Shah, Manish Theses Advised
Permanent URI for this collection
Browse
Recent Submissions
Item Structural and Functional Basis of Drug Binding to Human Cytochrome P450 2C9*14 Genetic Variant(Albany College of Pharmacy and Health Sciences Theses, 2022-12) Edara, Sreeja '22Cytochrome P450 (CYP) enzymes are a group of enzymes encoded by P450 genes and are expressed as membrane-bound proteins mostly found in the endoplasmic reticulum of the liver cells. About 17 of the 57 CYP proteins have a significant role in drug metabolism. CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 are the major enzymes involved in drug metabolism. The highly polymorphic CYP2C9 is responsible for the metabolism of up to 15% of clinical drugs that include the anticoagulant S-Warfarin, the anti-hypertensive losartan, the anti-diabetic tolbutamide, and the analgesic ibuprofen, and others. The CYP2C9*14 allele, which results in the amino acid substitution from arginine to histidine at position 125 (R125H), is found in 3-5% of population, predominantly southeast Asians. Recent studies have illustrated that the CYP2C9*14 variant results in decreased enzyme function leading to the \342\200\234poor metabolizer\342\200\235 phenotype. For example, patients with this allele required a lower S-Warfarin dose compared to those with the normal or wild type allele. However, the implications of the CYP2C9*14 allele on the metabolism of losartan, an anti-hypertensive drug substrate of CYP2C9 is less clear. In this study, we aim to determine the impact of the amino acid change R125H (CYP2C9*14) on the binding of losartan using structural, functional, and biophysical analysis that include X-ray crystallography, enzymatic assays, and isothermal titration calorimetry, respectively. The results of this research will help advance our understanding of the effect of genetic polymorphisms and inter-individual differences in losartan response and may help implementing clinical guideline for patients on losartan that possesses the *14 allelic variation.Item Crystal Structure of the Genetic Variant of Human Cytochrome P450 2C9 (*27): Insights into the Effect of Single Nucleotide Polymorphism(Albany College of Pharmacy and Health Sciences Theses, 2022-12) Deodhar, Shruti '22Cytochrome P450 (CYP) enzymes are heme-thiolate monooxygenases involved in the metabolism of xenobiotics. Most of the currently available drugs are bio-transformed by the CYP 1, 2, and 3 families. CYP2C9 is a major drug-metabolizing enzyme with more than 80 single nucleotide polymorphisms, several of which significantly affect drug metabolism. This study aims to characterize the CYP2C9*27 allele that represents an amino acid substitution Arg150Leu (arginine to leucine at position 150) in the presence or absence of various drug substrates. The change from Arg150Leu is predominantly found in the Japanese population. In this study, we elucidated the impact of distal variation on the binding of drug substrate in the active site. The CYP2C9*27 enzyme was expressed in E. coli, purified, and crystallized with the antihypertensive drug substrate, losartan. For data collection and structure determination, crystals were sent to Stanford Synchrotron Radiation Light Source. The structure of the CYP2C9*27 protein in complex with losartan is similar in conformation to the previously solved structure of the CYP2C9 wild-type-losartan complex but differed in the number of losartan bound. There were two molecules of losartan bound in the *27 complex, one in the active site and another at the periphery in an orientation identical to that observed in the wild-type complex. This was in contrast to the wild-type complex that demonstrated the binding of three molecules, with an additional losartan in the access channel that was not observed in the *27 structure. In a reconstituted enzymatic assay that also included the redox partner P450 reductase, the CYP2C9*27 showed lower consumption of NADPH than the wild type enzyme suggesting reduced capacity of the variant to turn-over losartan. This was further corroborated by isothermal titration calorimetry studies that illustrated significantly reduced binding affinity of losartan with the *27 variant than for the wildtype. Furthermore, various other angiotensin II receptor blockers were characterized to elucidate differences and role of substrate selectivity in binding to CYP2C9. Overall, the results obtained provide insights into the effect of single nucleotide polymorphism on losartan binding that could be clinically relevant in patients carrying the *27 allele.Item Crystal structure of the human