Increase In EC50 Research Articles

Toxicity of antimony to Daphnia magna: Influence of environmental factors, development of biotic ligand approach and biochemical response at environmental relevant concentrations

Acute toxicity of antimony pentavalent to neonatal Daphnia magna and the influence of water quality parameters were investigated, and enzymatic activities of organisms at environmentally relevant levels of antimony were determined. EC50 values of antimony to neonatal D. magna were 90.3 and 63.8 mg/L at 24 and 48 h of exposure, respectively. Dissolved organic matter (FA and HA) and cation (Ca2+, Mg2+ or Na+) had significant protective effects on D. magna against antimony toxicity. With increasing pH in the range from 7.4 to 8.5, increase of EC50 were observed due to the competition of OH- on biotic ligands. Based on the biotic ligand model (BLM) concept, stability constants for the binding of Sb(OH)6- and OH- to the biotic ligand were estimated, and the LogKXSb(OH)6- and LogKXOH- were 3.137 and 2.859, respectively. Moreover, antimony exposure in low concentrations significantly increased MDA levels and maybe exert oxidative stress to the organisms. Antimony can also induce toxicity in D. magna by affecting oxidative stress and neurotransmitter systems. The relatively comprehensive toxicological data can contribute to the toxicity prediction and ecological risk assessments of antimony.

PMON148 ACQUIRED TEMOZOLOMIDE RESISTANCE IN CORTICOTROPE PITUITARY TUMOR CELLS

Abstract Introduction Temozolomide (TMZ) treatment is an effective treatment for aggressive pituitary tumors, leading to tumor regression or stabilization of tumor progression in the majority of cases. In cases of disease recurrence, however, TMZ treatment does not control tumour regrowth. The expression of O6-methyl-guanine-DNA-methyltransferase (MGMT) protein and mismatch repair components (MMR) are implicated in the mechanism of TMZ resistance. In the present study, we aimed to develop an in vitro model of acquired TMZ resistance and to describe its mechanism. Methods The mouse AtT20 corticotrope pituitary tumor cell line was used. Two protocols were developed, one based on high dose of TMZ (high TMZ-challenge) and another one using lower TMZ concentrations (low TMZ-challenge) that are clinically achievable after a standard TMZ schedule. Both protocols consisted in pulse exposures to increasing concentrations of TMZ for 24rhs. TMZ concentrations in the high TMZ-challenge were 100, 200 and 300 µM while in the low TMZ-challenge were 20, 40, 60 and 80 µM. AtT20 cells treated with challenges of the vehicle dimethyl sulfoxide (vehicle-challenged cells) were used as control. Results The TMZ challenges induced a significant increase in EC50 (decreased sensitivity) of the growth inhibitory effect of TMZ in TMZ-challenged cells compared to vehicle-challenged cells in both protocols (high TMZ-challenge, EC50 = 123 µM vs 19 µM, p<0.001; low TMZ-challenge, EC50 = 69 µM vs 21 µM, p<0.001). The TMZ-challenged cells remained resistant compared to vehicle-challenged cells even after 9 weeks following the last TMZ challenge. The apoptotic cell death was higher in vehicle-challenged cells compared to TMZ-challenged cells in both protocols, albeit not statistically significant in the low TMZ-challenge protocol (high TMZ-challenge, p=0.003; low TMZ-challenge, p=0.169). TMZ-induced changes in cell cycle phases were more significant in vehicle-challenged cells compared to TMZ-challenged cells. Differences in cell morphology were observed between vehicle- and TMZ-challenged cells in the high dose protocol. In vehicle-challenged cells, small rounded and fusiform-shaped cells were observed, whereas in the TMZ-challenged cells only rounded cells were detected in culture. mRNA expression of MGMT was significantly higher in TMZ-challenged cells compared to vehicle-challenged cells. MMR components expression was not affected by TMZ challenges. Conclusion The present study describes an acquired TMZ-resistant corticotrope pituitary tumor cell model. TMZ resistance was demonstrated in both protocols by a sustained increase in TMZ EC50, small TMZ-induced cell cycle changes, and lower increase in TMZ-induced apoptosis. Additionally, TMZ challenges affected AtT20 cell morphology. This was evident only in the high TMZ-challenge protocol. Acquired TMZ resistance was associated with a strong increase in MGMT expression. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Abstract 2094: Conjugates of TLR9 and STING agonists achieved profound synergistic effects in vitro and in vivo

