PK Inhibitors Research Articles

An improved synthetic strategy for the multigram-scale synthesis of DNA–PK inhibitor AZD7648

An improved synthetic strategy for the multigram-scale synthesis of DNA–PK inhibitor AZD7648

Abstract 213: Assessing the role of X-ray radiation in combination with a DNA-PK inhibitor on cellular activity and adhesion using an in vitro HER2-positive breast cancer model

Abstract Introduction: Breast cancer (BC) is the most common cancer for women regardless of ethnicity, and the leading cause of cancer-related deaths in women. Radiation therapy (XRT) plays a vital role in multidisciplinary treatment approaches. Although XRT has been found to reduce breast cancer recurrence and mortality rate, like all other therapeutics, XRT contains some risks. Radiation can increase the mortality rate due to the risk of ischemic heart diseases. As part of an overall effort to understand the interaction between different types of radiations (e.g., radiopharmaceutical therapy (RPT) with β or α-particle emitters) and DNA double-strand break repair (DSB) repair pathways, we observed that impairment of DNA DSB by DNA-PKcs inhibitor (NU7441) fragmented HER2-positive BC spheroids in the presence of XRT. This could be possibly due to the modulation of cellular adhesion protein mediated by DNA-PK inhibition. In this study, we aim to assess the spheroids fragmentations and also measure the cell viability and levels of a cell adhesion protein, β1-integrin in irradiated HER2-positive monolayer cells, to get a mechanistic view for XRT and NU7441 combinatorial action so as to compare with β- or α-emitter RPT. Methods: In brief, NT2.5 BC spheroids were formed in agarose-coated 96-well plates. For both spheroids and monolayer cells, half of the media was replaced with fresh media containing 1 nM-5μM of NU7441 every other day. A colorimetric cell viability kit was used to measure cell viability and western blot analysis was performed to measure the β1-integrin level, which was normalized against β-actin. Results: Microscopic analysis suggested that XRT increased spheroid fragmentations in the presence of NU7441 compared to the XRT alone. Interaction between XRT and NU7441 (6.68% of the total variation, P<0.0001) was found for the cell viability data, where XRT (74.07% of the total variation, P<0.0001) and NU7441 (5.07% of the total variation, P<0.0001) both regulated cell viability on their own. Western blot data suggest that β1-integrin level was reduced for 8 Gy XRT and 5 μM NU7441 combination compared to the no XRT and vehicle combination. Moreover, 2-way ANOVA analysis revealed that XRT plays a role to regulate the β1-integrin level in NT2.5 monolayer cells (57.61% of the total variation, P<0.0001). Conclusion: XRT increased BC spheroids fragmentations when DNA damage was inhibited by a small molecule inhibitor. XRT and NU7441 both regulated NT2.5 cell viability with an interaction between them. Furthermore, XRT reduced β1-integrin level in monolayer NT2.5 cells. The XRT and NU7441 combination may induce fragmentations by decreasing cell viability and adhesion proteins. Future studies assessing the mechanisms of XRT and DNA PK inhibitor in BC spheroids and in vivo are warranted to confirm these results. Citation Format: Mahmud Hasan, Ioanna Liatsou, George Sgouros. Assessing the role of X-ray radiation in combination with a DNA-PK inhibitor on cellular activity and adhesion using an in vitro HER2-positive breast cancer model [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 213.

DNA-PK inhibition plus immune adjuvants promotes CD8 TIL infiltration, neoantigen presentation, and diversifies the tumor-reactive TCRβ repertoire in B16 melanoma

Abstract Despite significant improvements in cancer immunotherapies, enhancing immunogenicity of non-responsive tumors warrants further investigation. A prior drug screen of ~3,000 compounds identified NU7441, a DNA PK inhibitor, as an effective compound that promotes immunogenicity of various melanoma lines in vitro. In this study, we hypothesized that in vivo combination therapy NU7441, STING-L, and CD40 agonist will enhance tumor immunogenicity resulting in both expansion and increased cytotoxic activity of CD8+TCRβ tumor-reactive TILs in B16 melanoma models. Results obtained by flow cytometry demonstrated that combination treatment 1) significantly increased the ratio of CD8/CD4 TILs, 2) expanded several tumor-reactive TCRβ clones, 3) increased granzyme B production and expression of 4-1BB and PD-1, 4) increased ratio of DC/MDSC infiltration, and 5) potentially identified a novel cytotoxic CD8+CD11c+GR-1+ population. Additionally, the expansion of tumor-reactive TCRs was attributed to DNA-PKi’s ability to both expand and diversify the number of neoantigen transcripts resulting in a broader neoantigen expression profile. RNA-seq identified 27 unique neoantigens as potential novel targets in melanoma immunotherapy. TILs isolated from B16 tumors were co-cultured with dendritic cells transfected with tandem-mini genes, each encoding ~10 neoantigens, and T cells were analyzed by flow cytometry to quantify the TCRβ repertoire and functional response to neoantigens. We demonstrate combination treatment with NU7441, STING-L and CD40 agonist enhance antitumor responses through increased myeloid cell infiltration and sensitization of tumor cells to T cell-mediated killing from an expanded CD8+TCRβ repertoire. Supported by R01CA207913 NCI BX004935-01, VA, Merit Award P30CA046934, NCI Leukemia and Lymphoma Society Department of Defense Gates Grubstake University of Colorado Anschutz Medical Campus Cancer League of Colorado

