The history of silver applications started from its use in coins and jewelry. Women loved to decorate themselves with various trinkets of silver, but it is less well known that this metal can be an excellent metallotherapeutic agent. In the human body, silver (Ag) is not acting as an endogenous metal and exhibits relatively low toxicity. Ag(I) coordination compounds with a variety of ligands having nitrogen, phosphorus, and/or sulfur donor atoms have large applications in medicinal and analytical chemistry [
Human serum albumin (HSA) is responsible for about 60% of the plasma protein in humans and is accountable for nearly 80% of the osmotic pressure of the blood, and it plays a prominent role in drug disposition and efficacy [
Norharmane, 9H-pyrido[3,4-b]indole, is a rather unconventional ligand, belonging to an alkaloid family called
The structures of the ligand 9H-pyrido[3,4-b]indole (Hnor) and the four used Ag(I) compounds.
In a previous study, some of us have shown that compounds of composition [Ag(Hnor)2](anion) display significant anticancer activity for the anions, namely, NO3−, ClO4−, BF4−, and PF6−, with the ClO4− compound being even comparable to cisplatin in two different cancer cell lines [
AgNO3, AgClO4, AgBF4, AgPF6, DL-tryptophan, and formaldehyde were purchased from Sigma-Aldrich. Human serum albumin (HSA; ≥99%, Sigma, USA) was essentially fatty-acid free and globulin free, purchased from Sigma, and used as received. We have synthesized the four Ag(I) compounds as described in before [
The samples of HSA were prepared in 20 mM phosphate buffer (pH 7.4), whereas silver complexes (1 mM) stock solution were prepared in DMSO and further diluted in 20 mM phosphate buffer to reach the desired concentration. In all the samples, the final concentration of DMSO was not more than 1%. The concentration of HSA was determined using the Beer–Lambert law with the molar extinction coefficient of 36500 M−1·cm−1 at 280 nm. To study structural changes in HSA by the addition of [Ag(I)(Hnor)2](anion) compounds, the UV absorption spectra were measured, by variation of the concentration of the [Ag(I)(Hnor)2](anion) compounds, while keeping the concentration of HSA constant. UV absorption spectra, from 240 nm to 320 nm, were recorded on a Perkin–Elmer Lambda 45 spectrophotometer at 25°C. Quartz cuvettes of 1 cm path length were used for the measurements. Fluorescence measurements were performed on a Hitachi spectrofluorometer (Model F 7000) equipped with a PC. The fluorescence spectra were collected at 25°C with a path length cell of 1 cm. The slit width used was 5 nm with a protein concentration of 5
CD measurements were carried out on a Jasco spectropolarimeter (Model J-815) equipped with a microcomputer. The instrument was calibrated with D-10-camphorsulfonic acid. All the CD measurements were performed at 25°C with a thermostatically controlled cell holder, attached to a Neslab RTE-110 water bath with an accuracy of ±0.1°C. Spectra were collected with a scan speed of 0.2 nm/min and a response time of 1 s. Each spectrum was taken as the average of three scans. The far-UV CD spectra were measured at a protein concentration of 20
All the spectra were recorded after equilibration of the reaction mixture for 5 min.
