Shah, Manish Publications
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Item Structural and biophysical analysis of cytochrome P450 2C9*14 and *27 variants in complex with losartan.(Journal of Inorganic Biochemistry, 2024-09) Parikh, Sonia J*; Edara, Sreeja*; Deodhar, Shruti*; Singh, Ajit K; Maekawa, Keiko; Zhang, Qinghai; Glass, Karen C; Shah, Manish BThe human cytochrome P450 (CYP) 1, 2 and 3 families of enzymes are responsible for the biotransformation of a majority of the currently available pharmaceutical drugs. The highly polymorphic CYP2C9 predominantly metabolizes many drugs including anticoagulant S-warfarin, anti-hypertensive losartan, anti-diabetic tolbutamide, analgesic ibuprofen, etc. There are >80 single nucleotide changes identified in CYP2C9, many of which significantly alter the clearance of important drugs. Here we report the structural and biophysical analysis of two polymorphic variants, CYP2C9*14 (Arg125His) and CYP2C9*27 (Arg150Leu) complexed with losartan. The X-ray crystal structures of the CYP2C9*14 and *27 illustrate the binding of two losartan molecules, one in the active site near heme and another on the periphery. Both losartan molecules are bound in an identical conformation to that observed in the previously solved CYP2C9 wild-type complex, however, the number of losartan differs from the wild-type structure, which showed binding of three molecules. Additionally, isothermal titration calorimetry experiments reveal a lower binding affinity of losartan with *14 and *27 variants when compared to the wild-type. Overall, the results provide new insights into the effects of these genetic polymorphisms and suggests a possible mechanism contributing to reduced metabolic activity in patients carrying these alleles.Item Four Decades of Cytochrome P450 2B Research: From Protein Adducts to Protein Structures and Beyond.(Drug Metabolism and Disposition, 2023-01) Roberts, Arthur G; Stevens, Jeffrey C; Szklarz, Grazyna D; Scott, Emily E; Kumar, Santosh; Shah, Manish B; Halpert, James RThis article features selected findings from the senior author and colleagues dating back to 1978 and covering approximately three-fourths of the 60 years since the discovery of cytochrome P450. Considering the vast number of P450 enzymes in this amazing superfamily and their importance for so many fields of science and medicine, including drug design and development, drug therapy, environmental health, and biotechnology, a comprehensive review of even a single topic is daunting. To make a meaningful contribution to the 50th anniversary of , we trace the development of the research in a single P450 laboratory through the eyes of seven individuals with different backgrounds, perspectives, and subsequent career trajectories. All co-authors are united in their fascination for the structural basis of mammalian P450 substrate and inhibitor selectivity and using such information to improve drug design and therapy. An underlying theme is how technological advances enable scientific discoveries that were impossible and even inconceivable to prior generations. The work performed spans the continuum from: 1) purification of P450 enzymes from animal tissues to purification of expressed human P450 enzymes and their site-directed mutants from bacteria; 2) inhibition, metabolism, and spectral studies to isothermal titration calorimetry, deuterium exchange mass spectrometry, and NMR; 3) homology models based on bacterial P450 X-ray crystal structures to rabbit and human P450 structures in complex with a wide variety of ligands. Our hope is that humanizing the scientific endeavor will encourage new generations of scientists to make fundamental new discoveries in the P450 field. SIGNIFICANCE STATEMENT: The manuscript summarizes four decades of work from Dr. James Halpert's laboratory, whose investigations have shaped the cytochrome P450 field, and provides insightful perspectives of the co-authors. This work will also inspire future drug metabolism scientists to make critical new discoveries in the cytochrome P450 field.Item Inhibition of CYP2C8 by Acyl Glucuronides of Gemfibrozil and Clopidogrel: Pharmacological Significance, Progress and Challenges.(Biomolecules, 9/1/2022) Shah, Manish BThe lipid-regulating drug gemfibrozil is a useful medication for reducing high cholesterol and triglycerides in the blood. In addition to oxidation, it undergoes extensive glucuronidation to produce gemfibrozil acyl glucuronide, which is a known mechanism-based inactivator of cytochrome P450 (CYP) 2C8. Such selective and time-dependent inhibition results in clinically important drug-drug interactions (DDI) with the drugs metabolized by CYP2C8. Similarly, the acyl glucuronide of clopidogrel, a widely used antiplatelet agent, is a potent time-dependent inhibitor of CYP2C8 that demonstrated significant DDI with the substrates of CYP2C8. Current progress in atomic-level understanding mostly involves studying how different drugs bind and undergo oxidation in the active site of CYPs. It is