Targeted delivery of antigen processing inhibitors to antigen presenting cells via mannose receptors.
Improved chemical inhibitors are required to dissect the role of specific antigen processing enzymes and to complement genetic models. In this study we explore the in vitro and in vivo properties of a novel class of targeted inhibitor of aspartic proteinases, in which pepstatin is coupled to mannosylated albumin (MPC6), creating an inhibitor with improved solubility and the potential for selective cell tropism. Using these compounds, we have demonstrated that MPC6 is taken up via mannose receptor facilitated endocytosis, leading to a slow but continuous accumulation of inhibitor within large endocytic vesicles within dendritic cells and a parallel inhibition of intracellular aspartic proteinase activity. Inhibition of intracellular proteinase activity is associated with reduction in antigen processing activity, but this is epitope-specific, preferentially inhibiting processing of T cell epitopes buried within compact proteinase-resistant protein domains. Unexpectedly, we have also demonstrated, using quenched fluorescent substrates, that little or no cleavage of the disulfide linker takes place within dendritic cells. This does not appear to affect the activity of MPC6 as an inhibitor of cathepsins D and E in vitro and in vivo. Finally, we have shown that MPC6 selectively targets dendritic cells and macrophages in spleen in vivo. Preliminary results suggest that access to nonlymphoid tissues is very limited in the steady state but is strongly enhanced at local sites of inflammation. The strategy adopted for MPC6 synthesis may therefore represent a more general way to deliver chemical inhibitors to cells of the innate immune system, especially at sites of inflammation.
Raiber
Eun-Ang
EA
Dept of Chemistry, UCL, London, UK.
Tulone
Calogero
C
Division of Infection and Immunity, UCL, London, UK.
Zhang
Yanjing
Y
Division of Infection and Immunity, UCL, London, UK.
Martinez-Pomares
Luisa
L
School of Molecular Medical Sciences, University of Nottingham, UK.
Steed
Emily
E
Dept of Chemistry, UCL, London, UK.
Sponaas
Anna M
AM
Division of Parasitology, National Institute for Medical Research, London, UK.
Langhorne
Jean
J
Division of Parasitology, National Institute for Medical Research, London, UK.
Noursadeghi
Mahdad
M
Division of Infection and Immunity, UCL, London, UK.
Chain
Benjamin M
BM
Division of Infection and Immunity, UCL, London, UK.
Tabor
Alethea B
AB
Dept of Chemistry, UCL, London, UK.
D000818:Animals; D017951:Antigen Presentation / Q000187:drug effects* / Q000276:immunology; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002402:Cathepsin D / Q000037:antagonists & inhibitors; D003713:Dendritic Cells / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte / Q000378:metabolism; D047468:Immunoprecipitation; D037181:Lectins, C-Type / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000276:immunology / Q000378:metabolism; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010436:Pepstatins / Q000138:chemical synthesis / Q000276:immunology / Q000494:pharmacology; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism; D013154:Spleen / Q000166:cytology / Q000276:immunology / Q000378:metabolism
D018984:Epitopes, T-Lymphocyte; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D010436:Pepstatins; D011480:Protease Inhibitors; D011956:Receptors, Cell Surface; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D; C533923:Ctsd protein, mouse; D008358:Mannose; C031375:pepstatin