Potent mTOR Inhibitor Research Articles

Induction of Melanogenesis by Rapamycin in Human MNT-1 Melanoma Cells

BackgroundMelanogenesis is one of the characteristic parameters of differentiation in melanocytes and melanoma cells. Specific inhibitors of phosphatidylinositol 3-kinase (PI3K), such as wortmannin and LY294002, stimulate melanin production in mouse and in human melanoma cells, suggesting that PI3K and mammalian target of rapamycin (mTOR) might be involved in the regulation of melanogenesis.ObjectiveThe involvement of the mTOR pathway in regulating melanogenesis was examined using human MNT-1 melanoma cells, and the effects of the potent inhibitor of mTOR, rapamycin, in the presence or absence of α-melanocyte-stimulating hormone (α-MSH) were evaluated.MethodsIn cells treated with rapamycin, cell viability, melanin content, and tyrosinase (TYR) activity were measured and compared with untreated controls. Protein levels of TYR, tyrosinase-related protein (TYRP)-1, TYRP-2, and microphthalmia-associated transcription factor (MITF) were also analyzed by Western blot.ResultsIn rapamycin-treated cells, the melanin content increased concomitantly with an elevation in TYR activity, which plays a major role in melanogenesis. There was also an up-regulation of TYR, TYRP-1, and MITF proteins. Combined treatment with rapamycin or wortmannin and α-MSH increased melanogenesis more strongly than α-MSH alone.ConclusionRapamycin-induced melanin formation may be mediated through the up-regulation of TYR protein and activity. Furthermore, rapamycin and wortmannin, inhibitors of mTOR and PI3K, respectively, have co-stimulatory effects with α-MSH in enhancing melanogenesis in melanocyte cells.

PKI-587 and Sorafenib Targeting PI3K/AKT/mTOR and Ras/Raf/MAPK Pathways Synergistically Inhibit HCC Cell Proliferation

Deregulated Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PKI-587 and sorafenib as single agents or in combination on HCC (Huh7 cell line) proliferation. (3)H-thymidine incorporation and MTT assay were used to assess Huh7 cell proliferation. Phosphorylation of the key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways was detected by Western blot. We found that PKI-587 is a more potent PI3K/mTOR inhibitor than PI-103. Combination of PKI-587 and sorafenib was a more effective inhibitor of Huh7 proliferation than the combination of PI-103 and sorafenib. Combination of PKI-587 and sorafenib synergistically inhibited epidermal growth factor (EGF)-stimulated Huh7 proliferation compared with monodrug therapy. EGF increased phosphorylation of Ras/Raf downstream signaling proteins MEK and ERK; EGF-stimulated activation was inhibited by sorafenib. However, sorafenib, as a single agent, increased AKT (Ser473) phosphorylation. EGF-stimulated AKT (ser473) activation was inhibited by PKI-587. PKI-587 is a potent inhibitor of AKT (Ser473), mTOR (Ser2448), and S6K (Thr389) phosphorylation; in contrast, rapamycin stimulated mTOR complex 2 substrate AKT(Ser473) phosphorylation although it inhibited mTOR complex 1 substrate S6K phosphorylation. PKI-587, as a single agent, stimulated MEK and ERK phosphorylation. However, when PKI-587 and sorafenib were used in combination, they inhibited all the tested kinases in the Ras/Raf /MAPK and PI3K/AKT/mTOR pathways. The combination of PKI-587 and sorafenib has the advantage over monodrug therapy on inhibition of HCC cell proliferation by blocking bothPI3K/AKT/mTOR and Ras/Raf/MAPK signaling pathways.

Everolimus in Combination with Rituximab Induces Complete Responses in Heavily Pretreated Diffuse Large B-Cell Lymphoma

