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Fragment-Based Drug Discovery
From fragment hit to lead compound
Graduate Lecture Series
Lecture 2
Dr Anthony Coyne
(anthony.g.coyne@gmail.com)
Outline
Recap of Lecture 1
Lecture 2 – From fragment hit to lead compound
Hit rate Challenging targets
Fragment library design
and composition
Fragment Growing
Cyclin Dependant Kinase (CDK)
Astex
Fragment Merging
Cytochrome P450 (CYP121)
Abell Group
Fragment Linking
Replication Protein A (RPA70A)
Fesik Group
Fragment Development
Target Protein
Secondary Screening
NMR Spectroscopy
Binding Affinity
ITC / SPR/ FP
Primary Screening
Thermal Shift / SPR / NMR
X-Ray
Recap of Lecture 1
Thermal Shift
SPR
FP
ITC
NMR
Christina Spry
Fragment Based Drug Discovery - the concept
High-throughput Screening (HTS) Fragment-based drug discovery (FBDD)
Libraries typically > 100,000
Molecular Weight > 300Da
Coverage of chemical space can be poor
Broader range of targets including whole-cell screening
approaches
Affinities typically in the mM range
Can be difficult to optimise hits as the structures can be
complex.
Libraries typically < 5000
Molecular Weight < 300Da
Requires well characterised targets
Affinities typically in the mM range
Iterative step-by-step optimisation possible to increase
the size of the molecule and potency
X-ray crystallography or 2D-NMR guided is critical for
optimisation
Biophysical methods tend to be low-medium throughput
Typically HTS screens are ran in parallel with a FBDD screen
Fragment Based Drug Discovery - Why is there the need for new methodology?
While HTS generally works for most enzyme classes in some cases this does not work
The limitations of HTS was highlighted by researchers from GSK who examined success rates in antibiotic drug discovery over a
five year period. Of the 70 campaigns (67 target based, 3 whole screening) only 5 leads were found
The reason for this failure was that the physicochemical properties of compounds that bind to anti-bacterials are different
(higher MW and lower logP) than other drug targets so HTS libraries are not suitable
This trend is also showing up with some protein-protein interaction targets (AZ – 15 targets and no hits)
FBDD the way forward with these targets?
E.coli ZipA (interacts with FtsZ)
Fragment Based Drug Discovery - Academia can make a impact on this area
Stephen W. Fesik (Vanderbildt University)
Cancer research drug discovery
Seth Cohen (UC San Diego)
Metalloproteins
Iwan De Esch (VU Amsterdam)
NMR Screening
Damien Young (Broad Institute/Baylor)
3D Fragments
Rod Hubbard (University of York/Vernalis)
Kinases, proteases
Paul Wyatt (University of Dundee (DDU))
Neglected diseases
Rob van Montford (ICR Sutton)
Cancer research drug discovery
Chris Abell (University of Cambridge)
Cancer research drug discovery
TB drug discovery
While HTS screening is done primarily in the realms of the pharmaceutical industry, fragment based drug discovery is within the
budgets of many academic research groups. This is an ever expanding list
Fragment Based Drug Discovery - Screening Cascade
Target Protein Fragment Library
Secondary Screening
NMR Spectroscopy
X-Ray
Binding Affinity
ITC / SPR
Molecular Design
Fragment analoging, Docking
Chemical Synthesis
Fragment Growing, Fragment Linking
Fragment Merging
Primary Screening
Thermal Shift / SPR / NMR
Other Assays
Enzymatic / FP
Lecture 1
Lecture 2
Iterative Development Cycle
A typical screening of a fragment library through
primary and secondary screening can take 1 - 6
months depending on the system.
Typically fragments will be found to bind with
potencies in the region of 10 mM to 5 mM.
Normally these are in the mid-micromolar region
With the stronger binding fragments these can be
quicker to progress although this is very much
target dependent.
The fragment elaboration step is very much
dependent on the information obtained from the
initial screening cascade.
How is fragment screening carried out?
Fragment Libraries
Typically fragment libraries are put together in-house (Pharma) or are purchased from
suppliers such as Maybridge, Enamine (Academia)
In-house libraries offer the possibility to include scaffolds and fragments that are
not present in commercial fragment libraries
In some cases commercial libraries can be biased to a specific scaffold (e.g. indole or
pyridine) or functional group (COOH)
Current focus of fragment library development is to include as
a diverse set of fragments so that the chemical space covered
Another focus has been to develop 3D fragments
(3DFrag.org). The aim of this is again to increase the diversity
and expand the chemical space of the fragment library.
3D fragments are available from some companies however at
a cost
1 mg ~£40
Some synthetic organic chemistry research groups are
developing methodology that can be applied to the synthesis
of these fragments (e.g. Damien Young (Baylor/Broad) and
James Bull (Imperial))
Expanding area of research
Target Type
What is meant by the term ‘challenging target’?
Initial fragment screening campaigns focused on kinases
These have clearly defined ATP pockets and are
considered more druggable
Typical hit rate: 5-10%
Protein-protein interactions are more difficult to target as
these do not have clearly defined pockets. These tend to
have ‘hot-spots’ on the protein surface where binding
occurs
Typical hit rate: 0.1-4%
CYP121
(Metalloprotein)
Hit rate 3.9%
CDK
(Kinase)
Hit rate 8.7%
RAD51-BRCA2
(Protein-Protein Interaction)
Hit rate 0.2 %
Hit Rate and Ligand Efficiency
Typical fragment screen – the numbers
Target Protein Fragment Library
Secondary Screening
NMR Spectroscopy
ITC / SPR
Primary
Thermal Shift / SPR / NMR
Development cycle
800 Fragments
90 Fragments
(11% Hit rate)
28 Fragments
(3.5% Hit rate)
~ 5 Fragments
The hit rate is dependent on the library size and composition. It also depends upon the type of target where the more
‘’challenging’ the target the fewer hits that arise from a fragment screening
Ligand Efficiency is one of a number of metrics used to look at fragment development (Lecture 1)
(Binding energy per atom in a ligand)
LE = DG/NHA
NHA = number of heavy atoms
DG = Gibbs free energy of binding (from KD)
Typically no more than 5 fragments are taken forward for development
LE 0.25-0.50
Murray, C.A.. et al, ACS Med. Chem. Lett., 2014, asap article
The elaboration of fragment hits into chemical probes or drugs aims to improve the affinity from mM to mM and eventually to
nM
Different strategies are employed
What happen when you get a confirmed fragment hit?
Fragment Growing
Fragment Merging
Fragment Linking
How is the potency of a fragment increased?
Fragment Elaboration
This is the most frequent method of increasing potency for a fragment and a number of successful fragment campaigns
have been carried out using this strategy
Typically a single fragment in a binding pocket is ‘grown’ using chemical synthesis to pick up further interactions with the
protein.
This is the case that is the most likely to arise where a single fragment binds to protein or multiple fragments bind to a
specific area of the binding pocket
Structural information on how the ligand binds to the protein is key to guiding fragment development
Enzyme
Enzyme
Fragment A
Fragment Growing
Fragment Growing –Kinases (CDK2)
Human Kinome
ATP
ADP
General phosphorylation reaction catalysed by kinases
The first targets that were screened using a fragment
based approach were kinases.
