Improvement of native structure-based peptides as efficient inhibitors of protein-protein interactions of SARS-CoV-2 spike protein and human ACE2.

New pathogens responsible for novel human disease outbreaks in the last two decades are mainly the respiratory system viruses. Not different was the last pandemic episode, caused by infection of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One of the extensively explored targets, in the recent scientific literature, as a possible way for rapid development of COVID-19 specific drug(s) is the interaction between the receptor-binding domain of the virus' spike (S) glycoprotein and human receptor angiotensin-converting enzyme 2 (hACE2). This protein-protein recognition process is involved in the early stages of the SARS-CoV-2 life cycle leading to the host cell membrane penetration. Thus, disrupting this interaction may block or significantly reduce the infection caused by the novel pathogen. Previously we have designed (by in silico structure-based analysis) three very short peptides having sequences inspirited by hACE2 native fragments, which effectively bind to the SARS-CoV-2 S protein and block its interaction with the human receptor. In continuation of the above mentioned studies, here we presented an application of molecular modeling approach resulting in improved binding affinity of the previously proposed ligand and its enhanced ability to inhibit meaningful host-virus protein-protein interaction. The new optimized hexapeptide binds to the virus protein with affinity one magnitude higher than the initial ligand and, as a very short peptide, has also great potential for further drug development. The peptide-based strategy is rapid and cost-effective for developing and optimizing efficient protein-protein interactions disruptors and may be successfully applied to discover antiviral candidates against other future emerging human viral infections.

On the use of adenovirus dodecahedron as a carrier for glycoconjugate vaccines.

Virus-Like Particles (VLPs) have been used as immunogenic molecules in numerous recombinant vaccines. VLPs can also serve as vaccine platform to exogenous antigens, usually peptides incorporated within the protein sequences which compose the VLPs or conjugated to them. We herein described the conjugation of a synthetic tetrasaccharide mimicking the Streptococcus pneumoniae serotype 14 capsular polysaccharide to recombinant adenoviral type 3 dodecahedron, formed by the self-assembling of twelve penton bases and investigated the induced immune response when administered subcutaneously (s.c.). Whether formulated in the form of a dodecahedron or disassembled, the glycoconjugate induced an anti-protein response after two and three immunizations equivalent to that observed when the native dodecahedron was administered. On the other hand, the glycoconjugate induced a weak anti-IgM response which diminishes after two doses but no IgM-to-IgG switch was observed in mice against the serotype 14 capsular polysaccharide. In definitive, the whole conjugation process preserved both particulate nature and immunogenicity of the adenoviral dodecahedron. Further studies are needed to fully exploit adenoviral dodecahedron potential in terms of plasticity towards sequence engineering and of its capacity to stimulate the immune system via the intranasal route of administration as well as to shift the response to the carbohydrate antigen by playing both with the carbohydrate to protein ratio and the length of the synthetic carbohydrate antigen.

Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase.

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

Cholesterol and phosphatidylserine are engaged in adenoviral dodecahedron endocytosis.

Adenoviral dodecahedron is a virus-like particle composed of twelve penton base proteins, derived from the capsid of human adenovirus type 3. Due to the high cell penetration capacity, it was used as a vector for protein, peptide and drug delivery. Two receptors are known to be involved in the endocytic dodecahedron uptake, namely αv integrins and heparan sulfate proteoglycans. Since it has been observed, that dodecahedron efficiently penetrates a wide range of cancer cells, it suggests that other cellular compounds may play a role in the particle endocytosis. To shed some light onto the interactions with membrane lipids and their potential role in dodecahedron entry, we performed a series of experiments including biochemical assays, fluorescence confocal imaging of giant unilamellar vesicles and surface plasmon resonance, which indicated specific preference of the particle to anionic phosphatidylserine. Experiments performed on cholesterol-depleted epithelial cells showed that cholesterol is essential in the endocytic uptake, however a direct interaction was not observed. We believe that the results will allow to better understand the role of lipids in dodecahedron entry and to design more specific dodecahedron-based vectors for drug delivery to cancer cells.

