Release of immunomodulatory peptides at bacterial membrane interfaces as a novel strategy to fight microorganisms.

Cationic and amphiphilic peptides can be used as homing devices to accumulate conjugated antibiotics to bacteria-enriched sites and promote efficient microbial killing. However, just as important as tackling bacterial infections, is the modulation of the immune response in this complex microenvironment. In the present report, we designed a peptide chimaera called Chim2, formed by a membrane-active module, an enzyme hydrolysis site and a formyl peptide receptor 2 (FPR2) agonist. This molecule was designed to adsorb onto bacterial membranes, promote their lysis, and upon hydrolysis by local enzymes, release the FPR2 agonist sequence for activation and recruitment of immune cells. We synthesized the isolated peptide modules of Chim2 and characterized their biological activities independently and as a single polypeptide chain. We conducted antimicrobial assays, along with other tests aiming at the analyses of the cellular and immunological responses. In addition, assays using vesicles as models of eukaryotic and prokaryotic membranes were conducted and solution structures of Chim2 were generated by 1H NMR. Chim2 is antimicrobial, adsorbs preferentially to negatively charged vesicles while adopting an α-helix structure and exposes its disorganized tail to the solvent, which facilitates hydrolysis by tryptase-like enzymes, allowing the release of the FPR2 agonist fragment. This fragment was shown to induce accumulation of the cellular activation marker, lipid bodies, in mouse macrophages and the release of immunomodulatory interleukins. In conclusion, these data demonstrate that peptides with antimicrobial and immunomodulatory activities can be considered for further development as drugs.

Smart probes for optical imaging of T cells and screening of anti-cancer immunotherapies.

T cells are an essential part of the immune system with crucial roles in adaptive response and the maintenance of tissue homeostasis. Depending on their microenvironment, T cells can be differentiated into multiple states with distinct functions. This myriad of cellular activities have prompted the development of numerous smart probes, ranging from small molecule fluorophores to nanoconstructs with variable molecular architectures and fluorescence emission mechanisms. In this Tutorial Review, we summarize recent efforts in the design, synthesis and application of smart probes for imaging T cells in tumors and inflammation sites by targeting metabolic and enzymatic biomarkers as well as specific surface receptors. Finally, we briefly review current strategies for how smart probes are employed to monitor the response of T cells to anti-cancer immunotherapies. We hope that this Review may help chemists, biologists and immunologists to design the next generation of molecular imaging probes for T cells and anti-cancer immunotherapies.

Imaging Proteins Sensitive to Direct Fusions Using Transient Peptide-Peptide Interactions.

Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to ∼200 nm, which is 2 orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs.

Enzyme-Activatable Near-Infrared Hemicyanines as Modular Scaffolds for in vivo Photodynamic Therapy.

Photodynamic therapy is an anti-cancer treatment that requires illumination of photosensitizers to induce local cell death. Current near-infrared organic photosensitizers are built from large and non-modular structures that cannot be tuned to improve safety and minimize off-target toxicity. This work describes a novel chemical platform to generate enzyme-activatable near-infrared photosensitizers. We optimized the Se-bridged hemicyanine scaffold to include caging groups and biocompatible moieties, and generated cathepsin-triggered photosensitizers for effective ablation of human glioblastoma cells. Furthermore, we demonstrated that enzyme-activatable Se-bridged hemicyanines are effective photosensitizers for the safe ablation of microtumors in vivo, creating new avenues in the chemical design of targeted anti-cancer photodynamic therapy agents.

Activatable Fluorophores for Imaging Immune Cell Function.

