presented by dr. shazzad hosain asst. prof. eecs, nsu the protein

Presented By Dr. Shazzad Hosain Asst. Prof. EECS, NSU The Protein

Proteins Proteins do the nitty-gritty jobs of every living cell. Proteins are made of long strings of individual building blocks known as amino acids.

Connection between DNA and Protein Exon Intron

DNA Transcription and Translation mRNA is read in triplets, called codon Four different nucleotides, thus 4 3 = 64 possible codon However, there are only 20 different amino acids Thus genetic code is degenerate i.e. multiple codon produce same protein mRNA tRN A

The Genetic Code of RNA The codon AUG for Methionine acts as start codon

Protein Functions

Proteins Make up about 15% of the cell Have many functions in the cell Enzymes Structural Transport Motor Storage Signaling Receptors Gene regulation Special functions

Folded proteins are placed into two general categories Fibrous proteinsglobular proteins

Fibrous proteins have polypeptide chains organized in long fibers or sheets Water insoluble Very tough physically, may be stretchy

Functions of fibrous proteins Structural proteins function in support Insects and spiders use silk fibers to make cocoons and webs Collagen and elastin are used in animal tendons and ligaments Keratin is the protein in hairs, horns and feathers

Functions of fibrous proteins Contractile proteins function in movement Actin and myosin contract to create the cleavage furrow and to move muscles Contractile proteins move cilia and flagella

Globular proteins have their chains folded into compact, rounded shapes Easily water soluble

Functions of globular proteins Storage proteins function in the storage of amino acids Ovalbumin is the protein in egg whites Casein is the protein in milk, source of amino acids for baby mammals

Functions of globular proteins Transport proteins function in the movement of other substances Hemoglobin, the iron containing protein in blood, transport oxygen from lungs to other parts of the body (C 3032 H 4816 O 872 N 780 S 9 Fe 4 ) Membrane transport proteins such as channels for potassium and water

Functions of globular proteins Hormone proteins function as cellular messenger molecules that help maintain homeostasis Insulin: sends message allow sugar into cells (when blood glucose levels are high, cells will transport glucose into the cells for use or storage) Glucagon: sends message we need more sugar in the blood (when blood glucose is too low, cells will release glucose)

Functions of globular proteins Receptor proteins allow cells to respond to chemical stimuli Growth factor receptors initiate the signal transduction pathway when a growth hormone attaches

Functions of globular proteins Protective proteins function as protection against disease Antibodies combat bacteria and viruses

Functions of globular proteins Enzymes speed up chemical reactions Amylase and other digestive enzymes hydrolyze polymers in food Catalase converts hydrogen peroxide H 2 O 2 into water and oxygen gas during cellular respiration

Protein Structures

Peptide Bonds join amino acids Its a condensation reaction (meaning that H 2 0 (or some other small molecule) is released when the bond is formed). Two amino acids form a DI-PEPTIDE POLYPEPTIDES are formed from more than two amino acids bonded together

Polar R groups make the amino acid hydrophilic Non-polar R groups make the amino acid hydrophobic

Ionic R groups make the amino acid hydrophilic

Polar vs Nonpolar Amino Acids Hydrophilic Hydrophobic

There are 20 commonly occurring amino acids that are found in proteins alanine - ala - A arginine - arg - R *** asparagine - asn - N aspartic acid - asp - D cysteine - cys - C glutamine - gln - Q glutamic acid - glu - E glycine - gly - G histidine - his - H *** isoleucine - ile - I leucine - leu - L lysine - lys - K methionine - met - M phenylalanine - phe - F proline - pro - P serine - ser - S threonine - thr - T tryptophan - trp - W tyrosine - tyr - Y valine - val - V Essential Amino Acids are those that must be ingested in the diet (our body cant make them) *** essential in certain cases

Proteins have four levels of organization

Primary structure is the amino acid sequence

The amino acid sequence is coded for by DNA and is unique for each kind of protein

The amino acid sequence determines how the polypeptide will fold into its 3D shape

Even a slight change in the amino acid sequence can cause the protein to malfunction For example, mis-formed hemoglobin causes sickle cell disease

Secondary structure results from hydrogen bonding between the oxygen of one amino acid and the hydrogen of another

Red = oxygen Black = Carbon Blue = Nitrogen Green = R

The alpha helix is a coiled secondary structure due to a hydrogen bond every fourth amino acid

The beta pleated sheet is formed by hydrogen bonds between parallel parts of the protein

