TREX: a universal tool for the transfer and expression of biosynthetic pathways in bacteria.

Secondary metabolites represent a virtually inexhaustible source of natural molecules exhibiting a high potential as pharmaceuticals or chemical building blocks. To gain broad access to these compounds, sophisticated expression systems are needed that facilitate the transfer and expression of large chromosomal regions, whose genes encode complex metabolic pathways. Here, we report on the development of the novel system for the transfer and expression of biosynthetic pathways (TREX), which comprises all functional elements necessary for the delivery and concerted expression of clustered pathway genes in different bacteria. TREX employs (i) conjugation for DNA transfer, (ii) randomized transposition for its chromosomal insertion, and (iii) T7 RNA polymerase for unimpeded bidirectional gene expression. The applicability of the TREX system was demonstrated by establishing the biosynthetic pathways of two pigmented secondary metabolites, zeaxanthin and prodigiosin, in bacteria with different metabolic capacities. Thus, TREX represents a valuable tool for accessing natural products by allowing comparative expression studies with clustered genes.

Peptide-based targeting of the platelet-derived growth factor receptor beta.

PURPOSE: The aim of this work is to identify new ligands targeting the platelet-derived growth factor receptor beta (PDGFRß). PROCEDURES: Biopanning was carried out with a 12-amino-acid phage display library against the recombinant extracellular domain of PDGFRß. The identified peptide PDGFR-P1 was chemically synthesized and labeled with (125)I or (131)I. In vitro studies were performed on the PDGFRß-expressing cell lines BxPC3 and MCF7 and on PDGFRß-transfected HEK cells in comparison to negative control wtHEK293 and CaIX-transfected HEK cells. Biodistribution experiments were performed in Balb/c nude mice, carrying subcutaneously BxPC3 tumors. RESULTS: In vitro studies demonstrated a higher binding to BxPC3, MCF7, and PDGFRß-tr-HEK cells in comparison to negative control cell lines. Binding was inhibited up to 90% by the unlabeled PDGFR-P1 peptide. Organ distribution studies revealed a higher accumulation in BxPC3 tumors than in most organs. CONCLUSIONS: PDGFR-P1 is a promising candidate for targeting human PDGFRß.

Combination of phage display and molecular grafting generates highly specific tumor-targeting miniproteins.

Frankenstein's peptide: the grafting of the binding domain from miniprotein Min-23 into the sunflower trypsin inhibitor (SFTI-I) peptide scaffold preserved its in vitro and in vivo binding specificity and proteolytic stability. The combination of these peptides was shown to be tumor-specific with a good binding affinity for delta-like ligand 4 (Dll4) protein. The use of SFTI-I as a peptide scaffold is ideal for hit-to-lead development.

Antitumor effects of proteasome inhibition in anaplastic thyroid carcinoma.

UNLABELLED: The ubiquitin-proteasome pathway has been identified as a potential molecular target for cancer therapy. In this study, we investigated the effect of the proteasome inhibitor bortezomib on anaplastic thyroid carcinoma (ATC) characterized by complete refractoriness to multimodal therapeutic approaches. METHODS: The ATC cell lines C643 and SW1736 were treated with bortezomib (1 nM to 1 µM) for 12-72 h. Thereafter, growth inhibition was analyzed by thymidine uptake experiments and determination of the viable cell number. Apoptosis was measured and a cell cycle analysis was done. Using gene chip analysis and the real-time quantitative PCR system, we measured transcriptional changes. The activity of the nuclear factor (NF)-κB and p53 signal transduction pathways was monitored using the reporter constructs pNF-κB-TA-Luc and pp53-TA-Luc in the luciferase activity assay. Uptake measurements using (3)H-FDG, (14)C-aminoisobutyric acid, and Na(125)iodide were performed to investigate metabolic changes and iodide symporter activity in vitro. Moreover, the (18)F-FDG uptake was evaluated in ATC tumor-bearing nude mice 1 or 2 d after treatment with bortezomib. RESULTS: Bortezomib induced growth inhibition, apoptosis, and G(2)-M cell cycle arrest associated with upregulation of p21(CIP1/WAF1) expression in SW1736 and C643 cells. Moreover, the glucose metabolism and aminoisobutyric acid uptake significantly decreased in vitro in both of the ATC cell lines in vivo only in SW1736 tumors at 2 d after the bortezomib treatment. The transcriptional profile in bortezomib-treated SW1736 and C643 cells revealed increased expression of genes involved in stress response, apoptosis, regulation of the cell cycle, and differentiation. Using real-time quantitative PCR for the quantification of gene expression, we additionally noticed upregulation of the tumor necrosis factor-related apoptosis-inducing ligand and the thyroid-specific transcription factors Pax8 and TTF-1, leading to expression of the thyroid-specific target genes thyroglobulin, sodium iodide symporter, thyroperoxidase, and thyroid-stimulating hormone receptor and to a moderate accumulation of iodide in ATC cells. CONCLUSION: On the basis of our data, bortezomib represents a promising antineoplastic agent for the treatment of ATC. To improve the clinical outcome, further investigation into the potential of bortezomib therapy of thyroid cancer is clearly warranted.

