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Supplementary tables 1-6 summarise the kinase panel screen data (1) mouse PK parameters (2) and further details of the anti-tumour activity (5) conferred by ASTX029, plus details of cell lines used in this study (3 and 4) and further details of the effects of ASTX029 and other MAPK inhibitors in models of MAPK resistance (6).

Supplementary figures 1-7 show the effects of AST029 on ERK signalling in RAS-mutant cell lines (1) and tumour xenograft tissue (3), ASTX029 plasma PK linearity (2), ASTX029 activity in a cell line panel showing MAPK mutation status (4) the effects of ASTX029 on mouse body weight (5), PKPD effects of ASTX029 following b.i.d dosing to Colo205 tumour-bearing mice (6) and PD effects of ASTX029 following qd dosing to A375R tumour-bearing mice (7).

The NRF2-mediated cytoprotective response is central to cellular homoeostasis, and there is increasing interest in developing small-molecule activators of this pathway as therapeutics for diseases involving chronic oxidative stress. The protein KEAP1, which regulates NRF2, is a key point for pharmacological intervention, and we recently described the use of fragment-based drug discovery to develop a tool compound that directly disrupts the protein-protein interaction between NRF2 and KEAP1. We now present the identification of a second, chemically distinct series of KEAP1 inhibitors, which provided an alternative chemotype for lead optimization. Pharmacophoric information from our original fragment screen was used to identify new hit matter through database searching and to evolve this into a new lead with high target affinity and cell-based activity. We highlight how knowledge obtained from fragment-based approaches can be used to focus additional screening campaigns in order to de-risk projects through the rapid identification of novel chemical series.

The MAPK signaling pathway is commonly upregulated in human cancers. As the primary downstream effector of the MAPK pathway, ERK is an attractive therapeutic target for the treatment of MAPK-activated cancers and for overcoming resistance to upstream inhibition. ASTX029 is a highly potent and selective dual-mechanism ERK inhibitor, discovered using fragment-based drug design. Because of its distinctive ERK-binding mode, ASTX029 inhibits both ERK catalytic activity and the phosphorylation of ERK itself by MEK, despite not directly inhibiting MEK activity. This dual mechanism was demonstrated in cell-free systems, as well as cell lines and xenograft tumor tissue, where the phosphorylation of both ERK and its substrate, ribosomal S6 kinase (RSK), were modulated on treatment with ASTX029. Markers of sensitivity were highlighted in a large cell panel, where ASTX029 preferentially inhibited the proliferation of MAPK-activated cell lines, including those with BRAF or RAS mutations. In vivo, significant antitumor activity was observed in MAPK-activated tumor xenograft models following oral treatment. ASTX029 also demonstrated activity in both in vitro and in vivo models of acquired resistance to MAPK pathway inhibitors. Overall, these findings highlight the therapeutic potential of a dual-mechanism ERK inhibitor such as ASTX029 for the treatment of MAPK-activated cancers, including those which have acquired resistance to inhibitors of upstream components of the MAPK pathway. ASTX029 is currently being evaluated in a first in human phase I–II clinical trial in patients with advanced solid tumors (NCT03520075).

In China, the correlation between environmental space and drug use by gay male sex workers remains an under-researched public health concern. Drugs are commonly used to enhance sexual performance a...

Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.

