[Show abstract][Hide abstract]ABSTRACT: Background:
Searching for two-dimensional (2D) structural similarities is a useful tool to identify new active compounds in drug-discovery programs. However, as 2D similarity measures neglect important structural and functional features, similarity by 2D might be underestimated. In the present study, we used combined 2D and three-dimensional (3D) similarity comparisons to reveal possible new functions and/or side-effects of known bioactive compounds.
We utilised more than 10,000 compounds from the SuperTarget database with known inhibition values for twelve different anti-cancer targets. We performed all-against-all comparisons resulting in 2D similarity landscapes. Among the regions with low 2D similarity scores are inhibitors of vascular endothelial growth factor receptor (VEGFR) and inhibitors of poly ADP-ribose polymerase (PARP). To demonstrate that 3D landscape comparison can identify similarities, which are untraceable in 2D similarity comparisons, we analysed this region in more detail. This 3D analysis showed the unexpected structural similarity between inhibitors of VEGFR and inhibitors of PARP. Among the VEGFR inhibitors that show similarities to PARP inhibitors was Vatalanib, an oral "multi-targeted" small molecule protein kinase inhibitor being studied in phase-III clinical trials in cancer therapy. An in silico docking simulation and an in vitro HT universal colorimetric PARP assay confirmed that the VEGFR inhibitor Vatalanib exhibits off-target activity as a PARP inhibitor, broadening its mode of action.
In contrast to the 2D-similarity search, the 3D-similarity landscape comparison identifies new functions and side effects of the known VEGFR inhibitor Vatalanib.
[Show abstract][Hide abstract]ABSTRACT: Resistance to cell death is the major cause of chemotherapy failure in most kinds of cancers, including Burkitt lymphoma (BL). When analyzing therapy resistance in Burkitt lymphoma (BL), we discovered a link between apoptosis resistance and ploidy control. We therefore studied systematically a panel of 15 BL lines for apoptosis induction upon treatment with microtubule inhibitors and compared three types of microtubule toxins, i.e., paclitaxel, nocodazole and vincristine. We found an inverse relationship between apoptosis sensitivity and ploidy control. Thus, cells resistant to paclitaxel- or nocodazole-induced apoptosis underwent mitotic catastrophe and developed polyploidy (>4N). Mechanistically, apoptosis resistance was linked to failure of caspase activation, which was most pronounced in cells lacking the pro-apoptotic multidomain Bcl-2 homologs Bax and Bak. Pharmacological caspase inhibition promoted polyploidy upon exposure to paclitaxel and nocodazole supporting the relationship between resistance to apoptosis and polyploidization. Of note, vincristine induced persistent mitotic arrest but no loss of ploidy control. Considering targets to facilitate Bax/Bak-independent cell death and to avoid drug-induced mitotic catastrophe and consecutive mitotic catastrophe should be of great importance to overcome therapy resistance and therapy-related events that result in ploidy changes and tumor progression.
Inverse relation of apoptosis and polyploidy induction by paclitaxel or nocodazole in BL. Resistant cells undergo mitotic catastrophe and develop polyploidy. Lack of Bax/Bak confers resistance and leads to induction of polyploidy in BL. Intact apoptosis response protects from polyploidy as a result of mitotic catastrophe.
Full-text Article · Dec 2014 · Journal of Molecular Medicine
[Show abstract][Hide abstract]ABSTRACT: TRAIL is a promising anticancer agent, capable of inducing apoptosis in a wide range of treatment-resistant tumor cells. In 'type II' cells, the death signal triggered by TRAIL requires amplification via the mitochondrial apoptosis pathway. Consequently, deregulation of the intrinsic apoptosis-signaling pathway, for example, by loss of Bax and Bak, confers TRAIL-resistance and limits its application. Here, we show that despite resistance of Bax/Bak double-deficient cells, TRAIL-treatment resulted in caspase-8 activation and complete processing of the caspase-3 proenzymes. However, active caspase-3 was degraded by the proteasome and not detectable unless the XIAP/proteasome pathway was inhibited. Direct or indirect inhibition of XIAP by RNAi, Mithramycin A or by the SMAC mimetic LBW-242 as well as inhibition of the proteasome by Bortezomib overcomes TRAIL-resistance of Bax/Bak double-deficient tumor cells. Moreover, activation and stabilization of caspase-3 becomes independent of mitochondrial death signaling, demonstrating that inhibition of the XIAP/proteasome pathway overcomes resistance by converting 'type II' to 'type I' cells. Our results further demonstrate that the E3 ubiquitin ligase XIAP is a gatekeeper critical for the 'type II' phenotype. Pharmacological manipulation of XIAP therefore is a promising strategy to sensitize cells for TRAIL and to overcome TRAIL-resistance in case of central defects in the intrinsic apoptosis-signaling pathway.
