Source: OHIO STATE UNIVERSITY submitted to
NOVEL THERAPEUTIC LEADS; DEMONSTRATION OF EFFICACY, SAFETY AND APPLICABILITY OF ANTI-APEC MOLECULES IN CHICKENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1022675
Grant No.
2020-67015-31401
Cumulative Award Amt.
$500,000.00
Proposal No.
2019-05383
Multistate No.
(N/A)
Project Start Date
Sep 1, 2020
Project End Date
Aug 31, 2024
Grant Year
2020
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
OARDC Food Animal Health
Non Technical Summary
APEC is a leading cause of morbidity and mortality in poultry and results in multi-million dollar annual losses for all facets of the poultry industry. Currently, APEC infections in poultry are controlled by antibiotics medication and vaccination. However, APEC isolates are becoming more resistant to antibiotics and the currently available vaccines are only effective against homologous serotype, O78. There is a critical need for developing novel anti-APEC therapeutics to improve the control of this key endemic avian disease. Through our recent innovative small molecules based studies, we have identified promising anti-APEC leads [growth inhibitors (GI-7, GI-10) and quorum sensing inhibitors (QSI-5 and QSI-10)]. These lead molecules in small pilot studies reduced the APEC induced mortality of chickens, reduced the APEC load in chicken internal organs and lessen the severity of APEC lesions. In addition, these leads are effective against diverse APEC serotypes, antibiotic-resistant APECs and against biofilm protected APEC. Here, we will evaluate the therapeutic potential of these lead molecules under experimental and natural setup as well as elucidate their mechanisms of action. The demonstration of the efficacy, safety and applicability of these lead molecules in chickens is vital to advance them as novel anti-APEC therapeutics. Our study will help to promote sustainable poultry production worldwide together with improved food security and food safety.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31132201100100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3220 - Meat-type chicken, live animal;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Overall goal:Demonstration of efficacy, safety and applicability of lead anti-APEC small molecules (GI-7, GI-10, QSI-5, and QSI-10) under experimental as well as natural setup using large number of chickens and to elucidate their mechanisms of action. This will be accomplished via conducting the studies described in the following objectives:Objective 1: Determine the efficacy, safety and applicability of GI-7, GI-10, QSI-5, and QSI-10 in broiler chickens under experimental setup and under conditions mimicking the natural APEC infection.Objective 2: Elucidate the mechanisms of action of anti-APEC leads using metabolomics and affinity based pull-down approaches.
Project Methods
Objective 1.1: Determine the optimum doses of lead anti-APEC small molecules (GI-7, GI-10, QSI-5 and QSI-10) in drinking water of chickens.Dose determination in drinking water: GI-10 will be tested at 20, 40 and 60 mg/L doses; whereas, QSI-10 will be tested at 1, 5 and 10 mg/L doses. SMs will be administered in drinking water continuously (changed daily) for 7 days as above. Chickens (n=10/dose) will be raised in cages, infected and efficacy will be measured by assessing mortality, APEC load and lesion severity.Objective 1.2: Efficacy, safety and applicability of lead anti-APEC small molecules (GI-7, GI-10, QSI-5 and QSI-10) in chickens under experimental conditions.Efficacy assessment: We will test the efficacy of GI-7, GI-10, QSI-5, and QSI-10 to control APEC infection in chickens. A total of 60 chickens will be allocated in each group; among them, 30 will be used for efficacy assessment and other 30 will be used for safety/performance assessment. Chickens will be infected s/c at 7 days of age with Rifr APEC O78 (1 x 107 CFU/chicken in 100 µL PBS) using 1 mL syringe. GI's/QSI's will be administered in drinking water continuously for 7 days at their optimum doses starting one day before the infection (day 6) to 6 dpi (day 12). Sulfadimethoxine will be administered at its therapeutic dose (0.05%). Clinical signs and mortality will be recorded until 8 dpi (day 15). After 8 dpi (at day 15), half of the live chickens from each group will be necropsied to assess lesions and APEC load in internal organs. To measure the efficacy of GI's/QSI's, mortality, APEC load and APEC lesions in GI's/QSI's treated groups will be compared to infected non-treated and infected antibiotic-treated groups. The remaining half of live chickens will be raised until 42 days (slaughter age) to assess the safety of these SMs.Safety assessment:A) Impact on production performance: For this purpose, feed intake and body weight will be measured weekly (days 7, 14, 21, 28, 35, and 42). BWG and FCR (feed intake/BWG) will be determined weekly. Overall BWG and FCR will be calculated at the end of the experiment (day 42) for all SMs treated groups and compared with non-infected and non-treated, infected and non-treated and infected and antibiotic-treated groups.B) Evaluation of resistance to GI's/QSI's: APEC colonies isolated from different tissues of infected and GI's/QSI's treated chickens at day 15 (Ist necropsy) or day 42 (IIIrd necropsy) (n=20 isolates/treatment group, including 5 isolates from each tissue) will be used for testing whether APEC acquire resistance to GI's/QSI's. The MIC and MBC of GI's/QSI's against isolated colonies will be determined and compared with the original MIC/MBC of GI's/QSI's.C) Impact on microbiota of chickens: Tissue samples from 10 chickens (n=30/group) will be collected from each treatment group at Ist necropsy (day 15 for assessing immediate impact) and IIIrd necropsy (day 42 for assessing long-term impact). Genomic DNA will be extracted from cecal contents, lung and trachea. Impact of GI's/QSI's will be assessed by metagenomics analysis of 16S rRNA gene amplicons byV4-V5 gene amplicons sequencing.D) Bioaccumulation of GI's/QSI's in chicken's tissues: We will quantify the GI's/QSI'sresidues in muscle, liver, and kidney at day 15 (Ist necropsy; 2 days post last treatment) and day 17 (IInd necropsy; 5 days post last treatment) to determine the drug withdrawal period and day42 (IIIrd necropsy; at slaughter age) to confirm the absence of GI's/QSI's residues. At each necropsy, liver (n=5/group), muscle (n=5/group), and kidney (n=5/group) will be collected and shipped to Mass Spectrometry and Proteomics Facility (MS&P) at the OSU to quantify the GI's/QSI's in the tissues using targeted metabolomics (HPLC-MS triple quadrupole (QQQ) Quantiva).PK, PD and Stability assessment: For PK study, chickens will be sacrificed and blood (2 mL, n=4 chickens each time point) will be collected at 0, 1, 2, 4, 8, 12, 