Biology subjects

Jochmans, D.

Publications and source records attributed to Jochmans, D..

At least 19 recordsLinked to original sources

bioRxiv2025

Discovery of orally bioavailable Zika and West Nile Virus antiviral compounds targeting the NS2B-NS3 protease

Flaviviruses are a class of pathogenic viruses with pandemic potential that are typically transmitted via infected arthropods. In addition, Zika virus is sexually transmissible and causes congenital malformations if infection occurs during pregnancy. Although over 1.5 million people were infected during the 2015-2016 Zika outbreak, to date, there are no clinical-stage vaccines or antivirals. Herein, we report the discovery of potent inhibitors of the Zika virus NS2B-NS3 protease that also show activity against the West Nile virus NS2B-NS3 protease. Starting from a crystallographic fragment screen, we employed a pharmacophore approach coupled with high-throughput library chemistry to elaborate fragments in the active site. Potent, metabolically stable, non-covalent, non-peptidomimetic inhibitors were identified with antiviral activity in vitro. The lead compound was progressed to a three-day Zika virus challenge study in AG129 mice, where it displayed significant viral RNA load reduction in both mouse plasma and spleen.

microbiology↗

bioRxiv2025

Open-science discovery of DNDI-6510, a compound that addresses genotoxic and metabolic liabilities of the COVID Moonshot SARS-CoV-2 Mpro lead inhibitor

The 2020 SARS-CoV-2 coronavirus pandemic highlighted the urgent need for novel small molecule antiviral drugs. (S)-x38 DNDI-6510 is a non-covalent SARS-CoV-2 main protease inhibitor developed by the open science collaboration COVID Moonshot. Here, we report on the metabolic and toxicologic optimization of the lead series previously disclosed by the COVID Moonshot Initiative, leading up to the selection of (S)-x38 DNDI-6510 as the preclinical candidate. We describe the thorough profiling of the series, identifying key risks such as formation of genotoxic metabolites and high clearance, which were successfully addressed during lead optimization. In addition, we disclose the in vitro and in vivo evaluation of (S)-x38 DNDI-6510 in pharmacokinetic and pharmacodynamic models, exploring multiple approaches to ameliorate rodent-specific metabolic clearance, and show that both co-dosing of (S)-x38 DNDI-6510 with an ABT inhibitor and utilizing a metabolically humanized mouse model (8HUM) achieve significant improvements in exposure. Through comparisons of ABT co-dosing and humanized mouse models in efficacy experiments, we demonstrate that continuous exposure over cellular EC90 is required for SARS-CoV-2 antiviral efficacy in vivo in an antiviral model using a mouse-adapted SARS-CoV-2 strain. Finally, (S)-x38 DNDI-6510 was assessed in maximum tolerated dose experiments in two species, demonstrating significant in vivo PXR-linked auto-induction of metabolism, leading to the discontinuation of this compound. In summary, we report the successful effort to overcome series-specific AMES liabilities in a lead development program. Downstream optimization of existing series will require in-depth optimization of rodent-specific liabilities and metabolic induction profile.

pharmacology and toxicology↗

bioRxiv2025

Human Myelin Spheres for In Vitro Oligodendrocyte Maturation, Myelination and Neurological Disease Modeling

Demyelinating diseases, such as multiple sclerosis, damage the protective myelin sheaths of the central nervous system. The development of effective therapies has been hampered by the lack of models that accurately replicate human myelin biology. Here we present a novel method to generate human myelin spheres (MyS) by coculturing of hPSC-derived neuronal and oligodendrocyte precursor cells, to create myelinated neurons. Using multimodal analyses including confocal and (electron)microscopy, single-nuclei transcriptomics, lipidomics, and electrophysiology, we demonstrate myelination in MyS as early as six weeks into coculture. These myelinated structures mature over time into multilamellar and compacted myelin sheaths with lipid compositions and transcriptomic profiles mirror the temporal dynamics of in vivo human oligodendrocyte development and neuronal myelination, resembling those of late fetal/postnatal oligodendrocytes. By employing lysolecithin-induced demyelination and Rabies virus infection experiments, we demonstrate the potential of MyS as an innovative, physiologically relevant platform for studying myelin-related neurodegeneration and neuroinfection.

