Protein Tyrosine Phosphatase Inhibition by Metals and Metal Complexes

Abstract

Significance: Protein tyrosine phosphatases (PTPs) play essential roles in controlling cell proliferation, differentiation, communication, and adhesion. The dysregulated activities of PTPs are involved in the pathogenesis of a number of human diseases such as cancer, diabetes, and autoimmune diseases. Recent Advances: Many PTPs have emerged as potential new targets for novel drug discovery. PTP inhibitors have attracted much attention. Many PTP inhibitors have been developed. Some of them have been proven to be efficient in lowering blood glucose levels in vivo or inhibiting tumor xenograft growth. Critical Issues: Some metal ions and metal complexes potently inhibit PTPs. The metal atoms within metal complexes play an important role in PTP binding, while ligand structures influence the inhibitory potency and selectivity. Some metal complexes can penetrate the cell membrane and selectively bind to their targeting PTPs, enhancing the phosphorylation of the related substrates and influencing cellular metabolism. PTP inhibition is potentially involved in the pathophysiological and toxicological processes of metals and some PTPs may be cellular targets of certain metal-based therapeutic agents. Future Directions: Investigating the structural basis of the interactions between metal complexes and PTPs would facilitate a comprehensive understanding of the structure–activity relationship and accelerate the development of promising metal-based drugs targeting specific PTPs. Antioxid. Redox Signal. 20, 2210–2224.

Introduction

Metals play important roles in living organisms. They are usually divided into two groups, essential metals and nonessential metals. Metal ions such as copper, zinc, iron, and manganese are the essential metals, while others are nonessential metals. Essential metals are components of proteins. More than one-third of all proteins need essential metal ions to function. Essential metal ions act as a catalytic center in metalloenzymes. They also serve as structural components in some proteins. Cells have mechanisms to acquire essential metal ions from their extracellular environment; many proteins are involved in their uptake, transport, distribution, and regulation (Fig. 1) (51, 69). However, these metal ions become toxic whenever excessive intracellular accumulation occurs. Thus, their concentrations are strictly controlled in cells. Dysregulation of these metals results in severe neurodegenerative diseases such as Alzheimer's, Parkinson's, Wilson's, and Menkes diseases. Nonessential metals can be transported by hijacking the transporters available for essential metals as well as other transporters or channels (Fig. 1). The uptake of nonessential metals may disrupt the physiological homeostasis of essential metals and disturb cellular metabolism. Metals can also enter cells in the form of metal complexes by passive diffusion, with the help of transport proteins, or by endocytosis (85). When humans are exposed to metals by using metal industrial products, eating metal-contaminated foods, dinking metal-contaminated water, or through occupational exposure to metals and metal conjugates, metals are taken up in excess. The excess metals react with specific systems to produce teratogenic, neurotoxic, cardiotoxic, and/or nephrotoxic effects, resulting in various diseases such as cancer. Because of their toxicity, metals are efficient therapeutic agents. Indeed, metals have been used in the treatment of a variety of diseases for thousands of years. Since the discovery of the anticancer activity of cisplatin (88), investigations of metal-based therapeutic agents have attracted great interest. Some metal complexes containing ruthenium, gold, titanium, copper, rhodium, vanadium, and cobalt are tested for their anticancer activity and several promising candidates have undergone (pre)clinical evaluation (14, 25, 39, 100, 124). Although metals interfere with cellular metabolism in living organisms, little is known about the physiological processing of metals to date. One better understood mechanism is the disruption of the redox balance. Excess metals and metal complexes increase intracellular reactive oxygen species (ROS), resulting in the oxidation of proteins, the peroxidation of lipids, and DNA damage (47). Recently, investigations into the interactions between proteins and metals or metal complexes have attracted increasing attention. Metals and metal complexes have been observed to tightly bind to some proteins or enzymes that are not metalloproteins or metalloenzymes, and influence cellular metabolism (33, 49, 54, 71). Some metal complexes even exhibit selective inhibition against certain enzymes (71). These studies have deepened our understanding of metals in living organisms and present proteins or enzymes as possible targets for metal-based drugs.

FIG. 1.

