Patent Application
20250019399
This application contains a sequence listing having the filename 1959919-00008 Sequence Listing.xml, which is 26 KB in size, and was created on Sep. 16, 2024. The entire content of this sequence listing is incorporated herein by reference.
The emergence of SARS-COV-2 and its variants has posed a massive healthcare problem. In this regard, pharmacological targeting of the main protease (Mpro; 3CLpro) appears to be a key therapeutic strategy to reduce the COVID-19 symptoms and therefore, there is a significant need for the development of such inhibitors. Therefore, a number of assays have been developed to aid in the discovery of such inhibitors. Recently, a genetically encoded, Bioluminescence Resonance Energy Transfer (BRET)-based Mpro biosensor has been reported.
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-COV-2), a member of the beta-coronavirus family and the causative agent of COVID-19 (Coronavirus Disease of 2019), has emerged as a major health and economic threat in the current times. The infection cycle of the virus is initiated by the binding of the virus to the host receptor, angiotensin-converting enzyme 2 (ACE2) expressed on the epithelial cell membranes of the human host in a multitude of organs including the lungs, nose, and the gut. The viral binding is mediated by the S1 spike protein present on the viral surface. The binding of the virus is followed by endocytosis-mediated internalization and proteolytic cleavage of the S1 spike protein by the host protease TMPRSS2, making the virus competent for fusion with the host membrane. Following viral-host cell membrane fusion, the viral genome is released in the host cytosol where the positive-sense RNA-genome gets translated into viral proteins. These include both structural as well as non-structural proteins that are essential for viral replication and further infection. Of particular interests are the Open Reading Frame 1a (ORF1a) and ORF 1b that together represent two-thirds of the SARS-COV-2 genome. These ORFs are translated into two polyproteins referred to as polyprotein 1a (pp1a) and pp1ab, respectively. These polyproteins consist of the non-structural proteins (nsp) 1 to 11 and 1 to 16, respectively. These polyproteins are proteolytically processed to generate functionally active proteins that are necessary for viral replication by the viral proteases, main protease Mpro (also referred to nsp5 or 3-chymotrypsin-like cysteine protease (3CLpro) or main proteinase) and papain-like protease (PLpro). Importantly, Mpro is a cysteine protease that cleaves the pp1ab at 11 conserved sites (recognition site—LQ↓SAG), including its own autocleavage at the N-and C-termini. It is active in a homodimeric form, and each protomer has three domains-domain I, II and III. The substrate-binding site with Cys145-His41 catalytic dyad is present in a wide cleft formed between the domains I and II whereas domain III is responsible for protein dimerization.
The tremendous threat posed by COVID-19 has resulted in numerous efforts to develop novel therapeutic strategies to control and eradicate this pandemic. These include both the development of novel pharmacological anti-SARS-COV-2 agents and repurposing clinically approved drugs that may either directly or indirectly reduce viral infections. Additionally, significant efforts have been directed towards effective vaccine development using through the traditional methods such as using either whole attenuated virus or viral structural proteins as well as novel RNA-based strategies. However, the frequent emergence of SARS-COV-2 variants/lineages with mutations in the SI spike or other proteins providing increased infection and immune evasion potential to the virus has raised concerns regarding their efficacy. Therefore, alternatives to vaccines are suggested to be required for the successful elimination of the pandemic. In this regard, Mpro is considered an appealing target for designing anti-SARS-COV-2 agents since none of the human proteases have similar cleavage sequence specificity. Indeed, a number of structural, biophysical, and biochemical studies have reported the specific pharmacological targeting of Mpro using both de novo drug discovery as well as repurposing of already available drugs, although with limited success. In this regard, the development of facile technologies will accelerate further drug discovery programs against Mpro, and thus, SARS-COV-2.
