Discovery of selective fragment-sized immunoproteasome inhibitors.

Proteasomes contribute to maintaining protein homeostasis and their inhibition is beneficial in certain types of cancer and in autoimmune diseases. However, the inhibition of the proteasomes in healthy cells leads to unwanted side-effects and significant effort has been made to identify inhibitors specific for the immunoproteasome, especially to treat diseases which manifest increased levels and activity of this proteasome isoform. Here, we report our efforts to discover fragment-sized inhibitors of the human immunoproteasome. The screening of an in-house library of structurally diverse fragments resulted in the identification of benzo[d]oxazole-2(3H)-thiones, benzo[d]thiazole-2(3H)-thiones, benzo[d]imidazole-2(3H)-thiones, and 1-methylbenzo[d]imidazole-2(3H)-thiones (with a general term benzoXazole-2(3H)-thiones) as inhibitors of the chymotrypsin-like (ß5i) subunit of the immunoproteasome. A subsequent structure-activity relationship study provided us with an insight regarding growing vectors. Binding to the ß5i subunit was shown and selectivity against the ß5 subunit of the constitutive proteasome was determined. Thorough characterization of these compounds suggested that they inhibit the immunoproteasome by forming a disulfide bond with the Cys48 available specifically in the ß5i active site. To obtain fragments with biologically more tractable covalent interactions, we performed a warhead scan, which yielded benzoXazole-2-carbonitriles as promising starting points for the development of selective immunoproteasome inhibitors with non-peptidic scaffolds.

Discovery of Ethyl Ketone-Based Highly Selective HDACs 1, 2, 3 Inhibitors for HIV Latency Reactivation with Minimum Cellular Potency Serum Shift and Reduced hERG Activity.

We describe the discovery of histone deacetylase (HDACs) 1, 2, and 3 inhibitors with ethyl ketone as the zinc-binding group. These HDACs 1, 2, and 3 inhibitors have good enzymatic and cellular activity. Their serum shift in cellular potency has been minimized, and selectivity against hERG has been improved. They are also highly selective over HDACs 6 and 8. These inhibitors contain a variety of substituted heterocycles on the imidazole or oxazole scaffold. Compounds 31 and 48 stand out due to their good potency, high selectivity over HDACs 6 and 8, reduced hERG activity, optimized serum shift in cellular potency, and good rat and dog PK profiles.

Characterization of inthomycin biosynthetic gene cluster revealing new insights into carboxamide formation.

Inthomycins are polyketide antibiotics which contain a terminal carboxamide group and a triene chain. Inthomycin B (1) and its two new analogues 2 and 3 were isolated from the crude extract of Streptomyces pactum L8. Identification of the gene cluster for inthomycin biosynthesis as well as the 15N-labeled glycine incorporation into inthomycins are described. Combined with the gene deletion of the rare P450 domain in the NRPS module, a formation mechanism of carboxamide moiety in inthomycins was proposed via an oxidative release of the assembly chain assisted by the P450 domain.

Identification of Adenosine Deaminase Inhibitors by Metal-binding Pharmacophore Screening.

Adenosine deaminase (ADA) is a human mononuclear Zn2+ metalloenzyme that converts adenosine to inosine. ADA is a validated drug target for cancer, but there has been little recent work on the development of new therapeutics against this enzyme. The lack of new advancements can be partially attributed to an absence of suitable assays for high-throughput screening (HTS) against ADA. To facilitate more rapid drug discovery efforts for this target, an in vitro assay was developed that utilizes the enzymatic conversion of a visibly emitting adenosine analogue to the corresponding fluorescent inosine analogue by ADA, which can be monitored via fluorescence intensity changes. Utilizing this assay, a library of ∼350 small molecules containing metal-binding pharmacophores (MBPs) was screened in an HTS format to identify new inhibitor scaffolds against ADA. This approach yielded a new metal-binding scaffold with a Ki value of 26±1 µM.

Solvent-Induced Protein Precipitation for Drug Target Discovery on the Proteomic Scale.

