Inhibition of protein–protein interactions among tubulins in vitro and in cellulo using C-terminal-binding nanoparticles

Protein–protein interaction inhibitors in the human proteome: lessons from 117 targeted interactions

A framework for accelerating the development of next-generation PPI therapeutics is provided by connecting interface architecture with optimal inhibitor modality and discovery strategy, revealing clear links between interface topology and inhibitor discovery strategies.

Chemoproteomics identifies a pyrimido[4,5-d]pyrimidine analogue as a tubulin-tyrosine ligase binder

Minimal N-methylated and stapled peptide inhibitors of the autophagy protein GABARAP

The LC3/GABARAP protein family is a promising target for selective inhibition of autophagy. Further, LC3/GABARAP ligands have been used as targeted degraders of soluble proteins, protein aggregates, mitochondria, lipid droplets, and RNA. However, the small molecules used for such applications have poor binding affinity and known off-target effects. LC3/GABARAP proteins are challenging targets for small-molecule drug development due to their long, shallow binding grooves. In this work, we evaluate multiple approaches to stabilizing the extended structure of the native binding motif, producing N-methylated peptides and stapled peptides with low nanomolar affinity. A crystal structure and molecular dynamics simulations support a model where the N-methylation pre-organizes the motif into an extended, strand-like structure. N-methylation allowed minimization of the binding motif to a tetrapeptide that retained sub-micromolar affinity while minimizing charge and overall molecular weight. The truncated, N-methylated tetrapeptide showed passive permeability in artificial membrane and cell-based transwell assays. These results highlight new drug-like space for LC3/GABARAP ligands with high affinity and subfamily selectivity. Table of Contents Graphic Drawn and developed by Mollie McGibbon and Joshua Kritzer

Chemocarrier-Enabled Peptide Inhibition of MYC-Cofactor Interactions in Plants.

Direct chemical inhibition of transcription factor (TF)-cofactor interactions in plants remains challenging because their large protein-protein interfaces are poorly suited to conventional small molecules, while peptide-based inhibitors are limited by inefficient intracellular delivery. In our previous work, we demonstrated that peptide inhibitors can disrupt MYC transcription factor complexes in plants, but only at relatively high concentrations. Here, we show that a chemically defined chemocarrier overcomes this limitation and enables low-dose functional activity of peptide inhibitors in planta. To access TF-cofactor interfaces, we established a multi-step, target-guided screening platform that links in vitro high-throughput assays with functional validation in plants. This workflow integrates primary and secondary AlphaScreen assays using full-length and truncated MYC constructs with a tertiary assay targeting the interaction between MYCs and the MED25 coactivator. Screening of an approved-drug library followed by structure-activity optimization led to the identification of a low-toxicity aromatic derivative with potent cell-penetrating activity in Arabidopsis. Conjugation with this chemocarrier markedly enhanced the intracellular delivery and functional efficacy of multiple MYC-targeting peptide scaffolds, including cyclic and stapled peptides, resulting in over two orders of magnitude improvement in potency. This enabled robust inhibition of MYC-dependent transcriptional programs at substantially reduced concentrations, converting peptide-based inhibition from a high-dose proof-of-concept into a low-dose, functionally tractable strategy. These findings establish a practical chemical modality for accessing transcription factor-cofactor interfaces in plants.

Induced ubiquitination of the partially disordered estrogen receptor alpha via a 14-3-3 directed molecular glue-PROTAC

Proteins lacking defined ligandable pockets remain challenging drug targets. Here, we develop a molecular glue-based PROTAC (MGPROTAC) approach that chemically conjugates a molecular glue stabilizer to a VHL-recruiting ligand to capture and ubiquitinate the 14-3-3/Estrogen receptor α (ERα) complex. Our designed MGPROTACs engage a composite interface between 14-3-3 and the disordered F-domain of ERα, promoting cooperative complex formation and targeted ubiquitination. Biophysical characterization revealed distinct linker-dependent cooperativities across the MGPROTAC series, which influenced both cellular permeability and ubiquitination efficiency. Cryo-EM of the most cooperative MGPROTAC uncovered de novo VHL–14-3-3ζ contacts, while molecular dynamics simulations rationalize the stabilizing interactions underlying cooperativity. Strikingly, fine-tuning linker design enables selective ubiquitination of distinct complex subunits. These findings establish a structural and mechanistic framework for integrating molecular glue and PROTAC principles, expanding the scope of drug discovery to previously intractable protein complexes. Proteins lacking defined ligandable pockets are challenging drug targets. Here the authors combine molecular glue-stabilizer and PROTAC strategies to enable ubiquitination of the 14-3-3/estrogen receptor α complex, revealing cooperative linker-dependent activity and a structural basis for targeted E3 ligase recruitment.

Structure-Based Design of First Ndc80 Loop-Targeting Peptide Disrupting Ndc80–Nuf2 Interaction for Hepatocellular Carcinoma Therapy

Ndc80 is essential for kinetochore–microtubule attachment, but its broad protein–protein interaction interface remains difficult to target. Here, we used structure-guided peptide optimization to develop Peptide-4, a loop-directed peptide designed to modulate the Ndc80–Nuf2 interaction. In contrast to reported Ndc80-directed compounds acting through the Hec1–Nek2 axis or the calponin homology domain, Peptide-4 addresses an underexplored loop region. Peptide-4 bound Ndc80 with a Kd of 0.45 ± 0.01 nM and inhibited the Ndc80–Nuf2 interaction with an IC50 of 0.76 ± 0.03 nM. In Huh7 cells, Peptide-4 altered microtubule organization, suppressed proliferation, clonogenic growth, migration, and invasion, and induced G2/M-phase accumulation and apoptosis. Ndc80 depletion attenuated its antiproliferative effect, consistent with a contribution of Ndc80 to its cellular activity. Peptide-4 also reduced tumor growth in Huh7 xenografts without apparent toxicity under the tested conditions. These findings support the Ndc80 loop as an underexplored peptide-addressable site and identify Peptide-4 as a lead for further optimization.