Abstract
Lysosomal cysteine peptidase cathepsin B, involved in multiple processes associated with tumor progression, is validated as a target for anti-cancer therapy. Nitroxoline, a known antimicrobial agent, is a potent and selective inhibitor of cathepsin B, hence reducing tumor progression in vitro and in vivo. In order to further improve its anti-cancer properties we developed a number of derivatives using structure-based chemical synthesis. Of these, the 7-aminomethylated derivative (compound 17) exhibited significantly improved kinetic properties over nitroxoline, inhibiting cathepsin B endopeptidase activity selectively. In the present study, we have evaluated its anti-cancer properties. It was more effective than nitroxoline in reducing tumor cell invasion and migration, as determined in vitro on two-dimensional cell models and tumor spheroids, under either endpoint or real time conditions. Moreover, it exhibited improved action over nitroxoline in impairing tumor growth in vivo in LPB mouse fibrosarcoma tumors in C57Bl/6 mice. Taken together, the addition of a 2-(ethylamino)acetonitrile group to nitroxoline at position 7 significantly improves its pharmacological characteristics and its potential for use as an anti-cancer drug.
Highlights
The proteolytic potential of lysosomal cysteine peptidases is highly relevant for cancer progression, initiating and assisting invasion related proteolytic cascades at the invasive edges of tumors
The ability of compound 17 to reduce tumor cell invasion was evaluated on the human glioma cell line U-87 MG and on the mouse fibrosarcoma cell line LPB-1
Invasion was monitored in real time using the xCELLigence system [34]. This system measures invasion of cells through Matrigel, a model of extracellular matrix (ECM), by monitoring the impedance, expressed as cell index (CI) (Figure 1A), across microelectrodes integrated in the membrane between top and bottom compartments of the CIM-plate 16
Summary
The proteolytic potential of lysosomal cysteine peptidases is highly relevant for cancer progression, initiating and assisting invasion related proteolytic cascades at the invasive edges of tumors. Its dual activity is due to the presence of an extra structural element termed the occluding loop [14]. This ~20 amino acid long occluding loop blocks binding of longer endopeptidase substrates, as it covers the S2’ and S3’ subsites of the active site cleft [15]. In the acidic milieu two salt bridges (Asp22-His110 and Asp224-Arg116) bind the loop to the body of the enzyme, limiting access of extended substrates to primed sites of the active site cleft, thereby favoring its dipeptidyl carboxypeptidase activity [14,15,16,17]. It has been proposed that the endopeptidase activity is involved predominantly in ECM degradation and tumor progression, recent results suggest the contribution of exopeptidase activity in tumor progression [19]
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