Available Technology
Delivery of bioactive compounds using a transporter domain of engineered bacterial proteins
Technology:
Delivery technology
Markets Addressed
This novel technology allows for the efficient delivery of bioactive molecules containing non-natural amino acids, as well as novel chemical entities, into the cytosol of mammalian cells for therapeutic purposes:
• Delivery of non-natural bioactive molecules: The delivery of bioactive molecules with non-natural components into the cytosol of cells, includes: peptides and proteins comprising one or more D-amino acids, cyclic peptides and proteins, side-chain modified amino acids, backbone modifications, stapled peptides, and other variants containing a range of non-natural amino acids.
• Delivery of novel chemical entities: Novel chemical entities can be covalently attached to the toxin using native chemical ligation and/or sortase tagging. Once within the cytosol, molecules can be released from the toxin by proteolytic cleavage. This delivery process is very efficient, using only nanomolar concentrations of reagents.
• Delivery using other AB toxins: In addition to anthrax toxin, this technology may be applied to other AB toxins, since AB toxins all have a similar mechanism of action (e.g. cholera toxin, botulinum neurotoxin, diphtheria toxin, shiga toxin and exotoxin A).
• Cell-type specific targeting: Specific cell types may be targeted by modifying the receptor binding domain of the respective toxin.
• Stimulation of a protective immune response to antigens containing non-natural amino acids: Chemical entities containing novel biomarkers that contain non-natural amino acids including glycopeptides can be used to stimulate antigen-specific CD4(+) T cells and antigen-specific CD8(+).
Innovations and Advantages
While there are a number of methods for the delivery of bioactive peptides and proteins into mammalian cells, there is a need for methods to deliver (1) larger molecules that cannot traverse the plasma membrane by a simple diffusive process (e.g. peptides, proteins, small molecules), and (2) agents that are non-naturally occurring (e.g. modified peptides, D-peptides and other organic molecules not normally trafficked into a cell). In addition, current technologies used to gain therapeutic access to the cytosol are limited in that they require large quantities of sample, have limited selectivity and tend to not escape the endosome.
Many pathogenic bacteria have evolved protein machinery that efficiently delivers enzymes to the cytosol of mammalian cells. A major class of bacterial toxins, termed AB toxins, uses a transporter protein (B unit) that actively translocates enzymes (A unit) into cells. Examples of AB toxins include anthrax toxin, botulinum neurotoxin, diphtheria toxin, shiga toxin, exotoxin A and cholera toxin. In the case of the anthrax lethal toxin, the transporter protein is protective antigen (PA), a receptor-binding/pore-forming moiety, and the enzyme is lethal factor (LF), a zinc protease. LF binds to PA pores via its N-terminal domain with nanomolar binding affinity, and this domain alone can be used for translocation of compounds.
Delivery of non-natural bioactive agents: Dr. Collier’s laboratory developed a method for the efficient delivery of bioactive peptide and protein molecules containing non-natural amino acids, as well as novel chemical entities, into the cytosol of eukaryotic cells. Specifically, they discovered that modified versions of bacterial toxins, such as anthrax toxin or other AB toxins, can be used in efficient delivery of these agents. Their novel results demonstrate that a naturally existing transport system can be used for the delivery of non-naturally occurring proteins and peptides, an unexpected finding since most protein-protein interactions are based on highly stereochemical arrangements.
The technology uses the N-terminal domain of LF (LFN) and the transporter protein PA, which when added to cells, form a nanomachine that delivers functional bioactive molecules to the cytosol of a target cell. In particular, the Collier lab modified LFN by semi-synthesis to probe how structural and electrostatic changes affected protein translocation through PA:
• LFN variant using D amino acids: The LFN domain functioned equally well when the segment was built from D amino acids as from L amino acids, both in its ability to inhibit ion conductance through PA and to be translocated through the pores. This finding indicates that the LFN does not adopt a conformation that interacts with the pore in a stereospecific manner during protein translocation.
• LFN variant with alternating Lys-Glu sequence: This modified LFN domain demonstrated no significant change versus wild-type LFN in the ability of the protein to block ion conductance or to be translocated. This finding demonstrates the independence of translocation from strict sequence.
• LFN variants with DTA fusion proteins: For the variants described above, the lab also prepared them with a DTA (diphtheria toxin A chain) fusion protein using chemical ligation or the transpeptidase, sortase A. These variants behaved essentially identically to those without the fusion protein, indicating that covalent attachment of chemical entities does not affect translocation.
• LFN variant with cysteic acid: The Collier lab replaced selected acidic residues with cysteic acid, which has a negatively charged side chain, to test whether charge affected translocation. As predicted, this variant strongly inhibited translocation.
Additional Information
This technology is available for worldwide, exclusive licensing and/or a collaborative research program with the Collier laboratory.
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Inventor(s):
Collier, R. John
Pentelute, Brad L.
Categories:
For further information, please contact:
Michal Preminger, Director of Business Development
(617) 432-0920
Reference Harvard Case #4018