David Carroll

Preclinical characterization of Aurora kinase inhibitor R763/AS703569 identified through an image-based phenotypic screen

PurposeAurora kinases play a key role in mitotic progression. Over-expression of Aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. Therefore, Aurora kinase inhibitors should be useful in the treatment of cancers.MethodsCell-based screening methods have an advantage over biochemical approaches because hits can be optimized to inhibit targets in the proper intracellular context. We developed a novel Aurora kinase inhibitor R763/AS703569 using an image-based phenotypic screen. The anti-proliferative effect was examined in a panel of tumor cell lines and primary cells. The efficacy was determined in a broad panel of xenograft models.ResultsR763/AS703569 inhibits Aurora kinases, along with a limited number of other kinases including FMS-related tyrosine kinase 3 (FLT3), and has potent anti-proliferative activity against many cell types accompanying unique phenotypic changes such as enlarged cell size, endoreduplication and apoptosis. The endoreduplication cycle induced by R763/AS703569 was irreversible even after the compound was withdrawn from the culture. Oral administration of R763/AS703569 demonstrated marked inhibition of tumor growth in xenograft models of pancreatic, breast, colon, ovarian, and lung tumors and leukemia. An acute myeloid leukemia cell line MV4-11, which carries a FLT3 internal tandem duplication mutation, is particularly sensitive to R763/AS703569 in vivo.ConclusionsR763/AS703569 is a potent inhibitor of Aurora kinases and exhibited significant anti-proliferative activity against a wide range of tumor cells both in vitro and in vivo. Inhibition of Aurora kinases has the potential to be a new addition to the treatment of cancers.

Novel Immunosuppression : R348, a JAK3-and Syk-Inhibitor Attenuates Acute Cardiac Allograft Rejection

Background. Janus kinase (JAK)3 is crucial for signal transduction downstream of various cytokine receptors in immune cells. This is the first report on the novel JAK3 inhibitor R348. Methods. (1) Detailed pharmacokinetic data were obtained in rats; (2) multiple in vitro enzyme inhibition assays were performed to characterize the drug; (3) prevention of acute rejection was investigated in animals treated with different doses of R348 or rapamycin for 5 days; and (4) cardiac allograft survival after a 10-day treatment period was studied for various regimens of R348, tacrolimus, or rapamycin; combination indices were calculated to evaluate drug interactions. Results. (1) Plasma levels of R348s active metabolite R333 sustained high for 8 hr or more, depending on the dose. (2) In vitro enzyme assays showed potent inhibition of JAK3- and Syk-dependent pathways. (3) R348 40 mg/kg preserved graft function, significantly reduced graft infiltration, and decreased histologic ISHLT rejection scores on postoperative day 5. Results were similar to those of rapamycin 3 mg/kg. Likewise, both drugs significantly reduced the cellular Th1 and Th2 immune responses, as determined by enzyme-linked immunosorbent assays. Intragraft inflammatory cytokine upregulation was similarly suppressed by R348 and rapamycin. R348 10 mg/kg was subtherapeutic. (4) Allograft survival was similar for R348 20 and 40 mg/kg, which was comparable with therapeutically dosed tacrolimus or rapamycin. In combination regimens, R348 demonstrated highly beneficial synergistic interactions with tacrolimus. Conclusions. R348 is a promising novel JAK3/Syk-inhibitor with favorable pharmacokinetics and biological activity. It effectively diminishes acute cardiac allograft rejection and is suitable for combination regimens with tacrolimus.

A novel JAK3 inhibitor, R348, attenuates chronic airway allograft rejection.