Whole genome analysis of a novel species of enterococci, Enterococcus lacertideformus, causing multi-systemic and invariably fatal disease in critically endangered Christmas Island reptiles was undertaken to determine the genetic elements and potential mechanisms conferring its pathogenic nature, biofilm-forming capabilities, immune recognition avoidance, and inability to grow in vitro. Comparative genomic analyses with related and clinically significant enterococci were further undertaken to infer the evolutionary history of the bacterium and identify genes both novel and absent. The genome had a G + C content of 35.1%, consisted of a circular chromosome, no plasmids, and was 2,419,934 bp in length (2,321 genes, 47 tRNAs, and 13 rRNAs). Multi-locus sequence typing (MLST), and single nucleotide polymorphism (SNP) analysis of multiple E. lacertideformus samples revealed they were effectively indistinguishable from one another and highly clonal. E. lacertideformus was found to be located within the Enterococcus faecium species clade and was closely related to Enterococcus villorum F1129D based on 16S rDNA and MLST house-keeping gene analysis. Antimicrobial resistance (DfreE, EfrB, tetM, bcrRABD, and sat4) and virulence genes (Fss3 and ClpP), and genes conferring tolerance to metals and biocides (n = 9) were identified. The detection of relatively few genes encoding antimicrobial resistance and virulence indicates that this bacterium may have had no exposure to recently developed and clinically significant antibiotics. Genes potentially imparting beneficial functional properties were identified, including prophages, insertion elements, integrative conjugative elements, and genomic islands. Functional CRISPR-Cas arrays, and a defective prophage region were identified in the genome. The study also revealed many genomic loci unique to E. lacertideformus which contained genes enriched in cell wall/membrane/envelop biogenesis, and carbohydrate metabolism and transport functionality. This finding and the detection of putative enterococcal biofilm determinants (EfaAfs, srtC, and scm) may underpin the novel biofilm phenotype observed for this bacterium. Comparative analysis of E. lacertideformus with phylogenetically related and clinically significant enterococci (E. villorum F1129D, Enterococcus hirae R17, E. faecium AUS0085, and Enterococcus faecalis OG1RF) revealed an absence of genes (n = 54) in E. lacertideformus, that encode metabolic functionality, which potentially hinders nutrient acquisition and/or utilization by the bacterium and precludes growth in vitro. These data provide genetic insights into the previously determined phenotype and pathogenic nature of the bacterium.

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

Background In patients with right iliac fossa pain, the need for surgery is largely determined by the likelihood of appendicitis. Patients often undergo ultrasound scanning despite a low diagnostic accuracy for appendicitis. This study aimed to determine the feasibility of a larger trial of computed tomography in the evaluation of patients with atypical right iliac fossa pain. Materials and methods A single-centre, unblinded, parallel randomised controlled trial of computed tomography in the assessment of patients with atypical right iliac fossa pain. After a retrospective evaluation, standard care was defined as serial examination with or without ultrasound. Atypical right iliac fossa pain was defined as no firm diagnosis after initial senior review. Simple descriptions of the risks and benefits of computed tomography were devised for patients to consider. Primary objectives were to assess feasibility and acceptability of the study procedures. Results A total of 71 patients were invited to participate and 68 were randomised. Final analysis included 31 participants in the standard care arm and 33 in the computed tomography arm, with comparable demographics. Computed tomography was associated with superior diagnostic accuracy, with 100% positive and negative predictive value. The proportion of scans that positively influenced management was 73% for computed tomography and 0% for ultrasound. In the computed tomography arm, there was a trend towards a shorter length of stay (2.3 vs 3.1 days) and a lower negative laparoscopy rate (2 of 11 vs 4 of 9). Conclusion A large randomised trial to evaluate the use of unenhanced computed tomography in atypical right iliac fossa pain appears feasible and justified.

Introduction: Group therapeutic singing (GTS) may be an effective treatment for voice and respiratory impairments in persons with Parkinson's disease (PD). However, it remains unknown if GTS can be effectively delivered through telemedicine. Methods: Participants with PD from rural areas were recruited to complete a prerecorded GTS program, once a week for 8 weeks. Voice and respiratory outcome measures were collected 1 week before and 1 week after intervention. Results: Ten participants were enrolled in the study. One participant dropped at week 3. Five participants attended all eight sessions, two participants completed seven sessions, and one participant completed six sessions for 93.75% compliance. Statistical analysis for the data collected from the eight participants enrolled in the study revealed that voice outcome measures improved but did not reach significance. However, respiratory outcome measures significantly improved. Discussion: Results suggest that prerecorded GTS is feasible. Moreover, results are in keeping with results from a previous study using the exact same intervention with an in person therapist. Thus, this pilot work suggests that the use of prerecorded GTS may be a viable treatment option for those with limited access to care.

This study describes general synthesis aspects of fragments for FBDD, as illustrated by the dihydroisoquinolones 1-3. Previous Rh(III) methodology is extended to incorporate amines, heteroatoms (N and S), and substituents (halogen, ester) as potential binding groups and/or synthetic growth points for fragment-to-lead elaboration.

