Christoph G. W. Gertzen

Synthesis, Biological Evaluation and Molecular Modeling of Substituted Indeno[1,2-b]indoles as Inhibitors of Human Protein Kinase CK2.

Due to their system of annulated 6-5-5-6-membered rings, indenoindoles have sparked great interest for the design of ATP-competitive inhibitors of human CK2. In the present study, we prepared twenty-one indeno[1,2-b]indole derivatives, all of which were tested in vitro on human CK2. The indenoindolones 5a and 5b inhibited human CK2 with an IC50 of 0.17 and 0.61 µM, respectively. The indeno[1,2-b]indoloquinone 7a also showed inhibitory activity on CK2 at a submicromolar range (IC50 = 0.43 µM). Additionally, a large number of indenoindole derivatives was evaluated for their cytotoxic activities against the cell lines 3T3, WI-38, HEK293T and MEF.

A Membrane-proximal, C-terminal α-Helix Is Required for Plasma Membrane Localization and Function of the G Protein-coupled Receptor (GPCR) TGR5

Background: TGR5 is a G protein-coupled bile acid receptor that modulates the immune response, glucose homeostasis, and liver regeneration. Results: Secondary structure of the receptor C terminus determines plasma membrane trafficking. Conclusion: TGR5 plasma membrane content and responsiveness to extracellular ligands depends on C-terminal α-helix formation. Significance: This provides insights into the structure-function relationship of TGR5, which is a potential drug target for metabolic diseases. The C terminus of G protein-coupled receptors (GPCRs) is important for G protein-coupling and activation; in addition, sorting motifs have been identified in the C termini of several GPCRs that facilitate correct trafficking from the endoplasmic reticulum to the plasma membrane. The C terminus of the GPCR TGR5 lacks any known sorting motif such that other factors must determine its trafficking. Here, we investigate deletion and substitution variants of the membrane-proximal C terminus of TGR5 with respect to plasma membrane localization and function using immunofluorescence staining, flow cytometry, and luciferase assays. Peptides of the membrane-proximal C-terminal variants are subjected to molecular dynamics simulations and analyzed with respect to their secondary structure. Our results reveal that TGR5 plasma membrane localization and responsiveness to extracellular ligands is fostered by a long (≥ 9 residues) α-helical stretch at the C terminus, whereas the presence of β-strands or only a short α-helical stretch leads to retention in the endoplasmic reticulum and a loss of function. As a proof-of-principle, chimeras of TGR5 containing the membrane-proximal amino acids of the β2 adrenergic receptor (β2AR), the sphingosine 1-phosphate receptor-1 (S1P1), or the κ-type opioid receptor (κOR) were generated. These TGR5β2AR, TGR5S1P1, or TGR5κOR chimeras were correctly sorted to the plasma membrane. As the exchanged amino acids of the β2AR, the S1P1, or the κOR form α-helices in crystal structures but lack significant sequence identity to the respective TGR5 sequence, we conclude that the secondary structure of the TGR5 membrane-proximal C terminus is the determining factor for plasma membrane localization and responsiveness towards extracellular ligands.

Structural assemblies of the di- and oligomeric G-protein coupled receptor TGR5 in live cells: an MFIS-FRET and integrative modelling study.

TGR5 is the first identified bile acid-sensing G-protein coupled receptor, which has emerged as a potential therapeutic target for metabolic disorders. So far, structural and multimerization properties are largely unknown for TGR5. We used a combined strategy applying cellular biology, Multiparameter Image Fluorescence Spectroscopy (MFIS) for quantitative FRET analysis, and integrative modelling to obtain structural information about dimerization and higher-order oligomerization assemblies of TGR5 wildtype (wt) and Y111 variants fused to fluorescent proteins. Residue 111 is located in transmembrane helix 3 within the highly conserved ERY motif. Co-immunoprecipitation and MFIS-FRET measurements with gradually increasing acceptor to donor concentrations showed that TGR5 wt forms higher-order oligomers, a process disrupted in TGR5 Y111A variants. From the concentration dependence of the MFIS-FRET data we conclude that higher-order oligomers – likely with a tetramer organization - are formed from dimers, the smallest unit suggested for TGR5 Y111A variants. Higher-order oligomers likely have a linear arrangement with interaction sites involving transmembrane helix 1 and helix 8 as well as transmembrane helix 5. The latter interaction is suggested to be disrupted by the Y111A mutation. The proposed model of TGR5 oligomer assembly broadens our view of possible oligomer patterns and affinities of class A GPCRs.

