RRM1 and RRM2 subunits of Ribonucleotide reductase interact with Chk1 and Polα.

<p>(a) At 30 h after siRNA transfections of the luciferase, RRM1, and RRM2 siRNA duplexes, extracts were prepared for immunoprecipitations. Cells transfected with Luciferase were incubated with 1 mM HU for 30 min. Chk1 was immunoprecipitated from luciferase (positive control), Luciferase + HU, RRM1, and RRM2 depleted cells with Chk1 antibodies (MAb58D7) cross-linked to protein A and were immunoblotted as indicated. Chk1 immunoprecipitations were peptide blocked as a negative control. (b) Polα was immunoprecipitated from luciferase (positive control), luciferase +HU, RRM1, and RRM2 depleted cells depleted cells with Polα antibody (SJK132-20) cross-linked to protein G and Western blots were immunoblotted as indicated. (c) Whole cell extracts were immunoblotted as indicated.</p

Percentage of cells in G1, S, or G2 phase.

<p>Percentage of cells in G1, S, or G2 phase.</p

% H2AX phosphorylation.

<p>Data represents 2-independent experiments performed in triplicates.</p><p>± denotes standard deviation.</p><p>% H2AX phosphorylation.</p

RRM1/Chk1 and RRM2/Chk1 co-depletion enhances the accumulation of DNA damage, apoptotic response and effects cell proliferation in U2OS cells.

<p>Cells were transfected with Chk1, RRM1, RRM2, RRM1/Chk1, RRM2/Chk1, and Luciferase control (untreated or treated with 1 mM HU for the last 8 h) before harvesting at 30 h. At 30 h after siRNA transfections, (a) extracts were prepared and immunoblotted with the indicated antibodies. (b) DNA damage was assessed for γ-H2AX phosphorylation using flow cytometry. (c) Cells were collected at indicated time points indicated and analyzed for activated caspases. Data performed in duplicates. Error bars represent SD between experiments. (d) Cell proliferation was assessed with clonogenicity assay.</p

Quantitation of dNTPs and γ-H2AX phosphorylation in U20S cells following depletion of RRM1 and RRM2 subunits of Ribonucleotide reductase.

<p>Cells were transfected with RRM1, RRM2 and Luciferase control (untreated or treated with 1 µM GEM or 1 µM CAFdA for the last 2 h) before harvesting at 30 h. At 30 h after siRNA transfections, (a) NTP/dNTP extractions were prepared and quantified. (b) DNA damage was assessed for γ-H2AX phosphorylation using flow cytometry. Data performed in triplicates. Error bars represent standard deviations (SD) between experiments.</p

A Functional Approach Reveals a Genetic and Physical Interaction between Ribonucleotide Reductase and CHK1 in Mammalian Cells

<div><p>Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. In this study, we show that ablation of RRM1 and RRM2 by siRNA induces G1/S phase arrest, phosphorylation of Chk1 on Ser345 and phosphorylation of γ-H2AX on S139. Combinatorial ablation of RRM1 or RRM2 and Chk1 causes a dramatic accumulation of γ-H2AX, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts. These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation.</p></div

Quantitation (AUC) of dNTP & NTP pools in the U20S cells.

<p>Analysis was done in triplicate.</p><p>± denotes standard deviation.</p><p>nd  =  not detected.</p><p>Units of measurement are AUC (Area under the curve).</p><p>Quantitation (AUC) of dNTP & NTP pools in the U20S cells.</p

Structure-Based Drug Design of Novel Potent and Selective Tetrahydropyrazolo[1,5‑<i>a</i>]pyrazines as ATR Inhibitors

A saturation strategy focused on improving the selectivity and physicochemical properties of ATR inhibitor HTS hit <b>1</b> led to a novel series of highly potent and selective tetrahydropyrazolo­[1,5-<i>a</i>]­pyrazines. Use of PI3Kα mutants as ATR crystal structure surrogates was instrumental in providing cocrystal structures to guide the medicinal chemistry designs. Detailed DMPK studies involving cyanide and GSH as trapping agents during microsomal incubations, in addition to deuterium-labeled compounds as mechanistic probes uncovered the molecular basis for the observed CYP3A4 TDI in the series

Structure-Based Drug Design of Novel, Potent, and Selective Azabenzimidazoles (ABI) as ATR Inhibitors

Compound <b>13</b> was discovered through morphing of the ATR biochemical HTS hit <b>1</b>. The ABI series was potent and selective for ATR. Incorporation of a 6-azaindole afforded a marked increase in cellular potency but was associated with poor PK and hERG ion channel inhibition. DMPK experiments established that CYP P450 and AO metabolism in conjunction with Pgp and BCRP efflux were major causative mechanisms for the observed PK. The series also harbored the CYP3A4 TDI liability driven by the presence of both a morpholine and an indole moiety. Incorporation of an adjacent fluorine or nitrogen into the 6-azaindole addressed many of the various medicinal chemistry issues encountered