Activity In Cell-based Assays Research Articles

Abstract LB-305: Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP

Abstract Background Evasion of cell death is a major cause of resistance to anti-cancer therapies, making proteins that regulate cell death clinically relevant therapeutic targets. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers, including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and has been shown by us and others to be a major mediator of drug resistance. FLIP and procaspase-8 form complexes with the adaptor protein FADD in response to a variety of clinically relevant stimuli, including ligation of death receptors, such as TRAIL-R1 and R2, and treatment with chemotherapeutic agents. In these complexes, FLIP modulates the activation of procaspase-8, and thereby apoptosis and necroptosis - two major cell death mechanisms. We have found that there are important differences between FLIP and procaspase-8 in terms of their preferred modes of interaction with FADD that are potentially therapeutically exploitable [1]. Herein, we report the development and pre-clinical characterization of first-in-class inhibitors of FLIP. Methods Molecular modelling of the FLIP-FADD interaction; cell-free screening assays; cell-based activity assays; biophysical binding assays; on-target mechanism-of-action studies, in vivo studies. Results Molecular modelling of the FLIP-FADD interaction identified a putative drug-binding pocket on FLIP against which a virtual small-molecule screen was carried out. Subsequent biochemical screening of selected compounds using a FLIP-FADD protein-protein interaction assay identified hits with on-target activity. Medicinal chemistry optimization of these hits identified a series of compounds that are able to disrupt FLIP’s interaction with the DISC and display nanomolar activity in NSCLC and CRC cell-based assays (i.e. caspase activation, cell death and cell survival), which is in line with their binding affinity in an orthogonal biophysical assay (isothermal calorimetry). The pro-apoptotic effects of these FLIP inhibitors were enhanced upon addition of death ligands, such as TRAIL, and lead-molecules have been shown to potentiate the effects of the standard-of-care chemotherapeutics such as e.g. cisplatin (NSCLC) or 5FU (CRC). FLIP overexpression and CRISPR-mediated procaspase-8 deletion abrogated the effects of these novel inhibitors consistent with the expected mechanism-of-action. In addition, using peripheral blood mononuclear cells (PBMCs), we demonstrate that FLIP inhibitors have selectivity against cancer cells. Finally, we identified lead molecules with ADME profiles suitable for in vivo evaluation and using these compounds, single-agent anti-tumor effects have been demonstrated in xenograft models. Conclusions The first-in-class inhibitors of FLIP developed in this study have the potential for broad application in treatment of cancer, either as monotherapy or in combination with other agents. Acknowledgements This work was supported by a Seeding Drug Discovery award from the Wellcome Trust. 1. Majkut, J., et al., Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly. Nat Commun, 2014. 5: p. 3350. Citation Format: Joanna Majkut, Catherine Higgins, Jennifer Fox, Greti E. Fiedler, Timothy Harrison, Daniel B. Longley. Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-305. doi:10.1158/1538-7445.AM2017-LB-305

Anemone rivularis inhibits pyruvate dehydrogenase kinase activity and tumor growth