24, 60, 108 and 180 h post administration of SMs. Plasma will be separated and GI's/QSI's concentrations will be quantitated using HPLC-MS triple QQQ Quantiva at MS&P facility, OSU. For PD study, the collected plasma at different time points as above will be examined for effect on APEC growth and QS inhibition. For the stability study, samples (2 mL, n=3 at each time point) of drinking water will be collected at 0, 4, 8, 12, 16, 20 and 24 h post administration of GI's/QSI's in drinking water of chickens. The concentrations of the GI's/QSI's in drinking water will be quantitated using HPLC-MS triple QQQ Quantiva as above. The growth and QS inhibitory activity of the collected water samples will also be examined. For the other 30 chickens, body weight and feed intake will be measured weekly (day 7, 14, 21, 28, 35, and 42) as described above to determine the impact of GI's/QSI's alone on the BWG and FCR of chickens.Objective 1.3: Efficacy, safety and applicability of 2 best anti-APEC small molecules (from Obj. 1.2) alone or in combination under conditions mimicking the natural APEC infection.We will use 6 groups of commercial broiler chickens (n=70 per group). The groups will be divided as follows: Group 1: non-infected and non-treated group, Group 2: infected and non-treated group, Group 3: infected and best molecule 1 treated group, Group 4: infected and best molecule 2 treated group, Group 5: infected and best molecule 1 + best molecule 2 treated group, and Group 6: infected and antibiotic (sulfadimethoxine) treated group. Chickens will be challenged with Rifr APEC O78 at day 7 orally (1 x 108 CFU/chicken). GI's/QSI's will be administered at their optimal doses in drinking water continuously (changed daily) for 7 days starting at day 8 and end at day 14. Clinical signs and mortality will be recorded for 35 dpi (day 42). After 35 dpi, all live chickens from each group will be euthanized, necropsied, lesions and APEC load in internal organs, including cecum, will be assessed. Mortality, APEC load and APEC lesions in GI's/QSI's treated groups will be compared to control groups to measure the efficacy of GI's/QSI's and their combination. The impact of GI's/QSI's on production performance of chickens will be also assessed by measuring BWG and FCR as described above.Objective 2: Elucidate the mechanisms of action of lead anti-APEC small molecules using metabolomics and affinity based pull-down approaches.Metabolomics approach: APEC O78 grown overnight will be sub-cultured to logarithmic phase (OD600 ~0.6) in M63 media. Fifty ml cultures will be exposed to bacteriostatic and bactericidal concentrations of GI's and 100 µM concentration of QSI's in replicates for 15, 30, 60 and 120 mins. Unexposed APEC culture will be included as control. Following exposure, extracellular and intracellular metabolites of the bacteria will be extracted and untargeted metabolomics will be performed using ThermoFisher QE Plus (Orbitrap) HPLC-MS or Agilent 4650 HPLC-MS Q-TOF. Bioinformatics analysis of mass spectral data will identify significantly altered metabolites specific to GI's/QSI's treatment which will provide insights on GI's/QSI's MOAs.Affinity based pull-down approach: In addition to metabolomics approach, affinity-based pull-down assays will be conducted to identify specific targets of GI's/QSI's. The classical approach to this strategy is the synthesis of biotin-linked probes which can be captured after binding of the drug region to proteins in the cell lysate through treatment with streptavidin. Sequencing of the proteins then reveals potential targets for validation.

Progress 09/01/20 to 07/16/24

Outputs
Target Audience:Veterinarians, Veterinary Daignosticians, Poultry Practitioners, Poultry Producers, Researchers, and Veterinary, Bacteriologists, Pharmaceutical companies, and Immunologists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One post-doctoral researcher and two graduate students have been involved during the project period. The project provided learning opportunities in drug discovery, infectious diseases, animal experiments, molecular biology and related disciplines. Furthermore, this helped in developing collaborations and bringing in multi-disciplinary expertise to navigate the research. This research also contributed to the professional development in the form of attendance to conferences and meetings aiding in fostering new connections, sharing the research output and impact to scientific community and public speaking. How have the results been disseminated to communities of interest?The results from this study were shared through multiple conferences, publications, and scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Determine the efficacy, safety, and applicability of GI-7, GI-10, QSI-5, and QSI-10 in broiler chickens under experimental setup and under conditions mimicking the natural APEC infection. Determined the optimal therapeutic doses of GI-7 and QSI-5 in drinking water: The SMs were administered daily for 24 h (final concentration of 20, 40, and 60 mg/L) in drinking water. GI-7: 60 mg/L treatment reduced 84.7 % mortality, 2.5 logs APEC load in internal organs, and up to 58 % reduction in the severity of APEC lesions; whereas QSI-5: 1 mg/L showed 58 % reduction in mortality compared to the control group; whereas a 25% and 50% reduction was observed in QSI-5: 5 mg/L and QSI-5: 10 mg/L treated groups, respectively. Based on these studies we conclude that the treatment of chickens with GI-7 at 60 mg/L and QSI-5 at 1 mg/L in drinking water resulted in a significant reduction in mortality, lesion scores, and APEC load in chickens. Standardization of oral APEC challenge model: We infected the chickens orally (n=14/group) with APEC either at day 2 or day 7 of age using two different challenge doses (2.5 x 109and 2.5 x 108 CFU/chicken). Results showed that challenge of APEC (109 CFU/chicken) at day 2 resulted in mortality, APEC lesions, and APEC colonization in the cecum and internal organs of chickens as similar to field conditions. Evaluated the efficacy of small molecules GI-7 and QSI-5 individually and in combination against APEC infection in chickens: APEC oral challenge model was used to evaluate the efficacy of small molecules GI-7 and QSI-5 individually and in combination (GI7+ QSI-5) and to perform the comparative evaluation with sulfadimethoxine (SDM), an antibiotic currently used to treat APEC. Our results showed that GI-7, QSI-5, and GI-7+QSI-5 possessed better efficacy against APEC than SDM at lower doses when administered in drinking water. Safety, pharmacokinetic (PK), pharmacodynamics (PD), and stability of GI-7 and QSI-5 under experimental conditions: We evaluated the pharmacokinetic (PK) efficacy, safety, PD, and stability of the optimized doses for QSI-5 (1 mg/L) and GI-7 (60 