Alkoxyurea-Based Histone Deacetylase Inhibitors Increase Cisplatin Potency in Chemoresistant Cancer Cell Lines

The synthesis and biological evaluation of potent hydroxamate-based dual HDAC1/6 inhibitors with modest HDAC6 preference and a novel alkoxyurea connecting unit linker region are described. The biological studies included the evaluation of antiproliferative effects and HDAC inhibitory activity in the human ovarian cancer cell line A2780, the human squamous carcinoma cell line Cal27, and their cisplatin resistant sublines A2780CisR and Cal27CisR. The three most potent compounds 1g-i showed IC50 values in the low μM and sub-μM range. 1g-i revealed low nM IC50 values for HDAC6 with up to 15-fold preference over HDAC1, >3500-fold selectivity over HDAC4, and >100-fold selectivity over HDAC8. Furthermore, their ability to enhance cisplatin sensitivity was analyzed in Cal27 and Cal27CisR cells. Notably, a 48 h preincubation of 1g-i significantly enhanced the antiproliferative effects of cisplatin in Cal27 and Cal27CisR. 1g-i interacted synergistically with cisplatin. These effects were more pronounced for the cisplatin resistant subline Cal27CisR.

Interaction of Ochratoxin A and Its Thermal Degradation Product 2′R-Ochratoxin A with Human Serum Albumin

Ochratoxin A (OTA) is a toxic secondary metabolite produced by several fungal species of the genus Penicillium and Aspergillus. 2′R-Ochratoxin A (2′R-OTA) is a thermal isomerization product of OTA formed during food processing at high temperatures. Both compounds are detectable in human blood in concentrations between 0.02 and 0.41 µg/L with 2′R-OTA being only detectable in the blood of coffee drinkers. Humans have approximately a fifty-fold higher exposure through food consumption to OTA than to 2′R-OTA. In human blood, however, the differences between the concentrations of the two compounds is, on average, only a factor of two. To understand these unexpectedly high 2′R-OTA concentrations found in human blood, the affinity of this compound to the most abundant protein in human blood the human serum albumin (HSA) was studied and compared to that of OTA, which has a well-known high binding affinity. Using fluorescence spectroscopy, equilibrium dialysis, circular dichroism (CD), high performance affinity chromatography (HPAC), and molecular modelling experiments, the affinities of OTA and 2′R-OTA to HSA were determined and compared with each other. For the affinity of HSA towards OTA, a logK of 7.0–7.6 was calculated, while for its thermally produced isomer 2′R-OTA, a lower, but still high, logK of 6.2–6.4 was determined. The data of all experiments showed consistently that OTA has a higher affinity to HSA than 2′R-OTA. Thus, differences in the affinity to HSA cannot explain the relatively high levels of 2′R-OTA found in human blood samples.

