Effect of GNP functionalisation and multiple N ‐methylation of β ‐amyloid residue (32–37) on Gram‐positive bacterium

Abstract

In the previous report, the authors showed the gold nanoparticle (GNP) functionalised multiple N ‐methylated fragments of the residue (32–37) of beta (β)‐amyloid protein (1–42), CGGIGLMVG and CGGGGGIGLMVG toward disruption of β ‐amyloid (1–42), the predominant component of senile plaques. Herein the in vitro antimicrobial activities of both normal and multiple N ‐methylated sequences of CGGIGLMVG and CGGGGGIGLMVG were screened and it was found that all the eight sequences including four (non‐functionalised with GNP) to possess activity against both Gram‐positive [Staphylococcus aureus (ATCC 43300) and Enterococcus faecalis (ATCC 5129)] and Gram‐negative [Escherichia coli (ATCC 35218), Pseudomonas aeruginosa (ATCC 27853) and Klebsiella pneumoniae (ATCC 700603)] bacteria. Among them, N ‐methylated sequences CGGIGLMVG and CGGGGGIGLMVG shown remarkable activity against Gram‐positive bacteria.

1 Background

The development of small molecules targeting microbial infections and cancers has been the subject of drug discovery for many years [1]. An approach aimed at improving the selectivity of target drugs is the current interest of medicinal and synthetic chemists [2]. One way to do so is through chemical modifications of existing drugs, yet the struggle to construct efficient molecules for various diseases by incorporating suitable and effective ligands or metals making the drug more efficient [3] still persistent. Peptide chemistry has matured over the past five decades, which enables scientist to synthesise libraries of peptides, small‐to‐medium size proteins and with various post‐translational modifications [4]. Peptides and proteins are the fundamental contributors of almost all physiological activities of biological systems as they involve in a variety of physiological and pathological processes and play critical roles in modulating various cell functions. Compared with small chemical entity drugs, peptide‐based drugs possess certain favourable characteristics in terms of higher potency and high selectivity [5]. Peptide drugs have been successfully applied in treating certain human diseases, for example, breast cancer, prostate cancer [6], multiple sclerosis [7], Alzheimer's disease [8] and type II diabetes [9] are the common ones. The synthesis of peptide drugs was made easy by the introduction of solid‐phase peptide synthesis (SPPS) in the early 1960s, various characterisation techniques [high‐performance liquid chromatography (HPLC), electrospray ionisation mass spectrometry], capping with various nanoparticles, and also in inclusion of amino acids into bio‐active molecules to enhance the possible activity of the drug candidates [10]. At present, smaller sequences dominate in the market with over 100 approved peptide‐based therapeutics [11]. Besides many advantages, peptides consisting of natural 20 amino acids alone are relatively not suitable drugs since they are quickly degraded by proteases and excreted from the body [12]. Therefore, it is necessary to synthesise the peptides with desired modification to the original sequence to compensate for the disadvantages associated with existing therapeutic peptides or to improve the activity, stability to proteolytic degradation and increase serum half‐life.

On the other hand, N ‐methylated amino acids are also important building blocks of several drugs. It was reported that peptides containing single/multiple N ‐methylated amino acids are successful in inhibiting amyloidosis [13]. N ‐methyl endothelin hexapeptide antagonists have shown enhanced proteolytic stability and cellular permeability [14]. Gorden et al. [15] demonstrated that alternative N ‐methyl amino acids in the sequence NH₂ ‐K(Me‐L)V(Me‐F)F(Me‐A)E‐CONH₂, Aβ 16–22 m to be an excellent inhibitor of fibrillogenesis of Aβ. Human amylin (20–29) is found to disrupt β‐ sheet formations, and it is interesting to note that N ‐alkylation retards fibrillogenesis [16]. N ‐methylated peptides have also been used as therapeutic agents, because of their ability to easily diffuse through the blood brain barrier (BBB). For example, N ‐methylated peptides have been used as BBB shuttles to carry L ‐Dopa and baicalin drugs through the Parallel artificial membrane permeability assay membrane across BBB models by passive diffusion [17]. It should also be noted that N ‐methylated peptides improve pharmacological properties such as lipophilicity, proteolytic stability, bio‐availability and conformational rigidity [18]. Multiple N ‐methylations can drastically improve the metabolic stability and intestinal permeability of peptides (1, Fig. 1), and also increase oral bio‐availability and selectivity [19]. N ‐methylating the amide (NH) groups of amino acids and peptides at the outer edges of β ‐sheet prevents intermolecular hydrogen bonding and β ‐sheet formation of the α ‐peptides, thus preventing aggregation, also it was shown that, N ‐methylation blocks potential hydrogen bonding on the backbone conformation of bio‐active cyclic peptides. Tri‐N ‐methylation of Veber–Hirschmann peptide analogue (2, Fig. 1) has proved to enhance 10% oral bio‐availability and selectivity [19]. It is also important to note that, while enhancing the pharmacokinetic parameters by N ‐methylation, the active peptides retained their biological activities [20]. There is therefore a scope in designing N ‐methylated amino acids and using them as therapeutic agents. Fjell et al. [21] have reported in their review that antimicrobial peptides (APs) and amyloidogenic peptides are predominant sequences of cytolytic peptides. Butterfield and Lashuel [22] have studied and well documented that APs to induce toxicity toward bacterial cells and amyloidogenic peptides toward neuron cells and both the peptides capable of disrupting membrane under similar mechanism to induce cell toxicity.

