Emergent properties of melanin-inspired peptide/RNA condensates.

Most biocatalytic processes in eukaryotic cells are regulated by subcellular microenvironments such as membrane-bound or membraneless organelles. These natural compartmentalization systems have inspired the design of synthetic compartments composed of a variety of building blocks. Recently, the emerging field of liquid-liquid phase separation has facilitated the design of biomolecular condensates composed of proteins and nucleic acids, with controllable properties including polarity, diffusivity, surface tension, and encapsulation efficiency. However, utilizing phase-separated condensates as optical sensors has not yet been attempted. Here, we were inspired by the biosynthesis of melanin pigments, a key biocatalytic process that is regulated by compartmentalization in organelles, to design minimalistic biomolecular condensates with emergent optical properties. Melanins are ubiquitous pigment materials with a range of functionalities including photoprotection, coloration, and free radical scavenging activity. Their biosynthesis in the confined melanosomes involves oxidation-polymerization of tyrosine (Tyr), catalyzed by the enzyme tyrosinase. We have now developed condensates that are formed by an interaction between a Tyr-containing peptide and RNA and can serve as both microreactors and substrates for tyrosinase. Importantly, partitioning of Tyr into the condensates and subsequent oxidation-polymerization gives rise to unique optical properties including far-red fluorescence. We now demonstrate that individual condensates can serve as sensors to detect tyrosinase activity, with a limit of detection similar to that of synthetic fluorescent probes. This approach opens opportunities to utilize designer biomolecular condensates as diagnostic tools for various disorders involving abnormal enzymatic activity.

Curcumin-QingDai Combination for Patients With Active Ulcerative Colitis: A Randomized, Double-Blinded, Placebo-Controlled Trial.

BACKGROUND & AIMS: We evaluated the efficacy of herbal combination of curcumin-QingDai (CurQD) in active ulcerative colitis (UC). METHODS: Part I was an open-label trial of CurQD in patients with active UC, defined by a Simple Clinical Colitis Activity Index score of 5 or higher and a Mayo endoscopic subscore of 2 or higher. Part II was a placebo-controlled trial conducted in Israel and Greece, randomizing active UC patients at a 2:1 ratio to enteric-coated CurQD 3 g/d or placebo for 8 weeks. The co-primary outcome was clinical response (reduction in the Simple Clinical Colitis Activity Index of ≥3 points) and an objective response (Mayo endoscopic subscore improvement of ≥1 or a 50% fecal calprotectin reduction). Responding patients continued either maintenance curcumin or placebo alone for an additional 8 weeks. Aryl-hydrocarbon receptor activation was assessed by cytochrome P450 1A1 (CYP1A1) mucosal expression. RESULTS: In part I, 7 of 10 patients responded and 3 of 10 achieved clinical remission. Of 42 patients in part II, the week 8 co-primary outcome was achieved in 43% and 8% of CurQD and placebo patients, respectively (P = .033). Clinical response was observed in 85.7% vs 30.7% (P < .001), clinical remission in 14 of 28 (50%) vs 1 of 13 (8%; P = .01), a 50% calprotectin reduction in 46.4% vs 15.4% (P = .08), and endoscopic improvement in 75% vs 20% (P = .036) in the CurQD and placebo groups, respectively. Adverse events were comparable between groups. By week 16, curcumin-maintained clinical response, clinical remission, and clinical biomarker response rates were 93%, 80%, and 40%, respectively. CurQD uniquely up-regulated mucosal CYP1A1 expression, which was not observed among patients receiving placebo, mesalamine, or biologics. CONCLUSIONS: In this placebo-controlled trial, CurQD was effective for inducing response and remission in active UC patients. The aryl-hydrocarbon receptor pathway may merit further study as a potential UC treatment target. CLINICALTRIALS: gov ID: NCT03720002.

A duplex structure of SARM1 octamers stabilized by a new inhibitor.

