Novel computational methods for biomechanistic and metallodrug discovery

The growth of molecular modelling and free-energy calculations for drug discovery and development have provided a platform for the work undertaken in this thesis. The known affinity of certain metals for biological targets, such as gold/thiol affinity, has guided this research on the characterisation of interactions involved in both protein function inhibition and non-canonical DNA sequence stabilisation. The ability to calculate microsecond timeframes of our biological targets has allowed for greater understanding of these mechanisms at a molecular level. The research in this thesis is organised into two sections. The first part covers the mechanism of permeation and inhibition of AQP3 via a number of molecular dynamic techniques, such as umbrella sampling and metadynamics. The effects of a Au(III) complex on the protein conformation of AQP3 upon metal binding were investigated using steered molecular dynamics. The pH induced gating mechanism of AQP7 and its effect on both water and glycerol permeation was studied to understand the protein conformational changes involved. The second part investigates the stabilising effect of Au(I) N-heterocyclic carbenes (NHCs) on G-quadruplex DNA structures. The metadynamics calculated free-energy results were then compared to FRET melting assay results to determine the mechanism of interaction. Throughout this work we have shown how the use of in silico methods can enhance our mechanistic knowledge and understanding of biological systems, helping validate and explain experimental results, and vice versa. We have used state of the art molecular dynamics techniques, either rarely or not yet used for such complex systems, thus, furthering the knowledge of our group, and through published work, the wider scientific communit

Aquaporin-driven hydrogen peroxide transport: a case of molecular mimicry?

Aquaporins (AQPs) are membrane proteins that have evolved to control cellular water uptake and efflux, and as such are amongst the most ancient biological “devices” in cellular organisms. Recently, using metadynamics, we have shown that water nanoconfinement within aquaporin channels results into bidirectional water movement along single file chains, extending previous investigations. Here, the elusive mechanisms of H2O2 facilitated transport by the human ‘peroxiporin’ AQP3 has been unravelled via a combination of atomistic simulations, showing that while hydrogen peroxide is able to mimic water during AQP3 permeation, this comes at a certain energy expense due to the required conformational changes within the channel. Furthermore, the intrinsic water dynamics allows for host H2O2 molecule solvation and transport in both directions, highlighting the fundamental role of water nanoconfinement for successful transduction and molecular selection. Overall, the bidirectional nature of the water flux under equilibrium conditions along with the mimicking behavior of hydrogen peroxide during a conductance event introduce a new chemical paradigm never reported so far in any theoretical paper involving any aquaporin isoform

Preclinical musculoskeletal junction testbed: Optimisation of a reproducible skeletal muscle construct [Abstract]

Preclinical musculoskeletal junction testbed: Optimisation of a reproducible skeletal muscle construct [Abstract

Small-scale skeletal muscle constructs for in-vitro musculoskeletal junction preclinical testbed [Abstract]

Small-scale skeletal muscle constructs for in-vitro musculoskeletal junction preclinical testbed [Abstract

A review of the current state of the art of physiologically-based tests for measuring human dermal in vitro bioavailability of polycyclic aromatic hydrocarbons (PAH) in soil

Polycyclic Aromatic Hydrocarbons are classed as Persistent Organic Pollutants, a large group of compounds that share similar characteristics. They are lipophilic, resistant to degradation in the environment and harmful to human and environmental health. Soil has been identified as the primary reservoir for Polycyclic Aromatic Hydrocarbons in the United Kingdom. This study reviews the literature associated with, or is relevant to, the measurement and modelling of dermal absorption of Polycyclic Aromatic Hydrocarbons from soils. The literature illustrates the use of in vivo, in vitro and in silico methods from a wide variety of scientific disciplines including occupational and environmental exposure, medical, pharmaceutical and cosmetic research and associated mathematical modelling. The review identifies a number of practical shortcomings which must be addressed if dermal bioavailability tests are to be applied to laboratory analysis of contaminated soils for human health risk assessment

Unveiling the mechanisms of aquaglyceroporin-3 water and glycerol permeation by metadynamics

Water and glycerol permeation via human AQP3 are described exploiting advanced metadynamics approaches, which enabled to both explore the free energies involved in pore permeation, as well as to achieve a description of the mechanisms with an atomistic level of detail. Moreover, thanks to this technique, a basic water transport mechanism, single‐filed and bidirectional in nature was retrieved, on which glycerol molecules are inserted during channel permeation. Therein, activation energies result from static and dynamic effects involving hydrogen bonds within the water filament and among substrate molecules and pore internal residues

Preclinical musculoskeletal junction testbed: co-culture of 3D tissue engineered skeletal muscle and bone constructs [Abstract]

Preclinical musculoskeletal junction testbed: co-culture of 3D tissue engineered skeletal muscle and bone constructs [Abstract

The mechanism of aquaporin inhibition by gold compounds elucidated by biophysical and computational methods

The inhibition of water and glycerol permeation via human aquaglyceroporin-3 (AQP3) by gold(III) complexes has been studied by stopped-flow spectroscopy and, for the first time, its mechanism has been described using molecular dynamics (MD), combined with density functional theory (DFT) and electrochemical studies. The obtained MD results showed that the most effective gold-based inhibitor, anchored to Cys40 in AQP3, is able to induce shrinkage of pores preventing glycerol and water permeation. Moreover, the good correlation between the affinity of the Au(III) complex to Cys binding and AQP3 inhibition effects was highlighted, while no influence of the different oxidative character of the complexes could be observed

Soil-sebum partition coefficients for high molecular weight polycyclic aromatic hydrocarbons (HMW-PAH)

In vitro high molecular weight polycyclic aromatic hydrocarbons (HMW-PAH) soil-sebum partition coefficients (KSS) were measured for twelve soils collected from former UK gasworks. Concentrations of ∑16 USEPA PAH in the soils ranged from 51 to 1440 mg/kg. Time series extractions (0.5, 1, 2, 4, 8 and 24 h) at skin temperature (32 °C) of HMW-PAH from sebum to soil for two samples were conducted to determine the maximum release time-step. The maximum HMW-PAH release time-step was determined as 4 h, which was subsequently used as the extraction time for the remaining samples. The 4 h extraction for all samples showed HMW-PAH KSS ranging from 0.06 to 1.4 (median = 0.59; mean = 0.59; standard deviation = 0.27; median absolute deviation = 0.29). Evaluation of KSS data for the 4 h extractions showed that soil type and selected HMW-PAH properties (literature based molecular weight and octanol-carbon partition coefficients) affect the amount of HMW-PAH released from soil into sebum