Fig. 1.

(1) Intestine permeable N‐methyl‐cyclic peptide and (2) Veber–Hirschmann peptide

On the other hand, synthesis and use of soluble gold (Au) was initiated in 290 AD for photography and artwork [23]. Researchers are attracted to explore the synthesis and use of Au nanoparticles (GNPs) after Michael Faraday's experiment on the reduction of chloroaurate by phosphorous in carbon disulphide to obtain deep red colloidal solutions of Au [24]. Since then, thousands of research articles are reported for the synthesis, properties and assembly of nanoparticles employing numerous possible ways and materials [23]. The technology of utilising nanomaterials has now been extending its major role in all possible areas of human needs and the use of nanomaterials to catalyse organic reactions is one of the most important applications [25]. Consequent impacts lead the synthesis of organic molecules by homogeneous and heterogeneous catalysis under milder reaction conditions and separations of reaction mixture with easy and less polluting processes [26]. Silver and GNPs are the centre of attraction among other nanoparticles, and have become important tool for chemists and biologists owing to their wide spectra of applications in constructing bio‐molecules and even as prime agents for drug delivery [27, 28]. By varying the size and shape of silver and GNPs can make them express various colours by surface plasmon absorption leading plethora of varied biological and chemical applications [29]. Therefore, synthesis of GNPs and the discovery of their applications are critical and of current interest.

While thorough screening of the sequences of APs database (APD) and the collection of APD containing 2330 Anti Microbial Peptidess, it was found that the sequences contain LACIVFWM hydrophobic amino acids, STNQY polar amino acids and KREDHGP charged amino acids. In our previous effort to disrupt amyloid protein using the sequences CGGIGLMVG and CGGGGGIGLMVG, we have discovered the GNP functionalised multiple N ‐methylations of these peptides to be effective in dis‐aggregation of β ‐amyloid (1–42) [30]. On the basis of some common features between the APs and amyloid peptides, it was planned to test the possible antimicrobial activity of CGGIGLMVG and CGGGGGIGLMVG and the multiple N ‐methylated sequences of these peptides against both Gram‐positive (Staphylococcus aureus and Enterococcus faecalis) and Gram‐negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) bacteria. It was found that, normal peptide and the multiple N ‐methylated peptide sequences of CGGIGLMVG and CGGGGGIGLMVG have shown activity against both Gram‐positive and Gram‐negative pathogens in moderately. However, the GNP functionalised N ‐methylated sequences showed remarkable activity against Gram‐positive bacteria.

