A novel PET probe to selectively image heat shock protein 90α/β isoforms in the brain

doi:10.21203/rs.3.rs-3601041/v1

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Research Article

A novel PET probe to selectively image heat shock protein 90α/β isoforms in the brain

https://doi.org/10.21203/rs.3.rs-3601041/v1

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published 04 Mar, 2024

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Background

Heat shock proteins (HSPs), ubiquitously found proteins called molecular chaperones in the brain, play important functional roles in the neuropathological processes of Alzheimer’s disease and Lewy body disease. Of many HSP subtypes, HSP90 has been shown to play a central role in the process. Many studies have shown that drugs that inhibit HSP90 activity have beneficial effects in the neurodegenerative diseases. Therefore, HSP90 PET imaging ligand, an in vivo biomarker of HSP90, can be used effectively to study HSP90 in neurodegenerative diseases. Among four HSP90 isoforms, two cytosolic isoforms (HSP90α and HSP90β) thought to be involved in the structural homeostasis of the proteins related to the neurodegenerative diseases. Currently, no useful PET imaging ligands selectively targeting the two cytosolic isoforms of HSP90 have been available yet.

Results

In this study, we developed a novel PET imaging ligand, [¹¹C]NCGG801, selectively targeting HSP90α and HSP90β by radiolabeling BIIB021, an inhibitor with a high affinity for and selectivity to HSP90α and HSP90β. [¹¹C]NCGG801 was synthesized with a high yield, molar activity and radiochemical purity. [¹¹C]NCGG801 showed a high binding affinity for rat brain homogenate as well as human recombinant HSP90α and HSP90β proteins. This radioligand was well taken up into the rat brain (SUV 1.4) and showed clear specific binding in PET imaging of healthy rats and autoradiography of healthy rat and human brain sections.

Conclusions

Our data suggested that [¹¹C]NCGG801 has a potential to be a new imaging biomarker of HSP90α/β in the brain. Further studies appear warranted to evaluate [¹¹C]NCGG801 in humans.

HSP90

heat shock protein

positron emission tomography

brain

Neurodegenerative diseases are one of the health care challenges in the aging society. Neuropathological features in the two major neurodegenerative diseases, Alzheimer's disease (AD) and Lewy body disease (LBD) are characterized by aggregation of abnormal proteins, including both amyloid-β (senile plaques) and phosphorylated tau (neurofibrillary tangles) in AD, and α-synuclein (Lewy bodies) in LBD. Recently it has been found that ubiquitously found proteins in the brain, called molecular chaperones, play important functional roles in these neuropathological processes.

Molecular chaperones are proteins that assist the conformational folding or unfolding of large proteins or macromolecular protein complexes. There are many families of molecular chaperones depending on the way they assist in folding macro proteins. For example, one family called heat shock proteins (HSPs) become highly expressed in the experimentally induced heat stress. When tissue cells are exposed to stresses such as heat shock, oxidative stress, and inflammation, regenerated intracellular proteins may become structurally misfolded and accumulated as abnormal proteins. Molecular chaperones such as heat shock proteins are activated by these stresses and form a protein complex by combination of chaperones and other proteins (chaperome) to enhance their functions [1]. In response to stressful conditions, chaperomes can either restore or decompose misfolded proteins, acting as the first line of defense against stress [2]. Due to its high metabolic demands, the central nervous system (CNS) is exposed to high stresses including the oxidative stress. For example, CNS is particularly vulnerable to the effects of aging. One of aging effects is an imbalance between chaperon and neuronal activity levels, resulting in an inability to restore/decompose misfolded proteins and subsequently leading to toxic protein accumulation and the development of neurodegenerative diseases. [3] [4].