Abstract Introduction: Both Toll-like receptor 9 (TLR9) and STING pathways are two important pathways involved in immune activation. We reasoned that concurrent activation of TLR9 and STING pathways could activate immunity with an efficacy and safety profile that could not be achieved by activating either pathway alone, and we designed a series of conjugates where TLR9 and STING agonists were linked together by a releasable or stable linker. The conjugates have been evaluated in in vitro assays and in vivo mouse models in comparison with TLR9 agonist or STING agonist alone and combination of the two agonists. Methods: Cellular potency was assessed using THP-1 monocyte cells and human PBMC. In vivo efficacy was evaluated in mouse tumor models, where tumor cells were inoculated into either the right flank or both flanks of C57BL/6 or BALB/c mice, test compounds were injected intratumorally into the right flank three times at every three days when tumors reached a volume of 90-100 mm3, muDX400 was dosed via IP injection six times at every four days. In MC38 murine model, compounds-treated mice free from tumor for 30 days were re-challenged with MC38 tumor cells. Results: Conjugates CS-2554, 2571 and 2600 showed five to ten folds improvement in potency over the corresponding STING agonist alone or a mixture of the corresponding STING and TLR9 agonists (1/1molar ratio) in THP1 assay, a similar fold of increase in EC50 was observed in human PBMC assay, where induction of IFNα, IFNβ, TNFα and IL6 were measured. In MC38 murine model, CS-2554 at the dose of 9 ug/injection resulted in CR in all treated mice; whereas no CR was obtained when a mixture of an equivalent amount of the corresponding STING agonist and TLR9 agonist was used. The tumor-free mice remained tumor-free after re-challenge study. In MC38 dual tumor model, when combined with aPD-1, a mixture of 50 ug of TLR9 agonist and 5 ug of STING agonist resulted in CR in 38% treated mice, whereas the same level was achieved with only 4 ug of the corresponding conjugate CS-2571. Comparisons of a conjugate and either the corresponding agonist alone or the two in combination were also carried out in CT26 dual tumor model. On day 13, for the injected tumor, the tumor growth inhibition rate (TGI) of an equivalent amount of a TLR9 agonist, a STING agonist, a mixture of the two, and the corresponding conjugate CS-2600 was 29.65%, 31.3%, 61.00%, and 91.93%, respectively. For the distal tumor, only the conjugate CS-2600 showed significant inhibition of tumor growth with 57% TGI, whereas no tumor growth inhibition was observed with a TLR9 agonist, a STING agonist, or a mixture of the two. Conclusions: The conjugates harnessed significant synergy between TLR9 and STING pathways in vitro and in vivo and resulted in over ten times enhancement of potency in certain assays over either agent alone or a combination of the two. The preclinical studies of the conjugates support clinical evaluation of this class of novel compounds. Citation Format: Yuntao Song, Anrong Li, Hui Li, Xianfeng Li, Zeao Huang, Junbao Yang, Yi Ding, Grace Szu. Conjugates of TLR9 and STING agonists achieved profound synergistic effects in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2094.

Role of SERCA3-dependent calcium signaling in mouse aortic function

Calcium homeostasis has a major role in vascular reactivity. The Sarco/Endoplasmic Reticulum Ca2+-ATPases (SERCAs) contribute to generate intracellular Ca2+-stores that are mobilized during functional responses. The SERCA2 family is described as the most important in vascular cells, but SERCA3 has been reported to be co-expressed, with SERCA3a mainly at the endothelial level and SERCA3b at the smooth muscle level. Our objective is to evaluate the role of the SERCA3-dependent calcium pathway in vascular reactivity. For experiments, aortic rings were collected from wild-type (WT) and SERCA3-deficient (SERCA3-KO) mice (2–3 month-old male) and mounted in an organ bath system. Contractile and relaxant agents were applied to compare the in vitro aortic reactivity in both animal groups. The contractile responses to a depolarizing high-potassium solution or to cumulative increasing concentrations of serotonin (a 5-HT agonist, 10-9 to 10-5 M) or U46619 (a TXA2 analog, 10-10 to 3.10-6 M) were similar in SERCA3-KO and WT aorta. By contrast the response to phenylephrine (an α1-adrenergic agonist, 10-9 to 3.10-5 M) was significantly decreased in SERCA3-KO compared to WT aorta (N = 8-8; P < 0.05, 2-way ANOVA). However, in aorta denuded from endothelium, the contractile response to phenylephrine was similar in SERCA3-KO and WT vessels. In phenylephrine-precontracted aorta, the relaxant responses to acetylcholine (a muscarinic agonist, 10-9 to 3.10-5 M) or to isoproterenol (a b-adrenergic agonist, 10-10 to 10-5 M) were similar in both groups. The concentration-response curve to DEA-NOate (a NO donor, 10-11 to 10-5 M) was shifted to the right in SERCA3-KO compared to WT aortas, with a slight increase in EC50 (N = 9-8; P < 0.05, t -test). When the endothelium was removed, the effect to DEA-NOate was similar in both groups. We observed that SERCA3-KO aorta exhibit a major decrease in the amplitude of the α1-adrenergic contractile response, and in the sensitivity to the relaxant NO donor. These alterations are abolished in the absence of a functional endothelium. These preliminary data suggest that there is an influence of SERCA3 protein on the NO signaling pathway in mouse aorta.

Differential voltage-dependent modulation of the ACh-gated K+ current by adenosine and acetylcholine.

Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, including the 'relaxation' property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC50 increase with depolarization) and in the IKACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the IKACh relaxation is consequence of this property.

