ABSTRACT
Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. One-Sentence SummaryWe designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.
ABSTRACT
The COVID-19 pandemic has demonstrated the need for exploring different diagnostic and therapeutic modalities to tackle future viral threats. In this vein, we propose the idea of sentinel cells, cellular biosensors capable of detecting viral antigens and responding to them with customizable responses. Using SARS-CoV-2 as a test case, we developed a live cell sensor (SARSNotch) using a de novo-designed protein binder against the SARS-CoV-2 Spike protein. SARSNotch is capable of driving custom genetically-encoded payloads in immortalized cell lines or in primary T lymphocytes in response to purified SARS-CoV-2 Spike or in the presence of Spike-expressing cells. Furthermore, SARSNotch is functional in a cellular system used in directed evolution platforms for development of better binders or therapeutics. In keeping with the rapid dissemination of scientific knowledge that has characterized the incredible scientific response to the ongoing pandemic, we extend an open invitation for others to make use of and improve SARSNotch sentinel cells in the hopes of unlocking the potential of the next generation of smart antiviral therapeutics.
ABSTRACT
Despite the introduction of public health measures and spike protein-based vaccines to mitigate the COVID-19 pandemic, SARS-CoV-2 infections and deaths continue to rise. Previously, we used a structural design approach to develop picomolar range miniproteins targeting the SARS-CoV-2 receptor binding domain. Here, we investigated the capacity of modified versions of one lead binder, LCB1, to protect against SARS-CoV-2-mediated lung disease in human ACE2-expressing transgenic mice. Systemic administration of LCB1-Fc reduced viral burden, diminished immune cell infiltration and inflammation, and completely prevented lung disease and pathology. A single intranasal dose of LCB1v1.3 reduced SARS-CoV-2 infection in the lung even when given as many as five days before or two days after virus inoculation. Importantly, LCB1v1.3 protected in vivo against a historical strain (WA1/2020), an emerging B.1.1.7 strain, and a strain encoding key E484K and N501Y spike protein substitutions. These data support development of LCB1v1.3 for prevention or treatment of SARS-CoV-2 infection.
ABSTRACT
We used two approaches to design proteins with shape and chemical complementarity to the receptor binding domain (RBD) of SARS-CoV-2 Spike protein near the binding site for the human ACE2 receptor. Scaffolds were built around an ACE2 helix that interacts with the RBD, or de novo designed scaffolds were docked against the RBD to identify new binding modes. In both cases, designed sequences were optimized first in silico and then experimentally for target binding, folding and stability. Nine designs bound the RBD with affinities ranging from 100pM to 10nM, and blocked bona fide SARS-CoV-2 infection of Vero E6 cells with IC50 values ranging from 35 pM to 35 nM; the most potent of these -- 56 and 64 residue hyperstable proteins made using the second approach -- are roughly six times more potent on a per mass basis (IC50 ~ 0.23 ng/ml) than the best monoclonal antibodies reported thus far. Cryo-electron microscopy structures of the SARS-CoV-2 spike ectodomain trimer in complex with the two most potent minibinders show that the structures of the designs and their binding interactions with the RBD are nearly identical to the computational models, and that all three RBDs in a single Spike protein can be engaged simultaneously. These hyperstable minibinders provide promising starting points for new SARS-CoV-2 therapeutics, and illustrate the power of computational protein design for rapidly generating potential therapeutic candidates against pandemic threats.
ABSTRACT
Naturally occurring allosteric protein switches have been repurposed for developing novel biosensors and reporters for cellular and clinical applications 1, but the number of such switches is limited, and engineering them is often challenging as each is different. Here, we show that a very general class of allosteric protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which binding of a peptide key triggers biological outputs of interest 2. Using broadly applicable design principles, we allosterically couple binding of protein analytes of interest to the reconstitution of luciferase activity and a bioluminescent readout through the association of designed lock and key proteins. Because the sensor is based purely on thermodynamic coupling of analyte binding to switch activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We demonstrate the modularity of this platform by creating biosensors that, with little optimization, sensitively detect the anti-apoptosis protein Bcl-2, the hIgG1 Fc domain, the Her2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac Troponin I and an anti-Hepatitis B virus (HBV) antibody that achieve the sub-nanomolar sensitivity necessary to detect clinically relevant concentrations of these molecules. Given the current need for diagnostic tools for tracking COVID-19 3, we use the approach to design sensors of antibodies against SARS-CoV-2 protein epitopes and of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The latter, which incorporates a de novo designed RBD binder, has a limit of detection of 15pM with an up to seventeen fold increase in luminescence upon addition of RBD. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.
