Measurement of Aromatisation by a Urine Technique Suitable for the Evaluation of Aromatase Inhibitors in Vivo

Abstract

By modification of a recently developed method for separation of radio-labelled urinary oestrogens we were able to separate oestrogen metabolites and measure their isotope ratios in urine following injections of [³H]Δ⁴-androstenedione and [¹⁴C]oestrone. This method provides a useful tool for studying in vivo aromatisation of Δ⁴-androstenedione into oestrone in breast cancer patients before and during treatment with aromatase inhibitors.     

Evidence for an Elevated Aspartate pKa in the Active Site of Human Aromatase

Aromatase (CYP19A1), the enzyme that converts androgens to estrogens, is of significant mechanistic and therapeutic interest. Crystal structures and computational studies of this enzyme shed light on the critical role of Asp³⁰⁹ in substrate binding and catalysis. These studies predicted an elevated pK_a for Asp³⁰⁹ and proposed that protonation of this residue was required for function. In this study, UV-visible absorption, circular dichroism, resonance Raman spectroscopy, and enzyme kinetics were used to study the impact of pH on aromatase structure and androstenedione binding. Spectroscopic studies demonstrate that androstenedione binding is pH-dependent, whereas, in contrast, the D309N mutant retains its ability to bind to androstenedione across the entire pH range studied. Neither pH nor mutation perturbed the secondary structure or heme environment. The origin of the observed pH dependence was further narrowed to the protonation equilibria of Asp³⁰⁹ with a parallel set of spectroscopic studies using exemestane and anastrozole. Because exemestane interacts with Asp³⁰⁹ based on its co-crystal structure with the enzyme, its binding is pH-dependent. Aromatase binding to anastrozole is pH-independent, consistent with the hypothesis that this ligand exploits a distinct set of interactions in the active site. In summary, we assign the apparent pK_a of 8.2 observed for androstenedione binding to the side chain of Asp³⁰⁹. To our knowledge, this work represents the first experimental assignment of a pK_a value to a residue in a cytochrome P450. This value is in agreement with theoretical calculations (7.7–8.1) despite the reliance of the computational methods on the conformational snapshots provided by crystal structures.     

Molecular basis of the recognition of arachidonic acid by cytochrome P450 2E1 along major access tunnel

Cytochrome P450 2E1 is widely known for its ability to oxidize both low molecular weight xenobiotics and endogenous fatty acids (e.g., arachidonic acid (AA)). In this study, we investigated the structural features of the AA‐bound CYP2E1 complex utilizing molecular dynamics (MD) and found that the distinct binding modes for both AA and fatty acid analog are conserved. Moreover, multiple random acceleration MD simulations and steered MD simulations uncovered the most possible tunnel for fatty acids. The main attractions are derived from three key residues, His107, Ala108, and His109, whose side chains reorient to keep ligands bound via hydrogen bonds during the initial unbinding process. More importantly, based on the calculated binding free energy results, we hypothesize that the hydrogen bonds between the receptor and the ligand are the most important contributors involved in the binding affinity. Thus, it is inferred that the hydrogen bonds between these three residues and the ligand may help offer insights into the structural basis of the different ligand egress mechanisms for fatty acids and small weight compounds. Our investigation provides detailed atomistic insights into the structural features of human CYP2E1–fatty acid complex structures. Furthermore, the ligand‐binding characteristics obtained in the present study are helpful for both experimental and computational studies of CYPs and may allow future researchers to achieve desirable changes in enzymatic activities. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 53–66, 2015.     

Interactions of selected indole derivatives with COX-2 and their in silico structure modifications towards the development of novel NSAIDs

Cyclooxygenase-2 (COX-2) is an important enzyme responsible for the formation of potent inflammatory mediators like prostaglandins, prostacyclin and thromboxane. Hence, inhibition of COX-2 is one of the best ways to control the inflammation. Non-steroidal anti-inflammatory drugs can control inflammation by inhibiting Cyclooxygenase. Selective inhibition of COX-2 is preferable over the inhibition of COX-1 because of the fewer adverse effects produced. Molecular modeling and docking of 134 selected indole compounds were done against COX-2. The pharmacophore-based in silico structural modifications of the best scored compounds were carried out in order to enhance the binding affinity and selectivity. The modification resulted in derivatives with better binding energies than that of known COX-2 inhibitors. The four best derivatives in terms of the binding energies were selected and their binding stabilities were studied by molecular dynamics simulation methods.     

