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Deconstruction of a bicyclic tetrahydroquinoline-6-carboxylic acid scaffold into aminosalicylic acids yielded dual MCL-1/BCL-xL inhibitors from an MCL-1 selective lead.

Introduction: The chromatin factor BRD9 is a genetic dependency in some cancers, often referred to as SMARCB1-perturbed cancers. Two types of genetic alterations result in SMARCB1 perturbation: SS18-SSX gene fusion and SMARCB1 loss-of-function mutations. In synovial sarcoma, a rare and aggressive soft tissue malignancy comprising approximately 10% of all soft tissue sarcomas, the presence of the SS18-SSX fusion gene drives the disruption of SMARCB1 function and leads to a synthetic lethal dependence on BRD9. In SMARCB1-null solid tumors, for example malignant rhabdoid tumors (MRT), poorly differentiated chordomas, and epithelioid sarcomas, the absence of SMARCB1 protein results in a similar BRD9 dependence. Thus, in SMARCB1-perturbed cancers, including synovial sarcoma and SMARCB1-null cancers, degradation of BRD9 is hypothesized to result in an anticancer effect. CFT8634 is an orally bioavailable heterobifunctional degrader that induces ternary complex formation with BRD9 and an E3 ligase, leading to the ubiquitination of BRD9 and its subsequent degradation by the proteasome. Results: Here we describe the chemical structure of CFT8634 and an overview of the medicinal chemistry path leading to its discovery. In vitro, CFT8634 promotes rapid, potent, deep, and selective degradation of BRD9 with a half-maximal degradation concentration (DC50) of 2 nM in a synovial sarcoma cell line. In long-term growth assays, CFT8634 is effective at impairing cell growth in a concentration-dependent manner specifically in SMARCB1-perturbed contexts. In vivo, oral dosing of CFT8634 in xenograft models of SMARCB1-perturbed cancers leads to robust and dose-dependent degradation of BRD9, which translates to significant and dose-dependent inhibition of tumor growth in preclinical xenograft models. Conclusion: The preclinical data presented herein support the clinical development of CFT8634 for the treatment of synovial sarcoma and SMARCB1-null tumors. Citation Format: Katrina L. Jackson, Roman V. Agafonov, Mark W. Carlson, Prasoon Chaturvedi, David Cocozziello, Kyle Cole, Richard Deibler, Scott J. Eron, Andrew Good, Ashley A. Hart, Minsheng He, Christina S. Henderson, Hongwei Huang, Marta Isasa, R. Jason Kirby, Linda Lee, Michelle Mahler, Moses Moustakim, Christopher G. Nasveschuk, Michael Palmer, Laura L. Poling, Roy M. Pollock, Matthew Schnaderbeck, Stan Spence, Gesine K. Veits, Jeremy L. Yap, Ning Yin, Rhamy Zeid, Adam S. Crystal, Andrew J. Phillips, Stewart L. Fisher. The discovery and characterization of CFT8634: A potent and selective degrader of BRD9 for the treatment of SMARCB1-perturbed cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND09.

The BRAF kinase is a critical node in the MAPK signaling pathway and is mutated in approximately 8% of human cancers including melanoma (~60%), thyroid (~60%), and lung adenocarcinoma (~10%). The most common mutation in BRAF is V600E (Class I), occurring in half of malignant melanomas. This mutation hyperactivates ERK and signals as a RAF inhibitor-sensitive monomer. BRAF inhibitors including vemurafenib, dabrafenib and encorafenib have produced impressive responses in V600X patients, however resistance usually emerges within a year, including RAS mutation, BRAFV600E amplification, and BRAFV600E intragenic deletion or splice variants. These inhibitors are also ineffective against non-V600 BRAF mutants (Class II & III). To address some of these limitations we have developed CFT1946, a bifunctional degradation activating compound (BiDAC™) degrader comprising a BRAF kinase domain targeting ligand linked to a cereblon ligand. CFT1946 is capable of degrading BRAF V600E (Class I), G469A (Class II), G466V (Class III) mutations, and the p61-BRAFV600E splice variant while maintaining exquisite selectivity against the proteome including WT BRAF and CRAF. In A375 cells, CFT1946 potently degraded BRAFV600E (Emax = 26%; DC50 = 14nM at 24hr) and, inhibited ERK phosphorylation (IC50 = 11nM at 24hr) and cell growth (GI50 = 94nM at 96hr) while having no effect in the mutant KRAS driven cell line HCT116. In A375 xenografts, oral delivery of CFT1946 resulted in deeper tumor regressions when dosed at 10 mg/kg PO BID and compared favorably to a clinically relevant dose of encorafenib. We further evaluated CFT1946 in an engineered A375-BRAFV600E/NRASQ61K double mutant model of BRAF inhibitor resistance. CFT1946 was able to degrade BRAFV600E in these cells and was much more effective than encorafenib at inhibiting viability in vitro. In this model, in vivo dosing of single agent CFT1946 caused robust tumor growth inhibition and combination with the MEK inhibitor, trametinib, resulted in tumor regressions. The combination of encorafenib and trametinib showed no activity in the same model. Next, we demonstrated that CFT1946 was able to degrade additional BRAF mutant proteins including G469A (Class II), G466V (Class III), and the p61-BRAFV600E splice variant using heterologous expression in HEK293T cells. Additionally, we also showed that CFT1946, but not encorafenib, inhibited proliferation of the BRAFG466V heterozygous lung tumor cell line H1666. Based on its activity in preclinical models, including models of BRAF inhibitor resistance, and its drug-like properties we are progressing CFT1946 as a candidate for clinical development in patients with solid tumors bearing BRAF V600X mutations. Further, given CFT1946’s activity on non-V600 BRAF mutations, we are continuing to explore CFT1946 and related BiDAC degraders as therapeutic options for patients bearing Class II or Class III BRAF mutations. Citation Format: Mathew E. Sowa, Bridget Kreger, Joelle Baddour, Yanke Liang, Jeffrey R. Simard, Laura Poling, Ping Li, Robert Yu, Ashley Hart, Roman V. Agafonov, Grace Sarkissian, Joe Sahil Patel, Richard Deibler, Kyle S. Cole, Scott Eron, David Cocozziello, Fazlur Rahman, Moses Moustakim, Christopher G. Nasveschuk, Katrina L. Jackson, Mark Fitzgerald, Victoria Garza, Morgan O’Shea, Gesine Veits, Jeremy L. Yap, Andrew J. Phillips, Elizabeth Norton, Adam S. Crystal, Stewart L. Fisher, Roy M. Pollock. Preclinical evaluation of CFT1946 as a selective degrader of mutant BRAF for the treatment of BRAF driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2158.

