Amiloride and Other Blockers of Electrolyte Flux As Inhibitors of Progesterone-Stimulated Meiotic Maturation In Xenopus Oocytes

ABSTRACT Amiloride and seven of its analogues, as well as seven substances of other structural types, all of which control electrolyte transport, were tested for their effectiveness for inhibiting the progesterone-stimulated meiotic maturation cycle of oocytes from the amphibian Xenopus luevis. Data were also collected on the ability of the oocytes to recover from the drug's inhibitory effect and on the toxicity of each drug. the data revealed that up to a 14-fold difference exists in the inhibitory concentration 50% between substances, that recovery from the drug's inhibitory effect ranged from non-reversible to almost complete reversibility and that toxicity, as measured by failure to exclude trypan blue, ranged from 0 to 57%. In some, but not all cases, the failure to recover from the drug's inhibitory effects could be correlated to the drug's toxic effects. Amiloride which has been shown to be a reversible inhibitor of cell proliferation in rapidly dividing mammalian cell populations has similar properties on the oocyte maturation division cycle of Xenopus.     

How does the novel T315L mutation of breakpoint cluster region-abelson (BCR-ABL) kinase confer resistance to ponatinib: a comparative molecular dynamics simulation study

Abstract

Acute lymphocytic leukemia (ALL) is one of the most dangerous types of leukemia, and about 40% of them is Philadelphia chromosome-positive acute lymphocytic leukemia (Ph?+?ALL). Ph?+?ALL is caused by the fusion of the breakpoint cluster region (BCR) and the Ableson (ABL) genes, named the BCR-ABL fused gene that codes for an autonomously active tyrosine kinase. Tyrosine kinase inhibitors (TKIs) are among the first-line therapeutic agents for the treatment of Ph?+?ALL. Drug resistance are the major obstacle, limiting their clinical utility. The latest third-generation TKIs, ponatinib, can tackle most abnormal BCR-ABL kinases, including the T315I mutant that is resistant to first- and second-generations TKIs such as imatinib. However, drug resistance still emerges with the novel T315L mutation and the underlying mechanisms remain elusive. Here, using molecular dynamics (MD) simulations, we explored into the detailed interactions between ponatinib and BCR-ABL in the wild-type (WT), T315I, and T315L systems. The simulations revealed the significant conformational changes of ponatinib in its binding site due to the T315L mutation and the underlying structural mechanisms. Binding free energy analysis unveiled that the affinity of ponatinib to BCR-ABL decreased upon T315L mutation, which resulted in its unfavorable binding and drug resistance. Key residues responsible for the unfavored unbinding were also identified. This study elucidates the detailed mechanisms for the resistance of ponatinib in Ph?+?ALL triggered by the T315L mutation and will provide insights for future drug development and optimization.     

Targeting Wild-Type and Mutationally Activated FGFR4 in Rhabdomyosarcoma with the Inhibitor Ponatinib (AP24534)

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC₅₀ in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.     

Structural Mechanisms Determining Inhibition of the Collagen Receptor DDR1 by Selective and Multi-Targeted Type II Kinase Inhibitors

The discoidin domain receptors (DDRs), DDR1 and DDR2, form a unique subfamily of receptor tyrosine kinases that are activated by the binding of triple-helical collagen. Excessive signaling by DDR1 and DDR2 has been linked to the progression of various human diseases, including fibrosis, atherosclerosis and cancer. We report the inhibition of these unusual receptor tyrosine kinases by the multi-targeted cancer drugs imatinib and ponatinib, as well as the selective type II inhibitor DDR1-IN-1. Ponatinib is identified as the more potent molecule, which inhibits DDR1 and DDR2 with an IC₅₀ of 9 nM. Co-crystal structures of human DDR1 reveal a DFG-out conformation (DFG, Asp-Phe-Gly) of the kinase domain that is stabilized by an unusual salt bridge between the activation loop and αD helix. Differences to Abelson kinase (ABL) are observed in the DDR1 P-loop, where a β-hairpin replaces the cage-like structure of ABL. P-loop residues in DDR1 that confer drug resistance in ABL are therefore accommodated outside the ATP pocket. Whereas imatinib and ponatinib bind potently to both the DDR and ABL kinases, the hydrophobic interactions of the ABL P-loop appear poorly satisfied by DDR1-IN-1 suggesting a structural basis for its DDR1 selectivity. Such inhibitors may have applications in clinical indications of DDR1 and DDR2 overexpression or mutation, including lung cancer.     

