BID expression determines the apoptotic fate of cancer cells after abrogation of the spindle assembly checkpoint by AURKB or TTK inhibitors.

BACKGROUND: Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection. METHODS: A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice. RESULTS: Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors. CONCLUSIONS: The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.

Targeting BCL2 Overcomes Resistance and Augments Response to Aurora Kinase B Inhibition by AZD2811 in Small Cell Lung Cancer.

PURPOSE: Therapeutic resistance to frontline therapy develops rapidly in small cell lung cancer (SCLC). Treatment options are also limited by the lack of targetable driver mutations. Therefore, there is an unmet need for developing better therapeutic strategies and biomarkers of response. Aurora kinase B (AURKB) inhibition exploits an inherent genomic vulnerability in SCLC and is a promising therapeutic approach. Here, we identify biomarkers of response and develop rational combinations with AURKB inhibition to improve treatment efficacy. EXPERIMENTAL DESIGN: Selective AURKB inhibitor AZD2811 was profiled in a large panel of SCLC cell lines (n = 57) and patient-derived xenograft (PDX) models. Proteomic and transcriptomic profiles were analyzed to identify candidate biomarkers of response and resistance. Effects on polyploidy, DNA damage, and apoptosis were measured by flow cytometry and Western blotting. Rational drug combinations were validated in SCLC cell lines and PDX models. RESULTS: AZD2811 showed potent growth inhibitory activity in a subset of SCLC, often characterized by, but not limited to, high cMYC expression. Importantly, high BCL2 expression predicted resistance to AURKB inhibitor response in SCLC, independent of cMYC status. AZD2811-induced DNA damage and apoptosis were suppressed by high BCL2 levels, while combining AZD2811 with a BCL2 inhibitor significantly sensitized resistant models. In vivo, sustained tumor growth reduction and regression was achieved even with intermittent dosing of AZD2811 and venetoclax, an FDA-approved BCL2 inhibitor. CONCLUSIONS: BCL2 inhibition overcomes intrinsic resistance and enhances sensitivity to AURKB inhibition in SCLC preclinical models.

Safety, tolerability, and pharmacokinetics of Aurora kinase B inhibitor AZD2811: a phase 1 dose-finding study in patients with advanced solid tumours.

BACKGROUND: AZD2811 is a potent, selective Aurora kinase B inhibitor. We report the dose-escalation phase of a first-in-human study assessing nanoparticle-encapsulated AZD2811 in advanced solid tumours. METHODS: AZD2811 was administered in 12 dose-escalation cohorts (2-h intravenous infusion; 15‒600 mg; 21-/28-day cycles) with granulocyte colony-stimulating factor (G-CSF) at higher doses. The primary objective was determining safety and maximum tolerated/recommended phase 2 dose (RP2D). RESULTS: Fifty-one patients received AZD2811. Drug exposure was sustained for several days post-dose. The most common AZD2811-related adverse events (AEs) were fatigue (27.3%) at ≤200 mg/cycle and neutropenia (37.9%) at ≥400 mg/cycle. Five patients had dose-limiting toxicities: grade (G)4 decreased neutrophil count (n = 1, 200 mg; Days 1, 4; 28-day cycle); G4 decreased neutrophil count and G3 stomatitis (n = 1 each, both 400 mg; Day 1; 21-day cycle); G3 febrile neutropenia and G3 fatigue (n = 1 each, both 600 mg; Day 1; 21-day cycle +G-CSF). RP2D was 500 mg; Day 1; 21-day cycle with G-CSF on Day 8. Neutropenia/neutrophil count decrease were on-target AEs. Best overall responses were partial response (n = 1, 2.0%) and stable disease (n = 23, 45.1%). CONCLUSIONS: At RP2D, AZD2811 was tolerable with G-CSF support. Neutropenia was a pharmacodynamic biomarker. CLINICAL TRIAL REGISTRATION: NCT02579226.

