Biology subjects

Shoba, V. M.

Publications and source records attributed to Shoba, V. M..

3 recordsLinked to original sources

bioRxiv2023

Bifunctional small molecules that induce nuclearlocalization and targeted transcriptional regulation

The aberrant localization of proteins in cells is a key factor in the development of various diseases, including cancer and neurodegenerative disease. To better understand and potentially manipulate protein localization for therapeutic purposes, we engineered bifunctional compounds that bind to proteins in separate cellular compartments. We show these compounds induce nuclear import of cytosolic cargoes, using nuclear-localized BRD4 as a "carrier" for co-import and nuclear trapping of cytosolic proteins. We use this system to calculate kinetic constants for passive diffusion across the nuclear pore and demonstrate single-cell heterogeneity in response to these bifunctional molecules, with cells requiring high carrier to cargo expression for complete import. We also observe incorporation of cargoes into BRD4-containing condensates. Proteins shown to be substrates for nuclear transport include oncogenic mutant nucleophosmin (NPM1c) and mutant PI3K catalytic subunit alpha (PIK3CAE545K), suggesting potential applications to cancer treatment. In addition, we demonstrate that chemical-induced localization of BRD4 to cytosolic-localized DNA-binding proteins, namely, IRF1 with a nuclear export signal, induces target gene expression. These results suggest that induced localization of proteins with bifunctional molecules enables the rewiring of cell circuitry with significant implications for disease therapy.

biochemistry↗

bioRxiv2023

Development and applications of chimera platforms for tyrosine phosphorylation

Chimeric small molecules that induce post-translational modification (PTM) on a target protein by bringing it in proximity to a PTM-inducing enzyme are furnishing novel modalities to perturb protein function. Despite recent advances, such molecules are unavailable for a critical PTM, tyrosine phosphorylation. Furthermore, the contemporary design paradigm of chimeric molecules, formed by joining a non-inhibitory binder of the PTM-inducing enzyme with the binder of the target protein, prohibits the recruitment of most PTM-inducing enzymes as their non-inhibitory binders are unavailable. Here, we report two platforms to generate phosphorylation-inducing chimeric small molecules (PHICS) for tyrosine phosphorylation. We generate PHICS from both non-inhibitory binders (scantily available, platform 1) and kinase inhibitors (abundantly available, platform 2) using cysteine-based group transfer chemistry. PHICS triggered phosphorylation on tyrosine residues in diverse sequence contexts and target proteins (e.g., membrane-associated, cytosolic) and displayed multiple bioactivities, including initiation of a growth receptor signaling cascade and death of drug-resistant cancer cells. These studies provide an approach to induce biologically relevant PTM and lay the foundation for pharmacologic PTM editing (i.e., induction or removal) on target proteins using abundantly available inhibitors of PTM-inducing or erasing enzymes. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/531183v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@cdf529org.highwire.dtl.DTLVardef@19a70cdorg.highwire.dtl.DTLVardef@63dbf9org.highwire.dtl.DTLVardef@1febaaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Proteolysis Targeting Chimeras With Reduced Off-targets

Proteolysis Targeting Chimeras (PROTACs), a class of heterobifunctional molecules that recruit target proteins to E3 ligases, have gained traction for targeted protein degradation. However, pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other targets, such as zinc-finger (ZF) proteins, that hold key functions in normal development and disease progression. This off-target degradation of pomalidomide-based PROTACs raises concerns about their therapeutic applicability and long-term side effects. Therefore, there is a crucial need to develop rules for PROTAC design that minimize off-target degradation. In this study, we developed a high-throughput platform that interrogates the off-target degradation of ZF domains and discovered, using this platform, that PROTACs with the current design paradigm induce degradation of several ZF proteins. To identify new rules for PROTAC design, we generated a library of pomalidomide analogs that allowed systematic exploration of the impact of positional isomerism (e.g., C4 and C5 positions of the phthalimide ring), hydrogen bonding, steric and hydrophobic effects on propensities for ZF protein degradation. We found that modifications of appropriate size on the C5 position reduced off-target ZF degradation. We validated these results using immunoblotting, target engagement, and global mass spectrometric studies. We applied our newfound design principles on a previously developed ALK oncoprotein-targeting PROTAC and generated PROTACs with enhanced potency and minimal off-target degradation. We envision the reported off-target profiling platform and pomalidomide analogs will find utility in design of specific PROTACs.

pharmacology and toxicology↗