How To Design Selective Ligands for Highly Conserved Binding Sites: A Case Study Using N-Myristoyltransferases as a Model System

Under embargo until: 2020-08-19A model system of two related enzymes with conserved binding sites, namely N-myristoyltransferase from two different organisms, was studied to decipher the driving forces that lead to selective inhibition in such cases. Using a combination of computational and experimental tools, two different selectivity-determining features were identified. For some ligands, a change in side-chain flexibility appears to be responsible for selective inhibition. Remarkably, this was observed for residues orienting their side chains away from the ligands. For other ligands, selectivity is caused by interfering with a water molecule that binds more strongly to the off-target than to the target. On the basis of this finding, a virtual screen for selective compounds was conducted, resulting in three hit compounds with the desired selectivity profile. This study delivers a guideline on how to assess selectivity-determining features in proteins with conserved binding sites and to translate this knowledge into the design of selective inhibitors.acceptedVersio

Identification of a potential allosteric site of Golgi α-mannosidase II using computer-aided drug design

Golgi α-mannosidase II (GMII) is a glycoside hydrolase playing a crucial role in the N-glycosylation pathway. In various tumour cell lines, the distribution of N-linked sugars on the cell surface is modified and correlates with the progression of tumour metastasis. GMII therefore is a possible molecular target for anticancer agents. Here, we describe the identification of a non-competitive GMII inhibitor using computer-aided drug design methods including identification of a possible allosteric binding site, pharmacophore search and virtual screening.publishedVersio

Identification of a potential allosteric site of Golgi α-mannosidase II using computer-aided drug design

Golgi α-mannosidase II (GMII) is a glycoside hydrolase playing a crucial role in the N-glycosylation pathway. In various tumour cell lines, the distribution of N-linked sugars on the cell surface is modified and correlates with the progression of tumour metastasis. GMII therefore is a possible molecular target for anticancer agents. Here, we describe the identification of a non-competitive GMII inhibitor using computer-aided drug design methods including identification of a possible allosteric binding site, pharmacophore search and virtual screening

Design, Synthese und Testung potentieller Inhibitoren der Golgi-α-Mannosidase II

The enzyme Golgi-α-mannosidase II (GMII) plays a crucial role in the N-glycosylation pathway, which is involved in the metastatic process of several types of cancer. It provides essential precursors of glycosyl chains which are neccesary for cell recognition through interaction with selectins on the cell surface. In different cancer cells, modified glycosylation patterns were found. Their origin is believed to be the misregulation of protein expression which leads to unnatural high concentration of glycosly transferases. In clinical trials, the reduction of metastasis and tumor growth by inhibition oft he GMII has been shown. Hence the inhibition of this enzyme represents a promising target in cancer therapy. As a new class of active substances covalent reversible inhibitors are to be developed and tested. These should combine the advantages of covalent inhibitors omitting their disadvantages. These inhibitors require extremely precise adaption of the ligand structure to its receptor and in the reactivity of the inhibitor in order to form a covalent reversible adduct with the enzyme. To predict potential inhibitors molecular docking methods and quantum mechanics/molecular mechanics (QM/MM) methods were used. The main task of this thesis was the synthesis of these structures as well as the synthesis of fluorescent substrates to establish new assays with α-mannosidases and β-glucosidases. In total, 41 substances were synthesized. An overview of the individual subjects is shown in figure 1. In a docking assisted de-novo-design a structure (L039) was identified, to meet all identified requirements for a covalent reversible inhibition of GMII in QM/MM-calculation. In further docking studies, several structures from the substance class of monocyclic N,O-acetals were identified as potential covalent reversible inhibitors. Furthermore, investigations on a spiroacetal were carried out. In the context of this work, 25 monocyclic N,O-acetals, two derivatives of azamannose as potential building block, isofagomine and its 5-isomer, four potential inhibiors of a potential allosteric site of GMII and fragments of L039 were synthesized, purified and analysed spectrometroscopically. None of those compounds showed significant inhibition of dGMII. To investigate the activity of potential inhibitors, enzyme assays were carried out. For this task, two substrates were prepared in four- and five-steps synthesis, as well as a reference inhibitor of β-glucosidases (isofagomine) in a six-step synthesis to validate the assay systems.Die Golgi-α-Mannosidase II (GMII) spielt eine entscheidende Rolle im N-Glycosylierungsprozess. Dieser ist maßgeblich an der Metastasierung diverser Krebstumorarten beteiligt, da er essentielle Vorstufen der zur Zellerkennung notwendigen Glycosylketten bereitstellt, welche auf der Zelloberfläche mit Selectinen interagieren. Auf der Oberfläche verschiedener Krebszellen wurden modifizierte Glycosylierungsmuster nachgewiesen, als deren Ursache eine Fehlregulierung der Expression angenommen wird, die zu unnatürlich hohen Konzentrationen an Glycosyltransferasen führt. In klinischen Studien konnte die Reduzierung von Tumorwachstum und Metastasierung durch die Inhibition der GMII bereits nachgewiesen werden. Daher stellt die Inhibition dieses Enzyms einen vielversprechenden Angriffspunkt in der Krebstherapie dar. Als neue Wirkstoffklasse für die GMII sollten kovalent reversible Inhibitoren entwickelt und getestet werden, da sie die Vorteile kovalenter Inhibitoren durch Reversibilität ohne deren Nachteile kombinieren. Solche Inhibitoren bedürfen äußerst genauer Anpassung der Struktur an den Rezeptor und hinsichtlich der Reaktivität in Bezug auf die Reaktion mit dem Inhibitor zur Bildung eines kovalent reversiblen Addukts. Zur Vorhersage von potentiellen Inhibitoren wurden molekulares Docking durchgeführt sowie auf quantenmechanische und molekularmechanische (QM/MM) Simulationen zurückgegriffen. Der Kerninhalt dieser Arbeit war die Synthese dieser Verbindungen sowie die Synthese von fluorogenen Substraten für β-Glucosidasen und α-Mannosidasen. Insgesamt wurden 41 Verbindungen synthetisiert. Eine Übersicht der einzelnen Teilprojekte ist in Abbildung 1 dargestellt. In Docking-gestütztem De-Novo-Design wurde eine Struktur (L039) entwickelt, die allen zuvor durch QM/MM-Simulationen identifizierten Anforderungen an einen kovalent reversiblen Inhibitor genügte. In weiteren Dockingstudien wurden diverse Verbindungen aus der Substanzklasse der monocyclischen N,O-Acetale als poteniell kovalent reversible Inhibitoren identifiziert. Daneben wurden Untersuchungen an einem Spiroacetal durchgeführt. Im Rahmen dieser Arbeit wurden ein Spiroacetal, 25 monocyclische N,O-Acetale, zwei Derivate der Azamannose als potentielle Synthesebausteine, Isofagomin und dessen 5-Epimer, vier potentielle Inhibitoren für eine potentielle allosterische Bindetasche sowie Fragmente der Struktur L039 synthetisiert, aufgereinigt und spektroskopisch charakterisiert. Keine dieser Verbindungen zeigte signifikante Hemmung der dGMII. Zur Untersuchung der Aktivität der potentiellen Inhibitoren wurden Enzymassays durchgeführt. Zu diesem Zweck wurden zwei Substrate in vier- bzw. fünfstufiger Synthese für fluorometrische Assays sowie ein Referenz-Inhibitor der β-Glucosidase (Isofagomin) in sechstufiger Synthese zur Validierung des Assays-Systems dargestellt

