400 μM OdDHL treatment significantly increases necrosis of MDA-MB-231 cells in all conditions.

<p>(A) Differences in the annexin V-FITC staining mean gray value (MGV) for MDA-MB-231 cells treated with 400 μM OdDHL, normalized to 0.6% DMSO control for 3D/normoxia, 2D/hypoxia, and 2D/normoxia culture conditions (N = 3). (B) Differences in the propidium iodide staining MGV for MDA-MB-231 cells treated with 400 μM OdDHL, normalized to 0.6% DMSO control for 3D/normoxia, 2D/hypoxia, and 2D/normoxia culture conditions (N = 3); ** = p-value < 0.01, * = p-value < 0.05 based on least mean contrasts. (C) Differences in the annexin V-FITC staining MGV for MCF-DCIS.com cells treated with 400 μM OdDHL, normalized to 0.6% DMSO control for 3D/normoxia (N = 3), 2D/hypoxia (N = 4), and 2D/normoxia culture conditions (N = 4); *** = p-value < 0.001 based on least mean contrasts. (D) Differences in the propidium iodide staining MGV for MCF-DCIS.com cells treated with 400 μM OdDHL, normalized to 0.6% DMSO control for 3D/normoxia, 2D/hypoxia, and 2D/normoxia culture conditions (N = 3); ** = p-value < 0.01, * = p-value < 0.05 based on least mean contrasts. (E) Differences in the annexin V-FITC staining MGV for MCF-10A cells treated with 400 μM OdDHL, normalized to 0.6% DMSO control for 3D/normoxia, 2D/hypoxia, and 2D/normoxia culture conditions (N = 3); ** = p-value < 0.01 based on least mean contrasts. (F) Differences in the propidium iodide staining MGV for MCF-10A cells treated with 400 μM OdDHL, normalized to 0.6% DMSO control for 3D/normoxia, 2D/hypoxia, and 2D/normoxia culture conditions (N = 3); ** = p-value < 0.01 based on least mean contrasts. Error bars represent standard error of the mean.</p

Chemical Proteomic Platform To Identify Citrullinated Proteins

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and are routinely used for disease diagnosis. Protein citrullination is also increased in cancer and other autoimmune disorders, suggesting that citrullinated proteins may serve as biomarkers for diseases beyond RA. To identify these citrullinated proteins, we developed biotin-conjugated phenylglyoxal (biotin-PG). Using this probe and our platform technology, we identified >50 intracellular citrullinated proteins. More than 20 of these are involved in RNA splicing, suggesting, for the first time, that citrullination modulates RNA biology. Overall, this chemical proteomic platform will play a key role in furthering our understanding of protein citrullination in rheumatoid arthritis and potentially a wider spectrum of inflammatory diseases

Biological Studies and Target Engagement of the 2‑<i>C</i>‑Methyl‑d‑Erythritol 4‑Phosphate Cytidylyltransferase (IspD)-Targeting Antimalarial Agent (1<i>R</i>,3<i>S</i>)‑MMV008138 and Analogs

Malaria continues to be one of the deadliest diseases worldwide, and the emergence of drug resistance parasites is a constant threat. <i>Plasmodium</i> parasites utilize the methylerythritol phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are essential for parasite growth. Previously, we and others identified that the Malaria Box compound MMV008138 targets the apicoplast and that parasite growth inhibition by this compound can be reversed by supplementation of IPP. Further work has revealed that MMV008138 targets the enzyme 2-<i>C</i>-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD) in the MEP pathway, which converts MEP and cytidine triphosphate (CTP) to cytidinediphosphate methylerythritol (CDP-ME) and pyrophosphate. In this work, we sought to gain insight into the structure–activity relationships by probing the ability of MMV008138 analogs to inhibit <i>Pf</i>IspD recombinant enzyme. Here, we report <i>Pf</i>IspD inhibition data for fosmidomycin (FOS) and 19 previously disclosed analogs and report parasite growth and <i>Pf</i>IspD inhibition data for 27 new analogs of MMV008138. In addition, we show that MMV008138 does not target the recently characterized human IspD, reinforcing MMV008138 as a prototype of a new class of species-selective IspD-targeting antimalarial agents