Abstract Resistance to targeted therapies is a major challenge in oncology. Genetic mechanisms of resistance such as gatekeeper mutations in the primary drug target can be readily identified. However, drug targets responsible for non-genetic resistance mechanisms such as transcriptional reprogramming remain elusive. Here we describe an innovative phenotypic screening approach that led to the development of the clinical candidate TT125-802 and identification of its target CBP/p300 which we discovered as a novel regulator of transcriptional resistance to a range of targeted therapies. Targeted cancer therapies have been reported to induce profound transcriptional reprogramming leading to drug resistance. For instance, inhibition of oncogenic MAPK signaling using a BRAF/MEK inhibitor combination in BRAF-V600E mutated melanoma, triggers expression of the pluripotency transcription factor SOX2 along with broader expression of associated stemness and EMT genes. We used this system as a model to screen for small molecules that prevent transcriptional escape mechanisms to targeted cancer therapies. For this, we developed an automated high-throughput immunofluorescence staining and analysis method of SOX2 as a surrogate marker for resistance-conferring transcriptional reprogramming. A hit-like high diversity library of 16’000 small molecules was screened using an image-based multi-parameter readout with an integrated counter-screen for general cell toxicity. A battery of downstream assessments including patentability, synthetic tractability, physico-chemical properties, and dose-dependent modulation of transcriptional resistance signatures was used to select the chemical scaffold TT125 for further hit to lead optimization. Hit to lead optimization was initially target agnostic. To de-orphan the scaffold, a chemical probe of TT125 was developed in house and used in a chemoproteomics assay. The transcriptional co-activators CBP/p300 were identified as the target of TT125. Biochemical assays revealed that the TT125 scaffold binds specifically to the bromodomain of CBP/p300 but not the histone acetyl transferase domain. Moreover, TT125 was confirmed to bind highly selectively to the bromodomain of CBP/p300 but not other bromodomain-containing proteins such as BRD2 or BRD4. Structure-based drug design along with in vivo PD models using transcriptional short-term readouts were used for lead optimization that resulted in the development of the potent and highly selective clinical candidate TT125-802. Validating our phenotypic approach to drug discovery and development, TT125-802 is currently being evaluated in a phase I oncology trial. The modular setup of our phenotypic screening system has the potential to power the discovery of additional novel modulators of transcriptional drug resistance. Citation Format: Dorothea Gruber, Charles-Henry Fabritius, Thomas Bohnacker, Martin Schwill, Sara Laudato, Raquel Herrador, Katrin Westritschnig, Thushara Pattupara, Stefanie Flückiger-Mangual. An image-based phenotypic screen identified CBP/p300 as new cancer drug resistance target and enabled the development of the clinical candidate TT125-802 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5891.

Chemical probes have gained importance in the elucidation of signal transduction in biology. Insufficient selectivity and potency, lack of cellular activity and inappropriate use of chemical probes has major consequences on interpretation of biological results. The catalytic subunit of phosphoinositide 3-kinase α (PI3Kα) is one of the most frequently mutated genes in cancer, but fast-acting, high-quality probes to define PI3Kα's specific function to clearly separate it from other class I PI3K isoforms, are not available. Here, we present a series of novel covalent PI3Kα-targeting probes with optimized intracellular target access and kinetic parameters. On-target TR-FRET and off-target assays provided relevant kinetic parameters (k chem, k inact and K i) to validate our chemical probes. Additional intracellular nanoBRET tracer displacement measurements showed rapid diffusion across the cell membrane and extremely fast target engagement, while investigations of signaling downstream of PI3Kα via protein kinase B (PKB/Akt) and forkhead box O (FOXO) revealed blunted pathway activity in cancer cell lines with constitutively activated PI3Kα lasting for several days. In contrast, persistent PI3Kα inhibition was rapidly bypassed by other class I PI3K isoforms in cells lacking functional phosphatase and tensin homolog (PTEN). Comparing the rapidly-diffusing, fast target-engaging chemical probe 9 to clinical reversible PI3Kα-selective inhibitors alpelisib, inavolisib and 9r, a reversible analogue of 9, revealed 9's superior potency to inhibit growth (up to 600-fold) associated with sustained suppression of PI3Kα signaling in breast cancer cell lines. Finally, using a simple washout protocol, the utility of the highly-selective covalent PI3Kα probe 9 was demonstrated by the quantification of the coupling of insulin, EGF and CXCL12 receptors to distinct PI3K isoforms for signal transduction in response to ligand-dependent activation. Collectively, these findings along with the novel covalent chemical probes against PI3Kα provide insights into isoform-specific functions in cancer cells and highlight opportunities to achieve improved selectivity and long-lasting efficacy.