A single polypeptide may have portions with both types of secondary structure

Tertiary structure depends on the interactions among the R group side chains

Types of interactions Hydrophobic interactions: amino acids with nonpolar side chains cluster in the core of the protein, out of contact with water = charged = hydrophobic

Types of interactions Hydrogen bonds between polar side chains

Types of interactions Ionic bonds between positively and negatively charged side chains

Types of interactions Disulfide bridge (strong covalent bonds) between sulfur atoms in the amino acid cysteine Link to video

Keratin is a family of fibrous structural proteinsfibrous structural proteins

Quaternary structure results from interactions among separate polypeptide chains into a larger functional cluster

For example, hemoglobin is composed of 4 polypeptide chains

The folding of proteins is aided by other proteins, called chaperones Act as temporary braces as proteins fold into their final conformation Research into chaperones is a area of research in biology

Denaturation results in disruption of the secondary, tertiary, or quaternary structure of the protein

Denaturation may be due to changes in pH, temperature or various chemicals

Protein function is lost during denaturation, which is often irreversible

Protein Folding

What is Protein Folding ? Protein folding is the process by which a protein assumes its functional shape or conformation. Random Coil Native conformation

Why is the Protein Folding so important Most of the proteins should fold in order to function Misfolding cause some diseases. Cystic Fibrosis, affects lungs and digestive system and cause early death Alzheimerss and Parkinson's disease It may help us to understand the structure of proteins which has not been known

LEVINTHAL PARADOX Let have Protein composed of 100 amino acids. Assume that each amino acid has only 3 possible conformations. Total number of conformations = 3 100 ~= 5x10 47. If 100 psec (100x10 -12 sec) were required to convert from a conformation to another one, a random search of all conformations would require 5x10 47 x 10 -10 sec = 1.6 x 10 30 years. However, folding of proteins takes place in msec to sec order.

Forces that stabilize protein structure Interactions between atoms within the protein chain Interactions between the protein and the solvent Electrostatic Interactions Interaction of charged side chain with the opposite charged side chain. Hydrogen Bonds & van der Waals forces Hydrophobic interactions

The kinetic Theory of Protein Folding Folding proceeds through a definite series of steps or a Pathway. A protein does not try out all possible rotations of conformational angles, but only enough to find the pathway.

Energy Landscape

Protein folding models The Framework Model Hydrophobic collapse Nucleation Model

The Framework Model Local interactions are main determinants of protein structures unfolded state Transition state native state

Hydrophobic Collapse Hydrophobic core forms first. unfolded state collapse native state

Hydrophobic Collapse Formation of hydrophobic globule may hinder the reorganization of both side chains and whole protein

Nucleation Model Unites hydrophobic collapse and frame work model unfolded state formation of a nucleus native state

Nucleation Model Substantial expulsion of water from the burial of non polar surfaces Good correlation between decrease in hydrodynamic volume and increase in secondary structure

Disease caused by Protein Mis-folding

Diseases caused by the defect in protein folding: Cystic fibrosis: Defect in the folding of cystic fibrosis Tran membrane conductance regulator protein. Diseases caused by misfolding of Prion proteins: Kuru Disease Creutzfedlt-Jakob Disease Scrapie Disease in sheep Mad cow disease Misfolded prion protein act as infectious agents. They act as chaperons which can multiply by binding to normal PrP and folding it to dangerous form similar to itself. Mechanisms of the functions of normal prions and the dangerous ones are still not clear.

Stained section from the cerebral cortex from a patient with Creutzfedlt-Jakob disease indicating spongiform patahlogy

Non-local contacts = High contact order contacts between residues in the primary sequence: NEARBYFAR APART A B B A A B A B ordering many more residues at once = selecting from more conformational states -> How is aggregation avoidance encoded?

How do high CO structures form co-translationally? in vitro: B A A B in vivo: A What conformations does A adopt before B appears? How much native structure can be formed co-translationally? ribosome ordering many more residues at once = selecting from more conformational states -> How is aggregation avoidance encoded?

How are drugs discovered and developed? Drug Discovery

Choose a disease Choose a drug target Identify a bioassay bioassay = A test used to determine biological activity.

Find a lead compound lead compound = structure that has some activity against the chosen target, but not yet good enough to be the drug itself. If not known, determine the structure of the lead compound

Synthesize analogs of the lead Identify Structure-Activity-Relationships (SARs) Synthesize analogs of the lead Identify Structure-Activity-Relationships (SARs)

Identify the pharmacophore pharmacophore = the structural features directly responsible for activity Optimize structure to improve interactions with target

Determine toxicity and efficacy in animal models.

