Structure-Based Development of Isoform-Selective Inhibitors of Casein Kinase 1ε vs Casein Kinase 1δ.

Specific inhibition of a single kinase isoform is a challenging task due to the highly conserved nature of ATP-binding sites. Casein kinase 1 (CK1) δ and ε share 97% sequence identity in their catalytic domains. From a comparison of the X-ray crystal structures of CK1δ and CK1ε, we developed a potent and highly CK1ε-isoform-selective inhibitor (SR-4133). The X-ray co-crystal structure of the CK1δ-SR-4133 complex reveals that the electrostatic surface between the naphthyl unit of SR-4133 and CK1δ is mismatched, destabilizing the interaction of SR-4133 with CK1δ. Conversely, the hydrophobic surface area resulting from the Asp-Phe-Gly motif (DFG)-out conformation of CK1ε stabilizes the binding of SR-4133 in the ATP-binding pocket of CK1ε, leading to the selective inhibition of CK1ε. The potent CK1ε-selective agents display nanomolar growth inhibition of bladder cancer cells and inhibit the phosphorylation of 4E-BP1 in T24 cells, which is a direct downstream effector of CK1ε.

Targeting Casein Kinase 1 Delta Sensitizes Pancreatic and Bladder Cancer Cells to Gemcitabine Treatment by Upregulating Deoxycytidine Kinase.

Although gemcitabine is the cornerstone of care for pancreatic ductal adenocarcinoma (PDA), patients lack durable responses and relapse is inevitable. While the underlying mechanisms leading to gemcitabine resistance are likely to be multifactorial, there is a strong association between activating gemcitabine metabolism pathways and clinical outcome. This study evaluated casein kinase 1 delta (CK1δ) as a potential therapeutic target for PDA and bladder cancer, in which CK1δ is frequently overexpressed. We assessed the antitumor effects of genetically silencing or pharmacologically inhibiting CK1δ using our in-house CK1δ small-molecule inhibitor SR-3029, either alone or in combination with gemcitabine, on the proliferation and survival of pancreatic and bladder cancer cell lines and orthotopic mouse models. Genetic studies confirmed that silencing CK1δ or treatment with SR-3029 induced a significant upregulation of deoxycytidine kinase (dCK), a rate-limiting enzyme in gemcitabine metabolite activation. The combination of SR-3029 with gemcitabine induced synergistic antiproliferative activity and enhanced apoptosis in both pancreatic and bladder cancer cells. Furthermore, in an orthotopic pancreatic tumor model, we observed improved efficacy with combination treatment concomitant with increased dCK expression. This study demonstrates that CK1δ plays a role in gemcitabine metabolism, and that the combination of CK1δ inhibition with gemcitabine holds promise as a future therapeutic option for metastatic PDA as well as other cancers with upregulated CK1δ expression.

ßarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1.

Cellular DNA is constantly under threat from internal and external insults, consequently multiple pathways have evolved to maintain chromosomal fidelity. Our previous studies revealed that chronic stress, mediated by continuous stimulation of the ß2-adrenergic-ßarrestin-1 signaling axis suppresses activity of the tumor suppressor p53 and impairs genomic integrity. In this pathway, ßarrestin-1 (ßarr1) acts as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, MDM2. We sought to determine whether ßarr1 plays additional roles in the repair of DNA damage. Here we demonstrate that in mice ßarr1 interacts with p53-binding protein 1 (53BP1) with major consequences for the repair of DNA double-strand breaks. 53BP1 is a principle component of the DNA damage response, and when recruited to the site of double-strand breaks in DNA, 53BP1 plays an important role coordinating repair of these toxic lesions. Here, we report that ßarr1 directs 53BP1 degradation by acting as a scaffold for the E3-ubiquitin ligase Rad18. Consequently, knockdown of ßarr1 stabilizes 53BP1 augmenting the number of 53BP1 DNA damage repair foci following exposure to ionizing radiation. Accordingly, ßarr1 loss leads to a marked increase in irradiation resistance both in cells and in vivo. Thus, ßarr1 is an important regulator of double strand break repair, and disruption of the ßarr1/53BP1 interaction offers an attractive strategy to protect cells against high levels of exposure to ionizing radiation.

