XX TrxR Thioredoxin reductase
I. Introduction
3. Results and Discussion: Computational Design and Analysis of Potential COX-2 Selective
3.1. Exploring the Possibilities: Computational Analysis of Curcumin and Celecoxib
“Art matters because it is the one true great connector in a world that seems to be very unconnected, and it's important now more than ever to shine a huge light on that connectivity that we have, that we often forget.”
Josh Groban
“Drug design is a creative act of the same magnitude as composing, sculpting, or writing. The results can touch the lives of millions, but the creator is rarely one scientist and the rewards are distributed differently in the arts than in the sciences. The mechanisms of creativity are the same, i.e., incremental (plodding from darkness to dawn) or sudden (the “Eureka” effect) realization, but both are poorly understood. Creativity remains a human characteristic…There is beauty in the fusion of structure and function. As a creative enterprise, drug design is a synthesis of scientific knowledge, experience, intuition, and aesthetics. However, unlike the arts, this beauty has limited distribution; the general public is severely under-informed about the creative process whereby molecules are designed and created. Indeed, like artists, scientists are hard-pressed to enunciate their intuitive insights.” These opening paragraphs to Meyer, Swanson and Williams’ 2000 paper on molecular modeling and drug design succinctly covers the elation and frustration inherent to modern drug design.680 Inspiration (for lack of a better word) for drug design can be found in much the same way as an artist looks for ideas in the forms and functions of items, the play of light over a surface, the sounds at dawn or the church bells of a city in winter. While not as romantic as the artist’s inspirations, the muse of drug designers is often found in the more mundane – the novel compound in a rare sea sponge, or in the ingredients of traditional medicines.
With 2012 sales of over-the-counter (OTC) internal analgesics totaling over $3.9 billion in the United States alone, and not counting the other forms of analgesics such as rubs and sprays, the painkiller
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industry is the second largest contributor to the $23 billion OTC market.681 As the search for new anti-inflammatory, painkilling compounds without serious side effects is always ongoing, it is often a good idea to consult the wealth of knowledge contained in the various traditional medicines from around the world. One such compound is curcumin (Figure 3.1), one of the components of turmeric.
This spice has been used for thousands of years in Indian Ayurvedic and other traditional medicines and the extensive list of treatments include numerous entries where the antiseptic, anti-oxidant, antimalarial, analgesic and anti-inflammatory properties of turmeric have been exploited.329-331 Curcumin itself has been shown to have therapeutic potential against a number of diseases and disorders,330,333,351
and over 50 clinical trials utilizing curcumin are currently ongoing. However, curcumin has a number of drawbacks, including poor bioavailability and as-yet-unknown mechanisms of action, which limit its utilization in mainstream medicine, and therefore vast amounts of work have been carried out to reduce these negative aspects, including solubility enhancement and structural modifications.329-331
O O
O H
O
OH O
Figure 3.1. 2D structure of curcumin.
The interactions of curcumin with proteins in the solid state are notoriously difficult to study, as curcumin does not withstand X-ray irradiation.497 This protein-mediated decomposition of curcumin has prevented the acquisition of definitive answers to the question of how curcumin binds to proteins.
While technically a diketone, curcumin undergoes keto-enol tautomerization (Figure 3.2), and it is the enol form (1) which dominates in solution and the solid state,455-456 although whether the enol or the diketone form (2) bind to the protein is unknown at present. One possible explanation for the relative proportions of the tautomers is the simultaneous extension of conjugation throughout the molecule and the formation of a stabilizing internal hydrogen bond between the enol hydrogen atom and the keto- oxygen atom. The result of this internal hydrogen bond is a thermodynamically preferred 6-membered ring.682
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O O
O H
O
OH O
OH O
O H
O
OH O
Figure 3.2. Keto-enol tautomerization of curcumin.
Despite this uncertainty, curcumin has been the subject of much study, and one of the plethora of molecular targets of curcumin are the cyclooxygenase (COX) enzymes. These enzymes are largely responsible for the formation of prostanoids from arachidonic acid, including prostacyclin and thromboxane A (Figure 3.3). Prostacyclin is known to be an effective vasodilator and also inhibits platelet aggregation, while thromboxane A2, a vasoconstrictor, is important during injury and inflammation. At present, two main isoforms of COX are known: COX-1, which is constitutively expressed at low levels throughout the body, and the inducible COX-2, the production of which is stimulated by various inflammatory triggers.683
COOH
OH O COOH
O
OH
COOH
O H
O
+ 2O2 COX-1/COX-2
PGE Synthases
Figure 3.3. An example of prostaglandin synthesis: the formation of PGE2 from arachidonic acid.609
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Reduction of prostaglandin synthesis is the central mechanism upon which non-steroidal anti-inflammatory drugs (NSAIDs) act, and the discovery of COX-2 opened up the development of targeted NSAIDs which did not show the gastrointestinal toxicity of the non-selective NSAIDs. This lead to the development of COX-2 selective anti-inflammatory compounds including celecoxib (Celebrex®), rofecoxib (Vioxx®), etoricoxib (Arcoxia®) and lumiracoxib (Prexige®) (Figure 3.4). While effective as COX- 2 selective inhibitors or “coxibs”, there are a number of severe side effects associated with these compounds, including heart attacks and strokes, and these side effects have resulted in a number of these COX-2 selective compounds either being withdrawn from the market or not receiving approval for sale.
N N S
O N O H2
F F
F O
S O O
O
N N
Cl S
O O
NH
OH O Cl
F
Figure 3.4. 2D structures of the coxibs (clockwise from top left) celecoxib, rofecoxib, etoricoxib and lumiracoxib.