The rigid molecular docking studies were performed by using HEX 8.0.0 software [
Synthesis of the four Ag(I) compounds, namely, [Ag(Hnor)2](ClO4) (
The interaction between the small bioactive molecules and protein receptors is a fundamental step in the drug discovery process. Obtaining a thorough idea of the interaction of the protein with chemical entities plays a vital role in the etiology of several diseases. The protein-drug intermediate products involved in governing various biochemical phenomena in both normal and diseased cells are known to play a significant role in metabolizing therapeutic compounds and their transport [
The binding propensity of the drug candidate with the biomolecule was first studied by using the UV technique. The cumulative absorption of three aromatic amino acid residues gives rise to an absorption peak at 280 nm for human serum albumins (HSA) [
UV absorption difference spectra of HSA (5
With the concomitant increase in concentration of Ag(I) compounds, the absorbance of HSA increased, and shifts toward longer wavelengths were observed; the Ag(I) compounds give a definite pattern of the UV–Vis spectrum with weak absorbance at a higher concentration between 295 and 320 nm, ascribed to the ligand “Hnor.” The profound enhancement of UV absorbance (hyperchromism) with a redshift (bathochromic effect) of 7 nm (
The difference spectra of HSA have confirmed that the conformational changes to HSA are due to binding of the Ag(I) compounds (Figure
Fluorescence emission spectra of HSA (5
The interaction between metal compounds and proteins has been widely investigated by using fluorescence spectroscopies [
The Stern–Volmer plot of HSA fluorescence quenching by [Ag(I)(Hnor)2]ClO4 at 295 nm. Inset: plot of log (
A gradual decrease in the luminescence of HSA was observed upon increasing concentration of Ag(I) compounds showed significant redshift at the maximal emission wavelength of tyrosine(Tyr) and tryptophan(Trp) residues of 7 nm (
The interaction of the Ag compounds with HSA was quantified; the Stern–Volmer equation has been employed [
The value of tau(zero),
Fluorescence emission spectra of HSA (5
Stern–Volmer quenching constants and bimolecular quenching rate constant for the interaction of HSA with four Ag(I) Hnor compounds.
Complex |
|
|
|
|
∆ |
---|---|---|---|---|---|
|
5.37 | 53.7 | 3.74 | 1.1 | −37.50 |
|
5.34 | 53.4 | 3.64 | 1.0 | −37.44 |
|
4.53 | 45.3 | 0.55 | 1.2 | −32.73 |
|
3.97 | 39.7 | 1.14 | 1.1 | −34.55 |
Hnor | 2.95 | 29.5 | 0.33 | 1.0 | −31.40 |
AgNO3 | 3.10 | 31.0 | 1.94 | 1.1 | −35.88 |
Thus, these data ascertain the static quenching in the interaction of Ag(I) compounds with HSA, by the calculated value of
The values of
The extent of interaction of Ag(I) compounds with HSA was found in the order
Since it is known that HSA is a monomeric, three-domain, allosteric protein with only one free cysteine, Cys34, the Ag(I) ion could selectively bind at this site because it has a strong preference for S-donor atoms. Also, it can be assumed that the counteranions have facilitated the microenvironmental changes and may have contributed to the exposure of Cys34 from the subdomain of HSA and facilitated that the sulfur atom of cysteine will coordinate to the Ag(I) center of the compounds. Nevertheless, the known affinity of Ag(I) for Met residues and disulfide bridges and nitrogen atoms of HSA coordination on such sites cannot be ignored completely. This hypothesis is supported by the observation that the extent of microenvironmental changes of the protein is much higher in the presence of additional ClO4− and NO3− anions (see below), and which agrees with the trend in their binding parameter (Table
Given the fact that the highest anticancer activity and also the most substantial HSA interaction takes place in case of the perchlorate and nitrate salts, it was decided to add extra perchlorate (and also nitrate) for all cases and to study the effect on the binding affinity. So, we carried out two sets of experiments and studied the binding propensity of Ag(I) compounds with HSA. In one experiment, the extra ClO4− anion was added in a 1 : 4 ratio, compared to the Ag compound. In a second experiment, we used additional nitrate together with the Ag(I) compounds. Details are given in Figures
Stern–Volmer plot of HSA fluorescence quenching by (
Far-UV CD spectra of HSA-compound system (HSA = 20
To determine the role of only the anions (i.e., without Ag and Hnor), we also used KClO4 and KNO3 and studied their HSA binding by adding variable amounts of anions to the concentration maximum used in the experiments. We found that on the addition of KClO4 and KNO3 to HSA, only a negligible perturbance of the HSA structure has occurred. Hence, the effect of these anions (ClO4− and KNO3−) on HSA conformation can be neglected in comparison to the effect of Ag compounds and Ag compounds + additional anions on HSA conformation. When the extra anions + compounds were titrated, the results obtained did show an exponential increase in binding affinity of the Ag(I) compounds.
These findings can be attributed to the enhanced exposure of the cysteine sulfur atoms of HSA, most likely caused by the significant electrostatic effect of the additional anions. This behavior is indicative of the increased microenvironmental changes of the HSA, thereby allowing the more binding of the Ag(I) center to coordinate to the HSA binding site.
