The prosurvival BCL-2 family protein BCL-X(L) is often overexpressed in solid tumors and renders malignant tumor cells resistant to anticancer therapeutics. Enhancing apoptotic responses by inhibiting BCL-X(L) will most likely have widespread utility in cancer treatment and, instead of inhibiting multiple prosurvival BCL-2 family members, a BCL-X(L)-selective inhibitor would be expected to minimize the toxicity to normal tissues. We describe the use of a high-throughput screen to discover a new series of small molecules targeting BCL-X(L) and their structure-guided development by medicinal chemistry. The optimized compound, WEHI-539 (7), has high affinity (subnanomolar) and selectivity for BCL-X(L) and potently kills cells by selectively antagonizing its prosurvival activity. WEHI-539 will be an invaluable tool for distinguishing the roles of BCL-X(L) from those of its prosurvival relatives, both in normal cells and notably in malignant tumor cells, many of which may prove to rely upon BCL-X(L) for their sustained growth.

Apoptosis is an important part of the host's defense mechanism for eliminating invading pathogens. Some viruses express proteins homologous in sequence and function to mammalian pro-survival Bcl-2 proteins. Anti-apoptotic F1L expressed by vaccinia virus is essential for survival of infected cells, but it bears no discernable sequence homology to proteins other than its immediate orthologues in related pox viruses. Here we report that the crystal structure of F1L reveals a Bcl-2-like fold with an unusual N-terminal extension. The protein forms a novel domain-swapped dimer in which the a1 helix is the exchanged domain. Binding studies reveal an atypical BH3-binding profile, with sub-micromolar affinity only for the BH3 peptide of pro-apoptotic Bim and low micromolar affinity for the BH3 peptides of Bak and Bax. This binding interaction is sensitive to F1L mutations within the predicted canonical BH3-binding groove, suggesting parallels between how vaccinia virus F1L and myxoma virus M11L bind BH3 domains. Structural comparison of F1L with other Bcl-2 family members reveals a novel sequence signature that redefines the BH4 domain as a structural motif present in both pro-and anti-apoptotic Bcl-2 members, including viral Bcl-2-like proteins. In higher organisms, programmed cell death (apoptosis) is a prominent feature of the response to viral infection. The central role of the Bcl-2 protein family in maintaining cell survival or driving apoptosis, thereby removing infected, damaged or unwanted cells, is reflected by the expression of sequence, structural and functional orthologues of Bcl-2 by certain viruses. 1 The Bcl-2-related proteins share the presence of one or more of four Bcl-2 homology (BH) domains in their primary sequences and act either to promote cell survival or to counter this. 2 Pro-survival family members such as mammalian Bcl-2, Bcl-x L , Bcl-w, Mcl-1 and A1 block apoptosis until their protective effect is countered by binding of proapoptotic BH3-only proteins, such as Bim, Bad or Noxa. 2,3 Pro-survival Bcl-2 proteins contain multiple BH domains, whereas the distantly related BH3-only proteins contain only the a-helical BH3 domain, which binds a receptor-like groove on the pro-survival proteins, thereby inactivating them. 4,5 Upon activation, pro-apoptotic Bax and Bak, which are essential for apoptosis to proceed, 6 oligomerize to cause organellar damage.The viral Bcl-2-like proteins, including those expressed by adenovirus, Kaposi sarcoma-associated herpesvirus, Epstein-Barr virus (EBV) and g-herpesvirus 68, are all required for successful viral propagation and/or persistence. However, other viruses express anti-apoptotic proteins that are unrelated by sequence to any known cell death regulator. These include the myxoma virus M11L, 7 cytomegalovirus vMIA 8 and vaccinia virus F1L 9 and E3L. 10 Despite the lack of sequence similarity, M11L adopts a Bcl-2-like fold. 11 Moreover, the structure of M11L in complex with the BH3 peptide from Bak revealed that the canonical BH3-binding groove is utilized in...

An attractive approach for developing therapeutic peptides is to enhance binding to their targets by stabilizing their α-helical conformation, for example, stabilized BimBH3 peptides (BimSAHB) designed to induce apoptosis. Unexpectedly, we found that such modified peptides have reduced affinity for their targets, the pro-survival Bcl-2 proteins. We attribute this loss in affinity to disruption of a network of stabilizing intramolecular interactions present in the bound state of the native peptide. Altering this network may compromise binding affinity, as in the case of the BimBH3 stapled peptide studied here. Moreover, cells exposed to these peptides do not readily undergo apoptosis, strongly indicating that BimSAHB is not inherently cell permeable.