New insights into the mechanism of imposex induction in the dogwhelk Nucella lapillus

In an attempt to clarify the mechanism(s) of tributyltin-mediated imposex induction in females of the neogastropod Nucella lapillus, dogwhelks collected in an almost imposex free population were exposed to several treatments for a 3 month-period, and the effects on imposex induction and testosterone/estradiol levels were evaluated. As a positive control, tributyltin (50 ng TBT Sn/L) clearly induced imposex and led to a significant increase in the severity of the phenomenon. In contrast, although a selective P450 aromatase inhibitor (formestane at 0.3 mg/L) was capable of imposex induction, it failed to increase its severity. A vertebrate androgen receptor (AR) antagonist (cyproterone acetate at 1.25 mg/L) in combination with TBT completely blocked the imposex induction capacity of TBT. On the other hand, an estrogen receptor antagonist (tamoxifen at 0.3 mg/L) rendered no effect. The determination of steroid levels in female specimens revealed that TBT induces an elevation of free testosterone (but not the total amount, free+esterified), while the co-administration of the anti-androgen and TBT was able to rescue the increase of free testosterone levels. Despite a minor decrease in the amount of testosterone-fatty acid esters in the TBT group, significant differences in esterified testosterone were not found among treatments. On the contrary, free estradiol levels were elevated in the TBT, anti-androgens and TBT plus anti-androgens groups. These results indicate that free estradiol biosynthesis in TBT-exposed females does not seem to be affected. Overall, our results demonstrate that a selective aromatase inhibitor can induce imposex in N. lapillus but not to a similar extent of TBT, which may suggest the involvement of other mechanism in imposex induction, besides aromatase inhibition. Additionally, the study points to the involvement of AR receptors in imposex induction.     

Insight into the interactive residues between two domains of human somatic Angiotensin-converting enzyme and Angiotensin II by MM-PBSA calculation and steered molecular dynamics simulation

Angiotensin-converting enzyme (ACE), a membrane-bound zinc metallopeptidase, catalyzes the formation of Angiotensin-II (AngII) and the deactivation of bradykinin in the renin–angiotensin-aldosterone and kallikrein–kinin systems. As a hydrolysis product of ACE, AngII is regarded as an inhibitor and displays stronger competitive inhibition in the C-domain than the N-domain of ACE. However, the AngII binding differences between the two domains and the mechanisms behind AngII dissociation from the C-domain are rarely explored. In this work, molecular docking, Molecular Mechanics/Poisson–Boltzmann Surface Area calculation, and steered molecular dynamics (SMD) are applied to explore the structures and interactions in the binding or unbinding of AngII with the two domains of human somatic ACE. Calculated free energy values suggest that the C-domain–AngII complex is more stable than the N-domain–AngII complex, consistent with available experimental data. SMD simulation results imply that electrostatic interaction is dominant in the dissociation of AngII from the C-domain. Moreover, Gln106, Asp121, Glu123, and Tyr213 may be the key residues in the unbinding pathway of AngII. The simulation results in our work provide insights into the interactions between the two domains of ACE and its natural peptide inhibitor AngII at a molecular level. Moreover, the results provide theoretical clues for the design of new inhibitors.     

Insight into the structural requirements of thiophene-3-carbonitriles-based MurF inhibitors by 3D-QSAR,molecular docking and molecular dynamics study

The discovery of clinically relevant inhibitors against MurF enzyme has proven to be a challenging task. In order to get further insight into the structural features required for the MurF inhibitory activity, we performed pharmacophore and atom-based three-dimensional quantitative structure–activity relationship studies for novel thiophene-3-carbonitriles based MurF inhibitors. The five-feature pharmacophore model was generated using 48 inhibitors having IC₅₀ values ranging from 0.18 to 663?μm. The best-fitted model showed a higher coefficient of determination (R²?=?0.978), cross-validation coefficient (Q²?=?0.8835) and Pearson coefficient (0.9406) at four component partial least-squares factor. The model was validated with external data set and enrichment study. The effectiveness of the docking protocol was validated by docking the co-crystallized ligand into the catalytic pocket of MurF enzyme. Further, binding free energy calculated by the molecular mechanics generalized Born surface area approach showed that van der Waals and non-polar solvation energy terms are the main contributors to ligand binding in the active site of MurF enzyme. A 10-ns molecular dynamic simulation was performed to confirm the stability of the 3ZM6-ligand complex. Four new molecules are also designed as potent MurF inhibitors. These results provide insights regarding the development of novel MurF inhibitors with better binding affinity.     

