Tables S1-S3, Figures S1-S5

Flavaglines such as silvestrol (1) and rocaglamide (2) constitute an interesting class of natural products with promising anticancer activities. Their mode of action is based on inhibition of eukaryotic initiation factor 4A (eIF4A) dependent translation through formation of a stable ternary complex with eIF4A and mRNA, thus blocking ribosome scanning. Herein we describe initial SAR studies in a novel series of 1-aminomethyl substituted flavagline-inspired eIF4A inhibitors. We discovered that a variety of N-substitutions at the 1-aminomethyl group are tolerated, making this position pertinent for property and ADME profile tuning. The findings presented herein are relevant to future drug design efforts towards novel eIF4A inhibitors with drug-like properties.

Abstract The PI3K/AKT/mTOR pathway is often activated in lymphoma through alterations in PI3K, PTEN, and B-cell receptor signaling, leading to dysregulation of eIF4A (through its regulators, eIF4B, eIF4G, and PDCD4) and the eIF4F complex. Activation of eIF4F has a direct role in tumorigenesis due to increased synthesis of oncogenes that are dependent on enhanced eIF4A RNA helicase activity for translation. eFT226, which inhibits translation of specific mRNAs by promoting eIF4A1 binding to 5′-untranslated regions (UTR) containing polypurine and/or G-quadruplex recognition motifs, shows potent antiproliferative activity and significant in vivo efficacy against a panel of diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma models with ≤1 mg/kg/week intravenous administration. Evaluation of predictive markers of sensitivity or resistance has shown that activation of eIF4A, mediated by mTOR signaling, correlated with eFT226 sensitivity in in vivo xenograft models. Mutation of PTEN is associated with reduced apoptosis in vitro and diminished efficacy in vivo in response to eFT226. In models evaluated with PTEN loss, AKT was stimulated without a corresponding increase in mTOR activation. AKT activation leads to the degradation of PDCD4, which can alter eIF4F complex formation. The association of eFT226 activity with PTEN/PI3K/mTOR pathway regulation of mRNA translation provides a means to identify patient subsets during clinical development.

Dysregulation of protein translation is a key driver for the pathogenesis of many cancers. Eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase, is a critical component of the eIF4F complex, which regulates cap-dependent protein synthesis. The flavagline class of natural products (i.e., rocaglamide A) has been shown to inhibit protein synthesis by stabilizing a translation-incompetent complex for select messenger RNAs (mRNAs) with eIF4A. Despite showing promising anticancer phenotypes, the development of flavagline derivatives as therapeutic agents has been hampered because of poor drug-like properties as well as synthetic complexity. A focused effort was undertaken utilizing a ligand-based design strategy to identify a chemotype with optimized physicochemical properties. Also, detailed mechanistic studies were undertaken to further elucidate mRNA sequence selectivity, key regulated target genes, and the associated antitumor phenotype. This work led to the design of eFT226 (Zotatifin), a compound with excellent physicochemical properties and significant antitumor activity that supports clinical development.

Abstract Aberrant protein translation plays a role in the pathogenesis of multiple solid tumors and hematologic malignancies. The translation initiation factor eIF4E is essential for the translation of m7G-capped mRNA and is a key point of convergence for several signaling pathways, such as PI3K/mTOR and MAPK, which are intimately involved in tumor cell growth and survival. As such, eIF4E has generated intense interest as a target for anticancer drug discovery. We have designed a series of potent, selective, and orally available m7G cap-competitive inhibitors of eIF4E (eFT-4Ei) with favorable drug-like properties. These inhibitors bind free eIF4E, eIF4E-4EBP and eIF4E-eIF4F complexes within tumor cells. Ribosomal profiling of eIF4E inhibitor-treated tumor cells has identified a subset of translationally regulated target genes that overlap with mTORC1/2 regulated genes, but also include a larger set of unique translationally regulated target mRNAs that are enriched for 5'-TOP, PRTE and CERT sequence elements in their 5'-untranslated regions. eIF4E inhibition results in potent antiproliferative activity and induction of apoptosis in a subset of tumor cell lines. Consistent with this observation, our eIF4E inhibitors show some similarities, yet several important differences from existing mTORC1 or mTORC1/2 dual inhibitors in both cellular and physiologic assays. Finally, significant antitumor efficacy was observed with eIF4E inhibition in both solid tumor and hematologic xenografts in vivo. Taken together, these results highlight the potential of targeting eIF4E as a novel and differentiated therapeutic strategy to treat cancer. Citation Format: Gregory S. Parker, Ivy N.J. Hung, Jocelyn Staunton, Maria Barrera, Eric Sung, Ana Parra, Craig R. Stumpf, Joan Chen, Peggy A. Thompson, Andreas Nevarez, Christopher J. Wegerski, Jeff Clarine, Samuel Sperry, Alan Xiang, Christian Nilewski, Garrick K. Packard, Kaveri Urklalan, Takasuke Mukaiyama, Theo Michels, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich, Gary G. Chiang, Kevin R. Webster. Targeting PI3K/mTOR signaling with potent, selective and orally-available small-molecule inhibitors of eIF4E [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B33.

