Search

Histone demethylase LSDl (KDMlA) belongs to the flavin adenine dinucleotide (FAD) dependent family of monoamine oxidases and is vital in regulation of mammalian biology. Dysregulation and overexpression of LSD1 are hallmarks of a number of human diseases, particularly cancers that are characterized as morphologically poorly differentiated. As such, inhibitors of LSD1 have potential to be beneficial as a cancer therapy. The most clinically advanced inhibitors of LSDl are covalent inhibitors derived from tranylcypromine (TCP). Herein, we report the discovery of a novel series of reversible and selective LSDl inhibitors. Exploration of structure-activity relationships (SARs) and optimization of ADME properties resulted in the identification of clinical candidate CC-90011. CC-90011 exhibits potent on-target induction of cellular differentiation in acute myeloid leukemia (AML) and small cell lung cancer (SCLC) cell lines, and antitumor efficacy in patient-derived xenograft (PDX) SCLC models. CC-90011 is currently in phase 2 trials in patients with first line, extensive stage SCLC (ClinicalTrials.gov identifier: NCT03850067).

The bromodomain and extra-terminal (BET) family of epigenetic proteins has attracted considerable attention in drug discovery given its involvement in regulating gene transcription. Screening a focused small molecule library based on the bromodomain pharmacophore resulted in the identification of 2-methylisoquinoline-1-one as a novel BET bromodomain-binding motif. Structure guided SAR exploration resulted in >10,000-fold potency improvement for the BRD4-BD1 bromodomain. Lead compounds exhibited excellent potencies in both biochemical and cellular assays in MYC-dependent cell lines. Compound 36 demonstrated good physicochemical properties and promising exposure levels in exploratory PK studies.

The histone demethylase LSD1 is a key enzyme in the epigenetic regulation of gene transcription. Here we present our efforts to discover small molecule reversible inhibitors of LSD1 as an attractive approach to treat hematologic malignancies and certain solid tumors. Using structure-based drug design, we designed and synthesized a novel series of heteroaromatic imidazole inhibitors that demonstrate potent inhibition of the demethylase activity and low nanomolar cell-based activity. This novel LSD1 inhibitor series was further optimized by attenuating the hERG inhibition and improving oral bioavailability.

Histone lysine demethylases (KDMs) play a vital role in the regulation of chromatin-related processes. Herein, we describe our discovery of a series of potent KDM4 inhibitors that are both cell permeable and antiproliferative in cancer models. The modulation of histone H3K9me3 and H3K36me3 upon compound treatment was verified by homogeneous time-resolved fluorescence assay and by mass spectroscopy detection. Optimization of the series using structure-based drug design led to compound 6 (QC6352), a potent KDM4 family inhibitor that is efficacious in breast and colon cancer PDX models.

Accelerated proliferation of solid tumor and hematologic cancer cells is linked to accelerated transcription of rDNA by the RNA polymerase I (Pol I) enzyme to produce elevated levels of rRNA (rRNA). Indeed, upregulation of Pol I, frequently caused by mutational alterations among tumor suppressors and oncogenes, is required for maintenance of the cancer phenotype and forms the basis for seeking selective inhibitors of Pol I as anticancer therapeutics. 2-(4-Methyl-[1,4]diazepan-1-yl)-5-oxo-5H-7-thia-1,11b-diaza-benzo[c]fluorene-6-carboxylic acid (5-methyl-pyrazin-2-ylmethyl)-amide (CX-5461, 7c) has been identified as the first potent, selective, and orally bioavailable inhibitor of RNA Pol I transcription with in vivo activity in tumor growth efficacy models. The preclinical data support the development of CX-5461 as an anticancer drug with potential for activity in several types of cancer.

In this article we describe the preclinical characterization of 5-(3-chlorophenylamino) benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first orally available small molecule inhibitor of protein CK2 in clinical trials for cancer. CX-4945 was optimized as an ATP-competitive inhibitor of the CK2 holoenzyme (Ki = 0.38 nM). Iterative synthesis and screening of analogs, guided by molecular modeling, led to the discovery of orally available CX-4945. CK2 promotes signaling in the Akt pathway and CX-4945 suppresses the phosphorylation of Akt as well as other key downstream mediators of the pathway such as p21. CX-4945 induced apoptosis and caused cell cycle arrest in cancer cells in vitro. CX-4945 exhibited a dose-dependent antitumor activity in a xenograft model of PC3 prostate cancer model and was well tolerated. In vivo time-dependent reduction in the phosphorylation of the biomarker p21 at T145 was observed by immunohistochemistry. Inhibition of the newly validated CK2 target by CX-4945 represents a fresh therapeutic strategy for cancer.

