Mesorhizobium sp. The majority of the strains produced HCN and ammonia. The maximum indole acetic acid (IAA) (31.54 µM/mL) and cellulase (390 µM/mL) were produced by the isolate KSB (Grz). In contrast, the highest protease (525.12 µM/mL) and chitinase (5.20 µM/mL) activities were shown by standard strain Bacillus mucilaginosus and KSB (Gmr) isolate, respectively.Soil potassium (K) supplement depends intensively on the application of chemical fertilizers, which have substantial harmful environmental effects. However, some bacteria can act as inoculants by converting unavailable and insoluble K forms into plant-accessible forms. Such bacteria are an eco-friendly approach for enhancing plant K absorption and consequently reducing utilization of chemical fertilization. Therefore, the present research was undertaken to isolate, screen, and characterize the K solubilizing bacteria (KSB) from the rhizosphere soils of northern India. Overall, 110 strains were isolated, but only 13 isolates showed significant K solubilizing ability by forming a halo zone on solid media. They were further screened for K solubilizing activity at 0 °C, 1 °C, 3 °C, 5 °C, 7 °C, 15 °C, and 20 °C for 5, 10, and 20 days. All the bacterial isolates showed mineral K solubilization activity at these different temperatures. However, the content of K solubilization increased with the upsurge in temperature and period of incubation. The isolate KSB (Grz) showed the highest K solubilization index of 462.28% after 48 h of incubation at 20 °C. The maximum of 23.38 µg K/mL broth was solubilized by the isolate KSB (Grz) at 20 °C after 20 days of incubation. Based on morphological, biochemical, and molecular characterization (through the 16S rDNA approach), the isolate KSB (Grz) was identified as  The mination [3 . Bacillus sp. [Pseudomonas sp. [Paenibacillus sp. [Burkholderia sp. [Streptomyces sp. [Growing amounts of high-yielding fertilizer responsive crop varieties and the intensification of agriculture have led to the depletion of available reserves of various nutrients, including K, at an alarming rate. Imbalanced fertilizer application is also a prominent contributing factor to the K deficiency in agricultural soils. These issues have enabled a new research frontier focused on the discovery of an alternative natural, sustainable and safe method for improving K availability by exploring the plant-growth-promoting bacteria (PGPB) that stimulate plant growth through nitrogen (N) fixation, mineral solubilization , organic matter degradation, and phytohormone production ,19. Amonllus sp. ,14, Pseuonas sp. , Paeniballus sp. , Burkholeria sp. , Streptoyces sp.  have beeyces sp. .The Kashmir and Ladakh regions of Jammu and Kashmir State are unique in that they experience more diverse climatic conditions than other states in India. The soils of the extreme northern areas of this Himalayan state harbor cold-tolerant mineral solubilizing microbes, which can be applied for improving the nutrient availability for growing crops on an eco-friendly and long-term basis. The microbes that have attained the adaption of surviving and functioning under cold climatic conditions are referred to as cold-tolerant; such microbes isolated from the Arctic and Himalayas were found to exhibit plant growth-promoting (PGP) activity at a temperature range of 4–25 °C ,30. TherThe study area from which the K solubilizing bacteria have been isolated includes the Ladakh region and Kashmir valley of Jammu and Kashmir State, India. The Ladakh region, known as the cold desert, is located at altitudes of 2676 to 3500 m, with a mean annual temperature of 8.6 °C, and receives the scant annual rainfall of 102 mm to 318 mm. The soils are by and large sandy in texture with low organic matter content. Soil reaction is generally alkaline. Drass, a subdivision of the Ladakh region, is recognized as being the second coldest place globally, with mean annual winter and summer temperatures of −12 °C and 23 °C, respectively [The soil adhered to the roots of crop plants like maize, oats, wheat, barley, potato, brinjal, tomato, apple, pear, walnut, and almond were collected in sterile polythene bags. Samples from pastures, forests, and glacier sites were also collected from the study area between 2015–2016. The upper surface layer was scrapped, and the rhizosphere soil samples up to the depth of 10 cm were collected. All samples were collected in sterilized zip-locked polybags and stockpiled in an ice chest during transport, preserved at 4 °C under refrigeration conditions in the laboratory, and utilized within 12–24 h after collection for further analysis.Bacillus mucilaginosus used as a control was obtained from the Department of Soil Science and Agricultural Chemistry, VN Marathwada Agriculture University, Parbhani, Maharashtra, India.K solubilizing bacteria were isolated on Aleksandrov’s medium containing (g/L) 5.0 g/L glucose, 0.005 g/L magnesium sulphate, 0.1 g/L ferric chloride, 2.0 g/L calcium carbonate, 2.0 g/L K mineral (mica), 2.0 g/L calcium phosphate, 20.0 g/L agar, and 1000 mL distilled water, following modified standard methodology . Serial K solubilization potential of rhizobacterial isolates was qualitatively determined by spot inoculating pure bacterial colonies on Aleksandrov’s medium plates containing mica as insoluble K source and incubating for 20 days. The K solubilization potential was assessed by measuring the diameter of hollow zones formed on the medium. The colony diameter was also measured to work out the solubilization efficiency by using the following formula .Bacillus mucilaginosus were used as controls. The incubated bacterial suspension was centrifuged at 8000× g after different time intervals for 10 min to separate supernatant from cell mass, and insoluble K. Supernatant 1 mL was collected in a volumetric flask of 50 mL capacity, and volume was made up to 50 mL with dH2O and thoroughly mixed. This solution was fed to a flame photometer to assess the K content [To analyze the K solubilization of isolates, a loopful of 24 h old culture of each rhizobacterial cultures were inoculated into 25 mL of sterilized Aleksandrov’s broth in 50 mL flasks and incubated at 0, 1, 3, 5, 7, 15, and 20 °C for 5, 10 and 20 days on an incubating shaker at 100 rpm. Flasks inoculated with  content . WorkingA = absorbance of the solution m = slope of curve, andc = concentration of unknown solution 2CO3, 1 L of dH2O) was positioned inside the upper lid of the petri dish. The dishes were sealed adequately with the parafilm and incubated at 20 ± 1 °C for 48 h. Color change from yellow to different brownish shades was treated as a positive result for hydrogen cyanide production. Based on color change, concentration from yellow to light brown or reddish-brown was considered as weak (+), moderate (++), or strong (+++), accordingly [Qualitative production of hydrogen cyanide was assessed by streaking KSB isolates on King’s B medium augmented with 4.4 g/L glycine. Sterilized filter paper soaked in picric acid solution  for 4–5 days at 20 ± 2 °C for the determination of ammonia production . After ig for 5 min. To 1 mL of supernatant, 4 mL IAA reagent  was added, and a final volume of 5 mL was produced, followed by incubation at 20 ± 2 °C for 30 min. The development of a pink color was considered to be positive for IAA production. Using a UV spectrophotometer, absorbance at 530 nm was taken to determine the concentration of IAA in all culture tubes with the help a standard curve prepared with different concentrations of analytical grade IAA.IAA production was tested according to the modified standard protocol . Fresh cg for 10 min at 4 °C to collect the supernatant of crude extracellular enzyme, followed by determination of protease activity according to the modified protocol [w/v) in 200 µL (0.05 M) Tris–HCl buffer (pH 7.0) and 100 µL enzyme. For 30 min the reaction mixture was incubated at 20 °C followed by the addition of 300 µL of 10% (w/v) trichloroacetic acid (TCA) to stop the reaction. Afterwards, the solution was centrifuged at 16,128× g and 4 °C for 10 min, and supernatants (500 µL) were taken by pipetting into separate tubes to which Na2CO3  and Folin–Cio-Calteu reagent  were added. The solutions were incubated at 30 °C for 25 min and absorbance was measured at 660 nm. The protease activity was expressed in µg of tyrosine produced by 1 mL of enzyme in 60 min at 20 °C on tyrosine equivalent. One unit of protease activity was defined as the increase of 0.1-unit optic density at a one h incubation period.The overnight grown bacterial cultures in protease production media were centrifuged at 10,000× protocol . The reaThe chitinase activity of the bacterial isolates was determined using the modified protocol of . The reaCellulase activity of the bacterial strains was measured by the following method . The reaThe best KSB isolate was examined for colony morphology, cell shape, and gram reaction, and biochemical characterization according to Bergey’s Manual of Determinative Bacteriology. 2), and 37.5 μL of de-ionized water. Amplification was performed as follows: 30 cycles of 95 °C for 3 min, 94 °C for 30 s, 55 °C for 30 s, 72 °C for 50 s, 72 °C for 8 min. PCR products were sequenced at AgriGenome Lab, Pvt. Ltd., C°Chi, Kerala and the unknown sequence was then identified using the maximum aligned 16S rDNA sequences available in the Gen-Bank of NCBI through BLAST. The phylogenetic tree was built by using the neighbor-joining method.By using the colony-polymerase chain reaction (PCR) method, the molecular characterization of the selected bacterial isolate was carried out . The unip < 0.05).All experiments were performed in five replicates and the results were stated as means for each sample, including the control group (standard group). The SPSS 16.0 statistical package was used for performing all statistical analyses. One-way analysis of variance (ANOVA) and post hoc analysis (Duncan’s Multiple Range Test) were performed to compare mean values of the isolates and the standard  higher solubilization efficiency in comparison to the standard isolate (Bacillus mucilaginosus) (169.79%). However, the highest significant (p < 0.05) solubilization efficiency was displayed by isolate KSB (Grz) (462.28%). Solubilization of mineral K compounds by the abundant soil microorganisms is a common phenomenon through the secretion of organic acids and enzymes, and the production of capsular polysaccharides [All thirteen strains were evaluated for the potential qualitative K solubilization under in-vitro conditions. All the strains produced clear solubilization halo zones of different diameters on Aleksandrov’s agar medium, ranging from 3.10–5.27 cm after 48 h of incubation at 20 °C . The rescharides ,43. Othecharides . Moreovecharides .The quantitative K solubilization by all 13 KSB isolates was assessed by incubating them at different incubation periods viz. 5, 10, and 20 days, at incubation temperatures of 0 °C,1 °C, 3 °C, 5 °C, 7 °C, 15 °C, and 20 °C as shown in p < 0.05) higher quantity of K compared to standard at an incubation temperature of 20 °C. However, the maximum quantity (10.19 µg K/mL broth) of K was solubilized by isolate KSB (Grz), which is 136.4 % more than the standard. Mineral K solubilization by isolates even below sub-optimal temperature conditions supports the cold-tolerant nature of these isolates  and KSB (Trs) produced a significantly (isolates .Bacillus mucilaginosus) did not solubilize any quantity of K at 0 °C, 1 °C and 3 °C (p < 0.05) quantities of K from an insoluble source (mica), while the isolates KSBM, KSB(Sls), KSB(Trs) and KSB (Afr) produced significantly (p < 0.05) lower K quantities in comparison to standard at 20 °C incubation temperature. However, the maximum quantity (13.33 µg K/mL broth) of K was solubilized by isolate KSB(Grz), 63.9% more than that produced by the standard.K solubilization by different bacterial isolates after ten days of incubation time at 0 °C, 1 °C, 3 °C, 5 °C, 7 °C, 15 °C, and 20 °C incubation temperatures, exhibited that the standard (and 3 °C . HoweverBacillus mucilaginosus) exhibited solubilization activity only at and beyond 5 °C by producing 0.04, 0.97, 10.76 and 15.33 µg K/mL broth at 5 °C, 7 °C, 15 °C, and 20 °C, respectively (p < 0.05) higher K content than standard. However, a significant (p < 0.05) maximum of 23.38 µg K/mL broth was solubilized by KSB (Grz), which is 52.5% more than the quantity solubilized by the standard isolate 20 °C. All the K solubilizing isolates showed almost stabilized K solubilization at 15 °C and 20 °C after 20 days of incubation that could be attributed to their psychrotrophic nature, while the standard isolate did not express any stabilization at 20 °C after 20 days of incubation. The ability of KSB bacterial strains to show activity at lower temperatures could be attributed to their adaptability to these conditions [After twenty days of incubation at 0 °C, 1 °C, 3 °C, 5 °C, 7 °C, 15 °C and 20 °C incubation temperatures, the K solubilization pattern by various isolates revealed that the standard  higher IAA content, as compared to standard isolate, with KSB(Grz) producing the highest content of 31.54 µM/mL. The protease activity shown by all KSB isolates was non-significant in comparison to the standard. Only one isolate, KSB(Gmr), showed significantly (p < 0.05) higher (5.2 µM/mL) chitinase activity, similarly only two isolates, KSB(Grz) and KSB(Afr), showed significantly higher cellulase activities of 390 and 270 µM/mL, respectively. The potential of cold tolerant bacteria to produce cold active enzymes and plant growth promoting substances has been also documented in an earlier study [2 [Pseudomonas, Bacillus, Bradyrhizobium, KlebsiellaRhizobium sp. etc., were found to be involved in the biosynthesis of IAA through the formation of indole-3-pyruvic acid and indole-3-acetic aldehyde [All the K solubilizing isolates exhibited PGP activities such as the production of HCN, ammonia, and IAA . The isoer study . The capstudy [2 . Severalstudy [2 ,57,58. Maldehyde ,61,62,63aldehyde ,65,66,67Mesorhizobium sp.Based on morphological and biochemical features, the strain KSB-Grz was found to be rod-shaped and Gram-negative bacterium with white creamy, circular, entire margins, convex, smooth, and opaque colonies appearing on Aleksandrov’s agar media within 2–3 days of incubation at 20 °C. Biochemical analysis results showed that KSB-Grz is positive for catalase, motility, and casein hydrolysis, and negative for citrate utilization, indole, methyl red, and the Voges Proskauer test. The isolate was found to belong to the species of Mesorhizobium sp. within the GenBank database. The phylogenetic tree of the KSB-Grz strain depicts its position within the Mesorhizobium genus . All the KSB strains also possessed plant growth promoting activities by producing HCN, ammonia, IAA and lytic enzymes such as cellulase, protease and chitinase with the isolate KSB (Grz) producing IAA and cellulase significantly higher than those produced by the standard strain and the other strains, while the protease and chitinase were not significant. The potential isolate KSB (Grz) was identified as Mesorhizobium sp. via 16SrDNA sequencing. The results of the present study showed that out of a total 110 bacterial isolates, only 13 bacterial isolates showed the ability of potassium solubilization at lower temperature conditions  under in-vitro conditions. However, the isolate KSB (Grz) was outstanding with respect to having the most significant and highest K solubilization potential, both qualitatively and quantitatively, in comparison to other strains and the standard strain (


Tlr3 and Tlr7. Using anti-HA antibodies to detect endogenous tagged TLR3 and TLR7, we found that both TLRs display differential tissue expression and posttranslational modifications. C-terminal tagging did not impair TLR3 activity. However, it disrupted the interaction between TLR7 and myeloid differentiation primary response 88 (MYD88), the Tir domain-containing adaptor of TLR7, which blocked its downstream signaling necessary to trigger cytokine and chemokine expression. Our study demonstrates different properties for TLR3 and TLR7, and also provides useful mouse models for further investigation of these two RNA-sensing TLRs.Toll-like receptor (TLR) signaling is critical for defense against pathogenic infection, as well as for modulating tissue development. Activation of different TLRs triggers common inflammatory responses such as cytokine induction. Here, we reveal differential impacts of TLR3 and TLR7 signaling on transcriptomic profiles in bone marrow-derived macrophages (BMDMs). Apart from self-regulation, TLR3, but not TLR7, induced expression of other TLRs, suggesting that TLR3 activation globally enhances innate immunity. Moreover, we observed diverse influences of TLR3 and TLR7 signaling on genes involved in methylation, caspase and autophagy pathways. We compared endogenous TLR3 and TLR7 by using CRISPR/Cas9 technology to knock in a dual Myc-HA tag at the 3’ ends of mouse  Innate immunity is the first line of defense for eliminating pathogens. To detect diverse pathogens, hosts have evolved various pattern recognition receptors (PRRs) to recognize foreign molecules derived from pathogens , 2. TollOf the 13 recognized TLRs, TLR3, TLR7, TLR8 and TLR9 are known as nucleic acid-sensing TLRs , 6, 7. TTlr mRNAs, the trafficking chaperone Unc93b1 controls their transport from the endoplasmic reticulum (ER) to endosomal compartments where they are proteolyzed into N- and C-terminal fragments (NTF and CTF)  , 16, 17.and CTF) –20. Downand CTF) , 5. The pression , 5. In gpression , 21. InsOur previous study indicated that TLR3, TLR7 and TLR8 cell-autonomously control expression of distinct downstream target genes to regulate neuronal morphology , 21, 22.All mice were housed in the animal facility of the Institute of Molecular Biology, Academia Sinica, with a 12 h light/12 h dark cycle and controlled temperature and humidity. All animal experiments were performed with the approval of the Academia Sinica Institutional Animal Care and Utilization Committee and in strict accordance with its guidelines and those of the Council of Agriculture Guidebook for the Care and Use of Laboratory Animals (Protocol # 13-02-520). Both male and female mice were used randomly.–/–Tlr3 (–/yTlr7 (t/tTlr3 is used to represent homozygous tagged knockin mice), we adopted a CRISPR/Cas9-mediated knockin approach to insert the Myc-HA tag sequences into exon 7 of Tlr3 before the stop codon. A gRNA (5’-tgcaagtagcacttggatct) and a single-stranded DNA template (ssODN: 5′-catgatggacctttataaattggatctatccctttaccgactccaaatcttcaaatgagtttaAGCGTAATCTGGAACATCGTATGGGTAaccgttCAGATCCTCTTCTGAGATGAGTTTTTGTTCTCTAGAatgtgctgaattTcTagatccaagtgctacttgcaatttatgatgaaaggcatttatccgttctttct) were used, incorporating an XbaI site , as well as Myc  and HA  sequences.Tlr3–/–  and Tlr7, the Myc-HA tag sequences were inserted into exon 3 of Tlr7 before the stop codon by using CRISPR/Cas9-mediated knockin approach. The gRNA (5’-ggcttatagtcaaatgttca) and ssODN (5’- cagaccttcatttatggaaacctttatgaaaacttcaggtaccaaggcatgtcctaggtggtgacattcttcagagagctaAGCGTAATCTGGAACATCGTATGGGTAaccgttCAGATCCTCTTCTGAGATGAGTTTTTGTTCTCTAGAgactgtttccttgaacatttgactataagccacatgattgtctgtggtca) was used to incorporate an XbaI site , as well as Myc  and HA  sequences.To generate Myc-HA-tagged Tlr7 gene in t/yTlr7 or t/tTlr7 mice also contains two loxP sites to flank the exon 3 that was achieved by using CRISPR/Cas9D10A paired-nicking approach. Two pairs of gRNAs  and two ssODNs carried loxP sequences  and HaeII site (underlined)  were used along with Cas9D10A. The PAM sequences were mutated . The Cas9 mRNA, D10ACas9 mRNA, gRNA preparation, and mouse production protocols were as described previously (The eviously .Tlr3-tagged knockin and wild-type (WT) alleles were detected by PCR using Tlr3-Fw (5’-CACTCTGTTTGCGAAGAG-3’) and Tlr3-Rv (5’-CTATCCCTTTACCGACTC-3’) primers that amplify a 162 base pair (bp) fragment from the WT allele and a 232-bp fragment from the tagged knockin allele of Tlr3. To genotype t/yTlr7 and Tlr7t/t mice, we used Tlr7-Fw (5’-CATCAGAGGCTCCTGGATG-3’), Tlr7-Rv (5’-GCCCAGAGAACTTCTCAGTA-3’), and HA-Rv (5’-GCGTAATCTGGAACATCG-3’) to amplify a 708-bp fragment from the WT allele and a 293-bp fragment from the tagged knockin allele of Tlr7.For genotyping, the http://www.clcbio.com). Raw sequencing reads were trimmed of low-quality sequences  and of sequences with length < 25 bp. Sequencing reads were mapped to the mouse genome assembly (GRCm38.p6) from Ensembl with the following parameters: mismatches cost=2, insertion cost=3, deletion cost=3, minimum fraction length=0.9, minimum fraction similarity=0.9, and maximum hits per read=5. A Generalized Linear Model (GLM) and Wald Test were then used to analyze differential expression of genes and their statistical significance. The second system employed Hisat2 for sequence mapping (GRCm38.p6) with default settings, featureCounts to count reads, and edgeR (qlf model) for differential expression and statistical analysis. To select significantly altered genes, we first set a false discovery rate (FDR) of P < 0.05 and fold change > 1.5. To remove genes with background expression levels, the average transcripts per kilobase million (TPM) in one of the groups must be > 0.5. Genes meeting the criteria for both analysis pipelines (CLC and Hisat2-featureCounts-edgeR) were considered as significantly differentially-expressed