Global Reactivity Profiling of the Catalytic Lysine in Human Kinome for Covalent Inhibitor Development.

Advances in targeted covalent inhibitors (TCIs) have been made by using lysine-reactive chemistries. Few aminophiles possessing balanced reactivity/stability for the development of cell-active TCIs are however available. We report herein lysine-reactive activity-based probes (ABPs; 2-14) based on the chemistry of aryl fluorosulfates (ArOSO2 F) capable of global reactivity profiling of the catalytic lysine in human kinome from mammalian cells. We concurrently developed reversible covalent ABPs (15/16) by installing salicylaldehydes (SA) onto a promiscuous kinase-binding scaffold. The stability and amine reactivity of these probes exhibited a broad range of tunability. X-ray crystallography and mass spectrometry (MS) confirmed the successful covalent engagement between ArOSO2 F on 9 and the catalytic lysine of SRC kinase. Chemoproteomic studies enabled the profiling of >300 endogenous kinases, thus providing a global landscape of ligandable catalytic lysines of the kinome. By further introducing these aminophiles into VX-680 (a noncovalent inhibitor of AURKA kinase), we generated novel lysine-reactive TCIs that exhibited excellent in vitro potency and reasonable cellular activities with prolonged residence time. Our work serves as a general guide for the development of lysine-reactive ArOSO2 F-based TCIs.

A New Fluorescent Probe for Hydrogen Sulfide Detection in Solution and Living Cells.

Since Hydrogen Sulfide (H2S) was recognized as a gas transmitter, its detection and quantification have become a hot research topic among chemists and biologists. In this area, fluorescent probes have shown great advantages: fast and strong response, low detection limit and easy manipulation. Here we developed a new fluorescent probe that detected H2S selectively among various bioactive and inorganic salts. This probe was based on the core structure of fluorescein and reacted with H2S through azide-reduction. Great linearity was achieved correlating fluorescence intensity and H2S concentrations in solution. The detection of H2S in cancer cells was also achieved.

Fluorescent probes for bioimaging of potential biomarkers in Parkinson's disease.

Parkinson's disease (PD), as the second most common neurodegenerative disease, is caused by complex pathological processes and currently remains very difficult to treat. PD brings great distress to patients and imposes a heavy economic burden on society. The number of PD patients is growing as the aging population increases worldwide. Therefore, it is crucial to develop new tools for aiding the early diagnosis and treatment of PD. The significant pathological features involved in PD include the abnormal accumulation of α-synuclein, metal ion dyshomeostasis, oxidative stress, mitochondrial dysfunction and neurotransmitter deficiencies. In recent years, fluorescent probes have emerged as a powerful bioimaging tool with potential to help understand the pathological processes of PD via the detection and monitoring of pathological features. In this review, we comprehensively summarize the design and working mechanisms of fluorescent probes along with their applications in the detection of various PD biomarkers. We also discuss the current limitations of fluorescent probes and provide perspectives on how these limitations can be overcome to develop better fluorescent probes suitable for application in clinical trials in the future. We hope that this review provides valuable information and guidance for the development of new fluorescent probes that can be used clinically in the early diagnosis of PD and contributes to the development of efficient PD drugs in the future.

Engineered Cell-Penetrating Peptides for Mitochondrion-Targeted Drug Delivery in Cancer Therapy.

