Intro to Pharmacology and Toxicology
Topics
The activity of kinases and phosphatases may be manipulated by overexpression paradigms, upstream signaling, inhibitors and activators. Overexpression paradigms usually include the use of constitutively active enzyme or dead mutants (constitutively inactive enzyme). An example of upstream signaling is the use of forkolin (an activator of adenylyl cyclase) to activate cAMP-dependent protein kinases (PKAs).
General inhibitors are used when manipulating cell extracts and include metal chelators, fluoride and vanadate. Metal chelators like EDTA and EGTA block kinase activity by chelating Mg2+ and other divalent cations. Fluoride is a generic Ser/Thr phosphatase inhibitor. Vanadate is a generic Tyr phosphatase inhibitor.
There are several naturally occurring phosphatase inhibitors, but no natural kinase inhibitor has been found. Naturally occurring phosphatase inhibitors of PP1 and PP2A include:
Natural toxin inhibitors of PP1 and PP2A include:
There are many nonpeptide inhibitors of protein kinases, most of which target the ATP binding site. There are also peptide inhibitors that act as pseudosubstrates. No natural toxin inhibitor of kinases has been found. It is hard to show specificity of kinase inhibitors because it is a large gene family. In order to validate results, show that the effects of an inhibitor disappear when a drug resistant mutant of kinase is overexpressed. If a drug resistant mutant is not available, show that the effect occurs at the same concentration that prevents phosphorylation of a physiological substrate of kinase. Make sure that the same effect is observed with at leas two structurally unrelated inhibitors of known specificity.
Some experimental targets for phosphatase/kinase drug applications include: PTP1B (a Tyr phosphatase) and glycogen synthase kinase for the treatment of diabetes, and PKA as an exercise pill.
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1- List 4 ways of manipulating phosphorylation
overexpression
upstream signaling
inhibitors
activators
2- How can overexpression manipulate
phosphorylation?
By using constitutively active enzyme or dead mutants (constitutively inactive
enzyme).
3- List 1 example of upstream signaling
manipulation of phosphorylation.
Use of forkolin (an adenylyl cyclase activator) to activate PKA.
4- List 3 general inhibitors of phosphoproteins.
metal chelators (EDTA and EGTA)
fluoride
vanadate
5- How do metal chelators inhibit
phosphoproteins?
They block kinase activity by chelating Mg2+ and other divalent cations.
6- What is the activity of fluoride
on phosphoproteins?
Is a generic Ser/Thr phosphatase inhibitor.
7- What is the activity of vanadate
on phosphoproteins?
Is a generic Tyr phosphatase inhibitor.
8- List 8 naturally occurring (non-toxin)
phosphatase inhibitors.
PP2A-I1
PP2A-I2
Inhibitor 1
Inhibitor 2
DARPP32
PP1-I1
PP1-I2
NIPP1
9- What is the activity of PP2A-I1
and PP2A-I2?
Heat-stable inhibitors of all forms of PP2A at mM concentrations, are phosphorylated
in vivo with unknown results.
10- What is the common activity of
Inhibitor 1, DARPP32 and PP1-I1?
Heat-stable phosphatase inhibitors localized to the nucleus, do not require
phosphorylation.
11- What is the common activity of
Inhibitor 2 and PP1-I2?
Heat-stable phosphatase inhibitors, do not require phosphorylation.
12- List 6 natural toxin inhibitors
of phosphatases.
okadaic acid (from dinoflagellates)
tautomycin
microcystin
fostriecin
cantharidine
calyculin A (from sponges)
13- What is the activity of the
natural toxin phosphatase inhibitors?
They all inhibit PP1 and PP2A. Okadaic acid and microcystin also inhibit PP5.
14- What kinds of kinase inhibitors
are available?
Many nonpeptide inhibitors, most of which target ATP binding sites.
Many peptide inhibitors act as pseudosubstrates.
No naturally-occuring inhibitor has been found.
15- How can it be shown that a
kinase inhibitor is specific using overexpression?
Show that the effects of the inhibitor dissapear when a drug-resistant mutant
of the kinase is overexpressed. Make sure that the same effect is observed with
at least two structurally unrelated inhibitors of known specificity.
16- How can it be shown that a
kinase inhibitor is specific using a physiological substrate?
Show that the effect occurs at the same concentration that prevents phosphorylation
of a physiological substrate of the kinase. Make sure that the same effect is
observed with at least two structurally unrelated inhibitors of known specificity.
17- List 3 experimental targets
for phosphoprotein drug application.
PTP1B, a Tyrosine phosphatase, for diabetes treatment.
Glycogen synthase kinase, for diabetes treatment.
PKA as an excersise pill
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