Background: Cell and Molecular Biology

1. King Saud University College of Pharmacy Departments of Pharmaceutics PHT 560: Pharmaceutical Biotechnology Background: Cell and Molecular Biology Ibrahim A. Alsarra, Ph.D. Professor of Pharmaceutical Biotechnology

3. Outlines • Introduction • Concepts and Definitions • Historical Perspective • Molecular Biotechnology • Recombinant DNA Technology • Cell Cultures

4. Pharmaceutical Biotechnology ...Biotechnology …What exactly do we know …

5. Introduction In the past 10-15 years, dramatic changes have occurred in the processes used by the pharmaceutical industry to discover and develop new drugs, in the chemical nature of new drugs emerging from this industry, and in the way that these new drugs are used in medicine. The processes used to conduct drug discovery have changed significantly because of the introduction of new chemical (e.g. combinatorial chemistry), biological (e.g. genomics, proteomics), computational (e.g. bioinformatics), and mechanical (e.g. robotics) technologies.

6. Combinatorial Chemistry Finding of novel drug is a complex process. Historically, the main source of biologically active compounds used in drug discovery programs has been natural products, isolated from plant, animal or fermentation sources. Combinatorial chemistry is one of the important new methodologies developed by researchers in the pharmaceutical industry to reduce the time and costs associated with producing effective and competitive new drugs. By accelerating the process of chemical synthesis, this method is having a profound effect on all branches of chemistry, but especially on drug discovery. It is now possible to produce compound libraries to screen for novel bioactivities. This powerful new technology has begun to help pharmaceutical companies to find new drug candidates quickly, save significant money in preclinical development costs and ultimately change their fundamental approach to drug discovery.

7. Genomics Genomics is the study of the genomes of organisms. The field includes intensive efforts to determine the entire DNA sequence of organisms and fine-scale genetic mapping efforts. Proteomics Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells.

8. Bioinformatics Bioinformatics is the application of information technology to the field of molecular biology. Bioinformatics nowadays entails the creation and advancement of databases, algorithms, computational and statistical techniques, and theory to solve formal and practical problems arising from the management and analysis of biological data. Common activities in bioinformatics include mapping and analyzing DNA and protein sequences, aligning different DNA and protein sequences to compare them and creating and viewing 3-D models of protein structures.

9. g g c c g g g g g c g c g g g c g g g c t c g g c Advanced Genomics Will Accelerate Discovery of Next Generation Products Genome Gene map Gene sequence Gene expression Ag traits Pharma traits c Yield t Alzheimers Drought Breast Cancer a t t Disease Arthritis Stress t t t Stress a a t t Stress CV Disease a a t Aging Oil quality a a t t t Disease Obesity Vision Yield Aging Maturity a t t Arthritis Herbicide tolerance • Solving unmet needs in human health and food production

10. Introduction … cont. Since the discovery in 1800 that the human body is composed of cells and proteins that are susceptible to pathogenic microbes, many efforts have been made to develop biopharmaceuticals-biologically based therapeutic products. The considerable history of biopharmaceuticals, the refinement of our abilities to produce recombinant macromolecules and monoclonal antibodies is a recent achievement is a result of an exponential growth in the knowledge of biological processes and engineering advancements.

11. Concepts and Definitions • Biotechnology as integrated application of scientific and technical understanding of biological process or molecule to develop a useful product. • Biological processes of inertest include cellular activities such as protein synthesis, DNA replication, transcription (to RNA), protein processing…etc. • Involvement of many disciplines, including microbiology, biochemistry, genetics and bioengineering.

12. Concepts and Definitions… cont. • Biotechnology in general implies the use of microorganisms, plants and animals or parts thereof for the production of useful compounds. • One definition of biotechnology is "the deliberate manipulation of DNA molecules to produce commercial products from living organisms." • Pharmaceutical Biotechnology:biotechnological manufacturing of pharmaceutical products.

13. Concepts and Definitions… cont. • Pharmaceutical biotechnology is a growing field in which the principles of biotechnology are applied to the development of drugs. A majority of therapeutic drugs in the current market are bioformulations, such as antibodies, nucleic acid products and vaccines. • Such bioformulations are developed through several stages that include: understanding the principles underlying health and disease; the fundamental molecular mechanisms governing the function of related biomolecules; synthesis and purification of the molecules; determining the product shelf life, stability, toxicity and immunogenicity; drug delivery systems; patenting; and clinical trials.

14. Biotechnology's Impact on Human Health Careers

15. Biotechnology's Impact on Human Health Careers … Human insulin for the treatment of diabetes: • One of the first genetically engineered products to become commercially available, was marketed in 1982. Since then, biotechnologists have been working to develop new ways for health care professionals to detect and fight disease. Detecting and Treating Hereditary Diseases: • Many diseases, including some types of anemia, cystic fibrosis, Huntington's disease, and some blood disorders, are the result of a defective gene that parents pass to their children. • Biotechnologists are working to identify and locate where defects occur in genes that are related to hereditary diseases. Once the correct genetic code is known, health care professionals hope, in the future, to be able to replace the missing or defective genes to make the individual healthy.

