ABSTRACT
Within the last two decades, the methylotrophic yeast Pichia pastoris (Komagataella phaffii) has become an important alternative to E. coli or mammalian cell lines for the production of recombinant proteins. Easy handling, strong promoters, and high cell density cultivations as well as the capability of posttranslational modifications are some of the major benefits of this yeast. The high secretion capacity and low level of endogenously secreted proteins further promoted the rapid development of a versatile Pichia pastoris toolbox. This chapter reviews common and new "Pichia tools" and their specific features. Special focus is given to expression strains, such as different methanol utilization, protease-deficient or glycoengineered strains, combined with application highlights. Different promoters and signal sequences are also discussed.
Subject(s)
Escherichia coli , Pichia , Escherichia coli/metabolism , Pichia/genetics , Pichia/metabolism , Promoter Regions, Genetic , Recombinant Proteins/metabolism , SaccharomycetalesABSTRACT
Doxorubicin, a well-established chemotherapeutic agent, is known to accumulate in the cell nucleus. By using ICP-MS, we show that the conjugation of two small organometallic rhenium complexes to this structural motif results in a significant redirection of the conjugates from the nucleus to the mitochondria. Despite this relocation, the two bioconjugates display excellent toxicity toward HeLa cells. In addition, we carried out a preliminarily investigation of aspects of cytotoxicity and present evidence that the conjugates disrupt the mitochondrial membrane potential, are strong inhibitors of human Topoisomeraseâ II, and induce apoptosis. Such derivatives may enhance the therapeutic index of the aggressive parent drug and overcome drug resistance by influencing nuclear and mitochondrial homeostasis.
Subject(s)
Antibiotics, Antineoplastic/pharmacokinetics , Doxorubicin/pharmacokinetics , Mitochondria/drug effects , Organometallic Compounds/pharmacology , Rhenium/pharmacology , HeLa Cells , Humans , Microscopy, Fluorescence , Rhenium/chemistryABSTRACT
We present the combination of the clinically well-proven chemotherapeutic agent, Doxorubicin, and (99m)Tc, an Auger and internal conversion electron emitter, into a dual-action agent for therapy. Chemical conjugation of Doxorubicin to (99m)Tc afforded a construct which autonomously ferries a radioactive payload into the cell nucleus. At this site, damage is exerted by dose deposition from Auger radiation. The (99m)Tc-conjugate exhibited a dose-dependent inhibition of survival in a selected panel of cancer cells and an in vivo study in healthy mice evidenced a biodistribution which is comparable to that of the parent drug. The homologous Rhenium conjugate was found to effectively bind to DNA, inhibited human Topoisomerase II, and exhibited cytotoxicity in vitro. The collective in vitro and in vivo data demonstrate that the presented metallo-conjugates closely mimic native Doxorubicin.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Doxorubicin/chemistry , Doxorubicin/pharmacology , Technetium/chemistry , Technetium/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Cell Survival/drug effects , DNA Topoisomerases, Type II/metabolism , Doxorubicin/pharmacokinetics , Humans , Mice , Neoplasms/drug therapy , Technetium/pharmacokinetics , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/pharmacokinetics , Topoisomerase II Inhibitors/pharmacologyABSTRACT
Radiolabeling allows noninvasive imaging by single photon emission computed tomography (SPECT) or positron emission tomography (PET) for assessing the biodistribution of nanostructures. Herein, the synthesis of a new coating ligand for gold nanoparticles (AuNPs) and quantum dots (QDs) is reported. This ligand is multifunctional; it combines the metal chelate with conjugating functions to biological vectors. The concept allows the coupling of any targeting function to the chelator; an example for the prostate specific membrane antigen is given. Derivatized NPs can directly be labeled in one step with [(99m) Tc(OH2 )3 (CO)3 ](+) . AuNPs in particular are highly stable, a prerequisite for in vivo studies excluding misinterpretation of the biodistribution data. AuNPs with differing sizes (7 and 14â nm core diameter) were administered intravenously into nude NMRI mice bearing LNCaP xenografts. MicroSPECT images show for both probes rapid clearance from the blood pool through the hepatobiliary pathway. The 7â nm AuNPs revealed a significantly higher bone uptake than the 14â nm AuNPs. The high affinity towards bone mineral is further confirmed in vitro with hydroxyapatite.
Subject(s)
Gold/chemistry , Metal Nanoparticles/chemistry , Organotechnetium Compounds/pharmacokinetics , Quantum Dots/chemistry , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Gold/metabolism , Gold/pharmacokinetics , Ligands , Metal Nanoparticles/ultrastructure , Mice, Nude , Models, Molecular , Neoplasms/diagnosis , Organotechnetium Compounds/chemistry , Organotechnetium Compounds/metabolism , Quantum Dots/metabolism , Tissue Distribution , Tomography, Emission-Computed, Single-PhotonABSTRACT
Within the last two decades, the methylotrophic yeast Pichia pastoris has become an important alternative to E. coli or mammalian cell lines for the production of recombinant proteins. Easy handling, strong promoters, and high cell density cultivations as well as the capability of posttranslational modifications are some of the major benefits of this yeast. The high secretion capacity and low level of endogenously secreted proteins further promoted the rapid development of a versatile Pichia pastoris toolbox. This chapter reviews common and new "Pichia tools" and their specific features. Special focus is given to expression strains, such as different methanol utilization, protease-deficient or glycoengineered strains, combined with application highlights. Different promoters and signal sequences are also discussed.
Subject(s)
Genetic Engineering/methods , Pichia/genetics , Recombinant Proteins/biosynthesis , Gene Dosage/genetics , Genetic Vectors/genetics , Promoter Regions, Genetic/genetics , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Species SpecificityABSTRACT
We describe the syntheses of half-sandwich complexes of the type [(η(5)-Cp(CONH-R))M(CO)3] with M = Re or (99m)Tc. The R group represents different tri-peptides (tpe) which display high binding affinities for oligopeptide transporters PEPT2. The (99m)Tc complexes were prepared directly from [(99m)Tc(OH2)3(CO)3](+) and Diels-Alder dimerized, cyclopentadienyl derivatized peptides in water. This approach corroborates the feasibility of metal-mediated retro Diels-Alder reactions for the preparation of not only small molecules but also peptides carrying a [(η(5)-Cp)(99m)Tc(CO)3] tag. We synthesized the Diels-Alder product [(HCpCONH-tpe)2] from Thiele's acid [(η(5)-HCpCOOH)2] via double peptide coupling. The Re-complexes [(η(5)-CpCONH-tpe)Re(CO)3] were obtained by attaching [(Cp-COOH)Re(CO)3] directly to the N-terminus of peptides as received from SPPS. The authenticity of the (99m)Tc-complexes is confirmed by chromatographic comparison with the corresponding rhenium complexes, fully characterized by spectroscopic techniques.
Subject(s)
Cyclopentanes/chemistry , Oligopeptides/chemistry , Organometallic Compounds/chemical synthesis , Radiopharmaceuticals/chemical synthesis , Microwaves , Molecular Structure , Oligopeptides/chemical synthesis , Organometallic Compounds/chemistry , Radiopharmaceuticals/chemistry , Rhenium/chemistry , Technetium/chemistryABSTRACT
N-Alkylated derivatives of 1,3,5-triazacyclohexane (tach) are versatile, facially coordinating ligands. We present the syntheses and full characterization of two new complexes of the [(R(3)tach)ReO(3)](+) type. In these complexes, the tach ligand is readily substituted by bi- and tridentate ligands; hence, they can be considered as sources of the [ReO(3)](+) motif for Re(VII) complexes.
