Bicyclic benzofuran and indole-based salicylic acids as protein tyrosine phosphatase inhibitors

Protein tyrosine phosphatases (PTPs) constitute a large and structurally diverse family of signaling enzymes that control the cellular levels of protein tyrosine phosphorylation. Malfunction of PTP activity has significant implications in many human diseases, and the PTP protein family provides an exciting array of validated diabetes/obesity (PTP1B), oncology (SHP2), autoimmunity (Lyp), and infectious disease (mPTPB) targets. However, despite the fact that PTPs have been garnering attention as novel therapeutic targets, they remain largely an untapped resource. The main challenges facing drug developers by the PTPs are inhibitor specificity and bioavailability. Work over the last ten years has demonstrated that it is feasible to develop potent and selective inhibitors for individual members of the PTP family by tethering together small ligands that can simultaneously occupy both the active site and unique nearby peripheral binding sites. Recent results with the bicyclic salicylic acid pharmacophores indicate that the new chemistry platform may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. Structural analysis of PTP-inhibitor complexes reveals molecular determinants important for the development of more potent and selective PTP inhibitors, thus offering hope in the medicinal chemistry of a largely unexploited protein class with a wealth of attractive drug targets.     

Parallel synthesis of a library of bidentate protein tyrosine phosphatase inhibitors based on the alpha-ketoacid motif

Protein tyrosine phosphatases (PTPases) regulate intracellular signal transduction pathways by controlling the level of tyrosine phosphorylation in cells. These enzymes play an important role in a variety of diseases including type II diabetes and infection by the bacterium Yersinia pestis, which is the causative agent of bubonic plague. This report describes the synthesis, using parallel solution-phase methods, of a library of 104 potential inhibitors of PTPases. The library members are based on the bis(aryl alpha-ketocarboxylic acid) motif that incorporates a carboxylic acid on the central benzene linker. This carboxylic acid was coupled with a variety of different aromatic amines through an amide linkage. The aromatic component of the resulting amides is designed to make contacts with residues that surround the active site of the PTPase. The library was screened against the Yersinia PTPase and PTP1B. Based upon the screening results, four members of the library were selected for further study. These four compounds were evaluated against the Yersinia PTPase, PTP1B, TCPTP, CD45, and LAR. Compound 14 has an IC(50) value of 590nM against PTP1B and is a reversible competitive inhibitor. This affinity represents a greater than 120-fold increase in potency over compound 2, the parent structure upon which the library was based. A second inhibitor, compound 12, has an IC(50) value of 240nM against the Yersinia PTPase. In general, the selectivity of the inhibitors for PTP1B was good compared to LAR, but modest when compared to TCPTP and CD45.     

Hydroxamido vanadates: aqueous chemistry and function in protein tyrosine phosphatases and cell cultures

The protein tyrosine phosphatases (PTPases) are a group of regulatory enzymes that are critically important to a wide variety of cellular functions. A number of these PTPases have significant potential as targets for therapeutic intervention, for instance, in diabetes and autoimmune disease treatment. The hydroxylamine complex, bis(N,N-dimethylhydroxamido)hydroxooxovanadate (DMHAV), is an excellent inhibitor of the two PTPases, protein tyrosine phosphatase 1B (PTP1B) and leucocyte common antigen related phosphatase (LAR). However, because of the similarity of the active site architecture within the group of known PTPases, DMHAV is probably an effective inhibitor of most PTPases. Information gleaned from studies of the mechanism of inhibition of PTPases by peptide-derived inhibitors, together with information from comparative protein modelling and studies of the aqueous chemistry of DMHAV, has provided insights for the development of selective PTPase inhibitors. In cell cultures, DMHAV is effective in increasing phosphotyrosine levels on the insulin receptor and greatly facilitates glucose transport and glycogen synthesis. Selective PTPase inhibitors that are developed from the basis of the hydroxylamine motif may lead to effective vanadate-based complexes that have potential as therapeutic agents.     