Development of dual casein kinase 1δ/1ε (CK1δ/ε) inhibitors for treatment of breast cancer.

Casein kinase 1δ/ε have been identified as promising therapeutic target for oncology application, including breast and brain cancer. Here, we described our continued efforts in optimization of a lead series of purine scaffold inhibitors that led to identification of two new CK1δ/ε inhibitors 17 and 28 displaying low nanomolar values in antiproliferative assays against the human MDA-MB-231 triple negative breast cancer cell line and have physical, in vitro and in vivo pharmacokinetic properties suitable for use in proof of principle animal xenograft studies against human cancers.

Discovery of 2-arylquinazoline derivatives as a new class of ASK1 inhibitors.

The development of a new series of apoptosis signal-regulating kinase 1 (ASK1) inhibitors is described. Starting from purine, pyrimidine and quinazoline scaffolds identified by high throughput screening, we used tools of structure-based drug design to develop a series of potent kinase inhibitors, including 2-arylquinazoline derivatives 12 and 23, with submicromolar inhibitory activities against ASK1. Kinetic analysis demonstrated that the 2-arylquinazoline scaffold ASK1 inhibitors described herein are ATP competitive.

In Silico HTS and Structure Based Optimization of Indazole-Derived ULK1 Inhibitors.

We present the outcome of an in silico high throughput screen (HTS) and optimization of a small molecule Unc-51-Like Kinase 1 (ULK1) inhibitor hit, SR-17398, with an indazole core. Docking studies guided design efforts that led to inhibitors with increased activity vs ULK1 (IC50 < 50 nM). The most advanced molecules in this inhibitor series (3a and 3g) hold promise for further development into selective ULK1 molecular probes to interrogate the biology of ULK1 and to assess whether selectively targeting autophagy is an effective anticancer strategy.

Therapeutic targeting of casein kinase 1δ in breast cancer.

Identification of specific drivers of human cancer is required to instruct the development of targeted therapeutics. We demonstrate that CSNK1D is amplified and/or overexpressed in human breast tumors and that casein kinase 1δ (CK1δ) is a vulnerability of human breast cancer subtypes overexpressing this kinase. Specifically, selective knockdown of CK1δ, or treatment with a highly selective and potent CK1δ inhibitor, triggers apoptosis of CK1δ-expressing breast tumor cells ex vivo, tumor regression in orthotopic models of triple-negative breast cancer, including patient-derived xenografts, and tumor growth inhibition in human epidermal growth factor receptor 2-positive (HER2(+)) breast cancer models. We also show that Wnt/ß-catenin signaling is a hallmark of human tumors overexpressing CK1δ, that disabling CK1δ blocks nuclear accumulation of ß-catenin and T cell factor transcriptional activity, and that constitutively active ß-catenin overrides the effects of inhibition or silencing of CK1δ. Thus, CK1δ inhibition represents a promising strategy for targeted treatment in human breast cancer with Wnt/ß-catenin involvement.

Development of an HTS-Compatible Assay for the Discovery of Ulk1 Inhibitors.

A rapidly accumulating body of work suggests the autophagy pathway is an attractive therapeutic target for neurodegenerative diseases and cancer. To validate autophagy as an anticancer strategy and to assess if systemic inhibition of the pathway will have deleterious effects on normal tissues and physiology, highly selective autophagy inhibitors are needed. While several inducers and inhibitors of autophagy are known, all are nonspecific and none target the enzymes that execute the pathway. A central upstream regulator of the autophagy pathway is the serine/threonine kinase Ulk1 (UNC-51-like kinase-1). Selective molecular probes that function as Ulk1-specific inhibitors are needed to improve our understanding of the autophagy pathway. To identify inhibitors of Ulk1 kinase activity, we developed an HTS-compatible, homogeneous biochemical assay using AlphaScreen technology. This novel assay design uses purified stress-activated Ulk1 and monitors phosphorylation of its full-length native substrate, Atg13. This assay was optimized and validated in a 384-well format by screening the Sigma LOPAC library. Here we report that the Ulk1 AlphaScreen assay is robust and reproducible, with a Z' factor value of 0.83 ± 0.02 and a signal to background ratio of 20 ± 1.2. Thus, this assay can be used to screen large chemical libraries to discover novel inhibitors of Ulk1.

