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1.
Anal Chem ; 93(37): 12808-12816, 2021 09 21.
Article in English | MEDLINE | ID: covidwho-1402013

ABSTRACT

CRISPR-Cas systems integrated with nucleic acid amplification techniques improve both analytical specificity and sensitivity. We describe here issues and solutions for the successful integration of reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube under an isothermal condition (40 °C). Specific detection of a few copies of a viral DNA sequence was achieved in less than 20 min. However, the sensitivity was orders of magnitude lower for the detection of viral RNA due to the slow initiation of RPA when the complementary DNA (cDNA) template remained hybridized to RNA. During the delay of RPA, the crRNA-Cas12a ribonucleoprotein (RNP) gradually lost its activity in the RPA solution, and nonspecific amplification reactions consumed the RPA reagents. We overcame these problems by taking advantage of the endoribonuclease function of RNase H to remove RNA from the RNA-cDNA hybrids and free the cDNA as template for the RPA reaction. As a consequence, we significantly enhanced the overall reaction rate of an integrated assay using RT-RPA and CRISPR-Cas12a for the detection of RNA. We showed successful detection of 200 or more copies of the S gene sequence of SARS-CoV-2 RNA within 5-30 min. We applied our one-tube assay to 46 upper respiratory swab samples for COVID-19 diagnosis, and the results from both fluorescence intensity measurements and end-point visualization were consistent with those of RT-qPCR analysis. The strategy and technique improve the sensitivity and speed of RT-RPA and CRISPR-Cas12a assays, potentially useful for both semi-quantitative and point-of-care analyses of RNA molecules.


Subject(s)
COVID-19 , Reverse Transcription , COVID-19 Testing , Humans , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , Recombinases/genetics , SARS-CoV-2 , Sensitivity and Specificity , Technology
2.
RSC Med Chem ; 12(10): 1722-1730, 2021 Oct 20.
Article in English | MEDLINE | ID: covidwho-1392908

ABSTRACT

Tragically, the death toll from the COVID-19 pandemic continues to rise, and with variants being observed around the globe new therapeutics, particularly direct-acting antivirals that are easily administered, are desperately needed. Studies targeting the SARS-CoV-2 3CL protease, which is critical for viral replication, with different peptidomimetics and warheads is an active area of research for development of potential drugs. To date, however, only a few publications have evaluated the nitrile warhead as a viral 3CL protease inhibitor, with only modest activity reported. This article describes our investigation of P3 4-methoxyindole peptidomimetic analogs with select P1 and P2 groups with a nitrile warhead that are potent inhibitors of SARS-CoV-2 3CL protease and demonstrate in vitro SARS-CoV-2 antiviral activity. A selectivity for SARS-CoV-2 3CL protease over human cathepsins B, S and L was also observed with the nitrile warhead, which was superior to that with the aldehyde warhead. A co-crystal structure with SARS-CoV-2 3CL protease and a reversibility study indicate that a reversible, thioimidate adduct is formed when the catalytic sulfur forms a covalent bond with the carbon of the nitrile. This effort also identified efflux as a property limiting antiviral activity of these compounds, and together with the positive attributes described these results provide insight for further drug development of novel nitrile peptidomimetics targeting SARS-CoV-2 3CL protease.

3.
Vaccine ; 39(40): 5769-5779, 2021 09 24.
Article in English | MEDLINE | ID: covidwho-1392616

ABSTRACT

SARS-CoV-2 is the etiological agent of COVID19. There are currently several licensed vaccines approved for human use and most of them target the spike protein in the virion envelope to induce protective immunity. Recently, variants that spread more quickly have emerged. There is evidence that some of these variants are less sensitive to neutralization in vitro, but it is not clear whether they can evade vaccine induced protection. In this study, we tested SARS-CoV-2 spike RBD as a vaccine antigen and explored the effect of formulation with Alum/MPLA or AddaS03 adjuvants. Our results show that RBD induces high titers of neutralizing antibodies and activates strong cellular immune responses. There is also significant cross-neutralization of variants B.1.1.7 and B.1.351 and to a lesser extent, SARS-CoV-1. These results indicate that recombinant RBD can be a viable candidate as a stand-alone vaccine or as a booster shot to diversify our strategy for COVID19 protection.


Subject(s)
Antibodies, Neutralizing , COVID-19 , Antibodies, Viral , Humans , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics
4.
J Med Chem ; 65(4): 2905-2925, 2022 02 24.
Article in English | MEDLINE | ID: covidwho-1303733

ABSTRACT

Recurring coronavirus outbreaks, such as the current COVID-19 pandemic, establish a necessity to develop direct-acting antivirals that can be readily administered and are active against a broad spectrum of coronaviruses. Described in this Article are novel α-acyloxymethylketone warhead peptidomimetic compounds with a six-membered lactam glutamine mimic in P1. Compounds with potent SARS-CoV-2 3CL protease and in vitro viral replication inhibition were identified with low cytotoxicity and good plasma and glutathione stability. Compounds 15e, 15h, and 15l displayed selectivity for SARS-CoV-2 3CL protease over CatB and CatS and superior in vitro SARS-CoV-2 antiviral replication inhibition compared with the reported peptidomimetic inhibitors with other warheads. The cocrystallization of 15l with SARS-CoV-2 3CL protease confirmed the formation of a covalent adduct. α-Acyloxymethylketone compounds also exhibited antiviral activity against an alphacoronavirus and non-SARS betacoronavirus strains with similar potency and a better selectivity index than remdesivir. These findings demonstrate the potential of the substituted heteroaromatic and aliphatic α-acyloxymethylketone warheads as coronavirus inhibitors, and the described results provide a basis for further optimization.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Peptidomimetics/pharmacology , SARS-CoV-2/drug effects , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/metabolism , Coronavirus 3C Proteases/metabolism , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Glutamine/chemistry , Glutamine/pharmacology , Humans , Ketones/chemistry , Ketones/pharmacology , Microbial Sensitivity Tests , Molecular Structure , Peptidomimetics/chemistry , SARS-CoV-2/enzymology , Virus Replication/drug effects
5.
Eur J Med Chem ; 222: 113584, 2021 Oct 15.
Article in English | MEDLINE | ID: covidwho-1252810

