Week 1A - Enzymes and enzyme inhibitors + Drug development

Question 1

HC01: Parameters for enzyme substrate reaction

Answer 1

Interaction enzyme with substrate: Km
Bound substrate is converted: Vmax, kcat (turnover number)

Question 2

Michaelis Menten equation

Answer 2

v = (Vmax*[S])/(Km+[S])
Hyperbolic curve between v and [S]

Question 3

Reversible inhibitors with change parameters and examples

Answer 3

-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

Question 4

Irreversible inhibitors with change on MM parameters and examples

Answer 4

Irreversible: decreased Vmax, fewer active enzymes
> Acetylsalicyclic acid (aspirin)
-Mechanism based (suicide) inhibitors: penicillin

Question 5

Allosteric enzymes

Answer 5

Multidomain enzymes (regulatory and catalytic domain in subunit) or multisubunit enzymes

Question 6

Allosteric inhibitors (small metabolites)

Answer 6

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

Question 7

Types of allosteric inhibition

Answer 7

-Noncompetitive: binds to allosteric site inhibiting enzyme activity but the substrate can still bind.
-Competitive: prevents substrate from binding

Question 8

Suicide inhibitors

Answer 8

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

Question 9

CYP enzymes full name

Answer 9

Cytochrome P450

Question 10

Function cytochrome

Answer 10

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

Question 11

CYP enzyme groups

Answer 11

-Those that metabolize xenobiotic (foreign) molecules like drugs and pollutants
-Those that participate in key biosynthetic pathways (biosythesis of sterols or vitD)

Question 12

How many different CYP enzymes?

Answer 12

57 in 18 families

Question 13

Reaction mechanism CYP enzymes

Answer 13

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

Question 14

Name the prostethic group, co-enzyme, substrate and co-substrate of CYP enzymes?

Answer 14

Prosthetic group: hem
Co-enzyme: NADPH
Substrate: RH
Co-substrate: O2

Question 15

Full reaction CYP enzymes

Answer 15

RH + O2 + NADPH + H+ > ROH + H2O + NADP+

Question 16

Which CYP families for metabolizing xenobiotics and whch for alcohol (hydroxylase activity)?

Answer 16

CYP1,2,3,4 for xenobiotics
CYP2E1 for alcohol

Question 17

HC02: The structures of peicillin and ampicillin are similar. What do they share

Answer 17

A beta-lactam ring, but also a benzene ring and overall structure except extra amino group on ampicillin

Question 18

Approaches drug discovery

Answer 18

-Compound > physiological effect > molecular target
-Target > compound > physiological effect

Question 19

Active bit of the penicillin

Answer 19

Beta-lactam ring
> carbon atoms, NH and C=O group and single N.

Question 20

Function penicillin

Answer 20

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

Question 21

Function Sildenafil, initial target and temporary use

Answer 21

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

Question 22

Smooth muscle relaxation mechanism

Answer 22

GTP -> cGMP by guanylate cyclase (which is allosterically upregulated by NO)
cGMP > GMP by phosphodiesterase 5, inhibited by the drug sildenafil

Question 23

Enzyme inhibitors can be used as drug against HIV-1. Explain the mechanism of HIV-1

Answer 23

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

Question 24

Name for combinations of different anti-retroviral drugs for treatment aids?

Answer 24

Highly active anti-retroviral therapy (HAART)
> for example two nucleoside analogs which inhibit HIV cDNA synthesis and one HIV protease inhibitor

Question 25

HIV protease inhibitor

Answer 25

Indinavir: competitive inhibitor HIV protease which resembles gag-pol peptide

Question 26

HIV protease structure

Answer 26

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

Question 27

Indinavir function

Answer 27

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

Question 28

Anti-retroviral drig to treat influenza virus H1N1

Answer 28

Oseltamivir

Question 29

Influenze virus structure

Answer 29

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)

Question 30

Oseltamivir function

Answer 30

Transition-state analog of influenza neuraminidase (of the sialic acid) > competitive inhibitor
> mimics the ring oxinium ion in reaction of viral enzyme neuraminidase

Question 31

ADME properties of drugs

Answer 31

-Absorption
-Distribution
-Metabolism
-Excretion

Question 32

The concentration of a drug at its target compartment depends on its …

Answer 32

ADME properties

Question 33

ADME: absorption

Answer 33

-Ideally drug as smal oral tablet
> depends on oral bioavailability: ability to be absorbed
-Lipinskis Rules of 5 for drugs taken up by diffusion

Question 34

Lipinski’s Rules of 5

Answer 34

-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

Question 35

Do the O of the OH group count as a hydrogen bond acceptor, and is the H of the OH a donor?

Answer 35

Yes and yes
> just a -H > no donor

Question 36

ADME: distribution

Answer 36

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

Question 37

ADME: metabolism

Answer 37

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

Question 38

Ibuprofen belongs to the …

Answer 38

non-steroidal anti-inflammatory drugs (NSAIDs) > relieve fever

Question 39

Oxidation ibuprofen (phase I transformation)

Answer 39

Ibuprof + NADPHH + H+ + O2 > Ibuprof-ox (OH group extra) + NADP+ + H2O (by CYP enzymes in liver)

Question 40

Phase II transformation of drugs

Answer 40

Conjugation: Addition of one of these groups to xenobiotic compound
> glutathione
> glucoronic acid
> sulfate

Question 41

Is phase I and phase II oxidation a rigid order of drug metabolism

Answer 41

Often yes, first oxidation then conjugation

Question 42

ADME: excretion, two pathways

Answer 42

-Absorption through kidneys and excretion in urine
-Active transport by liver into the bile and excretion into intestines and the stool

Question 43

Enterohepatic cycling

Answer 43

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

Question 44

Half-life of drug excretion

Answer 44

Time until the concentration of the drug is halved of its value.

Question 45

Different stages of drug development

Answer 45

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

Question 46

Split pool synthesis

Answer 46

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

Question 47

Induced fit model for enzyme substrate binding

Answer 47

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

Question 48

Using 3D structure for drugs

Answer 48

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

Question 49

Characteristics drugs

Answer 49

-IC50
-log(P)
-cmax

Question 50

IC50, log(P) and cmax

Answer 50

IC50: inhibitory concentration at 50% inhibition
log(P) partition coefficient
cmax: maximal concentration in bloodstream

Question 51

Ideal characteristics drug

Answer 51

Low IC50, low log(P), high cmax (important, has to be transported in blood)

Question 52

Clinical trial phases

Answer 52

-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

Question 53

Efficacy of the dose out of Phase II and III trials

Answer 53

Number of patients who report improvement
> the power of placebo effect should not be underestimated.

Question 54

Human genome to discover drug targets

Answer 54

New targets through protein kinases and 7TM receptors by looking at genome
> genome: 22000 proteins
> > 500 distinct kinases, 800 distinct 7TM receptors

Question 55

Effective dose

Answer 55

the dose or concentration of a drug that produces a biological response.
> measure efficacy and determine effective dose