In a recent study posted to the bioRxiv* preprint server, researchers discovered compounds able to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain 1 (Mac1) and adenosine diphosphate (ADP)-ribose binding via high throughput screening.
In the last two decades, the emergence of various zoonotic viruses, especially the SARS-CoV-2 outbreak, has revealed the scarcity of antiviral medication therapy for viruses with pandemic potential. To date, coronavirus disease 2019 (COVID-19) has claimed 6 million lives and caused 446 million cases worldwide.
Our capacity to respond quickly to new CoVs and various other viruses with pandemic potential will be dependent on developing a broader antiviral drug portfolio. Hence there is an urgent need for devising broad-spectrum antiviral therapies directed towards a wide span of CoVs to combat severe illness during the ongoing SARS-CoV-2 pandemic and future zoonotic outbreaks.
About the study
In the present study, the researchers analyzed some molecules that could inhibit binding between SARS-CoV-2's conserved Mac1 and ADP-ribose. For this, the team optimized a luminescent-based high throughput assay named AlphaScreenTM (AS) and a novel fluorescence polarization (FP) assay.
Further, they used AS assay to screen approximately 38,000 small molecules to detect their ability to hinder SARS-CoV-2 Mac1-ADP-ribose binding. The researchers also determined the direct binding of these compounds to the Mac1. The ability of the compounds to restrain Mac1-ADP-ribose binding was compared between Mac1s of SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), SARS-CoV, and human macrodomain MDO2.
The results show that the Mac1 from SARS-CoV-2, MERS-CoV, and SARS-CoV, and the human macrodomain MDO2 bound to ADP-ribosylated control peptides better than non-ADP-ribosylated peptides in the AS and FP assays. The human MDO2 had a heightened affinity to peptides in dose-dependent AS and FP experiments relative to SARS-CoV-2, MERS-CoV, and SARS-CoV Mac1s. The supplementation of free ADP-ribose, but not adenosine triphosphate (ATP), decreased CoV Mac1 and human macrodomain binding to ADP-ribosylated peptides in the AS and FP assays.
The half-maximal inhibitory concentration (IC50) values for free ADP-ribose varied between 0.24 µM to 1.5 µM with SARS-CoV-1 and SARS-CoV-2, respectively, in the AS assay. In addition, the IC50 values ranged from 2.3 µM to 9.74 µM for SARS-CoV-1 and COV-2, respectively, in the FP assay. This observation proves that these assays could be utilized to find macrodomain binding inhibitors.
Of the 406 original hits from the Peptidomimetics, three-dimensional (3D) BioDiversity, and Analyticon libraries, 26 compounds hindered the ADP-ribose-SARS-CoV-2 Mac1 binding in an exposure-response manner in the AS assay, and six compounds inhibited Mac1 binding in FP and AS assays. Compounds 1, 2, 6, 7, 10, and 11. Compounds 11, 10, and 1 also displayed inhibitory potential towards Mac1 of MERS-CoV, although lower than that towards SARS-CoV-2's Mac1. Further, only compound 2 hindered MDO2, suggesting that the other compounds were selective towards viral macrodomains.
The supplementation of free ADP-ribose demonstrated a dose-dependent elevation of about 4℃ in the melting temperature of SARS-CoV-2 Mac1, whereas ATP had no impact in the differential scanning fluorimetry (DSF) assay. The hike in melting temperature of Mac1 was linked to the binding of the compound to Mac1. The melting temperature of Mac1 changed in a dose-dependent manner in compounds 11, 10, 7, 6, and 1.
Compounds 7, 6, and 1 demonstrated a dose-dependent hindrance of Mac1 ADP-ribosylhydrolase activity in a gel-based Mac1 ADP-ribosylhydrolase experiment. In contrast, all six compounds showed dose-dependent inhibition of ADP-ribosylhydrolase activity in the recently published high-throughput luminescence-based ADP-ribosylhydrolase test. However, compound 6 was the most efficient inhibitor of ADP-ribosylhydrolase activity versus others.
Out of 16 distinct viral and human macrodomains in the recently established fluorescence resonance energy transfer (FRET)-based assay, compound 6 substantially inhibited the SARS-CoV-2 Mac1. This inference implies the selectivity of compound 6 towards the Mac1 protein of SARS-CoV-2. Further, the five compounds can be stratified into three chemotypes based on their structures.
The study findings identified five drug molecules among three chemotypes that hinder SARS-CoV-2 Mac1-ADP-ribose binding in several experiments with IC50 values less than 100μM, restraining ADP-ribosylhydrolase activity, and proof of direct Mac1 binding. These chemotypes have a good chance of being derivatized and optimized into very potent SARS-CoV-2 Mac1 inhibitors. Compounds 1 and 6 exhibited IC50 values of around 10μM in the AS assay and directly bonded with Mac1. Further, compound 6 has remarkable selectivity for SARS-CoV-2 over human macrodomains, indicating that it should be developed further.
Collectively, the study identified compounds that can be developed into potent Mac1 inhibitors and therapeutical agents for COVID-19 and other CoVs of interest.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.