Early Stage Efficacy and Toxicology Screening for Antibiotics and Enzyme Inhibitors

The rise in organisms resistant to existing drugs has added urgency to the search for new antimicrobial agents. Aspartate β-semialdehyde dehydrogenase (ASADH) catalyzes a critical step in an essential microbial pathway that is absent in mammals. Our laboratory is using fragment library screening to identify efficient and selective ASADH inhibitors. These preliminary agents are then tested to identify compounds with desired antimicrobial properties for further refinement. Towards this end, we have established a microplate-based, dual assay approach using a single reagent to evaluate antibiotic activity and mammalian cell toxicity during early stage screening. The bacterial assay utilizes non-pathogenic bacteria to allow efficacy testing without a dedicated microbial laboratory. Toxicity assays are performed with a panel of mammalian cells derived from representative susceptible tissues. These assays can be adapted to target other microbial systems, such as fungi and biofilms, and additional mammalian cell lines can be added as needed. Application of this screening approach to antibiotic standards demonstrates the ability of these assays to identify bacterial selectivity and potential toxicity issues. Tests with ASADH inhibitors show some compounds with antibiotic activity, but as expected most of these early agents display higher than desired mammalian cell toxicity

Early Stage Efficacy and Toxicology Screening for Antibiotics and Enzyme Inhibitors

The rise in organisms resistant to existing drugs has added urgency to the search for new antimicrobial agents. Aspartate β-semialdehyde dehydrogenase (ASADH) catalyzes a critical step in an essential microbial pathway that is absent in mammals. Our laboratory is using fragment library screening to identify efficient and selective ASADH inhibitors. These preliminary agents are then tested to identify compounds with desired antimicrobial properties for further refinement. Towards this end, we have established a microplate-based, dual assay approach using a single reagent to evaluate antibiotic activity and mammalian cell toxicity during early stage screening. The bacterial assay utilizes non-pathogenic bacteria to allow efficacy testing without a dedicated microbial laboratory. Toxicity assays are performed with a panel of mammalian cells derived from representative susceptible tissues. These assays can be adapted to target other microbial systems, such as fungi and biofilms, and additional mammalian cell lines can be added as needed. Application of this screening approach to antibiotic standards demonstrates the ability of these assays to identify bacterial selectivity and potential toxicity issues. Tests with ASADH inhibitors show some compounds with antibiotic activity, but as expected most of these early agents display higher than desired mammalian cell toxicity

1 Molecular Docking and Enzymatic Evaluation to Identify Selective Inhibitors of Aspartate Semialdehyde Dehydrogenase

Microbes that have gained resistance against antibiotics pose a major emerging threat to human health. New targets must be identified that will guide the development of new classes of antibiotics. The selective inhibition of key microbial enzymes that are responsible for the biosynthesis of essential metabolites can be an effective way to counter this growing threat. Aspartate semialdehyde dehydrogenases (ASADHs) produce an early branch point metabolite in a microbial biosynthetic pathway for essential amino acids and for quorum sensing molecules. In this study, molecular modeling and docking studies were performed to achieve two key objectives that are important for the identification of new selective inhibitors of ASADH. First, virtual screening of a small library of compounds was used to identify new core structures that could serve as potential inhibitors of the ASADHs. Compounds have been identified from diverse chemical classes that are predicted to bind to ASADH with high affinity. Next, molecular docking studies were used to prioritize analogs within each class for synthesis and testing against representative bacterial forms of ASADH from Streptococcus pneumoniae and Vibrio cholerae. These studies have led to new micromolar inhibitors of ASADH, demonstrating the utility of this molecular modeling and docking approach for the identification of new classes of potential enzyme inhibitors

