The Influence of Dimerization on the Pharmacokinetics and Activity of an Antibacterial Enzyme Lysostaphin

Abstract

The increasing prevalence of antibiotic-resistant strains of pathogenic bacteria is a major healthcare problem. Antibacterial lysins are enzymes that cleave the peptidoglycan of the bacterial cell wall. These proteins hold potential as a supplement or an alternative to traditional antibiotics since they are active against antibiotic resistant strains. However, antibacterial lysins are rapidly eliminated from the systemic circulation, which limits their application. Dimerization of an anti-pneumococcal lysin Cpl-1 has been demonstrated to decrease the clearance rate of this protein in mice. In the present work, we constructed a dimer of an anti-staphylococcal lysin lysostaphin by fusing it with an anti-parallel α-helical dimerization domain. Lysostaphin dimer had a more favorable pharmacokinetic profile with increased terminal half-life and area under the curve (AUC) values compared to monomeric lysostaphin. However, the staphylolytic activity of dimerized lysostaphin was decreased. This decrease in activity was likely caused by the dimerization; since the catalytic efficacy of lysostaphin dimer towards pentaglycine peptide was unaltered. Our results demonstrate that, although dimerization is indeed beneficial for the pharmacokinetics of antibacterial lysins, this approach might not be suitable for all lysins, as it can negatively affect the lysin activity.

Engineering of Long-Circulating Peptidoglycan Hydrolases Enables Efficient Treatment of Systemic Staphylococcus aureus Infection

“…Previous studies were able to show improvements in the PGH pharmacokinetic ( 36 – 38 , 41 , 45 ) or pharmacodynamic ( 37 ) properties but not the therapeutic implications of a prolonged half-life. The presumable reason for that was either a complete loss ( 44 ) or at least a significant decrease in the activity of the modified PGHs ( 36 , 37 , 41 , 45 ), in which a prolonged half-life would likely have not been able to compensate for lost activity. By altering the intramolecular position of the ABD in the context of the PGH (i.e., at an N- or C-terminal position), as well as by the introduction of a specific endolysin-originating ( 52 ) connecting linker sequence, we were able to provide constructs that retained significant staphylolytic activity in the presence of HSA.…”

“…To date, half-life extension of PGHs had been attempted via multiple approaches, including dimerization ( 38 , 45 ), PEGylation ( 36 ), and ABD fusion ( 37 , 41 ). Previous studies were able to show improvements in the PGH pharmacokinetic ( 36 – 38 , 41 , 45 ) or pharmacodynamic ( 37 ) properties but not the therapeutic implications of a prolonged half-life.…”

“…To date, half-life extension of PGHs had been attempted via multiple approaches, including dimerization ( 38 , 45 ), PEGylation ( 36 ), and ABD fusion ( 37 , 41 ). Previous studies were able to show improvements in the PGH pharmacokinetic ( 36 – 38 , 41 , 45 ) or pharmacodynamic ( 37 ) properties but not the therapeutic implications of a prolonged half-life. The presumable reason for that was either a complete loss ( 44 ) or at least a significant decrease in the activity of the modified PGHs ( 36 , 37 , 41 , 45 ), in which a prolonged half-life would likely have not been able to compensate for lost activity.…”

“…As mentioned above, the ABD fusion strategy to extend PGH serum circulation half-life ( 37 , 41 ) appears superior to other approaches, such as PEGylation ( 36 , 44 ) and dimerization ( 38 , 45 ). Both of the latter strategies also increase the protein’s molecular radius, thereby reducing the kidney excretion rate and, consequently, the elimination rate.…”

“…Elimination of proteins from the organism occurs through a combination of processes, which include general proteolysis, endocytosis and lysosomal degradation, renal filtration, and hepatic elimination ( 42 ). Elimination through renal filtration could be controlled by increasing the size of proteins above 70 kDa, which is reported to be the molecular weight cutoff value of the renal filters ( 43 ), e.g., by PEGylation ( 36 , 44 ) or dimerization ( 38 , 45 ). Another strategy that aims to bypass both endocytosis followed by lysosomal degradation and renal filtration involves fusion to serum proteins, which feature a long half-life due to the recycling pathway operating via the neonatal Fc receptor (FcRn) ( 46 ).…”