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. 2025 Jul 4;4(1):64–78. doi: 10.1021/cbmi.5c00053

Natural Selection-Guided ACE2-Targeted Molecular Imaging: A New Paradigm for PET Tracer Development

Rou Li †, Jie Li ‡, Shuai Xue §, Danni Li †, Zixin Yang ‡, Tao Wang †,*, Wangxi Hai ∥,*, Xiao Li §,‡,*
PMCID: PMC12848671  PMID: 41613763

Abstract

Angiotensin-converting enzyme 2 (ACE2) has been identified as a biomarker and a promising therapeutic target in several diseases. The noninvasive visualization of in vivo ACE2 mapping is urgent for disease guidance and treatment assessment. A radioimmuno assay method adapting to the RBD peptide library was proposed here to screen a high ACE2-specific peptide sequence as the targeting molecule. Derived from the constructed RBD peptide libraries of the dominant Omicron variants (BA.1, BA.2, and BA.5), the superior peptide was high-throughput screened through the binding rate to the HEK293-hACE2 cell and verified by the molecular docking with ACE2. Further, biodistribution studies were conducted through 125I-based SPECT imaging. The DOTA-modified derivant was labeled with Ga-68 to enable ACE2-targeted PET imaging. The peptide 505HQPYRVVVLSFELLH519 (named as Omi-X) showed a superior ACE2 binding via molecular docking and cellular assays. 125I-labeled Omi-X SPECT imaging demonstrated the high ACE2-specificity and binding retention in K18-hACE2 mice, together with an ideal performance in labeling stability and flexibility. The ACE2-targeted PET imaging tracers68Ga-DOTA-Omi-X realized the ACE2 mapping and further applicability in RAAS-related diseasescardiac hypertrophy, intuitively reflecting the ACE2 expression and regulating role. This natural evolution-guided approach not only enabled a noninvasive visualization of ACE2 but also established a paradigm for developing targeted therapies by leveraging viral-host adaptation mechanisms. Our work bridged natural evolution and target molecule screening and offered imaging tools for RAAS pathophysiology, investigation, and precision diagnostics.

Keywords: natural evolution, ACE2, molecular imaging, RBD peptide library, renin-angiotensin-aldosterone system

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Introduction

Angiotensin converting enzyme 2 (ACE2) has re-entered the stage as a chief regulatory protein and a potential therapeutic target in the remodeling processes of numerous diseases, especially following the outbreak of coronavirus disease 2019 (COVID-19). − ACE2 takes effect in physiological regulation, including glycometabolism and lipid metabolism, and pathological remodeling of diseases, particularly in terms of counter-regulation of the overactivation of local and systemic renin-angiotensin-aldosterone system (RAAS). − Thus, ACE2 has been identified as a key regulator in COVID-19 and its sequelae, cardiovascular disorders, diabetes, hypertension, cancer, obesity, and other diseases. − The counter-regulatory RAAS axis, the ACE2-Ang (1–7)-MasR axis, counteracts the effect of the ACE-Ang II-AT1R axis and has been found to reverse organ damage in several diseases. , However, the relationship between ACE2 and RAAS (including anti-RAAS drugs) is not simply linear, rather complex, dynamic, and multidimensional, and it is commonly confounded by age, BMI, and underlying diseases. Therefore, organ- or lesion-specific ACE2 mapping is useful for exploring RAAS-related disease progression and guiding disease treatment options, which will especially benefit from a timely, noninvasive, and whole-body molecular-imaging solution.

Molecular imaging with radio-nuclide-labeled agent targeting ACE2 using PET or SPECT can meet the aforementioned needs, especially for the identification of the regulation mode of the RAAS system under pathophysiological conditions. , Hopefully, in our previous research, it has been preliminarily demonstrated that ACE2-targeting PET molecular imaging, based on the known ACE2 inhibitor DX600 as a ligand, could achieve the visualization and quantification of ACE2 both in animal models and in humans. , Even though existing ACE2-targeted PET agents have been initially implemented in monitoring some diseases, including cancer and COVID-19, some issues need to be addressed. , For DX600-derived tracers, a high binding rate to blood ACE2 and the consequently slow blood clearance yield a relatively low target-to-background imaging ratio and hence limit the translation to clinical practice.

A screening tracer that is highly targeted and can avoid interference with the body’s immune system and enzyme system to prolong the retention is the core of targeted molecular imaging. The ongoing mutations of SARS-CoV-2, particularly the predominant Omicron variants, resulted in a huge boost in improving the targeting specificity of the axis between the binding molecule-ACE2 of the host cell. , After continuous propagation and replication processes, Omicron has accumulated a large number of mutations, and 15 of 30 mutation sites are positioned in the receptor binding domain (RBD). , The RBD is located at the termini of the ectodomain of the spike glycoprotein that specifically recognizes and binds to the host receptor ACE2. , Mutations have resulted in a nearly 10-fold increase in binding affinity and specificity to ACE2 compared to the original strain of the novel coronavirus. − Widely known, derived from BA.1, the sublineages BA.2 and BA.5 have stronger immune-evasion capabilities and thus resist clearance by the body’s immune system. − Benefiting from this evolution, the naturally selected Omicron variants with a higher ACE2 binding affinity along with enhanced immune-evasion capabilities provide impressive natural resources for screening ACE2-targeting molecules. The critical and core part of the viral RBD that interacts with the ACE2 protein is an excellent candidate for developing ACE2-binding inhibitors and molecular imaging tracers.

Here, leveraging natural evolution and subsequent screening based on RBD-ACE2 interactions, we constructed RBD peptide libraries of representative Omicron strains (sublineages BA.1, BA.2, and BA.5) to screen the sequence with superiority in ACE2-binding. We also aimed to develop novel ACE2-specific PET radiotracers. By establishing RAAS-related disease models with cardiac hypertrophy and depending on PET scans at multiple time points, we assessed the feasibility of monitoring the RAAS status and variation via molecular imaging. This novel design holds great potential in screening naturally evolved biomolecules and developing targeting imaging tracers.