Of many HSP subtypes, HSP90 has been shown to play a central role in the pathophysiology of neurodegenerative disease. Recently, HSP90 has become therapeutic targets for neurodegenerative diseases, because it is involved in the structural homeostasis of proteins including amyloid-β, tau, and α-synuclein [5]. HSP90 inhibition studies have shown that the amount of tau aggregates and α-synuclein-induced toxicity are reduced in AD and LBD model rats, respectively [6] [7]. In the stressed brain, the expression of HSP90 is expected to be increased. However, HSP90 levels in vitro appear complex and inconsistent between AD and LBD brains. In AD brains, HSP90 levels are paradoxically decreased by ~ 20–30%, whereas in LBD brains, HSP90 levels are increased [8][9]. The reason(s) for these discrepant in vitro findings is currently not well understood. However, considering the potential beneficial effects of drugs that inhibit HSP90 activity in these neurodegenerative diseases, the availability of in vivo biomarkers of HSP90, for example, HSP90 PET imaging ligands, may allow us to track changes in HSP90 levels in these diseases and they could also be an additional ligand to the arsenal currently used for pathophysiological studies of neurodegenerative diseases in general.

In fact, several Hsp90-targeting PET imaging ligands have already been reported [10] [11] [12] [13] (Fig. 1). These ligands have been developed based on the known HSP90 inhibitors. HSP90 has four isoforms: two isoforms (HSP90α and HSP90β) in the cytosol, one (TRAP1) in the mitochondria, and one (GRP94) in the endoplasmic reticulum [14]. The cytosolic isoforms are fold nascent polypeptide and thought to be involved in the structural homeostasis of amyloid-β, tau, and α-synuclein [5] [15]. Currently available PET ligands bind to all three subtypes of HSP90 isoforms except for [¹¹C]SNX-ab [16]. However this ligand failed to show sufficient brain uptake in mice [16].

In the current study, we report a novel PET imaging ligand [¹¹C]NCGG801 (Scheme 1) to selectively measure HSP90α/β in the brain of patients with neurodegenerative diseases. As a lead compound for the synthesis of HSP90α/β-selective PET imaging ligand, we focused on a different HSP90α/β specific inhibitor than one used for the previously described study [16], namely BIIB021 (NCGG801, Scheme 1), a moderately selective purine-based compound (K_i = 2 nM for HSP90α, 4 nM for HSP90β, 176 nM for GRP94, and 62 nM for TRAP1) [17]. NCGG801 has a methoxy group on the pyridine ring, allowing ¹¹C-labeling at this site. When calculated using ChemDraw® Professional version 16.0.1.4(77), [¹¹C]NCGG801 has a molecular weight of 319 and a polar surface area of 92 Å, considered to be suitable for brain PET imaging. NCGG801 is expected to have good brain uptake, considering its structure with two hydrogen donors, seven hydrogen acceptors, and a clogP of 1.9. Considering these features, we decided to synthesize an ¹¹C-labeled PET imaging ligand selective to HSP90α/β, [¹¹C]NCGG801, and evaluate its usefulness in vitro and in vivo.

Chemicals.

Methanol were HPLC grade from Merck (Darmstadt, Germany). Citric acid monohydrate and sodium acetate from Kanto Chemical (Tokyo, Japan), ethylenediaminetetraacetic acid (EDTA) from Dojindo (Kumamoto, Japan), and Sodium octane sulfonate (SOS) from FUJIFILM Wako Pure Chemical (Osaka, Japan). All other chemicals were of analytical grade and were used without any further pretreatment.

General synthetic procedure

All commercially available reagents and solvents were used without further purification. Normal-phase thin layer chromatography (TLC) was carried out on Silica gel 60 F254 (Merck, 1.05715.0009) using reagent grade solvents. TLC was detected by the absorption of UV light (254 nm) or using a visualization reagent (molybdophosphoric acid). Column chromatography was performed on silica gel (Silica Gel 60, Kanto Chemical co., Inc. Tokyo, Japan) with mixed solvents as described. ¹H and ¹³C NMR spectra were obtained for samples in the indicated solution at 25°C utilizing the JNM-ECA500 spectrometer (JEOL. Ltd. Tokyo, Japan) at 500 MHz frequency for ¹H or the JNM-AL400 spectrometer at 400 MHz frequency for ¹H in CD₃OD or deuterated dimethylsulfoxide (DMSO)-d₆ with tetramethylsilane as an internal standard. ¹H NMR chemical shifts are reported in terms of the chemical shift (δ, ppm) relative to the singlet corresponding to tetramethylsilane at 0 ppm. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Coupling constants are reported in Hz. ¹³C NMR spectra were fully decoupled and are reported in terms of the chemical shift (δ, ppm) relative to a septet at δ = 39.5 ppm corresponding to DMSO-d₆ or a septet at δ = 49.0 ppm corresponding to CD₃OD. Electrospray ionization-mass (ESI)-mass spectrometry were carried out on LC-MS spectrometer (LCMS2020, Shimadzu Industrial Systems Co., Ltd.).

