Search

Baseline gene expression analysis of B cell lymphoma cell lines with higher (IC50 < 200 nM) or lower sensitive (IC50 > 400 nM) to PQR309.

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.

Supplementary figures, table legends, Table S1, S9 Figure S1. In vitro antitumor activity of the dual PI3K/mTOR inhibitor apitolisib and its correlation with PQR309. Figure S2. Antitumor In vivo activity of PQR309 in the treatment of RI-1 and SU-DHL-6 xenograft model. Figure S3. Apoptosis is induced by co-treatment of PQR309 with venetoclax or panobinostat in primary cells and in the DLBCL SU-DHL-6 cell line. Figure S4. Specific baseline gene expression signatures are associated with higher or lower sensitivity to PQR309. Figure S5. In vitro antitumor activity of the PI3K� inhibitor idelalisib and its correlation with PQR309. Figure S6. PQR309 induced decrease in phosphorylation of AKT-Ser473 and p70S6K-Thr389 in DLBCL cell lines. Figure S7. PQR309 reduces AKT-Ser473 phosphorylation in lymphoma cell lines. Figure S8. Transcriptional expression signature of ABC DLBCL cell lines induced by PQR309. Figure S9. ABC and GCB DLBCL PQR309-treated signatures were highly overlapping. Figure S10. Transcript expression levels of PQR309-treated samples. Figure S11. Changes in protein phosphorylation and RNA expression differently contribute to PQR309 affected biologic pathways in ABC DLBCL. Figure S12. PQR309 can largely regulate the same genes affected by the BTK inhibitor ibrutinib, the PI3K� idelalisib, the dual PI3K�/� inhibitor duvelisib (A), the dual PI3K�/� inhibitor AZD8835 or the AKT inhibitor AZD5363 (B). Figure S13. BCR pathway signature is similarly affected after ibrutinib, idelalisib and duvelisib treatments in ABC DLBCL cell lines. Figure S14. The dual PI3K/mTOR inhibitor PQR309 and the PIM inhibitor AZD1208 synergize in DLBCL cell lines. Supplementary Table 1. List of phosphoresidues investigated by Carna Bioscience to perform RPPA analysis. Supplementary Table 9. Transcripts differentially expressed in ABC DLBCL cell lines treated with ibrutinib (A), idelalisib (B) or duvelisib (C) versus DMSO.

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.

A new, scalable, rapid, high yielding, and practical synthesis of 4-(difluoromethyl)pyridin-2-amine provides a key intermediate for the preparation of numerous protein kinase inhibitors and clinica...

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.

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 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 (

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.

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.

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.

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.

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.

Selektive Dimerisierungsreagentien (CIDs; chemical inducers of dimerization) wurden entwickelt, um die Protein-Dimerisierung und -Translokation chemisch zu steuern. Wir stellen hier ein neues, photospaltbares CID (MeNV-HaXS) vor, das HaloTag- und SNAP-tag-Fusionsproteine bindet und eine exzellente Selektivitat und intrazellulare Reaktivitat besitzt. Die Anregung bei 360 nm spaltet die Methyl-6-nitroveratryl-Gruppe von MeNV-HaXS, und das “Dimere”, wieder in die beiden Proteine. MeNV-HaXS verknupft HaloTag- und SNAP-tag-Fusionsproteine und erlaubt ihre zielgerichtete Verschiebung an Membranen und Zellorganellen, z. B. Plasmamembran, Endosomen, Lysosomen, Golgi, Mitochondrien und das Aktin Zytoskeleton. Die photolytische Spaltung von MeNV-HaXS setzt Zielproteine frei und ermoglicht die optische Manipulation der Proteinlokalisation mit hoher subzellularer Prazision in Raum und Zeit. MeNV-HaXS ermoglicht so kinetische Studien, Manipulation der Proteindynamik und der subzellularer Enzymaktivitat. Demonstriert wurde die Anwendung von MeNV-HaXS fur die zielgerichtete Protein-Translokation an den Golgi und die kinetische Erhebung von Importprozessen in den Zellkern.

A practical method for the formation of thiophosphonates bearing functionalized monocyclic, fused bicyclic and spirocyclic residues is presented. The procedure requires the easily available terminal alkynes as starting materials as well as commercially and readily available reagents such as diethyl thiophosphite. The experimental procedure consists of a one-pot process without any slow addition of one of the reagents.

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.