The efficacy of the recently approved drug fingolimod (FTY720) in multiple sclerosis patients results from the action of its phosphate metabolite on sphingosine-1-phosphate S1P1 receptors, while a variety of side effects have been ascribed to its S1P3 receptor activity. Although S1P and phospho-fingolimod share the same structural elements of a zwitterionic headgroup and lipophilic tail, a variety of chemotypes have been found to show S1P1 receptor agonism. Here we describe a study of the tolerance of the S1P1 and S1P3 receptors toward bicyclic heterocycles of systematically varied shape and connectivity incorporating acidic, basic, or zwitterionic headgroups. We compare their physicochemical properties, their performance in in vitro and in vivo pharmacokinetic models, and their efficacy in peripheral lymphocyte lowering. The campaign resulted in the identification of several potent S1P1 receptor agonists with good selectivity vs S1P3 receptors, efficacy at

In the field of visual art, metaphor is a way to communicate meaning to the viewer. We present a computational system for communicating visual metaphor that can identify adjectives for describing an image based on a low-level visual feature representation of the image. We show that the system can use this visual-linguistic association to render source images that convey the meaning of adjectives in a way consistent with human understanding. Our conclusions are based on a detailed analysis of how the system's artifacts cluster, how these clusters correspond to the semantic relationships of adjectives as documented in WordNet, and how these clusters correspond to human opinion.

Using X-ray crystallographic screening, fragments 4 and 6 were identified as inhibitors of hematopoietic prostaglandin D2 synthase (H-PGDS). Both fragments induced a small protein movement in the X-ray crystal structure relative to the apo structure, where the highly polar nature of the ligand complemented the induced protein conformation. The manuscript describes the fragment optimisation of 4 and 6 followed by fragment growth to lead molecule 10. This showed favourable physicochemical properties and evidence of oral activity in blocking PGD2 generation in vivo.

Creativity is an important component of human intelligence, and imbuing artificially intelligent systems with creativity is an interesting challenge. In particular, it is difficult to quantify (or even qualify) creativity. Recently, it has been suggested that conditions for attributing creativity to a system include: appreciation, imagination, and skill. We demonstrate and describe an original computer system (called DARCI) that is designed to produce images through creative means. We present methods for evaluating DARCI and other artificially creative systems with respect to appreciation, imagination, and skill, and use these methods to show that DARCI is arguably a creative system.

A series of α7 nicotinic acetylcholine receptor full-agonists with a 1,3,4-oxadiazol-2-amine core has been discovered. Systematic exploration of the structure-activity relationships for both α7 potency and selectivity with respect to interaction with the hERG channel are described. Further profiling led to the identification of compound 22, a potent full agonist showing efficacy in the novel object recognition model of cognition enhancement.

The RAS-RAF-MEK-ERK signalling cascade is activated through mutations in RAS or RAF in over 30% of cancers. The successful development of inhibitors of BRAF and MEK kinases has led to effective treatment particularly of melanomas whose tumor growth is driven by activating mutations in BRAF such as V600E. Despite these successes, resistance emerges after several months, leading to increased signaling through ERK1/2. This has prompted the development of direct inhibitors of ERK1/2, several of which are in early clinical trials. The majority of clinical ERK1/2 inhibitors are ATP competitive, blocking ERK1/2 catalytic phosphorylation of downstream substrates such as RSK, but do not modulate phosphorylation of ERK1/2 by MEK. Crystal structural studies performed by us and others on the pERK1/2 modulating inhibitor SCH772984 suggested that it induces a conformational change in the glycine-rich loop of ERK2, which leads to Tyr36 becoming tucked under the loop and creating a new binding pocket. We hypothesized that this binding mode might underlie the ability of SCH772984 to block the phosphorylation of ERK1/2, and initiated a fragment-based approach to develop novel, orally bioavailable inhibitors that elicit a similar conformational change and also modulate the phosphorylation of ERK1/2. Using screening methods including high-throughput X-ray crystallography and biophysical assays, we identified fragments binding to both the hinge and the inducible pocket of ERK2. Progressive rounds of structure-guided fragment optimization and growing led to an understanding of inhibitor structure determinants required to induce the conformational change in ERK2. These efforts, together with iterative optimization in a screening cascade including measurement of pRSK and pERK levels and antiproliferative activity in RAS and BRAF mutant cells, led to the discovery of a novel series of pERK modulating ERK1/2 inhibitors. The lead compound shows low nanomolar potency in biochemical ERK1/2 assays and an excellent kinome selectivity profile. In BRAF and RAS mutant cell lines, the lead shows low nanomolar cell proliferation IC50 values, while sparing cell lines not driven by the MAPK pathway. The lead exhibits robust antitumor activity upon oral dosing in a range of subcutaneous xenograft models including the mutant BRAF colorectal line Colo205, providing a promising basis for further optimization towards clinical pERK1/2 modulating ERK1/2 inhibitors. Citation Format: Tom D. Heightman, Valerio Berdini, Hannah Braithwaite, Ildiko Buck, Megan Cassidy, Juan Castro, Aurélie Courtin, James Day, Charlotte East, Lynsey Fazal, Brent Graham, Charlotte Griffiths-Jones, John Lyons, Vanessa Martins, Sandra Muench, Joanne Munck, David Norton, Marc O'Reilly, Nick Palmer, Puja Pathuri, Mike Reader, David Rees, Sharna Rich, Caroline Richardson, Harpreet Saini, Neil Thompson, Nicola Wallis, Hugh Walton, Nicola Wilsher, Alison Woolford, Chris Murray. Fragment-based discovery of a highly potent, orally bioavailable ERK1/2 inhibitor that modulates the phosphorylation and catalytic activity of ERK1/2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B161.