Resistance to targeted therapies is a major challenge in oncology. Disease progression is caused by multiple resistance mechanisms. Besides pre-existing and acquired genetic alternations, adaptive non-mutational reprogramming as well as modulation of phenotypic plasticity have emerged as drivers of disease progression. For example, in epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer (NSCLC) patients who were treated with osimertinib, ~50% of progressive disease could not be attributed to genetic mutations (Leonetti et al., BJC 2019). Similarly, in ~ 40% of patients suffering from KRAS G12C-mutated NSCLC and colorectal cancer (CRC), disease progressed under sotorasib treatment without identifiable acquired mutations (Zhao et al., Nature 2021). Thus, targeting non-genetic adaptive resistance mechanism such as drug-induced transcriptional reprogramming might be of great therapeutic benefit. Here we identified small molecules, that interfere with cancer drug-induced transcriptional escape mechanisms using a phenotypic screen based on an SRY-Box Transcription Factor 2 (SOX2) reporter system. The screen led to the development of TT125-802, a highly specific and potent, orally available small molecule inhibitor of the bromodomain of the transcriptional and epigenetic regulator CBP [cyclic adenosine monophosphate response element binding protein (CREB) binding protein] and its paralogue p300. TT125-802 dose-dependently prevented osimertinib resistance development in EGFR-mutated NSCLC cell lines HCC827 and HCC4006, as well as sotorasib resistance development in KRAS G12C-mutated NCI-H358 (NSCLC), SW837 and SNU-1411 (CRCs) as assessed by label-free long-term live microscopy assays. Data generated in mouse xenograft studies confirmed the ability of TT125-802 increasing response rates and prolonging the duration of response to osimertinib and sotorasib in vivo. In cells and tumours which were already resistant to osimertinib or sotorasib, TT125-802 could still delay cell or tumor growth. Complementary and longitudinal analysis of transcriptional changes using RNA sequencing in vitro and in vivo identified several early adaptive and late acquired resistance signatures that were reversed by TT125-802. A first-in-human study of TT125-802 in cancer patients is on track to start in 2023. Citation Format: Thomas Bohnacker, Dorothea Gruber, Sara Laudato, Martin Schwill, Charles-Henry Fabritius, Raquel Herrador, Katrin Westritschnig, Thushara Pattupara, Vikram Ayinampudi, Stefanie Flückiger-Mangual. Targeting adaptive resistance to EGFR and KRAS G12C inhibitors by TT125-802, a novel and specific CBP/p300 bromodomain inhibitor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3907.

