Person: Luccarelli, James
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Luccarelli
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James
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Luccarelli, James
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Publication Direct Inhibition of Oncogenic KRAS by Hydrocarbon-Stapled SOS1 Helices(National Academy of Sciences, 2015-02-10) Leshchiner, Elizaveta S.; Parkhitko, Andrey; Bird, Gregory; Luccarelli, James; Bellairs, Joseph A.; Escudero, Silvia; Opoku-Nsiah, Kwadwo; Godes, Marina; Perrimon, Norbert; Walensky, LorenActivating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequence-specific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85DV12 activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.Publication Allosteric Sensitization of Pro-Apoptotic BAX(2017) Pritz, Jonathan R.; Wachter, Franziska; Lee, Susan; Luccarelli, James; Wales, Thomas E.; Cohen, Daniel; Coote, Paul; Heffron, Gregory; Engen, John R.; Massefski, Walter; Walensky, LorenBAX is a critical apoptotic regulator that can be transformed from a cytosolic monomer into a lethal mitochondrial oligomer, yet drug strategies to modulate it are underdeveloped due to longstanding difficulties in conducting screens on this aggregation-prone protein. Here, we overcame prior challenges and performed an NMR-based fragment screen of full-length human BAX. We identified a compound that sensitizes BAX activation by binding to a pocket formed by the junction of the α3/α4 and α5/α6 hairpins. Biochemical and structural analyses revealed that the molecule sensitizes BAX by allosterically mobilizing the α1–α2 loop and BAX BH3 helix, two motifs implicated in the activation and oligomerization of BAX, respectively. By engaging a region of core hydrophobic interactions that otherwise preserve the BAX inactive state, the identified compound informs fundamental mechanisms for conformational regulation of BAX and provides a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit.Publication Small Molecule Modulators of Apoptosis(2017-05-12) Luccarelli, JamesControl of cell survival relies on a delicate balance between pro-apoptotic and anti-apoptotic signalling. In humans, the key regulatory proteins are those of the BCL-2 family, which include effector proteins such as BAX and BAK, anti-apoptotic proteins including BCL-2 and MCL-1, and pro-apoptotic proteins including BID and BIM. Dysregulation of apoptosis is among the Hallmarks of Cancer, and modulation of apoptosis holds promise as an effective therapeutic strategy for a range of malignancies. This thesis advances new strategies for modulating apoptosis using small molecules. The first section explores the properties of stapled peptides. These molecules incorporate two non-natural amino acids with olefin sidechains that are then covalently linked. The resulting “staple” modifies the biophysical properties of the molecule. This chapter shows how stapling results in greater serum stability of the peptides, improves binding affinity for anti-apoptotic targets, and allows for facile transformation of a native sequence into an improved peptide, as demonstrated by stapling a SOS1 peptide to target KRAS. The second section targets MCL-1, an antiapoptotic BCL-2 family protein that has emerged as a major pathogenic factor in human cancer. MCL-1 bears a surface groove whose function is to sequester the BH3 killer domains of proapoptotic BCL-2 family members, but successful drugging of this groove has not been achieved. This chapter develops an alternative strategy using a small molecule that covalently modifies C286 at a novel interaction site distant from the BH3-binding groove. This allosteric mechanism results in reduced BH3 binding capacity of MCL-1 and impairs the oncogenic anti-apoptotic activity of the protein. The final chapter targets BAX, a critical executioner protein in the apoptotic pathway whose oligomerization causes permeabilization of the mitochondrial outer membrane. Using STD-NMR, a library of nearly 1,000 fragments was screened for binding to full-length BAX. This resulted in the discovery of a compound BIF-44 that sensitizes BAX by engaging a noncanonical hydrophobic pocket formed by the junction of the α3-α4 and α5-α6 hairpins. Biochemical and structural analyses indicate that the molecule sensitizes BAX by allosterically mobilizing the α1-α2 loop, a mechanism implicated in the initiation of BH3-mediated direct BAX activation. The identified compound thus informs the mechanism for initiation of BAX activation, and provides a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit.