ABSTRACT
Due to their low binding affinities, detecting small-molecule fragments bound to protein structures from crystallographic datasets has been a challenge. Here, we report a trove of 65 new fragment hits for PTP1B, an "undruggable" therapeutic target enzyme for diabetes and cancer. These structures were obtained from computational analysis of data from a large crystallographic screen, demonstrating the power of this approach to elucidate many (â¼50% more) "hidden" ligand-bound states of proteins. Our new structures include a fragment hit found in a novel binding site in PTP1B with a unique location relative to the active site, one that links adjacent allosteric sites, and, perhaps most strikingly, a fragment that induces long-range allosteric protein conformational responses. Altogether, our research highlights the utility of computational analysis of crystallographic data, makes publicly available dozens of new ligand-bound structures of a high-value drug target, and identifies novel aspects of ligandability and allostery in PTP1B.
Subject(s)
Allosteric Site , Protein Binding , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Crystallography, X-Ray , Humans , Ligands , Catalytic Domain , Models, Molecular , Allosteric Regulation , Binding Sites , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/metabolism , Small Molecule Libraries/chemistry , Protein ConformationABSTRACT
Vitamin B12 is a group of biologically active cobalamin compounds. In this study, we investigated the inhibitory effects of methylcobalamin (MeCbl) and hydroxocobalamin acetate (OHCbl Acetate) on protein tyrosine phosphatase 1B (PTP1B). MeCbl and OHCbl Acetate exhibited an IC50 of approximately 58.390 ± 2.811 µM and 8.998 ± 0.587 µM, respectively. The Ki values of MeCbl and OHCbl Acetate were 25.01 µM and 4.04 µM respectively. To elucidate the inhibition mechanism, we conducted a 500 ns Gaussian accelerated molecular dynamics (GaMD) simulation. Utilizing PCA and tICA, we constructed Markov state models (MSM) to examine secondary structure changes during motion. Our findings revealed that the α-helix at residues 37-42 remained the most stable in the PTP1B-OHCbl Acetate system. Furthermore, upon binding of OHCbl Acetate or MeCbl, the WPD loop of PTP1B moved inward to the active pocket, forming a closed conformation and potentially obstructs substrate entry. Protein-ligand interaction analysis and MM-PBSA showed that OHCbl Acetate exhibited lower binding free energy and engaged in more residue interactions with PTP1B. In summary, our study confirmed the substantial inhibitory activity of OHCbl Acetate against PTP1B, with its inhibitory potency notably surpassing that of MeCbl. We demonstrated potential molecular mechanisms of OHCbl Acetate inhibiting PTP1B.
Subject(s)
Molecular Dynamics Simulation , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Vitamin B 12 , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Vitamin B 12/chemistry , Vitamin B 12/analogs & derivatives , Vitamin B 12/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Molecular Docking Simulation , Protein Binding , Kinetics , Structure-Activity RelationshipABSTRACT
Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for obesity, diabetes, and certain types of cancer. In particular, allosteric inhibitors hold potential for therapeutic use, but an incomplete understanding of conformational dynamics and allostery in this protein has hindered their development. Here, we interrogate solution dynamics and allosteric responses in PTP1B using high-resolution hydrogen-deuterium exchange mass spectrometry (HDX-MS), an emerging and powerful biophysical technique. Using HDX-MS, we obtain a detailed map of backbone amide exchange that serves as a proxy for the solution dynamics of apo PTP1B, revealing several flexible loops interspersed among more constrained and rigid regions within the protein structure, as well as local regions that exchange faster than expected from their secondary structure and solvent accessibility. We demonstrate that our HDX rate data obtained in solution adds value to estimates of conformational heterogeneity derived from a pseudo-ensemble constructed from ~200 crystal structures of PTP1B. Furthermore, we report HDX-MS maps for PTP1B with active-site versus allosteric small-molecule inhibitors. These maps suggest distinct and widespread effects on protein dynamics relative to the apo form, including changes in locations distal (>35 Å) from the respective ligand binding sites. These results illuminate that allosteric inhibitors of PTP1B can induce unexpected changes in dynamics that extend beyond the previously understood allosteric network. Together, our data suggest a model of BB3 allostery in PTP1B that combines conformational restriction of active-site residues with compensatory liberation of distal residues that aid in entropic balancing. Overall, our work showcases the potential of HDX-MS for elucidating aspects of protein conformational dynamics and allosteric effects of small-molecule ligands and highlights the potential of integrating HDX-MS alongside other complementary methods, such as room-temperature X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations, to guide the development of new therapeutics.
