Biology subjects

Schols, D.

Publications and source records attributed to Schols, D..

9 recordsLinked to original sources

bioRxiv2023

C-terminal truncation of CXCL10 attenuates inflammatory activity but retains angiostatic properties

Interferon-{gamma}-inducible protein of 10 kDa (IP-10/CXCL10) is a dual-function CXC chemokine that coordinates chemotaxis of activated T cells and natural killer (NK) cells via interaction with its G protein-coupled receptor (GPCR), CXC chemokine receptor 3 (CXCR3). As a consequence of natural posttranslational modifications, human CXCL10 exhibits a high degree of structural and functional heterogeneity. However, the biological effect of natural posttranslational processing of CXCL10 at the carboxy (C)-terminus has remained partially elusive. The truncated CXCL10 proteoform CXCL10(1-73), lacking the four endmost C-terminal amino acids, was previously identified in human cell culture supernatant. To further explore the functioning of CXCL10(1-73), we optimized its production in this study through Fmoc-based solid phase peptide synthesis (SPPS) and propose an SPPS strategy to efficiently generate human CXCL10 proteoforms. Compared to intact CXCL10(1-77), CXCL10(1-73) had diminished affinity for glycosaminoglycans including heparin, heparan sulfate and chondroitin sulfate A. Moreover, CXCL10(1-73) exhibited an attenuated capacity to induce CXCR3A-mediated signaling, as evidenced in calcium mobilization assays and through quantification of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) and protein kinase B/Akt. Furthermore, CXCL10(1-73) incited reduced primary human T lymphocyte chemotaxis in vitro and evoked less peritoneal ingress of CXCR3+ T lymphocytes in mice receiving intraperitoneal chemokine injections. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on spontaneous and/or FGF-2-induced migration, proliferation, wound healing, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells. Thus, C-terminally truncated CXCL10 has attenuated inflammatory properties, but preserved anti-angiogenic capacity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/548382v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@10c1207org.highwire.dtl.DTLVardef@173bc5eorg.highwire.dtl.DTLVardef@153d6a2org.highwire.dtl.DTLVardef@1302a76_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Cellular electrical impedance to profile SARS-CoV-2 fusion inhibitors and to assess the fusogenic potential of spike mutants

Despite the vaccination campaigns for COVID-19, we still cannot control the spread of SARS-CoV-2, as evidenced by the ongoing circulation of the Omicron variants of concern. This highlights the need for broad-spectrum antivirals to further combat COVID-19 and to be prepared for a new pandemic with a (re-)emerging coronavirus. An interesting target for antiviral drug development is the fusion of the viral envelope with host cell membranes, a crucial early step in the replication cycle of enveloped viruses. In this study, we explored the use of cellular electrical impedance (CEI) to quantitatively monitor morphological changes in real time, resulting from cell-cell fusion elicited by SARS-CoV-2 spike. The impedance signal in CEI-quantified cell-cell fusion correlated with the expression level of SARS-CoV-2 spike in transfected HEK293T cells. For antiviral assessment, we validated the CEI assay with the fusion inhibitor EK1 and measured a concentration-dependent inhibition of SARS-CoV-2 spike mediated cell-cell fusion (IC50 value of 0.13 M). In addition, CEI was used to confirm the fusion inhibitory activity of the carbohydrate-binding plant lectin UDA against SARS-CoV-2 (IC50 value of 0.55 M), which complements prior in-house profiling activities. Finally, we explored the utility of CEI in quantifying the fusogenic potential of mutant spike proteins and in comparing the fusion efficiency of SARS-CoV-2 variants of concern. In summary, we demonstrate that CEI is a powerful and sensitive technology that can be applied to studying the fusion process of SARS-CoV-2 and to screening and characterizing fusion inhibitors in a label-free and non-invasive manner. ImportanceDespite the success of the vaccines against SARS-CoV-2, new variants of the virus are still emerging and spreading, underlining the need for additional effective antiviral countermeasures. An interesting antiviral target for enveloped viruses is the fusion of the viral envelope with host cell membranes, a crucial early step in the life cycle of coronaviruses like SARS-CoV-2. Here, we present a sensitive impedance-based method to monitor in real-time cell-cell fusion elicited by the SARS-CoV-2 spike protein. With this technique we can profile entry inhibitors and determine the inhibitory potential of fusion inhibitors for SARS-CoV-2. In addition, with cellular electrical impedance we can evaluate the fusogenic properties of new emerging SARS-CoV-2 variants. Overall, the impedance technology adds valuable information on the fusion process of circulating coronaviruses and helps unravel the mode of action of new antivirals, opening new avenues for the development of next generation fusion inhibitors with improved antiviral activity.

