Discovery of N-(2-chloro-5-(3-(pyridin-4-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)pyridin-3-yl)-4-fluorobenzenesulfonamide (FD274) as a highly potent PI3K/mTOR dual inhibitor for the treatment of acute myeloid leukemia.

PI3K-Akt-mTOR pathway is a highly activated signal transduction pathway in human hematological malignancies and has been validated as a promising target for acute myeloid leukemia (AML) therapy. Herein, we designed and synthesized a series of 7-azaindazole derivatives as potent PI3K/mTOR dual inhibitors based on our previously reported FD223. Among them, compound FD274 showed excellent dual PI3K/mTOR inhibitory activity, with IC50 values against PI3Kα/ß/γ/δ and mTOR of 0.65 nM, 1.57 nM, 0.65 nM, 0.42 nM, and 2.03 nM, respectively, superior to compound FD223. Compared to the positive drug Dactolisib, FD274 exhibited significant anti-proliferation of AML cell lines (HL-60 and MOLM-16 with IC50 values of 0.092 µM and 0.084 µM, respectively) in vitro. Furthermore, FD274 demonstrated dose-dependent inhibition of tumor growth in the HL-60 xenograft model in vivo, with 91% inhibition of tumor growth at an intraperitoneal injection dose of 10 mg/kg and no observable toxicity. All of these results suggest that FD274 has potential for further development as a promising PI3K/mTOR targeted anti-AML drug candidate.

Discovery of a high potent PIM kinase inhibitor for acute myeloid leukemia based on N-pyridinyl amide scaffold by optimizing the fragments toward to Lys67 and Asp128/Glu171.

Despite the recent development of PIM inhibitors based on N-(pyridin-3-yl)acetamide scaffold for acute myeloid leukemia (AML), the structural-activity relationship (SAR) associated with the effects of positional isomerization of N toward to Lys67 and freedom of solvent fragment toward to Asp128/Glu171 still remains an open question. In this work, a structurally novel compound based on N-pyridinyl amide was designed by fragment hybridization and then our SAR exploration revealed that the positional isomerization would lead to a decrease in activity, while increase of the freedom of solvent fragment by breaking the intramolecular hydrogen bond unprecedentedly leads to an increase in activity. These studies finally resulted in the screening out of a potent PIM inhibitor FD1024 (compound 24) which exerts strong antiproliferative activity against the tested AML cell lines and achieves profound antitumor efficacy in mice at well-tolerated dose schedules.

A pyridinesulfonamide derivative FD268 suppresses cell proliferation and induces apoptosis via inhibiting PI3K pathway in acute myeloid leukemia.

Aberration of PI3K signaling pathway has been confirmed to be associated with several hematological malignancies including acute myeloid leukemia (AML). FD268, a pyridinesulfonamide derivative characterized by the conjugation of 7-azaindole group, is a newly identified PI3K inhibitor showing high potent enzyme activity at nanomole concentration. In this study, we demonstrated that FD268 dose-dependently inhibits survival of AML cells with the efficacy superior to that of PI-103 (pan-PI3K inhibitor) and CAL-101 (selective PI3Kδ inhibitor) in the tested HL-60, MOLM-16, Mv-4-11, EOL-1 and KG-1 cell lines. Further mechanistic studies focused on HL-60 revealed that FD268 significantly inhibits the PI3K/Akt/mTOR signaling pathway, promotes the activation of pro-apoptotic protein Bad and downregulates the expression of anti-apoptotic protein Mcl-1, thus suppressing the cell proliferation and inducing caspase-3-dependent apoptosis. The bioinformatics analysis of the transcriptome sequencing data also indicated a potential involvement of the PI3K/Akt/mTOR pathway. These studies indicated that FD268 possesses high potent activity toward AML cells via inhibition of PI3K/Akt/mTOR signaling pathway, which sheds some light on the pyridinesulfonamide scaffold for further optimization and investigation.

Integrated multi-omics analysis and microbial recombinant protein system reveal hydroxylation and glycosylation involving nevadensin biosynthesis in Lysionotus pauciflorus.

