Losartan Binding Research Articles

Abstract 14132: The Crystal Structures Of Human Cytochrome P450 2C9 Provide Insights Into The Effect Of Genetic Variation And The Binding Of Losartan

The human cytochrome P450 2C9 (CYP2C9) enzyme metabolizes many of the currently available medications that include anti-coagulant warfarin, anti-hypertensive losartan, anti-diabetics tolbutamide and glimepiride. Genetic variations in CYP2C9 that include the common alleles *2 and *3 have resulted in reduced enzyme activity leading to altered metabolism of various drug substrates compared with the wild-type (WT). The CYP2C9*2 genetic variant represent an amino acid change from arginine to cysteine at position 144. The crystal structure of CYP2C9 WT and the *2 variant complexed with losartan were recently published. However, the structure of these enzymes in the absence of any ligand or losartan is not available. Thus, the aim was to determine the ligand free structure of these enzymes and assess the hypothesis that the drug binding orchestrates the conformational change and leads to reorientation of amino acid sidechains to interact with losartan in the active site. The data for the CYP2C9 WT and the CYP2C9*2 crystals in the absence of any ligands was collected remotely at the Stanford Synchrotron Radiation Light Source and the structures were solved using x-ray crystallography. The ligand-free structure of the *2 variant revealed markedly different conformation compared to the recently published structure in complex with losartan. The changes in the F, F’, G and G’ helices indicate an open conformation in the absence of losartan compared to the losartan complex. Whereas the ligand-free structure of the WT illustrated similarity in the overall conformation to the previously published structures with losartan bound. Comparison of the ligand free structure of the CYP2C9 WT or the *2 variant with the losartan bound structure identified the amino acid sidechains important for interaction with the drug in the active site. Furthermore, the structure of the *2 variant in the absence or presence of losartan provided a useful framework to investigate the effect of amino acid substitution that leads to altered binding of the drug. In conclusion, the structural characterization of the CYP2C9 WT and the *2 variant yield insights into the conformational changes that occur upon binding of losartan and help understand the effect of genetic polymorphisms on drug metabolism.

Insights into the Genetic Variations of Human Cytochrome P450 2C9: Structural Analysis, Characterization and Comparison.

Cytochromes 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.

Biochemical and Biophysical Characterization of Pharmacogenetic Variants of Human Cytochrome P450 2C9

Cytochromes P450 (CYP) are a superfamily of heme-containing enzymes essential in the metabolism of various xenobiotics. The drug metabolizing CYP2C9 enzyme metabolizes up to 15-20% of the medications that include warfarin, losartan, tolbutamide, and glimepiride. Genetic variations in CYP2C9 gene that include the more prevalent *2, *3, and *8, as well as the less common *28 and *30 alleles, have resulted in reduced enzyme activity leading to altered metabolism of various drug substrates compared with the wild-type (WT). The CYP2C9*2 and *3 genetic variants represent an amino acid change from arginine to cysteine at position 144 (R144C) and isoleucine to leucine at position 159 (I359L), respectively. Whereas, the CYP2C9*8, *28 and *30 represents amino acid substitution from arginine to histidine at positions 150 (R150H), glutamine to leucine at 214 (Q214L) and alanine to threonine at 477 (A477T), respectively. These CYP2C9 variants were recombinantly expressed and purified with an aim to characterize the binding of the drug substrate losartan using enzymatic and thermodynamic approaches. The activity of the enzyme was measured in the reconstituted system containing P450 reductase and losartan by the consumption of NADPH or change in absorbance at 340 nm. The *3 variant demonstrated a significantly reduced ability to turn over the losartan substrate compared to the WT and the other variants. Furthermore, differential scanning fluorimetry confirmed lower thermal stability of the *2 and *3 variants in the presence or absence of losartan compared to the WT or *8 variant. This was consistent to the weaker binding affinity of losartan to these two variants as elucidated by isothermal titration calorimetry. The average apparent dissociation constant (KD) for losartan binding to CYP2C9*3 was more than three-fold lower than the WT enzyme. Whereas the KD value for losartan binding to CYP2C9*8 did not show any significant difference. Overall, functional characterization of these variants provide insights into the enzyme activity and binding of drug substrates to CYP2C9, and help improve our understanding of the effect of amino acid substitutions on drug metabolism.

