Abstract
PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.
Highlights
It was shown that membrane associated guanylate kinase (MAGUK) proteins have an architecture in which the PDZ1-2 and PDZ3-src homology 3 (SH3)-guanylate kinase (GK) domains, respectively, form two independent supramodules
We demonstrated that a trimeric ligand (17) containing a very long PEG28 linker had a affinity than a dimeric ligand (11) to PDZ1-2-3 of PSD-95, representing a proof of concept for trivalent binding to PSD-95
When examining the trimeric ligands at PDZ1-2-3 of PSD-93, synapse associated protein-97 (SAP-97) and SAP-102, we generally found that affinities were similar to those of PSD-95
Summary
Trimeric Ligands Targeting PSD-95 classes of PPI domains in the human genome, with an estimated 256 PDZ domains in 142 different proteins[3] that facilitates several vital PPIs.[4,5] One PDZ-domain containing protein family, the discs large (dlg) membrane associated guanylate kinase (MAGUK) family, comprises four members, PSD-93, PSD-95, synapse associated protein-97 (SAP-97) and SAP-102. They share the same overall topology of three PDZ domains, one src homology 3 (SH3) domain and one inactive guanylate kinase (GK) domain. The MAGUK proteins are involved in receptor trafficking and scaffolding of primarily post-synaptic signaling complexes through their PDZ domains and have been implicated as potential drug targets for the treatment of neurological diseases such as stroke, chronic pain and Alzheimer’s disease.[4,6,7,8,9] In particular, PSD-95 has been shown to function as a scaffolding protein, bridging the N-methyl-D-aspartate (NMDA) receptor and neuronal NO synthase (nNOS) via the PDZ1 and PDZ2 domains. [10,11] A 20-mer peptide ligand that disrupts this ternary complex, Tat-NR2B9c (NA-1), has been shown to alleviate neuropathic pain in animal models,[12] reduce ischemic brain damage, [13,14,15,16,17] and has recently successfully passed phase II clinical trials for iatrogenic stoke.[18,19]
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