Design, synthesis and high-throughput assay of inhibitors for p97-cofactor binding

• Main Author: Sudlow, James
• Other Authors: Leatherbarrow, Robin; Freemont, Paul
• Published: Imperial College London 2014
• Subjects: 540
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.656807

p97 is an abundant protein in human cells and is essential for many forms of life. Its putative mechanism of action is the binding of adaptor proteins, which mediate its functions, and subsequent transfer of energy from ATP hydrolysis through the adaptor to substrate proteins. The unfolding or degradation of these substrates is known to regulate a diverse number of processes and the malfunction of p97 in many of these has been linked to a range of diseases. Developing inhibitors for p97-cofactor binding may help uncover further functions, as well as potentially providing a treatment for a number of the diseases in which p97 is implicated. A high-throughput assay based on Förster Resonance Energy Transfer (FRET) was developed, which allowed the high-throughput analysis of candidate compounds. Using this assay, the affinity of p97 for several of its partner proteins was measured. The results were found to be similar to literature values and the assay was measured to have a high Z' factor. Analysis of the hot-spot interactions between p97 and the adaptor protein, p47, enabled the design of novel peptide inhibitors of p97-Cofactor binding. A range of small molecules were also identified through a computer modelling approach known as scaffold hopping. A peptide that was designed to closely mimic the S3/S4 loop within p47 was found to bind to p97 and inhibit its interaction with p47. No inhibition, however, was detected from a second generation of peptides. Two molecules from the virtual ligand screen were found to inhibit p97 binding. Of these, one of the compounds was an unnatural tripeptide, amenable to the rapid synthesis of multiple variations. A second generation of compounds based on this original hit was analysed and another compound was identified with an improved level of inhibition.