Materials and Methods

Chemicals and Consumable Items

Sodium l-ascorbate and acetonitrile (CH3CN) were bought from AcroSeal. The [125I]­NaI solution was purchased from Shanghai Xinke Pharmaceutical Co., Ltd. Reversed-phase extraction C18 Sep-Pak cartridges were acquired from Waters Co., Ltd. and pretreated with acetonitrile and water before use. The syringe filter (0.22 μm) was obtained from Sinopharm Chemical Reagent Co., Ltd. The glass microfiber chromatography paper impregnated with silica gel (iTLC-SG) was purchased from Agilent Technologies. The glass tube with 100 μg 1,3,4,6-tetra chloro-3α,6α-diphenyl glycouril (Iodogen) coated at the bottom was purchased from Nice-labeling Co., Ltd.

Construction of Peptide Libraries

Three peptide libraries, respectively, based on the reported amino acid sequence of receptor binding domain of Omicron sublineages BA.1, BA.2, and BA.5 were generated by Shanghai QYAOBIO Biotechnology Co., Ltd. ,− Each peptide library included 36 peptides to fully cover the RBD sequence, for 108 peptides in total, theoretically. Specifically, each peptide comprised 15 amino acids that overlapped with nine amino acids with the end of the previous peptide sequence, as well as the beginning of the following one. − Moreover, to ensure that the terminal peptide was covering 15 amino acids in length, it had 11 amino acids overlapping with the previous peptide. After the exclusion of a repeated sequence without mutations, 52 peptides derived from BA.1, BA.2, and BA.5 were built to screen the core sequence of ACE2-binding.

Radio-Iodination of Peptides and Quality Control

For the customized sequence derived from the Omicron variants, each peptide contained 15 amino acids, among which nine amino acids separately duplicated the prior and subsequent peptides. Notably, for the customized sequence without tyrosine, an extra one was modified to the N-terminus as the iodinating site.

Phenolic hydroxyl groups of tyrosine in each peptide provided the 125I-labeling site. A classical iodination protocol using Iodogen as a catalyst was utilized in 125I-labeling. In detail, 10 μL of peptide solution at 0.5 mM was added to the Iodogen-coated glass tube and then 10 μL of 0.9% saline containing 3.7 MBq Na125I was added in the tube. The mixture was gently vibrated at 37 °C for 10 min.

The mixture was removed from the tube to end the reaction. One microliter was dropped on the one side of iTLC-SG, which was then analyzed via thin-layer chromatography with saline as the mobile phase. The migration on the plate was recorded with a mini-Scan TLC (B-MS-1000F) equipped with a radiation detector (Flow-Count, Eckert & Ziegler). The radio-TLC spectrum was used to characterize the labeling efficiency, where 125I– moved to the front end and the labeled peptide remained at the starting point.

Cellular Level Screening

High-throughput screening of key peptides highly targeting ACE2 at the cellular level was performed by a radioimmunoassay utilizing 125I-labeled peptide derived from RBD. HEK293T-ACE2 cells stably expressing hACE2 (HEK293-hACE2) purchased from Shanghai Heyuan Biotechnology Co., Ltd. were utilized for the screening of target peptides. In order to measure the target binding efficiency of peptides, all 125I-labeled peptides (1 μg in 10 μL 0.01 M PBS for each tube) were, respectively, placed into a centrifuge tube with HEK293-hACE2 cells (at a density of 2 × 106 cells/mL, 0.5 mL), gently vortexed, and then cocultured at 4 °C for 120 min (three repetitions per set). Cells remained in full contact with the iodinated peptide by being placed on a rotary shaker (OM12101287, LICHEN Technology) that was maintained at 20 rpm. The supernatants were collected and cells were washed three times with 0.01 M cold PBS. The radioactivity count of 125I determined using a γ-counter (GC-2101γ, Beijing Pet Technology Co., Ltd.) in the supernatant and in cells were, respectively, recorded to calculate the ACE2-binding rate of each iodinated peptide. Under the same conditions, the ACE2-binding rate of DX600 was determined as a reference.

Molecular Docking Simulation

Molecular docking studies were carried out using AutoDock software (AutoDock 4.2) to clarify the spatial information on the ligand-target protein binding. According to the results of cellular high-throughput screening, the peptides with a high affinity to HEK293-hACE2 cells were preliminarily screened out as candidates. AutoDock 4.2 was utilized to simulate the binding mode of candidate peptides to ACE2 proteins; meanwhile, a molecular docking simulation of the known ACE2 inhibitor DX600 that we previously utilized in designing a radiotracer was performed as a comparison.

The sequence, structure, and functional information on human ACE2 protein (ACE2_HUMAN, protein data bank (PDB) ID: 1R42) were obtained from the Protein Data Bank. First, the candidate peptides were converted to a protein data bank (PDB) format using the online web server Simplified Molecular Input Line Entry System (SMILES) tool, and molecular docking was then conducted using the Lamarckian genetic algorithm in AutoDock v 4.2. Docking simulations were analyzed between ACE2 protein and candidate peptides or DX600 by comparing the AutoDock-offered automated docking capabilities to predict the binding site and the binding affinity (ΔG). Comprehensively considering the results at the cellular and molecular levels, the superior peptides with a higher binding affinity to ACE2 were selected (named Omi-X hereafter).

Biosafety of Omicron-Derived Peptide

The cell-division rate was assessed using Carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) Cell Proliferation Assay and Tracking Kit (Beyotime Biotechnology) according to the manufacturer’s protocol. Briefly, 5 × 106 cells were incubated with 2 mL of CFDA SE in a 15 mL centrifuge tube for 15 min at 37 °C. Then, 10 mL of a medium containing 10% FBS was added to the tube, and the cells were centrifuged at 1000 g for 5 min. After repeating this process twice, approximately 1 × 105 HEK293-hACE2 cells were placed in a 3.5 cm Petri dish and treated with Omi-X (three concentrations: the low dose was 0.544 μM (1 μg/mL), the medium dose was 5.441 μM (10 μg/mL)), and the high dose was 54.410 μM (100 μg/mL) for 36 h. A fluorescence microscope (Nexcope, China) was used to observe and capture fluorescence images. The samples were then analyzed using flow cytometry (NovoCyte 2060R, Agilent), and the data were analyzed with NovoExpress 1.6.2.

