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Chem390 > Proteins As Drug Targets > Flashcards

Proteins As Drug Targets Flashcards

0
Q

What are the 5 main components of a protein amino acid?

A 
Amino group
Alpha carbon
Alpha hydrogen (attached to alpha carbon)
Carboxylic acid group
Variable side chain
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1
Q

What are amino acids?

A

The building blocks of proteins

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2
Q

Which amino acid is achiral?

A

Glycine as its r group is a H

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3
Q

Which amino acids are natural?

A

L amino acid and D amino acid

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4
Q

Which amino acids are not found in proteins?

A

Beta amino acid where the amino and carboxy groups differ by two C
Gamma amino acid where the amino and carboxy groups differ by three C

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5
Q

What are zwitterions?

A

Dipolar ions.
Proteins exist in this state in physiological pH

The cationic form exists in pH less than 7 (acidic environments)
The anionic form exists in pH greater than 7 (basic environments)

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6
Q

What are the different types of amino acids side chains?

A

Hydrophobic: where the R group is either a methyl or cyclic compound. E.g. Alanine (methyl group), phenylalanine (methyl+benzene ring)

Polar neutral: where the side group has no charge but is polar. E.g. Asparagine (amide group), serine (methanol group)

Polar charged: where the side group is polar AND has a charge. E.g. Aspartic acid (charged carboxyl group), Lysine (charged amino group)

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7
Q

How are the amino acid side chains significant?

A

The hydrophobic side chains cluster to avoid aqueous environment. (Val, Leu, Ile, Met)

The aromatic side chains can undergo stacking (Phe, Trp, Tyr)

Side chains can be chemically reactive undergoing glycosylation, disulphides bonds. (Cys, Ser, Thr)

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8
Q

What is significant about proline?

A

Restricts conformation, and is commonly found in bends and kinks in proteins.
Proline is the only amino acid which is a secondary amide. This allows it to form a cis peptide bond (the energy difference between cis and trans peptide is not as significant in peptide)

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9
Q

What are the roles of polar neutral side chains?

A

They are often found on the protein surface as well as in the interior, and in the active site.

They mostly remain hydrogen bonded

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10
Q

What are the roles of polar charged side chains?

A

These are often found in the active site of enzymes
The COOH terminal of Asp and Glu are nearly always deprotonated.
The NH terminal of Lys, Arg are nearly always protonated

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11
Q

Why is histidine in particular, likely to be found in the active site of enzymes?

A

The pKa of histidine is similar to the pH of physiological pH and can therefore switch between the charged and uncharged state easily

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12
Q

What are peptides?

A

Alpha amino acids which are linked in a defined order and linked by a secondary peptide bond which is formed via a condensation reaction

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13
Q

What is the direction of the peptide chain?

A

From the n terminus to the c terminus

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14
Q

What does peptide synthesis require?

A

The specific formation of amide bonds
Because there are lots of amino and carboxy groups to react with, this could result in a mixture of products.

Thus we need to protect the amino and carboxy groups we don’t want to react with with protecting groups.

The reaction then needs to be activated and coupled.

The product is deprotected and forms the amide

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15
Q

How is Ala-Val made?

A

Protect the NH2 group of alanine and the carboxyl group of valine
Add in a coupling agent which facilitates the condensation reaction to form the new amide bond.
Then deprotect the product

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16
Q

What are the two main types of peptide synthesis in solution?

A

Linear/stepwise synthesis

Fragment condensation/convergent synthesis

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17
Q

What is linear synthesis?

A

The incremental addition of one amino acid at a time
The two amino acids are first protected, activated and coupled and then deprotected. This cycle continues as each amino acid is added to the chain

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19
Q

What is fragment condensation?

A

Construction of the target structure by assembly of separately made intermediate fragments

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20
Q

What are the advantages and disadvantages of linear chain synthesis?

A

+ smaller risk of racemisation

  • Low net yield of final product
  • Slow as you have to add amino acids one at a time.
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21
Q

What are the advantages and disadvantages of fragment condensation synthesis?

A

+ Better overall yield
+ faster synthesis
- poor solubility of larger protected intermediate segments
- Increased risk of racemisation (chiral centres present.)
- low coupling rate with a concurrent risk of side reactions.

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22
Q

What are the two main classes of protecting groups?