not clear how an acyl glucuronide metabolite of the drug gemfibrozil or clopidogrel interacts in the active site of CYP2C8 and selectively inhibit the enzyme. This mini-review summarizes the current knowledge on some of the important clinical DDI caused by gemfibrozil and clopidogrel due to the inhibition of CYP2C8 by acyl glucuronide metabolites of these drugs. Importantly, it examines recent developments and potential applications of structural biology tools to elucidate the binding and orientation of gemfibrozil acyl glucuronide and clopidogrel acyl glucuronide in the active site near heme that contributes to the inhibition and inactivation of CYP2C8.Item Insights into the Genetic Variations of Human Cytochrome P450 2C9: Structural Analysis, Characterization and Comparison.(International Journal of Molecular Sciences, 9/22/2021) Parikh, Sonia J; Kamat, Sumit*; Phillips, Margaret; Boyson, Samuel P; Yarbrough, Thomas*; Davie, Dylan*; Zhang, Qinghai; Glass, Karen C; Shah, Manish BCytochromes P450 (CYP) are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics. The CYP2C9 enzyme is responsible for the metabolism of a wide range of clinical drugs. More than sixty genetic variations have been identified in CYP2C9 with many demonstrating reduced activity compared to the wild-type (WT) enzyme. The CYP2C9*8 allele is predominantly found in persons of African ancestry and results in altered clearance of several drug substrates of CYP2C9. The X-ray crystal structure of CYP2C9*8, which represents an amino acid variation from arginine to histidine at position 150 (R150H), was solved in complex with losartan. The overall conformation of the CYP2C9*8-losartan complex was similar to the previously solved complex with wild type (WT) protein, but it differs in the occupancy of losartan. One molecule of losartan was bound in the active site and another on the surface in an identical orientation to that observed in the WT complex. However, unlike the WT structure, the losartan in the access channel was not observed in the *8 complex. Furthermore, isothermal titration calorimetry studies illustrated weaker binding of losartan to *8 compared to WT. Interestingly, the CYP2C9*8 interaction with losartan was not as weak as the CYP2C9*3 variant, which showed up to three-fold weaker average dissociation constant compared to the WT. Taken together, the structural and solution characterization yields insights into the similarities and differences of losartan binding to CYP2C9 variants and provides a useful framework for probing the role of amino acid substitution and substrate dependent activity.Item Structure of Cytochrome P450 2C9*2 in Complex with Losartan: Insights into the Effect of Genetic Polymorphism.(Molecular Pharmacology, 2020-11) Parikh, Sonia J; Evans, Chiara M*; Obi, Juliet O*; Zhang, Qinghai; Maekawa, Keiko; Glass, Karen C; Shah, Manish BThe human CYP2C9 plays a crucial role in the metabolic clearance of a wide range of clinical therapeutics. The *2 allele is a prevalent genetic variation in CYP2C9 that is found in various populations. A marked reduction of catalytic activity toward many important drug substrates has been demonstrated by CYP2C9*2, which represents an amino acid variation at position 144 from arginine to cysteine. The crystal structure of CYP2C9*2 in complex with an antihypertensive drug losartan was solved using X-ray crystallography at 3.1-Å resolution. The Arg144Cys variation in the *2 complex disrupts the hydrogen-bonding interactions that were observed between the side chain of arginine and neighboring residues in the losartan complex of CYP2C9 and the wild-type (WT) ligand-free structure. The conformation of several secondary structural elements is affected, thereby altering the binding and orientation of drug and important amino acid side chains in the distal active site cavity. The new structure revealed distinct interactions of losartan in the compact active site of CYP2C9*2 and differed in occupancy at the other binding sites previously identified in the WT-losartan complex. Furthermore, the binding studies in solution using losartan illustrated lower activity of the CYP2C9*2 compared with the WT. Together, the findings yield valuable insights into the decreased hydroxylation activity of losartan in patients carrying CYP2C9*2 allele and provide a useful framework to investigate the effect of a single-nucleotide polymorphism that leads to altered metabolism of diverse drug substrates. SIGNIFICANCE STATEMENT: The *2 allele of the human drug-metabolizing enzyme CYP2C9 is found in different populations and results in significantly reduced activity toward various drug substrates. How the CYP2C9*2 variant induces altered drug metabolism is poorly understood given that the Arg144Cys variation is located far away from the active site. This work yield insight into the effect of distal variation using multitude of techniques that include X-ray crystallography, isothermal titration calorimetry, enzymatic characterization, and computational studies.