Full-text Article · May 2013 · Cell Death & Disease
[Show abstract][Hide abstract]ABSTRACT: Nonsense peptides and peptides A4 and B1 do not relocate mutant dysferlin to the sarcolemma. C2C12 cells were transfected with either missense-mutated dysferlin cDNA DYSF p.G299R (upper lane) or p.L1341P (lower lane). Transfected cells were treated with TAT-labeled dysferlin-peptides. Bar: 10 µm.
[Show abstract][Hide abstract]ABSTRACT: Relocated dysferlin abuts to the basal lamina. Primary human myotubes carrying the dysferlin missense mutation DYSF p.G299R in (A) and immortalized human myotubes carrying the DYSF p.L1341P mutation in (B) were treated with the dysferlin-peptides. Dysferlin was detected by anti-dysferlin ab (left column). Co-staining with WGA as a marker of the basal lamina was performed (middle column). Merge is shown at the right column. Arrows indicate the sarcolemmal reallocation of dysferlin by the 10mer mutant dysferlin-peptides. Bar: 10 µm.
[Show abstract][Hide abstract]ABSTRACT: DYSF p.L1341P does not stably bind corresponding dysferlin peptides. Protein binding studies were performed by surface plasmon resonance analysis. As analyte the recombinant C2E domain harboring DYSF p.L1341P expressed in E.coli was used. The kinetics of interaction, the rates of association and dissociation between the C2E and the corresponding peptides B1, B2 and B3 were tested. There is no evidence for binding between C2E and dysferlin peptides.
[Show abstract][Hide abstract]ABSTRACT: Life cell imaging. ATTO-495-ME-labeled and TAT conjugated dysferlin-peptide B2 in DYSF p.L1341P primary human myotubes. Time lapse is indicated in the video.
[Show abstract][Hide abstract]ABSTRACT: Mutations in the dysferlin gene cause the most frequent adult-onset limb girdle muscular dystrophy, LGMD2B. There is no therapy. Dysferlin is a membrane protein comprised of seven, beta-sheet enriched, C2 domains and is involved in Ca(2+)dependent sarcolemmal repair after minute wounding. On the protein level, point mutations in DYSF lead to misfolding, aggregation within the endoplasmic reticulum, and amyloidogenesis. We aimed to restore functionality by relocating mutant dysferlin. Therefore, we designed short peptides derived from dysferlin itself and labeled them to the cell penetrating peptide TAT. By tracking fluorescently labeled short peptides we show that these dysferlin-peptides localize in the endoplasmic reticulum. There, they are capable of reducing unfolded protein response stress. We demonstrate that the mutant dysferlin regains function in membrane repair in primary human myotubes derived from patients' myoblasts by the laser wounding assay and a novel technique to investigate membrane repair: the interventional atomic force microscopy. Mutant dysferlin abuts to the sarcolemma after peptide treatment. The peptide-mediated approach has not been taken before in the field of muscular dystrophies. Our results could redirect treatment efforts for this condition.
[Show abstract][Hide abstract]ABSTRACT: ER stress is reduced by TAT-labeled dysferlin-peptides. (A) In human myotubes harboring the DYSF p.G299R mutation specific peptides reduce the relative gene expression of the ER stress mediators ATF6 (ATF6), EIF2AK3 (PERK) and DDIT3 (CHOP). The nonsense peptide serves as control. Data represent median + SEM, n = 9/group. P values are listed in (C). (B) The 15mer mutant peptide A4 also effectively reduces ER stress, whereas both wildtype peptides A1 and A3 do not have an equivalent effect on the relative gene expression of ATF6, EIF2AK3 and DDIT3. Data represent median + SEM, n = 3/group. (C) Supplementary statistics to Fig. 6 and Fig. S5A. The fold induction of each sample condition (treatment with peptide A2 or nonsense peptide) is compared to untreated controls DYSF p.G299R. Median ± SEM for each condition is listed and p values are indicated in the table; n = 9/group.