Mitochondrion is a promising target in cancer therapy. However, gaining access to this organelle is difficult due to the obstacles to cross the complicated mitochondrial membrane. Cell-penetrating peptides (CPPs) with mitochondrion-targeting ability, named mitochondrion-targeting peptides (MTPs), are efficient tools to deliver exogenous therapeutics into mitochondria. Herein, we report several new MTPs, which can be readily synthesized via resin-based solid-phase peptide synthesis. In particular, MTP3 (compound 5), consisting of three positively charged arginines and two D- and L- alternating naphthylalanines, demonstrated excellent mitochondrion-targeting ability with high Pearson's correlation coefficient, suggesting that MTP3 has good potential for mitochondrion-targeted drug delivery. As proof-of-concept, the feasibility of MTP3 was validated by the preparation of a mitochondrion-targeting prodrug (compound 17, doxorubicin-based prodrug). This prodrug was subsequently confirmed to be specifically transported to the mitochondria of tumor cells, where it was able to release the native doxorubicin upon intracellular GSH activation, leading to mitochondrial depolarization and eventually cell death. Importantly, compound 17 showed good cytotoxicity against human tumor cells while negligible toxicity towards normal cells, indicating its potential as a potent mitochondrial medicine for targeted cancer therapy. Our study thus opens a way for engineered CPPs to be used to deliver bioactive cargos in mitochondrion-targeted cancer therapy.

Cell-Penetrating Mitochondrion-Targeting Ligands for the Universal Delivery of Small Molecules, Proteins and Nanomaterials.

Mitochondria are key organelles that perform vital cellular functions such as those related to cell survival and death. The targeted delivery of different types of cargos to mitochondria is a well-established strategy to study mitochondrial biology and diseases. Of the various existing mitochondrion-transporting vehicles, most suffer from poor cytosolic entry, low delivery efficiency, limited cargo types, and cumbersome preparation protocols, and none was known to be universally applicable for mitochondrial delivery of different types of cargos (small molecules, proteins, and nanomaterials). Herein, two new cell-penetrating, mitochondrion-targeting ligands (named MitoLigand ) that are capable of effectively "tagging" small-molecule drugs, native proteins and nanomaterials are disclosed, as well as their corresponding chemoselective conjugation chemistry. Upon successful cellular delivery and rapid endosome escape, the released native cargos were found to be predominantly localized inside mitochondria. Finally, by successfully delivering doxorubicin, a well-known anticancer drug, to the mitochondria of HeLa cells, we showed that the released drug possessed potent cell cytotoxicity, disrupted the mitochondrial membrane potential and finally led to apoptosis. Our strategy thus paves the way for future mitochondrion-targeted therapy with a variety of biologically active agents.

A CE-FL based method for real-time detection of in-capillary self-assembly of the nanoconjugates of polycysteine ligand and quantum dots.

Small molecules with free thiol groups always show high binding affinity to quantum dots (QDs). However, it is still highly challenging to detect the binding capacity between thiol-containing molecules and QDs inside a capillary. To conquer this limitation, a capillary electrophoresis with fluorescence detection (CE-FL) based assay was proposed and established to investigate the binding capacity between QDs and a poly-thiolated peptide (ATTO 590-DDSSGGCCPGCC, ATTO-C4). Interestingly, the results showed that interval time had a great influence on QDs and ATTO-C4 self-assembly, which can be attributed to longer interval time benefitting the binding of QDs to ATTO-C4. The stability assays on ATTO-C4-QD assembly indicated that high concentration of imidazole or GSH had a high capability of competing with the bound ATTO-C4, evidenced by dramatically dropping of S 625/S 565 ratio from 0.78 to 0.30 or 0.29. Therefore, all these results above suggested that this novel CE-FL based detection assay could be successfully applied to the binding studies between QDs and thiol-containing biomolecules.

Syntheses of arabinose-derived pyrrolidine catalysts and their applications in intramolecular Diels-Alder reactions.

Six chiral hydroxylated pyrrolidine catalysts were synthesized from commercially available D-arabinose in seven steps. Various aromatic substituents α to the amine can be introduced readily by a Grignard reaction, which enables facile optimization of the catalyst performance. The stereoselectivities of these catalysts have been assessed by comparing with those of MacMillan's imidazolidinone in a known intramolecular Diels-Alder (IMDA) reaction of a triene. Two additional IMDA reactions of symmetrical dienals with concomitant desymmetrisation further established the potential use of these novel amine catalysts. These pyrrolidines are valuable catalysts for other synthetic transformations.

Rationally designed BCR-ABL kinase inhibitors for improved leukemia treatment via covalent and pro-/dual-drug targeting strategies.