Vinyl radicals have a propensity to rearrange via intramolccular 1,5-hydrogen transfer to the more-stable alkyl radicals, which themselves possess a suitable structure to undergo rapid 5-exo-trig cyclizations. This tandem radical translocation-cyclization process represents a useful tool for the preparation of a wide range of five-membered rings. Moreover, this reaction is highly selective and, due to its radical nature, tolerates a variety of functional groups. During the last decade, tin-free procedures have emerged, rendering this process very attractive for the synthesis of natural products and their analogues. Selected examples of this strategy illustrating the scope of the method are described. 1 Introduction 2 Vinyl Radicals from Vinyl Halides 3 Vinyl Radicals Generated by Addition to Alkynes and Allenes 3.1 Addition of Carbon-Centered Radicals 3.1.1 Intermolecular Addition of Carbon-Centered Radicals 3.1.2 Intramolecular Addition of Carbon-Centered Radicals 3.2 Addition of Tin-Centered Radicals 3.3 Addition of Oxygen-Centered Radicals 3.4 Addition of Nitrogen-Centered Radicals 3.5 Addition of Sulfur-Centered Radicals 3.6 Addition of Phosphorus-Centered Radicals 4 Translocation-Cyclization Processes Involving a Stereoselective Hydrogen Atom Transfer 5 Conclusion

SummaryThe 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.

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.

A thiophenol-mediated method for cyclization of alkynes is described. The reaction cascade involves the intermolecular addition of a phenylthiyl radical to a terminal triple bond generating an alkenyl radical, followed by a 1,5-hydrogen atom transfer and a 5-exo-trig radical cyclization. This very efficient tin-free procedure allows one to prepare highly functionalized cyclopentane derivatives as well as fused bicyclic and spirocyclic compounds from easily available precursors. During this cyclization process, a phenylthio moiety is incorporated into the final cyclized products. This functionalization is particularly attractive for further transformation of the products.

The PI3K/AKT/mTOR signaling pathway plays a fundamental role in cell proliferation, growth and survival and aberrant activation of this signaling pathway has been shown to drive the progression of malignant tumors.[1] Drugs targeting the pathway at multiple points, such as dual PI3K/mTOR inhibitors appear to have the broadest activity profile to address cancer therapeutic strategies and are currently being explored in numerous clinical studies. Recently, we presented PQR309, a novel, brain-penetrant pan-PI3K/mTOR inhibitor, which entered phase II clinical trials in 2016.[2] Here, we report the lead optimization of PQR530, a potent and brain-penetrant follow-up compound as pan-PI3K/mTORC1/2 inhibitor. The development of a follow-up compound concentrated on the improvement of both, the potency and the selectivity for all targeted kinases, namely the class IA PI3K isoforms as well as mTOR. We present a detailed ligand-based structure-activity relationship study which was obtained by systematic modifications of the hinge region as well as the affinity binding substituents. This study led to the identification of PQR530, a dual pan-PI3K/mTORC1/2 inhibitor showing excellent activities in cellular assays as well as in PI3Kα and mTOR enzymatic binding assays. In A2058 melanoma cells PQR530 inhibited protein kinase B (PKB, pSer473) and ribosomal protein S6 (pS6, pSer235/236) phosphorylation with IC50 values of 0.07 µM. PQR530 showed excellent selectivity over a wide panel of kinases, as well as excellent selectivity versus unrelated receptor enzymes and ion channels. Moreover, PQR530 displayed potency in a panel of 44 cancer cell lines (NTRC OncolinesTM) to prevent cancer cell growth (mean value for GI50 of 426 nM). Oral application of PQR530 to mice resulted in a dose-proportional PK and demonstrated good oral bioavailability and excellent brain penetration.[3] An optimized, robust synthetic route allowed rapid access to multi-gram quantities of PQR530 for pre-clinical development in only 4 steps. In conclusion, PQR530 inhibits all PI3K isoforms and the mammalian target of rapamycin (mTOR) complexes C1/2 potently and selectively, and shows anti-tumor effects in vitro and in vivo. [1] M. P. Wymann, M. Zvelebil, M. Laffargue (2003). Phosphoinositide 3-kinase signalling – which way to target? Trends Pharmacol Sci.; 24, 366-376. [2] V. Cmiljanovic et. al. “PQR309: Structure-Based Design, Synthesis and Biological Evaluation of a Novel, Selective, Dual Pan-PI3K/mTOR Inhibitor” presented at AACR Annual Meeting 2015, April 18-22, Philadelphia, Pennsylvania, USA. [3] P. Hillmann et al. “Pharmacological Characterization of the Selective, Orally Bioavailable, Potent Dual PI3K/mTORC1/2 Inhibitor PQR530” abstract submitted for AACR Annual Meeting 2017, April 1-5, Washington, D. C., USA. Citation Format: Denise Rageot, Florent Beaufils, Anna Melone, Alexander M. Sele, Thomas Bohnacker, Marc Lang, Jürgen Mestan, Petra Hillmann, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Discovery and biological evaluation of PQR530, a highly potent dual pan-PI3K/mTORC1/2 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 140. doi:10.1158/1538-7445.AM2017-140

Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases, produce PtdIns(3,4,5)P3 and trigger intracellular signaling pathways that are vital to cell growth, proliferation, survival and migration. Constitutive activation of PI3K is frequently observed in many tumor types, which defines PI3K as a valuable drug target in oncology.1 Numerous PI3K inhibitors in clinical development contain a morpholine moiety that mediates hinge region binding in the ATP pocket of PI3K by a hydrogen bond with the active site valine backbone nitrogen (Val851 in PI3Kα)2. We present here novel pyrimidinopyrrolo-oxazines related to the clinically advanced, pyridinylmorpholine and triazinylmorpholine derived pan-PI3K/mTOR inhibitors BKM120 and PQR309. The novel fused tricyclic core of these compounds contains two morpholine moieties of which one is conformationally restricted by the introduction of a methylene bridge that links the pyrimidine core with one of the two morpholine moieties. This modification leads to the generation of two regioisomers, each existing as a set of enantiomers. We investigated the influence of this conformational restriction on PI3K inhibitory activity and analyzed the distinct selectivity profiles and potencies of the respective stereo- and regio-isomers. The design and preparation of specific compounds in combination with biological assays (phosphorylation of PKB and S6, binding affinity to p110α), structure-activity relationship (SAR) and molecular modelling studies allowed us to understand the binding mode of these compounds and acquire valuable information that potentially lead to the development of derivatives with a distinct selectivity profile (e.g. PI3K versus mTOR). A selection of compounds demonstrated inhibition of protein kinase B (pSer473) and ribosomal protein S6 (pSer235/236) phosphorylation with IC50 values in the nanomolar range and high inhibitory potency of all PI3K isoforms (Ki(p110α) > 40 nM). Single p.o. administration of our lead compound to SD rats resulted in good oral bioavailability as well as excellent brain penetration. Furthermore, mechanism of action-based increases in glucose levels and insulin levels have been observed. In conclusion, we present here the development, optimization, preparation and biological evaluation of a novel class of potent, orally available and brain-penetrant pan-PI3K inhibitors that represent an innovative extension to known pyrimidinomorpholine derived PI3K inhibitors. Moreover, our results add to the understanding of how introducing specific structural and conformational modifications can lead to the development of optimized, selective PI3K and mTOR inhibitors. [1] Thorpe, L. M.; Yuzugullu, H.; Zhao, J. J. Nat. Rev. Cancer 2015, 15, 7-24. [2] Andrs, M.; Korabecny, J.; Jun, D.; Hodny, Z.; Bartek, J.; Kuca, K. J. Med. Chem. 2015, 58, 41-71. Citation Format: Alexander M. Sele, Denise Rageot, Florent Beaufils, Anna Melone, Thomas Bohnacker, Eileen Jackson, Jean-Baptiste Langlois, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Tricyclic fused pyrimidinopyrrolo-oxazines reveal conformational preferences of morpholine for PI3K hinge region binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 153. doi:10.1158/1538-7445.AM2017-153

Introduction: The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a fundamental role in many cellular processes like growth, survival, proliferation, differentiation and motility. In cancers several mutations have been identified that lead to constitutive activation of PI3K. PQR530 is a novel, ATP site directed inhibitor of all PI3K isoforms and the mammalian target of rapamycin (mTOR) complexes C1/2 that is currently in pre-clinical development. PQR530 potently binds to its targets, inhibits cell proliferation and shows excellent selectivity versus related and unrelated kinases [1]. Results: PQR530 inhibits PI3K signaling in stimulated MCF7 cells as detected by PathScan analysis. Excellent tolerability has been found for PQR530 during GLP toxicological testing in rats and dogs. Increase in insulin and blood glucose, a treatable class effect of PI3K inhibitors, has been observed after PQR530 administration to mice. Investigation of mutagenicity and hERG binding resulted in a clean profile. PQR530 exhibited dose-proportional pharmacokinetics (PK) in male C57BL/6J mice. A maximum concentration (Cmax) in plasma and brain was reached after 30 minutes (7.8 μg/ml and 112.6 μg/ml, respectively) indicating that efficacious concentrations were reached in both tissues. The calculated half-life (t1/2) for plasma and brain was approximately 5 hours. PQR530 potently inhibited PI3K signaling in vivo for several hours after administration of a single oral dose of 50 mg/kg. Tumor growth was significantly decreased in SUDHL-6 lymphoma, RIVA lymphoma and OVCAR-3 ovarian cancer mouse xenografts using daily, oral administration. Conclusion: PQR530 is a potent, ATP competitive pan-PI3K and mTORC1/2 inhibitor. The physico-chemical properties of PQR530 result in good oral bioavailability and excellent brain penetration. PQR530 is well tolerated and efficiently inhibits tumor growth in xenograft models. Preclinical data allow for further development of the compound. [1] Rageot D, et al., Discovery and biological evaluation of PQR530, a highly potent dual pan-PI3K/mTORC1/2 inhibitor, abstract submitted for AACR Annual Meeting 2017, April 1-5, Washington, D. C., USA. Citation Format: Petra Hillmann, Denise Rageot, Florent Beaufils, Anna Melone, Alexander Sele, Robert A. Ettlin, Jürgen Mestan, Vladimir Cmiljanovic, Marc Lang, Elisabeth Singer, Carolin Walter, Hoa HP Nguyen, Paul Hebeisen, Matthias P. Wymann, Doriano Fabbro. Pharmacological characterization of the selective, orally bioavailable, potent dual PI3K/mTORC1/2 inhibitor PQR530 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 159. doi:10.1158/1538-7445.AM2017-159

