Discovery of potent small molecule PROTACs targeting mutant EGFR.

Epidermal growth factor receptor (EGFR) is an important therapeutic target for the treatment of non-small cell lung cancer. A number of efficacious EGFR tyrosine kinase inhibitors have been developed. However, acquired drug resistance largely encumbered their clinical practicability. Therefore, there is an urgent need to develop new therapeutic regime. Herein, we designed and synthesized a set of EGFR-targeting small molecule PROTACs which showed promising efficacy. In particular, VHL-recruiting compound P3 showed potent anti-proliferative activity against HCC827 and H1975 cell lines with IC50 values of 0.83 and 203.01 nM, respectively. Furthermore, both EGFRdel19 and EGFRL858R/T790M could be significantly induced to be degraded under treatment of P3 with DC50 values of 0.51 and 126.2 nM, respectively. Compound P3 was able to dramatically suppress EGFR pathway signal transduction. Moreover, compound P3 could significantly induce cell apoptosis, arrest cell cycle and suppress cell colony formation. In addition, we identified that ubiquitination was indispensable in the degradation process, and found that the degradation was related to autophagy. Our work would provide an alternative approach for development of potentially effective EGFR degraders and give a new clue for investigation of PROTAC-induced protein degradation.

W941, a new PI3K inhibitor, exhibits preferable anti-proliferative activities against nonsmall cell lung cancer with autophagy inhibitors.

The PI3K pathway is aberrantly activated in many cancers and plays a critical role in tumour cell proliferation and survival, making it a rational therapeutic target. In the present study, the effects and the underlying mechanism of a new PI3K inhibitor, W941, were investigated in non-small-cell lung cancer (NSCLC). The results of this study showed that W941 inhibited the growth of A549 and Hcc827 cells with IC50 values of 0.12 and 0.23 µM, respectively, and that W941 markedly inhibited the growth of A549 xenograft tumours in a nude mouse model without decreasing body weight. Western blotting assays showed that W941 inhibited the phosphorylation of downstream proteins in the PI3K pathway (AKT, mTOR, p70S6K and 4EBP1) in both A549 and Hcc827 cells. In addition, after W941 treatment, a dose-dependent increase in the ratio of the LC3-II/I ratio was observed. When cells were pre-treated with chloroquine or bafilomycin A1, W941 increased the LC3-II/I ratio, suggesting that W941 acted as an autophagy inducer. Moreover, autophagy blockers enhanced apoptosis after W941 treatment, indicating that W941-induced autophagy actually protected the cells against its cytotoxicity. Our findings suggest that the combination of a PI3K inhibitor with an autophagy inhibitor might be a novel option for NSCLC treatment.

The dual luciferase reporter system and RT-qPCR strategies for screening of MicroRNA-21 small-molecule inhibitors.

The therapeutic potential of microRNA-21 (miR-21) small-molecule inhibitors has been of particular interest to medicinal chemists. Moreover, the development of more facile screening methods is lacking. In the present study, two potential screening strategies for miR-21 small-molecule inhibitor including the stem-loop reverse transcription-quantitative PCR and dual luciferase reporter assay system were demonstrated and discussed in detail. A pmirGLO-miR21cswt plasmid and its two different mutants were constructed for dual luciferase reporter assay system. In addition, the sensitivity and specificity of these two methods were validated. Our results demonstrated that both strategies are decent choices for the screening of small-molecule inhibitors for miR-21 and possibly other miRNAs. Eventually, we applied our optimized strategy to discover and characterize several promising compounds such as azobenzene derivate A, enoxacin, and norfloxacin for their potential impact on intracellular miR-21 concentration.

Discovery of 2-(aminopyrimidin-5-yl)-4-(morpholin-4-yl)-6- substituted triazine as PI3K and BRAF dual inhibitor.

