Biotherapeutics: A Deep Dive into Biological Pharmaceuticals

Biotherapeutics: Biopharmaceuticals are pharmaceuticals inherently biological in nature due to their method of manufacture; specifically those using biotechnology and involving the use of live organisms." • Peptides • Proteins • Antibodies • Nucleic acids • Vaccines • Gene Products Erythropoesis Levels of Protein Structure • Water is a polar molecule. • Water molecules are polar because hydrogen and oxygen atoms have substantially different electronegativities (affinities for electrons). Structure of Water Molecules • Electronegativity O = 3.5, H = 2.1 • Because electrons are shared unequally, the hydrogens have less electron density and are partially positive and the oxygen has more electron density and is partially negative • This feature plus the fact that 2 unshared pairs of electrons are located within the sp3 orbitals of O atoms creates a net dipole moment 1 / 21 • Each water molecule in ice forms four hydrogen bonds to its neighbours, creating a regular tetrahedral crystal lattice. • In liquid water each water molecule forms hydrogen bonds with an average of 3.4 other water molecules. • Due to the more expanded crystal lattice structure of ice, ice is less dense than liquid water, and ice floats on water. • Hydrogen bonds account for the relatively high melting point of ice and boiling Hydrogen Bonding between Water Mole- point of liquid water. cules • Hydrogen bonds are noncovalent interactions occurring between the H atom of a dipolar molecule such as water, and the unshared electron pair of another atom (i.e., O or N). • These bonds represent the primary way in which water molecules interact with themselves and many types of biomolecules. • Hydrogen bonds are relatively short-lived (lifetime = 1- 20 picoseconds). 2 / 21 Water and Hydrogen Bonding Hydrogen Bonding Hydrophobic Effect Due to the hydrophobicity of their R groups, they often cluster together within the interior of proteins, stabilizing protein structure via hydrophobic interactions. Sidechains cannot Hbond, therefore Amino Acids sidechains are found in hydrophobic arThe amino acids in this group lack polar eas on the inside away from aqueous functional groups in their side chains medium L= NH3 on the left D= NH3 on the right how to know d/l isomers for amino acids 3 / 21 The side-chains of the aromatic amino acids, phenylalanine, tyrosine, and tryptophan, overall are very hydrophobic. The R group of tyrosine also contains a polar hydroxyl group that can participate in H bonding interactions. Aromatic Amino Acids The R groups of tyrosine, and particularly tryptophan, absorb ultraviolet light at a maximum of 280 nm wavelength. The R groups of the polar, uncharged amino acids all contain polar functional groups that can hydrogen bond with water. Polar Uncharged Amino acids Serine and threonine contain hydroxyl groups, asparagine and glutamine contain amide groups, and cysteine contains a sulfhydryl group. The sulfhydryl group of the cysteine side chain is a weak acid (pKa = 8.2). The cysteine side chain therefore is mostly uncharged at neutral pH. oxidation of SH to form disulphide bridges Disulphides 4 / 21 The most hydrophilic R groups are those that are either positively or negatively charged. The side-chains of lysine and arginine are fully positively charged at neutral pH. In lysine, a primary amino group is attached to the e carbon of the side-chain. Positively Charged Hydrophilic In arginine, the guanidinium group of the side-chain is positively charged. The histidine R group contains an aromatic imidazole group that is partially positively charged at neutral pH •The R groups of aspartate and glutamate contain carboxyl groups that are fully negatively charged at neutral pH (pKa of 3.65 and 4.25). Negatively Charged Amino acids • In aspartate, the carboxyl group is attached to the ²carbon of the amino acid backbone. •In glutamate, the carboxyl group is attached to the ³carbon. 5 / 21 Five Groups of Amino acids The non-ionic and zwitterionic forms of a simple amino acid such as alanine are shown . The zwitterionic form predominates at neutral pH. Charged Amino acids The non-ionic form does not occur in significant amounts in aqueous solution at any pH. A zwitterion can act as either an acid (proton donor) or a base (proton acceptor) •Each amino acid has a characteristic isoelectric point which is the pH at which the positive equals the negative charge. This varies based on the side chain. Isoelectric Point •For amino acid without ionisable side chains (non-polar), the Isoelectric Point (equivalence point, pI) is At this point, the net charge is zero. The AA is least soluble in water and the AA does not migrate in electric field (impor6 / 21 tant in electrophoretic separation of peptides) At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; such ions are called Zwitterions Ionisation At alkaline pH, the amino group is neutral -NH2 and the amino acid is in the anionic form. The R groups also gets protonated. This varies from amino acid to amino acid. Thus different amino acids have different pKa. Amino acids with uncharged side-chains, such as glycine, have two pKa values: Glycine Ionisation The pKa of the ±carboxyl group is 2.34 The pKa of the ±amino group is 9.6 It can act as a buffer in two pH regimes. 