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Chemistry and Disease > Modern approaches to drug discovery > Flashcards

Modern approaches to drug discovery Flashcards

1
Q

What is the full name for COX enzymes and what reaction to they catalyse?

A

Cycloxygenases, catalyse the production of prostaglandin precursors from arachidonic acid.

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2
Q

What do prostaglandin hormones mediate?

A

A range of physiological effects, eg inflammation and stomach lining renewal.

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3
Q

Describe the differences between the 3 COX isoforms

A

COX1: constitutive, found in most mammalian cells.
COX2: induced mainly in activated macrophages at the site of inflammation- most important for therapeutics.
COX3: recently discovered in the nervous system.

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4
Q

Name and explain some drugs which bind to the COX isoforms.

A

Aspirin and ibuprofen- non-selective bind to all COX isoforms, problems with prolonged use (stomach lining degradation).
Paracetamol- main action through COX-3 nervous system (only recently discovered).

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5
Q

Identify the main general steps for the drug discovery process

A

Decide on a disease, identify a target, find hits, optimise the hits, prepare for clinical trials, clinical trials, recover the investment!!

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6
Q

Describe some ideal properties of a drug

A

Stable (long shelf life), convenient (a pill), distribution all around the body, excretion (after an appropriate time), specificity, good affinity, tolerability, works against resistant bacteria, bioavailability (ADME).

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7
Q

Define: Ki, Kd, IC50 and GI50

A 
Ki= inhibition constant (Michaelis-Menton derivation).
Kd= dissociation constant.
IC50= inhibition constant- conc of ligand where you get 50% inhibition.
GI50= growth inhibition.
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8
Q

What does ADME stand for and what term covers these effects?

A

ABSORBED into system.
Gets to the site of action- DISTRIBUTION.
Remains active- METABOLISM.
Is circulated not EXCRETED.

Pharmacokinetics

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9
Q

What takes place during phenotypic screening?

A

Set up an assay to see if the compound has an effect on the growth of the organism.

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10
Q

Define angiogenesis

A

The formation of new blood vessels (crucial to the growth of solid tumours).

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11
Q

Name the natural product compound isolated from Aspergillus fumagatus which has anti-cancer activity

A

Fumagillin

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12
Q

Describe and explain 3 genetic tools for target ID

A

Knock out animals: generate animals with a gene missing- problems with lethal knock outs and development adjustment.
RNA: introduce ds mRNA into cell- induces mechanisms that remove the message, temporal and sometimes spacial knock-down in mature organism (essentially selectively removes RNA for a particular protein).
CRISPR: a new technique where a gene (or RNA) is marked for destruction exploiting an internal ‘clean up’ process.

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13
Q

Describe the steps involved when cells are infected by the influenza virus

A

Virus moves through mucus in respiratory tract, virus (encounters receptor) enters cell, virus breaks up to reveal RNA, host cell replication machinery produces copies of RNA and viral proteins using viral error prone polymerase (replication and reassembly), new virus breaks out of the cell and goes on to infect other cells.

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14
Q

What are some potential drug targets in inflluenza?

A

Immunisation (vaccination), cell entry (inhibit haemagluttinin on virus- recognises cell surface receptor- shallow recognition site so difficult to block), virus breakup (large cavity which is difficult to block specifically, m2 ion channel responds to pH change), viral RNA polymerase, virus release from cell (key enzyme= neuraminidase, also helps virus move through mucus).

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15
Q

Name 2 locations where sialic acid is cross-linked?

A

In mucus and on glycoproteins.

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16
Q

How does the human and avian sialic acid/galactose linkage differ?

A

Human= alpha 2,6 and avian= alpha 2,3

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17
Q

What position does neuraminidase hydrolyse on sialic acid?

A

The 2 position.

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18
Q

Describe the structure of neuraminidase

A

Beta-sheets in a barrel structure, binding site with key amino acids for hydrolysis is buried in a cavity in the protein which is too small for antibodies to recognise (can recognise the surface but not the key amino acids).

