Structure-based design and discovery of potent and selective KDM5 inhibitors

Histone lysine demethylases (KDMs) play a key role in epigenetic regulation and KDM5A and KDM5B have been identified as potential anti-cancer drug targets. Using structural information from known KDM4 and KDM5 inhibitors, a potent series of pyrazolylpyridines was designed. Structure-activity relationship (SAR) exploration resulted in the identification of compound 33, an orally available, potent inhibitor of KDM5A/5B with promising selectivity. Potent cellular inhibition as measured by levels of tri-methylated H3K4 was demonstrated with compound 33 in the breast cancer cell line ZR-75-1.     

Structural characterization of the GSK-3beta active site using selective and non-selective ATP-mimetic inhibitors

GSK-3beta is a regulatory serine/threonine kinase with a plethora of cellular targets. Consequently, selective small molecule inhibitors of GSK-3beta may have a variety of therapeutic uses including the treatment of neurodegenerative diseases, type II diabetes and cancer. In order to characterize the active site of GSK-3beta, we determined crystal structures of unphosphorylated GSK-3beta in complex with selective and non-selective ATP-mimetic inhibitors. Analysis of the inhibitors' interactions with GSK-3beta in the structures reveals how the enzyme can accommodate a number of diverse molecular scaffolds. In addition, a conserved water molecule near Thr138 is identified that can serve a functional role in inhibitor binding. Finally, a comparison of the interactions made by selective and non-selective inhibitors highlights residues on the edge of the ATP binding-site that can be used to obtain inhibitor selectivity. Information gained from these structures provides a promising route for the design of second-generation GSK-3beta inhibitors.     

The discovery of potent and selective 4-aminothienopyridines as B-Raf kinase inhibitors

A series of novel, potent 4-aminothienopyridine B-Raf kinase inhibitors was designed and synthesized using knowledge-based design. Compounds 5f, and 6k exhibited not only excellent potency in both enzyme assay (IC₅₀ = 5.1, 16.6 nM) and cellular assay (IC₅₀ = 0.2, 0.2 μM), but also had an outstanding selectivity profile against other kinases.     

Novel selective and non-selective optical detection of micro-organisms

A new instrument, capable of detecting metabolic changes due to microbiological activity, is described. Optical changes in growth media are monitored in a semi-fluid zone that separates the liquid medium containing the sample. Data demonstrate that common media can be utilized in conjunction with this rapid automated technology. Nutrient broth with the pH dye indicator bromocresol purple was suitable for total counts. Selective media containing dyes were utilized to assess the presence or absence of specific groups of organisms. Biochemical reactions, such as lysine decarboxylase activity, were identified by the unique generated patterns, and specific enzymatic cleavage reactions with chromogenic substrates, such as 5-bromo-4 chloro-3 indolyl-β- D -glucuronic acid (X-GLUC), were monitored.     

NSAID inhibition of RGM1 gastric monolayer wound re-epithelialization: comparison of selective Cox-2 versus non-selective Cox inhibitors

Clinical studies indicate that specific cyclooxygenase-2 (Cox-2) inhibitors are less ulcerogenic than their non-selective predecessors (e.g. indomethacin). However, Cox-2 inhibitors may also interfere with ulcer healing. Re-epithelialization is a crucial factor in both gastrointestinal mucosal injury and ulcer healing. This study was aimed to compare the effects of selective Cox-2 inhibitor (NS398) versus non-selective Cox inhibitor (indomethacin) on basal and basic fibroblast growth factor (bFGF) - stimulated gastric wound re-epithelialization. In-vitro epithelial wounds were created in confluent monolayers of RGM1 rat gastric epithelial cells by a razor blade scrape. Following wounding there was a significant re-epithelialization by 24 hrs. Indomethacin (0.25 mM and 0.5 mM) significantly inhibited basal wound re-epithelialization in a dose dependent manner. In contrast, selective Cox-2 inhibitor NS398 did not inhibit the basal re-epithelialization process. Basic FGF treatment produced significant enhancement of wound re-epitheliazation at the various concentrations [10, 20, 30, 40, 50 and 70 ng/ml] studied. Both indomethacin and NS398 inhibited bFGF stimulated wound re-epithelialization, with indomethacin having a greater inhibitory effect. The extent of NS398 inhibition was limited to the bFGF-stimulated component, whereas indomethacin inhibition extended to both the bFGF-stimulated and the basal re-epithelialization components. These findings indicate that specific Cox-2 inhibitor (NS398) does not interfere with the basal re-epithelialization but significantly inhibits the bFGF - stimulated re-epithelialization, whereas indomethacin interferes with both the basal as well as the bFGF-stimulated wound re-epithelialization.     

