An all-in-one tetrazine reagent for cysteine-selective labeling and bioorthogonal activable prodrug construction.

The prodrug design strategy offers a potent solution for improving therapeutic index and expanding drug targets. However, current prodrug activation designs are mainly responsive to endogenous stimuli, resulting in unintended drug release and systemic toxicity. In this study, we introduce 3-vinyl-6-oxymethyl-tetrazine (voTz) as an all-in-one reagent for modular preparation of tetrazine-caged prodrugs and chemoselective labeling peptides to produce bioorthogonal activable peptide-prodrug conjugates. These stable prodrugs can selectively bind to target cells, facilitating cellular uptake. Subsequent bioorthogonal cleavage reactions trigger prodrug activation, significantly boosting potency against tumor cells while maintaining exceptional off-target safety for normal cells. In vivo studies demonstrate the therapeutic efficacy and safety of this prodrug design approach. Given the broad applicability of functional groups and labeling versatility with voTz, we foresee that this strategy will offer a versatile solution to enhance the therapeutic range of cytotoxic agents and facilitate the development of bioorthogonal activatable biopharmaceuticals and biomaterials.

Insight into small-molecule inhibitors targeting extracellular nucleotide pyrophosphatase/phosphodiesterase1 for potential multiple human diseases.

Extracellular nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as a type II transmembrane glycoprotein. It plays a crucial role in various biological processes, such as bone mineralization, cancer cell proliferation, and immune regulation. Consequently, ENPP1 has garnered attention as a promising target for pharmacological interventions. Despite its potential, the development of clinical-stage ENPP1 inhibitors for solid tumors, diabetes, and silent rickets remains limited. However, there are encouraging findings from preclinical trials involving small molecules exhibiting favorable therapeutic effects and safety profiles. This perspective aims to shed light on the structural properties, biological functions and the relationship between ENPP1 and diseases. Additionally, it focuses on the structure-activity relationship of ENPP1 inhibitors, with the intention of guiding the future development of new and effective ENPP1 inhibitors.

Comprehensive Insights that Targeting PIM for Cancer Therapy: Prospects and Obstacles.

Proviral integration sitea for Moloney-murine leukemia virus (PIM) kinases are a family of highly conserved serine/tyrosine kinases consisting of three members, PIM-1, PIM-2, and PIM-3. These kinases regulate a wide range of substrates through phosphorylation and affect key cellular processes such as transcription, translation, proliferation, apoptosis, and energy metabolism. Several PIM inhibitors are currently undergoing clinical trials, such as a phase I clinical trial of Uzanserti (5) for the treatment of relapsed diffuse large B-cell lymphoma that has been completed. The current focus encompasses the structural and biological characterization of PIM, ongoing research progress on small-molecule inhibitors undergoing clinical trials, and evaluation analysis of persisting challenges in this field. Additionally, the design and discovery of small-molecule inhibitors targeting PIM in recent years have been explored, with a particular emphasis on medicinal chemistry, aiming to provide valuable insights for the future development of PIM inhibitors.

Sequential activation strategy of triazinyl resorufin for high selectivity fluorescence GSH detection.

The abnormally elevated expression level of glutathione (GSH) has been observed in various human cancer cells and tissue. Thus, effective methods for glutathione detection are of great importance in early diagnosis of cancer. However, many fluorescent probes for GSH detection suffer from the interference of the abundantly existent nucleophilic biomolecules in biological environment. In this work, we propose a sequential activation strategy to overcome this problem by designing and synthesizing a series of 1,3,5-triazinyl resorufin turn-on fluorescent probe (Probes 1-3). As two electrophilic sites are presented in probes, GSH sequentially reacts with the resorufin and the triazine moiety, resulting in significant fluorescence augmentation (up to 165.0-fold). Designed probes possess low limit of detection as low as 1.8 µM). Cellular fluorescent imaging has been successfully applied to selectively detect GSH in several living cells.

Small-molecule LRRK2 inhibitors for PD therapy: Current achievements and future perspectives.

Leucine-rich repeat kinase 2 (LRRK2) is a multifunctional protein that orchestrates a diverse array of cellular processes, including vesicle transport, autophagy, lysosome degradation, neurotransmission, and mitochondrial activity. Hyperactivation of LRRK2 triggers vesicle transport dysfunction, neuroinflammation, accumulation of α-synuclein, mitochondrial dysfunction, and the loss of cilia, ultimately leading to Parkinson's disease (PD). Therefore, targeting LRRK2 protein is a promising therapeutic strategy for PD. The clinical translation of LRRK2 inhibitors was historically impeded by issues surrounding tissue specificity. Recent studies have identified LRRK2 inhibitors that have no effect on peripheral tissues. Currently, there are four small-molecule LRRK2 inhibitors undergoing clinical trials. This review provides a summary of the structure and biological functions of LRRK2, along with an overview of the binding modes and structure-activity relationships (SARs) of small-molecule inhibitors targeting LRRK2. It offers valuable references for developing novel drugs targeting LRRK2.

Development and therapeutic potential of adaptor-associated kinase 1 inhibitors in human multifaceted diseases.

