Bioorthogonal Radiolabeling of Azide-Modified Bacteria Using [18F]FB-sulfo-DBCO.

Purpose: This study was motivated by the need for better positron emission tomography (PET)-compatible tools to image bacterial infection. Our previous efforts have targeted bacteria-specific metabolism via assimilation of carbon-11 labeled d-amino acids into the bacterial cell wall. Since the chemical determinants of this incorporation are not fully understood, we sought a high-throughput method to label d-amino acid derived structures with fluorine-18. Our strategy employed a chemical biology approach, whereby an azide (-N3) bearing d-amino acid is incorporated into peptidoglycan muropeptides, with subsequent "click" cycloaddition with an 18F-labeled strained cyclooctyne partner. Procedures: A water-soluble, 18F-labeled and dibenzocyclooctyne (DBCO)-derived radiotracer ([18F]FB-sulfo-DBCO) was synthesized. This tracer was incubated with pathogenic bacteria treated with azide-bearing d-amino acids, and incorporated 18F was determined via gamma counting. In vitro uptake in bacteria previously treated with azide-modified d-amino acids was compared to that in cultures treated with amino acid controls. The biodistribution of [18F]FB-sulfo-DBCO was studied in a cohort of healthy mice with implications for future in vivo imaging. Results: The new strain-promoted azide-alkyne cycloaddition (SPAAC) radiotracer [18F]FB-sulfo-DBCO was synthesized with high radiochemical yield and purity via N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB). Accumulation of [18F]FB-sulfo-DBCO was significantly higher in several bacteria treated with azide-modified d-amino acids than in controls; for example, we observed 7 times greater [18F]FB-sulfo-DBCO ligation in Staphylococcus aureus cultures incubated with 3-azido-d-alanine versus those incubated with d-alanine. Conclusions: The SPAAC radiotracer [18F]FB-sulfo-DBCO was validated in vitro via metabolic labeling of azide-bearing peptidoglycan muropeptides. d-Amino acid-derived PET radiotracers may be more efficiently screened via [18F]FB-sulfo-DBCO modification.

Introducing the Role of Genotoxicity in Neurodegenerative Diseases and Neuropsychiatric Disorders.

Decades of research have identified genetic and environmental factors involved in age-related neurodegenerative diseases and, to a lesser extent, neuropsychiatric disorders. Genomic instability, i.e., the loss of genome integrity, is a common feature among both neurodegenerative (mayo-trophic lateral sclerosis, Parkinson's disease, Alzheimer's disease) and psychiatric (schizophrenia, autism, bipolar depression) disorders. Genomic instability is associated with the accumulation of persistent DNA damage and the activation of DNA damage response (DDR) pathways, as well as pathologic neuronal cell loss or senescence. Typically, DDR signaling ensures that genomic and proteomic homeostasis are maintained in both dividing cells, including neural progenitors, and post-mitotic neurons. However, dysregulation of these protective responses, in part due to aging or environmental insults, contributes to the progressive development of neurodegenerative and/or psychiatric disorders. In this Special Issue, we introduce and highlight the overlap between neurodegenerative diseases and neuropsychiatric disorders, as well as the emerging clinical, genomic, and molecular evidence for the contributions of DNA damage and aberrant DNA repair. Our goal is to illuminate the importance of this subject to uncover possible treatment and prevention strategies for relevant devastating brain diseases.

Genomic stress in diseases stemming from defects in the second brain.

This review discusses the less-explored realm of DNA damage and repair within the enteric nervous system (ENS), often referred to as the "second brain." While the central nervous system has been extensively studied for its DNA repair mechanisms and associated neuropathologies, the ENS, which can autonomously coordinate gastrointestinal function, experiences unique challenges and vulnerabilities related to its genome integrity. The susceptibility of the ENS to DNA damage is exacerbated by its limited protective barriers, resulting in not only endogenous genotoxic exposures, such as oxidative stress, but also exogenous threats, such as ingested environmental contaminants, local inflammatory responses, and gut dysbiosis. Here, we discuss the evidence for DNA repair defects in enteric neuropathies, most notably, the reported relationship between inherited mutations in RAD21 and LIG3 with chronic intestinal pseudo-obstruction and mitochondrial gastrointestinal encephalomyopathy disorders, respectively. We also introduce the lesser-recognized gastrointestinal complications in DNA repair syndromes, including conditions like Cockayne syndrome. The review concludes by pointing out the potential role of DNA repair defects in not only congenital disorders but also aging-related gut dysfunction, as well as the crucial need for further research to establish direct causal links between DNA damage accumulation and ENS-specific pathologic phenotypes.