ABSTRACT
<p><b>OBJECTIVE</b>To review the current knowledge about the pathophysiological mechanisms, preclinical models, novel contributors and potential therapies of cardiorenal syndrome.</p><p><b>DATA SOURCES</b>The literature concerning cardiorenal syndrome in this review was collected from PubMed published in English up to January 2014.</p><p><b>STUDY SELECTION</b>Original articles and critical reviews related to cardiorenal syndrome were selected and carefully analyzed.</p><p><b>RESULTS</b>Cardiorenal syndrome is a condition characterized by kidney and heart failure where failure of one organ worsens the function of the other thus further accelerating the progressive failure of both organs. The pathophysiology of cardiorenal syndrome is not fully understood, but may be caused by a complex combination of neurohormonal system activation, endothelial dysfunction, proteinuria, oxidative stress, uremic toxins and other factors. Managing cardiorenal syndrome is still a major therapeutic challenge in clinical practice because many of the drugs used to control heart failure can worsen renal function, and vice versa. Non-dialyzable uremic toxins, such as indoxyl sulfate, causing detrimental effects on the heart and kidney as well as stimulation of inflammatory responses, may be an effective therapeutic target for cardiorenal syndrome.</p><p><b>CONCLUSIONS</b>Suitable disease models of cardiorenal syndrome are urgently needed to investigate the pathophysiology and effective therapeutic approaches to the condition. Non-dialyzable protein-bound uremic toxins that may have cardiac and renal effects may provide therapeutic benefit to cardiorenal syndrome patients.</p>
Subject(s)
Female , Humans , Male , Cardio-Renal Syndrome , Metabolism , Toxins, Biological , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To explore the relationship between coronary collateral circulation following percutaneous coronary intervention (PCI) for a single left anterior descending artery and the recovery of cardiac function.</p><p><b>METHODS</b>A total of 625 patients with coronary heart disease were retrospectively analyzed, who received selective coronary angiography demonstrating lesions involving a single left anterior descending artery and underwent stent placement between January, 2010 and December, 2012. According to Rentrop's classification, the patients were divided into group A (n=280) with Rentrop grades 1-3 and group B (n=325) with Rentrop grade 0. Group A were further divided into 3 subgroups according to the source of collateral circulation, namely group A1 (n=200) with contralateral collateral circulation, group A2 (n=44) with contralateral+ ipsilateral collateral circulation, and group A3 (n=36) with ipsilateral collateral circulation. The outcomes of cardiac function recovery were compared between groups A and B and between the 3 subgroups in group A.</p><p><b>RESULTS</b>Compared with patients without collateral circulation, patients with collateral coronary circulation showed greater left ventricular ejection fraction increment and reduction in brain natriuretic peptide and red cell volume distribution width with also lower expansion left ventricular end-diastolic volume. Among the 3 subgroups in group A, cardiac function improvement was the most obvious in patients with contralateral+ ipsilateral collateral circulation (group A2) followed by those in group A3, and was the worst in group A1.</p><p><b>CONCLUSION</b>The presence of collateral coronary circulation promotes cardiac function recovery in patients receiving PCI for lesions involving a single left anterior descending artery. Patients with contralateral+ipsilateral collateral circulation have the best cardiac function improvement followed by those with contralateral collateral circulation.</p>
Subject(s)
Aged , Female , Humans , Male , Middle Aged , Collateral Circulation , Coronary Disease , Therapeutics , Percutaneous Coronary Intervention , Retrospective StudiesABSTRACT
<p><b>OBJECTIVE</b>To review the advances in studies on renal denervation.</p><p><b>DATA SOURCES</b>References concerning renal denervation and resistant hypertension cited in this review were collected from PubMed published in English and those of renal denervation devices from official websites of device manufacturers up to January 2014.</p><p><b>STUDY SELECTION</b>Articles with keywords "renal denervation" and "resistant hypertension" were selected.</p><p><b>RESULTS</b>Renal and systemic sympathetic overactivity plays an important role in pathology of hypertension as well as other diseases characterized by sympathetic overactivity. Renal denervation is a new, catheter based procedure to reduce renal and systemic sympathetic overactivity by disruption of renal sympathetic efferent and afferent nerves through radiofrequency or ultrasound energy delivered to the endoluminal surface of both renal arteries. Although several studies have shown the efficacy and safety of renal denervation in the treatment of resistant hypertension and the potential benefit of the procedure in other diseases, Symplicity HTN 3 study, the most rigorous clinical trial of renal denervation to date, failed to meet its primary endpoint. The procedure also has other limitations such as the lack of long term, efficacy and safety data and the lack of the predictors for the blood pressure lowering response and nonresponse to the procedure. An overview of current renal denervation devices holding Conformité Européenne mark is also included in this review.</p><p><b>CONCLUSIONS</b>Renal denervation is a promising therapeutic approach in the management of resistant hypertension and other diseases characterized by sympathetic overactivity. In its early stage of clinical application, the efficacy of the procedure is still controversial. Large scale, blind, randomized, controlled clinical trials are still necessary to address the limitations of the procedure.</p>
