A Screening of 10,240 NatureBank Fractions Identifies Nematicidal Activity in Agelasine-Containing Extracts from Sponges.

Nematode infections affect a fifth of the human population, livestock, and crops worldwide, imposing a burden to global public health and economies, particularly in developing nations. Resistance to commercial anthelmintics has increased over the years in livestock infections and driven the pursuit for new drugs. We herein present a rapid, cost-effective, and automated assay for nematicide discovery using the free-living nematode Caenorhabditis elegans to screen a highly diverse natural product library enriched in bioactive molecules. Screening of 10,240 fractions obtained from extracts of various biological sources allowed the identification of 7 promising hit fractions, all from marine sponges. These fractions were further assayed for nematicidal activity against the sheep nematode parasite Haemonchus contortus and for innocuity in zebrafish. The most active extracts against parasites and innocuous toward vertebrates belong to two chemotypes. High-performance liquid chromatography (HPLC) coupled with nuclear magnetic resonance (NMR) revealed that the most abundant compound in one chemotype is halaminol A, an aminoalcohol previously identified in a small screen against H. contortus. Terpene-nucleotide hybrids known as agelasines predominate in the other chemotype. This study reinforces the power of C. elegans for nematicide discovery from large collections and the potential of the chemical diversity derived from marine invertebrate biota.

Novel Kinetic Resolution of Thiazolo-Benzimidazolines Using MAO Enzymes.

The kinetic resolution of racemic 1H,3H-thiazolo[3,4-a]benzimidazoline (TBIM) heterocycles was achieved using E. coli whole cells expressing the MAO-N D11 enzyme. Several cosolvents were screened using TBIM 2a as the substrate. DMF was the best cosolvent, affording the pure enantiomer (+)-2a in 44% yield, 94% ee. The stereochemistry of TBIM was predicted by means of ab initio calculations of optical rotation and circular dichroism spectra. The reaction scope was investigated for 11 substituted (±) TBIM using an optimized protocol. The best yield and % ee were obtained for the nonsubstituted 2a. Among the substituted compounds, the 5-substituted-TBIM showed better % ee than the 4-substituted one. The small electron donor group (Me) led to better % ee than the electron-withdrawing groups (-NO2 and -CO2Et), and the bulky naphthyl group was detrimental for the kinetic resolution. Docking experiments and molecular dynamics (MD) simulations were employed to further understand the interactions between MAO-N D11 and the thiazolo-benzimidazoline substrates. For 2a, the MD showed favorable positioning and binding energy for both enantiomers, thus suggesting that this kinetic resolution is influenced not only by the active site but also by the entry tunnel. This work constitutes the first report of the enzymatic kinetic resolution applied to TBIM heterocycles.

Structure-Based Bioisosterism Design, Synthesis, Biological Evaluation and In Silico Studies of Benzamide Analogs as Potential Anthelmintics.

A recent screen of 67,012 compounds identified a new family of compounds with excellent nematicidal activity: the ortho-substituted benzamide families Wact-11 and Wact-12. These compounds are active against Caenorhabditis elegans and parasitic nematodes by selectively inhibiting nematode complex II, and they display low toxicity in mammalian cells and vertebrate organisms. Although a big number of benzamides were tested against C. elegans in high-throughput screens, bioisosteres of the amide moiety were not represented in the chemical space examined. We thus identified an opportunity for the design, synthesis and evaluation of novel compounds, using bioisosteric replacements of the amide group present in benzamides. The compound Wact-11 was used as the reference scaffold to prepare a set of bioisosteres to be evaluated against C. elegans. Eight types of amide replacement were selected, including ester, thioamide, selenoamide, sulfonamide, alkyl thio- and oxo-amides, urea and triazole. The results allowed us to perform a structure-activity relationship, highlighting the relevance of the amide group for nematicide activity. Experimental evidence was complemented with in silico structural studies over a C. elegans complex II model as a molecular target of benzamides. Importantly, compound Wact-11 was active against the flatworm Echinococcus granulosus, suggesting a previously unreported pan-anthelmintic potential for benzamides.

Increased sensitivity of an infrared motility assay for nematicide discovery.

Parasitic nematodes constitute a health problem for humans, livestock and crops, and cause huge economic losses to developing-country economies. Due to the spread of nematicide resistance, there is an urgent need for new drugs. C. elegans is now recognized as a cost-effective alternative for the screening of compound libraries with potential nematicidal activity, as parasitic organisms are hard to maintain under laboratory conditions. Here we describe an adaptation of a previously reported high throughput (HTP) infrared-based motility assay that leads to increased sensitivity. The modified assay uses L1 instead of L4 stage worms and matches the sensitivity reported by Burns et al. (2015) for the anthelmintic benzamides Wact11 and Wact11p. In addition, this method presents practical advantages over Burns et al. (2015) and other image-based protocols and provides a robust assay with a fast and simple readout ideal for HTP drug discovery.