Pavla Simerska

Peptides As Therapeutics with Enhanced Bioactivity

The development of techniques for efficient peptide production renewed interest in peptides as therapeutics. Numerous modifications for improving stability, transport and affinity profiles now exist. Several new adjuvant and carrier systems have also been developed, enhancing the immunogenicity of peptides thus allowing their development as vaccines. This review describes the established and experimental approaches for manufacturing peptide drugs and highlights the techniques currently used for improving their drug like properties.

Chemical methods for peptide and protein production.

Since the invention of solid phase synthetic methods by Merrifield in 1963, the number of research groups focusing on peptide synthesis has grown exponentially. However, the original step-by-step synthesis had limitations: the purity of the final product decreased with the number of coupling steps. After the development of Boc and Fmoc protecting groups, novel amino acid protecting groups and new techniques were introduced to provide high quality and quantity peptide products. Fragment condensation was a popular method for peptide production in the 1980s, but unfortunately the rate of racemization and reaction difficulties proved less than ideal. Kent and co-workers revolutionized peptide coupling by introducing the chemoselective reaction of unprotected peptides, called native chemical ligation. Subsequently, research has focused on the development of novel ligating techniques including the famous click reaction, ligation of peptide hydrazides, and the recently reported α-ketoacid-hydroxylamine ligations with 5-oxaproline. Several companies have been formed all over the world to prepare high quality Good Manufacturing Practice peptide products on a multi-kilogram scale. This review describes the advances in peptide chemistry including the variety of synthetic peptide methods currently available and the broad application of peptides in medicinal chemistry.

Modern lipid-, carbohydrate-, and peptide-based delivery systems for peptide, vaccine, and gene products.

Research related to peptide, vaccine, and gene delivery has grown exponentially over the last decade. In this review, we discuss the development of delivery systems for peptides, gene and vaccine products. Special focus is given to different lipidation and glycosylation strategies to improve the metabolic stability and membrane permeability of therapeutics, and their targeting to specific sites. The synthetic methods for preparation of the systems are also described.

Characterization of a Serine Protease Homologous to House Dust Mite Group 3 Allergens from the Scabies Mite Sarcoptes scabiei

The scabies mite, Sarcoptes scabiei var. hominis, infests human skin, causing allergic reactions and facilitating bacterial infection by Streptococcus sp., with serious consequences such as rheumatic fever and rheumatic heart disease. To identify a possible drug target or vaccine candidate protein, we searched for homologues of the group 3 allergen of house dust mites, which we subsequently identified in a cDNA library. The native protein, designated Sar s 3, was shown to be present in the mite gut and excreted in fecal pellets into mite burrows within the upper epidermis. The substrate specificity of proteolytically active recombinant rSar s 3 was elucidated by screening a bacteriophage library. A preference for substrates containing a RS(G/A) sequence at the P1-P2′ positions was revealed. A series of peptides synthesized as internally quenched fluorescent substrates validated the phage display data and high performance liquid chromatography/mass spectrometry analysis of the preferred cleaved substrate and confirmed the predicted cleavage site. Searches of the human proteome using sequence data from the phage display allowed the in silico prediction of putative physiological substrates. Among these were numerous epidermal proteins, with filaggrin being a particularly likely candidate substrate. We showed that recombinant rSar s 3 cleaves human filaggrin in vitro and obtained immunohistological evidence that the filaggrin protein is ingested by the mite. This is the first report elucidating the substrate specificity of Sar s 3 and its potential role in scabies mite biology.

Development of a liposaccharide-based delivery system and its application to the design of group A streptococcal vaccines.

Group A streptococcus (GAS) is associated with many human diseases, ranging in severity from benign to life-threatening. A promising strategy for developing vaccines against GAS involves the use of carbohydrates as carriers for peptide antigens. This study describes the optimized synthesis of d-glucose and d-galactose derived carriers, bearing an adipate linker and four tert-butoxycarbonyl protected aminopropyl groups. Prophylactic GAS vaccine candidates were synthesized by conjugating multiple copies of a single GAS M protein derived peptide antigen (either J8 or J14) onto the carbohydrate carriers. These antigens contain peptide sequences, which are highly conserved and offer the potential to prevent infections caused by up to 70% of GAS strains. Lipophilic amino acids were also conjugated to the d-glucose anomeric carbon to produce a self-adjuvanting liposaccharide vaccine. High serum IgG antibody titers against each of the incorporated peptide epitopes were detected following subcutaneous immunization of B10.BR (H-2 (k)) mice with the liposaccharide vaccine candidates.

