Drug Discovery Flashcards
What the general characteristics of interactions between a drug and its target?
However?
Weak and non-covalent
Can sometimes be covalent
Describe the mechanism of aspirin
Hint - Has covalent interactions involved
Acetylates a serine in prostaglandin synthase
Acetylated serine blocks hydrophobic tunnel of enzyme, preventing the substrate from reaching active site
Kd - ?
Give the definition
Equation?
Dissociation Constant
Measure of the strength of interaction between drug candidates and their target
Kd = [R][L]/[RL]
Lower Kd means what?
Stronger interaction
Meaning of EC50 and EC90 in drug potency?
EC50 - Ligand concentration required to elicit 50% response
EC90 - Ligand concentration required to elicit 90% response
When is IC50 analogous to EC50?
When the drug target is an enzyme or binding partner
Meaning of IC50 and IC90?
What factors is this dependent on?
IC50 - Inhibitor (drug candidate) concentration required to decrease activity by 50%
IC90 - Inhibitor concentration required to decrease activity by 90%
Concentration of drug required for inhibition is dependent on [S] and Km
What is Km?
Substrate concentration at half the Vmax
What is Ki?
Inhibition constant (analogous to Kd) - Dissociation constant for the binding of inhibitor in absence of substrate
With IC50 = Ki (1+[S]/KM) in mind:
- How does small Km and high [S] affect IC50?
- How does large Km and low [S] affect IC50?
IC50 = High
IC50 = Low
What is the selectivity equation?
(Kd binding of drug to other molecules (off target))
/
(Kd binding of drug to target molecule)
What is ADME?
What does it stand for?
The important factors for a drug to be effective
Absorption - Distribution - Metabolism - Excretion
Which part of ADME does Lipinski’s Rule of 5 concern?
How many rules
A - Absorption
4 rules
What are Lipinski’s rules of 5 and what do they mean?
Poor absorption is likely when:
1. Molecular weight > 500
2. Number of H-bond donors > 5
3. Number of H-bond acceptors > 10
4. Partition Coefficient, log(P) > 5
Partition coefficient (logP) equation?
Log(P) = log10 ([Ligand (1-octanol)]/[Ligand (aq)])
Why is it important drugs are distributed to the correct location?
They may cause damage or unwanted side effects as they are not designed for these locations
How are drugs that are insoluble in the bloodstream distributed?
Human serum albumin
How can the distribution of drugs be visualised?
Give an example of a molecule with a structure enabling this?
Through probes
Fluconazole has Fluorine which can be probed
What can happen to xenobiotic drugs in the free/non-bound state?
What are the 2 stages of this process?
They can be metabolised by the body
Oxidation and Conjugation
What isozymes catalyse oxidation of foreign compounds?
Cytochrome P450 isozymes
What occurs during conjugation?
At what site does it occur?
Example of ‘conjugator’?
A functional group is added so the drug can be recognised; Acts as a ‘tag’
Occurs at oxidation site after oxidation has occurred
Glutathione
What can be generated by metabolism which is sometimes taken advantage of by the drug?
Metabolism sometimes generates the active form of the drug
What are the 2 pathways of excretion and which comes first?
What happens to compounds which avoid the first pathway?
Kidneys (1st) and Enterohepatic Cycling (2nd)
If a compound avoids filtration by the kidneys, they can be actively transported into bile and then the intestine (2nd pathway)
- Further metabolism can occur OR the drug can be reabsorbed
Why can drug toxicity limit drug effectiveness?
What happens if too much paracetamol is taken?
Can have off target effects
Toxic metabolic by-products
Sudden drop in molecules used for tagging e.g. glutathione
Other toxic compounds cannot then be removed
What is LD50?
What is the therapeutic index?
Lethal dose - Amount required to kill 50% of the animals in a test
LD50/EC50
How can promising ‘lead’ drugs (drug leading to final drug) be improved?
What can the effects of this be? (4 listed effects)
Through fluorination i.e. Substituting C-H for C-F
Alter lipophilicity - Usually increase
Improve metabolic stability
Increased bioavailability
All often with no adverse effects for binding affinity of ligand to target
Why is fluorine so effective in improving drugs? (2 reasons - elaborate too)
Good mimic of hydrogen; Similar Van der Waals radius and C bond length
Fluorine is very electronegative; Can shift pKa of neighbouring carboxylate groups
- More fluorine –> pKa decreases
When does fluorination decrease lipophilicity (absorption) and why?
When there is a fluorine adjacent to an oxygen
Possibly due to polarisation of the oxygen leading to stronger H-bonds with water
How does Fluorination affect distribution?
Modulates binding of ligand to HAS; Improving bioavailability
How does Fluorination affect metabolism?
How does this link to bioavailability?
C-F is stronger than C-H; Oxidation of C-F by cytochrome P450 is more difficult
Reduced metabolism of drug, therefore increasing bioavailability
How can fluorination affect binding efficiency?
