Biology subjects

Van Horn, W. D.

Publications and source records attributed to Van Horn, W. D..

8 recordsLinked to original sources

bioRxiv2026

TRPV1 antagonism occurs through diverse structural mechanisms

Transient receptor potential vanilloid 1 (TRPV1) ion channel mediates thermosensation and pain and is a target for non-addictive analgesics; however, clinical candidates have failed due to thermoregulatory side effects. Limited structural data for human TRPV1 (hTRPV1) bound to clinically relevant antagonists has constrained mechanistic insight. Using chemoinformatics-informed cryo-EM and BRET assays, we define the structural basis of antagonism across diverse chemotypes, including failed clinical compounds. A structure of hTRPV1 bound to 6-iodo-dihydrocapsaicin shows how a single substitution converts an agonist into an antagonist. Additional structures with Asivatrep, Mavatrep, and JNJ-17203212 reveal vanilloid pocket plasticity and divergent interaction networks, including lipid co-binding. Despite this diversity, antagonists converge on a conserved inhibited state, showing high potency is maintained across flexible binding modes. These findings redefine our understanding of hTRPV1 antagonism and illustrate how chemically diverse ligands stabilize an inhibited state in polymodal ion channels, laying groundwork for next-generation analgesics with improved safety.

biophysics↗

bioRxiv2025

Pharmacological Tools to Modulate Ordered Membrane Domains and Order-Dependent Protein Function

Ordered membrane nanodomains colloquially known as "lipid rafts" have many proposed cellular functions. However, pharmacological tools to modulate protein affinity for rafts and to manipulate raft formation are currently lacking. We screened 24,000 small molecules for compounds that impact the raft affinity of a known raft-preferring protein, peripheral myelin protein 22 (PMP22), in giant plasma membrane vesicles (GPMVs). Hits were counter-screened against another raft protein, MAL, and also tested for their impact on raft stability. We identified three chemically distinct tools for manipulating lipid rafts. Two compounds were seen to both decrease PMP22 raft partitioning and to destabilize ordered domains (VU0607402 and VU0519975) while a third (primaquine diphosphate) increased PMP22 partitioning and stabilized ordered domains. While discovered in a PMP22-focused screen, all three were seen to modulate raft formation in a protein-independent manner by altering lipid-lipid interactions and membrane fluidity. Acute treatment of live cells with the raft destabilizing compound, VU0607402 was seen to modulate TRPM8 channel function, highlighting the utility of this compound in live-cell experiments for dissecting the role that membrane order and fluidity play in cell signaling. These compounds provide novel pharmacological tools for probing lipid raft properties and function in biophysical experiments and in living cells.

biophysics↗

bioRxiv2025

Neurobiological and Chemical Characterization of the Cyanobacterial Metabolite Veraguamide E

Ver Es structure was validated by {superscript 1}H NMR, HRMS, and molecular networking analyses. Computational docking and NMR titration confirmed direct, saturable, and tight binding of Ver E to the human Sigma-2 receptor/transmembrane protein 97 ({sigma}2R/TMEM97). Functional calcium imaging in primary mouse sensory neurons revealed that Ver E increases intracellular Ca{superscript 2} levels without modulating store-operated calcium entry (SOCE). Multi-well microelectrode array experiments using human induced pluripotent stem cell (hiPSC) derived nociceptors showed that Ver E significantly reduced neuronal activity at physiological temperatures, but not under heat-stress conditions. Ver E exhibited no cytotoxicity at concentrations up to 30 {micro}M in HEK293 cells, and immunocytochemistry confirmed that it does not alter phosphorylated eIF2 (p-eIF2) expression, indicating a mechanism distinct from integrated stress response modulators. Collectively, these findings position Ver E as a non-toxic compound capable of selectively modulating neuronal excitability, thereby advancing the development of novel therapeutics for pain management. SignificanceNatural products have long been recognized as a rich source of therapeutics, accounting for over 60% of currently approved small-molecule drugs and underscoring their pivotal role in drug discovery. Marine cyanobacteria produce structurally diverse secondary metabolites with a wide array of biological activities. Among these are the veraguamides, a family of depsipeptides that have shown promise as future therapeutics in our recent studies. This work presents a detailed biological and chemical characterization of veraguamide E (Ver E), isolated from a Panamanian marine cyanobacterial collection. The {sigma}2R/TMEM97 system has been identified as a promising target to address unmet need for non-opioid therapeutics which can modulate neuronal excitability in the context of chronic pain. Discovery and identification of novel compounds which modulate this system can help us better understand its function as well as allow us to develop future therapeutics targeting this pathway. HighlightsO_LIVeraguamide E specifically binds {sigma}2R/TMEM97 receptor with high affinity. C_LIO_LIComputational docking and NMR confirm a distinct binding mechanism. C_LIO_LIVer E modulates calcium signaling in mouse DRG neurons and human iPSC-derived nociceptors. C_LIO_LIVer E demonstrates no detectable cytotoxicity in human cell lines. C_LI