genes. In addition, noncoding RNAs were removed before gene ontology (GO) analysis using Metascape (http://metascape.org/gp/index.html#/main/step1). The raw datasets of the Tlr3 and Tlr7 target genes have been deposited online . Up- and down-regulated target genes of TLR3 and TLR7 are summarized in BMDMs were treated with poly(I:C) or CL075 for 6 h. RNA-seq was performed as described previously , 25. Brit/tTlr3, t/yTlr7, and t/tTlr7 mice at postnatal day 2 (P2). The cells were cultured for 1 to 2 weeks in DMEM containing 10% fetal calf serum (FCS), penicillin and streptomycin. For BMDMs, bone marrow cells were collected from tibias and femurs of adult WT, t/tTlr3, t/yTlr7, and t/tTlr7 mice. The cells were then cultured in DMEM containing 10% fetal bovine serum (FBS), penicillin and streptomycin, and 50 μM β-mercaptoethanol plus 20% conditioned medium of L929 cells for 6 days. Poly(I:C) , CL075  and R848  were used to treat cells for TLR3 and TLR7 activation.Neuronal culture containing both hippocampi and dorsal cortices was prepared at mouse embryonic day E16.5-17.5 as described previously , 22, 26.Tlr3 cDNA encoding TLR3 protein (a.a. 1-905) was amplified by using the primer pair Tlr3-1Fw 5’ GGGATCCATGAAAGGGTGTTCC 3’ and Tlr3-2715Rv 5’ GCTGGTCGACATGTGCTGAATTCCG 3’, and then subcloned into pGw1-cMyc vector at the BglII and SalI sites to generate a TLR3-Myc expression construct. The mouse Tlr7 cDNA encoding TLR7 protein (a.a. 1-1050) was amplified by using the primer pair Tlr7-1Fw 5’ GGGATCCGCCACCATGGTGTTTTCG 3’ and Tlr7-3150Rv 5’ GCTGGTCGACGACTGTTTCCTTGAAC 3’, and then subcloned into pGw1-cMyc vector at the BglII and SalI sites to generate a TLR7-Myc expression construct. All software used in this report is listed in Detailed information on the antibodies used in this report is summarized in t/tTlr3 and t/yTlr7 mouse brains and spleens were harvested and homogenized using a dounce homogenizer (20 passes) in ice-cold Tris-buffered saline , 150 mM NaCl, 1 mM β-glycerophosphate, 1 mM PMSF, 1 μg/ml Aprotinin, 1 μg/ml Leupeptin and 1 μg/ml pepstatin). Proteins were further extracted by adding 0.5% Triton X-100 and gentle mixing at 4°C for 30 min. After removing cell debris by centrifugation, 250 μg lysate was incubated with 1 μg TLR3 (PaT3), TLR7 (A94B10), or HA antibody and 10 μl protein A/G slurry (1:1) at 4°C for 2-3 h. The resulting protein A/G resin was washed three times with TBS containing 0.5% Triton X-100 and then twice with 50 mM Tris (pH7.4). The IP complex was eluted by adding 1X SDS-sample buffer and boiling for 5 min. The samples were then subjected to immunoblotting analysis. To examine TLR7 signaling complex in BMDMs, WT and t/yTlr7 BMDMs were treated with 0.5 μg/ml R848 for 30 and 60 min at 37°C. After rinsing with cold PBS, the BMDMs were lysed with cold TBS containing 0.5% Triton X-100 and kept on ice for 10 min. After centrifugation, the BMDM extract was then used to carry out IP using TLR7 antibody (A94B10), as described above. Immunoblotting was performed as described previously (For immunoprecipitation (IP), WT, eviously . All fulImmunostaining of BMDMs and mixed glial cells was performed as described previously . Immunoft/tTlr3 mice received an intraperitoneal injection of saline or 5 mg/kg poly(I:C). Six hours later, mouse spleens were harvested for total RNA extraction using TRIzol according to the manufacturer’s instructions (Invitrogen), followed by DNase I digestion (New England BioLabs). Reverse transcription and quantitative RT-PCR analysis was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche) with an oligo(dT)18 primer and the Universal ProbeLibrary probes  system. Primer sets and probe numbers for selected genes from the results of RNA-seq and internal control Hprt are listed in Hprt measured at the same time on the same reaction plate. Brain and spleen of adult WT and t/yTlr7 mice were also harvested and subjected to RNA extraction to enable quantitative RT-PCR analysis of Tlr7 and Tlr8 expression levels.To detect gene expression in BMDMs and glial cells, primary cell cultures were treated with 10 μg/ml poly(I:C) or 4 μM CL075 for 6 h before harvesting using TRIzol reagent. To quantify gene expression in spleen, adult WT and post hoc Bonferroni correction was applied. For two-factor experiments, two-way ANOVA with post hoc Bonferroni correction was used. Data are presented as mean ± SEM. P values < 0.05 were considered significant.Statistical analyses were performed using GraphPad Prism 8.0 software. Experiments were performed blind by relabeling the samples with the assistance of other laboratory members. The majority of our data met the assumptions  for statistical tests. For two-group experiments, an unpaired t-test was used. For experiments with more than two groups, one-way ANOVA with Our previous study has indicated that TLR3 and TLR7 regulate expressions of distinct downstream genes to control cell morphology in neurons , 22. SinNext, we performed Ingenuity Pathway Analysis (IPA) to further dissect the involvement of common target genes in canonical signaling pathways   or t/tTlr7  mice .To overcome the antibody issue and to allow us to investigate expression and functions of the TLR3 and TLR7 proteins in vivo and in vitro.Next, we examined the expression and distribution of TLR3 and TLR7 in our Myc-HA-tagged knockin mouse lines using anti-HA and anti-Myc tag antibodies. Hereafter, we denote these dual-tagged TLR3 and TLR7 proteins as TLR3-MH and TLR7-MH, respectively. We tested commercially available anti-HA tag antibodies—namely rabbit monoclonal C29F4, rat monoclonal 3F10 and mouse monoclonal 16B12 antibodies—as well as one anti-Myc tag antibody , to detect these recombinant proteins  treatment, but the proteolysis and glycosylation patterns were different from that in poly(I:C)-treated BMDMs  treatment significantly increased levels in t/tTlr3 mice of Tlr3 RNA as well as of different cytokines, including Il-6, Tnfα and Ifnβ  stimulation. First, we intraperitoneally injected poly(I:C) or vehicle control into and Ifnβ 2. Consit/yTlr7  and/or t/tTlr7  mice. Similar to our observations for TLR3-MH, we detected FL and the cleaved CTF fragment of TLR7-MH proteins in different tissues and BMDM and glial cell primary cultures via immunoblot analyses  and MYD88 antibodies. We found that only WT TLR7 coimmunoprecipitated with MYD88 , Tir domain-containing adaptors (except Sarm1), and inflammation-related transcriptional factors (except Irf3)  but not Trif. For TLR7 activation, both Myd88 and Trif were increased around 1 to 2 fold , which triggered subsequent expression of cytokines and chemokines. Thus, TLR3-MH is a functional protein and our Tlr2, Tlr4, Tlr5, Tlr7 and Tlr9 with a view to assessing TLR expression , and mouse BMDMs , 43, 44,https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE163347.Data is available at The animal study was reviewed and approved by Academia Sinica Institutional Animal Care and Utilization Committee.Conceptualization, C-YC, T-FW and M-ZL and Y-PH. Methodology and investigation, C-YC, Y-FH, C-YT, Y-CS, T-FC, T-FW and M-ZL. Writing, C-YC, Y-FH, and Y-PH. Funding acquisition, C-YT and Y-PH. Supervision and project administration, Y-PH. All authors contributed to the article and approved the submitted version.This work was supported by grants from Academia Sinica .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lujo virus (LUJV), a highly pathogenic arenavirus, was first identified in 2008 in Zambia. To aid the identification of effective therapeutics for LUJV, we developed a recombinant reporter virus system, confirming reporter LUJV comparability with wild-type virus and its utility in high-throughput antiviral screening assays. Using this system, we evaluated compounds with known and unknown efficacy against related arenaviruses, with the aim of identifying LUJV-specific and potential new pan-arenavirus antivirals. We identified six compounds demonstrating robust anti-LUJV activity, including several compounds with previously reported activity against other arenaviruses. These data provide critical evidence for developing broad-spectrum antivirals against high-consequence arenaviruses. Lujo virus (LUJV), the first highly pathogenic arenavirus identified in Africa in over 40 years, was recognized after a cluster of severe viral hemorrhagic fever (VHF) cases in Southern Africa in September and October of 2008 ,2. The iMammarenavirus, Reptarenavirus, and Hartmanivirus [Mammarenavirus species in both these groups can cause severe VHF disease in humans. Highly pathogenic Old World complex viruses such as LUJV and Lassa virus (LASV) are found in Africa, whereas the New World pathogens such as Junin (JUNV), Guanarito (GTOV), Machupo, and Chapare viruses are found in South America [The family Arenaviridae encompasses three genera: anivirus . With thanivirus ,5. Infecanivirus . Mammareanivirus . Several America . Treatme America , althoug America . While c America .Here, we describe the development and rescue of a novel recombinant ZsGreen1 (ZsG)-expressing reporter LUJV, termed rLUJV/ZsG. By incorporating rLUJV/ZsG into a high-throughput antiviral compound screening assay, we were able to rapidly evaluate potential therapeutic candidates that were effective against LUJV. A panel of compound candidates was selected based on: (1) demonstrated activity against other arenaviruses; (2) demonstrated activity against other negative-sense RNA viruses; and (3) preliminary data from large in-house screening assays against multiple virus species. Of the 83 compounds tested, 20 demonstrated robust anti-LUJV activity; 6 were further confirmed to potently inhibit wild-type (wt) LUJV infection. Using LUJV in these assays provides critical support for antiviral activity of previously identified and novel compounds against re-emerging and emerging highly pathogenic arenaviruses.All work with the infectious virus was conducted in a biosafety level 4 (BSL-4) laboratory at the Centers for Disease Control and Prevention  following established BSL-4 standard operating procedures approved by the Institutional Biosafety Committee. All recombinant virus work was approved by the Centers for Disease Control and Prevention Institutional Biosafety Committee.v/v) fetal calf serum, non-essential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin.Vero-E6, Huh7 , and BSR-T7/5 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% (50) assays, using either immunofluorescence (wt virus) or ZsG expression (reporter virus) [Recombinant reporter LUJV expressing the green fluorescent protein ZsGreen1 (rLUJV/ZsG) was generated using a previously described reverse genetics system . Brieflyr virus) .RNA was extracted from cell culture supernatants (clarified