2 Experimental Section

2.1 Peptide synthesis

Peptides were synthesised by standard Fmoc SPPS procedures. Fmoc–Wang resins (Sigma–Aldrich) were swelled in dimethylformamide (DMF) (Sigma–Aldrich) followed by washings with dichloromethane (DCM). Amino acids (Sigma–Aldrich) were dissolved in DMF (Sigma–Aldrich) along with coupling agent benzotriazole tetramethyluronium hexafluorophosphate (2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate) (Sigma–Aldrich). About 0.5 M solution of N ‐diisopropylethylamine (Sigma–Aldrich) was used as the activator base. Deprotection of amino acids was performed by two washing cycles of 20 min each using a 20% v/v piperidine in DMF. The peptides were cleaved from the resin with 20 ml of cleaving mixture [20% v/v trifluoroacetic acid (TFA), 1% v/v trisisopropylsilan and 1% v/v 1,2‐ethanedithiol and 1% v/v thioanisole in DCM at 20°C for 2 h], the resin was washed twice with DCM, TFA was removed under a stream of nitrogen followed by vacuum drying. The crude peptide was purified by preparative reverse‐phase HPLC (Shimadzu‐HPLC‐ACE 5 C 18 Signal to noice ratio A 38892, 150×21.2 mm²) on a module. Eluents used were 0.1% formic acid in water (v/v; buffer A) and 0.1% formic acid in methanol (v/v; buffer B) with a flow rate of 20 ml/min. The preparative HPLC fractions dried under high‐vacuum rotavap, frozen in liquid nitrogen and lyophilised to get pure peptides which were analysed on Shimadzu Liquid Chromatography‐Mass Spectrometry‐2020, matrix‐assisted laser desorption/ioniser.

2.2 Synthesis of GNPs and conjugating with peptides

Aqueous chloroaurate was reduced by sodium citrate to prepare GNPs. Glassware used to prepare GNPs were thoroughly washed with aquaregia followed by double‐distilled water, rinsed several times with Millipore water (18 MΩ using a Milli‐Q water system) and dried in an oven overnight prior to use. HAuCl₄  × 3H₂ O (25 µl) was added to 47.5 ml Millipore water, and the yellow coloured aqueous Au solution was brought to boil. Sodium citrate (2.5 ml) solution in water (1% w/v) was then added and the dispersion refluxed with stirring for 20 min. The complete reduction of HAuCl₄  × 3H₂ O was observed by the appearance of a clear wine‐red colour which was confirmed by ultraviolet–visible. The respective peptides (2.5 mM) were added to the resulting aqueous solution of mono‐dispersed GNPs and efficiently stirred for 5–6 min, during the attachment of peptide onto GNPs, the wine‐red colour of the aqueous solution of GNPs turned blue which were centrifuged for 20 min at (8 × 1000 RPM) and drained. The peptide‐coated GNPs were added to 10 ml of 10 mM sodium phosphate solution and utilised for further experiments.

2.3 Characterisation of GNPs by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses

The functionalised GNPs have been thoroughly characterised by SEM and TEM represented in Figs. 2 and 3. The AuNPs were repeatedly centrifuged and re‐dispersed in sterile distilled water prior to SEM and TEM analyses to remove any unreacted material. The results obtained by SEM analyses have confirmed that the particles are of nano‐size. The spherical shape of the nanoparticles is shown in the SEM images. The energy dispersive X‐ray spectroscopy (EDX) analysis confirmed that the particles were composed of element Au. TEM analysis showed that GNPs were poly‐dispersed and predominantly spherical or polyhedral in shape. The majority of the GNPs were 14 ± 1 nm in size and the embedded figure clearly shows the morphology of GNPs. The selected area electron diffraction pattern (SAED) clearly indicated that the AuNPs formed are polycrystalline in nature with clear lattice fringes, characteristic of crystalline nature of the obtained nanoparticle. After characterisation of the AuNPs and functionalisation with respective peptides, we were excited to know their efficiency as possible antibacterial activity.

Fig. 2.

Characterisation of GNPs by TEM and SEM analyses

Fig. 3.

Clockwise from top left

(a) High‐resolution TEM micrograph, (b) Histogram, (c) SAED pattern, (d) Lattice fringes of AuNPs