Subject(s)
Humans , Blood Pressure , Physiology , Denervation , Methods , Hypertension , Kidney , Neurosurgical Procedures , Sympathetic Nervous System , MetabolismABSTRACT
Inflammation plays a pivotal role in coronary heart disease. Dendritic cells (DCs) are principal players in inflammation and atherosclerosis. Although the percentage of circulating DC precursors in coronary heart disease have been investigated, circulating myeloid DC (mDC) and plasmacytoid DC (pDC) precursors have not been extensively studied, particularly in relation to the severity of coronary artery lesions in patients with coronary heart disease. In this study, we recruited controls (n = 29), patients with stable angina pectoris (SAP, n = 30), patients with unstable angina pectoris (UAP, n = 56), and patients with acute myocardial infarction (AMI, n = 50). The severity and extent of coronary artery lesions was evaluated by Gensini score, following coronary angiograms. The percentage of circulating mDC and pDC precursors was determined by fluorescence-activated cell sorting (FACS). Plasma levels of MCP-1 and MMP-9, which correlate with atherosclerosis and DC migration, were also measured. The percentage of circulating mDC precursors was reduced in patients with AMI and UAP compared with control and SAP patients, respectively (p < 0.01 for AMI vs. SAP and Control, p < 0.05 for UAP vs. SAP and Control). The percentage of circulating pDC precursors was not significant changed. The levels of plasma MMP-9 and MCP-1 and Genisi score were all increased in patients with AMI and UAP, compared to control and SAP patients, respectively (p < 0.01 for AMI vs. SAP and control, p < 0.05 for UAP vs. SAP and control). Overall, the percentage of circulating mDC precursors was negatively correlated with MCP-1 (p < 0.001), MMP-9 (p < 0.001) and Genisi scores (p < 0.001). Genisi scores were positively correlated with the levels of MCP-1 (p < 0.001) and MMP-9 (p < 0.001). Our study suggested that the percentage of circulating mDC precursors is negatively correlated with the severity and extent of coronary artery lesions in patients with coronary heart disease.
Subject(s)
Coronary Artery Disease/diagnosis , Coronary Vessels/pathology , Dendritic Cells/immunology , Plaque, Atherosclerotic , Adult , Aged , Aged, 80 and over , Angina, Stable/diagnosis , Angina, Stable/immunology , Angina, Stable/pathology , Angina, Unstable/diagnosis , Angina, Unstable/immunology , Angina, Unstable/pathology , Biomarkers/blood , Case-Control Studies , Cell Count , Cell Separation/methods , Chemokine CCL2/blood , Chi-Square Distribution , Coronary Angiography , Coronary Artery Disease/blood , Coronary Artery Disease/immunology , Coronary Artery Disease/pathology , Coronary Vessels/diagnostic imaging , Disease Progression , Female , Flow Cytometry , Humans , Male , Matrix Metalloproteinase 9/blood , Middle Aged , Myocardial Infarction/diagnosis , Myocardial Infarction/immunology , Myocardial Infarction/pathology , Predictive Value of Tests , Severity of Illness IndexABSTRACT
<p><b>OBJECTIVE</b>To assess the association between peripheral blood dendritic cells subtype distribution and plasma monocyte chemoattractant protein 1 (MCP-1) concentration in patients with coronary heart disease (CHD).</p><p><b>METHODS</b>Sixty consecutive CHD patients admitted in our department during the period from November, 2010 to December, 2011 were enrolled, including 10 with stable angina pectoris (SAP), 25 with unstable angina pectoris (UAP), and 25 with acute myocardial infarction (AMI), with 28 healthy volunteers as normal controls. All the subjects underwent routine tests and coronary angiography. The percentages of peripheral blood myeloid dendritic cells (mDCs) and plasma cell-like dendritic cells (pDCs) in peripheral blood mononuclear cells were detected by flow cytometry, and plasma MCP-1 levels were detected using enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>The percentage and absolute quantity of mDCs and pDCs were significantly lower in AMI and UAP groups than in the normal control and SAP groups (P<0.001). In the CHD patients, the plasma MCP-1 level was significantly higher than that in the normal control group (P<0.001) with an inverse correlation with the percentage of peripheral mDCs.</p><p><b>CONCLUSION</b>MCP-1 may promote the migration of mDCs into atherosclerotic plaques and mediate the local immune and inflammatory responses to aggravate plaque instability in CHD patients.</p>
