Hydrocarbon double-stapling remedies the proteolytic instability of a lengthy peptide therapeutic

Abstract

The pharmacologic utility of lengthy peptides can be hindered by loss of bioactive structure and rapid proteolysis, which limits bioavailability. For example, enfuvirtide (Fuzeon, T20, DP178), a 36-amino acid peptide that inhibits human immunodeficiency virus type 1 (HIV-1) infection by effectively targeting the viral fusion apparatus, has been relegated to a salvage treatment option mostly due to poor in vivo stability and lack of oral bioavailability. To overcome the proteolytic shortcomings of long peptides as therapeutics, we examined the biophysical, biological, and pharmacologic impact of inserting all-hydrocarbon staples into an HIV-1 fusion inhibitor. We find that peptide double-stapling confers striking protease resistance that translates into markedly improved pharmacokinetic properties, including oral absorption. We determined that the hydrocarbon staples create a proteolytic shield by combining reinforcement of overall alpha-helical structure, which slows the kinetics of proteolysis, with complete blockade of peptide cleavage at constrained sites in the immediate vicinity of the staple. Importantly, double-stapling also optimizes the antiviral activity of HIV-1 fusion peptides and the antiproteolytic feature extends to other therapeutic peptide templates, such as the diabetes drug exenatide (Byetta). Thus, hydrocarbon double-stapling may unlock the therapeutic potential of natural bioactive polypeptides by transforming them into structurally fortified agents with enhanced bioavailability.

Fig. 1.

Design and synthesis of SAH-gp41 peptides. (A) Schematic of the HIV-1 viral fusion mechanism and its disruption by a decoy HR2 helix. (B) A series of reported next-generation gp41 HR2 peptides (18, 20), which contain natural amino acid substitutions, helix-promoting alanine residues, and/or (i, i + 4) salt bridges, exhibited rapid proteolytic degradation upon exposure to chymotrypsin. (C) SAH-gp41 peptides were designed based on gp41 HR2 domain sequences 626–662 (a variant of T649). X, substitution sites for crosslinking nonnatural amino acids; B, norleucine (substituted for methionine to optimize activity of the ruthenium catalyst). (D) Upon exposure to chymotrypsin, the singly stapled SAH-gp41 peptides exhibited 6–8-fold longer half-lives compared to the unmodified peptide, whereas double-stapling conferred a 24-fold enhancement in protease resistance. (E) Singly and doubly stapled SAH-gp41 peptides were also constructed based on the enfuvirtide peptide sequence (638–673). (F) Like SAH-gp41_(626–662) peptides, the singly stapled SAH-gp41_(638–673) derivatives showed enhanced chymotrypsin resistance compared to the template peptide, and the doubly stapled peptide was strikingly more resistant to proteolysis. Fraction intact, mean ± s.d.

Fig. 2.

Mechanistic analysis of peptide fortification by hydrocarbon double-stapling. (A) T649v and its synthetic derivatives contain the identical number of chymotrypsin cleavage sites. (B) CD demonstrated that T649v is predominantly a random coil in solution, whereas SAH-gp41_(626–662) peptides exhibited varying degrees of increased α-helical content. UAH- and SAH-gp41_(626–662) (A, B) displayed similar α-helical content, which was intermediate to that of the singly stapled peptides. (C) UAH-gp41_(626–662)(A, B) exhibited an 8-fold shorter half-life than its doubly stapled counterpart. Fraction intact, mean ± s.d. (D) UAH- and SAH-gp41_(626–662)(A, B) displayed similar melting profiles, with T_m values of 27 and 22 °C, respectively. Temperature-dependent unfolding was reversible for both peptides, as evidenced by the overlapping repeat melting curves. (E) Comparative chymotrypsin degradation patterns of T649v-based peptides. Of note, the N-terminal staple uniquely prevented proteolytic hydrolysis of the cleavage site flanked by the staple, with no corresponding M + 18 species observed by LC/MS.

Fig. 3.

Enhanced antiviral activity of SAH-gp41_(626–662)(A, B). (A) The gp41 HR2-derived peptides all blocked HXBc2 infectivity, with the T649v-based constructs showing slightly superior activity compared to enfuvirtide. (B) Singly and doubly stapled SAH-gp41_(626–662) peptides demonstrated enhanced blockade of a neutralization-resistant virus that contains the YU2 envelope glycoproteins, with SAH-gp41_(626–662)(A, B) exhibiting the most potent antiviral activity. SAH-gp41_(626–662)(A, B) outperformed enfuvirtide and T649v in HR1/HR2 complex assembly (C, D) and infectivity assays (E, F) that respectively employed peptides and recombinant HIV-1 constructs bearing HR1 enfuvirtide-resistance mutations. % viral infection, mean ± s.e.m; IC50, mean ± s.d.

Fig. 4.

Effect of double-stapling on pharmacokinetic behavior, acid protease resistance, and oral absorption. (A) Plasma concentrations of intact SAH-gp41_(626–662)(A, B) and T649v in mice treated by intravenous bolus injection. (B) CD revealed that SAH-gp41_(626–662)(A, B) exhibits even greater α-helicity at pH 2, surpassing T649v. (C) Upon exposure to pepsin at pH 2, T649v was rapidly degraded but SAH-gp41_(626–662)(A, B) was strikingly resistant. (D) Orally administered SAH-gp41_(626–662)(A, B) achieved measurable and dose-dependent plasma concentrations, whereas T649v was not detectable. Plasma concentration, mean ± s.d.; fraction intact, mean ± s.d.