Ethnopharmacological relevanceAnemone rivularis Buch.-Ham. ex DC. (Ranunculaceae) have been used as a traditional remedy for treatment of inflammation and cancer. However, there is no report demonstrating experimental evidence on anti-tumor action of A. rivularis. Aim of studyThe Warburg's effect, preference of aerobic glycolysis rather than oxidative phosphorylation (OXPHOS) even in oxygen rich condition, is focused as one of major characteristics of malignant tumor. Thus, we investigated the effect of A. rivularis on the Pyruvate dehydrogenase (PDH) kinases (PDHKs), a major molecular targets for reducing aerobic glycolysis. Materials and methodsThe ethanol extract of whole plant of A. rivularis (ARE), fingerprinted by high performance liquid chromatography (HPLC), was applied to in vitro and cell-based PDHK activity assays. The effect of ARE on cell viabilities of several tumor cells was estimated by MTT assay. The expression of phosphor-PDH, PDH and PDHK1 were measured by Western blot analysis. The production of reactive oxygen species (ROS) and apoptosis was measured by fluorescence-activated cell sorting analysis, using 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) and Annexin V/propidium iodide (PI) staining, respectively. Mitochondrial membrane potential was examined by tetramethylrhodamine methyl ester (TMRM) staining. In vivo anti-tumor efficacy of ARE was estimated by means of tumor volume and weight using allograft injection of murine Lewis lung carcinoma (LLC) cells to dorsa of C57BL/6 mice. ResultsARE inhibited the viabilities of several cancer cells, including MDA-MB321, K562, HT29, Hep3B, DLD-1, and LLC. ARE suppressed PDHK activity in in vitro kinase assay, and also inhibited aerobic glycolysis by reducing phosphorylation of PDHA in human DLD-1 colon cancer and murine LLC cells. The expression of PDHK1, a major isoform of PDHKs in cancer, was not affected by ARE treatment. Moreover, ARE increased the both ROS production and mitochondrial damage. In addition, ARE suppressed the in vitro tumor growth through mitochondria-mediated apoptosis. The growth rates of allograft LLC cells were also reduced by ARE treatment. ConclusionsHere, we firstly report that ARE inhibits PDHK activity and growth of tumor in both in vitro and in vivo experiments. Therefore, we suggest ARE as a potential candidate for developing anti-cancer drugs.

Clinical immunogenicity of the d-amino acid peptide therapeutic etelcalcetide: Method development challenges and anti-drug antibody clinical impact assessments

The immunogenicity risk assessment and bioanalytical strategy for novel therapeutics should account for both unique biophysical properties and potential consequences of immunogenicity. When assessing the immunogenicity risk of etelcalcetide, a peptide agonist of the calcium-sensing receptor, we considered the potential that the d-amino acid ‘backbone’ and biotransformation of etelcalcetide could allow the drug to act as a hapten. As a consequence, we validated and implemented a surface plasmon resonance immunoassay platform with both etelcalcetide and etelcalcetide-‘carrier’ surfaces to detect anti-drug antibodies (ADA). No evidence of in-vitro neutralizing activity with surrogate controls was detected despite multiple immunization approaches and a sensitive cell-based activity assay. Therefore, a neutralizing assay was not implemented for clinical support. We conducted an integrated analysis of immunogenicity data pooled from two pivotal placebo-controlled trials to define the clinical impact of anti-etelcalcetide antibodies. While both pre-existing and developing anti-etelcalcetide antibodies were detected, we show here that they have no consequences for clinical exposure, efficacy, or safety of etelcalcetide.

Novel Treatment with Intraperitoneal MOC31PE Immunotoxin in Colorectal Peritoneal Metastasis: Results From the ImmunoPeCa Phase 1 Trial

MOC31PE immunotoxin was developed to rapidly kill cells expressing the tumor-associated epithelial cell adhesion molecule, which is highly expressed in colorectal cancer. Although cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) may offer long-term survival to patients with peritoneal metastasis from colorectal cancer (PM-CRC), most patients experience disease relapse and novel therapeutic options are needed. On this basis, MOC31PE is being developed as a novel therapeutic principle to target PM-CRC. This was a dose-escalating phase I trial to evaluate the safety and toxicity (primary endpoint), pharmacokinetic profile, and neutralizing antibody response (secondary endpoints) upon intraperitoneal administration of MOC31PE in patients with PM-CRC undergoing CRS-HIPEC with Mitomycin C. Fifteen patients received the study drug at four dose levels (3+3+3+6), administered intraperitoneally as a single dose the day after CRS-HIPEC. No dose-limiting toxicity was observed, and the maximum tolerated dose was not reached. There was negligible systemic absorption of the study drug. Drug concentrations in peritoneal fluid samples were in the cytotoxic range and increased in a dose-dependent manner. MOC31PE recovered from peritoneal cavity retained its cytotoxic activity in cell-based assays. All patients developed neutralizing antibodies. Intraperitoneal administration of MOC31PE was safe and well tolerated, and combined with low systemic uptake, MOC31PE seems ideal for local intraperitoneal treatment. The drug will be further evaluated in an ongoing phase II expansion cohort.

Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology

Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl-acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (1011 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.

Identification of Inonotus obliquus polysaccharide with broad-spectrum antiviral activity against multi-feline viruses

Inonotus obliquus polysaccharides (IOPs) are a potential drug for the prevention and treatment of cancer, cardiopathy, diabetes, AIDs, pancreatitis and other diseases. In this study, we found that IOP can act as a broad-spectrum antiviral drug against feline viruses in the in vitro experiment. Using cell models of feline calicivirus (FCV), we demonstrated that IOP treatment was capable of exhibiting anti-FCV strain F9 activity in cell-based assays and also showed low cytotoxicity. Investigation of the mechanism of action of the compound revealed that IOP treatment induces its inhibitory actions directly on virus particles through blocking viral binding/absorpting. The inhibitory activity against other FCV isolates from China was also identified. More importantly, we found that IOP exhibited broad-spectrum antiviral activity against the feline herpesvirus 1, feline influenza virus H3N2 and H5N6, feline panleukopenia virus and feline infectious peritonitis virus that can contribute to respiratory and gastrointestinal diseases in cats. These findings suggest that IOP may be a potential broad-spectrum antiviral drug against feline viruses.

Functionomics of NCC mutations in Gitelman syndrome using a novel mammalian cell-based activity assay.

Gitelman syndrome (GS) is an autosomal recessive salt-wasting tubular disorder resulting from loss-of-function mutations in the thiazide-sensitive NaCl cotransporter (NCC). Functional analysis of these mutations has been limited to the use of Xenopus laevis oocytes. The aim of the present study was, therefore, to analyze the functional consequences of NCC mutations in a mammalian cell-based assay, followed by analysis of mutated NCC protein expression as well as glycosylation and phosphorylation profiles using human embryonic kidney (HEK) 293 cells. NCC activity was assessed with a novel assay based on thiazide-sensitive iodide uptake in HEK293 cells expressing wild-type or mutant NCC (N59I, R83W, I360T, C421Y, G463R, G731R, L859P, or R861C). All mutations caused a significantly lower NCC activity. Immunoblot analysis of the HEK293 cells revealed that 1) all NCC mutants have decreased NCC protein expression; 2) mutant N59I, R83W, I360T, C421Y, G463R, and L859P have decreased NCC abundance at the plasma membrane; 3) mutants C421Y and L859P display impaired NCC glycosylation; and 4) mutants N59I, R83W, C421Y, C731R, and L859P show affected NCC phosphorylation. In conclusion, we developed a mammalian cell-based assay in which NCC activity assessment together with a profiling of mutated protein processing aid our understanding of the pathogenic mechanism of the NCC mutations.

Design of nucleotide-mimetic and non-nucleotide inhibitors of the translation initiation factor eIF4E: Synthesis, structural and functional characterisation

