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C2 > Molecules of life > Flashcards

Molecules of life Flashcards

1
Q

what is fundamental for interactions with class A GPCRs?

A

common basic Nitrogen (amine)

amines also have region of aromaticity

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2
Q

whats an inverse agonist?

A

Inverse agonist: drug which binds to same receptor as agonist BUT induces opposite pharmacological response to agonist

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3
Q

effect of zwitterions on BBB

A

zwitterions = overall neutral, do not cross BBB, only act on peripheral level. side effects associated with central and H1 antagonism such as sedation and drowsiness are not caused.

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4
Q

common pkas:

A

5: cooh
10: sp2 OH
15: sp3 OH

Amine: pKa 10.

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5
Q

what drugs generally cross BBB? example and affect?

A

highly lipophilic drugs.
e.g. first gen antihistamine
cause drowsiness

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6
Q

Whats KI (nm)- what does it tell you?

A

inhibitor constant- potency of inhibitor

want lower value = more potent

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7
Q

whats Ic50 and whats a good value range? high/low

A

Ic50= lower is better as more potent (same with KI)

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8
Q

What composes a nucleoside?

A

a heterocyclic base + a sugar

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9
Q

What composes a nucleotide?

A

a heterocyclic base + a sugar + a phosphate

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10
Q

What is the difference between an RNA monomer and a DNA monomer?

A

RNA: sugar is ribose, where there is an OH at position 2 of base ring

DNA: sugar is deoxyribose, where there is a H at position 2 of base ring

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11
Q

What is a pyrimidine?

A

A heterocyclic ring with 2 N atoms

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12
Q

What bases are pyrimidines?

A
  • cytosine
  • thymine (DNA)
  • uracil (RNA)
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13
Q

What is a purine?

A

a bicyclic ring with 4 N atoms

2 rings

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14
Q

What bases are purines?

A
  • adenine

- guanine

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15
Q

What is the difference between the bases found in RNA and DNA?

A

RNA contains uracil instead of thymine

both still pyrimidines

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16
Q

What are polymers of nucleotides known as?

A

Nucleic acids

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17
Q

What do the properties of nucleic acids depend on? (3)

A
  • the nature of the monomer (nucleotide base) A/C/G/T
  • specific order of monomers (DNA sequence)
  • the 3D structure (double helix)
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18
Q

Describe the nature of DNA. What links the monomers and where?

A
  • long unbranched chain of DNA monomers (nucleotides)
  • monomers linked by phosphodiester bond
  • bond links phosphate group of one nucleotide and sugar of next nucleotide
  • 5’ to 3’
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19
Q

Is DNA always linear?

A

no, the ends can be linked in other organisms to give circular DNA

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20
Q

What does Watson-Crick pairing describe?

A

the nature of bonding between base pairs:

A = T (2 H bonds)

C ≡ G (3 H bonds)

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21
Q

DNA is a _ _ structure

A

double helix

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22
Q

Describe the structure of DNA (chains? phosphate? grooves)?

A
  • double helix chains twist on a common axis
  • phosphate backbone
  • major and minor grooves
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23
Q

Where are the major grooves in DNA?

A

the wider regions

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24
Q

What are base pairs to one another on a DNA strand?

A

complementary

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25
Q

How do the nucleotide chains of DNA appear against each other?

A

antiparallel

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26
Q

Where are the minor grooves in DNA?

A

the narrower regions, between base pairs

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27
Q

How are bases directed to each other? Why?

A
  • inwards

- allows for complementary base pairing and H bond formation

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28
Q

What types of interactions exist between sequential bases?

A

hydrophobic

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29
Q

How are base pairs positioned relative to the helix axis?

A

perpendicular

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30
Q

How many degrees is the twist between consecutive base pairs? What is the consequence in the structure?

A
  • 36°

- every 10 base pairs there’s a complete 360° twist of the DNA helix

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31
Q

What are the 3 stages of the cell cycle? (DNA replication)

A
  • interphase
  • mitosis
  • cytokinesis
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32
Q

In the interphase of the cell cycle, what are the 3 stages of DNA replication?

A
  • initiation
  • elongation
  • termination
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33
Q

Where and What occurs in the initiation stage of DNA replication? What enzyme is involved?

A

in DNA regions called ‘origins’

DNA double helix is unzipped - DNA helicase breaks the H bonds between base pairs

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34
Q

What occurs in the elongation stage of DNA replication? What enzyme is involved?

A

DNA polymerase catalyses the formation of the new (leading) strand from the parent (template) strand via an esterification reaction

5’->3’ direction

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35
Q

Each parent stand acts as a __ __ for new DNA

A

template strand

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36
Q

What are the new strands compared to the parent stands?

A

complementary

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37
Q

What type of reaction results in the formation of new DNA strands during DNA replication?

A

esterification

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38
Q

What occurs in the termination stage of DNA replication?

A

terminus site binding proteins block the replication fork

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39
Q

What is the replication fork? (3. termination step of interphase)

A

the two fork-like structure of DNA following unzipping by DNA helicase

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40
Q

What are the building blocks of DNA?

A

nucleotide triphosphates

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40
Q

In the esterification of the new strand by DNA polymerase , what part of the nucleotriphosphate is susceptible to nucleophilic attack?

A

the triphosphate anhydrides

e.g. -OH group on sugar of the adjacent nucelotide

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41
Q

Why is the esterification reaction for DNA chain extension possible? (hint - sketch mechanism for nucleophilic attack)

A

diphosphate anhydride is a good leaving group

from phosphod bond/backbone

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42
Q

How are the nucleoside triphosphates of DNA named? (hint: dCTP)

A

2’-deoxy[cytosine]-triphosphate for cytosine - replace cytosine with name of base

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43
Q

What are the 4 key differences between RNA and DNA?

A
  • DNA is deoxyribonucleic acid (H at 2’ position), RNA is ribonucleic acid (OH at 2’ position)
  • DNA: double-stranded; RNA: single-stranded
  • DNA: thymine as its base, RNA: uracil as its base
  • DNA stores genetic info, RNA takes genetic info and uses it in processes
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44
Q

What is uridine?

A

a uracil attached to a ribose sugar

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45
Q

difference between thymidine and uridine.

A

DNA: thymidine

  • has H at 2 position of ring
  • methyl group at ortho of base

RNA: Uridine

  • has OH at 2 position of ring
  • NO methyl group at ortho of base
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46
Q

What are the 3 major forms of RNA?

A
  • mRNA
  • tRNA
  • rRNA
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47
Q

What is a gene?

A

the specific segment of DNA that codes for a protein

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48
Q

What are the 2 strands of DNA known as?

A
  • sense strand

- antisense strand

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49
Q

What is the sense strand also known as?

A

coding

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50
Q

What is the antisense strand also known as?

A

template

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51
Q

Describe how mRNA is transcribed from DNA in the nucleus (3)

A
  • DNA unwound
  • RNA polymerase constructs mRNA strand using antisense (template) strand to build it using codons (transcription)
  • mRNA leaves the nucleus
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52
Q

Why must mRNA be transcribed from the antisense strand?

A
  • the antisense strand is the template strand

- this ensures mRNA is the same as the coding strand

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53
Q

What is the difference between DNA polymerase and RNA polymerase?

A

RNA polymerase only replicates single-stranded mRNA

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54
Q

What are the building blocks of mRNA?

A

codons

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55
Q

What is a codon?

A

a triplet of 3 bases that codes for a specific amino acid

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56
Q

What is the difference between the mRNA strand and the coding strand?

A

every T is replaced with a U

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57
Q

After mRNA is transcribed, where does it go and what happens?

A
  • leaves the nucleus and goes to ribosome

- translated into a peptide

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58
Q

What is each amino acid specified by on an mRNA stand?

A

a codon

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59
Q

How many triplet codes does each amino acid have?

A

2-3

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60
Q

How many START codons are there? What is it?

A

1 - AUG - methionine

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61
Q

How many STOP codons are there?

A

3

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62
Q

What is the structure of ribosomes and where in the cell are they found? What are they made of?

A
  • 2 subunits: 30S and 50S in humans
  • found in cytoplasm
  • made of rRNA and proteins
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63
Q

What does tRNA do?

A

transfers amino acids to ribosome

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64
Q

names of the arms of a tRNA molecule

A

D arm
T?C arm
Variable loop
Anticodon region

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65
Q

What part of a tRNA molecule is most important and why?

A

the anticodon region; brings the amino acid and is complementary to the codon on the mRNA strand

(bottom)

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66
Q

What does a tRNA sequence always end in?

A

CCA-3’

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67
Q

What process does tRNA undergo to attach amino acids?

A

acylation

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68
Q

What is involved in the reaction that tRNA uses to attach free amino acids?

A

ATP

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69
Q

ATP is required as energy source in process of attaches a.as to tRNA. what the eqn?

A

ATP-> AMP + PPi

inorganic phosphate

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70
Q

Describe the 3 stages of acylation that tRNA uses to attach amino acids

A
  • free amino acid O- attacks triphosphate in ATP, causing diphosphate to leave and single phosphate anhydride to attach to amino acid
    PPi lost
  • (AA bound to AMP)… tRNA attacks carbonyl C of amino acid, causing phosphate anhydride to leave (rest of AMP)
    AMP lost
  • tRNA now has amino acid loaded on
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71
Q

In what direction is mRNA read by the ribosome during translation?
(peptide synthesis)
(What prime and what terminal?)

