protein structure, penicillin, drug delivery and sulphonamides Flashcards
1
Q
What makes up the cell membrane?
A
Phosphoglycerides and key proteins
2
Q
What are the components of phosphoglycerides?
A
Polar phosphate and glycerol head group, and hydrophobic tails
3
Q
What are amines?
A
Derivatives of carboxylic acids where the OH group is replaced by an amine
4
Q
What is the difference between primary, secondary, and tertiary amides?
A
Primary has no R groups (O=NH2), secondary has one R group (O=NH-R), tertiary has two R groups (O=NR1-R2)
5
Q
Why are amides fairly chemically inert?
A
The lone pair on the nitrogen is delocalised into the carbonyl group, giving a resonance structure with the negative charge on the oxygen and a positive, double-bonded nitrogen
6
Q
Why are amides planar?
A
delocalisation of the N lone pair gives a high energy barrier for C-N bond rotation
7
Q
Why do secondary amides adopt the trans configuration?
A
due to steric repulsion
8
Q
What configuration are all naturally occurring amino acids in?
A
S-configuration
9
Q
What is a dipeptide?
A
Two amino acids joined where the OH of one of the amino acids is
10
Q
What is the difference between amides and amino acids?
A
Amino acids have an OH group as well as a =O
11
Q
How are peptides and proteins formed?
A
by formation of amide bonds between alpha-amino acids
12
Q
How many common naturally occurring alpha-amino acids are there?
A
20
13
Q
What is the general formula for amino acids?
A
H2N-R-OOH
14
Q
How are peptides different to proteins?
A
Peptides usually contain less than fifty amino acids and a poorly defined structure.
Proteins are much larger and have a well-defined tertiary structure
15
Q
How do you calculate the possible number of combinations of an aa chain?
A
20 possible aa so use 20 to the power of the chain length number
16
Q
What does mer mean?
A
refers to the length of the chain e.g. 5-mer = chain of 5
17
Q
What are the secondary structures of proteins?
A
alpha helix and beta-pleated sheet
18
Q
Describe the alpha helix
A
coiling of a peptide chain, structure held together by hydrogen bonds and aa residues sticking out at right angles
19
Q
Describe the beta pleated sheet
A
layering of peptide chains on top of each other, hydrogen bonds hold the structure together, aa residues at right angles to the sheets
20
Q
How do aa residues in a primary sequence interact?
A
by repulsive and attractive forces, the system is dynamic and constantly trying to find energy minima
21
Q
What is the most stable conformation of any protein?
A
with aa on the outer surface where favourable bonding interactions can take place with water, in the middle the aa with non-polar resides reside in the hydrophobic centre
22
Q
What are 5 important drug targets?
A
enzymes, receptors, carrier proteins, ion channels, DNA
23
Q
What are the four key bonding interactions for forming tertiary protein structures in order of bond strength, strongest first?
A
Covalent, ionic, hydrogen, van der waals
24
Q
What is a key example of covalent bonding for forming protein tertiary structures?
A
disulfide bridge = 2 oxidised thiol residues connected, and covalent bonds usually occur between 2 cysteine residues
25
How is ionic bonding used for forming tertiary protein structures?
only available for 4 of the 20 aa, used for forming salt bridges (bond between anion and cation to join two residues)
26
How many aa have residues capable of H bonding?
8
27
How many aa have residues capable of vdw bonding?
8
28
Describe vdw bonding
control the overall shape of the tertiary structure, attraction due to uneven electron density in aromatic residues = temporary dipoles interacting
29
What are the interactions where a drug binds to a receptor?
ionic, hydrogen, vdw, dipole-dipole/ion-dipole, hydrophobic, covalent
30
Describe hydrophobic interactions
a non-polar drug and a hydrophobic part of a receptor interact and there is an increase in entropy of the surrounding water as there is increased disorder
31
Describe dipole-dipole interactions
Higher electronegative atoms bound with C (N, O, Cl) give the C-X bond an uneven distribution of e-, giving a dipole. the dipole of a particular functional group in a drug can be attracted to dipoles in the protein active site
32
Describe ion-dipole interactions
a charged ion residue in the protein active site interacts with the dipole of the drug
33
What is the equation for reversible drug interactions?
drug + receptor -> (kf) complex
34
What is Kf?
rate constant for association
35
What is Kr?
rate constant for dissociation
36
What is the equation of Gibbs free energy using K?