Identification of a conserved interface of HIV-1 and FIV Vifs with Cullin 5

ABSTRACT Members of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 [A3]) family of DNA cytidine deaminases are intrinsic restriction factors against retroviruses. In felids such as the domestic cat (Felis catus), the A3 genes encode the A3Z2, A3Z3, and A3Z2Z3 antiviral cytidine deaminases. Only A3Z3 and A3Z2Z3 inhibit viral infectivity factor (Vif)-deficient feline immunodeficiency virus (FIV). The FIV Vif protein interacts with Cullin (CUL), Elongin B (ELOB), and Elongin C (ELOC) to form an E3 ubiquitination complex to induce the degradation of feline A3s. However, the functional domains in FIV Vif for the interaction with Cullin are poorly understood. Here, we found that the expression of dominant negative CUL5 prevented the degradation of feline A3s by FIV Vif, while dominant negative CUL2 had no influence on the degradation of A3. In coimmunoprecipitation assays, FIV Vif bound to CUL5 but not CUL2. To identify the CUL5 interaction site in FIV Vif, the conserved amino acids from positions 47 to 160 of FIV Vif were mutated, but these mutations did not impair the binding of Vif to CUL5. By focusing on a potential zinc-binding motif (K175-C161-C184-C187) of FIV Vif, we found a conserved hydrophobic region (174IR175) that is important for the CUL5 interaction. Mutation of this region also impaired the FIV Vif-induced degradation of feline A3s. Based on a structural model of the FIV Vif-CUL5 interaction, the 52LW53 region in CUL5 was identified as mediating binding to FIV Vif. By comparing our results to the human immunodeficiency virus type 1 (HIV-1) Vif-CUL5 interaction surface (120IR121, a hydrophobic region that is localized in the zinc-binding motif), we suggest that the CUL5 interaction surface in the diverse HIV-1 and FIV Vifs is evolutionarily conserved, indicating a strong structural constraint. However, the FIV Vif-CUL5 interaction is zinc independent, which contrasts with the zinc dependence of HIV-1 Vif. IMPORTANCE Feline immunodeficiency virus (FIV), which is similar to human immunodeficiency virus type 1 (HIV-1), replicates in its natural host in T cells and macrophages that express the antiviral restriction factor APOBEC3 (A3). To escape A3s, FIV and HIV induce the degradation of these proteins by building a ubiquitin ligase complex using the viral protein Vif to connect to cellular proteins, including Cullin 5. Here, we identified the protein residues that regulate this interaction in FIV Vif and Cullin 5. While our structural model suggests that the diverse FIV and HIV-1 Vifs use conserved residues for Cullin 5 binding, FIV Vif binds Cullin 5 independently of zinc, in contrast to HIV-1 Vif.Members of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC3 [A3]) family of DNA cytidine deaminases are intrinsic restriction factors against retroviruses. In felids such as the domestic cat (Felis catus), the A3 genes encode the A3Z2, A3Z3, and A3Z2Z3 antiviral cytidine deaminases. Only A3Z3 and A3Z2Z3 inhibit viral infectivity factor (Vif)-deficient feline immunodeficiency virus (FIV). The FIV Vif protein interacts with Cullin (CUL), Elongin B (ELOB), and Elongin C (ELOC) to form an E3 ubiquitination complex to induce the degradation of feline A3s. However, the functional domains in FIV Vif for the interaction with Cullin are poorly understood. Here, we found that the expression of dominant negative CUL5 prevented the degradation of feline A3s by FIV Vif, while dominant negative CUL2 had no influence on the degradation of A3. In coimmunoprecipitation assays, FIV Vif bound to CUL5 but not CUL2. To identify the CUL5 interaction site in FIV Vif, the conserved amino acids from positions 47 to 160 of FIV Vif were mutated, but these mutations did not impair the binding of Vif to CUL5. By focusing on a potential zinc-binding motif (K175-C161-C184-C187) of FIV Vif, we found a conserved hydrophobic region (174IR175) that is important for the CUL5 interaction. Mutation of this region also impaired the FIV Vif-induced degradation of feline A3s. Based on a structural model of the FIV Vif-CUL5 interaction, the 52LW53 region in CUL5 was identified as mediating binding to FIV Vif. By comparing our results to the human immunodeficiency virus type 1 (HIV-1) Vif-CUL5 interaction surface (120IR121, a hydrophobic region that is localized in the zinc-binding motif), we suggest that the CUL5 interaction surface in the diverse HIV-1 and FIV Vifs is evolutionarily conserved, indicating a strong structural constraint. However, the FIV Vif-CUL5 interaction is zinc independent, which contrasts with the zinc dependence of HIV-1 Vif. IMPORTANCE Feline immunodeficiency virus (FIV), which is similar to human immunodeficiency virus type 1 (HIV-1), replicates in its natural host in T cells and macrophages that express the antiviral restriction factor APOBEC3 (A3). To escape A3s, FIV and HIV induce the degradation of these proteins by building a ubiquitin ligase complex using the viral protein Vif to connect to cellular proteins, including Cullin 5. Here, we identified the protein residues that regulate this interaction in FIV Vif and Cullin 5. While our structural model suggests that the diverse FIV and HIV-1 Vifs use conserved residues for Cullin 5 binding, FIV Vif binds Cullin 5 independently of zinc, in contrast to HIV-1 Vif.

Design, multicomponent synthesis and anticancer activity of a focused histone deacetylase (HDAC) inhibitor library with peptoid-based cap groups

In this work, we report the multicomponent synthesis of a focused histone deacetylase (HDAC) inhibitor library with peptoid-based cap groups and different zinc-binding groups. All synthesized compounds were tested in a cellular HDAC inhibition assay and an MTT assay for cytotoxicity. On the basis of their noteworthy activity in the cellular HDAC assays, four compounds were further screened for their inhibitory activity against recombinant HDAC1-3, HDAC6, and HDAC8. All four compounds showed potent inhibition of HDAC1-3 as well as significant inhibition of HDAC6 with IC50 values in the submicromolar concentration range. Compound 4j, the most potent HDAC inhibitor in the cellular HDAC assay, revealed remarkable chemosensitizing properties and enhanced the cisplatin sensitivity of the cisplatin-resistant head-neck cancer cell line Cal27CisR by almost 7-fold. Furthermore, 4j almost completely reversed the cisplatin resistance in Cal27CisR. This effect is related to a synergistic induction of apoptosis as seen in the combination of 4j with cisplatin.

