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1.
Eur J Med Chem ; 248: 115038, 2023 Feb 15.
Article in English | MEDLINE | ID: mdl-36634458

ABSTRACT

Upregulation of mechanistic target of rapamycin (mTOR) signaling drives various types of cancers and neurological diseases. Rapamycin and its analogues (rapalogs) are first generation mTOR inhibitors, and selectively block mTOR complex 1 (TORC1) by an allosteric mechanism. In contrast, second generation ATP-binding site inhibitors of mTOR kinase (TORKi) target both TORC1 and TORC2. Here, we explore 3,6-dihydro-2H-pyran (DHP) and tetrahydro-2H-pyran (THP) as isosteres of the morpholine moiety to unlock a novel chemical space for TORKi generation. A library of DHP- and THP-substituted triazines was prepared, and molecular modelling provided a rational for a structure activity relationship study. Finally, compound 11b [5-(4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)-6-(tetrahydro-2H-pyran-4-yl)-1,3,5-triazin-2-yl)-4-(difluoromethyl)pyridin-2-amine] was selected due its potency and selectivity for mTOR kinase over the structurally related class I phosphoinositide 3-kinases (PI3Ks) isoforms. 11b displayed high metabolic stability towards CYP1A1 degradation, which is of advantage in drug development. After oral administration to male Sprague Dawley rats, 11b reached high concentrations both in plasma and brain, revealing an excellent oral bioavailability. In a metabolic stability assay using human hepatocytes, 11b was more stable than PQR620, the first-in-class brain penetrant TORKi. Compound 11b also displayed dose-dependent anti-proliferative activity in splenic marginal zone lymphoma (SMZL) cell lines as single agent and when combined with BCL2 inhibition (venetoclax). Our results identify the THP-substituted triazine core as a novel scaffold for the development of metabolically stable TORKi for the treatment of chronic diseases and cancers driven by mTOR deregulation and requiring drug distribution also to the central nervous system.


Subject(s)
Neoplasms , TOR Serine-Threonine Kinases , Rats , Animals , Male , Humans , Rats, Sprague-Dawley , TOR Serine-Threonine Kinases/metabolism , Mechanistic Target of Rapamycin Complex 1 , Morpholines/pharmacology , Morpholines/chemistry , Sirolimus/pharmacology , Sirolimus/therapeutic use , Neoplasms/drug therapy , Pyrans/therapeutic use , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use
2.
J Med Chem ; 63(22): 13595-13617, 2020 11 25.
Article in English | MEDLINE | ID: mdl-33166139

ABSTRACT

The mechanistic target of rapamycin (mTOR) pathway is hyperactivated in cancer and neurological disorders. Rapalogs and mTOR kinase inhibitors (TORKi) have recently been applied to alleviate epileptic seizures in tuberous sclerosis complex (TSC). Herein, we describe a pharmacophore exploration to identify a highly potent, selective, brain penetrant TORKi. An extensive investigation of the morpholine ring engaging the mTOR solvent exposed region led to the discovery of PQR626 (8). 8 displayed excellent brain penetration and was well-tolerated in mice. In mice with a conditionally inactivated Tsc1 gene in glia, 8 significantly reduced the loss of Tsc1-induced mortality at 50 mg/kg p.o. twice a day. 8 overcomes the metabolic liabilities of PQR620 (52), the first-in-class brain penetrant TORKi showing efficacy in a TSC mouse model. The improved stability in human hepatocytes, excellent brain penetration, and efficacy in Tsc1GFAPCKO mice qualify 8 as a potential therapeutic candidate for the treatment of neurological disorders.


