Receptor-Binding-Motif-Targeted Sanger Sequencing: a Quick and Cost-Effective Strategy for Molecular Surveillance of SARS-CoV-2 Variants

Chaki, Sankar P.; Kahl-McDonagh, Melissa; Neuman, Benjamin W.; Zuelke, Kurt A.

doi:10.1128/spectrum.00665-22

Cited by 10 publications

(11 citation statements)

References 38 publications

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“…Samples were processed and RT-qPCR was performed as per protocol established previously. 54 Samples were diluted 1:1 in 2× Tris-borate-EDTA [TBE] containing 1% Tween-20 and heated at 95°C for 15 min to lyse and inactivate virions. RT-qPCR screening was performed using the CDC N1 oligonucleotide pair/FAM probe (CDC N1-F, 5′-GACCCCAAAATCAGCGAAAT-3′; CDC N1-R, 5′-TCTGGTTACTGCCAGTTGAATCTG-3′; and Probe CDC N1, 5′ FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1 3′) and the Luna Universal Probe one-step RT-qPCR kit (catalog no.…”

Section: Methodsmentioning

confidence: 99%

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease

Khatua¹,

Alugubelli²,

Yang³

et al. 2023

Preprint

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Main protease (MPro) of SARS-CoV-2, the viral pathogen of COVID-19, is a crucial nonstructural protein that plays a vital role in the replication and pathogenesis of the virus. Its protease function relies on three active site pockets to recognize P1, P2, and P4 amino acid residues in a substrate and a catalytic cysteine residue for catalysis. By converting the P1 C(alpha) atom in an MPro substrate to nitrogen, we showed that a large variety of azapeptide inhibitors with covalent warheads targeting the MPro catalytic cysteine could be easily synthesized. Through the characterization of these inhibitors, we identified several highly potent MPro inhibitors. Specifically, one inhibitor, MPI89 that contained an aza-2,2-dichloroacetyl warhead, displayed a 10 nM EC50 value in inhibiting SARS-CoV-2 from infecting ACE2+ A549 cells and a selectivity index of 875. The crystallography analyses of MPro bound with 6 inhibitors, including MPI89, revealed that inhibitors used their covalent warheads to covalently engage the catalytic cysteine and the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 represents one of the most potent MPro inhibitors developed so far, suggesting that further exploration of the azapeptide platform and the aza-2,2-dichloroacetyl warhead is needed for the development of potent inhibitors for the SARS-CoV-2 MPro as therapeutics for COVID-19.

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Section: Methodsmentioning

confidence: 99%

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease

Khatua¹,

Alugubelli²,

Yang³

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Samples were processed and RT-qPCR was performed as per protocol established previously. 62 Samples were diluted 1:1 in 2× Tris-borate-EDTA [TBE] containing 1% Tween-20 and heated at 95°C for 15 min to lyse and inactivate virions. RT-qPCR screening was performed using the CDC N1 oligonucleotide pair/FAM probe (CDC N1-F, 5′-GACCCCAAAATCAGCGAAAT-3′; CDC N1-R, 5′-TCTGGTTACTGCCAGTTGAATCTG-3′; and Probe CDC N1, 5′ FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1 3′) and the Luna Universal Probe one-step RT-qPCR kit (catalog no.…”

Section: Methodsmentioning

confidence: 99%

“…Samples were processed and RT-qPCR was performed as per protocol established previously. 62 In Vitro Metabolic Stability in Human Liver Microsomes. This metabolic stability measurements were based on previous publications and modified as described below.…”

Section: Methodsmentioning

confidence: 99%

Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease

Alugubelli,

Xiao,

Khatua

et al. 2023

Preprint

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We have witnessed three coronavirus (CoV) outbreaks in the past two decades, including the COVID-19 pandemic caused by SARS-CoV-2. Main protease (MPro) is a highly conserved and essential protease that plays key roles in viral replication and pathogenesis among various CoVs, representing one of the most attractive drug targets for antiviral drug development. Traditional antiviral drug development strategies focus on the pursuit of high-affinity binding inhibitors against MPro. However, this approach often suffers from issues such as toxicity, drug resistance, and a lack of broad-spectrum efficacy. Targeted protein degradation represents a promising strategy for developing next-generation antiviral drugs to combat infectious diseases. Here we leverage the proteolysis targeting chimera (PROTAC) technology to develop a new class of small-molecule antivirals that induce the degradation of SARS-CoV-2 MPro. Our previously developed MProinhibitors MPI8 and MPI29 were used as MProligands to conjugate a CRBN E3 ligand, leading to compounds that can both inhibit and degrade SARS-CoV-2 MPro. Among them, MDP2 was demonstrated to effectively reduce MProprotein levels in 293T cells (DC50= 296 nM), relying on a time-dependent, CRBN-mediated, and proteasome-driven mechanism. Furthermore, MPD2 exhibited remarkable efficacy in diminishing MProprotein levels in SARS-CoV-2-infected A549-ACE2 cells, concurrently demonstrating potent anti-SARS-CoV-2 activity (EC50= 492 nM). This proof-of-concept study highlights the potential of PROTAC-mediated targeted protein degradation of MProas an innovative and promising approach for COVID-19 drug discovery.

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“…On the basis of the genomic sequences, different techniques have been developed for the detection of variants. The main features of three groups of techniques, including sequencing [ [93] , [94] , [95] , [96] , [97] ], mutation-specific RT-PCR [ [98] , [99] , [100] ], and CRISPR-based methods, are summarized and compared ( Table S1 ). In general, sequencing-based methods can provide high-resolution information of the whole genome or a particular gene, enabling the identification of new variants.…”

Section: Sars-cov-2 Variants Of Concern (Alpha Beta Gamma Delta and O...mentioning

confidence: 99%

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern

Xiao

Huang

et al. 2023

TrAC Trends in Analytical Chemistry

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Receptor-Binding-Motif-Targeted Sanger Sequencing: a Quick and Cost-Effective Strategy for Molecular Surveillance of SARS-CoV-2 Variants

Abstract: The coronavirus disease 2019 (COVID-19) pandemic resulted in 427 million infections and 5.9 million deaths globally as of 21 February 2022. SARS-CoV-2, the causative agent of the COVID-19 pandemic, frequently mutates and has developed into variants of major public health concerns.

Cited by 10 publications

References 38 publications

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease

An Azapeptide Platform in Conjunction with Covalent Warheads to Uncover High-Potency Inhibitors for SARS-CoV-2 Main Protease

Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease

CRISPR techniques and potential for the detection and discrimination of SARS-CoV-2 variants of concern

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