BACKGROUND: Chronic Myeloid Leukemia (CML) is a blood cancer that remains challenging to cure due to drug resistance and side effects from current BCR-ABL inhibitors. There is an urgent need for novel and more effective BCR-ABL targeting inhibitors and therapeutic strategies to combat this deadly disease. METHOD: We disclose an "OH-implant" strategy to improve a noncovalent BCR-ABL inhibitor, PPY-A, by adding a hydroxyl group to its scaffold. By taking advantage of this OH "hot spot", we designed a panel of irreversible covalent kinase inhibitors and hypoxia-responsive pro-/dual-drugs, and their biological activities were studied in vitro, in cellulo and in vivo. RESULT: The resulting compound B1 showed enhanced solubility and biological activity. B4 achieved sustained BCR-ABL inhibition by forming a stable covalent bond with ABL kinase. Hypoxia-responsive prodrug P1 and dual-drugs D1/D2/D3 demonstrated significant anti-tumor effects under hypoxic conditions. The in vivo studies using K562-xenografted mice showed that B1 displayed superior antitumor activity than PPY-A, while P1 and D3 offered better safety profiles alongside significant tumor control. CONCLUSION: We have successfully developed a chemical biology approach to convert a known noncovalent BCR-ABL inhibitor into more potent and safer inhibitors through covalent and pro-/dual-drug targeting strategies. Our "OH-implant" approach and the resulting drug design strategies have general applicability and hold promise for improvement the performance of various other reported drugs/drug candidates, thereby providing advanced medicines for disease treatment.

Amplifying the efficacy of ALA-based prodrugs for photodynamic therapy using nanotechnology.

5-aminolevulinic acid (ALA) is a clinically approved prodrug involved in intracellular Heme biosynthesis to produce the natural photosensitizer (PS) Protoporphyrin IX (PpIX). ALA based photodynamic therapy (PDT) has been used to treat various malignant and non-malignant diseases. However, natural ALA has disadvantages such as weak lipophilicity, low stability and poor bioavailability, greatly reducing its clinical performance. The emerging nanotechnology is expected to address these limitations and thus improve the therapeutic outcomes. Herein, we summarized important recent advances in the design of ALA-based prodrugs using nanotechnology to improve the efficacy of PDT. The potential limitations and future perspectives of ALA-based nanomedicines are also briefly presented and discussed.

Recent Advances on PKM2 Inhibitors and Activators in Cancer Applications.

Metabolic reprogramming of cells, from the normal mode of glucose metabolism named glycolysis, is a pivotal characteristic of impending cancerous cells. Pyruvate kinase M2 (PKM2), an important enzyme that catalyzes the final rate-limiting stage during glycolysis, is highly expressed in numerous types of tumors and aids in development of favorable conditions for the survival of tumor cells. Increasing evidence has suggested that PKM2 is one of promising targets for innovative drug discovery, especially for the developments of antitumor therapeutics. Herein, we systematically summarize the recent advancement on PKM2 modulators including inhibitors and activators in cancer applications. We also discussed the classifications of pyruvate kinases in mammals and the biological functions of PKM2 in this review. We do hope that this review would provide a comprehensive understanding of the current research on PKM2 modulators, which may benefit the development of more potent PKM2-related drug candidates to treat PKM2-associated diseases including cancers in future.

A novel cascaded energy conversion system inducing efficient and precise cancer therapy.

