Tetraciclinas quimicamente modificadas como inibidores de metaloproteinases de matriz (MMPS): um estudo de estrutura, reatividade, dinâmica e implicações biológicas

Abstract

Matrix Metalloproteinases (MMPs) play a critical role in physiological processes and pathological conditions such as tumor progression and metastasis. In recent years, a number of MMP inhibitors (MMPIs) have been proposed, including the chemically modified tetracyclines (CMTs), which have been evaluated in preclinical cancer models showing promising results. However, until present there were no studies on the structure-activity relationship for these compounds. In this sense, the present thesis focuses on the direct interaction of CMTs with the active site of the MMPs. Density Functional Theory (DFT), molecular docking and molecular dynamics (MD) simulations were accomplished for seven CMT derivatives. Firstly the structure and stability of mimetic molecular models of active site of MMP-2 represented by: [Zn(LHn)(H2O)2]2+x (n = 0, 1, 2 and x = -2, -1, 0) and [Zn(L)(His)3], where L=CMT-1, CMT-3, CMT-4, CMT-7 and CMT-8, were calculated at DFT level. The solvent and pH effects were also evaluated. The results suggested that the coordination at the O11-O12 site (site VI) was quite favorable to CMT-3 (log β = 15,9) and CMT-8 (log β = 21,3). This initial analysis showed that the activity of CMTs as inhibitors of MMP is directly related to its structure. In the second stage of the study, we investigated the interaction of CMTs directly with the active site of MMP-2 (1QIB). New sets of parameters were proposed for structural and catalytic zinc atoms in order to study MMPs and their complexes by means of the AMBER force field. The CMTs ligands were optimized at the B3LYP/6-31G(d) and docked onto the active site of MMP-2. The best solutions were minimized and subjected to 12ns MD simulation. The binding free energy was calculated using the methods of thermodynamic integration and MM-PBSA. The main results showed that the CMTs bind to the catalytic zinc of the MMP-2 enzyme through the O11-O12 moiety (site VI) as proposed experimentally. The exception was the CMT-3 analogue which is involved in hydrophobic contacts and van der Waals interactions with amino acids of the S1’ pocket in the MMP-2 enzyme and also interacts with the Zn (II) by O11. The binding energy calculated in solution predicts the CMT-3 complexes as the most favorable, followed by the CMT-7 and CMT-8 analogues, whose energy difference is within 3 kcal mol-1. These results suggest that the occupancy of S1’ pocket is an important molecular feature for biological response of CMT-3, while the other CMTs do not enter the hydrophobic tunnel of the enzyme due to the steric hindrance. The results presented in this thesis are the first step towards understanding the mechanism of CMTs as MMP inhibitors at a molecular level and could be a significant contribution to the understanding of experimental observations, especially the biological tests and the CMTs action mechanism as MMP inhibitors, allowing the design of new analogues with enhanced biological potency.