Kinetic Analysis of Fosmidomycin Analogs

According to the World Health Organization, in 2022 approximately 1.3 million people worldwide died from tuberculosis, making the disease the second-leading infectious killer known to man. For new strains of Mycobacterium tuberculosis (Mtb), the bacterium responsible for the disease, multidrug therapy consisting of isoniazid, rifampin, pyrazinamide, and ethambutol has replaced the now-resistant single-drug protocol. However, the emergence of multidrug-resistant (MDR) strains presents an urgent need for new antibiotics. The 2C-methyl-D-erythritol 4-phosphate (MEP) pathway is an outstanding target for novel drug development due to its essentiality in M. tuberculosis and its absence in humans. This pathway is a seven-step mechanism that results in the synthesis of isoprenoids; cell wall components and other biomolecules essential to the pathogen. The first step of the MEP pathway is the reduction and isomerization of 1-deoxy-D-xylulose 5-phosphate (DXP) via the enzyme 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr or IspC). Fosmidomycin is a natural product known to inhibit IspC, but has poor drug-like properties. Thus, rationally designed inhibitors based on the backbone structure of fosmidomycin have been developed. In this study, we analyzed the effectiveness of several fosmidomycin analogs via a kinetic enzymatic assay buffered at either pH 6.5 or 7.8. Results indicate that the fosmidomycin analogs are especially potent in the more acidic environment (pH 6.5), relative to pH 7.8, which is particularly important since the intracellular pH of M. tuberculosis is 6.5.

Hi. My name is Quintin Carroll, and today we’re gonna be talking about my research on the kinetic analysis of those Fosmidomycin analogs. So starting off, let’s talk about what is tuberculosis. Tuberculosis is one of the deadliest infectious diseases known to man. In twenty eighteen, roughly one point five million people died from its infection, and less than sixty percent of patients who undergo treatment are successfully treated. Thus new treatment options are height of heightened urgency to address the disease. Currently, tuberculosis is treated with a multi drug protocol consisting of rifapentine, isoniazid, pyrazinamide and ethambutol. However, the emergence of drug-resistant strains call for new treatment options of the disease. Mycobacteria tuberculosis is the causative agent of tuberculosis. And it is characterized by a fatty acid outer membrane. This outer membrane plays a significant role in antibiotic impermeability. To develop drugs that target Mtb, we look to the methylerythritol phosphate pathway or Mep pathway for short. This is an outstanding target because it does not exist in humans, thus therapies are highly specific to enzymes in this pathway. Fosmidomycin a natural product has been shown to inhibit ISPC in the Mep pathway. However, poor drug like properties such as unfavorable membrane permeability and poor half life make it an unsuitable antibiotic, thus analogs that can that can address these poor drug like properties or paramount interests. For our methodology, we used recombinant protein expression and isolated and purified our target enzyme. And from there, we perform kinetic analysis using an assay mixture that is buffered to six point five pH. From there, we can incubate for ten to fifteen minutes afterwards, we can monitor the oxidation of nadph to produce a time trace that is pictured above or to my left rather. Previously research has used similar methods, but with a more basic assay mixture at pH seven point eight. Our results show that there are much more potent inhibition at pH six point five compared to pH seven point eight. As we can see from the chart, there is much more compounds that are below the dotted line compared to this bar graph over here, which has many above. In terms of compounds, we have two graphs that display the IC fifty value. Or in other words, the concentration that gives us half maximal and enzymatic activity. analyzing the graphs we can see that the n-acyl moiety on the FR nine zero zero nine eight compound proved to be a positive structural change compared to Fosmidomycin In conclusion, results of this research demonstrate that seven compounds exhibit pH dependent activity. Likely from protonation effects influencing inhibitor binding. However, additional investigation is being done to validate this hypothesis. Furthermore, experiments in the form of antimicrobial susceptibility assays are warranted to test these compounds whole cell Mtb to determine their effectiveness against the fatty acid cell wall.