Comprehensive Fragment Screening of the SARS‐CoV‐2 Proteome Explores Novel Chemical Space for Drug Development

SARS‐CoV‐2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti‐virals. Within the international Covid19‐NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR‐detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure‐based drug design against the SCoV2 proteome.

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development.

SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.

Binding Adaptation of GS-441524 Diversifies Macro Domains and Downregulates SARS-CoV-2 de-MARylation Capacity.

Viral infection in cells triggers a cascade of molecular defense mechanisms to maintain host-cell homoeostasis. One of these mechanisms is ADP-ribosylation, a fundamental post-translational modification (PTM) characterized by the addition of ADP-ribose (ADPr) on substrates. Poly(ADP-ribose) polymerases (PARPs) are implicated in this process and they perform ADP-ribosylation on host and pathogen proteins. Some viral families contain structural motifs that can reverse this PTM. These motifs known as macro domains (MDs) are evolutionarily conserved protein domains found in all kingdoms of life. They are divided in different classes with the viral belonging to Macro-D-type class because of their properties to recognize and revert the ADP-ribosylation. Viral MDs are potential pharmaceutical targets, capable to counteract host immune response. Sequence and structural homology between viral and human MDs are an impediment for the development of new active compounds against their function. Remdesivir, is a drug administrated in viral infections inhibiting viral replication through RNA-dependent RNA polymerase (RdRp). Herein, GS-441524, the active metabolite of the remdesivir, is tested as a hydrolase inhibitor for several viral MDs and for its binding to human homologs found in PARPs. This study presents biochemical and biophysical studies, which indicate that GS-441524 selectively modifies SARS-CoV-2 MD de-MARylation activity, while it does not interact with hPARP14 MD2 and hPARP15 MD2. The structural investigation of MD•GS-441524 complexes, using solution NMR and X-ray crystallography, discloses the impact of certain amino acids in ADPr binding cavity suggesting that F360 and its adjacent residues tune the selective binding of the inhibitor to SARS-CoV-2 MD.

NMR study of human macroPARPs domains: 1H, 15N and 13C resonance assignment of hPARP14 macro domain 2 in the free and the ADPr bound state.

hPARP14 is a human ADP-ribosyl-transferase (ART) that belongs to the macroPARPs family, together with hPARP9 and hPARP15. It contains a tandem of three macro domains (MD) while each of them has different properties. The first one, namely MD1, has not been reported to exhibit a high binding affinity for ADP-ribose (ADPr) in contrast to the following two (MD2 and MD3). All three MDs exhibit an α/ß/α sandwich-like fold as reported by the deposited crystallographic structures. MD2 and MD3 recognize mono-ADP-ribosylated (MARylated) but not poly-ADP-ribosylated (PARylated) substrates and thus they allow hPARP14 to bind its targets, which can be potentially MARylated by its catalytic domain (CD). hPARP14 participates in DNA damage repair process and immune response against viruses like SARS-CoV-2, which also harbors an MD fold. Furthermore, hPARP14 like the other two macroPARPs (hPARP9 and hPARP15), is implicated in numerous types of cancer, such as B-aggressive lymphoma and sarcoma, rendering its MDs as potential important drug targets. Herein, we report the complete NMR backbone and side chain assignment (1H, 13C, 15N) of hPARP14 MD2 in the free and ADPr bound states and the NMR chemical shift-based prediction of its secondary structure elements. This is the first reported NMR study of a hPARP macro domain, paving the way to screen by NMR chemical compounds which may alter the ability of hPARP14 to interact with its substrates affecting its function.

NMR study of macro domains (MDs) from betacoronavirus: backbone resonance assignments of SARS-CoV and MERS-CoV MDs in the free and the ADPr-bound state.

SARS-CoV and MERS-CoV Macro Domains (MDs) exhibit topological and conformational features that resemble the nsP3b macro (or "X") domain of SARS-CoV-2. Indeed, all the three domains (SARS-CoV-2, SARS-CoV and MERS-CoV MDs) fold in a three-layer α/ß/α sandwich structure, as reported by crystallographic structural investigation of SARS-CoV MD and MERS-CoV MD. These viral MDs are able to bind ADP-ribose as many other MDs from different kingdoms. They have been characterized also as de-ADP-ribosylating enzymes. For this reason, these viral macrodomains recently emerged as important drug targets since they can counteract antiviral ADP-ribosylation mediated by poly-ADP-ribose polymerase (PARPs). Even in presence of the 3D structures of SARS-CoV MD and of MERS-CoV MD, we report herein the almost complete NMR backbone (1H, 13C, 15N) of SARS-CoV MD and MERS-CoV proteins in the free and ADPr bound forms, and the NMR chemical shift-based prediction of their secondary structure elements. These NMR data will help to further understanding of the atomic-level conformational dynamics of these proteins and will allow an extensive screening of small molecules as potential antiviral drugs.

