It is certified that the work described in this thesis, entitled: "Xanthine-based inhibitors for therapeutic agents targeting phosphodiesterase 9A", carried out by Ms Nivedita Singh (Roll no. for the award of the degree of Doctor of Philosophy is an authentic record of the results obtained from the research work carried out under my supervision in the Department of Biosciences & Bioengineering, Indian Institute of Technology Guwahati, India and this work has not been submitted elsewhere for a degree It is certified that the work described in this thesis entitled: “Xanthine-based inhibitors for therapeutic agents targeting phosphodiesterase 9A”, carried out by Nivedita Singh for the award of the degree of Doctor of Philosophy, is an authentic record of the results obtained from the research work carried out under my co-supervision in the Department of Chemistry, Indian Institute of Technology Guwahati, India and this work has not been submitted elsewhere for a degree.
Importance of uniqueness of PDE9A structure in drug development
Molecular diversification of ligands in PDE9A drug development process
Identification of potent xanthine based inhibitors targeting Phosphodiesterase 9A
- Virtual screening for selection of specific inhibitors for PDE9A
- Comparative studies of top four screened compounds with other PDEs
- Molecular Dynamic Simulation of the best compound obtained from screening against ZINC database
- Pre-docking screening of extracted compounds from ZINC database
- Comparative binding studies of ZINC62579975 with existing xanthine derivative
Molecular Dynamics Simulation of the best compound obtained from screening against ZINC database obtained from screening against ZINC database.
Chapter 3: Development of Xanthine Based Inhibitors, Targeting 85-127
Comparative analysis of pharmaceutical properties of selected compounds
Chemical synthesis of selected xanthine derivatives 129-167
Development of new routes for synthesis of xanthine derivatives
Biological studies of selected compounds 169-209
Spectrophotometric Malachite green activity assay of selected PDEs
Structure activity relationship analysis of selected compounds from virtual screening and manual designing
LIST OF TABLES
Comparative analysis of docking of compound 34 and 40 with various members of PDE superfamily
- Depiction of the surface view of compound 40 when it interacts with PDE9A
- Interaction of Compound 40 with PDE8A - inhibitor bound surface view (left) and empty active site view (right)
- Comparative interaction pattern of IBMX (left) and compound 28 (right) with PDE9A
- Comparative interaction pattern of BAY73-6691 and compound 28 with PDE9A
The role of xanthine derivatives as inhibitors to control the catalytic activity of PDE9A in a pathophysiological state.
LIST OF ACRONYMS
List of Amino acid residues and codes
Code Amino acid residues
Introduction and Literature Review
Prologue
Introduction
In the brain, most of the cellular signaling pathways are conducted through cGMP (Andreeva et al., 2001; Singh and Patra, 2014). Xanthine derivatives are widely known for their non-specific inhibitory properties against phosphodiesterase enzymes (Ogawa et al., 1989).
Cell signaling pathway- an overview
- Role of cGMP in Signal Transduction
- Synthesis of cGMP
- Molecular targets of cGMP
In mammalian cells, cGMP is well known to activate protein kinase G (PKG), which is involved in various cGMP-dependent intracellular cellular functions (Wall et al., 2003). a) Activation of protein kinase G by cGMP. PKG has two functional domains - a regulatory domain containing two cGMP binding sites and a catalytic domain (Hofmann et al., 2000).
Cyclic nucleotide phosphodiesterase enzyme - an overview
These specificities are determined by the catalytic domain, but the mechanism of specific substrate recognition by phosphodiesterases is still a question [Ke et al., 2011;. Whereas in case of dual specificity, the side chain of glutamine can switch between two orientations, resulting in specificity towards both cyclic nucleotides (Jeon et al., 2005; Zhang et al., 2004).
- Catalytic mechanism of PDE9A
- Distribution and localization of PDE9A in mammals
- Cloning, expression and characterization of PDE9A
- Architecture of PDE9A gene
- Importance of catalytic domain of PDE9A
- Therapeutic disease targets of PDE9A
- Importance of uniqueness of PDE9A structure in drug development
PDE9A sammuu fuulduraa bu’uuraa, sammuu fi bulbula urgooftuu keessatti baay’ee ibsama (Andreeva et al., 2001). Bara walfakkaatutti, garaagarummaa ispilaayisii afur (PDE9A1, PDE9A2, PDE9A3 fi PDE9A4) Guipponi fi kkfn gabaafameera.
Molecular diversification of ligands in PDE9A drug development process
TYR424 further increases the polarity of the active site and may act as active residue for inhibitor selectivity (Huai et al., 2004). With appropriate substitution over the xanthine scaffold according to the requirement according to the composition of the active site and the size of PDE9A, a specifically designed inhibitor can be obtained.
