Biology subjects

Romigh, T.

Publications and source records attributed to Romigh, T..

5 recordsLinked to original sources

bioRxiv2025

Fragment-Based Development of NSP14 Exonuclease Inhibitors Confounded by Batch-to-Batch Variability

Point mutations in the exonuclease (ExoN) site of non-structural protein 14 (NSP14) compromise the fitness of betacoronaviruses like SARS-CoV-2, implicating NSP14 ExoN inhibition as an antiviral strategy. However, there are no advanced compounds that inhibit NSP14s ExoN activity. Building upon the reported crystal structures of two fragments bound to NSP14s ExoN site, we identified a series of 3,5-disubsituted pyrazoles that bound to and inhibited NSP14 ExoN. However, upon resynthesis, we discovered that these putative leads were false positives, perhaps due to contaminating divalent cations which potently inhibit NSP14 ExoN. Our results provide a cautionary tale to the field about the sensitivity of NSP14 to divalent cations and illustrate the challenges associated with directly targeting the NSP14 ExoN site via fragment merging.

biochemistry↗

bioRxiv2025

Development and Characterization of Triazole-Based WDR5 Inhibitors for the Treatment of Glioblastoma

Glioblastoma (GBM) cancer stem cells (CSCs) contribute to tumor recurrence, treatment resistance, and dismal clinical outcomes. Genetic and pharmacological evidence suggests that the nuclear scaffolding protein WD-repeat containing protein 5 (WDR5) is a therapeutic vulnerability of the CSC population. However, previously reported WDR5 inhibitors display low permeability and are unable to penetrate the blood-brain barrier (BBB), limiting their utility in GBM. Herein, we report the structure-guided development of a novel series of triazole-based WDR5 WIN-site inhibitors designed to increase passive brain penetration. We identified triazole-based WDR5 inhibitors that are potent, passively permeable, and in some cases more brain penetrant than other scaffolds. We phenotypically assessed our novel WDR5 inhibitors in a panel of patient-derived CSC models and uncovered unique WDR5-regulated metabolic genes in GBM. We also evaluated their antiproliferative activity against CSCs both in vitro and in vivo. Finally, to identify novel combination opportunities, we screened a 2,100-compound chemical probe library and identified that the ATAD2 inhibitor BAY-850 synergizes with WDR5 inhibitors to enhance CSC killing. Our work diversifies the chemical matter targeting WDR5, clarifies the in vitro consequences of WIN-site inhibition in CSCs, and encourages the future development of next-generation WDR5 inhibitors with the potential to achieve in vivo efficacy in the brain.

cancer biology↗

bioRxiv2025

Mechanism and Structure-Guided Optimization of SLC1A1/EAAT3-Selective Inhibitors in Kidney Cancer

Renal Cell Carcinomas (RCCs) depend metabolically on the trimeric sodium-coupled aspartate and glutamate transporter, SLC1A1/EAAT3; however, pharmacologically targeting SLC1A1 is challenging. We determined a cryo-EM structure of human SLC1A1 bound to compound 3e, a recently described SLC1A1-selective bicyclic imidazo[1,211]pyridine-3-amine (BIA) inhibitor. 3e binds a membrane-embedded allosteric pocket accessible only in the apo state, when SLC1A1 is unbound to substrate and sodium, and likely prevents sodium and substrate binding. Moreover, by wedging between the trimerization domain and the substrate-binding transport domain, alongside a cholesterol moiety from the lipid bilayer, 3e blocks SLC1A1s elevator-like movements that are essential for transport. Mutations in this binding pocket abolish 3e interaction and counteract 3es cytotoxicity in RCC cells, confirming on-target activity and explaining SLC1A1 selectivity. A structure-guided medicinal chemistry effort yielded two new SLC1A1-selective BIA derivatives, PBJ1 and PBJ2, with enhanced cytotoxicity resulting from the inhibition of SLC1A1-dependent aspartate, glutamate, and cysteine metabolism.

cancer biology↗

bioRxiv2025

XPC loss-of-function triggers melanomagenesis through CDKN2A downregulation

We identified a novel XPC variant, c.2420+5G>A (XPCvar), in siblings with multiple melanomas, inherited alongside c.779+1G>T, which results in an absent or disrupted protein. However, they did not exhibit significantly higher nucleotide excision repair (NER) deficits compared to their unaffected parents, suggesting an NER-independent tumor suppressor function for XPC. XPC knockdown increased cell proliferation and tumorigenicity in vitro without affecting NER. Single-cell RNA sequencing revealed lower Cdkn2a in Xpcvar/var mouse melanocytes. Additionally, XPC knockdown downregulated CDKN2A in vitro. Furthermore, patient fibroblasts showed decreased p16INK4a, which was rescued by XPC overexpression. ChIP-PCR and luciferase assays confirmed XPC binding to the CDKN2A promoter, initiating transcription. Premature stop codon read-through, with gentamicin, restored XPC and p16INK4A in patient fibroblasts. These suggest that XPC regulates CDKN2A and XPC loss might promote melanomagenesis by downregulating CDKN2A, independent of its NER function. We provide preclinical evidence for potential preventive and/or therapeutic strategies for similarly affected individuals. STATEMENT OF SIGNIFICANCECharacterizing a novel XPC variant identified in patients with melanoma revealed a non-canonical role for XPC in regulating CDKN2A transcription. XPC binding to the CDKN2A promoter is crucial for CDKN2A expression and might suppress melanomagenesis. This expands our understanding of melanomagenesis and suggests potential therapies targeting XPC-mediated regulation of CDKN2A.

cancer biology↗

bioRxiv2021

Redefining the PTEN Promoter: Identification of Two Upstream Transcription Start Regions

Germline mutation of PTEN is causally observed in Cowden syndrome (CS) and is one of the most common genetic causes of autism spectrum disorder (ASD). However, the majority of individuals who present with CS-like clinical features are found to be PTEN-mutation negative. Reassessment of PTEN promoter regulation may help explain abnormal PTEN dosage, as only the minimal promoter and coding regions are currently included in diagnostic PTEN mutation analysis. We reanalyzed the architecture of the PTEN promoter using next-generation sequencing datasets. Specifically, run-on sequencing assays identified two additional TSRs at -2052 and -1907 basepairs from the start of PTEN, thus redefining the PTEN promoter and extending the PTEN 5UTR. The upstream TSRs described are active in cancer cell lines and human cancer and normal tissue. Further, these TSRs can produce novel PTEN transcripts due to the introduction of new splice sites. Evaluation of transcription factor binding specific to the upstream TSRs shows overrepresentation of TFs involved in inflammatory processes. Together, these data suggest that potentially clinically relevant promoter variants upstream of the known promoter may be overlooked in indivduals considered PTEN germline mutation-negative and may also explain lack of PTEN expression in sporadic neoplasias without PTEN somatic structural defects.

genetics↗