In recent years, there has been growing interest in SARM1 as a potential breakthrough drug target for treating various pathologies of axon degeneration. SARM1-mediated axon degeneration relies on its TIR domain NADase activity, but recent structural data suggest that the non-catalytic ARM domain could also serve as a pharmacological site as it has an allosteric inhibitory function. Here, we screened for synthetic small molecules that inhibit SARM1, and tested a selected set of these compounds in a DRG axon degeneration assay. Using cryo-EM, we found that one of the newly discovered inhibitors, a calmidazolium designated TK106, not only stabilizes the previously reported inhibited conformation of the octamer, but also a meta-stable structure: a duplex of octamers (16 protomers), which we have now determined to 4.0 Å resolution. In the duplex, each ARM domain protomer is engaged in lateral interactions with neighboring protomers, and is further stabilized by contralateral contacts with the opposing octamer ring. Mutagenesis of the duplex contact sites leads to a moderate increase in SARM1 activation in cultured cells. Based on our data we propose that the duplex assembly constitutes an additional auto-inhibition mechanism that tightly prevents pre-mature activation and axon degeneration.

Joint Action of Pb1 and Pb2 Provides Dominant Complementary Resistance Against New Races of Peronospora belbahrii (Basil Downy Mildew).

Sweet basil (Ocimum basilicum, 2n = 4x = 48) is susceptible to downy mildew caused by Peronospora belbahrii. The Pb1 gene exhibits complete resistance to the disease. However, Pb1 became prone to disease because of occurrence of new virulent races. Here, we show that Zambian accession PI 500950 (Ocimum americanum var. pilosum) is highly resistant to the new races. From an interspecies backcross between PI 500950 and the susceptible 'Sweet basil' we obtained, by embryo rescue, a population of 131 BC1F1 plants. This population segregated 73 resistant (58) and susceptible (1:1; P = 0.22) plants, suggesting that resistance is controlled by one incompletely dominant gene called Pb2. To determine whether allelic relationship exists between Pb1 and Pb2, we used two differential races: race 0, which is avirulent to both PI 500945 (Pb1) and PI 500950 (Pb2), and race 1, which is virulent to PI 500945 but avirulent to PI 500950. F1 plants obtained from '12-4-6' (BC6F3 derived from PI 500945) and '56' (BC3F3 derived from PI 500950) showed resistant superiority to both races through dominant complementary interaction. F2 plants segregated to race 0 as follows: 12:3:1 (immune/incomplete resistant/susceptible) as opposed to 9:3:4 to race 1, indicating that Pb1 and Pb2 are not alleles. Because joint action is contributed in F1 plants and in advanced [BC3F3(56) × BC6F3(12-4-6) F4] populations that carry both genes, it can be assumed that both accessions carry two unlinked genes but share a common signal transduction pathway, which leads to dominant complementation superiority of the resistance against different races of basil downy mildew.

Phenyl-imidazolo-cytidine analogues: structure-photophysical activity relationship and ability to detect single DNA mismatch.

To expand the arsenal of fluorescent cytidine analogues for the detection of genetic material, we synthesized para-substituted phenyl-imidazolo-cytidine ((Ph)ImC) analogues 5a-g and established a relationship between their structure and fluorescence properties. These analogues were more emissive than cytidine (λem 398-420 nm, Φ 0.009-0.687), and excellent correlation was found between Φ of 5a-g and σp(-) of the substituent on the phenyl-imidazolo moiety (R(2) = 0.94). Calculations suggested that the dominant tautomer of (Ph)ImC in methanol solution is identical to that of cytidine. DFT calculations of the stable tautomer of selected (Ph)ImC analogues suggested a relationship between the HOMO-LUMO gap and Φ and explained the loss of fluorescence in the nitro analogue. Incorporation of the CF3-(Ph)ImdC analogue into a DNA probe resulted in 6-fold fluorescence quenching of the former. A 17-fold reduction of fluorescence was observed for the G-matched duplex vs ODN(CF3-(Ph)ImdC), while for A-mismatched duplex, only a 2-fold decrease was observed. Furthermore, since the quantum yield of ODN(CF3-(Ph)ImdC):ODN(G) was reduced 17-fold vs that of a single strand, whereas that of ODN(CF3-(Ph)ImdC):ORN(G) was reduced only 3.8-fold, ODN(CF3-(Ph)ImdC) appears to be a DNA-selective probe. We conclude that the ODN(CF3-(Ph)ImdC) probe, exhibiting emission sensitivity upon single nucleotide replacement, may be potentially useful for DNA single nucleotide polymorphism (SNP) typing.

