Lead structure discovery mainly focuses on the identification of noncovalently binding ligands. Covalent linkage, however, is an essential binding mechanism for a multitude of successfully marketed drugs, although discovered by serendipity in most cases. We present a concept for the design of fragments covalently binding to proteases. Covalent linkage enables fragment binding unrelated to affinity to shallow protein binding sites and at the same time allows differentiated targeted hit verification and binding location verification through mass spectrometry. We describe a systematic and rational computational approach for the identification of covalently binding fragments from compound collections inhibiting enteroviral 3C protease, a target with high therapeutic potential. By implementing reactive groups potentially forming covalent bonds as a chemical feature in our 3D pharmacophore methodology, covalent binders were discovered by high-throughput virtual screening. We present careful experimental validation of the virtual hits using enzymatic assays and mass spectrometry unraveling a novel, previously unknown irreversible inhibition of the 3C protease by phenylthiomethyl ketone-based fragments. Subsequent synthetic optimization through fragment growing and reactivity analysis against catalytic and noncatalytic cysteines revealed specific irreversible 3C protease inhibition.

Lead structure discovery mainly focuses on the identification of noncovalently binding ligands. Covalent linkage, however, is an essential binding mechanism for a multitude of successfully marketed drugs, although discovered by serendipity in most cases. We present a concept for the design of fragments covalently binding to proteases. Covalent linkage enables fragment binding unrelated to affinity to shallow protein binding sites and at the same time allows differentiated targeted hit verification and binding location verification through mass spectrometry. We describe a systematic and rational computational approach for the identification of covalently binding fragments from compound collections inhibiting enteroviral 3C protease, a target with high therapeutic potential. By implementing reactive groups potentially forming covalent bonds as a chemical feature in our 3D pharmacophore methodology, covalent binders were discovered by high-throughput virtual screening. We present careful experimental validation of the virtual hits using enzymatic assays and mass spectrometry unraveling a novel, previously unknown irreversible inhibition of the 3C protease by phenylthiomethyl ketone-based fragments. Subsequent synthetic optimization through fragment growing and reactivity analysis against catalytic and noncatalytic cysteines revealed specific irreversible 3C protease inhibition.

Lead structure discovery mainly focuses on the identification of noncovalently binding ligands. Covalent linkage, however, is an essential binding mechanism for a multitude of successfully marketed drugs, although discovered by serendipity in most cases. We present a concept for the design of fragments covalently binding to proteases. Covalent linkage enables fragment binding unrelated to affinity to shallow protein binding sites and at the same time allows differentiated targeted hit verification and binding location verification through mass spectrometry. We describe a systematic and rational computational approach for the identification of covalently binding fragments from compound collections inhibiting enteroviral 3C protease, a target with high therapeutic potential. By implementing reactive groups potentially forming covalent bonds as a chemical feature in our 3D pharmacophore methodology, covalent binders were discovered by high-throughput virtual screening. We present careful experimental validation of the virtual hits using enzymatic assays and mass spectrometry unraveling a novel, previously unknown irreversible inhibition of the 3C protease by phenylthiomethyl ketone-based fragments. Subsequent synthetic optimization through fragment growing and reactivity analysis against catalytic and noncatalytic cysteines revealed specific irreversible 3C protease inhibition.

Novel and sensitive electrochemical sensor was fabricated for the assay of anti-HCV ledipasvir (LEDV) in differentmatrices. The designed sensor was based on 3D spinel ferromagnetic NiFe2O4 nanospheres and reduced graphene oxide (RGO) supported by morpholinium acid sulphate (MHS), as an ionic liquid (RGO/NSNiFe2O4/ MHS). This sensor design was assigned to synergistically tailor the unique properties of nanostructured ferrites,RGO, and ionic liquid to maximize the sensor response. Electrode modification prevented aggregation of NiFe2O4, increasing electroactive surface area and allowed remarkable electro-catalytic oxidation of LEDV with an enhanced oxidation response. Differential pulse voltammetry was used for detection LEDV in complex matrices whereas; cyclic voltammetry and other techniques were employed to characterize the developed sensor properties. All experimental factors regarding sensor fabrication and chemical sensing properties were carefully studied and optimized. Under the optimum conditions, the designated sensor displayed a wide linear range (0.4–350 ng mL-1) with LOD of 0.133 ng mL-1. Additionally, the proposed sensor demonstrated good selectivity,stability and reproducibility, enabling the quantitative detection of LEDV in Harvoni® tablets, human plasma and in a pharmacokinetic study. Our findings suggest that the developed sensor is a potential prototype material for fabrication of high-performance electrochemical sensors.