Optical imaging has become an essential tool to study biomolecular processes in live systems with unprecedented spatial resolution. New fluorescent technologies and advances in optical microscopy have revolutionized the ways in which we can study immune cells in real time. For example, activatable fluorophores that emit signals after target recognition have enabled direct imaging of immune cell function with enhanced readouts and minimal background. In this Account, we summarize recent advances in the chemical synthesis and implementation of activatable fluorescent probes to monitor the activity and the role of immune cells in different pathological processes, from infection to inflammatory diseases or cancer. In addition to the contributions that our group has made to this field, we review the most relevant literature disclosed over the past decade, providing examples of different activatable architectures and their application in diagnostics and drug discovery. This Account covers the imaging of the three major cell types in the immune system, that is, neutrophils, macrophages, and lymphocytes. Attracted by the tunability and target specificity of peptides, many groups have designed strategies based on fluorogenic peptides whose fluorescence emission is regulated by the reaction with enzymes (e.g., MMPs, cathepsins, granzymes), or through Förster resonance energy transfer (FRET) mechanisms. Selective imaging of immune cells has been also achieved by targeting different intracellular metabolic routes, such as lipid biogenesis. Other approaches involve the implementation of diversity-oriented fluorescence libraries or the use of environmentally sensitive fluorescent scaffolds (e.g., molecular rotors). Our group has made important progress by constructing probes to image metastasis-associated macrophages in tumors, apoptotic neutrophils, or cytotoxic natural killer (NK) cells against cancer cells, among other examples. The chemical probes covered in this Account have been successfully validated in vitro in cell culture systems, and in vivo in relevant models of inflammation and cancer. Overall, the range of chemical structures and activation mechanisms reported to sense immune cell function is remarkable. However, the emergence of new strategies based on new molecular targets or activatable mechanisms that are yet to be discovered will open the door to track unexplored roles of immune cells in different biological systems. We anticipate that upcoming generations of activatable probes will find applications in the clinic to help assessing immunotherapies and advance precision medicine. We hope that this Account will evoke new ideas and innovative work in the design of fluorescent probes for imaging cell function.

Charting the Chemical Reaction Space around a Multicomponent Combination: Controlled Access to a Diverse Set of Biologically Relevant Scaffolds.

Charting the chemical reaction space around the combination of carbonyls, amines, and isocyanoacetates allows the description of new multicomponent processes leading to a variety of unsaturated imidazolone scaffolds. The resulting compounds display the chromophore of the green fluorescent protein and the core of the natural product coelenterazine. Despite the competitive nature of the pathways involved, general protocols provide selective access to the desired chemotypes. Moreover, we describe unprecedented reactivity at the C-2 position of the imidazolone core to directly afford C, S, and N-derivatives featuring natural products (e.g. leucettamines), potent kinase inhibitors, and fluorescent probes with suitable optical and biological profiles.

Small Fluorogenic Amino Acids for Peptide-Guided Background-Free Imaging.

The multiple applications of super-resolution microscopy have prompted the need for minimally invasive labeling strategies for peptide-guided fluorescence imaging. Many fluorescent reporters display limitations (e.g., large and charged scaffolds, non-specific binding) as building blocks for the construction of fluorogenic peptides. Herein we have built a library of benzodiazole amino acids and systematically examined them as reporters for background-free fluorescence microscopy. We have identified amine-derivatized benzoselenadiazoles as scalable and photostable amino acids for the straightforward solid-phase synthesis of fluorescent peptides. Benzodiazole amino acids retain the binding capabilities of bioactive peptides and display excellent signal-to-background ratios. Furthermore, we have demonstrated their application in peptide-PAINT imaging of postsynaptic density protein-95 nanoclusters in the synaptosomes from mouse brain tissues.

Fluorogenic Granzyme A Substrates Enable Real-Time Imaging of Adaptive Immune Cell Activity.

Cytotoxic immune cells, including T lymphocytes (CTLs) and natural killer (NK) cells, are essential components of the host response against tumors. CTLs and NK cells secrete granzyme A (GzmA) upon recognition of cancer cells; however, there are very few tools that can detect physiological levels of active GzmA with high spatiotemporal resolution. Herein, we report the rational design of the near-infrared fluorogenic substrates for human GzmA and mouse GzmA. These activity-based probes display very high catalytic efficiency and selectivity over other granzymes, as shown in tissue lysates from wild-type and GzmA knock-out mice. Furthermore, we demonstrate that the probes can image how adaptive immune cells respond to antigen-driven recognition of cancer cells in real time.