Synthesis and biological evaluation of urea derivatives as highly potent and selective rho kinase inhibitors.

RhoA and its downstream effector ROCK mediate stress fiber formation and cell contraction through their effects on the phosphorylation of myosin light chain (MLC). Inhibition of the RhoA/ROCK pathway has proven to be a promising strategy for several indications such as cardiovascular disease, glaucoma, and inflammatory disease. In 2010, our group reported urea-based ROCK inhibitors as potential antiglaucoma agents. These compounds showed potent IC50 values in enzymatic and cell-based assays and significant intraocular pressure (IOP)-lowering effects in rats (∼7 mmHg). (22) To develop more advanced ROCK inhibitors targeting various potential applications (such as myocardial infarction, erectile dysfunction, multiple sclerosis, etc.) in addition to glaucoma, a thorough SAR for this urea-based scaffold was studied. The detailed optimization process, counter-screening, and in vitro and in vivo DMPK studies are discussed. Potent and selective ROCK inhibitors with various in vivo pharmacokinetic properties were discovered.

Amino acid derived quinazolines as Rock/PKA inhibitors.

SAR and lead optimization studies for Rock inhibitors based on amino acid-derived quinazolines are described. Studies demonstrated that these amino acid derived quinazolinones were mainly pan-Rock (I & II) inhibitors. While selectivity against other kinases could be achieved, selectivity for most of these compounds against PKA was not achieved. This is distinct from Rock inhibitors based on non-amino acid derived quinazolinones, where high selectivity against PKA could be obtained.(22) The inhibitors presented here in some cases possessed sub-nanomolar inhibition of Rock, nanomolar potency in ppMLC cell based assays, low to fair cytochrome P-450 inhibition, and good human microsomal stability.

Discovery and optimization of indole and 7-azaindoles as Rho kinase (ROCK) inhibitors (part-II).

Therapeutic interventions with Rho kinase (ROCK) inhibitors may effectively treat several disorders such as hypertension, stroke, cancer, and glaucoma. Herein we disclose the optimization and biological evaluation of potent novel ROCK inhibitors based on substituted indole and 7-azaindole core scaffolds. Substitutions on the indole C3 position and on the indole NH and/or amide NH positions all yielded potent and selective ROCK inhibitors (25, 42, and 50). Improvement of aqueous solubility and tailoring of in vitro and in vivo DMPK properties could be achieved through these substitutions.

Discovery and optimization of indoles and 7-azaindoles as Rho kinase (ROCK) inhibitors (part-I).

Rho kinase (ROCK) inhibitors are potential therapeutic agents to treat disorders such as hypertension, multiple sclerosis, cancers, and glaucoma. Here, we disclose the synthesis, optimization, biological evaluation of potent indole and 7-azaindole based ROCK inhibitors that have high potency on ROCK (IC(50)=1 nM) with 740-fold selectivity over PKA (47). Moreover, 47 showed very good DMPK properties making it a good candidate for further development. Finally, docking studies with a homology model of ROCK-II were performed to rationalize the binding mode of these compounds and showed the compounds bound in both orientations to take advantage to H-bonds with Lys-121 of ROCK-II.

A stress response pathway regulates DNA damage through ß2-adrenoreceptors and ß-arrestin-1.

