Advances in Metabolism and Metabolic Toxicology of Quinoxaline 1,4-Di-N-oxides.

Quinoxaline 1,4-di-N-Oxides (QdNOs) have been used as synthetic antimicrobial agents in animal husbandry and aquaculture. The metabolism and potential toxicity have been also concerns in recently years. The metabolism investigations showed that there were 8 metabolites of Carbadox (CBX), 34 metabolites of Cyadox (CYA), 33 metabolites of Mequindox (MEQ), 35 metabolites of Olaquindox (OLA), and 56 metabolites of Quinocetone (QCT) in different animals. Among them, Cb3 and Cb8, M6, and O9 are metabolic residual markers of CBX, MEQ and OLA, which are associated with N → O reduction. Toxicity studies revealed that QdNOs exhibited severe tumorigenicity, cytotoxicity, and adrenal toxicity. Metabolic toxicology showed that toxicity of QdNOs metabolites might be related to the N → O group reduction, and some metabolites exhibited higher toxic effects than the precursor, which could provide guidance for further research on the metabolic toxicology of QdNOs and provide a wealth of information for food safety evaluation.

The inhibitory mechanism of 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx) formation by ultraviolet-gallic acid (UV-GA) during the oil-frying process of squid.

The inhibitory effect of ultraviolet-gallic acid (UV-GA) on carbonyl valence and intermediates and precursors of 2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (MeIQx) was investigated to futher clarify the inhibitory mechanism for safety control the quality of oil-fried squid. Ultraviolet C-treated gallic acid (UVC-GA) and ultraviolet B-treated gallic acid (UVB-GA) were produced by ultraviolet 225 nm of band C and 300 nm of band B, respectively. The MeIQx contents in oil-fried squid were significantly higher, and UVC-GA and UVB-GA could significantly inhibit the MeIQx formation and the formation rates of carbonyl valence and precursors (threonine (Thr), creatinine, and glucose). The UVB-GA inhibited formaldehyde formation, while UVC-GA significantly reduced the formaldehyde, acetaldehyde, and 2,5-dimethyl pyrazine contents. In conculsion, UV-GA reduced carbonyl produced from the lipid oxidation to further weaken the catalysis of carbonyl, rendering the MeIQx precursor degrading into the intermediates during Strecker degradation. Thus, the MeIQx formation was inhibited.

Crystal structure of the α1B-adrenergic receptor reveals molecular determinants of selective ligand recognition.

α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.

Substituent effect of benzyl moiety in nitroquinoxaline small molecules upon DNA binding: Cumulative destacking of DNA nucleobases leading to histone eviction.

Cooperative disruption of Watson-Crick hydrogen bonds, as well as base-destacking, is shown to be triggered by a quinoxaline-based small molecule consisting of an N,N-dimethylaminopropyl tether, and a para-substituted benzyl moiety. This events lead to superstructure formation and DNA condensation as evident from biophysical experiments and classical molecular dynamics simulations. The DNA superstructure formation by mono-quinoxaline derivatives is highly entropically favored and predominantly driven by hydrophobic interactions. Furthermore, oversupercoiling of DNA and base-destacking cumulatively induces histone eviction from in-vitro assembled nucleosomes at lower micromolar concentrations implicating biological relevance. The DNA structural modulation and histone eviction capacity of the benzyl para-substituents are in the order: -I > -CF3> -Br > -Me > -OMe > -OH, which is largely guided by the polarity of benzyl para-substituent and the resulting molecular topology. The most hydrophobic derivative 3c with para-iodo benzyl moiety causes maximal disruption of base pairing and generation of superstructures. Both these events gradually diminish as the polarity of the benzyl para-substituent increases. On the other hand, quinoxaline derivatives having heterocyclic ring instead of benzyl ring, or in the absence of N,N-dimethylamino head-group, is incapable of inducing any DNA structural change and histone eviction. Further, the quinoxaline compounds displayed potent anticancer activities against different cancer cell lines which directly correlates with the hydrophobic effects of the benzyl para-substituents. Overall, the present study provides new insights into the mechanistic approach of DNA structural modulation driven histone eviction guided by the hydrophobicity of synthesized compounds leading to cellular cytotoxicity towards cancer cells.

