Discover the most talked about and latest scientific content & concepts.

Concept: Inhibitor


Kinase inhibitors are effective cancer therapies, but tumors frequently develop resistance. Current strategies to circumvent resistance target the same or parallel pathways. We report here that targeting a completely different process, autophagy, can overcome multiple BRAF inhibitor resistance mechanisms in brain tumors. BRAF(V600E)mutations occur in many pediatric brain tumors. We previously reported that these tumors are autophagy-dependent and a patient was successfully treated with the autophagy inhibitor chloroquine after failure of the BRAF(V600E) inhibitor vemurafenib, suggesting autophagy inhibition overcame the kinase inhibitor resistance. We tested this hypothesis in vemurafenib-resistant brain tumors. Genetic and pharmacological autophagy inhibition overcame molecularly distinct resistance mechanisms, inhibited tumor cell growth, and increased cell death. Patients with resistance had favorable clinical responses when chloroquine was added to vemurafenib. This provides a fundamentally different strategy to circumvent multiple mechanisms of kinase inhibitor resistance that could be rapidly tested in clinical trials in patients with BRAF(V600E) brain tumors.

Concepts: Medicine, Cancer, Oncology, Brain tumor, Tumor, Neoplasm, Enzyme inhibitor, Inhibitor


The primary defence against mosquitoes and other disease vectors is often the application of a repellent. Despite their common use, the mechanism(s) underlying the activity of repellents is not fully understood, with even the mode of action of DEET having been reported to be via different mechanisms; e.g. interference with olfactory receptor neurones or actively detected by olfactory receptor neurones on the antennae or maxillary palps. In this study, we discuss a novel mechanism for repellence, one of P450 inhibition. Thirteen essential oil extracts from Colombian plants were assayed for potency as P450 inhibitors, using a kinetic fluorometric assay, and for repellency using a modified World Health Organisation Pesticide Evaluations Scheme (WHOPES) arm-in cage assay with Stegomyia (Aedes) aegypti mosquitoes. Bootstrap analysis on the inhibition analysis revealed a significant correlation between P450-inhibition and repellent activity of the oils.

Concepts: Mosquito, Enzyme inhibitor, Inhibitor, Aedes aegypti, Aedes, Xanthine oxidase inhibitor, Insect repellent, Oil


Activated RAS promotes dimerization of members of the RAF kinase family. ATP-competitive RAF inhibitors activate ERK signalling by transactivating RAF dimers. In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumour-specific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E). However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.

Concepts: Gene expression, Transcription factor, Enzyme inhibitor, Dimer, Monomer, Inhibitor, Xanthine oxidase inhibitor, Alternative splicing


A library of 367 protein kinase inhibitors, the GSK Published Kinase Inhibitor Set (PKIS), which has been annotated for protein kinase family activity and is available for public screening efforts, was assayed against the commonly used luciferase reporter enzymes from the firefly, Photinus pyralis (FLuc) and marine sea pansy, Renilla reniformis (RLuc). A total of 22 compounds (∼6% of the library) were found to inhibit FLuc with 10 compounds showing potencies ≤1 µM. Only two compounds were found to inhibit RLuc, and these showed relatively weak potency values (∼10 µM). An inhibitor series of the VEGFR2/TIE2 protein kinase family containing either an aryl oxazole or benzimidazole-urea core illustrate the different structure activity relationship profiles FLuc inhibitors can display for kinase inhibitor chemotypes. Several FLuc inhibitors were broadly active toward the tyrosine kinase and CDK families. These data should aid in interpreting the results derived from screens employing the GSK PKIS in cell-based assays using the FLuc reporter. The study also underscores the general need for strategies such as the use of orthogonal reporters to identify kinase or non-kinase mediated cellular responses.

Concepts: Signal transduction, Enzyme, Protein kinase, Enzyme inhibitor, Inhibitor, Protein kinase inhibitor, Luciferase, Sea pansy


