Lecture 10 Flashcards
Drug Design and Regulation
Influenza Virus: The Design of Relenza, an anti-flu medicine
The reaction catalyzed by neuraminidase
Proposed mechanism of the neuraminidase catalyzed
glycoside hydrolysis reaction
Sialic acid = N-ACETYL-NEURAMINIC ACID
(You do not need to know the chemical formula of Sialic Acid or of the TS, but you need to know that in the TS four atoms (C-2 and its surrounding three atoms) are in a plane)
Design of Transition State Analog neuraminidase
inhibitors
TSA = transition state analog
Transition State Features:
• Planarity at C2 (sp2)
• Buildup of + charge
DANA
• Transition state analogue
• Ki = 10-6 M
• Also inhibits human neuraminidase
Influenza Virus Neuraminidase in complex with 9-amino-DANA
View of two key Neuraminidase residues near the 4-OH of 9-amino-DANA
View of two key Neuraminidase residues near the 4-OH of 9-amino-DANA
Two negatively charged carboxylates are quite close to the 4-OH !!!!!
Compound made: 4-guanidino-DANA
A guanidino substituent at the 4-position instead of a hydroxyl
Does it indeed live up to the expectations? i.e. of being a better inhibitor than DANA?
Inhibitory Properties of modified TSA’s
Based on the structure of the TSA DANA in complex with influenza virus neuraminidase, the compound
4-guanidino-DANA was designed and synthesized.
The Ki-values (in M) were as follows:
DANA Flu= 1 x 10-6 human= 1.2 x 10-5
4-guanidino-DANA flu= 2 x 10-10 flu= 1 x 10-3
By changing one single functional group:
• The affinity for the target flu enzyme was enhanced by a factor of ~10,000.
• The affinity for the human homologous enzyme was decreased by a factor of ~100.
• Hence, the selectivity was improved by a factor of ~1,000,000!!!
Medicines have to fulfill many requirements
Drugs are very precious compounds
For orally available medicines a fine balance is required between :
(i) Sufficient capacity to cross membranes
(so it can be taken up from the digestive tract)
(ii) Sufficient water solubility
(so it can reach the site of action in sufficient concentrations)
Some other requirements of an ideal medicine are:
(iii) Not being converted to an inactive substance by human enzymes
(iv) Not being cleared rapidly from the blood
(v) No teratogenicity
(vi) No mutagenicity
(vii) No toxicity
Hence, it is not really a surprise that it is a major challenge to make a new safe, effective, orally available, affordable medicine.
Enzyme Regulation
Essential for Life
Three examples of enzyme regulation follow, showing some of the ways enzymes are regulated:
• Make inactive enzyme precursors (“zymogens”) and activate when needed
• Use inhibitory proteins to inactivate enzymes
• Use substrate/product binding (“substrate-level” control)
- inhibition by an enzyme’s product (e.g., hexokinase)
- or through feedback loops in a metabolic pathway
Other methods (not discussed today) include: • Covalent modification (phosphorylation/dephosphorylation)
Make First Inactive Proenzymes and only activate these when and where needed.
Zymogens are the inactive precursors of enzymes.
This term is generally applied to proteases. Proenzyme is a synonym.
zymogens of enzymes
Zymogens are the inactive precursors of enzymes.
This term is generally applied to proteases. Proenzyme is a synonym.
Pepsinogen --> Pepsin Chymotrypsinogen -->Chymotrypsin Trypsinogen -->Trypsin Procarboxypeptidase -->Carboxypeptidase Proelastase--> Elastase
Activation of chymotrypsinogen
After transport of the inactive chymotrypsinogen to the duodenum:
Trypsin activates the zymogen chymotrypsinogen in multiple steps to obtain active forms of chymotrypsin.
After the “nick” between residues 15 and 16, a conformational change occurs near the active site such that the active “π-chymotrypsin” is obtained.
Subsequent removal of two dipeptides leads to the also active “α-chymotrypsin”.
Enzyme Activity Control by an Inhibiting Protein
Pancreatic trypsin inhibitor (PTI) is a protein which binds very tightly to one specific enzyme to form a stable, inactive complex.
The dissociation constant Kd = 10-13 M (!)
Key to the inhibition is Lys15 of PTI which fits perfectly into the “specificity pocket” of Trypsin where Asp189 “rules”. The reaction cannot proceed since the inhibitor is bound so tightly.
Proteases wreak havoc if they escape their controls.
Organisms have evolved a battery of protease inhibitors to counter this threat.
This complex is formed in the pancreatic cells which produce trypsin.
Allosteric Regulation of Metabolic Pathways
Aspartate Transcarbamoylasse (ATCase)
This is called “feedback inhibition” : BY THE END PRODUCT OF THIS PATHWAY.
Purpose: If there is plenty CTP in the cell, there is no need to make more CTP.
ATCase catalyzes the first committed step in pyrimidine synthesis (CTP & UTP)
- In addition, ATP stimulates ATCase, one of the ways to balance the ATP and CTP concentrations in the cell since these two compounds are both pretty important for nucleic acid synthesis.
- Both substrates, as well as CTP and ATP, affect the activity of ATCase in an allosteric manner
- That is, by affecting an active site away from the compound binding site
The end product of the pathway, CTP, inhibits ATCase, which catalyzes the pathway’s first step
Kinetics of ATCase (I)
Note how the curves are sigmoidal and not hyperbolic.
• The Michaelis-Menten model would have predicted a hyperbolic curve.
• We have seen an analogous case before for hemoglobin, and is the result of
cooperativity.
ATCase is a multimer with six Catalytic (C) and six Regulatory subunits (R).
Lets first look at substrates alone (pink curve):
• Substrates aspartate and carbamoyl phosphate bind cooperatively to the enzyme
• Substrate binding to one catalytic subunit increases substrate binding and catalytic activity of the other five subunits.
• Substrate binding is accompanied by conformational changes (quaternary structure)