The discovery of 6-amino nicotinamides as potent and selective histone deacetylase inhibitors

This communication highlights the development of a nicotinamide series of histone deacetylase inhibitors within the benzamide structural class. Extensive exploration around the nicotinamide core led to the discovery of a class I selective HDAC inhibitor that possesses excellent intrinsic and cell-based potency, acceptable ancillary pharmacology, favorable pharmacokinetics, sustained pharmacodynamics in vitro, and achieves in vivo efficacy in an HCT116 xenograft model.     

The discovery and synthesis of highly potent subtype selective phosphodiesterase 4D inhibitors

The SAR study of a series of 6-aryloxymethyl-8-aryl substituted quinolines is described. Optimization of the series led to the discovery of compound 26b, a highly potent (IC₅₀ = 0.6 nM) and selective PDE4D inhibitor with a 75-fold selectivity over the A, B, and C subtypes and over 18,000-fold selectivity against other PDE family members. Rat pharmacokinetics and tissue distribution are also summarized.     

The discovery of tetrahydropyridine analogs as hNav1.7 selective inhibitors for analgesia

hNav1.7 small molecular inhibitors have attracted lots of attention by its unique analgesic effect. Herein, we report the design and synthesis of a novel series of tetrahydropyridine analogs as hNav1.7 inhibitors for analgesia. Detail structural–activity relationship (SAR) studies were undertaken towards improving hNav1.7 activity, in vitro ADME, and in vivo PK profiles. These efforts resulted in the identification of compound (?)-15h, a highly potent and selective hNav1.7 inhibitor with good ADME and PK profiles.     

Effect of selective and non-selective cysteine protease inhibitors on the intracellular processing of interleukin 6 by HepG2 cells

Summary The effects were measured and compared of three nonselective cysteine cathepsin inhibitors (leupeptin, trans-Epoxysuccinyl-l-Leucylamido(4-guanidino)-butane (E-64), and Z-Phe-Ala-CH₂F) and a selective cathepsin B inhibitor, CA074Me, on the intracellular processing of ¹²⁵I-labeled human recombinant Interleukin 6 (IL-6) by HepG2 cells. The uptake and processing of ¹²⁵I-IL-6 by cells treated with inhibitors was followed over a 7-h period. All inhibitors caused an increased residence time of IL-6 inside the cell and a corresponding decrease in the output of non-trichloroacetic acid-precipitable fragments of radiolabeled protein. Maximal effect was achieved with leupeptin at 200 μM, with which the rate of IL-6 digestion was reduced to 50% that of control cells. The specific inhibitor CA074Me was the least effective in slowing the intracellular processing of IL-6. The effects of all of the inhibitors on the production of haptoglobin, either stimulated by IL-6 or basal, was negligible over a similar time period, indicating continued cell viability. The data from this model suggest that cathepsin inhibitors would not interfere with lysosomal processing to an extent which would prohibit the development of selective and potent cathepsin inhibitors for the treatment of diseases in which individual cysteine cathepsins play clearly pathophysiological roles.     

