Anna Fredriksson

Exploiting the 21st amino acid—purifying and labeling proteins by selenolate targeting

Selenium is essential to human life and occurs in selenoproteins as selenocysteine (Sec), the 21st amino acid. The selenium atom endows selenocysteine with unique biochemical properties, including a low pKa and a high reactivity with many electrophilic agents. Here we describe the introduction of selenocysteine into recombinant non-selenoproteins produced in Escherichia coli, as part of a small tetrapeptide motif at the C terminus. This selenocysteine-containing motif could subsequently be used as a protein tag for purification of the recombinant protein, selenolate-targeted labeling with fluorescent compounds or radiolabeling with either γ-emitting 75Se or short-lived positron emitters such as 11C. The results presented here thus show how a wide range of biotechnological applications can be developed starting from the insertion of selenocysteine into proteins.

IN VIVO EVALUATION OF THE BIODISTRIBUTION OF 11C-LABELED PD153035 IN RATS WITHOUT AND WITH NEUROBLASTOMA IMPLANTS

The biodistribution of 11C-labeled 4-(3-bromoanilino)-6,7-dimethoxyquinazoline, an inhibitor of the epidermal growth factor (EGF) receptor tyrosine kinase, has been evaluated in vivo in rats using positron emission tomography (PET). Time-activity data obtained after i.v. administration in one rat revealed that the radiotracer rapidly cleared from plasma with subsequent uptake in major organs of the body (brain, heart, liver, gastrointestinal tract and bladder). Uptake in proliferating tissue in rats with human neuroblastoma xenografts indicate that [O-11C-methyl]PD153035 shows promise as a new agent for in vivo imaging of tumors with PET.

Synthesis of [methoxy-11C]PD153035, a selective EGF receptor tyrosine kinase inhibitor

[Methoxy- 11 C]PD153035, a potent and specific inhibitor of the EGF receptor tyrosine kinase, was prepared by O-alkylation of O-desmethyl PD153035 with [ 11 C]methyl iodide in DMF. The radiochemical incorporation of [ 11 C]CH 3 I was on the order of 45%. The mean specific activity obtained at end-of-synthesis (EOS) was 26 GBq/μmol (n=3; range 20-36 GBq/μmol) and total synthesis time was 45-50 minutes including formulation.

Selenolthiol and dithiol C-terminal tetrapeptide motifs for one-step purification and labeling of recombinant proteins produced in E. coli

We have previously shown that a redox‐active selenocysteine‐containing tetrapeptide—Sel‐tag (Gly‐Cys‐Sec‐Gly)—can be used as a C‐terminal fusion motif for recombinant proteins produced in Escherichia coli. This Sel‐tag allows selenolate‐targeted one‐step purification, as well as fluorescent labeling or radiolabeling either with gamma emitters (75Se) or with positron‐emitting radionuclides (11C). Here we have analyzed four different redox‐active C‐terminal motifs, carrying either dithiol (Gly‐Cys‐Cys‐Gly or Ser‐Cys‐Cys‐Ser) or selenolthiol (Gly‐Cys‐Sec‐Gly or Ser‐Cys‐Sec‐Ser) motifs. Utilizing these different functional motifs with the same recombinant protein (Fel d 1), we were able to assess their relative reactivities and potential usefulness for biotechnological applications. We found that all four redox‐active tags could be utilized for efficient one‐step purification to provide pure protein from a crude bacterial lysate through reversible binding to phenylarsine oxide sepharose, with yields and purities comparable to those obtained for a His‐tagged protein purified by the more common approach with use of a Ni2+ column. For labeling with electrophilic fluorescent or radioactive compounds, however, the selenolthiol motifs were considerably more efficient than their dithiol counterparts. The results thus show that both the selenolthiol‐ and the dithiol‐containing tags can serve as efficient alternatives to His‐tags for protein purification, while the selenolthiol motifs offer additional and unique potential for Sec‐targeted labeling. It should therefore be possible to utilize these multifunctional tetrapeptide motifs to develop a wide range of novel biotechnological applications based on Sec targeting with electrophilic compounds.

Microwaving in F-18 Chemistry: Quirks and Tweaks

Since the late 1980s, microwave dielectric heating has been used to speed up chemical transformations, also in radiolabeling tracers for positron emission tomography. In addition to shorter reaction times, higher yields, cleaner product mixtures and improved reproducibility have also been obtained for reactions involving polar components that require heating at elevated temperatures. The conditions used in microwave chemistry can differ considerably from those in conventional heating. Understanding the factors that influence the interaction of the electromagnetic field with the sample is critical for the successful implementation of microwave heating. These parameters are discussed here and exemplified with radiolabelings with fluorine-18.

Cerebral uptake of [ethyl-11C]vinpocetine and 1-[11C]ethanol in cynomolgous monkeys: a comparative preclinical PET study

PET provides the potential to quantify the distribution of radiolabelled drugs in the human body. In cases when radiolabelled compounds undergo metabolic transformation after administration in vivo, it is necessary to examine the kinetics and distribution of both the labeled mother compound and labeled metabolites. The objective of this study was to assess the extent by which 11C-labeled ethanol, the product arising from the de-esterification of the neuroprotective drug vinpocetine (ethyl-apovincaminate), might contribute to the regional cerebral radioactivity measured by PET after the administration of [ethyl-11C]vinpocetine. In three cynomolgous monkeys PET measurements were made after intravenous bolus injection of both [11C]vinpocetine and 1-[11C]ethanol. There was a marked difference between the regional time-activity curves of [11C]ethanol and [11C]vinpocetine. The distribution pattern obtained with [11C]ethanol was similar to that observed with blood flow tracers such as [15O]water and [15O]butanol. The study shows that although [11C]ethanol may moderately contribute to the brain radioactivity distribution pattern of [11C]vinpocetine, the rapid degradation of [11C]ethanol makes it unlikely that the contribution of this metabolite is of importance. The distinct distribution patterns and kinetics of [11C]vinpocetine and [11C]ethanol also support the view, obtained from our previous observations, that vinpocetine may bind to specific sites in the monkey and human brain, especially in the thalamus.

In vivo biodistribution and pharmacokinetics of 18F-labeled human C-peptide: evaluation in monkeys using positron emission tomography

The recently observed beneficial effects exerted by C-peptide in insulin-dependent diabetes patients (IDDM) have instigated research into the mechanisms of C-peptide action as well as the location for it. Here we report in vivo biodistribution studies performed in monkeys using positron emission tomography (PET) and C-peptide labeled in the N-terminal with fluorine-18. Following iv injection of the radiotracer, dynamic decay data were collected over the chest and/or abdomens of the monkeys. The radioactivity distributed mainly to the kidneys, less to the heart and to some extent to the liver. Excretion of radioactivity into the urinary bladder was observed. Brain uptake was not detected in a static emission scan of the head performed at late times. Accumulation of radioactivity in the skeleton as a result of in vivo defluorination was not observed. Pharmacokinetic modeling of the regional concentrations of radioactivity over time resulted, for most organs, in two-compartment models. The organs with the highest radioactivity concentrations have been identified, enabling dose estimations for studies in humans with low or no C-peptide.