18 research outputs found
Hemoglobin-Based Oxygen Carrier Microparticles: Synthesis, Properties, and In Vitro and In Vivo Investigations
Bovine hemoglobin microparticles (Hb-MPs) as suitable
oxygen carriers
are fabricated easily by three key steps: coprecipitation of Hb and
CaCO<sub>3</sub> to make Hb-CaCO<sub>3</sub>-microparticles (Hb-CaCO<sub>3</sub>-MPs), cross-linking by glutaraldehyde (GA) to polymerize
the Hb and dissolution of CaCO<sub>3</sub> template to obtain pure
Hb-MPs. The Hb entrapment efficiency ranged from 8 to 50% corresponding
to a hemoglobin quantity per Hb-MP of at least one-third of that in
one erythrocyte. The Hb-MPs are spherical, with an average diameter
of 3.2 μm and high oxygen affinity. The methemoglobin level
was increased after preparation, but can be reduced to less than 7%
with ascorbic acid. Phagocytosis assays showed low immunogenicity
of Hb-MPs if the particles were cross-linked with low concentration
of GA and treated with sodium borohydride. Magnetite-loaded Hb-MPs
circulated up to 4 days after intravenous application
Development of Selective CBP/P300 Benzoxazepine Bromodomain Inhibitors
CBP (CREB (cAMP responsive element
binding protein) binding protein
(CREBBP)) and P300 (adenovirus E1A-associated 300 kDa protein) are
two closely related histone acetyltransferases (HATs) that play a
key role in the regulation of gene transcription. Both proteins contain
a bromodomain flanking the HAT catalytic domain that is important
for the targeting of CBP/P300 to chromatin and which offeres an opportunity
for the development of protein–protein interaction inhibitors.
Here we present the development of CBP/P300 bromodomain inhibitors
with 2,3,4,5-tetrahydro-1,4-benzoxazepine backbone, an <i>N</i>-acetyl-lysine mimetic scaffold that led to the recent development
of the chemical probe I-CBP112. We present comprehensive SAR of this
inhibitor class as well as demonstration of cellular on target activity
of the most potent and selective inhibitor TPOP146, which showed 134
nM affinity for CBP with excellent selectivity over other bromodomains
Development of Selective CBP/P300 Benzoxazepine Bromodomain Inhibitors
CBP (CREB (cAMP responsive element
binding protein) binding protein
(CREBBP)) and P300 (adenovirus E1A-associated 300 kDa protein) are
two closely related histone acetyltransferases (HATs) that play a
key role in the regulation of gene transcription. Both proteins contain
a bromodomain flanking the HAT catalytic domain that is important
for the targeting of CBP/P300 to chromatin and which offeres an opportunity
for the development of protein–protein interaction inhibitors.
Here we present the development of CBP/P300 bromodomain inhibitors
with 2,3,4,5-tetrahydro-1,4-benzoxazepine backbone, an <i>N</i>-acetyl-lysine mimetic scaffold that led to the recent development
of the chemical probe I-CBP112. We present comprehensive SAR of this
inhibitor class as well as demonstration of cellular on target activity
of the most potent and selective inhibitor TPOP146, which showed 134
nM affinity for CBP with excellent selectivity over other bromodomains
Development and Characterization of Type I, Type II, and Type III LIM-Kinase Chemical Probes
LIMKs are important regulators of actin and microtubule
dynamics,
and they play essential roles in many cellular processes. Deregulation
of LIMKs has been linked to the development of diverse diseases, including
cancers and cognitive disabilities, but well-characterized inhibitors
known as chemical probes are still lacking. Here, we report the characterization
of three highly selective LIMK1/2 inhibitors covering all canonical
binding modes (type I/II/III) and the structure-based design of the
type II/III inhibitors. Characterization of these chemical probes
revealed a low nanomolar affinity for LIMK1/2, and all inhibitors 1 (LIMKi3; type I), 48 (TH470; type II), and 15 (TH257; type III) showed
excellent selectivity in a comprehensive scanMAX kinase selectivity
panel. Phosphoproteomics revealed remarkable differences between type
I and type II inhibitors compared with the allosteric inhibitor 15. In phenotypic assays such as neurite outgrowth models
of fragile X-chromosome, 15 showed promising activity,
suggesting the potential application of allosteric LIMK inhibitors
treating this orphan disease
Development and Characterization of Type I, Type II, and Type III LIM-Kinase Chemical Probes
LIMKs are important regulators of actin and microtubule
dynamics,
and they play essential roles in many cellular processes. Deregulation
of LIMKs has been linked to the development of diverse diseases, including
cancers and cognitive disabilities, but well-characterized inhibitors
known as chemical probes are still lacking. Here, we report the characterization
of three highly selective LIMK1/2 inhibitors covering all canonical
binding modes (type I/II/III) and the structure-based design of the
type II/III inhibitors. Characterization of these chemical probes
revealed a low nanomolar affinity for LIMK1/2, and all inhibitors 1 (LIMKi3; type I), 48 (TH470; type II), and 15 (TH257; type III) showed
excellent selectivity in a comprehensive scanMAX kinase selectivity
panel. Phosphoproteomics revealed remarkable differences between type
I and type II inhibitors compared with the allosteric inhibitor 15. In phenotypic assays such as neurite outgrowth models
of fragile X-chromosome, 15 showed promising activity,
suggesting the potential application of allosteric LIMK inhibitors
treating this orphan disease
Development, Optimization, and Structure–Activity Relationships of Covalent-Reversible JAK3 Inhibitors Based on a Tricyclic Imidazo[5,4‑<i>d</i>]pyrrolo[2,3‑<i>b</i>]pyridine Scaffold
Janus
kinases are major drivers of immune signaling and have been
the focus of anti-inflammatory drug discovery for more than a decade.
