Biology subjects

Kieffer, C.

Publications and source records attributed to Kieffer, C..

8 recordsLinked to original sources

bioRxiv2025

Integrative in silico and experimental identification of non-covalent UBE2N inhibitors enhancing PARP inhibitor sensitivity

UBE2N, an E2 ubiquitin-conjugating enzyme, has emerged as a compelling therapeutic target in oncology due to its critical roles in DNA damage repair and NF-{kappa}B signalling. While covalent inhibitors have shown preclinical promise, non-covalent inhibitors offer potential advantages in terms of selectivity and reduced off-target effects. However, structural and mechanistic data for non-covalent UBE2N inhibitors remain scarce. To address this gap, we implemented a dual in silico strategy combining structure-based molecular docking and ligand-based 3D pharmacophore modelling. Screening a home library of [~]19,000 compounds targeting both the ubiquitin-binding and cofactor interfaces of UBE2N, we identified 22 candidates suitable for biological evaluation. Among these, two compounds, CERMN-2 and CERMN-16, emerged as promising non-covalent inhibitors. CERMN-16, structurally related to the natural compound Variabine B (identified through 3D pharmacophore screening), significantly reduced SKOV-3 ovarian cancer cell viability and enhanced their sensitivity to the PARP inhibitor Olaparib. CERMN-2, identified through docking, also demonstrated a synergistic effect with Olaparib and showed low toxicity in normal ovarian epithelial cells. Molecular dynamics simulations indicated distinct binding modes for each compound, consistent with their targeted binding sites. Biophysical experiments revealed weak binding of CERMN-16 to UBE2N, whereas CERMN-2 bound UBE2N in two orthogonal assays (Microscale thermophoresis and Nano differential scanning fluorimetry). CERMN-16, and more importantly CERMN-2, therefore represent promising leads for the development of selective, non-natural, non-covalent UBE2N inhibitors. These results provide new insights into UBE2N inhibition and support further investigation of their mechanisms of action and therapeutic potential in combination cancer therapies. HIGHLIGHTSO_LIDual in silico screening (docking and 3D pharmacophore) identified new non-covalent UBE2N inhibitor candidates. C_LIO_LITwo compounds, CERMN-2 and CERMN-16, displayed synergistic activity with Olaparib in ovarian cancer cells. C_LIO_LIMD simulations revealed distinct, site-specific binding modes for both compounds. C_LIO_LIBiophysical assays confirmed UBE2N binding for CERMN-2, identifying it as a promising non-natural, non-covalent lead. C_LI

cancer biology↗

bioRxiv2024

Seasonal influenza A virus lineages exhibit divergent abilities to antagonize interferon induction and signaling

The circulation of seasonal influenza A viruses (IAVs) in humans relies on effective evasion and subversion of the host immune response. While the evolution of seasonal H1N1 and H3N2 viruses to avoid humoral immunity is well characterized, relatively little is known about the evolution of innate immune antagonism phenotypes in these viruses. Numerous studies have established that only a small subset of infected cells is responsible for initiating the type I and type III interferon (IFN) response during IAV infection, emphasizing the importance of single cell studies to accurately characterize the IFN response during infection. We developed a flow cytometry-based method to examine transcriptional changes in IFN and interferon stimulated gene (ISG) expression at the single cell level. We observed that NS segments derived from seasonal H3N2 viruses are more efficient at antagonizing IFN signaling but less effective at suppressing IFN induction, compared to the pdm2009 H1N1 lineage. We compared a collection of NS segments spanning the natural history of the current seasonal IAV lineages and demonstrate long periods of stability in IFN antagonism potential, punctuated by occasional phenotypic shifts. Altogether, our data reveal significant differences in how seasonal and pandemic H1N1 and H3N2 viruses antagonize the human IFN response at the single cell level.

microbiology↗

bioRxiv2024

Monocyte to macrophage differentiation and changes in cellular redox homeostasis promote cell type-specific HIV latency reactivation

