title: Identification of novel small molecule inhibitors of adenovirus gene transfer using a high throughput screening approach.
abstract: Due to many favourable attributes adenoviruses (Ads) are the most extensively used vectors for clinical gene therapy applications. However, following intravascular administration, the safety and efficacy of Ad vectors are hampered by the strong hepatic tropism and induction of a potent immune response. Such effects are determined by a range of complex interactions including those with neutralising antibodies, blood cells and factors, as well as binding to native cellular receptors (coxsackie adenovirus receptor (CAR), integrins). Once in the bloodstream, coagulation factor X (FX) has a pivotal role in determining Ad liver transduction and viral immune recognition. Due to difficulties in generating a vector devoid of multiple receptor binding motifs, we hypothesised that a small molecule inhibitor would be of value. Here, a pharmacological approach was implemented to block adenovirus transduction pathways. We developed a high throughput screening (HTS) platform to identify small molecule inhibitors of FX-mediated Ad5 gene transfer. Using an in vitro fluorescence and cell-based HTS, we evaluated 10,240 small molecules. Following sequential rounds of screening, three compounds, T5424837, T5550585 and T5660138 were identified that ablated FX-mediated Ad5 transduction with low micromolar potency. The candidate molecules possessed common structural features and formed part of the one pharmacophore model. Focused, mini-libraries were generated with structurally related molecules and in vitro screening revealed novel hits with similar or improved efficacy. The compounds did not interfere with Ad5:FX engagement but acted at a subsequent step by blocking efficient intracellular transport of the virus. In vivo, T5660138 and its closely related analogue T5660136 significantly reduced Ad5 liver transgene expression at 48 h post-intravenous administration of a high viral dose (1×10¹¹ vp/mouse). Therefore, this study identifies novel and potent small molecule inhibitors of the Ad5 transduction which may have applications in the Ad gene therapy setting.
authors.affiliation: British Heart Foundation, Glasgow Cardiovascular Research Centre, Institute of Cardiovascular and Medical Sciences, University of Glasgow, 126 University Place, Glasgow G12 8TA, UK.
authors.forename: Margaret R
authors.initials: MR
authors.lastname: Duffy
authors.forename: Alan L
authors.initials: AL
authors.lastname: Parker
authors.forename: Eric R
authors.initials: ER
authors.lastname: Kalkman
authors.forename: Katie
authors.initials: K
authors.lastname: White
authors.forename: Dmytro
authors.initials: D
authors.lastname: Kovalskyy
authors.forename: Sharon M
authors.initials: SM
authors.lastname: Kelly
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Baker
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D045744:Cell Line, Tumor; D005170:Factor X / Q000037:antagonists & inhibitors*; D005822:Genetic Vectors; D057166:High-Throughput Screening Assays; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D054852:Small Molecule Libraries; D014161:Transduction, Genetic
chemical_list: D054852:Small Molecule Libraries; D005170:Factor X; D008156:Luciferases