A hallmark of cancer is the evasion of apoptosis, which is often associated with the upregulation of the antiapoptotic members of the Bcl-2 family of proteins. The prosurvival function of the antiapoptotic Bcl-2 proteins is manifested by capturing and neutralizing the proapoptotic Bcl-2 proteins via their BH3 death domains. Accordingly, strategies to antagonize the antiapoptotic Bcl-2 proteins have largely focused on the development of low-molecular-weight, synthetic BH3 mimetics (“magic bullets”) to disrupt the protein–protein interactions between anti- and proapoptotic Bcl-2 proteins. In this way, apoptosis has been reactivated in malignant cells. Moreover, several such Bcl-2 family inhibitors are presently being evaluated for a range of cancers in clinical trials and show great promise as new additions to the cancer armamentarium. Indeed, the selective Bcl-2 inhibitor venetoclax (Venclexta) recently received FDA approval for the treatment of a specific subset of patients with chronic lymphocytic leukem...

The disruption of aberrant protein-protein interactions (PPIs) with synthetic agents remains a challenging goal in contemporary medicinal chemistry but some progress has been made. One such dysregulated PPI is that between the anti-apoptotic Bcl-2 proteins, including myeloid cell leukemia-1 (Mcl-1), and the α-helical Bcl-2 homology-3 (BH3) domains of its pro-apoptotic counterparts, such as Bak. Herein, we describe the discovery of small-molecule inhibitors of the Mcl-1 oncoprotein based on a novel chemotype. Particularly, re-engineering of our α-helix mimetic JY-1-106 into 2,6-di-substituted nicotinates afforded inhibitors of comparable potencies but with significantly decreased molecular weights. The most potent inhibitor 2-(benzyloxy)-6-(4-chloro-3,5-dimethylphenoxy)nicotinic acid (1 r: Ki =2.90 μm) likely binds in the p2 pocket of Mcl-1 and engages R263 in a salt bridge through its carboxylic acid, as supported by 2D (1) H-(15) N HSQC NMR data. Significantly, inhibitors were easily accessed in just four steps, which will facilitate future optimization efforts.

Overexpression of anti-apoptotic proteins belonging to the B cell lymphoma (Bcl)-2 family is observed in numerous cancer types and has been postulated to promote cancer cell survival and chemotherapy resistance. Bcl-extra large (xL)/myeloid cell leukemia sequence (Mcl)-1 was demonstrated to be expressed at relatively high levels in clinically aggressive basal-like cancers and inhibiting Bcl-xL overexpression could potentially provoke cell death. A molecule able to target Bcl-xL/Mcl-1, JY-1-106, is herein under investigation. It is also known that vitamin A-derived compounds exhibit antitumor activity in a variety of in vitro experimental models, promoting their effects via nuclear receptor isoforms including retinoic acid receptors (RARs). Pre-clinical observation highlighted that triple negative (estrogen receptor/progesterone receptor/human epidermal growth factor receptor)-breast cancer cells displayed resistance to retinoids due to the RARγ high expression profile. The present study used the triple-negative human breast cancer cell line, MDA-MB-231, to analyze the effects of the Bcl-xL/Mcl-1 synthetic inhibitor, JY-1-106, alone or in combination with retinoids on cell viability. The results revealed a synergistic effect in reducing cell viability primarily by using JY-1-106 with the selective RARγ antagonist SR11253, which induces massive autophagy and necrosis. Furthermore, the results highlighted that JY-1-106 alone is able to positively influence the gene expression profile of p53 and RARα, providing a therapeutic advantage in human triple-negative breast cancer treatment.

