Fluorescence detecting glycopeptide antibiotics via a dynamic molecular switch.

BACKGROUND: Glycopeptide antibiotics (GPAs) represented by vancomycin (VAN) are clinically used as a first-line treatment for serious infections caused by Gram-positive pathogens. The use and dosing methods of GPAs are rigorously managed for safety considerations, which calls for fast and accurate quantification approaches. RESULT: A new sort of fluorescent probes for GPAs has been proposed, each of which was integrated by a fluorescein-based reporter and a GPAs' recognition peptide D-alanyl-D-alanine (D-Ala-D-Ala). These probes work as dynamic molecular switches, which mainly exist as non-fluorescent spirolactam forms in the absence of GPAs. GPAs binding with the dipeptide regulates the dynamic balance between fluorescence OFF lactam form and fluorescence ON ring-opened form, rendering these probes capable of GPAs detecting. The most promising one P1 exhibits excellent sensitivity and selectivity towards GPAs detection. SIGNIFICANCE: Different to previous developments, P1 consists of a single fluorophore without the need of a fluorescence-quenching group or a secondary dye, which is the smallest fluorescent probe for GPAs up to now. P1 realizes direct VAN quantification from complex biological samples including real serums, dispensing with additional drug extraction. More interestingly, both P1 and P6 can distinguish GPAs with different peptide backbones, which has not been achieved previously.

Proposing anti-counterfeiting pharmacopoeia quality markers for Shenlingbaizhu granule based on UHPLC-Q-orbitrap-MS identification.

INTRODUCTION: Shenlingbaizhu granule, a Traditional Chinese Medicine prescription comprising Renshen, Gancao, and Shanyao, is widely consumed in China nowadays. OBJECTIVE: The study tries to propose pharmacopoeia quality markers (Q-markers) to prevent counterfeiting involving Renshen, Gancao, and Shanyao. METHODOLOGY: A novel strategy, that is, library-based ultra-high-performance liquid chromatography-quadrupole-orbitrap mass spectrometry, was used to analyse the lyophilised aqueous powder of Shenlingbaizhu granule. Subsequently, quantum chemistry calculation and UV-vis spectra scanning were also performed through theoretical or experimental approaches. RESULT: Thirty-two isomers have been strictly distinguished, especially positional isomeric isochlorogenic acid B versus isochlorogenic acid C, positional isomeric schaftoside versus isoschaftoside, positional isomeric ginsenoside Rg2 versus 20S-ginsenoside Rg3, and stereoisomeric 20S-ginsenoside Rg3 versus 20R-ginsenoside Rg3. Seventeen compounds were unexpectedly observed, particularly scoparone and pectolinarigenin, while a total of 76 bioactive compounds have been putatively identified in the study. The quantum chemistry calculation and UV-vis spectra scanning results revealed that glycyrrhizic acid, ginsenoside Re, ginsenoside Rb1, and diosgenin displayed different dipole moment values and maximum absorption wavelengths from each other. CONCLUSION: The study recommends glycyrrhizic acid, ginsenoside Re, ginsenoside Rb1, and diosgenin as four anti-counterfeiting Q-markers for the pharmacopoeia. The anti-counterfeiting Q-markers can be detected using conventional HPLC. The observation of 17 unexpected compounds updates our knowledge regarding the bioactives of Shenlingbaizhu granule.

Natural products in pursuing novel therapies of nonalcoholic fatty liver disease and steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are hepatic manifestations of systemic metabolic dysfunction, which affect one-quarter of the adult population worldwide as estimated, and exhibit high risk in progressing to hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. Current drug discovery focuses on modifying homeostasis of lipids, carbohydrates, and cholesterol, as well as inhibiting inflammation and fibrogenesis. Many natural products show promising activities on various molecular targets involving these mechanisms; however, they have not been fully exploited. Since some compounds are components of healthy food, they may be employed in chemoprevention as adjuvants to lifestyle modification, while natural products such as alkaloids and sesquiterpenoids could serve as promising starting points for structural modifications and deserve further development.