Adaptor-Associated Kinase 1 (AAK1), a Ser/Thr protein kinase, responsible for regulating clathrin-mediated endocytosis, is ubiquitous in the central nervous system (CNS). AAK1 plays an important role in neuropathic pain and a variety of other human diseases, including viral invasion, Alzheimer's disease, Parkinson's syndrome, etc. Therefore, targeting AAK1 is a promising therapeutic strategy. However, although small molecule AAK1 inhibitors have been vigorously developed, only BMS-986176/LX-9211 has entered clinical trials. Simultaneously, new small molecule inhibitors, including BMS-911172 and LP-935509, exhibited excellent druggability. This review elaborates on the structure, biological function, and disease relevance of AAK1. We emphatically analyze the structure-activity relationships (SARs) of small molecule AAK1 inhibitors based on different binding modalities and discuss prospective strategies to provide insights into novel AAK1 therapeutic agents for clinical practice.

Targeting FGFRs for tumor therapy: current status and novel strategies.

The FGF receptors (FGFRs) belong to a family of receptor tyrosine kinases. Abundant evidence shows that FGFRs are closely related to tumor cell invasion and angiogenesis. Hence, targeted modulation of FGFRs has become an effective strategy for cancer treatment. Recently, the development of small-molecule inhibitors targeting FGFRs has been extensively studied, and three inhibitors have been approved for marketing. Based on the clinical problems with the current inhibitors, there is a need to develop novel inhibitors and technologies to address the pitfalls. This review summarizes recent advances in small-molecule inhibitors targeting FGFRs, focusing on structure-activity relationships. Moreover, recent progress of novel technologies are summarized to provide a reference for promoting the application of drugs targeting FGFRs in tumor therapy.

Bioorthogonal Cleavage of Tetrazine-Caged Ethers and Esters Triggered by trans-Cyclooctene.

In this study, we report the bioorthogonal cleavage of physiologically stable methylene tetrazines bearing an ether or ester linkage in the presence of trans-cyclooctene. Based on this approach, molecules with phenol or carboxylic acid moieties were efficiently released in a controlled manner, which can be effectively applied in living cells. We expect this bioorthogonal cleavage approach can be applied to several biomedical applications, including the development of antibody-drug conjugates, pretargeted prodrug release, and protein activation.

A smart probe for simultaneous imaging of the lipid/water microenvironment in atherosclerosis and fatty liver.

A smart lipid droplet specific fluorescent probe (LDs-DM) with dual-emission in water (706 nm) and oil (535 nm) under single-excitation was prepared for revealing the ultra-structure of the aqueous and lipidic microenvironment in living cells, fatty liver tissues and atherosclerotic plaques without fluorescence emission crosstalk. This work is expected to provide inspiration for designing a novel probe with color switching ability.

Strategic Design of Amyloid-ß Species Fluorescent Probes for Alzheimer's Disease.

Alzheimer's disease (AD) is a high mortality and high disability rates neurodegenerative disease characterized by irreversible progression and poses a significant social and economic burden throughout the world. However, currently approved AD therapeutic agents only alleviate symptoms and there is still a lack of practical therapeutic regimens to stop or slow the progression of this disease. Thus, there is urgently needed novel diagnosis tools and drugs for early diagnosis and treatment of AD. Among several AD pathological hallmarks, amyloid-ß (Aß) peptide deposition is considered a critical initiating factor in AD. In recent years, with the advantages of excellent sensitivity and high resolution, near-infrared fluorescence (NIRF) imaging has attracted the attention of many researchers to develop Aß plaque probes. This review mainly focused on different NIRF probe design strategies for imaging Aß species to pave the way for the future design of novel NIRF probes for early diagnosis AD.

Overcoming Spectral Dependence: A General Strategy for Developing Far-Red and Near-Infrared Ultra-Fluorogenic Tetrazine Bioorthogonal Probes.

Bioorthogonal fluorogenic dyes are indispensable tools in wash-free bioimaging of specific biological targets. However, the fluorogenicity of existing tetrazine-based bioorthogonal probes deteriorates as the emission wavelength shifts towards the NIR window, greatly limiting their applications in live cells and tissues. Herein, we report a generalizable molecular design strategy to construct ultra-fluorogenic dyes via a simple substitution at the meso-positions of various far-red and NIR fluorophores. Our probes demonstrate significant fluorescence turn-on ratios (102 -103 -fold) in the range 586-806 nm. These results will greatly expand the applications of bioorthogonal chemistry in NIR bioimaging and biosensing.

Stereochemically altered cephalosporins as potent inhibitors of New Delhi metallo-ß-lactamases.

