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BIOT3002 > Lecture 1: Drug targets, discovery, and screening > Flashcards

Lecture 1: Drug targets, discovery, and screening Flashcards

1
Q

Natural Products

A

A substance produced by a living organism

e.g. caffeine, nicotine, penicillin

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2
Q

Secondary metabolites

A

are biologically active small molecules that are not required for viability but which provide a competitive advantage to the producing organism.

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3
Q

Pharmacognosy

A

“deals with natural products used as drugs of for the production and discovery of drugs”

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4
Q

Why do organisms make secondary metabolites?

A
  • Defense – insecticidal/antifeedant compounds
  • Offense - antimicrobials
  • Competition - antifoulants
  • Reproduction - pheromones
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5
Q

Taxol (Paclitaxel)

A

Isolated from bark of the Pacific yew

Used to treat breast and ovarian
cancer

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6
Q

Artemisinin

A

An antimalarial
* Isolated from the sweet wormwood

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7
Q

Why are natural products important?

A
  • NPs possess enormous structural and chemical diversity that is unsurpassed by any synthetic libraries.
  • NPs are evolutionarily optimised as drug-like molecules.
  • The bioactivity of natural products stems from the hypothesis that
    essentially all natural products have some receptor-binding activity; the
    problem is to find which receptor a given natural product is binding to
  • A long history of traditional medicine
  • Massive untapped resource
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8
Q

Challenges of natural products in drug discovery

A
  • Low yields.
  • Limited supply of source material.
  • The Rio Convention - Convention on Biodiversity
  • Complex structures precluding practical synthesis
  • Taxol – only 9 labs have reported a total synthesis. ~40 steps, yield <1%
  • Complex structures posing enormous difficulty for structural
    modifications.
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9
Q

Extraction of molecules from source

A
  • Grind up material (could be dry or wet)
  • Mix with one or more solvents
  • typically varying in hydrophobicity
  • May include solvent partitioning steps
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10
Q

Fractionation of the crude extract

A
  • Separation of extracts into less complex mixtures
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11
Q

Purification of individual compounds

A
  • Chromatography – separate compounds based on
    * Hydrophobicity
    * Size
    * Ionic interactions
  • Often coupled with activity assay
    * Bioassay-guided fractionation
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12
Q

Describe the concept of bioassay-guided fractionation.

A

tbc

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13
Q

Structure determination

A

A chemical structure of the drug lead is required for chemical
synthesis, medicinal chemistry, structure-based design

Chemical tests
* Detects particular functional groups or classes of molecules
* Elemental analysis (ratios of different elements)

Mass spectrometry
* Exact molecular weight and formula

Nuclear Magnetic Resonance spectroscopy
* Use NMR data to determine structure
* Non-destructive and in solution

X-ray Crystallography
* Need to be able to crystallise your compound
* Provides structure and stereochemistry

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14
Q

Chemical Space

A

The “space” spanned by all possible molecules with MW<500 Da.

Vast, > 1060 (Proteins ~10390)

Biologically relevant chemical space
- Only a small fraction of chemical space

Accessible chemical space
- Only a minute fraction of chemical space

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15
Q

Chemogenomics

A

Aims to discover active and/or selective ligands for biologically related targets in a systematic way

Ideal World
* Screen all possible compounds against all possible targets.

Real World
* Screening compound classes, enriched compound collections or focused libraries against target families (e.g. GPCRs, protein kinases, proteases)

A target family approach
* Identify small molecules that interact via a specific molecular recognition mode with a target family

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16
Q

Privileged scaffolds

A

Molecular frameworks that are capable of being ligands for a diverse
range of receptors

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17
Q

Drug-like molecules – Lipinski’s rule of 5

A
  • Not more than 5 hydrogen bond donors
  • Not more than 10 hydrogen bond acceptors
  • A molecular mass lees that 500 daltons
  • An octanol/water partition coefficient log P not greater than 5

Rules are meant to be broken
* Cyclosporine – immunosuppresant
* MW = 1203 Da
* 11 H-bond acceptors
* 27% oral bioavailability

18
Q

PAINS (and IMPS)

A
  • Pan Assay Interference Compounds
  • Promiscuous compounds that give false positives in biochemical
    assays
  • substance to avoid (??)
19
Q

Antagonists vs. Agonists

A

Agonists – bind to and activate the receptor to produce a response.

Antagonists – bind to the receptor but do not produce a response and they
block the action of the agonist

As a general rule, it is more common and easier to develop drugs that are
antagonists to receptors, or enzyme inhibitors, rather than drugs which
activate cells, i.e. agonists.

20
Q

Drug Targets

A

Enzymes
Intracellular receptors
* cytoplasmic
* Nuclear
Cell surface receptors
* G-coupled proteins (GCPRs)
* Ion-channels
Nucleic Acid
* DNA binding cancer drugs
Microbial membranes/cell walls

21
Q

Who does DDD?

