What is new in synthetic lung surfactant protein technology?

Lung surfactant is essential for mammalian breathing. This mixture of phospholipids and surfactant proteins reduces surface tension in the lung and thereby prevents lung collapse during expiration. Dipalmitoyl phosphatidylcholine (DPPC) and phosphatidylglycerol (PG) are the main phospholipids in surfactant. Surfactant proteins B and C (SP-B and SP-C) are important for the ordering of the phospholipids at the air-water interface in the alveoli and surface tension reduction, whereas surfactant proteins A and D (SP-A and SP-D) play an important role in innate immunity in the lung [1]. Presence of SP-B in lung surfactant mixtures is pivotal and its deficiency by inherited mutations is often lethal in affected newborn infants [2]. Surfactant deficiency due to lung immaturity may lead to respiratory failure (RDS: respiratory distress syndrome) in preterm infants and require respiratory support and intratracheal treatment with animal-derived lung surfactant.

Respiratory support of preterm infants with intubation and mechanical ventilation may result in lung injury and chronic lung disease (bronchopulmonary dysplasia, BPD) despite improvement of lung function with surfactant treatment. The high incidence of BPD in very preterm infants exposed to invasive ventilatory assistance has resulted in increasing use of non-invasive respiratory approaches such as nasal continuous positive airway pressure (nCPAP) [3] and nasal intermittent positive pressure ventilation (NIPPV) [4] in combination with less invasive surfactant administration (LISA) [5] via a thin catheter inserted into the trachea. Simultaneously, limited availability and high production costs of animal-derived surfactant and advances in synthetic lung surfactant protein technology have led to the search for efficient synthetic lung surfactant formulations, consisting of SP-B and SP-C peptide mimics in a lipid mixture, that can replace animal-derived surfactants and be administered non-invasively in combination with nCPAP.

In a previous review [6] we reported that the availability of highly functional SP-B and SP-C peptide analogs had led to the development of potential third generation synthetic lung surfactants. CHF5633 (Chiesi Farmaceutici, Parma, Italy) is a new synthetic surfactant for intratracheal administration and consists of 0.2% Mini-B and 1.5% SP-C33leu in a 1:1 dipalmitoyl phosphatidylcholine (DPPC):palmitoyl-oleoyl-phosphoglycerol (POPG) mixture. Mini-B is a 34-amino acid peptide that incorporates the N-terminal alfa-helix (~ amino acids 8–25) and C-terminal alfa-helix (~ amino acids 63–78) of native SP-B, joined with a customized turn and cross-linked with two vicinal disulfide bonds (i.e., Cys-8 to Cys-77 and Cys-11 to Cys-71) to form a helix hairpin (Fig. 1A, 2A). SP-C33leu is a 33-amino acid peptide in which the palmitoylated cysteines in the N-terminal are replaced with serine and the valines in the transmembrane helix by leucines to ensure alfa helicity (Fig. 1B, 3B). MiniSurf (Molecular Express Inc., Rancho Dominguez, CA) contains 3% Super Mini-B (SMB) in 5:3:2 (mole:mole) DPPC:palmitoyl-oleoyl-phosphocholine (POPC):POPG lipid mixture (Fig.1C, 2B). SMB is Mini-B to which the hydrophobic N-terminal insertion sequence of SP-B (i.e., amino acids 1–7) has been added. In contrast with MiniSurf, CHF5633 contains a relatively small percentage of SP-B peptide mimic and depends functionally upon its SP-C peptide mimic, whereas MiniSurf relies solely on a higher concentration of the SP-B peptide mimic.

Figure 1. Surfactant SP-B and SP-C amino acid sequences.

(A) Mini-B and (C) Super Min-B amphipathic amino acid sequences with Cysteine disulfide linked residues highlighted in orange. (B) SP-C33 analog amino acid sequence with vicinal serine residues shown in orange. (D) B-YL amino acid sequence with tyrosine pi-pi interactive residues in orange. (E) Bovine SP-C amino acid sequence with thioester linked cysteine palmitic acid residues shown in orange. (F) Canine SP-C ion-lock surfactant peptide construct amino acid sequence with vicinal phenylalanine residues in orange and ion lock glutamic acid – lysine pair highlighted in red. (G) Mini-Bleu – SP-C33leu recombinant combination construct with amphipathic SP-B N-terminal sequence, mid linkers sequence GSG in orange and C-terminal hydrophobic poly-leucine SP-C mimic sequence.

Figure 2. Molecular illustrations of SP-B mimic peptide N-terminal – C-terminal amphipathic helix hairpin structures.