2-(chloromethyl)-3,5-dimethylpyridin-4-ol (2)

2-(Chloromethyl)-4-methoxy-3,5-dimethylpyridine Hydrochloride (1 ,1.11 g, 5 mmol) in toluene (0.9 mL) was stirred and refluxed under Ar. After 19 hours, the reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure to give 2-(chloromethyl)-3,5-dimethylpyridin − 4-ol (2) was obtained in 465 mg (54%). LRMS (ESI+): m/z [M + H]⁺: 186.

6-Chloro-9-(4-methoxy-3,5-dimethylpyridin-2-ylmethyl)-9 H -purin-2-ylamine (NCGG801) [18].

A suspension of 2-(Chloromethyl)-4-methoxy-3,5-dimethylpyridine Hydrochloride (4, 169.6 mg, 1.0 mmol), potassium carbonate (276 mg, 387 mmol), sodium iodide (15.0 mg, 0.1 mmol), and compound 2 (222.1 g, 1.0 mmol) in DMF (5 mL) was heated at 40°C with stirring under Ar. After 15 h, the reaction mixture was cooled, and the inorganic solids were filtered and washed with DMF. Dilution with 10 mL of water induced the crystallization of the desired isomer 1 (NCGG801, 199.4 mg, 63%). Rf 0.20 (EtOAc); ¹H NMR (DMSO-d₆) δ = 8.09 (s, 1H), 8.03 (s, 1H), 6.85 (s, 2H), 5.37 (s, 2H), 3.75 (s, 3H), 2.31 (s, 3H), 2.17 (s, 3H).

2-(2-Amino-6-chloropurin-9-ylmethyl)-3,5-dimethylpyridin-4-ol (Dm-NCGG801) [18].

A suspension of 2-amino-6-chloropurine 4 (459.5 mg, 2.71 mmol), potassium carbonate (1.12 g, 8.13 mmol), sodium iodide(40.6 mg, 0.27 mmol), and compound 3 (464.8 mg, 2.71 mmol) in DMF (5 mL) was heated at 40°C with stirring under Ar. After 14 h, the reaction mixture was cooled, and the inorganic solids were filtered and washed with DMF. Dilution with 10 mL of water induced the crystallization of the desired isomer 5 (362.4 mg, 44%). ¹H NMR (400 MHz, DMSO-d₆) δ = 10.96 (s, 1H), 8.10 (s, 1H), 7.52 (s, 1H), 6.94 (s, 2H), 5.26 (s, 2H), 2.04 (s, 3H), 1.90 (s, 3H).

Preparation of [ ¹¹ C]NCGG801.

Radioactive ¹¹C was generated by the ¹⁴N (p, α) ¹¹C nuclear reaction using the cyclotron. ¹¹C-methylation of 5 to [¹¹C]NCGG801, HPLC purification and formulation were achieved automatically using specially designed equipment (Sumitomo Heavy Industries, Co. Ltd, Japan) The [¹¹C]iodomethane obtained was trapped in 250 µL of anhydrous DMF containing 0.5–5 mg of 5 (1.64–16.4 µmol) and 10 mg of potassium carbonate (2.3 µmol) at -15 ℃ to -20 ℃, and then the reaction mixture was heated to 100 ℃ for 10 min. The radioactive mixture containing [¹¹C]NCGG801 was diluted with 1 mL of HPLC mobile phase, and transferred onto a column (10 mm I.D. × 250 mm, CAPCELL PAK C₁₈, SHISEIDO, Tokyo, Japan) attached to the JACSO HPLC system. Elution with 20:80 v/v acetonitrile /2 M ammonium formate (in sterile water) at a flow rate of 5 mL/min gave a radioactive fraction corresponding to pure [¹¹C]NCGG801 (Supplementary Figure S1, retention time: 7.6 min). The fraction was collected in a rotary evaporator with 25% ascorbic acid solution 50 µL and evaporated to dryness at about 75 ℃ under reduced pressure. The residue was dissolved in 3 mL of sterile tween-saline, and filtered through a 0.22 µm Millex^®-GV filter (Merck, Darmstadt, Germany). At the end of synthesis, 1.43–2.94 GBq of [¹¹C]NCGG801 was obtained with the molar activity of 52.6–144.1 GBq/µmol. The calculation of the molar activity of [¹¹C]NCGG801 was performed using the HPLC calibration curve shown in SupplementaryFigureS3.

