Week 1A - Enzymes and enzyme inhibitors + Drug development Flashcards
Chapter 7 pg 210-235 Chapter 25.3 pg 780-783 Chapter 12.4 pg 362-369 Chapter 32 pg 977-1001 Chapter 7, 25.3, 12.4, 32 + HC02/03
HC01: Parameters for enzyme substrate reaction
Interaction enzyme with substrate: Km
Bound substrate is converted: Vmax, kcat (turnover number)
Michaelis Menten equation
v = (Vmax*[S])/(Km+[S])
Hyperbolic curve between v and [S]
Reversible inhibitors with change parameters and examples
-Competitive inhibitors: substrate and inhibitor bind substrate binding site
> apparent Km increased, Vmax unchanged
>Ibuprofen, statins, bortezomib, azoles
> transition-state analogs: osaltamivir
-Noncompetitive inhibitors bind allosteric sites
> decreased apparant Vmax but Km unchanged
> echinocandines
Irreversible inhibitors with change on MM parameters and examples
Irreversible: decreased Vmax, fewer active enzymes
> Acetylsalicyclic acid (aspirin)
-Mechanism based (suicide) inhibitors: penicillin
Allosteric enzymes
Multidomain enzymes (regulatory and catalytic domain in subunit) or multisubunit enzymes
Allosteric inhibitors (small metabolites)
Bind allosteric enzymes reversibly at an allosteric site (not active site) and change the enzyme conformation from a relaxed (R) state to a less active tense (T) state.
> sigmoid curve, no MM kinetics
Types of allosteric inhibition
-Noncompetitive: binds to allosteric site inhibiting enzyme activity but the substrate can still bind.
-Competitive: prevents substrate from binding
Suicide inhibitors
Modified substrates that modify the catalytic residue of an enzyme covalently.
> first reversible binding
catalytic mechanism creates reactive intermediate which covalently binds the catalytic residue
CYP enzymes full name
Cytochrome P450
Function cytochrome
protein that transfers electrons, using heme as its prosthetic group
> the iron ion of cytochrome alternates between reduced 2+ and oxidized 3+ state during electron transport
CYP enzyme groups
-Those that metabolize xenobiotic (foreign) molecules like drugs and pollutants
-Those that participate in key biosynthetic pathways (biosythesis of sterols or vitD)
How many different CYP enzymes?
57 in 18 families
Reaction mechanism CYP enzymes
Enzyme with Fe3+ binds the substrate: RH
> Adrenodoxin (reductant) donates electron and Fe2+ form in CYP
(regeneration adrenodoxin by reducing FP with an NADPH. FPred reduces the adrenodoxin)
> Fe2+ can bind molecular oxygen (O2): Addition Fe2+-O=O
> Adrenodoxin donates another electron: Fe3+-O-O (2-)
> One oxygen is removed using H+: Fe4+=O and H2O creation
> Addition oxygen in substrate: RH > ROH
> CYP with prostethic heme group with ferric iron (3+)
> cycle
Name the prostethic group, co-enzyme, substrate and co-substrate of CYP enzymes?
Prosthetic group: hem
Co-enzyme: NADPH
Substrate: RH
Co-substrate: O2
Full reaction CYP enzymes
RH + O2 + NADPH + H+ > ROH + H2O + NADP+
Which CYP families for metabolizing xenobiotics and whch for alcohol (hydroxylase activity)?
CYP1,2,3,4 for xenobiotics
CYP2E1 for alcohol
HC02: The structures of peicillin and ampicillin are similar. What do they share
A beta-lactam ring, but also a benzene ring and overall structure except extra amino group on ampicillin
Approaches drug discovery
-Compound > physiological effect > molecular target
-Target > compound > physiological effect
Active bit of the penicillin
Beta-lactam ring
> carbon atoms, NH and C=O group and single N.
Function penicillin
Inhibit crosslinking of peptido-glycan chains, such that the bacterial cell wall weakens and bacterial cell lyse
> suicide inhibitor: resembles D-Ala-D-Ala bond
> transpeptidase
Function Sildenafil, initial target and temporary use
Initial target: treat angina pectoris (chest pain) by relaxing smooth muscle.
> Increase of cGMP results in relaxation of smooth muscle cells in blood veins
> Sildenafil inhibits phosphodiesterase 5 as competitive inhibitor (catalyzes hydrolysis of cGMP to GMP.
> effect: relaxation smooth muscle cell in corpus caverosum > inflow blood: erection
> Viagra
Smooth muscle relaxation mechanism
GTP -> cGMP by guanylate cyclase (which is allosterically upregulated by NO)
cGMP > GMP by phosphodiesterase 5, inhibited by the drug sildenafil
Enzyme inhibitors can be used as drug against HIV-1. Explain the mechanism of HIV-1
Fusion with cell, reverse transcriptase
> provirus (DNA from virus integated in nucleus human DNA) makes mega protein which needs to be cut by a viral protease
> drug: protease inhibitor for HIV protease
Name for combinations of different anti-retroviral drugs for treatment aids?
