Bone-Seeking Matrix Metalloproteinase Inhibitors for the Treatment of Skeletal Malignancy

Matrix metalloproteinases (MMPs) are a family of enzymes involved at different stages of cancer progression and metastasis. We previously identified a novel class of bisphosphonic inhibitors, selective for MMPs crucial for bone remodeling, such as MMP-2. Due to the increasing relevance of specific MMPs at various stages of tumor malignancy, we focused on improving potency towards certain isoforms. Here, we tackled MMP-9 because of its confirmed role in tumor invasion, metastasis, angiogenesis, and immuno-response, making it an ideal target for cancer therapy. Using a computational analysis, we designed and characterized potent MMP-2/MMP-9 inhibitors. This is a promising approach to develop and clinically translate inhibitors that could be used in combination with standard care therapy for the treatment of skeletal malignancies.


Expression and Purification of the Protein
The truncated form M80-G242 of the catalytic domain of MMP-8 was expressed in E. coli BL21 (DE3). The culture was induced with 0.5 mM IPTG when OD600 = 0.5 -0.6 and further incubated at 310 K for 3 hours. Inclusion bodies isolated and purified from harvested E. coli cells were resuspended in Tris 20 mM, pH 8.5, Urea 6 M, β-mercaptoethanol 100 mM and incubated overnight at room temperature under shaking to extract the solubilized collagenase. This extract was centrifuged for 30' at 40000 rpm, and the supernatant was loaded onto a Mono Q-Sepharose column (GE Healthcare) previously equilibrated with the denaturating buffer.
Elution of the collagenase was carried out by applying a linear gradient of NaCl 0 -1 M in the same buffer at a flow rate of 1 mL/min. The truncated form of MMP-8 was eluted at a salt concentration of 100 mM NaCl and could be purified to apparent homogeneity. A further step of purification was carried out by gel filtration using a Superdex 75 10/300 GL column (GE Healthcare) equilibrated with Tris 20 mM, pH 8.5, Urea 6 M, DTT 10 mM at a flow rate of 0.5 mL/min. The collected protein was then refolded onto a Superdex 75 10/300 GL column in buffer MES/NaOH 3 mM, pH 6.0, NaCl 100mM, CaCl2 5 mM, ZnCl2 0.5 mM, NaN3 0.02% at a flow rate of 0.5 mL/min.

Protein Crystallization
The inhibitor (stock solution 50 mM in DMSO) was immediately added to the fraction containing the refolded protein in the ratio 3:1 (final concentration of DMSO 1%) in order to prevent autoproteolysis during concentration. The MMP-8 protein with the inhibitor was then concentrated with Amicon-Ultra-15 (Millipore), to a final concentration of 6 mg/mL. Crystallization was performed by hanging-drop vapor diffusion method at 20°C. Hanging droplets were made by mixing 2 L of protein/inhibitor solution with 5 L of PEG solution (PEG6000 10% w/v, MES/NaOH 0.2 M, pH 6.0, NaN3 0.02%). Droplets were concentrated against a reservoir buffer containing Sodium Phosphate 1.0-2.0 M, pH 6.0, NaN3 0.02%. Crystals appeared after few days.

Data Collection and Processing
X-ray data were collected under cryogenic conditions (100 K) at the ID29 beamline of ESRF, Grenoble, using a wavelength of 0.976 Å and a Pilatus 6M_F detector. The crystals were flash-frozen in the nitrogen stream after transferring them for few seconds into the mother solution containing 35% PEG400. Data were integrated and scaled using the programs MOSFLM and Scala. 1 The statistics of collection is given in Table S1.

Structure Solution and Refinement
Structure solution was performed with AMoRe 2 using the coordinates of the complex between MMP-8 and a non-zinc chelating inhibitor (PDB entry 3DPE) 3 as the starting model. The coordinates were then refined with CNS. 4 The statistics of refinement is summarized in Table S1.   Proteins are represented as grey cartoon, the catalytic zinc ion is a magenta sphere, residues at 5 Å from the ligands are represented as thin sticks. Ligands and protein residues are almost perfectly superposed; main differences reside in the Ile159 and Arg222 positions.