Superior Inhibitory Activity Research Articles

Design, synthesis of novel celastrol derivatives and study on their antitumor growth through HIF-1α pathway.

Four series of hypoxia-inducible factor-1 alpha (HIF-1α) functioning derivatives stemming from modifications to the C-29 carboxyl group of celastrol were designed and synthesized, and their anticancer activities were evaluated. To address the structure and activity relationship of each derivative, extensive structural changes were made. HRE luciferase reporter assay demonstrated that 12 modified compounds showed superior HIF-1α inhibitory activity. Among them, compound C6 exhibited the best features: firstly, the strongest HIF-1α inhibitory activity (IC50=0.05μM, 5-fold higher than that of celastrol); secondly, lower cytotoxicity (22-fold lower, C6-16.85μM vs celastrol-0.76μM). Thus, the safety factor of C6 was about 112 times higher than that of celastrol. Western blot assay indicated that C6 may inhibit the expression of HIF-1α protein in cells. Additionally, C6 hindered tumor cell cloning, migration and induced cell apoptosis. It is worth mentioning that in the mouse tumor xenograft model, C6 (10mg/kg) displayed good antitumor activity invivo, showing a better inhibition rate (74.03%) than the reference compound 5-fluorouracil (inhibition rate, 59.58%). However, the celastrol treatment group experienced collective death after four doses of the drug. Moreover, C6 minimally affected the mouse weight, indicating that its application invivo has little toxic effect. H&E staining experiments show that it could also exacerbate the degree of tumor cell damage. The results of water solubility experiment show that the solubility of C6 is increased by 1.36 times than that of celastrol. In conclusion, C6 is a promising antitumor agent through HIF-1α pathway.

Identification and optimization of 3-bromo-N'-(4-hydroxybenzylidene)-4-methylbenzohydrazide derivatives as mTOR inhibitors that induce autophagic cell death and apoptosis in triple-negative breast cancer.

Triple negative breast cancer (TNBC) has a worse prognosis than other types of breast cancer due to its special biological behavior and clinicopathological characteristics. TNBC cell proliferation and progression to metastasis can be suppressed by inducing cytostatic autophagy. mTOR is closely related to autophagy and is involved in protein synthesis, nutrient metabolism and activating mTOR promotes tumor growth and metastasis. In this paper, we adopted the strategy of structure simplification, aimed to look for novel small-molecule inhibitors of mTOR by pharmacophore-based virtual screening and biological activity determination. We found a lead compound with 3-bromo-N'-(4-hydroxybenzylidene)-4-methylbenzohydrazide for rational drug design and structural modification, then studied its structure-activity relationship. After that, compound 7c with the best TNBC cells inhibitory activities and superior mTOR enzyme inhibitory activity was obtained. In addition, we found that compound 7c could induce autophagic cell death and apoptosis in MDA-MB-231 and MDA-MB-468cell lines. In conclusion, these findings provide new clues for our 3-bromo-N'-(4-hydroxybenzylidene)-4-methylbenzohydrazide derivatives, which are expected to become drug candidates for the treatment of TNBC in the future.

New Mixed-Ligand Complexes of Cytosine and Its Silver Nanoparticles: Spectral, Analytical, Theoretical and Biological Activity Studies