Mimicry of two faces of an α-helix might yield more potent and more selective inhibitors of dysregulated, helix-mediated protein–protein interactions (PPI). Herein, we demonstrate that a 2,6,9-tri-substituted purine is capable of disrupting the Mcl-1–Bak-BH3 PPI through effective mimicry of key residues on opposing faces of the Bak-BH3 α-helix.

The acute promyelocytic leukemia (APL) subtype of acute myeloid leukemia (AML) is characterized by chromosomal translocations that result in fusion proteins, including the promyelocytic leukemia-retinoic acid receptor, alpha fusion protein (PML-RARα). All-trans retinoic acid (atRA) treatment is the standard drug treatment for APL yielding cure rates > 80% by activating transcription and proteasomal degradation of retinoic acid receptor, alpha (RARα). Whereas combination therapy with As2O3 has increased survival further, patients that experience relapse and are refractory to atRA and/or As2O3 is a clinically significant problem. BCL-2 family proteins regulate apoptosis and over-expression of anti-apoptotic B-cell leukemia/lymphoma 2 (BCL-2) family proteins has been associated with chemotherapeutic resistance in APL including impairment of the ability of atRA to induce growth arrest and differentiation. Here we investigated the novel BH3 domain mimetic, JY-1-106, which antagonizes the anti-apoptotic BCL-2 family members B-cell lymphoma-extra large (BCL-xL) and myeloid cell leukemia-1 (MCL-1) alone and in combination with retinoids including atRA, AM580 (RARα agonist), and SR11253 (RARγ antagonist). JY-1-106 reduced cell viability in HL-60 cells alone and in combination with retinoids. The combination of JY-1-106 and SR11253 had the greatest impact on cell viability by stimulating apoptosis. These studies indicate that dual BCL-xL/MCL-1 inhibitors and retinoids could work cooperatively in leukemia treatment.

A hallmark of cancer is the evasion of apoptosis, which is often associated with the upregulation of the antiapoptotic members of the Bcl-2 family of proteins. The prosurvival function of the antiapoptotic Bcl-2 proteins is manifested by capturing and neutralizing the proapoptotic Bcl-2 proteins via their BH3 death domains. Accordingly, strategies to antagonize the antiapoptotic Bcl-2 proteins have largely focused on the development of low-molecular-weight, synthetic BH3 mimetics ("magic bullets") to disrupt the protein-protein interactions between anti- and proapoptotic Bcl-2 proteins. In this way, apoptosis has been reactivated in malignant cells. Moreover, several such Bcl-2 family inhibitors are presently being evaluated for a range of cancers in clinical trials and show great promise as new additions to the cancer armamentarium. Indeed, the selective Bcl-2 inhibitor venetoclax (Venclexta) recently received FDA approval for the treatment of a specific subset of patients with chronic lymphocytic leukemia. This review focuses on the major developments in the field of Bcl-2 inhibitors over the past decade, with particular emphasis on binding modes and, thus, the origins of selectivity for specific Bcl-2 family members.

Structure-based drug design was utilized to develop novel, 1-hydroxy-2-naphthoate-based small-molecule inhibitors of Mcl-1. Ligand design was driven by exploiting a salt bridge with R263 and interactions with the p2 and p3 pockets of the protein. Significantly, target molecules were accessed in just two synthetic steps, suggesting further optimization will require minimal synthetic effort. Molecular modeling using the Site-Identification by Ligand Competitive Saturation (SILCS) approach was used to qualitatively direct ligand design as well as develop quantitative models for inhibitor binding affinity to Mcl-1 and the Bcl-2 relative Bcl-xL as well as for the specificity of binding to the two proteins. Results indicated hydrophobic interactions with the p2 pockets dominate the affinity of the most favourable binding ligand (3bl: Ki = 31 nM). Compounds were up to 20-fold selective for Mcl-1 over Bcl-xL. Selectivity of the inhibitors was driven by interactions with the deeper p2 pocket in Mcl-1 versus Bcl-xL. The SILCS-based SAR of the present compounds represents the foundation for the development of Mcl-1 specific inhibitors with the potential to treat a wide range of solid tumours and hematological cancers, including acute myeloid leukaemia.

3-Substituted, 2,6-dichloropyridines have featured in the syntheses of small molecule inhibitors of a wide variety of biological targets. Hence, the regioselective displacement of the chlorines is of significant interest. Through conducting an extensive solvent study, we have found that non-polar, aprotic solvents of low hydrogen bond basicities favour substitution of the chlorine ortho to the 3-substituent by alkali metal alkoxides. We present convincing evidence that coordination of the alkali metal counter-ion to the 3-substituent (nitro, ester, amide) is the origin of the ortho-selectivity to give a cyclic, six-membered transition state. Excellent ortho-selectivities (⩾98:2) for secondary and tertiary alkoxides were realized with the sodium counter-ion, whereas the more reactive primary alkoxides required the harder, more Lewis acidic lithium counter-ion.

