Theranostic Fluorescent Probes.

The ability to gain spatiotemporal information, and in some cases achieve spatiotemporal control, in the context of drug delivery makes theranostic fluorescent probes an attractive and intensely investigated research topic. This interest is reflected in the steep rise in publications on the topic that have appeared over the past decade. Theranostic fluorescent probes, in their various incarnations, generally comprise a fluorophore linked to a masked drug, in which the drug is released as the result of certain stimuli, with both intrinsic and extrinsic stimuli being reported. This release is then signaled by the emergence of a fluorescent signal. Importantly, the use of appropriate fluorophores has enabled not only this emerging fluorescence as a spatiotemporal marker for drug delivery but also has provided modalities useful in photodynamic, photothermal, and sonodynamic therapeutic applications. In this review we highlight recent work on theranostic fluorescent probes with a particular focus on probes that are activated in tumor microenvironments. We also summarize efforts to develop probes for other applications, such as neurodegenerative diseases and antibacterials. This review celebrates the diversity of designs reported to date, from discrete small-molecule systems to nanomaterials. Our aim is to provide insights into the potential clinical impact of this still-emerging research direction.

Targeting chemokine receptors from the inside-out: discovery and development of small-molecule intracellular antagonists.

Ever since the first biologically active chemokines were discovered in the late 1980s, these messenger proteins and their receptors have been the target for a plethora of drug discovery efforts in the pharmaceutical industry, as well as in academia. Owing to the publication of several chemokine receptor X-ray crystal structures, a highly druggable, intracellular, allosteric binding site which partially overlaps with the G protein binding site was discovered. This intriguing, new approach for chemokine receptor antagonism has captured researchers around the world, pushing the exploration of this intracellular binding site and new antagonists thereof. In this review, we have highlighted the past two decades of research on small-molecule chemokine receptor antagonists that modulate receptor function at the intracellular binding site.

Picomolar-sensitive ß-amyloid fibril fluorophores by tailoring the hydrophobicity of biannulated π-elongated dioxaborine-dyes.

The pathological origin of Alzheimer's disease (AD) is still shrouded in mystery, despite intensive worldwide research efforts. The selective visualization of ß-amyloid (Aß), the most abundant proteinaceous deposit in AD, is pivotal to reveal AD pathology. To date, several small-molecule fluorophores for Aß species have been developed, with increasing binding affinities. In the current work, two organic small-molecule dioxaborine-derived fluorophores were rationally designed through tailoring the hydrophobicity with the aim to enhance the binding affinity for Aß1-42 fibrils -while concurrently preventing poor aqueous solubility-via biannulate donor motifs in D-π-A dyes. An unprecedented sub-nanomolar affinity was found (K d = 0.62 ± 0.33 nM) and applied to super-sensitive and red-emissive fluorescent staining of amyloid plaques in cortical brain tissue ex vivo. These fluorophores expand the dioxaborine-curcumin-based family of Aß-sensitive fluorophores with a promising new imaging agent.

Calix[n]triazolium based turn-on fluorescent sensing ensemble for selective adenosine monophosphate (AMP) detection.

A novel calix[n]triazolium was synthesized and exhibited excellent selectivity for AMP. The binding between calix[n]triazolium and chromenolate anions forms a non-fluorescent complex and the resulting supramolecular ensemble selectively detects AMP in water and induces "turn-on" fluorescence. The sensing platform is the first macrocyclic system to discriminate AMP from ADP and ATP through fluorescence changes.

A Small Molecule Strategy for Targeting Cancer Stem Cells in Hypoxic Microenvironments and Preventing Tumorigenesis.