Optimization of a novel peptide ligand targeting human carbonic anhydrase IX.

BACKGROUND: Carbonic anhydrase IX (CA IX) is a hypoxia-regulated transmembrane protein over-expressed in various types of human cancer. Recently, a new peptide with affinity for human carbonic anhydrase IX (CaIX-P1) was identified using the phage display technology. Aim of the present study is to characterize the binding site in the sequence of CaIX-P1, in order to optimize the binding and metabolic properties and use it for targeting purposes. METHODOLOGY/PRINCIPAL FINDINGS: Various fragments of CaIX-P1 were synthesized on solid support using Fmoc chemistry. Alanine scanning was performed for identification of the amino acids crucial for target binding. Derivatives with increased binding affinity were radiolabeled and in vitro studies were carried out on the CA IX positive human renal cell carcinoma cell line SKRC 52 and the CA IX negative human pancreatic carcinoma cell line BxPC3. Metabolic stability was investigated in cell culture medium and human serum. Organ distribution and planar scintigraphy studies were performed in Balb/c nu/nu mice carrying subcutaneously transplanted SKRC 52 tumors. The results of our studies clearly identified amino acids that are important for target binding. Among various fragments and derivatives the ligand CaIX-P1-4-10 (NHVPLSPy) was found to possess increased binding potential in SKRC 52 cells, whereas no binding capacity for BxPC3 cells was observed. Binding of radiolabeled CaIX-P1-4-10 on CA IX positive cells could be inhibited by both the unlabeled and the native CaIX-P1 peptide but not by control peptides. Stability experiments indicated the degradation site in the sequence of CaIX-P1-4-10. Biodistribution studies showed a higher in vivo accumulation in the tumor than in most healthy tissues. CONCLUSIONS: Our data reveal modifications in the sequence of the CA IX affine ligand CaIX-P1 that might be favorable for improvement of target affinity and metabolic stability, which are necessary prior to the use of the ligand in clinical approaches.

A T7 RNA polymerase-based toolkit for the concerted expression of clustered genes.

Bacterial genes whose enzymes are either assembled into complex multi-domain proteins or form biosynthetic pathways are frequently organized within large chromosomal clusters. The functional expression of clustered genes, however, remains challenging since it generally requires an expression system that facilitates the coordinated transcription of numerous genes irrespective of their natural promoters and terminators. Here, we report on the development of a novel expression system that is particularly suitable for the homologous expression of multiple genes organized in a contiguous cluster. The new expression toolkit consists of an Ω interposon cassette carrying a T7 RNA polymerase specific promoter which is designed for promoter tagging of clustered genes and a small set of broad-host-range plasmids providing the respective polymerase in different bacteria. The uptake hydrogenase gene locus of the photosynthetic non-sulfur purple bacterium Rhodobacter capsulatus which consists of 16 genes was used as an example to demonstrate functional expression only by T7 RNA polymerase but not by bacterial RNA polymerase. Our findings clearly indicate that due to its unique properties T7 RNA polymerase can be applied for overexpression of large and complex bacterial gene regions.

Engineering and functionalization of the disulfide-constrained miniprotein min-23 as a scaffold for diagnostic application.

Miniproteins are scaffolds for the development of alternative non-immunoglobin binding agents for medical applications. This peptide format features high tolerance to sequence mutagenesis, excellent proteolytic stability, and fast blood pool clearance. Herein we present the total chemical synthesis of the disulfide-constrained scaffold Min-23 and its functionalization for in vitro and in vivo application. Optimized solid-phase peptide chemistry and oxidative folding strategies were developed to engineer this miniprotein with native-like disulfide configuration. High levels of serum stability and proteolytic resistance, as well as a beneficial pharmacokinetic profile for diagnostic imaging, were determined by using radiolabeling techniques such as positron emission tomography. The reported achievements highlight Min-23 as a promising scaffold for the development of novel recognition molecules for medical application.

Biotechnology techniques for the development of new tumor specific peptides.

Peptides, proteins and antibodies are promising candidates as carriers for radionuclides in endoradiotherapy. This novel class of pharmaceuticals offers a great potential for the targeted therapy of cancer. The fact that some receptors are overexpressed in several tumor types and can be targeted by small peptides, proteins or antibodies conjugated to radionuclides has been used in the past for the development of peptide endoradiotherapeutic agents such as (90)Y-DOTATOC or radioimmunotherapy of lymphomas with Zevalin. These procedures have been shown to be powerful options for the treatment of cancer patients. Design of new peptide libraries and scaffolds combined with biopanning techniques like phage and ribosome display may lead to the discovery of new specific ligands for target structures overexpressed in malignant tumors. Display methods are high throughput systems which select for high affinity binders. These methods allow the screening of a vast amount of potential binding motifs which may be exposed to either cells overexpressing the target structures or in a cell-free system to the protein itself. Labelling these binders with radionuclides creates new potential tracers for application in diagnosis and endoradiotherapy. This review highlights the advantages and problems of phage and ribosome display for the identification and evaluation of new tumor specific peptides.