Probing the molecular basis for potent and selective protein-tyrosine phosphatase 1B inhibition

Protein-tyrosine phosphatases (PTPs) are important for the control of proper cellular tyrosine phosphorylation. Despite the large number of PTPs encoded in the human genome and the emerging roles played by PTPs in human diseases, a detailed understanding of the role played by PTPs in normal physiology and in pathogenic conditions has been hampered by the absence of PTP-specific inhibitors. Such inhibitors could serve as useful tools for determining the physiological functions of PTPs and may constitute valuable therapeutics in the treatment of several human diseases. However, because of the highly conserved nature of the active site, it has been difficult to develop selective PTP inhibitors. By taking an approach to tether together two small ligands that can interact simultaneously with the active site and a unique proximal noncatalytic site, we have recently acquired Compound 2 (see Fig. 1), the most potent and selective PTP1B inhibitor identified to date, which exhibits several orders of magnitude selectivity in favor of PTP1B against a panel of PTPs. We describe an evaluation of the interaction between 2 and its analogs with PTP1B and its site-directed mutants selected based on hydrogen/deuterium exchange of PTP1B backbone amides in the presence and absence of 2. We have established the binding mode of Compound 2 and identified 12 PTP1B residues that are important for the potency and selectivity of Compound 2. Although many of the residues important for Compound 2 binding are not unique to PTP1B, the combinations of all contact residues differ between PTP isozymes, which suggest that the binding surface defined by these residues in individual PTPs determines inhibitor selectivity. Our results provide structural information toward understanding of the molecular basis for potent and selective PTP1B inhibition and further establish the feasibility of acquiring potent, yet highly selective, PTP inhibitory agents.     

Structure-based design of selective and potent inhibitors of protein-tyrosine phosphatase beta

Protein-tyrosine phosphatases (PTPs) are considered important therapeutic targets because of their pivotal role as regulators of signal transduction and thus their implication in several human diseases such as diabetes, cancer, and autoimmunity. In particular, PTP1B has been the focus of many academic and industrial laboratories because it was found to be an important negative regulator of insulin and leptin signaling, and hence a potential therapeutic target in diabetes and obesity. As a result, significant progress has been achieved in the design of highly selective and potent PTP1B inhibitors. In contrast, little attention has been given to other potential drug targets within the PTP family. Guided by x-ray crystallography, molecular modeling, and enzyme kinetic analyses with wild type and mutant PTPs, we describe the development of a general, low molecular weight, non-peptide, non-phosphorus PTP inhibitor into an inhibitor that displays more than 100-fold selectivity for PTPbeta over PTP1B. Of note, our structure-based design principles, which are based on extensive bioinformatics analyses of the PTP family, are general in nature. Therefore, we anticipate that this strategy, here applied to PTPbeta, in principle can be used in the design and development of selective inhibitors of many, if not most PTPs.     

The structural basis for the selectivity of benzotriazole inhibitors of PTP1B

Protein tyrosine phosphatase 1B (PTP1B) has been implicated in the regulation of the insulin signaling pathway and represents an attractive target for the design of inhibitors in the treatment of type 2 diabetes and obesity. Inspection of the structure of PTP1B indicates that potent PTP1B inhibitors may be obtained by targeting a secondary aryl phosphate-binding site as well as the catalytic site. We report here the crystal structures of PTP1B in complex with first and second generation aryldifluoromethyl-phosphonic acid inhibitors. While all compounds bind in a previously unexploited binding pocket near the primary binding site, the second generation compounds also reach into the secondary binding site, and exhibit moderate selectivity for PTP1B over the closely related T-cell phosphatase. The molecular basis for the selectivity has been confirmed by single point mutation at position 52, where the two phosphatases differ by a phenylalanine-to-tyrosine switch. These compounds present a novel platform for the development of potent and selective PTP1B inhibitors.     

Calixarene-based phosphinic acids as inhibitors of protein tyrosine phosphatases

In the present work, the derivatives of calix[4]arene, thiacalix[4]arene, and sulfonylcalix[4]arene bearing four methylene(phenyl)phosphinic acid groups on the upper rim of the macrocycle were synthesized and studied as inhibitors of human protein tyrosine phosphatases. The inhibitory capacities of the three compounds towards PTP1B were higher than those for protein tyrosine phosphatases TC–PTP, MEG1, MEG2, and SHP2. The most potent sulfonylcalix[4]arene phosphinic acid displayed K_i value of 32?nM. The thiacalix[4]arene phosphinic acid was found to be a low micromolar inhibitor of PTP1B with selectivity over the other PTPs. The kinetic experiments showed that the inhibitors compete with the substrate for the active site of the enzyme. Molecular docking was performed to explain possible binding modes of the calixarene-based phosphinic inhibitors of PTP1B.     