Cellular metal transport. The mechanisms of cellular handling of both essential and nonessential metals. Transporters whose primary functions are to transport metals are bolded, while those whose primary functions are not the transport of metals are italicized. The essential metals are represented in green and the nonessential metals in red [from ref Martinez-Finley et al. (69)]. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Protein tyrosine phosphatases (PTPs) constitute a large family of signaling enzymes that counteract protein kinases by removing phosphate moieties on tyrosine residues of the target proteins. They play essential roles in intracellular signal transduction, cell growth and differentiation, cell migration, gene transcription, ion-channel activity, immune response, and cell apoptosis (20, 79, 80, 86, 105, 107). In the human genome, there are at least 107 PTP genes, and 81 PTPs of them are active protein phosphatases with the ability to dephosphorylate phosphotyrosine residues (1). PTP activities are dysregulated in many human diseases, including diabetes, obesity, cancer, immune disorders, and osteoporosis (40, 46, 55, 96, 116). Protein tyrosine phosphatase 1B (PTP1B) was the first identified and characterized PTP. It takes part in the negative regulation of signaling pathways mediated by insulin receptors (IRs) and leptin receptors (5, 123). The PTP1B gene null mice exhibit increased insulin sensitivity and obesity resistance with normal phenotypic characteristics, even on a high-fat diet (23), establishing PTP1B as a potential therapeutic target in the treatment of type 2 diabetes and obesity. In addition, leukocyte common related antigen (LAR), PTPα, leukocyte common antigen 45 (CD45), receptor protein tyrosine phosphatase ɛ (RPTPɛ), Src homology phosphatase 1 (SHP-1), T-cell protein tyrosine phosphatase (TCPTP), and Src homology phosphatase 2 (SHP-2) have been conformed to be involved in diabetes mellitus (5, 21, 26). So far, at least 37 PTPs, including SHP-2, phosphatase of regenerating liver-3 (PRL-3), cell division cycle 25 (Cdc25), PTP1B, cell division cycle 14 (Cdc14), and low molecular weight PTP, have been implicated in human cancers (38, 41, 55, 98). Some PTPs (CD45, leukocyte common antigen 148 [CD148], lymphoid tyrosine phosphatase [LYP], SHP-1, TCPTP, and PTP with proline/glutamate/serine/threonine-rich domains [PTP-PEST]) have been linked to lymphocyte activation and autoimmunity (72, 86). Alzheimer's disease is a progressive disease associated with memory loss and impaired cognitive function and characterized by the cortical accumulation of amyloid plaques and neurofibrillary tangles. Amyloid-β accumulation has been demonstrated to be associated with an increase in striatal-enriched PTP levels and activity that in turn disrupts glutamate receptor trafficking to and from the neuronal membrane (115). Because PTPs are implicated in the pathophysiology of various disorders, many of them have been identified as potential new targets for novel drug discovery. A number of PTP inhibitors have been developed (6, 9, 45, 68, 81, 93). Some of them have been proven to be efficient in lowering blood glucose levels in vivo or in inhibiting tumor xenograft growth (93). Although PTPs do not contain any metals, their activities are potently inhibited by some metal ions and metal complexes (65). In this review, we will focus on the recent developments in metal-based PTP inhibitors.

PTP Inhibition by Metal Ions

Metal ions are known to strongly inhibit PTPs. Brautigan et al. originally identified Zn²⁺ as a PTP inhibitor in 1981 (10). They found that Zn²⁺ completely inhibited the dephosphorylation of phosphotyrosine residues on an unidentified membrane protein at concentrations of 10 μM, while other divalent metal cations had no substantial effect. Zn²⁺ strongly inhibits many PTPs in vitro (19, 30, 42, 106, 112, 126). The IC₅₀ values of Zn²⁺ inhibition of some PTPs are in the nanomolar range, ∼17 nM for PTP1B, 200 nM for TCPTP, 98 nM for SHP-1, and 1–2 μM for SHP-2 (35, 36). The study by Maret and colleagues demonstrated that Zn²⁺ is a reversible inhibitor of the receptor PTPβ, with a remarkably low K_i of 21 pM. The cellular free or loosely bound zinc concentrations in various cultured cells are in the range of tens to hundreds of picomolar, suggesting that cellular Zn²⁺ may act as a physiological regulator of PTPs and modulates signal transduction (114). Cellular studies have shown that high concentrations of Zn²⁺ (>100 μM) cause an increase in the tyrosine phosphorylation of proteins such as the epidermal growth factor receptor (EGFR), IR/IGR, c-Src kinase, and extracellular signal-regulated kinase (Erk) (35, 36, 90, 91, 99).

Choi's group investigated the effects of various metal ions on the activity of the vaccinia H1-related protein tyrosine phosphatase (VHR) (50). Among the various metal ions examined, Fe³⁺, Cu²⁺, Zn²⁺, and Cd²⁺ were found to inactivate VHR, and Cu²⁺ was the most potent inactivator. The inactivation effect of Cu²⁺ ion on VHR can be restored by ethylenediamine tetraacetic acid (EDTA). High concentrations of GSH effectively decrease the inactivation effects. The loss of VHR activity is correlated with a decreased ¹⁴C-iodoacetate labeling of active-site cysteine. They thus concluded that the highly potent Cu²⁺ inactivation of VHR is a consequence of the oxidation of the active-site cysteine. Cu²⁺ also potently inhibits PTP1B, TCPTP, megakaryocyte protein-tyrosine phosphatase (PTP-MEG2), and SHP-1 with IC₅₀ values of ∼0.14, 0.15, 0.20, and 0.18 μM, respectively, but almost does not inactivate SHP-2 (127).

