Biology subjects

Hsu, K.-L.

Publications and source records attributed to Hsu, K.-L..

7 recordsLinked to original sources

bioRxiv2026

Liposomal DAGLβ Inhibitor Shows Substantial Antinociceptive Potency Mediated by AMPK in a Neuropathic Pain Mouse Models

Diacylglycerol lipase-beta (DAGL{beta}), a biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG), is highly expressed in macrophages and serves as a potential contributor to inflammatory and hypernociceptive responses. Moreover, the DAGL{beta} inhibitor KT109 produces antinociceptive effects in various rodent pain models, and its liposome formulation dramatically increases its antinociceptive potency in a lipopolysaccharide mouse model of inflammatory pain compared to free KT109. Here, we further optimized the liposomal KT109 formulation through adjustment of lipid composition and improved processing, achieving 100-fold higher drug encapsulation efficiency than the previous formulation, uniform size distribution, and excellent storage stability. Further, the second generation liposomal KT109 showed remarkable potency (ED50 [95% CL] = 0.226 [0.113-0.454] mg/kg) in a mouse model of paclitaxel-induced neuropathic pain, compared to free KT109 (ED50 [95% CL] = 31 [21-46] mg/kg). Liposomal KT109 (0.4 mg/kg) fully reversed paclitaxel-induced mechanical hypersensitivity for up to 24 hours, and its antinociceptive effects were sustained following a week of daily injections, with no detectable tolerance. In addition, the adenosine monophosphate-activated protein kinase (AMPK) inhibitor, dorsomorphin, prevented the antinociceptive effects of KT109, which is consistent with previous work demonstrating AMPK activation mediates the antinociceptive phenotype of DAGL{beta} knockout mice.

pharmacology and toxicology↗

bioRxiv2026

High-Speed AFM Reveals Ligand-Dependent Supramolecular Switching of Human Phosphofructokinase-1

Phosphofructokinase-1 (PFK-1) catalyzes the ATP-dependent conversion of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F1,6BP), the first committed step of glycolysis. Beyond classical allostery, the liver isoform PFKL forms higher-order assemblies, but how ligand binding redirects intermolecular interactions remains unclear. Here we use high-speed atomic force microscopy (HS-AFM), topology-based AFM image simulations, and molecular dynamics (MD) simulations to define ligand-dependent assembly switching of human PFKL. Wild-type PFKL (PFKL WT) forms lattice-like assemblies under APO and ATP conditions, whereas coordinated ATP and F6P loading redirects assembly toward filaments in a ligand-order-dependent manner. APO-PFKL WT also forms lattice-like assemblies on or near membrane-supported surfaces, suggesting that interfacial environments influence where assembly initiates. The filament-defective N702T mutant forms ordered double-layer lattices and preserves this geometry under APO, ATP, and ATP+F6P conditions. MD simulations suggest that lattice stabilization arises from distributed inter-tetramer contacts. These findings define a structural framework for ligand-dependent supramolecular regulation of glycolysis.

biophysics↗

bioRxiv2026

In Cellulo pharmacological profiling and genomic editing reveals paralog-specific targets for PA generation during PLC signaling

Phosphatidic acid (PA) is an essential intermediate generated during phospholipase C (PLC) signaling, but its regulation is complex. PA can be generated by ten different diacylglycerol kinase paralogs (DGKs) and two different phospholipase D paralogs (PLDs) in mammals. Because these enzymes are activated under diverse conditions and at various membranes, understanding paralog-specific contributions to PA production is critical for therapeutic development of drugs that modulate the PLC pathway. To address this, we aimed to characterize the paralog specificity of the DGK inhibitors R59022 and BMS-502 against individual endogenously-tagged DGK paralogs in live cells. We found that R59022 and BMS-502 both recruited endogenous DGKalpha to the plasma membrane, and inhibited the catalytic fragment of DGKalpha when ectopically localized to the mitochondrial outer membrane. However, at its effective dose, R59022 paradoxically increased PA levels, while BMS-502 functioned as a potent inhibitor. Live-cell imaging experiments using BMS-502 with carbachol stimulation of endogenous muscarinic receptors showed that inhibition of both DGKalpha and the PLDs is needed to substantially reduce PA levels during PLC activation. Our findings both identify paralog-specific druggable targets for modulating PLC signaling events, and establish a new platform for characterizing DGK and PLD activity in living cells.

biochemistry↗

bioRxiv2026

Re-Engineering P(V) Chemical Warfare: Harnessing Stereogenic Phosphorus-Azoles for Protein Ligand Discovery In Vivo