In many cases a key chemotype mimicking the
aminopurine ring typically comes out these fragment
screens
Typically the hit-rate for kinases are high due to the nature
of the ATP binding pocket
A major problem in targeting kinases is selectivity
(over 500 in human genome)
CDK2
Fragment Library
500 Fragments
Primary Screening
X-Ray crystallography
(Cocktails of 4 fragments)
X-Ray Crystallography
Isothermal titration calorimetry (ITC)
500 Fragments
>30 Fragments
4 Fragments
With companies such as Astex the screening
is carried out using X-ray crystallography
where the fragment are screened in cocktails
With this type of screening it is important to
ensure when cocktailing there is sufficient
fragment difference to ensure that when the
hits are deconvoluted that the fragment can
be identified
In some cases fragment libraries containing
Br modified fragments is used
Fragment Growing – CDK2 (Astex)
The fragment library was composed of a focused
kinase set, a drug fragment set and compounds
identified by virtual screening against a structure of
CDK 2
Small fragment library size
Fragment Screening Cascade - CDK2
Fragment Screening – X-ray crystallography
How are these fragments binding to
CDK2?
Fragment Growing - Kinases
%I 64% (1 mM)%I 54% (1 mM)
IC50 0.185 mM IC50 0.120 mM
Fragment Growing – CDK Series 1 and 2
Series 1
%I 64% (1 mM)
IC50 7 mM
IC50 1.9 mM
Series discontinued as optimisation below low micromolar is not
straightforward (LE not maintained through optimisation)
Series 2
%I 54% (1 mM)
IC50 1.6 mM
IC50 30nM
Series discontinued as while it showed good cellular activity did
not show good in-vivo activity
Fragment Growing – CDK Series 3
IC50 185 mM
LE 0.57
IC50 3 mM
LE 0.42
IC50 97 mM
LE 0.39
IC50 3 nM
LE 0.45
IC50 47 nM
LE 0.40
AT7519
Fragment growing of the initial indazole hit led to a compound with a 50 fold increase in potency. Removal of the phenyl
ring of the indazole offered a new startpoint and this was subsequently elaborated to a compound with a IC50 of 47 nM
with only a small drop in LE (AT7519)
Interestingly the piperidine is protruding out of the pocket toward solvent and the two chlorine atoms in the 2 and 6
position of the phenyl ring fill small hydrophobic pockets on the protein
AT7519 is currently in Phase II clinical trials and has shown good indications against a range of human tumor cell lines
The structure of AT7519 makes amenable to scale-up which is important in the later stage clinical trials
Series 3
Fragment Growing – Pros and Cons
Pros Cons
 Fragment growing is one of the most used methods
for increasing potency of a fragment.
 Choosing the right fragment is important and this is
driven by synthetic tractability and other medicinal
chemistry considerations. With well developed
chemistry the fragments can be elaborated with
ease.
 Multiple series can be taken forward using a
fragment growing strategy. The fragment
development is carried out in a stepwise manner
 Other successful fragment merging strategies
DNA Ligase – Astex
NAMPT – Genentech
b-Secretase – numerous companies
CDK4/CDK6 - Astex
 X ray-crystallography is key to determine the
position of the fragments in the binding pocket
Without X-ray information fragment growing can be
difficult.
This is where a number of fragments bind to a protein and bind in a similar region
Using structural information the overlap of the fragments can be combined using chemical synthesis to increase the
potency.
This is the case that is the most likely to arise where there is a common scaffold with variation on the substitution pattern
is observed
Structural information on how the ligand binds to the protein is key to guiding fragment development
Fragment Elaboration
Enzyme Enzyme
Fragment A
Fragment B
Fragment Merging
Merged
fragment
Fragment Merging – CYP’s (M. tuberculosis)
Cytochrome P450’s in M. tuberculosis
M. Tuberculosis contains 20 CYP’s of which the function of only five has been fully characterized.
This is an unusually high number of CYPs for the size of the genome
Human: 57 CYP’s (3234 Mb)
M. Tuberculosis: 20 CYP’s (4.4 Mb H37Rv)
E. Coli: 0 CYP’s ( 4.6 Mb)
Other organisms such as E. Coli do not contain any CYP’s and the M.tb genome contains a 200-fold gene density compared to
the Human genome
High density of CYP’s suggests importance in M. tuberculosis survival
.
Hudson S.A. et al, Biochem J., 2014, 57, 2455-2461
CYPome (M.tb)
CYP121
CYP125
CYP51
Fragment Merging – CYP121 (M. tuberculosis)
Sterol 14 a-demethylase
(40% sequence similarity with HsCYP51B1)
Cholesterol oxidase
(Other associated M. tb CYP’s CYP124 and CYP142)
Cyclodipeptide synthetases
(unique reaction to M. tb – no Hs comparison)
cYY Mycocyclosin
Fragment Merging – CYP121 (M. tuberculosis)
CYP121
Fragment Library
665 Fragments
Secondary Screening
NMR Spectroscopy
(WaterLOGSY, CPMG and STD)
Primary Screening
Thermal Shift (Hit > 0.8oC)
X-Ray Crystallography
Isothermal titration calorimetry (ITC)
665 Fragments
66 Fragments
(55 Fragments NMR)
9.9 % Hit rate
26 Fragments
(cYY Displaced)
(3.9% Hit rate)
5 Fragments
KD = 0.40 mM
LE = 0.39
KD = 1.60 mM
LE = 0.29
KD = 0.27 mM
LE = 0.35
KD = 3.0 mM
LE = 0.26
KD = 1.70 mM
LE = 0.32
Fragment screening against CYP121 yielded 5 fragments that were chosen to be
carried forward for elaboration
The KD of these fragments were in the range of between 0.27-3.0 mM which is
typically expected for fragment binding
The ligand efficiency (LE) of these fragments was good
How are these fragments binding to CYP121?
Hudson S.A. et al, Angew. Chem. Int. Ed, 2012, 51, 9311-9316
Fragment Merging – CYP121 (X-Ray Crystallography)
Heme binder through
NH2
Difficult to merge with
other fragments
Heme binder through NH2
Fragment Merging of
the two compounds
together
Non-heme binder
however shows two
distinct binding poses in
the X-Ray crystal
structure
Fragment Merging
Non-heme binder.
Merge with the
triazole fragment
Two distinct binding regions in CYP121 where fragments bind to the heme iron or further up the
pocket. There are a number of possible fragment merging strategies possible to increase potency
Hudson S.A. et al, Angew. Chem. Int. Ed, 2012, 51, 9311-9316
Fragment Merging – CYP121 (M. tuberculosis)
Strategy 1 (Heme binders)
Strategy 2 (Non-heme binders)
KD = 0.40 mM
LE = 0.39
KD = 1.60 mM
LE = 0.29 KD = 28 mM
LE = 0.39
Increase in potency when the two fragments are merged together.
Overlap in X-ray crystal structure on merged compound shows almost identical overlay with original fragments.
Ligand efficiency is maintained
KD = 3.0 mM
LE = 0.26
KD = 1.7 mM
LE = 0.32
No binding observed
Merging of these two compounds gave no increase in potency and had the opposite effect where no binding was
observed for the merged compounds.
Unsuccessful merging strategy
Hudson S.A. et al, Angew. Chem. Int. Ed, 2012, 51, 9311-9316
Fragment Merging – CYP121 (M. tuberculosis)
Strategy 3 (Non-heme binder)
KD = 1.7 mM
LE = 0.32
KD = 2.8mM
LE < 0.20
KD = 0.50 mM
LE = 0.24
KD = 40 mM
LE = 0.30
Merging of the two poses of the 1,2,4-triazole into a 1,5 disubstituted 1,2,3-triazole gave a compound which bound in a
similar pose as the initial fragment hit however the potency was much poorer
Further elaboration of the triazole ring to a pyrazole and subsequently an aminopyrazole had a significant effect on the
potency where this increased to 40 mM with a slight drop in ligand efficiency.
