Lecture 5: Biological Targets and Their Modulation 2

Types of protein Targets for drugs

• Enzymes

  • Drugs usually inhibit the enzyme

  • Effect: block or reduce formation of the enzyme product

• Receptors:

  • Drugs can activate (agonist) or block (antagonist) the receptor

  • Some drugs can act as both

  • Effect: amplify or suppress receptor signaling

• Ion channels

  • Drugs can open or close the channel

  • Some drugs can perform both functions

  • Effect: amplify or suppress channel activity

• Structural proteins (less common)

  • Drugs interfere with assembly or disassembly of protein structure

Biological mechanism of top 50 drugs

• Enzyme inhibitors: 38%

• Receptor antagonists: 24%

• Receptor agonists: 12%

• Ion channel modulators: 8%

• Unknown/miscellaneous: 18%

Enzymes catalyze reactions

• Provide a designer solvent for the transition state of a reactions

• Enzymes bind very tightly to transition states 

Designer solvent for Transition state

• Enzymes stabilize the reaction transition state

• Best binding occurs at the transition state, not the substrate or product

Enzyme Active site is Relatively small

• The active site is a tiny pocket where catalysis occurs

• The rest of the enzyme functions mainly as a scaffold

Only a small part of the enzyme touches the substrate

Enzyme (E) binds to substrate (S)

• Binding forms the enzyme-substrate complex (ES)

• The process is reversible (equilibrium)

• Enzyme changes shape to better fit the substrate

• The substrate also adjusts its shape during binding

Reaction occurs

• In the enzyme-substrate complex (ES), the substrate is positioned correctly for reaction

• The substrate is converted into product within the active site

• The enzyme may undergo shape changes during catalysis

Product is released

• After catalysis, the product is formed in the active site

• The product has lower affinity for the enzyme that the transition state or substrate

• Product is released from the enzyme

• The enzyme returns to its original form, ready to catalyze another reaction

THeories of Enzymatic conformational change

• Lock and key (older model)

  • Active site is a rigid picket already shaped to fir the substrate exactly

• Induced fir(current model)

  • Substrate binding causes the enzyme to change shape

  • Conformational change can “activate” or “deactivate” the enzyme

  • Remember that the substrate is also flexible and will change conformation during binding

Molecules are not static 

• Often drawn as fixed shapes, but this is an oversimplification

• Enzymes and substrate are constantly moving and flexing

• Shape changes are gradual, not instant and often asymmetrical 

Michaelis-Menten Kinetics

Michaelis-Menten Kinetics

Kcat and Km

• Kcat = turnover rate

  • Measure of how efficiently ES complex produces product

  • Larger Kcat = easier reaction

• Km= [S] at 1/2 Vmax

  • Ratio of Kcat/Km used to index enzyme efficiency

  • High ratio = more efficient enzyme

  • Also used as a specificity constant

  • Compare ration for different substrates

• Kd = dissociation constant = Kr/kf

  • Measure of binding between E and S

  • Small Kd = tight binding

Rearranging the equation gives a linear relationship 

Enzyme inhibition

• Competitive inhibition 

  • Inhibitor competes with substrate for the active store

  • Binding occurs at the active site

  • Substrate is blocked from binding

• Non-competitive inhibition

  • Inhibitor binds to enzyme but not at the active site (allosteric site)

  • Substrate may still bind to the active site

  • Inhibitor binding prevents catalysis (disrupt enzyme function or conformation change)

• Uncompetitive inhibition (very rare)

  • Inhibitors binds only to ES complex 

  • Binding prevents the reaction (destroys catalytic ability)

Competitive inhibitor

• Inhibitor molecule competes with substrate molecule for active site

• Bindind is in the active site

  • Substrate cannot bind

Disulfiram(Antabuse) Is competitive

• Blocks aldehyde dehydrogenase

• Generates rapid and severe hangover when drinking 

Non- Competitive Inhibitor

• Inhibitor binds to enzyme but not in active site

• Substrate binds to active site

  • Inhibitor binding preventing ES complex from forming (prevents or alters conformational change)

