Crystal structures and inhibitor identification for PTPN5, PTPRR and PTPN7: a family of human MAPK-specific protein tyrosine phosphatases

Abstract

Protein tyrosine phosphatases PTPN5, PTPRR and PTPN7 comprise a family of phosphatases that specifically inactivate MAPKs (mitogen-activated protein kinases). We have determined high-resolution structures of all of the human family members, screened them against a library of 24000 compounds and identified two classes of inhibitors, cyclopenta[c]quinolinecarboxylic acids and 2,5-dimethylpyrrolyl benzoic acids. Comparative structural analysis revealed significant differences within this conserved family that could be explored for the design of selective inhibitors. PTPN5 crystallized, in two distinct crystal forms, with a sulphate ion in close proximity to the active site and the WPD (Trp-Pro-Asp) loop in a unique conformation, not seen in other PTPs, ending in a 3(10)-helix. In the PTPN7 structure, the WPD loop was in the closed conformation and part of the KIM (kinase-interaction motif) was visible, which forms an N-terminal aliphatic helix with the phosphorylation site Thr66 in an accessible position. The WPD loop of PTPRR was open; however, in contrast with the structure of its mouse homologue, PTPSL, a salt bridge between the conserved lysine and aspartate residues, which has been postulated to confer a more rigid loop structure, thereby modulating activity in PTPSL, does not form in PTPRR. One of the identified inhibitor scaffolds, cyclopenta[c]quinoline, was docked successfully into PTPRR, suggesting several possibilities for hit expansion. The determined structures together with the established SAR (structure-activity relationship) propose new avenues for the development of selective inhibitors that may have therapeutic potential for treating neurodegenerative diseases in the case of PTPRR or acute myeloblastic leukaemia targeting PTPN7.

Figure 1. Sequence alignment of PTP catalytic domains and ribbon diagram of their three-dimensional structures

(A) PTPN5, PTPRR, PTPN7 and PTP1B were aligned using the program ICM Pro 3.3 (Molsoft LLC). Secondary-structure elements described in the text are labelled in the same way as in the ribbon diagrams showing the three-dimensional structure of (B) PTPN5(1) in complex with a sulphate ion shown; (C) PTPRR and (D) PTPN7 in complex with phosphate ions.

Figure 2. Conformation of the WPD loops

(A) Structural overlay of WPD loops found in PTP1B (PDB code 1SUG; purple) in the closed conformation; PTPN7 (black); PTPRR (green); PTPSL (PDB code 1JLN; red); PTP1B (PDB code 2HNP; blue) in the open conformation and PTPN5 (yellow). (B) Structural details of the WPD loop of PTPN5. In order to differentiate the WPD loop from other residues, carbon atoms of WPD loop residues are coloured pink. The 3₁₀ helix η6 is coloured magenta. Side-chain conformations were identical in both PTPN5 structures, except for Arg⁴⁴³ which has a slightly different conformation in PTPN5(1). Asp⁴³⁷ is largely disordered in PTPN5(1), but was visible in the second structure determined. The sulphate moiety found in both crystal structures is shown in a ball-and-stick representation. (C) Structural details of the WPD loop of PTPRR. (D) Structural details of the WPD loop of PTPN7. The phosphate moieties are shown in a ball-and-stick representation.

Figure 3. Docking of compound 1a in the active site of PTPRR

Representation of the active-site surface is shown as well as predicted hydrogen bonds formed by the carboxy acid group of compound 1a to PTPRR residues. Unoccupied regions in the PTPRR active-site cavity offer multiple possibilities for expansion of this compound class.