Subject(s)
Hydrogen Deuterium Exchange-Mass Spectrometry , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Allosteric Regulation , Humans , Molecular Dynamics Simulation , Protein Conformation , Models, Molecular , Catalytic DomainABSTRACT
Tyrosine protein phosphatase non-receptor type 1 (PTP1B; also known as protein tyrosine phosphatase 1B) is a member of the protein tyrosine phosphatase (PTP) family and is a soluble enzyme that plays an essential role in different physiological processes, including the regulation of metabolism, specifically in insulin and leptin sensitivity. PTP1B is crucial in the pathogenesis of type 2 diabetes mellitus and obesity. These biological functions have made PTP1B validated as an antidiabetic and anti-obesity, and potentially anticancer, molecular target. Four main approaches aim to inhibit PTP1B: orthosteric, allosteric, bidentate inhibition, and PTPN1 gene silencing. Developing a potent and selective PTP1B inhibitor is still challenging due to the enzyme's ubiquitous expression, subcellular location, and structural properties. This article reviews the main advances in the study of PTP1B since it was first isolated in 1988, as well as recent contextual information related to the PTP family to which this protein belongs. Furthermore, we offer an overview of the role of PTP1B in diabetes and obesity, and the challenges to developing selective, effective, potent, bioavailable, and cell-permeable compounds that can inhibit the enzyme.
Subject(s)
Diabetes Mellitus, Type 2 , Enzyme Inhibitors , Hypoglycemic Agents , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/genetics , Humans , Hypoglycemic Agents/pharmacology , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/therapeutic use , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/therapeutic use , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/genetics , Animals , Obesity/drug therapy , Obesity/metabolism , Obesity/enzymology , Obesity/geneticsABSTRACT
Dynamics-driven allostery provides important insights into the working mechanics of proteins, especially enzymes. In this study, we employ this paradigm to answer a basic question: in enzyme superfamilies, where the catalytic mechanism, active sites, and protein fold are conserved, what accounts for the difference in the catalytic prowess of the individual members? We show that when subtle changes in sequence do not translate to changes in structure, they do translate to changes in dynamics. We use sequentially diverse PTP1B, TbPTP1, and YopH as representatives of the conserved protein tyrosine phosphatase (PTP) superfamily. Using amino acid network analysis of group behavior (community analysis) and influential node dominance on networks (eigenvector centrality), we explain the dynamic basis of the catalytic variations seen between the three proteins. Importantly, we explain how a dynamics-based blueprint makes PTP1B amenable to allosteric control and how the same is abstracted in TbPTP1 and YopH.
Subject(s)
Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases , Catalytic Domain , Protein Tyrosine Phosphatases/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistryABSTRACT
Overexpression of protein tyrosine phosphatase 1B (PTP1B) is the major cause of various diseases such as diabetes, obesity, and cancer. PTP1B has been identified as a negative regulator of the insulin signaling cascade, thereby causing diabetes. Numerous anti-diabetic medications based on thiazolidinedione have been successfully developed; however, 2,4-thiazolidinedione (2,4-TZD) scaffolds have been reported as potential PTP1B inhibitors for the manifestation of type 2 diabetes mellitus involving insulin resistance. In the present study, we have employed amalgamated approach involving MD-simulation studies (100 ns) as well as Gaussian field-based 3D-QSAR to develop a pharmacophoric model of 2,4-TZD as potent PTP1B inhibitors. MD simulation studies of the most potent compound in the PTP1B (PDB Id: 2QBS) binding pocket revealed that compound 43 was stable in the binding pocket and demonstrated excellent binding efficacy within the active site pocket. MM/GBSA results revealed that compound 43, bearing C-5 arylidine substitution, strongly bound to the target as compared to rosiglitazone with ΔGMM/GBSA difference of -11.13 kcal/mol. PCA, Rg, RMSF, RMSD, and SASA were analyzed from the complex's trajectories to anticipate the simulation outcome. We have suggested a series of 2,4-TZD as possible PTP1B inhibitors based on the results of MD simulation and 3D-QSAR studies.