Non-competitive inhibitor changes the slope and intercept

• Km appears unaltered

• Slope and intercept change

Fluconazole is non-competitive

• Blocks cytochrome P450

• Does not bind to active site

Uncompetitive Inhibition (very rare)

• Inhibitor binds to ES complex

  • Binding destroys catalytic ability of ES complex 

Un-competitive inhibitor changes the intercept

• Vmax and Km decreased

Irreversible inhibitors

• Also known as suicide inhibtors

• Bond covalent to enzyme

• Inhibit by altering conformational or disabling functional groups

• Specific inhibitors usually bond in active site

Penicillin is an irreversible inhibitor

• Blocks transpeptidase, enzyme used to finish cell wall construction

Penicillin reacts covalently with transpeptidase

Messengers work by inducing shape changes

• Chemical interactions between messenger(ligand) and receptor change the shape of the receptor 

Chemical messaging via binding

• Chemical interactions between messenger and receptor change the shape of the receptor 

• Shape change allows a second messenger to bind or to be released

Chemical messaging via catalysis

• Chemical interactions between messenger and receptor change the shape of the receptor

• Shape change allows creates/destroys catalytic function

Agonists “SImulate” The normal messenger

• Agonist binding induces shape change that results in transmission of a signal

• Usually bind at same location (Active site) as messenger

• Some agonists bind to other locations (allosteric sites)

  • Allosteric modulators

Asthma drugs are adrenaline agonists

• Adrenaline receptors in lungs stimulate bronchial opening when activated

• Bing to active site of the noradrenaline receptor 

Benzodiazepines are Allosteric Modulators

• Binds allosterically to GABA ion channel

  • GABA is an inhibitory neurotransmitter

  • In presence of benzodiazepines the channel opens more readily and stays open longer )lower concentration of GABA opens the channel)

Antagonists block normal receptor function

• Antagonist binding induces abnormal shape change that results in no signal transmission

• May bind at same location (active site) as messenger

• May bind at other locations (allosteric sites)

Tagamet is an active site antagonist

• Binds to histamine binding site

• Distance between main binding domains is larger in histamine

  • Stretches the active site and produces antagonism 

Allosteric Antagonists

• Can bind near or even partly inside the active site

• Can bind in a distal location

Partial agonists (2 possibilities)

• Agonist binds to receptor and produces non-ideal conformational change

  • Weak signal is sent (short duration)

• Agonist capable of binding to receptor in more than one way

  • One binding mode gives agonism

  • Other binding mode gives antagonism

Buprenorphine is a partial agonist of opioid receptor

• Used to block the effects of opioid poisoning

Inverse agonist

• Some receptors have a background activity

• Receptor shows weak function in absence of messenger

• Antagonist changes normal confirmation, shutting off this background activity

  • GABA agonists produce relaxation

  • GABA inverse agonists produce agitation

Clozapie shows inverse agonist behaviour

• Originally thought to be a weak D2 (dopamine receptor) antagonist

• Recently found to be an inverse agonist of D2

Measuring drug beahviour

• Use a biological assay

• Qualitative (yes/no)

• Quantitative (number)

• In vitro 

  • Using biological chemicals, cells or tissues

• In vivo

  • Tests done in living animals

• In vitro methods are ALWAYS preferred

General Assay types

• HTS (qualitative)

• Routine SAR (quantitative)

• Kinetics or special studies (quantitive)

• Cell based

  • Antibiotics, antivirals, anti-cancer, metabolism studies

  • Result of several properties

• Tissue based

  • Permeability

  • Complex

HTS

• Emphasis on speed 

• Usually qualitative (yes/no at set concentration)

• Fully automated

• Preferred detection method is fluorescence

• Some assays may use radioactivity to measure effects

• Usually performed on 384 or 1536 well plates

Assays for routine work

• Emphasis on accuracy

• Usually quantitative (concentration or rate)

• Semi-automated or manual

• Preferred detection method is fluorescence

• Some assays may use radioactivity to measure effects

Cell-based assays

• Usually quantitative

• Required skilled technicians

• Usually slow

• SOmetimes difficult to interpret results

  • Black box

• Industry uses some standardized cell-based assays