mg/L) and compared these with the therapeutic dose of sulfadimethoxine (SDM) (495.3 mg/L) (0.05%). Our results showed that QSI-5 and GI-7 were rapidly absorbed = compared to the rate of absorption of SDM. Similarly, our SMs excreted by 24 h, whereas no excretion of SDM was observed until 24 h. In addition, the GI-7 and QSI -5 residues were below the limits permitted by the FDA for antibiotics. The safety of the GI-7 and QS-5 was evaluated by analyzing the cecal microbiota of the treated chickens. The Lactobacillaceae population in GI-7-treated chickens at the family level. Specifically, the abundance of two Lactobacillus species (L. zeae, and L. helveticus,) increased in GI-7-treated chickens compared to the NC. QSI-5 increased Ruminiclostridium and Erysipelatoclostridium; while it reduced the abundance of Enterococcus compared to the NC group. Investigating the impact of SMs on the serum metabolites of chickens: Principal-component analysis (PCA) for GI-7 treated showed no distinct clustering of metabolome profiles between the treatment groups, whereas QSI-5-treated chickens clustered separately from the PC (P = 0.04) and NC groups (P = 0.0007). GI-7 treatment significantly elevated the level of oleate (2-fold; P < 0.05). The QSI-5 treatment significantly reduced the level of 5′-methylthioadenosine (7 - fold; P = 0.0009) involved in the methionine metabolism pathway and spermidine and spermine biosynthesis pathway compared to the positive control group. Synthesis and characterization of derivatives/analogues to enhance the efficacy, safety, and potency of the lead compounds: To enhance the efficacy, potency, and oral bioavailability of the lead compounds we developed derivatives of GI-7 and QSI-5.Analogues were generated utilizing our previously established chemistry and generated intermediates. GI-7 analogues included modifications at the methoxy position, pyrrolidine ring, and alternate substitutions of the N-benzyl group, resulting in the formation of 10 synthetic analogues. The analogues were tested to evaluate their bactericidal effect on APEC O78 and compound KAG-49 with a benzyloxy group instead of the methoxy group in GI-7 exhibited increased activity with a MIC of 50 μM, when compared to the GI-7 with a MIC of 100 μM. The structural modification in the QSI-5 molecule resulted in the formation of 12 analogues, where derivatization focused on alternations within each of the terminal rings and linkage to the piperazine ring. Compounds OA4-108 and OA4-109 displayed improved potency compared to QSI-5, where OA4-108 contains a naphthyl group in place of the 4-methyl phenyl in QSI-5 and OA4-109 contains a 5-chlorobenzothiophene heterocyclic moiety in place of the same 4-methyl phenyl. Both compounds were non-toxic to wax moth larvae and greatly enhanced the survival of infected wax moth larvae (60%-65% survival at 50 μM) with 6-8 log reduction in the APEC O78. These molecules are being extensively studied to identify their mechanism of action. Objective 2: Elucidate the mechanism of action of anti-APEC leads using metabolomics and affinity based pull-down approaches. Gene and protein expression analysis of GI-7 treated APEC: Bacterial cytological profiling of GI-7 treated APEC demonstrated the accumulation of membrane-like material in the form of knobs inside the cells, similar to previous reports, and the commonality of the Lpt inhibitors (responsible for membrane proteins). So, we characterized the potential target of GI-7, LptD through gene expression studies, measuring the levels of Lpt proteins in the membrane and by LC-MS/MS analysis. We also performed in silico docking studies using Autodock and predicted that GI-7 binding to LptD. To validate this further, we cloned the lptD and lptE genes and co-expressed the proteins in E. coli, and purified the proteins using affinity chromatography to perform in-vitro interaction studies with GI-7. We plan to perform protein-ligand interaction studies using isothermal titration calorimetry. Thermal proteome profiling to identify the potential targets for GI-7 and QSI-5: We performed thermal proteome profiling to understand other targets for the SMs in APEC. We identified 55 and 266 differentially enriched proteins from GI-7 and QSI-5, respectively. For GI-7, upregulated proteins in the treated (versus untreated) condition were RecQ, SeqA, and ABC transporter ATP-binding protein at room temperature, and FdhD. For QSI-5, bifunctional ADP-dependent NAD(P)H-hydrate dehydratase/NAD(P)H-hydrate epimerase, DcuA, murein peptide amidase A, DeoR/GlpR family transcriptional regulator, two-component sensor histidine kinase, biotin synthase, hypothetical protein AW108_03710, glutathione S-transferase, and MepM hypothetical protein AW108_01520 were enriched. Synthesis of regioisomeric biotin-linked probes for GI-7 and QSI-5: We performed a pull-down assay using PierceTM Biotinylated Protein Interaction Pull-Down Kit to identify the antibacterial target of GI-7. However, it did not yield the anticipated results. Therefore, we synthesized a series of regioisomeric biotin-linked probes that will be utilized in pull-down experiments with cell lysates to identify the protein target of GI-7 and QSI-5. We identified unique bands of protein in the SDS-PAGE gel in the probe treated wells with different molecular weights; 20-25 kDa, 50-75 kDa, and at 150kDa. These bands are now submitted for LC-MS analysis to identify the potential protein targets of QSI-5. Metabolomic study to identify the altered cellular pathways on exposure to QSI-5, OA4-108, and OA4-109: Intracellular and extracellular metabolite after exposure of the compounds were extracted to identify the potential cellular MOA of QSI-5, OA4-108, and OA4-109. The extracted samples have been submitted for untargeted metabolomics using LC-MS analysis.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: 1. Helmy, Y. A., Kathayat, D., Closs Jr, G., Galgozy, K., Fuchs, J. R., & Rajashekara, G. (2023). Efficacy of quorum sensing and growth inhibitors alone and in combination against avian pathogenic Escherichia coli infection in chickens. Poultry Science, 102(4), 102543.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 2. Helmy, Y. A., Kathayat, D., Deblais, L., Srivastava, V., Closs Jr, G., Tokarski, R. J., ... & Rajashekara, G. (2022). Evaluation of novel quorum sensing inhibitors targeting auto-inducer 2 (AI-2) for the control of avian pathogenic Escherichia coli infections in chickens. Microbiology Spectrum, 10(3), e00286-22.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 3. Kathayat, D., Helmy, Y. A., Deblais, L., Srivastava, V., Closs Jr, G., Khupse, R., & Rajashekara, G. (2021). Novel small molecule growth inhibitor affecting bacterial outer membrane reduces extraintestinal pathogenic Escherichia coli (ExPEC) infection in avian model. Microbiology Spectrum, 9(2), e00006-21.