Final Report Summary - OESTROMETAB (Quantum chemical and combined quantum mechanical/molecular mechanical (QM/MM) modelling of oestrogen biosynthesis and metabolism)
Estrogens are essential steroid hormones in the human body regulating the reproductive system and influencing among others brain functioning and bone homeostasis. Low levels of estrogens in women in menopause lead to numerous unpleasant symptoms which have long been treated by increasing the estrogen level via hormone-replacement therapies. However, there is a clear indication for the carcinogenic effect of estrogens, especially in causing breast cancer. This dual, essential and still carcinogenic, character of estrogens, have prompted our research work.
The aim of our project was to obtain a better understanding of the cytochrome P450 enzyme-dependent biosynthesis and metabolism of estrogens. Biosynthesis is responsible for the natural increase of estrogen levels in the body and the aromatase enzyme catalysis the last step of this process, the aromatisation of androgens to estrogens. Although, there are drugs on the market to inhibit this enzyme and help in the treatment of breast cancer patients, the catalytic mechanism of this enzyme is not known in detail. On the other hand, cytochrome P450 enzyme dependent metabolism of estrogens facilitates their excretion from the body, but simultaneously may lead to toxic by-products as well. In order to get detailed insight into these processes at the atomic level, we used modelling tools, high-level quantum chemical and combined quantum chemical calculations and molecular dynamic simulations.
In the first part of the project major constituents of Premarine, a frequently used hormone replacement therapy medication was studied. These included estrone and its conjugated derivatives, equiline and equilenine, which differ only in the degree of unsaturation of ring B. Two-hydroxylation of estrogens is a benign metabolic pathway, while four-hydroxylation leads to carcinogenic products. Using quantum chemical calculations we could show that the degree of unsaturation in ring B is directly linked to the ratio of two versus four-hydroxylation pathways, as conjugation between the aromatic ring A and ring B will fundamentally influence the nucleophilic character of positions two and four. This in turn will affect to the tendency of these positions to undergo hydroxilation catalysed by cytochrome P450 enzymes. Increasing conjugation between ring A and B in the estrone, equiline and equilenine order changes the predominantly two-hydroxylation pathway observed for estrone to exclusive four-hydroxylation for equilenine. These results imply that the harmful nature of hormone replacement therapies could be reduced by the removal of conjugated estrone derivatives.
In order to maximise the benefit of the reintegration grant collaborations have also been set up within Hungary and with Belgium. These collaborations further ensured the success of knowledge transfer from the United Kingdom (UK) to Hungary, helped in the reintegration of the research fellow into the host institute and into Hungarian research circles. These collaborations were in most cases interdisciplinary therefore they promoted a combined, experimental and theoretical, approach to research.
1. We studied the mechanism of hydride transfer in the cytochrome P450nor enzyme with Dr D. Menyhárd (Eötvös Lóránd University). We showed that hydride transfer to the nitrogen of nitric oxide is by far much more favoured than hydride transfer to the oxygen end of nitric oxide, therefore the former reaction is the most likely to occur. Our results imply that for quantum mechanical/molecular mechanical (QM/MM) calculations it is very much advised to use co-crystallised enzyme-substrate structures instead of docked models, as the latter ones are not able to capture those processes which lead to changes in the ternary structure of the protein upon ligand binding and which in turn will influence the reliability of the calculations.
2. With Prof. Nyulászi's group we showed that a new class of catalysts might be formed from oxazol-ylidines as they possess different nucleophilic and electrophilic properties than the presently used imidazole-ylidine catalysts.
3. We analysed the hydrogen bond topology in water and methanol at various temperatures, using molecular dynamics simulations and a statistical analysis of several network-theory based descriptors (local cyclic and bonding coefficients, ring size distribution etc.) in cooperation with Prof. Bakó at the Research Centre for Natural Sciences. We showed that liquid water primarily contains hydrogen-bonded five and six membered rings of water molecules and that the distribution of the ring structures changes with temperature.
4. We prepared homology models of two constructs of the cytidil-choline transferase enzyme for Prof. Vértessy's research group Budapest University of Technology and Economics (BUTE) and Research Centre for Natural Sciences. This enzyme plays a vital role in the lipid biosynthesis pathway of the pathogen of malaria and its potential inhibition may lead to new types of drugs to treat malaria. The homology models served as a good starting point for explaining the similarities between the two enzyme constructs prepared by the experimental group and helps to explain the minor role of a lysine-rich segment found in one of the constructs. Molecular dynamics simulations have also been carried out to study the product binding properties of the constructs and of several mutant structures.
5. Molecular dynamics simulations on the AraF enzyme of E. Coli were carried out to identify the residues possibly involved in the deprotonation and re-protonation of Cys 64 in collaboration with Dr Goedele Roos (Vrije Universiteit Brussel). We showed that Asp 90 is a likely candidate for accepting the proton of Cys 64 and that the hydrogen bond network around Cys 64 can very easily rearrange to stabilise the thiolate anion.
6. We studied the mechanism of hydride transfer in the isopropyl-malate dehydrogenase enzyme in collaboration with the experimental group of Prof. M. Vas (Research Centre for Natural Sciences). The molecular dynamics simulations confirm that the crystal structure obtained by the experimental group contains the two substrates in a favourable orientation for the hydride transfer reaction to occur. Calculations performed in the vacuum predict the activation energy of the reaction to be around 15 kcal/mol and suggests that the hydride transfer reaction and carbon-carbon bond cleavage leading to decarboxylation occurs in a non-synchronous but concerted process. However, it is also likely that within the enzyme the reaction follows a step-wise process.