(A) Mini-B peptide with the longer N-terminal amphipathic alpha helix in red highlight ribbon and the shorter C-terminal amphipathic helix as red ribbon with cysteine disulfide linkages shown in yellow (Protein Data Bank accession code: PDB 2DWF). Bend and disordered structures shown as green tube. (B) Super Mini-B peptide with longer amphipathic helix in red ribbon connected to N-terminal insertion sequence represented as a blue tubular structure. Bend connecting N-terminal helix to C-terminal alpha helix shown as a green tube with cysteine disulfide linkages in yellow (ModelArchive accession code: ma-abz44). (C) B-YL construct with N-terminal insertion sequence as blue tube structure with N-terminal helix in red connected by bend domain shown as green tubular structure to the C-terminal amphipathic helix in red ribbon. The tyrosine side chain pi-pi interactions that stabilize the helical hairpin structure are illustrated in yellow highlight (ModelArcive accession code: ma-vilb7).

Figure 3. Schematic molecular representation of SP-C protein and mimic peptides in cross-sections of a bilayer – monolayer ensemble.

(A) Native bovine SP-C protein with hydrophobic poly-valine alpha helix in red ribbon inserted in a bilayer of lipid with the aqueous interfaces shown as red and blue spheres. The N-terminal sequence shown a green tubular structure with thioester linked palmitic acid side chains covalently attached vicinal cysteine residues that interact with the bilayer adjacent monolayer represented as a blue-green spheres (Protein Data Bank accession code: PDB 1SPF). (B) SP-C33leu peptide with hydrophobic poly-leucine in red ribbon inserted in bilayer with the N-terminal vicinal serine residues in orange highlight interacting with adjacent monolayer represented by blue-green spheres (Protein Data Bank accession code: PDB 5NDA). (C) SP-C ion-lock construct inserted into bilayer lipid ensemble. The glutamic acid – lysine pair that stabilizes the hydrophobic alpha helical structure in a trans-bilayer orientation is denoted by blue highlight. Tubular bend components of the structure are in green while the vicinal phenylalanine residues that are surrogates for cysteine-palmitate residues are shown in orange highlight and facilitate the cross-talk of the peptide in the bilayer with the adjacent monolayer so to provide bilayer-monolayer continuity [26]. Proteins were inserted and oriented into simulated surfactant bilayer with the OPM server [30].

CHF5633 has undergone both phase I and II clinical studies. In the multicenter phase I study, two groups of 20 infants from 27⁺⁰ to 33⁺⁶ weeks of gestation with RDS who were supported with nCPAP and required a fraction of inspired oxygen (FiO₂) ≥0.35, were treated with a single dose of 100 or 200 mg/kg of CHF5633 within 48 hours after birth. CHF5633 treatment was safe, tolerated well, and efficient [7]. Reassessment of the subjects at 2 years of corrected age did not detect surfactant-related adverse neurodevelopmental, respiratory, or health outcomes [8]. A subsequent phase II multicenter, double-blind, randomized, single dose, and active-controlled clinical trial in 56 very preterm infants with moderate-to severe RDS treated with CHF5633 and 57 infants treated with the porcine surfactant poractant alfa (Curosurf) demonstrated similar efficacy and safety as the phase I trial [9], but the lack of a sample size calculation adversely affects this study conclusion [10]. Animal studies with CHF5633 demonstrated its efficacy in meconium aspiration syndrome in ventilated newborn rabbits [11] and in severe acute respiratory distress syndrome (ARDS) in adult rabbits [12]. After completion of the phase II trial, CHF5633 was withdrawn by the manufacturer because of high costs and concerns about the low SP-B mimic content in the mixture.

The SP-B peptide mimic SMB (~41 amino acids; Fig. 1C, 2B) has a hairpin structure that is stabilized by two intramolecular disulfide bonds and requires an oxidation step to produce the surface-active, alfa-helical hairpin. To omit the oxidation step, we developed the B-YL peptide (Fig. 1D, 2C), a sulfur-free, non-covalent, aromatic ring side chain interaction stabilized SMB variant that has its four cysteine and two methionine residues replaced by tyrosine and leucine, respectively [13, 14]. Liquid surfactant phospholipid formulations with Mini-B, SMB or B-YL have been tested in ventilated, lavaged, surfactant-deficient rabbits and preterm lambs and are as efficient in improving oxygenation and lung compliance as animal-derived surfactants [13, 15].

Increased use of nCPAP instead of mechanical ventilation in clinical neonatal intensive care and affirmation of the correlation between intratracheal intubation and the incidence of BPD has led to a research focus on aerosol delivery of synthetic lung surfactant as this might phase out intratracheal placement of a tube or catheter for surfactant treatment. However, newborn infants have a 2–3 times higher respiratory rate than adults and a relatively small tidal volume, requiring redesign of delivery devices that can cooperate and not interfere with nCPAP function. We have tested aerosol delivery of various highly surface active spray-dried synthetic surfactant formulations (composed of SMB or B-YL peptide, mixed in 2 or 3 phospholipids (DPPC:POPG or DPPC:POPC:POPG), and lactose or trehalose and NaCl as excipients) in surfactant-deficient rabbits and preterm lambs, supported with mechanical ventilation or nCPAP, and shown the potential of this approach [16–20]. A combination of these novel dry powder synthetic surfactants and aerosol delivery systems with breath synchronization resulted in even more efficient lung dosing in preterm infants with RDS [21]. The surfactant group of Hindle and Longest at Virginia Commonwealth University has added to this development by adding mannitol and NaCl as hygroscopic excipients and l-leucine or trileucine as dispersion enhancer to the B-YL-lipid mixture. This excipient enhanced growth (EEG) surfactant showed efficient aerosol delivery from a low dispersion air volume dry powder inhaler [22].

