Discovery of 2-(3-Benzamidopropanamido)thiazole-5-carboxylate Inhibitors of the Kinesin HSET (KIFC1) and the Development of Cellular Target Engagement Probes.

The existence of multiple centrosomes in some cancer cells can lead to cell death through the formation of multipolar mitotic spindles and consequent aberrant cell division. Many cancer cells rely on HSET (KIFC1) to cluster the extra centrosomes into two groups to mimic the bipolar spindle formation of non-centrosome-amplified cells and ensure their survival. Here, we report the discovery of a novel 2-(3-benzamidopropanamido)thiazole-5-carboxylate with micromolar in vitro inhibition of HSET (KIFC1) through high-throughput screening and its progression to ATP-competitive compounds with nanomolar biochemical potency and high selectivity against the opposing mitotic kinesin Eg5. Induction of the multipolar phenotype was shown in centrosome-amplified human cancer cells treated with these inhibitors. In addition, a suitable linker position was identified to allow the synthesis of both fluorescent- and trans-cyclooctene (TCO)-tagged probes, which demonstrated direct compound binding to the HSET protein and confirmed target engagement in cells, through a click-chemistry approach.

Discovering cell-active BCL6 inhibitors: effectively combining biochemical HTS with multiple biophysical techniques, X-ray crystallography and cell-based assays.

By suppressing gene transcription through the recruitment of corepressor proteins, B-cell lymphoma 6 (BCL6) protein controls a transcriptional network required for the formation and maintenance of B-cell germinal centres. As BCL6 deregulation is implicated in the development of Diffuse Large B-Cell Lymphoma, we sought to discover novel small molecule inhibitors that disrupt the BCL6-corepressor protein-protein interaction (PPI). Here we report our hit finding and compound optimisation strategies, which provide insight into the multi-faceted orthogonal approaches that are needed to tackle this challenging PPI with small molecule inhibitors. Using a 1536-well plate fluorescence polarisation high throughput screen we identified multiple hit series, which were followed up by hit confirmation using a thermal shift assay, surface plasmon resonance and ligand-observed NMR. We determined X-ray structures of BCL6 bound to compounds from nine different series, enabling a structure-based drug design approach to improve their weak biochemical potency. We developed a time-resolved fluorescence energy transfer biochemical assay and a nano bioluminescence resonance energy transfer cellular assay to monitor cellular activity during compound optimisation. This workflow led to the discovery of novel inhibitors with respective biochemical and cellular potencies (IC50s) in the sub-micromolar and low micromolar range.

Femoral Nerve Block Intervention in Neck of Femur Fracture (FINOF): a randomised controlled trial.

OBJECTIVE: Fractured neck of femur is a severely painful condition with significant mortality and morbidity. We investigated whether early and continuous use of femoral nerve block can improve pain on movement and mobility after surgery in older participants with fragility neck of femur fracture. DESIGN: Prospective single-centre, randomised controlled pragmatic trial. SETTING: Secondary care, acute National Health Service Trust, UK. PARTICIPANTS: Participants admitted with a history and examination suggesting fractured neck of femur. INTERVENTION: Immediate continuous femoral nerve block via catheter or standard analgesia. OUTCOME MEASURES: Primary outcome measures were Cumulative Dynamic Pain score and Cumulated Ambulation Score from surgery until day 3 postoperatively. Secondary outcome measures included pain scores at rest, cumulative side effects (nausea and constipation), quality of life (measured by EuroQOL 5 D instrument (EQ-5D) score) at day 3 and day 30, and rehabilitation outcome (measured by mobility score). RESULTS: 141 participants were recruited, with 23 excluded. No significant difference was detected between Cumulative Dynamic Pain Score (standard care (n=56) vs intervention (n=55) 20 (IQR 15-24) vs 20 (15-23), p=0.51) or Cumulated Ambulation Score (standard care vs intervention 6 (5-9) vs 7 (5-10), p=0.76). There were no statistically different differences in secondary outcomes except cumulative pain at rest: 5 (0.5-6.5) in the standard care group and 2 (0-5) in the intervention group (p=0.043). CONCLUSIONS: Early application of continuous femoral nerve block compared with standard systemic analgesia did not result in improved dynamic pain score or superior postoperative ambulation. This technique may provide superior pain relief at rest. Continuous femoral nerve block did not delay initial control of pain or mobilisation after surgery. TRIAL REGISTRATION NUMBER: ISRCTN92946117; Pre-results.

Privileged Structures and Polypharmacology within and between Protein Families.