The binding strength was evaluated by calculating the Stern–Volmer constant (
Parameters for the interaction of HSA with Ag(I) compounds with the addition of extra anion, namely, ClO4− and NO3−.
Complex |
|
|
|
∆ |
---|---|---|---|---|
|
||||
|
2.21 | 17.2 | 1.77 | −64.12 |
|
1.29 | 6.05 | 1.58 | −57.48 |
|
0.84 | 0.46 | 1.55 | −55.13 |
|
1.85 | 1.19 | 1.69 | −61.52 |
|
||||
|
2.06 | 3.64 | 1.64 | −60.26 |
|
1.24 | 0.66 | 1.53 | −56.05 |
|
0.80 | 0.08 | 1.41 | −51.01 |
|
1.38 | 0.38 | 1.48 | −54.67 |
The effect of anions was also analyzed on the basis of binding constant (
CD spectroscopy is an ideal technique for monitoring the conformational changes of proteins. As shown in Figure
Binding of compounds (a)
The CD signal, expressed in millidegree, obtained over the wavelength range of 190–250 nm, was converted to a mean residue ellipticity (MRE, θ), using the following conversion:
The
With increasing concentrations of the Ag(I) compounds, the CD signal exhibits significant changes in ellipticity, and this change corroborates with the binding of the compounds with the HSA backbone (spectra 2–6, Figure
The results confirm that the compound binds to the amino acid residues of the primary polypeptide chain in HSA, which has distorted its hydrogen-bonding networks. Interestingly, the decrease in the
Effect of the Ag(I) compounds on the
Concentration (mM) | % | |||
---|---|---|---|---|
|
|
|
| |
0 | 67.03 | 67.03 | 67.03 | 67.03 |
20 | 64.07 | 62.85 | 63.57 | 65.25 |
40 | 62.23 | 61.61 | 62.57 | 63.82 |
60 | 58.59 | 58.41 | 60.37 | 61.53 |
80 | 50.18 | 52.02 | 56.31 | 56.66 |
100 | 35.63 | 42.73 | 47.18 | 47.52 |
To provide further and deeper insight into the interactions of HSA with compounds
(a) Molecular docked model of compound
Noncovalent interactions of compounds (a)
Noncovalent interactions of compound
Name | Distance (Å) | Category | Type |
---|---|---|---|
ARG257: H11-Complex |
2.81 | Hydrogen bond | Conventional |
HIS288: H1-compound |
1.68 | ||
Compound |
3.24 | ||
Compound |
3.63 | Electrostatic | Attractive charge |
GLN196-compound |
3.32 | Hydrophobic |
|
HIS242-compound |
4.78 |
| |
HIS242-compound |
3.92 |
| |
Compound |
5.38 |
| |
Compound |
4.19 |
| |
Compound |
4.75 |
| |
Compound |
5.03 |
| |
Compound |
5.25 |
| |
Compound |
4.23 |
| |
Compound |
4.92 |
|
The resulting docked pattern (Figure
Noncovalent interactions of compound
Name | Distance (Å) | Category | Type |
---|---|---|---|
Compound |
2.73 | Hydrogen bond | Conventional |
LYS190-compound |
4.88 | Electrostatic |
|
Compound |
3.39 | Electrostatic |
|
TYR161-compound |
4.29 | Hydrophobic |
|
TYR161-compound |
4.72 |
| |
Compound |
3.91 |
| |
Compound |
3.64 |
| |
Compound |
4.98 |
| |
Compound |
5.43 |
| |
Compound |
4.91 |
| |
Compound |
4.11 |
| |
Compound |
3.09 |
| |
Compound |
3.21 |
|
To find out the effect of counterion in the HSA binding, we also perform the docking of the individual counterion (nitrate ion and perchlorate ion) with the HSA (Figure
Human serum albumin
Circular dichroism
Cysteine
Methionine
9H-pyrido[3,4-b]indole (norharmane).
The authors declare that there are no conflicts of interest.
Ali Alsalme and Rais Ahmad Khan contributed equally to this work.
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through the research group number RG-1438-006.
Figure S1: UV absorption spectra of HSA in the absence and presence of compounds