medchem Flashcards
1
Q
how is polarisation of a neuron created and how does depolarisation occur
A
the natural eqm flow of potassium ions out of the cell creates a electric potential across the neuron cell membrane = polarisation.
depolarisation occurs when certain neurotransmitters bind to post-synaptic cell and open the sodium ion channels.
sodium ions flow -> cell and the internal electric potential becomes less negative and results in stimulation of the neuron.
2
Q
why is ACE2 an important enzyme in humans
A
ACE-2 is membrane-bound enzyme with a homeostatic role. Present in most organs; including airway epithelial cells and vascular endothelial cells.
3
Q
describe the actions of voltage-gated sodium and potassium ion channels in the axon leading to an action potential
A
they are controlled by the electric potential of the cell membrane.
when the post-synaptic cell body is depolarised, the sodium ion channels of the axon open also.
the stronger the cell body depolarisation, the more sodium channels open and the likelihood of reaching the threshold to generate a signal increases.
4
Q
infection phase of HIV life cycle
A
HIV surface proteins gp120 and gp41 and T-cell surface proteins CD4, CCR5 and CXCR4 are crucial
5
Q
integration phase of HIV life cycle
A
Integration into T-cell DNA: HIV capsid disintegrates (viral protease); viral RNA converted into proviral DNA by viral reverse transcriptase; proviral DNA incorporated into host DNA by viral integrase
6
Q
release phase of HIV life cycle
A
New gp120 and gp 41 incorporate into cell membrane, other viral proteins and RNA congregate on inner membrane surface; budding occurs to release new viruses
7
Q
processing phase of HIV life cycle
A
Transcription produces more viral RNA, some is incorporated into new virions, some translated into viral proteins
8
Q
ADME
A
Absorption, Distribution Metabolism, Excretion
9
Q
what happens if an oral drug is too lipophilic
A
instead of crossing through membranes drug will become stuck in fat tissue
10
Q
what happens if an oral drug is too polar
A
it will be excreted by kidneys
11
Q
what happens if an oral drug is too anionic or cationic
A
too cationic = bind nucleic acids
too anionic = bind plasma proteins
12
Q
requirements for orally administrated drug
A
- water soluble to dissolve in GI tract & blood
- lipophilic to cross membranes/BBB
- resistant to stomach acid & enzymes
13
Q
3 measurable parameters of pharmacokinetics
A
plasma concentration
half-life
rate constants
14
Q
generally, how are drugs metabolized?
A
via various routes, enzyme controlled reactions that convert drugs into biologically inert metabolites that are easily excreted
Phase I and Phase 2
15
Q
Phase 1 metabolism
A
-introduce or reveal functional groups important in Phase 2
-products are more water soluble than original drug
16
Q
what makes a drug more excretable?
A
increased water solubility
i.e. polarity
17
Q
Phase 2 metabolism
A
-products much more water soluble than original drug
-products form bulk of inactive excreted metabolites
18
Q
methods to study drug metabolism
A
-radiolabeling the drug
-chromatography (mainly HPLC)
-NMR and/or MS
19
Q
biological factors affecting drug metabolism
A
-dose level
-route of administration
-species differences
-sex
-age
-disease
-other drugs
-genetics
20
Q
why does dose level matter when designing drugs?
A
dose level increasing will saturate enzyme capability leading to alternative pathways & toxic products
21
Q
why does route of administration matter when designing drugs?
A
the concentration of effective drug decreases if drug is metabolized in organs before reaching circulation
22
Q
why does variation between species matter when designing drugs?
A
rates, dose etc may be different between model organism & human
sex of rats is important for drug efficacy but not in humans unless pregnant
23
Q
why does age matter when designing drugs?
A
liver function decreases with age
24
Q
why does disease matter when designing drugs?
A
liver disease has greatest effect
25
why do other drugs matter when designing drugs?
one drug can affect the metabolism of another
26
why does genetics matter when designing drugs?
isoenzymes
27
drug metabolism occurs in...
all tissues and most biological fluids
most reactions occur in liver
selective reactions can occur in kidneys, lungs, brain, placenta
28
6 main types of Phase 1 metabolic reactions
Oxidation
Reduction
Hydrolyses
Hyrdations
Deacteylations
Isomerisations
29
3 main types Phase 2 metabolic reactions
acylation
sulfate formation
conjugation
--mainly occur in liver and gut wall
30
How to increase resistance to metabolism?
steric shields
bioisosteres (electronic effect)
combination of the 2 ^
metabolic blockers
31
how & why may it be necessary to decrease lifetime of a drug?
too resistant to metabolism
add metabolically labile groups
32
what is a prodrug
biologically inert but converted to an active drug in the body after metabolism
33
what group could be added to reduce lifetime of drug?
CH3 is readily oxidized > CH2OH and CO2H
34
6 benefits of prodrugs
improve membrane permeability
prolong drug activity
mask toxicity
increase/decrease water solubility
target tumors (selectivity by only being activated inside tumor)
35
how can you sterically shield a drug? example
e.g. tertiary butyl group
a big bulky group that prevents enzyme from accessing it's target functional group
36
how can you metabolically block metabolism of a drug to increase lifetime?
adding a functional group to block site of metabolism on the drug
37
what functional groups can you add to improve membrane permeability?
hydrophobic groups
e.g. (long) alkyl chains
38
what functional groups can you add to prolong drug activity?
fatty side chain will embed prodrug in membrane and guarantees slow release into blood where it is rapidly hydrolyzed
39
how can you mask drug toxicity?
add functional group that slows release of high dose
prevents saturation of metabolic enzymes which decreases side effects caused by alternate metabolism
40
what kind of functional group should be added to increase water solubility of a drug?
polar/hydrophilic
e.g. lysine ester
41
what is the most common functional group added to decrease water solubility?
most examples involve esters
42
what is ADEPT
antibody directed enzyme prodrug therapy
43
antitumor mechanism of prodrug indolequinone
metabolized inside cancer cell to active form by NQO1
becomes a powerful electrophile which intercalates and irreversibly alkylates DNA
44
what is GDEPT
gene directed enzyme prodrug therapy
45
functional groups most prone to Phase 1 oxidation
N-methyl
aromatic rings
terminus of alkyl chains
least hinderered position of acicyclic ring
46
functional groups most prone to Phase 1 reduction by reductases
nitro
azo
carbonyl
47
functional group most prone to Phase 1 hydrolysis by esterases
ester
48
functional group most prone to Phase 1 hydrolysis by peptidases
amides
49
how do cytochrome P450 enzymes oxidise a drug
split molecular oxygen
add O to C, N, P or S atom
H2O and NADP+ produced
requires NADPH and H+
50
the most likely C atoms to be oxidised in Phase 1 are
exposed or activated
51
oxidation of saturated carbon centres produce what functional groups
epoxide
52
lipinski's rule of 5
-HBD < 5
-HBA < 10
-MW < 500 d
-(Clog P) < 5
53
pharmacophore
the important functional groups required for activity and their relative positions in space.
54
hydrophobicity
hydrophobic character important for crossing biological membranes & receptor interactions
55
eqn partition coefficient
[drug] in octanol/ [drug] in aqueous solution
56
hydrophobic compounds have _ partition coefficient
high
57
polar surface area
surface associated with heteroatoms & polar H atoms
58
exceptions to Lipinksi's rule
Antibiotics, antifungals, vitamins and cardiac glycosides
act as substrates for naturally occurring transporters
natural products
drugs not required to be orally bioavailable
59
types of minor changes to determine SAR
the size and shape of the carbon skeleton
the nature and degree of substitution
the stereochemistry of the lead
60
SAR approach
attempts to remove element of luck from drug design by establishing mathematical relationship:
eqn between biological activity and measurable physicochemical parameters.