microbiology↗

bioRxiv2022

Carbohydrate-Binding Protein from Stinging Nettle as Fusion Inhibitor for SARS-CoV-2 Variants of Concern

Urtica dioica agglutinin (UDA) is a carbohydrate-binding small monomeric protein isolated from stinging nettle rhizomes. It inhibits replication of a broad range of viruses, including coronaviruses, in multiple cell types, with appealing selectivity. In this work, we investigated the potential of UDA as a broad-spectrum antiviral agent against SARS-CoV-2. UDA potently blocks entry of pseudotyped SARS-CoV-2 in A549.ACE2+-TMPRSS2 cells, with IC50 values ranging from 0.32 to 1.22 {micro}M. Furthermore, UDA prevents viral replication of the early Wuhan-Hu-1 strain in Vero E6 cells (IC50 = 225 nM), but also the replication of SARS-CoV-2 variants of concern, including Alpha, Beta and Gamma (IC50 ranging from 115 to 171 nM). In addition, UDA exerts antiviral activity against the latest circulating Delta and Omicron variant in U87.ACE2+ cells (IC50 values are 1.6 and 0.9 {micro}M, respectively). Importantly, when tested in Air-Liquid Interface (ALI) primary lung epithelial cell cultures, UDA preserves antiviral activity against SARS-CoV-2 (20A.EU2 variant) in the nanomolar range. Surface plasmon resonance (SPR) studies demonstrated a concentration-dependent binding of UDA to the viral spike protein of SARS-CoV-2, suggesting interference of UDA with cell attachment or subsequent virus entry. Moreover, in additional mechanistic studies with cell-cell fusion assays, UDA inhibited SARS-CoV-2 spike protein-mediated membrane fusion. Finally, pseudotyped SARS-CoV-2 mutants with N-glycosylation deletions in the S2 subunit of the spike protein remained sensitive to the antiviral activity of UDA. In conclusion, our data establish UDA as a potent and broad-spectrum fusion inhibitor for SARS-CoV-2.

microbiology↗

bioRxiv2022

Cryo-EM structures of Doravirine and Rilpivirine with HIV-1 Reverse Transcriptase/DNA Aptamer: Nonnucleoside Inhibitor Resistance by E138K and M184I Mutations

Structures trapping a verity of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles in understanding the mechanisms of catalysis, inhibition and drug resistance, and in driving drug design. However, structures of several desired complexes of RT could not be obtained even after many crystallization or crystal soaking experiments. The ternary complexes of doravirine and rilpivirine with RT/DNA are such examples. Structural study of HIV-1 RT by single-particle cryo-EM has been challenging due to the enzymes relatively smaller size and higher flexibility. We optimized a protocol for rapid structure determination of RT complexes by cryo-EM and determined six structures of wild-type and E138K/M184I mutant RT/DNA in complexes with the nonnucleoside inhibitors rilpivirine, doravirine, and nevirapine. RT/DNA/rilpivirine and RT/DNA/doravirine complexes have structural differences between them and from the representative conformation of RT/DNA/nevirapine (or efavirenz); the primer grip in the RT/DNA/doravirine and the YMDD motif in the RT/DNA/rilpivirine complexes have large shifts. The DNA primer 3-end in the doravirine-bound structure is positioned at the active site, but the complex is in a non-productive state. In the mutant RT/DNA/rilpivirine structure, I184 is stacked with the DNA such that their relative positioning can influence rilpivirine in the pocket. Simultaneously, E138K mutation widens the NNRTI-binding pocket entrance, potentially contributing to a faster rate of rilpivirine dissociation by E138K/M184I mutant RT, as reported by earlier kinetic studies. These structural differences have implications for drug design and for understanding molecular mechanisms of drug resistance.