Genomic stress and impaired DNA repair in Alzheimer disease.

Alzheimer disease (AD) is the most prominent form of dementia and has received considerable attention due to its growing burden on economic, healthcare and basic societal infrastructures. The two major neuropathological hallmarks of AD, i.e., extracellular amyloid beta (Aß) peptide plaques and intracellular hyperphosphorylated Tau neurofibrillary tangles, have been the focus of much research, with an eye on understanding underlying disease mechanisms and identifying novel therapeutic avenues. One often overlooked aspect of AD is how Aß and Tau may, through indirect and direct mechanisms, affect genome integrity. Herein, we review evidence that Aß and Tau abnormalities induce excessive genomic stress and impair genome maintenance mechanisms, events that can promote DNA damage-induced neuronal cell loss and associated brain atrophy.

Imaging the Granzyme Mediated Host Immune Response to Viral and Bacterial Pathogens In Vivo Using Positron Emission Tomography.

Understanding how the host immune system engages complex pathogens is essential to developing therapeutic strategies to overcome their virulence. While granzymes are well understood to trigger apoptosis in infected host cells or bacteria, less is known about how the immune system mobilizes individual granzyme species in vivo to combat diverse pathogens. Toward the goal of studying individual granzyme function directly in vivo, we previously developed a new class of radiopharmaceuticals termed "restricted interaction peptides (RIPs)" that detect biochemically active endoproteases using positron emission tomography (PET). In this study, we showed that secreted granzyme B proteolysis in response to diverse viral and bacterial pathogens could be imaged with [64Cu]Cu-GRIP B, a RIP that specifically targets granzyme B. Wild-type or germline granzyme B knockout mice were instilled intranasally with the A/PR/8/34 H1N1 influenza A strain to generate pneumonia, and granzyme B production within the lungs was measured using [64Cu]Cu-GRIP B PET/CT. Murine myositis models of acute bacterial (E. coli, P. aeruginosa, K. pneumoniae, and L. monocytogenes) infection were also developed and imaged using [64Cu]Cu-GRIP B. In all cases, the mice were studied in vivo using mPET/CT and ex vivo via tissue-harvesting, gamma counting, and immunohistochemistry. [64Cu]Cu-GRIP B uptake was significantly higher in the lungs of wild-type mice that received A/PR/8/34 H1N1 influenza A strain compared to mice that received sham or granzyme B knockout mice that received either treatment. In wild-type mice, [64Cu]Cu-GRIP B uptake was significantly higher in the infected triceps muscle versus normal muscle and the contralateral triceps inoculated with heat killed bacteria. In granzyme B knockout mice, [64Cu]Cu-GRIP B uptake above the background was not observed in the infected triceps muscle. Interestingly, live L. monocytogenes did not induce detectable granzyme B on PET, despite prior in vitro data, suggesting a role for granzyme B in suppressing their pathogenicity. In summary, these data show that the granzyme response elicited by diverse human pathogens can be imaged using PET. These results and data generated via additional RIPs specific for other granzyme proteases will allow for a deeper mechanistic study analysis of their complex in vivo biology.

Protocol for producing hyperpolarized 13C-bicarbonate for clinical MRI of extracellular pH in aggressive tumors.

Tumor acidosis is one of the hallmarks indicating the initiation and progression of various cancers. Here, we present a protocol for preparing a hyperpolarized (HP) 13C-bicarbonate tissue pH MRI imaging contrast agent to detect aggressive tumors. We describe the steps for the formulation and polarization of a precursor molecule 13C-glycerol carbonate (13C-GLC), the post-dissolution reaction, and converting HP 13C-GLC to an injectable HP 13C-bicarbonate solution. We then detail procedures for MRI data acquisition to generate tumor pH maps for assessing tumor aggressiveness. For complete details on the use and execution of this protocol, please refer to Mu et al.1.

