Conformational Restriction of the NPF Motif to Target EHD1 and Endocytic Recycling
Abstract: The development of specific inhibitors for the long-loop recycling
pathway is critical for the study of vesicle trafficking and its involvement in the spread
of metastasized cancer cells. Many of the proteins involved in this pathway are difficult
to target due to the large and shallow protein-protein interactions they require to
function. EHD1, a C-terminal EH domain-containing prot... read moreein, is a promising target for this
process due to its involvement in surface receptor recycling. In addition, many of its
interacting proteins do so through an asparagine-proline-phenylalanine (NPF) motif, which
forms a type 1 β-turn in the hydrophobic binding pocket of the EH domain of EHD1 (EHD1-EH).
In the work described in this thesis, we have developed inhibitors of EHD1 by synthesizing
a cyclic peptide with an NPF sequence. The incorporation of the NPF motif into a backbone
macrocyclic peptide appeared to stabilize its naturally occurring β-turn, and the cyclic
peptide inhibitors bound EHD1-EH more tightly than the corresponding linear controls. We
then developed our best inhibitor of the first design series, cNPF1, into a fluorescent
probe to further study EH domain binding and screen for stronger EH domain inhibitors. Our
newest series of inhibitors utilize the NPF motif in conjunction with a new synthetic
pathway to explore a variety of linkers with a series of binding epitopes. The
bis-alkylation of thiol-containing peptides allowed for many linkers to be examined in the
context of the same peptide sequence. We search for more beneficial binding conformations,
while ideally improving passive cell penetration through the incorporation of a hydrophobic
linker. These higher affinity inhibitors will help us gather a better understanding of the
effects of EHD1 in the context of cancer cell invasion.
Thesis (Ph.D.)--Tufts University, 2017.
Submitted to the Dept. of Chemistry.
Advisor: Joshua Kritzer.
Committee: David Walt, Clay Bennett, and Robert Hammer.
Keywords: Chemistry, Biochemistry, and Biophysics.read less