Development of a Lymphoid Organ-Chip to Evaluate Covid Vaccine Boosting Strategies

Background: Secondary lymphoid organs provide the adequate microenvironment for the development of antigen (Ag)-specific immune responses. The tight collaboration between CD4+ T cells and B cells in germinal centers is crucial to shape B cell fate and optimize antibody maturation. Dissecting these immune interactions remains challenging in humans, and animal models do not always recapitulate human physiology. To address this issue, we developed an in vitro 3D model of a human lymphoid organ. The model relies on a microfluidic device, enabling primary human cells to self-organize in an extracellular matrix (ECM) under continuous fluid perfusion. We applied this Lymphoid Organ-Chip (LO chip) system to the analysis of B cell recall responses to SARS-CoV-2 antigens. Method(s): We used a two-channel microfluidic Chip S1 from Emulate, where the top channel is perfused with antigen (spike protein or SARS-CoV-2 mRNA vaccine), while the bottom channel contains PBMC (n = 14 independent donors) seeded at high-density in a collagen-based ECM. Immune cell division and cluster formation were monitored by confocal imaging, plasmablast differentiation and spike-specific B cell amplification by flow cytometry, antibody secretion by a cell-based binding assay (S-flow). Result(s): Chip perfusion with the SARS-CoV-2 spike protein for 6 days resulted in the induction CD38hiCD27hi plasmablast maturation compared to an irrelevant BSA protein (P< 0.0001). Using fluorescent spike as a probe, we observed a strong amplification of spike-specific B cell (from 3.7 to 140-fold increase). In line with this rapid memory B cell response, spike-specific antibodies production could be detected as early as day 6 of culture. Spike perfusion also induced CD4+ T cell activation (CD38+ ICOS+), which correlated with the level of B cell maturation. The magnitude of specific B cell amplification in the LO chip was higher than in 2D and 3D static cultures at day 6, showing the added value of 3D perfused culture for the induction of recall responses. Interestingly, the perfusion of mRNA-based SARS-CoV-2 vaccines also led to strong B cell maturation and specific B cell amplification, indicating that mRNA-derived spike could be expressed and efficiently presented in the LO chip. Conclusion(s): We developed a versatile Lymphoid Organ-Chip model suitable for the rapid evaluation of B cell recall responses. The model is responsive to protein and mRNA-encoded antigens, highlighting its potential in the evaluation of SARS-CoV-2 vaccine boosting strategies.

Targeting Spike Glycans to Inhibit SARS-CoV-2 Viral Entry

SARS-CoV-2 Spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the Spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the Spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical tools such as SEC chromatography, dynamics light scattering, fluorescence binding assays, and electron microscopy, we demonstrate that the interaction is avidity driven and that BOA crosslinks the Spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV-1, establishing that glycan-targeting molecules have the potential to be pan-coronavirus inhibitors.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Towards New Antimicrobials - Targeting Bacterial Response Regulators

Purpose of Study: Antibiotic resistance remains one of the largest healthcare and public health challenges. Several studies have documented that the spread of antibiotic resistant bacteria in nosocomial settings has been exacerbated worldwide due to increased rates of hospitalization and intubation in the wake of the COVID-19 pandemic. One way to address antibiotic resistance is to identify novel compounds that inhibit essential microbial processes. Two-component regulatory systems are important mediators of signal transduction that allow bacteria to communicate with and respond to changes in their environment. The WalRK system is a two-component system that is conserved and essential for viability in many Gram-positive human pathogens. We hypothesize that a ligand that specifically binds with the DNA-interaction surface of the WalR protein can lead to cell death and can serve as a lead compound for future drug development efforts. Methods Used: We describe the development process of an assay to identify WalR binding compounds. In silico molecular dynamics docking approaches were utilized to identify potential WalR binding compounds from virtual compound libraries. To assess their WalR-binding capacity in vitro, overexpression strains for several WalR recombinant constructs were engineered and protein constructs were purified to homogenicity. Isothermal titration calorimetry (ITC) is a technique that measures heat release or absorption when two molecules interact. A MicroCal PEAQ ITC instrument was utilized to develop a WalR-binding assay. Summary of Results: WalR is a two-domain protein featuring a regulatory and a DNA-binding domain. Two constructs, a truncated DNA-binding domain and a full-length protein construct proved soluble, and pure quantities necessary to conduct ITC measurements could be successfully obtained (12 mg full-length protein and 23 mg truncated protein). These proteins were amenable to ITC experiments. We found that experiments were best run with at least a two-fold increase of ligand concentration to protein concentration supplied in identical buffer conditions over nineteen injections. We are currently assessing the binding affinities of our in silico hit compounds. Conclusion(s): Our results show that ITC enables the detailed, rapid, and reproducible characterization of the binding relationship between the DNA-binding domain of the WalR protein and any potential ligands. The protocol discussed herein will enable further drug discovery studies on the WalR response regulator protein to identify and characterize inhibitors, providing insight towards the development of novel antimicrobial compound.