Development of CD46 targeted alpha theranostics in prostate cancer using 134Ce/225Ac-Macropa-PEG4-YS5.

Rationale: 225Ac, a long-lived α-emitter with a half-life of 9.92 days, has garnered significant attention as a therapeutic radionuclide when coupled with monoclonal antibodies and other targeting vectors. Nevertheless, its clinical utility has been hampered by potential off-target toxicity, a lack of optimized chelators for 225Ac, and limitations in radiolabeling methods. In a prior study evaluating the effectiveness of CD46-targeted radioimmunotherapy, we found great therapeutic efficacy but also significant toxicity at higher doses. To address these challenges, we have developed a radioimmunoconjugate called 225Ac-Macropa-PEG4-YS5, incorporating a stable PEGylated linker to maximize tumoral uptake and increase tumor-to-background ratios. Our research demonstrates that this conjugate exhibits greater anti-tumor efficacy while minimizing toxicity in prostate cancer 22Rv1 tumors. Methods: We synthesized Macropa.NCS and Macropa-PEG4/8-TFP esters and prepared Macropa-PEG0/4/8-YS5 (with nearly ~1:1 ratio of macropa chelator to antibody YS5) as well as DOTA-YS5 conjugates. These conjugates were then radiolabeled with 225Ac in a 2 M NH4OAc solution at 30 °C, followed by purification using YM30K centrifugal purification. Subsequently, we conducted biodistribution studies and evaluated antitumor activity in nude mice (nu/nu) bearing prostate 22Rv1 xenografts in both single-dose and fractionated dosing studies. Micro-PET imaging studies were performed with 134Ce-Macropa-PEG0/4/8-YS5 in 22Rv1 xenografts for 7 days. Toxicity studies were also performed in healthy athymic nude mice. Results: As expected, we achieved a >95% radiochemical yield when labeling Macropa-PEG0/4/8-YS5 with 225Ac, regardless of the chelator ratios (ranging from 1 to 7.76 per YS5 antibody). The isolated yield exceeded 60% after purification. Such high conversions were not observed with the DOTA-YS5 conjugate, even at a higher ratio of 8.5 chelators per antibody (RCY of 83%, an isolated yield of 40%). Biodistribution analysis at 7 days post-injection revealed higher tumor uptake for the 225Ac-Macropa-PEG4-YS5 (82.82 ± 38.27 %ID/g) compared to other conjugates, namely 225Ac-Macropa-PEG0/8-YS5 (38.2 ± 14.4/36.39 ± 12.4 %ID/g) and 225Ac-DOTA-YS5 (29.35 ± 7.76 %ID/g). The PET Imaging of 134Ce-Macropa-PEG0/4/8-YS5 conjugates resulted in a high tumor uptake, and tumor to background ratios. In terms of antitumor activity, 225Ac-Macropa-PEG4-YS5 exhibited a substantial response, leading to prolonged survival compared to 225Ac-DOTA-YS5, particularly when administered at 4.625 kBq doses, in single or fractionated dose regimens. Chronic toxicity studies observed mild to moderate renal toxicity at 4.625 and 9.25 kBq doses. Conclusions: Our study highlights the promise of 225Ac-Macropa-PEG4-YS5 for targeted alpha particle therapy. The 225Ac-Macropa-PEG4-YS5 conjugate demonstrates improved biodistribution, reduced off-target binding, and enhanced therapeutic efficacy, particularly at lower doses, compared to 225Ac-DOTA-YS5. Incorporating theranostic 134Ce PET imaging further enhances the versatility of macropa-PEG conjugates, offering a more effective and safer approach to cancer treatment. Overall, this methodology has a high potential for broader clinical applications.

Peptidoglycan-Targeted [18F]3,3,3-Trifluoro-d-alanine Tracer for Imaging Bacterial Infection.

