Compositions and methods for deactivating enveloped viruses such as SARS-CoV-2

FIELD OF THE INVENTION

This invention relates generally to therapeutic methods and compositions for treating and/or preventing viral infections in humans and animals, and more specifically, to protein-based therapeutic methods and compositions for treating and/or preventing infection caused by the SARS-CoV-2 virus.

BACKGROUND OF THE INVENTION

The current pandemic produced by the SARS-CoV-2 virus, the causal agent of COVID-19, has compromised the global healthcare system. Enormous efforts at a global scale are being made to produce new antiviral compounds, to repurpose FDA-approved drugs, and to obtain new vaccines that can be used to treat this disease. However, traditional methods to treat infectious diseases have serious limitations, mostly due to the highly evolution and recombination rates of viruses. For instance, the antivirals developed against a single strain of the virus are expected to decrease in effectiveness as new strains appear, which might require complete reformulation. Moreover, as these strains appear, vaccines may only partially protect against the disease or could become completely infective, as had been reported in the case of seasonal vaccines against the common flu. Furthermore, the development of new drugs is time-consuming, extremely expensive, and it is difficult to predict if the outcome would be positive.

In contrast to targeting virus-dependent processes, human-derived processes are attractive alternatives, as the human enzymes would not mutate in a time frame that would allow viruses to become resistant. The “Spike” protein of SARS-CoV-2 coronavirus, responsible for the attachment to the host receptor and the fusion of the viral envelope with the host cell membrane, is heavily N-glycosylated by the host, which helps the virus evade the immune system and infect human cells. There is an urgent need for developing new compositions and treatment to inactivate SARS-CoV-2 coronavirus by impeding the virus invading mechanism to host cells.

SUMMARY OF THE INVENTION

The invention provides new compositions and methods for preventing and treating virus infection, in particular, for treating the SARS-CoV-2 virus which has caused the COVID-19 pandemic.

The invention includes a pharmaceutical formulation for inactivating a virus having fully or partially glycosylated surface glycoproteins. The pharmaceutical formulation includes at least an active portion of a glycosidase and a pharmaceutically acceptable carrier configured to deliver the active portion to an infecting virus within an infected human or animal. The active portion retains enzymatic activity and is adapted to remove glycans from the surface glycoproteins. Exemplary glycosidase is selected from the group consisting of endo- and exo-glycosidases, including: peptide N-glycosidase (PNGase), endo-beta-N-acetylglucosaminidase (ENGase), fucosidase, mannosidase, sialidase, alpha- or beta-galactosidase, neuraminidase, hexosaminidase, aspartyl-glucosaminidase and combinations thereof. In embodiments of this invention, the active portion of the glycosidase is bound to a binding domain corresponding to the surface glycoprotein of the virus. The connecting domain can connect to the active portion and the binding domain.

Embodiments of the invention include compounds having a therapeutic domain, a binding domain, and a connecting domain. The therapeutic domain comprises of an inhibitory activity that prevents or impedes the infection of a virus. The therapeutic domain includes a glycosidase or at least one catalytic domain of a glycosidase. Again, exemplary glycosidases include isoforms of endo- and exo-glycosidases, including: peptide N-glycosidase (PNGase), endo-beta-N-acetylglucosaminidase (ENGase), fucosidase, mannosidase, sialidase, alpha- or beta-galactosidase, neuraminidase, hexosaminidase, aspartyl-glucosaminidase and combinations thereof.

The binding domain binds the therapeutic domain described above to the surface protein of targeted virus. Examples of such a binding domain include the angiotensin-converting enzyme 2 (ACE2) receptor or a soluble domain of the ACE2 receptor, preferably being substantially homologous to human ACE2 receptor. The connecting domain connects the therapeutic and binding domains, increasing contact between the therapeutic domains and targeted virus proteins.

Embodiments of this invention further include an affinity-enhancing domain, which is configured to bind tightly to complex glycans and enhance a catalytic effect of the catalytic domain.

In one aspect, the invention provides a fusion protein for preventing and treating virus infection. One example of such fusion protein is referred to as “CROWNase” herein, which comprises parts of the human PNGase in the therapeutic domain and comprises ACE2 receptor in the binding domain.

In another aspect, the invention includes a glycosidase or an active portion of a glycosidase. Embodiments of the invention include the catalytic domain of a glycosidase but otherwise less than the entire glycosidase sequence. Such embodiments can include other protein sequences such as functional domains derived from other proteins. Certain mutations can be engineered to enhance the catalytic and therapeutic functions of the proteins.