Novel mixed-ligands complexes of cytosine (M-cytH-isaH, where isaH denotes isatin) and cytosine capped silver nanoparticles (cytH-AgNPs) are synthesized. The complexes are characterized by different spectroscopic techniques and thermal analyses. Cytosine exhibits different binding modes with the metal ions through the ring nitrogen N(3) and/or C=O forming square planar or octahedral geometries. The ESR spectra of the Cu(II) complexes point to the existence of spin exchange interaction between the metal centers. For all complexes, the thermal decomposition of the inner sphere starts at T ~ 200 °C indicating their high thermal stability. Structure of cytH-AgNPs is investigated by proper microscopic techniques; SEM, TEM, EDX and XRD. The crystal pattern of AgNPs is FCC symmetry with spherical shape and an average nanoparticle size of 5.72–16.31 nm. Biological screening of the synthesized compounds shows that Cu(II) complex, [Cu2(cyt)(isa)2Cl], displays the highest antibacterial activity with inhibition zone diameter of 21 mm/mg sample similar to that of ampicillin. Furthermore, only AgNPs exhibits significant antifungal activity against A. flavus where its smaller size is assumed to play an important role in blocking cell communication mechanisms. Density functional theory, DFT, and molecular docking studies are performed to evaluate the quantum chemical parameters and to shed light on the sites of interaction with the selected protein 1bqb of S. aureus. The scoring energy of [Cu2(cyt)(isa)2Cl] is in good agreement with the superior inhibition activity measured experimentally for this complex.

Antimicrobial activity of Spondias mombin L. aqueous and hydroethanolic extracts on Enterococcus faecalis and Pseudomonas aeruginosa - an in vitro study

Objective: To chemically characterize Spondias mombin L. extracts and to evaluate the in vitro antimicrobial activity of these extracts on Enterococcus faecalis and Pseudomonas aeruginosa. Material and methods: Both extracts were submitted to phytochemical prospecting and thin layer chromatography (TLC) analysis prior to determination of Minimum Inhibitory Concentration (MIC), Minimum Inhibitory Adhesion Concentration (MIAC) and Bactericidal Kinetics (BK). The Student’s t-test was used to compare MIC results at the 5% significance level. The chemical characterization, MIAC and BK data were analyzed descriptively. Results: Phenolic compounds, flavonoids, tannins and saponins were detected in the extracts. The hydroethanolic extracts in different concentrations showed statistically superior inhibitory bacterial growth activity (p <0.05) compared to the control against the two investigated microorganisms, whereas the aqueous extracts (also in different concentrations) was statistically superior (p <0.05) to the control only against Pseudomonas aeruginosa. The anti-adherent effect of both extracts was superior to that of the control for P. aeruginosa, and similar to that for E. faecalis. In their crude and diluted concentrations, aqueous and hydroethanolic extracts showed bactericidal action within the first two to four hours of contact with the bacteria. Conclusion: Spondias mombin L. extracts in different concentrations showed superior antimicrobial activity than 0.12% chlorhexidine digluconate against the microorganisms with this activity associated to the chemical characterization of these extracts.

Design, synthesis and biological evaluation of novel benzoxaborole derivatives as potent PDE4 inhibitors for topical treatment of atopic dermatitis.

In this work, a series of structurally novel benzoxaborole derivatives were designed, synthesized and biologically evaluated as PDE4 inhibitors for battling atopic dermatitis (AD). Among them, the majority exhibited superior PDE4B inhibitory activities to that of the lead compound Crisaborole, an approved PDE4 inhibitor. In particular, 72, the most potent PDE4B inhibitor throughout this series, displayed 136-fold improved enzymatic activity (IC50=0.42nM) as compared to Crisaborole (IC50=57.20nM), along with favorable isoform specificity. In the phorbol ester (PMA)-induced mouse ear oedema model, 72 exerted remarkably greater efficacy than Crisaborole at the same dosage (P<0.05). Moreover, the ointment of 72 exerted dramatically enhanced therapeutic potency than the ointment of Crisaborole (P<0.05) in the calcipotriol-induced mouse AD model. In addition to the potent invitro and invivo activity, 72 displayed favorable safety in the repeated oral dose toxicity study and did not exhibit phototoxicity. With the above attractive biological performance, 72 is worthy of further functional investigation as a novel anti-AD therapeutic agent.

