Mechanisms
M
h i
off Molecular
M l
l Interaction
I t
ti in
i
Biology and Medicine
Prof. Antonio Macchiarulo
Dipartimento di Scienze Farmaceutiche,
U i
University
it off P
Perugia
i
Perugia, June 6-10 2016
Mechanisms of Molecular Interaction in
Biology and Medicine
Mechanisms of
Interaction
Ligand
Mechanisms of
Interaction ?
Target
Target
Ligands
Mechanisms of Molecular Interaction in
Biology and Medicine
Definition of Ligands
Definition of Biological Targets
Mechanisms of Molecular Recognition
- Theory of Molecular Recognition
- Specificity of Molecular Recognition
- Promiscuity of Molecular Recognition
What is a Ligand ?
g
Small-molecules
Drug-like
like molecules
- Drug
- Lead-like molecules
- Nature
Nature’s
s compounds
Peptides
Oligonucleotides
Therapeutic Proteins
The Chemical Space
Chemical space is the unlimited set of all chemical compounds.
In one of the more highly cited estimates,
R i B
Regine
Bohacek
h
k considers
id
creating
ti a lilinear
compound from scratch, choosing a carbon,
oxygen or nitrogen atom to form molecules of
30 members. Adding any stable chemical
group onto
t the
th free
f
bonds,
b d and
d considering
id i
aspects that would produce greater chemical
diversity (branching, recyclization and
stereochemistry), gives an estimate in excess
of 10 60 possible
ibl molecules
l
l .
Chemical
Space
(1060)
Bohacek, R. S.; et al. Med Res Rev 1996, 16, 3-50
Petit-Zeman:
Petit
Zeman: 1018 – 10200
Nat. Rev. Drug Discov. 2004, 3, 375
Geysen et al.: 1014 –
Nat
Nat.
Rev Drug Discov
Discov. 2004,
2004 3,
3 375
1030Rev.
Kirkpatrick: 10200
Kirkpatrick, P.; Ellis, C., Nature 2004, 432, 823-823
…peptides, oligonucleotides and proteins rise the extension of the chemical space.
The Chemical Space
Not all of the Chemical Space is biologically meaningful
If we consider
id as potential
t ti l biologically
bi l i ll
actives those molecules with molecular
masses in the same range of small
molecules
l
l presentt iin bi
biological
l i l systems
t
(about 500 daltons), then the theoretical
extension of the biological space is
estimated around 1018 small molecules
Chemical
S
Space
Unlimited
Active
Ligands
n°1018
The Chemical Space
Estimated
st ated > o
of 10
060 molecules
o ecu es
Need of criteria to identify “promising planets” of the chemical space
wherein biologically active molecules are located
located.
The Drug-Like
g
Molecules
Christopher A. Lipinski, Ph.D.
In 1997,
1997 Christopher Lipinski suggested four parameters to identify small
smallmolecules with high chance to become successful marketed drugs (drug-likeness).
The Drug-Like
g
Molecules
Analyzing a dataset containing data on the physicochemical properties of
2.245 small-molecule drugs, Lipinski was able to identify some conserved
properties:
ti
89% small-molecules endowed with Molecular Weight (MW) < 500
90% small-molecules endowed with Partition Coeff. Octanol / Water
(LogP) < 5.0
92% small-molecules endowed with a sum of hydrogen bond donating
groups
g
p ((O-H, N-H)) < 5
88% small-molecules endowed with a sum of hydrogen bond
accepting groups(O, N) < 10
Being all of these parameters related to the «magic number 5», the
criteria was named:
“Rule of Five”
The Drug-Likeness
g
Criteria
Drug-like compounds are those small molecules compliant
t the
to
th following
f ll i criteria
it i or ‘‘rule
l off fifive’:
’
MW < 500 dalton
LogP < 5
Hydrogen bond donor groups (sum of ‘O-H’ and ‘N-H’) < 5
Hydrogen bond acceptor groups (sum of ‘O’
O and ‘N’)
N ) < 10
The Drug-Like
g
Molecules
Some good examples of drug-like small-molecules…
1-[2-dimethylamino-1-(4-methoxyphenyl)ethyl]cyclohexan-1-ol (EFFEXOR)
Disease: Depression
Wyeth
Income = $2.6
$2 6 billion
N
O
MW = 277
LogP = 2.81
Acc. = 3
Don. = 1
HO
O
O
N
N
S
N
H
O
Esomeprazole (NEXIUM)
Disease: Gastric Ulcera
AstraZeneca
Income = $4.4 billion
MW = 345
LogP = 2.17
Acc. = 5
Don. = 1
Source: IMS Health, a healthcare information company. December 2005
The ((non)) Drug-Like
g
Molecules
Some bad examples of drug-like small-molecules…
O
OH
N
Ph
C2H5
NH
N
HO
H
MeO
Me
N
OCOMe
H
CO
Me
2
Me HO
Me
Me
HO
O
Me
O
O
MeO
H
OCOPh OCOMe
Vinblastine and Taxol are antimitotic
agents being currently used for treating
a variety of cancers.