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19
Q

Describe the mode of action of relenza

A

Sialic acid substrate analogue (neu-5Ac-2-en), mimics the transition state in hydrolysis (lost O groups) and is a weak non-selective inhibitor (binds to any sialic acid enzyme).

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20
Q

What is the aim of lead optimization?

A

Improve drug like properties: affinity for target (nm), specificity for target, physico-chemical properties, ADME properties, resistance issues.

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21
Q

What are the 2 main non-covalent interactions?

A

van der Waals (attractive and repulsive) and electrostatics.

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22
Q

How do you do a protein GRID calculation and what is the aim?

A

Construct a grid over the protein active site, place a probe (with vdW andcharge) at each grid point and calculate energy of intraction, store the energy on the grid, contour to highlight the regions of space at which particular functional groups/atoms may want to be.
Want to probe the surface of the protein for hotspots of functionality.

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23
Q

Describe the development of the design of relenza

A

The crystal structure of relenza bound to neuraminidase identified a large negatively charged pocket at the back of the sit which could have a good interaction with a positive group. Changed to 4-amino Neu-5Ac-2-en which had sub micromolar affinity. Changed to 4-guanidino Neu-5Ac-2-en which had nm affinity, was selectivity and inhibited the rate of infection in vivo.

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24
Q

What are the 2 types of resistance?

A

Bacterial and viral.

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25
Q

Why is relenza good against resistant bacteria?

A

As the mechanism is not affected by the resistance mutation.

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26
Q

What are the bioavailability issues with relenza?

A

Very polar so can’t be administered orally- diskhaler. Needs to be used within 48 hours of infection- is difficult to diagnose flu that quickly, when do you know you have the flu?

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27
Q

How does tamiflu differ from relenza and what are the advantages of tamiflu?

A

Removed/substituted OH groups hence less polar so can be administered orally.

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28
Q

What was so deadly about the 1918 Spanish Flu and why was this?

A

Was particularly virulent and triggered anaphalactic shock- lungs filled up with fluid so you essentially drowned. The 1918 haemagluttinin is avian in character but had mutations to allow binding to alpha 2,6 ie human cells.

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29
Q

What is the general function of aspartyl proteases?

A

Selectively degrades proteases.

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30
Q

What does activation of the angiotensin2 receptor lead to?

A

Causes vasoconstriction (and other effects) which leads to hypertension.

31
Q

What does AIDS stand for?

A

Acquired immune deficiency syndrome.

32
Q

Describe the life cycle of HIV

A

Virus moves through the blood stream to lymphocytes, virus enters cell and breaks up to reveal RNA, RNA is reverse transcribed to DNA (rather than replicated to make new viruses), ‘viral’ DNA is integrated into host DNA and the cell enters the dormant phase. Some event causes ‘viral’ DNA to be transcribed, host cell replication machinery produces copies of RNA and viral polyprotein, new virus breaks out of the cell, virus matures to become INFECTIVE and goes on to infect other cells.

33
Q

Describe some of the potential targets in HIV

A

Immunisation- innoculate with ‘dead’ portions of the virus (but the enzymes mutate rapidly).
Cell entry- block inhibition between gp120 and CD4 (large recognition site, difficult to block).
Reverse transcriptase- has a number of different binding sites that can be inhibited.
Integrase (integrates DNA into the host).
Viral maturation- done by HIV protease.

34
Q

Explain the mutation which led to a sub-population of prostitutes not contracting AIDS

A

They had an inactive CCR5 protein (single mutation)- a chemokine receptor involved in viral entry,

35
Q

What are some of the disadvantages of high-throughput screening (HTS)?

A

Large and expensive process that depends on having the right compound in the library.

36
Q

What are GPCRs?

A

G protein-coupled receptors- largest group of receptors that are involved with transmitting signals from outsdie to inside the cell.

37
Q

What is Ozanimod currently in clinical trials for?

A

Multiple sclerosis (ulcerative colitis).

38
Q

Describe the hallmarks of cancer

A

Self-sufficiency in growth signals (doesn’t require external signal), insensitive to anti-growth signals, limitless replication, evading apoptosis, sustained angiogenesis, reprogramming energy metabolism, evading immune destruction, tissue invasion and metastasis.

