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Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target. However, eEF2K siRNA was ineffective against cancer cells including those sensitive to NH125. To test if pharmacological intervention differs from siRNA interference, we identified a highly selective small molecule eEF2K inhibitor A-484954. Like siRNA, A-484954 had little effect on cancer cell growth. We carefully examined the effect of NH125 and A-484954 on phosphorylation of eEF2, the known cellular substrate of eEF2K. Surprisingly, NH125 increased eEF2 phosphorylation, whereas A-484954 inhibited the phosphorylation as expected for an eEF2K inhibitor. Both A-484954 and eEF2K siRNA inhibited eEF2K and reduced eEF2 phosphorylation with little effect on cancer cell growth. These data demonstrated clearly that the anticancer activity of NH125 was more correlated with induction of eEF2 phosphorylation than inhibition of eEF2K. Actually, induction of eEF2 phosphorylation was reported to correlate with inhibition of cancer cell growth. We compared several known inducers of eEF2 phosphorylation including AMPK activators and an mTOR inhibitor. Interestingly, stronger induction of eEF2 phosphorylation correlated with more effective growth inhibition. We also explored signal transduction pathways leading to NH125-induced eEF2 phosphorylation. Preliminary data suggested that NH125-induced eEF2 phosphorylation was likely mediated through multiple pathways. These observations identified an opportunity for a new multipathway approach to anticancer therapies.

First generation BET inhibitors, including ABBV-075, bind with nearly equimolar affinity to two highly conserved bromodomains (BDI and BDII) in the N-terminus of BRD2, BRD3, BRD4, and BRDt. These “pan” inhibitors compete with the natural acetylated lysine substrates of BDI and BDII to displace BET family proteins from chromatin. In so doing, pan-BET inhibitors generally block transcription and induce potent anti-tumor activity across a wide range of pre-clinical models. Consequently, numerous phase I clinical trials have been initiated with pan-BET inhibitors. The initial reports of responses in the leading indication of AML have been encouraging; however, thrombocytopenia and other dose limiting toxicities may prevent realization of a therapeutic dose in most solid tumor indications. We hypothesized that selective inhibition of BDII but not BDI of BET family proteins would improve tolerability while retaining efficacy. Medicinal chemistry efforts produced ABBV-744, a potent inhibitor specific for BDII of BRD2/3/4, with >250x differential binding preference for BDII over BDI and excellent drug-like properties. In striking contrast to the broad range of cell growth inhibition effected by pan BET inhibitor ABBV-075, BDII-selective ABBV-744 anti-proliferative activity was largely but not exclusively restricted to AML and androgen receptor (AR) positive prostate cancer cell line models, including models of Enzalutamide resistance. RNA-Seq and qPCR revealed that ABBV-744 was a potent and selective inhibitor of the AR transcription pathway. ChIP-Seq and ChIP-qPCR revealed a BDII-inhibitor displacement of BRD4 from AR-occupied enhancers and promoters, consistent with a BRD4 BDII-specific dependency of AR to sustain an oncogenic gene expression program. In vivo, doses of ABBV-744 at fractions of its MTD in LNCaP and MDA-PCa-2b xenograft models induced tumor growth inhibition that was comparable to that observed with ABBV-075 when dosed at its MTD. Together, these in vitro and in vivo results provide proof of concept that selective BDII BET family inhibitors may have improved tolerability relative to pan-BET inhibitors while maintaining tumor growth inhibition in AR positive prostate cancer. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Emily J. Faivre, Denise Wilcox, Mai Ha-Bui, Paul Hessler, Vasudha Sehgal, Xin Lu, Tamar Uziel, Gaurav Mehta, Daniel H. Albert, Keith McDaniel, Warren Kati, Yu Shen. First-in-class, highly BDII-selective BET family inhibitor ABBV-744 displays potent anti-tumor activity in androgen receptor positive prostate cancer models and an improved tolerability profile [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 4960.