Mpro has been typically studied in vitro using fluorescently labeled cognate peptides that show reduced fluorescence resonance energy transfer (FRET) upon cleavage of the peptide is cleaved by the enzyme. Such assays have been utilized for the identification of Boceprevir, GC376, and calpain inhibitors II, XII as potent inhibitors of SARS-COV-2 Mpro13. Mpro activity has been determined through direct visualization of cleaved proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While convenient, these assays cannot be implemented in living cells. Additionally, double labeling of the peptide during synthesis is significantly costly. These have led to the development of genetically encoded biosensors that can be used in living cells with a significantly reduced assay cost and possible gain in sensitivity. Genetically encoded biosensors, either based on fluorescence or bioluminescence, have seen significant usage in assessing a variety of cellular processes including cellular signaling events, protein conformational changes, and protein-protein interactions including oligomerization in living cells. Specifically, biosensors have been utilized for detecting a number of viruses including HIV, Hepatitis, Zika, Dengue, Ebola, and Influenza viruses as well as SARS-COV and MERS-COV19. Further, split protein-based biosensors, using either luciferase or GFP as reporters, have also been developed to monitor Mpro activity in live cells. In these biosensors, Mpro-mediated cleavage of the split protein results in the activation of the reporter protein (luciferase or GFP), which is then measured in microplate readers or quantified from microscopic images. Additionally, Mpro activity has also been monitored through gene expression changes as well as through nuclear localization of reporter protein upon proteolytic cleavage.
A need therefore exists for a BRET-based Mpro biosensor with an increased rate of cleavage.
Example systems, methods, and apparatus are disclosed herein for a BRET-based Mpro biosensor with an increased rate of cleavage.
The disclosed invention includes synthetic Mpro cleavage sequences to be utilized for generating BRET-based Mpro biosensors displaying increased Mpro-mediated proteolytic cleavage rate and show that a biosensor containing 2×cleavage site performs better as a Mpro substrate. These sequences included 2×, 4× and 8× repeats of the Mpro N-terminal (Nter) cleavage sequences and a combination of Mpro cleavage sequences containing a total of 12 cleavage sites. Secondary structure predictions as well three-dimensional structural modeling indicated predominantly alpha-helical structure for both the synthetically designed sequences as well as the original Mpro Nter cleavage (1×) sequence. Gaussian accelerated molecular dynamics (GaMD) simulations of the synthetic Mpro cleavage sequences revealed a dynamic nature of the cleavage sequences, which is critical for their efficient cleavage, and a relatively short end-to-end distances, which is required for high BRET. Live cell and in vitro assays revealed a cleavage sequence length-dependent resonance energy transfer, except for the 12×-syn cleavage site and an enhanced rate of cleavage for the Mpro biosensor containing 2× cleavage sequences. The inclusion of a Mpro-binding, but non-inhibiting, NB2E3 nanobody at the N-terminal further increased the cleavage rate of the 2× cleavage sequence containing the Mpro biosensor. The 2× Mpro biosensor will find utility in both the development Mpro proteolytic activity assays that can be employed for drug discovery applications as well as functional genomics application in characterization impact of mutations in the Mpro protein.
In light of the disclosure herein, and without limiting the scope of the invention in any way, in a first aspect of the present disclosure, which may be combined with any other aspect listed herein unless specified otherwise, a BRET-based Mpro biosensor including an mNeonGreen (mNG) reporter protein, a NanoLuc (NLuc) reporter protein, and x repeats of an N-terminal autocleavage peptide sequence of Mpro. The x repeats of an N-terminal autocleavage peptide sequence of Mpro are located between the mNG reporter protein and the NLuc reporter protein.
In a second aspect which may be combined with any other aspect listed herein unless specified otherwise, the x repeats of an N-terminal autocleavage peptide sequence of Mpro are two.
In a third aspect which may be combined with any other aspect listed herein unless specified otherwise, the x repeats of an N-terminal autocleavage peptide sequence of Mpro are four.
In a fourth aspect which may be combined with any other aspect listed herein unless specified otherwise, the x repeats of an N-terminal autocleavage peptide sequence of Mpro are eight.
In a fifth aspect which may be combined with any other aspect listed herein unless specified otherwise, the N-terminal autocleavage peptide sequence of Mpro includes a peptide sequence AVLQSGFR (SEQ ID NO:15).
In a sixth aspect which may be combined with any other aspect listed herein unless specified otherwise, the x repeats of an N-terminal autocleavage peptide sequence of Mpro are twelve.
In a seventh aspect which may be combined with any other aspect listed herein unless specified otherwise, amino acid residues C-terminal to a critical Gln residue are varied.
