Fragments vs VE-PTP: biophysics in action

Protein kinases attach a phosphate group onto amino acid side chains in proteins. Phosphorylation regulates myriad aspects of cell signaling, and thus kinases are common drug targets. Indeed, roughly one third of fragment-derived clinical compounds target kinases. Protein phosphatases remove phosphate groups and thus also make potentially valuable drug targets. Unfortunately, they are very difficult to selectively inhibit, and indeed no fragment-based drugs have entered the clinic. A new paper in Biochemistry from Wataru Asano, Yoshiji Hantani, and colleagues at Japan Tobacco takes the first steps towards rectifying this.

 

Phosphatases are so difficult to drug because most of them have small, highly charged active sites that have evolved to bind phosphate. This moiety and strongly anionic analogs are not very cell permeable or orally bioavailable. Moreover, the small size of the active site makes selectivity challenging, and the fact that many phosphatases contain an active-site cysteine makes them particularly susceptible to assay artifacts.

 

The researchers were interested in vascular endothelial protein tyrosine phosphatase (VE-PTP), which plays a role in vascular homeostasis and angiogenesis. They chose 25,000 fragment-sized molecules (with < 20 heavy atoms) from their HTS collection, all with aqueous solubility > 300 µM, and screened these at 250 µM in a mass-spectrometry-based functional assay. Those that inhibited enzyme activity by at least 40% were retested in dose-response format and also characterized by SPR. Many highly acidic compounds such as sulfonic acids were found, but the researchers were particularly intrigued by Cpd-1, which is only modestly acidic with a calculated pKa of 3.9.

 

Cpd-1 inhibited VE-PTP, but although SPR showed binding, this was not saturable. Thus, the researchers turned to NMR, using multiple protein-observed as well as ligand-observed methods to demonstrate that the molecule binds to the active site of the enzyme. This was confirmed with a crystal structure, which also revealed an “unhappy” water molecule nearby, leading to Cpd-2. This molecule was characterized by crystallography, SPR, and ITC. The molecule proved to be unexpectedly selective for VE-PTP over four other PTPs. The researchers hypothesize that binding to PTPs is often dominated by conserved electrostatic contacts, and because Cpd-2 is less highly charged it relies on other, more specific interactions.

 

This is a nice example of using a variety of biophysical techniques to find and advance fragments. The researchers do a good job of describing the strengths and weaknesses; for example, it was impossible to determine the dissociation constant of Cpd-1 by SPR due to non-specific binding with the protein, reminiscent of a Pin1 story from several years ago.

 

There is still a long way to go, with no cell activity or permeability described for Cpd-2. Still, the paper ends boldly: “we believe that this compound will be developed as a potential drug for VE-PTP-related diseases.” Here’s wishing them success.