(chan) 2. Medicines Design Flashcards
What is PFK-1 and its characteristic?
- Primary control enzyme of glycolysis
- Allosteric enzyme composed of 4 subunits
- ATP is both substrate and allosteric effector
- High activity: Michaelis-Menten Kinetics
- Low activity, Sigmoidal Kinetics
What are the inhibitors and activators of PFK-1 enzyme?
Inhibitor
- ATP, citrate, H+
Activator
- AMP
Describe TCA(Tricarboyxlic acid) cycle
- Oxaloacetate and Acetyl-CoA are condensed
- Citrate is rearranged to isocitrate by cis-aconitase
- Isocitrate is oxidised and decarboxylates
- 2-Oxoglutarate is decarboxylased by 2-OG dehydrogenase complex
- Succinyl-CoA is converted to succinate
- Desaturation and formation of FADH2
- Hydration
- Oxidation and formation of NADH
How is the TCA cyle regulated?
- Primary control enzyme is pyruvate dehydrogenase complex
- High energy signals and products of TCA cycle are inhibitors
- ADP increases activity of isocitrate dehydrogenase
What is anaplerotic reaction regarding TCA cycle?
- ‘Filling up’ Process
- TCA cycle is used to provide starting materials for biosynthesis
- This results in depletion of oxaloacetate
- Acetyle-CoA accumulates
- High levels of Acetyl-CoA ↓ pyruvate dehydrogenase complex and ↑ pyruvate carboxylase
- Net effect is to rebalance oxaloacetate and acetyl-CoA levels
- Production of oxaloacetate is also important for gluconeogenesis
What is the Cori cycle?
- Recycling R-lactate to glucose
- Lactate is transported from muscle to liver in blood
- Lactate dehydrogenase in liver converts R-lactate to pyruvate
- Pyruvate is converted to glucose by gluconeogenesis
- Process requires input of 6 x ATP per glucose molecule
What are glycolysis and gluconeogenesis regulated by?
- Both glycolysis and gluconeogenesis take place in the cytosol
Glycolysis and Gluconeogensis reciprocally controlled by 2 processes
- Energy levels in the cell
: Signalled by ATP, ADP, AMP, citrate
- Hormonal control (glucagon)
: Signalled by fructose-2,6-bis-phosphate levels
What is Glucagon and its role?
- Hormone secreted in response to low blood glucose levels
- Binding to a receptor initiates cAMP-mediated signalling
- This activates protein kinase A (PKA)
- PFK-2 is phosphorylated by PKA
- F-6-P kinase activity is reduced
- F-2,6-BP phosphorylase activity is increased
- pyruvate kinase is phosphorylated, reducing activity
- Activity of the glycolysis pathway is reduced
What is insulin and its role?
- Hormone secreted in response to high blood glucose levels by the pancreas
- Has opposite effect to glucagon
- In fed state, it increases the GLUT4 concentration enabling uptake of glucose into muscle and adipose cells
- It counteracts the inhibitory effect of glucagon by reducing phosphorylation PFK-2 / phosphatase and pyruvate kinase
What is the effect of adrenaline on glucose metabolism?
- Adrenaline is a hormone produced in response to stress and exercise
- Stress response requires glucose to be mobilised to produce ATP
- Therefore glycogen breakdown is stimulated and glycogen synthesis is inhibited in a similar manner to glucagon
Describe Electron transport system regarding ATP synthesis
- Under anaerobic conditions, the reduced electron acceptors in the transport system are not re-oxidised
- Terminal acceptor for the system is dioxygen
- This means that entry of pyruvate and flux through TCA cycle is reduced
- Pyruvate is converted to lactate instead to maintain NAD+ level in the cytosol
- Lactate is recycled via the cori cycle
How does glucagon induce increase in glucose?
