TY - JOUR T1 - Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library. JF - Cell Chem Biol Y1 - 2021 A1 - Emery, Amy A1 - Hardwick, Bryn S A1 - Crooks, Alex T A1 - Milech, Nadia A1 - Watt, Paul M A1 - Mithra, Chandan A1 - Kumar, Vikrant A1 - Giridharan, Saranya A1 - Sadasivam, Gayathri A1 - Mathivanan, Subashini A1 - Sudhakar, Sneha A1 - Bairy, Sneha A1 - Bharatham, Kavitha A1 - Hurakadli, Manjunath A A1 - Prasad, Thazhe K A1 - Kamariah, Neelagandan A1 - Muellner, Markus A1 - Coelho, Miguel A1 - Torrance, Christopher J A1 - McKenzie, Grahame J A1 - Venkitaraman, Ashok R AB -

Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.

ER - TY - JOUR T1 - Structure-Guided Synthesis and Evaluation of Small-Molecule Inhibitors Targeting Protein-Protein Interactions of BRCA1 tBRCT Domain. JF - ChemMedChem Y1 - 2019 A1 - Kurdekar, Vadiraj A1 - Giridharan, Saranya A1 - Subbarao, Jasti A1 - Nijaguna, Mamatha B A1 - Periasamy, Jayaprakash A1 - Boggaram, Sanjana A1 - Shivange, Amol V A1 - Sadasivam, Gayathri A1 - Padigaru, Muralidhara A1 - Potluri, Vijay A1 - Venkitaraman, Ashok R A1 - Bharatham, Kavitha AB -

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.

VL - 14 IS - 18 ER - TY - JOUR T1 - Targeting Phosphopeptide Recognition by the Human BRCA1 Tandem BRCT Domain to Interrupt BRCA1-Dependent Signaling. JF - Cell Chem Biol Y1 - 2018 A1 - Periasamy, Jayaprakash A1 - Kurdekar, Vadiraj A1 - Jasti, Subbarao A1 - Nijaguna, Mamatha B A1 - Boggaram, Sanjana A1 - Hurakadli, Manjunath A A1 - Raina, Dhruv A1 - Kurup, Lokavya Meenakshi A1 - Chintha, Chetan A1 - Manjunath, Kavyashree A1 - Goyal, Aneesh A1 - Sadasivam, Gayathri A1 - Bharatham, Kavitha A1 - Padigaru, Muralidhara A1 - Potluri, Vijay A1 - Venkitaraman, Ashok R AB -

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.

VL - 25 IS - 6 ER -