Abstract 1635 Background:Patients with diffuse large B cell lymphoma (DLBCL) who are primarily refractory to, or relapse after initial therapy with R-CHOP have a poor outcome and novel therapies are needed in these clinical scenarios. Pre-clinical data suggests that inhibition of mammalian target of rapamycin (mTOR) induces cell cycle arrest in DLBCL cell lines. We sought to investigate the activity of everolimus, a potent inhibitor of mTOR, combined with rituximab in this population Methods:We conducted a phase II study employing a Simon’s optimal two-stage design. Eligible subjects had previously received ≥1 prior line of chemotherapy and had either relapsed after autologous stem cell transplantation or were not candidates for transplant. Everolimus was administered orally once daily at 10 mg on days 1–28 of a 28-day cycle. Rituximab 375mg/m2 was administered IV weekly during the first cycle, then on Day 1 of cycles 2–6. Subjects received up to 6 cycles of treatment in the absence of disease progression. Patients without disease progression after 6 cycles continued everolimus monotherapy for up to 6 additional cycles in the absence of disease progression. The primary end point was overall response rate (ORR) as defined by the Revised International Workshop Response Criteria (2007). Secondary endpoints included progression free survival (PFS) at 1 year, duration of response, toxicity, and correlation of response with baseline tissue AKT, pAKT, and p70s6 kinase expression and p70s6 kinase expression from PBMC during treatment. The study was designed with 90% power to show a 30% ORR, with a 10% ORR considered unworthy for further study. Results:Between July 2009 and June 2010, 25 eligible subjects were enrolled. The median age was 65 (range 33–87). The median number of prior therapies was 4 (range 1–7), median time from prior therapy was 31 months (range 4–261), and 5 (20%) of subjects had undergone prior autologous stem cell transplant. Nineteen (76%) patients presented with advanced stage at relapse, 16 (64%) had an elevated LDH and 10 (40%) had greater than 1 extranodal site at relapse. The median number of cycles completed was 2 (range 0–8) and 5 (20%) subjects received more than 6 cycles. The ORR was 24% (90% CI [11%-42%] with 2 subjects having a CR and 4 with a PR. The median duration of response was 11 months. One subject with a CR remained on therapy a total of 12 cycles and is alive and free of disease 8 months post treatment. A second subject with a relapse after allogeneic transplant obtained CR to everolimus and remains in remission after donor lymphocyte infusion. A third subject with a PR completed 6 cycles followed by a full intensity unrelated allogeneic transplant and is alive and well at 19 months post transplant. At a median follow of 4.4 months (range 1.8–15), 29% of patients remained progression-free (95%CI [11%,47%]). The median progression-free survival was 2.6 months (95% CI [1.7, 3.8]). Fourteen patients remain alive. Treatment was well tolerated with 10 episodes of grade 3/4 neutropenia but only 1 episodes of febrile neutropenia. There were 3 episodes of grade 3 hypertriglyceridemia and no episodes of hyperglycemia. Correlative studies including pharmacodynamic evidence of mTOR inhibition and baseline activation of the mTOR pathway are ongoing. Conclusions:These data indicate that the combination of everolimus with rituximab is well tolerated and able to induce responses in a proportion of subjects with relapsed DLBCL, some of which are sustained and may serve as a bridge to allogeneic stem cell transplantation. Ongoing correlative studies are assessing predictive biomarkers of response. Disclosures:Off Label Use: Everolimus is not FDA-approved for treatment of DLBCL. Hochberg:Genentech: Consultancy. Fisher:Genentech: Consultancy. Abramson:Novartis: Consultancy; Genentech: Consultancy.

L-Tryptophan-mediated Enhancement of Susceptibility to Nonalcoholic Fatty Liver Disease Is Dependent on the Mammalian Target of Rapamycin

Nonalcoholic fatty liver disease is one of the most common liver diseases. l-Tryptophan and its metabolite serotonin are involved in hepatic lipid metabolism and inflammation. However, it is unclear whether l-tryptophan promotes hepatic steatosis. To explore this issue, we examined the role of l-tryptophan in mouse hepatic steatosis by using a high fat and high fructose diet (HFHFD) model. l-Tryptophan treatment in combination with an HFHFD exacerbated hepatic steatosis, expression of HNE-modified proteins, hydroxyproline content, and serum alanine aminotransaminase levels, whereas l-tryptophan alone did not result in these effects. We also found that l-tryptophan treatment increases serum serotonin levels. The introduction of adenoviral aromatic amino acid decarboxylase, which stimulates the serotonin synthesis from l-tryptophan, aggravated hepatic steatosis induced by the HFHFD. The fatty acid-induced accumulation of lipid was further increased by serotonin treatment in cultured hepatocytes. These results suggest that l-tryptophan increases the sensitivity to hepatic steatosis through serotonin production. Furthermore, l-tryptophan treatment, adenoviral AADC introduction, and serotonin treatment induced phosphorylation of the mammalian target of rapamycin (mTOR), and a potent mTOR inhibitor rapamycin attenuated hepatocyte lipid accumulation induced by fatty acid with serotonin. These results suggest the importance of mTOR activation for the exacerbation of hepatic steatosis. In conclusion, l-tryptophan exacerbates hepatic steatosis induced by HFHFD through serotonin-mediated activation of mTOR.