H
N
O
Me
HO
O
Taxol
(MW: 854)
OH
HO
OH
Me
O
C2H5
Vinblastine
(MW: 811)
MeO
O
Me
N
H
MeO2C
HO
MeOCO
O Me
Ph
Me
O
O
OMe
Rapamycin
(MW: 915)
Rapamycin is a cell cycle arresting
agent studied for anticancer activity.
Nature’s Molecules ((Natural Products))
Charting
Ch
ti Bi
Biologically
l i ll Relevant
R l
t Chemical
Ch i l
Space with Natural Products
Natural
Products
Chemical
S
Space
(1060)
Active
Ligands
(1018)
> 40% of New Chemical Entities
(NCEs) approved from 1981 to 2006
are related to Natural Products (NPs).
NPs are considered as privileged by
nature since they have been optimized
for optimal interactions with biological
targets through evolution.
It ensues that a large component of
biologically relevant chemical space is
occupied by natural products (NPs).
(NPs)
Dobson C. M. Nature 432, 824-8 (2004).
Lipinski, C. & Hopkins, A. Nature 432, 855-61 (2004).
Harvey, A. L. Drug Discovery Today 13, 894
894–901
901 (2008).
Newman DJ et al. J. Nat. Prod. 70, 461-477 (2007)
Rosén J, et al. J. Med. Chem., (2009).
Nature’s Molecules ((Natural Products))
Natural products show greater structural diversity and complexity than
synthetic
h i d
drugs
They contain a greater proportion of oxygen than nitrogen heteroatoms
They have stereogenic centers (increase of complexity).
O
O
H
N
O
H3CO
Me
Me
O
Me
N
O
H3CO
OCH3
Colchicine
N
HN
Me
Me
O
N
N
H
Me
N
O
Me HO
O
O
O
N
N
H
N
CH3
Me
NH
N
H
O
OCH3
Cyclosporin
(MW: 1203)
O
Nature’s Molecules
(N t
(Natural
lP
Products)
d t )
Synthetic Compounds
Drugs
Natural Products
Feher M. et al. J. Chem. Inf. Comput. Sci. 43:218-227 (2003).
Natural Product Screening
The isolation of many bioactive products from natural sources
has led to the systematic screening of plant and animal
extracts for activity.
y
Historical Drugs from Nature’s Molecules
H
N
O
S
N
O
Penicillin G
Discovery (Fleming), 1928
Marketed in 1942
CH3
CH3
COO-
Current Drugs from Nature’s
Nature s Molecules.
In December 1992, FDA approved Taxol (Paclitaxel)
for treatment of breast cancer that has not
responded to standard chemotherapy
chemotherapy.
Taxus brevifolia
Taxol
Paclitaxel
Current Best-Selling Drugs Derived
from Nature’s Molecules.
O
HO
O
N
H
O
O
OH
N
OH
CO2H
O
F
Lovastatin
Aspergillus terreus
Liptor (Atorvastatin)
Lovastatin was isolated from a strain of Aspergillus terreus and it was the first
pp
by
y the FDA ((August
g
1987).
)
statin approved
Lovastatin provided the pharmacophore for the current leader in the statin class,
the fullyy synthetic
y
compound
p
atorvastatin,, which was the world's best-selling
g drug
g
in 2003.
Natural Product Screening
Problems with natural product screening
Isolation of an active component present in a very small amount
can be problematic given a large amount of background “rubbish”.
The mixtures are often very complex and contain many large
macromolecules. These can often “hide” biological activity.
Compound isolation and structure determination difficult.
Chemical structures often complex, therefore difficult to synthesise
and to scale-up for commercial purposes.
Mechanisms of Molecular Interaction in
Biology and Medicine
Definition of Ligands
Definition of Biological Targets
Mechanisms of Molecular Recognition
- Theory of Molecular Recognition
- Specificity of Molecular Recognition
- Promiscuity of Molecular Recognition
What is a Target ?
What is a Target ?
Biological macromolecule or molecular complex that is
critical for the disease
– e.g.
e g an enzyme that is required for the growth of the
infecting bacterium
Peptidoglycan:
N-Acetyl-D-Glucosamine (NAG) + N-acetyl-Muramic Acid (NAM)
Transglycolase
Transpeptidase
What is a Druggable Target ?
