Signatures and mechanisms of efficacious therapeutic ribonucleotides against SARS-CoV-2 revealed by analysis of its replicase using magnetic tweezers

SummaryCoronavirus Disease 2019 (COVID-19) results from an infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the third coronavirus outbreak to plague humanity this century. Currently, the most efficacious therapeutic against SARS-CoV-2 infection is the Remdesivir (RDV), an adenine-like ribonucleotide analogue that is very efficiently incorporated by the SARS-CoV-2 replicase. Understanding why RDV is so well incorporated will facilitate development of even more effective therapeutics. Here, we have applied a high-throughput, single-molecule, magnetic-tweezers platform to study thousands of cycles of nucleotide addition by the SARS-CoV-2 replicase in the absence and presence of RDV, a Favipiravir-related analog (T-1106), and the endogenously produced ddhCTP. Our data are consistent with two parallel catalytic pathways of the replicase: a high-fidelity catalytic (HFC) state and a low-fidelity catalytic (LFC) state, the latter allowing the slow incorporation of both cognate and non-cognate nucleotides. ddhCTP accesses HFC, T-1106 accesses LFC as a non-cognate nucleotide, while RDV efficiently accesses both LFC pathway. In contrast to previous reports, we provide unequivocal evidence against RDV functioning as a chain terminator. We show that RDV incorporation transiently stalls the replicase, only appearing as termination events when traditional, gel-based assays are used. The efficiency of ddhCTP utilization by the SARS-CoV-2 replicase suggests suppression of its synthesis during infection, inspiring new therapeutic strategies. Use of this experimental paradigm will be essential to the development of therapeutic nucleotide analogs targeting polymerases.

Mre11 exonuclease activity removes the chain-terminating nucleoside analog gemcitabine from the nascent strand during DNA replication

The Mre11 nuclease is involved in early responses to DNA damage, often mediated by its role in DNA end processing. MRE11 mutations and aberrant expression are associated with carcinogenesis and cancer treatment outcomes. While, in recent years, progress has been made in understanding the role of Mre11 nuclease activities in DNA double-strand break repair, their role during replication has remained elusive. The nucleoside analog gemcitabine, widely used in cancer therapy, acts as a replication chain terminator; for a cell to survive treatment, gemcitabine needs to be removed from replicating DNA. Activities responsible for this removal have, so far, not been identified. We show that Mre11 3′ to 5′ exonuclease activity removes gemcitabine from nascent DNA during replication. This contributes to replication progression and gemcitabine resistance. We thus uncovered a replication-supporting role for Mre11 exonuclease activity, which is distinct from its previously reported detrimental role in uncontrolled resection in recombination-deficient cells.

Halting coronavirus polymerase

The nucleotide analogue remdesivir is an investigational drug for the treatment of human coronavirus infection. Remdesivir is a phosphoramidate prodrug and is known to target viral RNA-dependent RNA polymerases. In this issue, Gordon et al. identify that remdesivir acts as a delayed RNA chain terminator for MERS-CoV polymerase complexes.

Optimization of Polydimethylsiloxane Synthesized Parameters as Vitreous Humour Substitutes

Polydimethylsiloxane (PDMS) with two type viscosity that commonly used as vitreous humour substitutes in vitreoretinal surgery has been successfully synthesized by ring-opening polymerization. Optimization of synthesized parameters such as ratio of monomer and chain terminator, the amount of initiator, mixing temperature and reaction time was carried out to obtain PDMS materials having similar properties with commercial product. PDMS with viscosity of 1.17 Pas that very close to viscosity of commercial PDMS of 1.08 Pas, was successfully synthesized with a ratio of 26:10 monomer and terminator chain, 0.58 M of initiator KOH, 170 oC of mixing temperature and 35 minutes of reaction time. By changing reaction time to 40 minutes, PDMS was successfully synthesized with viscosity of 3.42 that similar to that of commercial one.

Stimuli-responsively porating gels by condensation

A polyurethane (PU) resin derived from glycerol and hexamethylene diisocyanate and an imidazolium bromide ionic liquid chain terminator yield a stimuli-responsive resin that reversibly porates as a solvation response.