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XIAP antagonism measurements in HEK293-XIAP-Caspase-9 xenografts

XIAP antagonism in A375 cells in the presence or absence of TNF-α

Preparation of 2-chloro-1-{6-[(4-fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl}ethan-1-one

Fold induction in secreted cytokine concentrations in PBMC supernatants after IAP antagonist treatment

We describe here the identification and characterization of 2 novel inhibitors of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases. The compounds exhibit selective inhibition of FGFR over the closely related VEGFR2 receptor in cell lines and in vivo. The pharmacologic profile of these inhibitors was defined using a panel of human tumor cell lines characterized for specific mutations, amplifications, or translocations known to activate one of the four FGFR receptor isoforms. This pharmacology defines a profile for inhibitors that are likely to be of use in clinical settings in disease types where FGFR is shown to play an important role. Mol Cancer Ther; 10(9); 1542–52. ©2011 AACR.

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines in vitro, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I–II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. Mol Cancer Ther; 17(7); 1381–91. ©2018 AACR.

Comparison of XIAP activity, cIAP1 activity and selectivity of clinical IAP antagonists

IncuCyte images from neutralization of TNF-α activity with anti-TNF-α experiment in MDA-MB-231 cells

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Additional data from ASTX669 in vivo xenograft studies

Summary Acoustic velocity is one of the key thermodynamic properties that can supplement phase behavior or pressure/volume/temperature (PVT) measurements of pure substances and mixtures. Several important fluid properties are relatively difficult to obtain through traditional measurement techniques, correlations, or equation of state (EOS) models. Acoustic measurements offer a simpler method to obtain some of these properties. In this work, we used an experimental method based on ultrasonic pulse-echo measurements in a high-pressure/high-temperature (HP/HT) cell to estimate acoustic velocity in fluid mixtures. We used this technique to estimate related key PVT parameters (such as compressibility), thereby bridging gaps in essential data. In particular, the effect of dilution with methane (CH4) and carbon dioxide (CO2) at pressures from 15 to 62 MPa and temperatures from 313 to 344 K is studied for two reservoir fluid systems to capture the effect of the gas/oil ratio (GOR) and density variations on measured viscosity and acoustic velocity. Correlative analysis of the acoustic velocity and viscosity data were then performed to develop an empirical correlation that is a function of GOR. Such a correlation can be useful for improving the interpretation of the sonic velocity response and the calibration of viscosity changes when areal fluid properties vary with GOR, especially in disequilibrium systems. In addition, under isothermal conditions, the acoustic velocity of a live oil decreases monotonically with decreasing pressure until the saturation point where the trend is reversed. This observation can also be used as a technique to estimate the saturation pressure of a live oil or as a byproduct of the target experiments. It supplements the classical pressure/volume measurements to determine the bubblepoint pressure.

Chemical Enhanced Oil Recovery (EOR) methods have been implemented in a West Texas fractured carbonate. Due to the partially oil-wet nature of Yates field and slightly viscous oil (5-7 cP), surfactant injection was implemented to alter wettability and polymer was injected in the waterflood area to improve displacement efficiency, respectively. Single well huff-n-puff (HnP) surfactant treatments (late 1980's-today) and well-to-well pilots (1990's-2000's) have increased incremental oil production relative to base decline. Optimum surfactant chemicals were chosen based on laboratory results, reservoir performance, and economic viability. Polymer injection was carried out over a 6 year span (1983-1989) in which 55+ million pounds of polymer was injected; however the interpretation and analysis was complicated due to concurrent drilling, workover activities, and no prior waterflood development.Design parameters key to the surfactant implementation included: surfactant type and concentration, Critical Micelle Concentration (CMC), fluid saturations, oil composition, formation water salinity, fracture intensity, and treatment soak timing. Laboratory experiments included interfacial tension, contact angle, adsorption, fluid phase stability, Amott tests, and coreflooding. Numerical models were developed to help understand the sensitivity of each parameter on EOR performance and guide the design of treatments.Field implementation of surfactant included different surfactant types: anionic, non-ionic, and cationic. HnP treatments were followed by a soak period before returning the well to production and conducting flow back water analysis. Overall, HnP treatments using cationic surfactant resulted in the highest efficiency in terms of barrels of oil per kilogram of surfactant. Well-to-well tests were only conducted with non-ionic surfactants and showed mixed results.Design parameters for polymer injection such as fluid viscosity, concentration, adsorption and molecular weight were determined through coreflooding and fluid viscosity experiments. Two polymer types, high and low molecular weight, were studied and manufactured in-field and used in 200 or more injectors either continuously or alternating with produced water. Polymer injection was not effective in improving displacement efficiency in the water flood area of Yates reservoir and was suspended in 1989.The scale of field implementation and analysis of the impact of chemical injection on oil production in a massive, densely fractured carbonate field has provided valuable insight and learnings for future development and will be discussed. Other chemical EOR methods currently under investigation such as foam and other wettability altering technologies will also be discussed.