Breast cancer consists of heterogenic subpopulations, which determine the prognosis and response to chemotherapy. Among these subpopulations, a very limited number of cancer cells are particularly problematic. These cells, known as breast cancer stem cells (BCSCs), are thought responsible for metastasis and recurrence. They are thus major contributor to the unfavorable outcomes seen for many breast cancer patients. BCSCs are more prevalent in the hypoxic niche. This is an oxygen-deprived environment that is considered crucial to their proliferation, stemness, and self-renewal but also one that makes BCSCs highly refractory to traditional chemotherapeutic regimens. Here we report a small molecule construct, AzCDF, that allows the therapeutic targeting of BCSCs and which is effective in normally refractory hypoxic tumor environments. A related system, AzNap, has been developed that permits CSC imaging. Several design elements are incorporated into AzCDF, including the CAIX inhibitor acetazolamide (Az) to promote localization in MDA-MB-231 CSCs, a dimethylnitrothiophene subunit as a hypoxia trigger, and a 3,4-difluorobenzylidene curcumin (CDF) as a readily released therapeutic payload. This allows AzCDF to serve as a hypoxia-liable molecular platform that targets BCSCs selectively which decreases CSC migration, retards tumor growth, and lowers tumorigenesis rates as evidenced by a combination of in vitro and in vivo studies. To the best of our knowledge, this is the first time a CSC-targeting small molecule has been shown to prevent tumorigenesis in an animal model.

Data driven evaluation of healthy volunteer characteristics at screening for phase I clinical trials to inform on study design and optimize screening processes.

Protocols for clinical trials describe inclusion and exclusion criteria based on general and compound-specific considerations to ensure subject safety and data quality. In phase I clinical trials, healthy volunteers (HVs) are screened against these criteria that often specify predefined eligibility ranges for vital signs, electrocardiogram, and laboratory tests. HVs are excluded if baseline parameters deviate from these ranges even though this may not indicate underlying pathology, which could delay trial execution. Data from 3365 HVs participating in 9670 screening visits for 94 phase I HV trials, conducted between December 2008 and May 2019 at the Janssen Clinical Pharmacology Unit, were retrospectively analyzed. Commonly predefined protocol ranges were overlaid with HV data to estimate predicted screen failure rates (SFRs). Of the overall population, 91% was White and 64% were men with mean age of 42.8 ± 12.5 years. High predicted SFRs are related to cardiovascular/metabolic (body mass index, heart rate [HR], blood pressure [BP], and corrected QT Fridericia's formula [QTcF]), renal (estimated glomerular filtration rate [eGFR]), liver (alanine aminotransferase [ALT], and total bilirubin), and coagulation (prothrombin time [PT]) parameters. Predicted SFRs increased with age for high systolic and diastolic BP, QTcF interval, and eGFR. In contrast, lower SFRs in the older age groups were seen for low diastolic BP, liver function test, ALT, PT, and total bilirubin. This analysis can be used to inform on study design, protocol inclusion and exclusion criteria, and to optimize the screening process. Data-driven critical appraisal of proposed inclusion and exclusion criteria using a risk-based approach may significantly reduce screen failure rates without compromising subjects' safety.

Human Glioblastoma Visualization: Triple Receptor-Targeting Fluorescent Complex of Dye, SIWV Tetra-Peptide, and Serum Albumin Protein.

Fluorescence guided surgery (FGS) has been highlighted in the clinical site for guiding surgical procedures and providing the surgeon with a real-time visualization of the operating field. FGS is a powerful technique for precise surgery, particularly tumor resection; however, clinically approved fluorescent dyes have often shown several limitations during FGS, such as non-tumor-targeting, low in vivo stability, insufficient emission intensity, and low blood-brain barrier penetration. In this study, we disclose a fluorescent dye complex, peptide, and protein for the targeted visualization of human glioblastoma (GBM) cells and tissues. Our noble triple receptor-targeting fluorescent complex (named BSA-OXN-SIWV) consists of (i) dipolar oxazepine dye (OXN), which has high stability, low cytotoxicity, bright fluorescence, and two-photon excitable, (ii) tetra-peptide (SIWV) for the targeting of the caveolin-1 receptor, and (iii) bovine serum-albumin (BSA) protein for the targeting of albondin (gp60) and secreted protein acidic and rich in cysteine receptor. The photophysical properties and binding mode of BSA-OXN-SIWV were analyzed, and the imaging of GBM cell lines and human clinical GBM tissues were successfully demonstrated in this study. Our findings hold great promise for the application of BSA-OXN-SIWV to GBM identification and the surgery at clinical sites, as a new FGS agent.

Mitochondrial relocation of a common synthetic antibiotic: A non-genotoxic approach to cancer therapy.