The MAPK pathway is commonly hyper-activated in human cancers due to the occurrence of oncogenic mutations in RAF, RAS and the upregulation of RTKs. The therapeutic potential of MAPK pathway inhibition has been demonstrated by the clinical efficacy of RAF and MEK1/2 (MEK) inhibitors in the treatment of BRAF-mutant melanoma. However, response to such agents is short-lived due to the onset of resistance mechanisms, which in the majority of cases result in the reactivation of ERK1/2 (ERK) signalling. Therefore, the direct targeting of ERK is an attractive therapeutic approach to overcoming the limitations of RAF or MEK inhibitors. Here, we describe a novel, potent, and selective ERK inhibitor, which inhibits both ERK catalytic activity and also the phosphorylation of ERK by MEK. Using fragment-based drug discovery we have developed a selective ERK inhibitor, which inhibits in vitro ERK catalytic activity with a low nM IC50 value. This lead compound has strong antiproliferative effects in a wide range of MAPK-activated cell lines, including the BRAF-mutant cell lines A375 (melanoma) and Colo205 (colorectal), the KRAS-mutant cell lines HCT116 (colorectal), Calu6 (lung) and Panc05.04 (pancreatic), and the NRAS-mutant cell line Ma-mel-27 (melanoma). The lead compound potently inhibits ERK cell signalling. The potent (nM) inhibition of RSK phosphorylation (a direct ERK substrate) was confirmed in A375 (BRAF-mutant melanoma) cells, using MSD analysis. In addition to inhibiting downstream ERK signalling, we demonstrated by ELISA and Western blotting that the lead compound confers a decrease in phospho-ERK levels in both BRAF-mutant and KRAS-mutant cell lines. We investigated the biochemical mechanism of the modulation of ERK phosphorylation in vitro and demonstrated that the compound prevents the phosphorylation of ERK by MEK (at key ERK activation loop residues, T202/Y204), without directly inhibiting MEK activity. The compound was profiled in a range of subcutaneous xenograft models including A375 (BRAF-mutant melanoma) and Calu-6 (KRAS-mutant lung). Once-daily oral dosing of the lead compound conferred significant antitumor activity in a range of in vivo efficacy studies. The compound potently inhibited the phosphorylation of downstream ERK substrates (including RSK) in tumor xenograft tissue. There was a clear relationship between in vivo compound concentrations and the modulation of ERK substrate phosphorylation. Furthermore, as was demonstrated in vitro, we confirmed that in addition to inhibiting ERK catalytic activity the compound potently inhibited the phosphorylation of ERK itself, in both KRAS and BRAF-mutant tumor xenografts. Here, we characterize a novel, highly potent, selective ERK inhibitor, which inhibits both ERK catalytic activity and also the upstream phosphorylation of ERK by MEK. These data support the further optimization of this series of compounds for clinical development. Citation Format: Joanne M. Munck, Valerio Berdini, Luke D. Bevan, Hannah Braithwaite, Ildiko M. Buck, Megan Cassidy, Juan Castro, Aurelie Courtin, James E. Day, Charlotte East, Lynsey Fazal, Brent Graham, Charlotte M. Griffiths-Jones, Tom D. Heightman, Chris J. Hindley, Birikiti Kidane, Justyna Kucia-Tran, John F. Lyons, Vanessa Martins, Sandra Muench, Chris W. Murray, David Norton, Marc O'Reilly, Nick Palmer, Puja Pathuri, Mike Reader, David C. Rees, Sharna J. Rich, Caroline J. Richardson, Harpreet K. Saini, Alpesh Shah, Lukas Stanczuk, Neil T. Thompson, Hugh Walton, Nicola E. Wilsher, Alison J. Woolford, Nicola G. Wallis. Characterization of a novel ERK1/2 inhibitor, which modulates the phosphorylation and catalytic activity of ERK1/2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B154.