by low-speed centrifugation) using the MagMAX-96 Total RNA Isolation Kit  on a 96-well Roche MagMAX extraction platform with a DNase-I treatment step according to manufacturer’s instructions. RNA sequencing was performed using KAPA RNA HyperPrep kit  for library preparation and analyzed on the MiniSeq system .4 cells/well in a 96-well plate 16–20 h prior to treatment with compounds diluted in dimethyl sulfoxide . For experiments measuring fluorescence reduction, 1 h post-treatment, cells were infected with rLUJV/ZsG ; 72 h post-infection (hpi), ZsG fluorescence was determined using a BioTek Synergy reader . For experiments measuring the reduction in wt virus titers, 1 h post-treatment, cells were infected with LUJV ; 72 hpi, supernatants were collected and viral titers determined by TCID50 in Vero-E6 cells. Cell viability (ATP content) was determined in parallel in compound-treated, mock-infected cells using CellTiter-Glo 2.0 . All experiments were performed in quadruplicate and repeated at least three times.Compounds were obtained from Selleckchem , Tocris , or MedChemExpress , with the exception of favipiravir, which was obtained from BOC Sciences . Huh7 cells were seeded at 1 × 10v/v) for 10 min, and blocked with PBS with 5% bovine serum albumin (v/v) for 30 min. Primary antibodies were α-LUJV HMAF , and secondary antibodies were labeled with AlexaFluor488 .ZsG fluorescence in infected cells was imaged directly using an EVOS digital inverted microscope . For immunofluorescence, cells were fixed in 10% formalin for 30 min, permeabilized in phosphate-buffered saline (PBS) containing 0.1% Triton-X100 (50) were interpolated. Cell viability was similarly calculated in compound-treated, mock-infected cells to determine the 50% cell cytotoxicity concentration (CC50) of each compound. The selectivity index was calculated by dividing CC50 by EC50. Data analysis was performed using GraphPad Prism v9 . Suitability for high-throughput screening was determined using the Z prime (Z′) score, a measure of statistical effect size, calculated as Z′ = 1 − (3 × (σP + σN)/|µP − µN|, where σP = standard deviation of positive control, σN = standard deviation of negative control, µP = mean of positive control, and µN = mean of negative control. Z′ values between 0.5 and 1.0 were considered acceptable. Signal-to-noise ratio was determined as (µP − µB)/σB, where µP = mean signal of positive control, µB = mean background signal, and σB = standard deviation of background signal. Significance was calculated using a one-sample t-test.ZsG fluorescence values for compound-treated infected cells were normalized to mock-treated (DMSO only), infected cells and used to fit a 4-parameter equation to semi-log plots of the concentration-response data. From this, the compound concentrations inhibiting ZsG expression by 50% (ECLUJV contains two ambisense genome segments termed large (L) and small (S) with respect to their relative nucleotide length A. Each gPrevious attempts to generate mammarenaviruses expressing reporter plasmids have either incorporated additional genome segments, as done with tri-segmented recombinant lymphocytic choriomeningitis virus (LCMV) , or intrTo establish the suitability of rLUJV/ZsG as a surrogate for wt LUJV, we first investigated viral growth kinetics. Huh7 or Vero-E6 cells were infected with either wt LUJV or rLUJV/ZsG at MOI 0.1, and titers were determined at 12, 24, 48, 72, and 96 hpi. In both cell lines, growth kinetics of wt LUJV and rLUJV/ZsG were comparable at each time point assessed. In cells infected with rLUJV/ZsG, strong ZsG expression was observed in monolayers of both cell types at 96 hpi C. To ass50 value of 11.13 ± 1.4 µM and CC50 of >50 µM , with cell viability confirmed concurrently . Twenty The 20 compounds demonstrating robust anti-LUJV activity can be broadly grouped based on known or predicted modes of antiviral action as: (1) inhibitors of viral replication; (2) inhibitors of viral entry, intracellular trafficking, or virus egress; (3) protein kinase inhibitors; (4) selective estrogen receptor modulators (SERMs); or (5) miscellaneous compounds not falling into the four previous groups.50 values > 50 µM, and EC50 values of 0.54 ± 0.21 µM and 2.95 ± 0.69 µM, respectively. 2-dFC has shown similar potency against LASV [50 values of 0.20 ± 0.09 µM and 0.24 ± 0.1 µM, respectively, although MPA’s CC50 value of 2.0 ± 0.4 µM resulted in a reduced SI of 8.6. MPA has previously demonstrated antiviral effects against a wide range of RNA viruses including influenza [50 0.109 ± 0.02 µM, CC50 > 12.5 µM, SI > 115), a selective inhibitor of the enzyme dihydroorotate dehydrogenase. This compound is believed to act by blocking de novo pyrimidine biosynthesis, and recent studies have shown brequinar inhibits other arenaviruses such as LASV, JUNV, and LCMV [Some of the most potent anti-LUJV compounds and those with the highest SI in our screens were compounds predicted to target viral replication . The nucnst LASV , the nainst LASV , and thenfluenza , dengue nfluenza , Zika vinfluenza , CCHFV [nfluenza , and hannfluenza . AVN-944nfluenza . The besand LCMV , as welland LCMV  and HIV-and LCMV .50 0.085 ± 0.029 µM, CC50 0.272 ± 0.034 µM, SI = 3.2) with a sub-micromolar EC50 value  demonstrated greater inhibition than isavucanazole . Interestingly, the niclosamide values for rLUJV/ZsG inhibition are very similar to the reported EC50 of 0.08 µM for LASV inhibition in a pseudovirus screening assay; however, the same study reported an EC50 of 0.05 µM for apilimod against LASV, which is considerably lower than EC = 1.44 µM for rLUJV/ZsG [50 1.2 µM, CC50 > 30 µM) in an HIV-luc pseudovirus screen, but did not inhibit LUJV in the same screen [50 4.90 ± 0.51 µM, CC50 13.63 ± 1.51 µM, SI = 2.8) is a cationic amphiphilic drug (CAD) that can accumulate within acidic intracellular vesicles, inhibiting membrane fusion and ribonucleoprotein release [The compounds apilimod, niclosamide, and isavucanazole have all been shown to inhibit LASV entry in vitro ,32. Of t50 value A. Of theLUJV/ZsG . Isavuca release . It can  release , and has release , SARS-Co release , and hep release . While t50 0.60 ± 0.27 µM, CC50 9.91 ± 1.09 µM) performed the best, with an SI value of 16.6. AR-12  and afatinib  both inhibited rLUJV/ZsG, but their cytotoxicity reduced their SI values compared to BX-795   demonstrated a similar EC50 value, but was associated with significant cytotoxicity, which reduced the SI values. Both tamoxifene citrate  and toremifene citrate  inhibited rLUJV/ZsG, but the associated cytotoxicity again led to a very narrow window of antiviral effect.Four of the compounds in the screen were SERMs, a class of compounds that are also CADs and have antiviral effects against several viral species, including EBOV . The SERI = 6.2) A. Bazedo50 5.14 ± 1.52 µM, CC50 > 25, SI > 4.9) is a neurotransmitter inhibitor (anticholinergic); clemastine fumarate  acts primarily as an H1 histamine antagonist; and loperamide HCl  is an opioid receptor that targets μ-opioid receptors in the large intestine and acts as an antidiarrheal agent by decreasing intestinal movement. All three were able to moderately inhibit rLUJV/ZsG and had SI values between 3 and 5. The final compound, obatoclax  inhibits the Bcl-2 family of proteins and has been investigated as an experimental anti-cancer drug. It inhibited rLUJV/ZsG at sub-micromolar EC50 value, but was cytotoxic at a relatively low concentrations, which reduced SI to 4.2.Four of the compounds selected from the initial screen did not fit into the four previous groups based on predicted mode of action B. Three 50 value), with cell viability determined concurrently. All compounds evaluated inhibited wt LUJV titers in a concentration-dependent manner, and all compounds reduced viral titers by approximately 4 logs while retaining >75% cell viability  were selected to be screened using wt LUJV; these compounds were 2-dFC, apilimod, AVN-944, brequinar, BX-795, and favipiravir. These six drugs were tested using a shortened concentration curve , which has previously been shown to inhibit LASV, JUNV, and LCMV at similar sub-micromolar EC50 values [Given the genetic diversity among species, the large number of different arenavirus host reservoirs, and the geographic range of these pathogens, the emergence of new arenaviruses pathogenic to humans remains an ongoing public health concern . Importa0 values .50 8.1 µM) [50 16.6 µM), and SARS-CoV (EC50 21.6 µM) [50 5.4 µM) [50 0.95 µM), and SARS-CoV-1 (EC50 6.6 ± 1.48 µM) [There is notable value in identifying efficacious compounds that have previously been approved for clinical use. Several of the compounds with anti-LUJV efficacy are already approved for other indications. Benztropine (Congentin) is used as an anti-tremor medication to treat Parkinson’s disease, and has also been shown to have mild antiviral effects against EBOV (EC 8.1 µM) , MERS-Co21.6 µM) . Clemast 5.4 µM) , SARS-Co1.48 µM) . While n1.48 µM)  as well 1.48 µM) . The abiThe six most promising candidates with both high efficacy and low cytotoxicity fell within three broadly defined groups based on modes of action: inhibiting viral replication; viral entry and trafficking; or protein kinases. The majority (four out of six) of these with the highest SI values (and therefore the most promising therapeutic candidates) were inhibitors of viral replication. Several other compounds that were identified demonstrated potent anti-LUJV activity, but had associated cytotoxicity, leading to low SI values and a narrow therapeutic window. Although this would likely make these compounds unattractive as single-drug treatment options, combination therapy using compounds with distinct modes of action may synergistically inhibit LUJV ,27,55,56Here we describe the development of a recombinant LUJV engineered to express the fluorescent protein ZsGreen1 and validate its use in high-throughput screening assays for large or complex screens containing multiple compounds at various concentrations. We identified several highly efficacious anti-LUJV compounds with multiple predicted modes of action. Future studies are planned to investigate if potential synergistic actions increase the effectiveness of these compounds, as well as to advance the most promising candidates to studies using the available animal models of disease to evaluate in vivo effectiveness. Given the genetic diversity of mammarenaviruses and the high-consequence nature of their infections, it is promising to report that newly emerging species such as LUJV are similarly inhibited by several previously reported compounds. Identifying pan-arenavirus inhibiting compounds serves as a key step in the preparedness against new and emerging pathogenic threats.


