In this work, we develop recombinant human cationic ferritin (rHCF) as a contrast agent to detect glomeruli in the kidney using positron emission tomography (PET). We first expressed recombinant human ferritin (rHF) in E. coli and then functionalized and radiolabeled it with Copper-64 (64Cu) to form Cu-64-rHCF. Intravenously injected Cu-64-rHCF bound to kidney glomeruli and was detected by PET. A subchronic toxicity study after an intravenous injection of rHCF revealed no significant toxicity. The development of rHCF is an important step toward the potential clinical translation of CF to detect the nephron number in humans.

Cationic ferritin (CF) has been developed as a multimodal, targeted imaging tracer to directly detect and map nephrons in the kidney in vivo. Direct detection of functional nephrons provides a unique, sensitive biomarker to predict or monitor kidney disease progression. CF has been developed to map functional nephron number from magnetic resonance imaging (MRI) or positron emission tomography (PET). Previous preclinical imaging studies have used non-human-derived ferritin and commercial formulations that must still be developed for translation to clinical use. Here we describe the reproducible formulation of CF (either derived from horse or from human recombinant ferritin) optimized for intravenous injection and radiolabeling by PET. The human recombinant heteropolymer ferritin is spontaneously assembled in liquid culture (Escherichia coli, E. coli) and modified to form human recombinant cationic ferritin (HrCF) to mitigate potential immunologic reactions for use in humans.

Prior work employing functional analysis, photolabeling, and X-ray crystallography have identified three distinct binding sites for potentiating steroids in the heteromeric GABAA receptor. The sites are located in the membrane-spanning domains of the receptor at the β-α subunit interface (site I) and within the α (site II) and β subunits (site III). Here, we have investigated the effects of mutations to these sites on potentiation of the rat α1β2γ2L GABAA receptor by the endogenous neurosteroid allopregnanolone (3α5αP). The mutations were introduced alone or in combination to probe the additivity of effects. We show that the effects of amino acid substitutions in sites I and II are energetically additive, indicating independence of the actions of the two steroid binding sites. In site III, none of the mutations tested reduced potentiation by 3α5αP, nor did a mutation in site III modify the effects of mutations in sites I or II. We infer that the binding sites for 3α5αP act independently. The independence of steroid action at each site is supported by photolabeling data showing that mutations in either site I or site II selectively change steroid orientation in the mutated site without affecting labeling at the unmutated site. The findings are discussed in the context of linking energetic additivity to empirical changes in receptor function and ligand binding. SIGNIFICANCE STATEMENT: Prior work has identified three distinct binding sites for potentiating steroids in the heteromeric γ-aminobutyric acid type A receptor. This study shows that the sites act independently and additively in the presence of the steroid allopregnanolone and provide estimates of energetic contributions made by steroid binding to each site.

Aliphatic diazirine analogues of cholesterol have been used previously to elaborate the cholesterol proteome and identify cholesterol binding sites on proteins. Cholesterol analogues containing the trifluoromethylphenyl diazirine (TPD) group have not been reported. Both classes of diazirines have been prepared for neurosteroid photolabeling studies and their combined use provided information that was not obtainable with either diazirine class alone. Hence, we prepared cholesterol TPD analogues and used them along with previously reported aliphatic diazirine analogues as photoaffinity labeling reagents to obtain additional information on the cholesterol binding sites of the pentameric Gloeobacter ligand-gated ion channel (GLIC). We first validated the TPD analogues as cholesterol substitutes and compared their actions with those of previously reported aliphatic diazirines in cell culture assays. All the probes bound to the same cholesterol binding site on GLIC but with differences in photolabeling efficiencies and residues identified. Photolabeling of mammalian (HEK) cell membranes demonstrated differences in the pattern of proteins labeled by the two classes of probes. Collectively, these date indicate that cholesterol photoaffinity labeling reagents containing an aliphatic diazirine or TPD group provide complementary information and will both be useful tools in future studies of cholesterol biology.