Tyrp1 Mutant Variants Associated with OCA3: Computational Characterization of Protein Stability and Ligand Binding.

Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the TYRP1 gene. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally to understand Tyrp1 protein stability and catalytic activity. Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Consistent with the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. SDS-PAGE and Western blot analysis of the purified recombinant proteins confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability. Further, the mutant variant structures were built and simulated for 100 ns to generate free energy landscapes and perform docking experiments. Free energy landscapes formed by Y362, N378, and T391 indicate that the binding clefts of C30R and H215Y mutants are larger than the wild-type Tyrp1. In docking simulations, the hydrogen bond and salt bridge interactions that stabilize DHICA in the active site remain similar among Tyrp1, D308N, and R326H. However, the strengths of these interactions and stability of the docked ligand may decrease proportionally to mutation severity due to the larger and less well-defined natures of the binding clefts in mutants. Mutational perturbations in mutants that are not unfolded may result in allosteric alterations to the active site, reducing the stability of protein-ligand interactions.

Discovery of Quinoxaline-Based P1-P3 Macrocyclic NS3/4A Protease Inhibitors with Potent Activity against Drug-Resistant Hepatitis C Virus Variants.

The three pan-genotypic HCV NS3/4A protease inhibitors (PIs) currently in clinical use-grazoprevir, glecaprevir, and voxilaprevir-are quinoxaline-based P2-P4 macrocycles and thus exhibit similar resistance profiles. Using our quinoxaline-based P1-P3 macrocyclic lead compounds as an alternative chemical scaffold, we explored structure-activity relationships (SARs) at the P2 and P4 positions to develop pan-genotypic PIs that avoid drug resistance. A structure-guided strategy was used to design and synthesize two series of compounds with different P2 quinoxalines in combination with diverse P4 groups of varying sizes and shapes, with and without fluorine substitutions. Our SAR data and cocrystal structures revealed the interplay between the P2 and P4 groups, which influenced inhibitor binding and the overall resistance profile. Optimizing inhibitor interactions in the S4 pocket led to PIs with excellent antiviral activity against clinically relevant PI-resistant HCV variants and genotype 3, providing potential pan-genotypic inhibitors with improved resistance profiles.

Anti-MRSA drug discovery by ligand-based virtual screening and biological evaluation.

S. aureus resistant to methicillin (MRSA) is one of the most-concerned multidrug resistant bacteria, due to its role in life-threatening infections. There is an urgent need to develop new antibiotics against MRSA. In this study, we firstly compiled a data set of 2,3-diaminoquinoxalines by chemical synthesis and antibacterial screening against S. aureus, and then performed cheminformatics modeling and virtual screening. The compound with the Specs ID of AG-205/33156020 was discovered as a new antibacterial agent, and was further identified as a Gyrase B (GyrB) inhibitor. In light of the common features, we hypothesized that the 6c as the representative of 2,3-diaminoquinoxalines also inhibited GyrB and eventually proved it. Via molecular docking and molecular dynamics simulations, we identified binding modes of AG-205/33156020 and 6c to the ATPase domain of GyrB. Importantly, these GyrB inhibitors inhibited the MRSA strains and showed selectivity to HepG2 and HUVEC. Taken together, this research work provides an effective ligand-based computational workflow for scaffold hopping in anti-MRSA drug discovery, and discovers two new GyrB inhibitors that are worthy of further development.

Chemical Screen for Vancomycin Antagonism Uncovers Probes of the Gram-Negative Outer Membrane.