The HEK293-hACE2 cells were seeded in a 3.5 cm Petri dish and incubated overnight. Cultured cells were treated with Omi-Xs (three concentrations: the low dose was 0.544 μM (1 μg/mL), the medium dose was 5.441 μM (10 μg/mL), and the high dose was 54.410 μM (100 μg/mL)) for another 36 h and were harvested using trypsin, washed in PBS, and fixed in 75% ethanol at 4 °C overnight. The fixed cells were then centrifuged to remove ethanol and washed with PBS twice. Cells were resuspended in 0.5 mL of a staining solution containing 25 μL of propidium iodide (20×) and 10 μL of RNase A (50×) and then incubated for 30 min at room temperature. The samples were analyzed using flow cytometry (NovoCyte 2060R, Agilent), and the data were analyzed with NovoExpress 1.6.2.

All animal experimental protocols for this research were approved and guided by the Ethics Committee of Shanghai Changhai Hospital (Approval No.: 2023-MS-DS-08). K18-hACE2 mice (humanized ACE2 mice driven by the human keratin K18 promoter, Suzhou Cyagen Biosciences Inc., male, 4 months old, n = 10) were intraperitoneally injected with the screened peptide at a dosage of 10 μg/day, for 5 days in total. The serum was separated from the blood at 4 °C and 3000 g for 20 min for electrochemiluminescence (SQ120, Meso Scale Discovery) analysis using SULFO-TAGTM (K15048D-X, Meso Scale Discovery) as the labeling marker. IFN-γ, IL-10, IL-13, IL-1β, IL-4, IL-5, IL-6, KC/GRO, and TNF-α were the primary immune-related data.

Chemical Stability of Omicron-Derived Peptide

To evaluate the chemical stability, we dissolved Omi-X in diluted HCl (pH = 2) at 1 mg/mL, and then stored the resolution at 37 °C to mimic an in vivo acid environment for a period of up to 24 h. The chemical purity was assessed with analytical HPLC (IdealChrom 910, Elay-tech) equipped with a Kromasil 100-5C18 column with the parameters as follows: mobile phase A, acetonitrile with 0.1% TFA; mobile phase B, water with 0.1% TFA; gradient, 65-40-0% B over 0-20-20.1 min; flow rate: 1 mL/min; column temperature, room temperature.

In Vivo ACE2-Specific Distribution of Omi-X

K18-hACE2 mice and C57BL/6 mice (Suzhou Cyagen Biosciences Inc., male, 4 months old, n = 5) were anesthetized with isoflurane (1.5%–2% isoflurane induction, 1%–1.5% maintenance) before imaging, and then each mouse was administered 3.28 ± 0.42 MBq of 125I-Omi-X via the lateral tail vein. SPECT images were acquired at 1 and 6 h postinjection using a SPECT/CT scanner (γ-CUBE & X-CUBE, Molecubes, Belgium). SPECT images (energy peak, 35 keV; algorithm of reconstruction, Maximum Likelihood Expectation Maximization; isometric voxel size, 500 μm) were acquired for 28 min, and CT images were subsequently acquired (tube voltage: 50 kV; tube current: 500 μA; exposure time, 300 ms; algorithm of reconstruction, iterative; isometric voxel size, 200 μm).

Image analysis was conducted with VivoQuant software (Invicro) to obtain hybrid SPECT images in the transverse, corona, and sagittal planes. The organ-specific uptake (counts/mm3) of the heart, lungs, liver, and gastrointestinal tract was quantified via the drawing region of interest manually in a three-dimensional view.

Preparation of Omi-X-Derived Precursor and Characterization

For the selected Omi-X with a higher affinity to ACE2, the chemical modification was conducted to suit the radio-labeling. 1,4,7,10-tetraazacyclododecane-N,N′,N,N′-tetraacetic acid (DOTA) was conjugated to the N-terminal to supply a chelator for 68Ga-labeling via a chelating reaction.

The purity of Omi-X and its derivatives was assessed by HPLC and electrospray mass spectrometry (EMS). For the LC–MS performed on a Waters ZQ 2000 equipped with Kromasil 100-5C18 (4.6 mm × 250 mm, 5 μm), the HPLC detection conditions were as follows: mobile phase A, acetonitrile with 0.1% TFA; mobile phase B, water with 0.1% TFA; gradient, 65-40-0% B over 0-20-20.1 min; flow rate, 1 mL/min; column temperature, 50 °C. The EMS conditions were as follows: capillary voltage, ± (2500–3000) V; dissolvent flow rate, 800 L/h; desolvation temperature, 450 °C; cone voltage, 15–30 V; and run time, 1 min.

Synthesis and Characterization of 68Ga-DOTA-Omi-X

In order to develop a PET tracer with an easier synthesis protocol, the generator-eluted Ga-68 and macrocyclic chelating agent DOTA were chosen for the Omi-X-derived ACE2 tracer. Ga-68 labeling to DOTA-Omi-X was carried out in a mixture (pH = 4) of 100 μL 1 M NaOAc buffer and 1.5 mL 68Ga3+ elution in 0.05 M HCl, and the reaction system was heated to 100 °C and maintained for 10 min.

The radiochemical purity (RCP) of 68Ga-DOTA-Omi-X was analyzed via thin-layer chromatography. For radio-TLC, a mixture of 1 M NH4OAc/methyl alcohol (1:1) was the mobile phase, and iTLC-SG was the stationary phase. The retardation factor (R f) of 68Ga3+ was 0–0.1, and the Rf of 68Ga-DOTA-Omi-X was 0.75–1. 68Ga-DOTA-Omi-X was used immediately after the confirmation of quality control, and the injected amount for each mouse was controlled to about a 0.1 μg level.

Binding Efficiency between 68Ga-DOTA-Omi-X and 68Ga-cyc-DX600 to Serum ACE2

We strictly followed our published literature, and the specific activity of 68Ga-cyc-DX600 and 68Ga-DOTA-Omi-X was controlled as the same in the following comparison.

The serum was separated from the blood at 4 °C, amounting to 3000 g for 20 min. The freshly prepared 68Ga-DOTA-Omi-X and 68Ga-cyc-DX600 with the same specific activity were cocultured separately with 100 μL of the serum at 37 °C for 10 min. The mixture was separated with a 3K filtration tube twice at 4 °C, 12,000 rpm, and the binding rate was assessed via quantifying the radioactivity attached to serum ACE2 (maintained in the filtration tube) and the radioactivity in liquid waste.