Novel benzothiazole-based dual VEGFR-2/EGFR inhibitors targeting breast and liver cancers: Synthesis, cytotoxic activity, QSAR and molecular docking studies

A novel series of benzothiazole-based derivatives linked to various amino acids and their corresponding ethyl ester analogues were prepared and were initially evaluated for their anticancer activity againstMCF-7 and HepG-2 and were further assessed as VEGFR-2 inhibitors. All the newly synthesized benzothiazole derivatives showed promising cytotoxic activities against the tested cell lines. Derivatives exhibited potent cytotoxic and VEGFR-2 inhibitory activities were then evaluated further as anticancer agents against the resistant MDA-MB-231 and as EGFR inhibitors. The carboxylic acid derivatives 10–12 and their ester analogues 21–23 displayed the highest anticancer activities with IC50 of 0.73–0.89 µM, against MCF-7 and IC50 of 2.54–2.80 µM, against HepG-2; compared to doxorubicin (IC50 = 1.13 and 2.75 µM, respectively); also they showed safety towards the normal cell line, the ethyl ester derivatives 21–23 showed a potent activity against the resistant MDA-MB-231 cell line with IC50 of 5.45–7.28 µM, relative to doxorubicin (IC50 = 7.46 µM) surpassing their carboxylic acid analogues 10–12 (IC50 of 8.88–11.02 µM). Furthermore, the promising derivatives 10–12 and 21–23 displayed promising VEGFR-2 inhibitory activity (IC50 = 0.15–0.19 µM) comparable to that of sorafenib (IC50 = 0.12 µM). Against EGFR, the ethyl ester derivatives 21–23 showed superior inhibitory activity relative to the used reference standard, erlotinib, with IC50 of 0.11–0.16 vs. 0.18 µM, respectively. The QSAR study revealed that the molecular bulkiness and molecular partial charge distribution govern the kinase inhibition potency in this series. Furthermore, the molecular docking study in VEGFR-2 active site showed that the novel synthesized benzothiazole derivatives adopted the common binding pattern of type II PK inhibitors.

A Phase 1 Dose Escalation Study of Dual PI3K and DNA PK Inhibitor, BR101801 in Adult Patients with Advanced Hematologic Malignancies

