Site-specific Isopeptide Bridge Tethering of Chimeric gp41 N-terminal Heptad Repeat Helical Trimers for the Treatment of HIV-1 Infection

Abstract

Peptides derived from the N-terminal heptad repeat (NHR) of HIV-1 gp41 can be potent inhibitors against viral entry when presented in a nonaggregating trimeric coiled-coil conformation via the introduction of exogenous trimerization motifs and intermolecular disulfide bonds. We recently discovered that crosslinking isopeptide bridges within the de novo helical trimers added exceptional resistance to unfolding. Herein, we attempted to optimize (CCIZN17)3, a representative disulfide bond-stabilized chimeric NHR-trimer, by incorporating site-specific interhelical isopeptide bonds as the redox-sensitive disulfide surrogate. In this process, we systematically examined the effect of isopeptide bond position and molecular sizes of auxiliary trimeric coiled-coil motif and NHR fragments on the antiviral potency of these NHR-trimers. Pleasingly, (IZ14N24N)3 possessed promising inhibitory activity against HIV-1 infection and markedly increased proteolytic stability relative to its disulfide-tethered counterpart, suggesting good potential for further development as an effective antiviral agent for treatment of HIV-1 infection.

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

“…a 5 °C increase in melting temperature); the origin of this disparity was not explored in detail. Jiang, Liu, and coworkers 88 formed an isopeptide bond between each of three identical e -position Glu residues within a helix-bundle trimer and a g -position Lys from the previous heptad on an adjacent helix. The resulting triply-stapled helix bundle had no cooperative thermal unfolding transition below 90 °C and was resistant to aggregation and proteolysis, though no comparison with its non-stapled counterpart was reported.…”

“…22 These Cys-centric approaches are generally limited to side-chain/side-chain crosslinks; in contrast, other approaches facilitate stapling in both side-chain and backbone contexts. 23 For example, lactam staples can be prepared via conventional peptide coupling chemistry [24][25][26][27][28][29] or by diverse chemoselective strategies, including the Ugi reaction; 30,31 direct thioester aminolysis; 32 native chemical ligation; [33][34][35] KAHA ligation; 36 traceless Staudinger ligation; 37 and a variety of enzymatic methods. [38][39][40] Other creative stapling strategies include C-H activation; [41][42][43] the Petasis reaction; 44 the Glaser reaction; 45 oxime 46,47 or hydrazone 48 formation; the copper(I)catalyzed azide-alkyne cycloaddition (CuAAC); [49][50][51][52][53][54][55] and olefin metathesis.…”

A protein tertiary structure mimetic modulator of the Hippo signalling pathway

“…In these cases, more complex protein mimetics would be required to facilitate the spatial arrangement of multiple secondary structures. The feasibility of stabilizing such complex folds has been shown for multi-helix arrangements which required the use of multiple tailor-made intra-helical crosslinks, but those entities have not been used for the targeting of proteins so far [8][9][10][11] .…”

Proteomimetics as protein-inspired scaffolds with defined tertiary folding patterns

“…Early examples involve the use of cyclic β-hairpins 28 or porphyrins 29 as scaffolds for the covalent attachment of four helical peptides resulting in folded higher-order structures. In later work, natural and de novo-designed coiled coils 30,31 served as starting point for the design of cross-linked helix bundles [32][33][34] . Dimeric coiled coils (Fig.…”

“…2c) have been stabilized using various linkages such as ethylene glycol ( 7) and dibenzyl ether-based structures (8) installed via biselectrophilic cross-linkers and targeting two cysteine side chains 32,33 . Analogously, cross-linked trimeric helix bundles were created, revealing that robust assembly relies on carefully designed cross-linkers and supporting interhelical hydrophobic contacts 32,34 . Hydrophobic bis-and triselectrophiles, previously used for the generation of mono-and bicyclic peptides 35,36 , have also been utilized to create covalent linkages in the hydrophobic core of designed artificial tertiary folds (termed the CovCore approach) 37 .…”