Subject(s)
Brain/metabolism , Morpholines/administration & dosage , Morpholines/metabolism , Nervous System Diseases/metabolism , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism , Administration, Oral , Animals , Brain/drug effects , Dogs , Female , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Madin Darby Canine Kidney Cells , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Nude , Morpholines/chemistry , Nervous System Diseases/drug therapy , Rats , Rats, Sprague-Dawley
3.
Neuropharmacology ; 162: 107812, 2020 01 01.
Article in English | MEDLINE | ID: mdl-31622602

ABSTRACT

One of the pathological hallmarks of Huntington disease (HD) is accumulation of the disease-causing mutant huntingtin (mHTT), which leads to the disruption of a variety of cellular functions, ultimately resulting in cell death. Induction of autophagy, for example by the inhibition of mechanistic target of rapamycin (mTOR) signaling, has been shown to reduce HTT levels and aggregates. While rapalogs like rapamycin allosterically inhibit the mTOR complex 1 (TORC1), ATP-competitive mTOR inhibitors suppress activities of TORC1 and TORC2 and have been shown to be more efficient in inducing autophagy and reducing protein levels and aggregates than rapalogs. The ability to cross the blood-brain barrier of first generation catalytic mTOR inhibitors has so far been limited, and therefore sufficient target coverage in the brain could not be reached. Two novel, brain penetrant compounds - the mTORC1/2 inhibitor PQR620, and the dual pan-phosphoinositide 3-kinase (PI3K) and mTORC1/2 kinase inhibitor PQR530 - were evaluated by assessing their potential to induce autophagy and reducing mHTT levels. For this purpose, expression levels of autophagic markers and well-defined mTOR targets were analyzed in STHdh cells and HEK293T cells and in mouse brains. Both compounds potently inhibited mTOR signaling in cell models as well as in mouse brain. As proof of principle, reduction of aggregates and levels of soluble mHTT were demonstrated upon treatment with both compounds. Originally developed for cancer treatment, these second generation mTORC1/2 and PI3K/mTOR inhibitors show brain penetrance and efficacy in cell models of HD, making them candidate molecules for further investigations in HD.


Subject(s)
Azabicyclo Compounds/pharmacology , Enzyme Inhibitors/pharmacology , Huntingtin Protein/drug effects , Huntington Disease/metabolism , Morpholines/pharmacology , Neurons/drug effects , Protein Aggregates/drug effects , Pyridines/pharmacology , Triazines/pharmacology , Animals , Autophagy/drug effects , Blood-Brain Barrier , Cell Line , Corpus Striatum/cytology , HEK293 Cells , Humans , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Huntington Disease/genetics , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 2/antagonists & inhibitors , Mice , Neurons/metabolism , Phosphatidylinositol 3-Kinases , Phosphoinositide-3 Kinase Inhibitors/pharmacology
4.
ACS Med Chem Lett ; 10(10): 1473-1479, 2019 Oct 10.
Article in English | MEDLINE | ID: mdl-31620236

ABSTRACT

The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is a critical regulator of cell growth and is frequently hyperactivated in cancer. Therefore, PI3K inhibitors represent a valuable asset in cancer therapy. Herein we have developed a novel anticancer agent, the potent pan-PI3K inhibitor PQR514 (4), which is a follow-up compound for the phase-II clinical compound PQR309 (1). Compound 4 has an improved potency both in vitro and in cellular assays with respect to its predecessor compounds. It shows superiority in the suppression of cancer cell proliferation and demonstrates significant antitumor activity in an OVCAR-3 xenograft model at concentrations approximately eight times lower than PQR309 (1). The favorable pharmacokinetic profile and a minimal brain penetration promote PQR514 (4) as an optimized candidate for the treatment of systemic tumors.

5.
J Med Chem ; 62(18): 8609-8630, 2019 09 26.
Article in English | MEDLINE | ID: mdl-31465220

ABSTRACT

The mechanistic target of rapamycin (mTOR) plays a pivotal role in growth and tumor progression and is an attractive target for cancer treatment. ATP-competitive mTOR kinase inhibitors (TORKi) have the potential to overcome limitations of rapamycin derivatives in a wide range of malignancies. Herein, we exploit a conformational restriction approach to explore a novel chemical space for the generation of TORKi. Structure-activity relationship (SAR) studies led to the identification of compound 12b with a ∼450-fold selectivity for mTOR over class I PI3K isoforms. Pharmacokinetic studies in male Sprague Dawley rats highlighted a good exposure after oral dosing and a minimum brain penetration. CYP450 reactive phenotyping pointed out the high metabolic stability of 12b. These results identify the tricyclic pyrimido-pyrrolo-oxazine moiety as a novel scaffold for the development of highly selective mTOR inhibitors for cancer treatment.


