Cargo-selective and adaptive delivery of nucleic acid therapeutics by bola-amphiphilic dendrimers.

Nucleic acid therapeutics are becoming an important drug modality, offering the unique opportunity to address "undruggable" targets, respond rapidly to evolving pathogens, and treat diseases at the gene level for precision medicine. However, nucleic acid therapeutics have poor bioavailability and are chemolabile and enzymolabile, imposing the need for delivery vectors. Dendrimers, by virtue of their well-defined structure and cooperative multivalence, represent precision delivery systems. We synthesized and studied bola-amphiphilic dendrimers for cargo-selective and on-demand delivery of DNA and small interfering RNA (siRNA), both important nucleic acid therapeutics. Remarkably, superior performances were achieved for siRNA delivery with the second-generation dendrimer, yet for DNA delivery with the third generation. We systematically studied these dendrimers with regard to cargo binding, cellular uptake, endosomal release, and in vivo delivery. Differences in size both of the dendrimers and their nucleic acid cargos impacted the cooperative multivalent interactions for cargo binding and release, leading to cargo-adaptive and selective delivery. Moreover, both dendrimers harnessed the advantages of lipid and polymer vectors, while offering nanotechnology-based tumor targeting and redox-responsive cargo release. Notably, they allowed tumor- and cancer cell-specific delivery of siRNA and DNA therapeutics for effective treatment in different cancer models, including aggressive and metastatic malignancies, outperforming the currently available vectors. This study provides avenues to engineer tailor-made vectors for nucleic acid delivery and precision medicine.

Synthesis, Antifungal, and Antibacterial Activities of Novel Benzoylurea Derivatives Containing a Pyrimidine Moiety.

To explore more efficient and less toxic antibacterial and antifungal pesticides, we utilized 2,6-difluorobenzamide as a starting material and ultimately synthesized 23 novel benzoylurea derivatives containing a pyrimidine moiety. Their structures were characterized and confirmed by 1H NMR, 13C NMR, 19F NMR, and HRMS. The bioassay results demonstrated that some of the title compounds exhibited moderate to good in vitro antifungal activities against Botrytis cinerea in cucumber, Botrytis cinerea in tobacco, Botrytis cinerea in blueberry, Phomopsis sp., and Rhizoctonia solani. Notably, compounds 4j and 4l displayed EC50 values of 6.72 and 5.21 µg/mL against Rhizoctonia solani, respectively, which were comparable to that of hymexazol (6.11 µg/mL). Meanwhile, at 200 and 100 concentrations, the target compounds 4a-4w exhibited lower in vitro antibacterial activities against Xanthomonas oryzae pv. oryzicola and Xanthomonas citri subsp. citri, respectively, compared to those of thiodiazole copper. Furthermore, the molecular docking simulation demonstrated that compound 4l formed hydrogen bonds with SER-17 and SER-39 of succinate dehydrogenase (SDH), providing a possible explanation for the mechanism of action between the target compounds and SDH. This study represents the first report on the antifungal and antibacterial activities of novel benzoylurea derivatives containing a pyrimidine moiety.

Self-assembling supramolecular dendrimer nanosystem for PET imaging of tumors.

Bioimaging plays an important role in cancer diagnosis and treatment. However, imaging sensitivity and specificity still constitute key challenges. Nanotechnology-based imaging is particularly promising for overcoming these limitations because nanosized imaging agents can specifically home in on tumors via the "enhanced permeation and retention" (EPR) effect, thus resulting in enhanced imaging sensitivity and specificity. Here, we report an original nanosystem for positron emission tomography (PET) imaging based on an amphiphilic dendrimer, which bears multiple PET reporting units at the terminals. This dendrimer is able to self-assemble into small and uniform nanomicelles, which accumulate in tumors for effective PET imaging. Benefiting from the combined dendrimeric multivalence and EPR-mediated passive tumor targeting, this nanosystem demonstrates superior imaging sensitivity and specificity, with up to 14-fold increased PET signal ratios compared with the clinical gold reference 2-fluorodeoxyglucose ([18F]FDG). Most importantly, this dendrimer system can detect imaging-refractory low-glucose-uptake tumors that are otherwise undetectable using [18F]FDG. In addition, it is endowed with an excellent safety profile and favorable pharmacokinetics for PET imaging. Consequently, this dendrimer nanosystem constitutes an effective and promising approach for cancer imaging. Our study also demonstrates that nanotechnology based on self-assembling dendrimers provides a fresh perspective for biomedical imaging and cancer diagnosis.