Highly active anti-retroviral therapy (HAART)
> for example two nucleoside analogs which inhibit HIV cDNA synthesis and one HIV protease inhibitor
HIV protease inhibitor
Indinavir: competitive inhibitor HIV protease which resembles gag-pol peptide
HIV protease structure
It cleaves multidomain viral proteins into active forms
> inhibition prevents HIV from being infectious
> homodimer
> after substrate binding, the flexible flaps close over the internal substrate binding site: high affinity in enzyme
Indinavir function
Indinavir contains a peptide bond: recognized as substrate, and a group which enhances affinity and makes it more affinity than the actual substrate.
> Indinavir is able to close the flaps of HIV protease
Anti-retroviral drig to treat influenza virus H1N1
Oseltamivir
Influenze virus structure
Carries two surface proeins: hemagglutinin (H) and neuraminidase (N)
> only certain H and N types are infectious for humans
- Influenza virus targets PM of cells through binding hemagglutinin to sialic acids residues (sugar chains)
-Later stage: neuraminidase cleaves off the sialic acis residues to release the viral particle. (after assembly and budding of new virus, for infecting other cells)
Oseltamivir function
Transition-state analog of influenza neuraminidase (of the sialic acid) > competitive inhibitor
> mimics the ring oxinium ion in reaction of viral enzyme neuraminidase
ADME properties of drugs
-Absorption
-Distribution
-Metabolism
-Excretion
The concentration of a drug at its target compartment depends on its …
ADME properties
ADME: absorption
-Ideally drug as smal oral tablet
> depends on oral bioavailability: ability to be absorbed
-Lipinskis Rules of 5 for drugs taken up by diffusion
Lipinski’s Rules of 5
-Molecular weight < 500
-Number of H-bond donors (OH/NH) <5 (too many, too hydrophilic)
-Number H-bond acceptors (N/O) < 5
-Partition coefficient log(P) < 5
– Log(P): log10 ratio of drug concentration in octanol to the concentration in water
Do the O of the OH group count as a hydrogen bond acceptor, and is the H of the OH a donor?
Yes and yes
> just a -H > no donor
ADME: distribution
Hydrophobic compounds do no dissolve freely in blood but bind abundant proteins like serum albumin
> Compounds distribute over various body fluids and tissues (compartments)
> many compounds unable to pass blood-brain barrier
> for target organs
ADME: metabolism
Body has defense against foreign/xenobiotic compounds by modifying them through drug metabolism
> oxidation (Phase I transformation)
> conjugation (Phase II transformation)
-become more water soluble > easily recognized as foreign
Ibuprofen belongs to the …
non-steroidal anti-inflammatory drugs (NSAIDs) > relieve fever
Oxidation ibuprofen (phase I transformation)
Ibuprof + NADPHH + H+ + O2 > Ibuprof-ox (OH group extra) + NADP+ + H2O (by CYP enzymes in liver)
Phase II transformation of drugs
Conjugation: Addition of one of these groups to xenobiotic compound
> glutathione
> glucoronic acid
> sulfate
Is phase I and phase II oxidation a rigid order of drug metabolism
Often yes, first oxidation then conjugation
ADME: excretion, two pathways
-Absorption through kidneys and excretion in urine
-Active transport by liver into the bile and excretion into intestines and the stool
Enterohepatic cycling
Some compounds are recycled after excretion
> from intestine to blood to liver and via bile to intestine
-The cycling decreases the rate of drug excretion
Half-life of drug excretion
Time until the concentration of the drug is halved of its value.
Different stages of drug development
Research institutes: target identification, validation, and in vitro assay
Pharmaceutical companies: High throughput assay, lead compound, combinatorial chemistry, candidate drug
Clinical trial: phase I, II, III, and then Registration for new drug at EMA or FDA
Split pool synthesis
Reactions performed on beads which are pooled after the reaction
> all compounds on a single bead are identical, and different from those on other beads
Induced fit model for enzyme substrate binding
Enzymes are flexible and shape of active site can change upon substrate binding to make complementary shape when substrate is bind for higher affinity binding
Using 3D structure for drugs
Compound with a part with good inhibition but poor solubility and part with better solubility by combining the parts which were two compounds
> soluble and high affinity
Characteristics drugs
-IC50
-log(P)
-cmax
IC50, log(P) and cmax
IC50: inhibitory concentration at 50% inhibition
log(P) partition coefficient
cmax: maximal concentration in bloodstream
Ideal characteristics drug
Low IC50, low log(P), high cmax (important, has to be transported in blood)
Clinical trial phases
-Phase I: small cohort of healthy volunteers
-Phase II: small controlled study of drug vs placebo (double blind and different doses tested)
-Phase III: large population of subjects, thousands patients, look for side effects, is expensive
Efficacy of the dose out of Phase II and III trials
Number of patients who report improvement
> the power of placebo effect should not be underestimated.
Human genome to discover drug targets
New targets through protein kinases and 7TM receptors by looking at genome
> genome: 22000 proteins
> > 500 distinct kinases, 800 distinct 7TM receptors
Effective dose
the dose or concentration of a drug that produces a biological response.
> measure efficacy and determine effective dose
