Chlorinated Dehydrocurvularins and Alterperylenepoxide A from <i>Alternaria</i> sp. AST0039, a Fungal Endophyte of <i>Astragalus lentiginosus</i>

Investigation of <i>Alternaria</i> sp. AST0039, an endophytic fungus obtained from the leaf tissue of <i>Astragalus lentiginosus</i>, led to the isolation of (−)-(10<i>E</i>,15<i>S</i>)-4,6-dichloro-10­(11)-dehydrocurvularin (<b>1</b>), (−)-(10<i>E</i>,15<i>S</i>)-6-chloro-10­(11)-dehydrocurvularin (<b>2</b>), (−)-(10<i>E</i>,15<i>S</i>)-10­(11)-dehydrocurvularin (<b>3</b>), and alterperylenepoxide A (<b>4</b>) together with scytalone and α-acetylorcinol. Structures of <b>1</b> and <b>4</b> were established from their spectroscopic data, and the relative configuration of <b>4</b> was determined with the help of nuclear Overhauser effect difference data. All metabolites were evaluated for their cytotoxic activity and ability to induce heat-shock and unfolded protein responses. Compounds <b>2</b> and <b>3</b> exhibited cytotoxicity to all five cancer cell lines tested and increased the level of the pro-apoptotic transcription factor CHOP, but only <b>3</b> induced the heat-shock response and caused a strong unfolded protein response

Arsenic Compromises Both p97 and Proteasome Functions

Exposure to arsenic is a worldwide problem that affects more than 200 million people. The underlying mechanisms of arsenic toxicity have been difficult to ascertain due to arsenic’s pleotropic effects. A number of recent investigations have shown that arsenic can compromise protein quality control through the ubiquitin proteasome system (UPS) or the endoplasmic reticulum associated protein degradation (ERAD) pathway. In this article, a link between arsenic and protein quality control is reported. Biochemical and cellular data demonstrate a misregulation of the ATPase cycle of the ATPase associated with various cellular activities (AAA+) chaperone, p97. Interestingly, the loss of p97 activity is due to the increased rate of ATP hydrolysis, which mimics a collection of pathogenic genetic p97 lesions. Cellular studies, using a well characterized reporter of both the proteasome and p97, show the proteasome to also be compromised. This loss of both p97 and proteasome functions can explain the catastrophic protein quality control issues observed in acute, high level arsenic exposures

Oxaspirol B with p97 Inhibitory Activity and Other Oxaspirols from <i>Lecythophora</i> sp. FL1375 and FL1031, Endolichenic Fungi Inhabiting <i>Parmotrema tinctorum</i> and <i>Cladonia evansii</i>

A new metabolite, oxaspirol D (<b>4</b>), together with oxaspirols B (<b>2</b>) and C (<b>3</b>) were isolated from <i>Lecythophora</i> sp. FL1375, an endolichenic fungus isolated from Parmotrema tinctorum, whereas <i>Lecythophora</i> sp. FL1031 inhabiting the lichen Cladonia evansii afforded oxaspirols A (<b>1</b>), B (<b>2</b>), and C (<b>3</b>). Of these, oxaspirol B (<b>2</b>) showed moderate p97 ATPase inhibitory activity. A detailed characterization of all oxaspirols was undertaken because structures proposed for known oxaspirols have involved incomplete assignments of NMR spectroscopic data leading only to their planar structures. Thus, the naturally occurring isomeric mixture (<b>2a</b> and <b>2b</b>) of oxaspirol B was separated as their diacetates (<b>5a</b> and <b>5b</b>) and the structures and absolute configurations of <b>1</b>, <b>2a</b>, <b>2b</b>, <b>3</b>, and <b>4</b> were determined by the application of spectroscopic techniques including two-dimensional NMR and the modified Mosher’s ester method. Oxaspirol B (<b>2</b>) and its diacetates <b>5a</b> and <b>5b</b> were evaluated for their ATPase inhibitory activities of p97, p97 mutants, and other ATP-utilizing enzymes, and only <b>2</b> was found to be active, indicating the requirement of some structural features in oxaspirols for their activity. Additional biochemical and cellular assays suggested that <b>2</b> was a reversible, non-ATP competitive, and specific inhibitor of p97

HPTN 067 participant enrollment location and gender information.

<p>HPTN 067 participant enrollment location and gender information.</p

Previously unreported <i>AK2</i> missense variants detected in clinical trial participants located in Bangkok, Thailand, Cape Town, South Africa, and New York City, USA.

<p>Previously unreported <i>AK2</i> missense variants detected in clinical trial participants located in Bangkok, Thailand, Cape Town, South Africa, and New York City, USA.</p

Previously unreported <i>CKM</i> missense variants detected in clinical trial participants located in Bangkok, Thailand, Cape Town, South Africa, and New York City, USA.

<p>Previously unreported <i>CKM</i> missense variants detected in clinical trial participants located in Bangkok, Thailand, Cape Town, South Africa, and New York City, USA.</p

Naturally occurring genetic variants of AK2 and the resulting impact on TFV phosphorylation.

<p>Wild-type (WT) AK2 and variants were expressed in and purified from <i>E</i>. <i>coli</i> in order to evaluate their effects on TFV phosphorylation in vitro. Proteins were incubated with 1 mM TFV in assay buffer for 2.5 h before reaction was quenched. A saturating concentration of TFV was used in order to ensure that substrate depletion would not play a causal role in differences observed in the formation of phosphates in these activity assays. TFV-MP and TFV-DP were detected by uHPLC-MS/MS where signal to noise ratio was used to establish the corresponding bar graph. Error bars represent standard deviation; n = 3. A two-tailed unpaired <i>t</i> test and significance was set as follows: *, p≤0.05; **, p≤0.01; ***, p≤0.001.</p

Previously unreported <i>PKLR</i> missense variants detected in clinical trial participants located in Thailand, South Africa, and the USA.

<p>Previously unreported <i>PKLR</i> missense variants detected in clinical trial participants located in Thailand, South Africa, and the USA.</p

Distribution of individuals enrolled at the Bangkok, Cape Town, and New York City study sites carrying genetic variants in TFV-activating kinases.

<p>Each rectangle is representative of a TFV-activating kinase that was sequenced: <i>AK2</i> in blue, <i>CKM</i> in pink, <i>PKM</i> in orange and <i>PKLR</i> in green. Numerical values indicate the number of individuals detected to carry a single nucleotide variation or deletion. Overlapping regions of each rectangle indicate the number of individuals with genetic variants in more than one kinase.</p

Withaferin A Analogs That Target the AAA+ Chaperone p97

Understanding the mode of action (MOA) of many natural products can be puzzling with mechanistic clues that seem to lack a common thread. One such puzzle lies in the evaluation of the antitumor properties of the natural product withaferin A (WFA). A variety of seemingly unrelated pathways have been identified to explain its activity, suggesting a lack of selectivity. We now show that WFA acts as an inhibitor of the chaperone, p97, both <i>in vitro</i> and in cell models in addition to inhibiting the proteasome <i>in vitro</i>. Through medicinal chemistry, we have refined the activity of WFA toward p97 and away from the proteasome. Subsequent studies indicated that these WFA analogs retained p97 activity and cytostatic activity in cell models, suggesting that the modes of action reported for WFA could be connected by proteostasis modulation. Through this endeavor, we highlight how the parallel integration of medicinal chemistry with chemical biology offers a potent solution to one of natures’ intriguing molecular puzzles