Successful merging strategy however further elaboration needed in order to increase the potency
With the three strategies in CYP121 only one gave an increase in potency where the fragments were directly
merged
Hudson S.A. et al, Angew. Chem. Int. Ed, 2012, 51, 9311-9316
Hudson, S.A., et al, ChemMedChem, 2013, 8, 1451-1455
Fragment Merging – Pros and Cons
Pros Cons
 X ray-crystallography is key to determine the
position of the fragments in the binding pocket
Without X-ray information fragment merging is
difficult.
 In some cases there is a potential overlap between
the fragments however the strategy might fail due to
the number/difficulty of synthetic steps.
 Where a merged compound is synthesised in some
cases no in binding affinity is observed possibly due
to subtle electronic/steric changes in the merged
molecule
 In many cases there is more than one fragment
that binds into the pocket and overlap is easy to
see using X-ray crystallography.
 The synthetic chemistry to synthesise the
fragments can be facile especially where the
fragment scaffolds are well studied (e.g. indoles,
pyridines)
 Other successful fragment merging strategies
- Nicotonamide phosphoribosyltransferase
(NAMPT) (Genentech)
- Chymase (Boehringer Ingelheim)
- Mcl-1 (Fesik – Vanderbildt)
- AmpC (Shoichet – UCSF)
- PI3 Kinase (Pfizer)
- AChBP (De Esch – VU Amsterdam)
Fragment Elaboration
This is where a number of fragments bind to a protein and in different regions of the binding pockets or on the surface of
a protein
Using structural information the fragments can be linked using chemical synthesis to increase the potency.
This is the most difficult approach to increasing potency as there has to be an optimal linker as well as ensuring the
binding interactions of the fragments are maintained
Only a handful of sucessful examples in the literature especially against protein-protein interaction targets
Enzyme Enzyme
Fragment B
Fragment A Fragment A
Fragment B
Fragment Linking
Fragment Linking – Protein-Protein Interactions
p53-HDM2
Bcl-BAD RAD51-BRC4
Protein-Protein interactions (PPI’s) are found throughout biological
systems. Typically these are defined as difficult targets as success
rates in targeting these has been low especially using HTS approaches.
Unlike conventional targets they do not have distinct binding pockets
however they have what is known as ‘hot-spots’ typically on the surface
of the protein
FBDD has been used successfully against a number of these
targets however none to date have been approved as drugs
although in a number of cases there are compounds in Phase I/II
development.
Why protein-protein interactions as targets?
Fragment Linking – Protein-Protein Interaction Inhibitors (FBDD)
Bcl-XL – Fragment Linking Approaches (Fesik (Abbott))
1st site
2nd site
1st site 2nd site
KD = 0.3 mM
1st site binder
KD = 4.3 mM
2nd site binder
Ki = 1.4 mM Ki = 36 nM
ABT263
Phase II
Ki < 0.5 nM
MW 973
One of the first successful examples of fragment linking against Bcl-XL where the initial fragment linking with an alkene gave a
significant drop in potency. Second site binder discovered through ‘SAR by NMR’
Subsequent elaboration led to the development of ABT273 which has a Ki <0.5 nM although the molecular weight of this
compound is large (MW 973). Looking at this structure there are still some components of the initial fragment hits present.
Do PPI inhibitors need to be higher in molecular weight due to the nature of the PPI interface?
Fragment Linking – Replication Protein A (RPA70N)
Replication Protein A: Stephen W. Fesik (Vanderbildt) PhD Connectut
PostDoc – Yale
Abbott (20 years)
Currently at Vanderbildt
University
Replication Protein A (RPA) is essential for eukaryotic DNA replication,
damage response and repair
The N-terminal domain of the RPA70 subunit (RPA70N) interacts with a wide
range of DNA processing proteins.
Small molecule inhibitors of these protein-protein interactions are of interest
as they have the potential as anticancer drugs in conjunction with
radiotherapy or chemotherapeutic agents
A number of X-ray crystal structures have been solved of RPA70N and show
distinct binding regions for both small molecules and peptides
Apo structure
(RPA70N)
Overlay of structure showing interaction
with the P53N fragment (Green)
Small molecule (VU079104) binding in a
site adjacent to P53 binding site
(Orange) (RPA70N)
RPA70N
Fragment Library
14976 Fragments
149 Fragments
(Hit Rate 1%)
Fragment Linking – Replication Protein A (RPA70N)
Site 1 Binders
52
KD 0.63-5 mM
LE up to 0.35
Site 1 and Site 2 Binders
81
Site 2 Binders
16
KD 0.49-5 mM
LE up to 0.28
Primary Screening
1H-15N HSQC 2D Protein Based NMR
Site 1 Binders (1H-15N HSQC 2D NMR)
Site 2 Binders (1H-15N HSQC 2DNMR)
The fragment library was screened in cocktails of 12
fragments and at a concentration of 20 mM.
Once a hit was obtained the mixture was deconvoluted.
Fragment Linking – Replication Protein A (RPA70N)
KD = 0.64 mM
LE = 0.24
Site 1 Binders Site 1 and Site 2 BindersSite 2 Binders
KD = 1.85 mM
LE = 0.31
KD = 0.71 mM (S1)
LE = 0.29
KD = 1.4 mM (S2)
LE = 0.26
KD = 0.58 mM (S1)
LE = 0.22
KD >2.0 mM (S2)
KD = 1.12 mM
LE = 0.28
KD = 1.62 mM
LE = 0.23
Rotation of the
phenyl ring off
the furan
A number of fragment-linking strategies are possible
Fragment Linking – Replication Protein A (RPA70N)
KD = 1.4 mM (Site 2)
LE = 0.26
KD = 0.58 mM (Site 1)
LE = 0.22
NMR KD = 26 mM
FP KD = 20 mM
(good agreement)
NMR KD = 1.9 mM
Fragment Linking
Fragment Linking – Replication Protein A (RPA70N) – Further applications
NMR KD = 1.9 mM
FITC-DFTADDLEEWFALAS-NH2
FITC-DFTADDLEEWZALLL---NH2
FP KD = 4.8 mM
FP KD = 220 nM
Fragment Linked Compound
Modified Stapled Peptide
While the fragment linked compound gave a KD (1.9 mM) with a ligand efficiency of 0.23 a further study by Fesik and co-
workers used the information from the fragment screening to develop a modified peptide which incorporated the
dichlorophenyl unnatural amino acid and this gave a peptide which bound with a KD of 220 nM
Fesik, S.W. et al, J. Med. Chem., 2014, 57, 2455-2461
Fesik, S.W. et al, J. Med. Chem., 2013, 56, 9242-9250
Fesik, S.W. et al, Biochemistry., 2013, 52, 6515-6524
Fesik, S.W. et al, ACS Med.Chem. Lett., 2013, 4, 601-605
With the study of RPA70N the proximity of the fragments makes introducing a linker seem facile however this is not always
the case.
Is there an easier methodology available for fragment linking?