Alpha-ketoglutarate inhibits glutamine degradation and enhances protein synthesis in intestinal porcine epithelial cells

α-Ketoglutarate (AKG) is a key intermediate in glutamine metabolism. Emerging evidence shows beneficial effects of AKG on clinical and experimental nutrition, particularly with respect to intestinal growth and integrity. However, the underlying mechanisms are unknown. Intestinal porcine epithelial cells (IPEC-1) were used to test the hypothesis that AKG inhibits glutamine degradation and enhances protein synthesis. IPEC-1 cells were cultured for 3 days in Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing 0, 0.2, 0.5 or 2 mM of AKG. At the end of the 3-day culture, cells were used to determine L-[U-14C]glutamine utilization, protein concentration, protein synthesis, and the total and phosphorylated levels of the mammalian target of the rapamycin (mTOR), ribosomal protein S6 kinase-1 (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1). Compared with 0 mM of AKG (control), 0.2 and 0.5 mM of AKG dose-dependently reduced (P<0.05) glutamine degradation and the production of glutamate, alanine and aspartate in IPEC-1 cells. Addition of 0.5 and 2 mM of AKG to culture medium enhanced protein synthesis (P<0.05) by 78 and 101% without affecting protein degradation, compared to the control group. Rapamycin (50 nM; a potent inhibitor of mTOR) attenuated the stimulatory effect of AKG on protein synthesis. Consistent with these metabolic data, the addition of 0.5 or 2 mM of AKG to culture medium increased (P<0.05) the phosphorylated levels of mTOR, S6k1 and 4E-BP1 proteins. Collectively, these results indicate that AKG can spare glutamine and activate the mTOR signaling pathway to stimulate protein synthesis in intestinal epithelial cells.

Structure–Activity Relationships of Phosphoinositide 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitors: Investigations of Various 6,5-Heterocycles to Improve Metabolic Stability

N-(6-(6-Chloro-5-(4-fluorophenylsulfonamido)pyridin-3-yl)benzo[d]thiazol-2-yl)acetamide (1) is a potent and efficacious inhibitor of PI3Kα and mTOR in vitro and in vivo. However, in hepatocyte and in vivo metabolism studies, 1 was found to undergo deacetylation on the 2-amino substituent of the benzothiazole. As an approach to reduce or eliminate this metabolic deacetylation, a variety of 6,5-heterocyclic analogues were examined as an alternative to the benzothiazole ring. Imidazopyridazine 10 was found to have similar in vitro potency and in vivo efficacy relative to 1, while only minimal amounts of the corresponding deacetylated metabolite of 10 were observed in hepatocytes.

3D-QSAR and Docking Studies of 4-morpholinopyrrolopyrimidine Derivatives as Potent mTOR Inhibitors

3D-QSAR and Docking Studies of 4-morpholinopyrrolopyrimidine Derivatives as Potent mTOR Inhibitors

Discovery and Optimization of a Series of Benzothiazole Phosphoinositide 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitors

Phosphoinositide 3-kinase α (PI3Kα) is a lipid kinase that plays a key regulatory role in several cellular processes. The mutation or amplification of this kinase in humans has been implicated in the growth of multiple tumor types. Consequently, PI3Kα has become a target of intense research for drug discovery. Our studies began with the identification of benzothiazole compound 1 from a high throughput screen. Extensive SAR studies led to the discovery of sulfonamide 45 as an early lead, based on its in vitro cellular potency. Subsequent modifications of the central pyrimidine ring dramatically improved enzyme and cellular potency and led to the identification of chloropyridine 70. Further arylsulfonamide SAR studies optimized in vitro clearance and led to the identification of 82 as a potent dual inhibitor of PI3K and mTOR. This molecule exhibited potent enzyme and cell activity, low clearance, and high oral bioavailability. In addition, compound 82 demonstrated tumor growth inhibition in U-87 MG, A549, and HCT116 tumor xenograft models.