16. Biotechnology's Impact on Human…cont. Heart Disease: • Heart attacks occur when a blood clot enters one of the coronary arteries and cuts off blood flow to a portion of the heart. If the artery is not reopened quickly, severe damage to the heart can occur.Doctors can now prescribe a genetically engineered drug called tissue plasminogen activator(TPA) that travels to the blood clot and breaks it up within minutes, restoring blood flow to the heart and lessening the chance of permanent damage. Cancer: • Medical professionals are using biotechnology to treat cancer in several ways. Genetically engineered proteins called lymphokines seem to work with the body's immune system to attack cancer cells and growth inhibitor proteins seem to slow the reproduction of cancer cells. Highly specific and purified antibodies can be loaded with poisons that locate and destroy cancer cells.

17. Biotechnology's Impact on Human…cont. AIDS: • Genetic engineering has produced several substances that show promise in the treatment of AIDS. These substances stimulate the body's own immune system to fight the disease. Other Diseases: • Many other diseases can be treated with genetically engineered products. Doctors can use a genetically engineered vaccine to treat human hepatitis B or a growth hormone to help children with dwarfism. Vaccines: • Most vaccines are made from viruses or bacteria that have been weakened or killed. Monoclonal Antibodies: • Antibodies are produced naturally by animals when invaded by a disease-causing organism. Each type of antibody is very specific-it recognizes and attacks only one particular disease organism.

18. Historical Prospective -Today biotechnology-based pharmaceutical companies have invested millions of dollars in biotechnology business. - These companies spend more that 20% of revenues in research and development (R&D).

19. Major Players

20. Historical Prospective …cont. • The application of biological processes to develop useful products is as old as Mendel’s pea experiment,which he conducted in 1866. As a result of his avocation, Mendel developed the principle of heredity, and thereby laid the basis of modern genetics. • The fermentation technology we use today for production of recombinant proteins was first used in World War I to ferment corn starch to produce acetone for manufacturing of explosives.

21. Historical Prospective …cont. • An enhanced understanding of protein structure, a detailed elucidation of cell replication and protein synthesis, and isolation of DNA replication enzymes including restriction enzymes and polymerase led to the rapid development of recombinant DNA technology. • Almost all of the biopharmaceuticals available today, other than vaccines, are proteins or peptides. • Recently, considerable attention was given to monoclonal antibodies.

23. Molecular Biotechnology • Although biotechnology does not exclusively make use of cells but also of complete organisms or cell constituents, knowledge of basic cell biology is required to understand biotechnology to its full extent.

24. Molecular Biotechnology …cont. The Cell: • Cells from all sorts of organisms are used in biotechnology. Not only prokaryotic cells like simple unicellular bacteria are used, but also eukaryotic cells, like cells of higher microorganisms, plants and animals, are exploited.

26. Molecular Biotechnology …cont. Prokaryotic Cell: • The prokaryotes, to which the bacteria belong, represent the simplest cells in nature. Such as cell is in fact no more than cytoplasm surrounded by some surface layers. There are two main type of organisms: Gram-positive or Gram-negative based on different staining behavior in a classical cell staining technique. • The cell envelop consists of a cytoplasmic membrane called the peptidoglycan layer. The cells ofGram-positive organisms are multilayered with peptidoglycan while one or two of such layers are found in cells of Gram-negative organisms.

27. Molecular Biotechnology …cont. Prokaryotic Cell: • Aclearest distinction between the two types of bacterial cells is that the Gram-negative organisms, the cells is surrounded by a very specific extra membrane layer, called outer membrane (OM). The OM is a permeation barrier for substances that are transported into or out of the cell. • A very particular chemical constituent of the OM is the compound named lipopolysacchride (LPS). Biopharmaceutical products gained from Gram-negative organisms must be extensively purified especially when they are used a pharmaceuticals for man or animal, since LPS lead to sever toxic effects to man and animals.

28. Molecular Biotechnology …cont. Eukaryotic Cell: • The eukaryotic, e.g. plant cell, cell has a very complex structure, not only by the presence of cell organelles like nucleus, mitochondria and chloroplast (exclusively found in plant cells), but also by the presence of specific internal membrane. • Initial phase of modern biotechnology simple bacterial cells, easier to handle and more simple to modify, were used. Nowadays, molecular biotechnologists use all sorts of eukaryotic cells.

30. Molecular Biotechnology …cont. Gene Expression: • Genetic information, chemically determined by the DNA structure, is transferred to daughter cells by DNA replication and expressed by transcription(conversion of DNA into RNA) followed by translation (conversion of RNA into protein). The pioneers of molecular biology called that series of events the “central dogma” of biology. It was found later that retroviruses, a special class of animal RNA viruses encodes an enzyme that catalyses the conversion of RNA into complementary DNA. This enzyme called the reverse transcription which enables an information flow from RNA into DNA. DNA ↔ RNA → protein

31. Molecular Biotechnology …cont. Complementary DNA: • In genetics, a double-stranded DNA or RNA strand consists of two complementary strands of base pairs, which are non-covalently connected via two or three hydrogen bonds. • Since there is only one complementary base for any of the bases found in DNA/RNA, one can reconstruct a complementary strand for any single strand. This is essential for DNA replication. • For example, the complementary strand of the DNA sequence: A G T C A T Gis T C A G T A C A= adenine; G= Guanine; T= thynine; C= cytosine; U= uracil

36. Molecular Biotechnology …cont. RNA Splicing: • In some cases the RNA derived by transcription of eukaryotic DNA segment is subject to a process called splicing before it leaves the nucleus. During this process certain parts, the introns, of nascent RNA molecules are removed, after which other parts (the exons) are linked together to form the effective RNA for protein synthesis.