Background: BR101801 not only blocks the signaling responsible for cell growth caused by PI3K, but also efficiently induces cell cycle arrest and apoptosis through inhibition of DNA-PK activation and stimulates decreasing stability of the oncogenic protein, c-Myc(AACR2020 abstract #655). This phase I study evaluated safety, tolerability, pharmacokinetics and preliminary activity of the BR101801 (PI3Kγ/δ and DNA PK inhibitor) in patients with advanced hematologic malignancies.Method: This is a Phase I, multi-center, open-label, first-in-human study. The Phase Ia (dose escalation) part of the study was designed to determine the maximum tolerated dose (MTD)/recommended dose for expansion (RDE) of BR101801. BR101801 was administered orally once daily in 28-day cycles. The dose escalation part was initiated with a dose titration in the initial cohort, followed by a 3 + 3 design.Results: 11 patients were enrolled and have been treated at 4 dose levels: 50mg, 100mg, 200mg, 325mg and expanded 200mg through fifth cohort escalation. Pathological subtypes include 7 PTCL, 2 DLBCL, 1 MZBL and 1 composite CTCL/MF. 3 females and 8 males have been treated to date. Median age is 58 (range 30-71) and ECOG PS is in the range of 0-1. All patients had taken at least one prior chemotherapy. 10 of total patients have completed at least one cycle except 1 premature drop-out case due to disease progression. First interim analysis after completion of cycle 3 of the last patient of 200mg QD cohort had been conducted, which was to include 5 patients (1 DLBCL and 4 PTCLs). No DLT had been identified in Cohorts 1-3, and 2 patients discontinued the study treatment due to adverse event (G4 thrombocytopenia, not related to IP) and disease progression, respectively. The PK values from multiple dosing range of 50mg to 200mg cohort resulted in an approximate 2.92-fold and 4.97 fold increase in exposure based on Cmax and AUCtau, respectively. BR101801 PK profile showed that the exposure of concentration increased in a dose dependent manner and there was no accumulation observed in the dose range of 50mg to 200mg. 2 DLTs was observed at 325mg QD cohort. The dose was de-escalated to the previous lower dose level (200mg QD) and was expanded to 3 additional patients. The expansion cohort is ongoing at present. 2 of 11 patients had G3 skin reaction and 3 had G3 hepatotoxicites. All adverse effects were manageable and recovered to grade 0-1 upon BR1010801 discontinuation. Total 5 patients have been currently ongoing. For overall tumor response assessment, 4 SDs and 2 PRs have been obsereved.Summary/Conclusion: 200 mg QD of BR101801 was shown to provide target exposure for clinical efficacy with the tolerable and safe profiles. BR101801 was well tolerated and showed preliminary signs of activity in patients with relapsed or refractory hematologic malignancies. The phase Ib/II study of BR101801 is warranted in relapsed/refractory NHL. This study is registered at clinicaltrials.gov identifier NCT04018248. DisclosuresKim: AstraZeneca-KHIDI: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; GI CELL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Hanmi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Boryung: Consultancy, Membership on an entity's Board of Directors or advisory committees; BeyondBIO: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; AstraZeneca/MedImmune: Consultancy, Membership on an entity's Board of Directors or advisory committees. Curran: Servier pharmaceuticals and Amgen: Consultancy. Kim: Boryung pharmaceuticals: Current Employment.

Abstract 328: DNA damage inhibitors with topotecan in a complex spheroid model in PDM and established cell lines

Abstract Many solid tumor treatment regimens include combination chemotherapy. Over thirty-five human tumor cell lines, some recently developed and some well-established, were grown as complex spheroids, tumor cells mixed with HUVEC and MSC, to better model human solid tumors. The complex spheroids were established for 3 days, then the drug(s) and/or investigational agent(s) were added. After 7 days, the experiment was terminated with CellTiterGlo 3D. Cell viability was determined relative to a vehicle treated control and IC50s were calculated from concentration response curves. The response to topotecan was determined over a concentration range achievable in patients alone and in combination with nedisertib (M3814) or VX984, DNA PK inhibitors, M6620 or BAY-1895344, ATR inhibitors, MK-1775, a Wee-1 inhibitor, AZD-1390, an ATM inhibitor or olaparib or talazoparib, PARP inhibitors. The clinical Cmax of topotecan is 0.15 uM and the topotecan concentration range tested was 0.001 to 0.3 uM. Topotecan IC50's spanned more than 2 logs from 0.003 uM to >>0.3 uM. The ATRi inhibitors were tested over the concentration range from 0.01 to 10 uM. The M6620 IC50's covered 2 logs from 0.015 to >>1 uM and the BAY-189-5344 IC50 range was similar. Combinations of topotecan and M6620 produced greater than additive cytotoxicity in Rh28 and Rh30 rhabdomyosarcoma and 328373-195-R-J1 squamous cell carcinoma with >1 log increase in cell killing. BAY-1895344 in combination with topotecan was greater than additive in the COR L88 SCLC line and the 287954-098-R-J1 Ewing sarcoma line. The combination of topotecan and M6620 resulted in approximately additive cell killing in 929823-356-R-J2 squamous carcinoma, and NCI-H196 and NCI-H1618 SCLC with 4-6-fold increase in cell killing. Topotecan along with the ATM inhibitor AZD-1390, produced greater than additive cell killing in the 287954-098-R-J1 Ewing sarcoma line at the lower concentrations of topotecan (0.003 and 0.01 uM) tested. Primarily additive effects of the ATM inhibitor and topotecan were seen in most other cell lines tested. The combination of topotecan and the Wee-1 inhibitor MK1775 was tested in complex spheroids of the same cell lines. Only in the NCI-H196 SCLC line did topotecan and MK1775 produce greater than additive cell killing. There was a 1.5- to 4-fold increase in cell killing with topotecan and MK1775 in the RH28 and Rh30 rhabdomyosarcomas, 929823-356-R-J2 and 328373-195-R-J1 squamous cell carcinomas and the NCI-H1618 SCLC which was additive to sub-additive. The combination of topotecan with the DNA PK inhibitor M3814 produced little or no increase in cell killing (1.2- to 2-fold) compared with topotecan alone in complex spheroids. These findings may provide guidance regarding selection of agent combinations worthy of further investigation. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261200800001E. Citation Format: Beverly A. Teicher, Austin Doyle, John Wright, Charles Kunos, Thomas Silvers, Rene Delosh, Julie Laudeman, Russell Reinhart, Chad Ogle, Nathan Coussens, Joel Morris, James Doroshow. DNA damage inhibitors with topotecan in a complex spheroid model in PDM and established cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 328.