Novel and sensitive electrochemical sensor was fabricated for the assay of anti-HCV ledipasvir (LEDV) in differentmatrices. The designed sensor was based on 3D spinel ferromagnetic NiFe2O4 nanospheres and reduced graphene oxide (RGO) supported by morpholinium acid sulphate (MHS), as an ionic liquid (RGO/NSNiFe2O4/ MHS). This sensor design was assigned to synergistically tailor the unique properties of nanostructured ferrites,RGO, and ionic liquid to maximize the sensor response. Electrode modification prevented aggregation of NiFe2O4, increasing electroactive surface area and allowed remarkable electro-catalytic oxidation of LEDV with an enhanced oxidation response. Differential pulse voltammetry was used for detection LEDV in complex matrices whereas; cyclic voltammetry and other techniques were employed to characterize the developed sensor properties. All experimental factors regarding sensor fabrication and chemical sensing properties were carefully studied and optimized. Under the optimum conditions, the designated sensor displayed a wide linear range (0.4–350 ng mL-1) with LOD of 0.133 ng mL-1. Additionally, the proposed sensor demonstrated good selectivity,stability and reproducibility, enabling the quantitative detection of LEDV in Harvoni® tablets, human plasma and in a pharmacokinetic study. Our findings suggest that the developed sensor is a potential prototype material for fabrication of high-performance electrochemical sensors.

A series of novel compounds carrying pyrazino[1,2-a]indol-1(2H)-one scaffold (5a-g) and their reaction intermediates, indole-2-carboxamides, (3a-g) were synthesized and evaluated for their ability to inhibit reactive oxygen species (ROS) generation, antioxidant activity and anticancer activity against a panel of cancer cell lines using MTT assay. The results showed that these compounds can inhibit ROS generation during the metabolic phase of phagocytosis in a dose-dependent manner where compounds 5d and 5e were the most potent samples with higher inhibitory activities (IC50 values 3.3 and 1.4 µM, respectively) than that of the reference acetylsalicylic acid (IC50 ¼ 9.7 µM). Results for the determination of potential antioxidant properties of the synthesized compounds showed that compounds 5d and 5e containing pyrazino[1,2-a]indol-1-one backbone were the most acive and even comparable to Trolox. Compounds 3d-f and 5d-f with the least IC50 values in MTT assay were tested against three known anticancer targets EGFR, BRAF and Tubulin. Histopathological and immunohistochemical study were performed to determine the consequence of exposure to chronic low dose of chlorpyrifos on the testis of male mice and results revealed that these effects can be ameliorated by co-treatment with the most active antioxidant compounds 5d and 5e. Finally, molecular docking studies were performed to explore the binding mode of the most active compounds against EGFR and BRAF kinases.

A series of novel compounds carrying pyrazino[1,2-a]indol-1(2H)-one scaffold (5a-g) and their reaction intermediates, indole-2-carboxamides, (3a-g) were synthesized and evaluated for their ability to inhibit reactive oxygen species (ROS) generation, antioxidant activity and anticancer activity against a panel of cancer cell lines using MTT assay. The results showed that these compounds can inhibit ROS generation during the metabolic phase of phagocytosis in a dose-dependent manner where compounds 5d and 5e were the most potent samples with higher inhibitory activities (IC50 values 3.3 and 1.4 µM, respectively) than that of the reference acetylsalicylic acid (IC50 ¼ 9.7 µM). Results for the determination of potential antioxidant properties of the synthesized compounds showed that compounds 5d and 5e containing pyrazino[1,2-a]indol-1-one backbone were the most acive and even comparable to Trolox. Compounds 3d-f and 5d-f with the least IC50 values in MTT assay were tested against three known anticancer targets EGFR, BRAF and Tubulin. Histopathological and immunohistochemical study were performed to determine the consequence of exposure to chronic low dose of chlorpyrifos on the testis of male mice and results revealed that these effects can be ameliorated by co-treatment with the most active antioxidant compounds 5d and 5e. Finally, molecular docking studies were performed to explore the binding mode of the most active compounds against EGFR and BRAF kinases.