61
IC50
concentration required to achieve 50% inhibition
62
ED50
mean effective dose required to produce a therapeutic effect in 50% of test sample
63
main properties of a drug that influence its activity are
Lipophilicity
Electronic effects within the molecule
Size and shape of the molecule
64
eqn: relationship between P and drug activity
log(1/C) = K1log(P) + K2
y= mx + c
65
increasing hydrophobicity does not directly translate to biological activity because...
a drug can be so hydrophobic it gets trapped in fat deposits
66
eqn: Hansch parabolic relationship
log(1/C) = K1log(P)^2 + K2logP + K3
67
optimisation of lead compound: vary accessible substituents
variation of alkyl group i.e. length
68
optimisation of lead compound: increase activity and/or decrease side effects
increase rigidity i.e. add ring or double/triple bond to inhibit bond rotation
69
optimisation of lead compound: simplify synthesis
strip away non-essential regions after SAR
70
optimisation of lead compound: increase selectivity and/or activity
ring fusion
71
optimisation of lead compound: add additional binding group
aromatic ring > heteroaromatic ring
heteroaromatic ring > different heteroaromatic ring (different ring size or position of heteroatom)
72
optimisation of lead compound: make use of unused (extra) binding pocket
extending the structure
73
optimisation of lead compound: confer selectivity
varying bulk (increasing may confer specificity to a receptor with larger binding pocket, and rule out binding to a receptor with a smaller binding pocket)
74
bioisosteres for -OH
-NHCOR
-NHSO(2)R
-CH(2)OH
-NHCONH(2)
-NHCN
-CHCN(2)
75
bioisosteres for halogens
-CF(3)
-CN
-NCN(2)
-CCN(3)
76
bioisosteres of thiourea
-NHC(=S)-NH(2)
-NHC(=NCN)NH(2)
-NHC(=CHNO(2))NH(2)
77
11 optimisations of a lead compound
- vary alkyl substituents
- increase bulk/chain length (hydrophobic interactions)
- vary bulk (selectivity)
- extension of structure (unused binding region)
- ring expansion/contraction
- ring variation (heteroatoms etc.)
- ring fusions
- simplification
- rigidification
- conformation blockers
- isosteres
78
optimisation of lead compound: improve binding in a hydrophobic pocket
increasing bulk or chain length
varying ring structure/ring positions
79
how does an ester eliminate any H bonding (SAR hydroxyl)
electronically: lone pair delocalised (weaker acceptor)
sterically: ester group is bulky and block H bond formation
80
SAR test of hydroxyl groups (hydrogen bonding)
test if H bonding is important to activity
R-OH > R-O-Me removes HBD activity
however, still has HBA activity, so form ester
R-OH > R-O-C(=O)-Me which has neither activity
81
SAR test of amino groups (hydrogen or ionic bonding)
test if H bonding and ionic bonding is important to activity
amine > amide stops lone pair taking part in H bonding
also inhibits protonation because lp delocalises > carbonyl, so no ionic bonds
82
SAR test of aromatic rings (v.d.w)
flat ring has v. strong v.d.w because of proximity to binding site
hydrogenate to remove flatness, weakens/decreases v.d.w because the ring cannot bind as close
83
SAR test of alkenes (v.d.w & hydrophobic interactions)
hydrogenate to alkene, cannot bind as close, decreasing number of interactions
84
SAR test of ketones (H-bonding & dipole-dipole)
reduce > alcohol
carbonyl group is flat, proximity increases interactions
alcohol is tetrahedral, distance decreases interactions
also flips dipole
85
SAR test of amides (H bonding)
hydrolysis > split into amine + carb acid (see if amide is necessary for carbonyl activity)
reduction to amine > disrupts H bonding of carbonyl (removed)
(see if carbonyl is necessary)
86
SAR test: bioisosteres
replace with atom of same valency
87
what does C mean w.r.t QSAR
concentration of drug required to reach defined level of biological activity
88
IC50
concentration required to achieve 50% inhibition
89
ED50
mean effective dose required to produce a therapeutic effect in 50% of test sample
90
main properties of a drug that appear to influence its activity are
lipophilicity
electronic effects
size & shape
91
hydrophobicity
how easily a drug can cross the cell membrane
92
partition coefficient eqn
P = [drug] in octanol / [drug] in water
93
hydrophobic compounds have _ P values
high
94
straight line eqn for relationship between P value and drug activity
log (1/C) = k1logP + k2
95
relationship between log(1/C) and logP is
parabolic
96
typical logP value for CNS active drug
1.75
97
LogD
logP at a particular pH
98
why is logP pH independant?
only takes into account [neutral species]
99
hydrophobic parameter
pi symbol
contribution of each substituent to hydrophobicity
100
P(H) =
partition coefficient for the standard compound
101
P(X) =
partition coefficient for the standard compound with the substituent.
102
solid phase synthesis requires
-inert cross-linked insoluble polymeric support
-anchor or linker
-stable bond between linker & substrate
-simple efficient removal of product from linker
-protecting groups
103
in 2012, how many drugs violated at least 1 of lipinski's rules and how many failed more than 2?
1 = 16%
>2 = 6%
104
wang resin used for
peptide synthesis
105
mix and split process
1. pool of beads > separate reaction vessels, each for a different reactant attached
2. each vessel sees a different 2' reactant
3. filter/wash beads and mix all into one
4. split into 3 equal portions, treat each with a different 3' reactant
5. this gives 9 products. repeat.
106
deconvolution
isolating and identifying the most active component in a mixture
107
micromanipulation
each bead in a mixture only contains one structural product; separate beads individually and test
108
recursive deconvolution
test compounds at each stage of synthesis.
test if activity is only detected after addition of a specific component
109
sequential release
split a library by bead into smaller components
110
tagging (QSAR)
2 molecules of interest are built up on the same bead
1 is compound of interest, the other acts as a code for each step.
111
parallel synthesis
reaction carried out in a series of wells to generate a library of chemicals
112
what is parallel synthesis used for?
focused lead optimisation studies
113
dynamic combinatorial synthesis
alternative to split and mix
synthesis and screen in vivo
identify active compounds by stopping reaction
convert eqm products into stable compounds that cannot > starting materials
114
spider approach (QSAR)
attach multiple different functional groups to a central scaffold
increases of finding a compound that will interact with target binding site
115
fragment based lead discovery
design lead compound by identifying epitopes (SM) that bind to regions of active site
link several epitopes together to give lead compound
116
vebers rules (additional to RO5)
rotatable bonds < 10
polar surface area < 140 Å
HBA + HBD <12
117
what kinds of drugs are exceptions to RO5?
antifungals, antibiotics, vitamins and cardiac glycosides
118
parabolic (Hansch) eqn for log (1/C) and P
log (1/C) = -k1(logP)^2 + k2logP + k3
119
hydrophobic paramater eqn
pi,x = logPx - logPh
120
if the hydrophobic parameter is a +ve value
the substituent is hydrophobic
121
if the hydrophobic parameter is a -ve value
the substituent is hydrophilic
122
the electronic effect
if X is EWD it will stabilise the anion, eqm > RHS and K increases
123
advantages of QSAR
Quantifying the relationship between structure and activity provides an under-standing of these effects, not available from raw data
Allows informed predictions, particularly regarding the synthesis of new compounds. Can easily interpolate the data, but must not extrapolate beyond the range of the data set.
124
disadvantages of QSAR
-False correlations may arise through too heavy reliance on biological data (error prone)
-Data set may be incomplete
-Some physicochemical properties are already cross-correlated. Should ideally use independent variables in QSAR.
125
drug repurposing
Use old drugs for a new purpose (indication)
Recycle a molecule, a pathway, a biological activity
126
advantages of drug repurposing
-low cost
-skip animal studies > phase 2
-compounds already availible
-large scale production already established
127
disadvantages of drug repurposing
-intellectual property
-target identification can be more challenging
-SAR to improve potency loses repurposing potential
-effective [drug] often higher in vitro than is viable in vivo
128
non-specific drug
no defined molecular target
act outside of cells
different structures can illicit a similiar pharmacological response
129
examples of non-specific drugs
general anaesthetics
130
specific drugs
direct interaction between drug and target
minor changes in structure can have major effects on activity
131
describe the structure of Valinomycin
circular (micelle like)
hydrophobic on outside, isopropyl groups
hydrophilic on inside, carbonyl groups
132
give the mechanism of valinomycin
slots into membrane and creates a pore for leakage
leaky cell = dead cell
133
describe the structure and give the mechanism of Gramicidin A
15 aa peptide
helical, forms dimers long enough to span the membrane to make a pore
pore for K+
leaky cell = dead cell
134
is the sugar phosphate backbone of DNA +ve or -vely charged?