A molecular toolbox for ADP-ribosyl binding proteins.

Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or viral infections, making them attractive drug targets. We present a robust and accessible assay applicable to both hydrolyzing or non-hydrolyzing binders of mono- and poly-ADP-ribosyl groups. This technology relies on a C-terminal tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups or analogs. By fusing the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we generate robust FRET partners and confirm the interaction with 22 known ADP-ribosyl binders. The applicability for high-throughput screening of inhibitors is demonstrated with the SARS-CoV-2 nsp3 macrodomain, for which we identify suramin as a moderate-affinity yet non-specific inhibitor. High-affinity ADP-ribosyl binders fused to nanoluciferase complement this technology, enabling simple blot-based detection of ADP-ribosylated proteins. All these tools can be produced in Escherichia coli and will help in ADP-ribosylation research and drug discovery.

Investigating novel thiazolyl-indazole derivatives as scaffolds for SARS-CoV-2 MPro inhibitors

COVID-19 is a global pandemic caused by infection with the SARS-CoV-2 virus. Remdesivir, a SARS-CoV-2 RNA polymerase inhibitor, is the only drug to have received widespread approval for treatment of COVID-19. The SARS-CoV-2 main protease enzyme (MPro), essential for viral replication and transcription, remains an active target in the search for new treatments. In this study, the ability of novel thiazolyl-indazole derivatives to inhibit MPro is evaluated. These compounds were synthesized via the heterocyclization of phenacyl bromide with (R)-carvone, (R)-pulegone and (R)-menthone thiosemicarbazones. The binding affinity and binding interactions of each compound were evaluated through Schrodinger Glide docking, AMBER molecular dynamics simulations, and MM-GBSA free energy estimation, and these results were compared with similar calculations of MPro binding various 5-mer substrates (VKLQA, VKLQS, VKLQG) and a previously identified MPro tight-binder X77. From these simulations, we can see that binding is driven by residue specific interactions such as pi-stacking with His41, and S/pi interactions with Met49 and Met165. The compounds were also experimentally evaluated in a MPro biochemical assay and the most potent compound containing a phenylthiazole moiety inhibited protease activity with an IC50 of 92.9 muM. This suggests that the phenylthiazole scaffold is a promising candidate for the development of future MPro inhibitors.Copyright © 2022 The Authors

Highlights of the 13th Japan Bioanalysis Forum Symposium.

Approximately 280 people from pharmaceutical industries, contractors, academic institutions and regulatory authorities attended the 13th Japan Bioanalysis Forum Symposium. The symposium was held via web to prevent the spread of COVID-19 from the 28 February to 2 March 2022. The theme of the symposium was 'All for One Goal', and the event has provided an opportunity for open discussion among researchers with different backgrounds but who share a common goal: "to deliver more effective and safe pharmaceuticals to patients as quickly as possible". The speakers focused on hot topics in bioanalysis, including chromatography, biomarker analysis, cell and gene therapy, COVID-19 and antidrug antibody. This symposium provided a great opportunity for the participants to have meaningful discussions, even though 'on the web' was a limited space.