High-throughput drug discovery is highly dependent on the targets available to accelerate the process of candidates screening. Traditional chemical proteomics approaches for the screening of drug targets usually require the immobilization/modification of the drug molecules to pull down the interacting proteins. Recently, energetics-based proteomics methods provide an alternative way to study drug-protein interaction by using complex cell lysate directly without any modification of the drugs. In this study, we developed a novel energetics-based proteomics strategy, the solvent-induced protein precipitation (SIP) approach, to profile the interaction of drugs with their target proteins by using quantitative proteomics. The method is easy to use for any laboratory with the common chemical reagents of acetone, ethanol, and acetic acid. The SIP approach was able to identify the well-known protein targets of methotrexate, SNS-032, and a pan-kinase inhibitor of staurosporine in cell lysate. We further applied this approach to discover the off-targets of geldanamycin. Three known protein targets of the HSP90 family were successfully identified, and several potential off-targets including NADH dehydrogenase subunits NDUFV1 and NDUFAB1 were identified for the first time, and the NDUFV1 was validated by using Western blotting. In addition, this approach was capable of evaluating the affinity of the drug-target interaction. The data collectively proved that our approach provides a powerful platform for drug target discovery.

Discovery and evaluation of novel nitrodihydroimidazooxazoles as promising anti-tuberculosis agents.

New analogues of antitubercular drug Delamanid were prepared, seeking drug candidates with enhanced aqueous solubility and high efficacy. The strategy involved replacement of phenoxy linker proximal to the 2-nitroimidazooxazole of Delamanid by piperidine fused 5 or 6-membered ring heterocycles (ring A). The new compounds were all more hydrophilic than Delamanid, and several class of analogues showed remarkable activities against M. bovis. And among these series, the tetrahydro-naphthyridine-linked nitroimidazoles displayed excellent antimycobacterial activity against both replicating (MABA) and nonreplicating (LORA) M. tb H37Rv and low cytotoxicity. Compared to Delamanid, these new compounds (6, 7, 45) demonstrated dramatically improved physicochemical properties and are suitable for further in vitro and in vivo evaluation.

Fe3O4 MNPs as a green catalyst for syntheses of functionalized [1,3]-oxazole and 1H-pyrrolo-[1,3]-oxazole derivatives and evaluation of their antioxidant activity.

In the present study, iron oxide magnetic nanoparticles (Fe3O4 MNPs) were synthesized in a green biosynthetic manner using aqueous extract of clover leaves. Fe3O4 MNPs were applied as a magnetically separable nanocatalyst for the green syntheses of functionalized [1,3]-oxazoles 1(a-e) and 1H-pyrrolo-[1,3]-oxazoles 4(a-i) as promising antioxidant compounds in excellent yields at 50 °C and room temperature, respectively. The antioxidant activities of the most stable compounds (1a, 1b, 4a, and 4b) were evaluated by both 2,2-diphenyl-1-picrylhydrazyl radical scavenging and ferric reduction activity potential assays. Compound 1b was shown a remarkable radical scavenging activity, and 4a was shown very good reducing activity relative to standards (BHT and TBHQ).

Two goitrogenic 1,3-oxazolidine-2-thione derivatives from Brassicales taxa: Challenging identification, occurrence and immunomodulatory effects.

1,3-Oxazolidine-2-thione derivatives are glucosinolate-related food constituents known to impart (thyreo)toxic properties to some cruciferous vegetables. In this work, 5,5-dimethyl-1,3-oxazolidine-2-thione and (-)-(R)-5-phenyl-1,3-oxazolidine-2-thione, known goitrogens, were isolated from Draba lasiocarpa Rochel (Brassicaceae) and Reseda luteola L. (Resedaceae), respectively, and were fully spectrally characterized. Subsequently, the occurrence of the two 1,3-oxazolidine-2-thiones was verified in six additional taxa out of in total 78 screened Serbian Brassicales taxa. The stereochemistry of 5-phenyl-1,3-oxazolidine-2-thione was inferred from nuclear magnetic resonance experiments with a chiral lanthanide-shift reagent, employed in this work for the first time for this type of compounds. Unexpectedly, during gas chromatography, 5-phenyl-1,3-oxazolidine-2-thione underwent an unreported thermal core isomerization (1,3-oxazolidine-2-thione to 1,3-thiazolidine-2-one). These goitrogenic volatile glucosinolate products were tested for their effect on rat macrophage viability (three assays) and nitric oxide production. It was shown that the compounds displayed different levels of cytotoxicity. All tested compounds caused a significant lactate dehydrogenase leakage, but only (R)-5-phenyl-1,3-oxazolidine-2-thione statistically significantly reduced macrophage mitochondrial activity, whereas the racemic 5-phenyl-1,3-oxazolidine-2-thione and 5,5-dimethyl-1,3-oxazolidine-2-thione had little or no effect. Again only (R)-5-phenyl-1,3-oxazolidine-2-thione exerted nitric oxide production-inhibiting properties, suggesting the higher immunomodulatory potential of this enantiomer compared with its antipode and racemic mixture.

Identification and biochemical characterization of DC07090 as a novel potent small molecule inhibitor against human enterovirus 71 3C protease by structure-based virtual screening.