Overall, 1 is chemically stable compared to parent 14-3-3 PPI inhibitors, and thus emerged as a confirmed hit for further development.14-3-3 are regulatory proteins that through protein–protein interactions (PPI) with numerous binding partners could be involved in several human diseases, including cancer, neurodegenerative disorders, and pathogens infections. Following our research interest in the development of 14-3-3 PPI inhibitors, here we exploited the privileged 4-aminoantipyrine scaffold in the design and synthesis of some derivatives endowed with antiproliferative activity against K-562 cells, and capable of binding to recombinant 14-3-3σ as evidenced by NMR spectroscopy. The binding mode was further explored by molecular modelling, while coupling confocal microscopy with intensitometric analysis showed that compound  To date, more than 500 partners of 14-3-3 have been identified through biochemical and bioinformatics studies, most of which are disease-relevant phosphorylated proteins sharing the optimal binding sequence R(X)XpS/TXPBV02, BV02 has been used as tool compound in multiple biological studies aimed at investigating the pharmacological inhibition of 14-3-3 PPI. Notwithstanding, the molecule suffers from chemical instability in aqueous buffer, which leads to the phthalimide derivative 9 by means of a hydration/dehydration pathway  pathway 11. Thus,BV02/9 but endowed with a suitable chemical stability profile, here we designed and synthesised three derivatives of the 14-3-3 PPI inhibitor 9. Interaction with 14-3-3σ was monitored by NMR spectroscopy and molecular modelling, while the mechanism of action and anticancer potential were assayed in in vitro cancer cell models.To further shed light on the mechanism of 14-3-3 PPI inhibition by the privileged 4-aminoantipyrine scaffold, and to identify novel derivatives endowed with pharmacological properties comparable to 1–3 were synthesised using the synthetic route outlined in 9 was obtained in quantitative yield after stirring at room temperature (r.t.) for 72 h. Then, it was treated with AcOH and H2SO4 in the presence of tin powder, which selectively reduces the carbonyl group of the phthalimide moiety, to afford compound 1 in a high yield (79%). To afford compound 2, acid chloride was prepared by reacting compound 1 with oxalyl chloride and subsequently methylated by the addition of MeOH into the reaction mixture. Synthesis of compound 3 was completed after the reduction of the carboxylic acid3.Compounds 9, chemical stability at physiological conditions was remarkably improved by removing one of the carbonyl groups from the phthalimide moiety (compound 1), as observed by NMR spectroscopy  corresponding to that present in the highest concentration of the compounds. At the end point, cell number and viability were evaluated by automatic cell counter, and mean number of viable cells was expressed as percentage with respect to control cells  previously described for BV02 and compound 93 showed indeed the weakest antiproliferative activity.Compounds ol cells . Compoun 15.8 µM . Compare1–3 to the crystallographic structure of 14-3-3σ was investigated by molecular docking simulations, which were carried out as described previouslyThe possible binding mode of compounds 1–3 share a highly consistent binding mode, which is also comparable to that already described for the parent compound 9, with only a small difference in the orientation of the N-methyl group of the pyrazolone ring. This is indeed a consequence of the possibility to flip pyramidal N atoms in GOLD docking program and does not affect significantly the overall binding mode of 1–3.It is worth mentioning that compounds 1 and 2 to recombinant 14-3-3σ was preliminarily assessed by screening techniques such as fluorescence polarisation (FP) and surface plasmon resonance (SPR) (data not shown). However, results were difficult to interpret, most likely because of the relatively weak affinity of the molecules for 14-3-3σ. Thus, we decided to monitor the interaction between recombinant 14-3-3σ and compounds 1 and 2 through 1H NMR spectroscopy by running transferred nuclear overhauser effect (Tr-NOE) experiments. This approach is well established and relies on the slow tumbling of the ligand when it is bound to specific protein partners. In fact, ligand–protein interaction greatly enhances the efficiency of the proton-proton NOE cross relaxation between protons of the ligandThe direct binding of 1 and 2 have negative NOE correlations (positive NOEs enhancements) while 14-3-3-σ has positive NOE correlations (negative NOE enhancements), as highlighted in Because of their size, 1 and 2 changes from negative to zero, upon 14-3-3σ addition. This behaviour is consistent with the interaction between the two molecules and 14-3-3σ. The zero intensities of NOEs might be explained by considering the occurrence of positive NOE peaks due to the presence of small fractions of either 1 or 2 bound to 14-3-3σ.In the presence of the ligand–protein interaction, if the ligand off-rate is fast compared to the bound NOE cross relaxation rate, the Tr-NOESY signal intensities are the weighted average between the bound and free cross-peaks intensities1H spectra of either 1 or 2 does not reveal any significant variations after protein addition. In fact, the concentration of the protein-ligand adduct is too small to induce any perturbation in NMR signals. In addition, it is consistent with the results obtained by NOESY experiments that support the formation of complexes with fast ligand off‐rates and KD values > 1 μM,The analysis of 1 D BV02 inhibitor of 14-3-3/c-Abl interaction is able to promote c-Abl translocation into the nucleus and to provide antiproliferative effects also in CML cells expressing the Imatinib-resistant T315I Bcr-Abl construct1, using HeLa adenocarcinoma cells stably overexpressing EGFP-tagged c-Abl (HeLa EGFP-Abl) as a reporter system1, compared to control   , confirm1–3 showed antiproliferative effects against K-562 erythroleukemia cell line at low micromolar concentrations. Molecular modeling suggested a possible binding mode of these compounds that clearly emphasises the role of the carboxylic function. NMR spectroscopy further confirmed the direct binding of 1 and 2 to recombinant 14-3-3σ, while the most promising hit 1 was found to enhance the nuclear translocation of c-Abl in a dose-dependent manner by confocal microscopy and intensitometric analysis.Modulation of 14-3-3 proteins is a valuable tool to probe the regulatory functions of eukaryotic cells, and to develop effective therapeutic strategies against a number of human diseases. By exploiting the 4-aminoantipyrine scaffold of 14-3-3σ PPI inhibitors previously developed by our research group, here we designed and synthesised a number of derivatives endowed with enhanced chemical stability at physiological conditions. Particularly, compounds 1 as a valuable hit that is chemically stable at physiological conditions, and is suitable for further development.Overall, this study further confirms the relevance of the 4-aminoantipyrine scaffold in the inhibition of 14-3-3σ PPI, and highlights compound 1H NMR and 13C NMR were recorded at 400 and 100 MHz, respectively, on a Bruker AC200F spectrometer . Proton shift for 1H NMR are reported in parts per million  and internally referenced to the CDCl3 or DMSO signal, at 7.26 and 2.50 ppm, respectively. Chemical shifts for carbon are reported in parts per million  and referenced to the carbon resonances of the solvent (CDCl3 at δ 77.16 and DMSO at δ 39.52 ppm). Data are shown as following: chemical shift, multiplicity , coupling constant (J) in Hertz (Hz), and integration. Mass spectra (MS) data were obtained using an Agilent 1100 LC/MSD VL system (G1946C)  by direct injection with a 0.4 ml min−1 flow rate using a binary solvent system of MeOH:H2O (95:5). UV detection was monitored at 254 nm. MS were acquired in positive scanning mode over the mass range m/z 50–1500.All commercially available chemicals and solvents were used as purchased. THF was dried over sodium and benzophenone prior to use. Anhydrous reactions were run under positive pressure of dry nitrogen. TLC was carried out using Merck TLC plates silica gel 60 F254 . 2O (20 ml) was added and the solution was stirred 20 min until a white precipitate appeared. The white solid was filtered and washed with H2O and hexane.1,2,4-benzenetricarboxylic anhydride  and DMAP  were added to a solution of 4-aminoantipyrine  in DMF dry (15 ml), giving a yellow colour. The mixture was stirred at r.t. for 72 h. After this time, HYield: quantitative1H NMR  (ppm): 13.83 ; 8.43 ; 8.32 ; 8.10 ; 7.55 ; 7.40 ; 3.32 ; 2.26 .MS (ESI) m/z: 377.8 [M + H]9  was dissolved in a mixture of concentrated HCl (5 ml) and glacial acetic acid (5 ml). Sn  was added to the solution, and the mixture was stirred at 80 °C for 24 h before being cooled to r.t. and filtered through celite. The solution was extracted with AcOEt (30 ml × 3). The combined organic phases were washed with H2O and brine, and dried over anhydrous Na2SO4. The solvent was removed under vacuum to afford 92 mg of a white solid.Compound Yield: 79%1H NMR  (ppm): 8.22 ; 7.78 ; 7.51 ; 7.38 ; 4.87 ; 3.17 ; 2.23 . 13C NMR  (ppm): 167.13, 166.48, 161.55, 153.09, 147.40, 135.07, 133.13, 132.36, 131.39, 129.67, 127.25, 124.64, 124.36, 107.04, 51.09, 36.03, 11.67. MS (ESI) m/z: 363.8 [M + H], 385.8 [M + Na]1  in THF (4 ml) at 0 °C. The mixture was then stirred for 15 min, and anhydrous DMF (0.1 ml) was added dropwise. The solution was stirred for 1 h at r.t. and then cooled again to 0 °C. MeOH was added to the reaction and left under stirring for 15 min. The reaction was quenched by the addition of NaOH 2 N, and then extracted with AcOEt (15 ml × 3). The organic layer was washed with NaHCO3, brine, dried over Na2SO4 and evaporated under reduced pressure to obtain 49 mg of a brown oil. The mixture was purified by column chromatography (DCM/MeOH 95:5) to give a yellow solid.Oxalyl chloride  was slowly added to a solution of compound Yield: 47%1H NMR  (ppm): 8.23 ; 7.80 ; 7.51 ; 7.37 ; 4.87 ; 3.89 ; 3.16 ; 2.22 .13C NMR  (ppm): 166.72, 166.30, 161.44, 151.07, 146.88, 134.47, 132.92, 132.37, 130.41, 129.39, 129.26, 127.16, 125.47, 124.50, 123.18, 108.11, 52.34, 50.31, 35.86, 12.08. MS (ESI) m/z: 377.9 [M + H], 399.8 [M + Na]1  in dry DMSO. The mixture was stirred overnight at r.t. and then quenched with K2CO3. The solution was extracted with DCM (15 ml × 3). The combined organic layer wasw washed with HCl 1 N, brine, dried over Na2SO4 and concentrated.Borane dimethyl sulphide 1 M  was added dropwise to a solution at 0 °C of compound Yield: quantitative1H NMR  (ppm): 7.74 ; 7.59 ; 7.52 ; 7.38 ; 5.42 ; 4.77 ; 4.63; 3.16 ; 2.22 .13C NMR  (ppm): 167.42, 161.73, 153.18, 143.38, 141.43, 135.14, 131.83, 130.67, 129.65, 127.16, 124.53, 123.71, 121.35, 107.53, 62.98, 