Crystal Structure of CYP2C9*8 Genetic Variant Bound to an Anti‐Hypertensive Drug Losartan.

Cytochrome P450 (CYP) enzymes are one of the major xenobiotic metabolizing enzymes with increasing importance in pharmacogenetics, and more recently to pharmacogenomics. The human CYP2C9 is involved in the metabolism of over 15% of clinical drugs that include warfarin, losartan, tolbutamide, etc. More than eighty genetic variations have been identified in CYP2C9, and many of these have demonstrated significantly reduced activity compared with the wild‐type (WT) enzyme. The CYP2C9*8 allele, prevalent among African‐American population 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 Å 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. The region near the access channel was more compact than the WT enzyme. Furthermore, the losartan turn‐over rates measured using enzymatic assays differed significantly, with the variant demonstrating marked reduction in activity than the WT enzyme. Together, our findings from multiple techniques suggest an alternate mechanism may be involved in reduced activity of this variant located on the surface that leads to differences in binding of losartan near the active site. The results yield insights into the role of simultaneous binding of multiple substrate molecules, orientation of important amino acid side chains and altered hydroxylation profile of losartan with this variant.Support or Funding InformationThe X‐ray data collection at the Stanford Synchrotron Radiation Lightsource (SSRL) was done remotely. Use of the SSRL, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE‐AC02076SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the NIH, NIGMS (P41GM103393). The research was funded by the start‐up funds from the Albany College of Pharmacy and Health Sciences.

Crystal Structure of CYP2C9*2 Genetic Variant in Complex with an Angiotensin II Receptor Blocker Drug Losartan

Cytochrome P450 (CYP)‐dependent monooxygenases are a superfamily of heme‐containing enzymes involved in the metabolism of various xenobiotics. The drug metabolizing CYP2C9 enzyme metabolizes up to 20% of the clinical drugs that include losartan, warfarin, ibuprofen, tolbutamide, and glimepiride. The CYP2C9*2 and *3 are the two prevalent genetic variants that demonstrate significant reduction of the catalytic activity compared to the wild‐type (WT) enzyme for many important CYP2C9 substrates. Despite the abundant structural information available regarding various CYPs, the structural information about the effect of genetic polymorphisms on drug binding has remained scant. The CYP2C9*2 variant represents an amino acid change from arginine to cysteine at position 144, also referred to as R144C. To understand how genetic variation in CYP2C9 affects drug binding and oxidation, we solved the crystal structure of CYP2C9*2 in complex with the antihypertensive drug losartan using X‐ray crystallography. The prodrug losartan is an angiotensin II receptor antagonist that is predominantly metabolized by CYP2C9 and CYP3A4. The overall structure of the CYP2C9*2‐losartan complex illustrate remarkable differences compared to the WT complex. These include altered conformation of important secondary structural elements and residue side chains previously shown to be responsible for binding interactions with different substrates. Furthermore, the new structure revealed significant changes in binding of losartan compared to the previously solved losartan complexes of the CYP2C9 WT, *3 and *30. The structure highlights how single nucleotide polymorphism affects drug binding and yield insights into altered metabolism. Overall, the structural complex provides a useful framework to study, computationally as well as biophysically, the binding of different substrates to genetic variants of CYP2C9 and understand the effect of amino acid substitution in decreased hydroxylation activity of the enzyme.Support or Funding InformationThe authors are grateful to Albany College of Pharmacy and Health Sciences for funding.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigation into the interaction of losartan with human serum albumin and glycated human serum albumin by spectroscopic and molecular dynamics simulation techniques: A comparison study

The interaction between losartan and human serum albumin (HSA), as well as its glycated form (gHSA) was studied by multiple spectroscopic techniques and molecular dynamics simulation under physiological conditions. The binding information, including the binding constants, effective quenching constant and number of binding sites showed that the binding partiality of losartan to HSA was higher than to gHSA. The findings of three-dimensional fluorescence spectra demonstrated that the binding of losartan to HSA and gHSA would alter the protein conformation. The distances between Trp residue and the binding sites of the drug were evaluated on the basis of the Förster theory, and it was indicated that non-radiative energy transfer from HSA and gHSA to the losartan happened with a high possibility. According to molecular dynamics simulation, the protein secondary and tertiary structure changes were compared in HSA and gHSA for clarifying the obtained results.

