Advances in synthetic lung surfactant protein technology

Abstract

Lung surfactant is a mixture of phospholipids and proteins that is synthesized by alveolar type 2 cells and secreted into the alveoli to reduce surface tension at the air-liquid interface. Mammalian lung surfactant consists of approximately 80% phospholipids, 10% neutral lipids and 10% proteins. The discovery of surfactant deficiency as the cause of neonatal respiratory distress syndrome (RDS) in preterm infants in 1959 by Avery and Mead [1] has been a starting point for the development of synthetic lung surfactants for intratracheal instillation in preterm infants. The first generation of synthetic lung surfactants included phospholipid mixtures, such as ALEC®, a mixture of dipalmitoylphosphatidylcholine (DPPC) and egg phosphatidylglycerol (PG), and Exosurf®, a mixture of DPPC with hexadecanol and tyloxapol. However, these pure phospholipid-based preparations had a limited clinical efficacy because they lacked surfactant proteins B and C (SPB and SP-C) that enhance the surface-active properties of surfactant phospholipids. The second generation of synthetic lung surfactants therefore contained SP-B and/or SP-C peptide mimics, such as the SP-B analog KL4 (Surfaxin®) and recombinant human surfactant protein C (rhSP-C) (Venticute®). These synthetic lung surfactants were quickly overshadowed by the introduction of natural surfactants prepared by organic solvent extraction of bovine (Survanta®, Infasurf®, Alveofact®) or porcine (Curosurf®) lungs and consisting of phospholipids and small quantities (~1 wt%) of SP-B and SP-C. Clinical surfactants have had a tremendous effect on the outcome of preterm infants with RDS. In combination with antenatal corticosteroid therapy and advanced modes of ventilatory support, intratracheal administration of animal-derived lung surfactants has boosted survival and decreased morbidity of preterm infants born at the margins of viability. Potential side-effects of clinical surfactants due to cross-species transfer of infectious agents did not materialize, but high production costs and batch-to-batch variability have led to renewed interest in synthetic lung surfactants and the creation of third generation synthetic lung surfactants, using advanced SP-B and SP-C peptide mimics. Both SP-B and SP-C have been sequenced and genetic studies have shown that hereditary SP-B deficiency causes fatal neonatal RDS [2] and that SP-C mutations can lead to childhood interstitial lung disease [3]. Discovery of the structures of SP-B and SP-C 30 years ago and advances in peptide synthesis promised a quick development of functional peptide mimics and synthetic lung surfactant for clinical use. However, the complex 3-D structure of native SP-B and the propensity of SP-C for amyloid formation of its transmembrane region have been quite a challenge in designing and producing highly functional, stable peptide mimics of SP-B and SP-C. Human SP-B is an amphipathic, 79 amino-acid (MW ~8.7 kDa) saposin-like protein, consisting of 4–5 α-helices with three intramolecular disulfide bonds (i.e. Cys-8 to Cys-77, Cys-11 to Cys-71 and Cys-35 to Cys-46). The helical bundle for SP-B is folded into two leaves and held together with disulfide bridges, with one leaf having α-helices 1 (N-terminal helix), 5 (C-terminal helix) and 4 and the second composed of α-helices 2 and 3 [4,5]. Using truncated synthetic peptides (e.g. SP-B1–25, ~residues 1-25; SP-B4949–66, ~residues 49-66) that encompassed α-helices predicted from 3D-saposin homology comparisons, we found that Nand C-terminal peptides not only interacted with phospholipids similarly to that of native SP-B but also partially mimicked in vitro and in vivo activities of the parent protein [6]. Refinement of this approach led to the more advanced Mini-B and Super Mini-B mimics [4]. For example, Mini-B is a 34-residue, ‘short-cut’ peptide that incorporates the N-terminal α-helix (~residues 8–25) and C-terminal α-helix (~residues 63–78) of native SP-B, joined with a customized turn and cross-linkedwith two vicinal disulfide bonds (i.e. Cys-8 to Cys-77 and Cys-11 to Cys-71) to form an αhelix hairpin. Mini-B shows high surface activity in vitro and in animal models of surfactant deficiencies, which may be due to this mimic accurately reproducing the topology of the Nand C-terminal domains in the native SP-B [5]. Adding the hydrophobic N-terminal insertion sequence of SP-B (i.e. residues 1–7) to Mini-B led to ‘Super Mini-B’ [7]. Super Mini-B demonstrates excellent in vitro and in vivo surface activity after formulation with phospholipids [8], which may be a consequence of both the cross-linked α-helix hairpin and deeper insertion of the peptide into the lipid bilayer (Figure 1). Revak and Cochrane imitated the α-helical structure of SP-B with a 21-residue peptide alternating one lysine residue with a sequence of 4 leucine residues (KL4) [9]. KL4-surfactant (Surfaxin®) received FDA approval for the prevention of RDS in 2012, but production was discontinued in 2015. Except that it was not approved for treatment of preterm infants with neonatal RDS, Surfaxin® had a practical problem because it