μM EC50 Research Articles

Revisiting Coley's Toxins: Immunogenic Cardiolipins from Streptococcus pyogenes.

Coley's toxins, an early and enigmatic form of cancer (immuno)therapy, were based on preparations of Streptococcus pyogenes. As part of a program to explore bacterial metabolites with immunomodulatory potential, S. pyogenes metabolites were assayed in a cell-based immune assay, and a single membrane lipid, 18:1/18:0/18:1/18:0 cardiolipin, was identified. Its activity was profiled in additional cellular assays, which showed it to be an agonist of a TLR2-TLR1 signaling pathway with a 6 μM EC50 and robust TNF-α induction. A synthetic analog with switched acyl chains had no measurable activity in immune assays. The identification of a single immunogenic cardiolipin with a restricted structure-activity profile has implications for immune regulation, cancer immunotherapy, and poststreptococcal autoimmune diseases.

Novel HIV-1 RNA biogenesis inhibitors identified by virtual pharmacophore-based screening

The complex between the Rev protein of HIV-1 and the Rev Recognition Element (RRE) within the virus RNA promotes nuclear export of unspliced or incompletely spliced viral transcripts and is required for virus transmission. Here, we have screened a virtual collection of compounds using a pharmacophore based on the chemical similarity of previously characterized inhibitors to identify new chemical scaffolds blocking the RRE-Rev interaction. The best molecules discovered with this strategy inhibited the complex by binding to the RRE and exhibited substantial antiretroviral activity (between 0.582 and 11.3 μM EC50 values) likely associated to inhibitory actions on viral transcription and Rev function. These results have allowed us to identify structural features required for RRE-Rev inhibition as well as to add new compounds to the pool of possible candidates for developing antiretroviral agents based on blockage of HIV-1 RNA biogenesis.

Synthesis of Pogostone-Inspired Dehydroacetic Acid Derivatives and Their Antifungal Activity against Sclerotinia sclerotiorum (Lib.) de Bary.

In an effort to exploit the natural antifungal pogostone, its simplified scaffold dehydroacetic acid (DHA) was used as a lead compound to semi-synthesize 56 DHA derivatives (I1-48, II, III, and IV1-6). Among them, compound IV4 exhibited the most potent antifungal activity with 11.0 μM EC50 against mycelial growth of Sclerotinia sclerotiorum (Lib.) de Bary whose sclerotia production was also completely suppressed at this concentration. Furthermore, IV4 could completely inhibit infection cushion formation of S. sclerotiorum on rape leaves and achieved a preventive efficacy of 90.2 % at 500 μM, which was on the same level as that of commercial boscalid at 30 μM (88.7 %). The results of physiological and ultrastructural studies indicated that IV4 might disrupt the cell membrane permeability or induce the imbalance of mitochondrial membrane potential homeostasis to exert the antifungal mode of action. Besides, the robust and predicative three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed and discussed herein.

Abstract 6194: A bifunctional inhibitor of PARP and HDAC enzymes with activity in Ewing sarcoma 3D spheroid and metastasis models

