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Cryo-EM disclosed how the D614G mutation changes SARS-CoV-2 spike protein structure.

Reporter: Dr. Premalata Pati, Ph.D., Postdoc

SARS-CoV-2, the virus that causes COVID-19, has had a major impact on human health globally; infecting a massive quantity of people around 136,046,262 (John Hopkins University); causing severe disease and associated long-term health sequelae; resulting in death and excess mortality, especially among older and prone populations; altering routine healthcare services; disruptions to travel, trade, education, and many other societal functions; and more broadly having a negative impact on peoples physical and mental health.

It’s need of the hour to answer the questions like what allows the variants of SARS-CoV-2 first detected in the UK, South Africa, and Brazil to spread so quickly? How can current COVID-19 vaccines better protect against them?

Scientists from the Harvard Medical School and the Boston Children’s Hospital help answer these urgent questions. The team reports its findings in the journal “Science a paper entitled Structural impact on SARS-CoV-2 spike protein by D614G substitution. The mutation rate of the SARS-CoV-2 virus has rapidly evolved over the past few months, especially at the Spike (S) protein region of the virus, where the maximum number of mutations have been observed by the virologists.

Bing Chen, HMS professor of pediatrics at Boston Children’s, and colleagues analyzed the changes in the structure of the spike proteins with the genetic change by D614G mutation by all three variants. Hence they assessed the structure of the coronavirus spike protein down to the atomic level and revealed the reason for the quick spreading of these variants.


This model shows the structure of the spike protein in its closed configuration, in its original D614 form (left) and its mutant form (G614). In the mutant spike protein, the 630 loop (in red) stabilizes the spike, preventing it from flipping open prematurely and rendering SARS-CoV-2 more infectious.

Fig. 1. Cryo-EM structures of the full-length SARS-CoV-2 S protein carrying G614.

(A) Three structures of the G614 S trimer, representing a closed, three RBD-down conformation, an RBD-intermediate conformation and a one RBD-up conformation, were modeled based on corresponding cryo-EM density maps at 3.1-3.5Å resolution. Three protomers (a, b, c) are colored in red, blue and green, respectively. RBD locations are indicated. (B) Top views of superposition of three structures of the G614 S in (A) in ribbon representation with the structure of the prefusion trimer of the D614 S (PDB ID: 6XR8), shown in yellow. NTD and RBD of each protomer are indicated. Side views of the superposition are shown in fig. S8.

IMAGE SOURCE: Bing Chen, Ph.D., Boston Children’s Hospital, https://science.sciencemag.org/content/early/2021/03/16/science.abf2303

The work

The mutant spikes were imaged by Cryo-Electron microscopy (cryo-EM), which has resolution down to the atomic level. They found that the D614G mutation (substitution of in a single amino acid “letter” in the genetic code for the spike protein) makes the spike more stable as compared with the original SARS-CoV-2 virus. As a result, more functional spikes are available to bind to our cells’ ACE2 receptors, making the virus more contagious.


Fig. 2. Cryo-EM revealed how the D614G mutation changes SARS-CoV-2 spike protein structure.

IMAGE SOURCE:  Zhang J, et al., Science

Say the original virus has 100 spikes,” Chen explained. “Because of the shape instability, you may have just 50 percent of them functional. In the G614 variants, you may have 90 percent that is functional. So even though they don’t bind as well, the chances are greater and you will have an infection

Forthcoming directions by Bing Chen and Team

The findings suggest the current approved COVID-19 vaccines and any vaccines in the works should include the genetic code for this mutation. Chen has quoted:

Since most of the vaccines so far—including the Moderna, Pfizer–BioNTech, Johnson & Johnson, and AstraZeneca vaccines are based on the original spike protein, adding the D614G mutation could make the vaccines better able to elicit protective neutralizing antibodies against the viral variants

Chen proposes that redesigned vaccines incorporate the code for this mutant spike protein. He believes the more stable spike shape should make any vaccine based on the spike more likely to elicit protective antibodies. Chen also has his sights set on therapeutics. He and his colleagues are further applying structural biology to better understand how SARS-CoV-2 binds to the ACE2 receptor. That could point the way to drugs that would block the virus from gaining entry to our cells.

In January, the team showed that a structurally engineered “decoy” ACE2 protein binds to SARS-CoV-2 200 times more strongly than the body’s own ACE2. The decoy potently inhibited the virus in cell culture, suggesting it could be an anti-COVID-19 treatment. Chen is now working to advance this research into animal models.

