Biology subjects

Amblard, F.

Publications and source records attributed to Amblard, F..

5 recordsLinked to original sources

bioRxiv2025

Broad-Spectrum Antiviral Efficacy of 7-Deaza-7-Fluoro-2'-C-Methyladenosine Against Multiple Coronaviruses In Vitro and In Vivo

The Coronaviridae family has been implicated in several major epidemics over the past two decades, including those caused by SARS-CoV, MERS-CoV, and, most recently, SARS-CoV-2. The COVID-19 pandemic, driven by SARS-CoV-2, has led to over seven million deaths worldwide and has been associated with prolonged symptoms, chronic sequelae, and substantial socioeconomic disruptions. The limited availability of effective antiviral treatments, coupled with the ability of coronaviruses to mutate and evade immune defenses, underscores the urgent need for innovative antiviral agents. This study explores the efficacy of the nucleoside analogue DFMA as a potential antiviral agent against multiple Coronaviridae family members, including SARS-CoV-2 and two strains of murine hepatitis viruses (MHV-3 and MHV-A59). In vitro analyses demonstrated that DFMA effectively reduced the viral load in the supernatant of infected cells and enhanced cell viability for both MHV-3 and MHV-A59. Against SARS-CoV-2, DFMA showed a significant reduction in viral load, with a calculated Selectivity Index (SI) of 6.2. In vivo investigations further confirmed the antiviral potential of DFMA. In three distinct murine models--a severe COVID-19 model using MHV-3, a mild COVID-19 model employing MHV-A59, and a transgenic K18-hACE2 mouse model infected with SARS-CoV-2--DFMA administration significantly reduced viral loads in the lungs of infected mice. Additionally, DFMA mitigated inflammatory responses in all models by lowering levels of key inflammatory mediators, such as CXCL1, CCL2, and IL-6. These findings suggest that DFMA possesses broad-spectrum antiviral activity against coronaviruses and may serve as a promising therapeutic candidate for current and future coronavirus outbreaks. Further research is warranted to elucidate its mechanism of action and evaluate its efficacy in clinical settings. ImportanceCoronaviruses have caused significant outbreaks over the past two decades. Since 2020, COVID-19 has resulted in millions of deaths and lasting global impacts. The limited availability of effective antivirals and the viruss ability to mutate and evade vaccines and monoclonal antibody therapy emphasize the urgent need for new treatments. This study investigates DFMA, a promising antiviral candidate, targeting SARS-CoV-2 and two related coronaviruses.Our promising results demonstrated significant antiviral activity of DFMA, not only against SARS-CoV-2 but also against other similar coronaviruses, indicating potential future use against COVID-19 and other possible coronavirus-related diseases.

microbiology↗

bioRxiv2024

The structural and mechanistic bases for the viral resistance to allosteric HIV-1 integrase inhibitor pirmitegravir

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are investigational antiretroviral agents which potently impair virion maturation by inducing hyper-multimerization of IN and inhibiting its interaction with viral genomic RNA. The pyrrolopyridine-based ALLINI pirmitegravir (PIR) has recently advanced into Phase 2a clinical trials. Previous cell culture based viral breakthrough assays identified the HIV-1(Y99H/A128T IN) variant that confers substantial resistance to this inhibitor. Here, we have elucidated the unexpected mechanism of viral resistance to PIR. While both Tyr99 and Ala128 are positioned within the inhibitor binding V-shaped cavity at the IN catalytic core domain (CCD) dimer interface, the Y99H/A128T IN mutations did not substantially affect direct binding of PIR to the CCD dimer or functional oligomerization of full-length IN. Instead, the drug-resistant mutations introduced a steric hindrance at the inhibitor mediated interface between CCD and C-terminal domain (CTD) and compromised CTD binding to the CCDY99H/A128T + PIR complex. Consequently, full-length INY99H/A128T was substantially less susceptible to the PIR induced hyper-multimerization than the WT protein, and HIV-1(Y99H/A128T IN) conferred >150- fold resistance to the inhibitor compared to the WT virus. By rationally modifying PIR we have developed its analog EKC110, which readily induced hyper-multimerization of INY99H/A128T in vitro and was [~]14-fold more potent against HIV-1(Y99H/A128T IN) than the parent inhibitor. These findings suggest a path for developing improved PIR chemotypes with a higher barrier to resistance for their potential clinical use. IMPORTANCEAntiretroviral therapies save the lives of millions of people living with HIV (PLWH). However, evolution of multi-drug-resistant viral phenotypes is a major clinical problem, and there are limited or no treatment options for heavily treatment-experienced PLWH. Allosteric HIV-1 integrase inhibitors (ALLINIs) are a novel class of antiretroviral compounds which work by a unique mechanism of binding to the non-catalytic site on the viral protein and inducing aberrant integrase multimerization. Accordingly, ALLINIs potently inhibit both wild type HIV-1 and all drug-resistant viral phenotypes that have so far emerged against currently used therapies. Pirmitegravir, a highly potent and safe investigational ALLINI, is currently advancing through clinical trials. Here we have elucidated structural and mechanistic bases behind the emergence of HIV-1 integrase mutations in infected cell that confer resistance to pirmitegravir. In turn, our findings allowed us to rationally develop an improved ALLINI with substantially enhanced potency against the pirmitegravir resistant virus.