A

1) intermediary

2) semipermanent

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23
Q

What are intermediary protecting groups mainly for?

A

protecting amine and carboxyl functions

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24
Q

what are semipermanent protecting groups mainly for?

A

protecting side chains of certain amino acids

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25
Q

Why are protecting groups used?

A

enables selective cleavage
prevents racemisation
allows favourable stability and characterisation of intermediates
allows favourable solubility of protected amino acid components

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26
Q

What are examples of protecting groups used for amines?

A

Tert-butyloxycarbonyl (Boc) This can easily be removed with acids like trifluoroacetic acid (TFA)

9-Fluorenylmethyloxycarbonyl (Fmoc) which can be easilyremoved with an organic base

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27
Q

Why is carboxyl protection necessary?

A

The carboxyl group must be unreactive during peptide bond formation, but easily removable for chain elongation.

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28
Q

How does carboxyl protection work?

A

It is standard ester formation achieved with mild hydrolysis (NaOH and acid) or catalytic hydrogenation (Hydrogen and Pd)for benzyl esters

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29
Q

What does the choice of protection of side chain groups depend on?

A

Whether Boc or Fmoc is used to protect the alpha amino group
e.g. if the alpha amino acid is protected by Boc, the side chains should be protected by Fmoc as the Boc will need to be removed to extend the chain

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30
Q

Why are coupling reagents used?

A

to enhance the reactivity of the carboxylic acid group by facilitating amide bond formation
e.g. Dicyclohexlcarbodiimide or DCC

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31
Q

What is solid phase peptide synthesis?

A

pioneered by Merrifield.
primarily for the product of large polypeptides

The C terminus is linked to the solid phase and the peptide is constructed from the C to the N terminus on chloromethylated polystyrene beads.

Merrifield originally used Boc chemistry as protecting groups

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32
Q

Why has Boc chemistry in the solid phase synthesis been replaced?

A

Boc requires harzardous reagents like HF so nowadays we use Fmoc chemistry as we can cleave this with TFA and it is easy and more convenient

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33
Q

Why is solid phase synthesis used?

A

There may be other side reactions and products which can be hard to separate. This will require purification of the intermediates, making solution phase synthesis very time consuming. In solid phase synthesis you can just wash off the side products you don’t want.

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34
Q

Is DCC the only coupling reagent used?

A

No there are other types, but DCC is the most common

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35
Q

What must be done at the end of solid phase synthesis?

A

The peptide must be cleaved from the solid phase which is another reaction. This is the fmoc reaction we have replaced boc with.

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36
Q

What is the process of solid phase synthesis?

A

The process is similar to incremental linear addition except that this is on the solid phase. Both amino acids are initially protected with Fmoc. This is then removed from aa1 while aa2 is activated

The C terminus of aa1 is attached to the Resin. It is then mixed with aa2 as well as an activator. Fmoc is then removed from the now dipeptide.

Side products are soluble and filtered off by washing.

aa3 with fmoc and activator attached is then reacted with the dipeptide. Then fmoc and activator is removed. The resin is then also cleaved off.

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37
Q

What are the different levels of protein structure?

A

Primary - sequence of amino acids
secondary - regular arrangements of polypeptide backbone stablised by intramolecular hydrogen bonds
tertiary - assembly of secondary structural elements
quaternary - proteins consisting of more than one poly peptide chain like haemoglobin

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38
Q

What is the primary structure and why is it important for a protein?

A

the amino acid sequence listed sequentially from the n terminus to the c terminus in either the 1 or 3 letter codes

contains all of the info necessary for folding the petpide chain into its native structure

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39
Q

What is secondary structure and why is it important for a protein?

A

arrangements of the amino acids into a stable polypeptide backbone. This is held together by hydrogen bonds

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40
Q

What is the nature of the peptide bond like?

A

It is planar and exists mostly in the tans conformation (except for proline which exists in the cis peptide bond)

There is a partial double bond character, hence there is restricted rotation around the N-Ca and Ca-C bonds

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41
Q

Why is proline in cis conformation?

A

Steric hindrance between R groups disfavours the cis peptide bond except for proline as it has a cyclic R group so it exists in cis conformation

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42
Q

What is special about the peptide bond?

