Diamagnetic Imaging Agents with a Modular Chemical Design for Quantitative Detection of β‑Galactosidase and β‑Glucuronidase Activities with CatalyCEST MRI

Imaging agents for the noninvasive in vivo detection of enzyme activity in preclinical and clinical settings could have fundamental implications in the field of drug discovery. Furthermore, a new class of targeted prodrug treatments takes advantage of high enzyme activity to tailor therapy and improve treatment outcomes. Herein, we report the design and synthesis of new magnetic resonance imaging (MRI) agents that quantitatively detect β-galactosidase and β-glucuronidase activities by measuring changes in chemical exchange saturation transfer (CEST). Based on a modular approach, we incorporated the enzymes’ respective substrates to a salicylate moiety with a chromogenic spacer via a carbamate linkage. This furnished highly selective diamagnetic CEST agents that detected and quantified enzyme activities of glycoside hydrolase enzymes. Michaelis–Menten enzyme kinetics studies were performed by monitoring catalyCEST MRI signals, which were validated with UV–vis assays

Arsenite-induced phenotypic changes in BEAS-2B.

<p>A) Representative images of soft agar growth over the course of 52 weeks of constant arsenite (1 µM) exposure. B) Colony counts in soft agar. Bars represent mean, 1 standard deviation, from 3 experimental replicates. C) Immunoblot analysis of HIF-1A and E-cadherin (E-cad) in BEAS-2B over the course of 52 weeks of constant arsenite (1 µM) exposure. D) Lactate levels (percent control) in BEAS-2B over the course of 52 weeks of constant arsenite (1 µM) exposure. Absolute lactate production in vector control: 0.733±0.017 µmol/10⁶cells/hr) Bars represent mean +1 standard deviation, from 3 experimental replicates. E) Percentage aneuploid cells in BEAS-2B treated with 1 µM arsenite for 0–52 weeks. Bars represent mean, +1 standard deviation, from 3 experimental replicates. *p<0.05.</p

Effect of suppressed HIF-1A expression on arsenite mediated transformation.

<p>A) Immunoblot analysis of HIF-1A knockdown in BEAS-2B, short immunoblot exposure shown for MG132-treated samples; long immunoblot exposure shown for MG132-untreated samples. B) QPCR for HIF-1A mRNA. Bars represent mean, +1 standard deviation, from 5 experimental replicates. C) Lactate levels (percent control) in arsenite-exposed (denoted “As”, exposed for 8 weeks) and unexposed control (denoted “Ct”) BEAS-2B stably transfected with scrambled control shRNA (denoted “Vector”) or with shRNA targeting HIF1A (denoted “shHIF1A”) expression. Absolute lactate production in vector control: 0.696±0.04 µmol/10⁶cells/hr). Bars represent mean, +1 standard deviation, from 3 experimental replicates. D) Colony count of soft agar assay from BEAS-2B cells treated as described above in panel C. Bars represent mean, +1 standard deviation, from 3 experimental replicates. *p<0.05.</p

Arsenite causes HIF-1A accumulation/translocation in BEAS-2B.

<p>A) Immunoblot analysis of HIF-1A in BEAS-2B treated with 0–8 µM arsenite for 48 hours. B) Immunoblot analysis of HIF-1A in BEAS-2B treated with 1 µM arsenite for 0–48 hours. C) Immunoblot analysis of nuclear and cytosolic fractions of BEAS-2B, control or treated with 1 µM arsenite for 2 weeks, probed for HIF-1A, Lamin A (a nuclear marker) and tubulin (a cytosolic marker). D) Immunofluorescence staining of HIF-1A in BEAS-2B, control or treated with 1 µM arsenite for 2 weeks, arrows show HIF-1A nuclear accumulation. E) QPCR of HIF-1A mRNA in BEAS-2B treated with 1 µM arsenite for 0–4 weeks, bars represent mean, 1 standard deviation. F) Half-life measurement of HIF-1A in BEAS-2B, control or treated with 1 µM arsenite for 2 weeks, protein synthesis blocked with cycloheximide (CHX) for 0–10 min, followed by HIF-1A immunoblot. G) Quantification of HIF-1A protein half-life (t1/2). Densitometry of HIF-1A normalized to Tubulin was used for calculation. Points represent mean, +/− 1 standard deviation, 3 independent replicates. *p<0.05.</p

Glycolysis induction by HIF-1A overexpression in BEAS-2B.

<p>A) Immunoblot analysis of HIF-1A in BEAS-2B, vector control and transiently transfected with degradation-resistant HIF-1A mutant. B) Lactate levels (percent vector control) in cells described in 2A (Absolute lactate production in vector control: 0.729±0.054 µmol/10⁶cells/hr). Bars represent mean, 1 standard deviation, from 3 independent replicates. *p<0.05. C) Intracellular metabolite concentration (percent control BEAS-2B) of 1 µM arsenite-exposed (2 weeks) BEAS-2B cells. Bars represent mean, 1 standard deviation, from 4 experimental replicates. For each metabolite, levels in arsenite-exposed BEAS-2B are significantly different compared to control (p<0.05).</p