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Hydrocarbon Double-Stapling Remedies the Proteolytic Instability of a Lengthy Peptide Therapeutic

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Hydrocarbon Double-Stapling Remedies the Proteolytic Instability of a Lengthy Peptide Therapeutic Hydrocarbon double-stapling remedies the proteolytic instability of a lengthy peptide therapeutic Gregory H. Birda,b, Navid Madanic,d, Alisa F. Perrya,b, Amy M. Princiottoc, Jeffrey G. Supkoe, Xiaoying Hee, Evripidis Gavathiotisa,b, Joseph G. Sodroskic,f, and Loren D. Walenskya,b,1 aDepartment of Pediatric Oncology, Dana–Farber Cancer Institute and Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115; bProgram in Cancer Chemical Biology, Dana–Farber Cancer Institute, Boston, MA 02115; cDepartment of Cancer Immunology and AIDS, Dana–Farber Cancer Institute, Division of AIDS, Harvard Medical School, Boston, MA 02115; dDepartment of Pathology, Harvard Medical School, Boston, MA 02115; eClinical Pharmacology Laboratory, Department of Medicine, Massachusetts General Hospital, Boston, MA 02114; and fDepartment of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115 Edited* by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved June 18, 2010 (received for review March 3, 2010) The pharmacologic utility of lengthy peptides can be hindered by on this helical bundle assembly mechanism to infect host cells, loss of bioactive structure and rapid proteolysis, which limits bioa- highlighting the potential of this pharmacologic strategy to yield vailability. For example, enfuvirtide (Fuzeon, T20, DP178), a 36-ami- both preventive and suppressive antiviral agents (8, 9). Despite no acid peptide that inhibits human immunodeficiency virus type 1 successful validation of this targeted anti-HIV-1 therapy in (HIV-1) infection by effectively targeting the viral fusion apparatus, humans (7), enfuvirtide has remained a treatment option of last has been relegated to a salvage treatment option mostly due to resort mostly due to cost, lack of oral bioavailability, accelerated poor in vivo stability and lack of oral bioavailability. To overcome systemic clearance from in vivo proteolysis, and the development the proteolytic shortcomings of long peptides as therapeutics, we of drug resistance (10, 11). Whereas peptide-based inhibition of examined the biophysical, biological, and pharmacologic impact of viral fusion is mechanistically feasible and clinically effective, the inserting all-hydrocarbon staples into an HIV-1 fusion inhibitor. biophysical, biological, and pharmacologic liabilities of fusion- We find that peptide double-stapling confers striking protease re- inhibiting peptides have thwarted their optimal therapeutic sistance that translates into markedly improved pharmacokinetic application. properties, including oral absorption. We determined that the We sought to optimize HIV-1 fusion inhibitor peptides by BIOCHEMISTRY hydrocarbon staples create a proteolytic shield by combining rein- generating SAH of gp41 (SAH-gp41) through all-hydrocarbon forcement of overall α-helical structure, which slows the kinetics covalent crosslinking of a variant of T649 (628–663), which we of proteolysis, with complete blockade of peptide cleavage at con- refer to as T649v (626–662). T649 peptides contain conserved strained sites in the immediate vicinity of the staple. Importantly, HIV-1 gp41 residues that contribute to the stability of the six- double-stapling also optimizes the antiviral activity of HIV-1 fusion helix bundle and thus exhibit improved antiviral potency com- peptides and the antiproteolytic feature extends to other therapeu- pared to enfuvirtide (638–673) (12). Because the intramolecular tic peptide templates, such as the diabetes drug exenatide (Byetta). binding interfaces of fusion complexes are large, shallow, and Thus, hydrocarbon double-stapling may unlock the therapeutic hydrophobic, complex peptides such as those derived from the potential of natural bioactive polypeptides by transforming them HR regions of gp41 are more effective inhibitory ligands than into structurally fortified agents with enhanced bioavailability. small molecules. Thus, for the last fifteen years, there has been an intensive and ongoing effort to develop synthetic constraints ∣ ∣ ∣ ∣ fusion inhibitor HIV-1 protease resistance stapled peptide alpha-helix (13–17) or mutagenesis approaches (18–24) that fortify the natural helical structure of gp41 peptide sequences (9, 25–27) to maximize arnessing bioactive peptides to develop highly selective and the pharmacologic potential of inhibiting viral fusion. To