Determine pharmacodynamics and pharmacokinetics of the drug. Pharmacodynamics explores what a drug does to the body, whereas pharmacokinetics explores what the body does to the drug.

Patent the drug Continue to study drug metabolism Continue to test for toxicity

Design a manufacturing process Carry out clinical trials Market the drug

Choosing a Disease Pharmaceutical companies are commercial enterprises Pharmaceutical companies will, therefore, tend to avoid products with a small market (i.e. a disease which only affects a small subset of the population)

Choosing a Disease Pharmaceutical companies will also avoid products that would be consumed by individuals of lower economic status (i.e. a disease which only affects third world countries)

Choosing a Disease (cont.) Most research is carried out on diseases which afflict first world countries: (e.g. cancer, cardiovascular diseases, depression, diabetes, flu, migraine, obesity).

The Orphan Drug Act The Orphan Drug Act of 1983 was passed to encourage pharmaceutical companies to develop drugs to treat diseases which affect fewer than 200,000 people in the US

Under this law, companies who develop such a drug are entitled to market it without competition for seven years. This is considered a significant benefit, since the standards for patent protection are much more stringent.

Identifying a Drug Target Drug Target = specific macromolecule, or biological system, which the drug will interact with Sometimes this can happen through incidental observation

Identifying a Drug Target (cont.) Example: In addition to their being able to inhibit the uptake of noradrenaline, the older tricyclic antidepressants were observed to incidentally inhibit serotonin uptake. Thus, it was decided to prepare molecules which could specifically inhibit serotonin uptake. It wasn t clear that this would work, but it eventually resulted in the production of fluoxetine (Prozac).

The mapping of the human genome should help! In the past, many medicines (and lead compounds) were isolated from plant sources. Since plants did not evolve with human beings in mind, the fact that they posses chemicals which results in effects on humans is incidental.

Having the genetic code for the production of an enzyme or a receptor may enable us to over- express that protein and determine its structure and biological function. If it is deemed important to the disease process, inhibitors (of enzymes), or antagonists or agonists of the receptors can be prepared through a process called rational drug design.

Simultaneously, Chemistry is Improving! This is necessary, since, ultimately, plants and natural sources are not likely to provide the cures to all diseases. In a process called combinatorial chemistry large numbers of compounds can be prepared at one time. The efficiency of synthetic chemical transformations is improving.

Selectivity is Important! e.g. targeting a bacterial enzyme, which is not present in mammals, or which has significant structural differences from the corresponding enzyme in mammals

The Standards are Being Raised More is known about the biological chemistry of living systems For example: Targeting one subtype of receptor may enable the pharmaceutical chemist to avoid potentially troublesome side effects.

Problems can arise Example: The chosen target, may over time, lose its sensitivity to the drug Example: The penicillin-binding-protein (PBP) known to the the primary target of penicillin in the bacterial species Staphylococcus aureus has evolved a mutant form that no longer recognizes penicillin.

Choosing the Bioassay Definitions: In vitro: In an artificial environment, as in a test tube or culture media In vivo: In the living body, referring to tests conductedin living animals Ex vivo: Usually refers to doing the test on a tissue taken from a living organism.

Choosing the Bioassay (cont.) In vitro testing Has advantages in terms of speed and requires relatively small amounts of compound Speed may be increased to the point where it is possible to analyze several hundred compounds in a single day (high throughput screening) Results may not translate to living animals

Choosing the Bioassay (cont.) In vivo tests More expensive May cause suffering to animals Results may be clouded by interference with other biological systems

Finding the Lead Screening Natural Products Plants, microbes, the marine world, and animals, all provide a rich source of structurally complex natural products.

It is necessary to have a quick assay for the desired biological activity and to be able to separate the bioactive compound from the other inactive substances Lastly, a structural determination will need to be made

Finding the Lead (cont.) Screening synthetic banks Pharmaceutical companies have prepared thousands of compounds These are stored (in the freezer!), cataloged and screened on new targets as these new targets are identified

Finding the Lead (cont.) Using Someone Elses Lead Design structure which is similar to existing lead, but different enough to avoid patent restrictions. Sometimes this can lead to dramatic improvements in biological activity and pharmacokinetic profile. (e.g. modern penicillins are much better drugs than original discovery).

