Kinya
Toda
a,
Junichi
Goto
a and
Noriaki
Hirayama
*b
aComputational Science Department, Ryoka Systems Inc., 1-28-38 Shinkawa, Chuo-ku, Tokyo 104-0033, Japan
bTokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1143, Japan. E-mail: hirayama@is.icc.u-tokai.ac.jp; Tel: +81 463 93 1121
First published on 14th October 2010
Three-dimensional structures of drug target molecules are extremely useful for designing novel ligands. We have developed a novel structure-based de novo design method by the combination of a novel concept of pseudomolecular probe(PMP) and alpha spheres aiming to make the utmost use of the three-dimensional structures of the target molecules. Alpha spheres generated at the binding site give good clues to place molecular fragments at the site. A PMP consisting of a functional group and a supporting group can determine the most appropriate position of the functional group at the binding site. The results have indicated that the strategy works reasonably well in reproducing the structures of the original ligands at the binding sites and have proved that this method can become one of the target-based de novo design methods.
Structure-based de novo molecule-design software has emerged to help medicinal chemists to take advantage of structural information of target molecules to design promising molecules. Several dozen de novo design programs have been published1 and some of them have been successfully applied in generating novel compounds. Most of the currently available de novo design algorithms are based on the construction of novel molecules from multiple molecular fragments. The first and most important step of assembling of fragments is placing suitable molecular fragments at each interaction center in the binding site.
Mainly two strategies are taken for this purpose, i.e. the link/grow and lattice strategies. For the link/grow strategies employed in software such as LUDI,2 mostly rule-based methods are applied to place chemical groups at the biding site. Since they are based on the empirical rules, the results are heavily dependent on the rules regarded as allowable. The performance of grid strategy3 employed in software such as LUDI crucially depends on the resolution of the grid. In addition, many meaningless grids must be taken in account.
A concave portion at the surface of a target molecule where a ligand is supposed to bind can be identified as a collection of spheres by use of the modified Delaunay triangulation.4 The sphere is designated as ‘alpha sphere.’ We have already confirmed that a set of the alpha spheres generated at the binding site is a very useful clue to place docking poses.5 As the set of the alpha spheres can represent physico-chemical properties and shape of the binding site, it can be used to place molecular fragments at the binding site. In this study we have applied the alpha spheres to find appropriate positions of the molecular fragments at the binding site. Since the alpha-sphere-based approach is not based on the empirical rules and does not depend on the resolution of the grid it is expected to be reasonably accurate and fast.
One of the most important steps to build novel molecules is disposition of functional groups at appropriate sites in a concavity of the target protein. We have introduced a novel concept of pseudomolecular probe (PMP) for this purpose. A PMP consists of a functional group and a supporting group. A supporting group helps the functional group properly position at the binding site. The supporting group should not be too large and should be rigid enough in order to determine the position of the functional group at the binding site ambiguously and efficiently. We found COMPOUND LINKS
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Download mol file of compound3-methylcyclopropene (Fig. 1) is a suitable supporting group to anchor the functional group at the proper position of the binding site.
Fig. 1 The structure of a pseudomolecular probe. |
De novo design software in general is inherently confronted with a virtually infinite search space. An essential problem underlying is combinatorial explosion. From the practical point of view, the search space for structure generation should be reduced in some way to avoid this NP-hard problem. Since X-ray structure of a ligand bound in a target molecule is crucially important to start the optimization or de novo design procedure, we make utmost use of this structural information in this study to avoid this problem. Ten target systems for which X-ray structures of protein-ligand complexes are available were used in the validation of this method. The results have indicated that the strategy works reasonably well in reproducing the original ligands at the binding sites of the target molecules and have proved that this method can become one of the target-based de novo design methods.