A
  • 5’ to 3’

- N to C terminal

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72
Q

Describe the stages of translation (5)

A
  • first (empty) tRNA bound at P site
  • next tRNA bound at A site
  • new peptide bond forms between 2 aas close together
  • peptide now attached at A site
  • tRNA leaves at E site

(Sites: E,P,A)

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73
Q

In translation, what ensures the correct tRNA is selected?

A

H bonding between the (mRNA) codon and (tRNA) anti-codon

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74
Q

how do alkylating agents disrupt DNA replication / transcription and potentially trigger cell death

A

Alkylating agents form new bonds with DNA, thus disrupting DNA replication / transcription and potentially triggering cell death

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75
Q

Nitrogen mustard agents form WHAT between DNA bases?

A

Nitrogen mustard agents form covalent bonds between DNA bases (intra-strand or inter-strand crosslinks)

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76
Q

Nitrosoureas and triazene agents decompose (spontaneously or enzymatically) to form what?
and affect?

A

form reactive species which can alkylate DNA

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77
Q

DNA repair enzymes can contribute to ? how?

A

to drug resistance (by repairing alkylation)

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78
Q

What is the therapeutic use of alkylating agents?

A

cancer therapy

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79
Q

What kind of bonds do alkylating agents form with DNA?

A

covalent bonds

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80
Q

By forming covalent bonds with DNA, what is the effect on DNA?

A
  • the metabolism is disrupted

- cannot transcript or replicate DNA

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81
Q

By forming covalent bonds with DNA, what is the effect on the cell?

A

cell apoptosis is triggered

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82
Q

What are examples of types alkylating agents?(3)

A
  • nitrogen mustards
  • nitrosoureas
  • organoplatinum agent
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83
Q

Nitrogen mustards are what type of agents apart from alkylating?

A

crosslinking agents

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84
Q

What are the different ways that nitrogen mustards can work? (3)

A
  • can crosslink bases on the SAME DNA strand (intra-crosslink)
  • can crosslink bases on two DIFF DNA strands (inter-crosslink)
  • can form covalent bond between nucleotide AND amino acid, forming DNA-protein complex (tertiary)
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85
Q

Draw the general structure of a nitrogen mustard agent.

A

Cl \ / \ N / \ / Cl
I
R

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86
Q

How many DNA bases can one molecule of nitrogen mustard alkylate (join together)?

A

2

learn mechanism

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87
Q

What is the first step in how a nitrogen mustard reacts with guanine?
whats formed and whats lost?

A

lp from N of nitrogen mustard curly arrow to C closest to end, curly arrow to Cl.
Cl = good LG.
forms 3 membered ring w N- v reactive!
high polarisation of Cs on ring

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88
Q

whats the name for first step of nitrogen mustard mechanism?

i.e. lp of N causing Cl to leave

A

intramol cyclisation

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89
Q

nitrogen mustard step 2: 3 memb ring, high polarisation of Cs on ring causes ….?

A

nucleophilic attack from lp on DNA base

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90
Q

3rd step of nitrogen mustard mech?

then what?

A

DNA base and N.mustard bond.

AND like first step, now lp of N causes other Cl to leave

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91
Q

final step of N mustard reac between 2 bases leads to 2 DNA bases which are now…

A

alkylated

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92
Q

What governs the mechanism of action of nitrogen mustards?

A

the reactivity of the nitrogen mustard and N from the DNA base

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93
Q

Order the following in decreasing reactivity with a nitrogen mustard agent:
N3 (guanine), N7 (guanine), N3 (adenine), N7 (adenine), N1 (adenine), N3 (pyrimidine)

A

N7(G) > N3(A) > N7(A) > N3(G) > N1 A > N3 pyrimidine

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94
Q

The reactivity of the N atom from a base with a nitrogen mustard can be modulated by what?

A

sterics, electronics and hydrogen bonding

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95
Q

What is an enol?

A

alkene to alcohol

R - C= C- OH
I I
R R

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96
Q

When you alkylate the N7 guanine (lone N on the attached 5 memb ring), what ion does it form?
What is the consequence?

A
  • ammonium ion
  • makes the base more acidic
  • this pushes the tautomeric equilibrium from the keto to the enol form
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97
Q

What is the difference between the keto and enol form of guanine?
How is this useful?

A
  • keto binds to C (normal)
  • enol binds to T (abnormal)
  • changes the base that guanine binds to introducing errors into the DNA
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98
Q

What is the difference between the keto and enol form of guanine in terms of curly arrows?

A

before: H-> N = -> O
now: H-> O =-> N

backwards/reverse, coming onto N, inside ring instead of out onto =O

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99
Q

what can stabilise the + charge: keto and enol form of guanine

A

aromaticity of the 6-memb rings

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100
Q

Name the nitrogen mustard agent used to treat advanced Hodgkin’s lymphoma.

A

Chlormethine (Mustargen)

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101
Q

What is the R group of chlormethine N mustard? How does this help its reactivity?

A
  • CH3

- electron-donating, so increases the nucleophilicity of the N lone pair

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102
Q

Can chlormethine (N. mustard) be administered orally? Why?

A
  • no
  • the N atom is too reactive due to the CH3 R group
  • the three-membered intermediate ring will be too reactive and toxic
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103
Q

What is the version of chlormethine (N mustard w CH3 R group) that can be orally administered? Why is this?

A
  • chlorambucil
  • CH3 R group is replaced by aromatic ring with COOH
  • stabilises N lone pair by resonance, reducing the toxicity
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104
Q

How do nitrosoureas work?

A

they’re unstable in water so decompose (in situ) to form an alkylating agent

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105
Q

What type of agent is carmustine?

State the R1 and R2 groups for carmustine.

A

nitrosourea

R1: CH2CH2Cl

R2: CH2CH2Cl

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106
Q

What type of agent is lomustine?

State the R1 and R2 groups of lomustine?

A

nitrosourea
R1: cyclohexyl

R2: CH2CH2Cl

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107
Q

What type of tumour are nitrosoureas used to treat? Why?

A
  • brain tumours

- the R1 and R2 groups are used to increase the lipophilicity so it can cross the blood brain barrier

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108
Q

What are nitrosoureas commonly associated with?

A

severe side-effects e.g. bone marrow toxicity

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109
Q

How is resistance to nitrosoureas mediated?

example and MoA

A

through special class of DNA repair enzymes: O6-guanine DNA methyltransferase- can move methyl group off C-> damage by methyl. agent

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110
Q

How do triazenes work?

A

undergo spontaneous or enzymatic degradation to form a reactive species

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111
Q

Draw a triazene group.

A

N = N
\
N

may be present in ring or not

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112
Q

What are two examples of triazenes?

A
  • decarbazine

- temozolomide

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113
Q

What is the reactive species generated from a triazene?

what can it do?

A

a methyldiazonium ion

N ≡ N+

v reactive, can do alkylations

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114
Q

How do triazenes overcome the resistance mediated for nitrosoureas?

A

they’re able to methylate O6-guanine methyltransferase by generating a reactive CH3 species

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115
Q

Draw the equilibrium that allows triazenes to overcome the resistance mediated for nitrosoureas.

A

[(-N ≡ N+ ) N2 + +CH3]

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116
Q

like nitrosureas, how can DNA repair enzymes contribute to drug resistance?

A

by repairing alkylation

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117
Q

topoisomerase inhibitors:

role of topoisomerase enzymes?

A

regulate DNA supercoiling during replication and transcription (regulating access to the genetic code)

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118
Q

what are the 2 types of topoisomerase enzymes?

A

type I: regulate SINGLE stranded DNA

type II: regulate DOUBLE stranded DNA

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119
Q

3 steps in the action of topoisomerase?

A
  • they cleave DNA strands by catalysing a transesterification reac
  • cleaving DNA introduces ‘breaks’ in DNA, exposing genetic code(not whole DNA) for replication/transcription
  • when complete, breaks in DNA are repaired by same enz- topoisomerase (via reverse transesterification)
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120
Q

what do topoisomerase enz interfere with and what affect does this have?

A

DNA repair, thus leaving DNA irreversibly damaged

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121
Q

3 main classes of topoisomerase inhibitors: categorise them as Type I inhibitor (SS-DNA) or Type II (DS-DNA)

  • Camptothecin analogues
  • Epipodophyllotoxins
  • Anthracyclines
A

Type I
- Camptothecin analogues

Type II

  • Epipodophyllotoxins
  • Anthracyclines
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122
Q

what is -Camptothecin?

A

class of Type I topoisomerase enz,
plant alkoid
natural product
limited solubility

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123
Q

name a semi-synthetic analogue developed of Camptothecin and reason?

A

e.g. Irinotecan, to imporve PK profile

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124
Q

how is PK profile improved in the Camptothecin analogue: Irinotecan?

A

carbamate ester (COON) added = hydrolysis causes bioactivation in liver, can admin IV

quinolone added to N in carbamate ester- has basic side chain within: improves solubility

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125
Q

Whats Epipodophyllotoxins?

semi-synthetic derivatives of what and vary in nature of…?