-ve
135
width of minor DNA groove (Cai 2009)
10 A (Cai 2009)
136
width of major DNA groove (Cai 2009)
24 A (Cai 2009)
137
how do proteins bind the grooves of DNA
through H bonds and non-specific binding interactions. (Cai 2009)
138
2 modes of action of groove binders
reversible inhibition of DNA function &
induce permanent DNA damage
139
how does the curved structure of Distamycin and Netropsin help their function.
why are they curved?
they mimic the curve of DNA and so bonding interactions are consistently strong through the complex with DNA
curved bc of the ox. state of the atoms
140
how are minor groove binders that bind AT sequences advantageous
have antimicrobial and antitumor activity
specific to AT islands, so do not cause damage to healthy cells
141
C =
minimum concentration required to cause a specific biological response
142
bioactivity =
A (hydrophobic parameter) + B (electronic parameter) + C (steric parameter) + D (other parameters) + E
143
Hansch proposed that drug action can be split into 2 stages
1. transport of drug to the site of action
2. binding of the drug to the target site
144
P =
partition coefficient
145
D =
distribution coeffient
146
pi =
hydrophobic parameter
147
sigma =
electronic parameter (Hansch substitution constant)
148
why are steric effects important in drug design?
drugs that bind to receptors or enzymes must have a specific size or shape
149
Es =
taft steric parameter
150
MR =
(steric) molar refractivity
151
if logP values are limited to a short range the eqn will be
linear
152
if logP values are across a greater range the eqn will be
parabolic
153
accuracy of Hansch eqn will depend on 3 things:
1. number of analogues used (greater the no., higher probability of obtaining an accurate eqn)
2. accuracy of biological data used in derivation of eqn
3. choice of parameter
154
n =
no. of analogues
155
craig plot shows that there is no relationship between
pi and sigma
156
6 steps to planning a QSAR study
1. decide physiochemical properties to investigate, using data tables and Craig plot
2. synthesise enough compounds to make results statistically significant
3. best to avoid ionisable substituents which many confuse results
4. best avoid groups that are easily metabolised
5. dont vary too much at once, consider nature and position
6. apply state-of-art 3D-QSAR and other computer methods
157
positive sigma =
EWD
158
negative sigma =
ED
159
positive pi =
hydrophobic
160
negative pi =
electron donor
161
what are common structural and chemical features of intercalators that relate to their function/mode of action?
all have a flat (usually aromatic) region that slides between base pairs and a charged group
162
4 main classes of DNA alkylating agents
nitrogen mustards
ethyleneimines (arizidines)
methanosulfonates
platinum complexes
163
how to improve selectivity of alkylating agents
lowering reactivity by substitution of groups that contribute to electrophile character
i.e. replace N-methyl with N-aryl : lone pair of N interacts with ring pi system so is less able to displace Cl- (nitrogen mustards).
only strong nucleophiles like guanine can then react with the drug
164
what % of drugs act on receptors?
40%
165
agonist
drug which binds to receptor and stimulates same response as endogenous chemical messenger
166
antagonist
drug which binds to receptor and stimulates no response, but blocks agonist
167
inverse agonist
drug which acts as antagonist, but also eliminates any resting activity associated with a receptor
168
function of receptors
-mediate an intracellular response to an extracellular messenger
-the ligand does not react with the receptor
169
where are receptors usually located in a cell?
plasma membrane/exterior cell surface
170
specificity of receptor and consequences for drug design
very specific proteins wrt shape and binding sites so there must be specific complementary interactions between receptor and drug for activity
171
4 main types of receptor targeted by drugs
1) Ion channels
2) G-protein coupled receptors (GPCRs)
3) Kinase-linked receptors
4) Intracellular receptors (aka nuclear hormone receptors)
172
2 main types of ion channels
voltage-gated ion channel
ligand-gated ion channel
173
function of ion channels
membranes are hydrophobic so ions cannot freely diffuse through membranes
ion channels allow rapid movement of ions across membranes after binding to external stimulus
transmembrane proteins
174
voltage-gated ion channels respond to and are found in what kind of cells
electrical stimulus
excitable cells e.g. nerves
175
ligand-gated ion channels respond to and are found in what kind of cells
chemical stimulus
ubiquitous, therefore common drug targets
176
what happens when a ligand binds to a ligand-gated ion channel?
- Specific chemical messenger binds to ion channel protein
- Induced fit of binding causes shape change in protein
- Ion channel opens, specific ions pass through
- occurs in milliseconds because the direct consequence of ligand binding is the channel opening
177
synapse
point of communication between a neuron and another cell
178
acetylcholine interactions with neurons and ligand-gated ion channels
- common synaptic neurotransmitter
- Released into synapse from presynaptic neuron
- Binds to nicotinic acetylcholine receptors (nAChR) on post-synaptic cell membrane
- Post-synaptic cell becomes permeable to cations, movement of Na+, K+ and Ca2+ which causes post-synaptic response
- Acetylcholine broken down rapidly; nerves staying “on” is a bad thing
179
how do neuromuscular blockers interact with nicotinic acetylcholine receptors and what is the outcome?
- Nicotinic acetylcholine receptor antagonists are able to compete with ACh for nAChR binding sites
- Prevent neuromuscular function - i.e. cause muscle paralysis
180
origin and clinical use of nicotinic acetylcholine receptor antagonists.
- Widely used with general anaesthesia in surgery
- Original source compound: tubocurarine - S. American arrow-tip poison
- Modern analogues: pancuronium amongst others
181
receptor activated by adrenaline and noradrenaline
adrenergic receptor/adrenoreceptor
182
are all receptors that are activated by the same neurotransmitter the same across the body? what does this mean for drug design
no, they may have distinct amino acid sequences etc.
can target drug to a specific sub-type of receptor based on its features that are distinct from the other sub-types
i.e. nicotinic vs muscarinic cholinergic receptor
183
describe the induced fit model of receptor-signal binding
- binding site contains specific binding regions which contain functional groups that are complimentary to the binding groups of the messenger
- messenger fits into the binding site and binding groups interact weakly but fit is not perfect.
- receptor protein alters shape to bring the groups to obtain a stronger interaction.
- bonding forces must be large enough to change the shape of the binding site, but not so strong that the messenger is unable to leave.
184
the ion selectivity of an ion channel is based on
the amino acids lining the ion channel
185
structure of an ion channel lock-gate and how it is opened?
- lock gate is made up of five kinked α-helices (the 2-TM region) where one helix is contributed by each of the five protein subunits
- closed state = kinks are pointing towards each other
- conformational change induced by ligand binding causes each of these helices to rotate such that the kink points the other way, opening up the pore
186
where are voltage-gated ion channels located in the neuron?
axon
(Intro to Med Chem P. L. Graham, 2017)
187
function of voltage-gated ion channels
react to neuron depolarization/polarisation to produce an action potential -> signal
(Intro to Med Chem P. L. Graham, 2017)
188
describe the mechanism of ligand-gated sodium channels and potassium channels in neurons
resting state :
potassium ion channels = open
sodium ion channels = closed
excited :
sodium ion channels = open
189
what are g-protein coupled receptors?
are transmembrane proteins which activate an intracellular signalling protein called a G-protein (in response to stimulus).
slower than ion channels, mechanism takes minutes.
190
function of GPCRs
- Mediate response to hormones and slow-acting neurotransmitters
- e.g. glutamic acid, GABA, noradrenaline, dopamine, acetylcholine, serotonin, prostaglandins, adenosine, opiates, angiotensin, bradykinin, thrombin
191
function of a G-protein
initiate a signalling cascade, involving a variety of enzymes
192
structure of GPCRs
- protein chain winds 7 seven through membrane
- each transmembrane section is helical and hydrophobic
- G protein binding site on intracellular side receptor, signal binding site on extracellular side
- each signal molecule has a distinct binding site location
193
GCPR mechanism
1. Specific chemical messenger binds to GPCR, induces conformational change
2. Conformational change of intramembrane domain affects gamma subunit, results in a swap of GDP for GTP
3. Dissociation of subunits Ga // BY to distinct membrane-embedded effector molecules elicits signaling cascades
4. Signaling cascade activates a 2nd messenger molecule which activates a target enzyme.
194
2 main signal transduction pathways regulated by GPCRs
cyclic adenosine monophosphate (cAMP) signal pathway
phosphatidylinositol signal pathway
195
how does GPCR initiate cAMP pathway
- Gα-subunit binds to membrane-bound enzyme adenylate cyclase
- AMP becomes activated, and catalyses synthesis of cAMP from ATP. Chops off 2 phosphate groups and forms ring between O and OH
- cAMP moves into cytoplasm and activates protein kinase A (PKA)
- PKA phosphorylates and activates further enzymes with specific function for the cell in question
196
2 main groups of GPCR adrenergic receptors
- α (smooth muscle contraction, inhibition of insulin release)
- β (increase cardiac output, act via cAMP)
197
example of β-blocker and clinical uses
propranolol
- antagonist
- Opposes the action of adrenaline on β-receptors, so used to treat high blood pressure, angina and various dysrhythmias, and used in recovery post- myocardinal infarction
198
3 types of GPCRs
adrenergic
muscarinic
opioid
199
3 sub-families of GPCRs
- class A rhodopsin-like receptors
- class B secretin-like receptors
- class C metabotropic glutamate-like and pheromone receptors
(Intro to Med Chem P. L. Graham, 2017)
200
why is the existence of receptor subtypes important?