A phase 1/2 study of the combination of acalabrutinib and vistusertib in patients with relapsed/refractory B-cell malignancies.

In a phase 1b study of acalabrutinib (a covalent Bruton tyrosine kinase (BTK) inhibitor) in combination with vistusertib (a dual mTORC1/2 inhibitor) in patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL), multiple ascending doses of the combination as intermittent or continuous schedules of vistusertib were evaluated. The overall response rate was 12% (3/25). The pharmacodynamic (PD) profile for acalabrutinib showed that BTK occupancy in all patients was >95%. In contrast, PD analysis for vistusertib showed variable inhibition of phosphorylated 4EBP1 (p4EBP1) without modulation of AKT phosphorylation (pAKT). The pharmacokinetic (PK)/PD relationship of vistusertib was direct for TORC1 inhibition (p4EBP1) but did not correlate with TORC2 inhibition (pAKT). Cell-of-origin subtyping or next-generation sequencing did not identify a subset of DLBCL patients with clinical benefit; however, circulating tumor DNA dynamics correlated with radiographic response. These data suggest that vistusertib does not modulate targets sufficiently to add to the clinical activity of acalabrutinib monotherapy. Clinicaltrials.gov identifier: NCT03205046.

AZD0364 Is a Potent and Selective ERK1/2 Inhibitor That Enhances Antitumor Activity in KRAS-Mutant Tumor Models when Combined with the MEK Inhibitor, Selumetinib.

The RAS-regulated RAF-MEK1/2-ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.

Venous air embolism: ultrasonographic diagnosis and treatment with hyperbaric oxygen therapy.

A man with neuromuscular respiratory failure requiring intubation and ventilation suffered a venous air embolism during inadvertent administration of 5 ml of air. Ultrasound (US) imaging confirmed an air embolus in the left subclavian vein, which was only partially treated by US-guided aspiration. The embolus completely resolved on US imaging during hyperbaric oxygen therapy, and the patient recovered with no complications secondary to the embolism. Venous air embolism is under-recognised, and can cause siginificant neurological morbidity and death if untreated. When available, urgent hyperbaric oxygen therapy appears to be an effective approach.

Discovery and Optimization of Pyrrolopyrimidine Inhibitors of Interleukin-1 Receptor Associated Kinase 4 (IRAK4) for the Treatment of Mutant MYD88L265P Diffuse Large B-Cell Lymphoma.

Herein we report the optimization of a series of pyrrolopyrimidine inhibitors of interleukin-1 receptor associated kinase 4 (IRAK4) using X-ray crystal structures and structure based design to identify and optimize our scaffold. Compound 28 demonstrated a favorable physicochemical and kinase selectivity profile and was identified as a promising in vivo tool with which to explore the role of IRAK4 inhibition in the treatment of mutant MYD88L265P diffuse large B-cell lymphoma (DLBCL). Compound 28 was shown to be capable of demonstrating inhibition of NF-κB activation and growth of the ABC subtype of DLBCL cell lines in vitro at high concentrations but showed greater effects in combination with a BTK inhibitor at lower concentrations. In vivo, the combination of compound 28 and ibrutinib led to tumor regression in an ABC-DLBCL mouse model.

Osteopontin binds and modulates functions of eosinophil-recruiting chemokines.