The outer membrane of Gram-negative bacteria is a formidable permeability barrier which allows only a small subset of chemical matter to penetrate. This outer membrane barrier can hinder the study of cellular processes and compound mechanism of action, as many compounds including antibiotics are precluded from entry despite having intracellular targets. Consequently, outer membrane permeabilizing compounds are invaluable tools in such studies. Many existing compounds known to perturb the outer membrane also impact inner membrane integrity, such as polymyxins and their derivatives, making these probes nonspecific. We performed a screen of ∼140 000 diverse synthetic compounds, for those that antagonized the growth inhibitory activity of vancomycin at 15 °C in Escherichia coli, to enrich for chemicals capable of perturbing the outer membrane. This led to the discovery that liproxstatin-1, an inhibitor of ferroptosis in human cells, and MAC-0568743, a novel cationic amphiphile, could potentiate the activity of large-scaffold antibiotics with low permeation into Gram-negative bacteria at 37 °C. Liproxstatin-1 and MAC-0568743 were found to physically disrupt the integrity of the outer membrane through interactions with lipopolysaccharide in the outer leaflet of the outer membrane. We showed that these compounds selectively disrupt the outer membrane while minimally impacting inner membrane integrity, particularly at the concentrations needed to potentiate Gram-positive-targeting antibiotics. Further exploration of these molecules and their structural analogues is a promising avenue for the development of outer membrane specific probes.

Computational drug repurposing study elucidating simultaneous inhibition of entry and replication of novel corona virus by Grazoprevir.

Outcomes of various clinical studies for the coronavirus disease 2019 (COVID-19) treatment indicated that the drug acts via inhibition of multiple pathways (targets) is likely to be more successful and promising. Keeping this hypothesis intact, the present study describes for the first-time, Grazoprevir, an FDA approved anti-viral drug primarily approved for Hepatitis C Virus (HCV), mediated multiple pathway control via synergistic inhibition of viral entry targeting host cell Angiotensin-Converting Enzyme 2 (ACE-2)/transmembrane serine protease 2 (TMPRSS2) and viral replication targeting RNA-dependent RNA polymerase (RdRP). Molecular modeling followed by in-depth structural analysis clearly demonstrated that Grazoprevir interacts with the key residues of these targets. Futher, Molecular Dynamics (MD) simulations showed stability and burial of key residues after the complex formation. Finally, Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analysis identified the governing force of drug-receptor interactions and stability. Thus, we believe that Grazoprevir could be an effective therapeutics for the treatment of the COVID-19 pandemic with a promise of unlikely drug resistance owing to multiple inhibitions of eukaryotic and viral proteins, thus warrants further clinical studies.

New Quinoxaline Derivatives as Dual Pim-1/2 Kinase Inhibitors: Design, Synthesis and Biological Evaluation.

Proviral integration site for Moloney murine leukemia virus (Pim)-1/2 kinase overexpression has been identified in a variety of hematologic (e.g., multiple myeloma or acute myeloid leukemia (AML)) and solid (e.g., colorectal carcinoma) tumors, playing a key role in cancer progression, metastasis, and drug resistance, and is linked to poor prognosis. These kinases are thus considered interesting targets in oncology. We report herein the design, synthesis, structure-activity relationships (SAR) and in vitro evaluations of new quinoxaline derivatives, acting as dual Pim1/2 inhibitors. Two lead compounds (5c and 5e) were then identified, as potent submicromolar Pim-1 and Pim-2 inhibitors. These molecules were also able to inhibit the growth of the two human cell lines, MV4-11 (AML) and HCT-116 (colorectal carcinoma), expressing high endogenous levels of Pim-1/2 kinases.

Binding of a negative allosteric modulator and competitive antagonist can occur simultaneously at the ionotropic glutamate receptor GluA2.

Ionotropic glutamate receptors are ligand-gated ion channels governing neurotransmission in the central nervous system. Three major types of antagonists are known for the AMPA-type receptor GluA2: competitive, noncompetitive (i.e., negative allosteric modulators; NAMs) used for treatment of epilepsy, and uncompetitive antagonists. We here report a 4.65 Å resolution X-ray structure of GluA2, revealing that four molecules of the competitive antagonist ZK200775 and four molecules of the NAM GYKI53655 are capable of binding at the same time. Using negative stain electron microscopy, we show that GYKI53655 alone or ZK200775/GYKI53655 in combination predominantly results in compact receptor forms. The agonist AMPA provides a mixed population of compact and bulgy shapes of GluA2 not impacted by addition of GYKI53655. Taken together, this suggests that the two different mechanisms of antagonism that lead to channel closure are independent and that the distribution between bulgy and compact receptors primarily depends on the ligand bound in the glutamate binding site. DATABASE: The atomic coordinates and structure factors from the crystal structure determination have been deposited in the Protein Data Bank under accession code https://doi.org/10.2210/pdb6RUQ/pdb. The electron microscopy 3D reconstruction volumes have been deposited in EMDB (EMD-4875: Apo; EMD-4920: ZK200775/GYKI53655; EMD-4921: AMPA compact; EMD-4922: AMPA/GYKI53655 bulgy; EMD-4923: GYKI53655; EMD-4924: AMPA bulgy; EMD-4925: AMPA/GYKI53655 compact).