G = -RT lnK
37
What is the rate constant using the constants for association and dissociation?
K = Kf/Kr
38
What is the equation of Gibbs free energy using T?
G = change in standard H - T*(change in S)
39
What drugs are used in chemotherapy?
agents that form covalent bonds at enzymes or receptors
40
What is the target and use of acetylcholine?
muscarinic and/or nicotinic receptors
used for cholinergic transmission
41
What type of drug is acetylcholine?
agonist
42
What bond types are involved in acetylcholine?
ASN (asparagine) H-bonds, ionic, hydrophobic
43
What is the target and use of cyclopentolate?
muscarinic receptor, opthalmic inspection
44
What type of drug is cyclopentolate?
antagonist
45
What bond types are involved in cyclopentolate?
ASN (asparagine) H-bonds, ionic, hydrophobic
46
How is acetylcholine removed from the binding pocket?
by hydrolysis of the ester group
47
What is the target and use of methotrexate?
Dihydrofolate reductase, used in cancer/chemotherapy and to shut down folate synthesis
48
What type of drug is methotrexate?
enzyme
49
What bond types are involved in methotrexate?
ionic is the most important
50
What is the target and use of dyflos?
acetyl cholinesterase enzyme (dyflos breaks down acetyl cholinesterase) / glaucoma chemical warfare
51
What bond types are involved in dyflos?
covalent
52
What is stage 1 in the drug discovery process?
- A disease of importance to the western world is chosen.
- A drug target is chosen
- Bioassay identified
- A hit compound is found from natural sources or a combinatorial library
- The hit compound is isolated and purified
- The structure of the hit/lead compound is determined
53
What is stage 2 in the drug discovery process?
- Identifying structure-activity relationships (SARs)
- Identifying the pharmacophore
- Improving pharmacokinetic properties
- Patenting the drug
- Studying drug metabolism (ADME)
- Testing for toxicity in vitro
54
What does ADME stand for?
Absorption Distribution Metabolism Excretion
55
What is stage 3 in the drug discovery process?
- Designing a manufacturing process
- Carrying out clinical trials
- Marketing the drug
56
What does In Vitro testing involve?
isolated tissue, cells or enzymes
57
How are enzyme inhibitors tested in vitro?
on the purified enzyme in solution
58
How are receptor agonists or antagonists tested in vitro?
on isolated tissues or cells that express the target receptor on the surface
59
How are genetic techniques used in vitro?
a gene that codes for an enzyme or receptor can be cloned and then overexpressed in cells such as bacteria or yeast - giving more of a target receptor/enzyme than by conventional methods of isolation
60
How is in vitro activity expressed?
as an IC50 or EC50 concentration
61
What is the difference between IC50 and EC50?
IC50 = Inhibitory, EC50 = effective
62
What is IC50 usually for a good hit compound?
10mg/ml
63
What does high throughput screening involve?
automated testing of thousands of compounds against 30 and 50 biochemical targets, the test must be easily measured e.g. by colour change, so combinatorial chemistry is usually coupled to HTS
64
What does In vivo testing involve? How can the drug be assessed?
inducing a clinical condition in an animal to produce observable symptoms, the drug can then be assessed either by its ability to treat the condition, or to prolong survival
65
What are transgenic animals?