- Glucagon binds to its receptor, which produces cAMP
- This activates protein kinase A which phosphorylates target enzymes
: 1. Phosphorylase kinase is activated
- This activates glycogen phosphorylase
- Glycogen synthase is inhibited
- More glucose is produced
What is the key process regulating fatty acid β-oxidation and biosynthesis?
- Synthesis of malonyl-CoA
- High level of malonyl-CoA allow fatty acid biosynthesis
- Malonyl-CoA inhibits import of fatty acids into mitochondria for degradation
What is the overall effect of ‘Glucagon’? (Summary)
Increases blood glucose level by cAMP-mediated acitvation of PKA which causes
- Phosphorylation of glycogen synthase and glycogen phosphorylase, releasing glucose from glycogen
- Phosphorylation of PFK-2 and pyruvate kinase reducing glycolysis
- Increase in fructose-1,6-biphosphatase and glucose-6-phosphatase in the cell inccreases gluconeogenesis
- Activatio nof triacyl-glycerol lipase, releasing fatty acids for β-oxidation
- Phosphorylation of acetyl-CoA carboxylase which lowers malonyl-CoA level reducing fatty acid biosynthesis
- Increased acetyl-CoA levels activate pyruvate carboxylase in TCA cycle anaplerotic reaction, increasing oxaloacetate for gluconeogenesis
What is the overall effect of ‘Insulin’? (Summary)
Secreted from the pancreas in response to high blood glucose levels which causes
- Increased GLUT transporters in muscle and adipose tissue allowing glucose uptake
- reduced phosphorylation of glycogen synthase and glycogen phosphorylase promoting glycogen synthesis
- reduced phosphorylation of PFK-2 and pyruvaate kinase increasing glycolysis
- Increased level of glycolytic enzymes and reduced gluconeogenic enzymes
- Decreased phosphorylation of acetyl-CoA carboxylase, increasing malonyl-CoA. This increases fatty acid biosynthesis
What is the effect of glucocorticoids on glucose and lipid metabolism?
- High levels of glucocorticoids promote biosynthesis of gluconeogenic enzymes and suppresses production of glycolytic enzymes
How is ketone bodies formed in diabetic patients?
- In diabetes or starvation glucose is scare
- Brain and erythrocytes are particularly dependent on glucose for energy
- Fatty acids are degraded to acetyl-CoA to make up the short-fall
- Three molecules of acetyle-CoA are used to acetoacetate and 3-hydroxybutyrate by the mevalonic acid pathway
- These are distributed by the blood and can be used as fuels instead of glucose
- Acetoacetate is easily converted to acetone hence the solvent smell on the breath
(JUST READ)
Summary of Overview of carbohydrate and lipid metabolism and hormonal control
- The metabolism of glucose, fatty acids is coordinated to maintain the required levels of ATP
- Control enzymes usually exist at the concomitant point (where pathway becomes unique)
- Increasing and decreasing activity changes flux of metabolites through various pathways
- Activity of control enzymes is controlled by cellular signals and hormones
- Glucagon produces its effect by cAMP-mediated activation of Protein kinase. Phosphorylation of control enzymes changes their activity
- Insulin blocks glucagon signaling and reverses its effects (causes dephosphorylation)
What is a Glycation reactions?
- non-enzymatic reaction of sugars(glucose) with nucleophilic groups
- Reaction is at various sites
- Derivatives can further react to produce advanced glycation products (AGP)
- Glycation of haemoglobin often used to measure glucose control in diabetic patients
What is Hexose monosaccharides?
- Monosaccharide with 6 carbons
- Glucose is the most abundant hexose
- Galactose and Mannose are epimers of glucose
How is anomers formed?
- Addition of alcohol group to aldehyde forms a hemiacetal
- Presence of OH group on carbon-5 means a 6 membered ring is formed
- A new chiral centre is formed on hemiacetal formation, which can have one of two configurations (α- or β-)
- These new isomers are called anomers
Glycation can only occur with reducing sugars. True or False?