Chromium supplementation either improves glycemic control or reduces insulin requirements in patients with type 2 diabetes mellitus (2 –4). An in vitro study demonstrated that 0.1 mM CrCl₃ inhibits PTP1B (rat and human) activity to 21%–33% of control activity. The treatment of cultured rat hepatoma cells with 0.1 mM CrCl₃ increased the insulin-stimulated tyrosine phosphorylation of the high M_r IR substrate (IRS) proteins by 49% to 7.3-fold over basal phosphorylation levels, without altering basal IR or IRS tyrosine phosphorylation or insulin-stimulated receptor autophosphorylation (31).

Gallium nitrate inhibits membrane PTPs both in vitro and from cells with the IC₅₀ values of ∼6.0 μM (7).

These studies indicate that some metal ions are potent inhibitors of PTPs. Particularly, copper and zinc inhibit most PTPs, with IC₅₀ values in the nanomolar range. Copper and zinc are essential metals. Total cellular copper or zinc concentrations are in the several to a few hundred micromolar range, which is usually considered physiologically significant. The nanomolar IC₅₀ values of the copper and zinc required for inhibiting most PTPs are obviously lower than the concentrations of physiological significance, implying that a minor increase in cellular copper or zinc ions will efficiently inhibit PTPs and increase cellular phosphorylation levels, thereby disturbing metabolic processes. Thus, PTP inhibition is possibly involved in the pathophysiological and toxicological processes of metals. Furthermore, the cellular free zinc may act as a physiological regulator of PTPs to modulate signal transduction.

PTP Inhibition by Metal Complexes

Many metal complexes have been utilized for curing diseases. For example, auranofin is a therapeutic agent used clinically to improve the symptoms of rheumatoid arthritis, and cisplatin is used to treat cancers, especially for testicular and ovarian cancers. Indeed, metal complexes have a wide range of biological effects, such as anticancer, antibacterial, antimicrobial, antifungal, and antiparasitic activities. They have also been tested in the treatment of heart diseases, diabetes, rheumatoid arthritis, and ulcers (25, 27 –29, 47, 60, 73, 113). However, their cellular targets are not well known, impeding the design of more efficient metal-based drugs. With the development of proteomics in the last two decades, the interactions between metal complexes and proteins have attracted more and more interest. Metal complexes have been shown to potently inhibit many enzymes, such as protein kinases, matrix metalloproteinases, telomerases, topoisomerase II, glutathione-S- transferases, histone deacetylases, and the chymotrypsin-like activity of the proteasome and so on (11, 14, 24, 49, 54, 71, 83). These enzymes have become possible targets for a novel metal-based drug design.

The activities of PTPs are potently inhibited by metal complexes. Some metal complexes have been shown to be more efficient than metal ions in affecting cellular tyrosine phosphorylation.

Vanadium complexes

Vanadium mimics the effects of insulin. Organic vanadium complexes exhibit high insulin-mimetic potential with few side effects compared with inorganic vanadium (102, 103, 113). A widely accepted mechanism for this action is the inhibition of PTPs.

An earlier study indicated that pervanadate and peroxovanadium complexes display a higher insulin-mimetic activity and PTP inhibitory activities (8, 58). They inhibit the in situ dephosphorylation of autophosphorylated IRs and EGFRs activating the IR kinase and other kinases in cells (82, 94). The increased PTP inhibition activity is partially related to their ability to induce intracellular oxidation (52). An in vitro study has shown that pervanadate inhibits PTP1B by an irreversible oxidation of the catalytic cysteine-SH group to -SO₃H (43).