Embodiments of the invention further include a pharmaceutical formulation including said compounds and a pharmaceutically acceptable carrier configured to deliver the active portion of the compound to an infecting virus within an infected human or animal. The pharmaceutical formulation can be formulated in different forms, including nasal sprays, inhalants, tablets, suspensions, solutions for injection or for oral administration, and drops or ointment for eyes. Preferred embodiments of the invention for treating and preventing SARS-CoV-2 are in the formulations of a spray or an inhalant.

Embodiments of the invention also include methods of using a glycosidase or an active portion of a glycosidase to remove glycans from a viral surface protein, comprising contacting the compounds including such glycosidase or the active portion of the glycosidase with the targeted viral surface proteins.

Embodiments of the invention further include a method for treating or preventing viral infection, including applying therapeutic effective amount of the compounds to the targeted subjects. The subject being treated can be human or animal. Preferably, the pharmaceutical composition is applied by the use of a spray or an inhalant.

Other embodiments, objects, and advantages of the invention will be apparent to those skilled in the art from the following detailed description taken in conjunction with the appended claims and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic depiction of the entry mechanism of SARS-CoV-2.

FIG. 1B depicts a structure of a Spike protein.

FIG. 1C depicts four most common glycans attached the Spike protein (▪; N-acetylglucosamine, ●; mannose, ▴; fucose, ●; galactose, ♦; sialic acid).

FIG. 2A depicts a domain structure of mammalian PNGase with the PUB, TG and PAW domains.

FIG. 2B depicts a tridimensional structure of mouse PNGase core (PDB ID: 2F4O), showing the TG domain and the zinc-binding domain. The structure contains the covalently-attached caspase inhibitor Z-VAD-FMK (carboxy-benzyloxy-Val-Ala-Asp-a-fluoromethyl ketone), in the location associated with peptide binding.

FIG. 2C depicts an oligosaccharide removal reaction catalyzed by PNGase.

FIG. 3 depicts a delivery of an example of the invention (CROWNase) to the lungs of COVID-19 patients by inhaler. CROWNase binds tightly to the glycosylated Spike protein, through the ACE2 receptor, blocking virus attachment.

FIG. 4A depicts a model of N-linked glycan with and without fucose in h-PNGase isoform 1.

FIG. 4B shows results from SDS-PAGE of Spike protein treated with PNGase.

FIG. 4C shows other results from SDS-PAGE of Spike protein treated with h-PNGase.

FIG. 4D shows results from an immunohistochemistry experiment of Vero E6 cells transformed with liposomes containing the Spike protein treated with PNGase.

FIG. 4E shows results from Western blot experiment of Vero E6 cells transformed with liposomes containing the Spike protein treated with PNGase.

FIG. 4F is another example of immunohistochemistry of Calu-3 cells transformed with liposomes containing the Spike protein and the GAPDH gene.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated. With regard to this specification, any time a definition of a term as defined herein, differs from a definition given for that same term in an incorporated reference, the definition explicitly defined herein is the correct definition of the term.

The words “a” and “an” denote one or more, unless specifically noted.

“Polypeptide” and “protein” are used interchangeably herein and mean any peptide-linked chain of amino acids, regardless of length or post-translational modification.

Abbreviations used herein are of common usage in the art, e.g., “PNGase” refers to peptide N-glycosidase, “ACE2” refers to angiotensin-converting enzyme 2.

“Fusion protein” is a protein comprising amino acid sequences from at least two different sources. A fusion protein can comprise amino acid sequence that is derived from a naturally occurring protein or is substantially homologous to all or a portion of a naturally occurring protein, and in addition can comprise amino acids that are synthetic sequences. “Substantially similar” or “substantially homologous” refers to at least 50% sequence identity, desirably more that 50% sequence identity, such as each 1% increment over 50%, and desirably at least 60%, 70%, 80%, or 90%, and preferably greater than 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

Coronaviruses are enveloped positive single-stranded RNA viruses. Members of this family are among the largest RNA viruses known, with virions ranging from 118 to 136 nm in diameter and genome sizes from 25 to 32 kb (49). Virions are spherical and have a large glycoprotein protruding from the envelope, the Spike protein, giving the appearance of a crown under the microscope. The genome size of the SARS-CoV-2 virus is approximately 30 kb, encoding 15 non-structural proteins, 4 structural proteins, and 8 auxiliary proteins (50). The structural proteins are the Spike glycoprotein, small envelope glycoprotein E, membrane glycoprotein M, and the nucleocapsid protein. The three glycoproteins are embedded or tightly associated to the membrane. Glycoprotein M has roles in determining the shape of the virus envelope and stabilizing the nucleocapsid (51). Glycoprotein E is involved in production and maturation of the virus (52) and the N protein binds to the RNA genome, forming the nucleocapsid (51). The Spike glycoprotein is particularly important, as this is the viral component involved both in host cell recognition and cell infection, through the fusion of the viral envelope with the cell membrane (18).

