In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection.

Iron is an essential metal for all organisms, yet disruption of its homeostasis, particularly in labile forms that can contribute to oxidative stress, is connected to diseases ranging from infection to cancer to neurodegeneration. Iron deficiency is also among the most common nutritional deficiencies worldwide. To advance studies of iron in healthy and disease states, we now report the synthesis and characterization of iron-caged luciferin-1 (ICL-1), a bioluminescent probe that enables longitudinal monitoring of labile iron pools (LIPs) in living animals. ICL-1 utilizes a bioinspired endoperoxide trigger to release d-aminoluciferin for selective reactivity-based detection of Fe2+ with metal and oxidation state specificity. The probe can detect physiological changes in labile Fe2+ levels in live cells and mice experiencing iron deficiency or overload. Application of ICL-1 in a model of systemic bacterial infection reveals increased iron accumulation in infected tissues that accompany transcriptional changes consistent with elevations in both iron acquisition and retention. The ability to assess iron status in living animals provides a powerful technology for studying the contributions of iron metabolism to physiology and pathology.

A reactivity-based probe of the intracellular labile ferrous iron pool.

Improved methods for studying intracellular reactive Fe(II) are of significant interest for studies of iron metabolism and disease-relevant changes in iron homeostasis. Here we describe a highly selective reactivity-based probe in which a Fenton-type reaction with intracellular labile Fe(II) leads to unmasking of the aminonucleoside puromycin. Puromycin leaves a permanent and dose-dependent mark on treated cells that can be detected with high sensitivity and precision using a high-content, plate-based immunofluorescence assay. Using this new probe and screening approach, we detected alteration of cellular labile Fe(II) in response extracellular iron conditioning, overexpression of iron storage and/or export proteins, and post-translational regulation of iron export. We also used this new tool to demonstrate that labile Fe(II) pools are larger in cancer cells than in nontumorigenic cells.

Toward a Ferrous Iron-Cleavable Linker for Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are antigen-targeted therapeutics that employ antibodies to deliver potent, cytotoxic effectors to cells with potentially high specificity. While promising clinical results have been achieved, significant pitfalls remain including internalization of ADCs in nontargeted cells expressing target antigen, which can limit therapeutic windows. Novel ADC linkers that are cleaved selectively in cancer cells but not in normal cells could minimize collateral damage caused by ADC uptake in nontargeted tissues. Here, we describe a prototypical ADC linker based on an Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX) that by itself has demonstrated tumor-selective activity in preclinical cancer models. We prepared TRX-linked ADCs by site-selective conjugation to two sites in trastuzumab and compared their activity in Her2 positive and negative cells to ADC controls based on established linker chemistry. Our results confirm that the TRX moiety efficiently releases its payload following ADC uptake, affording picomolar potencies in antigen-positive cells. We also identified a destabilizing interaction between these initial TRX linkers and nearby antibody residues and suggest an approach to improve upon these prototypical designs.

Development of a bioorthogonal fluorescence-based assay for assessing drug uptake and delivery in bacteria.

Bioorthogonal chemistry can facilitate the development of fluorescent probes that can be used to sensitively and specifically detect the presence of biological targets. In this study, such an assay was developed to evaluate the uptake and delivery of antimicrobials into Escherichia coli, building on and extending previous work which utilised more resource intensive LCMS detection. The bacteria were genetically engineered to express streptavidin in the periplasmic or cytoplasmic compartments, which was used to localise a bioorthogonal probe (BCN-biotin). Azido-compounds which are delivered to these compartments react with the localised BCN-biotin-streptavidin in a concentration-dependent manner via a strain-promoted alkyne-azide cycloaddition. The amount of azido-compound taken up by bacteria was determined by quantifying unreacted BCN-biotin-streptavidin via an inverse electron demand Diels-Alder reaction between remaining BCN-biotin and a tetrazine-containing fluorescent dye. Following optimisation and validation, the assay was used to assess uptake of liposome-formulated azide-functionalised luciferin and cefoxitin. The results demonstrated that formulation into cationic liposomes improved the uptake of azide-functionalised compounds into the periplasm of E. coli, providing insight on the uptake mechanism of liposomes in the bacteria. This newly developed bioorthogonal fluorescence plate-reader based assay provides a bioactivity-independent, medium-to-high throughput tool for screening compound uptake/delivery.

The Survival Benefit of Postoperative Bacterial Infections in Patients With Glioblastoma Multiforme: Myth or Reality?

Glioblastoma multiforme (GBM), the most common malignant brain tumor, universally carries a poor prognosis. Despite aggressive multimodality treatment, the median survival is ~18-20 months, depending on molecular subgroups. A long history of observations suggests antitumor effects of bacterial infections against malignant tumors. The present review summarizes and critically analyzes the clinical data providing evidence for or against the survival benefit of post-operative bacterial infections in GBM patients. Furthermore, we explore the probable underlying mechanism(s) from basic science studies on the topic. There are plausible explanations from immunobiology for the mechanism of the "favorable effect" of bacterial infections in GBM patients. However, available clinical literature does not provide a definitive association between postoperative bacterial infection and prolonged survival in GBM patients. The presently available, single-/multi-center and national database retrospective case-control studies on the topic provide conflicting results. A prospective randomized study on the subject is clearly not possible. Immunobiology literature supports development of genetically modified bacteria as part of multimodal regimen against GBM.

Cognitive Impairment After Intracerebral Hemorrhage: A Systematic Review and Meta-Analysis.

