Synthesis and Evaluation of 11C- and 18F-Labeled SOAT1 Inhibitors as Macrophage Foam Cell Imaging Agents.

PD-132301, an inhibitor of sterol O-acyltransferase 1 (SOAT1; also known as acyl-coenzyme A:cholesterol acyltransferase-1, ACAT1), is under clinical investigation for numerous adrenal disorders. Radiolabeled SOAT1 inhibitors could support drug discovery and help diagnose SOAT1-related disorders, such as atherosclerosis. We synthesized two radiolabeled SOAT1 inhibitors, [11C]PD-132301 and fluorine analogue [18F]1. Rat biodistribution studies were conducted with both agents and, as the most selective tracer, [11C]PD-132301 was advanced to preclinical positron emission tomography studies in (atherosclerotic) ApoE-/- mice. The uptake of [11C]PD-132301 in SOAT1-rich tissue warrants further investigation into the compound as an atherosclerosis and adrenal imaging agent.

Synthesis and evaluation of NLRP3-inhibitory sulfonylurea [11C]MCC950 in healthy animals.

The diaryl sulfonylurea MCC950/CRID3 is a potent NLRP3 inhibitor (IC50 = 8 nM) and, in animal models, MCC950 protects against numerous NLRP3-related neurodegenerative disorders. To evaluate the brain uptake and investigate target engagement of MCC950, we synthesised [11C-urea]MCC950 via carrier added [11C]CO2 fixation chemistry (activity yield = 237 MBq; radiochemical purity >99%; molar activity = 7 GBq/µmol; radiochemical yield (decay-corrected from [11C]CO2) = 1.1%; synthesis time from end-of-bombardment = 31 min; radiochemically stable for >1 h). Despite preclinical efficacy in neurodegeneration studies, preclinical positron emission tomography (PET) imaging studies in mouse, rat and rhesus monkey revealed poor brain uptake of low molar activity [11C]MCC950 and rapid washout. In silico prediction tools suggest efflux transporter liabilities for MCC950 at microdoses, and this information should be taken into account when developing next generation NLRP3 inhibitors and/or PET radiotracers.

MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition.

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and specific small-molecule inhibitor of the NLRP3 pathway, but its molecular target is not defined. Here, we show that MCC950 directly interacts with the Walker B motif within the NLRP3 NACHT domain, thereby blocking ATP hydrolysis and inhibiting NLRP3 activation and inflammasome formation.

Fishing for Drug Targets: A Focus on Diazirine Photoaffinity Probe Synthesis.

Target identification is a high-priority, albeit challenging, aspect of drug discovery. Diazirine-based photoaffinity probes (PAPs) can facilitate the process by covalently capturing transient molecular interactions. This can help identify target proteins and map the ligand's interactome. Diazirine probes have even been incorporated by cellular machinery into proteins. Embarking on the synthesis of customized PAPs, containing either an aliphatic or trifluoromethyl phenyl diazirine, can be a considerable endeavor, particularly for medicinal chemists and chemical biologists new to the field. This review takes a synthetic focus, aiming to summarize available routes, propose new avenues, and illuminate recent advances in diazirine synthesis. Select examples of diazirine photoaffinity labeling applications have been included throughout to provide instructive definition of the advantages and limitations of the technology while simultaneously highlighting how these reagents can be applied in a practical sense.

Sulfonylureas as Concomitant Insulin Secretagogues and NLRP3 Inflammasome Inhibitors.

Insulin-secretory sulfonylureas are widely used, cost-effective treatments for type 2 diabetes (T2D). However, pancreatic ß-cells are continually depleted as T2D progresses, thereby rendering the sulfonylurea drug class ineffective in controlling glycaemia. Dysregulation of the innate immune system via activation of the NLRP3 inflammasome, and the consequent production of interleukin-1ß, has been linked to pancreatic ß-cell death and multiple inflammatory complications of T2D disease. One proposed strategy for treating T2D is the use of sulfonylurea insulin secretagogues that are also NLRP3 inhibitors. We report the synthesis and biological evaluation of nine sulfonylureas that inhibit NLRP3 activation in murine bone-marrow- derived macrophages in a potent, dose-dependent manner. Six of these compounds inhibited NLRP3 at nanomolar concentrations and can also stimulate insulin secretion from a murine pancreatic cell line (MIN6). These novel compounds possess unprecedented dual modes of action, paving the way for a new generation of sulfonylureas that may be useful as therapeutic candidates and/or tool compounds in T2D and its associated inflammatory complications.

The Endoplasmic Reticulum-Mitochondrion Tether ERMES Orchestrates Fungal Immune Evasion, Illuminating Inflammasome Responses to Hyphal Signals.