7 / 21 Side Chain Ionisation Formation of a peptide bond by condensation. The ±amino group of one amino acid (with R2 group) acts as a nucleophile to displace the hydroxyl group of another amino acid (with R1 group), forming a peptide bond (shaded in yellow). Amino groups are good nucleophiles, but the hydroxyl group is a poor leaving group and is not readily displaced. Peptide Bond Formation At physiological pH, the reaction shown here does not occur to any appreciable extent. Peptide bonds are generated via a dehydration synthesis reaction (condensation reaction) 8 / 21 inhibits inflammation of tissue regulates the body retention of water , it is released in response to stress Therapeutic Peptides induces labour and stimulates milk production Peptide Bond The lone pair of electrons on the nitrogen is delocalised onto the carbonyl oxygen: Peptide Bond Resonance • Nitrogen is not basic • Carbonyl is not reactive • Nitrogen - carbon - oxygen is planer •The carbonyl oxygen has a partial negative charge and the amide nitrogen a partial positive charge, setting up a small electric dipole. Peptide Resonance 2 where is the dipole what is the usual configuration, and what •Virtually all peptide bonds in proteins is the exception? occur in this trans configuration, exceptionally proline is cis Peptide Bond Rotation - Rotation around the peptide bond is not permitted - Rotation around bonds connected to the alpha carbon is permitted - Phi(Æ) angle around the alpha carbon amide nitrogen bond 9 / 21 - Psi (È) angle around the alpha carbon carbonyl carbon bond - Three bonds separate sequential alpha carbons in a polypeptide chain. The C bonds can rotate, deN—C±and C±— scribed by dihedral angles designated ¦ and ¨,respectively. Planar Peptide Bond - The peptide C—N bond is not free to rotate. - Other single bonds in the backbone may also be rotationally hindered, depending on the size and charge on the R group What is Ramachandran plot? - It is a way to visualise dihedral angles Psi against Phi of amino acid residues in Protein structures - It is the way by which we can identify secondary structures of a protein depending upon the rotation or degree of freedom of the dihedral angles - Secondary structures of protein are either alpha helix or Beta pleated sheets 1. 4 quadrants 2. Y axis is the Psi angle 3. X axis is the Phi angle 4. Angles range between -180 to 180 Characteristics of Ramachandran plot: degrees 5. Definite position in the plot will indicate definite secondary structure of protein 6. The plot contains of both allowed and disallowed region 10 / 21 what does dihedral angle rotation depend upon? - The R group determines how much rotation can occur - bulkier means less rotation In computing Ramachandran plot, atoms are treated as hard spheres where dimensions correspond to their van der Waals radii Any angle that results into collisions of how to know if a confirmation is sterically spheres are regarded as sterically unfavourable and hence such confirmation unfavourable is not allowed Graph is divided into three regions: 1. Most allowed region - has no steric hinderance 2. Allowed region - confirmation could be possible if slightly shorter van der waals radii is used 3. Disallowed region - sterically unfavourable due to presence of very bulky groups 11 / 21 Protein Interactions • 1-Hydrophobic Interactions- Hydrophobic R groups are brought together in close proximity because the exclude water, highly ordered water, increase entropy - major driving force for protein folding. • 2-Electrostatic interactions- strongest electrostatic interaction occur between ionic groups of opposite charges for example aspartate and lysine • 3-Hydrogen bonds - hydrogen shared between electronegative atoms (N,O) • 4-Covalent bonds- chemical reactions that alter polypeptide structure, disulphide bridhes and post translational modifications • 5-Hydration - structured water is an important stabilising feature of protein structure. The dynamic hydration shell that forms around the protein also contributes to the flexibility required. 12 / 21 Alpha Helix This is the hydrogen bonding pattern: •In the ±helix, the carbonyl oxygen of residue i forms a hydrogen bond with the amide of residue i+4 •Although each hydrogen bond is relatively weak in isolation, the sum of the hydrogen bonds in a relic makes it quite stable •The propensity of a peptide for forming an ±helix also depends on the sequence - The N terminal is PINK - I+1 is the next residue as in the sequence of amino acids it is one residue towards the C terminus - The H on the i+4 residue donates its hydrogen to the i residue forming a hydrogen bond, which is the bonding pattern seen in all alpha helix of all amino acids - The hydrogen bond occurs every 3.6 amino acids - Helical backbone is held together by hydrogen bonds between the nearby backbone amides - Right-handed helix with 3.6 residues (5.4 Å, 0.54 nm) per turn - Peptide bonds are aligned roughly parallel with the helical axis - Side chains point out and are roughly perpendicular with the helical axis 13 / 21 Ala and leu are... proline is a... glycine acts as a ... ²SHEET •Not all residues will adopt a helical structure • Small hydrophobic residues such as Ala and Leu are strong helix formers •Pro is a helix breaker - because rotation around N-C±is not possible •Gly acts a helix breaker because there is no R group (R=H) which supports other conformations - Two or more poly peptides line up side by side - Strands are fully extended - ²-pleated sheets are stabilised by hydrogen bonds that form between polypeptide backbone NH and carbonyl of adjacent chains - ²pleated sheets - either parallel or antiparallel - Parallel - H-bonds in the the polypeptide are arranged in the same direction - Anti-parallel - H bonds are arranged in the opposite direction 14 / 21 An antiparallel ² -sheet emphasizing the