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Target identification and selection
(Drug development science)
Péter Arányi, DSc
Contents
• Introduction: general landscape
• Molecular mechanisms – molecular
targets
• Target selection and validation
• Examples
• Screening and structure optimization:
Development Candidate (DC) properties
• Examples
TÁRSADALMI
IGÉNY
52
RESEARCH
PRE-
CLINICAL
DEVELOP-
MENT
CLINICAL
DEVELOP-
MENT
PHASE I.
CLINICAL
DEVELOP-
MENT
PHASE II.
CLINICAL
DEVELOP-
MENT
PHASE III.
VALUE CHAIN OF DRUG DEVELOPMENT
P
R
O
D
U
C
T
UNMET NEED
FDA új gyógyszer regisztrációk
Mullard, 2015
FDA approved drugs 1930-2013
All indications 1453
Infectious
diseases
299
Cardiovascular
diseases
211
Oncology 193
Autoimmunity/
Inflammation
180
(Kinch, 2014)
FDA approved oncology drugs
(Kinch, 2014)
A LARGE PART OF DEVELOPMENT
COMPOUNDS FAIL
Lemorzsolódási görbe
0
2
4
6
8
10
12
preklinika I. Fázis II. Fázis III.Fázis NDA launch
Attrition curve
Reasons for attrition
The Innovative Medicines Initiative (IMI)
Research Agenda (2008)
Phase II failures 2008-2010
(Arrowsmith, 2011)
Phase II success rate ~ 20%
Phase III failures 2007-2010
83 (~50%) of submissions failed
Way to improve: avoid wishful thinking and rely on high quality science
(Arrowsmith, 2011)
EVOLUTION OF A DRUG
PREPA- RATION
AND SUBMISSION
OF NEW DRUG
APPLICATION
DOSSIER
MARKETING CLINICAL EVALUATION
IN MAN
PHASE I PHASE II PHASE III
P H A R M A C O V I G I L A N C E
Tolerance and
pharmaco-
kinetics
Biological activity and
research of a therapeutic
effect
Determination of the optimal
dose
Confirmation of the
therapeutic effect and
tolerance
1
RESEARCH DEVELOPMENT BIRTH
1 to 2 years 1 to 2 years 1 to 2 yrs Contin. 6 to 8 years
LCM
50 3 10 5 000
molecules
RESEARCH INTO
CHEMICAL STARTING
POINTS
OPTIMIZATION PRECLINICAL EVALUATION IN VITRO AND IN
ANIMALS
• Conception
• Synthesis
SCREENING
• Intrinsic activity
• Selectivity
• Oral absoprtion
• Duration of action
1 to 2 years
P
R
O
D
U
C
T
I
O
N
• Chemical Process
• General Pharma- cology Profile
• Analytical Methods
• Stability
• Safety
• Metabolism and Pharmacokinetics
• Formulation
LIFE
100000 50 1 10 3
Project mangement
• The course of drug development is
unpredictible, have realistic expectations for any
given project
• Running a drug development project is
Inherently difficult
• The larger the project the more numerous are
the problems
• Account for the fctors that may prevent success
• Take an objective view of the strategy
What kind of new drug do we
want?
Molecular pathomechanism
• What does it mean
• How does it help drug discovery
• Examples
Haemophilia - thrombosis
Direct anticoagulants
Clot-bound factor Xa activity was
resistant to inhibition by antithrombin,
suggesting that the ability to directly
inhibit clot-associated factor Xa, with no
requirement for a cofactor, could provide
an effective and highly localized
approach to the prevention of thrombus
growth.
Apixaban, a direct Factor Xa inhibitor
Warfarin, a vitamin K antagonist
• Antagonizes γ-carboxylation of prothrombin and Factor VII
• (Strongly variable effect, difficult dosing)
Cell types involved in inflammation
How do we proceed?
New target
Internal
Literature
Validation
Selected Target
TSS & LG plan
HTS/MTS
Pre-program A2L Program DC
Use rational approaches and structural information to enhance
and facilitate Drug discovery and optimisation
Discovery approach
• „Biology driven”
• Exploiting an original biological target with
probable role in the pathomechanism
• NME should be found, which interacts with
the molecular target, defining the clinical
indication at the same time
Validating the target
• Full validation is only possible with clinical
results
• Partial validation on the basis of indirect
data (literature and experimental) – Association of genetic polymorphism with disease
– Gene expression in healthy and diseased tissues
– Dependence of cellular functions on the level of gene
expression
– Transgenic and GM animals
– Gene silencing (RNAi)
– Preliminary pharmacology
Technology intensive research
HPLC, GC, /MS
Bioanalyzer
Microarray scanner
Real-time PCR, PCR
Druggabbility
• Postulate: since the binding sites on
biological molecules are complementary with
their ligands in terms of volume, topology
and physicochemical properties, then only
certain binding sites on putative drug targets
will be compatible with high-affinity binding
to compounds with drug-like properties.