The human mind and body respond to stress, a state of perceived threat to homeostasis, by activating the sympathetic nervous system and secreting the catecholamines adrenaline and noradrenaline in the 'fight-or-flight' response. The stress response is generally transient because its accompanying effects (for example, immunosuppression, growth inhibition and enhanced catabolism) can be harmful in the long term. When chronic, the stress response can be associated with disease symptoms such as peptic ulcers or cardiovascular disorders, and epidemiological studies strongly indicate that chronic stress leads to DNA damage. This stress-induced DNA damage may promote ageing, tumorigenesis, neuropsychiatric conditions and miscarriages. However, the mechanisms by which these DNA-damage events occur in response to stress are unknown. The stress hormone adrenaline stimulates ß(2)-adrenoreceptors that are expressed throughout the body, including in germline cells and zygotic embryos. Activated ß(2)-adrenoreceptors promote Gs-protein-dependent activation of protein kinase A (PKA), followed by the recruitment of ß-arrestins, which desensitize G-protein signalling and function as signal transducers in their own right. Here we elucidate a molecular mechanism by which ß-adrenergic catecholamines, acting through both Gs-PKA and ß-arrestin-mediated signalling pathways, trigger DNA damage and suppress p53 levels respectively, thus synergistically leading to the accumulation of DNA damage. In mice and in human cell lines, ß-arrestin-1 (ARRB1), activated via ß(2)-adrenoreceptors, facilitates AKT-mediated activation of MDM2 and also promotes MDM2 binding to, and degradation of, p53, by acting as a molecular scaffold. Catecholamine-induced DNA damage is abrogated in Arrb1-knockout (Arrb1(-/-)) mice, which show preserved p53 levels in both the thymus, an organ that responds prominently to acute or chronic stress, and in the testes, in which paternal stress may affect the offspring's genome. Our results highlight the emerging role of ARRB1 as an E3-ligase adaptor in the nucleus, and reveal how DNA damage may accumulate in response to chronic stress.

Synthesis and biological evaluation of 4-quinazolinones as Rho kinase inhibitors.

Rho kinase (ROCK) is an attractive therapeutic target for various diseases including glaucoma, hypertension, and spinal cord injury. Herein, we report the development of a series of ROCK-II inhibitors based on 4-quinazolinone and quinazoline scaffolds. SAR studies at three positions of the quinazoline core led to the identification of analogs with high potency against ROCK-II and good selectivity over protein kinase A (PKA).

Tetrahydroisoquinoline derivatives as highly selective and potent Rho kinase inhibitors.

Rho kinase (ROCK) is a promising drug target for the treatment of many diseases including hypertension, multiple sclerosis, cancer, and glaucoma. The structure-activity relationships (SAR) around a series of tetrahydroisoquinolines were evaluated utilizing biochemical and cell-based assays to measure ROCK inhibition. These novel ROCK inhibitors possess high potency, high selectivity, and appropriate pharmacokinetic properties for glaucoma applications. The lead compound, 35, had subnanomolar potency in enzyme ROCK-II assays as well as excellent cell-based potency (IC(50) = 51 nM). In a kinase panel profiling, 35 had an off-target hit rate of only 1.6% against 442 kinases. Pharmacology studies showed that compound 35 was efficacious in reducing intraocular pressure (IOP) in rats with reasonably long duration of action. These results suggest that compound 35 may serve as a promising agent for further development in the treatment of glaucoma.

Discovery of Potent and Selective Urea-Based ROCK Inhibitors and Their Effects on Intraocular Pressure in Rats.

A series of urea-based Rho kinase (ROCK) inhibitors were designed and evaluated. The discovered compounds had excellent enzyme and cellular potency, high kinase selectivity, high aqueous solubility, good porcine corneal penetration, and appropriate DMPK profiles for topical applications as antiglaucoma therapeutics.

Benzothiazoles as Rho-associated kinase (ROCK-II) inhibitors.

A series of benzothiazole derivatives as ROCK inhibitors have been discovered. Compounds with good biochemical and cellular potency, and sufficient kinase selectivity have been identified.

Characterisation of a fatty acid and retinol binding protein orthologue from the hookworm Ancylostoma ceylanicum.