Eukaryotic translation initiation factor 4E (eIF4E) is considered as the corner stone in the cap-dependent translation initiation machinery. Its role is to recruit mRNA to the ribosome through recognition of the 5′-terminal mRNA cap structure (m7GpppN, where G is guanosine, N is any nucleotide). eIF4E is implicated in cell transformation, tumourigenesis, and angiogenesis by facilitating translation of oncogenic mRNAs; it is thus regarded as an attractive anticancer drug target. We have used two approaches to design cap-binding inhibitors of eIF4E by modifying the N7-substituent of m7GMP and replacing the phosphate group with isosteres such as squaramides, sulfonamides, and tetrazoles, as well as by structure-based virtual screening aimed at identifying non-nucleotide cap-binding antagonists. Phosphomimetic nucleotide derivatives and highly ranking virtual hits were evaluated in a series of in vitro and cell-based assays to identify the first non-nucleotide eIF4E cap-binding inhibitor with activities in cell-based assays, N-[(5,6-dihydro-6-oxo-1,3-dioxolo[4,5-g]quinolin-7-yl)methyl]-N′-(2-methyl-propyl)-N-(phenyl-methyl)thiourea (14), including down-regulation of oncogenic proteins and suppression of RNA incorporation into polysomes. Although we did not observe cellular activity with any of our modified m7GMP phosphate isostere compounds, we obtained X-ray crystallography structures of three such compounds in complex with eIF4E, 5′-deoxy-5′-(1,2-dioxo-3-hydroxycyclobut-3-en-4-yl)amino-N7-methyl-guanosine (4a), N7-3-chlorobenzyl-5′-deoxy-5′-(1,2-dioxo-3-hydroxy-cyclobut-3-en-4-yl)amino-guanosine (4f), and N7-benzyl-5′-deoxy-5′-(trifluoromethyl-sulfamoyl)guanosine (7a). Collectively, the data we present on structure-based design of eIF4E cap-binding inhibitors should facilitate the optimisation of such compounds as potential anticancer agents.

Abstract 3011: Development of selective small-molecule inhibitors of cellular MALT1 protease activity

Abstract Background: The aim of this study was to develop potent, selective and cell-permeable inhibitors of MALT1 protease activity. MALT1 is an Arg-specific protease that cleaves multiple substrates, acting as a key component in T- and B-cell receptor signaling in lymphocytes. MALT1 protease activity is reported to mediate normal and disease-associated lymphocyte proliferation and survival as part of the CARMA-1/BCL-10/MALT1 (CBM) signaling complex, making MALT1 an attractive therapeutic target. Constitutive MALT1 activity is a hallmark of Diffuse Large B Cell Lymphoma of the activated B cell type (ABC-DLBCL). This aberrant activation is observed as a consequence of mutations in CARMA-1/CARD11, and in multiple regulatory proteins upstream of the CBM complex. Methods: MALT1 enzyme assay with Ac-Leu-Arg-Ser-Arg-AMC as substrate was used to measure Ki. Selectivity towards other Arg-specific proteases was measured by inhibition of Trypsin and Thrombin. For determination of cellular potency the ABC-DLBCL cell line OCI-LY3, carrying the L251P mutation of CARMA-1/CARD11, was used. A MALT1 cell assay to measure A20 substrate cleavage was adapted to capillary electrophoresis and quantification. MALT1 down-stream signaling was measured by inhibition of IL-6 expression. Results: Published inhibitors of MALT1 were profiled for potency, selectivity, DMPK properties and activity in cell-based assays, and showed several drawbacks such as low MALT1 activity, low cell permeability and/or selectivity. We developed several novel reversible small molecule inhibitors of MALT1 with Ki <20 nM and high selectivity against Trypsin and Thrombin. Compounds show inhibition of intracellular MALT1 activity with potencies <300nM in the A20 cleavage assay, as well as inhibition of IL-6 expression. Intracellular inhibition of MALT1 activity requires both potent inhibition of MALT1 and cell permeability (estimated by Caco-2 permeability), e.g. compound A with MALT1 Ki = 16nM, Trypsin Ki>100μM, Thrombin Ki>100μM, and Caco-2 Papp = 1×10−6cm/s shows intracellular A20 cleavage IC50 = 1100nM, whereas compound B with MALT1 Ki = 47nM and Caco-2 Papp = 5.6×10−6cm/s, displays intracellular A20 cleavage IC50 = 600nM. We observe a clear dissociation between intracellular inhibition of MALT1 and inhibition of cell proliferation of DLBCL cell lines of ABC-type with constitutive MALT1 activity, e.g. compound C (MALT1 Ki = 35nM, Trypsin Ki>100μM, Thrombin Ki>100μM and intracellular A20 cleavage IC50 = 260nM) inhibits the proliferation of OCI-LY3 cells and TMD8 cells with CC50 values of 73μM and 96μM respectively, after 5 days exposure. Conclusion: Inhibition of MALT1 protease activity did not translate into significant anti-proliferative effects in DLBCL cell lines. These new MALT1 inhibitors provide the opportunity to test other therapeutic hypotheses involving the targeting of MALT1 protease activity. Citation Format: Fredrik G. Oberg, Sofia Karlstrom, Ian Henderson, Ellen Hewitt, Anders Kallin, Susanne Sedig, Jimmy Lindberg, Anna-Karin Sohlenius-Sternbeck, Richard Bethell, Mark Albertella. Development of selective small-molecule inhibitors of cellular MALT1 protease activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3011.