When animal genes are replaced by human genes and the genes produce the human receptor or enzyme in the host
66
List methods for discovering a lead compound
- Medical folklore
- random screening
- existing drugs - 'me too' drugs
- starting from the natural ligand (beta2 agonists)
- combinatorial chemistry
- computer aided design
- computer bases searching of structural data banks
- screening natural materials
67
What are 'me too' drugs also known as?
patent busting drugs
68
What enzyme does penicillin target and how does this affect the cell?
transpeptidase enzyme, is responsible for bacterial cell wall synthesis so penicillin inhibits bacterial cell synthesis - leads to holes developing in the cell wall so cell content leaks and causes death of the bacteria
69
Describe the mechanism for penicillin inhibiting transpeptidase
the enzyme-OH attacks the =O in the beta-lactam ring, irreversible acylation occurs, and the structure formed has the bond broken between the =O and the N, forming NH instead of N, and the O-enzyme attached to the =O
70
What is the structure of penicillin?
Acetyl ring (R-=O-NH), attached to beta lactam ring (square with =O in bottom left corner and N bottom right), top right of square connected to (thiazolidine ring) an S, joining the the bottom N in a pentagon, CO2H in bottom right of pentagon and two Me joined to top right of pentagon
71
What were the desirable properties of penicillin?
- active against gram positive and some gram negative bacteria
- not toxic and very selective - very safe drug
72
What were the undesirable properties of penicillin?
- not active over a wide range (spectrum) of bacteria
- ineffective when taken orally
- sensitive to all known beta-lactamases (bacterial resistance)
73
Why is penicillin ineffective when taken orally?
due to stomach decomposition
74
What is the structure activity relationship (SAR) for penicillin? Which parts can be changed?
- the bicyclic ring is important as it puts strain on the beta lactam ring, making the analogue more potent (however too much strain makes it chemically unstable)
- the beta lactam is essential as it covalently modifies the target bacteria
- the amide, cis stereochemistry, and free carboxylic acid are also essential so can't be changed
- only minor variations in the nucleus and the R group side chain can be changed
75
What are parts of a molecule which are crucial for activity called?
known collectively as the pharmacophore/pharmacophoric groups
76
What are the two problems with penicillin that had to be resolved?
acid sensitivity, and penicillin analogue sensitivity to beta-lactamases
77
Why is penicillin G acid sensitive?
- the bicyclic ring system gives large angle and torsional strain which is relieved with acid catalysed ring opening, which opens up a more highly strained 4 membered ring (=no longer penicillin)
- the carbonyl group (=O) in beta-lactams is very reactive
- the side chain can act to cleave the beta-lactam ring (neighbouring group participation) - self-destructing penicillin
78
What is NGP?
neighbouring group participation
79
What is the solution to penicillin G acid sensitivity?
nothing can be done in regards to the beta-lactam ring bc its essential for activity, so only self-destruction of the beta-lactam can be reduced. this is by chemical substitution in the acyl side-chain with an ewg to reduce the nucleophilicity of the carbonyl oxygen
80
What was the impact of solving the acid sensitivity of penicillin G?
- more stable
| - survives stomach acid so can be taken orally
81
Describe the mechanism for acid catalysed ring opening of penicillin
- H2O attacks the beta-lactam carbonyl and ed is moved onto the O, adding an OH in a new bond too
- The negative charge on the O moves back into the O bond, while a H+ is attacked by the N to be added
- this forms =O and -OH where the B-lactam carbonyl was, and the B-lactam ring is broken as the N is NH instead
82
How was penicillin analogue sensitivity to B-lactamases solved?
by incorporation of a large side-chain to the R group (for penicillin G = two R on either side of benzene bond to acetyl group) to act as a shield to prevent cleavage by lactamases (steric blockage)
83
What large side chains were used to tackle B-lactamase sensitivity in penicillin and why?
- methicillin (resists lactamase cleavage but still acid sensitive so given as IV)
- Oxacillin and flucloxacillin (not as active as methicillin but acid stable and resists lactamase cleavage)
84
What is the R group on penicillin G?
benzene
85
How is pharmacology used in the treatment of disease?
selecting the biological effect/efficacy
86
How is clinical pharmacology used in treatment of disease?
determining the safe and effective use in man
87
What is epidemiology?