Structure–activity relationship analysis in a series of 3-(5-((2-oxoindolin-3-ylidene)methyl)furan-2-yl)amides identified compound 13, a pan-Pim kinases inhibitor with excellent biochemical potency and kinase selectivity. Compound 13 exhibited in vitro synergy with chemotherapeutics and robust in vivo efficacy in two Pim kinases driven tumor models.

Herein we chronicle the discovery of CX-4945 (25n), a first-in-class, orally bioavailable ATP-competitive inhibitor of protein kinase CK2 in clinical trials for cancer. CK2 has long been considered a prime cancer drug target because of the roles of deregulated and overexpressed CK2 in cancer-promoting prosurvival and antiapoptotic pathways. These biological properties as well as the suitability of CK2's small ATP binding site for the design of selective inhibitors, led us to fashion novel therapeutic agents for cancer. The optimization leading to 25n (K(i) = 0.38 nM) was guided by molecular modeling, suggesting a strong binding of 25n resulting from a combination of hydrophobic interactions, an ionic bridge with Lys68, and hydrogen bonding with the hinge region. 25n was found to be highly selective, orally bioavailable across species (20-51%) and efficacious in xenograft models. The discovery of 25n will allow the therapeutic targeting of CK2 in humans for the first time.

We describe the discovery of novel potent substituted pyrimido[4,5-c]quinoline ATP-competitive inhibitors of protein kinase CK2. A binding model of the inhibitors with the protein was elaborated on the basis of SAR and revealed various modes of interaction with the hinge region. Representative analog 14k (CK2 IC(50)=9 nM) showed anti-viral activity at nanomolar concentrations against HIV-1. Orally available compound 7e (CK2 IC(50)=3 nM) reduced pain in the phase II of a murine formalin model. These preliminary data confirm that properly optimized CK2 inhibitors may be used for anti-viral and pain therapy.

Abstract 2897 Specific inhibition of signaling elements essential for chronic lymphocytic leukemia (CLL) cell survival offers great promise for the design of improved therapies against this still incurable malignancy. The serine/threonine protein kinase CK2 is frequently upregulated in cancer, and mounting evidence implicates CK2 in tumorigenesis. Here, we evaluated whether CK2 is a valid target for therapeutic intervention in CLL, by testing the efficacy of CX-4945, a potent and highly specific orally available ATP-competitive inhibitor of CK2 that is undergoing phase I clinical trials for solid tumors and multiple myeloma. We previously showed that CK2 phosphorylates and thereby inactivates PTEN in primary T-ALL and CLL cells, leading to the hyperactivation of PI3K signaling pathway, and consequently promoting leukemia cell survival (Silva et al, JCI 2008; Martins et al, Blood 2010). Therefore, we first analyzed the impact of CX-4945 on PTEN phosphorylation and PI3K pathway activation. Incubation of CLL cells with 20 μM CX-4945 for 2h resulted in striking downregulation of PTEN phosphorylation, indicative of increased PTEN activity, and a concomitant decrease in the activity of PI3K downstream targets Akt and PKC, as determined by Akt (S473), PKCβ (S660) and PKCδ (T550) phosphorylation in both MO1043 and primary CLL cells collected and isolated to >90% purity from the peripheral blood of untreated patients. Importantly, we confirmed that Akt phosphorylation on the CK2 direct target site (S129) was also inhibited by CX-4945. Next, we evaluated the functional impact of the CK2 inhibitor on CLL cell viability. Primary CLL cells (n=11) were cultured with 10 and 20 μM CX-4945. Both drug concentrations exerted clear pro-apoptotic effects in all cases (P The promising results obtained in vitro prompted us to assess the impact of CX-4945 on CLL tumor development in vivo. We implanted MO1043 CLL cells subcutaneously into Swiss nude mice. At day 3, all animals presented palpable tumor masses of approximately 150 mm3, and were randomly assigned into 4 groups (n=6 per group) to receive either CX-4945 alone (75mg/kg, bid, p.o.), fludarabine alone (34mg/kg, i.p., 5 days + 2 days rest, every week), the combination of both drugs, or vehicle control. A significant delay in tumor growth was observed in all of the treatment groups when compared to the control group (P Overall, our data indicate that pharmacological inhibition of CK2 is a promising therapeutic strategy in CLL that may be of special benefit to patients with aggressive and advanced stage disease. Moreover, our studies pave the way to the development of clinical trials using CX-4945 or other CK2 antagonists to manage CLL. Disclosures: Stansfield: Cylene Pharmaceuticals Inc.: Employment. Drygin:Cylene Pharmaceuticals Inc.: Employment.

