Probing the target-specific inhibition of sensitized protein tyrosine phosphatases with biarsenical probes

Abstract

Selective control of enzyme activity is critical for elucidating the roles of specific proteins in signaling pathways. One potential means for developing truly target-specific inhibitors involves the use of protein engineering to sensitize a target enzyme to inhibition by a small molecule that does not inhibit homologous wild-type enzymes. Previously, it has been shown that protein tyrosine phosphatases (PTPs) can be sensitized to inhibition by a biarsenical probe, FlAsH-EDT2, which inhibits PTP activity by specifically binding to cysteine residues that have been introduced into catalytically important regions. In the present study, we developed an array of biarsenical probes, some newly synthesized and some previously reported, to investigate for the first time the structure-activity relationships for PTP inhibition by biarsenicals. Our data show that biarsenical probes which contain substitutions at the 2' and 7' positions are more effective than FlAsH-EDT2 at inhibiting sensitized PTPs. The increased potency of 2',7'-substituted probes was observed when PTPs were assayed with both para-nitrophenylphosphate and phosphopeptide PTP substrates and at multiple probe concentrations. The data further indicate that the enhanced inhibitory properties are the result of increased binding affinity between the 2',7'-substituted biarsenical probes and sensitized PTPs. In addition we provide previously unknown physicochemical and stability data for various biarsenical probes.

Figure 1

Biarsenical probe acting as an allosteric inhibitor. (A) Spatially arranged cysteines are introduced (light green) in the WPD loop of a protein tyrosine phosphatase (PTP). The mutation causes only limited changes in the catalytic efficiency of the enzyme. However, when the biarsenical probe FlAsH is conjugated, the enzyme is strongly inhibited. (B) Amino acid sequences of wild type and cysteine mutants of TCPTP and HePTP used in this study. Red letters highlight amino acid residues substituted or added to the wild type protein sequence. Protein model was based on structure of TCPTP (PBD: 1L8K).

Figure 2

Structures of biarsenical EDT-capped probes based on different parent fluorophores used in this work.

Figure 3

Schematic pathway for the synthesis of biarsenical probes built on the fluorescein platform. Stages include the synthesis of parent the fluorophore, its subsequent mercurization, transmetalation with AsCl₃, and final capping with EDT. It should be noted that first reaction results in forming of two isomers, where Z is located at position 5 or 6 in the product. Since the probes used in this study are single isomers the Z was placed at position 5.

Figure 4

Synthesis of 5-(N-butylamide)-fluorescein, a parent platform used for the synthesis of 5-BuCrAsH-EDT₂.

Figure 5

Decomposition of biarsenical probes in aqueous pH buffered solution. 25 μM biarsenical probes were incubated for 1, 3 and 6 h in 50 mM Na+-HEPES buffer, 100 mM NaCl, pH 7.4. The percentage of monoarsenical species was determined using analytical HPLC. Details can be found in Table S1 (ESI).

Figure 6

Michaelis-Menten plot for TCPTP 4C incubated without biarsenical probe (black), with FlAsH-EDT₂ (blue) or Et2FlAsH-EDT₂ (red). Assay was performed at various concentrations of para-nitrophenylphosphate (pNPP) using 100 nM enzyme or enzyme-biarsenical complex as described in Materials and Methods. Data was fitted to the Michaelis-Menten equation.

Figure 7

Graphical representation of catalytic efficiency (k_cat/K_m, s⁻¹ mM⁻¹) values for PTP mutants in the presence of 4 equivalents of biarsenical probes and assayed with pNPP. Blue color demonstrate the typical enzyme activity (not inhibited) and red complete inhibition of activity. Gradient color is directly proportional to the degree of inhibition of the enzyme. k_cat, K_m and k_cat/K_m values are listed with their errors in Table S2–S4 (ESI).

Figure 8

Dose-dependent inhibition of the biarsenical-responsive TCPTP mutants. The indicated enzyme (final concentration 1 nM) was incubated for 2.5 h with 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 4 or 8 molar equivalents of biarsenical probe – FlAsH-EDT₂ (black), F2FlAsH-EDT₂ (red), Cl2FlAsH EDT₂ (magenta). Blue demonstrates the activity of wild-type TCPTP incubated with F2FlAsH-EDT₂ for the comparison. The activity was measured using fluorogenic phosphorylated peptide substrate MCA-Gly-Asp-Ala-Glu-Tyr(PO₃H₂)-Ala-Ala-Lys(DNP)-Arg-amide.