Three new series of 5-aminosalicylic acid derivatives; series I (14, 16–18), series II (19–30) and series III (31–41) were synthesized as potential dual COX-2/5-LOX inhibitors. Their chemical structures were confirmed using spectroscopic tools including IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analyses. The anti-inflammatory activity for all target compounds was evaluated in vivo using carrageenan-induced paw edema. Compound 36 showed the highest anti-inflammatory activity (114.12%) relative to reference drug indomethacin at 4 h interval. Selected derivatives were evaluated in vitro to inhibit ovine COX-1, human recombinant COX-2 and 5-LOX enzymes. Compounds 34 & 35 exhibited significant COX-2 inhibition (IC50 = 0.10 µM) with significant COX-2 selectivity indices (SI = 135 & 145 respectively) approximate to celecoxib (IC50 = 0.049 µM, SI = 308.16) and exceeding indomethacin (IC50 = 0.51 µM, SI = 0.08). Interestingly, all compounds showed superior 5-LOX inhibitory activity about 2–5 times relative to zileuton. Compound 16 was the superlative 5-LOX inhibitor that revealed (IC50 = 3.41 µM) relative to zileuton (IC50 = 15.6 µM). Compounds 34, 35, 36 and 41 showed significant dual COX-2/5-LOX inhibitions. The gastric ulcerogenic effect of compound 36 was examined on gastric mucosa of albino rats and they showed superior GI safety profile compared with indomethacin. Molecular docking studies of the compounds into the binding sites of COX-1, COX-2 and 5-LOX allowed us to shed light on the binding mode of these novels dual COX and 5-LOX inhibitors.

Three new series of 5-aminosalicylic acid derivatives; series I (14, 16–18), series II (19–30) and series III (31–41) were synthesized as potential dual COX-2/5-LOX inhibitors. Their chemical structures were confirmed using spectroscopic tools including IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analyses. The anti-inflammatory activity for all target compounds was evaluated in vivo using carrageenan-induced paw edema. Compound 36 showed the highest anti-inflammatory activity (114.12%) relative to reference drug indomethacin at 4 h interval. Selected derivatives were evaluated in vitro to inhibit ovine COX-1, human recombinant COX-2 and 5-LOX enzymes. Compounds 34 & 35 exhibited significant COX-2 inhibition (IC50 = 0.10 µM) with significant COX-2 selectivity indices (SI = 135 & 145 respectively) approximate to celecoxib (IC50 = 0.049 µM, SI = 308.16) and exceeding indomethacin (IC50 = 0.51 µM, SI = 0.08). Interestingly, all compounds showed superior 5-LOX inhibitory activity about 2–5 times relative to zileuton. Compound 16 was the superlative 5-LOX inhibitor that revealed (IC50 = 3.41 µM) relative to zileuton (IC50 = 15.6 µM). Compounds 34, 35, 36 and 41 showed significant dual COX-2/5-LOX inhibitions. The gastric ulcerogenic effect of compound 36 was examined on gastric mucosa of albino rats and they showed superior GI safety profile compared with indomethacin. Molecular docking studies of the compounds into the binding sites of COX-1, COX-2 and 5-LOX allowed us to shed light on the binding mode of these novels dual COX and 5-LOX inhibitors.

Three new series of 5-aminosalicylic acid derivatives; series I (14, 16–18), series II (19–30) and series III (31–41) were synthesized as potential dual COX-2/5-LOX inhibitors. Their chemical structures were confirmed using spectroscopic tools including IR, 1H NMR, 13C NMR, mass spectroscopy and elemental analyses. The anti-inflammatory activity for all target compounds was evaluated in vivo using carrageenan-induced paw edema. Compound 36 showed the highest anti-inflammatory activity (114.12%) relative to reference drug indomethacin at 4 h interval. Selected derivatives were evaluated in vitro to inhibit ovine COX-1, human recombinant COX-2 and 5-LOX enzymes. Compounds 34 & 35 exhibited significant COX-2 inhibition (IC50 = 0.10 µM) with significant COX-2 selectivity indices (SI = 135 & 145 respectively) approximate to celecoxib (IC50 = 0.049 µM, SI = 308.16) and exceeding indomethacin (IC50 = 0.51 µM, SI = 0.08). Interestingly, all compounds showed superior 5-LOX inhibitory activity about 2–5 times relative to zileuton. Compound 16 was the superlative 5-LOX inhibitor that revealed (IC50 = 3.41 µM) relative to zileuton (IC50 = 15.6 µM). Compounds 34, 35, 36 and 41 showed significant dual COX-2/5-LOX inhibitions. The gastric ulcerogenic effect of compound 36 was examined on gastric mucosa of albino rats and they showed superior GI safety profile compared with indomethacin. Molecular docking studies of the compounds into the binding sites of COX-1, COX-2 and 5-LOX allowed us to shed light on the binding mode of these novels dual COX and 5-LOX inhibitors.