ABSTRACT
FLT3 kinase is a potential drug target in acute myeloid leukemia (AML). Patients with FLT3 mutations typically have higher relapse rates and worse outcomes than patients without FLT3 mutations. In this study, we investigated the suitability of various heterocycles as central cores of FLT3 inhibitors, including thieno[3,2-d]pyrimidine, pyrazolo[1,5-a]pyrimidine, imidazo[4,5-b]pyridine, pyrido[4,3-d]pyrimidine, and imidazo[1,2-b]pyridazine. Our assays revealed a series of imidazo[1,2-b]pyridazines with high potency against FLT3. Compound 34f showed nanomolar inhibitory activity against recombinant FLT3-ITD and FLT3-D835Y (IC50 values 4 and 1 nM, respectively) as well as in the FLT3-ITD-positive AML cell lines MV4-11, MOLM-13, and MOLM-13 expressing the FLT3-ITD-D835Y mutant (GI50 values of 7, 9, and 4 nM, respectively). In contrast, FLT3-independent cell lines were much less sensitive. In vitro experiments confirmed suppression of FLT3 downstream signaling pathways. Finally, the treatment of MV4-11 xenograft-bearing mice with 34f at doses of 5 and 10 mg/kg markedly blocked tumor growth without any adverse effects.
Subject(s)
Antineoplastic Agents , Leukemia, Myeloid, Acute , Pyridazines , Humans , Mice , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyridazines/pharmacology , Pyridazines/therapeutic use , Leukemia, Myeloid, Acute/pathology , Pyrimidines/pharmacology , fms-Like Tyrosine Kinase 3/genetics , Mutation , ApoptosisABSTRACT
Mutation of FLT3 protein kinase is often associated with deregulated cell proliferation in acute myeloid leukemia and the inhibition of this kinase is a potential therapeutic strategy. We report a novel series of 3,5,7-trisubstituted pyrazolo[1,5-a]pyrimidines prepared in an effort to study their biological activity particularly toward FLT3-ITD and its downstream regulators as well as toward CDK2 and CDK9. Derivative 10b was capable to strongly inhibit all kinases and its selectivity in FLT3-ITD expressing cell lines MOLM13 and MV4-11 was in line with FLT3-ITD inhibition. Further biochemical analyses and molecular docking confirmed FLT3 as a cellular target of 10b.
Subject(s)
Leukemia, Myeloid, Acute , Pyrimidines , Humans , Cell Line, Tumor , Pyrimidines/chemistry , Molecular Docking Simulation , Protein Kinase Inhibitors/chemistry , Mutation , fms-Like Tyrosine Kinase 3/genetics , Leukemia, Myeloid, Acute/drug therapy , Apoptosis , Cell ProliferationABSTRACT
Considering its very short elimination half-life, the approved oxacillin dosage might not be sufficient to maintain the pharmacokinetic/pharmacodynamics (PK/PD) target of time-dependent antibiotics. This study aimed to describe the population pharmacokinetics of oxacillin and to explore the probability of PK/PD target attainment by using various dosing regimens with oxacillin in staphylococcal infections. Both total and unbound oxacillin plasma concentrations retrieved as a part of routine therapeutic drug-monitoring practice were analyzed using nonlinear mixed-effects modeling. Monte Carlo simulations were used to generate the theoretical distribution of unbound oxacillin plasma concentration-time profiles at various dosage regimens. Data from 24 patients treated with oxacillin for staphylococcal infection have been included into the analysis. The volume of distribution of oxacillin in the population was 11.2 L, while the elimination rate constant baseline of 0.73 h-1 increased by 0.3 h-1 with each 1 mL/s/1.73 m2 of the estimated glomerular filtration rate (eGFR). The median value of oxacillin binding to plasma proteins was 86%. The superiority of continuous infusion in achieving target PK/PD values was demonstrated and dosing according to eGFR was proposed. Daily oxacillin doses of 9.5 g, 11 g, or 12.5 g administered by continuous infusion have been shown to be optimal for achieving target PK/PD values in patients with moderate, mild, or normal renal function, respectively.
