Enzymes as Drug Targets - Done Flashcards
What is the drug development process? - How many compounds, cost, etc.
10,000 - 250 - 5 - 1
Healthy volunteers - toxicity
Some patients - dosage
Many patients - efficacy / adverse effects
12 year process
> 1 billion Euro
Define Medicinal Chemistry
Chemistry discipline concerned with the design, development and synthesis of pharmaceutical drugs.
Give 2 properties of reversible inhibitors.
Bind through many non-covalent interactions
DO NOT undergo chemical reaction when bound
What are competitive inhibitors?
They reversibly bind more strongly than the natural substrate
What are statins? And what do they contain?
They are competitive inhibitors that lower cholesterol synthesis and reduce LDL conc in blood
They contain an extra hydrophobic region allowing them to bind more strongly than HMG-SCoA
Give a Type 1 and Type 2 Statin example respectively
Type 1 are derived from fungi metabolites and usually very toxic - Simvastatin IC50 = 11nM
Type 2 are fully synthetic- Atorvastatin IC50 = 8nM
What are ACE Inhibitors? What do they treat?
Angiotensin-converting enzyme inhibitors are anti-hypertensive drugs used to treat high blood pressure. They are competitive and reversible
What are ACEs a member of?
ACE is a member of the Zn(II) metalloproteinases - they catalyse the hydrolysis of a dipeptide
Give a named example of an ACE inhibitor and its binding interactions
Enalaprilate IC50 = 1.2nM
Benzene ring fills hydrophobic pocket
COO- nearest benzene anchors at Zn2+
Other COO- binds to Arginine
H-bond on ketone
What are transition state analogues?
A drug designed to resemble the transition state for the catalysed reaction.
Bind so strongly they are effectively irreversible
Where do anti-hypertensive drugs inhibit in angiotensin conversion pathway?
They inhibit the first step in the synthesis of angiotensin 2.
Provide the mechanism of renin-catalysed hydrolysis of Angiotensin to Angiotensin 1
What key functional group does Aliskren contain?
Hydroxyethylene moiety - stable to hydrolysis as no leaving group
How do allosteric inhibitors work?
By causing a conformational change which deforms the active site of the enzyme.
What drug is used to treat leukemia and inhibits the first enzyme involved in the synthesis of purines - hence blocking DNA synthesis?
6-Mercaptopurine
It is an allosteric inhibitor
How do uncompetitive inhibitors work and can you overcome them by increasing substrate conc?
They bind to the enzyme when the substrate is already bound to the active site.
Increasing substrate concentration will not overcome inhibition.
How do Non-competitive inhibitors work?
They bind to an allosteric binding site sufficiently to prevent catalytic mechanism, but have no effect on the binding process.
How do irreversible inhibitor differ from reversible inhibitors?
They form covalent bonds permanently blocking the active site. Usually serine or cysteine affected due to nucleophilic groups.
Give three examples of irreversible inhibitor functional groups.
Alkyl halides
Epoxides
A/B-Unsaturated ketones
Fluorophosphonates
B-Lactams
Lactones
What does Disulfiram treat and how?
Alcoholism, irreversible inhibitor preventing oxidation of alcohol leading to a build up of acetaldehyde - causing nauseous sensation.
What drug is used to treat obesity?
Orlistat - inhibits pancreatic lipase which catalyses digestion of fats.
Define suicide substrates.
Agents which are converted to irreversible inhibitors by the enzyme-catalysed reaction.
What is a clinical use of transaminase enzymes?
Levels are commonly measured as bio markers for liver health
How does transaminase inhibition differ to natural substrate?
3 Fluorine’s replacing the methyl group on alanine
These result in nucleophilic attack from a group off the enzyme, which locking it to the structure
Preventing the release
Why was Tienilic acid withdraw from the market?
It acted as a suicide substrate for cytochrome P450 enzymes.
How does Tienilic acid covalently bind to the P450 enzyme?
The P450 enzymes convert tienilic acid to thiophene sulfoxide - highly electrophilic. Thiol group becomes alkylated and loss of water restores the thiophene ring.
What are isozymes?
Where polypeptide subunits of enzymes differ
What does MAO stand for and do?
Monoamine oxidase - metabolises neurotransmitters
What are MAO-A and MAO-B selective for respectively?
MAO-A selective for noradrenaline and serotonin
MAO-B selective for dopamine
What reaction do MAO isozymes catalyse?
R-CH2NH2 —————————> R-CHO
Flavin coenzyme
Give an example of MAO-A inhibitor
Clorgiline - antidepressant
Give an example of an MAO-B inhibitor. Administered with what? What does this protect and treat?
Selegiline - administered with Levodopa to protect it from metabolism
Treats Parkinson’s disease
What are both Cyclooxygenase isozymes (COX-1 COX-2) involved in?
Prostaglandin biosynthesis
Which cyclooxygenase isozyme is active under normal conditions? And when does the other become active?
COX-1 - COX-2 becomes active in diseases such as arthritis
What drug is used to treat arthritis? What are its side effects?
Indometacin - but inhibits both isozymes (COX-1 and COX-2)
Also inhibits beneficial prostaglandins in gastrointestinal tract and kidney.
Give an example of a COX-2 selective drug
Celecoxib
Valdecoxib
Rofecoxib (**CPR)
What is the key difference in binding sites that allows for selectivity of COX-1 and COX-2?
Isoleucine present in COX-1 binding site
Valine present in COX-2 binding site
(Size of the molecule means can’t fit)
What assumptions are made in the Michaelis-Menton equation?
That enzyme combines with all of substrate to form ES complex
ES complex can then dissociate back or go on to form product - irreversible
What is Michaelis constant KM and its significance?
it is the substrate concentration at which the reaction rate is half its max value
Significant as this is the substrate concentration at which half the active sites of the enzyme are filled
What problem is there with the Michaelis-Menton plot?