Kinase profiling of liposarcomas using RNAi and drug screening assays identified druggable targets

Background

Liposarcoma, the most common soft tissue tumor, is understudied cancer, and limited progress has been made in the treatment of metastatic disease. The Achilles heel of cancer often is their kinases that are excellent therapeutic targets. However, very limited knowledge exists of therapeutic critical kinase targets in liposarcoma that could be potentially used in disease management.

Methods

Large RNAi and small-molecule tyrosine kinase inhibitor screens were performed against the proliferative capacity of liposarcoma cell lines of different subtypes. Each small molecule inhibitor was either FDA approved or in a clinical trial.

Results

Screening assays identified several previously unrecognized targets including PTK2 and KIT in liposarcoma. We also observed that ponatinib, multi-targeted tyrosine kinase inhibitor, was the most effective drug with anti-growth effects against all cell lines. In vitro assays showed that ponatinib inhibited the clonogenic proliferation of liposarcoma, and this anti-growth effect was associated with apoptosis and cell cycle arrest at the G0/G1 phase as well as a decrease in the KIT signaling pathway. In addition, ponatinib inhibited in vivo growth of liposarcoma in a xenograft model.

Conclusions

Two large-scale kinase screenings identified novel liposarcoma targets and a FDA-approved inhibitor, ponatinib with clear anti-liposarcoma activity highlighting its potential therapy for treatment of this deadly tumor.

     

Vitamin D and cancer

Vitamin D, a steroid hormone and exerts its biological effects through its active metabolite 1alpha, 25 dihydroxyvitamin D3 [1,25(OH)2D3]. Like steroid hormones, 1,25(OH)2D3 is efficacious at very low concentrations and serves as a ligand for vitamin D receptors (VDR), associating with VDR very high affinity. Despite its potent property as a differentiating agent, its use in the clinical practice is hampered by the induction of hypercalcemia at a concentration required to suppress cancer cell proliferation. Therefore nearly 400 structural analogs of vitamin D3 have been synthesized and evaluated for their efficacy and toxicity. Among these analogs, relatively less toxic but highly efficacious analogs, EB1089, RO24-5531, 1alpha-hydroxyvitamin D5 and a few others have been evaluated in a preclinical toxicity and in Phase I clinical trials for dose tolerance in advanced cancer patients. Clinical trials using vitamin D analogs for prevention or therapy of cancer patients are still in their infancy. Vitamin D mediates its action by two independent pathways. Genomic pathway involves nuclear VDR and induces biological effects by interactions with hormone response elements and modulation of differential gene expressions. Evidence also suggests that vitamin D analogs also interact with steroid hormone(s) inducible genes. The non-genomic pathway is characterized by rapid actions of vitamin D. It involves interactions with membrane-VDR interactions and its interactions with protein kinase C and by altering intracellular calcium channels. Thus, the development of nontoxic analogs of vitamin D analogs and understanding of their molecular mechanism(s) of action are of significant importance in the prevention and treatment of cancer by vitamin D.     

Goldilocks’ Determination of What New In Vivo Data are “Just Right” for Different Common Drug Development Scenarios,Part 1

As alternative models and scientific advancements improve the ability to predict developmental toxicity, the challenge is how to best use this information to support safe use of pharmaceuticals in humans. While in vivo experimental data are often expected, there are other important considerations that drive the impact of developmental toxicity data to human risk assessment and product labeling. These considerations include three key elements: (1) the drug's likelihood of producing off‐target toxicities, (2) risk tolerance of adverse effects based on indication and patient population, and (3) how much is known about the effects of modulating the target in pregnancy and developmental biology. For example, there is little impact or value of a study in pregnant monkeys to inform the risk assessment for a highly specific monoclonal antibody indicated for a life‐threatening indication against a target known to be critical for pregnancy maintenance and fetal survival. In contrast, a small molecule to a novel biological target for a chronic lifestyle indication would warrant more safety data than simply in vitro studies and a literature review. Rather than accounting for innumerable theoretical possibilities surrounding each potential submission's profile, we consolidated most of the typical situations into eight possible scenarios across these three elements, and present a discussion of these scenarios here. We hope that this framework will facilitate a rational approach to determining what new information is required to inform developmental toxicity risk of pharmaceuticals in context of the specific needs of each program while reducing animal use where possible.     

