Novel inhibitors of the bacterial de novo purine biosynthesis enzymes, n5-carboxyaminoimidazole ribonucleotide synthetase and mutase

Antibiotic resistance has seen a significant increase during the past decade. The increasing frequency of the drug-resistant bacterial infections has amplified the need for novel antimicrobial agents. De novo purine biosynthesis is one area that has great potential for antibacterial drug development because this pathway is different in microorganisms versus humans. The difference in the pathway is centered on the synthesis and utilization of the purine intermediate N5-carboxy-5-aminoimidazole ribonucleotide (N5-CAIR). Previous studies have shown that N5-CAIR is a key intermediate in purine biosynthesis in bacteria, yeast and fungi, but not in humans. N5-CAIR is synthesized from 5-aminoimidazole ribonucleotide (AIR) by the enzyme N5-CAIR synthetase and it is utilized by N5-CAIR mutase to produce the intermediate 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). In our laboratory we explored both enzymes as potential targets for the design of novel de novo purine biosynthesis inhibitors. Previous studies suggested that the isatin-based inhibitors were promising low micromolar inhibitors of N5-CAIR synthetase. Here, the biological verification of the isatin compounds as potential hits and their kinetic analysis are presented. The second project involves the discovery, kinetic evaluation, molecular modeling, and exploratory synthesis of the first known, selective inhibitor of N5-CAIR mutase

Futureproofing [18F]Fludeoxyglucose manufacture at an Academic Medical Center

Abstract Background We recently upgraded our [18F]fludeoxyglucose (FDG) production capabilities with the goal of futureproofing our FDG clinical supply, expanding the number of batches of FDG we can manufacture each day, and improving patient throughput in our nuclear medicine clinic. In this paper we report upgrade of the synthesis modules to the GE FASTLab 2 platform (Phase 1) and cyclotron updates (Phase 2) from both practical and regulatory perspectives. We summarize our experience manufacturing FDG on the FASTLab 2 module with a high-yielding self-shielded niobium (Nb) fluorine-18 target. Results Following installation of Nb targets for production of fluorine-18, a 55 μA beam for 22 min generated 1330 ± 153 mCi of [18F]fluoride. Using these cyclotron beam parameters in combination with the FASTLab 2, activity yields (AY) of FDG were 957 ± 102 mCi at EOS, corresponding to 72% non-corrected AY (n = 235). Our workflow, inventory management and regulatory compliance have been greatly simplified following the synthesis module and cyclotron upgrades, and patient wait times for FDG PET have been cut in half at our nuclear medicine clinic. Conclusions The combination of FASTlab 2 and self-shielded Nb fluorine-18 targets have improved our yield of FDG, and enabled reliable and repeatable manufacture of the radiotracer for clinical use.https://deepblue.lib.umich.edu/bitstream/2027.42/145727/1/41181_2018_Article_48.pd

Phospholipids impact the protective effects of HDL-mimetic nanodiscs against lipopolysaccharide-induced inflammation - supplementary dataset

Aim: The impacts of synthetic high-density lipoprotein (sHDL) phospholipid components on anti-sepsis effects were investigated. Methods: sHDL composed with ApoA-I mimetic peptide (22A) and different phosphatidylcholines were prepared and characterized. Anti-inflammatory effects were investigated in vitro and in vivo on lipopolysaccharide (LPS)-induced inflammation models. Results: sHDLs composed with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (22A-DMPC) most effectively neutralizes LPS, inhibits toll-like receptor 4 recruitment into lipid rafts, suppresses nuclear factor κB signaling and promotes activating transcription factor 3 activating. The lethal endotoxemia animal model showed the protective effects of 22A-DMPC. Conclusion: Phospholipid components affect the stability and fluidity of nanodiscs, impacting the anti-septic efficacy of sHDLs. 22A-DMPC presents the strongest LPS binding and anti-inflammatory effects in vitro and in vivo, suggesting a potential sepsis treatment.Plain language summary: Sepsis is triggered by endotoxins released by bacteria. These endotoxins trigger an exaggerated inflammatory response, leading to widespread inflammation and organ damage. Synthetic high-density lipoprotein (sHDL) is a potential treatment of sepsis by neutralizing endotoxins and regulating inflammatory responses. The phospholipid components of sHDL may affect the effectiveness of sHDL against sepsis. In this study, we prepared sHDLs with different phospholipids and compared their anti-septic effects on cells and in animal models. We found that sHDL made from DMPC presented the best anti-septic effects, possibly because DMPC-sHDL had the best fluidity at body temperature.</p

Synthesis and Evaluation of [¹⁸F]RAGER: A First Generation Small-Molecule PET Radioligand Targeting the Receptor for Advanced Glycation Endproducts

The receptor for advanced glycation endproducts (RAGE) is a 35 kDa transmembrane receptor that belongs to the immunoglobulin superfamily of cell surface molecules. Its role in Alzheimer’s disease (AD) is complex, but it is thought to mediate influx of circulating amyloid-β into the brain as well as amplify Aβ-induced pathogenic responses. RAGE is therefore of considerable interest as both a diagnostic and a therapeutic target in AD. Herein we report the synthesis and preliminary preclinical evaluation of [¹⁸F]­RAGER, the first small molecule PET radiotracer for RAGE (<i>K</i>_d = 15 nM). Docking studies proposed a likely binding interaction between RAGE and RAGER, [¹⁸F]­RAGER autoradiography showed colocalization with RAGE identified by immunohistochemistry in AD brain samples, and [¹⁸F]­RAGER microPET confirmed CNS penetration and increased uptake in areas of the brain known to express RAGE. This first generation radiotracer represents initial proof-of-concept and a promising first step toward quantifying CNS RAGE activity using PET. However, there were high levels of nonspecific [¹⁸F]­RAGER binding <i>in vitro</i>, likely due to its high log <i>P</i> (experimental log <i>P</i> = 3.5), and rapid metabolism of [¹⁸F]­RAGER in rat liver microsome studies. Therefore, development of second generation ligands with improved imaging properties would be advantageous prior to anticipated translation into clinical PET imaging studies

