The effects of oil adjuvant on the degradation of spirotetramat and its metabolites in apple cultivation.

Spirotetramat is a frequently used insecticide in integrated pest management (IPM) strategies against rosy apple aphid (Dysaphis plantaginea) and woolly apple aphid (Eriosoma lanigerum) in apple cultivation. It is known that paraffin oil adjuvants increase the effect of spirotetramat against aphids. In contrast, there is a knowledge gap regarding the effects of co-applied paraffin (mineral or petroleum) oil on the degradation of spirotetramat and its metabolites (B-enol, B-keto, B-mono, and B-glu), which has not been previously investigated. Spirotetramat combined with formulated paraffin oil was tested against two aphid species and the residues of spirotetramat and its metabolites on leaves and apples were quantified using Liquid Chromatography coupled with tandem Mass Spectrometry (LC-MS/MS). The results showed that spirotetramat is highly effective against D. plantaginea, decreasing infestation by almost 100%. Furthermore, spirotetramat was shown to be effective against E. lanigerum, the reduction in infestation ranged between 67.9 and 97.7% during the last validation date. The addition of paraffin oil increased the effect of spirotetramat and affected its degradation, indicating that its efficacy could be connected to its metabolites. B-mono and B-glu were the most persistent metabolites in apple fruit overall when paraffin oil was applied.

Biosynthesis of Atypical Angucyclines Unveils New Ring Rearrangement Reactions Catalyzed by Flavoprotein Monooxygenases.

Six new angucycline structures, including spirocyclione A (1), which contains an unusual oxaspiro[5.5]undecane architecture, and its ring-A-cleaved product spirocyclione B (2), were discovered by heterologous expression of a type II polyketide biosynthetic gene cluster captured from a marine actinomycete strain Streptomyces sp. HDN155000. Three flavoprotein monooxygenases are confirmed to be responsible for the oxidative carbon skeleton rearrangements in the biosynthesis of compounds 1 and 2. The obtained compounds showed promising cytotoxicity against different types of cancer cells.

Identification and Functional Characterization of Carboxylesterase Genes Involved in Spirodiclofen Resistance in Panonychus citri (McGregor).

Overexpression of carboxyl/cholinesterase (CCE) genes has been reported to be associated with many cases of pesticide resistance in arthropods. However, it has been rarely documented that CCE genes participate in spirodiclofen resistance in Panonychus citri. In previous research, we found that spirodiclofen resistance is related to increased P450 and CCE enzyme activities in P. citri. In this study, we identified two CCE genes, PcCCE3 and PcCCE5, which were significantly upregulated in spirodiclofen-resistant strain and after exposure to spirodiclofen. RNA interference of PcCCE3 and PcCCE5 increased the spirodiclofen susceptibility in P. citri. In vitro metabolism indicated that PcCCE3 and PcCCE5 could interact with spirodiclofen, but metabolites were detected only in the PcCCE3 treatment. Our results indicated that PcCCE3 participates in spirodiclofen resistance through direct metabolism, and PcCCE5 may be involved in the spirodiclofen resistance by passive binding and sequestration, which provides new insights into spirodiclofen resistance in P. citri.

From Intercalation to External Binding: Ru(II) Complexes with a Spiro Ligand for TAR RNA Selective Binding and HIV-1 Reverse Transcriptase Inhibition.

As a typical RNA virus, the genetic information on HIV-1 is entirely stored in RNA. The reverse transcription activity of HIV-1 reverse transcriptase (RT) plays a crucial role in the replication and transmission of the virus. Non-nucleoside RT inhibitors (NNRTIs) block the function of RT by binding to the RNA binding site on RT, with very few targeting viral RNA. In this study, by transforming planar conjugated ligands into a spiro structure, we convert classical Ru(II) DNA intercalators into a nonintercalator. This enables selective binding to HIV-1 transactivation response (TAR) RNA on the outer side of nucleic acids through dual interactions involving hydrogen bonds and electrostatic attraction, effectively inhibiting HIV-1 RT and serving as a selective fluorescence probe for TAR RNA.

Overexpression of a nuclear receptor HR96 contributes to spirodiclofen susceptibility in Panonychus citri (McGregor).

