Desenvolvimento e aplicação de técnicas de amostragem aumentada dependente do pH na optimização da tecnologia pHLIP como marcador tumoral

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In silico tumor-targeting technologies for the evasion of acidity-induced multidrug resistance

Publication . Silva, Tomás F.D.; Machuqueiro, Miguel Ângelo dos Santos; Viçosa, Diogo Ruivo dos Santos Vila

The physiology of tumors is tied to MDR mechanisms that hamper chemotherapeutic effects, particularly passive membrane crossing compounds, like hydrophobic Lewis base drugs. Although the lysosomal entrapment phenomena remains to be fully understood, this pH-dependent MDR mechanism induces drug sequestration in the acidic lysosomal lumen. Overcoming the MDR requires multi-pronged therapies, which often overlook an ubiquitous tumor trait: the extracellular acidity of the tumor microenvironment (TME). To address this, pHLIP peptides have emerged as an acidity-selective technology for tumor-targeting drug delivery. We focused on refining our protocols with enhanced sampling techniques and tumor-like features to improve the predictive abilities of the CpHMD-L methodology and augment the realism of these biomolecular models, thus bridging the gap to in vivo and cellular conditions. The optimized protocol coupled the CpHMD-L method with a pHRE scheme, providing a robust baseline. Then, we applied the protocol to study the diverging therapeutic efficiency of the wt and an over-performing Var3 peptide. A novel implementation of a pH gradient CpHMD-L method successfully reproduced experimental performances, thus elucidating pivotal residues electrostatic networks that dictate peptides thermodynamic stability in TME conditions. A multi-peptide study highlighted the remarkable effects of permuting arginines in modulating the local vicinity of key aspartates. These findings heavily correlate with their tumor-targeting performance, supporting more rational and in silico-based approaches to peptide design. Finally, the pH-dependent mechanism of lysosomal entrapment was modelled, hinting at the important role of acidity in Lewis base drugs membrane intercalation. Additional pH-dependent permeability calculations, using a novel US-CpHMD method, identified the TME acidity as an additional MDR defense mechanism that impairs clinical efficiency. It also revealed an intrinsic flaw of these compounds, since they preferably target healthy cells. These findings have important implications in rational drug design, especially of conjugated therapies with pHLIP-like drug delivery systems to overcome these challenges.

2023-01Tese de doutoramento

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Fundação para a Ciência e a Tecnologia

Número da atribuição

SFRH/BD/140886/2018