BACKGROUND: Allergic asthma is characterized by eosinophilic inflammation and airway obstruction. There is also an increased risk of pulmonary infection caused by Streptococcus pneumoniae, in particular during severe asthma where high levels of the glycoprotein, osteopontin (OPN), are present in the airways. Eosinophils can be recruited by chemokines activating the receptor CCR3 including eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, RANTES/CCL5, and MEC/CCL28. In addition to inducing chemotaxis, several of these molecules have defensin-like antibacterial properties. This study set out to elucidate the functional consequences of OPN binding to eosinophil-recruiting chemokines. METHODS: Antibacterial activities of the chemokines were investigated using viable count assays and electron microscopy. Binding studies were performed by means of surface plasmon resonance. The potential interference of OPN with antibacterial, receptor-activating, and lipopolysaccharide-neutralizing abilities of these chemokines was investigated. RESULTS: We found that OPN bound all eosinophil-recruiting chemokines with high affinity except for CCL5. The eosinophil-recruiting chemokines all displayed bactericidal activity against S. pneumoniae, but only CCL26 and CCL28 retained high antibacterial activity in the presence of sodium chloride at physiologic concentrations. Preincubation of the chemokines with OPN strongly inhibited their antibacterial activity against S. pneumoniae but did not affect their ability to activate CCR3. All chemokines investigated showed LPS-neutralizing activity that was impaired by OPN only in the case of CCL24. CONCLUSIONS: The data suggest that OPN may impair host defense activities of the chemokines without affecting their eosinophil-recruiting properties. This could be one mechanism explaining the increased vulnerability to acquire pneumococcal infection in parallel with sustained allergic inflammation in asthma.

Eotaxin-3 (CCL26) exerts innate host defense activities that are modulated by mast cell proteases.

BACKGROUND: During bacterial infections of the airways, a Th1-profiled inflammation promotes the production of several host defense proteins and peptides with antibacterial activities including ß-defensins, ELR-negative CXC chemokines, and the cathelicidin LL-37. These are downregulated by Th2 cytokines of the allergic response. Instead, the eosinophil-recruiting chemokines eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 are expressed. This study set out to investigate whether these chemokines could serve as innate host defense molecules during allergic inflammation. METHODS: Antibacterial activities of the eotaxins were investigated using viable count assays, electron microscopy, and methods assessing bacterial permeabilization. Fragments generated by mast cell proteases were characterized, and their potential antibacterial, receptor-activating, and lipopolysaccharide-neutralizing activities were investigated. RESULTS: CCL11, CCL24, and CCL26 all showed potent bactericidal activity, mediated through membrane disruption, against the airway pathogens Streptococcus pneumoniae, Staphylococcus aureus, Nontypeable Haemophilus influenzae, and Pseudomonas aeruginosa. CCL26 retained bactericidal activity in the presence of salt at physiologic concentrations, and the region holding the highest bactericidal activity was the cationic and amphipathic COOH-terminus. Proteolysis of CCL26 by chymase and tryptase, respectively, released distinct fragments of the COOH- and NH2 -terminal regions. The COOH-terminal fragment retained antibacterial activity while the NH2 -terminal had potent LPS-neutralizing properties in the order of CCL26 full-length protein. An identical fragment to NH2 -terminal fragment generated by tryptase was obtained after incubation with supernatants from activated mast cells. None of the fragments activated the CCR3-receptor. CONCLUSIONS: Taken together, the findings show that the eotaxins can contribute to host defense against common airway pathogens and that their activities are modulated by mast cell proteases.

Expanding medicinal chemistry space.

Clinically useful drugs target a relatively small number of proteins that lie within a clearly defined and chemically accessible space. However, many high value biological targets lie outside this chemical space, and an ability to access such 'intractable' targets not amenable to traditional small molecule intervention would expand treatment options and be a major boost for patients and the pharmaceutical industry. To date, success has been limited but new technologies and approaches are beginning to emerge that could provide novel lead generation capabilities that enable access to new drug target classes. We review these new approaches and their ability to provide the novel leads needed to tackle a new generation of biological targets.

Molecular requirements for inhibition of the chemokine receptor CCR8--probe-dependent allosteric interactions.