Background. This study aimed at investigating the role of a novel JAK3 inhibitor, R348, in the prevention of chronic airway allograft rejection. Methods. The heterotopic rat trachea transplant model was used. Recipients were treated daily with R348 (10, 20, 40, 80 mg/kg) or rapamycin (0.75 or 3 mg/kg). Blood levels of R348 and of its active metabolite R333 were measured. Grafts were harvested after 28 days to analyze epithelial morphology, mononuclear infiltration, and luminal obliteration. Plasma levels of circulating donor strain-reactive IgG antibodies were quantified. Results. R348 was well tolerated at up to 40 mg/kg, but was toxic at 80 mg/kg. Blood levels of R333 at 2 and 24 hr were consistently 10 to 15 times higher than those of R348. Airway luminal obliteration after 28 days was significantly inhibited by R348 at 40 mg/kg (20.6%±13.2%, P<0.05) and 80 mg/kg (15.7%±7.6%, P<0.05) and by rapamycin at 3 mg/kg (11.6%±6.7% P<0.001) versus untreated controls (100%). R348 is more than or equal to 40 mg/kg but neither dose of rapamycin preserved the physiologic epithelial coverage with its prominent goblet cells population (8.8±1.5 goblet cells/&mgr;m circumference in syngeneic grafts and 8.0±0.9 and 4.3±1.2 with R348 80 mg/kg and 40 mg/kg, respectively). Peritracheal graft mononuclear infiltration was most effectively suppressed by R348 is more than or equal to 40 mg/kg (P<0.05) and rapamycin 3 mg/kg (P<0.01). Donor strain-reactive IgG antibodies were significantly decreased by R348 is more than or equal to 40 mg/kg (P≤0.05) and rapamycin 3 mg/kg (P<0.001). Animals treated with R348 is more than or equal to 40 mg/kg showed elevated alanine transferase (P<0.05), whereas hypercholesterolemia was only found in animals receiving rapamycin 3 mg/kg (P<0.05). Conclusions. R348 is an effective drug, and it is expected to be introduced into clinical transplant pharmacology soon.

Activity of a tamoxifen–Raloxifene hybrid ligand for estrogen receptors at an AP-1 Site

To test the effect of ligand flexibility on the selective transcriptional activities of ERalpha and ERbeta from an AP-1 site, an analogue of raloxifene was made that removed the ketone functionality and made the ligand more planar and conformationally more similar to 4-hydroxytamoxifen. Desketoraloxifene was found to be a much stronger activator at an AP-1 site with ERalpha than with ERbeta, mimicking 4-hydroxytamoxifen more than raloxifene.

Developing structure-activity relationships from an HTS hit for inhibition of the Cks1-Skp2 protein-protein interaction.

Structure-activity relationships have been developed around 5-bromo-8-toluylsulfonamidoquinoline 1 a hit compound in an assay for the interaction of the E3 ligase Skp2 with Cks1, part of the SCF ligase complex. Disruption of this protein-protein interaction results in higher levels of CDK inhibitor p27, which can act as a tumor suppressor. The results of the SAR developed highlight the relationship between the sulfonamide and quinoline nitrogen, while also suggesting that an aryl substituent at the 5-position of the quinoline ring contributes to the potency in the interaction assay. Compounds showing potency in the interaction assay result in greater levels of p27 and have been shown to inhibit cell growth of two p27 sensitive tumor cell lines.

Design, synthesis of diaminopyrimidine inhibitors targeting IgE- and IgG-mediated activation of Fc receptor signaling.

Using cultured human mast cells (CHMC) the optimization of 2,4-diaminopyrimidine compounds leading to 22, R406 is described. Compound 22 is a potent upstream inhibitor of mast cell degranulation and its mechanism of action is via inhibition of Syk kinase. Compound 22 has significant activity in inhibiting both IgE- and IgG-mediated activation of Fc receptor (FcR) in mast cells and basophils, and in addition inhibits Syk kinase-dependent activity of FcR-mediated activation of monocytes, macrophages, neutrophils, and B cell receptor (BCR)-mediated activation of B lymphocytes. Overall, the biological activity of 22 suggests that it has potential for application as a novel therapeutic for the treatment of an array of autoimmune maladies and hematological malignancies.

Application of cultured human mast cells (CHMC) for the design and structure-activity relationship of IgE-mediated mast cell activation inhibitors.

Here we report the optimization of small molecule inhibitors of human mast cell degranulation via anti-IgE-mediated tryptase release following cross-linking and activation of IgE-loaded FcεR1 receptors. The compounds are selective upstream inhibitors of FcεR1-dependent human mast cell degranulation and proved to be devoid of activity in downstream ionomycin mediated degranulation. Structure-activity relationship (SAR) leading to compound 26 is outlined.