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For disorders of the skin, eyes, ears, and respiratory tract, topical drugs, delivered directly to the target organ, are a therapeutic option. Compared with systemic oral therapy, the benefits of topical treatments include a faster onset of action, circumventing the liver first pass drug metabolism, and reducing systemic side effects. Nevertheless, some systemic absorption still occurs for many topical agents resulting in systemic side effects. One way to prevent these would be to develop drugs that are instantly degraded upon entry into the bloodstream by serum esterases. Because topical β-blockers are used in glaucoma and infantile hemeangioma and cause systemic side effects, the β-adrenoceptor system was used to test this hypothesis. Purified liver esterase reduced the apparent affinity of esmolol, an ester-containing β-blocker used in clinical emergencies, for the human β-adrenoceptors in a concentration and time-dependent manner. However, purified serum esterase had no effect on esmolol. Novel ester-containing β-blockers were synthesized and several were sensitive to both liver and serum esterases. Despite good in vitro affinity, one such compound, methyl 2-(3-chloro-4-(3-((2-(3-(3-chlorophenyl)­ureido)­ethyl)­amino)-2-hydroxypropoxy)­phenyl)­acetate, had no effect on heart rate when injected intravenously into rats, even at 10 times the equipotent dose of esmolol and betaxolol that caused short and sustained reductions in heart rate, respectively. Thus, ester-based drugs, sensitive to serum esterases, offer a mechanism for developing topical agents that are truly devoid of systemic side effects. Furthermore, differential susceptibility to liver and serum esterases degradation may also allow the duration of systemic availability for other drugs to be fine-tuned.

The combined antiretroviral therapy (cART) can efficiently suppress HIV replication, but the cessation of cART usually results in viral rebound, mostly due to the presence of viral reservoirs. The mesenteric lymphatic system, including mesenteric lymph nodes (MLNs), is an important viral reservoir into which antiretroviral drugs poorly penetrate. In this work, we proposed a novel lipophilic ester prodrug approach, combined with oral lipid-based formulation, to efficiently deliver lopinavir (LPV) to the mesenteric lymph and MLNs. A series of prodrugs was designed using an in-silico model for prediction of affinity to chylomicrons (CMs), and then synthesized. The potential for mesenteric lymphatic targeting and bioconversion to LPV in physiologically relevant media was assessed in vitro and ex vivo. Subsequently, LPV and selected prodrug candidates were evaluated for their in vivo pharmacokinetics and biodistribution in rats. Oral co-administration of lipids alone could not facilitate the delivery of unmodified LPV to the mesenteric lymphatic system and resulted in undetectable levels of LPV in these tissues. However, a combination of the lipophilic prodrug approach with lipid-based formulation resulted in efficient targeting of LPV to HIV reservoirs in mesenteric lymph and MLNs. The maximum levels of LPV in mesenteric lymph were 1.6- and 16.9-fold higher than protein binding-adjusted IC90 (PA-IC90) of LPV for HIV-1 (140 ng/mL) following oral administration of simple alkyl ester prodrug and activated ester prodrug, respectively. Moreover, the concentrations of LPV in MLNs were 1.1- and 7.2-fold higher than PA-IC90 following administration of simple alkyl ester prodrug and activated ester prodrug, respectively. Furthermore, the bioavailability of LPV was also substantially increased following oral administration of activated ester prodrug compared to unmodified LPV. This approach, especially if can be translated to other antiretroviral drugs, has potential for reducing the size of HIV reservoirs within the mesenteric lymphatic system.

Polymorphisms in the region of the calmodulin-dependent kinase isoform D (CaMK1D) gene are associated with increased incidence of diabetes, with the most common polymorphism resulting in increased recognition by transcription factors and increased protein expression. While reducing CaMK1D expression has a potentially beneficial effect on glucose processing in human hepatocytes, there are no known selective inhibitors of CaMK1 kinases that can be used to validate or translate these findings. Here we describe the development of a series of potent, selective, and drug-like CaMK1 inhibitors that are able to provide significant free target cover in mouse models and are therefore useful as in vivo tool compounds. Our results show that a lead compound from this series improves insulin sensitivity and glucose control in the diet-induced obesity mouse model after both acute and chronic administration, providing the first in vivo validation of CaMK1D as a target for diabetes therapeutics.

