The copper binding properties of metformin – QCM-D, XPS and nanobead agglomeration

TL;DR: Results of a quantitative investigation of interactions between metformin and L-cysteine-copper complexes suggest that met formin could interact with biological copper, which plays a key role in mitochondrial function.

About: This article is published in Chemical Communications.The article was published on 2015-11-24 and is currently open access. It has received 24 citations till now.

Summary

Summary

• Study of the copper binding properties of metformin is important for revealing its mechanism of action as a first-line type-2 diabetes drug.
• A quantitative investigation of interactions between metformin and L-cysteine-copper complexes was performed.
• The results suggest that metformin could interact with biological copper, which plays a key role in mitochondrial function.
• This is one of the common copper binding domains in copper containing proteins.
• 10 Metformin-copper complex has been shown to form electron delocalised planar ring structures 11, 12 (illustrated in Scheme 1(a)) and the determined stability constant (pK) varies between 14.3 and 18.5.

Figures

Fig. 3 V20 spectra of 200 nm MNB suspensions (1 mg ml 1) incubated in the Cu formed L-cysteine–copper complex (1) (a) and complex (2) (b), respectively, and subsequently metformin solutions of the indicated concentrations measured after incubation in a strong magnetic field.

Fig. 2 (a) Cu 2p XPS spectra for different metformin incubation times. (b) Integrated area of C 2p and S 2p peaks as function of metformin incubation time. The error bars represent the standard deviation for triplicate measurements.

Fig. 1 QCM-D frequency and dissipation data (overtone 7): (step A) perfusion with PBS; (step B) addition of 100 mM buffered Cu(NO3)2 solution; (step C) rinsing with PBS; (step D) injection of 10 mM buffered metformin solution; (step E) rinsing with PBS.

Frequently asked questions

Q1. Why did the authors use streptavidin functionalized magnetic nanobeads?

Because of the favoured formation of strongly bound thiolates on gold, the authors used streptavidin functionalized magnetic nanobeads (MNBs) to form the L-cysteine–copper complex (2). 

Q2. What is the effect of metformin on copper?

During incubation with metformin, the abstraction of copper ions breaks the clusters, resulting in an increase in the signal amplitude due to an increased quantity of free MNBs. 

Q3. What was used to evaluate the quantitative change in the copper and sulphur species?

X-ray photoelectron spectroscopy(XPS) was applied for elemental analysis to evaluate the quantitative change in the copper and sulphur species during the process. 

Q4. What is the effect of increased incubation time on the integrated area of the S peaks?

With an increased incubation time, the integrated area of the S peaks remains relatively stable regardless of the incubation time, being indicative of a stable L-cysteine SAM. 

Q5. What is the way to measure the surface change of the functionalized QCM chip?

Monitoring of the surface change of the functionalized QCM chip allows an effective investigation of the extent to which metformin can abstract copper from surface bound L-cysteine– copper complexes. 

Q6. What was the process of forming the L-cysteine–copper complex?

the binding sites of the amino acids were saturated with copper ions to form the L-cysteine–copper complex followed by exposure to metformin solution. 

Q7. What is the extent of copper abstraction from the sulphur coordinated complex?

the extent of copper abstraction from the sulphur coordinated complex, which is analogous to the protein bound copper in the biological systems, was similar to the abstraction from L-cysteine–copper complexes coordinated by carboxyl/amino groups. 

Q8. How long did the MNBs remain in the disc?

40 Metformin has been added to the MNBs and Cu mixture (volume ratio 2 : 1) and incubated for another 2 minutes followed by loading in the disc and incubation in between permanent magnets of 3 minutes. 

Q9. What is the DD of the copper ions?

Since no significant change in DD was observed during the copper binding process, the L-cysteine– copper(II) complex forms a compact film. 

Q10. What is the effect of increased incubation time on the integrated area of the Cu peaks?

On the other hand, a dramatic decrease (B75%) in the integrated area of the Cu peaks was observed with increased incubation time from 0 to 90 min, demonstrating the extent to which L-cysteine bound copper was removed from the surface due to the interaction with metformin. 

Q11. What is the way to monitor the abstraction of copper?

For monitoring the abstraction of copper, the authors applied an agglutination type assay scheme33,34 with a novel readout.35 As illustrated in Scheme 1(c), the MNBs were modified with biotinylated L-cysteine followed by incubation in copper solution to form the complex, which results in the clustering of the MNBs.