Synthesis and in vitro evaluation of anti-inflammatory activity of ester and amine derivatives of indoline in RAW 264.7 and peritoneal macrophages.

UNLABELLED: A prolonged increase in pro-inflammatory cytokines, TNF-α and IL-6 occurs in inflammatory diseases. Although existing therapies like steroids and TNF-α antagonists are effective they may cause serious adverse effects. We describe the preparation and evaluation for anti-inflammatory activity of 11 novel derivatives of indoline carbamates with a propionic ester, 2-aminoethyl, 3-aminopropyl 2-(dimethylamino)ethyl or 3-(dimethylamino)propyl group in positions 3 or 1. Compounds 25, 26 and 29 were previously shown to inhibit acetylcholinesterase with IC50s ranging from 0.4 to 55µM and to prevent cytotoxicity induced by reactive oxygen species in a concentration range of 100pM-1µM. Compounds 25, 26, 29, 9, 10, 17 and 18, reduced NO, TNF-α and IL-6 at concentrations of 1-10pM in LPS-activated RAW-264.7 and mouse peritoneal macrophages. The reduction in cytokines by compound 25 was associated with an increase in IκBα degradation and a decrease in the phosphorylation of p38 but not that of ERK. CONCLUSION: Indoline derivatives substituted at position 3 with chains carrying ester or amino groups may have potential for the treatment of chronic inflammatory and neurodegenerative diseases.

Treating late-onset Tay Sachs disease: Brain delivery with a dual trojan horse protein.

Tay-Sachs (TS) disease is a neurodegenerative disease resulting from mutations in the gene encoding the α-subunit (HEXA) of lysosomal ß-hexosaminidase A (HexA). We report that (1) recombinant HEXA alone increased HexA activity and decreased GM2 content in human TS glial cells and peripheral mononuclear blood cells; 2) a recombinant chimeric protein composed of HEXA linked to two blood-brain barrier (BBB) entry elements, a transferrin receptor binding sequence and granulocyte-colony stimulating factor, associates with HEXB in vitro; reaches human cultured TS cells lysosomes and mouse brain cells, especially neurons, in vivo; lowers GM2 in cultured human TS cells; lowers whole brain GM2 concentration by approximately 40% within 6 weeks, when injected intravenously (IV) to adult TS-mutant mice mimicking the slow course of late-onset TS; and increases forelimbs grip strength. Hence, a chimeric protein equipped with dual BBB entry elements can transport a large protein such as HEXA to the brain, decrease the accumulation of GM2, and improve muscle strength, thereby providing potential treatment for late-onset TS.

Rules for the design of highly fluorescent nucleoside probes: 8-(substituted cinnamyl)-adenosine analogues.

Currently, there are no tools that can help the design of useful fluorescent analogues. Hence, we synthesized a series of 8-(substituted cinnamyl)-adenosine analogues, 5-17, and established a relationship between their structure and fluorescence properties. We attempted to find a correlation between maximum emission wavelengths (λ(em)) of 5-17 or their quantum yields (φ), and Hammett constants (σ(p) and σ(m)) of the substituent on the cinnamyl moiety. A linear correlation was observed at low-medium σ values, but not at high σ values (≥0.7). Next, we explored correlation between λ(em) and φ of 5-17 and computed HOMO and LUMO energy levels of fragments of 5-17, i.e., 8-vinyl 9-Me-adenine (fluorescent molecule), 18, and substituted toluene rings (fluoresence modulators), 19-30. High φ correlated with relatively close LUMO levels of 19-30 and 18 (-0.076 to -0.003 eV). The electron density of LUMO of nitro analogues 9 and 15 is localized on the aryl ring only, which explains their low φ. Calculation of HOMO-LUMO gap of 5-17 enables accurate prediction of the λ(abs) for a planned analogue, and LUMO levels of an aryl moiety vs 8-vinyl 9-Me-adenine, allows the prediction of high or low φ. These findings lay the ground for prediction of fluorescence properties of additional analogues having a similar structure.

