What is a fragment? FBDD Size of chemical space: 10 7 or 10 60 ? Ligand efficiency, LE HCS...
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What is a fragment? OH FBDD Size of chemical space: 10 7 or 10 60 ? Ligand efficiency, LE HCS Disadvantages? Advantages? Drug-like compounds (rule of 5) lead-like compounds (rule of 3) Group efficiency, LE Ligand-Lipophilicity Efficiency, LLE Drug attrition, promiscuit and side-effects No of compounds to screen Chance of finding a hit (complexity Evolving fragments Joining fragments Screening technologies Role of computation
What is a fragment? FBDD Size of chemical space: 10 7 or 10 60 ? Ligand efficiency, LE HCS Disadvantages? Advantages? Drug-like compounds (rule of 5)
What is a fragment? FBDD Size of chemical space: 10 7 or 10 60
? Ligand efficiency, LE HCS Disadvantages? Advantages? Drug-like
compounds (rule of 5) lead-like compounds (rule of 3) Group
efficiency, LE Ligand-Lipophilicity Efficiency, LLE Drug attrition,
promiscuit and side-effects No of compounds to screen Chance of
finding a hit (complexity) Evolving fragments Joining fragments
Screening technologies Role of computation
Slide 3
What is a fragment? (A) (B) K d = 400 M K d = 2 mM (C) K I <
0.5 nM (C) Is a Bcl-XL inhibitor that has been in phase I/IIa
cancer clinical trials; it was developed from fragments (A) and
(B). This is a full-size drug-like/lead-like compound, not a
fragment. (Aside: M = 10 -6 M; nM = 10 -9 M; mM = 10 -3 M)
Fragments (e.g. (A), (B)) have: low molecular mass (~100-200 Da)
typically low binding affinities (> 100 M)
Slide 4
Fragment based drug design: 2 key ideas 1 st key idea: The size
of chemical space Lipinksis rule of 5: a drug should have a
Molecular mass less than about 500 g mol -1. How many isomers are
there of C2H6 ___ C4H10 ___ C6H14 ___ C7H16?___ How many potential
drug-like molecules are there, i.e. with MM < 500 g mol -1 and
therefore with less than ~30 heavy atoms (i.e. excluding H) ? i.e.
how big is chemical space for drug-like compounds? How many
potential fragments are there with < MM < 250 Da (g mol -1 )
~12 heavy atoms? i.e. how big is chemical space for fragment-like
compounds? 1 2 4 7, including optical isomers 10 60 10 7 It is
feasible to screen a far greater proportion of fragment space
compared to chemical space for drug-like compounds.
Slide 5
Fragment based drug design: 2 key ideas 2nd key idea: Binding
efficiency Because fragments are much smaller, they will bind to
their target protein with lower affinity, typically M mM (rather
than M nM for drug-like compounds that can form far more
interactions). Consequently, the screening techniques employed in
FBDD must be more sensitive than those used in a HTS assay.
Nevertheless, the binding energy per atom can be as high as that
for good drug-like hits. Generally, sensitive biophysical
techniques are required (a) to detect weak binding and (b) to
determine the binding interactions. X-ray crystallography and NMR
are excellent for detecting low affinity binding of fragments;
X-ray crystallography has the advantage that it also shows how the
fragment bind. Surface plasmon reasonance (Biacore) (c.f. CARs
BS133 lecture notes) is also used. Although good fragment hits bind
weakly, they can still form good quality interactions with the
target enzyme. Once weakly binding fragments have been identified,
it is necessary to develop them into lead molecules, which will
naturally be much more drug- like in size.
Slide 6
FBDD motivation Success A number of biotech companies have used
FBDD to develop clinical candidates Failures The optimism of HTS
and combinatorial chemistry has not yielded the new drugs that had
been expected in the 1990s companies are looking for other
approaches and FBDD is one of those actively being considered
Disadvantages Difficult to screen low affinity compounds:
biophysical approaches required. High investment in structural
biology required to develop fragment hits into lead compounds as
this requires understanding of molecular interactions and of the
binding modes within the active site; also to eliminate false
positives. Some targets are not amenable to 3D- structure
determination. Advantages The need to screen only a small number of
compounds means that small biotechnology companies and universities
can do FBDD. (Universities are often good at biophysics).