P(V) electrophiles such as tabun, sarin, soman, and VX are notorious for their lethality and nefarious intent in chemical warfare. Consequently, these deadly agents have largely been abandoned except for fluorophosphonate tool compounds that were repurposed for activity-based protein profiling (ABPP). Stereogenic P(V) centers hold strong potential as enabling scaffolds for synthetic and medicinal chemistry due to their inherent chirality and favorable bioavailability but are limited principally by potent off-target toxicity. Herein, we developed phosphorus-azole exchange (PhAzE) chemistry for tuning reactivity of the stereogenic P(V) pharmacophore to increase selectivity and mitigate off-target activity in cells and animal models. We demonstrate ultrapotent (300 pM in cells, 1 mg kg-1 in mice), enantioselective, covalent inhibition of the serine hydrolases DPP8/9 with PhAzE ligand in cells and in vivo; no overt toxicity was detected in mice treated daily over the course of a week. These finding show the P(V) electrophile can potently and enantioselectively engage a target protein without a deadly outcome, charting a path towards broader adoption of these agents in laboratory and industry settings.

biochemistry↗

bioRxiv2025

A Chemoproteomic Atlas of the Human Purine Interactome for Regioselective Ligand Discovery

Purines are essential bioactive molecules that interact with a large fraction of the human proteome. Despite their importance, the scope of actionable purine-binding pockets for ligand discovery remains limited. Here, we developed a quantitative chemoproteomics platform using sulfonyl-purine (SuPUR) chemistry to produce a massive and functional map of the human purine interactome. The SuPUR platform captured 31,000+ targetable tyrosine and lysine sites, representing the most comprehensive beyond cysteine chemoproteomics database for enabling protein ligand discovery. SuPUR ligands that bind through a regioselective fashion serve as enabling starting points for developing potent (nanomolar) and proteome-wide-selective modulators of enzymatic and protein-protein interaction function. Phenotypic screening identified a site-specific (Y237) and regioselective SuPUR ligand of ACAT2 to reveal an unexpected metabolic dependency in cancer cells. A crystal structure of SuPUR ligand-bound ACAT2 revealed the purine group binds deep in the CoA pocket forming key interactions with catalytic residues via a water bridge to guide future structure-based ligand design.

biochemistry↗

bioRxiv2025

A Covalent PFKL Activator Suppresses Tumor Growth

Glycolysis fuels vital cellular functions and its dysregulation is implicated in cancer, neurodegeneration, antibiotic resistance and diabetes. The glycolytic dependency of cancer, known as the Warburg effect, presents a key vulnerability for developing targeted anticancer agents but remains challenging due to metabolic heterogeneity and resistance. Here, we developed a first-in-class covalent phosphofructokinase-1 liver type (PFKL) activator that induces metabolic imbalance coupled to delivery of a cytotoxic payload to cancer cells in vitro and in vivo. The electrophile-drug conjugate (EDC) site-specifically and proteome-wide selectively modifies K677 in the allosteric effector site to stabilize the R-state tetramer of PFKL and destabilize cell metabolism. We introduce EDCs as a new delivery mechanism analogous to antibody-drug conjugates but differentiated by selective covalent targeting of intracellular proteins.

biochemistry↗

bioRxiv2019

Automated Spatially Targeted Optical Micro Proteomics (autoSTOMP) to determine protein complexity of subcellular structures

Spatially Targeted Optical Micro Proteomics (STOMP) is a method to study region-specific protein complexity of a biological specimen. STOMP uses a confocal microscope to both visualize structures of interest and to tag the proteins within those structures by a photo-driven crosslinking reaction so that they can be affinity purified and identified by mass spectrometry1. STOMP has the potential to perform discovery proteomics on sub-cellular structures in a wide range of primary cells types and biopsy-scale tissue samples. However, two significant limitations have prevented the broad adoption of this technique by the scientific community. First, STOMP is performed across two software platforms written in different languages, which requires user operation at each field of view. Up to 48 hours of microscope time is necessary to tag sufficient protein ([~]1 g) for mass spectrometry making STOMP prohibitively time and labor-consuming for many researchers. Second, the original STOMP protocol uses a custom photo-crosslinker that limits the accessibility of the technique for some user. To liberate the user, we developed a protocol that automates communication between Zeiss Zen Black imaging software and FIJI image processing software using a customizable code in SikuliX. To fully automate STOMP (autoSTOMP), this protocol includes a tool to make tile array, autofocus and capture images of fields of view across the sample; as well as a method to modify the file that guides photo-tagging so that subsets of the structures of interest can be targeted. To make this protocol broadly accessible, we implemented a commercially available biotin-benzophenone crosslinker as well as a procedure to block endogenous biotin and purify tagged proteins using magnetic streptavidin beads. Here we demonstrate that autoSTOMP can efficiently label, purify and identify proteins that belong to structures measuring 1-2 m in diameter using human foreskin fibroblasts or mouse bone marrow-derived dendritic cells infected with the protozoan parasite Toxoplasma gondii (Tg). The autoSTOMP platform can easily be adapted to address a range of research questions using Zeiss Zen Black microscopy systems and LC-MS protocols that are standard in many institutional research cores.

biochemistry↗