Introduction Curcumin EF-24 β -Lactoglobulin Binding efficacy of different polyphenolic Human Serum Albumin phytochemicals with β -Lactoglobulin Methodology and Human Serum Albumin: Docking with Auto-Dock Implication for therapeutics against neurodegenerative diseases Auto-Dock Algorithms Auto-Dock Docking . . . Rene Duran Home Page Andres Ortiz Title Page Departments of Chemistry & Mathematical Sciences ◭◭ ◮◮ University of Texas at El Paso El Paso, Texas 79968, USA ◭ ◮ rlduran@miners.utep.edu, aortiz19@miners.utep.edu Page 1 of 22 supervised by Go Back Vladik Kreinovich (vladik@utep.edu) & Mahesh Narayan (narayan@utep.edu) Full Screen This work is supported in part by NSF grant DUE-0926721, NIH grant 5G12RR008124-18, ADRF, and the Undergraduate Participation in Bioinformatics Training Close Quit
Introduction Curcumin 1. Introduction EF-24 • Nitrosative stress has recently been demonstrated as a β -Lactoglobulin crucial causal factor in the pathogenesis of Parkinson’s Human Serum Albumin (PD) and Alzheimer’s (AD) diseases. Methodology Docking with Auto-Dock • Specifically, increased levels of NO disrupt the redox Auto-Dock Algorithms activity of protein-disulfide isomerase, a key endoplas- Auto-Dock Docking . . . mic reticulum-resident chaperone by S-nitroso modifi- Home Page cation of its redox-active cysteines. Title Page • This leads to aggregation of misfolded proteins in AD and PD. ◭◭ ◮◮ ◭ ◮ Page 2 of 22 Go Back Full Screen Close Quit
Introduction Curcumin 2. Curcumin EF-24 • Derivative of turmeric (Indian spice). β -Lactoglobulin Human Serum Albumin • Small ligand molecule. Methodology • Well known free radical scavenger. Docking with Auto-Dock • Low bioavailability. Auto-Dock Algorithms Auto-Dock Docking . . . Home Page Title Page ◭◭ ◮◮ ◭ ◮ Page 3 of 22 Go Back (1E,6E)-1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6- Full Screen heptadiene-3,5-dione Close (Curcumin). Quit
Introduction Curcumin 3. EF-24 EF-24 • Curcumin analog. β -Lactoglobulin Human Serum Albumin • Small ligand molecule. Methodology • High bioavailability. Docking with Auto-Dock • Potent nitrosative stress scavenger. Auto-Dock Algorithms Auto-Dock Docking . . . Home Page Title Page ◭◭ ◮◮ ◭ ◮ 3, 5-bis (2-flurobenzylidene) piperidin-4-one (EF-24). Page 4 of 22 Go Back Full Screen Close Quit
Introduction Curcumin β -Lactoglobulin 4. EF-24 β -Lactoglobulin Human Serum Albumin Methodology Docking with Auto-Dock Auto-Dock Algorithms Auto-Dock Docking . . . Home Page Title Page β Lactoglobulin structure (PDB: 1B8E). ◭◭ ◮◮ ◭ ◮ Page 5 of 22 Go Back Full Screen Close Quit
Introduction Curcumin 5. Human Serum Albumin EF-24 β -Lactoglobulin Human Serum Albumin Methodology Docking with Auto-Dock Auto-Dock Algorithms Auto-Dock Docking . . . Home Page Title Page ◭◭ ◮◮ ◭ ◮ Page 6 of 22 Go Back Full Screen Close Quit
6. Methodology Introduction Curcumin EF-24 β -Lactoglobulin Human Serum EF24-2.5mL stock Human Serum Albumin Albumin (30mg/40ml solution of 3mM Methodology or 13.4 µ M) dissolved was first prepared Docking with Auto-Dock in 200 mM Tris-HCl, in water/ethanol Auto-Dock Algorithms 1mM EDTA buffer 50% and then Auto-Dock Docking . . . (pH 8) (50mL of diluted to 0.500 µ M, β -Lactoglobulin 1mM, and 2mM in prepared the same). water/ethanol 50%. Home Page Title Page ◭◭ ◮◮ UV/Vis Spec- Charcoal Treatment trophotometry tests ◭ ◮ Similarly, a stock (fatty acid removal) were conducted solution 2.5mL of 30 minutes of ultra- to determine con- Page 7 of 22 curcumin solution centrifugation at max centration (Beer’s was prepared. speed (4000rpm/rcf). law was applied to Go Back the absorbances). Full Screen Close Quit
Introduction Curcumin Fluorescence Spec- troscopy (DM-45 EF-24 Spectrofluo- β -Lactoglobulin rimeter [Olis]). Human Serum Albumin Methodology Docking with Auto-Dock Data Collection Raw Auto-Dock Algorithms data incorporated Auto-Dock Docking . . . with Sigma Plot. Home Page Title Page ◭◭ ◮◮ ◭ ◮ Results interpreted. Page 8 of 22 Go Back Full Screen Close Quit
Introduction Curcumin 7. Docking with Auto-Dock EF-24 • Energy scoring function β -Lactoglobulin Human Serum Albumin Methodology Docking with Auto-Dock Auto-Dock Algorithms Auto-Dock Docking . . . Home Page Title Page ◭◭ ◮◮ ◭ ◮ ∆ G = ( V L − L bound − V L − L unbound ) + ( V P − P bound − V P − P unbound ) Page 9 of 22 +( V L − P bound − V L − P unbound + ∆ S conf ) . Go Back Full Screen Close Quit
