Biology subjects

Pokhrel, R.

Publications and source records attributed to Pokhrel, R..

5 recordsLinked to original sources

bioRxiv2026

Single-Cell Transcriptomic Mapping of PD-L1/TLR4 Remodeling Informs Topical Immunoprevention Timing in Skin Carcinogenesis

Cutaneous squamous cell carcinoma (cSCC) represents a growing public health burden, with incidence projected to increase 23-29% over the coming decade. Topical immunoprevention strategies targeting the PD-L1/PD-1 and TLR4 axes have demonstrated preclinical efficacy, yet optimal intervention timing in humans remains undefined. To address this gap, single-cell RNA sequencing was performed on matched sun-protected (SP), sun-damaged (SD), and actinic keratosis (AK) biopsies from the same individuals, along with independent cSCC cases. Immune checkpoint and innate inflammatory signals were detectable as early as SD skin, prior to histologically confirmed dysplasia. Monotonically increasing expression of CD274 (PD-L1), CTLA4, PDCD1, CD27, and STAT1, alongside progressive TLR4-MYD88 innate immune signaling, was revealed through pseudobulk data analysis, with earliest upregulation at the SD stage. Fuzzy c-means trajectory clustering identified cell-typespecific programs across dendritic cells, macrophages, T cells, fibroblasts, endothelial cells, and keratinocytes. Dendritic cells shifted from early inflammatory antigen-presenting programs toward late PD-L1/IFN-regulatory states; macrophages showed monotonically increasing TLR4-associated myeloid activation; and T cells defined a "hot but exhausted" microenvironment in established cSCC. These findings identify SD and AK as biologically active stages for topical immunoprevention and provide a cellular roadmap for PD-L1/PD-1 and TLR4 blockade strategies.

cancer biology↗

bioRxiv2026

Monocyte Migration Emerges from a Divergent Chemokine Signaling Network

Migration of leukocytes in the context of immune homeostasis or inflammatory diseases is regulated by activation of chemokine receptors by chemokine ligands. To elucidate how these interactions give rise to cell migration, we mapped the chemokine-stimulated signal transduction network in monocytic THP-1 cells. Global phosphoproteomics revealed 630 time-resolved changes in phosphorylated proteins downstream of the chemokine receptor CCR2. We used the "PHONEMeS" network modeling algorithm to generate the most parsimonious signal transduction network consistent with the observed protein phosphorylation data. The CCR2 signaling network is highly divergent, acting via multiple branches to regulate proteins required for cell migration. We validated this model using kinase inhibitors targeting different branches of the network and successfully blocked chemokine-stimulated cell migration. Thus, chemotaxis is an emergent property resulting from an integrated cellular response to divergent signaling pathways. This paradigm suggests that physiological regulation or pharmacological blockade of chemokine-driven inflammation could potentially be achieved by inhibiting any of the divergent pathways within the network.

systems biology↗

bioRxiv2026

Targeting of subcellular metabotropic glutamate receptor 5 signaling to modulate pain transmission

Metabotropic glutamate receptor 5 (mGlu5) is a class C GPCR crucial for neuronal development and synaptic transmission. mGlu5 is a potential therapeutic target in pain management and modulates pain-associated gene expression and signaling pathways. Although mGlu5 inhibitors have shown promise in treating pain, none have translated to the clinic. Up to 90% of neuronal mGlu5 expression is intracellular, although the precise locations and function of different mGlu5 intracellular pools remains unclear. Building on recent evidence showing the importance of endosome-mediated nociceptive signaling by other GPCRs, we hypothesized that endosomal pools of mGlu5 contribute to pain transmission, and that targeted inhibition of intracellular mGlu5 signaling results in superior analgesia. Using calcium mobilization assays and genetically encoded resonance energy transfer biosensors, we report that upon its activation mGlu5 recruits Gq/11 and Gs to the plasma membrane. Conversely, internalized mGlu5 in endosomes recruits only Gq/11 proteins. mGlu5 signaling is highly dependent on receptor trafficking to endosomes, with sustained nuclear ERK1/2 signaling requiring both receptor internalization and active glutamate transport into the cell. We generated pH responsive nanoparticles loaded with the mGlu5 negative allosteric modulator VU0366058 (DIPMA-VU058), enabling endosome-targeted inhibition of mGlu5. Nanoparticle encapsulation of VU0366058 enhanced inhibition of both acute and sustained nuclear ERK1/2 signaling, and significantly reduced neuronal excitability in nociceptive circuits in spinal cord slices from rats with neuropathic pain. Intrathecal administration of DIPMA-VU058 achieved superior analgesia in both inflammatory and neuropathic models of pain in mice compared to free VU0366058 and the reference compound fenobam. These studies demonstrate the importance of endosome-associated receptors for the complete mGlu5 signaling response. Furthermore, we show that manipulating the cellular distribution of an allosteric modulator can engender location-biased pharmacological effects. Together, we have revealed new and unappreciated roles for endosome-specific mGlu5 signaling and demonstrate that endosome-selective targeting may offer an alternative therapeutic approach for modulating mGlu5 activity.

pharmacology and toxicology↗

bioRxiv2025

Identification of novel human topoisomerase III beta inhibitors

Human topoisomerase III beta (TOP3B) is a type IA topoisomerase that can change the topology of DNA and RNA substrates via a phosphotyrosine covalent intermediate. TOP3B has been shown to be required for the efficient replication of certain positive-sense ssRNA viruses including Dengue. We applied molecular dynamics simulation combined with docking studies to identify potential inhibitors of TOP3B from a library comprised of drugs that are FDA-approved or undergoing clinical trials for potential drug repurposing. Topoisomerase activity assay of the top virtual screening hits showed that bemcentinib, a compound known to target the AXL receptor tyrosine kinase, can inhibit TOP3B relaxation activity. Additional small molecules that share the N5,N3-1H-1,2,4-triazole-3,5-diamine moiety of bemcentinib were synthesized and tested for inhibition of TOP3B relaxation activity. Five of these molecules showed comparable IC50 as bemcentinib for inhibition of TOP3B. However, these five molecules had less selectivity towards TOP3B inhibition versus bemcentinib when inhibition of the type IB human topoisomerase I was com-pared. These results suggest that exploration of tyrosine kinase inhibitors and their analogs may allow the identification of novel topoisomerase inhibitors.

biochemistry↗

bioRxiv2021

Molecular assembly of measles and Nipah virus: specific lipid binding drives conformational change and matrix polymerization

Measles virus, Nipah virus, and multiple other paramyxoviruses cause disease outbreaks in humans and animals worldwide. The paramyxovirus matrix (M) protein mediates virion assembly and budding from host cell membranes. M is thus a key target for antivirals, but few high-resolution structures of paramyxovirus M are available, and we lack the clear understanding of how viral M proteins interact with membrane lipids to mediate viral assembly and egress needed to guide antiviral design. Here, we reveal that M proteins associate with phosphatidylserine and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane. Using X-ray crystallography, electron microscopy, and molecular dynamics we demonstrate that PI(4,5)P2 binding induces conformational and electrostatic changes in the M protein surface that trigger membrane deformation, matrix layer polymerization, and virion assembly.

microbiology↗