Subject(s)
Oxazines/chemistry , Protein Kinase Inhibitors/chemistry , Pyrimidinones/chemistry , Pyrroles/chemistry , TOR Serine-Threonine Kinases/antagonists & inhibitors , Adenosine Triphosphate/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Dogs , Drug Design , Humans , Inhibitory Concentration 50 , Kinetics , Male , Mice , Molecular Conformation , Neoplasms/drug therapy , Oxazines/pharmacokinetics , Protein Kinase Inhibitors/pharmacokinetics , Pyrimidinones/pharmacokinetics , Pyrroles/pharmacokinetics , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , TOR Serine-Threonine Kinases/chemistry
6.
Cancers (Basel) ; 11(6)2019 Jun 04.
Article in English | MEDLINE | ID: mdl-31167506

ABSTRACT

The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling cascade is an important therapeutic target for lymphomas. Rapamycin-derivates as allosteric mTOR complex 1 (TORC1) inhibitors have shown moderate preclinical and clinical anti-lymphoma activity. Here, we assessed the anti-tumor activity of PQR620, a novel brain penetrant dual TORC1/2 inhibitor, in 56 lymphoma cell lines. We observed anti-tumor activity across 56 lymphoma models with a median IC50 value of 250 nM after 72 h of exposure. PQR620 was largely cytostatic, but the combination with the BCL2 inhibitor venetoclax led to cytotoxicity. Both the single agent and the combination data were validated in xenograft models. The data support further evaluation of PQR620 as a single agent or in combination with venetoclax.

7.
J Med Chem ; 62(13): 6241-6261, 2019 07 11.
Article in English | MEDLINE | ID: mdl-31244112

ABSTRACT

The phosphoinositide 3-kinase (PI3K)/mechanistic target of rapamycin (mTOR) pathway is frequently overactivated in cancer, and drives cell growth, proliferation, survival, and metastasis. Here, we report a structure-activity relationship study, which led to the discovery of a drug-like adenosine 5'-triphosphate-site PI3K/mTOR kinase inhibitor: (S)-4-(difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530, compound 6), which qualifies as a clinical candidate due to its potency and specificity for PI3K and mTOR kinases, and its pharmacokinetic properties, including brain penetration. Compound 6 showed excellent selectivity over a wide panel of kinases and an excellent selectivity against unrelated receptor enzymes and ion channels. Moreover, compound 6 prevented cell growth in a cancer cell line panel. The preclinical in vivo characterization of compound 6 in an OVCAR-3 xenograft model demonstrated good oral bioavailability, excellent brain penetration, and efficacy. Initial toxicity studies in rats and dogs qualify 6 for further development as a therapeutic agent in oncology.


Subject(s)
Aminopyridines/pharmacology , Antineoplastic Agents/pharmacology , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors/pharmacology , Pyridines/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Triazines/pharmacology , Aminopyridines/chemical synthesis , Aminopyridines/metabolism , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Brain/metabolism , Cell Line, Tumor , Dogs , Female , Humans , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Microsomes, Liver/metabolism , Molecular Docking Simulation , Molecular Structure , Morpholines/chemical synthesis , Morpholines/metabolism , Phosphoinositide-3 Kinase Inhibitors/chemical synthesis , Phosphoinositide-3 Kinase Inhibitors/metabolism , Protein Binding , Pyridines/chemical synthesis , Pyridines/metabolism , Rats, Wistar , Structure-Activity Relationship , TOR Serine-Threonine Kinases/metabolism , Triazines/chemical synthesis , Triazines/metabolism , Xenograft Model Antitumor Assays
8.
J Med Chem ; 61(22): 10084-10105, 2018 11 21.
Article in English | MEDLINE | ID: mdl-30359003

