abstract
A series of fluorinated tripodal tris-thioureas function as highly active anion transporters across lipid bilayers and cell membranes. Here, we investigate their mechanism of action using anion transport assays in cells and synthetic vesicles and molecular modelling of transporter-lipid interactions. When compared with non-fluorinated analogues, fluorinated compounds demonstrate a different mechanism of membrane transport because the free transporter cannot effectively diffuse through the membrane. As a result, in H+/Cl- cotransport assays, fluorinated transporters require the presence of oleic acid to form anionic oleate complexes for recycling of the transporter, whereas non-fluorinated analogues readily diffuse through the membrane as free transporters and show synergistic transport with the proton transporter gramicidin. Molecular dynamics simulations revealed markedly stronger transporter-lipid interactions for fluorinated compounds compared with non-fluorinated analogues and hence, higher energy barriers for fluorinated compounds to cross the membrane as free transporters. With use of appropriate proton transporters to ensure measurement of the correct rate-limiting steps, the transport rates determined in synthetic vesicle assays show excellent agreement with the anion transport rates determined in cell-based assays. We conclude that integration of computational and experimental methods provides a strategy to optimise transmembrane anion transporter design for biomedical applications.
keywords
LIPOPHILICITY; CELL
subject category
Chemistry
authors
Spooner, MJ; Li, HY; Marques, I; Costa, PMR; Wu, X; Howe, ENW; Busschaert, N; Moore, SJ; Light, ME; Sheppard, DN; Felix, V; Gale, PA
our authors
Igor Oliveira Marques
Researcher
Pedro Costa
Research Fellowship
Vítor Félix
Associate Professor with AggregationProjects
Anion transmembrane transport promoted by drug-like molecules: building a library of anion carriers inspired in Ataluren (PTC124) (PTDC/QEQ-SUP/4283/2014)
CICECO - Aveiro Institute of Materials (UID/CTM/50011/2013)
acknowledgements
We thank A. S. Verkman for the generous gift of YFP-expressing FRT cells and A. P. Davis and colleagues for help and advice. This work was supported by EPSRC grants EP/J009687/1 and EP/J00961X/1. P. A. G. thanks the University of Sydney and the Australian Research Council (DP180100612) for funding. V. F. was supported by projects PTDC/QEQ-SUP/4283/2014 (POCI-01-0145-FEDER-016895), CICECO - Aveiro Institute of Materials (UID/CTM/50011/2013), financed by National Funds through the FCT/MEC and co-financed by QREN-FEDER through COMPETE under the PT2020 Partnership Agreement. M. J. S. was supported by an EPSRC DTG studentship and a Doctoral Prize Award from Southampton University and the EPSRC (EP/N509747/1) and I. M. by FCT PhD scholarship SFRH/BD/87520/2012. EPSRC-funded data in this manuscript are available through the University of Bristol data repository (data.bris).