68Ga-DOTA-Omi-X PET Imaging (for Mice)

High-resolution images were acquired by using a Siemens Inveon Micro PET/CT system. 68Ga-DOTA-Omi-X (3.7 MBq) was injected into each healthy hACE2 mouse (C57BL/6J-Tgtn­(CAG-human ACE2-IRES-Luciferase-WPRE-polyA)­Smoc mice, male, 10 months old, weight 35–40 g, n = 3) via the tail vein. For dynamic scans (3–122 min P.I.), mice were fixed in the prone position in the center of FOV and kept under continuous anesthesia with 1.5% isoflurane at an oxygen flow rate of 2 L/min. Image acquisition was performed using Inveon Acquisition Workplace (IAW) 1.5.0.28, which established a new acquisition process prior to data acquisition, including CT acquisition, CT reconstruction, PET acquisition, PET histogram generation, and PET reconstruction. CT data acquisition, following the dynamic PET data acquisition, was performed at 80 kV tube voltage, with 500 μA tube current, and 1100 ms exposure time for 10 min (5 min for each bed position).

As the comparison, the wild-type mice (C57BL/6 with the same gender, similar age, and body weight) were scanned at 2 h post injection of 68Ga-DOTA-Omi-X (3.7 MBq). The image acquisition parameters were as follows: for CT, tube voltage: 80 kV; tube current: 500 μA; exposure time 1100 ms; for PET, acquisition of whole-body images for each bed was 5 min, two beds at all.

Images were reconstructed by an Three-Dimensional Ordered Subsets Expectation Maximum (OSEM3D) algorithm, followed by Maximization/Maximum a Posteriori (MAP). Three-dimensional regions of interest were drawn over the target organ guided by CT images, the corresponding SUVmax was subsequently quantified, and the values were taken as the log 10 logarithm, defined as SUV-bw. The curves of SUV-bw values over time were generated. For each experimental set, three mice were used as parallel samples.

Establishment of Cardiac Hypertrophy Model

Based on prior studies, we were able to create a model of pressure overload-induced cardiac hypertrophy through the constriction of the ascending aorta. Consequently, three 10-month-old male Bama pigs (body weight 30–40 kg) were administered sedation with Zorel 50 (comprising tiprazole, tiramin hydrochloride, and zolazepam hydrochloride) at a dosage of 5 mg/kg via intramuscular injection, followed by tracheal intubation and controlled ventilation, with supplemental oxygen provided to keep arterial blood gas levels within the normal range. Sevoflurane at a concentration of 3.0% was utilized to sustain sedation. A limited thoracotomy was conducted in the left third intercostal space to expose the pulmonary artery and ascending aorta. The segment of the ascending aorta, positioned 3 cm above the coronary Ostia, was meticulously isolated and gently constricted using a band, resulting in a peak systolic pressure gradient ranging from 55 to 70 mmHg.

Cardiac MR scans were used to validate the cardiac-hypertrophy models. All pig models underwent cardiac MR using a 3.0-T MR imaging unit (Siemens Medical Systems, Germany). The scan sequences were as follows: T2_BLADE_FS_TRA (T2WI) and T1_VIBE_DIXON_TRA (T1WI). ECG-gated steady-state free-precession (SSFP)-segmented cine images were acquired in the short axis, two- and four-chamber views, using a SSFP precession sequence with the following imaging parameters: repetition time ms/echo time ms, approximately 3.8/1.6 (minimized); matrix size, 256 × 205; field of view, 360 × 360 mm; spatial resolution, 1.4 × 1.7 mm; section thickness, 8 mm; number of phases, 40.

68Ga-DOTA-Omi-X and 18F-FDG PET for Myocardial Imaging (for Pigs)

Bama pigs with cardiac hypertrophy and healthy pigs (male, body weight 30–40 kg, per group n = 3) all underwent 68Ga-DOTA-Omi-X PET/CT and 18F-FDG PET/CT scan successively, with an interval of 8 h on 1 day. For each pig, 68Ga-DOTA-Omi-X (1.85 MBq/kg body weight) was intravenously injected, then anesthetized, and scanned at 45 min postinjection (min P.I.) using a PET/CT scanner (Biograph64, Siemens, Germany). The scan range included the region from the origin of the aorta to the apex of the heart. PET/CT scans started with a low-dose CT scan with a tube voltage: 120 kV; tube current: 170 mA; pitch: 1.0; reconstructed layer thickness: 1 mm; and followed by the PET scan of whole-body within one bed for 3 min. The SUVmax of the left-ventricular myocardium and left-ventricular blood pool were measured in reconstructed PET/CT images.

Glucose loading administration was needed before 18F-FDG PET myocardial metabolism imaging. All pig models were required to fast for more than 6 h, and the blood-glucose concentration was controlled to be < 7.8 mmol/L. Each pig was fed with 50% glucose 30–50 g, and blood glucose was tested 45 min after glucose loading (>7.8 mmol/L). Then, insulin (0.05–0.1 U/kg) was administered intravenously. Blood glucose had to be controlled <7.8 mmol/L 30 min after insulin injection. Subsequently, 18F-FDG was intravenously injected at 3.7 MBq/kg body weight, and images were acquired using a PET/CT scanner (Biograph64, Siemens, Germany) at 45 min P.I. The image acquisition parameters were set as follows: for CT, tube voltage: 120 kV; tube current: 170 mA; pitch: 1.0; reconstructed layer thickness: 1 mm; for PET, acquisition of whole-body images for each bed was 3 min. After image reconstruction, the SUVmax of the left-ventricular myocardium and the left-ventricular blood pool was quantified.

Hematoxylin and Eosin Staining, Immunocytochemistry and Picrosirius Red Staining of Ex-Vivo Tissues

Heart sections embedded in paraffin were dewaxed and dehydrated before being rinsed with distilled water. Following this, the sections underwent staining with a hematoxylin solution for 3 min, were subsequently washed with running tap water for 5 min, and then counterstained with eosin alcoholic solution for 1 min. Lastly, the sections were dehydrated using progressively higher concentrations of ethyl alcohol and were cleared in xylene for a duration of 2 min. Photographs were captured by using a microscope.