What is a Druggable Target ?
Druggable Target is defined as the property of a biological target to
be modulated by...:
Marketed small-molecule drugs
Small-molecules with drug-like properties (compliant to
Lipinski'ss rule of five);
Lipinski
Lipinski, C. A.; et al. Advanced Drug Delivery Review 2001, 46, 3-26.
Hopkins, A.; et al. Nat Rev Drug Discov 2002, 1, 727-730.
Different Targets and Mechanisms of Molecular Interaction
TARGET
Mechanisms of Molecular Interaction
Enzymes
Reversible & Irreversible Inhibitors
R
Receptors
t
A
Agonist
i t and
dA
Antagonists
t
i t
Viral Surface
Blocking the Entry to Cell
Proteins Cross-talk
Blockers and Enhancers
Ion Channels
Uptake Inhibitors or Enhancers
Transporters
Alkylating Agents, Binders,
DNA, RNA
Wrong Substrates (trojan horses)
How Many Targets Do Exist ?
How Many Targets Do Exist ?
# genes
% of genes
with inferred
f
function
ti
Completion
date of
genome
E. coli
4288
60
1997
yeastt
6 600
6,600
40
1996
C. elegans
19,000
40
1998
Drosophila
12-14K
25
1999
Arabadopsis
25,000
40
2000
mouse
~30,000
10-20
2002
human
~30,000
30,000
10-20
10
20
2000
Organism
How Many Druggable Targets Do Exist ?
60% of the drugs marketed today act upon certain targets (druggable targets)
How Many Druggable Targets Do Exist ?
How many of these “druggable”
druggable targets are present and unexploited
in the Human Genome ?
Human Genome Druggable Targets
gg
g
Novel Gene Families
Other 119 gene families
and singleton
targets 44%
GPCRs 25%
ST/Y kinases 10%
Zn peptidases 4%
NHRs 3%
Cys proteases 2%
Gated ion-channel 2%
PDEs 3%
CYP enzymes 2%
Cation channels 2%
Ser proteases 3%
(trypsin)
Marketed Small‐Molecules Acting on Human Genome Druggable Targets
Druggable Targets
Farmaci disponibili per 100 membri della famiglia GPCRs
100
N° of targets
g
in a gene family
targeted by
marketed
smallmolecules
~11%
~15%
~27%
~15%
50
~25%
700 memb
bri della famiglia
a GPCRs nel Ge
enoma Umano
GPCRs
Ser/Thr Protein Kinases
Tyr Protein Kinases
Cys Proteases
I Channel
Ion
Ch
l
Tyr Phosphatases
Otheri Targets
~14%
1
1
10
100
1,000
Popolation of the gene family in the human genome Nature Rev. Drug Disc., 2002, 1(9), 727
Any Drug Target Left ?
Any Drug Target Left ?
• Human genome: ca. 30,000 predicted genes
• Currently known drug targets: ca. 500
Mechanisms of Molecular Interaction in
Biology and Medicine
Definition of Ligands
Definition of Biological Targets
Mechanisms of Molecular Recognition
- Theory of Molecular Recognition
- Specificity of Molecular Recognition
- Promiscuity of Molecular Recognition
Theory of Molecular Recognition:
The Lock and Key Model (1890)
The target contains the binding site
wherein the ligand binds like a key fitting its
complementary lock.
Ligand
Ligand
Target
Target
Emil Fischer
Theory of Molecular Recognition:
The “Magic Bullet” (1911)
Notion
ot o of
o Magic
ag c Bullet:
u et
Attempts to develop effective treatments
for diseases by discovering drugs
selective for single molecular targets
O drug,
One
d
one ttarget,
t one disease
di
Paul Ehrlich
((1854-1915))
Nobel Price for Medicine
((1908))
Ehrlich,P. FoliaSerologica 7,697–714 (1911)
Theory of Molecular Recognition:
The “Induced Fit” Model (1958)
Ligand
Glutamine Binding Protein
Target
Daniel Koshland
Theory of Molecular Recognition:
The “Induced Fit” Model (1958)
Li
Ligand
d
Glutamine Binding Protein
Target
Daniel Koshland
Specificity of Molecular Recognition
Specificity is the hallmark of molecular recognition in biology.
Complementary of shape between ligand and target’s
binding site
A. Conformational aspects of ligand
B. Configurational aspects of ligand
Complementary
C
l
t
off interactions
i t
ti
b
between
t
liligand
d and
d ttarget’s
t’
binding site
A. Covalent Interactions.
B. Non covalent interactions.
Specificity of Molecular Recognition
Complementary of shape between ligand
and target’s
target s active site
site.