Subject(s)
Diabetes Mellitus, Type 2 , Thiazolidinediones , Humans , Molecular Dynamics Simulation , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/metabolism , Quantitative Structure-Activity Relationship , Enzyme Inhibitors/chemistry , Thiazolidinediones/pharmacology , Thiazolidinediones/therapeutic use , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Molecular Docking SimulationABSTRACT
Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin and leptin signaling pathways, making it a highly attractive target for the treatment of type II diabetes. For PTP1B to perform its enzymatic function, a loop referred to as the "WPD loop" must transition between open (catalytically incompetent) and closed (catalytically competent) conformations, which have both been resolved by X-ray crystallography. Although prior studies have established this transition as the rate-limiting step for catalysis, the transition mechanism for PTP1B and other PTPs has been unclear. Here we present an atomically detailed model of WPD loop transitions in PTP1B based on unbiased, long-timescale molecular dynamics simulations and weighted ensemble simulations. We found that a specific WPD loop regionâthe PDFG motifâacted as the key conformational switch, with structural changes to the motif being necessary and sufficient for transitions between long-lived open and closed states of the loop. Simulations starting from the closed state repeatedly visited open states of the loop that quickly closed again unless the infrequent conformational switching of the motif stabilized the open state. The functional importance of the PDFG motif is supported by the fact that it is well conserved across PTPs. Bioinformatic analysis shows that the PDFG motif is also conserved, and adopts two distinct conformations, in deiminases, and the related DFG motif is known to function as a conformational switch in many kinases, suggesting that PDFG-like motifs may control transitions between structurally distinct, long-lived conformational states in multiple protein families.
Subject(s)
Diabetes Mellitus, Type 2 , Phosphoric Monoester Hydrolases , Humans , Phosphoric Monoester Hydrolases/metabolism , Kinetics , Molecular Dynamics Simulation , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Catalysis , Protein ConformationABSTRACT
Fragment-based drug discovery has led to six approved drugs, but the small sizes of the chemical fragments used in such methods typically result in only weak interactions between the fragment and its target molecule, which makes it challenging to experimentally determine the three-dimensional poses fragments assume in the bound state. One computational approach that could help address this difficulty is long-timescale molecular dynamics (MD) simulations, which have been used in retrospective studies to recover experimentally known binding poses of fragments. Here, we present the results of long-timescale MD simulations that we used to prospectively discover binding poses for two series of fragments in allosteric pockets on a difficult and important pharmaceutical target, protein tyrosine phosphatase 1b (PTP1b). Our simulations reversibly sampled the fragment association and dissociation process. One of the binding pockets found in the simulations has not to our knowledge been previously observed with a bound fragment, and the other pocket adopted a very rare conformation. We subsequently obtained high-resolution crystal structures of members of each fragment series bound to PTP1b, and the experimentally observed poses confirmed the simulation results. To the best of our knowledge, our findings provide the first demonstration that MD simulations can be used prospectively to determine fragment binding poses to previously unidentified pockets.
Subject(s)
Molecular Dynamics Simulation , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Crystallography, X-Ray , Retrospective Studies , Drug Discovery/methods , Protein Binding , Binding SitesABSTRACT
Much of our current understanding of how small-molecule ligands interact with proteins stems from X-ray crystal structures determined at cryogenic (cryo) temperature. For proteins alone, room-temperature (RT) crystallography can reveal previously hidden, biologically relevant alternate conformations. However, less is understood about how RT crystallography may impact the conformational landscapes of protein-ligand complexes. Previously, we showed that small-molecule fragments cluster in putative allosteric sites using a cryo crystallographic screen of the therapeutic target PTP1B (Keedy et al., 2018). Here, we have performed two RT crystallographic screens of PTP1B using many of the same fragments, representing the largest RT crystallographic screens of a diverse library of ligands to date, and enabling a direct interrogation of the effect of data collection temperature on protein-ligand interactions. We show that at RT, fewer ligands bind, and often more weakly - but with a variety of temperature-dependent differences, including unique binding poses, changes in solvation, new binding sites, and distinct protein allosteric conformational responses. Overall, this work suggests that the vast body of existing cryo-temperature protein-ligand structures may provide an incomplete picture, and highlights the potential of RT crystallography to help complete this picture by revealing distinct conformational modes of protein-ligand systems. Our results may inspire future use of RT crystallography to interrogate the roles of protein-ligand conformational ensembles in biological function.
