11 research outputs found
Protein Degradation by In-Cell Self-Assembly of Proteolysis Targeting Chimeras
Selective
degradation of proteins by proteolysis targeting chimeras
(PROTACs) offers a promising potential alternative to protein inhibition
for therapeutic intervention. Current PROTAC molecules incorporate
a ligand for the target protein, a linker, and an E3 ubiquitin ligase
recruiting group, which bring together target protein and ubiquitinating
machinery. Such hetero-bifunctional molecules require significant
linker optimization and possess high molecular weight, which can limit
cellular permeation, solubility, and other drug-like properties. We
show here that the hetero-bifunctional molecule can be formed intracellularly
by bio-orthogonal click combination of two smaller precursors. We
designed a tetrazine tagged thalidomide derivative which reacts rapidly
with a <i>trans</i>-cyclo-octene tagged ligand of the target
protein in cells to form a cereblon E3 ligase recruiting PROTAC molecule.
The in-cell click-formed proteolysis targeting chimeras (CLIPTACs)
were successfully used to degrade two key oncology targets, BRD4 and
ERK1/2. ERK1/2 degradation was achieved using a CLIPTAC based on a
covalent inhibitor. We expect this approach to be readily extendable
to other inhibitor-protein systems because the tagged E3 ligase recruiter
is capable of undergoing the click reaction with a suitably tagged
ligand of any protein of interest to elicit its degradation
Highly Potent Clickable Probe for Cellular Imaging of MDM2 and Assessing Dynamic Responses to MDM2-p53 Inhibition
MDM2
is a key negative regulator of the p53 tumor suppressor. Direct
binding of MDM2 to p53 represses the proteinās transcriptional
activity and induces its polyubiquitination, targeting it for degradation
by the proteasome. Consequently, small molecule inhibitors that antagonize
MDM2-p53 binding, such as RG7388, have progressed into clinical development
aiming to reactivate p53 function in <i>TP53</i> wild-type
tumors. Here, we describe the design, synthesis, and biological evaluation
of a trans-cyclooctene tagged derivative of RG7388, RG7388-TCO, which
showed high cellular potency and specificity for MDM2. The in-cell
reaction of RG7388-TCO with a tetrazine-tagged BODIPY dye enabled
fluorescence imaging of endogenous MDM2 in SJSA-1 and T778 tumor cells.
RG7388-TCO was also used to pull down MDM2 by reaction with tetrazine-tagged
agarose beads in SJSA-1 lysates. The data presented show that RG733-TCO
enables precise imaging of MDM2 in cells and can permit a relative
assessment of target engagement and MDM2-p53 antagonism in vitro
Synthesis and Biological Evaluation of JAHAs: Ferrocene-Based Histone Deacetylase Inhibitors
<i>N</i><sup>1</sup>-Hydroxy-<i>N</i><sup>8</sup>-ferrocenyloctanediamide, JAHA (<b>7</b>), an organometallic analogue of SAHA containing a ferrocenyl group as a phenyl bioisostere, displays nanomolar inhibition of class I HDACs, excellent selectivity over class IIa HDACs, and anticancer action in intact cells (IC<sub>50</sub> = 2.4 Ī¼M, MCF7 cell line). Molecular docking studies of <b>7</b> in HDAC8 (a,b) suggested that the ferrocenyl moiety in <b>7</b> can overlap with the aryl cap of SAHA and should display similar HDAC inhibition, which was borne out in an in vitro assay (IC<sub>50</sub> values against HDAC8 (Ī¼M, SD in parentheses): SAHA, 1.41 (0.15); <b>7</b>, 1.36 (0.16). Thereafter, a small library of related JAHA analogues has been synthesized, and preliminary SAR studies are presented. IC<sub>50</sub> values as low as 90 pM toward HDAC6 (class IIb) have been determined, highlighting the excellent potential of JAHAs as bioinorganic probes
3,5-Dimethylisoxazoles Act As Acetyl-lysine-mimetic Bromodomain Ligands
Histoneālysine acetylation is a vital chromatin post-translational modification involved in the epigenetic regulation of gene transcription. Bromodomains bind acetylated lysines, acting as readers of the histone-acetylation code. Competitive inhibitors of this interaction have antiproliferative and anti-inflammatory properties. With 57 distinct bromodomains known, the discovery of subtype-selective inhibitors of the histoneābromodomain interaction is of great importance. We have identified the 3,5-dimethylisoxazole moiety as a novel acetyl-lysine bioisostere, which displaces acetylated histone-mimicking peptides from bromodomains. Using X-ray crystallographic analysis, we have determined the interactions responsible for the activity and selectivity of 4-substituted 3,5-dimethylisoxazoles against a selection of phylogenetically diverse bromodomains. By exploiting these interactions, we have developed compound <b>4d</b>, which has IC<sub>50</sub> values of <5 Ī¼M for the bromodomain-containing proteins BRD2(1) and BRD4(1). These compounds are promising leads for the further development of selective probes for the bromodomain and extra C-terminal domain (BET) family and CREBBP bromodomains
Fragment-Based Discovery of 6āAzaindazoles As Inhibitors of Bacterial DNA Ligase
Herein we describe the application
of fragment-based drug design
to bacterial DNA ligase. X-ray crystallography was used to guide structure-based
optimization of a fragment-screening hit to give novel, nanomolar,
AMP-competitive inhibitors. The lead compound <b>13</b> showed
antibacterial activity across a range of pathogens. Data to demonstrate
mode of action was provided using a strain of <i>S. aureus</i>, engineered to overexpress DNA ligase
Discovery of a Potent Nonpeptidomimetic, Small-Molecule Antagonist of Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) and XāLinked Inhibitor of Apoptosis Protein (XIAP)
XIAP and cIAP1 are
members of the inhibitor of apoptosis protein
(IAP) family and are key regulators of anti-apoptotic and pro-survival
signaling pathways. Overexpression of IAPs occurs in various cancers
and has been associated with tumor progression and resistance to treatment.