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 4. Kathayat, D., Lokesh, D., Ranjit, S., & Rajashekara, G. (2021). Avian pathogenic Escherichia coli (APEC): an overview of virulence and pathogenesis factors, zoonotic potential, and control strategies. Pathogens, 10(4), 467.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: 5. Kathayat D, Antony L, Deblais L, Helmy YA, Scaria J, Rajashekara G. (2020). Small molecule adjuvants potentiate colistin activity and attenuate resistance development against Escherichia coli by affecting pmrAB system. Infection & Drug Resistance 2020;13:2205-2222.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 1. Thenissery A, Ayinde OR, Galgozy K, Fuchs JR, Rajashekara G. Enhancing potency against avian pathogenic Escherichia coli: In vitro and in vivo characterization of synthetic analogues of a lead quorum sensing inhibitor. CFAES Annual Research Forum, April 9, 2024, College of Food, Agriculture and Environmental Sciences, The Ohio State University. (Poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: 2. Thenissery A, Lokesh D, Helmy YA, Kathayat D, Closs G, Galgozy K, Fuchs JR, Rajashekara G. Investigating efficacy and mechanism of action of novel small molecule inhibitors of avian pathogenic E. coli. Conference of Research Workers in Animal Diseases, January 20 -23, 2024, Chicago, Illinois. (Poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: 3. Rajashekara G, Helmy YA, Kathayat D, Lokesh D, Ayinde OR, Galgozy K, Duran DA, Foster M, Fuchs JR. Novel Therapeutic Leads; Demonstration of efficacy, safety, and applicability of anti-APEC molecules in chickens. Conference of Research Workers in Animal Diseases, January 20th  24th , 2023, Chicago. (Poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: 4. Galgozy K, Lokesh D, Bhandari M, Kathayat D, Rajashekara G, Fuchs JR. Synthesis and Biological Evaluation of Novel Antibacterial Small Molecules and Peptides Against APEC. ACS Spring 2023, March 26 -30, Indianapolis, IN. (Poster)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: 5. Lokesh D, Kathayat D, Rajashekara G. Elucidate of mechanism of action of GI7, a novel small molecule inhibitor of avian pathogenic E coli. Conference of Research Workers in Animal Diseases, Chicago, Dec 5, 2021, IL (Poster).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 6. Kathayat D, Helmy YA, Deblais L, Srivastava V, Closs Jr G, Rajashekara G. Small molecule targeting outer membrane lipopolysaccharide transporter complex (LptD/E) reduces avian pathogenic Escherichia coli (APEC) infection in poultry. CFAES Annual Research Forum, April 21, 2020, College of Food, Agriculture and Environmental Sciences, The Ohio State University. (virtual-poster).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: 7. Helmy YA, Kathayat D, Deblais L, Closs Jr G, Srivastava V, Rajashekara G. Novel anti-virulence compound to control avian pathogenic Escherichia coli (APEC) infections in poultry. CFAES Annual Research Forum, April 21, 2020, College of Food, Agriculture and Environmental Sciences, The Ohio State University. (virtual-poster).


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Veterinarians, Veterinary Daignosticians, Poultry Practitioners, Poultry Producers, Researchers, and Veterinary, Bacteriologists, Pharmaceutical companies, and Immunologists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student and one post-doc arecurrently working on this project. The graduate student and postdoc are being trained in drug discovery, animal experiments and avian health, microbiology, and related disciplines. How have the results been disseminated to communities of interest?The results from this study are being disseminated through conferences, publications, and meetings. What do you plan to do during the next reporting period to accomplish the goals?Objective 1:Determine the efficacy, safety and applicability of GI-7, GI-10, QSI-5, and QSI-10 in broiler chickens under experimental setup and under conditions mimicking the natural APEC infection. We will determine the optimum therapeutic dose of GI-10 and QSI-10 in the drinking water of chickens. We will further characterize the potent QSI-5 derivatives through in-vitro assays and identify the mechanism of action of the same. Objective 2:Elucidate the mechanisms of action of anti-APEC leads using metabolomics and affinity based pull-down approaches. We will do pull-down assays with the biotinylated GI-7 and QSI-5. We will further purify the LptDE using gel filtration chromatography and perform in-vitro interaction studies

Impacts
What was accomplished under these goals? Objective 1: Determine the efficacy, safety and applicability of GI-7, GI-10, QSI-5, and QSI-10 in broiler chickens under experimental setup and under conditions mimicking the natural APEC infection. Evaluated the efficacy of small molecules GI-7 and QSI-5 individually and in combination against avian pathogenic Escherichia coli (APEC) infection in chickens. An APEC oral challenge model was developed to evaluate the efficacy of GI-7 and QSI-5 individually and in combination (GI7+ QSI-5) and to perform the comparative evaluation with sulfadimethoxine (SDM), an antibiotic currently used to treat APEC. Our results showed that GI-7, QSI-5, and GI-7+QSI-5 possessed better efficacy against APEC than SDM at lower doses when administered in drinking water. Overall, GI- 7 and QSI-5 have promising effects as a potential antibiotic-independent approach to control APEC infections in chickens in treated groups compared to the positive control infected and not treated group (PC). Based on these studies, we conclude that when used in combination, the SMs can potentiate each other's anti-APEC activities, where GI-7 can kill APEC while QSI-5 can make APEC avirulent. Therefore, the SM combination can reduce APEC infections in chickens by simultaneously lowering the APEC burden and rendering APEC non-pathogenic. Safety, pharmacokinetic (PK), pharmacodynamics (PD), and stability of GI -7 and QSI-7 under experimental conditions: We evaluated the pharmacokinetic (PK) efficacy, safety, PD, and stability of the optimized doses for QSI-5 (1 mg/L) and GI-7 (60 mg/L) and compared these with the therapeutic dose of sulfadimethoxine (SDM) (495.323 mg/L) (0.05%). For PK efficacy, we administered these compounds to chickens orally as a single dose, and blood was collected from five chickens per group at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h). Plasma proteins were analyzed by LC-MS. Our results showed that QSI-5 and GI-7 were rapidly absorbed (0.5-h post-treatment) compared to the rate of absorption of SDM (2 h). Similarly, our SMs excreted by 24 h, whereas no excretion of SDM was observed until 24 h. We assessed