neuroscience↗

bioRxiv2025

The combination of GS-441524 (remdesivir) and ribavirin results in a potent antiviral effect against human parainfluenza virus 3 infection in human airway epithelial cell cultures and in a mouse infection model

Human parainfluenza virus type 3 (HPIV-3) can cause severe respiratory diseases, particularly in young children, the elderly and immunocompromised. There are no approved antiviral drugs against this virus. We report that the combination of ribavirin with either remdesivir or its parent nucleoside GS-441524 results in a pronounced antiviral effect against HPIV-3 in LLC-MK2 cells and in human airway epithelial cells grown at the air-liquid interface. In AG129 mice intranasally inoculated with HPIV-3, the combined treatment of ribavirin and GS-441524 decreases infectious viral lung titers by >2.5 log10 to undetectable levels in 4 out of 11 mice and by 1.6 log10 in the remaining 7 mice as compared with vehicle. The lungs of all mice that received the combined treatment appear histologically normal or virtually normal, whereas 8 of 11 vehicle-treated mice present with bronchopneumonia. By contrast, ribavirin alone does not result in a reduction of infectious viral lung titers; GS-441524 alone reduces infectious viral lung titers by 1.2 log10. Moreover, several mice in the single-treatment groups exhibit severe lung pathology. These findings may warrant exploring this combination in patients with severe HPIV-3 infections and possibly also against infections with other viruses that are in vitro susceptible to these two drugs.

microbiology↗

bioRxiv2024

The triple combination of Remdesivir (GS-441524), Molnupiravir and Ribavirin is highly efficient in inhibiting coronavirus replication in human nasal airway epithelial cell cultures and in a hamster infection model

The use of fixed dose-combinations of antivirals with different mechanisms of action has proven a key in the successful treatment of infections with HIV and HCV. For the treatment of infections with SARS-CoV-2 and possible future epi-/pandemic coronaviruses, it will be important to explore the efficacy of combinations of different drugs, in particular to avoid resistance development, such as in patients with immunodeficiencies. As a first effort, we studied the antiviral potency of combinations of antivirals. To that end, we made use of primary human airway epithelial cell (HAEC) cultures grown at the air-liquid interface that were infected with the beta coronavirus OC43. We found that the triple combination of GS-441524 (parent nucleoside of remdesivir), molnupiravir, and ribavirin resulted in a more pronounced antiviral efficacy than what could be expected from a purely additive antiviral effect. The potency of this triple combination was next tested in SARS-CoV-2 infected hamsters. To that end, for each of the drugs, intentionally suboptimal or even ineffective doses were selected. Yet, in the lungs of all hamsters that received triple prophylactic therapy with suboptimal/inactive doses of GS-441524, molnupiravir, and ribavirin, no infectious virus was detectable. Our finding indicate that co-administration of approved drugs for the treatment of coronavirus infections should be further explored but also against other families of viruses with epidemic and pandemic potential for which no effective antiviral treatment is available.

microbiology↗

bioRxiv2024

PIP4K2C inhibition reverses autophagic flux impairment induced by SARS-CoV-2

In search for broad-spectrum antivirals, we discovered a small molecule inhibitor, RMC-113, that potently suppresses the replication of multiple RNA viruses including SARS-CoV-2 in human lung organoids. We demonstrated selective dual inhibition of the lipid kinases PIP4K2C and PIKfyve by RMC-113 and target engagement by its clickable analog. Advanced lipidomics revealed alteration of SARS-CoV-2-induced phosphoinositide signature by RMC-113 and linked its antiviral effect with functional PIP4K2C and PIKfyve inhibition. We discovered PIP4K2Cs roles in SARS-CoV-2 entry, RNA replication, and assembly/egress, validating it as a druggable antiviral target. Integrating proteomics, single-cell transcriptomics, and functional assays revealed that PIP4K2C binds SARS-CoV-2 nonstructural protein 6 and regulates virus-induced impairment of autophagic flux. Reversing this autophagic flux impairment is a mechanism of antiviral action of RMC-113. These findings reveal virus-induced autophagy regulation via PIP4K2C, an understudied kinase, and propose dual inhibition of PIP4K2C and PIKfyve as a candidate strategy to combat emerging viruses.