Structure-based drug design (SBDD) guided by structural information
from X-ray crystallography, computational studies, and NMR solution
conformational analysis was successfully applied to a fragment-derived
lead resulting in AT-IAP, a potent, orally bioavailable, dual antagonist
of XIAP and cIAP1 and a structurally novel chemical probe for IAP
biology
Optimization of Sphingosine-1-phosphateā1 Receptor Agonists: Effects of Acidic, Basic, and Zwitterionic Chemotypes on Pharmacokinetic and Pharmacodynamic Profiles
The efficacy of the recently approved
drug fingolimod (FTY720)
in multiple sclerosis patients results from the action of its phosphate
metabolite on sphingosine-1-phosphate S1P<sub>1</sub> receptors, while
a variety of side effects have been ascribed to its S1P<sub>3</sub> receptor activity. Although S1P and phospho-fingolimod share the
same structural elements of a zwitterionic headgroup and lipophilic
tail, a variety of chemotypes have been found to show S1P<sub>1</sub> receptor agonism. Here we describe a study of the tolerance of the
S1P<sub>1</sub> and S1P<sub>3</sub> receptors toward bicyclic heterocycles
of systematically varied shape and connectivity incorporating acidic,
basic, or zwitterionic headgroups. We compare their physicochemical
properties, their performance in <i>in vitro</i> and <i>in vivo</i> pharmacokinetic models, and their efficacy in peripheral
lymphocyte lowering. The campaign resulted in the identification of
several potent S1P<sub>1</sub> receptor agonists with good selectivity
vs S1P<sub>3</sub> receptors, efficacy at <1 mg/kg oral doses,
and developability properties suitable for progression into preclinical
development
Structure of the Epigenetic Oncogene MMSET and Inhibition by <i>N</i>āAlkyl Sinefungin Derivatives
The members of the NSD subfamily
of lysine methyl transferases
are compelling oncology targets due to the recent characterization
of gain-of-function mutations and translocations in several hematological
cancers. To date, these proteins have proven intractable to small
molecule inhibition. Here, we present initial efforts to identify
inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal
structural methods. On the basis of 2D NMR experiments comparing NSD1
and MMSET structural mobility, we designed an MMSET construct with
five point mutations in the N-terminal helix of its SET domain for
crystallization experiments and elucidated the structure of the mutant
MMSET SET domain at 2.1 Ć
resolution. Both NSD1 and MMSET crystal
systems proved resistant to soaking or cocrystallography with inhibitors.
However, use of the close homologue SETD2 as a structural surrogate
supported the design and characterization of <i>N</i>-alkyl
sinefungin derivatives, which showed low micromolar inhibition against
both SETD2 and MMSET
Monoacidic Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2āRelated Factor 2 (KEAP1:NRF2) ProteināProtein Interaction with High Cell Potency Identified by Fragment-Based Discovery
KEAP1
is the key regulator of the NRF2-mediated cytoprotective
response, and increasingly recognized as a target for diseases involving
oxidative stress. Pharmacological intervention has focused on molecules
that decrease NRF2-ubiquitination through covalent modification of
KEAP1 cysteine residues, but such electrophilic compounds lack selectivity
and may be associated with off-target toxicity. We report here the
first use of a fragment-based approach to directly target the KEAP1
KelchāNRF2 interaction. X-ray crystallographic screening identified
three distinct āhot-spotsā for fragment binding within
the NRF2 binding pocket of KEAP1, allowing progression of a weak fragment
hit to molecules with nanomolar affinity for KEAP1 while maintaining
drug-like properties. This work resulted in a promising lead compound
which exhibits tight and selective binding to KEAP1, and activates
the NRF2 antioxidant response in cellular and <i>in vivo</i> models, thereby providing a high quality chemical probe to explore
the therapeutic potential of disrupting the KEAP1āNRF2 interaction
Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2
Aberrant activation of the MAPK pathway
drives cell proliferation
in multiple cancers. Inhibitors of BRAF and MEK kinases are approved
for the treatment of BRAF mutant melanoma, but resistance frequently
emerges, often mediated by increased signaling through ERK1/2. Here,
we describe the fragment-based generation of ERK1/2 inhibitors that
block catalytic phosphorylation of downstream substrates such as RSK
but also modulate phosphorylation of ERK1/2 by MEK without directly
inhibiting MEK. X-ray crystallographic and biophysical fragment screening
followed by structure-guided optimization and growth from the hinge
into a pocket proximal to the C-Ī± helix afforded highly potent
ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant
cells, the lead compound suppresses pRSK and pERK levels and inhibits
proliferation at low nanomolar concentrations. The lead exhibits tumor
regression upon oral dosing in BRAF mutant xenograft models, providing
a promising basis for further optimization toward clinical pERK1/2
modulating ERK1/2 inhibitors