Although pervanadate and peroxovanadium complexes display higher insulin-mimetic and PTP inhibitory activities, they are more toxic due to their higher valence (92). Vanadate and vanadium complexes are potent PTP inhibitors and efficiently lower blood glucose levels, such as bis(ethylmaltolato)oxovanadium(IV) (BEOV), a bis(maltolato)oxovanadium(IV) (BMOV) derivative, selected for phase 1 and 2 clinical trials (92, 102, 103, 113). The model of PTP inhibition by vanadium compounds is reversible rather than an irreversible oxidation of the catalytic cysteine-SH group (37, 43, 61, 64, 78, 120). The inhibitory abilities of most vanadium complexes (Fig. 2) are shown in Table 1. These data show that vanadium complexes inhibit a great variety of PTPs. BMOV 1 competitively inhibits several PTPs in vitro, and exhibits a four- to eightfold selectivity for human receptor protein tyrosine phosphatase beta (HRPTPβ) as opposed to PTP1B, human low molecular weight cytoplasmic protein tyrosine phosphatase (HCPTPA), or SHP-2 (78). It increases the phosphorylation of IRs in cells. Because PTP1B plays key roles in the negative regulation of signaling pathways mediated by insulin and leptin receptors and regulates insulin sensitivity in humans (5, 75, 97, 123), it is an ideal pharmaceutical target for the treatment of type 2 diabetes and obesity. Vanadium complexes that selectively inhibit PTP1B are desirable for use as potential antidiabetic drugs with lower toxicity. Recently, we compared the ability of different vanadium complexes (Fig. 2) to inhibit several nonreceptor PTPs that have varying degrees of homology. For example, the catalytic domains of TCPTP and PTP1B are ∼74% identical and have very similar active sites (44), while there is a low sequence identity (38%) between the catalytic domains of SHP-1 and PTP1B (119). As shown in Table 1, the mixed-ligand vanadium complexes 2–7 are the most potent PTP1B inhibitors reported so far, with IC₅₀ values at several tens of nM. They also exhibit some selectivity over PTP1B. Their inhibitory potency on PTP1B is about four- to ninefold stronger than on SHP-1, and about twofold greater than on TCPTP (120, 121). The vanadium complexes 12 and 15 with the Schiff base ligands from the condensation of 5-substituted-2-hydroxylbenzaldehyde and 2-substitutedbenzenamine show better selectivity against PTP1B, although their inhibitory effects are not the strongest. The ability of these complexes to inhibit PTP1B is ∼5-, 3-, 8-, 20-fold or 3-, 4-, 10-, 50-fold stronger than their ability to inhibit TCPTP, PTP-MEG2, SHP-1, and SHP-2, respectively (37). Some complexes, such as 8 and 16, display almost no or very little difference in the inhibition of PTP1B, TCPTP, hematopoietic protein tyrosine phosphatase (HePTP), PTP-MEG2, SHP-1, or SHP-2 (37, 64). The different levels of inhibition show that the structures of vanadium complexes influence their inhibitory ability and selectivity. These results also show that almost all of the vanadium complexes have the strongest inhibitory effect on PTP1B, and that some of them display a certain selectivity for PTP1B inhibition, suggesting that the screening of vanadium-based PTP1B selective inhibitors is feasible with proper modifications of the organic ligand moieties.

FIG. 2.

Selected vanadium complexes with known protein tyrosine phosphatase (PTP) inhibition activity.

Table 1.

The Inhibition of Protein Tyrosine Phosphatases by Vanadium Complexes (IC₅₀ , μ M)

Complex	PTP1B	TCPTP	SHP-1	SHP-2	MEG2	HePTP	HRPTPβ	HCPTPA	Ref.
1	0.109	—	—	0.201	—	—	0.026	0.126	(78)
2	0.030	0.054	0.259	—	—	—	—	—	(120)
3	0.041	0.072	0.162	—	—	—	—	—	(121)
4	0.042	0.082	0.214	—	—	—	—	—	(121)
5	0.044	0.086	0.290	—	—	—	—	—	(121)
6	0.060	0.136	0.411	—	—	—	—	—	(121)
7	0.068	0.087	0.410	—	—	—	—	—	(121)
8	0.29	0.24	0.21	—	—	0.37	—	—	(64)
9	0.086	0.109	2.38	—	—	0.218	—	—	(63)
10	0.105	0.129	0.813	—	—	0.191	—	—	(63)
11	0.157	0.152	2.80	—	—	0.413	—	—	(63)
12	0.21	0.19	3.0	49	0.97	—	—	—	(37)
13	0.23	1.3	1.9	4.2	0.6	—	—	—	(37)
14	0.8	8.3	1.0	2.3	3.6	—	—	—	(37)
15	0.6	1.9	6.5	35	2.9	—	—	—	(37)
16	0.93	1.4	2.5	2.6	3.2	—	—	—	(37)

HCPTPA, human low molecular weight cytoplasmic protein tyrosine phosphatase; HePTP, hematopoietic protein tyrosine phosphatase; HRPTPβ, human receptor protein tyrosine phosphatase beta; PTP, protein tyrosine phosphatase; PTP1B, protein tyrosine phosphatase 1B; SHP-1 Src homology phosphatase 1; SHP-2 Src homology phosphatase 2; TCPTP, T-cell protein tyrosine phosphatase.