Correction to "The SARS-COV-2 Spike Protein Binds Sialic Acids, and Enables Rapid Detection in a Lateral Flow Point of Care Diagnostic Device".

[This corrects the article DOI: 10.1021/acscentsci.0c00855.].

1H,13C and 15N chemical shift assignments of the SUD domains of SARS-CoV-2 non-structural protein 3c: "The SUD-M and SUD-C domains".

SARS-CoV-2 RNA, nsP3c (non-structural Protein3c) spans the sequence of the so-called SARS Unique Domains (SUDs), first observed in SARS-CoV. Although the function of this viral protein is not fully elucidated, it is believed that it is crucial for the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral "components" with the host cell; thus, it is essential for the entire viral life cycle. The first two SUDs, the so-called SUD-N (the N-terminal domain) and SUD-M (domain following SUD-N) domains, exhibit topological and conformational features that resemble the nsP3b macro (or "X") domain. Indeed, they are all folded in a three-layer α/ß/α sandwich structure, as revealed through crystallographic structural investigation of SARS-CoV SUDs, and they have been attributed to different substrate selectivity as they selectively bind to oligonucleotides. On the other hand, the C-terminal SUD (SUD-C) exhibit much lower sequence similarities compared to the SUD-N & SUD-M, as reported in previous crystallographic and NMR studies of SARS-CoV. In the absence of the 3D structures of SARS-CoV-2, we report herein the almost complete NMR backbone and side-chain resonance assignment (1H,13C,15N) of SARS-CoV-2 SUD-M and SUD-C proteins, and the NMR chemical shift-based prediction of their secondary structure elements. These NMR data will set the base for further understanding at the atomic-level conformational dynamics of these proteins and will allow the effective screening of a large number of small molecules as binders with potential biological impact on their function.

1H,13C and 15N chemical shift assignments of the SUD domains of SARS-CoV-2 non-structural protein 3c: "the N-terminal domain-SUD-N".

Among the proteins encoded by the SARS-CoV-2 RNA, nsP3 (non-structural Protein3) is the largest multi-domain protein. Its role is multifaceted and important for the viral life cycle. Nonetheless, regarding the specific role of each domain there are many aspects of their function that have to be investigated. SARS Unique Domains (SUDs), constitute the nsP3c region of the nsP3, and were observed for the first time in SARS-CoV. Two of them, namely SUD-N (the first SUD) and the SUD-M (sequential to SUD-N), exhibit structural homology with nsP3b ("X" or macro domain); indeed all of them are folded in a three-layer α/ß/α sandwich. On the contrary, they do not exhibit functional similarities, like ADP-ribose binding properties and ADP-ribose hydrolase activity. There are reports that suggest that these two SUDs may exhibit a binding selectivity towards G-oligonucleotides, a feature which may contribute to the characterization of their role in the formation of the replication/transcription viral complex (RTC) and of the interaction of various viral "components" with the host cell. While the structures of these domains of SARS-CoV-2 have not been determined yet, SUDs interaction with oligonucleotides and/or RNA molecules may provide a platform for drug discovery. Here, we report the almost complete NMR backbone and side-chain resonance assignment (1H,13C,15N) of SARS-CoV-2 SUD-N protein, and the NMR chemical shift-based prediction of the secondary structure elements. These data may be exploited for its 3D structure determination and the screening of chemical compounds libraries, which may alter SUD-N function.

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

The SARS-COV-2 Spike Protein Binds Sialic Acids and Enables Rapid Detection in a Lateral Flow Point of Care Diagnostic Device.

There is an urgent need to understand the behavior of the novel coronavirus (SARS-COV-2), which is the causative agent of COVID-19, and to develop point-of-care diagnostics. Here, a glyconanoparticle platform is used to discover that N-acetyl neuraminic acid has affinity toward the SARS-COV-2 spike glycoprotein, demonstrating its glycan-binding function. Optimization of the particle size and coating enabled detection of the spike glycoprotein in lateral flow and showed selectivity over the SARS-COV-1 spike protein. Using a virus-like particle and a pseudotyped lentivirus model, paper-based lateral flow detection was demonstrated in under 30 min, showing the potential of this system as a low-cost detection platform.