Recent studies have shown that the Spike protein binds tightly to the angiotensin-converting enzyme 2 receptor (ACE2), found in the membrane of the host cell (53, 54), the same receptor used by the SARS-CoV-1 virus to infect cells. Remarkably, the binding of SARS-CoV-2 to this receptor is 10-20 times stronger, explaining the very high infectivity of COVID-19 virus (54, 55). The Spike protein is homotrimeric and highly glycosylated (56, 57). Each monomer has an ectodomain, comprising the majority of the protein, a single transmembrane helix and a short internal C-terminal tail (56). The ectodomain contains two smaller regions (also known as subunits), the receptor binding S1 subunit and the membrane-fusion S2 subunit. Upon attachment of the receptor binding domain in S2 to the ACE2 receptor, the spike protein is cleaved at the S1/S2 boundary region and at the S2′ site by TMPRSS2 and other proteases found in the host membrane (56). The proteolytic cleavage of the spike protein triggers the formation of a six-helix bundle fusion core complex by the Heptad repeat 1 and 2 domains in the S2 subunit, which brings the virus and host membrane together and mediate membrane fusion, allowing the release of the genetic material into the host cell, completing the infection process (19, 56, 58) (FIG. 1A).

As indicated previously, the N-glycosylation of the Spike protein is a critical structural factor that allows the virus to escape the immune system. Moreover, several studies indicate the N-linked glycans play an essential role in cell entry and that their removal halts SARS-CoV-2 infection. For instance, site-directed mutagenesis of predicted N-glycosylation sites N212D, N276D and N283D of the spike protein of infectious bronchitis virus (IBV) coronavirus resulted in inhibition of a cell-cell fusion model of Vero cells (32). Remarkably, mutations of these three N-glycosylation sites completely stop IBV viral replication (32). Two of these sites, N282 and N343 (located in the N terminal domain and receptor binding domain respectively) are conserved in SARS-CoV1 and SARS-CoV-2 (FIG. 1B), indicating that they play similar roles.

PNGase (peptide N-glycosidase) catalyzes the hydrolysis of the amino bond between the first N-acetylglucosamine residue of N- attached glycans and a conserved asparagine residue in the protein, producing aspartate and a free glycan with an amino group attached to the proximal N-acetyl glucosamine (NAG) (FIG. 2C). The amino group is spontaneously hydrolyzed, producing the free glycan with two β (1-4) NAG residues (i.e. N—N-diacetylchitobiose) in the reducing end (70). PNGase catalytic activity is highly variable and depending on the species, it can process high mannose, hybrid, or complex oligosaccharides (70-73), and native or denatured glycosylated peptides (74, 75). SARS-CoV, MERS-CoV, and SARS-CoV-2 spike protein glycans are for the most part complex, carrying NAG as the second sugar residue in the branches and an α (1-6) fucose residue in the proximal NAG residue (57, 59) (FIG. 1C). PNGase is a very important part of the protein homeostasis mechanisms in the cell, processing misfolded or nonfunctional glycosylated proteins. The non-functional proteins are retrotransported from the endoplasmic reticulum to the cytoplasm. In the cytoplasm, PNGase removes the glycans, allowing the degradation of the peptide by the proteasome and the oligosaccharide by diverse glycosidases (76, 77).

In contrast with bacteria, unicellular fungi, and plants, which contain the active core (Transglutaminase; TG) of the enzyme as the only domain, the human enzyme contains two additional domains: the PUB (PNGase and UBiquitin related) and the PAW (PNGase And other Worm proteins) domains (70). FIG. 2B shows the structure of the mammalian TG core domain, composed of a central highly curved β-sheet with six antiparallel strands, stabilized by eight a-helices. The TG core domain binds the β (1-6) NAG dimer (N—N-diacetylchitobiose) (78) and the peptide region carrying the glycosylated asparagine (79), and also carries the catalytic triad of conserved cysteine, histidine, and aspartate residues involved in the de-glycosylation reaction (80, 81). The TG domain also includes a three-stranded beta-sheet zinc binding domain, which forms the “lid” of the peptide binding site (79, 82). The PAW domain is also able to bind oligosaccharides, in particular high-mannose glycans with three branches (70, 83). The PUB domain appears to be regulatory and binds to several proteins involved in the proteasome function (79, 84, 85).