biophysics↗

bioRxiv2022

Live biotherapeutic throat spray for respiratory virus inhibition and interferon pathway induction

Respiratory viruses such as influenza viruses, respiratory syncytial virus (RSV), and coronaviruses initiate infection at the mucosal surfaces of the upper respiratory tract (URT), where the resident respiratory microbiome has an important gatekeeper function. In contrast to gut-targeting administration of beneficial bacteria against respiratory viral disease, topical URT administration of probiotics is currently underexplored, especially for the prevention and/or treatment of viral infections. Here, we report the selection and formulation of a broad-acting throat spray with live lactobacilli which induce interferon regulatory pathways and are able to inhibit respiratory viruses. Rational selection of Lactobacillaceae strains was based on safety, applicability, and potential antiviral and immunostimulatory efficacy in the URT. Three strains, Lacticaseibacillus casei AMBR2, Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum WCFS1 significantly reduced the cytopathogenic effects of RSV, influenza A/H1N1 and B viruses, and HCoV-229E coronavirus in co-culture models with bacteria, virus and host cells. Subsequently, these strains were formulated in a throat spray and human monocytes were employed to confirm the formulation process did not reduce the interferon regulatory pathway-inducing capacity. Administration of the throat spray in healthy volunteers revealed that the lactobacilli were capable of temporary colonization of the throat in a metabolically active form.

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

Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket - Targeting P-pocket by fragment screening

HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography and identified binding of two to P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drugresistance mutations. Analogs of a fragment were synthesized of which two showed noticeable RT inhibition. An engineered RT/DNA aptamer complex trapped the transient P-pocket in solution. Structures of the RT/DNA complex were determined with a fragment and a synthesized analog bound at P-pocket by single-particle cryo-EM. Identification of P-pocket and the devised structurebased platform provide an opportunity for designing new types of polymerase inhibitors.

biophysics↗

bioRxiv2021

Simultaneous inhibition of human CD4 and 4-1BB biogenesis suppresses cytotoxic T lymphocyte proliferation

The small molecule cyclotriazadisulfonamide (CADA) down-modulates the human CD4 receptor, an important factor in T cell activation. Here, we addressed the immunosuppressive potential of CADA using in vitro activation models. CADA inhibited lymphocyte proliferation in a mixed lymphocyte reaction, and when human PBMCs were stimulated with CD3/CD28 beads or phytohemagglutinin. The immunosuppressive effect of CADA involved both CD4+ and CD8+ T cells but was, surprisingly, most prominent in the CD8+ T cell subpopulation where it inhibited cell-mediated lympholysis. We discovered a direct down-modulatory effect of CADA on 4-1BB (CD137) expression, a survival factor for activated CD8+ T cells. More specifically, CADA blocked 4-1BB protein biosynthesis by inhibition of its co-translational translocation across the ER membrane in a signal peptide-dependent way. This study demonstrates that CADA, as potent down-modulator of human CD4 and 4-1BB, has promising in vitro immunomodulatory characteristics for future in vivo exploration as immunosuppressive drug.

immunology↗

bioRxiv2020

STAT2 signaling as double-edged sword restricting viral dissemination but driving severe pneumonia in SARS-CoV-2 infected hamsters

Introductory paragraphSince the emergence of SARS-CoV-2 causing COVID-19, the world is being shaken to its core with numerous hospitalizations and hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that productive SARS-CoV-2 infection in the lungs of mice is limited and restricted by early type I interferon responses. In contrast, we show that Syrian hamsters are highly permissive to SARS- CoV-2 and develop bronchopneumonia and a strong inflammatory response in the lungs with neutrophil infiltration and edema. Moreover, we identify an exuberant innate immune response as a key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Finally, we assess SARS-CoV- 2-induced lung pathology in hamsters by micro-CT alike used in clinical practice. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients.

microbiology↗