To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

Here, we describe the identification of a clinical candidate via structure-based optimization of a ligand efficient pyrazole-benzimidazole fragment. Aurora kinases play a key role in the regulation of mitosis and in recent years have become attractive targets for the treatment of cancer. X-ray crystallographic structures were generated using a novel soakable form of Aurora A and were used to drive the optimization toward potent (IC(50) approximately 3 nM) dual Aurora A/Aurora B inhibitors. These compounds inhibited growth and survival of HCT116 cells and produced the polyploid cellular phenotype typically associated with Aurora B kinase inhibition. Optimization of cellular activity and physicochemical properties ultimately led to the identification of compound 16 (AT9283). In addition to Aurora A and Aurora B, compound 16 was also found to inhibit a number of other kinases including JAK2 and Abl (T315I). This compound demonstrated in vivo efficacy in mouse xenograft models and is currently under evaluation in phase I clinical trials.

We describe the structure-guided optimization of the molecular fragments 2-amino-3-benzyloxypyridine 1 (IC(50) 1.3 mM) and 3-(2-(4-pyridyl)ethyl)indole 2 (IC(50) 35 microM) identified using X-ray crystallographic screening of p38alpha MAP kinase. Using two separate case studies, the article focuses on the key compounds synthesized, the structure-activity relationships and the binding mode observations made during this optimization process, resulting in two potent lead series that demonstrate significant increases in activity. We describe the process of compound elaboration either through the growing out from fragments into adjacent pockets or through the conjoining of overlapping fragments and demonstrate that we have exploited the mobile conserved activation loop, consisting in part of Asp168-Phe169-Gly170 (DFG), to generate significant improvements in potency and kinase selectivity.

A ubiquitously expressed chaperone, heat shock protein 90 (HSP90) is of considerable interest as an oncology target because tumor cells and oncogenic proteins are acutely dependent on its activity. AT13387 (2,4-dihydroxy-5-isopropyl-phenyl)-[5-(4-methyl-piperazin-1-ylmethyl)-1,3-dihydro-isoindol-2-yl] methanone, L-lactic acid salt) a novel, high-affinity HSP90 inhibitor, which is currently being clinically tested, has shown activity against a wide array of tumor cell lines, including lung cancer cell lines. This inhibitor has induced the degradation of specific HSP90 client proteins for up to 7 days in tumor cell lines in vitro. The primary driver of cell growth (mutant epidermal growth factor receptors) was particularly sensitive to HSP90 inhibition. The long duration of client protein knockdown and suppression of phospho-signaling seen in vitro after treatment with AT13387 was also apparent in vivo, with client proteins and phospho-signaling suppressed for up to 72 h in xenograft tumors after treatment with a single dose of AT13387. Pharmacokinetic analyses indicated that while AT13387 was rapidly cleared from blood, its retention in tumor xenografts was markedly extended, and it was efficacious in a range of xenograft models. AT13387's long duration of action enabled, in particular, its efficacious once weekly administration in human lung carcinoma xenografts. The use of longer-acting HSP90 inhibitors, such as AT13387, on less frequent dosing regimens has the potential to maintain antitumor efficacy as well as minimize systemic exposure and unwanted effects on normal tissues. (Cancer Sci 2012; 103: 522-527) T he super-chaperone system is involved in the folding and maturation of newly synthesized proteins.(1,2) HSP90 in particular aids in the folding and maturation of a distinct subset of proteins, which includes kinases, cell surface receptors and transcription factors. (3,4) The N-terminal domain ATPase activity of HSP90 is essential for this function.(5) Inhibition of this domain induces remodeling of the HSP90 chaperone complex, resulting in the recruitment of ubiquitin ligases, polyubiquitination and subsequent proteasomal degradation of HSP90 client proteins.(6,7) Through this mechanism, the inhibition of a single target enzyme can have a wide effect on the stability and, hence, the function of a large set of client proteins. As many oncogenic proteins are HSP90 clients, HSP90 inhibition has been found to have broad antitumor effects.(8-10) In contrast to more recent targeted therapies, where the appearance of new driver mutations or resistance mutations result in a loss of efficacy, client protein mutation increases dependence on HSP90 chaperoning activity as these mutations tend to render the proteins less stable. (11)(12)(13) Previous studies have also demonstrated that the constitutively activated mutant forms of EGFR are particularly dependent on HSP90 both in vitro and in vivo, (14)(15)(16) indicating an HSP90 inhibitor may be particularly efficacious in mutant EGFR tumors.After HSP90 inhibiti...

