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SUMMARYDeeper understanding of the mechanism of the action of insulin and insulin-degrading enzyme (IDE) is a central theme in research into physiology and the pathophysiology of type 2 diabetes mellitus. Despite significant progress regarding the substrate recruitment, unfolding, digestion, and release by IDE, the structure and function of the insulin hexamer during the degradation cycle of IDE remain to be fully characterized. In the present study, we have characterized the behavior of human insulin hexamer in the absence of zinc. Using cryo-electron microscopy, we also observed that these hexamers represented a structure similar to that of T6insulin. More interestingly, we also observed complexes in which some of their monomeric insulin components are partially distorted at their hexametric symmetry. This ensures that insulin determines the kinetics of its degradation by IDE without the requirement for zinc. These findings provide new information regarding the molecular events in insulin assembly and disassembly that permit its selective digestion by IDE.

Glucagon is a peptide hormone which posits a significant potential as a therapeutic molecule for various human diseases. One of the major challenges hampering medicinal application of glucagon, however, is its insoluble and aggregation-prone property. Although glucagon is dissolvable, it aggregates easily and forms amyloid fibrils. To date, despite many studies to understand how glucagon aggregates and fibrillizes, its detailed amyloidogenesis mechanism is still elusive, particularly due to insufficient structural information of glucagon amyloid fibrils. Here we report the novel structure of a glucagon amyloid fibril, which was determined with cryo-electron microscopy (cryo-EM) to a 3.8-Å resolution. Our model features with tight and extensive inter-monomer interactions, which efficiently stabilize the entire chain of full-length glucagon into the V-shape conformation, clamping the other monomer to construct a dimeric architecture orthogonal to the fibril axis. Notably, the current structure significantly differs from the previous solid-state NMR model, which gives direct evidence for multiple and dynamic amyloidogenesis mechanisms of glucagon. In addition, our results are expected to provide important insights to appreciate the molecular details of glucagon in its initial amyloidogenesis and fibril elongation processes.

Photosystem I (PSI) is a light driven electron pump transferring electrons from Cytochrome c6 (Cyt c6) to Ferredoxin (Fd). An understanding of this electron transfer process is hampered by a paucity of structural detail concerning the binding of Fd and the bound Cyt c6. Here we describe the high resolution cryo-EM structure of Thermosynechococcus elongatus BP-1 PSI in complex with Fd and Cyt c6. In our structure Cyt c6 is loosely bound in a non-productive site on the periphery of the complex. Side chain interactions at the PSI:Fd interface are visualized in detail. The structure explains the properties of mutants of PsaE and PsaC and suggests a molecular switch for the dissociation of Fd upon reduction. Calorimetry-based thermodynamic analyses confirms a single binding site for Fd and demonstrates that PSI:Fd complexation is purely driven by entropy. A reaction cycle for the efficient transfer of electrons from Cyt c6 to Fd via PSI is proposed.

Aggregation of intrinsically disordered amyloid β (Aβ) is a hallmark of Alzheimer’s disease. Although complex aggregation mechanisms have been increasingly revealed, structural ensembles of Aβ monomers with heterogeneous and transient properties still hamper detailed experimental accesses to early events of amyloidogenesis. We herein developed a new mathematical tool based on multiple linear regression to obtain the reasonable ensemble structures of Aβ monomer by using the solution nuclear magnetic resonance (NMR) and molecular dynamics simulation data. Our approach provided the best-fit ensemble to two-dimensional NMR chemical shifts, also consistent with circular dichroism and dynamic light scattering analyses. The major monomeric structures of Aβ including β-sheets in both terminal and central hydrophobic core regions and the minor partially-helical structures suggested initial structure-based explanation on possible mechanisms of early molecular association and nucleation for amyloid generation. A wide-spectrum application of the current approach was also indicated by showing a successful utilization for ensemble structures of folded proteins. We propose that multiple linear regression in combination to experimental results will be highly promising for studies on protein misfolding diseases and functions by providing a convincing template structure.Graphic abstract