Immunoglobulin M (IgM) Inhibits Replication of SARS-CoV-2 In Vero E6 Cells In Vitro and Delays Disease in K18-hACE2 Mice Infected with SARSCoV-2 In Vivo

Purpose: To determine if IgM has a direct effect in preventing SARS-CoV-2 replication in Vero E6 cells, and delaying or preventing disease in infected K18- hACE2 mice. Method(s): 1) Vero E6 cells, grown to confluence in 12 well plates, were used to test the effect of IgM in reducing the number of plaque-forming units (PFU).There were 4 groups: a) 25PFU WA-1 SARS-CoV-2 was combined with 20, 5 or 0.8mug IgM in growth medium, and incubated for 1hr in a final volume of 500ul. 100mul was added to Vero E6 cells in replicate wells and incubated for 1hr;b) 100mul of 20, 5 or 0.8mug IgM was added to Vero E6 cells and incubated. Media was aspirated and the cells were then inoculated with 25PFU WA-1 and incubated for 1hr;c) Virus control - as above, but with no IgM;d) No virus or IgM. FBS growth medium containing Avicel was overlain in the wells and incubated for 48 hours. Virus replication was stopped by incubating with 10% buffered formalin. Following removal of formalin, plates were stained with Giemsa violet, dried, and photographed. 2) A COVID -19 Spike- ACE2 binding assay kit was used to determine if IgM (2ug, 4.5ug, 20ug, 45ug IgM) inhibits the interaction between the Spike-receptor binding domain (S-RBD) and Angiotensin I ConvertingEnzyme 2 (ACE2) receptor. 3) K18-hACE2 mice were divided into 3 groups based on treatment regimen;Group 1: with IgM, No virus;2: with Saline, with virus;3: with IgM, with virus. 35ug IgM was injected intraperitoneal in a single dose, 2 days prior to infection. Mice were innoculated intranasally with 1250 pfu of HK SARS-CoV-2. Result(s): 1) Exposure of 25PFU SARS-CoV-2 to IgM (at all concentrations) prior to incubation with Vero E6 cells, inhibited its replication in Vero E6 cells. When Vero E6 cells were incubated with IgM prior to infection, no plaques were seen in wells with 20ug and 5ug IgM but were observed in wells with 0.8ug IgM. Plaques were also observed in the Virus alone group, but none were seen in the 'No IgM-No virus' group. 2) 45ug IgM/100uls inhibited the binding of S-RBD to ACE2 by ~94-100%, 20ug IgM/100uls inhibited it by ~80%, and 2 or 4.5ug/100ul by ~70-75%. Control without IgM did not inhibit the S-RBD-ACE-2 binding. 3) Pretreatment with a single low dose IgM injection delayed weight loss and mortality. Conclusion(s): IgM inhibits the replication of SARS-CoV-2 in Vero cells in vitro. It also inhibits the interaction between S-RBD that is present on the viral surface and the ACE2 receptor, by binding to S-RBD. A single low dose of IgM given prechallenge delayed disease in infected mice. The discovery that IgM interferes with the formation of the S-RBD-ACE2 complex, and that a single low dose can delay disease, indicates its translational potential as a vaccine/therapeutic to prevent or treat COVID-19.

Anti-platelet factor 4 pathologic antibodies after chadox1 ncov-19 vaccination mauriziano hospital diagnosis and findings

Introduction.Vaccine induced immune thrombocytopenia and thrombosis (VITT) following ChAdOx1 nCOV-19 vaccine has been described, associated with unusual site thrombosis, thrombocytopenia, raised D-dimer and high titre immunoglobulin-G (IgG) class anti-Platelet Factor 4 (PF4) antibodies. Laboratory management of suspected cases begins with a sensitive anti PF4 antibodies binding assay (PF4-ELISA). If the PF4 binding assay is negative, this patient does not have Heparin induced Thrombocytopenia (HIT) or VITT. If the PF4 binding assay is positive, the positivity should be confirmed with one or multiple HIT functional assays as available, such as the serotonin release assay (SRA), heparin-induced platelet activation assay, platelet aggregation (PAT) test, flow cytometry test.Methods.We summarized clinical and laboratory findings of 7 patients in Piedmont who developed thrombosis and thrombocytopenia following AZD1222 vaccination. Plasma from all patients was used to test for anti PF4 antibodies by 2 different ELISA assays (Immucor and Stago) and by 2 different HIT functional assays, PAT and flow cytometry (HIT alert test) both performed in the presence of heparin, PF4 or both.Results.The 7 patients [6 males and 1 female, median age: 38 (range:31-76)] presented with thrombosis 2 to 17 days post vaccination: 5 males had deep vein thrombosis not in unusual sites, 1 male had stroke and the female had cerebral venous thrombosis (CVT). None had received heparin prior symptoms onset. Only 2 out of 7 patients tested positive for anti PF4 ELISA antibodies with both assays: the men with stroke showed low positivity (OD = 0,56 and 0,41) and the female with CVT strong positivity (OD = 3,2 and 3,87). Only the female patient with CVT tested positive with both HIT functional assays, PAT and HIT alert cytometry test in the presence of PF4 independently of heparin. Both assays were inhibited by high concentrations of heparin.Conclusions. In our limited experience VITT demonstrated to be an extremely rare event in the context of AZD1222 COVID-19 vaccination even in the subset of patients with thrombosis and thrombocytopenia.

