Cell-permeant and photocleavable chemical inducer of dimerization.

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.

Investigation of morpholine isosters for the development of a potent, selective and metabolically stable mTOR kinase inhibitor.

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.

Brain-penetrant PQR620 mTOR and PQR530 PI3K/mTOR inhibitor reduce huntingtin levels in cell models of HD.

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.

4-(Difluoromethyl)-5-(4-((3R,5S)-3,5-dimethylmorpholino)-6-((R)-3-methylmorpholino)-1,3,5-triazin-2-yl)pyridin-2-amine (PQR626), a Potent, Orally Available, and Brain-Penetrant mTOR Inhibitor for the Treatment of Neurological Disorders.

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.

Site-specific, covalent labeling of recombinant antibody fragments via fusion to an engineered version of 6-O-alkylguanine DNA alkyltransferase.

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.

Total syntheses of amphidinolides B1, B4, G1, H1 and structure revision of amphidinolide H2.

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.

An engineered protein tag for multiprotein labeling in living cells.

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.

Preclinical Development of PQR514, a Highly Potent PI3K Inhibitor Bearing a Difluoromethyl-Pyrimidine Moiety.

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.

A Conformational Restriction Strategy for the Identification of a Highly Selective Pyrimido-pyrrolo-oxazine mTOR Inhibitor.

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.

The Novel TORC1/2 Kinase Inhibitor PQR620 Has Anti-Tumor Activity in Lymphomas as a Single Agent and in Combination with Venetoclax.

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.

(S)-4-(Difluoromethyl)-5-(4-(3-methylmorpholino)-6-morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530), a Potent, Orally Bioavailable, and Brain-Penetrable Dual Inhibitor of Class I PI3K and mTOR Kinase.

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.

The novel, catalytic mTORC1/2 inhibitor PQR620 and the PI3K/mTORC1/2 inhibitor PQR530 effectively cross the blood-brain barrier and increase seizure threshold in a mouse model of chronic epilepsy.

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.

Discovery and Preclinical Characterization of 5-[4,6-Bis({3-oxa-8-azabicyclo[3.2.1]octan-8-yl})-1,3,5-triazin-2-yl]-4-(difluoromethyl)pyridin-2-amine (PQR620), a Highly Potent and Selective mTORC1/2 Inhibitor for Cancer and Neurological Disorders.

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.

PQR309 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in Lymphomas as a Single Agent and in Combination Therapy.

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.

Thiophenol-mediated 1,5-hydrogen transfer for the preparation of pyrrolizidines, indolizidines, and related compounds.

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.

A concise synthesis of the Pennsylvania Green fluorophore and labeling of intracellular targets with O6-benzylguanine derivatives.

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.

5-(4,6-Dimorpholino-1,3,5-triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309), a Potent, Brain-Penetrant, Orally Bioavailable, Pan-Class I PI3K/mTOR Inhibitor as Clinical Candidate in Oncology.

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.

Deconvolution of Buparlisib's mechanism of action defines specific PI3K and tubulin inhibitors for therapeutic intervention.

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.

Highly diastereoselective formation of spirocyclic compounds via 1,5-hydrogen transfer: a total synthesis of (-)-erythrodiene.

[reaction: see text] A highly stereoselective synthesis of (-)-erythrodiene starting from 4-isopropylcyclohexanone is described. The key reactions are an asymmetric methoxycarbonylation of the starting ketone and a highly diastereoselective radical cascade involving addition of a phenylthiyl radical to a terminal alkyne followed by a 1,5-hydrogen transfer and a 5-exo-cyclization.

Thiophenol-mediated hydrogen atom abstraction: an efficient tin-free procedure for the preparation of cyclopentane derivatives.

[reaction: see text] An efficient procedure for running a cascade reaction involving 1,5-abstraction of a hydrogen atom followed by a radical cyclization is reported. Alkenyl radicals are generated from easily available terminal alkynes and thiophenol. This procedure eliminates the need of using the toxic tributyltin hydride and gives a greater amount of radical translocation products.