Comparative study of the binding mode between cytochrome P450 17A1 and prostate cancer drugs in the absence of haem iron

Abstract

According to the X-ray crystal structures of CYP17A1 (including its complexes with inhibitors), it is shown that a hydrogen bond exists between CYP17A1 and its inhibitors (such as abiraterone and TOK-001). Previous short MD simulations (50?ns) suggested that the binding of abiraterone to CYP17A1 is stronger than that of TOK-001. In this work, by carrying out long atomistic MD simulations (200?ns) of CYP17A1 and its complexes with abiraterone and TOK-001, we observed a binding mode between CYP17A1 and abiraterone, which is different from the binding mode between CYP17A1 and TOK-001. In the case of abiraterone binding, the unfilled volume in the active site cavity increases the freedom of movement of abiraterone within CYP17A1, leading to the collective motions of the helices G and B′ as well as the breaking of hydrogen bond existing between the 3β-OH group of abiraterone and N202 of CYP17A1. However, the unfilled volume in the active site cavity can be occupied by the benzimidazole ring of TOK-001, restraining the motion of TOK-001. By pulling the two inhibitors (abiraterone and TOK-001) out of the binding pocket in CYP17A1, we discovered that abiraterone and TOK-001 were moved from their binding sites to the surface of protein similarly through the channels formed by the helices G and B′. In addition, based on the free energy calculations, one can see that it is energetically favorable for the two inhibitors (abiraterone and TOK-001) to enter into the binding pocket in CYP17A1.     

细胞色素P450基因AsCYP6Z2在中华按蚊溴氰菊酯抗性维持中的作用

【目的】探究细胞色素P450基因AsCYP6Z2是否与中华按蚊Anopheles sinensis抗拟除虫菊酯有关。【方法】克隆中华按蚊AsCYP6Z2基因的编码区。采用实时定量PCR(real-time quantitative PCR, qPCR)检测该基因在中华按蚊溴氰菊酯敏感品系(WX-LS)和抗性品系(YN-LR)雌蚊不同发育阶段的表达和在敏感品系雌蛹不同组织中的表达。雌蛹腹部后端分别在25 mg/mL溴氰菊酯溶液和纯丙酮溶液(对照)中体外培养后,采用qPCR检测AsCYP6Z2在溴氰菊酯处理组和丙酮对照组中的表达差异。于化蛹后10 h分别注射dsAsCYP6Z2(RNAi组)和dsEGFP(对照组),采用qPCR检测AsCYP6Z2在RNAi组和对照组中的表达差异;采用生物测定方法测定RNAi组和对照组中雌成蚊接触0.05%溴氰菊酯药膜1 h内的击倒率及雌成蚊接触0.05%溴氰菊酯药膜1 h并恢复24 h时的死亡率。通过分子对接预测该基因与溴氰菊酯相互作用的氨基酸残基。【结果】克隆获得了AsCYP6Z2基因(GenBank登录号： MT840336)的编码区,其开放阅读框(ORF)长1 251 bp,编码416个氨基酸,无信号肽和跨膜结构域。发育表达谱表明,AsCYP6Z2基因主要在化蛹30 h至成蚊3 h期间高表达,且抗性品系的基因表达量明显高于敏感品系的;组织表达谱显示该基因在腹部后端的表达量较高,其次是在腹部前端和胸部,在头部的表达量最低。雌蛹腹部后端在25 mg/mL溴氰菊酯溶液中培养12 h和24 h后,处理组中AsCYP6Z2的表达量相比于对照组(纯丙酮培养组)分别上调4倍和13倍。RNAi干扰AsCYP6Z2后,RNAi组中AsCYP6Z2基因的表达量相较于对照组(dsEGFP注射组)下调了约80%。雌成蚊接触0.05%溴氰菊酯药膜1 h后,RNAi组中的个体比对照组中的约提前20 min出现明显的击倒现象,且击倒率明显高于对照组;雌成蚊接触0.05%溴氰菊酯药膜1 h并恢复24 h后,RNAi组中的个体死亡率相比对照组增加了17%, 表明沉默AsCYP6Z2基因导致蚊虫对溴氰菊酯的敏感度显著增加。溴氰菊酯与AsCYP6Z2预测蛋白的分子对接研究结果显示,溴氰菊酯可以进入AsCYP6Z2蛋白的结合口袋,并且与Cys-155形成Pi-硫相互作用以及产生较稳定的氢键,侧链也可与AsCYP6Z2的Ala-165, Val-72, Leu-76, Leu-82和Ala-24等氨基酸残基形成稳定的疏水相互作用网络。【结论】研究结果表明AsCYP6Z2基因参与中华按蚊溴氰菊酯抗性表型的维持,这为进一步探究该基因在拟除虫菊酯类杀虫剂代谢过程中的分子机理奠定了前期基础。     