Rocaglates, rocaglamides, and related flavagline natural products exert their remarkable anticancer activity through inhibition of eukaryotic initiation factor 4A (eIF4A) but generally display suboptimal drug-like properties. In our efforts to identify potent drug-like eIF4A inhibitors, we developed synthetic strategies for diastereoselectively functionalizing the C1 position of aza-rocaglamide scaffolds (cf. 14 and 18), which proceed via retention or inversion of configuration at C1 depending on the C2 substituent (cf. 15 and 21) and ultimately enabled the discovery of novel and potent eIF4A inhibitors such as 25.

Aberrant protein translation plays a role in the pathogenesis of multiple solid tumors and hematologic malignancies. The translation initiation factor eIF4E is essential for the translation of m7G-capped mRNA and is a key point of convergence for several signaling pathways, such as PI3K/mTOR and MAPK, which are intimately involved in tumor cell growth and survival. As such, eIF4E has generated intense interest as a target for anticancer drug discovery. We have designed a series of potent, selective, and orally available m7G cap-competitive inhibitors of eIF4E (eFT-4Ei) with favorable drug-like properties. These inhibitors bind free eIF4E, eIF4E-4EBP and eIF4E-eIF4F complexes within tumor cells. Ribosomal profiling of eIF4E inhibitor-treated tumor cells has identified a subset of translationally regulated target genes that overlap with mTORC1/2 regulated genes, but also include a larger set of unique translationally regulated target mRNAs that are enriched for 59-TOP, PRTE and CERT sequence elements in their 59-untranslated regions. eIF4E inhibition results in potent antiproliferative activity and induction of apoptosis in a subset of tumor cell lines. Consistent with this observation, our eIF4E inhibitors show some similarities, yet several important differences from existing mTORC1 or mTORC1/2 dual inhibitors in both cellular and physiologic assays. Finally, significant antitumor efficacy was observed with eIF4E inhibition in both solid tumor and hematologic xenografts in vivo. Taken together, these results highlight the potential of targeting eIF4E as a novel and differentiated therapeutic strategy to treat cancer. Citation Format: Gregory S. Parker, Ivy N.J. Hung, Jocelyn Staunton, Maria Barrera, Eric Sung, Ana Parra, Craig R. Stumpf, Joan Chen, Peggy A. Thompson, Andreas Nevarez, Christopher J. Wegerski, Jeff Clarine, Samuel Sperry, Alan Xiang, Christian Nilewski, Garrick K. Packard, Kaveri Urklalan, Takasuke Mukaiyama, Theo Michels, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich, Gary G. Chiang, Kevin R. Webster. Targeting PI3K/mTOR signaling with potent, selective and orally-available small-molecule inhibitors of eIF4E [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B33.