In an eight aspect which may be combined with any other aspect listed herein unless specified otherwise, the twelve repeats of an N-terminal autocleavage peptide sequence of Mpro comprises SEQ ID NO: 12.
In a nine aspect which may be combined with any other aspect listed herein unless specified otherwise, a BRET-based Mpro biosensor including an mNeonGreen (mNG) reporter protein, a NanoLuc (NLuc) reporter protein, x repeats of an N-terminal autocleavage peptide sequence of Mpro, a first nanobody, and a second nanobody. The x repeats of an N-terminal autocleavage peptide sequence of Mpro are located between the mNG reporter protein and the NLuc reporter protein.
In a tenth aspect which may be combined with any other aspect listed herein unless specified otherwise, the first nanobody is NB1D10.
In an eleventh aspect which may be combined with any other aspect listed herein unless specified otherwise, the first nanobody is a C-terminal fusion.
In a twelfth aspect which may be combined with any other aspect listed herein unless specified otherwise, the first nanobody is an N-terminal fusion.
In a thirteenth aspect which may be combined with any other aspect listed herein unless specified otherwise, the second nanobody is NB2E3.
In a fourteenth aspect which may be combined with any other aspect listed herein unless specified otherwise, the second nanobody is a C-terminal fusion.
In a fifteenth aspect which may be combined with any other aspect listed herein unless specified otherwise, the second nanobody is an N-terminal fusion.
In a sixteenth aspect which may be combined with any other aspect listed herein unless specified otherwise, the x repeats of an N-terminal autocleavage peptide sequence of Mpro are two.
In a seventeenth aspect which may be combined with any other aspect listed herein unless specified otherwise, the N-terminal autocleavage peptide sequence of Mpro comprises a peptide sequence AVLQSGFR.
In an eighteenth aspect which may be combined with any other aspect listed herein unless specified otherwise, a method of creating a BRET-based Mpro biosensor including placing x repeats of an N-terminal autocleavage peptide sequence of Mpro between an mNeonGreen (mNG) reporter protein and a NanoLuc (NLuc) reporter protein.
In a nineteenth aspect which may be combined with any other aspect listed herein unless specified otherwise, further including inserting a first nanobody between the nNG reporter protein and the x repeats of an N-terminal autocleavage peptide sequence of Mpro, and inserting a second nanobody between the x repeats of an N-terminal autocleavage peptide sequence of Mpro and the Nluc reporter protein.
In a twentieth aspect which may be combined with any other aspect listed herein unless specified otherwise, the x repeats of an N-terminal autocleavage peptide sequence of Mpro are two.
In a twenty-first aspect of the present disclosure, any of the structure, functionality, and alternatives disclosed in connection with any one or more of
In light of the present disclosure and the above aspects, it is therefore an advantage of the present disclosure to provide users with a BRET-based Mpro biosensor with an increased rate of cleavage.
Additional features and advantages are described in, and will be apparent from, the following Detailed Description and the Figures. The features and advantages described herein are not all-inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the figures and description. Also, any particular embodiment does not have to have all of the advantages listed herein and it is expressly contemplated to claim individual advantageous embodiments separately. Moreover, it should be noted that the language used in the specification has been selected principally for readability and instructional purposes, and not to limit the scope of the inventive subject matter.
Methods, systems, and apparatus are disclosed herein for a BRET-based Mpro biosensor with an increased rate of cleavage.
While the example methods, apparatus, and systems are disclosed herein as BRET-based Mpro biosensor with an increased rate of cleavage, it should be appreciated that the methods, apparatus, and systems may be operable with other explorers.
Building on recently reported Bioluminescence Resonance Energy Transfer (BRET)-based Mpro biosensor, the disclosed invention generates and characterizes a number of additional biosensor constructs in order to achieve a higher a rate of cleavage. Specifically, the disclosed invention generated biosensors containing 2×, 4× and 8× copies of the Mpro N-terminal autocleavage sequence, which has been shown to possess the highest catalytic efficiency for the N-terminal autocleavage sequence, in between mNeonGreen (mNG) and NanoLuc (NLuc) reporter proteins. The disclosed invention follows the principle that a greater number of cleavage sites in the biosensor will lead to an increased effective concentration and therefore, rate of cleavage.
