Interaction of the oncoprotein transcription factor MYC with its chromatin cofactor WDR5 is essential for tumor maintenance.

The oncoprotein transcription factor MYC is overexpressed in the majority of cancers. Key to its oncogenic activity is the ability of MYC to regulate gene expression patterns that drive and maintain the malignant state. MYC is also considered a validated anticancer target, but efforts to pharmacologically inhibit MYC have failed. The dependence of MYC on cofactors creates opportunities for therapeutic intervention, but for any cofactor this requires structural understanding of how the cofactor interacts with MYC, knowledge of the role it plays in MYC function, and demonstration that disrupting the cofactor interaction will cause existing cancers to regress. One cofactor for which structural information is available is WDR5, which interacts with MYC to facilitate its recruitment to chromatin. To explore whether disruption of the MYC-WDR5 interaction could potentially become a viable anticancer strategy, we developed a Burkitt\u27s lymphoma system that allows replacement of wild-type MYC for mutants that are defective for WDR5 binding or all known nuclear MYC functions. Using this system, we show that WDR5 recruits MYC to chromatin to control the expression of genes linked to biomass accumulation. We further show that disrupting the MYC-WDR5 interaction within the context of an existing cancer promotes rapid and comprehensive tumor regression in vivo. These observations connect WDR5 to a core tumorigenic function of MYC and establish that, if a therapeutic window can be established, MYC-WDR5 inhibitors could be developed as anticancer agents

Discovery of Quinazolines That Activate SOS1-Mediated Nucleotide Exchange on RAS.

Proteins in the RAS family are important regulators of cellular signaling and, when mutated, can drive cancer pathogenesis. Despite considerable effort over the last 30 years, RAS proteins have proven to be recalcitrant therapeutic targets. One approach for modulating RAS signaling is to target proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). Here, we report hit-to-lead studies on quinazoline-containing compounds that bind to SOS1 and activate nucleotide exchange on RAS. Using structure-based design, we refined the substituents attached to the quinazoline nucleus and built in additional interactions not present in the initial HTS hit. Optimized compounds activate nucleotide exchange at single-digit micromolar concentrations in vitro. In HeLa cells, these quinazolines increase the levels of RAS-GTP and cause signaling changes in the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway

ChemMedChem

Replication protein\u2005A (RPA) is an essential single-stranded DNA (ssDNA)-binding protein that initiates the DNA damage response pathway through protein-protein interactions (PPIs) mediated by its 70N domain. The identification and use of chemical probes that can specifically disrupt these interactions is important for validating RPA as a cancer target. A high-throughput screen (HTS) to identify new chemical entities was conducted, and 90 hit compounds were identified. From these initial hits, an anthranilic acid based series was optimized by using a structure-guided iterative medicinal chemistry approach to yield a cell-penetrant compound that binds to RPA70N with an affinity of 812\u2005nm. This compound, 2-(3- (N-(3,4-dichlorophenyl)sulfamoyl)-4-methylbenzamido)benzoic acid (20\u2009c), is capable of inhibiting PPIs mediated by this domain.F32ES021690-01/ES/NIEHS NIH HHS/United StatesDP1 CA174419/CA/NCI NIH HHS/United StatesF32 CA174315/CA/NCI NIH HHS/United StatesF32CA174315-02/CA/NCI NIH HHS/United StatesR01 GM065484/GM/NIGMS NIH HHS/United StatesR01M065484/PHS HHS/United States8DP1A174419/DP/NCCDPHP CDC HHS/United StatesRC2 CA148375/CA/NCI NIH HHS/United StatesDP1 OD006933/OD/NIH HHS/United StatesR01 CA174887/CA/NCI NIH HHS/United States5RC2A148375/RC/CCR NIH HHS/United States5DP1OD006933/OD/NIH HHS/United StatesP01 CA092584/CA/NCI NIH HHS/United StatesF32 ES021690/ES/NIEHS NIH HHS/United StatesT32 ES007028/ES/NIEHS NIH HHS/United StatesP01CA092584/CA/NCI NIH HHS/United States11566/Cancer Research UK/United Kingdom2017-04-19T00:00:00Z26748787PMC4838552vault:1854

siRNA-mediated off-target gene silencing triggered by a 7 nt complementation

A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three ‘top hits’ from our screen have been demonstrated to result from off-target gene silencing. Two of the three ‘siRNA hits’ were found to directly trigger down-regulation of hif-1α mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1α mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1α was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3′-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving ‘hits-follow-up’ strategies for future large-scale knockdown experiments

Peak-tracking for stepwise perturbed NMR spectra: development of a new analysis method

Stepwise perturbed NMR spectra analysis is a powerful tool capable of describing kinetic, thermodynamic, structural aspects of proteins at a residue level and of following the physical and chemical changes of the system. The analysis of an NMR spectrum still offers compelling challenges to the automatic identification of the chemical shift evolution. We designed and developed a data-analysis method which allows automatic peak detection in every spectrum, peak tracking between spectra and peak reconstruction for BLUU-Tramp sessions, a stepwise isotopic exchange experiment producing few hundreds of 2D NMR spectra. The method has been named TinT (Trace in Track), referring to the idea that a gaussian decomposition traces peaks within the tracks recognized through 3D mathematical morphology. TinT is capable of determining the evolution of the chemical shifts, intensity and linewidths of each tracked peak. The performances obtained in term of track reconstruction and correct assignment on realistic synthetic spectra were high above 90% when a noise level similar to that of experimental data were considered. TinT was applied successfully to several protein systems during a temperature ramp in isotope exchange experiments. The comparison with a state-of-the-art algorithm showed very good results for great numbers of spectra and low signal to noise ratios, when the graduality of the perturbation is appropriate. In the thesis, in addition to the description of the current version of TinT, some observations and considerations that can allow future revisions or improvements on BLUU-Tramp protocol or its analysis are also described.Co-supervisore: Andrea Fusiello - Dottorato di ricerca cofinanziato FSE: progetto S.H.A.R.M. per la realizzazione di attività di ricerca in collaborazione con imprese; Impresa tutor: Bruker Italia S.R.L.openDottorato di ricerca in Scienze biomediche e biotecnologicheopenBanelli, Tommas