Inhibition and Structural Determination of Dihydrofolate Reductase for Targeting Lymphatic Filariasis
Presentation Type
Poster
Faculty Advisor
Nina Goodey
Access Type
Event
Start Date
26-4-2024 12:45 PM
End Date
26-4-2024 1:44 PM
Description
Lymphatic filariasis, commonly known as elephantiasis, is a parasitic disease that attacks the lymphatic system, causing severe inflammation leading to the enlargement of limbs and/or genitalia. Although currently there are no cures for lymphatic filariasis, new drug targets like dihydrofolate reductase (DHFR) are being researched. Dihydrofolate reductase is a crucial enzyme to DNA synthesis and folate metabolism in the parasitic worm W. bancrofti, as well as in humans. When looking at known inhibitors of DHFR, like methotrexate, little is known about the structure and interaction of residues between the enzyme and inhibitor. Crystalizing the structure of DHFR with bound inhibitors can provide insight into the functional roles of specific amino acids and lead to the development of more potent and selective inhibitors towards W. bancrofti DHFR over the human DHFR. The process involves expressing and purifying DHFR protein that is produced from W. bancrofti, grown in a two day procedure involving terrific broth culture tubes, optical density monitoring at 600 nm, and the addition of isopropyl beta-D-1-thiogalactopyranoside to induce protein expression. After centrifugation, the pellet is sonicated to release protein, followed by methotrexate affinity and nickel-nitriloacetic column chromatography for collection of purified protein. The purified protein undergoes dialysis, concentration determination at A280 nm, and storage in a freezer for further experimentation, such as inhibition studies using the microplate reader and protein crystallization.
Inhibition and Structural Determination of Dihydrofolate Reductase for Targeting Lymphatic Filariasis
Lymphatic filariasis, commonly known as elephantiasis, is a parasitic disease that attacks the lymphatic system, causing severe inflammation leading to the enlargement of limbs and/or genitalia. Although currently there are no cures for lymphatic filariasis, new drug targets like dihydrofolate reductase (DHFR) are being researched. Dihydrofolate reductase is a crucial enzyme to DNA synthesis and folate metabolism in the parasitic worm W. bancrofti, as well as in humans. When looking at known inhibitors of DHFR, like methotrexate, little is known about the structure and interaction of residues between the enzyme and inhibitor. Crystalizing the structure of DHFR with bound inhibitors can provide insight into the functional roles of specific amino acids and lead to the development of more potent and selective inhibitors towards W. bancrofti DHFR over the human DHFR. The process involves expressing and purifying DHFR protein that is produced from W. bancrofti, grown in a two day procedure involving terrific broth culture tubes, optical density monitoring at 600 nm, and the addition of isopropyl beta-D-1-thiogalactopyranoside to induce protein expression. After centrifugation, the pellet is sonicated to release protein, followed by methotrexate affinity and nickel-nitriloacetic column chromatography for collection of purified protein. The purified protein undergoes dialysis, concentration determination at A280 nm, and storage in a freezer for further experimentation, such as inhibition studies using the microplate reader and protein crystallization.