Animals.

F344/NSlc rats (male, 8.3 week old, Japan SLC, Inc., Hamamatsu, Japan) and wild type TgF344-19 rat (male, 14 month old, obtained from Rat Resource and Research Center, MO, USA and bred inhouse) [19] were used for the current study. The animals used here were maintained and handled in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals and our institutional guidelines. Protocols for the present animal experiments were approved by the Animal Ethics Committee of the National Center for Gelotology and Geriatrics.

Binding Assays of rat brain homogenate

Frozen brain powder derived from wild type TgF344-19 rat (14-month-old) were homogenized in 50 mM Tris-HCl buffer, pH 7.4 (at 25°C), containing protease inhibitor cocktail (cOmplete™, EDTA-free; Roche). The homogenate were incubated with 0.3–40 nM [¹¹C]NCGG801 (molar activity: 144.1 GBq/µmol) in the absence or presence of unlabelled NCGG801 at 10 µM in Tris-HCl buffer, pH 7.4 (at 4°C), for 30 min at 30°C. Non-specific binding of [¹¹C]NCGG801 was determined in the presence of 10 µM BIIB021. The dissociation constant (K_D) was estimated with a one-site total binding model performed using GraphPad Prism (version 9 for Mac, GraphPad Software, www.graphpad.com).

Binding Assays of human recombinant HSP90α and β protein

Human recombinant HSP90α and β protein with a His-tag (20 pmol BPS Bioscience, USA) were separately mixed with µMACS Anti-His Tag MicroBeads (50 µL, Miltenyi Biotec, USA) and incubated at 0°C for 30 min (total volume was 400 µL). Then, the mixture was incubated with 0.35–45 nM [¹¹C]NCGG801 (molar activity: 99.4 or 102.8 GBq/µmol, respectively) with or without unlabeled NCGG801 in the reaction buffer (20 mM HEPES, pH 7.4, 5 mM MgCl₂, 50 mM KCl, 20 mM Na₂MoO₄, 500 µL) at 30°C for 30 min. Each reaction solution was applied to µMACS columns (Multi-8 columns, Miltenyi Biotec, USA) placed in a magnetic field of the MultiMACS M96 Separator (Miltenyi Biotec, USA) and washed three times with the buffer (100 µL x 2, 500 µL x 1). Then, the buffer (500 µL) was applied to the µMACS columns placed outside the magnetic field. Radioactivity of the elution was measured with a gamma counter (AccuFLEX γ7000, ALOKA, Japan). The free concentration of NCGG801 was corrected to account for ligand depletion based on the radioactivity in the flow-through liquid after applying the reaction solution to the columns. K_D values were calculated in the same manner as for the rat brain homogenate binding assay.

Off-target Protein Binding Assays

Off-target protein binding screens were conducted by Sekisui Medical Inc. Binding inhibition effects of 10 µM NCGG801 were evaluated in competitive radioligand assays against 60 commonly expressed proteins including neurotransmitter receptors, ion channels, and transporters in the brain. Inhibition ratio (%) for each protein was calculated following the reported method (Okamura et al., 2013. J Nucl Med.)

