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Irreversible TGS was pioneered by Benkovic and coworkers [106]. They developed multisubstrate adduct inhibitors by an irreversible reaction, in which the enzyme
glycinamide ribonucleotide transformylase templated the alkylation of its substrate
glycinamide ribonucleotide thiol analog with a folate-derived bromide. Wilson and
coworkers described an enzyme-templated epoxide opening reaction of folatederived epoxide with glycinamide ribonucleotide to generate a tight-binding inhibitor [107]. Huc and Nguyen described a similar approach in which inhibitors of
carbonic anhydrase (CAII) were generated via a reaction of a thiol with different
a-chloroketones in the presence of the Zn(II) metalloenzyme [108].
To date, the most popular in situ click chemistry, developed by Sharpless et al.,
is the 1,3-dipolar cycloaddition reaction, also known as Huisgen cycloaddition [109] between an azide and an alkyne to afford the 1,2,3-triazole moiety [93].
Totally different from the other highly reactive reagents mentioned previously
(e.g., aldehydes, hydrazine, thiols, epoxides, and a-chloroketones), the azide and
alkyne groups are low-reactive functional groups in organic chemistry and are
inert to biological molecules and living systems. The cycloaddition reaction of an
azide and an alkyne is extremely slow at room temperature. However, the
maximally unsaturated azide and alkyne bonds store a large amount of energy.
When these two groups are arranged in close proximity, such as in the active site
of a target protein [110], the 1,3-dipolar cycloaddition reaction is accelerated
remarkably.
Besides the reliability of the linking reaction between the azide and alkyne groups,
the newly formed 1,2,3-triazole ring has favorable physicochemical properties for
the biological system, which has been regarded as a nonclassical bioisotere of the
amide group. The 1,2,3-triazole ring possesses a large dipole moment of 5 Debye,
and the nitrogen atoms on the triazole ring are weak hydrogen bond acceptors. The
successful application of in situ azide and alkyne click chemistry has been reported for
the inhibitor design of acetylcholinesterase [111], carbonic anhydr ase [112], and HIV
protease [113].
11.3 CASE STUDIES OF FRAGMENT-BASED SCREENING FOR
BETTER BIOAVAILABILITY
11.3.1 Adenosine Kinase
Adenosine kinase [114] is a 39 kDa protein that is primarily responsible for the
intracellular metabolism of adenosine. Inhibitors of adenosine kinase have potential
use as anticonvulsant and antinociceptive agents. Compound 1 has poor solubility.
On the basis of known SARs, 2 was chosen as an anchoring pharmacophore, and
NMR-based screening (SAR by NMR) was used to identify alternative companion
fragments that bound to adenosine kinase in the presence of 1 mM 1 (Scheme 11.1).
Indole 3 was found bound to the bromobenzene-binding pocket with a K
d
of 3 mM.
The merging of 3 with 1 generated 4, which showed lower potency than 1, but had
better pharmacokinetic properties.
CASE STUDIES OF FRAGMENT-BASED SCREENING FOR BETTER BIOAVAILABILITY 431
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11.3.2 Leukocyte Function-Associated Antigen-1
Leukocyte function-associated antigen-1 (LFA-1) [115] is a heterodimeric transmembrane glycoprotein expressed on all leukocytes. An interaction between LFA-1
and its counterreceptors, the intracellular adhesion molecules (ICAM)-1, -2, and -3,
cause immune responses. Therapeutic prevention of LFA-1 from binding to ICAMs
has the potential for the treatment of inflammatory diseases and graft rejection after
transplantation. Diaryl sulfide lead compound 5 (IC
50
¼ 44 nM) was found by NMR
spectroscopy to bind to an allosteric site on the I domain of LFA-1. However, the
solubility of this compound is very low (0.9 mg/mL), and this compound shows no oral
bioavailability (Scheme 11.2). NMR-based fragment screening (SAR by NMR) was
used to identify new fragments in the presence of the substructure of 5 (i.e., 6,
IC
50
¼ 80 mM). A number of fragments were found to coexist with 6. Two of them,
7 and 8, have K
d
values of 300 mM and 10 mM, respectively. The merging of 7 or 8
with 6 generated 9 (IC
50
¼ 20 nM) or 10 (IC50¼ 40 nM). The solubility of 10 is
fourfold better than 5 and shows oral bioavailability.