Relevance of N-terminal residues for amyloid-β binding to platelet integrin α IIb β 3 , integrin outside-in signaling and amyloid-β fibril formation

A pathological hallmark of Alzheimers disease (AD) is the aggregation of amyloid-β peptides (Aβ) into fibrils, leading to deposits in cerebral parenchyma and vessels known as cerebral amyloid angiopathy (CAA). Platelets are major players of hemostasis but are also implicated in AD. Recently we provided strong evidence for a direct contribution of platelets to AD pathology. We found that monomeric Aβ40 binds through its RHDS sequence to integrin αIIbβ3, and promotes the formation of fibrillar Aβ aggregates by the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin (CLU) from platelets. Here we investigated the molecular mechanisms of Aβ binding to integrin αIIbβ3 by using Aβ11 and Aβ16 peptides. These peptides include the RHDS binding motif important for integrin binding but lack the central hydrophobic core and the C-terminal sequence of Aβ. We observed platelet adhesion to truncated N-terminal Aβ11 and Aβ16 peptides that was not mediated by integrin αIIbβ3. Thus, no integrin outside-in signaling and reduced CLU release was detected. Accordingly, platelet mediated Aβ fibril formation was not observed. Taken together, the RHDS motif of Aβ is not sufficient for Aβ binding to platelet integrin αIIbβ3 and platelet mediated Aβ fibril formation but requires other recognition or binding motifs important for platelet mediated processes in CAA. Thus, increased understanding of the molecular mechanisms of Aβ binding to platelet integrin αIIbβ3 is important to understand the role of platelets in amyloid pathology.

The secondary structure of the TGR5 membrane-proximal C-terminus determines plasma membrane localization and responsiveness towards extracellular ligands

