Protein Structure Inspired Drug Discovery.

Drug discovery starts with known function, either of a compound or a protein, in-turn prompting investigations to probe 3D structure of the compound-protein interface. As protein structure determines function, we hypothesized that unique 3D structural motifs represent primary information denoting unique function that can drive discovery of novel agents. Using a physics-based protein structure analysis platform developed by us, designed to conduct computationally intensive analysis at supercomputing speeds, we probed a high-resolution protein x-ray crystallographic library developed by us. We selected 3D structural motifs whose function was not otherwise established, that offered environments supporting binding of drug-like chemicals and were present on proteins that were not established therapeutic targets. For each of eight potential binding pockets on six different proteins we accessed a 60 million compound library and used our analysis platform to evaluate binding. Using eight-day colony formation assays acquired compounds were screened for efficacy against human breast, prostate, colon and lung cancer cells and toxicity against human bone marrow stem cells. Compounds selectively inhibiting cancer growth segregated to two pockets on separate proteins. The compound, Dxr2-017, exhibited selective activity against human melanoma cells in the NCI-60 cell line screen, had an IC50 of 19 nM against human melanoma M14 cells in our eight-day assay, while over 2100-fold higher concentrations inhibited stem cells by less than 30%. We show that Dxr2-017 induces anoikis, a unique form of programmed cell death in need of targeted therapeutics. The predicted target protein for Dxr2-017 is expressed in bacteria, not in humans. This supports our strategy of focusing on unique 3D structural motifs. It is known that functionally important 3D structures are evolutionarily conserved. Here we demonstrate proof-of-concept that protein structure represents high value primary data to support discovery of novel therapeutics. This approach is widely applicable.

Itraconazole-Induced the Activation of Adenosine 5'-Monophosphate (Amp)-Activated Protein Kinase Inhibits Tumor Growth of Melanoma via Inhibiting ERK Signaling.

Itraconazole, an effective broad-spectrum antifungal drug, has been well established for its anticancer activity in cancers including melanoma. However, details concerning its underlying mechanism in melanoma are unclear. This work investigated the function of itraconazole-induced 5'-monophosphate (AMP)-activated protein kinase alpha (AMPKα) in melanoma progression through ERK signaling. The AMPKα level in melanoma tissues and cells was assessed by RT-qPCR and western blot. Survival analysis of patients with melanoma based on the AMPKα expression level was performed according to TCGA database. Melanoma cell proliferation, migration, and invasion were examined using CCK-8, colony formation, wound healing, and Transwell assays. A xenograft tumor model was established to examine the effect of itraconazole on tumor growth in vivo. The AMPKα mRNA and protein levels were reduced in melanoma tissues and cells. A low expression of AMPKα indicated a poor prognosis. Functionally, itraconazole restrained melanoma cell proliferation, migration, and invasion by upregulating AMPKα. Itraconazole activated AMPK signaling and inhibited ERK signaling in melanoma cells. Activation of ERK signaling reversed the effect of itraconazole on cellular process in melanoma. Moreover, itraconazole-induced AMPKα inhibited melanoma tumor growth in vivo by inhibiting ERK signaling. Itraconazole-induced AMPKα inhibits the progression of melanoma by inhibition of ERK signaling.

Functional Attributes and Anticancer Potentialities of Chico (Pachycereus Weberi) and Jiotilla (Escontria Chiotilla) Fruits Extract.

Mexico has a great diversity of cacti, however, many of their fruits have not been studied in greater depth. Several bioactive compounds available in cacti juices extract have demonstrated nutraceutical properties. Two cactus species are interesting for their biologically active pigments, which are chico (Pachycereus weberi (J. M. Coult.) Backeb)) and jiotilla (Escontria chiotilla (Weber) Rose)). Hence, the goal of this work was to evaluate the bioactive compounds, i.e., betalains, total phenolic, vitamin C, antioxidant, and mineral content in the extract of the above-mentioned P. weberi and E. chiotilla. Then, clarified extracts were evaluated for their antioxidant activity and cytotoxicity (cancer cell lines) potentialities. Based on the obtained results, Chico fruit extract was found to be a good source of vitamin C (27.19 ± 1.95 mg L-Ascorbic acid/100 g fresh sample). Moreover, chico extract resulted in a high concentration of micronutrients, i.e., potassium (517.75 ± 16.78 mg/100 g) and zinc (2.46 ± 0.65 mg/100 g). On the other hand, Jiotilla has a high content of biologically active pigment, i.e., betaxanthins (4.17 ± 0.35 mg/g dry sample). The antioxidant activities of clarified extracts of chico and jiotilla were 80.01 ± 5.10 and 280.88 ± 7.62 mg/100 g fresh sample (DPPH method), respectively. From the cytotoxicity perspective against cancer cell lines, i.e., CaCo-2, MCF-7, HepG2, and PC-3, the clarified extracts of chico showed cytotoxicity (%cell viability) in CaCo-2 (49.7 ± 0.01%) and MCF-7 (45.56 ± 0.05%). A normal fibroblast cell line (NIH/3T3) was used, as a control, for comparison purposes. While jiotilla extract had cytotoxicity against HepG2 (47.31 ± 0.03%) and PC-3 (53.65 ± 0.04%). These results demonstrated that Chico and jiotilla are excellent resources of biologically active constituents with nutraceuticals potentialities.

