Chose a target and find hit/ lead compounds I

Question 1

1) Choose a disease

Answer 1

need for new drug

economical factor (market strategies)

Question 2

2) Choose a drug target

Answer 2

Biomacromolecules involved (proteins, DNA/RNA, etc)

Question 3

advantages of choosing a disease and a drug target?

Answer 3

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

Question 4

3) Identify a Bioassay - simple, quick, relevant

Answer 4

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…)

Question 5

In vivo (Pre-clinical Phase)

Answer 5

• Induce a clinical condition in the animal
• Transgenic animals
• slow and expensive
• sometimes result are invalid
• variability according to the species

Question 6

RECAP; in vitro/ in vivo

Answer 6

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.

Question 7

In vitro tests methods:

Answer 7

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

Question 8

NMR screening steps (4)

Answer 8

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

Question 9

in vitro tests methods…

Answer 9

Isothermal titration calorimetry (ITC):
determine the thermodynamic proper- ties of binding between a drug and its protein target— the binding affinity and enthalpy change

Question 10

What is a Hit compound?

Answer 10

compound active on the target,with low cytotoxicity (synthetic ornatural)

Question 11

I. Screening of natural products

Answer 11

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

Question 12

II. Screening synthetic compound libraries

Answer 12

Compounds or synthetic intermediate which have been previously synthesised

Question 13

III. Existing Drugs

Answer 13

‘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

Question 14

IV. Starting from the natural ligand or modulator

Answer 14

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

Question 15

V. Serendipity

Answer 15

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)

Question 16

advantages of III) existing drugs

Answer 16

faster

cheaper

only need to redo, testing drug on human model

already tested

Question 17

VI. Computer-aided design (CADD)

Answer 17

se computers to discover hit molecules

Study the target 3D structure using a computer

“Rise of PDB”: X-ray crystallography, NMR spectroscopy, cryo-EM

Question 18

Finding a Hit compound-using CADD

Answer 18

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)

Question 19

Active as a homodimer, essential for the viral replication:

Answer 19

produces the active form of the viral non-structural proteins

Question 20

Ligand =

Answer 20

peptide-base inhibitor

Question 21

Target analysis

Answer 21

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

Question 22

Intermolecular bonding forces

Answer 22

Ionic bonds
H-bonds
Van der Waals
Induced-dipole
Ion-dipole
Dipole-dipole

Question 23

Available software:
analyse interactions between proteins and bound ligands

Answer 23

> we can see where (and how) we can modify the ligand structure

Peptide Inhibitor

NB: the full structure should always be inspected

Question 24

Analysis of the structure of HIV protease led to the development of potent inhibitors which became drugs (HAART).

Answer 24

The first was saquinavir, followed by others, such as nelfinavir

Question 25

Nelfinavir

Answer 25

non-peptidic inhibitor with KI of 2 nM, and an EC50 of 14 nM against HIV replication

Question 26

nelfinavir v peptide inhibitor

Answer 26

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

Question 27

Pharmacophores and pharmacophore modelling

Answer 27

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

Question 28

Pharmacophore

Answer 28

binding group types essential for activity and their 3-D arrangement (relative position) in the active molecule

Question 29

Pharmacophoric models, used for what?

Answer 29

design new active molecules or optimise existing ones (potency)

Question 30

Virtual Screening

Answer 30

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

Question 31

Structure-based virtual screening

Answer 31

molecular docking

Question 32

Given a target site, the software takes each virtual compound and explore its possible conformations (docking poses) within the target site>

Answer 32

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)

Question 33

equation for delta gibbs bind

Answer 33

〖∆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

Question 34

Ligand-based virtual screening: shape similarity

Answer 34

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

Question 35

Ligand-based virtual screening are usually associated with ______ (actual) hit rates

Answer 35

higher

Question 36

“Mixed” virtual screening:

Answer 36

Pharmacophoric Search

Question 37

Virtual compound libraries can also be screened for their matching of a pharmacophoric query.