microbiology↗

bioRxiv2024

Stably-Inverted Apical-Out Human Upper Airway Organoids for SARS-CoV-2 Infection and Therapeutic Testing

While breakthroughs with organoids have emerged as next-generation in vitro tools, standardization for drug discovery remains a challenge. This work introduces human airway organoids with reversed biopolarity (AORBs), cultured and analyzed in a high-throughput, single-organoid-per-well format, enabling milestones towards standardization. AORBs exhibit a spatio-temporally stable apical-out morphology, facilitating high-yield direct intact-organoid virus infection. Single-cell RNA sequencing and immunohistochemistry confirm the physiologically relevant recapitulation of differentiated human airway epithelia. The cellular tropism of five severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains along with host response differences between Delta, Washington, and Omicron variants, as observed in transcriptomic profiles, also suggest clinical relevance. Dose-response analysis of three well-studied SARS-CoV-2 antiviral compounds (remdesivir, bemnifosbuvir, and nirmatrelvir) demonstrates that AORBs efficiently predict human efficacy, comparable to gold-standard air-liquid interface cultures, but with higher throughput ([~]10-fold) and fewer cells ([~]100-fold). This combination of throughput and relevance allows AORBs to robustly detect false negative results in efficacy, preventing irretrievable loss of promising lead compounds. While this work leverages the SARS-CoV-2 study as a proof-of-concept application, the standardization capacity of AORB holds broader implications in line with regulatory efforts to push alternatives to animal studies.

bioengineering↗

bioRxiv2023

N4-Hydroxycytidine/Molnupiravir Inhibits RNA-Virus Induced Encephalitis by Producing Mutated Viruses with Reduced Fitness

A diverse group of RNA viruses including Rabies, Polio, La Crosse, West Nile, Zika, Nipah, Eastern and Western equine encephalitis, Venezuelan equine encephalitis, Japanese encephalitis, and tick-borne encephalitis viruses have the ability to gain access to and replicate in the central nervous system (CNS), causing severe neurological disease. Current treatment for these patients is generally limited to supportive care. To address the need for a generalizable antiviral, we utilized a strategy of mutagenesis to limit virus replication. We evaluated ribavirin (RBV), favipiravir (FAV) and N4-hydroxycytidine (NHC) against La Crosse virus (LACV) which is the primary cause of pediatric arboviral encephalitis cases in North America. NHC was more potent than RBV or FAV in neuronal cells. Oral administration of molnupiravir (MOV), the 5-isobutyryl prodrug of NHC, decreased neurological disease development by 32% following intraperitoneal (IP) infection of LACV. MOV also reduced disease by 23% when virus was administered intranasally (IN). NHC and MOV produced less fit viruses by incorporating predominantly G-to-A or C-to-U mutations. Furthermore, NHC also inhibited two other orthobunyaviruses, Jamestown Canyon virus and Cache Valley virus. Collectively, these studies indicate that NHC/MOV has therapeutic potential to inhibit virus replication and subsequent neurological disease caused by this neurotropic RNA virus.

microbiology↗

bioRxiv2021

Dynlt3, a novel fundamental regulator of melanosome movement, distribution, maturation and transfer

Skin pigmentation is dependent on cellular processes including melanosome biogenesis, transport, maturation and transfer to keratinocytes. However, how the cells finely control these processes in space and time to ensure proper pigmentation remains unclear. Here, we show that a component of the cytoplasmic dynein complex, Dynlt3, is required for efficient melanosome transport, maturation and transfer. In melanocytes with decreased levels of Dynlt3, pigmented melanosomes undergo a more directional (convective) motion leading to their peripheral location in the cell, and are not fully matured. Stage IV melanosomes are more acidic, but still heavily pigmented, resulting in a less efficient melanosome transfer. Finally, the level of Dynlt3 is dependent on {beta}-catenin activity, revealing a novel function of the Wnt/{beta}-catenin signalling pathway during melanocyte and skin pigmentation, by coupling the transport, position and maturation of melanosomes required for their transfer.

cell biology↗