MEET US AT AMERICAN PEPTIDE SYMPOSIUM, ORLANDO, USA, JUNE 20-25, 2015
MODIFIED PEPTIDES – FIFTH SPRING SYMPOSIUM BY BACHEM
Six presentations covered different areas of peptide and protein modifications.
Prof. Stephen Kent from the University of Chicago gave an insight into his research on “Chemical Protein Synthesis for the Systematic Development of a D-Protein Therapeutic”.
Pointing out the advantages like protease resistance, non-immunogenicity, precision modification and the possibility for chemical synthesis, he described a way to get to this class of compounds.
Prof. John Robinson from the University of Zürich covered “Synthetic Protein Epitope Mimetics”. He described ß‐Hairpin mimetic design using macrocyclic peptides and applications.
Chemical glycosylation is an innovative approach for peptide modifications to improve drug properties, e.g. pharmacokinetics, efficacy, solubility.
Prof. Yasuhiro Kajihara from Osaka University and Dr. Michael Haller from GlyTech, Inc, held two interesting talks in this regards. Glycosylation is a post-translational process naturally taking place in mammalian cells. By now, glycosylated peptides are usually obtained by using recombinant cells. A synthetic approach leads to more homogenous glycosylated products.
We have an e-brochure available.
Describing Tau as a promising target for a therapeutic vaccine for Alzheimer’s disease, Prof. Andrea Pfeifer from AC Immune SA, presented the company’s platform SupraAntigenTM they used to obtain an anti-pTau vaccine. A first clinical trial has been started.
Dr. Ralph Schönleber from Bachem presented the “Synthesis of T3, a highly palmitoylated phosphopeptide”. He described the route selection and successful process development for this extremely challenging peptide synthesis, leading to an improved API purity from 80% to >95%.
Please contact us if you are interested in additional information. We are happy to provide you deeper insights into recent research topics.
CELL PERMEABLE PEPTIDES (CPP)
The cellular uptake of therapeutic agents has always been proved to be a challenge, especially in the delivery of large molecules. The plasma membrane acts as a barrier to the direct translocation of hydrophilic macromolecules, preventing efficient and controlled intracellular delivery. A drug must be either highly lipophilic or very small to stand a chance of cellular internalization. The existing methods for delivery of macromolecules, such as viral vectors and membrane perturbation techniques, can result in high toxicity, immunogenicity and low delivery yield.
A class of peptides called “cell-permeable peptides” (also known as cell-penetrating peptides) has attracted considerable interest in recent years due to their ability to translocate through the cellular plasma membrane on their own. They are also known as protein transduction domains (PTDs), membrane translocating sequences (MTSs) and Trojan peptides. They are generally amphipathic sequences containing 5-40 amino acids and often contain cationic amino acid side chains. Due to their ability to cross cell membrane, cell-permeable peptides (CPPs) constitute a promising tool for the cellular import of drug cargos and have been successfully applied for in vitro and in vivo delivery of a variety of therapeutic molecules including plasmids, DNA, oligonucleotide, siRNA, PNA, proteins, peptides, liposomes, low molecular weight drugs and nanoparticles.
In 1988, Frankel and Pablo observed the remarkable ability of HIV-Tat protein to enter cells and translocate into the nucleus. Soon after, in 1991, the group of Prochiantz demonstrated that Drosophila Antennapedia homeodomain could be internalized by neuronal cells. This discovery subsequently led to the identification of a 16-amino acid peptide, penetratin, derived from the third helix of the homeodomain of Antennapedia. Based on mutagenesis studies, the biological activity and penetrating capability of Tat protein, it was found that the region between residues 47-57 (sequence: YGRKKRRQRRR) was important for cellular uptake. Since then, the number of known natural and synthetic peptides with cell- permeable capabilities has continued to grow. Such peptides and proteins with a membrane transduction domain are derived as partial sequences from transcription factors, bacterial or viral surface proteins, toxins, amphipathic helix-forming peptides and from ligands of membrane-bound receptors or adhesion proteins. CPPs can be broadly classified as protein-derived, chimeric –derived from two or more genes which are coded for separate proteins – and synthetic.
Examples of various CPPs
TAT (48-60): GRKKRRQRRRQC (Protein-derived)
Penetratin: RQIKIWFQNRRMKWKK-NH2 (Protein-derived)
pVEC: LLIILRRRIRKQAHAHSK-NH2 (Protein-derived)
MPG8: AFLGWLGAWGTMGWSPKKKRK-cya (Chimeric)
Transportan: GWTLNSAGYLLGKINLKALAALAKKIL-NH2 (Chimeric)
Transportan10: AGYLLGKINLKALAALAKKIL-NH2 (Chimeric, modified)
PepFect3: AGYLLGKINLKALAALAKKIL-NH2 (Chimeric, modified)
PepFect: AGYLLGK(eNHa)INLKALAALAKKIL-NH2 (Chimeric, modified)
Polyarginine: Rn (n = 6-12) (Synthetic)
Stearyl polyarginine: Stearyl-Rn (n = 6-12) (Synthetic)
Pep-1: Ac-KETWWETWWTEWSQPKKKRKVcya (Synthetic)
Pep-3: KWFETWFTEWPKKRK-cya (Synthetic)
CADY: Ac-GLWRALWRLLRSLWRLLWRA-cya (Synthetic)
YTA2: YTAIAWVKAFIRKLRK-NH2 (Synthetic)
YTA4: IAWVKAFIRKLRKGPLG-NH2 (Synthetic)
SynB1: RGGRLSYSRRRFSTSTGR (Protein-derived)
SynB3: RRLSYSRRRF (Protein-derived)
Maurocalcine: GDCLPHLKLCKENKDCCSKKCKRRGTNIEKRCR (Protein-derived)
PTD4: YARAAARQARA (Protein-derived)
Attaching cargos to CPPs
The Tat peptide derived from HIV-Tat protein transduction domain is one of the most widely investigated cell permeable peptides. A major breakthrough in the CPP field came from the first proofs-of-concept of their in vivo application, by Dowdy et al, for the delivery of small peptides and large proteins and of Langel et al, for delivery of peptide-nucleic acids (PNAs) using the chimeric peptide Transportan, derived from the N-terminal fragment of the neuropeptide galanin, linked to mastoparan, a wasp venom peptide.