Xanthine- as potential scaffold in drug development
- Insight to molecular structure of Xanthine
- Overview of existing xanthine derivatives
- Xanthine derivatives in therapeutics
- Role of xanthine derivatives in pharmacology
- Mechanism of action of xanthine derivatives in mammals
Natural xanthine derivatives are commonly present in cocoa, tea and coffee plants (Baraldi et al., 2007). The phosphodiesterase inhibitory properties of xanthine derivatives have been linked to their tracheal relaxant activities (Ogawa et al., 1989). However, xanthine derivatives such as doxofylline and enprofilin are weak adenosine antagonists and do not stimulate gastric acid and pepsin secretion (Lazzaroni et al., 2007).
Xanthine derivatives such as caffeine, theophylline, etc. are reported for their effective role in reducing the synthesis of leukotrienes (Lee et al., 2014).
Potential of xanthine derivatives to regulate the PDE9A regulated signal transduction pathway
Under such conditions, further signal processing may be hindered by a further decrease in cGMP levels due to normal PDE9A activity. All of these can affect the normal functioning of the pathway (Andreeva et al., 2001; Singh and Patra, 2014). Here, xanthine derivatives as inhibitors play a necessary role in regulating the catalytic activity of PDE9A (Singh and Patra, 2014).
Scope of xanthine derivatives as potential scaffold for PDE9A inhibitor
Research Approach for the present study
Conclusion
Therefore, inhibition of PDE9A is necessary to maintain the consistency of the cGMP-dependent cell signaling pathway. This chapter covers an overview of the signal transduction process in general and details of the structure, function, mechanism of action, tissue distribution, genetics, therapeutic role and current status of drugs developed specifically for PDE9A. This chapter further explores the enormous potential of 'xanthine' in the drug development process.
Thus, the present study has tried to open a new page in xanthine-based research.
Identification of Potent Xanthine Based Inhibitors Targeting
Phosphodiesterase 9A
Introduction
Although researchers have revealed the structure and mechanism of action of the enzyme, its structural uniqueness in terms of substrate selectivity remains to be explored (Huai et al., 2004; Liu et al., 2008). The presence of some unique amino acid residues such as GLU406 and TYR424; which plays a key role in inhibitor selectivity has not yet been exploited (Hou et al., 2011; Huai et al., 2004). Previous studies on the development of drugs targeting phosphodiesterases have shown that xanthine derivatives are nonspecific PDE inhibitors (Glennon et al., 1981; Maurice et al., 2014; Tanaka et al., 1991; Wong and Ooi, 1985 ).
One of the known xanthine derivatives IBMX has been crystallized with PDE9A and submitted to the protein database (Huai et al., 2004).
Materials and Methods
- Structural analysis of active site of PDE9A for in silico studies
- Initial screening of xanthine based derivatives from ZINC database using Lipinski rule of five
- Macromolecule files preparation for virtual screening and other in silico studies
- Virtual Screening for selection of specific inhibitors for PDE9A
- Molecular docking of screened compounds obtained from virtual screening
- Comparative studies of top four screened compounds with other PDEs
- Molecular Dynamic Simulation of the best compound obtained from screening against ZINC database
- Drug likeness and ADMET properties of screened inhibitors
Thus, the current research work screens potent and selective inhibitors for PDE9A using xanthine as a base from ZINC database through virtual screening and docking. The virtual screening files were generated using raccoon, a graphical interface for handling ligand libraries in different formats (PDB, multi-structure MOL2 and PDBQT), multiple receptor conformations (e.g. relaxed complex experiments) and flexible residues. The protein ‘pdb’ files were prepared in Swiss-pdb Viewer, which helped in thoroughly analyzing the protein and preparing the macromolecule file for virtual screening and docking by removing heteroatoms including ligands and water molecules.
The virtual screening was performed in CentOS Linux system using scripts to generate grid .glg file and docking .dlg file.
Results and Discussion
- Structure analysis of Phosphodiesterase 9A
- Docking based virtual screening of xanthine derivatives
- Docking studies of top 10 hits with PDE9A
- Comparative binding studies of ZINC62579975 with various members of PDE superfamily
- Comparative binding studies of ZINC62579975 with existing xanthine derivative
- Comparative binding studies of ZINC62579975 with known inhibitors of PDE9A
- Molecular dynamics simulations of ZINC62579975
- Drug likeness properties of ZINC62579975
This study revealed the best conformation interaction pattern of inhibitors in the PDE9A active site pocket. The presence of a substituent at the N1 position of ZINC12231988, ZINC12231966 and ZINC12232141 results in a change in the orientation of the compounds when they interact in the active site pocket of PDE9A. The difference in the orientation of ZINC62579975 in the active site was also due to the presence of an unsubstituted -NH group.
The compound formed only one H-bond with ASP764 at the entrance of the PDE5A active site.
Xanthine scaffold for future PDE9A drug development
Conclusion
Development of Xanthine Based Inhibitors Targeting
Introduction
These factors have been an obstacle in the progress of the development of specific drugs targeting PDE9A (Meng et al., 2012). The use of the limited available scaffold has been a major obstacle in the development of specific PDE9A inhibitor. Xanthine" may have the potential to act as a new scaffold in the drug development process for
Only the pre-screened compounds will be on the final list of compounds to be synthesized.
