8 Mar 2019

'Roofies' Conversion by Cyp3A4 is Altered by Ethanol and Caffeine

Justice Spriggs '19

Flunitrazepam (FNTZ) is a benzodiazepine that is prescribed to treat insomnia throughout Europe. In North America, FNTZ is more commonly known by the name "Roofies" and has been associated with drug-facilitated sexual assault. Generally, the beverages that FNTZ is placed in contain some combination of alcohol and caffeine. Cytochrome P450 3A4 (Cyp3A4) is the main enzyme responsible for drug degradation in the human body. To look at the effect of caffeine and alcohol on the degradation of FNTZ by Cyp3A4, steady-state kinetics was measured in the presence and absence of caffeine and/or ethanol. Steady-state kinetics were measured via High-Performance Liquid Chromatography (HPLC) analysis. Reactions of different concentrations of inhibitor were initiated by the addition of NADPH and incubated for 30 minutes. Product formation was fitted using the Michaelis-Menten equation, and the manifolds of inhibition were analyzed via replot. Preliminary steady-state kinetic analysis shows a significant decrease in the production of the two main metabolites of FNTZ in the presence of caffeine. We hypothesize that caffeine binds allosterically to Cyp3A4, where it affects enzyme kinetics. Additionally, preliminary studies show that caffeine and ethanol show a synergistic effect on the production of the main metabolites. Further kinetic analysis, as well as structural studies by NMR Spectroscopy, will be pursued.

Hexosaminidase Beta gene sequencing (Exons 1-7) in a family with possible LOTS

Elizabeth Johnson '19

Late-Onset Tay-Sachs (LOTS) is a lysosomal storage disorder that affects certain demographics including the Ashkenazi Jewish population. It is a similar disease to infantile Tay-Sachs which has a carrier rate of 1 in 35 in the Ashkenazi Jewish population according to researchers Mark et al. Tay-Sachs causes neurodegenerative symptoms ranging from mental illness to loss of motor function and many patients have multiple symptoms. It is believed that this disease is due to a deficiency in the Hexosaminidase A protein by researchers Navon et al. There is no biological test for this disease and diagnoses are based on symptoms. It is not yet understood how LOTS mutations are passed on through generations. In 1985 a patient, SG, passed away from complications due to symptoms that we now associate with LOTS. The participants of this study are SG’s offspring, spouse, and a control from the same demographic, Ashkenazi Jewish. The hypothesis for this study is that the patient SG was misdiagnosed and had LOTS. Previously this study sequenced the participants for mutations in the HexA gene that may have been inherited from SG. In this part of the study we moved onto the HexB gene because Hexosaminidase A is a heterodimer using both HexA and HexB to make the protein. PCR and gene sequencing was done on the participants DNA for the HexB gene. No mutations were found that could affect the functionality of Hexosaminidase A.

Searching for Genetic Markers in Canine Periodontal Disease Susceptibility: Interleukin-1 Genes

Katie Carlson '19

Genetic disorders may be due to a single gene or multiple genes. The latter case brings complexity to predicting and treating genetic diseases. These diseases exhibit differences in their heritability and their contributing genes, so each disease is unique in their genetic composition. Periodontal disease, a disease of the mouth caused by inflammation, is a multi-gene disorder that has been linked to various genes, including those involved in the regulation of the immune system. In this study by Albuquerque et al., the inflammatory gene family, interleukin-1, is analyzed for genetic markers of periodontal susceptibility in canines. Six new variants were identified, two of which showed statistical significance in the frequency between healthy and diseased dogs. One variant was associated with increased risk, while the other was associated with decreased risk for disease. However, these variants are not causal, and are merely pieces in the whole genetic composition of the disease. Recent developments in genetic analysis, such as haplotyping and genome wide association studies, can help to identify more variants and genetic relationships between genes for not only periodontal disease, but other genetic disorders. With a better understanding of what gene variations are important and the contributions of each gene, we can gain a better understanding and treatment of these diseases.