Effects of the CK2 Inhibitors CX-4945 and CX-5011 on Drug-Resistant Cells

CK2 is a pleiotropic protein kinase, which regulates many survival pathways and plays a global anti-apoptotic function. It is highly expressed in tumor cells, and is presently considered a promising therapeutic target. Among the many inhibitors available for this kinase, the recently developed CX-4945 and CX-5011 have proved to be very potent, selective and effective in inducing cell death in tumor cells; CX-4945 has recently entered clinical trials. However, no data are available on the efficacy of these compounds to overcome drug resistance, a major reasons of cancer therapy failure. Here we address this point, by studying their effects in several tumor cell lines, each available as variant R resistant to drug-induced apoptosis, and normal-sensitive variant S. We found that the inhibition of endogenous CK2 was very similar in S and R treated cells, with more than 50% CK2 activity reduction at sub-micromolar concentrations of CX-4945 and CX-5011. A consequent apoptotic response was induced both in S and R variants of each pairs. Moreover, the combined treatment of CX-4945 plus vinblastine was able to sensitize to vinblastine R cells that are otherwise almost insensitive to this conventional antitumor drug. Consistently, doxorubicin accumulation in multidrug resistant (MDR) cells was greatly increased by CX-4945.In summary, we demonstrated that all the R variants are sensitive to CX-4945 and CX-5011; since some of the treated R lines express the extrusion pump Pgp, often responsible of the MDR phenotype, we can also conclude that the two inhibitors can successfully overcome the MDR phenomenon.     

CX-4945: the protein kinase CK2 inhibitor and anti-cancer drug shows anti-fungal activity

CX-4945 is a selective inhibitor of protein kinase CK2 exhibiting clinical significance. Its antitumor properties arise from the abrogation of CK2-mediated pro-survival cellular pathways. The presented data reveal the influence of CX-4945 on the growth of yeast cells showing variable potency against Saccharomyces cerevisiae deletion strains with different contents of CK2 subunits. The catalytic subunit CK2α appears to sensitize yeast to the CX-4945 action. Moreover, the compound suppresses hyphal growth and cell adhesion of Candida albicans, thereby abolishing some hallmarks of invasiveness of the pathogen. It is known that cancer patients are more prone to fungal infections. Our data unveil the dual-activity of CX-4945; when used in anti-cancer therapy, it may simultaneously prevent cancer-associated candidiasis.     

The protein kinase 2 inhibitor CX-4945 regulates osteoclast and osteoblast differentiation In vitro

Drug repositioning can identify new therapeutic applications for existing drugs, thus mitigating high R&D costs. The Protein kinase 2 (CK2) inhibitor CX-4945 regulates human cancer cell survival and angiogenesis. Here we found that CX-4945 significantly inhibited the RANKL-induced osteoclast differentiation, but enhanced the BMP2-induced osteoblast differentiation in a cell culture model. CX-4945 inhibited the RANKL-induced activation of TRAP and NFATc1 expression accompanied with suppression of Akt phosphorylation, but, in contrast, it enhanced the BMP2-mediated ALP induction and MAPK ERK1/2 phosphorylation. CX-4945 is thus a novel drug candidate for bone-related disorders such as osteoporosis.     

Synthesis and characterization of a potent and selective protein tyrosine phosphatase inhibitor, 2

The synthesis and biological activity of a series of 2-[(4-methylthiopyridin-2-yl)methylsulfinyl]benzimidazoles are described. These compounds have potent inhibitory effects against the protein tyrosine phosphatase activity of CD45. Enzymatic analysis with several phosphatases revealed that compound 5a had high specificity for CD45 compared with serine/threonine phosphatases (PP1, PP2A), tyrosine phosphatases (LAR, PTP1B and PTP-S2) and dual phosphatase (VHR).     

Identification of a selective small molecule inhibitor of breast cancer stem cells

A high-throughput screen (HTS) with the National Institute of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) compound collection identified a class of acyl hydrazones to be selectively lethal to breast cancer stem cell (CSC) enriched populations. Medicinal chemistry efforts were undertaken to optimize potency and selectivity of this class of compounds. The optimized compound was declared as a probe (ML239) with the NIH Molecular Libraries Program and displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control line (HMLE_sh_GFP).     