This is important, because one receptor subtype may be prevalent in one part of the body (e.g. the gut) while a different receptor subtype is prevalent in another part (e.g. the heart)
(Intro to Med Chem P. L. Graham, 2017)
201
why is the convergent evolution of GPCRs important? including example
greater similarities between receptors which bind different ligands and have evolved from the same branch of the tree
>>
similarities between subtypes of receptors which bind the same ligand.
e.g histamine H1 receptor resembles muscarinic receptor > histamine H2 receptor.
- important consequences in drug design : increased possibility that a drug aimed at a muscarinic receptor may also interact with a histamine H1 receptor and lead to unwanted side effects.
(Intro to Med Chem P. L. Graham, 2017)
202
how is the active conformation of kinase receptor different from inactive conformation?
active = intracellular domain is phosphorylated
203
what is the effect of kinase receptor dimerisation and why is this important?
Dimerisation means primary messenger (signal) must be dimerized too.
Dimerization is important as the enzyme on one side of the dimer phosphorylates the tyrosine of the other enzyme (and vice-versa).
(Intro to Med Chem P. L. Graham, 2017)
204
mechanism of kinase-linked receptors
- minutes to respond
- Activated by polypeptide hormones, growth factors and cytokines i.e. important in endocrine regulation
- messenger binds causing shape in protein conformation
- dimerisation allows each active protein to phosphorylate the other
- intracellular proteins can now bind to phosphorylated kinase domain
205
kinase-linked receptor structure
- receptor N terminus (transmembrane) and enzyme C terminus (intracellular)
- Active conformation functions as intracellular kinase enzyme (i.e. signal transduction by phosphorylation)
- ATP cofactor
- often tyrosine-linked
206
example of a kinase-linked receptor that is an important anti-cancer drug target
Epidermal growth factor receptors (EGFRs)
Inhibition of kinases is possible by blocking the ATP binding site (as kinases use ATP for phosphorylation)
207
how do EGFRs work in cancer?
- EGFRs are a type of tyrosine kinase-linked receptor that are over-expressed and/or abnormal in many cancer types
- Consist of extracellular EGF binding site and intracellular tyrosine kinase
- Over-expression of EGFR leads to uncontrolled proliferation of cells
208
how would you target EGFRs with anti-cancer drugs
- Inhibition of kinases is possible by blocking the ATP binding site (as kinases use ATP for phosphorylation)
- However, targeting an ATP binding site, ubiquitous across all cells, you create many side effects.
209
example of anti-cancer drug targeting EGFRs
gefitinib is a competitive inhibitor of ATP binding site
antagonist- lack of phosphate groups
210
difference between type 1 and type 2 inhibitors of kinase-linked receptors
- Type I inhibitors bind to the active conformation of the enzyme, whereas type II inhibitors bind to the inactive conformation.
- exceptions: Sunitinib and dasatinib are able to bind to both active and inactive forms of the same kinase enzyme and could be defined as type I or type II.
(Intro to Med Chem P. L. Graham, 2017)
211
SAR of lapatinib type 2 inhibitor of EGFRs
- binds to inactive conformation, allows fluorobenzyloxy substituent to interact with hydrophobic pocket that isn't exposed in active form
- chain containing the amine and the sulphonyl group increases aqueous solubility, located in region of active site that's exposed to solvent
- potent activity for another receptor tyrosine kinase ErbB-2 (HER2), structurally related to EGFR. ergo, dual-action inhibitor used for cancers that overexpress both EGFR and ErbB-2.
(Intro to Med Chem P. L. Graham, 2017)
212
mechanism of kinase-linked receptor
- active site closed in resting conformation
- binding of signal to extracellular region cause conformational change and active site opens
- tyrosine residues on protein substrate are phosphorylated OR enzyme dimerizes and each monomer phosphorylates the other; and protein substrates dock on phospho-tyrosine
- ATP cofactor
- several phosphorylation's occur so signal is amplified
(Intro to Med Chem P. L. Graham, 2017)
213
mechanism of intracellular receptors
- Binding of chemical messenger causes shape change
- active conformation binds DNA and modulates transcription for particular protein
= transcription factor
- Slower response than other receptors as rely upon transcription (hours-days)
- responds to steroid hormones, thyroid hormones and retinoids
214
mechanism of intracellular estrogen receptor
- binds estrogen
- conformational change opens up AF-2 region and dimerization occurs to allow coactivator to bind, allowing DNA complex to form and (increased) transcription.
commonly overexpressed in breast cancer
- DNA binding region: zinc finger domain recognizes particular DNA sequence
215
tamifoxen as anti-cancer drug targeting estrogen receptors
Tamoxifen has similar hydrophobicity and size to estradiol but has a protonated (at physiological pH) N so forms an H bond with the receptor.
Prevents transcription from estradiol-responsive genes.
takes 5-10 years to eradicate cancer.
216
raloxifene important functional groups
- synthetic agent binds to the binding site without activating the receptor, prevents estradiol from binding
- 2 phenol groups which mimic the phenol and alcohol group of estrogen
- skeleton also hydrophobic and matches hydrophobic character of estrogens tetracyclic skeleton
- side chain = antagonism; contains an amino group which is protonated and forms a hydrogen bond to Asp-351—an interaction that does not take place with estrogen
- side chain protrudes from the binding pocket and prevents the receptor helix H12 folding over as a lid so AF-2 binding region is not exposed, coactivator can't bind and transcription factor can't form
- side chain must contain amine group of the correct basicity such that it ionizes and forms the interaction with Asp-351
(Intro to Med Chem P. L. Graham, 2017)
217
raloxifene as anti-cancer drug targeting estrogen receptors
antagonist of the estrogen receptor and is used for the treatment of hormone-dependent breast cancer
- side chain = antagonism; contains an amino group which is protonated and forms a hydrogen bond to Asp-351—an interaction that does not take place with estrogen
(Intro to Med Chem P. L. Graham, 2017
218
HIV structure
- single strand RNA
- two copies of genome
- enzymes: proteases, reverse transcriptase, integrase
- cell membrane and protein capsid
219
HIV life cycle
- infects T cells
1. Infection: HIV surface proteins gp120 and gp41 and T-cell surface proteins CD4, CCR5 and CXCR4 are crucial
2. Integration into T-cell DNA: HIV capsid disintegrates (viral protease); viral RNA converted into proviral DNA by viral reverse transcriptase; proviral DNA incorporated into host DNA by viral integrase
3. Processing of viral DNA: Transcription produces more viral RNA, some is incorporated into new virions, some translated into viral proteins
4. Release of new viruses: New gp120 and gp 41 incorporate into cell membrane, other viral proteins and RNA congregate on inner membrane surface; budding occurs to release new viruses
220
obvious drug targets for anti-HIV drugs and why
viral proteins/enzymes because they are completely distinct from eukaryotic proteins/enzymes.
221
HAART
highly active antiretroviral therapy uses combinations of Nucleoside reverse transcriptase inhibitors (NRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Protease inhibitors (PIs), Integrase inhibitors
222
structure and function of NRTIs
nucleoside pro-drugs
compete with cellular nucleotides for reverse transcriptase
generally effective but relatively toxic
223
mechanism of NRTIs
- competitive inhibition of reverse transcriptase
- halt DNA synthesis when incorporated into growing DNA chain as they are missing 3'-OH
- ergo no site for further chain extension so DNA synthesis halts
224
type of inhibition of NNRTIs
- Bind allosteric site adjacent to active site
- non-competitive, reversible
225
mechanism of NNRTIS
- Binding of NNRTI causes conformational change which inactivates active site
- No accessible active site = no viral DNA synthesis
226
disadvantages of NNRTIs
- rapid drug resistance
- Mutations in NNRTI binding site stop drugs interacting with RT
- Most common mutations: replacement of Lys-103 with Asn (mutant = K103N)
and replacement of Tyr-181 with Cys (mutant = Y181C)
-resistance reduced when initially used with NRTIs
227
describe the conformational changes that occur to RT hand structure during catalysis
palm contains polymerase active site and non-nucleoside-binding pocket, separated by ~10 Å. T
(i) thumb lifts up to bind nucleic acid
(ii) fingers fold down to capture dNTP substrates in the presence of nucleic acid
(iii) non-nucleoside binding leads to thumb hyperextension.