May not be sufficient data points to determine the curve has reached its max value or not - hence values for max rate and KM are likely to be inaccurate.
What are the axis of the Michaelis-Menton plot?
X axis = [S]
Y axis = rate of reaction
What are the axis of a Lineweaver-Burke plot?
X axis = 1/[S]
Y axis = 1/rate
What is the gradient of a Lineweaver-Burke plot?
M = KM / rate max
How does competitive inhibition look graphically?
Max rate is unaffected
Gradient differs - with inhibitor is steeper
KM is also higher - hence higher substrate conc needed for the same rate max / 2
How does uncompetitive inhibition look graphically?
Max rate is reduced
Gradients are the same
How does non-competitive inhibition look graphically?
Lines have same y-intercept (KM is unaffected)
Max rate is reduced
DOESN’T APPLY TO ALLOSTERIC BINDING INHIBITION
Define EC50
The concentration of inhibitor required to reduce a particular cellular effect by 50%
Give 3 medicinal uses of enzyme inhibitors
Enzyme inhibitors used against microorganisms.
Enzyme inhibitors used against viruses.
Enzyme inhibitors used against the body’s own enzymes.
Other uses include treating parasitic infections.
The enzyme MUST be ESSENTIAL for microorganism and not present in humans - specificity.
What prokaryotic characteristic is often exploited by medicinal chemists?
The Cell Wall
Provide 1 example of a fungal metabolite that acts as a bacterial enzyme inhibitor
Cephalosporin C
How do sulphonamides carry out inhibition?
They inhibit cell metabolism
Competitive enzyme inhibitors of dihydropteroate synthetase
Hence blocking biosynthesis of tetrahydrofolate - key metabolite in bacteria
List the current uses of sulphonamides
Treatment of UTIs
Eye lotions
Mucous membrane infections
Gut infections
What 3 functional properties are REQUIRED in sulphonamides?
Amino group must be at position 4
Both amino and sulphonamide group must be directly bonded to the aromatic ring
R2 (on sulphonamide) is the only site that can be varied
What is the Mechanism of Action of penicillins?
Inhibition of bacterial cell wall synthesis
Irreversible inhibition of transpeptidase - preventing final cross coupling reaction.
Give 2 examples of penicillins and a structural difference between the two.
Amoxicillin - contains phenol
Ampicillin - contains phenyl NOT phenol group
How do penicillins act as irreversible inhibitors?
B - Lactam group reacts with nucleophilic (serine or cysteine) residue off of enzyme forming an ester linkage
Creating a steric shield against substrate or water
Provide the mechanism for penicillins irreversible inhibition
Nucleophilic attacks electrophilic Carbon - pushing e- up to oxygen
Oxygen then reform O=C double bond
This causes the ring (C-N bond) to break and N picks up a proton from solution
How does the B-lactamase enzyme provide penicillin resistance in bacteria?
The serine residue attacks the electrophilic carbon, the oxygen also reforms the O=C so the ring (C-N bond) breaks
The ester bond is then hydrolysed to release the inactivated penicilllin and free up the serine group - catalytic
How does clavulanic acid prevent resistance to the penicillin used?
It enters the binding site and fits similar to penicillins
The acyl-enzyme intermediate then reacts further with NH2 - creating irreversible binding.
What is the name clavulanic acid is marketed under?
Augmentin
Give the full mechanism of clavulanic acid.
Week 6 GN8 Enzymes as drug targets Page 12/13
What amino acid would need to be present in the active site of B-lactamase for clavulanic acid to work?
Histidine - due to constant loss & gain of protons through mechanism
Give 4 structures found in a virus
Capsid - contains genetic material
Viral envelope - made of fatty lipid molecules taken from host cells
Surface proteins - recognition and binding
Virus genetic material (DNA/RNA) - info for making new copies of virus.
What drug inhibits DNA polymerase in viruses?
Aciclovir
How is aciclovir specific to viral DNA polymerase?
Viral thymidine kinase is 100 times more efficient at converting aciclovir - hence only active when virus is present.
Give 2 current use of aciclovir clinically.
Herpes infections
Varicella-zoster viruses - such as Chicken pox and Shingles
What drug inhibits HIV protease?
Saquinavir
Why has saquinavir use reduced over time?
Poor bioavailability and drug resistance
Several improved analogues are now available
What does MMP stand for?
Matrix Metalloproteinases
What do MMPs depend on and what’s their importance?
Zinc - important role in the invasiveness and metastasis of cancer cells
How do MMP inhibitors work?
They inhibit the breakdown of the extra-cellular matrix
Making it difficult for cancer cells to escape and metastasize.
What general properties do MMP (peptide-based) inhibitors possess?
Replacement of susceptible peptide bond - OH instead, creating a Transition-state isostere/analogue
At least one functional group capable of H-bonding to enzyme backbone
Group capable of strong interactions with zinc ion co-factor - Marimastat
- Thiol
- Carboxylate
- Hydroxamic acid ( —C(O)NHOH )
What is the function of kinases?
To catalyse the phosphorylation of proteins, and have an important role in signalling pathways within cells.
What is the use of halide groups attached to a drug
The prevent metabolism
Give the main properties 4 - anilinoquinazolines developed by Astra Zeneca
Electron-donating substituents
Small lipophilic group (CH3) - fat solubility
Secondary amine
OH group removes anti-cancer properties
Where do kinase inhibitors act?
Epidermal growth factor receptor (EGF-R)
What is the benefit of adding a morpholine ring to a drug like Getifinib?
Improves water solubility with no effect on target binding as it protrudes from the binding site - no energy penalty for desolvation
