Pharmaceutical Biotechnology Mid Exam Slides PDF
Document Details
Uploaded by FastestGrowingVoice1759
Al-Ahliyya Amman University
Tags
Summary
These slides cover the fundamentals of pharmaceutical biotechnology. They discuss topics such as DNA and RNA structures, protein folding, and the role of proteins in drug response. The slides also give an introduction to the human genome and gene regulation.
Full Transcript
Pharmaceutical Biotechnology Introduction Most traditional pharmaceuticals are low molecular weight organic chemicals. However, a range of pharmaceutical substances (e.g. hormones and blood products) are produced or extracted from biological sources >>>> products of biotechnolo...
Pharmaceutical Biotechnology Introduction Most traditional pharmaceuticals are low molecular weight organic chemicals. However, a range of pharmaceutical substances (e.g. hormones and blood products) are produced or extracted from biological sources >>>> products of biotechnology. Introduction In pharmaceutical circles, ‘biologic’ generally refers to medicinal products derived from blood, as well as vaccines, toxins and allergen products. ‘Biotechnology’ refers to the use of biological systems (e.g. cells or tissues) or biological molecules (e.g. enzymes or antibodies) for/in the manufacture of commercial products. Although the majority of biopharmaceuticals or biotechnology products now approved or in development are proteins produced via genetic engineering (insulin). Introduction Molecular biology describes the study of biology at a molecular level, but focuses in particular upon the structure, function and interaction/relationship between DNA, RNA and proteins. Genetic engineering describes the process of manipulating genes (outside of a cell’s/organism’s normal reproductive process). It generally involves the isolation, manipulation and subsequent reintroduction of stretches of DNA into cells and is usually undertaken in order to confer on the recipient cell the ability to produce a specific protein, such as a biopharmaceutical. What is recombinant DNA (rDNA) technology? is a piece of DNA artificially created in vitro which contains DNA (natural or synthetic) obtained from two or more sources. Advantages of Biotechnology 1. It overcomes the problem of source availability. 2. It facilitates the generation of engineered therapeutic proteins displaying some clinical advantage over the native protein product. The Human Genome and Gene Regulation The structure of Human Genome The size of human genome, in haploid (half), is 3100 million base pairs?!!! Only 1-1.5% constitutes coding DNA or protein-encoding genes. Human has around 30,000 genes. 20% of the human genome consists of intron, untraslated regions of the genes and psaudogenes. THINK: what is psaudogene???! The remaining (75%) of total human genome is extragenic. 55% of the genome comprises of repeated sequences!!! The structure of Human Genome This repetitive sequences composed of transposons that can insert multiple copies of themselves in the genome in scattered location. Nucleic acid the carrier of genetic information. Nucleic acid is in 2 types: a) DNA: Deoxyribonucleic acid b) RNA: Ribonucleic acid Both consist of suger-phosphate backbone together with nitrogenous bases. The structure of Human Genome DNA is deoxyribose Is DNA water soluble? WHY? The structure of Human Genome In DNA, the sager is deoxyribose, whereas in RNA it is ribose. The nitrogenous bases are attached to the 1’ position of the suger. The backbone is formed by the linking of 3’ and 5’ hydroxyl groups by a phosphate. The nitrogenous bases are: a) Pyrimidine bases: Thymine (T) and cytosine (C). b) Purine bases: Adenine (A) and guanine (G). RNA contains uracil (U) instead of thymine (T). The structure of Human Genome Which is more stable binding, G:C OR A:T ? WHY? The structure of Human Genome DNA composed of 2 strands, which are coiled clockwise around each another. The are a double helix, with the 2 chains running in opposite directions (5’ to 3’ for one and 3’ to 5’ for the other). The strands are held together by hydrogen bonds between them. The strands are complementary to one another, in A:T and G:C pairing, which is obligatory. DNA’a unit of length is the base piar (bp). Gene Structure The gene consists of the following regions: a) Exon: Protein-encoding DNA sequence. b) Intron: Noncoding segment which is removed in the mature RNA through splicing proteins. It is involved in the regulation of the expression of the gene. c) 5’ flanking sequence: Contains promotor region which controls the expression of the gene. d) 3’ flanking sequence: Regulates the expression of the gene. Gene Structure Around 30,000 protein-encoding genes. There are several thousands of genes that code for RNA molecules but do not encode polypeptides. These RNA genes include at least 727 ribosomal RNA and 131 transfer RNA genes. Generally, RNA molecules are generally involved in the regulation of gene expression. These include microRNA genes, small cytoplasmic RNA, small nuclear RNA and small nucleolar RNA, which is processed from