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
Structure-based virtual screening
molecular docking
Given a target site, the software takes each virtual compound and explore its possible conformations (docking poses) within the target site>
The software scores and ranks each generated pose according to a mathematical evaluation (scoring function) of the predicted free energy change upon binding (docking score)
equation for delta gibbs bind
〖∆𝑮〗_𝒃𝒊𝒏𝒅=〖∆𝑮〗_𝒊𝒏𝒕𝒆𝒓𝒂𝒄𝒕+〖∆𝑮〗_𝒂𝒔𝒔𝒐𝒄+〖∆𝑮〗_𝒄𝒐𝒏𝒇+〖∆𝑮〗_𝒓𝒐𝒕+〖∆𝑮〗_𝒗𝒊𝒃+〖∆𝑮〗_𝒔𝒐𝒍𝒗
〖∆𝑮〗_𝒊𝒏𝒕𝒆𝒓𝒂𝒄𝒕 = new protein-ligand interactions formed
〖∆𝑮〗_𝒄𝒐𝒏𝒇 = altered conformation of both ligand and protein
〖∆𝑮〗_𝒔𝒐𝒍𝒗 = changes in salvation
Ligand-based virtual screening: shape similarity
Virtual compound libraries can be screened for their shape-similarity with known active molecules.
All the possible conformations of the screened molecules are explored and compared for their shape and functional group matching with a query molecule
Ligand-based virtual screening are usually associated with ______ (actual) hit rates
higher
“Mixed” virtual screening:
Pharmacophoric Search
Virtual compound libraries can also be screened for their matching of a pharmacophoric query.
All the possible conformations of the screened molecules are explored and evaluated for their fitting to a given pharmacophoric model
These pharmacophores can be ligand-based, structure-based or a mixture of both
Ligand-based elements
“Exclusion volumes”
VII. Fragment-based hit discovery
Rather than screening large, high-affinity molecules, small molecular fragments are screened against a given target
due to size and bind weakly
once multiple active fragments are identified (mM-high uM IC50), crystal structures of their complexes with target are resolved, link different fragments together > optimised high-affinity molecule
By linking the fragments together, we usually obtain bigger molecules with very good affinity for the given target =
good hit compounds for further optimisation
Fragments used in fragment-based drug discovery usually obey “rule of 3”:
Relative molecular mass <300 (and >150)
Number of H-bond donating groups ≤ 3
Number of H-bond accepting groups ≤ 3
ClogP ≤ 3 (NB: calculated!)
Number of rotatable bonds ≤ 3
Big advantage: / finding a hit
it saves a lot of efforts related to the synthesis (and the screening) of large molecules only those ones very likely to be active are considered
NB: Software for in silico fragment-based drug design available
SAR by NMR
epitope mapping
Screening small fragments (epitopes) and elaborating them into a potent hit using NMR
Binding of fragments to protein sub-sites: detected by a shift in specific protein amide signals (protein labelled with 15N or 13C)
>
monitor IF binding is taking place and WHERE
Process repeated to find small ligands for each sub-region (usually binding of 2nd ligand in the presence of 1st ligand to ensure different binding regions)
The small fragments are then optimised and ______
LINKED
if drug is not water soluble
not effective
Hit to Lead process
Lead = compound highly active on the target, effective in the disease model, amenable to synthetic modifications, drug-like properties
Easily synthesised & chemically stable
Hit to Lead process
Lead = compound highly active on the target, effective in the disease model, amenable to synthetic modifications, drug-like properties
Easily synthesised & chemically stable
Improve Pharmacodynamic properties:
ability of a compound to selectively bind to a target andproduce a desired pharmacological effect. Low side effects, non-toxic.
Improve Pharmacokinetic properties:
range of factors which affect a compound’s ability to reach its target in living systems. PK properties and life-time
Pharmacodynamics and pharmacokinetics should have ______ priority in influencing drug design strategies and determining which analogues are synthesised
equal
Once an Hit is found >
synthesis of analogues: iterative, systematic modification of the structure to investigate effects on activity (1 modification at a time)
Produce list of tolerated and non-tolerated modifications and allow for pharmacophore identification
3-D arrangement of those components of the structure which are essential for activity
Drug-like properties
- Water Solubility
- LogP = balance between the two
- molecular weight
Presence of PAINs:
Pan-Assay Interference compounds interfere with biological screening assays by acting through a range of mechanisms (toxoflavin, isothiazolones, hydroxyphenyl hydrazones, curcumin, phenol-sulfonamides, rhodanines, enones, quinones, and catechols
Presence of Toxicophoric groups:
functional groups with the potential of inducing toxicity. Very reactive groups: aromatic amine, sites of ring hydroxylation, aldehydes, Michael acceptors, aromatic nitro group, bromoarenes, hydrazines, hydroxylamines, or polyhalogenated groups, etc…
What is Lipinski’s Rule of five?