The spike-ACE2 binding assay: An in vitro platform for evaluating vaccination efficacy and for screening SARS-CoV-2 inhibitors and neutralizing antibodies.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 has become a worldwide pandemic, and there is a pressing need for the rapid development of novel therapeutic strategies. SARS-CoV-2 viral entry is mediated by interaction between the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein and host cellular receptor, human angiotensin converting enzyme 2 (ACE2). The lack of a high throughput screening (HTS) platform for candidate drug screening means that no targeted COVID-19 treatments have been developed to date. To overcome this limitation, we developed a novel, rapid, simple, and HTS binding assay platform to screen potential inhibitors of the RBD-ACE2 complex. Three "neutralizing" mouse monoclonal antibodies capable of blocking the RBD-ACE2 interaction were identified using our binding assay and pseudovirus neutralization assay followed by further validation with the Focus Reduction Neutralization Test (FRNT), which analyzes the neutralization capacity of samples in the presence of live SARS-CoV-2. Furthermore, the consistency of our binding assay and FRNT results (R2 = 0.68) was demonstrated by patients' serum, of which were COVID-19 positive (n = 34) and COVID-19 negative (n = 76). Several small molecules selected for their potential to inhibit the Spike-ACE2 complex in silico were also confirmed with the binding assay. In addition, we have evaluated vaccine efficacy using binding assay platform and validated through pseudovirus neutralization assay. The correlation between binding assay & psuedovirus assay of the post vaccinated serum showed well correlated (R2 = 0.09) Moreover, our binding assay platform successfully validated different Spike RBD mutants. These results indicate that our binding assay can be used as a platform for in vitro screening of small molecules and monoclonal antibodies, and high-throughput assessment of antibody levels after vaccination. When conducting drug screening, computer virtual screening lacks actual basis, construction of pseudoviruses is relatively complicated, and even FRNT requires a P3 laboratory. There are few methods to determine the competitiveness of the target drug and SRBD or ACE2. Our binding assay can fill this gap and accelerate the process and efficiency of COVID-19 drug screening.

Expression, purification and characterization of SARS-CoV-2 spike RBD in ExpiCHO cells.

Reliable diagnosis is critical to identify infections of SARS-CoV-2 as well as to evaluate the immune response to virus and vaccines. Consequently, it becomes crucial the isolation of sensitive antibodies to use as immunocapture elements of diagnostic tools. The final bottleneck to achieve these results is the availability of enough antigen of good quality. We have established a robust pipeline for the production of recombinant, functional SARS-CoV-2 Spike receptor binding domain (RBD) at high yield and low cost in culture flasks. RBD was expressed in transiently transfected ExpiCHO cells at 32 °C and 5% CO2 and purified up to 40 mg/L. The progressive protein accumulation in the culture medium was monitored with an immunobinding assay in order to identify the optimal collection time. Successively, a two-step chromatographic protocol enabled its selective purification in the monomeric state. RBD quality assessment was positively evaluated by SDS-PAGE, Western Blotting and Mass Spectrometry, while Bio-Layer Interferometry, flow cytometer and ELISA tests confirmed its functionality. This effective protocol for the RBD production in transient eukaryotic system can be immediately extended to the production of RBD mutants.