intron sliced out from other genes. DNA replication, Repairing, mutations and Genetic Polymorphisms DNA Replication DNA is replicated in the cell cycle of mitotic cell division. DNA replication occurs during the S (synthesis) phase. DNA polymerase is responsible for copying of the DNA templates are only able to read the sequence in the 3’ to 5’ and it synthesizes the new DNA strand in the 5’ to 3’ (antiparallel) direction. At least 5 eukaryotic DNA polymerases have been identified. They are designated by Greek letters. DNA Replication The 2 newly formed DNA strands must grow in opposite direction: 1) One in 5’ to 3’ direction toward the replication fork (leading strand) 2) One in the 5’ to 3’ direction away from the replication fork (lagging strand). The leading strand is synthesized continuously while the lagging strand is synthesized discontinuously (in fragments started near the replication fork). WHAT ARE ORIGIN OF REPLICATION AND REPLICATION FORK? DNA Replication RNA primer DNA polymerase can not initiate synthesis of complementary starnd of DNA unless there is an RNA primer (which is a short sequence, approximately 10 nucleotide along, of RNA paired with the initial starting sequence of DNA). The RNA primer provides a free hydroxyl group on the 3’-end of the RNA strand. The RNA primer is synthesized by a specific RNA polymerase called primase. DNA Replication DNA Repair Despite the proofreading system employed during DNA synthesis, errors- including incorrect base-pairing or insertion of one to a few extra nucleotides-can occur. In addition, DNA is exposed to environmental harsh conditions, such as ultraviolet light and free radicals, which may cause alteration or removal of nucleotide bases. UV light can fuse 2 pyrimidines adjacent to each other in the DNA. High energy radiation can cause double-strand breaks. If the damage is not repaired >>>>a permanent mutation may be introduced >>>>>may cause harmful effects and diseases such as cancer. DNA Repair Most of the repairing systems involve: 1) Recognition of the damage (lesion) on DNA. 2) Removal or excision of the damage. 3) Replacement or filling the gap left by excision using the sister strand as a template for DNA synthesis. 4) Lastly, ligation which means joining of the newly sysnthesized nucleotides with the old nucleotides in a covalent bond. DNA Repair There are different mechanisms of DNA repairing: a) Methyl-directed mismatch repair: When a mismatch occurs, the Mu proteins identifies the mispaired nucleotide(s) and discriminates between the correct and strand with error. (HOW?) This discrimination is based on the degree of methylation. This methylation is not done immediately after synthesis. So, the newly synthesized starnd is not methylated in the same way of the old (parental) strand. The methylated strand is assumed to be the correct. Then, the mismatched nucleotide is removed by exonuclease enzyme. DNA Repair B) Repair of damage caused by UV light: Exposure to UV light causes a dimer between 2 adjacent pyrimidines (usually thymine), which prevents DNA polymerase in DNA replication. This dimer is recognized by UV-specific endonuclease (uvrABC excinuclease), then removed, filled and ligated. C) Correction of base alterations (base excision repair): The bases of DNA can be altered, as the case of cytosine, which undergoes deamination to form uracil. Abnormal bases (uracil) are recognized by specific glycolyases that cleave them from the suger-phosphate backbone of the strand. This leaves an apyrimidinic site. DNA Repair D. Repair of double-strand breaks: High energy radiation or free radicals can cause double-strand breaks in DNA. This double strand break can not be repaired by the previous mechanisms. The 2 ends of the double stranded DNA fragments are brought together by a group of proteins that cause re-ligation >>>> this repair system may induce harmful errors and mutations >>> cancer and immunedeficiency diseases. GENOMIC VARIATION Genetic differences in individuals within and between populations are responsible for human diversity, including traits such as eye and skin color and even disease susceptibility. Intra- and interpopulation variation has arisen over time as a result of, for example, mutation, recombination, and natural selection. Natural selection may occur, for instance, when alleles or sequences confer selective advantages for individuals carrying such Different types of variation. Genetic Variations can cause phenotypic variation, both pathogenic and nonpathogenic, by altering expression levels, protein function, or protein efficacy. Single-Nucleotide Polymorphisms Single-nucleotide polymorphisms (SNPs) are single base pair substitutions. SNPs are mostly result from replication errors not repaired by DNA repair machinery. SNPs can be located throughout the genome, but they are more frequently found in noncoding regions than in coding regions (WHY?) because of selective pressure on protein-coding domains. Single-Nucleotide Polymorphisms SNPs in a coding region can be synonymous; that is, that they do not result in a change in amino