AIM: The discovery and development of novel agents simultaneously targeting PI3K/AKT/mammalian target of rapamycin and Ras/RAF/MEK, two signaling pathways, are urgent to improve the curative effect of kinase inhibitors and overcome acquired resistance. METHODS/RESULTS: In the present study, 2-(2-aminopyrimidin-5-yl)-4-(morpholin-4-yl)-6-(N-cyclopropyl-N- (1-benzoylpiperidin-4-yl))triazines/pyrimidines were designed as PI3K and BRAF dual inhibitors. The synthesized 20 compounds exhibited potent antiproliferative effects in vitro against HCT116, A375, MCF-7, Colo205, A549 and LOVO cancer cell lines. The tested compounds A6, A7, A9 and A11 remarkably displayed inhibitory activities toward both PI3Kα and BRAFV600E. CONCLUSION: These results indicated that our design compounds can serve as potent PI3Kα and BRAFV600E dual inhibitors and effective antiproliferative agents, which can be further optimized to discover more potent PI3Kα and BRAFV600E dual inhibitors.

Sodium selenite induces apoptosis and inhibits autophagy in human synovial sarcoma cell line SW982 in vitro.

The present study aimed to examine the effects of sodium selenite on the SW982 human synovial sarcoma cell line in relation to cell viability, apoptosis and autophagy. The results indicated that sodium selenite reduced cell viability and induced apoptosis by activating caspase­3 and members of the poly (ADP­ribose) polymerase and Bcl­2 protein families in SW982 cells. Furthermore, autophagy was also suppressed by sodium selenite treatment in SW982 cells, and apoptosis was upregulated in cells co­treated with sodium selenite and the autophagy inhibitor 3­methyladenine. By contrast, apoptosis was downregulated when sodium selenite was combined with rapamycin, an inducer of autophagy. The results indicated that autophagy may protect cells from the cytotoxicity of sodium selenite. The present study results demonstrated that sodium selenite induced apoptosis and inhibited autophagy and autophagy­protected cells from death by antagonizing sodium selenite­induced apoptosis in SW982 cells in vitro.

[Bidirectional regulation of Pam3CSK4?induced inflammatory response by ATP?binding cassette transporter A1 knockdown in mouse mononuclear macrophages in vitro].

OBJECTIVE: To investigate the regulatory effect of ATP?binding cassette transporter A1 (ABCA1) knockdown on inflammatory response induced by Pam3CSK4 in mouse mononuclear macrophage RAW264.7 cell line. METHODS: A mouse mononuclear macrophage RAW264.7 cell line with stable ABCA1 knockdown was constructed and stimulated with Toll?like receptor 2 (TLR2) ligand Pam3CSK4, and the changes in the transcriptional levels of the proinflammatory and anti-inflammatory cytokines were analyzed in this cell model. RESULTS: In RAW264.7 cells, ABCA1 knockdown significantly up-regulated Pam3CSK4 stimulation?induced expressions of IL?1ß, TNF?α and IL?6 and also enhanced the expression of transcription factor cAMP?dependent transcription factor 3 (ATF3) without obviously affecting the expressions of the transcription factors ATF1, ATF2, ATF4 or ATF5. CONCLUSION: ABCA1 knockdown in macrophages may have both proinflammatory and anti?inflammatory effects. ABCA1 knockdown up?regulates the transcription of ATF3 possibly through a mechanism that is different from that for the other members of the ATF protein family.

Synthesis and antitumor activity evaluation of PI3K inhibitors containing 3-substituted quinazolin-4(3H)-one moiety.

In present study, a series of N-(2-methoxy-5-(3-substituted quinazolin-4(3H)-one-6-yl)-pyridin-3-yl)phenylsulfonamide were synthesized. Their antiproliferative activities in vitro were evaluated via MTT assay against HCT116 and MCF-7 cancer cell lines. The SAR of title compounds was discussed. The compounds (S)-C5 and (S)-C8 displayed potent inhibitory activity against PI3Ks and mTOR, especially against PI3Kα. In addition, compound (S)-C5 can efficaciously inhibit tumor growth in a mice S-180 model. These findings suggest that our designed compounds can serve as potent PI3K inhibitors and effective anticancer agents.

Discovery of 4-benzoylamino-N-(prop-2-yn-1-yl)benzamides as novel microRNA-21 inhibitors.

