First generation EGF receptor tyrosine kinase inhibitors (EGFR TKIs) provide significant clinical benefit in patients with advanced EGFR mutant (EGFRm+) non-small cell lung cancer (NSCLC). Patients ultimately develop disease progression, often driven by acquisition of a second T790M EGFR TKI resistance mutation. AZD9291 is a novel oral, potent and selective third generation irreversible inhibitor of both EGFRm+ sensitizing and T790M resistance mutants that spares wild-type EGFR. This monoanilino-pyrimidine compound is structurally distinct from other third generation EGFR TKIs and offers a pharmacologically differentiated profile from earlier generation EGFR TKIs. Pre-clinically, the drug potently inhibits signaling pathways and cellular growth in both EGFRm+ and EGFRm+/T790M mutant cell lines in vitro, with lower activity against wild-type EGFR lines, translating into profound and sustained tumor regression in EGFR mutant tumor xenograft and transgenic models. The treatment of two patients with advanced EGFRm T790M+ NSCLC is described as proof of principle.

A novel series of small-molecule inhibitors has been developed to target the double mutant form of the epidermal growth factor receptor (EGFR) tyrosine kinase, which is resistant to treatment with gefitinib and erlotinib. Our reported compounds also show selectivity over wild-type EGFR. Guided by molecular modeling, this series was evolved to target a cysteine residue in the ATP binding site via covalent bond formation and demonstrates high levels of activity in cellular models of the double mutant form of EGFR. In addition, these compounds show significant activity against the activating mutations, which gefitinib and erlotinib target and inhibition of which gives rise to their observed clinical efficacy. A glutathione (GSH)-based assay was used to measure thiol reactivity toward the electrophilic functionality of the inhibitor series, enabling both the identification of a suitable reactivity window for their potency and the development of a reactivity quantitative structure-property relationship (QSPR) to support design.

Off-target pharmacology may contribute to both adverse and beneficial effects of a new drug. In vitro pharmacological profiling is often applied early in drug discovery; there are fewer reports addressing the relevance of broad profiles to clinical adverse effects. Here, we have characterized the pharmacological profile of the active metabolite of fostamatinib, R406, linking an understanding of drug selectivity to the increase in blood pressure observed in clinical studies. R406 was profiled in a broad range of in vitro assays to generate a comprehensive pharmacological profile and key targets were further investigated using functional and cellular assay systems. A combination of traditional literature searches and text-mining approaches established potential mechanistic links between the profile of R406 and clinical side effects. R406 was selective outside the kinase domain, with only antagonist activity at the adenosine A3 receptor in the range relevant to clinical effects. R406 was less selective in the kinase domain, having activity at many protein kinases at therapeutically relevant concentrations when tested in multiple in vitro systems. Systematic literature analyses identified KDR as the probable target underlying the blood pressure increase observed in patients. While the in vitro pharmacological profile of R406 suggests a lack of selectivity among kinases, a combination of classical searching and text-mining approaches rationalized the complex profile establishing linkage between off-target pharmacology and clinically observed effects. These results demonstrate the utility of in vitro pharmacological profiling for a compound in late-stage clinical development.

The Notch signaling pathway has been implicated in cell fate determination and differentiation in many tissues. Accumulating evidence points toward a pivotal role in blood vessel formation, and the importance of the Delta-like ligand (Dll) 4-Notch1 ligand-receptor interaction has been shown in both physiological and tumor angiogenesis. Disruption of this interaction leads to a reduction in tumor growth as a result of an increase in nonfunctional vasculature leading to poor perfusion of the tumor. MEDI0639 is an investigational human therapeutic antibody that targets Dll4 to inhibit the interaction between Dll4 and Notch1. The antibody crossreacts to cynomolgus monkey but not mouse species orthologues. In vitro MEDI0639 inhibits the binding of Notch1 to Dll4, interacting via a novel epitope that has not been previously described. Binding to this epitope translates into MEDI0639 reversing Notch1-mediated suppression of human umbilical vein endothelial cell growth in vitro. MEDI0639 administration resulted in stimulation of tubule formation in a three-dimensional (3D) endothelial cell outgrowth assay, a phenotype driven by disruption of the Dll4-Notch signaling axis. In contrast, in a two-dimensional endothelial cell-fibroblast coculture model, MEDI0639 is a potent inhibitor of tubule formation. In vivo, MEDI0639 shows activity in a human endothelial cell angiogenesis assay promoting human vessel formation and reducing the number of vessels with smooth muscle actin-positive mural cells coverage. Collectively, the data show that MEDI0639 is a potent modulator of Dll4-Notch signaling pathway.

