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Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.

The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM (“SAR by X-ray Poses Quickly”) platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or “undruggable” targets, allows for (i) hit generation; (ii) the mapping of protein–ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.

CRISPR-Cas9 gene editing can induce a p53 mediated damage response. Here the authors investigate the possibility of selection of pre-existing cancer driver mutations during CRISPR-Cas9 knockout based gene editing and identify KRAS mutants that may confer a selected advantage to edited cells.

Aging is characterized by the accumulation of damage to macromolecules and cell architecture that triggers a proinflammatory state in blood and solid tissues, termed inflammaging. Inflammaging has been implicated in the pathogenesis of many age-associated chronic diseases as well as loss of physical and cognitive function. The search for mechanisms that underlie inflammaging focused initially on the hallmarks of aging, but it is rapidly expanding in multiple directions. Here, we discuss the threads connecting cellular senescence and mitochondrial dysfunction to impaired mitophagy and DNA damage, which may act as a hub for inflammaging. We explore the emerging multi-omics efforts that aspire to define the complexity of inflammaging — and identify molecular signatures and novel targets for interventions aimed at counteracting excessive inflammation and its deleterious consequences while preserving the physiological immune response. Finally, we review the emerging evidence that inflammation is involved in brain aging and neurodegenerative diseases. Our goal is to broaden the research agenda for inflammaging with an eye on new therapeutic opportunities.

Targeted nanotheranostic systems offer significant benefits due to the integration of diagnostic and therapeutic functionality, promoting personalized medicine. In recent years, prostate-specific membrane antigen (PSMA) has emerged as an ideal theranostic target, fueling multiple new drug approvals and changing the standard of care in prostate cancer (PCa). PSMA-targeted nanosystems such as self-assembled nanoparticles (NPs), liposomal structures, water-soluble polymers, dendrimers, and other macromolecules are under development for PCa theranostics due to their multifunctional sensing and therapeutic capabilities. Herein, we discuss the significance and up-to-date development of “PSMA-targeted nanocarrier systems for radioligand imaging and therapy of PCa”. The review also highlights critical parameters for designing nanostructured radiopharmaceuticals for PCa, including radionuclides and their chelators, PSMA-targeting ligands, and the EPR effect. Finally, prospects and potential for clinical translation is discussed.

Tools for noninvasive detection of bacterial pathogens are needed but are not currently available for clinical use. We have previously shown that para-aminobenzoic acid (PABA) rapidly accumulates in a wide range of pathogenic bacteria, motivating the development of related PET radiotracers. In this study, 11C-PABA PET imaging was used to accurately detect and monitor infections due to pyogenic bacteria in multiple clinically relevant animal models. 11C-PABA PET imaging selectively detected infections in muscle, intervertebral discs, and methicillin-resistant Staphylococcus aureus–infected orthopedic implants. In what we believe to be first-in-human studies in healthy participants, 11C-PABA was safe, well-tolerated, and had a favorable biodistribution, with low background activity in the lungs, muscles, and brain. 11C-PABA has the potential for clinical translation to detect and localize a broad range of bacteria.

Androgen receptor (AR) mediated transcription is critical to prostate tumorigenesis and development. Here, utilising clinical whole genome sequencing data, the authors show that the non-coding AR binding sites on DNA are frequently mutated in prostate cancer potentially due to faulty base excision repair mechanisms

Polymerase eta (or Pol η or POLH) is a specialized DNA polymerase that is able to bypass certain blocking lesions, such as those generated by ultraviolet radiation (UVR) or cisplatin, and is deployed to replication foci for translesion synthesis as part of the DNA damage response (DDR). Inherited defects in the gene encoding POLH (a.k.a., XPV) are associated with the rare, sun-sensitive, cancer-prone disorder, xeroderma pigmentosum, owing to the enzyme’s ability to accurately bypass UVR-induced thymine dimers. In standard-of-care cancer therapies involving platinum-based clinical agents, e.g., cisplatin or oxaliplatin, POLH can bypass platinum-DNA adducts, negating benefits of the treatment and enabling drug resistance. POLH inhibition can sensitize cells to platinum-based chemotherapies, and the polymerase has also been implicated in resistance to nucleoside analogs, such as gemcitabine. POLH overexpression has been linked to the development of chemoresistance in several cancers, including lung, ovarian, and bladder. Co-inhibition of POLH and the ATR serine/threonine kinase, another DDR protein, causes synthetic lethality in a range of cancers, reinforcing that POLH is an emerging target for the development of novel oncology therapeutics. Using a fragment-based drug discovery approach in combination with an optimized crystallization screen, we have solved the first X-ray crystal structures of small novel drug-like compounds, i.e., fragments, bound to POLH, as starting points for the design of POLH inhibitors. The intrinsic molecular resolution afforded by the method can be quickly exploited in fragment growth and elaboration as well as analog scoping and scaffold hopping using medicinal and computational chemistry to advance hits to lead. An initial small round of medicinal chemistry has resulted in inhibitors with a range of functional activity in an in vitro biochemical assay, leading to the rapid identification of an inhibitor to advance to subsequent rounds of chemistry to generate a lead compound. Importantly, our chemical matter is different from the traditional nucleoside analog-based approaches for targeting DNA polymerases.

