Direct inhibition of transcription factor complexes remains a central challenge in the discipline of ligand discovery. In general, these proteins lack surface involutions suitable for high-affinity binding by small molecules. Here we report the design of synthetic, cell-permeable, stabilized α-helical peptides that target a critical protein-protein interface in the NOTCH transactivation complex. We demonstrate that direct, high-affinity binding of the hydrocarbon-stapled peptide SAHM1 prevents assembly of the active transcriptional complex. Inappropriate NOTCH activation is directly implicated in the pathogenesis of several disease states, including T-cell acute lymphoblastic leukaemia (T-ALL). The treatment of leukaemic cells with SAHM1 results in genome-wide suppression of NOTCH-activated genes. Direct antagonism of the NOTCH transcriptional program ©2009 Macmillan Publishers Limited. All rights reservedCorrespondence and requests for materials should be addressed to J.E.B. (james_bradner@dfci.harvard.edu) or G.L.V. (gregory_verdine@harvard.edu). Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Author Contributions R.E.M., G.L.V. and J.E.B. conceptualized the study, designed the experiments, interpreted data, and wrote the manuscript. Design, synthesis and biological characterization of SAHM peptides was performed by R.E.M. C.D.B., J.C.A. and S.C.B. contributed key reagents and analysed data. R.E.M., M.C., T.N.D., J.C.A., A.L.K., D.G.G. and J.E.B. established the bioluminescent T-ALL model, designed and performed in vivo experiments and analysed data.Author Information All microarray data has been deposited to the Gene Expression Omnibus at the National Center for Biotechnology Information under accession numbers GSE18198 and GSE18351. Reprints and permissions information is available at www.nature.com/reprints. The authors declare competing financial interests: details accompany the full-text HTML version of the paper at www.nature.com/nature. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 October 7. Ligand binding to the extracellular domain of NOTCH1 initiates sequential proteolytic processing events catalysed respectively by an ADAM family metalloprotease and a γ-secretase complex, resulting in cytoplasmic release of the intracellular domain of NOTCH1 (ICN1)4 -6. ICN1 then translocates to the nucleus and loads onto the DNA-bound transcription factor CSL7. The engagement of ICN1 with CSL creates a long, shallow groove along the interface of the two proteins that serves as a binding surface for coactivator proteins of the mastermind-like (MAML) family8 , 9. The resulting ICN-CSL-MAML ternary complex then recruits the core transcription machinery, effecting activation of NOTCH-dependent target genes.The duration and strength of NOTCH signalling is normally tightly controlled. Whereas lossof-function mut...

High grade serous carcinoma (HGSC) has a poor prognosis primarily due to its early dissemination throughout the abdominal cavity. Genomic and proteomic approaches have provided snapshots of the proteogenomics of ovarian cancer (OvCa)1,2, but a systematic examination of both the tumor and stromal compartments is critical to understanding OvCa metastasis. We developed a label-free proteomic workflow to analyze as few as 5,000 formalin-fixed, paraffin embedded cells microdissected from each compartment. The tumor proteome was stable during progression from in situ lesions to metastatic disease; however, the metastasis-associated stroma was characterized by a highly conserved proteomic signature, prominently including the methyltransferase nicotinamide N-methyltransferase (NNMT) and several proteins it regulates. Stromal NNMT expression was necessary and sufficient for functional aspects of the cancer associated fibroblast (CAF) phenotype, including the expression of CAF markers and the secretion of cytokines and oncogenic extracellular matrix. Stromal NNMT expression supported OvCa migration, proliferation, and in vivo growth and metastasis. Expression of NNMT in CAFs led to a depletion of S-adenosyl methionine (SAM) and a reduction in histone methylation associated with widespread gene expression changes in the tumor stroma. This work supports the use of ultra-low input proteomics to identify candidate drivers of disease phenotypes. NNMT is a central, metabolic regulator of CAF differentiation and cancer progression in the stroma that may be therapeutically targeted.

A systematic study on cell penetration by stapled peptides.

Aberrant activation of signaling by the Wnt pathway is strongly implicated in the onset and progression of numerous types of cancer. Owing to the persistent dependence of these tumors on Wnt signaling for growth and survival, inhibition of this pathway is considered an attractive mechanism-based therapeutic approach. Oncogenic activation of Wnt signaling can ensue from a variety of distinct aberrations in the signaling pathway, but most share the common feature of causing increased cellular levels of β-catenin by interfering with its constitutive degradation. β-Catenin serves as a central hub in Wnt signaling by engaging in crucial protein–protein interactions with both negative and positive effectors of the pathway. Direct interference with these protein–protein interactions is a biologically compelling approach toward suppression of β-catenin hyperactivity, but such interactions have proven intransigent with respect to small-molecule targeting. Hence β-catenin remains an elusive target for translational cancer therapy. Here we report the discovery of a hydrocarbon-stapled peptide that directly targets β-catenin and interferes with its ability to serve as a transcriptional coactivator for T-cell factor (TCF) proteins, the downstream transcriptional regulators of the Wnt pathway.

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The post-translational modification (PTM) of proteins and their allosteric regulation by metabolites represent conserved regulatory mechanisms in biology. At the confluence of these two processes, we report that the primary glycolytic intermediate 1,3-bisphosphoglycerate reacts with select lysine residues in proteins to form 3-phosphoglyceryl-lysine (pgK). This reaction, which does not require enzyme catalysis, but rather exploits the electrophilicity of 1,3-bisphosphoglycerate, was found by proteomic profiling to be enriched on diverse classes of proteins and prominently in or around the active sites of glycolytic enzymes. pgK modifications inhibit glycolytic enzyme activities, and, in cells exposed to high glucose, accumulate on these enzymes to create a potential feedback mechanism that contributes to the buildup and redirection of glycolytic intermediates to alternate biosynthetic pathways.

Creating first-in-class medications to treat human disease is an extremely challenging endeavor. While genome sequencing and genetics are making direct connections between mutations and human disorders at an unprecedented rate, matching molecular target(s) with a suitable therapeutic indication must ultimately be achieved by pharmacology. Here, we will discuss how the integration of chemical proteomic platforms, such as activity-based protein profiling, into the earliest stages of the drug discovery process has the potential to greatly expand the scope of proteins that can be pharmacologically evaluated in living systems, and, through doing so, promote the identification and prioritization of new therapeutic targets.

Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free co-factor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium catalyzed transformations, including N- H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.