Abstract 894: Discovery of YAP-TEAD protein-protein interaction (PPI) inhibitors for cancer therapy

Abstract The Hippo pathway is emerging as a critical player in several processes involved in cancer progression, including cell proliferation and EMT. YAP mediates the downstream effects of Hippo signaling, and high nuclear expression of YAP has been documented in many tumors types. YAP translocates to the nucleus, binds to TEAD transcription factor and drives the expression of growth factors, including CTGF, Cyr61 and surviving, suggesting that therapies targeting YAP-TEAD interaction are likely to have clinical impact for the treatment of cancer patients. However, due to the challenging nature of protein-protein interactions, a potent inhibitor that surpasses the affinity of the YAP-TEAD interactions has not been developed. All the critical residues for the YAP-TEAD interaction belong to interface S3. In order to identify interface S3 binders able to disrupt the YAP-TEAD interaction, we started a drug discovery program based on combined FBS/HTS strategy. TEAD protein drugabbility was assessed by fragment screen using NMR and SPR technologies. This approach, supported by assigned HSQC protein NMR information, provided fragment hits which were used as starting points for the identification of more potent binders. We also designed an AlphaLisa assay to validate the ability of our compounds to disrupt the YAP-TEAD interaction. 240,000 compounds of our proprietary library were screened using AlphaLisa assay. Several hits in the μM range were identified and further confirmed using SPR. Successful chemistry optimization generated compounds showing inhibitory activity in the AlphaLisa assay at 50-100 nM for the best compounds. To confirm the validity of our inhibitors, we constructed plasmids encoding TEAD protein fused to Gal4 DNA-binding domain. We transfected HEK293 cells with these plasmids together with Gal4-driven luciferase reporter plasmid in the presence of YAP S127A/S397A. Our compounds showed marked inhibition of cell-based transactivation assay at 3-10 μM. In order to establish a panel of cancer cell lines whose proliferation and target gene expression were either dependent or independent of YAP/TEAD, we selected several cell lines based on their mutational status, YAP nuclear localization, and their proliferative response to a siRNA targeting either YAP or TEAD. Our data show that YAP-TEAD inhibitors block both cell proliferation and target gene expression only in YAP/TEAD-dependent cell lines. In conclusion, we have successfully demonstrated that YAP-TEAD PPI can be inhibited by small molecules, and our compounds show activity in cell-based assays. Medicinal chemistry optimization on various series is ongoing to improve potency and DMPK parameters for further in vivo evaluation in cancer xenograft models. Citation Format: Anne Soude, Martine Barth, Stephanie Bocart, Frederic Thoreau, Philippe Masson, Isabelle Braccini, Christian Montalbetti, Pierre Broqua, Claudia Fromond. Discovery of YAP-TEAD protein-protein interaction (PPI) inhibitors for cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 894.

Discovery of Manassantin A Protein Targets Using Large-Scale Protein Folding and Stability Measurements.