The c-Myc (Myc) oncoprotein is a high-value therapeutic target given that it is deregulated in multiple types of cancer. However, potent small molecule inhibitors of Myc have been difficult to identify, particularly those whose mechanism relies on blocking the association between Myc and its obligate heterodimerization partner, Max. We have recently reported a structure-activity relationship study of one such small molecule, 10074-G5, and generated an analog, JY-3-094, with significantly improved ability to prevent or disrupt the association between recombinant Myc and Max proteins. However, JY-3094 penetrates cells poorly. Here, we show that esterification of a critical para-carboxylic acid function of JY-3-094 by various blocking groups significantly improves cellular uptake although it impairs the ability to disrupt Myc-Max association in vitro. These pro-drugs are highly concentrated within cells where JY-3-094 is then generated by the action of esterases. However, the pro-drugs are also variably susceptible to extracellular esterases, which can deplete extracellular reservoirs. Furthermore, while JY-3-094 is retained by cells for long periods of time, much of it is compartmentalized within the cytoplasm in a form that appears to be less available to interact with Myc. Our results suggest that persistently high extracellular levels of pro-drug, without excessive susceptibility to extracellular esterases, are critical to establishing and maintaining intracellular levels of JY-3-094 that are sufficient to provide for long-term inhibition of Myc-Max association. Analogs of JY-3-094 appear to represent promising small molecule Myc inhibitors that warrant further optimization.

The rational design of inhibitors of the bHLH-ZIP oncoprotein c-Myc is hampered by a lack of structure in its monomeric state. We describe herein the design of novel, low-molecular-weight, synthetic α-helix mimetics that recognize helical c-Myc in its transcriptionally active coiled-coil structure in association with its obligate bHLH-ZIP partner Max. These compounds perturb the heterodimer’s binding to its canonical E-box DNA sequence without causing protein–protein dissociation, heralding a new mechanistic class of “direct” c-Myc inhibitors. In addition to electrophoretic mobility shift assays, this model was corroborated by further biophysical methods, including NMR spectroscopy and surface plasmon resonance. Several compounds demonstrated a 2-fold or greater selectivity for c-Myc–Max heterodimers over Max–Max homodimers with IC50 values as low as 5.6 μM. Finally, these compounds inhibited the proliferation of c-Myc-expressing cell lines in a concentration-dependent manner that correlated with the loss of expression of a c-Myc-dependent reporter plasmid despite the fact that c-Myc–Max heterodimers remained intact. © 2015 American Chemical Society.This document is the Accepted Manuscript version of a Published Work that appeared in final form after peer review and technical editing by the publisher. To access the final edited and published work see dx.doi.org/10.1021/jm501440q

c-Myc is a bHLH-ZIP transcription factor that is responsible for the transcription of a wide range of target genes involved in many cancer-related cellular processes, such as proliferation, differentiation, apoptosis and metabolism. Over-expression of c-Myc has been observed in, and directly contributes to, a variety of human cancers including those of the hematopoietic system, lung, prostate and colon. To become transcriptionally active, c-Myc must first dimerize with Max via its own bHLH-ZIP domain. A proven strategy towards the inhibition of c-Myc oncogenic activity is to interfere with the structural integrity of the c-Myc–Max heterodimer. The small-molecule 10074-G5 is an inhibitor of c-Myc–Max dimerization (IC50 = 146 μM) that operates by binding and stabilizing c-Myc in its monomeric form. Herein, we report on our on-going efforts to optimize the c-Myc–Max inhibitory activity of 10074-G5-related molecules in vitro and in cancer cells that over-express c-Myc. Specifically, we have identified a congener of 10074-G5, termed 3jc48-3, that is about five times as potent (IC50 = 34 μM) at inhibiting c-Myc–Max dimerization as the parent compound. In addition, 3jc48-3 exhibited an approximate two-fold selectivity for c-Myc–Max heterodimers over Max–Max dimers, suggesting that, like its predecessor, its mode of action is through binding c-Myc. 3jc48-3 inhibited the proliferation of c-Myc-over-expressing HL60 and Daudi cells with single-digit micromolar IC50 values by causing growth arrest at the G0/G1 phase. Furthermore, co-immunoprecipitation studies indicated that 3jc48-3 inhibits c-Myc–Max dimerization in cells, which was further substantiated by the specific silencing of a c-Myc-driven luciferase reporter gene. Finally, unlike previously described 10074-G5 analogues, which are rapidly released and/or metabolized by cells following their uptake, 3jc48-3’s intracellular half-life was >17 h. Collectively, these data demonstrate 3jc48-3 to be one of the most potent cellularly active c-Myc inhibitors reported to date.