Fatty acid binding proteins (FABPs), in particular FABP5 and FABP7, have recently been identified by us as intracellular transporters for the endocannabinoid anandamide (AEA). Furthermore, animal studies by others have shown that elevated levels of endocannabinoids resulted in beneficial pharmacological effects on stress, pain and inflammation and also ameliorate the effects of drug withdrawal. Based on these observations, we hypothesized that FABP5 and FABP7 would provide excellent pharmacological targets. Thus, we performed a virtual screening of over one million compounds using DOCK and employed a novel footprint similarity scoring function to identify lead compounds with binding profiles similar to oleic acid, a natural FABP substrate. Forty-eight compounds were purchased based on their footprint similarity scores (FPS) and assayed for biological activity against purified human FABP5 employing a fluorescent displacement-binding assay. Four compounds were found to exhibit approximately 50% inhibition or greater at 10 µM, as good as or better inhibitors of FABP5 than BMS309403, a commercially available inhibitor. The most potent inhibitor, γ-truxillic acid 1-naphthyl ester (ChemDiv 8009-2334), was determined to have K(i) value of 1.19±0.01 µM. Accordingly a novel α-truxillic acid 1-naphthyl mono-ester (SB-FI-26) was synthesized and assayed for its inhibitory activity against FABP5, wherein SB-FI-26 exhibited strong binding (K(i) 0.93±0.08 µM). Additionally, we found SB-FI-26 to act as a potent anti-nociceptive agent with mild anti-inflammatory activity in mice, which strongly supports our hypothesis that the inhibition of FABPs and subsequent elevation of anandamide is a promising new approach to drug discovery. Truxillic acids and their derivatives were also shown by others to have anti-inflammatory and anti-nociceptive effects in mice and to be the active component of Chinese a herbal medicine (Incarvillea sinensis) used to treat rheumatism and pain in humans. Our results provide a likely mechanism by which these compounds exert their effects.

Concepts: Pharmacology, Fatty acid, Enzyme inhibitor, Drug discovery, Inhibitor, Oleic acid, Linoleic acid, Fatty acid-binding protein


Neuron-microglia co-cultures treated with pro-inflammatory agents are a useful tool to study neuroinflammation in vitro, where to test the potential neuroprotective effect of anti-inflammatory compounds. However, a great diversity of experimental conditions can be found in the literature, making difficult to select the working conditions when considering this approach for the first time. We compared the use of neuron-primary microglia and neuron-BV2 cells (a microglial cell line) co-cultures, using different neuron:microglia ratios, treatments and time post-treatment to induce glial activation and derived neurotoxicity. We show that each model requires different experimental conditions, but that both neuron-BV2 and neuron-primary microglia LPS/IFN-γ-treated co-cultures are good to study the potential neuroprotective effect of anti-inflammatory agents. The contribution of different pro-inflammatory parameters in the neurotoxicity induced by reactive microglial cells was determined. IL-10 pre-treatment completely inhibited LPS/IFN-γ-induced TNF-α and IL-6 release, and COX-2 expression both in BV2 and primary microglial cultures, but not NO production and iNOS expression. However, LPS/IFN-γ induced neurotoxicity was not inhibited in IL-10 pre-treated co-cultures. The inhibition of NO production using the specific iNOS inhibitor 1400 W totally abolished the neurotoxic effect of LPS/IFN-γ, suggesting a major role for NO in the neurotoxic effect of activated microglia. Consequently, among the anti-inflammatory agents, special attention should be paid to compounds that inhibit NO production.

Concepts: Enzyme inhibitor, Inhibitor, Glial cell, Glial cells, Microglia


The anti-alcoholism medication, disulfiram (Antabuse), decreases cocaine use in humans regardless of concurrent alcohol consumption and facilitates cocaine sensitization in rats, but the functional targets are unknown. Disulfiram inhibits dopamine β-hydroxylase (DBH), the enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic neurons. The goal of this study was to test the effects of chronic genetic or pharmacological DBH inhibition on behavioral responses to cocaine using DBH knockout (Dbh -/-) mice, disulfiram, and the selective DBH inhibitor, nepicastat. Locomotor activity was measured in control (Dbh +/-) and Dbh -/- mice during a 5 day regimen of saline+saline, disulfiram+saline, nepicastat+saline, saline+cocaine, disulfiram+cocaine, or nepicastat+cocaine. After a 10 day withdrawal period, all groups were administered cocaine, and locomotor activity and stereotypy were measured. Drug-naïve Dbh -/- mice were hypersensitive to cocaine-induced locomotion and resembled cocaine-sensitized Dbh +/- mice. Chronic disulfiram administration facilitated cocaine-induced locomotion in some mice and induced stereotypy in others during the development of sensitization, while cocaine-induced stereotypy was evident in all nepicastat-treated mice. Cocaine-induced stereotypy was profoundly increased in the disulfiram+cocaine, nepicastat+cocaine, and nepicastat+saline groups upon cocaine challenge after withdrawal in Dbh +/- mice. Disulfiram or nepicastat treatment had no effect on behavioral responses to cocaine in Dbh -/- mice. These results demonstrate that chronic DBH inhibition facilitates behavioral responses to cocaine, although different methods of inhibition (genetic vs. non-selective inhibitor vs. selective inhibitor) enhance qualitatively different cocaine-induced behaviors.