Protein kinase CK2 regulates multiple pathways that play key roles in carcinogenesis, including cell cycle progression, suppression of apoptosis, hypoxia, angiogenesis, inflammation and DNA damage repair. Overexpression of CK2 in various cancer tissues has been frequently linked to poor prognosis. All this makes targeting of CK2 highly attractive for both single agent and combination therapies. To date therapeutic targeting of CK2 proved to be challenging, with only one compound, CX-4945, entering clinical development. In phase I clinical trials CX-4945 was well tolerated, inhibited CK2 activity in PBMC, modulated the PI3K-AKT-mTOR oncogenic pathway, reduced tumor-related serum IL-6 and IL-8 biomarkers, resulted in stable disease for at least 16 weeks in 20% of treated patients, and caused single agent tumor shrinkage. Building on these successes with the first generation CK2 inhibitor, we sought to design a second generation CK2 inhibitor with distinct pharmacologic properties. The application of a focused screening cascade and iterative medicinal chemistry efforts generated CX-8184, a selective single digit nanomolar inhibitor of the CK2 enzyme that demonstrated desirable biologic properties. When evaluated against a panel of 32 cancer cell lines, CX-8184 demonstrated broad antiproliferative activity with median IC50 = 145 nM. CX-8184 suppressed NF-κB, Hif-1α and β-catenin driven transcription at concentrations of 100-300 nM, and inhibited IL-6 expression with IC50 = 30 nM. Since CK2 is known to potentiate AKT signaling, treatment of cancer cells with CX-8184 decreased phosphorylation of AKT, as well as its substrates. The Hsp90/Cdc37 machinery is regulated by CK2 and plays an important role in activation of various kinases, including members of the ErbB RTK family. CX-8184 rapidly suppressed phosphorylation of Cdc37 by CK2, leading to the deactivation of ErbB family members. In combination with erlotinib or lapatinib, CX-8184 produced synergistic killing of lung cancer and breast cancer cells, respectively. CX-8184 also inhibited the phosphorylation of XRCC1, a scaffold protein involved in DNA strand break repair that plays an important role in sensitivity of cancer cells to DNA damaging agents. Consequently, the treatment of ovarian cancer cells pre-challenged with gemcitabine and/or cisplatin with 300 nM CX-8184 led to synergistic induction of cell death. In vivo, CX-8184 demonstrated favorable PK properties with oral bioavailability and a half-life amenable to once daily dosing in mice. CX-8184 exhibited potent anti-tumor activity and was well tolerated in A431 xenograft model. The compound has also showed an acceptable pharmacological profile as evidenced by CYP, hERG and Ames testing. The pharmacokinetic and pharmacological properties and the biological activity of CX-8184, both as single agent or in combination, support its further development as a preclinical candidate.9 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3765. doi:1538-7445.AM2012-3765