Hand, foot and mouth disease (HFMD) is a serious, highly contagious disease. HFMD caused by Enterovirus 71 (EV71), results in severe complications and even death. The pivotal role of EV71 3Cpro in the viral life cycle makes it an attractive target for drug discovery and development to treat HFMD. In this study, we identified novel EV71 3Cpro inhibitors by docking-based virtual screening. Totally 50 compounds were selected to test their inhibitory activity against EV71 3Cpro. The best inhibitor DC07090 exhibited the inhibition potency with an IC50 value of 21.72 ± 0.95 µM without apparent toxicity (CC50 > 200 µM). To explore structure-activity relationship of DC07090, 15 new derivatives were designed, synthesized and evaluated in vitro enzyme assay accordingly. Interestingly, four compounds showed inhibitory activities against EV71 3Cpro and only DC07090 inhibited EV71 replication with an EC50 value of 22.09 ± 1.07 µM. Enzyme inhibition kinetic experiments showed that the compound was a reversible and competitive inhibitor. The Ki value was determined to be 23.29 ± 12.08 µM. Further molecular docking, MD simulation and mutagenesis studies confirmed the binding mode of DC07090 and EV71 3Cpro. Besides, DC07090 could also inhibit coxsackievirus A16 (CVA16) replication with an EC50 value of 27.76 ± 0.88 µM. Therefore, DC07090 represents a new non-peptidyl small molecule inhibitor for further development of antiviral therapy against EV71 or other picornaviruses.

Physical compatibility of tedizolid phosphate with selected i.v. drugs during simulated Y-site administration.

PURPOSE: The physical compatibility of commonly used agents that could be coadministered in the clinical setting with tedizolid phosphate during Y-site administration was evaluated. METHODS: Tedizolid phosphate vials were reconstituted to a final concentration of 0.8 mg/mL. All other drugs were prepared according to manufacturers' recommendations and diluted with 0.9% sodium chloride injection (where applicable) to the highest standard concentrations used clinically. Y-site conditions were simulated in culture tubes by mixing 5 mL of tedizolid phosphate solution with 5 mL of the test drug solutions. The physical characteristics, turbidity, and pH of all admixtures were examined immediately after mixing and at 15, 60, and 120 minutes. Incompatibility was defined as gross precipitation, a positive Tyndall beam test, color changes, or increases in turbidity. RESULTS: With simulated Y-site administration, tedizolid phosphate was compatible with 69 of 86 drugs in 0.9% sodium chloride injection, including 24 of 31 antimicrobial agents. Of note, incompatibility was observed immediately after mixing except with ceftaroline and diphenhydramine, whose incompatibility with tedizolid phosphate was apparent after 15 and 60 minutes, respectively. Among the drug classes tested, tedizolid phosphate was compatible only with 1 aminoglycoside (amikacin) and incompatible with 1 echinocandin (caspofungin) and 1 cephalosporin (ceftaroline). In addition, tedizolid phosphate was incompatible with divalent cations (calcium chloride, calcium gluconate, and magnesium sulfate), probably due to precipitation with the phosphate component. A pH change of >1 unit occurred only with epinephrine (at 120 minutes). CONCLUSION: Tedizolid phosphate 0.8 mg/mL in 0.9% sodium chloride injection was physically compatible with 69 of 86 study drugs during simulated Y-site administration.

2-pentadecyl-2-oxazoline: Identification in coffee, synthesis and activity in a rat model of carrageenan-induced hindpaw inflammation.

N-acylethanolamines (NAEs) comprise a family of bioactive lipid molecules present in animal and plant tissues, with N-palmitoylethanolamine (PEA) having received much attention owing to its anti-inflammatory, analgesic and neuroprotective activities. 2-Pentadecyl-2-oxazoline (PEA-OXA), the oxazoline of PEA, reportedly modulates activity of N-acylethanolamine-hydrolyzing acid amidase (NAAA), which catabolizes PEA. Because PEA is produced on demand and exerts pleiotropic effects on non-neuronal cells implicated in neuroinflammation, modulating the specific amidases for NAEs (NAAA in particular) could be a way to preserve PEA role in maintaining cellular homeostasis through its rapid on-demand synthesis and equally rapid degradation. This study provides the first description of PEA-OXA in both green and roasted coffee beans and Moka infusions, and its synthesis. In an established model of carrageenan (CAR)-induced rat paw inflammation, PEA-OXA was orally active in limiting histological damage and thermal hyperalgesia 6h after CAR intraplantar injection in the right hindpaw and the accumulation of infiltrating inflammatory cells. PEA-OXA appeared to be more potent compared to ultramicronized PEA given orally at the same dose (10mg/kg). PEA-OXA markedly reduced also the increase in hindpaw myeloperoxidase activity, an index of polymorphonuclear cell accumulation in inflammatory tissues. NAAA modulators like PEA-OXA may serve to maximize availability of NAEs (e.g. PEA) while providing for recycling of the NAE components for further resynthesis.