Metabolism and disposition in rats, dogs, and humans of erdafitinib, an orally administered potent pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor.

This article describes in vivo biotransformation and disposition of erdafitinib following single oral dose of 3H-erdafitinib and 14C-erdafitinib to intact and bile duct-cannulated (BC) rats (4 mg/kg), 3H-erdafitinib to intact dogs (0.25 mg/kg), and 14C-erdafitinib to humans (12 mg; NCT02692677). Peak plasma concentrations of total radioactivity were achieved rapidly (Tmax: animals, 1 h; humans, 2-3 h). Recovery of drug-derived radioactivity was significantly slower in humans (87%, 384 h) versus animals (rats: 91-98%, 48 h; dogs: 81%, 72 h). Faeces was the primary route of elimination in intact rats (95%), dogs (76%), and humans (69%); and bile in BC rats (48%). Renal elimination of radioactivity was relatively low in animals (2-12%) versus humans (19%). Unchanged erdafitinib was major component in human excreta (faeces, 17%; urine, 11%) relative to animals. M6 (O-desmethyl) was the major faecal metabolite in humans (24%) and rats (intact, 46%; BC, 11%), and M2 (O-glucuronide of M6) was the prevalent biliary metabolite in rats (14%). In dogs, besides M6, majority of radioactive dose in faeces was composed of multiple minor metabolites. In humans, unchanged erdafitinib was the major circulating entity. O-demethylation of erdafitinib was the major metabolic pathway in humans and animals.

Investigating the Potential of Phosphatidylcholine-Based Nano-Sized Carriers in Boosting the Oto-Topical Delivery of Caroverine: in vitro Characterization, Stability Assessment and ex vivo Transport Studies.

PURPOSE: Drug delivery into the inner ear across the intact tympanic membrane (TM) has been a challenge in the treatment of inner ear disorders. In this study, nano-sized carriers were formulated for improving the non- invasive oto-topical delivery of caroverine for the treatment of tinnitus. METHODS: Caroverine was loaded into two types of phospholipid-containing systems, namely, nano elastic vesicles (EVs) and phosphatidylcholine-based liquid crystalline nano-particles (PC-LCNPs). The prepared formulations were characterized for their drug loading, particle size, polydispersity index, zeta potential, morphological features by transmission electron microscopy (TEM), and physicochemical stability. In addition, comparative ex vivo transport study was carried out using rabbits' TM for both types of formulations. RESULTS: The findings show a significant superiority of PC-LCNPs over the EVs formulations in the drug payload (1% and 0.25%, respectively), physical stability and the efficiency of permeation across rabbits' TM. The results showed a more than twofold increase in the cumulative drug flux values of PC-LCNPs (699.58 ± 100 µg/cm2) compared to the EVs (250 ± 45 µg/cm2) across the TM. CONCLUSION: The current study revealed the smart physicochemical properties of PC-LCNPs demonstrating the potential of this carrier as a new attractive candidate for improving the non-invasive oto-topical delivery of caroverine.

NHD2-15, a novel antagonist of Growth Factor Receptor-Bound Protein-2 (GRB2), inhibits leukemic proliferation.