Isolation, Identification and Molecular Characterization of Pathogenic Fungus causing bud Rot Disease to Coconut: Its Inhibition using Silkworm Fecal Matter Mediated Synthesized Silver and Copper Nanoparticles

Coconut is one the major economic crop in India; a considerable amount of crop will be lost every year due to bud rot disease. Phytophthora palmivora is an omnipresent pathogen which causes many different diseases on a wide range of plants including bud rot of coconut. In the present investigation, we have successfully isolated bud rot disease causing fungus from the infected coconut plant samples. The isolated fungus was primarily identified by observing in microscopy further the same sample was sent for molecular identification. The presence of Phytophthora palmivora was confirmed in 18s rRNA sequencing. The growth of isolated fungus was effectively inhibited using biosynthesized Silver and Copper nanoparticles. The inhibition effects of nanoparticles against Phytophthora palmivora were observed excellent in dose-dependent manner. The silver nanoparticles synthesized using Silkworm fecal matter was shown superior inhibition activity towards Phytophthora palmivora compare to standard fungicide Fluconazole. Hence, these silver Nano particles could be successfully used in inhibiting the pathogenic fungus causing bud rot disease to coconut.

Andrographolide-loaded solid lipid nanoparticles enhance anti-cancer activity against head and neck cancer and precancerous cells.

Increasing evidence indicates that andrographolide (ADG) exhibits anti-cancer activity against various cancer cell lines. However, its high hydrophobicity and poor bioavailability restrict its clinical application as a chemopreventative agent. Previously, we have shown that ADG-loaded solid lipid nanoparticles (SLNs) significantly enhanced the bioavailability and anti-hyperlipidemic activity of ADG. We aimed to investigate whether ADG-SLN enhanced the bioavailability and anti-cancer efficacy of ADG in the human immortalized oral epithelial (HIOEC), precancerous leukoplakia (Leuk1), HN6, and HN30 cells that represented an in vitro model of stepwise head and neck squamous cell carcinoma development. The 50% inhibitive concentration (IC50) of ADG-SLN was significantly lower than that of free ADG against HIOEC, Leuk1, and HN6 and HN30 cells. Moreover, ADG-SLN was more effective than free ADG in promoting cell cycle arrest and apoptosis. Importantly, intracellular absorption of ADG was significantly higher in HN6 cells treated with ADG-SLN compared with free ADG-treated cells. Our preliminary study demonstrates that ADG-SLN exhibits superior inhibitory activity against head and neck cancer and precancerous cells compared with free ADG. This effect is due to the higher efficiency of cellular uptake and intracellular absorption by ADG-SLN.

Synthesis and Anti-HCV Activities of 18β-Glycyrrhetinic Acid Derivatives and Their In-Silico ADMET Analysis

Licorice is widely used as a hepatoprotective herb for thousands of years in Traditional Chinese Medicine, and its main chemical constituent glycyrrhizin (GL) is used as a treatment for chronic hepatitis in Japan for over 20 years. 18β-Glycyrrhetinic acid (GA) is the main active metabolite of GL. Series of GA derivatives were designed and synthesized, and their anti-HCV activities were screened to investigate the structure-activity relationship (SAR). Besides, their in-silico ADMET properties were analyzed to search for a promising lead compound for further identification of anti-HCV terpenoid candidates. GA derivatives were synthesized via reactions of oxidation, oxime, rearrangement, esterification and acylation. In vitro anti-HCV activity of derivatives was tested on the HCV cell culture (HCVcc) system. In-silico ADMET properties analysis was performed via "pkCSM" and "SwissADME" platforms. Eighteen GA derivatives were synthesized, and their structures were confirmed by MS and NMR spectrums. All compounds exhibited superior HCV inhibitory activity to that of GA. Compound 2 possessed the most potent anti-HCV activity with an IC50 value of 0.79 μM, which is nearly 58 times potent than SA (a previously reported potent anti-HCV terpenoids) and >200 times than GA. SAR revealed that the introduction of 3-oxo, short-chain (C1-C3) aliphatic alcohols or cyclic aliphatic amines is conducive to improving anti-HCV activity. In-silico ADMET prediction demonstrated most of the potent compounds possessed favorable ADMET properties. Structural modification of GA at 3-position and 30-position is an effective approach to searching for potent anti-HCV agents. Compound 2, with the most potent anti-HCV activity and favorable in-silico ADMET properties, is a promising lead compound for further identification of anti-HCV terpenoid candidates.

