Untitled

Funder

Organizational Unit

Publications

Tackling solvation and permeability using computational methods

Publication . Fortuna, Andreia; Costa, Paulo Jorge Ferreira de Matos; Florindo, Pedro Ricardo da Rocha

Noncovalent interactions are essential for several biological processes, such as solvation and drug absorption. In this context, halogen bonds (XBs) are often overlooked despite their relevance in biological systems. Indeed, halogenation is a common strategy to improve drug pharmacokinetics, with many halogenated compounds in clinical use; however, the anisotropic nature of halogen is often not considered in in silico approaches. In this work, the impact of noncovalent interactions in solvation, with a special focus on XBs, was studied using two approaches: an implicit (faster) and an explicit solvent (more detailed) model. We provided optimized parameters, namely halogen PB radii (ropt), and validated the use of extra-points (EP), a simple strategy to account for the halogen anisotropy using empirical force fields, for MD simulations. This work showed that describing the halogen anisotropy is important even for the weakest XB donors, leading to an improvement of the calculated hydration free energies. Since desolvation plays an important role in protein-ligand systems, the use of optimized parameters was also assessed in the determination of binding free energies, with the description of halogen anisotropy in combination with ropt, achieving higher correlations with the experimental values. In the context of membrane permeability and noncovalent interactions, the literature is biased towards hydrogen bonds (HBs). To tackle this issue, the study of other noncovalent interactions was assessed for six currently used halogenated drugs, providing valuable insights about the use of EPs and the determination of the membrane permeability coefficients, showing that the use of this charge model does not impair the sampling of HBs. In addition, the synthesis of a library of structurally identical compounds was undertaken aiming to study the substituent effect on membrane permeability. The validation of a proper procedure to measure their permeability in Caco-2 cells is ongoing.

2024-12-18Doctoral thesis

Embargoed access

Expanding the toolbox of four-membered rings to study Serine hydrolases

Publication . Félix, Rita; Moreira, Rui Ferreira Alves; Afonso, Carlos Alberto Mateus; Schofield, Christopher Joseph

Serine hydrolases comprise nearly 1% of all proteins in mammalian cells and play vital roles in relevant pathologies such as cancer and chronic inflammatory diseases. Human Neutrophil Elastase (HNE), a serine hydrolase expressed in polymorphonuclear neutrophiles, is present in the tumour microenvironment and recent studies have shown it promotes tumour proliferation and metastasis. The role of HNE in the tumour microenvironment strongly suggests that this enzyme is an important therapeutic target for cancer therapy, and it has also the potential to be used as a biomarker. Thus, is of utmost importance to develop chemical tools capable of rigorously analyse the expression of HNE in tissues. This project was designed to address this goal by preparing activity-based probes (ABPs) that react covalently with the enzyme target through a reactive chemical group (also defined as warhead). By incorporating different tags, ABPs can be used for detection by fluorescence or protein separation for proteomic studies. In particular, we focused on quenched activity-based probes (qABPs). These are also called Förster resonance energy transfer (FRET) probes, in which the activation by the enzymatic machinery increases the quencher and fluorophore distance enormously and produces an intensiometric readout useful to develop “turn-on” sensors. In the present work novel qABPs with different “turn-on” mechanisms were designed and synthesised, and the ability of the probes to detect and target HNE in biological media was also analysed. Using two different strategies, we started by optimising a synthetic strategy to obtain a series of qABPs using a β-lactam ring as a warhead that promotes the release of the quencher through a suicide-type mechanism of activation. The photophysical properties of these compounds were evaluated and the fluorescence quantum yield was calculated before and after activation. In general, the probes presented low fluorescence that increases significantly after activation. All qABPs showed displayed IC50 against HNE lower than 0.5 µM. The probes were incubated with proteomes of two different cell lines that were spiked with HNE to simulate an overexpression and type II probes could selectively target HNE even when the concentration of the spiked was decreased. Moreover, the probe that presented the best photophysical and biological properties was incubated with human neutrophils, and it was possible to observe that the probe was internalized into the cells and showed a fluorescence signal, probably by reaction with HNE. While the structural requirements for acylation and sulfonylation of catalytic amino acid residues by four-membered ring probes, phosphonylation remains largely unexplored. The second part of this thesis reports the efforts to develop ABPs based on phospholactam warheads. Four-membered phospholactams proved to be extremely challenging from a synthetic point-of-view, suggesting that these probes would be too reactive to develop useful ABPs. In contrast, we were able to synthesise a γ-phospholactam probe containing a “clickable” alkyne handle for chemical biology applications. This compound will be further tested against serine hydrolases to understand the potency and selectivity. Also, crystallographic studies will be performed to elucidate the phosphonylation of PPE by a γ-phospholactam. In summary, this thesis reports the synthesis of novel qABPs with different activation mechanisms, different quenchers, fluorophores, and linkers. Analysis of the fluorescence properties and the selectivity observed in-gel experiments showed that the synthesised compounds have excellent potential to detect HNE in biological matrices and to understand the role of this target in the tumour microenvironment. Moreover, we also report the synthesis of completely novel probes using as a warhead a phospholactam ring.

2024-12Doctoral thesis

Embargoed access