[Das et al., 2012]
228
advantages of NNRTIs
- Higher selectivity for reverse transcriptase over DNA polymerases than NRTIs hence less toxic / fewer side effects
229
Nevirapine NNRTI structure/interactions
- hydrophobic, as allosteric site has hydrophobic residues
- one half - interaction with tyrosine residues (aromatic)
- other half - interaction with leucine and valine residues (aliphatic)
- additional electrostatic interactions with Lys-103
230
Azidomythine and Lamivudine NRTIs activation mechanism and affinity
- Prodrugs phosphorylated intracellularly
- negative phosphate groups would inhibit diffusion across hydrophobic cell membrane.
- Drug also has quite high affinity for host DNA polymerases so induces toxic side effects on host cells.
231
how is Efavirenz suited to reduce resistance to NNRTIs
- Small: can shift binding position when mutation occurs
- Reduced emphasis on Tyr-181 binding; active vs Y181C
- Able to interact with main peptide chain of binding site
via hydrogen bonding
- Still suffers from lower activity vs K103N
232
how is Capravirine suited to reduce resistance to NNRTIs
- Active vs K103N and Y181C variants
- Forms 3 H-bonds to main peptide chain in
active site (in addition to aromatic interactions)
- Conformationally flexible; can bend itself to fit
even in mutated binding sites
- Failed in phase II trials
233
how is Etravirine suited to reduce resistance to NNRTIs
- As above: conformational flexibility means
drug can adapt to mutants
- Lots of different interactions, so loss of one
interaction through a mutation is less
critical to overall binding
- Used where NNRTI resistance to other
drugs has developed
234
What are HIV protease inhibitors?
- example of structure-based drug design
- Structures designed to mimic reaction transition-state:
TS is usually more tightly bound to enzyme than substrate or product, so a TS mimic should also display strong binding
235
mechanism of HIV protease
- 2 Aspartic acid residues, one from each subunit. one is protonated and the other deprotonated
- Amide hydrolysis proceeds via tetrahedral intermediate by asp.acid
- Selectively cleaves peptides between proline and aromatic residues (Phe or Tyr)
236
Structure of HIV protease
- Dimer; active site at interface of subunits
- Aspartyl protease: active site contains aspartic acid residue crucial to catalytic mechanism
237
what makes HIV proteases good drug targets
- Peptide cleavage next to Pro is rare; doesn’t occur in mammalian proteases = selectivity
- Symmetrical; mammalian proteases aren’t
238
how is hydroxyethylamine a good inhibitor of viral proteases
- Inhibitor design requires transition-state isostere:
- OH mimics OH in TS and binds to Asp in active site
- Chiral centre; R-configuration preferred
239
tipranavir use and resistance profile
- Non-peptide protease inhibitor
- Resistance is slow to arise; used in patients with resistance to other treatments
240
saquinavir disadvantages
- Quite low oral bioavailability and 98% bound to plasma proteins
- High dose required to maintain therapeutically high plasma levels
- Taken with ritonavir: reduces saquinavir metabolism so higher plasma conc.
- Simpler PIs developed; lower molecular weight, less peptide character (see text book)
241
saquinavir optimisation from pentapeptide
- Simplify to Phe-Pro dipeptide with hydroxyethylamine link (also weak inhibitor)
- Add Asn: greater activity than pentapeptide (S2-S2’= key section)
- Vary Z-group: important S3 interaction with large hydrophobic pocket so large quinoline added
- Tweak Pro structure (go bigger) and tBu ester (swap for amide: more stable) = final structure
242
HIV protease-substrate interactions
- Substrate is viral polypeptide; cleavage occurs between Pro and Phe / Tyr
- 8 binding subsites per enzyme; 4 per subunit (only 6 shown)
- Numbered S1-S4 on one monomer, S1’-S4’ on the other
- Each enzyme subsite, accepts an amino acid residue of the substrate
- N and O of each peptide bond in substrate involved in H-bond with enzyme
243
why are peptides bad drugs?
- Poor pharmacokinetics (i.e. poor absorption, metabolic susceptibility, rapid excretion, limited access to CNS, high plasma protein binding)
- Particular impact on administration: poor oral bioavailability
244
what an HIV protease inhibitor should be, an exception
- Short peptide mimic of viral substrate
- Linked via transition-state isostere (instead of peptide bond)
- Tipranavir
245
mechanism of HIV DNA integrase
- Integrase picks up viral DNA (synthesised by reverse transcriptase) and other cellular cofactors to form preintegration complex (PIC)
- Integrase exposes reactive OH groups on viral DNA 3’-terminus
- PIC enters nucleus, binds to host cell DNA; integrase reacts with host DNA to expose 5’-phosphate groups
- Viral DNA couples with host DNA (3’-OH reacts with 5’-phosphate)
246
Function of HIV integrase inhibitors
Target the splicing of viral DNA into host DNA by HIV integrase
i.e. swapped for 5 base pairs of host DNA
247
overview of SARS-CoV-2 3 step life cycle
attachment and entry
replication and synthesis
viral assembly and release
248
SARS-CoV-2 mechanism of attachment and entry
1. Viral spike protein mediates binding and attachment to peptidase domain of host ACE-2 enzyme.
2. Once virus is bound, a 2nd host cell surface protein, TMPRSS2, cuts off N-terminus of spike protein, allowing fusion and entry of virus into host cell.
249
spike protein features
Sugar chains mask virus from immune system.
membrane bound glycosylated trimer that mediates attachment and entry into host cell
2 subunits
250
important spike protein mutations
Alpha = type of H bond interaction changes and addition of aromatic ring when asp-501-tyr.
Delta and Gamma = asp-501-tyr; and glu-484-lys which swaps –ve to +ve charge.
Delta = leu-452-arg, swapping a hydrophobic amino acid to a hydrogen bond forming amino acid. Allows escape from antibodies and increased infectivity as receptor affinity is increased.
251
SARS-CoV-2 mechanism of replication and synthesis
1. Uncoating releases viral genome.
2. RNA immediately translated into protein by ribosomes in cytoplasm.
252
importance of double membrane vesicles in HIV life cycle
create protective micro environment for the viral replication and transcription complex.
253
SARS-CoV-2 mechanism of viral assembly and release
Post-translational modification occurs here.
1. Viral genome delivered and bound to nucleocapsid protein.
2. Membrane-bound proteins are packaged around capsid.
3. Vesicles encapsulating new virions combine with host cell plasma membrane and released by exocytosis.
4. Host cell is killed by virus hijacking resources, dampening vital host processes or final exocytosis.
254
mRNA vaccines disadvantages
- Unintended effects
- Delivery (free mRNA in the body is quickly degraded)
- Storage (requires lower temperatures than other vaccines)
255
mRNA vaccines advantages
- Safety (no infectious agents produced)
- Efficacy (generate reliable immune response with few side effects)
- Faster and ‘cheaper’ to produce
256
how is the SARS-CoV-2 vaccine made in vitro
1. In vitro transcription from a DNA template, with promoter (often T7)
2. Utilises RNA polymerase (often T7), rNTPs, buffer
3. 5’ cap added by enzymes
257
Two methods of overcoming immune response to mRNA vaccine
Purification (removal of dsRNA)
Introduction of modified bases
258
why is the SARS-CoV-2 mRNA vaccine made in vitro
Exogenous mRNA is inherently immunostimulatory, as it is recognized by a variety of cell surface, endosomal and cytosolic innate immune receptors
259
contents of SARS-CoV-2 mRNA vaccine
5’ cap
3’ poly-adenosine (Poly-A) Tail
5' untranslated region (UTR)
3' untranslated region (UTR)
Coding Sequence (CDS) (for SARS-CoV-2 spike protein)
260
5 common targets of antibacterial drugs
1. inhibition of cell metabolism
2. inhibition of bacterial cell wall synthesis
3. interaction with plasma membrane
4. disruption of protein synthesis
5. inhibition of nucleic acid transcription and replication
261
how are drugs which target prokaryotic cell metabolism specific?
target bacterial metabolic pathways that are not present in animal cells
262
function of drugs which inhibit prokaryotic cell metabolism
- Inhibit bacterial metabolism, but not metabolism of host
263
example of a sulfonamide
prontosil
- pro-drug of active sulfanilamide
- azo group readily metabolised
- Effective against Gram-positive bacteria (e.g. pneumococci and meningococci) but not against enterobacteria/Gram-negative (e.g. Salmonella)
264
sulfonamide mechanism
- competitive inhibitors of dihydropteroate synthase
- enzyme converts PABA -> tetrahydrofolate
- block tetrahydrofolate biosynthesis, stop DNA synthesis hence stop bacterial cell growth and replication
- Amine group N lone pair nucleophilic-ly attack (SN2) slightly +ve carbon adjacent to the phosphate group of target molecule.
