The antitumor agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine, TPZ, 1) gains medicinal activity through its ability to selectively damage DNA in the hypoxic cells found inside solid tumors. This occurs via one-electron enzymatic reduction of TPZ to yield an oxygen-sensitive drug radical (2) that leads to oxidatively generated DNA damage under hypoxic conditions. Two possible mechanisms have been considered to account for oxidatively generated DNA damage by TPZ. First, homolysis of the N-OH bond in 2 may yield the well known DNA-damaging agent, hydroxyl radical. Alternatively, it has been suggested that elimination of water from 2 generates a benzotriazinyl radical (4) as the ultimate DNA-damaging species. In the studies described here, the TPZ analogue 3-methyl-1,2,4-benzotriazine 1,4-dioxide (5) was employed as a tool to probe the mechanism of DNA damage within this new class of antitumor drugs. Initially, it was demonstrated that 5 causes redoxactivated, hypoxia-selective oxidation of DNA and small organic substrates in a manner that is completely analogous to TPZ. This suggests that 5 and TPZ damage DNA by the same chemical mechanism. Importantly, the methyl substituent in 5 provides a means for assessing whether the putative benzotriazinyl intermediate 7 is generated following one-electron reduction. Two complementary isotopic labeling experiments provide evidence against the formation of the benzotriazinyl radical intermediate. Rather, a mechanism involving the release of hydroxyl radical from the activated drug radical intermediates can explain the DNA-cleaving properties of this class of antitumor drug candidates.The compound 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine, TPZ, 1, Scheme 1) is currently undergoing a variety of phase I, II, and III clinical trials for the treatment of human cancers. 1 TPZ gains medicinal activity from its ability to selectively damage DNA in the oxygen-poor (hypoxic) cells found inside solid tumors. [2][3][4][5][6][7][8] This DNA-damage process begins with intracellular enzymatic reduction of TPZ to yield the drug radical intermediate (2 , Scheme 1). [9][10][11][12] In normally-oxygenated cells, 2 undergoes relatively harmless oxidation back to the parent drug (Scheme 1), 9,10,13 while, under hypoxic conditions, the drug radical intermediate 2 leads to oxidatively generated DNA damage including hydroxylation of the nucleobases 22,23 and strand breaks initiated by the abstraction of hydrogen atoms from the sugar-phosphate backbone of DNA. [7][8][9][24][25][26][27][28] In the recent literature, two mechanisms have been considered to explain TPZ-mediated DNA damage. We have presented evidence [22][23][24]26,29 supporting a mechanism involving homolysis of the N-OH bond in the neutral drug radical (2) to yield the mono-N-oxide metabolite 3 and the well known DNA-damaging agent hydroxyl radical (Scheme 1, upper branch). 30 This *To whom correspondence should be addressed: gatesk@missouri.edu; phone: (573) FAX: (573) NIH-PA Author ManuscriptNIH-PA ...

Chromium was proposed to be an essential trace element over 50 years ago and has been accepted as an essential element for over 30 years. However, the studies on which chromium's status are based are methodologically flawed. Whether chromium is an essential element has been examined for the first time in carefully controlled metal-free conditions using a series of purified diets containing various chromium contents. Male Zucker lean rats were housed in specially designed metal-free cages for 6 months and fed the AIN-93G diet with no added chromium in the mineral mix component of the diet, the standard AIN-93G diet, the standard AIN-93G diet supplemented with 200 μg Cr/kg, or the standard AIN-93G diet supplemented with 1,000 μg Cr/kg. The chromium content of the diet had no effect on body mass or food intake. Similarly, the chromium content of the diet had no effect on glucose levels in glucose tolerance or insulin tolerance tests. However, a distinct trend toward lower insulin levels under the curve after a glucose challenge was observed with increasing chromium content in the diet; rats on the supplemented AIN-93G diets had significantly lower areas (P < 0.05) than rats on the low-chromium diet. The studies reveal that a diet with as little chromium as reasonably possible had no effect on body composition, glucose metabolism, or insulin sensitivity compared with a chromium-"sufficient" diet. Together with the results of other recent studies, these results clearly indicate that chromium can no longer be considered an essential element.

Chromatin acetylation, a critical regulator of synaptic plasticity and memory processes, is thought to be altered in neurodegenerative diseases. Here, we demonstrate that spatial memory and plasticity (LTD, dendritic spine formation) deficits can be restored in a mouse model of tauopathy following treatment with CSP‐TTK21, a small‐molecule activator of CBP/p300 histone acetyltransferases (HAT). At the transcriptional level, CSP‐TTK21 re‐established half of the hippocampal transcriptome in learning mice, likely through increased expression of neuronal activity genes and memory enhancers. At the epigenomic level, the hippocampus of tauopathic mice showed a significant decrease in H2B but not H3K27 acetylation levels, both marks co‐localizing at TSS and CBP enhancers. Importantly, CSP‐TTK21 treatment increased H2B acetylation levels at decreased peaks, CBP enhancers, and TSS, including genes associated with plasticity and neuronal functions, overall providing a 95% rescue of the H2B acetylome in tauopathic mice. This study is the first to provide in vivo proof‐of‐concept evidence that CBP/p300 HAT activation efficiently reverses epigenetic, transcriptional, synaptic plasticity, and behavioral deficits associated with Alzheimer's disease lesions in mice.

Protein arginine methyltransferases (PRMTs) are proved to play vital roles in chromatin remodeling, RNA metabolism and signal transduction. Aberrant regulation of PRMT activity is associated with various pathological states such as cancer and cardiovascular disorders. Development and application of small molecule PRMT inhibitors will provide new avenues for therapeutic discovery. We combined pharmacophore-based virtual screening methods with radioactive methylation assays, six hits were identified as inhibitors against the predominant arginine methyltransferase PRMT1 within micromolar potency. Two potent compounds, A9 and A36, exhibitting the inhibitory effect by directly targeting substrate H4 other than PRMT1 and displayed even higher inhibition activity than the well-known PRMT inhibitors AMI-1 and stilbamidine. A9 significantly inhibits proliferation of castrate-resistant prostate cancer cells. Together, A9 may be a potential inhibitor against advanced hormone-independent cancers and the work will provide clues for the future development of specific compounds that block the interaction of PRMTs with their targets.

Summary Protein arginine methylation regulates multiple biological processes. Deregulation of protein arginine methyltransferase (PRMT) activities has been observed in many disease phenotypes. Small molecule probes that target PRMTs with strong affinity and selectivity can be used as valuable tools to dissect biological mechanisms of arginine methylation and establish the role of PRMT proteins in a disease process. In this work, we report synthesis and evaluation of a class of carbocyanine compounds containing indolium, benz[e]indolium or benz[c,d]indolium heterocyclic moieties that bind to the predominant arginine methyltransferase PRMT1 and inhibit its methyltransferase activity at low micromolar potencies. In particular, the developed molecules have long wavelength colorimetric and fluorometric photoactivities, which can be used for optical and near-infrared fluorescence imaging in cells or biological tissues. Together, these new chemical probes have potential application in PRMT studies both as enzyme inhibitors and as fluorescent dyes for microscope imaging.