An immunohistochemical examination was conducted on organs to assess the expression levels of ACE2. The sections, after being deparaffinized, were heated in a 1 × target retrieval solution for a duration of 20 min. Following a blocking step with a 5% normal goat serum diluted in 0.01 M PBS, the sections were treated with the anti-ACE2 antibody (ab108209, Abcam) overnight at 4 °C. Subsequently, the sections were rinsed three times with 0.01 M PBS, and a secondary antibody was applied for incubation at room temperature for 30 min. After another round of washing with 0.01 M PBS, the sections underwent staining with DAB, were counterstained with hematoxylin, dehydrated, cleared, and then mounted. Finally, the slides were examined under a 200× objective and captured by using a color camera attached to the microscope. ImageJ software facilitated the quantification and analysis of the images.

Heart sections were dewaxed in xylene for 15 min (twice), dehydrated in gradient ethanol, and washed with running tap water for 30 s. Then, the sections were immersed in a 0.1% picrosirius red stain for 90 min and washed in running tap water for 10 s. After being dyed, sections were treated with 0.5% acetic acid for 20 s, dehydrated in ethanol, and cleared in xylene. The sections were sealed with resin before a light microscope observation.

Statistical Analysis

GraphPad Prism version 9.3.0 and Origin version 10.1.0.164 were used for statistical analysis of all data and curve fitting. All analyses were quantitatively performed with at least three samples per group or per test. All experiments were conducted at least in triplicate and are presented as the mean ± SD unless otherwise stated. Data sets with only two independent groups were analyzed for statistical significance using unpaired, two-tailed Student’s t-test. P values of less than 0.05 were considered as statistical significance.

Results and Discussion

Screening of Predominant Functional Peptides (Omi-X)

Following existing reports, we constructed RBD peptide libraries of BA.1, BA.2, and BA.5, with 36 peptides for each RBD library containing 15 amino acids per peptide, resulting in a theoretical total of 108 peptides. A radio-immuno assay utilizing 125I-labeled peptide derived from RBD was first performed between alternative peptides and HEK293-hACE2 cells that expressed humanized ACE2 (Figure A). Radio-iodination was achieved by labeling native or terminally modified tyrosine residues with 125I (Figure S1), preserving the core chemical structures.

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High-throughput screening of the dominant peptide binding to ACE2. (A) Strategy for the construction of peptide libraries and screening at the cellular level. (B) The radioactive uptake rate of each 125I-labeled peptide (sublineages BA.1) in HEK293-hACE2 cells, preliminarily peptide Omi-A (505HQPYRVVVLSFELLH519) and Omi-B (523TVCGPKKSTNLVKNK537) stood out. (C,D) Molecular docking predicted the ligand (Omi-A, Omi-B and DX600)-ACE2 protein binding structure and binding energy.

High-throughput screening was performed to identify ACE2-binding peptides, focusing primarily on the BA.1 sublineage. Besides mostly the same peptides, there were 11 peptides with mutations in Omicron BA.2 compared with Omicron BA.1, and five peptides with mutations in Omicron BA.5 compared with the Omicron BA.2 libraries, which were also singled out for screening. Specifically, among the 36 peptides in the Omicron BA.1 peptide library, two peptides exhibited an outstanding radioactive uptake in HEK293-hACE2 cells, in which 125I-labeled peptide 505HQPYRVVVLSFELLH519 (named Omi-A) and 523TVCGPKKSTNLVKNK537 (named Omi-B) were of absolute predominance in cell binding. After 2 h of coculture, Omi-A and Omi-B showed significantly higher uptake rates (15.23% ± 3.21% and 15.04% ± 2.98%, respectively) compared to other peptides (1.88%–7.89%, Figure B). Besides, for the DX600, the uptake rates were 10.33% ± 2.01%, approximately 67.5% of that of Omi-A.

Molecular docking simulation was executed to intuitively visualize the spatial space structure of these candidate peptide-ACE2 protein interactions and predicted the binding sites and their binding affinity (ΔG). The detailed views of molecular docking Omi-A, Omi-B, and DX600 to ACE2 are shown in Figure C. In peptide Omi-A, amino acids His and Tyr were the most effective and actively involved in ACE2 binding, and they formed hydrogen bonds with Asp206, Ser511, Arg514, and Lys562 of ACE2, respectively. Peptide Omi-B formed hydrogen bonds with Tyr202, Asn394, Arg514, and Asp509 of ACE2 via the amino acids Lys and Asn (Figure C). Moreover, the results showed that the binding sites of these two functional peptides with ACE2 were all located in the extracellular peptidase domain of the transmembrane ACE2 protein. When compared with the widely used DX600 in ACE2 interactions, this value corresponded to the results at the cellular level, where Omi-A-ACE2 and DX600-ACE2 complexes were of comparable ΔG values of −7.6 and −7.7 kcal/mol, respectively, while the Omi-B-ACE2 complex had a value of −6.4 kcal/mol (Figure D).

Peptide Omi-A and Omi-B also demonstrated an outstanding binding rate to ACE2 among the RBD peptide libraries of BA.2 and BA.5 (Figure A,B). For a long time afterward, Omicron BA.2 remained the predominant strain globally, and many of the mainstream subvariants in subsequent years were derived subtypes of BA.2 or derivatives of its branch, including BQ.1, XBB, BF.7, BA.2.86, JN.1, etc. Notably, these two peptides with the highest ACE2 affinity remained invariable without mutations not only in Omicron sublineages BA.2 and BA.5 but also in subsequent mainstream strains of Omicron (Figure C). They maintained predominance in ACE2-binding, strongly suggesting that these two peptides are the main and stable functional regions of the RBD targeting ACE2. Consequently, combining the cellular- and molecular-level results, we determined that peptide Omi-A was first chosen as the predominant functional peptide (termed Omi-X hereafter) for the further exploration of the ACE2-specific tracer.

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Dominant peptides were free of mutations appearing in Omicron BA.2, BA.5, and subsequent mainstream strains. (A,B) The cellular-uptake rate of 125I-labeled peptides in the peptide libraries of Omicron BA.2 (A) and Omicron BA.5 (B). The mutations in the Omicron BA.2 compared with Omicron BA.1 were highlighted in red, showing that existing six mutation sites appeared in 11 peptides. On the basis of Omicron BA.2, there were three mutation sites (highlighted in green) appearing in five peptides. Same as Omicron BA.1, the peptide 505HQPYRVVVLSFELLH519 (named as Omi-A) and 523TVCGPKKSTNLVKNK537 (named as Omi-B) were of superior ACE2 affinity in Omicron BA.2 and BA.5 libraries, and there remained no mutation site. (C) The RBD sequence of representative Omicron strains that were once prevalent (BQ.1, XBB, BF.7, BA.2.86, and JN.1) compared with Omicron BA.2, and the mutations were highlighted.