Leukocytes and drug-resistant cancer cells are targets for intracellular delivery by adenoviral dodecahedron.

One of the major factors limiting the effectiveness of cancer chemotherapy is inefficient drug delivery. Systems enabling efficient delivery and enhanced intracellular uptake appear particularly promising in this respect. Virus-like particle, adenoviral dodecahedron (Dd), employs receptor-mediated endocytosis for cell penetration and is able to deliver intracellularly dozens of cargo molecules attached to one particle. We focused on studying Dd properties in the context of cancer treatment, showing that intratumoral injection of Dd, assessed in mouse xenograft model, results in vector accumulation in tumor without spreading in off-target organs. Moreover, we demonstrated that Dd is a promising vector targeting leukocytes and drug-resistant cancer cells. Dd uptake by human blood cells analyzed in vitro indicated the preference for leukocytes in comparison to red blood cells and platelets. Furthermore, internalization of Dd-doxorubicin conjugate by drug-resistant cells leads to increased nuclear accumulation of doxorubicin and significant enhancement of cytotoxicity against target cancer cells.

Virus-like particle-mediated intracellular delivery of mRNA cap analog with in vivo activity against hepatocellular carcinoma.

Adenovirus dodecahedron (Dd), a nanoparticulate proteinaceous biodegradable virus-like particle (VLP), was used as a vector for delivery of an oncogene inhibitor to hepatocellular carcinoma (HCC) rat orthotopic model. Initiation factor eIF4E is an oncogene with elevated expression in human cancers. Cell-impermeant eIF4E inhibitor, cap structure analog (cap) and anti-cancer antibiotic doxorubicin (Dox) were delivered as Dd conjugates. Dd-cap and Dd-dox inhibited cancer cell culture proliferation up to 50 and 84%, respectively, while with free Dox similar results could be obtained only at a 5 times higher concentration. In animal HCC model the combination treatment of Dd-cap/Dd-dox caused 40% inhibition of tumor growth. Importantly, the level of two pro-oncogenes, eIF4E and c-myc, was significantly diminished in tumor sections of treated rats. Attachment to Dd, a virus-like particle, permitted the first demonstration of cap analog intracellular delivery and resulted in improved doxorubicin delivery leading to statistically significant inhibition of HCC tumor growth.

Virus-like particles as vaccine.

This review presents data on commercial and experimental virus-like particle (VLP) vaccines, including description of VLP vaccines against influenza. Virus-like particles are multimeric, sometimes multiprotein nanostructures assembled from viral structural proteins and are devoid of any genetic material. VLPs present repetitive high-density displays of viral surface proteins. Importantly, they contain functional viral proteins responsible for cell penetration by the virus, ensuring efficient cell entry and thus tissue-specific targeting, determined by the origin of the virus. The foremost application of VLPs is in vaccinology, where they provide delivery systems that combine good safety profiles with strong immunogenicity and constitute a safe alternative to inactivated infectious viruses. These stable and versatile nanoparticles display excellent adjuvant properties capable of inducing innate and cognate immune responses. They present both, high-density B-cell epitopes, for antibody production and intracellular T-cell epitopes, thus inducing, respectively, potent humoral and cellular immune responses. Uptake of VLPs by antigen-presenting cells leads to efficient immune responses resulting in control of pathogenic microorganisms.

Self-adjuvanting influenza candidate vaccine presenting epitopes for cell-mediated immunity on a proteinaceous multivalent nanoplatform.

We exploit the features of a virus-like particle, adenoviral dodecahedron (Ad Dd), for engineering a multivalent vaccination platform carrying influenza epitopes for cell-mediated immunity. The delivery platform, Ad Dd, is a proteinaceous, polyvalent, and biodegradable nanoparticle endowed with remarkable endocytosis activity that can be engineered to carry 60 copies of a peptide. Influenza M1 is the most abundant influenza internal protein with the conserved primary structure. Two different M1 immunodominant epitopes were separately inserted in Dd external positions without destroying the particles' dodecahedric structure. Both kinds of DdFluM1 obtained through expression in baculovirus system were properly presented by human dendritic cells triggering efficient activation of antigen-specific T cells responses. Importantly, the candidate vaccine was able to induce cellular immunity in vivo in chickens. These results warrant further investigation of Dd as a platform for candidate vaccine, able to stimulate cellular immune responses.