Effect of Synthetic High Density Lipoproteins Modification with Polyethylene Glycol on Pharmacokinetics and Pharmacodynamics

Synthetic high density lipoprotein nanoparticles (sHDLs) capable of mobilizing excess cholesterol from atherosclerotic arteries and delivering it to the liver for elimination have been shown to reduce plaque burden in patients. Unfortunately, sHDLs have a narrow therapeutic index and relative to the endogenous HDL shorter circulation half-life. Surface modification with polyethylene glycol (PEG) was investigated for its potential to extend sHDL circulation <i>in vivo</i>. Various amounts (2.5, 5, and 10%) and different chain lengths (2 and 5 kDa) of PEG-modified lipids were incorporated in sHDL’s lipid membrane. Incorporating PEG did not reduce the ability of sHDL to facilitate cholesterol efflux, nor did it inhibit cholesterol uptake by the liver cells. By either adding more PEG or using PEG of longer chain lengths, the circulation half-life was extended. Addition of PEG also increased the area under the curve for the phospholipid component of sHDL (<i>p</i> < 0.05), but not for the apolipoprotein A-I peptide component of sHDL, suggesting sHDL is remodeled by endogenous lipoproteins <i>in vivo</i>. The extended phospholipid circulation led to a higher mobilization of plasma free cholesterol, a biomarker for facilitation of reverse cholesterol transport. The area under the cholesterol mobilization increased about 2–4-fold (<i>p</i> < 0.05), with greater increases observed for longer PEG chains and higher molar percentages of incorporated PEGylated lipids. Mobilized cholesterol was associated primarily with the HDL fraction, led to a transient increase in VLDL cholesterol, and returned to baseline 24 h postdose. Overall, PEGylation of sHDL led to beneficial changes in sHDL particle pharmacokinetic and pharmacodynamic behaviors

Targeting Metal-Aβ Aggregates with Bifunctional Radioligand [¹¹C]L2‑b and a Fluorine-18 Analogue [¹⁸F]FL2‑b

Interest in quantifying metal-Aβ species <i>in vivo</i> led to the synthesis and evaluation of [¹¹C]­L2-b and [¹⁸F]­FL2-b as radiopharmaceuticals for studying the metallobiology of Alzheimer’s disease (AD) using positron emission tomography (PET) imaging. [¹¹C]­L2-b was synthesized in 3.6% radiochemical yield (nondecay corrected, <i>n</i> = 3), >95% radiochemical purity, from the corresponding desmethyl precursor. [¹⁸F]­FL2-b was synthesized in 1.0% radiochemical yield (nondecay corrected, <i>n</i> = 3), >99% radiochemical purity, from a 6-chloro pyridine precursor. Autoradiography experiments with AD positive and healthy control brain samples were used to determine the specificity of binding for the radioligands compared to [¹¹C]­PiB, a known imaging agent for β-amyloid (Aβ) aggregates. The <i>K</i>_d for [¹¹C]­L2-b and [¹⁸F]­FL2-b were found to be 3.5 and 9.4 nM, respectively, from those tissue studies. Displacement studies of [¹¹C]­L2-b and [¹⁸F]­FL2-b with PiB and AV-45 determined that L2-b binds to Aβ aggregates differently from known radiopharmaceuticals. Finally, brain uptake of [¹¹C]­L2-b was examined through microPET imaging in healthy rhesus macaque, which revealed a maximum uptake at 2.5 min (peak SUV = 2.0) followed by rapid egress (<i>n</i> = 2)

Synthetic high-density lipoprotein nanoparticles for the treatment of Niemann–Pick diseases

Abstract Background Niemann–Pick disease type C is a fatal and progressive neurodegenerative disorder characterized by the accumulation of unesterified cholesterol in late endosomes and lysosomes. We sought to develop new therapeutics for this disorder by harnessing the body’s endogenous cholesterol scavenging particle, high-density lipoprotein (HDL). Methods Here we design, optimize, and define the mechanism of action of synthetic HDL (sHDL) nanoparticles. Results We demonstrate a dose-dependent rescue of cholesterol storage that is sensitive to sHDL lipid and peptide composition, enabling the identification of compounds with a range of therapeutic potency. Peripheral administration of sHDL to Npc1 I1061T homozygous mice mobilizes cholesterol, reduces serum bilirubin, reduces liver macrophage size, and corrects body weight deficits. Additionally, a single intraventricular injection into adult Npc1 I1061T brains significantly reduces cholesterol storage in Purkinje neurons. Since endogenous HDL is also a carrier of sphingomyelin, we tested the same sHDL formulation in the sphingomyelin storage disease Niemann–Pick type A. Utilizing stimulated Raman scattering microscopy to detect endogenous unlabeled lipids, we show significant rescue of Niemann–Pick type A lipid storage. Conclusions Together, our data establish that sHDL nanoparticles are a potential new therapeutic avenue for Niemann–Pick diseases.https://deepblue.lib.umich.edu/bitstream/2027.42/152143/1/12916_2019_Article_1423.pd