This study examines how site-specific binding to three identified neurosteroid-binding sites in the α1β3 GABAA receptor (GABAAR) contributes to neurosteroid allosteric modulation. We found that the potentiating neurosteroid, allopregnanolone, but not its inhibitory 3β-epimer epi-allopregnanolone, binds to the canonical β3(+)–α1(-) intersubunit site that mediates receptor activation by neurosteroids. In contrast, both allopregnanolone and epi-allopregnanolone bind to intrasubunit sites in the β3 subunit, promoting receptor desensitization and the α1 subunit promoting effects that vary between neurosteroids. Two neurosteroid analogues with diazirine moieties replacing the 3-hydroxyl (KK148 and KK150) bind to all three sites, but do not potentiate GABAAR currents. KK148 is a desensitizing agent, whereas KK150 is devoid of allosteric activity. These compounds provide potential chemical scaffolds for neurosteroid antagonists. Collectively, these data show that differential occupancy and efficacy at three discrete neurosteroid-binding sites determine whether a neurosteroid has potentiating, inhibitory, or competitive antagonist activity on GABAARs.

Nephron number varies widely in humans. A low nephron endowment at birth or a loss of functioning nephrons is strongly linked to increased susceptibility to chronic kidney disease. In this work, we developed a contrast agent, radiolabeled cationic ferritin (RadioCF), to map functioning glomeruli in vivo in the kidney using positron emission tomography (PET). PET radiotracers can be detected in trace doses (<30 nmol), making them useful for rapid clinical translation. RadioCF is formed from cationic ferritin (CF) and with a radioisotope, Cu-64, incorporated into the ferritin core. We showed that RadioCF binds specifically to kidney glomeruli after intravenous injection in mice, whereas radiolabeled noncationic ferritin (RadioNF) and free Cu-64 do not. We then showed that RadioCF-PET can distinguish kidneys in healthy wild-type (WT) mice from kidneys in mice with oligosyndactylism (Os/+), a model of congenital hypoplasia and low nephron mass. The average standardized uptake value (SUV) measured by PET 90 min after injection was 21% higher in WT mice than in Os/+ mice, consistent with the higher glomerular density in WT mice. The difference in peak SUV from SUV at 90 min correlated with glomerular density in male mice from both WT and Os/+ cohorts (R-2 = 0.98). Finally, we used RadioCF-PET to map functioning glomeruli in a donated human kidney. SUV within the kidney correlated with glomerular number (R-2= 0.78) measured by CF-enhanced magnetic resonance imaging in the same locations. This work suggests that RadioCF-PET appears to accurately detect nephron mass and has the potential for clinical translation.

The ρ1 GABAA receptor is prominently expressed in the retina and is present at lower levels in several brain regions and other tissues. Although the ρ1 receptor is insensitive to many anesthetic drugs that modulate the heteromeric GABAA receptor, it maintains a rich and multifaceted steroid pharmacology. The receptor is negatively modulated by 5β-reduced steroids, sulfated or carboxylated steroids, and β-estradiol, whereas many 5α-reduced steroids potentiate the receptor. In this study, we analyzed modulation of the human ρ1 GABAA receptor by several neurosteroids, individually and in combination, in the framework of the coagonist concerted transition model. Experiments involving coapplication of two or more steroids revealed that the receptor contains at least three classes of distinct, nonoverlapping sites for steroids, one each for the inhibitory steroids pregnanolone (3α5βP), 3α5βP sulfate, and β-estradiol. The site for 3α5βP can accommodate the potentiating steroid 5αTHDOC. The findings are discussed with respect to receptor modulation by combinations of endogenous neurosteroids. SIGNIFICANCE STATEMENT The study describes modulation of the ρ1 GABAA receptor by neurosteroids. The coagonist concerted transition model was used to determine overlap of binding sites for several inhibitory and potentiating steroids.