The adsorption behavior of n-pentane and cyclohexane in mica slits at room temperature has been studied as a function of chemical potential and gap width with multiple-beam interferometry. The measured film thicknesses close to saturation for large slit widths (effectively isolated surfaces) range up to 7 nm with n-pentane (at a relative vapor pressure of 0.9996) and 3 nm with cyclohexane (at a relative vapor pressure of 0.995). The thickness of these adsorbed wetting films is slightly larger than that predicted by van der Waals theory. The difference may be accounted for by thermal fluctuations of the adsorbed liquid-vapor interface. At smaller slit widths a capillary condensation transition occurs as the slit fills up with liquid. The separation at which this occurs is in good agreement with a film-thickening mechanism due to van der Waals forces across the gap only for the thickest films (t g 6 nm). For thinner films the capillary condensation transition occurs at larger than expected slit widths, and the deviations are large for t e 3 nm. We speculate that these larger-than-expected condensation separations are related to a fluctuation-enhanced film thickness in this regime. The work demonstrates the utility of measurements in a system consisting of a single slit-pore, without the complications of polydispersity and connectivity of pore networks. The results show that vapor adsorption isotherms can be measured with multiple-beam interferometry, i.e., in the surface force apparatus.

BackgroundNasopharyngeal carcinoma (NPC) is an epithelial malignancy strongly associated with Epstein-Barr virus (EBV). AT13387 is a novel heat shock protein 90 (Hsp90) inhibitor, which inhibits the chaperone function of Hsp90 and reduces expression of Hsp90-dependent client oncoproteins. This study aimed to evaluate both the in vitro and in vivo antitumor effects of AT13387 in the EBV-positive NPC cell line C666-1.ResultsOur results showed that AT13387 inhibited C666-1 cell growth and induced cellular senescence with the downregulation of multiple Hsp90 client oncoproteins EGFR, AKT, CDK4, and restored the protein expression of negative cell cycle regulator p27. We also studied the ability of AT13387 to restore p27 expression by downregulation of AKT and the p27 ubiquitin mediator, Skp2, using AKT inhibitor and Skp2 siRNA. In the functional study, AT13387 inhibited cell migration with downregulation of a cell migration regulator, HDAC6, and increased the acetylation and stabilization of α-tubulin. We also examined the effect of AT13387 on putative cancer stem cells (CSC) by 3-D tumor sphere formation assay. AT13387 effectively reduced both the number and size of C666-1 tumor spheres with decreased expression of NPC CSC-like markers CD44 and SOX2. In the in vivo study, AT13387 significantly suppressed tumor formation in C666-1 NPC xenografts.ConclusionAT13387 suppressed cell growth, cell migration, tumor sphere formation and induced cellular senescence on EBV-positive NPC cell line C666-1. Also, the antitumor effect of AT13387 was demonstrated in an in vivo model. This study provided experimental evidence for the preclinical value of using AT13387 as an effective antitumor agent in treatment of NPC.

Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpointcompetent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.

We investigated the stability of an octadecyltriethoxysilane (OTE) monolayer self-assembled on plasmatreated and untreated mica using a surface forces apparatus by measuring the thickness of the water layer that is adsorbed from vapor. The OTE monolayers are initially highly hydrophobic, but contact angle hysteresis indicates that water interacts favorably with the monolayers on prolonged exposure. Defects in the monolayer most likely make it possible for the water to reach the hydrophilic region between the silane headgroups and the mica. This explains why there has been very little success in measuring hydrophobic forces between OTE-coated mica surfaces. Hydrophobic forces between OTE surfaces have been successfully measured only with silica as the substrate. Even though these monolayers are not suitable for studies of the so-called hydrophobic force, they are ideal for studies designed to probe the interactions between the silane headgroups and mica. For a given relative humidity, the water film thickness is always less if the surfaces are plasma-treated before the monolayer is deposited. In the untreated case, water penetrates into the hydrophilic region between the monolayer and the mica. This does not occur in the plasma-treated case because the monolayer is more firmly anchored, most likely through covalent bonding.