Vasoactive Effects of Acute Ergot Exposure in Sheep

Ergotism is a common and increasing problem in Saskatchewan’s livestock. Chronic exposure to low concentrations of ergot alkaloids is known to cause severe arterial vasoconstriction and gangrene through the activation of adrenergic and serotonergic receptors on vascular smooth muscles. The acute vascular effects of a single oral dose with high-level exposure to ergot alkaloids remain unknown and are examined in this study. This study had two main objectives; the first was to evaluate the role of α1-adrenergic receptors in mediating the acute vasocontractile response after single-dose exposure in sheep. The second was to examine whether terazosin (TE) could abolish the vascular contractile effects of ergot alkaloids. Twelve adult female sheep were randomly placed into control and exposure groups (n = 6/group). Ergot sclerotia were collected and finely ground. The concentrations of six ergot alkaloids (ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, and ergotamine) were determined using HPLC/MS at Prairie Diagnostic Services Inc., (Saskatoon, SK, Canada). Each ewe within the treatment group received a single oral treatment of ground ergot sclerotia at a dose of 600 µg/kg BW (total ergot) while each ewe in the control group received water. Animals were euthanized 12 h after the treatment, and the pedal artery (dorsal metatarsal III artery) from the left hind limb from each animal was carefully dissected and mounted in an isolated tissue bath. The vascular contractile response to phenylephrine (PE) (α1-adrenergic agonist) was compared between the two groups before and after TE (α1-adrenergic antagonist) treatment. Acute exposure to ergot alkaloids resulted in a 38% increase in vascular sensitivity to PE compared to control (Ctl EC50 = 1.74 × 10−6 M; Exp EC50 = 1.079 × 10−6 M, p = 0.046). TE treatment resulted in a significant dose-dependent increase in EC50 in both exposure and control groups (p < 0.05 for all treatments). Surprisingly, TE effect was significantly more pronounced in the ergot exposed group compared to the control group at two of the three concentrations of TE (TE 30 nM, p = 0.36; TE 100 nM, p < 0.001; TE 300 nM, p < 0.001). Similar to chronic exposure, acute exposure to ergot alkaloids results in increased vascular sensitivity to PE. TE is a more potent dose-dependent antagonist for the PE contractile response in sheep exposed to ergot compared to the control group. This study may indicate that the dry gangrene seen in sheep, and likely other species, might be related to the activation of α1-adrenergic receptor. This effect may be reversed using TE, especially at early stages of the disease before cell death occurs. This study may also indicate that acute-single dose exposure scenario may be useful in the study of vascular effects of ergot alkaloids.

Differential impact of various substitutions at codon 715 in region II of HSV-1 and HCMV DNA polymerases

This study aimed at understanding the impact of different substitutions at codon 715 localized in the region II of the palm domain of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) DNA polymerases (pol). Here, we report a new theoretical mutation V715S that confers resistance of HSV-1 to foscarnet/acyclovir (5.6- and 9.2-fold increases EC50 values compared to wild type, respectively) and of HCMV to foscarnet/ganciclovir (2.8- and 2.9-fold increases in EC50 values compared to wild type, respectively). To further analyze the importance of this amino acid, we investigated the impact of the already known mutations V715M and V715G on the replicative capacities and drug susceptibilities of both viruses as well as on the activity and drug inhibition of the DNA pol. The V715G recombinant HSV-1 mutant was resistant to foscarnet and acyclovir (3.4- and 4.6-fold EC50 increase, respectively) whereas the V715M mutant was susceptible to foscarnet and resistant to acyclovir (3.4-fold EC50 increase). The V715G recombinant HCMV mutant did not grow and the V715M mutant was resistant to foscarnet (3.7-fold EC50 increase) and susceptible to ganciclovir. Finally, we showed by three-dimensional modeling that the differential impact of these mutations on the viral replicative capacity and drug resistance profile was related to different hydrophobic local environments for V715 in the DNA pol of the two viruses. Furthermore, we hypothesize that the DNA pol of HSV-1 is more tolerant to changes at this residue compared to that of HCMV because of a more hydrophobic environment stabilizing the region.

In-situ treatment of herbicide-contaminated groundwater–Feasibility study for the cases atrazine and bromacil using two novel nanoremediation-type materials

Two injectable reactive and sorption-active particle types were evaluated for their applicability in permeable reaction zones for in-situ removal of herbicides (“nanoremediation”). As model substances, atrazine and bromacil were used, two herbicides frequently occurring in groundwater. In order to provide recommendations for best use, particle performance was assessed regarding herbicide degradation and detoxification. For chemical reduction, Carbo-Iron® was studied, a composite material consisting of zerovalent iron and colloidal activated carbon. Carbo-Iron reduced bromacil with increased activity compared to nanoscale zerovalent iron (nZVI). The sole reaction product, 3-sec-butyl-6-methyluracil, showed 500-fold increase in half-maximal-effect concentration (EC50) towards the chlorophyte Scendesmus vacuolatus compared to the parent compound. The detoxification based on dehalogenation confirmed the dependency of the specific mode-of-action on the carbon-halide bond. For atrazine, neither nZVI nor Carbo-Iron showed significant degradation under the conditions applied. As novel subsurface treatment option, Trap-Ox® zeolite FeBEA35 was studied for generation of in-situ permeable oxidation barriers. Both adsorbed atrazine and bromacil underwent fast unselective oxidation. The transformation products of the Fenton-like reaction were identified, and oxidation pathways derived. For atrazine, a 300-fold increase in EC50 for S. vacuolatus was found over the duration of the reaction, and a loss of phytotoxicity to non-detectable levels for bromacil.