Metabolic plasticity is an emerging hallmark of cancer, and increased glycolysis is often observed in transformed cells. Small molecule inhibitors that target driver oncogenes can potentially inhibit the glycolytic pathway. Osimertinib (AZD9291) is a novel EGFR tyrosine kinase inhibitor (TKI) that is potent and selective for sensitising (EGFRm) and T790M resistance mutations. Clinical studies have shown osimertinib to be efficacious in patients with EGFRm/ T790M advanced NSCLC who have progressed after EGFR-TKI treatment. However experience with targeted therapies suggests that acquired resistance may emerge. Thus there is a need to characterize resistance mechanisms and to devise ways to prevent, delay or overcome osimertinib resistance. We show here that osimertinib suppresses glycolysis in parental EGFR-mutant lung adenocarcinoma lines, but has not in osimertinib-resistant cell lines. Critically, we show osimertinib treatment induces a strict dependence on mitochondrial oxidative phosphorylation (OxPhos), as OxPhos inhibitors significantly delay the long-term development of osimertinib resistance in osimertinib-sensitive lines. Accordingly, growth conditions which promote a less glycolytic phenotype confer a degree of osimertinib resistance. Our data support a model in which the combination of osimertinib and OxPhos inhibitors can delay or prevent resistance in osimertinib-naïve tumour cells, and represents a novel strategy that warrants further pre-clinical investigation.

Inhibition of VEGFR-2 signaling reduces angiogenesis and retards tumor growth. Current biotherapeutics that inhibit VEGFR-2 signaling by either sequestering VEGF ligand or inhibiting VEGF binding to VEGFR-2 may be compromised by high VEGF concentrations. Here we describe a biotherapeutic that targets VEGFR-2 signaling by binding to Ig domains 4-7 of VEGFR-2 and therefore has the potential to work independently of ligand concentration. 33C3, a fully human VEGFR-2 antibody, was generated using XenoMouse technology. To elucidate the mechanism of action of 33C3, we have used a number of competition and binding assays. We show that 33C3 binds VEGFR-2 Ig domains 4-7, has no impact on VEGF-A binding to VEGFR-2, and does not compete with an antibody that interacts at the ligand binding site. 33C3 has a high affinity for VEGFR-2 (KD < 1 nmol/L) and inhibits VEGF-A induced phosphorylation of VEGFR-2 with an IC50 of 99 ± 3 ng/mL. In vitro, in a 2D angiogenesis assay, 33C3 potently inhibits both tube length and number of branch points, and endothelial tubule formation in a 3D assay. In vivo, 33C3 is a very effective inhibitor of angiogenesis in both a human endothelial angiogenesis assay and in a human skin chimera model. These data show targeting VEGFR-2 outside of the ligand binding domain results in potent inhibition of VEGFR-2 signaling and inhibition of angiogenesis in vitro and in vivo. Mol Cancer Ther; 10(5); 770–83. ©2011 AACR.

Vascular Endothelial Growth Factor Receptor (VEGFR) mediated signalling drives angiogenesis. This is predominantly attributed to the activity of VEGFR-2 following binding of VEGF-A. Whether other members of the VEGFR and ligand families such as VEGFR-1 and its ligand Placental Growth Factor (PlGF) can also contribute to developmental and pathological angiogenesis is less clear. We explored the function of PlGF in VEGF-A dependent angiogenesis using an in vitro co-culture assay in which endothelial cells are cultured on a fibroblast feeder layer. In the presence of 2% FS MCDB media (containing limited growth factors) in vitro endothelial tube formation is driven by endogenous angiogenic stimuli which are produced by the fibroblast and endothelial cells. Under these conditions independent sequestration of either free VEGF-A or PlGF with polyclonal and monoclonal antibodies inhibited tube formation suggesting that both ligands are required to drive an angiogenic response. Endothelial tube formation could only be driven within this assay by the addition of exogenous VEGF-A, VEGF-E or VEGF-A/PlGF heterodimer, but not by PlGF alone, implying that activation of either VEGFR-2/VEGFR-1 heterodimers or VEGFR-2 homodimers were responsible for eliciting an angiogenic response directly, but not VEGFR-1 homodimers. In contrast to results obtained with an endogenous angiogenic drive, sequestration of PlGF did not affect endothelial tube formation when the assay was driven by 1 ng/ml exogenous VEGF-A. These data suggest that although neutralising PlGF can be shown to reduce endothelial tube formation in vitro, this effect is only observed under restricted culture conditions and is influenced by VEGF-A. Such data questions whether neutralising PlGF would have a therapeutic benefit in vivo in the presence of pathological concentrations of VEGF-A.

Mixed micelles of lipoic acidchitosan-poly(ethylene glycol) and distearoylphosphatidylethanolamine-poly(ethylene glycol) for tumor delivery. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Phasci(2017Phasci( ), doi: 10.1016Phasci( / j.ejps.2017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T 2 AbstractMany chemotherapeutics suffer from poor aqueous solubility and tissue selectivity.Distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG) micelles are a promising formulation strategy for the delivery of hydrophobic anticancer drugs. However, storage and in vivo instability restrict their use. The aim of this study was to prepare mixed micelles, containing a novel polymer, lipoic acid-chitosan-poly(ethylene glycol) (LACPEG), and DSPE-PEG, to overcome these limitations and potentially increase cancer cell internalisation. Drug-loaded micelles were prepared with a model tyrosine kinase inhibitor and characterized for size, surface charge, stability, morphology, drug