Acid-Resistant BODIPY Amino Acids for Peptide-Based Fluorescence Imaging of GPR54 Receptors in Pancreatic Islets.

The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.

Miniaturized Chemical Tags for Optical Imaging.

Recent advances in optical bioimaging have prompted the need for minimal chemical reporters that can retain the molecular recognition properties and activity profiles of biomolecules. As a result, several methodologies to reduce the size of fluorescent and Raman labels to a few atoms (e.g., single aryl fluorophores, Raman-active triple bonds and isotopes) and embed them into building blocks (e.g., amino acids, nucleobases, sugars) to construct native-like supramolecular structures have been described. The integration of small optical reporters into biomolecules has also led to smart molecular entities that were previously inaccessible in an expedite manner. In this article, we review recent chemical approaches to synthesize miniaturized optical tags as well as some of their multiple applications in biological imaging.

Enzyme-Activatable Chemokine Conjugates for In Vivo Targeting of Tumor-Associated Macrophages.

Increased levels of tumor-associated macrophages (TAMs) are indicators of poor prognosis in most cancers. Although antibodies and small molecules blocking the recruitment of macrophages to tumors are under evaluation as anticancer therapies, these strategies are not specific for macrophage subpopulations. Herein we report the first enzyme-activatable chemokine conjugates for effective targeting of defined macrophage subsets in live tumors. Our constructs exploit the high expression of chemokine receptors (e.g., CCR2) and the activity of cysteine cathepsins in TAMs to target these cells selectively over other macrophages and immune cells (e.g., neutrophils, T cells, B cells). Furthermore, we demonstrate that cathepsin-activatable chemokines are compatible with both fluorescent and therapeutic cargos, opening new avenues in the design of targeted theranostic probes for immune cells in the tumor microenvironment.

A Bivalent Activatable Fluorescent Probe for Screening and Intravital Imaging of Chemotherapy-Induced Cancer Cell Death.

The detection and quantification of apoptotic cells is a key process in cancer research, particularly during the screening of anticancer therapeutics and in mechanistic studies using preclinical models. Intravital optical imaging enables high-resolution visualisation of cellular events in live organisms; however, there are few fluorescent probes that can reliably provide functional readouts in situ without interference from tissue autofluorescence. We report the design and optimisation of the fluorogenic probe Apotracker Red for real-time detection of cancer cell death. The strong fluorogenic behaviour, high selectivity, and excellent stability of Apotracker Red make it a reliable optical reporter for the characterisation of the effects of anticancer drugs in cells in vitro and for direct imaging of chemotherapy-induced apoptosis in vivo in mouse models of breast cancer.

Rational Design of Phe-BODIPY Amino Acids as Fluorogenic Building Blocks for Peptide-Based Detection of Urinary Tract Candida Infections.

Fungal infections caused by Candida species are among the most prevalent in hospitalized patients. However, current methods for the detection of Candida fungal cells in clinical samples rely on time-consuming assays that hamper rapid and reliable diagnosis. Herein, we describe the rational development of new Phe-BODIPY amino acids as small fluorogenic building blocks and their application to generate fluorescent antimicrobial peptides for rapid labelling of Candida cells in urine. We have used computational methods to analyse the fluorogenic behaviour of BODIPY-substituted aromatic amino acids and performed bioactivity and confocal microscopy experiments in different strains to confirm the utility and versatility of peptides incorporating Phe-BODIPYs. Finally, we have designed a simple and sensitive fluorescence-based assay for the detection of Candida albicans in human urine samples.

A Functional Chemiluminescent Probe for in Vivo Imaging of Natural Killer Cell Activity Against Tumours.