Design, Synthesis, and Biological Evaluations of Pyridyl 4,5,6,7-Tetrahydro-4,7-Methanobenzo[d]isoxazoles as Potent and Selective Inhibitors of 11ß-Hydroxylase.

Inhibition of CYP11B1 is a promising therapy for severe diseases caused by excessive cortisol. Enantiomer discrimination provides clues to achieve selectivity that CYP11B1 and homologous CYP11B2 were selectively bound by S- and R-fadrozole, respectively, in distinct binding modes. Pyridyl 4,5,6,7-tetrahydro-4,7-methanobenzo[d]isoxazoles showing a similar binding mode to S-fadrozole in CYP11B1 were designed as potent and selective CYP11B1 inhibitors. Compound 7aa exhibited a highly potent CYP11B1 inhibition similar to that of the drug osilodrostat (IC50's of 9 and 6 nM, respectively) but was 1500-fold more selective over CYP11B2 compared to osilodrostat (selectivity factors of 125 versus 0.08, respectively). Strong reductions of plasma cortisol concentrations by compound 7aa were demonstrated in rats without interference in aldosterone production after oral application. It showed no inhibition against a panel of steroidogenic and hepatic CYP enzymes. Exhibiting a good pharmacokinetic profile, compound 7aa was considered as a drug candidate for further development.

Sinomenine Derivatives: Synthesis, Antitumor Activity, and Apoptotic Induction in MCF-7 Cells via IL-6/PI3K/Akt/NF-κB Signaling Pathway.

Natural products have been widely considered as an important resource for new drugs or lead compounds. Sinomenine (SIN) and its derivatives exert antitumor activity via regulation of inflammatory mediators. For these reasons we synthesized three series of SIN derivatives (compounds 4 a-i, 7 a-c and 11 a-c) as antitumor agents from this natural product. All compounds were prepared by modification at the C1 and C4 positions of the A ring, the C4 position of the A ring, and the C6 and C7 positions of the C ring, respectively. All the derivatives were subjected to in vitro antitumor activity against HeLa, A549, HepG-2, MCF-7 and HT-29 cell lines. To observe the apoptotic induction of SIN derivatives and its mechanism, fluorescent staining and western blot assays were carried out for active compound against MCF-7. Based on the screening results, most of the SIN derivatives showed better antitumor activity than SIN. Some of them were found to possess broad-spectrum antitumor activity. Most notably, 11 c exhibited obvious antitumor activity in both cell lines with IC50 values less than 11 µM. Besides, 11 c induced apoptosis of MCF-7 in a dose-dependent manner. Western blot assay demonstrated that 11 c inhibited IL-6-mediated activation of PI3K/Akt pathway. A docking study revealed that 11 c had stronger binding interaction with the residues of IL-6 than SIN. All these results indicate that 11 c may be a potential anti-breast cancer agent by directly targeting IL-6.

Manipulations of phenylnorbornyl palladium species for multicomponent construction of a bridged polycyclic privileged scaffold.

Hexahydromethanocarbazole is a privileged scaffold in the discovery of new drugs and photoactive organic materials due to its good balance between structural complexity and minimized entropy penalty upon receptor binding. To address the difficulty of synthesizing this highly desirable bridged polycyclic scaffold, we designed a convenient multicomponent reaction cascade as intercepted Heck addition/C-H activation/C-palladacycle formation/electrophilic attack of ANP/N-palladacycle formation/Buchwald amination. A distinguishing feature of this sophisticated strategy is the successive generation of two key phenylnorbornyl palladium species to control the reaction flow towards desired products. DFT calculations further reveal the crucial roles of Cs2CO3 and 5,6-diester substitutions on the norbornene reactant in preventing multiple side-reactions. This innovative method exhibits a broad scope with good yields, and therefore will enable the construction of natural-product-like compound libraries based on hexahydromethanocarbazole.