The paralogous lysine acetyltransferases CREB-binding protein (CBP) and p300 are key epigenetic regulators involved in diverse signaling pathways in cancer. The bromodomain (BRD) of CBP/p300 serves as an acetyl-lysine “reader” that allows CBP/p300 to bind chromatin at acetylated histone and non-histone proteins leading to the regulation of gene transcription. Indeed, CBP/p300 are critical co-activators of nuclear receptors, including the androgen receptor (AR) in castration resistant prostate cancer (CRPC). Thus, inhibition of CBP and p300 is an emerging therapeutic strategy to block the transactivation activity of the AR in CRPC. In addition, inhibition of the CBP/p300 BRD has been described as a potential therapeutic strategy to treat multiple myeloma (MM) through transcriptional suppression of interferon regulatory factor 4 (IRF4) and concomitant repression of its target genes MYC and MYB. TT125-802 is a highly selective and potent, oral small molecule inhibitor of the BRD of CBP/p300. In a BROMOscan assay against a panel of 40 BRD-containing proteins, TT125-802 revealed the unique selectivity to the BRD of CBP/p300 with a minimal off-target binding to all other BRDs, including BET proteins.TT125-802 shows selective anti-proliferative activity in AR-dependent prostate cancer cell lines (22Rv-1, C4-2, and LNCaP) and inhibits AR target gene expression (KLK2, KLK3, TMPRSS2, and MYC) in a dose-dependent manner (IC50 of 2 to 33 nM). AR-negative prostate cancer cell lines (DU-145 and PC-3) are insensitive to TT125-802 in vitro, pointing to an AR-selective mode of action. In an in vivo model of CRPC, TT125-802 inhibited tumor growth when administered orally to human C4-2 xenograft-bearing mice. Daily dosing of TT125-802 was well tolerated with stable bodyweights and platelet counts. In addition, preclinical studies in CRPC patient-derived xenograft (PDX)-bearing mice showed that the combination treatment of TT125-802 and enzalutamide had a synergistic effect on tumor growth inhibition compared to single agent treatments. TT125-802 reduced mRNA expression of the AR-target genes in tumor samples and decreased plasma PSA levels compared to enzalutamide alone, and the combination reduced levels even further. In a preclinical model of MM (OPM2), TT125-802 had a dose-dependent effect on tumor growth, inducing tumor regressions at the highest dose. Target genes such as MYC, MYB, and IRF4 were potently downregulated in tumors. We conclude that TT125-802 is a novel, highly selective inhibitor of the BRD of CBP/P300. It has therapeutic potential as monotherapy in prostate cancer and multiple myeloma and in combination with next-generation AR inhibitors for patients with lethal prostate cancer. A FIH study of TT125-802 in cancer patients is on track to start in 2023. Citation Format: Sara Laudato, Dorothea Gruber, Thomas Bohnacker, Martin Schwill, Charles-Henry Fabritius, Raquel Herrador, Katrin Westritschnig, Thushara Pattupara, Vikram Ayinampudi, Stefanie Flückiger-Mangual. TT125-802 is a potent and highly selective CBP/p300 bromodomain inhibitor for the treatment of castration resistant prostate cancer and haematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6268.

Mast cells are the major effector cells in immunoglobulin E (IgE)-mediated allergy. The high affinity IgE receptor FcεRI, as well as G protein-coupled receptors (GPCRs) on the mast cell surface signals to phosphoinositide 3-kinase γ (PI3Kγ) to initiate degranulation, cytokine release, and chemotaxis. PI3Kγ is therefore considered as a target for treatment of allergic disorders. However, leukocyte PI3Kγ is key to many functions in innate and adaptive immunity, and attenuation of host defense mechanisms is an expected adverse effect that complicates treatment of chronic illnesses. PI3Kγ operates as a p110γ/p84 or p110γ/p101 complex, where p110γ/p84 requires Ras activation. Here we investigated if modulation of Ras-isoprenylation could target PI3Kγ activity to attenuate PI3Kγ-dependent mast cell responses without impairment of macrophage functions. In murine bone marrow-derived mast cells, GPCR stimulation triggers activation of N-Ras and H-Ras isoforms, which is followed by the phosphorylation of protein kinase B (PKB/Akt) relayed through PI3Kγ. Although K-Ras is normally not activated in Ras wild-type cells, it is able to compensate for genetically deleted N- and H-Ras isoforms. Inhibition of Ras isoprenylation with farnesyltransferase inhibitor FTI-277 leads to a significant reduction of mast cell degranulation, cytokine production, and migration. Complementation experiments expressing PI3Kγ adaptor proteins p84 or p101 demonstrated a differential sensitivity towards Ras-inhibition depending on PI3Kγ complex composition. Mast cell responses are exclusively p84-dependent and were effectively controlled by FTI-277. Similar results were obtained when GTP-Ras was inactivated by overexpression of the GAP-domain of Neurofibromin-1 (NF-1). Unlike mast cells, macrophages express p84 and p101 but are p101-dominated and thus remain functional under treatment with FTI-277. Our work demonstrates that p101 and p84 have distinct physiological roles, and that Ras dependence of PI3Kγ signaling differs between cell types. FTI-277 reduces GPCR-activated PI3Kγ responses in p84-expressing but not p101-containing bone marrow derived cells. However, prenylation inhibitors have pleiotropic effects beyond Ras and non-tolerable side-effects that disfavor further clinical validation. Statins are, however, clinically well-established drugs that have previously been proposed to block mast cell degranulation by interference with protein prenylation. We show here that Simvastatin inhibits mast cell degranulation, but that this does not occur via Ras-PI3Kγ pathway alterations.