Subject(s)
Crystallography , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Allosteric Site , Binding Sites , Ligands , Temperature , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistryABSTRACT
Protein tyrosine phosphatase 1B (PTP1B), a negative regulator of the insulin signaling pathway, has gained attention as a validated druggable target in the management of type 2 diabetes mellitus (T2DM). The lack of clinically approved PTP1B inhibitors has continued to prompt research in plant-derived therapeutics possibly due to their relatively lesser toxicity profiles. Flavonoid C-glycosides are one of the plant-derived metabolites gaining increased relevance as antidiabetic agents, but their possible mechanism of action remains largely unknown. This study investigates the antidiabetic potential of flavonoid C-glycosides against PTP1B in silico and in vitro. Of the seven flavonoid C-glycosides docked against the enzyme, three compounds (apigenin, vitexin, and orientin) had the best affinity for the enzyme with a binding score of -7.3 kcal/mol each, relative to -7.4 kcal/mol for the reference standard, ursolic acid. A further probe (in terms of stability, flexibility, and compactness) of the complexes over a molecular dynamics time study of 100 ns for the three compounds suggested orientin as the most outstanding inhibitor of PTP1B owing to its overall -34.47 kcal/mol binding energy score compared to ursolic acid (-19.24 kcal/mol). This observation was in accordance with the in vitro evaluation result, where orientin had a half maximal inhibitory concentration (IC50) of 0.18 mg/ml relative to 0.13 mg/ml for the reference standard. The kinetics of inhibition of PTP1B by orientin was mixed-type with V max and K m values of 0.004 µM/s and 0.515 µM. Put together, the results suggest orientin as a potential PTP1B inhibitor and could therefore be further explored in the management T2DM as a promising therapeutic agent.
Subject(s)
Diabetes Mellitus, Type 2 , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Diabetes Mellitus, Type 2/drug therapy , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Flavonoids/pharmacology , Flavonoids/therapeutic use , Glycosides/pharmacology , Glycosides/therapeutic use , Humans , Hypoglycemic Agents/pharmacology , Hypoglycemic Agents/therapeutic use , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolismABSTRACT
Three complex polyoxygenated diterpenoids possessing uncommon tetradecahydro-2,13:6,9-diepoxybenzo[10]annulene scaffold, namely ximaoornatins A-C (1-3), one new eunicellin-type diterpene, litophynin K (4), and a related known compound, litophynol B (5) were isolated from the South China Sea soft coral Sinularia ornata. The structures and absolute configurations of 1-4 were established by extensive spectroscopic analysis, X-ray diffraction analysis, and/or modified Mosher's method. A plausible biosynthetic relationship of 1 and its potential precursor 4 was proposed. In a bioassay, none of the isolated compounds showed obvious anti-inflammatory activity on LPS-induced TNF-α release in RAW264.7 macrophages and PTP1B inhibitory effects.
Subject(s)
Anthozoa/chemistry , Biological Products , Diterpenes , Animals , Anthozoa/metabolism , Biological Products/chemistry , Biological Products/isolation & purification , Biological Products/metabolism , Diterpenes/chemistry , Diterpenes/isolation & purification , Diterpenes/metabolism , Lipopolysaccharides/pharmacology , Mice , Models, Molecular , Molecular Structure , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , RAW 264.7 Cells , Tumor Necrosis Factor-alpha/metabolismABSTRACT
T-Cell Protein Tyrosine Phosphatase (TCPTP, PTPN2) is a non-receptor type protein tyrosine phosphatase that is ubiquitously expressed in human cells. TCPTP is a critical component of a variety of key signaling pathways that are directly associated with the formation of cancer and inflammation. Thus, understanding the molecular mechanism of TCPTP activation and regulation is essential for the development of TCPTP therapeutics. Under basal conditions, TCPTP is largely inactive, although how this is achieved is poorly understood. By combining biomolecular nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and chemical cross-linking coupled with mass spectrometry, we show that the C-terminal intrinsically disordered tail of TCPTP functions as an intramolecular autoinhibitory element that controls the TCPTP catalytic activity. Activation of TCPTP is achieved by cellular competition, i.e., the intrinsically disordered cytosolic tail of Integrin-α1 displaces the TCPTP autoinhibitory tail, allowing for the full activation of TCPTP. This work not only defines the mechanism by which TCPTP is regulated but also reveals that the intrinsically disordered tails of two of the most closely related PTPs (PTP1B and TCPTP) autoregulate the activity of their cognate PTPs via completely different mechanisms.