The YRD motif is a major determinant of substrate and inhibitor specificity in T-cell protein-tyrosine phosphatase

We have studied T-cell protein-tyrosine phosphatase (TCPTP) as a model phosphatase in an attempt to unravel amino acid residues that may influence the design of specific inhibitors. Residues 48--50, termed the YRD motif, a region that is found in protein-tyrosine phosphatases, but absent in dual-specificity phosphatases was targeted. YRD derivatives of TCPTP were characterized by steady-state kinetics and by inhibition studies with BzN-EJJ-amide, a potent inhibitor of TCPTP. Substitution of Asp(50) to alanine or Arg(49) to lysine, methionine, or alanine significantly affected substrate hydrolysis and led to a substantial decrease in affinity for BzN-EJJ-amide. The influence of residue 49 on substrate/inhibitor selectivity was further investigated by comparing subsite amino acid preferences of TCPTP and its R49K derivative by affinity selection coupled with mass spectrometry. The greatest effect on selectivity was observed on the residue that precedes the phosphorylated tyrosine. Unlike wild-type TCPTP, the R49K derivative preferred tyrosine to aspartic or glutamic acid. BzN-EJJ-amide which retains the preferred specificity requirements of TCPTP and PTP1B was equipotent on both enzymes but greater than 30-fold selective over other phosphatases. These results suggest that Arg(49) and Asp(50) may be targeted for the design of potent and selective inhibitors of TCPTP and PTP1B.     

Isoxazole carboxylic acids as protein tyrosine phosphatase 1B (PTP1B) inhibitors

Guided by X-ray crystallography, we have extended the structure-activity relationship (SAR) study on an isoxazole carboxylic acid-based PTP1B inhibitor (1) and more potent and equally selective (>20-fold selectivity over the highly homologous T-cell PTPase, TCPTP) PTP1B inhibitors were identified. Inhibitor 7 demonstrated good cellular activity against PTP1B in COS 7 cells.     

Investigations of linker structure on the potency of a series of bidentate protein tyrosine phosphatase inhibitors

Protein tyrosine phosphatases (PTPases) and protein tyrosine kinase (PTKases) regulate the phosphorylation and dephosphorylation of tyrosine residues in proteins, events that are essential for a variety of cellular functions. PTPases such as PTP1B and the Yersinia PTPase play an important role in diseases including type II diabetes and bubonic plague. A library of 67 bidentate PTPase inhibitors that are based on the alpha-ketocarboxylic acid motif has been synthesized using parallel solution-phase methods. Two aryl alpha-ketocarboxylic acids were tethered to a variety of different diamine linkers through amide bonds. The compounds were assayed in crude form against the Yersinia PTPase, PTP1B, and TCPTP. Six compounds were selected for further evaluation, in purified form, against the Yersinia PTPase, PTP1B, TCPTP, LAR, and CD45. These compounds had IC50 values in the low micromolar range against the Yersinia PTPase, PTP1B, and TCPTP, showed good selectivity for PTP1B over LAR, and modest selectivity over CD45. The correlation between linker structure and inhibitor activity shows that aromatic groups in the linker can play an important role in determining binding affinity in this class of inhibitors.     

Development of a robust scintillation proximity assay for protein tyrosine phosphatase 1B using the catalytically inactive (C215S) mutant

Protein tyrosine phosphatases are a class of enzymes that function to modulate tyrosine phosphorylation of cellular proteins and play an essential role in regulating cell function. PTP1B has been implicated in the negative regulation of the insulin signaling pathway by dephosphorylating the activated insulin receptor. Inhibiting this phosphatase and preventing the insulin-receptor downregulation has been suggested as a target for the treatment of Type II diabetes. A high-throughput screen for inhibitors of PTP1B was developed using a scintillation proximity assay (SPA) with GST-- or FLAG--PTP1B((1-320)) and a potent [(3)H]-tripeptide inhibitor. The problem of interference from extraneous oxidizing and alkylating agents which react with the cysteine active-site nucleophile was overcome by the use of the catalytically inactive C215S form of the native enzyme (GST--PTP1B(C215S)). The GST--PTP1B was linked to the protein A scintillation bead via GST antibody. The radiolabeled inhibitor when bound to the enzyme gave a radioactive signal that was competed away by the unknown competitive compounds. Further utility of PTP1B(C215S) was demonstrated by mixing in the same well both the catalytically inactive GST--PTP1B(C215S) and the catalytically active FLAG--CD45 with an inhibitor. Both a binding and kinetic assay was then performed in the same 96-well plate with the inhibition results determined for the PTP1B(C215S) (binding assay) and CD45 (activity assay). In this way inhibitors could be differentiated between the two phosphatases under identical assay conditions in one 96-well assay plate. The use of a mutant to reduce interference in a binding assay and compare with activity assays is also amenable for most cysteine active-site proteases.     

