Biology subjects

Seiple, I. B.

Publications and source records attributed to Seiple, I. B..

5 recordsLinked to original sources

bioRxiv2026

Ninhydrin as a covalent warhead for chemical proteomic-enabled discovery and selective engagement of reactive arginines

Covalent molecules have emerged as next-generation therapeutics and as powerful tools for perturbing fundamental biological processes. Chemical proteomic methods to screen for reactive proteinaceous amino acids have transformed small-molecule discovery pipelines, but their application remains mostly limited to sites where reactive cysteines and lysines are present. Here we report a ninhydrin-based warhead that selectively modifies arginine residues, thus expanding the repertoire of amino acids targetable by covalent molecules. Specifically, we developed alkyne-functionalized variants of ninhydrin to establish an arginine-specific chemical proteomics platform, enabling the classification of more than 6,800 unique reactive arginines. These studies uncovered potential modification sites on disease-relevant proteins, including reactive arginines within catalytic sites that are essential for function. By endowing a reversible small molecule inhibitor of cyclophilin A with a ninhydrin warhead, we achieved selective, covalent engagement, and attenuation of enzymatic activity, highlighting the potential for targeting arginines in future therapeutic development campaigns. These findings establish ninhydrin as a warhead for studying arginine reactivity and modulating protein function.

biochemistry↗

bioRxiv2025

Initial leads to combat streptogramin resistance generated from X-ray fragment screening against VatD

Streptogramins, a potent antibiotic class targeting bacterial ribosomes, are effective against severe infections such as those caused by vancomycin-resistant Staphylococcus aureus (VRSA) and Enterococcus faecium (VRE). The synergistic binding of group A and B streptogramins to the peptidyl transferase center (PTC) and nascent peptide exit tunnel (NPET), respectively, yields a bactericidal effect. However, the efficacy of streptogramins is compromised by resistance mechanisms, including the enzymatic acetylation of group A streptogramins by Virginiamycin Acetyl Transferase (Vat) enzymes, which reduce antibiotic affinity for the bacterial ribosome. Here, we applied fragment-based drug discovery (FBDD) to identify starting points for the development of inhibitors of VatD. X-ray crystallography screening revealed three primary fragment-binding sites on VatD. In the acetyl-CoA binding site, fragments bound to stabilize distinct conformational states in critical residues, His82 and Trp121, within the acetyl subsite. In the antibiotic binding site, two fragments were identified that form unique hydrogen/hydrophobic bonds, suggesting specific interactions that could be leveraged for competitive inhibition by disrupting streptogramin modification. Elaborations of these fragments showed weak inhibition of VatDs acetyltransferase activity, indicating the potential for further optimization. These findings establish initial hits that could restore streptogramin efficacy by targeting VatD directly, providing a structural foundation for inhibitor development against resistant bacterial strains.

biophysics↗

bioRxiv2023

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

biochemistry↗

bioRxiv2020

Selective Targeting of Ras Mutant Cancers via a New Small Molecule

Mutations in the Ras family of oncogenes are implicated in 33% of human cancers, making Ras an intensely pursued target in drug discovery. As an alternative to direct pharmacological inhibition of Ras, we looked for sensitivities in RAS mutant cells. Using a small molecule screen in cell lines with mutations in Ras and its effector Raf, we discovered 249C as a Ras-mutant selective cytotoxic agent against a spectrum of RAS-mutant cancers. By combining CRISPR chemical-genetic screening, comparative profiling and chemoproteomics, we identified that 249C binds to a unique subunit on vacuolar (V)-ATPase with nanomolar affinity, inhibiting its biochemical activity and, unexpectedly, altering V-ATPase translocation in Ras-induced macropinocytosis. Via binding to V-ATPase, 249C prevents lysosomal acidification and inhibits autophagy and macropinocytosis pathways that several Ras-driven cancers rely on for survival. In characterizing 249Cs mechanism, we show that potency varies with the identity of the RAS driver mutation highlighting a mutant-specific dependence on autophagy and macropinocytosis. Indeed, 249C potently inhibits tumor growth without adverse side effects in a mouse xenograft model of KRAS-driven non-small cell lung cancer. These data establish proof-of-concept for targeting V-ATPase as a way to indirectly target specific Ras mutants, and provide a fundamental link between V-ATPase localization and specific Ras mutant tumor-related activity.

cancer biology↗

bioRxiv2019

Label-retention expansion microscopy

Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with super-resolution Stochastic Optical Reconstruction Microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

bioengineering↗