ARIMIDEX: a potent and selective aromatase inhibitor for the treatment of advanced breast cancer

Aromatase inhibitors have been available for a number of years and their ability to reduce circulating estradiol levels has been shown to produce clinical benefit in women with advanced breast cancer. Until recently, the only commercially available aromatase inhibitor was aminoglutethimide. Although aminoglutethimide has been shown to be efficacious in the treatment of advanced breast cancer, it does cause significant toxicity and requires the use of concomitant hydrocortisone therapy. Anastrozole is one of a new class of potent aromatase inhibitors able to suppress estradiol to the limit of detection of sensitive assays without suppressing adrenal steroidal synthesis. Two large clinical trials (n = 764) conducted in the U.S.A. and in Europe evaluated two doses of anastrozole, 1 and 10 mg a day, compared to megesterol acetate, 40 mg four times a day, in postmenopausal women who had progressed while on tamoxifen. Response rates and time to progression with anastrozole were similar to those of megesterol acetate. Objective responses (CR + PR) were 10.3%, 8.9% and 7.9% in the 1 and 10 mg of anastrozole and megesterol acetate treatment groups, respectively. Another 25.2%, 22.6% and 26.1% had stable disease for over 24 weeks on 1, 10 mg anastrozole and megesterol acetate, respectively. Anastrozole and megesterol acetate were well tolerated; however, more patients had significant weight gain on megesterol acetate than with anastrozole treatment. The weight gain seen with megesterol acetate continued to increase over time. Anastrozole has a better therapeutic index (fewer side-effects) and has recently been approved by the FDA and a number of other regulatory agencies around the world for the treatment of advanced breast cancer.     

Identification of a small molecule SIRT2 inhibitor with selective tumor cytotoxicity

As a member of the class III histone deacetylases, Sirtuin-2 (SIRT2) is critical in cell cycle regulation which makes it a potential target for cancer therapeutics. In this study, we identified a novel SIRT2 inhibitor, AC-93253, with IC₅₀ of 6 μM in vitro. The compound is selective, inhibiting SIRT2 7.5- and 4-fold more potently than the closely related SIRT1 and SIRT3, respectively. AC-93253 significantly enhanced acetylation of tubulin, p53, and histone H4, confirming SIRT2 and SIRT1 as its cellular targets. AC-93253 as a single agent exhibited submicromolar selective cytotoxicity towards all four tumor cell lines tested with a therapeutic window up to 200-fold, comparing to any of the three normal cell types tested. Results from high content analysis suggested that AC-93253 significantly triggered apoptosis. Taken together, SIRT2 selective inhibitor AC-93253 may serve as a novel chemical scaffold for structure-activity relationship study and future lead development.     

Discovery of a potent and selective small molecule hGPR91 antagonist

GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC₅₀: 0.8 μM)—originally synthesized in Merck for Bradykinin B₁ Receptor (BK₁R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5g (IC₅₀: 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC₅₀: 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay.     

Discovery of TAK-733, a potent and selective MEK allosteric site inhibitor for the treatment of cancer

A novel 5-phenylamino-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione series of MEK inhibitors has been developed using structure-based drug design. Lead optimization of this series led to the discovery of TAK-733. This was advanced to Phase I clinical studies for cancer treatment.     

Discovery of potent and selective spiroindolinone MDM2 inhibitor,RO8994, for cancer therapy

The field of small-molecule inhibitors of protein–protein interactions is rapidly advancing and the specific area of inhibitors of the p53/MDM2 interaction is a prime example. Several groups have published on this topic and multiple compounds are in various stages of clinical development. Building on the strength of the discovery of RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound, we have developed additional scaffolds that provide opportunities for future development. Here, we report the discovery and optimization of a highly potent and selective series of spiroindolinone small-molecule MDM2 inhibitors, culminating in RO8994.     