Systematic review of artesunate pharmacokinetics: Implication for treatment of resistant malaria

BackgroundArtesunate (ART) is an artemisinin derivative used as monotherapy for the treatment of severe malaria and in combination with a partner drug for non-severe malaria. Resistance of malaria parasites to artemisinins have emerged in Southeast Asia. Adjustment of drug regimen may be an option to prevent therapeutic failures considering the relative favourable safety profile of ART high doses. MethodsFor that purpose, a systematic review was done using PubMed, Scopus and Web of Science databases. All studies on ART and DHA pharmacokinetic post-administration of artesunate in human patients or volunteers were included. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist 2009 was used. FindingsFifty studies exploring oral, intravenous, rectal, and intramuscular route (1470 persons, volunteers and patients) were included. Correlations between artesunate doses and Cmax or AUC0-∞ of dihydroartemisinin (DHA) and DHA+ART were evaluated. This correlation was good (R2>0.9) using intravenous (IV) route. DHA and ART+DHA average concentrations (Cav) were well above estimated in vivo half-maximal effective concentration (EC50) for intravenous route, but this was not the case for oral route. InterpretationThe favorable Cav/EC50 ratio for IV route provides evidence that IV ART will remain efficient even in the case of increased resistance level, whereas for the oral route, a two-fold increase in EC50 may lead to therapeutic failures, thus providing a rationale for oral dose escalation. Considering the inter-individual variability of ART pharmacokinetic, Therapeutic Drug Monitoring through antimalarial stewardship activities is needed to optimize drug exposure and avoid resistance development.

A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors

Cys-loop receptors are pentameric ligand-gated ion channels that facilitate communication within the nervous system. Upon neurotransmitter binding, these receptors undergo an allosteric activation mechanism connecting the binding event to the membrane-spanning channel pore, which expands to conduct ions. Some of the earliest steps in this activation mechanism are carried out by residues proximal to the binding site, the relative positioning of which may reflect functional differences among members of the Cys-loop family of receptors. Herein, we investigated key side-chain interactions near the binding site via mutagenesis and two-electrode voltage-clamp electrophysiology in serotonin-gated 5-HT3A receptors (5-HT3ARs) and nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus laevis oocytes. We found that a triad of residues aligning to Thr-152, Glu-209, and Lys-211 in the 5-HT3AR can be exchanged between the homomeric 5-HT3AR and the muscle-type nAChR α-subunit with small functional consequences. Via triple mutant cycle analysis, we demonstrated that this triad forms an interdependent network in the muscle-type nAChR. Furthermore, nAChR-type mutations of the 5-HT3AR affect the affinity of nicotine, a competitive antagonist of 5-HT3ARs, in a cooperative manner. Using mutant cycle analyses between the 5-HT3A triad, loop A residues Asn-101 and Glu-102, β9 residue Lys-197, and the channel gate at Thr-257, we observed that residues in this region are energetically linked to the channel gate and are particularly sensitive to mutations that introduce a net positive charge. This study expands our understanding of the differences and similarities in the activation mechanisms of Cys-loop receptors.

BTKCys481Ser Mutation Drives Ibrutinib Resistance through ERK1/2 Hyperactivation, and Can Confer a Protective Effect on Bystander Waldenstrom's Macroglobulinemia and ABC DLBCL Cells through Paracrine Mediated Pro-Survival Signaling