Using Acetone/Water Binary Solvent to Enhance the Stability and Bioavailability of Spray Dried Enzalutamide/HPMC-AS Solid Dispersions.

We demonstrated a facile approach, by adjusting the solvent ratio of water/acetone binary mixture, to alter the intermolecular interactions between Enzalutamide (ENZ) and hydroxypropyl methylcellulose acetate succinate (HPMC-AS) for spray drying process, which can be readily implemented to produce spray-dried dispersions (SDD) with enhanced stability and bioavailability. The prepared SDD of ENZ/HPMC-AS were examined systematically in terms of particle size, morphology, dissolution, solubility, stability, and bioavailability. Our results show that the introduction of water (up to 30% volume fraction) can effectively reduce the hydrodynamic diameter of HPMC-AS from approximately 220 nm to 160 nm (a reduction of c.a. 20%), which increases the miscibility of the drug and polymer, delaying or inhibiting the crystallization of ENZ during the spray drying process, resulting in a homogeneous amorphous phase. The benefits of using acetone/water binary mixture were subsequently evidenced by an increased specific surface area, improved dissolution profile and relative bioavailability, enhanced stability, and elevated drug release rate. This fundamental finding underpins the great potential of using binary mixture for spray drying process to process active pharmaceutical ingredients (APIs) that are otherwise challenging to handle.

Chimera induced protein degradation: PROTACs and beyond.

Ubiquitin-proteasome system, autophagy-lysosome pathway and N-end rule pathway are crucial protein quality control mechanisms in human body. Hijacking these endogenous protein degrading measures by chimera degraders could be a revolutionary strategy for the discovery of small-molecule drugs. As the most advanced chimera degraders, PROTACs have demonstrated the potential by delivering two drug candidates into clinical trials. The development of chimera degraders exploiting these three pathways are reviewed, a focus is given on the chemical structures and their influences on biological effects from a viewpoint of medicinal chemistry.

Pharmaceutical Inhibition of Neddylation as Promising Treatments for Various Cancers.

BACKGROUND: Neddylation is an important post-translational modification of proteins, in which a NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is covalently introduced onto the substrate proteins to regulate their functions and homeostasis. As neddylation is frequently up-regulated in various cancers, its interference was proposed as a promising therapy of related diseases. OBJECTIVE: The recent advances in developing neddylation interfering agents were summarized to provide an overview of current achievements and perspectives for future development. METHODS: Reports on neddylation interfering agents were acquired from Pubmed as well as the EPO and clinicaltrials.gov websites, which were subsequently analyzed and summarized according to targets, chemical structures and biological activities. RESULTS: Neddylation as a sophisticated procedure comprises proteolytic processing of NEDD8 precursor, deploying conjugating enzymes E1 (NAE), E2 (UBE2M and UBE2F) and various E3, as well as translocating NEDD8 along these conjugating enzymes sequentially and finally to substrate proteins. Among these nodes, NAE, UBE2M and the interaction between UBE2M-DCN1 have been targeted by small molecules, metal complexes, peptides and RNAi. A NAE inhibitor pevonedistat (MLN4924) is currently under evaluation in clinical trials for the treatment of various cancers. CONCLUSION: With multiple inhibitory approaches of neddylation being introduced, the development of neddylation interference as a novel cancer therapy is significantly boosted recently, although its efficacy and the best way to achieve that are still to be demonstrated in clinical trials.

Benzophenones as xanthone-open model CYP11B1 inhibitors potentially useful for promoting wound healing.