Imaging is increasingly used to detect and monitor bacterial infection. Both anatomic (X-rays, computed tomography, ultrasound, and MRI) and nuclear medicine ([111In]-WBC SPECT, [18F]FDG PET) techniques are used in clinical practice but lack specificity for the causative microorganisms themselves. To meet this challenge, many groups have developed imaging methods that target pathogen-specific metabolism, including PET tracers integrated into the bacterial cell wall. We have previously reported the d-amino acid derived PET radiotracers d-methyl-[11C]-methionine, d-[3-11C]-alanine, and d-[3-11C]-alanine-d-alanine, which showed robust bacterial accumulation in vitro and in vivo. Given the clinical importance of radionuclide half-life, in the current study, we developed [18F]3,3,3-trifluoro-d-alanine (d-[18F]-CF3-ala), a fluorine-18 labeled tracer. We tested the hypothesis that d-[18F]-CF3-ala would be incorporated into bacterial peptidoglycan given its structural similarity to d-alanine itself. NMR analysis showed that the fluorine-19 parent amino acid d-[19F]-CF3-ala was stable in human and mouse serum. d-[19F]-CF3-ala was also a poor substrate for d-amino acid oxidase, the enzyme largely responsible for mammalian d-amino acid metabolism and a likely contributor to background signals using d-amino acid derived PET tracers. In addition, d-[19F]-CF3-ala showed robust incorporation into Escherichia coli peptidoglycan, as detected by HPLC/mass spectrometry. Based on these promising results, we developed a radiosynthesis of d-[18F]-CF3-ala via displacement of a bromo-precursor with [18F]fluoride followed by chiral stationary phase HPLC. Unexpectedly, the accumulation of d-[18F]-CF3-ala by bacteria in vitro was highest for Gram-negative pathogens in particular E. coli. In a murine model of acute bacterial infection, d-[18F]-CF3-ala could distinguish live from heat-killed E. coli, with low background signals. These results indicate the viability of [18F]-modified d-amino acids for infection imaging and indicate that improved specificity for bacterial metabolism can improve tracer performance.

Prostate-Specific Membrane Antigen Targeted StarPEG Nanocarrier for Imaging and Therapy of Prostate Cancer.

The tumor uptake of large non-targeted nanocarriers primarily occurs through passive extravasation, known as the enhanced permeability and retention (EPR) effect. Prior studies demonstrated improved tumor uptake and retention of 4-arm 40 kDa star polyethylene glycol (StarPEG) polymers for cancer imaging by adding prostate-specific membrane antigen (PSMA) targeting small molecule ligands. To test PSMA-targeted delivery and therapeutic efficacy, StarPEG nanodrugs with/without three copies of PSMA-targeting ligands, ACUPA, are designed and synthesized. For single-photon emission computed tomography (SPECT) imaging and therapy, each nanocarrier is labeled with 177Lu using DOTA radiometal chelator. The radiolabeled nanodrugs, [177Lu]PEG-(DOTA)1 and [177Lu]PEG-(DOTA)1(ACUPA)3, are evaluated in vitro and in vivo using PSMA+ PC3-Pip and/or PSMA- PC3-Flu cell lines, subcutaneous xenografts and disseminated metastatic models. The nanocarriers are efficiently radiolabeled with 177Lu with molar activities 10.8-15.8 MBq/nmol. Besides excellent in vitro PSMA binding affinity (kD = 51.7 nM), the targeted nanocarrier, [177Lu]PEG-(DOTA)1(ACUPA)3, demonstrated excellent in vivo SPECT imaging contrast with 21.3% ID/g PC3-Pip tumors uptake at 192 h. Single doses of 18.5 MBq [177Lu]PEG-(DOTA)1(ACUPA)3 showed complete resolution of the PC3-Pip xenografts observed up to 138 days. Along with PSMA-targeted excellent imaging contrast, these results demonstrated high treatment efficacy of [177Lu]PEG-(DOTA)1(ACUPA)3 for prostate cancer, with potential for clinical translation.

CD46-Targeted Theranostics for PET and 225Ac-Radiopharmaceutical Therapy of Multiple Myeloma.