Materials and methods
- Manual designing of xanthine based ligands
- Molecular docking of manually designed xanthine derivatives with PDE9A
- Comparative analysis of pharmaceutical properties of selected compounds
- Comparative binding study of selected compounds with existing xanthine derivatives
- Comparative inhibition study of selected compounds and known PDE9A inhibitor
Replacement of xanthine derivatives with fragments according to the amino acid composition of the active site of the target protein can lead to the formation of compounds with a higher affinity for PDE9A. Therefore, in this study, N1, N3, C8, and N9 positions were chosen for the initial phase of xanthine scaffold modifications. PreADMET software was used to calculate the drug similarity, ADMET and toxicity properties of the selected ones.
A comparative study of the reported inhibitor with selected compounds for PDE9A was performed by comparative analysis.
Results and Discussion
- Manual designing of xanthine based ligands targeting PDE9A
- Interaction studies of manually designed xanthine derivatives to find out specific inhibitors for PDE9A
- Interaction study of set-1 compounds with PDE9A
- Interaction study of set-2 compounds with PDE9A
- Comparative interaction study of the best compounds from set-1 and set-2 with various members of PDE superfamily
- Analysis of predicted pharmaceutical properties of set-1 and set-2 compounds
- Comparative binding study of set-1 compounds and existing xanthine derivative
- Comparative binding study of set-1 compounds and known PDE9A inhibitor
All these compounds showed corresponding occupancy at the active site of PDE9A in a similar manner. In this way, the modification at the N1 and C8 positions appropriately accommodated the compound in the active site pocket of PDE9A. Both compounds formed two H-bonds with GLN453, showing a similar interaction pattern in the active site pocket of PDE9A.
BAY73-6691 formed two H-bonds with the side chain of GLN453 in the active site pocket of PDE9A.
Conclusion
Group-1 compounds were replaced with aliphatic fragment in the N3 position, while in set-2 compounds, the N3 position was replaced with aromatic fragments. Due to the presence of the aromatic fragment at the N3 position, set-2 compounds showed better binding strength to PDE9A than set-1 compounds. However, the presence of the aromatic fragment at the N3 position in set-2 compounds imparted polarity as well as rigidity to the compounds which may be a major reason behind their weak pharmaceutically active nature.
Thus, with these concluding remarks, eight set-1 compounds were selected for chemical synthesis and further biological studies.
Chemical Synthesis of Selected Xanthine Derivatives
Introduction
One of the most validated targets of these derivatives is the phosphodiesterase superfamily (Allwood et al., 2007). In the last few decades, based on the natural xanthine derivatives, number of xanthine-based inhibitors have been developed synthetically and reported as therapeutic molecule (Glennon et al., 1981; Wong and Ooi, 1985). Most of the previous researches proposed the synthetic route for ring closure and classical condensation route for the synthesis of the xanthine derivatives (Allwood et al., 2007; Bandyopadhyay et al., 2012; Bussi et al., 2006).
The search for a new synthetic route has been the most challenging task for researchers working with xanthine derivatives (Bandyopadhyay et al., 2012; Hayallah et al., 2002).
Material and Methods
- Chemicals
- Selection of compounds for chemical synthesis
- Chemical synthesis of xanthine derivatives
After completion of the reaction, the product mixture was allowed to cool at room temperature and filtered. Hydrochloric acid (10%) was added dropwise to neutralize the product mixture followed by the addition of water. After completion of the reaction, the reaction mixture was allowed to cool on ice for 10 minutes.
After the reaction was complete, the product mixture was allowed to cool to room temperature, 10 ml of water was added and stirred for 10 minutes.
Results and Discussion
- Selection of starting material
- Development of new routes for synthesis of xanthine derivatives
Selective substitution at predetermined xanthine positions using xanthine as a starting material for the synthesis of xanthine derivatives was the most challenging task due to the presence of three -NH groups at the N1, N3 and N7 positions of xanthine. Both acid deprotection and catalytic hydrogenation methods were used for deprotection at the N3 and N7 positions, but these methods worked differently at the N3 and N7 positions of xanthine derivatives. Catalytic deprotection of benzyl group at N7 position of xanthine derivatives in scheme-I and scheme-II.
Scheme-II Step-6b: Acid deprotection of p-methoxy benzyl (PMB) group at N3 position of xanthine derivative in Scheme-II.
Conclusion
Due to these advantages, the above proposed schemes are better alternatives compared to existing methods used worldwide to date for the synthesis of xanthine derivatives. Acid deprotection method was the best method for selective deprotection of N3, while catalytic deprotection method was for selective deprotection of benzyl group at N7 position. Alkylation was an important step for substitution at N1 and N3 positions while, for selective arylation, Suzuki coupling method was applied.
Thus, the two schemes (Scheme-I and Scheme-II) developed in the present study will have great long-term significance for synthesizing a diverse library of xanthine-based compounds.
Biological Studies of Selected Compounds
