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Introduction

What are Minibinders?

These mini proteins target the interaction between ACE2 and COVID-19 spike protein ^[3]. The mini binders are small proteins carefully designed to bind to the COVID-19 spike protein with a greater affinity than ACE2 ^[3]. These mini binders were able to reduce the viral burden of SARS-CoV-2 in mice ^[4]. 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 ^[3]. 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 ^[5]. 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 ^[6]. The activated spike protein is able to cleave itself into S1 and S2 subunits ^[6]. The S2 subunit is in charge of viral entry and does this through conformational changes ^[6]. 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 ^[6]. 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 ^[6].

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 ^[3]. 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 ^[3]. Targeting this specific interaction between the COVID-19 spike protein has proven effective and is hopeful target for future therapeutics to treat the virus ^[6]. LCB1 proved to be quite effective at weakening the immune response, compared to the other mini binders, which can be explained by the between the spike protein and LCB1 ^[3].

SARS-COV-2 Spike Protein

The of SARS-COV-2 is a symmetric trimer featuring 3 spike glycoprotein chains (UNIPROT: P0DTC2). Each monomer of the spike is called a spike glycoprotein, and the total assembly contains 2 main parts: The and subunits^[6]. However, the native spike protein does not exist in this state prior to infection. The protein is actually inactive initially, but is later activated by proteases cleaving the inactive S protein into its two active subunits^[6]. The S1 subunit contains the . The RBD is responsible for on the surface of the target cell, as well as neutralizing antibodies. The NTD, CTD, and their relevant interfaces actually play much larger roles in the binding of the spike protein to ACE2 than the RBD does due to their larger surface areas^[6]. The S2 subunit is responsible for viral fusion and entry. Once bound to ACE2, and after the different domains in S2 have anchored to the membrane as well as delivered the viral envelope, the S2 subunit then changes conformation from the pre-hairpin to postfusion-hairpin conformation^[6]. The S2 subunit contains a fusion peptide domain (FP), heptapeptide repeat sequences 1 and 2 (HR1 & HR2), TM domain, and cytoplasmic fusion domain (CT). Full information about the location and structures of these domains within the S2 subunit can be found in references 1 and 3^[6][7]. For the purpose of this article about the minibinders, attention will be directed to the S1 subunit and its binding properties with ACE2.

Figure 1. Spike protein shown in "B-Factor"; depicting mobility and flexibility of different portions. Depicted in red are the most mobile, whilst dark blue are the least mobile. The 2 red portions depict RBDs, which correspond to 1-up and 2-up conformational states.

Throughout the entire process, the spike protein has 3 main conformations. An conformation; an active, "open" conformation; and a conformation mentioned previously^[6][8][7]. In the closed conformation, the RBDs of each monomer are tucked inwards, preventing interaction. In the open conformation, however, 1 or more of these RBDs can be in the "up" conformation, meaning they are exposed and able to interact within the extracellular space. Mainly, there exits a "" and "" conformation in this phase^[8][7]. Depicted in Figure 1, the RBDs of the spike protein have the highest mobility, which further support the many conformational changes in which they are involved. Most of the depictions of the minibinders bound to the spike protein show the spike protein in the 2-up conformation.

ACE2

is a carboxypeptidase present on cell surfaces that is responsible for the degradation of angiotensin II. It is a critical enzyme in the suppression of the renin-angiotensin system. This improves both cardiovascular and renal systems, as well as abates acute respiratory distress syndrome (ARDS). It does the 2 former via the RAS System's role in the regulation of blood pressure, renal function, water homeostasis, electrolyte balance, and/or inflammation^[9]. The critical role that this enzyme plays in the regulation of this system is what results in the adverse symptomology observed in victims of the SARS-COV-2 virus. The ACE2 receptor is considered the only essential receptor in the SARS-COV-2 viral mechanism, and thus the collateral debilitation of ACE2 results in the adverse respiratory effects including ARDS, pulmonary edema, destruction of alveolar structures, and others^[9]. This relationship was further proven when ACE-2 deficient mice had developed these effects at higher rates compared to the wild type^[10].

As mentioned previously, all of the S1 subunit domains play important roles in the . The surface area of the NTD and CTD are particularly important, along with the direct interactions observed in the RBD. Whilst ACE2 is not the focus of this article, understanding its role in the infection pathway of COVID 19, as well as how it binds to the spike protein will assist in understanding the design and functional processes of the minibinders.

Minibinders

Structure

Function

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 ^[3]. Using Rotamer Interaction Field (RIF) docking, the proteins were able to make the most efficient bonding using the ACE2 spike protein binding interface ^[3]. 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 ^[11]. 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 ^[3].

Implications

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