New Fragment Linking Approaches – In-situ Click Chemistry
Huisgen (1968)
Sharpless and Fokin (2002)
Sharpless (2004)
1,4 and 1,5 isomer formed in a 1:1
ratio. Need to be heated over 80oC
Cu - 1,4-isomer
Ru - 1,5-isomer
Azide chemistry has undergone a renaissance with the advent of the CuAAC, RuAAC and SPAAC by Sharpess, Fokin, Meldal
and Bertozzi
In-situ click chemistry approach has
no metals present and the formation
of the product is templated by the
protein. The 1,4 or the 1,5 isomer can
be formed. Only select number of
examples have been reported
Acetylcholine esterase (AChE) (Sharpless et al (2004))
Enzymes as reaction vessels
Catalytic
binding site
Peripheral
binding site
Narrow ‘gorge’
between the two sites
Acetylcholine esterase (AChE) is a key component of neurological function and is a known drug target
This has two distinct binding sites, catalytic binding site and a peripheral binding site with a narrow ‘gorge’ between them.
There has been a number of inhibitors developed against the catalytic binding site and these have extended into the
narrow ‘gorge’
This was used as a test case to look at the ‘in-situ’ approach to linking the two active sites.
New Fragment Linking Approaches – In-situ Click Chemistry
PhD. Stanford University
(E.E. Van Tamelen)
PostDoc – Stanford
University and Harvard
Acetylcholine esterase (Sharpless et al (2004))
Tacrine (catalytic side binder, KD = 18 nM) and propidium (peripherial site binder, KD = 1.1 mM) were used as a test
cases where each has been appended with an azide or alkyne
A library of 8 tacrine and 8 propidium (8 x 8 array) derivatives were synthesised with variation in the alkyl chain length
which were then incubated with the AChE in pairs (azide/alkyne)
The 1,5-isomer (syn) was selectively synthesised in-situ where the potency was measured to be 14 pM and the 1,5-
isomer was not observed.
Why is there a difference in the potency of these isomers?
syn (1,5-isomer)
99 fM
anti (1,4-isomer)
14 pM
140 fold drop in potency
New Fragment Linking Approaches – In-situ Click Chemistry
Only isomer observed with
‘in-situ’ approach
Synthesised using CuAAC
New Fragment Linking Approaches – In-situ Click Chemistry
In-situ Fragment Linking Concept
Acetylcholine esterase (Sharpless et al (2004))
Has been applied to targeting other proteins – Abl tyrosine kinase, Carbonic anhydrase, Histone deacetylase 8, Nictonic
acetylcholine receptors, EthR.
Conventional fragment screening
Append fragments with ‘reactive’ functional groups – guided by X-ray
crystallography. Incubate an array of these modified fragments with the
protein and allow the protein to choose the optimal fragment linker length.
This strategy could be applied to find an linker by allowing the enzyme to choose the optimal length
Fragment Linking – Pros and Cons
Pros Cons
 One of the most difficult strategies to carry out as
there are not many cases where different fragments
bind into different regions of the enzyme
 The ideal linker can be difficult to find
 X ray-crystallography is key to determine the
position of the fragments in the binding pocket
Without X-ray information fragment linking is
difficult.
 This is seen as one of the best ways to increase
potency of two or more fragments binding to an
enzyme.
 In theory a compound derived from linking
fragments with an ideal linker is expected to
have a Gibbs free energy of bonding better than
the sun of the individual binding fragments
(superadditivity)
 Other successful fragment linking strategies
- Pantothenate synthetase (Abell -
Cambridge)
- EthR – (Abell – Cambridge)
- Bcl-Xl (Fesik – Abbott)
- Chitinase (Omura – Tokyo)
- LDHA (Astra Zeneca)
- HSP90 (Abbott)
Fragment Elaboration Strategies – A Comparison
Fragment
Growing
Fragment
Merging
Fragment
Linking
Enzyme
Fragment
Fragment elaboration strategies
Fragment growing: easiest option however
structural information is required in order to
grow the fragments
Fragment merging: Where fragments overlap
this is a good option however structural
information is key. In some cases the merged
compounds can be difficult to synthesise
Fragment linking: Observing two or more
fragments binding in separate parts of the
binding pocket is rare. Linking fragments
together optimally is very difficult. Structural
information is key
Fragment Based Drug Discovery - Where are we with? (2013)
Phase I Phase II Phase III
Approved
Vemurafenib
(BRAF Kinase)
AT13387
(HSP90 Astex)
AT7519
CDK2
AT9283
(Aurora, Astex)
AUY922
(HSP90 Vernalis)
Indeglitazar
(Plexxikon)
ABT8693
(VGEF, Abbott)
Navitoclax
(ABT263)
LY2886721
(BACE1, Lilly)
LY517717
(Fxa, Lilly)
PLX3397
(FMS, Plexxikon)
ABT518
ABT737
AZD3839
AZD5363
DG-051
IC776
JNJ-42756493
LEE011
LP-261
LY2811376
PLX5568
SGX-393
SGX-523
SNS-314
MK-8931
(BACE1, Merck)
Many of the drugs in Phase II/III are from smaller pharma companies. There is the distinct lack of compounds derived from a
fragment based approach in development from the big two – GSK and Pfizer
Future Directions
What does the future hold for fragment-based drug discovery?
Fragment-based drug discovery is here to stay and has become common place alongside HTS as a means for finding
compounds that bind to a target.
Fragment library design to expand the coverage of chemical space is an active area of research however these
fragments need to be synthetically accessible (synthetic organic chemistry)
Developments in fragment screening capabilities are key where the screening time needs to shortened and the amount
of protein used needs to be minimised.
Fragment elaboration strategies need to be faster and the application of methodologies such as ‘in-situ’ click chemistry
needs to be developed
Further drugs to be approved for clinical use
Key References
A three stage biophysical screening cascade for
fragment-based drug discovery
Mashalidis, E.H., Sledz, P., Lang, S., Abell, C
Nature Protocols, 2013, 8(11), 2309-2324
Fragment-based approaches in drug discovery and
chemical biology
Scott, D.E, Coyne, A.G., Hudson S.A., Abell, C
Biochemistry, 2012, 51(25), 4990-5003
Recent developments in fragment-based drug
discovery
Congreve, M., Chessari, G., Tisi, D., Woodhead, A.J.,
J. Med Chem., 2008 51 (13), 3661-3680
Structural biology in fragment-based drug design
Murray, C.W., Blundell, T.L.
Curr. Opin, Struct. Biol., 2010 20 (4), 497-507
Drugging challenging targets using fragment-based
approaches
Coyne, A.G., Scott, D.E, Abell, C
Curr. Opin. Chem. Biol, 2010, 14 (3), 299-307
Fragment based drug discovery and X-ray
crystallography (Topics in Current Chemistry)
Davis, T.G, Hyvönen, M,. (Eds)
Springer, 2012
ISBN: 3642275397
Fragment based drug discovery : A practical approach
Zartler, E., Shapiro, M (Eds)
Wiley-Blackwell, 2012
ISBN: 0470058137
Fragment based approaches in drug discovery : 34
(Methods and principles in Medicinal Chemistry)
Jahnke, W., Erlansson, D.A., Mannhold, R., Kubinyi, H. (Eds)
Wiley-VCH 2006
ISBN: 3527312919
http://practicalfragments.blogspot.co.uk
gives an up to date overview of what research is been carried
out in both academia and industry
Reaching the high-hanging fruit in drug discovery at
protein-protein interfaces
Wells, J.A., McClendon, C. L.
Nature, 2007, 450 (13), 1001-1009
Modulators of protein-protein interactions
Milroy, L-G., Grossmann, T.N. Hennig, S., Brunsved, L.,
Ottmannm C.