Many small-molecule inhibitors that target both bromodomains of the BET family proteins (pan BET inhibitors) are undergoing studies in clinical trials. Emerging data are beginning to suggest that clinical responses to these pan BET inhibitors in subsets of hematologic malignancies may be modest and short lived, perhaps due, at least in part, to tolerability issues that limit dosing levels. We hypothesized that selective inhibition of four of the eight bromodomains in BET family proteins might retain the anticancer activities in certain tumor subsets while alleviating some of the tolerability liabilities of pan BET inhibitors, thus possibly providing better therapeutic benefits. ABBV-744 is a highly selective inhibitor for the second bromodomain (BDII) of the four BET family proteins, exhibiting greater than 300-fold more potent binding affinity to the BDII bromodomain of BRD4 relative to the first bromodomain (BDI) of BRD4. In contrast to the broad antiproliferative activities observed with pan BET inhibitors, ABBV-744 only displayed significant antiproliferative activities in a limited number of cancer cell lines, including AML and androgen receptor (AR)-positive prostate cancer. Studies in AML xenograft models demonstrated antitumor efficacy for ABBV-744 that was comparable to the pan-BET inhibitor ABBV-075 but with improved tolerability. Taken together, these results suggest that ABBV-744 could be a promising second-generation BET inhibitor for AML therapy. Affiliation: Oncology Discovery, AbbVie Inc., 1 North Waukegan Rd., North Chicago, IL 60064. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: Xiaoyu Lin, Xiaoli Huang, Richard Bellin, Emily Faivre, Paul Hessler, Lloyd Lam, Mai Ha Bui, Denise Wilcox, Tamar Uziel, Debra C. Ferguson, Terrance J. Magoc, Daniel H. Albert, Keith F. McDaniel, Warren Kati, Yu Shen. ABBV-744, a first-in-class and highly selective inhibitor of the second bromodomain of BET family proteins, displays robust activities in preclinical models of acute myelogenous leukemia [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 800.

The BET family of proteins consists of BRD2, BRD3, BRD4 and BRDT, with each of these proteins containing two distinct bromodomains (BDI and BDII). ABBV-075, like other first generation BET family bromodomain inhibitors currently under clinical development, binds to each of the 8 bromodomains with similar affinity and inhibits the proliferation of cancer cells that represent a wide range of tumor types. We hypothesized that selectively targeting specific subsets of the BET bromodomain might abolish this broad spectrum profile such that only the tumor types that are highly addicted to the subtype specific BET bromodomain-mediated transcription would remain sensitive to these selective agents. Both BDI and BDII are highly conserved across BET family members (> 70% identity), suggesting that the generation of compounds that are selective for the BDI bromodomains or the BDII bromodomains might be achievable. Structure-based design targeting the Asp144/His 437 and Ile146/Val439 sequence differences (BRD4 BDI/BDII numbering) led to the identification of structural analogs of ABBV-075 demonstrating greater than 100X selectivity for BRD4 BDII over BRD4 BDI. Further elaboration led to compounds with improved BDII-selectivity and oral bioavailability and the identification of the clinical asset ABBV-744. Disclosures: All authors are employees or former employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Citation Format: George S. Sheppard, Le Wang, Steven D. Fidanze, Lisa A. Hasvold, Dachun Liu, John K. Pratt, Chang H. Park, Mai-Ha Bui, Emily J. Faivre, Xiaoli Huang, Xiaoyu Lin, Denise M. Wilcox, Yu Shen, Daniel H. Albert, Terrance J. Magoc, Ganesh Rajaraman, Warren M. Kati, Keith F. McDaniel. Discovery of ABBV-744, a first-in-class highly BDII-selective BET bromodomain inhibitor [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 931.

A new series of potent macrocyclic urea-based Chk1 inhibitors are described. A detailed SAR study on the 4-position of the phenyl ring of the 14-member macrocyclic ureas 1a and d led to the identification of the potent Chk1 inhibitors 2, 5-7, 10, 13, 14, 19-21, 25, 27, and 31-34. These compounds significantly sensitize tumor cells to the DNA-damaging antitumor agent doxorubicin in a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and camptothecin-induced S checkpoints, indicating that the potent biological activities of these compounds are mechanism-based through Chk1 inhibition. Kinome profiling analysis of a representative macrocyclic urea 25 against a panel of 120 kinases indicates that these novel macrocyclic ureas are highly selective Chk1 inhibitors. Preliminary PK studies of 1a and b suggest that the 14-member macrocyclic inhibitors may possess better PK properties than their 15-member counterparts. An improved synthesis of 2 and 20 by using 2-(trimethylsilyl)ethoxycarbonyl (Teoc) to protect the amino group not only readily provided the desired compounds in pure form but also facilitated the scale up of potent compounds for various biological studies.

