In this study we used high-throughput chemical library screening in a first step to generate hits for VHR, which were further prioritized through Michaelis-Menten kinetic studies and counter screens against a number of structurally related phosphatases. The resulting lead compound 1 competitively inhibited VHR with a Ki value of 0.81 μM and exhibited a promising degree of selectivity for VHR among other PTPs. In silico docking of 1 into the active site of VHR and SAR analysis suggested a binding mode, in which the sulfonic acid moiety of 1 binds through a network of hydrogen bond interactions into the phosphate binding pocket, whereas the thiazolidine heterocycle interacts with the rim of the pocket and the phenyl-allyl moiety makes van der Waals interactions with side chains of hydrophobic amino acids that flank the active site.
The sulfonic acid head group is an ideal chemical structure to mimic a phosphate group, without undergoing any hydrolysis reaction. Phosphorus and sulfur atoms have similar atomic and van der Waals radii, and are coordinated by 4 (phosphate) or 3 (sulfonate) oxygen atoms, respectively. However, under physiological pH conditions, a phosphate group carries two negative charges whereas the sulfonate has only one, making it easier for compounds carrying the latter to penetrate cell membranes. We therefore decided to keep the oxo-thioxothiazolidinyl-ethanesulfonic acid moiety of 1 as pharmacophore and applied a structure-based approach to optimize 1 in terms of both potency and selectivity.
Examination of the surface area that surrounds the active site in VHR revealed several distinct hydrophobic regions that were exploited in the design of more potent and selective inhibitors. A search for compounds that would still bind to the catalytic pocket, and additionally could interact with not just one but multiple hydrophobic areas in a multidentate fashion resulted in five molecules with significant lower IC50 values than lead structure 1. These compounds contain a thioxothiazolidinyl-ethanesulfonic acid moiety, representing the defined pharmacophore in 1. In addition, a pyrazole ring functions as a linker from which two hydrophobic entities branch off – a phenyl ring in 1-position and a more variable group at 3-position. Co-crystallization of VHR with one of these compounds, SA3, yielded a crystal structure in which clear electron density of SA3 was observed for the thioxothiazolidinyl-ethanesulfonic acid moiety. As predicted by the docking studies, the sulfonic acid group was found to bind tightly into the catalytic pocket, mimicking the phosphate group of the natural substrate. No clear electron density was observed for the two hydrophobic entities of SA3, which is most likely due to some degree of flexibility in the interactions between these parts of the molecule and the hydrophobic amino acid residues of the protein. Nonetheless, modeling of SA3 into the visible electron density leaves no doubt about the bidentate fashion that SA3 employs to bind to distinct hydrophobic patches at the surface that flanks the catalytic pocket in VHR. Besides highly improved potency against VHR with IC50 values as low as 18 nM, these multidentate inhibitors were at least one order of magnitude less potent for any other PTP tested, including HePTP and MKP-1, which share the same physiological substrate with VHR. This result provides a good example for a general applicable concept, in which targeting unique surface features outside of the catalytic pocket can generate selective small-molecule inhibitors for individual members of the PTP family.
We also present several lines of evidence strongly suggesting that our VHR inhibitors are able to pass cell membrane barriers and target VHR in cultured cells. In particular, we tested our compounds in cervical cancer cells, which were shown earlier to express higher levels of endogenous VHR compared to non-cancerous cells of the cervix. Indeed, incubation of the cancer cell lines HeLa and CaSki with the inhibitors at 20 μM induced a very significant inhibition of cell proliferation after an incubation period as short as 24 h. This inhibition of cell growth was not due to cell death, and is also in accordance with our previous work, demonstrating that loss of VHR using RNAi induces a dramatic decrease of HeLa cell numbers and proliferation. It is interesting to note that SA3 was more effective in inhibiting proliferation than SA1, although its IC50 value is 4times higher in vitro. SA3 also exhibited greater antiproliferative effects than SA2 and SA4, both of which share similar IC50 values with SA3 in vitro. These results suggest a beneficial role for the fluorine substituent (which is only present in SA3), maybe by facilitating better membrane permeability. The latter could be a limiting factor for these compounds, considering the substantially higher concentration that is needed to see clear effects in cells vs. inhibition of recombinant protein. Nonetheless, the fact that our inhibitors are not toxic to cells with low levels of endogenous VHR, such as primary normal keratinocytes, these compounds may well be a starting point to develop drugs for the treatment of cervical cancer and perhaps other cancers. Indeed, our results provide first evidence that pharmacological inhibition of VHR could be beneficial in treating such diseases. However, additional studies will be necessary to get better insights into the role of VHR phosphatase in cell cycle regulation and cancer, and to test the activity of these compounds in vivo, using mouse models.