Abstract
Hsp70 is a protein that refolds misfolded proteins and has housekeeping functions which regulate apoptosis and other cellular activities. Hsp70 consists of a nucleotide-binding domain which binds ATP and a substrate binding domain that binds misfolded proteins. The substrate-binding domain contains a peptide-binding pocket which is covered by an a-helical lid that consists of helices A-E and a tail. The movement of the lid is important for the biological activity of Hsp70.|In cancer cells, the Hsp70-1 isotype helps to inhibit apoptosis in response to stress and treatment with drugs. The depletion or inhibition of Hsp70-1 results in the apoptosis of only cancerous cells. This makes Hsp70-1 a good target for the inhibition of cancer growth. |The design of peptides that specifically bind to the lid of human Hsp70-1 and restrict the movement of the lid, would make good Hsp70-1 specific inhibitors. To this end peptides were designed through the modification of the structure of Pyr, an antimicrobial peptide that binds to the lid of DnaK and the screening of a combinatorial peptide library. Ten Pyr(1-10)-NH2 analogs and a combinatorial library of 274,360 tri- and tetra-peptides were docked to the lid of the SBD of Hsp70-1 using Glide and Molegro molecular docking software, respectively. The MD simulations of the Pyr(1-10)-NH2 analog – Hsp70-1 complexes showed that Pyr(1-10)-NH2 and [Ala3]Pyr(1-10)-NH2 bound to the lid of Hsp70-1, were able to stabilize the structure of the lid and restrict lid movement. When these analogs were synthesized they had a significant inhibitory effect on the ATPase activity of Hsp70-1, but not Hsp70-8. The docking of the combinatorial libraries to the lid of Hsp70-1 and the MD simulations of the peptide – Hsp70-1 complexes identified peptides that formed a stable complex with the lid and restricted lid movement. Of these peptides, Arg-Glu-Val was shown to inhibit the ATPase activity of Hsp70-8 in addition to Hsp70-1, indicating it was not Hsp70-1 specific.|The peptides that the docking calculations predicted would bind to the lid of Hsp70-1 and restrict lid movement in the MD simulations, were shown experimentally to inhibit the ATPase activity of Hsp70-1. This work has laid a foundation for the further development and refinement of Hsp70-1 inhibitors through the computational screening of peptides.