Computer-aided, rational design of a potent and selective small peptide inhibitor of cyclooxygenase 2 (COX2)

Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are the mediators of inflammation. This enzyme exists mainly in two isoforms, COX1 and COX2. Prostaglandins responsible for the inflammatory process could be sufficiently controlled with the conventional non-steroidal anti-inflammatory drugs (NSAIDs). These drugs, however, had adverse gastrointestinal side-effects and, therefore, drugs that selectively inhibit COX2, such as the coxibs, were developed. Recent reports on the harmful cardiovascular and renal side-effects of the conventional NSAIDs as well as the COX2 selective inhibitors valdecoxib and rofecoxib have once again led to the quest for a novel class of COX2 selective inhibitors. Keeping this in mind, we have used the available X-ray crystal structures of the complexes of COX1 and COX2 with the known inhibitors to carry out a structure-based, rational, molecular modeling approach to design a small peptide inhibitor, which is both potent and selective for COX2. Docking studies using SYBYL 6.81 (Tripos, Inc.) and AutoDock 3.0, indicate that the designed peptides inhibit COX2 with potency in the nanomolar range. Furthermore, it is found to be a million-fold selective for COX2 as compared with COX1. Thus, the small peptide inhibitor is a suitable lead compound for the design of a new class of anti-inflammatory drugs.     

MEK Inhibitor PD-0325901 Overcomes Resistance to CK2 Inhibitor CX-4945 and Exhibits Anti-Tumor Activity in Head and Neck Cancer

The serine-threonine kinase CK2 exhibits genomic alterations and aberrant overexpression in human head and neck squamous cell carcinomas (HNSCC). Here, we investigated the effects of CK2 inhibitor CX-4945 in human HNSCC cell lines and xenograft models. The IC_50''s of CX-4945 for 9 UM-SCC cell lines measured by MTT assay ranged from 3.4-11.9 μM. CX-4945 induced cell cycle arrest and cell death measured by DNA flow cytometry, and inhibited prosurvival mediators phospho-AKT and p-S6 in UM-SCC1 and UM-SCC46 cells. CX-4945 decreased NF-κB and Bcl-XL reporter gene activities in both cell lines, but upregulated proapoptotic TP53 and p21 reporter activities, and induced phospho-ERK, AP-1, and IL-8 activity in UM-SCC1 cells. CX-4945 exhibited modest anti-tumor activity in UM-SCC1 xenografts. Tumor immunostaining revealed significant inhibition of PI3K-Akt-mTOR pathway and increased apoptosis marker TUNEL, but also induced p-ERK, c-JUN, JUNB, FOSL1 and proliferation (Ki67) markers, as a possible resistance mechanism. To overcome the drug resistance, we tested MEK inhibitor PD-0325901 (PD-901), which inhibited ERK-AP-1 activation alone and in combination with CX-4945. PD-901 alone displayed significant anti-tumor effects in vivo, and the combination of PD-901 and CX-4945 slightly enhanced anti-tumor activity when compared with PD-901 alone. Immunostaining of tumor specimens after treatment revealed inhibition of p-AKT S129 and p-AKT T308 by CX-4945, and inhibition of p-ERK T202/204 and AP-1 family member FOSL-1 by PD-901. Our study reveals a drug resistance mechanism mediated by the MEK-ERK-AP-1 pathway in HNSCC. MEK inhibitor PD-0325901 is active in HNSCC resistant to CX-4945, meriting further clinical investigation.     

A small molecule inhibitor selective for a variant ATP-binding site of the chaperonin GroEL

The chaperonin GroEL is a megadalton-sized molecular machine that plays an essential role in the bacterial cell assisting protein folding to the native state through actions requiring ATP binding and hydrolysis. A combination of medicinal chemistry and genetics has been employed to generate an orthogonal pair, a small molecule that selectively inhibits ATPase activity of a GroEL ATP-binding pocket variant. An initial screen of kinase-directed inhibitors identified an active pyrazolo-pyrimidine scaffold that was iteratively modified and screened against a collective of GroEL nucleotide pocket variants to identify a cyclopentyl carboxamide derivative, EC3016, that specifically inhibits ATPase activity and protein folding by the GroEL mutant, I493C, involving a side chain positioned near the base of ATP. This orthogonal pair will enable in vitro studies of the action of ATP in triggering activation of GroEL-mediated protein folding and might enable further studies of GroEL action in vivo. The approach originated for studying kinases by Shokat and his colleagues may thus also be used to study large macromolecular machines.     