Antibiotic resistance caused by ß-lactamases, particularly metallo-ß-lactamases, has been a major threat to public health globally. New Delhi metallo-ß-lactamase-1 (NDM-1) represents one of the most important metallo-ß-lactamases; the production of NDM-1 in bacterial pathogen significantly reduces the efficacy of ß-lactam antibiotics, including life-saving carbapenems. Herein, we have demonstrated stereochemically altered cephalosporins as potent inhibitors against NDM-1, as well as mutants of NDM. The structure and activity relationship (SAR) study on over twenty cephalosporin analogues discloses the stereochemistry and the substituents on 7-position and 3'-position of cephalosporin are critical to suppress the activity of NDM-1 and the optimal compound 1u exhibited an IC50 of 0.13 µM. Furthermore, a crystal complex of NDM-1 and 1u has been obtained, suggesting this cephalosporin derivative inhibits enzyme activity by the formation of a relatively stable hydrolytic product-NDM-1 intermediate. The discovery in this study may pave the way to turn cephalosporin, a natural substrate of ß-lactamase, into an effective NDM-1 inhibitor to combat antibiotic resistance.

Isonitrile induced bioorthogonal activation of fluorophores and mutually orthogonal cleavage in live cells.

Fluorophores with different emission wavelengths were efficiently quenched by a tert-butyl terminated tetrazylmethyl group and activated by an isonitrile-tetrazine click-to-release reaction. Nucleic acid templated chemistry significantly accelerated this bioorthogonal cleavage. Moreover, two mutually orthogonal fluorogenic cleavage reactions were simultaneously conducted in live cells for the first time.

A General Strategy to Design Highly Fluorogenic Far-Red and Near-Infrared Tetrazine Bioorthogonal Probes.

Highly fluorogenic tetrazine bioorthogonal probes emitting at near-infrared wavelengths are in strong demand for biomedical imaging applications. Herein, we have developed a strategy for forming a palette of novel Huaxi-Fluor probes in situ, whose fluorescence increases hundreds of times upon forming the bioorthogonal reaction product, pyridazine. The resulting probes show large Stokes shifts and high quantum yields. Manipulating the conjugate length and pull-push strength in the fluorophore skeleton allows the emission wavelength to be fine-tuned from 556 to 728 nm. The highly photo-stable and biocompatible probes are suitable for visualizing organelles in live cells without a washing step and for imaging of tumors in live small animals to depths of 500 µm by two-photon excitation.

Organocatalytic and Scalable Syntheses of Unsymmetrical 1,2,4,5-Tetrazines by Thiol-Containing Promotors.

Despite the growing application of tetrazine bioorthogonal chemistry, it is still challenging to access tetrazines conveniently from easily available materials. Described here is the de novo formation of tetrazine from nitriles and hydrazine hydrate using a broad array of thiol-containing catalysts, including peptides. Using this facile methodology, the syntheses of 14 unsymmetric tetrazines, containing a range of reactive functional groups, on the gram scale were achieved with satisfactory yields. Using tetrazine methylphosphonate as a building block, a highly efficient Horner-Wadsworth-Emmons reaction was developed for further derivatization under mild reaction conditions. Tetrazine probes with diverse functions can be scalably produced in yields of 87-93 %. This methodology may facilitate the widespread application of tetrazine bioorthogonal chemistry.

A carbapenem-based fluorescence assay for the screening of metallo-ß-lactamase inhibitors.

Reported herein is a fluorescence assay for the rapid screening of metallo-ß-lactamase (MBL) inhibitors. This assay employs a fluorogenic carbapenem CPC-1 as substrate and is compatible with all MBLs, including B1, B2 and B3 subclass MBLs. The efficiency of this assay was demonstrated by the rapid inhibition screening of a number of molecules against B2 MBL CphA and 2,3-dimercaprol was identified as a potent CphA inhibitor.

A Carbapenem-Based Off-On Fluorescent Probe for Specific Detection of Metallo-ß-Lactamase Activities.

Metallo-ß-lactamase is one of the major clinical threats because this ß-lactam-hydrolyzing enzyme confers significant resistance to most ß-lactam antibiotics, including carbapenems, among bacterial pathogens. Reported herein is a novel fluorogenic sensor for the specific detection of metallo-ß-lactamase activities. This carbapenem-based reagent exhibits excellent selectivity to metallo-ß-lactamase over other serine-ß-lactamases, including serine carbapenemases. The usefulness of this probe was further demonstrated in the rapid identification of metallo-ß-lactamase-expressing pathogenic bacteria.

Specific Detection of Extended-Spectrum ß-Lactamase Activities with a Ratiometric Fluorescent Probe.

The dissemination of antimicrobial resistance around the world is one of the biggest threats to global public health. The acquisition and expression of extended-spectrum ß-lactamases (ESBLs) in pathogenic bacterial are mainly responsible for bacterial resistance to third-generation cephalosporins. Reported herein is a ratiometric fluorescent probe for the detection of the activity of ESBLs. This imaging reagent adopts the core structure of cefotaxime as an enzymatic recognition moiety, and exhibits excellent selectivity to ESBLs over other ß-lactamases. The specific activation of this sensor by ESBLs can lead to over 2500-fold changes in the fluorescent ratio, which is independent of the concentration of the probe and environmental conditions. Further experiments have demonstrated that this ratiometric fluorescent probe can distinguish bacteria with extended-spectrum antibiotic resistance from a group of clinically important pathogens within a short period of time.