True
- Reducing sugars reduce Cu2+ and Ag+ ions in basic solution and are themselves oxidised
What is the Amadori reaction?
- Rearrangement mediated by protonation/deprotonation
- Reversible
- Further oxidative and non-oxidative reactions can occur to give Advanced Glycation Products (AGPs)
What is the consequences of Glycation?
- Recognised as a major cause of Diabetic secondary complications including nephropathy, retinopathy and atherosclertosis
- Glycated proteins are difficult to degrade so there is a build-up of damaged proteins in the cell
- Damage results in production of auto-antibodies, resulting increased level of inflammation
What techniques can be used to measure glycation products?
- Ion-exchane chromatography
- Affinity chromatography or gel electrophoresis
- Isoelectric focussing (detecting changes in isoelectric point of the protein)
- Immunoassays such as ELISA which detect particular antigen
- Inflammation causes production of several inflammatory markers
(JUST READ)
Summary of Glycation & Monitoring
- Reducing sugars such as glucose contain highly reactive aldehyde groups
- These aldehydes can react with amines to form imines condensatio n products
- Rearrangement of the imine gives the Amadori product
- Further reaction of the Amadori product by oxidative and non-oxidative pathways give rise to Advanced Glycation Products (AGPs)
- The most important protein residues in terms of glycation are internal lysines
- Reaction with other protein residues and other biomolecules occur
- Glycation is thought to be responsible for the majority of diabetic complications and there are a number of analytical methods to measure this
- There is no proven therapy to reduce glycation, but amines such as pyridoxamine, reducing agents and plant extracts and drugs such as penicillamine may be of use
Describe Classical Potential Energy Methods
- Energy of a molecule calculated using a potential energy function which generally only considered how close non-bonded atoms are to each other
What is the aim of Conformational Searching via Classical potential energy methods?
- To locate low energy conformations as it is unlikely that high energy conformers are common enough to be involved in biological processes due to having Boltzman distribution of energies
- Low energy conformations are frequently used as starting points for more sophiscated calculations
What does Potential Energy Map show?
- illustrates the charge distributions of molecules three dimensionally
- These maps allow us to visualize variably charged regions of a molecule
Describe Molecular Mechanics Optimisation
- Optimising the structure of a single conformation of a molecule by altering the geometry of the molecule slight so that a more realistic structure is generated
What is a Force Field?
- Overall potential energy of the molecule calculated through a combination of energy functions
What is Molecular Mechanics Minimisation?
- Mathematical process whereby the structure obtained by a round of calculation processes is compared to a previous structure
- The structure is modified to make it more consistent with the parameter information within the program
common methods)
- steepest descent
- Newton-Raphson method
- simplex method
- Fletcher-Powell method
- combination of methods
Describe Optimisation Process
- Components contributing to the energy are calculated
- Those making the greatest contribution to the energy are changed in order to make the energy fall
Force fields used for Optimisation are essentially divided into two classes. What are they?
1 ‘all atom’
- The first is for use with small molecules with all atoms including hydrogen atoms
- essential atoms only
- for biological molecules
- majority of hydrogen atoms are removed to decrease computational time
Best-known molecular mechanics for 1. small molecules and 2. large molecules?
- small molecule
- MM2 - large molecule
- AMBER
What is the problem of Local minima?
- Major problem with molecular mechanics calculations is that they converge on the nearest local minimum which is not necessarily the global minimum
Describe Molecular Dynamics
- Method using the Newtonian equations of motion, a potential energy function and associated force field to follow the displacement of atoms in a molecule over a certain period of time, temperature and pressure.
What is Simulated Annealing?
- Special type of dynamics where the molecule is heated and then cooled very slowly so that conformational changes taking place will lead to a local minimum being located
What is Monte Carlo Methods?
- Moving molecules randomly to a new geometry/conformation
What is Crystal regarding molecular modelling?