PURPOSE: Multiple myeloma is a plasma cell malignancy with an unmet clinical need for improved imaging methods and therapeutics. Recently, we identified CD46 as an overexpressed therapeutic target in multiple myeloma and developed the antibody YS5, which targets a cancer-specific epitope on this protein. We further developed the CD46-targeting PET probe [89Zr]Zr-DFO-YS5 for imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of prostate cancer. These prior studies suggested the feasibility of the CD46 antigen as a theranostic target in multiple myeloma. Herein, we validate [89Zr]Zr-DFO-YS5 for immunoPET imaging and [225Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of multiple myeloma in murine models. EXPERIMENTAL DESIGN: In vitro saturation binding was performed using the CD46 expressing MM.1S multiple myeloma cell line. ImmunoPET imaging using [89Zr]Zr-DFO-YS5 was performed in immunodeficient (NSG) mice bearing subcutaneous and systemic multiple myeloma xenografts. For radioligand therapy, [225Ac]Ac-DOTA-YS5 was prepared, and both dose escalation and fractionated dose treatment studies were performed in mice bearing MM1.S-Luc systemic xenografts. Tumor burden was analyzed using BLI, and body weight and overall survival were recorded to assess antitumor effect and toxicity. RESULTS: [89Zr]Zr-DFO-YS5 demonstrated high affinity for CD46 expressing MM.1S multiple myeloma cells (Kd = 16.3 nmol/L). In vitro assays in multiple myeloma cell lines demonstrated high binding, and bioinformatics analysis of human multiple myeloma samples revealed high CD46 expression. [89Zr]Zr-DFO-YS5 PET/CT specifically detected multiple myeloma lesions in a variety of models, with low uptake in controls, including CD46 knockout (KO) mice or multiple myeloma mice using a nontargeted antibody. In the MM.1S systemic model, localization of uptake on PET imaging correlated well with the luciferase expression from tumor cells. A treatment study using [225Ac]Ac-DOTA-YS5 in the MM.1S systemic model demonstrated a clear tumor volume and survival benefit in the treated groups. CONCLUSIONS: Our study showed that the CD46-targeted probe [89Zr]Zr-DFO-YS5 can successfully image CD46-expressing multiple myeloma xenografts in murine models, and [225Ac]Ac-DOTA-YS5 can effectively inhibit the growth of multiple myeloma. These results demonstrate that CD46 is a promising theranostic target for multiple myeloma, with the potential for clinical translation.

Discovery and In Vivo Efficacy of AZ-PRMT5i-1, a Novel PRMT5 Inhibitor with High MTA Cooperativity.

PRMT5, a type 2 arginine methyltransferase, has a critical role in regulating cell growth and survival in cancer. With the aim of developing MTA-cooperative PRMT5 inhibitors suitable for MTAP-deficient cancers, herein we report our efforts to develop novel "MTA-cooperative" compounds identified through a high-throughput biochemical screening approach. Optimization of hits was achieved through structure-based design with a focus on improvement of oral drug-like properties. Bioisosteric replacement of the original thiazole guanidine headgroup, spirocyclization of the isoindolinone amide scaffold to both configurationally and conformationally lock the bioactive form, and fine-tuning of the potency, MTA cooperativity, and DMPK properties through specific substitutions of the azaindole headgroup were conducted. We have identified an orally available in vivo lead compound, 28 ("AZ-PRMT5i-1"), which shows sub-10 nM PRMT5 cell potency, >50-fold MTA cooperativity, suitable DMPK properties for oral dosing, and significant PRMT5-driven in vivo efficacy in several MTAP-deficient preclinical cancer models.

Biology of Stress Responses in Aging.

On April 28th, 2022, a group of scientific leaders gathered virtually to discuss molecular and cellular mechanisms of responses to stress. Conditions of acute, high-intensity stress are well documented to induce a series of adaptive responses that aim to promote survival until the stress has dissipated and then guide recovery. However, high-intensity or persistent stress that goes beyond the cell's compensatory capacity are countered with resilience strategies that are not completely understood. These adaptative strategies, which are an essential component of the study of aging biology, were the theme of the meeting. Specific topics discussed included mechanisms of proteostasis, such as the unfolded protein response (UPR) and the integrated stress response (ISR), as well as mitochondrial stress and lysosomal stress responses. Attention was also given to regulatory mechanisms and associated biological processes linked to age-related conditions, such as muscle loss and regeneration, cancer, senescence, sleep quality, and degenerative disease, with a general focus on the relevance of stress responses to frailty. We summarize the concepts and potential future directions that emerged from the discussion and highlight their relevance to the study of aging and age-related chronic diseases.