Tumor recurrence as a result of therapy-induced nuclear DNA lesions is a major issue in cancer treatment. Currently, only a few examples of potentially non-genotoxic drugs have been reported. Mitochondrial re-localization of ciprofloxacin, one of the most commonly prescribed synthetic antibiotics, is reported here as a new approach. Conjugating ciprofloxacin to a triphenyl phosphonium group (giving lead Mt-CFX), is used to enhance the concentration of ciprofloxacin in the mitochondria of cancer cells. The localization of Mt-CFX to the mitochondria induces oxidative damage to proteins, mtDNA, and lipids. A large bias in favor of mtDNA damage over nDNA was seen with Mt-CFX, contrary to classic cancer chemotherapeutics. Mt-CFX was found to reduce cancer growth in a xenograft mouse model and proved to be well tolerated. Mitochondrial relocalization of antibiotics could emerge as a useful approach to generating anticancer leads that promote cell death via the selective induction of mitochondrially-mediated oxidative damage.

Visualizing mitochondria and mouse intestine with a fluorescent complex of a naphthalene-based dipolar dye and serum albumin.

We have explored a new research field of fluorophores through the manipulation of fluorophore-binding proteins. The development of a new imaging agent for tracing a specific organelle or a particular site within a living organism has been of great interest in the field of basic science as well as translational medicine. In this work and for the first time, we will disclose a new naphthalene-based dipolar dye and its complex, with serum albumin (SA), and show their applicability for the selective imaging of mitochondria in cells and the intestine in a mouse. The SA-binding dipolar dye, IPNHC, was synthesized straightforwardly, and we identified its photophysical properties and binding mode with SA. IPNHC-SA complex showed a bright emission in the blue wavelength range with a high quantum yield and stability. In the fluorescence imaging study, bright fluorescence images of mouse intestines were observed under a UV light, as well as two-photon (TP) deep tissue imaging after intravenous injection of IPNHC and IPNHC-SA complex. The present findings hold great promise for the application of the fluorescent complex for use in the tracing and tracking of intestine-related diseases at clinical sites.

Combining viscosity-restricted intramolecular motion and mitochondrial targeting leads to selective tumor visualization.

We report a novel fluorescent molecular conjugate, V-M1, enabling an accurate visualization of tumor tissues. The emission wavelength of V-M1 exceeds 650 nm, which is well within the near-infrared therapeutic window. Tumor accumulation of this cationic dye allows the visualization of cancerous cells as a function of mitochondrial viscosity.

STORM imaging of mitochondrial dynamics using a vicinal-dithiol-proteins-targeted probe.

Stochastic optical reconstruction microscopy (STORM) is a promising method for the visualization of ultra-fine mitochondrial structures. However, this approach is limited to monitoring dynamic intracellular events owing to its low temporal resolution. We developed a new strategy to capture mitochondrial dynamics using a compressed sensing STORM algorithm following raw data pre-treatments by a noise-corrected principal component analysis and K-factor image factorization. Using STORM microscopy with a vicinal-dithiol-proteins targeting probe, visualizing mitochondrial dynamics was attainable with spatial and temporal resolutions of 45 nm and 0.8 s, notably, dynamic mitochondrial tubulation retraction of ~746 nm in 1.2 s was monitored. The labeled conjugate was observed as clusters (radii, ~90 nm) distributed on the outer mitochondrial membranes, not yet reported as far as we know. This strategy is promising for the quantitative analysis of intracellular behaviors below the optical diffraction limit.

MDM2-Associated Clusterization-Triggered Emission and Apoptosis Induction Effectuated by a Theranostic Spiropolymer.

Heteroatom-containing spiropolymers were constructed in a facile manner by a catalyst-free multicomponent spiropolymerization route. P1a2b as the most potent of these spiropolymers, demonstrates cluster-triggered emission resulting from strong interactions with the MDM2 protein. By preventing the anti-apoptotic p53/MDM2 interaction, P1a2b triggers apoptosis in cancerous cells, while demonstrating a good biocompatibility and non-toxicity in non-cancerous cells. The combined results from solution and cell-based cluster-triggered emission studies, docking, protein expression experiments and cytotoxicity data strongly support the MDM2-binding hypothesis and indicate a potential application as a fluorescent cancer marker as well as therapeutic for this spiropolymer.

Crown ether-appended calix[2]triazolium[2]arene as a macrocyclic receptor for the recognition of the H2PO4- anion.

