Evaluation of Novel Inhibitors of Tryptophan Dioxygenases for Enzyme and Species Selectivity Using Engineered Tumour Cell Lines Expressing Either Murine or Human IDO1 or TDO2

Indoleamine 2, 3-dioxygenase 1 (IDO1) is commonly expressed by cancers as a mechanism for evading the immune system. Preclinical and clinical studies have indicated the potential of combining IDO1 inhibitors with immune therapies for the treatment of cancer, strengthening an interest in the discovery of novel dioxygenase inhibitors for reversing tumour-mediated immune suppression. To facilitate the discovery, development and investigation of novel small molecule inhibitors of IDO1 and its hepatic isozyme tryptophan dioxygenase (TDO2), murine tumour cells were engineered to selectively express either murine or human IDO1 and TDO2 for use as tools to dissect both the species specificity and isoenzyme selectivity of newly discovered inhibitors. Lewis lung carcinoma (LLTC) lines were engineered to express either murine or human IDO1 for use to test species selectivity of the novel inhibitors; in addition, GL261 glioma lines were engineered to express either human IDO1 or human TDO2 and used to test the isoenzyme selectivity of individual inhibitors in cell-based assays. The 20 most potent inhibitors against recombinant human IDO1 enzyme, discovered from a commissioned screening of 40,000 compounds in the Australian WEHI compound library, returned comparable IC50 values against murine or human IDO1 in cell-based assays using the LLTC-mIDO1 and LLTC-hIDO1 line, respectively. To test the in vivo activity of the hits, transfected lines were inoculated into syngeneic C57Bl/6 mice. Individual LLTC-hIDO1 tumours showed variable expression of human IDO1 in contrast to GL261-hIDO1 tumours which were homogenous in their IDO1 expression and were subsequently used for in vivo studies. W-0019482, the most potent IDO1 inhibitor identified from cell-based assays, reduced plasma and intratumoural ratios of kynurenine to tryptophan (K:T) and delayed the growth of subcutaneous GL261-hIDO1 tumours in mice. Synthetic modification of W-0019482 generated analogues with dual IDO1/TDO2 inhibitory activity, as well as inhibitors that were selective for either TDO2 or IDO1. These results demonstrate the versatility of W-0019482 as a lead in generating all three subclasses of tryptophan dioxygenase inhibitors which can be applied for investigating the individual roles and interactions between IDO1 and TDO2 in driving cancer-mediated immune suppression

Synthesis and Biological Activity of Azido Analogues of 5,6-Dimethylxanthenone-4-acetic Acid for Use in Photoaffinity Labeling

5,6-Dimethylxanthenone-4-acetic acid (1) is scheduled for phase III clinical trials as a vascular disrupting agent. However, its biochemical receptor(s) have yet to be identified. In this report, the synthesis of azido analogues of 1 that could be used for photoaffinity labeling of proteins as an approach toward identifying its molecular targets is described. While 5-azidoxanthenone-4-acetic acid (2) and 5-azido-6-methylxantheone-4-acetic acid (3) were found to have biological activities similar to that of 1, 6-azido-5-methylxanthenone-4-acetic acid (4) was unstable and could not be evaluated. Both azido compounds 2 and 3 activated NF-kappaB, induced the production of tumor necrosis factor in cultured mouse splenocytes, and induced hemorrhagic necrosis of colon 38 tumors in mice. Photoreaction of lysates from spleen cells with tritiated 2 resulted in two radiolabeled protein bands at 50 and 14 kDa that could be competitively inhibited with cold 1 and cold 2. The azido compounds 2 and 3 exhibit all the requirements for use in photoaffinity labeling of potential receptor(s) for 1

Access to and Timeliness of Lung Cancer Surgery, Radiation Therapy, and Systemic Therapy in New Zealand: A Universal Health Care Context

PURPOSELung cancer is the biggest cancer killer of indigenous peoples worldwide, including Māori people in New Zealand. There is some evidence of disparities in access to lung cancer treatment between Māori and non-Māori patients, but an examination of the depth and breadth of these disparities is needed. Here, we use national-level data to examine disparities in access to surgery, radiation therapy and systemic therapy between Māori and European patients, as well as timing of treatment relative to diagnosis.METHODSWe included all lung cancer registrations across New Zealand from 2007 to 2019 (N = 27,869) and compared access with treatment and the timing of treatment using national-level inpatient, outpatient, and pharmaceutical records.RESULTSMāori patients with lung cancer appeared less likely to access surgery than European patients (Māori, 14%; European, 20%; adjusted odds ratio [adj OR], 0.82 [95% CI, 0.73 to 0.92]), including curative surgery (Māori, 10%; European, 16%; adj OR, 0.72 [95% CI, 0.62 to 0.84]). These differences were only partially explained by stage and comorbidity. There were no differences in access to radiation therapy or systemic therapy once adjusted for confounding by age. Although it appeared that there was a longer time from diagnosis to radiation therapy for Māori patients compared with European patients, this difference was small and requires further investigation.CONCLUSIONOur observation of differences in surgery rates between Māori and European patients with lung cancer who were not explained by stage of disease, tumor type, or comorbidity suggests that Māori patients who may be good candidates for surgery are missing out on this treatment to a greater extent than their European counterparts

Labeling of Oxidizable Proteins with a Photoactivatable Analog of the Antitumor Agent DMXAA: Evidence for Redox Signaling in Its Mode of Action12

The signaling pathway(s) and molecular target(s) for 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a tumor vascular disrupting agent in late stages of clinical development, are still undefined. As an approach toward identifying potential targets for DMXAA, a tritiated azido-analog of DMXAA was used to probe for cellular binding proteins. More than 20 cytosolic proteins from murine splenocytes, RAW 264.7 cells, and the HECPP immortalized endothelial cells were photoaffinity-labeled. Although no protein domain, fold, or binding site for a specific ligand was found to be shared by all the candidate proteins, essentially all were noted to be oxidizable proteins, implicating a role for redox signaling in the action of DMXAA. Consistent with this hypothesis, DMXAA caused an increase in concentrations of reactive oxygen species (ROS) in RAW264.7 cells during the first 2 hours. This increase in ROS was suppressed in the presence of the antioxidant, N-acetyl-l-cysteine, which also suppressed DMXAA-induced cytokine production in the RAW 264.7 cells with no effects on cell viability. Short interfering RNA (siRNA)-mediated knockdown of one of the photoaffinity-labeled proteins, superoxide dismutase 1, an ROS scavenger, resulted in an increase in tumor necrosis factor-α production by RAW 264.7 cells in response to DMXAA compared with negative or positive controls transfected with nontargeting or lamin A/C-targeting siRNA molecules, respectively. The results from these lines of study all suggest that redox signaling plays a central role in cytokine induction by DMXAA