Identification of 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one scaffolds as potent Lck inhibitors as anti-cancer agents.

Lymphocyte-specific protein tyrosine kinase (Lck) plays vital roles in the T-cell receptor- mediated development, function, and differentiation of T-cells. Given its substantial involvement in T cell signaling, irregularities in the expression and functionality of Lck may lead to various diseases, including cancer. In this study, we found that compound 12a exerted significant inhibitory potency against Lck with an IC50 value of 10.6 nM. In addition, 12a demonstrated high efficacy in various colon cancer cell lines as indicated by GI50 values ranging from 0.24 to 1.26 µM. Notably, 12a inhibited the phosphorylation of Lck in Colo201 cells. Overall, the anti-proliferative effects of 12a on diverse cancer cell lines highlights its potential application for the treatment of various cancer types.

Discovery of Orally Bioavailable Phthalazinone Analogues as an ENPP1 Inhibitor for STING-Mediated Cancer Immunotherapy.

A lack of the T cell-inflamed tumor microenvironment limits the efficacy of immune checkpoint inhibitors (ICIs). Activation of stimulator of interferon genes (STING)-mediated innate immunity has emerged as a novel therapeutic approach in cancer therapy. 2',3'-Cyclic GMP-AMP (cGAMP) is a natural STING agonist; however, cGAMP is subjected to endogenous degradation by ecto-nucleotide pyrophosphatase phosphodiesterase 1 (ENPP1). To improve the ICI response rate, we developed 29f, a novel ENPP1 inhibitor with phthalazin-1(2H)-one as the core scaffold. 29f inhibited the cGAMP hydrolysis by ENPP1 in vitro (IC50 = 68 nM) and enhanced the STING-mediated type I interferon response in both immune and tumor cells. 29f demonstrated excellent metabolic stability and bioavailability (F = 65%). Orally administered 29f promoted tumor growth inhibition in a CT26 syngeneic model and increased the anti-PD-L1 response. Furthermore, 29f-induced immunological memory prevented the tumor relapse against tumor rechallenge, suggesting the promising therapeutic potential of 29f.

Identification of novel pyrrolopyrimidine and pyrrolopyridine derivatives as potent ENPP1 inhibitors.

In an effort to discover novel scaffolds of non-nucleotide-derived Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) inhibitors to stimulate the Stimulator of Interferon Genes (STING) pathway, we designed and synthesised pyrrolopyrimidine and pyrrolopyridine derivatives and performed structure-activity relationship (SAR) study. We found 18p possessed high potency (IC50 = 25.0 nM) against ENPP1, and activated STING pathway in a concentration dependent manner. Also, in response to STING pathway activation, cytokines such as IFN-ß and IP-10 were induced by 18p in a concentration dependent manner. Finally, we discovered that 18p causes inhibition of tumour growth in 4T1 syngeneic mouse model. This study provides new insight into the designing of novel ENPP1 inhibitors and warrants further development of small molecule immune modulators for cancer immunotherapy.

Real-time monitoring of glucose-6-phosphate dehydrogenase activity using liquid droplet arrays and its application to human plasma samples.

Glucose-6-phosphate dehydrogenase (G6PD) regulates nicotinamide adenine dinucleotide phosphate (NADPH) levels and is related to the pathogenesis of various diseases, including G6PD deficiency, type 2 diabetes, aldosterone-induced endothelial dysfunction, and cancer. Therefore, a highly sensitive array-based assay for determining quantitative G6PD activity is required. Here, we developed an on-chip G6PD activity assay using liquid droplet fluorescence arrays. Quantitative G6PD activity was determined by calculating reduced resorufin concentrations in liquid droplets. The limit of detection (LOD) of this assay was 0.162 mU/ml (2.89 pM), which is much more sensitive than previous assays. We used our activity assay to determine kinetic parameters, including Michaelis-Menten constants (Km) and maximum rates of enzymatic reaction (Vmax) for NADP(+) and G6P, and half-maximal inhibitory concentrations (IC50). We successfully applied this new assay to determine G6PD activity in human plasma from normal healthy individuals (n=30) and patients with inflammation (n=30). The inflammatory group showed much higher G6PD activities than did the normal group (p<0.001), with a high area under the curve value of 0.939. Therefore, this new activity assay has the potential to be used for diagnosis of G6PD-associated diseases and utilizing kinetic studies.