date, a Hpotent pharmacologic agents for targeting pathologic pro- synthetic design that accomplishes structural stabilization, neutral teins and their interactions remains an appealing strategy for drug and acid protease resistance, enhanced antiviral efficacy, activity “ ’ ” discovery. Despite the allure of applying nature s solution to against resistant strains, and improved pharmacokinetic behavior protein targeting, peptides are typically administered by injection that includes oral absorption, has not been achieved. Here, we because of their inherently poor oral bioavailability and they are report the application of hydrocarbon double-stapling to redress rapidly cleared as a consequence of peptidase-mediated metabo- the biophysical and pharmacologic liabilities of a lengthy peptide lism. Nevertheless, therapeutic antibodies, enzymes, hormones, therapeutic. and other peptides, whose chemical architecture is based on the amide bond, represent an essential and growing component of Results the pharmaceutical repertoire. By inserting an all-hydrocarbon “ ” – Hydrocarbon Double-Stapling Fortifies Natural Peptide Therapeutics. staple within short peptide sequences (1 3), we previously de- Synthetic optimization of the HR2 fusion domain of gp41 has re- veloped stabilized alpha helices (SAH) of BCL-2 domains (4, 5) mained a pressing research goal since the successful development and p53 (6) that exhibit sturdy α-helical structure, protease resis- of enfuvirtide as an HIV-1 pharmaceutical (18–20). Whereas clin- tance, cell penetrance, and targeted biological activity. Here, we explored the potential of hydrocarbon double-stapling to structu- rally fortify lengthy bioactive peptides and remedy their proteo- Author contributions: G.H.B., N.M., J.G. Supko, J.G.Sodroski, and L.D.W. designed research; lytic vulnerability in vitro and in vivo. We chose the peptidic fusion G.H.B., N.M., A.F.P., A.M.P, J.G. Supko, X.H., E.G., J.G. Sodroski, and L.D.W. performed inhibitors of human immunodeficiency virus type 1 (HIV-1) as research; G.H.B., N.M., E.G., J.G. Sodroski, and L.D.W. contributed new reagents/analytic tools; G.H.B., N.M., A.F.P., A.M.P., J.G. Supko, E.G., J.G. Sodroski, and L.D.W. analyzed data; the template for our study because their biophysical shortcomings and G.H.B., N.M., J.G. Supko, J.G. Sodroski, and L.D.W. wrote the paper. reflect the practical challenges for broader application of lengthy Conflict of interest statement: L.D.W. is a consultant and scientific advisory board member peptide therapeutics. for Aileron Therapeutics. Enfuvirtide, the first fusion inhibitor shown to block HIV-1 *This Direct Submission article had a prearranged editor. α entry in humans, is a decoy -helix that disrupts assembly of 1To whom correspondence should be addressed. E-mail: Loren_Walensky@ the six-helix bundle viral fusion apparatus by mimicking the hep- dfci.harvard.edu. tad repeat 2 (HR2) oligomerization domain of the gp41 envelope This article contains supporting information online at www.pnas.org/lookup/suppl/ glycoprotein (7) (Fig. 1A). A wide variety of viral families rely doi:10.1073/pnas.1002713107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1002713107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 29, 2021 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 þ 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 chymotryp- sin, the singly stapled SAH-gp41 peptides exhibited 6–8-fold longer half-lives compared to the unmodi- fied peptide, whereas double-stapling conferred a 24-fold enhancement in protease resistance. (E) Singly and doubly stapled SAH-gp41 peptides were also con- structed based on the enfuvirtide peptide sequence (638–673).(F)LikeSAH-gp41ð626–662Þ peptides,thesingly stapled SAH-gp41ð638–673Þ derivatives showed en- hanced chymotrypsin resistance compared to the tem- plate peptide, and the doubly stapled peptide was strikingly more resistant to proteolysis. Fraction intact, mean Æ s:d: ical enthusiasm for fusion inhibitors has waned due to the phar- plored the synthetic feasibility and biophysical impact of “double- macologic liabilities, the emergence of resistance, and their repla- stapling” by inserting chemical staples at both N- and C-terminal cement by more effective therapeutic options, the mechanistic positions (Fig. 1C). To achieve this synthetic goal, two pairs of elegance of viral fusion inhibition and the potential broad appli- (i, i þ 4) substitutions were made, ensuring a sufficient distance cation to preventing a host of viral infections, have continued to between nonnatural amino
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