Investigations on Key Principles of PTP1B Selectivity

Protein Tyrosine Phosphatase 1B (PTP1B) is a validated drug target for the treatment of diabetes type 2 and obesity. Until now, development of suitable modulators has been hampered by the polarity of the binding site and related bioavailability issues of the molecules. The design of selective inhibitors of PTP1B against the closely related T-Cell protein tyrosine phosphatase (TC-PTP), which was associated to severe side effects in animal studies, proved even more challenging. Over the years progress wasmade, but known PTP1B inhibitors only achieved atmaximummoderate selectivity over TC-PTP. This study aims to break the traditional boundaries of PTP1B selectivity by deliberately exploiting structural differences of both proteins. Due to their high similarity this requires thorough analysis of their static structures as well as their flexible behavior. The goal was therefore pursued with two different approaches: In Part I of the study a detailed analysis of protein complexes with selective ligands was performed including their flexible behavior as determined frommolecular dynamics simulations. Since common analysismethods were not able to explain the selectivity of the investigated ligands, a newmethod was developed which is able to assess parameters of ligand affinity that are not covered by currently available methods: steric complementarity of the ligand to the protein together with ligand strain. The developed tool allows to assess those properties on high numbers of molecular dynamics frames to calculate ligand shape fit in a flexible context. It further enables to trace back the ligand atoms or parts responsible for good or bad shape fit. In Part II of this study the flexible behavior of the apoproteins was studied. Since surface properties are highly similar in both proteins, the analysis focused on binding site shapes. For this, a novel approach was chosen that translates binding site shapes from molecular dynamics simulations into point maps and subsequently uses clustering and difference calculations to find a PTP1B conformationmost unlike to all discovered TC-PTP conformations. Theworkflowincludes calculation of a selectivity map consisting of points in the binding site where highest and most relevant differences in PTP1B and TC-PTP conformations occur. This map can be visualized and used for screening of selective PTP1B frames, which can then be used for protein structure based virtual screening for potentially selective PTP1B inhibitors. Results of Part I indicate shape fit as the previously undiscovered reason for selectivity of some known PTP1B inhibitors. They further suggest that selectivity can be achieved by interactions in the catalytic cavity as well as in previously suggested areas (the B and C site) of the binding site. Additionally, the discovered reduced ligand strain in PTP1B for one of the analyzed ligands, while almost maintaining same occurences of protein-ligand interaction features, lead to the assumption that PTP1B possesses a higher ability to conformationally adapt to the ligand than TC-PTP. This assumption is corroborated by the results of Part II: The selectivitymap indicates the catalytic cavity and the YRD-loop (C site) as areas of different flexible behavior. Especially the YRD-loop shows increased flexibility in PTP1B compared to TC-PTP. Additionally, ligands that have a high likelihood of exploiting the discovered conformational differences while still showing sufficient activity in PTP1B could be found in databases of commercially available molecules. Overall, the two innovative approaches to discover key factors of PTP1B selectivity did not only lead to interesting findings, but could also be adapted to promote other drug design projects where selectivity is crucial but hard to achieve.Das Enzym Protein Tyrosin Phosphatase 1B (PTP1B) ist ein validiertes Wirkstoffziel für die Behandlung von Diabetes Typ 2 und Übergewicht. Die Entwicklung passender Modulatoren wurde immer wieder durch die Polarität der Bindetasche und damit verbundene Bioverfügbarkeitsprobleme der Moleküle zurückgeworfen. Noch schwieriger ist es aber, die Moleküle so zu modifizieren, dass sie Selektivität gegenüber dem nahe verwandten Enzym T-Zell Protein Tyrosin Phosphatase (TC-PTP) erhalten, was aufgrund