Hookworms, bloodfeeding intestinal nematodes, infect nearly one billion people in resource limited countries and are a leading cause of anaemia and malnutrition. Like other nematodes, hookworms lack the capacity to synthesise essential fatty acids de novo and therefore must acquire those from exogenous sources. The cDNA corresponding to a putative Ancylostoma ceylanicum fatty acid and retinol binding protein-1 (AceFAR-1) was amplified from adult hookworm mRNA. Studies using quantitative reverse transcriptase real-time PCR demonstrate that AceFAR-1 transcripts are most abundant in the earliest developmental stages of the parasite, and greater in females than males. Using in vitro assays, the recombinant AceFAR-1 (rAceFAR-1) was shown to bind individual fatty acids with equilibrium dissociation constants in the low micromolar range. The pattern of fatty acid uptake by live adult worms cultured ex vivo was similar to the in vitro binding profile of rAceFAR-1, raising the possibility that the native protein may be involved in acquisition of fatty acids by A. ceylanicum. Animals vaccinated orally with rAceFAR-1 and the mucosal adjuvant cholera toxin exhibited a statistically significant (40-47%) reduction in intestinal worm burden compared with controls immunized with antigen or adjuvant alone. Together, these data suggest a potential role for AceFAR-1 in hookworm biology, making it a potentially valuable target for drug and vaccine development.

Protease activation during in vivo pancreatitis is dependent on calcineurin activation.

The premature activation of digestive proenzymes, specifically proteases, within the pancreatic acinar cell is an early and critical event during acute pancreatitis. Our previous studies demonstrate that this activation requires a distinct pathological rise in cytosolic Ca(2+). Furthermore, we have shown that a target of aberrant Ca(2+) in acinar cells is the Ca(2+)/calmodulin-dependent phosphatase calcineurin (PP2B). In this study, we hypothesized that PP2B mediates in vivo protease activation and pancreatitis severity. To test this, pancreatitis was induced in mice over 8 h by administering hourly intraperitoneal injections of the cholecystokinin analog caerulein (50 microg/kg). Treatment with the PP2B inhibitor FK506 at 1 and 8 h after pancreatitis induction reduced trypsin activities by greater than 50% (P < 0.005). Serum amylase and IL-6 was reduced by 86 and 84% relative to baseline (P < 0.0005) at 8 h, respectively. Histological severity of pancreatitis, graded on the basis of pancreatic edema, acinar cell vacuolization, inflammation, and apoptosis, was reduced early in the course of pancreatitis. Myeloperoxidase activity from both pancreas and lung was reduced by 93 and 83% relative to baseline, respectively (P < 0.05). These data suggest that PP2B is an important target of the aberrant acinar cell Ca(2+) rise associated with pathological protease activation and pancreatitis.

Cyclic AMP accelerates calcium waves in pancreatic acinar cells.

Cytosolic Ca(2+) (Ca(i)(2+)) flux within the pancreatic acinar cell is important both physiologically and pathologically. We examined the role of cAMP in shaping the apical-to-basal Ca(2+) wave generated by the Ca(2+)-activating agonist carbachol. We hypothesized that cAMP modulates intra-acinar Ca(2+) channel opening by affecting either cAMP-dependent protein kinase (PKA) or exchange protein directly activated by cAMP (Epac). Isolated pancreatic acinar cells from rats were stimulated with carbachol (1 muM) with or without vasoactive intestinal polypeptide (VIP) or 8-bromo-cAMP (8-Br-cAMP), and then Ca(i)(2+) was monitored by confocal laser-scanning microscopy. The apical-to-basal carbachol (1 muM)-stimulated Ca(2+) wave was 8.63 +/- 0.68 microm/s; it increased to 19.66 +/- 2.22 microm/s (*P < 0.0005) with VIP (100 nM), and similar increases were observed with 8-Br-cAMP (100 microM). The Ca(2+) rise time after carbachol stimulation was reduced in both regions but to a greater degree in the basal. Lag time and maximal Ca(2+) elevation were not significantly affected by cAMP. The effect of cAMP on Ca(2+) waves also did not appear to depend on extracellular Ca(2+). However, the ryanodine receptor (RyR) inhibitor dantrolene (100 microM) reduced the cAMP-enhancement of wave speed. It was also reduced by the PKA inhibitor PKI (1 microM). 8-(4-chloro-phenylthio)-2'-O-Me-cAMP, a specific agonist of Epac, caused a similar increase as 8-Br-cAMP or VIP. These data suggest that cAMP accelerates the speed of the Ca(2+) wave in pancreatic acinar cells. A likely target of this modulation is the RyR, and these effects are mediated independently by PKA and Epac pathways.