Mobility of oxathiapiprolin in and between tomato plants.

BACKGROUND: Oxathiapiprolin (OXPT; FRAC code 49) is a new piperidinyl-thiazole isooxazoline anti-oomycete fungicide that targets oxysterol-binding proteins. The fungicide is known to translocate acropetally from root to shoot to protect plants against fungal attack. RESULTS: OXPT is ambimobile. It can also translocate basipetally from shoot to root. OXPT exhibits an unprecedented capacity for trans-plant protection. When two tomato plants are grown in one pot, and one is treated with OXPT (on the stem, leaves or apex), while the other plant and soil surface are adequately covered, both plants become protected against late blight caused by Phytophthora infestans. CONCLUSION: Trans-plant systemic protection induced by OXPT involves translocation of the fungicide from the shoot of the treated plant to its root, exudation into the soil and uptake by the root of the neighboring untreated plant to protect it against the disease. Liquid chromatography-tandem mass spectrometry analyses confirmed the occurrence of OXPT in root exudates of OXPT-treated tomato plants in quantities sufficient to protect detached tomato leaves and intact plants against P. infestans. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Structure-function analysis of ceTIR-1/hSARM1 explains the lack of Wallerian axonal degeneration in C. elegans.

Wallerian axonal degeneration (WD) does not occur in the nematode C. elegans, in contrast to other model animals. However, WD depends on the NADase activity of SARM1, a protein that is also expressed in C. elegans (ceSARM/ceTIR-1). We hypothesized that differences in SARM between species might exist and account for the divergence in WD. We first show that expression of the human (h)SARM1, but not ceTIR-1, in C. elegans neurons is sufficient to confer axon degeneration after nerve injury. Next, we determined the cryoelectron microscopy structure of ceTIR-1 and found that, unlike hSARM1, which exists as an auto-inhibited ring octamer, ceTIR-1 forms a readily active 9-mer. Enzymatically, the NADase activity of ceTIR-1 is substantially weaker (10-fold higher Km) than that of hSARM1, and even when fully active, it falls short of consuming all cellular NAD+. Our experiments provide insight into the molecular mechanisms and evolution of SARM orthologs and WD across species.

Electrostatically guided dynamics--the root of fidelity in a promiscuous terpene synthase?

Terpene cyclases are responsible for the initial cyclization cascade in the multistep synthesis of more than 60,000 known natural products. This abundance of compounds is generated using a very limited pool of substrates based on linear isoprenoids. The astounding chemodiversity obtained by terpene cyclases suggests a tremendous catalytic challenge to these often promiscuous enzymes. In the current study we present a detailed mechanistic view of the biosynthesis of the monoterpene bornyl diphosphate (BPP) from geranyl diphosphate by BPP synthase using state of the art simulation methods. We identify the bornyl cation as an enzyme-induced bifurcation point on the multidimensional free energy surface, connecting between the product BPP and the side product camphene. Chemical dynamics simulations suggest that the active site diphosphate moiety steers reaction trajectories toward product formation. Nonetheless, chemical dynamics is not precise enough for exclusive product formation, providing a rationale for the lack of fidelity in this promiscuous terpene cyclase.

Multifunctional 5-aminolevulinic acid prodrugs activating diverse cell-death pathways.