Surface-decorated S. cerevisiae for flow cytometric array immunoassay.

Our laboratory had developed a cell-based bio-bead for protein quantification. However, the selection of antibody in the above immunoassay is limited. This study describes a surface-decorated Saccharomyces cerevisiae for flow cytometric array immunoassay. S. cerevisiae was labeled with fluorescein isothiocyanate (FITC) and oxidized by sodium periodate, in which the saccharide group on the cytoderm outer layer was converted to an aldehyde group. In succession, adipic dihydrazide was bio-conjugated to the aldehyde group and glutaraldehyde bound to the hydrazide group. Phycoerythrin (PE)-labeled goat anti-mouse polyclonal antibody was used to assess the conjugation of mouse anti-human monoclonal antibody to surface-decorated S. cerevisiae. Cytokeratin 19 fragment (Cyfra21-1) and neuron-specific enolase (NSE) antigens were also employed to evaluate the flow cytometric array immunoassay based on surface-decorated S. cerevisiae. Flow cytometry demonstrated that FITC-barcoded S. cerevisiae as two legible populations. PE-labeled polyclonal antibody validated the coating of surface-decorated S. cerevisiae with the monoclonal antibody. The flow cytometric array immunoassays for Cyfra21-1 and NSE documented that the limit of detection (LOD) was at least 0.4 ng/mL. Precision and accuracy assessments appeared that the relative standard deviation (R.S.D.) was <15%, and the relative error (R.E.) ranged from 0.9 to 1.1. The correlation coefficient between this immunoassay and electrochemiluminescence immunoassay was 0.9622 for serum Cyfra21-1 and 0.9918 for serum NSE. In conclusion, the surface-decorated S. cerevisiae may be of use in flow cytometric array immunoassay.

Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study.

[Establishment and identification of the near-infrared fluorescence labeled exosomes in breast cancer cell lines].

Exosomes, a population of extracellular membrane vesicles of 30-100 nm in diameter, play important roles in cell biological functions, intercellular signal transduction and especially in cancer diagnosis and therapy. To better apply exosomes in mechanistic study of breast cancer signal transduction, we constructed recombinant eukaryotic expression vector expressing the near-infrared fluorescence protein and CD63 fusion protein through cloning iRFP682 gene and exosomal marker protein CD63 gene into plasmid containing the ITR of AAV. The constructed plasmids were co-transfected with helper plasmid in AAV-293 cell lines and were packaged into rAAV. After titer measurement, the recombinant plasmids were transfected into breast cancer cell lines. The cell lines that stably expressing near-infrared fluorescence protein were selected by fluorescence. Through isolation, purification and identification, we finally obtained a new biomarker: iRFP682 labeled exosomes secreted by breast cancer cell lines, which could be used in further studies of the distribution and signal transduction of exosomes in breast cancer microenvironment.

Establishment of a forward primers-superposed amplification analysis for accurate aspirin pharmacogenomic measurement.

Genotyping of gDNA rs12041331 (PEAR1), rs6065 (GP1BA), and rs730012 (LTC4S) can provide systematic guidance on the use of aspirin. However, an accurate, reliable and economical approach to simultaneous detection of the above single nucleotide polymorphisms (SNPs) is not reported. Herein, we designed and substantiated an allele-specific (AS) forward primer-superposed amplification analysis for measurement of the SNPs in PEAR1, GP1BA and LTC4S genes, in which the values of ∆Cq (differences in threshold cycles between the wild-type forward primer-based assay and the mutated-type forward primer-based assay) were employed to decide genotype. Mismatch AS forward primers were screened with the singleplex amplification analysis. Moreover, Cq extension optimized by AS forward primer superposition was observed in the selected forward primer-based triplex analysis. Further, robustness assessment of the triplex analysis showed the amplification efficiency ranging from 0.9 to 1.1. Precision test demonstrated the coefficient of variation of less than 2%. And the detective results of 189 DNA samples was completely concordant with that of commercial Sanger sequencing. In summary, we developed a simple, accurate and economical approach to genotyping of rs12041331 (PEAR1), rs6065 (GP1BA) and rs730012 (LTC4S) to provide a valuable pharmacogenomics tool for guidance of aspirin delivery.

Screening, Confirmation and Validation of Mismatch AS F-Primers for Hypertension-Related Genotyping.