Human Immunodeficiency Virus (HIV) latency regulation in monocytes and macrophages can vary according to signals directing differentiation, polarization, and function. To investigate these processes, we generated an HIV latency model in THP-1 monocytes and showed differential levels of HIV reactivation among clonal populations. Monocyte-to-macrophage differentiation of HIV-infected primary human CD14+ and THP-1 cells induced HIV reactivation and showed that virus production increased concomitant with macrophage differentiation. We applied the HIV-infected THP-1 monocyte-to- macrophage (MLat) model to assess the biological mechanisms regulating HIV latency dynamics during monocyte-to-macrophage differentiation. We pinpointed PKC signaling pathway activation and Cyclin T1 upregulation as inherent differentiation mechanisms that regulate HIV latency reactivation. Macrophage polarization regulated latency, revealing pro-inflammatory M1 macrophages suppressed HIV reactivation while M2 macrophages promoted HIV reactivation. Because macrophages rely on reactive- oxygen species (ROS) to exert numerous cellular functions, we disrupted redox pathways and discovered that inhibitors of the thioredoxin (Trx) system acted as latency promoting agents (LPAs) in T-cells and monocytes, but opposingly acted as latency reversing agents (LRAs) in macrophages. We explored this mechanism with Auranofin, a clinical candidate for reducing HIV reservoirs, and demonstrated Trx reductase (TrxR) inhibition led to ROS induced NF-{kappa}B activity, which promoted HIV reactivation in macrophages, but not in T-cells and monocytes. Collectively, cell type-specific differences in HIV latency regulation could pose a barrier to HIV eradication strategies.

microbiology↗

bioRxiv2022

High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike

Designing prefusion-stabilized SARS-CoV-2 spike is critical for the effectiveness of COVID-19 vaccines. All COVID-19 vaccines in the US encode spike with K986P/V987P mutations to stabilize its prefusion conformation. However, contemporary methods on engineering prefusion-stabilized spike immunogens involve tedious experimental work and heavily rely on structural information. Here, we established a systematic and unbiased method of identifying mutations that concomitantly improve expression and stabilize the prefusion conformation of the SARS-CoV-2 spike. Our method integrated a fluorescence-based fusion assay, mammalian cell display technology, and deep mutational scanning. As a proof-of-concept, this method was applied to a region in the S2 domain that includes the first heptad repeat and central helix. Our results revealed that besides K986P and V987P, several mutations simultaneously improved expression and significantly lowered the fusogenicity of the spike. As prefusion stabilization is a common challenge for viral immunogen design, this work will help accelerate vaccine development against different viruses.

biochemistry↗

bioRxiv2022

Probing the biophysical constraints of SARS-CoV-2 spike N-terminal domain using deep mutational scanning

Increasing the expression level of the SARS-CoV-2 spike (S) protein has been critical for COVID-19 vaccine development. While previous efforts largely focused on engineering the receptor-binding domain (RBD) and the S2 subunit, the N-terminal domain (NTD) has been long overlooked due to the limited understanding of its biophysical constraints. In this study, the effects of thousands of NTD single mutations on S protein expression were quantified by deep mutational scanning. Our results revealed that in terms of S protein expression, the mutational tolerability of NTD residues was inversely correlated with their proximity to the RBD and S2. We also identified NTD mutations at the interdomain interface that increased S protein expression without altering its antigenicity. Overall, this study not only advances the understanding of the biophysical constraints of the NTD, but also provides invaluable insights into S-based immunogen design.

biochemistry↗

bioRxiv2020

In vitro characterization of engineered red blood cells as potent viral traps against HIV-1 and SARS-CoV-2

Engineered red blood cells (RBCs) expressing viral receptors could be used therapeutically as viral traps as RBCs lack nuclei and other organelles required for viral replication. Here we show that the combination of a powerful erythroid-specific expression system and transgene codon optimization yields high expression levels of the HIV-1 receptors CD4 and CCR5, as well as a CD4-glycophorin A (CD4-GpA) fusion protein on enucleated RBCs. Engineered RBCs expressing CD4 and CCR5 were efficiently infected by HIV-1, but CD4 or CD4-GpA expression in the absence of CCR5 was sufficient to potently neutralize HIV-1 in vitro. To facilitate continuous large-scale production of engineered RBCs, we generated erythroblast cell lines stably expressing CD4-GpA or ACE2-GpA fusion proteins, which produced potent RBC viral traps against HIV-1 and SARS-CoV-2. Our results suggest that this approach warrants further investigation as a potential treatment against viral infections.

bioengineering↗

bioRxiv2020

Deletion of intestinal SHP impairs short-term response to cholic acid challenge in mice.