▪Activating mutations in MYD88 promote NF-kB survival signaling in WM and ABC DLBCL cells through BTK and HCK, both targets of ibrutinib (Yang et al, Blood 2013; 2016). These findings likely explain the high rates of response observed in MYD88 mutated WM and ABC DLBCL patients (Treon et al, NEJM 2015; Wilson et al, Nature Med 2015). Resistance to ibrutinib is increasingly being recognized with prolonged therapy in patients with various B-cell malignancies. Mutations at the BTKCys481 site are observed in half of WM patients with disease progression following initial response (Xu et al, Blood 2017), and are also associated with clinical resistance in patients with CLL and MCL. However, in many WM and CLL cases, BTKCys481 mutations are subclonal and their relevance to clinical disease progression remains unclear. Moreover, the signaling mechanisms that promote ibrutinib resistance remain to be clarified. We therefore performed lentiviral transduction studies in MYD88 mutated WM (BCWM.1, MWCL-1) and ABC DLBCL (TMD-8, HBL-1) cells with vector alone, wild-type BTK (BTKWT), and the BTK variant most frequently observed in ibrutinib resistant WM and CLL cases (BTKCys481Ser). BTKCys481Ser but not BTKWT or vector only transduced WM and ABC DLBCL cells uniformly demonstrated persistent BTK and PLCγ2 activation in the presence of ibrutinib (100, 500 nM), along with a 1-3 log fold increase in EC50 to ibrutinib. Western blot analysis showed persistent BTK signaling in BTKCys481Ser expressing cells that was accompanied by ERK1/2 hyperactivation. Use of highly selective and potent ERK1/2 inhibitors (ulixertinib, GDC-0994) induced apoptosis of BTKCys481Ser expressing WM and ABC DLBCL cells, and showed synergistic tumor cell killing with ibrutinib by CellTiter-Glo® Luminescent Cell Viability Assays using Calcasyn, as well as enhanced apoptosis by Annexin V staining. Using a multiplex cytokine assay and confirmed by TaqMan® quantitative RT-PCR assay for cytokine mRNA levels, we identified that ERK1/2 activation in ibrutinib treated BTKCys481Ser WM and ABC DLBCL cells was accompanied by persistent release of pro-inflammatory and growth enhancing cytokines including CXCL13, IL-2R, IL-6, IL-10, IL-12p40, and TNF-a that was blocked by ulixertinib. In contrast, ibrutinib blocked their release in BTKWT WM and ABC DLBCL cells. We next sought to clarify if cytokine release by ibrutinib treated BTKCys481Ser WM and ABC DLBCL could impact survival of bystander tumor cells. We performed experiments using a Transwell system in which BTKCys481Ser or BTKWT transduced WM or ABC DLBCL cells were co-cultured with their native counterpart cells in the presence or absence of ibrutinib (Figures 1A, B). These studies showed that native WM or ABC DLBCL cells were rescued in the presence of ibrutinib when co-cultured with their BTKCys481Ser but not BTKWT transduced counterparts. Finally, use of IL-6 and IL-10 blocking antibodies abolished the protective effect on native tumor cells conferred by co-culture with BTKCys481Ser expressing cells in the presence of ibrutinib (Figures 1C, D).Our findings show that BTKCys481Ser mutation drives ibrutinib resistance through hyperactivation of ERK1/2, and that paracrine mediated ERK1/2 dependent pro-survival signaling by BTKCys481Ser expressing cells can confer a protective effect on bystander Waldenstrom's Macroglobulinemia and ABC DLBCL cells in the presence of ibrutinib. [Display omitted] DisclosuresCastillo:Janssen: Consultancy, Research Funding; Abbvie: Research Funding; Pharmacyclics: Consultancy, Research Funding; Millennium: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

Antiviral Activity of Bictegravir and Cabotegravir against Integrase Inhibitor-Resistant SIVmac239 and HIV-1.

Animal models are essential to study novel antiretroviral drugs, resistance-associated mutations (RAMs), and treatment strategies. Bictegravir (BIC) is a novel potent integrase strand transfer inhibitor (INSTI) that has shown promising results against HIV-1 infection in vitro and in vivo and against clinical isolates with resistance against INSTIs. BIC has a higher genetic barrier to the development of resistance than two clinically approved INSTIs, termed raltegravir and elvitegravir. Another clinically approved INSTI, dolutegravir (DTG) also possesses a high genetic barrier to resistance, while a fourth compound, termed cabotegravir (CAB), is currently in late phases of clinical development. Here we report the susceptibilities of simian immunodeficiency virus (SIV) and HIV-1 integrase (IN) mutants containing various RAMs to BIC, CAB, and DTG. BIC potently inhibited SIV and HIV-1 in single cycle infection with 50% effective concentrations (EC50s) in the low nM range. In single cycle SIV infections, none of the E92Q, T97A, Y143R, or N155H substitutions had a significant effect on susceptibility to BIC (≤4-fold increase in EC50), whereas G118R and R263K conferred ∼14-fold and ∼6-fold increases in EC50, respectively. In both single and multiple rounds of HIV-1 infections, BIC remained active against the Y143R, N155H, R263K, R263K/M50I, and R263K/E138K mutants (≤4-fold increase in EC50). In multiple rounds of infection, the G140S/Q148H combination of substitutions decreased HIV-1 susceptibility to BIC 4.8-fold compared to 16.8- and 7.4-fold for CAB and DTG, respectively. BIC possesses an excellent resistance profile in regard to HIV and SIV and could be useful in nonhuman primate models of HIV infection.

Acquisition of BTK C481S Produces Resistance to Ibrutinib in MYD88 Mutated WM and ABC DLBCL Cells That Is Accompanied By ERK1/2 Hyperactivation, and Is Targeted By the Addition of the ERK1/2 Inhibitor Ulixertinib