Abstract Mechanistic target of rapamycin (mTOR), a key player in cell proliferation, growth and survival, is overactivated in tumors and neurological disorders.[1] Rapalogs have recently been explored to alleviate epileptic seizures in Tuberous Sclerosis Complex (TSC).[2] Herein, we combined pharmacophore features of PQR620[3], the first-in-class brain penetrant ATP-competitive mTOR kinase inhibitor showing efficacy in a TSC mouse model, and PQR617[4], a potent, highly selective mTOR inhibitor. An extensive chemical exploration of the morpholine ring led to the discovery of PQR626, a highly potent, selective, brain penetrant inhibitor of mTORC1/2 kinase. In male Sprague Dawley rats and female C57BL/6J mice, PQR626 displayed an excellent brain penetration compared to everolimus, which possessed a limited ability to cross the blood-brain-barrier (brain/plasma levels ~ 1.4:1 vs 1:92). An additional pharmacokinetic comparative study with everolimus and AZD2014 confirmed the superiority of PQR626 [brain/plasma levels: ~ 1:25 (AZD2014), ~ 1:61 (everolimus), ~ 1.8:1 (PQR626)]. PQR626 showed very good tolerability in mice (MTD 100-150 mg/kg). Efficacy studies, using mice with conditional inactivation of the Tsc1 gene primarily in glia (Tsc1GFAPCKO mice), were performed in a dose-range finding study. PQR626 (50 mg/kg, BID - twice a day) showed a significant effect on survival and significantly prevented/decreased mortality as compared to the vehicle. On the basis of its favorable pharmacological parameters, excellent brain penetration, safety profile and efficacy in Tsc1GFAPCKO mice, PQR626 qualifies as a novel mTOR inhibitor with potential application in the treatment of epilepsy and neurological disorders. [1] Wymann M. P. and Schneiter R. Nat. Rev. Mol. Cell Biol. 2008, 9, 162-176. [2] Krueger D. A. et. al. Neurology 2016, 87, 2408-2415. [3] Rageot D. et. al. J Med Chem. 2018, 61 (22), 10084-10105. [4] Borsari C. et. al. J Med Chem. 2019, 62 (18), 8609-8630. Citation Format: Chiara Borsari, Erhan Keles, Denise Rageot, Anna Melone, Thomas Bohnacker, Lucinda Kate Batchelor, Martina De Pascale, Paul Hebeisen, Petra Hillmann, Doriano Fabbro, Matthias Wymann. Discovery and preclinical characterization of PQR626: A potent, orally available, and brain-penetrant mTOR inhibitor for the treatment of tuberous sclerosis complex [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 665.

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.