11.3.3 Matrix Metalloproteinase 3 (Stromelysins)
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endoproteinases.
The inhibitors of MMPs-3 (also called stromelysins) have been implicated in the
treatment of cancer. Potent pept ide-based inhibitors designed on the basis of substrate
specificity had been discovered; however, many of these compounds exhibit poor
bioavailability. A previous HTS of 115,000 compounds had failed to identify any
nonpeptide inhibitors with a potency better than 10 mM. Acetohydroxamic acid (11)
was selected as an initial fragment based on its known ability to serve as a zinc chelator
N
N
N
Br
N N
O
NH
2
1
N
N
N
N
O
NH
2
N
H
2
3
N
N
N
HN
N
N
O
NH
2
HN
4
IC
50
nM1.7=
IC
50
nM10=
KdmM3=
Scheme 11.1 NMR-based fragment screening for adenosine kinase inhibitors.
432
FRAGMENT-BASED DRUG DESIGN: CONSIDERATIONS FOR GOOD ADME PROPERTIES
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(Scheme 11.3). A NMR-based screening (SAR by NMR) was conducted in the
presence of 11, and the biphenyl binders 12 and 13 were identified. The linking of 11
with 12 or 13 generated 14 (IC
50
¼ 25 nM) or 15 (IC50¼ 15 nM). However, due to
the lability of the hydroxamate moiety to hydrolysis, 14 and 15 lack oral
bioavailability [116].
Further structural modifications targeting to the hydroxamate analogs were made.
A SAR by NMR screen in the presence of 12 and 13 identified 16 with a K
d
of 50 mM.
The merging of 16 to 14 and 15 led to 17 and 18, having IC
50
values with stromelysin
of 340 and 62 nM, respectively. Compound 17 exhibits a C
max
of 28 mM and a t
1/2
of
about 2 h [117].
11.3.4 Protein Tyrosine Phosphatase 1B
Protein tyrosine phosphatase 1B (PTP1B) regulates phosphorylation of the insulin
receptor and is a promising target for diabetes and obesity therapy. However, finding
small-molecule inhibitors for this target has been recognized to be challenging.
PTP1B has two adjacent phosphotyrosine-binding sites. One is the catalytic
pocket. The other one is on the substrate recognition surface. NMR-based screening
(SAR by NMR) was used to identify a potential lead series that occupies both
sites (Scheme 11.4). Compound 19 was identified as a weak binder that showed
inhibition of PTP1B with a K
i
¼ 293 mM. A simple optimization led to 20, which was
shown to be a competitive and reversible inhibitor (K
i
¼ 39 mM). Structure-based
optimization led to 21 (K
i
¼ 1.1 mM), which extended a pentyl chain toward the
5
IC
50
=80 µM
6
S
N
O
N
NO
2
O
N
O
N
NO
2
O
O
O
N
H
+
+
S
N
O
N
Cl O
N
H
S
N
O
N
Cl O
Kd= 300 µM
7
K
d
=10mM
8
O
O
IC50=20nM
9
IC
50
=40nM
so lub ility: 4.1µg/mL
F%: 12.4
IC
50
= 44 nM
solubility: 0.9µg/mL
F%: 0
10
Scheme 11.2 NMR-based fragment screening for leukocyte function-associated antigen-1
inhibitors.
CASE STUDIES OF FRAGMENT-BASED SCREENING FOR BETTER BIOAVAILABILITY 433
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second phosphotyrosine-binding site. NMR-based screening to the second phosphotyrosine-binding site identified a weak binder (22). A closely related analog of 22 was
appended to 21 to generate 23, which exhibited a K
i
of 22 nM [118].
