Functional and druggability analysis of the SARS-CoV-2 proteome

Abstract

The infectious coronavirus disease (COVID-19) pandemic, caused by the coronavirus SARS-CoV-2, appeared in December 2019 in Wuhan, China, and has spread worldwide. As of today, more than 46 million people have been infected and over 1.2 million fatalities. With the purpose of contributing to the development of effective therapeutics, we performed an in silico determination of binding hot-spots and an assessment of their druggability within the complete SARS-CoV-2 proteome. All structural, non-structural, and accessory proteins have been studied, and whenever experimental structural data of SARS-CoV-2 proteins were not available, homology models were built based on solved SARS-CoV structures. Several potential allosteric or protein-protein interaction druggable sites on different viral targets were identified, knowledge that could be used to expand current drug discovery endeavors beyond the currently explored cysteine proteases and the polymerase complex. It is our hope that this study will support the efforts of the scientific community both in understanding the molecular determinants of this disease and in widening the repertoire of viral targets in the quest for repurposed or novel drugs against COVID-19.

Keywords: Binding hot-spots; COVID-19; Coronavirus; Drug discovery; Druggability; SARS-CoV-2.

Graphical abstract

Fig. 1

Schematic representation of a SARS-CoV-2 viral particle and key steps in virus entry. (A) The N, S, E and M proteins are represented in their oligomeric state. N protein dimers bind +ssRNA, forming the nucleocapsid. The nucleocapsid is surrounded by the viral membrane that contains S, E and M proteins. The M protein is shown interacting with the S, E and N proteins. (B) Domain localization of the S protein, showing the S1 and S2 fragments; S1 contains the receptor binding domain (RBD), and S2 the fusion peptide (FP). (C) Angiotensin I converting enzyme 2 (ACE2) recognition by RBD, and the subsequent S proteolytic activation by a Furine-like protease or TMPRSS2. (D) Viral and host membrane fusion induction by the exposed FP.

Fig. 2

SARS-CoV-2 genome organization. (A) Open reading frames (ORF) distribution in SARS-CoV-2 genome. (B) Non-structural proteins (nsp's) distribution in orf1a and orf1ab, detailing multidomain organization of nsp3, nsp12, and nsp14; red and blue arrows indicate PL^pro and M^pro cleaving sites, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Representative steps during the synthesis of a complementary RNA strain. Priming of the complementary strain, in this case, a -ssRNA, is catalyzed by nsp8 (red letters and arrows). Primer dependent RNA extension, catalyzed by nsp7-nsp8-nsp12 complex (blue lines and arrows). Magenta asterisk (*) and arrow represent a mis-incorporated nucleotide or nucleotide analog, and nonobligate RNA chain termination, respectively. Orange lines and arrows represent nucleotides forming dsRNA or structured RNA, and extension inhibition, respectively. Light green arrow represents nucleotide excision by nsp14 3′-5′ exonuclease (ExoN). Dark green arrow represents the unwinding activity of nsp13 helicase. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Viral RNA CAP synthesis. The capping of viral RNA has 4 enzymatic steps. i) Removal of the first phosphate of the 5′-triphosphate end of RNA by a RNA 5′-triphosphatase (RTPase), probably nsp13 (red arrow); ii) Addition of a GMP molecule to the 5′RNA by an unconfirmed guanylyltransferase (GTase) (blue); iii) Methylation of GpppN-RNA at the N7 position by nsp14 N7-methyltransferase (N7-MTase), forming the cap-0 (green arrow); iv) Methylation at the 2′-O position of the first nucleotide's ribose by the 2′-O-methyltransferase (2′-O-Mtase) (nsp16), yielding the cap-1 (orange arrow). Dotted or arrow lines indicate unconfirmed enzymes. Pi: phosphate, Ppi: pyrophosphate, SAM: S-Adenosyl methionine, SAH: S-Adenosyl homocysteine, m: methyl group. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Structure and binding sites of SARS-CoV-2 PL^pro. (A) Complex of PL^pro with inhibitor VIR251 covalently bound to C111 within the catalytic binding site (PDB 6XW4). (B) Potential binding site (yellow mesh) on SARS-CoV-2 PL^pro (ivory surface). Ubiquitin (red) and ISG15 (green) are displayed in ribbon representation. The N-terminus of these two proteins are inserted within the catalytic site. A small-molecule binding to the predicted site would interfere with ubiquitin binding, but not with ISG15. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

SARS-CoV-2 main protease M^pro binding sites. (A) M^pro (grey ribbon) in complex with peptide inhibitor N3 (PDB 6LU7). The protein subsites S1, S2, S4 and S1′ are labeled. The molecular surface of the other protomer is shown in light green. (B) A cryptic site (brown) on M^pro with a CS site nearby lined up by residues T199, Y237, Y239, L271, L272, G275, M276, and A285-L287 (yellow surface). M^pro is represented by a green molecular surface. N3 is also displayed within the catalytic site (light yellow carbons). The other protomer is represented in magenta ribbon. A small-molecule binding to this potential site might interfere with homodimerization. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