ABSTRACT

Mechanistic target of rapamycin (mTOR) promotes cell proliferation, growth, and survival and is overactivated in many tumors and central nervous system disorders. PQR620 (3) is a novel, potent, selective, and brain penetrable inhibitor of mTORC1/2 kinase. PQR620 (3) showed excellent selectivity for mTOR over PI3K and protein kinases and efficiently prevented cancer cell growth in a 66 cancer cell line panel. In C57BL/6J and Sprague-Dawley mice, maximum concentration ( Cmax) in plasma and brain was reached after 30 min, with a half-life ( t1/2) > 5 h. In an ovarian carcinoma mouse xenograft model (OVCAR-3), daily dosing of PQR620 (3) inhibited tumor growth significantly. Moreover, PQR620 (3) attenuated epileptic seizures in a tuberous sclerosis complex (TSC) mouse model. In conclusion, PQR620 (3) inhibits mTOR kinase potently and selectively, shows antitumor effects in vitro and in vivo, and promises advantages in CNS indications due to its brain/plasma distribution ratio.


Subject(s)
Azabicyclo Compounds/pharmacology , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 2/antagonists & inhibitors , Pyridines/pharmacology , Seizures/drug therapy , Triazines/pharmacology , Animals , Azabicyclo Compounds/metabolism , Azabicyclo Compounds/therapeutic use , Blood-Brain Barrier/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Mice , Models, Molecular , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Protein Conformation , Pyridines/metabolism , Pyridines/therapeutic use , Rats , Triazines/metabolism , Triazines/therapeutic use
9.
Neuropharmacology ; 140: 107-120, 2018 09 15.
Article in English | MEDLINE | ID: mdl-30081001

ABSTRACT

The mTOR signaling pathway has emerged as a possible therapeutic target for epilepsy. Clinical trials have shown that mTOR inhibitors such as everolimus reduce seizures in tuberous sclerosis complex patients with intractable epilepsy. Furthermore, accumulating preclinical data suggest that mTOR inhibitors may have anti-seizure or anti-epileptogenic actions in other types of epilepsy. However, the chronic use of rapalogs such as everolimus is limited by poor tolerability, particularly by immunosuppression, poor brain penetration and induction of feedback loops which might contribute to their limited therapeutic efficacy. Here we describe two novel, brain-permeable and well tolerated small molecule 1,3,5-triazine derivatives, the catalytic mTORC1/C2 inhibitor PQR620 and the dual pan-PI3K/mTOR inhibitor PQR530. These derivatives were compared with the mTORC1 inhibitors rapamycin and everolimus as well as the anti-seizure drugs phenobarbital and levetiracetam. The anti-seizure potential of these compounds was determined by evaluating the electroconvulsive seizure threshold in normal and epileptic mice. Rapamycin and everolimus only poorly penetrated into the brain (brain:plasma ratio 0.0057 for rapamycin and 0.016 for everolimus). In contrast, the novel compounds rapidly entered the brain, reaching brain:plasma ratios of ∼1.6. Furthermore, they significantly decreased phosphorylation of S6 ribosomal protein in the hippocampus of normal and epileptic mice, demonstrating effective mTOR inhibition. PQR620 and PQR530 significantly increased seizure threshold at tolerable doses. The effect of PQR620 was more marked in epileptic vs. nonepileptic mice, matching the efficacy of levetiracetam. Overall, the novel compounds described here have the potential to overcome the disadvantages of rapalogs for treatment of epilepsy and mTORopathies directly connected to mutations in the mTOR signaling cascade.