Erythropoietin therapy in patients with chronic renal failure.

Symptomatic anemia is a common complication of chronic renal failure. Treatment is now possible with the availability of recombinant human erythropoietin (epoetin alfa). Previous experimental studies have suggested that correcting the anemia of chronic renal failure may be harmful in that renal failure may be accelerated. Although experience with this drug has been primarily restricted to its use in patients with end-stage renal disease, several recent trials have been reported in patients with varying degrees of chronic renal failure. We review these studies with particular reference to the progression of renal failure and the drug''s reported side effects. We conclude that the use of epoetin is beneficial and well tolerated and that there is no compelling evidence for the acceleration of renal failure associated with its use in patients.     

Immunotherapy of Crohn's disease.

Although the initiating events of Crohn''s disease are unknown, models of experimental colitis have provided new insights in the immunologically mediated pathways of mucosal inflammation. In Crohn''s disease activated mucosal T lymphocytes produce proinflammatory cytokines within the mucosal compartment. With this understanding, there has been a shift in past years from the use of unspecific anti-inflammatory agents (corticosteroids, aminosalicylates) to the use of immunomodulatory drugs (azathioprine, methotrexate). Moreover, novel strategies have been designed for specific targets in Crohn''s disease, in particular T lymphocytes and cytokines. In an open label study treatment of steroid-refractory Crohn''s disease with anti- CD4+ antibodies was well tolerated and showed clinical benefit. However, a sustained depletion of the CD4+ cells precluded further clinical trials. In controlled clinical studies, anti-tumour necrosis factor (TNF-alpha) antibodies induced complete remissions and few side effects were observed. One study suggested efficacy in active Crohn''s disease of recombinant interleukin-10. Long term treatment studies will have to answer questions about the indications for use, benefit and toxicity. Altogether, these results hold promise for future management of Crohn''s disease, where disease-modifying interventions and strategies that effectively maintain disease remission will play a key role.     

The catalytic activity of Abl1 single and compound mutations: Implications for the mechanism of drug resistance mutations in chronic myeloid leukaemia

BackgroundAbl1 is a protein tyrosine kinase whose aberrant activation due to mutations is the culprit of several cancers, most notably chronic myeloid leukaemia. Several Abl1 inhibitors are used as anti-cancer drugs. Unfortunately, drug resistance limits their effectiveness. The main cause for drug resistance is mutations in the kinase domain (KD) of Abl1 that evolve in patients. The T315I mutation confers resistance against all clinically-available inhibitors except ponatinib. Resistance to ponatinib can develop by compound (double) mutations.MethodsKinetic measurements of the KD of Abl1 and its mutants were carried out to examine their catalytic activity. Specifically, mutants that lead to drug resistance against ponatinib were considered. Molecular dynamics simulations and multiple sequence analysis were used for explanation of the experimental findings.ResultsThe catalytic efficiency of the T315I pan-resistance mutant is more than two times lower than that of the native KD. All ponatinib resistant mutations restore the catalytic efficiency of the enzyme. Two of them (G250E/T315I and Y253H/E255V) have a catalytic efficiency that is more than five times that of the native KD.ConclusionsThe measurements and analysis suggest that resistance is at least partially due to the development of a highly efficient kinase through subsequent mutations. The simulations highlight modifications in two structurally important regions of Abl1, the activation and phosphate binding loops, upon mutations.General significanceExperimental and computational methods were used together to explain how mutations in the kinase domain of Abl1 lead to resistance against the most advanced drug currently in use to treat chronic myeloid leukaemia.     

Ponatinib Is a Pan-BCR-ABL Kinase Inhibitor: MD Simulations and SIE Study

BCR-ABL kinase domain inhibition can be used to treat chronic myeloid leukemia. The inhibitors such as imatinib, dasatinib and nilotinib are effective drugs but are resistant to some BCR-ABL mutations. The pan-BCR-ABL kinase inhibitor ponatinib exhibits potent activity against native, T315I, and all other clinically relevant mutants, and showed better inhibition than the previously known inhibitors. We have studied the molecular dynamics simulations and calculated solvated interaction energies of native and fourteen mutant BCR-ABL kinases (M244V, G250E, Q252H, Y253F, Y253H, E255K, E255V, T315A, T315I, F317L, F317V, M351T, F359V and H396P) complexed with ponatinib. These studies revealed that the interactions between ponatinib and individual residues in BCR-ABL kinase are also affected due to the remote residue mutations. We report that some residues, Met244, Lys245, Gln252, Gly254, Leu370 and Leu298 do not undergo any conformational changes, while the fluctuations in residues from P-loop, β3-, β5- strands and αC- helix are mainly responsible for ponatinib binding to native and all mutant BCR-ABL kinases. Our work provides the molecular mechanisms of native and mutant BCR-ABL kinases inhibition by ponatinib at atomic level that has not been studied before.     