der mit diesem Protein assoziierten starken unerwünschten Wirkungen notwendig erscheint. Trotz einiger Fortschritte in den letzten Jahren erreichen bekannte PTP1B-Inhibitoren bis jetzt maximal moderate Selektivität gegenüber TC-PTP. Diese Arbeit hat es sich zum Ziel gemacht die durch bisherige Forschung gesetzten Grenzen der PTP1B-Selektivität zu durchbrechen, indem systematisch strukturelle Unterschiede der beiden Proteine ausgenutzt werden. Aufgrund ihrer sehr großen Ähnlichkeit erfordert dies eine genaue Analyse der statischen Proteinstrukturen sowie ihres dynamischen Verhaltens. Um dieses Ziel zu erreichen, wurden zwei Ansätze gewählt: In Teil I der Arbeit wurde eine detaillierte Analyse von Protein-Komplexen mit selektiven Liganden und deren dynamischen Verhaltens mithilfe von Moleküldynamiksimulationen durchgeführt. Da verfügbare Analysemethoden nicht in der Lage waren die beobachtete Selektivität zu erklären, wurde eine neue Methode entwickelt, welche es ermöglicht zusätzliche Faktoren für Ligandenaffinität zu erfassen: sterische Komplementarität und konformationelle Energie des Liganden. Die entwickelte Methode ermöglicht es diese Faktoren an einer großen Anzahl von Moleküldynamik-Schritten zu erfassen, um Verteilungen für die sterische Komplementarität am flexiblen Protein-Liganden-Komplex zu erhalten. Zusätzlich können die Anteile eines jeden Ligandenatoms an der Gesamtkomplementarität berechnet und dargestellt werden. In Teil II der Arbeit wurde das flexible Verhalten der Apoproteine näher untersucht. Da die Oberflächeneigenschaften beider Proteine kaum Unterschiede zeigen, konzentrierte sich diese Analyse auf die Form der Bindetaschen. Dafür wurde ein neuartiger Ansatz gewählt, der die Form der Bindetasche aus Moleküldynamiksimulationen extrahiert und in Punktwolken übersetzt. Diese werden anschließend geclustert, woraufhin mithilfe von Distanzberechnungen PTP1B-Konformationen ermittelt werden, die den größtmöglichen Unterschied zu allen ermittelten TC-PTP-Konformationen aufweisen. Der dafür verwendete Workflow berechnet zusätzlich einen Selektivitätsfilter bestehend aus denjenigen Punkten der Punktwolke, die die größten beziehungsweise relevantesten Unterschiede indenPTP1B- und TC-PTP-Konformationen zeigen. Dieser Filter dient einerseits dazu die Selektivitätsrelevanten Bindetaschenareale zu visualisieren, kann aber auch zum Filtern nach selektiven PTP1B-Konformationen aus Moleküldynamiksimulationen dienen, welche anschließend zum strukturbasierten virtuellen Screening nach potentiell selektiven PTP1B-Inhibitoren genutzt werden können. Die Ergebnisse von Teil I deuten darauf hin, dass die sterische Komplementarität den bisher unbekannten Grund für die Selektivität einiger bekannter PTP1B-Inhibitoren darstellen könnte. Außerdem geht aus den Analysen hervor, dass Selektivität womöglich auch durch Interaktionen mit der katalytischen Tasche hervorgerufen werden kann, neben Interaktionen mit schon in früheren Studien vorgeschlagenen Arealen der Bindetasche (B- und C-Seite). Zusätzlich zeigt einer der selektiven Liganden eine teilweise reduzierte konformationelle innere Energie, obwohl kaum Unterschiede im Vorkommen der Häufigkeiten der Protein-Liganden-Bindungen zu erkennen sind. Hier kann die Vermutung aufgestellt werden, dass dies durch eine erhöhte Fähigkeit von PTP1B im Vergleich zu TC-PTP verursacht wird seine Konformation an den Liganden anzupassen. Diese Vermutung wird durch die Ergebnisse aus Teil II der Arbeit gestützt: Der Selektivitätsfilterweist einerseits auf die katalytische Bindetasche, andererseits auf den YRD-loop (C-Seite) als Areale unterschiedlicher Flexibilität hin. Insbesondere der YRD-loop zeigt erhöhte Flexibilität in PTP1B im Vergleich zu TC-PTP. Zusätzlich konnten in diesem Teil der Arbeit Liganden in Datenbanken käuflich erwerbbarer Moleküle gefunden werden, welche nach unserem Modell eine große Wahrscheinlichkeit haben die ermittelten konformationellen Unterschiede bei zusätzlich guter Aktivität in PTP1B gezielt auszunutzen. Insgesamt führten die zwei innovativen Ansätze zur Ermittlung von Schlüsselfaktoren der PTP1B-Selektivität nicht nur zu äußerst interessanten Ergebnissen, sondern könnten auch angepasst werden um Wirkstoffdesignprojekte voranzutreiben, bei denen Selektivität von großer Wichtigkeit, aber schwer zu erreichen ist