Starting with fragment 19, structure-based optimization led to 24 and 25, with K
i
values of 1.2 and 1.5 mM respectively [119]. NMR-based screening (SAR by NMR) of
the second phosphotyrosine-binding site identified 26. The linking of 26 to 24 led to
27 with a K
i
of 42 nM. The methyl ester (28) exhibited better potency [120].
Unfortunately, 23, 27, and 28 show no cell permeability. To increase the cell
permeability of the inhibitors, heterocyclic monocarboxylic acids were screened
by NMR spectroscopy, and fragment 29 was found to bind to the active site
(Scheme 11.5). Limited optimization led to 30. The linking of modified 30 and 26
generated 31, which displayed low micromolar potency; however, it displayed
cell permeability in a Caco-2 permeability assay and cell-based activity in
a phosphorylation assay [121].
Mass spectrometry-based approach (tethering) was also used to search monocarboxylic acid fragments that can bind to the catalytic binding site [122]. Because
the catalytic binding site of PTP1B is deep and highly conserved, the introduction of
Kd =20µM
IC
50
=0.34µM
IC
50
= 62 nM
12
IC
50
=25nM
IC
50
=15nM
14
+
K
d
=17mM
K
d
= 20 µ M
K
d
= 50 µ M
11
HO
N
H
O
HO CN
HO
CN
13
O CN
O
NH
HO
15
O
O
NH
HO
CN
O NH
OH
16
17
O CN
O
18
S
O CN
ONHOH
O
O
ONHOH
Scheme 11.3 NMR-based screening for matrix metalloproteinase 3 (MMP-3, stromelysis)
inhibitors.
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FRAGMENT-BASED DRUG DESIGN: CONSIDERATIONS FOR GOOD ADME PROPERTIES
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a cysteine mutation within the active site itself is not desirable. A technology called
breakaway tethering was developed [122]. In this method, a cysteine is introduced
outside the active site and alkylated with a spacer that has a thiol group positioned
toward the catalytic site (Figure 11.3). This thiol group is used to interrogate
fragment libraries. In the case of PTP1B, an R47C mutation was introduced, and
a 2-mercaptoethylcarbamoyl spacer group was appended (Figure 11.3). Fragment 32
was identified by breakaway tethering. It is a competitive inhibitor with a K
i
of
4.1 mM. The binding affinity for 32 is comparable to pTyr (K
m
¼ 4.9 mM). An X-ray
crystal structure of PTP1B with 32 showed that 32 binds to the catalytic site. This
fragment forces a loop that lines the active site to adopt a new open conformation,
which can be utilized to design selective inhibitors.
An X-ray crystallography-based method (Pyramid) was also used to search
monocarboxylic acid fragments to solve the problem that PTP1B generally recognizes
+
K
d
800=µM
29
NH
2
CO2H
OH
K
d
mM1.2=
26
Br
N
O
CO2H
N
O
CO2H
Ki148=µM
30
N
H
O
N
O
CO2H
O
F
CO2Me
OH
Ki6.9=µM
31
Scheme 11.5 NMR-based screening for protein tyrosine phosphatase 1B (PTP1B) inhibitors
with improved Caco-2 permeability.
Kd>1mM
22
+
K
d
=100µM
K
i
=293µM
19
20
N
N
O
O
O
OH
CO2H
O
O
CO
2
H
CO
2
H
CO
2
H
CO2H
CO
2
H
CO2R
HO
2
C
HO
2
C
CO
2
H
O
OH
OH
N
H
H
N
O
O
Ki=1.1µM
21
S
N
O
O
O
OH
N
H
H
N
O
O
K
d
=26µM
K
i
=39
µ
M
Ki=22nM
23
N
O
O
OH
R
N
H
H
N
O
O
24 R=CH
3
, Ki=1.2µM
25 R=CH
2
OH,Ki=1.5µ M
NH
2
N
O
O
OH
OH
OH
N
H
H
N
O
O
Kd=1.2mM
26
27
R=H, Ki=42nM
28 R=CH
3
, Ki=18nM
Scheme 11.4 NMR-based screening for protein tyrosine phosphatase 1B (PTP1B) inhibitors.
