CG
this is a very detailed and important paper regarding the molecular structure of SARS 2 and drug options. Here are some extracts for this audience that I thought relevant. Bold and underlined are mine.
'Although the optimal strategy to control this disease would be through vaccination capable of generating a long-lasting and protective immune response, there is an urgent need for the development of treatment for this disease
In terms of antiviral strategies, while no specific therapeutics are available, approved or experimental drugs developed for other diseases are being tested in different clinical trials, in an effort to rapidly find accessible treatments with already established safety profiles, through drug repurposing strategies [18-23]. So far, most of these strategies are focused in targeting the two viral proteases, and the RNA-dependent RNA polymerase (RdRp) complex. However, protease inhibitors might bind to host proteases, thus resulting in cell toxicity and side effects [24]; the effect of nucleoside inhibitors targeting RdRp is decreased by the highly efficient SARS-CoV-2 proofreading machine, and even limitations in targeting the S protein have been pointed out [25]. Therefore, alternative therapeutic options to fight COVID-19 are badly needed.
We do hope our contribution will help in the development of fast and effective SARS-CoV2-centered therapeutic options, which considering the high similarity among CoVs, might also be effective against related viruses. Moreover, since it is unlikely that SARS-CoV-2 will be the last CoV to threaten global health, those therapeutics might be instrumental to fight future epidemics.
The last step in the viral cycle is the assembly of the viral particles and the release of the viral progeny. During this step, several N proteins bind and compact each copy of the genomic +ssRNA, forming the nucleocapsid. Proteins S, E, and M, accumulated in the ER-Golgi intermediate
compartment (ERGIC) [48], bind the nucleocapsid, by direct M-N protein interactions [49], and then through the action of E and M proteins on the ERGIC membrane a viral particle is formed [50] in a secretory compartment [51]. Finally, the viral progeny particle or virion is released by the exocytosis pathway [2], and the viral cycle is completed.
E Protein
It is a small homopentameric membrane protein (75 amino-acids per protomer) [180], which has been shown to be essential for viral particle assembly in SARS-CoV [51]. The N-terminal region of the protein spans the lipid bilayer twice, while the C-terminal is exposed to the interior of the virus [181,182]. In IBV and SARS-CoV, the E protein interacts with the viral M protein through an undefined region [183,184]; and also with the host Protein Associated with Lin Seven 1 (PALS1), a factor associated to the pathogenesis [185], thought the E protein C-terminal. Also, in many CoVs,
the E protein works as an ion-channelling viroporin [180], which affects the production of cytokines, and in consequence the inflammatory response [186]. While there is no structural data available for the SARS-CoV-2 E protein, the structure of the TM region of the SARS-CoV homopentamer is available (residues 8-65) (PDB 5X29). Within that region, the corresponding E proteins share 91% sequence identity, and 98% sequence similarity. The homology model of the SARS-CoV-2 E protein is shown in Figure S12. Based on structural and functional considerations, the E protein channel would be the primary target site for drug development. In fact, hexamethylene amiloride (HMA) has been reported to bind to the SARS-CoV E protein homopentamer, but not to an isolated protomer [187]
Orf3a/X1/U274 (I've included this section about ORF3a as it too is a viroporin)
Orf3a was characterized as a potassium ion channel in SARS-CoV, involved in inducing caspase dependent apoptosis under different pathologic conditions [194]. It is included in the virion [195], and also interacts specifically with the M, E, and S structural proteins, as well as with Orf7a/U122
[196]. In SARS-CoV, orf3a expression increases the mRNA levels of all three subunits of fibrinogen, thus promoting fibrosis, one of the serious pathogenic aspects of SARS [197], and the expression of NFκB, IL8, and JNK, all involved in inflammatory responses [198]. Thus, since orf3a is responsible for inducing two of the serious pathological affections induced by SARS-CoV, design therapeutics that suppress its function could be very important; moreover, the presence of similar proteins to orf3a other β-CoVs (SARS-CoV-2 orf3a shares ~73% identity and ~85% similarity with its SARS-CoV counterpart) and in α-CoVs and suggests that drugs targeting orf3a might be a therapeutic option against a broad range of CoV-related diseases [199]
Considering that SARS-CoV orf3a has been identified as an emodin-sensitive potassium-permeable cation channel, the narrow size of the pore in the SARS-CoV2 orf3a structure strongly suggests that the latter is in the closed or inactive conformation [199]...... Since conformational changes of the TMs are needed for channel opening, we could hypothesize that a small-molecule binding within this site would interfere with this rearrangement, or directly block the channel
SARS-CoV-2 orf7a is a transmembrane protein of 121 amino acids (106 if only the mature protein is considered, excluding the signalling peptide), with 86% identity and 94% similarity with respect to its SARS-CoV counterpart......... Like orf3a, orf7a is also included in the virion, and both proteins have been shown to interact with each other in SARSCoV [196,202] and in SARS-CoV-2 [166]. In SARS-CoV, orf7a expression increases the expression of NFκB, IL8 and JNK, all involved in inflammatory responses [198], and the deletion of orf7a reduces the virus titer in 30-fold. Orf7a appears to be unique to SARS CoVs, showing no significant similarity to any other protein, either viral or non-viral
While the functionality and interaction with partners of orf7a are not clearly known, we identified two hot-spots on the surface of orf7a. Both sites might have functional roles, and further studies are needed to confirm this hypothesis, and whether small molecules binding to any of them might interfere with the viral mechanism.
In SARS-CoV, orf9b is a non-essential dimeric membrane protein, produced from an alternative start codon to N-orf9a, with a lipid-binding-like structure [207], and with the ability to bind several other viral proteins [208], including structural proteins, which allows orf9b to be incorporated into the virion [209]. In SARS-CoV, its action is used in interfering with mitochondrial factors that limit IFN responses [210] and in regulating apoptosis, which is a mechanism associated with the immune response [211].......... In this context, a small-molecule which could bind to the central cavity would impede membrane attachment by competing with the lipid tails.
We are convinced that our work will contribute to the quick development of an effective SARS-CoV-2 antiviral strategy, which in view of the high similarity among CoVs, it might be useful to fight related viruses. Moreover, these therapeutic options might be instrumental in fighting different CoV-associated diseases that could threaten global health in the future.
How's that for optimism !