37. Molecular Biotechnology …cont. DNA Replication: • Strands of DAN are composed of four specific building elements, the deoxyribonucleotides dATP, dCTP, dGTP, dTTP liked by phsophodiester bounds. The two strands in the DAN helix are held together through hydrogen bound between the nucleotides in the various strands. DNA Structure

38. Molecular Biotechnology …cont. DNA Replication … cont.: • The DNA strands in the helix are held complementary in their nucleotide composition: an A in one strand is always facing a T in the other one, while a C is always facing a G. Moreover, the strands in the double stranded DNA run antiparallel: the 5’-P end of one strand faces the 3’-OH end of the complementary strand of the other way around. DNA Structure

39. Molecular Biotechnology …cont. DNA Replication … cont.: • During DNA replication process: 1- Transferal of genetic information. 2- Each DNA strand is copied.

40. Molecular Biotechnology …cont. Forms of DNA Replication: A. DNA replication (general); b. Closed circle; c. Rolling circle model.

41. Molecular Biotechnology …cont. Transcription: • Genetic information is located in the genes formed by discrete segments of the cellular DNA. In a process called transcription, genes are copied into a complementary length of ribonucleic acid (RNA) by the enzyme RNA polymerase. Most of the RNA molecules, the Messenger RNAs (mRNAs), specify the amino acid composition of the cellular protein. Other RNA molecules derived by transcription, Ribosomal RNA (rRNA) and Transfer RNA (tRNA) participates as an auxiliary molecules for translation.

42. Molecular Biotechnology …cont. Transcription …cont.: • Transcription starts: 1- The binding of the enzyme RNA polymerase at a specific site, called promoter. 2- Immediately upstream from a gene or from a set of genes transcribed as an operational unit. 3- After binding of the RNA polymerase, the DNA helix is partially unwound and subsequently the transcription process starts.

43. Molecular Biotechnology …cont. Transcription …cont.: • RNA synthesis runs antiparallel in the direction of 5’ to 3’ and processed in a complementary way. This implies that a G in the matrix DNA leads to a C in the RNA, a C leads to a G, a T t an A while A in the DNA shows up a U in the RNA.

45. Molecular Biotechnology …cont. Translation: • A very distinction between pro- and eukaryotes as regards to this process, in which: In prokaryotic cell, mRNA is already available for the ribosome while it is still in the process of transcription. In the eukaryotic cell, the mRNA is only available for translation after it is completely synthesized and after it is transported through the translation.

46. Recombinant DNA Technology • Investigators tried to manipulate the genetic properties of all kind of cells. To achieve this, they simply added foreign DNA to microbial cells. All these attempts failed. Reasons: - Only a limited number of bacteria species is able to take up DNA spontaneously. - Foreign DNA, if taken up at all, is in general not maintained in the receptor cell. DNA brought into a cell from outside will only be maintained if it is able to replicate autonomously, or if it is integrated in the recipient genome. In all other cases foreign DNA will not be propagated and will eventually be degraded through the activity of cellular nucleases.

47. Recombinant DNA Technology …cont. • Recombinant DNA technology enables the fusion of any DNA fragment to DNA molecules able to maintain themselves by autonomous replication (such molecules are called replicons). • Replicons used as carrier for foreign DNA fragments are termed vectors. The vectors exploited in the DNA technology include plasmids from bacteria or yeast, or DNA from bacteriophages, animals viruses or plant viruses. • Recombinant DNA technology or DNA cloning technology: fusion of foreign DNA to the isolated plasmid in order to create a recombinant DNA molecule.

48. Recombinant DNA Technology …cont. • Plasmid with only one recognition site for restriction enzyme. • The double stranded DNA is then cut. • The isolated foreign DNA is also cut. Principle of cloning a foreign DNA fragment.

49. Recombinant DNA Technology …cont. • DNA fragments are brought together, the various single stranded ends may recombine due to the presence of a complementary bases. • The enzyme DNA ligase, able to catalyze the formation of phosphodiester bonds, is used to create a closed circular recombinant DNA molecule. Principle of cloning a foreign DNA fragment.

50. Recombinant DNA Technology …cont. • Some bacterial cells are able to take up DAN under physiological conditions. This process is described as natural transformation. • Other method involves the transfer of bacterial cells to package DNA in a bacteriophae capsid and then mimic the normal bacteriophae infectionprocedures. Phage as a mediator for transfer of recombinant DNA.