SummaryCell 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.

Introduction: The mammalian target of rapamycin (mTOR) signaling pathway is an integrating factor in cell physiology that influences many processes like growth, metabolism and proliferation. mTOR signaling is constitutively activated in many cancers. Rapamycin is an allosteric inhibitor of mTOR that targets a subset of mTOR functions via inhibition of the mTORC1 complex. An ATP site-directed mTORC1/2 inhibitor that fully blocks all mTOR functions is desirable as cancer therapeutic. PQR620 is a novel, ATP site directed inhibitor of mTOR that is currently in pre-clinical development. PQR620 potently binds to its target (Kd = 6 nM) and shows excellent selectivity versus related and unrelated kinases [1]. Results: PQR620 inhibits mTOR signaling in stimulated MCF7 cells as detected by PathScan analysis. Excellent tolerability has been observed in mice (MTD = 150 mg/kg). A 14 day GLP toxicological study in rats showed very good tolerability (MTD = 30 mg/kg). Only minor toxicities such as dose-related changes in body weight and blood count were observed. PQR620 was administered to male C57BL/6J mice for a pharmacokinetic (PK) and pharmacodynamics (PD) evaluation. After oral application PQR620 exhibited dose-proportional PK, a maximum concentration (Cmax) in plasma and brain was reached after 30 minutes (4.8 μg/ml and 7.7 μg/ml, respectively). In muscle, Cmax (7.6 μg/ml) was reached after 2 hours. The calculated half-life (t1/2) for plasma and brain was approximately 5 hours. After 8 hours, the total exposure (expressed as AUC0-tz (area under the curve)) was 20.5 μg*h/ml in plasma, while it was approximately 30% higher in both, brain and thigh muscle (30.6 and 32.3 μg*h/ml, respectively). PQR620 potently inhibited mTOR signaling in vivo after administration of a single oral dose of 50 mg/kg. Importantly, no effect on plasma insulin levels was observed. In an OVCAR-3, ovarian carcinoma mouse xenograft, PQR620 effectively attenuated tumor growth using daily, oral dosing. Conclusion: PQR620 potently inhibits mTORC1/2 in vitro and in vivo. The physico-chemical properties of PQR620 result in good oral bioavailability and excellent brain penetration. PQR620 is well tolerated and efficiently inhibits tumor growth in xenograft models. Preclinical data allow for further development of the compound. [1] Beaufils F, Rageot D, et al., Structure-Activity Relationship Studies, Synthesis and Biological Evaluation of PQR620, a Highly Potent and Selective mTORC1/2 Inhibitor, AACR annual meeting 2016 Citation Format: Florent Beaufils, Denise Rageot, Anna Melone, Alexander Sele, Marc Lang, Juergen Mestan, Robert A. Ettlin, Petra Hillmann, Vladimir Cmiljanovic, Carolin Walter, Elisabeth Singer, Hoa HP Nguyen, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 393A.