50.72, 36.09, 11.67. MS (ESI) m/z: 371.9 [M + Na]1H NMR spectra were recorded after dilution of 10 mg of compound 1 in a mixture of DMSO/D2O 70:30. The chemical shift variation of the aromatic protons was used to monitor the behaviour of 1 in solution. NMR spectra were recorded after 2 h and 24 h. The chemical identity of compound 1 was confirmed by mass spectrometry after 24 h in solution (MS (ESI) m/z: 363.8 [M + H], 385.8 [M + Na]).in vitro experiments on 14-3-3 PPI inhibitors were carried out using human erythroleukemia cell line K-5624 cells/ml, and treated with increasing concentrations of selected compounds. Control cells were treated with the vehicle of the experimental point containing the highest percentage of DMSO. Cell cultures were maintained at 37 °C in 5% v/v CO2 for 72 h. Cell number and vitality were evaluated on cell suspension using the automatic cell counter NucleoCounter® . Each experiment was performed at least three times and results were expressed as mean and standard deviation (±SD).As in a previous exercise, NMR spectra were performed at 14.1 T with a Bruker Avance 600 Spectrometer (Bruker) operating at controlled temperature (± 0.2 K) and using a 5 mm SEI probe. Chemical shifts were referenced to external 3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TMSP-d4). NOESY spectra were obtained by using standard pulse sequences. NOESY spectra were acquired with a mixing time of 320 ms.,,The crystallographic structure of 14-3-3σ in complex with a phosphopeptide coded by PDB ID: 1YWT was used as rigid receptor in molecular docking simulations, upon removal of the coordinates of the phosphopeptide and crystallographic water molecules− 1 G418  for 3 weeks. HeLa EGFP-Abl were maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 10,000 Uml−1 penicillin, and 10 mg ml−1 streptomycin and were constantly kept under selective pressure with 2 mgml−1 G418. Selective medium was replaced with regular growth medium on the day before experiments.HeLa cells stably expressing EGFP-Abl (HeLa EGFP-Abl) were obtained by transfection of HeLa cells with the pCEFL EGFP ABL wild-type expression vectorp values) was assessed by t-test. Asterisks were attributed for the following significance values: *****p < .0001.Cells were washed with PBS, then fixed with 4% paraformaldehyde in PBS for 20 min and permeabilised with 0.1% Triton X-100 in PBS for 30 min. Nuclei were stained with a solution of 6 μm of 4′,6-diamidino-2-phenylindole  in PBS for 10 min. Coverslips were mounted in fluorescence mounting medium . Samples were visualised on a TSC SP5 confocal microscope , installed on an inverted LEICA DMI 6000CS (10741320) microscope and equipped with an oil immersion PlanApo 40 × 1.25 NA objective. Images were acquired using LAS AF acquisition software . Intensitometric analysis of fluorescence was performed using the Quantitation Module of the Volocity software (PerkinElmer Life Science). Briefly, total nuclear EGFP fluorescence, defined as EGFP signal co-staining with DAPI nuclear dye, was measured in seven representative confocal fields for each experimental condition. The resulting mean values ± SEM are expressed as a percentage of nuclear EGFP fluorescence. Seven representative fields were acquired and analysed for each sample. Significance (6-tag) between the BamHI and NotI restriction site, was a kind gift from Prof. C. Ottmann . The expression plasmid was introduced by thermal shock in the Escherichia coli strain BL21(DE3). Transformants were selected on LB-agar plates added by 100 mgl−1 ampicillin. Bacterial cells were cultured in LB medium, supplemented with 100 mgl−1 ampicillin at 37 °C under vigorous aeration. When OD600nm reached values ranging from 0.6 to 0.8, protein over-expression was induced by adding 0.5 mM IPTG to the culture that was then incubated at 25 °C for 16 h (under vigorous aeration). Cells, harvested by centrifugation, were resuspended in buffer A  added by 20 mM imidazole and lysozyme (0.5 mg ml−1) and disrupted by sonication after 1 h of incubation on ice. The soluble cellular fraction, clarified by centrifugation , was purified by taking advantage of the N-terminal His6-tag using nickel affinity chromatography . The purification protocol relied on a three-step gradient imidazole concentration (in the same buffer) that showed elution of the His6-tag h14-3-3σ at imidazole concentrations ranging from 75 to 250 mM. Fractions containing the target protein (identified by SDS-PAGE) were pooled and dialysed in 3 mM Tris-HCl, pH 8 at 4 °C. The final protein yield resulted of 87 mg l−1 bacterial culture.The gene encoding for human 14-3-3σ (h14–3-3σ) cloned in the pPROEX-HTb vector (including a TEV-cleavable His


Derivative 8 revealed the maximum antitumor activity toward both tumor cell lines, with an SI value of about 26 and IC50 value of about 5.9 μg/mL. The effect of these derivatives  upon the expression of 5 tumor regulating genes was studied through quantitative real-time PCR, where its interaction with these genes was simulated through the molecular docking study. Furthermore, the antimicrobial activity results revealed that compounds 2, 7, 8, and 9 have a potential antimicrobial activity, with maximum broad-spectrum activity through compound 3 against the three tested pathogens: Streptococcus mutans, Pseudomonas aeruginosa, and Candida albicans. The newly prepared compounds also revealed anti-biofilm formation activity with maximum activity against Streptococcus mutans, Pseudomonas aeruginosa, and Candida albicans, respectively.The current study was directed toward developing a new series of fused heterocycles incorporating indazolylthiazole moiety. The newly synthesized compounds were characterized through elemental analysis and spectral data . The cytotoxic effect of the newly synthesized compounds was evaluated against normal human cells (HFB-4) and cancer cell lines (HepG-2 and Caco-2). Among the synthesized compounds, derivatives  Recent studies have shown that hepatocellular carcinoma and colorectal cancer are among the most common and fatal cancer types3. Hepatocellular carcinoma (HCC) is a worldwide health care issue that accounts for a high rate of mortality4. Many factors are associated with HCC development, including chronic infections with hepatitis viruses (hepatitis B and C), aflatoxin exposure, obesity, prolonged and/or heavy alcohol consumption6. Currently, the only FDA-approved medication for HCC treatment is Sorafenib (a multikinase inhibitor), which can only extend patient survival for a brief period7. Though the extrahepatic invasion of HCC outside liver cells is uncommon, growing evidence for the rare colon metastasis with HCC is treated only with surgical resection8. In the same context, colorectal cancer (CRC) characterized by a high mortality rate, is the second leading cause of death among cancer-affected patients9. The current procedures applied for cancer prevention and treatment including radiation, chemotherapy, and surgery are usually combined with immunosuppression for treated patients, which makes them susceptible to microbial infections10. In addition, none of the applied treatments satisfy the requirements for selectivity and high efficacy toward cancer cells and posttreatment side-effects are many and serious. As a result, ongoing research to develop highly selective new antitumor candidates with effective antimicrobial properties is a pressing demand toward an effective treatment for cancer patients11.Cancer, one of the fundamental challenges for human health, represents the second causative agent of human mortality, following cardiovascular diseases12. Heterocyclic derivatives, which contain at least two different elements, have received a great deal of attention in the development of pharmacologically active molecules and advanced organic materials14 and account for more than 75% of the materials currently used clinically15. Due to their broad biological and pharmacological applications, Sulfur, nitrogen, and/or oxygen-containing heterocyclic compounds such as thiophene and pyrazole are always of interest to medicinal chemists and researchers18. Thiophene has been dubbed the "wonder heterocycle" due to its diverse biological activities, which include anticancer21, antimicrobial24, antioxidant25, and anti-inflammatory26. The name “thiophene” was derived from the Greek words ‘theion’ for sulfur and ‘phaino’ for shining27. Tiquizium Bromides, Tipepidine, Tioconazole, and Citizolam among many marketed pharmaceutical drugs that contain thiophene nucleus32. Nevertheless, thiophene's low water solubility, combined with its hepatotoxicity, limited its wide widespread application and even forces the removal of many thiophene-containing medicines from the drug market12. On the other hand, Pyrazole is well known as a five-membered heterocyclic compound that has two neighboring nitrogen atoms, C3H3N2H. Pyrazole derivatives have been used for many years in agrochemicals as herbicides33 and in the pharmaceuticals field as antimicrobial34, anti-inflammatory35, anticonvulsant37, and anticancer38, with pyrazole's recent application as a selective analgesic and anti-inflammatory drug (COX-2 inhibitor) attracting more attention in medicinal chemistry33. With the recent advances in molecular hybridization strategies, new structures could be developed toward a novel antitumor drug with lower cytotoxicity. This strategy is based upon combining pharmacophoric moieties of various bioactive compounds to develop a new potent hybrid compound with higher activity compared to the parent’s structures39. According to the preceding information, the present study concerns the synthesis and characterization of novel furan, thiophene, pyrazole, pyran, and pyridine derivatives from indazolylthiazolidinone moieties. In addition, their biological potential, including anti-tumor and anti-microbial activities, was evaluated.In the last few decades, anticancer drugs have been developed from many chemically synthesized compounds11. The molecular hybridization strategy of combining the active moieties of different compounds represents a promising tool for developing new structures with higher biological activity than their starting precursors39. In order to continue our search for novel heterocyclic chemistry-based anticancer agents, we used indazolylthiazolidin-4-one (1), which was prepared using the reported procedure by reacting indazole-2-carbothioamide and chloroacetic acid in dimethylformamide40. Reactivity of the thiazolidine moiety was tested by the reaction of compound 1 with different reagents, as depicted in    in compound (1) in their IR spectra. The 1H-NMR spectra of 5, and 6 in DMSO-d6 are characterized by singlet signal in the regions of 6.95 ppm and 7.80 ppm, which indicates the presence of thiophene moiety NH2 protons exchangeable with D2O. In addition to the characteristic singlet signal assigned to