Site-Directed Mutagenesis of the Gerbil and Human Angiotensin II AT1Receptors Identifies Amino Acid Residues Attributable to the Binding Affinity for the Nonpeptidic Antagonist Losartan

Gerbil angiotensin II AT(1) receptors have more than 90% amino acid sequence homology with human AT(1) receptors and similar affinity for the natural peptide agonist angiotensin II. However, their binding affinity for the biphenylimidazole AT(1) receptor antagonist losartan is greatly reduced compared with the hAT(1) receptor (400 times lower for the gAT(1A) receptor and 40 times lower for the gAT(1B) receptor cloned here). Gain- and loss-of-function site-directed mutagenesis revealed that in gerbil and human AT(1) receptors, the amino acid most important for losartan binding is located in position 108, followed by 107, both in transmembrane (TM) III. In both gerbil and human AT(1) receptors, the effect of G107S and I108V mutants is cumulative. Mutation L195M in TM V is very important, when combined with mutations G107S and I108V, for both gerbil and human AT(1) receptors. In the gerbil, less important amino acids are located in positions 150/151 (TM IV) and 177 in the extracellular loop 2. The study of gerbil natural mutants allowed us to advance our understanding of amino acids selectively involved in the determination of antagonist affinity for gerbil and, most importantly, for human angiotensin II AT(1) receptors.

Influence of Estrogen and/or Progesterone on Isolated Ovariectomized Rat Uterus

Steroids hormones can influence several functions of the uterus, including agonist-induced contractions. The aim of this work is to study the influence of hormonal replacement on angiotensin II (Ang II) and losartan responsiveness on the isolated rat uterus. The Ang II pD₂ values are: 9.69 ± 0.07 for vehicle-treated animals, 8.85 ± 0.06 for estrogen-treated animals, 10.12 ± 0.03 for progesterone-treated animals and 8.90 ± 0.03 for estrogen-and-progesterone-treated animals. The losartan pD₂’ values are: 8.43 ± 0.03 for vehicle-treated animals, 8.21 ± 0.03 for estrogen-treated animals, 7.83 ± 0.05 for progesterone-treated animals and 8.70 ± 0.09 for estrogen-and-progesterone-treated animals. There is not a correlation between Ang II pD₂ and losartan pD₂’ values, suggesting that the hormones affect Ang II and losartan binding by different mechanisms in rat uterus.

Functional interactions of L-162,313 with angiotensin II receptor subtypes and mutants.

A nonpeptide ligand, L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-is obutyl-3-thienyl]phenyl]methyl]imidazo[4,5,6]pyridine) was characterized on the angiotensin II receptors. This compound displaced [125I][Sar1]angiotensin II from rat angiotensin AT1A, AT1B or AT2 receptor individually expressed in COS-7 cells (Ki = 207 nM, 226 nM and 276 nM, respectively). In monkey kidney cells expressing angiotensin AT1A or AT1B receptors, it stimulated inositol phosphate accumulation, but the maximal response was 34.9 and 23.3%, respectively, of those of angiotensin II. Furthermore, an antagonist effect of L-162.313 was observed in response to angiotensin II. Single-point substitutions in the second and third transmembrane domains of the rat angiotensin AT1A receptor, which impaired the binding of losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1[(1H-tetrazol-5-yl)biphenyl-4 -yl)methyl]imidazole), also affected the binding of L-162,313. Losartan and L-162,313 require some common structural determinants for non-peptide recognition on the angiotensin AT1 receptor. Furthermore, some of these substitutions, which impaired the inositol phosphate accumulation in response to angiotensin II, also impaired the response to L-162,313. Angiotensin II and L-162,313 require common critical residues for angiotensin AT1 receptor activation.