Subject(s)
Integrin alpha1/chemistry , Intrinsically Disordered Proteins/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 2/chemistry , Amino Acid Sequence , Binding Sites , Biocatalysis , Cloning, Molecular , Enzyme Activation , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Integrin alpha1/genetics , Integrin alpha1/metabolism , Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/metabolism , Kinetics , Models, Molecular , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Tyrosine Phosphatase, Non-Receptor Type 1/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
In the present study, we investigated the structure-activity relationship of naturally occurring hesperetin derivatives, as well as the effects of their glycosylation on the inhibition of diabetes-related enzyme systems, protein tyrosine phosphatase 1B (PTP1B) and α-glycosidase. Among the tested hesperetin derivatives, hesperetin 5-O-glucoside, a single-glucose-containing flavanone glycoside, significantly inhibited PTP1B with an IC50 value of 37.14 ± 0.07 µM. Hesperetin, which lacks a sugar molecule, was the weakest inhibitor compared to the reference compound, ursolic acid (IC50 = 9.65 ± 0.01 µM). The most active flavanone hesperetin 5-O-glucoside suggested that the position of a sugar moiety at the C-5-position influences the PTP1B inhibition. It was observed that the ability to inhibit PTP1B is dependent on the nature, position, and number of sugar moieties in the flavonoid structure, as well as conjugation. In the kinetic study of PTP1B enzyme inhibition, hesperetin 5-O-glucoside led to mixed-type inhibition. Molecular docking studies revealed that hesperetin 5-O-glucoside had a higher binding affinity with key amino residues, suggesting that this molecule best fits the PTP1B allosteric site cavity. The data reported here support hesperetin 5-O-glucoside as a hit for the design of more potent and selective inhibitors against PTP1B in the search for a new anti-diabetic treatment.
Subject(s)
Enzyme Inhibitors/chemistry , Hesperidin/analogs & derivatives , Molecular Docking Simulation , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Hesperidin/chemistry , Humans , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Structure-Activity RelationshipABSTRACT
The T-cell protein tyrosine phosphatase (TCPTP/PTPN2) targets a broad variety of substrates across different subcellular compartments. In spite of that, the structural basis for the regulation of TCPTP's activity remains elusive. Here, we investigated whether the activity of TCPTP is regulated by a potential allosteric site in a comparable manner to its most similar PTP family member (PTP1B/PTPN1). We determined two crystal structures of TCPTP at 1.7 and 1.9 Å resolutions that include helix α7 at the TCPTP C-terminus. Helix α7 has been functionally characterized in PTP1B and was identified as its allosteric switch. However, its function is unknown in TCPTP. Here, we demonstrate that truncation or deletion of helix α7 reduced the catalytic efficiency of TCPTP by â¼4-fold. Collectively, our data supports an allosteric role of helix α7 in regulation of TCPTP's activity, similar to its function in PTP1B, and highlights that the coordination of helix α7 with the core catalytic domain is essential for the efficient catalytic function of TCPTP.
Subject(s)
Protein Tyrosine Phosphatase, Non-Receptor Type 2/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism , Allosteric Regulation , Allosteric Site/genetics , Amino Acid Sequence , Amino Acid Substitution , Biophysical Phenomena , Catalytic Domain/genetics , Crystallography, X-Ray , Humans , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation, alpha-Helical , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal TransductionABSTRACT
Protein tyrosine phosphorylation is one of the major post-translational modifications in eukaryotic cells and represents a critical regulatory mechanism of a wide variety of signaling pathways. Aberrant protein tyrosine phosphorylation has been linked to various diseases, including metabolic disorders and cancer. Few years ago, protein tyrosine phosphatases (PTPs) were considered as tumor suppressors, able to block the signals emanating from receptor tyrosine kinases. However, recent evidence demonstrates that misregulation of PTPs activity plays a critical role in cancer development and progression. Here, we will focus on PTP1B, an enzyme that has been linked to the development of type 2 diabetes and obesity through the regulation of insulin and leptin signaling, and with a promoting role in the development of different types of cancer through the activation of several pro-survival signaling pathways. In this review, we discuss the molecular aspects that support the crucial role of PTP1B in different cellular processes underlying diabetes, obesity and cancer progression, and its visualization as a promising therapeutic target.