Allosteric inhibition of protein tyrosine phosphatase 1B

Obesity and type II diabetes are closely linked metabolic syndromes that afflict >100 million people worldwide. Although protein tyrosine phosphatase 1B (PTP1B) has emerged as a promising target for the treatment of both syndromes, the discovery of pharmaceutically acceptable inhibitors that bind at the active site remains a substantial challenge. Here we describe the discovery of an allosteric site in PTP1B. Crystal structures of PTP1B in complex with allosteric inhibitors reveal a novel site located approximately 20 A from the catalytic site. We show that allosteric inhibitors prevent formation of the active form of the enzyme by blocking mobility of the catalytic loop, thereby exploiting a general mechanism used by tyrosine phosphatases. Notably, these inhibitors exhibit selectivity for PTP1B and enhance insulin signaling in cells. Allosteric inhibition is a promising strategy for targeting PTP1B and constitutes a mechanism that may be applicable to other tyrosine phosphatases.     

Potent,selective inhibitors of protein tyrosine phosphatase 1B

We have previously reported a novel series of oxalyl-aryl-amino benzoic acid-based, catalytic site-directed, competitive, reversible protein tyrosine phosphatase 1B (PTP1B) inhibitors. With readily access to key intermediates, we utilized a solution phase parallel synthesis approach and rapidly identified a highly potent PTP1B inhibitor (19, K(i)=76 nM) with moderate selectivity (5-fold) over T-cell PTPase (TCPTP) through interacting with a second phosphotyrosine binding site (site 2) in the close proximity to the catalytic site.     

Small molecule peptidomimetics containing a novel phosphotyrosine bioisostere inhibit protein tyrosine phosphatase 1B and augment insulin action

Protein tyrosine phosphatase 1B (PTP1B) attenuates insulin signaling by catalyzing dephosphorylation of insulin receptors (IR) and is an attractive target of potential new drugs for treating the insulin resistance that is central to type II diabetes. Several analogues of cholecystokinin(26)(-)(33) (CCK-8) were found to be surprisingly potent inhibitors of PTP1B, and a common N-terminal tripeptide, N-acetyl-Asp-Tyr(SO(3)H)-Nle-, was shown to be necessary and sufficient for inhibition. This tripeptide was modified to reduce size and peptide character, and to replace the metabolically unstable sulfotyrosyl group. This led to the discovery of a novel phosphotyrosine bioisostere, 2-carboxymethoxybenzoic acid, and to analogues that were >100-fold more potent than the CCK-8 analogues and >10-fold selective for PTP1B over two other PTP enzymes (LAR and SHP-2), a dual specificity phosphatase (cdc25b), and a serine/threonine phosphatase (calcineurin). These inhibitors disrupted the binding of PTP1B to activated IR in vitro and prevented the loss of tyrosine kinase (IRTK) activity that accompanied PTP1B-catalyzed dephosphorylation of IR. Introduction of these poorly cell permeant inhibitors into insulin-treated cells by microinjection (oocytes) or by esterification to more lipophilic proinhibitors (3T3-L1 adipocytes and L6 myocytes) resulted in increased potency, but not efficacy, of insulin. In some instances, PTP1B inhibitors were insulin-mimetic, suggesting that in unstimulated cells PTP1B may suppress basal IRTK activity. X-ray crystallography of PTP1B-inhibitor complexes revealed that binding of an inhibitor incorporating phenyl-O-malonic acid as a phosphotyrosine bioisostere occurred with the mobile WPD loop in the open conformation, while a closely related inhibitor with a 2-carboxymethoxybenzoic acid bioisostere bound with the WPD loop closed, perhaps accounting for its superior potency. These CCK-derived peptidomimetic inhibitors of PTP1B represent a novel template for further development of potent, selective inhibitors, and their cell activity further justifies the selection of PTP1B as a therapeutic target.     

Probing protein-tyrosine phosphatase substrate specificity using a phosphotyrosine-containing phage library

Protein tyrosine phosphatases (PTPs) play important, highly dynamic roles in signaling. Currently about 90 different PTP genes have been described. The enzymes are highly regulated at all levels of expression, and it is becoming increasingly clear that substrate specificity of the PTP catalytic domains proper contributes considerably to PTP functionality. To investigate PTP substrate selectivity, we have set up a procedure to generate phage libraries that presents randomized, phosphotyrosine-containing peptides. Phages that expressed suitable substrates were selected by immobilized, substrate-trapping GST-PTP fusion proteins. After multiple rounds of selection, positive clones were confirmed by SPOT analysis, dephosphorylation by wild-type enzyme, and Km determinations. We have identified distinct consensus substrate motifs for PTP1B, Sap-1, PTP-beta, SHP1, and SHP2. Our results confirm substrate specificity for individual PTPs at the peptide level. Such consensus sequences may be useful both for identifying potential PTP substrates and for the development of peptidomimetic inhibitors.     