PET imaging

To investigate in vivo specific binding of [¹¹C]NCGG801, a baseline and blocking PET study was performed on two F344/NSlc rats (male, 8.3 weeks old, 167 and 169 g). Rats were scanned for 120 min on the small animal PET scanner (FX3200, TriFoil Imaging, CA, USA) after the injection of [¹¹C]NCGG801 (14.5 and 14.6 MBq, equivalent to 0.195 and 0.196 nmol) via the tail vein under the isoflurane anesthesia (~ 2.0%). BIIB021 (1.0 mg/kg) were intravenously injected to a rat as a blocking agent 30 min before the injection of [¹¹C]NCGG801. All PET images were reconstructed with the three-dimensional ordered subset expectation maximization method (4 subsets and 20 iterations; voxel size: 0.6 × 0.5 × 0.5 mm with the resolution of 0.92 mm full width at half maximum at the center of view). Time-activity curves were generated using data extracted from PET images. A template of preset volumes of interest was applied to the PET images to extract time-activity curves for the following ROIs: whole brain, amygdala, striatum, cingulate, thalamus, hippocampus, and cerebellum. All image analyses were performed using PMOD version 4.3 (PMOD Technologies Ltd., Zurich, Switzerland)

Autoradiography

Wild-type F344Tg rat (14-month-old) brains were removed rapidly and then frozen immediately in powdered dry ice. Postmortem healthy human brains were obtained from autopsies carried out at the Brain Research Institute, Niigata University on a subject with colon cancer with liver metastases but without any significant brain pathologies. In vitro autoradiography was performed using 20-µm thick fresh frozen sagittal sections for rat whole brain and 7-µm-thick for the frontal section of the human subject. Sections were first pre-incubated only in bovine serum for 30 min and then incubated in bovine serum containing [¹¹C]NCGG801 (15.7 nM for rat samples and 5.1 nM for human samples) at 30℃ for 30 min without or with BIIB021 (10 µM) as a blocking agent. The samples were then rinsed with the serum (4°C) twice for 2 min each, and dipped into water (4°C) for 10 sec. The sections were subsequently dried by treating with cold air and were then exposed to an imaging plate (BAS-SR2040, Fuji Film, Tokyo, Japan). The imaging plate was scanned with a Typhoon FLA9500 (GE Healthcare, IL, USA) to acquire autoradiograms.

Statistics

Results are expressed as the mean ± standard deviation of experiments conducted multiple times. Multiple group comparisons were performed by one-way analysis of variance with Dunnet’s post hoc tests. Statistical analyses were performed utilizing Prism v9 software (GraphPad Software Inc., La Jolla, CA). Differences were considered significant at p values < 0.05.

Synthesis

The synthesis of the standard and precursor compounds was conducted based on the method of Kasibhatlaet et al [18] (Scheme 1). The standard compound was obtained in a moderate yield (78%) by the S_N2 reaction where methoxypyridine 1 was coupled with 2-amino-6-chloropurine. The precursor compound was synthesized in two steps. First, methoxypyridine 1 was demethylated to afford hydroxypyridine 2 in a moderate yield (38༅). Then the precursor compound Dm-NCGG801 was obtained in a moderate yield (44༅) by the same SN2 reaction as the standard compound.

[¹¹C]NCGG801 was synthesized by reacting Dm-NCGG801 with [¹¹C]iodomethane under a basic condition (Scheme 2). Initially, we found that [¹¹C]NCGG801 was partially radiolytically degraded in the post-preparative HPLC process, and radiochemical purity was about 72%. Therefore, we decided to add 50 µL of a 25% aq ascorbic acid solution as a radical scavenger before removing the fraction at reduced pressure. The radiochemical purity was improved to > 99% (0.33% yield, molar activity of 113.1 ± 34.3 GBq/µmol at the end of radiosyntheses, n = 6). The chemical identity of [¹¹C]NCGG801 was confirmed to have the identical HPLC retention time with that of the standard compound (Supplementary Fig. 2).

Binding Assays of rat brain homogenate and human recombinant HSP90α and β protein

To evaluate the binding affinity of [¹¹C]NCGG801, a homologous binding assay was performed using brain homogenate of a healthy rat and human recombinant HSP90α and HSP90β protein. The curve fitting of the total binding gave a stable estimation of K_D value of 2.1 nM for rat brain homogenates (Fig. 2A). The stable estimated K_D values were 2.0 nM for HSP90α and 15 nM for HSP90β, indicating that [¹¹C]NCGG801 preferably targets HSP90α compared to HSP90β (Fig. 2B, C). The previously reported affinity measurements were K_i = 2 nM for HSP90α, 4 nM for HSP90β, 176 nM for GRP94, and 62 nM for TRAP1 [17]. Thus, NCGG801 appears to selectively bind to HSP90α and HSP90β isoforms.