The SARS-CoV-2 RdRp complex (nsp12-nsp8-nsp7). (A) Structure of the nsp12-nsp7-nsp8 complex. The channel in the middle corresponds to the catalytic active site. Color code: nsp7, white ribbon; nsp8-2, light blue ribbon; nsp8-1, yellow ribbon. The nsp12 domains are colored as follows: palm, yellow; fingers, tans; palm, red; interface, pale green ribbon; NiRAN, magenta ribbon. (B) RdRp complex bound to RNA. Nsp12 is displayed as a green surface. Color code: Primer RNA, blue; template RNA, red; nsp7, yellow ribbon; nsp8-1, grey ribbon. (C) Molecule of ADP-Mg²⁺ within the NiRAN domain of nsp12. Interacting residues are shown, in what may constitute a druggable binding site. (D) Target site in nsp8 (light yellow surface). The predicted binding site is represented using a blue mesh representation, and nsp12 is shown as grey ribbon. Nsp12 residues N386–K391 are displayed (though not labeled) to highlight that a small molecule binding to these potential sites might interfere with PPIs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

The SARS-CoV-2 helicase/NTPase/RTPase (nsp13). (A) The ADP-Mg²⁺ bound within nsp13. This site has been identified as being involved in NTP hydrolysis in SARS-CoV, and could constitute a druggable site. (B) Two potential borderline druggable binding sites identified in nsp13. The structure of RNA (blue) was modeled based on the yeast Upf1-RNA complex structure (PDB 2XZL). Nsp13 is represented as a green ribbon, but nsp13 domains 1A and 2A are displayed as grey and tan molecular surfaces, respectively. The potential sites are shown in yellow. Based on our model, molecules binding to these sites might interfere with RNA binding. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

The ExoN/MTase complex (nsp14-nsp10) (A) The ExoN nsp14 domain (in green) and the MTase domain (in red) are connected by the hinge loop F286-G300 (yellow). Nsp10 is shown in dark grey ribbon, and a S-adenosyl-L-methionine (SAM) molecule (light yellow carbons) is displayed within the catalytic site. (B) A model of SAM (light yellow carbons) within the catalytic site of the MTase domain of SARS-CoV-2 nsp14. A molecule of guanosine-P3-adenosine-5′,5′-triphosphate (GpppA) (green carbons) is added as reference. (C) Potential druggable (allosteric) binding site in the vicinity of the hinge region F286-G300 (in yellow), including Y296 and P297. The linked nsp14 domains ExoN and MTase are displayed in green and magenta, respectively. (D) Druggable site (yellow surface) within a cryptic site on nsp10 (colored in lighter or darker brown according to the cryptic score). The ExoN domain of nsp14 is shown as green ribbon. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10

The SARS-CoV-2 RNA nucleoside-2′O-methyltransferase complex (nsp16-nsp10). (A) Structure of the 2′O-MTase (nsp16, green molecular surface) heterodimer with nsp10 (magenta ribbon). Molecules of SAM (light yellow carbons) and m7GpppA (cyan carbons) are shown within the catalytic site. (B) Catalytic site of nsp16 with methyl donor SAM (light yellow carbons) and methyl acceptor m7GpppA (cyan carbons). The nucleotide binding site flexible loops (D26-K38, M131-N138) are colored in blue. The highly conserved KDKE motif (K46, D130, K170, E203) for methyl-transfer, found in many 2′O-MTases, is highlighted in magenta, and oxygen water molecules are displayed in red. (C) Binding hot-spot (transparent yellow) identified with FTMap in the vicinity of residues L57, T58, A188, C209, N210, and S276, on the surface of nsp16, within an extended cryptic site identified in the same region using CryptoSite (brown colored surface). Small-molecules bound within that site (taken from crystal structures) are also displayed (light yellow carbon atoms): [adenosine, 2-(n-morpholino)-ethanesulfonic acid, β-D-fructopyranose, 7-methyl-guanosine-5′-triphosphate, and 7-methyl-guanosine-5′-diphosphate]. This site lies on the opposite side of the catalytic site, ~25 Å away from it, in what thus could be an allosteric site. (D) Extension of the RNA groove in nsp16 towards nsp10. Five RNA nucleotides are shown (light yellow carbon atoms), which correspond to those of the human mRNA 2′O-MTase (PDB 4N48), after structural superposition of the binding site residues. SAM is displayed in grey carbon atoms. The consensus site identified with FTMap is shown in yellow mesh. Nsp16 and nsp10 are colored according to their electrostatic potential (blue, positively charged; red, negatively charged; white, neutral). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 11

Structure and potential binding sites of the SARS-CoV-2 accessory protein orf3a. (A) Ribbon representation of the orf3a homodimer (cyan and red). Residues W131, R134, K136, H150, T151, N152,C153, and D155, which might be involved in homo-tetramerization, are displayed (not labeled for the sake of clarity). (B) Potential druggable site within the orf3a homodimer interface (blue surface). The neighboring residues are displayed, and labeled for one protomer. (C) Tetramerization interface and a partially overlapping potentially druggable site (green molecular surface). Residue labels have been colored red (binding site), blue (tetramerization interface), black (common residues). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)