Chem. Rev, 2014, asap article (doi 10.1021/cr400698c)
Fragment-based approaches to finding novel small molecules that bind to proteins are now firmly established in drug
discovery and chemical biology. Initially developed primarily in a few centers in the biotech and pharma industry, this
methodology has now been adopted widely in both the pharmaceutical industry and academia. After the initial success
with kinase targets, the versatility of this approach has now expanded to a broad range of different protein classes such
as metalloproteins and protein-protein interactions. In the course of these two lectures we will explore the different
strategies for finding a fragment hit and the subsequent elaboration strategies used in order to increase potency to
develop a lead compound.

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Fragment Based Drug Discovery

  • 1. Fragment-Based Drug Discovery From fragment hit to lead compound Graduate Lecture Series Lecture 2 Dr Anthony Coyne (anthony.g.coyne@gmail.com)
  • 2. Outline Recap of Lecture 1 Lecture 2 – From fragment hit to lead compound Hit rate Challenging targets Fragment library design and composition Fragment Growing Cyclin Dependant Kinase (CDK) Astex Fragment Merging Cytochrome P450 (CYP121) Abell Group Fragment Linking Replication Protein A (RPA70A) Fesik Group Fragment Development
  • 3. Target Protein Secondary Screening NMR Spectroscopy Binding Affinity ITC / SPR/ FP Primary Screening Thermal Shift / SPR / NMR X-Ray Recap of Lecture 1 Thermal Shift SPR FP ITC NMR Christina Spry
  • 4. Fragment Based Drug Discovery - the concept High-throughput Screening (HTS) Fragment-based drug discovery (FBDD) Libraries typically > 100,000 Molecular Weight > 300Da Coverage of chemical space can be poor Broader range of targets including whole-cell screening approaches Affinities typically in the mM range Can be difficult to optimise hits as the structures can be complex. Libraries typically < 5000 Molecular Weight < 300Da Requires well characterised targets Affinities typically in the mM range Iterative step-by-step optimisation possible to increase the size of the molecule and potency X-ray crystallography or 2D-NMR guided is critical for optimisation Biophysical methods tend to be low-medium throughput Typically HTS screens are ran in parallel with a FBDD screen
  • 5. Fragment Based Drug Discovery - Why is there the need for new methodology? While HTS generally works for most enzyme classes in some cases this does not work The limitations of HTS was highlighted by researchers from GSK who examined success rates in antibiotic drug discovery over a five year period. Of the 70 campaigns (67 target based, 3 whole screening) only 5 leads were found The reason for this failure was that the physicochemical properties of compounds that bind to anti-bacterials are different (higher MW and lower logP) than other drug targets so HTS libraries are not suitable This trend is also showing up with some protein-protein interaction targets (AZ – 15 targets and no hits) FBDD the way forward with these targets? E.coli ZipA (interacts with FtsZ)
  • 6. Fragment Based Drug Discovery - Academia can make a impact on this area Stephen W. Fesik (Vanderbildt University) Cancer research drug discovery Seth Cohen (UC San Diego) Metalloproteins Iwan De Esch (VU Amsterdam) NMR Screening Damien Young (Broad Institute/Baylor) 3D Fragments Rod Hubbard (University of York/Vernalis) Kinases, proteases Paul Wyatt (University of Dundee (DDU)) Neglected diseases Rob van Montford (ICR Sutton) Cancer research drug discovery Chris Abell (University of Cambridge) Cancer research drug discovery TB drug discovery While HTS screening is done primarily in the realms of the pharmaceutical industry, fragment based drug discovery is within the budgets of many academic research groups. This is an ever expanding list
  • 7. Fragment Based Drug Discovery - Screening Cascade Target Protein Fragment Library Secondary Screening NMR Spectroscopy X-Ray Binding Affinity ITC / SPR Molecular Design Fragment analoging, Docking Chemical Synthesis Fragment Growing, Fragment Linking Fragment Merging Primary Screening Thermal Shift / SPR / NMR Other Assays Enzymatic / FP Lecture 1 Lecture 2 Iterative Development Cycle A typical screening of a fragment library through primary and secondary screening can take 1 - 6 months depending on the system. Typically fragments will be found to bind with potencies in the region of 10 mM to 5 mM. Normally these are in the mid-micromolar region With the stronger binding fragments these can be quicker to progress although this is very much target dependent. The fragment elaboration step is very much dependent on the information obtained from the initial screening cascade. How is fragment screening carried out?
  • 8. Fragment Libraries Typically fragment libraries are put together in-house (Pharma) or are purchased from suppliers such as Maybridge, Enamine (Academia) In-house libraries offer the possibility to include scaffolds and fragments that are not present in commercial fragment libraries In some cases commercial libraries can be biased to a specific scaffold (e.g. indole or pyridine) or functional group (COOH) Current focus of fragment library development is to include as a diverse set of fragments so that the chemical space covered Another focus has been to develop 3D fragments (3DFrag.org). The aim of this is again to increase the diversity and expand the chemical space of the fragment library. 3D fragments are available from some companies however at a cost 1 mg ~£40 Some synthetic organic chemistry research groups are developing methodology that can be applied to the synthesis of these fragments (e.g. Damien Young (Baylor/Broad) and James Bull (Imperial)) Expanding area of research
  • 9. Target Type What is meant by the term ‘challenging target’? Initial fragment screening campaigns focused on kinases These have clearly defined ATP pockets and are considered more druggable Typical hit rate: 5-10% Protein-protein interactions are more difficult to target as these do not have clearly defined pockets. These tend to have ‘hot-spots’ on the protein surface where binding occurs Typical hit rate: 0.1-4% CYP121 (Metalloprotein) Hit rate 3.9% CDK (Kinase) Hit rate 8.7% RAD51-BRCA2 (Protein-Protein Interaction) Hit rate 0.2 %
  • 10. Hit Rate and Ligand Efficiency Typical fragment screen – the numbers Target Protein Fragment Library Secondary Screening NMR Spectroscopy ITC / SPR Primary Thermal Shift / SPR / NMR Development cycle 800 Fragments 90 Fragments (11% Hit rate) 28 Fragments (3.5% Hit rate) ~ 5 Fragments The hit rate is dependent on the library size and composition. It also depends upon the type of target where the more ‘’challenging’ the target the fewer hits that arise from a fragment screening Ligand Efficiency is one of a number of metrics used to look at fragment development (Lecture 1) (Binding energy per atom in a ligand) LE = DG/NHA NHA = number of heavy atoms DG = Gibbs free energy of binding (from KD) Typically no more than 5 fragments are taken forward for development LE 0.25-0.50 Murray, C.A.. et al, ACS Med. Chem. Lett., 2014, asap article
  • 11. The elaboration of fragment hits into chemical probes or drugs aims to improve the affinity from mM to mM and eventually to nM Different strategies are employed What happen when you get a confirmed fragment hit? Fragment Growing Fragment Merging Fragment Linking How is the potency of a fragment increased?