Copper complexes

Copper complexes have been widely explored as therapeutic agents (22, 76). Many new copper complexes have been demonstrated to have great anticancer potentials and relatively low side effects compared with traditional platinum-based drugs (70, 89, 100). Copper complexes decrease the levels of the Alzheimer disease amyloid-β peptide by activating cell signaling pathways, having potentially neuroprotective effects (12, 17). As potential therapeutics, copper complexes are generally thought to generate ROS and damage macromolecules such as DNA (34, 47, 101). Some investigations have indicated that proteasome inhibition is involved in the apoptosis of cancer cells induced by copper complexes (15, 16, 18, 104). Copper complexes have been designed as human immunodeficiency virus type 1 (HIV-1) protease inhibitors (56, 57). Thus, cellular proteins are possible targets of therapeutic copper complexes.

We found that copper complexes (Fig. 3) can potently inhibit PTPs (Table 2) in vitro. Kinetic studies indicated that these complexes inhibit PTPs by a reversible inhibition model. They bind to PTPs at the active site or allosteric sites. This inhibition may not involve ROS, but may be related to the oxidation state of copper, because monovalent copper complexes exert weaker inhibition (110, 111). The structure of copper complexes affects the potency and selectivity of their inhibition of PTPs. The copper complexes 17–22 with Schiff base ligands containing 3,5-substituted-4-salicylideneamino-3,5-dimethyl-1,2,4- triazole exhibit the strongest and most selective inhibition against PTP1B. They have IC₅₀ values ranging from 0.038 to 0.091 μM, approximately three- to fourfold stronger than their inhibition of TCPTP and PTP-MEG2, and between three- and ninefold stronger than their inhibition of SHP-1 (67). Binuclear copper complexes with phosphono-containing multidentate ligands 23–25 display similar inhibition against PTP1B and TCPTP, with IC₅₀ values ranging from 0.15 to 0.24 μM, ∼10-, 30-, and more than 100-fold stronger inhibition than their inhibition of SHP-1, SHP-2, and PTP-MEG2 (111). The dinuclear copper complexes with organic claw ligands 26–28 exhibit almost the same inhibition of PTP1B and SHP-1, with IC₅₀ values in a range of 0.15–0.31 μM, ∼2-fold stronger inhibition than their inhibition of PTP-MEG2 and 10-fold stronger than their inhibition of TCPTP (66). Although the copper complexes with multibenzimidazoles 29–33 have almost the same inhibitory effects on PTP1B, TCPTP, and PTP-MEG2, with IC₅₀ values from 0.12 to 0.25 μM, they display about a 10-fold weaker inhibition of SHP-1 (61). In contrast, the dinuclear copper complex 34 potently and selectively inhibits TCPTP, with an IC₅₀ of 0.03 μM, ∼10-fold stronger than its inhibition of PTP1B, 41-fold stronger than its inhibition of HePTP, 34-fold stronger than its inhibition of SHP-2, and 3 orders of magnitude stronger than its inhibition of SHP-1. Complex 34 is the copper complex that possesses the strongest inhibitory ability and the best selectivity for TCPTP in our investigation so far, clearly distinguishing TCPTP from the highly homologous PTP1B. Detailed kinetic analyses indicated that complex 34 inhibits TCPTP by a noncompetitive model, suggesting that it binds to TCPTP at an allosteric site. Usually, the key residues at an allosteric site are less conserved and present an opportunity to develop selective inhibitors. Thus, the further characterization of this allosteric site is expected to assist in distinguishing between the highly homologous TCPTP and PTP1B and in identifying TCPTP or PTP1B selective inhibitors.

FIG. 3.

Selected copper complexes with known PTP inhibition activity.

Table 2.

The Inhibition of Protein Tyrosine Phosphatases by Copper Complexes (IC₅₀ , μ M)

Complex	PTP1B	TCPTP	PTP-MEG2	SHP-1	SHP-2	HePTP	Ref.
17	0.043	0.14	0.26	0.27	>100	—	(67)
18	0.038	0.12	0.17	0.32	>100	—	(67)
19	0.089	0.069	0.14	0.29	>100	—	(67)
20	0.075	0.10	0.12	0.25	>100	—	(67)
21	0.052	0.20	0.19	0.31	>100	—	(67)
22	0.091	0.23	0.21	0.50	>100	—	(67)
23	0.16	0.18	26.4	1.2	6.6	—	(110, 111)
24	0.20	0.24	22.7	1.1	6.2	—	(111)
25	0.20	0.22	38.3	1.2	5.7	—	(111)
26	0.15	1.81	0.35	0.23	>100	—	(66)
27	0.17	1.89	0.47	0.16	>100	—	(66)
28	0.31	3.05	0.68	0.26	>100	—	(66)
29	0.12	0.26	0.14	3.9	>100	—	(61)
30	0.21	0.20	0.81	1.4	>100	—	(61)
31	0.13	0.17	0.17	1.8	>100	—	(61)
32	0.14	0.31	0.17	1.2	>100	—	(61)
33	0.24	0.25	0.73	1.4	>100	—	(61)
34	0.03	0.29	—	1.06	29.6	1.23	(122)

PTP-MEG2, megakaryocyte protein-tyrosine phosphatase.