In this study, screening, confirmation and validation of mismatch allele-specific (AS) forward (F)-primers are executed to establish a quadruplex amplification analysis (real-time PCR) for discrimination of CYP2D6*10, ADRB1, NPPA and CYP3A5*3 genotypes associated with hypertensive pharmacogenomics. To significantly distinguish heterozygote and homozygote, ΔCq (differences in threshold cycles between the wild-type F-primer amplification assay and the mutant-type F-primer amplification assay) was utilized to determine outcomes. Detection of plasmid by uniplex real-time PCR was used to screen the mismatch AS F-primers. Robustness assessment and agreement analysis were employed to confirm and validate initially selected F-primers, respectively. Robustness assessment confirmed that except of ADRB1 (0.7-0.9), amplification efficiency ranged from 0.9 to 1.1. No statistically significant difference was found between the analysis and NGS. Therefore, the optimized F-primer as polymorphism recognition molecules can benefit the genotyping guiding drug delivery in anti-hypertension treatment.

Design, Synthesis, and Bioactivities of Novel Trifluoromethyl Pyrimidine Derivatives Bearing an Amide Moiety.

Twenty-three novel trifluoromethyl pyrimidine derivatives containing an amide moiety were designed and synthesized through four-step reactions and evaluated for their antifungal, insecticidal, and anticancer properties. Bioassay results indicated that some of the title compounds exhibited good in vitro antifungal activities against Botryosphaeria dothidea (B. dothidea), Phompsis sp., Botrytis cinereal (B. cinerea), Colletotrichum gloeosporioides (C. gloeosporioides), Pyricutaria oryzae (P. oryzae), and Sclerotinia sclerotiorum (S. sclerotiorum) at 50 µg/ml. Meanwhile, the synthesized compounds showed moderate insecticidal activities against Mythimna separata (M. separata) and Spdoptera frugiperda (S. frugiperda) at 500 µg/ml, which were lower than those of chlorantraniliprole. In addition, the synthesized compounds indicated certain anticancer activities against PC3, K562, Hela, and A549 at 5 µg/ml, which were lower than those of doxorubicin. Notably, this work is the first report on the antifungal, insecticidal, and anticancer activities of trifluoromethyl pyrimidine derivatives bearing an amide moiety.

Small Activating RNA Modulation of the G Protein-Coupled Receptor for Cancer Treatment.

G protein-coupled receptors (GPCRs) are the most common and important drug targets. However, >70% of GPCRs are undruggable or difficult to target using conventional chemical agonists/antagonists. Small nucleic acid molecules, which can sequence-specifically modulate any gene, offer a unique opportunity to effectively expand drug targets, especially those that are undruggable or difficult to address, such as GPCRs. Here, the authors report  for the first time that small activating RNAs (saRNAs) effectively modulate a GPCR for cancer treatment. Specifically, saRNAs promoting the expression of Mas receptor (MAS1), a GPCR that counteracts the classical angiotensin II pathway in cancer cell proliferation and migration, are identified. These saRNAs, delivered by an amphiphilic dendrimer vector, enhance MAS1 expression, counteracting the angiotensin II/angiotensin II Receptor Type 1 axis, and leading to significant suppression of tumorigenesis and the inhibition of tumor progression of multiple cancers in tumor-xenografted mouse models and patient-derived tumor models. This study provides not only a new strategy for cancer therapy by targeting the renin-angiotensin system, but also a new avenue to modulate GPCR signaling by RNA activation.

Synthesis and Antifungal Activity of Pyrimidine Derivatives Containing an Amide Moiety.

In this study, 17 novel pyrimidine derivatives containing an amide moiety were synthesized. Then their in vitro antifungal activities against Botryosphaeria dothidea (B. dothidea), Phomopsis sp., and Botrytis cinereal (B. cinereal) were determined. A preliminary biological test showed that compounds 5-bromo-2-fluoro-N-(2-((2-methyl-6-(trifluoromethyl)pyrimidin-4-yl)oxy)phenyl)benzamide (5f) and 5-bromo-2-fluoro-N-(3-((2-methyl-6-(trifluoromethyl)pyrimidin-4-yl)oxy)phenyl)benzamide (5o) exhibited higher antifungal activity against Phomopsis sp., with an inhibition rate of 100% compared to that of Pyrimethanil at 85.1%. In particular, compound 5o exhibited excellent antifungal activity against Phompsis sp., with the EC50 value of 10.5 µg/ml, which was even better than that of Pyrimethanil (32.1 µg/ml). As far as we know, this is the first report on the antifungal activities against B. dothidea, Phomopsis sp., and B. cinereal of this series of pyrimidine derivatives containing an amide moiety.

