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.

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.

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.

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.

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.

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 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.

Fluorizoline-induced apoptosis requires prohibitins in nematodes and human cells.

We previously showed that fluorizoline, a fluorinated thiazoline compound, binds to both subunits of the mitochondrial prohibitin (PHB) complex, PHB1 and PHB2, being the expression of these proteins required for fluorizoline-induced apoptosis in mouse embryonic fibroblasts. To investigate the conservation of this apoptotic mechanism, we studied the effect of PHB downregulation on fluorizoline activity on two human cell lines, HEK293T and U2OS. Then, we asked whether PHBs mediate the effect of fluorizoline in a multicellular organism. Interestingly, reduced levels of PHBs in the human cells impaired the induction of apoptosis by fluorizoline. We observed that fluorizoline has a detrimental dose-dependent effect on the development and survival of the nematode model Caenorhabditis elegans. Besides, such effects of fluorizoline treatment in living nematodes were absent in PHB mutants. Finally, we further explored the apoptotic pathway triggered by fluorizoline in human cell lines. We found that the BH3-only proteins NOXA, BIM and PUMA participate in fluorizoline-induced apoptosis and that the induction of NOXA and PUMA is dependent on PHB expression.

Cytokine and chemokine profiles in episodes of persistent high-risk febrile neutropenia in children with cancer.

BACKGROUND: In children with cancer and persistent high-risk febrile neutropenia (HRFN), cytokines/chemokines profiles can guide the differentiation of febrile neutropenia (FN) due to infections and episodes of unknown origin (FN-UO). METHODS: A prospective, multicenter study in Santiago, Chile included patients ≤ 18 years with cancer and HRFN. Clinical and microbiological studies were performed according to validated protocols. Serum levels of 38 cytokines/chemokines were determined on day 4 of persistent HRFN. We performed comparisons between i) HRFN episodes with a detected etiological agent (FN-DEA) and FN-UO, and ii) bacterial versus viral infections. ROC curves were used to assess the discriminatory power of the analytes. RESULTS: 110 HRFN episodes were enrolled (median age 8 years, 53% female). Eighty-four patients were FN-DEA: 44 bacterial, 32 viral, and 8 fungal infections. Twenty-six cases were categorized as FN-UO. Both groups presented similar clinical and laboratory characteristics. Nineteen out of 38 analytes had higher concentrations in the FN-DEA versus FN-UO group. G-CSF, IL-6, and Flt-3L showed the highest discriminatory power to detect infection (AUC 0.763, 0.741, 0.701). Serum levels of G-CSF differentiated bacterial infections and IP-10 viral agents. A combination of G-CSF, IL-6, Flt-3L, and IP-10 showed an AUC of 0.839, 75% sensitivity, and 81% specificity. CONCLUSION: A specific immune response is present on day four of persistent HRFN in children with cancer. We propose a combined measure of serum concentrations of G-CSF, IL-6, IP-10, and Flt-3L, in order to predict the presence of an infectious agent as compared to an episode of FN with unknown origin.

Activation of the Integrated Stress Response and ER Stress Protect from Fluorizoline-Induced Apoptosis in HEK293T and U2OS Cell Lines.

The prohibitin (PHB)-binding compound fluorizoline as well as PHB-downregulation activate the integrated stress response (ISR) in HEK293T and U2OS human cell lines. This activation is denoted by phosphorylation of eIF2α and increases in ATF4, ATF3, and CHOP protein levels. The blockage of the activation of the ISR by overexpression of GRP78, as well as an increase in IRE1 activity, indicate the presence of ER stress after fluorizoline treatment. The inhibition of the ER stress response in HEK293T and U2OS led to increased sensitivity to fluorizoline-induced apoptosis, indicating a pro-survival role of this pathway after fluorizoline treatment in these cell lines. Fluorizoline induced an increase in calcium concentration in the cytosol and the mitochondria. Finally, two different calcium chelators reduced fluorizoline-induced apoptosis in U2OS cells. Thus, we have found that fluorizoline causes increased ER stress and activation of the integrated stress response, which in HEK293T and U2OS cells are protective against fluorizoline-induced apoptosis.

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.

Respiratory Syncytial Virus Genotypes, Host Immune Profiles, and Disease Severity in Young Children Hospitalized With Bronchiolitis.