ABSTRACT
OBJECTIVES: The objective of this study was to develop a population pharmacokinetic model of meropenem in a heterogeneous population of patients with a serious bacterial infection in order to propose dosing optimisation leading to improved achievement of the pharmacokinetic/pharmacodynamic (PK/PD) target. METHODS: A total of 174 meropenem serum levels obtained from 144 patients during therapeutic drug monitoring were analysed using a non-linear mixed-effects modelling approach and Monte Carlo simulation was then used to compare various dosing regimens in order to optimise PK/PD target attainment. RESULTS: The meropenem volume of distribution of the patient population was 54.95 L, while clearance started at 3.27 L/hour and increased by 0.91 L/hour with each 1 mL/s/1.73 m2 of estimated glomerular filtration rate. Meropenem clearance was also 0.31 L/hour higher in postoperative patients with central nervous system infection. Meropenem administration by continuous infusion showed a significantly higher probability of attaining the PK/PD target than a standard 30 min infusion (95.3% vs 49.5%). CONCLUSIONS: A daily meropenem dose of 3 g, 6 g and 10.5 g administered by continuous infusion was shown to be accurate for patients with moderate to severe renal impairment, normal renal function to mild renal impairment and augmented renal clearance, respectively.
ABSTRACT
Deregulation of protein kinases is often associated with uncontrolled cell proliferation in various tumours and the inhibition of kinase activity remains an important target for anti-tumour drug development. Here, we report a novel series of 2-aminocyclohexylamino-6-(substituted benzylamino/anilino)-9-cyclopentylpurine derivatives conjugated with putrescine, spermidine or spermine moiety in an effort to expand library of highly potent 2,6,9-trisubstituted purine kinase inhibitors. Presented aniline-type conjugates exhibit significant cytotoxic activity in MV4-11 and EOL-1 cell lines which correlates with FLT3-ITD and PDGFRα inhibition. Furthermore, 6-anilinopurines affected MAPK and STAT pathways in the treated MV4-11 cells and induced cell cycle arrest in the G1 phase. 6-Benzylaminopurines showed comparable CDK2 inhibitory activity to 6-anilinopurines, however, the PDGFRα and FLT3-ITD inhibition was strongly suppressed. Our results show that novel compounds containing aniline in the structure can be involved in the development of potent tyrosine kinase inhibitors with strong activity toward acute myeloid leukemia or chronic eosinophilic leukemia.
Subject(s)
Antineoplastic Agents/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Purines/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Purines/chemical synthesis , Purines/chemistry , Structure-Activity RelationshipABSTRACT
BACKGROUND: Karrikins (KARs) are recently described group of plant growth regulators with stimulatory effects on seed germination, seedling growth and crop productivity. So far, an analytical method for the simultaneous targeted profiling of KARs in plant tissues has not been reported. RESULTS: We present a sensitive method for the determination of two highly biologically active karrikins (KAR1 and KAR2) in minute amounts of plant material (< 20 mg fresh weight). The developed protocol combines the optimized extraction and efficient single-step sample purification with ultra-high performance liquid chromatography-tandem mass spectrometry. Newly synthesized deuterium labelled KAR1 was employed as an internal standard for the validation of KAR quantification using a stable isotope dilution method. The application of the matrix-matched calibration series in combination with the internal standard method yields a high level of accuracy and precision in triplicate, on average bias 3.3% and 2.9% RSD, respectively. The applicability of this analytical approach was confirmed by the successful analysis of karrikins in Arabidopsis seedlings grown on media supplemented with different concentrations of KAR1 and KAR2 (0.1, 1.0 and 10.0 µmol/l). CONCLUSIONS: Our results demonstrate the usage of methodology for routine analyses and for monitoring KARs in complex biological matrices. The proposed method will lead to better understanding of the roles of KARs in plant growth and development.
