Liposomal FRET Assay Identifies Potent Drug-Like Inhibitors of the Ceramide Transport Protein (CERT).

Ceramide transfer protein (CERT) mediates non-vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate-limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non-homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Förster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT-mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96-well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis.

Design, Synthesis, Antitumor Activity and Molecular Docking Study of Novel 5-Deazaalloxazine Analogs.

Protein tyrosine kinases (PTKs) are the most potential therapeutic targets for cancer. Herein, we present a sound rationale for synthesis of a series of novel 2-(methylthio), 2-(substituted alkylamino), 2-(heterocyclic substituted), 2-amino, 2,4-dioxo and 2-deoxo-5-deazaalloxazine derivatives by applying structure-based drug design (SBDD) using AutoDock 4.2. Their antitumor activities against human CCRF-HSB-2, KB, MCF-7 and HeLa have been investigated in vitro. Many 5-deazaalloxazine analogs revealed high selective activities against MCF-7 tumor cell lines (IC50: 0.17-2.17 µM) over HeLa tumor cell lines (IC50 > 100 µM). Protein kinase profiling revealed that compound 3h induced multi- targets kinase inhibition including -43% against (FAK), -40% against (CDKI) and -36% against (SCR). Moreover, the Annexin-V/PI apoptotic assay elucidate that compound 3h showed 33% and potentially 140% increase in early and late apoptosis to MCF-7 cells respectively, compared to the control. The structure-activity relationship (SAR) and molecular docking study using PTK as a target enzyme for the synthesized 7-deazaalloaxazine derivatives were investigated as potential antitumor agents. The AutoDock binding affinities of the 5-deazaalloxazine analogs into c-kit PTK (PDB code: 1t46) revealed reasonable correlations between their AutoDock binding free energy and IC50.

Sphingolipid-Transporting Proteins as Cancer Therapeutic Targets.

The understanding of the role of sphingolipid metabolism in cancer has tremendously increased in the past ten years. Many tumors are characterized by imbalances in sphingolipid metabolism. In many cases, disorders of sphingolipid metabolism are also likely to cause or at least promote cancer. In this review, sphingolipid transport proteins and the processes catalyzed by them are regarded as essential components of sphingolipid metabolism. There is much to suggest that these processes are often rate-limiting steps for metabolism of individual sphingolipid species and thus represent potential target structures for pharmaceutical anticancer research. Here, we summarize empirical and biochemical data on different proteins with key roles in sphingolipid transport and their potential role in cancer.

Structural-based design, synthesis, and antitumor activity of novel alloxazine analogues with potential selective kinase inhibition.

Protein kinases are promising therapeutic targets for cancer therapy. Here, we applied multiple approaches to optimize the potency and selectivity of our reported alloxazine scaffold. Flexible moieties at position 2 of the hetero-tricyclic system were incorporated to fit into the ATP binding site and extend to the adjacent allosteric site and selectively inhibit protein kinases. This design led to potential selective inhibition of ABL1, CDK1/Cyclin A1, FAK, and SRC kinase by 30-59%. Cytotoxicity was improved by ∼50 times for the optimized lead (10b; IC50 = 40 nM) against breast cancer (MCF-7) cells. Many compounds revealed potential cytotoxicity against ovarian (A2780) and colon carcinoma (HCT116) cells of ∼10-30 time improvement (IC50 5-17 nM). The results of the Annexin-V/PI apoptotic assay demonstrated that many compounds induced significantly early (89-146%) and a dramatically late (556-1180%) cell death in comparison to the vehicle control of MCF-7 cells. SAR indicated that 5-deazaalloxazines have a higher selectivity for Abl-1 and FAK kinases than alloxazines. The correlations between GoldScore fitness into FAK and SRC kinases and IC50 against MCF-7 and A2780 cells were considerable (R2: 0.86-0.98).