Exploratory studies on soluble small molecule CD4 mimics as HIV entry inhibitors.

Several small molecule CD4 mimics, which inhibit the interaction of gp120 with CD4, have been developed. Original CD4 mimics such as NBD-556, which has an aromatic ring, an oxalamide linker and a piperidine moiety, possess significant anti-HIV activity but with their hydrophobic aromatic ring-containing structures are poorly soluble in water. We have developed derivatives with a halopyridinyl group in place of the phenyl group, such as KKN-134, and found them to have excellent aqueous solubility. Other leads that were examined are YIR-821, a compound with a cyclohexane group in a spiro attachment to a piperidine ring and a guanidino group on the piperidine nitrogen atom, and its PEGylated derivative, TKB-002. YIR-821 and TKB-002 retain potent anti-HIV activity. Here, new CD4 mimics, in which the phenyl group was replaced by a halopyridinyl group with the halogen atoms in different positions, their derivatives without a cyclohexane group on the piperidine ring and their hybrid molecules with PEG units were designed and synthesized. Some of these compounds show significantly higher aqueous solubility with maintenance of certain levels of anti-HIV activity. The present data should be useful in the future design of CD4 mimic molecules.

Serine/threonine ligation-assisted chemical synthesis of HMGA1a protein with site-specific post-translational modifications.

Dissecting the function of proteins' post-translational modifications (PTMs) is seriously hindered by the difficulty in obtaining the homogeneous protein with the PTMs of interest. Chemical protein synthesis offers a great potential to overcome this limitation. Here, a detailed protocol is introduced for chemical synthesis of HMGA1a protein with site-specific modifications via Ser/Thr ligation strategy, by which we can systematically study the function of the triple phosphorylation (3pSer) in the HMGA1a acidic tail. For complete details on the use and execution of this protocol, please refer to Wei et al. (2021).

Spray-Dried Inhalable Powder Formulations of Therapeutic Proteins and Peptides.

Respiratory diseases are among the leading causes of morbidity and mortality worldwide. Innovations in biochemical engineering and understanding of the pathophysiology of respiratory diseases resulted in the development of many therapeutic proteins and peptide drugs with high specificity and potency. Currently, protein and peptide drugs are mostly administered by injections due to their large molecular size, poor oral absorption, and labile physicochemical properties. However, parenteral administration has several limitations such as frequent dosing due to the short half-life of protein and peptide in blood, pain on administration, sterility requirement, and poor patient compliance. Among various noninvasive routes of administrations, the pulmonary route has received a great deal of attention and is a better alternative to deliver protein and peptide drugs for treating respiratory diseases and systemic diseases. Among the various aerosol dosage forms, dry powder inhaler (DPI) systems appear to be promising for inhalation delivery of proteins and peptides due to their improved stability in solid state. This review focuses on the development of DPI formulations of protein and peptide drugs using advanced spray drying. An overview of the challenges in maintaining protein stability during the drying process and stabilizing excipients used in spray drying of proteins and peptide drugs is discussed. Finally, a summary of spray-dried DPI formulations of protein and peptide drugs, their characterization, various DPI devices used to deliver protein and peptide drugs, and current clinical status are discussed.

Vaccine formulations in clinical development for the prevention of severe acute respiratory syndrome coronavirus 2 infection.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to an unprecedented effort toward the development of an effective and safe vaccine. Aided by extensive research efforts into characterizing and developing countermeasures towards prior coronavirus epidemics, as well as recent developments of diverse vaccine platform technologies, hundreds of vaccine candidates using dozens of delivery vehicles and routes have been proposed and evaluated preclinically. A high demand coupled with massive effort from researchers has led to the advancement of at least 31 candidate vaccines in clinical trials, many using platforms that have never before been approved for use in humans. This review will address the approach and requirements for a successful vaccine against SARS-CoV-2, the background of the myriad of vaccine platforms currently in clinical trials for COVID-19 prevention, and a summary of the present results of those trials. It concludes with a perspective on formulation problems which remain to be addressed in COVID-19 vaccine development and antigens or adjuvants which may be worth further investigation.

Recombinant Antibodies in Basic Neuroscience Research.