Subject(s)
Metabolic Diseases/etiology , Metabolic Diseases/metabolism , Neoplasms/etiology , Neoplasms/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1/metabolism , Signal Transduction , Animals , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/etiology , Diabetes Mellitus, Type 2/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Humans , Metabolic Diseases/drug therapy , Neoplasms/drug therapy , Obesity/drug therapy , Obesity/etiology , Obesity/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Signal Transduction/drug effectsABSTRACT
The new copper(II) complex dichloridobis(4-{[3-(pyridin-2-yl-κN)-1H-pyrazol-1-yl-κN2]methyl}benzoic acid)copper(II) methanol sesquisolvate hemihydrate, [CuCl2L2]·1.5CH3OH·0.5H2O, (1), has been synthesized from CuCl2·2H2O and the ligand 4-{[3-(pyridin-2-yl)-1H-pyrazol-1-yl]methyl}benzoic acid (L, C15H11N3O2). The complex was characterized by elemental analysis, Fourier transform IR spectroscopy, electrospray ionization mass spectrometry and single-crystal X-ray diffraction. Two chloride ligands and two bidentate L ligands coordinate the CuII centre in 1 in a Jahn-Teller-distorted octahedral geometry of rather unusual configuration: a chloride substituent and a pyrazole N atom of an N,N'-chelating ligand occupy the more distant axial positions. Classical O-H...O hydrogen bonds and O-H...Cl interactions link neighbouring complex molecules and cocrystallized methanol molecules into chains that propagate parallel to the b direction. The title compound shows intriguing bioactivity: the effects of 1 on the enzymatic activity of protein tyrosine phosphatase 1B (PTP1B) and on the viability of human breast cancer cells of cell line MCF7 were evaluated. Complex 1, with an IC50 value of 0.51â µM, can efficiently inhibit PTP1B activity. An enzyme kinetic assay suggests that 1 inhibits PTP1B in a noncompetitive manner. A fluorescence titration assay indicates that 1 has a strong affinity for PTP1B, with a binding constant of 4.39 × 106 M-1. Complex 1 may also effectively decrease the viability of MCF7 cells in an extent comparable to that of cisplatin (IC50 = 6.3â µM). The new copper complex therefore represents a promising PTP1B inhibitor and an efficient antiproliferation reagent against MCF7 cells.
Subject(s)
Copper/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Crystallography, X-Ray , Humans , Hydrogen Bonding , Ligands , MCF-7 Cells , Magnetic Resonance Spectroscopy , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistryABSTRACT
Diabetes mellitus (DM) represents a group of metabolic disorders that leads to acute and long-term serious complications and is considered a worldwide sanitary emergence. Type 2 diabetes (T2D) represents about 90% of all cases of diabetes, and even if several drugs are actually available for its treatment, in the long term, they show limited effectiveness. Most traditional drugs are designed to act on a specific biological target, but the complexity of the current pathologies has demonstrated that molecules hitting more than one target may be safer and more effective. The purpose of this review is to shed light on the natural compounds known as α-glucosidase and Protein Tyrosine Phosphatase 1B (PTP1B) dual-inhibitors that could be used as lead compounds to generate new multitarget antidiabetic drugs for treatment of T2D.