Background: Data on how respiratory syncytial virus (RSV) genotypes influence disease severity and host immune responses is limited. Here, we characterized the genetic variability of RSV during 5 seasons, and evaluated the role of RSV subtypes, genotypes, and viral loads in disease severity and host transcriptional profiles. Methods: A prospective, observational study was carried out, including a convenience sample of healthy infants hospitalized with RSV bronchiolitis. Nasopharyngeal samples for viral load quantitation, typing, and genotyping, and blood samples for transcriptome analyses were obtained within 24 hours of hospitalization. Multivariate models were constructed to identify virologic and clinical variables predictive of clinical outcomes. Results: We enrolled 253 infants (median age 2.1 [25%-75% interquartile range] months). RSV A infections predominated over RSV B and showed greater genotype variability. RSV A/GA2, A/GA5, and RSV B/BA were the most common genotypes identified. Compared to GA2 or BA, infants with GA5 infections had higher viral loads. GA5 infections were associated with longer hospital stay, and with less activation of interferon and increased overexpression of neutrophil genes. Conclusions: RSV A infections were more frequent than RSV B, and displayed greater variability. GA5 infections were associated with enhanced disease severity and distinct host immune responses.

Intravenous palivizumab in respiratory syncytial virus infection after hematopoietic stem cell transplant in children.

Respiratory syncytial virus (RSV) infection can cause lower respiratory tract disease and mortality in pediatric hematopoietic stem cell transplant (HSCT) recipients. We report two children who underwent HSCT and developed RSV infection simultaneously at the Bone Marrow Transplant Unit. The treatment with intravenous palivizumab was provided and sequential viral loads were measured in nasopharyngeal (NP) and whole blood samples. To our knowledge, this is the first report where RSV loads were measured in parallel (NP and blood), before and after palivizumab, in correlation with a favorable clinical outcome in both cases.

Genomic Loads and Genotypes of Respiratory Syncytial Virus: Viral Factors during Lower Respiratory Tract Infection in Chilean Hospitalized Infants.

The clinical impact of viral factors (types and viral loads) during respiratory syncytial virus (RSV) infection is still controversial, especially regarding newly described genotypes. In this study, infants with RSV bronchiolitis were recruited to describe the association of these viral factors with severity of infection. RSV antigenic types, genotypes, and viral loads were determined from hospitalized patients at Hospital Roberto del Río, Santiago, Chile. Cases were characterized by demographic and clinical information, including days of lower respiratory symptoms and severity. A total of 86 patients were included: 49 moderate and 37 severe cases. During 2013, RSV-A was dominant (86%). RSV-B predominated in 2014 (92%). Phylogenetic analyses revealed circulation of GA2, Buenos Aires (BA), and Ontario (ON) genotypes. No association was observed between severity of infection and RSV group (p = 0.69) or genotype (p = 0.87). After a clinical categorization of duration of illness, higher RSV genomic loads were detected in infants evaluated earlier in their disease (p < 0.001) and also in infants evaluated later, but coursing a more severe infection (p = 0.04). Although types and genotypes did not associate with severity in our children, higher RSV genomic loads and delayed viral clearance in severe patients define a group that might benefit from new antiviral therapies.

Constrained Cyclopeptides: Biaryl Formation through Pd-Catalyzed C-H Activation in Peptides-Structural Control of the Cyclization vs. Cyclodimerization Outcome.

A series of short tryptophan-phenylalanine peptides containing an iodo substituent on the phenyl ring was subjected to Pd-catalyzed CH activation reactions to give the corresponding aryl-indole coupled products. Two types of adducts were generated: cyclomonomer and cyclodimeric peptides; no evidence of oligo- or polymerization products was detected. Contrary to standard peptide macrocyclizations, the factors controlling the fate of the reaction are the number of amino acids between the aromatic residues and the regiochemistry of the parent iodo derivative, independent of both the concentration and the cyclization mode. The method is general and allows access to novel biaryl peptidic topologies, which have been fully characterized.

Enhanced antimicrobial activity of a peptide derived from human lysozyme by arylation of its tryptophan residues.

Antimicrobial peptides are valuable agents to fight antibiotic resistance. These amphipatic species display positively charged and hydrophobic amino acids. Here, we enhance the local hydrophobicity of a model peptide derived from human lysozyme (107RKWVWWRNR115) by arylation of its tryptophan (Trp) residues, which renders a positive effect on Staphylococcus aureus and Staphylococcus epidermidis growth inhibition. This site-selective modification was accessed by solid-phase peptide synthesis using the non-proteinogenic amino acid 2-aryltryptophan, generated by direct C-H activation from protected Trp. The modification brought about a relevant increase in growth inhibition: S. aureus was fully inhibited by arylation of Trp 112 and by only 10% by arylation of Trp 109 or 111, respect to the non-arylated peptide. On the other hand, S. epidermidis was fully inhibited by the three arylated peptides and the parent peptide. The minimum inhibitory concentration was significantly reduced for S. aureus depending on the arylation site.