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==Design==
==Design==
These mini binders, <scene name='10/1075249/Lcb1_fullspike_rotating/1'>LCB1</scene> and AHB2, were designed from “scratch” (de novo) with the intention to mimic the binding of ACE2 to spike protein <ref name="Longxing">PMID:32907861</ref>. Using Rotamer Interaction Field (RIF) docking, the proteins were able to make the most efficient bonding using the ACE2 spike protein binding interface <ref name="Longxing">PMID:32907861</ref>. Using Site Saturation Mutagenesis (SSM), every residue in the minibinder’s helix scaffold will be substituted with each of the 20 amino acids, one at a time <ref name="Valleti">PMID:24970191</ref>. Forming SSM libraries, each of the libraries converged on a small number of closely related sequences, and from these libraries, the design was selected for LCB1 and AHB2 to find the sequence that yields a protein with a high affinity for the spike proteins receptor binding domain  <ref name="Longxing">PMID:32907861</ref>.  
These mini binders, <scene name='10/1075249/Lcb1_fullspike_rotating/1'>LCB1</scene> and <scene name='10/1075249/Ahb2_fullspikeprotein/1'>AHB2</scene>, were designed from “scratch” (de novo) with the intention to mimic the binding of ACE2 to spike protein <ref name="Longxing">PMID:32907861</ref>. Using Rotamer Interaction Field (RIF) docking, the proteins were able to make the most efficient bonding using the ACE2 spike protein binding interface <ref name="Longxing">PMID:32907861</ref>. Using Site Saturation Mutagenesis (SSM), every residue in the minibinder’s helix scaffold will be substituted with each of the 20 amino acids, one at a time <ref name="Valleti">PMID:24970191</ref>. Forming SSM libraries, each of the libraries converged on a small number of closely related sequences, and from these libraries, the design was selected for LCB1 and AHB2 to find the sequence that yields a protein with a high affinity for the spike proteins receptor binding domain  <ref name="Longxing">PMID:32907861</ref>.  




Revision as of 19:15, 10 April 2025

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This Sandbox is Reserved from March 18 through September 1, 2025 for use in the course CH462 Biochemistry II taught by R. Jeremy Johnson and Mark Macbeth at the Butler University, Indianapolis, USA. This reservation includes Sandbox Reserved 1828 through Sandbox Reserved 1846.
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Structure

Introduction

What are Minibinders?

These mini proteins target the interaction between ACE2 and COVID-19 spike protein [1]. The mini binders are small proteins carefully designed to bind to the COVID-19 spike protein with a greater affinity than ACE2 [1]. These mini binders were able to reduce the viral burden of SARS-CoV-2 in mice [2]. These proteins were de novo (from scratch) designs to mimic the ACE2 helix, but have a lower dissociation constant, yielding a greater affinity for the spike protein [1]. The binding Region between spike protein and ACE2 can give a better explanation as to how these proteins were designed.

COVID-19 Disease Pathway

Understanding the pathway of the COVID-19 virus is essential to understanding the mechanism in which the virus’ surface proteins attach to the mini binders. The COVID-19 virus has spike proteins on its surface that bind to the host cell receptor, known as ACE2, and this allows the virus to remain anchored to the host for viral entry [3]. When the spike protein binds to the receptor, ACE2 for example, the cell membrane-associated protease, protease serine 2 TMPRSS2 promotes viral entry by activating the spike protein [4]. The activated spike protein is able to cleave itself into S1 and S2 subunits [4]. The S2 subunit is in charge of viral entry and does this through conformational changes [4]. The S2 subunit will insert it's FP domain into the host cell's membrane, and this will trigger an interaction with the HR2 domain and HR1 trimer to form the 6-helical bundle to bring the viral envelope and cell membrane in close enough distance for viral fusion and ultimately viral entry [4]. Once the virus is within the host cell, it is able to translate viral proteins, eliciting an immune response and spreading the viral particles throughout the body [4].

COVID-19 Viral Infection Interruption

The primary goal of the mini binders is to prevent the spike proteins from binding to ACE2, and when the mini binders are bound to the spike protein, the virus is unable to anchor itself to the host protein [1]. Because the mini binders have a greater binding affinity than ACE2 for the spike protein, they are able to effectively prevent the entry of the virus and ultimately prevent an immune response [1]. Targeting this specific interaction between the COVID-19 spike protein has proven effective and is hopeful target for future therapeutics to treat the virus [4]. LCB1 proved to be quite effective at weakening the immune response, compared to the other mini binders, which can be explained by the LCB1 between the spike protein and LCB1 [1].

Design

These mini binders, LCB1 and AHB2, were designed from “scratch” (de novo) with the intention to mimic the binding of ACE2 to spike protein [1]. Using Rotamer Interaction Field (RIF) docking, the proteins were able to make the most efficient bonding using the ACE2 spike protein binding interface [1]. Using Site Saturation Mutagenesis (SSM), every residue in the minibinder’s helix scaffold will be substituted with each of the 20 amino acids, one at a time [5]. Forming SSM libraries, each of the libraries converged on a small number of closely related sequences, and from these libraries, the design was selected for LCB1 and AHB2 to find the sequence that yields a protein with a high affinity for the spike proteins receptor binding domain [1].


Caption for this structure

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LCB1 AND 7JZU

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  1. ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Cao L, Goreshnik I, Coventry B, Case JB, Miller L, Kozodoy L, Chen RE, Carter L, Walls AC, Park YJ, Strauch EM, Stewart L, Diamond MS, Veesler D, Baker D. De novo design of picomolar SARS-CoV-2 miniprotein inhibitors. Science. 2020 Oct 23;370(6515):426-431. PMID:32907861 doi:10.1126/science.abd9909
  2. ↑ Case JB, Chen RE, Cao L, Ying B, Winkler ES, Johnson M, Goreshnik I, Pham MN, Shrihari S, Kafai NM, Bailey AL, Xie X, Shi PY, Ravichandran R, Carter L, Stewart L, Baker D, Diamond MS. Ultrapotent miniproteins targeting the SARS-CoV-2 receptor-binding domain protect against infection and disease. Cell Host Microbe. 2021 Jul 14;29(7):1151-1161.e5. PMID:34192518 doi:10.1016/j.chom.2021.06.008
  3. ↑ Sang P, Chen YQ, Liu MT, Wang YT, Yue T, Li Y, Yin YR, Yang LQ. Electrostatic Interactions Are the Primary Determinant of the Binding Affinity of SARS-CoV-2 Spike RBD to ACE2: A Computational Case Study of Omicron Variants. Int J Mol Sci. 2022 Nov 26;23(23):14796. PMID:36499120 doi:10.3390/ijms232314796
  4. ↑ 4.0 4.1 4.2 4.3 4.4 4.5 Huang Y, Yang C, Xu XF, Xu W, Liu SW. Structural and functional properties of SARS-CoV-2 spike protein: potential antivirus drug development for COVID-19. Acta Pharmacol Sin. 2020 Sep;41(9):1141-1149. doi: 10.1038/s41401-020-0485-4., Epub 2020 Aug 3. PMID:32747721 doi:https://dx.doi.org/10.1038/s41401-020-0485-4
  5. ↑ Valetti F, Gilardi G. Improvement of biocatalysts for industrial and environmental purposes by saturation mutagenesis. Biomolecules. 2013 Oct 8;3(4):778-811. PMID:24970191 doi:10.3390/biom3040778
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