Because of the invariable colocalization of JAK1 and JAK3 at cytokine
receptors, the question if selective JAK3 inhibition is sufficient
to effectively block downstream signaling has been highly controversial.
Recently, we discovered the covalent-reversible JAK3 inhibitor FM-381
(<b>23</b>) featuring high isoform and kinome selectivity. Crystallography
revealed that this inhibitor induces an unprecedented binding pocket
by interactions of a nitrile substituent with arginine residues in
JAK3. Herein, we describe detailed structure–activity relationships
necessary for induction of the arginine pocket and the impact of this
structural change on potency, isoform selectivity, and efficacy in
cellular models. Furthermore, we evaluated the stability of this novel
inhibitor class in <i>in vitro</i> metabolic assays and
were able to demonstrate an adequate stability of key compound <b>23</b> for <i>in vivo</i> use
Development, Optimization, and Structure–Activity Relationships of Covalent-Reversible JAK3 Inhibitors Based on a Tricyclic Imidazo[5,4‑<i>d</i>]pyrrolo[2,3‑<i>b</i>]pyridine Scaffold
Janus
kinases are major drivers of immune signaling and have been
the focus of anti-inflammatory drug discovery for more than a decade.
Because of the invariable colocalization of JAK1 and JAK3 at cytokine
receptors, the question if selective JAK3 inhibition is sufficient
to effectively block downstream signaling has been highly controversial.
Recently, we discovered the covalent-reversible JAK3 inhibitor FM-381
(<b>23</b>) featuring high isoform and kinome selectivity. Crystallography
revealed that this inhibitor induces an unprecedented binding pocket
by interactions of a nitrile substituent with arginine residues in
JAK3. Herein, we describe detailed structure–activity relationships
necessary for induction of the arginine pocket and the impact of this
structural change on potency, isoform selectivity, and efficacy in
cellular models. Furthermore, we evaluated the stability of this novel
inhibitor class in <i>in vitro</i> metabolic assays and
were able to demonstrate an adequate stability of key compound <b>23</b> for <i>in vivo</i> use
Discovery of a Chemical Tool Inhibitor Targeting the Bromodomains of TRIM24 and BRPF
TRIM24 is a transcriptional regulator
as well as an E3 ubiquitin
ligase. It is overexpressed in diverse tumors, and high expression
levels have been linked to poor prognosis in breast cancer patients.
TRIM24 contains a PHD/bromodomain offering the opportunity to develop
protein interaction inhibitors that target this protein interaction
module. Here we identified potent acetyl-lysine mimetic benzimidazolones
TRIM24 bromodomain inhibitors. The best compound of this series is
a selective BRPF1B/TRIM24 dual inhibitor that bound with a <i>K</i><sub>D</sub> of 137 and 222 nM, respectively, but exerted
good selectivity over other bromodomains. Cellular activity of the
inhibitor was demonstrated using FRAP assays as well as cell viability
data
Structure Enabled Design of BAZ2-ICR, A Chemical Probe Targeting the Bromodomains of BAZ2A and BAZ2B
The bromodomain containing proteins
BAZ2A/B play essential roles
in chromatin remodeling and regulation of noncoding RNAs. We present
the structure based discovery of a potent, selective, and cell active
inhibitor <b>13</b> (BAZ2-ICR) of the BAZ2A/B bromodomains through
rapid optimization of a weakly potent starting point. A key feature
of the presented inhibitors is an intramolecular aromatic stacking
interaction that efficiently occupies the shallow bromodomain pockets. <b>13</b> represents an excellent chemical probe for functional studies
of the BAZ2 bromodomains in vitro and in vivo
[1,2,4]Triazolo[4,3‑<i>a</i>]phthalazines: Inhibitors of Diverse Bromodomains
Bromodomains
are gaining increasing interest as drug targets. Commercially
sourced and de novo synthesized substituted [1,2,4]ÂtriazoloÂ[4,3-<i>a</i>]Âphthalazines are potent inhibitors of both the BET bromodomains
such as BRD4 as well as bromodomains outside the BET family such as
BRD9, CECR2, and CREBBP. This new series of compounds is the first
example of submicromolar inhibitors of bromodomains outside the BET
subfamily. Representative compounds are active in cells exhibiting
potent cellular inhibition activity in a FRAP model of CREBBP and
chromatin association. The compounds described are valuable starting
points for discovery of selective bromodomain inhibitors and inhibitors
with mixed bromodomain pharmacology