Absolute Configuration of Acremoxanthone C, a Potent Calmodulin Inhibitor from <i>Purpureocillium lilacinum</i>

Bioassay-guided fractionation of an extract prepared from the culture medium and mycelium of <i>Purpureocillium lilacinum</i> allowed the isolation of two calmodulin (CaM) inhibitors, namely, acremoxanthone C (<b>1</b>) and acremonidin A (<b>2</b>). The absolute configuration of <b>1</b> was established as 2<i>R</i>, 3<i>R</i>, 1′<i>S</i>, 11′<i>S</i>, and 14′<i>R</i> through extensive NMR spectroscopy and molecular modeling calculations at the DFT B3LYP/DGDZVP level, which included the comparison between theoretical and experimental specific rotation, ³<i>J</i>_C,H, and ³<i>J</i>_H,H values. Compounds <b>1</b> and <b>2</b> bind to the human calmodulin (<i>h</i>CaM) biosensor <i>h</i>CaM M124C-<i>mBBr</i>, with dissociation constants (<i>K</i>_d) of 18.25 and 19.40 nM, respectively, 70-fold higher than that of chlorpromazine (<i>K</i>_d = 1.24 μM), used as positive control. Docking analysis using AutoDock 4.2 predicted that <b>1</b> and <b>2</b> bind to CaM at a similar site to that which KAR-2 binds, which is unusual. Furthermore, a novel, sensible, and specific fluorescent biosensor of <i>h</i>CaM, i<i>.</i>e<i>., h</i>CaM T110C-<i>mBBr</i>, was constructed; this device is labeled at a site where classical inhibitors do not interact and was successfully applied to measure the interaction of <b>1</b> with CaM. This is the first report of xanthone–anthraquinone heterodimers in species of <i>Paecilomyces</i> or <i>Purpureocillium</i> genera

(+)-Ascosalitoxin and Vermelhotin, a Calmodulin Inhibitor, from an Endophytic Fungus Isolated from <i>Hintonia latiflora</i>

Chemical investigation of the endophytic MEXU 26343, isolated from the medicinal plant <i>Hintonia latiflora</i>, yielded the known polyketide vermelhotin (<b>1</b>) and a new salicylic aldehyde derivative, namely, 9<i>S</i>,11<i>R</i>-(+)-ascosalitoxin (<b>2</b>). The structure and absolute configuration of the new compound were established through extensive NMR spectroscopy and molecular modeling calculations at the DFT B3LYP/DGDZVP level, which included the comparison between theoretical and experimental optical rotation values. In addition, chemical transformations of <b>2</b> yielded suitable derivatives for NOESY and ¹H–¹H NMR coupling constant analyses, which reinforce the stereochemical assignment. The potential affinity of <b>1</b> and <b>2</b> with (Ca²⁺)₄-<i>h</i>CaM in solution was measured using the fluorescent biosensor <i>h</i>CaM M124C-<i>mBBr</i>. The results showed that <b>1</b> bound to the protein with a dissociation constant (<i>K</i>_d) of 0.25 ± 0.04 μM, close to that of chlorpromazine (<i>K</i>_d = 0.64 ± 0.03 μM), a classical CaM inhibitor. The stoichiometry ratio of <b>1</b> to (Ca²⁺)₄-<i>h</i>CaM was 1:4, similar to other well-known CaM ligands

Comparison of Three Antihapten VHH Selection Strategies for the Development of Highly Sensitive Immunoassays for Microcystins

Owing to their reproducibility, stability, and cost-effective production, the recombinant variable domains of heavy-chain-only antibodies (VHHs) are becoming a salient option as immunoassay reagents. Recently, there have been several reports describing their application to the detection of small molecules (haptens). However, lacking the heavy-light chain interface of conventional antibodies, VHHs are not particularly apt to bind small analytes and failures are not uncommon. Here we describe the construction of a VHH phage display library against the cyanobacterial hepatotoxin microcystin LR and its selection using competitive panning and two novel panning strategies. The outcome of each strategy was evaluated by a large-scale screening using <i>in vivo</i> biotinylated nanobodies. The three methods selected for different nonoverlapping subsets of VHHs, allowing one to optimize the immunodetection of the toxin. The best results were obtained by promoting the isolation of VHHs with the slowest <i>k</i>_off (off-rate selection). Among these, the biotinylated nanobody A2.3 performed in ELISA with excellent recovery and high sensitivity, IC50 = 0.28 μg/L, with a limit of detection that is well below the most rigorous guidelines for the toxin. While it may be case-specific, these results highlight the importance of exploring different panning strategies to optimize the selection of antihapten nanobodies

Optimization of Antitrypanosomatid Agents: Identification of Nonmutagenic Drug Candidates with in Vivo Activity

Chagas disease, caused by <i>Trypanosoma cruzi</i> parasite, was described thousands of years ago. Currently, it affects millions of people, mostly in Latin America, and there are not suitable drugs for treating it. As an attempt to find appropriate drugs to deal with this problem, we report here on the design, synthesis, and characterization of 82 new compounds. Trypanosomicidal behavior in vitro showed more than 20 outstanding derivatives with anti-<i>Trypanosoma cruzi</i> activity. Furthermore, we studied the nonspecific toxicity against mammalian cells determining their selectivity and also performed mutagenicity studies. Proof of concept, in vivo studies, was conducted with two of the most promising derivatives (<b>77</b> and <b>80</b>). They were identified as candidates because they have (i) very simple and cost-effective syntheses; (ii) activity against different stages and strains of the parasite showing excellent in vivo behavior during the acute phase of Chagas disease; and (iii) neither nonspecific toxicity nor mutagenic activity

Acute kidney disease beyond day 7 after major surgery: a secondary analysis of the EPIS-AKI trial

Purpose: Acute kidney disease (AKD) is a significant health care burden worldwide. However, little is known about this complication after major surgery. Methods: We conducted an international prospective, observational, multi-center study among patients undergoing major surgery. The primary study endpoint was the incidence of AKD (defined as new onset of estimated glomerular filtration rate (eCFR) &lt; 60&nbsp;ml/min/1.73&nbsp;m2 present on day 7 or later) among survivors. Secondary endpoints included the relationship between early postoperative acute kidney injury (AKI) (within 72&nbsp;h after major surgery) and subsequent AKD, the identification of risk factors for AKD, and the rate of chronic kidney disease (CKD) progression in patients with pre-existing CKD. Results: We studied 9510 patients without pre-existing CKD. Of these, 940 (9.9%) developed AKD after 7&nbsp;days of whom 34.1% experiencing an episode of early postoperative-AKI. Rates of AKD after 7&nbsp;days significantly increased with the severity (19.1% Kidney Disease Improving Global Outcomes [KDIGO] 1, 24.5% KDIGO2, 34.3% KDIGO3; P &lt; 0.001) and duration (15.5% transient vs 38.3% persistent AKI; P &lt; 0.001) of early postoperative-AKI. Independent risk factors for AKD included early postoperative-AKI, exposure to perioperative nephrotoxic agents, and postoperative pneumonia. Early postoperative-AKI carried an independent odds ratio for AKD of 2.64 (95% confidence interval [CI] 2.21-3.15). Of 663 patients with pre-existing CKD, 42 (6.3%) had worsening CKD at day 90. In patients with CKD and an episode of early AKI, CKD progression occurred in 11.6%. Conclusion: One in ten major surgery patients developed AKD beyond 7&nbsp;days after surgery, in most cases without an episode of early postoperative-AKI. However, early postoperative-AKI severity and duration were associated with an increased rate of AKD and early postoperative-AKI was strongly associated with AKD independent of all other potential risk factors