Copper complexes have been found to inhibit PTPs and increase phosphorylation in cells (84, 122). Treatment with bis(thiosemicarbazonato) copper complexes substantially enhances phosphorylation of EGFR in U87MG-EGFR (human astroglial U87MG cell line transfected with the epidermal growth factor receptor) and HeLa cells (cells derived from Henrietta Lacks). However, Cu²⁺ alone does not have this function. The PTP activity inhibited by bis(thiosemicarbazonato)copper complexes at lower concentrations (5 μM) can be partly restored by EDTA or dithiothreitol, but not at higher concentrations (25 μM). The authors consider that the inactivity of PTPs is possibly due to oxidative damage of the enzymes. The proposed mechanism (Fig. 4) is that once the complex enters the cell and encounters the intracellular reducing environment, the Cu²⁺ ion is reduced and dissociated from the complex, releasing Cu⁺ that inhibits PTP1B phosphatase activity and leads to sustained phosphorylation and activation of EGFR, which elicits the release of growth factor and cytokine (84).

FIG. 4.

The proposed bis(thiosemicarbazonato) copper-mediated activation of epidermal growth factor receptor (EGFR) by the inhibition of protein tyrosine phosphatase 1B (PTP1B) [from Price et al . (84) with minor revisions]. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Because complex 34 potently and selectively inhibits TCPTP in vitro, its cellular efficacy has been assessed in C6 rat glioma cells (122). Previous studies have indicated that PTP1B can activate c-Src by dephosphorylating the negative regulatory pTyr529 in the C-terminus of c-Src, while TCPTP is capable of dephosphorylating pSrc 418 (62, 108, 125). Treatment of the cells with complex 34 resulted in an obvious increase in Src418 phosphorylation, whereas no significant change was observed in Src529 phosphorylation (Fig. 5). These data indicate that 34 is also a potent and selective TCPTP inhibitor in cells, as suggested by in vitro studies. Cu²⁺ alone does not increase in Src418 phosphorylation in C6 rat glioma cells (Fig. 5). This result demonstrates that when complex 34 enters the cells, its structure is maintained, thus it selectively binds to TCPTP and not PTP1B, leading to the selective inhibition of TCPTP and enhanced phosphorylation of c-Src 418. This process is different from that of bis(thiosemicarbazonato)copper complexes. The proposed scheme is shown in Figure 6.

FIG. 5.

The effects of different concentrations of complex 34 on Src phosphorylation at Tyr418 and 529 in C6 rat glioma cells. From left to right, lane 1: Cu(NO₃)₂ (2 mM); lane 2: ligands (3 mM 1,10-phen and 1 mM IDA); lanes 3–5: complex 34 ; lane 6: control.

FIG. 6.

The proposed model of selective inhibition of T-cell protein tyrosine phosphatase (TCPTP) by complex 34 and its effect in enhancing the phosphorylation of c-Src 418. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Gold complexes

Gold complexes have been used for many years in the treatment of autoimmune and inflammatory diseases, including rheumatoid and psoriatic arthritis, pemphigus vulgaris, and bronchial asthma (95). They also display anticancer, anti-HIV, and anti-AIDS, as well as antiparasitic properties. Their cellular targets are involved in DNA, thioredoxin reductase, proteasome, and topoisomerase I (73, 87, 95, 117, 118). In 1997, Wang et al. first investigated the PTP inhibitory activity of disodium aurothiomalate 35 (Fig. 7), a complex employed in clinical practice (109). Disodium aurothiomalate competitively inhibits CD45 and PTP1B in the low micromolar range in vitro (Table 3). CD45 that has been inhibited by disodium aurothiomalate can be reactivated by the addition of dithiothreitol in excess, suggesting that disodium aurothiomalate interacts with the essential cysteine residue of the PTP's active site in the catalytic domain.

FIG. 7.

Gold complexes with known PTP inhibition activity.

Table 3.