Epidermal growth factor receptor: a promising therapeutic target for colorectal cancer

The epidermal growth factor receptor is a 170,000-kd transmembrane glycoprotein involved in signaling pathways affecting cellular growth, differentiation, and proliferation. An abnormal expression of the epidermal growth factor receptor (EGFR) has been described in many human tumors and implicated in the development and prognosis of malignancies, thus representing not only a possible prognostic marker, but primarily a rational molecular target for a new class of anticancer agents. The aim of this analysis is to review the available data about the biology of the EGFR and its use as a target for a new class of anticancer agents for colorectal cancer. Several clinical trials have been reported with the use of EGFR-targeted monoclonal antibodies and tyrosine kinase inhibitors, mainly in combination with chemotherapy for advanced colorectal cancer patients. Results available so far demonstrated a manageable and acceptable toxicity profile and a promising level of activity. Many critical issues are yet unresolved, such as the optimal chemotherapy regimen to combine with anti-EGFR treatment and the most adequate patient setting. Moreover, the biological selection of colorectal tumors more likely to benefit from this treatment approach is still to be defined.     

Drug screening of α-amylase inhibitors as candidates for treating diabetes

In the present study, the identification of potential α-amylase inhibitors is explored as a potential strategy for treating type-2 diabetes mellitus. A computationally driven approach using molecular docking was employed to search for new α-amylase inhibitors. The interactions of potential drugs with the enzyme's active site were investigated and compared with the contacts established by acarbose (a reference drug for α-amylase inhibition) in the crystallographic structure 1B2Y. For this active site characterization, both molecular docking and molecular dynamics simulations were performed, and the residues involved in the α-amylase–acarbose complex were considered to analyse the potential drug's interaction with the enzyme. Two potential α-amylase inhibitors (AN-153I105594 and AN-153I104845) have been selected following this computational strategy. Both compounds established a large number of interactions with key binding site α-amylase amino acids and obtained a comparable docking score concerning the reference drug (acarbose). Aiming to further analyse candidates' properties, their ADME (absorption, distribution, metabolism, excretion) parameters, druglikeness, organ toxicity, toxicological endpoints and median lethal dose (LD₅₀) were estimated. Overall estimations are promising for both candidates, and in silico toxicity predictions suggest that a low toxicity should be expected.     

Evolving concepts in cancer therapy through targeting sphingolipid metabolism

Traditional methods of cancer treatment are limited in their efficacy due to both inherent and acquired factors. Many different studies have shown that the generation of ceramide in response to cytotoxic therapy is generally an important step leading to cell death. Cancer cells employ different methods to both limit ceramide generation and to remove ceramide in order to become resistant to treatment. Furthermore, sphingosine kinase activity, which phosphorylates sphingosine the product of ceramide hydrolysis, has been linked to multidrug resistance, and can act as a strong survival factor. This review will examine several of the most frequently used cancer therapies and their effect on both ceramide generation and the mechanisms employed to remove it. The development and use of inhibitors of sphingosine kinase will be focused upon as an example of how targeting sphingolipid metabolism may provide an effective means to improve treatment response rates and reduce associated treatment toxicity. This article is part of a Special Issue entitled Tools to study lipid functions.     

Prostate cancer diagnostics: Clinical challenges and the ongoing need for disruptive and effective diagnostic tools

The increased incidence and the significant health burden associated with carcinoma of the prostate have led to substantial changes in its diagnosis over the past century. Despite technological advancements, the management of prostate cancer has become progressively more complex and controversial for both early and late-stage disease. The limitations and potential harms associated with the use of prostate-specific antigen (PSA) as a diagnostic marker have stimulated significant investigation of numerous novel biomarkers that demonstrate varying capacities to detect prostate cancer and can decrease unnecessary biopsies. However, only a few of these markers have been approved for specific clinical settings while the others have not been adequately validated for use. This review systematically and critically assesses ongoing issues and emerging challenges in the current state of prostate cancer diagnostic tools and the need for disruptive next generation tools based on analysis of combinations of these biomarkers to enhance predictive accuracy which will benefit clinical diagnostics and patient welfare.     

