Robert R. Flavell

Expressed protein ligation (EPL) in the study of signal transduction, ion conduction, and chromatin biology.

Expressed protein ligation (EPL) is a semisynthetic technique in which a recombinant protein thioester, generated by thiolysis of an intein fusion protein, is reacted with a synthetic or recombinant peptide with an N-terminal cysteine to produce a native peptide bond. This method has been used extensively for the incorporation of biophysical probes, unnatural amino acids, and post-translational modifications in proteins. In the 10 years since this technique was developed, the applications of EPL to studying protein structure and function have grown ever more sophisticated. In this Account, we review the use of EPL in selected systems in which substantial mechanistic insights have recently been gained through the use of the semisynthetic protein derivatives. EPL has been used in many studies to unravel the complexity of signaling networks and subcellular trafficking. Herein, we highlight this application to two different systems. First, we describe how phosphorylated or otherwise modified proteins in the TGF-beta signaling network were prepared and how they were applied to understanding the complexities of this pathway, from receptor activation to nuclear import. Second, Rab-GTPases are multiply modified with lipid derivatives, and EPL-based techniques were used to incorporate these modifications, allowing for the elucidation of the biophysical basis of membrane association and dissociation. We also review the use of EPL to understand the biology of two other systems, the potassium channel KcsA and histones. EPL was used to incorporate d-alanine and an amide-to-ester backbone modification in the selectivity filter of the KcsA potassium channel, providing insight into the mechanism of selectivity in ion conduction. In the case of histones, which are among the most heavily post-translationally modified proteins, the modifications play a key role in the regulation of gene transcription and chromatin structure. We describe how native chemical ligation and EPL were used to generate acetylated, phosphorylated, methylated, and ubiquitylated histones and how these modified histones were used to interrogate chromatin biology. Collectively, these studies demonstrate the utility of EPL in protein science. These techniques and concepts are applicable to many other systems, and ongoing advances promise to extend this semisynthetic technique to increasingly complex biological problems.

Site-Specific 18F-Labeling of the Protein Hormone Leptin Using a General Two-Step Ligation Procedure

The protein hormone leptin acts to regulate body fat and energy expenditure. Resistance to this hormone is implicated in human obesity and its pathophysiological consequences. In order to gain insight into the mechanism of leptin resistance, an (18)F-labeled derivative was developed to study the biodistribution of the hormone using positron emission tomography (PET). A two-step, site specific ligation approach was developed for this purpose, in which an aminooxy-reactive group was incorporated at the C-terminus of leptin using expressed protein ligation (EPL), which was subsequently derivatized with [ (18)F]fluorobenzaldehyde using an aniline-accelerated radiochemical oximation reaction. The modified hormone was shown to be biologically active in vitro and in vivo, and it was applied to PET imaging in ob/ ob mice. These protocols will allow for the routine production of site-specifically (18)F radiolabeled leptin, as well as other proteins, for use in PET imaging in systems from mouse to man.

PET Imaging of Leptin Biodistribution and Metabolism in Rodents and Primates

We have determined the systemic biodistribution of the hormone leptin by PET imaging. PET imaging using (18)F- and (68)Ga-labeled leptin revealed that, in mouse, the hormone was rapidly taken up by megalin (gp330/LRP2), a multiligand endocytic receptor localized in renal tubules. In addition, in rhesus monkeys, 15% of labeled leptin localized to red bone marrow, which was consistent with hormone uptake in rodent tissues. These data confirm a megalin-dependent mechanism for renal uptake in vivo. The significant binding to immune cells and blood cell precursors in bone marrow is also consistent with prior evidence showing that leptin modulates immune function. These experiments set the stage for similar studies in humans to assess the extent to which alterations of leptins biodistribution might contribute to obesity; they also provide a general chemical strategy for (18)F labeling of proteins for PET imaging of other polypeptide hormones.

Caged [18F]FDG Glycosylamines for Imaging Acidic Tumor Microenvironments Using Positron Emission Tomography

Solid tumors are hypoxic with altered metabolism, resulting in secretion of acids into the extracellular matrix and lower relative pH, a feature associated with local invasion and metastasis. Therapeutic and diagnostic agents responsive to this microenvironment may improve tumor-specific delivery. Therefore, we pursued a general strategy whereby caged small-molecule drugs or imaging agents liberate their parent compounds in regions of low interstitial pH. In this manuscript, we present a new acid-labile prodrug method based on the glycosylamine linkage, and its application to a class of positron emission tomography (PET) imaging tracers, termed [(18)F]FDG amines. [(18)F]FDG amines operate via a proposed two-step mechanism, in which an acid-labile precursor decomposes to form the common radiotracer 2-deoxy-2-[(18)F]fluoro-d-glucose, which is subsequently accumulated by glucose avid cells. The rate of decomposition of [(18)F]FDG amines is tunable in a systematic fashion, tracking the pKa of the parent amine. In vivo, a 4-phenylbenzylamine [(18)F]FDG amine congener showed greater relative accumulation in tumors over benign tissue, which could be attenuated upon tumor alkalinization using previously validated models, including sodium bicarbonate treatment, or overexpression of carbonic anhydrase. This new class of PET tracer represents a viable approach for imaging acidic interstitial pH with potential for clinical translation.

[11C]Ascorbic and [11C]dehydroascorbic acid, an endogenous redox pair for sensing reactive oxygen species using positron emission tomography

Here we report the radiosynthesis of an endogenous redox pair, [(11)C]ascorbic acid ([(11)C]VitC) and [(11)C]dehydroascorbic acid ([(11)C]DHA), the reduced and oxidized forms of vitamin C, and their application to ROS sensing. These results provide the basis for in vivo detection of ROS using positron emission tomography (PET).