The invention provides compositions and methods of using the TG catalytic core and PAW domains to remove SARS-CoV-2 glycan shield. Embodiments of the invention include a fusion protein (referred to herein as “CROWNase”) that can remove the glycan shield of SARS-CoV-2 virus, stopping the entry of the virus into the cell and exposing the antigenic components of the virus to the immune system, allowing the quick development of an appropriate immune response by the body.

Embodiments of this invention include CROWNase, including domains of h-PNGase and improves the performance of h-PNGase. Mutation sites found in highly active homologous enzymes that stabilize high-energy conformations of the substrate and lead to an improved catalytic rate are introduced to catalytic site. The PAW domain of h-PNGase, which is involved in substrate affinity and specificity, is modified to improve the affinity for the types of glycans found in the Spike protein. The PAW domain can be also substituted for an affinity-enhancing domain which include another protein (e.g. galectin) that can bind more tightly to the complex. CROWNase further adds the ACE2 receptor to the engineered h-PNGase. FIG. 3 demonstrates the example of delivery of CROWNase to the lungs of COVID-19 patients. CROWNase binds tightly to the glycosylated Spike protein, through the ACE2 receptor, blocking virus attachment. This enzyme removes the virus glycan layer through the activity of engineered h-PNGase. CROWNase further includes a connecting domain that comprises a highly flexible loop of about 10 amino acid residues to connect both h-PNGase domain and ACE2 receptor, to allow h-PNGase to reach to the virus and carry out its function. Examples of a connecting domain can include polyglycine or sequences from the protein itself, e.g., the sequence between the TG and PAW domains. To stabilize the construct for improved delivery through an inhaler, solvent-accessible and hydrophobic regions of the protein can be replaced with hydrophilic residues.

Embodiments of the invention include a protein compound that has PNGase activity, catalyzing the cleavage of a beta-aspartyl-glucosamine and the amide side chain of Asn, converting Asn to Asp and free glycan. Such embodiments can also include a protein compound that has endo-beta-N-acetylglucosaminidase (ENGase) activity, cleaving the beta-1,4-glycosidic bond of an N,N′-diacetylchitobiose core. Other examples of embodiments include compounds removing the alpha glycosidic bond between the proximal NAG and fucose (e.g., fucosidase), compounds removing the alpha glycosidic bond between sialic acid and the terminal antennary galactose residue (e.g. Sialidase or neuraminidase, compounds removing the alpha bonds between galactose molecules at the base of the antenna and the beta bond between the second NAG and the galactose molecule (e.g., alpha and beta-manosidases), compounds removing the beta glycosidic bond between the antennary mannose and NAG residues (e.g., hexosaminidase), and compounds removing the beta bond between the NAG and galactose molecule in the antenna (e.g., beta-galatosidase).

Embodiments of the invention include compounds formulated as pharmaceutical compositions, which include a therapeutically effective amount of the compound. Depending on the target cell, the compounds of the invention can be formulated as tablets, capsules for oral administration, solutions, suspensions, inhalants or nasal sprays. The therapeutically effective amount of the compound depends on the route of administration, condition and physical characteristics of the patients, and are tailored to achieve a desired therapeutic effect. The compounds can be designed for human use or animal use. The invention further includes the method of treating and preventing infection by viruses such as SARS-CoV-2 by applying the therapeutically effective amount of the compounds. In the case of SARS-CoV-2 virus, as it primarily infects the upper respiratory tract of the patient, the preferred embodiment is applying the compound in the form of nasal spray or inhalants. Embodiments of the invention can be also administered orally or delivered topically to the eyes in the forms of drops, sprays or ointments to prevent and treat the infection.

The invention also includes methods of inactivating viruses by glycosidases, which remove the glycan (sugar) coating from the surface proteins. Examples of such glycosidases include PNGase, endo-beta-N-acetylglucosaminidase (ENGase), fucosidase, mannosidase sialidaseglatactosidase, neuramidiase, hexosaminidase, aspartylglucosaminidase and their combinations. Examples of viruses include SARS-CoV-2, other known coronaviruses, and enveloped viruses in general, which depend on N-glycosylation for attachment and infection.

The present invention is described in further detail in connection with the following examples which illustrate or simulate various aspects involved in the practice of the invention. It is to be understood that all changes that come within the spirit of the invention are desired to be protected and thus the invention is not to be construed as limited by these examples.