BACKGROUND: Karst-adapted plant, Lysionotus pauciflours accumulates special secondary metabolites with a wide range of pharmacological effects for surviving in drought and high salty areas, while researchers focused more on their environmental adaptations and evolutions. Nevadensin (5,7-dihydroxy-6,8,4'-trimethoxyflavone), the main active component in L. pauciflours, has unique bioactivity of such as anti-inflammatory, anti-tubercular, and anti-tumor or cancer. Complex decoration of nevadensin, such as hydroxylation and glycosylation of the flavone skeleton determines its diversity and biological activities. The lack of omics data limits the exploration of accumulation mode and biosynthetic pathway. Herein, we integrated transcriptomics, metabolomics, and microbial recombinant protein system to reveal hydroxylation and glycosylation involving nevadensin biosynthesis in L. pauciflours. RESULTS: Up to 275 flavonoids were found to exist in L. pauciflorus by UPLC-MS/MS based on widely targeted metabolome analysis. The special flavone nevadensin (5,7-dihydroxy-6,8,4'-trimethoxyflavone) is enriched in different tissues, as are its related glycosides. The flavonoid biosynthesis pathway was drawn based on differential transcripts analysis, including 9 PAL, 5 C4H, 8 4CL, 6 CHS, 3 CHI, 1 FNSII, and over 20 OMTs. Total 310 LpCYP450s were classified into 9 clans, 36 families, and 35 subfamilies, with 56% being A-type CYP450s by phylogenetic evolutionary analysis. According to the phylogenetic tree with AtUGTs, 187 LpUGTs clustered into 14 evolutionary groups (A-N), with 74% being E, A, D, G, and K groups. Two LpCYP82D members and LpUGT95 were functionally identified in Saccharomyces cerevisiae and Escherichia coli, respectively. CYP82D-8 and CYP82D-1 specially hydroxylate the 6- or 8-position of A ring in vivo and in vitro, dislike the function of F6H or F8H discovered in basil which functioned depending on A-ring substituted methoxy. These results refreshed the starting mode that apigenin can be firstly hydroxylated on A ring in nevadensin biosynthesis. Furthermore, LpUGT95 clustered into the 7-OGT family was verified to catalyze 7-O glucosylation of nevadensin accompanied with weak nevadensin 5-O glucosylation function, firstly revealed glycosylation modification of flavones with completely substituted A-ring. CONCLUSIONS: Metabolomic and full-length transcriptomic association analysis unveiled the accumulation mode and biosynthetic pathway of the secondary metabolites in the karst-adapted plant L. pauciflorus. Moreover, functional identification of two LpCYP82D members and one LpUGT in microbe reconstructed the pathway of nevadensin biosynthesis.

Discovery of cinnoline derivatives as potent PI3K inhibitors with antiproliferative activity.

Cinnoline is a potential pharmacophore which has rarely been reported for uses as PI3K inhibitors. In this study, a series of cinnoline derivatives were developed as PI3K inhibitors and evaluated for enzymatic and cellular activities. Most compounds displayed nanomolar inhibitory activities against PI3Ks, among which 25 displayed high LLE and micromolar inhibitory potency against three human tumor cell lines (IC50 = 0.264 µM, 2.04 µM, 1.14 µM).

Bioisosteric replacements of the indole moiety for the development of a potent and selective PI3Kδ inhibitor: Design, synthesis and biological evaluation.

Based on indole scaffold, a potent and selective phosphoinositide 3-kinase delta (PI3Kδ) inhibitor, namely FD223, was developed by the bioisosteric replacement drug discovery approach and studied for the treatment of acute myeloid leukemia (AML). In vitro studies revealed that FD223 displays high potency (IC50 = 1 nM) and selectivity (29-51 fold over other PI3K isoforms) against PI3Kδ, and exhibits efficient inhibition of the proliferation of AML cell lines (MOLM-16, HL-60, EOL-1 and KG-1) by suppressing p-AKT Ser473 thus causing G1 phase arrest during the cell cycle. Further given the favorable pharmacokinetic (PK) profiles of FD223, in vivo studies were evaluated using xenograft model in nude mice, confirming its significant antitumor efficacy meanwhile with no observable toxicity. All these results are comparable to the positive group of Idelalisib (CAL-101), indicating that FD223 has potential for further development as a promising PI3Kδ inhibitor for the treatment of leukemia such as AML.

