Recent Advances in RNA-Targeted Cancer Therapy.

Ribonucleic acid (RNA) plays a pivotal role in gene regulation and protein biosynthesis. Interfering the physiological function of key RNAs to induce cell apoptosis holds great promise for cancer treatment. Many RNA-targeted anti-cancer strategies have emerged continuously. Among them, RNA interference (RNAi) has been recognized as a promising therapeutic modality for various disease treatments. Nevertheless, the primary obstacle in siRNA delivery-escaping the endosome and crossing the plasma membrane severely impedes its therapeutic potential. Thus far, a variety of nanosystems as well as carrier-free bioconjugation for siRNA delivery have been developed and employed to enhance the drug delivery and anti-tumor efficiency. Besides, the use of small molecules to target specific RNA structures and disrupt their function, along with the covalent modification of RNA, has also drawn tremendous attention recently owing to high therapeutic efficacy. In this review, we will provide an overview of recent progress in RNA-targeted cancer therapy including various siRNA delivery strategies, RNA-targeting small molecules, and newly emerged covalent RNA modification. Finally, challenges and future perspectives faced in this research field will be discussed.

Detection of Microcystin-LR in the Cells and Natural Lake Water Samples by A Unique Fluorescence-Based Method.

Microcystin-LR (MC-LR) is widely distributed in natural lakes and could strongly inhibit protein phosphatase activity; it is also a potent liver tumor promoter. Over the last two decades, tremendous efforts have been devoted to enhance the detection of MC-LR in water samples. However, the traditional method is complex and costly, and achieving the fast, sensitive, and accurate determination of MC-LR in the cells and natural lakes by using fluorescence signal changes is fairly difficult. Our work explores novel fluorescent probes that are capable of concurrently analyzing and detecting MC-LR in the cells and water. In this study, we introduce, for the first time, 5-AF and 6-AF as small-molecule fluorescent probes suitable for MC-LR detection in the cells and water samples based on fluorescence signal changes. We titrated 5-AF and 6-AF with MC-LR in pure water, scanned the fluorescence of the sample, and then obtained the equation the fluorescence intensity versus MC-LR concentration curve. MC-LR in lake water samples was crudely purified, and then 5-AF was added to measure its fluorescence peak. The fluorescence intensity of 5-AF is significantly enhanced with increasing MC-LR concentration. This enhancement trend is stable and could be mathematically modeled. We also comprehensively analyzed the mechanism and recognition principle of the probe response to MC-LR in natural lake water. Moreover, we believe that 5-AF may be capable of detecting exogenous MC-LR in cells. The results of this study reveal that these unique fluorescent probes may be applied to construct near-infrared fluorescent probes that could detect MC-LR levels in vivo.

Visualizing Borrelia burgdorferi Infection Using a Small-Molecule Imaging Probe.

In vivo diagnostic imaging of bacterial infections is currently reliant on targeting their metabolic pathways, an ineffective method to identify microbial species with low metabolic activity. Here, we establish HS-198 as a small-molecule fluorescent conjugate that selectively targets the highly conserved bacterial protein HtpG (high-temperature protein G), within Borrelia burgdorferi, the bacterium responsible for Lyme disease. We describe the use of HS-198 to target morphologic forms of B. burgdorferi in both the logarithmic growth phase and the metabolically dormant stationary phase as well as in inactivated spirochetes. Furthermore, in a murine infection model, systemically injected HS-198 identified B. burgdorferi as revealed by imaging in postnecropsy tissue sections. These findings demonstrate how small-molecule probes directed at conserved bacterial protein targets can function to identify the microbe using noninvasive imaging and potentially as scaffolds to deliver antimicrobial agents to the pathogen.

Probing the Compatibility of an Enzyme-Linked Immunosorbent Assay toward the Reprogramming of Nonribosomal Peptide Synthetase Adenylation Domains.