Challenges in optimizing a prostate carcinoma binding peptide, identified through the phage display technology.

The transfer of peptides identified through the phage display technology to clinical applications is difficult. Major drawbacks are the metabolic degradation and label instability. The aim of our work is the optimization of DUP-1, a peptide which was identified by phage display to specifically target human prostate carcinoma. To investigate the influence of chelate conjugation, DOTA was coupled to DUP-1 and labeling was performed with ¹¹¹In. To improve serum stability cyclization of DUP-1 and targeted D-amino acid substitution were carried out. Alanine scanning was performed for identification of the binding site and based on the results peptide fragments were chemically synthesized. The properties of modified ligands were investigated in in vitro binding and competition assays. In vivo biodistribution studies were carried out in mice, carrying human prostate tumors subcutaneously. DOTA conjugation resulted in different cellular binding kinetics, rapid in vivo renal clearance and increased tumor-to-organ ratios. Cyclization and D-amino acid substitution increased the metabolic stability but led to binding affinity decrease. Fragment investigation indicated that the sequence NRAQDY might be significant for target-binding. Our results demonstrate challenges in optimizing peptides, identified through phage display libraries, and show that careful investigation of modified derivatives is necessary in order to improve their characteristics.

Therapy of thyroid carcinoma with the histone deacetylase inhibitor MS-275.

PURPOSE: Dysregulation of histone acetylation associated with an up-regulation of histone deacetylase (HDAC) activity is common in malignant tumours. Therefore, HDAC inhibitors were developed whose effects on proliferation and apoptosis have been shown in different tumour entities. Since non-iodide-concentrating thyroid carcinomas represent a therapeutic problem, this study addressed the effects of the HDAC inhibitor MS-275 on thyroid carcinoma cells. METHODS: After the antiproliferative effect of MS-275 had been proven in different human and rat thyroid carcinoma cell lines, FRO82-2, SW1736 and FTC133 cells were further investigated with respect to changes in apoptosis, cell cycle and metabolism by the annexin V/propidium iodide assay, FACS analysis and uptake experiments employing 3-O-methyl-D-(3H)glucose, fluoro-2-deoxy-D-glucose2 [5,6-(3)H] and 14C-aminoisobutyric acid (AIB). The induction of iodide transport and gene expression were investigated in 125iodide uptake experiments and real-time polymerase chain reaction (PCR). RESULTS: MS-275 induced a concentration- and time-dependent inhibition of proliferation in the thyroid carcinoma cell lines with varying IC50 values. In FRO82-2, SW1736 and FTC133 cells characterized by low, moderate and high sensitivity an up-regulation of p21CIP/WAF1 expression and G1 and/or G2 phase arrest were observed upon MS-275 exposure corresponding to the sensitivity of individual cell lines. In addition, high MS-275 concentrations increased the apoptotic cell fraction of FTC133 and SW1736 cells, whereas resistance to apoptosis and simultaneous up-regulation of Bcl-2 gene expression were observed in FRO82-2 cells. MS-275 treatment also mediated a concentration-dependent decrease of 3H-FDG uptake and an increased 3-O-methyl-D-(3H)glucose uptake in all thyroid carcinoma cell lines after 24 h, an increased uptake of both tracers in FTC133 cells after 48 h, and restored the functional activity of the sodium-iodide symporter in SW1736 and FTC133 cells up to 20- and 45-fold. CONCLUSION: MS-275 exerts dose-dependent antiproliferative effects including growth arrest, differentiation and apoptosis in some thyroid carcinoma cell lines and might, therefore, be considered for the treatment of anaplastic and non-iodide-concentrating thyroid carcinomas.

A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology.

UNLABELLED: Carbonic anhydrase IX (CAIX) is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy. METHODS: Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC). Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR. RESULTS: In vitro binding experiments of (125)I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney. CONCLUSIONS: These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.

A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus.

The functional expression of heterologous genes using standard bacterial expression hosts such as Escherichia coli is often limited, e.g. by incorrect folding, assembly or targeting of recombinant proteins. Consequently, alternative bacterial expression systems have to be developed to provide novel strategies for protein synthesis exceeding the repertoire of the standard expression host E. coli. Here, we report on the construction of a novel expression system that combines the high processivity of T7 RNA polymerase with the unique physiological properties of the facultative photosynthetic bacterium Rhodobacter capsulatus. This system basically consists of a recombinant R. capsulatus T7 expression strain (R. capsulatus B10S-T7) harboring the respective polymerase gene under control of a fructose inducible promoter. In addition, a set of different broad-host-range vectors (pRho) was constructed allowing T7 RNA polymerase dependent and independent target gene expression in R. capsulatus and other Gram-negative bacteria. The expression efficiency of the novel system was studied in R. capsulatus and E. coli using the yellow fluorescent protein (YFP) as model protein. Expression levels were comparable in both expression hosts and yielded up to 80mg/l YFP in phototrophically grown R. capsulatus cultures. This result clearly indicates that the novel R. capsulatus-based expression system is well suited for the high-level expression of soluble proteins.