Mammalian target of rapamycin (mTOR) signaling pathway plays a fundamental role in cell proliferation, differentiation, growth and survival.[1] As a consequence, various tumors and central nervous system (CNS) disorders share aberrant activation of the mTOR pathway. Drugs targeting the mTOR pathway represent therefore a valuable path to address multiple therapeutic areas.[1-2] Here, we report the lead optimization of PQR620, a novel potent and selective brain penetrant inhibitor of mTORC1/2. The development of selective mTOR inhibitors is particularly challenging due to extensively conserved amino acid residues in the ATP binding pocket within the PI3K and PI3K-related protein kinase family. Here, we present a detailed ligand-based structure activity relationship study allowing selective targeting of mTOR kinase activity without the interference of other PI3K family members. Systematic variation of the hinge region and affinity binding motifs led to the identification of PQR620, a morpholino-triazinyl derivative, as potent and selective mTOR inhibitor. Substitution of the morpholine binding to the hinge region and introduction of a 2-aminopyridine, substituted with a difluoromethyl group, induced a >1000-fold selectivity towards mTOR over PI3Kα in enzymatic binding assays. In A2058 melanoma cells PQR620 demonstrated inhibition of protein kinase B (pSer473) and ribosomal protein S6 (pSer235/236) phosphorylation with IC50 values of 0.2 μM and 0.1 μM, respectively. The physico-chemical properties of PQR620 result in good oral bioavailability and excellent brain penetration. PQR620 showed excellent selectivity over a wide panel of kinases, as well as excellent selectivity versus unrelated receptor enzymes and ion channels. Moreover, PQR620 demonstrated its potency to prevent cancer cell growth in an NTRC 44 cancer cell line panel, resulting in a 10log(IC50) of 2.86 (nM). Further pharmacological properties and in vivo efficacy of PQR620 are presented in detail in Ref. [3]. The preparation of PQR620 was optimized towards a robust synthetic route involving only 4 steps, allowing for a rapid access to quantities required for pre-clinical testing. In conclusion, PQR620 inhibits mTOR potently and selectively, and shows anti-tumor effects in vitro and in vivo. PQR620 is currently in pre-clinical development. [1] M. Laplante, D. Sabatini, Cell 2012, 149, 274-293. [2] Z. Z. Chong, Y. C. Shang, L. Zhang, S. Wang, K. Maiese, Oxid. Med. Cell. Longev. 2010, 3, 374–391. [3] F. Beaufils, D. Rageot, A. Melone, A. M. Sele, M. Lang, J. Mestan, R. A. Ettlin, P. Hillmann, V. Cmiljanovic, C. Walter, E. Singer, H. P. Nguyen, P. Hebeisen, D. Fabbro, M. P. Wymann, “Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620” presented at AACR Annual Meeting 2016, April 16-20, New Orleans, Louisiana, USA. Citation Format: Florent Beaufils, Denise Rageot, Anna Melone, Marc Lang, Jürgen Mestan, Vladimir Cmiljanovic, Petra Hillmann, Paul Hebeisen, Doriano Fabbro, Matthias P. Wymann. Structure-activity relationship studies, synthesis, and biological evaluation of PQR620, a highly potent and selective mTORC1/2 inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1336.

Microtubules are dynamic polymers and integral components of the cytoskeleton. They play important roles in the regulation of cellular signaling, motility, maintenance of cell shape, secretion, intercellular transport and spindle formation during mitosis.1 As a consequence, small molecules that interfere with the dynamics of microtubules have been recognized as powerful tools for the treatment of cancer.2, 3 A systematic structure-activity relationship (SAR) study starting from morpholino-substituted biheteroaryls with moderate microtubule disrupting activities allowed the optimizations of biological activity, metabolic stability, and drug-like properties. Interestingly, the type of core (aryl, pyridine or pyrimidine) was of importance, as well as the regioisomeric arrangement of the pyrimidine nitrogens. Pyrimidines substituted with four- or five-membered N-heterocycles proved to be superior both in terms of biological activity and metabolic stability. Finally, optimization of the heteroaryl substituent of the pyrimidine derivatives culminated in the identification of a novel series of substituted 4-(pyrimidin-2-yl)morpholines targeting microtubule polymerization in the nM range. Selected compounds potently inhibit cellular microtubule polymerization with EC50 values of 20-90 nM. This was confirmed by phosphorylation of Histone3, nuclear DNA condensation, and cell cycle arrest in G2/M or induction of cell death across multiple cell lines. Moreover, substituted 4-(pyrimidin-2-yl)morpholines were shown to be poor substrates for P-gp multi-drug resistance pumps, and therefore caused efficiently mitotic arrest and cell death in colchicine-resistant cells. The co-crystal structure of tubulin with selected compounds showed that 4-(pyrimidin-2-yl)morpholines bind to the colchicine pocket located between the α and β subunits of the αβ-tubulin dimer. Relevant inhibitor contact residues include Lys352, Met259, Ala316, Leu248, Val238, Tyr202 and Cys241 of β-tubulin. Moreover, two water molecules link the morpholine oxygen to the β-tubulin bound GTP. Conformational changes induced by inhibitor binding suggest that free or plus end β-tubulin is targeted by this compound series. Pre-clinical studies characterized a lead compound selection with excellent stability in human hepatocytes, and human, mouse and rat microsomes. Overall, these compounds qualify as a novel class of microtubule destabilizing agents that target the colchicine-binding site, and which warrant further investigations currently in progress (PK studies, xenograft tumor mouse models). 1) Walczak, C. E, Curr. Opin. Cell Biol. 2000, 12, 52-56. 2) Risinger, A. L.; Giles, F. J.; Mooberry, S. L, Cancer Treat. Rev. 2009, 35, 255-261. 3) Downing, K. H.; Nogales, E., Curr. Opin. Struct. Biol. 1998, 8, 785-791. Citation Format: Alexander M. Sele, Denise Rageot, Thomas Bohnacker, Florent Beaufils, Andrea E. Prota, Michel O. Steinmetz, Matthias P. Wymann. Novel 4-(pyrimidin-2-yl)morpholines targeting the colchicine-binding site of tubulin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1364.

