Table 3.

Examples of the use of crosslinking strategy in designing peptide binders.

Starting point	Target	Results
Linear peptide known to bind in the pocket	TLE1	Kd as low as $3.5\pm 1.9$ nM [75]
Originally designed antagonist	C5a receptor	IC50 as low as 0.2 nM (cyclization combined with mutagenesis) [76]
Mutagenesis study of native protein-protein interactions	CK2 subunit interaction	IC50 as low as 3.0 nM [77]
Antibody loops	Influenza hemagglutinin	Kd as low as $17\pm 4$ nM and IC50 as low as 30 nM (after mutagenesis) [74]
Original helical motifs	NHR2-binding (N2B) motif of E-proteins	Kd as low as 53 ± 20 μM [78]
Structure of B-hairpin at the binding site	CdiI	Kd of 13 ± 2 μM [79]
Structure of bovine immunodeficiency virus Tat with TAR RNA	HIV TAR	Kd as low as 1 nM (disulfide cyclization and mutations) [80]
Structure of LXXLL motif (NR box)	Estrogen receptor	KD as low as 0.075 μM for ER $\alpha$ and 0.155 μM for ER $\beta$[81]
Crystal structure of 14-3-3ζ in complex with ESp	14-3-3	Kd as low as 0.25 $\pm$ 0.01 after stapling [82]
Structure of similar CYFIP1 to homologus WASF1	CYFIP1	Showed inhibition in cells [83]
EED binding domain of EZH2	EED	Kd as low as 264 nM after stapling [84]
Helical domain from Ras interaction with sos	Ras	nucleotide-free Ras with a KD of $28\pm 4.8$ μM and GDP-bound Ras with a KD of $158\pm 16$ μM [85]
Previously known binder, StRIP3 [86]	Rab8a	Kd as low as 7.8 μM after double stapling and mutagenesis [87]
Helical domain of HIF1a interacting with p300	p300	$K_{\text{i}}=3.5\pm 1.2$ μM [88], kd as low as $420\pm 35$ nM [89]
Loop identified by LoopFinder	Stonin2 and Eps15 interactions	Kd as low as $0.33\pm 0.01$ μM and IC50 as low as $2.2\pm 0.3$ μM by crosslinking the identified hot loop with Kd of $18.2\pm 3.4$ μM and $\text{I}{\text{C}}_{50}>100$ μM [68]
LC3 interacting region	LC3B	Kd of $0.12\pm 0.004$ μM after stapling and mutational optimization [69]
10mer hot segment identified at the interface	TLR4	Synergistic activation of TLR signaling was observed for cyclized peptides [71]
Short helical segment from TRF1TRFH-Fbx4G structure	Ubiquitin E3 ligase SCFFbx4	kD as low as $23.3\pm 12.8$ μM and IC50 of 31.3 μM after mutagenesis [52]
BH3 domain mimics	BCL-2, Bcl-xL	Dissociation constants as low as $21\pm 1$ nM [90], Kd of 38.8 nM [91], IC50 as low as $5.5\pm 0.8$ nM with stapling and $\beta$-amino acid [92]
BCL9 helix	$\beta$-Catenin	Ki as low as $0.13\pm 0.05$ μM [93]
Helix segment from HIV-1 gp41 structure	gp41	Kd as low as $7.50\pm 1.70$ nM after stapling [94], half life enhanced by an order of magnitude after double stapling [95], IC50 as low as 10 nM and Kd of 17 nM after stapling peptides containing the core binding residues [96]
Structure of p53 helical domain interacting with MDM2/MDMX	MDM2/MDMX	IC50 as low as 2$.2\pm 1.1$ nM for MDM2 and $3.1\pm 1.2$ nM for MDMX [97], MDM2 ki as low as $3.21\pm 0.38$ nM [98], IC50 as low as $6.2\pm 1.5$ nM for MDM2 and as low as $210\pm 23$ nM for MDMX using a photoinduced cycloaddition [99], Kd of $160\pm 80$ nM for MDM2 [100]