We present numerical and experimental investigation on the existence of diffusion-induced compositional variations in a reservoir fluid in the absence of convective mixing. New pressure-decay experimental data are presented for ethane in contact with stock tank oil, and the values of diffusivity and solubility parameters are obtained by using the one-dimensional transient diffusion model and integral-based regression method. We show the variation in oil composition through composition-height measurements at the end of the experiment. Finally, the diffusion model with estimated parameters is used to predict the compositional variations, where comparisons show excellent agreement with the measurements, validating the parameter estimation technique and quantifying the compositional variations in the oil phase caused by diffusion.

Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP–XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate 27 (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423).

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines in vitro, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I–II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. Mol Cancer Ther; 17(7); 1381–91. ©2018 AACR.

XIAP and cIAP1 are members of the inhibitor of apoptosis protein (IAP) family and are key regulators of anti-apoptotic and pro-survival signaling pathways. Overexpression of IAPs occurs in various cancers and has been associated with tumor progression and resistance to treatment. Structure-based drug design (SBDD) guided by structural information from X-ray crystallography, computational studies, and NMR solution conformational analysis was successfully applied to a fragment-derived lead resulting in AT-IAP, a potent, orally bioavailable, dual antagonist of XIAP and cIAP1 and a structurally novel chemical probe for IAP biology.

Summary In terms of experimentation, acoustic velocity can be measured with a high degree of accuracy. Several thermodynamic properties related to acoustic velocity such as density, isothermal compressibility, and heat capacity can be extracted from measured data. In this study, technical improvements are implemented in an effort to develop a technique for fast and reliable determination of fluid properties on the basis of acoustic velocity measurements over an expanded range of pressures. The potential use of this device as a quality-control tool in typical pressure/volume/temperature (PVT) measurements is demonstrated. Baseline measurements matched to published literature verify the suitability of the device. Results of tests on three recombined oil samples containing dissolved gas, with prescribed gas/oil ratios (GORs), and one bitumen sample are presented. A sharp change in the acoustic velocity trend near the gas/liquid-saturation point is evidence of gas evolution during depressurization. Strong attenuation complicates measurement of acoustic velocity on the heavy fluids used in this study. Blending bitumen with a midrange-molecular-weight hydrocarbon mixture enables estimation of the undiluted-fluid acoustic velocity by extrapolation. By use of the measured acoustic velocity data available, a methodology is developed to estimate and quality check measured isothermal compressibility (κT) values. This is especially important for low-compressibility systems. Heat-capacity data for simple alkanes (CH4 to n-C10) and toluene helps to define a reasonable range of heat-capacity ratio (γ) expected for typical reservoir fluids. For the typical values of acoustic velocity encountered in the pressure and temperature range of interest, the isothermal compressibility can be calculated and/or quality checked by use of estimated values of γ. In addition, by use of various data sets and by performing graphical error analysis, we have shown the reasons that the methodology works. Available data for n-decane and n-hexadecane along with measured data for a live oil and numerical work on calibrated data sets in this study are used to develop the methodology.

Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP-XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate 27 (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423).

With a high degree of accuracy, acoustic velocity has been utilized in phase behavior measurements of pure substances and mixtures to supplement other PVT (Pressure-Volume-Temperature) measurements or as a standalone measurement. Several key fluid property parameters are challenging to obtain through traditional measurement techniques, correlations, or equation of state models. In this work, a high-pressure high-temperature (HPHT) tool is utilized to obtain acoustic velocity data, which is correlated with key PVT parameters to bridge gaps in the required data. The experimental method is based on pulse-echo response utilizing ultrasonic acoustic transducers in a HPHT fluid cell. Compression wave travel times are measured within 0.2% and a special mixing mechanism allows for in-situ homogenization of fluids mixtures. Effect of dilution with methane is studied to capture the impact of GOR/density variations on acoustic velocity and viscosity response. Dilution with CO2 is investigated to interpret CO2-oil interactions for EOR projects. An electromagnetic viscometer is used to measure viscosity which is correlated with acoustic velocity values over a range of pressure, temperature and methane/CO2 concentration levels. Experimental acoustic velocity and viscosity data for two fluid systems with varying concentrations of methane, and CO2 at pressures from 15 – 62MPa and temperatures from 313K – 344K are presented. Under isothermal conditions, acoustic velocity of a live oil decreases monotonically with decreasing pressure until the saturation point where the trend is reversed. This observation can also be utilized as a technique to determine saturation pressure, at least as a byproduct of the target experiments, or simply to substitute the isothermal classical pressure-volume measurements. In the current work, we have measured the acoustic velocity, viscosity and density of live oil systems diluted with a gas (methane or CO2) at HTHP conditions. Most importantly, acoustic velocity data are correlated with dynamic viscosity to develop an empirical correlation using the extent of dilution that are relates to density and GOR. Such correlation can be utilized in interpreting the sonic velocity responses and calibration of viscosity changes when areal fluid properties vary with GOR, especially in disequilibrium systems. Novelty of the work includes new acoustic velocity and viscosity data for live reservoir fluids with varying methane/CO2 concentrations. To the author's knowledge, this is the first time an experiment-based empirical correlation is developed to estimate dynamic viscosity from acoustic velocity for fluid mixtures at various temperature, pressure and dilution conditions. When limited viscosity-measurements are available to calibrate fluid viscosities with GOR variations, such correlation can be very useful in establishing the bounds for viscosity, especially in the context of field-scale fluid distributions/reservoir initialization and the interpretation of the acoustic responses.

