This proposal leverages bacterial evolution to improve targeted delivery of therapeutics to cancer cells. Over time, bacterial proteins have evolved to excel at host cell targeting and cargo delivery. This proposal harnesses the advantageous targeting and delivery mechanisms of bacterial proteins to address key problems with cancer treatment: low efficacy and off-target toxicity. For aggressive types of cancer such as acute myeloid leukemia (AML), systemic treatments like chemotherapy are effective but lead to chronic, treatment-related complications and even death. Targeted therapeutics will mitigate the tradeoff between effective treatment and debilitating side effects. Newly designed therapeutic peptides target CREB (cAMP Response-Element Binding Protein), a transcription factor that regulates AML cell proliferation and survival. By disrupting interactions between CREB and CREB-binding protein, therapeutic peptides specifically inhibit AML cell proliferation and improve patient prognosis. Despite being a promising treatment, delivery of CREB-inhibiting peptides remains challenging. Traditional delivery of therapeutics using endocytic pathways traps therapeutic molecules in endosomes, which are isolated from the rest of the cell. To overcome endosomal sequestration, treatment requires a high concentration of peptides, resulting in a reduced overall therapeutic potency. 

Instead of relying on endocytosis, this collaborative proposal explores a new delivery pathway, where cargo is directly translocated across cell membranes into the cytosol. A non-endocytic delivery mechanism increases therapeutic potency to make CREB-inhibiting peptides a more viable clinical candidate. Our approach harnesses natural myeloid cell recognition to target cancerous cells, reducing off-target toxicity. Leveraging expertise in protein engineering, we will design a protein delivery mechanism that releases peptides in response to endogenous stimuli, enabling nuclear localization and increasing therapeutic potency. Using high-resolution structural insights into native bacterial protein translocation, we will establish the design criteria for larger and more charged cargo. Combined, this proposal will demonstrate the viability of bacteria-inspired translocation of therapeutic cargo and lay the groundwork for a modular therapeutic delivery mechanism.