ABSTRACT
Activating JAK2 point mutations are implicated in the pathogenesis of myeloid and lymphoid malignancies, including high-risk B-cell acute lymphoblastic leukemia (B-ALL). In preclinical studies, treatment of JAK2 mutant leukemias with type I JAK2 inhibitors (e.g., Food and Drug Administration [FDA]-approved ruxolitinib) provided limited single-agent responses, possibly due to paradoxical JAK2Y1007/1008 hyperphosphorylation induced by these agents. To determine the importance of mutant JAK2 in B-ALL initiation and maintenance, we developed unique genetically engineered mouse models of B-ALL driven by overexpressed Crlf2 and mutant Jak2, recapitulating the genetic aberrations found in human B-ALL. While expression of mutant Jak2 was necessary for leukemia induction, neither its continued expression nor enzymatic activity was required to maintain leukemia survival and rapid proliferation. CRLF2/JAK2 mutant B-ALLs with sustained depletion or pharmacological inhibition of JAK2 exhibited enhanced expression of c-Myc and prominent up-regulation of c-Myc target genes. Combined indirect targeting of c-Myc using the BET bromodomain inhibitor JQ1 and direct targeting of JAK2 with ruxolitinib potently killed JAK2 mutant B-ALLs.
Subject(s)
Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/physiopathology , Animals , Antineoplastic Agents/pharmacology , Azepines/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Gene Knockdown Techniques , Humans , Male , Mice , Mutation , Nitriles , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Pyrimidines , RNA Interference , Receptors, Cytokine/genetics , Transcriptome , Triazoles/pharmacologyABSTRACT
The ability of a drug or probe to cross a biological barrier has historically been viewed to be a function of its intrinsic physical properties. This view has largely restricted drug design and selection to agents within a narrow log P range. Molecular transporters offer a strategy to circumvent these restrictions. In the case of guanidinium-rich transporters (GRTs), a typically highly water-soluble conjugate is found to readily pass through the non-polar membrane of a cell and for some across tissue barriers. This activity opens a field of opportunities for the use of GRTs to enable delivery of polar and non-polar drugs or probes as well as to enhance uptake of those of intermediate polarity. The field of transporter enabled or enhanced uptake has grown dramatically in the last decade. Some GRT drug conjugates have been advanced into clinical trials. This review will provide an overview of recent work pertinent to the design and mechanism of uptake of GRTs.
Subject(s)
Drug Carriers/chemistry , Drug Design , Guanidine/chemistry , Pharmaceutical Preparations/chemistry , Animals , Cell Membrane/metabolism , Cell Membrane Permeability , Humans , Models, Biological , Molecular Structure , Pharmaceutical Preparations/administration & dosage , Pharmaceutical Preparations/metabolismABSTRACT
A major challenge confronting the further advancement of using molecular transporters conjugated to small molecular weight therapeutics in the clinic is the development of linkers that would allow for the controllable release of a free drug/probe only after cell entry. Development of assays that would allow for the rapid real-time quantification of transporter conjugate uptake and cargo release in cells and animals would greatly help in their development. In this chapter, we describe a imaging method that quantitatively measures transporter conjugate uptake and cargo release in real-time in both cell culture and animal models.
Subject(s)
Drug Carriers/chemistry , Drug Carriers/metabolism , Firefly Luciferin/chemistry , Firefly Luciferin/metabolism , Molecular Imaging/methods , Peptides/chemistry , Administration, Topical , Animals , Biological Transport , Calibration , Cell Line, Tumor , Cyclization , Drug Carriers/administration & dosage , Firefly Luciferin/administration & dosage , Hydrolysis , Injections, Intradermal , Intracellular Space/metabolism , Kinetics , Luciferases/genetics , Luciferases/metabolism , Luminescent Measurements , Membrane Potentials , Mice , Mice, Transgenic , Oxidation-Reduction , Polylysine/chemistryABSTRACT
The daphnane diterpene orthoesters constitute a structurally fascinating family of natural products that exhibit a remarkable range of potent biological activities. Although partial activity information is available for some natural daphnanes, little information exists for non-natural congeners or on how changes in structure affect mode of action, function, potency or selectivity. A gateway strategy designed to provide general synthetic access to natural and non-natural daphnanes is described and utilized in the synthesis of two novel members of this class. In this study, a commercially available tartrate derivative was elaborated through a key late-stage diversification intermediate into B-ring yuanhuapin analogues to initiate exploration of the structure-function relationships of this class. Protein kinase C was identified as a cellular target for these agents, and their activity against human lung and leukaemia cell lines was evaluated. The natural product and a novel non-natural analogue exhibited significant potency, but the epimeric epoxide was essentially inactive.
Subject(s)
Cell Division , Diterpenes/chemical synthesis , Protein Kinase C/metabolism , Diterpenes/metabolismABSTRACT
Many therapeutic leads fail to advance clinically because of bioavailability, selectivity, and formulation problems. Molecular transporters can be used to address these problems. Molecular transporter conjugates of otherwise poorly soluble or poorly bioavailable drugs or probes exhibit excellent solubility in water and biological fluids and at the same time an enhanced ability to enter tissues and cells and with modification to do so selectively. For many conjugates, however, it is necessary to release the drug/probe cargo from the transporter after uptake to achieve activity. Here, we describe an imaging method that provides quantification of transporter conjugate uptake and cargo release in real-time in animal models. This method uses transgenic (luciferase) reporter mice and whole-body imaging, allowing noninvasive quantification of transporter conjugate uptake and probe (luciferin) release in real time. This process effectively emulates drug-conjugate delivery, drug release, and drug turnover by an intracellular target, providing a facile method to evaluate comparative uptake of new transporters and efficacy and selectivity of linker release as required for fundamental studies and therapeutic applications.
Subject(s)
Firefly Luciferin/metabolism , Firefly Luciferin/pharmacokinetics , Genes, Reporter , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Animals , Biological Transport, Active , Disulfides/chemistry , Dose-Response Relationship, Drug , Firefly Luciferin/chemistry , Injections, Intradermal , Luminescence , Luminescent Measurements , Membrane Transport Proteins/chemistry , Mice , Mice, Transgenic , Molecular Structure , Whole Body ImagingABSTRACT
The design, synthesis, and evaluation of conjugates of arginine-rich transporters and luciferin are described that release luciferin only after entry into cells that are stably transfected with luciferase. Each molecule of free luciferin that is released after entry generates a photon that can be measured allowing for real-time quantification of uptake and release in cells. The process provides a method to assay uptake and release of free luciferin as a function of variations in the releasable linker and in the transporter.