Fragment-based discovery of selective inhibitors of the Mycobacterium tuberculosis protein tyrosine phosphatase PtpA

The development of low μM inhibitors of the Mycobacterium tuberculosis phosphatase PtpA is reported. The most potent of these inhibitors (K_i = 1.4 ± 0.3 μM) was found to be selective when tested against a panel of human tyrosine and dual-specificity phosphatases (11-fold vs the highly homologous HCPtpA, and >70-fold vs all others tested). Modeling the inhibitor-PtpA complexes explained the structure–activity relationships observed in vitro and revealed further possibilities for compound development.     

Divergent synthesis of kinase inhibitor derivatives,leading to discovery of selective Gck inhibitors

We accomplished divergent synthesis of potent kinase inhibitor BAY 61-3606 (1) and 27 derivatives via conjugation of imidazo[1,2-c]pyrimidine and indole ring compounds with aromatic (including pyridine) derivatives by means of palladium-catalyzed cross-coupling reaction. Spleen tyrosine kinase (Syk) and germinal center kinase (Gck, MAP4K2) inhibition assays showed that some of the synthesized compounds were selective Gck inhibitors.     

Resilience of an Australian savanna grassland to selective and non-selective perturbations

Abstract Using a randomized experimental design, plots of a savanna grassland were subjected to two levels of grass tuft removal (50% and 90%) in two ways; non-selective (all species removed in proportion to abundance) and selective (tufts of the most palatable species removed first, then the next most palatable, etc.). The plots were maintained in their cleared states for three years, then monitored for the next five. In general, the sward was resilient to the disturbance except for the 90% selectively cleared treatment, in which a dominant, palatable species (Themeda triandra) failed to recover (though die most palatable species, Sorghum plumosum, did recover). The recovery patterns were dependent on post-disturbance conditions, and markedly influenced by a particular rainy season and a fire during one of the dry seasons. In addition to species effects, the treatments induced changes in spatial patterning and associated micro-scale hydrology. These effects persisted in the 90% removal treatment. In this regard the results are scale-dependent, and the same percentage removals at different scales (e. g. 5 × 5 m patches rather than tuft × tuft scale) would lead to differences in ability to recover. In terms of value to livestock the selective 90% removal treatment was in a poor state at the end of die experiment. In all treatments die trajectory of species changes was back towards the controls, but the selective 90% plots were fully re-vegetated before this could be achieved. In these plots, the final steps to complete recovery will occur only after death of established new tufts.     

The discovery of potent and selective pyridopyrimidin-7-one based inhibitors of B-RafV600E kinase

Herein we describe the discovery of a novel series of ATP competitive B-Raf inhibitors via structure based drug design (SBDD). These pyridopyrimidin-7-one based inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. Optimization led to the identification of compound 17, a potent, selective and orally available agent with excellent pharmacokinetic properties and robust tumor growth inhibition in xenograft studies.     

Protease inhibitors and proteolytic signalling cascades in insects

Proteolytic signalling cascades control a wide range of physiological responses. In order to respond rapidly, protease activity must be maintained at a basal level: the component zymogens must be sequentially activated and actively degraded. At the same time, signalling cascades must respond precisely: high target specificity is required. The insects have a wide range of trapping- and tight-binding protease inhibitors, which can regulate the activity of individual proteases. In addition, the interactions between component proteases of a signalling cascade can be modified by serine protease homologues. The suicide-inhibition mechanism of serpin family inhibitors gives rapid turnover of both protease and inhibitor, but target specificity is inherently broad. Similarly, the TEP/macroglobulins have extremely broad target specificity, which suits them for roles as hormone transport proteins and sensors of pathogenic virulence factors. The tight-binding inhibitors, on the other hand, have a lock-and-key mechanism capable of high target specificity. In addition, proteins containing multiple tight-binding inhibitory domains may act as scaffolds for the assembly of signalling complexes. Proteolytic cascades regulated by combinations of different types of inhibitor could combine the rapidity of suicide-inhibitors with the specificity lock-and-key inhibitors. This would allow precise control of physiological responses and may turn out to be a general rule.     