Natural killer (NK) cells are immune cells that can kill certain types of cancer cells. Adoptive transfer of NK cells represents a promising immunotherapy for malignant tumours; however, there is a lack of methods to validate anti-tumour activity of NK cells in vivo. Herein, we report a new chemiluminescent probe to image in situ the granzyme B-mediated killing activity of NK cells against cancer cells. We have optimised a granzyme B-specific construct using an activatable phenoxydioxetane reporter so that enzymatic cleavage of the probe results in bright chemiluminescence. The probe shows high selectivity for active granzyme B over other proteases and higher signal-to-noise ratios than commercial fluorophores. Finally, we demonstrate that the probe can detect NK cell activity in mouse models, being the first chemiluminescent probe for in vivo imaging of NK cell activity in live tumours.

A Palette of Minimally Tagged Sucrose Analogues for Real-Time Raman Imaging of Intracellular Plant Metabolism.

Sucrose is the main saccharide used for long-distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.

BODIPY-Labeled Cyclobutanes by Secondary C(sp3 )-H Arylations for Live-Cell Imaging.

Arylated cyclobutanes were accessed by a versatile palladium-catalyzed secondary C(sp3 )-H activation, exploiting chelation assistance by modular triazoles. The C-H arylation led to cyclobutane natural product derivatives in a highly regioselective fashion, setting the stage for the easy access to novel fluorogenic boron-dipyrrin (BODIPY)-labeled probes for live-cell imaging.

Fluorescent peptides for imaging of fungal cells.

Fungal infections, especially with the advent of antimicrobial resistance, represent a major burden to our society. As a result, there has been an increasing interest in the development of new probes that accelerate the study of fungi-related biological processes and facilitate novel clinical diagnostic and treatment strategies. Fluorescence-based reporters can provide dynamic information at the molecular level with high spatial resolution. However, conventional fluorescent probes for microbes often suffer from low specificity. In the last decade, numerous studies have been reported on the chemical design and application of fluorescent peptides for both in vitro and in vivo imaging of fungal cells. In this article, we review different strategies used in the preparation of fluorescent peptides for pathogenic fungi as well as some of their applications in medical imaging and in mode-of-action mechanistic studies.

Natural product-inspired profluorophores for imaging NQO1 activity in tumour tissues.

Herein we designed a collection of trimethyl-lock quinone profluorophores as activity-based probes for imaging NAD(P)H:quinone oxidoreductase (NQO1) in cancer cells and tumour tissues. Profluorophores were prepared via synthetic routes from naturally-occurring quinones and characterised in vitro using recombinant enzymes, to be further validated in cells and fresh frozen canine tumour tissues as potential new tools for cancer detection and imaging.

Quinone-based fluorophores for imaging biological processes.

Quinones are privileged chemical structures playing crucial roles as redox and alkylating agents in a wide range of processes in cells. The broad functional array of quinones has prompted the development of new chemical approaches, including C-H bond activation and asymmetric reactions, to generate probes for examining their activity by means of fluorescence imaging. This tutorial review covers recent advances in the design, synthesis and applications of quinone-based fluorescent agents for visualizing specific processes in multiple biological systems, from cells to tissues and complex organisms in vivo.

A Fluorescent Activatable AND-Gate Chemokine CCL2 Enables In Vivo Detection of Metastasis-Associated Macrophages.

We report the novel chemical design of fluorescent activatable chemokines as highly specific functional probes for imaging subpopulations of immune cells in live tumours. Activatable chemokines behave as AND-gates since they emit only after receptor binding and intracellular activation, showing enhanced selectivity over existing agents. We have applied this strategy to produce mCCL2-MAF as the first probe for in vivo detection of metastasis-associated macrophages in a preclinical model of lung metastasis. This strategy will accelerate the preparation of new chemokine-based probes for imaging immune cell function in tumours.