Introduction Curcumin – Semi-empirical force field EF-24 β -Lactoglobulin Human Serum Albumin � A ij − B ij � � V = W vdw r 12 r 6 Methodology ij ij i,j Docking with Auto-Dock Auto-Dock Algorithms � C ij � − C ij � + W hbond E ( t ) r 12 r 10 Auto-Dock Docking . . . ij ij i,j Home Page q i q j � + W elec Title Page ǫ ( r ij ) r ij i,j ◭◭ ◮◮ � ( S i V j + S j V i ) e − r 2 ij / 2 σ 2 . ◭ ◮ + W sol i,j Page 10 of 22 Go Back Full Screen Close Quit
Introduction Curcumin 8. Auto-Dock Algorithms EF-24 • Algorithms β -Lactoglobulin Human Serum Albumin – Simulated annealing Methodology – Local search Docking with Auto-Dock – Genetic algorithm Auto-Dock Algorithms – Lamarckian algorithm. Auto-Dock Docking . . . Home Page Title Page ◭◭ ◮◮ ◭ ◮ Page 11 of 22 Go Back Full Screen Close Quit
Introduction Curcumin 9. Auto-Dock Docking Procedure EF-24 • Obtaining PDB files for • Preparing ligand β -Lactoglobulin protein and ligand. Human Serum Albumin – Specifying rotatable Methodology bonds. • Preparing protein file Docking with Auto-Dock • Specifying flexible Auto-Dock Algorithms – Deleting waters and resides (if known). extra atoms Auto-Dock Docking . . . Home Page • Grid parameters. – Adding hydrogens. Title Page • Docking parameters. ◭◭ ◮◮ ◭ ◮ Page 12 of 22 Go Back Full Screen Close Quit
Introduction Curcumin EF-24 β -Lactoglobulin Human Serum Albumin Methodology Docking with Auto-Dock Auto-Dock Algorithms Auto-Dock Docking . . . Home Page Title Page ◭◭ ◮◮ ◭ ◮ Page 13 of 22 Go Back Full Screen Close Quit
Introduction Curcumin 10. Docking with Glide EF-24 • Energy scoring function (Chemscore) β -Lactoglobulin Human Serum Albumin – Empirically based Methodology � ∆ G bind = C 0 + C lipo f ( r lr ) Docking with Auto-Dock Auto-Dock Algorithms � � + C hbond g (∆ r ) h (∆ α )+ C metal f ( r lm )+ C rotb H rot . Auto-Dock Docking . . . Home Page • Docking algorithm Title Page – Conformation generation ◭◭ ◮◮ – Initial screening of ◭ ◮ ligand poses Page 14 of 22 – Energy minimization Go Back using molecular mechanics scoring Full Screen function. Close Quit
Introduction Curcumin 11. Glide docking procedure EF-24 • Importing PDB file. β -Lactoglobulin Human Serum Albumin • Preparing protein (Protein preparation wizard). Methodology • Preparing ligands (LigPrep). Docking with Auto-Dock • Grid parameters. Auto-Dock Algorithms Auto-Dock Docking . . . • Docking parameters. Home Page Title Page ◭◭ ◮◮ ◭ ◮ Page 15 of 22 Go Back Full Screen Close Quit
Introduction Curcumin 12. Results to Date EF-24 β -Lactoglobulin Human Serum Albumin Methodology Docking with Auto-Dock Auto-Dock Algorithms Auto-Dock Docking . . . Home Page Title Page ◭◭ ◮◮ ◭ ◮ Page 16 of 22 Go Back Full Screen Close Quit
Introduction Curcumin EF-24 β -Lactoglobulin Human Serum Albumin Methodology Docking with Auto-Dock Auto-Dock Algorithms Auto-Dock Docking . . . Home Page Title Page ◭◭ ◮◮ ◭ ◮ Page 17 of 22 Go Back Full Screen Close Quit
Introduction Curcumin EF-24 β -Lactoglobulin Human Serum Albumin Methodology Docking with Auto-Dock Auto-Dock Algorithms Auto-Dock Docking . . . Home Page Title Page ◭◭ ◮◮ ◭ ◮ Page 18 of 22 Go Back Full Screen Close Quit
Introduction Curcumin EF-24 β -Lactoglobulin Human Serum Albumin Methodology Docking with Auto-Dock Auto-Dock Algorithms Auto-Dock Docking . . . Home Page Title Page ◭◭ ◮◮ ◭ ◮ Page 19 of 22 Go Back Full Screen Close Quit
Introduction Curcumin 13. Conclusions EF-24 • By conducting extensive spectrofluorimetry experiments, β -Lactoglobulin we have determined the binding efficacy of curcumin to Human Serum Albumin β -Lactoglobulin and curcumin to human serum albu- Methodology min, which is formidable. Docking with Auto-Dock Auto-Dock Algorithms • Similarly, the same spectrofluorimeter experiments were Auto-Dock Docking . . . carried out and we have determined that the binding Home Page efficacy of this EF-24 to β -Lactoglobulin and human serum albumin is exceptional. Title Page • Using docking results on binding affinities we will be ◭◭ ◮◮ able to reproduce a binding curve and validate by com- ◭ ◮ paring to experimental results. Page 20 of 22 Go Back Full Screen Close Quit
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