Chose a target and find hit/ lead compounds I Flashcards
1) Choose a disease
need for new drug
economical factor (market strategies)
2) Choose a drug target
Biomacromolecules involved (proteins, DNA/RNA, etc)
advantages of choosing a disease and a drug target?
Target specificity/selectivity between species (i.e. antimicrobial agents, antivirals, etc…)
Target specificity/selectivity within the body (i.e. selectivity among various isoenzymes, etc…)
Targeting specific organs/tissues (i.e. 𝛽1 in the heart and 𝛽2 in the lungs, etc..)
Multi-target drugs: combination therapy (i.e. cancer, HIV) and multi-target direct ligand (promiscuous ligand or dirty drugs)
Target Validation: disease association, cell-based models, protein interactions, etc
3) Identify a Bioassay - simple, quick, relevant
In vitro
- specific tissues, cells, enzymes
- use bacteria and yeast = produce enzymes (HIV protease) IC50, competitive/non-competitive
- Isolated tissues or cells expressing a receptor
- Intracellular and extracellular events
- PK properies
- (i.e. Caco-2 cells, microsomes, etc…)
In vivo (Pre-clinical Phase)
- Induce a clinical condition in the animal
- Transgenic animals
- slow and expensive
- sometimes result are invalid
- variability according to the species
RECAP; in vitro/ in vivo
in vitro study occurs in a controlled environment, such as a test tube or petri dish. In vivo is Latin for “within the living.” It refers to tests, experiments, and procedures that researchers perform in or on a whole living organism, such as a person, laboratory animal, or plant.
In vitro tests methods:
High-throughput screening (HTS):
automated test of large number of compounds (several thousands) against a large number of targets (30-50); efficient to hit identification; false-positive hits (promiscuous inhibitors and PAINS-Pan-assay interference compounds);
Screening by NMR: detect whether a compound binds to a protein target; screen a mixture of compounds; 1000 small-molecules a day; detect weak binding; no false-positive
NMR screening steps (4)
1) NMR spectrum of the drug is taken
2)Protein is added and the spectrum is re-run (protein signals are not detected)
3)Drug not binding: its NMR spectrum will still be detected
4)Drug binding: no NMR spectrum will be detected
in vitro tests methods…
Isothermal titration calorimetry (ITC):
determine the thermodynamic proper- ties of binding between a drug and its protein target— the binding affinity and enthalpy change
What is a Hit compound?
compound active on the target,with low cytotoxicity (synthetic ornatural)
I. Screening of natural products
Active Principle
Complicated structures (chiral centres, strange bonds): extraction
Plants Source: morphine, cocaine, taxol, etc..
Microorganisms: bacteria, fungi (antimicrobial agents-cephalosporins)
Marine sources: coral, sponges, fishes, marine microorganisms
Animal sources, Venoms and toxins
II. Screening synthetic compound libraries
Compounds or synthetic intermediate which have been previously synthesised
III. Existing Drugs
‘Me too and “me better’ drugs: use established drugs as hit compounds in order to design a drug that gives them a foothold in the same market area.
Modify the structure sufficiently such that it avoids patent restrictions, retains activity, and, ideally, has improved therapeutic properties
Selective optimisation of side activities (SOSA): enhance the desired side effect and to eliminate the major biological activity of existing drugs
Repurposing: screening existing drugs, compounds that are either in clinical use or have reached late-stage clinical trials against new disease/targets
IV. Starting from the natural ligand or modulator
Natural ligands for receptors: used as hit to design agonists (i.e. adrenaline and noradrenaline); design of antagonists (i.e. histamine);
Natural substrate for enzymes: used as hit to design inhibitors (i.e. natural substrate HIV protease -development of the first HIV protease inhibitor)
Enzyme products as hit compounds: used as hit to design inhibitors (i.e. carboxypeptidase inhibitors)
Natural modulators as hit compounds: receptors and enzymes are under allosteric control. The natural or endogenous chemicals that exert this control (modulators) could also serve as hit compounds
V. Serendipity
Hit compounds found by chance
Research to improve a drug can have unexpected and beneficial spin offs (i.e. Propanolol and Practolol)
Research projects carried out in a totally different field (i.e tolbutamide)
advantages of III) existing drugs
faster
cheaper
only need to redo, testing drug on human model
already tested
VI. Computer-aided design (CADD)
se computers to discover hit molecules
Study the target 3D structure using a computer
“Rise of PDB”: X-ray crystallography, NMR spectroscopy, cryo-EM
Finding a Hit compound-using CADD
Target analysis - Examination of the target structure allows to rationally design compounds that can bind to it
Easier if there are co-crystallised ligands > we know where the active/binding site is (or we can use software for binding site prediction)
Active as a homodimer, essential for the viral replication:
produces the active form of the viral non-structural proteins
Ligand =
peptide-base inhibitor
Target analysis
The residues forming the active/binding site can be analysed to identify key interactions (more difficult if we do not have a ligand bound) to design new ligands/inhibitors
Intermolecular bonding forces
Ionic bonds
H-bonds
Van der Waals
Induced-dipole
Ion-dipole
Dipole-dipole
Available software:
analyse interactions between proteins and bound ligands
> we can see where (and how) we can modify the ligand structure
Peptide Inhibitor
NB: the full structure should always be inspected
Analysis of the structure of HIV protease led to the development of potent inhibitors which became drugs (HAART).
The first was saquinavir, followed by others, such as nelfinavir
Nelfinavir
non-peptidic inhibitor with KI of 2 nM, and an EC50 of 14 nM against HIV replication
nelfinavir v peptide inhibitor
nelfinavir much smaller and more compact
binding by improved fit into the hydrophobic regions of the enzyme rather than increased hydrogen bonding, which makes nelfinavir a better inhibitor
Pharmacophores and pharmacophore modelling
Software (or rational methods) can be used to “extract” information on the essential functional group types, and their 3-D spatial arrangements, required for activity on a given target (essential interactions) = pharmacophore
This information can be used to design new drugs, or simplify/optimise the structures of existing ones
Pharmacophore
binding group types essential for activity and their 3-D arrangement (relative position) in the active molecule
Pharmacophoric models, used for what?
design new active molecules or optimise existing ones (potency)
Virtual Screening
instead of testing large number of compounds in the lab (HTS), we can rely on computer simulations which can predict if a compound is good or not, e.g. to bind a given target