An important challenge in natural product biosynthesis is the biosynthetic design and production of artificial peptides. One of the most promising strategies is reprogramming adenylation (A) domains to expand the substrate repertoire of nonribosomal peptide synthetases (NRPSs). Therefore, the precise detection of subtle structural changes in the substrate binding pockets of A domains might accelerate their reprogramming. Here we show that an enzyme-linked immunosorbent assay (ELISA) using a combination of small-molecule probes can detect the effects of substrate binding pocket residue substitutions in A-domains. When coupled with a set of aryl acid A-domain variants (total of nine variants), the ELISA can analyze the subtle differences in their active-site architectures. Furthermore, the ELISA-based screening was able to identify the variants with substrate binding pockets that accepted a non-cognate substrate from an original pool of 45. These studies demonstrate that ELISA is a reliable platform for providing insights into the active-site properties of A-domains and can be applied for the reprogramming of NRPS A-domains.

[Label-free small molecule probe and target discovery of traditional Chinese medicine].

Target discovery is the core of elucidating the mechanism of traditional Chinese medicine( TCM),and it is also the key to correlate the chemical composition and pharmacological action of TCM. The traditional target screening methods such as the activitybased probe profiling,affinity chromatography,and protein microarray are commonly used in the past,however,which are limited in TCM due to the complexity of small molecules existed in the herbal medicine. The label-free small molecule probe is a recently well-applied technology in the target discovery of natural products,which is characterized by discovering the small molecule-protein ligation without any structural modification at the ligands,and is therefore suitable to the complex chemical constituents in TCM. Furthermore,this method is conducted on the basis of proteome,which is advanced in the discovery of new or multiple target proteins of TCM. Owing to the potential of label-free probe in the target discovery of TCM,its analytical principle,application status,and general protocol were reviewed in this paper. The label-free probe technology is anticipated to accelerate the mechanism-uncovering of TCM.

Design, synthesis, and initial evaluation of affinity-based small molecular probe for detection of WDR5.

WDR5, a subunit of the SET/MLL complex, plays critical roles in various biological progresses and are abnormally expressed in many cancers. Here we report the design, synthesis, and biochemical characterization of a new chemical tool to capture WDR5 protein. The probe is a biotinylated version of compound 30 that is a potent WDR5 inhibitor we previously reported. Importantly, the probe displayed high affinity to WDR5 protein in vitro binding potency and showed the ability in specifically and real time monitoring WDR5 protein. Further, the biotinylated tag of the probe enabled selectively "chemoprecipitation" of WDR5 from whole cell lysates of MV4-11. This probe provided a new approach to identify the overexpressed WDR5 protein in different cancer cells and applications to proteomic analysis of WDR5 and WDR5-binding partners.

Design of a phosphinate-based bioluminescent probe for superoxide radical anion imaging in living cells.

Superoxide radical anion (O2 ˙- ) as an important member of reactive oxygen species (ROS) plays a vital role both in physiology and pathology. Herein we designed and synthesized a novel phosphinate-based bioluminescence probe for O2 ˙- detection in living cells, which exhibited good sensitivity for capturing O2 ˙- at the nanomole level and high selectivity against other ROS. The probe was further found to be of low toxicity for living cells and was then successfully employed for sensing endogenous O2 ˙- by using phorbol-12-myristate-13-acetate (PMA) as a traditional O2 ˙- stimulator in Huh7 cells. Moreover, the increasing production and use of nanoparticles, has given rise to many concerns and debates among the public and scientific authorities regarding their safety and final fate in biological systems. Herein it was found that mondisperse polystyrene particles could stimulate O2 ˙- generation in Huh7 cells. Overall, the probe was demonstrated to have a great potential as a novel bioluminescent sensor for detecting O2 ˙- in living cells. To our knowledge, this is the first small-molecule phosphinate-based bioluminescence probe that will open up great opportunities for unlocking the mystery of O2 ˙- in human health and disease.