To the best of our knowledge, the exploration and design of targeting molecules is a decisive factor in molecular imaging, influencing the specificity, affinity, and stability in biological processes of target binding. These labeled molecules or materials can represent the functions of their target molecules in vivo or in vitro and can generate image signals for recognition. Today, active targeting agents can be developed from diverse sources. Among them, natural or artificial sugar derivatives, peptide derivatives, biogenic amines, nucleotide analogues, biomimetic nanoparticles, and amino-acid nanoparticles/polymers are common and abundant sources for ligand design. , In recent years, methodologies, including computer virtual screening technology, nucleic-acid aptamer, artificial-intelligence in-depth analysis/generative models, and multiomics data integration and analysis have gradually emerged, allowing versatility in ligand design, synthesis, and screening. , Regarding ACE2-specific molecular imaging, already available small-molecule inhibitors and analogues, such as DX600 and MLN4760 have been developed as targeting ligands in the early stages, but the two differ in terms of the amount and mode of administration. ,

Innovatively, instead of using known inhibitors as imaging agents, we honed in on the natural binding process of the Omicron RBD to ACE2, and then peptide libraries of RBD of the dominant Omicron variants (BA.1, BA.2, and BA.5) were built as a resource for screening the ACE2-targeting sequence. Omicron BA.1 was designated after the Delta variants, and then continuous surveillance of Omicron evolution showed that BA.1 was overtaken by BA.2, which became the mainstream SARS-CoV-2 strain until mid-2022 but was subsequently replaced by BA.4, BA.5, and newer sublineages, including BA.2.86, XBB, BQ.1, and JN.1. − Any mutation may affect the virus’s infection tropism and immunogenicity, which occurring in RBD can be crucial for the binding affinity to ACE2 on the host. Consequently, RBD peptide libraries covering mutational properties represent a natural, advantageous, and ideal resource for identifying core and key peptide sequences in the drug design and development process. Notably, during the tracing process of the mainstream Omicron strains, we found that of the two dominant peptides we screened through a high-throughput screening of RBD peptide libraries and molecular simulation analysis, the Omi-A and Omi-B were stable and constant in the above prevalent variants, with no mutation reported. Omi-A was demonstrated to be the optimal probe candidate (Omi-X) through preliminary high-throughput screening and molecular modeling. These in vivo analysis studies supported this choice, pending for further in vivo verification of biosafety and biodistribution.

Biosafety and Stability of Omi-X

The Omi-X was derived from Omicron variants, herein, the biosafety and stability of Omi-X were indispensable before being engineered as a molecular probe. Cytotoxicity experiments through Carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) show that the cell-division rate of HEK293-hACE2 cells were of no significant difference when cocultured with low-dose (concentration, 0.544 μM (1 μg/mL)) and medium-dose (concentration, 5.441 μM (10 μg/mL)) Omi-X for 36 h (Figure A). At the concentration up to 54.410 μM (100 μg/mL), the cell-division rate appeared to be slightly inhibited. Similarly, there was no significant effect of Omi-X on the cell cycle distribution, even at a concentration of 36.27 μM (Figure B). More detailed fluorescence images of cell proliferation and cell cycle are in Figures S2 and S3. Besides, no immunological stress reaction was seen in K18-hACE2 mice after a 10 μg/day intravenous injection of Omi-X for 5 days (Table S1). The HPLC spectrum of chemical stability of Omi-X in diluted HCl (pH = 2) at 37 °C is shown in Figure C. In biomimetic conditions (37 °C, 24 h), the peptide sequence was chemically stable in weakly acid conditions. The chemical stability of the peptide sequence met a requirement of >95% in a weakly acidic environment. In the reaction simulating labeling conditions (95 °C, pH = 4, 24 h), the derivant was less stable, as shown in Figure S4.

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Biosafety and chemical stability. (A) The cytotoxicity characterized by the cell division rate that was determined using the CFDA SE staining of HEK293-hACE2 cells treated with Omi-X. (B) The cell cycle analysis of HEK293-hACE2 cells treated with different concentrations of Omi-X. The percentage of cells in different phases was presented as bar graphs. (C) HPLC spectrum of chemical stability of Omi-X.

In Vivo Distribution and Metabolism of Omi-X in Mice

To investigate the in vivo metabolism and ACE2-specificity of Omi-X, 125I-based SPECT imaging was used for early and long-term monitoring. For 125I-Omi-X (Figure A), radiochemical purity met the requirement of >95% (Figure B).

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In vivo distribution of Omi-X characterized by 125I-based SPECT imaging. (A) The chemical structure of 125I-Omi-X. (B) The typical radio-TLC spectrum of 125I-Omi-X. (C) SPECT imaging 1 and 6 h after intravenous administration of 125I-Omi-X in K18-hACE2 mice and C57BL/6 mice. (D) The unit uptake in the region of interest. (E) Representative immunohistochemistry staining for ACE2 in the tissue harvested from K18-hACE2 mice.

ACE2-dependent in vivo distribution was observed in C57BL/6-derived K18-hACE2 mice; when compared with those of the wild-type C57BL/6 mice, ACE2-targeting and metabolic characteristics of 125I-Omi-X were observed via SPECT/CT imaging (Figure C). For K18-hACE2 mice, 125I-Omi-X was rapidly cleared from circulation and notably accumulated in the organs highly expressing ACE2, including the lung, heart, liver, and spleen. Further, unbound 125I-Omi-X was primarily excreted through the urinary system. Five hours later, after initial in vivo distribution and clearance, 125I-Omi-X was still primarily concentrated in ACE2-related organs, including the lung, heart, and liver, and slightly in the gastrointestinal tract (Figure D). Specifically, the characteristics of 125I-Omi-X in vivo ACE2 targeting were consistent with ACE2 immunohistochemistry staining in the RAAS-related organs of K18-hACE2 mice (Figure E), revealing an indirect manifestation of the distribution and abundance of ACE2 in organs. In sharp contrast, although 125I-Omi-X had a preliminary in vivo distribution in WT C57BL/6 mice at 1 h P.I., most agents passed through intestinal tracts or were excreted via the urinary system and the uptake in RAAS-related organs was greatly reduced by 6 h P.I. (Figure C,D). Considerable differences in the distribution and metabolic pattern of 125I-Omi-X in mice with entirely different ACE2 expression types were also observed, verifying the favorable ACE2 targeting of the screened dominant peptides Omi-X. Moreover, the unbound agent was effectively cleared to reduce the “noisy” background, realizing a high target-to-background ratio.