EXAMPLES
De-Glycosylation of SARS-CoV-2 Spike Protein by PNGase

A tridimensional model of the TG core domain of isoform 1 of h-PNGase was built using the I-TASSER server (88-90), mostly based on crystal structures of mouse PNGase. A Val-Ala-Asn peptide was placed into the model based on the crystallographic position of the inhibitor Z-VAV-FMK. An oligosaccharide carrying an a fucose residue in the proximal NAG residue, similar to the complex-type glycans found in the spike protein, was N-linked to the peptide using the glycoprotein builder on the GLYCAM server (91) (FIG. 1C). As shown in FIG. 4A, the predicted site of h-PNGase isoform 1 can accommodate the glycan in a pose that would allow the catalytic triad to perform the de-glycosylation. To corroborate that PNGase is able to remove the complex oligosaccharides in the native Spike protein, the 6×-His tagged h-PNGase isoform 1 was cloned and purified with a purity of 80%. Tests with commercially available PNGase F from the bacterium Elizabethkingia meningoseptica (New England Biolabs) were carried out. The native glycosylated Spike protein, containing the S1, S2, transmembrane, and internal domains was purchased from Fisher Scientific (catalog number: 50-196-3983) and was passed once through gel filtration chromatography to eliminate impurities. PNGase F or h-PNGase (0.1 mg/ml) were incubated for 1 h at 37° C. with the spike protein trimer (2 mg/ml) in Gamble's saline medium (pH 7.4), which resembles the composition of the extracellular liquid found in the lungs (92). The reaction was stopped with the addition of 0.1% SDS and the samples were run under denaturing conditions in SD S-PAGE. The molecular weight of the fully glycosylated spike protein monomer is 220-250 kDa and upon removal of all glycans its molecular weight decreases to the predicted size of the polypeptide, 170 kDa (93). The commercially available Spike protein appears to be a population of species with different molecular weights, with a relatively diffused band spanning 200-260 kDa (FIG. 4B). The incubation with PNGase F produces two diffuse bands of 230 and 180 kDa, indicating that the enzyme is able to remove the glycan shield. The incubation with human PNGase isoform 1 also produces species of lower molecular weights but with a different pattern, with minor bands of 200 and 180 kDa and with a seemingly lower activity. The lower activity could be due in part to the presence of the 6×-His Tag. These results indicate that both the human and commercially available PNGase are able to remove the glycan shield of the native Spike protein.

Inactivation of Spike Protein-Mediated Membrane Fusion by PNGase

To test that the de-glycosylation of the spike protein by PNGase inactivates virus entry, a virus-free assay is developed with the spike protein incorporated into artificial proteoliposomes. As reported extensively in the literature, the Spike protein solely carries out binding to the receptor and entry into the cell (56). In this method, the commercially-available full-length Spike protein was resuspended in Gamble's buffer containing 0.3% b-D-dodecylmaltoside, a gentle non-detergent that preserves the structure of membrane proteins. The solubilized spike protein was mixed with membrane lipid extract from liver (Avanti Polar Lipids) with 0.5 M octyl-glucoside. The slow removal of detergents by Riorad BioBeads allows the formation of tightly sealed artificial proteoliposomes. This technique was extensively used to reconstitute large bacterial membrane complexes into impermeable and stable liposomes (94-96). In this case, the solution with the lipids and the Spike protein contained Myc-tagged human GADPH gene into the pCMV-Myc plasmid, which is incorporated into the liposomes serving as a reporter of the ability of the liposomes to be fused with mammalian cells. The proteoliposomes were incubated with Vero cells, which carry the ACE2 receptor and TMPRSS2 protease and can be infected by SARS-CoV-2 (97, 98). The cells were fixed and an anti-Myc antibody was used to visualize transformed cells by immunohistochemistry. FIG. 4C shows another example of inactivation of the Spike protein by CROWNase, with SDS-PAGE of Spike protein treated with h-PNGase.

As shown in FIG. 4D, proteoliposomes with the reconstituted Spike protein were able to effectively deliver the GAPDH gene to the cells and those without the spike protein delivered the recombinant gene with a low efficiency, as expected. Remarkably, proteoliposomes carrying the spike protein that were incubated with the human and bacterial PNGases for 2 hours showed a significant decrease (30% and 60%, respectively) in the ability to deliver the GAPDH gene. These effects were confirmed by western blot against the Myc-tag (FIG. 4E). The results demonstrate that h-PNGase is able to inactivate the spike protein-mediated fusion by removing the glycan coating. FIG. 4F is another example of immunohistochemistry of Calu-3 cells transformed with liposomes containing the Spike protein and the GAPDH gene after a 20 min treatment with PNGase treatment. DNA was stained with Hoescht, GAPDH stained with anti-myc (grey).