Where the disease occurs and the groups that it effects
88
What is pathology?
which organs/tissues are damaged
89
How are potency and dosage balanced?
if a drug is potent it can be given at a low dosage so there's a lower likelihood of adverse events
90
How does molecular recognition work?
Small molecules interact with specific amino acids in the target protein or bases in DNA - involves irreversible bonds (NON-COVALENT)
91
What encompasses molecular response?
enzyme inhibition, receptor - (if agonist = response, if antagonist = blocked response), ion channel (open or closed), human cells = change of function
92
What are sulphonamides?
antibacterial agents, ineffective against salmonella (responsible for typhoid), block the biosynthesis of tetrahydrofolate in bacterial cells - preventing their DNA synthesis
93
What is the mechanism of sulphonamide action?
Dihydropteroate synthetase and para-aminobenzoic acid (PABA) are in a reaction together, but the sulphonamide prevents PABA from binding as the sulphonamide is similar in structure to PABA) (irreversible inhibition), instead, L-glutamate reacts with dihydropteroate to form folic acid.
Folic acid then reacts with NADP to form tetrahydrofolic acid (Coenzyme F)
94
What is PABA?
para-aminobenzoic acid: one of the normal substrates for dihydropteroate synthetase
95
What is the structure of dihydropteroate synthetase?
Benzene ring with N in positions 4 and 6 instead of carbon, (counting C1 at the top of the ring), in positions 1 there is an OH and in position 5 NH2.
Another ring is joined alongside the first ring -
this ring has no double bonds and has N at the top and bottom, replacing the C. CH2OOP'P' is joined to the second ring in position 2
96
What does P with a circle around it mean (P') for the purposes in these cards?
= O-P-O-P-O
with =O at the top of both of the Ps
and -O(minus) at the bottom of both of the Ps
97
What is the structure of PABA?
benzene ring with H2N at the top, and CO2H at the bottom
98
What is the structure of L-Glu?
H2N-CH-CO2H with CH2CH2CO2H also attached to the CH
99
What is the structure of folic acid?
The structure of dihydropteroate synthetase (minus the OP'P') connected to PABA (with the OH removed), connected to L-Glu (with one H removed from H2N)
100
What is the structure of sulphonamides?
same as PABA but S with 2(=O) on top and bottom, and NHR connected to the S too
101
How do sulphonamides form toxic metabolites?
hydroxylamine metabolites, the amino group is metabolised by P450 enzymes, one of the Hs is converted to OH, however the amino group can't be removed as it is essential for activity (and is only toxic in some patients)
102
What are the effects of asthma?
narrowing of the tubes carrying air into the lungs, as a result of muscle constriction (bronchospasm)
103
What is the target for treating asthma?
beta2-adrenoceptor (if stimulated dilates the vessels in the lungs = bronchodilation)
104
What is the chemical transmitter for asthma?
noradrenaline
105
What is the structure of noradrenaline?
benzene with 2(OH) in positions 3&4, and in position 1 CH-OH with CH2-NH2 also connected to the CH in the row (the OH is beneath the CH)
106
What happens if noradrenaline interacts with the alpha1 receptor?
increases heart rate if noradrenaline is directly administered
107
What is the drug which stimulates both B1 and B2 adrenoreceptors?
isoprenaline (= noradrenaline with H3C-CH-CH3 in place of one of the Hs on NH2) - bulky substituent
108
Does isoprenaline affect alpha1- receptors?
no
109
What is the drug which is B2-adrenoreceptor selective?
salbutamol (=same as isoprenaline but extra CH2 between the top OH and the benzene ring) = a lung-specific agonist
110
What factors are the effectiveness of a drug dependent on?
route of administration, disposition, metabolism, excretion, efficacy at drug target
111
What is the pharmaceutical phase?
routes of drug administration
112
What are the forms of drug administration?