the safety of the compounds by measuring the level of drug residue in the muscle, kidney, and liver using LC-MS. The compounds were administered to chickens in drinking water daily for seven days. Tissue samples were collected individually from chickens at their slaughter age at 2, 5, and 31 days post-last treatment (DPLT). The GI-7 residues were below the limits permitted by the FDA for antibiotics. The safety of the GI-7 and QS-5 was evaluated by analyzing the cecal microbiota of the treated chickens. For the cecal microbiota of chickens, 16S rRNA-based meta-barcoding sequencing was performed, and the resulting data was analyzed with QIIME 2. At the class level, the Bacilli abundance (4% to 14%) at order level Lactobacillales (4% to 14%), at the family level. The Lactobacillaceae population (1% to 11%) in GI-7-treated chickens at the family level. Specifically, the abundance of two Lactobacillus species (L. zeae, 0% to 7%, and L. helveticus, 0% to 4%) increased in GI-7-treated chickens compared to the NC. QSI-52.1%), Ruminiclostridium 5 (0%to 3.3%), and Erysipelatoclostridium (7.2% to 10.8%); while it reduced the abundance of Enterococcus (4% to 0%; P <0.05) compared to the NC group. Investigating the impact of SMs on the serum metabolites of chickens: The untargeted serum metabolome profile was determined using (LC-MS). Principal-component analysis (PCA) for GI-7 treated showed no distinct clustering of metabolome profiles between the treatment groups, whereas QSI-5-treated chickens clustered separately from the PC (P = 0.04) and NC groups (P = 0.0007). GI-7 treatment significantly elevated the level of Oleate (2-fold; P < 0.05). The QSI-5 treatment significantly reduced the level of 5′-methylthioadenosine (7 - fold; P = 0.0009) involved in the methionine metabolism pathway and spermidine and spermine biosynthesis pathway compared to the positive control group. Synthesis of derivatives/analogues to enhance the efficacy, safety, and potency of the lead compounds: To enhance the efficacy, potency, and oral bioavailability of the lead compounds we developed derivatives of the lead compounds. We developed 6 derivatives of the QSI-5 by introducing structural modification in the molecule, which is expected to increase its potency. These compounds have been subjected to series of in-vitro tests to assess their efficacy, and safety profiles. Two compounds have been shown to inhibit autoinducer-2 productions at 50 µM, when compared to QSI-5 which has been shown to be effective at 100 µM in in-vitro assays. These compounds have also proven to be non-hemolytic to chicken and sheep red blood cells and non-toxic to wax moth larvae. Objective 2: Elucidate the mechanisms of action of anti-APEC leads using metabolomics and affinity based pull-down approaches. Elucidate the mechanisms of action of anti-APEC leads using metabolomics and an affinity-based pull-down approach:Synthesis of regioisomeric biotin-linked probes for GI-7 and QSI-5: As mentioned in the proposal, we performed a pull-down assay using PierceTM Biotinylated Protein Interaction Pull-Down Kit to identify the antibacterial target of GI-7. However, it did not yield the anticipated results. Therefore, we synthesized a series of regioisomeric biotin-linked probes that will be utilized in pull-down experiments with cell lysates to identify the protein target of GI-7 and QSI-5. These developed probes have been rigorously tested to ensure their efficacy in retaining the interaction capability of the lead molecule with the target pathway, verified through in-vitro assays using biotinylated probes. Gene and protein expression analysis of GI-7 treated APEC: Bacterial cytological profiling of GI-7 treated APEC demonstrated the accumulation of membrane-like material in the form of knobs inside the cells, similar to previous reports, and the commonality of the Lpt inhibitors (responsible for membrane proteins). So, we characterized the potential target of GI-7, LptD through gene expression studies, measuring the levels of Lpt proteins in the membrane and by LC-MS/MS analysis of the excised ∼21.4-kDa (expected size of LptE protein) gel fragment. We also performed in silico docking studies using Autodock and predicted that GI-7 binding to LptD. To validate this further, we cloned the lptD and lptE genes and co-expressed the proteins in E. coli, and purified the proteins using affinity chromatography to perform in-vitro interaction studies with GI-7. We plan to perform protein-ligand interaction studies using isothermal titration calorimetry. Thermal proteome profiling approach to identify the potential targets for GI-7 and QSI-5: We performed thermal proteome profiling to understand other targets for the SMs in APEC. Protein-drug interactions increase the thermal tolerance of the proteins. Thus, treating the bacteria/whole cell lysate with the drug and subjecting it to temperatures helps to identify the targets of the drug. We identified 55 and 266 differentially enriched proteins from GI-7 and QSI-5, respectively. For GI-7, upregulated proteins in the treated (versus untreated) condition were RecQ, SeqA, and ABC transporter ATP-binding protein at room temperature, and FdhD. For QSI-5, bifunctional ADP-dependent NAD(P)H-hydrate dehydratase/NAD(P)H-hydrate epimerase, DcuA, murein peptide amidase A, DeoR/GlpR family transcriptional regulator, two-component sensor histidine kinase, biotin synthase, hypothetical protein AW108_03710, glutathione S-transferase, and MepM hypothetical protein AW108_01520 were enriched.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Helmy YA, Kathayat D, Closs G Jr, Galgozy K, Fuchs JR, Rajashekara G. Efficacy of quorum sensing and growth inhibitors alone and in combination against avian pathogenic Escherichia coli infection in chickens. Poult Sci. 2023 Apr;102(4):102543. doi: 10.1016/j.psj.2023.102543. Epub 2023 Feb 1. PMID: 36863122; PMCID: PMC10011511.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Gireesh Rajashekara, Yosra A. Helmy, Dipak Kathyat, Dhanashree Lokesh, Oluwatosin R. Ayinde, Katie Galgozy, Antonia D. Duran, Mark Foster, James Fuchs. Novel Therapeutic Leads; Demonstration of efficacy, safety, and applicability of anti-APEC molecules in chickens. CRWAD, January 20th  24th , Chicago, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: 1. Katherine A. Galgozy, Dhanashree, Menuka Bhandari, Dipak Kathayat, Gireesh Rajashekara, James R. Fuchs Synthesis and Biological Evaluation of Novel Antibacterial Small Molecules and Peptides Against APEC. ACS Spring 2023, Indianapolis, IN March 26 -30.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Dhanashree Lokesh, Menuka Bhandari, Gireesh Rajashekara LGG-derived Antimicrobial Peptides As E!ective Antibiotic Alternatives To Treat ExPEC. ASM Microbe, June 15-19, 2023, in Houston.