A

peptides are flexible and adopt a large number of conformations in solution. This flexibility arises due to the freedom of rotation around the N-Ca and Ca-C bonds.

The backbone conformation is determined by those torsion angles which in turn define secondary struture

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43
Q

What are the most common secondary structures?

A

alpha helix and beta sheet. These have characterisic values of the torsion angles

44
Q

What are torsion angles?

A

The angles of rotation or freedom of rotation around the N-Ca bond and Ca-C bond.
Φ (phi) = rotation around N-Ca bond
Ψ (psi) = rotation around Ca-C bond

45
Q

When are possible conformations about the a-C between 2 peptide planes forbidden?

A

When Φ=0 degrees and Ψ=180 degrees due to steric crowding between the carbonyl oxygens.

46
Q

What happens in forbidden conformations?

A

any non bonding interatomic distance is less than its corresponding van der waals distance.

47
Q

What is the Ramachandran plot?

A

A plot of Φ and Ψ from known protein structures. The sterically favourable combinations are the basis for preferred secondary structures

Light grey region - no steric hindrance (this is when both Φ and Ψ are around 60 degrees)
Dark grey region - some steric hndrance but still possible to bring about angles
White regions/all other areas - completely forbidden

48
Q

What are the torsion angles for a right handed alpha helix?

A

Φ -57 and Ψ -47

49
Q

What are the torsion angles for left handed alpha helix?

A

Φ ~+60 and Ψ ~+60

50
Q

What are the torsion angles for a 3 10 helix?

A

Φ -49 and Ψ -26

51
Q

What are the torsion angles for a pi helix?

A

Φ -57 and Ψ -70

52
Q

What are the torsion angles for an antiparallel betasheet?

A

Φ -139 and Ψ +135

53
Q

What are the torsion angles for a parallel betasheet?

A

Φ -119 and Ψ +113

54
Q

What are the torsion angles for a hypothetically fully extended structure?

A

Φ and Ψ both = +180

55
Q

What does secondary protein structure arise from?

A

The rigidity of the amide bond and

The conformational preference of amino acid residues which are near each other in the linear sequence.

The ability of amino acid residues to form hydrogen bonds between donor and acceptor atoms within the peptide chain

56
Q

How does the rigidity of the amide bond contribute to secondary protein structure?

A

The amide bond has partial double bond character and is planar, and nearly always in trans configuration

57
Q

How does the conformational preference amino acid residues affect secondary protein structure?

A

This is associated with the side chain of the amino acid. Met prefers alpha helix, while Pro prefers beta turn

58
Q

How does the ability of amino acid residues to form hydrogen bonds affect secondary protein struture?

A

The most common secondary structures in protes are the alpha helix, the beta sheet and the beta turn.

These elements satisfy a strong hydrogen bonded network within the geometric constraints of the bond angles

59
Q

What is the alpha helix?

A

This is characterised by hydrogen bonds between the CO of the ith residue and NH of the i+4th residue.
This results in 13 atoms joined together in a hydrogen bonded network.

Always right handed (clockwise) turning alpha helices are found in proteins

60
Q

What is the beta sheet?

A

Actually composed of 2 or more beta strands and occurs when the polypeptide chain adopts an almost fully extended zig zag conformation

Beta sheets can be formed by parallel or antiparallel arrangement of the individual beta strands

61
Q

Which beta sheet arrangement is more common?

A

Antiparallel.
The strands have the opposite orientation with respect to their N and C termini

Whereas in parallel arrangements (less common) the strands have the same orientation with respect to their N and C termini)

62
Q

What is the beta turn?

A

Most proteins have compact globularshapes
These arise due to reversals in the direction of polypeptide chains which are mediated by the beta turn structural unit.

The C=O residue of i is hydrogen bonded to the N-H group residue of i+3

63
Q

What is the tertiary structure and how is it important to the protein shape?

A

The 3d spatial arrangement of all the secondary structural units in a protein.
Here amino acids from far apart in the primary sequences can interact

Stabilised by relatively weak non covalent bonds.

64
Q

What are the differences in the interactions between the secondary and tertiary structures of amino acids?

A

In secondary structures, the polypeptide chain is determined by the short range interactions and structural relationship of amino acid residues

whereas in tertiary structure the polypeptide chains are conferred by longer range interactions

65
Q

If the interactions between amino acids that stabilise tertiary structure are weak, how does tertiary structure remain together?