39
Q

Describe some of the potential targets in cancer

A

Non-specific DNA replication (radiation therapy and chemotherapy), targetted therapies (antibodies with warheads), specific pathways (Bcr-Abl chromosomal abnormality in CML, block growth factor receptor from outside, EGFR kinase, cell cycle control), pleiotropic treatments- hits a number of pathways (hsp90).

40
Q

What is hsp90 and how is it important in cancer cells?

A

Molecular chaperone involved in protein folding/protection, is important for cells under stress. Cancer cells are under stress due to lots of unstable, mutated proteins, so hsp90 in overexpressed in tumours to help unstable proteins remain folded.

41
Q

Name a natural product inhibitor of hsp90

A

Geldanamycin

42
Q

What is virtual screening?

A

Take compounds and do calculations to see whether the compound will fit into the active site and bind with favourable energy.

43
Q

What are some of the limitations of virtual screening?

A

Need a crystal structure, potential conformational change on ligand binding (best with rigid binding site), potential solvent molecule interactions, only approximate calculations, need right compounds in virtual library.

44
Q

What is a key part of the N-terminal domain of hsp90?

A

ATP binding site, network of key, solvent-mediated hydrogen bonds.

45
Q

What is a potency handle?

A

A chemically trackable VECTOR for optimisation.

46
Q

What are pharmacodynamic markers used for and give an example

A

Used to check the compound is working on the target ie what the drug does to the cell.
Example= anti-hsp90 compound: would expect a decrease in client proteins, inhibition of chaperone leads to unfolded client which is degraded.

47
Q

How do you run a Western blot?

A

Generate an antibody against the target protein of interest, run down cell content to get to separate by molecular weight, stain the gel with the antibody- modify to get a readout.

48
Q

What are some of the issues with the hsp90 lead series?

A

Potency, selectivity, low solubility, poor pharmacokinetics, p450 inhibition (particularly 3A4).

49
Q

Describe the structure-guided lead optimisation of hsp90

A 
Structure of hits in binding site used to guide optimisation, retained key hydrogen bonds, increased interactions with the protein (additional H-bonds), pyrazole to isoxazole modification to enhance efficacy (no longer H-bond donor, only acceptor, changes solvent interaction). 
MORPHILINO added (solubiliser/permeabiliser) to increase solubility whilst giving a good balance of hydrophobic/philic properties so can also cross membranes.
Isopropyl added on- modulates glucuronidation and increases affinity.
50
Q

What is a biophysical method for measuring thermodynamics?

A

Isothermal titration calorimetry

51
Q

What is a biophysical method for measuring kinetics?

A

Surface plasmon resonance (SPR)

52
Q

Give an example of a virtual screening product for hsp90

A

AUY922

53
Q

What are the advantages of using fragments for screening (rather than HTS)?

A

Hits from fragments: find small parts that bind, then grow/merge fragments to create hit compound.

54
Q

Name and describe the ligand-based NMR used for detecting fragments

A

Saturation transfer difference (STD): measure 1D spectrum of ligands, saturate protein with ligands- saturation energy transfers by spin diffusion to any compound that transiently touches the protein, 1D spectrum of such ligands reduces- TAKE DIFFERENCE- aka binding to another site on the protein.
Essentially looking at changes in the NMR spectrum (which signals have been reduced) upon saturating the protein with ligands.

55
Q

Name 2 of the fragment hits identified from virtual screening with hsp90

A

Resorcinol andamino-triazine

56
Q

What methods can be used for finding fragments?

A

Enzyme assays (usually can’t withstand high conc needed for weak binding), SPR, protein based NMR (15N labelled protein- each peak is an amide- detect change in protein environment.

57
Q

Describe the role of the Bcl-2 family of proteins

A

Major role in mediating apoptosis- the ‘intrinsic’ apoptosis pathway is via the proteins Bax/Bak: localised in the mitochondrial membrane and remain inactivated through binding to other Bcl-2 family members. The BH3 peptides are produced when a cell receives signals to enter apoptosis- such as unstable oncogenic cells; these BH3 peptides bind to Vdac2 etc =, releasing Bax/Bak to form pores which release cytochrome c into the cell, which with APAF1 forms apoptosome which activates caspase cascade, leading to cell destruction.