348 Objectives: Head and neck squamous cell carcinoma (HNSCC) accounts for ca. 4% of malignancy worldwide. Although it is curable in the early stages, the survival rate of patients with advanced cancer has not significantly improved. Hence, the current unmet clinical need is to develop diagnostic tools to detect the disease at the early stages. The previous studies revealed the upregulation of chemokine receptor 2 (CCR2) in HNSCC. In this study, we assessed the performance of CCR2 PET detecting HNSCC in patient-derived xenograft (PDX) mouse models and human tissues and translated this radiotracer in humans. Methods: The novel CCR2-targeted PET tracer, 64Cu-DOTA-ECL1i, was employed for PET imaging. Multiple-time-point PET imaging was performed in HNSCC PDX models in order to assess the correlation between tracer uptake and HNSCC progression. Pathophysiological analysis and autoradiography studies of human HNSCC tissues were carried out to confirm CCR2 upregulation and binding of 64Cu-DOTA-ECL1i to human CCR2. Competitive receptor blocking studies were conducted to verify CCR2 specific binding of the radiotracer. 64Cu-DOTA-ECL1i PET/CT in healthy volunteers demonstrated acceptable radiation dosimetry. PET/CT imaging in head and neck cancer patients is underway. Results: Tumor uptake was gradually increased in HNSCC PDX mice from 0.87 ± 0.14 %ID/g at 9 weeks post tumor implantation to 1.70 ± 0.35 %ID/g at 13 weeks. Competitive receptor blocking showed significantly decreased tumor uptake (p < 0.005), indicating the targeting specificity of the tracer. IHC showed CCR2 overexpression in PDX tumors and human HNSCC tissues. Autoradiography studies demonstrated the binding of 64Cu-DOTA-ECL1i to human CCR2. Human radiation dosimetry in healthy volunteers showed 0.064 ± 0.011 rem/mCi of gender average effective dose (0.07 rem/mCi for females and 0.0575 rem/mCi for males). The dose-limiting organ is the urinary bladder wall due to rapid renal clearance. PET healthy volunteers showed minimal tracer uptake in the head and neck area. Conclusions: CCR2 is a promising imaging biomarker for human HNSCC imaging. The sensitivity and specificity of 64Cu-DOTA-ECL1i warrant further investigation in HNSCC patients.

Nanoparticles have been widely used for preclinical cancer imaging. However, their successful clinical translation is largely hampered by potential toxicity, unsatisfactory detection of malignancy at early stages, inaccurate diagnosis of tumor biomarkers, and histology for imaging-guided treatment. Herein, a targeted copper nanocluster (CuNC) is reported with high potential to address these challenges for future translation. Its ultrasmall structure enables efficient renal/bowel clearance, minimized off-target effects in nontargeted organs, and low nonspecific tumor retention. The pH-dependent in vivo dissolution of CuNCs affords minimal toxicity and potentially selective drug delivery to tumors. The intrinsic radiolabeling through the direct addition of 64Cu to CuNC (64Cu-CuNCs-FC131) synthesis offers high specific activity for sensitive and accurate detection of CXCR4 via FC131-directed targeting in novel triple negative breast cancer (TNBC) patient-derived xenograft mouse models and human TNBC tissues. In summary, this study not only reveals the potential of CXCR4-targeted 64Cu-CuNCs for TNBC imaging in clinical settings, but also provides a useful strategy to design and assess the translational potential of nanoparticles for cancer theranostics.