ABSTRACT
Pharmacological inhibition of cyclin-dependent kinases has emerged as a possible treatment option for various cancer types. We recently identified substituted imidazo[1,2-c]pyrimidin-5(6H)-ones as inhibitors of cyclin-dependent kinase 2 (CDK2). Here, we report the synthesis of derivatives modified at positions 2, 3, 6 or 8 prepared using Suzuki-Miyaura cross-coupling, halogenation, Dimroth-type rearrangement and alkylation as the main synthetic methods. The compounds displayed micro- to submicromolar inhibition of CDK2/cyclin E activity. Binding of the most potent compound 3b to CDK2 was determined using isothermal titration calorimetry. The co-crystal structure of 3b in complex with fully active CDK2 was solved, revealing the binding mode of 3b in the ATP pocket and a hydrogen bonding interaction with hinge region residue Leu83. Evaluation against leukaemia cell lines revealed low cytotoxicity, which is in line with the high selectivity towards CDK2. This study demonstrates that substituted imidazo[1,2-c]pyrimidines can be exploited for future kinase inhibitor development.
Subject(s)
Cyclin-Dependent Kinase 2/antagonists & inhibitors , Imidazoles/chemistry , Protein Kinase Inhibitors/chemical synthesis , Pyrimidines/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Binding Sites , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Cyclin-Dependent Kinase 2/metabolism , Humans , Hydrogen Bonding , Imidazoles/metabolism , Imidazoles/pharmacology , Molecular Dynamics Simulation , Protein Binding , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Pyrimidines/metabolism , Pyrimidines/pharmacology , Structure-Activity RelationshipABSTRACT
Receptor tyrosine kinase PDGFRα is often constitutively activated in various tumours and is regarded as a drug target. Here, we present a collection of 2,6,9-trisubstituted purines with nanomolar potency against PDGFRα and strong and selective cytotoxicity in the human eosinophilic leukaemia cell line EOL-1 that expresses the FIP1L1-PDGFRA oncogene. In treated EOL-1â¯cells, the example compound 14q inhibited the autophosphorylation of PDGFRα and the phosphorylation of STAT3 and ERK1/2. Interestingly, we observed pronounced and even increased effects of 14q on PDGFRα and some of its downstream signalling pathways after drug washout. In accordance with suppressed PDGFRα signalling, treated cells were arrested in the G1 phase of the cell cycle and eventually underwent apoptosis. Our results show that substituted purines can be used as specific modulators of eosinophilic leukaemia.
Subject(s)
Antineoplastic Agents/pharmacology , Hypereosinophilic Syndrome/drug therapy , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Receptor, Platelet-Derived Growth Factor alpha/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Hypereosinophilic Syndrome/metabolism , Hypereosinophilic Syndrome/pathology , Molecular Structure , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Purines/chemical synthesis , Purines/chemistry , Receptor, Platelet-Derived Growth Factor alpha/genetics , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
BACKGROUND: Karrikins (KARs) are plant growth regulators that promote seed germination and the subsequent growth and development of seedlings of many plant species. In nature they are generated and released by combustion of plant material and promote the restoration of burned ecosystems. Smoke water can be artificially prepared as a saturated extract of all substances in smoke produced by burning plants, and it has various horticultural and agricultural applications. RESULTS: We have developed, validated and applied the first fast, specific and sensitive method, based on ultra-high performance liquid chromatography-tandem mass spectrometry, for quantifying KARs in smoke water. To assist these efforts and further analyses, standards of the main KARs (which are not commercially available) were synthesized. Due to the complex matrix of smoke waters, two quantification approaches (standard dilution with a structural KAR analogue and standard addition) were compared. The standard addition method allowed absolute quantification of KARs in six of eight smoke water samples of diverse origins and ages. CONCLUSIONS: Our findings reveal differences in both total and relative levels of KARs in smoke water, and indicate that differences in its KAR composition may be linked to variations in its bioactivity.