Polypharmacology is often a key contributor to the efficacy of a drug, but is also a potential risk. We investigated two hits discovered via a cell-based phenotypic screen, the CDK9 inhibitor CCT250006 (1) and the pirin ligand CCT245232 (2), to establish methodology to elucidate their secondary protein targets. Using computational pocket-based analysis, we discovered intrafamily polypharmacology for our kinase inhibitor, despite little overall sequence identity. The interfamily polypharmacology of 2 with B-Raf was used to discover a novel pirin ligand from a very small but privileged compound library despite no apparent ligand or binding site similarity. Our data demonstrates that in areas of drug discovery where intrafamily polypharmacology is often an issue, ligand dissimilarity cannot necessarily be used to assume different off-target profiles and that understanding interfamily polypharmacology will be important in the future to reduce the risk of idiopathic toxicity and in the design of screening libraries.

Demonstrating In-Cell Target Engagement Using a Pirin Protein Degradation Probe (CCT367766).

Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be particularly challenging for poorly studied and noncatalytic proteins, as robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical probe 1 (CCT251236) binds the putative transcription factor regulator pirin in living cells, we developed a heterobifunctional protein degradation probe. Focusing on linker design and physicochemical properties, we generated a highly active probe 16 (CCT367766) in only three iterations, validating our efficient strategy for degradation probe design against nonvalidated protein targets.

Discovery of a Chemical Probe Bisamide (CCT251236): An Orally Bioavailable Efficacious Pirin Ligand from a Heat Shock Transcription Factor 1 (HSF1) Phenotypic Screen.

Phenotypic screens, which focus on measuring and quantifying discrete cellular changes rather than affinity for individual recombinant proteins, have recently attracted renewed interest as an efficient strategy for drug discovery. In this article, we describe the discovery of a new chemical probe, bisamide (CCT251236), identified using an unbiased phenotypic screen to detect inhibitors of the HSF1 stress pathway. The chemical probe is orally bioavailable and displays efficacy in a human ovarian carcinoma xenograft model. By developing cell-based SAR and using chemical proteomics, we identified pirin as a high affinity molecular target, which was confirmed by SPR and crystallography.

A fragment-based approach applied to a highly flexible target: Insights and challenges towards the inhibition of HSP70 isoforms.

The heat shock protein 70s (HSP70s) are molecular chaperones implicated in many cancers and of significant interest as targets for novel cancer therapies. Several HSP70 inhibitors have been reported, but because the majority have poor physicochemical properties and for many the exact mode of action is poorly understood, more detailed mechanistic and structural insight into ligand-binding to HSP70s is urgently needed. Here we describe the first comprehensive fragment-based inhibitor exploration of an HSP70 enzyme, which yielded an amino-quinazoline fragment that was elaborated to a novel ATP binding site ligand with different physicochemical properties to known adenosine-based HSP70 inhibitors. Crystal structures of amino-quinazoline ligands bound to the different conformational states of the HSP70 nucleotide binding domain highlighted the challenges of a fragment-based approach when applied to this particular flexible enzyme class with an ATP-binding site that changes shape and size during its catalytic cycle. In these studies we showed that Ser275 is a key residue in the selective binding of ATP. Additionally, the structural data revealed a potential functional role for the ATP ribose moiety in priming the protein for the formation of the ATP-bound pre-hydrolysis complex by influencing the conformation of one of the phosphate binding loops.

Exploiting Protein Conformational Change to Optimize Adenosine-Derived Inhibitors of HSP70.

HSP70 is a molecular chaperone and a key component of the heat-shock response. Because of its proposed importance in oncology, this protein has become a popular target for drug discovery, efforts which have as yet brought little success. This study demonstrates that adenosine-derived HSP70 inhibitors potentially bind to the protein with a novel mechanism of action, the stabilization by desolvation of an intramolecular salt-bridge which induces a conformational change in the protein, leading to high affinity ligands. We also demonstrate that through the application of this mechanism, adenosine-derived HSP70 inhibitors can be optimized in a rational manner.

Pre-Operative nutrition In Neck of femur Trial (POINT)--carbohydrate loading in patients with fragility hip fracture: study protocol for a randomised controlled trial.