Dysregulated messenger RNA (mRNA) translation drives the pathogenesis of multiple hematological malignancies. In lymphoma this includes the upregulation of key driver oncogenes and anti-apoptotic proteins (e.g., MYC, CCND1/3, BCL2 and MCL1) that contain a highly structured 5’-untranslated region (UTR) in their mRNA requiring enhanced eIF4A helicase activity for translation. eIF4A is a component of the eIF4F translation initiation complex and catalyzes the ATP-dependent unwinding of RNA duplexes and facilitates 43S ribosome scanning within the 5’-UTR. The activation of oncogenic signaling pathways, including RAS and PI3K, enhance eIF4A activity through phosphorylation of eIF4B, eIF4G and PDCD4 which facilitates formation of eIF4F and full activation of eIF4A. The PI3K/AKT/mTOR pathway is frequently activated in lymphoma, promoting the translation of oncogenes with complex 5’-UTRs that are required for tumor cell proliferation, survival and metastasis. eFT226 is a potent and sequence selective eIF4A1 inhibitor that promotes eIF4A1 binding to specific 5’-UTR polypurine and/or G-quadraplex recognition motifs leading to a selective block in ribosome mRNA scanning. The sequence dependency of eFT226 translational inhibition was evaluated in cell-based reporter assays demonstrating >100-fold greater sensitivity for reporter constructs containing a polypurine motif in the 5’-UTR (IC50 ~2 nM). Direct binding studies also confirmed the formation of a stable ternary complex with increased drug residence time between eFT226, eIF4A1 and RNA oligonucleotides containing polypurine motifs. The ability of eFT226 to inhibit MYC or MCL1 expression was found to be dependent on the presence of their respective 5’-UTR supporting a translational regulation mechanism dependent on recognition elements within the 5’-UTR. eFT226 shows potent anti-proliferative activity (GI50 < 15 nM) against a panel of B-cell lymphoma cell lines. Treatment with eFT226 leads to coordinated inhibition of MYC, CCND1/3, BCL2 or MCL1 protein expression resulting in significant anti-tumor activity. eFT226 has good pharmacokinetic properties and exhibits significant in vivo activity across a panel of diffuse large B cell lymphoma (DLBCL), and Burkitt lymphoma tumor models with ≤1 mg/kg/week IV administration. Further evaluation of predictive markers of sensitivity or resistance has shown that tumors with mTOR mediated activation of eIF4A are most sensitive to eFT226. In addition, tumors with PTEN mutations do not exhibit activated eIF4A and are generally resistant to induction of apoptosis by eFT226, resulting in reduced in vivo efficacy. The association of eFT226 activity with PI3K/mTOR pathway activation and mutational status provides a means to identify patient subsets during clinical development. Clinical trials in patients with lymphoma and other malignancies are planned. Citation Format: Peggy A. Thompson, Boreth Eam, Nathan P. Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Eric Sung, Ana Parra, Jocelyn Staunton, Gary G. Chiang, Christopher J. Wegerski, Andres Nevarez, Jeff Clarine, Samuel Sperry, Alan Xiang, Chinh Tran, Christian Nilewski, Garrick K. Packard, Theodore Michels, Paul A. Sprengeler, Justin T. Ernst, Siegfried H. Reich, Kevin R. Webster. eFT226, a potent and selective inhibitor of eIF4A, is efficacious in preclinical models of lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2698.