We have discovered a class of PI3Kγ inhibitors exhibiting over 1,000-fold selectivity over PI3Kα and PI3Kβ. On the basis of X-ray crystallography, hydrogen-deuterium exchange–mass spectrometry and surface plasmon resonance experiments we propose that the cyclopropylethyl moiety displaces the DFG motif of the enzyme away from the adenosine tri-phosphate binding site, inducing a large conformational change in both the kinase- and helical domains of PI3Kγ. Site directed mutagenesis explained how the conformational changes occur. Our results suggest that these cyclopropylethyl substituted compounds selectively inhibit the active state of PI3Kγ, which is unique to these compounds and to the PI3Kγ isoform, explaining their excellent potency and unmatched isoform selectivity that were confirmed in cellular systems. This is the first example of a Class I PI3K inhibitor achieving its selectivity by affecting the DFG motif in a manner that bears similarity to DFG in/out for type II protein kinase inhibitors.

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 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 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.

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.

Background. Diffuse large B-cell lymphoma (DLBCL) is the commonest lymphoma in both humans and dogs. Canine DLBCL (cDLBCL) is considered an ideal comparative model for drug development, but a complete genomic characterization of this tumor is still lacking. In this study, we report an integrated analysis to comprehensively define the molecular mechanisms of cDLBCL and possible associations with clinical outcome. Methods. Fifty cDLBCLs were analyzed by RNA-Seq, methyl-CpG-binding sequencing and array comparative genomic hybridization. Normal B-cells derived from lymph nodes of 11 healthy dogs were used as controls.Additionally, immunohistochemistry, in vitroand in vivoexperiments were performed as validation analyses. Results.Compared to normal B-cells, cDLBCL showed a marked up-regulation of genes involved in the PI3K/mTOR and NF-κB pathways, including several TLRs in association with MYD88, indicating mechanisms similar to the human activated B cell-like subtype DLBCL. Both RNA-Seq and methylation sequencing led to the identification of two groups of cDLBCLs bearing different clinical outcome. The two groups did not overlap with the human germinal center B-cell (GCB) and the activated B-cell-like (ABC) DLBCL subtypes or the human DLBCL consensus clusters. The dogs with the poorest outcome presented a signature largely defined by markers of T-cell-mediated immune responses, with a high expression of PDL-1, PD-1 and CTLA-4, also validated in an independent cohort of cDLBCL by immunohistochemistry. These data provide a strong rationale for the use of cDLBCL to study immune checkpoint modulators. The observed high expression of PI3K/mTOR pathway genes was confirmed and validated achieving a clear anti-tumor activity with the use of the PI3K-delta inhibitor idelalisib and of the novel dual PI3K/mTOR inhibitor bimiralisib in the cDLBCL cell line CLBL-1. The cDLBCLs showed an up-regulation of MYC and of its targets, sustained by recurrent gains in the chromosome 13, where the oncogene is located, in approximately half of the cases. Thus, we have exposed the cDLBCL cell line CLBL-1 to the BET inhibitor birabresib (OTX015) and to the BRD4 degrader MZ1. Both compounds caused a significant reduction in the proliferation of tumor cells, and this effect was stronger especially with the second compound. Exposure to MZ1 determined an important downregulation of MYC and also of LIN28B, the most overexpressed transcript in cDLBCL when compared to controls. While LIN28B does not seem to be a relevant gene for human DLBCL, its overexpression causes murine T-cell lymphomas (Beachy et al, Blood 2011), and there is a direct association of MYC with LIN28B promoter resulting in transcriptional transactivation (Chang et al, PNAS 2009). Here, LIN28B genetic silencing in the CLBL-1 lead to a reduction in cell growth, opening new therapeutic target perspectives in canine lymphoma. Conclusions. We have reported the first large next generation sequencing study investigating the cDLBCL transcriptome, methylome and the genome-wide CNVs. We identified deregulated pathways and individual transcripts providing therapeutic targets, including an immune-related signature affecting the outcome of a subgroup of cDLBCL. Our data sustain the use of cDLBCL as comparative models for human DLBCL but also highlight differences that must be kept in consideration. Hillmann: PIQUR Therapeutics AG: Employment. Wymann:PIQUR Therapeutics AG: Employment, Equity Ownership, Patents & Royalties.

Abstract 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

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.

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