Brief Article pubs.acs.org/jmc
A Fragment-Based Approach to Identifying S‑Adenosyl‑L‑methionine -Competitive Inhibitors of Catechol O‑Methyl Transferase (COMT). Marion Lanier,*,† Geza Ambrus,‡ Derek C. Cole,† Richard Davenport,# Jonathan Ellery,∇ Richard Fosbeary,∇ Andy J. Jennings,‡ Akito Kadotani,⊥ Yusuke Kamada,⊥ Ruhi Kamran,§ Shin-Ichi Matsumoto,⊥ Atsushi Mizukami,⊥ Shoichi Okubo,⊥ Kengo Okada,⊥ Kumar Saikatendu,‡ Louise Walsh,# Haihong Wu,∥ and Mark S. Hixon*,§ †
Medicinal Chemistry, ‡Structural Biology, §Discovery Biology, ∥Analytical Chemistry, Takeda California Inc., 10410 Science Center Drive San Diego California 92121, United States ⊥ Biomolecular Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd., 26-1 Muraoka-Higashi 2-chome, Fujisawa, 251-8555, Kanagawa, Japan # Medicinal Chemistry, ∇In Vivo Pharmacology, Takeda Cambridge Inc., 418 Cambridge Science Park, Cambridge CB4 0PA, United Kingdom S Supporting Information *
ABSTRACT: Catechol O-methyl transferase belongs to the diverse family of S-adenosyl-L-methionine transferases. It is a target involved in the treatment of Parkinson’s disease. Here we present a fragment-based screening approach to discover noncatechol derived COMT inhibitors which bind at the SAM binding pocket. We describe the identification and characterization of a series of highly ligand efficient SAM competitive bisaryl fragments (LE = 0.33−0.58). We also present the first SAM-competitive small-molecule COMT co-complex crystal structure.
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INTRODUCTION
The S-adenosyl-L-methionine (SAM)-dependent methyl-transferases are a diverse and biologically important class of enzymes.1,2 They are involved in a wide array of biological processes including signal transduction, gene transcription, and cell proliferation. SAM-dependent methyl-transferases utilize the ubiquitous methyl donor SAM as a co-substrate to methylate proteins, small molecules, lipids, and nucleic acids.2,3 One methyl transferase of particular interest is catechol O-methyl transferase (COMT). Inhibitors of COMT are used for the treatment of Parkinson’s disease,4,5 making COMT an important pharmacological target. COMT is a bisubstrate enzyme catalyzing the methyl transfer from S-adenosyl-L-methionine to the hydroxyl groups of dopamine. The catalytic site contains distinct catechol and SAM binding pockets in close proximity connected by a narrow channel through which methyl transfer occurs. Marketed COMT inhibitors6 (Figure 1) are catechol-derived and alleviate Parkinson’s disease symptoms by preventing the catabolism of dopamine, thus increasing its half-life and alleviating symptoms resulting from dopamine level fluctuation. However, they are linked to liver toxicity and increased dyskinesia frequency.7 Efforts to develop superior COMT inhibitors have met with limited success, due to issues of selectivity, pharmacokinetics, pharmacodynamics, clinical efficacy, or safety.5,7,8 © 2014 American Chemical Society
Figure 1. Structures of catechol-competitive COMT inhibitors 1−3.
To avoid the potential complications of catechol-derived pharmaceuticals, we focused our research on the discovery of COMT inhibitors which bind in the SAM pocket. There have been limited reports of SAM competitive inhibitors. In the 1970s, the Borchardt lab published a series of papers on COMT inhibition by structural analogues of S-adenosyl-LReceived: March 25, 2014 Published: May 21, 2014 5459
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homocysteine (SAH).9−14 The key recognition elements of SAH binding appear to be the amino and carboxy termini of Shomocysteine, the ribose 2-hydroxy group, and the adenine 6amino group. Deletion of any one of these four interactions results in a complete loss of potency. More recently, the Diederich lab has explored a series of catechol−SAM bisubstrate analogues where an adenosine moiety is bound to a nitrocatechol with various linkers.15−17 Bisusbstrate analogues optimized to low nanomolar potency required an unmodified SAM portion. For an alternative path to discover SAM pocket binders, we chose the build-up approach of fragment-based drug discovery (FBDD). In addition, FBDD provides a rapid exploration of chemical diversity space and by focusing on ligand efficiency permits rapid evolution from hit to lead.18 Ligand efficiency (LE = ligand efficiency = 1.36 × pIC50/heavy atoms)19 refers to the binding energy contributed by each heavy atom of a molecule. The lipophilic ligand efficiency (LLE = lipophilic ligand efficiency = pIC50 − clogP)20 is a measure of potency relative to a compound’s lipophilicity. Employing a combination of orthogonal fragment screening techniques maximizes the opportunity to identify diverse hits and also allows rapid confirmation of hits identified by multiple methods.