BACKGROUND: Trauma such as hip fracture initiates a neurohumoral stress response that changes the balance between anabolism and catabolism resulting in muscle breakdown and reduced mobilisation. Various studies have demonstrated a reduction in catabolism with pre-operative carbohydrate loading but only in an elective setting. METHODS/DESIGN: This is a two-centre, randomised double-blinded trial in the United Kingdom. Sample size will be 30 patients (approximately 15 from each centre). Randomisation will be web based using computer-generated concealed tables. Both participants and investigators will be blinded to group allocation. Participants will be >70 years of age, cognitively intact (Abbreviated Mental Score ≥ 7), able to give informed consent, and admitted directly through the emergency department with fractured neck of femur requiring hemiarthroplasty. Intervention will consist of two carbohydrate drinks (Nutricia pre-Op) given the night before, and the morning of the surgery. The control will receive two placebo drinks of equal volume. All participants will receive standard hospital care at the discretion of the clinical team. The primary outcome is the difference between groups in insulin resistance calculated by a glucose tolerance test administered pre-operatively and 24 hours postoperatively. Secondary endpoints will be changes in muscle carbohydrate metabolism (biopsy), mobility (Cumulative Ambulation Score) and subjective measures of tolerability. DISCUSSION: This is a small-scale pilot study, investigating the benefits and tolerability of carbohydrate loading in an emergency setting in a frail elderly group with known high morbidity and mortality. Positive findings will provide the basis for a larger scale study. TRIAL REGISTRATION: Current Controlled Trials ISRCTN91109766 (7 April 2014); NRES ref: 13/EM/0214Trial Sponsor: University of Nottingham Ref.13036.

Femoral nerve block Intervention in Neck of Femur fracture (FINOF): study protocol for a randomized controlled trial.

BACKGROUND: Hip fractures are very painful leading to lengthy hospital stays. Conventional methods of treating pain are limited. Non-steroidal anti-inflammatories are relatively contraindicated and opioids have significant side effects.Regional anaesthesia holds promise but results from these techniques are inconsistent. Trials to date have been inconclusive with regard to which blocks to use and for how long. Interpatient variability remains a problem. METHODS/DESIGN: This is a single centre study conducted at Queen's Medical Centre, Nottingham; a large regional trauma centre in England. It is a pragmatic, parallel arm, randomized controlled trial. Sample size will be 150 participants (75 in each group). Randomization will be web-based, using computer generated concealed tables (service provided by Nottingham University Clinical Trials Unit). There is no blinding. Intervention will be a femoral nerve block (0.5 mls/kg 0.25% levo-bupivacaine) followed by ropivacaine (0.2% 5 ml/hr-1) infused via a femoral nerve catheter until 48 hours post-surgery. The control group will receive standard care. Participants will be aged over 70 years, cognitively intact (abbreviated mental score of seven or more), able to provide informed consent, and admitted directly through the Emergency Department from their place of residence. Primary outcomes will be cumulative ambulation score (from day 1 to 3 postoperatively) and cumulative dynamic pain scores (day 1 to 3 postoperatively). Secondary outcomes will be cumulative dynamic pain score preoperatively, cumulative side effects, cumulative calorific and protein intake, EUROQOL EQ-5D score, length of stay, and rehabilitation outcome (measured by mobility score). DISCUSSION: Many studies have shown the effectiveness of regional blockade in neck of femur fractures, but the techniques used have varied. This study aims to identify whether early and continuous femoral nerve block can be effective in relieving pain and enhancing mobilization.Trial registration. TRIAL REGISTRATION: The trial is registered with the European clinical trials database Eudract ref: 2010-023871-25. (17/02/2011). ISRCTN: ISRCTN92946117. Registered 26 October 2012.

The effect of intravenous iron on postoperative transfusion requirements in hip fracture patients: study protocol for a randomized controlled trial.

BACKGROUND: Anaemia following hip fracture is common. Approximately 30 to 45% of patients have haemoglobin concentrations below population norms on admission, and around 10% are severely anaemic. Anaemia on admission, and in the postoperative period, is associated with poor outcomes with regard to mobility, postoperative mortality and readmission. There is currently no clear consensus on the optimal method of managing perioperative anaemia in this group of frail patients with frequent comorbidity. Liberal red cell transfusion in the postoperative period does not appear to improve outcome, whereas tranexamic acid appears to reduce transfusion rate at the expense of increased cardiovascular morbidity. There are encouraging results from one centre with the use of agents to stimulate red cell production, including intravenous iron and erythropoietin. UK practice differs significantly from these patients and these studies, and it is not clear whether these promising results will translate to the UK population. METHODS/DESIGN: This is a single-centre randomized controlled parallel group trial, in a British university hospital.Randomization is achieved using a website and computer-generated concealed tables. Participants are 80 patients 70 years or over with acute hip fracture undergoing operative repair. The intervention group receive three daily infusions of 200 mg iron sucrose, starting within 24 hours of admission. The control group receive standard hospital care at the discretion of the clinical team. Red cell transfusions for each group are given in accordance with standard clinical triggers. The primary outcome is an increase in mean reticulocyte count in the intervention group at day 7. Secondary outcome measures include haemoglobin concentrations, early and late transfusion rates, infectious and cardiovascular complications, mobility and 30-day mortality. DISCUSSION: This is a pilot study to demonstrate haematopoietic efficacy of intravenous iron in this setting. Hence, we have chosen to measure change in reticulocyte count rather than the more clinically relevant differences in haemoglobin concentration or transfusion rate. If our results are positive, the study will provide the necessary information for development of a full-scale trial of intravenous iron. TRIAL REGISTRATION: Current Controlled Trials ISRCTN76424792; UK Medicines and Healthcare products Regulatory Authority (EuDRACT: 2011-003233-34).