Binding of valsartan to mammalian angiotensin AT 1 receptors

The binding characteristics of the angiotensin AT 1 receptor antagonist valsartan were investigated in different animal species and tissues. Using [ 125I](Sar 1,Ile 8)angiotensin II as radioligand, affinity constants were determined in liver and adrenal of rat and marmoset, human adrenal and in rat aortic smooth muscle cells. In all tissues tested, valsartan had a greater affinity for the AT 1 receptor than losartan (on average 5-fold). The affinities of both antagonists were up to 30 times weaker in the dog tissues. [ 3H]Valsartan bound with high affinity ( K d 1.44 nmol/l) to the rat aortic smooth muscle cell AT 1 receptor. Binding was saturable and reversible. Non-specific binding was low (10%). Reports that [ 3H]losartan binds to a non-angiotensin II binding site in rat liver and in other tissues could be confirmed. [ 3H]Valsartan on the other hand bound only to the AT 1 receptor. Using a competition binding assay with [ 3H]losartan on rat liver membranes it could be shown that valsartan can bind to the ‘losartan binding site’, but at a 10,000-fold less affinity than for the AT 1 receptor. Valsartan is therefore a highly specific and selective antagonist of the AT 1 receptor. Due to its high affinity and low non-specific binding it is a suitable radioactive antagonist for the study of the distribution and function of the angiotensin AT 1 receptor.

Human Plasma Protein Binding of the Angiotensin II Receptor Antagonist Losartan Potassium (DuP 753/MK 954) and Its Pharmacologically Active Metabolite EXP3174

The in vitro protein binding characteristics of the prototypical angiotensin II receptor antagonist losartan potassium (DuP 753/MK 954) and its pharmacologically active metabolite EXP3174 were determined by ultrafiltration with plasma from naive donors, volunteers dosed with losartan, and purified human plasma proteins. The binding of losartan was high, with a percent unbound (free) of 1.4 +/- 0.2% to 1.2 +/- 0.1% at concentrations ranging from 0.5 to 5.0 micrograms/mL; that is, approximately 98.6 to 98.8% bound. EXP3174 was more highly bound than losartan (P < .05) with 0.2 +/- 0.0% free at concentrations ranging from 0.1 to 10.0 micrograms/mL; or, greater than 99.7% bound. The binding in the plasma from volunteers given oral losartan was similar to that determined with plasma from naive donors, with 1.5 +/- 0.3 versus 1.4 +/- 0.1% free for losartan, and 0.5 +/- 0.1 versus 0.4 +/- 0.0% for EXP3174, respectively. This extensive plasma binding of both acidic compounds occurs primarily to albumin, with negligible binding to the alpha 1-acid glycoprotein. Although highly bound, neither losartan nor EXP3174 were displaced in vitro by pharmacologically relevant concentrations of non-steroidal antiinflammatory drugs (NSAIDs), warfarin, or diazepam; however, suprapharmacologic concentrations of the NSAIDs increased the free fraction of both compounds. These data show that the angiotensin II receptor antagonists losartan and EXP3174 are highly bound to plasma albumin in humans, although clinically significant drug interactions due to displacement from binding sites are unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential structural requirements for specific binding of nonpeptide and peptide antagonists to the AT1 angiotensin receptor. Identification of amino acid residues that determine binding of the antihypertensive drug losartan

The biphenylimidazole derivative losartan exemplifies a novel class of nonpeptide antagonists that selectively inhibit angiotensin II binding to the mammalian AT1 receptor and have potentially wide application as anti-hypertensive agents. In contrast to mammalian AT1 receptors, which have high affinity for both peptide antagonists and losartan, amphibian and avian angiotensin II receptors are pharmacologically distinct and recognize peptide but not nonpeptide antagonists. Mutant rat AT1 receptors in which non-conserved amino acids were replaced by the corresponding amphibian residues were constructed to identify specific sites in the AT1 receptor that determine losartan binding. Only minor changes in binding affinity for peptide antagonists were observed in COS-7 cells transiently expressing mutant receptors, indicating that the structural integrity of the receptor was maintained. However, analysis of receptors with single point and combined mutations revealed that specific residues in transmembrane domains III, IV, V, VI, and VII are involved in binding of the nonpeptide antagonist to the mammalian AT1 receptor. The most marked attenuation of losartan binding (IC50 > 50 microM) was observed in a mutant receptor containing amino acid substitutions at Val108, Ala163, Thr198, Ser252, Leu300, and Phe301. These results have demonstrated that the binding site of a potent nonpeptide antagonist of the AT1 receptor is defined by epitopes located within the membrane-spanning regions of the receptor and is distinct from the site at which peptide antagonists bind to the AT1 receptor.

