Lecture 4: Targeting Enzymes: High selectivity Flashcards
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
Drug selectivity problem
-try to design small molecule that binds to subtilisn serine protease, but NOT chymotrypsin serine protease
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
COX-1
constitutive enzyme
-inhibition undesirable bc of gastrointestinal side effects
COX-2
inducible and produced in response to inflammation
-inhibition stops inflammation
Nonspecific inhbition of COX effects
gastrointestinal bleeding
COX-2 inducers
-IL-1, TNF, growth factors
COX-2 selective inhibitors
-designed to avoid gassy side effects from COX-1 inhibition
-Celecoxib (celebrex)
-Rofecoxib (vioxx)
-bind same region as aspirin
If COX-1 protein was mutated at position 523 to Asp instead of Ile,
the selectivity of celecoxib would still favor COX-2
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
Steric clash region
-part of COX-2 inhibitor that reduces affinity towards COX-1
Quantifying selective inhibitors
IC50(COX-2) / IC50(COX-1) ratio defines selectivity
IC50
total concentration of inhibitor needed to reach 50% inhibition
IC50 ratio > 1
DEcreased COX-2 specificity
IC50 ratio < 1
INcreased COX-2 specificity
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
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
Allosteric inhibitor of PTP4A
-JMS-053 designed and is allosteric and selective
-interacts with catalytic residues making them unavailable for catalysis
PTP4A (protein tyrosine phosphatase)
-overexpressed in human cancers
-potential target
How do allosteric compounds provide the opportunity for enzyme
activation, not just inhibition?
-cooperativity in kinetics
-multimeric enzyme
-detect cooperativity in velocity curves
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