Oral vaccine delivery - New strategies and technologies

Although most commercial vaccines are delivered by injection, there is an increasing interest in needle-free vaccine delivery for reasons including the ability to elicit immune responses at mucosal surfaces, ease of administration, and the ability to administer vaccines without the need for trained medical professionals. This review summarizes strategies and technologies that are being used to improve oral vaccine absorption. Peptides and proteins, which comprise important vaccine components, exhibit unfavorable physicochemical properties including degradation in the gastrointestinal tract, and poor transport across the intestinal wall, which hinder oral vaccine development. Approaches to overcome these obstacles aim to provide new vaccines and delivery systems that are capable of eliciting protective immune responses, and are making an impact on current vaccine development.

α-Galactosidases and their applications in biotransformations

α-d-Galactosidases (α-d-galactoside galactohydrolase, E.C. 3.2.1.22) are exoglycosidases, which in vivo catalyze the hydrolysis of simple and complex oligo- and polysaccharides containing terminal α-d-galactosyl groups. A number of industrial applications of α-d-galactosidases are known, mainly in the sugar industry, pulp and paper industry or medicine. Although α-d-galactosidases occur in all living systems, microbial and possibly plant enzymes are preferred for practical applications. The regioselectivity of glycosidases is somewhat poor. Selective protection of oligosaccharides and development of new modified donors/acceptors is a promising method to overcome this problem. Genetic engineering has enabled the creation of glycosynthases from the hydrolytic glycosidases, which are highly complementary to glycosyltransferases. The present review summarizes recent advances in the application of α-d-galactosidases in synthetic and biotransformation applications.

Synthesis and in vivo studies of carbohydrate-based vaccines against group A Streptococcus

Carbohydrates, as carriers, providing numerous attachment points for the conjugation of peptide antigens and their optimal orientation for the recognition by cells of the immune system, reducing degradation of the attached peptide antigens and many other advantages make carbohydrate-based vaccine highly promising approach. Multiple copies of a single group A streptococcal (GAS) M protein derived specific peptide antigens (J8 or J14) were coupled onto carbohydrate cores (D-glucose and D-galactose) linked to lipophilic amino acids to produce a self-adjuvanting liposaccharide vaccine against GAS strains. In vivo experiments showed high serum IgG antibody titers against each of the incorporated peptide epitopes, J8 or J14.

Application of selectively acylated glycosides for the alpha-galactosidase-catalyzed synthesis of disaccharides

Abstract4-Nitrophenyl α-d-galactopyranosyl-(1→3)-6-O-acetyl-α-d-galactopyranoside was prepared in a transglycosylation reaction catalyzed by α-d-galactosidase fromTalaromyces flavus using 4-nitrophenyl α-d-galactopyranoside as a glycosyl donor and 4-nitrophenyl 6-O-acetyl-α-d-galactopyranoside as an acceptor. 4-Nitrophenyl 6-O-acetyl-α-d-galactopyranoside and 4-nitrophenyl 6-O-acetyl-β-d-galactopyranoside were prepared in a regioselective enzymic transesterification in pyridine-acetone catalyzed by the lipase PS fromBurkholderia cepacia. A series of water-miscible organic solvents (acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, 2-methoxyethanol, pyridine, 2-methylpropan-2-ol, tetrahydrofuran, propargyl alcohol) were used as co-solvents in this enzymic reaction. Their influence on the activity and stability of the α-galactosidase fromT. flavus was established. 2-Methylpropan-2-ol and acetone (increasing the solubility of the modified substrate acceptors and displaying the minimum impairment of the activity and stability of the enzyme) were used as co-solvents in transglycosylation reactions.

Pro-apoptotic activity of lipidic α-amino acids isolated from Protopalythoa variabilis

Lipidic α-amino acids (LAAs) have been described as non-natural amino acids with long saturated or unsaturated aliphatic chains. In the continuing prospect to discover anticancer agents from marine sources, we have obtained a mixture of two cytotoxic LAAs (1a and 1b) from the zoanthid Protopalythoa variabilis. The anti-proliferative potential of 14 synthetic LAAs and 1a/1b were evaluated on four tumor cell lines (HCT-8, SF-295, MDA-MB-435, and HL-60). Five of the synthetic LAAs showed high percentage of tumor cell inhibition, while 1a/1b completely inhibited tumor cell growth. Additionally, apoptotic effects of 1a/1b were studied on HL-60 cell line. 1a/1b-treated cells showed apoptosis morphology, loss of mitochondrial potential, and DNA fragmentation.