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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5701_Библиотеки_им_академика_М_И_Перельмана
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136
Allosteric Inhibition ATP-noncompetitive inhibitors exert their effects by binding
to allosteric sites on the MAPK protein rather than competing with ATP for the
active site. This mechanism offers the advantage of potentially higher selectivity
and reduced risk of cross-reactivity with other ATP-binding proteins.
Binding Specicity These inhibitors may bind to regions of the kinase outside the
ATP-binding pocket, which allows for greater selectivity in targeting specic iso-
forms or conformations of MAPKs.
Therapeutic Potential ATP-noncompetitive MAPK inhibitors hold promise for
therapeutic applications in diseases where kinase signaling is dysregulated, such as
cancer, metabolic disorders, neurodegenerative disorders, and inammatory condi-
tions. By modulating MAPK activity through noncompetitive mechanisms, these
inhibitors offer alternative strategies for therapeutic intervention.
Challenges Despite their potential benets, ATP-noncompetitive MAPK inhibi-
tors may face challenges related to specicity, pharmacokinetics, and off-target
effects. Optimization of these compounds is necessary to enhance their therapeutic
efcacy and safety prole.
4.2 Small Peptides asATP-Noncompetitive Inhibitors
ofMAPK 8, 9, 10
The rst ATP-noncompetitive inhibitor is a minimal JIP1 peptide, corresponding to
a C-terminal 11-amino acid peptide (JIP1 154–163) [1–6], which is able to inhibit
JNK activity with a K
d
value of 420nM against JNK1, compound in Fig.4.1 and
Table4.1. In order to improve peptide cell permeability and stability, researchers
designed the D-JNK1 peptide which is a retro-inverso D-peptide with a linked cell
permeable TAT sequence. These peptide JNK inhibitors bind with a JIP site which
were conrmed by X-ray crystallography (PDB code: 1UKH) and kinetics studies
[4, 5]. Kaoud etal. reported several peptide-based JNK inhibitors using JIP1 peptide
with or without TAT sequence [6]. They found a potent JNK2 inhibitor with an IC
50
value of 92nM (Table4.1). This peptide consists of a JIP-10 mer (JIP144–153,
PKRPTTLNLF), linker 6-aminohexanoyl and inverso HIV-TAT sequence. This
peptide inhibits phosphorylation of c-Jun and cell growth in a breast cancer cell line.
Similarly, a potent JNK inhibitor was obtained using JIP-10 mer, the same linker,
and nine arginines. These two peptides are very selective over JNK inhibition, more
than 160 times selective against p38α and 1700 times selective against ERK2.
However, peptide’s instability and poor cell permeability may interfere with the
advance stage development of JNK inhibitors from the JIP1 peptide.
4 Non-ATP Site Inhibitors andBi-dentate Inhibitors
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Fig. 4.1 Adapted from Yan etal. with permission from the American Chemical Society publica-
tions (ref 18) [Cartoon diagram of the JNK1 structure highlighting the location of the L-pepJIP1
(red and sticks) (PDB code: 1UKI) and the sequences of the scaffolding protein (L-pepJIP1)]. The
G loop is indicated in cyan, the MAP kinase insert in yellow, αC helix in brown, the activation loop
in magenta, the catalytic loop in blue, the loop connecting β5 and β6, and loop 16in gray. The
secondary structures are labeled based on reference
Table 4.1 JIP-1 site peptide binders
Peptide Sequence
JNK1,
IC
50
(μM)
JNK2,
IC
50
(μM)
JNK3,
IC
50
(μM) References
pepJIP
10 mer
Ac-PKRPTTLNLF-amide 1.05 2.15 2.67 Heo etal. [4]
D-JNK-1 dqsrpvqnlttprkprpprrrqrrkkG 0.05 0.025 0.05 Bonny etal. [33]
L-PYC71N FFWRLNKIFYFID Ngoei etal. [2]
L-PYC98 LAYRVIIPCFRKRYFCFL N.R. N.R. N.R. Ngoei etal. [3]
D-PYC98-
TAT
lfcfyrkrfcpiivryalGrrrqrrkkrG N.R. N.R. N.R. Ngoei etal. [3]
JIP
10
-
Δ-TAT
(inverso)
Ac-PKRPTTLNLF-
aminohexanoyl- RRRQRRKKRG-
amide
1.21 0.092 1.27 Kaoud etal. [6]
JIP
10
- Δ-R9 PKRPTTLNLF-aminohexanoyl-
RRRRRRRRR
1.13 0.89 1.29 Kaoud etal. [6]
N.R. not reported
4.2 Small Peptides asATP-Noncompetitive Inhibitors ofMAPK 8, 9, 10
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138
The ASK1-JNK3 signaling network has an important role in Parkinson’s disease.
