ABSTRACT
Emerging variants of SARS-CoV-2 still threaten the effectiveness of currently deployed vaccines, and antivirals can prove to be an effective therapeutic option for attenuating it. The papain-like protease (PLpro) is an attractive target due to its sequence conservation and critical role in the replication and pathogenesis of SARS-CoV-2. PLpro also plays very important role in modulation of host immune responses by deubiquitinating (DUBs) or deISGylating host proteins. Thus, targeting PLpro serves as a two-pronged approach to abate SARS-CoV-2. Due to its structural and functional similarities with the host DUB enzymes, an in-house library of DUB inhibitors was constituted in this study. Five promising compounds exhibiting high binding affinities with the substrate binding site of PLpro were identified from a library of 81 compounds with in silico screening, docking, and simulation studies. Interestingly, lithocholic acid, linagliptin, teneligliptin, and flupenthixol significantly inhibited the proteolytic activity of PLpro. Each of these compounds abrogated in vitro replication of SARS-CoV-2 with EC50 values in the range of 5-21 M. In addition, crystal structure of SARS-CoV-2 PLpro and its complex with inhibitors have been determined that revealed their inhibitory mechanism. The findings of this study provide the proof-of-principle that the DUB inhibitors hold high potential as a new class of therapeutics against SARS-CoV-2. Additionally, this is the first study that has opened a new avenue towards not only targeting PLpro active site but also simultaneously directing towards restoration of antiviral immune response of the host for deterring SARS-CoV-2. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/516107v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@90c3fborg.highwire.dtl.DTLVardef@32b4daorg.highwire.dtl.DTLVardef@ff858dorg.highwire.dtl.DTLVardef@198fce7_HPS_FORMAT_FIGEXP M_FIG C_FIG
ABSTRACT
SARS-CoV-2 nucleocapsid protein (N-protein) is a virus specific multitasking protein, responsible for recognition and encapsidation of the viral genome. The N-terminal domain (NTD) of N-protein has a major role of packaging viral RNA genome into a long helical nucleocapsid structure. In this study, using structure-based drug repurposing strategy, small molecules from a FDA approved, natural product, and LOPAC1280 libraries have been virtually screened against the RNA binding pocket of SARS-CoV-2 NTD and twelve candidate molecules with high binding affinity were identified. Highly sensitive isothermal titration calorimetry (ITC) method was utilized to confirm binding of these molecules to purified NTD protein. In vitro cell-based SARS-CoV-2 antiviral assays demonstrate that nine of these identified molecules are highly efficacious in inhibiting virus replication with half maximal effective concentration (EC50) ranging from 0.98 M-10 M. FDA approved drugs: Telmisartan, an angiotensin II type 1 (AT1) receptor antagonist used in the management of hypertension and Bictegravir, an HIV-1 integrase inhibitor showed significant inhibitory activity against SARS-CoV-2 with a EC50 values of 1.02 M and 8.11 M respectively. Additionally, Bisdemethoxycurcumin, a natural analogue of curcumin and MCC-555, an anti-diabetic drug exerted antiviral activity with EC50 values of 1.64 M and 4.26 M, respectively. Taken together, this is the first report of drug molecules targeting the NTD of SARS-CoV-2 N-protein and the data presented in this study exhibit high potential for development of COVID-19 therapy based on drug repurposing.
