Major differences include (a) that both prior studies employed 125I-labelled rhTIMP-1 to follow distribution and clearance while we used an ELISA with high specificity for human TIMP-1, and (b) that the prior studies administered much lower doses (mg/kg rather than mg/kg). Figure 7. MMP inhibitory activity of PEG20K-TIMP-1 in fibroblast cultures and orthotopic tumors. (A) In situ zymography of mammary fibroblast cultures developed with (right panel) or without (left panel) 500 nM PEG20K-TIMP-1 show decreased gelatinase activity in the treated cells, as indicated by a reduction in green fluorescent signal produced by cleavage of quenched fluorescent substrate DQ-gelatin. (B) In situ zymography of tumor sections from mammary tumor-bearing mice injected 24 hours prior to sacrifice with saline (left) or 2 mg/kg PEG20K-TIMP-1 (right) show reduced gelatinase activity in the tumor from the mouse receiving PEG20K-TIMP-1. would detect partially degraded polypeptide fragments of rhTIMP-1 as well. Indeed, electrophoretic analysis of 125I-labelled rhTIMP-1 in the rat ischemia-reperfusion model showed that the signal for intact protein peaked at 1.5 h and was much diminished by 3 h [62]. By contrast, we would anticipate that the ELISA used in our experiment would provide a more specific readout for intact biologically active TIMP-1, and therefore a more meaningful measurement from the perspective of therapeutic utility. Our pharmacokinetic study showed gradual elimination of PEG20KTIMP-1 over the course of several days (Fig. 5); this is consistent with our in situ zymography results (Fig. 7B), which suggest that substantial inhibitory activity is retained in vivo even after 24 h. TIMP-1 and its modified derivatives may be particularly well suited for therapeutic targeting of MMP-9, a potential drug target in many pathological processes. MMP-9 has been implicated in atherosclerotic plaque rupture, tissue damage after acute myocardial infarction, and breakdown of the blood-brain barrier and development of brain edema after cerebral ischemia and in other CNS conditions [29]. It also plays significant roles promoting cancer invasion, metastasis, and angiogenesis, as well as inflam matory diseases including osteoarthritis, rheumatoid arthritis, multiple sclerosis, chronic obstructive pulmonary disease (COPD) and other conditions of pulmonary inflammation and fibrosis [29,63,64]. Among TIMPs, native TIMP-1 has the strongest preexisting affinity for MMP-9 [50,65,66], and possesses the unique ability to bind to proMMP-9 through an interaction between the C-terminal domain of TIMP-1 and the C-terminal hemopexin (PEX) domain of the proenzyme [49,50]. This highaffinity interaction substantially accelerates the kinetics of MMP9/TIMP-1 association [50], likely contributing to selectivity of TIMP-1 toward MMP-9 in vivo. The N-terminal domain of TIMP1 in the proMMP-9/TIMP-1 complex remains available and competent for inhibiting other active MMP molecules including MMP-3 [67,68], a physiological activator of MMP-9 [69,70]; the interaction with TIMP-1 therefore protects proMMP-9 from enzymatic activation in vitro [67,71] and in vivo [72]. While native TIMP-1 therefore offers special advantages where targeting of MMP-9 is desired, we found that the PEG20K-TIMP-1 preparation inhibited MMP-9 somewhat less effectively than it inhibited MMP-3cd. This was possibly due to steric incompatibility between one or more sites of TIMP-1 lysine PEGylation and the MMP-9 PEX domain.
For retention of optimal activity toward MMP-9, it may be advantageous for future studies to further pursue approaches for more regioselective PEGylation of rhTIMP-1. Although we did not find success with PEGylation of an introduced Cys residue, the most common approach toward sitespecific PEGylation, another possible approach might pursue PEGylation on the two glycosyl groups of TIMP-1, both of which lie within the N-terminal domain and well removed from the inhibitory site. Yet another possibility could involve mutational studies to identify which of the TIMP-1 Lys residues interferes with MMP-9 association upon PEGylation, and then specifically removing that site of modification by mutagenesis. TIMP-1 offers a potential biopharmaceutical MMP inhibitor but is rapidly eliminated; our results indicate that PEGylation is one feasible approach to improve its pharmacokinetic profile while preserving activity. Several recent publications have suggested other biopharmaceutical approaches to MMP inhibition. Nanoparticles loaded with mouse TIMP-1 were shown to provide neuroprotection in an organotypic hippocampal slice culture model [73]. A fusion protein formed from human TIMP-2 and human serum albumin was found to provide an improved pharmacokinetic profile and biodistribution in a tumor model and to provide an antiangiogenic effect [74]. Furthermore, TIMPs are not the only biomolecules to be investigated as scaffolds for development of MMP inhibitors. An inhibitory human antibody targeting MMP-14, developed using phage display technology, has shown in vivo activity in mouse xenograft tumor models [75]. Mouse monoclonal antibodies, raised against a synthetic antigen that mimics the MMP catalytic site, were shown to inhibit gelatinases via a TIMP-like binding mechanism, and to show therapeutic promise in a mouse model of inflammatory bowel disease [76]. The methods tested and developed in the present work will contribute to the developing biotechnological arsenal for creating next-generation MMP inhibitors.
Abstract
PTPs is a dual-domain receptor type protein tyrosine phosphatase (PTP) with physiologically important functions which render this enzyme an attractive biological target. Specifically, loss of PTPs has been shown to elicit a number of cellular phenotypes including enhanced nerve regeneration following spinal cord injury (SCI), chemoresistance in cultured cancer cells, and hyperactive autophagy, a process critical to cell survival and the clearance of pathological aggregates in neurodegenerative diseases. Owing to these functions, modulation of PTPs may provide therapeutic value in a variety of contexts. Furthermore, a small molecule inhibitor would provide utility in discerning the cellular functions and substrates of PTPs. To develop such molecules, we combined in silico modeling with in vitro phosphatase assays to identify compounds which effectively inhibit the enzymatic activity of PTPs. Importantly, we observed that PTPs inhibition was frequently mediated by oxidative species generated by compounds in solution, and we further optimized screening conditions to eliminate this effect. We identified a compound that inhibits PTPs with an IC50 of 10 mM in a manner that is primarily oxidation-independent. This compound favorably binds the D1 active site of PTPs in silico, suggesting it functions as a competitive inhibitor. This compound will serve as a scaffold structure for future studies designed to build selectivity for PTPs over related PTPs.