Fig. 2 Comparison of pseudomolecular probes with cyclopentenyl (green) and cyclobutenyl (blue) groups. The functional group of the ligand determined by X-ray analysis is shown in red. |
Fig. 3 Anchoring points in a pseudo molecular probe (a) and a linker (b). |
Two points of α and β in PMP's are superposed to the corresponding α and β points in a linker. Here, the α and β points are pseudo-atoms with the distance between α and β points being set to the typical Csp3–Csp3 bond length of 1.54 Å. In the linking procedures, the allowance errors of superposition at the anchoring points were set to 0.2 and 0.3 Å, for α and β points, respectively. Since taking all 831 linkers into account is not practical and certain linkers are inherently unsuitable to link the particular PMP's, a simple selection rule is applied. The van der Waals volume (VL) of the linker moiety of the X-ray structure of the ligand is used to determine the size of the linkers to be selected. In this study, the linkers whose volumes range between 0.9VL and 1.1VL were selected. Through this procedure we could eliminate unsuitable linkers preliminarily.
Udock = Uele + Uvdw + Ustrain. |
Fig. 4 Chemical structures of the ligands used for the validation. Squares are put around the functional groups which were used for de novo design. |
PDB code | Ligand identifier | Number of linkers | Number of generated molecules (number of conformations) | U dock/kcal mol−1 of the original ligand | The ranking of the original ligand | rmsd/Å | V m/Udock |
---|---|---|---|---|---|---|---|
a
N-(phosphonacetyl)-L-aspartic acid.
b Donepedil.
c Reverse hydroxamate inhibitor.
d
N-methyl-4-{[(2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl] amino} benzenesulfonamide.
e Ganciclovir.
f
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundN-(phosphonoacetyl)-L-ornithine. g Sitagliptin. h Rosiglitazon. i COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundIbuprofen. j COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundZanamivir. |
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1EKX | PALa | 11 | 76(284) | −548.3 | 3 | 1.348 | 2 |
1EVE | E20b | 17 | 411(2123) | −36.2 | 990 | 2.611 | 498 |
1GKC | BUM + STNc | 59 | 1953(9627) | −150.0 | 139 | 0.232 | 22 |
1KE5 | LS1d | 19 | 136(313) | −48.5 | 52 | 0.496 | 27 |
1KI2 | GA2e | 12 | 158(1338) | −62.4 | 13 | 1.161 | 13 |
1OTH | PAOf | 20 | 108(255) | −377 | 1 | 0.349 | 1 |
1X70 | 715g | 61 | 1184(4943) | −92.9 | 34 | 2.055 | 26 |
2PRG | BRLh | 186 | 6202(24602) | −34.5 | 3320 | 2.000 | 1777 |
2WD9 | COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundIBPi |
17 | 222(1208) | −37.2 | 589 | 1.727 | 214 |
3B7E | ZMRj | 60 | 5462(81496) | −199.5 | 176 | 2.042 | 13 |
Here, Uele and Uvdw mean electrostatic and van der Waals interaction energies, respectively, between the target and the ligand molecules. Ustrain refers to the difference between the conformation energy of a docked ligand and the conformation energy of the energy minimum conformation nearest to that of the docked ligand. The results are summarized in Table 1. As the linkers similar in size to the original linker are used to connect the PMP's, the number of linkers is different for each system. Since the original ligand was reconstructed, the number of generated molecules minus one is the number of the novel molecules. Multiple conformations were generated for each de novo molecule and the number of conformations given in the parenthesis means the total number of generated conformations for all de novo molecules. The total number of conformations depends on the number of linkers used and the flexibility of the generated molecule. The ranking of the original ligand means the ranking of the regenerated original molecule in the order of Udock values. The rmsd (Å) means the root mean square deviation between the non-hydrogen atoms of the original ligand molecule and those of the regenerated molecule by docking simulation. From the standpoint of making maximum use of the X-ray structure of the complex, the space occupied by the ligand at the binding site should be taken into account. The volume (Vcom) commonly occupied by the alpha spheres and the original ligand can be a simple index to judge how well a generated molecule corresponds to the original ligand. Suppose the common volume between the alpha spheres and a generated molecule is V, we can select the utmost similar molecules to the ligand by picking up the molecules whose V values range between 0.9Vcom and 1.1Vcom. The rankings of the regenerated ligands by Udock after this filtering are generally higher as shown in the last column (Vm/Udock) of Table 1. From the practical point, this filtering may be useful. Although the original ligands are highly ranked in all systems, the results indicate some de novo molecules with higher predicted binding affinity than the original ligand were generated. The exception is 1OTH. In this case, the original ligand came in first.
