Theranostic Near-Infrared Monoamine Oxidase Inhibitor (NMI) Protein Binding Interactions with MAOA and Albumin

Abstract

Purpose: The protein binding interactions of near-infrared monoamine oxidase inhibitor (NMI) are reported here.

Methods: NMI-bound proteins were examined by fluorescent SDS-PAGE and mass spectrometry using tumor tissues from brain and colon cancer mouse models.

Results: This study shows protein interactions with NMI, a chemical conjugate of MAOA inhibitor clorgyline and tumor-seeking dye, MHI-148. NMI fluorescence in MAOA knock-out (KO) mice was significantly lower compared to WT mice, including whole animal, organs, and tissue lysates which indicated that NMI binds to MAOA. Pure recombinant MAOA protein was detectable as a single fluorescent band that migrated at ~ 65kD. NMI inhibited MAOA activity (IC₅₀ 1-5 µM). In a glioma mouse model, NMI targeted specifically to tumor with high contrast to adjacent normal brain, shown by a 65 kD protein band. Recent studies demonstrated heptamethine cyanine dyes (e.g., MHI-148) interact with serum albumin, contributing to tumor uptake and cancer cell internalization. Our study shows NMI binds to albumin but highly prefers MAOA, providing a plausible mechanism for systemic drug delivery via serum albumin to the tumor target and subsequent MAOA inhibition. Further studies in a colon cancer mouse model found the ~ 65 kD SDS-PAGE band, bound to NMI, contained both MAOA and albumin proteins by mass spectrometry.

Conclusion: NMI was shown to interact with MAOA and the blood carrier protein, albumin. This study provides insights for drug delivery and protein target specificity of NMI to image and treat cancer.

Keywords: MAOA; albumin; colorectal cancer; glioblastoma; monoamine oxidase; theranostic.

Fig. 1

NMI was administered as single bolus dose (5 mg/kg) by intravenous (i.v.) route to C576BL/6 J (WT) and MAOA knock-out (KO) mice and imaged with IVIS system in near-infrared range to determine differences in uptake and biodistribution. (a) Whole animal in vivo comparison of NMI biodistribution in WT and KO mice at 1 h and 24 h. (b) Quantitation of total flux (photons/s) of total ventral (belly) fluorescent region of interest (ROI) at 1 h and 24 h (n = 3 per group). (c) Brain and liver organ comparison at 24 h. (d) Quantitation of total flux (photons/s) of organs ex vivo after 24 h (n = 3 per group). Brain and liver tissues were separated on SDS-PAGE gel and imaged with iBright. (e) Coomassie total protein stain and molecular size marker. (f) Near-infrared iBright image of gel showed major band at ~ 65 kD. (g) Black and white image of same gel.

Fig. 2

C576BL/6 J (WT) mice bearing intracranial glioma GL26 tumors were treated with NMI (5 mg/kg), normal adjacent brain and tumor samples were collected 1 week after a single dose. Protein lysates were separated by SDS-PAGE and imaged with iBright at NIR wavelength for analysis. (a) Coomassie total protein stain and molecular size marker. (b) Near-infrared iBright image of gel showed a major band at ~ 65 kD. Normal brain tissue surrounding the tumor (Lane 1) showed relatively no visible NMI band. Intracranially implanted GL26 glioblastoma tumor tissue (Lane 2) displayed a high-intensity NMI band. The fastest migrating band at gel front was attributed to ~ 1 kD representing the free NMI-amide molecule (formula weight 980.5) that separated from the protein sample during SDS-PAGE conditions. (c) Black and white image of same gel.

Fig. 3

Purified recombinant human MAOA (7 µg) was incubated with NMI (10 µM in PBS vs. PEG) at 37 ºC for 1 h. After 1 h, 5 µg protein was loaded on SDS-PAGE. (a) Near-infrared iBright image of gel showed major band at ~ 60–65 kD which demonstrated MAOA and NMI binding. (b) Black and white image of same gel. (c) Purified recombinant human MAOA glo activity assay inhibition by NMI in PEG formulation improved solubility and improved potency (IC₅₀ = 1.6 µM; black squares) of NMI in MAO glo activity assay compared to PBS formulation (IC₅₀ 5.87 µM; red circles). As a control, the irreversible selective MAOA inhibitor clorgyline (IC₅₀ 2.8 nM) was measured under the same conditions (not shown). The inhibitor potency of NMI was enhanced almost fourfold by improving NMI solubility in PEG-400 in aqueous reaction, mean IC₅₀ of 3–5 separate experiments, (p < 0.05). (d) Visual representation of NMI serial-diluted range of 1 × 10^–3 M to 1 × 10^–6 M NMI in PBS (top) compared to NMI in PEG-400 66% (bottom) working solutions that were then tenfold diluted in assay buffer for MAO glo assay.

Fig. 4

Albumin (3 µg in 24 µl) and NMI (1 µM) or MHI-148 (1 µM) were incubated for 5 min, 30 min, 1 h and 3 h at 37 ºC. After the incubation, 2 µg albumin protein was loaded on SDS-PAGE. Gel was imaged using iBright at NIR wavelength. (a) Near-infrared iBright image of gel showed major band at ~ 65 kD (monomer) and ~ 135 kD (dimer) with increased intensity at 3 h. (b) Black and white image of same gel. (c) The chemical structure of NMI (FW 980.55) and its NIR dye moiety MHI-148 (FW 764.23) and color photograph of their serial dilutions from a stock solution of 1 × 10^–2 M in DMSO to a range of working solutions, 2.5 × 10^–3 M to 1 × 10^–6 M in PBS.

Fig. 5

NMI (1 µM), MAOA (1 µM) and albumin (1 µM) were incubated in different combinations to understand the binding affinities between them. (a) Near-infrared iBright image of gel showed major band at ~ 65 kD which demonstrated NMI-protein binding interactions at NMI 1 µM. Lane 1 shows NMI and albumin incubated together for 24 h at 37 ºC, Lane 2 shows NMI, MAOA and albumin, all added at the same time and incubated for 24 h and lane 3 shows NMI and albumin pre-incubated for 30 min and then MAOA added to incubate for 24 h. (b) Black and white image of same gel. The amount of each protein added to the well is 1 µg. Gel was imaged using iBright at 750 nm (NIR wavelength) with exposure time 10 s for analysis.

Fig. 6

Tumor tissue lysates were prepared from mice bearing MC-38 subcutaneous colon cancer tumor treated with NMI (5 mg/kg, every other day; IP) for 21 d. Tumor tissue lysate (20 µg) was separated on SDS-PAGE gel. Gel was imaged using iBright at 750 nm (NIR wavelength) for exposure time 10 s for analysis. (a) Near-infrared iBright image of gel shows a major band at ~ 65 kD. (b) Coomassie stain gel image with band excised for LC/MS/MS protein identification.