7 research outputs found
Synthesis of the DEF-Ring Spirocyclic Core of Cyclopamine
A stereoselective
synthesis of the DEF-ring spirocyclic core of
cyclopamine was accomplished using commercially available materials.
The key steps in the synthesis were (i) the enantioselective vinylogous
Mannich reaction, followed by lactamization to generate the piperidine
F ring, and (ii) intramolecular oxidative dearomative spiroetherification
to construct the DEF-ring spirocyclic core of cyclopamine. We found
that the stereochemistry of the spirocyclization was controlled by
the configuration of the methyl group (C-20) in the substrate
Protein Cross-Linking Capillary Electrophoresis for Protein–Protein Interaction Analysis
Capillary
electrophoresis (CE) has been identified as a useful
platform for detecting, quantifying, and screening for modulators
of protein–protein interactions (PPIs). In this method, one
protein binding partner is labeled with a fluorophore, the protein
binding partners are mixed, and then, the complex is separated from
free protein to allow direct determination of bound to free ratios.
Although it possesses many advantages for PPI studies, the method
is limited by the need to have separation conditions that both prevent
protein adsorption to capillary and maintain protein interactions
during the separation. In this work, we use protein cross-linking
capillary electrophoresis (PXCE) to overcome this limitation. In PXCE,
the proteins are cross-linked under binding conditions and then separated.
This approach eliminates the need to maintain noncovalent interactions
during electrophoresis and facilitates method development. We report
PXCE methods for an antibody–antigen interaction and heterodimer
and homodimer heat shock protein complexes. Complexes are cross-linked
by short treatments with formaldehyde after reaching binding equilibrium.
Cross-linked complexes are separated by electrophoretic mobility using
free solution CE or by size using sieving electrophoresis of SDS complexes.
The method gives good quantitative results; e.g., a lysozyme–antibody
interaction was found to have <i>K</i><sub>d</sub> = 24
± 3 nM by PXCE and <i>K</i><sub>d</sub> = 17 ±
2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70)
in complex with bcl2 associated athanogene 3 (Bag3) was found to have <i>K</i><sub>d</sub> = 25 ± 5 nM by PXCE which agrees with <i>K</i><sub>d</sub> values reported without cross-linking. Hsp70–Bag3
binding site mutants and small molecule inhibitors of Hsp70–Bag3
were characterized by PXCE with good agreement to inhibitory constants
and IC<sub>50</sub> values obtained by a bead-based flow cytometry
protein interaction assay (FCPIA). PXCE allows rapid method development
for quantitative analysis of PPIs
Comparative Structural and Functional Studies of 4‑(Thiazol-5-yl)-2-(phenylamino)pyrimidine-5-carbonitrile CDK9 Inhibitors Suggest the Basis for Isotype Selectivity
Cyclin-dependent kinase 9/cyclin T, the protein kinase
heterodimer
that constitutes positive transcription elongation factor b, is a
well-validated target for treatment of several diseases, including
cancer and cardiac hypertrophy. In order to aid inhibitor design and
rationalize the basis for CDK9 selectivity, we have studied the CDK-binding
properties of six different members of a 4-(thiazol-5-yl)-2-(phenylamino)Âpyrimidine-5-carbonitrile
series that bind to both CDK9/cyclin T and CDK2/cyclin A. We find
that for a given CDK, the melting temperature of a CDK/cyclin/inhibitor
complex correlates well with inhibitor potency, suggesting that differential
scanning fluorimetry (DSF) is a useful orthogonal measure of inhibitory
activity for this series. We have used DSF to demonstrate that the
binding of these compounds is independent of the presence or absence
of the C-terminal tail region of CDK9, unlike the binding of the CDK9-selective
inhibitor 5,6-dichlorobenzimidazone-1-β-d-ribofuranoside
(DRB). Finally, on the basis of 11 cocrystal structures bound to CDK9/cyclin
T or CDK2/cyclin A, we conclude that selective inhibition of CDK9/cyclin
T by members of the 4-(thiazol-5-yl)-2-(phenylamino)Âpyrimidine-5-carbonitrile
series results from the relative malleability of the CDK9 active site
rather than from the formation of specific polar contacts
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Exploration of Benzothiazole Rhodacyanines as Allosteric Inhibitors of Protein–Protein Interactions with Heat Shock Protein 70 (Hsp70)
Cancer
