Preclinical evaluation of AGTR1-Targeting molecular probe for colorectal cancer imaging in orthotopic and liver metastasis mouse models.

Despite advancements in colorectal cancer (CRC) treatment, the prognosis remains unfavorable for patients with distant liver metastasis. Fluorescence molecular imaging with specific probes is increasingly used to guide CRC surgical resection in real-time and treatment planning. Here, we demonstrate the targeted imaging capacity of an MPA-PEG4-N3-Ang II probe labeled with near-infrared (NIR) fluorescent dye targeting the angiotensin II (Ang II) type 1 receptor (AGTR1) that is significantly upregulated in CRC. MPA-PEG4-N3-Ang II was highly selective and specific to in vitro tumor cells and in vivo tumors in a mouse CRC xenograft model. The favorable ex vivo imaging and in vivo biodistribution of MPA-PEG4-N3-Ang II afforded tumor-specific accumulation with low background and >10 contrast tumor-to-colorectal values in multiple subcutaneous CRC models at 8 h following injection. Biodistribution analysis confirmed the probe's high uptake in HT29 and HCT116 orthotopic and liver metastatic models of CRC with signal-to-noise ratio (SNR) values of tumor-to-colorectal and -liver fluorescence of 5.8 ± 0.6, 5.3 ± 0.7, and 2.7 ± 0.5, 2.6 ± 0.5, respectively, enabling high-contrast intraoperative tumor visualization for surgical navigation. Given its rapid tumor targeting, precise tumor boundary delineation, durable tumor retention and docking study, MPA-PEG4-N3-Ang II is a promising high-contrast imaging agent for the clinical detection of CRC.

Visualize intracellular ß-galactosidase using an asymmetric near-infrared fluorescent probe with a large Stokes shift.

ß-galactosidase (ß-Gal) is an important biomarker of cell senescence and primary ovarian cancer. Therefore, it is of great significance to construct a near-infrared fluorescent probe with deep tissue penetration and a high signal-to-noise ratio for visualization of ß-galactosidase in biological systems. However, most near-infrared probes tend to have small Stokes shifts and low signal-to-noise ratios due to crosstalk between excitation and emission spectra. Using d-galactose residues as specific recognition units and near-infrared dye TJ730 as fluorophores, a near-infrared fluorescence probe SN-CR with asymmetric structure was developed for the detection of ß-Gal. The probe has a fast reaction equilibrium time (<12 min) with ß-Gal, excellent biocompatibility, near-infrared emission (738 nm), low detection limit (0.0029 U/mL), and no crosstalk between the excitation spectrum and emission spectrum (Stokes shifts 142 nm) of the probe. Cell imaging studies have shown that SN-CR can visually trace ß-Gal in different cells and distinguish ovarian cancer cells from other cells.

Clickable, selective, and cell-permeable activity-based probe of human cathepsin B - Minimalistic approach for enhanced selectivity.

Human cathepsin B is a cysteine-dependent protease whose roles in both normal and diseased cellular states remain yet to be fully delineated. This is primarily due to overlapping substrate specificities and lack of unambiguously annotated physiological functions. In this work, a selective, cell-permeable, clickable and tagless small molecule cathepsin B probe, KDA-1, is developed and kinetically characterized. KDA-1 selectively targets active site Cys25 residue of cathepsin B for labeling and can detect active cellular cathepsin B in proteomes derived from live human MDA-MB-231 breast cancer cells and HEK293 cells. It is anticipated that KDA-1 probe will find suitable applications in functional proteomics involving human cathepsin B enzyme.

Asialoglycoprotein receptor targeted optical and magnetic resonance imaging and therapy of liver fibrosis using pullulan stabilized multi-functional iron oxide nanoprobe.

Early diagnosis and therapy of liver fibrosis is of utmost importance, especially considering the increased incidence of alcoholic and non-alcoholic liver syndromes. In this work, a systematic study is reported to develop a dual function and biocompatible nanoprobe for liver specific diagnostic and therapeutic applications. A polysaccharide polymer, pullulan stabilized iron oxide nanoparticle (P-SPIONs) enabled high liver specificity via asialogycoprotein receptor mediation. Longitudinal and transverse magnetic relaxation rates of 2.15 and 146.91 mM-1 s-1 respectively and a size of 12 nm, confirmed the T2 weighted magnetic resonance imaging (MRI) efficacy of P-SPIONs. A current of 400A on 5 mg/ml of P-SPIONs raised the temperature above 50 °C, to facilitate effective hyperthermia. Finally, a NIR dye conjugation facilitated targeted dual imaging in liver fibrosis models, in vivo, with favourable histopathological results and recommends its use in early stage diagnosis using MRI and optical imaging, and subsequent therapy using hyperthermia.