The 52 fragment hits were further characterized using LC− MS/MS to identify and eliminate any chemically reactive hits and SPR to eliminate SAM noncompetitive binders. A series of three structurally related fragments hits (4−6, Figure 2 and
Figure 2. Structures of fragment hits.
Table 1. COMT pKi and pKD, Values Measured on hCOMT Clone, and LE and LLE Values for Compounds 4−7
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compd
RESULTS AND DISCUSSION A priori, the COMT SAM binding site appears to be an attractive pocket to exploit for inhibitor design. The binding motif is similar to the Rossmann fold, a common feature of many nucleotide binding proteins.21 An in silico druggability assessment of this pocket using Fpocket22 gives a calculated pocket volume of 585 Å3, a polar solvent accessible surface area of 120 Å2 (of the total solvent accessible surface area of 365 Å2), and a druggability score of 0.661 (Table S1, Supporting Information (SI)). These values are consistent with a tractable protein pocket for drug discovery. To identify starting points for lead optimization, our fragment-based screening (FBS) approach included both activity-based and biophysical assays to identify and characterize SAM-competitive fragments and is summarized in Scheme 1. Using an enzyme activity assay, Takeda’s 11000 member
4 5 6 7
pKi (Ki) 3.6 4.8 4.6 5.1
(251 μM) (16 μM) (25 μM) (7.9 μM)
pKD (SPR)
LE/LLE (clogP)
3.6 4.5 4.2 4.6
0.33/1.7 0.54/3.9 0.57/3.9 0.58/3.9
Table 1) which bound stoichiometrically to COMT and competed with SAM were selected for further studies. Similarity searches in our corporate database led to the identification of a fourth active fragment (7, Figure 2) with similar potency and LE (Table 1). The SAM competitive fragment hits (4−7) were characterized further using several orthogonal techniques including NMR, differential scanning fluorimetry (DSF), and co-crystallization. An NMR-based saturation transfer difference26 (STD) assay was used to confirm the small-molecule binding to hCOMT and possibly reveal which protons strongly engage the binding pocket. A 1D proton NMR of 7 alone was first run as reference (Figure 3A) then 7 was added to a solution of hCOMT and the NMR run (Figure 3B). The two peaks that produced a large change in intensity were attributed to the two methyls, implying that they are in close contact with the protein. The same experiment was also performed in the presence of Tolcapone (Figure 3C). STD-NMR signals of 7 with hCOMT increased by 3-fold after addition of Tolcapone, suggesting that 7 does not compete with Tolcapone for the catechol pocket but binds in the SAM pocket and is stabilized (binds cooperatively) by Tolcapone. Differential scanning fluorescence (DSF) or thermal shift experiments27 detect the ability of a ligand to stabilize a protein’s native state. Stabilization is quantified by measuring an increase in the target protein’s denaturation (melting) temperature (Tm) with compound bound compared to the unbound protein. In the absence of ligand, hCOMT melts at 48.6 ± 1.0 °C. When 7 was added at 0.2 mM and at 1 mM, hCOMT melting temperature increased by 2.4 and 4.2 °C, respectively (Table 2). Similar shifts were observed with compounds 5 and 6, confirming that these also bind and stabilize hCOMT. The binding mode of 5 was confirmed by obtaining a cocrystal complex bound to mouse COMT (Figure 4).28 This is
Scheme 1. SAM Competitive Fragment Identification and Characterization Strategy
fragment library23 was screened at 100 μM concentration. In contrast to the in silico druggability assessment, the screen produced only 52 fragments with inhibition above 15% at 100 μM (hit rate = 0.5%). This hit rate is significantly lower than a typical fragment screens where hit rates are frequently in the 3−10% range.24,25 5460
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interactions are also observed in the SAM−COMT co-complex (Figure 5). The backbone NH of Ser169 (Ser162 in mouse)
Figure 3. NMR spectra of compound 7. (A) Reference spectrum of 7 (0.2 mM) alone; (B) STD spectrum of 7 (0.2 mM) and hCOMT(2.5 μM); (C) STD spectrum of 7 (0.2 mM), hCOMT (2.5 μM), and Tolcapone (0.1 M). Solvent peaks denoted by *.