Can decrease binding efficiency; Increased EC50 and IC50
What is a chiral centre?
How does this affect enzyme binding?
Atom bound to 4 different groups; Usually a carbon
Enzyme only binds to one enantiomer
2 types of stereoisomers and their characteristics?
Enantiomers - Mirror images and optically active
Diastereoisomers - Not mirror images; Cis-trans isomerism or 2 chiral centres
How to assign R/S chirality?
- Assign priority to the 4 substituent groups on the central carbon
- Look from the centre of the chiral carbon towards the group with least priority
- Draw an imaginary circle through the other 3 groups in the priority order 1, 2, 3
If the circle runs clockwise the chirality is R
If the circle runs anticlockwise the chirality is S
What are the 3 factors in drug discovery?
Serendipity - Unplanned fortunate discovery
Screening
Design
Give example of serendipity in drug discovery
Penicillin - β-Lactam inhibits transpeptidase of peptidoglycan synthesis
Give example of screening in drug discovery
Aspirin - Salicylic acid derivatives screened for more efficacy
Acetylsalicylic acid found
How can a drug be designed using biochemical knowledge to effectively target an enzyme and a receptor?
Enzyme - Substrate or transition state mimic
Receptor - Natural ligand mimic or monoclonal antibody
What is Cmax?
Maximum serum concentration that a drug achieves in a specified compartment
What is the purpose of trying different functional groups in drug design?
To find the optimum drug characteristics such as IC50 or EC50, log(P) etc.
How do proteases act in HIV?
How do drugs for HIV proteases work? (hint - functional group)
Proteases cleave 2 large polypeptides into smaller ones in order to control cell
Have a secondary alcohol which mimics transition state
2 types of inhibitor
Competitive/Orthosteric - Can bind at the same site
Allosteric - Can bind at a different site
Does an allosteric inhibitor enhance or inhibit activity?
Can both enhance and inhibit enzyme activity
What happens when competitive inhibitor is bound to an enzyme?
That enzyme becomes temporarily ‘unavailable’
How do you reach 50% of E in the ES state when you add more inhibitor?
More substrate must be added to compete with increased inhibitor concentration
Why can you force ES over EI by making [S] high enough?
Inhibitor will become unbound over time, so a high [S] means the active site is more likely to be filled by S
Ki equation
Ki = ([E][I])/([EI])
How does increasing [I] (competitive) affect Vmax?
Vmax remains unaffected, however a larger [S] is required to reach it
Vmax equation?
Total Enzyme x Kcat
What is Kcat?
The rate at which an enzyme converts substrate into product
Rate equation
Km(apparent equation) - Just need to know how to interpret
Total E x Kcat x [S]/([S] + Km(apparent))
Km(apparent) = Km x (1 + [I]/Ki)
How can a drug target bacteria which deformylate their peptides in post-translational modification?
Target and inhibit the peptide deformylase (PDF) enzyme to interrupt the pathway
How can the binding of an allosteric inhibitor affect an enzyme? (3 ways)
Substrate binding site is different when inhibitor is bound
Enzyme may hold onto substrate worse when inhibitor is bound
Enzyme may take longer to achieve chemical reaction when inhibitor is bound
How can an allosteric inhibitor affect an enzyme in the case of ACTIVATION? (2 ways + 1 way which goes both ways)
Active site conformation changes when inhibitor is bound, allowing better binding of substrate
Enzyme may take less time to achieve chemical reaction when inhibitor is bound
May be cases where the enzyme interacts with substrate better when inhibitor binds but does the chemistry worse
How does binding of allosteric inhibitor affect Vmax, Km and Kcat?
Vmax reduced as no matter [S], available enzyme is reduced
Km slightly increased
Kcat massively decreased
How is the release of a signal from a receptor facilitated?
Endogenous agonist binds to receptor at orthosteric site and signal is released
How can another ligand affect the binding of an endogenous agonist to a receptor? (2 ways)
- Give characteristics
Ligand can compete for same binding site as agonist
- Antagonist - Doesn’t produce signal; Blocks pathway
- Inverse agonist - Gives opposite signal
Allosteric ligand binds at allosteric site - Can enhance or reduce activity
How is receptor-ligand binding different to enzyme-substrate binding? (hint - ratios)
In receptors, there doesn’t have to be a 1:1 correspondence with ligand and signal
- 1 ligand binding could release many signal molecules
How does the R140Q mutant of Isocitrate Dehydrogenase 2 (IDH2) enzyme affect the cell?
This mutant has a gain of function allowing it to reduce α-ketoglutarate to 2-hydroxyglutarate; Build up of 2HG blocks cell differentiation, causing cancer
What does the Ag-221 inhibitor do to the IDH2 mutant?
The inhibitor binds between the 2 subunits of IDH2 (allosteric site)
This stabilises the open form of IDH2, preventing conversion of αKG to 2HG