As most of the coxibs are essentially celecoxib derivatives, these coxib compounds show remarkable structural similarity. All of these molecules contain a central ring with a 1,2-substitution pattern, and, apart from lumiracoxib - a diclofenac derivative - two additional phenyl rings. Another feature common to the coxibs is a p-sulfuryl group, be it a sulfonamide as seen in celecoxib and valdecoxib or a sulfone as
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found in rofecoxib and etoricoxib. This p-sulfuryl group is vitally important to the selectivity of coxibs for COX-2 over COX-1, as it is this moiety which interacts with the secondary pocket present in COX-2 (Figure 3.5). This interaction of celecoxib with the secondary pocket is highlighted when the structures of celecoxib and arachidonic acid from the X-ray crystal structures of COX-2 (PDB files 3LN1684 and 3HS6685 respectively) are superimposed (Figure 3.6).
Figure 3.5. Ligand interaction diagram of celecoxib with COX-2 showing the interactions of the p-sulfonamide group of celecoxib with the secondary pocket present in COX-2, taken from the PDB file 3LN1.684 The red line indicates the presence of a π-cation interaction between the ligand and the protein, the solid purple lines a presence of an H-bond between the ligand and the backbone of the protein, and the dashed purple line an H-bond interaction between the ligand and a side chain.
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Figure 3.6. X-ray crystal structures of celecoxib (PDB file 3LN1),684 shown with green carbon atoms, and arachidonic acid (PDB file 3HS5),685 shown with blue carbon atoms.
In order to test the applicability of the docking conditions, the native celecoxib ligand was removed from the protein structure, prepared using LigPrep686 and redocked using Glide XP687 into the active site of COX-2, which had separately undergone preparation (Epik688) and the active site defined using the Receptor Grid Generation application of the Schrödinger Maestro suite.689 The poses generated were then compared to the X-ray crystal structure of celecoxib, and the root-mean-square deviation (RMSD) calculated between the generated pose and the original PDB coordinates. A final RMSD value of 0.44 Å was calculated between the best scoring docking pose generated for celecoxib and the original structure (Figure 3.7), validating the docking method used.
Figure 3.7. The generated pose of celecoxib with carbon atoms shown in red overlapping with the pose of celecoxib bound to COX-2, shown with green carbon atoms (PDB file 3LN1).684
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Despite being a COX-2 selective compound, celecoxib is also known to bind to COX-1, and an X-ray crystal structure of celecoxib bound to COX-1 (PDB file 3KK6) was obtained in 2010 by Rimon, et al.690 While the docking procedure used for celecoxib and COX-2 showed good correlation, the same procedure was used in order to determine the applicability of this procedure for the docking of compounds into COX-1. An RMSD value of 0.57 Å between the docked pose and the crystal structure (Figure 3.8) again confirmed the suitability of the docking procedure towards COX-1.
Figure 3.8. The generated pose of celecoxib, carbon atoms shown in red, overlapping with the pose of celecoxib bound to COX-1, carbon atoms shown in green (PDB file 3KK6).690
In an effort to understand the possible binding of curcumin to COX-1 and more importantly to COX-2, curcumin was subjected to the same docking process as used for celecoxib. Both 1 and 2 were docked into the protein (Table 3.1), as while 1 dominates in solution,455-456 it is possible for 2 to exist in solution and interact with the protein rather than the predominant enol. As MM-GBSA scores are considered better points for comparison than Glide XP scores,691 the docked poses of celecoxib and the two isomers of curcumin were rescored using the Prime689 function of Schrödinger, and these results were used for comparison, along with visual inspection of the docking poses.
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Table 3.1. Glide XP and Prime scores for celecoxib and the two isomers of curcumin.
Entry Compound
COX-2 COX-1
Glide XP /kCal.mol-1
Prime /kCal.mol-1
Glide XP /kCal.mol-1
Prime /kCal.mol-1
1 Celecoxib -10.650 -84.269 -11.605 -86.676
2 1 -7.888 -86.566 -10.180 -71.117
3 2 -7.371 -79.410 -11.361 -70.784
At first glance, 1 shows an improvement in binding to COX-2 and a reduction in the binding to COX-1 as compared to celecoxib, while 2 shows a reduction in binding to both proteins. However, on further inspection of the docked poses, 1 shows fewer interactions with the protein (Figure 3.9), while 2 appears to fold in such a manner as to mimic the pose and a few of the interactions found between celecoxib and COX-2 (Figure 3.10), while neither of the curcumin tautomers show the presence of π- cation interactions, as shown in Figure 3.5.
Figure 3.9. Ligand interaction diagram of 1 with COX-2 showing the interactions present between the protein and the ligand.
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Figure 3.10. Ligand interaction diagram (left) of 2 with COX-2 and the overlap of the generated pose with the X-ray crystal structure of celecoxib in COX-2 (right).684
While the docking results show that curcumin could interact with both COX-1 and COX-2 proteins, a glaringly obvious piece of information gleaned from these docking studies is that 1 does not interact with the secondary pocket of COX-2 as desired, and based on these results, it is not expected to be selective for COX-2 over COX-1. While 2 does appear to occupy the secondary pocket, the amount of 2 present in solution is essentially zero,692 which hinders the applicability of curcumin to wider use as a COX-2 selective compound. Consequently, a COX-2 selective compound based on curcumin would need to contain other functional groups and/or structural modifications which would enable the selective inhibition of COX-2 through contact with the secondary pocket. Another vital piece of information obtained from this study is that the pose of the compound under study is important, and that the numbers obtained from the various analyses and calculations cannot be the sole basis for judging the suitability of a ligand for a protein.
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