  • 12. Fragment Elaboration This is the most frequent method of increasing potency for a fragment and a number of successful fragment campaigns have been carried out using this strategy Typically a single fragment in a binding pocket is ‘grown’ using chemical synthesis to pick up further interactions with the protein. This is the case that is the most likely to arise where a single fragment binds to protein or multiple fragments bind to a specific area of the binding pocket Structural information on how the ligand binds to the protein is key to guiding fragment development Enzyme Enzyme Fragment A Fragment Growing
  • 13. Fragment Growing –Kinases (CDK2) Human Kinome ATP ADP General phosphorylation reaction catalysed by kinases The first targets that were screened using a fragment based approach were kinases. In many cases a key chemotype mimicking the aminopurine ring typically comes out these fragment screens Typically the hit-rate for kinases are high due to the nature of the ATP binding pocket A major problem in targeting kinases is selectivity (over 500 in human genome)
  • 14. CDK2 Fragment Library 500 Fragments Primary Screening X-Ray crystallography (Cocktails of 4 fragments) X-Ray Crystallography Isothermal titration calorimetry (ITC) 500 Fragments >30 Fragments 4 Fragments With companies such as Astex the screening is carried out using X-ray crystallography where the fragment are screened in cocktails With this type of screening it is important to ensure when cocktailing there is sufficient fragment difference to ensure that when the hits are deconvoluted that the fragment can be identified In some cases fragment libraries containing Br modified fragments is used Fragment Growing – CDK2 (Astex) The fragment library was composed of a focused kinase set, a drug fragment set and compounds identified by virtual screening against a structure of CDK 2 Small fragment library size Fragment Screening Cascade - CDK2 Fragment Screening – X-ray crystallography How are these fragments binding to CDK2?
  • 15. Fragment Growing - Kinases %I 64% (1 mM)%I 54% (1 mM) IC50 0.185 mM IC50 0.120 mM
  • 16. Fragment Growing – CDK Series 1 and 2 Series 1 %I 64% (1 mM) IC50 7 mM IC50 1.9 mM Series discontinued as optimisation below low micromolar is not straightforward (LE not maintained through optimisation) Series 2 %I 54% (1 mM) IC50 1.6 mM IC50 30nM Series discontinued as while it showed good cellular activity did not show good in-vivo activity
  • 17. Fragment Growing – CDK Series 3 IC50 185 mM LE 0.57 IC50 3 mM LE 0.42 IC50 97 mM LE 0.39 IC50 3 nM LE 0.45 IC50 47 nM LE 0.40 AT7519 Fragment growing of the initial indazole hit led to a compound with a 50 fold increase in potency. Removal of the phenyl ring of the indazole offered a new startpoint and this was subsequently elaborated to a compound with a IC50 of 47 nM with only a small drop in LE (AT7519) Interestingly the piperidine is protruding out of the pocket toward solvent and the two chlorine atoms in the 2 and 6 position of the phenyl ring fill small hydrophobic pockets on the protein AT7519 is currently in Phase II clinical trials and has shown good indications against a range of human tumor cell lines The structure of AT7519 makes amenable to scale-up which is important in the later stage clinical trials Series 3
  • 18. Fragment Growing – Pros and Cons Pros Cons  Fragment growing is one of the most used methods for increasing potency of a fragment.  Choosing the right fragment is important and this is driven by synthetic tractability and other medicinal chemistry considerations. With well developed chemistry the fragments can be elaborated with ease.  Multiple series can be taken forward using a fragment growing strategy. The fragment development is carried out in a stepwise manner  Other successful fragment merging strategies DNA Ligase – Astex NAMPT – Genentech b-Secretase – numerous companies CDK4/CDK6 - Astex  X ray-crystallography is key to determine the position of the fragments in the binding pocket Without X-ray information fragment growing can be difficult.
  • 19. This is where a number of fragments bind to a protein and bind in a similar region Using structural information the overlap of the fragments can be combined using chemical synthesis to increase the potency. This is the case that is the most likely to arise where there is a common scaffold with variation on the substitution pattern is observed Structural information on how the ligand binds to the protein is key to guiding fragment development Fragment Elaboration Enzyme Enzyme Fragment A Fragment B Fragment Merging Merged fragment
  • 20. Fragment Merging – CYP’s (M. tuberculosis) Cytochrome P450’s in M. tuberculosis M. Tuberculosis contains 20 CYP’s of which the function of only five has been fully characterized. This is an unusually high number of CYPs for the size of the genome Human: 57 CYP’s (3234 Mb) M. Tuberculosis: 20 CYP’s (4.4 Mb H37Rv) E. Coli: 0 CYP’s ( 4.6 Mb) Other organisms such as E. Coli do not contain any CYP’s and the M.tb genome contains a 200-fold gene density compared to the Human genome High density of CYP’s suggests importance in M. tuberculosis survival . Hudson S.A. et al, Biochem J., 2014, 57, 2455-2461 CYPome (M.tb)
  • 21. CYP121 CYP125 CYP51 Fragment Merging – CYP121 (M. tuberculosis) Sterol 14 a-demethylase (40% sequence similarity with HsCYP51B1) Cholesterol oxidase (Other associated M. tb CYP’s CYP124 and CYP142) Cyclodipeptide synthetases (unique reaction to M. tb – no Hs comparison) cYY Mycocyclosin
  • 22. Fragment Merging – CYP121 (M. tuberculosis) CYP121 Fragment Library 665 Fragments Secondary Screening NMR Spectroscopy (WaterLOGSY, CPMG and STD) Primary Screening Thermal Shift (Hit > 0.8oC) X-Ray Crystallography Isothermal titration calorimetry (ITC) 665 Fragments 66 Fragments (55 Fragments NMR) 9.9 % Hit rate 26 Fragments (cYY Displaced) (3.9% Hit rate) 5 Fragments KD = 0.40 mM LE = 0.39 KD = 1.60 mM LE = 0.29 KD = 0.27 mM LE = 0.35 KD = 3.0 mM LE = 0.26 KD = 1.70 mM LE = 0.32 Fragment screening against CYP121 yielded 5 fragments that were chosen to be carried forward for elaboration The KD of these fragments were in the range of between 0.27-3.0 mM which is typically expected for fragment binding The ligand efficiency (LE) of these fragments was good How are these fragments binding to CYP121? Hudson S.A. et al, Angew. Chem. Int. Ed, 2012, 51, 9311-9316
  • 23. Fragment Merging – CYP121 (X-Ray Crystallography) Heme binder through NH2 Difficult to merge with other fragments Heme binder through NH2 Fragment Merging of the two compounds together Non-heme binder however shows two distinct binding poses in the X-Ray crystal structure Fragment Merging Non-heme binder. Merge with the triazole fragment Two distinct binding regions in CYP121 where fragments bind to the heme iron or further up the pocket. There are a number of possible fragment merging strategies possible to increase potency Hudson S.A. et al, Angew. Chem. Int. Ed, 2012, 51, 9311-9316
  • 24. Fragment Merging – CYP121 (M. tuberculosis) Strategy 1 (Heme binders) Strategy 2 (Non-heme binders) KD = 0.40 mM LE = 0.39 KD = 1.60 mM LE = 0.29 KD = 28 mM LE = 0.39 Increase in potency when the two fragments are merged together. Overlap in X-ray crystal structure on merged compound shows almost identical overlay with original fragments. Ligand efficiency is maintained KD = 3.0 mM LE = 0.26 KD = 1.7 mM LE = 0.32 No binding observed Merging of these two compounds gave no increase in potency and had the opposite effect where no binding was observed for the merged compounds. Unsuccessful merging strategy Hudson S.A. et al, Angew. Chem. Int. Ed, 2012, 51, 9311-9316