OBJECTIVE: The present systematic review and meta-analysis analyzes the available clinical literature on post-intracerebral hemorrhage (ICH) cognitive impairment. METHODS: We conducted a systematic review with meta-analysis following PRISMA guidelines. A search of bibliographic databases up to July 31, 2020 yielded 2155 studies. Twenty articles were included in our final qualitative systematic review and 18 articles in quantitative meta-analysis. RESULTS: Based on analysis of data from 18 studies (3270 patients), we found prevalence of post-ICH cognitive impairment to be 46% (confidence interval, 35.9-55.9), with a follow-up duration ranging from 8 days to 4 years. The estimated pooled prevalence of cognitive decline decreased over longitudinal follow-up, from 55% (range, 37.7%-71.15%) within 6 months of ICH to 35% (range, 27%-42.7%) with >6 months to 4 years follow-up after ICH. The modalities used to evaluate cognitive performance after ICH in studies varied widely, ranging from global cognitive measures to domain-specific testing. The cognitive domain most commonly affected included nonverbal IQ, information processing speed, executive function, memory, language, and visuoconstructive abilities. Prognostic factors for poor cognitive performance included severity of cortical atrophy, age, lobar ICH location, and higher number of hemorrhages at baseline. CONCLUSIONS: The prevalence of post-ICH cognitive impairment is high. Despite the heterogeneity among studies, the present study identified cognitive domains most commonly affected and predictors of cognitive impairment after ICH. In future, prospective cohort studies with larger sample sizes and standardized cognitive domains testing could more accurately determine prevalence and prognostic factors of post-ICH cognitive decline.

A Unified Framework for the Incorporation of Bioorthogonal Compound Exposure Probes within Biological Compartments.

Compartmentalization is a crucial facet of many biological systems, and key aspects of cellular processes rely on spatial segregation within the cell. While many drug targets reside in specific intracellular compartments, the tools available for assessing compound exposure are generally limited to whole-cell measurements. To address this gap, we recently developed a bioorthogonal chemistry-based method to assess compartment-specific compound exposure and demonstrated its use in Gram-negative bacteria. To expand the applicability of this approach, we report here novel bioorthogonal probe modalities which enable diverse probe incorporation strategies. The probes we developed utilize a cleavable thiocarbamate linker to connect localizing elements such as metabolic substrates to a cyclooctyne moiety which enables the detection of azide-containing molecules. Adducts between the probe and azide-bearing compounds can be recovered and affinity purified after exposure experiments, thus facilitating the mass-spectrometry based analysis used to assess compound exposure. The bioorthogonal system reported here thus provides a valuable new tool for interrogating compartment-specific compound exposure in a variety of biological contexts while retaining a simple and unified sample preparation and analysis workflow.

Molecular Probes for the Determination of Subcellular Compound Exposure Profiles in Gram-Negative Bacteria.

The Gram-negative cell envelope presents a formidable barrier to xenobiotics, and achieving sufficient compound exposure inside the cell is a key challenge for the discovery of new antibiotics. To provide insight on the molecular determinants governing compound exposure in Gram-negative bacteria, we developed a methodology leveraging a cyclooctyne-based bioorthogonal probe to assess compartment-specific compound exposure. This probe can be selectively localized to the periplasmic or cytoplasmic compartments of Gram-negative bacteria. Once localized, the probe is used to test azide-containing compounds for exposure within each compartment by quantifying the formation of click-reaction products by mass spectrometry. We demonstrate this approach is an accurate and sensitive method of determining compartment-specific compound exposure profiles. We then apply this technology to study the compartment-specific exposure profiles of a small panel of azide-bearing compounds with known permeability characteristics in Gram-negative bacteria, demonstrating the utility of the system and the insight it is able to provide regarding compound exposure within intact bacteria.

A Novel Tumor-Activated Prodrug Strategy Targeting Ferrous Iron Is Effective in Multiple Preclinical Cancer Models.

Here we describe a new approach for tumor targeting in which augmented concentrations of Fe(II) in cancer cells and/or the tumor microenvironment triggers drug release from an Fe(II)-reactive prodrug conjugate. The 1,2,4-trioxolane scaffold developed to enable this approach can in principle be applied to a broad range of cancer therapeutics and is illustrated here with Fe(II)-targeted forms of a microtubule toxin and a duocarmycin-class DNA-alkylating agent. We show that the intrinsic reactivity/toxicity of the duocarmycin analog is masked in the conjugated form and this greatly reduced toxicity in mice. This in turn permitted elevated dosing levels, leading to higher systemic exposure and a significantly improved response in tumor xenograft models. Overall our results suggest that Fe(II)-dependent drug delivery via trioxolane conjugates could have significant utility in expanding the therapeutic index of a range of clinical and preclinical stage cancer chemotherapeutics.

Drug delivery to the malaria parasite using an arterolane-like scaffold.

Antimalarial agents artemisinin and arterolane act via initial reduction of a peroxide bond in a process likely mediated by ferrous iron sources in the parasite. Here, we report the synthesis and antiplasmodial activity of arterolane-like 1,2,4-trioxolanes specifically designed to release a tethered drug species within the malaria parasite. Compared with our earlier drug delivery scaffolds, these new arterolane-inspired systems are of significantly decreased molecular weight and possess superior metabolic stability. We describe an efficient, concise and scalable synthesis of the new systems, and demonstrate the use of the aminonucleoside antibiotic puromycin as a chemo/biomarker to validate successful drug release in live Plasmodium falciparum parasites. Together, the improved drug-like properties, more efficient synthesis, and proof of concept using puromycin, suggests these new molecules as improved vehicles for targeted drug delivery to the malaria parasite.

HNF4α antagonists discovered by a high-throughput screen for modulators of the human insulin promoter.

Hepatocyte nuclear factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic ß cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and were proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.