ImportanceThe antigen-based rapid diagnostic test (Ag-RDT), using saliva specimens, is fast, non-invasive and suitable for SARS-CoV-2 self-testing, unlike nasopharyngeal swab (NPS) testing.ObjectiveTo assess the diagnostic sensitivity of a novel Beanguard gargle™ (BG)-based virus detection method for early diagnosis of COVID-19.DesignThis clinical trial was conducted at Gunsan Medical Center, Namwon Medical Center, and Jeonbuk National University Hospital, between May 7 and July 7, 2021.SettingPaired NPS and BG-based saliva specimens collected from COVID-19 patients and healthy individuals were analyzed using NPS-RT-PCR, BG-RT-PCR, and BG-Ag-RDTs.ParticipantsThe study comprised 102 COVID-19-positive patients hospitalized after governmental screening process and 100 healthy individuals. Forty-five COVID-19 patients were sampled within 6 days of illness and 57 within 7–15 days; 27 were categorized as asymptomatic and 75, as symptomatic. Eight and 2 patients carried the SARS-CoV-2 Alpha and Delta variants, respectively.InterventionThe diagnostic performances of BG-Ag-RDT, BG-RT-PCR, and NPS-RT-PCR for detecting SARS-CoV-2 were compared.Main outcomesThe sensitivities of BG-Ag-RDT and BG-RT-PCR towards salivary viral detection were highly concordant, with no discrimination between symptomatic, asymptomatic, or SARS-CoV-2 variant cases.ResultsAmong total participants (mean age, 43.7 years), 51% were women. BG-Ag-RDTs showed high sensitivity (97.8%, [95% CI, 88.4% to 99.6%]) and specificity (100%, [95% CI, 96.3% to 100%) in 45 patients within 6 days of illness and could detect all cases of SARS-CoV-2 Alpha and Delta variants. In 11 asymptomatic early-stage cases, both BG-Ag-RDTs and BG-RT-PCR showed excellent sensitivity and specificity of 100% (95% CI, 74.1% to 100% and 95% CI, 20.7% to 100%, respectively). The interaction between SARS-CoV-2 spike proteins and truncated canavalin, an active ingredient from bean extract (BE) and the ultrastructural features of SARS-CoV-2 particles coated with BE were observed. The detachment of the SARS-CoV-2 receptor-binding domain from hACE2 increased as the BE concentration increased, allowing the release of the virus from hACE2 for early diagnosis.Conclusions and RelevanceUsing BG-based saliva remarkably enhances the Ag-RDT diagnostic performance as an alternative to NPS and enables rapid and accurate COVID-19 self-testing and mass screening, supporting efficient COVID-19 management.Trial RegistrationKCT0006438Key PointsQuestionHow can we collect SARS-CoV-2 from oral cavity to improve the sensitivity of antigen-based rapid diagnostic test (Ag-RDT)?FindingsIn this clinical study involving 102 hospitalized COVID-19 patients, the Ag-RDT test using Beanguard gargle™-based saliva specimens showed significantly enhanced sensitivity and specificity towards detection of SARS-CoV-2 along with Alpha and Delta variants in all patients tested within 6 days of illness.MeaningOur self-testing method represents an attractive alternative to nasopharyngeal swab RT-PCR for the early diagnosis of symptomatic and asymptomatic COVID-19 cases.

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global crisis. There is no therapeutic treatment specific for COVID-19. It is highly desirable to identify potential antiviral agents against SARS-CoV-2 from existing drugs available for other diseases and, thus, repurpose them for treatment of COVID-19. In general, a drug repurposing effort for treatment of a new disease, such as COVID-19, usually starts from a virtual screening of existing drugs, followed by experimental validation, but the actual hit rate is generally rather low with traditional computational methods. Here we report a new virtual screening approach with accelerated free energy perturbation-based absolute binding free energy (FEP-ABFE) predictions and its use in identifying drugs targeting SARS-CoV-2 main protease (Mpro). The accurate FEP-ABFE predictions were based on the use of a new restraint energy distribution (RED) function designed to accelerate the FEP-ABFE calculations and make the practical FEP-ABFE-based virtual screening of the existing drug library possible for the first time. As a result, out of twenty-five drugs predicted, fifteen were confirmed as potent inhibitors of SARS-CoV-2 Mpro. The most potent one is dipyridamole (Ki=0.04 μM) which has showed promising therapeutic effects in subsequently conducted clinical studies for treatment of patients with COVID-19. Additionally, hydroxychloroquine (Ki=0.36 μM) and chloroquine (Ki=0.56 μM) were also found to potently inhibit SARS-CoV-2 Mpro for the first time. We anticipate that the FEP-ABFE prediction-based virtual screening approach will be useful in many other drug repurposing or discovery efforts.Significance StatementDrug repurposing effort for treatment of a new disease, such as COVID-19, usually starts from a virtual screening of existing drugs, followed by experimental validation, but the actual hit rate is generally rather low with traditional computational methods. It has been demonstrated that a new virtual screening approach with accelerated free energy perturbation-based absolute binding free energy (FEP-ABFE) predictions can reach an unprecedently high hit rate, leading to successful identification of 16 potent inhibitors of SARS-CoV-2 main protease (Mpro) from computationally selected 25 drugs under a threshold of Ki = 4 μM. The outcomes of this study are valuable for not only drug repurposing to treat COVID-19, but also demonstrating the promising potential of the FEP-ABFE prediction-based virtual screening approach.

Amyloid aggregation of human islet amyloid polypeptide (hIAPP) is linked to insulin-producing islet cell death in type II diabetes. Previous studies have shown that Zinc (Zn(II)) and insulin, co-secreted with hIAPP, have an inhibition effect on hIAPP aggregation. Lipid membranes have also been shown to significantly influence the aggregation kinetics of hIAPP. An increasing number of studies report the importance of developing small molecule inhibitors to suppress the hIAPP’s aggregation and subsequent toxicity. The ability of epigallocatechin-gallate (EGCG) to inhibit aggregation of a variety of amyloid peptide/proteins initiated numerous studies as well as the development of derivative compounds to potentially treat amyloid diseases. In this study, a combination of Thioflavin- T fluorescence kinetics, transmission electron microscopy, isothermal titration calorimetery, circular dicrosim and nucelar magnetic resonance experiments were used to demonstrate a significant enhancement in EGCG’s efficiency when complexed with Zn(II). We demonstrate that the Zn-EGCG complex is able to significantly suppress hIAPP’s amyloid aggregation both in presence and absence of lipid membrane. Circular dichroism experiments indicate the formation and stabilization of a helical structure of hIAPP in presence of the EGCG:Zn(II) complex. Our results also reveal the ability of EGCG or EGCG:Zn(II) to efficiently suppress hIAPP’s cellular toxicity. We believe that the reported results could be useful to develop strategies to trap hIAPP intermediates for further biophysical and structural studies, and also to devise approaches to abolish amyloid aggregation and cellular toxicity.