A

Topoisomerase enz Type II

semi-synthetic derivatives of podophyllotoxin, vary in nature of the glycosidic substituent

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126
Q

despite the into of glycoside, both Etoposide and Teniposide (Epipodophyllotoxins) are..?

A

highly water insoluble

think ab administration

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127
Q

3: what are Anthracyclines, what do they contain?

A

Topoisomerase inhib Type II,

struct related to tetracycline antibiotics and contain sugar + organic portion- that intercalated DNA

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128
Q

In Anthracyclines, whats the role of:

a) the sugar
b) the organic portion?

A

a) binds to DNA minor groove, drive sequence specificity. Amine = protonated at physiological pH (NH3+) doesnt make contact with double helix (repelled by DNA backbone)
b) rings intercalate DNA

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129
Q

2 side effects of 3:Anthracyclines?

A

acute (reversible) cardiotoxicity due to formation of free radicals (ROS)

chronic cardiotoxicity due to interference w Ca2+ homeostasis

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130
Q

how does: acute cardiotoxicity due to formation of free radicals (ROS) of 3:Anthracyclines occur?

A

P450 reduction of quinone ring -> dihydroxy form
(ring w 2x =O –> ring w 2x -OH)

ALSO: 02 oxidised to O2- at same time
= superoxide radical (ROSP) V REACTIVE
…specific cardiotox as cardiac tissues dont have enough enz dedicated to disposing ROS

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131
Q

how does: chronic cardiotoxicity due to interference w Ca2+ homeostasis of 3:Anthracyclines occur?

A

reduction: aldo-keto reductase

large R group next to ketone slows rate of reduction, limiting side effects
sterics… hard for enz to get close

metabolite accumulates in cardiomyocytes, interferes w Ca2+ homeostasis = congestive HF that resists treatment

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132
Q

what is the term dubbed to nucleoside analogues?

A

‘false’ substrates

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133
Q

what are the precursors to new DNA strands and what are these susceptible to?

A

nucleoside triphosphates,, nucleophilic attack

correct base delected due to its H-bonding properties with other DNA strands

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134
Q

how do nucleoside analgoes become incorporated into enz active site? what allows them to? and why dont they just have same effect as the enz then?

A

sufficiently similar to a true nucleotide… but diff enough to disrupt the process

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135
Q

consequences of disrupting DNA replication?

A

trigger apoptosis (prog. cell death) in cancer cells

other chemotherpay drugs/ immune sysem have better chance of overcoming the malignancy!

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136
Q

case study: whats Gemcitabine and whats its use?

A

a false nucleotide- similar to the true: 2’deoxycytidine

the 2x F attached to 2 position of ring will block DNA synthesis (Di-fluoro).

prevent chain elongation

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137
Q

how are Gemcitabine di and tri phosphate (the two active and false metabolites) formed?

A

through kinase reactions G- G mono- G di- G tri

each time adding a Phosphate

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138
Q

how do the ‘false’ nucleeotides/ active metabolites: Gemcitabine di and tri phosphate disrupt DNA synthesis and elongation?

A

G di:
- inhib enz ribonucleotide reductase (converts ribonucleotides into 2’deoxyribonucs) i.e. inhib DNA synthesis

G tri:
- causes apoptosis through inhibition of DNA elongation- cant add any more chains

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139
Q

challenges in the bio-activation of Gemcitabine (prodrug)

and consequences?

A
  • cytosine deamination:
    NH2-C=N –> O=C-NH
    (C and last N: in ring)
    impacts nucleobase pairing
  • inefficient phosphorylation
    limits/slows drug efficacy particularly the first phosphorylation

cant admin G mono directly - compromised PK profile

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140
Q

how to overcome challenges in the bio-activation of Gemcitabine (prodrug)

A

created next gen prodrug of G mono, add AA ester and aryl motif (control logP, solubilities)

MoA exploits enzymatic machinery of inside cells to obtain the active metabolite

G mono formed inside cell

(mehcanism!!)

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141
Q

antiviral nucleoside analogues:

DNA targeting drugs are selective for what type of cells? (2)

A

cells that lack DNA repair mechanisms/ are rapidly replicating
…. but other organisms store genetic info in other ways

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142
Q

what 2 types of RNA/DNA can viruses have?

A

ss single stranded

ds double stranded

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143
Q

what does The Baltimore Classification system describe?

A

7 virus classes based on genetic material and replication strategy:

1 and 2: DNA viruses ss/ds
3,4,5: RNA viruses
6 and 7: reverse transcribing viruses

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144
Q

in class 6 of baltimore virus classification, what viral enzyme is envolved and its role?

A

reverse transcriptase: reverse transcribes RNA into DNA

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145
Q

what happens to DNA reverse transcribed from RNA in class 6 viruses?

A

new DNA then integrates into host cells genome (becomes infected w viral genetic code -> expression of viral proteins)

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146
Q

how can we target the reverse transcriptase enz? (responsible for RNA->DNA in viruses) class 6

A

with inhibitor drugs.

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147
Q

similarities that exist between anti cancer drugs and antivirals?

A

nucleoside analogues (antimetabolites) - disrupt process by changing building blocks

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148
Q

whats the structure of Nucleoside reverse transcriptase inhibitors (NTRIs)?
and what are they?

A

= class of nucleoside analogue that lack the hydroxyl (OH) group at the 3’ position of the deoxyribose sugar- where usually extended

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149
Q

what do NTRIs do? MoA

A

once incorporated into DNA, further chain extension is blocked i.e. NTRIs are a chain terminator of viral DNA synthesis! :)

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150
Q

example NTRI: AZT (Zidovudine)- use and effect?

A

orig: anticancer to inhibit cellular DNA synth
but: too toxic, had high affinity and selectivity for HIV-RT.

dose limiting bone marrow toxicity (why…)

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151
Q

RNA replicase inhibitors

What do class 3-4 viruses use to store genetic info?

A

RNA rather than DNA.

replication + trans carried out without resorting to any DNA intermed i.e. RNA used as template for RNA synthesis

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152
Q

suitable targets of RNA replicase inhibitors and why? (class 3-4 viruses)

A

no analogous human process, so the enz RNA polymerase/RNa replicase are suitable targets

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153
Q

nucleoside RNA replicase inhibitors often introde small substituents at?

A

2’ position of the sugar

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154
Q

antiviral nucleoside analogues
case study: Molnupiravir- what is it?
how does it wokr?

A

RNA replication inhibitor prodrug used to treat COVID19

  1. hydrolyse ester -> OH.
    active nucleoside analogue formed -> 2 possible tautomers promotes mutation during viral RNA replication by RNA polymerase
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155
Q

antiviral nucleoside analogues
case study: Molnupiravir

the 2 possible tautomers result in….?
and why- what 2 things do they mimic?

A

base mismatching and mutations
as bind w wrong base!!

one mimics cytidine, one uridine

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156
Q

how is coding DNA sequence transcribed to

a) template
b) then mRNA
c) then anti-codon

d) straight to anti codon?

A

a) template= opposite direction (3’-5’ instead) flip
change all A to T, C to G…

b) HAS URACIL: change all A to U, T to A, C to G….
c) all A - U, C- G… still no T.
d) just change all T to U. only

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157
Q

when nitrogen mustard undergoes intramolecular cyclisation and folds in on itself, what key intermediate is created?

what are the key features of its behaviour (angle)

A

Aziridium ion (N- epoxide like)

60 degrees in triangle: compressed :(
unhappy in 3 memb ring system =breaks and attacked

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158
Q

what type of reaction is it both tyoes when N mustard agent folds in on itself (curly arrows part and Cl goes)?

A

sub = Sn2’ like

as 2 arrows, LG and nucleophile on SAME MOL

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159
Q

where do curly arrows usually go to and form?

A

from lp –> delta +

delta + –> delta -

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160
Q

why N mustards used for oncology, cancer treatments?

A

react quickly in body - aziridium ion (N- epoxide like v reactive)…

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161
Q

how does N mustard join 2 base pairs?

A

can form 2x N triangle = Aziridium ions

then broken by G and A to attach.

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162
Q

2 types of links that N mustards can create between 2 base pairs?
what does it lead to?

A

interstrand cross link: bases on opposite strands on DNA

intrastrand cross link: bases on same strand on DNA

covalent bond formed between bases- changes DNA code
apoptosis and cell death :D if cancer.
mRNA approaches, cycle stopped

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163
Q

N mustard with what as R group will be most potent?

A

R= Me (attached on N)

alkyl- inductive - inc e- density on N = reacts quicker!

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164
Q

N mustard with what as R group will be least reactive?

A

R = benzene-COOH

direct attach so more stable caarboxylate ion @pH7.4 = higher aq sol in body - easier to travel
also got aromatic resonance = stable:)

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165
Q

affect of adding to a compound (spec N mustards) more: aromatic groups

A

stabilises charge
can be given orally
lp on N can be stabl. by resonance

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166
Q

affect of adding to a compound (spec N mustards) more: alkyl chains

A

lp less available - sterics

Me = inc nucleophilic nature of N lp

167
Q

affect of adding to a compound (spec N mustards) more: aryl substituent (phenyl- ring)

A

electron withsrawing

limits nucleophilicity of N.