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Veterinarians, Veterinary Daignosticians, Poultry Practitioners, Poultry Producers, Researchers, and Veterinary, Bacteriologists, Pharmaceutical companies, and Immunologists Changes/Problems:Due to COVID-19 the scheduled animal experiments have been delayed which also delayed our chicken studies. Meantime, we are exploring different novel approaches for uncovering the mechanisms of action of lead small molecules from our studies. What opportunities for training and professional development has the project provided?One graduate student and one post-doc are currently working on this project. How have the results been disseminated to communities of interest?The results from this study are being disseminated through conferences, publications and meetings. What do you plan to do during the next reporting period to accomplish the goals? We will determine the optimum therapeutic dose of GI-10 and QSI-10 in the drinking water of chickens. We will do pull-down assays with the biotinylated GI-7 and QSI-5 We will further purify the LptDE using gel filtration chromatography and perform in-vitro interaction studies

Impacts
What was accomplished under these goals? Evaluated the efficacy of small molecules GI-7 and QSI-5 individually and in combination against avian pathogenicEscherichia coli(APEC) infection in chickens: To evaluate the efficacy of small molecules (SMs) GI-7 and QSI-5 individually and in combination (GI7+ QSI-5) and to perform the comparative evaluation with sulfadimethoxine (SDM), an antibiotic currently used to treat APEC. Our results showed that GI-7, QSI-5, and GI-7+QSI-5 possessed better efficacy against APEC than SDM at lower doses when administered in drinking water. Overall, GI- 7 and QSI-5 have promising effects as a potential antibiotic-independent approach to control APEC infections in chickens in treated groups compared to the positive control (PC). Based on these studies, we conclude that when used in combination, the SMs can potentiate each other's anti-APEC activities, where GI-7 can kill APEC while QSI-5 can make APEC avirulent. Therefore, the SM combination can reduce APEC infections in chickens by simultaneously lowering the APEC burden and rendering APEC non-pathogenic. Safety, Pharmacokinetic (PK),Pharmocodynamics (PD), and stability of GI -7 and QSI-7 under experimental conditions: We evaluated the pharmacokinetic (PK) efficacy, safety, PD, and stability of the optimized doses for QSI-5 (1 mg/L) and GI-7 (60 mg/L) and compared these with the therapeutic dose of sulfadimethoxine (SDM) (495.323 mg/L) (0.05%). For PK efficacy, we administered these compounds to chickens orally as a single dose, and blood was collected from five chickens per group at different time points (0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h). Plasma proteins were analyzed by LC-MS. Our results showed that QSI-5 and GI-7 were rapidly absorbed (0.5-h post-treatment) compared to the rate of absorption of SDM (2 h). Similarly, our SMs excreted by 24 h, whereas no excretion of SDM was observed until 24 h. We assessed the safety of the compounds by measuring the level of drug residue in the muscle, kidney, and liver using LC-MS. The compounds were administered to chickens in drinking water daily for seven days. Tissue samples were collected individually from chickens at their slaughter age at 2, 5, and 31 days post-last treatment (DPLT). The GI-7 residues were below the limits permitted by the FDA for antibiotics. The safety of the GI-7 and QS-5 were evaluated by analyzing the cecal microbiota of the treated chickens. For the cecal microbiota of chickens, 16S rRNA-based meta-barcoding sequencing was performed, and the resulting data was analyzed with QIIME 2. At the class level, the Bacilli abundance (4% to 14%) at order level Lactobacillales (4% to 14%), at the family level. The Lactobacillaceae population (1% to 11%) in GI-7-treated chickens at the family level. Specifically, the abundance of twoLactobacillusspecies (L. zeae, 0% to 7%, andL. helveticus, 0% to 4%) increased in GI-7-treated chickens compared to the NC. QSI-5 treatment increased the abundance of Ruminococcus (torques group) (9% to 6%), Flavonifractor (2.9% to 6%), Lactobacillus (0% to 1.1%), Clostridium sensu stricto 1 (0% to 2.1%), Ruminiclostridium 5 (0%to 3.3%), and Erysipelatoclostridium (7.2% to 10.8%); while it reduced the abundance of Enterococcus (4% to 0%; P <0.05) compared to the NC group. Investigating the impact of SMs on the serum metabolites of chickens: The untargeted serum metabolome profile was determined using (LC-MS).Principal-component analysis (PCA) for GI-7 treated showed no distinct clustering of metabolome profiles between the treatment groups, whereas QSI-5-treated chickens clustered separately from the PC (P = 0.04) and NC groups (P = 0.0007). GI-7 treatment significantly elevated the level of Oleate (2-fold; P < 0.05). The QSI-5 treatment significantly reduced the level of 5′-methylthioadenosine (7 - fold; P = 0.0009) involved in the methionine metabolism pathway and spermidine and spermine biosynthesis pathway compared to the positive control group. Elucidate the mechanisms of action of anti-APEC leads using metabolomics and an affinity-based pull-down approach: Synthesis of regioisomeric biotin-linked probes for GI-7 and QSI-5:As mentioned in the proposal, we performed a pull-down assay using PierceTMBiotinylated Protein Interaction Pull-Down Kit to identify the antibacterial target of GI-7. However, it did not yield the anticipated results. Therefore, we synthesized a series of regioisomeric biotin-linked probes that will be utilized in pull-down experiments with cell lysates to identify the protein target of GI-7 and QSI-5. We also synthesized alcohol and propargyl derivatives of QSI-5 that showed improvements in anti-QS activity. Additionally, we determined the aqueous solubility of QSI-5 and its analogs using a kinetic solubility assay and found that certain derivatives of QSI-5 had better solubility. In our effort to continue identifying anti-QS agents for APEC, the complete characterization of QSI-10, another potent anti-QS agent discovered from the high-throughput screen, will be performed. For this reason, we synthesized quantities of QSI-10 required for in vivo assessment of the molecule. We also plan to synthesize a series of QSI-5 derivatives to optimize their potency and physicochemical properties. Gene and protein expression analysis of GI-7 treated APEC: Bacterial cytological profiling of GI-7 treated APEC demonstrated the accumulation of membrane-like material in the form of knobs inside the cells, similar