Small-molecule inhibitors of the protein methyltransferase SET7/9 identified in a high-throughput screen.

Aberrant expression of chromatin-modifying enzymes (CMEs) is associated with a range of human diseases, including cancer. CMEs are now an important target area in drug discovery. Although the role that histone and protein (lysine) methyltransferases (PMTs) play in the regulation of transcription and cell growth is increasingly recognized, few small-molecule inhibitors of this class of enzyme have been reported. Here we describe an assay suitable for primary compound screening for the identification of PMT inhibitors. The assay followed the methylation of histones in the presence of the PMT SET7/9 and the radioactive cofactor S-adenosyl-methionine using scintillating microplates (FlashPlate) and was used to screen approximately 65 000 compounds (% coefficient of variation = 10%; Z' = 0.6). The hits identified from a library of more than 63 000 diverse small molecules included a series of rhodanine compounds with micromolar activity. A screen of the National Cancer Institute Diversity Set (2000 compounds) identified an orsein derivative that inhibited SET7/9 (~20 µM) and showed modest growth inhibition associated with the expected cellular phenotype of reduced histone methylation in a human tumor cell line. The assay represents a useful tool for the identification of inhibitors of PMT activity.

Targeting the Hsp90 molecular chaperone with novel macrolactams. Synthesis, structural, binding, and cellular studies.

A series of resorcylic acid macrolactams, nitrogen analogues of the naturally occurring macrolactone radicicol, have been prepared by chemical synthesis and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. The synthesis involves, as key steps, ring opening of an isocoumarin intermediate, followed by a ring-closing metathesis reaction to form the macrocycle. Subsequent manipulation of the ester group into a range of amides allows access to a range of new macrolactams following deprotection of the two phenolic groups. These new resorcylic acid lactams exhibit metabolic stability greater than that of related lactone counterparts, while co-crystallization of three macrolactams with the N-terminal domain ATP site of Hsp90 confirms that they bind in a similar way to the natural product radicicol and to our previous synthetic lactone analogues. Interestingly, however, in the case of the N-benzylamide, additional binding to a hydrophobic pocket of the protein was observed. In biological assays, the new macrocyclic lactams exhibit a biological profile equivalent or superior to that of the related lactones and show the established molecular signature of Hsp90 inhibitors in human colon cancer cells.

Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response.

Ire1 (Ern1) is an unusual transmembrane protein kinase essential for the endoplasmic reticulum (ER) unfolded protein response (UPR). Activation of Ire1 by association of its N-terminal ER luminal domains promotes autophosphorylation by its cytoplasmic kinase domain, leading to activation of the C-terminal ribonuclease domain, which splices Xbp1 mRNA generating an active Xbp1s transcriptional activator. We have determined the crystal structure of the cytoplasmic portion of dephosphorylated human Ire1α bound to ADP, revealing the 'phosphoryl-transfer' competent dimeric face-to-face complex, which precedes and is distinct from the back-to-back RNase 'active' conformation described for yeast Ire1. We show that the Xbp1-specific ribonuclease activity depends on autophosphorylation, and that ATP-competitive inhibitors staurosporin and sunitinib, which inhibit autophosphorylation in vitro, also inhibit Xbp1 splicing in vivo. Furthermore, we demonstrate that activated Ire1α is a competent protein kinase, able to phosphorylate a heterologous peptide substrate. These studies identify human Ire1α as a target for development of ATP-competitive inhibitors that will modulate the UPR in human cells, which has particular relevance for myeloma and other secretory malignancies.

Discovery of 2-(6-{[(6-fluoroquinolin-2-yl)methyl]amino}bicyclo[3.1.0]hex-3-yl)-N-hydroxypyrimidine-5-carboxamide (CHR-3996), a class I selective orally active histone deacetylase inhibitor.