Subject(s)
Anticonvulsants , Azabicyclo Compounds , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Enzyme Inhibitors/pharmacology , Epilepsy/complications , Epilepsy/drug therapy , Morpholines , Pyridines , Seizures/complications , Seizures/prevention & control , Triazines , Animals , Anticonvulsants/blood , Anticonvulsants/pharmacokinetics , Anticonvulsants/pharmacology , Anticonvulsants/therapeutic use , Azabicyclo Compounds/blood , Azabicyclo Compounds/pharmacokinetics , Azabicyclo Compounds/pharmacology , Azabicyclo Compounds/therapeutic use , Catalysis/drug effects , Electroshock , Everolimus/blood , Everolimus/pharmacokinetics , Everolimus/pharmacology , Female , Hippocampus/drug effects , Hippocampus/metabolism , Levetiracetam/pharmacology , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 2/antagonists & inhibitors , Mice , Morpholines/blood , Morpholines/pharmacokinetics , Morpholines/pharmacology , Morpholines/therapeutic use , Phenobarbital/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Pyridines/blood , Pyridines/pharmacokinetics , Pyridines/pharmacology , Pyridines/therapeutic use , Ribosomal Proteins/metabolism , Sirolimus/blood , Sirolimus/pharmacokinetics , Sirolimus/pharmacology , Triazines/blood , Triazines/pharmacokinetics , Triazines/pharmacology , Triazines/therapeutic use
10.
Clin Cancer Res ; 24(1): 120-129, 2018 01 01.
Article in English | MEDLINE | ID: mdl-29066507

ABSTRACT

Purpose: Activation of the PI3K/mTOR signaling pathway is recurrent in different lymphoma types, and pharmacologic inhibition of the PI3K/mTOR pathway has shown activity in lymphoma patients. Here, we extensively characterized the in vitro and in vivo activity and the mechanism of action of PQR309 (bimiralisib), a novel oral selective dual PI3K/mTOR inhibitor under clinical evaluation, in preclinical lymphoma models.Experimental Design: This study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination, validation experiments on in vivo models and primary cells, proteomics and gene-expression profiling, and comparison with other signaling inhibitors.Results: PQR309 had in vitro antilymphoma activity as single agent and in combination with venetoclax, panobinostat, ibrutinib, lenalidomide, ARV-825, marizomib, and rituximab. Sensitivity to PQR309 was associated with specific baseline gene-expression features, such as high expression of transcripts coding for the BCR pathway. Combining proteomics and RNA profiling, we identified the different contribution of PQR309-induced protein phosphorylation and gene expression changes to the drug mechanism of action. Gene-expression signatures induced by PQR309 and by other signaling inhibitors largely overlapped. PQR309 showed activity in cells with primary or secondary resistance to idelalisib.Conclusions: On the basis of these results, PQR309 appeared as a novel and promising compound that is worth developing in the lymphoma setting. Clin Cancer Res; 24(1); 120-9. ©2017 AACR.


Subject(s)
Antineoplastic Agents/pharmacology , Lymphoma/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Drug Resistance, Neoplasm , Humans , Lymphoma/drug therapy , Lymphoma/genetics , Lymphoma/pathology , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Purines , Quinazolinones , Xenograft Model Antitumor Assays
11.
J Med Chem ; 60(17): 7524-7538, 2017 09 14.
Article in English | MEDLINE | ID: mdl-28829592

ABSTRACT

Phosphoinositide 3-kinase (PI3K) is deregulated in a wide variety of human tumors and triggers activation of protein kinase B (PKB/Akt) and mammalian target of rapamycin (mTOR). Here we describe the preclinical characterization of compound 1 (PQR309, bimiralisib), a potent 4,6-dimorpholino-1,3,5-triazine-based pan-class I PI3K inhibitor, which targets mTOR kinase in a balanced fashion at higher concentrations. No off-target interactions were detected for 1 in a wide panel of protein kinase, enzyme, and receptor ligand assays. Moreover, 1 did not bind tubulin, which was observed for the structurally related 4 (BKM120, buparlisib). Compound 1 is orally available, crosses the blood-brain barrier, and displayed favorable pharmacokinetic parameters in mice, rats, and dogs. Compound 1 demonstrated efficiency in inhibiting proliferation in tumor cell lines and a rat xenograft model. This, together with the compound's safety profile, identifies 1 as a clinical candidate with a broad application range in oncology, including treatment of brain tumors or CNS metastasis. Compound 1 is currently in phase II clinical trials for advanced solid tumors and refractory lymphoma.