A crown ether-appended calix[2]triazolium[2]arene, which exhibits excellent selectivity for H2PO4- compared to other anions, has been designed and synthesized. The selectivity of the prepared receptor for H2PO4- is caused by the stabilization of H2PO4- by the neighboring triazolium hydrogen bond donors and crown ether hydrogen bond acceptors.

In Vitro Activation of Cytochrome P450 46A1 (CYP46A1) by Efavirenz-Related Compounds.

Cytochrome P450 46A1 (CYP46A1) is a central nervous system-specific enzyme, which catalyzes cholesterol 24-hydroxylation. Currently CYP46A1 is being evaluated in a clinical trial for activation by small doses of the anti-HIV drug efavirenz. Eight efavirenz-related compounds were investigated for CYP46A1 activation in vitro, induction of a CYP46A1 spectral response, spectral Kd values, interaction with the P450 allosteric sites, and a model of binding to the enzyme active site. We gained insight into structure-activity relationships of efavirenz for CYP46A1 activation and found that the investigated efavirenz primary metabolites are stronger and better activators of CYP46A1 than efavirenz. We also established that CYP46A1 is activated by racemates and that a conformational-selection mechanism is operative in CYP46A1. The results suggest structural modifications of efavirenz to further increase CYP46A1 activation without inhibition at high compound concentrations. It is possible that not only efavirenz but its metabolites activate CYP46A1 in vivo.

Nanomolar detection of adenosine triphosphate (ATP) using a nanostructured fluorescent chemosensing ensemble.

We report a novel nanostructured chemosensing ensemble PyNp-C13/UD, obtained by self-assembling uranine dye (UD) and an amphiphilic pyridinium salt PyNp-C13. The ensemble was developed for the fluorescence turn-on sensing of ATP in aqueous solutions and inside living cells. The assembly operates via an indicator displacement assay (IDA) method with an ultra-low detection limit of 6.8 nM.

Revealing Protein Aggregates under Thapsigargin-Induced ER Stress Using an ER-Targeted Thioflavin.

Endoplasmic reticulum-thioflavin T (ER-ThT), a thioflavin T-based fluorescent chemosensor, was developed to detect protein aggregates in the endoplasmic reticulum (ER) and was applied to live cells under various forms of ER stress. Upon dithiothreitol (DTT)-induced reductive denaturation of lysozyme and albumin, the intensity was increased in a protein concentration-dependent way, following a nonfluorescent lag phase. ER-ThT detects protein aggregates rather than unfolded proteins in solution, and the protein aggregation can be visualized in the presence of lipid membranes or native proteins. Within live HeLa cells, ER-ThT is localized in the ER and its fluorescence was dramatically increased upon ER stress induction by DTT, Thapsigargin, or Brefeldin A. Moreover, in the presence of ER stress modulators (tauroursodeoxycholic acid, trimethylamine N-oxide, or 4-phenylbutyric acid), also known as chemical chaperones, the fluorescence under Thapsigargin treatment was suppressed to the level of the control group. Thus, ER-ThT is capable of detecting the accumulation of protein aggregates under ER stress in living cells and acts as an in vitro screening tool for ER stress modulators, putative prodrugs against ER-related proteopathy. Overall, the results strongly suggest that protein aggregation is intricately involved in the activation of the unfolded protein response following ER stress.

Enhanced sensitivity of fluorescence-based Fe(ii) detection by freezing.

The first example of combining the fluorescent probe-based freeze concentration effect with N-oxide chemistry is reported for the highly sensitive and selective detection of ferrous ion (Fe(ii)). Interestingly, our preliminary results demonstrated that the fluorescence intensity of Fe(ii) was markedly enhanced upon freezing, and the location of Fe(ii) in the freezing state was visualized by confocal microscopy using a cryostage.

A colorimetric and fluorescent lighting-up sensor based on ICT coupled with PET for rapid, specific and sensitive detection of nitrite in food.

An anthracene carboxyimide derivative was synthesized as a colorimetric and fluorogenic sensor to determine NO2- with a rapid response (<4 min), excellent selectivity and a low detection limit (84 nM). Paper strips containing the sensor were applied to visually determine the NO2- content in food.