Pan etal. discovered a 21-amino acid peptide named JNK3-N-TAT as a JNK3 non-
ATP site binding inhibitor [30]. The JNK3-N-TAT sequence is Tyr-Gly-Arg-Lys-
Lys- Arg-Arg-Gln-Arg-Arg-Arg-Cys-Ser-Glu-Pro-Thr-Leu-Asp-Val-Lys-Ile. It is a
10-amino acid sequence of the C9–18 segment of the N-terminus of JNK3 conju-
gated with the HIV-TAT sequence. This peptide inhibits the binding of β-arrestin 2
to the N-terminus of JNK3. JNK3-N-TAT peptide shows an improvement of mito-
chondrial function and reduces MPP
+
/MPTP-induced damage in both SH-SY5Y
cellular assays and PD mouse models.
Mitochondrial JNK signaling exhibits invitro and invivo DNA damage causing
various diseases. The JNK is activated by phosphorylation of two upstream kinases
(MKK4 and MKK7) that causes a small population of JNK to migrate to mitochon-
dria. To inhibit JNK translocation to the mitochondria selectively, investigators at
Scripps Research developed a peptide of mitochondrial membrane protein Sab with
small interfering RNAs (siRNAs) and a cell-permeable peptide to the KIM1 domain
of Sab [31]. This retro-inverso peptide consists of 11 amino acids corresponding to
the phosphorylated JNK binding site of Sab (AVVRPGSLDLR), conjugated with an
HIV-Tat motif to improve cell permeability. This peptide disrupts JNK translocation
to the mitochondria and reduces the phosphorylation of Bcl-2 without any remark-
able effect on c-Jun phosphorylation or AP-1 transcription in the nuclear. Regular
JIP1 peptide prevents these three functions; there is no selectivity. Tat-SabKIM1
peptide selectively prevents Bcl-2 phosphorylation, cell death, loss of mitochondrial
membrane potential, and superoxide generation in anisomycin- stressed HeLa cells.
Nevertheless, this peptide protects against 6-OHDA-induced oxidative stress, mito-
chondrial dysfunction, and neurotoxicity invitro and invivo by selectively blocking
the JNK mitochondrial translocation.
Laughlin etal. reported three crystal structures [32] for JNK3 bound to JIP1-
derived peptide (pepJIP1: RPKRPTTLNLF, PDB code: 4H39, 2.0Å), Sab-derived
peptide (pepSAB: AVVRPGSLDLP, PDB code: 4H3B, 2.1Å), and a substrate tar-
get, ATF2 (pepATF2: KHKHEMTLKFG, PDB code: 4H36, 3.0Å), respectively.
These three are different 11-mer D-motif peptides and bind to the C-terminal lobe
of JNK3. The crystal structure pepJIP1 with JNK3 reveals that peptide binds to the
carboxy-terminal and moves a rotation of the amino-terminal N-lobe. The Arg160
interacts with the residue Glu367 of JNK3 making a salt bridge. The Arg160 also
forms hydrogen bonding interactions with JNK3 Tyr168, the backbone of Trp362,
and a network of water. Thr162 and Thr163 form hydrogen bonding networks with
Arg165 of JNK3. PepJIP1 forms van der Waals interactions with Tyr171, Asp364,
Val156, and Val197 of JNK3. These interactions are not observed with pepATF2.
For the binding mode of pepSAB, Arg343 (JIP1 Arg160, ATF2 His49) makes an
electrostatic interaction with Glu367 of JNK3 and considerably shifts the L-16
loop and G-loop. The Arg160 of pepJIP1 is important for binding afnity. From
ITC, K
d
values are for pepJIP1 0.8μM, pepSAB 17.2μM, and pepATF2>10μM
respectively.