The pathogenic yeast Candida albicans escapes macrophages by triggering NLRP3 inflammasome-dependent host cell death (pyroptosis). Pyroptosis is inflammatory and must be tightly regulated by host and microbe, but the mechanism is incompletely defined. We characterized the C. albicans endoplasmic reticulum (ER)-mitochondrion tether ERMES and show that the ERMES mmm1 mutant is severely crippled in killing macrophages despite hyphal formation and normal phagocytosis and survival. To understand dynamic inflammasome responses to Candida with high spatiotemporal resolution, we established live-cell imaging for parallel detection of inflammasome activation and pyroptosis at the single-cell level. This showed that the inflammasome response to mmm1 mutant hyphae is delayed by 10 h, after which an exacerbated activation occurs. The NLRP3 inhibitor MCC950 inhibited inflammasome activation and pyroptosis by C. albicans, including exacerbated inflammasome activation by the mmm1 mutant. At the cell biology level, inactivation of ERMES led to a rapid collapse of mitochondrial tubular morphology, slow growth and hyphal elongation at host temperature, and reduced exposed 1,3-ß-glucan in hyphal populations. Our data suggest that inflammasome activation by C. albicans requires a signal threshold dependent on hyphal elongation and cell wall remodeling, which could fine-tune the response relative to the level of danger posed by C. albicans. The phenotypes of the ERMES mutant and the lack of conservation in animals suggest that ERMES is a promising antifungal drug target. Our data further indicate that NLRP3 inhibition by MCC950 could modulate C. albicans-induced inflammation. IMPORTANCE The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies.

Home blood pressure monitoring and self-titration of antihypertensive medications: Proposed patient selection criteria.

BACKGROUND AND PURPOSE: Recent studies have demonstrated that home blood pressure monitoring (HBPM), coupled with self-titration of medications is a viable intervention to control hypertension. There are currently no established criteria to evaluate patients for inclusion in such a program. The purpose of this discussion is to propose criteria for determining if a patient is appropriate to participate in a program of HBPM and self-titration. METHODS: Inclusion criteria for two self-titration trials were examined, and additional factors in clinical practice were identified and discussed. Additional selection criteria were proposed to support the decision to enroll a patient in an antihypertensive self-titration program. CONCLUSIONS: Inclusion criteria from self-titration trials provide a reasonable starting point for choosing appropriate patients in clinical practice, but additional research is necessary. Adaptation of these criteria and consideration of the identified factors can be used to develop decision support instruments. Such instruments should be evaluated for effectiveness and reliability prior to use in clinical practice. IMPLICATIONS FOR PRACTICE: HBPM combined with self-titration is an effective patient-centered approach for hypertension management. Decision support instruments to determine appropriate patients are necessary for safe and effective use in clinical practice.

Toxicity of a quinocarmycin analog, DX-52-1, in rats and dogs in relation to clinical outcome.

PURPOSE: Quinocarmycin analog DX-52-1 is a cyanated derivative of quinocarmycin, a compound isolated from cultures of Streptomyces melanovinaceus. DX-52-1 was selected for preclinical development because it showed efficacy against melanoma cell lines in the NCI human tumor cell screen and melanoma xenografts in mice. This report describes studies in rats and dogs to determine the maximum tolerated dose (MTD) and identify dose-limiting toxicities (DLT) in each species in different regimens to establish a safe starting dose and potential target organs of DX-52-1 for phase I clinical trials. METHODS: DX-52-1 was administered to Fischer 344 rats using repeated intravenous (i.v.) slow bolus injections following q3hx3 and q3hx3,q7dx3 regimens, and to beagle dogs using a single injection, 6-h continuous i.v. infusion (c.i.v.) and weekly 6-h c.i.v. for 3 weeks. Endpoints evaluated included clinical observations, body weights, hematology, serum clinical chemistry, and microscopic pathology of tissues. RESULTS: The MTD of DX-52-1 was a total dose of 18 mg/m(2) body surface area for q3hx3 administration in rats and 30 mg/m(2) for a single c.i.v. administration in dogs. The total dose MTD for rats on a weekly (q3hx3,q7dx3) regimen was 54 mg/m(2), and for dogs on the weekly x3 (6-h c.i.v.) infusion was 60 mg/m(2). In rats, significant elevations in blood urea nitrogen and creatinine were observed together with acute renal tubular necrosis histologically. Modest increases in liver enzymes were also observed, as were decreases in reticulocytes that were unaccompanied by histologic changes in liver and bone marrow. In dogs, adverse signs included vomiting/retching, diarrhea, and transient hypothermia; also red blood cells, hemoglobin, hematocrit, and lymphocytes were decreased. Histologic evaluation of tissues from dogs revealed necrosis and cellular depletion of the bone marrow, and extensive damage to the entire gastrointestinal tract, including marked cellular necrosis of the mucosa and lymphoid necrosis of the gastrointestinal associated lymphoid tissue. Destruction of the mucosal lining of the intestinal tract was likely responsible for dehydration, toxemia, septicemia, and shock seen in moribund dogs. CONCLUSIONS: The MTD values were comparable between rats and dogs given roughly similar dose regimens (single dose or weekly) and both species tolerated a higher total dose with weekly administration. However, the principal target organ responsible for DLT in rats was the kidney, whereas in dogs, the most severe effects were on the gastrointestinal tract and bone marrow. Both renal and gastrointestinal toxicities were reported in patients after 6-h c.i.v. infusions in a limited phase I clinical trial, indicating that neither animal model alone was predictive of DX-52-1-induced toxicity in humans, and that both species were required to define human toxicity.