The citrus red mite, Panonychus citri, is one of the most notorious and devastating citrus pests around the world that has developed resistance to multiple chemical acaricides. In previous research, we found that spirodiclofen-resistant is related to overexpression of P450, CCE, and ABC transporter genes in P. citri. However, the regulatory mechanisms of these detoxification genes are still elusive. This study identified all hormone receptor 96 genes of P. citri. 8 PcHR96 genes contained highly conserved domains. The expression profiles showed that PcHR96h was significantly upregulated in spirodiclofen resistant strain and after exposure to spirodiclofen. RNA interference of PcHR96h decreased expression of detoxification genes and increased spirodiclofen susceptibility in P. citri. Furthermore, molecular docking, heterologous expression, and drug affinity responsive target stability demonstrated that PcHR96h can interact with spirodiclofen in vitro. Our research results indicate that PcHR96h plays an important role in regulating spirodiclofen susceptibility and provides theoretical support for the resistance management of P. citri.

Elucidation of Palmarumycin Spirobisnaphthalene Biosynthesis Reveals a Set of Previously Unrecognized Oxidases and Reductases.

Spirobisnaphthalenes (SBNs) are a class of highly oxygenated, fungal bisnaphthalenes containing a unique spiroketal bridge, that displayed diverse bioactivities. Among the reported SBNs, palmarumycins are the major type, which are precursors for the other type of SBNs structurally. However, the biosynthesis of SBNs is unclear. In this study, we elucidated the biosynthesis of palmarumycins, using gene disruption, heterologous expression, and substrate feeding experiments. The biosynthetic gene cluster for palmarumycins was identified to be distant from the polyketide synthase gene cluster, and included two cytochrome P450s (PalA and PalB), and one short chain dehydrogenase/reductase (PalC) encoding genes as key structural genes. PalA is an unusual, multifunctional P450 that catalyzes the oxidative dimerization of 1,8-dihydroxynaphthalene to generate the spiroketal linkage and 2,3-epoxy group. Chemical synthesis of key intermediate and in vitro biochemical assays proved that the oxidative dimerization proceeded via a binaphthyl ether. PalB installs the C-5 hydroxy group, widely found in SBNs. PalC catalyzes 1-keto reduction, the reverse 1-dehydrogenation, and 2,3-epoxide reduction. Moreover, an FAD-dependent oxidoreductase, encoded by palD, which locates outside the cluster, functions as a 1-dehydrogenase. These results provided the first genetic and biochemical evidence for the biosynthesis of palmarumycin SBNs.

Non-Heme Iron Enzymes Catalyze Heterobicyclic and Spirocyclic Isoquinolone Core Formation in Piperazine Alkaloid Biosynthesis.

We report the discovery and biosynthesis of new piperazine alkaloids-arizonamides, and their derived compounds-arizolidines, featuring heterobicyclic and spirocyclic isoquinolone skeletons, respectively. Their biosynthetic pathway involves two crucial non-heme iron enzymes, ParF and ParG, for core skeleton construction. ParF has a dual function facilitating 2,3-alkene formation of helvamide, as a substrate for ParG, and oxidative cleavage of piperazine. Notably, ParG exhibits catalytic versatility in multiple oxidative reactions, including cyclization and ring reconstruction. A key amino acid residue Phe67 was characterized to control the formation of the constrained arizonamide B backbone by ParG.

Pathway elucidation and microbial synthesis of proaporphine and bis-benzylisoquinoline alkaloids from sacred lotus (Nelumbo nucifera).

Sacred lotus (Nelumbo nucifera) has been utilized as a food, medicine, and spiritual symbol for nearly 3000 years. The medicinal properties of lotus are largely attributed to its unique profile of benzylisoquinoline alkaloids (BIAs), which includes potential anti-cancer, anti-malarial and anti-arrhythmic compounds. BIA biosynthesis in sacred lotus differs markedly from that of opium poppy and other members of the Ranunculales, most notably in an abundance of BIAs possessing the (R)-stereochemical configuration and the absence of reticuline, a major branchpoint intermediate in most BIA producers. Owing to these unique metabolic features and the pharmacological potential of lotus, we set out to elucidate the BIA biosynthesis network in N. nucifera. Here we show that lotus CYP80G (NnCYP80G) and a superior ortholog from Peruvian nutmeg (Laurelia sempervirens; LsCYP80G) stereospecifically convert (R)-N-methylcoclaurine to the proaporphine alkaloid glaziovine, which is subsequently methylated to pronuciferine, the presumed precursor to nuciferine. While sacred lotus employs a dedicated (R)-route to aporphine alkaloids from (R)-norcoclaurine, we implemented an artificial stereochemical inversion approach to flip the stereochemistry of the core BIA pathway. Exploiting the unique substrate specificity of dehydroreticuline synthase from common poppy (Papaver rhoeas) and pairing it with dehydroreticuline reductase enabled de novo synthesis of (R)-N-methylcoclaurine from (S)-norcoclaurine and its subsequent conversion to pronuciferine. We leveraged our stereochemical inversion approach to also elucidate the role of NnCYP80A in sacred lotus metabolism, which we show catalyzes the stereospecific formation of the bis-BIA nelumboferine. Screening our collection of 66 plant O-methyltransferases enabled conversion of nelumboferine to liensinine, a potential anti-cancer bis-BIA from sacred lotus. Our work highlights the unique benzylisoquinoline metabolism of N. nucifera and enables the targeted overproduction of potential lotus pharmaceuticals using engineered microbial systems.