Individualized screening trial of innovative glioblastoma therapy (INSIGhT).

TPS2079 Background: Patient with glioblastoma (GBM) with unmethylated MGMT promoters derive limited benefit from temozolomide (TMZ) and have dismal outcome. Prioritizing the numerous available therapies and biomarkers for late stage testing requires an efficient clinical testing platform. INSIGhT (NCT02977780) is a biomarker-based, Bayesian adaptively randomized, multi-arm phase II platform screening trial for patients with newly diagnosed GBM and unmethylated MGMT promoters. Methods: INSIGhT compares experimental arms to a common control of standard concurrent TMZ and radiation therapy (RT) followed by adjuvant TMZ. The primary endpoint is overall survival (OS). Patients with newly diagnosed, unmethylated GBM that are IDH R132H mutation negative, and with genomic data available or who consent to whole exome sequencing through the ALLELE companion study for biomarker grouping are eligible. Two experimental arms consist of concurrent RT/TMZ followed by adjuvant neratinib (EGFR, HER2, and HER4 inhibitor) or abemaciclib CDK 4/6 inhibitor), respectively, in place of TMZ. The other experimental arm is CC-115 (TORC1/2 and DNA PK inhibitor), which replaces TMZ in both the concurrent and adjuvant phases. Biomarker groups include: EGFR + patients with EGFR amplification/mutation; PI3K + patients with PIK3CA mutation/amplification, PIK3R1 mutation, AKT3amplification, PIK3C2B > 1 copy gain, or PTEN dual loss; CDK: + patients with wild-type RB1 and CDK4 amplification, CDK6 amplification, or CDKN2A > 1 copy loss. Given the lack of pretrial biomarker data and the anticipated overlap of the groups, randomization will initially be equal. As the trial progresses, randomization probabilities will be adapted based on the Bayesian estimation of the probability of treatment impact on progression-free survival (PFS). These randomization probabilities can vary among the biomarker groups so predictive biomarkers will be identified and utilized if present. Treatment arms may drop due to low probability of treatment impact on OS, and new arms may be added. Experimental arms are compared only with control and should be thought of as discrete experimental questions, with INSIGhT being open to new investigators with proposed therapeutic hypotheses. Clinical trial information: NCT02977780.

2017 Clinical trials update in new treatments of β‐thalassemia

The underlying basis of β-thalassemia pathology is the diminished β-globin synthesis leading to α-globin accumulation and premature apoptotic destruction of erythroblasts, causing oxidative stress-induced ineffective erythropoiesis, bone marrow hyperplasia, splenomegaly, and increased intestinal iron absorption with progressive iron overload. Better understanding of the molecular mechanisms underlying this disease led to the recognition of new targets with potential therapeutic utility. Agents such as JAK2 inhibitors and TGF-β ligand traps that reduce the ineffective erythropoiesis process are already being tested in clinical trials with promising results. Other agents that aim to reduce oxidative stress (activators of Foxo3, HRI-eIF2aP, Prx2, Hsp70, and PK anti-oxidant systems and inhibitors of HO-1) and to decrease iron overload (hepcidin agonists, erythroferrone inhibitors and exogenous transferrin) are also under experimental investigation. Significant progress has also been made in the area of allogeneic hematopoietic stem cell transplantation with several ongoing clinical trials examining new condition regimens as well as different donor selection and stem cell source options. Gene therapy has reached a critical point and phase 1 clinical trials have recently been launched to examine the effectiveness and especially long term safety. Epigenetic manipulation and genomic editing of the γ- or β-globin gene are novel and promising experimental gene therapy approaches for β-thalassemia giving hope for cure for this chronic disease. This review outlines the key points of the molecular mechanisms underlying β-thalassemia in relation to the development of new therapies and an update is given both at the pre-clinical and clinical level. Am. J. Hematol. 91:1135-1145, 2016. © 2016 Wiley Periodicals, Inc.