ABSTRACT
Rho-associated serine/threonine kinases (ROCKs) are principal regulators of the actin cytoskeleton that regulate the contractility, shape, motility, and invasion of cells. We explored the relationships between structure and anti-ROCK2 activity in a group of purine derivatives substituted at the C6 atom by piperidin-1-yl or azepan-1-yl groups. Structure-activity relationship (SAR) analyses suggested that anti-ROCK activity is retained, and may be further increased, by substitution of the parent compounds at the C2 atom or by expansion of the C6 side chain. These inhibitors of ROCK can reach effective concentrations within cells, as demonstrated by a decrease in phosphorylation of the ROCK target MLC, and by inhibition of the ROCK-dependent invasion of melanoma cells in the collagen matrix. Our study may be useful for further optimization of C6-substituted purine inhibitors of ROCKs and of other sensitive kinases identified by the screening of a broad panel of protein kinases.
Subject(s)
Antineoplastic Agents/pharmacology , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , rho-Associated Kinases/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Humans , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Purines/chemical synthesis , Signal Transduction/drug effects , Structure-Activity RelationshipABSTRACT
This study compares alternative approaches for analyzing phytocannabinoids in different plant materials. Three chromatographic analytical methods (ultra-high-performance liquid chromatography with tandem mass spectrometric detection and gas chromatography with mass spectrometric and flame ionization detection) were evaluated regarding selectivity, sensitivity, analytical accuracy, and precision. The performance of the methods was compared and all three methods were demonstrated to be appropriate tools for analyzing phytocannabinoids in cannabis. Gas chromatography coupled with mass spectrometric detection showed slightly better accuracy in determining phytocannabinoid acids, which are often difficult to quantify owing to their limited stability. Aspects of sample preparation, such as material homogenization and extraction, were also considered. A single ultrasonic-assisted ethanolic extraction of dried and powdered plant samples of cannabis was shown to be exhaustive for extracting the samples prior to analysis.
Subject(s)
Cannabinoids/analysis , Cannabis/chemistry , Chromatography, High Pressure Liquid/methods , Flame Ionization/methods , Gas Chromatography-Mass Spectrometry/methods , Laboratories/organization & administration , Tandem Mass Spectrometry/methods , Limit of Detection , Reproducibility of ResultsABSTRACT
Chronic lymphocytic leukemia (CLL) treatment is improving; however, some patients do not respond to therapy. Due to the high heterogeneity in disease development, there is an urgent need for personalization of therapy. In the present study, the response of leukemic mononuclear cells to anticancer drugs used for CLL treatment (cladribine + mafosfamide; CM or CM combined with rituximab; RCM) was compared with the response to new cyclindependent kinase (CDK) inhibitors: BP14 and BP30. Viable apoptotic and necrotic cells were quantified by flow cytometry using propidium iodide and YoPro stains. CDK inhibitors were studied in several doses to determine the reduction of necrosis and simultaneous increase of apoptosis in leukemic cell incubations with anticancer agents. The distinct cell response to applied doses/anticancer agents was observed. Results obtained in the current manuscript confirmed that modulation of doses is important. This was particularly indicated in results obtained at 24 h of cells incubation with anticancer agent. While an important time for analysis of anticancer response efficacy (monitoring of apoptosis induction potential) seems to be 48 h of cells exposition to anticancer agents. High variability in response to the drugs revealed that both the nature and the dose of the anticancer agents could be important in the final effect of the therapy. The present findings support the thesis that personalized medicine, before drug administration in the clinic, could be important to avoid the application of ineffective therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Antineoplastic Agents/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Female , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Male , Protein Kinase Inhibitors/administration & dosage , Tumor Cells, CulturedABSTRACT
Cyclin-dependent kinases (CDKs) are an important and emerging class of drug targets for which many small-molecule inhibitors have been developed. However, there is often insufficient data available on the selectivity of CDK inhibitors (CDKi) to attribute the effects on the presumed target CDK to these inhibitors. Here, we highlight discrepancies between the kinase selectivity of CDKi and the phenotype exhibited; we evaluated 31 CDKi (claimed to target CDK1-4) for activity toward CDKs 1, 2, 4, 5, 7, 9 and for effects on the cell cycle. Our results suggest that most CDKi should be reclassified as pan-selective and should not be used as a tool. In addition, some compounds did not even inhibit CDKs as their primary cellular targets; for example, NU6140 showed potent inhibition of Aurora kinases. We also established an online database of commercially available CDKi for critical evaluation of their utility as molecular probes. Our results should help researchers select the most relevant chemical tools for their specific applications.
