Borrelidin modulates the alternative splicing of VEGF in favour of anti-angiogenic isoforms

Woolard, Jeanette; Vousden, William; Moss, Steven J.; Krishnakumar, Arjun; Gammons, Melissa V.; Nowak, David G; Dixon, Neil; Micklefield, Jason; Spannhoff, Astrid; Bedford, Mark T.; Gregory, Matthew A.; Martin, Christine; Leadlay, Peter F.; Zhang, Ming Q; Harper, Steven J.; Bates, David O.; Wilkinson, Barrie

doi:10.1039/c0sc00297f

Cited by 25 publications

(19 citation statements)

References 33 publications

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“…The K i of borrelidin for human ThrRS is 5 nM, whereas the K i for secondary targets such as splicing factor FBP21 or CDKs, are more than 1000-fold higher, approximately of 10 μM (26,58). Moreover, borrelidin exhibits comparable potencies in the HUVEC (human umbilical vein endothelial cells) tube formation and chicken chorionic allantoic membrane models, the concentration and composition of extracellular proteins of which is vastly different (26).…”

Section: Discussionmentioning

confidence: 90%

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo

Novoa

Camacho

Tor

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.aminoacyl-tRNA synthetase | malaria | drug design | borrelidin | plasmodium

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Section: Discussionmentioning

confidence: 90%

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo

Novoa

Camacho

Tor

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Other anti-tumorigenic effects of borrelidin, including inhibition of osteosarcoma cell migration, induction of apoptosis in acute lymphoblastic leukemia and reduction of metastasis in a mouse model of melanoma may thus also be related to the extracellular functions of TARS121323. Prior to this report, it was suggested that borrelidin's angiostatic effects may be the consequence of inhibiting or activating secondary targets or through promoting anti-angiogenic isoforms of VEGF2425. In those studies, effects on the secondary targets required μM concentrations of borrelidin, known to induce cell toxicity, thus the effects may be related to effects on intracellular TARS function or through an apoptotic response.…”

Section: Discussionmentioning

confidence: 95%

Secreted Threonyl-tRNA synthetase stimulates endothelial cell migration and angiogenesis

Williams

Mirando

Wilkinson³

et al. 2013

Sci Rep

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Aminoacyl-tRNA synthetases classically regulate protein synthesis but some also engage in alternative signaling functions related to immune responses and angiogenesis. Threonyl-tRNA synthetase (TARS) is an autoantigen in the autoimmune disorder myositis, and borrelidin, a potent inhibitor of TARS, inhibits angiogenesis. We explored a mechanistic link between these findings by testing whether TARS directly affects angiogenesis through inflammatory mediators. When human vascular endothelial cells were exposed to tumor necrosis factor-α (TNF-α) or vascular endothelial growth factor (VEGF), TARS was secreted into the cell media. Furthermore, exogenous TARS stimulated endothelial cell migration and angiogenesis in both in vitro and in vivo assays. The borrelidin derivative BC194 reduced the angiogenic effect of both VEGF and TARS, but not a borrelidin-resistant TARS mutant. Our findings reveal a previously undiscovered function for TARS as an angiogenic, pro-migratory extracellular signaling molecule. TARS thus provides a potential target for detecting or interdicting disease-related inflammatory or angiogenic responses.

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“…Human ARPE19 [24], PC3 [25], A375 [26], human lung fibroblasts [27], conditionally immortalised podocytes [13], colonic adenoma cell lines AA/C1 and the in-vitro transformed adenoma cell line 10C cells [28] were maintained as described previously. Primary human RPE cells were derived from donors from the Bristol eye bank, as previously described [14].…”

Section: Methodsmentioning

confidence: 99%

Detection of VEGF-Axxxb Isoforms in Human Tissues

et al. 2013

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Vascular Endothelial Growth Factor-A (VEGF-A) can be generated as multiple isoforms by alternative splicing. Two families of isoforms have been described in humans, pro-angiogenic isoforms typified by VEGF-A165a, and anti-angiogenic isoforms typified by VEGF-A165b. The practical determination of expression levels of alternative isoforms of the same gene may be complicated by experimental protocols that favour one isoform over another, and the use of specific positive and negative controls is essential for the interpretation of findings on expression of the isoforms. Here we address some of the difficulties in experimental design when investigating alternative splicing of VEGF isoforms, and discuss the use of appropriate control paradigms. We demonstrate why use of specific control experiments can prevent assumptions that VEGF-A165b is not present, when in fact it is. We reiterate, and confirm previously published experimental design protocols that demonstrate the importance of using positive controls. These include using known target sequences to show that the experimental conditions are suitable for PCR amplification of VEGF-A165b mRNA for both q-PCR and RT-PCR and to ensure that mispriming does not occur. We also provide evidence that demonstrates that detection of VEGF-A165b protein in mice needs to be tightly controlled to prevent detection of mouse IgG by a secondary antibody. We also show that human VEGF165b protein can be immunoprecipitated from cultured human cells and that immunoprecipitating VEGF-A results in protein that is detected by VEGF-A165b antibody. These findings support the conclusion that more information on the biology of VEGF-A165b isoforms is required, and confirm the importance of the experimental design in such investigations, including the use of specific positive and negative controls.

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Borrelidin modulates the alternative splicing of VEGF in favour of anti-angiogenic isoforms

Cited by 25 publications

References 33 publications

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo

Secreted Threonyl-tRNA synthetase stimulates endothelial cell migration and angiogenesis

Detection of VEGF-Axxxb Isoforms in Human Tissues

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