Publications by authors named "Gayathri Sadasivam"

7 Publications

  • Page 1 of 1

Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library.

Cell Chem Biol 2021 Jun 1. Epub 2021 Jun 1.

Medical Research Council Cancer Unit, University of Cambridge, Hills Road, Cambridge CB2 0XZ, UK; Center for Chemical Biology & Therapeutics, inStem & NCBS, Bellary Road, Bangalore 560065, India; PhoreMost Ltd., Babraham Research Campus, Cambridge CB22 3AT, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chembiol.2021.05.009DOI Listing
June 2021

Discovery of a Potent and Selective PI3Kδ Inhibitor ()-2,4-Diamino-6-((1-(7-fluoro-1-(4-fluorophenyl)-4-oxo-3-phenyl-4-quinolizin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile with Improved Pharmacokinetic Profile and Superior Efficacy in Hematological Cancer Models.

J Med Chem 2020 12 1;63(23):14700-14723. Epub 2020 Dec 1.

Novel Drug Discovery and Development, Lupin Ltd., Lupin Research Park, Survey No. 46 A/47A, Village Nande, Taluka Mulshi, Pune 412115, India.

PI3Kδ inhibitors have been approved for B-cell malignancies like CLL, small lymphocytic lymphoma, and so forth. However, currently available PI3Kδ inhibitors are nonoptimal, showing weakness against at least one of the several important properties: potency, isoform selectivity, and/or pharmacokinetic profile. To come up with a PI3Kδ inhibitor that overcomes all these deficiencies, a pharmacophoric expansion strategy was employed. Herein, we describe a systematic transformation of a "three-blade propeller" shaped lead, 2,3-disubstituted quinolizinone , through a 1,2-disubstituted quinolizinone to a novel "four-blade propeller" shaped 1,2,3-trisubstituted quinolizinone . Compound has excellent potency, isoform selectivity, metabolic stability across species, and exhibited a favorable pharmacokinetic profile. Compound also demonstrated a differentiated efficacy profile in human germinal center B and activated B cell-DLBCL cell lines and xenograft models. Compound qualifies for further evaluation as a candidate for monotherapy or in combination with other targeted agents in DLBCLs and other forms of iNHL.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01264DOI Listing
December 2020

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

ChemMedChem 2019 09 9;14(18):1620-1632. Epub 2019 Sep 9.

Center for Chemical Biology & Therapeutics, InStem, Bellary Road, Bangalore, Karnataka, 560065, India.

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.
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http://dx.doi.org/10.1002/cmdc.201900300DOI Listing
September 2019

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

Cell Chem Biol 2018 06 29;25(6):677-690.e12. Epub 2018 Mar 29.

Center for Chemical Biology & Therapeutics, InSTEM, Bellary Road, Bangalore, Karnataka 560065, India; Medical Research Council Cancer Unit, University of Cambridge, Hills Road, Cambridge CB2 0XZ, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chembiol.2018.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015222PMC
June 2018

Tyrosine phosphatases such as SHP-2 act in a balance with Src-family kinases in stabilization of postsynaptic clusters of acetylcholine receptors.

BMC Neurosci 2007 Jul 2;8:46. Epub 2007 Jul 2.

Brain Research Institute, University of Zurich, Zurich, Switzerland.

Background: Development of neural networks requires that synapses are formed, eliminated and stabilized. At the neuromuscular junction (NMJ), agrin/MuSK signaling, by triggering downstream pathways, causes clustering and phosphorylation of postsynaptic acetylcholine receptors (AChRs). Postnatally, AChR aggregates are stabilized by molecular pathways that are poorly characterized. Gain or loss of function of Src-family kinases (SFKs) disassembles AChR clusters at adult NMJs in vivo, whereas AChR aggregates disperse rapidly upon withdrawal of agrin from cultured src-/-;fyn-/- myotubes. This suggests that a balance between protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) such as those of the Src-family may be essential in stabilizing clusters of AChRs.

Results: We have analyzed the role of PTPs in maintenance of AChR aggregates, by adding and then withdrawing agrin from cultured myotubes in the presence of PTP or PTK inhibitors and quantitating remaining AChR clusters. In wild-type myotubes, blocking PTPs with pervanadate caused enhanced disassembly of AChR clusters after agrin withdrawal. When added at the time of agrin withdrawal, SFK inhibitors destabilized AChR aggregates but concomitant addition of pervanadate rescued cluster stability. Likewise in src-/-;fyn-/- myotubes, in which agrin-induced AChR clusters form normally but rapidly disintegrate after agrin withdrawal, pervanadate addition stabilized AChR clusters. The PTP SHP-2, known to be enriched at the NMJ, associated and colocalized with MuSK, and agrin increased this interaction. Specific SHP-2 knockdown by RNA interference reduced the stability of AChR clusters in wild-type myotubes. Similarly, knockdown of SHP-2 in adult mouse soleus muscle by electroporation of RNA interference constructs caused disassembly of pretzel-shaped AChR-rich areas in vivo. Finally, we found that src-/-;fyn-/- myotubes contained elevated levels of SHP-2 protein.