Activating mutations in MYD88 mutations trigger BTK (Yang et al, Blood 2013; Wilson et al, Nat. Med. 2015) and HCK (Yang et al, Blood 2016) activation in Waldenström’s macroglobulinemia (WM) and ABC-DLBCL, which activate multiple downstream pro-survival signaling cascades that include NFkB, AKT, and ERK1/2. Ibrutinib targets BTK and HCK, and shows high levels of activity in MYD88 mutated WM and ABC DLBCL. Resistance to ibrutinib has been observed in CLL, MCL, and in WM (Xu et al, submitted) due to acquisition of mutations that impact the binding of ibrutinib to BTK at Cysteine 481, which include the C481S mutation. To explore the functional impact of the BTK C481S mutation on ibrutinib treatment in MYD88 mutated WM and ABC DLBCL cells, we transduced MYD88 L265P mutated BCWM.1 and MWCL-1 WM, and TMD-8 and HBL-1 ABC DLBCL cell lines with either lentiviral vector alone, or lentiviral vectors expressing BTK wild-type (BTK WT) or BTK with C481S (BTK C481S) mutation. Transduction with BTK C481S led to a 1-3 log fold increase in EC50 for ibrutinib versus vector only or BTK WT transduced cells. BTK C481S expressing cells showed hyperphosphorylation of PLCγ2 (Tyr 1217) which is immediately downstream of BTK, and ERK1/2 (Thr 202/Tyr 204) versus vector only or BTK WT cells in all four MYD88 mutated cell lines following ibrutinib (0.1, 0.5 uM) treatment for 2 hrs. In contrast, no changes in the phospho-IKBa (Ser 32), the gatekeeper of NFkB, nor phospho-AKT (Ser 473) or p38MAPK (Thr 180/Tyr 182) were observed between vector only, BTK WT or BTK C481S transduced cells following ibrutinib treatment. Given the hyperactivation of ERK1/2 in BTK C481S transduced cells treated with ibrutinib, we next evaluated the activity of ulixertinib (BVD-523, VRT752271), a highly selective ERK1/2 inhibitor that is currently under clinical investigation. Ulixertinib blocked ERK1/2 activity as evidenced by reduction of phospho-p90RSK (Ser 380) by western blot analysis in BTK C481S transduced BCWM.1 and TMD-8 cells. The combination of ulixertinib with ibrutinib produced higher levels of tumor cell killing than either agent alone, and showed synergistic interactions with combination index (CI) values below 1.0 at pharmacologically relevant concentrations. Our data indicate that acquisition of BTK C481S produces resistance to ibrutinib in MYD88 mutated WM and ABC DLBCL cells that is accompanied by ERK1/2 hyperactivation. The addition of the ERK1/2 inhibitor ulixertinib to ibrutinib at pharmacologically relevant concentrations produced synergistic tumor cell killing in BTK C481S ibrutinib resistant cells. The findings provide rationale for the investigation of ERK1/2 inhibitors in ibrutinib resistant MYD88 driven WM and ABC DLBCL disease mediated by BTK mutations. DisclosuresCastillo:Pharmacyclics: Honoraria; Janssen: Honoraria; Millennium: Research Funding; Biogen: Consultancy; Otsuka: Consultancy; Abbvie: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy.

Human polymorphisms in nicotinic receptors: a functional analysis in iPS-derived dopaminergic neurons.

Tobacco smoking is a public health problem, with ∼5 million deaths per year, representing a heavy burden for many countries. No effective therapeutic strategies are currently available for nicotine addiction, and it is therefore crucial to understand the etiological and pathophysiological factors contributing to this addiction. The neuronal α5 nicotinic acetylcholine receptor (nAChR) subunit is critically involved in nicotine dependence. In particular, the human polymorphism α5D398N corresponds to the strongest correlation with nicotine dependence risk found to date in occidental populations, according to meta-analysis of genome-wide association studies. To understand the specific contribution of this subunit in the context of nicotine addiction, an efficient screening system for native human nAChRs is needed. We have differentiated human induced pluripotent stem (iPS) cells into midbrain dopaminergic (DA) neurons and obtained a comprehensive characterization of these neurons by quantitative RT-PCR. The functional properties of nAChRs expressed in these human DA neurons, with or without the polymorphism in the α5 subunit, were studied with the patch-clamp electrophysiological technique. Our results in human DA neurons carrying the polymorphism in the α5 subunit showed an increase in EC50, indicating that, in the presence of the polymorphism, more nicotine or acetylcholine chloride is necessary to obtain the same effect. This human cell culturing system can now be used in drug discovery approaches to screen for compounds that interact specifically with human native and polymorphic nAChRs.-Deflorio, C., Blanchard, S., Carisì, M. C., Bohl, D., Maskos, U. Human polymorphisms in nicotinic receptors: a functional analysis in iPS-derived dopaminergic neurons.

Cerebrovascular dysfunction following subfailure axial stretch

Cerebral blood vessels are vital to maintaining the health of the brain. Traumatic brain injury (TBI) commonly results in autoregulatory dysfunction and associated failure of cerebral vessels to maintain homeostasis in the brain. While post-injury changes to brain biochemistry are known to contribute to this dysfunction, tissue deformation may also directly alter vascular smooth muscle cell (SMC) function. As a first step toward understanding stretch-induced dysfunction, this study investigates the effect of overstretch on the contractile behavior of SMCs in middle cerebral arteries (MCAs). We hypothesized that vessel function is altered above a threshold of stretch and strain rate.Twenty-four MCAs from Sprague Dawley rats were tested. Following development of basal SMC tone, vessels were subjected to increasing levels of isosmotic extracellular potassium (K+). Samples were then subjected to an axial overstretch of either 1.2*λIV or 1.3*λIV at strain rates of 0.2 or 20s−1. Following overstretch, SMC contractile behavior was measured again, both immediately and 60min after overstretch. Control vessels were subjected to the same protocol but without overstretch. SMC contractile behavior was characterized using both percent contraction (%C) relative to the fully dilated inner diameter and the K+ dose required to evoke the half maximal contractile response (EC50). Control vessels exhibited increased sensitivity to K+ in successive characterization tests, so all effects were quantified relative to the time-matched control response.Samples exhibited the typical biphasic response to extracellular K+, dilating and contracting in response to small and large K+ concentrations, respectively. As hypothesized, axial overstretch altered SMC contractile behavior, as seen in a decrease in %C for sub-maximal contractile K+ doses (p<0.05) and an increase in EC50 (p<0.01), but only for the test group stretched rapidly to 1.3*λIV. While the change in %C was only significantly different immediately after overstretch, the change to EC50 persisted for 60min. These results indicate that deformation can alter SMC contractile behavior and thus potentially play a role in cerebrovascular autoregulatory dysfunction independent of the pathological chemical environment in the brain post-TBI.