Development of 68Ga-DOTA-Omi-X

For the sequence of 505HQPYRVVVLSFELLH519 with superiority in binding to ACE2 (Figure S5), 1,4,7,10-tetraazacyclododecane-N,N′,N,N′-tetraacetic acid was conjugated to the N-terminal to provide a chelator for 68Ga-labeling via a chelating reaction, and it was named as DOTA-Omi-X (Figure S6). The derivant tracer was named as 68Ga-DOTA-Omi-X. The schemes of radiolabeling that began with the introduction of nuclides are shown in Figure A. The probe 68Ga-DOTA-Omi-X (Figure B) was acquired in an overall radiochemical yield of 65% from aqueous [68Ga]­GaCl3 (nondecay corrected) and 98.55% ± 0.50% radiochemical purity without purification (Figure C, TLC spectrum with a radioactive signal). The specific activity of the probe increased to 160 GBq/μmol by the end of the synthesis. The time-dependent stability in 0.01 M PBS and 10% FBS all met requirement (Figure D,E). The radiochemical stability (4 h) in 0.01 M PBS (%) was 96.10 ± 0.75, and the radiochemical stability (4 h) in 10% FBS (%) was 94.10 ± 1.55. The typical TLC spectrum of stabilities is shown in Figure S7.

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Development and characterization of 68Ga-DOTA-Omi-X. (A) The schemes of radiolabeling of Ga-68 to DOTA-Omi-X via chelating. (B) The chemical structure and sequence of 68Ga-DOTA-Omi-X. (C) Radio-TLC spectrum of labeling system of 68Ga-DOTA-Omi-X in 0.01 M PBS at 25 °C. (D,E) The time-dependent stability in 0.01 M PBS and 10% FBS. 68Ga-DOTA-Omi-X-based PET imaging.

Due to the high labeling rate and convenient preparation conditions, 68Ga-DOTA-Omi-X PET is suitable for translational research. For the tracer-binding efficiency to serum ACE2 that was separated from hACE2 mice with or without previous UDCA-blocking, 68Ga-DOTA-Omi-X exhibited a bigger drop in the binding rate when compared with 68Ga-cyc-DX600, hinting at a more sensitive response in recording ACE2 fluctuation (Figure A).

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The in vivoACE2-specificity of 68Ga-DOTA-Omi-X. (A) The binding rate to serum ACE2 with or without previous UDCA-blocking between 68Ga-DOTA-Omi-X and 68Ga-cyc-DX600. (B) The 68Ga-DOTA-Omi-X PET images at 2 h P.I. of wild type C57BL/6 mice and hACE2 mice. (C) The organ-specific uptake of 68Ga-DOTA-Omi-X between hACE2 mice and wild-type C57BL/6 mice at 2 h P.I. (D) The representative cross-sectional views at the level of the heart, liver, and kidneys of hACE2 mice in 68Ga-DOTA-Omi-X PET. (E) The time-dependent (5 ∼ 115 min P.I.) organ-specific tracer uptake. 68Ga-DOTA-Omi-X PET in cardiac hypertrophy.

The performance of the ACE2-specific distribution of 68Ga-DOTA-Omi-X was initially compared in hACE2 mice (C57BL/6J-Tgtn­(CAG-human ACE2-IRES-Luciferase-WPRE-polyA)­Smoc mice) with the ACE2 expression throughout the body and wild-type C57BL/6 mice. The represented MIP images 2 h after the injection of 68Ga-DOTA-Omi-X are shown in Figure B, reflecting a high uptake spread almost all over the body of hACE2 mice, mainly in the salivary glands, heart, lung, and intestines, and the quantitative information is summarized in Figure C. Figure D,E displays the time-dependent (5 ∼ 115 min P.I.) organ-specific tracer uptake of 68Ga-DOTA-Omi-X in hACE2 mice. The long-term retention of 68Ga-DOTA-Omi-X revealed favorable ACE2-specific targeting properties. The tracer was mainly cleared through the kidneys and subsequently increased in the bladder over time. As expected, with the largest difference from wild-type C57BL/6 mice, 68Ga-DOTA-Omi-X accumulated in the liver and spleen of hACE2 mice, which is consistent with the majority of small-molecule peptide imaging agents metabolized and cleared through the liver-spleen pathways. However, 68Ga-DOTA-Omi-X did not appear to show significant ACE2-specific targeting in wild-type C57BL/6 mice. Metabolic details and more representative images of dynamic scans are shown in Figures S8–S10.

The capacity of 68Ga-DOTA-Omi-X PET to record and monitor the patterns of ACE2 fluctuations during the process of cardiac hypertrophy involving RAAS regulation were administered in models of healthy pigs and pigs with cardiac hypertrophy (Figure A), which were previously confirmed as cardiac hypertrophy in CT and MR images (Figure B). The final autopsy reports after imaging, including hematoxylin and eosin staining and picrosirius red staining of heart tissues, also verified the formation of myocardial hypertrophy and myocardial fibrosis (Figure C,D). Morphologically, compared to normal, myocardial fibers show marked disorganization and an irregular arrangement when myocardial hypertrophy occurred. Varying degrees of fibrosis could be seen in the myocardial interstitium, appearing in the picrosirius red staining of heart tissues. In 18F-FDG PET/CT imaging of healthy pigs, the left ventricular shape, myocardial thickness and chamber size were standard (Figure E). In contrast, there was ventricular disproportion in pigs with cardiac hypertrophy, where the left ventricular myocardium was significantly hypertrophied and the chamber was smaller. The ratio of the 18F-FDG uptake of the left-ventricular myocardium and left-ventricular blood pool (M/B) also exhibited significant differences, suggesting higher metabolic activity in the hypertrophied left-ventricular myocardium. Consistent with 18F-FDG PET/CT results, a higher 68Ga-DOTA-Omi-X uptake was observed in the hypertrophied left-ventricular myocardium compared with the healthy-pig models (Figure F). The regulation of ACE2 is a systematic and dynamic process, which may appear as a heterogeneously increased 68Ga-DOTA-Omi-X uptake in the myocardium when cardiac hypertrophy has occurred. From the imaging perspective, although the increased uptake of 68Ga-DOTA-Omi-X in the hypertrophied myocardium was not as marked as that of FDG visually, there was also a statistical difference in the ratio of M/B, which could reflect the upregulation of ACE2 in myocardial remodeling and guide the follow-up treatment (Figure G,H). Pressure overload-induced cardiac hypertrophy and remodeling were generally accompanied by reactive fibrosis, where the ACE2 overexpression could alleviate and reverse myocardial fibrosis (Figure D).

