Identification of a critical horseshoe-shaped region in the nsp5 (Mpro, 3CLpro) protease interdomain loop (IDL) of coronavirus mouse hepatitis virus (MHV)

Abstract

26Human coronaviruses are enveloped, positive-strand RNA viruses which cause respiratory 27 diseases ranging in severity from the seasonal common cold to SARS and COVID-19. Of the 7 28 human coronaviruses discovered to date, 3 emergent and severe human coronavirus strains 29 (SARS-CoV, MERS-CoV, and SARS-CoV-2) have recently jumped to humans in the last 20 30 years. The COVID-19 pandemic spawned by the emergence of SARS-CoV-2 in late 2019 has 31 highlighted the importance for development of effective therapeutics to target emerging 32 coronaviruses. Upon entry, the replicase genes of coronaviruses are translated and subsequently 33 proteolytically processed by virus-encoded proteases. Of these proteases, nonstructural protein 5 34 (nsp5, Mpro, or 3CLpro), mediates the majority of these cleavages and remains a key drug target 35 for therapeutic inhibitors. Efforts to develop nsp5 active-site inhibitors for human coronaviruses 36 have thus far been unsuccessful, establishing the need for identification of other critical and 37 conserved non-active-site regions of the protease. In this study, we describe the identification of 38 an essential, conserved horseshoe-shaped region in the nsp5 interdomain loop (IDL) of mouse 39 hepatitis virus (MHV), a common coronavirus replication model. Using site-directed 40 mutagenesis and replication studies, we show that several residues comprising this horseshoe-41 shaped region either fail to tolerate mutagenesis or were associated with viral temperature-42 sensitivity. Structural modeling and sequence analysis of these sites in other coronaviruses, 43 including all 7 human coronaviruses, suggests that the identified structure and sequence of this 44 horseshoe regions is highly conserved and may represent a new, non-active-site regulatory 45 region of the nsp5 (3CLpro) protease to target with coronavirus inhibitors. 46 47 48 49 In December 2019, a novel coronavirus (SARS-CoV-2) emerged in humans and triggered a 50 pandemic which has to date resulted in over 8 million confirmed cases of COVID-19 across 51 more than 180 countries and territories (June 2020). SARS-CoV-2 represents the third emergent 52 coronavirus in the past 20 years and the future emergence of new coronaviruses in humans 53 remains certain. Critically, there remains no vaccine nor established therapeutics to treat cases of 54 COVID-19. The coronavirus nsp5 protease is a conserved and indispensable virus-encoded 55 enzyme which remains a key target for therapeutic design. However, past attempts to target the 56 active site of nsp5 with inhibitors have failed stressing the need to identify new conserved non-57 active-site targets for therapeutic development. This study describes the discovery of a novel 58 conserved structural region of the nsp5 protease of coronavirus mouse hepatitis virus (MHV) 59 which may provide a new target for coronavirus drug development. 60 61 Introduction 62 63 Coronaviruses (CoVs) are enveloped, positive-strand RNA viruses which encode among the 64 largest RNA virus genomes on the planet ...

Essential Oil of Cestrum diurnum L.: GC/MS Analysis, in Vitro and in Silico Anti‐HCoV‐229E Effects and Inhibitory Activity against LPS‐Induced Inflammation

“…2ZU3 consists of one peptide chain, a native peptido‐mimetic inhibitor (TG‐0204998), N ‐[(benzyloxy)carbonyl]‐3‐[(2,2‐dimethylpropanoyl)amino]‐L‐alanyl‐ N ‐[(1 R )‐4‐oxo‐1‐{[(3 S )‐2‐oxopyrrolidin‐3‐yl]methyl}pentyl]‐L‐leucinamide and water molecules. TG‐0204998 occupies a site near the catalytic center of 2ZU3 surrounded by the key catalytic amino acid residues HIS 40 and CYS 147 which indicates the binding specificity [23] . Therefore, TG‐0204998 was used to validate the docking protocol.…”

“…TG-0204998 occupies a site near the catalytic center of 2ZU3 surrounded by the key catalytic amino acid residues HIS 40 and CYS 147 which indicates the binding specificity. [23] Therefore, TG-0204998 was used to validate the docking protocol. The results revealed 2D-interactions of co-crystallized TG-0204998 and its re-docked conformational form through the involvement of: (i) key amino acid residues HIS 40 and CYS 147 (ii) amino acid residues: VAL 162, GLY 164, THR 142, HIS 161, GLY 145 as hydrogen bond donors or acceptors and ASN 165 as H-pi interaction (iii) two water molecules (Figure 3).…”

Structural leitmotif and functional variations of the structural catalytic core in (chymo)trypsin-like serine/cysteine fold proteinases

“…Furthermore, the presence of the C-terminal extension near the active site explains the fact that proteinases of 27R and 29R&OH Groups have very short 102T-Zone consisting of the fragment 4-6 amino acids long (Table S1, rows numbered 117-118, 129-133 and 158-169). For example, in 3CLpro from SARS-CoV-2, the conservative loop Val186-Gln192 [43] actually compensates for the short 102T-Zone by including the Asp187 in the active site (Figure 1D). The Val186-Gln192 loop is part of the interdomain loop (IDL), which is compact in all 11 coronavirus proteases (Table S1, rows numbered 159-168).…”