from identical genes, e.g., through alternative gene splicing or post-translational modifications, or very similar genes found in gene families. Protein isoforms can have either overlapping or unique functions and traditional chemical, biochemical, and genetic techniques are often limited in their ability to differentiate between isoforms due to their high similarity. This is particularly true in the context of highly dynamic cell signaling cascades, which often require acute spatiotemporal perturbation to assess mechanistic details. To that end, we describe a method for the selective perturbation of the individual protein isoforms of the mitogen-activated protein kinase (MAPK) p38. The genetic installation of a photocaging group at a conserved active site lysine enables the precise light-controlled initiation of kinase signaling, followed by investigation of downstream events. Through optical control, we have identified a novel point of crosstalk between two major signaling cascades: the p38/MAPK pathway and the extracellular signal-regulated kinase (ERK)/MAPK pathway. Specifically, using the photoactivated p38 isoforms, we have found the p38γ and p38δ variants to be positive regulators of the ERK signaling cascade, while confirming the p38α and p38ß variants as negative regulators.

Visible light-induced specific protein reaction delineates early stages of cell adhesion.

Light is well established for control of bond breakage, but not for control of specific bond formation in complex environments. We previously engineered diffusion-limited reactivity of SpyTag003 peptide with its protein partner SpyCatcher003 through spontaneous transamidation. This system enables precise and irreversible assembly of biological building blocks, with applications from biomaterials to vaccines. Here, we establish a system for rapid control of this amide bond formation with visible light. We have generated a caged SpyCatcher003, which allows light triggering of covalent bond formation to SpyTag003 in mammalian cells. Photocaging is achieved through site-specific incorporation of an unnatural coumarin-lysine at the reactive site of SpyCatcher003. We showed uniform specific reaction in cell lysate upon light activation. We then used the spatiotemporal precision of a 405 nm confocal laser for uncaging in seconds, probing the earliest events in mechanotransduction by talin, the key force sensor between the cytoskeleton and extracellular matrix. Reconstituting talin induced rapid biphasic extension of lamellipodia, revealing the kinetics of talin-regulated cell spreading and polarization. Thereafter we determined the hierarchy of recruitment of key components for cell adhesion. Precise control over site-specific protein reaction with visible light creates diverse opportunities for cell biology and nanoassembly.

Peroxide-Mediated Release of Organophosphates from Boron-Containing Phosphotriesters: A New Class of Organophosphate Prodrugs.

Phosphate mono- and diesters can be liberated efficiently from boryl allyloxy (BAO) and related phosphotriesters by H2O2. This protocol was applied to the release of a phosphorylated serine derivative and the nucleotide analogue AZT monophosphate. Nucleotide release in the presence of ATP and a kinase provides a diphosphate, demonstrating that this method can be applied to biological processes.

Cell Surface Labeling and Detection of Protein Tyrosine Kinase 7 via Covalent Aptamers.

Covalent aptamers are novel biochemical tools for fast and selective transfer of labels to target proteins. Equipped with cleavable electrophiles, these nucleic acid probes enable the installation of functional handles onto native proteins. The high affinity and specificity with which aptamers bind their selected targets allows for quick, covalent labeling that can compete with nuclease-mediated degradation. Here, we introduce the first application of covalent aptamers to modify a specific cell surface protein through proximity-driven label transfer. We targeted protein tyrosine kinase 7 (PTK7), a prominent cancer marker, and demonstrated aptamer-mediated biotin transfer to specific lysine residues on the extracellular domain of the protein. This allowed for tracking of PTK7 expression, localization, and cellular internalization. These studies validate the programmability of covalent aptamers and highlight their applicability in a cellular context, including protein and small molecule delivery.

Genetic Encoding of Arylazopyrazole Phenylalanine for Optical Control of Translation.

An arylazopyrazole was explored for its use as an enhanced photoswitchable amino acid in genetic code expansion. This new unnatural amino acid was successfully incorporated into proteins in both bacterial and mammalian cells. While photocontrol of translation required pulsed irradiations, complete selectivity for the trans-configuration by the pyrrolysyl tRNA synthetase was observed, demonstrating expression of a gene of interest selectively controlled via light exposure.

Antisense Oligonucleotide Activation via Enzymatic Antibiotic Resistance Mechanism.

The structure and mechanism of the bacterial enzyme ß-lactamase have been well-studied due to its clinical role in antibiotic resistance. ß-Lactamase is known to hydrolyze the ß-lactam ring of the cephalosporin scaffold, allowing a spontaneous self-immolation to occur. Previously, cephalosporin-based sensors have been developed to evaluate ß-lactamase expression in both mammalian cells and zebrafish embryos. Here, we present a circular caged morpholino oligonucleotide (cMO) activated by ß-lactamase-mediated cleavage of a cephalosporin motif capable of silencing the expression of T-box transcription factor Ta (tbxta), also referred to as no tail a (ntla), eliciting a distinct, observable phenotype. We explore the use of ß-lactamase to elicit a biological response in aquatic embryos for the first time and expand the utility of cephalosporin as a cleavable linker beyond targeting antibiotic-resistant bacteria. The addition of ß-lactamase to the current suite of enzymatic triggers presents unique opportunities for robust, orthogonal control over endogenous gene expression in a spatially resolved manner.