Figure 5. Crystal structure of mCOMT (salmon) with 5 (yellow) overlaid with hCOMT (gray) with SAM (green). Ile 139 and the three residues (Tyr118, Ile141, and Trp193) which undergo the most significant movement are highlighted.
Table 2. Melting Temperatures of hCOMT in the Presence of 0, 0.2, and 1 mM Compound 5−7 5
6
7
conc (mM)
Tm (°C)
ΔTm (°C)
Tm (°C)
ΔTm (°C)
Tm (°C)
ΔTm (°C)
0 0.2 1
48.6 ± 1.0 54.3 ± 0.6 54.7 ± 0.2
5.7 6.1
48.6 ± 1.0 52.7 ± 0.2 55.2 ± 0.5
4.1 6.6
48.6 ± 1.0 51.0 ± 0.3 52.8 ± 0.8
2.4 4.2
forms a hydrogen bond with the 1-N of the SAM purine ring; a lipophilic packing between Ile141 (Ile134 in mouse) and the purine ring is present. Finally Glu140 (Glu133 in mouse) forms two hydrogen bonds with the two sugar hydroxylic groups. This COMT−fragment co-structure allowed us to identify essential protein ligand interactions and also suggested vectors for fragment growth and lead optimization. In contrast to catechol-based inhibitors, SAM competitive fragments 4−7 displayed significant interspecies differences in affinity. For example, the pKi of 5 varies from 3.8 to 5.2 from rat to mouse and human COMT, while the pKi of the catechol inhibitor 3 was unchanged throughout the species (Table 3). Table 3. pKi (Ki) for 3 and 5 in Human, Mouse, and Rat COMT 3 (pKi/Ki) (μM)
5 (pKi/Ki) (μM)
8.0/0.0010 7.8/0.0015 7.8/0.0015
5.2/6.3 4.2/63 3.8/158
human COMT mouse COMT rat COMT
Table 4. Human, Rat, and Mouse COMT Amino Acid Residue Differences in the SAM Binding Pocket Figure 4. Crystal structure of mCOMT (mouse COMT) showing the interaction between 5 and the SAM pocket.
human rat mouse
the first reported cocrystal structure of COMT with a SAM competitive small-molecule inhibitor (PDB code 4P58). Noteworthy binding interactions include lipophilic packing with Ile134, an edge−face interaction with Trp186 and edge− face interaction with Tyr111, a hydrogen bond between the pyrazole NH and side chain of Glu133, a hydrogen bond between the unprotonated pyrazole nitrogen and the backbone NH of Ile 134, and a hydrogen bond between dimethylpyrazole nitrogen and the backbone NH of Ser162. These three
Ile139 Met132 Met132
Ile141 Met134 Ile134
Cys145 Tyr138 Tyr138
Comparing human and mouse sequences of COMT (Table 4) and inspection of an overlay (Figure 4) of SAM co-complexed in human COMT and 5 crystallized in mouse COMT provides insight about the similarities and differences between these two species and how fragment 5 forms critical interactions with residues responsible for binding the purine rings of SAM. There are two nonconserved amino acids in close proximity to 5: Ile139 and Cys145 in human COMT. Their homologues in 5461
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fluorimetry; SPR surface plasmon resonance; STD-NMR saturation transfer difference-nuclear magnetic resonance
mouse COMT are Met132 and Tyr138, respectively. The side chain of Cys145/Tyr138 is directed toward the solvent having little apparent interaction with 5. On the other hand, Ile139/ Met132 is positioned deep in the pocket and more likely responsible for the variable binding affinity. The pyrazole 3methyl fills nicely the pocket in the human construct (when Ile139 is present). In mouse COMT, Ile139 is replaced by Met132 and opens a small cavity that is not optimally filled by the ligand thus potentially explaining the loss in potency.