Subject(s)
Cell Cycle/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , HCT116 Cells , Humans , Quinolines/pharmacology , Thiazoles/pharmacologyABSTRACT
N2-(4-Amino-cyclohexyl)-9-cyclopentyl-N6-(6-furan-2-yl-pyridine-3-ylmethyl)-9H-purine-2,6-diamine (BP-14) and 2-(5-{[2-(4-amino-cyclohexylamino)-9-cyclopentyl-9H-purine-6-ylamino]-methyl}-pyridine-2-yl)-phenol (BP-20) are novel cyclin-dependent kinase inhibitors, structurally related to roscovitine, with significant biological activity. A simple, selective and sensitive liquid chromatography - tandem mass spectrometry method for determining them in rat plasma, using roscovitine as an internal standard, was developed and validated. Chromatographic separation was performed in reversed phase mode on Acquity BEH C18 column (100â¯×â¯2.1â¯mm, 1.7⯵m) by gradient elution with mobile phases composed of 15â¯mM ammonium formate pHâ¯4.0 and methanol at flow rate 0.25â¯mL/min at 40⯰C. The analytes were detected based on their characteristic multiple reaction monitoring transitions in positive electrospray ionization mode m/z 473.07â¯>â¯157.93 for BP-14, m/z 499.62â¯>â¯184.2 for BP-20 and m/z 355.5â¯>â¯90.86 for internal standard. In plasma the method provided good linearity within the entire concentration range: 1-10,000â¯nmol/L (r2â¯=â¯0.9989) for BP-14 and 10-25,000â¯nmol/L (r2â¯=â¯0.9994) for BP-20; the limit of detection was 0.6â¯nmol/L for BP-14 and 6.1â¯nmol/L for BP-20. Validation was also performed in bile and urine. The results of validation fit within the acceptance limits following European Medicines Agency guidelines. The method was applied in a pharmacokinetic study of BP-14 and BP-20 in vivo in rats following intravenous and intraduodenal administration including plasma pharmacokinetics, tissue distribution and excretion (renal and biliary). Both compounds showed low bioavailability after intraduodenal administration (0.630 and 1.58% for BP-14 and BP-20, respectively). Distribution into all the analyzed tissues (brain, lungs, liver, kidney, spleen, muscle, adipose tissue) was observed 3â¯h after single dose administration, the highest and lowest concentrations being reached in the adipose tissue and brain, respectively. The biliary excretion of the parent BP-14 and BP-20 compounds accounted for 4.81% and 10.6% of the doses, respectively, and renal excretion for <0.5% in both cases. The obtained results represent pilot knowledge for further development of a new generation of compounds with strong anticancer activities.