A novel 3-(4,5-diphenyl-1,3-oxazol-2-yl)propanal oxime compound is a potent Transient Receptor Potential Ankyrin 1 and Vanilloid 1 (TRPA1 and V1) receptor antagonist.

Transient Receptor Potential Ankyrin 1 and Vanilloid 1 (TRPA1, TRPV1) ion channels expressed on nociceptive primary sensory neurons are important regulators of pain and inflammation. TRPA1 is activated by several inflammatory mediators including formaldehyde and methylglyoxal that are products of the semicarbazide-sensitive amine-oxidase enzyme (SSAO). SZV-1287 is a new 3-(4,5-diphenyl-1,3-oxazol-2-yl)propanal oxime SSAO inhibitor, its chemical structure is similar to other oxime derivatives described as TRPA1 antagonists. Therefore, we investigated its effects on TRPA1 and TRPV1 receptor activation on the cell bodies and peripheral terminals of primary sensory neurons and TRPA1 or TRPV1 receptor-expressing cell lines. Calcium influx in response to the TRPA1 agonist allyl-isothiocyanate (AITC) (200 µM) and the TRPV1 stimulator capsaicin (330 nM) in rat trigeminal neurons or TRPA1 and TRPV1 receptor-expressing cell lines was measured by microfluorimetry or radioactive (45)Ca(2+) uptake experiments. Calcitonin gene-related peptide (CGRP) release as the indicator of 100 µM AITC - or 100 nM capsaicin-induced peripheral sensory nerve terminal activation was measured by radioimmunoassay. SZV-1287 (100, 500 and 1000 nM) exerted a concentration-dependent significant inhibition on both AITC- and capsaicin-evoked calcium influx in trigeminal neurons and TRPA1 or TRPV1 receptor-expressing cell lines. It also significantly inhibited the TRPA1, but not the TRPV1 activation-induced CGRP release from the peripheral sensory nerve endings in a concentration-dependent manner. In contrast, the reference SSAO inhibitor LJP 1207 with a different structure had no effect on TRPA1 or TRPV1 activation in either model system. This is the first evidence that our novel oxime compound SZV-1287 originally developed as a SSAO inhibitor has a potent dual antagonistic action on TRPA1 and TRPV1 ion channels on primary sensory neurons.

Evaluation of radiofrequency safety by high temperature resolution MR thermometry using a paramagnetic lanthanide complex.

PURPOSE: The current practice of calculating the specific absorption rate (SAR) relies on local temperature measurements made using temperature probes. For an accurate SAR measurement, a temperature imaging method that provides high temperature sensitivity is desirable, because acceptable levels of SAR produce small temperature changes. MR thermometry using paramagnetic lanthanide complexes can be used to obtain absolute temperature measurements with sub-degree temperature and sub-millimeter spatial resolution. The aim of this study was to develop and evaluate a high temperature resolution MR technique to determine SAR. METHODS: MR thermometry using a paramagnetic lanthanide complex thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis (methylene phosphonate) (TmDOTP(5-)), which has an almost 10(2) times stronger chemical shift temperature dependence than water, was used to develop a novel method for SAR measurement. Three-dimensional temperature and SAR images were calculated using MR images acquired with a conventional gradient recalled echo sequence and SAR-intensive T1ρ sequence. Effects of the presence of conducting wire and increasing T1ρ spin-lock pulse duration were also examined. RESULTS: SAR distribution could be visualized clearly and surges associated with conducting wires and increasing pulse duration were identified clearly in the computed high spatial resolution SAR images. CONCLUSION: A novel method with high temperature sensitivity is proposed as a tool to evaluate radiofrequency safety in MRI.

Further insights into the pharmacology of the human trace amine-associated receptors: discovery of novel ligands for TAAR1 by a virtual screening approach.

Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor that is expressed in brain and periphery and responds to a class of compounds called trace amines, such as ß-phenylethylamine (ß-PEA), tyramine, tryptamine, octopamine. The receptor is known to have a very rich pharmacology and could be also activated by different classes of compounds, including dopaminergic, adrenergic and serotonergic ligands. It is expected that targeting hTAAR1 could provide a novel pharmacological approach for several human disorders, such as schizophrenia, depression, attention deficit hyperactivity disorder, Parkinson's disease and metabolic diseases. Only recently, a small number of selective hTAAR1 agonists (among which RO5166017 and T1 AM) and antagonist (EPPTB), have been reported in literature. With the aim to identify new molecular entities able to act as ligands for this target, we used an homology model for the hTAAR1 and performed a virtual screening procedure on an in-house database of compounds. A number of interesting molecules were selected and by testing them in an in vitro assay we found several agonists and one antagonist, with activities in the low micromolar range. These compounds could represent the starting point for the development of more potent and selective TAAR1 ligands.

Discovery of oral and inhaled PDE4 inhibitors.

The optimization of oxazole-based PDE4 inhibitor 1 has led to the identification of both oral (compound 16) and inhaled (compound 34) PDE4 inhibitors. Selectivity against PDE10/PDE11, off target screening, and in vivo activity in the rat are discussed.

Discovery of novel oxazolidinedione derivatives as potent and selective mineralocorticoid receptor antagonists.

Novel oxazolidinedione analogs were discovered as potent and selective mineralocorticoid receptor (MR) antagonists. Structure-activity relationship (SAR) studies were focused on improving the potency and microsomal stability. Selected compounds demonstrated excellent MR activity, reasonable nuclear hormone receptor selectivity, and acceptable rat pharmacokinetics.

An enzymatic route to selenazolines.

Ringing the changes: Selenazolines have applications in medicinal chemistry, but their synthesis is challenging. We report a new convenient and less toxic route to these heterocycles that starts from commercially available selenocysteine. The new route depends on a heterocyclase enzyme that creates oxazolines and thiazolines from serines/threonines and cysteines.

Stereodivergent SN2@P reactions of borane oxazaphospholidines: experimental and theoretical studies.

The stereodivergent ring-opening of 2-phenyl oxazaphospholidines with alkyl lithium reagents is reported. N-H oxazaphospholidines derived from both (+)-cis-1-amino-2-indanol and (-)-norephedrine provide inversion products in a highly stereoselective process. In contrast, N-Me oxazaphospholidines yield ring-opening products with retention of configuration at the P center, as previously reported by Jugé and co-workers. As a result, from a single amino alcohol auxiliary, both enantiomers of key P-stereogenic intermediates could be synthesized. Theoretical studies of ring-opening with model oxazaphospholidines at the DFT level have elucidated the streochemical course of this process. N-H substrates react in a single step via preferential backside S(N)2@P substitution with inversion at phosphorus. N-methylated substrates react preferentially via a two-step frontside S(N)2@P, yielding a ring-opened product in which the nucleophilic methyl binds to P with retention of configuration. DFT calculations have shown that the BH3 unit is a potent directing group to which the methyl lithium reagent coordinates via Li in all the reactions studied.

On the structure of aspongopusin recently isolated from Aspongopus chinensis.

The 2,5-disubstituted oxazole recently proposed for aspongopusin, a natural product isolated from Aspongopus chinensis, was synthesized through an unambiguous route. The synthetic sample showed (1)H and (13)C NMR entirely different from those in the literature, revealing that the initially assigned structure was incorrect. The spectroscopic data for the given structure are thus made available for the first time.

Synthesis of 2-phenyl-4,5-substituted oxazoles by copper-catalyzed intramolecular cyclization of functionalized enamides.

An efficient two-step synthesis of 2-phenyl-4,5-substituted oxazoles involving intramolecular copper-catalyzed cyclization of highly functionalized novel ß-(methylthio)enamides as the key step has been reported. These enamides are obtained by nucleophilic ring-opening of newly synthesized 4-[(methylthio)hetero(aryl)methylene]-2-phenyl-5-oxazolone precursors by alkoxides, amines, amino acid esters and aryl/alkyl Grignard reagents, thus leading to the introduction of an ester, N-substituted carboxamide or acyl functionalities at 4-position of the product oxazoles. Synthesis of two naturally occurring 2,5-diaryloxazoles, i.e., texamine and uguenenazole, via two-step hydrolysis-decarboxylation of the corresponding 2,5-diaryloxazole-4-carboxylates has also been described. Similarly, three of the serine-derived oxazole-4-carboxamides were elaborated to novel trisubstituted 4,2'-bisoxazoles through DAST/DBU-mediated cyclodehydration-dehydrohalogenation sequence. The present protocol is complementary and an improvement to our previously reported silver carbonate-induced cyclization of ß-bis(methylthio)enamides to 2-phenyl-5-(methylthio)-4-substituted oxazoles.