Enhancement of Tumour-Specific Immune Responses In Vivo by ‘MHC Loading-Enhancer’ (MLE)

BACKGROUND:Class II MHC molecules (MHC II) are cell surface receptors displaying short protein fragments for the surveillance by CD4+ T cells. Antigens therefore have to be loaded onto this receptor in order to induce productive immune responses. On the cell surface, most MHC II molecules are either occupied by ligands or their binding cleft has been blocked by the acquisition of a non-receptive state. Direct loading with antigens, as required during peptide vaccinations, is therefore hindered. PRINCIPAL FINDINGS:Here we show, that the in vivo response of CD4+ T cells can be improved, when the antigens are administered together with 'MHC-loading enhancer' (MLE). MLE are small catalytic compounds able to open up the MHC binding site by triggering ligand-release and stabilizing the receptive state. Their enhancing effect on the immune response was demonstrated here with an antigen from the influenza virus and tumour associated antigens (TAA) derived from the NY-ESO-1 protein. The application of these antigens in combination with adamantane ethanol (AdEtOH), an MLE compound active on human HLA-DR molecules, significantly increased the frequency of antigen-specific CD4+ T cells in mice transgenic for the human MHC II molecule. Notably, the effect was evident only with the MLE-susceptible HLA-DR molecule and not with murine MHC II molecules non-susceptible for the catalytic effect of the MLE. CONCLUSION:MLE can specifically increase the potency of a vaccine by facilitating the efficient transfer of the antigen onto the MHC molecule. They may therefore open a new way to improve vaccination efficacy and tumour-immunotherapy

Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling

<div><p>Diastolic dysfunction is increasingly prevalent in our ageing society and an important contributor to heart failure. The giant protein titin could serve as a therapeutic target, as its elastic properties are a main determinant of cardiac filling in diastole. This study aimed to develop a high throughput pharmacological screen to identify small molecules that affect titin isoform expression through differential inclusion of exons encoding the elastic PEVK domains. We used a dual luciferase splice reporter assay that builds on the titin splice factor RBM20 to screen ~34,000 small molecules and identified several compounds that inhibit the exclusion of PEVK exons. These compounds belong to the class of cardenolides and affect RBM20 dependent titin exon exclusion but did not affect RBFOX1 mediated splicing of FMNL3. We provide evidence that cardenolides do not bind to the RNA interacting domain of RBM20, but reduce RBM20 protein levels and alter transcription of select splicing factors that interact with RBM20.</p><p>Cardenolides affect titin isoform expression. Understanding their mode of action and harnessing the splice effects through chemical modifications that suppress the effects on ion homeostasis and more selectively affect cardiac splicing has the potential to improve cardiac filling and thus help patients with diastolic heart failure, for which currently no targeted therapy exists.</p></div

Identification and validation of splice active compounds by a semi-automated high-throughput screen.

<p><b>(a)</b> Optimization of the splicing reporter to RBM20 ratio by co-transfection of HEK293 cells. The assay is saturated at a 5-fold excess of RBM20 (N = 8). <b>(b)</b> Assay kinetics with maximum effect after 60 hours of incubation (N = 8). Polypyrimidine Tract Binding Protein 1 (PTBP1) served as negative control not leading to exon exclusion of the splice reporter. <b>(c)</b> Assay suitability for a high-throughput approach—z’ values >0.5 are adequate. <b>(d)</b> Screening strategy to identify splice active compounds. The DLR assay was adapted to the 384-well format followed by the pilot screen with >34,000 compounds at 10 μM. Potentially active compounds were re-evaluated in 9 serial dilutions. Resulting candidates were validated manually in the 96-well format leading to the identification of 7 inhibitors that belong to the group of cardenolides. <b>(e)</b> Number of compounds (cpds) passing the different steps of the screening procedure. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 versus CTRL (Dunnett’s post-test). Data are presented as mean ±SD.</p

Use of a sequential high throughput screening assay to identify novel inhibitors of the eukaryotic SRP-Sec61 targeting/translocation pathway.