The Inhibition of Protein Tyrosine Phosphatases by Gold(I) Complexes (IC₅₀ , μ M)

Complex	LYP	PTP-PEST	CD45	HePTP	TCPTP	YopH	PTP1B	Ref
35	—	—	1.2	—	—	—	3.6	(109)
36	200	9	>1000	150	>1000	>1000	>400	(53)
37	150	34	>400	275	>400	>400	76	(53)
38	11	7	>150	22	>150	>150	40	(53)
39	10	9	>150	22	∼150	>150	26	(53)
40	2	70	>70	40	—	—	—	(48)
41	3.5	70	>100	>50	—	—	—	(48)
42	5.0	76	>150	35	—	—	—	(48)
43	2.0	39	26	16	—	—	—	(48)
44	3.5	>80	>70	>50	—	—	—	(48)
45	1.5	15	23	13	—	—	—	(48)
46	25	45	>70	20	—	—	—	(48)
47	33	>150	33	120	—	—	—	(48)

CD45, leukocyte common antigen 45; LYP, lymphoid tyrosine phosphatase; PTP-PEST, PTP with proline/glutamate/serine/threonine-rich domains.

Barrios' group investigated the inhibitory activities of many gold(I) complexes on PTPs (48, 53). Like vanadium and copper complexes, the structures of gold(I) complexes obviously affect their inhibitory potency and selectivity on PTPs. However, their potency seems weaker than vanadium and copper complexes, usually in the micromolar range in vitro. Investigations into the model of inhibition indicate that the complexes reversibly and competitively inhibit PTPs. A comparison of the inhibition activities of gold(I) complexes (Fig. 7) on several PTPs is shown in Table 3. Four gold(I) N-heterocyclic carbine complexes 36–39 exhibit maximum inhibitory potency on PTP-PEST. Complex 36 is a very potent and highly selective PTP-PEST inhibitor. Complex 37 has a slightly weaker potency and selectivity for PTP-PEST than 36 . Complexes 38 and 39 exhibit strong inhibition of PTP-PEST, LYP, PTP1B, and HePTP, with a slight selectivity for PTP-PEST and LYP, but weak inhibition of CD45, TCPTP, and YopH (53). Most gold(I) phosphine complexes 40–45 show stronger and better selectivity for LYP as opposed to PTP-PEST, CD45, and HePTP, while 46 displays similar inhibition of LYP and HePTP, and 47 displays similar inhibition of LYP and CD45 (48). It seems that most gold(I) complexes exhibit better selectivity for LYP or PTP-PEST.

Cellular investigations of PTP inhibition by gold(I) complexes in a Jurkat T antigen human T-cell line and primary mouse thymocytes revealed that the complexes 36–39 cause increased phosphorylation of lymphocyte-specific protein tyrosine kinase (Lck) on Tyr394, a substrate of LYP and PTP-PEST. However, they do not affect phosphorylation of the Lck kinase at Tyr505, which is dephosphorylated by CD45 (Fig. 8). These results imply that these gold(I) complexes have a lower potency on CD45, but are good inhibitors of LYP and (or) PTP-PEST in cells, correlating well with the selectivity observed in vitro. Similar results were obtained for complexes 42, which inhibits LYP and PTP-PEST, but not CD45 (Fig. 8), and in addition, consistent with activity and selectivity in vitro. When the cells are treated with complex 47 , the phosphorylation levels at both the Tyr394 and Tyr505 residues of Lck are increased (Fig. 8), suggesting that complex 47 inhibits cellular LYP and CD45 as it does in vitro (48). These cellular studies demonstrate that, like copper complex 34 , the gold(I) complexes that have entered cells are able to conserve their structures and maintain the inhibitory and selectivity activities on the PTPs that they display in vitro.

FIG. 8.

Intracellular inhibition of lymphoid tyrosine phosphatase (LYP) by gold complexes (from Karver et al. (48) and Krishnamurthy et al. (53) with minor changes). For complexes 36–39 , the phosphorylation of the pTyr394 site of lymphocyte-specific protein tyrosine kinase (Lck) was determined using anti-pSrc (Y416) antibodies, which selectively recognize the phosphorylated Tyr416 in Src, and cross-reacts with the equivalent Lck pTyr394 site in T-cell lysates.

Ruthenium complexes

In the last four decades, many ruthenium complexes have been synthesized and tested for antitumor properties (59, 60). Similar to cisplatin, ruthenium complexes are expected to reduce tumor growth by a mechanism of interaction with cellular DNA (60). Recent studies suggest that ruthenium complexes interact with enzymes such as protein kinases, topo II, and glutathione S-transferases (33, 49, 71). A cocrystal structure of a ruthenium complex and GST P1-1 demonstrated that the complex binds to the enzyme at the dimer interface, close to the thiol of Cys101. Some ruthenium complexes were designed as selective inhibitors of PTPs.

Chatterjee et al. found that [Ru^III(EDTA)(OH₂/OH)]^1−/2− 48 (Fig. 9) inhibits a recombinant Yersinia enterocoliticia PTP at physiological pH levels (13). This inhibition is substantially reduced by the addition of GSH to the reaction system. They presumed that the [Ru^III(EDTA)(OH₂)]⁻ complex binds the Cys residue in the catalytic domain of the PTP through a rapid aqua substitution reaction to inhibit its phosphatase activity.