Main Source:

Abstract

Substitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 appears to facilitate rapid viral spread. The G614 strain and its recent variants are now the dominant circulating forms. We report here cryo-EM structures of a full-length G614 S trimer, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain. A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity, and to modulate structural rearrangements for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.

https://science.sciencemag.org/content/early/2021/03/16/science.abf2303?rss=1

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Miniproteins against the COVID-19 Spike protein may be therapeutic

Reporter: Stephen J. Williams, PhD

https://pharmaceuticalintelligence.com/2020/09/30/miniproteins-against-the-covid-19-spike-protein-may-be-therapeutic/

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Coronavirus mutation-does it matter?

Reporter : Irina Robu, PhD

Soon after SARS-CoV-2 was detected in China, scientists began analyzing viral sample and posting the genetic codes online. Mutations allowed researchers to track the spread by linking closely related viruses to understand how SARS-CoV-2 infects humans.  They recognized that SARS-CoV-2 encode their genome in RNA and tends to pick up mutations quickly as they are copied inside their hosts.  Yet,  sequencing data suggest that coronaviruses change more slowly than most RNA viruses, probably because of a proofreading enzyme that corrects fatal copying mutations.  In spite of the virus slow mutation rate, scientists have been able to classified more than 12,000 mutations in SARS-CoV-2 genomes.

Many scientists such as David Montefiori, a virologist who spent much of his career studying how chance mutations in HIV helps it evade the immune system thought that COVID-19 might cause the same thing.  His laboratory in collaboration with Dr. Bette Korber investigated several thousands of coronavirus sequences for mutations that might have changed virus properties around the world.

Compared to HIV, SARS-CoV-2 seems to be changing slower than it spreads, but one mutation is obvious. That mutation  includes a gene encoding the spike protein, which helps the virus particles penetrate cells. According to Korber, the 614th amino acid position of the spike protein, the amino acid aspartate was replaced by glycine, because of a mutation, D614G that altered a single nucleotide in the virus’s 29,903-letter RNA code.

To observe whether D614G  mutation made the virus more transmissible, Montefiori evaluated its effects under laboratory conditions but he couldn’t study the natural SARS-CoV-2 virus in his lab, because of the biosafety containment required. So, he studied a genetically modified form of HIV that used the SARS-CoV-2 spike protein to infect cells. Such ‘pseudo virus’ particles are a workhorse of virology labs: they enable the safe study of deadly pathogens such as the Ebola virus, and they make it simpler to test the effects of mutations.

The strongest sign that D614G has a consequence on the spread of SARS-CoV-2 in humans comes from an ambitious UK effort called the COVID-19 Genomics UK Consortium, which has analyzed genomes of around 25,000 viral samples. From these data, researchers have identified more than 1,300 instances in which a virus entered the United Kingdom and spread, including examples of D- and G-type viruses.

What is clearly known is that D614G is an adaptation that helps the virus infect cells or compete with viruses that don’t carry the change, while at the same time altering a bit of information about how SARS-CoV-2 spreads between people and through a population.  Some scientists believe that D614G mutation should explain how SARS-CoV-2 fuses with cells and can use that process to develop a more efficient vaccine. 

At the present time, the evidence suggests that D614G doesn’t stop the immune system’s neutralizing antibodies from recognizing SARS-CoV-2, partly because the mutation is not in the spike protein’s receptor-binding domain.

SOURCE

https://www.nature.com/articles/d41586-020-02544-6?utm_source=Nature+Briefing

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Miniproteins against the COVID-19 Spike protein may be therapeutic

Reporter: Stephen J. Williams, PhD

Computer-designed proteins may protect against coronavirus

At a Glance

  • Researchers designed “miniproteins” that bound tightly to the SARS-CoV-2 spike protein and prevented the virus from infecting human cells in the lab.
  • More research is underway to test the most promising of the antiviral proteins.

 

 

 

 

 

 

 

An artist’s conception of computer-designed miniproteins (white) binding coronavirus spikes. UW Institute for Protein Design

The surface of SARS-CoV-2, the virus that causes COVID-19, is covered with spike proteins. These proteins latch onto human cells, allowing the virus to enter and infect them. The spike binds to ACE2 receptors on the cell surface. It then undergoes a structural change that allows it to fuse with the cell. Once inside, the virus can copy itself and produce more viruses.