To evaluate the possibility of off-target protein binding of NCGG801, we studied 60 other commonly expressed proteins in the brain. Of these, melatonin receptor 1 showed the highest inhibition rate at 25.71% inhibition at 10 µM NCGG801(Supplementary Table S1). Thus, NCGG801 does not appeasers to bind other protein than HSP90α and HSP90β.

PET imaging

PET imaging was conducted in two healthy rats after intravenous administration of [¹¹C]NCGG801 in baseline and blocking experiments with pre-administration of the standard compound (2 mg/kg, NCGG801). In the baseline condition, brain radioactivity peaked in 4 minutes (SUV 1.4), followed by a slow washout (Fig. 3). Radioactivity was the highest in the thalamus and relatively low in the cerebellum, amygdala, and hippocampus. In the blocking condition, the time-activity curves showed a faster washout and smaller regional differences indicating the presence of specific binding in the whole brain.

Autoradiography

Autoradiography was performed on brain sections of a healthy rat and human (Fig. 4). Radioactivity accumulated in the brain with higher activity in the gray matter and lower activity in the white matter of the rat brain section and human frontal section. That radioactivity accumulation was blocked with addition of non-labeled NCGG801 indicating the presence of specific binding in the healthy rat and human brains.

In this study, we developed a novel PET imaging ligand, [¹¹C]NCGG801, selectively targeting HSP90α and HSP90β by radiolabeling BIIB021, an inhibitor with a high affinity for and selectivity to HSP90α and β. [¹¹C]NCGG801 was synthesized with a high yield, molar activity and radiochemical purity. [¹¹C]NCGG801 showed a high binding affinity for rat brain homogenate as well as human recombinant HSP90α and HSP90β proteins. This radioligand was well taken up into the rat brain (SUV 1.4) and showed clear specific binding in PET imaging of healthy rats and autoradiography of healthy rat and human brain sections.

Radiosynthesis

We synthesized [¹¹C]NCGG801 according to the method of Kasibhatlaet et al. [16]. The standard and precursor compounds, NCGG801 and Dm-NCGG801 respectively, were obtained in a moderately high yield as reported previously [16]. Then, we successfully synthesized [¹¹C]NCGG801 by [11C]methylation of the phenolic hydroxyl group in Dm-NCGG801 under basic conditions by adding potassium carbonate, and stabilyzed with the addition of a radical scavenger to prevent radiolysis.

In vitro and vivo studies

In the binding assays, we confirmed the high binding affinity of [¹¹C]NCGG801 for HSP90α and HSP90β in binding assays, showing the K_D values of 2.0 nM for human recombinant HSP90α and 15 nM for HSP90β. The higher binding affinity of this ligand for HSP90α than HSP90β makes [¹¹C]NCGG801 advantageous for PET imaging because it is the HSP90α that increases more significantly by stress than does HSP90β [20]. Although we did not confirm this ligand’s affinity for other HSP90 isoforms in this study, other studies suggested that this ligand does not have any significant affinity for other HSP90 isoforms [17]. Additionally, we did confirm that this ligand does not have any significantly high affinity for other commonly present proteins in the brain.

In the autoradiography, we confirmed specific binding of this tracer in both rat and human brain sections with its uptake distribution consistent with the known distribution of HSP90α, where this protein is mainly expressed in the gray matter but also in the white matter to a lesser extent [21].

In the PET imaging, we observed specific binding in the rat brain. However, gray/white matter distinction in the rat brain was unclear due to limited spatial resolution of our small animal PET system. In human PET studies, considering this ligand’s good brain uptake, we expect to visualize regional distribution of HSP90α/β more.

We developed a novel PET imaging ligand [¹¹C]NCGG801 selectively targeting HSP90α/β. We have been successful in synthesizing [¹¹C]NCGG801 and in vitro imaging HSP90. Our data suggest that [¹¹C]NCGG801 has the potential to be a new imaging biomarker of HSP90α/β in the brain. Further studies appear warranted to evaluate [¹¹C]NCGG801 in humans.

