Identification of Two Non-Peptidergic Small Molecule Inhibitors of CBX2 Binding to K27 Trimethylated Oligonucleosomes.

The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively.

CMV specific cytokine release assay in whole blood is optimized by combining synthetic CMV peptides and toll like receptor agonists.

BACKGROUND: Interferon gamma release assays (IGRAs) are widely used to detect pathogen specific cellular immunity. Cytomegalovirus (CMV) is the foremost problematic viral infection in immunocompromised patients such as transplant or HIV infected patients. CMV antibody ELISAs are not able to predict CMV specific cellular immunity during immunosuppression. We developed a CMV specific IGRA comparing synthetic CMV peptides, native lysate and recombinant antigen. In addition, TLR agonists were tested to enhance CMV antigen immunogenicity. METHODS: 397 healthy controls (HC) were stratified according to CMV IgM and IgG serostatus and subsequently tested for IFNγ- and IL2-secretion in whole blood after challenge with synthetic, native or recombinant CMV antigens and TLR agonists by ELISA. The selected TLR agonists were lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN), zymosan (Zym), polyinosinic-polycytidylic acid (Poly(I:C)), flagellin (Fla), R848, loxoribine (Lox) and bropirimine (Bro). RESULTS: Synthetic pp65 peptides elicited strong IFNγ responses in CMV seropositive, but not seronegative HC (6418 vs. 13 pg/ml). Native lysates and recombinant pp65 induced equally high IFNγ responses in seropositive (35,877 and 26,428 pg/ml) and increased background IFNγ expression in seronegative HC (43 and 1148 pg/ml). Diagnostic sensitivity and specificity with regard to anti-CMV serology reached 100% for synthetic pp65 and native CMV lysate, but 57% and 100% for recombinant pp65, respectively. TLR agonists LTA and Poly(I:C) augmented IFNγ responses after challenge with synthetic pp65 peptide, native lysate or recombinant pp65 in seropositive HC. Seronegative HC remained unaffected. IL2 production was negligible compared to IFNγ. CONCLUSION: IGRAs using synthetic CMV peptides or native lysate showed the best cytokine signal to noise ratio compared to recombinant antigen and TLR agonists LTA and Poly(I:C) constitute potential costimulating reagents.