GLP-1 Agonists Inhibit ox-LDL Uptake in Macrophages by Activating Protein Kinase A

Oxidized low-density lipoprotein (ox-LDL) uptake by monocytes/macrophages plays a pivotal role in atherogenesis. This study was designed to examine the effect of glucagon-like peptide-1 (GLP-1) agonists on ox-LDL uptake in macrophages. Human primary monocytes/macrophages were incubated with native GLP-1 (nGLP-1) or GLP-1 agonist liraglutide to evaluate their effect on ox-LDL uptake and the expression of scavenger receptors (SRs), such as SR-A, CD36, and lectin-like ox-LDL SR-1, in this process. Our study showed a decrease in ox-LDL uptake and CD36 expression in macrophages treated with nGLP-1 or liraglutide. However, nGLP-1 and liraglutide did not affect the expression of other SRs SR-A and lectin-like ox-LDL SR-1. Simultaneously, there was an increase in the expression of activated protein kinase A (PKA). To examine the role of PKA in the effects of nGLP-1 or liraglutide, we treated macrophages with PK inhibitor (6-22) amide, a PKA inhibitor, followed by treatment with nGLP-1 or liraglutide. Inhibition of PKA activation markedly reversed the effect of nGLP-1 or liraglutide on ox-LDL uptake and enhanced the expression of CD36. Our results suggest that GLP-1 agonism inhibits ox-LDL uptake through PKA/CD36 pathway in macrophages. This study provides a novel insight in the mechanism of foam cell formation and the role by GLP-1 agonists therein.

THU0097 Systematic review of the safety of protein kinase inhibitors in clinical trials in rheumatoid arthritis

BackgroundThe safety profile of the protein kinase inhibitors (PKI) in trials for Rheumatoid Arthritis (RA) is unknownObjectivesTo describe the safety profile of PKI in trials for RA.MethodsSystematic review of all...

Novel Role for Pendrin in Orchestrating Bicarbonate Secretion in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-expressing Airway Serous Cells

In most HCO(3)(-)-secreting epithelial tissues, SLC26 Cl(-)/HCO(3)(-) transporters work in concert with the cystic fibrosis transmembrane conductance regulator (CFTR) to regulate the magnitude and composition of the secreted fluid, a process that is vital for normal tissue function. By contrast, CFTR is regarded as the only exit pathway for HCO(3)(-) in the airways. Here we show that Cl(-)/HCO(3)(-) anion exchange makes a major contribution to transcellular HCO(3)(-) transport in airway serous cells. Real-time measurement of intracellular pH from polarized cultures of human Calu-3 cells demonstrated cAMP/PKA-activated Cl(-)-dependent HCO(3)(-) transport across the luminal membrane via CFTR-dependent coupled Cl(-)/HCO(3)(-) anion exchange. The pharmacological and functional profile of the luminal anion exchanger was consistent with SLC26A4 (pendrin), which was shown to be expressed by quantitative RT-PCR, Western blot, and immunofluorescence. Pendrin-mediated anion exchange activity was confirmed by shRNA pendrin knockdown (KD), which markedly reduced cAMP-activated Cl(-)/HCO(3)(-) exchange. To establish the relative roles of CFTR and pendrin in net HCO(3)(-) secretion, transepithelial liquid secretion rate and liquid pH were measured in wild type, pendrin KD, and CFTR KD cells. cAMP/PKA increased the rate and pH of the secreted fluid. Inhibiting CFTR reduced the rate of liquid secretion but not the pH, whereas decreasing pendrin activity lowered pH with little effect on volume. These results establish that CFTR predominately controls the rate of liquid secretion, whereas pendrin regulates the composition of the secreted fluid and identifies a critical role for this anion exchanger in transcellular HCO(3)(-) secretion in airway serous cells.