A

There are many interactions, and this large number provides the stabilising force which holds the protein together in its folded 3D structure

66
Q

Why is the tertiary structure referred to as the protein’s native conformation?

A

The folded structure is the biologically active form of the protein

67
Q

What is present on the surface of a protein generally?

A

Polar and hydrophilic amino acids which allow the protein to interact with the aqueous environment of the cell

68
Q

What is present in the interior of a protein generally?

A

Hydrophobic amino acids, and usually also contains the protected microenvironments like the active site (if eznyme) or the binding domain (if receptor)

69
Q

What is quarternary structure?

A

confined to proteims that are assembled from a number of protein subunits

70
Q

which level of structure is most important for drug action?

A

tertiary

71
Q

What are the spectroscopic techniques used to characterise peptides and proteins?

A 
Circular Dichroism 
Nuclear Magnetic resonance
Mass spectrometry
Analytical ultracentrifugation
Xray crystallography (for solid state structure)
72
Q

What is Circular Dichroism?

A

Plane polarised, composed of two circularly polarised vectorial components (Right handed is polarised clockwise and left handed is polarised anticlockwise) are shown onto the sample and may not be equally absorbed by an optically active sample .

absorbance is method and an equation is used to calculate the extinction coefficient

CD is essentially the difference between the extinction oeffcieints for left and right circularly polarised light.

CD is normally reported in molar ellipticity
Useful for polypeptide secondary structure determination

73
Q

What is nuclear magnetic resonance?

A

developement of high field NMR methods enables structure determination of moderately sized proteins (<30kDa) in solution

H1 is the most common nucleus studied

There are several useful parameters to note: chemical shift, coupling constant, through-bond and through-space connectivities

74
Q

How is NMR used to determine protein structure?

A

Resonances in NMR spectrum are assigned to individual protons
This can determine which protons are close to each other in space ( from the nuclear overhauser effect)

75
Q

what are the two types of proton NMR used and what can they tell us?

A

One dimensional proton NMR: gives info about chemical shift dispersion, line broadening, coupling constant.

Two dimensional proton NMR: (homo and heter nuclear)
Gives info about through bond (COSY, TOCSY) and through space connectivities (NOESY)

76
Q

what is proton NMR useful to study for?

A

structure-activity relationships, ligand binding etc.

77
Q

What is mass spectrometry used for?

A

It is highly sensitive and enables the identification of trace amounts of chemicals
exhibits both molecular and fragment ions.

78
Q

Where does fragmentation primarily occur?

A

Fragmentation occurs primarily at peptide or glycosidic bonds, generating a ladder of fragments
y-ions are C terminal fragments, b-ions are N terminal fragments

79
Q

What does the mass difference between certain peaks correspond to?

A

THe mass difference between certain peaks correspond to the loss of a single amino acid

80
Q

What can the peptide sequence be reconstituted by?

A

a ladder walk. This is done by measuring mass difference between successive masses for specific types of ions

81
Q

How are bigger proteins identified?

A

by analysing their tryptic fragments. This is known as the peptide mass fingerprint, followed by database searches however the protein must be very pure

82
Q

What potential therapeutics are instore for peptides?

A

Peptides generally contain <50 amino acids and are less than 5000 Da.
They have a high degree of secondary structure but lack a defined tertiary structure.

They have potential antimicrobial properties and some are also biolgoically active

83
Q

What are examples of antimicrobial peptides?

A

components of the innate immune system (in vertebrates, plants, microbes)

short, linear, helical, cationic (most abundant)

e.g. Magainin, Melitin, Defensin

84
Q

What are other biologically active peptides?

A

Neuropeptide Y, Substance P, Oxytocin, Vasopressin, Enkephalins

85
Q

what are the advantages of peptide use for therapeutics?

A

increased specificity
lower toxicity
smaller size therefore better storage and tissue penetration

86
Q

What are the disadvantages of using peptides for therapeutics?

A

Lack in vivo stability due to protease sensitivity
Conformational flexibility - not rigid enough to retain shape
Hard to cross blood brain barrier

87
Q

Why must peptide structure be controlled?

A

Peptide-protein non covalent interactions control and modulate cellular function, intracellular communication, immune response and info-transduction pathways.