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(1) (a) Copeland, R. A.; Solomon, M. E.; Richon, V. M. Protein methyltransferases as a target class for drug discovery. Nature Drug Discovery 2009, 8, 724−732. (b) Loenen, W. A. M. S-Adenosylmethionine: jack of all trades and master of everything? Biochem. Soc. Trans. 2006, 34, 330−333. (2) Martin, J. L.; McMillan, F. M. SAM (dependent) I AM: the Sadenosylmethionine-dependent methyltransferase fold. Curr. Opin. Struct. Biol. 2002, 12, 783−793. (3) Miller, D. J.; Ouellette, N.; Evdokimova, E.; Savchenko, A.; Edwards, A.; Anderson, W. F. Crystal complexes of a predicted Sadenosylmethionine-dependent methyltransfe rase reveal a typical AdoMet binding domain and a substrate recognition domain. Protein Sci. 2003, 12, 1432−1442. (4) Kaakkola, S. Clinical pharmacology, therapeutic use and potential of COMT inhibitors in Parkinson’s disease. Drugs 2000, 59, 1233− 1250. (5) Bonifácio, M. J.; Palma, P. N.; Almeida, L.; Soares-da-Silva, P. Catechol-O-methyltransferase and its inhibitors in Parkinson’s Disease. CNS Drug Rev. 2007, 13, 352−379. (6) Mannisto, P. T.; Kaakkola, S. Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of the new selective COMT inhibitors. Pharm. Rev. 1999, 51, 593−628. (7) Kaakkola, S. Problems with the present inhibitors and a relevance of new and improved COMT inhibitors in Parkinson’s disease. Int. Rev. Neurobiol. 2010, 95, 207−225. (8) Almeida, L.; Rocha, J. F.; Falcão, A.; Palma, P. N.; Loureiro, A. I.; Pinto, R.; Bonifácio, M. J.; Wright, L. C.; Nunes, T.; Soares-da-Silva, P. Pharmacokinetics, pharmacodynamics and tolerability of Opicapone, a novel catechol-O-methyltransferase inhibitor, in healthy subjects: prediction of slow enzyme−inhibitor complex dissociation of a short-living and very long-acting inhibitor. Clin. Pharmacokinet. 2013, 52, 139−151. (9) Borchardt, R. T.; Wu, Y. S. Potential inhibitors of Sadenosylmethionine-dependent methyl transferases. 1. Modification of the amino acid portion of S-adenosylhomocysteine. J. Med. Chem. 1974, 17, 862−868. (10) Borchardt, R. T.; Huber, J. A.; Wu, Y. S. Potential inhibitors of S-adenosylmethionine-dependent methyltransferases. 2. Modification of the base portion of S-adenosylhomocysteine. J. Med. Chem. 1974, 17, 868−873. (11) Borchardt, R. T.; Wu, Y. S. Potential inhibitors of Sadenosylmethionine-dependent methyl transferases. 3. Modifications of the sugar portion of S-adenosylhomocysteine. J. Med. Chem. 1975, 18, 300−304. (12) Borchardt, R. T.; Huber, J. A.; Wu, Y. S. Potential inhibitors of S-adenosylmethionine-dependent methyltransferases. 4. Further modifications of the amino acid and base portions of S-adenosylhomocysteine. J. Med. Chem. 1976, 19, 1094−1099. (13) Borchardt, R. T.; Wu, Y. S. Potential inhibitors of Sadenosylmethionine-dependent methyl transferases. 6. Structural modifications of S-adenosylhomocysteine. J. Med. Chem. 1976, 19, 1104−1110. (14) Borchardt, R. T.; Wu, Y. S.; Wu, B. S. Potential inhibitors of Sadenosylmethionine-dependent methyl transferases. 7. Role of the ribosyl moiety in enzymatic binding of S-adenosylhomocysteine. J. Med. Chem. 1978, 21, 1307−1310. (15) Lerner, C.; Ruf, A.; Gramlich, V.; Masjost, B.; Jakob-Roetne, R.; Borroni, E.; Diederich, F. X-ray crystal structure of a bisubstrate inhibitor bound to the enzyme catechol-O-methyltransferase: a dramatic effect of inhibitor preorganization on binding affinity. Angew. Chem., Int. Ed. 2001, 40, 4040−4042. (16) Paulini, R.; Trindler, C.; Lerner, C.; Brandli, L.; Schweizer, W. B.; Jakob-Roetne, R.; Zurcher, G.; Borroni, E.; Diederich, F. Bisubstrate inhibitors of catechol O-methyltransferase (COMT): the
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CONCLUSION Our goal was to identify COMT SAM competitive fragments that could lead to a new class of drugs for the treatment of Parkinson’s disease. Using a FBDD approach, we identified a series of four fragments structurally unrelated to SAM. Orthogonal assays confirmed their competitive nature. Their affinities (Ki < 10 μM) and high efficiencies (LE > 0.4) make them ideal starting points as fragment leads. The co-crystal complex 6-COMT provided insight into the binding interactions within the SAM pocket and potential vectors for chemical elaboration. This co-crystallized 6−COMT complex is the first report of a non-SAM analogue cocrystallized in COMT or in any SAM binding pocket. It reinforces the power of combining a FBDD approach with orthogonal methods such as crystallography to identify new chemical matter for elusive targets.