Partial Loss of Function of the GHRH Receptor Leads to Mild Growth Hormone Deficiency.

Recessive mutations in GHRHR are associated with severe isolated growth hormone deficiency (IGHD), with a final height in untreated patients of 130 cm ± 10 cm (-7.2 ± 1.6 SDS; males) and 114 ± 0.7 cm (-8.3 ± 0.1 SDS; females). We hypothesized that a consanguineous Pakistani family with IGHD in three siblings (two males, one female) would have mutations in GH1 or GHRHR. Two novel homozygous missense variants [c.11G>A (p.R4Q), c.236C>T (p.P79L)] at conserved residues were identified in all three siblings. Both were absent from control databases, aside from pR4Q appearing once in heterozygous form in the Exome Aggregation Consortium Browser. The brothers were diagnosed with GH deficiency at 9.8 and 6.0 years (height SDS: -2.24 and -1.23, respectively), with a peak GH of 2.9 μg/liter with low IGF-1/IGF binding protein 3. Their sister presented at 16 years with classic GH deficiency (peak GH <0.1 μg/liter, IGF-1 <3.3 mmol/liter) and attained an untreated near-adult height of 144 cm (-3.0 SDS); the tallest untreated patient with GHRHR mutations reported. An unrelated Pakistani female IGHD patient was also compound homozygous. All patients had a small anterior pituitary on magnetic resonance imaging. Functional analysis revealed a 50% reduction in maximal cAMP response to stimulation with GHRH by the p.R4Q/p.P79L double mutant receptor, with a 100-fold increase in EC50. We report the first coexistence of two novel compound homozygous GHRHR variants in two unrelated pedigrees associated with a partial loss of function. Surprisingly, the patients have a relatively mild IGHD phenotype. Analysis revealed that the pP79L mutation is associated with the compromise in function, with the residual partial activity explaining the mild phenotype.

Preclinical Characterization of the Novel HCV NS3 Protease Inhibitor GS-9256

GS-9256 is an inhibitor of HCV NS3 protease with a macrocyclic structure and novel phosphinic acid pharmacophore. Key preclinical properties of GS-9256 including in vitro antiviral activity, cross-resistance and pharmacokinetic properties were investigated in non-human species. In genotype (GT) 1b Huh-luc cells with a replicon encoding luciferase, GS-9256 had a mean 50% effective concentration (EC50) value of 20.0 nM, with minimal cytotoxicity. Antiviral activity was similar in a number of additional GT1b and GT1a replicon cell lines. Similar potency was observed in chimeric replicons encoding the NS3 protease of GT1 clinical isolates. GS-9256 was less active in GT2a replicon cells (14.2-fold increase in EC50). Additive to synergistic in vitro antiviral activity was observed when GS-9256 was combined with other agents including interferon-α, ribavirin, NS5B polymerase inhibitors GS-6620 and tegobuvir, as well as the NS5A inhibitor ledipasvir. GS-9256 retained wild-type activity against all tested NS5B and NS5A inhibitor resistance mutations. GS-9256 was metabolically stable in microsomes and hepatocytes of tested species, including rodents, dogs and humans. GS-9256 had high bioavailability in mice (near 100%) and moderate bioavailability in rats (14%), dogs (21%) and monkeys (14%). Elimination half-lives were approximately 2 h in mice, 0.6 h in rats, 5 h in dogs and 4 h in monkey. A study in bile duct-cannulated rats indicated that the major route of elimination is through biliary excretion of unmetabolized GS-9256. GS-9256 showed a favourable preclinical profile supportive of clinical development for the treatment of chronic HCV infection in GT1 patients.

Involvement of β adrenergic receptors in spasmolytic effect of caulerpine on guinea pig ileum

Previously, we demonstrated that caulerpine has spasmolytic effect on guinea pig ileum. The aim of this study was to investigate pathways of its spasmolytic action. We test caulerpine against phasic contractions induced by carbachol in the circular layer of guinea pig ileum and this alkaloid did not inhibit these contractions, indicating that caulerpine did not interfering with the mobilisation of Ca2+ from intracellular stores. Additionally, the spasmolytic effect of caulerpine did not involve K+ channels. Furthermore, we observed that α2-adrenergic receptors were not involved in the spasmolytic effect of caulerpine, since the relaxation curve induced by caulerpine was not shifted in the presence of yohimbine (α2-adrenergic antagonist). However, in the presence of propranolol (β-adrenergic antagonist), the relaxation curve induced by caulerpine was right-shifted, resulting in a fivefold increase in EC50. Thus, a possible mechanism for the spasmolytic action of caulerpine is the activation of β-adrenergic receptors.

