Lecture 4: Targeting Enzymes: High selectivity Flashcards by Brooke Thornburg

binding selectivity of drug

-affinity for binding one receptor relative to the affinity for a second receptor
-how well dosage produces desired effect vs side effects
-KD vs KD or IC50 vs IC50

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Drug selectivity problem

-try to design small molecule that binds to subtilisn serine protease, but NOT chymotrypsin serine protease

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NSAIDs

-Non-Steroidal Anti-Inflammatory Drug
-inhibit COX-1 and COX-2 nonspecifically
-block prostaglandin production
-irreversible or reversible
-aspirin, ibuprofen, naproxen
-Selectivity is beneficial

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COX-1

constitutive enzyme
-inhibition undesirable bc of gastrointestinal side effects

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COX-2

inducible and produced in response to inflammation
-inhibition stops inflammation

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Nonspecific inhbition of COX effects

gastrointestinal bleeding

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COX-2 inducers

-IL-1, TNF, growth factors

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COX-2 selective inhibitors

-designed to avoid gassy side effects from COX-1 inhibition
-Celecoxib (celebrex)
-Rofecoxib (vioxx)
-bind same region as aspirin

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If COX-1 protein was mutated at position 523 to Asp instead of Ile,

the selectivity of celecoxib would still favor COX-2

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COX-2 inhibitors: structure

-COX-2 has a VALINE residue (V523) near active site that is smaller than isoleucine (I523) in COX-1
-reduced affinity for COX-1 due to steric hindrance

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Steric clash region

-part of COX-2 inhibitor that reduces affinity towards COX-1

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Quantifying selective inhibitors

IC50(COX-2) / IC50(COX-1) ratio defines selectivity

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IC50

total concentration of inhibitor needed to reach 50% inhibition

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IC50 ratio > 1

DEcreased COX-2 specificity

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IC50 ratio < 1

INcreased COX-2 specificity

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Targeting allosteric sites

-discover/develop ligands that bind enzymes at allosteric sites and alter enzyme activity in a desired manner
-potentially greater selectivity
-possibility of controlling enzyme activity

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Allosteric effector (inhibitor)

-acts at a distance
-binds target protein at a site other than where substrates and cofactors bind (not modified chemically during reaction
-typically binds E and ES (mixed-inhibition)
-inhibition kinetics with lineweaver plots that intersect on X-axis

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Allosteric inhibitor of PTP4A

-JMS-053 designed and is allosteric and selective
-interacts with catalytic residues making them unavailable for catalysis

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PTP4A (protein tyrosine phosphatase)

-overexpressed in human cancers
-potential target

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How do allosteric compounds provide the opportunity for enzyme
activation, not just inhibition?

-cooperativity in kinetics
-multimeric enzyme
-detect cooperativity in velocity curves

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sigmoidal curve

cooperativity

hyperbola curve

no cooperativity

multimeric enzyme

activity of one subunit affects activity of another
=subunits not independent

Effectors

-inhibitors(-)/activators(+) that affect activity of allosteric enzymes
-alter affinity (KM) and/or reactivity (Vmax)

Effector Binding

-most often multiple subunits -conformational change that propogates to other subunits via contacts at subunit interface -hetero vs homo

inhibition

increases KM

heterotropic

ligands in effector site not same as substrate ligand

homotropic

same ligand as substrate

Positive effector (activate)

changes both subunits to higher affinity form

Negative effector (inhibit)

binding changes both subunits to low affinity form

Cooperativity graph

-sigmoidal curve -velocity vs substrate concentration -top is nearly fully active -increases with slope (measured by Hill coefficient) -consider range of concentrations needed to vary from active to inactive

Misregulation causes disease: GOUT

-inflammatory disease from overproduction of uric acid (from purine degradation) -could be due to overproduction of purines from abnormal enzyme activity -likely PRPP synthetase culprit

PRPP synthetase

-catalyzes rate limiting step in nucleotide synthesis -excessive activity leads to uric acid overproduction

Gout Case Study

-increased PRPP levels consistent with PRPP synthetase disfunction -BUT PRPP synthetase had normal Km and Vmax and normal cell levels

PRPP synthetase activity

-regulated by phosphate ion, divalent metal cations and ADP -inhibited by ADP

Gout Case conclusion

-PRPP synth normally inhibited by ADP -patient's increase in PRPP came from ADP not inhibiting the synthase =possibility of mutation in allosteric site lead to failure of allosteric control

Highly selective drugs

-bind receptor (enzyme) with higher affinity than structurally similar proteins/isoforms -reduce side efffects from binding similar shaped proteins

Increase drug selectivity

-increase target protein affinity -decrease affinity with off-target proteins

Selectivity quantification

-ratio of affinity (KI) or activity (IC) of targeted receptor to off-target receptor -either can be in numerator or denominator

Selectivity of binding

-structure based -shape and chemical complementarity for target but not for similar proteins

Allosteric sites

-explored for therapeutics -high selectivity -can theoretically be used to gain control (+/-) by following principles of cooperativity

Enzyme Cooperativity

-sigmoidal curves -Hill coefficient -allosteric (homo/hetero) effector molecules activate/inhibit

Allosteric effectors

-homo/heterotropic -dont bind in catalytic site -bind at oligomeric interfaces of the enzyme (ex PRPP synthetase)

Improper regulation and activity

=disease

The basis for the selectivity of certain NSAIDS, such as celecoxib, for COX-2 over COX-1 was discussed in class. Selectivity was achieved by

disrupting the spatial complementarity between the inhibitor and COX-1

DFP has the second highest affinity for COX-2 but is the most selective of all listed compounds

High affinity

-low IC50 -low KI

High selectivity

-low target to off-target ratio -target IC50 / off-target IC50 <1

Allosteric inhibitor increases Km

shifts graph to right

low KI

less amount of ligand needed to inhibit partner