Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

Combining High‐Throughput Synthesis and High‐Throughput Protein Crystallography for Accelerated Hit Identification

“…While the exponential increase in speed of structure determination is not reflected in the largely bystander role of PC,i ntegration of HT PX with HT synthesis of more complex molecules than fragments could help to advance early drug discovery.W es howed how HT synthesis can advantageously synergize with HT crystallography to yield single digit mMhits for the SARS-CoV-2 main protease with avariety of warheads involved in covalent bond formation, derived from newly synthesized libraries.T his is conceptually different from the mostly performed approach of cocrystallizing or soaking existing static fragment libraries of low complexity. [10,11] Fort his,w ed eveloped an ovel, general, highly diverse multicomponent method for the onepot synthesis of acyl amides and esters,based on the building blocks acrylic acid, ammonia, aldehydes,a nd isocyanides. Noteworthy many substrates with different functional groups are compatible with the reaction and show good product formation.…”

“…[10] HT soaking of static fragment libraries is nowadays routinely offered to clients at synchrotrons (Figure 1B). [10,11] These advances have been driven by the work of thousands of members of the structural biology community globally. [12] On the other hand there are tremendous developments in miniaturization and automation of synthetic chemistry.…”

NAD+ Degrading Enzymes, Evidence for Roles During Infection

“…Some viral families, mainly Coronaviruses, Togaviruses and Herpesviridae, possess macrodomain proteins that bind both MAR and PAR units, hydrolysing them. Studies have since corroborated the functional significance in tandem with the structural characterization of these macrodomains in modulating PARP catalytic activity to mediate viral replication and pathogenesis (Li et al, 2016;Abraham et al, 2018;Grunewald et al, 2019a,b;Alhammad and Fehr, 2020;Rack et al, 2020;Schuller et al, 2021). These studies not only strongly implicate PARPs as a modulatory target that can be utilised by pathogens during infection to evade immune response and drive replication, they also underscore the significance of these viral macrodomains as targets for pioneering therapeutic strategies to combat pathogenicity, particularly in the context of SARS-CoV2 which has been recently presented (Rack et al, 2020;Schuller et al, 2021).…”

Structural biology of SARS-CoV-2: open the door for novel therapies

“…In the processes of viral infection, the Mac1 domain counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling via its ADP-ribosyl hydrolase activity. 164 , 165 Accordingly, catalytic null mutations of the Mac1 domain render viruses nonpathogenic, 164 – 166 and the Mac1 domain is a promising drug target for disrupting the viral life cycle. The Mac1 domain adopts a conserved three-layered α/β/α sandwich fold, in which there is a central seven-stranded β-sheet (β1-β2-β7-β6-β3-β5-β4), and six α-helices are located on the outside.…”