TGR5 is a structurally unknown bile acid sensing [1] G protein-coupled receptor (GPCR). It is located in different non-parenchymal cells of the liver [2]. Previous studies demonstrated that the deletion of the 35 C-terminal amino acids results in ER retention of the mutated receptor. The membrane proximal C-terminus of GPCRs has been shown to be important for membrane localization of these receptors because it can contain a sorting motif (e.g., the F(X)6LL motif). Since the N-terminal part of the C-Terminus of TGR5 contains no known sorting motif, we hypothesize that it must be the secondary structure of the C-terminus that determines TGR5 trafficking [3]. Up to 18 amino acids of the membrane proximal C-terminus of TGR5 wildtype as well as of 8 different substitution and deletion variants within this region were subjected to molecular dynamics (MD) simulations of 600 ns length each using the Amber 11 suite of programs [4]. The starting structure was a straight peptide chain in each case to exclude any bias from prefolded structures. Of the 600 ns of the simulations, the last 500 ns were used for further analyses, respectively. For each variant, the generated conformations were analyzed with respect to the secondary structure sequence by the program “dssp”. All secondary structure sequences of all variants were pooled and hierarchically clustered with the program “R”. Functional analyses of the variants were done using a cAMP reporter gene assay. Membrane localization was determined via FACS analysis. The clustering shows that clusters 1 and 5 are mainly comprised of the wildtype (WT) and variants 285-290G, 291-279A, and Δ291-297 (Figure ​(Figure1A).1A). These variants show intermediate to high functionality up to 174% of the WT and a membrane localization of more than 70% (Figure ​(Figure1B).1B). The conformations in these clusters show a predominant α-helix formation. Cluster 2 contains the variants 285-290A and 291-297G (Figure ​(Figure1A).1A). These variants show a functionality < 13% of the WT and a membrane localization < 53% (Figure ​(Figure1B).1B). The most frequently occurring secondary structure in cluster 2 is a β-sheet. Finally, clusters 3 and 4 contain the variants 285-290P, Δ285-290, and 291-297P. These variants show a low to intermediate functionality (11% to 37% of the WT) and membrane localization (41% to 77%) (Figure ​(Figure1B).1B). The most frequently occurring secondary structure in clusters 3 and 4 is a loop formation. These results show that an α-helix formation in the variants C-terminus is associated with a high functionality and membrane localization, as can be seen for clusters 1 and 5. In contrast, β-sheet or loop formation is associated with reduced functionality and membrane localization, as can be seen for clusters 2 to 4. Overall, this suggests that mere secondary structure content rather than a specific amino acid sequence of the membrane-proximal part of the C-terminus determine PM localization and function of TGR5. Figure 1 A Clustering results of the TGR5 C-terminus WT and mutants according to the secondary structure sequence. The numbers indicate how many percent of the conformations of each variant are found in the respective cluster. B Membrane localization and function ... As a proof of concept, we constructed a chimera of TGR5 with the 13 membrane-proximal amino acids of the C-terminus replaced by respective amino acids of the C-terminus of the β2-adrenergic receptor (β2AR). These β2AR residues form an α-helix in the β2AR crystal structure and show no sequence identity to the exchanged amino acids from TGR5. As expected, this chimera was correctly sorted to the plasma membrane and showed a similar functional activity as the TGR5 wildtype. As the constructed chimera does contain a F(X)6LL sorting motif within the β2AR part, however, we also investigated the influence of the sorting motif by mutation studies and subsequent experimental and computational characterization of the variants. The TGR5β2AR chimera and its F287Y variant both show a membrane localization of approx. 87%, and the mutant a functionality of > 95% of the chimera WT (Figure ​(Figure1C).1C). Both variants also show the highest α-helix formation, averaged over all residues, with > 23% over the course of the MD simulations but no β-sheet formation (Figure ​(Figure1C).1C). Variants F287Y//LL294/5VV, L294V, and L295V show functionalities between 86% and 123% of the chimera WT and membrane localizations of approx. 88% (Figure ​(Figure1C).1C). While the α-helix content of these variants is reduced to 5% to 11%, their β-sheet content is still < 0.6% (Figure ​(Figure1C).1C). In contrast, variant LL294/5VV shows a β-sheet content of 6% but only an α-helicality of 2%, which is accompanied by a reduced membrane localization of 84% (Figure ​(Figure1C).1C). Along these lines, the variants F287A//LL294/5AA and LL294/5AA show a low functionality between 30% and 43% of the chimera WT and a reduced membrane localization of < 79%. Their respective α-helicality is 8% and 0.9%, but their respective β-sheet content is as high as 4.6% and 8%. Taken together, the TGR5β2AR variants reveal a reduced functionality and membrane localization if the α-helicality decreases and the β-sheet content increases in the membrane-proximal part of their C-termini. These results are consistent with our observations on the TGR5 variants and further strengthen our hypothesis that an α-helix formation in that region fosters membrane localization of TGR5. For a final validation, we created the F291A mutant of the LL294/5AA variant of TGR5β2AR. This was done because our MD simulations had identified a strong hydrophobic contact between F291 and A295 in the LL294/5AA variant, leading to a β-sheet formation between these two residues for 60% of the simulated time. In contrast, MD simulations predicted that this contact is broken in the triple alanine mutant F291A//LL294/5AA, consequently restoring α-helicality (to 3.9%) and reducing the β-sheet content (to 1.6%) (Figure ​(Figure1C).1C). Experimental testing confirmed our expectation in that the triple alanine mutant shows an increased membrane localization (85.1%) and functionality (91.6% of the chimera WT) (Figure ​(Figure1C).1C). Thus, we were able to rescue a variant with impaired functionality and membrane localization by introducing another mutation that had been predicted to restore α-helicality in the membrane-proximal part of their C-termini. In summary, we first demonstrated that an α-helix in the membrane proximal C-terminus of TGR5 fosters membrane localization of the receptor while β-sheet or loop formation in this region leads to ER retention and a loss of function. Second, a chimera with a known α-helical C-terminus of β2AR showed a membrane localization and function similar to the TGR5 WT. Additionally, variants of the TGR5β2AR chimera with mutations in the β2AR sorting motif showed a likewise relationship between secondary structure content and membrane localization and function as observed for the variants of the TGR5 WT. Finally, we demonstrated that a non-functional chimera variant can be rescued by introducing an additional mutation that restores α-helicality. We thus conclude that the secondary structure of the TGR5 membrane-proximal C-terminus, which forms an α-helix according to our MD simulations, is the determining factor for plasma membrane localization and responsiveness towards extracellular ligands.

High-precision FRET analysis of the G-protein coupled receptor TGR5 in live cells

Background TGR5 is a widely expressed and highly conserved G protein coupled receptor. Its activity and functionality is commonly modulated by bile acids, especially by lithocholic acid. As true for all ligand activated G protein coupled receptors a G protein subunit is released from TGR5 after ligand binding and initiates a signaling cascade resulting in a cell type specific response. Current investigations suggest an involvement of TGR5 in bile homeostasis, inflammatory responses and hepatobiliary diseases. Therefore a targeted therapy involving site specific inhibition of TGR5 is of immense interest. However, up to date no solved structure of TGR5 exists and oligomerization properties are largely unknown. To determine structural changes and oligomerization properties of TGR5 we designed a three way strategy including TGR5 plasmids coupled with (I) fluorescent proteins (FPs) at the C-terminus or (II) a N-terminal peptide tag (ACP) for subsequent labeling with a fluorescent dye and (III) unnatural amino acids (UAAs) for site specific extracellular labeling.