MicroRNA-21, as an oncogenic miRNA, has caught great attention for medicinal chemists to develop its novel inhibitors for cancer therapy. In the present study, we designed 4-benzoylamino-N-(prop-2-yn-1-yl)benzamides as miR-21 inhibitor candidates on the basis of scaffold hopping. Eighteen compounds were synthesized. The inhibitory activities of synthesized compounds against the expression of miR-21 were evaluated using stem loop RT-qPCR and compound 1j was discovered as the most potent compound, which displayed a time and concentration dependent inhibition manner. In addition, various functional assays such as the expression of miR-21 target gene detected by Western blotting and the cell growth and apoptosis detected by flow cytometric analysis were checked in Hela (human epithelioid cervix carcinoma) and U-87 MG (human glioblastoma) cells to confirm its activity. The results indicate that compound 1j can enhance apoptosis, retard proliferation, and up-regulate PDCD4, a target protein of miR-21. In addition, the compound 1j does not influence the expression of multiple miRNAs and the genes that participate in miRNA universal biosynthesis pathway. These results strongly support the assumption that title compounds can serve as a small molecule inhibitor of miR-21.

Synthesis and anticancer effects evaluation of 1-alkyl-3-(6-(2-methoxy-3-sulfonylaminopyridin-5-yl)benzo[d]thiazol-2-yl)urea as anticancer agents with low toxicity.

As a PI3K and mTOR dual inhibitor, N-(2-chloro-5-(2-acetylaminobenzo[d]thiazol-6-yl)pyridin-3-yl)-4-fluorophenylsulfonamide displays toxicity when orally administrated. In the present study, alkylurea moiety replaced the acetamide group in the compound and a series of 1-alkyl-3-(6-(2,3-disubstituted pyridin-5-yl)benzo[d]thiazol-2-yl)urea derivatives were synthesized. The antiproliferative activities of the synthesized compounds in vitro were evaluated against HCT116, MCF-7, U87 MG and A549 cell lines. The compounds with potent antiproliferative activity were tested for their acute oral toxicity and inhibitory activity against PI3Ks and mTORC1. The results indicate that the compound attached a 2-(dialkylamino)ethylurea moiety at the 2-positeion of benzothiazole can retain the antiproliferative activity and inhibitory activity against PI3K and mTOR. In addition, their acute oral toxicity reduced dramatically. Moreover, compound 2f can effectively inhibit tumor growth in a mice S180 homograft model. These findings suggest that 1-(2-dialkylaminoethyl)-3-(6-(2-methoxy-3-sulfonylaminopyridin-5-yl)benzo[d]thiazol-2-yl)urea derivatives can serve as potent PI3K inhibitors and anticancer agents with low toxicity.

Synthesis and antitumor activities evaluation of m-(4-morpholinoquinazolin-2-yl)benzamides in vitro and in vivo.

In the present study, a series of m-(4-morpholinoquinazolin-2-yl)benzamides were designed, synthesized and characterized. The antiproliferative activities of the synthesized compounds were evaluated against two human cell lines (HCT-116 and MCF-7). Compounds with IC50 values below 4 µM were further evaluated against U-87 MG and A549 cell lines. Among these evaluated compounds, compound T10 displayed a remarkable antiproliferative effect in vitro. The hoechst staining assay showed that compound T10 caused morphological changes. The cell cycle and apoptosis assay further indicated that compound T10 can arrest HCT-116 cells in G2/M and G0/G1 phase and induce apoptosis. PI3K enzyme assays indicated that compounds T7 and T10 selectively inhibit PI3Kα. A Western bolt assay further suggested that compound T10 can block the PI3K/Akt/mTOR pathway. Moreover, compound T10 inhibited tumor growth on a mice S180 homograft model. These findings directly identify m-(4-morpholinoquinazolin-2-yl)benzamide derivatives as novel anticancer agents.

Design, synthesis and antiproliferative activity evaluation of m-(4-morpholinyl-1,3,5-triazin-2-yl)benzamides in vitro.

In the present study, a series of m-(4-morpholino-1,3,5-triazin-2-yl)benzamides were designed, synthesized and characterized. Their antiproliferative activities against HCT-116 cell and MCF-7 cell at 10µM were evaluated by MTT assay. Compounds T6, T10, T11, T12 and T19 exhibited potent antiproliferative activities. Thus, their IC50 values against HCT-116 cell, MCF-7 cell, Hela cell, U-87 MG cell and A549 cell were measured. The SAR of the target compounds was preliminary discussed. The Western bolt assay suggested that compound T11 can block the PI3K/Akt/mTOR pathway. Hoechst staining assay indicated that compound T11 can cause morphological changes and induce apoptosis of HCT-116 cells. These findings directly identify the m-(4-morpholinyl-1,3,5-triazin-2-yl)benzamide derivatives as novel antiproliferative agents and further verify the value of the benzamide fragment in drug design.