cytochrome P450 2C9*8 genetic variant(Albany College of Pharmacy and Health Sciences Theses, 2020-07) Kamat, Sumit S. '20Cytochrome P450 (CYP) enzymes are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics. The human CYP2C9 enzyme that metabolizes over 15% of clinical drugs including warfarin, losartan, tolbutamide, etc. is highly polymorphic with more than eighty genetic variations identified thus far. Many of these variants have demonstrated significantly reduced activity compared with the wild-type (WT) enzyme. The CYP2C9*8 allele, prevalent among different populations and predominantly found in African-Americans with a frequency of around 0.06, is associated with altered clearance of several drug substrates of CYP2C9. The *8 represents an amino acid variation from arginine to histidine at position 150 (R150H). The R150H variant was generated using CYP2C9 WT construct by site-directed mutagenesis, and the enzyme was expressed in E. coli followed by protein purification and crystallization. The CYP2C9*8 was crystallized in the presence of the drug substrate losartan and the structure was determined using X-ray crystallography at 2.3 \303\205 resolution. The R150H, found on the surface of the protein on D-helix that is distal from the active site, illustrates minimal effect on the overall conformation of the protein compared to the WT. Despite subtle changes in the structure itself, there were clear differences in the binding of losartan compared to the previously solved CYP2C9 WT complex. One molecule of losartan was bound in the active site and one on the surface, consistent to that observed in the WT complex. However, unlike the WT complex, the losartan in the access channel was not observed in the *8 complex. In addition, the losartan turn-over rates measured using enzymatic assays differed significantly, with the variant demonstrating marked reduction in activity than the WT enzyme. The results yield insight into the role of simultaneous binding of multiple substrate molecules that may suggest a possible role of cooperativity and altered hydroxylation profile of losartan with this variant. Further studies are warranted to determine the role of such distal variation.Item Structure-Function Studies of Human Cytochrome P450 2C9*30 Genetic Variant(Albany College of Pharmacy and Health Sciences Theses, 2020-07) Cho, Sunghwan '20Cytochrome P450 (CYP) enzymes are a superfamily of heme-containing proteins found on the membrane of the endoplasmic reticulum. CYPs constitute the major enzyme family in drug metabolism with increasing importance in pharmacogenetics. The human CYP2C9 enzyme, which is responsible for the metabolism of over 20% of available clinical drugs, is highly polymorphic. More than 80 genetic variants of this enzyme have been identified, with many exhibiting significantly altered activities toward various medications compared to the wild type (WT). Despite the extensive functional and structural studies of various CYPs from different species, the fundamental basis of genetic polymorphisms is scant. The CYP2C9*30 is a rare allelic variant, which is found in Japanese population. It represents an amino acid substitution at position 477 from alanine to threonine that has resulted in significantly altered activity of the enzyme towards various substrates compared to the WT. We have expressed and purified the CYP2C9*30 variant in E.coli to the optimal quality. The in-vitro enzymatic assays revealed reduced activity and substrate turn-over by CYP2C9*30 compared to the WT. The previously solved crystal structure of the CYP2C9*30-losartan complex demonstrated the reorientation of amino acid side-chain of glutamine at 214 or Q214 that interacted with the polar substitution threonine at 477 or A477T in the *30 structure. The goal is to investigate the activity of CYP2C9*30, using losartan as a substrate, and determine the role of Q214 side chain in the binding of the drug substrates near the heme in the active site using computational docking. A total of 5 important substrates (Glyburide, Irbesartan, Tolbutamide, S-warfarin, and Losartan) of CYP2C9 were studied via molecular docking to characterize the effect of variation to threonine at 477 on drug metabolism. The data suggest that the reorientation of Q214 side chain to interact with T477 in CYP2C9*30 may affect binding of drugs near the heme in the active site, which can lead to altered metabolism profile compared to the wild-type. Overall, the results yield insights into the reduced catalytic activity of the CYP2C9*30 and may help understand the differences in drug metabolism.