Evaluation of selective and non-selective enrichment PCR procedures for Salmonella detection

AIMS: To compare PCR combined with enrichment media with the standard microbiological techniques (SMT) and to determine the most sensitive method for the detection of Salmonella and the identification of Salm. typhimurium (ST), Salm. enteritidis (SE), Salm. gallinarum (SG) and Salm. pullorum (SP). METHODS AND RESULTS: We analysed 87 samples from poultry using PCR and SMT, PCR being performed from non-selective (NS) and Rappaport-Vassiliadis (RV) media. PCR-NS was less sensitive than PCR-RV and SMT for the detection and identification of Salmonella. PCR-RV detected more positive samples of Salmonella sp. than SMT but both these methods showed similar sensitivity regarding the identification of Salmonella serovars. CONCLUSIONS: PCR-RV was more sensitive and decreased the time necessary to detect and identify Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-RV is a powerful tool for the rapid and accurate detection and identification of Salmonella and can be implemented in diagnostic and food analysis laboratories.     

Hormonal control of renal functions in insects

Insect Malpighian tubules secrete an isosmotic, KCl-rich primary urine containing low concentrations of most other blood solutes. Neuropeptide diuretic hormones (DH), possibly related to vasopressin, stimulate tubular fluid secretion by 2- to 200-fold in response to water loading, e.g., feeding. DH acts on tubules through cyclic AMP (cAMP) to stimulate salt transport without measurable change in osmotic permeability. Changes in composition of tubular secretion after stimulation and the possible control of DH release are discussed. Most of the water, ions, and metabolites in tubular secretion are normally reabsorbed by active mechanisms in the rectum, where the urine may finally become either hyposmotic or strongly hyperosmotic to the blood. A newly discovered neuropeptide, chloride transport-stimulating hormone, controls (via cAMP) reabsorption of the principal salt by stimulating K-dependent, electrogenic transport of Cl- across the apical cell border. Passive net absorption of K+ is thereby enhanced. Diuretic and antidiuretic factors may control osmotic permeability of the rectal wall and thereby influence the osmotic concentrations of the rectal absorbate and final urine. The increased recycling of a KCl-rich fluid through the Malpighian tubule-rectal system after feeding probably serves to clear the body of unwanted substances ingested with, and produced by, metabolism of the meal.     

Effects of selective and non-selective COX inhibitors on antigen-induced release of prostanoid mediators and bronchoconstriction in the isolated perfused and ventilated guinea pig lung

The contribution of cycloxygenase (COX)-1 and COX-2 in antigen-induced release of mediators and ensuing bronchoconstriction was investigated in the isolated perfused guinea pig lung (IPL). Antigen challenge with ovalbumin (OVA) of lungs from actively sensitised animals induced release of thromboxane (TX)A(2), prostaglandin (PG)D(2), PGF(2)(alpha), PGI(2) and PGE(2), measured in the lung effluent as immunoreactive TXB(2), PGD(2)-MOX, PGF(2)(alpha), 6-keto PGF(1)(alpha) and PGE(2), respectively. This release was abolished by the non-selective COX inhibitor flurbiprofen (10 microM). In contrast, neither the selective COX-1 inhibitor FR122047 nor the selective COX-2 inhibitor celecoxib (10 microM each) significantly inhibited the OVA-induced bronchoconstriction or release of COX products, except for PGD(2). Another non-selective COX inhibitor, diclofenac (10 microM) also significantly inhibited antigen-induced bronchoconstriction. The data suggest that both COX isoenzymes, COX-1 and COX-2 contribute to the immediate antigen-induced generation of prostanoids in IPL and that the COX-1 and COX-2 activities are not associated with different profiles of prostanoid end products.     

Serendipity in discovery of proteasome inhibitors

Among its various catalytic activities, the 'chymotrypsin-like' activity of the proteasome, a large multicatalytic proteinase complex has emerged as the focus of drug discovery efforts in cancer therapy. Herein, a series of first generation (2S, 3R)-2-amino-3-hydroxybutyric acid derived proteasome inhibitors that were discovered serendipitously en route to original goal of generating a series of sterically constrained oxazoline derivatives has been reported.