Anacardic Acids from Knema hookeriana as Modulators of Bcl-xL/Bak and Mcl-1/Bid Interactions Charlotte Ge ́ ny, † Gwladys Rivie ̀ re, † Je ́ rome Bignon, † Nicolas Birlirakis, † Eric Guittet, † Khaljah Awang, ‡ Marc Litaudon, † Fanny Roussi,* ,† and Vincent Dumontet* ,† † Institut de Chimie des Substances Naturelles, CNRS-ICSN UPR2301, Universite ́ Paris-Saclay, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France ‡ Department of Chemistry, Faculty of Science, University Malaya, Kuala Lumpur 50603, Malaysia * S Supporting Information ABSTRACT: Proteins of the Bcl-2 family are key targets in anticancer drug discovery. Disrupting the interaction between anti- and pro-apoptotic members of this protein family was the approach chosen in this study to restore apoptosis. Thus, a biological screening on the modulation of the Bcl-xL/Bak and Mcl-1/Bid interactions permitted the selection of Knema hookeriana for further phytochemical investigations. The ethyl acetate extract from the stem bark led to the isolation of six new compounds, three acetophenone derivatives (1-3) and three anacardic acid derivatives (4-6), along with four known anacardic acids (7-10) and two cardanols (11, 12). Their structures were elucidated by 1D and 2D NMR analysis in combination with HRMS experiments. The ability of these compounds to antagonize Bcl-xL/Bak and Mcl-1/Bid association was determined, using a protein-protein interaction assay, but only anacardic acid derivatives (4-10) exhibited significant binding properties, with K i values ranging from 0.2 to 18 μM. Protein-ligand NMR experiments further revealed that anacardic acid 9, the most active compound, does not interact with the anti-apoptotic proteins Bcl-xL and Mcl-1 but instead interacts with pro-apoptotic protein Bid. T he Bcl-2 family of proteins, comprising both anti- and pro- apoptotic members, are key players in apoptosis. The anti- apoptotic proteins disable the pro-apoptotic ones by binding in a hydrophobic cleft through protein-protein interactions. The overexpression of anti-apoptotic proteins such as Bcl-xL and Mcl-1 plays a decisive role in cancer development and can be correlated with resistance to cancer therapeutics. 1 These proteins are considered to be challenging targets for the development of novel anticancer treatments, but it was shown that selective inhibition of Bcl-xL results in apoptosis “escape” through the Mcl-1 pathway. 2 Thus, identification of small molecules capable of binding to the hydrophobic cleft of both anti-apoptotic proteins Bcl-xL and Mcl-1, releasing the pro- apoptotic proteins such as Bax, Bad, or Bid, and ultimately restoring apoptosis, is a promising but challenging strategy in the fight against cancer. Recently, an extensive biological screening with 9000 plant extracts was conducted to investigate small molecules modulating Bcl-xL/Bak interactions, leading to the isolation of new active compounds such as meiogynin A 3 and kingianin G. 4 A second screening on Mcl-1/Bid interactions of the most active extracts on Bcl-xL/Bak interactions, along with 480 additional plant extracts led to the isolation of the dual inhibitors ferrugineic acids B, C, and J and a biologically active ethyl acetate extract from the Knema hookeriana Warb. (Myristicaceae). 5 The stem bark extract of this species displayed high-affinity binding to Bcl-xL and Mcl-1 (97% and 92% at 10 μg/mL, respectively) and prompted the search for secondary metabolites that regulate those anti-apoptotic proteins. Knema hookeriana Warb. (Myristicaceae) is a tree that can be found in Indonesia, Malaysia, Singapore, and Thailand. Traditionally, the leaves of this plant are used as a stomach remedy, and the sap is also useful for dying casting nets and cloths. 6 Additionally, this plant is used as a preservative surface coating material for valuable wooden, porcelain, and metallic wares. 6 Only one chemical investigation to find antinematodal components has been published. 6 However, other Knema Received: October 14, 2015 Article pubs.acs.org/jnp © XXXX American Chemical Society and American Society of Pharmacognosy A DOI: 10.1021/acs.jnatprod.5b00915 J. Nat. Prod. XXXX, XXX, XXX-XXX