Cancer therapies based on energy conversion, such as photothermal therapy (PTT, light-to-thermal energy conversion) and photodynamic therapy (PDT, light-to-chemical energy conversion) have attracted extensive attention in preclinical research. However, the PTT-related hyperthermia damage to surrounding tissues and shallow penetration of PDT-applied light prevent further advanced clinical practices. Here, we developed a thermoelectric therapy (TET) based on thermoelectric materials constructed p-n heterojunction (SrTiO3/Cu2Se nanoplates) on the principle of light-thermal-electricity-chemical energy conversion. Upon irradiation and natural cooling-induced the temperature gradient (35-45 oC), a self-build-in electric field was constructed and thereby facilitated charges separation in bulk SrTiO3 and Cu2Se. Importantly, the contact between SrTiO3 (n type) and Cu2Se (p type) constructed another interfacial electric field, further guiding the separated charges to re-locate onto the surfaces of SrTiO3 and Cu2Se. The formation of two electric fields minimized probability of charges recombination. Of note, high-performance superoxide radicals and hydroxyl radicals' generation from O2 and H2O under catalyzation by separated electrons and holes, led to intracellular ROS burst and cancer cells apoptosis without apparent damage to surrounding tissues. Construction of bulk and interfacial electric fields in heterojunction for improving charges separation and transfer is also expected to provide a robust strategy for diverse applications.

Mesopore-encaged active MnOx in nano-silica selectively suppresses lung cancer cells by inducing autophagy.

Autophagy induced by nanomaterials is one of the intracellular catabolic pathways that degrade and recycle the biomacromolecules and damaged organelles in cells and has emerged as a very promising pharmacological target critical to future drug development and anti-cancer therapy. Herein, we developed mesopore-encaged highly-dispersed active cluster-like MnOx in nanosilica entitled MnO-MS, with a size of around 130 nm. Our studies show that MnO-MS could not only obviously induce autophagy in both stable GFP-LC3 HeLa cells and GFP-LC3-mCherry HeLa cells but also could selectively inhibit lung cancer A549 cell growth at 11.19 µg mL-1 (IC50) while exhibiting little cytotoxicity in normal cells. Encouraged by these interesting results, a further mechanistic study reveals that reactive oxygen species (ROS) were excited by the active MnOx in nanosilica, leading to the disruption of mitochondrial membrane potential (MMP), enhancement of ATG5A/ATG16L/ATG4B/Beclin1, and finally, inhibition of the mTOR signaling pathways. Collectively, these findings indicate that MnO-MS-induced cell death via autophagy pathways in cancer cells. Furthermore, MnO-MS significantly inhibited tumor growth with minimal side effects in vivo, and it is envisioned that MnO-MS can be further developed as a potential autophagy inducer for the treatment of lung cancers.

Multitarget inhibitors/probes that target LRRK2 and AURORA A kinases noncovalently and covalently.

Herein, we report a salicylaldehyde-based, reversible covalent inhibitor (A2) that possesses moderate cellular activity against AURKA with a prolonged residence time and shows significant non-covalent inhibition towards LRRK2. Our results indicated that this multitarget kinase inhibitor may be used as the starting point for future development of more potent, selective and dual-targeting covalent kinase inhibitors against AURKA and LRRK2 for mitophagy.

Biodegradable silica nanocapsules enable efficient nuclear-targeted delivery of native proteins for cancer therapy.

Cell nucleus is the desired subcellular organelle of many therapeutic drugs. Although numerous nanomaterial-based methods have been developed which could facilitate nuclear-targeted delivery of small-molecule drugs, few are known to be capable of delivering exogenous native proteins. Herein, we report a convenient and highly robust approach for effective nuclear-targeted delivery of native proteins/antibodies by using biodegradable silica nanocapsules (BSNPs) that were surface-modified with different nuclear localization signals (NLS) peptides. We found that, upon gaining entry to mammalian cells via endocytosis, such nanocapsules (protein@BSNP-NLS) could effectively escape from endolysosomal vesicles with the assistance of an endosomolytic peptide (i.e., L17E), accumulate in cell nuclei and release the encapsulated protein cargo with biological activities. Cloaked with HeLa cell membrane, DNase@BSNP-NLS/L17E-M (with L17E encapsulated) homologously delivered functional proteins to cancer cell nuclei in tumor-xenografted mice. In vitro and in vivo anti-tumor properties, such as long blood circulation time and effective tumor growth inhibition, indicate that the nuclear-targeted cell-membrane-cloaked BSNPs (DNase@BSNP-NLS/L17E-M) platform is a promising therapeutic approach to nuclear related diseases.