DEVELOPMENT of COVID-19 MONOCLONAL ANTIBODIES and RECOMBINANT PROTEINS AS REAGENTS for BIOMEDICAL RESEARCH and DIAGNOSTIC TESTS

Since SARS-COV-2 virus spread worldwide and COVID-19 turned rapidly into a pandemic illness, the necessity for vaccines and diagnostic tests became crucial. The viral surface is decorated with Spike, the major antigenic determinant and main target for vaccine development. Within Spike, the receptor binding domain (RBD), constitutes the main target of highly neutralizing antibodies found in COVID-19 convalescent plasma. Besides vaccination, another important aspect of Spike (and RBD) is their use as immunogen for the development of poli- and monoclonal antibodies (mAbs) for therapeutic and diagnostic purposes. Here we report the development and preliminary biochemical characterization of a set of monoclonal antibodies against the Spike RBD domain along with the recombinant expression of two mayor COVID-19 protein reagents: the viral Spike RBD domain and the extracellular domain of the human receptor ACE2. RBD and the extracellular domain of ACE2 (aa 1-740) were obtained through transient gene transfection (TGE) in two different mammalian cell culture systems: HEK293T adherent monolayers and Expi293F™ suspension cultures. Due to its low cost and ease scale-up, all transfections were carried with polyethyleneimine (PEI). Expressed proteins were purified from culture supernatants by immobilized metal affinity chromatography. Anti-RBD mAbs were developed from two different immunization schemes: one aimed to elicit antibodies with viral neutralizing potential, and the other with the ability to recognize denatured RBD for routine lab immunoassays. To achieve this, the first group of mice was immunized with RBD in aluminum salts (RBD/Al) and the other with RBD emulsified in Freunds adjuvant (RBD/FA). Polyclonal and monoclonal antibody reactivities against native or denatured RBD forms were then assessed by ELISA. Complete RBD denaturation was followed by intrinsic fluorescence spectral changes upon different physicochemical stress treatments. As expected, RBD/Al immunized mice developed an antibody response shifted to native RBD while those immunized with RBD/FA showed a high response against both forms of the protein. In accordance with the observed polyclonal response, RBD/FA derived mAbs recognize both, native and denatured RBD. On the contrary, hybridomas generated from the RBD/Al protocol mostly recognize RBD in its native state. Further ELISA binding assays revealed that all RBD/FA derived mAbs can form a trimeric complex with ACE2 and RBD, denoting they would not have viral neutralizing activity. ELISA competition assays with the RBD/ACE2 complex aimed to determine the neutralization potential of the RBD/Al derived mAbs are under way. Overall, the anti-Spike RBD mAbs and the recombinant RBD and ACE2 proteins presented here constitute valuable tools for diverse COVID-19 academic research projects and local immunity surveillance testing.

Investigating Novel Thiazolyl-Indazole Derivatives as Scaffolds for SARS-Cov-2 MPro Inhibitors

COVID-19 is a global pandemic caused by infection with the SARS-CoV-2 virus. Remdesivir, a SARS-CoV-2 RNA polymerase inhibitor, is the only drug to have received widespread approval for treatment of COVID-19. The SARS-CoV-2 main protease enzyme (MPro), essential for viral replication and transcription, remains an active target in the search for new treatments. In this study, the ability of novel thiazolyl-indazole derivatives to inhibit MPro is evaluated. These compounds were synthesized via the heterocyclization of phenacyl bromide with (R)-carvone, (R)-pulegone and (R)-menthone thiosemicarbazones. The binding affinity and binding interactions of each compound were evaluated through Schrödinger Glide docking, AMBER molecular dynamics simulations, and MM-GBSA free energy estimation, and these results were compared with similar calculations of MPro binding various 5-mer substrates (VKLQA, VKLQS, VKLQG) and a previously identified MPro tight-binder X77. From these simulations, we can see that binding is driven by residue specific interactions such as π-stacking with His41, and S/π interactions with Met49 and Met165. The compounds were also experimentally evaluated in a MPro biochemical assay and the most potent compound containing a phenylthiazole moiety inhibited protease activity with an IC50 of 92.9 μM. This suggests that the phenylthiazole scaffold is a promising candidate for the development of future MPro inhibitors.

Novel Small-Molecule Inhibitors of the SARS-CoV-2 Spike Protein Binding to Neuropilin 1.