to previous reports, and the commonality of the Lpt inhibitors (responsible for membrane proteins). So, we characterized the potential target of GI-7, LptD through gene expression studies, measuring the levels of Lpt proteins in the membrane and by LC-MS/MS analysis of the excised∼21.4-kDa (expected size of LptE protein) gel fragment. We also performed in silico docking studies using Autodock and predicted that GI-7 binding to LptD. To validate this further, we cloned the lptD and lptE genes and co-expressed the proteins inE. coli, and purified the proteins using affinity chromatography to perform in-vitro interaction studies with GI-7. We plan to perform protein-ligand interaction studies using isothermal titration calorimetry. Thermal proteome profiling approach to identify the potential targets for GI-7 and QSI-5:We performed thermal proteome profiling to understand other targets for the SMs in APEC. Protein-drug interactions increase the thermal tolerance of the proteins. Thus, treating the bacteria/whole cell lysate with the drug and subjecting it to temperatures helps to identify the targets of the drug. We identified 55 and 266 differentially enriched proteins from GI-7 and QSI-5, respectively. For GI-7, upregulated proteins in the treated (versus untreated) condition were RecQ, SeqA, and ABC transporter ATP-binding protein at room temperature, and FdhD. For QSI-5, bifunctional ADP-dependent NAD(P)H-hydrate dehydratase/NAD(P)H-hydrate epimerase, DcuA, murein peptide amidase A, DeoR/GlpR family transcriptional regulator, two-component sensor histidine kinase, biotin synthase, hypothetical protein AW108_03710, glutathione S-transferase, and MepM hypothetical protein AW108_01520 were enriched.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: D. Lokesh, D. Kathayat, G. Rajashekara. Elucidate of mechanism of action of GI7, a novel small molecule inhibitor of avian pathogenic E coli. Conference of Research Workers in Animal Diseases, Chicago, IL, Dec 5, 2021 (poster).
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: 1. Helmy, Yosra A., Dipak Kathayat, Loic Deblais, Vishal Srivastava, Gary Closs Jr, Robert J. Tokarski, Oluwatosin Ayinde, James R. Fuchs, and Gireesh Rajashekara. "Evaluation of Novel Quorum Sensing Inhibitors Targeting Auto-Inducer 2 (AI-2) for the Control of Avian Pathogenic Escherichia coli Infections in Chickens." Microbiology Spectrum (2022): e00286-22.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: 2. Kathayat, Dipak, Yosra A. Helmy, Loic Deblais, Vishal Srivastava, Gary Closs Jr, Rahul Khupse, and Gireesh Rajashekara. "Novel Small Molecule Growth Inhibitor Affecting Bacterial Outer Membrane Reduces Extraintestinal Pathogenic Escherichia coli (ExPEC) Infection in Avian Model." Microbiology spectrum 9, no. 2 (2021): e00006-21


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Veterinarians, Veterinary Daignosticians, Poultry Practitioners, PoultryProducers, Researchers, and Veterinary, Bacteriologists, Pharmaceutical companies, and Immunologists, Changes/Problems:The scheduled animal experiments have been postponed due to COVID-19 which will delay our animal studies. We are exploring different novel approaches for uncovering the mechanisms of action of lead small molecules. What opportunities for training and professional development has the project provided?One graduate student and one post-doc are currently working on this project. How have the results been disseminated to communities of interest?The results from this study are being disseminated through conferences, publications and meetings. What do you plan to do during the next reporting period to accomplish the goals? We will determine the optimum therapeutic dose of GI-10 and QSI-10 in the drinking water of chickens We will determine the efficacy, safety, PK, PD, and stability of GI's/QSI's under experimental conditions We will use a Thermal proteome profiling assay for quorum sensing molecule, QSI-5 Full characterization of each intermediate (13C NMR and mass spectrometry) as well as purity determination of batches of GI-7 using HPLC. We will chemically add linker and then biotin to GI-7 to prepare probes to perform the pulldown assay. We will also perform a metabolomic approach to predict the Mechanism of action.

Impacts
What was accomplished under these goals? Objective 1:Determine the efficacy, safety and applicability of GI-7, GI-10, QSI-5 and QSI-10 Determined optimal therapeutic doses of GI-7 and QSI-5 in drinking water:We performed two chicken experiments for both growth (GI-7) and quorum sensing (QSI-5) inhibitors based on the preliminary results obtained via oral gavage.In our first chicken experimentchickens (n=11/group) were treated with GI-7 and QSI-5 from day 3 for 7 consecutive days. On day 4, chickens were infected (s/c) with rifampicin resistant (Rifr) APEC O78 (1 x 107CFU/chicken). The SMs were administered daily for 24 h (final concentration of 20, 40, and 60 mg/L) in drinking water. GI-7: 60 mg/L treatment reduced 84.7 % mortality, 2.5 logs APEC load in internal organs, and up to 58 % reduction in the severity of APEC lesions; whereas, QSI-5: 20 mg/L treatment only reduced 69.7 % mortality, 1.5 logs APEC load in internal organs and up to 40.9 % reduction in the severity of APEC lesions. In addition, we found increasing efficacy of QSI-5 with decreasing dose, and QSI-5 displayed higher efficacy at 1 mg/L when orally gavaged in our pilot experiment. So, we repeated the dose response study for QSI-5 (1, 5, and 10 mg/L) in another chicken experiment. Chickens treated with QSI-5: 1 mg/L showed 58 % reduction in mortality compared to the control group; whereas, a 25% and 50% reduction was observed in QSI-5: 5 mg/L and QSI-5: 10 mg/L treated groups, respectively. Based on these studies we conclude that the treatment of chickens with GI-7 at 60 mg/L and QSI-5 at 1 mg/L in drinking water resulted in a significant reduction in mortality, lesion scores, and APEC load in chickens. Standardization of oral APEC challenge model:We infected the chickens orally (n=14/group) with APEC either at day 2 or day 7 of age using two different challenge doses (2.5 x 109and 2.5 x 108CFU/chicken). The mortality of chickens was monitored until 7 DPI. At 8 DPI, chickens were euthanized, the severity of the pathological lesions in internal organs was scored, and the APEC load in the cecum and internal organs was determined by plating. Mortality of 50 % and 14 % were observed in chickens challenged at day 2 with 109and 108CFU, respectively; whereas, 7 % and 0 % mortality were observed in chickens challenged at day 7 with 109and 108CFU, respectively. Average APEC load of 