Sublingual, oral, rectal, cutaneous, inhalation, injection
113
What are pro-drugs?
When compounds are converted into pharmacologically active drugs in the body - used if the active drug is relatively unstable
114
What is sublingual drug administration?
drug absorbed through the anal cavity, dissolved under the tongue, used for rapid response and when drug is unstable in the stomach/when metabolised
115
What is oral drug administration?
absorption occurs largely in the intestine, drugs with low bioavailability need to be administered in higher doses or via an alternative route
116
What is bioavailability?
Indicates the proportion of drug that passes into the systemic circulation following oral administration
117
What could reduce bioavailability?
If the drug is incompletely absorbed from the gut or inactivated in the liver (first-pass effect - after metabolism)
118
Why is rectal drug administration used?
to produce a local effect
119
Why is cutaneous drug administration?
used for a localised effect on skin
120
What is required for skin absorption of drugs?
the drug requires high lipid solubility to get through the skin barrier
121
Why is inhalation used for drug administration?
for volatile gases, can be inhaled as an aerosol e.g. salbutamol
122
How is salbutamol administered?
inhalation as aerosol
123
When is injection used for drug administration?
to avoid first pass metabolism, iv = fastest and most certain route of drug administration, subcutaneous = faster route than oral but can be slowed by speed of diffusion through muscle/skin and local blood flow
124
What is a parenteral route of administration?
those avoiding the gi tract, e.g. injections, dermal delivery systems and inhalors
125
What is an enteral route of administration?
drugs absorbed through the alimentary canal, e.g. oral
126
What are factors to consider when designing a drug?
extent of transport across membranes (absorption), transit to the site of action (distribution), the nature of compliance, age and physical ability of the patient (e.g. a child can't swallow a tablet)
127
Why does the dose administered have to be higher than that which would be needed?
because for most absorbed drugs a percentage of compound is excreted and/or metabolised or bound to non-pharmacologically active target sites
128
Why are dosing regimens designed?
to maintain a concentration of drug within the therapeutic window at the site of action for the period of time that is required for a therapeutic effect
129
What is the therapeutic window?
the concentration where there's a pharmacological effect with no toxicity
130
What can improve therapeutic failure?
increasing the dose/doses per day, altering the site of administration, altering the formulation
131
What is the pharmacokinetic phase of drug action?
absorption, distribution, metabolism, and elimination of a drug
132
What is absorption?
the passage of the drug through biological membranes from its site of administration into the plasma after administration
133
What is distribution?
the transport of the drug from its point of administration (or absorption) to its site of action
134
Why is distribution a very fast process?
bc it mainly occurs through blood
135
How does albumin affect drugs?
drugs bound to protein have no pharmacological effect, albumin binds to many acidic drugs
136
What proteins bind to basic drugs?
beta globulin, acid glycoprotein
137
What does the amount of bound drug depend on?
the free drug conc, its affinity for binding sites, the protein conc
138
What influences GI absorption?
solubility, formulation and particle size, gut pH, motility, perfusion, stomach emptying time
139
What is required for good drug absorption?
water solubility for in solution, and lipid solubility for fat
140
Why is absorption good in the small intestine?
increased surface area bc of microvilli and high blood flow
141
What is volume of distribution? What affects it being high/low?
the volume of plasma that would contain the total body content of a drug equal to that found in plasma, e.g. if highly protein bound Vd will be low, if lipid soluble so readily crosses plasma membranes Vd will be higher
142
What are the two methods of drug translocation?
bulk flow transfer (in blood), and diffusional transfer
143
How does bulk flow transfer work?
v rapid, independent of chemical characteristics, the cardiovascular system supports continuous movement
144
How does diffusional transfer work?
across non-aqueous (lipid) cell membranes, dependent on chemical characteristics, because most drugs have a similar MW variations in aqueous diffusion only have a small effect on overall diffusion rate
145
What chemical characteristics affect diffusion?