FIG. 9.

Ruthenium complexes with known PTP inhibition activity.

Ong et al. synthesized a series of organoruthenium complexes containing difluoromethyl- phosphonate functional groups and evaluated their inhibition activity on PTPs (74). Only compounds containing the phosphonic acid moiety were efficacious. Among them, organoruthenium complexes, [(η⁶-cymene)Ru(benzimidazole-phosphonic acid)Cl]Cl 49 (Fig. 9) and [(η⁶-TIPB)Ru(benz-imidazole-phosphonic acid)Cl]Cl 50 (Fig. 9), exhibited ∼7–10-fold more effective inhibition of PTP1B (IC₅₀ of ∼15 μM) than TCPTP, while uncoordinated phenyldifluoromethylphosphonic acid (PFP) ligands were relatively nonselective. They postulated that the improved selectivity may be due to favorable interactions of the Ru(II) scaffold with amino acid residues surrounding the active site of PTP1B, thus leading to more efficient binding of the complexes in PTP1B.

Stibium complexes

Sodium stibogluconate is a drug used in the treatment of leishmaniasis. It selectively inhibits SHP-1 with 10-fold stronger potency than against SHP-2 and PTP1B, but does not inhibit dual specificity protein phosphatase 1/mitogen-activated kinase phosphatase 1 (DUSP1/MKP-1) phosphatase. The concentration completely inhibiting the SHP-1 activity is about 10 μg/ml. In Baf3 cells, the complex augments IL-3-induced Janus family kinase 2/Stat5 tyrosine phosphorylation and proliferation, consistent with its inhibition of SHP-1 in vitro (77).

Iron complexes

Recently, Olivier's group investigated the ability of a mononuclear dicitrate iron complex [Fe(cit)₂H_4-x]^(1−x)− to inhibit the PTP activity in macrophages (32). Their results demonstrated that the complex alone was able to inhibit the total PTP activity in macrophages, whereas the inorganic salts FeSO₄ or FeCl₃ did not inhibit the PTP activity in macrophages. The inhibition of the PTP activity in macrophages by this complex was not related to the generation of ROS via Habber Weiss and Fenton chemistry. The complex inhibited recombinant SHP-1, TCPTP, PTP1B, and PTP-PEST. Treatment with the complex led to protein hyperphosphorylation and enhanced mitogen-activated protein kinase (MAPK) signaling in response to LPS (Escherichia coli, serotype 0111:B4) stimulation.

Gallium complexes

Gallium complexes, digallium trisulfate, gallium tris(acetylacetonate), and gallium tris(D-gluconato) are potent PTP inhibitors. The IC₅₀ values for their inhibition of membrane PTPs are ∼8.4, 21.7, and 60.1 μM, respectively (7).

Innovation

Protein tyrosine phosphatases (PTPs) are involved in the pathogenesis of a number of human diseases and are recognized as potential new targets for drug discovery. The inhibition of PTPs by metal complexes demonstrates that metal complexes, especially the V, Cu, and Au complexes, are potent PTP inhibitors. Some copper and gold complexes are good selective inhibitors of one or two specific PTPs and selectively improve the phosphorylation of the corresponding substrates in cells. The data suggest that the exploitation of metal-based therapeutic agents targeting specific PTPs is promising.

Conclusions and Future Directions

The in vitro studies of PTP inhibition demonstrate that some metal ions and metal complexes are potent PTP inhibitors. Metal atoms in metal complexes play an important role in the binding of PTP, while ligand structures influence their inhibitory potency and selectivity. Although metal complexes are usually expected to exchange their metal ion in the cell, some metal complexes are stable enough to penetrate the cell membrane and enter cells, selectively binding to their targeting PTPs and influencing the related cell signaling cascades. These findings suggest that PTP inhibition is possibly implicated in the pathophysiological and toxicological processes of metals, and that PTPs may be cellular targets for metal-based therapeutic agents. However, how metal complexes bind to PTPs and what roles the metals and their ligands play in this binding are still not known. Therefore, using structural biology methods, such as X-ray crystallography, to study the structural basis of the interactions between metal complexes and PTPs will be important to facilitate a comprehensive understanding of the structure–activity relationship and help us to design novel metal-based therapeutic agents targeting specific PTPs.

Footnotes

Acknowledgments

This work was supported financially by the National Natural Science Foundation of China (21171109 and 21271121), SRFDP (20111401110002 and 20121401110005), the Natural Science Foundation of Shanxi Province of China (2010011011-2 and 2011011009-1), and the Research Project supported by Shanxi Scholarship Council of China (2012-004 and 2013-026).

Abbreviations Used

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