cells rely on the chaperone heat shock protein 70 (Hsp70)
for survival and proliferation. Recently, benzothiazole rhodacyanines
have been shown to bind an allosteric site on Hsp70, interrupting
its binding to nucleotide-exchange factors (NEFs) and promoting cell
death in breast cancer cell lines. However, proof-of-concept molecules,
such as JG-98, have relatively modest potency (EC<sub>50</sub> ≈
0.7–0.4 μM) and are rapidly metabolized in animals. Here,
we explored this chemical series through structure- and property-based
design of ∼300 analogs, showing that the most potent had >10-fold
improved EC<sub>50</sub> values (∼0.05 to 0.03 μM) against
two breast cancer cells. Biomarkers and whole genome CRISPRi screens
confirmed members of the Hsp70 family as cellular targets. On the
basis of these results, JG-231 was found to reduce tumor burden in
an MDA-MB-231 xenograft model (4 mg/kg, ip). Together, these studies
support the hypothesis that Hsp70 may be a promising target for anticancer
therapeutics
Exploration of Benzothiazole Rhodacyanines as Allosteric Inhibitors of Protein–Protein Interactions with Heat Shock Protein 70 (Hsp70)
Cancer
cells rely on the chaperone heat shock protein 70 (Hsp70)
for survival and proliferation. Recently, benzothiazole rhodacyanines
have been shown to bind an allosteric site on Hsp70, interrupting
its binding to nucleotide-exchange factors (NEFs) and promoting cell
death in breast cancer cell lines. However, proof-of-concept molecules,
such as JG-98, have relatively modest potency (EC<sub>50</sub> ≈
0.7–0.4 μM) and are rapidly metabolized in animals. Here,
we explored this chemical series through structure- and property-based
design of ∼300 analogs, showing that the most potent had >10-fold
improved EC<sub>50</sub> values (∼0.05 to 0.03 μM) against
two breast cancer cells. Biomarkers and whole genome CRISPRi screens
confirmed members of the Hsp70 family as cellular targets. On the
basis of these results, JG-231 was found to reduce tumor burden in
an MDA-MB-231 xenograft model (4 mg/kg, ip). Together, these studies
support the hypothesis that Hsp70 may be a promising target for anticancer
therapeutics
Substituted 4‑(Thiazol-5-yl)-2-(phenylamino)pyrimidines Are Highly Active CDK9 Inhibitors: Synthesis, X‑ray Crystal Structures, Structure–Activity Relationship, and Anticancer Activities
Cancer cells often have a high demand for antiapoptotic
proteins
in order to resist programmed cell death. CDK9 inhibition selectively
targets survival proteins and reinstates apoptosis in cancer cells.
We designed a series of 4-thiazol-2-anilinopyrimidine derivatives
with functional groups attached to the C5-position of the pyrimidine
or to the C4-thiazol moiety and investigated their effects on CDK9
potency and selectivity. One of the most selective compounds, <b>12u</b> inhibits CDK9 with IC<sub>50</sub> = 7 nM and shows over
80-fold selectivity for CDK9 versus CDK2. X-ray crystal structures
of <b>12u</b> bound to CDK9 and CDK2 provide insights into the
binding modes. This work, together with crystal structures of selected
inhibitors in complex with both enzymes described in a companion paper, provides a rationale for the observed SAR. <b>12u</b> demonstrates potent anticancer activity against primary
chronic lymphocytic leukemia cells with a therapeutic window 31- and
107-fold over those of normal B- and T-cells
Exploration of Benzothiazole Rhodacyanines as Allosteric Inhibitors of Protein–Protein Interactions with Heat Shock Protein 70 (Hsp70)
Cancer
cells rely on the chaperone heat shock protein 70 (Hsp70)
for survival and proliferation. Recently, benzothiazole rhodacyanines
have been shown to bind an allosteric site on Hsp70, interrupting
its binding to nucleotide-exchange factors (NEFs) and promoting cell
death in breast cancer cell lines. However, proof-of-concept molecules,
such as JG-98, have relatively modest potency (EC<sub>50</sub> ≈
0.7–0.4 μM) and are rapidly metabolized in animals. Here,
we explored this chemical series through structure- and property-based
design of ∼300 analogs, showing that the most potent had >10-fold
improved EC<sub>50</sub> values (∼0.05 to 0.03 μM) against
two breast cancer cells. Biomarkers and whole genome CRISPRi screens
confirmed members of the Hsp70 family as cellular targets. On the
basis of these results, JG-231 was found to reduce tumor burden in
an MDA-MB-231 xenograft model (4 mg/kg, ip). Together, these studies
support the hypothesis that Hsp70 may be a promising target for anticancer
therapeutics