In order to mimic amphipathic α-helices, a novel scaffold based on a 1,2-diphenylacetylene was designed. NMR and computational modeling confirmed that an intramolecular hydrogen bond favors conformations of the 1,2-diphenylacetylene that allow for accurate mimicry of the i, i + 7 and i + 2, i + 5 side chains found on opposing faces of an α-helix.

// Huabo Wang 1 , Jay Chauhan 2 , Angela Hu 1 , Kelsey Pendleton 1 , Jeremy L. Yap 2 , Philip E. Sabato 3 , Jace W. Jones 2 , Mariarita Perri 4 , Jianshi Yu 2 , Erika Cione 4 , Maureen A. Kane 2,5 , Steven Fletcher 2,5 and Edward V. Prochownik 1,6,7 1 Section of Hematology/Oncology, Children’s Hospital of Pittsburgh of UPMC 2 Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, USA 3 PharmD Program, University of Maryland School of Pharmacy, Baltimore, MD, USA 4 Department of Pharmaco-Biology, University of Calabria, Rende (CS), Italy 5 University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD, USA 6 Department of Microbiology and Molecular Genetics, The University of Pittsburgh School of Medicine 7 The University of Pittsburgh Cancer Institute, Pittsburgh, PA Correspondence: Edward V. Prochownik, email: // Steven Fletcher, email: // Keywords : 10058-F4, 10074-G5, intrinsically disordered proteins, JQ-1, BRD4 Received : May 31, 2013 Accepted : June 21, 2013 Published : June 22, 2013 Abstract The c-Myc (Myc) oncoprotein is a high-value therapeutic target given that it is deregulated in multiple types of cancer. However, potent small molecule inhibitors of Myc have been difficult to identify, particularly those whose mechanism relies on blocking the association between Myc and its obligate heterodimerization partner, Max. We have recently reported a structure-activity relationship study of one such small molecule, 10074-G5, and generated an analog, JY-3-094, with significantly improved ability to prevent or disrupt the association between recombinant Myc and Max proteins. However, JY-3094 penetrates cells poorly. Here, we show that esterification of a critical para -carboxylic acid function of JY-3-094 by various blocking groups significantly improves cellular uptake although it impairs the ability to disrupt Myc-Max association in vitro . These pro-drugs are highly concentrated within cells where JY-3-094 is then generated by the action of esterases. However, the pro-drugs are also variably susceptible to extracellular esterases, which can deplete extracellular reservoirs. Furthermore, while JY-3-094 is retained by cells for long periods of time, much of it is compartmentalized within the cytoplasm in a form that appears to be less available to interact with Myc. Our results suggest that persistently high extracellular levels of pro-drug, without excessive susceptibility to extracellular esterases, are critical to establishing and maintaining intracellular levels of JY-3-094 that are sufficient to provide for long-term inhibition of Myc-Max association. Analogs of JY-3-094 appear to represent promising small molecule Myc inhibitors that warrant further optimization.

We herein report on the pharmacophore determination of the ERK docking domain inhibitor (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione, which has led to the discovery of compounds with greater selectivities for inhibiting the proliferation of melanoma cells containing active ERK signaling.

Background It has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-2/Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. We designed the BH3 α-helix mimetic JY-1-106 to engage the hydrophobic BH3-binding grooves on the surfaces of both Bcl-xL and Mcl-1. Methods JY-1-106–protein complexes were studied using molecular dynamics (MD) simulations and the SILCS methodology. We have evaluated the in vitro effects of JY-1-106 by using a fluorescence polarization (FP) assay, an XTT assay, apoptosis assays, and immunoprecipitation and western-blot assays. A preclinical human cancer xenograft model was used to test the efficacy of JY-1-106 in vivo. Results MD and SILCS simulations of the JY-1-106–protein complexes indicated the importance of the aliphatic side chains of JY-1-106 to binding and successfully predicted the improved affinity of the ligand for Bcl-xL over Mcl-1. Ligand binding affinities were measured via an FP assay using a fluorescently labeled Bak-BH3 peptide in vitro. Apoptosis induction via JY-1-106 was evidenced by TUNEL assay and PARP cleavage as well as by Bax–Bax dimerization. Release of multi-domain Bak from its inhibitory binding to Bcl-2/Bcl-xL and Mcl-1 using JY-1-106 was detected via immunoprecipitation (IP) western blotting. At the cellular level, we compared the growth proliferation IC50s of JY-1-106 and ABT-737 in multiple cancer cell lines with various Bcl-xL and Mcl-1 expression levels. JY-1-106 effectively induced cell death regardless of the Mcl-1 expression level in ABT-737 resistant solid tumor cells, whilst toxicity toward normal human endothelial cells was limited. Furthermore, synergistic effects were observed in A549 cells using a combination of JY-1-106 and multiple chemotherapeutic agents. We also observed that JY-1-106 was a very effective agent in inducing apoptosis in metabolically stressed tumors. Finally, JY-1-106 was evaluated in a tumor-bearing nude mouse model, and was found to effectively repress tumor growth. Strong TUNEL signals in the tumor cells demonstrated the effectiveness of JY-1-106 in this animal model. No significant side effects were observed in mouse organs after multiple injections. Conclusions Taken together, these observations demonstrate that JY-1-106 is an effective pan-Bcl-2 inhibitor with very promising clinical potential.