- periodic arrangement of uni cells in a lattice
- The unit cell is repeated in a 3-D lattice to make a crystal
What is X-ray crystallography?
- method determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and scatters into many different directions
- From the angles and intensities of these scattered beams, a crystallographer can produce a 3D picture of the density of electrons within the crystal
What is Homology Modelling?
- Suppose you want to know 3D structure of a target protein and have the unknown sequence and a sufficiently similar protein (50% or better identity)
- You can use software that aaranges the backbone of your sequence identically to the template
What are the 3 items of input needed for Homology Modelling?
- Target sequence
- 3D template sequence
- Alignment between target sequence and template sequence
What are the examples of lead compound o drug discovery?
- Natural receptor ligands, enzyme substrates
- Collections of synthetic compounds
- Existing drugs
- Natural products
- Computer-aided rational design
What does “SWOT” stand for in the analysis finding peptide and protein therapeutics?
S: Strength
- good efficacy
W: Weakness
- Chemical and physical stability
Opportunities
- Discovery of new peptides
Threats
- Immunogenicity
What does British Pharmacopoeia Monograph provide?
- description of a pharmaceutical
- specification of required level of purity
- descriptions of tests to identify the substance and monitor possible impurities
- Specify an assay to determine the amount of substance
Which peptide and protein analysis method is particularly suitable for peptides?
- Reversed Phase HPLC (RP-HPLC)
What does insulin consist of?
2 Chain peptide consisting of 51 amino acids
- Chain A = 21 amino acids
- Chain B = 30 amino acids
- 2 x interchain disulfide bonds (A7→ B7; A20→ B19)
- 1 x interchain disulfide (A6 → A11)
What is Peptide Mapping?
- Identity test for peptides and proteins
- chemical or enzymatic cleavage of the peptide backbone in specific locations generates a set of fragments, which upon separation and identification provide a ‘fingerprint’
What is incretins?
- gastrointestinal hormones that increase insulin release from the pancreas in response to elevated serum glucose levels
(JUST READ)
Summary of ‘Development of Peptide and Peptide Analogues’
Development of non-peptide and peptide antidiabetic agents and the importance of physicochemical - lead compounds of both class derived from a range of sources; Lipinski properties related to typical mode of administration
Identification and characterisation of therapeutic peptides
- peptide mapping as a tool to distonguish closely related synthetic analogues
Incretin mimetics as antidiabetic agents
- stabilising natural peptides vs inactivation by DPP-4
Patients with T2DM produce normals levels of GIP (glucose-dependent insulinotropic polypeptide) but show lower sensitivity, while GLP-1(Glucagon Like Peptide-1) maintains its full efficacy, but is produced to a lesser extent
What are the 2 therapeutic strategies?
- Develop incretin mimetics (analogues of GLP-1) with extended plasma t1/2
- Prevent the inactivation of both incretins (GIP and GLP-1)
Most proteases may be divided into 4 distinct groups according to the chemical nature of their active sites.
What are they?
- Serine Protease
eg) DDP-4 aka dipeptidyl-peptidase-4 - Cysteine protease
eg) papain, cathepsin) - Aspartic protease
eg) pepsin, renin, HIV-1 protease - Metalloprotease
eg) ACE, MMPs
What is the role of SGLT2 (Sodium-Glucose cotransporter 2) regarding glucose level?
- controls glucose homeostasis in the kidney
- Inhibition of SGLT2 therefore recognised as a potential therapeutic target for treatment of T2DM
(JUST READ)
Summary of ‘Development and SAR of DPP4 inhibitors (gliptins) and SGLT2 inhibitors
‘
Principles involved in developing an inhibitor of a specific class of protease - design of inhibitors of serine protease
Design of DPP-4 inhibitors as antidiabetic agents
- design of covalent and non-covalent inhibitors
Development of current SGLT2 inhibitors and their pharmacophore
- natural product-inspired antidiabetic agents