neuroscience↗

bioRxiv2025

TRPM8 protein dynamics correlates with ligand structure and cellular function

Protein dynamics have emerged as a key feature associated with function in various systems. Here, NMR-based studies coupled with computational cheminformatics and cellular function are leveraged to identify a relationship between the human cold and menthol receptor TRPM8 dynamics, chemical structure, and cellular potency. TRPM8 is a validated target for a variety of pain indications but generally has been clinically limited by on-target side effects impacting thermosensing and thermoregulation. This study shows that cheminformatic analysis of a TRPM8 regulating small molecule ligand library correlates with cellular function. Electrophysiology studies further validate the relationship and show a correlation between chemical structure and functional features such as compound potency. Solution NMR studies of the TRPM8 voltage sensing-like domain, which houses the canonical menthol ligand binding site, show that ligand binding conformationally selects NMR-detected TRPM8 dynamics in a manner that quantitatively correlates with chemical structure. The relationship between chemical structure and protein dynamics can be used predictively, where a chemical structure is predictive of dynamics in a latent reduced dimensionality space. Moreover, the robustness of the conformational selection of the dynamic ensemble is evaluated by varying related and divergent chemotypes, signal-to-noise sensitivity, and sample bias. Taken together, this study identifies that protein dynamics can serve as a quantifiable bridge between chemical structure and cellular function, which has implications for drug discovery in difficult systems.

biophysics↗

bioRxiv2024

Small-molecule modulators of lipid raft stability and protein-raft partitioning

Development of an understanding of membrane nanodomains colloquially known as "lipid rafts" has been hindered by a lack of pharmacological tools to manipulate rafts and protein affinity for rafts. We screened 24,000 small molecules for modulators of the affinity of peripheral myelin protein 22 (PMP22) for rafts in giant plasma membrane vesicles (GPMVs). Hits were counter-screened against another raft protein, MAL, and tested for impact on raft, leading to two classes of compounds. Class I molecules altered the raft affinity of PMP22 and MAL and also reduced raft formation in a protein-dependent manner. Class II molecules modulated raft formation in a protein-independent manner. This suggests independent forces work collectively to stabilize lipid rafts. Both classes of compounds altered membrane fluidity in cells and modulated TRPM8 channel function. These compounds provide new tools for probing lipid raft function in cells and for furthering our understanding of raft biophysics. TeaserCompounds have been discovered that modulate the affinity of membrane proteins for lipid rafts as well as raft formation.

biophysics↗

bioRxiv2022

Uracil-DNA glycosylase efficiency is modulated by substrate rigidity

Uracil DNA-glycosylase (UNG) is a base excision repair enzyme that removes the highly mutagenic uracil lesion from DNA by a base flipping mechanism. UNG excision efficiency depends on DNA sequence, yet the underlying principles that dictate UNG substrate specificity have remained elusive. Here, we show that UNG efficiency is dictated by the intrinsic local deformability of the substrate sequence around the uracil. UNG specificity constants (kcat/KM) and DNA flexibilities were measured for an engineered set of DNA substrates containing AUT, TUA, AUA, and TUA motifs. Time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations of the bare DNA indicated significant differences in substrate flexibilities. A strong correlation between UNG efficiency and substrate flexibility was observed, with higher kcat/KM values measured for more flexible strands. DNA bending and base flipping were observed in simulations, with more frequent uracil flipping observed for the more bendable sequences. Experiments show that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and resultant UNG activity. The finding that substrate flexibility controls UNG efficiency has implications in diverse fields, including the genesis of mutation hotspots, molecular evolution, and understanding sequence preferences of emerging base editors.

biophysics↗

bioRxiv2021

Delivery of recombinant SARS-CoV-2 envelope protein into human cells

SARS-CoV-2 envelope protein (S2-E) is a conserved membrane protein that is essential to coronavirus assembly and budding. Here, we describe the recombinant expression and purification of S2-E into amphipol-class amphipathic polymer solutions. The physical properties of amphipols underpin their ability to solubilize and stabilize membrane proteins without disrupting membranes. Amphipol delivery of S2-E to pre-formed planar bilayers results in spontaneous membrane integration and formation of viroporin ion channels. Amphipol delivery of the S2-E protein to human cells results in membrane integration followed by retrograde trafficking to a location adjacent to the endoplasmic reticulum-to-Golgi intermediate compartment (ERGIC) and the Golgi, which are the sites of coronavirus replication. Delivery of S2-E to cells enables both chemical biological approaches for future studies of SARS-CoV-2 pathogenesis and development of "Trojan Horse" anti-viral therapies. This work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.

biochemistry↗

bioRxiv2019

Evidence that the TRPV1 S1-S4 Membrane Domain Contributes to Thermosensing

Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. However, the role of the S1-S4 domain in thermosensing is unclear. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, Far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.

biochemistry↗