Background It has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. Mcl-1 is a critical survival protein in a variety of cell lineages and is critically regulated via ubiquitination. Methods The Mcl-1, Bcl-xL and USP9X expression patterns in human lung and colon adenocarcinomas were evaluated via immunohistochemistry. Interaction between USP9X and Mcl-1 was demonstrated by immunoprecipitation-western blotting. The protein expression profiles of Mcl-1, Bcl-xL and USP9X in multiple cancer cell lines were determined by western blotting. Annexin-V staining and cleaved PARP western blotting were used to assay for apoptosis. The cellular toxicities after various treatments were measured via the XTT assay. Results In our current analysis of colon and lung cancer samples, we demonstrate that Mcl-1 and Bcl-xL are overexpressed and also co-exist in many tumors and that the expression levels of both genes correlate with the clinical staging. The downregulation of Mcl-1 or Bcl-xL via RNAi was found to increase the sensitivity of the tumor cells to chemotherapy. Furthermore, our analyses revealed that USP9X expression correlates with that of Mcl-1 in human cancer tissue samples. We additionally found that the USP9X inhibitor WP1130 promotes Mcl-1 degradation and increases tumor cell sensitivity to chemotherapies. Moreover, the combination of WP1130 and ABT-737, a well-documented Bcl-xL inhibitor, demonstrated a chemotherapeutic synergy and promoted apoptosis in different tumor cells. Conclusion Mcl-1, Bcl-xL and USP9X overexpression are tumor survival mechanisms protective against chemotherapy. USP9X inhibition increases tumor cell sensitivity to various chemotherapeutic agents including Bcl-2/Bcl-xL inhibitors.

A structure–activity relationship (SAR) study of the c-Myc (Myc) inhibitor 10074-G5 (N-([1,1′-biphenyl]-2-yl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine, 1) – which targets a hydrophobic domain of the Myc oncoprotein that is flanked by arginine residues – was executed in order to determine its pharmacophore. Whilst the 7-nitrobenzofurazan was found to be critical for inhibitory activity, the ortho-biphenyl could be replaced with a para-carboxyphenyl group to furnish the new inhibitor JY-3-094 (3q). Around five times as potent as the lead with an IC50 of 33 μM for disruption of the Myc–Max heterodimer, JY-3-094 demonstrated excellent selectivity over Max–Max homodimers, with no apparent effect at 100 μM. Importantly, the carboxylic acid of JY-3-094 improves the physicochemical properties of the lead compound, which will facilitate the incorporation of additional hydrophobicity that might enhance Myc inhibitory activity further still.

By conducting a structure-activity relationship study of the backbone of a series of oligoamide-foldamer-based α-helix mimetics of the Bak BH3 helix, we have identified especially potent inhibitors of Bcl-x(L). The most potent compound has a K(i) value of 94 nM in vitro, and single-digit micromolar IC(50) values against the proliferation of several Bcl-x(L)-overexpressing cancer cell lines.