168
Q

affect of adding to a compound (spec N mustards) more: carboxylate substituent

A

improves solubility but e- on N are so stabilised by delocalisation that resulting compound = v weak alkylator

169
Q

affect of adding to a compound (spec N mustards) : methylene units (C) between e- withdrawing carboxylate and aromatic ring?

A

incre reac

but, each -CH2- inc LogP and introduces a rotatable bond.

170
Q

role of adding a halogen (F) to C2 in an antimetabolite ring?
Chlofarabine

A

makes mol more resistant to metabolism (adenosine deaminase)

lot of drug elim unchanged via kidneys

171
Q

What within the DNA defines the sequence of amino acids?

A

the sequence of bases within the genetic code of DNA

172
Q

How does a gene turn into a protein?

A
  • transcribed into mRNA in the nucleus

- translated into a protein in the ribosome

173
Q

How do the primary structure of proteins compare between species?

A

they can be different e.g. human vs mouse

174
Q

where are proteins found and do they move about or stay there?

A

localised in all of the cells comptmts and can move from one part of cell to another and have targeted pharmac.

175
Q

What defines the sequence of amino acids?

A

the sequences of bases within a gene of DNA

176
Q

What reaction occurs between two amino acids to form a dipeptide?
whats lost and whats made?

A

dehydration reaction

H2O lost = amide made

177
Q

What do amino acids make up?

A

proteins/peptides

Peptides <40 AA
proteins > 40AA

178
Q

How many amino acids are there?

A

22

179
Q

How many groups can amino acids be divided into, and based on what?

A

3, based on the nature of the R groups

180
Q

9 amino acids are considered essential. What is meant by this?

A

they need to be obtained from diet and cannot be synthesised in the body

181
Q

How many of the 22 amino acids are used by cells to synthesise proteins?

A

only 20

182
Q

If only 20 out of 22 amino acids are used to synthesise proteins, what are the other two used for? What are these?

A
  • post-translational modification
  • cystine and hydroxyproline

made after the polypep chain

183
Q

How many amino acids have a chiral centre?

A

all except glycine

184
Q

For amino acids that have a chiral centre, how do they exist?

A

as a mixture of 2 enantiomers

185
Q

the following AA sometimes corresponds to what enantiomer?
L
D

A

L - S

D- R

186
Q

What is the configuration of all naturally occurring amino acids? Why?

A

L - they are S at the alpha carbon centre

187
Q

What is the configuration of all naturally occurring amino acids in microorganisms?

A

D

188
Q

What is the exception to the naturally occurring amino acids in terms of configuration? Why?

A
  • cysteine
  • sulphur atom from the SH changes the numbering of groups around the alpha carbon
  • this means that while it is L, it is R!! at the alpha carbon centre
189
Q

In protein synthesis, what configuration of amino acids are used? When are the others used?

A
  • L

- D are used for post-translational modification

190
Q

Which compound is used as the reference for determining the configuration of an amino acid?

A

glyceraldehyde

191
Q

how to assign R or S?

A

number the priorities: H=4,… C=3.. NH=1

arrow from 1-2-3

if H facing forwards (wedge) fine else flip if dash

clockwise R
anti: S

192
Q

Where in microorganisms are D-amino acids especially found?

A

the peptidoglycan of their cell walls

193
Q

What is an alpha amino acid?

A

the carboxylic acid and amine group are attached to the same carbon

i.e. the normal struc of aa

194
Q

What is a beta amino acid?

A

the carboxylic acid and amine group are attached to different carbons

i.e. norm but with an extra C in between

195
Q

Out of alpha and beta amino acids, which are more common?

A

alpha

196
Q

Why are beta amino acids useful?

A

beta amino acids used to make beta peptides are more stable to proteases, so can be used in drug design

not very common though

197
Q

Why are amino acids able to be ionised?

A

they have an acidic COOH group and basic NH2 group

198
Q

In solid state, how do amino acids exist?

A

as a dipolar ion/ zwitterion

199
Q

At a basic pH, what form of an amino acid exists?

A

the anionic form (COOH is deprotonated)

COO-

200
Q

At an acidic pH, what form of an amino acid exists?

A

the cationic form (NH2 is protonated)

NH3+

201
Q

At a neutral pH, what form of an amino acid exists?

A

the zwitterion/dipolar ion form

both: NH3+ and COO-

202
Q

When alanine is in an acidic environment, what form will it exist in?

A

cationic form

COOH same but NH3+ protonated

203
Q

How does the pKa of the carboxylic acid in alanine compare to the pKa in propanoic acid? Why?

A
  • alanine’s COOH pKa is more acidic
  • receives electron-withdrawing effect from ammonium ion NH3+ (prop doesnt have this)
  • this stabilises the -ve charge of the carboxylate
204
Q

Why can the dipolar form of an amino acid be considered a potential acid?

A

the NH3+ could donate a proton

205
Q

How do the pKas for glycine and acetic acid compare? Why?

A
  • glycine’s COOH pKa is more acidic

- receives electron-withdrawing from ammonium ion that stabilises COO-

206
Q

How do the pKas for glycine and glycine methyl ester compare?

A

glycine’s amine pKa is more basic

207
Q

What is the isoelectric point (pI) of a molecule?

A

the pH at which the molecule has no net charge

208
Q

What equation is applied to calculate the isoelectric point?

A

the Henderson-Hasselbach equation

pH = pKa + log [A-]/[HA]

A-: conj base
HA: molecule

209
Q

What equation is used to calculate the isoelectric point of amino acids with no charged side chains?

A

pI = (pK1 + pK2)/2

halfway of pKs

210
Q

Using glycine’s pKa values (2.35, 9.78), calculate the isoelectric point.

A

2.35 + 9.78/2 = 6.07

211
Q

Aspartic acid and Glutamic acid
have an acidic side chain.
What pKas are important and used to calculate the isoelectric point?

A

the acidic pKas of the side chain and the COOH group

212
Q

For arginine, histidine and lysine, what are the important dissociation constants? (all have charged, basic side chains)

A

the basic pKas for the side chains and amine/amino group

213
Q

What is the isoelectric point of peptides?

A

the average of the 2 pKas at both sides of the neutral form of the peptide (table!!)

214
Q

What is the general method for calculating the isoelectric point of peptides?

A
  • sketch peptide label any side-chain pKas
  • sketch a table w column 1:pH, next columns corresp to pKas of the relevant charged side chains and amine and COOH groups
  • label the last column as overall charge
  • write a range of pH values between 0 and 12 in the rows under the pH column
  • for each row, write whether the group will be +, 0 or - and write the overall charge at the end
  • at the point where the charge has changed from -1, 0 to +1, check which groups have changed signs
  • these are the important pKas - use them to find the isoelectric point
215
Q

What reagents are needed to form an amino acid in the first stage of Strecker synthesis?

A 
- aldehyde with desired R group (RCOH)
   R  
      \
       C = O
      /
  H
- NH3: nucleophile
  • HCN

lp of N-> C in aldehyde, then arrow from = onto O.

216
Q

What is the product formed from the first stage of Strecker synthesis?

A

alpha-amino nitrile

  R  
      \
       C - CN
      /
  NH2
217
Q

What reagents are needed to form an amino acid in the second stage of Strecker synthesis (include product from first stage)?

A
  • alpha amino nitrile
  • acid (H3O+)
  • heat
  • water (H2O)
218
Q

What is the product formed from the second stage of Strecker synthesis?

A

an alpha amino acid

from alpha amino nitrile

219
Q

What type of reaction is the second stage of Strecker synthesis?

A

a hydrolysis reaction (cyanide to carboxylic acid)

220
Q

What different functional groups can you react with a primary or secondary amine to generate an amide?

A
  • acyl chloride
  • carboxylic anhydride
  • ester
221
Q

acid chloride reacting with an amine: word eqn?

A

acid chloride + amine –> amide + HCL

222
Q

What compounds do you need to react to readily synthesise an acid chloride?

A

thionyl chloride (SOCl2) + carboxylic acid (RCOOH)

223
Q

Draw the mechanism for reacting the synthesis of an acid chloride.

A

PRACTISE!

224
Q

amide bonds can be made form amine (NH2) plus…(2)?

A

carboxylic anhydride or ester

225
Q

cannot synthesise amides easily from the carboxylic acid SM because?

A

it is too stable to do directly,

need to activate instead:
in situ with a coupling agent OR use derivative used instead (ester. carb anhydride)

226
Q

what do coupling agents e.g. carbodiimide (DCC) do?

A

activate carb acid in situ so it can react w amine and form aa

227
Q

when DCC coupling agent react w COOH, what side products created?

A

protonate DCC: curly arrow from N on DCC to OH on COOH then from O-H onto O.

stable urea side product… thermodynamic driving force

228
Q

2 ways amides undergo hydrolysis (breakage of C- NH2 bond)

and difference between the two types?

