Phorate (O,O-diethyl S-ethylthiomethyl phosphorodithioate) is a toxic organophosphate anticholinesterase insecticide. Organophosphate insecticides can cause respiratory depression and seizures due to acetylcholinesterase inhibition. Inhibited acetylcholinesterase cannot break down the neurotransmitter, acetylcholine; thus, causing an overload of acetylcholine in synapses and neuromuscular junctions. Oxidative desulfuration, from metabolism by cytochrome P450 enzymes, converts the P=S phosphorothionate group on phorate to the P=O oxon group. Electrophilic oxon groups attack the active site on acetylcholinesterase, inducing the toxicity associated with organophosphate insecticides. Possible further bioactivation to phorate-oxon-sulfoxide and phorate-oxon-sulfone near the site of acetylcholinesterase in the brain may increase acetylcholinesterase inhibitory potency. Adult male Sprague-Dawley rat brain and liver microsomes were used to determine the proportions of the phorate metabolites formed through bioactivation. Phorate-sulfoxide was produced in much greater proportion than any other metabolite, which may contribute to the delay observed in phorate toxicity as it takes longer to produce phorate-oxon, phorate-oxon-sulfoxide, or phorate-oxon-sulfone metabolites.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Clomazone is a herbicide used for grass weed control in paddy rice. However, label rate may not provide efficient control for some weed species. For this reason many times it is necessity to increase the rate to improve weed control efficiency. Clomazone selectivity in rice is affected for soil type, the rate of the herbicide and the cultivar used, been necessary the use of safener to protect the plants when high rates are used. Though, it is necessary to adjust those factors for better use of this technology. For this reason, it were conducted two greenhouse experiments, the first with the objective of quantify the selectivity of clomazone in sand and clay soils, to quantify the protection of the dietholate on rice plants in these soil conditions. The second experiment had the objective of verify if phorate provides similar protective effect as dietholate provides for clomazone. The results allowed to conclude that the cultivars IRGA 409 and IRGA 417 are equally sensible to clomazone. In regards of soil type, clomazone cause higher injury to rice cultivated in sandy soil. The application of dietholate allows the increase of dose of clomazone. As much in the sandy soil as in the clay soil, dietholate as treatment of seeds allowed increase of the dose of clomazone in up to three times the dose in relation to the treatment without dietholate. Organophosphate phorate is capable to act as safener for rice when clomazone is applied, with similar safening ability as dietholate, conferring to the crop capability to support higher rates of clomazone. / O herbicida clomazone é utilizado para controle de poáceas na cultura do arroz irrigado. Porém, sua dose de registro para essa cultura não proporciona controle eficiente para determinadas espécies de plantas daninhas. Assim, há a necessidade de elevar sua dose para melhorar a eficácia sobre estas espécies. A seletividade do clomazone no arroz irrigado está condicionada ao tipo de solo, à dose do herbicida e a cultivar semeada, necessitando-se o uso de protetor de plantas quando se praticam altas doses de clomazone. Entretanto, há a necessidade de adequar esses fatores para melhor uso da tecnologia. Em vista do exposto, instalaram-se dois experimentos em casa de vegetação. O primeiro, com o objetivo de quantificar a seletividade de doses de clomazone em solo com característica arenosa e argilosa e verificar a ação do protetor de plantas dietholate sobre cultivares de arroz. O segundo experimento objetivou verificar se o organofosforado phorate proporciona efeito protetor semelhante àquele proporcionado pelo dietholate. Os resultados permitiram concluir que as cultivares IRGA 409 e IRGA 417 são igualmente sensíveis ao clomazone, e respondem igualmente aos tratamentos com dietholate. Tanto no solo arenoso como no solo argiloso, o dietholate como tratamento de sementes permitiu aumento da dose de clomazone em até três vezes a dose em relação ao tratamento sem dietholate. O phorate apresenta papel protetor na cultivar de arroz IRGA 417, contra a ação do herbicida clomazone, de forma equivalente ao protetor dietholate.

Organophosphates are insecticides which result in acute adverse signs when exposed at toxic doses by animals and lead to death if left untreated. The current treatment for organophosphate toxicity includes atropine and the federally approved oxime 2-PAM. However, 2-PAM is not very effective at crossing the blood brain barrier which results in prolonged inactivation of acetylcholinesterase, which is the primary target of organophosphates, in the brain even after administration. The novel oximes, Oxime 15 and Oxime 20, are able to cross the blood brain barrier and reactivate the inhibited acetylcholinesterase. In this experiment with six animal species frequently used in toxicity studies, they were proven to be just as effective and sometimes better than 2-PAM at reactivating acetylcholinesterase or butyrylcholinesterase inhibited by paraoxon, chlorpyrifos-oxon, phorate-oxon, or dicrotophos. The detoxication enzymes butyrylcholinesterase, carboxylesterase, and paraoxonase were also studied as potential influences of the toxicity of the organophosphates in these different species.

In this thesis, we have employed two computational methods, molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) MD simulations with umbrella sampling (US), to gain insights into the molecular mechanism governing the molecular recognition and reactivity in several protein-ligand complexes. Three systems involving protein-ligand interactions are examined in this dissertation utilizing well-established computational methodologies and mathematical modeling. The three proteins studied here are acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1). These enzymes are known to interact with a variety of ligands. AChE dysfunction caused by organophosphorus (OP) chemicals is a severe hazard since AChE is a critical enzyme in neurotransmission. Oximes are chemical compounds that can reactivate inhibited AChE; hence in the development of better oximes, it is critical to understand the mechanism through which OPs block AChE. We have described the covalent inhibition mechanism between AChE and the OP insecticide phorate oxon and its more potent metabolites and established their free energy profiles using QM/MM MD-US for the first time. Our results suggest a concerted mechanism and provide insights into the challenges in reactivating phorate oxon inhibited AChE. Reactivating BChE is another therapeutic approach to detoxifying circulating OP molecules before reaching the target AChE. We explored the covalent modification of BChE with phorate oxon and its metabolites using hybrid quantum mechanics/molecular mechanics (QM/MM) umbrella sampling simulations (PM6/ff14SB) for the inhibition process. Our results reveal that the mechanism is distinct between the inhibitors. The PM6 methodology is a good predictor of these compounds' potency, which may efficiently help study OPs like phorate oxon with larger leaving groups. Finally, we investigated the interactions between Peptidyl-prolyl isomerase (PPIase), which consists of a peptidyl isomerase (PPIase) domain flexibly tethered to a smaller Trp-Trp (WW) protein-binding domain, and chimeric peptides based on the human histone H1.4 sequence (KATGAApTPKKSAKW), as well as the effects on inter-domain dynamics. Using explicit solvent MD simulations, simulated annealing, and native contact analysis, our modeling sugget that the residues in the N-terminal immediate to the pSer/Thr Pro site connect the PPIase and WW domains via a series of hydrogen bonds and native contacts.