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Plasma Protein Binding Structure Activity Relationship Related to the N-Terminus of Daptomycin

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Plasma Protein Binding Structure Activity Relationship Related to the N-Terminus of Daptomycin Monash Institute of Pharmaceutical P1318 Sciences ECCMID 2017, Plasma protein binding structure activity relationship related to the N-terminus of daptomycin 381 Royal Parade, Parkville, VIC 3052 Melbourne, Australia Vienna 1 2 3 2 4 1 Tel: +61 3 9903 9539 Elena K. Schneider , Johnny X. Huang , Vincenzo Carbone , Matthew A. Cooper , Jian Li *, Tony Velkov * [email protected] 1Drug Development and Innovation, Drug Delivery, Disposition and Dynamics. Monash Institute of Pharmaceutical Sciences, Monash University, Australia. 2Institute for Molecular Bioscience, The University of Queensland St Lucia QLD 4072. 3Animal Nutrition and Health, Ag Research Limited, Grasslands Research Centre, Palmerston North, New Zealand. 4Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Australia. Abstract Methods Results Background: Daptomycin is a lipopeptide antibiotic that is highly bound to plasma proteins. To date the plasma components and • Flourometric binding assay: • SPR: structure-activity relationships responsible for the plasma protein binding profile of daptomycin remain uncharacterized. We used the site selective fluorescent probes . Native daptomycin binds in order of decreasing Methods: dansylamide (site 1) and dansylsarcosine affinity to: HSA >> α-1-antitrypsin > low density (site 2) on HSA. As the control we employed lipoprotein (LDL) ≥ haemoglobin > sex hormone • In the present study we have employed surface plasmon resonance (SPR) and fluorescence displacement assay to Nifedipine for site 1 and ibuprofen for site 2. binding globulin (SHBG) > α-1-acid glycoprotein investigate the plasma protein binding structure activity relationships of daptomycin. Fluorescence was measured using a Cary (AGP) > hemopexin > fibrinogen > α2- • Three compounds were investigated (1) native daptomycin which displays an N-terminal n-decanoyl fatty acid side chain, and Eclipse Fluorescence spectrophotometer (Ex: macroglobulin > β2-macroglobulin > high density two analogs with modifications to the N-terminal fatty acyl chain, (2) des-acyl daptomycin and (3) acetyl-daptomycin. 350 nm; Em: 400-650 nm). lipoprotein (HDL) > fibronectin > haptoglobulin > transferrin > immunoglobulin G (IgG). Results: . For probe displacement experiments daptomycin stock solutions (6.25 mM, 12.5 . Acetyl-daptomycin the decreasing rank order • The SPR data showed the binding profile of native daptomycin was ranking as follows: human serum albumin>> α-1- mM, 25 mM and 50 mM) were titrated at 2 was: haemoglobin > fibronectin > hemopexin > antitrypsin> low density lipoprotein ≥ hemoglobin> sex hormone binding globulin> α-1-acid-glycoprotein> hemopexin> min intervals into a quartz cuvette (1.5 µM HAS > α2-macroglobulin > fibrinogen > IgG > fibrinogen> α-2-macroglobulin> β2-microglobulin> high density lipoprotein > fibronectin> haptoglobulin> transferrin> SHBG > LDL > β2-macroglobulin > haptoglobulin immunoglobulin G. HSA- 25 µM probe complex). > α-1-antitrypsin > transferrin > AGP > HDL. • Fluorometric assay results with site-selective probes indicated that the native daptomycin preferentially binds to drug binding . Displacement of probe was measured as the . Des-acyl daptomycin only displayed appreciable site 1 on delipidated human serum albumin. corresponding decrease in fluorescence at each successive drug titration. binding to HSA. • In vitro microbiological assays show that humans serum albumin, α-1-antitrypsin, low density lipoprotein, sex hormone binding globulin, α-1-acid-glycoprotein and hemopexin are responsible for the majority of the sequestering activity in human • SPR: Immobilization levels of each protein were as • Fluorometric assay: following: HSA 14000 RU, immunoglobulin G (IgG) plasma. The fluorimetric binding assay revealed daptomycin binds to site 1 of 17000 RU, AGP 4500 RU, holo-transferrin 17000 the HSA, with a stronger preference for delipidated HSA (Figure 3). Conclusion: In the development of next-generation daptomycin lipopeptides, tailored modifications to the N-terminal fatty acyl RU, fibrinogen 11000RU, α1-antitrypsin 8000 RU, domain should yield novel daptomycin lipopeptides (better plasma protein binding profiles to increase the levels of free drug in haptoglobin 11000 RU, hemoglobin 14000 RU, α2- • Molecular model: plasma and improved in vivo activity. macroglobulin 20000 RU, β-microglobulin 12000 RU, hemopexin 17000 RU, sex hormone binding . Daptomycin-human serum albumin A complex showed the pivotal role of the N-terminal fatty acyl Background globulin (SHBG) 4000 RU, LDL 7000 RU and HDL chain of daptomycin for binding to drug site 1 of human serum albumin. 