The majority of chronic myeloid leukemia (CML) cases are caused by a chromosomal translocation linking the breakpoint cluster region (BCR) gene to the Abelson murine leukemia viral oncogene-1 (ABL1), creating the mutant fusion protein BCR-ABL1. Downstream of BCR-ABL1 is growth factor receptor-bound protein-2 (GRB2), an intracellular adapter protein that binds to BCR-ABL1 via its src-homology-2 (SH2) domain. This binding constitutively activates growth pathways, downregulates apoptosis, and leads to an over proliferation of immature and dysfunctional myeloid cells. Utilizing novel synthetic methods, we developed four furo-quinoxaline compounds as GRB2 SH2 domain antagonists with the goal of disrupting this leukemogenic signaling. One of the four antagonists, NHD2-15, showed a significant reduction in proliferation of K562 cells, a human BCR-ABL1+ leukemic cell line. To elucidate the mode of action of these compounds, various biophysical, in vitro, and in vivo assays were performed. Surface plasmon resonance (SPR) assays indicated that NHD2-15 antagonized GRB2, binding with a KD value of 119 ± 2 µM. Cellulose nitrate (CN) assays indicated that the compound selectively bound the SH2 domain of GRB2. Western blot assays suggested the antagonist downregulated proteins involved in leukemic transformation. Finally, NHD2-15 was nontoxic to primary cells and adult zebrafish, indicating that it may be an effective clinical treatment for CML.

Substantial Cellular Penetration of Fluorescent Imidazoquinoxalines.

Fluorescent tools have revolutionized our capability to visualize, probe, study, and understand the biological cellular properties, processes and dynamics, enabling researchers to improve their knowledge for example in cancer field. In this paper, we use the peculiar properties of our Imiqualines derivatives to study their cellular penetration and distribution in a human melanoma cell line A375 using confocal microscopy. Preliminary results on colocalization with the potent protein target c-Kit of our lead are also described.

Rational modification, synthesis and biological evaluation of 3,4-dihydroquinoxalin-2(1H)-one derivatives as potent and selective c-Jun N-terminal kinase 3 (JNK3) inhibitors.

The c-Jun N-terminal kinase 3 (JNK3) plays key roles in a wide range of diseases, including neurodegeneration diseases, inflammation diseases, cancers, cardiovascular diseases, and metabolic disorders. Previously, we have identified a lead compound, (Z)-3-(2-(naphthalen-1-yl)-2-oxoethylidene)-3,4-dihydroquinoxalin-2(1H)-one (J46), which contains a 3,4-dihydroquinoxalin-2(1H)-one core structure as a key fragment to inhibit JNK3. However, compound J46 displayed high DDR1 and EGFR (T790M, L858R) inhibition and poor physicochemical properties, especially clogD and water-solubility, in its biological studies. Herein, we optimized compound J46 by structure-based drug design and exploiting the selectivity and physicochemical properties of various warhead groups to obtain compound J46-37, which not only exhibited a potent inhibition against JNK3 but also showed more than 50-fold potency better than DDR1 and EGFR (T790M, L858R). Furthermore, the selectivity and structure-activity relationship of novel synthesized 3,4-dihydroquinoxalin-2(1H)-one derivatives were analyzed by molecular docking and molecular dynamics simulation. Overall, compound J46-37, as a highly selective inhibitor of JNK3 with well physicochemical properties, is worth developing as therapies for the treatment of diseases related to JNK3.

Cometabolic biodegradation of quizalofop-p-ethyl by Methylobacterium populi YC-XJ1 and identification of QPEH1 esterase

