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Mapping Aldehyde Dehydrogenase 1A1 Activity Using an [18F]Substrate-Based Approach Raul Pereira,[A] Thibault Gendron,[B] Chandan Sanghera,[A] Hannah E

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Mapping Aldehyde Dehydrogenase 1A1 Activity Using an [18F]Substrate-Based Approach Raul Pereira,[A] Thibault Gendron,[B] Chandan Sanghera,[A] Hannah E CORE Metadata, citation and similar papers at core.ac.uk Provided by Birkbeck Institutional Research Online DOI: 10.1002/chem.201805473 Full Paper & Biochemistry Mapping Aldehyde Dehydrogenase 1A1 Activity using an [18F]Substrate-Based Approach Raul Pereira,[a] Thibault Gendron,[b] Chandan Sanghera,[a] Hannah E. Greenwood,[a] Joseph Newcombe,[b, c] Patrick N. McCormick,[a] Kerstin Sander,[b] Maya Topf,[c] Erik rstad,[b] and Timothy H. Witney*[a] Abstract: Aldehyde dehydrogenases (ALDHs) catalyze the focused library of compounds evaluated, N-ethyl-6-(fluoro)- oxidation of aldehydes to carboxylic acids. Elevated ALDH N-(4-formylbenzyl)nicotinamide 4b was found to have excel- expression in human cancers is linked to metastases and lent affinity and isozyme selectivity for ALDH1A1 in vitro. poor overall survival. Despite ALDH being a poor prognostic Following 18F-fluorination, [18F]4b was taken up by colorectal factor, the non-invasive assessment of ALDH activity in vivo tumor cells and trapped through the conversion to its 18F-la- has not been possible due to a lack of sensitive and transla- beled carboxylate product under the action of ALDH. In vivo tional imaging agents. Presented in this report are the syn- positron emission tomography revealed high uptake of thesis and biological evaluation of ALDH1A1-selective chemi- [18F]4b in the lungs and liver, with radioactivity cleared cal probes composed of an aromatic aldehyde derived from through the urinary tract. Oxidation of [18F]4b, however, was N,N-diethylamino benzaldehyde (DEAB) linked to a fluorinat- observed in vivo, which may limit the tissue penetration of ed pyridine ring either via an amide or amine linkage. Of the this first-in-class radiotracer. Introduction There are currently 20 known functional human ALDHs[2] that mediate the metabolism of aldehydes generated during oxida- Aldehyde dehydrogenases (ALDHs) are a family of enzymes tive stress,[3] amino acid and biogenic amine metabolism,[4] reti- that catalyze the NAD(P)+-dependent oxidation of a wide vari- noic acid biosynthesis,[5] and ethanol metabolism.[3a] In addi- ety of aldehydes to their corresponding carboxylic acids.[1] tion, ALDHs control the detoxification of exogenous reactive aldehydes and therapeutic drugs such as cyclophosphamide.[6] Aberrant expression of ALDH is associated with many diseases, [a] Dr. R. Pereira, C. Sanghera, H. E. Greenwood, Dr. P. N. McCormick, including cancer, with increased expression and activity of Dr. T. H. Witney Centre for Advanced Biomedical Imaging ALDH shown to be a predictor of metastatic potential and [7] University College London poor overall survival. In particular, the ALDH1A1 isozyme is a Paul O’Gorman Building, 72 Huntley Street well-characterized marker of cancer stem cells, which are London WC1E 6DD (UK), known for their tumor-initiating properties and resistance to and [8] Current address: Department of Imaging Chemistry & Biology conventional therapy. Studies have shown resistance to che- King’s College London, St. Thomas’ Hospital motherapy and poor prognosis is associated with high London SE1 7EH (UK) ALDH1A1 activity in breast,[9] ovarian,[10] prostate,[11] colon[12] E-mail: [email protected] and lung[13] cancer. As a consequence, ALDH1A1 has been se- [b] Dr. T. Gendron, J. Newcombe, Dr. K. Sander, Prof. Dr. E. rstad lected as a target for anti-cancer therapy, with ALDH inhibitors Department of Chemistry University College London shown to reverse chemoresistance in a range of preclinical [14] 20 Gordon Street tumor models. London WC1H 0AJ (UK) Given the causal link between ALDH expression and cancer [c] J. Newcombe, Prof. Dr. M. Topf drug resistance, the non-invasive identification of ALDH-ex- Department of Biological Sciences pressing tumors is of great clinical importance. The measure- Birkbeck, University of London Malet Street ment of chemoresistance through ALDH imaging could poten- London WC1E 7HX (UK) tially enable the clinician to select the most suitable therapeu- Supporting information and the ORCID identification number(s) for the au- tic intervention for the individual patient (e.g. chemotherapy thor(s) of this article can be found under: versus immunotherapy) with the possibility to improve out- https://doi.org/10.1002/chem.201805473. comes and reduce unnecessary treatment. Currently, the in 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. vitro assessment of ALDH activity has been restricted to fluo- This is an open access article under the terms of the Creative Commons At- [15] tribution License, which permits use, distribution and reproduction in any rescence-based assays. Despite these commercially available medium, provided the original work is properly cited. imaging agents being widely-adopted for the isolation of Chem. Eur. J. 2019, 25,1–8 1 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim && These are not the final page numbers! ÞÞ Full Paper ALDH-positive cells in cell culture, the poor tissue penetration was rapidly converted to the carboxylate product under the of the fluorescent signal currently limits their in vivo utility. In action of ALDH1A1 (Figure S1). In order to assess the enzyme order to circumvent these inherent limitations, we propose the kinetics of the compounds in this study, we examined the use of positron emission tomography (PET) as an alternative to effect of substrate concentration on the initial enzyme velocity fluorescence-based assays.