3 Results and discussion

Since it is known that the common features between AP and anti‐amyloid peptides and we had already worked on synthesis of extended N ‐methylated fragments (32–37) of Aβ and successful demonstration of dis‐aggregation of amyloid protein (1–42), we wanted to utilise the sequence for possible antimicrobial activities. IGLMVG is the (32–37) residue of DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA expected active part of the sequence from the previous study. The sequence GVMLGI was extended with two glycines in one example and five glycines in other to act as spacers, and next to spacer glycines cysteine was attached to provide sulphur terminal to facilitate conjugation with GNPs. Therefore, CGGIGLMVG and CGGGGGIGLMVG are the parent sequences of this paper and varied methylations have been done to improve the activity of the sequences as shown in the structures of peptides in Fig. 4. Therefore, out of four peptides, first two are the parent sequences (a) Cys–Gly–Gly–Ile–Gly–Leu–Met–Val–Gly and (b) Cys–Gly–Gly–Gly–Gly–Gly–Ile–Gly–Leu–Met–Val–Gly. Other two are N ‐methylated sequences of (a) and (b) [(c) Cys–Gly–Gly–N ‐methIle–N ‐methGly–N ‐methLeu–Met–N ‐methVal–N ‐methGly and (d) Cys–Gly–Gly–Gly–Gly–Gly–N ‐methIle–N ‐methGly–N ‐methLeu–Met–N ‐methVal–N ‐methGly]. All four sequences were conjugated with GNP and evaluated along with non‐conjugated peptides for their activities.

Fig. 4.

Sequences of both normal and N‐methylated peptides (a–d)

3.1 Antibacterial activity

The GNP functionalised normal and multiple N ‐methylated peptides (a)–(d) were screened for their in vitro antibacterial activity against both Gram‐positive and Gram‐negative bacteria. The bacterial test cultures used in this paper were S. aureus (ATCC 43300), E. faecalis (ATCC 5129), E. coli (ATCC 35218),P. aeruginosa (ATCC 27853) and K. pneumoniae (ATCC 700603) bacteria. The disc diffusion method was followed to study the antimicrobial activity of all normal and N ‐methylated peptides under similar conditions at pH 7.4. Stock solutions of all peptides diluted in dimethyl sulphoxide (1% DMSO in Millipore water (18 MΩ using a Milli‐Q water system) to give the concentration of 500 μg/ml. Gentamycin and streptomycin were used as reference drugs dissolving 500 μg/ml in 1% DMSO in Millipore water. The incubation was carried out at 37 ± 1°C for 24 h. The zones of inhibition produced by each compound were measured in millimetres and all tests have been carried out in triplicate and average is given in Table 1. It is very clear from Table 1 that GNP functionalised N ‐methylated peptides have more activity against both Gram‐positive and Gram‐negative bacteria compared with that of normal peptides. It is also worth noting that comparative results between naked peptides and GNP functionalized peptides are very interesting.

Table 1.

Antimicrobial activity of GNP functionalised normal and N ‐methylated peptides CGGIGLMVG and CGGGGGIGLMVG and comparative study of same sequences without GNPs

Bacteriaa	Percentage inhibition GNP functionalised peptides		Naked peptides		standard drugs
	a/b	c/d	a/b	c/d	Gentamycin	Streptomycin
S. aureus	6.3/15.7	6.2/15.8	3.2/8.6	3.2/8.8	26.5	4.8
E. faecalis	5.2/14.4	5.4/14.3	2.4/6.5	2.5/6.5	25.4	20.6
E. coli	5.4/7.3	5.5/7.2	4.1/5.5	4.3/5.7	20.1	19.8
P. aeruginosa	6.1/8.8	5.9/9.1	3.8/4.4	3.8/4.6	27.8	20.7
K. pneumoniae	7.3/9.2	7.2/9.3	3.4/5.1	3.4/5.3	22.7	19.4

^a Both Gram‐positive and Gram‐negative bacteria have been used to test the efficiency of all the peptides.

For our observations, GNP functionalised peptides showed more activity compared with naked peptides of same sequences. These results are not surprising to us since GNPs are best in attaching with bacteria facilitating the activity of the peptides. It is very important to note that there was no significant effect on glycine chain length. As can be seen from Table 1, there is no difference in activity between CGGIGLMVG and CGGGGGIGLMVG irrespective of they are normal or N ‐methylated. Therefore, it is confirmed that the active species in the sequence is IGLMVG and the efficacy improved by N ‐methylation and more effective when conjugated with GNPs.

4 Conclusions

In conclusion, the efficacy of normal and multiple N ‐methylated peptides of β ‐amyloid fragment IGLMVG against both the Gram‐positive and Gram‐negative pathogens is demonstrated. It was found that N ‐methylation is attributed to improve the activity of the sequence. The results also indicated that N ‐methylated peptides are more active against Gram‐positive bacterium. This report will definitely be of much interest to the peptide chemists and biologists in designing APs and to have N ‐methylated sequences for future exploration.