Combination of 2-methoxy-3-phenylsulfonylaminobenzamide and 2-aminobenzothiazole to discover novel anticancer agents.

The fragment of 2-substituted-3-sulfonylaminobenzamide has been proposed to replace the fragment of 2-substituted-3-sulfonylaminopyridine in PI3K and mTOR dual inhibitors to design novel anticancer agents based on bioisostere. The combination of the fragment of 2-substituted-3-sulfonylaminobenzamide with the fragment of 2-aminobenzothiazole or 2-aminothiazolo[5,4-b]pyridine, or 2-amino[1,2,4]triazolo[1,5-a]pyridine produced the novel structures of anticancer agents. As a result, nineteen target compounds were synthesized and characterized. Their antiproliferative activities in vitro were evaluated via MTT assay against four human cancer cell lines including HCT-116, A549, MCF-7 and U-87 MG. The SAR of target compounds was preliminarily discussed. Compound 1g with potent antiproliferative activity was examined for its effect on the AKT and p-AKT(473). The anticancer effect of 1g was evaluated in established nude mice HCT-116 xenograft model. The results suggested that compound 1g can block PI3K/AKT/mTOR pathway and significantly inhibit tumor growth. These findings strongly support our assumption that the fragment of benzamide can replace the pyridine ring in some PI3K and mTOR dual inhibitor to design novel anticancer agents.

Discovery of 2-aryl-8-hydroxy (or methoxy)-isoquinolin-1(2H)-ones as novel EGFR inhibitor by scaffold hopping.

2-Aryl-8-hydroxy (or methoxy)-isoquinolin-1(2H)-one has been proposed as a novel scaffold of EGFR inhibitor based on scaffold hoping. In the present study, a series of 2-aryl-8-hydroxy (or methoxy)-isoquinolin-1(2H)-one derivatives were synthesized. Their antiproliferative activities in vitro were evaluated via MTT assay against two human cancer cell lines, including A431 and A549. The SAR of the title compounds was preliminarily discussed. The compounds with ideal inhibition were evaluated through ELISA-based EGFR-TK assay. Compound 6c showed the best activity against A431 and EGFR tyrosine kinase. These findings suggest that title compounds are EGFR inhibitors with novel structures.

Synthesis and anticancer activity evaluation of a series of [1,2,4]triazolo[1,5-a]pyridinylpyridines in vitro and in vivo.

A series of [1,2,4]triazolo[1,5-a]pyridinylpyridines were synthesized and characterized. Their antiproliferative activities in vitro were evaluated by MTT against three human cancer cell lines including HCT-116, U-87 MG and MCF-7 cell lines. The SAR of target compounds was preliminarily discussed. The compounds 1c and 2d with potent antiproliferative activities were tested for their effects on the AKT and p-AKT(473). The anticancer effect of 1c was evaluated in mice bearing sarcoma S-180 model. The results suggest that the title compounds are potent anticancer agents.

Correlation of AIM2 expression in peripheral blood mononuclear cells from humans with acute and chronic hepatitis B.

The AIM2 (absent in melanoma 2) protein promotes host defenses against invading viruses and pathogenic bacteria through corresponding adapter molecules leading to the initiation of innate immune responses. We investigated the expression of AIM2 in peripheral blood mononuclear cells (PBMCs) from patients with acute hepatitis B (AHB) and chronic hepatitis B (CHB) during different clinical phases, and analyzed the correlation between AIM2 and clinical profiles in these groups. This study indicated that there is higher expression of AIM2, IL-1ß, and IL-18 in AHB compared with expression in CHB. The expression of AIM2 mRNA was significantly negatively correlated with serum hepatitis B virus (HBV) load, HBeAg, and significantly positively correlated with IL-1ß and IL-18 in AHB patients and CHB patients with immune clearance, which suggests that AIM2 expression is correlated with the immune clearance of HBV in the host. We summarized that there is a higher immune status in AHB, and a lower immune response in CHB. This suggests that the down-regulation of AIM2 may be associated with the chronic development of HB.