The inhibition of steroidogenic cytochrome P450 enzymes has been shown to play a central role in the management of life-threatening diseases such as cancer, and indeed potent inhibitors of CYP19 (aromatase) and CYP17 (17α hydroxylase/17,20 lyase) are currently used for the treatment of breast, ovarian and prostate cancer. In the last few decades CYP11B1 (11-ß-hydroxylase) and CYP11B2 (aldosterone synthase), key enzymes in the biosynthesis of cortisol and aldosterone, respectively, have been also investigated as targets for the identification of new potent and selective agents for the treatment of Cushing's syndrome, impaired wound healing and cardiovascular diseases. In an effort to improve activity and synthetic feasibility of our different series of xanthone-based CYP11B1 and CYP11B2 inhibitors, a small series of imidazolylmethylbenzophenone-based compounds, previously reported as CYP19 inhibitors, was also tested on these new targets, in order to explore the role of a more flexible scaffold for the inhibition of CYP11B1 and -B2 isoforms. Compound 3 proved to be very potent and selective towards CYP11B1, and was thus selected for further optimization via appropriate decoration of the scaffold, leading to new potent 4'-substituted derivatives. In this second series, 4 and 8, carrying a methoxy group and a phenyl ring, respectively, proved to be low-nanomolar inhibitors of CYP11B1, despite a slight decrease in selectivity against CYP11B2. Moreover, unlike the benzophenones of the first series, the 4'-substituted derivatives also proved to be selective for CYP11B enzymes, showing very weak inhibition of CYP19 and CYP17. Notably, the promising result of a preliminary scratch test performed on compound 8 confirmed the potential of this compound as a wound-healing promoter.

Accelerated skin wound healing by selective 11ß-Hydroxylase (CYP11B1) inhibitors.

Previous studies have shown that inhibition of cortisol biosynthesis in skin leads to accelerated wound healing. Here, pyridylmethyl pyridine type 11ß-hydroxylase (CYP11B1) inhibitors were optimized for topical application to avoid systemic side effects. The resulting very potent, non-toxic CYP11B1 inhibitor 14 (IC50 = 0.8 nM) exhibited good selectivity over 11ß-HSD1, CYP17A1 and CYP19A1. The compound showed high stability toward human plasma (t1/2= > 150 min, as a substitute for wound fluid) and low stability toward HLS9 (t1/2 = 19 min) for rapid metabolic clearance after absorption. Compound 14 was able to accelerate wound healing in human skin.

Drifting of heme-coordinating group in imidazolylmethylxanthones leading to improved selective inhibition of CYP11B1.

An abnormal increase in glucocorticoid levels is responsible for pathological disorders affecting different organs and systems, and the selective inhibition of appropriate steroidogenic enzymes represents a validated strategy to restore their physiological levels. In continuing our studies on CYP11B inhibitors, in this paper a small series of 6-substituted 3-imidazolylmethylxanthones was designed and synthesized, according to the data acquired from previously reported series of derivatives and from a purposely-performed docking study. The new compounds proved to be potent inhibitors of CYP11B isoforms, being effective on CYP11B1 in the low nanomolar range and improving selectivity with respect to CYP11B2, compared to previously reported related compounds. These data further confirmed that a suitable mutual arrangement of the imidazolylmethyl pharmacophore and a properly selected substituent on the xanthone core allows a fine tuning of the activity towards the different CYPs and further corroborate the role of the xanthone scaffold as a privileged structure in this field.

Lead Optimization Generates CYP11B1 Inhibitors of Pyridylmethyl Isoxazole Type with Improved Pharmacological Profile for the Treatment of Cushing's Disease.

Cushing's disease, characterized by elevated plasma cortisol levels, can be controlled by inhibition of 11ß-hydroxylase (CYP11B1). The previously identified selective and potent CYP11B1 inhibitor 5-((5-methylpyridin-3-yl)methyl)-2-phenylpyridine Ref 7 (IC50= 2 nM) exhibited promutagenic potential as well as very low oral bioavailability in rats (F = 2%) and was therefore modified to overcome these drawbacks. Successful lead optimization resulted in similarly potent and selective 5-((5-methoxypyridin-3-yl)methyl)-3-phenylisoxazole 25 (IC50 = 2 nM, 14-fold selectivity over CYP11B2), exhibiting a superior pharmacological profile with no mutagenic potential. Furthermore, compound 25 inhibited rat CYP11B1 (IC50 = 2 µM) and showed a high oral bioavailability (F = 50%) and sufficient plasma concentrations in rats, providing an excellent starting point for a proof-of-principle study.

