The complex structure of GRL0617 and SARS-CoV-2 PLpro reveals a hot spot for antiviral drug discovery

Fu, Ziyang; Huang, Bin; Tang, Jessica; Liu, Shuyan; Liu, Ming; Ye, Yuxin; Liu, Zhihong; Xiong, Yuxian; Zhu, Wenning; Cao, Ding; Li, Jihui; Niu, Xiaogang; Zhou, Huan; Zhao, Yong; Zhang, Guoliang; Huang, Hao

doi:10.1038/s41467-020-20718-8

Cited by 241 publications

(400 citation statements)

References 61 publications

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“…Towards developing potent inhibitors, we developed a robust high-throughput assay for SARS-CoV-2 PLpro using Z-RLRGG-AMC to screen chemical libraries including FDAapproved drugs and molecules in clinical trials (15,370 molecules). Consistent with contemporary reports, 38,40,47 an extremely low hit rate was observed, which makes repurposing of approved drugs as therapeutically useful PLpro inhibitors problematic. As recent reports have noted, the experimental data is not in accordance with in silico repurposing predictions: 48,49 for example, isotretinoin reportedly in clinical trials for COVID-19 as a PLpro inhibitor, 46 was inactive in our screen.…”

Section: Discussionsupporting

confidence: 80%

“…Compounds were screened against PLpro at a final compound concentration of 20 µM, which is a more stringent threshold than other contemporary screens of PLpro. 38 Assay of PLpro in the presence of 5 mM DTT, as reducing agent and electrophile trap, strongly biases against reactive (electrophilic and redox) hits. The 28 hit compounds from HTS were counter-assayed to remove false positives associated with signal interference and then further pruned to remove frequent hitters and known redox cyclers (Figure 1 & Figure S2A, B).…”

Section: Resultsmentioning

confidence: 99%

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Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures

Shen

Ratia

Cooper

et al. 2021

Preprint

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Antiviral agents blocking SARS-CoV-2 viral replication are desperately needed to complement vaccination to end the COVID-19 pandemic. Viral replication and assembly are entirely dependent on two viral cysteine proteases: 3C-like protease (3CLpro) and the papain-like protease (PLpro). PLpro also has deubiquitinase (DUB) activity, removing ubiquitin (Ub) and Ub-like modifications from host proteins, disrupting the host immune response. 3CLpro is inhibited by many known cysteine protease inhibitors, whereas PLpro is a relatively unusual cysteine protease, being resistant to blockade by such inhibitors. A high-throughput screen of biased and unbiased libraries gave a low hit rate, identifying only CPI-169 and the positive control, GRL0617, as inhibitors with good potency (IC50 < 10 lower case Greek μM). Analogues of both inhibitors were designed to develop structure-activity relationships; however, without a co-crystal structure of the CPI-169 series, we focused on GRL0617 as a starting point for structure-based drug design, obtaining several co-crystal structures to guide optimization. A series of novel 2-phenylthiophene-based non-covalent SARS-CoV-2 PLpro inhibitors were obtained, culminating in low nanomolar potency. The high potency and slow inhibitor off-rate were rationalized by newly identified ligand interactions with a 'BL2 groove' that is distal from the active site cysteine. Trapping of the conformationally flexible BL2 loop by these inhibitors blocks binding of viral and host protein substrates; however, until now it has not been demonstrated that this mechanism can induce potent and efficacious antiviral activity. In this study, we report that novel PLpro inhibitors have excellent antiviral efficacy and potency against infectious SARS-CoV-2 replication in cell cultures. Together, our data provide structural insights into the design of potent PLpro inhibitors and the first validation that non-covalent inhibitors of SARS-CoV-2 PLpro can block infection of human cells with low micromolar potency.