Pyrvinium, a potent small molecule Wnt inhibitor, promotes wound repair and post-MI cardiac remodeling

Wnt signaling plays an important role in developmental and stem cell biology. To test the hypothesis that temporary inhibition of Wnt signaling will enhance granulation tissue and promote angiogenesis in tissue repair, we employed a recently characterized small molecule Wnt inhibitor. Pyrvinium is an FDA-approved drug that we identified as a Wnt inhibitor in a chemical screen for small molecules that stabilize β-catenin and inhibit Axin degradation. Our subsequent characterization of pyrvinium has revealed that its critical cellular target in the Wnt pathway is Casein Kinase 1α. Daily administration of pyrvinium directly into polyvinyl alcohol (PVA) sponges implanted subcutaneously in mice generated better organized and vascularized granulation tissue; this compound also increased the proliferative index of the tissue within the sponges. To evaluate its effect in myocardial repair, we induced a myocardial infarction (MI) by coronary artery ligation and administered a single intramyocardial dose of pyrvinium. Mice were evaluated by echocardiography at 7 and 30 days post-MI and treatment; post mortem hearts were evaluated by histology at 30 days. Pyrvinium reduced adverse cardiac remodeling demonstrated by decreased left ventricular internal diameter in diastole (LVIDD) as compared to a control compound. Increased Ki-67+ cells were observed in peri-infarct and distal myocardium of pyrvinium-treated animals. These results need to be further followed-up to determine if therapeutic inhibition of canonical Wnt may avert adverse remodeling after ischemic injury and its impact on myocardial repair and regeneration.     

Discovery of a potent and selective c-Kit inhibitor for the treatment of inflammatory diseases

A potent and selective c-Kit inhibitor 20 was identified through a structure–activity relationship study. In an in vivo mouse model of mast cell activation, 20 blocked the SCF-induced histamine release with an EC₅₀ of 26 nM.     

Pharmacological characterization of a small molecule inhibitor of c-Jun kinase

c-Jun NH(2)-terminal kinase (JNK) plays an important role in insulin resistance; however, identification of pharmacologically potent and selective small molecule JNK inhibitors has been limited. Compound A has a cell IC(50) of 102 nM and is at least 100-fold selective against related kinases and 27-fold selective against glycogen synthase kinase-3beta and cyclin-dependent kinase-2. In C57BL/6 mice, compound A reduced LPS-mediated increases in both plasma cytokine levels and phosphorylated c-Jun in adipose tissue. Treatment of mice fed a high-fat diet with compound A for 3 wk resulted in a 13.1 +/- 1% decrease in body weight and a 9.3 +/- 1.5% decrease in body fat, compared with a 6.6 +/- 2.1% increase in body weight and a 6.7 +/- 2.1% increase in body fat in vehicle-treated mice. Mice pair fed to those that received compound A exhibited a body weight decrease of 7 +/- 1% and a decrease in body fat of 1.6 +/- 1.3%, suggesting that reductions in food intake could not account solely for the reductions in adiposity observed. Compound A dosed at 30 mg/kg for 13 days in high-fat fed mice resulted in a significant decrease in phosphorylated c-Jun in adipose tissue accompanied by a decrease in weight and reductions in glucose and triglycerides and increases in insulin sensitivity to levels comparable with those in lean control mice. The ability of compound A to reduce the insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) von Ser307 and partially reverse the free fatty acid inhibition of glucose uptake in 3T3L1 adipocytes, suggests that enhancement of insulin signaling in addition to weight loss may contribute to the effects of compound A on insulin sensitization in vivo. Pharmacological inhibition of JNK using compound A may therefore offer an effective therapy for type 2 diabetes mediated at least in part via weight reduction.