NSP15 in SARS-CoV-2: ADPRP macrodomain regulation and small-molecule inhibition

SARS-CoV-2 endoribonuclease NSP15 (NendoU) requires tight regulation within the replication-transcription complex (RTC); however, no viral protein has been described to date that regulates its activity. We demonstrate here that NSP3 ADP-ribose phosphatase, also known as ADPRP (Mac1), binds directly to NSP15 and stimulates its endoribonuclease activity. Surface plasmon resonance revealed moderate-affinity binding (KD = 1.59 ± 0.4 µM), with Mn²⁺ significantly enhancing binding compared to metal-free conditions. FRET assays confirmed close molecular association between the fluorophore-labeled proteins, while pull-down experiments revealed a stable complex formation with ∼51% ADPRP retention. Functionally, ADPRP increased NSP15-mediated cleavage of an 18-mer RNA substrate from 51-52% to 90-98%, with Mn²⁺ further potentiating maximal catalytic efficiency. Kinetic analysis showed an increase in k_cat with no significant change in K_m, indicating increased catalytic turnover rather than altered substrate affinity. EMSA and Denaturing PAGE showed that ADPRP neither bound nor cleaved RNA, indicating an indirect mode of regulation. AlphaFold modeling predicted binding in the vicinity of the NSP15 N-terminal oligomerization region. Normal Mode Analysis of the AlphaFold Complex revealed a dominant low-frequency collective motion consistent with intrinsic conformational flexibility relevant to interactions and regulation. Molecular docking using the Surflex-Dock program available in SYBYL-X v2.1 identified AW00832 and HTS00094 as the two computationally predicted hits and candidate inhibitors of NSP15. Overall, our results identified ADPRP as the first putative viral regulator of NSP15 activity and established NSP15-ADPRP as a regulatory pathway that can be therapeutically exploited through direct inhibition of NSP15.