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A dual inhibitor of matrix metalloproteinases and a disintegrin and metalloproteinases, [ 18 F]FB-ML5, as a molecular probe for

Scheme 1: Synthesis of the building block 9 Figure 1: Structure and design of ML5

4. Materials and methods 1 General

4.20 Metabolite analysis (plasma) of [ 18 F]FB-ML5 in a HT1080 xenograft mouse model

Metabolite analysis was performed on plasma collected after the ex vivo biodistri-bution study. 750 µL of ACN was added to 250 µL of plasma, the mixture was then centrifuged for 3 min at 3000 rpm. The supernatant was passed through a Millex Filter (0.22 µm), diluted with 600 µL acetonitrile and 600 µL H2O, and analysed by semi-preparative HPLC. Fractions of the eluate were collected every minute and radioactivity in these fractions was then determined with a gammacounter (LKB Wallac, Turku, Finland).

5. Acknowledgements

The authors wish to thank the Dutch Technology Foundation (STW) for financial support (project 08008).

Supplementary information

Molecular modeling of ML5 and FB-ML5

The MolDock Scores for ML5 and FB-ML5 for MMP-2, MMP-9, MMP-12 and ADAM-17 are summarized in [Table 1]. MolDock score is described as the fitness of pose into the binding site by evaluating the intermolecular interaction energy between the ligand and the enzyme, and the intramolecular interaction energy of the enzyme. Docking results of ML5 and FB-ML5 into MMPs shows alignment to the molecule design concept. The substituent construct on ML5 and FB-ML5 which are P1’, P2’ and P3’, correspond with MMPs and ADAM-17 pockets S1’, S2’, and S3’. The hydroxamic acid group was in position to form binding coordination with Zn2+. The detail of the ML5 and FB-ML5 docking results on MMPs and ADAM-17 are described below.

ML5 with MMP-2

The distances between the hydroxamic acid group and Zn2+ are respectively 2.22 and 2.26 Å.

The isopropyl chain is positioned inside the S1’ pocket, whereas the phenyl ring is in the S2’ pocket (S1’ and S2’ are hydrophobic cavities which contain non-polar residues (valine and leucine at S1’; isoleucine, alanine, proline and leucine at S2’).

The lysine side chain of ML5 is solvent exposed.

Several hydrogen bonds are formed with ML5.

FB-ML5 with MMP-2

The distances between the hydroxamic acid group and Zn2+ are respectively 2.15 and 2.27 Å.

The isopropyl chain is positioned inside the S1’ pocket.

Both phenyl and para-chain are solvent exposed. The fluorobenzoyl-lysine cannot adopt the same pose as the fluorobenzoyl-lysine on ML5 since it has a longer chain and is bulky.

The phenyl side chain does not fit into the S2’ pocket, possibly due to the fluoroben-zoyl-lysine side chain which pulls the molecule to be solvent exposed. Moreover, the aromatic interaction between the tyrosine residue and the phenyl ring could contribute to the affinity of FB-ML5.



ML5 with MMP-9

The distances between the hydroxamic acid group and Zn2+ are respectively 2.11 and 2.26 Å.

The isopropyl chain is positioned inside the S1’ pocket.

The phenyl ring is solvent exposed.

The lysine chain of ML5 fits into the S3’ pocket.

Several hydrogen bonds are formed, which suggests that ML5 can bind tightly to the active site of MMP-9.

FB-ML5 with MMP-9

The distances between the hydroxamic acid group and Zn2+ are respectively 2.10 and 2.18 Å.

The isopropyl chain is positioned inside the S1’ pocket.

The phenyl ring is solvent exposed.

The fluorobenzoyl-lysine side chain fits into the S3’ pocket and is solvent exposed.

ML5 with MMP-12

The lysine side chain of ML5 fits into the S3’ pocket, a little space is left.

FB-ML5 with MMP-12

The fluorobenzoyl-lysine side chain is too large for the S3’ pocket and as a result, the group bends to another direction.

ML5 with ADAM-17

The benzene group is solvent exposed and forms a hydrophobic interaction with isoleucine.

The lysine side chain of ML5 fits nicely into the S3’ pocket and forms hydrogen bonds with alanine.

FB-ML5 with ADAM-17

The fluorobenzoyl-group goes to the S3’ pocket but as it is a big structure, it cannot go deep into this pocket.

The lysine side chain pulls out to the solvent and as a result, less hydrogen bonds can be formed by the backbone of ML5.


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MicroPET evaluation of a hydroxamate-based MMP inhibitor,