Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

Mickolajczyk, Keith J.; Shelton, Patrick M. M.; Grasso, Michael; Cao, Xiaocong; Warrington, Sara; Aher, Amol; Liu, Shixin; Kapoor, Tarun M.

doi:10.1016/j.bpj.2020.11.2276

Cited by 44 publications

(33 citation statements)

References 58 publications

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“…We do not describe an active base separating mechanism for NSP13 consistent with recent biochemical and single molecule studies of NSP13. In the RNA unwinding reaction, NSP13 was a predominantly passive helicase (advancing upon the spontaneous opening of base pairs), with a peak step size of 2 base pairs (interpreted to be 2 rate-limiting ATP binding events) and a strong force-dependent stimulation of activity that suggests mechanoragulation by the RNA polymerase NSP12 8 . Single molecule FRET studies of DNA unwinding by NSP13 show larger step sizes of up to 4–9 base pairs depending on nucleotide used which the authors suggest may indicate a “spring loaded” unwinding mechanism with the flexible 1B and Stalk domains 30 .…”

Section: Resultsmentioning

confidence: 99%

“…NSP13 is a 67 kDa protein that belongs to the helicase superfamily 1B, it utilizes the energy of nucleotide triphosphate hydrolysis to catalyze the unwinding of double-stranded DNA or RNA in a 5′ to 3′ direction 7 . Although NSP13 is believed to act on RNA in vivo enzymatic characterization shows a significantly more robust activity on DNA in in vitro assays with relatively weak non processive helicase activity when compared to other superfamily 1B enzymes 8 , 9 . NSP13 has been shown to interact with the viral RNA-dependent RNA polymerase NSP12 10 , 11 , and acts in concert with the replication-transcription complex (NSP7/NSP8/NSP12) 12 .…”

Section: Introductionmentioning

confidence: 99%

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Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase

et al. 2021

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There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two “druggable” pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase

et al. 2021

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“…To determine the optimal concentration of substrates for best sensitivity in nsp13 inhibitor screening, we determined the Michaelis-Menten constants (K m ) for ATP and nucleic acid substrates. It has been shown by others that coronavirus helicases lack nucleic acid specificity, being capable of unwinding DNA and RNA [18][19][20][21]. We synthesized an RNA substrate of identical sequence as its DNA counterpart and tested nsp13 activity.…”

Section: Development Of a Fret-based Helicase Assaymentioning

confidence: 99%

“…Once expressed in the host cell, pp1a and pp1ab are processed by virus-encoded proteases into 16 non-structural proteins (nsps) [15,16]. Coronavirus nsp13, one of the non-structural proteins, is a superfamily 1B (SF1B) helicase that can unwind DNA or RNA in an NTP-dependent manner with a 5 0 to 3 0 polarity [17][18][19][20][21]. Moreover, nsp13 harbours RNA 5 0 -triphosphatase activity that could play a role in viral 5 0 RNA capping [18,20,22].…”

Section: Introductionmentioning

confidence: 99%

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase

Zeng

Weissmann

Bertolin

et al. 2021

Biochemical Journal

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The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.

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“…It's there for a cofactor of nsp14 and nsp16 which enhance their activities [56] . Nsp 13 is the 1 helicase superfamily with plays an essential role in viral replication and conservation across all CoV species [57] . Nsp15 is a nidoviral RNA uridylate-specific endoribonuclease (NendoU) belonging to Endo U enzymes family.…”

Section: Pathophysiologymentioning

confidence: 99%

Human Coronaviruses: Genetics, Virulence Factors and Pathophysiology, Diseases’ Epidemiology

Katawa

Gambogou

Nguepou

et al. 2021

ijirms

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Coronaviruses are pathogens for both animals and humans. The recent Coronavirus Disease (Covid-19), caused by a novel Coronavirus (SARS-CoV-2), outbreak that took place in Hubei (Wuhan, China) from December 2019 aroused numerous questions regarding the nature and the origin of the SARS-CoV-2. Literature review of the main publications from scientific data bases was undertaken. These data revealed similarities of symptoms and identity of genome with the SARS-CoV. In this review, the genetics and virulence factors of coronaviruses, the disease pathophysiology and epidemiology were examined.

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Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

Cited by 44 publications

References 58 publications

Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase

Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase

Human Coronaviruses: Genetics, Virulence Factors and Pathophysiology, Diseases’ Epidemiology

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