Subject(s)
Aminopyridines/therapeutic use , Antineoplastic Agents/therapeutic use , Morpholines/therapeutic use , Neoplasms/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/therapeutic use , TOR Serine-Threonine Kinases/antagonists & inhibitors , Administration, Oral , Aminopyridines/administration & dosage , Aminopyridines/pharmacokinetics , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Brain/drug effects , Brain/metabolism , Cell Proliferation/drug effects , Dogs , Humans , Mice , Models, Molecular , Morpholines/administration & dosage , Morpholines/pharmacokinetics , Neoplasms/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacokinetics , Rats , Rats, Nude , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism
12.
Nat Commun ; 8: 14683, 2017 03 09.
Article in English | MEDLINE | ID: mdl-28276440

ABSTRACT

BKM120 (Buparlisib) is one of the most advanced phosphoinositide 3-kinase (PI3K) inhibitors for the treatment of cancer, but it interferes as an off-target effect with microtubule polymerization. Here, we developed two chemical derivatives that differ from BKM120 by only one atom. We show that these minute changes separate the dual activity of BKM120 into discrete PI3K and tubulin inhibitors. Analysis of the compounds cellular growth arrest phenotypes and microtubule dynamics suggest that the antiproliferative activity of BKM120 is mainly due to microtubule-dependent cytotoxicity rather than through inhibition of PI3K. Crystal structures of BKM120 and derivatives in complex with tubulin and PI3K provide insights into the selective mode of action of this class of drugs. Our results raise concerns over BKM120's generally accepted mode of action, and provide a unique mechanistic basis for next-generation PI3K inhibitors with improved safety profiles and flexibility for use in combination therapies.


Subject(s)
Aminopyridines/pharmacology , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Tubulin Modulators/pharmacology , Tubulin/metabolism , Aminopyridines/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Crystallography, X-Ray , HCT116 Cells , Humans , Molecular Structure , Morpholines/chemistry , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Tubulin/chemistry , Tubulin Modulators/chemistry
13.
Angew Chem Int Ed Engl ; 53(18): 4717-20, 2014 Apr 25.
Article in English | MEDLINE | ID: mdl-24677313

ABSTRACT

Chemical inducers of dimerization (CIDs) have been developed to orchestrate protein dimerization and translocation. Here we present a novel photocleavable HaloTag- and SNAP-tag-reactive CID (MeNV-HaXS) with excellent selectivity and intracellular reactivity. Excitation at 360 nm cleaves the methyl-6-nitroveratryl core of MeNV-HaXS. MeNV-HaXS covalently links HaloTag- and SNAP-tag fusion proteins, and enables targeting of selected membranes and intracellular organelles. MeNV-HaXS-mediated translocation has been validated for plasma membrane, late endosomes, lysosomes, Golgi, mitochondria, and the actin cytoskeleton. Photocleavage of MeNV-HaXS liberates target proteins and provides access to optical manipulation of protein relocation with high spatiotemporal and subcellular precision. MeNV-HaXS supports kinetic studies of protein dynamics and the manipulation of subcellular enzyme activities, which is exemplified for Golgi-targeted cargo and the assessment of nuclear import kinetics.


Subject(s)
Cell Membrane Permeability , Green Fluorescent Proteins/metabolism , Light , Photosensitizing Agents/pharmacology , Protein Multimerization/drug effects , Recombinant Fusion Proteins/metabolism , Cell Membrane/metabolism , Cytosol/metabolism , Endocytosis/physiology , Endocytosis/radiation effects , Golgi Apparatus/metabolism , Green Fluorescent Proteins/genetics , Guanine/analogs & derivatives , Guanine/chemistry , Guanine/pharmacology , HeLa Cells , Humans , Kinetics , Lysosomes/metabolism , Mitochondria/metabolism , Photosensitizing Agents/chemistry , Protein Transport , Recombinant Fusion Proteins/genetics
14.
Chem Biol ; 20(4): 549-57, 2013 Apr 18.
Article in English | MEDLINE | ID: mdl-23601644

ABSTRACT

Cell activation initiated by receptor ligands or oncogenes triggers complex and convoluted intracellular signaling. Techniques initiating signals at defined starting points and cellular locations are attractive to elucidate the output of selected pathways. Here, we present the development and validation of a protein heterodimerization system based on small molecules cross-linking fusion proteins derived from HaloTags and SNAP-tags. Chemical dimerizers of HaloTag and SNAP-tag (HaXS) show excellent selectivity and have been optimized for intracellular reactivity. HaXS force protein-protein interactions and can translocate proteins to various cellular compartments. Due to the covalent nature of the HaloTag-HaXS-SNAP-tag complex, intracellular dimerization can be easily monitored. First applications include protein targeting to cytoskeleton, to the plasma membrane, to lysosomes, the initiation of the PI3K/mTOR pathway, and multiplexed protein complex formation in combination with the rapamycin dimerization system.