Identification of h-PNGase Isoforms that Inactivate SARS-CoV-2

1. Human PNGase Purification

It has been reported that PNGase is ubiquitous in the body, with an activity that varies enormously (by up to 2 orders of magnitude) in different organs, with the highest activity found in the liver and testes (99), suggesting that the isoforms could have very different kinetic properties and selectivity. The human genome contains a single gene coding for PNGase (NGLY1). This gene produces 8 isoforms (isoforms 1-4 and X1-X4) due to alternative splicing (100). cDNA for isoforms 1-4 is acquired (Genecopoeia) and specific primers for each of the isoforms are used to produce the PCR amplicons, which can be cloned into the pBAD-HisB expression vector with a 6xHis tag, to allow a quick one-step purification of the enzymes. The predicted h-PNGase isoforms X1-X4 can be synthesized (biomatik) and similarly cloned into pBAD/HisB. The 6xHis tag is located in the C-terminus, separated from the h-PNGase by a flexible 4xGly linker and by the TEV protease sequence (ENLYFQS), to eliminate the 6xHis tag (101), which has shown to decrease the activity of several proteins (102).

2. Spike Protein Purification

The full-length SARS-CoV-2 spike DNA sequence, with a C-terminal 4xGly linker and a 6xHis tag, can be synthesized from cDNA prepared from genomic RNA (ATCC) and cloned into the mammalian expression vector pαH, such as using methods described in the literature (31, 54). The FreeStyle 293-F cells are transfected with the pαH-S construct (54). These cells have high transfection efficiencies and can reaching high density in suspension, which allows the production of large amounts of recombinant protein. Previous reports indicate that the Spike proteins obtained from these cells carry their native fold and contain the expected glycosylation pattern (31, 54).

3. Activity Measurements

The activity of the isoforms can be tested using the Spike protein produced as described above. PNGase activity can be measured by Western blot, following the molecular weight of the spike protein, using rabbit polyclonal antibody against the SARS-CoV spike protein (SARS-Sm antibody, Abgent). However, this method has a limited resolution, is time-consuming, and it is only semiquantitative. To obtain steady-state kinetic rates, the recently developed 96-well plate colorimetric technique by Wang, T et al. (103) can be used, based on the reaction of the tetrazolium dye WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) with the reducing end of the polysaccharide produced by PNGase. Steady state activity rates are measured at different concentrations of the spike protein substrate, allowing the calculation of the kinetic parameters Vmax and Km for the eight isoforms of h-PNGase. The isoform that shows the highest Vmax and lowest Km (indicating high activity and affinity) can be selected for further tests, in particular for protein engineering experiments.

4. PNGase Crystallization

The h-PNGase isoform (or isoforms) that show the highest activities or affinities can be crystalized. There are currently only two eukaryotic PNGases crystallized, from yeast (PDB: 3ESW) and mouse (2F4O). The main objectives of the crystallization experiments are to obtain the tridimensional structure at high resolution (ideally <2.0 Å) of the whole protein and to solve the complex with the substrates (i.e., asparagyl-chitobiose) or substrate analogs, to reveal atomic-level interactions between the substrates and the protein. As a starting point, conditions previously reported for the mouse PNGase crystallization (82) are used. In addition, new conditions for protein crystallization can be screened, which could be obligatory for protein-substrate complexes. The protein-substrate complexes can be explored by direct co-crystallization and by protein-crystal soaking with various concentrations of the substrates. Initial screening for crystal growth can be performed for 100-400 nL protein-solution droplets by sitting-droplet vapor-diffusion method, using Mosquito crystallization robots and 96-well CystalQuick Greiner plates. If necessary, larger crystallization droplets can be set up manually. Once high-quality crystals are obtained, protein structures can be determined based on the previously used procedures (104). Diffraction data can be collected at 100 K at the 19-ID (105) and 19-BM beamlines of the Structural Biology Center at the ANL Advanced Photon Source (APS). The structures can be determined by molecular replacement (MR) method using available structures from Protein Data Bank (PDB) and the HKL3000 program suite (106) incorporating MOLREP (107), SOLVE/RESOLVE (108) and ARP/wARP (109) programs. Initial models can be completed by manual modeling using COOT program (110) and refined using the REFMAC program (111) from CCP4 suite (112). The stereochemistry of the structures can be validated with PHENIX suite (113) incorporating MOLPROBITY tools (114).