Basic neuroscience research employs antibodies as key reagents to label, capture, and modulate the function of proteins of interest. Antibodies are immunoglobulin proteins. Recombinant antibodies are immunoglobulin proteins whose nucleic acid coding regions, or fragments thereof, have been cloned into expression plasmids that allow for unlimited production. Recombinant antibodies offer many advantages over conventional antibodies including their unambiguous identification and digital archiving via DNA sequencing, reliable expression, ease and reliable distribution as DNA sequences and as plasmids, and the opportunity for numerous forms of engineering to enhance their utility. Recombinant antibodies exist in many different forms, each of which offers potential advantages and disadvantages for neuroscience research applications. I provide an overview of recombinant antibodies and their development. Examples of their emerging use as valuable reagents in basic neuroscience research are also discussed. Many of these examples employ recombinant antibodies in innovative experimental approaches that cannot be pursued with conventional antibodies. © 2020 Wiley Periodicals LLC.

Semisynthesis of site-specifically succinylated histone reveals that succinylation regulates nucleosome unwrapping rate and DNA accessibility.

Posttranslational modifications (PTMs) of histones represent a crucial regulatory mechanism of nucleosome and chromatin dynamics in various of DNA-based cellular processes, such as replication, transcription and DNA damage repair. Lysine succinylation (Ksucc) is a newly identified histone PTM, but its regulation and function in chromatin remain poorly understood. Here, we utilized an expressed protein ligation (EPL) strategy to synthesize histone H4 with site-specific succinylation at K77 residue (H4K77succ), an evolutionarily conserved succinylation site at the nucleosomal DNA-histone interface. We then assembled mononucleosomes with the semisynthetic H4K77succ in vitro. We demonstrated that this succinylation impacts nucleosome dynamics and promotes DNA unwrapping from the histone surface, which allows proteins such as transcription factors to rapidly access buried regions of the nucleosomal DNA. In budding yeast, a lysine-to-glutamic acid mutation, which mimics Ksucc, at the H4K77 site reduced nucleosome stability and led to defects in DNA damage repair and telomere silencing in vivo. Our findings revealed this uncharacterized histone modification has important roles in nucleosome and chromatin dynamics.

Nanoparticle-based immunotherapy of breast cancer using recombinant Helicobacter pylori proteins.

Breast cancer is the most common cancer in women worldwide and is associated with substantial medical and economic burden. We report the development of a hybrid immunotherapeutic system based on recombinant Nap protein from Helicobacter pylori (HP-Nap) for the treatment of breast tumors. Chitosan nanoparticles with pseudo-spherical morphology and positive zeta potential were synthesized as carriers for HP-Nap. In vitro study was performed on mouse breast cancer cell line (4T1) and human breast cancer cell lines (MCF7). In vivo study was done on 4T1 tomural mice. TUNEL assay and real time PCR test were performed on tumor mice receiving the nanoparticle treatment. The nanoparticle-protein complex induced apoptosis in vitro in cultured breast cancer cells. In-vivo studies on inbred, female BALB/c mice confirmed the shrinkage of tumor mass after administration of the nanoparticle complex containing HP-Nap. The TUNEL assay further confirmed apoptosis in extracted mouse breast cancer cells. A decrease in the expression of VEGF and MMP9 genes was observed in 4T1 cells as shown by real time PCR. Our data suggesting that the therapeutic nanocomplex may have led to decreased tumor growth in mice through changing the production rate of cytokines and increasing tumoricidal activities of the immune system.

Properties of Biomimetic Artificial Spider Silk Fibers Tuned by PostSpin Bath Incubation.

Efficient production of artificial spider silk fibers with properties that match its natural counterpart has still not been achieved. Recently, a biomimetic process for spinning recombinant spider silk proteins (spidroins) was presented, in which important molecular mechanisms involved in native spider silk spinning were recapitulated. However, drawbacks of these fibers included inferior mechanical properties and problems with low resistance to aqueous environments. In this work, we show that ≥5 h incubation of the fibers, in a collection bath of 500 mM NaAc and 200 mM NaCl, at pH 5 results in fibers that do not dissolve in water or phosphate buffered saline, which implies that the fibers can be used for applications that involve wet/humid conditions. Furthermore, incubation in the collection bath improved the strain at break and was associated with increased ß-sheet content, but did not affect the fiber morphology. In summary, we present a simple way to improve artificial spider silk fiber strain at break and resistance to aqueous solvents.

Development of a Novel Short Synthetic Antibacterial Peptide Derived from the Swallowtail Butterfly Papilio xuthus Larvae.

