A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo

Minimized β hairpins have provided additional data on the geometric preferences of Trp interactions in TW-loop-WT motifs. This motif imparts significant fold stability to peptides as short as 8 residues. High-resolution NMR structures of a 16- (KKWTWNPATGKWTWQE, ΔG_U²⁹⁸ ≥ +7 kJ/mol) and 12-residue (KTWNPATGKWTE, ΔG_U²⁹⁸ = +5.05 kJ/mol) hairpin reveal a common turn geometry and edge-to-face (EtF) packing motif and a cation-π interaction between Lys¹ and the Trp residue nearest the C-terminus. The magnitude of a CD exciton couplet (due to the two Trp residues) and the chemical shifts of a Trp Hε3 site (shifted upfield by 2.4 ppm due to the EtF stacking geometry) provided near-identical measures of folding. CD melts of representative peptides with the -TW-loop-WT- motif provided the thermodynamic parameters for folding, which reflect enthalpically driven folding at laboratory temperatures with a small ΔC_p for unfolding (+420 J K^-1/mol). In the case of Asx-Pro-Xaa-Thr-Gly-Xaa loops, mutations established that the two most important residues in this class of direction-reversing loops are Asx and Gly: mutation to alanine is destabilizing by about 6 and 2 kJ/mol, respectively. All indicators of structuring are retained in a minimized 8-residue construct (Ac-WNPATGKW-NH₂) with the fold stability reduced to ΔG_U²⁷⁸ = −0.7 kJ/mol. NMR and CD comparisons indicate that -TWXNGKWT- (X = S, I) sequences also form the same hairpin-stabilizing W/W interaction.

SARS-CoV-2 belongs to the family of enveloped, single-strand RNA viruses known as Betacoronavirus in Coronaviridae, first reported late 2019 in China. It has since been circulating world-wide, causing the COVID-19 epidemic with high infectivity and fatality rates. As of the beginning of April 2021, pandemic SARS-CoV-2 has infected more than 130 million people and led to more than 2.84 million deaths. Given the severity of the epidemic, scientists from academia and industry are rushing to identify antiviral strategies to combat the disease. There are several strategies in antiviral drugs for coronaviruses including empirical testing of known antiviral drugs, large-scale phenotypic screening of compound libraries and target-based drug discovery. To date, an increasing number of drugs have been shown to have anti-coronavirus activitiesin vitroandin vivo, but only remdesivir and several neutralizing antibodies have been approved by the US FDA for treating COVID-19. However, remdesivir’s clinical effects are controversial and new antiviral drugs are still urgently needed. We will discuss the current status of the drug discovery efforts against COVID-19 and potential future directions. With the ever-increasing movability of human population and globalization of world economy, emerging and reemerging viral infectious diseases seriously threaten public health. Particularly the past and ongoing outbreaks of coronaviruses cause respiratory, enteric, hepatic and neurological diseases in infected animals and human (Woo et al., 2009). The human coronavirus (HCoV) strains (HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1) usually cause common cold with mild, self-limiting upper respiratory tract infections. By contrast, the emergence of three deadly human betacoronaviruses, middle east respiratory syndrome coronavirus (MERS) (Zaki et al., 2012), severe acute respiratory syndrome coronavirus (SARS-CoV) (Lee et al., 2003), the SARS-CoV-2 (Jin et al., 2020a) highlight the need to identify new treatment strategies for viral infections. SARS-CoV-2 is the etiological agent of COVID-19 disease named by World Health Organization (WHO) (Zhu N. et al., 2020). This disease manifests as either an asymptomatic infection or a mild to severe pneumonia. This pandemic disease causes extent morbidity and mortality in the whole world, especially regions out of China. Similar to SARS and MERS, the SARS CoV-2 genome encodes four structural proteins, sixteen non-structural proteins (nsp) and accessory proteins. The structural proteins include spike (S), envelope (E), membrane (M), nucleoprotein (N). The spike glycoprotein directly recognizes and engages cellular receptors during viral entry. The four non-structural proteins including papain-like protease (PL^pro), 3-chymotrypsin-like protease (3CL^pro), helicase, and RNA-dependent RNA polymerase (RdRp) are key enzymes involved in viral transcription and replication. The spike and the four key enzymes were considered attractive targets to develop antiviral agents (Zumla et al., 2016). The catalytic sites of the four enzymes of SARS-CoV2 share high similarities with SARS CoV and MERS in genomic sequences (Morse et al., 2020). Besides, the structures of the key drug-binding pockets are highly conserved among the three coronaviruses (Morse et al., 2020). Therefore, it follows naturally that existing anti-SARS-CoV and anti-MERS drugs targeting these enzymes can be repurposed for SARS-CoV-2. Based on previous studies in SARS-CoV and MERS-CoV, it is anticipated a number of therapeutics can be used to control or prevent emerging infectious disease COVID-19 (Li and de Clercq, 2020;Wang et al., 2020c;Ita, 2021), these include small-molecule drugs, peptides, and monoclonal antibodies. Given the urgency of the SARS-CoV-2 outbreak, here we discuss the discovery and development of new therapeutics for SARS-CoV-2 infection based on the strategies from which the new drugs are derived.