Oxidative stress occurs at various phases of spinal cord injury (SCI), promoting detrimental processes such as free radical injury of proteins, nucleic acids, lipids, cytoskeleton, and organelles. Oxidative DNA damage is likely a major contributor to the pathogenesis of SCI, as a damaged genome cannot be simply turned over to avert detrimental molecular and cellular outcomes, most notably cell death. Surprisingly, the evidence to support this hypothesis is limited. There is some evidence that oxidative DNA damage is increased following SCI, mainly using comet assays and immunohistochemistry. However, there is great variability in the timing and magnitude of its appearance, likely due to differences in experimental models, measurement techniques, and the rigor of the approach. Evidence indicates that 8-oxodG is most abundant at 1 and 7 days post-injury (dpi), while DNA strand breaks peak at 7 and 28 dpi. The DNA damage response seems to be characterized by upregulation of PCNA and PARP1 but downregulation of APEX1. Significant improvements in the analysis of oxidative DNA damage and repair after SCI, including single-cell analysis at time points representative for each phase post-injury using new methodologies and better reporting, will uncover the role of DNA damage and repair in SCI.

IntroductionThe pentose phosphate pathway (PPP) is essential for NADPH generation and redox homeostasis in cancer, including glioblastomas. However, the precise contribution to redox and tumor proliferation of the second PPP enzyme 6-phosphogluconolactonase (PGLS), which converts 6-phospho-δ-gluconolactone to 6-phosphogluconate (6PG), remains unclear. Furthermore, non-invasive methods of assessing PGLS activity are lacking. The goal of this study was to examine the role of PGLS in glioblastomas and assess the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive imaging.MethodsTo interrogate the function of PGLS in redox, PGLS expression was silenced in U87, U251 and GS2 glioblastoma cells by RNA interference and levels of NADPH and reduced glutathione (GSH) measured. Clonogenicity assays were used to assess the effect of PGLS silencing on glioblastoma proliferation. Hyperpolarized δ-[1-13C]gluconolactone metabolism to 6PG was assessed in live cells treated with the chemotherapeutic agent temozolomide (TMZ) or with vehicle control. 13C 2D echo-planar spectroscopic imaging (EPSI) studies of hyperpolarized δ-[1-13C]gluconolactone metabolism were performed on rats bearing orthotopic glioblastoma tumors or tumor-free controls on a 3T spectrometer. Longitudinal 2D EPSI studies of hyperpolarized δ-[1-13C]gluconolactone metabolism and T2-weighted magnetic resonance imaging (MRI) were performed in rats bearing orthotopic U251 tumors following treatment with TMZ to examine the ability of hyperpolarized δ-[1-13C]gluconolactone to report on treatment response.ResultsPGLS knockdown downregulated NADPH and GSH, elevated oxidative stress and inhibited clonogenicity in all models. Conversely, PGLS expression and activity and steady-state NADPH and GSH were higher in tumor tissues from rats bearing orthotopic glioblastoma xenografts relative to contralateral brain and tumor-free brain. Importantly, [1-13C]6PG production from hyperpolarized δ-[1-13C]gluconolactone was observed in live glioblastoma cells and was significantly reduced by treatment with TMZ. Furthermore, hyperpolarized δ-[1-13C]gluconolactone metabolism to [1-13C]6PG could differentiate tumor from contralateral normal brain in vivo. Notably, TMZ significantly reduced 6PG production from hyperpolarized δ-[1-13C]gluconolactone at an early timepoint prior to volumetric alterations as assessed by anatomical imaging.ConclusionsCollectively, we have, for the first time, identified a role for PGLS activity in glioblastoma proliferation and validated the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive in vivo imaging of glioblastomas and their response to therapy.