Synthesis of the N‐methyl Derivatives of 2‐Aminothiazol‐4(5H)‐one and Their Interactions with 11βHSD1‐Molecular Modeling and in Vitro Studies

11β‐Hydroxysteroid dehydrogenase type 1 (11β‐HSD1) is an enzyme that affects the body's cortisol levels. The inhibition of its activity can be used in the treatment of Cushing's syndrome, metabolic syndrome and type 2 diabetes. In this study, we synthesized new derivatives of 2‐(methylamino)thiazol‐4(5H)‐one and tested their activity towards inhibition of 11β‐HSD1 and its isoform – 11β‐HSD2. The results were compared with the previously tested allyl derivatives. We found out that methyl derivatives are weaker inhibitors of 11β‐HSD1 in comparison to their allyl analogs. Due to significant differences in the activity of the compounds, molecular modeling was performed, which was aimed at comparing the interactions between 11β‐HSD1 and ligands differing by substituent at the amine group (allyl vs. methyl). Modeling showed that the absence of the allyl group can lead to the rotation of whole ligand molecule which affects its interaction with the enzyme.     

Computational identification and analysis of functional polymorphisms involved in the activation and detoxification genes implicated in endometriosis

Endometriosis is a complex disorder of the female reproductive system where endometrial tissue embeds and grows at extrauterine location leading to inflammation and pain. Hundreds of polymorphisms in several genes have been studied as probable risk factors of this debilitating disease. Bioinformatics tools have come a long way in augmenting the search for putative functional polymorphisms in human diseases. In this study we have explored 16 genes involved in the detoxification of xenobiotic chemicals that are implicated in endometriosis by utilising publically available programs like SIFT, Polyphen, Panther, FastSNP, SNPeffect and PhosSNP. The variations among different ethnic populations of the SNPs were studied. We then calculated the extent to which bioinformatics based predictions are concurrent with real world epidemiological, genotyping studies using a set of SNPs that have been studied in endometriosis case–control studies. Our study shows that there is a significant positive correlation (r = 0.569, p < 0.005) between the summary of the predicted scores taken from 4 different servers and the odds ratio found from epidemiological studies. This report has identified and catalogued various deleterious SNPs that could be important in endometriosis and could aid in further analysis by in vitro and in vivo methods for the better understanding of the disease pathophysiology.     

Exemestane metabolites suppress growth of estrogen receptor-positive breast cancer cells by inducing apoptosis and autophagy: A comparative study with Exemestane

Around 60–80% of all breast tumors are estrogen receptor-positive. One of the several therapeutic approaches used for this type of cancers is the use of aromatase inhibitors. Exemestane is a third-generation steroidal aromatase inhibitor that undergoes a complex and extensive metabolism, being catalytically converted into chemically active metabolites. Recently, our group showed that the major exemestane metabolites, 17β-hydroxy-6-methylenandrosta-1,4-dien-3-one and 6-(hydroxymethyl)androsta-1,4,6-triene-3,17-dione, as well as, the intermediary metabolite 6β-Spirooxiranandrosta-1,4-diene-3,17-dione, are potent aromatase inhibitors in breast cancer cells. In this work, in order to better understand the biological mechanisms of exemestane in breast cancer and the effectiveness of its metabolites, it was investigated their effects in sensitive and acquired-resistant estrogen receptor-positive breast cancer cells. Our results indicate that metabolites induced, in sensitive breast cancer cells, cell cycle arrest and apoptosis via mitochondrial pathway, involving caspase-8 activation. Moreover, metabolites also induced autophagy as a promoter mechanism of apoptosis. In addition, it was demonstrated that metabolites can sensitize aromatase inhibitors-resistant cancer cells, by inducing apoptosis. Therefore, this study indicates that exemestane after metabolization originates active metabolites that suppress the growth of sensitive and resistant breast cancer cells. It was also concluded that, in both cell lines, the biological effects of metabolites are different from the ones of exemestane, which suggests that exemestane efficacy in breast cancer treatment may also be dependent on its metabolites.