Week 4 Part 1 Flashcards
What is structural biology?
A branch of molecular biology, biochemistry and biophysics concerned with the molecular structure of biological marcomolecule, how they acquire the structure they have, and how alteration in their structure affect their functions
Where does structural biology fit in?
- Research and development stage
2. Very early on, when a target is determined
How long does it take to bring a drug to market?
~15 years
What is the average cost to bring a drug to market?
~$800 million
Why are new approached and methods for drug discovery being employed?
Reduce time
Expense of bringing a drug to market
Why is structural biology important?
- Try to understand 3D structure
- Understand in terms of function/inhibition
- By determining their atomic structure
What are the different experimental approaches for structural biology?
- X-ray crystallography
- NMR
- Cryo-electron microscopy
What are the computational approaches to structural biology?
- Homology modelling
- Ab initio modelling
- Molecular docking
What is Homology modelling?
Modelling a protein 3D structure using a known experimental structure of a homologous protein (the template)
Provides “low-resolution” structure - sufficient information about spatial arrangement of important residues in the protein
What is Ab initio modelling?
These methods attempt to identify structure with minimum free energy
What is the molecular docking?
- Model the interaction between a small molecule and a protein at the atomic level - characterise behaviour of small molecules in the binding side of target proteins
What are the 2 predictions of molecular docking?
- Prediction of the ligand conformation as well as its position and orientation within these sites
- Assessment of the binding affinity
What is the aim of molecular docking?
Give a prediction of the ligand-receptor complex structure using computation methods
How can molecular docking be achieved,
- Sampling confirmations of the ligand in the active site of the protein
- Ranking these confirmations via a scoring function
What is virtual screening?
Computational technique used in drug discovery to search libraries of small molecules in order to identify those structures which are most likely to bind to a drug target
What is De-novo design?
The three-dimensional structure of the receptor is used to design newer molecules
It involves structure determination of lead target complexes and the design of lead modifications using molecular modelling tools
What can de novo design be used to design?
New chemical classes of compound that present similar substituents to target using a template or scaffold
What is fragment-based drug discovery?
Identify small chemical fragments
Bind only weakly to biological target
Growing/combining them to produce a lead with higher affinity
What is protein-ligand models?
Predict the position and orientation of a ligand (or a small molecule) when it is bound to a protein receptor or enzyme
How can structural biology experiment guide lead identification and optimisation?
- Virtual screening
- De-Novo design
- Fragment-based drug discovery
- Protein-ligand models
For any experimental technique, what do we have to do?
Purify the protein
Where can protein purification be isolated from?
Natural sources
What is a feature of protein purification?
Recombinant overexpression
What are examples of recombinant overexpression?
- E.coli
- Insect
- Human cells
What is the most common form of recombinant overexpression?
E.coli
What is a common technique for purification step?
Use affinity tags
Why do you need a very homogenous sample of very pure protein?
Determine the structure of a single entity
How do crystals form?
Local concentration of protein goes up
How does local concentration of protein go up?
Dehydration
Why can we work with little small crystals now?
Have very bright beams which give us signal and resolution to achieve these structures
Have laser technique now
What are the steps to X-day crystallography?
- Crystallisation
- Data collection
- Solving the structure
Crystallisation of X-Ray crystallography
- Increase the protein concentration in solution until it precipitated in an ordered fashion - form crystals
- Crystals take from a few hours to weeks/months to grow
- 10mg/ml protein concentration and no molecular weight limitations
- can either be “co-crystallised” or “soaked” into existing crystals
What does the size and quality of crystal depend on?
- Exact details of the solvent
- Procedure used
Procedures must be optimised
Why are smaller crystals much more common now?
Advances in data collection methods
Data collection of X-ray crystallography?
- Crystals are placed in an X-ray beam and diffraction is observed
What is diffraction due to?
Interaction between radiation and the regular microscopic arrangement of molecules in the crystals
What does the angle of deflection depend on?
- Structure of the crystal
- Orientation relative to beam
- Wavelength of radiation
What can having a regular pattern of proteins in regular array tell?
That they diffract in the same manner
They all become additive
Get a diffraction pattern in terms of intensity and spacing
What makes use of data collected at synchrotrons?
90% of macromolecule structures solved by X-Ray crystallography
What is a synchrotron light a source of?
- Different electron accelerator type
- Includes a storage ring in which desired electromagnetic radiation is generated
- Radiation is used in experimental stations located in different beam lines
What is a synchrotron?
Extremely powerful source of X-rays
X-rays are produced by high energy electrons as they circulate around the synchrotron
What does a synchrotron machine exist to do?
Accelerate electrons to extremely high energy and then make them change direction periodically
Resulting X-rays are emitted as dozens of thin beams, each directed toward a beamline best to the accelerator
What do you get with a synchrotron?
Very high large single wavelength of monochromatic X-rah beams that have very high intensity that generate very good signals
What can diffraction pattern be used to generate?
Map of electron density within the crystal
What can be built into the map of electron density?
- Atoms and molecules
What can the electron density nap be used to reconstruct?
- Molecular structure
2. X,Y,Z coordinates of the atoms - tells you the position of atoms in the protein
What does the final structure of the protein represent?
- The confirmation that was captured in the crystal lattice
- Single ‘frozen’ conformation
What is crystallography now?
- High-throughout technique
2. Structure can be obtained within few days with well behaved data
What is resolution?
How confidently we have resulted the distance between 2 atoms in the protein structure
What does the diffraction pattern give?