Slide 7
HCS: high concentration screening HTS Can only detect compounds
that bind with a reasonable affinity and it will miss fragment hits
that bind with a lower affinity. HCS The advantages of a smaller
screening library can be partially obtained by using higher
concentrations for the ligands in the screen appropriate for
ligands up to 350 Da. The use of higher concentrations will make it
a little easier to detect the smaller hits that have lower
affinity. This is a half-way house towards FBDD.
Slide 8
Lead-like compounds Most drug-like compounds have a molecular
mass < 500 Da. But most lead compounds need to be modified to
increase potency; usually involves adding functional groups and so
increases the molecular mass. Starting a lead development programme
with a compound of mass ~500 Da leaves no room to develop the
compound (without first making it smaller). Starting with a mass of
around 350 Da makes this development easier. Indeed, it has been
observed that most lead-like hits have a molecular mass < 350
Da. (Note the rule of 3 for lead-like compounds: MM 300 Da, clogP
3, # H bond donors 3, # H bond acceptors 3, cf Lipinski rule of 5
for drug-like compounds) One strategy for following up a fragment
hit is to find lead-like compounds (e.g. with mass ~ 350 Da) that
are in some ways similar to the fragment hit screening ~500 such
compounds could be useful. (See the PC lab activities for ways of
checking if compounds are similar; NB clogP is a programme to
calculate log P)
Slide 9
Ligand efficiency, LE LE is a concept for comparing hits across
different series of compounds and for assessing the effectiveness
of compounds optimization. LE = -G / HAC -RTln(IC 50 ) / HAC Where
HAC is the number of heavy atoms (or non-hydrogen atoms) G is the
free energy of binding of the ligand; G will be ve if the drug
binds hence the ve sign to make sure LE is a +ve number The units
are kcal mol -1 (heavy atom) -1 the real world doesnt always use SI
units! and the literature often doesnt give them, hence my omission
below. For an oral drug with molecular mass < 500 Da (to fulfil
Lipinskis rules) and IC 50 < 10 nM, LE should be at least 0.3.
As the compound gets bigger during optimization, the idea is to
ensure that LE does not decrease too much but rather stays above
~0.3.
Slide 10
Calculating Ligand efficiency, LE IC 50 = 135 M LE = - 8.314 *
298 * -8.910 / (1000 *4.184* 11) = 0.48 ln(135 10 -6 ) = -8.910
(this is log to base e) (IC 50 = 135 M = 135 10 -6 M) R = 8.314, T
= 298, HAC = 11, by 1000 to covert from J to kJ and then by 4.184
to covert from kJ to kcal Using LE = -RTln(IC 50 ) / HAC
Slide 11
Group efficiency, GE (A) (B) IC 50 = 135 M IC 50 = 80 M The
group efficiency of the methyl group, 0.32, was determined from the
binding energy of the two fragments (A) and (B) that differ only in
a methyl group at this position. Assumptions: (i)that the total
binding free energy of the whole ligand is the sum of the binding
free energy of the groups this will not always be true. (ii)That
the fragments (A) and (B) bind to the target in the same way this
will not always be true (but can be checked by X-ray
crystallography). GE = -G / HAC, where G is the contribution of a
given group GE allows the estimation of an individual groups
contribution towards the overall free energy of binding. 0.28 0.54
0.42 1.5 0.32 1.6
Slide 12
Ligand-Lipophilicity Efficiency, LLE A significant part of
protein-ligand binding involves desolvation of the ligand.
Therefore, in general, provided that the shape of the drug is
favourable, the more lipophilic a ligand is, the more favourably it
will bind. A consequence of this is that lipophilic ligands could
bind to any hydrophobic binding pocket; this could be in the target
enzyme or conceivably in other enzymes. Thus, more hydrophobic
drugs tend to be more promiscuous (i.e. bind to targets that they
should not bind to. Analysis has shown that more lipophilic
compounds carry a higher risk of drug failure as this is associated
with nonspecific toxicity. Ligand-lipophicity efficiency is defined
as LLE = pIC 50 clog P ( or variants such as LLE = pK i clog P or
LLE = pK i log D ) where clogP is the log P calculated using the
clogP programme
Slide 13
Using Ligand-Lipophilicity Efficiency, LLE Inappropriate
physico-chemical properties are thought to be the main reason for
attrition (compound failure) in drug development. LLE can be used
to monitor a ligand during development to make sure that it does
not become too hydrophobic. The target value for LLE is around 5-7
or greater. Problems with hydrophobicity can be avoided by choosing
fragments that are not too hydrophobic. The mean clog P for recent
drugs from large pharma is ~ 3.5 4.2. The mean clog P for patented
compounds from Astex, a company based on FBDD was recently 2.4.