A series of 1,4-dihydroindeno[1,2-c]pyrazole compounds with a cyanopyridine moiety at the 3-position of the tricyclic pyrazole core was explored as potent CHK-1 inhibitors. The impact of substitutions at the 6 and/or 7-position of the core on pharmacokinetic properties was studied in detail. Compounds carrying a side chain with an ether linker at the 7-position and a terminal morpholino group, such as 29 and 30, exhibited much-improved oral biovailability in mice as compared to earlier generation inhibitors. These compounds also possessed desirable cellular activity in potentiating doxorubicin and will serve as valuable tool compounds for in vivo evaluation of CHK-1 inhibitors to sensitize DNA-damaging agents.

A novel series of 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-ones have been synthesized as potent and selective checkpoint kinase 1 (Chk1) inhibitors via structure-based design. Aided by protein X-ray crystallography, medicinal chemistry efforts led to the identification of compound 46d, with potent enzymatic activity against Chk1 kinase. While maintaining a low cytotoxicity of its own, compound 46d exhibited a strong ability to abrogate G2 arrest and increased the cytotoxicity of camptothecin by 19-fold against SW620 cells. Pharmacokinetic studies revealed that it had a moderate bioavailabilty of 20% in mice. Two important binding interactions between compound 46b and Chk1 kinase, revealed by X-ray cocrystal structure, were hydrogen bonds between the hinge region and the amide bond of the core structure and a hydrogen bond between the methoxy group and Lys38 of the protein.

Diffuse Large B-Cell Lymphoma (DLBCL) consists of activated B-cell-like (ABC), germinal center B cell-like (GCB), and primary mediastinal B cell lymphoma (PMBL) subtypes. Among these, the ABC subtype of DLBCL often harbors mutations that cause abnormal NF-κB activation, which subsequently activates genes important for cell survival and disease progression in ABC DLBCL. The bromodomain and extraterminal domain (BET) protein BRD4 binds acetylated NF-κB and regulates NF-κB dependent transcription. Accordingly, targeting BET family proteins using small molecule inhibitors such as ABBV-075 might provide therapeutic benefit in ABC DLBCL by blocking the NF-κB pathway. In this study, we examined the activity of ABBV-075 in both the ABC and GCB subtypes of DLBCL. As expected, ABBV-075 diminished NF-κB reporter activity, down-regulated NF-κB target genes, inhibited proliferation and survival of ABC DLBCL cells in vitro, and delayed the growth of ABC DLBCL tumors in vivo. Interestingly, ABBV-075 also exhibited robust anti-proliferative activities in GCB DLBCL cells in vitro and inhibited the growth of GCB DLBCL tumors in vivo. Further characterization of the responses of GCB DLBCL cells to ABBV-075 indicated that ABBV-075 induced strong apoptosis in GCB DLBCL cells that are resistant to the Bcl-2 inhibitor venetoclax (ABT-199), but rarely in venetoclax-sensitive GCB DLBCL cell lines. Our results indicate that ABBV-075 could be active against both the ABC and GCB subtypes of DLBCL. The complementary activity of ABBV-075 and ABT-199 further suggests that ABBV-075 maybe an interesting option for ABT-199 resistant/refractory DLBCL patients. Citation Format: Xiaoyu Lin, Xiaoli Huang, Aparna Sarthy, Terry Magoc, Daniel Albert, Lloyd Lam, Tamar Uziel, Xin Lu, Mai-Ha Bui, Emily Faivre, Denise Wilcox, Steven Elmore, Keith McDaniel, Warren Kati, Yu Shen. ABBV-075 exhibits robust in vitro and in vivo activities against the ABC and GCB subtypes of DLBCL. [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 4706.