Abstract A183: The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 exerts EB1 expression-dependent antitumor effects on glioblastoma stem-like cells in vitro and in vivo

Abstract Background: BAL101553 is the prodrug of the novel small molecule BAL27862, undergoing clinical evaluation in advanced cancer patients as an i.v. (phase 2a) and oral (phase 1) formulation. BAL27862 binds the colchicine site of tubulin with distinct effects on microtubule organization, resulting in activation of the ‘spindle assembly checkpoint’ and tumor cell death. BAL27862 has broad activity against diverse cancer models, including tumors refractory to conventional treatments. The drug efficiently distributes to tumor and to brain, with cytotoxic effects in glioblastoma (GBM) lines. We have shown that End-binding 1-protein (EB1) overexpression correlates with GBM progression and sensitizes GBM tumors to Vinca alkaloids. Moreover, EB1 is overexpressed in the GBM stem-like cells, GBM6, that display a high tumorigenicity with an infiltrative pattern of migration in vivo. Here, we investigate the activity of BAL27862/BAL101553 on GBM6 stem-like cells according to EB1 expression level in vitro and in orthotopically transplanted nude mice. Material and methods: Effects of BAL27862 on wild-type and EB1-downregulated GBM6 cells were analyzed using sulforhodamin B survival, clonogenic and transwell migration assays. Nude mice were orthotopically grafted with GBM6GFPSh0 (EB1+) and GBM6GFPShEB1 (EB1-) cells. BAL101553 (25mg/kg) or vehicle were administrated i.v. at D30, D33 and D36. Stem cell phenotype characterization, tumor volume and brain invasion were analyzed at D45, D75 and D105. Overall survivals were analyzed. Stem-like cell differentiation was analyzed by flow cytometry and real-time PCR by analyzing stem cell markers (A2B5, CD133), markers of differentiation (GFAP, β-III-tubulin and CNPase) and self-renewal assay. Results: BAL27862 inhibited GBM6 survival after 72h (half maximal effective concentration [EC50] = 20nM) with EB1-downregulation resulting in a ∼2-fold increase in EC50 (40nM). BAL27862 inhibited clonogenicity and cell migration in GBM6 cells, even at a low, non-cytotoxic concentration (6 nM). However, these effects were only detectable with cytotoxic concentrations (≥ 20 nM) in EB1 downregulated GBM6 cells. Just three BAL101553 administrations over a week provoked a mice survival increase of 69 and 32 days (vs. vehicle controls) after GBM6GFPSh0 and GBM6GFPShEB1 tumor grafting, respectively. Analysis of the pattern of invasion, and quantification of fluorescent tumor cells and A2B5+cells in brain, demonstrated that control cells were more invasive than EB1-downregulated cells, and that anti-invasive effects of BAL101553 were more potent in control cells than in EB1-downregulated cells. BAL27862 also acted on stem-like cells by inducing apoptosis and differentiation, as shown by the decrease in self renewal and stem cell markers. Conclusion: BAL27862 is the first tubulin-binding agent to show a strong inhibitory activity against stem-like cell proliferation and invasion. These results support further investigation of BAL101553 for the treatment of GBM patients, with EB1 expression a potential predictive biomarker for drug responders. Financial supports: (SIRIC label) INCa-DGOS-Inserm 6038, A*MIDEX project “Investissements d'Avenir” (No. ANR-11-IDEX-0001-02), the ITMO Cancer AVIESAN as part of the Cancer Plan and Basilea Pharmaceutica. Citation Format: Raphael Berges, Aurélie Tchoghandjian, Stephane Honore, Dominique Figarella-Branger, Felix Bachmann, Heidi Lane, Diane Braguer. The novel tubulin-binding ‘tumor checkpoint controller’ BAL101553 exerts EB1 expression-dependent antitumor effects on glioblastoma stem-like cells in vitro and in vivo. [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 A183.

Molecular simulation assisted identification of Ca(2+) binding residues in TMEM16A.

Calcium-activated chloride channels (CaCCs) play vital roles in a variety of physiological processes. Transmembrane protein 16A (TMEM16A) has been confirmed as the molecular counterpart of CaCCs which greatly pushes the molecular insights of CaCCs forward. However, the detailed mechanism of Ca(2+) binding and activating the channel is still obscure. Here, we utilized a combination of computational and electrophysiological approaches to discern the molecular mechanism by which Ca(2+) regulates the gating of TMEM16A channels. The simulation results show that the first intracellular loop serves as a Ca(2+) binding site including D439, E444 and E447. The experimental results indicate that a novel residue, E447, plays key role in Ca(2+) binding. Compared with WT TMEM16A, E447Y produces a 30-fold increase in EC50 of Ca(2+) activation and leads to a 100-fold increase in Ca(2+) concentrations that is needed to fully activate the channel. The following steered molecular dynamic (SMD) simulation data suggests that the mutations at 447 reduce the Ca(2+) dissociation energy. Our results indicated that both the electrical property and the size of the side-chain at residue 447 have significant effects on Ca(2+) dependent gating of TMEM16A.