An effective drug delivery system enables the release of the active ingredient where it is most needed to achieve the desired therapeutic effect. For cellular therapeutics to work well, the precise delivery of drugs is essential. Off-target effects of drugs are a significant hurdle in designing efficient therapies. Drug delivery technologies allowing the accurate targeting of disease-causing molecules or pathways hold the potential to minimize off-target effects during systematic drug administration.
For example, drugs administrated into blood vessels need to penetrate various biological barriers before reaching target sites. When administered, drugs encounter several obstacles, including enzymes, such as proteases and nuclease, renal filtration, sequestering by the mononuclear phagocyte system, or the endothelial barrier. Hence, delivery systems are needed to target specific tissues and cell compartments. Tissue- and cell compartment-specific drug delivery has the potential to avoid unnecessary toxicities resulting in the improvement of quality of life and patient well-being.
Peptide delivery into the nucleus
Loregian et al. in 1999 reported an intracellular peptide delivery system for targeting specific cellular compartments. The research group designed a chimeric protein (EtxB-Pol = EtxB-peptide) consisting of the nontoxic B subunit of Escherichia coli heat-labile enterotoxin.
The research group fused EtxB to a 27-mer peptide derived from the DNA polymerase of herpes simplex virus 1. The study revealed that the peptide dissociates the interaction complex between an accessory factor, UL42, encoded by the virus, and the DNA synthesis machinery. The chimeric protein enters cells through the acidic endosomal compartments where the Pol peptide is cleaved from the protein before translocating into the nucleus. After translocation, the antiviral peptide is localized in the nucleus. As a result, the nontoxic receptor-binding component of E. coli enterotoxin (EtxB) mediates the intracellular delivery to access the nucleus. The selected peptide corresponds to the C-terminal 27 amino acids of HSV-1 DNA polymerase (POL). This polymerase is involved in the interaction with UL42. The free peptide or EtxB alone does not affect virally infected cells; however, the EtxB-Pol fusion protein selectively inhibits HSV-1 replication.
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Figure 1: 2.2 Angstroms crystal structure of E. Coli heat-labile enterotoxin (Lt) With Bound Galactose [PDB ID 1LT5].
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Figure 2: Structure of the B subunit of human heat-labile enterotoxin from E. Coli carrying a peptide with anti-hsv activity [PDB ID 1LTR]
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Figure 3: B subunit of human heat-labile enterotoxin from E. Coli carrying a peptide with anti-hsv activity. The locations of the peptide in the complex are highlighted as van der Waals spheres [PDB ID 1LTR].
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Figure 4: Monomer of B subunit of the Escherichia coli heat-labile enterotoxin carrying a peptide with anti-herpes simplex virus type 1 activity (YAGAVVNDL) [PDB ID 1LTR]. The location of the peptide on the c-terminal end of the protein is highlighted as dots. Only the YAGAV part is visible in the crystal structure.
>pdb|1LTR|D Chain D, Heat-labile Enterotoxin
APQSITELCS EYHNTQIYTI NDKILSYTES MAGKREMVII TFKSGATFQV EVPGSQHIDS QKKAIERMKD TLRITYLTET KIDKLCVWNN KTPNSIAAIS MEKLYAGAVVNDL
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The delivery system described by Loregian et al. enables the delivery of peptides into the nucleus allowing the disruption of specific protein-protein interactions.
The B subunits of cholera toxin (CtxB) and Escherichia coli heat-labile enterotoxin (EtxB) are potent systemic and mucosal adjuvants. These adjuvants bind to their ganglioside receptor GM1 present in lipid rafts. After binding, the proteins undergo rapid cross-aggregation and internalization. This property of EtxB has led to its use as a carrier molecule for the intracellular delivery of exogenous peptides or antigens.
The heat-labile enterotoxin (EtxB) is a carrier molecule for the intracellular delivery of exogenous peptides or antigens.
Examples are the delivery of peptide sequences derived from ribonucleotide reductase and DNA polymerase of herpes simplex virus type 1 to intracellular compartments. The protein complex remained functionally intact.
This delivery system also allowed the delivery of peptides corresponding to known major histocompatibility complex (MHC) class I-restricted epitopes from the ovalbumin and influenza nucleoprotein to the MHC class I antigen processing and presentation pathway in murine dendritic cells.
Reference
Adepu S, Ramakrishna S. Controlled Drug Delivery Systems: Current Status and Future Directions. Molecules. 2021 Sep 29;26(19):5905. [PMC]
de Haan, L., A. R. Hearn, A. J. Rivett, and T. R. Hirst. 2002. Enhanced delivery of exogenous peptides into the class I antigen processing and presentation pathway. Infect. Immun.70:3249-3258. [PMC]
Intranuclear Delivery of an Antiviral Peptide Mediated by the B Subunit of Escherichia coli Heat-Labile Enterotoxin
Lencer, W. I., T. R. Hirst, and R. K. Holmes. 1999. Membrane traffic and the cellular uptake of cholera toxin. Biochim. Biophys. Acta1450:177-190. [ScienceDirect]
Loregian A, Papini E, Satin B, Marsden HS, Hirst TR, Palù G. Intranuclear delivery of an antiviral peptide mediated by the B subunit of Escherichia coli heat-labile enterotoxin. Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):5221-6. doi: 10.1073/pnas.96.9.5221. [PMC]
Marcello, A., A. Loregian, A. Cross, H. Marsden, T. R. Hirst, and G. Palu. 1994. Specific inhibition of herpes virus replication by receptor-mediated entry of an antiviral peptide linked to Escherichia coli enterotoxin B subunit. Proc. Natl. Acad. Sci. USA91:8994-8998. [PNAS]
Matković-Calogović D, Loregian A, D'Acunto MR, Battistutta R, Tossi A, Palù G, Zanotti G. Crystal structure of the B subunit of Escherichia coli heat-labile enterotoxin carrying peptides with anti-herpes simplex virus type 1 activity. J Biol Chem. 1999 Mar 26;274(13):8764-9. PMID: 10085117. [PDB ID 1LTR.]
Ong KW, Wilson AD, Hirst TR, Morgan AJ. The B subunit of Escherichia coli heat-labile enterotoxin enhances CD8+ cytotoxic-T-lymphocyte killing of Epstein-Barr virus-infected cell lines. J Virol. 2003 Apr;77(7):4298-305. doi: 10.1128/jvi.77.7.4298-4305.2003. [PMC]
Williams, N. A., T. R. Hirst, and T. O. Nashar. 1999. Immune modulation by the cholera-like enterotoxins: from adjuvant to therapeutic. Immunol. Today20:95-101. [PubMed]
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