1 Molecular Docking and Enzymatic Evaluation to Identify Selective Inhibitors of Aspartate Semialdehyde Dehydrogenase

Microbes that have gained resistance against antibiotics pose a major emerging threat to human health. New targets must be identified that will guide the development of new classes of antibiotics. The selective inhibition of key microbial enzymes that are responsible for the biosynthesis of essential metabolites can be an effective way to counter this growing threat. Aspartate semialdehyde dehydrogenases (ASADHs) produce an early branch point metabolite in a microbial biosynthetic pathway for essential amino acids and for quorum sensing molecules. In this study, molecular modeling and docking studies were performed to achieve two key objectives that are important for the identification of new selective inhibitors of ASADH. First, virtual screening of a small library of compounds was used to identify new core structures that could serve as potential inhibitors of the ASADHs. Compounds have been identified from diverse chemical classes that are predicted to bind to ASADH with high affinity. Next, molecular docking studies were used to prioritize analogs within each class for synthesis and testing against representative bacterial forms of ASADH from Streptococcus pneumoniae and Vibrio cholerae. These studies have led to new micromolar inhibitors of ASADH, demonstrating the utility of this molecular modeling and docking approach for the identification of new classes of potential enzyme inhibitors

Bilirubin binds to the ligand-binding pocket of PPARα.

<p><b>(A)</b> Bilirubin docked into PPARα binding pocket. <b>(B)</b> Bilirubin binds in the same site occupied by the known PPARα ligand GW735 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153427#pone.0153427.ref019" target="_blank">19</a>]. Bilirubin and the ligand are depicted in green and magenta carbon skeleton, respectively.</p

Bilirubin binds directly to PPARα to increase endogenous gene activity.

<p><b>(A)</b> Western of PPARα and HSP90 in lentiviral overexpression of PPARα and vector in 3T3-L1 cells. <b>(B)</b> Bilirubin or WY 14,643 linked sepharose resins were used to determine direct binding to PPARα. <b>(C)</b> Bilirubin or biliverdin linked sepharose resins were used to determine direct binding to PPARα. <b>(D)</b> The PPARα overexpression and vector 3T3-L1 cells were treated for 24 hours with biliverdin (BV) (50 μM), WY 14,643 (WY) (50 μM), or fenofibrate (Feno) (50 μM). RNA was extracted and CD36, CPT1, and FGF21 expression was measured by Real-time PCR. ***, <i>p</i> < 0.001 (<i>versus</i> veh 3T3-Vector); ^, <i>p</i> < 0.05 (<i>versus</i> veh 3T3-PPARα); ^^, <i>p</i> < 0.01 (<i>versus</i> veh 3T3-PPARα); ^^^, <i>p</i> < 0.001 (<i>versus</i> veh 3T3-PPARα); $, <i>p</i> < 0.05 (<i>versus</i> WY 3T3-PPARα);$ , p < 0.01 (versus WY 3T3-PPARα); #, p < 0.05 (versus BV 3T3-PPARα); (±S.E.; n = 3). (E) The mouse hepa1c1c7 liver cells overexpressing PPARα were treated in dialyzed FBS for 24 hours with biliverdin (BV) (50 μM), WY 14,643 (WY) (50 μM), or fenofibrate (Feno) (50 μM). RNA was extracted and mRNA expression was measured by Real-time PCR. ^, p < 0.05, ^^, p < 0.01, and ^^^, p < 0.001 (versus veh 3T3-PPARα); , <i>p</i> < 0.05, $$, <i>p</i> < 0.01, $$ $, <i>p</i> < 0.001,$ $$ $, <i>p</i> < 0.0001 (<i>versus</i> WY 3T3-PPARα); ###, <i>p</i> < 0.001, #, <i>p</i> < 0.01, ####, <i>p</i> < 0.0001 (<i>versus</i> BV 3T3-PPARα); (±S.E.; <i>n</i> = 3).</p

Bilirubin reduces body weight and body fat percentage.

<p>WT and PPARα KO mice were on a high fat diet for 6 weeks and treated with fenofibrate (FF) or bilirubin (BR) for seven days and body weight <b>(A)</b>, percent body fat <b>(B)</b>, and lean mass <b>(C)</b> were measured. a, <i>p</i> < 0.05 (KO <i>versus</i> WT Ctrl); b, <i>p</i> < 0.05 (WT FF or BR treated <i>versus</i> WT Ctrl) (±S.E.; <i>n</i> = 5).</p

Structural of PPARα ligands.

<p><b>(A)</b> Comparison of structures of WY 14, 643, fenofibrate and bilirubin. <b>(B)</b> Arachidonic acid is the precursor for CYP epoxygenase (2C and 2J) production of 5,6-, 8,9-, 11,12-, and 14, 15- epoxyeicosatrienoic acids (EETs).</p