  • 25. Fragment Merging – CYP121 (M. tuberculosis) Strategy 3 (Non-heme binder) KD = 1.7 mM LE = 0.32 KD = 2.8mM LE < 0.20 KD = 0.50 mM LE = 0.24 KD = 40 mM LE = 0.30 Merging of the two poses of the 1,2,4-triazole into a 1,5 disubstituted 1,2,3-triazole gave a compound which bound in a similar pose as the initial fragment hit however the potency was much poorer Further elaboration of the triazole ring to a pyrazole and subsequently an aminopyrazole had a significant effect on the potency where this increased to 40 mM with a slight drop in ligand efficiency. Successful merging strategy however further elaboration needed in order to increase the potency With the three strategies in CYP121 only one gave an increase in potency where the fragments were directly merged Hudson S.A. et al, Angew. Chem. Int. Ed, 2012, 51, 9311-9316 Hudson, S.A., et al, ChemMedChem, 2013, 8, 1451-1455
  • 26. Fragment Merging – Pros and Cons Pros Cons  X ray-crystallography is key to determine the position of the fragments in the binding pocket Without X-ray information fragment merging is difficult.  In some cases there is a potential overlap between the fragments however the strategy might fail due to the number/difficulty of synthetic steps.  Where a merged compound is synthesised in some cases no in binding affinity is observed possibly due to subtle electronic/steric changes in the merged molecule  In many cases there is more than one fragment that binds into the pocket and overlap is easy to see using X-ray crystallography.  The synthetic chemistry to synthesise the fragments can be facile especially where the fragment scaffolds are well studied (e.g. indoles, pyridines)  Other successful fragment merging strategies - Nicotonamide phosphoribosyltransferase (NAMPT) (Genentech) - Chymase (Boehringer Ingelheim) - Mcl-1 (Fesik – Vanderbildt) - AmpC (Shoichet – UCSF) - PI3 Kinase (Pfizer) - AChBP (De Esch – VU Amsterdam)
  • 27. Fragment Elaboration This is where a number of fragments bind to a protein and in different regions of the binding pockets or on the surface of a protein Using structural information the fragments can be linked using chemical synthesis to increase the potency. This is the most difficult approach to increasing potency as there has to be an optimal linker as well as ensuring the binding interactions of the fragments are maintained Only a handful of sucessful examples in the literature especially against protein-protein interaction targets Enzyme Enzyme Fragment B Fragment A Fragment A Fragment B Fragment Linking
  • 28. Fragment Linking – Protein-Protein Interactions p53-HDM2 Bcl-BAD RAD51-BRC4 Protein-Protein interactions (PPI’s) are found throughout biological systems. Typically these are defined as difficult targets as success rates in targeting these has been low especially using HTS approaches. Unlike conventional targets they do not have distinct binding pockets however they have what is known as ‘hot-spots’ typically on the surface of the protein FBDD has been used successfully against a number of these targets however none to date have been approved as drugs although in a number of cases there are compounds in Phase I/II development. Why protein-protein interactions as targets?
  • 29. Fragment Linking – Protein-Protein Interaction Inhibitors (FBDD) Bcl-XL – Fragment Linking Approaches (Fesik (Abbott)) 1st site 2nd site 1st site 2nd site KD = 0.3 mM 1st site binder KD = 4.3 mM 2nd site binder Ki = 1.4 mM Ki = 36 nM ABT263 Phase II Ki < 0.5 nM MW 973 One of the first successful examples of fragment linking against Bcl-XL where the initial fragment linking with an alkene gave a significant drop in potency. Second site binder discovered through ‘SAR by NMR’ Subsequent elaboration led to the development of ABT273 which has a Ki <0.5 nM although the molecular weight of this compound is large (MW 973). Looking at this structure there are still some components of the initial fragment hits present. Do PPI inhibitors need to be higher in molecular weight due to the nature of the PPI interface?
  • 30. Fragment Linking – Replication Protein A (RPA70N) Replication Protein A: Stephen W. Fesik (Vanderbildt) PhD Connectut PostDoc – Yale Abbott (20 years) Currently at Vanderbildt University Replication Protein A (RPA) is essential for eukaryotic DNA replication, damage response and repair The N-terminal domain of the RPA70 subunit (RPA70N) interacts with a wide range of DNA processing proteins. Small molecule inhibitors of these protein-protein interactions are of interest as they have the potential as anticancer drugs in conjunction with radiotherapy or chemotherapeutic agents A number of X-ray crystal structures have been solved of RPA70N and show distinct binding regions for both small molecules and peptides Apo structure (RPA70N) Overlay of structure showing interaction with the P53N fragment (Green) Small molecule (VU079104) binding in a site adjacent to P53 binding site (Orange) (RPA70N)
  • 31. RPA70N Fragment Library 14976 Fragments 149 Fragments (Hit Rate 1%) Fragment Linking – Replication Protein A (RPA70N) Site 1 Binders 52 KD 0.63-5 mM LE up to 0.35 Site 1 and Site 2 Binders 81 Site 2 Binders 16 KD 0.49-5 mM LE up to 0.28 Primary Screening 1H-15N HSQC 2D Protein Based NMR Site 1 Binders (1H-15N HSQC 2D NMR) Site 2 Binders (1H-15N HSQC 2DNMR) The fragment library was screened in cocktails of 12 fragments and at a concentration of 20 mM. Once a hit was obtained the mixture was deconvoluted.
  • 32. Fragment Linking – Replication Protein A (RPA70N) KD = 0.64 mM LE = 0.24 Site 1 Binders Site 1 and Site 2 BindersSite 2 Binders KD = 1.85 mM LE = 0.31 KD = 0.71 mM (S1) LE = 0.29 KD = 1.4 mM (S2) LE = 0.26 KD = 0.58 mM (S1) LE = 0.22 KD >2.0 mM (S2) KD = 1.12 mM LE = 0.28 KD = 1.62 mM LE = 0.23 Rotation of the phenyl ring off the furan A number of fragment-linking strategies are possible
  • 33. Fragment Linking – Replication Protein A (RPA70N) KD = 1.4 mM (Site 2) LE = 0.26 KD = 0.58 mM (Site 1) LE = 0.22 NMR KD = 26 mM FP KD = 20 mM (good agreement) NMR KD = 1.9 mM Fragment Linking
  • 34. Fragment Linking – Replication Protein A (RPA70N) – Further applications NMR KD = 1.9 mM FITC-DFTADDLEEWFALAS-NH2 FITC-DFTADDLEEWZALLL---NH2 FP KD = 4.8 mM FP KD = 220 nM Fragment Linked Compound Modified Stapled Peptide While the fragment linked compound gave a KD (1.9 mM) with a ligand efficiency of 0.23 a further study by Fesik and co- workers used the information from the fragment screening to develop a modified peptide which incorporated the dichlorophenyl unnatural amino acid and this gave a peptide which bound with a KD of 220 nM Fesik, S.W. et al, J. Med. Chem., 2014, 57, 2455-2461 Fesik, S.W. et al, J. Med. Chem., 2013, 56, 9242-9250 Fesik, S.W. et al, Biochemistry., 2013, 52, 6515-6524 Fesik, S.W. et al, ACS Med.Chem. Lett., 2013, 4, 601-605 With the study of RPA70N the proximity of the fragments makes introducing a linker seem facile however this is not always the case. Is there an easier methodology available for fragment linking?