Design and Evaluation of Rhein-Based MRI Contrast Agents for Visualization of Tumor Necrosis Induced by Combretastatin A-4 Disodium Phosphate.

PURPOSE: Visualization of tumor necrosis can determine tumor response to therapy. Our previous study showed that the rhein-based magnetic resonance imaging (MRI) contrast agent with alkane linker (GdL2) could clearly image tumor necrosis. However, its water solubility and cell safety needed to be improved. Herein, three rhein-based MRI agents with ether or lysine linkers were designed. PROCEDURES: Three rhein-based MRI agents were synthesized with a tetracarbon ether (GdP1), a hexacarbon ether (GdP2), and a lysine (GdP3) linker, respectively. Their octanol-water partition coefficients (log P) and cytotoxicity were determined. Necrosis avidity of the leading agent was explored on HepG2 cells and ischemia reperfusion-induced liver necrosis (IRLN) rats by MRI. The effect of visualization of tumor necrosis was tested on nude mice with W256 tumor treated by combretastatin-A4 phosphate (CA4P). DNA binding assays were applied to evaluate the possible necrosis-avidity mechanism of the leading agent. RESULTS: The log P of three agents (- 1.66 ± 0.09, - 1.74 ± 0.01, - 1.95 ± 0.01) decreased when compared with GdL2, indicating higher water solubility. GdP1 not only presented lower cytotoxicity and good necrotic affinity in vitro and in vivo, but also can be fast excreted by renal. According to MRI results of tumor, distinct visualization of tumor necrosis can be discernible from 3 to 4.5 h post-injection of GdP1. In DNA-binding assays, the fluorescence quenching constant KSV (1.00 × 104 M-1) and the ultraviolet binding constant Kb (1.11 × 104 M-1) suggested that GdP1 may bind to DNA through intercalation. CONCLUSION: GdP1 may serve as a potential candidate for early evaluation of tumor response to CA4P treatment.

Synthesis and Evaluation of Ga-68-Labeled Rhein for Early Assessment of Treatment-Induced Tumor Necrosis.

PURPOSE: This study aimed to synthesize a necrosis-avid agent using rhein as a precursor and labeled with gallium-68 (Ga-68) for positron emission tomography/computed tomography (PET/CT) imaging, to evaluate response to anticancer treatment in a mouse model. PROCEDURES: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated rhein was radiolabeled with Ga-68 to formulate [68Ga]DOTA-rhein. The in vitro stability of [68Ga]DOTA-rhein was assessed by radio-HPLC. Necrosis avidity was evaluated in a mouse model of muscle necrosis by microPET/CT imaging, biodistribution study, histochemical staining, and autoradiography studies. Murine tumor models with the subcutaneous implantation of S180 cell lines were generated for the evaluation of therapeutic effect. Tumor necrosis was induced by the treatment of combretastatin A4 disodium phosphate (CA4P), and microPET/CT imaging was performed at 1 h post tracer injection. DNA binding studies were conducted to explore the necrosis avidity mechanism of the tracer. RESULTS: [68Ga]DOTA-rhein exhibited a satisfactory yield, a radiochemical purity over 97 %, and a good serum stability. The uptakes of [68Ga]DOTA-rhein in necrotic muscles and tumors were significantly higher than those in normal muscles and tumors (P < 0.05). The results of autoradiography and histochemical staining were consistent with the selective uptake of the radiotracer in necrotic regions. MicroPET/CT images showed a high uptake of the tracer in necrotic muscles and necrotic tumors. DNA binding studies suggested that necrosis avidity correlated with DNA binding to a certain extent. CONCLUSIONS: Our results demonstrated that [68Ga]DOTA-rhein showed a prominent necrosis avidity and could be a useful probe for early assessment of response to anticancer therapy by PET/CT imaging.

Synthesis and Evaluation of Diindole-Based MRI Contrast Agent for In Vivo Visualization of Necrosis.