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4.3 Small Molecules asATP-Noncompetitive JNK (MAPK 8,
9, 10) Inhibitors
In 2008, investigators at Sanford-Burnham-Prebys reported a series of small mole-
cules as ATP-noncompetitive JNK inhibitors [7–10]. First small-molecule JIP site
inhibitor was discovered by a group of scientists from Sanford-Burnham-Prebys
coded BI-78D3 (compound 91) [7] which showed promising invitro and invivo
data. BI-78D3 inhibited JIP-JNK interaction with the IC
50
values of 500nM in
DELFIA assay and 280nM in kinase activity assays respectively. Molecular dock-
ing studies indicate that the benzodioxan group of BI-78D3 binds close to Arg127,
an extensive hydrogen bonding network, which occupies the JIP-1-JNK interaction
site. Liquid chromatography/mass spectrometry stability analysis suggests that
BI-78D3 has favorable microsome and plasma stability (T
1/2
=54min). BI-78D3 is
also a selective JNK inhibitor, almost inactive against p38α, PI-3K, mTOR, and
other kinases tested. BI-78D3 displays the inhibition of TNF-alpha-induced phos-
phorylation of c-Jun in a dose-dependent manner with an IC
50
value of 12.5μM in
HeLa cells. BI-78D3 abolishes ConA-induced liver damage as measured by the
release of the liver enzyme alanine-aminotransferase (ALT) to the circulation.
Furthermore, it restores insulin sensitivity compared to vehicle control in a mouse
mode of type-2 diabetes.
These molecules bind to JIP sites. The structures are shown in Fig.4.2. These
compounds should be more selective than ATP site inhibitors. Further optimization
of compound 91 resulted in compound 92 where the central core was replaced from
tetrazole to thiadiazole (Fig.4.2) [8]. Compound 92 was tested in a mouse model of
type-2 diabetes, 25mg/Kg dose, i.p., and showed a signicant reduction in blood
glucose levels compared to the vehicle control. Along the same line, researchers at
Sanford-Burnham reported the discovery of another molecule such as compound 93
that exhibited signicant inhibition of JIP-JNK activation invitro, in cell and favor-
able microsomal (T
1/2
=70min) and plasma stability (80% remaining after 1h) [9].
Further optimization of primary amine of compound 93 resulted in compound 94
(BI-90H9) as shown in Fig.4.3 as a secondary amine derivative, which had good
balance of potency from both JNK activity and DELFIA assays; improved cell per-
meability (76.3%) compared to BI-78D3 (30%, PAMPA % of ux), solubility, and
plasma stability (68% remaining after 1h [10]. These interesting results indicate
that targeting the protein-protein interaction between JNK and JIP with a small
molecule is a new and promising avenue for the development of novel therapeutic
drugs that inactivate the JNK pathway biology.
Investigators at Abbott Laboratories identied 68 compounds from an afnity-
based high throughput screening of a library of 500,000 small molecules [11]. They
extensively characterized allosteric binding site inhibitors using NMR spectros-
copy, Protein X-crystallography, surface plasmon resonance, direct enzymatic
activity, and activation cascade assays. However, they found many compounds
bound to the ATP site, conrmed by high-resolution NMR binding studies using
13
C-methyl-labeled inactive JNK1R1 (residues 1–364, Thr183-Glu, Tyr185Glu).
4.3 Small Molecules asATP-Noncompetitive JNK (MAPK 8, 9, 10) Inhibitors
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140
NN
N
SHO
O
O
S
N
NO
2
S
N
SS
N
NO
2
91 (BI-78D3) 92
IC
50
(JNK1) = 500 nM (DELFIA assay) IC
50
(JNK1) = 160 nM (DELFIA assa
y)
IC
50
(JNK1) = 280 nM (Kinase assay) IC
50
(JNK1) = 1800nM (Kinase assay
N
S
N
SS
N
NO
2
H
2
N
93
IC
50
(JNK1) = 239 nM (DELFIA assay)
IC
50
(JNK1) = 700 nM (Kinase assay)
JI
P-peptide: Arg-Pro-Lys-Arg-Pro-Thr-Thr-Leu-Asn-Leu-Phe
K
d
(JNK1) = 420 nM
Fig. 4.2 Recently reported JIP site inhibitors (non-ATP site or allosteric inhibitors)
S
N
N
S
N
H
O
S
N
NO
2
94 (BI-90H9)
JNK1 IC
50
= 160 nM (DELFIA)
JNK1 IC
50
= 4.8 µ
M (Kinase assay)
Fig. 4.3 Thiadiazole
derivative
Only two compounds (95, 96) bind to the non-ATP site (Fig.4.4) as these do not
exhibit any activity in biochemical assay using phosphorylated (e.g., active) JNK1
(IC
50
>50μM). These compounds (95, 96) bind to a different site on the inactive
form of the protein.