Neurological complications directly impact the lives of hundreds of millions of people worldwide. While the precise molecular mechanisms that underlie neuronal cell loss remain under debate, evidence indicates that the accumulation of genomic DNA damage and consequent cellular responses can promote apoptosis and neurodegenerative disease. This idea is supported by the fact that individuals who harbor pathogenic mutations in DNA damage response genes experience profound neuropathological manifestations. The review article here provides a general overview of the nervous system, the threats to DNA stability, and the mechanisms that protect genomic integrity while highlighting the connections of DNA repair defects to neurological disease. The information presented should serve as a prelude to the Special Issue “Genome Stability and Neurological Disease”, where experts discuss the role of DNA repair in preserving central nervous system function in greater depth.

High expression of Mcl-1 promotes tumorigenesis and resistance to anticancer therapies. Here they report a macrocyclic molecule with high selectivity and affinity for Mcl-1 that exhibits potent anti-tumor effects as single agent and in combination with bortezomib or venetoclax in preclinical models of multiple myeloma and acute myeloid leukemia.

The growing demand for metabolism-specific imaging techniques has rekindled interest in Deuterium (2H) Metabolic Imaging (DMI), a robust method based on administration of a substrate (glucose, acetate, fumarate, etc.) labeled with the stable isotope of hydrogen and the observation of its metabolic fate in three-dimensions. This technique allows the investigation of multiple metabolic processes in both healthy and diseased states. Despite its low natural abundance, the short relaxation time of deuterium allows for rapid radiofrequency (RF) pulses without saturation and efficient image acquisition. In this review, we provide a comprehensive picture of the evolution of DMI over the course of recent decades, with a special focus on its potential clinical applications.

FEN1 plays critical roles in long patch base excision repair (LP-BER), Okazaki fragment maturation, and rescue of stalled replication forks. In a clinical cohort, FEN1 overexpression is associated with aggressive phenotype and poor progression-free survival after platinum chemotherapy. Pre-clinically, FEN1 is induced upon cisplatin treatment, and nuclear translocation of FEN1 is dependent on physical interaction with importin β. FEN1 depletion, gene inactivation, or inhibition re-sensitizes platinum-resistant ovarian cancer cells to cisplatin. BRCA2 deficient cells exhibited synthetic lethality upon treatment with a FEN1 inhibitor. FEN1 inhibitor-resistant PEO1R cells were generated, and these reactivated BRCA2 and overexpressed the key repair proteins, POLβ and XRCC1. FEN1i treatment was selectively toxic to POLβ deficient but not XRCC1 deficient ovarian cancer cells. High throughput screening of 391,275 compounds identified several FEN1 inhibitor hits that are suitable for further drug development. We conclude that FEN1 is a valid target for ovarian cancer therapy.

Abstract Occult bacterial infections represent a worldwide health problem. Differentiating active bacterial infection from sterile inflammation can be difficult using current imaging tools. Present clinically viable methodologies either detect morphologic changes (CT/ MR), recruitment of immune cells (111In-WBC SPECT), or enhanced glycolytic flux seen in inflammatory cells (18F-FDG PET). However, these strategies are often inadequate to detect bacterial infection and are not specific for living bacteria. Recent approaches have taken advantage of key metabolic differences between prokaryotic and eukaryotic organisms, allowing easier distinction between bacteria and their host. In this report, we exploited one key difference, bacterial cell wall biosynthesis, to detect living bacteria using a positron-labeled D-amino acid. After screening several 14C D-amino acids for their incorporation into E. coli in culture, we identified D-methionine as a probe with outstanding radiopharmaceutical potential. Based on an analogous procedure to that used for L-[methyl-11C]methionine ([11C] L-Met), we developed an enhanced asymmetric synthesis of D-[methyl-11C]methionine ([11C] D-Met), and showed that it can rapidly and selectively differentiate both E. coli and S. aureus infections from sterile inflammation in vivo. We believe that the ease of [11C] D-Met radiosynthesis, coupled with its rapid and specific in vivo bacterial accumulation, make it an attractive radiotracer for infection imaging in clinical practice.