Unconventional amino acids in medicinal chemistry: First report on taurine merged within carbonic anhydrase inhibitors

This study reports the design, synthesis of a series of taurine containing benzenesulfonamide derivatives which were all screened in vitro against the physiological relevant human (h) expressed Carbonic Anhydrase (CA; EC 4.2.1.1) I, II, IX, XII isozymes. Compound 2, 5, 11–16 displayed superior inhibitory activities against the tumor associated hCA IX over the reference drug Acetazolamide (AAZ). Both hCA IX and XII isoforms were selectively inhibited only by compound 3, whereas the chloro-containing compound 12 was showed as the most selective and effective inhibitor profile for the CA IX isoforms. To the best of our knowledge the data reported herein are the first of this kind and introduce in the literature new compounds worth for future development within the Medicinal Chemistry field.

Design, synthesis, and biological evaluation of F-18-labelled 2, 4-diaminopyrimidine-type FAK-targeted inhibitors as potential tumour imaging agents

As a type of intracellular nonreceptor tyrosine kinase, focal adhesion kinase (FAK) can be highly expressed in most types of tumours and is thus regarded as a promising antitumour target. In this study, a series of novel 2,4-diaminopyrimidine FAK-targeted inhibitors were designed, synthesized and characterized by 1H NMR, 13C HNMR, and HRMS spectra. These compounds, with an IC50 range of 5.0–205.1 nM, showed superior inhibitory activity against FAK. Two compounds, [18F]Q-2 and [18F]Q-4, with respective IC50 values of 5.0 nM and 21.6 nM, were labelled by 18F, accompanied by evaluation of their biodistributions. For [18F]Q-2, at 30 min post-injection, promising target-to-nontarget ratios were observed, associated with tumour/blood, tumour/muscle, and tumour/bone ratios of 1.17, 2.99 and 2.19, respectively. The results indicated that [18F]Q-2 is a potential PET tracer for tumour diagnosis.

Discovery of a Novel Hybrid of Vorinostat and Riluzole as a Potent Antitumor Agent.

Vorinostat (suberoylanilide hydroxamic acid) was the first approved histone deacetylase (HDAC) inhibitor in a group of validated cancer therapeutic agents targeting epigenetics. Riluzole is a drug used to treat amyotrophic lateral sclerosis, the antitumor potency of which has been recently revealed. Herein, a novel hybrid of vorinostat and riluzole (compound 1) was rationally designed, synthesized, and evaluated. Compared with vorinostat, compound 1 exhibited superior total HDAC inhibitory activity and similar HDAC isoform selective profiles. The intracellular HDAC inhibition of compound 1 was confirmed by Western blot analysis. Moreover, compound 1 possessed more potent in vitro antiproliferative activity against all tested solid and hematological tumor cell lines than vorinostat. In vitro metabolic stability evaluation of compound 1 revealed better human plasma stability and comparable human liver microsomal stability than vorinostat. Additionally, compound 1 demonstrated more significant in vivo antitumor activity in a MDA-MB-231 xenograft model than vorinostat, which could be attributed to its superior in vitro antiproliferative activity and metabolic stability. Taken together, the results presented here support further research and development of compound 1 as a promising antitumor agent.

