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Federation, A. J.

Publications and source records attributed to Federation, A. J..

7 recordsLinked to original sources

Structure-free, site-resolved contrastive learningextends small-molecule discovery beyond the reachof structure-based modeling

Virtual screening asks which molecules, among an enormous space of drug-like chemistry, are worth synthesizing and testing against a protein target. Most modern methods answer by building and scoring an explicit three-dimensional pose through molecular docking, or the co-folding models that now approach experimental accuracy. Building these poses presumes a well-defined pocket, and the non-orthosteric, cryptic, and intrinsically disordered sites where unexplored ligandability lies offer none. There, these methods fail to generalize. Here we present Ptarmigan-1, a contrastive model that co-embeds the residues of a protein with candidate small molecules in a shared latent space, from sequence and two-dimensional chemistry alone, and without ever constructing a pose. Engagement reduces to the proximity of precomputed embeddings. Freed from the pose, Ptarmigan-1 trains directly on chemoproteomic and bioactivity data of mixed resolution, scores a compound in ten milliseconds rather than the tens of seconds a co-folding model demands, and resolves each prediction to the residues a compound engages. On well-folded, orthosteric targets it performs comparably to a collection of co-folding and docking models, and on covalent, cryptic, and disordered sites it matches or exceeds them. It localizes reversible and covalent inhibitors to the pockets they engage, even for targets withheld from training, and screens the entire human proteome against a library of 3.4 billion compounds in under a day. By decoupling molecular recognition from structure, Ptarmigan-1 recasts virtual screening as a reusable index that continuously improves as data accumulate.

molecular biology↗

CpG island density predicts CBP/p300 dependency across 3D chromatin clusters

RNA Polymerase II (Pol2) organizes transcription through higher-ordered chromatin clusters that integrate promoter and enhancer interactions to coordinate gene expression. Nevertheless, the features that distinguish unique classes of Pol2-mediated clusters remain to be defined. Here, we identify two distinct classes of Pol2-mediated clusters: one enriched for CpG islands, promoter-promoter interactions, and housekeeping gene expression, and another characterized by high CBP/p300 occupancy, enhancer-promoter looping, and lineage-defining (LD) transcriptional programs. Acute inhibition of CBP/p300 catalytic activity leads to rapid loss of acetylation at enhancers and preferential downregulation of LD genes, resulting in impaired cellular proliferation and activation of apoptotic programs. Integrative machine learning modeling reveals that cluster strength, RNA half-life, and CpG island content as strong predictors of genes sensitive to CBP/p300 inhibition. Together, these findings clarify enhancer-addiction and vulnerability to CBP/p300 inhibition.

cancer biology↗

Development of PROTACs for targeted degradation of oncogenic TRK fusions

Chromosomal translocations leading to the fusion of tropomyosin receptor kinases (TRK) with diverse partner proteins have been identified as oncogenic drivers in many adult and pediatric cancers. While first-generation TRK kinase inhibitors, such as entrectinib and larotrectinib, have shown positive responses in TRK fusion-positive cancers, resistance mutations against these inhibitors in the kinase domain limit their efficacy. Second-generation inhibitors are in clinical evaluation, highlighting a need for novel therapeutic modalities to achieve durable suppression of the oncogenic activity of TRK fusions. Here, we developed heterobifunctional small molecule degraders (PROTACs) to achieve targeted degradation of TRK fusions. By conjugating entrectinib to thalidomide, we identified JWJ-01-378 as a potent and selective CRBN-recruiting degrader of the TPM3-TRKA fusion. JWJ-01-378 induced TPM3-TRKA degradation through the ubiquitin-proteasome system and proteomics analysis confirmed the acute selectivity of JWJ-01-378 for achieving TPM3-TRKA degradation with minimal off-target effects. While JWJ-01-378 was also able to degrade wild-type TRK, it was unable to degrade TRK inhibitor resistant mutants and ALK fusions. Importantly, TPM3-TRKA degradation by JWJ-01-378 suppressed downstream signaling and reduced cancer cell viability, with improved responses compared to heterobifunctional control compounds that cannot degrade TPM3-TRKA. Together, our study expands the toolbox of compounds for evaluating targeted degradation of TRK fusions in cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/660465v1_ufig13.gif" ALT="Figure 13"> View larger version (19K): org.highwire.dtl.DTLVardef@6c78f7org.highwire.dtl.DTLVardef@179dcdeorg.highwire.dtl.DTLVardef@1939988org.highwire.dtl.DTLVardef@144c718_HPS_FORMAT_FIGEXP M_FIG C_FIG JWJ-01-378 recruits cereblon (CRBN) to induce potent and selective degradation of oncogenic TRK fusions, leading to a collapse in downstream signaling and loss of cancer cell viability. Graphical abstract was created using Biorender.com.

