Manipulating Active Structure and Function of Cationic Antimicrobial Peptide CM15 with the Polysulfonated Drug Suramin: A Step Closer to in Vivo Complexity

Abstract

Antimicrobial peptides (AMPs) kill bacteria by targeting their membranes through various mechanisms involving peptide assembly, often coupled with disorder-to-order structural transition. However, for several AMPs, similar conformational changes in cases in which small organic compounds of both endogenous and exogenous origin have induced folded peptide conformations have recently been reported. Thus, the function of AMPs and of natural host defence peptides can be significantly affected by the local complex molecular environment in vivo; nonetheless, this area is hardly explored. To address the relevance of such interactions with regard to structure and function, we have tested the effects of the therapeutic drug suramin on the membrane activity and antibacterial efficiency of CM15, a potent hybrid AMP. The results provided insight into a dynamic system in which peptide interaction with lipid bilayers is interfered with by the competitive binding of CM15 to suramin, resulting in an equilibrium dependent on peptide-to-drug ratio and vesicle surface charge. In vitro bacterial tests showed that when CM15⋅suramin complex formation dominates over membrane binding, antimicrobial activity is abolished. On the basis of this case study, it is proposed that small-molecule secondary structure regulators can modify AMP function and that this should be considered and could potentially be exploited in future development of AMP-based antimicrobial agents.

Keywords: IR spectroscopy; antimicrobial peptides; circular dichroism; folding; suramin.

Scheme 1

Structures of the compounds used in the study. A) Helical wheel diagram of CM15 (KWKLFKKIGAVLKVL‐amide), N and C stand for the N and C termini of the peptide. The helix plot was drawn with HELIQUEST.15 B) Chemical structure of suramin. C) Chemical structures of the lipid components used in model membranes built up of DOPC (PC) and DOPG (PG). For mimicking mammalian and bacterial cell membranes, pure DOPC and DOPC/DOPG (80:20, n/n%) were used throughout the study. In the chemical structures, oxygen and nitrogen atoms are coloured red and blue, highlighting negatively and positively charged parts, respectively.

Figure 1

Structural changes of CM15 in the presence and in the absence of model membranes and suramin, studied by CD spectroscopy. A) Far‐UV CD spectra were taken at peptide, suramin and lipid concentrations of 40, 40, and 635 μm, respectively. B) Effect of suramin on the helix content of free and membrane‐bound CM15. The peptide (40 μm) was titrated with suramin in the absence and in the presence of liposomes (635 μm total lipid) with use of CD buffer or PBS (for the compositions see the Experimental Section). Helix content was estimated with the aid of the BestSel online tool.30 Data are means± SEMs, two series of titrations were carried out with CD buffer, and a single titration in PBS was performed as a control.

Figure 2

Far‐UV CD spectra of CM15 in the presence and in the absence of model membranes and suramin. Spectra were collected at 40 μm peptide A), B) with PC, and C), D) with PC/PG liposomes (635 μm total lipid) at CM15/suramin ratios of A), C) 1:1, or B), D) 1:2. The order of addition in the three‐component system was varied so that the preincubation of the two compounds in parentheses was initially performed, with the third compound then being added.

Figure 3

Peptide–drug assembly in the two‐ and three‐component systems monitored by DLS. Correlation functions are shown for mixtures with CM15/suramin ratios of A), C) 1:1, and B), D) 1:2, in the absence and in the presence of A), B) PC, and C), D) PC/PG liposomes. Peptide, suramin and lipid concentrations are 20, 20 or 40, and 635 μm, respectively. For more details see Tables S2 and S3.

Figure 4

Morphology of the CM15⋅suramin complex imaged by TEM. Micrographs of the mixtures stained with phosphotungstic acid were taken at CM15/suramin ratios of 1:1 (left) and 1:2 (right). Peptide and suramin concentrations in the solutions prior to drying were 20 and 20 or 40 μm, respectively.

Figure 5

Fluorescence emission spectra of CM15 in the absence and in the presence of model membranes. Peptide and lipid concentrations were 1 and 100 μm, respectively.

Figure 6

Peptide fluorescence study of suramin binding to CM15 in the absence and in the presence of liposomes. A) Fluorescence emission spectra of CM15 (1 μm) upon addition of suramin. Arrow indicates increasing drug concentrations. B) Normalised maximum emission intensities of CM15 (1 μm) and the control NATA (1 μm) as a function of suramin concentration. Note that error bars for the peptide titration points are mostly smaller than symbol size (data are means±SEMs, n=2).

Figure 7

IR study of CM15 binding to model membranes and suramin. A) Amide I and II regions, as well as lipid carbonyl bands, recorded for CM15/liposome systems. B) Formation of CM15⋅suramin complex indicated by the appearance of an extra band at 1040 cm⁻¹.

Figure 8

IR study of CM15 interactions in the presence of model membranes and suramin. A), C) Amide I and II regions and lipid carbonyl band. B), D) Formation of CM15⋅suramin complex indicated by the appearance of an extra band at 1040 cm⁻¹. ATR FTIR spectra were collected for dry films produced from solutions containing CM15 (80 μm), suramin (160 μm) and PC or PC/PG liposome (1.3 mm lipid).

Figure 9

Antibacterial effect of CM15 in the presence and in the absence of suramin. A) E. coli treated with peptide/drug mixtures at various ratios. After 24 h incubation, no bacterial growth was detected in the CM15‐treated wells. In contrast, when CM15 was added with suramin, visible bacterial growth was observed. B) Relative bacterial counts after treatment with 5 μm CM15 and various concentrations of suramin. Values are means±SEMs (n=4).

Figure 10

Cytotoxic and haemolytic effects of CM15 in the presence and in the absence of suramin. Cytotoxicity was measured with MonoMac6 human monocytes, and haemolysis was assayed with a suspension of human RBCs (4 %, v/v). Data are means±SEMs (n=3). Note that error bars are often smaller than the symbol size, as well as the use of a logarithmic scale for the concentration. A) Significantly lower cytotoxicity was measured for CM15 administered together with suramin: IC₅₀ values for CM15 alone and for CM15/suramin at 1:1 and 1:2 ratio are (7.6±0.2), (19.6±4.3), and (66.9±4.5) μm, respectively, with p=0.0011 for CM15 versus CM15/Sur (1:1) and p<0.0001 for CM15 versus CM15/Sur (1:2). The effect was stronger when CM15 was mixed with suramin at higher (1:2) molar ratio. Suramin alone showed no cytotoxic effect (IC₅₀>100 μm). B) Haemolytic activity of CM15 [HC₅₀= (45.9±1.6) μm] was abolished in the presence of suramin at both ratios applied (HC₅₀>200 μm). Suramin alone showed no haemolytic effect (HC₅₀> 200 μm). Note that data points overlap for Sur, CM15/Sur (1:1) and CM15/ Sur (1:2).