The inhibitor-of-apoptosis proteins (IAPs) regulate programmed cell death by inhibiting members of the caspase family of enzymes. Recently, a mammalian protein called Smac (also named DIABLO) was identified that binds to the IAPs and promotes caspase activation. Although undefined in the X-ray structure, the amino-terminal residues of Smac are critical for its function. To understand the structural basis for molecular recognition between Smac and the IAPs, we determined the solution structure of the BIR3 domain of X-linked IAP (XIAP) complexed with a functionally active nine-residue peptide derived from the N terminus of Smac. The peptide binds across the third beta-strand of the BIR3 domain in an extended conformation with only the first four residues contacting the protein. The complex is stabilized by four intermolecular hydrogen bonds, an electrostatic interaction involving the N terminus of the peptide, and several hydrophobic interactions. This structural information, along with the binding data from BIR3 and Smac peptide mutants reported here, should aid in the design of small molecules that may be used for the treatment of cancers that overexpress IAPs.

Summary The dynamic and reversible acetylation of proteins catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) is a major epigenetic regulatory mechanism of gene transcription 1 associated with multiple diseases. While HDAC inhibitors are approved to treat certain cancers, progress on the development of drug-like HAT inhibitors has lagged 2. The HAT paralogs p300 and CBP (p300/CBP) are key transcriptional co-activators essential for a multitude of cellular processes and also implicated in human pathological conditions, including cancer 3. Current p300/CBP HAT domain inhibitors including natural products, 4 bi-substrate analogs (Lys-CoA) 5 and the widely utilized C646 6, 7 lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like p300/CBP catalytic inhibitor. We show the first high resolution (1.95Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 is acetyl-CoA competitive. A-485 selectively inhibited proliferation across lineage-specific tumor types, including several hematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen sensitive and castrate resistant prostate cancer and inhibited tumor growth in a castration resistant xenograft model. These results demonstrate the feasibility of selectively targeting the catalytic activity of histone acetyltransferases.

Inhibitor of apoptosis (IAP) proteins are overexpressed in many cancers and have been implicated in tumor growth, pathogenesis, and resistance to chemo- or radiotherapy. On the basis of the NMR structure of a SMAC peptide complexed with the BIR3 domain of X-linked IAP (XIAP), a novel series of XIAP antagonists was discovered. The most potent compounds in this series bind to the baculovirus IAP repeat 3 (BIR3) domain of XIAP with single-digit nanomolar affinity and promote cell death in several human cancer cell lines. In a MDA-MB-231 breast cancer mouse xenograft model, these XIAP antagonists inhibited the growth of tumors. Close structural analogues that showed only weak binding to the XIAP-BIR3 domain were inactive in the cellular assays and showed only marginal in vivo activity. Our results are consistent with a mechanism in which ligands for the BIR3 domain of XIAP induce apoptosis by freeing up caspases. The present study validates the BIR3 domain of XIAP as a target and supports the use of small molecule XIAP antagonists as a potential therapy for cancers that overexpress XIAP.

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The inhibitor-of-apoptosis (IAP) family of proteins, originally identified in baculoviruses, regulate programmed cell death in a variety of organisms. IAPs inhibit specific enzymes (caspases) in the death cascade and contain one to three modules of a common 70-amino-acid motif called the BIR domain. Here we describe the nuclear magnetic resonance structure of a region encompassing the second BIR domain (BIR2) of a human IAP family member, XIAP (also called hILP or MIHA). The structure of the BIR domain consists of a three-stranded antiparallel beta-sheet and four alpha-helices and resembles a classical zinc finger. Unexpectedly, conserved amino acids within the linker region between the BIR1 and BIR2 domains were found to be critical for inhibiting caspase-3. The absence or presence of these residues may explain the differences in caspase inhibition observed for different truncated and full-length IAPs. Our data further indicate that these residues may bind to the active site and that the BIR domain may interact with an adjacent site on the enzyme.

The molecular mechanism(s) that regulate apoptosis by caspase inhibition remain poorly understood. The main endogenous inhibitors are members of the IAP family and are exemplified by XIAP, which regulates the initiator caspase-9, and the executioner caspases-3 and -7. We report the crystal structure of the second BIR domain of XIAP (BIR2) in complex with caspase-3, at a resolution of 2.7 A, revealing the structural basis for inhibition. The inhibitor makes limited contacts through its BIR domain to the surface of the enzyme, and most contacts to caspase-3 originate from the N-terminal extension. This lies across the substrate binding cleft, but in reverse orientation compared to substrate binding. The mechanism of inhibition is due to a steric blockade prohibitive of substrate binding, and is distinct from the mechanism utilized by synthetic substrate analog inhibitors.

Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.

TRPA1 is an ion channel and has been proposed as a thermosensor across species. In invertebrate and ancestral vertebrates such as fly, mosquito, frog, lizard and snakes, TRPA1 serves as a heat receptor, a sensory input utilized for heat avoidance or infrared detection. However, in mammals, whether TRPA1 is a receptor for noxious cold is highly controversial, as channel activation by cold was observed by some groups but disputed by others. Here we attribute the discrepancy to species differences. We show that cold activates rat and mouse TRPA1 but not human or rhesus monkey TRPA1. At the molecular level, a single residue within the S5 transmembrane domain (G878 in rodent but V875 in primate) accounts for the observed difference in cold sensitivity. This residue difference also underlies the species-specific effects of menthol. Together, our findings identify the species-specific cold activation of TRPA1 and reveal a molecular determinant of cold-sensitive gating.