5. Spike Protein-Mediated Liposome Fusion

These experiments can be carried out to test the effects of different PNGase isoforms on the ability of Spike protein-containing proteoliposomes to deliver GAPDH to Vero E6 cells.

6. In Vitro SARS-CoV-2 Infectivity Test

To demonstrate that the de-glycosylation by PNGase inactivates the virus and prevents the infection by stopping membrane fusion, the effects of the incubation of h-PNGase with the SARS-CoV-2 virus can be tested. Calu-3 (lung carcinoma cell line) and Vero E6 cells are ideal to perform SARS-CoV-2 infection assays since they express the ACE2 receptor and produce high virus titers (97, 115). Polarized monolayers of Calu-3 and Vero E6 cells can be grown in Dulbecco minimal essential media (DMEM), supplemented with heat-inactivated fetal bovine serum (10%) and antibiotic/antimycotic mixture. Infection of Vero E6 and Calu-3 cells can be evaluated as the formation of plaques 48 hours post-infection (hpi) (115). The virus inoculum can be incubated for different times and with different concentrations of h-PNGase. Cells can be incubated at 37° C. for 2 hr and the unbound viral inoculum is washed with PBS. Cultures can be overlaid with culture media plus 0.8% agarose and after 48 hpi plaques be visually quantified by neutral red staining (116). Additionally, Western-blotting can be performed from total protein extracted from the infected cultures to detect the presence of the viral proteins using a rabbit polyclonal serum against the SARS-CoV-2 spike protein (40150-T52, Sino Biological).

7. Alternative Strategy

A construct taking the TG core domain of the isoform with the highest activity and the PAW domain of the isoform with the lowest Km can be made. The TG core carries the residues involved in catalysis (81) and thus it determines the Vmax, and the PAW domain determines the affinity and specificity (78, 83) of the enzyme and thus Km. Moreover, to identify if some of the isoforms have an intrinsic low activity, they can be tested using RNase, a preferred substrate of most PNGases (103). The tests can be conducted with the folded and heat-denatured RNase, since some PNGases act specifically on denatured samples (74). It is likely that some of the h-PNGase isoforms would not be highly active due to the steric hindrance of sugars interfering with the ability of the enzyme to find the binding site. If the de-glycosylation process is too slow, the effects of other human enzymes normally involved in oligosaccharide degradation can be tested. For instance, other commercially available human enzymes (New England Biolabs): 1) α-mannosidase Man2Cl, which removes the mannose branches from glycans in the cytosol (117, 118), 2) (β-Hexosaminidase A, which removes the distal NAG from oligosaccharide branches (119), and 3) Fucosidase, which removes the a (1-6) fucose reside in the proximal NAG (120). highly active isoforms that require no other glycosidase can be prioritized, but if these enzymes produce a significant effect (i.e., decrease of more than one order in magnitude in the inactivation time or PNGase concentration), they can be incorporated into the CROWNase chimera. It is also possible that some of the PNGase isoforms would not be stable during the purification. In this case, they can be co-expressed with the proteasome p97 protein, which has allowed the purification of mouse PNGase (83).

Protein Engineering and CROWNase Design

1. Chimera Construction

The CROWNase chimera desirably binds tightly to the SARS-CoV-2 virus. This can be achieved by including the soluble domain of the ACE2 receptor in the construct. It has been shown that the Spike protein has a very high affinity for the ACE2 receptor (nM range) (121). Moreover, studies with RBD fragments demonstrate that the glycosylation state of RBD does not modify its binding with ACE2 (122, 123), and thus de-glycosylation of the Spike protein by PNGase would not release CROWNase from the virus.

The human ACE2 fragment encoding for the soluble portion, residues 19-615 (124), can be amplified by PCR from a cDNA clone (NM_021804.2, GeneCopoeia) and cloned into a pBAD/HisB expression vector with a C-terminus 6x-His tag for purification. BL21 E. coli cells can be transformed with the constructs for protein expression. To verify that the purified ACE2 soluble domain retains its native fold and function, binding analysis with the Spike protein can be carried out by isothermal titration calorimetry. The chimera can be constructed by cloning both genes together in frame. A highly flexible loop of 5-20 amino acid residues connecting both domains can be introduced, to allow h-PNGase to reach to the virus and carry out its function. The activity of the chimera can be tested for its ability to de-glycosylate the Spike protein and to prevent liposome fusing. Depending on the kinetic parameters obtained the sequence of the linking peptide can be modified, to make it longer, more rigid or to modify its polarity. Chimeric proteins with high de-glycosylation activities can be tested against the SARS-CoV-2 virus in vitro, using the methods described above.

