Tumor cells employ a host of biochemical mechanisms in order to invade and metastasize. Many of these mechanisms are thought, in part, to involve proteases associated with cell membrane and extracellular matrix (ECM) molecules that are posited to initiate pro-angiogenic signaling cascades. Among cancer-associated proteases, matrix metalloproteases (MMPs), a class of zinc-dependent proteolytic enzymes, have been postulated to be used by cancer cells to dissolve ECM during neoplastic progression.1
In addition, numerous studies have documented a positive correlation between certain MMP expression levels and poor outcome in cancer patients.2
The importance of MMPs in tumor progression not only has guided the development of MMP inhibitors for therapy, it has also received particular attention as imaging target utilizing methods to detect tumor-associated proteolytic activity in vivo
The family of human MMPs contains 16 secreted and 7 membrane-tethered enzymes. 8
A subclass of the membrane-anchored proteinases, termed membrane type (MT) MMPs, plays dominant roles in controlling cancer cell behavior. 9,10
In particular, the up-regulation of the membrane-associated collagenase MMP-14 (MT1-MMP) correlates to the invasiveness of many different tumor types.2
MMP-14 not only promotes tumor growth through induction of angiogenesis and proteolysis of ECM, but it also acts as a critical regulatory switch in the activation of MMP-2 proenzyme.11
Clinical studies revealed that the expression of MMP-14 is associated with poor prognosis in patients with advanced neuroblastoma,12
small cell lung cancer (SCLC),13
tongue squamous cell carcinoma,14
head and neck carcinoma,15
bladder, and ovarian cancer.16,17
. MMP-14 has been detected in tumor cells and adjacent stromal cells in a variety of human tumors including breast. 9
Consequently, MMP-14 overexpression holds great promise as an early biomarker for invasive cancers.
In the past, several in vivo
optical imaging probes targeting various MMPs have been reported; the most successful of these efforts have been directed against MMP-2, -7, and -9.18–23
Attempts to image MMP activities by non-optical modalities (e.g., positron emission tomography (PET) or single photon emission computed tomography (SPECT)) using labeled substrates or inhibitors, however, have met with limited success in vivo
, in part due to the poor specificity and in vivo
stability of the radiolabeled probes.24–31
Our motivation, therefore, was to develop a sensitive nuclear probe for MMP-14 activities for early cancer detection. The success of such a probe would represent a significant advancement in preclinical and clinical imaging as it would be a tool able to locate and track the molecular evolution of malignant tissues for use in drug development.
A number of protease imaging strategies have been described previously. One particular class of probes comprising an “activatable” delivery mechanism has been developed by a number of research groups.21,23,32
These probes share a core structure consisting of a poly-d
-arginine cell-penetrating peptide (CPP) that is covalently tethered to a negatively charged attenuating peptide sequence through a proteolysis sensitive peptide (). The intact probe is believed to be prohibited from crossing the cell membrane due to the electrostatic interaction between the positively charged arginine and the tethered intramolecular negatively charged attenuator (I), but other mechanisms may also contribute. However, proteolytic cleavage (II) separates the polyarginine sequence from the negatively charged domain, thereby triggering uptake of the CPP (III). Protease-rich tissues may be imaged by tagging an imaging reporter group, such as a fluorophore, gadolinium chelate or radionuclide, to the CPP.32
As a result, the number of protease cleavage events may be correlated to the CPP concentration, and its associated tag, within targeted cells. Using agents directed against MMP-2/9 and -7, Rao and coworkers have selectively tagged cultured fibrosarcoma cells (HT-1080) with quantum dots, 23
while Tsien and co-workers successfully imaged cancers rich in MMP-2 and -9 in murine xenografts, using optical and magnetic resonance techniques.21
Figure 1 Outline of the probe structure and mechanism. The quenched probe (I) is able to freely circulate in vivo, until it encounters its protease target. Cleavage of the probe at a defined point by MMP-14 (II) releases a cell penetrating peptide, which can then (more ...)
This general strategy is attractive because the catalytic processing of more than one probe by each enzyme provides a robust mechanism for signal amplification. For the purpose of MMP-14 imaging, this is a particularly important point if one seeks to detect protease activity prior to the maturation of secondary downstream proteases, e.g., MMP-2 and -9.
The development of an “activatable” SPECT imaging probe specific for MMP-14 is reported herein. The probe design was undertaken realizing that attaching a large metal chelate for nuclear imaging may alter the topology of the MMP-14 selective peptide sequence and adversely affect the cleavage rate as well as attenuation characteristics of the basic probe platform. Therefore the combination of molecular modeling, parallel synthesis and bioassay screens were effectively utilized to optimize the imaging probe construct for MMP-14 activities. This work sheds new light on the intramolecular quenching and activation mechanisms of this class of imaging peptides, and demonstrates the value of computational chemistry relative to imaging probe development.