8.6 and 7.3 logs was quantitated in the cecum of chickens challenged at day 2 with 109and 108CFU, respectively; whereas, 7.7 and 7.1 logs were observed in chickens challenged at day 7 with 109and 108CFU, respectively. In internal organs (liver, heart, lung, and kidney), chickens challenged at day 2 with 109CFU had the highest APEC load (3.0 to 5.1 logs), followed by chickens challenged at day 2 with 108CFU (2.1 to 2.4 logs), chickens challenged at day 7 with 109CFU (0.4 to 1.1 logs), and chickens challenged at day 7 with 108CFU (up to 0.5 logs). Consistently, the cumulative lesions score was highest in chickens challenged at day 2 with 109 CFU (4.0), followed by chickens challenged at day 2 with 108CFU (2.6), chickenschallenged at day 7 with 109CFU (0.8), and chickens challenged at day 7 with 108CFU (0.2). So, we conclude that the challenge of APEC (109CFU/chicken) at day 2 resulted in mortality, APEC lesions, and APEC colonization in the cecum and internal organs of chickens as similar to field conditions. Objective 2:Elucidate the mechanisms of action of anti-APEC leads Thermal proteome profiling approach to identify the potential targets for GI-7:We performed a pulldown assay using PierceTM Biotinylated Protein Interaction Pull-Down Kit to identify the antibacterial target of GI-7. However, it did not yield anticipated results.Adding a linker to GI-7 helps with biotinylation and we are working on it. Meanwhile, we used a quantitative LC-MS-based thermal proteome profiling approach to identify potential targets for GI-7. Briefly, Bacteria were grown to the OD600of 0.1 and exposed to different drug concentrations for 20 mins. Then, cells were pelleted, washed with PBS, and 100 µl of 10 OD600treated and untreated cells were aliquoted into a 96-well PCR plate. Then, spun to remove the 80 µl of supernatant and, subject the plate containing 20 µl cells to temperature gradient ranges with 42 to 72 °C (n=11) for 3 mins followed by at 25 °C for 3 min. One set of both drug-treated and untreated samples were kept without subjecting to temperature gradient as temperature control. Cells were lysed followed by three freeze-thaw cycles in liquid nitrogen and room temperature with a quick vortex in between each cycle--separated the lysate containing soluble proteins by spinning down the debris, followed by filtration.Proteins were digested via STrap and subjected to capillary-liquid chromatography-nano spray tandem mass spectrometry (Capillary-LC/MS/MS) operated in positive ion mode. MS/MS data were acquired and searched using Mascot Daemon by Matrix Science version 2.7.0 (Boston, MA) via Proteome Discoverer (version 2.4 Thermo Scientific), and the database searched against the most recent Uniprot databases. A decoy database was also searched to determine the false discovery rate (FDR), and peptides were filtered at 1% FDR. Proteins identified with at least two unique peptides were considered as reliable identification. Any modified peptides are manually checked for validation. To understand the protein targets, we analyzed the untreated raw data, determined the total number of proteins, and calculated the fold change by comparing it with control. The low abundant proteins were excluded from the list by comparing with the room temperature control. In the presence of GI-7, the assay uncovered 472 targets from different temperature-treated pools with increased abundance out of 2155 total proteins obtained after spectral normalization. Synthesis of GI-7:The synthesis of GI-7 has not been previously reported in the literature. The relatively short synthetic sequence provides access to GI-7 in three synthetic steps from readily available commercial reagents. To this end, a solution of isovanillin1, epichlorohydrin2, and potassium carbonate in DMF and 2% H2O was utilized to prepare epoxide3. The original reaction conditions were based on work by Campbell et al.1and optimized to increase the yield. This optimization included heating the reaction to 60°C, doubling the equivalents of2, decreasing the concentration to 0.25 M, and adding 2% H2O to promote solubility of the inorganic base. The second step in this scheme was also based on the work of Campbell et al.,1which involves the addition of pyrrolidine4into the epoxide of3to smoothly provide5. The final step involves the reductive amination of5with 3-(trifluoromethyl)benzylamine6in the presence of sodium borohydride. This reaction is based on the conditions reported by Troelsen et al.,2although the amine was allowed to stir in the presence of aldehyde5for a longer period of time to promote complete imine formation prior to the addition of the reducing agent. Each intermediate as well as the final product was purified with normal phase silica gel column chromatography, and structures were confirmed using1H NMR. ?Synthesis of probe molecules for target identification studies: With the synthetic route to GI-7 established, we have also begun exploring the preparation of probe molecules for mechanism of action studies. The initial goal is to synthesize a series of biotin linked probes that will be utilized in pull-down experiments with cell lysates to identify the protein target of GI-7. The linker will be introduced at multiple locations on GI-7 since the binding pose of the compound is currently unknown and the attached linker may interfere with binding to the protein at certain positions. Biotin will then be connected through the Click reaction for the pull-down experiment. Prote The

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Kathayat D, Closs Jr G, Rajashekara G. 2020. Peptides affecting membrane phospholipid transport as novel therapeutics against avian pathogenic Escherichia coli (APEC). CRWAD, Dec. 5-8, 2020 (virtual-oral).
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Kathayat D, Antony L, Deblais L, Helmy YA, Scaria J, Rajashekara G. 2020. Small molecule adjuvants potentiate colistin activity and attenuate resistance development against Escherichia coli by affecting pmrAB system. Infection & Drug Resistance 2020;13:2205-2222.
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Kathayat D, Lokesh D, Ranjit S, Rajashekara G. Avian Pathogenic Escherichia coli (APEC): An Overview of Virulence and Pathogenesis Factors, Zoonotic Potential, and Control Strategies. Pathogens. 2021 Apr;10(4):467
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Kathayat D, Closs Jr G, Helmy YA, Lokesh D, Ranjit S, Rajashekara G. Peptides affecting outer membrane lipid asymmetry (MlaA-OmpC/F) system reduce avian pathogenic Escherichia coli (APEC) colonization in chickens. Applied and Environmental Microbiology. 2021 Jun 16:AEM-00567.