Herein we describe a series of multifunctional 5-aminolevulinic-acid (ALA) prodrugs for photodynamic dependent and independent cancer therapy (PDT). We studied the cell-death mechanisms in glioblastoma U251 cells treated with four ALA-prodrugs: (1) AlaAcBu, that releases ALA, acetaldehyde, and butyric acid; (2) AlaFaBu, that releases ALA, formaldehyde, and butyric acid; (3) AlaFaPi, that releases ALA, formaldehyde and pivalic acid (4) AlaAcPi that releases ALA, acetaldehyde and pivalic acid. We examined the light-activated and dark cell-death mechanisms of the active metabolites released from the prodrugs by unspecific cellular hydrolases. The active moieties accelerated biosynthesis of protoporphyrin IX (PpIX) due to upregulated porphobilinogen deaminase (PBGD) activity. AlaAcBu was found to be the superior prodrug for PDT due to its ability to induce the highest PpIX synthesis. Photo-irradiation of AlaAcBu-treated cells led to dissipation of the mitochondrial membrane potential and reduction in the mitochondria metabolic activities; apoptosis and necrosis. Electron microscopy analyses of these cells revealed mitochondrial and endoplasmic reticulum swelling, membrane blebbing, apoptotic bodies and necrotic cell rupture. The formaldehyde-releasing prodrugs AlaFaBu and AlaFaPi induced low PDT efficacy, moreover sequestering the formaldehyde with semicarbazide resulted in high PpIX synthesis, suggesting that formaldehyde inhibited its synthesis. ALA and AlaAcBu phototherapy resulted in a dramatic accumulation of ubiquitinated proteins due to reduced proteasome activity and expression. In conclusion, the PDT potency of the prodrugs was in the order: AlaAcBu, AlaAcPi > AlaFaBu ≥ ALA > AlaFaPi, and the superiority of AlaAcBu stems from lower molar concentrations of AlaAcBu and lower light intensity needed to activate cell death following PDT.

Spatiotemporal Control of Melanin Synthesis in Liquid Droplets.

Melanins are natural biopolymers that have remarkable properties including UV-protection, coloration, and antioxidant activity. Their biosynthesis is regulated both spatially and temporally and involves supramolecular templating and compartmentalization of enzymes and reactants within specialized organelles called melanosomes. In contrast, the laboratory-based bulk synthesis of melanin by tyrosine or dopamine oxidation is a poorly controlled process, resulting in materials with undefined properties. Inspired by the pigment's biosynthesis, we developed a methodology to spatiotemporally regulate melanin formation in liquid droplets. The spatial control is achieved by sequestration of the reaction in dextran-rich droplets of a polyethylene glycol/dextran aqueous two-phase system, where the use of a photocleavable protected tyrosine provides a temporal control over its enzymatic oxidation-polymerization. We show that the liquid droplets allow for confined local reactivity as they serve as reaction centers for melanin synthesis and compartmentalize the melanin product. This methodology opens tremendous opportunities for applications in skincare and biomedicine.

Synthesis and preliminary biological evaluation of gabactyzine, a benactyzine-GABA mutual prodrug, as an organophosphate antidote.

Organophosphates (OPs) are inhibitors of acetylcholinesterase and have deleterious effects on the central nervous system. Clinical manifestations of OP poisoning include convulsions, which represent an underlying toxic neuro-pathological process, leading to permanent neuronal damage. This neurotoxicity is mediated through the cholinergic, GABAergic and glutamatergic (NMDA) systems. Pharmacological interventions in OP poisoning are designed to mitigate these specific neuro-pathological pathways, using anticholinergic drugs and GABAergic agents. Benactyzine is a combined anticholinergic, anti-NMDA compound. Based on previous development of novel GABA derivatives (such as prodrugs based on perphenazine for the treatment of schizophrenia and nortriptyline against neuropathic pain), we describe the synthesis and preliminary testing of a mutual prodrug ester of benactyzine and GABA. It is assumed that once the ester crosses the blood-brain-barrier it will undergo hydrolysis, releasing benactyzine and GABA, which are expected to act synergistically. The combined release of both compounds in the brain offers several advantages over the current OP poisoning treatment protocol: improved efficacy and safety profile (where the inhibitory properties of GABA are expected to counteract the anticholinergic cognitive adverse effects of benactyzine) and enhanced chemical stability compared to benactyzine alone. We present here preliminary results of animal studies, showing promising results with early gabactyzine administration.