Manassantin A is a natural product that has been shown to have anticancer activity in cell-based assays, but has a largely unknown mode-of-action. Described here is the use of two different energetics-based approaches to identify protein targets of manassantin A. Using the stability of proteins from rates of oxidation technique with an isobaric mass tagging strategy (iTRAQ-SPROX) and the pulse proteolysis technique with a stable isotope labeling with amino acids in cell culture strategy (SILAC-PP), over 1000 proteins in a MDA-MB-231 cell lysate grown under hypoxic conditions were assayed for manassantin A interactions (both direct and indirect). A total of 28 protein hits were identified with manassantin A-induced thermodynamic stability changes. Two of the protein hits (filamin A and elongation factor 1α) were identified using both experimental approaches. The remaining 26 hit proteins were only assayed in either the iTRAQ-SPROX or the SILAC-PP experiment. The 28 potential protein targets of manassantin A identified here provide new experimental avenues along which to explore the molecular basis of manassantin A's mode of action. The current work also represents the first application iTRAQ-SPROX and SILAC-PP to the large-scale analysis of protein-ligand binding interactions involving a potential anticancer drug with an unknown mode-of-action.

Design, synthesis, and biological evaluation of anti-EV71 agents

Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease (HFMD), which can spread its infections to the central nervous and other systems with severe consequences. In this article, design, chemical synthesis, and biological evaluation of various anti-EV71 agents which incorporate Michael acceptors are described. Further SAR study demonstrated that lactone type of Michael acceptor provided a new lead of anti-EV71 drug candidates with high anti-EV71 activity in cell-based assay and enhanced mouse plasma stability. One of the most potent compounds (2K, cell-based anti-EV71 EC50=0.028μM), showed acceptable stability profile towards mouse plasma, which resulted into promising pharmacokinetics in mouse via IP administration.

Synthesis and Anti-Influenza Activity of Pyridine, Pyridazine, and Pyrimidine C-Nucleosides as Favipiravir (T-705) Analogues.

Influenza viruses are responsible for seasonal epidemics and occasional pandemics which cause significant morbidity and mortality. Despite available vaccines, only partial protection is achieved. Currently, there are two classes of widely approved anti-influenza drugs: M2 ion channel blockers and neuraminidase inhibitors. However, the worldwide spread of drug-resistant influenza strains poses an urgent need for novel antiviral drugs, particularly with a different mechanism of action. Favipiravir (T-705), a broad-spectrum antiviral agent, has shown potent anti-influenza activity in cell-based assays, and its riboside (2) triphosphate inhibited influenza polymerase. In one of our approaches to treat influenza infection, we designed, prepared, and tested a series of C-nucleoside analogues, which have an analogy to 2 and were expected to act by a similar antiviral mechanism as favipiravir. Compound 3c of this report exhibited potent inhibition of influenza virus replication in MDCK cells, and its triphosphate was a substrate of and demonstrated inhibitory activity against influenza A polymerase. Metabolites of 3c are also presented.

Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme.

Upregulation of neprilysin (NEP) to reduce Aβ accumulation in the brain is a promising strategy for the prevention of Alzheimer’s disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aβ(12–16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aβ(12–16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 μM. Moreover, qf-Aβ(12–16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(1–7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aβ(1–7)C and qf-Aβ(12–16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells.

Schizophrenia-Associated hERG channel Kv11.1-3.1 Exhibits a Unique Trafficking Deficit that is Rescued Through Proteasome Inhibition for High Throughput Screening.

The primate-specific brain voltage-gated potassium channel isoform Kv11.1-3.1 has been identified as a novel therapeutic target for the treatment of schizophrenia. While this ether-a-go-go related K + channel has shown clinical relevance, drug discovery efforts have been hampered due to low and inconsistent activity in cell-based assays. This poor activity is hypothesized to result from poor trafficking via the lack of an intact channel-stabilizing Per-Ant-Sim (PAS) domain. Here we characterize Kv11.1-3.1 cellular localization and show decreased channel expression and cell surface trafficking relative to the PAS-domain containing major isoform, Kv11.1-1A. Using small molecule inhibition of proteasome degradation, cellular expression and plasma membrane trafficking are rescued. These findings implicate the importance of the unfolded-protein response and endoplasmic reticulum associated degradation pathways in the expression and regulation of this schizophrenia risk factor. Utilizing this identified phenomenon, an electrophysiological and high throughput in-vitro fluorescent assay platform has been developed for drug discovery in order to explore a potentially new class of cognitive therapeutics.