[Image: see text] For disorders of the skin, eyes, ears, and respiratory tract, topical drugs, delivered directly to the target organ, are a therapeutic option. Compared with systemic oral therapy, the benefits of topical treatments include a faster onset of action, circumventing the liver first pass drug metabolism, and reducing systemic side effects. Nevertheless, some systemic absorption still occurs for many topical agents resulting in systemic side effects. One way to prevent these would be to develop drugs that are instantly degraded upon entry into the bloodstream by serum esterases. Because topical β-blockers are used in glaucoma and infantile hemeangioma and cause systemic side effects, the β-adrenoceptor system was used to test this hypothesis. Purified liver esterase reduced the apparent affinity of esmolol, an ester-containing β-blocker used in clinical emergencies, for the human β-adrenoceptors in a concentration and time-dependent manner. However, purified serum esterase had no effect on esmolol. Novel ester-containing β-blockers were synthesized and several were sensitive to both liver and serum esterases. Despite good in vitro affinity, one such compound, methyl 2-(3-chloro-4-(3-((2-(3-(3-chlorophenyl)ureido)ethyl)amino)-2-hydroxypropoxy)phenyl)acetate, had no effect on heart rate when injected intravenously into rats, even at 10 times the equipotent dose of esmolol and betaxolol that caused short and sustained reductions in heart rate, respectively. Thus, ester-based drugs, sensitive to serum esterases, offer a mechanism for developing topical agents that are truly devoid of systemic side effects. Furthermore, differential susceptibility to liver and serum esterases degradation may also allow the duration of systemic availability for other drugs to be fine-tuned.

The Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. The ISR is mediated by stress-induced phosphorylation of translation initiation factor 2 (eIF2) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, a bis-O-arylglycolamide, reverses the attenuation of eIF2B by phosphorylated eIF2, protecting mice from neurodegeneration and traumatic brain injury. We report on a cryo-electron microscopy-based structure of ISRIB-bound human eIF2B revealing an ISRIB-binding pocket at the interface between the β and δ regulatory subunits. CRISPR/Cas9 mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB congenersin vivoand ISRIB-bindingin vitro, thus providing chemogenetic support for the functional relevance of ISRIB binding at a distance from known eIF2-eIF2B interaction sites. Our findings point to a hitherto unexpected allosteric site in the eIF2B decamer exploited by ISRIB to regulate translation.

β-Blockers reduce mortality and improve symptoms in people with heart disease; however, current clinically available β-blockers have poor selectivity for the cardiac β1-adrenoceptor (AR) over the lung β2-AR. Unwanted β2-blockade risks causing life-threatening bronchospasm and reduced efficacy of β2-agonist emergency rescue therapy. Thus, current life-prolonging β-blockers are contraindicated in patients with both heart disease and asthma. Here, we describe NDD-713 and -825, novel highly β1-selective neutral antagonists with good pharmaceutical properties that can potentially overcome this limitation. Radioligand binding studies and functional assays that use human receptors expressed in Chinese hamster ovary cells demonstrate that NDD-713 and -825 have nanomolar β1-AR affinity >500-fold β1-AR vs. β2-AR selectivity and no agonism. Studies in conscious rats demonstrate that these antagonists are orally bioavailable and cause pronounced β1-mediated reduction of heart rate while showing no effect on β2-mediated hindquarters vasodilatation. These compounds also have good disposition properties and show no adverse toxicologic effects. They potentially offer a truly cardioselective β-blocker therapy for the large number of patients with heart and respiratory or peripheral vascular comorbidities.—Baker, J. G., Gardiner, S. M., Woolard, J., Fromont, C., Jadhav, G. P., Mistry, S. N., Thompson, K. S. J., Kellam, B., Hill, S. J., Fischer, P. M. Novel selective β1-adrenoceptor antagonists for concomitant cardiovascular and respiratory disease.

Pin1 is an emerging oncology target strongly implicated in Ras and ErbB2-mediated tumourigenesis. Pin1 isomerizes bonds linking phospho-serine/threonine moieties to proline enabling it to play a key role in proline-directed kinase signalling. Here we report a novel series of Pin1 inhibitors based on a phenyl imidazole acid core that contains sub-μM inhibitors. Compounds have been identified that block prostate cancer cell growth under conditions where Pin1 is essential.

Inhibition of the ATPase activity of the chaperone protein HSP90 is a potential strategy for treatment of cancers. We have determined structures of the HSP90α N-terminal domain complexed with the purine-based inhibitor, PU3, and analogs with enhanced potency both in enzyme and cell-based assays. The compounds induce upregulation of HSP70 and downregulation of the known HSP90 client proteins Raf-1, CDK4, and ErbB2, confirming that the molecules inhibit cell growth by a mechanism dependent on HSP90 inhibition. We have also determined the first structure of the N-terminal domain of HSP90β, complexed with PU3. The structures allow a detailed rationale to be developed for the observed affinity of the PU3 class of compounds for HSP90 and also provide a structural framework for design of compounds with improved binding affinity and drug-like properties.

Multiple co-crystal structures of an adenine-based series of inhibitors bound to the molecular chaperone Hsp90 have been determined. These structures explain the observed SAR for previously described compounds and new compounds, which possess up to 8-fold improved potency against the isolated enzyme. Anti-tumour cell potency and mechanism of action data is also described for the most potent compounds. These data should enable the design of more potent Hsp90 inhibitors.

