Tuberculosis (TB) is a disease of the lung caused by the bacterium, Mycobacterium tuberculosis (Mtb), and is the second leading cause of mortality from an infectious agent after SARS-CoV-2. Mtb is an opportunistic pathogen that benefits from a compromised immune system and is particularly deadly in HIV patients. New therapies targeting TB are necessary due to an increase in multi-drug resistant strains. Our collaborators at Texas Southern University identified novel bacterial methionine aminopeptidase (MtMetAP1) inhibitors. Genetic knockout studies support the lethality of the deletion of MtMetAP1 in Mtb. Further studies support the existence of an ortholog in Homo sapiens (HsMetAP1) that performs similar essential functions. Although these novel inhibitors are known to be effective against Mtb, their cytotoxicity in mammalian cells has yet to be established. We hypothesize that there will be low levels of cytotoxicity in cancerous and primary lung cell lines at varying concentrations of the MetAP novel inhibitor, UST-001. Using flow cytometry via a MUSE® Cell Count and Viability Kit, the cytotoxicity of UST-001 was assayed in H1299 cancer cells. The H1299 cells were exposed to concentrations of UST-001 ranging from 0.1μM to 750μM for 48 hours at 37℃and 5% CO2. Three negative controls were included in each replicate: DMSO (carrier), pure media, and Isoniazid (INH). INH is a common clinically effective TB treatment. The viability of H1299 cells was still over 60% at the highest inhibitor concentration tested. Trypan Blue Assays were performed on the same H1299 cells at each concentration and the controls, which corroborated flow cytometry results. In establishment of a dose-response curve, our data showed that at UST-001 concentrations below 100 μM, induced little to no cell death and cell viability of 90% or higher. Since cancerous cells have higher mitotic rates and more repair pathways, we hypothesize that we will be able to establish an IC50 curve in non-cancerous Mlg-2908 lung fibroblast cells at higher concentrations. Current flow cytometry testing on Mlg-2908 cells, using the same protocol as the H1299 cells, shows cell viability of 89% or higher in 0.1 μM to 10 μM UST-001 concentrations. Since cancerous cells have higher mitotic rates and more repair pathways, we hypothesize that we will be able to establish an IC50 curve in Mlg-2908 cells at higher concentrations. Further studies using a Muse Annexin V apoptosis assay will focus on the UST-001 mechanism of cell death. Our ultimate goal is to characterize the toxicity of this drug in mammalian systems, with hopes of potentially identifying a new class of antibiotics and pharmaceutical targets.
|Original language||English (US)|
|Journal||FASEB journal : official publication of the Federation of American Societies for Experimental Biology|
|State||Published - May 1 2022|
ASJC Scopus subject areas
- Molecular Biology