Design of Inhibitors of the Intrinsically Disordered Protein NUPR1: Balance between Drug Affinity and Target Function.

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their physiological ubiquity and their prevalence in various diseases, including cancer. NUPR1 is an IDP that localizes throughout the whole cell, and is involved in the development and progression of several tumors. We have previously repurposed trifluoperazine (TFP) as a drug targeting NUPR1 and, by using a ligand-based approach, designed the drug ZZW-115 starting from the TFP scaffold. Such derivative compound hinders the development of pancreatic ductal adenocarcinoma (PDAC) in mice, by hampering nuclear translocation of NUPR1. Aiming to further improve the activity of ZZW-115, here we have used an indirect drug design approach to modify its chemical features, by changing the substituent attached to the piperazine ring. As a result, we have synthesized a series of compounds based on the same chemical scaffold. Isothermal titration calorimetry (ITC) showed that, with the exception of the compound preserving the same chemical moiety at the end of the alkyl chain as ZZW-115, an increase of the length by a single methylene group (i.e., ethyl to propyl) significantly decreased the affinity towards NUPR1 measured in vitro, whereas maintaining the same length of the alkyl chain and adding heterocycles favored the binding affinity. However, small improvements of the compound affinity towards NUPR1, as measured by ITC, did not result in a corresponding improvement in their inhibitory properties and in cellulo functions, as proved by measuring three different biological effects: hindrance of the nuclear translocation of the protein, sensitization of cells against DNA damage mediated by NUPR1, and prevention of cancer cell growth. Our findings suggest that a delicate compromise between favoring ligand affinity and controlling protein function may be required to successfully design drugs against NUPR1, and likely other IDPs.

Novel triazole nucleoside analogues promote anticancer activity via both apoptosis and autophagy.

Novel nucleoside derivatives were developed using the strategy of "terminal N,N-dimethylation" to impart tertiary amines to a 1,2,4-triazole nucleoside. The obtained lead compounds displayed significantly improved anticancer activity with dual mechanisms of cell death via apoptosis and autophagy, offering a fresh perspective to searching for new anticancer candidates.

Targeting NUPR1 with the small compound ZZW-115 is an efficient strategy to treat hepatocellular carcinoma.

HCC is a highly lethal malignancy with Sorafenib as the only molecularly targeted drug. The multifunctional stress-associated protein, NUPR1, plays an essential role in controlling cell growth, migration, invasion and Sorafenib resistance in HCC. We report here that NUPR1 expression is absent in healthy liver and it is progressively upregulated in HCC premalignant lesions such as hepatitis and cirrhosis with a maximum expression in HCC samples, highlighting that NUPR1 is a potential drug target for HCC. We therefore assessed in this work, ZZW-115, a strong inhibitor of NUPR1, as a promising candidate for the treatment of HCC. We validated its extraordinary antitumor effect on HCC by using two HCC cell lines, HepG2-and Hep3B, both in cell based experiments and xenografted mice. We further revealed that ZZW-115 treatment induced cell death by apoptosis and necroptosis mechanisms, with a concomitant mitochondrial metabolism failure that triggers lower ATP production. Furthermore, the ATP depletion cannot be rescued by the apoptosis inhibitor Z-VAD-FMK and/or the necrosis inhibitor Necrostatin-1, indicating that ZZW-115 induces cell death through the mitochondrial failure.

ZZW-115-dependent inhibition of NUPR1 nuclear translocation sensitizes cancer cells to genotoxic agents.

Establishing the interactome of the cancer-associated stress protein Nuclear Protein 1 (NUPR1), we found that it binds to several hundreds of proteins, including proteins involved in nuclear translocation, DNA repair, and key factors of the SUMO pathway. We demonstrated that the NUPR1 inhibitor ZZW-115, an organic synthetic molecule, competes with importins for the binding to the NLS region of NUPR1, thereby inhibiting its nuclear translocation. We hypothesized, and then proved, that inhibition of NUPR1 by ZZW-115 sensitizes cancer cells to DNA damage induced by several genotoxic agents. Strikingly, we found that treatment with ZZW-115 reduced SUMOylation of several proteins involved in DNA damage response (DDR). We further report that the presence of recombinant NUPR1 improved the SUMOylation in a cell-free system, indicating that NUPR1 directly stimulates the SUMOylation machinery. We propose that ZZW-115 sensitizes cancer cells to genotoxic agents by inhibiting the nuclear translocation of NUPR1 and thereby decreasing the SUMOylation-dependent functions of key proteins involved in the DDR.