The PIM family of serine/threonine kinases are pro-proliferative kinases activated by multiple cytokines and growth factor signaling. The Pim kinases are unusual in that they are regulated primarily by transcription and not by membrane recruitment or phosphorylation like other serine/threonine kinases. Activated cytokine receptors recruit JAKs to induce STAT-dependent transcription of the Pim genes. They are proto-oncogenes and have been implicated in the process of lymphomagenesis and malignant transformation. Pim overexpression has been reported in diffuse B cell lymphoma, chronic lymphocytic leukemia, FLT3-mediated acute myelogenous leukemia and prostate cancer. Pim-2 is over expressed in leukemias and lymphomas, whereas Pim-3 overexpression has been observed in melanoma, pancreatic and gastric tumors. The recent reports of elevated levels of Pim-1 expression in human prostate tumor biopsies implicate the Pim family of protein kinases in the progression of human prostate tumors. Further, in transgenic animal models, Pim-1 expression has been shown to be elevated in prostate tumors that are caused by overexpression of the c-myc oncogene. Recent evidence reveals the overlapping and compensatory nature of Pim-1 and Pim-2 phosphorylation and highlights the importance of inhibiting all isoforms. The emerging role of the PIM kinase family in hematological malignancies and solid tumors and the druggable nature of their ATP binding pocket make them attractive targets for anticancer drug development. Utilizing a highly distinct molecular scaffold, CX-6258 was developed as a selective and potent small molecule pan-PIM kinase inhibitor. CX-6258 inhibits Pims 1, 2 and 3 with IC50 values in the low nanomolar range and high selectivity as evidenced in a screening panel of over 100 kinases. CX-6258 demonstrates potent in vitro antiproliferative activity, particularly in leukemia derived cell lines expressing the FLT3-ITD. Moreover, CX-6258 inhibits the phosphorylation of BAD and 4EBP1, known substrates for PIMs 1, 2 and 3. When delivered orally, this pan-Pim inhibitor is well tolerated and demonstrates potent antitumor activity in murine xenograft models of PIM driven cancer. Using CX-6258 as our “path finder” molecule, we have created four additional unique chemical scaffolds as pan-Pim inhibitors, and certain molecules from these scaffolds can inhibit Pims 1, 2 and 3 in the picomolar range while exhibiting no inhibitory activity of the Flt3 protein kinase. The in vivo and in vitro profiles of these chemically diverse series are indicative of an effective and potent anti-cancer mechanism mediated through the selective inhibition of PIM kinase activity. Together, these findings exemplify that we have created multiple proprietary chemical series of pan-Pim inhibitors exhibiting picomolar potency and discerning selectivity. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A246.

Abstract 1726 Poster Board I-752 The PIM family of serine/threonine kinases are pro-proliferative kinases activated by multiple cytokines and growth factor signaling. The Pim kinases are unusual in that they are regulated primarily by transcription and not by membrane recruitment or phosphorylation like other serine/threonine kinases. Activated cytokine receptors recruit JAKs to induce STAT-dependent transcription of the Pim genes. They are proto-oncogenes and have been implicated in the process of lymphomagenesis and malignant transformation. Pim overexpression has been reported in diffuse B cell lymphoma, chronic lymphocytic leukemia, FLT3-mediated acute myelogenous leukemia and prostate cancer. Pim-2 is over expressed in leukemias and lymphomas, whereas Pim-3 overexpression has been observed in melanoma, pancreatic and gastric tumors. The recent reports of elevated levels of Pim-1 expression in human prostate tumor biopsies implicate the Pim family of protein kinases in the progression of human prostate tumors. Further, in transgenic animal models, Pim-1 expression has been shown to be elevated in prostate tumors that are caused by overexpression of the c-myc oncogene. Recent evidence reveals the overlapping and compensatory nature of Pim-1 and Pim-2 phosphorylation and highlights the importance of inhibiting all isoforms. The emerging role of the PIM kinase family in hematological malignancies and solid tumors and the druggable nature of their ATP binding pocket make them attractive targets for anticancer drug development Utilizing a highly distinct molecular scaffold, CX-6258 was developed as a selective and potent small molecule pan-PIM kinase inhibitor. CX-6258 inhibits Pims 1, 2 and 3 with IC50 values in the low nanomolar range and high selectivity as evidenced in a screening panel of over 100 kinases. CX-6258 demonstrates potent in vitro antiproliferative activity, particularly in leukemia derived cell lines expressing the FLT3-ITD. Moreover, CX-6258 inhibits the phosphorylation of BAD and 4EBP1, known substrates for PIMs 1, 2 and 3. When delivered orally, this pan-Pim inhibitor is well tolerated and demonstrates potent antitumor activity in murine xenograft models of PIM driven cancer. Using CX-6258 as our “path finder” molecule, we have created four additional unique chemical scaffolds as pan-Pim inhibitors, and certain of these scaffolds can inhibit Pims 1, 2 and 3 in the picomolar range while exhibiting no inhibitory activity of the Flt3 protein kinase. The in vivo and in vitro profiles of these chemically diverse series are indicative of an effective and potent anti-cancer mechanism mediated through the selective inhibition of PIM kinase activity. Together, these findings exemplify that we have created multiple proprietary chemical series of pan-Pim inhibitors exhibiting picomolar potency and high selectivity. Disclosures No relevant conflicts of interest to declare.