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Section: Discussionsupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures

Shen

Ratia

Cooper

et al. 2021

Preprint

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“…As such, it has already been put in a spotlight by the scientific community. This includes research on its structure [24][25][26], functions [11,27], and similarity to PLpro from other related coronaviruses, most notably SARS-CoV [28]. The search for PLpro inhibitors has already begun.…”

Section: Introductionmentioning

confidence: 99%

SARS-CoV-2 Papain-Like Protease Potential Inhibitors—In Silico Quantitative Assessment

Stasiulewicz

Maksymiuk

Nguyen

et al. 2021

IJMS

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes the papain-like protease (PLpro). The protein not only plays an essential role in viral replication but also cleaves ubiquitin and ubiquitin-like interferon-stimulated gene 15 protein (ISG15) from host proteins, making it an important target for developing new antiviral drugs. In this study, we searched for novel, noncovalent potential PLpro inhibitors by employing a multistep in silico screening of a 15 million compound library. The selectivity of the best-scored compounds was evaluated by checking their binding affinity to the human ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), which, as a deubiquitylating enzyme, exhibits structural and functional similarities to the PLpro. As a result, we identified 387 potential, selective PLpro inhibitors, from which we retrieved the 20 best compounds according to their IC50 values toward PLpro estimated by a multiple linear regression model. The selected candidates display potential activity against the protein with IC50 values in the nanomolar range from approximately 159 to 505 nM and mostly adopt a similar binding mode to the known, noncovalent SARS-CoV-2 PLpro inhibitors. We further propose the six most promising compounds for future in vitro evaluation. The results for the top potential PLpro inhibitors are deposited in the database prepared to facilitate research on anti-SARS-CoV-2 drugs.

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“…18 Significantly, the deISGylating activity is 156-fold higher than the deubiquitinating activity, which is consistent with previous reports that the SARS-CoV-2 PL pro prefers ISG15 over ubiquitin as a substrate. 5-9…”

Section: Resultsmentioning

confidence: 99%

“…16 As SARS-CoV-2 and SARS-CoV PL pro share a sequence identity of 83% and similarity of 90%, GRL0617 was also repurposed for SARS-CoV-2 PL pro and it was reported to inhibit SARS-CoV-2 PL pro with IC 50 values of around 2 µM and the SARS-CoV-2 viral replication with EC 50 values around 20 µM from multiple studies. 8,9,15,17…”

Section: Introductionmentioning

confidence: 99%

Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay

Xia

Sacco

et al. 2021

Preprint

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The papain-like protease (PLpro) of SARS-CoV-2 is a validated antiviral drug target. PLpro is involved in the cleavage of viral polyproteins and antagonizing host innate immune response through its deubiquitinating and deISG15ylating activities, rendering it a high profile antiviral drug target. Through a FRET-based high-throughput screening, several hits were identified as PLpro inhibitors with IC50 values at the single-digit micromolar range. Subsequent lead optimization led to potent inhibitors with IC50 values ranging from 0.56 to 0.90 micromolar. To help prioritize lead compounds for the cellular antiviral assay against SARS-CoV-2, we developed the cell-based FlipGFP assay that is suitable for quantifying the intracellular enzymatic inhibition potency of PLpro inhibitors in the BSL-2 setting. Two compounds selected from the FlipGFP-PLpro assay, Jun9-53-2 and Jun9-72-2, inhibited SARS-CoV-2 replication in Caco-2 hACE2 cells with EC50 values of 8.89 and 8.32 micromolar, respectively, which were 3-fold more potent than GRL0617 (EC50 = 25.1 micromolar). The X-ray crystal structures of PLpro in complex with GRL0617 showed that binding of GRL0617 to SARS-CoV-2 induced a conformational change in the BL2 loop to the more closed conformation. Overall, the PLpro inhibitors identified in this study represent promising starting points for further development as SARS-CoV-2 antivirals, and FlipGFP-PLpro assay might be a suitable surrogate for screening PLpro inhibitors in the BSL-2 setting.

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The complex structure of GRL0617 and SARS-CoV-2 PLpro reveals a hot spot for antiviral drug discovery

Cited by 241 publications

References 61 publications

Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures

Potent, Novel SARS-CoV-2 PLpro Inhibitors Block Viral Replication in Monkey and Human Cell Cultures

SARS-CoV-2 Papain-Like Protease Potential Inhibitors—In Silico Quantitative Assessment

Discovery of SARS-CoV-2 papain-like protease inhibitors through a combination of high-throughput screening and FlipGFP-based reporter assay

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