Synthesis and Metabolic Fate of 4‐Methylthiouridine in Bacterial tRNA

Ribonucleic acid (RNA) is central to many life processes and, to fulfill its function, it has a substantial chemical variety in its building blocks. Enzymatic thiolation of uridine introduces 4-thiouridine (s(4)U) into many bacterial transfer RNAs (tRNAs), which is used as a sensor for UV radiation. A similar modified nucleoside, 2-thiocytidine, was recently found to be sulfur-methylated especially in bacteria exposed to antibiotics and simple methylating reagents. Herein, we report the synthesis of 4-methylthiouridine (ms(4)U) and confirm its presence and additional formation under stress inEscherichia coli. We used the synthetic ms(4)U for isotope dilution mass spectrometry and compared its abundance to other reported tRNA damage products. In addition, we applied sophisticated stable-isotope pulse chase studies (NAIL-MS) and showed its AlkB-independent removalin vivo. Our findings reveal the complex nature of bacterial RNA damage repair

Identification of a potential allosteric site of Golgi α-mannosidase II using computer-aided drug design

Golgi α-mannosidase II (GMII) is a glycoside hydrolase playing a crucial role in the N-glycosylation pathway. In various tumour cell lines, the distribution of N-linked sugars on the cell surface is modified and correlates with the progression of tumour metastasis. GMII therefore is a possible molecular target for anticancer agents. Here, we describe the identification of a non-competitive GMII inhibitor using computer-aided drug design methods including identification of a possible allosteric binding site, pharmacophore search and virtual screening

Concomitant statin use does not impair the clinical outcome of patients with diffuse large B cell lymphoma treated with rituximab-CHOP

Preclinical data indicated a detrimental effect of statins on the anti-lymphoma activity of rituximab. We evaluated the impact of concomitant statin medication on the response and survival of patients with diffuse large B cell lymphoma (DLBCL) receiving rituximab-cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) as first-line therapy. Medical histories of patients with DLBCL who were treated with R-CHOP as first-line therapy were assessed for concomitant statin use, response after completion of chemotherapy, event-free survival (EFS), and overall survival (OS). Furthermore, 2-[(18)F]fluor-2-deoxyglucose (FDG)-PET/CT results after completion of first-line therapy were compared between the groups. Overall, 145 patients with DLBCL treated with R-CHOP from January 2001 to December 2009 were analyzed. Twenty-one (15%) patients received statins throughout therapy. Five-year EFS was 67.3% in patients without statins compared with 79% in patients receiving statins during R-CHOP (HR, 0.47; 95% CI, 0.15-1.54, p = 0.2). Five-year OS was 81.4% for patients without statins compared with 93.3% for patients taking statins (HR, 0.58; 95% CI 0.07-4.55, p = 0.6). There were no statistically significant differences in the rates of complete remissions between the two groups (75% in the non-statin group versus 86% in the statin group, p = 0.45). A trend toward a lower rate of complete metabolic responses in FDG-PET/CT after chemotherapy was seen in patients without statin medication compared with the patients taking statins (84% versus 92%, p = 0.068). Concomitant statin use had no adverse impact on response to chemotherapy, EFS, and OS in patients treated with R-CHOP for DLBCL