Searching new structural scaffolds for BRAF inhibitors. An integrative study using theoretical and experimental techniques.

The identification of the V600E activating mutation in the protein kinase BRAF in around 50% of melanoma patients has driven the development of highly potent small inhibitors (BRAFi) of the mutated protein. To date, Dabrafenib and Vemurafenib, two specific BRAFi, have been clinically approved for the treatment of metastatic melanoma. Unfortunately, after the initial response, tumors become resistant and patients develop a progressive and lethal disease, making imperative the development of new therapeutic options. The main objective of this work was to find new BRAF inhibitors with different structural scaffolds than those of the known inhibitors. Our study was carried out in different stages; in the first step we performed a virtual screening that allowed us to identify potential new inhibitors. In the second step, we synthesized and tested the inhibitory activity of the novel compounds founded. Finally, we conducted a molecular modelling study that allowed us to understand interactions at the molecular level that stabilize the formation of the different molecular complexes. Our theoretical and experimental study allowed the identification of four new structural scaffolds, which could be used as starting structures for the design and development of new inhibitors of BRAF. Our experimental data indicate that the most active compounds reduced significantly ERK½ phosphorylation, a measure of BRAF inhibition, and cell viability. Thus, from our theoretical and experimental results, we propose new substituted hydroxynaphthalenecarboxamides, N-(hetero)aryl-piperazinylhydroxyalkylphenylcarbamates, substituted piperazinylethanols and substituted piperazinylpropandiols as initial structures for the development of new inhibitors for BRAF. Moreover, by performing QTAIM analysis, we are able to describe in detail the molecular interactions that stabilize the different Ligand-Receptor complexes. Such analysis indicates which portion of the different molecules must be changed in order to obtain an increase in the binding affinity of these new ligands.

New small-size peptides possessing antifungal activity.

The synthesis, in vitro evaluation, and conformational study of a new series of small-size peptides acting as antifungal agents are reported. In a first step of our study we performed a conformational analysis using Molecular Mechanics calculations. The electronic study was carried out using Molecular electrostatic potentials (MEPs) obtained from RHF/6-31G calculations. On the basis of the theoretical predictions three small-size peptides, RQWKKWWQWRR-NH(2), RQIRRWWQWRR-NH(2), and RQIRRWWQW-NH(2) were synthesized and tested. These peptides displayed a significant antifungal activity against human pathogenic strains including Candida albicans and Cryptococcus neoformans. Our experimental and theoretical results allow the identification of a topographical template which can serve as a guide for the design of new compounds with antifungal properties for potential therapeutic applications against these pathogenic fungi.

Structure-antifungal activity relationship of His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 and analogues.

The synthesis, in vitro evaluation and conformational study of His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH(2) and analogues acting as antifungal agents are reported. Among them, His-Phe-Lys-Trp-Gly-Arg-Phe-Val-NH(2) exhibited a moderate but significant antifungal activity against Cryptococcus neoformans, Candida albicans and Candida tropicalis. A theoretical study allows us to propose a biologically relevant conformation for these octapeptides acting as antifungal agents. In addition, these theoretical calculations allow us to determine the minimal structural requirements to produce the antifungal response and can provide a guide for the design of compounds with this biological activity.

Penetratin analogues acting as antifungal agents.

The synthesis, in vitro evaluation, and conformational study of penetratin analogues acting as antifungal agents are reported. Different peptides structurally related with penetratin were evaluated. Analogues of penetratin rich in Arg, Lys and Trp amino acids were tested. In addition, HFRWRQIKIWFQNRRM[O]KWKK-NH(2), a synthetic 20 amino acid peptide was also evaluated. These penetratin analogues displayed antifungal activity against human pathogenic strains including Candida albicans and Cryptococcus neoformans. In contrast, Tat peptide, a well-known cell penetrating peptide, did not show a significant antifungal activity against fungus tested here. We also performed a conformational study by means experimental and theoretical approaches (CD spectroscopic measurements and MD simulations). The electronic structure analysis was carried out from Molecular Electrostatic Potentials (MEP) obtained by using RHF/6-31G ab initio calculations. Our experimental and theoretical results permitted us to identify a topographical template which may provide a guide for the design of new peptides with antifungal effects.

Synthesis and antifungal activity of (Z)-5-arylidenerhodanines.

An efficient microwave-assisted synthesis of new (Z)-5-arylidenerhodanines under solvent-free conditions is described and their in vitro antifungal activity was evaluated following the CLSI (formerly NCCLS) guidelines against a panel of both standardized and clinical opportunistic pathogenic fungi. An analysis of the structure-activity relationship (SAR) along with computational studies showed that the most active compounds (F- and CF(3)-substituted rhodanines) possess high logP values and low polarizability. Mechanism-based assays suggest that active compounds neither would bind to ergosterol nor would produce a damage to the fungal membrane.