Additionally, the disclosed invention generated a biosensor containing 12× Mpro cleavage sites combining most of the 11 Mpro cleavage sites present in ppla and pp lab polyproteins in an overlapping fashion to reduce the total length and thus, distance between the BRET reporter proteins. This increases the number of cleavage sites and also allows sampling other cognate Mpro cleavage sequences.
Live cell and in vitro assays monitoring Mpro-mediated cleavage of the biosensors in the disclosed invention revealed highest rate of cleavage for the biosensor containing 2× Mpro N-terminal cleavage sites. Experiments with Mpro pharmacological inhibitors, GC376 and nirmatrelvir, revealed a decreased efficacy (higher IC50 values) of the inhibitors in Mpro-mediated cleavage of the 2× Mpro biosensor in living cells. Additionally, mutation of either the first or the second or both cleavage sites (Q to A) or replacement of the first or the second cleavage site with GS linker sequence revealed a requirement for both cleavage sites to be intact for the increased cleavage rate of the 2× Mpro biosensor. In an embodiment, including nanobodies that bind to Mpro but do not affect its activity in the 2× Mpro biosensor increases cleavage rate of the 2× Mpro biosensor when there is N-terminal fusion of the NB2E3 nanobody.
The BRET-based Mpro biosensor of the disclosed invention has direct utility in studies related to pharmacological targeting the SARS-COV-2 Mpro in living cells mimicking the in vivo conditions, in additional to functional genomics applications such as the determination of the effect of sequence variations in the Mpro that are observed in different viral isolates. Further, the biosensors with increased sensitivity can be deployed for point-of-care-testing (POCT) of live SARS-COV-2 infection in patient samples.
Structural modeling and MD simulation of Mpro cleavage sequences: Structural models of the 1×, 2×, 4× and 8× Mpro N-terminal and the 12×-syn cleavage sequences were generated using the trRosetta online server (https://yanglab.nankai.edu.cn/trRosetta/) 6, 29, 30 Quality of the structural models were checked using the available tools in the trRosetta server. Molecular dynamics simulations of the Mpro cleavage sequences were performed using the Nanoscale Molecular Dynamics (NAMD) software (version 2.13) with the CHARMM36(m) force field. A 2 fs time-step of integration was set for all simulations performed. First, energy minimization was performed on each system for 1000 steps (2 ps). Following energy minimization, the system was slowly heated from 60 K to 310 K at 1 K interval to reach the 310 K equilibrium temperature using a temperature ramp that runs 500 steps after each temperature increment. Following thermalization, temperature was maintained at 310 K using Langevin temperature control and at 1.0 atm using Nose-Hoover Langevin piston pressure control. The system was then equilibrated with 500000 steps (1 ns) using Periodic Boundary Conditions.
The NAMD output structure was then used as an input for Gaussian accelerated molecular dynamics (GaMD) simulation utilizing the integrated GaMD module in NAMD and its default parameters which included 2 ns of conventional molecular dynamics (cMD) equilibration run in GaMD, to collect potential statistics required for calculating the GaMD acceleration parameters, and another 50 ns equilibration run in GaMD after adding the boost potential, and finally GaMD production runs for 1000 ns. Both equilibration steps in GaMD were preceded by 0.4 ns preparatory runs. All GaMD simulations were run at the “dual-boost” level by setting the reference energy to the lower bound, i.e., E=Vmax. One boost potential is applied to the dihedral energetic term and the other to the total potential energetic term. The details for calculating the boost potentials including the equations used have been described previously. The upper limits of standard deviation (SD) of the dihedral and total potential boosts in GaMD were set to 6.0 kcal/mol. All GaMD simulations were performed using the same constant temperature and pressure parameters. For all simulations, short-range non-bonded interactions were defined at 12 Å cut-off with 10 A switching distance, while Particle-mesh Ewald (PME) scheme was used to handle long-range electrostatic interactions at 1 Å PME grid spacing. Trajectory frames were saved every 10,000 steps (20 ps) and trajectory analysis was performed using the available tools in Visual Molecular Dynamics (VMD) software 39. These included Cα root mean squared displacement (RMSD), Ca root mean squared fluctuation (RMSF) for each residue, radius of gyration (RoG), solvent accessible surface area (SASA), secondary structure and end-to-end distance measurements.