The Ras→Raf→MEK(mitogen-activated kinase kinase)→ERK (extracellular-signal-regulated kinase) signalling pathway is one of at least five mitogen-activated protein kinase (MAPK) pathways that control several fundamental cellular processes, driving proliferation, differentiation and cell survival.[1–3] Signal transduction through this particular pathway, which is depicted in Figure 1, is initiated by the binding of a wide variety of ligands, including hormones and growth factors, to receptor tyrosine kinases (RTKs). This leads to the activation of Ras proteins (H-, K- and N-Ras isoforms) previously anchored in the plasma membrane by earlier post-translational reactions, e.g. farnesylation. Subsequently, the Ras proteins are induced to exchange their bound GDP for GTP, which leads to a conformational change in Ras and the initiation of a three-stage phosphorylation cascade that climaxes with the activation of ERK1/2. First, the Raf family of kinases (A-, B- and Raf-1 isoforms), the best studied of which is Raf-1, is recruited to the plasma membrane. Upon its subsequent phosphorylation, Raf-1 then activates (phosphorylates) MAP / ERK kinase 1 and 2 (MEK1/2), which, in turn, activate (phosphorylate) ERK1 and ERK2 (p44 MAPK and p42 MAPK, respectively). ERK1/2 are activated through phosphorylation of both a threonine and a tyrosine residue, namely Thr202 and Tyr204 of ERK1 and Thr183 and Tyr185 of ERK2. MEK1/2 are the only known activators of ERK1/2 and are, thus, dual specificity kinases. Activated ERK1/2 then phosphorylate serine/threonine residues of more than 50 downstream cytosolic and nuclear substrates, leading to alterations in gene expression profiles and an increase in proliferation, differentiation and cell survival.[1–3] Figure 1 Schematic representation of the Ras→Raf→MEK1/2→ERK1/2 signalling pathway. GF = growth factor, RTK = receptor tyrosine kinase, Grb2 = growth factor receptor-bound protein 2; Sos = son of sevenless; P indicates a phosphorylated serine, ... There is now considerable evidence that links the dysregulation of the Ras→Raf→MEK→ERK pathway to the oncogenesis of human cancers. Ras is hyperactivated in around 30% of human cancers, most commonly the K-Ras isoform.[4] More specifically, Ras activating mutations have been reported in about 90% of pancreatic carcinomas, 50% of colon carcinomas, 30% of lung cancers and in around 30% of myeloid leukaemia cases.[4] Activating mutations of Raf have also been reported in around 7% of human cancers.[5,6] In particular, mutations of B-Raf have been observed in over 60% of melanomas, around 30% of ovarian cancer and in approximately 20% of colorectal carcinomas, as well as in several other malignancies at lower frequencies.[5,6] Constitutively activate MEK1/2 and ERK1/2 proteins are present in a relatively high number of human tumours, particularly those from the colon, lung, pancreas, ovary and kidney.[7] Since mutations of the MEK1/2 and ERK1/2 genes have not been observed in human tumours, it seems probable that the hyperactivity of these proteins is a consequence of their constitutive phosphorylation due to hyperactivation of upstream effectors, including receptors, Ras and B-Raf. In summary, the Ras→Raf→MEK1/2→ERK1/2 pathway is an appealing target for the development of potential anti-cancer therapeutics. Moreover, the pathway offers several junctures for signal transduction blockade; due to the converging functions of MEK1/2 and ERK1/2, specific inhibition of these proteins is particularly desirable. In this mini-review, some of the more prominent small molecule inhibitors of the ERK pathway will be presented, with a particular emphasis on those discovered within the last ten to fifteen years. In the first section, we shall discuss those inhibitors that target proteins upstream of ERK1/2, specifically Raf and MEK1/2. We will then shift to the main focus of this review, which is the direct inhibition of ERK1/2 through targeting either the ATP-binding site (ATP-competitive inhibitors) or the surface of ERK and blocking its protein–protein interactions with its substrates (non-ATP-competitive inhibitors).

The Ras→Raf→MEK(mitogen-activated kinase kinase)→ERK (extracellular-signal-regulated kinase) signalling pathway is one of at least five mitogen-activated protein kinase (MAPK) pathways that control several fundamental cellular processes, driving proliferation, differentiation and cell survival.[1–3] Signal transduction through this particular pathway, which is depicted in Figure 1, is initiated by the binding of a wide variety of ligands, including hormones and growth factors, to receptor tyrosine kinases (RTKs). This leads to the activation of Ras proteins (H-, K- and N-Ras isoforms) previously anchored in the plasma membrane by earlier post-translational reactions, e.g. farnesylation. Subsequently, the Ras proteins are induced to exchange their bound GDP for GTP, which leads to a conformational change in Ras and the initiation of a three-stage phosphorylation cascade that climaxes with the activation of ERK1/2. First, the Raf family of kinases (A-, B- and Raf-1 isoforms), the best studied of which is Raf-1, is recruited to the plasma membrane. Upon its subsequent phosphorylation, Raf-1 then activates (phosphorylates) MAP / ERK kinase 1 and 2 (MEK1/2), which, in turn, activate (phosphorylate) ERK1 and ERK2 (p44 MAPK and p42 MAPK, respectively). ERK1/2 are activated through phosphorylation of both a threonine and a tyrosine residue, namely Thr202 and Tyr204 of ERK1 and Thr183 and Tyr185 of ERK2. MEK1/2 are the only known activators of ERK1/2 and are, thus, dual specificity kinases. Activated ERK1/2 then phosphorylate serine/threonine residues of more than 50 downstream cytosolic and nuclear substrates, leading to alterations in gene expression profiles and an increase in proliferation, differentiation and cell survival.[1–3] Figure 1 Schematic representation of the Ras→Raf→MEK1/2→ERK1/2 signalling pathway. GF = growth factor, RTK = receptor tyrosine kinase, Grb2 = growth factor receptor-bound protein 2; Sos = son of sevenless; P indicates a phosphorylated serine, ... There is now considerable evidence that links the dysregulation of the Ras→Raf→MEK→ERK pathway to the oncogenesis of human cancers. Ras is hyperactivated in around 30% of human cancers, most commonly the K-Ras isoform.[4] More specifically, Ras activating mutations have been reported in about 90% of pancreatic carcinomas, 50% of colon carcinomas, 30% of lung cancers and in around 30% of myeloid leukaemia cases.[4] Activating mutations of Raf have also been reported in around 7% of human cancers.[5,6] In particular, mutations of B-Raf have been observed in over 60% of melanomas, around 30% of ovarian cancer and in approximately 20% of colorectal carcinomas, as well as in several other malignancies at lower frequencies.[5,6] Constitutively activate MEK1/2 and ERK1/2 proteins are present in a relatively high number of human tumours, particularly those from the colon, lung, pancreas, ovary and kidney.[7] Since mutations of the MEK1/2 and ERK1/2 genes have not been observed in human tumours, it seems probable that the hyperactivity of these proteins is a consequence of their constitutive phosphorylation due to hyperactivation of upstream effectors, including receptors, Ras and B-Raf. In summary, the Ras→Raf→MEK1/2→ERK1/2 pathway is an appealing target for the development of potential anti-cancer therapeutics. Moreover, the pathway offers several junctures for signal transduction blockade; due to the converging functions of MEK1/2 and ERK1/2, specific inhibition of these proteins is particularly desirable. In this mini-review, some of the more prominent small molecule inhibitors of the ERK pathway will be presented, with a particular emphasis on those discovered within the last ten to fifteen years. In the first section, we shall discuss those inhibitors that target proteins upstream of ERK1/2, specifically Raf and MEK1/2. We will then shift to the main focus of this review, which is the direct inhibition of ERK1/2 through targeting either the ATP-binding site (ATP-competitive inhibitors) or the surface of ERK and blocking its protein–protein interactions with its substrates (non-ATP-competitive inhibitors).

