Estudo farmacofórico sobre isomanídeos peptidomiméticos inibidores das calicreínas teciduais humanas 5 e 7

Abstract

The fifteen human tissue kallikrein (KLKs) are endopeptidases, whose genes belong to the largest uninterrupted cluster of enzymes in genome, and named 1-15 according to their order of discovery. Despite the common catalytic domain (serine protease), they have relatively little phylogenetic relationship with plasma kallikrein. They share the conserved catalytic triad residues of the family to which they belong, His57, Asp102 and Ser195. However, their activities (preference for different substrates) are classified according to the nature of the residue 189 (representative of the S1 subsite of the active site), as chymotrypsin-like or trypsin-like. Human KLKs can be subdivided into various tissue-specific groups, which fulfill numerous tasks throughout life. Disorders that occur due altered regulation of kallikreins activities vary according to where their genes are expressed. KLK5 and 7 present tryptic and quimotryptic activities, respectively. Both are usually found in many tissues, but they are specifically abundant in spinous and granular layers of the epidermis, where they degrade the proteins of corneodesmossomos, leading to desquamation. In addition to playing role in skin disorders such as atopic dermatitis, psoriasis and Netherton's syndrome, the two enzymes are able to digest multiple components of the extracellular matrix, leading to metastatic spread of tumor cells in various types of cancers such as epithelial ovarian carcinoma and pancreatic adenocarcinoma. KLKs are often maintained inhibited by a variety of endogenous inhibitors, whichare in fine equilibrium with the enzymatic activity, essential for tissue homeostasis. Several strategies have been applied in order to design inhibitors to serine proteases. Isomannides are compounds derived from D-mannitol dehydration, which possess a characteristic bicyclic structure. In our work, a computational pharmacophoric study was done with compounds derived from isomannide, divided into two series: amides and esters. In view of previous experimental results, it was known that the mechanism of inhibition of those compounds range from reversible competitive to reversible non-competitive. The search through CASTp online server pointed probable exosites for interaction in KLK5 and KLK7. Such exosites, in addition to the active sites, were used as receptor pockets for the compounds separately by molecular docking method. General positions of the docking inhibitors were not significantly close to those in the kinetic assays. That result was expected. Conformational analysis of torsion angles didn’t contribute to standardization of isomannides affinities to receptors, mainly due the large conformational freedom of those compounds. In other hand, it was observed that the non-competitive inhibitor, AM04 showed relatively high score in one exosite, topographically more likely to influence conformation disposition of the active site residues of KLK5. The other non-competitive compound, AM05, showed lower score for the exosite of greater identity with the active site, and score high for the others allosteric pockets. That result corroborates with probable presence of one or several allosteric sites with significant activity on the enzyme surface, whichcan be extended to other kallikreins and KLK7 through further studies. Additionally, electronic structure DFT calculations of inhibitors showed that, in KLK5, the HOMO of AM04 and LUMO of AM05 are favorably correlated to their interactions with allosteric sites. Other prediction methods approach, such as molecular dynamics, would give details about the influence of those ligands involvement in accommodation of other compounds in the active sites, primarily in KLK5. The development of inhibitors for KLK5 and KLK7 may also help elucidate the pathophysiological roles of kallikrein and the designing of new drugs.