acid. SNPs can be nonsynonymous of two types. A) Missense mutations cause a substitution of a single amino acid B) Nonsense mutations change an amino acid to a stop codon, thus truncating the protein. Nonsense mutations more likely to be pathogenic. Synonymous SNPs are more often neutral. The effect of missense mutations depends on the location of the altered amino acid in the protein as well as the nature of the substitution. Single-Nucleotide Polymorphisms SNPs in noncoding regions may still affect gene expression, particularly if located in a regulatory region, as this can affect the binding of regulatory subunits or transcriptional machinery. Also, SNPs located in introns may cause variants by altering gene splicing. Many small non-coding regions of the genome are transcribed and affect post-transcriptional regulation of gene. SNPs in these regions or their target regions in mRNAs can also cause variation in gene expression. Protein Folding, Stability and Modifications Secondary structures After synthesis of the primary structure of polypeptides, the backbone of most proteins and stretches of amino acids usually become evident. The most commonly observed secondary structural elements are termed the α-helix and β-strands. The α-helix and β-sheets are commonly formed because they maximize formation of stabilizing intramolecular hydrogen bonds and minimize steric repulsion between adjacent side chain groups, while also being compatible with the rigid planar nature of the peptide bonds. The α-helix structure of proteins β-sheets structure of proteins Tertiary structure A polypeptide’s tertiary structure refers to its exact three-dimensional structure, relating the relative positioning in space of all the polypeptide’s constituent atoms to each other. However, when the three-dimensional structure of many larger polypeptides is examined, the presence of two or more structural subunits within the polypeptide becomes apparent. These are termed domains. Domains, therefore, are (usually) tightly folded subregions of a single polypeptide, connected to each other by more flexible or extended regions. As well as being structurally distinct, domains often serve as independent units of function. Cell surface receptors, for example, usually contain one or more extracellular domains (some or all of which participates in ligand binding), a transmembrane domain (hydrophobic in nature and serving to stabilize the protein in the membrane) and one or more intracellular domains that play an effector function (e.g. generation of second messengers). Many therapeutic proteins also display several domains. Protein stability and folding Secondary structures are stabilized by short-range interactions between adjacent amino acid residues. Tertiary structure, on the other hand, is stabilized by interactions between amino acid residues that may be far apart from each other in terms of amino acid sequence, but which are brought into close proximity by protein folding. The major stabilizing forces of a polypeptide’s overall conformation are: hydrophobic interactions electrostatic attractions Covalent linkages (Disulfide bonds represent the major covalent bond type that can help stabilize a polypeptide’s three-dimensional structure. ) Protein modification (Post translational modifications PTM) It is estimated that the human proteome consists of ~300,000 different proteins, or about 10X more than the number of genes(!) - protein modifications - differential splicing Many polypeptides undergo covalent modification after (or sometimes during) their ribosomal assembly. Such modifications generally influence either the biological activity or the structural stability of the polypeptide. The majority of therapeutic proteins bear some form of PTM. Glycosylation Glycosylation (the attachment of carbohydrates) is one of the most common forms of PTM associated with eukaryotic proteins in general, particularly eukaryotic extracellular and cell surface proteins. Carboxylation and hydroxylation γ-Carboxylation and β-hydroxylation are PTMs characteristic of a limited number of proteins, mainly a subset of proteins that function in the haemostatic process. γ-Carboxylation entails the enzymatic conversion of the side chains of specific glutamate residues in target proteins, forming γ- carboxyglutamate β-Hydroxylation usually entails the hydroxylation of target aspartate (Asp) residues yielding β-hydroxyaspartate. Both PTMs help mediate the binding of calcium ions, which is important/essential to the effective functioning of blood factors VII, IX and X, as well as activated protein C and protein S of the anticoagulant system. Sulfation and amidation Sulfation and amidation are two additional PTMs characteristic of a small number of biopharmaceuticals. Sulfation entails the enzyme-catalysed attachment of sulfate (SO42-) groups to target polypeptides, usually via specific tyrosine side chains. Sulfation often plays a role in protein–protein interactions, and lack of sulfation tends to reduce a polypeptide’s activity. Amidation refers to the replacement of a protein’s