Concepts: Alcoholism, Nicotine, Enzyme inhibitor, Neurotransmitter, Inhibitor, Dopamine, Norepinephrine, Cocaine


A novel class of small molecule inhibitors for plasminogen activator inhibitor type 1 (PAI-1)(2), represented by AZ3976, was identified in a high through-put screening campaign. AZ3976 displayed an IC(50) value of 26 µM in an enzymatic chromogenic assay. In a plasma clot lysis assay, the compound was active with an IC(50) of 16 µM. Surprisingly, AZ3976 did not bind to active PAI-1, but bound to latent PAI-1 with a K(D) of 0.29 µM at 35 °C and a binding stoichiometry of 0.94, as measured by isothermal calorimetry. Reversible binding was confirmed by surface plasmon resonance (SPR) direct binding experiments. The X-ray structure of AZ3976 in complex with latent PAI-1 was determined to 2.4 Å resolution. The inhibitor was bound in the flexible joint region with the entrance to the cavity located between α-helix D and β-strand 2A. A set of SPR experiments revealed that AZ3976 inhibited PAI-1 by enhancing the latency transition of active PAI-1. Since AZ3976 only had measurable affinity for latent PAI-1, we propose that its mechanism of inhibition is based on binding to a small fraction in equilibrium with active PAI-1, a latent-like pre-latent form, from which latent PAI-1 is then generated more rapidly. This mode of action, with induced accelerated latency transition of active PAI-1 may, together with supporting X-ray data, provide improved opportunities for small molecule drug design in the hunt for therapeutically useful PAI-1 inhibitors.

Concepts: Fibrinolysis, Enzyme inhibitor, Inhibitor, Surface plasmon resonance, Plasminogen activator inhibitor-1, Plasma oscillation


Aldosterone synthase (CYP11B2) is a promising therapeutic target for the treatment of cardiovascular diseases related to abnormally high aldosterone levels. On the basis of our previously identified lead compounds I-III, a series of 3-pyridinyl substituted aliphatic cycles were designed, synthesized and tested as CYP11B2 inhibitors. Aromaticity abolishment of the core was successfully applied to overcome the undesired CYP1A2 inhibition. This study resulted in a series of potent and selective CYP11B2 inhibitors, with compound 12 (IC(50) = 21 nM, SF = 50) as the most promising one, which shows no inhibition toward CYP1A2 at 2 µM. The design conception demonstrated in this study can be helpful in the optimization of CYP inhibitor drugs regarding CYP1A2 selectivity.

Concepts: Aldosterone, Cytochrome P450, Enzyme inhibitor, Inhibitor, Xanthine oxidase inhibitor, Aldosterone synthase, Corticosterone, Zona glomerulosa


Ser/thr phosphatases dephosphorylate their targets with high specificity, yet the structural and sequence determinants of phosphosite recognition are poorly understood. Calcineurin (CN) is a conserved Ca/calmodulin-dependent ser/thr phosphatase and the target of immunosuppressants, FK506 and cyclosporin A (CSA). To investigate CN substrate recognition we used X-ray crystallography, biochemistry, modeling, and in vivo experiments to study A238L, a viral protein inhibitor of CN. We show that A238L competitively inhibits CN by occupying a critical substrate recognition site, while leaving the catalytic center fully accessible. Critically, the 1.7 Å structure of the A238L-CN complex reveals how CN recognizes residues in A238L that are analogous to a substrate motif, “LxVP.” The structure enabled modeling of a peptide substrate bound to CN, which predicts substrate interactions beyond the catalytic center. Finally, this study establishes that “LxVP” sequences and immunosuppressants bind to the identical site on CN. Thus, FK506, CSA, and A238L all prevent “LxVP”-mediated substrate recognition by CN, highlighting the importance of this interaction for substrate dephosphorylation. Collectively, this work presents the first integrated structural model for substrate selection and dephosphorylation by CN and lays the groundwork for structure-based development of new CN inhibitors.

Concepts: Protein structure, Enzyme, Enzyme inhibitor, Inhibitor, Immunosuppressants, Ciclosporin, Calcineurin, Nonribosomal peptide