Chemosensory Proteins Are Associated with Thiamethoxam and Spirotetramat Tolerance in Aphis gossypii Glover.

Chemosensory proteins (CSPs) are a class of transporters in arthropods. Deeper research on CSPs showed that CSPs may be involved in some physiological processes beyond chemoreception, such as insect resistance to pesticides. We identified two upregulated CSPs in two resistant strains of Aphis gossypii Glover. To understand their role in the resistance of aphids to pesticides, we performed the functional verification of CSP1 and CSP4 in vivo and in vitro. Results showed that the sensitivity of the thiamethoxam-resistant strain to thiamethoxam increased significantly with the silencing of CSP1 and CSP4 by RNAi (RNA interference), and the sensitivity of the spirotetramat-resistant strain to spirotetramat increased significantly with the silencing of CSP4. Transgenic Drosophila melanogaster expressing CSPs exhibited stronger resistance to thiamethoxam, spirotetramat, and alpha-cypermethrin than the control did. In the bioassay of transgenic Drosophila, CSPs showed different tolerance mechanisms for different pesticides, and the overexpressed CSPs may play a role in processes other than resistance to pesticides. In brief, the present results prove that CSPs are related to the resistance of cotton aphids to insecticides.

Molecular and Computational Bases for Spirofuranone Formation in Setosusin Biosynthesis.

The 3(2H)-furanone unit is observed in many biologically active natural products, as represented by the antifungal medication griseofulvin. Setosusin (1) is a fungal meroditerpenoid featuring a unique spiro-fused 3(2H)-furanone moiety; however, the biosynthetic basis for spirofuranone formation has not been investigated since its isolation. Therefore, in this study we identified the biosynthetic gene cluster of 1 in the fungus Aspergillus duricaulis CBS 481.65 and elucidated its biosynthetic pathway by heterologous reconstitution of related enzyme activities in Aspergillus oryzae. To understand the reaction mechanism to afford spirofuranone, we subsequently performed a series of in vivo and in vitro isotope-incorporation experiments and theoretical calculations. The results indicated that SetF, the cytochrome P450 enzyme that is critical for spirofuranone synthesis, not only performs the epoxidation of the polyketide portion of the substrate but also facilitates the protonation-initiated structural rearrangement to yield 1. Finally, a mutagenesis experiment using SetF identified Lys303 as one of the potential catalytic residues that are important for spirofuranone synthesis.

Theoretical Study on the Mechanism of Spirocyclization in Spiroviolene Biosynthesis.

Spiroviolene is a spirocyclic triquinane diterpene produced by Streptomyces violens. Recently, a biosynthetic pathway that includes secondary carbocation intermediates and a complicated concerted skeletal rearrangement was proposed for spiroviolene, based upon careful labeling experiments. On the basis of density functional theory (DFT) calculations, we propose a revised pathway for spiroviolene biosynthesis, involving a multistep carbocation cascade that bypasses the formation of unstable secondary carbocations by breaking the adjacent C-C bond to form a more stable tertiary carbocation (IM3) and by Wagner-Meerwein 1,2-methyl rearrangement (IM7).

Characterization of equine liver microsome-generated metabolites of growth hormone secretagogue small molecule ibutamoren.

