Reduced affinity of TraI36 for G151′C/C150′G and G144′C 17-base oligonucleotides

<p><b>Copyright information:</b></p><p>Taken from "Examination of an inverted repeat within the F factor origin of transfer: context dependence of F TraI relaxase DNA specificity"</p><p>Nucleic Acids Research 2006;34(2):426-435.</p><p>Published online 17 Jan 2006</p><p>PMCID:PMC1331984.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> TraI36 protein was titrated into 4 nM 3′-TAMRA-labeled WT (), G151′C/C150′G () or G144′C () 17-base oligonucleotides. Anisotropy and intensity (not shown) data were simultaneously fit with a 1:1 (protein:DNA) binding model using SPECTRABIND (,). Data points are shown as closed circles and the fit as solid lines

RMSF analysis of [Fe₄S₄]²⁺_{(open,substrate-free)} (black), [Fe₄S₄]²⁺/HMBPP_{(open,docked)} (red), and [Fe₄S₄]²⁺/HMBPP_(closed) (blue) aMD simulations.

<p>RMSF analysis of [Fe₄S₄]²⁺_{(open,substrate-free)} (black), [Fe₄S₄]²⁺/HMBPP_{(open,docked)} (red), and [Fe₄S₄]²⁺/HMBPP_(closed) (blue) aMD simulations.</p

Comparison of HMBPP-bound IspH structures from experiment and simulation.

<p>(A) shows the superposition of the [Fe₄S₄]²⁺_{(closed, HMBPP-bound)} IspH crystal structure (bronze, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003395#pcbi.1003395-Grawert4" target="_blank">[28]</a>) and a representative structure from the dominant closed cluster from [Fe₄S₄]²⁺/HMBPP_{(open,docked)} aMD simulations (purple). (B) and (C) correspond to the active site microenvironments of the crystal structure and the representative closed structure from aMD, respectively, while (D) illustrates the positions of the residues present in (B) and (C) in the [Fe₃S₄]⁺_{(open, substrate-free)} crystal structure <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003395#pcbi.1003395-Rekittke1" target="_blank">[23]</a>.</p

Reductive dehydroxylation of HMBPP affords the isoprenoid precursors, IPP and DMAPP.

<p>The relative positions of active site residues suggested to play a role in either substrate binding or catalysis are labeled.</p

(A) Use of the Kullback-Leibler (KL) divergence to highlight residues with distinct dihedral distributions between [Fe₄S₄]²⁺_{(open,substrate-free)} and [Fe₄S₄]²⁺/HMBPP_(closed) simulations of IspH.

<p>Visualization of residues with high “mutual divergence” in the IspH structure, ranging from blue (low) to red (high). (B) Illustration of the different dihedral distributions of the Asn-194 ψ-angles sampled in open (black) and closed (blue) conformations of IspH.</p

Plots of RMSD relative to the [Fe₃S₄]⁺_{(open, substrate-free)} crystal structure over the course of 3×100 ns aMD simulations of (A) [Fe₄S₄]²⁺_{(open,substrate-free)}, (B) [Fe₄S₄]²⁺/HMBPP_{(open,docked)}, and (C) [Fe₄S₄]²⁺/HMBPP_(closed) IspH.

<p>Plots of RMSD relative to the [Fe₃S₄]⁺_{(open, substrate-free)} crystal structure over the course of 3×100 ns aMD simulations of (A) [Fe₄S₄]²⁺_{(open,substrate-free)}, (B) [Fe₄S₄]²⁺/HMBPP_{(open,docked)}, and (C) [Fe₄S₄]²⁺/HMBPP_(closed) IspH.</p

Projections of (A) [Fe₄S₄]²⁺_{(open,substrate-free)}, (B) [Fe₄S₄]²⁺/HMBPP_{(open,docked)}, and (C) [Fe₄S₄]²⁺/HMBPP_(closed) trajectories onto principal component (PC) space constructed from [Fe₄S₄]²⁺_{(open,substrate-free)} and [Fe₄S₄]²⁺/HMBPP_{(open,docked)} aMD simulations.

<p>Crystal structures corresponding to [Fe₃S₄]⁺_{(open, substrate-free)} (white square, PDB ID: 3DNF) and [Fe₄S₄]²⁺_{(closed, HMBPP-bound)} IspH (white diamond, PDB ID: 3KE8) are also projected onto PC space <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003395#pcbi.1003395-Rekittke1" target="_blank">[23]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003395#pcbi.1003395-Grawert4" target="_blank">[28]</a>. Numbers in (A) correspond to POVME volumes (ų), as described in the text.</p

Residues with “mutual divergence” values greater than 1.0 and their respective sequence conservation scores, as computed by Shannon entropy [40], [45], [46].

<p>Residues with “mutual divergence” values greater than 1.0 and their respective sequence conservation scores, as computed by Shannon entropy <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003395#pcbi.1003395-Grant1" target="_blank">[40]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003395#pcbi.1003395-Mirny1" target="_blank">[45]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003395#pcbi.1003395-Shannon1" target="_blank">[46]</a>.</p

Superposition of [Fe₃S₄]⁺_{(open, substrate-free)} (bronze, [23]) and [Fe₄S₄]²⁺_{(closed, HMBPP-bound)} (purple, [28]) IspH crystal structures, viewed (A) head-on toward the binding site and (B) from a top-view highlighting the domain tilt of D3.

<p>Superposition of [Fe₃S₄]⁺_{(open, substrate-free)} (bronze, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003395#pcbi.1003395-Rekittke1" target="_blank">[23]</a>) and [Fe₄S₄]²⁺_{(closed, HMBPP-bound)} (purple, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003395#pcbi.1003395-Grawert4" target="_blank">[28]</a>) IspH crystal structures, viewed (A) head-on toward the binding site and (B) from a top-view highlighting the domain tilt of D3.</p

Optimization of Fused Bicyclic Allosteric SHP2 Inhibitors

SHP2 is a nonreceptor protein tyrosine phosphatase within the mitogen-activated protein kinase (MAPK) pathway controlling cell growth, differentiation, and oncogenic transformation. SHP2 also participates in the programed cell death pathway (PD-1/PD-L1) governing immune surveillance. Small-molecule inhibition of SHP2 has been widely investigated, including in our previous reports describing SHP099 (2), which binds to a tunnel-like allosteric binding site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional hit finding, evaluation, and structure-based scaffold morphing. These studies, reported here in the first of two papers, led to the identification of multiple 5,6-fused bicyclic scaffolds that bind to the same allosteric tunnel as 2. We demonstrate the structural diversity permitted by the tunnel pharmacophore and culminated in the identification of pyrazolopyrimidinones (e.g., SHP389, 1) that modulate MAPK signaling in vivo. These studies also served as the basis for further scaffold morphing and optimization, detailed in the following manuscript