A. Conformational aspects of ligand
Ha
90°
Hb
Ha
C
Torsional angle (diedral) of 90°
in a fragment HaC—CH
C CHb
1° plane C—C e C—Ha
2° p
plane C—C e C—Hb
Hb
X Y
C
X
X
X
Y
Y
Synplanar
=0° (±)
Synclinal
=60° (±)
Anticlinal
=120° (±)
Y
Antiplanar
=180° (±)
Specificity of Molecular Recognition
Complementary of shape between ligand and target’s
active site.
3
2
4
1
5
6
(Imatinib Gleevec®)
(Imatinib,
N 6 torsional angles may affect the shape of the molecule
N.6
molecule.
Theoretical expected conformations:
 360 

6 angles with 30° of torsional increment

 = 2.985.984 conformations
θ


N
Specificity of Molecular Recognition
Bioactive Conformation: The conformation adopted by the ligand to interact with the target
(highest complementary of shape). It is not necessarily the lowest energetical state of the
ligand. The energy required to stabilize the bioactive conformation of the ligand is furnished
by the interaction with the target
target.
O
N
N
Aryl
4
N
H
Het
NH
N
H
N
H
Imatinib
LP
N
Bi
Bioactive
ti C
Conformation
f
ti
CH3
Gauche (-sc)
Structure of Kinase TK-ABL2 in complex with Gleevec (3GVU)
Specificity of Molecular Recognition
Complementary of shape between ligand and target’s
active site
site.
B. Configurational aspects of ligand
Configuration: It is the positional correlation of
atoms or groups of atoms around a single atom.
CH2OH
NH2
HO
H
1850: Notion of Chirality in organic compounds.
Many molecule cannot overlap their mirror image
(Pasteur, Van’t Hoff).
Specificity of Molecular Recognition
1959: Tragedy of Thalidomide. A racemic drug is the mixture of
two isomers, being both potentially active !
O
N
*
O
N
O
O
H
Thalidomide
The isomer S of Thalidomide
was responsable of the
ansiolitic effect (therapeutic
effect)
ff t) whereas
h
the
th
enantiomer R was
responsable of the teratogenic
effect (toxic effect)
Specificity of Molecular Recognition
Complementary of interactions between ligand and target’s
bi di site.
binding
it
A Covalent Interactions: irreversible,
A.
i
ibl hi
high
h energy content.
t t
B. Non covalent interactions: reversible,
reversible low to medium energy
content.
• Electrostatic interactions.
• Hydrogen bond interactions.
• Aromatic - interactions.
• Van-der-Waals interactions.
• Hydrophobic interactions.
Energy
(KJ/ l)
(KJ/mol)
Specificity of Molecular Recognition
348
C
2 ‐5
C O
Covalent carbon-carbon interaction
irreversible
Hydrogen bond between C=O and H-N
reversible
ibl
H O
Hydrogen bond between C-O and H-O
reversible
H O
Salt bridge
reversible
Hydrogen bond between C=O and H-O
reversible
Attractive electrostatic interaction
reversible
Van-der-Waals interactions
reversible
Aromatic interaction (- stacking)
reversible
H d h bi iinteractions
Hydrophobic
t
ti
reversible
Dipole-Induced Dipole interactions
reversible
C
H N
H
C O
2 ‐5
2 ‐5
O
C
O-
2 ‐5
C O
C
O
H
O
>10
NH3+
0.3
CH 3
C
-O
CH 3
1.5
15
1.5
0.3 x CH3
> 10
H3C
H3C
CH CH3
CH CH3
H2C
H2C
NH
O
H3C
CH CH3
H2C
Metal interaction
reversible
Specificity of Molecular Recognition
Dipole-Induced
Dipole interactions
Hydrogen bond interactions
Hydrophobic interactions
- Interaction
Complementary of interactions
+ Hydrogen bonds
+ Hydrophobic Interactions
+ - Interaction
+ Dipole interactions
Complementary of shape
+ Bioactive conformation
+ Configuration
= Specificity of Molecular Recognition
Research Paradigms in the Classical Age of Drug Discovery
Notion of Magic Bullets:
Attempts to develop effective treatments for
diseases by discovering drugs selective for
single molecular targets
Paul Ehrlich
(1854-1915)
Nobel Price for Medicine
(1908)
O drug,
One
d
one target,
t
t one disease
di
Ehrlich,P. FoliaSerologica 7,697–714 (1911)
Mendelian Inheritance:
A change in observable features
(phenotype/disease) arises as a consequence of
mutations in one (dominant) or both (recessive)
copies of a gene.