We describe here the identification and characterization of 2 novel inhibitors of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases. The compounds exhibit selective inhibition of FGFR over the closely related VEGFR2 receptor in cell lines and in vivo. The pharmacologic profile of these inhibitors was defined using a panel of human tumor cell lines characterized for specific mutations, amplifications, or translocations known to activate one of the four FGFR receptor isoforms. This pharmacology defines a profile for inhibitors that are likely to be of use in clinical settings in disease types where FGFR is shown to play an important role. Mol Cancer Ther; 10(9); 1542–52. ©2011 AACR.

Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis and pro-survival signaling pathways whose deregulation is often associated with tumor genesis and tumor growth. IAPs have been proposed as targets for anticancer therapy, and a number of peptidomimetic IAP antagonists have entered clinical trials. Using our fragment-based screening approach, we identified nonpeptidic fragments binding with millimolar affinities to both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP). Structure-based hit optimization together with an analysis of protein-ligand electrostatic potential complementarity allowed us to significantly increase binding affinity of the starting hits. Subsequent optimization gave a potent nonalanine IAP antagonist structurally distinct from all IAP antagonists previously reported. The lead compound had activity in cell-based assays and in a mouse xenograft efficacy model and represents a highly promising start point for further optimization.

Abstract Acoustic velocity can be measured with a high degree of accuracy making it an attractive method for extracting related thermodynamic properties. Technical improvements were made to measure acoustic wave velocities in fluid samples over a range of temperatures, pressures, and fluid properties. The simplicity of this technique relative to conventional PVT measurements makes it a fast and reliable means for obtaining some of the data needed in petroleum engineering calculations. The primary objective in this study is to determine whether the designed apparatus is suitable for measuring compressional wave velocities in fluids commonly encountered in oil and gas operations. Additionally, the potential of the device to be used as a QC tool to accompany conventionally determined PVT properties was also studied. A robust experimental design allows for the determination of acoustic wave velocities at elevated pressures and temperatures and includes the ability to homogenize and recondition samples. For experiments involving heavy, viscous oils - especially those containing dissolved gas - this capability is particularly important. The following main points will be the focus of this study that also includes new data interpretation:Ultrasonic p-wave velocities in two heavy oil samples were measured up to 10,000 psig: A live fluid was diluted with solvent and the acoustic response was measured for two solvent dilution levels (8 and 20 percent by mass). Velocity decreased with increasing solvent concentration. A light hydrocarbon mixture was blended with a bitumen sample in a dilution range of 10 - 60% by mass. Velocity again decreased with increasing solvent concentration.