Reduced apoptosis correlates with enhanced autophagy in synovial tissues of rheumatoid arthritis.

OBJECTIVE: Defective apoptosis contributes to the massive synovial hyperplasia in rheumatoid arthritis (RA), but the mechanism is largely unknown. To investigate the reasons for the reduced apoptosis in RA synovium, we analyzed autophagy and its relationship to apoptosis in synovial tissues from RA and osteoarthritis (OA) patients. METHODS: Synovial tissues were obtained from seven RA and 12 OA patients undergoing knee replacement surgery. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and staining for p85 fragment of PolyADP-ribose polymerase (PARP). Autophagy was determined by immunoblotting for the autophagic markers Beclin-1 and LC3. MicroRNA-30a (miR-30a), which targets Beclin-1, was measured by real-time RT-PCR. The interplay between autophagy and apoptosis was determined via Spearman's correlation analysis. RESULTS: In comparison with OA, the synovial tissues from RA displayed decreased TUNEL-positive nuclei (P < 0.01). In contrast, Beclin-1 and LC3 were overexpressed in the synovial lining layers of RA, which was correlated with decreased levels of miR-30a. Moreover, there was a significant reverse relationship between apoptosis and autophagy in RA synovial tissues (P < 0.01 and r = -0.8937). CONCLUSION: The impaired apoptosis in RA synovium might result from increased autophagy, which in turn could be due to the deregulation of miRNA-30a.

Synthesis and biological evaluation of N-aryl salicylamides with a hydroxamic acid moiety at 5-position as novel HDAC-EGFR dual inhibitors.

A novel series of N-aryl salicylamides with a hydroxamic acid moiety at 5-position were synthesized efficiently. Their activities against EGFR kinase and HDACs were evaluated. All compounds displayed inhibitory activity against EGFR and HDACs. The antiproliferative activities of synthesized compounds were evaluated by MTT method against human cancer cell lines A431, A549 and HL-60. Compound 1o showed the most potent inhibitory activity against A431 and A549. Compounds 1k and 1n exhibited higher potency against HL-60 than gefitinib and SAHA. N-Aryl salicylamides with a hydroxamic acid moiety at 5-position is another new HDAC-EGFR dual inhibitors.

Infliximab alleviates inflammation and ex vivo airway hyperreactivity in asthmatic E3 rats.

Tumor necrosis factor-α (TNF-α) has been implicated in the pathogenesis of asthma, and neutralization of TNF-α is an effective therapy for inflammatory diseases. The present study tested the idea that a TNF-α antibody, infliximab, may be useful in the management of asthma. E3 rats were immunized with ovalbumin (OVA)/alum and received infliximab intra-peritoneally. Two weeks later, OVA-PBS was instilled intranasally daily for 7 days. Bronchoalveolar lavage fluids (BALFs), serum and lung homogenates were collected for analysis of cells and inflammatory mediators. Contractile responses of lobar-bronchus segments to agonists were functionally tested. Pulmonary tissues were investigated using histological examination. The results showed that the sensitized 'model E3 rats' exhibited an increase in the total amount of inflammatory cells, primarily eosinophils, in BALF and pulmonary tissue, as well as epithelial damage. Serum levels of IgE increased and so did the levels of nitric oxide, inducible nitric oxide synthase, TNF-α and IL-4, IL-5 and IL-13 in lung homogenate and serum. Furthermore, the contractile responses in bronchi induced by endothelin-1, sarafotoxin 6c and bradykinin increased and isoprenaline-induced relaxations decreased. All these changes induced by the sensitization procedure were reduced by the infliximab treatment. The results suggest that infliximab prevents the development of local airway inflammation and antagonizes changes of the bronchial smooth muscle receptor phenotype, thereby blocking the development of airway smooth muscle hyperreactivity of asthmatic rats.

Methotrexate ameliorates pristane-induced arthritis by decreasing IFN-γ and IL-17A expressions.

