Abstract
BackgroundCleavable linkers, which are specifically cleaved by defined conditions or enzymes, are powerful tools that can be used for various purposes. Amongst other things, they have been successfully used to deliver toxic payloads as prodrugs into target tissues. In this work novel linker sequences targeting meprin β, a metalloprotease expressed in the kidney brush-border membrane, were designed and included in the sequence of three radiolabelled exendin-4 derivatives. As radiolabelled exendin-4 derivatives strongly accumulate in the kidneys, we hypothesised that specific cleavage of the radiolabelled moiety at the kidney brush-border membrane would allow easier excretion of the activity into the urine and therefore improve the pharmacological properties of the peptide.ResultsThe insertion of a cleavable linker did not negatively influence the in vitro properties of the peptides. They showed a good affinity to the GLP-1 receptor expressed in CHL cells, a high internalisation and sufficiently high stability in fresh human blood plasma. In vitro digestion with recombinant meprin β rapidly metabolised the corresponding linker sequences. After 60 min the majority of the corresponding peptides were digested and at the same time the anticipated fragments were formed. The peptides were also quickly metabolised in CD1 nu/nu mouse kidney homogenates. Immunofluorescence staining of meprin β in kidney sections confirmed the expression of the protease in the kidney brush-border membrane. Biodistribution in GLP-1 receptor positive tumour-xenograft bearing mice revealed high specific uptake of the 111In-labelled tracers in receptor positive tissue. Accumulation in the kidneys, however, was still high and comparable to the lead compound 111In-Ex4NOD40.ConclusionIn conclusion, we show that the concept of cleavable linkers specific for meprin β is feasible, as the peptides are rapidly cleaved by the enzyme while retaining their biological properties.
Highlights
Over recent years cleavable linkers targeting specific physiologic environments or enzymes have proven to be a versatile tool for various medical applications
In this work we describe the characterisation of three peptides based on exendin-4 that contain a cleavable linker between the binding moiety and the In-labelled chelator that were designed as cleavable substrates for meprin β
The linker of PSI-CLNOD1 is based on a sequence that can be found within the binding sequence of exendin-4 spanning from Q13 to V19, which we have shown to be cleaved by meprin β
Summary
Over recent years cleavable linkers targeting specific physiologic environments or enzymes have proven to be a versatile tool for various medical applications. New near infrared (NIR) probes, highly sensitive tools for the diagnosis of such conditions, have two fluorescent, self-quenching dyes attached to the probe via a specific self-immolative linker. This linker is cleaved in the targeted tissue, thereby liberating the dye and resulting in a specific fluorescent signal [3,4]. Cleavable linkers, which are cleaved by defined conditions or enzymes, are powerful tools that can be used for various purposes Amongst other things, they have been successfully used to deliver toxic payloads as prodrugs into target tissues. As radiolabelled exendin-4 derivatives strongly accumulate in the kidneys, we hypothesised that specific cleavage of the radiolabelled moiety at the kidney brush-border membrane would allow easier excretion of the activity into the urine and improve the pharmacological properties of the peptide
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