Molecular Bidents with Two Electrophilic Warheads as a New Pharmacological Modality.

A systematic strategy to develop dual-warhead inhibitors is introduced to circumvent the limitations of conventional covalent inhibitors such as vulnerability to mutations of the corresponding nucleophilic residue. Currently, all FDA-approved covalent small molecules feature one electrophile, leaving open a facile route to acquired resistance. We conducted a systematic analysis of human proteins in the protein data bank to reveal ∼400 unique targets amendable to dual covalent inhibitors, which we term "molecular bidents". We demonstrated this strategy by targeting two kinases: MKK7 and EGFR. The designed compounds, ZNL-8162 and ZNL-0056, are ATP-competitive inhibitors that form two covalent bonds with cysteines and retain potency against single cysteine mutants. Therefore, molecular bidents represent a new pharmacological modality with the potential for improved selectivity, potency, and drug resistance profile.

1H, 13C and 15N backbone and sidechain assignment of the Burkholderia mallei acyl carrier protein.

Acyl carrier proteins (ACPs) are universally conserved proteins amongst different species and are involved in fatty acid synthesis. Bacteria utilize ACPs as acyl carriers and donors for the synthesis of products such as endotoxins or acyl homoserine lactones (AHLs), which are used in quorum sensing mechanisms. In this study, wehave expressed isotopically labeled holo-ACP from Burkholderia mallei in Escherichia coli to assign 100% of non-proline backbone amide (HN) resonances, 95.5% of aliphatic carbon resonances and 98.6% of aliphatic hydrogen sidechain resonances.

Structure-Based Design of Y-Shaped Covalent TEAD Inhibitors.

Transcriptional enhanced associate domain (TEAD) proteins together with their transcriptional coactivator yes-associated protein (YAP) and transcriptional coactivator with the PDZ-binding motif (TAZ) are important transcription factors and cofactors that regulate gene expression in the Hippo pathway. In mammals, the TEAD families have four homologues: TEAD1 (TEF-1), TEAD2 (TEF-4), TEAD3 (TEF-5), and TEAD4 (TEF-3). Aberrant expression and hyperactivation of TEAD/YAP signaling have been implicated in a variety of malignancies. Recently, TEADs were recognized as being palmitoylated in cells, and the lipophilic palmitate pocket has been successfully targeted by both covalent and noncovalent ligands. In this report, we present the medicinal chemistry effort to develop MYF-03-176 (compound 22) as a selective, cysteine-covalent TEAD inhibitor. MYF-03-176 (compound 22) significantly inhibits TEAD-regulated gene expression and proliferation of the cell lines with TEAD dependence including those derived from mesothelioma and liposarcoma.

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders.

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics1-3. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs4-6. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 Å cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.

On-Chip Preconcentration Microchip Capillary Electrophoresis Based CE-PRM-LIVE for High-Throughput Selectivity Profiling of Deubiquitinase Inhibitors.

The family of deubiquitinases (DUBs) comprises ∼100 enzymes that cleave ubiquitin from substrate proteins and thereby regulate key aspects of human physiology. DUBs have recently emerged as disease-relevant and chemically tractable, although currently there are no approved DUB-targeting drugs and most preclinical small molecules are low-potency and/or multitargeted. We paired a novel capillary electrophoresis microchip containing an integrated, "on-chip" C18 bed (SPE-ZipChip) with a TMT version of our recently described PRM-LIVE acquisition scheme on a timsTOF Pro mass spectrometer to facilitate rapid activity-based protein profiling of DUB inhibitors. We demonstrate the ability of the SPE-ZipChip to improve proteome coverage of complex samples as well as the quantitation integrity of CE-PRM-LIVE for TMT labeled samples. These technologies provide a platform to accurately quantify competitive binding of covalent and reversible inhibitors in a multiplexed assay that spans 49 endogenous DUBs in less than 15 min.

Open source fraction collector/MALDI spotter for proteomics.

We describe a complete open-source hardware/software solution for high performance thermostatted peptide fraction collection to support mass spectrometry experiments with complex proteomes. The instrument is easy to assemble using parts readily available through retail channels at a fraction of the cost compared to typical commercial systems. Control software is written in Python allowing for rapid customization. We demonstrate several useful applications, including the automated deposition of LC separated peptides for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) as well as collection and concatenation of peptide fractions from nanoflow HPLC separations.