cancer biology↗

Native, Spatiotemporal Profiling of the Global Human Regulome

The regulome, comprising transcription factors, cofactors, chromatin remodelers, and other regulatory proteins, forms the core machinery by which cells interpret signals and execute gene expression programs. Despite its central role in development, disease, and drug response, the regulome remains largely uncharted at scale due to its dynamic, low-abundance, and chromatin-associated nature. Here, we present a method for scalable, regulome profiling for global, compartment-resolved quantification of native regulome proteins. By enriching DNA- and chromatin-associated proteins and profiling them using high-throughput, label-free DIA mass spectrometry, regulome profiling captures chromatin-associated proteins across 36 human cell lines and thousands of perturbations. The resulting Regulome Atlas recovers nearly 60% of known human transcription factors, reveals lineage-specific TF localization, and distinguishes active nuclear engagement from latent, unbound states. We demonstrate that regulome profiles resolve acute immune pathway activation prior to transcriptional changes, identify previously unrecognized drug-induced regulome responses, and enable proteome-scale readouts of compound target engagement and complex remodeling. This work establishes a foundational resource for decoding the regulatory proteome and provides a blueprint for integrating regulome data into next-generation models of cellular behavior.

cell biology↗

A flexible workflow for building spectral libraries from narrow window data independent acquisition mass spectrometry data

Advances in library-based methods for peptide detection from data independent acquisition (DIA) mass spectrometry have made it possible to detect and quantify tens of thousands of peptides in a single mass spectrometry run. However, many of these methods rely on a comprehensive, high quality spectral library containing information about the expected retention time and fragmentation patterns of peptides in the sample. Empirical spectral libraries are often generated through data-dependent acquisition and may suffer from biases as a result. Spectral libraries can be generated in silico but these models are not trained to handle all possible post-translational modifications. Here, we propose a false discovery rate controlled spectrum-centric search workflow to generate spectral libraries directly from gas-phase fractionated DIA tandem mass spectrometry data. We demonstrate that this strategy is able to detect phosphorylated peptides and can be used to generate a spectral library for accurate peptide detection and quantitation in wide window DIA data. We compare the results of this search workflow to other library-free approaches and demonstrate that our search is competitive in terms of accuracy and sensitivity. These results demonstrate that the proposed workflow has the capacity to generate spectral libraries while avoiding the limitations of other methods.

bioinformatics↗

Quantification of nuclear protein dynamics reveals chromatin remodeling during acute protein degradation

Sequencing-based technologies cannot measure post-transcriptional dynamics of the nuclear proteome, but unbiased mass-spectrometry measurements of chromatin-associated proteins remain difficult. In this work, we have combined facile nuclear sub-fractionation approaches with data-independent acquisition mass spectrometry to improve detection and quantification of nuclear proteins in human cells and tissues. Nuclei are isolated and subjected to a series of extraction conditions that enrich for nucleoplasm, euchromatin, heterochromatin and nuclear-membrane associated proteins. Using this approach, we can measure peptides from over 70% of the expressed nuclear proteome. As we are physically separating chromatin compartments prior to analysis, proteins can be assigned into functional chromatin environments to illuminate systems-wide nuclear protein dynamics. The integrity of nuclear sub-compartments were validated with immunofluorescence, which confirms the presence of key markers during chromatin extraction. We then apply this method to study the nuclear proteome-wide response to pharmacological degradation of the BET bromodomain proteins. BET degradation leads to widespread changes in chromatin composition, and we discover global HDAC1/2-mediated remodeling of chromatin previously bound by BET bromodomains. In summary, we have developed a technology for reproducible, comprehensive characterization of the nuclear proteome to observe the systems-wide nuclear protein dynamics.

systems biology↗

Identification of candidate master transcription factors within enhancer-centric transcriptional regulatory networks

Regulation of gene expression through binding of transcription factors (TFs) to cis-regulatory elements is highly complex in mammalian cells. Genome-wide measurement technologies provide new means to understand this regulation, and models of TF regulatory networks have been built with the goal of identifying critical factors. Here, we report a network model of transcriptional regulation between TFs constructed by integrating genomewide identification of active enhancers and regions of focal DNA accessibility. Network topology is confirmed by published TF ChIP-seq data. By considering multiple methods of TF prioritization following network construction, we identify master TFs in well-studied cell types, and these networks provide better prioritization than networks only considering promoter-proximal accessibility peaks. Comparisons between networks from similar cell types show stable connectivity of most TFs, while master regulator TFs show dramatic changes in connectivity and centrality. Applying this method to study chronic lymphocytic leukemia, we prioritized several network TFs amenable to pharmacological perturbation and show that compounds targeting these TFs show comparable efficacy in CLL cell lines to FDA-approved therapies. The construction of transcriptional regulatory network (TRN) models can predict the interactions between individual TFs and predict critical TFs for development or disease.

systems biology↗