Discovery of potent and selective CDK8 inhibitors from an HSP90 pharmacophore.

Here we describe the discovery and optimization of 3-benzylindazoles as potent and selective inhibitors of CDK8, also modulating CDK19, discovered from a high-throughput screening (HTS) campaign sampling the Merck compound collection. The primary hits with strong HSP90 affinity were subsequently optimized to potent and selective CDK8 inhibitors which demonstrate inhibition of WNT pathway activity in cell-based assays. X-ray crystallographic data demonstrated that 3-benzylindazoles occupy the ATP binding site of CDK8 and adopt a Type I binding mode. Medicinal chemistry optimization successfully led to improved potency, physicochemical properties and oral pharmacokinetics. Modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8, was demonstrated in an APC-mutant SW620 human colorectal carcinoma xenograft model following oral administration.

Abstract C4: Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP for treatment of NSCLC

Abstract Background Evasion of cell death is a major cause of resistance to cancer therapy, making proteins that regulate cell death clinically relevant therapeutic targets. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers, including non-small cell lung cancer (NSCLC), and has been shown by us and others to be a major mediator of drug resistance. FLIP and procaspase-8 form complexes with the adaptor protein FADD in response to a variety of clinically relevant stimuli, including ligation of death receptors, such as TRAIL-R1 and R2, and treatment with chemotherapeutic agents. In these complexes, FLIP modulates the activation of procaspase-8, and thereby apoptosis and necroptosis - two major cell death mechanisms. We recently reported that there are important differences between FLIP and procaspase-8 in terms of both their binding affinities and preferred modes of interaction with FADD that are potentially therapeutically exploitable [1]. We now report our subsequent work leading to the development and pre-clinical characterisation of first-in-class inhibitors of FLIP. Methods Molecular modelling of the FLIP-FADD complex; virtual small molecule library screening; cell-free screening assays; cell-based activity assays; biophysical binding assays; in vivo anti-tumor studies. Results Molecular modelling of the FLIP-FADD complex identified a putative drug-binding pocket on FLIP against which a virtual small-molecule screen was carried out. Subsequent biochemical screening of selected compounds using a FLIP-FADD protein-protein interaction assay identified hits with on-target activity. Medicinal chemistry optimisation of these hits afforded lead and back-up series with nanomolar activity in cell-based assays (i.e. caspase activation, cell death and cell survival), which is in line with their binding affinity in an orthogonal biophysical assay (isothermal calorimetry). The pro-apoptotic effects of these FLIP inhibitors were enhanced upon addition of death ligands, such as TRAIL, and lead-molecules have been shown to potentiate the effects of the standard-of-care chemotherapeutic cisplatin. FLIP overexpression and procaspase-8 depletion abrogated the effects of these novel inhibitors consistent with the expected mechanism-of-action. Lead molecules have been identified with ADME profiles suitable for in vivo evaluation. Using these compounds, single-agent anti-tumor effects have been demonstrated in NSCLC xenograft models. Conclusions The novel first-in-class inhibitors of FLIP developed in this study have the potential for broad application in treatment of NSCLC, either as monotherapy or in combination with other agents. Acknowledgements This work was supported by a Seeding Drug Discovery award from the Wellcome Trust (reference: 099470). 1. Majkut, J., et al., Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly. Nat Commun, 2014. 5: p. 3350. Citation Format: Joanna Majkut, Catherine Higgins, Adnan Malik, Zsusannah Nemeth, Peter Blurton, Ray J. Boffey, Trevor R. Perrior, David Haigh, Timothy Harrison, Daniel B. Longley. Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP for treatment of NSCLC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C4.

The FDA-approved natural product dihydroergocristine reduces the production of the Alzheimer's disease amyloid-β peptides.