Abstract Introduction: Poly(ADP-ribose) polymerase (PARP) plays a major role in DNA repair and PARP inhibitors (PARPi) have shown promise in pre-clinical studies for the treatment of Ewing sarcoma (ES). While a clinical trial using olaparib as a single agent failed to show significant response against ES, combination therapies with PARPi have emerged as an area of interest. Deacetylation of histones, controlled by histone deacetylases (HDACs) is a key regulatory event in DNA repair and inhibition of HDACs has been shown to reduce ES tumor growth in vitro and in vivo. PARP inhibition combined with HDAC inhibition has demonstrated enhanced efficacy in pre-clinical studies in various tumor indications, and a clinical trial of olaparib and vorinostat combination therapy against metastatic breast cancer is currently ongoing. However, combination therapies can be limited in clinical utility due to overlapping toxicities and different pharmacokinetic profiles. Here, we report the efficacy of a novel bifunctional small-molecule compound, kt-3283, designed to have both PARP and HDAC inhibitory activities. Materials and methods: PARP1 and PARP2 activity were measured using Trevigen Universal Colorimetric PARP Assay Kit, BPS Bioscience PARP2 Colorimetric PARP2 Assay Kit, and PARylation assay. HDAC activity was measured using HeLa cell nuclear extracts and a fluorogenic peptide-based biochemical assay. Cell survival EC50s were determined using live cell imaging with an Incucyte® S3 system and CellTiter Glo viability assay. Cell cycle analysis was performed by flow cytometry with propidium iodide staining. DNA damage was investigated by western blot, immunofluorescence, and comet assay. Spheroid assays were performed using the Incucyte® S3 spheroid analysis module and inhibition of metastases was assessed in a PUMA ES mouse model. Results and discussion: Kt-3283 showed potent inhibition of PARP1/2 activity and PAR synthesis with IC50 values comparable to olaparib. Kt-3283 also showed inhibition of HDACs with an IC50 value in the low µM range. Cell survival EC50 values for the compound were also superior to those of olaparib and vorinostat in ES cell lines. Cell cycle and DNA damage analyses indicated S/G2/M cell cycle arrest and strong DNA damage upon treatment with kt-3283 at lower concentration range compared to olaparib and vorinostat. This compound also exhibited potent inhibition of 3D spheroid growth of ES cells with low µM EC50 values, and inhibited metastatic growth in a PUMA mouse model. Conclusion: Kt-3283 shows potent inhibition of PARP1/2 and HDAC activities. It induces S and G2/M cell cycle arrest and DNA damage, and inhibits 3D spheroid growth and metastatic potential of ES cells. Further investigation of this bifunctional single-molecule inhibitor may offer a novel treatment opportunity for ES and other solid tumors with limited responses to PARPi. Citation Format: Sarah Truong, Louise Ramos, Beibei Zhai, Jay Joshi, Fariba Ghaidi, Michael M. Lizardo, Taras Shyp, John Langlands, Dennis Brown, Jeffrey Bacha, Poul Sorensen, Wang Shen, Mads Daugaard. A bifunctional inhibitor of PARP and HDAC enzymes with activity in Ewing sarcoma 3D spheroid and metastasis models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6194.

Life sciences discovery and technology highlights

Life sciences discovery and technology highlights

Drug-like molecules with anti-trypanothione synthetase activity identified by high throughput screening

Trypanothione synthetase (TryS) catalyses the synthesis of N 1 ,N8 -bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC50 from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC50 values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC50 from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC50 against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS’s synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential.

Discovery of Small Molecule Adhesion GPCR Modulators

GPR56 (ADGRG1) is a member of a subset of G protein coupled receptors called adhesion GPCRs (AGPCRs). These GPCRs are unique in their possession of an autoproteolytic extracellular domain that cleaves the receptors as a prerequisite to activation. GPR56 couples to G12/13 family G proteins and we recently discovered that it functions as a platelet collagen receptor that is important for hemostasis. We hypothesize that GPR56 is an early-acting collagen sensor for platelets – enabling them to first recognize blood vessel injury and to initiate the platelet program that leads to repair. Despite being biologically relevant in a number of cellular processes and diseases, there is a distinct lack of available small molecule tool compounds for AGPCRs, including GPR56. We conducted a large-scale high-throughput screen to find agonists and antagonists of GPR56. Over 150,000 small molecules and extracts were screened at UM's Center for Chemical Genomics (CCG) using a cell-based serum response element (SRE) luciferase gene reporter assay in HEK293T cells. Primary screening yielded 47 initial candidate agonists that activated GPR56 in secondary cell-based reporter and biochemical GPCR reconstitution assays. GPR56 reconstitution assays with pure G proteins further revealed that 19 of these compounds caused at least a 5-fold increase over control in G protein GTPγS binding. A notable structural cluster was identified among these molecules with some members exhibiting a 10-fold increase in potency (~30 μM to ~3 μM EC50) and equivalent efficacy compared to the full agonist positive control, a synthetic peptide mimetic of the GPR56 tethered-peptide-agonist. Follow-up GPR56 activation assays investigating the structure-activity relationship (SAR) of these pharmacophores are being pursued in conjunction with orthogonal platelet activity assays. Knowledge of AGPCR mechanisms and pathologies is rapidly emerging. As such, the discovery of high affinity agonists and antagonists will be useful probes to explore their activities in endogenous contexts. These compounds may also serve as leads for the development of GPR56-targeted pro- or anticoagulants.