Neurosteroids positively modulate GABA-A receptor (GABAAR) channel activity by binding to a transmembrane domain intersubunit site. Understanding the interactions in this site that determine neurosteroid binding and its effect is essential for the design of neurosteroid-based therapeutics. Using photo-affinity labeling and an ELIC-α1GABAAR chimera, we investigated the impact of mutations (Q242L, Q242W and W246L) within the intersubunit site on neurosteroid binding. These mutations, which abolish the thermal stabilizing effect of allopregnanolone on the chimera, reduce neither photolabeling within the intersubunit site nor competitive prevention of labeling by allopregnanolone. Instead, these mutations change the orientation of neurosteroid photolabeling. Molecular docking of allopregnanolone in WT and Q242W receptors confirms that the mutation favors re-orientation of allopregnanolone within the binding pocket. Collectively, the data indicate that mutations at Gln242 or Trp246 that eliminate neurosteroid effects do not eliminate neurosteroid binding within the intersubunit site, but significantly alter the preferred orientation of the neurosteroid within the site. The interactions formed by Gln242 and Trp246 within this pocket play a vital role in determining the orientation of the neurosteroid that may be necessary for its functional effect.

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action.

1149 Introduction: Targeted PET imaging of chemokine receptor 2 in head and neck cancer and progression Objectives: The overall mortality rates of head and neck squamous cell carcinoma (HNSCC) have not significantly improved over the past several decades. A significant reason is the lack of tools, especially at the molecular level, to detect disease at early stages and to reliably assess presence of regional and/or distant metastasis. The expression of chemokine receptor 2 (CCR2) is increased in HNSCC. The goal of this study was to determine if changes in CCR2 expression with HNSCC progression could be imaged in patient derived xenografts (PDXs) tumor models with a novel targeted PET tracer, 64Cu-DOTA-ECL1i. Methods: The CCR2 binding peptide, ECL1i, was conjugated to a DOTA chelator and radiolabeled with 64Cu. In vivo pharmacokinetic evaluation was performed in wild-type C57 mice. PET imaging was carried out in HNSCC PDX models at multiple time points during progression of the malignancy. The imaging specificity was confirmed by competitive receptor blocking studies. The expression of CCR2 in PDX tumors was characterized by immunohistochemistry (IHC). De-identified human HNSCC tissues were also used to confirm the up-regulation of CCR2 and test the binding of 64Cu-DOTA-ECL1i to this receptor. Results: The integrity of DOTA-ECL1i was characterized with mass spectrometry. The high radiolabeling specific activity (1.04 mCi/nmol) led to tracer amount (10-100 pmol) administration for in vivo studies. Biodistribution of 64Cu-DOTA-ECL1i showed fast renal clearance with low accumulation in the liver. PET imaging in HNSCC PDX mice showed gradual increase in tumor uptake from 0.87 ± 0.14 %ID/g (n=8) at 9 weeks post tumor implantation to 1.70 ± 0.35 %ID/g (n=5) at 13 weeks. Competitive receptor blocking significantly decreased tumor uptake to 0.63 ± 0.08 %ID/g (n=3, p < 0.005), suggesting the targeting specificity. IHC of PDX tumors showed up-regulation of CCR2 in tumor cells, which was also confirmed in human HNSCC specimens. Autoradiography of 64Cu-DOTA-ECL1i in human HNSCC tumors demonstrated binding specificity and a similar profile to the immunostaining, indicating CCR2-mediated uptake. Conclusions: CCR2 was identified as a biomarker to target HNSCC in both PDX models and human malignant tissues. 64Cu-DOTA-ECL1i demonstrated high CCR2 imaging sensitivity and specificity in PDX tumor models. Its binding capability to human tissue indicated the great potential for future clinical translation.

There is a growing demand for diagnostic procedures including in vivo tumor imaging. Radiometal-based imaging agents are advantageous for tumor imaging because radiometals (i) have a wide range of half-lives and (ii) are easily incorporated into imaging probes via a mild, rapid chelation event with a bifunctional chelator (BFC). Microfluidic platforms hold promise for synthesis of radiotracers because they can easily handle minute volumes, reduce consumption of expensive reagents, and minimize personnel exposure to radioactive compounds. Here we demonstrate the use of a "click chip" with an immobilized Cu(I) catalyst to facilitate the "click chemistry" conjugation of BFCs to biomolecules (BMs); a key step in the synthesis of radiometal-based imaging probes. The "click chip" was used to synthesize three different BM-BFC conjugates with minimal amounts of copper present in reaction solutions (∼20 ppm), which reduces or obviates the need for a copper removal step. These initial results are promising for future endeavors of synthesizing radiometal-based imaging agents completely on chip.