• Extension of this concept to a whole genome
analysis leads to the identification of the
druggable genome. This is the expressed
proteome predicted to be amenable to
modulation by compounds with drug-like
properties.
Druggability concept (Vistoli et al 2008)
Classes of molecular targets
• Receptors
– GPCR
– Ion channel-bound
– RTK
– intracellular
• Ion channels
• Enzymes
– Protein kinases/phosphatases
– Hydrolases
– Enzymes metabolizing signal molecules
• Signal molecules
• Transcription factors
Results of target reseach
1996 2006
successful targets
(at least 1 marketed
drug)
120 268
research targets
(no product on market)
~380 1267
Potential targets
(human + microbial +
viral)
1700 - 3000
Zheng et al. (2006)
Gene family distribution of drug
targets
GPCR
Nuclear receptors
L-gated ion channel
V-gated ion channels
Penicllin binding
Myeloperoxidase.
Na-symporter
DNA topoisomerase
fibronectin
other
(Overington, 2006)
Molecular targets of FDA approved
drugs (2006)
GPCR Families (based on Analysis by Fredrikson et al; 2003)
CRHR2 CRF2
CALCRL CRLR
GRM2 mGlu2
GRM7 mGluR7
GLP1R GIPR
TM7XN1 GPR56 GPR97
PG, amine, opsin,
melatonin, EDF,
CB, MECA
Tachykinin, CCK,
NPY, AVP
SST, Gal,
Opioid, CC,
CXC
P2Y,
Thrombin,
LT, FSH
GPCR agonist drugs
• Morphine opioid analgesic
• Codeine opioid antitussive
• Salmeterol ß2 adrenerg asthma
• Buspirone 5HT1A (partial ag)
depression
• Sumatriptan 5HT1D migraine
Drug Receptor Indication
GPCR antagonist drugs
• Loratadin H1 allergy
• Cimetidine H2 ulcer
• Irbesartan AT1 hypertension
• Alfusosin α1 benign prostatic
hypertrophy
Drug Receptor Indication
(Levoye, 2007)
GPCR deorphanisation
An interesting target family: PKs
• 518 PKs coded in the human genome
• RTKs and intracellular signalling pathways
• Key role in cell proliferation and differentiated
cell functions
• Several oncogenes and cancer driver genes
code for PKs
• In vitro highly potent TK inhibitor series were
synthesized and are available
• The first TK inhibitor, Gleevec, approved in 2001
for CML and in 2002 for GIST
FDA approved cancer therapy
kinase inhibitors and mAbs
Kinase targeting oncology drugs
(Kinch, 2014)
FDA approved kinase inhibitors for
cancer therapy (examples)
Imatinib - Gleevec
Gefitinib - Iressa Afatinib - Gilotrif
FDA approved mAbs for cancer
Drug name, year of approval
2012 Perjeta (pertuzumab)
2013 Kadcyla (ado-
trastuzumab emtansine)
2013 Ganzyva (obinutu-
zumab)
2014 Cyramza
(ramucirumab)
2014 Keytruda
(pembrolizumab)
2014 Opdivo (nivolumab)
Indication
breast cancer
breast cancer
CLL
gastric cancer
melanoma
melanoma
Difficulties in developing PK
inhibitor drugs • Toxicity and side effects: selectivity issue
– Competition at the ATP binding site
– Type I (DFG in) inhibitors are generally less
selective than type II (DFG out) inhibitors
• Resistance
– Redundance of the signalling pathways
• Drug combinations or dual activity inhibitors
– New mutations occurring during treatment
• Inhibitors of mutant kinases
Dendrogram of 518 human protein
kinases
Manning et al, (2002)
Molecular targets in NSCLC
Class I PI3K signalling pathway
Liu et al, (2009)
Genetic alterations in the PI3K
pathway in cancer
Liu et al, (2009)
Signalling pathways in the heart
(Lal et al, 2013)
Signalling pathways in cancer
and heart
PI3-K
AKT
mTOR
RAF
MEK
ERK
nucleus
Plasma membrane RTK
cetuximab
erlotinib
idelalisib
Perifosine
everolimus
sorafenib
trametinib
preclinical
PD-1 signalling and effects
(Chinai et al 2015)
Efficacy of anti PD-1 antibodies
(pembrolizumab and nivolumab) shown
Orthogonal methods of target validation
• Genetic/epigenetic
– Differential gene expression in tissues/states • RNA
• protein
– KO gene
– Gene replacement by human ortholog gene
– Increased expression
– siRNA
Hardy & Peet, (2004)
Transcriptom map
(Caron et al 2001)
(11. human chromosome)
Proteomikai analízis (DIGE)
Orthogonal methods of target validation
• Chemical methods
– synthetic ligand
– mAB
– Aptamer
– Metabolic rescue (DHFR inhibitor trimetoprim
+ timidilát)
Hardy & Peet, (2004)
TECHNOLOGIES OF RESEARCH
HIGH THROUGHPUT SCREENING:
To cope with the high number of biological
targets provided by genomic
analysis and of compounds created by
combinatorial chemistry, it is
necessary to have rapid and efficient screening
tests.