Subject(s)
Cross-Linking Reagents/metabolism , Proteins/metabolism , Alkyl and Aryl Transferases/metabolism , Animals , Cell Line , Cross-Linking Reagents/chemistry , Cytoskeleton/metabolism , Dimerization , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Hydrolases/metabolism , Mice , NIH 3T3 Cells , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Protein Interaction Domains and Motifs , Proteins/chemistry , Proteins/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Signal Transduction , Substrate Specificity , TOR Serine-Threonine Kinases/chemistry , TOR Serine-Threonine Kinases/metabolism
15.
Bioconjug Chem ; 20(5): 1010-5, 2009 May 20.
Article in English | MEDLINE | ID: mdl-19388673

ABSTRACT

Recombinant antibodies are promising tools for a wide range of bioanalytical and medical applications. However, the chemical modification of such molecules can be challenging, which limits their broader utilization. Here we describe a universal method for the site-specific labeling of antibody fragments and protein ligands by genetically fusing them to an engineered version of the human DNA-repair enzyme O(6)-alkyllguanine DNA alkyltransferase (AGT), known as SNAP-Tag (1-3) . Substrates containing O(6)-benzylguanine are covalently bound to the fusion proteins via a stable thioether bond in a rapid and highly specific self-labeling reaction. The coupling is site-directed, allowing the design and synthesis of antibody conjugates with predefined stoichiometry. We cloned a series of ligand SNAP-Tag fusion proteins and expressed them in HEK 293T cells. The antibody/ligand-fusions were characterized by labeling with different fluorophores, labeling with biotin, or by coupling them to fluorescent nanobeads, followed by analysis by flow cytometry and confocal microscopy. All ligands retained their original antigen-binding properties when fused to the SNAP-Tag. The combination of recombinant antibodies or protein ligands with the SNAP-Tag facilitates simple and efficient covalent modification with a broad range of substrates, thus providing a useful and advantageous alternative to existing coupling strategies.


Subject(s)
O(6)-Methylguanine-DNA Methyltransferase/genetics , Protein Engineering/methods , Single-Chain Antibodies/genetics , Single-Chain Antibodies/metabolism , Animals , Binding Sites , Biotin/metabolism , CD30 Ligand/metabolism , CHO Cells , Cricetinae , Cricetulus , Flow Cytometry , Fluorescent Dyes/metabolism , Humans , Ki-1 Antigen/immunology , Ligands , Mice , Microscopy, Confocal , Microspheres , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Silicon Dioxide/chemistry , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/immunology , Staining and Labeling , Substrate Specificity
16.
Chemistry ; 15(16): 3983-4010, 2009.
Article in English | MEDLINE | ID: mdl-19241433