Subject(s)
2-Aminopurine/analogs & derivatives , Antineoplastic Agents/analysis , Antineoplastic Agents/chemistry , Cyclins/chemistry , Tandem Mass Spectrometry/methods , 2-Aminopurine/analysis , 2-Aminopurine/pharmacokinetics , Administration, Intravenous/methods , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Biological Availability , Calibration , Chromatography, High Pressure Liquid/methods , Hepatobiliary Elimination/drug effects , Humans , Limit of Detection , Male , Molecular Structure , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/analysis , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Rats , Rats, Wistar , Reproducibility of Results , Structure-Activity Relationship , Tissue Distribution/drug effectsABSTRACT
FLT3 tyrosine kinase is a potential drug target in acute myeloid leukemia (AML) because patients with FLT3-ITD mutations respond poorly to standard cytotoxic agents and there is a clear link between the disease and the oncogenic properties of FLT3. We present novel 2,6,9-trisubstituted purine derivatives with potent FLT3 inhibitory activity. The lead compound 7d displays nanomolar activity in biochemical assays and selectively blocks proliferation of AML cell lines harboring FLT3-ITD mutations, whereas other transformed and normal human cells are several orders of magnitude less sensitive. The MV4-11 cells treated with 7d suppressed the phosphorylation of FLT3 and its downstream signaling pathways, with subsequent G1 cell cycle arrest and apoptosis. Additionally, a single dose of 7d in mice with subcutaneous MV4-11 xenografts caused sustained inhibition of FLT3 and STAT5 phosphorylation over 48 h, in contrast to the shorter effect observed after administration of the reference FLT3 inhibitor quizartinib.
Subject(s)
Antineoplastic Agents/pharmacology , Diamines/pharmacology , Drug Discovery , Leukemia, Myeloid, Acute/pathology , Protein Kinase Inhibitors/pharmacology , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/genetics , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Cell Line, Tumor , Diamines/chemistry , Diamines/metabolism , Leukemia, Myeloid, Acute/genetics , Mice , Molecular Docking Simulation , Protein Conformation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Structure-Activity Relationship , Xenograft Model Antitumor Assays , fms-Like Tyrosine Kinase 3/chemistry , fms-Like Tyrosine Kinase 3/metabolismABSTRACT
Inhibition of protein kinases is a validated concept for pharmacological intervention in cancers. Many kinase inhibitors have been approved for clinical use, but their practical application is often limited. Here, we describe a collection of 23 novel 2,6,9-trisubstituted purine derivatives with nanomolar inhibitory activities against PDGFRα, a receptor tyrosine kinase often found constitutively activated in various tumours. The compounds demonstrated strong and selective cytotoxicity in the human eosinophilic leukemia cell line EOL-1, whereas several other cell lines were substantially less sensitive. The cytotoxicity in EOL-1, which is known to express the FIP1L1-PDGFRA fusion gene encoding an oncogenic kinase, correlated significantly with PDGFRα inhibition. EOL-1 cells treated with the compounds also exhibited dose-dependent inhibition of PDGFRα autophosphorylation and suppression of its downstream signaling pathways with concomitant G1 phase arrest, confirming the proposed mechanism of action. Our results show that substituted purines can be used as platforms for preparing tyrosine kinase inhibitors with specific activity towards eosinophilic leukemia.
Subject(s)
Antineoplastic Agents/pharmacology , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Receptor, Platelet-Derived Growth Factor alpha/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Purines/chemical synthesis , Purines/chemistry , Receptor, Platelet-Derived Growth Factor alpha/genetics , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Herein, we report an alternative synthetic approach for selected 2,6,9-trisubstituted purine CDK inhibitor conjugates with folic acid as a drug-delivery system targeting folate receptors. In contrast to the previously reported approaches, the desired conjugates were constructed stepwise using solid-phase synthesis starting from immobilized primary amines. The ability of the prepared conjugates to release the free drug was verified using dithiothreitol (DTT) and glutathione (GSH) as liberating agents. Finally, binding to the folate receptor (FOLR1) overexpressed in a cancer cell line was measured by flow cytometry using a fluorescent imaging probe.