Towards a novel influenza vaccine: engineering of hemagglutinin on a platform of adenovirus dodecahedron.

BACKGROUND: The production process for the current influenza vaccine takes about 6 months and its antigenic composition must be modified annually. In the attempt towards developing influenza vaccine production that would be faster, safer and cheaper we engineered an influenza vaccine in which multiple copies of hemagglutinin (HA) would be delivered by a vector, adenovirus dodecahedron (Ad Dd). Dd is a virus-like particle, formed by assembly of twelve copies of pentameric penton base (Pb) proteins responsible for virus penetration. In order to attach HA to the vector, an adaptor containing WW domains was used. The WW domain is a linear peptide fragment identified as a partner of proline-proline-x-tyrosine (PPxY) motif present at the N-terminal extremity of the Pb protein, which is a building block of Dd. That tandem of three WW domains in fusion with the protein of interest enables interaction with Dd and efficient translocation to the cytoplasm of cells in culture. RESULTS: Since HA is an oligomeric protein with complicated processing, we prepared six different constructs of HA (A/swan/Poland/467/2006(H5N1)) in fusion with the WW adaptor. Herein we report baculovirus expression and functional analysis of six HA-WW variants. The best behaving variant was successfully delivered into human cells in vitro. CONCLUSIONS: Engineering of a soluble complex of HA with Dd, a virus-like particle that serves as a vector, an adjuvant and as a multivalent presentation platform, is an important step toward a novel influenza vaccine.

The structural basis for the integrity of adenovirus Ad3 dodecahedron.

During the viral life cycle adenoviruses produce excess capsid proteins. Human adenovirus serotype 3 (Ad3) synthesizes predominantly an excess of free pentons, the complexes of pentameric penton base and trimeric fiber proteins, which are responsible for virus penetration. In infected cells Ad3 pentons spontaneously assemble into dodecahedral virus-like nano-particles containing twelve pentons. They also form in insect cells during expression in the baculovirus system. Similarly, in the absence of fiber protein dodecahedric particles built of 12 penton base pentamers can be produced. Both kinds of dodecahedra show remarkable efficiency of intracellular penetration and can be engineered to deliver several millions of foreign cargo molecules to a single target cell. For this reason, they are of great interest as a delivery vector. In order to successfully manipulate this potential vector for drug and/or gene delivery, an understanding of the molecular basis of vector assembly and integrity is critical. Crystallographic data in conjunction with site-directed mutagenesis and biochemical analysis provide a model for the molecular determinants of dodecamer particle assembly and the requirements for stability. The 3.8 Å crystal structure of Ad3 penton base dodecamer (Dd) shows that the dodecahedric structure is stabilized by strand-swapping between neighboring penton base molecules. Such N-terminal strand-swapping does not occur for Dd of Ad2, a serotype which does not form Dd under physiological conditions. This unique stabilization of the Ad3 dodecamer is controlled by residues 59-61 located at the site of strand switching, the residues involved in putative salt bridges between pentamers and by the disordered N-terminus (residues 1-47), as confirmed by site directed mutagenesis and biochemical analysis of mutant and wild type protein. We also provide evidence that the distal N-terminal residues are externally exposed and available for attaching cargo.

Certain protein transducing agents convert translocated proteins into cell killers.

The majority of proteins are unable to translocate into the cell interior. Hence for peptide- and protein-based therapeutics a direct intracytoplasmic delivery with the aid of transducing agents is an attractive approach. We wanted to deliver to the cell interior a putatively cytotoxic protein VPg. Protein transduction was achieved in vitro with three different commercial products. However, in our hands, delivery of various control proteins without known deleterious effects, as well as of protein VPg, always induced cell death. Finally, we used a novel transducing peptide Wr-T, which was not toxic to cultured cells, even in a quite large range of concentrations. Most importantly, control protein delivered to cells in culture did not display any toxicity while VPg protein exerted a strong cytotoxic effect. These data show that results obtained with cell-penetrating agents should be interpreted with caution.