Polyphosphoric acid-induced perylene probe self-assembly and label-free fluorescence turn-on detection of alkaline phosphatase.

A label-free fluorescence turn-on strategy for alkaline phosphatase (ALP) detection was established based on its enzymatic catalyzed hydrolysis of polyphosphoric acid (PPA, an anionic polymer) that had been utilized for aggregation with our homemade positively charged perylene derivative (Probe-1) via noncovalent interactions. The disaggregation caused turn-on fluorescence signal which was recovered by the released Proble-1 molecules whose original strong fluorescence in an aqueous buffer solution had been quenched due to their previous aggregation induced by PPA. Such method presents its great advantages of free labeling, convenience and simplicity, cost effectiveness, high selectivity, and high sensitivity, with a detection limit of 0.5 mU/mL of ALP. Graphical Abstract A label-free fluorescence turn-on strategy for alkaline phosphatase based on its enzymatic catalyzed hydrolysis of polyphosphoric acid that had been utilized for aggregation with our homemade positively charged perylene derivative via noncovalent interactions.

Detection of wood cell wall porosity using small carbohydrate molecules and confocal fluorescence microscopy.

A novel approach to nanoscale detection of cell wall porosity using confocal fluorescence microscopy is described. Infiltration of cell walls with a range of nitrophenyl-substituted carbohydrates of different molecular weights was assessed by measuring changes in the intensity of lignin fluorescence, in response to the quenching effect of the 4-nitrophenyl group. The following carbohydrates were used in order of increasing molecular weight; 4-nitrophenyl ß-D-glucopyrano-side (monosaccharide), 4-nitrophenyl ß-D-lactopyranoside (disaccharide), 2-chloro-4-nitrophenyl ß-D-maltotrioside (trisaccharide), and 4-nitrophenyl α-D-maltopentaoside (pentasaccharide). This technique was used to compare cell wall porosity in wood which had been dewatered to 40% moisture content using supercritical CO2, where cell walls remain fully hydrated, with kiln dried wood equilibrated to 12% moisture content. Infiltration of cell walls as measured by fluorescence quenching, was found to decrease with increasing molecular weight, with the pentasaccharide being significantly excluded compared to the monosaccharide. Porosity experiments were performed on blocks and sections to assess differences in cell wall accessibility. Dewatered and kiln dried wood infiltrated as blocks showed similar results, but greater infiltration was achieved by using sections, indicating that not all pores were easily accessible by infiltration from the lumen surface. In wood blocks infiltrated with 4-nitrophenyl α-D-maltopentaoside, quenching of the secondary wall was quite variable, especially in kiln dried wood, indicating limited connectivity of pores accessible from the lumen surface.

Small-Molecule Probe-Induced In Situ-Sensitized Photoelectrochemical Biosensor for Monitoring α-Glucosidase Activity.

Semiconductor-based photoelectrochemical (PEC) biosensors have garnered significant attention in the field of disease diagnosis and treatment. However, the recognition units of these biosensors are mainly limited to bioactive macromolecules, which hinder the photoelectric response due to their insulating characteristics. In this study, we develop an in situ-sensitized strategy that utilizes a small-molecule probe at the interface of the photoelectrode to accurately detect α-glucosidase (α-Glu) activity. Silane, a prototype small-molecule probe, was surface-modified on graphitic carbon nitride to generate Si nanoparticles upon reacting with hydroquinone, the enzymatic product of α-Glu. The in situ formed heterojunction enhances the light-harvesting property and photoexcited carrier separation efficiency. As a result, the in situ-sensitized PEC biosensor demonstrates excellent accuracy, a low detection limit, and outstanding anti-interference ability, showing good applicability in evaluating α-Glu activity and its inhibitors in human serum samples. This novel in situ sensitization approach using small-molecule probes opens up new avenues for developing simple and efficient PEC biosensing platforms by replacing conventional biorecognition elements.