41
Multiwell dish = microtiter plate
A
N
1 24
H
12
Screening flowchart
1st screen
Activity
2nd screen
selectivity
in vitro
metabolism
In vitro
absorption
In vivo pharma-
cology
In vivo pharma-
cology
In vivo pharma-
cology
passes
passes passes passes
passes
passes
passes
fails
fails
fails fails
New compounds
Prepared
By
combichem
New compounds
prepared
by parallel or
individual
synthesis
1 drug ≠ 1 target ≠ 1 phenotype
Screen systems – a comparison
High content screening
• Screen on living cells
• Fluorescent tags
• High resolution microscopy
• Parallel acquisition of data on different
properties
• Robotic handling
Predicting new targets for known
drugs • Drugs are not absolutely selective
• Polypharmacology defines 2nd (3rd) targets
• Similarity ensemble compares targets by the similarity of their ligands
• 3665 drugs compared against 65241ligands of 246 targets defined 184 new targets many of which unexpected
• 23 new drug-target associations experimentally confirmed
• The hallucinogen DMT known as σ1 ligand binds multiple serotonergic receptors with high affinity
Keiser et al, 2009
Targets for drugs
Chemical similarity between drugs
predicts affinities for unknown targets.
Some predictions verified experimentally:
ß1 antagonism by Prozac (transporter inhi-
bitor)
Inhibition of 5HT transporter by Vadilex
(NMDAR -ion channel - inhibitor)
H4 antagonism by Rescriptor (HIV reverse
transcriptase inhibitor)
Keiser, (2009)
Chemoinformatic drug repositioning may indicate new drug targets
Drug likeness (1/2)
• Various definitions of, and methods to predict,
drug-likeness have been proposed.
• Consensus is that drug-likeness is defined by
a range of molecular properties and
descriptors that can discriminate between
drugs and non-drugs for such characteristics
as oral absorption, aqueous solubility and
permeability. Computational property filters
can be used to rapidly assess the drug-
likeness of chemical libraries in silico before
purchase or synthesis.
Drug likeness (2/2)
• Orally administered drugs are likely to reside
in areas of chemical space defined by a limited
range of molecular properties.
• Lipinski’s ‘rule of five’. Historically, 90% of
orally absorbed drugs had
– fewer than five hydrogen-bond donors,
– less than ten hydrogen-bondacceptors,
– molecular masses of less than 500 daltons
– log P values (a measure of lipophilicity) of less than
five.
A DC should be • Active against target (in vitro)
• Selective
• Stable (as a substance)
• Efficient in vivo (both in acute a chronic
administration)
• Displaying acceptable ADME features
• Displaying an acceptable side effect profile
• Non toxic
• Patentable
• Better than the competitors
AND THAT REQUIRES…
MULTIDIMENSIONAL STRUCTURE OPTIMISATION
Biologicals • Proteins, peptides
– Replacement therapy (insulin)
– Antibodies (antiTNFα…)
– IL-1Ra (anakinra)
• Oligosacchrides
– Heparin and derivatives
• RNA
– Antisense
– siRNA
– miRNA
• Gene therapy
• Stem cells
Examples
Natural protein Humulin
GM fehérje Lantus
mAbs Orthoclone
OKT3
GM mAbs Remicade
Enzymes Pulmozyme
Vaccines TWINRIX
Aptamer Macugen
Diabetes
Diabetes
Kidney transplant
rejection
Crohn disease
Cystic fibrosis
Hepatitis A + rekombinant
hepatitis B
AR Macular degeneration
IgG1 antibody and related structures
H=heavy, L=light, VHH=camelid
IgNAR=shark ag receptor variable domain
ds=disulfid stabilizált
Antibody therapeutics, antibody
engineering, and the merits of
protein stability
S J Demarest* & S M Glaser (2008)
Popular mAb targets
Biopharmaceuticals in the
pipeline (2006)
2500 discovery phase
900 preclinical
1600 clinical phase
Drawbacks of biological drugs
• Limited therapeutic indications
• Parenteral administration (almost exclusively)
• Frequent unexpected AEs
• Safety and efficacy depends on production
process, may even change facility by facility
• High production cost/high price
• Expensive „generic” development: biosimilar
rather than generic
Biologicals – indispensable
therapeutics
• Substitution therapy for Proteins and/or
peptides
• Superior selectivity
• „non-druggable” biological targets
• Novel treatment options in case when the
available therapy falls short of need
Patenting
• Subject to the normal tests of
– 1) novelty,
– 2) inventive step and
– 3) industrial applicability.