Furin cleavage of the SARS-CoV-2 spike protein results in a polybasic terminal sequence termed the C-end rule (CendR), which is responsible for the binding to neuropilin 1 (NRP1), enhancing viral infectivity and entry into the cell. Here we report the identification of 20 small-molecule inhibitors that emerged from a virtual screening of nearly 950,000 drug-like compounds that bind with high probability to the CendR-binding pocket of NRP1. In a spike NRP1 binding assay, two of these compounds displayed a stronger inhibition of spike protein binding to NRP1 than the known NRP1 antagonist EG00229, for which the inhibition of the CendR peptide binding to NRP1 was also experimentally confirmed. These compounds present a good starting point for the design of small-molecule antagonists against the SARS-CoV-2 viral entry.

Nano-triciribine reduces sars-cov-2 infection by sequestering ace2 and the novel host factor LDLR

Introduction: People with previous CVD hospitalized for COVID-19 have elevated death rate. We reported that patients with diabetes and HF higher protein levels of the low density lipoprotein receptor (LDLR). We hypothesized that LDLR is a novel host factor for the SARS-CoV-2-Spike (S2S) protein that may be regulated by the Akt inhibitor Triciribine (TCN), a drug being tested in Phase III studies for breast cancer. We also hypothesized that nano-formulation of Triciribine (NanoTriciribine;NTCN) would enhance its efficacy and allow for intranasal delivery. Methods: Interactions between the recombinant proteins Spike-RBD (receptor binding domain), ACE2, LDLR and its ectodomains (EGFA-EFFB, C2-C5 and C2) were analyzed by binding assays and co-IP in HepG2, HK2, and 293T cells. Viral entry assays were performed with 2 S2S pseudoviruses using 293T cells + hACE2 and TMPRSS2 or Furin protease. The effect of NTCN or the LXR agonist GW-3965 on viral uptake (pseudotyped VSVΔG-GFP∗S2S or chimera VSV-S2SeGFP virus) was assessed. Akt, pAkt, ACE2, and LDLR levels were determined in 293T+hACE2 by flow cytometry. Assays were done in triplicates and 1-way-ANOVA with Tukey's correction was used for statistics. Results: RBD protein binds modestly to the human LDLR (EC50:10μM) and its C2-C5 ectodomain (EC50:13.8μM). Co-IP revealed a novel and strong LDLR-ACE2 interaction in several human cell lines. LDLR overexpression in human cells increased the uptake of VSVΔG-GFP∗S2S (FC=2.32;p<0.001) and chimera virus (FC=.33;p<.0001). NTCN and TCN each reduced pAkt/Akt ratio. 1μM TCN or NTCN reduced LDLR (7.2%;p<.01 &15.6%;p<0.0001) and ACE2 (32%;p<0.05 &44.7%;p<.01) cell surface expression, respectively. 1μM NTCN or GW-3965 reduced S2S viral entry by 64.2% (p<.0001) and 40.7% (p<.01), respectively, confirming a role for LDLR in S2S infection. In hACE2tg mice, chimera VSV-S2S caused significant lung infection as measured by qPCR, GFP expression in proximal and distal lung airway epithelial cells, and electron microscopy. Intranasal delivery of NTCN was well tolerated. Conclusions: LDLR enhanced S2S viral entry supporting the elevated COVID-19 susceptibility seen in patients with heart disease. NTCN is a promising candidate for prophylactic treatment against COVID-19.

PDZ-Containing Proteins Targeted by the ACE2 Receptor.

Angiotensin-converting enzyme 2 (ACE2) is a main receptor for SARS-CoV-2 entry to the host cell. Indeed, the first step in viral entry is the binding of the viral trimeric spike (S) protein to ACE2. Abundantly present in human epithelial cells of many organs, ACE2 is also expressed in the human brain. ACE2 is a type I membrane protein with an extracellular N-terminal peptidase domain and a C-terminal collectrin-like domain that ends with a single transmembrane helix and an intracellular 44-residue segment. This C-terminal segment contains a PDZ-binding motif (PBM) targeting protein-interacting domains called PSD-95/Dlg/ZO-1 (PDZ). Here, we identified the human PDZ specificity profile of the ACE2 PBM using the high-throughput holdup assay and measuring the binding intensities of the PBM of ACE2 against the full human PDZome. We discovered 14 human PDZ binders of ACE2 showing significant binding with dissociation constants' values ranging from 3 to 81 µM. NHERF, SHANK, and SNX27 proteins found in this study are involved in protein trafficking. The PDZ/PBM interactions with ACE2 could play a role in ACE2 internalization and recycling that could be of benefit for the virus entry. Interestingly, most of the ACE2 partners we identified are expressed in neuronal cells, such as SHANK and MAST families, and modifications of the interactions between ACE2 and these neuronal proteins may be involved in the neurological symptoms of COVID-19.