Faithful chaperones.

This review describes the properties of some rare eukaryotic chaperones that each assist in the folding of only one target protein. In particular, we describe (1) the tubulin cofactors, (2) p47, which assists in the folding of collagen, (3) α-hemoglobin stabilizing protein (AHSP), (4) the adenovirus L4-100 K protein, which is a chaperone of the major structural viral protein, hexon, and (5) HYPK, the huntingtin-interacting protein. These various-sized proteins (102-1,190 amino acids long) are all involved in the folding of oligomeric polypeptides but are otherwise functionally unique, as they each assist only one particular client. This raises a question regarding the biosynthetic cost of the high-level production of such chaperones. As the clients of faithful chaperones are all abundant proteins that are essential cellular or viral components, it is conceivable that this necessary metabolic expenditure withstood evolutionary pressure to minimize biosynthetic costs. Nevertheless, the complexity of the folding pathways in which these chaperones are involved results in error-prone processes. Several human disorders associated with these chaperones are discussed.

Purification of recombinant adenovirus type 3 dodecahedric virus-like particles for biomedical applications using short monolithic columns.

Adenovirus type 3 dodecahedric virus-like particles (Ad3 VLP) are an interesting delivery vector. They penetrate animal cells in culture very efficiently and up to 300,000 Ad3 VLP can be observed in one cell. The purification of such particles usually consists of several steps. In these work we describe the method development and optimization for the purification of Ad3 VLP using the Convective Interaction Media analytical columns (CIMac). Results obtained with the CIMac were compared to the already established two-step purification protocol for Ad3 VLP based on sucrose density gradient ultracentifugation and the Q-Sepharose ion-exchange column. Pure, concentrated and bioactive VLP were obtained and characterized by several analytical methods. The recovery of the Ad3 VLP was more than 50% and the purified fraction was almost completely depleted of DNA; less than 1% of DNA was present. The purification protocol was shortened from five days to one day and remarkably high penetration efficacy of the CIMac-purified vector was retained. Additionally, CIMac QA analytical column has proven to be applicable for the final and in-process control of various Ad3 VLP samples.

Adenovirus dodecahedron, as a drug delivery vector.

BACKGROUND: Bleomycin (BLM) is an anticancer antibiotic used in many cancer regimens. Its utility is limited by systemic toxicity and dose-dependent pneumonitis able to progress to lung fibrosis. The latter can affect up to nearly 50% of the total patient population, out of which 3% will die. We propose to improve BLM delivery by tethering it to an efficient delivery vector. Adenovirus (Ad) dodecahedron base (DB) is a particulate vector composed of 12 copies of a pentameric viral protein responsible for virus penetration. The vector efficiently penetrates the plasma membrane, is liberated in the cytoplasm and has a propensity to concentrate around the nucleus; up to 300000 particles can be observed in one cell in vitro. PRINCIPAL FINDINGS: Dodecahedron (Dd) structure is preserved at up to about 50 degrees C at pH 7-8 and during dialysis, freezing and drying in the speed-vac in the presence of 150 mM ammonium sulfate, as well as during lyophilization in the presence of cryoprotectants. The vector is also stable in human serum for 2 h at 37 degrees C. We prepared a Dd-BLM conjugate which upon penetration induced death of transformed cells. Similarly to free bleomycin, Dd-BLM caused dsDNA breaks. Significantly, effective cytotoxic concentration of BLM delivered with Dd was 100 times lower than that of free bleomycin. CONCLUSIONS/SIGNIFICANCE: Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP) results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs.

Virulence factor of potato virus Y, genome-attached terminal protein VPg, is a highly disordered protein.