Chemiluminescent probe for the detection of inverse electron demand Diels-Alder reaction between tetrazine and trans-Cyclooctene.

A chemiluminescent probe has been developed, consisting of phenoxy-dioxetane moiety covalently attached to trans-cyclooctene. The inverse electron demand Diels-Alder reaction with tetrazine produces a cycloaddition product which undergoes a series of spontaneous rearrangements resulting in emission of green light. The chemiluminescent probe can be applied to study bioconjugation chemistry with tetrazine-modified biomaterials, which have recently been shown to have great potential for anticancer drug delivery. This work describes in vitro studies, including NMR and spectroscopic investigation of chemiluminescence, which will pave way for future in vivo bioconjugation experiments.

Design of 99mTc-labeled zinc-chelating imaging probe for SPECT imaging of the pancreas.

Early and sensitive diagnosis of pancreatic diseases is a contemporary clinical challenge. Zinc level in pancreatic tissue and its secretion in pancreatic juice has long been considered a surrogate marker of pancreatic function. The objective of this study was to design a Zn-chelating imaging probe (ZCIP) which could be labeled with 99mTc radionuclide for imaging of pancreas using single photon emission tomography (SPECT). We synthesized ZCIP as a bifunctional chelate consisting of diethylene triamine pentaacetic acid for 99mTc-chelation at one end and bispicolylethylamine for Zn-complexation at the other end. ZCIP was labeled with 99mTc by standard Sn2+-based reduction method. The 99mTc-labeled ZCIP was studied in normal mice (0.3 mCi) for SPECT imaging. We found that ZCIP consistently labeled with 99mTc radionuclide with over 95% efficiency. Addition of ZCIP altered the spectrum of standard dithizone-Zn complex, indicating its ability to chelate Zn. SPECT data demonstrated the ability of 99mTc-ZCIP to image pancreas with high sensitivity in a non-invasive manner; liver and spleen were the other major organs of 99mTc-ZCIP uptake. Based on these results, we conclude that 99mTc-ZCIP presents as a novel radiotracer for pancreas imaging for diagnosis of diseases such as pancreatitis.

MMP-2-Activatable Photoacoustic Tumor Imaging Probes Based on Al- and Si-Naphthalocyanines.

Enzyme-activatable photoacoustic probes are powerful contrast agents to visualize diseases in which a specific enzyme is overexpressed. In this study, aluminum and silicon naphthalocyanines (AlNc and SiNc, respectively) conjugated with matrix metalloprotease-2 (MMP-2)-responsive PLGLAG peptide sequence and poly(ethylene glycol) (PEG) as an axial ligand were designed and synthesized. AlNc-peptide-PEG conjugates AlNc-pep-PEG formed dimeric species interacting with each other through face-to-face H-aggregation in water, while SiNc-based conjugates SiNc-pep-PEG hardly interacted with each other because of the two bulky hydrophilic axial ligands. Both conjugates formed spherical nanometer-sized self-assemblies in water, generating photoacoustic waves under near-infrared photoirradiation. The treatment of MNc-peptide-PEG conjugates (M = Al, Si) with MMP-2 smoothly induced the cleavage of the PLGLAG sequence to release the hydrophilic PEG moiety, resulting in the aggregation of MNcs. By comparing the PA signal intensity changes at 680 and 760 nm, the photoacoustic signal intensity ratios were shown to be enhanced by 3-5 times after incubation with MMP-2. We demonstrated that MNc-peptide-PEG conjugates (M = Al, Si) could work as activatable photoacoustic probes in the in vitro experiment of MMP-2-overexpressed cell line HT-1080 as well as the in vivo photoacoustic imaging of HT-1080-bearing mice.

Synthesis of L-[5-11 C]Leucine and L-α-[5-11 C]Methylleucine via Pd0 -mediated 11 C-Methylation and Microfluidic Hydrogenation: Potentiality of Leucine PET Probes for Tumor Imaging.

The efficient synthesis of L-[5-11 C]leucine and L-α-[5-11 C]methylleucine has been investigated using a continuous two-step sequence of rapid reactions consisting of Pd0 -mediated 11 C-methylation and microfluidic hydrogenation. The synthesis of L-[5-11 C]leucine and L-α-[5-11 C]methylleucine was accomplished within 40 min with a decay-corrected radiochemical yield of 15-38 % based on [11 C]CH3 I, radiochemical purity of 95-99 %, and chemical purity of 95-99 %. The Pd impurities in the injectable solution measured using inductively coupled plasma mass spectrometry met the international criteria for human use. Positron emission tomography scanning after an intravenous injection of L-[5-11 C]leucine or L-α-[5-11 C]methyl leucine in A431 tumor-bearing mice was performed. As a result, L-α-[5-11 C]methylleucine was found to be a potentially useful probe for visualizing the tumor. Tissue distribution analysis showed that the accumulation value of L-α-[5-11 C]methylleucine in tumor tissue was high [12±3% injected dose/g tissue (%ID/g)].