Subject(s)
Enzyme Inhibitors/chemistry , Hypoglycemic Agents/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , alpha-Glucosidases/drug effects , Diabetes Mellitus, Type 2/drug therapy , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/therapeutic use , Glycoside Hydrolase Inhibitors/chemical synthesis , Glycoside Hydrolase Inhibitors/chemistry , Glycoside Hydrolase Inhibitors/therapeutic use , Humans , Hypoglycemic Agents/chemical synthesis , Hypoglycemic Agents/therapeutic use , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/genetics , alpha-Glucosidases/chemistry , alpha-Glucosidases/geneticsABSTRACT
Diabetes mellitus is a multifactorial disease that affects both developing and developed countries and is a major public health concern. Many synthetic drugs are available in the market, which counteracts the associated pathologies. However, due to the propensity of side effects, there is an unmet need for the investigation of safe and effective drugs. This research aims to find a novel phytoconstituent having diminished action on blood glucose levels with the least side effects. Shikonin is a naturally occurring naphthoquinone dying pigment obtained by the roots of the Boraginaceae family. Besides its use as pigments, it can be used as an antimicrobial, anti-inflammatory, and anti-tumor agent. This research aimed to hypothesize the physicochemical and phytochemical properties of Shikonin's in silico interaction with protein tyrosine phosphate 1B, as well as it's in vitro studies, in order to determine its potential anti-diabetic impact. To do so, molecular docking experiments with target proteins were conducted to assess their anti-diabetic ability. Analyzing associations with corresponding amino acids revealed the significant molecular interactions between Shikonin and diabetes-related target proteins. In silico pharmacokinetics and toxicity profile of Shikonin using ADMET Descriptor, Toxicity Prediction, and Calculate Molecular Properties tools from Biovia Discovery Studio v4.5. Filter by Lipinski and Veber Rule's module from Biovia Discovery Studio v4.5 was applied to assess the drug-likeness of Shikonin. The in vitro studies exposed that Shikonin shows an inhibitory potential against the PTP1B with an IC50 value of 15.51 µM. The kinetics studies revealed that it has a competitive inhibitory effect (Ki = 7.5 M) on the enzyme system, which could be useful in the production of preventive and therapeutic agents. The findings of this research suggested that the Shikonin could be used as an anti-diabetic agent and can be used as a novel source for drug delivery.
Subject(s)
Enzyme Inhibitors/chemistry , Hypoglycemic Agents/chemistry , Molecular Docking Simulation , Naphthoquinones/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Allosteric Regulation , Humans , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Structure-Activity RelationshipABSTRACT
Regulating insulin and leptin levels using a protein tyrosine phosphatase 1B (PTP1B) inhibitor is an attractive strategy to treat diabetes and obesity. Glycyrrhetinic acid (GA), a triterpenoid, may weakly inhibit this enzyme. Nonetheless, semisynthetic derivatives of GA have not been developed as PTP1B inhibitors to date. Herein we describe the synthesis and evaluation of two series of indole- and N-phenylpyrazole-GA derivatives (4a-f and 5a-f). We measured their inhibitory activity and enzyme kinetics against PTP1B using p-nitrophenylphosphate (pNPP) assay. GA derivatives bearing substituted indoles or N-phenylpyrazoles fused to their A-ring showed a 50% inhibitory concentration for PTP1B in a range from 2.5 to 10.1 µM. The trifluoromethyl derivative of indole-GA (4f) exhibited non-competitive inhibition of PTP1B as well as higher potency (IC50 = 2.5 µM) than that of positive controls ursolic acid (IC50 = 5.6 µM), claramine (IC50 = 13.7 µM) and suramin (IC50 = 4.1 µM). Finally, docking and molecular dynamics simulations provided the theoretical basis for the favorable activity of the designed compounds.
Subject(s)
Enzyme Inhibitors , Glycyrrhetinic Acid , Indoles , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Pyrazoles , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Glycyrrhetinic Acid/analogs & derivatives , Glycyrrhetinic Acid/chemical synthesis , Glycyrrhetinic Acid/chemistry , Humans , Indoles/chemical synthesis , Indoles/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 1/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 1/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Structure-Activity RelationshipABSTRACT
The design of small molecules that inhibit disease-relevant proteins represents a longstanding challenge of medicinal chemistry. Here, we describe an approach for encoding this challenge-the inhibition of a human drug target-into a microbial host and using it to guide the discovery and biosynthesis of targeted, biologically active natural products. This approach identified two previously unknown terpenoid inhibitors of protein tyrosine phosphatase 1B (PTP1B), an elusive therapeutic target for the treatment of diabetes and cancer. Both inhibitors appear to target an allosteric site, which confers selectivity, and can inhibit PTP1B in living cells. A screen of 24 uncharacterized terpene synthases from a pool of 4464 genes uncovered additional hits, demonstrating a scalable discovery approach, and the incorporation of different PTPs into the microbial host yielded alternative PTP-specific detection systems. Findings illustrate the potential for using microbes to discover and build natural products that exhibit precisely defined biochemical activities yet possess unanticipated structures and/or binding sites.