Synthesis and Functional Characterization of 2-(2,5-Dimethoxyphenyl)-N-(2-fluorobenzyl)ethanamine (25H-NBF) Positional Isomers.

Serotonergic psychedelics, substances exerting their pharmacological action through activation of the serotonin 2A receptor (5-HT2AR), have continuously comprised a substantial fraction of the over 1000 reported New Psychoactive Substances (NPS) so far. Within this category, N-benzyl derived phenethylamines, such as NBOMes and NBFs, have shown to be of particular relevance. As these substances remain incompletely characterized, this study aimed at synthesizing positional isomers of 25H-NBF, with two methoxy groups placed on different positions of the phenyl group of the phenethylamine moiety. These isomers were then functionally characterized in an in vitro bioassay monitoring the recruitment of β-arrestin 2 to the 5-HT2AR through luminescent readout via the NanoBiT technology. The obtained results provide insight into the optimal substitution pattern of the phenyl group of the phenethylamine moiety of N-benzyl derived substances, a feature so far mostly explored in the phenethylamines underived at the N-position. In the employed bioassay, the most potent substances were 24H-NBF (EC50 value of 158 nM), 26H-NBF (397 nM), and 25H-NBF (448 nM), with 23H-NBF, 35H-NBF, and 34H-NBF yielding μM EC50 values. A similar ranking was obtained for the compounds' efficacy: taking as a reference LSD (lysergic acid diethylamide), 24H-, 26H-, and 25H-NBF had an efficacy of 106-107%, followed by 23H-NBF (96.1%), 34H-NBF (75.2%), and 35H-NBF (58.9%). The stronger activity of 24H-, 25H-, and 26H-NBF emphasizes the important role of the methoxy group at position 2 of the phenethylamine moiety for the in vitro functionality of NBF substances.

Glucose 6-Phosphate Dehydrogenase from Trypanosomes: Selectivity for Steroids and Chemical Validation in Bloodstream Trypanosoma brucei.

Glucose 6-phosphate dehydrogenase (G6PDH) fulfills an essential role in cell physiology by catalyzing the production of NADPH+ and of a precursor for the de novo synthesis of ribose 5-phosphate. In trypanosomatids, G6PDH is essential for in vitro proliferation, antioxidant defense and, thereby, drug resistance mechanisms. So far, 16α-brominated epiandrosterone represents the most potent hit targeting trypanosomal G6PDH. Here, we extended the investigations on this important drug target and its inhibition by using a small subset of androstane derivatives. In Trypanosoma cruzi, immunofluorescence revealed a cytoplasmic distribution of G6PDH and the absence of signal in major organelles. Cytochemical assays confirmed parasitic G6PDH as the molecular target of epiandrosterone. Structure-activity analysis for a set of new (dehydro)epiandrosterone derivatives revealed that bromination at position 16α of the cyclopentane moiety yielded more potent T. cruzi G6PDH inhibitors than the corresponding β-substituted analogues. For the 16α brominated compounds, the inclusion of an acetoxy group at position 3 either proved detrimental or enhanced the activity of the epiandrosterone or the dehydroepiandrosterone derivatives, respectively. Most derivatives presented single digit μM EC50 against infective T. brucei and the killing mechanism involved an early thiol-redox unbalance. This data suggests that infective African trypanosomes lack efficient NADPH+-synthesizing pathways, beyond the Pentose Phosphate, to maintain thiol-redox homeostasis.

Patent Highlights April–May 2019

Patent Highlights April–May 2019

Abstract 2623: Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death

Abstract Pediatric brain cancer medulloblastoma standard-of-care results in numerous comorbidities. Medulloblastoma is comprised of four molecular subgroups. Group 3 subgroup patients have the highest relapse rates and 20% survival after standard-of-care. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. We performed analysis of GABR and MYC expression in medulloblastoma tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional ‘druggable’ GABAAR in group 3 cells. Analysis of expression of 763 medulloblastoma tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, β3 and γ2 and 3. There are ~1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2x109 ions/sec. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine (‘KRM-II-08’) binds to the α5-GABAAR (0.8 μM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter (BAD) protein localization. GABR expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis. Citation Format: Laura Kallay, Havva Keskin, Alexandra Ross, Manali Rupji, Olivia A. Moody, Xin Wang, Guanguan Li, Taukir Ahmed, Farjana Rashid, Michael Rajesh Stephen, Kirsten A. Cottrill, Austin Nuckois, Maxwell Xu, Deborah E. Martinson, Frank Tranghese, Yanxin Pei, James M. Cook, Jeanne Kowalski, Michael D. Taylor, Andrew Jenkins, Daniel Pomeranz Krummel, Soma Sengupta. Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2623.

Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death

PurposePediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives.MethodsWe performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional ‘druggable’ GABAAR in group 3 cells.ResultsAnalysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, β3 and γ2 and 3. There are ~ 1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 109 ions/s. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine (‘KRM-II-08’) binds to the α5-GABAAR (0.8 µM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization.ConclusionGABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.

Improvement of Aqueous Solubility of Lapatinib-Derived Analogues: Identification of a Quinolinimine Lead for Human African Trypanosomiasis Drug Development.

Lapatinib, an approved epidermal growth factor receptor inhibitor, was explored as a starting point for the synthesis of new hits against Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). Previous work culminated in 1 (NEU-1953), which was part of a series typically associated with poor aqueous solubility. In this report, we present various medicinal chemistry strategies that were used to increase the aqueous solubility and improve the physicochemical profile without sacrificing antitrypanosomal potency. To rank trypanocidal hits, a new assay (summarized in a cytocidal effective concentration (CEC50)) was established, as part of the lead selection process. Increasing the sp3 carbon content of 1 resulted in 10e (0.19 μM EC50 against T. brucei and 990 μM aqueous solubility). Further chemical exploration of 10e yielded 22a, a trypanocidal quinolinimine (EC50: 0.013 μM; aqueous solubility: 880 μM; and CEC50: 0.18 μM). Compound 22a reduced parasitemia 109 fold in trypanosome-infected mice; it is an advanced lead for HAT drug development.

Small-Molecule Targeting of Musashi RNA-Binding Activity in Acute Myeloid Leukemia

RNA-binding proteins (RBPs) play critical roles in cell homeostasis by controlling gene expression post-transcriptionally, contributing to mRNA processing events (splicing, polyadenylation, localization, stability, export and translation). The involvement of RBPs to tumorigenesis, through genetic perturbation or epigenetic dysregulation, has been found in a variety of human cancers. The RBP MUSASHI-2 (MSI2) contributes to the pathogenesis of a spectrum of solid tumors and hematologic malignancies and predicts a worse clinical outcome in patients with myeloid and acute lymphoblastic leukemia (MDS, AML and ALL). Thus, MSI2 has been proposed as a putative biomarker for diagnosis as well as a potential therapeutic target for AML. However, there are currently no specific inhibitors for MSI.Previous work from our lab reported a Fluorescent Polarization (FP) screen with 6,208 compounds identifying small-molecules with MSI RNA-binding inhibition activity. Here, we characterize Ro 08-2750 (Ro), best FP screen hit, as a MSI RNA-competitive inhibitor. Electrophoresis Mobility Shift Assays (EMSA) demonstrated Ro inhibition of MSI2-RNA complexes formation. MicroScale Thermophoresis (MST) interaction studies showed that the compound interacts with MSI2 full-length and RNA-Recognition Motif 1 (RRM1) with μM affinity and with nearly 20-fold lower KD to an RBP control (SYNCRIP). We obtained the crystal structure of MSI2 RRM1 at 1.7Å and docking and mutagenesis validation confirmed K22, F66, F97 and R100 as crucial binding residues in the RNA-binding pocket. To further prove structure activity relationship, we used two chemical analogs: Ro-OH, an alcohol derivative of the Ro's aldehyde, showed 10-fold reduced activity and Ro-NGF, containing the Ro isoalloxazine scaffold, showed no binding or activity in vitro. Of note, in proliferation assays Ro EC50 was 2.6±0.1 μM in MLL-AF9 bone marrow cells and an average of 8.4±1.1 μM in MOLM13 and K562 human AML cells, whereas RoOH and RoNGF showed 10-fold or >50 μM EC50, respectively. Ro significantly reduced binding of MSI2 to its mRNA targets (such as cMYC, CDKN1A or SMAD3) in an RNA-IP and a direct effect in their protein translation in human leukemia cells. RNA-sequencing of 4h Ro treated MOLM13 and K562 AML cells resulted in gene expression changes that enriched for the gene expression profiling after shRNA mediated depletion of MSI2 in CML-BC and AML cell lines. Ro demonstrated a significant therapeutic index abolishing MLL-AF9+ BM colony formation at concentrations that did not affect the plating efficiency of normal Lin-Sca+cKit+ (LSK) cells. Similarly, Ro demonstrated differential sensitivity in three AML patient samples colony formation compared to normal human CD34+ cord blood cells. Finally, we sought to determine Ro in vivo activity by using an aggressive murine MLL-AF9 murine leukemia model. Acute treatment (4h and 12hr) with 13.75 mg/kg Ro in DMSO reduced c-KIT protein abundance and intracellular c-MYC. Administration of the same Ro dose every 3 days was well tolerated and showed a significant reduction in spleen weights, white blood cell counts and c-MYC levels compared to the controls. These data provide the feasibility that targeting MSI in vivo could have therapeutic efficacy in AML.This study identifies and characterizes Ro 08-2750 as a compound selectively inhibiting the oncogenic RNA-binding activity of MSI in myeloid leukemia. Ro targeting an RRM motif to block RNA activity represents a valuable proof of concept for the general inhibition of these class of RNA regulators. Overall, we provide a framework to identify and test novel RBP inhibitors thus validating this class of proteins as chemically “druggable” novel therapeutic targets in cancer. DisclosuresChodera:Schrödinger: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Use of a Fluorescent Analogue of a HBV Core Protein-Directed Drug To Interrogate an Antiviral Mechanism.