Development of a nano-luciferase based assay to measure the binding of SARS-CoV-2 spike receptor binding domain to ACE-2.

To identify drugs that could potentially be used to treat infection with SARS-CoV-2, a high throughput 384-well assay was developed to measure the binding of the receptor binding domain (RBD) of the viral S1 protein to its main receptor, angiotensin converting enzyme 2 (ACE2). The RBD was fused to both a HiBIT tag and an IL6 secretion signal to enable facile collection from the cell culture media. The addition of culture media containing this protein, termed HiBIT-RBD, to cells expressing ACE2 led to binding that was specific to ACE2 and both time and concentration dependant, Binding could be inhibited by both RBD expressed in E. coli and by a full length S1 - Fc fusion protein (Fc-fused S1) expressed in eukaryotic cells. The mutation of residues that are known to play a role in the interaction of RBD with ACE2 also reduced binding. This assay may be used to identify drugs which inhibit the viral uptake into cells mediated by binding to ACE2.

Markov state modeling reveals alternative unbinding pathways for peptide-MHC complexes.

Peptide binding to major histocompatibility complexes (MHCs) is a central component of the immune system, and understanding the mechanism behind stable peptide-MHC binding will aid the development of immunotherapies. While MHC binding is mostly influenced by the identity of the so-called anchor positions of the peptide, secondary interactions from nonanchor positions are known to play a role in complex stability. However, current MHC-binding prediction methods lack an analysis of the major conformational states and might underestimate the impact of secondary interactions. In this work, we present an atomically detailed analysis of peptide-MHC binding that can reveal the contributions of any interaction toward stability. We propose a simulation framework that uses both umbrella sampling and adaptive sampling to generate a Markov state model (MSM) for a coronavirus-derived peptide (QFKDNVILL), bound to one of the most prevalent MHC receptors in humans (HLA-A24:02). While our model reaffirms the importance of the anchor positions of the peptide in establishing stable interactions, our model also reveals the underestimated importance of position 4 (p4), a nonanchor position. We confirmed our results by simulating the impact of specific peptide mutations and validated these predictions through competitive binding assays. By comparing the MSM of the wild-type system with those of the D4A and D4P mutations, our modeling reveals stark differences in unbinding pathways. The analysis presented here can be applied to any peptide-MHC complex of interest with a structural model as input, representing an important step toward comprehensive modeling of the MHC class I pathway.

The inorganic polymer, polyphosphate, blocks binding of SARS-CoV-2 spike protein to ACE2 receptor at physiological concentrations.

Inorganic polyphosphate (polyP) is a morphogenetically active and metabolic energy-delivering physiological polymer that is released from blood platelets. Here, we show that polyP efficiently inhibits the binding of the envelope spike (S)-protein of the coronavirus SARS-CoV-2, the causative agent of COVID-19, to its host cell receptor ACE2 (angiotensin-converting enzyme 2). To stabilize polyP against the polyP-degrading alkaline phosphatase, the soluble polymer was encapsulated in silica/polyP nanoparticles. Applying a binding assay, soluble Na-polyP (sizes of 40 Pi and of 3 Pi units) as well as silica-nanoparticle-associated polyP significantly inhibit the interaction of the S-protein with ACE2 at a concentration of 1 µg/mL, close to the level present in blood. This inhibition is attributed to an interaction of polyP with a basic amino acid stretch on the surface of the receptor binding domain of S-protein. PolyP retains its activity in a flushing solution, opening a new strategy for the prevention and treatment of SARS-CoV-2 infection in the oropharyngeal cavity. The data suggest that supplementation of polyP might contribute to a strengthening of the human innate immunity system in compromised, thrombocytopenic COVID-19 patients.