Therapeutic compounds for Cushing's syndrome: a patent review (2012-2016).

INTRODUCTION: Endogenous Cushing's syndrome (CS) is a set of disorders caused by chronic exposure to excess glucocorticoids induced by neuroendocrine tumors in pituitary, adrenals, and infrequently other sites (ectopic ACTH syndrome). Due to various comorbidities, CS patients exhibit higher risks of cardiovascular diseases and thus increased mortality. Pharmaceutical therapy is an important constituent of treatment regimen. Areas covered: Patents published since 2012 are reviewed, which claim therapeutic compounds interfering with ACTH secretion and down-stream signal transduction, inhibiting cortisol biosynthesis and antagonizing glucocorticoid receptors. Advances focus on a) new analogues with improved efficacy and PK properties or less off-target toxicity; b) existing drugs (candidates) being repurposed to treat CS; and c) novel strategies such as selective inhibition of CYP11B1. Expert opinion: New compounds against established targets need to be developed because current drugs lack selectivity leading to off-target toxicity. Selective inhibition of CYP11B1 is a novel alternative strategy and is potentially versatile in controlling all types of hypercortisolism. Selective multi-targeting enzymes in steroidogenesis network is promising due to potential synergistic effects. However, doses toward each targets are not feasible to adjust because the corresponding intrinsic potencies are rigid. Targeting PRKACA mutations is promising in treating CS caused by adrenal adenomas.

Male Men1 heterozygous mice exhibit fasting hyperglycemia in the early stage of MEN1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited syndrome characterized by multiple tumors in the parathyroid glands, endocrine pancreas and anterior pituitary. Recent clinical studies have revealed a strong association between MEN1 syndrome and the risk of developing diabetes mellitus; however, the underlying mechanisms remain unknown. In this study, heterozygous Men1 knockout (Men1(+/-)) mice were used as MEN1 models to investigate MEN1-associated glucose metabolic phenotypes and mechanisms. Heterozygous deficiency of Men1 in 12-month-old male mice induced fasting hyperglycemia, along with increased serum insulin levels. However, male Men1(+/-) mice did not show insulin resistance, as evidenced by Akt activation in hepatic tissues and an insulin tolerance test. Increased glucose levels following pyruvate challenge and expression of key gluconeogenic genes suggested increased hepatic glucose output in the male Men1(+/-) mice. This effect could be partly due to higher basal serum glucagon levels, which resulted from pancreatic islet cell proliferation induced by heterozygous loss of Men1 Taken together, our results indicate that fasted male Men1(+/-) mice, in the early stage of development of MEN1, display glucose metabolic disorders. These disorders are caused not by direct induction of insulin resistance, but via increased glucagon secretion and the consequent stimulation of hepatic glucose production.

Specificity of anti-prostate cancer CYP17A1 inhibitors on androgen biosynthesis.

The orteronel, abiraterone and galeterone, which were developed to treat castration resistant prostate cancer, inhibit 17,20 lyase activity but little is known about their effects on adrenal androgen biosynthesis. We studied the effect of several inhibitors and found that orteronel was selective towards 17,20 lyase activity than abiraterone and galeterone. Gene expression analysis showed that galeterone altered the expression of HSD3B2 but orteronel did not change the expression of HSD3B2, CYP17A1 and AKR1C3. The CYP19A1 activity was not inhibited except by compound IV which lowered activity by 23%. Surprisingly abiraterone caused complete blockade of CYP21A2 activity. Analysis of steroid metabolome by gas chromatography - mass spectrometry revealed changes in steroid levels caused by different inhibitors. We can conclude that orteronel is a highly specific inhibitor of 17,20 lyase activity. The discovery of these specific drug actions on steroidogenic enzyme activities would be valuable for understanding the regulation of androgens.

Targeting Steroidogenic Cytochromes P450 (CYPs) with 6-Substituted 1-Imidazolylmethylxanthones.