Insects possess biological defense systems that can effectively combat the invasion of external microorganisms and viruses, thereby supporting their survival in diverse environments. Antimicrobial peptides (AMPs) represent a fast-acting weapon against invading pathogens, including various bacterial or fungal strains. A 37-residue antimicrobial peptide, papiliocin, derived from the swallowtail butterfly Papilio xuthus larvae, showed significant antimicrobial activities against several human pathogenic bacterial and fungal strains. Jelleines, isolated as novel antibacterial peptides from the Royal Jelly (RJ) of bees, exhibit broad-spectrum protection against microbial infections. In this study, we developed a novel antimicrobial peptide, PAJE (RWKIFKKPFKISIHL-NH2), which is a hybrid peptide prepared by combining 1-7 amino acid residues (RWKIFKK-NH2) of papiliocin and 1-8 amino acid residues (PFKISIHL-NH2) of Jelleine-1 to alter length, charge distribution, net charge, volume, amphipaticity, and improve bacterial membrane interactions. This novel peptide exhibited increased hydrophobicity and net positive charge for binding effectively to the negatively charged membrane. PAJE demonstrated antimicrobial activity against both gram-negative and gram-positive bacteria, with very low toxicity to eukaryotic cells and an inexpensive process of synthesis. Collectively, these findings suggest that this novel peptide possesses great potential as an antimicrobial agent.

Formulation and Characterization of Antithrombin Perfluorocarbon Nanoparticles.

Thrombin, a major protein involved in the clotting cascade by the conversion of inactive fibrinogen to fibrin, plays a crucial role in the development of thrombosis. Antithrombin nanoparticles enable site-specific anticoagulation without increasing bleeding risk. Here we outline the process of making and the characterization of bivalirudin and D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone (PPACK) nanoparticles. Additionally, the characterization of these nanoparticles, including particle size, zeta potential, and quantification of PPACK/bivalirudin loading, is also described.

High-Throughput Synthesis and Analysis of Intact Glycoproteins Using SAMDI-MS.

High-throughput quantification of the post-translational modification of many individual protein samples is challenging with current label-based methods. This paper demonstrates an efficient method that addresses this gap by combining Escherichia coli-based cell-free protein synthesis (CFPS) and self-assembled monolayers for matrix-assisted laser desorption/ionization mass spectrometry (SAMDI-MS) to analyze intact proteins. This high-throughput approach begins with polyhistidine-tagged protein substrates expressed from linear DNA templates by CFPS. Here, we synthesized an 87-member library of the E. coli Immunity Protein 7 (Im7) containing an acceptor sequence optimized for glycosylation by the Actinobacillus pleuropneumoniae N-glycosyltransferase (NGT) at every possible position along the protein backbone. These protein substrates were individually treated with NGT and then selectively immobilized to self-assembled monolayers presenting nickel-nitrilotriacetic acid (Ni-NTA) complexes before final analysis by SAMDI-MS to quantify the conversion of substrate to glycoprotein. This method offers new opportunities for rapid synthesis and quantitative evaluation of intact glycoproteins.

Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes.

BACKGROUND: Heterologous production of cold-adapted proteins currently represents one of the greatest bottlenecks in the ongoing bioprospecting efforts to find new enzymes from low-temperature environments, such as, the polar oceans that represent essentially untapped resources in this respect. In mesophilic expression hosts such as Escherichia coli, cold-adapted enzymes often form inactive aggregates. Therefore it is necessary to develop new low-temperature expression systems, including identification of new host organisms and complementary genetic tools. Psychrophilic bacteria, including Pseudoalteromonas haloplanktis, Shewanella and Rhodococcus erythropolis have all been explored as candidates for such applications. However to date none of these have found widespread use as efficient expression systems, or are commercially available. In the present work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes. RESULTS: We tested 12 bacterial strains, as well as available vectors, promoters and reporter systems. We used RNA-sequencing to determine the most highly expressed genes and their intrinsic promoters in A. wodanis. In addition we examined a novel 5'-fusion to stimulate protein production and solubility. Finally we tested production of a set of "difficult-to-produce" enzymes originating from various bacteria and one Archaea. Our results show that cold-adapted enzymes can be produced in soluble and active form, even in cases when protein production failed in E. coli due to the formation of inclusion bodies. Moreover, we identified a 60-bp/20-aa fragment from the 5'-end of the AW0309160_00174 gene that stimulates expression of Green Fluorescent Protein and improves production of cold-active enzymes when used as a 5'-fusion. A 25-aa peptide from the same protein enhanced secretion of a 25-aa-sfGFP fusion. CONCLUSIONS: Our results indicate the use of A. wodanis and associated genetic tools for low-temperature protein production and indicate that A. wodanis represents an interesting platform for further development of a protein production system that can promote further cold-enzyme discoveries.

Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level.