Answer 37

All the possible conformations of the screened molecules are explored and evaluated for their fitting to a given pharmacophoric model

Question 38

These pharmacophores can be ligand-based, structure-based or a mixture of both

Answer 38

Ligand-based elements “Exclusion volumes”

Question 39

VII. Fragment-based hit discovery

Answer 39

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

Question 40

By linking the fragments together, we usually obtain bigger molecules with very good affinity for the given target =

Answer 40

good hit compounds for further optimisation

Question 41

Fragments used in fragment-based drug discovery usually obey “rule of 3”:

Answer 41

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

Question 42

Big advantage: / finding a hit

Answer 42

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

Question 43

SAR by NMR

Answer 43

epitope mapping Screening small fragments (epitopes) and elaborating them into a potent hit using NMR

Question 44

Binding of fragments to protein sub-sites: detected by a shift in specific protein amide signals (protein labelled with 15N or 13C) >

Answer 44

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)

Question 45

The small fragments are then optimised and ______

Answer 45

LINKED

Question 46

if drug is not water soluble

Answer 46

not effective

Question 47

Hit to Lead process

Answer 47

Lead = compound highly active on the target, effective in the disease model, amenable to synthetic modifications, drug-like properties Easily synthesised & chemically stable

Question 48

Hit to Lead process

Answer 48

Lead = compound highly active on the target, effective in the disease model, amenable to synthetic modifications, drug-like properties Easily synthesised & chemically stable

Question 49

Improve Pharmacodynamic properties: 

Answer 49

ability of a compound to selectively bind to a target and produce a desired pharmacological effect. Low side effects, non-toxic.

Question 50

Improve Pharmacokinetic properties:

Answer 50

range of factors which affect a compound’s ability to reach its target in living systems. PK properties and life-time

Question 51

Pharmacodynamics and pharmacokinetics should have ______ priority in influencing drug design strategies and determining which analogues are synthesised

Answer 51

equal

Question 52

Once an Hit is found >

Answer 52

synthesis of analogues: iterative, systematic modification of the structure to investigate effects on activity (1 modification at a time)

Question 53

Produce list of tolerated and non-tolerated modifications and allow for pharmacophore identification

Answer 53

3-D arrangement of those components of the structure which are essential for activity

Question 54

Drug-like properties

Answer 54

- Water Solubility - LogP = balance between the two - molecular weight

Question 55

Presence of PAINs:

Answer 55

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

Question 56

Presence of Toxicophoric groups:

Answer 56

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…

Question 57

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:

Answer 57

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)

Question 58

Works best for synthetic compounds, which occasionally break one of the points, but rarely two

Answer 58

Several natural drugs break at least one of the points

Question 59

Hit to Lead process: drug optimisation Optimise the interactions of a drug with its target: gains to better.....

Answer 59

gain better activity/selectivity

Question 60

Variation of substituents (chemistry based):

Answer 60

1. 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 2. Drug optimisation by NMR 3. Drug optimisation using CADD (molecular modelling): docking, pharmacophore, etc.

Question 61

define Isosters:

Answer 61

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)

Question 62

define Bioisosters:

Answer 62

retain similar biological properties; useful in lead modification to improve ADMET properties (and potency in some cases)

Question 63

Characteristics of Bioisosteres?

Answer 63

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)

Question 64

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:

Answer 64

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

Question 65

Optimise access to the target

Answer 65

1. Optimise hydrophilic/hydrophobic properties Crucial in influencing solubility and ADME properties

Question 66

Drugs too polar or too hydrophilic:

Answer 66

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

Question 67

Drugs too hydrophobic:

Answer 67

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

Question 68

LogP ClogP logD

Answer 68

- calculate by software - relative distribution of all species - both ionised and unionised

Question 69

Optimise access to the target 1. Optimise hydrophilic/hydrophobic properties

Answer 69

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

Question 70

if drug is too hydrophilic

Answer 70

reduce hydrophilicity snd increase lipophilicity ORGANIC CHEMISTRY e.g. transform COOH to something less polar > ester remove H

Question 71

2. Making drugs more resistant to chemical and enzymatic degradation

Answer 71

this is the process to make drugs MORE resistant to hydrolysis and drug metabolism and prolong activity

Question 72

Optimise access tho the target 2. Making drugs more resistant to chemical and enzymatic degradation

Answer 72

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

Question 73

why is fluorine popular in medicinal chemistry?

Answer 73

although having similar size to H - increases lipophilicity and reduces pKa - reduces electron density - alters conformation

Question 74

Bioisostere of H with dramatic effects (not always beneficial) on:

Answer 74

potency, absorption, distribution, metabolism, excretion and toxicity

Question 75

characteristics and their properties - optimise access to target

Answer 75

potency - electronic properties conformation absorption/ distribution - lipiphilicity pKa metabolism - electronic properties excretion and toxicity - electrostatics, conformation, lipophilicity, pKa