Dissecting the Anticancer Mechanism of Trifluoperazine on Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with almost no curative chemotherapeutic treatment. Besides the development of new compounds, repurposing of approved drugs to treat cancer, alone or in combination, has become an attractive strategy, showing many therapeutic and economic advantages. However, it is necessary to improve our knowledge about the mechanism of cell death elicited by approved drugs itself, but also to rationally develop more powerful multidrug treatments. In this work, we focus our attention on determining the mechanism promoting cell death following trifluoperazine (TFP) treatment, which is an antipsychotic drug with strong anticancer activity in PDAC. We demonstrate that TFP induces cell death by apoptosis and necroptosis, which can be partially inhibited by Z-VAD-FMK as well as necrostatin-1, respectively. This cell death promotion is triggered by a poor ATP content, observed in TFP-treated cells as a consequence of a dramatic decrease in OXPHOS metabolism due to mitochondrial stress. Remarkably, mitochondrial homeostasis was seriously affected, and a loss of mitochondrial membrane potential and ROS overproduction was observed. Moreover, this mitochondrial stress was coupled with an ER stress and the activation of the endoplasmic-reticulum-associated protein degradation (ERAD) and the unf olded protein response (UPR) pathways. We took advantage of this information and inhibited this process by using the proteasome inhibitors MG-132 or bortezomib compounds in combination with TFP and found a significant improvement of the anticancer effect of the TFP on primary PDAC-derived cells. In conclusion, this study not only uncovers the molecular mechanisms that are triggered upon TFP-treatment but also its possible combination with bortezomib for the future development of therapies for pancreatic cancer.

Flavonoid-alkylphospholipid conjugates elicit dual inhibition of cancer cell growth and lipid accumulation.

Cancer development is often associated with lipid metabolic reprogramming, including aberrant lipid accumulation. We create novel paradigms endowed with dual functions of anticancer activity and inhibition of lipid accumulation by conjugating the natural product quercetin and synthetic alkylphospholipid drugs, and harnessing the biomedical effects of both. These conjugates offer fresh perspectives in the search for anticancer candidates.

Targeting the Stress-Induced Protein NUPR1 to Treat Pancreatic Adenocarcinoma.

Cancer cells activate stress-response mechanisms to adapt themselves to a variety of stressful conditions. Among these protective mechanisms, those controlled by the stress-induced nuclear protein 1 (NUPR1 ) belong to the most conserved ones. NUPR1 is an 82-residue-long, monomeric, basic and intrinsically disordered protein (IDP), which was found to be invariably overexpressed in some, if not all, cancer tissues. Remarkably, we and others have previously showed that genetic inactivation of the Nupr1 gene antagonizes the growth of pancreatic cancer as well as several other tumors. With the use of a multidisciplinary strategy by combining biophysical, biochemical, bioinformatic, and biological approaches, a trifluoperazine-derived compound, named ZZW-115, has been identified as an inhibitor of the NUPR1 functions. The anticancer activity of the ZZW-115 was first validated on a large panel of cancer cells. Furthermore, ZZW-115 produced a dose-dependent tumor regression of the tumor size in xenografted mice. Mechanistically, we have demonstrated that NUPR1 binds to several importins. Because ZZW-115 binds NUPR1 through the region around the amino acid Thr68, which is located into the nuclear location signal (NLS) region of the protein, we demonstrated that treatment with ZZW-115 inhibits completely the translocation of NUPR1 from the cytoplasm to the nucleus by competing with importins.

Ligand-based design identifies a potent NUPR1 inhibitor exerting anticancer activity via necroptosis.

Intrinsically disordered proteins (IDPs) are emerging as attractive drug targets by virtue of their prevalence in various diseases including cancer. Drug development targeting IDPs is challenging because they have dynamical structure features and conventional drug design is not applicable. NUPR1 is an IDP playing an important role in pancreatic cancer. We previously reported that Trifluoperazine (TFP), an antipsychotic agent, was capable of binding to NUPR1 and inhibiting tumors growth. Unfortunately, TFP showed strong central nervous system side-effects. In this work, we undertook a multidisciplinary approach to optimize TFP, based on the synergy of computer modeling, chemical synthesis, and a variety of biophysical, biochemical and biological evaluations. A family of TFP-derived compounds was produced and the most active one, named ZZW-115, showed a dose-dependent tumor regression with no neurological effects and induced cell death mainly by necroptosis. This study opens a new perspective for drug development against IDPs, demonstrating the possibility of successful ligand-based drug design for such challenging targets.