Eukaryotic elongation factor-2 kinase (eEF2K) relays growth and stress signals to protein synthesis through phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2). 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) is a widely accepted inhibitor of mammalian eEF2K and an efficacious anti-proliferation agent against different cancer cells. It implied that eEF2K could be an efficacious anticancer target. However, eEF2K siRNA was ineffective against cancer cells including those sensitive to NH125. To test if pharmacological intervention differs from siRNA interference, we identified a highly selective small molecule eEF2K inhibitor A-484954. Like siRNA, A-484954 had little effect on cancer cell growth. We carefully examined the effect of NH125 and A-484954 on phosphorylation of eEF2, the known cellular substrate of eEF2K. Surprisingly, NH125 increased eEF2 phosphorylation, whereas A-484954 inhibited the phosphorylation as expected for an eEF2K inhibitor. Both A-484954 and eEF2K siRNA inhibited eEF2K and reduced eEF2 phosphorylation with little effect on cancer cell growth. These data demonstrated clearly that the anticancer activity of NH125 was more correlated with induction of eEF2 phosphorylation than inhibition of eEF2K. Actually, induction of eEF2 phosphorylation was reported to correlate with inhibition of cancer cell growth. We compared several known inducers of eEF2 phosphorylation including AMPK activators and an mTOR inhibitor. Interestingly, stronger induction of eEF2 phosphorylation correlated with more effective growth inhibition. We also explored signal transduction pathways leading to NH125-induced eEF2 phosphorylation. Preliminary data suggested that NH125-induced eEF2 phosphorylation was likely mediated through multiple pathways. These observations identified an opportunity for a new multipathway approach to anticancer therapies.

An extensive structure-activity relationship study of the 3-position of a series of tricyclic pyrazole-based Chk1 inhibitors is described. As a result, 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-benzonitriles (4) and 4'-(1,4-dihydro-indeno[1,2-c]pyrazol-3-yl)-pyridine-2'-carbonitriles (29) emerged as new lead series. Compared with the original lead compound 2, these new leads fully retain the biological activity in both enzymatic inhibition and cell-based assays. More importantly, the new leads 4 and 29 exhibit favorable physicochemical properties such as lower molecular weight, lower Clog P, and the absence of a hydroxyl group. Furthermore, structure-activity relationship studies were performed at the 6- and 7-positions of 4, which led to the identification of ideal Chk1 inhibitors 49, 50, 51, and 55. These compounds not only potently inhibit Chk1 in an enzymatic assay but also significantly potentiate the cytotoxicity of DNA-damaging agents in cell-based assays while they show little single agent activity. A cell cycle analysis by FACS confirmed that these Chk1 inhibitors efficiently abrogate the G2/M and S checkpoints induced by DNA-damaging agent. The current work paved the way to the identification of several potent Chk1 inhibitors with good pharmacokinetics that are suitable for in vivo study with oral dosing.

Based on the crystallographic analysis of a urea-checkpoint kinase 1 (Chk1) complex and molecular modeling, a class of macrocyclic Chk1 inhibitors were designed and their biological activities were evaluated. An efficient synthetic methodology for macrocyclic ureas was developed with Grubbs metathesis macrocyclization as the key step. The structure-activity relationship studies demonstrated that the macrocyclization retains full Chk1 inhibition activity and that the 4-position of the phenyl ring can tolerate a wide variety of substituents. These novel Chk1 inhibitors exhibit excellent selectivity over a panel of more than 70 kinases. Compounds 5b, 5c, 5f, 15, 16d, 17g, 17h, 17k, 18d, and 22 were identified as ideal Chk1 inhibitors, which showed little or no single-agent activity but significantly potentiate the cytotoxicities of the DNA-damaging antitumor agents doxorubicin and camptothecin. These novel Chk1 inhibitors abrogate the doxorubicin-induced G2 and camptothecin-induced S checkpoint arrests, confirming that their potent biological activities are mechanism-based through Chk1 inhibition.

We have developed a PCR-oligonucleotide ligation assay to rapidly identify base substitutions in topoisomerase genes that are associated with quinolone resistance in clinical isolates of Streptococcus pneumoniae . Thirty-seven strains for which the ciprofloxacin MICs were ≥4 μg/ml and 16 strains for which the MICs were ≤2 μg/ml were assayed. Compared with sequence data, the assay correctly identified the DNA bases that encoded amino acids at the four positions most commonly associated with quinolone resistance (Ser79 and Asp83 of ParC and Ser81 and Glu85 of GyrA). Therefore, this procedure can rapidly distinguish single base substitutions associated with quinolone-resistant topoisomerases in S. pneumoniae .