Mpro biosensor plasmid construct generation: The BRET-based Mpro biosensor plasmid constructs were generated by synthesizing nucleotide sequences coding for the 1×, 2×, 4× and 8× repeats N-terminal autocleavage peptides (AVLQSGFR, SEQ ID NO:15) and 12×-syn amino acid residues and subcloning in between the mNG and NLuc genes present in the original Mpro biosensor, pmNG-Mpro-N-ter-auto-NLuc, plasmid. The 2× Mpro biosensor mutants were generated by mutating the critical Gln to Ala (from AVLQSGFR to AVLASGFR (SEQ ID NO: 16)) either in the first (Q262A) or second (Q270A) or both cleavage sequences (double mutant; DM). The 1GS and 2GS mutants of the 2× Mpro biosensor were generated by replacing the nucleotide sequence coding for AVLQSGFR with that of GSGSGSGS (SEQ ID NO:17) for the first (1GS) or the second (2GS) Mpro cleavage site.
The N- and C-terminal fusion of the NB1D10 and NB2E3 nanobodies in the 2× Mpro biosensor were generated by synthesizing gene fragments expressing amino acid residues of the respective nanobody as reported previously, codon optimized for mammalian cell expression, and subcloned into the pmNG-Mpro-Nter-auto-2×-NLuc plasmid. All clones were procured from GenScript (Singapore).
Cell culture and transfection: HEK293T cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, and 1% penicillin-streptomycin and grown at 37° C. in 5% CO2. Transfections were performed with polyethyleneimine (PEI) lipid according to manufacturers' protocol. Briefly, HEK 293T cells seeded onto 96-well white plates were transfected by combining the plasmid DNA (sensor and Mpro), Opti-MEM (Invitrogen; 31985088) and 1.25 μg/well of PEI lipid (Sigma-Aldrich; 408727-100mL) and incubating them at room temperature for 30 min.
Live cell Mpro biosensor proteolytic cleavage activity assays: Live cell Mpro proteolytic cleavage activity assays were performed by co-transfecting HEK 293T cells with the respective Mpro BRET sensor plasmid constructs along with pLVX-EF1alpha-SARS-COV-2-nsp5-2×Strep-IRES-Puro (Mpro WT) (Addgene plasmid #141370) in 96-well white flat bottom plates. Post 48 h (or otherwise indicated) of transfection, BRET measurements were performed at 37° C. by the addition of furimazine (Promega, Wisconsin, USA) at a dilution of 1:200. Experiments were performed in triplicate and repeated thrice.
Pharmacological inhibition of Mpro in live cells: HEK293T cells were seeded onto the 96-well white plate one day before transfection. The cells were co-transfected with either pmNG-Mpro-Nter-auto-NLuc (1×) or pmNG-Mpro-Nter-auto-2×-NLuc (2× WT) and pLVX-EF1alpha-SARS-COV-2-nsp5-2×Strep-IRES-Puro (Mpro WT) (Addgene plasmid #141370) at a ratio of 1:5. The transfected cells were concurrently treated with GC376 (GC376 Sodium; AOBIOUS-AOB36447; stock solution prepared in 50% DMSO at a concentration of 10 mM) or nirmatrelvir (Sigma; 4 mM stock solution in DMSO). BRET values were measured by adding furimazine (Promega, Wisconsin, USA) at a 1:200 dilution at 16 h post-transfection. The percentage activity was calculated after normalizing the BRET ratio with the negative control (No Mpro). The experiments were conducted in triplicates for each construct and repeated thrice.