BACKGROUND AND PURPOSE: Here we present a novel series of CCR8 antagonists based on a naphthalene-sulfonamide structure. This structure differs from the predominant pharmacophore for most small-molecule CC-chemokine receptor antagonists, which in fact activate CCR8, suggesting that CCR8 inhibition requires alternative structural probes. EXPERIMENTAL APPROACH: The compounds were tested as inverse agonists and as antagonists against CCL1-induced activity in Gα(i) signalling and chemotaxis. Furthermore, they were assessed by heterologous competition binding against two radiolabelled receptor ligands: the endogenous agonist CCL1 and the virus-encoded antagonist MC148. KEY RESULTS: All compounds were highly potent inverse agonists with EC(50) values from 1.7 to 23 nM. Their potencies as antagonists were more widely spread (EC(50) values from 5.9 to 1572 nM). Some compounds were balanced antagonists/inverse agonists whereas others were predominantly inverse agonists with >100-fold lower potency as antagonists. A correspondingly broad range of affinities, which followed the antagonist potencies, was disclosed by competition with [(125)I]-CCL1 (K(i) 3.4-842 nM), whereas the affinities measured against [(125)I]-MC148 were less widely spread (K(i) 0.37-27 nM), and matched the inverse agonist potencies. CONCLUSION AND IMPLICATIONS: Despite highly potent and direct effects as inverse agonists, competition-binding experiments against radiolabelled agonist and tests for antagonism revealed a probe-dependent allosteric effect of these compounds. Thus, minor chemical changes affected the ability to modify chemokine binding and action, and divided the compounds into two groups: predominantly inverse agonists and balanced antagonists/inverse agonists. These studies have important implications for the design of new inverse agonists with or without antagonist properties.

Unravelling the mechanisms underpinning chemokine receptor activation and blockade by small molecules: a fine line between agonism and antagonism?

Chemokines are a family of small basic proteins which induce the directed migration of cells, notably leucocytes, by binding to specific GPCRs (G-protein-coupled receptors). Both chemokines and their receptors have been implicated in a host of clinically important diseases, leading to the notion that antagonism of the chemokine-chemokine receptor network may be therapeutically advantageous. Consequently, considerable effort has been put into the development of small-molecule antagonists of chemokine receptors and several such compounds have been described in the literature. One curious by-product of this activity has been the description of several small-molecule agonists of the receptors, which are typically discovered following the optimization of lead antagonists. In this review we discuss these findings and conclude that these small-molecule agonists might be exploited to further our understanding of the molecular mechanisms by which chemokine receptors are activated.

Tails of the unexpected - an atypical receptor for the chemokine RANTES/CCL5 expressed in brain.

Chemokines and their receptors play a central role in the trafficking of leukocytes within the body, a process which is amenable to antagonism by small molecules and which holds promise as a treatment for clinically important diseases. In the issue of the British Journal of Pharmacology accompanying this commentary, Ignatov and colleagues describe an unexpected role for the chemokine RANTES/CCL5, namely an ability to signal via the orphan G protein-coupled receptor named GPR75. This receptor bears little homology to other chemokine receptors, most strikingly within the putative intracellular domains, with the third loop and C-terminal tail dwarfing those of other known chemokine receptors. This most likely accounts for the atypical pertussis toxin-insensitive signalling induced by RANTES. Intriguingly, this signalling is neuro-protective, inducing the survival of a hippocampal cell line following insult with the neurotoxic amyloid-beta peptide. Since this peptide is implicated in the pathogenesis of Alzheimer's disease, it may be that exploitation of this signalling pathway presents itself as a future therapeutic treatment.

Novel 4-anilinoquinazolines with C-6 carbon-linked side chains: synthesis and structure-activity relationship of a series of potent, orally active, EGF receptor tyrosine kinase inhibitors.

The structure-activity relationship of a novel subseries of 4-anilinoquinazoline EGFR inhibitors substituted at the C-6 position with carbon-linked side chains has been investigated. This exploration has led to the discovery of novel aminomethyl carboxamides with good biological, pharmacokinetic and physical properties.

Cyclin-dependent kinase 4 inhibitors as a treatment for cancer. Part 1: identification and optimisation of substituted 4,6-bis anilino pyrimidines.