CASE STUDIES OF FRAGMENT-BASED SCREENING FOR BETTER BIOAVAILABILITY 435
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doubly anionic groups in the phosphate-binding pocket [67] (Figure 11.4). Compound
33 was identified with a K
i
of 86 mM. This fragment can be used as a starting point to
design new lead structures with superior physical and pharmacokinetic properties.
11.3.5 b-Secretase (BACE-1)
b-Secretase is a membrane-associated aspartyl protease that cleaves the membraneassociated amyloid precursor protein (APP) at an extracellular membrane site.
Subsequently, g-secretase cleaves APP within the transmembrane site, which
generates the b-amyloid peptide. The aggregation of b-amyloid peptides forms
neurofibrillary plaques. The accumulation of amyloid plaq ues and neurofibrillary
tangles is thought to cause Alzheimer’sdisease. Since b-secretase cleavage is the ratelimiting step in the process from the membrane-associated APP to b-amyloid peptide,
b-secretase is considered an important target for the treatment of Alzheimer’s disease.
However, HTS of several different compound collections and traditional medicinal
chemistry to convert peptidic inhibitors to peptidomimetic or nonpeptidic inhibitors
failed to provide a suitable hit (31). An X-ray crystallography screen (Pyramid)
identified two initial fragment hits, 34 and 35, which exhibited approximately
30–40% inhibition at 1 mM (Scheme 11.6) [123]. Fragment evolution of 34 led to
36 with an IC
50
of 94 mM. Further optimization led to 37 with an IC50of 25 mM [124].
On the basis of the structure of 35, virtual screening of fragment librar ies generated 38
as a primary hit with an IC
50
of 310 mM. Further fragment evolution led to 39–41. The
introduction of an indole moiety led to 42 with an IC
50
of 9.1 mM. Further modi-
fication of 42 led to 43 with an IC
50
of 0.69 mM.
HS
HS
SH
H
O
HS
S
S
S
S
R47C
N
(spacer)
= = various fragments
PTP1B
PTP1B PTP1B
Figure 11.3 Breakaway tethering on PTP1B.
N
N
N
N
H
H
HO
O
O
O
S
OH
O
O
NH
H2N
32
33
K
i
= 86 µM
Ki = 4.1 mM
Figure 11.4 Two monocarboxylic acid inhibitors of PTP1B.
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FRAGMENT-BASED DRUG DESIGN: CONSIDERATIONS FOR GOOD ADME PROPERTIES
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Ligand-based NMR spectroscopy (waterLOGSY) was also used to screen frag-
ment libraries (Scheme 11.7). Fragment 45 was identified as a hit with a K
d
of
4.45 mM. Databa se mining around 45 resulted in the identification of 46, with
a sevenfold increase of affinity [125]. X-ray crystallography, SPR, and two highly
sensitive bioassay techniques, fluorescence resonance energy transfer (FRET) and
electrochemiluminescence-based assay (referred as IGEN), were used to drive the
optimization of the primary fragment hit. To increase solubility and exploit potential
hydrogen-bonding interactions with the enzyme, fragment evolution identified 47,
which had an IC
50
of 86 mM in the SPR assay, and an IC50of 130 mM in the FRET
assay. The X-ray crystal structure of b-secretase in complex with 46 suggested an
3
N-methylation, which led to 48. The SPR, FRET, and IGEN IC50values for 48 were
180, 220, and 150 mM, respectively. Further fragment evolution led to 50 with a SPR,
FRET, and IGEN IC
50
of 5.7, 5.9, and 5.3 mM, respectively. In consideration of the
possible metabolic oxidation of the dihyd roisocytosine ring of 50, 51 was designed
with the aid of the crystal structure, resulting in IC
50
values of 80 and 470 nM in the
FRET assay and cell-based assay [126].
Tethering also successfully identified new primary fragment hit 52 for b-secretase
where V332C was introduced as the tethering site (Scheme 11.8). The optimization of
the fragment hit generated 53. The conversion of tethered ligand to nonconvalent
inhibitors led to the nonpeptidic compound 55 with a K
i
of 74 m M [127].