RATIONALE: Interest in growth hormone secretagogues has intensified during the past several years based on capable, ever-widening investigational applications of recombinant growth hormone in animals and humans. Ibutamoren is a potent, long-acting, selective and orally active non-peptide growth hormone secretagogue, which has a great potential for abuse as a performance-enhancing agent in sports. METHODS: To support drug metabolism and pharmacokinetic studies of chiral pharmaceuticals, it is necessary to combine the resolving power of high-performance liquid chromatography with the sensitivity of mass spectrometric techniques. This paper describes the metabolic conversion of ibutamoren using equine liver microsomes and metabolite characterization using a QExactive high-resolution mass spectrometer. RESULTS: A total of 32 metabolites for ibutamoren (20 phase I and 12 phase II) were detected. The important findings of the current research are as follows: (1) the growth hormone secretagogue ibutamoren was prone to oxidation, resulting in corresponding hydroxylated metabolites; (2) in ibutamoren, the dissociation of the phenyl ring and 2-amino-2-methylpropanamide side chain was also observed; (3) the glucuronic acid conjugates of mono-, di- and trihydroxylated analogues were detected; and (4) no sulfonic acid conjugated metabolites were observed in this study of ibutamoren. CONCLUSIONS: The reported data help in the speedy detection of the growth hormone secretagogue ibutamoren and reveal its illegal use in competitive sports.

Structural basis of intron selection by U2 snRNP in the presence of covalent inhibitors.

Intron selection during the formation of prespliceosomes is a critical event in pre-mRNA splicing. Chemical modulation of intron selection has emerged as a route for cancer therapy. Splicing modulators alter the splicing patterns in cells by binding to the U2 snRNP (small nuclear ribonucleoprotein)-a complex chaperoning the selection of branch and 3' splice sites. Here we report crystal structures of the SF3B module of the U2 snRNP in complex with spliceostatin and sudemycin FR901464 analogs, and the cryo-electron microscopy structure of a cross-exon prespliceosome-like complex arrested with spliceostatin A. The structures reveal how modulators inactivate the branch site in a sequence-dependent manner and stall an E-to-A prespliceosome intermediate by covalent coupling to a nucleophilic zinc finger belonging to the SF3B subunit PHF5A. These findings support a mechanism of intron recognition by the U2 snRNP as a toehold-mediated strand invasion and advance an unanticipated drug targeting concept.

Atiprimod triggered apoptotic cell death via acting on PERK/eIF2α/ATF4/CHOP and STAT3/NF-ΚB axis in MDA-MB-231 and MDA-MB-468 breast cancer cells.

PURPOSE: The constitutive activation of STAT3 through receptor tyrosine kinases triggered breast cancer cell growth and invasion-metastasis. Atiprimod impacts anti-proliferative, anti-carcinogenic effects in hepatocellular carcinoma, lymphoma, multiple myeloma via hindering the biological activity of STAT3. Dose-dependent atiprimod evokes first autophagy as a survival mechanism and then apoptosis due to prolonged ER stress in pituitary adenoma cells. The therapeutic efficiency and mechanistic action of atiprimod in breast cancer cells have not been investigated yet. Thus, we aimed to modulate the pivotal role of ER stress in atiprimod-triggered apoptosis in MDA-MB-231 and MDA-MB-468 breast cancer cells. RESULTS: Dose- and time-dependent atiprimod treatment inhibits cell viability and colony formation in MDA-MB-468 and MDA-MB-231 breast cancer cells. A moderate dose of atiprimod (2 µM) inhibited STAT3 phosphorylation at Tyr705 residue and also suppressed the total expression level of p65. In addition, nuclear localization of STAT1, 3, and NF-κB was prevented by atiprimod exposure in MDA-MB-231 and MDA-MB-468 cells. Atiprimod evokes PERK, BiP, ATF-4, CHOP upregulation, and PERK (Thr980), eIF2α (Ser51) phosphorylation's. However, atiprimod suppressed IRE1α-mediated Atg-3, 5, 7, 12 protein expressions and no alteration was observed on Beclin-1, p62 expression levels. PERK/eIF2α/ATF4/CHOP axis pivotal role in atiprimod-mediated G1/S arrest and apoptosis via Bak, Bax, Bim, and PUMA upregulation in MDA-MB-468 cells. Moreover, atiprimod renders MDA-MB-231 more vulnerable to type I programmed cell death by plasmid-mediated increased STAT3 expression. CONCLUSION: Atiprimod induced prolonged ER stress-mediated apoptosis via both activating PERK/eIF2α/ATF4/CHOP axis and suppressing STAT3/NF-κB transcription factors nuclear migration in TBNC cells.

In vitro anti-Leishmania activity and molecular docking of spiro-acridine compounds as potential multitarget agents against Leishmania infantum.