Using a high-throughput screening campaign, we identified the 4,6-bis anilino pyrimidines as inhibitors of the cyclin-dependent kinase, CDK4. Herein we describe the further chemical modification and use of X-ray crystallography to develop potent and selective in vitro inhibitors of CDK4.

Cyclin-dependent kinase 4 inhibitors as a treatment for cancer. Part 2: identification and optimisation of substituted 2,4-bis anilino pyrimidines.

Through chemical modification and X-ray crystallography we identified the 2,4-bis anilino pyrimidines as potent inhibitors of CDK4. Herein, we describe the optimisation of this series.

Selective antagonism of the NPY Y5 receptor does not have a major effect on feeding in rats.

Neuropeptide Y (NPY) is thought to play a key role in stimulating feeding, thus making NPY receptors attractive appetite suppressant drug targets for treating obesity. Because the orexigenic effects of NPY have been ascribed to actions at the NPY Y5 receptor, we have determined the role of this receptor in feeding in rats, using a small molecule antagonist of this receptor. NPY5RA-972 is a selective and potent (<10 nmol/l) NPY Y5 receptor antagonist. This compound is central nervous system (CNS) penetrant, and an oral dose of 10 mg/kg NPY5RA-972 to rats produced concentrations in cerebrospinal fluid that greatly exceeded the in vitro IC(50) (inhibitory concentration 50%). Indeed, at doses to rats as low as 1 mg/kg, NPY5RA-972 inhibited feeding induced by intracerebroventricular (ICV) administration of a selective NPY Y5 agonist ([cPP(1-7),NPY(19-23),Ala(31),Aib(32),Gln(34)]-hPP). However, in the dose range 1-10 mg/kg, NPY5RA-972 had no significant effect on food intake in Wistar rats induced to feed by either ICV NPY or 24 h fasting or in free-feeding Wistar or obese Zucker rats. Chronic administration of NPY5RA-972 (10 mg/kg twice daily) had no effect on food intake or body weight in either free-feeding Wistar rats or dietary obese rats. These data indicate that NPY5RA-972 is a potent, selective, orally active, and CNS-penetrant antagonist of the NPY Y5 receptor that prevents feeding driven by activation of this receptor. The data obtained with this antagonist indicate that the NPY Y5 receptor is not a major regulator of feeding in the rat.

Discovery and optimization of a series of carbazole ureas as NPY5 antagonists for the treatment of obesity.

The hypothesis that antagonists of the neuropeptide Y5 receptor would provide safe and effective appetite suppressants for the treatment of obesity has prompted vigorous research to identify suitable compounds. We discovered a series of acylated aminocarbazole derivatives (e.g., 3a) that are potent and selective Y5 antagonists, representing interesting starting points but suffering from poor bioavailability and concerns about potential toxicity as a consequence of the embedded aminocarbazole fragment. It proved relatively easy to improve the drug metabolism and pharmacokinetic (DMPK) properties by variation of the side chain (as in 4a) but difficult to eliminate the aminocarbazole fragment. For compounds in this series to have the potential to be drugs, we believed that both the compound itself and the component aniline must be free of mutagenic activity. Parallel structure-activity relationship studies looking at the effects of ring substitution have proved that it is possible by incorporation of a 4-methyl substituent to produce carbazole ureas with potent Y5 activity, comprised of carbazole anilines that in themselves are devoid of mutagenic activity in the Ames test. Compound 4o (also known as NPY5RA-972) is highly selective with respect to Y1, Y2, and Y4 receptors (and also to a diverse range of unrelated receptors and enzymes), with an excellent DMPK profile including central nervous system penetration. NPY5RA-972 (4o) is a highly potent Y5 antagonist in vivo but does not block neuropeptide Y-induced feeding nor does it reduce feeding in rats, suggesting that the Y5 receptor alone has no significant role in feeding in these models.