It has been proven that it is possible to improve the performance of water flooding (WF hereafter) by chemically altering the brine/water composition. There are a number of compounds that can be added for this purpose and CO2 is one of them. Carbonated water flooding (CWF hereafter) is to inject CO2 saturated (or nearly saturated) water into reservoirs as the displacing fluid. CO2 stays in the water phase first and migrates into the oil phase afterwards, without forming an individual CO2 rich phase. Such mass transfer of CO2 from water into oil is substantial due to the fact that under the same pressure and temperature conditions, CO2 is more soluble in oil than in water. With the dissolution of CO2, oil viscosity is reduced, which makes mobility ratio between water and oil more favorable in the contacted zone, and oil volume expands (swelling effects), which increases the relative permeability of oil. Both effects result in improved ultimate recovery over conventional WF. Since miscibility is not sought during the CWF process, it has less restrictive requirements of reservoir conditions and oil types. In addition, ease of separation of CO2 water mixture at the production wells and less gas handling make CWF relatively easier to implement in fields with ongoing WF and/or in future candidates. Moreover, at the end of CWF cycle, it is still possible to implement other tertiary flooding techniques as in the case of conventional WF. CWF is an improved oil recovery (IOR hereafter) method that has been tested in the field [6][15][16][26][35]. Pilot programs and field trials were conducted in 1950's. Lab scale experiments were also active between 60's and 80's. During the past a few decades, however, little work was done on the topic. Literature data in this area are inconclusive in several aspects, for example the large variations of the incremental recovery and experimental conditions that can be represented in terms of dimensionless scaling factors (that control the displacement performance). This paper summarizes the results of a series of CWF experiments conducted using a specifically designed core flooding apparatus at our labs. These experiments consist of the Phase I study of a project designed to obtain a comprehensive understanding of CWF's displacement mechanism as well as the impact of several pre-identified sensitivity parameters, such as injection rate, salinity, etc. Eight sand pack experiments (divided into four sub groups) were completed under the same pressure, temperature, and CO2 saturation level, yet with different injection rates. The main contributions of this study are: Overall, CWF results in better oil recovery than its WF counterpart with improvement ranges from 35% (high speed, 15 PV/day) to 3% (low speed, 1 PV/day) of STOIIP, using a medium viscosity crude oil. Cross-comparison and interpretation of current and historical experimental results in terms of identification of flow regimes utilizing dimensionless numbers, such as gravity number, capillary number, etc. Identification of the optimal flow rate and the range of incremental recovery over WF. Providing new data for the literature.

Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFR) signalling as being key to the molecular pathology of cancer. FGFR is a receptor tyrosine kinase which activates the extracellular signal-regulated kinase / mitogen-activated protein kinase and the protein kinase B / Akt pathways which promote cell growth and survival. Amplification, over-expression or activating mutations of fibroblast growth factor receptors have been associated with bladder tumours, multiple myeloma, hormone-refractory prostate cancer and breast cancer. Multiple lead series of FGFR inhibitors were developed using Astex's fragment based medicinal chemistry approach, Pyramid™, linked to high throughput X-ray Crystallography. We describe here the characterisation of some examples of these lead molecules. In particular we detail the pharmacological profile of a compound from one of these lead series that demonstrated activity against FGFR 1-4 with an IC50 10 fold lower in cell lines lacking FGFR expression. We demonstrate inhibition of FGFR 2 and 3 phosphorylation in gastric and multiple myeloma cell lines respectively with associated inhibition of downstream signalling pathways. This lead molecule has an excellent pharmacokinetic profile and high oral bioavailibility in mice and rats. In xenograft models in mice where aberrant FGF signalling underlies tumour pathology, tumour growth inhibition is observed at doses of 100mg/kg /day orally for 21 days. This xenograft efficacy was observed in several models, with significantly lower activity in models where aberrant FGF signalling is not involved in tumour pathology. This suggests that the mechanism of action is consistent with FGFR inhibition. The pharmacological profile in these models is also distinct from other broader spectrum receptor tyrosine kinase inhibitors. The pre-clinical data shown here suggests that such compounds warrant further investigation pre-clinically and may benefit patients whose disease is driven by FGFR activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3626.

Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFr) signalling as being key to the molecular pathology of cancer. This poster will describe fragment based drug discovery using biophysical screening to identify initial fragments. Subsequently, in the fragments-to-leads stage a detailed structural understanding of the binding interactions between the fragment and its target protein utilised X-ray crystallography and NMR. Starting with different fragments allows several lead series to be identified, often by synthesizing only small numbers of compounds. A fragment screening campaign was conducted against the FGFr-1 to detect very low molecular weight compounds that bound to the hinge region of the kinase. The screening produced several fragment molecules (Molecular Weight This poster represents first disclosure of the structure of the lead series and illustrates how a fragment-based drug discovery approach can be efficiently used to discover compounds advanced nanomolar compounds with oral bioavailability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5778.

Among important surgical conditions, none has been given more thought and attention, in recent years, than acute intestinal obstruction. Its frequency and persistently high mortality account for this bad eminence. The work of Wangensteen1and others has stimulated efforts to place the management of this disease on a more scientific basis. The large public hospitals serve as unique clinical laboratories for the investigation of controversial medical and surgical treatments. Each surgeon bases his indications and procedures on his individual judgment and experience. Old and new methods are carried out simultaneously on identical groups of patients. Results of differing forms of treatment may be compared with a considerable degree of accuracy. Study of the literature presents confusing and often paradoxical mortality reports for this disease.2Most of the reports group together all the acute mechanical obstructions. This study is limited to obstructions due to bands and adhesions. By limiting