Biology subjects

Naesens, L. M.

Publications and source records attributed to Naesens, L. M..

3 recordsLinked to original sources

bioRxiv2024

Antiviral Mechanisms and Preclinical Evaluation of Amantadine Analogs that Continue to Inhibit Influenza A Viruses with M2 S31N-Based Drug Resistance

To better manage seasonal and pandemic influenza infections, new drugs are needed with enhanced activity against contemporary amantadine- and rimantadine-resistant influenza A virus (IAV) strains containing the S31N variant of the viral M2 ion channel (M2S31N). Here we tested 36 amantadine analogs against a panel of viruses containing either M2S31N or the parental, M2 S31 wild-type variant (M2WT). We found that several analogs, primarily those with sizeable lipophilic adducts, inhibited up to three M2S31N-containing viruses with activities at least 5-fold lower than rimantadine, without inhibiting M2S31N proton currents or modulating endosomal pH. While M2WT viruses in passaging studies rapidly gained resistance to these analogs through the established M2 mutations V27A and/or A30T, resistance development was markedly slower for M2S31N viruses and did not associate with additional M2 mutations. Instead, a subset of analogs, exemplified by 2-propyl-2-adamantanamine (38), but not 2-(1-adamantyl)piperidine (26), spiro[adamantane-2,2-pyrrolidine] (49), or spiro[adamantane-2,2-piperidine] (60), inhibited cellular entry of infectious IAV following pre-treatment and/or H1N1 pseudovirus entry. Conversely, an overlapping subset of the most lipophilic analogs including compounds 26, 49, 60, and others, disrupted viral M2-M1 protein colocalization required for intracellular viral assembly and budding. Finally, a pilot toxicity study in mice demonstrated that 38 and 49 were tolerated at doses approaching those of amantadine. Together, these results indicate that amantadine analogs act on multiple, complementary mechanisms to inhibit replication of M2S31N viruses. Highlights- Current IAVs have M2 mutations that confer resistance to amantadine and rimantadine - Several amantadine analogs inhibit these viruses without acting on M2 proton currents - Alternative antiviral targets include IAV entry and M2-M1 protein colocalization - Amantadine analogs are also tolerated in mice - Future amantadine antivirals could simultaneously act on multiple IAV mechanisms

microbiology↗

bioRxiv2024

A guanidine-based coronavirus replication inhibitor which targets the nsp15 endoribonuclease and combines a direct antiviral effect with subtle boosting of interferon

The approval of COVID-19 vaccines and antiviral drugs has been crucial to end the global health crisis caused by SARS-CoV-2. However, to prepare for future outbreaks from drug-resistant variants and novel zoonotic coronaviruses (CoVs), additional therapeutics with a distinct antiviral mechanism are needed. Here, we report a novel guanidine-substituted diphenylurea compound that suppresses CoV replication by interfering with the uridine-specific endoribonuclease (EndoU) activity of the viral non-structural protein-15 (nsp15). This compound, designated EPB-113, exhibits strong and selective cell culture activity against human coronavirus 229E (HCoV-229E) and also suppresses the replication of SARS-CoV-2. Viruses, selected under EPB-113 pressure, carried resistance sites at or near the catalytic His250 residue of the nsp15-EndoU domain. Although the best-known function of EndoU is to avoid induction of type I interferon (IFN-I) by lowering the levels of viral dsRNA, EPB-113 was found to mainly act via an IFN-independent mechanism, situated during viral RNA synthesis. Using a combination of biophysical and enzymatic assays with the recombinant nsp15 proteins from HCoV-229E and SARS-CoV-2, we discovered that EPB-113 enhances the EndoU cleavage activity of hexameric nsp15, while reducing its thermal stability. This mechanism explains why the virus escapes EPB-113 by acquiring catalytic site mutations which impair compound binding to nsp15 and abolish the EndoU activity. Since the EPB-113-resistant mutant viruses induce high levels of IFN-I and its effectors, they proved unable to replicate in human macrophages and were readily outcompeted by the wild-type virus upon co-infection of human fibroblast cells. Our findings suggest that antiviral targeting of nsp15 can be achieved with a molecule that induces a conformational change in this protein, resulting in higher EndoU activity and impairment of viral RNA synthesis. Based on the appealing mechanism and resistance profile of EPB-113, we conclude that nsp15 is a challenging but highly relevant drug target. Author summaryDespite major achievements in ending the global COVID-19 crisis through vaccines and antiviral drugs, SARS-CoV-2 infection remains a serious health threat for vulnerable individuals, such as elderly and immunocompromised patients. These populations could benefit from innovative treatments that target the virus from multiple angles. In this study, we focus on the nsp15 endoribonuclease (EndoU), a poorly understood coronavirus protein that aids in viral evasion from the host innate immune response and is also assumed to regulate viral RNA synthesis. Our research centers around a newly identified, first-in-class inhibitor of coronavirus nsp15, designated EPB-113. Although nsp15 is best-known for its function in interferon evasion, EPB-113 mainly acts via an interferon-independent mechanism that is situated during viral RNA synthesis. When incubated with nsp15 protein, EPB-113 induces a structural change and enhances its EndoU enzyme activity. EPB-113 selects for escape mutants that carry substitutions in the core of EndoU, leading to a severe replication defect. Hence, our study introduces a novel drug concept to disturb the multifunctional nsp15 protein and suppress coronavirus replication on multiple fronts.

microbiology↗

bioRxiv2019

Evidence for influenza B virus hemagglutinin adaptation to the human host: high cleavability, acid-stability and preference for cool temperature

Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared seasonal IAV (A/H1N1 and A/H3N2) and IBV viruses (B/Victoria and B/Yamagata lineage) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA, by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved present in human respiratory epithelium. Compared to IAV, the IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency. Accordingly, knockdown of a single protease, TMPRSS2, was sufficient to abrogate spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH proved similar for IBV and human-adapted IAVs (one exception being HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33{degrees}C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host-adaptation during prolonged circulation of this respiratory virus in the human population.\n\nImportanceInfluenza epidemics are caused by influenza A (IAV) and influenza B (IBV) viruses. IBV causes substantial disease, however it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We here resolve how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid-stability and a preference for 33{degrees}C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans, and may have broader relevance for understanding the biology and evolution of respiratory viruses.

microbiology↗