58
Q

Explain the up-regulation of Bcl-2 family members in various cancers

A

Sequester (bind to/soak up) BH3 peptides to prevent apoptosis, different selectivity of BH3 peptides for each Bcl-2 family member, a BH3-mimetic will release the BH3 peptides to induce apoptosis (usually measured as caspase induction or PARP cleavage).

59
Q

Describe the general structure of Bcl-2

A

Bundle of helices, protein surface is a long, hydrophobic cleft.

60
Q

What were the disadvantages with the 1st Bcl-2 clinical candidate ABT737?

A

Not very soluble so difficult to administer and not totally selective for Bcl-2- also inhibited another protein (Bcl-xL).

61
Q

Why are morphilino groups commonly added on to drugs?

A

To increase solubility.

62
Q

How can you monitor the cytochrome p450 metabolic pathway?

A

Individual enzyme assay, metabolite profiling- ie mass spec to show where bonds have been broken/where the compound has been oxidised.

63
Q

Where are the cytochrome p450 enzymes predominantly found in the body?

A

The liver

64
Q

What is the role of P-glycoprotein?

A

Is an ATP-dependent efflux pump with broad substrate specificity: natural role is dealing with toxic substances, up-regulated in response to drug administration reducing the effective concentration available.

65
Q

What is caco-2?

A

An immortalised cell line: a monolayer of cells is grown on a filter separating 2 stacked micro well plates and the permeability of drugs through the cells is determined after the introduction of a drug on one side of the filter. Is a widely used model for membrane permeability.

66
Q

What is important in plasma protein binding?

A

Need the right balance- strong enough to be transported but not too strong otherwise never leaves the plasma.

67
Q

Describe 2 different toxicology tests for in vitro toxicology testing

A

Ames test: looking for compounds that will induce mutations to see whether they’re potentially pathogenic.
Herg test: Herg channel (ion channel) is important for cardiovascular function, blocking the channel has an effect on the QT interval.

68
Q

What is the QT interval?

A

The QT interval begins at the onset of QRS complex and to the end of the T wave, it represents the time of ventricular depolarisation until ventricular repolarisation.

69
Q

Describe the optimisation of Maraviroc

A

1: issues with CYP2D6 activity and too lipophilic- replaced benzhydril for benzamide.
2: optimised activity and explored enantiomers- optimised amide region to cyclobutyl amide.
3: CYP2D6 still too high- replaced the aminopiperidine with tropane.
4: hERG activity had 99% inhibition- added an oygen bridgehead into the tropane.
5: still a hERG problem- reduced the polar surface area.
6: problems with bioavailability (pharmacokinetics)- reduced lipophilicity by replacing the benzimidazole with triazole.
7: hERG came back- identified as due to the cyclobutyl so replaced with cyclopentyl.
8: final optimisation- larger cyclohexyl and high polarity of fluoro substituents (changes polarity, common in drugs).
9: drug approved in 2007 as 1st CCR5 antagonist.

70
Q

What is Parkinson’s and treatments?

A

When your ability to generate/respond to dopamine is repressed, leading to problems with muscle movement. Treatment: stimulate the cells, inhibit the processes that damp down dopamine or to provide dopamine directly.

71
Q

What are the issues of using biologics in treatments?

A

Specificity of target, properties of antibody, cost of protein preparation.

72
Q

What are the 4 different classes of GPCRs?

A

Class A: small molecules such as adrenaline/neurotransmitters/odorant detection- major drug targets in CNS.
Class B: peptide hormones such as secretin.
Class C- glutamate receptors.
Others

73
Q

Describe the function and core structure of GPCRs

A

7 transmembrane helices, binding of ligand outside changes the dynamics/conformation and changes the binding to an internal G-protein, signalling then through the G-protein.

Chemistry and Disease (7 decks)

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