Heteroaryldihydropyrimidines (HAPs) are antiviral small molecules that enhance assembly of HBV core protein (Cp), lead to assembly of empty and defective particles, and suppress viral replication. These core protein allosteric modulators (CpAMs) bind to the pocket at the interface between two Cp dimers and strengthen interdimer interactions. To investigate the CpAM mechanism, we wanted to examine the cellular distributions of Cp and the CpAM itself. For this reason, we developed a fluorescently labeled CpAM, HAP-ALEX. In vitro, HAP-ALEX modulated assembly of purified Cp and at saturating concentrations induced formation of large structures. HAP-ALEX bound capsids and not dimers, making it a capsid-specific molecular tag. HAP-ALEX labeled HBV in transfected cells, with no detectable background with a HAP-insensitive Cp mutant. HAP-ALEX caused redistribution of Cp in a dose-dependent manner consistent with its 0.7 μM EC50, leading to formation of large puncta and an exclusively cytoplasmic distribution. HAP-ALEX colocalized with the redistributed Cp, but large puncta accumulated long before they appeared saturated with the fluorescent CpAM. CpAMs affect HBV assembly and localization; with a fluorescent CpAM both drug and target can be identified.

Prevalence and Prognostic Value of BCL2 and MYC Protein Expression within ABC and GCB Subtypes in Patients with Previously Untreated, Diffuse Large B-Cell Lymphoma: Analysis from the Phase III MAIN Study