The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs

Use of a sequential high throughput screening assay to identify novel inhibitors of the eukaryotic SRP-Sec61 targeting/translocation pathway

The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs.status: publishe

Cardenolides are potent inhibitors of RBM20 dependent titin splicing.

<p>Figure layout and panels as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198492#pone.0198492.g003" target="_blank">Fig 3</a>. <b>(a, b)</b> Additional cardenolides not present in the original library were selected to evaluate their potency to inhibit RBM20 dependent titin splicing. The structural analogs digoxin and digitoxin both efficiently inhibit RBM20 mediated splicing. (DLR N = 4; RNA N = 3). ***<i>P</i><0.001 versus CTRL (Dunnett’s post-test). Data are presented as mean ±SD.</p

Dual luciferase splicing reporter assay (DLR assay) to identify splicing modulators.

<p><b>(a)</b> Minigene containing titin PEVK exons 4–13. Boxes indicate exons, blue lines mature transcripts generated with and without RBM20. <b>(b)</b> PCR products of alternative transcripts produced from the PEVK minigene by RBM20. RBM20 increases the amount of transcripts lacking all alternative exons or only retaining alternative exon 5. <b>(c)</b> Quantitative PCR to relate inclusion of PEVK exon 8 to the constitutive exon 13 presented as percent spliced in (PSI). RBM20 reduces inclusion of exon 8 by ~70% (N = 3). <b>(d)</b> Dual luciferase splicing reporter with firefly luciferase (FLuc) integrated into exon 8 and renilla luciferase (RLuc) downstream of exon 13. The FLuc/RLuc ratio reflects the inclusion ratio of alternative exon 8. <b>(e)</b> RBM20 shifts alternative splicing of the dual luciferase reporter (DLR) construct to exclude all alternative exons. <b>(f, g)</b> Quantitative PCR (N = 3) and FLuc/RLuc activity (N = 8) produce similar readouts with increased sensitivity of the luciferase based assay. ***<i>P</i><0.001 versus CTRL (Dunnett’s post-test). Data are presented as mean ±SD.</p

Cardenolide effects on alternative splicing vs. ion homeostasis.

<p>Cardenolide effects on alternative splicing vs. ion homeostasis.</p

Specificity of the inhibitory effect of cardenolides on RBM20-mediated titin splicing.

<p>Figure layout and panels as described in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198492#pone.0198492.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198492#pone.0198492.g004" target="_blank">4</a>. <b>(a)</b> An independent minigene containing FMNL3 exons 25-26 was co-transfected with RBFOX1 and does not respond to digoxin until cell viability is below 30% (N = 3). <b>(b)</b> Digitonin does not affect the splice reporter readout until cell viability is below 20%. There is no effect on the mRNA level (N = 3). <b>(c)</b> Isoginkgetin treatment reduces alternative splicing and mRNA maturation (reduced PCR product at 507 bp in the control at 30 μM). <b>(d, e)</b> Digoxin treatment of co-transfected HEK293 cells leads to decreased expression of RBM20, which is not affected by addition of the proteasome inhibitor MG132. <b>(f)</b> Digoxin dependent splicing inhibition does not depend on the reporter to RBM20 ratio. High concentrations of RBM20 co-transfected with the reporter do not compensate the inhibitory effect. 2-WAY-ANOVA: interaction, column and row factor <0.0001, (N = 4). *<i>P</i><0.05, ***<i>P</i><0.001 versus CTRL (Dunnett’s post-test). Data are presented as mean ±SD.</p

Digitoxin affects mRNA processing and various signaling pathways related to cell division, signal transduction and cancer.

<p>Gene ontology according to WikiPathways analysis of transcripts differentially regulated by digitoxin in HEK293 suggest specific effects that include pathways related to growth and cell cycle as well as differential expression of > 60 genes involved in mRNA processing (adjusted <i>P</i> ≤ 0.05 [Benjamin-Hochberg], two-sided hypergeometric test).</p