The recent development of chemical and bio-conjugation techniques allows for the engineering of various protein polymers. However, most of the polymerization process is difficult to control. To meet this challenge, we develop an enzymatic procedure to build polyprotein using the combination of a strict protein ligase OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases 1) and a protease TEV (tobacco etch virus). We firstly demonstrate the use of OaAEP1-alone to build a sequence-uncontrolled ubiquitin polyprotein and covalently immobilize the coupled protein on the surface. Then, we construct a poly-metalloprotein, rubredoxin, from the purified monomer. Lastly, we show the feasibility of synthesizing protein polymers with rationally-controlled sequences by the synergy of the ligase and protease, which are verified by protein unfolding using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides a strategy for polyprotein engineering and immobilization.

Native Ion Mobility-Mass Spectrometry Reveals the Formation of ß-Barrel Shaped Amyloid-ß Hexamers in a Membrane-Mimicking Environment.

The mechanisms behind the Amyloid-ß (Aß) peptide neurotoxicity in Alzheimer's disease are intensely studied and under debate. One suggested mechanism is that the peptides assemble in biological membranes to form ß-barrel shaped oligomeric pores that induce cell leakage. Direct detection of such putative assemblies and their exact oligomeric states is however complicated by a high level of heterogeneity. The theory consequently remains controversial, and the actual formation of pore structures is disputed. We herein overcome the heterogeneity problem by employing a native mass spectrometry approach and demonstrate that Aß(1-42) peptides form coclusters with membrane mimetic detergent micelles. The coclusters are gently ionized using nanoelectrospray and transferred into the mass spectrometer where the detergent molecules are stripped away using collisional activation. We show that Aß(1-42) indeed oligomerizes over time in the micellar environment, forming hexamers with collision cross sections in agreement with a general ß-barrel structure. We also show that such oligomers are maintained and even stabilized by addition of lipids. Aß(1-40) on the other hand form significantly lower amounts of oligomers, which are also of lower oligomeric state compared to Aß(1-42) oligomers. Our results thus support the oligomeric pore hypothesis as one important cell toxicity mechanism in Alzheimer's disease. The presented native mass spectrometry approach is a promising way to study such potentially very neurotoxic species and how they could be stabilized or destabilized by molecules of cellular or therapeutic relevance.

Recombinant production of a bioactive peptide from spotless smooth-hound (Mustelus griseus) muscle and characterization of its antioxidant activity.

Bioactive peptides are short amino acid sequences with desirable health effects which are derived from animals, plants, and marine sources. In this study, recombinant production of a bioactive peptide (GIISHR) from spotless smooth-hound (Mustelus griseus) muscle and its antioxidant properties is discussed. A gene composed of 12 tandem copies of the peptide sequence was cloned in pET-28a and expressed as a His-tagged polypeptide in Escherichia coli. The recombinant polypeptide was then purified by Ni-NTA affinity chromatography, cleaved by Trypsin and purified by ultrafiltration. DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2'-azinobis-3-ethylbenzotiazoline-6-sulfonic acid) and hydroxyl radical scavenging activity assays, ferric reducing antioxidant power (FRAP) assay and ß-carotene bleaching test were used to characterize the antioxidant activity of the GIISHR. Liquid chromatography-mass spectrometry analysis revealed 60% purity for released bioactive peptide. Production yield was estimated as 60-80 mg GIISHR active peptide per 1 L bacterial culture. Antioxidant activity assays indicated that the antioxidant activity was increased with increase in peptide concentration. Though the DPPH radical scavenging activity, FRAP and ß-carotene bleaching power of the peptide were lower than those of the synthetic antioxidant tert-butylhydroquinone (TBHQ), the ABTS and hydroxyl radical scavenging activities of the peptide (at a concentration of 20 mg/mL) were similar to those of TBHQ (at a concentration of 0.1 mg/mL). The findings of the present study may be helpful in development of a process for production of the bioactive antioxidant peptides and its application in food industry.

Construction of a synthetic protein using PCR with a high essential amino acid content for nutritional purposes.

Ovalbumin is considered a protein of high nutritional value because it contains essential amino acids and is highly digestible. Therefore, it has a high biological value. Currently, the high food demand requires worldwide attention because food production is insufficient. Therefore, other alternatives are necessary to satisfy food demands, such as protein engineering. In this work, a protein with a high essential amino acid content similar to ovalbumin was synthesized by protein engineering, expressed, and digested in vitro. The assembly and sequential overlap extension PCR strategy was used to synthesize a 345-bp gene that encodes a high essential amino acid content protein (HEAAP). The 345-bp product was cloned into the vector pBAD TOPO®, and expressed in Escherichia coli BL21. PCR reactions and sequencing demonstrated the presence, orientation, and correct sequence of the insert. HEAAP expression was induced by L-arabinose and then purified using Ni-NTA affinity chromatography. The expression in E. coli was low and barely detected by Western blot assay. The in vitro multienzyme digestibility of HEAAP was around 79%, which suggests that the protein is potentially nutritious. Virtual analysis classifies the protein as unstable and hydrophilic, with a half-life in E. coli of 10 h. The recombinant HEAAP was successfully synthesized, but it is necessary to improve the digestibility and to optimize expression including selecting other expression models.