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141
N
N
N
NH
N
N
S
N
N
N
NH
N
N
N
95 96
IC
50
(JNK1) > 100 µM IC
50
(JNK1= 62 µM
IC
50
(MKK7-JNK1) = 7.8 µM IC
50
(MKK7-JNK1) = 7.7 µM
K
d
(JNK1-u) = 11 µM K
d
(JNK1-u) = 16 µM
K
d
(JNK1-a) > 50 µM K
d
(JNK1-a) > 50 µM
K
d
(p38-u) = 18 µM K
d
(p38-u)= 17 µM
K
d
(ERK2-u) = 18 µM K
d
(ERK2-u) = 32 µM
EC
50
(p-c-Jun) > 30 µM EC
50
(p-c-Jun) > 30 µM
OHHO
N
N
S
NS
N
F
HO
97 98
IC
50
(JNK1) > 100 µM IC
50
(JNK1) > 100 µM
IC
50
(MKK7-JNK1) = 2.9 µM IC
50
(MKK7-JNK1) =
3.8 µM
EC
50
(p-c-Jun) > 10 µM EC
50
(p-c-Jun) = 4 µM
Fig. 4.4 Non-ATP site binder
The X-ray crystallographic studies on compound 96 (Fig.4.5) in complex with
JNK1 (PDB code: 3O2M) reveal that it binds away from the ATP-binding site. It is
located in the MAP insertion region on the surface of the protein. The northern
binding pocket is occupied in the activation loop containing residues Ser179 to
4.3 Small Molecules asATP-Noncompetitive JNK (MAPK 8, 9, 10) Inhibitors
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142
F180
N
N
N
N
N
HN
N
Y230
I231
Y259
V256
Q253
Hinge
region
A
TP binding
site
MAP
insert re
gion
Faraway
Fig. 4.5 Non-ATP site binder
Glu185 which indicates a different conformation. The southern pocket is placed
with residues Gly242 to Ala267 and the western side pocket is occupied with resi-
dues Met242 to Trp234. The butyl group is inserted into a hydrophobic pocket close
to Trp234. The dimethyl pyrimidine moiety is accommodated between Ile231 and
Tyr230 and forms a ring-stacking interaction. The biaryl tetrazole moiety stays in
the activation loop. The tetrazole group does not make any specic interaction. Both
compounds are inactive in cytokine (TNFα)-stimulated JNK-1-active HepG2 cells.
In order to improve cellular potency, they made several analogs of this series such
as 97 and 98 by reducing molecular weight and replacing tetrazole moiety, while
maintaining lipophilicity and important interactions. Compound 98 inhibits the
c-Jun phosphorylation with an IC
50
value of 4μM.
The use of kinase inhibitors for treating non-oncology diseases requires great
selectivity of the target kinase.
Kauod et al. applied a new approach to nd non-ATP site binder [14]. They
screened a small molecule library from NCI diversity set using ligand-based virtual
screening with BI-78D3 as a reference compound. They found 11 hits (Fig.4.6)
from which natural product (−) zuonin A is the best. These compounds have some
structural features similar to BI-78D3, containing either a benzo[d][1,3]dioxole
moiety or nitro group or an aromatic ring with an electronegative substituent. The
key compound lignan (−)-zuonin A is a JNK-JIP site binder and exhibits a selectiv-
ity of 100-fold over ERK2 and p38 MAPKα.
