The discovery of potent small molecule cyclic urea activators of STING.

STING mediates innate immune responses that are triggered by the presence of cytosolic DNA. Activation of STING to boost antigen recognition is a therapeutic modality that is currently being tested in cancer patients using nucleic-acid based macrocyclic STING ligands. We describe here the discovery of 3,4-dihydroquinazolin-2(1H)-one based 6,6-bicyclic heterocyclic agonists of human STING that activate all known human variants of STING with high potency.

The discovery of potent small molecule activators of human STING.

The adaptor protein STING plays a major role in innate immune sensing of cytosolic nucleic acids, by triggering a robust interferon response. Despite the importance of this protein as a potential therapeutic target for serious unmet medical conditions including cancer and infectious disease there remains a paucity of STING ligands. Starting with a benzothiazinone series of weak STING activators (human EC50 ∼10 µM) we identified several chemotypes with sub-micromolar STING activity across all the major protein polymorphs. An example compound 53 based on an oxindole core structure demonstrated robust on-target functional activation of STING (human EC50 185 nM) in immortalised and primary cells and a cytokine induction fingerprint consistent with STING activation. Our study has identified several related series of potent small molecule human STING activators with potential to be developed as immunomodulatory therapeutics.

G10 is a direct activator of human STING.

The cGAS/STING pathway initiates an innate immune response when DNA is detected in the cytosol. DNA bound cGAS synthesizes cyclic dinucleotides which bind and activate the adaptor STING, leading to downstream secretion of Type I interferons and other pro-inflammatory NFκB pathway cytokines. In the mouse, the STING driven innate immune response is central to immune based clearance of various tumors and this has triggered a significant effort focused on the discovery of human STING agonists for the treatment of cancer. This report uses an in vitro kinase assay to show that G10, a previously identified STING pathway activator is actually a weak but direct STING agonist and identifies other more potent leads.

Overcoming compound fluorescence in the FLiK screening assay with red-shifted fluorophores.

In the attempt to discover novel chemical scaffolds that can modulate the activity of disease-associated enzymes, such as kinases, biochemical assays are usually deployed in high-throughput screenings. First-line assays, such as activity-based assays, often rely on fluorescent molecules by measuring a change in the total emission intensity, polarization state, or energy transfer to another fluorescent molecule. However, under certain conditions, intrinsic compound fluorescence can lead to difficult data analysis and to false-positive, as well as false-negative, hits. We have reported previously on a powerful direct binding assay called fluorescent labels in kinases ('FLiK'), which enables a sensitive measurement of conformational changes in kinases upon ligand binding. In this assay system, changes in the emission spectrum of the fluorophore acrylodan, induced by the binding of a ligand, are translated into a robust assay readout. However, under the excitation conditions of acrylodan, intrinsic compound fluorescence derived from highly conjugated compounds complicates data analysis. We therefore optimized this method by identifying novel fluorophores that excite in the far red, thereby avoiding compound fluorescence. With this advancement, even rigid compounds with multiple π-conjugated ring systems can now be measured reliably. This study was performed on three different kinase constructs with three different labeling sites, each undergoing distinct conformational changes upon ligand binding. It may therefore serve as a guideline for the establishment of novel fluorescence-based detection assays.

6-Substituted imidazo[1,2-a]pyridines: synthesis and biological activity against colon cancer cell lines HT-29 and Caco-2.

A range of 6-substituted imidazo[1,2-a]pyridines were synthesized using a multicomponent coupling reaction. Most of these compounds were found to exhibit excellent activity against the colon cancer cell lines HT-29 and Caco-2, whilst not showing significant toxicity against white blood cells. Our studies have shown that the proteolytic phase of apoptosis was initiated 2 h after treatment with these imidazo-[1,2-a]pyridines. The data suggests that the imidazo[1,2-a]pyridine-induced cell death in HT-29 and Caco-2 cells is mediated via pathway(s) that include the release of cytochrome c from the mitochondria to the cytosol and the activation of caspase 3 and caspase 8.

Naphtho[2,3-b][1,4]-thiazine-5,10-diones and 3-substituted-1,4-dioxo-1,4-dihydronaphthalen-2-yl-thioalkanoate derivatives: synthesis and biological evaluation as potential antibacterial and antifungal agents.

A series of 3-substituted-1,4-dioxo-1,4-dihydronaphthalen-2-yl-thio-alkanoate derivatives 3-21 and naphtho[2,3-b][1,4]-thiazine-5,10-diones 24 were synthesized and evaluated for their antibacterial and antifungal activities. The structure-activity relationships of these compounds were studied and the results show that the compound 24a exhibited better antibacterial activity than Gentamycin in vitro against Staphylococcus aureus. In addition 24a also imparted marked antifungal activity in vitro against Cryptococcus neoformans, Sporothrix schenckii, and Trichophyton mentagraphytes when compared with Fluconazole. Compounds 15, 18, 19, and 21 also exhibited significant antibacterial activity in vitro against S. aureus.

