Evaluating phosphodiesterase 7B inhibition by vasicine using Nuclear Magnetic Resonance Spectroscopy

 

Arthi Balasundaram*, Darling Chellathai David

Department of Pharmacology, Sri Ramachandra Medical College and Research Institute,

Sri Ramachandra Institute of Higher Education and Research (DU), Porur, Chennai - 116, Tamil Nadu, India.

 

ABSTRACT:

Background: The phosphodiesterase (PDE)7B, belonging to family PDE7 has high affinity and specificity for cyclic adenosine monophosphate (cAMP). This was reported to regulate a range of physiological functions. Inhibition of this enzyme was found to have therapeutic effects. The present study aims to evaluate the inhibition of PDE7B by vasicine, using Nuclear magnetic resonance (NMR) spectroscopy. Materials and Methods: The 1D NMR based time series experiment was performed; the initial and the final spectra were compared for the formation of cAMP in the presence and absence of the inhibitor. Results: The PDE7B was inhibited completely by vasicine and no formation of AMP was seen in the time series spectra. Conclusion: NMR spectroscopy method revealed the PDE7B inhibitory activity of vasicine which may be considered as a therapeutic agent for diseases regulated by cAMP.

 

 

 

INTRODUCTION: 

Phosphodiesterases (PDEs) modulates the intracellular cyclic adenosine monophosphate (cAMP). This cyclic nucleotide plays a significant role as second messenger in several physiological processes, like cardiac output, vascular resistance, immune response, vision, inflammation, neuroplasticity, visceral motility and reproduction. The range of physiological functions controlled by cAMP highlights the significance of PDEs for the appropriate function of the organism. PDEs consist of enzymes classified into 10 families. Usually, ≥60% amino acid sequence similarity is seen among the members of the PDE family, but this similarity drops to ≤40% between families¹. The PDEs differ from each other and can be identified by their protein structure, localization, specificity, sensitivity, and regulation. This diversity explains the regulation of various functions by members of PDE superfamily without overlapping, also with structural and physiological specificities. This makes PDEs as potential targets for the treatment of diseases regulated by cyclic AMP signalling.

 

Mammalian PDEs could promote the hydrolysis of cAMP in to inactive 5′-AMP to modulate various physiological functions. Many studies have demonstrated that PDEs were outstanding therapeutic targets for the development of drugs against various diseases^2,3. Phosphodiesterase 7 (PDE7) exists in two isoforms, PDE7A and PDE7B. They are cAMP specific enzymes. The PDE7B inhibition was represented for treatment of neurological and airway inflammatory diseases⁴. The importance of cyclic nucleotide in neurodegeneration⁵ and haematological disorders⁶ was also elaborated by previous studies. The present study evaluates vasicine for PDE7B inhibition, using Nuclear magnetic resonance (NMR) spectroscopy.

 

MATERIALS AND METHODS:

The PDE7B enzyme (10ug) was purchased from Signalchem (Canada). The vasicine (10mg) was purchased from Natural Remedies (Bangalore). cAMP was purchased from Sigma-Aldrich (India)

 

NMR sample preparation:

The NMR sample contained 1.5ug of PDE7B enzyme, Tris 50mM (pH 7.4), .002% Triton-X, magnesium chloride (MgCl2-50mM) and bovine serum albumin (BSA-3uM). The positive control experiment was carried out for PDE7B enzyme in the presence of cAMP (2mM). The inhibitor-based assay was carried out in the presence of vasicine (2mM) and the natural substrate cAMP (2mM).

 

NMR experiment:

Since the NMR sample contained 50mM of Tris in addition to water, a double solvent suppression-based pulse sequence was used to suppress both Tris and water. The offset of the peaks to be suppressed was changed prior to each of the W5 Watergate segment present in the default pulse sequence. The 1D NMR based time series experiment was performed; the initial and the final spectra were compared for the formation of cAMP in the presence and absence of the inhibitor. Control experiment was also performed for the substrate cAMP in the NMR buffer devoid of PDE7B, to ensure its stability over the time period of kinetic experiment.