Phloretin, an Apple Phytoalexin, Affects the Virulence and Fitness of Pectobacterium brasiliense by Interfering With Quorum-Sensing.

The effects of phloretin a phytoalexin from apple, was tested on Pectobacterium brasiliense (Pb1692), an emerging soft-rot pathogen of potato. Exposure of Pb1692 to 0.2 mM phloretin a concentration that does not affect growth, or to 0.4 mM a 50% growth inhibiting concentration (50% MIC), reduced motility, biofilm formation, secretion of plant cell wall-degrading enzymes, production of acyl-homoserine lactone (AHL) signaling molecules and infection, phenotypes that are associated with bacterial population density-dependent system known as quorum sensing (QS). To analyze the effect of growth inhibition on QS, the activity of ciprofloxacin, an antibiotic that impairs cell division, was compared to that of phloretin at 50% MIC. Unlike phloretin, the antibiotic hardly affected the tested phenotypes. The use of DH5α, a QS-negative Escherichia coli strain, transformed with an AHL synthase (ExpI) from Pb1692, allowed to validate direct inhibition of AHL production by phloretin, as demonstrated by two biosensor strains, Chromobacterium violaceaum (CV026) and E. coli (pSB401). Expression analysis of virulence-related genes revealed downregulation of QS-regulated genes (expI, expR, luxS, rsmB), plant cell wall degrading enzymes genes (pel, peh and prt) and motility genes (motA, fim, fliA, flhC and flhD) following exposure to both phloretin concentrations. The results support the inhibition of ExpI activity by phloretin. Docking simulations were used to predict the molecular associations between phloretin and the active site of ExpI, to suggest a likely mode of action for the compound's inhibition of virulence.

Chemical chaperones targeted to the endoplasmic reticulum (ER) and lysosome prevented neurodegeneration in a C9orf72 repeat expansion drosophila amyotrophic lateral sclerosis (ALS) model.

BACKGROUND: ALS is an incurable neuromuscular degenerative disorder. A familiar form of the disease (fALS) is related to point mutations. The most common one is an expansion of a noncoding GGGGCC hexanucleotide repeat of the C9orf72 gene on chromosome 9p21. An abnormal translation of the C9orf72 gene generates dipeptide repeat proteins that aggregate in the brain. One of the classical approaches for developing treatment against protein aggregation-related diseases is to use chemical chaperones (CSs). In this work, we describe the development of novel 4-phenylbutyric acid (4-PBA) lysosome/ER-targeted derivatives. We assumed that 4-PBA targeting to specific organelles, where protein degradation takes place, might reduce the 4-PBA effective concentration. METHODS: Organic chemistry synthetic methods and solid-phase peptide synthesis (SPPS) were used for preparing the 4-PBA derivatives. The obtained compounds were evaluated in an ALS Drosophila model that expressed C9orf72 repeat expansion, causing eye degeneration. Targeting to lysosome was validated by the 19F-nuclear magnetic resonance (NMR) technique. RESULTS: Several synthesized compounds exhibited a significant biological effect by ameliorating the eye degeneration. They blocked the neurodegeneration of fly retina at different efficacy levels. The most active CS was compound 9, which is a peptide derivative and was targeted to ER. Another active compound targeted to lysosome was compound 4. CONCLUSIONS: Novel CSs were more effective than 4-PBA; therefore, they might be used as a new class of drug candidates to treat ALS and other protein misfolding disorders.

Comparison of the tissue distribution and metabolism of AN1284, a potent anti-inflammatory agent, after subcutaneous and oral administration in mice.