Surface plasmon resonance was used to identify the primary fragment hit for
b-secretase (Scheme 11.9) [128]. The tyrosine metabolite tyramine 56 was found to
NH2N
NH2N
H
N
NH2N OMe
IC
50
=~2mM
IC
50
=94µM
IC
50
=25µM
34
36
N
approximately
30-4 0%
inhibition at 1mM
35
NH
2
37
N
H
N
NH
2
N
H
N
NH
2
N
R
38
39: R = H, IC
50
=100µM
40: R=OMe,IC
50
=40µM
41: R=OnPr, IC
50
=24µM
N
H
N
NH
2
HN
N
H
N
NH
2
HN
X
IC50=310µM
42
IC50=9.1µM
43: R =CH, IC
50
=0.69µM
44:R=N,IC
50
=4.2µM
Scheme 11.6 X-ray crystallography screen (pyramid) for b-secretase (BACE-1) inhibitors.
CASE STUDIES OF FRAGMENT-BASED SCREENING FOR BETTER BIOAVAILABILITY 437
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have a Kdof 2 mM for b-secretase. 2-Ethyl substitution (57) resulted in increased
binding affinity (K
d
¼ 660 mM). The replacement of the 2-ethyl group with a para-
toluene group (59) led to an increase of binding affinity by more than one order of
magnitude.
5352
N
HN
O
S S V
332
enzyme
C
N
HN
O
S S V
332
enzymeC
O
Cl
O
N
HN
O
O
Cl
O
S
R
H2N
O
O
54: R = H, Ki = 170µM
55: Me = Me, K
i
= 74µM
Scheme 11.8 Tethering strategy for b-secretase (BACE-1) inhibitors.
56
K
d
= 2 mM
57
K
d
= 660 µM
58
Kd = 350 µM
59
Kd = 60 µM
H2N
H
2
N
H
2
N
H
2
N
OH
OH
OH
OH
Scheme 11.9 Surface plasmon resonance for b-secretase (BACE-1) inhibitors.
N
HN
N
N
H2N
N
N
H
2
N
H
2
N
HN
N
H2N
Kd =4.45mM
45
K
d
=660 µM
46
SPR IC50 = 86 µM
FRET IC
50
= 130 µM
SPR IC
50
= 180 µM
FRET IC
50
= 220 µM
IGEN IC50 = 150 µM
SPR IC
50
= 5.7 µM
FRET IC
50
= 5.9 µM
IGEN IC50 = 5.3 µM
cell assay IC
50
=90 µM
FRET IC
50
= 29 µM
IGEN IC
50
= 30 µM
FRET IC
50
= 80 µM
Cell assay IC50 = 470 nM
51
47
48
O
O
O
H
N
O
O
N
N
H
2
N
O
N
N
H
2
N
O
49 50
O
O
Scheme 11.7 Ligand-based NMR spectroscopy (waterLOGSY) for b-secretase (BACE-1)
inhibitors.
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FRAGMENT-BASED DRUG DESIGN: CONSIDERATIONS FOR GOOD ADME PROPERTIES
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11.3.6 SH2 Domain of pp60Src [62, 129]
pp60
Src is involved in signal transduction and plays an essential role in bone
resorption. The selective binder of the SH2 domain of
pp60
Src can inhibit bone
resorption and could be useful for the treatment of osteoporosis. Structure-based
medicinal chemistry has identified 60 as an inhibitor with an IC
50
of 9 nM
(Scheme 11.10). However, the phosphate group of 60 has a high rate of hydrolysis
by phosphatases, and the high charge property of the phosphate group of 60 precludes
cell penetration. The t
1/2
in rat plasma of 60 is reported to be 0.6 h in rat plasma. X-ray
crystallography-based screening (SAR by X-ray) identified 61 with an IC
50
of
2.5 mM, which occupies the binding site of the phosphate group. The merging of 61
with 60 generated 62 with an IC
50
of 3 nM. This compound shows superior rat and
human plasma stability (it is stable in rat and human plasma over 24 h).