ABSTRACT

Nature is a pretty unselective "chemist" when it comes to making the highly cytotoxic amphidinolide macrolides of the B/G/H series. To date, 16 different such compounds have been isolated, all of which could now be approached by a highly convergent and largely catalysis-based route (see figure). This notion is exemplified by the total synthesis of five prototype members of this family.Dinoflagellates of the genus Amphidinium produce a "library" of closely related secondary metabolites of mixed polyketide origin, which are extremely scarce but highly promising owing to the exceptional cytotoxicity against various cancer cell lines. Because of the dense array of sensitive functionalities on their largely conserved macrocyclic frame, however, these amphidinolides of the B, D, G and H types elapsed many previous attempts at their synthesis. Described herein is a robust, convergent and hence general blueprint which allowed not only to conquest five prototype members of these series, but also holds the promise of making "non-natural" analogues available by diverted total synthesis. This notion transpires for a synthesis-driven structure revision of amphidinolide H2. The successful route hinges upon a highly productive Stille-Migita cross-coupling reaction at the congested and chemically labile 1,3-diene site present in all such targets, which required the development of a modified chloride- and fluoride-free protocol. The macrocyclic ring could be formed with high efficiency and selectivity by ring-closing metathesis (RCM) engaging a vinyl epoxide unit as one of the reaction partners. Because of the sensitivity of the targets to oxidizing and reducing conditions as well as to pH changes, the proper adjustment of the protecting group pattern for the peripheral -OH functions also constitutes a critical aspect, which has to converge to silyl groups only once the diene is in place. Tris(dimethylamino)sulfonium difluorotrimethylsilicate (TASF) turned out to be a sufficiently mild fluoride source to allow for the final deprotection without damaging the precious macrolides.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/chemical synthesis , Macrolides/chemistry , Macrolides/chemical synthesis , Marine Toxins/chemistry , Marine Toxins/chemical synthesis , Animals , Antineoplastic Agents/pharmacology , Catalysis , Combinatorial Chemistry Techniques , Dinoflagellida/chemistry , Macrolides/pharmacology , Marine Toxins/pharmacology , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Structure-Activity Relationship
17.
Chem Biol ; 15(2): 128-36, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18291317

ABSTRACT

The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O6-benzylguanine derivatives bearing a chemical probe. Here we report the generation of an AGT-based tag, named CLIP-tag, which reacts specifically with O2-benzylcytosine derivatives. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different molecular probes in living cells. We furthermore show simultaneous pulse-chase experiments to visualize different generations of two different proteins in one sample.


Subject(s)
Cell Survival , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/metabolism , Proteins/metabolism , Staining and Labeling/methods , Animals , Binding Sites , CHO Cells , Cricetinae , Cricetulus , Cytosine/analogs & derivatives , Cytosine/metabolism , Fluorescent Dyes/chemistry , Models, Molecular , Mutation , O(6)-Methylguanine-DNA Methyltransferase/genetics , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Protein Conformation , Substrate Specificity
19.
Org Lett ; 9(21): 4375-8, 2007 Oct 11.
Article in English | MEDLINE | ID: mdl-17887768

ABSTRACT

The efficient preparation of 1-azabicyclic alkanes is described. Highly functionalized skeletons are prepared in a concise manner using a radical tin-free 1,5-hydrogen transfer-cyclization process. The precursors for the radical reactions are readily assembled either from pyrrolidine/piperidine/hexahydro-1H-azepine or via condensation of a properly designed N-alkylimine with an allenylzinc species.


Subject(s)
Indolizines/chemical synthesis , Phenols/chemistry , Pyrrolizidine Alkaloids/chemical synthesis , Sulfhydryl Compounds/chemistry , Catalysis , Combinatorial Chemistry Techniques , Hydrogen/chemistry , Indolizines/chemistry , Molecular Structure , Pyrrolizidine Alkaloids/chemistry
20.
Org Lett ; 9(19): 3741-4, 2007 Sep 13.
Article in English | MEDLINE | ID: mdl-17705395

ABSTRACT

We report improved syntheses of the Pennsylvania Green and 4-carboxy-Pennsylvania Green fluorophores; the latter compound was prepared from methyl 4-iodo-3-methylbenzoate in a three-pot process (32% overall yield). Chinese hamster ovary cells expressing O6-alkylguanine-DNA alkyltransferase fusion proteins were treated with Pennsylvania Green and Oregon Green linked to O6-benzylguanine (SNAP-Tag substrates). Analysis of living cells by confocal microscopy revealed that Pennsylvania Green derivatives exhibit substantially higher cell permeability than analogous Oregon Green-derived molecular probes.


Subject(s)
Fluorescent Dyes/chemical synthesis , Guanine/analogs & derivatives , Alkyl and Aryl Transferases/chemistry , Alkyl and Aryl Transferases/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Fluorescent Dyes/chemistry , Genes, Reporter/genetics , Guanine/chemistry , Humans , Models, Molecular , Molecular Probes/chemistry , Molecular Structure
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