A

acidic hydrolysis:
reac w H3O+ in presence of H2O + heat
creat carb acid + NH4+

basic hydrolysis
reac w OH- in presence of H2O + heat
creat COO-+ NH3

Review cfp1 mechanism!!

and consider stability of the amide ebonds esp in stomach. with these hydrolysis conditions

229
Q

amide bonds made by reacting…

A

amide and carb acid DERIVATIVE

not COOH direct as too stable
- must be activates

230
Q

how is COOH activated, to then react w amine and form amide bonds? (2)

A
  • use derivative (convert it to acid anhydride (2 aas) or an acid (acyl) chloride)
  • activation in situ with a coupling reagent
231
Q

synthesis of a peptide or a protein is just about ….

A

making a series of amide bonds…

however slightly more complex as have bifunctional molecules

232
Q

why is a problem caused because/when both the amine and COOH groups of the aas you want to react, are free?

A

multiple possible reactions can take place!

e.g. Ala + Gly

get Ala+Gly :)
+ Ala-Ala
+Ala-Ala-Ala
+ Ala-Ala-Gly……..

decreased yield of desired Ala-Gly significantly and separation of product from all other compounds = difficult (consider diff in func group between these mols)

233
Q

creating peptides- how do we control the products generated?

A

must control reactivity of one of the nucleophilic groups/ nucleophile must be ‘protected’

234
Q

what nucleophilic group in aa is usually protected, to prevent unwanted products+reactions?

whens this done?

A

amino group of first aa (DO NOT want to react)

done before the COOH is activated on same molecule –> COO-

235
Q

what reaction do we get once we protect the amino grouop on first aa and react w activated COOH?

A

selective reaction between unprotected amine and activated COO-.

236
Q

Role of the amine protecting group on the aa?

A

converts it to another group that has lower nucleophilicity.

- to prevent reaction with reactive acyl derivatives

237
Q

why is choice of protecting groups important?

A

because once reaction over, need to remove it from synthesised peptide without disturbing newly formed amide bonds

238
Q

once amine group protected on aa, what is needed for next step of reaction?

A

SOCl2 and 2nd desired aa

239
Q

what are the 3 common protecting groups/ moieties

used to protect amino group of aas?

A

CBz

Boc

Fmoc

240
Q

behaviour of protected amines generated, towards acyl chlorides?

A

unreactive.

as sp3/sp2?

241
Q

what 2 protecting groups are good if protecting 2 things at once as can remove at diff times?

A

Fmoc and Boc …or perhaps Fmoc and Cbz

as Fmoc: OH-
and other 2:H+

242
Q

in what conditions are the following protecting groups removed?
Cbz
Boc
Fmocb

A

acidic (H+): Cbz and Boc

basic (OH-): Fmoc, often using piperidine

243
Q

how are Cbz and Boc protecting groups removed/ deprotonated?

A

both in H+ acidic conditions…

Cbz: by catalytic hydrogenation/ HBr in acetic acid
Boc: by treatment w HCl/ CF3COOH in acetic acid

244
Q

What is the nature of the bond between the C and O in a carbonyl? Why?

A
  • polarised

- the O atom is electronegative

245
Q

Because of the nature of the carbonyl bond, what is the nature of the carbon atom?

A

electrophilic (+ve)

246
Q

What is the nature of the O atom of a carbonyl group? Why? (hint: hybridisation)
what can happen as a result?

A
  • nucleophilic (lone pairs)
  • lone pairs are located in sp2 orbitals
  • these can attack an electrophile
247
Q

Because of the polarised nature of the carbonyl groups, they can have a role as both a.. or _.

A

Because of the polarised nature of the carbonyl groups, they can have a role as both a nucleophile or electrophile.

248
Q

What site of a carbonyl would be attacked by a nucleophile?

A

the delta-positive carbon

249
Q

What would the product of a nucleophilic addition to a carbonyl be?

A

a tetrahedral product

250
Q

What part of a carbonyl would attack an electrophile?

A

a lone pair from the O atom

251
Q

What makes a carbonyl being able to attack an electrophile possible?

A

resonance

252
Q

what is benzyl chloroformate?

A

Cbz protecting group + Cl on end

253
Q

amine + Cbz forms what amide bond + Cl- under what conditions?

A

OH-, 25 degrees

after tet intermediate collapses

254
Q

Cbz amide bond - Cbz group removed using what reagents? (2 options)

A

catalytic hydrogenation (H2/Pd)
of
HBr in acetic acid

(to give ring + CO2 + amine)

255
Q

amine + Boc forms what amide bond + CO2 + 2,2methyl-methanol under what conditions?

A

base, 25 degrees

256
Q

Boc amide bond - Boc group removed using what reagents?

A

HCL/ CF3COOH in acetic acid, 25 degreesC

257
Q

amine + Fmoc forms amide bond + CO2 + H2O under what conditions?

A

NaHCO3

H2O

258
Q

step 2 in reaction of amine + Fmoc?

Fmoc group removed using what reagents?

A

use piperidine in basic condition to remove

Deprotecttion mechanism!

259
Q

name groups added to SIDE CHAINS on AAs for protection…

A

O-tert-butyl (ether)
O-tert-butyl (ester)
Boc
Tritryl (Tr)

260
Q

automated peptide synthesis: Merrifield method uses Merrifield resin. what is it and why useful?

A

small insoluble polystyrene (PS) resin w CH2CL groups on surface

swells in solvent, can do chem reacs -> washed off at end, product recovered ☺

261
Q

6 steps of automated peptide synthesis

A
  1. Attach C-terminal of protected AA to (solid) resin
  2. purify resin w attached protected AA by washing
  3. remove prot group e.g. Boc
  4. purify by washing
  5. ass 2nd protected AA using coupling reagent e.g. DCC
  6. purify by washing
  7. remove protecting group e.g. Boc
  8. purify by washing

repeat 5-8 every time you add new AA! (eLab)

final step: detachment of completed polypeptide

262
Q

automated peptide synth- how is peptide sequence assembled?

A

assemble back to front!

263
Q

why do we keep washing to purify in automated peptide synthesis?

A

excess reagent removed and whatever reacted remains stuck

264
Q

automated peptide synth: whats left near end?

A

2 AAs attached to resin + a protecting group, can obvs carry on….

265
Q

after step 1 of automated peptide synth, step 2 = purify resin w attached protected AA by washing

what reagent used and why?

A

base plus minus H+

to mop up HCL because Merrifield resin has Cl which will be kicked off as LG

266
Q

final step of automated protein synthesis= detachment of completed polypeptide.

done under what conditions?

A

HBr in CF3COOH acid.
Br acts as nucleophile, attacks C in Merrifield resin, breaks off and leaves OH on end (COOH)
= peptide cleaved ☺

resin restored as – -/\Br + peptide made

267
Q

how is synthesis of proteins w <200 AAs done?

A

possible to make using small peptide fragments using powerful technology:
NATIVE CHEMICAL LIGATION

268
Q

NATIVE CHEMICAL LIGATION: allows/ consists of what? (2)

A

allows ligation of peptide sequences w unprotected AAs to yield the native peptide backbone

allows synthesis of long pep sequences which cant be made efficiently using automated pep synthesis

269
Q

NATIVE CHEMICAL LIGATION is an almost XX reaction.

A

quantitative

270
Q

how is NATIVE CHEMICAL LIGATION done?

why is S used?

A

convert AA to thioester: CO-SR
use cys residue: - C - NH2 - C - SH

S direct and allow reac to proceed

271
Q

automated synth can in theory be used instead of native chemical ligation, to synth peptides up to 200AAs but whats the problem?

A

as AA num inc, error rate inc.

272
Q

mechanism of NATIVE CHEMICAL LIGATION?

conditions/ reagents?

A

thiolate group (SH) of an N-terminal cysteine attacks the C- terminal thioester (COSR) of a 2nd unprotected peptide

aqueous buffer at pH 7.0,
20 degreesC < T< 37 degreesC

273
Q

first step of NATIVE CHEMICAL LIGATION involves reag of proton shuttle (plus/minus H+). whats the LG?

A

SR in the C-temrinal of 2nd unprotected peptide

274
Q

what type of intermediate created in NATIVE CHEMICAL LIGATION reaction mechanism? (last but 1 step)

A

tetrahedral intermediate: 5 membered ring!

275
Q

product of NATIVE CHEMICAL LIGATION?

A

cysteine group link

                   SH
    O          /
     II         I
... C - N - C  -  C - .....
          H           II
                       O
276
Q

how are peptide sequences determined?

A

using 2 protease enzymes:

Chymotrypsin and trypsin…
routinely used to cleave large peptide sequences into smaller fragments

277
Q

Chymotrypsin protease enzyme cleavage sites?

A

on C-terminal of aromatic AAs (F,Y,W)

and to a lesser extend L,M,N,Q

278
Q

Trypsin protease enzyme cleavage sites?

A

on C-terminal of basic AAs (R,K)

Lysine and Arginine

279
Q

define pI

A

isolelectric point
average of the 2 pKas that fall either side of pH where molecule is overall 0 charge.

tabulate answer! pKas/ diff pHs

280
Q

why may SOCl2 be added before joining 2 AAs?

A

make acid chloride from COOH, now less stable, better to react

281
Q

2 ways to make a peptide/ join 2 aas together?

what do they have in common?

