Angiotensin-I-converting enzyme (ACE), also known as peptidyl-dipeptidase A or kininase II, was first isolated in 1956. ACE is a chloride-dependent metalloenzyme. ACE cleaves a dipeptide from the carboxyl terminus of the decapeptide angiotensin I resulting in the potent vasopressor angiotensin II, a blood vessel constrictor.
In humans, two forms of the angiotensin-converting enzyme exist. The ubiquitous somatic ACE and the sperm-specific germinal ACE. The same gene encodes both proteins through transcription from alternative promoters. ACE regulates blood pressure as part of the renin-angiotensin-aldosterone and kallikrein-kinin systems as a physiological modulator of hematopoiesis. ACE cleaves angiotensin I (Ang I) to the vasoactive octapeptide angiotensin II (Ang II). Other peptide substrates are also cleaved by ACE. The vasodilator bradykinin, N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), and other bioactive peptides such as substance P, neurotensin, and enkephalin are included as well.
Angiotensin-converting enzyme 2 (ACE2) is a recently identified human homolog of ACE. ACE2 is a novel metallo-carboxy-peptidase with specificity, tissue distribution, and function different from those of ACE. Both, ACE2 and ACE are zinc metallopeptidases and angiotensin-converting enzymes existing in a membrane-associated and a secreted form. However, many differences exist between these two enzymes. For example, ACE2 contains a single zinc-binding catalytic domain, 42% identical to the human ACE active domain. ACE2 cleaves Ang I, Ang II, apelin-13, apelin-36, dynorphin A-(1–13), and des-Arg bradykinin. ACE2 appears to be important in cardiac function. ACE2 may play a role in the renin-angiotensin system by mediating cardiovascular and renal function.
Angiotensin-Converting Enzyme 2 is the Receptor for SARS-CoV S Protein
Researchers identified ACE 2 as the cellular receptor for SARS coronavirus (SARS-CoV) and the newly emerged coronavirus (SARS-CoV-2) that causes the epidemic COVID-19. The spike protein glycoprotein (S Protein) of the coronavirus is the key target for the development of vaccines, therapeutic antibodies as well as for diagnostics. Wrapp et al. have recently determined the structure of the 2019-nCoV S trimer structure at 3.5-angstroem-resolution using cryo-electron microscopy.
Figure 1: Different views of the Cryo-EM structure of the 209-nCoV spike protein [PBD ID 6VSB].
The spike protein of SARS coronavirus attaches the virus to the cellular receptor, ACE2. The receptor-binding domain (RBD) on the S protein mediates the interaction. The interaction between the S glycoprotein and ACE2 plays a critical role in SARS pathogenesis. Binding of S protein to ACE2 leads to the downregulation of the receptor resulting in the deregulation of the renin-angiotensin system and eventual lung injury.
Li et al. in 2005 determined the crystal structure at 2.0 Ångstroem resolution of the RBD bound with the peptidase domain of human ACE2. The structure of RBD is a scaffold for the design of coronavirus peptide vaccines.
Figure 2: Different views of the SARS coronavirus spike receptor-binding domain (RBD) in complex with the receptor ACE2. The interface between the two proteins illustrate residue that facilitate efficient cross-species infection and human-to-human transmission.
Since the spike protein mediates the entry of the coronavirus into cells, it is a valid target for the development of vaccines and antiviral agents such as viral inhibitor drugs. The S protein of SARS-CoV does not appear to be cleaved and is presumed to have two functional domains. The border between them has been suggested to be around amino acid 680. The receptor-binding domain (RBD) has been narrowed down to a 193 amino acid fragment (residues 318–510). This domain binds ACE2 with greater affinity than does a larger protein fragment representing the S1 domain (residues 12–672). Several amino acid residues were found to be important for binding the S glycoprotein.
Han et al., in 2006, performed alanine scanning mutagenesis analyses of ACE2. Cells were infected with non-replicating SARS pseudoviruses to study effects of mutations on viral entry. The study found that charged amino acids between residues 22 and 57 are important for virus infection. Three general regions on ACE2 are important for binding the S glycoprotein: (1) residues K31 and Y41 on α-helix 1; (2) M82, Y83 and P84 on loop 2; and (3) K353, D355 and R357 on β-sheet 5.
ACE2-derived peptides that bind S glycoprotein with high affinity are predicted to block the interaction between ACE2 and the S protein thereby inhibiting virus infection. However, the longer peptides P4 and P5 were found to be more active with approximately 50 % inhibitory concentrations (IC50) of 50 μM and 6 μM, respectively. The peptide P6 was found to be much more potent than peptides P4 or P5 peptides.
Table 1: Peptides studied
Han et al. also synthesized an artificial peptide P6 containing 31 amino acids. The research group joined the peptide fragments 22 to 44, and 351 to 357 to each other with a glycine. Peptide P6 inhibited SARS pseudovirus infection with an IC50 of approximately 0.1 μM. 60- and 500-fold lower than peptides P5 and P4, respectively.
The potent antiviral activity of P6 peptide suggested that its conformation may resemble the one in the ACE2 crystal structure when it is bound to S protein.
These findings suggest that the P6 peptide is a strong candidate for the design of therapeutic peptides that inhibit SARS-CoV infection and help prevent lethal lung failure. However, more research needs to be done before finding an efficient drug or vaccine for COV19.
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