hydrazide NH proton at 9.60 ppm, and another singlet signal at 4.30 ppm for hydrazide NH2 protons exchangeable with D2O) in case of compound 6  into the corresponding derivatives 5, and 6.The proposed mechanism and pathway for conversion of indazolylthiazolidin-4   and 4 (18.1 for HepG-2 and 23 for Caco-2), as indicated in Table 8 could be attributed to a thiazolyl–pyrazole moiety, which per the literature exhibits strong anticancer activity47. In addition, the presence of acetonitrile, incorporated into the pyrazole ring (at position 3), may reveal anticancer activity, as reported through other authors45.The antitumor activity of the prepared derivatives was evaluated in vitro against HepG-2 (hepatoma) and Caco-2 (colon cancer) cells in comparison with normal human HBF-4 cells. Herein, the IC4, 6, and 8) at different concentrations (4–16 μg/mL) were studied in a live-cell mode by inverted microscopy, and the effect of these derivatives on the tested tumor cells was elucidated. The obtained images show that compounds 4, 6, and 8 enhance obvious cell damage and stimulate a clear alteration of the cell morphology in a dose-dependent way  on five tumor regulating genes  was evaluated in both Caco-2 and HepG-2 cells using qRT_PCR and compared to 5-FU as a standard antitumor drug. Compounds 4, 6, and 8 significantly downregulated the gene expression of VEGF, MMP-9, and Bcl-2 compared to 5-FU and untreated cells  showing that the applied derivatives induce apoptosis in cancer cells.The β-catenin gene plays a major role in cell–cell adhesion in normal cells. Expression of this gene is under a strict regulation mechanism, where its overexpression is a main characteristic of cancer cells4, 6, and 8 for 48 h. Figure 8. Our significant results specify that the synthesized derivatives enhanced the cell cycle arrest of treated cells in both sub-G1 and S phases compared with untreated control.Arresting of cell cycle phases was studied for the treated Caco-2 cells with the most potent compounds to gain insight into their potential cellular mechanism to induce the anticancer effect. For this target, Caco-2 cells were treated with compounds 51. To this end, the interaction and affinity of the newly prepared potent anticancer compounds  toward five proteins, MMP-9, p53, β-catenin, Bcl-2, and VEGF, were simulated through molecular docking. The proteins were chosen based on the effect of the new derivatives on the expression levels of several tumor-regulating genes, including MMP-9, p53, β-catenin, Bcl-2, and VEGF, as demonstrated in the biological discussion. The PDB IDs of these proteins used in the docking study are 4XCT, 3ZME, 1JDH, 2W3LL, and 2XAC. The crystal structures were downloaded and prepared for docking of our compounds . We can see three essential residues to which the ligand binds. Figure 6 was found to be the most promising candidate. It exhibited a correct binding mode. In addition to its ability to form a hydrogen bond with the essential amino acid residue Asp228, it showed two further hydrogen bonds with Pro151 and Cys220 , the ligand showed one hydrogen bond with Asp228 and two arene-H interactions with Thr230 and Cys229. Compound 20 Figs. , 8. It a53. Hence these residues are effective residues to which the inhibitor should bind. Our compounds showed interactions with more than one essential residue with binding energies ranging from − 5.2 to − 5.9 kcal/mol. Hence they are expected to be effective inhibitors of β-catenin. Compound 8 was the best as it showed interactions with three effective residues; Lys435, Arg474, and His470. It formed hydrogen bonds with Lys435 and Arg474 and an arene-H interaction with His470. Moreover, it further exhibited a hydrogen bond with Asn430 and an arene-H interaction with Arg515 , it was reported that the β-catenin residues His260, Asn261, Lys292, Ile296, Asp299, Tyr306, Gly307, Lys312, Lys335, Lys345, Arg376, Arg386, Asn387, Asn426, Cys429, Lys435, Cys466, His470, Arg474, and Lys508 are the residues that interact with TCF4 to form a complex4, 6, and 8) are not able to bind to this protein effectively. Finally, the crystal structure of VEGF (PDB ID 2XAC) suggests that Gln27 is one of the effective residues involved in the binding of VEGF to VEGFR54. Compound 4, as well as compound 6, showed a hydrogen bond to Gln27. Compound 6 also demonstrated a further hydrogen bond with Gln55 and arene-H interaction with Pro28 , our compounds showed a binding manner that differed from that of the ligand. The ligand formed a hydrogen bond with Tyr67 to which none of our compounds bound, indicating that it is more likely that the studied compounds  was assessed against three pathogens, including Streptococcus mutans, Pseudomonas aeruginosa, and Candida albicans using a microliter-plate assay. The MIC results , comparable to that of the ampicillin MIC (13.5 µg/mL). For Gram-negative bacteria (Pseudomonas aeruginosa), derivatives 2 and 7 revealed moderate activities with maximum inhibition through derivative 3 with a MIC of 18.29 µg/mL, which is nearly the same as the applied reference ciprofloxacin MIC of 18.7 µg/mL. The newly prepared compounds revealed low to medium antifungal activity against Candida albicans with a maximum antifungal activity through compound 3 (MIC of 40.74 µg/mL), representing 36% clotrimazole activity. In the scope of this study, among the newly peppered series, compound 3 revealed potent broad-spectrum antibacterial activity with moderate antifungal activity. The broad-spectrum antibacterial activity of compound 3 may be attributed to the incorporated thiazolopyridine moiety, which is accordant to56. Khidre and Radini, reported the broad-spectrum potency of the thiazolopyridine nucleus and its derivative against many human pathogens, hypothesized that, the possible mechanisms of the thiazolopyridine nucleus antimicrobial activity is through targeting and inactivating vital microbial enzymes such as DNA gyrase57.The prevalence of drug-resistant pathogens highlights the need for novel antimicrobials with lower resistance induction potentialts Table  illustra58. As a result, compounds that interfere with biofilm formation improve microorganisms' susceptibility to therapeutic drugs. To this end, the efficacy of the newly prepared compounds (2–9) was evaluated for inhibition of microbial-biofilm formation using the TCP technique. The results , 5 (68%), 6 (61%), and 7 (66%) with a maximum biofilm inhibition recorded through compound 9 of nearly 79%.One of the key pathogenicity mechanisms for microbial pathogens is microbial-biofilm formation, which supports the persistence of infectionlts Fig.  revealedd 3 Fig. , whereas1H-NMR and 13C-NMR spectra were recorded on a Bruker 300 spectrometer, using DMSO as the solvent and tetramethylsilane (TMS) as an internal reference. Mass spectroscopy was carried out using direct inlet unit (DI-50) of Shimadzu GC/MS-QP5050 A.The melting point ranges were taken on a Gallenkamp electric melting point apparatus using the one-end open capillary method and were uncorrected. IR spectra were recorded (KBr discs) on a Shimadzu FT-IR 8201 PC spectrophotometer.1  containing a catalytic amount of triethylamine, and sulphanilamide  was heated under reflux for 12 h, cooled to room temperature (RT), and acidified by HCl. The solid formed was filtered off, washed with water, and purified by recrystallization from petroleum ether to give the required product 2 as a yellow powder, in a 53% yield; m. p: 120–123 °C; IR (KBr): ν (cm−1) 1154 SO2, 1598C=C, 1627C=N, 2933, 2857CH-Al, 3025CH-Ar, 3377, 3200 cm−1 of NH2; HRMS (m/z%): [M]+ calcd. for C22H23N5O2S2 453(100); found 453(14.16); analysis : C, H, N, S.An ethanolic solution of compound (2)  and arylidenemalononitrile  in ethanol (20 mL) containing a catalytic amount of triethylamine (0.5 mL) was heated under reflux for 12 h, cooled to RT, and acidified by HCl. The solid formed was filtered off, washed with water and purified by recrystallization from petroleum ether to give product 3 as a brown powder, with a 73% yield; m. p: 140–143 °C; IR (KBr): ν (cm−1) 1156 SO2, 1628 C=N, 2198 CN, 2933CH-Al, 3025CH-Ar, 3340, 3214 NH2; 1H-NMR : δ1.61–2.49 , 5.35, 7.27–7.90, 8.40; 13C-NMR : δ22.51, 25.32, 27.45, 112.70, 118.02, 122.00, 125.91, 126.66, 127.62, 128.26, 128.57, 128.70, 129.34, 138.05, 142.11, 154.35, 118.02, 161.50, 164.05; analysis : C, H, N, S.A mixture of 4-thiazol-4-yl)amino) benzenesulfonamide 1  and malononitrile  in dioxane (30 mL) containing a catalytic amount of triethylamine (1 mL) and/or in ethanol (25 mL) containing a catalytic amount of piperidine (0.5 mL) was heated under reflux for 5 h, cooled to RT, and acidified by HCl. The solid precipitate was filtered off, washed with distal water (3 times) and recrystallized from dioxane to give compound 4 as a brown powder in a 52% yield; m. p: 130–133 °C; IR (KBr): ν (cm−1) 1553C=C, 1627C=N, 2191 CN, 2935CH-Al, 3058CH-Ar, 3333, 3201NH2; 1H-NMR : δ1.66–2.49, 4.09, 6.87  exchangeable with D2O, 7.20–7.74, 8.15; 13C-NMR : δ21.25, 22.04, 25.84, 26.60, 98, 108, 115.53, 120.60, 126.82, 127.40, 128.23, 129.01, 129.40, 130.18, 142.93, 162.05; analysis : C, H, N, S.A mixture of derivative 1  containing a catalytic amount of piperidine (0.5 mL), malononitrile , and Sulphur element  was heated under reflux for 4 h, filtered off on hot to get rid of an excess of Sulphur, cooled to RT, and acidified by HCl. The separated solid was filtered off, washed with water, and purified by recrystallization from toluene to give derivative 5 as a brownish-red powder, in a 69% yield; m. p: 135–137 °C; IR (KBr): ν (cm−1) 1561 C = C, 1621C=N, 2190 CN, 2857, 2935CH-Al, 3025, 3057CH-Ar, 3324, 3193NH2; 1H-NMR : δ1.56–2.49 ; 4.25, 6.95 exchangeable with D2O, 7.12–7.97 , 8.45 . 