Mpro biosensor lysate preparation and in vitro assay: HEK 293T cells expressing the respective BRET sensor were washed with chilled Dulbecco's Phosphate-Buffered Saline (DBPS) 48 h post transfection and lysed in a buffer containing 50 mM HEPES (pH 7.5), 50 mM NaCl, 0.1% Triton X-100, 1 mM Dithiothreitol (DTT) & 1 mM ethylenediamine tetra acetic acid (EDTA) on ice. Cell lysates were collected in a 1.5 mL eppendorf tubes and centrifuged at 4° C. for 1 h at 14,000 rotations per min (RPM) following which supernatant was collected and stored at −80° C. until further usage. The lysate containing Nluc BRET sensor were mixed with recombinantly purified SARS-COV Mpro (3CL Protease, Recombinant from E. coli; NR-700; BEI Resources, NIAID, NIH; stock solution of the protein was prepared by dissolving the lyophilized protein in 50 μM in Tris-buffered saline (TBS) containing 10% glycerol) in 96-well white flat bottom plates. The effect of molecular crowding was monitored by incubating the sensor and the protease in the absence or presence of 25% (w/v) of polyethylene glycol (PEG; 8000 Da) (Sigma-Aldrich). BRET measurements were performed at 37° C. for 3 h by the addition of furimazine (Promega, Wisconsin, USA) at a dilution of 1:200.
BRET and fluorescence measurements: BRET measurements were performed using a Tecan SPARK® multimode microplate reader. For live cell assays, bioluminescence spectral scan was performed from 380 to 664 nm wavelengths with an acquisition time of 400 ms for each wavelength to determine relative emissions from Nluc (donor) and mNG (acceptor) and quantify BRET, which is expressed as a ratio of emissions at 533 nm and 467 nm. Total mNG fluorescence in the sensor expressing cells were measured by exciting the samples at 480 nm and emission acquired at a wavelength of 530 nm.
Data analysis and figure preparation: MD simulation data were plotted using Matplotlib in a Jupyter Notebook. GraphPad Prism (version 9 for MacOS, GraphPad Software, La Jolla California USA; www.graphpad.com) and Microsoft Excel will be used for data analysis and graph preparation. The results are presented as mean±SD obtained from a minimum of three experiments.
Designing BRET-based Mpro biosensors for increased cleavage rate: The Disclosed Invention is a highly specific, genetically encoded biosensor for monitoring SARS-COV-2 Mpro proteolytic activity in live cells using the BRET technology (
In order to engineer BRET-based Mpro biosensors with increased rate of cleavage compared to the originally designed biosensor containing a single Mpro N-terminal cleavage sequence, the disclosed invention includes a number of synthetic Mpro cleavage sequences. First, synthetic cleavage sequences containing either 2×, 4× and 8× repeats of the original Mpro N-terminal cleavage sequence were designed, which is expected to increase the cleavage rate by providing the indicated number of cleavage sequences per biosensor protein molecule (higher apparent concentration of the cleavage sequence;
Second, the disclosed invention includes a combination of Mpro cleavage sequences using the known Mpro cleavage sequences that contained a total of 12 cleavage sites while amino acid residues C-terminal to the critical Gln residue were varied (12×-syn-Mpro) (
To assess the structural dynamics and effective length (end-to-end distance) of the Mpro cleavage sequences, all atom, explicit solvent, GaMD simulations for each of the cleavage sequences were performed using NAMD (version 2.13) (
Characterization of Mpro biosensor constructs in live cells: To test the constructs for their ability to produce BRET signal and detect Mpro-mediated cleavage, the disclosed invention includes plasmid DNA constructs expressing 2×, 4×, 8× and 12×-syn Mpro cleavage sites (henceforth referred to as 2×, 4×, 8× and 12×-syn Mpro biosensors, respectively) and transfected them in HEK293T cells in addition to the 1× Mpro biosensor reported previously (
Increased rate of cleavage of the 2× and 4× Mpro biosensors in living cells: The temporal dynamics of Mpro-mediated cleavage of each biosensor construct in living cells were monitored. The cells were transfected with various Mpro biosensor constructs, either alone or along with Mpro, and BRET was determined at different time points (
To assess the rate of cleavage of the biosensors, the percentage change in BRET in the presence of Mpro, from that in the absence of Mpro, over time was determined. This analysis revealed a faster cleavage kinetics for the 2× and 4× Mpro biosensors (
Decreased Mpro inhibition efficacy of pharmacological inhibitors in cells expressing the 2× Mpro biosensor: Cells expressing either the 1× or the 2× Mpro biosensors were treated with a range of concentrations of the covalent inhibitor, GC376, and non-covalent inhibitor, nirmatrelvir. Total bioluminescence measurements revealed similar expression of the biosensors (
Mutational analysis reveals a requirement for both cleavage sites for the increased cleavage rate of the 2× Mpro biosensor: The underlying molecular requirements and basis for the observations made with the disclosed invention were determined. For this, first the critical Gln residue was mutated to an Ala residue (from AVLQSGFR to AVLASGFR) either in the first (Q262A) or the second (Q270A) or both (DM) cleavage sequences. Second, the first (1GS) or the second (2GS) cleavage sequence were replaced with a Gly-Ser linker of the same length (from AVLQSGFR to GSGSGSGS). Then HEK293T cells were transfected with the wild type (WT) and the mutant 2×, along with the original 1×, Mpro biosensor, expressing plasmids, either in the absence or in the presence of Mpro.