Finding the Lead (cont.) Enhance a side effect

Use structural similarity to a natural ligand

Computer-Assisted Drug Design If one knows the precise molecular structure of the target (enzyme or receptor), then one can use a computer to design a perfectly-fitting ligand. Drawbacks: Most commercially available programs do not allow conformational movement in the target (as the ligand is being designed and/or docked into the active site). Thus, most programs are somewhat inaccurate representations of reality.

Serendipity: a chance occurrence Must be accompanied by an experimentalist who understands the big picture (and is not solely focused on his/her immediate research goal), who has an open mind toward unexpected results, and who has the ability to use deductive logic in the explanation of such results. Example: Penicillin discovery Example: development of Viagra to treat erectile dysfunction

Finding a Lead (cont.) Sildenafil (compound UK-92,480) was synthesized by a group of pharmaceutical chemists working at Pfizer's Sandwich, Kent research facility in England. It was initially studied for use in hypertension (high blood pressure) and angina pectoris (a form of ischaemic cardiovascular disease). Phase I clinical trials under the direction of Ian Osterloh suggested that the drug had little effect on angina, but that it could induce marked penile erections.

Pfizer therefore decided to market it for erectile dysfunction, rather than for angina. The drug was patented in 1996, approved for use in erectile dysfunction by the Food and Drug Administration on March 27, 1998, becoming the first pill approved to treat erectile dysfunction in the United States, and offered for sale in the United States later that year. It soon became a great success: annual sales of Viagra in the period 19992001 exceeded $1 billion.

Finding a Lead (cont.)

Structure-Activity-Relationships (SARs) Once a lead has been discovered, it is important to understand precisely which structural features are responsible for its biological activity (i.e. to identify the pharmacophore)

The pharmacophore is the precise section of the molecule that is responsible for biological activity

This may enable one to prepare a more active molecule This may allow the elimination of excessive functionality, thus reducing the toxicity and cost of production of the active material This can be done through synthetic modifications Example: R-OH can be converted to R-OCH3 to see if O-H is involved in an important interaction Example: R-NH2 can be converted to R-NH-COR to see if interaction with positive charge on protonated amine is an important interaction

Next step: Improve Pharmacokinetic Properties Improve pharmacokinetic properties. pharmacokinetic = The study of absorption, distribution, metabolism and excretion of a drug (ADME). Video exercise=MedicationDistribution&title=Medication%20 Absorption,%20Distribution,%20Metabolism%20and% 20Excretion%20Animation&publication_ID=2450

Metabolism of Drugs The body regards drugs as foreign substances, not produced naturally. Sometimes such substances are referred to as xenobiotics Body has goal of removing such xenobiotics from system by excretion in the urine The kidney is set up to allow polar substances to escape in the urine, so the body tries to chemically transform the drugs into more polar structures.

Metabolism of Drugs (cont.) Phase 1 Metabolism involves the conversion of nonpolar bonds (eg C-H bonds) to more polar bonds (eg C-OH bonds). A key enzyme is the cytochrome P450 system, which catalyzes this reaction: RH + O 2 + 2H + + 2e ROH + H 2 O

Mechanism of Cytochrome P450

Phase I metabolism may either detoxify or toxify. Phase I reactions produce a more polar molecule that is easier to eliminate. Phase I reactions can sometimes result in a substance more toxic than the originally ingested substance. An example is the Phase I metabolism of acetonitrile

The Liver Oral administration frequently brings the drugs (via the portal system) to the liver

Metabolism of Drugs (cont.) Phase II metabolism links the drug to still more polar molecules to render them even more easy to excrete

Metabolism of Drugs (cont.) Another Phase II reaction is sulfation (shown below)

Phase II Metabolism Phase II reactions most commonly detoxify Phase II reactions usually occur at polar sites, like COOH, OH, etc.

Manufacture of Drugs Pharmaceutical companies must make a profit to continue to exist Therefore, drugs must be sold at a profit One must have readily available, inexpensive starting materials One must have an efficient synthetic route to the compound As few steps as possible Inexpensive reagents

The route must be suitable to the scale up needed for the production of at least tens of kilograms of final product This may limit the structural complexity and/or ultimate size (i.e. mw) of the final product In some cases, it may be useful to design microbial processes which produce highly functional, advanced intermediates. This type of process usually is more efficient than trying to prepare the same intermediate using synthetic methodology.