Blocking entry of SARS-CoV-2 into human cells can prevent infection. Researchers are testing monoclonal antibody therapies that bind to the spike protein and neutralize the virus. But these antibodies, which are derived from immune system molecules, are large and not ideal for delivery through the nose. They’re also often not stable for long periods and usually require refrigeration.

Researchers led by Dr. David Baker of the University of Washington set out to design synthetic “miniproteins” that bind tightly to the coronavirus spike protein. Their study was funded in part by NIH’s National Institute of General Medical Sciences (NIGMS) and National Institute of Allergy and Infectious Diseases (NIAID). Findings appeared in Science on September 9, 2020.

The team used two strategies to create the antiviral miniproteins. First, they incorporated a segment of the ACE2 receptor into the small proteins. The researchers used a protein design tool they developed called Rosetta blueprint builder. This technology allowed them to custom build proteins and predict how they would bind to the receptor.

The second approach was to design miniproteins from scratch, which allowed for a greater range of possibilities. Using a large library of miniproteins, they identified designs that could potentially bind within a key part of the coronavirus spike called the receptor binding domain (RBD). In total, the team produced more than 100,000 miniproteins.

Next, the researchers tested how well the miniproteins bound to the RBD. The most promising candidates then underwent further testing and tweaking to improve binding.

Using cryo-electron microscopy, the team was able to build detailed pictures of how two of the miniproteins bound to the spike protein. The binding closely matched the predictions of the computational models.

Finally, the researchers tested whether three of the miniproteins could neutralize SARS-CoV-2. All protected lab-grown human cells from infection. Candidates LCB1 and LCB3 showed potent neutralizing ability. These were among the designs created from the miniprotein library. Tests suggested that these miniproteins may be more potent than the most effective antibody treatments reported to date.

“Although extensive clinical testing is still needed, we believe the best of these computer-generated antivirals are quite promising,” says Dr. Longxing Cao, the study’s first author. “They appear to block SARS-CoV-2 infection at least as well as monoclonal antibodies but are much easier to produce and far more stable, potentially eliminating the need for refrigeration.”

Notably, this study demonstrates the potential of computational models to quickly respond to future viral threats. With further development, researchers may be able to generate neutralizing designs within weeks of obtaining the genome of a new virus.

—by Erin Bryant

Source: https://www.nih.gov/news-events/nih-research-matters/computer-designed-proteins-may-protect-against-coronavirus

Original article in Science

De novo design of picomolar SARS-CoV-2 miniprotein inhibitors

 

  1. View ORCID ProfileLongxing Cao1,2
  2. Inna Goreshnik1,2
  3. View ORCID ProfileBrian Coventry1,2,3
  4. View ORCID ProfileJames Brett Case4
  5. View ORCID ProfileLauren Miller1,2
  6. Lisa Kozodoy1,2
  7. Rita E. Chen4,5
  8. View ORCID ProfileLauren Carter1,2
  9. View ORCID ProfileAlexandra C. Walls1
  10. Young-Jun Park1
  11. View ORCID ProfileEva-Maria Strauch6
  12. View ORCID ProfileLance Stewart1,2
  13. View ORCID ProfileMichael S. Diamond4,7
  14. View ORCID ProfileDavid Veesler1
  15. View ORCID ProfileDavid Baker1,2,8,*

See all authors and affiliations

Science  09 Sep 2020:
eabd9909
DOI: 10.1126/science.abd9909

Abstract

Targeting the interaction between the SARS-CoV-2 Spike protein and the human ACE2 receptor is a promising therapeutic strategy. We designed inhibitors using two de novo design approaches. Computer generated scaffolds were either built around an ACE2 helix that interacts with the Spike receptor binding domain (RBD), or docked against the RBD to identify new binding modes, and their amino acid sequences designed to optimize target binding, folding and stability. Ten designs bound the RBD with affinities ranging from 100pM to 10nM, and blocked ARS-CoV-2 infection of Vero E6 cells with IC 50 values between 24 pM and 35 nM; The most potent, with new binding modes, are 56 and 64 residue proteins (IC 50 ~ 0.16 ng/ml). Cryo-electron microscopy structures of these minibinders in complex with the SARS-CoV-2 spike ectodomain trimer with all three RBDs bound are nearly identical to the computational models. These hyperstable minibinders provide starting points for SARS-CoV-2 therapeutics.