[16] Previous attempts to develop using recombinant human ALDH1A1, ALDH2 and ALDH3A1 en- ALDH1A1-specific radiotracers have so far failed due to the zymes, which are commonly expressed in human cancer. poor cellular retention of the carboxylate product, presumed ALDH2 is expressed in the mitochondrial matrix and plays a to be a consequence of its high hydrophobicity.[17] Here, we critical role in alcohol metabolism,[19] whereas ALDH3A1 is lo- report the synthesis and biological evaluation of 18F-fluorinated calized in both the nucleus and cytosol and functions to detox- aldehyde-based probes for the non-invasive detection of ify aldehydes formed during UV-induced lipid peroxidation.[20] ALDH1A1 activity in tumor cell models. Furthermore, these three isoforms exhibit three different rate- limiting steps: ALDH1A1’s being the cofactor dissociation,[21] the deacylation-step for ALDH2,[22] and hydride transfer for Results and Discussion ALDH3A1.[23] ALDH1A1 chemical probes were designed to have a) an alde- We made a single-point modification to 2, with the addition hyde that can serve as a substrate for ALDH1A1; b) contain a of a fluorine to the aromatic ring—an essential requirement 18 (radio)fluorine atom that would allow for detection via gamma for F-radiofluorine-based radiotracers—to give compounds counting/PET imaging; c) a suitable hydrophobic-hydrophilic 3a and 3b. The lithium/bromine exchange on 11 followed by balance which would allow for passive diffusion in and out of quenching with DMF afforded aldehyde 12. Stirring 12 with cells, and importantly; d) subsequent trapping of the in situ NaBH(OAc)3 and diethylamine in DCE afforded the crude re- generated carboxylic acid product within the cytosol as a ductive amination product which was directly treated with result of the acquired negative charge (Figure 1A). We took a aqueous hydrochloric acid in THF to furnish 3a (Scheme 1A). Reduction of nitrile 13 with diisobutylaluminium hydride clean- ly produced aldehyde 14 which was thereafter stirred with excess diethylamine in THF to yield aldehyde 3b (Scheme 1B). Amide 16 was prepared by reacting the acid chloride of 5-fluo- ronicotinic acid 15 with amine B (Scheme 1C). The acid-cata- lyzed cleavage of the acetal furnished compound 4a. LiAlH4 re- duction of the amide bond in 16 followed by the acid-cata- lyzed acetal cleavage afforded amine 5 (Scheme 1C). In a simi- lar manner, the acid chloride of 6-fluronicotinic acid 16 was re- acted with amine A to yield amide 18 which following an acid- catalyzed acetal cleavage furnished aldehyde 4b. The sulfona- mide 6 was accessed by reacting the commercially available 5- Figure 1. A) Schematic illustrating ALDH-mediated trapping of 18F-labeled al- fluoropyridine-3-sulfonyl chloride 19 with amine A following dehydes by conversion to their corresponding acid. B) Chemical structures of DEAB (1), an ALDH1A1 inhibitor, and 2, an ALDH1A1 substrate. an acid-catalyzed acetal cleavage (Scheme 1E). The benzylic amine 2 showed a higher affinity (lower KM) and catalytic efficiency (Vmax/KM) for ALDH1A1 over both substrate-based approach for the imaging of ALDH1A1 to pro- ALDH2 and ALDH3A1 (Table 1, entry 1 and Figure 2B, respec- vide a functional readout of enzymatic activity. Substrate- tively), indicating that the isozyme selectivity of DEAB was based radiotracers provide an advantage over radiolabeled in- maintained. Interestingly, 3a and 3b exhibited lower affinity hibitors which only report on enzyme expression. Moreover, for ALDH1A1 than the non-fluorinated analogue 2, with a KM multiple substrate molecules can be turned over by a single of 0.28 Æ0.12 mm, 0.26Æ 0.08 mm and 0.16 Æ0.03 mm, respec- enzyme, thereby increasing the sensitivity of detection when tively (Table 1, entries 1–3). compared to radiolabeled inhibitors. Given that fluorination proximal to the aldehyde moiety de- The starting point for our small molecule probe develop- creased affinity for ALDH1A1, we next explored compounds ment was N,N-diethylaminobenzaldehyde (DEAB) 1, which is a with fluorine atoms that were remote from the aldehyde. Com- m [18] well-known inhibitor of ALDH1A1 (Ki values of 10–40 n ). pounds 4a and 4b exhibited a five-fold increase in ALDH1A1 Given the advantages of substrate-based radiotracers, our ini- affinity over 2 (Table 1, entries 1, 4 and 5). Furthermore, the tial goal was to convert 1 in to a substrate whilst maintaining enzyme efficiency for 4a was 7-fold higher for ALDH1A1 than suitable ALDH1A1 selectivity over other commonly expressed ALDH2, with the enzyme efficiency for ALDH1A1 over 20-fold ALDH isozymes.
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