3000 RU. • MIC: • Additionally, we employed a molecular model of the daptomycin-human serum albumin A . Addition of physiological concentrations of HSA, α-1-antitrypsin, LDL, SHBG, AGP and hemopexin • Daptomycin is an amphipathic, acidic cyclic produced a 4- to 16-fold decrease in the MIC of daptomycin against S. aureus. No change in MIC lipopeptide (Streptomyces roseosporus). The N- • Minimum Inhibitory Concentration (MIC) were was observed in the presence of normal physiological concentrations of haptoglobulin, α2- terminus is acylated with an n-decanoyl fatty acid determined in accordance to the recommendations macroglobulin, holo-transferrin, β2-microglobulin, fibronectin and IgG; which were identified as side chain. of the Clinical and Laboratory Standards Institute. having poor daptomycin affinity by the SPR analysis (Figure 2). A 2-fold fluctuation commonly seen We employed a daptomycin sensitive strain (MIC with MIC measurements 0.5 mg/L), Staphylococcus aureus ATCC 29213. • Plasma protein binding has been implicated as a Figure 4: Top panel. Molecular docking model of daptomycin bound to HSA site 1. The bound myristate fatty acid molecules are major factor affecting the free concentration of Figure 2: Surface plasmon resonance response data for shown in sphere (yellow) representation and the HSA structure is shown in ribbon. The position of the crystallographic warfarin many clinically important antibiotics. Daptomycin the binding of 300 µM daptomycin, acetyl-daptomycin within site 1 is shown. Bottom right panel. The docked daptomycin and the cavity side chains are in stick form with the HSA surface displays a mean human plasma protein binding of and des-acyl daptomycin to the major proteins present in shown. The labelled side chains within the binding cavity represent contacts within 4 Å of the docked daptomycin molecule. >91.7. human plasma. Figure 3: Displacement of dansyl probes from HSA site Discussion and Conclusions • Previously, it has been reported that the extent of 1 (dansylamide) and site 2 (dansylsarcosine) by bacterial killing for daptomycin in supplemented daptomycin. Values are the normalized response to the • The plasma protein binding profile of highly bound antibiotics such as daptomycin can be very complex and diverse. media (albumin or plasma) reduces and delays its fluorescence intensity of the probe-HSA control bactericidal activity. (fluorescence intensity=100%) (n=3±SD). The insets . Native Daptomycin: HSA, α-1-antitrypsin, LDL, SHBG, AGP and hemopexin are predominantly responsible for the plasma binding. show the fluorescence emission spectra for the displacement of the dansyl probes from HSA by . Des-acyl Daptomycin: The removal of the decanoyl fatty acyl largely abolished plasma protein binding (remaining some affinity for HSA). Whereas, the truncation of the • Daptomycin has been shown to be 90-96% bound daptomycin. to human serum albumin (HSA) but other plasma decanoyl chain to an acetyl group did not abolish the plasma protein binding affinity, but instead altered the plasma protein binding specificity. protein targets remain uncharacterized. Acetyl-Daptomycin: Preferential binding to haemoglobin, fibronectin, hemopexin, HSA, α2-macroglobulin and fibrinogen. • We have shown that the plasma protein binding affinity and profile of daptomycin is driven largely by its N-terminal fatty acyl chain. Figure 1: Chemical structures of daptomycin and the • Careful chemical tailoring around the N-terminus of the daptomycin scaffold during the hit-to-lead optimization stages of drug discovery could lead to novel daptomycin analogues two N-terminal analogs used in this study, acetyl- daptomycin and des-acylated-daptomycin. The N- with improved plasma protein binding and pharmacokinetics profiles. terminal group on each structure is shaded red. Top right panel box: The three dimensional NMR structure of daptomycin is shown in surface representation (red References acidic: surface charge; blue: basic surface charge; white: non-polar surface). 1. Velkov, T., et al (2013) Future Microbiol 8, 711-724. 3. Li, J., et al (2006) Lancet Infect Dis 6, 589-601. 2. Velkov, T., et al (2010) J. Med. Chem. 53, 1898-1916. 4. Schneider EK., et al (2015) J Mol Recognit 28 (6), 339-48 Aim The focus of the present study was to characterize the plasma protein binding profile of daptomycin and to probe the Acknowledgement SAR using truncated N-terminal fatty acyl analogues. National Institute of Allergy and Infectious Diseases of the National Institutes of Health (R01 AI098771). EKS is an appointed ASM Young Ambassador (US) and supported by an Australian Postgraduate Award..
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