 

RESEARCH ARTICLE

De novo design of picomolar SARS-CoV-2 miniprotein inhibitors

  1. View ORCID ProfileLongxing Cao1,2
  2. Inna Goreshnik1,2
  3. View ORCID ProfileBrian Coventry1,2,3
  4. View ORCID ProfileJames Brett Case4
  5. View ORCID ProfileLauren Miller1,2
  6. Lisa Kozodoy1,2
  7. Rita E. Chen4,5
  8. View ORCID ProfileLauren Carter1,2
  9. View ORCID ProfileAlexandra C. Walls1
  10. Young-Jun Park1
  11. View ORCID ProfileEva-Maria Strauch6
  12. View ORCID ProfileLance Stewart1,2
  13. View ORCID ProfileMichael S. Diamond4,7
  14. View ORCID ProfileDavid Veesler1
  15. View ORCID ProfileDavid Baker1,2,8,*

See all authors and affiliations

Science  09 Sep 2020:
eabd9909
DOI: 10.1126/science.abd9909

Abstract

Targeting the interaction between the SARS-CoV-2 Spike protein and the human ACE2 receptor is a promising therapeutic strategy. We designed inhibitors using two de novo design approaches. Computer generated scaffolds were either built around an ACE2 helix that interacts with the Spike receptor binding domain (RBD), or docked against the RBD to identify new binding modes, and their amino acid sequences designed to optimize target binding, folding and stability. Ten designs bound the RBD with affinities ranging from 100pM to 10nM, and blocked ARS-CoV-2 infection of Vero E6 cells with IC 50 values between 24 pM and 35 nM; The most potent, with new binding modes, are 56 and 64 residue proteins (IC 50 ~ 0.16 ng/ml). Cryo-electron microscopy structures of these minibinders in complex with the SARS-CoV-2 spike ectodomain trimer with all three RBDs bound are nearly identical to the computational models. These hyperstable minibinders provide starting points for SARS-CoV-2 therapeutics.

 

SARS-CoV-2 infection generally begins in the nasal cavity, with virus replicating there for several days before spreading to the lower respiratory tract (1). Delivery of a high concentration of a viral inhibitor into the nose and into the respiratory system generally might therefore provide prophylactic protection and/or therapeutic benefit for treatment of early infection, and could be particularly useful for healthcare workers and others coming into frequent contact with infected individuals. A number of monoclonal antibodies are in development as systemic treatments for COVID-19 (26), but these proteins are not ideal for intranasal delivery as antibodies are large and often not extremely stable molecules and the density of binding sites is low (two per 150 KDa. antibody); antibody-dependent disease enhancement (79) is also a potential issue. High-affinity Spike protein binders that block the interaction with the human cellular receptor angiotensin-converting enzyme 2 (ACE2) (10) with enhanced stability and smaller sizes to maximize the density of inhibitory domains could have advantages over antibodies for direct delivery into the respiratory system through intranasal administration, nebulization or dry powder aerosol. We found previously that intranasal delivery of small proteins designed to bind tightly to the influenza hemagglutinin can provide both prophylactic and therapeutic protection in rodent models of lethal influenza infection (11).

Design strategy

We set out to design high-affinity protein minibinders to the SARS-CoV-2 Spike RBD that compete with ACE2 binding. We explored two strategies: first we incorporated the alpha-helix from ACE2 which makes the majority of the interactions with the RBD into small designed proteins that make additional interactions with the RBD to attain higher affinity (Fig. 1A). Second, we designed binders completely from scratch without relying on known RBD-binding interactions (Fig. 1B). An advantage of the second approach is that the range of possibilities for design is much larger, and so potentially a greater diversity of high-affinity binding modes can be identified. For the first approach, we used the Rosetta blueprint builder to generate miniproteins which incorporate the ACE2 helix (human ACE2 residues 23 to 46). For the second approach, we used RIF docking (12) and design using large miniprotein libraries (11) to generate binders to distinct regions of the RBD surface surrounding the ACE2 binding site (Fig. 1 and fig. S1).

 

 

 

 

 

 

 

 

 

 

 

Download high-res image

Fig. 1 Overview of the computational design approaches.

(A) Design of helical proteins incorporating ACE2 helix. (B) Large scale de novo design of small helical scaffolds (top) followed by rotamer interaction field (RIF) docking to identify shape and chemically complementary binding modes.

For full article please  go to Science at https://science.sciencemag.org/content/early/2020/09/08/science.abd9909

 

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