To View Your Biomolecule, Click inside the Cell.

The surging development of bioorthogonal chemistry has profoundly transformed chemical biology over the last two decades. Involving chemical partners that specifically react together in highly complex biological fluids, this branch of chemistry now allows researchers to probe biomolecules in their natural habitat through metabolic labelling technologies. Chemical reporter strategies include metabolic glycan labelling, site-specific incorporation of unnatural amino acids in proteins, and post-synthetic labelling of nucleic acids. While a majority of literature reports mark cell-surface exposed targets, implementing bioorthogonal ligations in the interior of cells constitutes a more challenging task. Owing to limiting factors such as membrane permeability of reagents, fluorescence background due to hydrophobic interactions and off-target covalent binding, and suboptimal balance between reactivity and stability of the designed molecular reporters and probes, these strategies need mindful planning to achieve success. In this review, we discuss the hurdles encountered when targeting biomolecules localized in cell organelles and give an easily accessible summary of the strategies at hand for imaging intracellular targets.

Development of Covalent, Clickable Probes for Adenosine A1 and A3 Receptors.

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A1 receptor (A1R) and adenosine A3 receptor (A3R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A1R and A3R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A2A and A2B adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A1R and A3R localization and trafficking in native cells and living systems.

Ratiometric Fluorescence Assay for Nitroreductase Activity: Locked-Flavylium Fluorophore as a NTR-Sensitive Molecular Probe.

Nitroreductases belong to a member of flavin-containing enzymes that can reduce nitroaromatic compounds to amino derivatives with NADH as an electron donor. NTR activity is known to be elevated in the cancerous environment and is considered an advantageous target in therapeutic prodrugs for the treatment of cancer. Here, we developed a ratiometric fluorescent molecule for observing NTR activity in living cells. This can provide a selective and sensitive response to NTR with a distinct increase in fluorescence ratio (FI530/FI630) as well as color changes. We also found a significant increase in NTR activity in cervical cancer HeLa and lung cancer A549 cells compared to non-cancerous NIH3T3. We proposed that this new ratiometric fluorescent molecule could potentially be used as a NTR-sensitive molecular probe in the field of cancer diagnosis and treatment development related to NTR activity.

Identifying Poly(ADP-ribose)-Binding Proteins with Photoaffinity-Based Proteomics.

Post-translational modification of proteins with poly(ADP-ribose) (PAR) is an important component of the DNA damage response. Four PAR synthesis inhibitors have recently been approved for the treatment of breast, ovarian, and prostate cancers. Despite the clinical significance of PAR, a molecular understanding of its function, including its binding partners, remains incomplete. In this work, we synthesized a PAR photoaffinity probe that captures and isolates endogenous PAR binders. Our method identified dozens of known PAR-binding proteins and hundreds of novel candidates involved in DNA repair, RNA processing, and metabolism. PAR binding by eight candidates was confirmed using pull-down and/or electrophoretic mobility shift assays. Using PAR probes of defined lengths, we detected proteins that preferentially bind to 40-mer versus 8-mer PAR, indicating that polymer length may regulate the outcome and timing of PAR signaling pathways. This investigation produces the first census of PAR-binding proteins, provides a proteomics analysis of length-selective PAR binding, and associates PAR binding with RNA metabolism and the formation of biomolecular condensates.

A "Three-in-One" Multifunctional Probe for Bcl-2/Mcl-1 Profiling and Visualizing in Situ.

Bcl-2 and Mcl-1, the two arms of the anti-apoptotic Bcl-2 family proteins, have been identified as key regulators of apoptosis and effective therapeutic targets of cancer. However, no small-molecular probe is capable of profiling and visualizing both Bcl-2 and Mcl-1 simultaneously in situ. Herein, we report a multifunctional molecular probe (BnN3 -OPD-Alk) by a "three-in-one" molecular designing strategy, which integrated the Bcl-2/Mcl-1 binding ligand, fluorescent reporter group and photoreactive group azido into the same scaffold. BnN3 -OPD-Alk exhibited sub-micromolar affinities to Bcl-2/Mcl-1 and bright green self-fluorescence. It was then successfully applied for Bcl-2/Mcl-1 labeling, capturing, enriching, and bioimaging both in vitro and in cells. This strategy could facilitate the precise early diagnosis and effective therapy of dual Bcl-2/Mcl-1-related diseases.