non-polar molecules diffuse easily, charged drugs remain in body compartments, so the degree of ionisation at physiological pH affects the degree of drug transfer through a cell membrane
146
What are the 4 ways drugs pass across cell membranes?
diffusion through lipid, diffusion across aqueous pores that traverse the lipid, combination with a carrier molecule, by pinocytosis (through vesicles)
147
How does fat affect drug delivery?
thiopentone is not sufficiently polar so absorbs into the large, non-polar compartment, creating a reservoir of drug that has no pharmacological action, fat also has low blood supply so drug delivery is slower
148
Outline the two phases of reactions in metabolism
phase 1: oxidation, reduction, elimination
| phase 2: conjugation
149
What do phase 1 reactions of metabolism involve?
products that are more reactive (or toxic) - often a functional group (hydroxyl, thiol, amino) is introduced as a point of attack for the conjugation of a larger substituent (in phase 2)
150
What do phase 2 reactions of metabolism involve?
conjugation of a large polar, water-soluble substituent (glucuronyl, sulphate, acetyl) following phase 1
151
How do phase 1 and phase 2 lead to metabolism?
decreased pharmacological activity, decreased lipid solubility (more water-soluble), increased MW. all give an increased rate of excretion
152
What type of drug is imipramine?
pro-drug
153
What is the key enzyme for catalysing phase 1 metabolism reactions? Where is it found?
cytochrome P450 found in the endoplasmic reticulum
154
What is the effect of cytochrome P450 for metabolism/
binds oxygen to the drug and forms an electron transfer chain, all P450 reactions start with a hydroxylation step catalysed by the P450 system
155
What is the enzyme for phase 1 metabolism of noradrenaline?
monoamine oxidase
156
What enzymes catalyse phase 2 metabolism reactions?
transferase enzymes
157
What is the first pass effect?
when there is extensive metabolism in the liver before reaching the systemic circulation, so the dose absorbed is less (important for aspirin and salbutamol)
158
What happens if phase 1 metabolism yields a highly reactive chemical?
it avoids phase 2 and covalently binds to cellular macromolecules, which if essential for function, can cause toxicity e.g. paracetamol hepatotoxicity
159
What is the pharmacodynamic phase?
the efficacy at the drug target
160
What are the main routes of excretion?
the kidneys, the hepato-biliary system (liver)
161
How do the kidneys undertake excretion?
effectively reabsorb lipophilic drugs, by glomerular filtration and active tubular secretion
162
How does glomerular filtration work?
allows all drug molecules with a MW below 20,000 to pass into the glomerular filtrate, while plasma and protein-bound drugs are held back
163
How does active tubular secretion work?
any remaining drug from glomerular filtration passes to the capillaries of the proximal tubule where carrier systems work against the electrochemical gradient to reduce plasma drug conc to virtually zero
164
How does the liver undertake excretion?
transfer substances from plasma to bile using transport systems, non-polar metabolites than concentrate in bile are hydrolysed in the intestine, giving free drug that can be reabsorbed (enterohepatic circulation) to prolong the action of the drug, drug eliminated in faeces
165
What is an agonist?
a drug that binds to a receptor and causes a pharmacological response
166
What is an antagonist?
a drug that binds to a receptor without causing a pharmacological response (can also inhibit the response of an agonist)
167
How is the ability of an agonist to stimulate a pharmacological response in a tissue measured?
with a dose-response curve
168
What is affinity?
the ability of a drug to bind to a receptor
169
What is efficacy?
the strength of a response stimulated by a drug-receptor interaction
170
How does the agonist dose-response curve change in the presence of a reversible competitive antagonist?
shifted to the right without a change in the maximum response (the slope doesn't change)
171
How does the agonist dose-response curve change in the presence of an irreversible competitive antagonist?
the strength of the maximum pharmacological response is reduced (curve = more shallow)