The resolution of the data collected
The higher the resolution
The smaller dots will be going outward
The smaller the resolution
The smaller dots keep coming in
What does he final resolution of data depend on?
- How well you can refine the structure in confidence
- How good are the procedures to get 3D structure
- Model building
What depends on a specific resolution?
Determination of specific features of the structure
What resolution is required to have a good definition of all secondary structure elements?
3.5 A and higher
Resolution less than 2.5-3.0 A and higher
- Details of the side chain conformation
2. Orientation of the peptide planes
What can NMR data be used to determine?
Structure of biomolecule in solution
No crystals are required
What does NMR measure?
The transition between energy levels of nuclei with a nuclear magnetic moment in external magnetic field
Study the nucleus rather than electrons
What can be measured by NMR?
Only some isotopes for a given element have a nuclear magnetic moment different from 0
What are the isotopes for biomolecules?
1H 2H 13C 15N 19F 31P
What are NMR application molecules?
Usually isotopicslly labelled
How can you resolve NMR better?
The higher the separation of recorded data
What is the consequence of protein becoming larger?
The peaks start broadening more
The separation is not good between the peaks
Resolving the structure in 3D becomes difficult
What is the limitations to using NMR?
- Limited to ~25KDa did the size of the protein
2. Impurities
Higher magnetic fields
Higher separation of recorded data
What does NMR signal depend on?
It’s electronic environment
Define chemical shifts
Measure differences between the frequency of the observed transition and the frequency of a reference standard
Measured in ppm
What does each chemical shift correspond to?
NMR visible isotope in the protein
Can be assigned using 3D experiments
What can chemical shift be used to reconstruct?
Secondary structure of a protein
What can certain nuclei exchange?
Excitation energy when they are close together
Nuclear Oberhauser Effect (NOE)
What is NOE?
Transfer of nuclear spin polarisation from one population of nuclear spins to another via cross-relaxation
What can the strength of NOE signal be used to indicate?
Distance between nuclei-distance restraints
If a structure cannot be determined
Functional data can still be derived
What is NMR?
Quite sensitive technique
Record both weak intermediate and tight interactions using NMR
Where is the protein-protein interaction?
Marco molar range
Role of deuterium labelling?
Reduces the speed of decay of NMR signal but can be coupled with more labelling
NMR spectra of proteins above ~25KDa
Become crowded
Signal become vastly reduced
Larger proteins - slower tumbling -broad lines
What can signal be increased by?
TROSY techniques at high magnetic field
TROSY experiment
Compensate for low sensitivity by using a high magnetic field strength
What does 2H labelling reduce?
The speed of decay of the NMR signal
Many experiments can not be performed
Coupled with selective methyl labelling of residues
Largest structure solved by NMR
Malate synthase G82 KdA
Not easy to determine
Largest protein interaction study
GroEL/GroES complex
~900 kDa - more standard now
Molecular docking
Class of computational methods used to fit or “dock” a molecule into its binding site
Can be manual or automatic
The interaction between molecule and target are calculated and used to rank the compounds
What is molecular docking used in?
Structure-based virtual screening to identify molecules likely to bind to a given target
What can automatic procedure used for?
Screen large numbers of compounds and avoid any bias
Higher ranking molecules
More likely to bind to target
What are the 3 majn types of docking approaches
- Rigid docking
- Flexible-ligand docking
- Flexible-protein docking
Rigid docking
The structures of both the ligand and the target are treated as rigid bodies
Acceptable only if the active conformation of ligand is known or ligand is a rigid cyclic structure
Flexible-ligand docking
The ligand is allowed to be flexible and different confirmations are explored
Flexible-protein docking
Both the ligand and protein are allowed to be flexible
Flexible-protein docking
Both the ligand and protein are allowed to be flexible
What can some of lead compounds extracted from natural sources be used for?
Directly for medical applications
In many cases leads need to be further modified (low activity, High toxicity or serious side effects)
What does lead tend to be?
- Smaller
- Less hydrophobic
- Less aromatic rings
- Less hydrogen bond acceptors
Ligand efficiency
Binding affinity
Number of non-hydrogen atoms
Ligand based
Screening based upon known Luganda of target
Structure based
Find ligands that complement features of 3D structure of biological target
Virtual screening: ligand based
- Uses known active molecules
- Identifies new ligands with similar properties - molecular shape, electrostatics
- Used virtual libraries of compounds
Virtual screening: structure based
- Uses X-Ray or NMR 3D structure of the target protein
- Uses virtual libraries of compounds
- Compounds from library are docked in the target using a molecular docking
If a lead cannot be discovered by screening
Designed from scratch
De-novo design - ligand based
- Uses the structure of known ligands to build the pharmacoporic models
- More than one ligand is usually required
- Drugs can be designed in such a way that they fit the pharmacophoric model
Pharmacophore
Summaries the important binding groups and their relative positions in space
De-novo design - structure based
- Start with the 3D structure of target protein
- Rational design process to construct molecules complementary in binding
- Molecular modelling
Protein and binding site
- A library of fragment is screened by NMR
- Binding of fragment A is detected
- Fragment A is optimised to increase interactions with target
- The fragment library is screened again in the presence of fragment A
- Binding of fragment B is detected
- Fragment B is optimised
- A linked is added to join 2 fragments
Advantages of fragment linking approach
- The optimisation stage is faster
- It is more likely to find fragment binding sites than leads binding the whole bind site
- Different combinations of fragments can be tested (high level of diversity)