Overall survival prediction of non-small cell lung cancer by integrating microarray and clinical data with deep learning.

Non-small cell lung cancer (NSCLC) is one of the most common lung cancers worldwide. Accurate prognostic stratification of NSCLC can become an important clinical reference when designing therapeutic strategies for cancer patients. With this clinical application in mind, we developed a deep neural network (DNN) combining heterogeneous data sources of gene expression and clinical data to accurately predict the overall survival of NSCLC patients. Based on microarray data from a cohort set (614 patients), seven well-known NSCLC biomarkers were used to group patients into biomarker- and biomarker+ subgroups. Then, by using a systems biology approach, prognosis relevance values (PRV) were then calculated to select eight additional novel prognostic gene biomarkers. Finally, the combined 15 biomarkers along with clinical data were then used to develop an integrative DNN via bimodal learning to predict the 5-year survival status of NSCLC patients with tremendously high accuracy (AUC: 0.8163, accuracy: 75.44%). Using the capability of deep learning, we believe that our prediction can be a promising index that helps oncologists and physicians develop personalized therapy and build the foundation of precision medicine in the future.

In vitro anti-inflammatory effects of curcumin on mast cell-mediated allergic responses via inhibiting FcεRI protein expression and protein kinase C delta translocation.

Allergy is a hypersensitivity reaction when exposed to certain environmental substances. It shows high relation between immunoglobulin E (IgE) binding to a specific receptor (FcεRI), pro-inflammatory cytokines, and mediators with allergic inflammation responses. Curcumin is a yellow pigment isolated from the turmeric. Curcumin possesses antioxidant and anti-inflammatory properties as well as exhibits significant chemopreventive activity. This study was aimed to investigate the in vitro assessment of the regulation of curcumin on allergic inflammatory responses on rat basophil leukemia (RBL)-2H3 and human pre-basophils (KU812) cell lines. Curcumin showed the activity against histamine and ß-hexosaminidase releases from both IgE-mediated and A23187-induced cells degranulation. The morphological observation also confirmed that curcumin inhibits cells degranulation. IgE-mediated allergic responses and significantly induced mast cells intracellular reactive oxygen species (ROS) production. Curcumin reduced ROS production from IgE-mediated or A23187-induced cells degranulation. Curcumin also successfully reduced FcεRI expressions and some pro-inflammatory cytokines, such as interleukin (IL)-4 and IL-13. Furthermore, curcumin inhibited protein kinase C (PKC)-δ translocation from cytosolic to particulate. These results suggested that curcumin can alleviate both the IgE-mediated and calcium ionosphere A23187-stimulated allergic responses through reducing the release of the allergic mediators.

Host heterogeneous ribonucleoprotein K (hnRNP K) as a potential target to suppress hepatitis B virus replication.

BACKGROUND: Hepatitis B virus (HBV) infection results in complications such as cirrhosis and hepatocellular carcinoma. Suppressing viral replication in chronic HBV carriers is an effective approach to controlling disease progression. Although antiviral compounds are available, we aimed to identify host factors that have a significant effect on viral replication efficiency. METHODS AND FINDINGS: We studied a group of hepatitis B carriers by associating serum viral load with their respective HBV genomes, and observed a significant association between high patient serum viral load with a natural sequence variant within the HBV enhancer II (Enh II) regulatory region at position 1752. Using a viral fragment as an affinity binding probe, we isolated a host DNA-binding protein belonging to the class of heterogeneous nuclear ribonucleoproteins--hnRNP K--that binds to and modulates the replicative efficiency of HBV. In cell transfection studies, overexpression of hnRNP K augmented HBV replication, while gene silencing of endogenous hnRNP K carried out by small interfering RNAs resulted in a significant reduction of HBV viral load. CONCLUSION: The evidence presented in this study describes a wider role for hnRNP K beyond maintenance of host cellular functions and may represent a novel target for pharmacologic intervention of HBV replication.

[Cellular ultrastructural changes of bone marrow of patients with hemorrhagic fever with renal syndrome].

BACKGROUND: To observe cytopathogenic effect of Hantaan virus (HV) on cultured human bone marrow cells. METHODS: Light and transmission electron microscopy and direct immunofluorescent technique were applied to study cellular structure especially ultrastructural changes of bone marrow cells from patients with Hantaan virus infection. Bone marrow cells of one healthy volunteer were also studied as control. RESULTS: The antigen of HV was found in bone marrow cells of 20 of 27 HFRS patients by the aid of direct immunofluorescent technique. It was found that the granulocytes had the highest percentage of HV antigen positive cells (76%), followed by monocytes (65%), lymphocytes (40%), megakaryocytes (20%) and the lowest was found in erythrocytes (3.7%). The injury of cell membrane after infection with HV was significantly more severe than that in the control group under the light and electron microscopy. CONCLUSION: This study demonstrated that HV could attack human bone marrow cells and cause cytopathogenic effect on them.