- Sulphanilamide binds to enzyme active site and blocks PABA preventing the product synthesis.
265
advantages of sulfonamides
- Human cells unaffected: tetrahydrofolate still vital, but acquired through diet from folic acid
- long lasting, 1 dose /week
266
what makes bacterial cell walls a good drug target
eukaryotic cells don't have cell walls
undergo lysis when cell wall doesn't form properly due to osmotic pressures
267
disadvantages of bacterial cell wall synthesis inhibitors
only act on bacteria that are stil synthesising cell wall
no activity on fully formed cell walls so immune system is also required to eradicate infection
268
how is penicillin made and its drug target
cell wall synthesis (usually Gram +ve)
biosynthesis of penicillin core from valine and cysteine
further analogues synthesised from 6-aminopenicillanic acid
through acetylation of 1' amine you can make many analogues
269
penicillin mechanism
- inhibits transpeptidase which catalyses final cross-linking step in peptidoglycan synthesis
- D-alanine--D-alanine -> D-alanine--glycine
- penicillin mimics D-ala--D-ala and reacts with serine in active site
- irreversibly blocks active site so enzyme is now useless, incomplete cell wall = leaky cell = dead cell
270
disadvantages of penicillin G and solution
- Acid sensitive (broken down in stomach; administered by injection only)
- Narrow spectrum of activity (poor activity vs Gram negative bacteria)
- Highly sensitive to b-lactamases
solution: synthesise analogues, varied in acylamino side-chain
271
penicillin G reasons for acid sensitivity and solutions
- ring strain (4 and 5 carbon rings) is relieved by acid-catalysed β-lactam ring-opening
- SAR: cannot remove ring strain or C=O, but can add EWD acylamide
e.g Penicillin V and Ampicillin
- β-lactam C=O highly reactive to nucleophiles (narrow spectrum of activity, only for Gram +ve)
- EWD group to remove e- density of C=O, reducing reactivity, add hydrophilic groups which increase activity against Gram -ve
- greatest effect if alpha to the C=O
- β-lactamase sensitivity
- sterically hindered penicillin derivatives won't fit in active site
- should also inhibit activity
272
give an example of a β-lactam antibiotic
cephalosporins
273
general structure of cephalosporins and advantages/disadvantages of this structure
6-membered ring reduces 4-membered ring strain
A
- greater acid stability
- greater β-lactamase resistance
D
- reduced antibacterial activity
274
how are cephalosporin analogues classified and what is the trend in activity?
classified by generation, each new generation has a broader spectrum of activity than the last
e.g 1st gen = only active against Gram +ve
4th gen = active against both Gram +ve & -ve with increased potency
275
what are β-lactamase inhibitors and their main advantage?
- weak antibiotics, but irreversible inhibitors of β-lactamase
- used in combo with penicillin
- allows reduced penicillin dose and increases activity spectrum
276
examples of β-lactamase inhibitors
Clavulanic acid
Sulbactam
Tazobactam
277
mechanism of β-lactamase inhibitor clavulanic acid
- nullifies serine OH group of B-lactamase.
- Inhibits water molecule approach and instigates a different reaction first.
- Lysine reacts at slight +ve charge next to O. N attacks to form C=O.
- Allows drug to do its job without interference from B-lactamase.
278
mechanism of glycopeptide antibiotics
- binds D-ala--D-ala in peptidoglycan
- caps growing glycan to stop cell wall synthesis
279
function of glycopeptide antibiotics and example
- inhibitors of bacterial cell wall synthesis
- specific, fixed conformation
- last resort for Gram +ve
e.g. vancomycin
280
how does vancomycin prevent cell wall synthesis
so large it surrounds/caps D-ala--D-ala to prevent access to glycine
281
what is the main issue with antibiotics that target the bacterial plasma membrane
all cells have a plasma membrane, so selectivity is low
282
what is the main aim of antibiotics that target the bacterial plasma membrane?
make the membrane and cell leaky
283
give 2 examples of antibiotics that target the bacterial plasma membrane by forming ion channels
valinomycin and gramicidin
284
give an example of antibiotics that target the bacterial plasma membrane by causing leakage of small molecules
polymyxin B
285
structure of polymyxin B
NP
relatively long hydrophobic chain for insertion into plasma membrane
large polar looped domain
286
function of polymyxin B
- selective (ish) for bacterial membranes so quite toxic
- causes leakage of small molecules like nucleosides
- ergo lack of nucleosides for DNA and RNA synthesis
- mostly used for drug resistant Gram -ve
287
how do cyclic peptides act on the bacterial plasma membrane and example
- inserts itself into the plasma membrane and aggregates to form hexamer
- causes general disruption of membrane leading to uncontrolled ion permeability and cell death
e.g. daptomycin
288
4 modes of action of drugs that impair bacterial protein synthesis
- prevent binding of subunits
- interfere with binding with tRNA
- prevent peptide bond formation between amino acids
- interfere with translocation of ribosome to next codon
289
how do Oxazolidinones inhibit bacterial protein synthesis?
- prevent ribosome formation by binding specific site on 50S subunit, preventing assembly with 30S subunit
- broad spectrum Gram +ve bacteriostatic
290
common motifs of Oxazolidinones
- aromatic ring with F substituent
- oxazolidinone ring
both form VdW and pi stacking interactions with nuclear bases of ribosomal RNA
291
2 examples of Oxazolidinones
Linezolid and Tedizolid
292
how do tetracyclines inhibit bacterial protein synthesis
- binds to 30s subunit, preventing tRNA from binding to the mRNA-ribosome complex
- aromatic ring forms pi stacking interactions
- many hydrophilic groups form H bond network with sugar phosphate backbone
- broad spectrum bacteriostatic
293
disadvantages of tetracyclines
- acid sensitive, cannot eat dairy as lactic acid prevents absorption in gut
- cannot go out in sunlight -> severe sunburn
- high resistance due to irresponsible use in agriculture
294
advantages of tetracyclines
- long lasting doses
- works against both Gram +ve and -ve
295
how do chloramphenicols inhibit bacterial protein synthesis and essential functional groups
- Prevents elongation of peptide chain by inhibiting peptide bond formation.
- Binds to ‘a’ site of larger subunit and acts by inhibiting movement of whole ribosome unit along mRNA.
- Nitrobenzyl group essential for binding: pi stacking of aromatic ring and cytosine ring of RNA.
- Hydroxyl group essential for H bonds with sugar phosphate backbone.
- Dichloroacetamide group is important but can be modified.
296
advantages and disadvantages of chloramphenicol
A
- cheap, broad spectrum bacteriostatic
- readily crosses BBB, used for brain abcesses
D
- side effects: rare and unpredictable aplastic anemia
297
activity-essential macrolide functional groups
- OH forms H bond to adenine base in 50S subunit. If this is mutated drug cannot bind.
- Large macrolide lactone ring forms VdW interactions in the ribosome, in a hydrophobic exit tunnel; ring sits there and blocks it (blocking translocation).
298
how do macrolides inhibit bacterial protein synthesis and spectrum of activity compared to penicillin
- bind 50s subunit to prevent translocation = shift of ribosome along mRNA to expose next codon for translation
- wider spectrum of activity against Gram +ve than penicillin
299
example of macrolide antibiotic and a related family
erythromycin
macrolides are polyketides, a related family is ketolides which are more potent, with a broader spectrum and active against macrolide-resistant strains
300
functional groups of erythromycin that make it acid-sensitive and solution to allow oral administration
- C=O at position 9
- OH at positions 12 and 6
- OH lone pairs act as nucleophiles and attack C=O to form ketyl structure which prevents binding to ribosome
solution: coated in strong shell allowing passage through stomach
301
structure of macrolide antibiotic
- large (14,15 or 16-membered) macrolide lactone ring decorated with sugars
- forms VdW interactions in the ribosome, in a hydrophobic exit tunnel; ring sits there and blocks it (blocking translocation).
- Hydroxyl and tertiary amine group are essential for activity.
- Hydroxyl forms H bond to adenine base in 50S subunit. If this is mutated, i.e. adenine is no longer present, the drug cannot bind.