pleat which gives a zigzag structure to the sheet Mixed Beta Sheet Less common secondary protein structure • ²-turns occur frequently whenever strands in b sheets change the direction • The 180° turn is accomplished over four amino acids • The turn is stabilised by a hydrogen bond from a carbonyl oxygen to amide proton three residues down the sequence Turns • Proline in position 2 or glycine in position 3 are common in ²-turns - Beta turn consists of 4 amino acid residues - Glycine and proline are most common in the beta turn structure (not found in helices as it causes bending but this is needed for a sheet) - Hydrogen bond between the i and i+2 residue 15 / 21 Steric Hinderance While many angles of rotation are possible, only a few are energetically favourable structures of ²turns. Trans and cis isomers of a peptide bond involving the imino nitrogen of proline. Of the peptide bonds between amino acid residues other than Pro, more than 99.95% are in the trans configuration. Cis Trans For peptide bonds involving the imino nitrogen of proline, however, about 6% are in the cis configuration; many of these occur at ²turns. - Proline contains a secondary amine group called an imine, instead of a primary amine group - Proline is called an imino acid amino acid propensity Tertiary Structure • Tertiary structure refers to the overall spatial arrangement of atoms in a polypeptide chain or in a protein • One can distinguish two major classes - fibrous proteins typically insoluble; made from a single secondary structure - globular proteins water-soluble globular proteins lipid-soluble membrane proteins 16 / 21 Tertiary Structure Insulin - Final 3D structure, stabilised by hydrogen, ionic an disulphide bridges along with hydrophobic/ hydrophilic interactions Protein structure summary Collagen Quaternary Structure Membrane Protein - Fibrous protein - Formed of 3 of the same polypeptide chains - triple helix - Coiled, insoluble, has a structural function - Rope like structure stabilised by hydrogen bonds - Combines multiple different polypeptides together - each polypeptide chain is a subunit - May contain a prosthetic group (a nonprotein group which activates the protein) - Glycoprotein/ glycolipids act as antigens molecules for cell recognition and as receptor molecules for cell signalling - Found in the bilayer too as channel or carrier proteins to transport molecules - Can be intrinsic or extrinsic 17 / 21 • Strong acids or bases. Changes in pH alter the protonation of side chains which alter the hydrogen bonding and electrostatic interactions. As a protein approaches its isoelectric point it becomes less soluble and may precipitate from solution • Organic Solvents - water soluble organic solvents such as alcohols interfere with hydrophobic interactions because they interact with non polar side chains and for hydrogen bonds with water and polar Loss of protein Structure- Denaturation protein groups. Non polar solvents also disrupt hydrophobic interaction • Detergents - disrupt hydrophobic interaction, proteins unfold • Reducing agents - convert disulphides into thiols • Salt - the protein molecule will aggregate and precipitate • Metal Ions, temperature, mechanical stress 18 / 21 Aka protease, breaks down protein Examples: Proteolytic Enzymes Globular Proteins - Trypsin cleaved peptides bonds on the carboxy side of basic side chains i.e.lysine and arginine - Chymotrypsin cleaves peptide bonds on the carboxy side aromatic side chains - Carboxypeptidase cleaves from the C-terminus - Aminopeptidase cleaves for the amino terminus • The globular shape allows for hydrophobic side-chains to be directed to the protein's interior (forming "water-free pockets"), while polar side-chains are oriented outward to form a hydrophilic exterior. The hydrophilic exterior allows globular proteins to be more easily dispersed in solutions (intercellular and extracellular). • Globular proteins function as enzymes, chemical signalling compounds, transporters of other compounds, and antibodies. Example: myoglobin and albumin 19 / 21 • They are much less compact than globular proteins. • The narrower shape makes it difficult for hydrophobic side-chains to be oriented toward the interior region of a fibrous protein, and results in a hydrophobic exterior. For this reason, fibrous proteins tend to be water-insoluble. Fibrous Proteins • Fibrous proteins play important roles in providing structural rigidity and in contractile movement (muscles). An example of a fibrous protein is collagen. Collagen is the most abundant protein in the body. Its function is to provide structural rigidity and stiffness. It is found in skin, ligaments, tendons, and other parts of the body. An illustration of the components of collagen are shown below. •immunoglobulins (Ig), are globular proteins. Immunoglobins • Antibodies are able to act as protective agents by binding to specific, usually harmful, objects - called antigens. Antigens are often foreign (nonself) objects such as harmful bacteria or viruses. When an antibody binds to an antigen, it either directly neutralizes the antigen, or marks it so that the antigen can be subsequently neutralized by other components of the immune system. • The smallest antibodies are composed of a single immunoglobulin unit, called an immunoglobulin monomer. An im20 / 21 munoglobulin monomer is shaped a bit like the letter "Y," and is often illustrated using a Y-shape, as shown on the right-hand side of the illustration. • An antigen binding site, called the paratope, binds to a particular part of an antigen called the epitope. 21 / 21