Leishmaniasis is an infectious disease with several limitations regarding treatment schemes. This work reports the anti-Leishmania activity of spiroacridine compounds against the promastigote (IC50 = 1.1 to 6.0 µg / mL) and amastigote forms of the best compounds (EC50 = 4.9 and 0.9 µg / mL) inLeishmania (L.) infantumand proposes an in-silico study with possible selective therapeutic targets for L. infantum. The substituted dimethyl-amine compound (AMTAC 11) showed the best leishmanicidal activity in vitro, and was found to interact with TryRandLdTopoI. comparisons with standard inhibitors were performed, and its main interactions were elucidated. Based on the biological assessment and the structure-activity relationship study, the spiroacridine compounds appear to be promisinganti-leishmaniachemotherapeutic agents to be explored.

Universal capability of 3-ketosteroid Δ1-dehydrogenases to catalyze Δ1-dehydrogenation of C17-substituted steroids.

BACKGROUND: 3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD's substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-ß-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. RESULTS: Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; - 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (- 8.40 kcal/mol) or diosgenone (- 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA = 23.7 µM) than to AD (KmA = 529.2 µM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA = 9.25∙106 M-1 s-1) is almost 20 times higher than measured for KSTD1 (kcat/KmA = 4.71∙105 M-1 s-1). CONCLUSIONS: We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.

Design, synthesis and biological evaluation of exiguamine A analogues as IDO1 inhibitors.

A series of exiguamine A analogues were designed and synthesized via 15 steps. Their inhibitory activities against IDO1 were tested and the structure-activity relationships were studied. Most compounds exhibited potent IDO1 inhibitory activities with IC50 values at the level of 10-7-10-8 M. Compound 21f was the most potent IDO1 inhibitor with an IC50 value of 65.3 nM, which was comparable with the positive control drug epacadostat (IC50 = 46 nM). Moreover, compound 21f showed higher selectivity for IDO1 over tryptophan 2,3-dioxygenase (TDO) and no cytotoxicity at its effective concentration, rending it justifiable for further optimization and evaluation.

Chemical Screen for Vancomycin Antagonism Uncovers Probes of the Gram-Negative Outer Membrane.

The outer membrane of Gram-negative bacteria is a formidable permeability barrier which allows only a small subset of chemical matter to penetrate. This outer membrane barrier can hinder the study of cellular processes and compound mechanism of action, as many compounds including antibiotics are precluded from entry despite having intracellular targets. Consequently, outer membrane permeabilizing compounds are invaluable tools in such studies. Many existing compounds known to perturb the outer membrane also impact inner membrane integrity, such as polymyxins and their derivatives, making these probes nonspecific. We performed a screen of ∼140 000 diverse synthetic compounds, for those that antagonized the growth inhibitory activity of vancomycin at 15 °C in Escherichia coli, to enrich for chemicals capable of perturbing the outer membrane. This led to the discovery that liproxstatin-1, an inhibitor of ferroptosis in human cells, and MAC-0568743, a novel cationic amphiphile, could potentiate the activity of large-scaffold antibiotics with low permeation into Gram-negative bacteria at 37 °C. Liproxstatin-1 and MAC-0568743 were found to physically disrupt the integrity of the outer membrane through interactions with lipopolysaccharide in the outer leaflet of the outer membrane. We showed that these compounds selectively disrupt the outer membrane while minimally impacting inner membrane integrity, particularly at the concentrations needed to potentiate Gram-positive-targeting antibiotics. Further exploration of these molecules and their structural analogues is a promising avenue for the development of outer membrane specific probes.

Chemistry and biology of spiroacetals from myxobacteria.

This review details the isolation, biosynthesis, biological activity and synthesis of spiroacetals from the myxobacterium Sorangium cellulosum. The strategies utilised to access the challenging structures and stereochemistry of these natural products are highlighted.

Exploring the putative mechanism of allosteric modulations by mixed-action kappa/mu opioid receptor bitopic modulators.

The modulation and selectivity mechanisms of seven mixed-action kappa opioid receptor (KOR)/mu opioid receptor (MOR) bitopic modulators were explored. Molecular modeling results indicated that the 'message' moiety of seven bitopic modulators shared the same binding mode with the orthosteric site of the KOR and MOR, whereas the 'address' moiety bound with different subdomains of the allosteric site of the KOR and MOR. The 'address' moiety of seven bitopic modulators bound to different subdomains of the allosteric site of the KOR and MOR may exhibit distinguishable allosteric modulations to the binding affinity and/or efficacy of the 'message' moiety. Moreover, the 3-hydroxy group on the phenolic moiety of the seven bitopic modulators induced selectivity to the KOR over the MOR.