C- terminal carboxyl group with an amide group (COOH → CONH2). This PTM is usually characteristic of peptides (very short chains of amino acids), as opposed to the longer polypeptides, but one therapeutic polypeptide (salmon calcitonin) is amidated, and amidation is required for full functional activity. Overall, the function(s) of amidation is not well understood, although in some cases at least it appears to contribute to peptide/polypeptide stability and/or activity. Phenotype and Population Genetics Introduction A major aspect of pharmacogenomics involves using DNA sequence data to predict drug response >>>> This requires an understanding of population genetics. Phenotype or trait: is the observable or measurable characteristic that is the target of genetic dissection. Genotype + Environmental factors =Phenotype In pharmacogenomics studies, phenotype can be measures of drug response (efficacy, side effects). Phenotype The prevalence of the phenotype in the population is calculated as the phenotype frequency, which is the ratio of the number of individuals with a particular phenotype to the total number of individuals in the population. Example: Three out of 100 patients developed myopathy after atorvastatin administration >>>> the prevelance of myopathy (side effect) induced by atorvastatin is (3/100) * 100 = 3% Phenotype A phenotype (depending on the influence of genotype on phenotype) can be simple or complex and the characteristics Simple phenotype have a monogenic basis >>>>due to a single gene with a large effect. Genotype + Environmental factors =Phenotype Simple phenotype can be expressed as dominant or recessive. Complex traits have a multifactorial etiology, and are due to numerous genes, environmental factors (Ex. Diabetes). Genotype + Environmental factors =Phenotype Phenotype Simple Phenotypes can be: Dominant: when the phenotype is expressed in the presence of one copy of the allele) or Recessive: when the trait is expressed only in homozygous form with two copies of the risk allele). ???????????? What is allele? Homozygous? Heterozygous?? ???????????? What is allele? Homozygous? Heterozygous?? Every person has 2 copies (alleles) of every gene: One from father (paternal) and one from mother (maternal). If the 2 alleles are non-mutated >>>>> no change in the phenotype >>>>> the genotype is WILD type; because most of the population have this genotype. If one allele is non-mutated and the other is mutated >>>>>> may cause a moderate alteration on the phenotype >>>> Heterozygous genotype. If the person inhireted 2 mutated alleles >>>> may cause significant or severe alteration on the phenotype >>>>> Homozygous genotype. Pharmacogenomics Dr. Yazun Jarrar Introduction Most of the physicians prescribe the drugs in empirical way, depending on the patients clinical sign, family history and probability that a certain drug will benefit the patients. Large randomized clinical trials, meta analysis and evidence-based medicine are used to deduce the probability that a drug will work for a specific condition. However, it is a rare for a drug to be safe or effective for everyone. Introduction The rate of drug response is 30 to 60% in most of common diseases, indicating that the common drug blockbuster strategy “one drug fits all” may need to be revised according to individual responses. 25% of cancer patients get beinifit from the drug treatment, while most of the patients suffer from the side effects without treating the cancer itself. Problem: Inter-individual Variation in Drug Response Introduction Inter-individual variation of drug response is caused by a combination of genetic and environmental factors, as well as, by the patients characteristics that can affect the drug’s pharmacokinetics and/or pharmacodynamics. Introduction Genetic factors affect the variation in the pharmacokinetics of the drugs by 20-95%. Data from twin and family studies showed higher inter-individual variability than intraindividual variability; indicating the role of shared inherited factors in determination of drug’s response. How can our genetics affect drug response? DNA >>>> mRNA >>>>> Protein Proteins play a key role in the pharmacokinetics and dynamics of the drugs. Proteins are responsible for absorption, metabolism and active secretion of the drugs. Many drug’s targets are proteins, such as receptors, enzymes and transporters. How can our genetics affect on drug response? Proteins play a key role in the pharmacokinetics and dynamics of the drugs. Proteins are responsible for absorption, metabolism and active secretion of the drugs. How can our genetics affect on drug response? Many drug’s targets are proteins, such as receptors, enzymes and transporters. How can our genetics affect on drug response? Genetic variant (mutation) >>>>>>> mutated protein >>>>> altered function >>>>>>> altered drug response (Less efficacy or more toxicity) Altered Drug response Altered protein structure, Genetic variation stability and function Pharmaco-genetics? Is the effect of our genetics on drug’s response. Drug’s response is either efficacy or toxicity.