An investigation on 4-thiazolidinone derivatives as dual inhibitors of aldose reductase and protein tyrosine phosphatase 1B, in the search for potential agents for the treatment of type 2 diabetes mellitus and its complications

Designed multiple ligands (DMLs), developed to modulate simultaneously a number of selected targets involved in etiopathogenetic mechanisms of a multifactorial disease, such as diabetes mellitus (DM), are considered a promising alternative to combinations of drugs, when monotherapy results to be unsatisfactory. In this work, compounds 1–17 were synthesized and in vitro evaluated as DMLs directed to aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two key enzymes involved in different events which are critical for the onset and progression of type 2 DM and related pathologies. Out of the tested 4-thiazolidinone derivatives, compounds 12 and 16, which exhibited potent AR inhibitory effects along with interesting inhibition of PTP1B, can be assumed as lead compounds to further optimize and balance the dual inhibitory profile. Moreover, several structural portions were identified as features that could be useful to achieve simultaneous inhibition of both human AR and PTP1B through binding to non-catalytic regions of both target enzymes

In Search for Multi-Target Ligands as Potential Agents for Diabetes Mellitus and Its Complications - A Structure-Activity Relationship Study on Inhibitors of Aldose Reductase and Protein Tyrosine Phosphatase 1B

Diabetes mellitus (DM) is a complex disease which currently affects more than 460 million people and is one of the leading cause of death worldwide. Its development implies numerous metabolic dysfunctions and the onset of hyperglycaemia-induced chronic complications. Multiple ligands can be rationally designed for the treatment of multifactorial diseases, such as DM, with the precise aim of simultaneously controlling multiple pathogenic mechanisms related to the disease and providing a more effective and safer therapeutic treatment compared to combinations of selective drugs. Starting from our previous findings that highlighted the possibility to target both aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two enzymes strictly implicated in the development of DM and its complications, we synthesised 3-(5-arylidene-4-oxothiazolidin-3-yl)propanoic acids and analogous 2-butenoic acid derivatives, with the aim of balancing the effectiveness of dual AR/PTP1B inhibitors which we had identified as designed multiple ligands (DMLs). Out of the tested compounds, 4f exhibited well-balanced AR/PTP1B inhibitory effects at low micromolar concentrations, along with interesting insulin-sensitizing activity in murine C2C12 cell cultures. The SARs here highlighted along with their rationalization by in silico docking experiments into both target enzymes provide further insights into this class of inhibitors for their development as potential DML antidiabetic candidates

IGF2 mRNA Binding Protein 2 Transgenic Mice Are More Prone to Develop a Ductular Reaction and to Progress Toward Cirrhosis

The insulin-like growth factor 2 (IGF2) mRNA binding proteins (IMPs/IGF2BPs) IMP1 and 3 are regarded as oncofetal proteins, whereas the hepatic IMP2 expression in adults is controversially discussed. The splice variant IMP2-2/p62 promotes steatohepatitis and hepatocellular carcinoma. Aim of this study was to clarify whether IMP2 is expressed in the adult liver and influences progression toward cirrhosis. IMP2 was expressed at higher levels in embryonic compared to adult tissues as quantified in embryonic, newborn, and adult C57BL/6J mouse livers and suggested by analysis of publicly available human data. In an IMP2-2 transgenic mouse model microarray and qPCR analyses revealed increased expression of liver progenitor cell (LPC) markers Bex1, Prom1, Spp1, and Cdh1 indicating a de-differentiated liver cell phenotype. Induction of these LPC markers was confirmed in human cirrhotic tissue datasets. The LPC marker SPP1 has been described to play a major role in fibrogenesis. Thus, DNA methylation was investigated in order to decipher the regulatory mechanism of Spp1 induction. In IMP2-2 transgenic mouse livers single CpG sites were differentially methylated, as quantified by amplicon sequencing, whereas human HCC samples of a human publicly available dataset showed promoter hypomethylation. In order to study the impact of IMP2 on fibrogenesis in the context of steatohepatitis wild-type or IMP2-2 transgenic mice were fed either a methionine-choline deficient (MCD) or a control diet for 2-12 weeks. MCD-fed IMP2-2 transgenic mice showed a higher incidence of ductular reaction (DR), accompanied by hepatic stellate cell activation, extracellular matrix (ECM) deposition, and induction of the LPC markers Spp1, Cdh1, and Afp suggesting the occurrence of de-differentiated cells in transgenic livers. In human cirrhotic samples IMP2 overexpression correlated with LPC marker and ECM component expression. Progression of liver disease was induced by combined MCD and diethylnitrosamine (DEN) treatment. Combined MCD-DEN treatment resulted in shorter survival of IMP2-2 transgenic compared to wild-type mice. Only IMP2-2 transgenic livers progressed to cirrhosis, which was accompanied by strong DR. In conclusion, IMP2 is an oncofetal protein in the liver that promotes DR characterized by de-differentiated cells toward steatohepatitis-associated cirrhosis development with poor survival