Although the present study proposes a novel method of de novo design, it is interesting to quick sketch of a few de novo molecules generated by this method. Human matrix metalloproteinase 9(MMP9) is one of important cardiovascular disease targets. Peptidic reverse hydroxamates such as shown in Fig. 4 are known to inhibit MMP9 and expected to be starting point for drug design. By use of the crystal structure of 1GKC,10 9,627 conformations of 1,952 novel molecules were generated. In this case 59 linkers were used. The original ligand ranked 139th in terms of the Udock value. The significant low rmsd of 0.232 Å has proven that the present de novo design method has succeeded in reconstruction and redocking of the original ligand. The ranking advanced from 139th to 22nd when the Vcom is taken into account. The reconstructed and original ligands are superposed in Fig. 5.
Fig. 5 The X-ray (grey) and the docked (green) ligand of 1GKC are superimposed. |
The chemical structure of the molecule with the lowest Udock value is shown in Fig. 6(a). In this novel molecule, the linker contains a relatively long hydrophobic chain with a carboxyl group at the end. The docked structure of the original and the novel molecules are superposed at the binding site in Fig. 7. The molecules with carbon atoms shown in green and yellow are the novel molecule and the original ligand, respectively. The hydroxamate groups, shown on the right side in this figure, share the same position at the binding site. The terminal carboxylic acid of the novel molecule which is located at the left side in Fig. 7 binds to the backbone nitrogen atom of Gly77. The N atom of the COMPOUND LINKS
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Download mol file of compoundN-methylamide group in the original ligand is hydrogen-bonded to the backbone oxygen atom of Gly77. The corresponding N atom in the novel molecule is, however, bonded to the backbone oxygen atom of Tyr136.
Fig. 6 The chemical structures of the novel molecule with the lowest Udock values. (a) 1GKC (b) 3B7E. |
Fig. 7 Comparison of the binding modes of the X-ray ligand and the novel molecule shown in Fig. 6(a) at the binding site of 1GKC. |
The influenza virus neuraminidase is one of the important targets of antiinfluenza drugs. Based on the crystal structure of the complex between a neuraminidase and an antiinfluenza drug of zanamivir(3B7E),11 81,496 conformations of 5,462 novel molecules were generated. In this case, 60 linkers were used. The original ligand ranked 176th in terms of the Udock value. The rmsd of non-hydrogen atoms is 2.042 Å. If the Vcom is considered to judge the results, the original ligand ranks 13th. The chemical structure of the novel molecule with the lowest Udock value is shown in Fig. 6(b). In the novel molecule, the ring system of COMPOUND LINKS
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Download mol file of compound3,4-dihydro-2H-pyran in COMPOUND LINKS
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Download mol file of compoundzanamivir is replaced by a COMPOUND LINKS
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Download mol file of compoundnaphthalene ring. In addition, two sulfonate groups are attached to the naphthalene ring. The superposition of zanamivir and the novel molecule at the binding site of neuraminidase is shown in Fig. 8. The molecules with carbon atoms shown in green and yellow are the novel molecule and the original ligand, respectively. The sulfonate groups are hydrogen bonded to the side chains of Asp151 and Glu227. In the complex structure of zanamivir, no corresponding hydrogen bonds are observed.
Fig. 8 Comparison of the binding modes of zanamivir and the novel molecule shown in Fig. 6(b) at the binding site of 3B7E. |
As these two examples have demonstrated, the de novo design method proposed in this study can generate novel molecules which are significantly different from the original ligands and possibly bind to the target molecules stronger than the original ligands. Therefore, the present method is expected to give medicinal chemists important clues to facilitate lead optimization.
The results of this method obviously depend on the libraries of linkers and functional groups. In this study, we have selected linkers and functional groups only from the database of drugs which are currently applied clinically in Japan. We understand the number of linkers and functional groups which can be applicable to drugs is strictly limited, but incorporation of novel chemical groups in generating novel molecules will be interesting. On the other hand, for specific targets the number of applicable linkers and functional groups may be more restrictive.
The present study has clearly indicated that a new and powerful strategy of de novo design has deployed in the methodology for in silico drug discovery.
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