Novel 1-hydroxy phenothiazinium-based derivative protects against bacterial sepsis by inhibiting AAK1-mediated LPS internalization and caspase-11 signaling.

Sepsis is a life-threatening syndrome with disturbed host responses to severe infections, accounting for the majority of death in hospitalized patients. However, effective medicines are currently scant in clinics due to the poor understanding of the exact underlying mechanism. We previously found that blocking caspase-11 pathway (human orthologs caspase-4/5) is effective to rescue coagulation-induced organ dysfunction and lethality in sepsis models. Herein, we screened our existing chemical pools established in our lab using bacterial outer membrane vesicle (OMV)-challenged macrophages, and found 7-(diethylamino)-1-hydroxy-phenothiazin-3-ylidene-diethylazanium chloride (PHZ-OH), a novel phenothiazinium-based derivative, was capable of robustly dampening caspase-11-dependent pyroptosis. The in-vitro study both in physics and physiology showed that PHZ-OH targeted AP2-associated protein kinase 1 (AAK1) and thus prevented AAK1-mediated LPS internalization for caspase-11 activation. By using a series of gene-modified mice, our in-vivo study further demonstrated that administration of PHZ-OH significantly protected mice against sepsis-associated coagulation, multiple organ dysfunction, and death. Besides, PHZ-OH showed additional protection on Nlrp3-/- and Casp1-/- mice but not on Casp11-/-, Casp1/11-/-, Msr1-/-, and AAK1 inhibitor-treated mice. These results suggest the critical role of AAK1 on caspase-11 signaling and may provide a new avenue that targeting AAK1-mediated LPS internalization would be a promising therapeutic strategy for sepsis. In particular, PHZ-OH may serve as a favorable molecule and an attractive scaffold in future medicine development for efficient treatment of bacterial sepsis.

Two-Photon Small-Molecule Fluorogenic Probes for Visualizing Endogenous Nitroreductase Activities from Tumor Tissues of a Cancer Patient.

Nitroreductase (NTR), a common enzymatic biomarker of hypoxia, is widely used to evaluate tumor microenvironments. To date, numerous optical probes have been reported for NTRs detection. Approaches capable of concisely guiding the probe design of NTRs suitable for deep-tissue imaging, however, are still lacking. As such, direct optical imaging of endogenous NTR activities from tumors derived from cancer patients is thus far not possible. Herein, aided by computational calculations, the authors have successfully developed a series of two-photon (TP) small-molecule fluorogenic probes capable of sensitively detecting general NTR activities from various biological samples; by optimizing the distance between the recognition moiety and the reactive site of NTRs from different sources, the authors have discovered and experimentally proven that X4 displays the best performance in both sensitivity and selectivity. Furthermore, X4 shows excellent TP excited fluorescence properties capable of directly monitoring/imaging endogenous NTR activities from live mammalian cells, growing zebrafish, and tumor-bearing mice. Finally, with an outstanding TP tissue-penetrating imaging property, X4 is used, for the first time, to successfully detect endogenous NTR activities from the liver lysates and cardia tissues of a cancer patient. The work may provide a universal strategy to design novel TP small-molecule enzymatic probes in future clinical applications.

Design, synthesis of symmetrical bivalent mimetics of annonaceous acetogenins and their cytotoxicities.

A new series of linear dimeric compounds mimicking naturally occurring annonaceous acetogenins have been synthesized by bivalent analogue design, and their cytotoxicities have been evaluated against the growth of cancer cells by MTT method. Most of these compounds show selective action favored to human cancer cell lines over normal cell lines, and compound 9 with bis-terminal benzoquinone functionality exhibits an IC(50)=0.40 µM against MCF7 cell lines. This work mentions that appropriate conformational constraints might be a useful optimizing tool for this unique class of anticancer compounds.

