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Principal Investigator: Stephen Leppla
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2024
Award: $933,913
Funding agency: National Institute of Allergy and Infectious Diseases
There is increasing recognition in the research and development community that the anthrax toxin proteins constitute a uniquely effective tool for delivering materials to the cytosol and nucleus of eukaryotic cells. Our own work with a non-toxic fusion of the N-terminal portion of lethal factor (LFn) to Cre recombinase was described several years ago (C. Merritt, et al., J Biol Chem 2021 Vol. 298, Pages 101467). When administered to cells or mice with appropriate Cre-activated fluorescent proteins (FP), this fusion protein can quantitatively change the FP expression, e.g., from red to green, etc. This system is being used in reporter (“Tomato”) mice to demonstrate the role of, and requirement for, activation of the delivery system by tumor cell surface proteases. We previously created protective antigen (PA) variants in which the amino acid loop normally cleaved by the furin protease is replaced by sequences susceptible to cleavage by either matrix metalloproteases (MMPs) or by urokinase plasminogen activator. During this 2024 reporting period, we used these PA variants along with the LFn-Cre fusion to verify that few if any mouse tissues normally express these cell surface proteases. This helps to explain and document that these PA variants are quite safe, and their targeting of tumor tissues (which do express these proteases) will not be accompanied by off-target toxicity to normal tissues.
We have also extended work during 2024 to improve the cargo delivered by the anthrax toxin proteins for targeting tumors. The native LF protein is a protease that cleaves all but one of the seven MEK proteins. Recognition of its MEK substrates by LF appears largely due to LF interaction through a region (“exosite”) that is distant from the catalytic center. We previously made several mutants of LF and confirmed work by others that an LF having a W271A mutation greatly prefers MKK1 to all other MKKs. To further define LF mutations that have specificity for particulr MKKs, we are working to purify a majority of the seven MKK proteins. MKKs 1,3,6, and 7 have been purified, while MKKs 4 and 5 have proved more difficult. Initial studies using isothermal calorimetry showed that LF binds several of the MKKs with affinities of 5-8 micromolar.
During this 2024 period, we have also initiated studies on delivery of peptides that are expected to bind and inhibit myc, a key transcriptional activator. Myc normally binds via a coiled-coil interaction with a similar protein named Max, and the complex activates expression of many genes. We are preparing LFn fusions to peptides have been reported to disrupt the Myc-Max complex. These will be tested in cultured cells, and those that prove active will be tested for anti-tumor activity in mice.
Terms: <Affinity><Amino Acids><Animals><Anthrax><Anthrax Vaccines><Anthrax disease><Antibodies><Antigens><Assay><B anthracis><B. anthracis><Bacillus anthracis><Bacteria><Bacterial Infections><Binding><Bioassay><Biochemical><Biological Assay><CRE Recombinase><Calorimetry><Cell Body><Cell Nucleus><Cell surface><Cells><Chimera Protein><Chimeric Proteins><Clinical Treatment Moab><Common Rat Strains><Community Developments><Complex><Cultured Cells><Cytosol><Data><Development and Research><Distant><Dropsy><Drugs><Edema><Engineering><Enterobacteria phage P1 Cre recombinase><Esteroproteases><Eukaryotic Cell><Fusion Protein><Genes><Genetic Alteration><Genetic Change><Genetic defect><Human><Hydrops><Infection><Infection prevention><Intervention><Intervention Strategies><LeTx><Lycopersicon esculentum><Lytotoxicity><MAP Kinase Kinase 1><MAP2K1><MAP2K1 gene><MAPK/ERK Kinase 1><MAPKK1><MEK-1><MEK1><MEKs><MKK1><Macrophage><Medication><Metallopeptidases><Metalloproteases><Metalloproteinases><Methods><Mice><Mice Mammals><Mitogen-Activated Protein Kinase Kinase-1><Modeling><Modern Man><Molecular Interaction><Monoclonal Antibodies><Murine><Mus><Mutation><Mφ><N-terminal><NH2-terminal><Names><Normal Tissue><Normal tissue morphology><Nucleus><PRKMK1><Pathogenicity Factors><Peptidases><Peptide Hydrolases><Peptides><Pharmaceutical Preparations><Predisposition><Prevent infection><Property><Protease Gene><Proteases><Proteinases><Proteins><Proteolytic Enzymes><R & D><R&D><Rat><Rats Mammals><Rattus><Reporter><Reporting><Role><Septic Toxemia><Solanum lycopersicum><Specificity><Susceptibility><Symptoms><System><Testing><Therapeutic><Therapeutic Index><Tomatoes><Toxemia><Toxic Actions><Toxic effect><Toxicities><Toxin><Transcription Activator><Transcription Coactivator><Transcription Factor Coactivator><Transcriptional Activator><Transcriptional Activator/Coactivator><Transcriptional Coactivator><Tumor Cell><Tumor Tissue><U-PA><U-Plasminogen Activator><Urinary Plasminogen Activator><Urokinase><Urokinase Plasminogen Activator><Urokinase-Type Plasminogen Activator><Vaccines><Variant><Variation><Virulence Factors><Work><aminoacid><anthracis><anthrax toxin><anticancer activity><bacteria infection><bacterial disease><bacteriophage P1 recombinase Cre><cell type><cytotoxicity><determine efficacy><drug/agent><efficacy analysis><efficacy assessment><efficacy determination><efficacy evaluation><efficacy examination><evaluate efficacy><examine efficacy><genome mutation><immunogen><improved><interventional strategy><lethal factor><lethal toxin><mAbs><monoclonal Abs><mutant><name><named><naming><neoplastic cell><pathogen><prevent><preventing><protein expression><research and development><social role><tool><transcription co-activator><transcriptional co-activator><tumor><vaccine candidate><vaccine efficacy>