2. h-PNGase Enzyme Engineering: Activity Enhancement

h-PNGase can be engineered to enhance the rate at which it can process complex glycans. The TG and PAW domains are designed to increase the activity and affinity of the enzyme, depending on the kinetic data obtained. If turnover is limited by the catalytic step, the core domain can be prioritized for engineering. If it is limited by substrate binding, then the PAW domain can be prioritized. Mutations in the core domain can be inspired by variations observed in other species, in particular by the PNGases from Aspergillus tubingensis and the commercially available PNGase F from E. meningoseptica, which are the most active enzymes (73, 125). Quantum mechanics/molecular mechanics (QM/MM) calculations (126) can be used to evaluate the extent to which variations of the core domain stabilize the tetrahedral transition state where the catalytic cysteine is covalently bonded to both the scissile carbonyl and the amine of the glycan. Variations that most substantially reduce the energetic barrier to transition are expected to provide the largest increases in activity.

Studies with mammalian PNGase show that the PAW domain determines the specificity and overall affinity of the protein (83). The PAW domain of the mouse enzyme binds 5 mannose residues at the branching point of the oligosaccharide. The crystal structure reveals that three of the mannose residues are bound tightly to this site through extensive H-bonding interactions. Interestingly, the most proximal and most distal mannoses are relatively loose (83), which might explain the low affinity observed in the enzymes from different organisms (81, 103, 125, 127). This domain does not appear optimal to bind the complex glycans observed on the SARS-CoV-2 spike. Mutations to residues in contact with the glycan, in particular those in the distal positions, can be prioritized for expression and biochemical assays based on molecular modeling (as a starting point) and on the tridimensional structure of the TG and PAW domains, when they are available. Mutants in which binding free energy calculations predict to have a reasonable affinity for the complex glycan can be prioritized for testing. Introducing residues in these positions that can form hydrogen bonds with the sugar likely can increase the affinity of the site.

3. h-PNGase Enzyme Engineering: Stability Enhancement

Major concerns with delivering protein therapeutics by inhalation are unfolding and aggregation due to nebulization stresses. These concerns are addressed by engineering the protein to increase its stability by making it more soluble. Common strategies to increase protein solubility include replacing hydrophobic surface residues with hydrophilic ones and shifting the pI away from the physiologic pH (128). Crystallographic studies have identified L431, 1435, L438, V439, and L442 of mouse PNGase (highly homologous to human) as a hydrophobic face of helix 12 that enables its interaction with the regulatory protein HR23 (82). Similarly, 154 is part of a hydrophobic pocket that mediates the interaction between PNGase and p9′7, a protein proposed to facilitate endoplasmic reticulum-associated protein degradation (129). As these protein-protein interactions of cytosolic PNGase are unnecessary for therapeutic purposes herein, these regions can be targeted for site-directed mutagenesis. Additional regions can be identified based on computer simulations of the enzyme's atomistic molecular dynamics. Molecular dynamics simulations can be analyzed to obtain the surface aggregation propensity (130) and identify hydrophobic patches on the enzyme. Molecular modelling can be used to prioritize mutants predicted to most significantly improve solubility. Initially, modelling can be based on the fast and computational inexpensive generalized Born implicit solvent model (131). As data on the stability of mutants can be accumulated, they can be used to refine computational predictions. If the generalized Born solvation model is unsuccessful at reproducing experimental stability trends, alternative strategies include: the domain decomposition Conductor-like Screening model (132), the quantum mechanical approach that accounts for polarization, or the free energy calculations for the ensemble of accessible protein conformations (133), or machine learning methods.

4. ACE2 Protein Engineering: Stability Enhancement

The stability of the PNGase-ACE chimera can be enhanced with guidance from the molecular modeling techniques to engineer PNGase. Regions that can be prioritized for substituting hydrophobic with hydrophilic residues include the interdomain areas and the linker between PNGase and ACE2.

Thus, the invention provides compositions and methods for treating viral infections. The invention illustratively disclosed herein suitably may be practiced in the absence of any element, part, step, component, or ingredient which is not specifically disclosed herein.

While in the foregoing detailed description this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.

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Compositions and methods for deactivating enveloped viruses such as SARS-CoV-2

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