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EXPERIMENTAL SECTION
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ASSOCIATED CONTENT
Compounds 1, 2, 5, and 7 are commercially available from SigmaAldrich, 4 and 6 from Ryan Scientific. 3 was prepared following a procedure described in the literature.15 Purity for all compounds was confirmed by LC−MS analysis to be above 95% by UV.
* Supporting Information S
Complete description of materials and methods, including enzyme preparation, assays, and crystallographic information (CIF). This material is available free of charge via the Internet at http://pubs.acs.org. Accession Codes
The PDB code for compound 6 bound to mouse COMT is 4P58.
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*For M.L.: phone, 1 858-731-3552; E-mail, marion.lanier@ takeda.com. *For M.S.H.: phone, 1 858-731-3578; E-mail, mark.hixon@ takeda.com. Author Contributions
All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED SAM S-adenosyl-L-methionine; COMT catechol O-methyl transferase; LE ligand efficiency; SAH S-adenosyl-L-homocysteine; FBDD fragment-based drug discovery; FBS fragmentbased screening; LC−MS/MS liquid chromatography−mass spectrometry/mass spectrometry; DSF differential scanning 5462
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crucial role of the ribose structural unit for inhibitor binding affinity. ChemMedChem 2006, 1, 340−357. (17) Ellermann, M.; Paulini, R.; Jakob-Roetne, R.; Lerner, C.; Borroni, E.; Roth, D.; Ehler, A.; Schweizer, W. B.; Schlatter, D.; Rudolph, M. G.; Diederich, F. Molecular recognition at the active site of catechol-O-methyltransferase (COMT): adenine replacements in bisubstrate inhibitors. Chem.Eur. J. 2011, 17, 6369−6381. (18) Congreve, M.; Chessari, G.; Tisi, D.; Woodhead, A. J. Recent developments in fragment-based drug discovery. J. Med. Chem. 2008, 51, 3661−3680. (19) Reynolds, C. H.; Tounge, B. A.; Bembenek, S. D. Ligand binding efficiency: trends, physical basis, and implications. J. Med. Chem. 2008, 51, 2432−2438. (20) Edwards, M. P.; Price, D. A. Role of physicochemical properties and ligand lipophilicity efficiency in addressing drug safety risks. Annu. Rep. Med. Chem. 2010, 45, 381−391. (21) Rao, S.; Rossmann, M. Comparison of super-secondary structures in proteins. J. Mol. Biol. 1973, 76, 241−256. (22) Le Guilloux, V.; Schmidtke, P.; Tuffery, P. F pocket: an open source platform for ligand pocket detection. BMC Bioinf. 2009, 10, 168−179 This method identifies pockets in a protein structure and calculates a number of metrics related to the characteristics of each one. A drugability score of 0.5 indicates that binding of druglike molecules is possible, and a value of 1.0 indicates that binding of druglike molecules is very likely.. (23) library is 66% “rule of three” compliant (i.e., molecular mass ≤ 300 amu, cLogP ≤ 3 H-bond donors and H-bond acceptors each ≤ 3). The remaining fragments violate no more than one rule. (24) Roughley, S. D.; Hubbard, R. H. How well can fragments explore accessed chemical space? A case study from heat shock protein 90. J. Med. Chem. 2011, 54, 3989−4005. (25) Hubbard, R. E.; Davis, B.; Chen, I.; Drysdale, M. J. The SeeDs approach: integrating fragments into drug discovery. Curr. Top. Med. Chem. 2007, 7, 1568−1581. (26) Coles, M.; Heller, M.; Kessler, H. NMR-based screening technologies. Drug Discovery Today 2003, 8, 803−810. (27) Fedorov, O.; Niesen, F. H.; Knapp, S. Kinase inhibitor selectivity profiling using differential scanning fluorimetry. Methods Mol. Biol. 2012, 795, 109−118. (28) At the start of the project, the PDB reported SAM and/or SAH only cocomplexed with mouse and rat, not human. Crystallization trials for SAM competitive fragments were first attempted in mouse and rat. A mouse cocomplex was obtained and deemed sufficient to drive the project.
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