Conclusion: Our data are the first to show that the fine balance between PTPs and SFKs is a key aspect in stabilization of postsynaptic AChR clusters. One phosphatase that acts in this equilibrium is SHP-2. Thus, PTPs such as SHP-2 stabilize AChR clusters under normal circumstances, but when these PTPs are not balanced by SFKs, they render clusters unstable.
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http://dx.doi.org/10.1186/1471-2202-8-46DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1924855PMC
July 2007

Cholesterol and lipid microdomains stabilize the postsynapse at the neuromuscular junction.

EMBO J 2006 Sep 24;25(17):4050-60. Epub 2006 Aug 24.

Department of Neurochemistry, Brain Research Institute, University of Zürich, Zürich, Switzerland.

Stabilization and maturation of synapses are important for development and function of the nervous system. Previous studies have implicated cholesterol-rich lipid microdomains in synapse stabilization, but the underlying mechanisms remain unclear. We found that cholesterol stabilizes clusters of synaptic acetylcholine receptors (AChRs) in denervated muscle in vivo and in nerve-muscle explants. In paralyzed muscles, cholesterol triggered maturation of nerve sprout-induced AChR clusters into pretzel shape. Cholesterol treatment also rescued a specific defect in AChR cluster stability in cultured src(-/-);fyn(-/-) myotubes. Postsynaptic proteins including AChRs, rapsyn, MuSK and Src-family kinases were strongly enriched in lipid microdomains prepared from wild-type myotubes. Microdomain disruption by cholesterol-sequestering methyl-beta-cyclodextrin disassembled AChR clusters and decreased AChR-rapsyn interaction and AChR phosphorylation. Amounts of microdomains and enrichment of postsynaptic proteins into microdomains were decreased in src(-/-);fyn(-/-) myotubes but rescued by cholesterol treatment. These data provide evidence that cholesterol-rich lipid microdomains and SFKs act in a dual mechanism in stabilizing the postsynapse: SFKs enhance microdomain-association of postsynaptic components, whereas microdomains provide the environment for SFKs to maintain interactions and phosphorylation of these components.
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http://dx.doi.org/10.1038/sj.emboj.7601288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1560359PMC
September 2006

Src-family kinases stabilize the neuromuscular synapse in vivo via protein interactions, phosphorylation, and cytoskeletal linkage of acetylcholine receptors.

J Neurosci 2005 Nov;25(45):10479-93

Department of Neurochemistry, Brain Research Institute, University of Zürich, CH-8057 Zürich, Switzerland.

Postnatal stabilization and maturation of the postsynaptic membrane are important for development and function of the neuromuscular junction (NMJ), but the underlying mechanisms remain poorly characterized. We examined the role of Src-family kinases (SFKs) in vivo. Electroporation of kinase-inactive Src constructs into soleus muscles of adult mice caused NMJ disassembly: acetylcholine receptor (AChR)-rich areas became fragmented; the topology of nerve terminal, AChRs, and synaptic nuclei was disturbed; and occasionally nerves started to sprout. Electroporation of kinase-overactive Src produced similar but milder effects. We studied the mechanism of SFK action using cultured src(-/-);fyn(-/-) myotubes, focusing on clustering of postsynaptic proteins, their interaction with AChRs, and AChR phosphorylation. Rapsyn and the utrophin-glycoprotein complex were recruited normally into AChR-containing clusters by agrin in src(-/-);fyn(-/-) myotubes. But after agrin withdrawal, clusters of these proteins disappeared rapidly in parallel with AChRs, revealing that SFKs are of general importance in postsynaptic stability. At the same time, AChR interaction with rapsyn and dystrobrevin and AChR phosphorylation decreased after agrin withdrawal from mutant myotubes. Unexpectedly, levels of rapsyn protein were increased in src(-/-);fyn(-/-) myotubes, whereas rapsyn-cytoskeleton interactions were unaffected. The overall cytoskeletal link of AChRs was weak but still strengthened by agrin in mutant cells, consistent with the normal formation but decreased stability of AChR clusters. These data show that correctly balanced activity of SFKs is critical in maintaining adult NMJs in vivo. SFKs hold the postsynaptic apparatus together through stabilization of AChR-rapsyn interaction and AChR phosphorylation. In addition, SFKs control rapsyn levels and AChR-cytoskeletal linkage.
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http://dx.doi.org/10.1523/JNEUROSCI.2103-05.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6725837PMC
November 2005