Known γ-secretase inhibitors or modulators display an undesirable pharmacokinetic profile and toxicity and have therefore not been successful in clinical trials for Alzheimer’s disease (AD). So far, no compounds from natural products have been identified as direct inhibitors of γ-secretase. To search for bioactive molecules that can reduce the amount of amyloid-beta peptides (Aβ) and that have better pharmacokinetics and an improved safety profile, we completed a screen of ~400 natural products by using cell-based and cell-free γ-secretase activity assays. We identified dihydroergocristine (DHEC), a component of an FDA- (Food and Drug Administration)-approved drug, to be a direct inhibitor of γ-secretase. Micromolar concentrations of DHEC substantially reduced Aβ levels in different cell types, including a cell line derived from an AD patient. Structure-activity relationship studies implied that the key moiety for inhibiting γ-secretase is the cyclized tripeptide moiety of DHEC. A Surface Plasmon Resonance assay showed that DHEC binds directly to γ-secretase and Nicastrin, with equilibrium dissociation constants (Kd) of 25.7 nM and 9.8 μM, respectively. This study offers DHEC not only as a new chemical moiety for selectively modulating the activity of γ-secretase but also a candidate for drug repositioning in Alzheimer’s disease.

Multiple spatially related pharmacophores define small molecule inhibitors of OLIG2 in glioblastoma.

Transcription factors (TFs) are a major class of protein signaling molecules that play key cellular roles in cancers such as the highly lethal brain cancer—glioblastoma (GBM). However, the development of specific TF inhibitors has proved difficult owing to expansive protein-protein interfaces and the absence of hydrophobic pockets. We uniquely defined the dimerization surface as an expansive parental pharmacophore comprised of several regional daughter pharmacophores. We targeted the OLIG2 TF which is essential for GBM survival and growth, we hypothesized that small molecules able to fit each subpharmacophore would inhibit OLIG2 activation. The most active compound was OLIG2 selective, it entered the brain, and it exhibited potent anti-GBM activity in cell-based assays and in pre-clinical mouse orthotopic models. These data suggest that (1) our multiple pharmacophore approach warrants further investigation, and (2) our most potent compounds merit detailed pharmacodynamic, biophysical, and mechanistic characterization for potential preclinical development as GBM therapeutics.

Shikonin Promotes Skin Cell Proliferation and Inhibits Nuclear Factor-κB Translocation via Proteasome Inhibition In Vitro.

Background:Shikonin is a major active chemical component extracted from Lithospermi Radix, an effective traditional herb in various types of wound healing. Shikonin can accelerate granulomatous tissue formation by the rat cotton pellet method and induce neovascularization in granulomatous tissue. The purpose of the study was to investigate its mechanism of action in human skin cells.Methods:MTS assay was used to measure cell growth. The collagen type I (COL1) mRNA expression and procollagen type I C-peptide (PIP) production were detected by real-time quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Immunofluorescence and western blot analyses were carried out to investigate nuclear factor-κB (NF-κB) signaling pathway. Cell-based proteasome activity assay was used to determine proteasome activity.Results:In this study, we found that 10 μmol/L shikonin stimulated the growth of normal human keratinocytes and 1 μmol/L shikonin promoted growth of human dermal fibroblasts. However, shikonin did not directly induce COL1 mRNA expression and PIP production in dermal fibroblasts in vitro. In addition, 1 μmol/L shikonin inhibited translocation of NF-κB p65 from cytoplasm to nucleus induced by tumor necrosis factor-α stimulation in dermal fibroblasts. Furthermore, shikonin inhibited chymotrypsin-like activity of proteasome and was associated with accumulation of phosphorylated inhibitor κB-α in dermal fibroblasts.Conclusions:These results suggested that shikonin may promote wound healing via its cell growth promoting activity and suppress skin inflammation via inhibitory activity on proteasome. Thus, shikonin may be a potential therapeutic reagent both in wound healing and inflammatory skin diseases.