α-Methylene-ß-Lactone Scaffold for Developing Chemical Probes at the Two Ends of the Selectivity Spectrum.

The utilities of an α-methylene-ß-lactone (MeLac) moiety as a warhead composed of multiple electrophilic sites are reported. We demonstrate that a MeLac-alkyne not only reacts with diverse proteins as a broadly reactive measurement probe, but also recruits reduced endogenous glutathione (GSH) to assemble a selective chemical probe of GSH-ß-lactone (GSH-Lac)-alkyne in live cells. Tandem mass spectrometry reveals that MeLac reacts with nucleophilic cysteine, serine, lysine, threonine, and tyrosine residues, through either Michael or acyl addition. A peptide-centric proteomics platform demonstrates that the proteomic selectivity profiles of orlistat and parthenolide, which have distinct reactivities, are measurable by MeLac-alkyne as a high-coverage probe. The GSH-Lac-alkyne selectively probes the glutathione S-transferase P responsible for multidrug resistance. The assembly of the GSH-Lac probe exemplifies a modular and scalable route to develop selective probes with different recognizing moieties.

Multifunctional rhodamine B appended ROMP derived fluorescent probe detects Al3+ and selectively labels lysosomes in live cells.

There a few reports of rhodamine-based fluorescent sensors for selective detection of only Al3+, due to the challenge of identifying a suitable ligand for binding Al3+ ion. The use of fluorophore moieties appended to a polymer backbone for sensing applications is far from mature. Here, we report a new fluorescent probe/monomer 4 and its ROMP derived polymer P for specific detection of Al3+ ions. Both monomer 4 and its polymer P exhibit high selectivity toward only Al3+ with no interference from other metal ions, having a limit detection of 0.5 and 2.1 µM, respectively. The reversible recognition of monomer 4 and P for Al3+ was also proved in presence of Na2EDTA by both UV-Vis and fluorometric titration. The experimental data indicates the behavior of 4 and P toward Al3+ is pH independent in medium conditions. In addition, the switch-on luminescence response of 4 at acidic pH (0 < 5.0), allowed us to specifically stain lysosomes (pH ~ 4.5-5.0) in live cells.

Macrocyclic Chelates Bridged by a Diaza-Crown Ether: Towards Multinuclear Bimodal Molecular Imaging Probes.

Bridged polymacrocyclic ligands featured by structurally different cages offer the possibility of coordinating multiple trivalent lanthanide ions, giving rise to the exploitation of their different physicochemical properties, e.g., multimodal detection for molecular imaging purposes. Intrigued by the complementary properties of optical and MR-based image capturing modalities, we report the synthesis and characterization of the polymetallic Ln(III)-based chelate comprised of two DOTA-amide-based ligands (DOTA-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bridged via 1,10-diaza-18-crown-6 (DA18C6) motif. The DOTA-amide moieties and the DA18C6 were used to chelate two Eu(III) ions and one Tb(III) ion, respectively, resulting in a multinuclear heterometallic complex Eu2LTb. The bimetallic complex without Tb(III), Eu2L, displayed a strong paramagnetic chemical exchange saturation transfer (paraCEST) effect. Notably, the luminescence spectra of Eu2LTb featured mixed emission including the characteristic bands of Eu(III) and Tb(III). The advantageous features of the complex Eu2LTb opens new possibilities for the future design of bimodal probes and their potential applicability in CEST MR and optical imaging.

Production of aptamers by cell-SELEX and their applications in cancer biomarker identification.

Cancer is a major cause of disease-related deaths worldwide, and early diagnosis involving detecting biomarkers of tumors can improve the cure rate and prognosis of patients. Biomarkers are signature proteins that can distinguish diseased cells from healthy cells, facilitating the diagnosis and treatment of diseases, especially cancer. Aptamers are single-stranded oligonucleotides that can recognize target proteins with high affinity and specificity. The development of biomarkers identified by aptamers has experienced extensive progress in current applications. The combinatorial strategy of cell-SELEX technology and proteomics analysis makes targeted protein identification more cost-effective and efficient and improves the success rate of discovery of multiple biomarkers simultaneously. In this methodology, biomarkers are identified via a series of operations such as screening of aptamers, separation, extraction, and analysis of target proteins, which has brought about the discovery of a number of new biomarkers of cancer. This review summarizes the current strategies, challenges, and potential applications for biomarker discovery using aptamers engendered by cell-SELEX.

Advances of aptamer-based clinical applications for the diagnosis and therapy of cancer.