Subject(s)
Fluorescent Dyes/chemistry , Folic Acid/chemistry , Protein Kinase Inhibitors/chemistry , Flow Cytometry , Folate Receptor 1/drug effects , Molecular Structure , Protein Kinase Inhibitors/pharmacologyABSTRACT
Naturally occurring cytokinins are adenine-based plant hormones. Although, the effect of various substituents at positions N1, C2, N3, N6, N7, or N9 on the biological activity of cytokinins has been studied, the C8-substituted compounds have received little attention. Here, we report the synthesis and in vitro biological testing of thirty-one cytokinin derivatives substituted at the C8 position of the adenine skeleton and twenty-seven compounds which served as their N9-tetrahydropyranyl protected precursors. The cytokinin activity of all the compounds was determined in classical cytokinin biotests (wheat leaf senescence, Amaranthus and tobacco callus assays). With some exceptions, the compounds with a N9-tetrahydropyranyl group were generally less active than their de-protected analogs. The latter were further tested for their ability to activate the Arabidopsis cytokinin receptors AHK3 and CRE1/AHK4 in bacterial receptor activation assays. Using this approach, we identified derivatives bearing short aliphatic chains and retaining high cytokinin activity. Such compounds are suitable candidates for fluorescence labeling or as protein-affinity ligands. We further found that some C8-substituted cytokinins exhibited no or lower cytotoxicity toward tobacco cells when compared to their parent compound. Therefore, we also present and discuss the cytotoxicity of all the compounds against three normal human cell lines.
Subject(s)
Arabidopsis/chemistry , Cytokinins , Adenine/analogs & derivatives , Adenine/chemistry , Cytokinins/chemical synthesis , Cytokinins/chemistry , Cytokinins/metabolism , Drug Screening Assays, Antitumor , Humans , Plant Growth Regulators/metabolism , Protein Kinases/metabolism , Structure-Activity RelationshipABSTRACT
Selective inhibitors of kinases that regulate the cell cycle, such as cyclin-dependent kinases (CDKs) and aurora kinases, could potentially become powerful tools for the treatment of cancer. We prepared and studied a series of 3,5,7-trisubstituted pyrazolo[4,3-d]pyrimidines, a new CDK inhibitor scaffold, to assess their CDK2 inhibitory and antiproliferative activities. A new compound, 2i, which preferentially inhibits CDK2, CDK5, and aurora A was identified. Both biochemical and cellular assays indicated that treatment with compound 2i caused the downregulation of cyclins A and B, the dephosphorylation of histone H3 at Ser10, and the induction of mitochondrial apoptosis in the HCT-116 colon cancer cell line. It also reduced migration as well as tube and lamellipodia formation in human endothelial cells. The kinase inhibitory profile of compound 2i suggests that its anti-angiogenic activity is linked to CDK5 inhibition. This dual mode of action involving apoptosis induction in cancer cells and the blocking of angiogenesis-like activity in endothelial cells offers possible therapeutic potential.
Subject(s)
Aurora Kinase A/antagonists & inhibitors , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 5/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Angiogenesis Inhibitors/chemical synthesis , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Cycle/drug effects , Cyclin-Dependent Kinase Inhibitor Proteins/chemical synthesis , Cyclin-Dependent Kinase Inhibitor Proteins/chemistry , Cyclin-Dependent Kinase Inhibitor Proteins/pharmacology , Drug Design , Drug Screening Assays, Antitumor , HCT116 Cells , Human Umbilical Vein Endothelial Cells , Humans , Molecular Docking Simulation , Protein Kinase Inhibitors/chemical synthesis , Structure-Activity RelationshipABSTRACT
Here we describe the leishmanicidal activities of a library of 2,6,9-trisubstituted purines that were screened for interaction with Cdc2-related protein kinase 3 (CRK3) and subsequently for activity against parasitic Leishmania species. The most active compound inhibited recombinant CRK3 with an IC50 value of 162 nM and was active against Leishmania major and Leishmania donovani at low micromolar concentrations in vitro. Its mode of binding to CRK3 was investigated by molecular docking using a homology model.