302
how do aminoglycosides inhibit bacterial protein synthesis
Block peptide translocation by binding 30S subunit.
inhibit protein synthesis
peptide chains are released before they are finished
many little hydrophobic peptides floating around, insert themselves into cell membranes and cause leaks
knock-on effect classifies aminoglycosides as bactericidal
303
why is mycobacterium TB difficult to kill
outer waxy coating not easily penetrated by drugs
304
how are aminoglycosides administered and why
intravenously, poorly absorbed by gut and are used for treatment of serious infections.
effective against most aerobic Gram -ve
305
examples of key aminoglycoside antibiotics
streptomycin
kanamycin
gentamicin
neomycin
306
how can antibiotics, e.g. kanamycin, be used for molecular cloning
- kill off bacteria that don't contain desired gene
- Usual marker are antibacterial resistance genes.
kanamycin:
- Hydroxyl group has key interaction.
- Gene product phosphorylates hydroxyl group to prevent binding to 30S subunit.
- Phosphate group and sugar phosphate backbone have repelling –ve charges.
- Stable over many temperatures, can be used with thermophilic bacteria.
- Works in both Gram +ve and –ve.
307
antibacterial resistance genes of kanamycin
neomycin phosphotransferase
308
antibacterial resistance genes of chloramphenicol
chloramphenicol acetyltransferase
309
antibacterial resistance gene product against tetracycline
tetracycline efflux transporter
310
antibacterial resistance genes of erythromycin
methylase
311
antibacterial resistance genes of ampicillin
beta lactamase
312
how do antibiotics inhibit bacterial nucleic acid transcription and replication
either
- act upon enzymes that manipulate bacterial DNA
- interact directly with bacterial DNA
313
how do aminoacridines inhibit bacterial nucleic acid transcription and replication
- DNA intercalators: large, flat heteroaromatic system
- distort helix, preventing replication and transcription
- not selective for bacterial DNA and so too toxic to use for systemic infections
314
how do quinolones and fluoroquinolones inhibit bacterial nucleic acid transcription and replication
- topoisomerase inhibitors
- bacterial topoisomerases are distinct enough from eukaryotic for selectivity
- bacteriostatic
315
4 examples of antibacterial quinolones and fluoroquinolones
nalidixic acid
enoxacin
ciprofloxacin
grepafloxacin
316
how do rifamycins inhibit bacterial nucleic acid transcription and replication
- Inhibition of bacterial DNA-dependent RNA polymerases prevents the start of bacterial RNA synthesis
- bind to a site on the bacterial enzyme not present on the respective mammalian enzyme, hence highly selective
- polyketide NP
317
what is a disadvantage of using rifamycin to treat TB
- TB is slow growing
- ergo transcription will be slow
- drugs must act over long timescale but most are heat/acid-sensitive
- treatment is long process
318
disadvantages of morphine use
- depression of respiratory centre
- tolerance and dependence (addiction)
- excitation and euphoria
- constipation (µ receptors in gut)
- nausea
- pupil constriction
319
advantages of morphine use
- analgesic and sedative
- one of the most effective painkillers
- elevates pain threshold (decreases brains awareness of pain)
- great control of constant pain
320
how does morphine act as an analgesic
- acts on CNS cells to disrupt pain signals
- via opioid receptors e.g. µ, κ and δ
- these receptors exist as we need control over pain
321
examples of opioid peptides/natural ligands of analgesic receptors
endorphins
endomorphins
dynorphins
enkephalins
322
similarities between µ, κ and δ-opioid receptors
- distribution: mostly CNS, µ also found in digestive tract and respiratory system
- receptor type: all GPCRs, morphine agnist against all 3
- analgesic activity mediated by various transduction mechanisms which links receptors to neuronal ion channels
323
effect of morphine agonism on µ receptor
- strongest sedative and analgesic effect due to tightest binding
- respiratory depression
- euphoria and addiction
- constipation
324
effect of morphine agonism on κ receptor
- lower levels of analgesia and sedation than µ
- no respiratory depression
- no addiction
- dysphoria and hallucinations
325
effect of morphine agonism on δ receptor
- analgesia without sedation
- evidence for respiratory depression is mixed (most likely at high doses)
- no addiction
326
how does morphine imitate natural ligands of opioid receptors
- natural ligands often have terminal tyr
- morphine imitates tyr OH substituted aromatic ring and tertiary amine
327
important structural features of morphine
- pentacyclic alkaloid with many stereocentres
- rigid, T shape
- many important chiral centres
328
how is morphine-derivative, pro-drug codeine orally available
one of the hydrophilic OH groups is masked
OH -> OMe
can cross BBB also
329
SAR morphine
6-OH
- Masking 6-OH with less polar group increases blood-brain barrier transfer, hence faster accumulation at greater concentration in brain, so greater activity
7-8 double bond
- Removal gives slight increase in analgesia over morphine
necessary groups:
- phenolic OH
- basic nitrogen of N-methyl
- aromatic ring
- stereochemistry
330
3 successful strategies of synthesising morphine analogues
- drug extension
- drug simplification
- rigidification
331
mechanism of kinase-linked receptors
- minutes to respond
- Activated by polypeptide hormones, growth factors and cytokines i.e. important in endocrine regulation
- messenger binds causing shape in protein conformation
- dimerisation allows each active protein to phosphorylate the othe
332
mechanism of kinase-linked receptors
- minutes to respond
- Activated by polypeptide hormones, growth factors and cytokines i.e. important in endocrine regulation
- messenger binds causing shape in protein conformation
- dimerisation allows each active protein to phosphorylate the other
333
erythromycin resistant genes and product
erm genes:
methylase
334
what is the aim of combinatorial chemistry?
find many analogues of a lead compound in one go
335
what is solid phase synthesis and why is it required for combichem?
what: starting material linked to solid support
why:
- range of starting materials attached to different beads but treated with same reagent
336
other advantages of solid phase synthesis
- excess reagent/unbound product easily removed so can use large excess of reagent to drive reaction forward
- intermediates do not need to be purified
- automation possible
337
5 essential requirements for solid phase synthesis
- Cross-linked insoluble polymeric support inert to the reaction
- Anchor or linker covalently attached to the support which has a reactive functional group that can used to attach a substrate
- Bond between the linker and substrate must be stable to the reaction conditions
- simple, efficient process for removal of product from linker
- Protecting groups for functional groups not involved in the synthetic route
338
5 common solid synthesis linkers
- merrifield
- wang
- rink amide
- sasrin
- tetrohydropyranyl
339
reaction conditions for removal of product from each of the 5 common solid synthesis linkers
- merrifield = HF
- wang = 50% TFA
- rink amide = 95% TFA
- sasrin = 1 % TFA
- tetrohyropyranyl = TFA
340
what is combichem deconvolution
the process of isolating and identifying the most active component in a mixture.
341
what is combichem micromanipulation
each bead in a mixture only contains one structural product; separate beads individually and test.
342
what is combichem recursive deconvolution
test compounds at each stage of synthesis (assuming solid support; alternatively remove some material at each stage for testing).
Test if activity only detected after addition of a specific component
343
what is combichem sequential release
Split a library, by bead, into smaller components
344
4 methods to isolate active component from combichem mix and split
deconvolution
micromanipulation
recursive deconvolution
sequential release
345
what is combichem tagging
two molecules are built up on the same bead. One is the compound of interest; the other acts as a code for each step
346
what aminos make up the Safety-Catch Acid-labile Linker
lysine and tryptophan
they each have a free amino group
347
what is the role of lysine and tryptophan in the Safety-Catch Acid-labile Linker?
tryptophan = free amino group is the foundation for target structure
lysine = after each stage of synthesis an amino acid is added to the growing peptide
348
how is the Safety-Catch Acid-labile linker removed?
cleaved by reducing the 2 sulphoxide groups and then treating with acid
349
what is combichem parallel synthesis
technique for focused lead optimisation studies where a reaction is carried out in a series of wells such that a library of single products is generated
350
for what purpose is a lollipop phase separator used
when parallel synthesis is conducted in liquid phase to separate the aqueous and organic solutions
351
what is the method of lollipop phase separator
1. pin inserted into mixture of aqueous/organic solution and rapidly cooled to -78 celcius
2. aqueous phase freezes to pin and be extracted from organic solution
352
how is dynamic combinatorial synthesis different to split and mix
- DCS synthesis and screen in situ
- Target is in reaction flask with building blocks
- Reactions should be reversible (amplification of active compound)
353
in dynamic combinatorial synthesis, how do you stop the reaction to identify active compounds?
by converting equilibrium products into stable compounds that cannot revert back to starting materials
354
what is the aim/main advantage of dynamic combinatorial synthesis
to synthesise a large library of compounds simultaneously in one flask and screen the for activity in situ as they form
much faster
355
discuss the key points of the dynamic combinatorial synthesis of imines from aldehydes and primary amines
- 3 different aldehydes and 4 different 1' amines = 12 possible imines
- building blocks mixed with target: carbonic anhydrase
- after time, sodium cyanoborohydride was added to reduce imines > 2' amines for identification
- HPLC shows which products were amplified and therefore which products were able to bind to target.