OBJECTIVE: This study was carried out to test the effects of methotrexate (MTX) and black seed oil (BSO) on pristane-induced arthritis (PIA) in rats. METHODS: Inbred dark agouti (DA) rats were induced by a single subcutaneous injection of pristane, and then treated with MTX or BSO. Arthritis severity was evaluated macroscopically and microscopically. Plasma nitric oxide (NO) concentration was determined by the Griess method and cytokine mRNA expression in the spleen was detected by the real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The clinical arthritis severity was decreased after MTX treatment, while the BSO groups did not show significant changes compared with the disease group. The plasma NO level of the MTX group was significantly decreased compared with the disease group, but the BSO groups showed no difference from the disease group in plasma NO levels. The interferon-γ (IFN-γ) and interleukin-17A (IL-17A) mRNA expressions in the spleens were significantly decreased in the MTX group, but only showed a declining trend in the BSO groups compared with the disease group. Neither MTX nor BSO had an effect on the mRNA expressions of IL-4, transforming growth factor ß (TGF-ß), and tumor necrosis factor-α (TNF-α) in the spleen. CONCLUSIONS: MTX, but not BSO, can reduce the arthritis severity and decrease the mRNA expressions of IFN-γ and IL-17A in pristane-induced arthritis of rats.

Analysis of correlation between blood biochemical indicators and bone mineral density of post-menopausal women.

Osteoporosis is a degenerative disease of the skeletal system, and its major complication is fracture that severely influences the living quality of the middle-aged and the aged. The purpose of this study was to investigate the significance of sex hormones and some biochemical indicators related to bone metabolism in the genesis and development of osteoporosis. The plasma samples were collected from 244 post-menopausal women of Xi'an urban area, and their plasma contents of testosterone, estradiol, calcitonin, osteocalcin and N-terminal propeptide of type I procollagen were detected by ELISA. The activity of tartrate-resistant acid phosphatase was determined by spectrophotometric method, and the content of nitric oxide was measured by Griess method. Bone mineral density (BMD) in lumbar vertebrae (L1-L4) and hips was measured by QDR-2000 dual energy X-ray absorptiometry. The concentrations of the biochemical indicators were compared among the three groups (normal bone mass group, osteopenia group and osteoporosis group), and Pearson correlation analysis was used to verify the correlations between the indicators and BMD. The comparison results of blood biochemical indicators of BMD-based groups showed that the plasma contents of estradiol (P = 0.006), testosterone (P = 0.038) and calcitonin (P = 0.042) decreased more significantly in the osteoporosis group, but the content of osteocalcin (P = 0.008) increased significantly in osteoporosis group than those in the other groups. The correlation analysis between BMD of different parts and the blood biochemical indicators showed that there was a significant positive correlation between estradiol and the BMD of lumber vertebra (r = 0.200, P = 0.002), femoral neck (r = 0.160, P = 0.013), and great trochanter (r = 0.204, P = 0.001). Significant positive correlations between calcitonin and BMD of lumber vertebra (r = 0.166, P = 0.018) and femoral great trochanter (r = 0.152, P = 0.041), and between testosterone and BMD of femoral great trochanter (r = 0.158, P = 0.014) were also observed. In addition, there existed significant negative correlations between osteocalcin and BMD of lumber vertebra (r = -0.220, P = 0.001), femoral neck (r = -0.259, P < 0.000), and great trochanter (r = -0.221, P = 0.001), and between the activity of tartrate-resistant acid phosphatase and BMD of femoral great trochanter (r = -0.135, P = 0.037). The partial correlation analysis also showed that there were significant correlations between estradiol (r = 0.160, P = 0.014), calcitonin (r = 0.240, P = 0.013), osteocalcin (r = -0.226, P = 0.023) and BMD when the influence of age was excluded. The Pearson correlation analysis of biochemical indicators showed there were positive correlations between the contents of testosterone and calcitonin, testosterone and osteocalcin, calcitonin and osteocalcin, calcitonin and PINP, calcitonin and NO, osteocalcin and NO, and PINP and NO, but negative correlations between the contents of testosterone and PINP, estradiol and calcitonin, estradiol and osteocalcin, and estradiol and NO. The blood contents of sex hormones and calcitonin significantly influence BMD and osteoporosis development, and the increase of osteocalcin contents could be used as a biomarker to indicate the degree of osteoporosis in post-menopausal women.