microbiology↗

bioRxiv2024

Yeast-based production platform for potent and stable heavy chain-only antibodies

Monoclonal antibodies are the leading drug of the biopharmaceutical market because of their high specificity and tolerability, but the current CHO-based manufacturing platform remains expensive and time-consuming leading to limited accessibility, especially in the case of diseases with high incidence and pandemics. Therefore, there is an urgent need for an alternative production system. In this study, we present a rapid and cost-effective microbial platform for heavy chain-only antibodies (VHH-Fc) in the methylotrophic yeast Komagataella phaffii (aka Pichia pastoris). We demonstrate the potential of this platform using a simplified single-gene VHH-Fc fusion construct instead of the conventional monoclonal antibody format, as this is more easily expressed in Pichia pastoris. We demonstrate that the Pichia-produced VHH-Fc fusion construct is stable and that a Pichia-produced VHH-Fc directed against the SARS-CoV-2 spike has potent SARS-CoV-2 neutralizing activity in vitro and in vivo. We expect that this platform will pave the way towards faster and cheaper development and production of broadly neutralizing single-chain antibodies in yeast.

microbiology↗

bioRxiv2022

Nirmatrelvir-resistant SARS-CoV-2 is efficiently transmitted in Syrian hamsters

The SARS-CoV-2 main protease (3CLpro) is one of the promising therapeutic target for the treatment of COVID-19. Nirmatrelvir is the only the 3CLpro inhibitor authorized for treatment of COVID-19 patients at high risk of hospitalization; other 3Lpro inhibitors are in development. We recently repored on the in vitro selection of a SARS-CoV2 3CLpro (L50F-E166A-L167F; short 3CLprores) virus that is cross-resistant with nirmatrelvir and yet other 3CLpro inhibitors. Here, we demonstrate that the resistant virus replicates efficiently in the lungs of intranassaly infected hamsters and that it causes a lung pathology that is comparable to that caused by the WT virus. Moreover, 3CLprores infected hamsters transmit the virus efficiently to co-housed non-infected contact hamsters. Fortunately, resistance to Nirmatrelvir does not readily develop (in the clinical setting) since the drug has a relatively high barrier to resistance. Yet, as we demonstrate, in case resistant viruses emerge, they may easily spread and impact therapeutic options for others. Therefore, the use of SARS-CoV-2 3CLpro protease inhibitors in combinations with drugs that have a different mechanism of action, may be considered to avoid the development of drug-resistant viruses in the future.

microbiology↗

bioRxiv2022

The substitutions L50F, E166A and L167F in SARS-CoV-2 3CLpro are selected by a protease inhibitor in vitro and confer resistance to nirmatrelvir

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with > 20x increase in EC50 values for ALG-097161, nirmatrelvir (PF-07321332) and PF-00835231. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6x to 72x). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. Abstract ImportancePaxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug resistant viruses. In order to guide the use of novel antivirals it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro. The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next generation SARS-CoV-2 3CLpro inhibitors.

microbiology↗

bioRxiv2022

A SCID mouse model to evaluate the efficacy of antivirals against SARS-CoV-2 infection