Design, synthesis, and biological evaluation of 1,2,3-trisubstituted-1,4-dihydrobenzo[g]quinoxaline-5,10-diones and related compounds as antifungal and antibacterial agents.

A series of (S)-N-(3-chloro-1,4-naphthoquinon-2-yl)-alpha-amino acid ethyl esters 3 and 1,2,3-trisubstituted-1,4-dihydrobenzo[g]quinoxaline-5,10-diones 6-23 were synthesized and evaluated for antifungal and antibacterial activities. The structure-activity relationship of these compounds was studied and the results show that the compounds 3a and 3b exhibited in vitro antifungal activity against Candida albicans, Cryptococcus neoformans, and Sporothrix schenckii whereas compounds 12 and 22 showed in vitro antibacterial activity against Klebsiella pneumoniae and Escherichia coli.

Synthesis and biological evaluation of novel (L)-alpha-amino acid methyl ester, heteroalkyl, and aryl substituted 1,4-naphthoquinone derivatives as antifungal and antibacterial agents.

A series of (S)-N-(1,4-naphthoquinon-2-yl)-alpha-amino acid methyl esters 3-9, 2-N,N-dialkylamino-1,4-naphthoquinones 10-11 and 2-hydroxy-3-(2'-mercaptoimidazolyl)-1,4-naphthoquinones and their cyclic analogs 12-15 were synthesized and evaluated for antifungal and antibacterial activities. The structure-activity relationships of these compounds were studied and the results show that the compounds 9b and 13c exhibited in vitro antifungal activity against Candida albicans, Cryptococcus neoformans, and Sporothrix schenckii, whereas compound 6a showed in vitro antibacterial activity against Streptococcus faecalis, K. pneumoniae, Escherichia coli, and Staphylococcus aureus.

Synthesis and biological evaluation of novel 1,4-naphthoquinone derivatives as antibacterial and antiviral agents.

A series of 1,4-naphthoquinone derivatives were synthesized and evaluated for antibacterial and antiviral activities. The structure-activity relationships of these compounds were also studied. The results suggest that compounds 9-22 showed in vitro marked antibacterial activity. Compounds 4c and 7a showed inhibitory effect against RNA dependent RNA polymerase induced poliovirus type 2 infected HeLa cells.

Synthesis of (1,4)-naphthoquinono-[3,2-c]-1H-pyrazoles and their (1,4)-naphthohydroquinone derivatives as antifungal, antibacterial, and anticancer agents.

A series of (1,4)-naphthoquinono [3,2-c]-1H-pyrazoles and their (1,4)-naphthohydroquinone derivatives 2-7 were synthesized and evaluated for antifungal, antibacterial, and anticancer activities. The structure-activity relationship of these compounds was studied and the results show that the compound 2b exhibited in vitro antifungal activity against Candida albicans and Cryptococcus neoformans, and also possessed antibacterial profile against Klebsiella pneumoniae and Escherichia coli whereas 1c showed anticancer activity against Walker 256 Carcinosarcoma in rats.

Synthesis and evaluation of novel 1,4-naphthoquinone derivatives as antiviral, antifungal and anticancer agents.

The synthesis and evaluation of some 2-substituted-1,4-naphthoquinones 2, S-(1,4-naphthoquinon-2-yl)-mercaptoalkanoic acid amides 4, related benzoquinone and naphthoquinone derivatives 6-9 and 2,3-disubstituted 1,4-naphthoquinones 10-11 were carried out. The antifungal, antibacterial, antiviral and anticancer activities were determined by using the standard assay. The results show that compounds 2b and 10a showed in vitro antiviral activity against Influenza-A Virus and Herpes Simplex Virus and possess pronounced antifungal profile whereas 4a showed anticancer activities against Lymphoid Leukaemia P 388.

Design, synthesis and evaluation of novel 1,4-naphthoquinone derivatives as antifungal and anticancer agents.

A series of 1,4-naphthoquinone derivatives were synthesized and tested for antifungal and antitumor activity against a number of fungal disease causative species and Walker 256 carcinoma cell lines. The results show that the compounds 8a,e and 11b possess pronounced antifungal profile where as 7b and f were found to be active against Walker 256 carcinoma cell lines. Moreover 7c and 11a showed inhibitory effect against reverse transcriptase enzyme from Rauscher Murine Leukemia Virus.