A
  1. SOCl2 method
  2. DCC coupling method
    both: need protecting groups
282
Q

describe peptide synthesis method 1: SOCl2 method

A
  • add boc to amine
  • react with SOCl2 to make COOH -> COCl, more reactive as less stable
  • join boc-COCl with amine of other AA, HCl lost
  • get boc-peptide
  • remove boc under acidic conditions (HCl)
283
Q

describe peptide synthesis method 1: DCC coupling reag method

A
  • start with boc on amine of BOTH aas
  • react N with OH of first aa, becomes boc-COO- (more reactive!) and DCC with N+
  • O- of aa attacks C in DCC… joins
  • lp on N of 2nd aa attacks first aa x DCC complex
  • proton shuttle… get Boc-peptide-Boc + urea (stable side product)
  • remove boc under acidic conditions (HCl)
284
Q

from SGT2-2, what form should COOH be in to react w NH2 and form amide bond?

A

COO- or COCl

285
Q

which amino acids will be protonated/ deprotonated at physiol. pH 7.4?

A

any aas in the peptide that:

  • have a side chain = side chain will be prot/dep
  • terminal aas = RHS: COO- and LHS: NH3+
286
Q

Why WONT any amino acids in middle of peptide (i.e. not the terminal ones) be protonated/ deprotonated at physiol. pH 7.4?

A

as the amide bond is neutral overall. has no charge.

287
Q

when will the following be protonated/ deprotonated?

a) amines NH2
b) COOH

A

a) protonated + when pH < pKa
b) deprotonated - when pH > pKa

consider AA side chains!
terminal AAs always affected

288
Q

what is TFA and use?

A

reagent: CF3COOH
Trifluoroacetic acid.

= H+, used to remove Boc (or Cbz/Z)

289
Q

What does tBu .. (ester/ether)?

side chain protecting groups

A

Tert-butyl ester

  • adding protecting group to COOH side chain (OH)
  • all amino acid RHS terminals in peptides before removal of PG

Tert-butyl ether
- adding protecting group to sec/ prim/ benzene OH

290
Q

whats tritryl (Tr) i.e. how made?

side chain PG

A

made by reacting trityl chloride (TrCl)/ 3 benz ring attached to C the Cl…
replace Cl w N/S from the NH/ SH youre adding it to to protect it.

291
Q

what to always remember if question asks to synthesis peptide ‘ using the solin resin support’ (merrifield resin)…..?

A

synthesise the peptide BACK TO FRONT

remember the 8? steps…

protect N side chains with Boc (and ed w Fmoc)

remove Fmoc throughout w piperidine

remove Boc at end w HBr (acidic, will do)

292
Q

what does it mean to ‘globally deprotect with HBr’ the side chain at end of protein peptide synthesis?

A

remove the Merrifield solid resin support/ Boc… acidic conditions

293
Q

when joining Gly and Lys, what is protected when 3rd aa: Glu is added?

A

tBu ester (side chain ester on end LHS with the merrifield resin)

SGT2-2

294
Q

do you still need to use SOCl2 on the COOH to destabilise it in pep synth if youre using Merrifield resin and Fmoc?

A

no because the MR will act as PG and attach to COOH part -> ester

removed at very end using HBr in CF3COOH (TFA)

to reveal/ cleave peptide from solid support

295
Q

in automated peptide synthesis, how is the finished peptide cleaved from solid support Merrifield resin?

A

using HBr in TFA (CF3COOH)

= resin restored + cleaved protein ☺

296
Q

whats DIPEA and when is it seen?

A

just base,

with reagent DCC, ‘DIPEA’

297
Q

CADD Pt 1: representing molecules

reason for doing this computationally?

A

money
time
resources (lab personell)

AI advances
Moore’s law
insight from detail
Safety: concerns/ unsafe routes avoid

298
Q

what is used as electronic databases of molecules?

A

Reaxys

Polypharmacology Browser: target prediction combining nearest neighbours w machine learning

299
Q

an elec database of molecules: Polypharmacology Browser predicts what?

A

possible targets of small molecule by similarity to compounds of known bioactivity using molec fingerprints (fps) describing composition, substructure, mol shape, pharmacophores

300
Q

drawing molecules electronically methods? (2)

A

SMILES notation: C1CCCCC1 = 6 memb ring, back to C1

ChemDraw:

301
Q

what can ChemDraw

a) calculate
b) predict

A

calculates

  • high accuracy MW
  • mass (isotope) distrib m/z
  • element analysis

predicts

  • H1 NMR
  • C13 NMR
302
Q

CADD Pt 2: modelling small mols

what are the 5 modelling systems?

A

(empirical force fields - v large systems, 1000s atoms: proteins, cell memb, liq, sol)
(QM/MM: small QM part, large MM env)

DFT
Hartree-Fock, MP2
Configuration interaction

303
Q

whats used in the following modelling systems? placed in order of accuracy and size…

DFT (Density functionla theory)
Hartree-Fock, MP2
Configuration interaction

A

DFT
- larger gas mols (small liq/solid systems)

Hartree-Fock, MP2
- small-med gas phase mols

Configuration interaction
- very small gas phase mols e.g. water

304
Q

what does molecular modelling require?

A

quantum mechanics, which provides mathem. description of motion/ interaction of subatomic particles (P,N,e-)

305
Q

name of methods used to ‘make’ new molecules in 3D using molec. modelling software?
what does it allow us to determine?

A

‘Ab initio’- from first principles

determine wavefunction, an object which depends on all 3n coordinates of the n electrons

306
Q

what is the schrodinger equation used for and describes?

A

\hat{H} x \Psi = E \Psi
\hat{H} = hamiltonian operator
\Psi = allowed wave function
E = energy

DONT NEED TO REMEMBER/USE

describes wavefunctions, used to calc properties of molecules (QM, MM software)

relates to nuclei, electrons

307
Q

what does DFT use as its fundamental input commpared to ab initio?
what do these simplifications allow?

A

electron density

.. simplifying things, reduces accuracy of model BUT electron density is only a function of 3 coordinated (x,y,z) per functional. in space

basically: DFT scales much slower than fully solving schrodingers eqn (ab initio)

308
Q

what 3 things is DFT often a good trade off between?

A

computational time
calculation accuracy
system size

309
Q

3 steps of studying a new mol/s using DFT?

A
  1. construct in software OR modify struc of existing x-ray crystal struck = good starting guess
  2. optimise struc geometry (shape in space) to its MINIMUM ENERGY
  3. calc output - models physioch props (gemetry, bonding, sol, spectra…) -electrostatic potential energy surface
310
Q

what does it mean in step 3 of stsudying mol using DFT: optimise struc geometry (shape in space) to its MINIMUM ENERGY?

A

what point are bonds in most favourable positions = lowest energy?

H-bonds shown at minimum energy

311
Q

DFT informing design of new drugs:

what does it predict/ provide? (3)

A
  • gives important 3D struc info
  • predicts inaccessible chem spaces
  • predicts many important physiochem props
312
Q

Why is DFT not practical to make large number of mols?

A
  • screening library sizes (can be around 10000 compounds)

many screens start w in silico screening before starting wet screenign

313
Q

how does DFT give important 3D struc info?

i.e. interactions and charges

A

can visualise localised charges- needed for modelling the potential interactions in target binding site
think ab geometry impact/ orientation of func groups

314
Q

how (when) does DFT predict inaccessible chem spaces?

whys this good?

A

rules out impossible/impractical chem strucs before starting out wet synthesis

optimise synthetic procedures…. save resources: time and money

315
Q

how does DFT predict many important physiochem properties?

i.e. which ones?

A
  • chem spectra (IR, UV-Vis, NMR)
  • acidity/basicity pKa, pKb

model a chem recation and check whether mechanism realistic or not
e..g is ester able to hydrolyse as expected

316
Q

case study: solubility of Ru(II) anticancer agents

good but limited by poor [water] solubility. how can this be increased?

A

using in silico DFT methods… ake model struc modifications

317
Q

case study: solubility of Ru(II) anticancer agents

how is modification to inc water sol done using DFT?

A

parent compound optimised (add R groups) –> get general struc for screening

18 possible struc… have to think?

  • synthesising in lab
  • purifying all
  • characterising all
  • determining LogP for all

is it worth it?

318
Q

DFT modelling shows EPS, whats this?

A

electrostatic potential energy surface..

for Ru(II) anticancer agents this is:

  • point localised charge of carboxylate, available for H-bonding
  • minimal electronic impact at Ru
319
Q

case study of Ru(II)…

how was Lead compound synthesised then confirmed to more water sol than parent comppound?

A

DFT…

confirm using relative hydrophobicity (HPLC)

320
Q

CADD part 3: modelling large molecules

what 2 methods/ approaches/ systems used for laregr molecules/proteins?

A

empirical force fields

QM/MM

321
Q

why is modelling of large molecules less accurate?

A
  • consider computational limits thus level of theory appropriate
    in sensible timescale
322
Q

what do we use instead of trying to solve structure using ab initio methods for drug targets?

-drug targets are often proteins and enzymes

A

big molecules
rely on experimental data as an input, then probe results using MM software and model atoms using simpler Newtonian mechanisms

(F=MA)

323
Q

how are large protein strucs understood using CADD?

what does it help us visualise?

