Hardcastle content L1-2 Flashcards by neve mcdougall

Describe the process from target to drug

target selection, hit identification, lead optimisation, clinical evaluation

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Define HIT

a hit is a compound that displays activity in a drug discovery assay

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Where do hits come from

searching literature

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Define assay

an assay is a way of measuring something

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What can assays be performed with

-isolated protein
-tissue slice
- cells in culture
- whole animals

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What is an assay

activity of a compound is measured at increasing concentrations is an assay. An activity vs concentration or inhibition vs concentration graph is plotted

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Define IC50

concentration that inhibits 50% of activity

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define GI50

concentration that inhibits 50% of growth

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Define LD90

concentration that kills 90% of cells

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Define MIC

minimum inhibitory concentration - lowest concentration of a chemical which prevents visible growth of a bacterium or bacteria

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What are the 4 stages of the make test cycle

1) design -look at an active compound and form a hypothesis for why it is active
2) Make - synthesise further compound
3) test - run various assays of the compound
4) analyse - look at results and test the hypothesis
Repeat

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What are the aims of Hit-to-lead

increase potency vs target
increase selectivity
improve metabolic profile
Provide a compound with in Vigo activity for lead optimisation

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What are the aims of lead optimisation

increase activity for chosen biological target
decrease activity for closely related targets and others
describe side-effects
optimise metabolism
improve pharmacological properties

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How do we know which changes to make to a hit molecule?

remove groups or part of structures to see if they are required for activity, introduce new groups to see if they increase activity, introduce substituents that modify existing interactions, balance physical properties

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What does SAR stand for

structure activity relationships

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Define SAR

study of how a given biological activity varies with chemical structure

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Why do we need SARs

helps us formulate new hypotheses to design improved compound by varying one substituents at a time

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How do we find the right R groups to use

using knowledge of
- binding interactions
- structure-based design
- computer-aided design

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In what ways can we vary size and shape to find the best structural activity

homologation
-ring chain switch
introduce unsaturation
chain homologation
introduce or remove rings

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Define homologation

increases the carbon chain length of a molecule

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How does changing character of ring chain and side chain change the molecule

Changing character of ring or side change changes 3D shape, interactions with biological targets and changes potency

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List the interactions between 2D structures

-lipophilic (Vdv, Pi-Pi)
- H - bond donors
- H - bond acceptors
- polar (dipole)
- ionic
- rotatable bonds
- stereochemistry

List the ligand 3D structure

-3D shape
-H-bond vectors
- steric size
- polarity
- lipophilicty
- electrostatic interactions

What is binding affinity

binding affinity is a combination of bulk properties and specific functional groups interactions

Describe pharmacophore investigation

1) molecular simplification 2) explore size and shape of carbon scaffold 3) functional group modifications 4) design and make sets of compounds

Describe how to design and make sets of compounds

choose the easiest to make first, vary one group at a time, explore key differences, investigate bioisosteres

Define enzymes

proteins that act as biological catalysts

Define substrate

molecules upon which enzymes act

Define products

molecules into which the enzymes converts the substrate

Define cofactors

additional molecules (non-proteins) requires by the enzyme to carry out the reaction

What is the two state binding model for enzymes

it describes an enzyme existing in two conformations which influence substrate binding and enzyme activity

What is deltaGd in the two state binding model for enzymes

deltaGd represents the energy difference between the two conformations of an enzyme influencing the equilibrium between them

What three components is the rate of an enzyme reaction made up of

Kon Koff Kcat The overall rate of reaction varies between enzymes

What is Kon

the rate of binding

What is Koff

the rate of dissociation

What is Kcat

the rate of conversion to product

What is the michaelis menten equation

see one note

What is the lineweaver-burn plots

the double reciprocal plot of the michaelis-menten equation See one note

For an enzyme what is IC50 dependent on

the substrate concentration IC50 = concentration [s] at 50% activity

When does care need to be taken in terms of different assay

iC50s gives useful comparison if the assay is the same, care needed comparing assays between different labs Smaller number = better inhibitor

What is Ki and what is the equation

inhibition constant Ki=[E][I]/[EI] Rate of enzyme reaction decreases with increasing inhibitor concentration

What is the Cheng-prussoff equation

IC50 = KI(1+[S]/Km)

How do you determine Ki

- multiple IC50 measurements at different inhibitor concentrations - Dixon plot of 1/Vo vs [I] - Ki at intersect lines for varying [S]

What are the three types of reversible inhibitors

- competitive - non-competitive - uncompetitive

What are the two types of irreversible inhibitors

- affinity labels - mechanism based

Describe reversible inhibitors

- equilibrium binding to enzyme - measures by equilibrium dissociation constant Kd - can be washed out of protein by increasing dilution - typically interact with protein via weak, non covalent interactions

Describe irreversible inhibitors

- non-equilibrium binding to enzyme - can not be washed out by increased dilution - often form strong covalent bonds to target in addition to weaker interactions

Describe the lineweaver-burn plots for competitive inhibition

Km increased, Vmax unaffected

Describe the lineweaver-burn plots for uncompetitive inhibition

Km reduced, Vmax reduced

Describe the lineweaver-burn plots for non competitive inhibition

Km unaffected, Vmax reduced

Describe competitive inhibitors

Compete with the substrate of the enzyme also described as orthosteric. Apparent potency diminished by increased substrate concentration. Most common form of enzyme inhibitor

Give an example of a competitive inhibitor

Ritonavir is a competitive inhibitor of HIV protease, used in HAART for the treatments of HIV/AIDs. Binds to the dimeric protein in the active site

Describe uncompetitive inhibitors (allosteric)

Do not block the substrate site, bind to an allosteric site, induces a conformational change that prevents the enzyme functioning. Apparent potency less dependent on substrate concentration.

Give an example of a noncompetitive inhibitor

Trametinib is a non-competitive inhibitor of kinases MEK1 and MEK2, used in the treatment of melanoma, binds next to the ATP-binding site.