Transcription factors are DNA-binding proteins that – usually in combination with other proteins to form the pre-initiation complex (PIC) – regulate the transcription of specific DNA sequences (genes) into mRNA by controlling the recruitment of RNA polymerase II. Constitutive activation of transcription factors can lead to a variety of cancers, and are, therefore, important therapeutic targets. However, in stark contrast to targeting enzyme active sites, disruption of protein–protein or protein–DNA interactions involved in the transcriptional machinery is particularly challenging owing to the large interfacial areas involved, a lack of obvious binding sites and often non-contiguous contact points. Especially problematic for the development of small-molecules is the need by such agents to overcome the large free energy of association between protein–protein and, to a lesser extent, protein–DNA interfaces. Nevertheless, recent years have seen considerable success in this area of medicinal chemistry, cementing the notion that disruption of such interactions is feasible with small-molecule, drug-like compounds. We discuss, in particular, the disruption of dimeric transcription factors, such as STAT3–STAT3, c-Myc–Max and c-Jun–c-Fos (AP-1), with small-molecules that block their protein–protein interactions or their interactions with DNA.

Extracellular signal-related kinase-1 and 2 (ERK1/2) are hyperactivated in several human cancers, predominantly due to gain-of function mutations in the upstream Ras and Raf proteins. However, its complete inhibition through targeting its active site is expected to be detrimental, since ERK is crucial in various “housekeeping” roles in normal cells. Therefore, our aim is to block specific substrate docking domains on the surface of ERK and, hence, inhibit functions involved in cancer cell proliferation, such as the phosphorylation (activation) of the transcription factor Elk-1 and the enzyme ribosomal S6 kinase-1 (RSK-1). We have previously used computer-aided drug design (CADD) to identify ERK docking domain inhibitors, which led to the discovery of (Z)-3-(2-aminoethyl)-5-(4-ethoxybenzylidene)thiazolidine-2,4-dione (referred to in the literature as 76) that is believed to target the groove in between the common docking (CD) and ED domains. A fluorescence quenching assay showed that 76 binds ERK2 with a KD of 5 μM, exhibits minimal selectivity over the structurally related p38α MAP kinase, and inhibits the proliferation of A375 melanoma cells, which harbor constitutive ERK pathway activation owing to a B-Raf mutation, with an IC50 value of about 20 μM. Through functionalization of the primary amino group of our lead to afford a focused library of amides, carbamates, sulfonamides and anilines, we have been able to improve binding affinities for ERK2 to as low as 70 nM. Importantly, selectivity for ERK2 over p38α was increased to more than 70-fold. Mutation studies suggested that several of our best compounds bind in the vicinity of Thr157 (located at the ED domain) and, to a lesser extent, Asp319 of the CD domain (a residue that was predicted by CADD to bind the primary amino group of 76). Immunoblot analysis demonstrated that new lead SF-2-054 (ERK2: KD = 170 nM) is a more potent inhibitor of Elk-1 phosphorylation than the parent compound 76. Furthermore, negligible effects on the levels of activated ERK2 suggested that SF-2-054 does not operate upstream of ERK2. Consistent with the mutation binding data, SF-2-054 exhibits minimal effects of the levels of phosphorylated Rsk-1, which is known to bind the CD site, opening the door for the development of substrate-specific ERK inhibitors. At the same time, it is noteworthy that this data suggests that SF-2-054 does not bind the catalytic domain of ERK2. In addition, SF-2-054 was found to inhibit proliferation of A375 cells with an IC50 value of < 5 μM, around 5 times more potent than in the HeLa cell line. In contrast to B-Raf (and MEK) inhibitors that afford complete blockade of the B-Raf/MEK/ERK signaling pathway, our approach demonstrates that only partial inhibition of ERK signaling function is necessary to inhibit the proliferation of melanoma cells. Moreover, it is envisaged that targeting ERK docking sites will lead to inhibitors with improved selectivities to which the development of resistance is less likely. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B132.