The determination of molecular dynamic properties of Novel 5-oxo-1,2,4-triazole phthalocyanines and investigation of their urease inhibition properties

In present study, the novel peripherally [3-methyl-5-oxo-4-(4-phenoxyphenyl)-4,5-dihydro-1H-1,2,4-triazol-1-yl] substituted Zn(II), Cu(II), Co(II) phthalocyanines were synthesized and characterized spectroscopically. The light absorption/emission behavior of the synthesized phthalocyanines were determined by UV-Vis and fluorescence spectroscopy. The fluorescence quantum yields of the novel phthalocyanine Zn(II) complex in different solvents were calculated. Besides, the urease inhibition activities of the novel compounds were investigated. All synthesized phthalocyanines have a superior inhibitory activity on urease enzyme, while novel phthalocyanines Cu(II) showed the best inhibitory effect against jack bean urease with IC50 values of 0.0035 ±0.0010 µM.

Thymidylate synthase inhibitor raltitrexed can induce high levels of DNA damage in MYCN-amplified neuroblastoma cells.

MYCN gene amplification is consistently associated with poor prognosis in patients with neuroblastoma, a pediatric tumor arising from the sympathetic nervous system. Conventional anticancer drugs, such as alkylating agents and platinum compounds, have been used for the treatment of high‐risk patients with MYCN‐amplified neuroblastoma, whereas molecule‐targeting drugs have not yet been approved. Therefore, the development of a safe and effective therapeutic approach is highly desired. Although thymidylate synthase inhibitors are widely used for colorectal and gastric cancers, their usefulness in neuroblastoma has not been well studied. Here, we investigated the efficacies of approved antifolates, methotrexate, pemetrexed, and raltitrexed (RTX), on MYCN‐amplified and nonamplified neuroblastoma cell lines. Cell growth‐inhibitory assay revealed that RTX showed a superior inhibitory activity against MYCN‐amplified cell lines. We found no significant differences in the protein expression levels of the antifolate transporter or thymidylate synthase, a primary target of RTX, among the cell lines. Because thymidine supplementation could rescue the RTX‐induced cell growth suppression, the effect of RTX was mainly due to the reduction in dTTP synthesis. Interestingly, RTX treatments induced single‐stranded DNA damage response in MYCN‐amplified cells to a greater extent than in the nonamplified cells. We propose that the high DNA replication stress and elevated levels of DNA damage, which are a result of deregulated expression of MYCN target genes, could be the cause of increased sensitivity to RTX.

Melanogenesis-inhibitory activities of limonoids and tricyclic diterpenoids from Azadirachta indica.

The chemical constituents of the roots and bark of Azadirachta indica were investigated, leading to the isolation of six tricyclic diterpenoids and four limonoids including a new compound, azadirachtin J (4). The structures were elucidated on the basis of NMR spectroscopic techniques, mass spectrometry as well as comparison with the literature. Furthermore, melanogenesis-inhibitory activities of the isolated compounds were evaluated. As a result, compounds 1-3 and 10 exhibited superior inhibitory activities against melanogenesis with no, or almost no, toxicity to the cells (86.5-105.1% cell viability). Western blot analysis showed that compounds 1 and 3 exhibited melanogenesis inhibitory activities in α-MSH-stimulated B16 melanoma cells due to, at least in part, inhibition of the expression of MITF, followed by a decrease in the expression of tyrosinase, TRP-1, and TRP-2. Compounds 1 and 3 exhibited tyrosinase inhibitory activities (IC50 values of 44.86μM and 69.85μM respectively). Docking results confirm that the active inhibitors strongly interact with tyrosinase residues.

Design and Synthesis of some new 2,4,6-trisubstituted quinazoline EGFR inhibitors as targeted anticancer agents.