AD: Alzheimer's disease

B max : Available receptor site concentration

CNS: Central nervous system

HPLC: High performance liquid chromatography

HSPs: Heat shock proteins

I.D.: Internal diameter

K_D : Equilibrium dissociation constant

LBD: Lewy body disease

NMR: Nuclear magnetic resonance

PET: Positron emission tomography

ppm: Parts per million

SUV: Standardized uptake value

UV: Ultraviolet

v/v : Volume per volume

Availability of data and materials

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests

Funding

This work was supported in part by JSPS KAKENHI Grant Number JP22K15874), and the Collaborative Research Project (22503) of the Brain Research Institute, Niigata University, Niigata, Japan, and Research Funding for Longevity Sciences (21–43) from National Center for Geriatrics and Gerontology, Obu, Japan.

Authors' contributions

For this manuscript, the 16 authors contributed profoundly. TS, AO and YK conceived and designed the study. TS, JA and HI synthesized the ligand; TS, TY, SH and YK acquired the data. TS and YK quantified the data. TS, HI, AO, SI and YK analyzed and interpreted the data; TS and YK wrote the manuscript; MI, SI, MT, AK, HK, MS, TK and KI critically edited and revised the manuscript. All authors read and approved the final manuscript.

Acknowledgment

We thank Akihiko Nishikimi (NCGG) and Yasuo Imai (NCGG) for the management of radiation protection, Noboru Ogiso (NCGG) and his team for the assistance in animal health and care, and Atsushi Watanabe (NCGG) for the management of common equipment.

Authors' information

¹Department of Clinical and Experimental Neuroimaging, Center for Development of Advanced Medicine for Dementia, National Center for Geriatrics and Gerontology (NCGG), Obu, Japan. ²Department of Pharmacy, Faculty of Pharmacy, Gifu University of Medical Science (GUMS), Kani, Japan. ³Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan. ⁴Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan.

Ethics declarations

Ethics approval

The protocols for the present animal experiments were approved by the Animal Ethics Committee of the National Center for Gelotology and Geriatrics. The study was carried out in compliance with the ARRIVE guidelines. The postmortem study was approved by the institutional review board of both Niigata University School of Medicine, Niigata, Japan and the National Center for Geriatrics and Gerontology, Obu, Japan.

Consent to participate

Written informed consent for autopsy including the use of tissue for research purposes was obtained from the next of kin.

Consent for publication

Not applicable

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Scheme 1 and 2 are available in the Supplementary Files section.

NCGG801SI20231110.docx
scheme1.tif
Scheme 1. Synthesis of standard (NCGG801) and precursor (Dm-NCGG801) compounds. (a) 2-amino-6-chloropurine, K₂CO₃, NaI, DMF, 40 °C, overnight; (b) Toluene, reflux, 19 h; (c) 2-amino-6-chloropurine, K₂CO₃, NaI, DMF, 40 °C, overnight.
scheme2.tif
Scheme 2. Radiosynthesis of [¹¹C]NCGG801. Dm-NCGG801 was methylated with [¹¹C]CH₃I with K₂CO₃ in DMF at 100 °C for 10 min.

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published 04 Mar, 2024

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Editorial decision: Major revision
11 Dec, 2023
Reviewers invited by journal
27 Nov, 2023
Reviewers agreed at journal
15 Nov, 2023
Editor assigned by journal
13 Nov, 2023
First submitted to journal
13 Nov, 2023

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A novel PET probe to selectively image heat shock protein 90α/β isoforms in the brain

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Methods

2-(chloromethyl)-3,5-dimethylpyridin-4-ol (2)

Binding Assays of rat brain homogenate

Binding Assays of human recombinant HSP90α and β protein

Off-target Protein Binding Assays

PET imaging

Autoradiography

Statistics

Results

Synthesis

Binding Assays of rat brain homogenate and human recombinant HSP90α and β protein

PET imaging

Autoradiography

Discussion

Radiosynthesis

Conclusion

Abbreviations

Declarations

References

Scheme

Supplementary Files

Status:

Journal Publication

Version 1