Development of Novel Phosphonodifluoromethyl-Containing Phosphotyrosine Mimetics and a First-In-Class, Potent, Selective, and Bioavailable Inhibitor of Human CDC14 Phosphatases

Abstract

Together with protein tyrosine kinases, protein tyrosine phosphatases (PTPs) control protein tyrosine phosphorylation and regulate numerous cellular functions. Dysregulated PTP activity is associated with the onset of multiple human diseases. Nevertheless, understanding of the physiological function and disease biology of most PTPs remains limited, largely due to the lack of PTP-specific chemical probes. In this study, starting from a well-known nonhydrolyzable phosphotyrosine (pTyr) mimetic, phosphonodifluoromethyl phenylalanine (F2Pmp), we synthesized 7 novel phosphonodifluoromethyl-containing bicyclic/tricyclic aryl derivatives with improved cell permeability and potency toward various PTPs. Furthermore, with fragment- and structure-based design strategies, we advanced compound 9 to compound 15, a first-in-class, potent, selective, and bioavailable inhibitor of human CDC14A and B phosphatases. This study demonstrates the applicability of the fragment-based design strategy in creating potent, selective, and bioavailable PTP inhibitors and provides a valuable probe for interrogating the biological roles of hCDC14 phosphatases and assessing their potential for therapeutic interventions.

Figure 1.

Design and synthesis of the novel phosphonodifluoromethyl-containing pTyr mimetics. (A) Design strategy for the novel pTyr Mimetics. (B) Structure of the 7 novel pTyr mimetics.

Figure 2.

Compound 9 competitively inhibited both hCDC14A and B with selectivity over the other 10 major mammalian PTPs. (A-B) Effect of compound 9 on hCDC14A (A) and hCDC14B (B) catalyzed DiFMUP hydrolysis. Compound 9 concentrations were 0 (●), 3 (■), 6 (▲), 9 (▼), and 12 (◆) μM, respectively. The Lineweaver-Burk plot displayed the characteristic intersecting line pattern, consistent with competitive inhibition. (C) Selectivity of compound 9 over a panel of 10 mammalian PTPs. IC₅₀ values of compound 9 tested against indicated PTPs. IC₅₀ values are mean ± standard deviation (SD) of the result from 3 independent experiments. (D) Putative binding pose of compound 9 (green stick) bound to hCDC14B (PDB 1OHC) colored by element with the nearby P+1 pocket shown in the transparent surface. Hydrogen bonds and ionic bonds are represented by yellow dashes. Cation-π interactions are shown with purple dashes. The 4-position of the dibenzofuran centroid points towards the P+1 pocket. (E) P-loop sequence alignment of hCDC14A, hCDC14B, and additional DUSPs (MKP5, Laforin, and VHZ). UniProt accession codes used for alignment are as follows: CDC14A – Q9UNH5; CDC14B – O60729; MKP5 – Q9Y6W6; Laforin – O95278; VHZ - Q9BVJ7. Key: red box with white text = identical residues; white box with black text = different residues; white box with red text = similar residues; text outlined with blue box = similar residues across the group. Figure was prepared using ESPript 3.0.

Figure 3.

Compound 15 is a potent and selective competitive inhibitor for human CDC14A and B. (A-B) Effect of compound 15 on hCDC14A (A) and hCDC14B (B) catalyzed DiFMUP hydrolysis. For CDC14A K_i measurement, compound 15 concentrations were 0 (●), 25 (■), 50 (▲), 75 (▼), and 100 (◆) nM, respectively. For CDC14B K_i measurement, compound 15 concentrations were 0 (●), 100 (■), 200 (▲), 300 (▼), and 400 (◆) nM, respectively. The Lineweaver-Burk plot displayed the characteristic intersecting line pattern, consistent with competitive inhibition. (C) Selectivity of compound 15 over a panel of 16 mammalian PTPs. IC₅₀ values are mean ± standard deviation (SD) of the result from 3 independent experiments. (D) DLS signal of 100 μM compound 15 dissolved in DMG assay buffer. (E) Thermal shift first-derivative curves of 5 μM hCDC14A (green) and 5 μM hCDC14A + 50 μM compound 15 (red).

Figure 4.

The docking pose of compound 15 bound to hCDC14B suggests engagement with the P+1 pocket. (A) Docking pose of compound 15 (cyan stick) with hCDC14B (PDB code 1OHC) colored by element. Hydrogen bonds and ionic bonds are depicted by yellow dashes, cation-π interactions are depicted by purple dashes, and the edge-to-face π-π stacking is shown with a red dash. (B) Docking pose of compound 15 (cyan stick) aligned to the cocrystal structure of hCDC14B (surface colored by element) and a phosphoserine peptide ligand (green stick) (PDB code 1OHE).

Figure 5.

Compound 15 is a cell active and bioavailable hCDC14A and B inhibitor. (A) Immunoblots of whole cell lysates from U2OS cells with indicated treatment for 24 hours. Compound 15 was able to further amplify Mitomycin C-induced p53 ser315 phosphorylation and stabilize p53 protein, whereas no effect on the negative control (compound 18) treated cells were observed. (B) In vivo pharmacokinetics data based on mass spectrometry quantification at 0, 0.5, 1, 3, 6, and 24 h from the time of intraperitoneal injection (I.P.) or oral administration (P.O.) of compound 15 with 50 mg/kg dose. Values are mean ± standard deviation (SD), n=3.

Scheme 1.

Synthesis of intermediates 2a-10a and pTyr mimetics 2–10^{a a}Reagents and conditions: (a) cadmium shot, DMA, argon, rt, 3 h. (b) compound 19, CuBr, argon, DMA, rt to 50 °C, 15 h. 63–74%. (c) TMSI, DCM, 0 °C, 4 h. 41–64%.

Scheme 2.

Synthesis of Ac-F2Pmp-OMe (1) ^{a a}Reagents and conditions: (a) compound 19, CuBr, argon, DMA, rt to 50 °C, 19 h. 77%. (b) 20% TFA/DCM, rt, 6h. (c) acetyl chloride, triethylamine, DCM 0 °C to rt, 4 h. 64% (b and c 2 steps). (c) TMSI, DCM, 0 °C, 4 h. 59%.

Scheme 3.

Synthesis of compound 11–18 ^{a a}Reagents and conditions: (a) Aryl boronic acid, K₂CO₃, Pd(PPh₃)₄, argon, DMF, H2O, 90°C, 16h, 39–49%. (b) ethynylbenzene, CuI, Pd(PPh₃)₄, triethylamine, argon, DMF, 90°C, 16h, 56%. (c) K₂CO_3, Pd(PPh₃)₄, argon, DMF, H₂O, 90°C, 16h, 71%.