The present study describes the synthesis of 6-bromo-2-(pyridin-3-yl)-4-substituted quinazolines starting from 4-chloro derivative VI via the reaction with either phenolic compounds to obtain VIIa-f, IXa-d, 2-amino-6-(un)substituted benzothiazole to produce VIIIa-c or hydrazine hydrate to give X. Reaction of the hydrazino functionality of X with appropriate acid anhydride, acid chloride or aldehyde affords XIa-c, XIIa-c and XIVa-i, respectively. The target compounds were screened for their efficacy as EGFR inhibitors compared to gefitinib. Compounds eliciting superior EGFR inhibitory activity were further screened for their in vitro cytotoxicity against two human cancer cell lines namely: MCF7 (breast) and A549 (lung), in addition to normal fibroblast cell WI38 relative to gefitinib as a reference. Furthermore, compounds that showed potent inhibitory activity on wild-type EGFR were screened against mutant EGFR and assayed for their cytotoxicity against mutant EGFR-expressing cell lines PC9 and HCC827. The unsubstituted benzothiazol-2-amine VIIa showing superior EGFR inhibition (IC50=0.096µM) and anticancer activity against MCF-7 cell line (IC50 =2.49µM) was subjected to cell cycle analysis and apoptotic assay. Moreover, a molecular docking study was performed to investigate the interaction of some representive compounds with the active site of EGFR- TK.

Semi-synthesis of C28-modified triterpene acid derivatives from maslinic acid or corosolic acid as potential α-glucosidase inhibitors.

Combining two bioactive moieties by covalent bond into a novel single hybrid biological entity in view of the principle of active splicing, twenty-two C28-modified derivatives of pentacyclic dihydroxytriterpene carboxylic acids with saturated nitrogen heterocycle segments (i.e. 1-deoxynojirimycin or piperazines) have been synthesized. The inhibitory activities of all final target compounds on α-glucosidase were evaluated in vitro. The results of α-glucosidase inhibition assay indicate that some derivatives (e.g. 4b: IC50=1468.4µM; 12b: IC50=499.6µM 12c: IC50=768.5µM, 13c: IC50=819.2µM) show superior inhibitory activity in α-glucosidase than that of the precursor maslinic acid (IC50=2540.6µM) or corosolic acid (IC50=1363.7µM), in which compound 12b (IC50=499.6µM) possesses stronger inhibitory activity than that of acarbose (IC50=606µM). In addition, the result of enzyme kinetics study reveals that the inhibitory mechanism of the compound 12b is non-competitive inhibition and the inhibition constant Ki is 570µM. The binding interaction between compounds with α-glucosidase are predicted by molecular docking simulation.

Synthesis and biological evaluation of nicotinamide derivatives with a diarylamine-modified scaffold as succinate dehydrogenase inhibitors.

Six novel nicotinamide derivatives bearing a diarylamine-modified scaffold with flexible heterocyclic patterns were designed, synthesized, and characterized in detail via Hydrogen nuclear magnetic resonance (1H-NMR), Carbon nuclear magnetic resonance (13C-NMR), and Electrospray ionization mass spectrometry (ESI-MS). Their fungicidal activities and succinate dehydrogenase (SDH) enzymatic inhibitory abilities were evaluated. Preliminary fungicidal bioassay results showed that some of the target compounds exhibited moderate fungicidal activity. Among them, compound 4a showed 40.54% inhibition against Botrytis cinerea fungi. An SDH enzymatic inhibition assay revealed that the IC50 of compound 4b was 3.18 µM. This result indicated that the enzymatic inhibition level of 4b was similar to that of boscalid. Compound 4f exhibited superior comprehensive fungicidal and SDH enzymatic inhibitory activities. Molecular docking results suggested that 4f could bind well to the substrate cavity and the entrance cavity of SDH (1YQ3). In particular, 4f could react with the key catalytic site Arg 297. This phenomenon implied that 4f could act as the lead compound for further optimization.