Immunogenicity of synthetic peptide constructs based on PvMSP9E795-A808, a linear B-cell epitope of the P. vivax Merozoite Surface Protein-9.

Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9E795-A808) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines.

Preparation and in vivo pharmacokinetics of rhGH-loaded PLGA microspheres.

Recombinant human growth hormone (rhGH) therapy must be administered as a daily injection due to its short half-life. To achieve sustained release of rhGH, the preparation of rhGH-loaded PLGA microspheres was investigated, and the influence of various factors on encapsulation efficiency was tested, including rhGH concentration, the ratio of internal phase to organic phase, stirring speed, PVA concentration, surrounding pH value, and the type of emulsifier and organic solvent. A pharmacokinetic study was performed by subcutaneous administration to explore the sustained release effect. It was found that rhGH-loaded PLGA microspheres were prepared with a narrow size distribution, and optimization of the formulation could enhance encapsulation efficiency. FTIR analysis indicated that the activity of rhGH was maintained after encapsulation. The pharmacokinetic behavior of rhGH solutions was consistent with a two-compartment model, which showed fast absorption and distribution. RhGH-loaded PLGA microspheres achieved a higher bioavailability and a long-term effective concentration by controlling the release, and PLGA 50/50 demonstrated favorable AUC compared with PLGA 75/25. Nevertheless, the higher bioavailability of rhGH-loaded PLGA microspheres lacking Span 80 did not predicate better sustained release behavior, indicating that further investigation is needed to explore the use of bioavailability as the standard in evaluating the sustained release characteristics and in vivo behavior of microspheres.

An explorative study towards the chemical synthesis of the immunoglobulin G1 Fc CH3 domain.

Monoclonal antibodies, fusion proteins including the immunoglobulin fragment c (Ig Fc) CH2-CH3 domains, and engineered antibodies are prominent representatives of an important class of drugs and drug candidates, which are referred to as biotherapeutics or biopharmaceuticals. These recombinant proteins are highly heterogeneous due to their glycosylation pattern. In addition, enzyme-independent reactions, like deamidation, dehydration, and oxidation of sensitive side chains, may contribute to their heterogeneity in a minor amount. To investigate the biological impact of a spontaneous chemical modification, especially if found to be recurrent in a biotherapeutic, it would be necessary to reproduce it in a homogeneous manner. Herein, we undertook an explorative study towards the chemical synthesis of the IgG1 Fc CH3 domain, which has been shown to undergo spontaneous changes like succinimide formation and methionine oxidation. We used Fmoc-solid-phase peptide synthesis (SPPS) and native chemical ligation (NCL) to test the accessibility of large fragments of the IgG1 Fc CH3 domain. In general, the incorporation of pseudoproline dipeptides improved the quality of the crude peptide precursors; however, sequences larger than 44 residues could not be achieved by standard stepwise elongation with Fmoc-SPPS. In contrast, the application of NCL with cysteine residues, which were either native or introduced ad hoc, allowed the assembly of the C-terminal IgG1 Fc CH3 sequence 371 to 450. The syntheses reported here show advantages and limitations of the chemical approaches chosen for the preparation of the synthetic IgG1 Fc CH3 domain and will guide future plans towards the synthesis of both the native and selectively modified full-length domain.

Synthesis and semisynthesis of selenopeptides and selenoproteins.

The versatile chemistry of the genetically encoded amino acid selenocysteine (Sec) is employed in Nature to expand the reactivity of enzymes. In addition to, its role in biology, Sec is used in protein engineering to modify folding, stability, and reactivity of proteins, to introduce conjugations and to facilitate reactions. However, due to limitations related to Sec's insertion mechanism in Nature, much of the production of Sec containing peptides and proteins relies on synthesis and semisynthesis. Here, we review recent advances that have enabled the assembly of complicated selenoproteins, including novel uses of protecting groups for solid phase peptide synthesis, rapid selenoester driven chemical ligations and versatile expressed protein ligations.