Abstract
BackgroundMany biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is one of the most important transport proteins in the cardiovascular system due to its great binding capacity and high physiological concentration. HSA has a preference for accommodating neutral lipophilic and acidic drug-like ligands, but is also surprisingly able to bind positively charged peptides. Understanding of how short cationic antimicrobial peptides interact with human serum albumin is of importance for developing such compounds into the clinics.ResultsThe binding of a selection of short synthetic cationic antimicrobial peptides (CAPs) to human albumin with binding affinities in the μM range is described. Competitive isothermal titration calorimetry (ITC) and NMR WaterLOGSY experiments mapped the binding site of the CAPs to the well-known drug site II within subdomain IIIA of HSA. Thermodynamic and structural analysis revealed that the binding is exclusively driven by interactions with the hydrophobic moieties of the peptides, and is independent of the cationic residues that are vital for antimicrobial activity. Both of the hydrophobic moieties comprising the peptides were detected to interact with drug site II by NMR saturation transfer difference (STD) group epitope mapping (GEM) and INPHARMA experiments. Molecular models of the complexes between the peptides and albumin were constructed using docking experiments, and support the binding hypothesis and confirm the overall binding affinities of the CAPs.ConclusionsThe biophysical and structural characterizations of albumin-peptide complexes reported here provide detailed insight into how albumin can bind short cationic peptides. The hydrophobic elements of the peptides studied here are responsible for the main interaction with HSA. We suggest that albumin binding should be taken into careful consideration in antimicrobial peptide studies, as the systemic distribution can be significantly affected by HSA interactions.
Highlights
Many biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective
We have previously found that Synthetic cationic antimicrobial peptide (CAP) bind to albumin in the low μM range, and when Human serum albumin (HSA) was included in cell-based assays at physiological concentrations the minimal inhibitory concentration (MIC) of the CAPs was increased by an order of magnitude [21]
CAP 1 and CAP 5 were included as controls as they contained either the positively charged arginines or one of the hydrophobic moieties necessary for activity [16]
Summary
Many biologically active compounds bind to plasma transport proteins, and this binding can be either advantageous or disadvantageous from a drug design perspective. Human serum albumin (HSA) is the most abundant transport protein present in blood plasma with a normal physiological concentration of 0.6 mM It is active as a monomer with 585 residues and a molecular weight of 66.5 kDa. HSA has a high overall binding capacity due to a number of diverse binding sites distributed over the whole protein, and binds numerous endogenous and exogenous compounds [1]. HSA is known to have a preference for accommodating neutral lipophilic and acidic drug-like ligands, which corresponds well with its main function as a transporter of fatty acids Exceptions to this generalization exist, and basic residues have been observed as HSA ligands, only a few experimental complexes have been published [1]. The experimental basic ligand HSA complexes comprise the drug site I fluorescence marker dansyl-L-arginine bound in drug site I, and the anaesthetic compound lidocaine located in a novel binding site [3,4]
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