BACKGROUND: Quizalofop-p-ethyl (QPE), a unitary R configuration aromatic oxyphenoxypropionic acid ester (AOPP) herbicide, was widely used and had led to detrimental environmental effects. For finding the QPEdegrading bacteria and promoting the biodegradation of QPE, a series of studies were carried out. RESULTS: A QPE-degrading bacterial strain YC-XJ1 was isolated from desert soil and identified as Methylobacterium populi, which could degrade QPE with methanol by cometabolism. Ninety-seven percent of QPE (50 mg/L) could be degraded within 72 h under optimum biodegradation condition of 35°C and pH 8.0. The maximum degradation rate of QPE was 1.4 mg/L/h, and the strain YC-XJ1 exhibited some certain salinity tolerance. Two novel metabolites, 2-hydroxy-6-chloroquinoxaline and quinoxaline, were found by high-performance liquid chromatography/mass spectroscopy analysis. The metabolic pathway of QPE was predicted. The catalytic efficiency of strain YC-XJ1 toward different AOPPs herbicides in descending order was as follows: haloxyfop-pmethyl ≈ diclofop-methyl ≈ fluazifop-p-butyl N clodinafop-propargyl N cyhalofop-butyl N quizalofop-p-ethyl N fenoxaprop-p-ethyl N propaquizafop N quizalofop-p-tefuryl. The genome of strain YC-XJ1 was sequenced using a combination of PacBio RS II and Illumina platforms. According to the annotation result, one α/ß hydrolase gene was selected and named qpeh1, for which QPE-degrading function has obtained validation. Based on the phylogenetic analysis and multiple sequence alignment with other QPE-degrading esterases reported previously, the QPEH1 was clustered with esterase family V. CONCLUSION: M. populi YC-XJ1 could degrade QPE with a novel pathway, and the qpeh1 gene was identified as one of QPE-degrading esterase gene.

Biomimetic asymmetric reduction of benzoxazinones and quinoxalinones using ureas as transfer catalysts.

Using ureas as transfer catalysts through hydrogen bonding activation, biomimetic asymmetric reduction of benzoxazinones and quinoxalinones with chiral and regenerable NAD(P)H models was described, giving chiral dihydrobenzoxazinones and dihydroquinoxalinones with high yields and excellent enantioselectivities. A key dihydroquinoxalinone intermediate of a BRD4 inhibitor was synthesized using biomimetic asymmetric reduction.

Glecaprevir and Maraviroc are high-affinity inhibitors of SARS-CoV-2 main protease: possible implication in COVID-19 therapy.

Due to the lack of efficient therapeutic options and clinical trial limitations, the FDA-approved drugs can be a good choice to handle Coronavirus disease (COVID-19). Many reports have enough evidence for the use of FDA-approved drugs which have inhibitory potential against target proteins of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we utilized a structure-based drug design approach to find possible drug candidates from the existing pool of FDA-approved drugs and checked their effectiveness against the SARS-CoV-2. We performed virtual screening of the FDA-approved drugs against the main protease (Mpro) of SARS-CoV-2, an essential enzyme, and a potential drug target. Using well-defined computational methods, we identified Glecaprevir and Maraviroc (MVC) as the best inhibitors of SARS-CoV-2 Mpro. Both drugs bind to the substrate-binding pocket of SARS-CoV-2 Mpro and form a significant number of non-covalent interactions. Glecaprevir and MVC bind to the conserved residues of substrate-binding pocket of SARS-CoV-2 Mpro. This work provides sufficient evidence for the use of Glecaprevir and MVC for the therapeutic management of COVID-19 after experimental validation and clinical manifestations.

Discovery of triazoloquinoxaline as novel STING agonists via structure-based virtual screening.

Stimulator of interferon genes (STING) is an endoplasmic reticulum adaptor facilitating innate immune signaling. Activation of STING leads to expression of interferons (IFNs) and pro-inflammatory cytokines which is associated with antiviral and antitumor responses. It is imperative to discovery potent compounds that precisely modulate STING. Herein, we describe the discovery of triazoloquinoxaline 1a as a novel STING agonist via Structure-based Virtual Screening. Specifically, biochemical and cell-based assays suggested that 1a stimulated concentration-dependently mRNA expression of IFNß, CXCL-10 and IL-6. Furthermore, 1a significantly induced phosphorylation of STING, TANK-binding kinases1 (TBK1) and interferon regulatory factor 3 (IRF3), suggesting the activation of STING and its downstream TBK1-IRF3 signaling axis. In addition, 1a activated secretion of secreted alkaline phosphatase (SEAP) in dose-dependent manner and EC50 was 16.77 ± 3.814 µM, which is comparable with EC50 of 2'3'-cGAMP (9.212 ± 2.229 µM). These studies revealed that 1a is a promising STING agonist possessing the potential to be further developed for antiviral and antitumor treatment.