Abnormally high corticosteroid levels are responsible for the onset of serious hormone-related diseases, and the inhibition of their biosynthesis by targeting cytochrome P450 (CYP) isoforms CYP11B1 and CYP11B2 has emerged as a promising strategy to restore healthy physiological levels of corticosteroids. With the aim of exploiting the xanthone scaffold as a privileged structure in medicinal chemistry and to further explore the chemical space of inhibitors of these CYPs, a small library of imidazolylmethylxanthones was designed based on the results of a previously described compound series. Assuming the capacity for an additional interaction with these enzymes, a properly selected substituent was introduced at position 6 of the xanthone core, maintaining the key imidazolylmethyl moiety at position 1. The 6-fluoro and 6-nitro derivatives [1-(1H-imidazol-1-yl)methyl-6-fluoro-9H-xanthen-9-one (1 a) and 1-(1H-imidazol-1-yl)methyl-6-nitro-9H-xanthen-9-one (1 d), respectively] proved to be active in the low nanomolar range, showing selectivity toward the related steroidogenic enzymes CYP19 and CYP17, even if the problem of selectivity between the two CYP11B isoforms remains unsolved. On the other hand, the 6-chloro derivative 1-(1H-imidazol-1-yl)methyl-6-chloro-9H-xanthen-9-one (1 b) was found to be a fairly potent and somewhat selective CYP19 inhibitor, confirming the versatility of the scaffold.

Exploiting the Chromone Scaffold for the Development of Inhibitors of Corticosteroid Biosynthesis.

The inhibition of corticosteroid biosynthesis could be considered as an emerging strategy to reduce their abnormally high levels, and in this framework CYP11B1 and CYP11B2 represent the most promising targets. In continuing our studies on flavonoid-like scaffolds as privileged structures in medicinal chemistry, in this paper we describe a small library of pyridyl- and imidazolylmethylchromones as potential inhibitors of these enzymes. Testing results proved that position 3 of the chromone scaffold is the most favorable for the introduction of the heme-coordinating heterocycles and, among them, the 4-imidazolyl moiety is the most convenient for the interaction with the heme iron of the selected cytochromes. A low nanomolar inhibitor of CYP11B1 (5c) was obtained, endowed with reasonable selectivity toward CYP11B2 and able to better discriminate with respect to CYP17 and CYP19.

Discovery of Triazole CYP11B2 Inhibitors with in Vivo Activity in Rhesus Monkeys.

Hit-to-lead efforts resulted in the discovery of compound 19, a potent CYP11B2 inhibitor that displays high selectivity vs related CYPs, good pharmacokinetic properties in rat and rhesus, and lead-like physical properties. In a rhesus pharmacodynamic model, compound 19 displays robust, dose-dependent aldosterone lowering efficacy, with no apparent effect on cortisol levels.

Identification of 4-(4-nitro-2-phenethoxyphenyl)pyridine as a promising new lead for discovering inhibitors of both human and rat 11ß-Hydroxylase.

The inhibition of 11ß-hydroxylase is a promising strategy for the treatment of Cushing's syndrome, in particular for the recurrent and subclinical cases. To achieve proof of concept in rats, efforts were paid to identify novel lead compounds inhibiting both human and rat CYP11B1. Modifications on a potent promiscuous inhibitor of hCYP11B1, hCYP11B2 and hCYP19 (compound IV) that exhibited moderate rCYP11B1 inhibition led to compound 8 as a new promising lead compound. Significant improvements compared to starting point IV were achieved regarding inhibitory potency against both human and rat CYP11B1 (IC50 values of 2 and 163 nM, respectively) as well as selectivity over hCYP19 (IC50 = 1900 nM). Accordingly, compound 8 was around 7- and 28-fold more potent than metyrapone regarding the inhibition of human and rat CYP11B1 and exhibited a comparable selectivity over hCYP11B2 (SF of 3.5 vs 4.9). With further optimizations on this new lead compound 8, drug candidates with satisfying profiles are expected to be discovered.