Introduction. Diffuse large B-cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma (NHL). Although more than half of patients are cured with standard immunochemotherapy (R-CHOP) in first-line therapy, the disease relapses or is refractory to R-CHOP in ~40% of cases, with limited options for second-line treatment. Molecular characteristics, including Cell-of-Origin (COO), BCL2 expression and concomitant BCL2/MYC expression, contribute to differences in outcome for DLBCL patients (Alizadeh, Nature 2000, Iqbal Clin Cancer Res. 2011, Johnson JCO. 2012). Understanding these molecular risk factors is potentially of value to guide treatment decisions and optimize therapy. Here, we used assays validated for formalin-fixed, paraffin-embedded (FFPE) specimens to retrospectively assess the prevalence and prognostic value of BCL2 and MYC in patients from MAIN, a Phase III trial that evaluated bevacizumab plus R-CHOP in frontline, CD20-positive DLBCL (NCT00486759). This analysis will help assess markers relevant for risk assessment and may guide use of new investigational agents in DLBCL. Methods. Tissue microarrays (TMAs) from FFPE tumor samples were evaluated using immunohistochemistry (IHC) for BCL2 (clone 124 DAKO), and MYC (clone Y69 Epitomics). FFPE cell pellets from 26 NHL cell lines were also stained as above. BCL2 staining was scored on a 0-3 intensity scale; the BCL2 cutoff was determined by the expression level that conferred sensitivity ( Results. Baseline tissue samples were available from 230/787 (29%) patients from MAIN, with baseline characteristics and survival similar to the overall ITT population. TMAs were available for IHC evaluation in 184/230 (80%) samples, 88/184 (48%) of which were BCL2 positive. BCL2-positivity rates in various sub-sets were: 58% of IPI high, poor prognostic group vs. 41% of IPI low, good prognostic group, and 64% of ABC vs. 40% of GCB samples (Figure 1). BCL2 expression appeared to associate with adverse PFS in the GCB subtype (HR adjusted for IPI, 3.89; 95% CI: 1.30-11.68, Figure 2a) but not in the ABC subtype (HR adjusted for IPI: 0.81; 95% CI: 0.3-2.2, Figure 2b). BCL2 mRNA expression or BCL2 gene rearrangements by FISH showed no association with PFS. Thirty-two of 174 (18%) samples evaluated for MYC and BCL2 by IHC were DP (95% CI: 13%-25%). PFS was similar in non-DP and DP group (HR adjusted by IPI, 0.77; 95% CI: 0.40-1.48), but the DP group appeared to have inferior OS (HR adjusted by IPI, 0.45; 95% CI: 0.22-0.93). These data should be interpreted cautiously due to the small number of PFS/OS events. BCL2 IHC, BCL2 rearrangements and MYC IHC data were available for 40 ABC and 59 GCB samples. DP by IHC was observed in 10/40 (25%) ABC subtype and 10/59 (17%) GCB subtype samples. BCL2 translocations were observed in 6/40 (15%) of ABC, vs. 19/59 (32%) of GCB samples. Conclusions. Using validated assays developed for FFPE tissue testing, we determined the prevalence and prognostic value of BCL2 in a frontline DLBCL population enrolled in a clinical trial. BCL2 expression appeared to be higher in the poor prognostic groups (high IPI and ABC), and appeared to be an independent prognostic factor in the GCB subtype. BCL2 expression, DP and COO may identify patients with DLBCL who could benefit from BCL2 inhibition; the assays employed here are currently in use to evaluate BCL2 as a predictive biomarker for the BCL2 inhibitor venetoclax in lymphoma trials. Disclosures Punnoose:Genentech, Inc.: Employment. Off Label Use: This abstract reports on biomarker analyses using samples taken from previously untreated patients with DLBCL who received bevacizumab in combination with R-CHOP in the MAIN study. Peale:Roche: Equity Ownership; Genentech, Inc.: Employment. Lei:Roche: Employment. Sandmann:F. Hoffmann-La Roche AG: Employment, Equity Ownership. Bourgon:Roche: Equity Ownership; Genentech, Inc.: Employment. Szafer-Glusman:Genentech, Inc.: Employment. Do:Genentech, Inc.: Employment. Salles:Celgene Corporation; Roche: Speakers Bureau; Celgene Corporation; Roche and Gilead Sciences: Research Funding; Calistoga Pharmaceuticals, Inc.; Celgene Corporation; Genentech, Inc.; Janssen Pharmaceutica Products, L.P.; Roche: Consultancy. Seymour:Roche: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Other: Travel support, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Incyte: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Infinity: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Phebra: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity9s Board of Directors or advisory committees, Other: Travel support, Research Funding, Speakers Bureau; Genentech, Inc.: Membership on an entity9s Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Trneny:Roche: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Research Funding. Munoz:Roche: Employment, Equity Ownership. Mundt:Roche: Employment.

Structure-activity relationship studies of indole-based compounds as small molecule HIV-1 fusion inhibitors targeting glycoprotein 41.

We previously described indole-containing compounds with the potential to inhibit HIV-1 fusion by targeting the hydrophobic pocket of transmembrane glycoprotein gp41. Here we report optimization and structure–activity relationship studies on the basic scaffold, defining the role of shape, contact surface area, and molecular properties. Thirty new compounds were evaluated in binding, cell–cell fusion, and viral replication assays. Below a 1 μM threshold, correlation between binding and biological activity was diminished, indicating an amphipathic requirement for activity in cells. The most active inhibitor 6j exhibited 0.6 μM binding affinity and 0.2 μM EC50 against cell–cell fusion and live virus replication and was active against T20 resistant strains. Twenty-two compounds with the same connectivity displayed a consensus pose in docking calculations, with rank order matching the biological activity. The work provides insight into requirements for small molecule inhibition of HIV-1 fusion and demonstrates a potent low molecular weight fusion inhibitor.