The project improved the representation of women in research and teaching positions at the Budapest BUTE, where the project was carried out and strengthened the research capacities of the department and led to various collaborative projects with experimental groups within and outside the university and it has strengthened the relationship between BUTE and the University of Bristol.
The aim of our project was to obtain a better understanding of the cytochrome P450 enzyme-dependent biosynthesis and metabolism of estrogens. Biosynthesis is responsible for the natural increase of estrogen levels in the body and the aromatase enzyme catalysis the last step of this process, the aromatisation of androgens to estrogens. Although, there are drugs on the market to inhibit this enzyme and help in the treatment of breast cancer patients, the catalytic mechanism of this enzyme is not known in detail. On the other hand, cytochrome P450 enzyme dependent metabolism of estrogens facilitates their excretion from the body, but simultaneously may lead to toxic by-products as well. In order to get detailed insight into these processes at the atomic level, we used modelling tools, high-level quantum chemical and combined quantum chemical calculations and molecular dynamic simulations.
In the first part of the project major constituents of Premarine, a frequently used hormone replacement therapy medication was studied. These included estrone and its conjugated derivatives, equiline and equilenine, which differ only in the degree of unsaturation of ring B. Two-hydroxylation of estrogens is a benign metabolic pathway, while four-hydroxylation leads to carcinogenic products. Using quantum chemical calculations we could show that the degree of unsaturation in ring B is directly linked to the ratio of two versus four-hydroxylation pathways, as conjugation between the aromatic ring A and ring B will fundamentally influence the nucleophilic character of positions two and four. This in turn will affect to the tendency of these positions to undergo hydroxilation catalysed by cytochrome P450 enzymes. Increasing conjugation between ring A and B in the estrone, equiline and equilenine order changes the predominantly two-hydroxylation pathway observed for estrone to exclusive four-hydroxylation for equilenine. These results imply that the harmful nature of hormone replacement therapies could be reduced by the removal of conjugated estrone derivatives.
In order to maximise the benefit of the reintegration grant collaborations have also been set up within Hungary and with Belgium. These collaborations further ensured the success of knowledge transfer from the United Kingdom (UK) to Hungary, helped in the reintegration of the research fellow into the host institute and into Hungarian research circles. These collaborations were in most cases interdisciplinary therefore they promoted a combined, experimental and theoretical, approach to research.
1. We studied the mechanism of hydride transfer in the cytochrome P450nor enzyme with Dr D. Menyhárd (Eötvös Lóránd University). We showed that hydride transfer to the nitrogen of nitric oxide is by far much more favoured than hydride transfer to the oxygen end of nitric oxide, therefore the former reaction is the most likely to occur. Our results imply that for quantum mechanical/molecular mechanical (QM/MM) calculations it is very much advised to use co-crystallised enzyme-substrate structures instead of docked models, as the latter ones are not able to capture those processes which lead to changes in the ternary structure of the protein upon ligand binding and which in turn will influence the reliability of the calculations.
2. With Prof. Nyulászi's group we showed that a new class of catalysts might be formed from oxazol-ylidines as they possess different nucleophilic and electrophilic properties than the presently used imidazole-ylidine catalysts.
3. We analysed the hydrogen bond topology in water and methanol at various temperatures, using molecular dynamics simulations and a statistical analysis of several network-theory based descriptors (local cyclic and bonding coefficients, ring size distribution etc.) in cooperation with Prof. Bakó at the Research Centre for Natural Sciences. We showed that liquid water primarily contains hydrogen-bonded five and six membered rings of water molecules and that the distribution of the ring structures changes with temperature.
4. We prepared homology models of two constructs of the cytidil-choline transferase enzyme for Prof. Vértessy's research group Budapest University of Technology and Economics (BUTE) and Research Centre for Natural Sciences. This enzyme plays a vital role in the lipid biosynthesis pathway of the pathogen of malaria and its potential inhibition may lead to new types of drugs to treat malaria. The homology models served as a good starting point for explaining the similarities between the two enzyme constructs prepared by the experimental group and helps to explain the minor role of a lysine-rich segment found in one of the constructs. Molecular dynamics simulations have also been carried out to study the product binding properties of the constructs and of several mutant structures.
5. Molecular dynamics simulations on the AraF enzyme of E. Coli were carried out to identify the residues possibly involved in the deprotonation and re-protonation of Cys 64 in collaboration with Dr Goedele Roos (Vrije Universiteit Brussel). We showed that Asp 90 is a likely candidate for accepting the proton of Cys 64 and that the hydrogen bond network around Cys 64 can very easily rearrange to stabilise the thiolate anion.
6. We studied the mechanism of hydride transfer in the isopropyl-malate dehydrogenase enzyme in collaboration with the experimental group of Prof. M. Vas (Research Centre for Natural Sciences). The molecular dynamics simulations confirm that the crystal structure obtained by the experimental group contains the two substrates in a favourable orientation for the hydride transfer reaction to occur. Calculations performed in the vacuum predict the activation energy of the reaction to be around 15 kcal/mol and suggests that the hydride transfer reaction and carbon-carbon bond cleavage leading to decarboxylation occurs in a non-synchronous but concerted process. However, it is also likely that within the enzyme the reaction follows a step-wise process.
The project improved the representation of women in research and teaching positions at the Budapest BUTE, where the project was carried out and strengthened the research capacities of the department and led to various collaborative projects with experimental groups within and outside the university and it has strengthened the relationship between BUTE and the University of Bristol.