1059 POSTER Sensitizing Hemopoietic Malignant Cells to Glucocorticoid Induced Apoptosis by PK Inhibitors

Background: The characterization of molecular alterations in each single tumour is at the basis of personalized anticancer approaches aimed to give each patient the most appropriate therapy. Adaptor/scaffold proteins are the key components of signalling networks involved in the control of cell physiology. In particular, by binding to numerous effector proteins adaptor/scaffold protein Ruk/CIN85 assembles multimeric complexes implicated in control of receptor tyrosine kinase signalling, neuronal and T cells apoptosis, adhesion and invasion. Material and Methods: Using Western-blot analysis, samples of uterine cervix, stomach, skin, thyroid, brain tumours and adjacent normal tissues were analyzed. Results: Increase of Ruk/CIN85 full-length form (p85) expression was revealed in uterine cervix, stomach and skin cancer samples in comparison with corresponding control samples. Down-regulation of p85 was revealed in the majority of thyroid tumour samples in comparison with adjacent normal tissue samples. Using anti-RukS Western-blot analysis, multiple molecular forms of CIN85/Ruk with molecular weight of 140, 130, 85, 70, 56, 50, 40 and 38 kDa were detected. Changes in expression level of some Ruk/CIN85 multiple molecular forms as well as up-regulation of low-molecular mass forms (40 and 34 kDa) were detected in skin cancer samples. Expression level up-regulation of high-molecular (140 and 130 kDa) and low-molecular (40 and 34 kDa) Ruk/CIN85 multiple molecular forms of thyroid tumour, renal tumour and glioblastoma samples in comparison with conditionally normal tissue were revealed. The additional feature of Ruk/CIN85 forms expression patterns in uterine as well as stomach malignancies was the high content of p70, p40 and p34 forms while control tissue samples were characterized by the predominant increase of high molecular forms (p140, p130, p100). Conclusions: The obtained results suggest that changes in the expression level of multiple molecular forms of CIN85/Ruk in tumour samples can lead to the loss of coordinated control of apoptosis and proliferation in the transformed cells. These data will offer new opportunities for the identification and validation of key molecular tumour targets to be exploited for novel therapeutic approaches.

Human Mre11/Human Rad50/Nbs1 and DNA Ligase IIIα/XRCC1 Protein Complexes Act Together in an Alternative Nonhomologous End Joining Pathway

Recent studies have implicated a poorly defined alternative pathway of nonhomologous end joining (alt-NHEJ) in the generation of large deletions and chromosomal translocations that are frequently observed in cancer cells. Here, we describe an interaction between two factors, hMre11/hRad50/Nbs1 (MRN) and DNA ligase IIIα/XRCC1, that have been linked with alt-NHEJ. Expression of DNA ligase IIIα and the association between MRN and DNA ligase IIIα/XRCC1 are altered in cell lines defective in the major NHEJ pathway. Most notably, DNA damage induced the association of these factors in DNA ligase IV-deficient cells. MRN interacts with DNA ligase IIIα/XRCC1, stimulating intermolecular ligation, and together these proteins join incompatible DNA ends in a reaction that mimics alt-NHEJ. Thus, our results provide novel mechanistic insights into the alt-NHEJ pathway that not only contributes to genome instability in cancer cells but may also be a therapeutic target.

Plasma Kallikrein Mediates Retinal Vascular Dysfunction and Induces Retinal Thickening in Diabetic Rats

OBJECTIVEPlasma kallikrein (PK) has been identified in vitreous fluid obtained from individuals with diabetic retinopathy and has been implicated in contributing to retinal vascular dysfunction. In this report, we examined the effects of PK on retinal vascular functions and thickness in diabetic rats.RESEARCH DESIGN AND METHODSWe investigated the effects of a selective PK inhibitor, ASP-440, and C1 inhibitor (C1-INH), the primary physiological inhibitor of PK, on retinal vascular permeability (RVP) and hemodynamics in rats with streptozotocin-induced diabetes. The effect of intravitreal PK injection on retinal thickness was examined by spectral domain optical coherence tomography.RESULTSSystemic continuous administration of ASP-440 for 4 weeks initiated at the time of diabetes onset inhibited RVP by 42% (P = 0.013) and 83% (P < 0.001) at doses of 0.25 and 0.6 mg/kg per day, respectively. Administration of ASP-440 initiated 2 weeks after the onset of diabetes ameliorated both RVP and retinal blood flow abnormalities in diabetic rats measured at 4 weeks’ diabetes duration. Intravitreal injection of C1-INH similarly decreased impaired RVP in rats with 2 weeks’ diabetes duration. Intravitreal injection of PK increased both acute RVP and sustained focal RVP (24 h postinjection) to a greater extent in diabetic rats compared with nondiabetic control rats. Intravitreal injection of PK increased retinal thickness compared with baseline to a greater extent (P = 0.017) in diabetic rats (from 193 ± 10 μm to 223 ± 13 μm) compared with nondiabetic rats (from 182 ± 8 μm to 193 ± 9 μm).CONCLUSIONSThese results show that PK contributes to retinal vascular dysfunctions in diabetic rats and that the combination of diabetes and intravitreal injection of PK in rats induces retinal thickening.