89Zr-labeled antibodies are being investigated in several clinical trials; however, the time requirement for synthesis of clinical doses can hinder patient throughput because of scheduling difficulties. Additionally, low specific activity due to poor labeling efficiency can require larger amounts of the radiopharmaceutical to be administered, possibly leading to adverse side effects. Here, we describe the design and evaluation of a microfluidic reactor capable of synthesizing a single clinical dose of 89Zr-labeled antibody. 89Zr-labeled trastuzumab was chosen for this validation because it is currently being evaluated in clinical trials for imaging human epidermal growth factor receptor 2–positive cancer patients. Methods: A microreactor fabricated from polydimethylsiloxane/glass was silanated with trimethoxy(octadecyl) silane to reduce antibody adsorption. Desferrioxamine-p-benzyl-isothiocyanate (DFO-Bz-NCS) was conjugated to trastuzumab in an 8:1 molar ratio following the literature procedures using aseptic techniques. Radiolabeling was performed by pumping 89Zr-oxalate and DFO-Bz-trastuzumab into the microfluidic reactor at a total rate of 20 μL/min in ratios varying from 1:37 to 1:592 mg:MBq at 37°C to achieve optimal labeling. Results: Silanated reactors showed low antibody adsorption in comparison to unmodified reactors (95% monoclonal antibody recovered vs. 0% recovered). Labeling of the modified trastuzumab was shown to be achievable at a specific activity above the reported literature value of 220 MBq/mg. A high radiochemical purity was achieved without an incubation period at specific activities of less than 148 MBq/mg; however, specific activities up to 592 MBq/mg could be achieved with an incubation period. Clinical doses were able to be prepared and passed all quality control guidelines set by the Food and Drug Administration. Samples were sterile, colorless, and radiochemically pure (100%); maintained the ability to bind to the intended receptor; formed a minimal amount of aggregates (1%–4%); and were completed within 45–60 min. Conclusion: 89Zr-labeled trastuzumab for use in a clinical setting was synthesized in a microfluidic reactor in under an hour while reducing the amount of handling required by a technician. Use of this compact platform not only could enable the use of radiolabeled antibodies to become a common practice, but also could spread the use of radiolabeled antibodies beyond locations with cyclotron facilities.

Ketamine is a psychotomimetic and antidepressant drug. Although antagonism of cell-surface NMDA receptors (NMDARs) may trigger ketamine’s psychoactive effects, ketamine or its major metabolite norketamine could act intracellularly to produce some behavioral effects. To explore the viability of this latter hypothesis, we examined intracellular accumulation of novel visualizable analogues of ketamine/norketamine. We introduced an alkyne “click” handle into norketamine (alkyne-norketamine, A-NK) at the key nitrogen atom. Ketamine, norketamine, and A-NK, but not A-NK-amide, showed acute and persisting psychoactive effects in mice. This psychoactivity profile paralleled activity of the compounds as NMDAR channel blockers; A-NK-amide was inactive at NMDARs, and norketamine and A-NK were active but ~4-fold less potent than ketamine. We incubated rat hippocampal cells with 10 μM A-NK or A-NK-amide then performed Cu 2+ catalyzed cycloaddition of azide-Alexa Fluor 488, which covalently attaches the fluorophore to the alkyne moiety in the compounds. Fluorescent imaging revealed intracellular localization of A-NK but weak A-NK-amide labeling. Accumulation was not dependent on membrane potential, NMDAR expression, or NMDAR activity. Overall, the approach revealed a correlation among NMDAR activity, intracellular accumulation/retention, and behavioral effects. Thus, we advance first generation chemical biology tools to aid in the identification of ketamine targets.