Total bioluminescence measurements revealed similar trends in the expression of all the biosensors, either in the absence (
Similarly, both the 1GS as well as the 2GS mutations in the 2× Mpro biosensor resulted in an increase in the t1/2 (7.727±0.622 and 7.928±0.492 h of the 1GS and 2GS mutant, respectively, vs. 6.420±0.186 h of the WT) and a decrease in the cleavage rate (0.090±0.007and 0.088±0.006 h−1 of the 1GS and 2GS mutant, respectively, vs. 0.108±0.003 h−1 of the WT), as compared to the WT 2× Mpro biosensor (
N-terminal NB2E3 nanobody containing 2× Mpro biosensor shows further increased cleavage rate: To further increase the cleavage rate of the 2× Mpro biosensor and thus, increase the sensitivity of Mpro-mediated cleavage of the biosensor, the local concentration of Mpro was increased through the fusion of Mpro-binding nanobodies to the 2× Mpro biosensor. Nanobodies are single-domain antibody fragments derived from the variable region of the heavy chain and are characterized by their efficient antigen-binding property, in addition to their small size (12-15 kD), long shelf-life, high stability, and solubility. In this regard, nanobodies against Mpro that can bind and inhibit Mpro activity as well as those that bind Mpro, but do not inhibit its activity were used. Specifically, the nanobodies NB1D10 and NB2E3 were chosen and generated both N-terminal (N-ter-NB1D10 and N-ter-NB2E3) as well as C-terminal (C-ter-NB1D10 and C-ter-NB2E3) fusions of the nanobodies with the 2× Mpro biosensor and transfected cells these nanobody-containing 2× Mpro biosensors, along with the 2× and 1× Mpro biosensors, either in the absence (
As previously noted, time-dependent, percentage change in BRET was determined (
Overall: 2×, 4×, 8× and 12×-syn Mpro cleavage sequences were designed for use in the development of a BRET-based Mpro biosensors increased cleavage rate compared to the 1× Mpro cleavage sequence. Following computational analysis, the biosensors were tested in live cells, both in terms of their BRET signal and cleavage efficiency, and it was found the 2× Mpro biosensor shows a high BRET, an increased cleavage rate and a decreased pharmacological inhibitor efficacy. Mutational analysis of the 2× Mpro biosensor showed a requirement for cleavage sites to be intact. Further, fusion of Mpro binding nanobodies to the 2× Mpro biosensor revealed the N-ter-NB2E3 2× Mpro biosensor to be the fastest cleaving Mpro biosensor. The N-ter-NB2E3 2× Mpro biosensor in the disclosed invention has application in the discovery of more potent pharmacological inhibitors of Mpro for COVID-19 treatment as well as in functional genomics applications such as characterizing mutations in the Mpro proteins that are observed in newly emerging SARS-COV-2 variants. Additionally, the increased sensitivity to Mpro-mediated cleavage of the N-ter-NB2E3 2× Mpro biosensor will be particularly useful in developing point-of-care testing (POCT) assays for detecting active SARS-COV-2 infection.
It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.
The present application claims priority to U.S. Provisional Patent Application No. 63/522,298, filed on Jun. 21, 2023, and entitled BRET-BASED MPRO BIOSENSOR WITH AN INCREASED RATE OF CLEAVAGE, the contents of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63522298 | Jun 2023 | US |