Role of gap junctions and protein kinase A during the development of oocyte maturational competence in Ayu ( Plecoglossus altivelis)

Meiotic resumption in teleost oocytes is induced by a maturation-inducing hormone (MIH). The sensitivity of oocytes to MIH, also known as oocyte maturational competence (OMC), is induced by LH via mechanisms that are not fully understood. A previous study of Ayu ( Plecoglossus altivelis) showed the presence of functional heterologous gap junctions (GJs) between oocytes and their surrounding granulosa cells. The objectives of this study were to determine the role of ovarian GJs and of protein kinase A (PKA) during the acquisition of OMC. We examined the effects of the specific GJ inhibitor carbenoxolone (CBX) and 18α-glycyrrhetinic acid (α-GA) on the LH-(hCG)-dependent acquisition of OMC and on MIH-(17,20β-dihydroxy-4-pregnen-3-one)-dependent meiotic resumption; measured the cAMP content of ovarian follicles during the hCG-dependent acquisition of OMC; and determined the effects of PK activators and inhibitors on hCG-dependent OMC. Production of follicular cAMP increased during the hCG-dependent acquisition of OMC. Both GJ inhibitors and the PKA inhibitor H8-dihydrochloride, but not the PKC inhibitor GF109203X, suppressed the hCG-dependent acquisition of OMC in a dose-dependent manner. The PKA activator forskolin induced OMC with a similar potency to hCG. Unlike previous observations with teleosts where disruption of heterologous GJ either blocks or stimulates meiotic resumption, treatment with GJ inhibitors did not affect MIH-dependent meiotic resumption in maturationally competent follicles of Ayu. These observations suggest that ovarian GJs are essential for LH-dependent acquisition of OMC but not for MIH-dependent meiotic resumption, and that the stimulation of OMC by LH is mediated by cAMP-dependent PKA. They are also consistent with the view that a precise balance between GJ-mediated signals (positive or negative) and oocyte maturational readiness is required for hormonally regulated meiotic resumption.

The Role of Protein Kinase A in Acute Ethanol-Induced Neurobehavioral Actions in Rats

cAMP-dependent protein kinase (PKA) signaling pathways are involved in the regulation of ethanol-induced sedative effects in knockout mouse models. In the present study, we examined the role of PKA on the behavioral action caused by ethanol in Sprague Dawley rats. A loss of righting reflex (LORR) test was used to study the acute sedative effects of intraperitoneally injected ethanol. Rotarod performance was used to study the motor impairment caused by ethanol. Convulsions induced by intracerebroventricular (ICV) N-methyl-d-aspartate (NMDA) were used to evaluate ethanol's effect on NMDA receptors. Western blot analysis was used to assay protein levels for NR1 and phosphoserine 897 on NR1 subnuits. ICV pretreatment with H-9 (a nonspecific PK inhibitor) or KT 5720 (a specific PKA inhibitor) dose-dependently attenuated ethanol-induced sleeping time as assessed by LORR. ICV KT 5720 did not reduce ketamine or pentobarbital-induced sleeping time. Pretreatment with forskolin (an activator of adenylyl cyclase) or chelerythrine (a selective PKC inhibitor) had no effect on ethanol-induced LORR. Ethanol-induced motor impairment was also attenuated after pretreatment with KT 5720. Ethanol significantly inhibited NMDA-induced convulsions; the inhibitory effects of ethanol were reduced by prior ICV KT 5720, which had no significant effects on the levels of phosphoserine 897 on NMDA NR1 subunits in the several brain areas we examined. Our results suggest that the PKA pathway may participate in ethanol-induced neurobehavioral changes and that NMDA receptors may be involved in the PKA regulation of ethanol's actions.

BDNF modulates GABAA receptors microtransplanted from the human epileptic brain to Xenopus oocytes.