We report the reactivity of new Cβ silylated ketenimines (4, 5, 6) and lithiated-silylated ketenimine 3 leasily generated from lithiated S-methyl-N-phenyl-trimethylsilylethanimidothioate 2] toward electrophilic reagents. Reactions of these ketenimines with strong electrophilic reagents such as sulfanyl chloride (PhSCl) gave access to new sulfanylated ketenimines. With less reactive species [benzenesulfinyl chloride (PhS(O)Cl), p-toluenesulfonyl chloride, trimethylhalosilanes, diisopropyl chlorophosphate, acetyl chloride and propylene oxide] and 3, the addition took place either on Cβ or on the nitrogen atom leading to relatively unstable functionalised ketenimines or new silylated ynamines respectively.

We report the reactivity of new Cβ silylated ketenimines (4, 5, 6) and lithiated-silylated ketenimine 3 leasily generated from lithiated S-methyl-N-phenyl-trimethylsilylethanimidothioate 2] toward electrophilic reagents. Reactions of these ketenimines with strong electrophilic reagents such as sulfanyl chloride (PhSCl) gave access to new sulfanylated ketenimines. With less reactive species [benzenesulfinyl chloride (PhS(O)Cl), p-toluenesulfonyl chloride, trimethylhalosilanes, diisopropyl chlorophosphate, acetyl chloride and propylene oxide] and 3, the addition took place either on Cβ or on the nitrogen atom leading to relatively unstable functionalised ketenimines or new silylated ynamines respectively.

Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe novel 2-aminothieno[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors, which were designed by combining structural elements of distinct low affinity hits generated from fragment-based and in silico screening exercises in concert with structural information from X-ray protein crystallography. Examples from this series have high affinity (IC50 = 50-100 nM) for Hsp90 as measured in a fluorescence polarization (FP) competitive binding assay and are active in human cancer cell lines where they inhibit cell proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Several examples (34a, 34d and 34i) caused tumor growth regression at well tolerated doses when administered orally in a human BT474 human breast cancer xenograft model.

We have designed four generations of a low molecular weight fragment library for use in NMR-based screening against protein targets. The library initially contained 723 fragments which were selected manually from the Available Chemicals Directory. A series of in silico filters and property calculations were developed to automate the selection process, allowing a larger database of 1.79 M available compounds to be searched for a further 357 compounds that were added to the library. A kinase binding pharmacophore was then derived to select 174 kinase-focused fragments. Finally, an additional 61 fragments were selected to increase the number of different pharmacophores represented within the library. All of the fragments added to the library passed quality checks to ensure they were suitable for the screening protocol, with appropriate solubility, purity, chemical stability, and unambiguous NMR spectrum. The successive generations of libraries have been characterized through analysis of structural properties (molecular weight, lipophilicity, polar surface area, number of rotatable bonds, and hydrogen-bonding potential) and by analyzing their pharmacophoric complexity. These calculations have been used to compare the fragment libraries with a drug-like reference set of compounds and a set of molecules that bind to protein active sites. In addition, an analysis of the overall results of screening the library against the ATP binding site of two protein targets (HSP90 and CDK2) reveals different patterns of fragment binding, demonstrating that the approach can find selective compounds that discriminate between related binding sites.

The mature petiole of celery is an organ with versatile sink/source capacities where sucrose and mannitol are unloaded from and reloaded into the phloem cells. Plasma-membrane vesicles were purified by twophase partitioning either from phloem strands isolated from mature petioles of celery (Apium graveolens L.) or from mature petioles devoid of vascular bundles. Both types of vesicle were comparable in purity (more than 86% of plasma-membrane origin), size (135 nm diameter) and orientation (72% right-side-out). Plasma-membrane vesicles from phloem tissues had a higher vanadate-sensitive ATPase activity than plasma-membrane vesicles from petioles. Plasma-membrane vesicles from phloem tissues accumulated mannitol and sucrose in response to an artificial proton-motive force, in agreement with the existence of proton/substrate carriers. Plasma-membrane vesicles from petioles devoid of vascular bundles accumulated only mannitol following application of an artificial proton-motive force. The data suggest the volvement of apoplasmic transport events. The pathway for sucrose uptake in storage parenchyma cells is discussed in the light of the available physiological data.

Resin beads with a loading of 200 nmol per bead have been prepared using a Generation 2.0 tris-based dendrimer to amplify resin loading sites.

The reactivity of stable silylated N-phenyl ketenimines towards some nucleophilic (organometallics, alcohols, thiols) and electrophilic reagents (PhSCI) were studied. Imino-coumarines (via tandem nucleophilic-electrophilic additions with Peterson olefination) and new phenylthio- and silyl-substituted ketenimines have been prepared.

The asymmetric version of the Claisen rearrangement (1) usually involves the preparation of a carboxylic ester of an enantiomerically pure allylic alcohol, its deprotonation and transposition either of the lithium enolate or its silyl ketene acetal.