A practical method for the formation of thiophosphonates bearing functionalized monocyclic, fused bicyclic and spirocyclic residues is presented. The procedure requires the easily available terminal alkynes as starting materials as well as commercially and readily available reagents such as diethyl thiophosphite. The experimental procedure consists of a one-pot process without any slow addition of one of the reagents.

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.

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.

The stereochemical outcome of intramolecular radical mediated hydrogen transfer (= radical translocation) is discussed. Low to excellent levels of stereocontrol are observed making such processes attractive for applications in target-oriented synthesis.

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.

PQR309 is a novel oral PI3K/mTOR inhibitor, being now evaluated as single agent in a phase I study for solid tumors patients (NCT01940133). Here, we present the activity of the compound in lymphoma pre-clinical models, also integrating response data with genomic features. Methods. IC50s were calculated with the MTT assay at 72h on 40 cell lines [27 diffuse large B-cell lymphoma (DLBCL), 10 mantle cell lymphoma (MCL), 3 splenic marginal zone lymphoma (SMZL)] treated with increasing doses of PQR309, a second dual PI3K/mTOR inhibitor (GDC0980) and the PI3Kdelta inhibitor Idelalisib. Gene expression profiling (GEP) was performed with Illumina HumanHT-12 Expression BeadChips. Results. PQR309 had potent anti-proliferative activity in most of the cell lines. Median IC50 were 166 nM in DLBCL (95%CI 128-343 nM), 235 in MCL (155-381), 214 in SMZL (188-304). Activated B-cell like (ABC) and germinal center B-cell like (GCB) DLBCL subtypes were equally sensitive. PQR309 and GDC0980 presented a highly correlated pattern of anti-proliferative activity (R = 0.9). Idelalisib appeared significantly less active, with a pattern of sensitive cell lines less correlated with PQR309 (R = 0.6) or GDC0980 (R = 0.6). Apoptosis after PQR309 (500 nM, 72h) was limited to 1/7 of cell lines. GEP was performed on 8 DLBCL cell lines (4 GCB, 4 ABC) after treatment with DMSO or PQR309 (1 mcM) for 4, 8 and 12h. PQR309 affected, in a time-dependent manner, relevant biologic pathways in both subtypes. Down-regulated transcripts were enriched of MYC targets, genes involved in NFKB/MYD88/BCR/IFN signaling, apoptosis, DNA damage and proteasome. Transcripts up-regulated were enriched of genes involved in cell cycle and senescence, up-regulated after MYD88 silencing, down-regulated by PI3K, involved in packaging of telomere, in autophagosome, up-regulated by inhibitors of HDAC, BET Bromodomain and JAK2. CXCR4, PIM1, PIM2, YPEL5 (up-regulated), LYAR, CCDC86, DDX21, HSPA8, STIP1, and PAK1IP1 (down-regulated) were among the genes changing after PQR309 treatment in more than one time-point or DLBCL cell-type. To identify markers of resistance we looked for transcripts differently regulated between cell lines with higher or lower sensitivity to PQR309 and we also compared baseline GEP between very sensitive (IC50 400 nM). Transcripts more expressed in sensitive cells were significantly enriched of genes involved in BCR pathway/signaling, kinases regulation, and immune system. Transcripts associated with less sensitive cells were enriched of members of proteasome pathway, oxidative phosphorylation, translation initiation. PQR309 (1 mcM) was able to inhibit IgM-stimulation induced p-AKT(Ser 473) in 3/3 DLBCL and 3/3 MCL cells. Conclusions. PQR309 showed strong anti-proliferative activity in lymphomas and GEP identified affected biologic pathways and features possibly associated with response to the molecule. Citation Format: Chiara Tarantelli, Eugenio Gaudio, Ivo Kwee, Andrea Rinaldi, Elena Bernasconi, Luciano Cascione, Petra Hillmann, Anastasios Stathis, Laura Carrassa, Massimo Broggini, Georg Stussi, Doriano Fabbro, Florent Beaufils, Anna Melone, Thomas Bohnacker, Matthias P. Wymann, Andreas Wicki, Emanuele Zucca, Vladimir Cmiljanovic, Francesco Bertoni. Pre-clinical activity and mechanism of action of the novel dual PI3K/mTOR inhibitor PQR309 in B-cell lymphomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2652. doi:10.1158/1538-7445.AM2015-2652

Phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling is key to the control of many physiological and pathophysiological processes, and promotes cancer and inflammatory disease. Therefore, targeting of PI3K and/or mTOR pathways is currently explored in numerous clinical studies. PQR309 is a novel, brain penetrant, potent and selective pan-PI3K/mTOR inhibitor with PK properties suitable for once a day oral dosing in humans. Structure activity relationship studies for PI3K and mTOR interactions are presented, including X-Ray analysis of PI3Kgamma co-crystal structures, modeling of PI3Kalpha and mTOR structures, and chemical derivatization. This led to the identification of PQR309 as a potent pan-PI3K and moderate mTOR inhibitor. PQR309 displays excellent selectivity versus PI3K-related lipid kinases (PIKKs) and protein kinases (KINOMEscan), as well as excellent selectivity versus unrelated targets (Cerep expresSProfile). PQR309 features excellent cell permeability, and was characterized as a BCS class II compound due to its limited water solubility (40 μM). Moreover, PQR309 is not a substrate for P-glycoprotein 1 (P-gp). In A2058 melanoma cells PQR309 demonstrated inhibition of protein kinase B (PKB/Akt; pS473) and ribosomal protein S6 (S6, pSer235/236) phosphorylation with IC50 values of 0.13 μM and 0.58 μM, respectively. In IGF-stimulated MCF7 breast cancer cells, PQR309 at 1 μM inhibited phosphorylation of downstream substrates of PI3K including PKB/Akt, S6, p70S6 kinase, GSK3 and Bad by 60-95%. PQR309 inhibited proliferation of all 58 cell lines of the NCI60 panel (GI50 from 50 to 3300 nM), of the NTRC Oncoline panel (44 cell lines, GI50 from 100-6700 nM) and of a lymphoma cell line panel (40 lymphoma cell lines, GI50 from 25-1740 nM). A concise 4-step synthetic process utilizing a novel protective group strategy provides a robust and scalable supply of PQR309 for clinical trials. In summary, PQR309 is a novel, potent, dual pan-PI3K/mTOR inhibitor with a balanced PI3K vs. mTOR profile, and displays excellent physico-chemical and pharmacological properties. The safety profile of PQR309 is currently addressed in Phase I clinical studies. Citation Format: Vladimir Cmiljanovic, Natasa Cmiljanovic, Romina Marone, Florent Beaufils, Xuxiao Zhang, Marketa Zvelebil, Paul Hebeisen, Marc Lang, Juergen Mestan, Anna Melone, Thomas Bohnacker, Eugenio Gaudio, Chiara Tarantelli, Francesco Bertoni, Reto Ritschard, Vincent Pretre, Andreas Wicki, Doriano Fabbro, Petra Hillmann, Roger Williams, Bernd Giese, Matthias P. Wymann. PQR309: Structure-based design, synthesis and biological evaluation of a novel, selective, dual pan-PI3K/mTOR inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2664. doi:10.1158/1538-7445.AM2015-2664

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is frequently activated in tumors and promotes oncogenic cell transformation, proliferation and tumor growth. PQR309, a novel dual inhibitor of PI3K and mTOR, is currently in Phase I clinical development in cancer patients. PQR309 binds potently and specifically to the ATP binding pocket of all PI3K class I isoforms and mTORC1/2, attenuates PI3K signaling and inhibits tumor cell growth. The preclinical pharmacological and toxicological characterization of PQR309 is presented here. Methods: PQR309 pharmacokinetics/-dynamics (PK/PD) were investigated in rats and mice. Tissue samples from plasma, brain and liver were analyzed by LC/MS detecting PQR309 distribution as well as blood insulin and glucose. Toxicological studies were performed in rats and dogs. Effects on neurological, hematopoietic, respiratory, lymphoid, reproductive and cardiovascular system as well as general health were monitored. The metabolic fate of PQR309 was analyzed in rat, dog and human hepatocytes. Results: PQR309 PK studies in rats, mice and dogs revealed dose-proportional PK, both PO and IV, with a half-life of 5-8 hours in plasma, brain and liver, allowing for once a day oral application. As on-target effect, increase of blood insulin and glucose could be observed within hours after oral dosage in rats, which makes both molecules suitable as PD markers. In in vivo PC-3 rat tumor xenograft models, PQR309 effectively inhibited PI3K signaling in tumors and reduced tumor growth at 10 mg/kg oral dosing. Preclinical toxicity testing showed no signs of cardiotoxicity (including lack of hERG binding), phototoxicity (3T3 NRU test) or mutagenicity (AMES test) for PQR309. No marked effect on CYP450 activity was observed making PQR309 a good combination partner in cancer therapy. As for other PI3K inhibitors, PQR309 leads at elevated doses to a fully reversible loss of body weight and appetite in rats and dogs. No further significant adverse events were observed when testing PQR309 for 28 days in these species. Conclusions: PQR309 potently inhibits class I PI3K isoforms and mTORC1/2 and shows anti-tumor effects in vitro and in vivo. The physico-chemical properties of PQR309 result in good oral bioavailability and equal distribution between plasma and brain. Pre-clinical data led to initiation of a Phase I clinical study of PQR309 in solid tumors. Citation Format: Vladimir Cmiljanovic, Robert A. Ettlin, Florent Beaufils, Walter Dieterle, Petra Hillmann, Juergen Mestan, Anna Melone, Thomas Bohnacker, Marc Lang, Natasa Cmiljanovic, Bernd Giese, Paul Hebeisen, Matthias P. Wymann, Doriano Fabbro. PQR309: A potent, brain-penetrant, dual pan-PI3K/mTOR inhibitor with excellent oral bioavailability and tolerability. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4514. doi:10.1158/1538-7445.AM2015-4514