Quaternary Ammonium Salt Based NIR-II Probes for in vivo Imaging.

Traditional luminescent materials including fluorescent probes suffer from notorious aggregation-caused quenching (ACQ) in aqueous solutions. Although several approaches such as the aggregation-induced emission (AIE) effect have been developed, it remains a significant challenge to identify an effective and efficient strategy to resolve this issue. Herein, quaternary ammonium salts Q8PBn and Q8PNap as a novel class of bright near infrared window II (NIR-II, 1,000 - 1,700 nm) probes were designed and synthesized, and the twisted intramolecular charge transfer (TICT) formation at the excited state can be effectively suppressed for the newly designed probes. Furthermore, Q8PNap complexation with fetal bovine serum (Q8PNap/FBS) significantly increased the quantum yield by ~ 32-fold compared with PEGylated tertiary amine Q8P, and Q8PNap/FBS was successfully used to achieve high spatial and temporal resolution imaging of hind limb vasculature, lymphatic system, and small tumor metastasis, as well as precise NIR-II imaging-guided tumor and lymph node surgery in small animal models for the first time.

Optical Imaging of Triple-Negative Breast Cancer Cells in Xenograft Athymic Mice Using an ICAM-1-Targeting Small-Molecule Probe.

PURPOSE: The development of early, accurate diagnostic strategies for triple-negative breast cancer (TNBC) remains a significant challenge. Intercellular adhesion molecule-1 (ICAM-1) overexpressed in human TNBC cells is a potential molecular target and biomarker for diagnosis. In this study, small-molecule probe (denoted as γ3-Cy5.5) constructed with a short 17-mer linear peptide (γ3) and near-infrared fluorescence (NIRF) dye cyanine 5.5 (Cy5.5) was used to detect the expression of ICAM-1 in vitro and in vivo, and to diagnose TNBC via NIRF imaging. PROCEDURES: Western blotting and flow cytometric analysis were used for the detection of ICAM-1 expression in MDA-MB-231 and MCF-7 cells. The cytotoxicity of the small-molecule probe γ3-Cy5.5 was detected using the CCK8 assay. The in vitro targeting of the small-molecule probe γ3-Cy5.5 was verified via flow cytometry and a laser scanning confocal microscope. Finally, the targeting of small-molecule probe in vivo and ex vivo was observed by NIRF imaging. RESULTS: Western blotting and flow cytometry demonstrate that ICAM-1 was highly expressed in the MDA-MB-231 TNBC cell line. Laser confocal microscopy and flow cytometry results show that TNBC cells have an increased cellular uptake of γ3-Cy5.5 compared to the control probe γ3S-Cy5.5. With in vivo NIRF, a significantly higher Cy5.5 signal appeared in the tumors of mice administered γ3-Cy5.5 than those treated with γ3S-Cy5.5. The target-to-background ratio observed on the NIRF images was significantly higher in the γ3-Cy5.5 group (10.2, 13.6) compared with the γ3S-Cy5.5 group (4.4, 4.0) at 1 and 2 h, respectively. CONCLUSIONS: This is the first report of the use of ICAM-1-specific small-molecule probe for in vivo NIRF optical imaging of TNBC. This method provides a noninvasive and specific strategy for the early diagnosis of TNBC.

Targeting Early Apoptosis in Acute Ischemic Stroke with a Small-Molecule Probe.