Combined chemical and biotechnological production of 20βOH-NorDHCMT, a long-term metabolite of Oral-Turinabol (DHCMT)

Anabolic androgenic steroids (AAS) are misused very frequently in sport competitions as performance enhancing agents. One of the doping compounds that has been detected with increased frequency in the last few years is dehydrochloromethyltestosterone (DHCMT, 4-chloro-17β-hydroxy-17α-methylandrosta-1,4-dien-3-one; brand name Oral Turinabol). The long-term DHCMT metabolite 20βOH-NorDHCMT (4-chloro-17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one) was reported earlier to be detectable in urine samples for more than 22 days after DHCMT administration; however, purified reference material was not available so far. In this study we demonstrate a successful combination of Wagner-Meerwein rearrangement of DHCMT to NorDHCMT (4-chloro-17,17-dimethyl-18-norandrosta-1,4,13-trien-3-one) and subsequent whole-cell biotransformation with a recombinant fission yeast strain expressing the human cytochrome P450 enzyme (CYP or P450) CYP21A2 for the synthesis of mg amounts of this metabolite. It was then used as reference for the analysis of a post administration urine of DHCMT. The availability of this reference compound will provide an incontestable proof for DHCMT abuse

Systematic Data Mining Reveals Synergistic H3R/MCHR1 Ligands

In this study, we report a ligand-centric data mining approach that guided the identification of suitable target profiles for treating obesity. The newly developed method is based on identifying target pairs for synergistic positive effects and also encompasses the exclusion of compounds showing a detrimental effect on obesity treatment (off-targets). Ligands with known activity against obesity-relevant targets were compared using fingerprint representations. Similar compounds with activities to different targets were evaluated for the mechanism of action since activation or deactivation of drug targets determines the pharmacological effect. <i>In vitro</i> validation of the modeling results revealed that three known modulators of melanin-concentrating hormone receptor 1 (MCHR1) show a previously unknown submicromolar affinity to the histamine H3 receptor (H₃R). This synergistic activity may present a novel therapeutic option against obesity

In Search for Multi-Target Ligands as Potential Agents for Diabetes Mellitus and Its Complications—A Structure-Activity Relationship Study on Inhibitors of Aldose Reductase and Protein Tyrosine Phosphatase 1B

Diabetes mellitus (DM) is a complex disease which currently affects more than 460 million people and is one of the leading cause of death worldwide. Its development implies numerous metabolic dysfunctions and the onset of hyperglycaemia-induced chronic complications. Multiple ligands can be rationally designed for the treatment of multifactorial diseases, such as DM, with the precise aim of simultaneously controlling multiple pathogenic mechanisms related to the disease and providing a more effective and safer therapeutic treatment compared to combinations of selective drugs. Starting from our previous findings that highlighted the possibility to target both aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two enzymes strictly implicated in the development of DM and its complications, we synthesised 3-(5-arylidene-4-oxothiazolidin-3-yl)propanoic acids and analogous 2-butenoic acid derivatives, with the aim of balancing the effectiveness of dual AR/PTP1B inhibitors which we had identified as designed multiple ligands (DMLs). Out of the tested compounds, 4f exhibited well-balanced AR/PTP1B inhibitory effects at low micromolar concentrations, along with interesting insulin-sensitizing activity in murine C2C12 cell cultures. The SARs here highlighted along with their rationalization by in silico docking experiments into both target enzymes provide further insights into this class of inhibitors for their development as potential DML antidiabetic candidates