The PI3K/mTOR pathway is commonly dysregulated in many hormone receptor-dependent tumors and plays a key role in promoting tumor growth and mediating drug resistance. In particular, PI3K and mTORC1/2 inhibitors have been intensively studied in the treatment of hormone receptor-dependent cancers and have shown benefit in some clinical settings. However, issues such as dose-limiting toxicities and emergent resistance limit the broader utility of these inhibitors. The translation initiation factor eIF4E is essential for the translation of m7G-capped mRNA and is a key point of convergence for both the PI3K/mTOR and MAPK signaling pathways. We have designed a series of potent, selective and orally-available m7G cap-competitive inhibitors of eIF4E (eFT-4Ei) with favorable drug-like properties. These inhibitors bind to eIF4E either as its free form or with eIF4E-4EBP and eIF4F complexes within tumor cells and downregulate hormone receptor-dependent signaling. Ribosomal profiling of eIF4E inhibitor-treated tumor cells identified a subset of translationally regulated target genes that overlap with mTORC1/2 regulated genes, but also a unique set of translationally regulated target mRNAs. Consistent with this observation, our eIF4E inhibitors show some similarities yet several important differences from existing mTORC1 or mTORC1/2 dual inhibitors in both cellular and physiological assays. Finally, significant anti-tumor efficacy was observed with eIF4E inhibition in vitro and in vivo. Taken together, these results highlight the potential for targeting eIF4E as a novel therapeutic strategy to treat hormone-receptor dependent cancers. Citation Format: Gary G. Chiang, Gregory S. Parker, Ivy N. Hung, Vikas K. Goel, Jocelyn Staunton, Maria Barrera, Eric Sung, Ana Parra, Craig R. Stumpf, Joan Chen, Peggy A. Thompson, Andreas Nevarez, Christopher J. Wegerski, Cody Parker, Jeff Clarine, Samuel Sperry, Alan Xiang, Christian Nilewski, Garrick K. Packard, Kaveri Urkalan, Takasuke Mukaiyama, Theo Michels, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich, Kevin R. Webster. Targeting hormone receptor-dependent cancers with potent, selective and orally-available small molecule inhibitors of eIF4E [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1302.

Abstract eFT226: A Selective and Highly Potent Inhibitor of Eukaryotic Initiation Factor 4A (eIF4A), a Novel Approach for the Treatment of Cancer Siegfried H Reich, Peggy A Thompson, Justin T Ernst, Boreth Eam, Nathan P Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Ana Parra, Eric Sung, Jocelyn Staunton, Ivy NJ Hung, Gregory S Parker, Gary G Chiang, Christopher J Wegerski, Andres Nevarez, Jeff Clarine, Samuel Sperry, Alan Xiang, Chinh Tran, Christian Nilewski, Garrick K Packard, Theodore Michels, Paul A Sprengeler, and Kevin R Webster Effector Therapeutics, San Diego, CA Oncoprotein expression is tightly controlled at the level of RNA translation which is largely regulated by the eukaryotic translation initiation factor 4F (eIF4F). eIF4A1, a component of the eIF4F complex, catalyzes the ATP dependent unwinding of RNA duplexes and facilitates 43S ribosome complex scanning within the 5'-untranslated region (UTR). eIF4A1 is required for efficient translation of key oncogenes that contain complex secondary structures within the 5'-UTR. eFT226 is a novel, potent and selective eIF4A small molecule inhibitor with excellent physicochemical and pharmaceutical properties. The design of eFT226 involved ab initio ligand-based methods coupled with small molecule crystal structure analysis. eFT226 inhibits eIF4A1 through a reversible sequence-selective enhancement of eIF4A1 binding to mRNA with specific polypurine motifs within the 5'-UTR. The formation of a stable ternary complex [eIF4A1/eFT226/mRNA] with specific sequence recognition motifs leads to a block in ribosome scanning of select mRNAs. Treatment of lymphoma, AML, breast, colorectal, lung and hepatocellular tumor cell lines with eFT226 led to a dose dependent translational down regulation (IC50 of ~5-20 nM) of key oncogenes that drive tumor cell survival and proliferation (i.e., c-MYC, CCND1, BCL2 and MCL-1). Oncogene down regulation results in potent inhibition of cellular proliferation (GI50 of ~2-30 nM) across a panel of tumor cell lines. Enhanced anti-tumor sensitivity and a rapid induction of apoptosis was observed in hematological cell lines including lymphoma and AML. Inhibition of tumor cell proliferation and survival with eFT226 treatment results in significant antitumor activity in vivo in multiple human tumor models of DLBCL, Burkitt's lymphoma, acute myeloid leukemia (AML), and solid tumors following ≤ 1 mg/kg/week IV administration. eFT226 regulates the protein expression of multiple metabolic markers (i.e. c-MYC, HK2, TXNIP and GLUT1) that result in inhibition of tumor glucose uptake supporting the use of 18F-FDG-PET imaging as a measure of eFT226 target engagement in the clinic. These data demonstrate that eFT226 is a sequence-selective translational repressor of key oncogenic drivers that are essential for tumor cell proliferation and survival and support the clinical development of eFT226 in cancer patients. Citation Format: Siegfried H. Reich, Peggy A. Thompson, Justin T. Ernst, Boreth Eam, Nathan P. Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Ana Parra, Eric Sung, Jocelyn Staunton, Ivy Nj Hung, Gregory S. Parker, Gary G. Chiang, Christopher J. Wegerski, Andres Nevarez, Jeff Clarine, Samuel Sperry, Alan Xiang, Chinh Tran, Christian Nilewski, Garrick K. Packard, Theodore Michels, Paul A. Sprengeler, Kevin R. Webster. eFT226: A selective and highly potent inhibitor of eukaryotic initiation factor 4A (eIF4A), a novel approach for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr DDT02-05.