A
  • protein data bank PDB
  • Pymol: probe experimental info
  • Maestro (Schrodinger, Inc.)- studying P-L ints in a 3D complex. visualise protein pocket and see AA ints
  • Ligplot+ (EMBL-EBI) -“ “
  • PLIP= tabulated interactions
  • binding sites
  • protein domains
  • sec, tertiary strucs
324
Q

case study: albumin binds to drug etoposide. whats albumin?

A

most abundant plasma protein in blood, known to bind and transport many drugs

etoposide drug looked for in PDB

325
Q

case study: albumin binds to drug etoposide.

once found in PDB using identifier code: 4LB9… why isnt it necessarily a true binding conformation?

(4LB9 = x-ray study of human serum albumin complexed with etoposide)

A

because drug is contained within structure HOWEVER it only represents where drug happened to co-crystallise in protein. not necessarily true binding conformation (of lowest energy)

326
Q

CADD part 4: combining small and large molecules

how do the 3 systems/ approaches of modelling large molecules compare w 2 systems/ approaches of modelling smaller mols?

A

larger = less accurate
(empriical FF, QM/MM)

smaller = more accurate
(DFT, Hartree-Fock MP2, configurational interaction)

327
Q

3 CADD system combinations used?

and differ based on what?

A
  • molecular docking and molecular dynamics
  • QMM/MM hybrid modelling
  • Full DFT (rare!)

depend on level of detail wanted

328
Q

what is the process of CADD combination method 1: molecular docking and dynamics (MD)?

A

least complex approach

treat P as rigid, fixed struc
allow drug mol to be conformationally flexible within defined grid (boundary)

flexibility (MDynamics) described using Newtonian mechs (not quantum mechs)

329
Q

molecular docking and dynamics (MD) ads and disads? 2/1

A

+ low computational demand
+ less time consuming

  • lower level of comp theory -> lower accuracy
330
Q

what is the process of CADD combination method 2: QMM/MM hybrid modelling?

A

calculate drug mol (+ active site) w higher level of theory = better accuracy (e.g. DFT) QM
treat remaining P using forcefield approach (MM)

final model = hybrid system

331
Q

QMM/MM hybrid modelling ads and disads? 1/2

A

+ info on non-covalenr and covalent intercations: can see whats happening

  • computat expensive
  • time consuming
    (as working at high level of com theory)
332
Q

Case study: DFT in COVID-19 drug modelling, allowed researched insight on…?

A

catalytic mechanism of SARS-CoV-2 enzymes

still cant do full DFT = modelled entire actuive site. specific detailed interactions

333
Q

part 5: peptides- functions and features

describe the 4 structures of protein complexity
- what does shape determine?

A

primary: AA residues, 2D
secondary: coiled alpha helix/ pleated B sheet
tertiary: 3D polypeptide chain
quat: assembles subunits

shape determines function

334
Q

where are disulphide bridges/ bonds found?

A

aa sequence of a protein -> primary struc

335
Q

how are disulphide bridges/ bonds broken down and what into?

A

using B-mercaptoethanol reducing agent.

  • S - S - reduced into -> - SH HS - (free thiols)

breaks covalent, restricted bond

336
Q

how are disulphide bridges/ bonds formed?

A
  • SH HS - (free thiols) oxidised [o] into -> - S - S -

restrict movement
covalent bond

337
Q

protein shape is influenced by what?

A

the backbone peptide bond

338
Q

affect of resonance on backbone peptide bond?

rigidity

A

resonance stabilises AMIDE BOND and creates partial double bond between N and C, preventing/ restricting free rotation… affecting backbone peptide bond by introducing rigidity!

339
Q

what is needed in the amide bonds in backbone peptide bond, in order for it to be stabilised by resonance?

A

p orbital of N must line up with carbonyl pi bond

340
Q

what structures in peptide will be affected by the retsricted rotation and rigidity introduced by resonance in amide bonds?

A

tertiary and quat strucs of peptide

341
Q

what can the partial double bond between N and C created by resonance, give rise to?

A

cis and trans configuartions.

Cis (Z), same
trans (E), different

342
Q

when will peptide bond adopt a cis/trans configuration?

A

in folded state- only 1 isomer favoured. (tert and quat strucs!)

(in unfolded: peptide bond free to isomerize and both cis and trans seen)

343
Q

what configuration: cis/ trans is energetically favoured in peptide bond resonated: C=N and why?

A

trans (diff) as more spaced otu, no steric clash between side chain groups as with cis

344
Q

will rotation still be possible even as amide bond introduces rigidity to the peptide structure?

A

yes- still possible around single bonds: C-NH-C-R

345
Q

what does secondary peptide structure result from?

A

(rotation freedom but)…

the coplanar CO and NH groups that occurs regularly in pep backbone allows for extensive H-bonds… sec struc

346
Q

what type of B sheets in secondary peptide strucs are better?

A

Anti-parallel

  • strong
  • less energy
  • O-NH are lined up for bonding

Parallel
- not lined up for bonding

347
Q

difference between the 2 types of B sheets in secondary peptide strucs?

A

Anti-parallel:
Carbonyl - Amino peptide linkages run in OPPOSITE directions

Parallel:
Carbonyl - Amino peptide linkages run in SAME directions

348
Q

is whole of parallel B sheet planar or not and why?

A

some groups = planar

BUT whole chain is not as pleated shape array = minimise interactions between side chain R groups

348
Q

what bonds join the [] chains in parallel B sheets?

  • different polypeptide chains
  • same polypeptide chains
A

diff: intermolecular H bonds
same: intermolecular bonds

349
Q

whats a B-turn in a polypeptide chain and what does it result in?

A

polypeptide chain is v long… allows peptide chain to bend back on itself

most common type of bending back a chain = B-turn, results in H- bonding between AA n and n+3

350
Q

why is proline AA unique in terms of its secondary struc?

A

destroys regular zigzag order and introduces bend in chain.
not: no NH group in proline! N in cycle instead

expect trand, get cis

351
Q

most common sec struc arrangement in proteins?

describe it and what its stabilised by

A

alpha helix: right handed helix, ordered coil array stabilised by H-bonding between CO and NH groups of SAME chain

352
Q

how many AAs does it take to complete and alpha helix?

A

3.6

353
Q

how do the R groups point/position on alpha helix?
what does this dictate?
whats the exception?

A

on outside … dictates function and interactions

exception = Proline as its often cis not trans.

overall folding of polypep chains into 3D shapes -> tertiary struc of proteins

354
Q

what determines the confirmation/ tertiary structure of a protein?

A

series of intramolecular interactions mediated by the AA side (R) groups
= non covalent ints through covalent disulfide links can also contribute

355
Q

in aq environments, where will hydrophobic (ints arising from R hydrocarbon groups of AAs) be locates and affect?

A

buried inside the protein, influence 3D shape

356
Q

what can tertairy struc of peptide side chains be? (2)

A

non-polar, hydrophobic (internalised in struc) side chains
- e.g. methyl groups, lots of CH3…

polar, hydrophilic side chains (solvated, pointing out)
- e.g. OH, CONH, SH….

357
Q

3 types of bonds in tertiary struc?

A

hydrophobic ints
H-bonds
ionic bonds

affect struc and function

358
Q

where do H- bonds play a role in peptide struc? (2)

A

Secondary: forming alpha helix and B sheets

T/Q: contribute to final shape of proteins

359
Q

what molecules can H bonds form between?

A

H—N/O/S

H-N: amide- carbonyl / amide- imidazole
H-O: hydroxyl- hydroxyl / hydroxyl- carbonyl / amide- hydroxyl
H-S: amide- sulphur (Met)

360
Q

in tertiary struc of proteins, what do ionic bonds form between, under what conditions?

A

positive and negatively charged groups on protein surface in proximity of water mols……
thus at diff pHs when they are prot/deprotonated
= stabilisedo

360
Q

in tert struc, do ionic bonds/ interactions only occur on protein surface?

A

no, also have interactions between oppositely charged groups interior of proteins

361
Q

when and how does quaternary strucs arise?

A

in some proteins… from 2+ protein chains aggregating by enzyme lock + key/ induced fit model.- enthalpy, entropy

consider sterics

362
Q

what are enzymes?

A

biological catalysts,

show high specificity towards rectaants (substrates) - gretaer than most chem reactions

363
Q

how much do enzymes speed up a reaction as opposed to uncatalysed?

A

by factors of 10^6-10^12

364
Q

lock and key of enzymes: where does specificity of enzyme (lock) and sobstrate cmoe from?

A

their geometrically complementary shapes

365
Q

describe enzyme lock and key process

A

specific complementary substrate enters and binds to active site of enzyme (protein)
induced fit (flexes), forms ES complex
sub-> productsm released from active site/pocket

366
Q

name of forces that bind substrate to active site, and what are they same as?

A

non-covalent… same forces that account for conformations of the proteins themselves:
H-bonds, Van der Walls, intercations….

367
Q

how are AAs located in active site?

A

arranged (around AS) so can interact specifically w substrate

368
Q

reactions catalysed by enzymes are _ _?
what does this mean?

and enzymes however vary in _ _… what does this mean?
linked

A
  • stereospecific

e.g. enz that bind proteins only bind L-amino acids
enz that metabolise sugars only bind D sugars

sensitive to AAs present.