This suggests that drugs derived from FBDD are likely to have fewer
failures in development (or post development) and are likely to
have fewer side effects. This may be an additional advantage of
FBDD.
Slide 14
Complexity The more complex a ligand is, the less likely it is
to bind to a the target enzyme because More chance of steric
clashes More chance of hydrogen bond mismatch Large ligands will
inevitably be complex. This reduces the chance of finding hits.
Fragments do not suffer from this complexity issue anywhere near as
badly as lead-like ligands or drug-like ligands simply because they
are so much smaller.
Slide 15
Optimization of fragments Evolving fragments Structural
information, e.g. from X-ray crystallography is used to design
larger molecules that make additional interactions. A requirement
is that the fragment anchor does not change binding mode during the
fragment evolution. (This can be checked by X-ray crystallography).
An example is the evolution of the -secretase lead molecule (C)
from (A) via (B). Note that LE should be monitored during fragment
optimization to ensure that it stays > 0.3. (A) (B) (C) ~1 mM IC
50 = 130 M IC 50 = 0.08 M
Slide 16
Optimization of fragments Combining fragments Sometimes
different fragments are found to bind to different parts of the
target enzyme. They can be linked using a suitable linker. The
difficulty is finding a suitable linker that allows both fragments
to bind in their original positions without too much strain in the
linker; the linker also needs to interaction favourably with the
enzyme. An example is fragments (A) and (B) linked and then
optimized to (D) before being developed into (C), a Bcl- XL
inhibitor in clinical trial. (A) (B) K d = 400 M K d = 2 mM (D) Kd
= 6.9 M (C) K I < 0.5 nM
Slide 17
Optimization of fragments Fragment tethering Uses the formation
of a disulfide bond between a chemically reactive fragment and a
cysteine residue in the target protein. Fragments with the greatest
affinity for the protein within the vicinity of the cysteine form
stable disulfide bonds. These are detected by mass Spectrometry.
(A) reacts with enzyme, Enz Fragments react with (A) bound to
enzyme, e.g. B) (B) (C) Developed from (B) IC 50 = 0.19 M (D)
caspase-1 inhibitor IC50=0.005 M Finally, (D) from (C)
Slide 18
Using fragments to identify new binding sites Heat Shock
Protein 90 (HSP 90) inhibitors (A) Was discovered using an
NMR-based screening approach A fragment library was screened with
(A) already bound (B) was found to bind tightly in presence of (A)
but weakly in absence of (A), showing cooperative effects. (C) Was
designed using structural information from NMR and X-ray to
effectively join the two fragments but note some improvements to
the part that came from (B). (A) Kd = 20 M (B) Kd = 150 M (with A)
Kd > 5000 M (without A) (C) Kd = 4 M
Slide 19
Computational approaches Used to design fragment libraries,
e.g. by analysing existing drugs and decomposing them into
constituent fragments Docking (virtual screening) to (a) identify
possible fragments that might bind (b) Suggest the binding mode of
fragments known to bind to generate ideas on how to grow the
fragment (c) To check the predicted binding mode of compounds
suggested by analysis of the X-ray structure. Docking programs were
developed to work on drug-like and lead-like compounds so do not
always work on fragments
Slide 20
Screening technologies Surface plasmon resonance For
methodology see BS133_CAR_audio_SPR_4A.ppt* Useful when there is no
structure. Requires smaller amounts of protein than X-ray
crystallography or NMR. Can determine enthalpy and entropy of
binding. Can also determine kinetics of binding e.g. this will show
if there is a slow off-rate for the ligand. NMR The NMR signal of a
ligand can be monitored it will change if the ligand is displaced
by a fragment. E.g. in HSP 90 it is possible for fragments to
displace ADP which binds with low affinity changes in the ADP NMR
signal indicate that a fragment has bound. Why is it important that
ADP binds weakly? X-ray-crystallography Expensive but essential for
confirming the fragment binding mode and for generating ideas for
fragment optimization. Generally carried out by soaking the
compounds into crystals. The fragment binds weakly so cannot
displace a ligand that binds tightly