Protein tyrosine phosphatases: prospects for therapeutics

Protein tyrosine phosphatases (PTPs) form a large family of enzymes that serve as key regulatory components in signal transduction pathways. Recent gene knockout studies in mice identify PTP1B as a promising target for anti-diabetes/obesity drug discovery. PTPs are also implicated in a wide variety of other disorders, including cancer. Significant progress has been made in identifying small molecules that simultaneously bind both the active site and a unique adjacent site that enables specific inhibition of individual PTP isoenzymes. As a consequence, there are compelling reasons to believe that PTP inhibitors may ultimately serve as powerful therapeutic weapons in our arsenal for battling human diseases.     

2-(oxalylamino)-benzoic acid is a general, competitive inhibitor of protein-tyrosine phosphatases

Protein-tyrosine phosphatases (PTPs) are critically involved in regulation of signal transduction processes. Members of this class of enzymes are considered attractive therapeutic targets in several disease states, e.g. diabetes, cancer, and inflammation. However, most reported PTP inhibitors have been phosphorus-containing compounds, tight binding inhibitors, and/or inhibitors that covalently modify the enzymes. We therefore embarked on identifying a general, reversible, competitive PTP inhibitor that could be used as a common scaffold for lead optimization for specific PTPs. We here report the identification of 2-(oxalylamino)-benzoic acid (OBA) as a classical competitive inhibitor of several PTPs. X-ray crystallography of PTP1B complexed with OBA and related non-phosphate low molecular weight derivatives reveals that the binding mode of these molecules to a large extent mimics that of the natural substrate including hydrogen bonding to the PTP signature motif. In addition, binding of OBA to the active site of PTP1B creates a unique arrangement involving Asp(181), Lys(120), and Tyr(46). PTP inhibitors are essential tools in elucidating the biological function of specific PTPs and they may eventually be developed into selective drug candidates. The unique enzyme kinetic features and the low molecular weight of OBA makes it an ideal starting point for further optimization.     

Novel benzofuran inhibitors of human mitogen-activated protein kinase phosphatase-1

Protein tyrosine phosphatases have a central role in the maintenance of normal cellular functionality. For example, PTP1B has been implicated in insulin-resistance, obesity, and neoplasia. Mitogen-activated protein kinase phosphatase-1 (MKP-1 or DUSP1) dephosphorylates and inactivates mitogen-activated protein kinase (MAPK) substrates, such as p38, JNK, and Erk, and has been implicated in neoplasia. The lack of readily available selective small molecule inhibitors of MKP family members has severely limited interrogation of their biological role. Inspired by a previously identified inhibitor (NSC 357756) of MKP-3, we synthesized seven NSC 357756 congeners, which were evaluated for in vitro inhibition against several protein phosphatases. Remarkably, none displayed potent inhibition against MKP-3, including the desamino NSC 357756 analog NU-154. Interestingly, NU-154 inhibited human PTP1B in vitro with an IC(50) value of 24 +/- 1 microM and showed little inhibition against Cdc25B, MKP-1, and VHR phosphatases. NU-126 [2-((E)-2-(5-cyanobenzofuran-2-yl)vinyl)-1H-indole-6-carbonitrile] inhibited MKP-1 and VHR in vitro but was less active against human MKP-3, Cdc25B, and PTP1B. The inhibition of MKP-1 by NU-126 was independent of redox processes. The benzofuran substructure represents a new potential scaffold for further analog development and provides encouragement that more selective and potent inhibitors of MKP family members may be achievable.     

Evans Blue and other dyes as protein tyrosine phosphatase inhibitors

Commonly used dyes including Evans Blue and Trypan Blue were examined for their inhibitory activities against protein tyrosine phosphatases (PTPases), all of them showed inhibition of PTPases with different potencies. Of the 13 dyes tested, four exhibited IC(50) value of less than 10 microM, Evans Blue lowest IC(50) of 1.3 microM against PTP1B. Care must be taken in the use of dyes for clinical or biochemical experiments to avoid unwanted side effects. Some of the low molecular weight dyes might be useful as lead compounds for the development of potent and selective PTPase inhibitors.