Improved Efficacy and Reduced Toxicity of Doxorubicin Encapsulated in Sulfatide-Containing Nanoliposome in a Glioma Model

As a glycosphingolipid that can bind to several extracellular matrix proteins, sulfatide has the potential to become an effective targeting agent for tumors overexpressing tenasin-C in their microenvironment. To overcome the dose-limiting toxicity of doxorubicin (DOX), a sulfatide-containing nanoliposome (SCN) encapsulation approach was employed to improve treatment efficacy and reduce side effects of free DOX. This study analysed in vitro characteristics of sulfatide-containing nanoliposomal DOX (SCN-DOX) and assessed its cytotoxicity in vitro, as well as biodistribution, therapeutic efficacy, and systemic toxicity in a human glioblastoma U-118MG xenograft model. SCN-DOX was shown to achieve highest drug to lipid ratio (0.5∶1) and a remarkable in vitro stability. Moreover, DOX encapsulated in SCN was shown to be delivered into the nuclei and displayed prolonged retention over free DOX in U-118MG cells. This simple two-lipid SCN-DOX nanodrug has favourable pharmacokinetic attributes in terms of prolonged circulation time, reduced volume of distribution and enhanced bioavailability in healthy rats. As a result of the improved biodistribution, an enhanced treatment efficacy of SCN-DOX was found in glioma-bearing mice compared to the free drug. Finally, a reduction in the accumulation of DOX in the drug''s principal toxicity organs achieved by SCN-DOX led to the diminished systemic toxicity as evident from the plasma biochemical analyses. Thus, SCN has the potential to be an effective and safer nano-carrier for targeted delivery of therapeutic agents to tumors with elevated expression of tenascin-C in their microenvironment.     

奈达铂的临床应用新进展

奈达铂是铂类第二代抗肿瘤药物,临床研究表明奈达铂对大部分实体瘤,如头颈部肿瘤、食管癌、肺癌、卵巢上皮癌和宫颈癌等有效,其可联合多种化疗药物并具有放疗增敏作用。并可作为新辅助化疗应用在癌症术前治疗中。奈达铂的抗肿瘤作用优于顺铂,并且毒性谱有差异。主要不良反应如恶心呕吐发生率明显低于顺铂,联合放疗治疗中晚期癌症能提高有效率及生存率,明显减轻不良。值得临床进一步推广。依据NDP的临床前研究及临床应用,本文就奈达铂在临床上的应用作一简要综述。     

Preclinical and Clinical Experience with a Doxorubicin-Liposome Preparation

Abstract

Entrapment of doxorubicin in liposomes results in increased drug concentrations in liver and spleen and decreased uptake by the heart muscle. these pharmacologic changes can be exploited to reduce the drug's toxicity and increase its therapeutic index in selected neoplastic conditions. We review here our preclinical and phase I clinical data with liposome-associated doxorubicin. these studies, together with preliminary observations on the pharmacokinetics of the liposome-associated drug and on the imaging of radiolabeled vesicles in patients, suggest that the maximal tolerated dosage is significantly increased over that of the free drug and that the reticuloendothelial system is responsible for the rapid and dominant pathway of liposome clearance. the implications of various pharmacologic aspects of liposome behavior in the circulation are also discussed.     

Recent advances in drug therapy for epilepsy.

Recent advances in drug therapy for epilepsy have contributed to the reduction in the proportion of persons whose epilepsy is uncontrolled. New knowledge of the pharmacokinetics of phenytoin has led to a better understanding of the drug''s bioavailability and uses. Carbamazepine has recently been introduced for the treatment of generalized tonic-clonic and partial seizures. Clonazepam has been found of particular benefit in the treatment of absence and myoclonic seizures. Valproic acid is a promising antiepileptic drug with broad-spectrum activity, and is particularly useful in the treatment of absence and myoclonic seizures, although further clinical experience is required before it can supplant ethosuximide as the preferred drug for the treatment of absence seizures. Monitoring of the plasma concentration of antiepileptic drugs has added greatly to the achievement of optimal drug therapy and the prevention of toxic effects.