Diblock and triblock copolymers were prepared from polyethylene glycol (PEG, M n = 2 000), L -lactide and D,L-lactide. A novel method was developed to prepare high-molecular-weight multiblock copolymers by transesterification of a PEG/succinic acid polyester with poly(L-lactide). Molecular weights were monitored by GPC and overall composition was checked by 1 H NMR. Crystallinity, bulk microstructure, and the glass transition were investigated by DSC and simultaneous SAXS/WAXS/DSC, and surface properties by measurement of advancing contact angle. It is found that the crystallization of the poly(ethylene oxide) (PEO) block is very much affected by the position of the PEO block in the copolymer and by the length and the state of the polylactide block. Both DSC and WAXS showed that the crystallization of PEG2000 block was very much suppressed when covalently bonding to polylactide (PLA) blocks. These methods also confirmed good miscibility between amorphous PLA and PEG blocks. The preparation of fiber by electrospinning demonstrates the target application of the multiblock copolymers in tissue engineering.

We describe an experimental closed bioreactor device for studying novel tissue engineered peripheral nerve conduits in vitro. The system integrates a closed loop system consisting of one, two, or three experimental nerve conduits connected in series or parallel, with the ability to study novel scaffolds within guidance conduits. The system was established using aligned synthetic microfiber scaffolds of viscose rayon and electrospun polystyrene. Schwann cells were seeded directly into conduits varying from 10 to 80 mm in length and allowed to adhere under 0 flow for 1 h, before being cultured for 4 days under static or continuous flow conditions. In situ viability measurements showed the distribution of live Schwann cells within each conduit and enabled quantification thereafter. Under static culture viable cells only existed in short conduit scaffolds (10 mm) or at the ends of longer conduits (20-80 mm) with a variation in viable cell distribution. Surface modification of scaffold fibers with type-1 collagen or acrylic acid increased cell number by 17% and 30%, respectively. However, a continuous medium flow of 0.8 mL/h was found to increase total cell number by 2.5-fold verses static culture. Importantly, under these conditions parallel viability measurements revealed a ninefold increase compared to static culture. Fluorescence microscopy of scaffolds showed cellular adhesion and alignment on the longitudinal axis. We suggest that such a system will enable a rigorous and controlled approach for evaluating novel conduits for peripheral nerve repair, in particular using hydrolysable materials for the parallel organization of nerve support cells, prior to in vivo study.

The dry etching characteristics of bulk single-crystal zinc- oxide (ZnO) and RF-sputtered indium-zinc-oxide (IZO) films have been investigated using an inductively coupled high- density plasma in Ar/IBr and Ar/BI3. In both plasma chemistries, the etch rate of ZnO is very similar to that of IZO, which indicates that zinc and indium atoms are driven by a similar plasma etching dynamics. IBr and BI3-based plasmas show no enhancement of the etch rate over pure physical sputtering under the same experimental conditions. Also, CH4/H2 -based discharges are attractive for dry etching of single crystal ZnO because of their non-corrosive nature. The C2H6/H2/Ar mixture provides a strong enhancement over pure Ar sputtering, in sharp contrast to the case of CH4/H2/Ar. The etched surface morphologies were smooth, independent of the chemistry and the Zn/O ratio in the near-surface region was unchanged within experimental error after etching with all chemistries.