Antimicrobial sodium alginate dressing immobilized with polydopamine-silver composite nanospheres

To endow antibacterial activity and maintain good porosity, oxidized sodium alginate (OSA) sponge was functionalized via polydopamine/silver composite nanospheres (PDA/AgNSs). It is found that uniform PDA spheres (around 1.7 μm) can be successfully coated on OSA sponge surface without compromise of the OSA sponge integrity and porosity using absolute ethanol as optimized solvent. Particularly, the newly-produced PDA spheres can crosslink with the OSA sponge via the Schiff-base condensation, leading to improved stability. On the other hands, those PDA spheres can be served as the active sites to realize the growth of silver nanoparticles (around 50–70 nm) on PDA spheres surface, leading to the formation of novel PDA/AgNSs. Further analysis indicates that our proposed OSA/PDA/AgNSs sponge shows high blood compatibility, low cell cytotoxicity, good hemostatic performance as well as superior inhibitory activity against P. aeruginosa, S. aureus and E. coli. In conclusions, our proposed strategy opens a new route for antimicrobial sodium alginate dressing development.

Structurally novel PI3Kδ/γ dual inhibitors characterized by a seven-membered spirocyclic spacer: The SARs investigation and PK evaluation

Herein, we communicate our recent medicinal chemistry efforts which have culminated in a series of PI3Kδ/γ dual inhibitors structurally featuring a seven-membered spirocyclic spacer. Compound 26, the most potent one among them, exhibited superior PI3Kδ inhibitory activity (IC50 = 1.0 nM) to that of the approved PI3Kδ inhibitor Idelalisib. Besides, it exerted remarkable anti-proliferative efficacy against human malignant B-cell line SU-DHL-6 with GI50 value of 33 nM. The biochemical assay against the other three class I PI3K isoforms identified compound 26 as a potent PI3Kδ/γ dual inhibitor with considerable selectivity over PI3Kα and PI3Kβ. In SU-DHL-6 cells, a dramatic down-regulation of PI3K signaling was observed following compound 26-treatment at the concentration as low as 10 nM. Inspiringly, the pharmacokinetic (PK) study in Sprague-Dawley (SD) rats revealed it was orally available with a favorable bioavailability (F = 87.5%). Overall, compound 26, a promising PI3Kδ/γ dual inhibitor, has the potential to emerge as a clinical candidate for the treatment of leukocyte-mediated malignancies after extensive functional investigation.

Screening of Host Specific Lactic Acid Bacteria Active Against Escherichia coli From Massive Sample Pools With a Combination of in vitro and ex vivo Methods.

A novel three-step combination of in vitro and ex vivo screening was established to massively screen host derived lactic acid bacteria (LAB) from the broiler chicken intestine with inhibitory activity against Escherichia coli. In a first step, a massive sample pool consisting of 7102 broiler-derived colonies from intestinal contents were established and sub-cultured. Supernatants thereof were incubated with an E. coli model strain to screen suitable isolates with inhibitory activity. A total of 76 isolates of interest were subsequently further studied based on either pH dependent or -independent activity in the second step of the assay. Here, in-depth growth inhibition of the E. coli model strain and the potential of isolates for lactic acid production as inhibitory substance were indexed for all isolates. Resulting scatter plots of both parameters revealed five isolates with exceptional inhibitory activity that were further studied under ex vivo condition in the third step of the assay. These isolates were taxonomically classified as strains of the species Lactobacillus agilis, Lactobacillus salivarius, and Pediococcus acidilactici. Samples from the broiler chicken intestine were inoculated with the Lactobacillus isolates and the E. coli model strain. After 8 and 24 h incubation, respectively, growth of the E. coli model strain was monitored by cultivation of the E. coli strain in antibiotic supplemented medium. By their superior inhibitory activity against the E. coli model strain, one L. agilis and one L. salivarius strain were selected and characterized for further application as probiotics in broiler chicken. Additionally, their antibiotic resistance patterns and resilience under gastric stress of isolates were also characterized. The results of this study demonstrate that the novel isolation procedure was able to efficiently and rapidly isolate and identify bacterial strains from a massive sample pool with inhibitory potential against specific types of bacteria (here E. coli). The introduction of the final ex vivo selection step additionally confirmed the inhibitory activity of the strains under conditions simulating the intestinal tract of the host. Furthermore, this method revealed a general potential for the isolation of antagonistic strains that active against other pathogenic bacteria with specific biomarker.