3 research outputs found
Fluorescence Resonance Energy Transfer Characterization of DNA Wrapping in Closed and Open <i>Escherichia coli</i> RNA Polymerase−λP<sub>R</sub> Promoter Complexes
Initial
recognition of promoter DNA by RNA polymerase (RNAP) is
proposed to trigger a series of conformational changes beginning with
bending and wrapping of the 40–50 bp of DNA immediately upstream
of the −35 region. Kinetic studies demonstrated that the presence
of upstream DNA facilitates bending and entry of the downstream duplex
(to +20) into the active site cleft to form an advanced closed complex
(CC), prior to melting of ∼13 bp (−11 to +2), including
the transcription start site (+1). Atomic force microscopy and footprinting
revealed that the stable open complex (OC) is also highly wrapped
(−60 to +20). To test the proposed bent-wrapped model of duplex
DNA in an advanced RNAP−λP<sub>R</sub> CC and compare
wrapping in the CC and OC, we use fluorescence resonance energy transfer
(FRET) between cyanine dyes at far-upstream (−100) and downstream
(+14) positions of promoter DNA. Similarly large intrinsic FRET efficiencies
are observed for the CC (0.30 ± 0.07) and the OC (0.32 ±
0.11) for both probe orientations. Fluorescence enhancements at +14
are observed in the single-dye-labeled CC and OC. These results demonstrate
that upstream DNA is extensively wrapped and the start site region
is bent into the cleft in the advanced CC, reducing the distance between
positions −100 and +14 on promoter DNA from >300 to <100
Ã…. The proximity of upstream DNA to the downstream cleft in the
advanced CC is consistent with the proposed mechanism for facilitation
of OC formation by upstream DNA
Potent GCN2 Inhibitor Capable of Reversing MDSC-Driven T Cell Suppression Demonstrates In Vivo Efficacy as a Single Agent and in Combination with Anti-Angiogenesis Therapy
General
control nonderepressible 2 (GCN2) protein kinase is a cellular
stress sensor within the tumor microenvironment (TME), whose signaling
cascade has been proposed to contribute to immune escape in tumors.
Herein, we report the discovery of cell-potent GCN2 inhibitors with
excellent selectivity against its closely related Integrated Stress
Response (ISR) family members heme-regulated inhibitor kinase (HRI),
protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase
(PERK), as well as good kinome-wide selectivity and favorable PK.
In mice, compound 39 engages GCN2 at levels ≥80%
with an oral dose of 15 mg/kg BID. We also demonstrate the ability
of compound 39 to alleviate MDSC-related T cell suppression
and restore T cell proliferation, similar to the effect seen in MDSCs
from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI)
as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced
by treatment with compound 39 demonstrating the complementarity
of these two mechanisms
Potent GCN2 Inhibitor Capable of Reversing MDSC-Driven T Cell Suppression Demonstrates In Vivo Efficacy as a Single Agent and in Combination with Anti-Angiogenesis Therapy
General
control nonderepressible 2 (GCN2) protein kinase is a cellular
stress sensor within the tumor microenvironment (TME), whose signaling
cascade has been proposed to contribute to immune escape in tumors.
Herein, we report the discovery of cell-potent GCN2 inhibitors with
excellent selectivity against its closely related Integrated Stress
Response (ISR) family members heme-regulated inhibitor kinase (HRI),
protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase
(PERK), as well as good kinome-wide selectivity and favorable PK.
In mice, compound 39 engages GCN2 at levels ≥80%
with an oral dose of 15 mg/kg BID. We also demonstrate the ability
of compound 39 to alleviate MDSC-related T cell suppression
and restore T cell proliferation, similar to the effect seen in MDSCs
from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI)
as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced
by treatment with compound 39 demonstrating the complementarity
of these two mechanisms