Structure-Guided Synthesis and Evaluation of Small-Molecule Inhibitors Targeting Protein-Protein Interactions of BRCA1 tBRCT Domain.

The tandem BRCT domains (tBRCT) of BRCA1 engage phosphoserine-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, the first small-molecule inhibitor of the BRCA1 tBRCT domain, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features responsible for Bractoppin's activity. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing K1702A, a key residue mediating phosphopeptide recognition, or F1662R or L1701K that adjoin the pSer-recognition site. However, the M1775R mutation, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding, confirming a binding mode distinct from the substrate phosphopeptide binding. We explored these structural features through structure-guided chemical elaboration and characterized structure-activity relationships (SARs) in biochemical assays. Two analogues, CCBT2088 and CCBT2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the activity of a novel inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target protein-protein interactions.

Targeting Phosphopeptide Recognition by the Human BRCA1 Tandem BRCT Domain to Interrupt BRCA1-Dependent Signaling.

Intracellular signals triggered by DNA breakage flow through proteins containing BRCT (BRCA1 C-terminal) domains. This family, comprising 23 conserved phosphopeptide-binding modules in man, is inaccessible to small-molecule chemical inhibitors. Here, we develop Bractoppin, a drug-like inhibitor of phosphopeptide recognition by the human BRCA1 tandem (t)BRCT domain, which selectively inhibits substrate binding with nanomolar potency in vitro. Structure-activity exploration suggests that Bractoppin engages BRCA1 tBRCT residues recognizing pSer in the consensus motif, pSer-Pro-Thr-Phe, plus an abutting hydrophobic pocket that is distinct in structurally related BRCT domains, conferring selectivity. In cells, Bractoppin inhibits substrate recognition detected by Förster resonance energy transfer, and diminishes BRCA1 recruitment to DNA breaks, in turn suppressing damage-induced G2 arrest and assembly of the recombinase, RAD51. But damage-induced MDC1 recruitment, single-stranded DNA (ssDNA) generation, and TOPBP1 recruitment remain unaffected. Thus, an inhibitor of phosphopeptide recognition selectively interrupts BRCA1 tBRCT-dependent signals evoked by DNA damage.

An Eighteen Serum Cytokine Signature for Discriminating Glioma from Normal Healthy Individuals.

Glioblastomas (GBM) are largely incurable as they diffusely infiltrate adjacent brain tissues and are difficult to diagnose at early stages. Biomarkers derived from serum, which can be obtained by minimally invasive procedures, may help in early diagnosis, prognosis and treatment monitoring. To develop a serum cytokine signature, we profiled 48 cytokines in sera derived from normal healthy individuals (n = 26) and different grades of glioma patients (n = 194). We divided the normal and grade IV glioma/GBM serum samples randomly into equal sized training and test sets. In the training set, the Prediction Analysis for Microarrays (PAM) identified a panel of 18 cytokines that could discriminate GBM sera from normal sera with maximum accuracy (95.40%) and minimum error (4.60%). The 18-cytokine signature obtained in the training set discriminated GBM sera from normal sera in the test set as well (accuracy 96.55%; error 3.45%). Interestingly, the 18-cytokine signature also differentiated grade II/Diffuse Astrocytoma (DA) and grade III/Anaplastic Astrocytoma (AA) sera from normal sera very efficiently (DA vs. normal-accuracy 96.00%, error 4.00%; AA vs. normal-accuracy 95.83%, error 4.17%). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using 18 cytokines resulted in the enrichment of two pathways, cytokine-cytokine receptor interaction and JAK-STAT pathways with high significance. Thus our study identified an 18-cytokine signature for distinguishing glioma sera from normal healthy individual sera and also demonstrated the importance of their differential abundance in glioma biology.

Definition of a serum marker panel for glioblastoma discrimination and identification of Interleukin 1ß in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein.

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. We profiled 724 cancer-associated proteins in sera of healthy individuals (n=27) and GBM (n=28) using antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p<0.0001. The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein was seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving CRP-FcγRIII signaling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1ß (IL1ß) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1ß neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Thus our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies. Biological significance A complex antibody microarray based serum marker profiling identified a three-marker panel - LYAM1, BHE40 and CRP as an accurate discriminator of glioblastoma sera from that of healthy individuals. CRP protein is seen in high levels without a concomitant increase of CRP transcripts in glioblastoma. Glioma-secreted IL6 induced hepatocytes to produce CRP in a JAK-STAT signaling dependent manner. CRP induced microglial cells to release IL1ß which in turn promoted endothelial cell survival. This study, besides defining a serum panel for glioblastoma discrimination, identified IL1ß as a potential candidate for developing targeted therapy.

Serum proteomics of glioma: methods and applications.

The prognosis of patients with glioblastoma, the most malignant adult glial brain tumor, remains poor in spite of advances in treatment procedures, including surgical resection, irradiation and chemotherapy. Genetic heterogeneity of glioblastoma warrants extensive studies in order to gain a thorough understanding of the biology of this tumor. While there have been several studies of global transcript profiling of glioma with the identification of gene signatures for diagnosis and disease management, translation into clinics is yet to happen. Serum biomarkers have the potential to revolutionize the process of cancer diagnosis, grading, prognostication and treatment response monitoring. Besides having the advantage that serum can be obtained through a less invasive procedure, it contains molecules at an extraordinary dynamic range of ten orders of magnitude in terms of their concentrations. While the conventional methods, such as 2DE, have been in use for many years, the ability to identify the proteins through mass spectrometry techniques such as MALDI-TOF led to an explosion of interest in proteomics. Relatively new high-throughput proteomics methods such as SELDI-TOF and protein microarrays are expected to hasten the process of serum biomarker discovery. This review will highlight the recent advances in the proteomics platform in discovering serum biomarkers and the current status of glioma serum markers. We aim to provide the principles and potential of the latest proteomic approaches and their applications in the biomarker discovery process. Besides providing a comprehensive list of available serum biomarkers of glioma, we will also propose how these markers will revolutionize the clinical management of glioma patients.