Potato virus Y (PVY) is a common potyvirus of agricultural importance, belonging to the picornavirus superfamily of RNA plus-stranded viruses. A covalently linked virus-encoded protein VPg required for virus infectivity is situated at the 5' end of potyvirus RNA. VPg seems to be involved in multiple interactions, both with other viral products and host proteins. VPgs of potyviruses have no known homologs, and there is no atomic structure available. To understand the molecular basis of VPg multifunctionality, we have analyzed structural features of VPg from PVY using structure prediction programs, functional assays, and biochemical and biophysical analyses. Structure predictions suggest that VPg exists in a natively unfolded conformation. In contrast with ordered proteins, PVY VPg is not denatured by elevated temperatures, has sedimentation values incompatible with a compact globular form, and shows a CD spectrum of a highly disordered protein, and HET-HETSOFAST NMR analysis suggests the presence of large unstructured regions. Although VPg has a propensity to form dimers, no functional differences were seen between the monomer and dimer. These data strongly suggest that the VPg of PVY should be classified among intrinsically disordered proteins. Intrinsic disorder lies at the basis of VPg multifunctionality, which is necessary for virus survival in the host.

The 100K-chaperone protein from adenovirus serotype 2 (Subgroup C) assists in trimerization and nuclear localization of hexons from subgroups C and B adenoviruses.

Recombinant hexons from subgroup C adenoviruses (Ad2 and Ad5) and from a member of subgroup B (Ad3) adenoviruses have been expressed in insect cells. When expressed alone, all three hexons were found to be insoluble and accumulated as inclusion bodies in the cytoplasm. However, co-expression of recombinant Ad2, Ad5 or Ad3 hexon with Ad2 L4-100K protein resulted in the formation of soluble trimeric hexons. EM analysis of hexons revealed that they were indistinguishable from native hexon capsomers isolated from Ad2-infected human cells, or released from partially disrupted adenovirions. This suggests that 100K acts as a chaperone for hexon folding and self-assembly into capsomer in insect cells. Since 100K protein assists in the trimerization of subgroup C hexon, and of subgroup B hexon protein, it implies that it functions in a manner that is both homo- and heterotypic. During the course of recombinant protein expression, the 100K protein was found in association with hexon monomers and trimers within the cytoplasm. In the nucleus, however, 100K was found in complexes with hexon trimers exclusively. EM observation of purified 100K protein samples showed a dumb-bell-shaped molecule compatible with a monomeric protein. EM analysis of hexon-100K protein complexes showed that interaction of hexon with the 100K protein occurred via one of the globular domains of the 100K protein molecule. Our data confirm the role of the 100K protein as a scaffold protein for hexon, and provide evidence suggesting its function in hexon nuclear import in insect cells.

Poneratoxin, a neurotoxin from ant venom. Structure and expression in insect cells and construction of a bio-insecticide.

Poneratoxin is a small neuropeptide found in the venom of the ant Paraponera clavata. It is stored in the venom reservoir as an inactive 25-residue peptide. Here we describe both chemically synthesized poneratoxin and poneratoxin obtained by expression in insect cells. When expressed in insect cells, poneratoxin was observed attached to cell membranes. Both synthetic and recombinant ponerotoxins were soluble below pH 4.5. The structure of synthetic poneratoxin was characterized by circular dichroism and solved by nuclear magnetic resonance. In an environment imitating a lipid bilayer, at pH within the range of insect hemolymph, synthetic poneratoxin has a V shape, with two alpha-helices connected by a beta-turn. Insect larvae were paralyzed by injection of either of the purified toxins, with the recombinant one acting faster. The recombinant toxin-producing baculovirus reduced the average survival time of the insect host by 25 h compared with unmodified virus. Mass spectrometry analysis showed that the recombinant toxin has an N-terminal 21-residue extension, possibly improving its stability and/or stabilizing the membrane-bound state. The potential use of poneratoxin for the construction of biological insecticide is discussed.

Effect of spatial position of uterine quail blastoderms cultured in vitro on bilateral symmetry formation.

A method of in vitro culture for uterine quail blastoderms has been developed, which allows them to develop from cleavage throughout gastrulation and further: stages 4-10 of Hamburger and Hamilton (1951). The method consists of cultivating the blastoderms on egg albumen in a vertical position; this permits about 50% of the blastoderms explanted before area pellucida formation to develop bilateral symmetry and to form normal primitive streak, somites and head structures. Development of the blastoderms explanted after their area pellucida was already formed, occurred normally and was not influenced by their spatial position in the culture.