Differential regulation of angiotensin II and losartan binding sites in glomeruli and mesangial cells

The aim of the present report was to examine the effect of several agents on angiotensin II (ANG II) and losartan receptors using 125I-[Sar1,Ala8]ANG II and [3H]losartan as radiolabeled ligand, respectively. ANG II receptors were downregulated in glomeruli from rats infused with ANG II during 3 wk or rats receiving losartan orally during 1 wk. The number of sites (Bmax) was reduced, but the dissociation constant (Kd) value was unchanged. Losartan receptors were downregulated in glomeruli from rats receiving losartan, but remained unchanged in glomeruli from rats infused with ANG II. Since in vivo administration of losartan results in increase of plasma ANG II and formation of metabolites, in vitro studies using human mesangial cells were performed to better analyze the present findings. Treatment of mesangial cells during 4 days by ANG II, losartan, or its metabolite, EXP-3174, also produced downregulation of 125I-[Sar1,Ala8]ANG II binding sites with a decreased Bmax and unchanged Kd value. Only treatment of mesangial cells by ANG II or EXP-3174 produced downregulation of [3H]losartan binding sites. In contrast, exposure of these cells to losartan resulted in upregulation of [3H]losartan binding sites. Under all conditions, only Bmax was modified. Whereas internalization of [3H]losartan in mesangial cells was negligible under all experimental conditions, there was an increase of the percentage of internalized 125I-[Sar1,Ala8]ANG II after exposure of the cells to ANG II or AT1 antagonists. No change was observed in mesangial cell AT1 receptor mRNA levels. This study demonstrates that 1) AT1 mRNA is expressed in human mesangial cells; 2) the characteristics of 125I-[Sar1,Ala8]ANG II and [3H]losartan binding sites in rat glomeruli and human mesangial cells are different, with Kd and Bmax values greater in both preparations when [3H]losartan was utilized; 3) both types of binding sites obey different regulations, and the effects of losartan in vivo are due in part to the associated increase in plasma ANG II levels and the transformation of the drug into its metabolite, EXP-3174; 4) downregulation of AT1 receptors does not depend on changes in mRNA expression but is associated with increased relative internalization.

Angiotensin II and non-angiotensin II displaceable binding sites for [ 3H]losartan in the rat liver

By virtue of the more than 1000-fold selectivity of losartan (DuP 753) for the AT 1 angiotensin II (AII) receptor subtype compared with the AT 2 subtype, [ 3H]losartan may be a useful radioligand for studies of the AT 1 receptor subtype. Comparison of B max values in the liver obtained from saturation isotherms using [ 3H]losartan ( B max = 194 pmol g tissue) and [ 125I]sarcosine 1,isoleucine 8 angiotensin II ( B max = 20 pmol g tissue) indicated that the AII receptor concentration was approximately 10% that of the [ 3H]losartan binding sites. In addition, AII at concentrations as high as 10 μM displaced less than one-third of specific [ 3H]losartan binding in the liver and less than 80% in the whole adrenal. The presence of non-AII displaceable [ 3H]losartan binding in the liver did not appear to result from metabolism of the radioligand since HPLC analysis of free and bound 3H revealed that greater than 90% of the 3H eluted at the same time as the parent [ 3H]losartan. This suggests that [ 3H]losartan binds with high affinity to a site(s) other than angiotensin II receptors in the rat liver.

Characterization of [ 3H]losartan receptors in isolated rat glomeruli

[ 3H]Losartan bound specifically to isolated rat glomeruli. Scatchard analysis revealed a single class of losartan binding sites with an apparent dissociation constant ( K D) of 6.2 nM and a density of receptor sites ( B max) of 1.2 pmol/mg protein. In comparison, [ 3H][Sar 1,Ala 8]angiotensin II binding sites exhibited the same K D value (4.3 nM), but a considerably lower B max (52 fmol/mg protein). Moreover whereas [ 125I][Sar 1,Ala 8]angiotensin II was almost equally displaced by angiotensin II, [Sar 1,Ala 8] angiotensin II and losartan, [ 3H]losartan was potently displaced by losartan only. Finally, [ 125I][Sar 1,Ala 8]angiotensin II but not [ 3H]losartan binding sites were sensitive to guanosine triphosphate (GTP) γS and dithiothreitol. These data, together with the recent demonstration of intrinsic effects of losartan, support the view that [ 3H]losartan does not label only the angiotensin II type 1 receptor (AT 1).