• Patent expires 20 years from filing date
– In Europe, SPC can extend term for up to additional 5 years (see later!)
– Patent Term Extensions can exist in relation to US patents as well
• Patent protection is subject to renewal fees
Patent types
• Product Patents – Molecular
• Generic formula
• Specific formula – Salt
– Hydrates/Solvates/Anhydrous
• Process Patent – Methods for preparing the API
– Methods for preparing the formulation
• Use patent – Medical use patent
– Second/further medical indication
Oncology approvals
0
2
4
6
8
10
12
14
16
18
20
years (2006-2013)
all PKI
Oncology drugs approved in 2013
PK inhibitors mAbs other
Afatinib (angiokinase) Obinutuzumab (CD20) trastuzumab emtanzine
Ibrutinib (BTK) Denosumab (RANKL) lenalomide
Trametinib (MEK) pomalidomide
Regorafenib (VEGFR2-TIE) mechlorethamine
Dabrafenib (BRAF V600E) Ra223 dichloride
Colorectal cancer
• 160000 new diagnosis, 57000 deaths in the US each year
• EGFR - Cetuximab therapy (RAS) BRAF V600E mutations activate the MAPK signalling pathway
• Activating mutations of PI3K 1/3 of colorectal cancers (or IRS2 and AKT activating mutations in the same pathway)
Melanoma drugs
• Lifetime risk of developing melanoma: 2% of
men and women will have melanoma
• Metastatic melanoma fatal: 1 yr survival ~50%:
• The cytotoxic T lymphocyte antigen 4 inhibitor
ipilimumab, which takes the brakes off T cell
activation and thereby lowers the immune
system’s threshold for attack,
• BRAF V600E or V600K mutations: vemurafenib,
trametinib, dabrafenib
Non small cell llung cancer
• Lung cancer is the most common type of
cancer worldwide
• It caused about 160000 deaths in 2012
worldwide (~75% NSCLC)
• Lung carcinogenesis is multistep process,
many oncogenes mutated and/or
overexpressed and tumor suppressor
genes are altered
Molecular targets in NSCLC
EGFR targeted drugs in NSCLC
• Erlotinib
• Gefitinib
• Lapatinib &Erb-B2
• Afatinib blocks EGFRT790M
• Vandetanib &VEGFR
• Cetuximab
• Panitumumab
Other signal transduction
pathways driving NSCLC
and respective drugs
• C-Met crizotinib
• C-Kit sunitinib, dasatinib, imatinib
• PDGFR sunitinib, dasatinib, imatinib
• VEGFR bevacizumab, sunitinib,
sorafenib
For optimal results mutation details and
cancer genetics should be known
Cell types involved in inflammation
Cell types and kinases involved in
inflammation
• Tissue cells – Cytokines, mediators of
inflammation
• Monocytes, macrophages
– Cytokines, antigen
presentation
• T-cells – Cytotoxicity, TNF, IFN
• B-cells – Abs
Kinases mediating
these diverse cell
functions: Syk and Jak
Inhibitors:
• Syk: fostamatinib
• Jak: tofacitinib,
masitinib
Inflammatory diseases
• Rheumatoid arthritis
• Psoriatic arthritis
• Multiple sclerosis
• COPD
• Crohn disease
• Ulcerative colitis,
• etc
RA biologic and TK inhibitor
therapies
• Eight biologic agents are approved as first-line or second-line therapy in RA in 2013,
• five (adalimumab, certolizumab, etanercept, golimu-mab and infliximab) targeting tumor necrosis factor (TNF)
• three (abatacept, rituximab and tocilizumab) targeting co-stimulation of T cells, B cells, and interleukin 6 (IL-6).
• Syk inhibitor: fostamatinib
• Jak inhibitor: tofacitinib
Experimental drugs for RA
treatment • ABT-494 selective JAK-1 inhibitor
• Tofacitinib nonselective JAK-1/2/3 inhibitor;
serious side effects
• Baricitinib JAK-1/2 inhibitor serious side effects
• Sirukumab anti-IL-6 mAb
• Sarilumab anti-IL-6 Receptor mAb
• Clazakizumab humanized anti-IL-6 mAb
• QAL964 PI-3K inhibitor
• LY3090106 bispecific anti-IL-17 anti-(BAFF) mAb
Serious side effects of signal
transduction therapies • Infections
• Cancers
• Acute coronary syndromes
• Heart failure
• Immune reactions to biologics
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