Small heterodimer partner (SHP) is a crucial regulator of bile acid (BA) transport and synthesis; however, its intestine-specific role is not fully understood. Here, we report that Intestine-specific Shp knockout (IShpKO) mice have higher intestinal and hepatic BAs, but not serum BAs when challenged with an acute (5-day) 1% cholic acid (CA) diet. Consistent with this finding, BA synthetic genes Cyp7A1 and Cyp8b1 are not repressed to the same extent in IShpKO compared to control mice post-CA challenge. Loss of intestinal SHP did not alter Fxr mRNA but increased Asbt (BA ileal uptake transporter) and Ost (BA ileal efflux transporter) expression even under chow-fed conditions. Surprisingly, the acute CA diet in IShpKO did not elicit the expected induction of Fgf15 but was able to maintain the suppression of Asbt, and Ost/{beta} mRNA levels. At the protein level, ASBT was downregulated, while OST/{beta} expression was induced and maintained regardless of diet. Examination of ileal histology in IShpKO mice challenged with acute CA diet revealed reduced villus length and goblet cell numbers. However, no difference in goblet cell number, villus morphology, crypt depth, and the expression of BA regulator and transporter genes was seen between f/f Shp and IShpKO mice after chronic (14-day) CA diet suggesting an adaptive response. We found the upregulation of the Ppar-Ugt axis, which can reduce the BA burden and compensate for the ileal SHP function. Thus, our study reveals that ileal SHP expression contributes to both overall intestinal structure and BA homeostasis.

physiology↗

bioRxiv2019

Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

Non-invasive bioluminescent imaging (NIBLI) of HIV-1 infection dynamics allows for real-time monitoring of viral spread and the localization of infected cell populations in living animals. In this report, we describe full-length replication-competent GFP and Nanoluciferase (Nluc) expressing HIV-1 reporter viruses from two clinical transmitted / founder (T/F) stains: TRJO.c and Q23.BG505. By infecting humanized mice with these HIV-1 T/F reporter viruses, we were able to directly monitor longitudinal viral spread at whole-animal resolution via NIBLI at a sensitivity of as few as 30-50 infected cells. Bioluminescent signal strongly correlated with HIV-1 infection and responded proportionally to virus suppression in vivo in animals treated daily with a combination antiretroviral therapy (cART) regimen. Longitudinal NIBLI following cART withdrawal visualized tissue-sites that harbored virus during infection recrudescence. Notably, we observed rebounding infection in the same lymphoid tissues where infection was first observed prior to ART treatment. Our work demonstrates the utility of our system for studying in vivo viral infection dynamics and identifying infected tissue regions for subsequent analyses.\n\nAuthor SummaryNon-invasive bioluminescent imaging (NIBLI) in small animals allows for in vivo longitudinal imaging of infection spread and pathogenesis. We have taken advantage of the small luciferase reporter protein, Nanoluciferase (Nluc), to generate a replication-competent HIV-1 reporter virus to allow for NIBLI of viral infection in humanize mice. NIBLI via Nluc enabled us to directly visualize longitudinal spreading patterns before, during, and after interruption of daily doses of combined antiretroviral therapy (cART). We observed that rebounding infection often emerged in tissue regions originally associated with infected cells prior to cART treatment. Thus, Nluc-based NIBLI of HIV-1 infection can be used as an experimental tool to study early events involved in viral dissemination and spread from initial sites of infection to draining lymphoid tissues as well as locate infected tissues for subsequent cellular characterization of HIV-1 infected cells.

microbiology↗