4.4 Dual Site Binding MAP Kinase 8, 9, 10 Inhibitors
More recently, Pzer reported a small molecule (compound 111) where a com-
pound can bind to the ATP and/or JIP site from their extensive kinetic experiments
[12]. This compound had no cellular activity due to poor cell permeability. Similarly,
researchers at Sanford-Burnham reported a dual JNK inhibitor [13] denoted as
BI-98F10 (112) as shown in Fig.4.7. These compounds are termed as dual site
inhibitors or mixed inhibitors. BI-98F10 was a selective JNK inhibitor as this
4 Non-ATP Site Inhibitors andBi-dentate Inhibitors
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143
N
N
N
N
N
O
O
O
O
,
O
O
O
O
O
,
O
O
O
O
O
99 100 101
NH
N
H
N
HN
HN
HN
HN
NH
N
N
Br
,
N
S
N
N
OMe
,
N
S
O
N
H
H
N
S
NH
2
O
O
102 103 104
O
O
O
H
N
HO
O
O
,
S
N
O
2
N
N
H
O
NO
2
,
O
O
O
O
HO
O
O
105 106 107
O
NO
2
NN
O
O
N
O
,
N
N
HN
NH
N
N
,
N
N
HN
NH
N
N
O
108
BI-78D3
109 110
NN
N
S
HO
S
N
NO
2
O
O
Fig. 4.6 Non-ATP site JNK inhibitors
4.4 Dual Site Binding MAP Kinase 8, 9, 10 Inhibitors
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N
S
N
H
N
H
N
O
NH
2
ONH
O
F
F
S
N
H
O
S
ONH
2
111 112 (BI-9F10)
IC
50
(JNK) = 3600 nM IC
50
(JNK) = 1300 nM
Fig. 4.7 Dual site JNK inhibitors
compound was tested over 56 kinases at 25μM, shown inactive against other kinases
including other MAP kinase families (Table4.2).
ATP-noncompetitive JNK inhibitors represent a promising avenue for therapeu-
tic intervention in diseases characterized by dysregulated JNK signaling. Their
unique mechanism of action and potential for selective targeting offers opportuni-
ties for the development of novel treatments with improved efcacy and safety
proles.
4.5 Bi-dentate Inhibitors ofMAP Kinase 8, 9, 10
Bi-dentate JNK inhibitors are a specic type of inhibitor that binds to both c-Jun
N-terminal kinases (JNKs) ATP site and JIP site (Fig. 4.8). These inhibitors are
designed to simultaneously engage multiple binding sites on the JNK protein,
enhancing their afnity and potency compared to mono-dentate inhibitors.
Here are some key characteristics and considerations regarding bi-dentate JNK
inhibitors:
Dual Binding Sites Bi-dentate JNK inhibitors typically consist of two pharmaco-
phores connected by a linker. Each pharmacophore targets a distinct binding site on
the JNK protein, allowing for simultaneous interaction with multiple regions of
the kinase.
Enhanced Binding Afnity By engaging multiple binding sites, bi-dentate inhibi-
tors can achieve higher binding afnity and potency compared to mono-dentate
inhibitors. This dual-binding approach enhances the stability of the inhibitor- protein
complex and improves the inhibitory efcacy against JNK activity.
Selective Targeting Bi-dentate inhibitors can be designed to selectively target spe-
cic isoforms or conformations of JNKs, offering opportunities for greater selectiv-
ity and specicity in modulating JNK signaling pathways.
Therapeutic Potential Bi-dentate JNK inhibitors hold promise for therapeutic
applications in diseases where dysregulated JNK signaling is implicated, such as
cancer, inammatory disorders, and neurodegenerative diseases. Their enhanced
potency and selectivity make them attractive candidates for drug development.
4 Non-ATP Site Inhibitors andBi-dentate Inhibitors
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Table 4.2 Small molecules JIP site binders or dual inhibitors
Inhibitor
JNK
IC
50
(μM)
Binding
assay
JNK
IC
50
(μM)
Activity
assay References
NN
N
S
HO
S
N
NO
2
O
O
0.5 0.28 Stebbins etal. [7]
NN
S
S
H
2
N
S
N
NO
2
0.23 0.70 De etal. [8]
N
S
S
S
N
NO
2
0.16 1.8 De etal. [10]
NN
S
SN
H
S
N
NO
2
O
0.16 4.8 De etal. [9]
N
N
H
NC
Cl
3
CCl
3
Et
2
N
EtO
5.7 N.R. Chen etal. [12]
SN
H
O
S
O
NH
2
4.6 1.3 De etal. [13]
O
N
S
O
H
2
N
H
N
O
HN
N
N
F
F
3.60 NR Chen etal. [12]
N
N
NH
N
NN
N
>100 7.8μM
(MKK7-
JNK)
Comess etal.
[11]
4.5 Bi-dentate Inhibitors ofMAP Kinase 8, 9, 10
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