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Biochem > Lecture 4: Targeting Enzymes: High selectivity > Flashcards

Lecture 4: Targeting Enzymes: High selectivity Flashcards

1
Q

binding selectivity of drug

A

-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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2
Q

Drug selectivity problem

A

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

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3
Q

NSAIDs

A

-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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4
Q

COX-1

A

constitutive enzyme
-inhibition undesirable bc of gastrointestinal side effects

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5
Q

COX-2

A

inducible and produced in response to inflammation
-inhibition stops inflammation

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6
Q

Nonspecific inhbition of COX effects

A

gastrointestinal bleeding

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7
Q

COX-2 inducers

A

-IL-1, TNF, growth factors

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8
Q

COX-2 selective inhibitors

A

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

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9
Q

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

A

the selectivity of celecoxib would still favor COX-2

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10
Q

COX-2 inhibitors: structure

A

-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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11
Q

Steric clash region

A

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

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12
Q

Quantifying selective inhibitors

A

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

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13
Q

IC50

A

total concentration of inhibitor needed to reach 50% inhibition

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14
Q

IC50 ratio > 1

A

DEcreased COX-2 specificity

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15
Q

IC50 ratio < 1

A

INcreased COX-2 specificity

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16
Q

Targeting allosteric sites

A

-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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17
Q

Allosteric effector (inhibitor)

A

-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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18
Q

Allosteric inhibitor of PTP4A

A

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

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19
Q

PTP4A (protein tyrosine phosphatase)

A

-overexpressed in human cancers
-potential target

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20
Q

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

A

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

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21
Q

sigmoidal curve

A

cooperativity

22
Q

hyperbola curve

A

no cooperativity

23
Q

multimeric enzyme

A

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

24
Q

Effectors

A

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

25
Q

Effector Binding

A

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

26
Q

inhibition

A

increases KM

27
Q

heterotropic

A

ligands in effector site not same as substrate ligand

28
Q

homotropic

A

same ligand as substrate

29
Q

Positive effector (activate)

A

changes both subunits to higher affinity form

30
Q

Negative effector (inhibit)

A

binding changes both subunits to low affinity form

31
Q

Cooperativity graph

A

-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

32
Q

Misregulation causes disease: GOUT

A

-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

33
Q

PRPP synthetase

A

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

34
Q

Gout Case Study

A

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

35
Q

PRPP synthetase activity

A

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

36
Q

Gout Case conclusion

A

-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

37
Q

Highly selective drugs

A

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

38
Q

Increase drug selectivity

A

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

39
Q

Selectivity quantification

A

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

40
Q

Selectivity of binding

A

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

41
Q

Allosteric sites

A

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

42
Q

Enzyme Cooperativity

A

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

43
Q

Allosteric effectors

A

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

44
Q

Improper regulation and activity

A

=disease

45
Q

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

A

disrupting the spatial complementarity between the inhibitor and COX-1

46
Q
A

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

47
Q

High affinity

A

-low IC50
-low KI

48
Q

High selectivity

A

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

49
Q

Allosteric inhibitor increases Km

A

shifts graph to right

50
Q

low KI

A

less amount of ligand needed to inhibit partner

Biochem (30 decks)

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