Current and future directions in mammalian target of rapamycin inhibitors development

Introduction: The mammalian target of the rapamycin (mTOR) signalling pathway has a central role in the regulation of cell growth, survival and angiogenesis and the frequent dysregulation of this pathway in tumor cells makes it a crucial target in the treatment of cancer. Temsirolimus and everolimus are approved for use in metastatic renal cell carcinoma and temsirolimus is also approved for mantle cell lymphoma. All three rapalogs, temsirolimus, everolimus and deforolimus, are currently being evaluated in Phase III studies in several tumors.Areas covered: This paper provides a review of the published literature on the mTOR pathway and related pathway signaling, analogs and novel mTOR inhibitors. The most recent and important data on the mTOR pathway, the role of mTOR inhibitors in cancer treatment and the current status of development of second-generation highly potent and selective mTOR inhibitors are overviewed.Expert opinion: The published data on new mTOR inhibitors are still limited, but the available preclinical results indicate that they have a potent antiproliferative activity against a broad panel of tumor cell lines, have a favorable safety profile, can obtain disease stabilization or even tumor regression and, in some cases, enhance the efficacy of other targeted or standard-of-care anticancer drugs when used in vivo in preclinical studies.

Abstract LB-70: In vitro anti-cancer activity of PF-04691502, a potent dual inhibitor of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) in a panel of human breast cancer cell lines

Abstract Introduction: Activating mutations in the PIK3CA gene (encoding for the P110 catalytic subunit of PI3K) are found in more than 25% of human breast cancers [Saal LH Cancer Res 2005]. PF-04691502 is a potent orally-available ATP-competitive and reversible dual inhibitor of PI3K and mTOR. This study evaluates the anti-proliferative and cytotoxic activity of PF-04691502 across a large panel of breast cancer cell lines that recapitulate the clinical spectrum of the disease. Methods: Over 40 human breast cancer cell lines were exposed in vitro to PF-0469102 over 9 concentrations to generate dose-response curves. These assays were performed over a 6-day incubation period using direct cell counts. The ability of PF-04691502 to inhibit proliferation and induce cell death was calculated using IC50 and LD50 values, respectively. PI3K mutation status was available from public databases. In addition, baseline gene expression profiles were generated using the Agilent platform. A subset of breast cancer cell lines with varying levels of sensitivity to the compound were incubated with PF-04691502 concentrations ranging from 10 to 1000 nM, and processed for Western blotting. Results: Twenty-two cell lines had IC50 values &amp;lt; 100 nM, with a trend towards increased sensitivity to PF-04691502 in breast cancer cell lines harboring PIK3CA mutations, and in those belonging to the luminal subtype. Lethality (LD50 &amp;lt; 1 M) was observed in 10 out of 12 breast cancer cell lines harboring PI3KCA mutations, and was prevalent in cell lines of luminal subtype. Inhibition of the downstream target pAKT was observed. Conclusions: PF-04691502 has anti-cancer activity across a spectrum of breast cancer cell lines, and demonstrates lethality against a subset of cell lines. A first-in-human phase I trial of PF-04691502 is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-70.

Abstract 3224: Preclinical PKPD modeling and human dose projection of PF-04691502, a PI3K/mTOR dual inhibitor

Abstract PF-04691502 is a potent dual inhibitor of PI3K and mTOR, which are involved in regulating cell growth, proliferation and survival. The main objective of this work was to develop a preclinical pharmacokinetic-pharmacodynamic (PKPD) model for the compound and project the human efficacious dose. The PK of PF-04691502 in mouse obtained following oral dosing was described by a linear 2-compartment model which was used to drive two separate PD models: 1) an exponential growth model describing the tumor growth inhibition (TGI) and 2) an indirect-response model describing changes in intratumoral pAKT-S473/AKT in the U87MG xenograft mouse model. Based on the PK model, the half-life calculated from the linear elimination constant (k = 0.306 hr−1) was 2.3 hr and the plasma-compartment volume of distribution was 8.72 L/kg. The inter-compartmental distribution rate constants were 0.282 hr−1 and 0.212 hr−1 for k12 and k21, respectively. The model estimated in-vivo potency (IC50) for pAKT-S473/AKT suppression was 38.3 nM which was within the range of the IC50 (23 nM) for TGI (as parameterized by the model). The Imax estimated from the model was 0.975 (or 97.5%), which indicated nearly complete suppression of pAKT-S473/AKT production rate by PF-04691502. Based on the PKPD model and the scaled human PK, a clinical dose of 10 mg QD was determined to be adequate for maintaining the level of pAKT-S473/AKT below 50% of baseline. At steady-state PK this dose yielded a range of 50-80% human pAKT-S473/AKT suppression over the 24 hr dosing interval. In conclusion, PF-04691502 was determined to have a robust PKPD relationship in the preclinical animal model and was projected to have a clinically achievable efficacious dose of approximately 10 mg QD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3224.