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The performance of 68Ga-DOTA-Omi-X PET imaging for pressure overload-induced cardiac hypertrophy. (A) The timeline of establishment of cardiac hypertrophy model and PET imaging. (B) The representative CT images (upper) and the T2WI MR (lower) of cardiac hypertrophy models. (C,D) Representative images of hematoxylin–eosin (HE) staining and ACE2 immunohistochemical and picrosirius red staining of hypertrophic left ventricular myocardium. (E) Representative 18F-FDG PET/CT images of healthy pigs and pig models of cardiac hypertrophy, and the ratio of the FDG uptake of left ventricular myocardium and left ventricular blood pool. (F) 68Ga-DOTA-Omi-X PET/CT of healthy pigs and pig models of cardiac hypertrophy, and the ratio of the 68Ga-DOTA-Omi-X uptake of left ventricular myocardium and left ventricular blood pool. (G) The corresponding ratio of SUVmax values between healthy pigs and pig models of cardiac hypertrophy in 18F-FDG PET/CT (upper) and 68Ga-DOTA-Omi-X PET/CT images (below). (H) The correlation analysis between IHC data of ACE2 and the SUVmax of 68Ga-DOTA-Omi-X.

68Ga-DOTA-Omi-X PET imaging showed a satisfactory diagnostic performance in RAAS-related cardiovascular diseases. In cardiac-hypertrophy models, we simultaneously explored the performance of glucose metabolism and the RAAS regulation during the pathological process of cardiac hypertrophy using two different imaging agents. Myocardial remodeling in response to a pressure overload represents an adaptation of the myocardial structure, as well as changes in the myocardial function, all of which could be visualized via molecular imaging. We demonstrated the increased 68Ga-DOTA-Omi-X uptake in a hypertrophied left ventricular myocardium, revealing the upregulation of ACE2 during myocardial remodeling. It is known that ACE2 is the key regulator in cardiovascular diseases, as an ACE2 overexpression can suppress Ang II-mediated myocardial hypertrophy and fibrosis and prevent cardiac dysfunction. Thus, ACE2 is frequently a target for cardiovascular disease treatment. − Compared with an enhanced glucose metabolism to compensate for an inadequate energy supply due to the impaired myocardial mitochondrial function, ACE2-based molecular imaging can help monitor the ongoing avid progression and identify the potential time point and location of intervention. Further, ACE2 PET has feasibility in providing support information for treatment decisions and guiding time windows of drug intervention, including for the RAAS inhibitor or ACE2 agonist.

All along, virus–host interactions have always served as an important basis and condition for exploring pathogenic mechanisms, designing drugs, identifying potential targets, and developing therapeutic strategies. − From a therapeutic perspective, each component and link of the virus–host interaction process is a therapeutic target for interfering with and blocking viral invasion, and this strategy is universally applicable to common viral infections and virus-induced tumors. As an example, multiple natural receptors on the surface of lung-cancer cells are often dysregulated, providing potential entry points for oncolytic viruses (OVs). , These receptors include the coxsackie-adenovirus receptor (CAR), herpes virus entry mediator, and CD46, which not only promote cancer-cell invasion and metastasis but are also natural targets for OV infection. − Through chemical coupling or genetic engineering, we can connect fluorescent molecules (e.g., enhanced GFP, mCherry, Cy5.5, ICG, and FITC) to the virus to prepare molecular-imaging probes that can specifically bind to the tumor cell CD46, CAR, and other receptors, achieving tumor localization and surgical-area delineation. For tracer development with more translational potential, nuclear medicine molecular imaging can be achieved by replacing fluorescent molecules with radionuclides to visualize the corresponding biological processes.

Conclusion

Here, we proposed a universal scheme for exploring the binding agent and derivatives utilizing the natural mutation of the virus/host interaction. On the basis of the RBD peptide library of Omicron, we leveraged the binding effect of viral RBD-ACE2 interactions via a radioimmunoassay, screened out 505HQPYRVVVLSFELLH519 as the superior peptide, and prepared the novel ACE2-targeted PET imaging tracer68Ga-DOTA-Omi-X. The preliminary application in the identification of diseased RAAS progression proved the feasibility of noninvasive ACE2 tracing in real time, lending strong support for exploring the inhibitors or derivatives from natural mutations and evolution.

Supplementary Material

im5c00053_si_001.pdf (1.2MB, pdf)

Acknowledgments

This research was funded by the Health Technology Project of the Pudong New Area health committee (PW2023D-06), the National Natural Science Foundation of China (Youth Program 82402423 and 82202216).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/cbmi.5c00053.

  • Radio-TLC spectrum of 125I-Omi-B; cytotoxicity of Omi-X characterized by the cell division rate, cell cycle analysis and multicytokine analysis of K18-hACE2 mice; chemical stability of DOTA-Omi-X; HPLC profiles and ESI-MS spectra of Omi-X and DOTA-Omi-X; radio-TLC spectrum of 68Ga-DOTA-Omi-X; time-dependent organ-specific uptake of 68Ga-DOTA-Omi-X in hACE2 mice; and represented MIP and tomographic images (sagittal, coronal, and transverse planes) of 68Ga-DOTA-Omi-X PET (PDF)

⊥.

R.L. and J.L. contributed equally to this work. X.L. designed the study; J.L., Z.Y., W.H., and X.L. synthesized and characterized the chemicals; R.L., S.X., D.L., and T.W. verified the ACE2-specificity in vitro and in vivo; R.L., and X.L. written the manuscript.

The authors declare no competing financial interest.

Ethics statement: All animal experimental protocols for this research were approved and guided by the Ethics Committee of Shanghai Changhai Hospital (Approval No.: 2023-MS-DS-08).

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