A

Programs in Drug Discovery and Development:
* Large pharmaceutical companies (big pharma, e.g. Pfizer, AstraZeneca,
Johnson & Johnson)
* Biotech companies (e.g. Genentech)
* Smaller start-up companies (Protagonist, Spinifex)
* Universities and Research Institutes

22
Q

Why Do Drug Design and Development?

A
  • Of huge importance to society:
  • Many important diseases are not adequately treated
  • need for discovery
  • Drugs could be more effective and free from side effects
  • need for improvement
  • New diseases
  • ageing population, new strains of bacteria/viruses
  • Limited patent life
  • need for replacement
  • To make money $$$
23
Q

Why make drugs

A

MONEY and SAVES LIVES

24
Q

Two major paradigms to discovery of
drugs:

A
  1. Physiology-based discovery
    - Follows physiological readouts
    - Compounds screened and profiled based purely on this readout
    - No initial target identification/validation
    Eg. natural compound discovery
    e.g Taxol (paclitaxel), Anti-cancer agent (ovarian, breast, lung)
  2. Target-based discovery
    - Begins with identifying the function of a possible therapeutic target and its role in disease
    - Drugs are then designed (often first in silico) on the basis of the target, using modern chemistry methods
    e.g. Omeprazole – proton pump inhibitor (PPI)
25
Q

Target Validation

A

Need to identify whether the “target” is valid for developing drugs against

Ideally:
* Clinical efficacy and safety data of existing drugs
against that target (2nd and 3rd generation drugs
are usually better)

Animal models of disease
- Inhibit target using non pharmacological methods (ie. transgenic mice)

26
Q

Estimated cost and time to develop a drug:

A

~10 – 15 years

27
Q

High throughput screening (HTS) - definition

A

High throughput screening (HTS) is the process by which large numbers of compounds can be tested, in an
automated fashion, for activity as inhibitors (antagonists) or activators (agonists) of a particular biological target, such as a cell surface receptor or a metabolic enzyme

28
Q

High throughput screening (HTS) - goal

A

Identify a molecular structure (= hit) that:
– Selectively binds to and modulates the activity of a biological target (e.g., a protein) of interest (target-based) OR
– Selectively induces a desired phenotype in a cell population or organism of interest (phenotype-based)

29
Q

High throughput screening (HTS) - terminology

A
  • Library – set of compounds to be screened
  • Target – protein/pathway for drug activity
  • Hit – compound with a signal above threshold
  • Lead – precursor of a drug
  • False negative – active against target but fails to score in assay
  • False positive – not active against target but scores as a hit in assay
  • Hit rate – number of ‘hits’ per screen
30
Q

(HTS) - workflow

A
  1. biological target
  2. assay development + optimisation
  3. primary screening + hit reconfirmation
  4. secondary screening
    5.medicinal chemistry optimisation
31
Q

HTS four sections (?)

A

Library
Assay
Automation
Data Analysis and Management

32
Q

Compound libraries

A

Types of molecule:
* Small molecules
* Proteins/peptides

Formats
* Solutions
* Fragments
* Beads

33
Q

Chemical space

A

the space spanned by all energetically stable stoichiometric combinations of electrons, atomic nuclei and topologies in molecules. Calculated to contain up to 1 x 1060 distinct molecules.

34
Q

Assay design and development considerations

A
  • Cost considerations
  • Access to sufficient quantities of proteins or cells of interest
  • DMSO resistance
  • Dynamic range optimisation
  • Miniaturisation
35
Q

Assay design – biochemical vs cell-based

A

Biochemical assays:
* Purified protein – enzyme or receptor
+ robust, direct readout
+ easy to validate and interpret
+ tend to be faster and higher throughput
- need lots of pure protein/enzyme/substrate
- hits need to be optimised for cell
permeability, toxicity profile etc.

Cell-based assays:
* 2nd-messenger assays, reporter gene
assays, cell proliferation, high content imaging, phenotypic
+ inbuilt permeability and toxicity screening
+ provide data in cellular context
- can be expensive and complicated
- need significant quantities of cells
- phenotypic assays tend to be lower
throughput

36
Q

Assay design – detection

A

Fluorescence
* Organic fluorophores
* Lanthanide cryptates
* Fluorescence polarisation
* FRET
* Genetically encoded fluorophores

Luminescence
* Second reporter assays

Radioactivity
* Scintillation proximity assays

37
Q

Assay examples

A

Scintillation proximity assays (SPA)
HTRF assays
Variation of FRET
Alphascreen
Fluorescence Polarization (FP)
Surface Plasmon Resonance imaging (SPRi)
Phenotypic assays

38
Q

Methods and software to:

A
  • Store data
  • Carry out statistical analyses
  • Evaluate the assay parameters
  • Identify false positives/negatives
  • Screen for PAINS
  • Deconvolute data
  • Link hits to chemical structures
39
Q

Data analysis and management - PAINS

A

PAINS – pan-assay interference compounds

PAINs fall into hundreds of chemical classes, but there are 8 main ones that should set off alarm bells if they are detected in the “hits” in drug screens

40
Q

HTS – the magic triangle

A

Time
Costs
Quality

BIOT3002 (13 decks)

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