Discovery of 2-ureidophenyltriazines bearing bridged morpholines as potent and selective ATP-competitive mTOR inhibitors

Incorporation of bridged morpholines in monocyclic triazine PI3K/mTOR inhibitors gave compounds with increased mTOR selectivity relative to the corresponding morpholine analogs. Compounds with ureidophenyl groups gave highly potent and selective mTOR inhibitors. Potency and selectivity was demonstrated both in vitro and in vivo through biomarker suppression studies. Select compounds exhibited potent inhibition of tumor growth in nude mouse xenograft assays upon PO and IV administration.

2-Arylureidophenyl-4-(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)triazines as highly potent and selective ATP competitive mTOR inhibitors: Optimization of human microsomal stability

Isosteric replacement of one of the 3,5-ethylene-bridged morpholines in 2-arylureidophenyl-4,6-di(3-oxa-8-azabicyclo[3.2.1]octan-8-yl)triazines led to significant improvements in human microsomal stability. 3- R-Me-morpholine and tetrahydropyran were identified as preferred isosteres for the bridged morpholine. Combination of tetrahydropyran substitution with an N-Me-piperazinophenylureido group led to 27, that selectively suppressed mTOR biomarkers in vivo and possessed excellent efficacy in a murine xenograft model.

Insulin Induces REDD1 Expression through Hypoxia-inducible Factor 1 Activation in Adipocytes

REDD1 (regulated in development and DNA damage responses) is essential for the inhibition of mTORC1 (mammalian target of rapamycin complex) signaling pathway in response to hypoxia. REDD1 expression is regulated by many stresses such as hypoxia, oxidative stress, and energy depletion. However, the regulation of REDD1 expression in response to insulin remains unknown. In the present study, we demonstrate that in murine and in human adipocytes, insulin stimulates REDD1 expression. Insulin-induced REDD1 expression occurs through phosphoinositide 3-kinase/mTOR-dependent pathways. Moreover, using echinomycin, a hypoxia-inducible factor 1 (HIF-1) inhibitor, and HIF-1alpha small interfering RNA, we demonstrate that insulin stimulates REDD1 expression only through the transcription factor HIF-1. In conclusion, our study shows that insulin stimulates REDD1 expression in adipocytes.

Immunohistochemical expression of mTOR protein in breast carcinoma tissues

Immunohistochemical expression of mTOR protein in breast carcinoma tissues

Abstract B138: SB2312, a novel and potent dual inhibitor of mTOR and PI3K with high target inhibition and antitumor efficacy

Abstract Background: Dysregulation of the PI3K signaling pathway, either through activating mutations, amplification of PI3K or deletion of PTEN has been linked to a wide range of hyperproliferative diseases. The development of small molecule modulators of key proteins in this signaling cascade has focused mainly on mTOR or PI3K. Recent investigations have shown a significant overlap in the activity of small molecule inhibitors against these two proteins. Inhibitors that are currently progressing through preclinical and early clinical development have been shown to affect both mTOR and PI3K to some extent. However, most reported inhibitors do not inhibit both of these kinases in a close dose range. Our drug discovery program has focused on equipotent inhibition of both targets. SB2312 is a novel compound with very favorable drug-like properties, giving rise to equivalent targeting of both mTOR and PI3K in cells and tumor tissue and resulting in a more efficient inhibition of this pathway compared to reference compounds currently under evaluation in clinical trials. Methods: In vitro-IC50 for mTOR and PI3K were determined using recombinant enzymes. Inhibitory activity of SB2312 on the mTOR/PI3K signaling pathway was determined by Western-blot analysis, Surefire assays, and proliferation assays in various cancer cell lines. Efficacious doses of SB2312 or reference compounds were selected after pharmacokinetic and biomarker analysis of PC3 tumor-bearing mice, treated with single oral doses. Anti-tumor efficacy of SB2312 and reference compounds was evaluated in mouse xenograft models after continuous daily oral dosing. Results: SB2312 is a potent and selective dual mTOR and PI3K inhibitor (IC50: 55 nM and 18 nM, respectively) and has no significant activity on a large panel of protein kinases or enzymes and receptors. Efficient and equivalent inhibition of mTOR and PI3K in human cell lines was demonstrated by blockade of the phosphorylation of S6KT389 (IC50= 70 nM) and AKTS473 (IC50= 21 nM). SB2312 inhibits the proliferation of a broad range of human tumor cell lines independently of their PI3K pathway mutation status (IC50= 112–882 nM) and is also effective against rapamycin-resistant cell lines. SB2312 has very favorable physicochemical and pharmacokinetic properties, with high oral bioavailability and tumor exposure. Efficient and sustained inhibition of PI3K and mTOR was demonstrated in tumor tissues from a PC3 tumor xenograft model, along with dose-dependent anti-tumor activity at very well tolerated doses. Tumor growth inhibition was 53%, 105%, and 117% at 11, 33, and 80 mg/kg SB2312, respectively, without any body weight loss at any of these doses. Summary: Herein, we report SB2312, a novel, potent and selective dual mTOR/PI3K inhibitor with excellent drug-like properties. Our studies show superior and sustained blockade of the mTOR/PI3K pathway and high antitumor efficacy at well tolerated doses. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B138.