Dynamic nuclear polarization of biocompatible 13C-enriched carbonates for in vivo pH imaging

A hyperpolarization technique using carbonate precursors of biocompatible molecules was found to yield high concentrations of hyperpolarized (13)C bicarbonate in solution. This approach enabled large signal gains for low-toxicity hyperpolarized (13)C pH imaging in a phantom and in vivo in a murine model of prostate cancer.

Somatostatin receptor PET/MRI for the evaluation of neuroendocrine tumors

With the introduction of simultaneous PET/MRI scanners, concurrent acquisition of PET and MRI data is feasible, allowing for improved patient convenience and decreased radiation dose. Although PET/MRI has been used in many settings, not all cancers benefit from the combined modality. With the availability of somatostatin receptor-targeted PET tracers such as 68Ga-DOTA-TOC and 68Ga-DOTA-TATE, imaging of NET patients has refocused on targeted imaging, particularly with the development of peptide receptor radiotherapy. Nonetheless, there are many patients who continue to benefit from dedicated MR imaging, such as those with liver-predominant disease. In these patients, SSR PET/MRI is an important option for optimal imaging. Both diffusion-weighted imaging and hepatobiliary phase imaging provide improved lesion detection compared to conventional MRI and CT, and the results can effect therapeutic decisions. Additionally, the use of motion correction techniques can be used to leverage the additional PET data acquired in dedicated liver PET/MRI to remove respiratory artifacts.

Incidental Detection of Head and Neck Squamous Cell Carcinoma on 68Ga-PSMA-11 PET/CT

We present a case of an incidentally detected squamous cell carcinoma of the oropharynx on Ga-PSMA-11 PET. A 71-year-old mans condition was diagnosed as prostate carcinoma after a year of rising serum prostate-specific antigen. The staging Ga-PSMA PET/CT demonstrated focal radiotracer uptake in the prostate corresponding to his known primary prostate cancer. However, a PSMA-avid 3.4-cm mass was incidentally found in the right tongue base that was biopsied, confirming squamous cell carcinoma.

Detecting Pulmonary Nodules in Lung Cancer Patients Using Whole Body FDG PET/CT, High-resolution Lung Reformat of FDG PET/CT, or Diagnostic Breath Hold Chest CT

RATIONALE AND OBJECTIVES Pulmonary nodules can be missed on the non-breath hold computed tomography (CT) portion of 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT), and for this reason prior studies have advocated for routinely performing dedicated breath hold CT of the chest in addition to PET/CT for routine staging of malignancy. We evaluated the rate of pulmonary nodule detection on standard CT images from whole body PET/CT studies (WB-PET/CT), high-resolution lung reconstruction CT images from PET/CT studies (HR-PET/CT), and diagnostic breath hold chest CT (BH-CT). MATERIALS AND METHODS A cohort of 25 patients was identified who had a history of lung cancer as well as a PET/CT staging or restaging scan and BH-CT within 30 days of each other. All PET/CTs included a set of CT images using a soft tissue algorithm filter and 3.75- to 5-mm slice thickness, as well as high-resolution reformats with a sharp reconstruction filter and 2-mm slice thickness. The CT images from WB-PET/CT, HR-PET/CT, and BH-CT were reviewed by three radiologists. Significance was analyzed by two-way repeated measures analysis of variance. RESULTS There were 2.84 nodules found per patient with WB-PET/CT, 3.85 nodules with HR-PET/CT, and 3.91 nodules with BH-CT. When only nodules less than or equal to 8 mm in size were considered, WB-PET/CT also demonstrated significantly fewer nodules (1.98) compared to the HR-PET/CT (2.94) or a BH-CT (2.86) (P < 0.001). No difference in detection rate was noted between the two higher resolution modalities. CONCLUSIONS More pulmonary nodules are detected on the CT portion of PET/CT studies when high-resolution reformatted images are created and reviewed. The ability to detect nodules with the reformatted images was indistinguishable from dedicated BH-CT. Overall, high-resolution reformats of PET/CT images of the lungs can increase the sensitivity for pulmonary nodule detection, approaching that of dedicated BH-CT. These data suggest that if HR-PET/CT reformats are used, additional dedicated BH-CT is unnecessary for routine staging of lung cancer.

[11C]Para-Aminobenzoic Acid: A Positron Emission Tomography Tracer Targeting Bacteria-Specific Metabolism

Imaging studies are frequently used to support the clinical diagnosis of infection. These techniques include computed tomography (CT) and magnetic resonance imaging (MRI) for structural information and single photon emission computed tomography (SPECT) or positron emission tomography (PET) for metabolic data. However, frequently, there is significant overlap in the imaging appearance of infectious and noninfectious entities using these tools. To address this concern, recent approaches have targeted bacteria-specific metabolic pathways. For example, radiolabeled sugars derived from sorbitol and maltose have been investigated as PET radiotracers, since these are efficiently incorporated into bacteria but are poor substrates for mammalian cells. We have previously shown that para-aminobenzoic acid (PABA) is an excellent candidate for development as a bacteria-specific imaging tracer as it is rapidly accumulated by a wide range of pathogenic bacteria, including metabolically quiescent bacteria and clinical strains, but not by mammalian cells. Therefore, in this study, we developed an efficient radiosynthesis for [11C]PABA, investigated its accumulation into Escherichia coli and Staphylococcus aureus laboratory strains in vitro, and showed that it can distinguish between infection and sterile inflammation in a murine model of acute bacterial infection.