Rule of thumb, formulated by Chris Lipinski of Pfizer in 1997, which provides predictors of drug-likeness for successful ORAL drugs:
1) Relative molecular mass <500
2) Number of H-bond donating groups (HBD) ≤ 5 (i.e. OH, NH, etc.)
3) LogP < 5 (log of partition coefficient between octanol and water: <0 favours water, >0 favours octanol; > Drugs need to be lipophilic to pass membranes and distribute to tissues, but not too lipophilic and not too hydrophilic)
Works best for synthetic compounds, which occasionally break one of the points, but rarely two
Several natural drugs break at least one of the points
Hit to Lead process: drug optimisation
Optimise the interactions of a drug with its target: gains to better…..
gain better activity/selectivity
Variation of substituents (chemistry based):
- alkyl substituents, substituents on aromatic or heteroaromatic rings, synergistic effect, extension of the structure, chain extension/contraction, ring expansion/contraction, ring variations ,ring fusions, isosteres and bio-isosteres, structure simplification, rigidification of the structure, conformational blockers
- Drug optimisation by NMR
- Drug optimisation using CADD (molecular modelling): docking, pharmacophore, etc.
define Isosters:
atoms or groups of atoms which share the same valency, size and which have chemical or physical similarities (SH, NH2, and CH3 are isosteres of OH)
define Bioisosters:
retain similar biological properties; useful in lead modification to improve ADMET properties (and potency in some cases)
Characteristics of Bioisosteres?
Groups/substituents with similar sizes or chemical/physical properties which produce roughly similar biological properties; useful in lead modification to improve ADMET properties (and potency in some cases)
What makes a good drug?
As a combination of what the drug does to the body (PD) and
what the body does to the drug (PK), a good drug is:
1) PHARMACODYNAMICS; effective targeting a disease process, not toxic
2) PHARMACOKINETICS; absorbed well by body, easily reach its target, not , modified/ removed too quickly from body
3) good PK/PD; efficacy/ potency and ADMe(T) properties
Optimise access to the target
- Optimise hydrophilic/hydrophobic properties
Crucial in influencing solubility and ADME properties
Drugs too polar or too hydrophilic:
cannot be used against intracellular targets as they will not cross cell membranes. Have polar functional groups which will make them prone to plasma protein binding, metabolic phase II conjugation reactions, and rapid excretion
Drugs too hydrophobic:
Orally, they are likely to be dissolved in fat globules in the gut and will be poorly absorbed.
Injected, they are poorly soluble in blood and are likely to be taken up by fat tissue, resulting in low circulating levels.
Toxic metabolites are more likely to be formed from hydrophobic drugs
LogP
ClogP
logD
- calculate by software
- relative distribution of all species
- both ionised and unionised
Optimise access to the target
- Optimise hydrophilic/hydrophobic properties
a. Masking polar functional groups to decrease polarity (i.e. OH or PhOH to ROR or COOR, COOH to COOR or CONH2, and RNH2 and R2NH to CONH2 or to R2NH or R3N
b. Adding or removing polar functional groups to vary polarity
c. Varying hydrophobic substituents to vary polarity (i.e include extra alkyl groups or remove them, methylene shuffle, halogen substituents)
d. Variation of N-alkyl substituents to vary pKa
e. Variation of aromatic substituents to vary pKa (i.e. EDG and EWG)c
f. Bioisosteres for polar groups (i.e. replacing COOH with 5-substituted tetrazole
if drug is too hydrophilic
reduce hydrophilicity snd increase lipophilicity
ORGANIC CHEMISTRY e.g. transform COOH to something less polar > ester
remove H
- Making drugs more resistant to chemical and enzymatic degradation
this is the process to make drugs MORE resistant to hydrolysis and drug metabolism and prolong activity
Optimise access tho the target
- Making drugs more resistant to chemical and enzymatic degradation
a. Steric shield: protect more susceptible groups (COOR and CONH2) adding steric shields (bulky groups), designed to hinder the approach of a nucleophile or an enzyme to the susceptible group
b. Electronic effects of bioisosteres: stabilise the labile functional group electronically using a bioisostere
c. Steric and electronic modifications
why is fluorine popular in medicinal chemistry?
although having similar size to H
- increases lipophilicity and reduces pKa
- reduces electron density
- alters conformation
Bioisostere of H with dramatic effects (not always beneficial) on:
potency, absorption, distribution, metabolism, excretion and toxicity
characteristics and their properties - optimise access to target
potency - electronic properties conformation
absorption/ distribution - lipiphilicity pKa
metabolism - electronic properties
excretion and toxicity - electrostatics, conformation, lipophilicity, pKa