  • geometric specificity
    an enz will only hydrolyse C-terminal peptide (example) from all polypeps as long as penultimate reside is not Arg/Lys/Pro and next is not Pro.
    enz + AAs are chiral
369
Q

enzymes III

enzymes vary in geometric specificity (enz + AAs are chiral)
chromotrypsin catalyses hydrolysis of what? (2)

A

peptide bonds and esters

370
Q

case example of enz specificity: protein kinases

what do they do? (explain mechanism)

A

catalyse attachment of a phosphate group to an AA residue in protein substrate

ATPin, ADP out

371
Q

role of phophatases?

in protein phosphorylation

A

reversible process,

enzymes that hydrolyse phosphate grousp from AAs (reverse of protein kinases)

372
Q

protein kinases play a key role in what biological processes?

A 
protein regulation
metabolism
cellular transport
cell signalling 
.....
373
Q

how many protein kinases does the human genome code for?

A

around 500

374
Q

where do protein kinases phosphorylate proteins?

what do they have in common

A

at 3 AA residues:

  • Serine
  • Threonine
  • Tyrosine

(also histidine but uncommon)

OH containing AAs

some kinases are AA/ dual AA specific!!

375
Q

what does phosphorylation of enzymes do in terms of enzymatic activity?

A

alter activity: siwtch on/off

376
Q

by what are kinases turned on/ off? (3)

A

phosphorylation
autophosphorylation
phosphorylation by another kinase

377
Q

whats another way (other than phosphorylation) that protein kinases can be activates?
key word A_ R_

A

by binding to another protein = common in regulating their activity too (allosteric regulation)

378
Q

role of receptor tyrosine kinases and what can they be activated by?

A

phosphorylation of AAs (add phosphate group)

activated by dimerisation

379
Q

what (and how) may cause protein kinases to change?

how is this fixed?

A

disease states- kinases become mutated

inhibition of the protein kinases -> disease treatment
work by switching off signalling cascade

380
Q

what are kinase inhibitors used for and whats the 2 types?

A

treating diseases - where kinases mutated.

work by switching off signalling cascade

  • ATP-competitive kinase inhibitors
  • allosteric inhibitors
381
Q

Part 8: neurotransmitters

NTs/ signalling molecules role regarding multicellular organisms?

A

array of diff molecules (small organic, small peptides, gas) allow coordinated functioning of multicellular orgs

382
Q

how are NTs/ signalling molecules classifies within cell?

4 types

A

based signalling distance from the emitting cell

intracrine:
autocrine:
juxtacrine:
paracrine:

383
Q

the 4 following signalling mols affect what? i.e. where and what signalling distances form emitting cell

intracrine:
autocrine:
juxtacrine:
paracrine:

A

intracrine: within cell
autocrine: affect producing cell via recpetors
juxtacrine: immediately adjacent cells
paracrine: immediate surroundings of cell Endocrine, distant cells, usually through circulation

384
Q

3 different types of signalling molecu;es?

A

cytokines

  • small proteins (5-20kDa)
  • regulate immune system (e.g. IL6)

hormones

  • made by glands, travel in circ
  • classed as AAs/peptides, eicosanoids, steroids

neurotransmitters

  • small, organic
  • released in synaptic cleft of neurone= allow intra communication
385
Q

2 examples of hormones

A

estradiol/ thromboxane A2

386
Q

how does neurotransmitter signalling go? 2 possibilities

3 examples

A

pre-synaptic -> post synaptic neuron

OR REVERSE: post -> pre
e.g. adenosine, GABA, Orexin

387
Q

where are NTs (small organic mols) released and whats their role?

A

rel in synaptic cleft

allow communication between neurons

388
Q

NTs: G protein-coupled receptors (GPCRs)

what are they and what do they consist of?

A

large superfamily of integral membrane proteins…
7 transmembrane hydrophobic helices

allow signal transduction (a, B, Y)

389
Q

how is the GPCR superfamily arranged?

what does it account for

A

6 subfamilies (A-F)

account for min 4% of coding DNA

390
Q

why are GPCRs important in use and development of drug?

A

approx. 34% of commercialised drugs bind to a GPCR

391
Q

order the compartments/ hierarchy: NTs, signalling mols, NTs interacting w Class A GPCRs

A

Signalling mols -> NTs -> NTs interacting w Class A GPCRs

392
Q

NTs interacting w Class A GPCRs are commonly what type of molecules?

A

biogenic amines
e.g. Ad, histamine, serotonine, dopamine, ACh
(simialr strucs)

393
Q

adrenaline (Ad) is a NT that does what and how is action exerted?

A 
stimulates sympathetic nervous system (fight/flight)
interacts w class A GPCRs calles adrenergic receptors
394
Q

what are adrenergic receptors?

A

class A GPCRs where Ad interacts and exerts effect

multiple subtypes: (a1, a2, B1, B2… )

395
Q

adrenergic transmission: how to acheive agonists and antagonists?

A

make mols that are selective for certain subtype = get tissue specific effects

  • have catechol
  • sec amine
  • specific stereoselectivity (chiral centre)
396
Q

NTs: GPCRs as drug targets

difference in affinity vs efficacy?
and what do both contribute to?

A

affinity = conc to acheive biological repsonse

efficacy = pharmacological effect

BOTH= contribute to potency

397
Q

what 4 agents affect % of adrenaline response, order them full -> lowest response at 10^-6

A

agonist
partial agonist
antagonist
inverse agonist

398
Q

adrenergic agonists: case study 1 Salbutamol

selective agonist for which receptors, and what causes selectivity?

A

B2 sleective agonist.
used as brochodilator in asthma treatment

tert-butyl group (4 C alkyl) with logP +0.7 (hydrophobic, lipophilic)

399
Q

adrenergic agonists: case study 2 Salmeterol

has increased selectivity towards what receptor?

A

same as salbutamol: B2

400
Q

adrenergic agonists: case studies..

Salbutamol and Salmeterol are same apart form a structure modification. no longer has tert-butyl alkyl bit, instead long alkyl change w benzene at end

how does this modification change therapeutic window?

A
  • slow onset as increased lipophilicity (LogP = +3.9)
  • extended duration

only [really] an ext. of alkyl chain … -> more useful drug!

401
Q

adrenergic agonists: case study 1 Pindolol

whats it commercialised for and what selectivity?

A

beta blocker: treat high BP/ angina

lipophilic/ lacks catechol HBAs/ HBDs

no selectivity between B1/B2 subtypes

402
Q

adrenergic agonists: case studies

Pindolol-> Atenolol.
next gen beta blocker is selective for what?
from what changes?

A

B1 subtype

treat high BP, angina, AND acute MI

struc change=–> haveHBA and HBD in aromatic ring

403
Q

GPCR therapeutic opportunities

what are the 2 NT/ enzyme therapeutic targets?

A

Serotonin (transportation)

MAOs

404
Q

therapeutic target 1: Serotonin transportation

what does serotonin do?

A

= an NT that has roles in central and peripheral nervous systems.

  • regulates mood, behaviour
  • other phyiological funcs
405
Q

therapeutic target 1: Serotonin transportation

effect of increasing the serotonergic tone?

A

useful for treating various conditions e.g. depression

406
Q

what 3 membrane proteins used in transport of Serotonin?

in serotonin transporter

A

K+
Na+
Cl-

407
Q

what is fluoxetine and how does it do its job?

A

SSRI
increases serotonergic tone by…
binding (blocking) activity of serotonin transporter, hormone stays for longer ☺

408
Q

where does fluoxetine (SSRI) get stuck and hows this beneficial?

A

in synaptic cleft as NT…
as reuptake blocked, cant enter serotonin transporter

effect of serotonin is enhanced and lasts longer

409
Q

therapeutic target 2: MAOs

what do mitochondrial membrane flavin-containing enzymes catalyse?

A

the oxidative deamination of monoamines (Ad, serotonin, dopamine….)

410
Q

what are the different isoforms of MAOs and how do they differ?

A

MAO-A: Ad, dopamine, serotonin
MAO-B: trace amines (e.g. tyramine)

each one with specific substrate specificity

411
Q

MAOs (therapeutic target 2)

whats FAD+ used as in the process and what happens to it?

A

in oxidative deamination process as a cofactor (H acceptor)

becomes reduced to FADH2

look at image on slide 206

412
Q

what are unusually high/ low levels of MAOs associated with?

A

pathological conditions: depression, schizophrenia, addiction, migraine….

413
Q

how were MAOs changed form 1st -> 2nd generation?

e.g. Nialamide -> Selegiline (for parkinsons)

A

1st gen: not specific for a particular MAO isoform

next gen -selective- inhibitors developed

414
Q

modelling small and large mols together (CADD)

3 differences in molecular dynamics vs QM/MM approaches?

A

molecular dynamics

  • faster as need less comp power
  • less accurate as using Newtonian mechanics
  • treats AS as grid on which substrate can move/ interact

QM/MM approaches

  • slower and expensive as need more comp power
  • more accurate as using quantum mechanics component
  • treats protein using MM and substrate (drug) using QM

C2 (5 decks)

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