Aptamers are short single-stranded oligonucleotides that have attracted considerable attention due to their favorable biological characteristics. Aptamers can specifically target and bind to proteins or tumor cells, achieving tumor diagnosis and therapy in vitro and in vivo. Following an introduction of methodologies of producing aptamers and the recent advances of aptamers being applied to clinical samples or xenograft tumors, tumor diagnosis using aptamers will be reviewed, including fluorescence imaging, radionuclide-based imaging, MRI, histochemical imaging, and multimodality imaging. Preclinical applications in tumor therapy in vivo will also be discussed, covering different kinds of treatment mechanisms, including aptamer therapeutics, chemotherapy, gene therapy, immunotherapy, and combination therapy. Safety and efficacy of tumor-targeting therapeutics via aptamers, as well as the current challenges and future perspectives about aptamers' clinical applications, will be summarized.

Synthesis and Evaluation of 68Ga- and 177Lu-Labeled (R)- vs (S)-DOTAGA Prostate-Specific Membrane Antigen-Targeting Derivatives.

Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer cells and therefore is an attractive target for prostate cancer diagnosis and radionuclide therapy. Recently, published results from clinical studies using a new PSMA-targeting PET imaging agent, [68Ga]Ga-PSMA-093 ([68Ga]Ga-HBED-CC-O-carboxymethyl-Tyr-CO-NH-Glu), support the development of this agent for the diagnosis of prostate cancer. In this study, the HBED-CC chelating group in PSMA-093 was replaced by stereoselective (R)- or (S)-DOTAGA. This chelating group serves not only for chelating 68Ga but is also amendable for complexing other radioactive metals for radionuclide therapy. The corresponding optically pure (R)- and (S)-[68Ga/177Lu]-DOTAGA derivatives, (R)-[68Ga/177Lu]-13 and (S)-[68Ga/177Lu]-13, were successfully prepared. Comparison of radiolabeling, binding affinity, cell uptake, and biodistribution between the two isomers was performed. Radiolabeling of (R)-[177Lu]Lu-13 and (S)-[177Lu]Lu-13 at 50 °C suggested that rates of complex formation were time-dependent and the formation of (S)-[177Lu]Lu-13 was distinctly faster. The rates of complex formation for the corresponding 68Ga agents were comparable between structural isomers. The natGa and natLu equivalents showed high binding PSMA affinity (IC50 = 24-111 nM), comparable to that of the parent agent, [natGa]Ga-PSMA-093 (IC50 = 34.0 nM). Results of cell uptake and biodistribution studies in PSMA-expressing PC3-PIP tumor-bearing mice appeared to show no difference between the labeled (R)- and (S)-isomers. This is the first time that a pair of [68Ga/177Lu]-(R)- and (S)-DOTAGA isomers of PSMA agents were evaluated. Results of biological studies between the isomers showed no noticeable difference; however, the distinctions on the rate of Lu complex formation should be considered in the development of new 177Lu-DOTAGA-based radionuclide therapy agents in the future.

Reactivity-Based Probe of the Iron(II)-Dependent Interactome Identifies New Cellular Modulators of Ferroptosis.

Ferroptosis is an iron-dependent form of cell death resulting from loss or inhibition of cellular machinery that protects from the accumulation of lipid hydroperoxides. Ferroptosis likely serves a tumor suppressing function in normal cellular homeostasis, but certain cancers exploit and become highly dependent on specific nodes of the pathway, presumably to survive under conditions of increased oxidative stress and elevated labile ferrous iron levels. Here we introduce Ferroptosis Inducing Peroxide for Chemoproteomics-1 (FIPC-1), a reactivity-based probe that couples Fenton-type reaction with ferrous iron to subsequent protein labeling via concomitant carbon-centered radical generation. We show that FIPC-1 induces ferroptosis in susceptible cell types and labels cellular proteins in an iron-dependent fashion. Use of FIPC-1 in a quantitative chemoproteomics workflow reproducibly enriched protein targets in the thioredoxin, oxidoreductase, and protein disulfide isomerase (PDI) families, among others. In further interrogating the saturable targets of FIPC-1, we identified the PDI family member P4HB and the functionally uncharacterized protein NT5DC2, a member of the haloacid dehalogenase (HAD) superfamily, as previously unrecognized modulators of ferroptosis. Knockdown of these target genes sensitized cells to known ferroptosis inducers, while PACMA31, a previously reported inhibitor of P4HB, directly induced ferroptosis and was highly synergistic with erastin. Overall, this study introduces a new reactivity-based probe of the ferrous iron-dependent interactome and uncovers new targets for the therapeutic modulation of ferroptosis.