 

RESULTS AND DISCUSSION:

This qualitative study evaluated PDE7B inhibitory property of vasicine using NMR spectroscopy. The cyclic AMP was analysed using NMR spectroscopy as done in previous studies^7-12. The proton spectrum contains the time series spectra of cAMP (2mM) in the presence of enzyme PDE7B, overlaid from 0 h, 8.5 h and 18.5 h. The blue arrow in spectrum indicates the position of AMP, the product formed from cAMP. The peaks of cAMP appear at 8.26ppm and 6.19ppm. The peaks of AMP appear at 8.59ppm and 6.19ppm (doublet) [Figure 1].

 

Similar kind of linearity for AMP was reported by other NMR studies¹³. The other spectrum contains the time series spectra of cAMP (2mM) and Vasicine (2mM) in the presence of enzyme PDE7B, overlaid from 0 h, 8.5 h and 17.5 h. The red arrow in spectrum indicates the position of peaks arising from vasicine. The PDE7B was inhibited completely by vasicine, so that no formation of AMP was seen in the time series spectra [Figure 2].

     

Figure 1: The proton NMR spectra containing the time series spectra of cAMP (2mM) in the presence of enzyme PDE7B (A). B- resolution of spectra [Standardization].

 

Figure 2: The proton NMR spectra containing the time series spectra of cAMP (2mM) and vasicine (2mM) in the presence of enzyme PDE7B.

Enzyme activities like modulation or synchronization of mechanistic processes prior or following the chemical reaction, may be based on kinetic or equilibrium changes in protein structure. Exchange of more open conformational states with more closed states of protein can influence enzyme activity^14-17. The NMR spectroscopy is commonly used to study the conformational dynamic processes in enzymes, because these events can be differentiated over multiple time scales with atomic site resolution. The NMR spectroscopy allows thorough description of the extent and time scales of protein conformational fluctuations linked to numerous stages of complex enzymatic reaction mechanisms¹⁸.

 

Vasicine/Peganine is a quinazoline type alkaloid mainly obtained from the plant Adhatoda Vasica (zeylanica). Few of the main chemical constituents of this plant are vasicine (derived from leaves), 2'-hydroxy-4- glucosyloxychalcone, vasicol (from leaves), vasicinone (from leaves, stem and roots), vasicinol (contained in stem and roots), and deoxyvasicinone (from leaves). It was first isolated from by Sen and Ghose in 1924¹⁹. Vasicine was proved safe by acute and chronic toxicity studies^20,21.

 

PDE7B activity in presence and absence of vasicine was analysed in-vitro using NMR proton spectroscopy by taking AMP formation as evaluating factor. It revealed the inhibitory action of vasicine. Many studies have found the activity of PDE’s through NMR spectroscopy. A study revealed the role of PDE in phospholipid sparing during ischemic stress²² and another study revealed the adulteration of plant/herbal products²³.

 

LIMITATIONS:

Since the present study was an exploratory qualitative analysis, IC₅₀ value was not established.

 

CONCLUSION:

The NMR based enzyme kinetic assay was developed and used to monitor the inhibition of PDE7B in the presence of vasicine. This study demonstrated that Vasicine inhibits PDE7B enzyme and prevents the formation of AMP from cAMP at substrate to inhibitor ratio of 1:1. Thus vasicine may be considered as a therapeutic agent for diseases regulated by cAMP.

 

ACKNOWLEDGEMENTS:

The authors are thankful to the Indian Council for Medical Research (ICMR), New Delhi for providing financial support in the form of TSS fellowship no. PhD (integrated) 32-F.T./I/2014.

 

CONFLICTS OF INTERESTS:

None.

FUNDING SOURCES:

ICMR TSS fellowship no. PhD (integrated) 32-F.T./I/2014.

 

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Accepted on 28.04.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(1):103-106.