Toxicity Toxicity standards are continually becoming tougher Must use in vivo (i.e. animal) testing to screen for toxicity Each animal is slightly different, with different metabolic systems, etc. Thus a drug may be toxic to one species and not to another

Example: Thalidomide Thalidomide was developed by German pharmaceutical company Grnenthal. It was sold from 1957 to 1961 in almost 50 countries under at least 40 names. Thalidomide was chiefly sold and prescribed during the late 1950s and early 1960s to pregnant women, as an antiemetic to combat morning sickness and as an aid to help them sleep. Before its release, inadequate tests were performed to assess the drug's safety, with catastrophic results for the children of women who had taken thalidomide during their pregnancies. Antiemetic = a medication that helps prevent and control nausea and vomiting

Birth defects caused by use of thalidomide

Example: Thalidomide From 1956 to 1962, approximately 10,000 children were born with severe malformities, including phocomelia, because their mothers had taken thalidomide during pregnancy. In 1962, in reaction to the tragedy, the United States Congress enacted laws requiring tests for safety during pregnancy before a drug can receive approval for sale in the U.S. Phocomelia presents at birth very short or absent long bones and flipper-like appearance of hands and sometimes feet.

Example: Thalidomide Researchers, however, continued to work with the drug. Soon after its banishment, an Israeli doctor discovered anti-inflammatory effects of thalidomide and began to look for uses of the medication despite its teratogenic effects. He found that patients with erythema nodosum leprosum, a painful skin condition associated with leprosy, experienced relief of their pain by taking thalidomide. Teratogenic = Causing malformations in a fetus

Thalidomide Further work conducted in 1991 by Dr. Gilla Kaplan at Rockefeller University in New York City showed that thalidomide worked in leprosy by inhibiting tumor necrosis factor alpha. Kaplan partnered with Celgene Corporation to further develop the potential for thalidomide. Subsequent research has shown that it is effective in multiple myeloma, and it is now approved by the FDA for use in this malignancy. There are studies underway to determine the drug's effects on arachnoiditis, Crohn's disease, and several types of cancers.

Clinical Trials Phase I: Drug is tested on healthy volunteers to determine toxicity relative to dose and to screen for unexpected side effects

Clinical Trials Phase II:Drug is tested on small group of patients to see if drug has any beneficial effect and to determine the dose level needed for this effect.

Clinical Trials Phase III: Drug is tested on much larger group of patients and compared with existing treatments and with a placebo

Clinical Trials Phase IV: Drug is placed on the market and patients are monitored for side effects

Assigned Reading Haffner Marlene E; Whitley Janet; Moses Marie Two decades of orphan product development. Nature reviews. Drug discovery (2002), 1(10), 821-5. LinkLink Franks Michael E; Macpherson Gordon R; Figg William D Thalidomide. Lancet (2004), 363(9423), 1802-11. LinkLink Abou-Gharbia, Magid. Discovery of innovative small molecule therapeutics. Journal of Medicinal Chemistry (2009), 52(1), 2-9. LinkLink Paul, S. M. et al. How to improve R&D productivity: the pharmaceutical industrys grand challenge. Nature Reviews Drug Discovery (2010), 9: 203-214. Jorgensen, W. L. The many roles of computation in drug discovery. Science (2004) 303: 1813-1818. Butcher, E. C. et al. Systems biology in drug discovery. Nature biotechnology (2004) 22(10): 1253-1259.

Optional Additional Reading Bartlett J Blake; Dredge Keith; Dalgleish Angus G The evolution of thalidomide and its IMiD derivatives as anticancer agents. Nature reviews. Cancer (2004), 4(4), 314-22. LinkLink Cragg, G. M.; Newman, D. J. Nature: a vital source of leads for anticancer drug development. Phytochemistry Reviews (2009), 8(2), 313-331. LinkLink Betz, U. A. K. et al. Genomics: success or failure to deliver drug targets? Current Opinion in Chemical Biology (2005), 9: 387-391 Sams-Dodd, F. Target-based drug discovery: is something wrong? Drug Discovery Today (2005) 10: 139-147.

Homework Questions What is an orphan drug. Why has the Orphan Drug Act been successful? Thalidomide is actually a mixture of two compounds. Draw their structures and list the physiological effects of each. What does ADMET stand for? List several possible reasons for poor efficacy of drug candidates in in vivo models. Explain how structure-based design was used to develop an inhibitor with improved selectivity for TACE over MMP-1 and MMP-9. How can the pharmaceutical industry increase the probability of technical success (p(TS))? What are the major causes of Phase II and III attrition?

Reference Mostly from Web

presented by dr. shazzad hosain asst. prof. eecs, nsu the protein

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