CPPs are usually connected via a covalent linkage to the cargo molecule. For example, proteins and peptides can be attached to CPPs through a disulfide bond (by modifying CPP and peptide/protein with cysteine) or through cross-linkers. Different strategies include cleavable disulfide, amide, thiazolidine, oxime and hydrazine linkages. Short interfering RNA (siRNA) can be covalently linked to transportan and penetratin by disulfide-linkage at the 5’-end of the sense strands of siRNA to target luciferase or enhanced Green fluorescent protein (eGFP) mRNA reporters. A stable covalent linkage between the cargo and CPP is not always necessary for translocation as simple mixing of two entities was shown to be efficient. In 1997, the first non-covalent CPP for delivery of nucleic acids, called methylpurine–DNA Glycosylase or MPG was designed by the group of Heitz and Divita closely followed by development of Pep-1 for non-covalent cellular delivery of proteins and peptides by Morris et al in 2001.
These non-covalent conjugates are formed through either electrostatic or hydrophobic interactions. With this method cargos such as nucleic acids and proteins could be efficiently delivered while maintaining full biological activity. MPG forms highly stable complexes with siRNA with a low degradation rate and can be easily functionalized for specific targeting, which are major advantages compared with the covalent CPP technology.
Recent progress in CPP based drug delivery
One of the limitations of CPPs has been the non-specific cellular uptake, thereby limiting the drug delivery to specific cellular targets such as tumour cells. Fortunately, in recent years some CPPs have shown high affinity for specific cell types or intracellular destinations. A recently discovered CPP known as ‘crotamine’ has shown unusually high affinity for actively proliferating cells. Another example is MPG, a synthetic CPP derived from the SV40 virus. Originally designed for nuclear delivery of siRNA, it has recently been altered to target the cytoplasm. Recently ‘activatable’ CPPs (ACCPs) were introduced to address the problem of tissue non-specificity of CPPs. Constructs have been described that are ‘activated’ either outside or inside the cells. ACPPs are polycationic CPPs whose adsorption and cellular uptake are minimised by a covalently attached polyanionic inhibitory domain. Cleavage of the linker connecting the polyanionic and polycationic domains by specific proteases (tumour associated matrix metalloproteases) dissociates the polyanion and enables the cleaved ACPP to enter cells.Nuclear localization sequences (NLSs) are special cases of cationic CPPs, which are short peptides based on lysine-, arginine- or proline-rich motifs that can be recognized by members of the Importin super family of nuclear transport proteins. Nuclear import efficiency could be improved through the direct or indirect attachment of CPPs with NLSs to DNA or gene carriers. The most well-known and extensively studied NLS sequence in the field of gene therapy is from the large tumor antigen of the simian virus 40 (SV40). Jun Yin et al explored expression of the genes transferred by a series of Co(II) complexes in the presence of NLS (PKKKRKV) in normal and cancer cell lines, whose results demonstrated that the Co(II) complexes in the presence of NLS facilitate cell internalization of the DNA condensates.Slightly acidic pH environment of a tumor cell can be exploited for improving cytoplasmic delivery of cargo molecules using CPPs. For example, Amit et al designed “Smart” TAT-modified liposomes, with PEG being coated to the surface via a pH-sensitive hydrazine bonds. The PEG chains, which shielded the surface-attached TATp, could be removed in the acidic environment of the tumor. This resulted in the exposure of the liposome-attached TATp residues, enhanced penetration of the liposomes into tumor cells and thus more effective intracellular delivery of gene.CPPs have also been investigated for: Non-invasive insulin delivery, CNS delivery of drugs due to their ability to cross blood-brain-barrier (BBB), encapsulation of protein therapeutics into red blood cells and delivery enhancers for vaccines etc.
1. Heitz F, et al., Br J Pharmacol. 2009; 157:195–206.2. Morris MC, et al., 2008; Biol Cell 100: 201–217.3. El-Andaloussi S, et al., Curr Pharm Design. 2005; 11: 3597–3611.4. Deshayes S, et al., Cell Mol Life Sci. 2005; 62:1839–1849.5. Simeoni, F, et al., Methods Mol Biol. 2005; 309: 251–264.
Jyothi,tell us what you do at American Peptide Company (APC)*?
I am Associate Director Marketing and together with my team we are taking care of the planning and implementing marketing strategies to faciliate peptide sales.
How long have you been with APC and what did you do before?
I have joined the Marketing Department at APC in 2012. Prior to APC I have served various capacities in industry and academia contributing to peptide chemistry for more than 12 years.
What is your academic background/degrees or training?
I have a Ph.D in Applied Chemistry and also performed postdoctoral training in the area of peptide chemistry.
Last but not least, what do you like to do outside of APC?
I am very passionate about learning and trying new things. I spend free time in travelling, hiking, photography and reading.
Thank you very much, Jyothi.
*A member of the Bachem Group