Due to its central role in growth, proliferation, survival and migration, phosphoinositide 3-kinase (PI3K) is considered as an important drug target in oncology (1). BKM120 is one of the clinically most advanced PI3K inhibitors (PI3Ki), and is currently listed in more than 80 clinical studies aimed at attenuating tumour progression. As an off-target effect, BKM120 was reported to disrupt microtubules (MT) at concentrations around 1 μM (2). Here, we elucidate in detail the structural factors defining PI3K- and tubulin-binding of BKM120, and present a pure PI3K inhibitor (PQR309) and a potent MT disruptor (MTD147) differing from BKM120 by only 1 Dalton. Separation of PI3Ki and MT disruption activities of BKM120 allowed profiling of BKM120 against PQR309 and MTD147: cellular growth profiles of PQR309 clustered with other PI3Ki such as GDC0941/GDC0980, while BKM120 matched MTD147. Both yielded a G2/M cell cycle arrest with typical histone3 phosphorylation. Accumulation of G2/M arrested cells was already evident at concentrations yielding 50% growth inhibition. Interestingly, BKM120 concentrations for 50% cell growth inhibition (with evident G2/M arrest) ranged below or within its reported AUC0-24 levels at day 8 in patient plasma (3,4). This result implies that the two activities of BKM120 cannot be separated, thus complicating the understanding of drug action and impacting on the rational of combination therapies at relevant drug doses. Using X-ray crystallography we found that BKM120 binds to the colchicine pocket on β-tubulin. This study further highlights the importance of the pyrimidine core orientation for tight tubulin binding. Interestingly, activities of regio-isomers of the pyrimidine core are inversed for PI3Ki and tubulin association, and modulate binding by a factor of >30x. Finally, a combination of biochemical, cellular and structural data suggests an inverted orientation of BKM120 in the catalytic cleft of PI3K as previously proposed (6). In summary, the dissection of BKM120 functions allows reassessment of its dominant activity, to increase drug safety, and to flexibly control PI3K and/or MT targeting in combination therapy. 1. M. P. Wymann, R. Schneiter, Nat Rev Mol Cell Biol 9, 162 (2008).; 2. S. M. Brachmann et al., Mol Cancer Ther 11, 1747 (2012). 3. J. C. Bendell et al., J Clin Oncol 30, 282 (2012). 4. C. Saura et al., Clin Cancer Res 20, 1935 (2014). 5. A. E. Prota et al., Science 339, 587 (2013). 6. S. M. Maira et al., Mol Cancer Ther 11, 317 (2012). Citation Format: Thomas Bohnacker, Florent Beaufils, Andrea E. Prota, John E. Burke, Anna Melone, Alison J. Inglis, Ludovico Fusco, Vladimir Cmiljanovic, Natasa Cmiljanovic, Denise Rageot, Katja Bargsten, Gonzalo Saez-Calvo, Olivier Pertz, Amol B. Aher, Anna Akhmanova, Fernando J. Diaz, Doriano Fabbro, Marketa Zvelebil, Roger L. Williams, Michel O. Steinmetz, Matthias P. Wymann. BKM120-mediated G2 arrest: Structural and functional segregation of off-target action and PI3K inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 671. doi:10.1158/1538-7445.AM2015-671

[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.

[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.

International audience

Highly cytotoxic amphidinolide macrolides have been prepared in a highly convergent and largely catalysis-based route. In their Full Paper on page 3983 ff., and two subsequent papers, A. Furstner et al. describe the total synthesis and biological evaluation of amphidinolide B1, B4, G1, H1, H2, V, X, and Y, as well as several analogues.