PURPOSE: Noninvasive imaging of cell necrosis can provide an early evaluation of tumor response to treatments. Here, we aimed to design and synthesize a novel diindole-based magnetic resonance imaging (MRI) contrast agent (Gd-bis-DOTA-diindolylmethane, Gd-DIM) for assessment of tumor response to therapy at an early stage. PROCEDURES: The oil-water partition coefficient (Log P) and relaxivity of Gd-DIM were determined in vitro. Then, its necrosis avidity was examined in necrotic cells in vitro and in rat models with microwave ablation-induced muscle necrosis (MAMN) and ischemia reperfusion-induced liver necrosis (IRLN) by MRI. Visualization of tumor necrosis induced by combretastatin A-4 disodium phosphate (CA4P) was evaluated in rats bearing W256 orthotopic liver tumor by MRI. Finally, DNA binding assay was performed to explore the possible necrosis-avidity mechanism of Gd-DIM. RESULTS: The Log P value and T1 relaxivity of Gd-DIM is - 2.15 ± 0.01 and 6.61 mM-1 s-1, respectively. Gd-DIM showed predominant necrosis avidity in vitro and in vivo. Clear visualization of the tumor necrosis induced by CA4P was achieved at 60 min after administration of Gd-DIM. DNA binding study indicated that the necrosis-avidity mechanism of Gd-DIM may be due to its binding to exposed DNA in necrotic cells. CONCLUSION: Gd-DIM may serve as a promising necrosis-avid MRI contrast agent for early assessment of tumor response to therapy.

Rhein-based necrosis-avid MRI contrast agents for early evaluation of tumor response to microwave ablation therapy.

PURPOSE: Early evaluation of tumor response to thermal ablation therapy can help identify untreated tumor cells and then perform repeated treatment as soon as possible. The purpose of this work was to explore the potential of rhein-based necrosis-avid contrast agents (NACAs) for early evaluation of tumor response to microwave ablation (MWA). METHODS: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to test the cytotoxicity of rhein-based NACAs against HepG2 cells. Rat models of liver MWA were used for investigating the effectiveness of rhein-based NACAs in imaging the MWA lesion, the optimal time period for post-MWA MRI examination, and the metabolic behaviors of 68 Ga-labeled rhein-based NACAs. Rat models of orthotopic liver W256 tumor MWA were used for investigating the time window of rhein-based NACAs for imaging the MWA lesion, the effectiveness of these NACAs in distinguishing the residual tumor and the MWA lesion, and their feasibility in early evaluating the tumor response to MWA. RESULTS: Gadolinium 2,2',2''-(10-(2-((4-(4,5-Dihydroxy-9,10-dioxo-9,10-dihydroanthracene-2-carboxamido)butyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (GdL2 ) showed low cytotoxicity and high quality in imaging the MWA region. The optimal time period for post-MWA MRI examination using GdL2 was 2 to 24 h after the treatment. During 2.5 to 3.5 h postinjection, GdL2 can better visualize the MWA lesion in comparison with gadolinium 2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetic acid (Gd-DOTA), and the residual tumor would not be enhanced. The tumor response to MWA as evaluated by using GdL2 -enhanced MRI was consistent with histological examination. CONCLUSION: GdL2 appears to be a promising NACA for the tumor response assessment after thermal ablation therapies.

Design and synthesis of NQO1 responsive fluorescence probe and its application in bio-imaging for cancer diagnosis.

As an over-expressed flavoprotein in several kinds of tumor cells, NAD(P)H: quinone oxidoreductase-1 (NQO1) is considered as a potent biomarker in early-stage cancer diagnosis. Developing a fast, selective and sensitive method of monitoring NQO1 on cellular level will greatly promote cancer diagnosis in clinical practice. In this paper, a fast NQO1 responsive fluorescence probe SYZ-30 containing quinone acid and 7-nitro-2,1,3-benzoxadiazole (NBD) fluorophore is constructed. The probe could selectively respond to NQO1 and rapidly emit strong fluorescence in vitro within only 5 min. Notably, the peak fluorescent intensities at 550 nm showed a linear relationship with NQO1 concentrations in the range of 3-30 ng/ml and limit of detection (LOD) was 0.0667 ng/ml. Furthermore, it was validated that the probe has good biocompatibility and could be applied for bio-imaging in NQO1 over-expressed cancer cells, together with its mitochondria targeting ability. Importantly, confocal fluorescence imaging confirmed the NQO1 detection ability on cellular level, which can be used for real-time detection of several cancer subtypes like adenocarcinoma. To conclude, the probe is rapidly responsive, highly sensitive and selective which will potentially become a practical tool for early cancer detection and diagnosis.