- a sulphonamide had significant amplification compared to control with no enzyme present
356
what is the aim of fragment based lead discovery?
to find small molecules (epitopes) that bind to specific part of active site.
357
what is the method of fragment based lead discovery?
- screen for small molecules that bind to avctive site of target enzyme
- epitopes have no biological activity themselves
- link several epitopes together to give the lead compound which will bind to the whole of the binding site.
358
how many fragment-derived drugs approved for use by FDA and what are their names?
[Wang et al., 2022]
4
Vemurafenib
venetoclax
erdafitinib
pexidartinib
[Wang et al., 2022]
359
what are the 2 major challenges of fragment-based lead discovery?
Identification of fragment binding to the active site
Subsequently linking/growing the fragments together to form viable ligand
360
rule of 3 for fragment-based lead discovery
MW < 300
< 3 HBD
< 3 HBA)
cLogP < 3
< 3 rotable bonds
polar surface area < 60 Å2
361
4 advantages of drug repurposing
- low cost
- skip animal studies > Phase 2
- compounds already available
- formulations and manufacturing chains already established for large-scale production
362
4 disadvantages of drug repurposing
- IP
- target identification can be more challenging
- SAR to improve potency loses repurposing potential
- effective concentrations in vitro too high for in vivo use
363
what is drug repurposing: same target, new virus
Antiviral drug which targets a specific viral or cellular function/pathway has activity against other viruses
364
example of drug repurposing: same target, new virus
Viral RNA polymerase inhibitors favipiravir (Ebola) and sofosbuvir (Zika)
365
what is drug repurposing: same target, new indication
established pharmacological target is found to be essential for another pathogenic process associated with a viral infection (e.g. protein or pathway that can be regulated by an approved drug)
366
example of drug repurposing: same target, new indication
Anticancer drug imatinib inhibits cellular Abelson kinase was also found to be active against MERS- and SARS-CoV
367
what is drug repurposing: new target, new indication
Approved drug with established bioactivity for a particular target is found to have another new molecular target.
368
example of drug repurposing: new target, new indication
antimicrobials that have been found to have a target in virus-infected cells: ivermectin (flaviviruses), azithromycin (Zika)
369
Imatinib effective against SARS-CoV but not SARS-CoV-2
- SARS-CoV relies on ABL2 kinase activity to infect host cells
- imatinib blocks coronavirus entry via preventing viral fusion with the cell membrane
- SARS-CoV-2 genome is 80 % homologous with SARS-CoV but imatinib is not effective at clinically appropriate doses
[Leukemia, 2020, 34:3085]
370
AZT to treat HIV
1st anti-HIV drug approved
mode of action: intercalates between bases. NRTI prevents replication.
SAR: flat aromatic region for VdW with bases, +ve charged azo nitrogen for ionic interactions to disrupt -ve backbone.
371
structure of ebola virus genome
Linear, single-stranded, negative sense RNA genome (19 kB)
372
chloroquine to treat Zika
- effective in reducing ZIKV vertical transmission in mice
- CQ suppressed in vitro ZIKV replication in Vero cells with an EC50 of 9.82 mM.
- CQ interferes in the fusion of envelope proteins with the endosome membrane
373
approved for pregnancy anti-zika drug
azithromycin
374
how does SARS-CoV-2 maintain efficient cell entry while evading immune surveillance?
The high hACE2 binding affinity of the RBD, furin preactivation of the spike, and hidden RBD in the spike
375
what was the oxford RECOVERY trial?
175 NHS hospitals
simple questions asked at enrolment
found 4 effective treatments of SARS-CoV-2
376
what was the WHO solidarity trial?
- > 12,000 patients
- currently evaluating 3 drugs: artesunate, infliximab and imatinib to reduce mortality
- initially investigated remdesivir, HCQ, lopinavir and interferon
- showed these 4 drugs were ineffective against rates of mortality, ventilation and duration of hospital stay
377
Ec50 of remdesivir against ebola vs SARS-CoV-2
ebola = 0.003-0.009 uM
SARS-CoV-2 = 0.77 uM
378
RECOVERY oxford trial results with tocilizumab
- improved survival
- regardless of amount of respiratory support - additional to the benefits of systemic corticosteroids.
- reduces mortality by 8%.
379
what was the primary clinical use of tocilizumab before SARS-CoV-2
- targets interleukin 6
- rheumatoid athritis
380
what were the adverse effects of treating patients with HCQ in the oxford RECOVERY trials
less likely to be discharged from hospital in 28 days
- higher frequency of invasive ventilation
- higher frequency of death
381
mechanism of action of CQ and HCQ against viruses
- increase pH of endosomes (used by viruses to enter host cell)
- HCQ interferes with glycosylation of ACE2 (required for SARS-CoV-2 binding)
382
what are lopinavir and why is it used in combination with ritonavir
lopinavir is HIV protease inhibitor
ritonavir increases plasma half-life of lopinavir
383
RECOVERY trial results of lopinavir and ritonavir
in vitro activity against COVID-19
no benefit found in humans
384
what is the mechanism of azithromycin antibiotic in reducing adverse immune reactions to coronaviruses
decreases production if inflammatory cytokines
inhibits neutrophil activation
Used in SARS and MERS
385
RECOVERY trial results with azithromycin
no effect on length of hospital stay or mortality with COVID-19
386
clinical uses and possible uses of ivermectin
broad spectrum in vivo against
- nematodes, arthropods, flavivirus, mycobacteria
in vitro activity against zika
inhibits dengue virus by blocking an essential viral RNA replication protein 5
contradicting evidence for use against COVID-19
387
anti-viral mechanism of molnupiravir
- targets viral RNA-dependent RNA polymerase
- incorporates into ssRNA genome of viruses and leads to accumulation of mutations by H bonding with nucleotides
- amino-M forms 3, imino-M forms 2
- mutations unrecognizable by viral proofreading
388
results of phase 2 trials of molnupiravir against COVID-19
72 structural nucleotide changes to the spike protein compared to 9 in placebo
389
major clinical disadvantage of using molnupiravir
targets viral genome so high risk of generating variant of concern
390
what is paxlovid made of and what was it created for
Mixture of Ritonavir and protease inhibitor
lead compound for SARS-CoV-2
391
drug target of paxlovid
3CL^Pro inhibitor
392
trial results of paxlovid
89% effective in patients at risk of serious illness
0/6 people died after admitted taking pax within 5 days of onset cf. placebo 10/41 admitted died
393
what are cell fusion inhibitors and what what are they used for
HIV drugs that prevent viral cell membrane from fusing with host cell membrane
394
enfuvirtide drug profile
anti-viral cell fusion inhibitor
structure: 36 residue peptide
mechanism: binds to viral protein gp41 involved in pulling host and viral cell together during fusion
- prevents proper gp41 assembly, inhibiting fusion
395
4 disadvantages of anti-viral cell fusion inhibitors
- Expensive: ~$25000 / year
- Requires injection twice daily
- Side effects
- Used as salvage therapy
396
maraviroc drug profile
anti-viral cell fusion inhibitor
mechanism: CCR5 antagonist.
- CCR5 is a GPCR co-receptor with host CD4 that mediates host entry
- bind to allosteric site, non-competitive inhibition of gp41-CCR5 binding
- gp41 polymer cannot assemble, inhibiting fusion
397
4 requirements for mRNA vaccine administration system
- Bind vaccine mRNA to form complexes
- Promote cellular uptake
- Protect vaccine mRNA from intracellular and extracellular nuclease degradation
- Enable the release of vaccine mRNA into the cytoplasm
398
5 components of lipid nanoparticles
- ionizable cationic lipid
- Lipid-linked polyethylene glycol (PEG)
- Naturally occurring phospholipid (DSPC)
- Cholesterol
- Buffers, salt, sugar
399
what are viral vector vaccines
a modified version of a different virus (other than the virus you're vaccinating against)
delivers mRNA vaccine sequence for the antigen to cells
vector itself should not illicit an immune response
400
how are adenoviruses modified to be used as mRNA viral vector vaccines
Engineered to remove replication genes (E1)
Propagated in a cell line that complements deletion in trans