A novel series of HDAC inhibitors demonstrating class I subtype selectivity and good oral bioavailability is described. The compounds are potent enzyme inhibitors (IC50 values less than 100 nM), and improved activity in cell proliferation assays was achieved by modulation of polar surface area (PSA) through the introduction of novel linking groups. Employing oral pharmacokinetic studies in mice, comparing drug levels in spleen to plasma, we selected compounds that were tested for efficacy in human tumor xenograft studies based on their potential to distribute into tumor. One compound, 21r (CHR-3996), showed good oral activity in these models, including dose-related activity in a LoVo xenograft. In addition 21r showed good activity in combination with other anticancer agents in in vitro studies. On the basis of these results, 21r was nominated for clinical development.

Design and synthesis of novel pyrimidine hydroxamic acid inhibitors of histone deacetylases.

Inhibition of histone deacetylase activity represents a promising new modality in the treatment of a number of cancers. A novel HDAC series demonstrating inhibitory activity in cell proliferation assays is described. Optimisation based on the introduction of basic amine linkers to effect good drug distribution to tumour led to the identification of a compound with oral activity in a human colon cancer xenograft study associated with increased histone H3 acetylation in tumour tissue.

Inhibition of Hsp90 with resorcylic acid macrolactones: synthesis and binding studies.

A series of resorcylic acid macrolactones, analogues of the natural product radicicol has been prepared by chemical synthesis, and evaluated as inhibitors of heat shock protein 90 (Hsp90), an emerging attractive target for novel cancer therapeutic agents. The synthesis involves acylation of an ortho-toluic acid dianion, esterification, followed by a ring-closing metathesis to form the macrocycle. Subsequent manipulation of the protected hydroxymethyl side chain allows access to a range of new analogues following deprotection of the two phenolic groups. Co-crystallization of one of the new macrolactones with the N-terminal domain of yeast Hsp90 confirms that it binds in a similar way to the natural product radicicol and to our previous synthetic analogues, but that the introduction of the additional hydroxymethyl substituent appears to result in an unexpected change in conformation of the macrocyclic ring. As a result of this conformational change, the compounds bound less favorably to Hsp90.

Detection of the ATPase activity of the molecular chaperones Hsp90 and Hsp72 using the TranscreenerTM ADP assay kit.

The molecular chaperone heat shock protein 90 (Hsp90) is required for the correct folding and stability of a number of client proteins that are important for the growth and maintenance of cancer cells. Heat shock protein 72 (Hsp72), a co-chaperone of Hsp90, is also emerging as an attractive cancer drug target. Both proteins bind and hydrolyze adenosine triphosphate (ATP), and ATPase activity is essential for their function. Inhibition of Hsp90 ATPase activity leads to the degradation of client proteins, resulting in cell growth inhibition and apoptosis. Several small-molecule inhibitors of the ATPase activity of Hsp90 have been described and are currently being evaluated clinically for the treatment of cancer. A number of methods for the measurement of ATPase activity have been previously used, but not all of these are ideally suited to screening cascades in drug discovery projects. The authors have evaluated the use of commercial reagents (Transcreener ADP) for the measurement of ATPase activity of both yeast and human Hsp90 (ATP K(m) approximately 500 microM) and human Hsp72 (ATP K(m) ~1 microM). The low ATPase activity of human Hsp90 and its stimulation by the co-chaperone Aha1 was measured with ease using reduced incubation times, generating robust data (Z' = 0.75). The potency of several small-molecule inhibitors of both Hsp90 and Hsp72 was determined using the Transcreener reagents and compared well to that determined using other assay formats.

Discovery of 4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamides as selective, orally active inhibitors of protein kinase B (Akt).

Protein kinase B (PKB or Akt) is an important component of intracellular signaling pathways regulating growth and survival. Signaling through PKB is frequently deregulated in cancer, and inhibitors of PKB therefore have potential as antitumor agents. The optimization of lipophilic substitution within a series of 4-benzyl-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidin-4-amines provided ATP-competitive, nanomolar inhibitors with up to 150-fold selectivity for inhibition of PKB over the closely related kinase PKA. Although active in cellular assays, compounds containing 4-amino-4-benzylpiperidines underwent metabolism in vivo, leading to rapid clearance and low oral bioavailability. Variation of the linker group between the piperidine and the lipophilic substituent identified 4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamides as potent and orally bioavailable inhibitors of PKB. Representative compounds modulated biomarkers of signaling through PKB in vivo and strongly inhibited the growth of human tumor xenografts in nude mice at well-tolerated doses.