Amantadine Analogs that Inhibit Mdck Cell Infection by Influenza a with M2(S31N)

A mutation of Ser31 to Asn has become dominant globally in human infections by influenza A since 2005, rendering the virus resistant to the FDA approved prophylactics, amantadine and rimantadine. Attempts to identify alternative M2 blockers have reportedly been futile so far. Here we report that infection of cultured Madin-Darby canine kidney cells by Influenza A/California/04/2009 (H1N1 swine flu), which bears the S31N mutation, is not blocked effectively by amantadine (115 μM EC50) nor rimantadine (56 μM EC50), but is effectively blocked by 11 amantadine variants. EC50s range from 1-37 μM. 8 of these 11 compounds were previously reported to block influenza A with wild type M2. As another control, an H3N2 strain of influenza A with wild type M2 (Strain Victoria from ATCC) was found to be effectively blocked by amantadine (3 μM EC50) and rimantadine, as expected. We suggest that the amantadine variants block viral reproduction by blocking the S31N strain of the M2 proton channel.

Effects of PGE2 in guinea pig colonic myenteric ganglia.

PGE(2) is a proinflammatory mediator that can influence many cell types. This study was conducted to determine whether PGE(2) alters the electrical activity of distal colonic myenteric neurons, because colitis is typically associated with altered motility and changes in neural signaling may be involved. The electrical properties of intact myenteric neurons were evaluated with intracellular microelectrodes. Acute application of PGE(2) elicited a prolonged depolarization in both AH and S neurons with little effect on input resistance or electrical excitability. PGE(2) effects were suppressed by tetrodotoxin (TTX) or neurokinin (NK) receptor antagonists, indicating that PGE(2) acts directly and indirectly to depolarize colonic neurons. PGE(2)-evoked depolarization was concentration dependent (approximately 3 microM EC(50)) and was attenuated by the E prostanoid (EP)1/2 receptor antagonist, AH-6809. When preparations were maintained for 48 h in the presence of the stable PGE(2) analog PGE(2)-ethanolamide (10 microM), neurons exhibited a significant membrane depolarization and enhanced excitability. These results suggest that PGE(2) can play a role in altered motility in colitis by evoking changes in the electrical properties of myenteric neurons.

Effects of adenosine, ATP, and UTP on chloride secretion by epithelia explanted from fetal rat lung.

Catecholamines trigger the switch from liquid secretion to absorption by perinatal lung, but regulation of Cl- and liquid secretion by pulmonary epithelia early in lung development (low [catecholamine]) is unknown. We looked for evidence for P1 and P2 receptors that mediate Cl- secretion in 14-d distal lungs and 14- and 18-d tracheas explanted from fetal rats (term = 22 d). We measured amiloride-insensitive transepithelial voltage changes induced by ATP, UTP, or adenosine. Explants were hyperpolarized by all three agonists and by terbutaline, a beta-adrenergic agonist and Cl- secretagogue. Whereas adenosine, ATP, or UTP injected into 14-d explant lumena, or adenosine added to the tracheal bath, induced hyperpolarization with EC50 of 2-15 microM EC50, values for all three agonists in the distal lung bath or ATP or UTP in the tracheal bath were five times greater. By 18 d, EC50 values for agonists in the bath were comparable to those for lumenal agonists (3-12 microM). In contrast, microinjection of terbutaline into all explant lumena (final concentration = 3 x 10(-5) M) induced minimal hyperpolarization, whereas the same concentration in the bath raised bioelectric potential difference maximally. We conclude that 1) beta-adrenergic receptors are present on the basolateral membranes of cells of the pulmonary epithelium early in lung development, and 2) adenosine, ATP, and UTP receptors are present in apical membranes throughout lung epithelial development, but basolateral receptors for these agonists in distal lung or ATP/UTP in trachea function later in gestation. The putative distribution of P1 and P2 receptors suggests a role for agonists released from pulmonary epithelial cells in the regulation of liquid secretion early in lung development.