Genetically Encoded Aminocoumarin Lysine for Optical Control of Protein-Nucleotide Interactions in Zebrafish Embryos.

The strategic placement of unnatural amino acids into the active site of kinases and phosphatases has allowed for the generation of photocaged signaling proteins that offer spatiotemporal control over activation of these pathways through precise light exposure. However, deploying this technology to study cell signaling in the context of embryo development has been limited. The promise of optical control is especially useful in the early stages of an embryo where development is driven by tightly orchestrated signaling events. Here, we demonstrate light-induced activation of Protein Kinase A and a RASopathy mutant of NRAS in the zebrafish embryo using a new light-activated amino acid. We applied this approach to gain insight into the roles of these proteins in gastrulation and heart development and forge a path for further investigation of RASopathy mutant proteins in animals.

Conditional control of universal CAR T cells by cleavable OFF-switch adaptors.

As living drugs, engineered T cell therapies are revolutionizing disease treatment with their unique functional capabilities. However, they suffer from limitations of potentially unpredictable behavior, toxicities, and non-traditional pharmacokinetics. Engineering conditional control mechanisms responsive to tractable stimuli such as small molecules or light is thus highly desirable. We and others previously developed "universal" chimeric antigen receptors (CARs) that interact with co-administered antibody adaptors to direct target cell killing and T cell activation. Universal CARs are of high therapeutic interest due to their ability to simultaneously target multiple antigens on the same disease or different diseases by combining with adaptors to different antigens. Here, we further enhance the programmability and potential safety of universal CAR T cells by engineering OFF-switch adaptors that can conditionally control CAR activity, including T cell activation, target cell lysis, and transgene expression, in response to a small molecule or light stimulus. Moreover, in adaptor combination assays, OFF-switch adaptors were capable of orthogonal conditional targeting of multiple antigens simultaneously following Boolean logic. OFF-switch adaptors represent a robust new approach for precision targeting of universal CAR T cells with potential for enhanced safety.

Small-Molecule Phosphine Activation of Protein Function in Zebrafish Embryos with an Expanded Genetic Code.

Conditional control of protein function in a living model organism is an important tool for studying the effects of that protein during development and disease. In this chapter, we walk through the steps to generate a small-molecule-activatable enzyme in zebrafish embryos through the incorporation of a noncanonical amino acid into the protein active site. This method can be applied to many enzyme classes, which we highlight with temporal control of a luciferase and a protease. We demonstrate that strategic placement of the noncanonical amino acid completely blocks enzyme activity, which is then promptly restored after addition of the nontoxic small molecule inducer to the embryo water.

Post-translational covalent assembly of CAR and synNotch receptors for programmable antigen targeting.

Chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors are engineered cell-surface receptors that sense a target antigen and respond by activating T cell receptor signaling or a customized gene program, respectively. Here, to expand the targeting capabilities of these receptors, we develop "universal" receptor systems for which receptor specificity can be directed post-translationally via covalent attachment of a co-administered antibody bearing a benzylguanine (BG) motif. A SNAPtag self-labeling enzyme is genetically fused to the receptor and reacts with BG-conjugated antibodies for covalent assembly, programming antigen recognition. We demonstrate that activation of SNAP-CAR and SNAP-synNotch receptors can be successfully targeted by clinically relevant BG-conjugated antibodies, including anti-tumor activity of SNAP-CAR T cells in vivo in a human tumor xenograft mouse model. Finally, we develop a mathematical model to better define the parameters affecting universal receptor signaling. SNAP receptors provide a powerful strategy to post-translationally reprogram the targeting specificity of engineered cells.

Protein labeling and crosslinking by covalent aptamers.

In this chapter, a new approach to the selective modification of native proteins is discussed, using electrophilic covalent aptamers. These biochemical tools are generated through the site-specific incorporation of a label-transferring or crosslinking electrophile into a DNA aptamer. Covalent aptamers provide the ability to transfer a variety of functional handles to a protein of interest or to irreversibly crosslink to the target. Methods for the aptamer-mediated labeling and crosslinking of thrombin are described. Thrombin labeling is fast and selective, in both simple buffer and in human plasma and outcompetes nuclease-mediated degradation. This approach provides facile, sensitive detection of labeled protein by western blot, SDS-PAGE, and mass spectrometry.

Targeted protein degradation through light-activated E3 ligase recruitment.

Optical control of protein function through proteasomal degradation benefits from the noninvasive nature and spatiotemporal precision of light as a trigger. In this chapter, light activation of protein degradation with an optically controlled degron, termed optoDeg, is discussed. This method utilizes genetic code expansion to insert a photocaged analog of lysine at the N-terminal position of a protein of interest for spatial and temporal control of the N-end pathway, inducing proteasomal degradation. Methods for the use of optoDeg for degradation of the fluorescent reporter EGFP and the kinase MEK1 are described. The system is fast, with complete degradation of proteins within minutes following irradiation, and highly specific, with genetically directed introduction of the light-activated degron.