This study is to compare the tissue distribution and metabolism of AN1284 after subcutaneous and oral administration at doses causing maximal reductions in IL-6 in plasma and tissues of mice. Anti-inflammatory activity of AN1284 and its metabolites was detected in lipopolysaccharide (LPS) activated RAW 264.7 macrophages. Mice were given AN1284 by injection or gavage, 15 min before LPS. IL-6 protein levels were measured after 4 h. Using a liquid chromatography/mass spectrometry method we developed, we showed that AN1284 is rapidly metabolized to the indole (AN1422), a 7-OH derivative (AN1280) and its glucuronide. AN1422 has weaker anti-inflammatory activity than AN1284 in LPS-activated macrophages and in mice. AN1284 (0.5 mg/kg) caused maximal reductions in IL-6 in the plasma, brain, and liver when injected subcutaneously and after gavage only in the liver. Similar reductions in the plasma and brain required a dose of 2.5 mg/kg, which resulted in 5.5-fold higher hepatic levels than after injection of 0.5 mg/kg, but 7, 11, and 19-fold lower ones in the plasma, brain, and kidneys, respectively. Hepatic concentrations produced by AN1284 were 2.5 mg/kg/day given by subcutaneously implanted mini-pumps that were only 12% of the peak levels seen after acute injection of 0.5 mg/kg. Similar hepatic concentrations were obtained by (1 mg/kg/day), administered in the drinking fluid. These were sufficient to decrease hepatocellular damage and liver triglycerides in previous experiments in diabetic mice. AN1284 can be given orally by a method of continuous release to treat chronic liver disease, and its preferential concentration in the liver should limit any adverse effects.

Challenges posed to bornyl diphosphate synthase: diverging reaction mechanisms in monoterpenes.

The simplest form of terpenoid chemistry is found for the monoterpenes, which give plants fragrance, flavor, and medicinal properties. Monoterpene synthases employ geranyl diphosphate as a substrate to generate an assortment of cyclic products. In the current study we present a detailed analysis of the multiple gas-phase reaction pathways in the synthesis of bornyl cation from geranyl diphosphate. Additionally, the fate of the proposed bornyl cation intermediate in the bornyl diphosphate synthase reaction is investigated by molecular dynamics simulations. We employ accurate density functional theory (DFT) methods after careful validation against high-level ab initio data for a set of model carbocations. The gas-phase results for the monoterpene reactions indicate a diverging reaction mechanism with multiple products in the absence of enzymatic control. This complex potential energy surface includes several possible bifurcation points due to the presence of secondary cations. Additionally, the suggested bornyl cation intermediate in the bornyl diphosphate synthase reaction is studied by molecular dynamics simulations employing a hybrid quantum mechanics (DFT)-molecular mechanics potential energy function. The simulations suggest that the bornyl cation is a transient species as in the gas phase and that electrostatic steering directs the formation of the final product, bornyl diphosphate.

A Novel Indoline Derivative Ameliorates Diabesity-Induced Chronic Kidney Disease by Reducing Metabolic Abnormalities.

Both diabetes and obesity (diabesity) contribute significantly to the development of chronic kidney disease (CKD). In search of new remedies to reverse or arrest the progression of CKD, we examined the therapeutic potential of a novel compound, AN1284, in a mouse model of CKD induced by type 2 diabetes with obesity. Six-week-old BKS Cg-Dock 7m+/+ Leprdb/J mice with type 2 diabetes and obesity were treated with AN1284 (2.5 or 5 mg kg-1 per day) via micro-osmotic pumps implanted subcutaneously for 3 months. Measures included renal, pancreatic, and liver assessment as well as energy utilization. AN1284 improved kidney function in BSK-db/db animals by reducing albumin and creatinine and preventing renal inflammation and morphological changes. The treatment was associated with weight loss, decreased body fat mass, increased utilization of body fat toward energy, preservation of insulin sensitivity and pancreatic ß cell mass, and reduction of dyslipidemia, hepatic steatosis, and liver injury. This indoline derivative protected the kidney from the deleterious effects of hyperglycemia by ameliorating the metabolic abnormalities of diabetes. It could have therapeutic potential for preventing CKD in human subjects with diabesity.