ABSTRACT

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (Mpro) to cleave viral proteins. Consequently, Mpro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of Mpro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-Mpro complexes reveal that an alternative binding pocket in Mpro, S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na+ counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for Mpro inhibitor design.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Pyrrolidines/pharmacology , SARS-CoV-2/drug effects , Sulfonic Acids/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Binding Sites , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/metabolism , Humans , Micelles , Microbial Sensitivity Tests , Molecular Structure , Protein Binding , Pyrrolidines/chemical synthesis , Pyrrolidines/metabolism , SARS-CoV-2/enzymology , Solubility , Structure-Activity Relationship , Sulfonic Acids/chemical synthesis , Sulfonic Acids/metabolism , Vero Cells
6.
Clin Transl Immunology ; 10(3): e1260, 2021.
Article in English | MEDLINE | ID: covidwho-1120050

ABSTRACT

OBJECTIVES: A major COVID-19 vaccine strategy is to induce antibodies that prevent interaction between the Spike protein's receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2). These vaccines will also induce T-cell responses. However, concerns were raised that aberrant vaccine-induced immune responses may exacerbate disease. We aimed to identify minimal epitopes on the RBD that would induce antibody responses that block the interaction of the RBD and ACE2 as a strategy leading to an effective vaccine with reduced risk of inducing immunopathology. METHODS: We procured a series of overlapping 20-amino acid peptides spanning the RBD and asked which were recognised by plasma from COVID-19 convalescent patients. Identified epitopes were conjugated to diphtheria-toxoid and used to vaccinate mice. Immune sera were tested for binding to the RBD and for their ability to block the interaction of the RBD and ACE2. RESULTS: Seven putative vaccine epitopes were identified. Memory B-cells (MBCs) specific for one of the epitopes were identified in the blood of convalescent patients. When used to vaccinate mice, six induced antibodies that bound recRBD and three induced antibodies that could partially block the interaction of the RBD and ACE2. However, when the sera were combined in pairs, we observed significantly enhanced inhibition of binding of RBD to ACE2. Two of the peptides were located in the main regions of the RBD known to contact ACE2. Of significant importance to vaccine development, two of the peptides were in regions that are invariant in the UK and South African strains. CONCLUSION: COVID-19 convalescent patients have SARS-CoV-2-specific antibodies and MBCs, the specificities of which can be defined with short peptides. Epitope-specific antibodies synergistically block RBD-ACE2 interaction.

7.
Anal Chem ; 92(24): 16204-16212, 2020 12 15.
Article in English | MEDLINE | ID: covidwho-947511

ABSTRACT

We have developed a single-tube assay for SARS-CoV-2 in patient samples. This assay combined advantages of reverse transcription (RT) loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) enzyme Cas12a. Our assay is able to detect SARS-CoV-2 in a single tube within 40 min, requiring only a single temperature control (62 °C). The RT-LAMP reagents were added to the sample vial, while CRISPR Cas12a reagents were deposited onto the lid of the vial. After a half-hour RT-LAMP amplification, the tube was inverted and flicked to mix the detection reagents with the amplicon. The sequence-specific recognition of the amplicon by the CRISPR guide RNA and Cas12a enzyme improved specificity. Visible green fluorescence generated by the CRISPR Cas12a system was recorded using a smartphone camera. Analysis of 100 human respiratory swab samples for the N and/or E gene of SARS-CoV-2 produced 100% clinical specificity and no false positive. Analysis of 50 samples that were detected positive using reverse transcription quantitative polymerase chain reaction (RT-qPCR) resulted in an overall clinical sensitivity of 94%. Importantly, this included 20 samples that required 30-39 threshold cycles of RT-qPCR to achieve a positive detection. Integration of the exponential amplification ability of RT-LAMP and the sequence-specific processing by the CRISPR-Cas system into a molecular assay resulted in improvements in both analytical sensitivity and specificity. The single-tube assay is beneficial for future point-of-care applications.


Subject(s)
CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Nucleic Acid Amplification Techniques , SARS-CoV-2/genetics , Humans , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction
8.
Nat Commun ; 11(1): 5409, 2020 Oct 20.
Article in English | MEDLINE | ID: covidwho-882896

ABSTRACT

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

9.
Nat Commun ; 11(1): 4282, 2020 08 27.
Article in English | MEDLINE | ID: covidwho-733525

ABSTRACT

The main protease, Mpro (or 3CLpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.


Subject(s)
Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus, Feline/drug effects , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , A549 Cells , Animals , Antiviral Agents/chemistry , Betacoronavirus/enzymology , Binding Sites , Chlorocebus aethiops , Coronavirus 3C Proteases , Coronavirus, Feline/enzymology , Crystallography, X-Ray , Cysteine Endopeptidases/chemistry , Cytopathogenic Effect, Viral/drug effects , Drug Repositioning , Humans , Inhibitory Concentration 50 , Molecular Structure , Prodrugs , Protease Inhibitors/chemistry , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , SARS Virus/drug effects , SARS Virus/enzymology , SARS-CoV-2 , Sulfonic Acids , Vero Cells , Viral Nonstructural Proteins/chemistry , Virus Replication/drug effects
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