MOLECULAR MODELING STUDY OF VARIOUS SYNTHETIC JHAS CONTAINING VARIED FUNCTIONALITY

ABSTRAC

Computational Analysis of Type 3 Iodothyronine Deiodinase: Potential Inhibitors, Substrate Binding, and Dimer Structure

Thyroid hormones (THs) in mammalian tissues are crucial for development and maintaining metabolic homeostasis. Iodothyronine deiodinases (Dios) remove iodines from THs by a selenocysteine (Sec) residue, which either activates or inactivates them. Halogen bonding (XB) has been proposed to describe the interaction between the Se and I atoms of the T4-Dio complex. Disruption of TH homeostasis by xenobiotics, such as polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) can cause deleterious effects on the endocrine system. Experimental studies have indicated that PBDEs and PCBs could disrupt TH homeostasis by inhibiting Dio through XB formation. However, no current quantitative study exists that compares the relative strengths of PBDE and PCB XB strengths. Trends in XB interactions of a small model of the active site (MeSe-) with THs and potential inhibitors PBDEs and PCBs are analyzed using density functional theory (DFT). In agreement with trends in XB, XB favorability follows in the order of THs \u3e PBDEs \u3e PCBs (i.e., I \u3e Br \u3e Cl). Highly brominated PBDEs show similar interaction energies to THs, suggesting possible inhibition and debromination of these compounds. Schweizer et al. solved the crystal structure of the monomeric catalytic domain of Dio3. However, Dio3 must dimerize in order to perform catalytic deiodination, and no structural data currently exists regarding a Dio3 dimer. A debate between two groups has been ongoing in the literature regarding how Dio3 undergoes dimerization. Proposed Dio3 dimer structures by Sagar and Schweizer may be attributed to the observance of A-type and B-type dimers within peroxiredoxins (Prxs). Sequential comparisons using Clustal Omega of Dio3 to known A-type and B-type dimers show the B-type dimerization dimerization is more plausible for Dio3. In silico protein-protein docking databases SymmDock and GalaxyRefineComplex were employed to successfully construct a Dio3 dimer based upon the B-type description. The refined Dio3 dimer was subject to MD simulations to test for the stability of the dimer. MMGBSA calculations show formation of the dimer is stable and interdimer interactions between the β-sheets and α-helices stabilize the dimer

Structure and Function Studies of Insect Carboxylesterases

Insect carboxylesterases (CBEs) have great importance in insect biology and play a crucial role in the ever-increasing occurrence of insecticide resistance. This resistance to insecticides (e.g. organophosphates) leads to downstream effects on agriculture and to human health (through reduced crop production and the spread of insect-borne diseases). Structural and functional studies of insect CBEs are vital for combating insecticide resistance, finding new potential targets and in providing insight into the evolution of new protein function. The work described in this thesis contributes to the overall understanding of the structure, function and evolution of insect CBEs. Chapter 1 is the introduction into the structure, function and evolution of insect CBEs. In particular, the classification of CBEs is discussed, as well, the current understanding of the role CBEs play in insect species. Chapter 2 describes the evolution of new oligomeric structure in the CBE αE7 from Lucilia cuprina and shows that higher order thermostable oligomers can be selected for during evolution. This work affirms the importance of oligomers in evolution to maintain or increase protein stability and demonstrates structure/activity trade-offs that are observed throughout enzyme evolution. Chapter 3 describes structural studies into the CBE esterase-6 (EST6) from Drosophila melanogaster - a CBE that has shown to be important for the reproductive success of Drosophila species. The enzyme has a unique active site entry for insect CBEs, which results in a narrow and shortened active site. Docking simulations in combination with kinetic analyses show that the enzyme is a probable odorant-degrading enzyme. It also indicates that EST6 does not directly interact with the sex pheromone 11-cis vaccenyl acetate (cVA), which contradicts the previously held belief that it is the main substrate of the enzyme. The evolution of CBEs involved in insecticide resistance in several species is discussed in Chapter 4. α-esterase orthologs from different dipteran species were analysed to discern important factors for the evolution of qualitative insecticide resistance and the signifiance of evolutionary contingency. A large variation in binding and turnover for the organophosphate compound was observed with the introduction of the Gly137Asp mutation into the orthologs. Given the similarity in the predicted structures of the orthologs, it suggests that second and third shell mutations are important in mediating the catalytic effects conferred by the Gly137Asp mutation in the orthologs. Chapter 5 discusses the development and testing of new inhibitors to treat the symptoms of Alzheimer’s disease, through molecular docking simulations. Galanthamine derivatives showed no significant binding to AChE, however, the tested marinoquinoline derivatives displayed a large variation in affinity for AChE. Chapter 6 summarises the conclusions that these studies have allowed us to make regarding the structures, functions and evolution of insect CBEs. The chapter finishes with a discussion of future work that could be undertaken to extend these findings and further develop our understanding of this important enzyme family

Scientific Opinion on the hazard assessment of endocrine disruptors: Scientific criteria for identification of endocrine disruptors and appropriateness of existing test methods for assessing effects mediated by these substances on human health and the environment

Upon request of the European Commission, the Scientific Committee (SC) of the European Food Safety Authority reviewed existing information related to the testing and assessment of endocrine active substances (EASs) and endocrine disruptors (EDs). This work was conducted by a working group of experts in endocrinology, risk assessment and toxicology, together with observers from other EU agencies, namely EMA, ECHA and EEA. To distinguish between EDs and other groups of substances with different modes of action, it was concluded that an ED is defined by three criteria: the presence of i) an adverse effect in an intact organism or a (sub)population; ii) an endocrine activity; and iii) a plausible causal relationship between the two. As scientific criteria for adversity have not been generally defined, specific criteria for endocrine disrupting effects could not be identified. Hence, expert judgement is required to assess on a case-by-case basis the (eco)toxicological relevance of changes at the molecular to individual and/or (sub)population level following exposure to an EAS. The SC concluded that a reasonably complete suite of standardised assays for testing the effects of EASs is (or will soon be) available for the oestrogenic, androgenic, thyroid and steroidogenic modalities in mammals and fish, with fewer tests for birds and amphibians. Shortcomings in current tests and for other endocrine modalities and species were reviewed. Critical effect, severity, (ir)reversibility and potency aspects are part of the hazard characterisation of EDs. To inform on risk and level of concern for the purpose of risk management decisions, risk assessment (taking into account hazard and exposure data/predictions) makes best use of available information. Levels of concern are not determined exclusively by risk assessment but also by protection goals set by the risk management