Inhibiting apoptotic cells helps ameliorate ischemic injury. Actually, only the apoptotic cells in early stage could be rescued. Molecular imaging of the early apoptosis would make sense in ischemic stroke; however, few of apoptosis molecular probes could specifically target early apoptosis. This study developed a small-molecule early apoptosis targeting probe CYS-F, which was synthesized by cystine with fluorescein isothiocyanate dyes. And the final molecular weight of CYS-F was only 1013 Da, which was much smaller than the traditional apoptosis marker annexin V. CYS-F showed excellent early apoptosis targeting ability both in vitro and in vivo. And CYS-F was cleared rapidly from the circulation with a blood half-life of 1.325 h. A favorable match was obtained between the images in fluorescence imaging and magnetic resonance imaging in stroke models. The target-to-background ratio of the lesions on 0 h was negative, which reflected the decreased blood flow. Multimodal molecular imaging showed the therapeutic effect of cystamine was dose dependence and CYS-F could also predict the outcome of ischemic stroke at an early stage. The versatility of CYS-F provides a comprehensive and convenient route for clinical decision-making in acute ischemic stroke.

Controlled self-assembly of small molecule probes and the related applications in bioanalysis.

Fluorescence spectroscopy is widely used in basic research, disease diagnosis, environmental monitoring, and the development of novel bioanalytical techniques. We mainly focus on the changes in fluorescence signal originated from the controlled self-assembly of small molecule probes, including aggregation caused quenching, aggregation induced emission, controlled turn-on of probe monomer emission, and the tunable monomer-excimer transition. Recent developments in the related bioanalytical techniques have been reviewed.

Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains.

A series of inhibitors have been designed based on 5'-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of L-Phe-AMS (L-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L-Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe.

Discovery of 8-Membered Ring Sulfonamides as Inhibitors of Oncogenic Mutant Isocitrate Dehydrogenase 1.

Evidence suggests that specific mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) are critical for the initiation and maintenance of certain tumor types and that inhibiting these mutant enzymes with small molecules may be therapeutically beneficial. In order to discover mutant allele-selective IDH1 inhibitors with chemical features distinct from existing probes, we screened a collection of small molecules derived from diversity-oriented synthesis. The assay identified compounds that inhibit the IDH1-R132H mutant allele commonly found in glioma. Here, we report the discovery of a potent (IC50 = 50 nM) series of IDH1-R132H inhibitors having 8-membered ring sulfonamides as exemplified by the compound BRD2879. The inhibitors suppress (R)-2-hydroxyglutarate production in cells without apparent toxicity. Although the solubility and pharmacokinetic properties of the specific inhibitor BRD2879 prevent its use in vivo, the scaffold presents a validated starting point for the synthesis of future IDH1-R132H inhibitors having improved pharmacological properties.

Synthesis, in vitro evaluation and in vivo imaging of a highly selective hydrazine bioluminescent probe based on luciferin-luciferase system / 药学学报

A highly sensitive and selective bioluminescent probe for hydrazine (BPH) was designed, synthesized and evaluated for detection of hydrazine in vitro and in vivo. BPH was designed to include a specific recognition group (acetyl) of hydrazine at an appropriate modification site of the optical reporter hydroxyluciferin (D-luciferin), which showed excellent performance both in selectivity and sensitivity to hydrazine. The results showed that the bioluminescent probe BPH developed in this study is an innovative and widely applicable tool for detecting hydrazine in complex natural environment or in animals.

Label-free small molecule probe and target discovery of traditional Chinese medicine / 中国中药杂志

Target discovery is the core of elucidating the mechanism of traditional Chinese medicine( TCM),and it is also the key to correlate the chemical composition and pharmacological action of TCM. The traditional target screening methods such as the activitybased probe profiling,affinity chromatography,and protein microarray are commonly used in the past,however,which are limited in TCM due to the complexity of small molecules existed in the herbal medicine. The label-free small molecule probe is a recently well-applied technology in the target discovery of natural products,which is characterized by discovering the small molecule-protein ligation without any structural modification at the ligands,and is therefore suitable to the complex chemical constituents in TCM. Furthermore,this method is conducted on the basis of proteome,which is advanced in the discovery of new or multiple target proteins of TCM. Owing to the potential of label-free probe in the target discovery of TCM,its analytical principle,application status,and general protocol were reviewed in this paper. The label-free probe technology is anticipated to accelerate the mechanism-uncovering of TCM.