Gregor Mendel
(1822-1884)
One gene, one target, one disease
Challenges in the Modern Age of Drug Discovery
Multifactorial Inheritance:
Manyy of current untreated diseases, including
g diabetes, asthma, and heart disease are
caused by mutations in more than one gene with a contribution from environmental factors.
Notion of Magic Shotguns:
Designing selectively nonselective drugs (that is, ‘magic
shotguns’)) that interact with
shotguns
several molecular targets will lead
to new and more effective
medications for a variety
of central nervous system
disorders.
Marketed Drugs and Their Molecular Targets
ChEMBL dataset:
d t
t 392 kknown orall d
drugs with
ith reported
t d activities
ti iti att any target.
t
t
8%
N >20
8%
N = 10-20
14%
29%
N=0
Weak modulators or
not disclosed activity
N = 5-10
N = 2-5
22%
N=1
18%
52% of marketed drugs shows a multi-target profile at or
below 1M potency threshold.
threshold
Gleeson MP, et a. Nat Rev Drug Discov. 2011, 10, 3, 197-208
Promiscuity of Molecular Recognition
Lack of Ligand Selectivity
- Ligand selectivity decreases with decreasing molecular weight.
Hopkins A. L., et al. Curr. Opin. Struct. Biol. 16, 127–136 (2006).
Morphy, R. & Rankovic, Z. Drug Discov. Today 12, 156–160 (2007).
- Ligand
Li
d selectivity
l ti it d
decreases with
ith increasing
i
i molecular
l
l weight.
i ht
Azzaoui, K. et al. Chem. Med. Chem. 2, 874 – 880 (2007).
Gleeson MP, et a. Nat Rev Drug Discov. 10, 3, 197-208 (2011).
- Ligand selectivity decreases with increasing conformational flexibility.
Stockwell, G.R. & Thornton, J.M. J. Mol. Biol. 356, 928–944 (2006).
Perola, E. & Charifson, P.S. J. Med. Chem. 47, 2499–2510 (2004).
- Ligand selectivity decreases with increasing lipophilicity and the
presence of a basic moiety
Leeson,, P. & Springthorpe,
p g
p , B. Nature Rev. Drug
g Discov. 6,, 881–890 ((2007).
)
Gleeson MP, et a. Nat Rev Drug Discov. 10, 3, 197-208 (2011).
- Ligand selectivity decreases with decreasing ligand complexity
(f
(few
sp3 carbon atoms; few
f
stereogenic elements).
)
Clemons PA, et al. Proc Natl Acad Sci U S A, 108, 17, 6817-22, (2011)
Promiscuity of Molecular Recognition
Target Promiscuity
- At the gene level.
Alternative splicing can result in protein variants with altered binding properties
- At the protein level.
Protein interacting with different ligand chemotypes and/or protein partners
- At the functional level
level.
Single protein involved in multiple signaling pathways
Nobeli I, et al. Nat Biotechnol., 27, 2, 157-67, (2009)
The Notion of Polypharmacology and Its Aspects
Th
Therapeutic
ti Polypharmacology
P l h
l
The treatment of multigenic, complex diseases by targeting multiple
targets with one or more drugs
drugs, in order to effectively reset the regulatory
pathways that are altered in the disease state.
Adverse Polypharmacology
The adverse, physiological effect caused by drug binding to protein targets
other than the therapeutic target or binding to the therapeutic target in non
target tissue.
Boran AD, Iyengar R. Curr Opin Drug Discov Devel. 13, 3, 297-309, (2010)
Promiscuity of Molecular Recognition
This result is still
underestimated
since the
probability of
additional targets
for a given
compound
increases as
further assays are
performed.
f
d
…but screening drugs against all the proteins of human genome is
currently unfeasible !!!
Paolini, G. V., et al. Nature Biotechnol. 24, 805–815 (2006).
In Silico Approaches to Investigate
the
h Promiscuity
P
i
i off Molecular
M l
l Recognition
R
ii
Ligand-based Approaches
- Chemical similarity
- Data
D t mining
i i
- Chemogenomic approaches
- Pharmacohoric models
Koutsoukas A, et al. J. Proteomics. 2011
Target-based Approaches
- Inverse docking
- Structure-based pharmacophore screening
- Binding site similarity
- Protein-ligand fingerprints
Rognan D. Mol. Inf. 2010;29:176–87.
Conclusion
Renè Magritte
The Human Condition
(1935)