The segmental premature aging disease Hutchinson-Gilford Progeria syndrome (HGPS) is caused by a truncated and farnesylated form of Lamin A called progerin. HGPS affects mesenchymal lineages, including the skeletal system, dermis, and vascular smooth muscle (VSMC). To understand the underlying molecular pathology of HGPS, we derived induced pluripotent stem cells (iPSCs) from HGPS dermal fibroblasts. The iPSCs were differentiated into neural progenitors, endothelial cells, fibroblasts, VSMCs, and mesenchymal stem cells (MSCs). Progerin levels were highest in MSCs, VSMCs, and fibroblasts, in that order, with these lineages displaying increased DNA damage, nuclear abnormalities, and HGPS-VSMC accumulating numerous calponin-staining inclusion bodies. Both HGPS-MSC and -VSMC viability was compromised by stress and hypoxia in vitro and in vivo (MSC). Because MSCs reside in low oxygen niches in vivo, we propose that, in HGPS, this causes additional depletion of the MSC pool responsible for replacing differentiated cells lost to progerin toxicity.

Drug target identification is a critical step toward understanding the mechanism of action of a drug, which can help one improve the drug's current therapeutic regime and expand the drug's therapeutic potential. However, current in vitro affinity-chromatography-based and in vivo activity-based protein profiling approaches generally face difficulties in discriminating specific drug targets from nonspecific ones. Here we describe a novel approach combining isobaric tags for relative and absolute quantitation with clickable activity-based protein profiling to specifically and comprehensively identify the protein targets of andrographolide (Andro), a natural product with known anti-inflammation and anti-cancer effects, in live cancer cells. We identified a spectrum of specific targets of Andro, which furthered our understanding of the mechanism of action of the drug. Our findings, validated through cell migration and invasion assays, showed that Andro has a potential novel application as a tumor metastasis inhibitor. Moreover, we have unveiled the target binding mechanism of Andro with a combination of drug analog synthesis, protein engineering, and mass-spectrometry-based approaches and determined the drugbinding sites of two protein targets, NF-B and actin. Molecular & Cellular Proteomics 13: 10.1074/mcp. M113.029793, 876-886, 2014.As most drugs exert pharmacological effects by interacting with their target proteins, the identification of these target proteins is a critical step in unraveling the mechanisms of drug action. It is also imperative for our understanding of the pharmacodynamics of a known drug, suggesting potentially unrevealed actions and thus refining future clinical applications of the substance. Traditional approaches used to identify protein targets of a drug typically utilize immobilized drug affinity chromatography coupled with mass spectrometry (MS) 1 (1, 2). These methods can be applied to cell lysates, but not in an in vivo setting, because of the requirement of a solid support. In vitro target profiling might not accurately reflect the drug's actions in the in vivo physiological environment. To overcome this limitation, several groups have used activity-based protein profiling (ABPP) combined with bio-orthogonal click chemistry to identify drug targets both in vitro and in vivo (supplemental Fig. S1) (3-15). ABPP probes exert their functions via covalent reactions with the target proteins or photoaffinity-based labeling via the incorporation of photoreactive groups. With the increasing sensitivity of modern MS platforms, low-abundance protein targets can be successfully identified. Although both conventional affinity chromatography and recent ABPP-based methods allow us to detect a set of candidate protein targets for a drug, it remains difficult to 1 The abbreviations used are: MS, mass spectrometry; ABPP, activity-based protein profiling; ICABPP, clickable activity-based protein profiling; iTRAQ, isobaric tags for relative and absolute quantitation; DMSO, dimethyl sulfoxide; Andro, androgr...

Few examples of [4 + 2] cycloaddition with unmasked ortho-benzoquinones (UMOBs) as carbodiene have been reported in complex molecule synthesis. Herein we report that this cycloaddition with podocarpane-type UMOB was developed and applied to construct fully functionalized bicyclo[2.2.2]octanes. Based on this methodology, divergent total syntheses of atisane-type diterpenoids, including (±)-crotobarin, crotogoudin, atisane-3β,16α-diol, and 16S,17-dihydroxy-atisan-3-one, were accomplished in 14, 14, 12, and 16 steps, respectively. Key elements in these total syntheses include: (1) FeCl3-catalyzed cationic cascade cyclization to construct podocarpane-type skeleton; (2) Mn(III)/Co(II)-catalyzed radical hydroxylation of alkene with high regio-, diastereo-, and chemoselectivities; (3) and a ketal-deprotection/lactone-opening/deprotonation/lactonization cascade. Additionally, the synthetic utility of the fully functionalized bicyclo[2.2.2]octane framework was further elucidated by applying ring distortion strategy to afford different skeleton-rearranged natural product-like compounds.

Scheme 1. Structures of leucosceptroids A-D. Scheme 2. Retrosynthetic analysis of leucosceptroid B. R = protecting group.