Translational control of oncoprotein expression has been implicated in the pathogenesis of multiple solid tumors and hematological malignancies. Protein synthesis is tightly regulated largely at the initiation stage through the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4A, an RNA helicase, is an essential component of the translation initiation complex and selectively regulates a subset of mRNAs based on the sequence and structure of the 5‘-untranslated region (UTR). B-cell receptor signaling activates eIF4A resulting in the selective upregulation of oncogenes involved in cell proliferation, survival and metastasis. B-cell malignancies are often associated with dysregulation of oncogenes or anti-apoptotic proteins (e.g., c-MYC, CCND1/3, BCL2 and MCL-1) that contain structured 5‘-UTRs and require enhanced eIF4A activity for translation.

In some patients who do not achieve an SVR, drug-resistant variants have been reported.A better understanding of the long-term persistence of these variants is needed.The aim of this study was to assess the prevalence of variants in patients both before treatment with telaprevir and at long term follow-up by using sensitive ultra-deep pyrosequencing (UDPS).Methods: Fourteen patients were recruited from 2 phase 1 clinical studies (VX101 and 103) of telaprevir.In these trials patients received either telaprevir monotherapy for 14 days or combination therapy with Peg-IFN.Previously well-described resistant variants at NS3 protease positions V36, T54, R155 and A156 were assessed at baseline and after a follow-up of 4±1.2 years by UDPS on the Roche 454 GS FLX platform.Resistant variants were tabulated at each time point and compared statistically between time points at each position using Fisher Exact tests, with Type I error controlled with the Bonferroni correction.Results: Median number of sequence reads per position ranged from 4677-13326.Prevalence of variants associated with resistance to telaprevir at baseline was found to be very low.The highest baseline prevalence was observed in a patient carrying a T54A variant (0.54%).No resistant variants were detected in 9 of 14 patients at baseline as compared to 8 of 14 at long-term followup.In 13 of 14 patients, there was no indication of significant enrichment of resistant variants at the long-term follow-up time point relative to baseline, despite the presence of resistant variants in the clinical trial in all patients.The remaining patient had both V36M and T54S significantly enriched at long-term follow-up (4.5% and 0.35%, respectively) relative to baseline, where neither variant was observed. Conclusion:In patients treated for 14 days with telaprevir monotherapy or in combination with Peg-IFN, highly sensitive sequencing methods indicated that resistant variants do not typically persist long-term.

We present Illuminator, a user-friendly web front end to computational models such as docking and 3D shape similarity calculations. Illuminator was specifically created to allow non-experts to design and submit molecules to computational chemistry programs. As such it provides a simple user interface allowing users to submit jobs starting from a 2D structure. The models provided are pre-optimized by computational chemists for each specific target. We provide an example of how Illuminator was used to prioritize the design of molecular substituents in the Anadys HCV Polymerase (NS5B) project. With 7500 submitted jobs in 1.5 years, Illuminator has allowed project teams at Anadys to accelerate the optimization of novel leads. It has also improved communication between project members and increased demand for computational drug discovery tools.

The discovery of 5,5'- and 6,6'-dialkyl-5,6-dihydro-1H-pyridin-2-ones as potent inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) is described. Several of these agents also display potent antiviral activity in cell culture experiments (EC50 <0.10 microM). In vitro DMPK data for selected compounds as well as crystal structures of representative inhibitors complexed with the NS5B protein are also disclosed.