In contrast with SP-B deficiency, SP-C (~35 amino acids; Fig. 1E, 3A) mutations are not lethal in the neonatal period, but may lead to interstitial lung disease later in life. Addition of a SP-C peptide mimic to a SP-B peptide surfactant enhances surface activity and stability of synthetic surfactant [23]. However, production of a stable SP-C peptide mimic is difficult because of its tendency to convert into beta-sheet aggregates. Antiparallel beta-sheet formation also occurs during storage of native SP-C protein when it loses the thioester linkages of the vicinal N-terminal cysteine residues that covalently link palmitoyl chains to the protein (24). Deacylation results in conversion of the hydrophobic alpha helix into antiparallel beta sheets that with time form inactive amyloid fibers and can be overcome by substituting amino acid residues that stabilize the hydrophobic helix. Strategies for SP-C helical stabilization include the substitution of valine by leucine residues that have a greater helical propensity to form stable helical conformations (SP-C33Leu; Fig. 1B, 3B) [25] and the substitution of polar residues ion-pairs in the hydrophobic poly-valine helix that form an ion-lock that stabilizes the alpha helical conformation of the protein (SP-C ion-lock, Fig. 1F) [26]. The canine sequence was taken as a reference to design this analog because one of the two palmitoylated cysteines of SP-C in most species is substituted by phenylalanine in the canine protein (also in the mink and seal sequences). This analog has the two cysteines substituted by phenylalanines (Fig. 1F).

Advancing expertise of chemical protein synthesis has thus led to a series of third generation, highly surface-active SP-B and SP-C peptide mimics. Recombinant production of these hydrophobic membrane proteins would offer a cheaper alternative for mass production, but is more complicated than chemical synthesis. Recently, Kronqvist et al. [27] were able to produce recombinant SP-C33Leu (Fig. 1B, 4) in E. coli by using a technique based on the high solubility of the N-terminal domain of spider silk proteins (NT). This rSP-C33Leu consists of a poly-leucyl transmembrane α-helix with a positively charged residue in the N-terminal of the helix to avoid oligomerization and replacement of a methionine residue by leucine to avoid inadvertent oxidation. Captive bubble surfactometry confirmed its efficacy in a mixture of 2% rSP-C33Leu and DPPC:POPG (68:31, w/w). However, rSP-C33Leu performance was less than of poractant alfa that contains both native SP-B and SP-C proteins. Basabe-Burgos et al. [28] went a step further by extending SP-C33Leu through a short linker with (Super) Mini-B mimics, creating a single recombinant polypeptide (Combo peptide) that fuses the essential characteristics of the structure and function of SP-B and SP-C analogs designed so far (Fig. 1G). Mikolka et al. [29] showed that Combo surfactant improves lung function in premature rabbits with RDS and in an acid instillation-induced ARDS adult rabbit model.

Figure 4. Molecular cross-sectional representation of Mini-Bleu – SP-C33leu combination B-C construct in bilayer-monolayer ensemble.

The poly-leucine hydrophobic SP-C trans-bilayer helical sequence is shown in red ribbon, while the amino acid sequence near the N-terminal domain of the SP-C33leu segment of the construct (residues PVHLK) is shown as a green helical ribbon. The Mini-Bleu SP-B mimic part of the construct is highlighted in orange ribbon that is interacting with the adjacent monolayer depicted as blue-green spheres. Bend and disordered domains are shown as green tubes. The predicted molecular structure of Mini-Bleu – SP-C33leu was approximated using A.I. based AlphaFold structure prediction program [31], while the insertion and orientation of the structure in the simulated surfactant lipid bilayer was estimated with the OPM server [30].

Synthetic lung surfactant protein technology is making progress towards a series of surfactant products that not only match structure and function of current clinically used, animal-derived surfactant formulations, but also complement and follow the technical advances in noninvasive respiratory support in preterm infants with RDS. Current (pre)clinical research indicates that we are close to formulation production that compares well with natural surfactants. Recombinant SP-B and SP-C analogs may overtake chemical peptide synthesis in the long run if amino acid hydrophobicity problems can be circumvented and may offer recyclable abilities like native surfactant proteins. Other applications, including surfactant inhibition in meconium aspiration syndrome, intrapulmonary drug delivery, and ARDS are also in the picture and may benefit from advances in synthetic lung surfactant technology.