Long-range PEG stapling: macrocyclization for increased protein conformational stability and resistance to proteolysis

Abstract

Long-range stapling of two Asn-linked PEG oligomers via olefin metathesis substantially increases the conformational stability of the WW and SH3 domain tertiary structures and the GCN4 coiled-coil quaternary structure.

Structural guidelines for stabilization of α-helical coiled coils via PEG stapling

“…[27][28][29][30][31][32][33] We recently showed that stapling via olefin metathesis vs. the copper-catalyzed azide-alkyne cycloaddition (CuAAC) provide similar increases in the conformational stability of WW, a βsheet miniprotein derived from the WW domain of the human protein Pin1. 34,35 We observed similar levels of stabilization for staples comprised of discrete polyethylene glycol oligomers (i.e., PEG staples) vs. conventional hydrocarbon staples. The most important determinant of PEG-staple-based stabilization in WW is that the two crosslinked groups be far apart in primary sequence but close together in the folded tertiary structure.…”

“…27–33 We recently showed that stapling via olefin metathesis vs. the copper-catalyzed azide–alkyne cycloaddition (CuAAC) provide similar increases in the conformational stability of WW, a β-sheet miniprotein derived from the WW domain of the human protein Pin1. 34,35 We observed similar levels of stabilization for staples comprised of discrete polyethylene glycol oligomers ( i.e. , PEG staples) vs. conventional hydrocarbon staples.…”

“…We then prepared PEG-stapled variant sd27e/29g'-z4x from d27e/29g'-z4x via CuAAC, which connects the azide of z4 to the alkyne of x via a triazole linkage (Figure 2C,D). 35 Our approach differs from that of Arora and coworkers 48,49 in the structure of the z4x staple and its placement between eand g'-positions that are not involved in a salt bridge with each other: in the structure of parent compound dA/B, Glu27 is involved in a salt bridge with Lys22', whereas Lys29' is involved in a salt bridge with Glu34.…”

Protein Macrocyclization for Tertiary Structure Stabilization

“…In particular, INCYPRO provides straight‐forward access to diverse linker structures. [81] In general, it can be expected that more cyclization strategies will be developed which allow the simultaneous introduction of an additional functionality (e. g. PEG chains [84] or photo‐switchable moieties[ 85 , 86 ]).…”

Genetically-Encoded Discovery of Perfluoroaryl-Macrocycles that Bind to Albumin and Exhibit Extended Circulationin-vivo

“…Interestingly, steric hindrance by these size-increasing moieties also protects against proteolytic degradation. [13][14][15] ; Class 2: increase in size via controlled oligomerization; 16 Class 3: covalent linking to long-living serum protein (e.g., FDA-approved drugs albiglutide and dulaglutide, exanatide that conjugated to albumin and the IfG4 Fc domain) [17][18][19] and Class 4: incorporation of moieties that bind non-covalently to serum proteins such as albumin, [20][21][22][23] immunoglobulin, 24,25 FcRn, 26 transthyretin, 27 and transferrin. 28,29 An important example in the last class is lipidation of peptides to allow interaction with serum albumin.…”