Florent Beaufils

Abstract 4514: PQR309: A potent, brain-penetrant, dual pan-PI3K/mTOR inhibitor with excellent oral bioavailability and tolerability

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

Abstract 2664: PQR309: Structure-based design, synthesis and biological evaluation of a novel, selective, dual pan-PI3K/mTOR inhibitor

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

Abstract 2652: Pre-clinical activity and mechanism of action of the novel dual PI3K/mTOR inhibitor PQR309 in B-cell lymphomas

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

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

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. HighlightsThe mTOR signaling pathway has emerged as a therapeutic target for epilepsy.However, clinically used mTOR inhibitors exhibit poor brain penetration and tolerability.Two novel inhibitors of mTORC1/2 or the PI3K/mTORC1/2 pathway are presented.Both compounds cross the blood‐brain barrier more effectively than everolimus.Both compounds increase seizure threshold in a mouse model of difficult‐to‐treat epilepsy.

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.

Abstract 153: Tricyclic fused pyrimidinopyrrolo-oxazines reveal conformational preferences of morpholine for PI3K hinge region binding

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

Abstract 1336: Structure-activity relationship studies, synthesis, and biological evaluation of PQR620, a highly potent and selective mTORC1/2 inhibitor

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, Jurgen 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.

Abstract 393A: Pharmacological characterization of the selective, orally bioavailable, potent mTORC1/2 inhibitor PQR620

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 (t 1/2 ) for plasma and brain was approximately 5 hours. After 8 hours, the total exposure (expressed as AUC 0-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.