Discovery of 3,6-dihydro-2 H-pyran as a morpholine replacement in 6-aryl-1 H-pyrazolo[3,4- d]pyrimidines and 2-arylthieno[3,2- d]pyrimidines: ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR)

The morpholine hinge-region binding group on a series of pyrazolopyrimidine and thienopyrimidine mammalian target of rapamycin (mTOR) inhibitors was replaced with 3,6-dihydro-2 H-pyran (DHP), giving compounds of equivalent potency and selectivity versus PI3K. These results establish the DHP group as a hinge-region binding motif for the preparation of highly potent and selective mTOR inhibitors.

Incorporation of water-solubilizing groups in pyrazolopyrimidine mTOR inhibitors: Discovery of highly potent and selective analogs with improved human microsomal stability

A series of highly potent and selective pyrazolopyrimidine mTOR inhibitors which contain water-solubilizing groups attached to the 6-arylureidophenyl moiety have been prepared. Such derivatives displayed superior potency to those in which these appendages were attached to alternative sites. In comparison to unfunctionalized arylureido compounds, these analogs demonstrated enhanced cellular potency and significantly improved stability towards human microsomes, resulting in an mTOR inhibitor with impressive efficacy in a xenograft model with an intermittent dosing regimen.

Discovery of 2-arylthieno[3,2-d]pyrimidines containing 8-oxa-3-azabi-cyclo[3.2.1]octane in the 4-position as potent inhibitors of mTOR with selectivity over PI3K

2-Aryl-4-morpholinothieno[3,2-d]pyrimidines are known PI3K inhibitors. This class of compounds also potently inhibited the homologous enzyme mTOR. Replacement of the morpholine group in these compounds with an 8-oxa-3-azabicyclo[3.2.1]octane group led to mTOR inhibitors with selectivity over PI3K. Optimization of the 2-aryl substituent led to the discovery of 2-(4-ureidophenyl)-thienopyrimidines as highly potent (IC(50) <1nM) mTOR inhibitors with excellent selectivity (up to >1000-fold) over PI3K and good potency in a cellular proliferation assay (IC(50) <50nM).

Discovery of Potent and Selective Inhibitors of the Mammalian Target of Rapamycin (mTOR) Kinase

The mammalian target of rapamycin (mTOR) is a central regulator of cell growth, metabolism, and angiogenesis and an emerging target in cancer research. High throughput screening (HTS) of our compound collection led to the identification of 3-(4-morpholin-4-yl-1-piperidin-4-yl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)phenol (5a), a modestly potent and nonselective inhibitor of mTOR and phosphoinositide 3-kinase (PI3K). Optimization of compound 5a, employing an mTOR homology model based on an X-ray crystal structure of closely related PI3Kgamma led to the discovery of 6-(1H-indol-5-yl)-4-morpholin-4-yl-1-[1-(pyridin-3-ylmethyl)piperidin-4-yl]-1H-pyrazolo[3,4-d]pyrimidine (5u), a potent and selective mTOR inhibitor (mTOR IC(50) = 9 nM; PI3Kalpha IC(50) = 1962 nM). Compound 5u selectively inhibited cellular biomarker of mTORC1 (P-S6K, P-4EBP1) and mTORC2 (P-AKT S473) over the biomarker of PI3K/PDK1 (P-AKT T308) and did not inhibit PI3K-related kinases (PIKKs) in cellular assays. These pyrazolopyrimidines represent an exciting new series of mTOR-selective inhibitors with potential for development for cancer therapy.