Ancestral SARS-CoV-2 lacks the intrinsic ability to bind to the mouse ACE2 receptor and therefore establishment of SARS-CoV-2 mouse models has been limited to the use of mouse-adapted viruses or genetically modified mice. Interestingly, some of the variants of concern, such as the beta B.1.351 variant, show an improved binding to the mouse receptor and hence better replication in different Wild type (WT) mice species. Here, we desribe the establishment of SARS-CoV-2 beta B.1.351 variant infection model in male SCID mice as a tool to assess the antiviral efficacy of potential SARS-CoV-2 small molecule inhibitors. Intranasal infection of male SCID mice with 105 TCID50 of the beta B.1.351 variant resulted in high viral loads in the lungs and moderate signs of lung pathology on day 3 post-infection (pi). Treatment of infected mice with the antiviral drugs Molnupiravir (200 mg/kg, BID) or Nirmatrelvir (300 mg/kg, BID) for 3 consecutive days significantly reduced the infectious virus titers in the lungs by 1.9 and 3.8 log10 TCID50/mg tissue, respectively and significantly improved lung pathology. Together, these data demonstrate the validity of this SCID mice/beta B.1.351 variant infection model as a convenient preclinical model for assessment of potential activity of antivirals against SARS-CoV-2. ImportanceUnlike the ancestral SARS-CoV-2 strain, the beta (B.1.351) VoC has been reported to replicate to some extent in WT mice (species C57BL/6 and BALB/c). We here demonstrate that infection of SCID mice with SARS-CoV-2 beta variant results in high viral loads in the lungs on day 3 post-infection (pi). Treatment of infected mice with the antiviral drugs Molnupiravir or Nirmatrelvir for 3 consecutive days markedly reduced the infectious virus titers in the lungs and improved lung pathology. The advantages of using this mouse model over the standard hamster infection models to assess the in vivo efficacy of small molecule antiviral drugs are (i) the use of a clinical isolate without the need to use mouse-adapted strains or genetically modified animals (ii) lower amount of the test drug is needed and (ii) more convenient housing conditions compared to bigger rodents such as hamsters.

microbiology↗

bioRxiv2022

Ivermectin does not protect against SARS-CoV-2 infection in the Syrian hamster model

Ivermectin, an FDA-approved antiparasitic drug, has been reported to have in vitro activity against SARS-CoV-2. An increasing off-label use of Ivermectin for COVID-19 has been reported. We here assessed the effect of Ivermectin in Syrian hamsters infected with the SARS-CoV-2 Beta (B.1.351) variant. Infected animals received a clinically relevant dose of Ivermectin (0.4 mg/kg subcutaneously dosed) once daily for four consecutive days after which the effect was quantified. Ivermectin monotherapy did not reduce lung viral load and even significantly worsened the SARS-CoV-2-induced lung pathology. Additionally, it did not potentiate the activity of Molnupiravir (Lagevrio) when combined with this drug. This study contributes to the growing body of evidence that Ivermectin does not result in a beneficial effect in the treatment of COVID-19. These findings are important given the increasing, dangerous off-label use of Ivermectin for the treatment of COVID-19.

microbiology↗

bioRxiv2022

Development and optimisation of a high-throughput screening assay for in vitro anti-SARS-CoV-2 activity: evaluation of 5676 phase 1 passed structures

Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19. As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging-based SARS-CoV-2 infection assay was developed in VeroE6-eGFP cells and was used to screen a library of 5676 compounds that passed phase 1 clinical trials. Eight candidates (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) with in vitro anti-SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines were identified. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment.

immunology↗

bioRxiv2021

Remdesivir, Molnupiravir and Nirmatrelvir remain active against SARS-CoV-2 Omicron and other variants of concern.

We assessed the in vitro antiviral activity of remdesivir and its parent nucleoside GS-441524, molnupiravir and its parent nucleoside EIDD-1931 and the viral protease inhibitor nirmatrelvir against the ancestral SARS-CoV2 strain and the five variants of concern including Omicron. VeroE6-GFP cells were pre-treated overnight with serial dilutions of the compounds before infection. The GFP signal was determined by high-content imaging on day 4 post-infection. All molecules have equipotent antiviral activity against the ancestral virus and the VOCs Alpha, Beta, Gamma, Delta and Omicron. These findings are in line with the observation that the target proteins of these antivirals (respectively the viral RNA dependent RNA polymerase and the viral main protease Mpro) are highly conserved.

microbiology↗

bioRxiv2021

Potent neutralizing anti-SARS-CoV-2 human antibodies cure infection with SARS-CoV-2 variants in hamster model