13C-NMR : δ 22.18, 23.65, 24.65, 26.33, 112.55, 115.00, 118.00, 125.23, 127.57, 128.20, 129.52, 143.05, 144.32, 161.55, 171.60; analysis : C, H, N, S.The ethanolic solution of derivative 1 , cyanoacetohydrazide , and Sulphur element  in dimethylformamide (20 mL) containing a catalytic amount of piperidine (0.5 mL), was heated under reflux for 12 h, filtered off at hot to get rid of an excess of Sulphur, cooled to RT, and acidified by HCl. The solid formed was filtered off, washed with water, and purified by recrystallization from petroleum ether to give product 6 as a brownish-red powder, in a 52% yield; m. p: 160–163 °C; IR (KBr): ν (cm−1) 1493 C=C, 1621 C=N, 2858, 2932 CH-Al, 3025, 3058CH-Ar, 3.350, 3195NH2; 1H-NMR : δ1.64–2.33, 4.30 exchangeable with D2O, 4.55, 7.15–7.50, 7.80 exchangeable with D2O, 9.75 exchangeable with D2O; analysis : C, H, N, S.A mixture of compound 1  and ethyl cyanoacetate  in dioxane (30 mL) containing a catalytic amount of piperidine (0.5 mL) was heated under reflux for 5 h, cooled to RT, and acidified by HCl. The separated solid was filtered off, washed with water and purified by recrystallization from benzene to give the required product 7 as a brown powder, with a 37% yield; m. p: 95–98 °C; IR (KBr): ν (cm−1) 1547C=C, 1626C=N, 1721C=O, δ-lactone, 2935 CH-Al, 3025CH-Ar, 3345 NH. 1H-NMR : δ1.67–2.49; 3.88; 7.22–7.52; 8.15, 8.40  with D2O exchangeable.13C-NMR: δ21.85, 23.59, 24.45, 26.33, 62, 94.40, 120.49, 12517, 125.66, 126.76, 127.19, 128.24, 129.30, 130.91, 131.98, 133.39, 135.77, 146.34, 151.44, 155.09, 156.20; analysis : C, H, N, S.A mixture of derivative 1  containing a catalytic amount of piperidine (0.5 mL) and cyanoacetohydrazide  was heated under reflux for 12 h, cooled to RT, and acidified by HCl. The solid formed was filtered off, washed with water, and purified by recrystallization from toluene to give product 8 as a pale brown powder, in a 70% yield; m. p: 170–173 °C; IR (KBr): ν (cm−1) 1493C=C, 1628C=N, 2213CN, 2857, 2933CH-Al, 3025, 3057CH-Ar, 3191NH; 1H-NMR : δ1.59–2.97, 3.39, 7.27–7.86, 10.63; analysis : C, H, N, S.An ethanolic solution of derivative 1 , ethyl chloroacetate , and carbon disulfide  in dry dioxane (30 mL) containing anhydrous K2CO3 (1.4 gm) was stirred at RT for 12 h, and filtered off to remove excess K2CO3. The filtrate was poured onto ice water. The precipitate was filtered out, washed three times with deionized water and purified by recrystallization from benzene to give the required product 9 (orange powder) in a 32% yield; m. p: 85–88 °C; IR(KBr): ν(cm−1)1028C-S, 1205C=S, 1583C=C, 1621C=N, 1731C=O, 2858, 2932CH-Al, 3025, 3057CH-Ar, 3411NH. 1H-NMR : δ1.18; 1.62–2.31; 3.99; 4.12 ; 4.50; 7.12–7.86 ; 8.55; analysis : C, H, N, S.A mixture of compound 59 against HFB-4 cells , HepG-2 cells (hepatoma), and Caco-2 cells (colon carcinoma). Normal and cancer cells (1.0 × 104) were cultured in sterile 96-well microplates, and incubated for 24 h in DMEM  supplemented with 10% fetal bovine serum  for HFB-4 and Caco-2 cells or in an RPMI-1640 medium  supplemented with 10% FBS for HepG-2 cells. After 24 h of incubation, different doses of the tested synthesized compounds (2–64 g/mL) were added in triplicate to all cells and incubated for another 48 h in 5% CO2. After washing the cells 3 times with fresh media, to remove dead cells and debris, a solution of MTT  was added to each well and further incubated for 2–5 h at 37 °C. Afterward, the MTT solution was decanted and 200 µL of DMSO was added. The optical density was measured at 570 nm using a microplate reader , where the cells’ relative viability (%) was estimated using the following equation: viability of cells (%) = [A1 − A0/AU − A0] × 100, where A1 is the absorbance of the test compound, A0 is the absorbance of the blank, and AU is the absorbance of untreated cells (control).The human melanocytes (HFB-4) cells, hepatoma (HepG-2) cells, and colon carcinoma (Caco-2) cells were obtained from American Type Culture Collection . The cytotoxicity of the newly synthesized compounds (at various concentrations) was determined using the 3--2, 5-diphenyltetrazolium bromide method (MTT assay)50 value  using Graph Pad Prism 6.0 software. The value of IC50 indicates the derivative concentration that causes 50% cell death, whereas the value of the selectivity index (SI), indicating the ratio of the IC50 value of normal cells versus the IC50 value of tumor cells, was also included as reported by61. Furthermore, the effect of the highly active antitumor derivatives  on the morphology of HepG-2 and Caco-2 cells was explored at different concentrations (4–16 μg/mL) by using phase-contrast microscopy  and compared to untreated cells (negative control).The antitumor activity of the synthesized compound was determined by calculating the IC4, 6, and 8) on the expression level of some tumor regulating genes were elucidated through quantitative real-time PCR (qRT- PCR) and compared to 5-fluorouracil (5-FU) as a reference antitumor drug. Five genes were assessed, including the tumor oncogene (Bcl-2), tumor suppressor gene (p53), matrix metalloproteinase gene (MMP-9), vascular endothelial growth factor gene (VEGF), and beta-catenin protein gene (β-catenin), in HepG-2 and Caco-2 cells, before and after treatment for 2 days with IC50 concentrations for compounds 4, 6, and 8. Following the extraction of total RNA from the tested cells, using the Gene JET RNA Purification Kit , cDNA synthesis was performed according to the cDNA Synthesis Kit protocol . qRT PCR was performed using the SYBR green kit and specific primers (Forward/Reverse) as follows: 5′-TCCGATCAGGAAGGCTAGAGTT-3'/5'-TCG GTCTCCTAAAAGCAGGC-3′ for the Bcl-2 gene, 5′-TAACAGTTCCTGCATG GG CG GC-3′/5′-AGGACAGGCACAAACACGCACC-3′ for p53, 5'-CTGCGTATTTCCATT CATC-3'/5'-CCTTGGGTCAGGTTTAGAG-3' for the MMP-9 gene, 5'-GGCTTTACT GCTGTACCTCC-3'/5'-CAAATGCTTTCTCCGCTCT-3' for the VEGF gene, and 5'-CATATGCGGCTGCTG TTCTA-3'/5'-CCGAAAG CCGTTTCTTGTAG-3' for the β-catenin gene. The upregulation and/or downregulation of the expression of the tested genes in Caco-2 and HepG-2 cells was determined by using the equation  2−ΔΔCT60.The effects of the potent antitumor derivatives  for 1 h at 37 °C. Then 10 μL of PI (Sigma-Aldrich) was added to cells at a final concentration of 1 mg/mL in deionized water and left at 4 °C until analysis in the dark. The cell cycle of Caco-2 cells before and after treatment was analyzed by FACS using Cell Quist and Mod Fit software by reading at 488 nm.The cell cycle arrest of treated Caco-2 cells was evaluated through flow cytometry  in comparison with untreated cells and treated cells with 5-FU as a positive control4, 6, and 8) into five tumor-regulating proteins, namely MMP-9, p53, β-catenin, Bcl-2, and VEGF, were simulated using Molecular Operating Environment (MOE) 2014) software. The three-dimensional structure (3D) of the selected proteins was downloaded from the PDB website. The water molecules and repeated chains were removed. Protons were added and the energy of the protein was minimized. The isolation of the pocket was then carried out. Validation of the downloaded structure was confirmed by re-docking the downloaded ligand into the isolated pocket. The obtained root mean square deviation (RMSD) was found to be lower than 1.5 Å. The preparation of potent antitumor derivatives  for docking was carried out by the construction of chemical structures at the MOE. Protons were then added to the 3D structure. Finally, the energy was minimized using Force Field MMFF94x. The prepared structures were added to the created database. MOE conducted the docking of the newly synthesized compounds, calculated the binding energies, and provided their binding modes of them11.The binding orientations and interactions of the potent antitumor derivatives ((2–9) was evaluated against three model pathogenic microorganisms as follows: Streptococcus mutans ATCC 25175 (Gram-positive bacterium); Pseudomonas aeruginosa ATCC 27853 (Gram-negative bacterium); and Candida albicans ATCC 10231 (fungi-like unicellular organism). After cultivating the three tested pathogens overnight in LB broth, 100 µL of each tested organism (106 CFU/mL) was added separately to 100 µL of the serially diluted test compounds (5–80 µg) in a 96-well tissue culture plate and incubated at 37 °C for 24 h. Eventually, microbial growth was measured at 600 nm, and the results are presented in the form of MIC  was inoculated separately (in triplicate) into 96-well tissue culture plates and incubated overnight at 37 °C. The free planktonic cells were removed, after the incubation period, by flipping the plate and washing each well three times with phosphate-buffered-saline (PBS), pH 7.1. To each well, 175 µL of fresh LB broth was added along with 25 µL of the tested compound . The plates were reincubated at 37 °C for another 24 h, washed with PBS buffer pH 7.1 three times, and dried for 5 min at 50 °C. The formed biofilm was stained with crystal violet solution (0.1% w/v) for 5 min. The excess stain was decanted, and the stained biofilm was solubilized with 200 µL of glacial acetic acid (30% v/v), where the developed blue color was measured at 590 nm. The results are expressed as relative inhibition (%) compared to untreated groups (control). Controls were prepared by culturing the three pathogens on LB medium without any tested compounds.The activity of the newly synthesized compounds against microbial biofilm formation was estimated through the tissue-culture plate technique (TCP) as follows: the three above-mentioned pathogens, p values < 0.05.All experiments were performed in triplicate (n = 3), and all data are expressed as the mean ± SEM. The significance of statistical analysis was evaluated by the multiple comparisons of Tukey’s post-hoc test of the one-way analysis of variance (ANOVA) using the SPSS16 program, and differences were considered statistically significant at This article does not contain any studies with human or animal subjects.50 values. The antitumor activity of the synthesized derivatives included obvious tumor cell damage and stimulated a clear alteration of the cell morphology in a dose-dependent manner. Among the tested compounds, derivatives 6 and 4 revealed potent antitumor activity, where derivative 8 showed the highest antitumor activity toward both tested tumor cells with SI values of approximately 26 and IC50 values of 5.9 μg/mL, attributed to the presence of a thiazolylpyrazole moiety, with acetonitrile, in the pyrazole ring. The gene expression level study confirmed apoptosis induction through upregulation of the p53 gene (2–eightfold) in both treated HepG-2 and Caco-2 cells. On the other hand, compound 3 revealed significant broad-spectrum antibacterial activity against Streptococcus mutans (MIC of 11.2 µg/mL) and Pseudomonas aeruginosa (MIC of 18.29 µg/mL), comparable to that of ampicillin MIC (13.5 µg/mL) and ciprofloxacin (18.7 µg/mL), which could be attributed to the incorporated thiazolopyridine ring. The newly prepared compounds revealed low to medium antifungal activity against Candida albicans with a maximum antifungal activity through compound 3 (36% clotrimazole activity). Many synthesized compounds revealed antibiofilm formation activities (58.5–79% inhibition) against the three applied pathogens. Collectively, the results confirmed the effectiveness of newly synthesized compounds as promising antitumor drugs with antimicrobial activity. The current study results encourage our research team to go deeper into the exact antitumor/antimicrobial mechanisms of the newly prepared potent derivatives and explore the structural–functional relationship.In this study, a new series of indazolylthiazole moieties was effectively generated and described in a new range of novel pyridine, pyran, furan, thiophene, and pyrazole-carrying compounds. The newly synthesized compounds showed great potency as selective anticancer drugs against both HepG-2 and Caco-2 cell lines, with high SI values and low ICSupplementary Information 1.Supplementary Information 2.