Broad-Spectrum Polymeric Nanoquencher as an Efficient Fluorescence Sensing Platform for Biomolecular Detection.

Colloidal inorganic nanostructures (metal, carbon, and silica) have been widely used as "nanoquenchers" for construction of nanosensors; however, inherent drawbacks such as insufficient fluorescence quenching efficiency, false positive signals, and uncertain long-term cytotoxicity have limited their further utility. Herein, by taking advantages of polymeric nanoparticles (PNPs) in terms of high loading capacity, facile surface modification chemistry, and good biocompatibility, we report a broad-spectrum (400-750 nm) polymeric fluorescence-quenching platform for sensor fabrication. Our newly developed polymeric nanoquenchers (qPNPs) were constructed by concurrently encapsulating various alkylated black-hole quenchers into nanoparticles made of poly(methyl methacrylate-co-methacrylic acid) and were found to have an excellent fluorescence quenching effect (>400-fold) on common fluorophores (FAM, TMR, and Cy5) together with high stability under physiological conditions. As a proof of concept, the feasibility of these qPNPs for fluorescence sensing was validated by successful construction of two nanosensors (FAMDEVD@qPNP and Cy5SurC@qPNP), which could be used as promising nanosensors for live-cell imaging of the apoptosis-related protease caspase-3 and cancer-related survivin mRNA, respectively. As expected, in the FAM channel, the FAMDEVD@qPNP showed fast and selective fluorescence responses toward caspase-3 in buffers and could be used to image the activation of drug-induced endogenous caspase-3. In the Cy5 channel, the Cy5SurC@qPNP could be used to distinguish normal cells (MCF10A) from cancer cells (HeLa) by quantitatively detecting the endogenous survivin mRNA level. It could be further used to monitor changes in the endogenous survivin mRNA expression levels in drug-treated HeLa cells. Altogether, by virtue of their high quencher loading and broad-spectrum quenching efficiency and good signal-to-background ratio, these qPNPs might be particularly attractive alternatives to other conventional nanoquenchers for the construction of more complex biosensors in the future.

In Vitro and In Vivo Demonstration of Ultraefficient and Broad-Spectrum Antibacterial Agents for Photodynamic Antibacterial Chemotherapy.

Increasing threats from both pathogenic infections and antibiotic resistance highlight the pressing demand for nonantibiotic agents and alternative therapies. Herein, we report several new phenothiazinium-based derivatives, which could be readily synthesized via fragment-based assembly, which exhibited remarkable bactericidal activities both in vitro and in vivo. Importantly, in contrast to numerous clinically and preclinically used antibacterial photosensitizers, these compounds were able to eliminate various types of microorganisms, including Gram-(+) Staphylococcus aureus (S. aureus), Gram-(-) Escherichia coli, multidrug-resistant S. aureus, and their associated biofilms, at low drug and light dosages (e.g., 0.21 ng/mL in vitro and 1.63 ng/cm2 in vivo to eradicate S. aureus at 30 J/cm2). This study thus unveils the potential of these novel phenothiaziniums as potent antimicrobial agents for highly efficient photodynamic antibacterial chemotherapy.

A Review of Light Sources and Enhanced Targeting for Photodynamic Therapy.

Photodynamic Therapy (PDT), as a clinically approved modality for the treatment of various disordered diseases including cancer, has received great advances in recent years. By preferentially accumulating non-toxic Photosensitizers (PSs) in the pathological area, and in situ generation of cytotoxic reactive oxygen species (ROS) under local irradiation by a light source with appropriate wavelength, PDT works in a dual-selective manner. Over the past decades, numerous studies and reviews on PDT mainly focused on activable PSs and the newly emerging PSs in PDT. However, to the best of our knowledge, there are few articles on the systematic introduction of light sources and limited reports about targeted strategies in PDT. This review comprehensively summarizes various light sources applied in PDT together with typical enhanced targeting strategies, and outlines their advantages and disadvantages, respectively. The clinical applications and future perspectives in light sources are also partly presented and discussed.