These are used by hormones, neurotransmitters, antigens, cytokines and growth factors.

Structure based drug design aims to combine biological and structural properties to create novel compounds with enhanced bioactivity

88
Q

What does peptide design involve?

A 
Use of unnatural amino acids
cyclisation
peptidomimetics
peptide bond and hydrogen bond surrogates
use of drug delivery technology.
89
Q

How is the conformational flexibility of proline overcome?

A

By adding the pyrolidineg ring which reduces the available conformational space of a proline containing peptide. This forms a bend (turn) in peptides and proteins
The torsian angle Ψ is reduced to -70+20 degrees

90
Q

What is alpha aminoisobutyric?

A

Where the alpha proton in alanine is replaced by a methyl group.
Φ and Ψ are retricted to -57,-47 and +57,+47
This is a strong helix promotor
Aib containing peptide is found to inhibit HIV-1 entry

91
Q

What are some higher homologues of Aib?

A

Cyclic and Acyclic side chains.
The cyclic chain favours helix formation whereas the acyclic favours exended structure,

There is increased steric hindrance in both and further restriction in the allowed Φ and Ψ values.

92
Q

what are N-alkylated amino acids?

A

The amide proton of an amino acid is substituted by an alkyl group
This changes the hydrogen bonding pattern. There is only a marginal energy difference between cis and trans geometry.
Φ and Ψ constraint produces peptides with high selectivity and potency

93
Q

What are alpha-beta dehyro amino acids?

A

Found in eznymes although infrequently.
Limited to experimental data on peptides/proteins with dehydro residues.
Does help to stabilise beta turns in short peptides and the helix in long peptides.

94
Q

What are D amino acids?

A

These occur in beta turns and help to enhance proteolytic stability. They are extensively used in the design of biologically active peptides.

95
Q

What are omega amino acids?

A

Where additional methylene groups are added between the amine and COOH groups.
This has the potential to enhance conformational flexibility and are exampels of peptides with increased biostability

96
Q

What are peptidomimetics?

A

Compounds (like Captopril) which mimc the biological activity of peptides while offering the advantages of increased bioavailabiltiy, biostability and bioselectivity against the natural biological target of the parent peptide.

A lead optimisation with the desired peptide as the lead compound.

Modifications to minimise or elminate the undesired properties.

97
Q

What sort of modifications are done to peptidomimetic compounds?

A

modify as much of the peptide backbone as possible with non peptide fragmentswhile maintaining the AA side chains

This makes the peptide more lipophilic

Amide bond isosteres like an ester, alkene or other suitable structures

enhances protease stability

98
Q

What are branched peptides?

A

Not naturally occurring but can be synthesised in the lab.

these are stable to proteases

99
Q

What are antimicrobial peptides?

A

Components of the innate immune system. Short, linear, helical, cationic with braod spectrum activity ( they protect against gram +ve and gram -ve bacteria, fungi, viruses etc.

They are classified into 5 major groups

100
Q

What are the 5 major groups which AMPs are classified into?

A 
alpha helices
beta sheets
composed of rare and modified amino acids
cysteine rich
rich in regular amino acids
101
Q

What are the mode of action of AMPs?

A

Disrupt cell membranes and cause cell lysis via two models:

Barrel Stave and Carpet model

102
Q

What is the barrel stave model?

A

The formation of transmembane channels or pores by bundles of AMP alpha helices

103
Q

What is the capret model?

A

Where AMPs bind the surface of the membrane and cover it in a carpet like manner

104
Q

What influences membrane interaction of AMPs?

A

secondary structure, overall charge and hydrophobicity

105
Q

What other modes of action are possible for AMPs?

A

inhibition of protein synthesis

degradation of proteins necessary for DNA replication

106
Q

Why are peptide dugs developed to target resistant bacterial biofilms?

A

There are matrix embedded microorganisms like pseudomonas aeruginosa which causes CF. they can grow on live or inert surfaces and are resistant to conventional antibiotics as they do not penetrate biofilms. Peptide drugs, if developed to target these resistant biofilms could provide a nice alternative

107
Q

What are examples of AMPs targeted at plant pathogens?

A

fire blight of pome fruits

PSA of kiwigold

Chem390 (21 decks)

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