Treatment with neutralizing monoclonal antibodies (mAbs) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contributes to COVID-19 management. Unfortunately, SARS-CoV-2 variants can escape several of these recently approved mAbs, highlighting the need for additional discovery and development. In a convalescent COVID-19 patient, we identified six mAbs, classified in four epitope groups, that potently neutralized SARS-CoV-2 Wuhan, alpha, beta, gamma and delta infection in vitro. In hamsters, mAbs 3E6 and 3B8 potently cured infection with SARS-CoV-2 Wuhan, beta and delta when administered post-viral infection at 5 mg/kg. Even at 0.2 mg/kg, 3B8 still reduced viral titers. Intramuscular delivery of DNA-encoded 3B8 resulted in in vivo mAb production of median serum levels up to 90 g/ml, and protected hamsters against delta infection. Overall, our data mark 3B8 as a promising candidate against COVID-19, and highlight advances in both the identification and gene-based delivery of potent human mAbs.

immunology↗

bioRxiv2021

The oral protease inhibitor (PF-07321332) protects Syrian hamsters against infection with SARS-CoV-2 variants of concern

There is an urgent need for potent and selective antivirals against SARS-CoV-2. Pfizer developed PF-07321332 (PF-332), a potent inhibitor of the viral main protease (Mpro, 3CLpro) that can be dosed orally and that is in clinical development. We here report that PF-332 exerts equipotent in vitro activity against the four SARS-CoV-2 variants of concerns (VoC) and that it can completely arrest replication of the alpha variant in primary human airway epithelial cells grown at the air-liquid interface. Treatment of Syrian Golden hamsters with PF-332 (250 mg/kg, twice daily) completely protected the animals against intranasal infection with the beta (B.1.351) and delta (B.1.617.2) SARS-CoV-2 variants. Moreover, treatment of SARS-CoV-2 (B.1.617.2) infected animals with PF-332 completely prevented transmission to untreated co-housed sentinels.

microbiology↗

bioRxiv2021

The Urtica Dioica Agglutinin Prevents Rabies Virus Infection in a Muscle Explant Model

Infection with the rabies virus (RABV) results, once symptoms develop, in a 100% lethal neurological disease. Post-exposure prophylaxis (PEP), consists of a combination of vaccination and anti-rabies immunoglobulins (RIGs); it is 100% effective if administered early after infection. Because of the limited availability, alternatives for RIGs are needed. To that end we evaluated a panel of 33 different lectins for their effect on RABV infection in cell culture. Several lectins, with either mannose or GlcNAc specificity elicited anti-RABV activity of which the GlcNAc specific Urtica dioica Agglutinin (UDA) was selected for further studies. UDA was found to prevent entry of the virus into the host cell. To further assess the potential of UDA, a physiologically relevant RABV infection muscle explant model was developed. Strips of dissected swine skeletal muscle that were kept in culture medium could be productively infected with the RABV. When the infection of the muscle strips was carried out in the presence of UDA, RABV replication was completely prevented. We thus developed a physiologically relevant RABV muscle infection model. UDA (i) may serve as a reference for further studies and (ii) holds promise as a cheap and simple to produce alternative for RIGs in PEP.

pathology↗

bioRxiv2021

Pan-ErbB inhibition protects from SARS-CoV-2 replication, inflammation, and injury

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

microbiology↗

bioRxiv2021

A robust SARS-CoV-2 replication model in primary human epithelial cells at the air liquid interface to assess antiviral agents

There are, besides remdesivir, no approved antivirals for the treatment of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HtAEC) and human small airway epithelial cells (HsAEC) grown at the air/liquid interface (ALI). These cultures were infected at the apical side with one of two different SARS-CoV-2 isolates. Each virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35{degrees}C than 37{degrees}C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. Remdesivir, GS-441524 (the parent nucleoside of remdesivir), EIDD-1931 (the parent nucleoside of molnupiravir) and IFN ({beta}1 and {lambda}1) all resulted in dose-dependent inhibition of viral RNA and infectious virus titers collected at the apical side. However, AT-511 (the free base form of AT-527 currently in clinical testing) failed to inhibit viral replication in these in vitro primary cell models. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.

microbiology↗