  • 35. New Fragment Linking Approaches – In-situ Click Chemistry Huisgen (1968) Sharpless and Fokin (2002) Sharpless (2004) 1,4 and 1,5 isomer formed in a 1:1 ratio. Need to be heated over 80oC Cu - 1,4-isomer Ru - 1,5-isomer Azide chemistry has undergone a renaissance with the advent of the CuAAC, RuAAC and SPAAC by Sharpess, Fokin, Meldal and Bertozzi In-situ click chemistry approach has no metals present and the formation of the product is templated by the protein. The 1,4 or the 1,5 isomer can be formed. Only select number of examples have been reported
  • 36. Acetylcholine esterase (AChE) (Sharpless et al (2004)) Enzymes as reaction vessels Catalytic binding site Peripheral binding site Narrow ‘gorge’ between the two sites Acetylcholine esterase (AChE) is a key component of neurological function and is a known drug target This has two distinct binding sites, catalytic binding site and a peripheral binding site with a narrow ‘gorge’ between them. There has been a number of inhibitors developed against the catalytic binding site and these have extended into the narrow ‘gorge’ This was used as a test case to look at the ‘in-situ’ approach to linking the two active sites. New Fragment Linking Approaches – In-situ Click Chemistry PhD. Stanford University (E.E. Van Tamelen) PostDoc – Stanford University and Harvard
  • 37. Acetylcholine esterase (Sharpless et al (2004)) Tacrine (catalytic side binder, KD = 18 nM) and propidium (peripherial site binder, KD = 1.1 mM) were used as a test cases where each has been appended with an azide or alkyne A library of 8 tacrine and 8 propidium (8 x 8 array) derivatives were synthesised with variation in the alkyl chain length which were then incubated with the AChE in pairs (azide/alkyne) The 1,5-isomer (syn) was selectively synthesised in-situ where the potency was measured to be 14 pM and the 1,5- isomer was not observed. Why is there a difference in the potency of these isomers? syn (1,5-isomer) 99 fM anti (1,4-isomer) 14 pM 140 fold drop in potency New Fragment Linking Approaches – In-situ Click Chemistry Only isomer observed with ‘in-situ’ approach Synthesised using CuAAC
  • 38. New Fragment Linking Approaches – In-situ Click Chemistry In-situ Fragment Linking Concept Acetylcholine esterase (Sharpless et al (2004)) Has been applied to targeting other proteins – Abl tyrosine kinase, Carbonic anhydrase, Histone deacetylase 8, Nictonic acetylcholine receptors, EthR. Conventional fragment screening Append fragments with ‘reactive’ functional groups – guided by X-ray crystallography. Incubate an array of these modified fragments with the protein and allow the protein to choose the optimal fragment linker length. This strategy could be applied to find an linker by allowing the enzyme to choose the optimal length
  • 39. Fragment Linking – Pros and Cons Pros Cons  One of the most difficult strategies to carry out as there are not many cases where different fragments bind into different regions of the enzyme  The ideal linker can be difficult to find  X ray-crystallography is key to determine the position of the fragments in the binding pocket Without X-ray information fragment linking is difficult.  This is seen as one of the best ways to increase potency of two or more fragments binding to an enzyme.  In theory a compound derived from linking fragments with an ideal linker is expected to have a Gibbs free energy of bonding better than the sun of the individual binding fragments (superadditivity)  Other successful fragment linking strategies - Pantothenate synthetase (Abell - Cambridge) - EthR – (Abell – Cambridge) - Bcl-Xl (Fesik – Abbott) - Chitinase (Omura – Tokyo) - LDHA (Astra Zeneca) - HSP90 (Abbott)
  • 40. Fragment Elaboration Strategies – A Comparison Fragment Growing Fragment Merging Fragment Linking Enzyme Fragment Fragment elaboration strategies Fragment growing: easiest option however structural information is required in order to grow the fragments Fragment merging: Where fragments overlap this is a good option however structural information is key. In some cases the merged compounds can be difficult to synthesise Fragment linking: Observing two or more fragments binding in separate parts of the binding pocket is rare. Linking fragments together optimally is very difficult. Structural information is key
  • 41. Fragment Based Drug Discovery - Where are we with? (2013) Phase I Phase II Phase III Approved Vemurafenib (BRAF Kinase) AT13387 (HSP90 Astex) AT7519 CDK2 AT9283 (Aurora, Astex) AUY922 (HSP90 Vernalis) Indeglitazar (Plexxikon) ABT8693 (VGEF, Abbott) Navitoclax (ABT263) LY2886721 (BACE1, Lilly) LY517717 (Fxa, Lilly) PLX3397 (FMS, Plexxikon) ABT518 ABT737 AZD3839 AZD5363 DG-051 IC776 JNJ-42756493 LEE011 LP-261 LY2811376 PLX5568 SGX-393 SGX-523 SNS-314 MK-8931 (BACE1, Merck) Many of the drugs in Phase II/III are from smaller pharma companies. There is the distinct lack of compounds derived from a fragment based approach in development from the big two – GSK and Pfizer
  • 42. Future Directions What does the future hold for fragment-based drug discovery? Fragment-based drug discovery is here to stay and has become common place alongside HTS as a means for finding compounds that bind to a target. Fragment library design to expand the coverage of chemical space is an active area of research however these fragments need to be synthetically accessible (synthetic organic chemistry) Developments in fragment screening capabilities are key where the screening time needs to shortened and the amount of protein used needs to be minimised. Fragment elaboration strategies need to be faster and the application of methodologies such as ‘in-situ’ click chemistry needs to be developed Further drugs to be approved for clinical use
  • 43. Key References A three stage biophysical screening cascade for fragment-based drug discovery Mashalidis, E.H., Sledz, P., Lang, S., Abell, C Nature Protocols, 2013, 8(11), 2309-2324 Fragment-based approaches in drug discovery and chemical biology Scott, D.E, Coyne, A.G., Hudson S.A., Abell, C Biochemistry, 2012, 51(25), 4990-5003 Recent developments in fragment-based drug discovery Congreve, M., Chessari, G., Tisi, D., Woodhead, A.J., J. Med Chem., 2008 51 (13), 3661-3680 Structural biology in fragment-based drug design Murray, C.W., Blundell, T.L. Curr. Opin, Struct. Biol., 2010 20 (4), 497-507 Drugging challenging targets using fragment-based approaches Coyne, A.G., Scott, D.E, Abell, C Curr. Opin. Chem. Biol, 2010, 14 (3), 299-307 Fragment based drug discovery and X-ray crystallography (Topics in Current Chemistry) Davis, T.G, Hyvönen, M,. (Eds) Springer, 2012 ISBN: 3642275397 Fragment based drug discovery : A practical approach Zartler, E., Shapiro, M (Eds) Wiley-Blackwell, 2012 ISBN: 0470058137 Fragment based approaches in drug discovery : 34 (Methods and principles in Medicinal Chemistry) Jahnke, W., Erlansson, D.A., Mannhold, R., Kubinyi, H. (Eds) Wiley-VCH 2006 ISBN: 3527312919 http://practicalfragments.blogspot.co.uk gives an up to date overview of what research is been carried out in both academia and industry Reaching the high-hanging fruit in drug discovery at protein-protein interfaces Wells, J.A., McClendon, C. L. Nature, 2007, 450 (13), 1001-1009 Modulators of protein-protein interactions Milroy, L-G., Grossmann, T.N. Hennig, S., Brunsved, L., Ottmannm C. Chem. Rev, 2014, asap article (doi 10.1021/cr400698c)
  • 44. Fragment-based approaches to finding novel small molecules that bind to proteins are now firmly established in drug discovery and chemical biology. Initially developed primarily in a few centers in the biotech and pharma industry, this methodology has now been adopted widely in both the pharmaceutical industry and academia. After the initial success with kinase targets, the versatility of this approach has now expanded to a broad range of different protein classes such as metalloproteins and protein-protein interactions. In the course of these two lectures we will explore the different strategies for finding a fragment hit and the subsequent elaboration strategies used in order to increase potency to develop a lead compound.

Hinweis der Redaktion

  1. Introduce fragment based approaches and talk about how CS talked about the initial screening and now the focus is going to be on the going from a fragment hit to a lead compound
  2. Expressible and stable protein