Mr.G.Thirupugal II M.Sc., Microbial Gene Technology Department of Microbial Technology

1. Designing a Peptide Aptamer to Unfold Pancreatic Cholesterol Esterase - Treatment of Obesity By Mr.G.Thirupugal II M.Sc., Microbial Gene Technology Department of Microbial Technology School of Biological Sciences Madurai Kamaraj University

2. Overweight and obesity result from an energy imbalance. This involves eating too many calories and not getting enough physical activity.  • Body weight is the result of genes, metabolism, behavior, environment, culture, and socioeconomic status.  • Behavior and environment play a large role causing people to be overweight and obese. These are the greatest areas for prevention and treatment actions INTRODUCTION

3. OBESITY • What is it? • How is it measured? • What causes it? • What are the health risks? • What can you do about it?

4. What Is Obesity? Overweight Overweight as an excess amount of body weight that includes muscle, bone, fat, and water. Obesity Obesity specifically refers to an excess amount of body fat. Some people, such as bodybuilders or other athletes with a lot of muscle, can be overweight without being obese.

5. How Is Obesity Measured? Measuring the exact amount of a person's body fat is not easy. The most accurate measures are to weigh a person underwater or to use an X-ray test called Dual Energy X-ray Absorptiometry (DEXA). In recent years, body mass index (BMI) has become the medical standard used to measure overweight and obesity.

6. What Is Body Mass Index BMI uses a mathematical formula based on a person's height and weight. BMI equals weight in kilograms divided by height in meters squared (BMI = kg/m2). A BMI of 25 to 29.9 indicates a person is overweight. A person with a BMI of 30 or higher is considered obese.

7. Calculate Your Risk

8. BMI Weight Status Below 18.5 Underweight 18.5 – 24.9 Normal 25.0 – 29.9 Overweight 30.0 Above Obese Body Mass Index for Adults Body Mass Index or BMI is a tool for indicating weight status in adults.It is a measure of weight for height. For adults over 20 years old, BMI falls into one of these categories:

9. What Causes Obesity In scientific terms, obesity occurs when a person consumes more calories than he or she burns. What causes this imbalance between calories in and calories out may differ from one person to another. • Genetic • Environmental • Psychological • Other Factors

10. Health Consequences • Approximately 280,000 adult deaths in the United States each year are related to obesity. • Several serious medical conditions have been linked to obesity, including • Type 2 Diabetes • Heart Disease • High Blood Pressure • Stroke • Cancer

11. Diseases Linked to Obesity • Gallbladder disease and gallstones • Liver disease • Osteoarthritis, a disease in which the joints deteriorate. This is possibly the result of excess weight on the joints • Gout, another disease affecting the joints • Pulmonary (breathing) problems, including sleep apnea in which a person can stop breathing for a short time during sleep • Reproductive problems in women, including menstrual irregularities and infertility

12. TREATMENT OF OBESITY

13. How Is Obesity Treated? The method of treatment depends on your level of obesity, overall health condition, and motivation to lose weight. Treatment may include a combination of diet, exercise, behavior modification, and weight-loss drugs. In some cases of severe obesity, gastrointestinal surgery may be recommended.

14. Weight Loss Drug Brand Name Dexfenfluramine Redux (withdrawn) Diethylpropion Tenuate, Dospan Fenfluramine Pondimin (withdrawn) Mazindol Sanorex, Mazanor Orlistat Xenical Phendimetrazine Bontril, Plegine, Prelu-2, X-Trozine Phentermine Adipex-P, Fastin, Ionamin, Oby-trim Sibutramine Meridia FDA-Approved Prescription Weight Loss Drugs

15. Appetite-suppressant medications Appetite-suppressant medications promote weight loss by decreasing appetite or increasing the feeling of being full. Inhibitors of Dietary Cholesterol absorption In 1999, the drug orlistat was approved by the Food and Drug Administration (FDA) as an obesity treatment. Orlistat works by reducing the body's ability to absorb dietary fat by about one third. Drugs For Obesity Treatment

16. Inhibitors of Dietary Cholesterol Absorption

17. PANCREATIC CHOLESTEROL ESTERASE

18. Pancreatic Cholesterol Esterase • Cholesterol esterase plays an important role in the dietary uptake of triacylglycerol and cholesterol esters. • It presents major lipolytic activity that is secreted from vertebrate pancreas into the intestinal tract. • Origin of the protein • Gene name - cel • synonyms- bal • EC 3.1.1.13 • Synonyms • Bile Salt activated lipase • Carboxylester lipase • Sterol esterase • Pancreatic lysophospholipase

19. Function of Pancreatic Cholesterol Esterase • Pancreatic cholesterol esterase has three proposed functions in the intestine. • To control the bioavailability of cholesterol from dietary cholesterol esters. • To contribute to incorporation of cholesterol into mixed micelles • To aid in transport of free cholesterol to the enterocytes

20. Catalytic Action of Pancreatic Cholesterol Esterase Hydrolysis of cholesterol ester in the lumen of the small intestine is catalyzed by cholesterol esterase, which liberates free cholesterol. Free cholesterol mixes with cholesterol contained in bile secretions to form the pool of absorbable cholesterol

21. Intestinal Cholesterol Absorption

22. Structure of Pancreatic Cholesterol Esterase • A crystal structure of the catalytic domain of human bile salt activated lipase or cholesterol esterase (CEL) contain 538 residues. • The crystal structure belongs to space group p212121. • The protein shows an / hydrolase fold. • The catalytic triad ser194 - His435- Asp320 • Surface Loops • 420 loop residues 423-433 • 270 loop residues 270-285 • 70 loop residues 65-75 • 120 loop residues 115-125 it is necessary for a cholestryl ester substrate to bind to cholesterol esterase

23. Structure of Pancreatic Cholesterol Esterase

24. Continue… PDB ID : 2BCE, 1F6W,1JMY 2BCE

25. In silico Analysis of Cholesterol Esterase • Secondary Structure Prediction • Multalign analysis • 3D Structure analysis • Cn3D 4.1 • UCSF Chimera • Chemera

26. Multalign Analysis of Bovine and Human Cholesterol Esterase

27. Analysis of Charge Distribution -Cholesterol Esterase The proteins positively charged residues in blue, negative in red and neutral in gray

28. Analysis of Hydrophobicity The proteins uses a standard hydrophobicity scale.The most hydrophobic residues in red to the least hydrophobic (most hydrophilic) in blue

29. Analysis of Temperature Uses a traditional temperature color cycle (blue-green-yellow-red-white) to indicate relative temperature factors for each atom where blue is lowest and white highest temperature

30. Target Screening

31. Analysis of Target Residues Target amino acid sequence SPYNKGLIRRAISQSGVALSPWVIQKNP Charge Hydrophobicity Temperature

32. Designing of an Aptamer Discovery: The Aptamers discovered by Tuerk & Gold and subsequentlyby Ellington & Szostak in 1990. But the therapeutic Aptamer has entered clinical evaluation just eight years after the inception of the technology (1998). Peptide Aptamers represents a distinct class of molecules that are binding to a target protein and can block its activity selectively. They thus represents a powerful alternative to traditional approaches e.g., Knockout or mutation of a gene.

33. Designing Of An Aptamer Overview of the SELEX Process

34. Thanks for Your Attention ?