11.3.7 Thrombin
Thrombin, a serine protease, which plays a central role in the blood coagulation
cascade, is an important target for thrombosis-related diseases such as deep vein
thrombosis, stroke, and myocardial infarction. The majority of the known thrombin
inhibitors are characterized by the presence of highly charged guanidine or benzamidine functionalities binding to the enzyme’sS1 pocket. While this featur e promotes
inhibitory potency, it is detrimental to oral bioavailability. One challenge for the
thrombin inhibitor design is the identification of nonbasic groups that can bind to
the thrombin S1 pocket. Ligand-based surface plasmon resonance screening was used
to identify new fragment hits that can bind to the thrombin S1 pocket. On-array
competition experiments with a known thrombin inhibitor showed that not only the
charged fragments 63 and 64 but also the nonbasic fragments 65–68 are among the top
ranked fragment hits (Figure 11.5)) [130].
The linking of 64 and 66 generated 69 with a K
i
of 200 mM (Scheme 11.11). X-ray
crystallography revealed that the 4-chlorophenylthioether moiety, but not the guanidinophenyl moiety, binds to the thrombin S1 pocket. Incorporation of a known
diphenylalanine proline dipeptide (70) scaffold led to 71 with a K
i
of 5 mM. On the
basis of computer modeling, cyclization of the thioether substructure with the
chlorophenyl ring generated 72 (K
i
¼ 2 nM). Incorporation of the pyrazinone acet-
amide scaffold from a known thrombin inhibitor led to 73 with a K
i
of 20 nM. The
HO2C CO
2
H
HO2C CO2H
O
HN
O
N
H
N
O
IC
50
=2.5mM
61
IC50=3nM
stable in rat and hu man plasma > 24 h
62
OPO3H
2
O
HN
O
N
H
N
O
+
IC
50
=9nM
t
1/2
=0.6hinratplasma
60
Scheme 11.10 X-ray crystallography-based screening for SH2 domain of
pp60
Src inhibitors.
CASE STUDIES OF FRAGMENT-BASED SCREENING FOR BETTER BIOAVAILABILITY 439
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substitution of the phenyl group in 73 with a pyridyl group led to 74 with an order of
magnitude increase in potency. However, intrinsically low aqueous solubility is an
inherent problem for the pyrazinone acetamide scaffold. When the amide group of 74
was changed to a secondary amine (75), the aqueous solubility was increased 10-fold
while retaining the potency at the same level (K
i
¼ 3 nM) [131].
X-ray crystallography-based fragment screening (Pyramid) was also used to
identify new fragment hits for thrombin (Scheme 11.12). Fragments 76–78 were
found to bind to the thrombin S1 pocket, where the guanidine or benza midine
functionalities of most thrombin inhibitors bind. The linking of 78 (IC
50
¼ 330 mM)
with 79 (IC
50
¼ 12 mM) led to 80 with an IC50of 1.4 nM [67, 132].
NH
NH
2
NH
2
NH
HN
HN
Br
Cl
Cl
Cl
S
S
O
O
O
O
O
N
64
65
66
67
68
63
Figure 11.5 Six fragment binders for thrombin identified by surface plasmon resonance.
Cl
S
O
66
O
N
NH
2
O
OH
HN
NH
2
NH
64
+
HN
H
2
N NH
N
H
O
O
SH2N
Cl
69
K
i
=200µM
O
N
NH
2
O
N
H
S
Cl
O
N
NH
2
O
N
H
S
Cl
70
71
Ki=5
µ
M
72
Ki=2nM
N
H
S
Cl
O
N
N
N
H
O
N
H
S
Cl
X
N
N
N
H
O
N
73
Ki=20nM
74 : X = O, Ki= 2 nM, solubility: 6.3
×
10-5mg/mL
75 : X = H
2
,
K
i
= 3 nM, solubility: 5.7
×
10-4mg/mL
Scheme 11.11 Surface plasmon resonance for thrombin inhibitors.
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