Cell membranes isolated from brain tissues, obtained surgically from six patients afflicted with drug-resistant temporal lobe epilepsy and from one nonepileptic patient afflicted with a cerebral oligodendroglioma, were injected into frog oocytes. By using this approach, the oocytes acquire human GABAA receptors, and we have shown previously that the "epileptic receptors" (receptors transplanted from epileptic brains) display a marked run-down during repetitive applications of GABA. It was found that exposure to the neurotrophin BDNF increased the amplitude of the "GABA currents" (currents elicited by GABA) generated by the epileptic receptors and decreased their run-down; both events being blocked by K252A, a neurotrophin tyrosine kinase receptor B inhibitor. These effects of BDNF were not mimicked by nerve growth factor. In contrast, the GABAA receptors transplanted from the nonepileptic human hippocampal uncus (obtained during surgical resection as part of the nontumoral tissue from the oligodendroglioma margins) or receptors expressed by injecting rat recombinant alpha1beta2gamma2 GABAA receptor subunit cDNAs generated GABA currents whose time-course and run-down were not altered by BDNF. Loading the oocytes with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester (BAPTA-AM), or treating them with Rp-8-Br-cAMP, an inhibitor of the cAMP-dependent PKA, did not alter the GABA currents. However, staurosporine (a broad spectrum PK inhibitor), bisindolylmaleimide I (a PKC inhibitor), and U73122 (a phospholipase C inhibitor) blocked the BDNF-induced effects on the epileptic GABA currents. Our results indicate that BDNF potentiates the epileptic GABAA currents and antagonizes their use-dependent run-down, thus strengthening GABAergic inhibition, probably by means of activation of tyrosine kinase receptor B receptors and of both PLC and PKC.

Changes in Bad phosphorylation are correlated with BCR-induced apoptosis of WEHI-231 immature B cells

Signaling through the B cell antigen receptor (BCR) is a key determinant in the regulation of B cell physiology. Depending on additional factors, such as microenvironment and developmental stage, ligation of the BCR can trigger B lymphocyte activation, proliferation, or apoptosis. The regulatory mechanisms determining B cell apoptosis and survival are not completely known. Using the murine B lymphoma cell line WEHI-231 as a model system, we investigated the role of Bad phosphorylation, a pro-apoptotic member of the Bcl-2 family, in anti-IgM mediated apoptosis. For apoptotic analysis we focused in particular on the mitochondrial potential (Δ Ψ m) collapse which has been reported as a rate-limiting step in the BCR-induced cell death of immature B lymphocytes. Bad phosphorylation at serine 112, 136 and 155 was found in WEHI-231 cell control cultures and its hypophosphorylation on the three sites correlated with the appearance of apoptosis when cross-linking surface IgM. Furthermore, treatment of cells with specific PK inhibitors known to be involved in serine phosphorylation of Bad (LY294002 for PI3K and H-89 for PKA) mimiced or enhanced BCR-induced cell death. These results strongly suggest that regulation of Bad phosphorylation plays an active role in mediating anti-IgM-induced apoptosis of immature B cells.

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans.

A multifunctional Ca2+/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca2+/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca2+/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca2+/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca2+/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca2+/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca2+/calmodulin-dependent protein kinase of Candida albicans.

Role of cAMP-PKA-PLC signaling cascade on dopamine-induced PKC-mediated inhibition of renal Na+-K+-ATPase activity

We studied the molecular events set into motion by stimulation of D(1)-like receptors downstream of Na(+)-K(+)-ATPase, while measuring apical-to-basal ouabain-sensitive, amphotericin B-induced increases in short-circuit current in opossum kidney (OK) cells. The D(1)-like receptor agonist SKF-38393 decreased Na(+)-K(+)-ATPase activity (IC(50), 130 nM). This effect was prevented by the D(1)-like receptor antagonist SKF-83566, overnight cholera toxin treatment, the protein kinase A (PKA) antagonist H-89, or the PKC antagonist chelerythrine, but not the mitogen-activated PK inhibitor PD-098059 or phosphatidylinositol 3-kinase inhibitors wortmannin and LY-294002. Dibutyryl cAMP (DBcAMP) and phorbol 12,13-dibutyrate (PDBu) both effectively reduced Na(+)-K(+)-ATPase activity. PKA downregulation abolished the inhibitory effects of SKF-38393 and DBcAMP but not those of PDBu. PKC downregulation abolished inhibition by PDBu, SKF-38393, and DBcAMP. The phospholipase C (PLC) inhibitor U-73122 prevented inhibition by SKF-38393 and DBcAMP. However, DBcAMP increased PLC activity. Although OK cells express both G(s)alpha and G(q/11)alpha proteins, D(1)-like receptors are coupled to G(s)alpha proteins only, as evidenced by studies in cells treated overnight with specific antibodies raised against G(s)alpha and G(q/11)alpha proteins. We conclude that PLC and Na(+)-K(+)-ATPase are effector proteins for PKA and PKC, respectively, after stimulation of D(1)-like receptors coupled to G(s)alpha proteins, in a sequence of events that begins with adenylyl cyclase-PKA system activation followed by PLC-PKC system activation.