RESUMO
Malaria is a life-threatening disease spread by mosquitoes. Plasmodium falciparum M1 alanyl aminopeptidase (PfM1-AAP) is a promising target for the treatment of malaria. The recently solved crystal structures of PfM1-AAP revealed that the buried active site can be accessed through two channel openings: a short N-terminal channel with the length of 8 Å and a long C-terminal channel with the length of 30 Å. It is unclear, however, how substrates and inhibitors migrate to the active site and a product of cleavage leaves. Here, we study the molecular mechanism of substrate and inhibitor migration to the active site and the product release using steered molecular dynamics simulations. We identified a stepwise passage of substrates and inhibitors in the C-terminal channel of PfM1-AAP, involving (I) ligand recognition at the opening of the channel, (II) ionic translation to the 'water reservoir', (III) ligand reorientation in the 'water reservoir' and (IV) passage in a suitable conformation into the active site. Endorsed by enzymatic analysis of functional recombinant PfM1-AAP and mutagenesis studies, our novel ligand-residue binding network analysis has identified the functional residues controlling ligand migration within the C-terminal channel of PfM1-AAP. Furthermore, from unbinding simulations of the Arg product we propose a charge repulsion as the driving force to expel the product out from the N-terminal channel of PfM1-AAP. Our work paves the way towards the design of a novel class of PfM1-AAP inhibitors based on preventing substrate entry to the active site.
Assuntos
Aminopeptidases , Simulação de Dinâmica Molecular , Plasmodium falciparum/enzimologia , Proteínas de Protozoários , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/química , Aminopeptidases/genética , Aminopeptidases/metabolismo , Domínio Catalítico , Biologia Computacional , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
INTRODUCTION: Accurate medical documentation is important in the perioperative period, ensuring the safe transfer of information between teams involved in the surgical patient's care. This has been highlighted by multiple standards of care guidelines within the United Kingdom. The use of standardized pre-templated documents has displayed significant success in minimizing errors during the admission and operative stages. The aim of this study is to evaluate whether a similar proforma for the post-operative stage is successful in orthopedic patients. METHODS: A retrospective review of 25 consecutive orthopedic elective patients was conducted during the first cycle. Exclusion criteria included patients who were under 16, day case procedures, and admission due to trauma. The second cycle consisted of a prospective review of 25 patients a month following the implementation of the new proforma. Both cycles were scored against 10 inclusion parameters as outlined by national guidelines. RESULTS: Implementation of the proforma resulted in a significant improvement in post-operative note compliance. A total of six parameters showed a statistically significant improvement (p<0.05). This included wound assessment (58.3%-100%, p<0.001), post-operative imaging (37.5%-92%, p<0.001), neurovascular assessment (83.3%-100%, p=0.017), National Early Warning Score (25.0%-100%, p<0.001), venous thromboembolism prophylaxis (29.2%-96.0%, p<0.001), and antibiotic administration (4.2%-84.0%, p<0.001). CONCLUSIONS: Monitoring of important clinical parameters significantly improved following the implementation of the post-operative proforma. These results will hopefully cause the introduction of other proformas in other surgical specialties and other units.